Aquaculture Research: from Cage to Consumption

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Aquaculture Research: From Cage to Consumption

Research Programmes AQUACULTURE – Production of Aquatic Organisms (2000 – 2005)

Aquaculture Research: From Cage to Consumption

Editorial Committee: Magny Thomassen, Norwegian University of Life Sciences Roar Gudding, National Veterinary Institute Birgitta Norberg, Institute of Marine Research Leif Jørgensen, Nord-Trondelag Research Institute

The Fishery and Aquaculture Research Fund (FHF) has financed or co-financed a number of projects under the Aquaculture programme, and has provided support for the publication of this book. © The Research Council of Norway 2007 The Research Council of Norway P.O. Box 2700 St. Hanshaugen NO–0131 Oslo, Norway Telephone: +47 22 03 70 00 Telefax: +47 22 03 70 01 [email protected] www.forskningsradet.no/english

The publication may be ordered at: www.forskningsradet.no/publikasjoner (Norwegian-language site)

or telefax: +47 80 08 30 01

English-language editorial team: Carol B. Eckmann, Victoria S. Coleman and Darren McKellep Design: Making Waves as/Design et cetera AS Cover photos/illustrations: Vidar Vassvik, Kerstin Mertens/Samfoto, P.G. Kvenseth and Norwegian Seafood Export Council Printing: PDC Tangen Number of copies: 1,400 Oslo, March 2007 ISBN 978-82-12-02408-3 (printed version) ISBN 978-82-12-02409-0 (pdf)

Photo credits for chapter introductory pages: Introduction: Main photo: Norwegian Seafood Export Council. Small photos, from left: Turid Mørkøre, Turid Mørkøre, Victor Øiestad Quality, Slaughter, Transport: Main photo: Norwegian Seafood Export Council. Small photos, from left: Anders Kiessling, Turid Mørkøre, Turid Mørkøre Production: Main photo: Geir Lasse Taranger. Small photos, from left: Institute of Marine Research, Synnøve Helland/Lars Thomas Poppe, Arne Duinker Health: All photos: T. Poppe Feed, Nutrition, Feeding: Main photo: EWOS. Small photos, from left: EWOS, anonymous, Karl Lukens Selective Breeding and Genetics: Main photo: AquaGen. Small photos, from left: AquaGen, Kjell Ingebrigtsen, Atle Mortensen Technology: All photos: SINTEF The Environment: Main photo: Anne Ditlefsen. Small photos, from left: Svein I. Opdal, Karin Boxaspen, Kjell Merok Appendix: Main photo: Jon Solberg. Small photos, from left: T. Poppe, Geir Lasse Taranger, AquaGen Foreword

As part of the effort to redesign the Research duction of more species, and Norway is home Council of Norway’s funding instruments and to a sizeable equipment industry that supplies programme structure from 2000 onward, a new goods and services to both the domestic and research programme, Aquaculture – Production international markets. Norwegian aquaculture of aquatic organisms, was established by the re- represents tremendous assets in terms of search board of the then Division for Biopro- exports, jobs and maintenance of coastal settle- duction and Processing. This new programme ment patterns. would incorporate the activities formerly en- Research and development, along with the compassed by the programmes Production of optimism and entrepreneurial spirit of many salmonids, Marine species in production, Fish Norwegian companies, have been key elements and animal health, and parts of the PUSH pro- behind the success of Norway’s aquaculture gramme1. The new programme was also to in- industry. These will also be essential to future corporate user-driven aquaculture research, efforts to make the most of new opportunities which had previously been part of a separate and promote the sustainable growth and advan- programme for user-driven research. cement of the industry. Needless to say, consolidating the research This book is part of the final report from the activities from several programmes into one Aquaculture programme and summarises rese- new programme was a major challenge. Profes- arch findings from the period 2000–2005. sor Sigurd Stefansson of the University of Ber- During this period, the Aquaculture pro- gen headed a planning group that in February gramme allocated more than NOK 470 million 1998 began drafting a work programme for the for R&D projects at research institutes, univer- new research programme. The new programme sities, university colleges, companies and board for the new programme assumed its industry organisations. responsibilities in February 1999, chaired by Many authors from a variety of research groups Professor Magny Thomassen of the Norwegian and companies deserve thanks for their contri- University of Life Sciences (UMB). butions to this book. Many thanks go to the The programme has now been in operation for editorial committee, comprised of Magny Tho- six years, from 2000 through 2005, financed massen (UMB), Roar Gudding (National Vete- from the outset by the Ministry of Fisheries and rinary Institute), Birgitta Norberg (Institute of Coastal Affairs and the Ministry of Agriculture Marine Research) and Leif Jørgensen (Nord- and Food. Since 2001 it has also received fun- Trondelag Research Institute). Last but not ding from the yield of the Fund for Research least, Anne Ditlefsen (Senior Adviser, Rese- and Innovation. The Fishery and Aquaculture arch Council of Norway) deserves recognition Industry Research Fund (FHF) has been a key for her efforts in organising the texts and seeing partner to the programme since its establish- this book through to its printed, finished pro- ment in 2001, contributing financing both to duct. thematic areas and individual projects. Innova- tion Norway has also contributed by co-finan- Oslo, March 2007 cing programme activities. Norway is one of the world’s largest producers of Atlantic salmon today. Much effort is also being directed into commercialising the pro- Anne Kjersti Fahlvik, Director, Division for Strategic Priorities 1.Programme for developing and promoting sea ranching Research Council of Norway

Foreword 3 Contents

Aquaculture – Production of Aquatic Organisms by Magny Thomassen ...... 8

Thematic area: Quality, Slaughter, Transport High-quality Seafood Products based on Ethical and Sustainable Production ...... 28 by Ragnar Nortvedt, Marit Espe, Ingrid S. Gribbestad, Leif Jørgensen, Ørjan Karlsen, Håkon Otterå, Mia B. Rørå, Lars Helge Stien and Nils Kristian Sørensen

From Cage to Table ...... 45 by Anders Kiessling, Marit Bjørnevik, Magny Thomassen, Mia B. Rørå, Turid Mørkøre, Bjørn Roth, Ulf Erikson and Odd Jordheim

Thematic area: Production Reproductive Physiology in Cultured Cold-water Marine Fish...... 66 by Birgitta Norberg, Geir Lasse Taranger and Helge Tveiten

From Extensive to Intensive Production of Marine Fish ...... 80 by Anders Mangor-Jensen, Victor Øiestad, Terje van der Meeren, Ingrid Lein, Torstein Harboe, Ingegjerd Opstad, Gunvor Øie, José Rainuzzo, Jan Ove Evjemo, Kjell Inge Reitan and Bendik Fyhn Terjesen

Water Quality – Salmonids ...... 101 by Sigurd Stefansson, Grete Bæverfjord, R. Nigel Finn, Sigurd Handeland, Bengt Finstad, Sveinung Fivelstad, Torstein Kristensen, Frode Kroglund, Trond Rosten, Bjørn Olav Rosseland, Brit Salbu, Hilde Toften, Olav Breck, Ellen Bjerkås, and Rune Waagbø

Effects of Intensive Production with Emphasis on On-growing Production: Fast Growth, Deformities and Production-related Diseases ...... 120 by Trygve T. Poppe, Grete Bæverfjord and Tom Hansen

A New Boost for the Norwegian Shellfish Sector ...... 136 by Stein Mortensen, Øivind Strand, Arne Duinker, Sissel Andersen and Tore Aune

Thematic area: Health New Diseases – Phenomena Developing into Problems ...... 156 by Atle Lillehaug and Aud Skrudland

Virulence Mechanisms...... 172 by Øystein Evensen, Nina Santi, Ann-Inger Sommer and Siri Mjaaland

The Fish Immune System ...... 185 by Børre Robertsen, Ivar Hordvik and Trond Jørgensen

4 Aquaculture Research: From Cage to Consumption Thematic area: Feed, Nutrition, Feeding Nutritional Biology in Farmed Fish ...... 200 by Olai Einen, Henriette Alne, Barbara Grisdale-Helland, Ståle J. Helland, Gro-Ingunn Hemre, Bente Ruyter, Ståle Refstie and Rune Waagbø

Nutritional Aspects – Marine Fish Larvae ...... 217 by Kristin Hamre, Ivar Rønnestad, José Rainuzzo, Yoav Barr and Torstein Harboe

Feed Resources – Feed Technology ...... 235 by Mette Sørensen, Rune Waagbø and Rolf Erik Olsen

Metabolism and Uptake of Carotenoids in Farmed Fish...... 252 by Bjørn Bjerkeng, Trine Ytrestøyl and Rolf Erik Olsen

Thematic area: Selective Breeding and Genetics Selective Breeding and Genetics – Atlantic Salmon...... 268 by Bjarne Gjerde, Anna Sonesson, Arne Storset and Morten Rye

Breeding and Genetics – New Species ...... 285 by Kjersti Turid Fjalestad, Svein-Erik Fevolden, Knut Jørstad and Ingrid Olesen

Thematic area: Technology Aquaculture Technology ...... 304 by Egil Lien, Leif Magne Sunde and Kjell Midling

Thematic area: The Environment Salmon Lice: Importance, Problem and Treatment ...... 322 by Karin Boxaspen, Peter Andreas Heuch, Pål Arne Bjørn, Bengt Finstad, Petter Frost and Kevin Glover

How Much Aquaculture Can the Norwegian Coast Tolerate? ...... 338 by Arne Ervik, Jan Aure and Pia Kupka Hansen

Genetic Interactions...... 347 by Øystein Skaala, Knut E. Jørstad and Reidar Borgstrøm

Appendix Members of the Aquaculture Programme Board...... 366 Publications ...... 367 Projects ...... 368

Contents 5 Introduction

■ Aquaculture – Production of Aquatic Organisms Programme board chair Magny S. Thomassen, Norwegian University of Life Sciences

Aquaculture – Production of Aquatic Organisms

In 1999, the Research Council’s various initiatives in the sphere of aquaculture of Nor- way were consolidated under a single research programme, Aquaculture – Production of aquatic organisms. The programme has been in operation for six years (2000–2005), and the time has come for the programme board to summarise the programme’s activities and – most importantly – to present the research findings from some 400 projects that received a total of NOK 470 million in funding. The programme has been financed by the Ministry of Fisheries and Coastal Affairs, the Ministry of Agriculture and Food, and the Fund for Research and Innovation. It has been a key instrument in the effort to follow up activities in marine research, which was defined as a thematic priority area in Report No. 39 to the Storting (1998–99) Research at the beginning of a new era. The Fishery and Aquaculture Industry Research Fund (FHF, which was established in 2001) has financed or cofinanced a number of projects. The Aquaculture programme has had a budget of just over NOK 470 million available for allocation during the programme period. During the same period, the programme board has received and assessed grant applications totalling some NOK 2.3 billion. Funding was thus allocated for roughly 20 per cent of the application mass, meaning that a great many proposals for interesting, scientifically sound projects went unfunded. How far have we come? The articles presented in this book indicate that a good deal of progress has been made. They offer valuable, and sometimes surprising, new knowledge, in relation to basic as well as applied fields. It is also important to note that many of the objectives set out by the programme board in 2000 have been met. How- ever, new questions are constantly emerging, reminding us that there is still much to be done. Aquaculture researchers will not lack challenges in the years ahead!

8 Aquaculture Research: From Cage to Consumption 145x100//Kap01-fig01.eps Programme board at work, from left to right: Oddbjørn Larsen, Birgitta Norberg, Leif Jørgensen, Aud Skrudland, Frode Meland, Magny Thomassen, Rolf Giskeødegård, Sigurd Stefansson, Børre Robertsen, Morten Lund and Ellen Gjøsteen. (Kari Morvik and Roar Gudding were not present.) (Photo: Siw Ellen Jacobsen)

Background and history 2000. The action plan has been updated on an As part of an overall effort to restructure fund- annual basis. ing instruments and research programmes starting in the year 2000, the research board of the The Aquaculture programme has primarily tar- then Division for Bioproduction and Processing geted universities, university colleges, research decided in December 1997 to establish a new institutes, companies and industry organisa- research programme, Aquaculture – Production tions. The programme was designed to encom- of aquatic organisms. The programme would pass the entire value chain, from basic research incorporate research activities formerly includ- and applied research, to user-driven research ed in the programmes Production of salmonids, that meets the needs of the production and sup- Marine species in culture, Health of fish and ply industries. animals and parts of the Programme for the De- velopment and Encouragement of Sea Ranching The Aquaculture programme has been financed (PUSH). User-driven projects previously organ- by the Ministry of Fisheries and Coastal Affairs ised under a separate programme were also to (FKD) and the Ministry of Agriculture and be incorporated. Food (LMD), as well as the Fund for Research and Innovation. The Fishery and Aquaculture The Aquaculture programme was launched in Industry Research Fund (FHF) has also contrib- 2000 and concluded in 2005. Its programme uted by financing or co-financing a number of board was appointed by the Research Council, projects. The Aquaculture programme has had a and included observers from Innovation Nor- budget of just over NOK 470 million available way, FHF and the Ministry of Fisheries and for allocation during the programme period. Coastal Affairs. The programme board assumed The annual total has risen from some NOK 60 its responsibilities in January 1999, and put to- million in 2000 to NOK 90 million in 2005, pri- gether an action plan for 2000, which was ap- marily due to increased funding from the Fund proved by the division research board and for Research and Innovation and increased served as the basis for a call for proposals for project support from FHF.

Introduction 9 Mill. NOK FKD 100 LMD FUND 80 FHF FHF 60 FUND 40 LMD 20 FKD

0 2000 2001 2002 2003 2004 2005

Figure 1: Overview of annual (figure to the left) and total allocations to the Aquaculture programme. (FKD – The Ministry of Fisheries and Coastal Affairs, LMD – The Ministry of Agriculture and Food, Fund – Fund for Research and Innovation, FHF – The Fishery and Aquaculture Industry Research Fund)

Assessment of applications and salmonid projects and less than 40 per cent to allocation of research funds marine species projects. Towards the end of the The annually updated action plans have formed period, some 30 per cent was allocated to the basis for the calls for proposals. In general, salmonid projects, 55 per cent to marine species separate calls for proposals have been issued for projects and the rest to non-species-specific researcher projects and user-driven projects. projects. Atlantic salmon has been given high The programme board has evaluated over 1,000 priority throughout the programme period. applications totalling some NOK 2.3 billion. Among the marine species, focus has been on About 20 per cent of the grant proposals re- Atlantic halibut (Hippoglossus hippoglossus), ceived funding. wolffish (Anarcichas spp.), shellfish and cod (Gadus morhua) – with particular attention In keeping with a recommendation from the Re- given to the latter towards the end of the pro- search Council administration, applications gramme period. were assessed in groups under the various sub- programmes. The activity level of all the sub- Projects have been assessed on the basis of sci- programmes has met the level stipulated in the entific merit, relevance to programme objec- action plan throughout the entire programme tives and commercial benefit. Importance has period. Figure 2 illustrates the proportional dis- also been attached to the inclusion of a recruit- tribution of funding among the individual sub- ment component and national and international programmes at the programme start in 2000. cooperation. External referees have been em- This distribution has largely been maintained ployed in the process of assessing the grant pro- throughout the programme period. The allocat- posals. The programme board has strictly ed funds have been further divided between re- adhered to the Research Council’s guidelines searcher projects and user-driven projects, as for impartiality and confidence. well as between projects on salmonids and projects on marine species. The distribution share between salmonids and marine species Have our research objectives has varied somewhat during the programme pe- been met? riod. At the beginning of the period, more than The Aquaculture programme was designed to 60 per cent of the funding was allocated to cover the entire value chain, and has thus en-

10 Aquaculture Research: From Cage to Consumption 1. Quality in Production 2. Slaughter, Transport and Distribution

3. Production and Operations 4. Health and Disease

5. Feed, Nutrition, Feeding, Feed Techn. 6. Breeding and Genetics

7. Technology and Equipment 8. The Environment

9. Diverse

145x100//Kap01-fig01.eps Figure 2: Proportional distribution of programme allocations by sub-programme (2000). compassed a very broad range of thematic ar- learned about handling-related stress and seda- eas, from genetics and breeding, keeping of tion procedures – and now the aquaculture in- broodstock and fry, and production of slaugh- dustry is rapidly implementing pre-rigor ter-sized fish, to health, the environment, nutri- filleting technology. A number of basic process- tion, and production technology, as well as es of sexual maturation and development of transport, slaughter, and quality control to meet eggs and germ cells in farmed fish have been market demands for product quality. The further elucidated, and the importance of proper projects were also intended to explore both ba- feeding regimens for the normal development sic and applied research questions. In retro- of halibut larvae has been recognised. Substan- spect, this was a formidable task, particularly tial progress has been made with regard to the given the limited financial framework within interplay between the aquatic environment and which the programme board and researchers the normal development of farmed fish, partic- were conducting their efforts. Nevertheless, ularly during the early life stages. The life cycle much has been accomplished during these six of salmon lice has been investigated closely, years. In the area of fish health and disease, new and recent research findings indicate that it may biotechnology methodologies have enhanced now be possible to develop a vaccine against the basic understanding of infectious agents, the this parasite. immune system of fish, and fish’s interaction with the environment. In the area of nutrition, These are merely a few examples of the consid- new knowledge about alternative feed ingredi- erable knowledge that has been generated dur- ents has been acquired. Of special importance is ing the programme period. Does this represent enhanced knowledge about the biological pos- enough to claim that the programme has sibilities and limitations of more extensive use achieved its objectives? It is hard to say. Many of feed ingredients for the most important questions remain unanswered. However, the ac- aquaculture species. Compared to six or seven tion plan for the programme has set a series of years ago, we now have a far better understand- verifiable objectives to provide a method of as- ing of the nutritional requirements of cod, its re- sessing the performance of the programme action to environmental conditions, and factors board and the researchers. The following sec- affecting product quality. A great deal has been tions will examine these in greater detail.

Introduction 11 Sub-programme 1: products. Some knowledge had been gained Quality in Production about the connections between treatment before The primary objective of this sub-programme slaughtering and important quality parameters was to strengthen communication between the in salmon and rainbow trout, such as fat content aquaculture industry and consumers by fostering and colour. Still, communication with different a common understanding of the term “quality” markets and an understanding of their quality and of the various quality parameters. The sub- criteria were somewhat lacking. Projects programme also sought to expand the knowledge funded during the programme period have base to improve control of product quality given these aspects far more attention; there is throughout production phase, and to develop bet- now an awareness of customers’ increasing ter measurement techniques and instruments for interest in fish welfare, ethics and food safety. research, quality sorting and grading. Cod emerged as a highly promising and important new farmed species, and focus was placed Verifiable objectives: on questions regarding quality parameters and • To have established which traits most possibilities for improving cod quality in the strongly characterise “quality” in Norwe- production phase. Factors such as colour, tex- gian aquaculture’s main species. ture and moisture retention have been identified • To have acquired knowledge for signifi- as vitally important, and ways to influence these cantly reducing the number of production during the production and slaughter phases has fish with problems of internal quality. been described to some extent. • To have generated knowledge for using non-destructive methods to sort fish to During the programme period, questions related avoid at least one important problem of to internal quality have mainly focused on defor- internal quality. mities. The level of knowledge in this area has risen significantly. Decreasing water temperature At the programme start, there was already a in the hatching and start-feeding periods and greater understanding in the aquaculture indus- controlling water quality have resulted in a sig- try of the importance of producing high-quality nificant reduction of deformity problems in salmon and rainbow trout. However, these problems have been observed in cod farming, and research in this area is only now being initiated.

Research into the use of near infrared (NIR) spectroscopy and picture-analysing techniques has shown promising results in connection with individual sorting of farmed salmonids based on fat content and colour, both in vivo and online at slaughter. The possibility of in vivo measurement of these quality characteristics is considered especially promising for future control of quality.

145x100//Kap01-fig01.eps Picture analyses are now used for rapid measurement of Performance: Good to very good. fillet colour. (Photo: Photofish)

12 Aquaculture Research: From Cage to Consumption Sub-programme 2: Slaughter, Systems for transferring fish from well-boats Transport and Distribution have been developed, resulting in reduced The objective of this sub-programme was to ac- handling stress by keeping the water level in the quire knowledge making it possible to develop boat constant. At slaughter the transfer of fish is new methods and improved technology for han- done either by pumping or transfer to net pens. dling and transport of important aquaculture Some knowledge about the effects of the new species. Special focus was placed on fish wel- handling methods on stress and product quality fare and high product quality. has been acquired, but there is still a great need for more research in this area. Verifiable objectives: • To have facilitated knowledge for desig- The importance of low water temperatures and ning effective and gentle transport solu- reduced stress before slaughter for the develop- tions for aquaculture species. ment of rigor and the quality of salmon fillets • To have clarified the effects of handling has been well documented during the pro- before slaughter and new slaughter met- gramme period. It has been shown that optimal hods on fish welfare, stress and product handling postpones by several hours the pro- quality. cesses in muscle tissue that lead to muscle rigor, • To have clarified the effects of temperature making it possible to fillet the salmon shortly and different packaging methods on pro- after slaughter. Such pre-rigor filleting has duct quality. already been implemented by large number of companies in the industry, creating possibilities of producing new super-fresh products of high quality.

Research questions relating to packaging methods lie in the interface between the Aquaculture programme and several other research programmes at the Research Council. Projects financed in collaboration with two other programmes have shown that package design significantly influences product sales. Cus- tomers like to see the core product before buying it. Much more research remains to be done in this area, which is considered very important to the aquaculture industry.

Performance: Medium-good to good.

145x100//Kap01-fig01.eps Traditional method of measuring rigor. (Photo: Turid Mørkøre)

Introduction 13 Sub-programme 3: Production and Operations A key objective of this sub-programme was to acquire more basic knowledge about the biology of salmonids in both freshwater and seawater as a foundation for efficient and safe production of pre-smolt, smolt and slaughter-sized fish of good quality. An additional focus was to enhance the knowledge base for the keeping of broodstock and fry of marine species in cultiva-

tion. Two other objectives were to increase the 145x100//Kap01-fig01.eps Continuous measurement of muscle pH in farmed understanding of how the water environment af- halibut. (Photo: Turid Mørkøre) fects the organism, and to describe in more detail the causes of production-related illnesses in intensive aquaculture systems. Lack of knowledge has, to a great extent, led to a trial and error approach to influencing sexual Verifiable objectives: maturation in culture and to feeding, feed com- • To have acquired a better quality of eggs position, the general handling of broodstock, and fry based on increased knowledge and seasonally independent spawning using about broodstock of important species. light treatment. Projects that aimed to generate • To have acquired improved knowledge about basic knowledge about the morphological, the influence of the environment on early life molecular and physiological changes through- stages and its consequences for further de- out maturation have therefore been given prior- velopment and growth in juvenile stages. ity. Genes important to the maturation process • To have generated knowledge that may yield have been identified in salmon, cod and halibut, improved growth without increased sexual and the distribution of different hypophyseal maturation and other quality problems. hormones has been described. The development • To have improved the welfare of cultured of germ cells has been studied in detail in male species through greater knowledge about fish during normal and light-manipulated matu- physiology and behaviour in relation to ration. High water temperature (12–14 °C) has new, modern systems for aquaculture and been shown to have negative effects on spawn- production strategies and methods. ing and gametal quality in cod and salmon. To • To have generated knowledge that may re- secure optimal content of nutritional com- duce the occurrence of production illnesses pounds in the eggs, a broodstock feed for hali- related to intensive production systems. but was developed containing higher levels of • To increase survival rates of marine larvae vitamin C, specific B-vitamins and fatty acids, from start-feeding to fry with normal pig- resulting in better egg quality. Halibut brood- mentation and complete morphological stock with high feed consumption produced development. groups of eggs with higher fecundity and sur- • To have generated knowledge that can vival, and thus a greater potential for yielding a shorten the period that marine larvae need higher number of halibut larvae of high quality. live feed.

14 Aquaculture Research: From Cage to Consumption Varying supply and quality of eggs is, however, ing fish invest more in gonadal development still a bottleneck for extensive halibut farming. than in body growth, resulting in decreased muscle quality, especially in male fish. The con- The interaction between the organism and its nections between growth and sexual maturation surroundings is one of the most important and in salmon and cod, and how environmental pa- challenging areas in biological research, both rameters such as light can stop or postpone mat- nationally and internationally. Development in uration, have been a priority area in the research fish is, even more than in mammals, influenced programme. In salmon farming, 24-hour con- by factors in the environment, and the appear- tinuous light treatment is regularly used to stop ance of fish can show a great variation between maturation and improve growth. Family back- individuals of almost identical genetic background and feeding also have an effect on the ground. Improved knowledge in this area is of number of maturing fish, as does probably an special importance to the aquaculture industry, array of other factors. In cod, light manipulation which is striving to create conditions that secure in net pens at sea is only partly effective; matu- normal development of functions vital to fur- ration is delayed but not stopped completely. ther growth. During the programme period, the Research has focused on understanding the pu- effects of environmental factors such as light, berty of cod. Effects of light intensity as well as temperature and water quality on organ devel- light colour have been studied, but much knowl- opment and function in salmon and marine spe- edge is still lacking about the physiological cies have been described, indicating negative mechanisms involved. effects of extremely rapid development in early life stages. Key genes for the development and A series of new physiological and molecular function of organs such as the muscles, skele- tools have been developed during the pro- ton, gills and heart have been partially de- gramme period, and will be of great help in scribed and studied, yielding important new learning more about the complex interplay of understanding. Deformities of the skeleton and factors that determines the onset of puberty heart seem to be influenced by both temperature among cultivated fish. and water quality, and the quality of salmon smolt is significantly affected by light condi- During the programme period, interest has in- tions. A good smolt can easily be destroyed by creased substantially for the welfare of animals even a brief deterioration in water quality. Mus- in general and fish in particular. This is a some- cle formation in fish is strongly influenced by what new and challenging research area, as environmental factors; consequently, the basis little basic knowledge exists. Still, questions are for later muscle growth, fillet yield and quality being debated about the ability of fish to feel is formed during the early life stages. A proper pain and to suffer, and how to evaluate the wel- physical/chemical environment is critical to fare of fish in culture. In spite of great challeng- normal development of the salmon brain. es, interesting results have been obtained during the programme period. Several factors related Rapid growth is a prerequisite for an economi- to cultivation threaten the welfare of fish, but cally sound aquaculture industry, but it often re- much more is now known about the effects of sults in more fish becoming sexually mature water quality, feeding, social interactions and before reaching optimal slaughter size. Matur- infections. Indicators that fish are thriving in

Introduction 15 their cultivation environment are high survival Sub-programme 4: Health and rate, optimum growth and good health. Welfare Disease is further connected to natural behavioural The objectives of this sub-programme were to needs and to what extent these can be met in acquire more knowledge about the basic mech- culture. Research has also added to our under- anisms for development of disease, as well as standing of fish stress physiology and behavi- about prevention and treatment of diseases in our. Good behavioural indicators of fish welfare salmonids and the most important marine spe- will be essential in putting knowledge to practi- cies. Focus was also placed on developing cal use; this important research topic is just be- methods and tools to improve diagnosis and ginning to emerge. preventive measures.

Proper feeding regimens have been shown to be Verifiable objectives: of key importance in the development of halibut • To have generated knowledge of import- larvae. Meal feeding in place of a continuous ance for controlling the spread of new feed supply resulted in good pigmentation and diseases. less feed loss. Eye migration in flatfish is relat- • To have gained expertise to improve con- ed to nutrition, and during the programme trol of viral diseases. period it has been shown that Artemia enriched • To have acquired knowledge that secures with nutrients over a longer period of time had continued low use of antibiotics. significant positive effects, probably related to • To have procured knowledge that reduces higher protein content and lower fat content. the use of anti-lice chemicals. Research on nutritional needs and digestibility • To have achieved at least one breakthrough have been prioritised, resulting in an enhanced in research on fish health and disease. knowledge base for developing formulated dry feed that can enable the industry to partially or In an attempt to “strike at the root of the evil” totally replace live feed. However, more re- there has been significant research activity search is needed before this becomes a reality. involving new diseases and diseases with somewhat limited spread. This has given the industry Performance: Very good. better diagnostic tools for infectious salmon anaemia (ILA), pancreas disease (PD), and heart and skeletal muscle inflammation (HSMB). New knowledge has also improved the basis on which management authorities take decisions and action – during outbreaks of the viral disease ILA, for example – as well as enhanced understanding of predisposing factors for outbreaks of the viral disease infectious pancreas necrosis (IPN).

At the programme start in 2000, the use of antibiotics in Norwegian salmon farming had already been greatly reduced and has remained

16 Aquaculture Research: From Cage to Consumption low ever since. The substantial growth in cod Sub-programme 5: Feed, farming has been accompanied by an increase Nutrition, Feeding and Feed in use of antibiotics, which the industry has not Technology yet been able to prevent. There is a great need The objectives of this sub-programme were to for more research in this area. We must avoid acquire basic knowledge in nutritional biology the kind of development in antibiotics use that as a foundation for developing feeds that pro- salmon farming underwent in the 1980s. vide cost-effective growth, normal development and defined quality, as well as safe production Research on salmon lice has been a prime topic of high-quality eggs and fry. In addition, focus during the programme period and has been of was placed on acquiring more knowledge con- major importance for the development of more cerning possibilities and limitations in the use effective treatments and possibilities for testing of alternative feed resources. resistance development. General knowledge about the salmon louse and its mechanisms of Verifiable objectives: infestation has delivered good results in the • To have acquired new knowledge about combating of this parasite. Research results the need for micro-nutrients in fish in have further raised hopes for the development intensive production. of vaccines against salmon lice. • To have enhanced existing knowledge in order to reduce pigmentation-related costs Research today is characterised by knowledge by 10 per cent. building that advances step by step rather than • To have cultivated expertise for control- by huge, revolutionary breakthroughs. Never- ling colouration in order to satisfy highly- theless, the detection of a new unknown virus as demanding markets. the basis for HSMB, as well as the very solid • To have acquired knowledge that can pro- characterisation work on the ILA virus done by vide a basis for selecting new ingredients Norwegian research groups may be counted as for feed production. an important breakthrough for disease preven- • To have acquired knowledge about formu- tion in aquaculture. The results provide a good lated feed for marine species that allows basis for developing vaccines against these dis- for commercial production. eases. Another milestone in the “Health and • To have cultivated expertise for reducing Disease” sub-programme is the identification of the feed coefficient. high-virulence and low-virulence varieties of the IPN virus. This may help in reducing the Classical estimations of the need for micro- loss of salmon due to IPN in future. nutrients have not been a focus for research during the programme period. Rather, studies of Performance: Good to very good. nutrient requirements have been related to specific problems. As an example, the need for phosphorous and zinc has been studied in salmon in connection with studies on bone deformities. The need for vitamin C for halibut fry has been studied. Results from other studies have shown that the need for vitamin A for hal-

Introduction 17 ibut fry can partly be met by metabolic transfor- ed, funded by the programme as well as other mation of cantaxanthin. Classical studies of sources, and have resulted in new knowledge. nutrient requirements are generally difficult to Effects of the use of various vegetable oils have perform on marine fry since enriching live feed been studied in detail, with special focus on the is difficult. A newly developed short but inten- fatty acid profile of the products. It has been sive enriching method will probably be very shown that a nutritionally unhealthy reduction helpful. Copepods have been shown to be good in the content of the n-3 fatty acids EPA and feed, and in studies of other live feeds in con- DHA that occurs with vegetable oil feeding can nection with the need for micro-nutrients, the generally be reversed by using marine oils dur- content of copepods is used as a positive refer- ing the last period before slaughter. The use of ence. other marine sources, such as Calanus finmar- cicus and krill, has been tested with promising There is still a need for pigment additives in results, as has the use of protein grown on Nor- salmon feed, which represents a significant por- wegian natural gas resources. These offer the tion of total production costs. Research activity industry new possibilities in future, but there is in this area has not been extensive during the still a great need for more research. programme period. Nevertheless, studies financed by the programme and by the industry During the programme period, new knowledge itself have yielded results that clearly indicate about the nutritional requirements and digestion that the total amount of pigment used through- of marine larvae was acquired, increasing the out the life cycle can be significantly reduced. possibility of developing formulated dry feed These studies have been built on knowledge ac- that can completely or partially replace live quired in other, more basic research projects feed. An important problem is the high leakage that focused on mechanisms for pigment uptake of nutritional compounds due to the very small and storage. A reduction in feed costs of 10 per feed particle size. There have been interesting cent or more seems within reach. The new new developments in this area, particularly with knowledge will allow pigmentation to be con- regard to different encapsulation techniques. trolled more closely, in order to satisfy market preferences. Feed coefficient (amount of feed per kg growth) has great influence on the economics of produc- At the programme start in 2000, it had already tion. In spite of this, the coefficient has not been recognised that the limited supply of ma- changed in commercial salmon farming in re- rine feed ingredients, especially marine lipids, cent years, possibly because cheaper feed with would be a factor curbing further growth of the a higher feed coefficient has been chosen rather aquaculture industry. Focus on this particular that more expensive feed that lowers the coeffi- problem increased significantly during the pro- cient. Research on optimising feed composition gramme period, with more and more resources for a further reduction in feed coefficient has being funnelled into research on use of alterna- not been given high priority during the pro- tive feed components from both land and sea. gramme period. The programme board put together a major report on possibilities and research needs (see Performance: Reduction in feed coefficient, Other activities). Several projects were initiat- poor; otherwise good to very good.

18 Aquaculture Research: From Cage to Consumption Sub-programme 6: Breeding As mentioned above, the interest in cod as a new and Genetics aquaculture species was renewed during the pro- Important objectives in this sub-programme gramme period, and a significant portion of the were to acquire knowledge to form the basis for research activity within this sub-programme was breeding programmes for new aquaculture spe- directed toward enhancing knowledge about cod. cies, as well as to further develop and improve ef- Systems were developed for secure production ficacy of existing programmes. Focus was also of a high number of full and half-sib families, an placed on creating new measurement techniques important basis for the start of breeding pro- and generating knowledge about how rapid de- grammes. Genetic parameters for a limited num- velopments in gene technology can be exploited. ber of traits were calculated, and genotype- environment interaction on the family group and Verifiable objectives: on strain level (coastal cod/North-east Arctic • To have acquired knowledge on which to cod) were evaluated and quantified. The research base new breeding programmes for hal- performed during these years has created the ba- ibut. sis for breeding programmes on cod, which have • To have built expertise for breeding at least already been initiated. one species in addition to halibut. • To have formed the basis for implementing Genetic markers tightly coupled to genes of a knowledge about genetic markers. quantitative trait are called Quantitative Trait • To have described the genetic basis for Loci (QTL). Finding such markers may enable increasing resistance against disease and marker-assisted selection. For salmon, genetic parasites. markers coupled to resistance against ILA have • To have procured information on non- been found, and potential markers for IPN and additive genetic variation. fillet colour are being studied. A basis for implementing such genetic markers in breeding At the programme start in 2000, greatest inter- programmes has been established through de- est was shown in halibut, in addition to salmo- velopment of statistical models and methods. nids, and the establishment of a breeding programme was expected within a short time. But the research in halibut farming faced challenges greater than foreseen, especially regarding fry production. At the same time, there was a renewed interest in cod farming; thus research activities in breeding of halibut slowed unexpectedly. Nevertheless, knowledge has been acquired during the programme period, especially in connection with the evaluation of existing family material. A plan on how to establish and run a family-based breeding programme has also been made, but such a programme has

145x100//Kap01-fig01.eps not yet been established. Programme board visiting fry cultivation facilities in Vigo, Spain. (Photo: Victor Øiestad)

Introduction 19 A significant genetic variation in resistance Sub-programme 7: Technology against several diseases has been documented and Equipment through controlled infection tests, and breeding The primary objective of this sub-programme programmes on salmon now include selection was to acquire knowledge that enables person- against furunculosis, ILA and IPN. In salmon, a nel in the Norwegian aquaculture industry to high genetic correlation exists between number develop technology and equipment reducing the of lice per fish during controlled and natural in- number of escaped farmed fish, improve work- fection. Statistical methods for obtaining more ing conditions and ensure cost-effective and reliable genetic parameters for disease resis- environment-friendly production, giving Nor- tance have been developed. wegian industry a competitive advantage in export markets. By the crossing of different river strains of salmon, low non-additive genetic variation in Verifiable objectives: growth had already been discovered, while a • To have gained knowledge reducing es- relatively high variation had been seen between capees by 50 per cent. fish within one population. For other traits such • To have developed land and sea-based far- variations have not been studied. During the ming systems for marine species. programme period, no studies on this topic have • To have developed technology for a com- been funded, and it is not known how such non- plete production line for marine fry that sa- additive genetic variation can be used in breed- tisfies the requirements of fry regarding ing programmes for fish. water quality and tank environment. • To have adapted Norwegian technology Performance: Non-additive genetic variation, for at least two warm-water species. poor; otherwise good to very good. • To have developed technology for separation of particulate material and waste handling to reduce discharged waste by 80 per cent compared to traditional systems.

Due to limited economic resources, activity within this sub-programme has been lower than planned. But activities related to development of technology have been carried out under several of the other sub-programmes. With respect to research on fry production, several projects have focused on technology and equipment for production of live feed, removing particulate material and cleaning of water in tanks for larvae and fry. In user-driven projects these technologies have also been utilised for warm-water species. Furthermore, research has been performed on particle separation and waste handling. In land- based production systems, the objective of an

20 Aquaculture Research: From Cage to Consumption 145x100//Kap01-fig01.eps Programme board studying mussel farming in Spain. (Photo: Victor Øiestad)

80 per cent reduction in particulate material in the wastewater has been achieved. Sub-programme 8: The Environment With knowledge gained from the offshore in- The objectives of this sub-programme were to dustry, work has been done on developing new acquire knowledge about the interactions be- solutions for seawater aquaculture construc- tween farmed and wild fish and to gather infor- tions able to withstand large waves and tough mation resulting in reducing negative effects conditions, while at the same time providing such as interactions between farmed and wild fish with a proper environment. The construc- salmon and spreading of salmon lice. Another tions also offer a safe working environment. To objective was to gain information that could be able to design such installations and to eval- help to reduce the effects of farming on the ma- uate their tolerance limits for waves and cur- rine environment, while improving the choices rent, methods have been developed for made regarding farm locations. computer-based calculations. The projects have shown that it is important to more realistically At start of the programme period, the focus was describe the wave movements by realising mainly on acquiring more knowledge about the three-dimensional effects and that waves have sustainability of farm locations. This know- special characteristics. With these methods the ledge has given the aquaculture industry a better calculated wave force can be reduced by almost understanding of the interrelationship between 60 per cent. A new net able to reduce the prob- environmental conditions and production re- lem of escaping fish has been developed and sults. The public authorities are demanding patented. more and more specific and rigorous environmental studies before a farm can be established, Performance: Poor, due to limited funding. as well as studies during production.

Introduction 21 145x100//Kap01-fig01.eps Mussel farming in Spain. (Photo: Victor Øiestad)

Verifiable objectives: preferences. A substantial amount of basic • To have acquired knowledge about envi- knowledge has been acquired and put into use ronmental parameters and criteria to form by industry, as well as by the authorities, to the basis for evaluating the sustainability combat lice problems. As described under the of a farm location or farming area. “Health and Disease” sub-programme, this has • To have generated knowledge to help to resulted in more effective anti-lice strategies. reduce the amount of salmon lice in farms and the infection of wild salmon smolt on The effect of escaped farmed fish on wild fish their way to the open sea. populations was studied and documented dur- • To have gained knowledge that can help to ing the programme period. But more knowl- reduce aquaculture’s use of environmen- edge is needed about the effects of chemicals tally harmful chemicals. used.

Salmon lice are looked upon as a serious envi- Due to limited economic resources, the environ- ronmental problem for farmed and wild fish mental activities have been integrated into sev- alike. The danger of infection to wild smolt on eral other sub-programmes. Grants have been their way to the open sea was of special con- allocated via the “Health and Disease” sub-pro- cern. Therefore, early in the programme period gramme, for instance, to projects involving re- the board decided to focus on salmon lice search on salmon lice; projects under the research. Projects have been financed to study “Technology and Equipment” sub-programme the louse’s biology, physiology, behaviour and have helped to develop more escape-preventive

22 Aquaculture Research: From Cage to Consumption In 2005, there were 38 doctoral fellowships associated with the programme – meeting the programme objective.

In recent years the Research Council has placed greater focus on post-doctoral fellowships, and has directed its allocations accordingly. In 2005, there were 17 post-doctoral fellowships associated with the programme.

Other activities The programme board initiated several Norwe- gian-language reports, which have formed the basis for development of the programme and for the establishment of the new Aquaculture – An industry in growth programme (see Appendix).

145x100//Kap01-fig01.eps Salmon lice – an important environmental problem. (Photo: Karin Boxaspen) Dissemination, conferences and collaboration net pens; and the sub-programme “Production The programme board has focused on dissemi- and Operations” has encompassed projects nation of information during the entire pro- focusing on water quality in both sea and land- gramme period, and has prepared and followed based installations. a dissemination plan. The board arranged three conferences as part of the programme’s dissem- Performance: Effects of harmful chemicals, ination efforts: one in Tromsø in 2002 (in col- poor; otherwise good to very good. laboration with the “Wild Salmon” programme), one at Gardermoen in 2004 and one in Bergen in 2006. Researcher recruitment A key objective of the Aquaculture programme Financial support for conferences and symposia was to support recruitment of young scientists arranged by other groups has also been an impor- and training of doctoral fellows. In 1999, 25–30 tant way of facilitating dissemination activities. doctoral fellowships were available in the field In addition, the programme board has shared of aquaculture research. Based on the expected findings by publishing information sheets and development in Norwegian aquaculture indus- regular editions of a programme newsletter, and try, an objective was set to increase this number by actively updating the programme’s homepage to 30–35, with the programme itself responsible (www.forskningsradet.no/havbruk). for a minimum of 20 doctoral fellowships and the rest funded through strategic institute pro- The programme board has been responsible for grammes and grants from FHF. scientific follow-up of relevant strategic insti-

Introduction 23 tute programmes (SIP). Toward the end of the grammes at the Research Council, there is good project period, annual allocations to SIPs com- reason to expect an even better funding situa- bined with allocations from other programmes tion in future. came to comprise a significant portion of the Research Council’s overall funding to aquacul- Progress in certain areas has not met expecta- ture research. In order to ensure the cohesive- tions. This is partly due to the fact that new is- ness of the programme, the board has sought to sues have arisen during the programme period, give due consideration to other activities as well such as the growing interest in cod as a new when awarding funding under the programme. aquaculture species. This has resulted in a change in focus within several sub-pro- The programme board has collaborated with grammes. In some areas progress was slower FHF since it was established in 2001. FHF has than expected, despite a relatively high level of been represented by an observer at board meet- activity. It should hardly come as a surprise that ings. Likewise, Innovation Norway has been an certain issues are more complex and require important partner during the programme peri- more research than previously assumed. od, and has also been represented by an observer at board meetings. Research related to So, what about the path ahead? It has been said markets, society and aquaculture was carried that good research generates more questions out under the MARKSAM (“Markets and Soci- than answers. So it is in aquaculture research as ety”) programme, with which the Aquaculture well. This programme summary has allowed us programme cooperated closely. to more easily identify areas that require further research, both basic and applied, that will bene- The programme board has increasingly focused fit the aquaculture industry in future. The art- on international cooperation through project icles in this book point out specific needs for support, participation in international symposia knowledge. In addition, questions raised by the and initiating contact with research groups in programme were thoroughly evaluated in con- other countries. nection with the creation of the work programme and action plan for the new Aquaculture – An industry in growth pro- Challenges – the road ahead gramme. Therefore, the programme board has As is evident from this summary of research ac- no further comments. Our hope for the future is tivities, verifiable objectives and performance, that each of us – after having assessed meter- much has been achieved during the programme high piles of applications – can contribute in a period. Performance in several areas has been personal way to making Norwegian aquaculture very good, and not quite so good in others. This “an industry in growth”. is due to a number of complex factors. Howev- er, limited funding is one that stands out. On These have been seven interesting years for the average, 20 per cent of the project proposals re- members of the programme board. At times it ceived funding, clearly indicating that there was has been hard work, at times it has been frustrat- no dearth of good ideas. With the establishment ing, due to financial limitations. But it has also of the new Aquaculture – An industry in growth been a very stimulating collaborative effort. It is programme, one of seven large-scale pro-

24 Aquaculture Research: From Cage to Consumption a bit sad that we members of the board are going We would also like to wish the new programme our separate ways. All we have left to say is: board of Aquaculture – An industry in growth and the aquaculture research community good Thank you everyone! luck in future.

Programme board, 1999–2006

Magny S. Thomassen, Professor, Norwegian Børre Robertsen, Professor, Norwegian College University of Life Sciences (chair) of Fishery Science, University of Tromsø Roar Gudding, Managing Director, National Aud Skrudland, Senior Inspector, Norwegian Veterinary Institute Food Safety Authority Leif Jørgensen, Senior Research Scientist, Sigurd Olav Stefansson, Professor, University Nord-Trondelag Research Institute of Bergen Oddbjørn Larsen, Sales Director, Joti Isoterm AS Morten Lund, Managing Director Åsen Sette- Secretariat: fisk AS Special adviser/coordinator: Rolf Giskeøde- Kari Morvik, Head of Section, Directorate of gård, Research Council of Norway Fisheries, Region West Consultant: Ellen Gjøsteen, Research Council Birgitta Norberg, Professor, Institute of Marine of Norway Research Special adviser: Frode Meland, Research Council of Norway

145x100//Kap01-fig01.eps Programme board for Aquaculture – Production of aquatic organisms (2000–2005), from left: Birgitta Norberg, Morten Lund, Magny Thomassen, Oddbjørn Larsen, Leif Jørgensen, Sigurd Stefansson, Børre Robertsen, Aud Skrudland. (Kari Morvik and Roar Gudding were not present.) (Photo: Siw Ellen Jacobsen)

Introduction 25 Quality, Slaughter, Transport

■ High-quality Seafood Products based on Ethical and Sustainable Production

■ From Cage to Table Ragnar Nortvedt1), Marit Espe2), Ingrid S. Gribbestad3), Leif Jørgensen4), Ørjan Karlsen5), Håkon Otterå5), Mia B. Rørå6), Lars Helge Stien1),5) and Nils Kristian Sørensen7) 1) University of Bergen, 2) NIFES (National Institute of Nutrition and Seafood Research), 3) Norwegian University of Science and Technology (NTNU), 4) North Trøndelag Research Institute, 5) Institute of Marine Research, 6) Akvaforsk – The Institute of Aquaculture Research, 7) Norwegian College of Fishery Science, University of Tromsø

High-quality Seafood Products based on Ethical and Sustainable Production

High-quality seafood is an important contributor to human health, regarding both malnutrition and obesity. The Norwegian vision should be to produce healthy seafood products by focusing on improved processing methods, new nutritious ingredients, and development of products from underutilised marine species such as blue whiting and krill. Even more marine resources should be utilised for human consumption by conversion into marine ingredients and by improved processing. The production of healthy and safe seafood has to be based on sustainable ethical principles. Seafood may become a substantial part of the daily diet of people who suffer from malnutrition in some parts of the world. At the same time there is likely a growing market in other areas for more tasty, trendy and nutritious seafood among school children and youngsters, as well as among the expanding group of single elderly. A positive and practical benefit of ethically based quality research on seafood is that ethical, sustainable fish farming also ensures better welfare and thus quality of the fish, which subsequently should lead to increased income for the fish farmer. When the market demands documentation of quality in all parts of the trade chain, the operators of catch- based and traditional fish farming, product development and further HACCP- based processing will all achieve added value. Norway enjoys a unique opportunity to supply the market with fresh products from clean waters.

28 Aquaculture Research: From Cage to Consumption Fresh seafood is attractive These are based today on fish captured on the All kinds of seafood are available in today’s high seas, frozen on-board as headed and gut- global food market. High product quality is im- ted, shipped to a country offering low-cost portant not only to consumers, but also to pro- labour for hand filleting and refreezing, then cessors, as it may open access to new markets sent to the international markets as a commodity and thereby increase profit opportunities. The product at medium quality and low price. The basic quality parameter for a food product is demand for these inexpensive frozen products is that it is safe to eat. In addition, the product can high as long as they are safe and offer accept- be of high or low quality, which most often is able quality and price. They have taken a large reflected in the sales price. One important trend share of the seafood volume in many supermar- today in quality-conscious markets, e.g. Europe kets. The Norwegian industry cannot compete and Japan, is that fresh or even live seafood with these low-price products. Its challenge is fetches very high prices. The supplier to these to satisfy the higher-paying segments demand- markets is dependent on raw materials from ing fresh, high-quality seafood offered as spe- nearby fishing grounds or products from aqua- cial cuts with traceable and documented high culture. In this respect Norway has a competi- quality and presented in elegant packaging. tive advantage, situated close to rich fishing Norwegian companies, with their skilled and grounds and having a highly developed aqua- experienced personnel, can accomplish this and culture industry. At the other end of the price offer value for money. Tailoring products to spectrum are the twice- frozen fillet products. special markets and customers can further

145x100//Kap01-fig01.eps Figure 1. Customers are asking for more documentation of quality. These fishmongers at the Torget fish market in Bergen improve both the visual appearance and the quality of their products by cooling fish and seafood in ice. (Photo: Ragnar Nortvedt)

Thematic area: Quality, Slaughter, Transport 29 develop this advantage. Such products can include seafood graded according to size, cut, fat content, fat distribution, skin and flesh colour, and pre- or post-rigor cut fillets offering special texture.

When choosing tailor-made and designed seafood, customers also ask for more documentation regarding raw materials and production processes, including traceability – now a prerequisite in many important markets (Figure 1). There is increasing focus on fish welfare, especially in aquaculture. The result is that ethical

145x100//Kap01-fig01.eps considerations related to feed production based Figure 2. Sea urchins are easily accessible along the on sustainable fish stocks, GMO-free feed, gene seashore. (Photo: Arne Duinker) technology, controlled use of medicine, density of fish in the pens, slaughtering methods and with defined quality, offering documented shelf possible use of additives are much-discussed life, and being based on ethical and sustainable among customers and consumers. Fish welfare production. Most important to the consumer, considerations are important for the industry, though, is that the seafood is tasty – a fancy from the fish farmer to the exporter marketing food that is “light” in calories and easily digest- the product. Most consumers are also very in- ible, whether eaten at home or in a restaurant terested in the good taste and other benefits (Figure 2). from eating high-quality seafood. Seafood provides high-quality protein, important vitamins and trace elements, in addition to the valuable Quality is multifaceted polyunsaturated fatty acids in the fatty fish spe- The quality of most products is defined accord- cies. During the past few years, research has ing to government regulations in the exporting been moving towards producing safe seafood and importing countries, in addition to market demands and consumer trends. The quality of 145x100//Kap01-fig01.eps our foods is of major concern to public health authorities and food producers. The basic re- Primary quality quirement for food is that the product must be • intrinsic/biological quality (species, size, safe to eat. season, health status) • workmanship in handling and production Quality can be defined in many ways. It can be • product quality (hygienic, sensory, tech- defined objectively, according to measurements nological, nutritional, ethical) related to composition, or subjectively, relating Secondary quality to what one prefers. In industry, quality can be • market quality (delivery according to defined as “supply of a product according to the specifications) demand and specifications of the customer”. • perceived quality (subjective – hedonic) Customers may have very different demands,

30 Aquaculture Research: From Cage to Consumption specifications and expectations of a product, depending on who they are, where they are and why they are purchasing seafood. It can therefore be of interest to connect the concept of quality for seafood to two main areas. Firstly, it is the seafood itself and its specific properties,

145x100//Kap01-fig01.eps from being alive (intrinsic quality) to becoming Figure 3: Aspects of seafood product quality. a product: in total, the primary quality. The second main area relates to what is offered together quality-conscious, demanding supply of correct with the seafood product to make the product quality, meaning according to specifications. special. This quality is related to delivery (of the The concept of primary and secondary quality product) according to contract, i.e. the customer can be described as being composed of five gets what he has ordered, on time. But it also in- main areas, constituting the overall quality of a volves the degree of satisfaction the consumer fish product: experiences when eating the product. This is the market and perceived quality, called the secon- Most consumers will assess the sensory part of dary quality. the product quality first, as it is most important in describing the eating quality of the seafood. The different sides of quality are dependent on The taste becomes the most important para- each other. Most important for the consumer to meter. As the English say, “The proof of the consider are aspects of product quality; i.e. its pudding is in the eating”. The important product sensory, hygienic, nutritional, technological quality is also the most difficult one to control. and ethical quality (Figure 3). It is not so much Over time, the biological material in the sea- of interest to know how these properties have food will change, whether alive or dead; this been built into the product. The consumer ex- cannot be stopped without altering the product pects a positive experience when eating fish, properties. confirming the message that fish is tasty, easy and quick to prepare, light in calories, easy to When the fish is alive, feed intake and sexual digest and a very healthy food. maturation will lead to changes in its quality with regard to muscle composition and colour Most consumers relate freshness to time after of skin and flesh, among other things. It is of in- capture or death of the fish. Freshness then terest to know that a farmed salmon should not means high eating quality and that the fish is a be sexually mature when sold, as it looks differ- “good” product that is highly appreciated. In ent from immature fish. In sport fishing in rivers other segments of the market, “good” quality is it is quite common to catch mature salmon, and the natural, real, clean, pure or biodynamic this catch is highly appreciated both as food and product. But a Japanese customer who is cele- as a trophy, although it has a more pronounced brating a special occasion may focus on size or exterior colour and less-bright flesh colour. It colour of the seafood rather than optimum can definitely be eaten, but it is different from freshness. Her interest is the service quality and the standards of farmed salmon, which should not only the taste of the product. This is “eating have a consistent quality with regard to exterior with the eyes”. Both types of customers are shape and colour.

Thematic area: Quality, Slaughter, Transport 31 When it comes to Atlantic cod, the mature fish Ethical and environmental is most appreciated, containing large amounts aspects related to quality of eggs (roe) or milt, in addition to liver, which Over the past ten years, ethical aspects have be- altogether is the basis for a perfect, traditional come an important part of the concept of quality meal. During maturation, the cod eats less and when producing food. Ethics came to the fore- the production of gonads leads to a reduced pro- front with the increasing focus on environ- tein content, increasing water content in the mental pollution, radiation, gene technology flesh. One main utilisation of the spawning At- and welfare when raising animals, including lantic cod, mainly caught in the Lofoten area, is fish. The concerned consumer is increasingly to make stockfish, i.e. drying the whole gutted interested in fish welfare. Fish farmers and ani- fish in open air over a period of several months mal welfare groups want to reduce unnecessary from catching during winter until late spring. stress and pain during the production and har- The catches of Lofoten cod at the start of the vesting of the fish. It is a challenge to handle season are not so mature, leading to a thick large numbers of fish rapidly and effectively in stockfish due to high protein content, while the an industrialised operation at slaughter with a late catches are mature fish with higher water minimum of stress and pain to the fish. Several content, resulting in a thin stockfish after dry- research projects have been started the last few ing. In the main market, Italy, the skinniest fish years related to fish welfare and ethics in fish fetches the highest prices in the north, while the farming. Much focus has been put on water thick and heavy stockfish has a better market in quality, density of fish in pens, medication, feed the south at a lower price per kilo. Quality is quality and methods of killing fish. One result definitely multifaceted, related to region, tradi- in Norway has been the introduction of new tion and use. regulations regarding slaughter of fish, demanding that the fish must be fully anaesthe- Product quality changes during the life of sea- tised before killing, which must be done by food, and during slaughter, handling, process- cutting the blood supply to the brain. An impor- ing, storage and distribution. Eating quality, tant side of ethical quality is related to the per- defined by the sensory aspect, is usually most ception of pain by the fish. It is not clear how important to the consumer, while the processor fish can feel pain, and even more important, is also very interested in the technological qual- whether the pain leads to suffering similar to the ity: how well the raw material is fit for a certain way we perceive pain. Anaesthesia may be used processing, be it fresh or frozen, or for cooking, before slaughter to reduce pain to a minimum. canning, slicing, smoking, etc. During the handling and processing steps, most critical is that Most people do not know the technology in- the product stays safe to eat, although it may volved during farming and slaughter of fish and lose characteristic quality criteria such as fla- are often afraid of unknown consequences. vour, taste, colour and nutritional value, due to Consumers should have the possibility to know storage, freezing or heating. how fish and other seafood are captured, farmed and treated before they end up as products on the shelves in a shop. Then the consumer is able to assess if the product is produced according to personal standards – he can buy the product or

32 Aquaculture Research: From Cage to Consumption refuse it. Lack of information from the farmer ditions must carry a higher price. Many custom- and processors may therefore have great finan- ers say they are willing to pay this price, but will cial implications if consumers do not believe in this willingness be expressed in practice? Prod- the product. Today, this type of information is ucts under ecological labels and labels referring available on labels and company homepages to sustainable production are usually found in and is most often part of a product traceability the market at a higher price. It is probable that system that is now demanded in many markets. this will be an interesting niche for some farmers and processors. They need to document that Some of the ethical questions that may be raised welfare and sustainability has been taken care by consumers regarding farming fish are: of throughout production by quality assurance • Was the fish fed enough and with correct systems, including HACCP (Hazard Analysis feed? of Critical Control Points) and traceability. • Was the feed produced from sustainable raw materials? In the slaughter process of both farm animals • Was the fish farmed with a minimum of neg- and aquaculture fish, stress and/or exhaustion ative environmental impact? level are important factors that affect biochemi- • Did the fish suffer from chronic stress – due cal reactions in the animal after death and may to high fish density, low water quality, pred- affect flesh quality. For years, industrial pro- ators, handling routines? ducers of pigs have been aware of the reduction • Was the fish harvested and slaughtered with in flesh quality if the animal is stressed at no unnecessary stress and pain? slaughter. The meat is then described as pale, soft and exudative (PSE meat) or dark, firm and When this type of information is available the dry (DFD meat), depending on level of stress or consumer can decide whether to buy the prod- exhaustion that is reflected in energy level and uct. The level of quality of a product is in this pH in the muscle and subsequent drip loss dur- way not only decided on the basis of taste, ap- ing processing if pH became too low. This has pearance, nutrition, microbiology and technical not been very clearly observed in farmed fish, properties, but also on the sustainability, wel- because the amount of connective tissue (col- fare and ethics in production. No farmer or fish- lagen) is much lower in fish than in land ani- erman is interested in making a product mals. The stress level influencing the energy resulting in low output or reduced quality. On level of the muscle in fish is most important the other hand they must produce with some when onset of rigor mortis is concerned. With profit, which may not allow farmers to keep the fish farming it is possible to start processing very highest focus on e.g. low density of fish in (e.g. filleting) immediately after slaughter. Tra- the pen. ditionally this has been difficult because the fish came into rigor mortis very quickly after death. For farmers and processors it is a challenge to This is due to quick reduction in energy level, combine the demand for welfare and ethics in measured by muscle pH. The energy reduction production with the demand for economical is closely correlated to muscle activity coming production, focussing on capacities and quanti- from slaughter stress that induces escape reac- ty produced. It may be that the product being tions, movements and in the final stage exhaus- produced under special welfare and ethical con- tion. If the fish is harvested in a rested state, the

Thematic area: Quality, Slaughter, Transport 33 energy levels are high and the onset of rigor safety and production of tailored fish to suit dif- may start as late as 20–30 hours after death. ferent markets. Food safety has mainly focused This delay in onset of rigor mortis allows on surveillance and ensures safe production of enough time for processing this very fresh fish, fish containing no harmful substances or sub- for example by pre-rigor filleting, and at the stances that might be converted to any harmful same time achieve a better bleeding out from substances in the fish. Fish tailoring mainly has the fillet. Then distribution can start immediate- focused on producing a fish of high nutritional ly and the shelf life of high quality fillets can be quality for the consumer as some ask for food extended. items that are healthy while others are more focused on the taste of the fish and the enjoyment This new concept of producing very fresh of eating fish and fish products. Profitability has farmed fish has gained much interest in industry generally been difficult for producers of niche and several ongoing projects have goals related products, but in recent years some have suc- to documenting and improving product quality, ceeded, so it is possible. utilising the by-products from filleting, reducing distribution costs and offering a fresher product to consumers. Healthy seafood and different market preferences Generally seafood is regarded as beneficial to Nutritional quality human health, mainly due to its content of the Nutritional quality may be defined as a food long-chained fatty acids EPA (eicosa pentaenic item containing components of high nutritional acid) and DHA (docosa hexaenoic acid) as well value for the human body and concomitantly as the content of zinc, selenium and iodid. Also not containing any harmful substances for hu- the fatty fish contains rather high amounts of man health. Aquaculture, including feeding the fat-soluble vitamins. In addition all seafood wild-caught fish at acceptable market sizes, contains proteins very well balanced in their provides an excellent opportunity to produce amino acid composition, as required by the fish of high quality as compared to wild-caught human body, and is highly digestible. fish. The quality of the fish may be affected by the genetic codes and thus may be selected for It is well known that the chemical composition different quality traits by genetic selection. of the fish is affected by the composition of the However, quality is also affected by feed com- feed offered to the fish. Thus feed composition positions as well as by the different feeding re- has typically been one of the main factors stud- gimes chosen. Thus upon choosing different ied when it comes to nutritional quality. Tradi- strategies the final quality may be tailored to the tionally, research has focused on maximising different qualities asked for by the different the growth and reducing the period of time markets. By producing niche products as re- spent until fish reach slaughter size. Historical- quested by the different markets, a higher price ly, this has been achieved by increasing the may be fetched for the fish as well. energy levels in the diets by increased addition of lipids while reducing the protein content, as In recent years research regarding fish qualities well as by changing feeding regimes and using has focused on two different main areas, food constant light in the grow-out seawater phase.

34 Aquaculture Research: From Cage to Consumption The increased dietary lipid content thus spares Since the catch of wild fish cannot be increased the protein for anabolic growth, and even if the to provide more lipids and proteins to the grow- fish grows fattier its increased lipid content is ing aquaculture industry, much research has fo- regarded as nutritionally beneficial due to the cused on alternative to fish meals and oils high content of omega-3 fatty acids. In the fish without jeopardising either the growth or the species containing high levels of lipids in the composition of production fish. It has been esti- muscle tissues, such as Atlantic salmon, it is mated that the supply of available fish oils will easy to increase the lipid-soluble components be limited before the supply of protein sources, by dietary treatments. On the other hand the but lately the available fish meal has been limit- non-lipid soluble components are not easy to ed, causing very high prices for fish meals. So tailor by dietary treatments. Due to this, re- for the aquaculture industry to grow, sustainable search has mainly focused on tailoring of total protein and lipid sources are needed. Much re- lipid content and fatty acid profiles as well as search has been done on replacing marine oil tailoring of the lipid-soluble vitamins, while re- and protein with plant proteins and oils. Plant search has been limited regarding the protein proteins generally contain anti-nutritional fac- and amino acids as well as water-soluble com- tors, though some of these might be inactivated ponents. upon processing prior to feed production. Still, the amino acids are not balanced to maximise While in the fatty fish species the fatty acid pro- growth. Therefore one has to mix different plant file in the muscle reflects the fatty acid profile proteins or add crystalline amino acids to fulfil of the diet, this is not the case with the lean fish the requirements of the fish. Furthermore, the species, such as Atlantic cod. Although the fatty fatty acid profile in plant oils differs from the acid profile of the phospholipids of the muscle fatty acid profile in the marine oils, and as will reflect the diet, the impact is low due to the dietary lipid profile determines the muscle pro- low total lipid content of the muscle. As Atlan- files in the fatty fish, this of course will have an tic cod stores excess lipids in hepatic tissues, the impact on the nutritional quality of the pro- lipid profile has been found to reflect the dietary duced fillets. lipid profile. Also, the size of the liver increased when the dietary lipid content increased. Several studies have been done regarding the effects of replacing the marine oils with plant oils The amino acids constituting the protein in fil- or mixtures of plant oils. All of these showed lets, of course, are determined by the genetic that neither the growth nor the quality of the code, and as amino acids cannot be stored, im- produced fish was significantly affected, with balanced dietary protein are catabolised and the exception of the fatty acid profile. Atlantic used for energy. To minimise catabolism and halibut can also be fed diets in which 50 per maximise protein deposition, diets balanced in cent of the dietary lipid content is soy oil, with amino acids for maximal protein deposition are no negative impact on sensory parameters or of utmost importance, also in reducing the storage properties. Since nutritionists as well as waste of nitrogen. When tailoring the minerals, markets might ask for fish containing high lev- research in recent years has shown that some els of omega-3 fatty acids due to their beneficial might be tailored while others are impossible to effects on human health, the possibilities of regulate. slaughter feeds containing a marine fatty acid

Thematic area: Quality, Slaughter, Transport 35 profile have been studied. Upon using such eral markets. Thus a study was done to evaluate slaughter feeds for different periods of time any chemical or quality differences in salmon prior to slaughter, the preferred marine fatty originating from three different countries (Nor- acid profile is achievable. way, Scotland and Ireland), but all smoked by French commercial smokehouses. Fish were During the programme period investigation be- collected every second month for a period of 12 gan into the possibilities of utilisation of the months, and at each sampling time the consum- Calanus and krill as alternative marine feed in- er test was also taken by about 100 respondents. gredients for production fish. However, to fully The conclusion of the test was that the typical utilise these rich marine resources, improved French consumer was able to detect differences technology for fishing, storage, processing and between the smoked salmon, but none of the utilisation of such feed items by the farmed fish fish were preferred over the others. Nor had the need to be studied in more detail. country of origin a high impact on the chemical composition of the smoked salmon. Generally Several studies have involved how the largest the fish from Ireland was redder and contained European markets for Atlantic salmon accept more cantaxanthine than the Norwegian salm- the different quality of produced fish, especially on, while the Norwegian contained more of the the fatty content of fillets. A study in which highly unsaturated fatty acids. The Scottish fish German and French consumers tested several was always between the Norwegian and the different tailored qualities of colour, consisten- Irish fish. All of the fish analysed was of high cy and lipid content of Atlantic salmon showed quality and none contained any harmful sub- that colour was the most important quality stances. The study thus could not find any parameter to consumers, although increased chemical differences or preferences by the gen- lipid content was also regarded as positive. Ger- eral consumer that should support any differ- man consumers appreciated salmon if it was ences in the price of the smoked salmon. soft and lipid-rich or hard and lean. The big and lipid-rich Atlantic halibut was also regarded as One may conclude then that several aspects in- better than the smaller and leaner halibut in a fluence the final product quality of seafood, all study by chefs in four Norwegian cities. Whole of which need to be studied in more detail. In Atlantic halibut stored for a period of 21 days addition, the quality of both the marine proteins was judged better in taste as well as smell when as well as the lipids clearly has impact on hu- compared to vacuum-packed stored halibut. man health and as such may contribute to im- The bigger fish (5.4 kg) were considered tastier proving health status in all parts of the world. as well as having a better texture than the smaller halibut (1.8 kg). The eating quality thus was good in the big halibut even after being stored Seafood and health for 21 days, while the smaller ones were not so Fish and seafood contain a well-balanced amino acceptable. acid composition as well as the long-chained fatty acids of high nutritional value for human Smoked Atlantic salmon is generally marked by beings. In addition, these components have a country of origin, and a different pricing system positive effect on human health, a fact which often occurs due to the country of origin in sev- has received particular attention during the past

36 Aquaculture Research: From Cage to Consumption decade. When it comes to beneficial health effects of fish and seafood, focus has been put on 7.0 the effects of the lipid content and fatty acid 6.0 profiles of Atlantic salmon on different health 5.0 aspects. Feed manufacturers have cooperated * 4.0 with medical and fish research institutions on * investigating how consumption of fish as well 3.0 as its chemical composition affect different pa- 2.0 tient groups or persons at risk for developing

Plasma cholesterol (mmol/l) lifestyle-related diseases such as coronary heart 1.0 disease, lipidemia and also psychology related diseases. As the fatty acid composition in lipid- FPHSoy Casein rich fish such as Atlantic salmon reflects feed Protein source composition, it has been important to investi- 145x100//Kap01-fig01.eps Figure 4. Total plasma cholesterol level as a function of gate which components in the fish have the different protein sources, such as fish protein hydrolysate greatest impact on human health. It has been (FPH), soy protein or milk protein (casein). (Modified after shown that consumption of Atlantic salmon by Wergedahl et al. 2004) humans with health problems results in positive health effects, even if a significant part of the tial high water activity (Aw >> 0.6), a prerequi- dietary fish oils has been replaced with vegeta- site for microbial activity that makes the fish ble oils. Thus other parts of the fish muscle more prone to putrefication as compared to besides the fatty acids, such as its protein or many other foodstuffs. Furthermore, the opti- minerals and vitamins, also contribute to the mal activities of the enzymes in cold-water fish health benefits of consuming fish. This under- are adapted to low water temperature, which re- lines the significance of the multivariable inter- sults in high autolytic activity that starts soon action between nutritional components in fish after slaughter and continues through cold stor- and seafood. Within the food and health sector, age and processing. The bacterial flora in newly there are opportunities to create niche products caught/slaughtered fish depends on water puri- for specific customer segments, including func- ty, and bacteria associated with water will also tional food – where fish is used as a means of be present on the fish, though in low numbers. supplying humans with desired components Slaughter of the fish initiates enzymatic and from fish feed, or for the significant effects of chemical reactions that result in loss of muscle fish protein in e.g. lowering blood cholesterol freshness. Firstly, due to oxygen depletion, lac- levels in humans (Figure 4). tic acid is produced and will lower the pH depending on the amount of glycogen available in the muscle tissues; thereafter the autolytic pro- Food safety cesses start, and further storage may result in Microbiology and hygiene growth of microorganisms that limit shelf life Fish contains nutritional components that are upon refrigeration or vacuum storage. easily digested and consequently more easily attacked by microorganisms. Fish also has a To avoid potential contamination during pro- high water content, which may induce a poten- cessing aimed at increasing shelf life, hygiene

Thematic area: Quality, Slaughter, Transport 37 throughout processing has major implications harmful pollutants present in farmed fish, and and should be very strict. In salmonids, the feed components. Several potentially harmful microorganism Listeria monocytogenes, which substances exist in the marine environment that may be introduced during processing, grows may pose a risk for food safety, such as non- even at low temperatures as well as in vacuum degradable organic compounds and metals. products. In both Atlantic salmon and rainbow Whether these are potentially harmful or not de- trout, this microorganism has been studied dur- pends on the amount present in the food, their ing smoking and fermentation methods and chemical structure, and whether they are ab- subsequent vacuum-packing. During fermenta- sorbed by the fish or not. Chemical contami- tion it is particularly important to keep the stor- nants may be added to the fish through age temperature constant, both to prevent processing, for instance PAH (poly aromatic growth of potential L. monocytogenes and to hydrocarbons) during the smoking process, or improve the sensory characteristica of the food. PCB (poly chlorinated biphenyls) from plastics The occurrence of L. monocytogenes and total or from contamination of feed ingredients. Pol- bacterial count in smoked salmon collected in a lution of the aquatic ecosystem may also con- hypermarket in France was investigated; bacte- tribute to contamination (for instance PCB, rial content varied with time of year as well as dioxin, bromated flame retardants) and accu- with different smoking facilities, while the con- mulation in the food chain, in particular in lipid- tent and variation in the raw material showed rich parts of the fish. Future research thus needs little variation. This indicated that treatment to focus on optimising the methods to separate during processing was the most important fac- these from feeds and fish to secure pure feed in- tor for contamination with L. monocytogenes. gredients for fish feed production. Research regarding bioavailability and toxicity of such components is also needed. Pollution Fish live in the oceans, and over the past 100 Metals occurring in the marine environment years pollutants have routinely been accumulat- have a natural origin from geological activity ed into the oceans by human activities. Since such as erosion and volcanic activity, but they pollutants know no borders and are distributed may also originate from industrial processes, by wind and ocean currents, pollutant levels for instance from metallurgic industries, from may be rising all over the planet. Different pol- galvanisation or from battery waste, e.g zinc, lutants may accumulate in the oceans, some cadmium, lead, arsene and mercury. In contrast with the possibility of accumulating in wild fish to the organic pollutants, most of the metals are used for human consumption or for producing water-soluble and are bound to the protein frac- fish feeds, thus appearing in farmed fish as well. tion of the fish muscle. Bacteria in marine and By using only feed ingredients free of harmful freshwater environments transform organic pollutants, producers can avoid food-borne pol- mercury into methylated mercury, which is lutants in their farmed fish. However, those ar- more poisonous than the inorganic forms. riving from the water are harder to avoid. Due to Methylated mercury leads to damage in the cen- increased concerns over food safety in the past tral nervous system in both animals and 5–10 years, special attention has been paid to humans. The mercury level in smoked farmed the occurrence – and the legal limits – of any

38 Aquaculture Research: From Cage to Consumption fish is fortunately far below the limits set by The freezing temperature as well as the period legislation. the fish is kept frozen affect the development of rancidity in fillets of fattier fish such as Atlantic salmon. To study this in more detail, gutted At- Rancidity problems in farmed fish lantic salmon were frozen at two temperatures Fish such as Atlantic salmon that contain high (-20 and -30 °C) at two different storage periods levels of polyunsaturated fatty acids also con- (one and four months), thereafter thawed, fil- tain high levels of EPA and DHA, which are leted and refrozen for a period of months. The more prone to oxidise post-slaughter, as com- product quality was assessed using a trained pared to fish containing less of these fatty acids. sensory panel, a consumer panel of 45 partici- The oxidation of the fatty acids may result in in- pants, and with chemical analysis after filleting creased rancidity, which is documented in the and after refreezing. One of the main conclu- smoked salmon market. This is due to that the sions was that salmon that was thawed and re- fact that fatty acid oxidation increases exponen- frozen performed well with only minor effect tially with the number of double bonds in the on the final quality. Increased storage time at fatty acids. In addition, some lipid oxidation the higher temperature (-20 °C) reduced prod- may occur in vivo, but the organisms have pro- uct quality, although the reduction was small, as tective mechanisms to handle this smaller-scale stored samples had acceptable quality after the oxidation. These mechanisms, however, do not treatment. Chemical analysis confirmed that no function post-slaughter and during storage of dramatic changes appeared in the salmon, the fish, since no new generations of antioxi- though prolonged storage at the higher tempe- dants are produced and meanwhile the antioxi- rature (-20 °C) resulted in slightly elevated ran- dative enzymes lose their efficiency. The degree cidity compared to those stored at the lower of oxidation post mortem is therefore due to the temperature (-30 °C). status of antioxidants in the muscle at slaughter, additions of antioxidants through processing, and factors such as the presence of oxygen and New methods in quality blood residues during processing and storage of research the product. Measuring rancidity of the product The most widely used variables applied as mea- is complicated and generally not sensitive, since sures of fish quality are condition factor, chem- there are many products that may increase or ical composition, microbiological criteria, decrease depending on how far along the degra- yield, bleeding, shelf life, fillet and skin colour, dation process is. One can measure rancidity in pH, rigor-development, fillet gaping, water- primary products, such as peroxide levels (num- holding capacity, texture, smell and taste. Tradi- ber of hydroxyperoxide groups present) or in tionally these quality criteria have been mea- secondary products, such as aldehyds (anisidine sured by means of labour-intensive and levels or TBARS that measure thiobarbituric re- destructive methods such as manual observa- active substances). Level of rancidity is also tion and chemical analysis. The demand for often expressed as total oxidation, based upon more detailed, more objective and faster met- both anisidine and peroxidation level (anisidine hods (primarily online) have led to significant + 2*peroxide). efforts in the programme period to develop new measuring methods for fish quality. Projects

Thematic area: Quality, Slaughter, Transport 39 under the Aquaculture programme have put par- digital cameras is an efficient and non-destruc- ticular emphasis on the quality index method tive way to measure quality attributes in fish. (QIM), digital image analysis, magnetic reso- There is no need for direct contact between the nance (MR), computer tomography (CT) and camera and the product. Image analysis can also near infrared spectroscopy (NIR/NIT). be very quick and give a description of a product in milliseconds. Another advantage is that image analysis gives an objective result, in con- The Quality Index Method (QIM) trast to visual judgement of colour, for example. The Quality Index Method represents a “retro culture” to instrumentation relying on tradi- Automatic image analysis methods for describ- tional manual inspection of selected character- ing various quality-related parameters of fish istics in stored fish. The different attributes, e.g. have been developed in the programme period. the shape and the colour of the eyes, the smell, Image analysis methods for describing rigor the colour and the mucus of the gills and the contraction in pre-rigor-filleted fish fillets in texture of the fish, are given scores 0–3 accord- various fish species are one example. The out- ing to a documented scale, where 0 indicates a put from this analysis is graphs showing how fresh fish. A maximum acceptable score after the fillets change in shape during rigor mortis ice storage of each single species is achieved (Figures 6 and 7). Image analysis methods have before the fish is no longer rated as fresh and also been developed for quantifying fat percent- must be discarded. Efforts have also been made age, colour, peritoneum area and melanin spots to calibrate the manual observations to instru- in salmon fillets. These methods have potential mental methods that do not rely on traditional

16 chemical methods and thus contribute to a 9 future latent quality index. In the programme 14 8 period researchers have evaluated how the QIM 12 7 score of Atlantic cod (Gadus morhua) can be 10 6 5 influenced by different slaughtering methods. It 8 4 is possible to calculate instant shelf life status 6 3 Sensory score 4 according to QIM score determination cassation limit 2 2 (Figure 5). This method has also become a Quality index (QIM points) 1

practical way to determine quality at inter- 2 468 10 12 14 national fish auctions, and hopefully the fish- Days in ice

mongers will implement this method as well in 145x100//Kap01-fig01.eps Figure 5. Principle for calculation of remaining shelf life, their daily quality assurance routines. based on the Quality Index Method (QIM). A linear relation (blue line) is tailored to the accumulated QIM vs. number of days in ice. By tasting prepared fish, an expert panel can Image analysis report the sensory score (green line) and determine the date Access to increasingly advanced digital camer- of cassation, which is 12–14 days for Atlantic cod. By as, faster computers and smarter image analysis inspecting the fish in the refrigerator, a fishmonger or an experienced customer may define the QIM score, find the techniques has made image analysis an impor- corresponding nos. of days in ice on the x-axis and thus also tant tool for the fishing industry and research in- calculate the remaining shelf life compared to the theoretic stitutions alike. Application of images from date of cassation.

40 Aquaculture Research: From Cage to Consumption a) b)

Figure 6. Pre-rigor cod fillets (a) without bones, i.e. without “reinforcement”, contract substantially until they end up in rigor mortis (b). (Photo: Lars Stien)

145x100//Kap01-fig01.eps for implementation in the fish industry to mea- variables that previously have been practically sure the quality in fish fillets in real time as they impossible to quantify in large scale. These pass by on a conveyor belt. Another significant variables include the size of the dorsal fat de- observation has been that image analysis of dig- posit, the size of the ventral fat deposits and the ital pictures of salmon cutlets taken by a com- size of the loins. The conclusion therefore is mon table scanner can be used to estimate the that automatic image analysis has great poten- fat percentage and colour of the muscle, thus tial both in the fish industry and as a research representing a good alternative to far more ex- tool. pensive methods. From scanned pictures of salmon cutlets it is also possible to measure Magnetic resonance spectroscopy (MR) MR is a method used both for imaging (MRI) and for studies of chemical composition (MRS) in fish. MR makes it is possible to look into the fish and get images at all levels. The contrast in the MR images can be changed by choosing different recording options. Lately MRI has been applied to study the distribution of water and lipids in fish, salt distribution in fillets and anatomical deformities. This technique has been applied in the Aquaculture programme to study 145x100//Kap01-fig01.eps Figure 7. Percentage of original fillet length = 100*current different freezing regimes and to see how the length/original length measured during the rigor-process cold front moves into the fish at different tem- for four different treatments of farmed cod: stressed (filled peratures. MR has also been used to study the squares) and unstressed (open squares) stored at 4 ˚C (dotted lines) and at 20 ˚C (solid lines). The predefined formation of ice crystals during the freezing biological time of initiation of rigor (b.t.2), during rigor process. (b.t.3–5) and full contraction (b.t.6) are marked in succession along the continuous graphs. (Modified after Stien et al. 2005).

Thematic area: Quality, Slaughter, Transport 41 145x100//Kap01-fig01.eps Figure 8. The shelf life of tempting high-quality seafood may in future be instantaneously determined by a handheld non- destructive NIR probe at the fish market. (Photo: Ragnar Nortvedt)

MRS has proved to be a good method for mea- and easily accessible calculation power in the suring chemical compounds in fish. It gives a 1980s, not to mention the software developed, broad picture of the biochemical composition. these methods achieved their breakthrough. The method has been applied on muscle extracts, fillets and whole fish. MRS gives infor- Calibrations between a range of traditional chem- mation on changes in metabolites in fish ical methods and quality of different food prod- through the rigor process. ucts were developed before the methods were applied on fish. The quantification of protein in grains by NIR has been established as a standard Near infrared and Raman spectroscopy method in the USA. At the end of the 1980s and Measured reflectance (NIR) or transmission beginning of the 1990s, calibrations were estab- (NIT) of light irradiated on a sample at a broad lished between fat, protein and dry matter in spectrum of wavelengths is the rough principle minced fish for several species. Once the calibra- for Raman and near infrared spectroscopy. tions were established the method became simple Some of the irradiated light at each specific to use and several variables (fat, protein, water, wavelength is absorbed by the sample, and the salt, pH etc.) could be determined simultaneously energy is converted to vibration and stretching in a new sample in seconds. Both NIR and Raman energy for specific molecules in specific parts have been successfully used for predicting the to- of the spectrum. The energy can be measured tal content of saturated, mono- and polyunsaturat- indirectly by reflection, transmission or by a ed fatty acids in complex mixtures of fat, protein combination of the two, called transflection. and water. The measuring principle for NIR is, Common to all these principles is that they gen- however, limited by not measuring specific erate spectra with a range of close peaks from micronutrients or quantities below percentage several similar wavelengths. No peaks stand out levels. The methods have nevertheless become as the specific response for a particular mole- more advanced during the programme period cule. Multivariate methods need to be applied to since basic research has made it possible to mea- calibrate this multivariate information against sure directly through the fish skin and establish other quantifying methods. Following the intro- the respective calibrations. Raman spectroscopy duction of personal computers with increased shows even higher chemical selectivity than NIR.

42 Aquaculture Research: From Cage to Consumption Fate Function

Resources Molecular and comparative nutrition Processing

Human health Quality

HealthHealth Biomarkers for fate and function

145x100//Kap01-fig01.eps Figure 9. Fate and function of the biomarkers along the entire value chain, from raw materials through processing to quality documented products. The results of scientific testing in comparative nutritive studies (cell cultures, animal models and human intervention studies) may contribute to improved human health.

Raman spectroscopy has thus also been success- described QIM. This activity opens the door for fully applied to quantify carotenoids at mg/Kg an appealing future application where a handheld concentration levels. This leads to the very inter- NIR probe can measure remaining shelf life di- esting opportunity of gently netting live fish from rectly during transport, during storage or at a fish a net pen, sedating it by ethical routines and mea- market (Figure 8). In this way modern instrumen- suring it by these spectroscopic methods, giving tal technology and traditional manual evaluations an instantaneous picture of its water, fat and pig- may be combined in a practical, handy documen- ment content before returning it live to the net tation tool for quality of seafood. pen. In this way the scientist and the fish farmer can predict the expected body composition in different dietary feeding regimes before slaughter- Future quality focus ing. The spectroscopic methods cannot be more Based on the quality-oriented topics described accurate than the traditional chemical methods in this chapter, future research is predicted to they are calibrated against but they have proved to turn more attention towards ethical aspects and be more precise than the traditional methods and further development of instrumental methods. It even more accurate than the Torry Fatmeter. In is also expected that more resources will be de- the final phase of the programme period, one voted to cooperative research across institutions project has also started to establish calibration along the entire value chain from raw materials models between NIR records and manually through processing to the final products. One determined quality, according to the previously should test the human health effects from intake

Thematic area: Quality, Slaughter, Transport 43 of marine ingredients through comparative nu- multivariate modelling, in such a way that it is tritional studies of both cell cultures and animal possible to identify and follow both the fate and models (fish included) as a basis for conducting function of certain biomarkers along the value intervention studies on humans. This kind of re- chain (Figure 9). The biomarkers may in turn be search would demand more than ever that the used to predict how specific raw materials and scientists apply the whole toolbox of methods ingredients may be utilised to benefit human from biology, chemistry, process technology, health – in a global perspective. medicine, molecular biology, informatics and

References

Bjørnevik, M., Karlsen, Ø., Johnston, I.A. and AKVAFORSK and Dep. of Animal Science, Agri- Kiessling, A., 2003. Effect of sustained exercise on cultural University of Norway, ISBN 82–575– white muscle structure and flesh quality in farmed 0564–1. cod (Gadus morhua L.). Aquaculture Research, Solberg, C., Saugen, E., Swenson, L.-P., Bruun, L. & 34(1), 55−64. Isaksson, T. 2003. Determination of fat in live Espe, M., Ruohonen, K., Bjørnevik, M., Frøyland, L., farmed Atlantic salmon using non-invasive NIR Nortvedt, R. and Kiessling, A., 2004. Interactions techniques. J. Sci. Food Agric., 83, 692−696. between ice storage time, collagen composition, Stien, L.H., Hirmas, E., Bjørnevik, M., Karlsen, Ø., gaping and textural properties in farmed salmon Nortvedt, R., Rørå, A.M.B., Sunde, J., Kiessling, muscle harvested at different times of the year. A., 2005. The effects of stress and storage tempera- Aquaculture, 240, 489−504 ture on the colour and texture of pre-rigor filleted Martinez, I., Bathen, T., Standal I.B., Halvorsen, J, farmed cod (Gadus morhua L.). Aquaculture Re- Aursand, M, and Gribbestad, I.S., 2005. Composi- search, 36, 1197−1206. tional analyses of cod (Gadus morhua) and Atlantic Stien, L.H., Amundsen, A.H., A., Mørkøre, T., Økland, salmon (Salmo salar) by high resolution 1H MR: S.N., Nortvedt, R., 2006. Instrumental colour anal- Application to authentication analyses. Proceed- ysis of Atlantic salmon (Salmo salar L.) muscle. ings, 34th WEFTA meeting, 12−15 September WEFTA 2005 Proceedings, Accepted. 2004, Lübeck-Germany. Stien, L. H., 2006. Application of machine vision to Mørkøre, T., 2002. Texture, fat content and product quantify rigor development, colour and fat content yield of salmonids. Dr.Scient. thesis, Akvaforsk and in slaughtered fish. Dissertation for the degree Dep. of Animal Science, Agricultural University of philosophiae doctor (PhD) at Department of Biolo- Norway, 50 p. + V papers. gy, University of Bergen, Norway. 111 p. + V pa- Nortvedt, R. 2000 (Ed.) Kunnskapsstatus for produk- pers. sjon av laksefisk. Rapport fra Område Bioproduk- van de Vis, H., Kestin, S., Robb, D., Oehlenschläger, J., sjon og Foredling, Research Council of Norway, Lambooij, B., Münkner, W., Kuhlmann, H., Kloost- ISBN 82–12–01 369–3, 71 pp. erboer, K., Tejada, M., Huidobro, A., Otterå, H., Otterå, H., Garatun-Tjeldstø, O., Julshamn, K. and Aus- Roth, B., Sørensen, N.K., Akse, L., Byrne, H. and treng, E., 2003. Feed preferences in juvenile cod es- Nesvadba, P., 2004. Is humane slaughter of fish pos- timated by inert lanthanid markers – effects of sible for industry? Aquaculture Research, 34(3), moisture content in the feed. Aquaculture Interna- 211−220. tional, 11(1−2), 217−224. Wergedahl, H., Liaset, B., Gudbrandsen, O.A., Lied, E., Roth, B., 2003. Electrical stunning of Atlantic salmon Espe, M., Muna, Z., Mørk, S. and Berge R.K., 2004. (Salmo salar). Dr.Scient. thesis, Dep. of Fisheries Fish Protein Hydrolysate Reduces Plasma Total and Marine Biology, University of Bergen, Norway, Cholesterol, Increases the Proportion of HDL Cho- 47 p. + IV papers + Errata paper IV. lesterol, and Lowers Acyl-CoA:Cholesterol Acyl- Rørå, A.M.B., 2003. Raw material characteristics and transferase Activity in Liver of Zucker Rats. J. treatment-effects on yield and quality of cold- Nutr.,134 (6), 1320−1327. smoked Atlantic salmon. Dr.Scient. thesis,

44 Aquaculture Research: From Cage to Consumption Anders Kiessling1), Marit Bjørnevik2), Magny Thomassen1), Mia B. Røra3), Turid Mørkøre3), Bjørn Roth4), Ulf Erikson5) and Odd Jordheim6) 1) Norwegian University of Life Sciences, 2) Bodø University College, 3) Akvaforsk – The Institute of Aquaculture Research, 4) University of Bergen, 5) The SINTEF Group, 6) Norwegian Seafood Centre

From Cage to Table

The concept of “good quality” in fish can have as many meanings as there are consumers. Everyone has an opinion about what tastes good. People also associate safe food with quality. Scientists prefer to deal with objective criteria and properties that can be measured and evaluated, but such criteria are not always relevant for consumers and their taste experiences. Cooks, who in their daily lives transform raw materials into tasty meals, acquire a great deal of experience with the way differences in raw materials affect the end results of their labours. When a structured quality researcher who wishes to document differences meets a chef who may well be emotionally involved in his work, bridges can be built between research data and consumer experience. This chapter’s point of departure is that research data can be used to steer fish production in the direction of obtaining good-quality fish in the form of firmer flesh, a reasonable fat content, fat with a healthy profile, red- dish colour in the case of salmonids, and several other factors. This chapter will follow the fish from the farm, via slaughter and post-harvest processing, to the multitude of products and types of packaging produced by the Norwegian seafood industry to satisfy the demands of new markets and new consumer groups. Today, salmon are the driving force of the Norwegian aquaculture sector. One question to be posed is whether this species will continue to play this role in the future, or whether other species are waiting in the wings for their great breakthrough. Nor- wegian salmon is still the most common salmon in the world, but many other countries have learned from this development and are eager to compete. Strong efforts on the part of Norwegian scientists to reach an understanding of what makes a high-quality salmon that is good and safe to eat have led to “Norwegian Salmon” being recognised as a sign of both quantity and quality. Maintaining this position is a challenge for the future.

Thematic area: Quality, Slaughter, Transport 45 The production phase is dietary vegetable oils on the nutritional quality essential for product quality of the fillets, but it is possible that altered fatty Different markets have different requirements acid composition of the fillet also influences, regarding quality-related characteristics. So for example, storage stability, taste and smell, knowledge that enables the aquaculture indus- as well as colour and texture characteristics. try to tailor fish products according to different market demands is essential. During the growth One question that has been asked concerns the phase, it is possible to control certain parame- possibility of interactions between modified ters of quality in fish through feed and environ- fatty acid composition in the cellular mem- mental conditions. Starvation produces a branes of the fish and the pattern of processes somewhat slimmer salmon, and in cod and hal- related to the development of rigor mortis and ibut it can be seen that starvation results in a subsequent muscle breakdown. In connection higher proportion of connective tissue, which in with research on pre-rigor processing of salmon turn leads to less gaping in fillets. Water tem- and cod, comparative studies have been carried perature in the egg and fry phases affects out on salmon fed on fish oil and rapeseed oil. muscle fibre recruitment and growth, which in A tendency towards more rapid onset of rigor in turn are important for texture and gaping. The the rapeseed group was observed, but the differ- time of release of salmon smolt to seawater, ences were small and not statistically signifi- whether in spring or autumn, is important for cant (2). However, in a similar study of cod, the growth and fat accumulation in the seawater onset of rigor was much more rapid in fish fed phase. soya oil (Mørkøre 2006). The underlying mechanisms are still unknown, but these observa- During the past few years, growing awareness tions underline the importance of knowledge of omega-3 fatty acids for human health has and control of all aspects of product quality in emphasised the importance of optimising the aquaculture. content of these beneficial fatty acids in farmed fish. At the same time, however, the limited Recent years have seen a great deal of attention availability of marine oils has led to the use of being paid to the size and number of muscle vegetable oils in aquaculture feeds. This has led cells and muscle fibres in fish. Fish muscle is to changes in the fillet fat composition, as the structured somewhat differently than in mam- fatty acid profile in the diet is reflected in the mals. Like ours, it has rapid (sprinter) and slow fish fillet. The first study that demonstrated the (marathon) types of fibres, but in fish these two effects of graded admixtures of rapeseed oil and types lie in separate layers. In mammals they lie soya oil was carried out as early as 1988 (1), but within the same muscles, but to different ex- only during the past few years has this problem tents, depending on how we train and what our aroused the interest of the fish-farming industry. genes permit. Figures 1 and 2 show how fish A number of studies of these and other veget- muscle is built up. able oils have been carried out in Norway and elsewhere. This is also discussed in the chapter In a Scottish study, two groups of salmon were entitled “High-quality seafood products based kept at two different temperatures throughout on ethical and sustainable production”. Most at- the freshwater phase, before being released into tention has been focused on the significance of seawater and followed up until slaughter. The

46 Aquaculture Research: From Cage to Consumption 145x100//Kap03-fig01.eps Figure 1: The schematic drawing to the left shows a cross-section of a fish muscle, with the distribution of red (highly aerobic, slow-contracting), pink (highly aerobic, highly glycolytic and rapid-contracting) and white (anaerobic, highly glycolytic and rapid-contracting) muscle fibres (from Bjørnevik et al., 2003). The upper right-hand image is a histo- chemical cross-section of bovine muscle stained to show different types of muscle fibre (rapid, slow and intermediate). (Photo: Karl Heintz Kiessling). The photomicrograph to the lower right is a cross-section of a fish muscle stained to illustrate its aerobic capacity, and showing the localisation of red and white muscle. (From Kiessling and Ostrowski, 1997. Photo: Anders Kiessling) group that had been kept at the lower temperature always had more, and thinner, muscle fibres. A number of studies have been carried out on salmon to find out whether there is a relationship between fillet firmness and muscle fibre size. Several studies now suggest that salmon with thinner muscle fibres have firmer flesh than those with thicker fibres, although the relationship is not unambiguous. A number of factors probably determine the texture of fillets, in which connective tissue, fat content, pH and muscle fibre all play a part.

Fish has much less connective tissue than does Figure 2: White muscle of rainbow trout stained for glycogen (PAS stain), showing the difference in glycogen mammalian meat. Nevertheless, collagen has a content of small (young) and large (old) fibres. (Photo: significant effect on the texture of both raw and Anders Kiessling)

145x100//Kap03-fig01.eps

Thematic area: Quality, Slaughter, Transport 47 tractive appearance, and problems arise when the skin is being removed and the fillets are cut into smaller portions or sliced. Gaping may take the form of rips between blocks of muscle. These can range from small cracks in the flesh to the extent that the musculature comes apart all the way through to the skin. Gaping can also occur when rigor contraction is particularly

145x100//Kap03-fig01.eps strong, which may happen if the fish is subject- Figure 3: Schematic image of texture measurement and of how different fibre thicknesses are believed to affect the ed to stress or high temperature during the texture of fish muscle. slaughtering process. Live-chilled, unstressed fish that have been filleted pre-rigor thus dis- cooked flesh. Raw fish muscle that contains 1–2 play much less muscle gaping. We see a similar per cent collagen is soft, whereas fish containing outcome in well-nourished cod, whose post- above 8 per cent collagen has a very tough tex- mortem muscle pH falls rapidly due to their ture. In cooked fish, the connective tissue con- large glycogen stores (see Figure 4). tributes less to the texture since collagen denatures long before it reaches the boiling point. It turns out that the connective tissue that keeps Figure 3 illustrates the principle of mechanical the muscle fibres together becomes weaker as measurement of texture and how we believe that the pH falls, and this may lead to more muscle different fibre thicknesses affect texture. gaping. Farmed cod have large stores of glycogen year-round, leading to a higher susceptibil- Fillet gaping is an economic problem for the ity to muscle gaping. Recent studies have fish-processing industry. Fillets have a less at- demonstrated that starving farmed cod for a few

7 7,4 Glycogen 7,2 6 Lactate pH 7,0 5 6,8 4

ate content (mg/g) 6,6

3 6,4 Muscle pH 6,2 2 6,0 1 5,8 0 5,6 048 Muscle glycogen and lact Days post mortem

145x100//Kap03-fig01.eps Figure 4: Relationships between pH, lactic acid and glycogen levels in cod muscle post mortem (Førde-Skjærvik et al. 2006, Aquaculture, 252, 409–420)

48 Aquaculture Research: From Cage to Consumption weeks before slaughter produces markedly less a muscle with many thin fibres will therefore gaping, but without loss of weight1. This is also contain more connective tissue than one that the result when cod are starved during the sum- consists of thicker fibres. Any relationship be- mer. The reduction in gaping in fillets of starved tween muscle fibre and gaping is probably a re- cod is most likely to be due to higher connective sult of a higher proportion of connective tissue tissue strength, as post-mortem pH remained rather than of thinner fibres. It has been shown constant during up to two months of starvation. in Atlantic salmon that levels of total collagen Previous studies have shown that starving cod and insoluble collagen are negatively correlated results in thicker, stronger connective tissue. with gaping frequency, while more soluble col- Histological studies of halibut indicate that in lagen has been found in salmon with a greater this species, too, starvation increases the tendency towards gaping (5, 6). amount of connective tissue between individual muscle fibres2. Appearance is an important quality parameter in fish. This is particularly true of salmonids, Contradictory results have been obtained with whose flesh is preferred to be red. Colour is also regard to the question of muscle fibre thickness important in whitefish. A pure white colour and fillet gaping. Johnston et al. (3) indicate that such as is seen in cod is regarded as more invit- thinner muscle fibres in fillets also produce less ing than the grey flesh colour of saithe, a factor gaping, while other studies have described a that also affects relative prices. Colour can be negative relationship between muscle fibre regarded as a function of light reflection and ab- thickness and gaping (8). Each individual mus- sorption by the muscle and we can therefore ex- cle fibre is surrounded by connective tissue, and pect to find a relationship between colour, fibre thickness and texture. A number of studies have 1. C. Solberg, personal communication 2. Ørjan Hagen, personal communication attempted to demonstrate such a relationship.

Figure 5: A salmon fillet can be discriminated from its background with the aid of digital imaging. In the future, this type of analysis will allow quality assessment of fillets as they pass along a production line for factors such as colour, fat content (amount of white fat), gaping (connective tissues split up in the white lines of fat between the muscle fibres), shape, and spots of blood (note the dark fleck on the ventral surface). (Photo and data analysis: Lars Stien)

Thematic area: Quality, Slaughter, Transport 49 Relationships between fibre density and flesh discharging, some boats have recently installed colour has been found in some studies (7, 8) movable bulkheads with video monitoring of while others have failed to demonstrate any cor- the behaviour of the fish when they are being relations (4). Fillet colour has also been report- crowded together. Some vessels are able to ed to vary with texture. It has been shown that pressurise the fish hold. In both cases, the fish the toughness of salmon fillets correlates posi- can be unloaded without lowering the water tively with light and negatively with red colour- level in the fish hold. This should help to reduce ing (8). In cod a negative relationship between the level of handling stress to which the fish are texture and green colouring is found (4). Tex- exposed. ture is determined by a number of different factors, including the amount of connective tissue, Transport of salmon for slaughter in closed sys- fat content, water content, muscle-fibre size, tems (closed valves) produces certain advant- pH, etc., so that any relationship between the ages, such as reducing the risk of infection colour and texture of a fillet is due to its chemi- when the transport passes close by certain fish cal and structural composition. farms, as well as cooling/calming the fish before they are delivered to the slaughter plant. During long journeys, however, closed systems Fish transport, storage and may involve a serious risk of mortality as water slaughter – what is the quality gradually deteriorates. From an ethical relationship with quality? point of view, we are not yet certain whether Transport transport in closed systems is an acceptable so- Transport by well-boat lution. General use of the concept for long trips The method of transporting fish to slaughter by probably ought to require the installation of on- well-boat is one that functions well enough for board water-treatment systems (scum separator, routine transport operations (9, 10). In order to etc.) and not least, a great deal of experience on remove the contents of the digestive system the part of the crew. A particularly important as- (food remains), reduce the oxygen require- pect of water quality is ammonia (NH4+/NH3) ments of the fish and calm them down before build-up. As is well known, it is primarily NH3 transport, a minimum of two to three days of that is toxic to fish, and the proportion of NH3 fasting is necessary, depending on water tem- is dependent on the pH of the water. Under un- perature. Siphon loading (with biomass/fish favourable conditions (pH > approx. 8), even counter) has turned out to function well. Water small amounts of ammonia can lead to acute quality during transport is good because trans- fish mortality. However, carbon dioxide pro- port takes place with open seawater valves, i.e. duced by the fishes’ own metabolism lowers the the water quality is virtually the same as pure pH of the water, thus considerably reducing the seawater. During the summer, the water is often risk of ammonia poisoning. We therefore need oxygenated when large quantities of fish are to be careful about ventilating the water (to re- being carried. Typical transports operate with move CO2) and mixing in fresh seawater with a fish densities of 80–200 kg/m3, although some higher pH. Transport in closed systems also transport operators have employed even higher leads to the production of large quantities of densities. Tests have shown that this can be scum (probably from glycoproteins in the slime done without lowering fillet quality (10). For of the fish). Transport in closed systems is not

50 Aquaculture Research: From Cage to Consumption currently practised in Norway, but the method their gills can be cut and they die from loss of has been successfully employed in a short (two- blood in the bleeding basin. In other species hour) transport by well-boat (10). such as eels, ammonia or sodium hydroxide have been employed. The problem with these Crowding and pumping traditional anaesthetisation methods is that they At the slaughter plant, transfer from the well- are relatively inefficient. Removing the blood boat to the plant takes place either by pumping supply to the brain means that the process of dy- the fish directly into the slaughtering pens or by ing may take anywhere from five minutes to transferring them to holding pens. In the latter several hours, depending on species and tem- case, the fish will usually be allowed to rest for perature. Similarly, a range of flight reactions 12–24 hours before slaughter, when they are (anaerobic swimming activity) during anaesthe- crowded together to a density of 200–300 kg/ tisation and bleeding can have a number of neg- m3 before pumping. ative effects on fillet quality (11–17). In order to satisfy demands for better fish welfare and to The use of holding pens has advantages and dis- meet future legislative requirements, a number advantages vis-à-vis slaughtering directly from of new methods have been adopted, including the well-boat. The greatest advantage of hold- electric anaesthetisation and killing by a blow to ing pens is that this method saves money, since the head. the well-boat does not have to be paid for waiting by the quayside, while the plant is able to Live chilling slaughter fish from several different producers The most common method of anaesthetising on the same day. It is unlikely that keeping fish salmon and trout used to be to anaesthetise them in holding pens will improve their quality. The in a relatively small tank containing carbon most important disadvantage is probably the dioxide, before placing them in a refrigerated greater risk of disease and of escapes associated sea water (RSW) bleeding basin (1–5 oC), pos- with keeping fish in sea cages. Using holding sibly in combination with chilling after gutting pens may be more difficult in the case of cod and washing in a tank of RSW (0–4 oC). Live than with salmon. Cod are less well adapted to chilling in RSW gradually arrived on the mar- water surface temperatures as they are farmed at ket. This method had two objectives: 1) live fish greater depths, and they are more inclined to try can be chilled more rapidly than dead fish to avoid being pumped, so that they need to be because their blood circulation and gill arch forced together more severely. Little documen- surfaces act as heat exchangers, and 2) the fish tation is available regarding pumping and how it are sedated in the tank by rapid chilling. In the affects the welfare, stress and quality of the fish. process line, the live chilling tanks used to be located before the CO2 tank. However, this Anaesthetisation and sacrifice turned out to be an unfortunate method, because One of the greatest challenges when fish are to sedated fish from the live-chilling tank tended be anaesthetised and slaughtered is that of strik- to waken again when they were transferred to ing a balance between fish welfare, quality and the CO2 tank, and they were – as before – com- process-related costs. In the case of salmon, pletely exhausted before they were anaesthe- CO2 or large quantities of ice water have tradi- tised. Subsequently, carbon dioxide and oxygen tionally been utilised to calm fish down so that were added directly to the live-chilling tank. By

Thematic area: Quality, Slaughter, Transport 51 2005, most large Norwegian slaughter plants was decided to change to adding the gas directly were utilising this method to sedate and (partly) to the live-chilling tank. It is important to be chill their salmonids. aware that given that the chilling tank is a (partly) closed system, carbon dioxide will A number of different technical methods for always accumulate in the tank whether gas is RSW live chilling exist. Fish are usually added or not. The point of adding a certain pumped from the well-boat or holding pen into amount of this gas is to maintain its concentra- the RSW tank, which may be divided into a tion at a given level, sufficient to anaesthetise number of rotating chambers. A certain number the fish. At the same time, oxygen is added (70– of fish are pumped into each chamber. After a 100 per cent saturation) to ensure that the fish given period of time, which depends on rotation have sufficient oxygen. It is also important to speed, the chambers are emptied and the sed- avoid high oxygen super-saturation, as this is ated fish are sent on to be bled. One method is capable of leading to abnormal behaviour based on the fish and the RSW passing in oppo- (unethical) and a potential reduction in quality site directions. Carbon dioxide and oxygen are (“soft” muscle). then mixed into the chilled water before it enters the tank. It is important to realise that when Depending on the proportion of RSW that is re- such large volumes of water (20–40 m3 per circulated, and how often water is drawn off to tank) are mixed, a large proportion of the water cleanse the tank, the water quality will deterio- is recirculated, and this affects water quality rate to a greater or lesser extent. This will result (see below). In another live-chilling method, an in the water gradually becoming less transpar- open tank (not divided into separate chambers) ent as a greater amount of biomass passes is employed, in which the supply and holding through the tank. The quantity of organic mat- time of the fish are controlled by a movable grid ter, mainly slime, also increases significantly. which is moved back and forth along the long The water in the live-chilling tank is often red- axis of the tank. Another concept has been de- dish, probably due to an accumulation of blood. veloped in which the fish are gradually chilled We can assume that this blood is due to injuries by transferring them from the well-boat to on- incurred by individual fish during pumping shore reception tanks (2 x 1000 m3) in which (from valve gates in the pressure/vacuum they are allowed to recover before being slaugh- pump) from the well-boat or holding pen to the tered. When the fish are to be slaughtered they live-chilling tank. Significant quantities of am- are pumped into 2 x 150 m3 chilling tanks at monia from the metabolism of the fish also ac- -1 oC before being sent on to the slaughter line cumulate in the tank. Because of the low pH (2). (<6.4, thanks to the added CO2), it does not accumulate in its toxic NH3 form (18). Water quality In order to anaesthetise large salmonids by car- Sedation bon dioxide, it is estimated that 200–400 mg Fish are usually anaesthetised in the course of CO2/l are required. It gradually became appar- 2–3 minutes. Carbon dioxide is typically the ent that this was a source of considerable stress primary agent of anaesthetisation. The sedative for the fish. The recommended level was there- effect of rapid chilling (hypothermia) is prob- fore lowered to around 80–150 mg/l when it ably slight because the difference between the

52 Aquaculture Research: From Cage to Consumption acclimation temperature of the fish (tempera- the water in the tank. It is important to remem- ture of the seawater in the sea cage) before ber, however, that as the fish gradually cools slaughter and the temperature of the water in the down, T, i.e. the rate of heat exchange, will live-chilling tank is usually small (T <10 oC). gradually decrease. In a process line, with its Hypothermia can be an efficient method of need to maintain a rapid, regular flow of pro- anaesthetising warm-water fish (T >10 oC). In duction, little will be gained in practice by keep- late summer Norway can have high water tem- ing the fish too long in the tank. At high water peratures (18–20 oC), so rapid cooling to temperatures (summer) live chilling may be an 0–2 oC can then have a sedative effect (used in efficient method, but during the winter the chill- o conjunction with CO2). If the processing line ing effect is slight (typically 2–4 C). During from the holding pen or well-boat to bleeding periods of low seawater temperature, the live- operates satisfactorily, the initial muscle pH of chilling tanks act primarily as anaesthetic tanks. the anaesthetised fish will be around 7.4±0.1 in Company chilling strategy should therefore also unstressed fish. As a matter of comparison it is be considered in connection with other chilling worth mentioning that completely exhausted methods: RSW bleeding tanks, RSW buffer fish have an initial pH of 6.7±0.1, while the tanks, or other chilling methods such as ice final pH of salmon flesh (measured one day slush. post mortem) is typically 6.3±0.1 (18). Slush is a relatively inefficient method of anaes- Chilling thetising and killing warm-water species such Depending on the circumstances, Atlantic as turbot. Behavioural and blood-gas measure- salmon begin to die at -0.7 to -1.7 oC (19, 20). ments in turbot raised at 14 oC have shown that Thus, in order to obtain the highest possible T this species responds to tactile stimuli at deep- for chilling (the “driving force” for heat trans- body temperatures as low as 0.25 oC. Deep- port from the fish to the chilled water), the body temperatures below 1 oC result in muscle recommended temperature of the water in the contraction in most individuals, which prevents live-chilling tank should be held at about the fish from expressing responses, while blood 0.0±0.5 oC. One also needs to remember that measurements indicate that metabolism contin- salmon and rainbow trout react differently to ues down to -0.5 oC. No fish died as a result of rapid chilling, in that chilling to 0.5 oC leads to chilling to -0.5 oC, but one fish died within 12 a severe stress reaction in rainbow trout. A hours of this treatment (21). higher water temperature should therefore be employed for this species. When rainbow trout Fish welfare are subjected to rapid chilling, as many as 25 If the cessation of brain activity can be taken as per cent of the fish may suffer water-filling of the criterion of good fish welfare, the use of the stomach, which can raise stress levels, mea- CO2 is a matter of dispute, as carbon dioxide sured as higher plasma osmolality. In order to anaesthetises the fish too slowly (minimum 2–3 lower the body temperature of the fish suffi- min) (22). How this circumstance will affect the ciently, it is important that the fish remain long current use of RSW live chilling as an industrial enough in the tank. The appropriate holding method of chilling and anaesthesiology remains time will depend on seawater temperature, bio- to be seen. mass, flow conditions and the temperature of

Thematic area: Quality, Slaughter, Transport 53 Slaughter by percussion of time, a procedure that will have effects on the Percussion machines are potentially very inter- onset of rigor (15). The use of electric current as esting as a slaughter method for salmonids and an instantaneous method of anaesthetisation cod. However, the efficiency of anaesthetising appears to be difficult for salmonids, but unpub- and slaughtering fish with a blow to the head lished data on cod and flatfish show extremely depends on the design of the percussion head promising results. Trials using 50 Hz current and the pressure of the blow (22, 23). Since the have shown that these species do not suffer pressure required for efficient anaesthetisation injury. For such species as eel, turbot and possi- is in the same range as that which can result in bly halibut, electrical anaesthetisation is a eye injury, the industry will need to tolerate a potential method of slaughter in which the fish, certain incidence of eye injuries if this method once it has been anaesthetised, is exposed to a is to be employed more safely. However, the weak current and pulsed frequencies for 2–5 force needed to immobilise a salmonid perma- min (27, 28). nently is not in the same range as that which can produce serious eye injuries such as eye pop- Bleeding ping. Where the efficiency of the design of a The most common method of slaughter is still particular hammer is concerned, a flat or to stop blood circulation by cutting the gills. rounded hammerhead is the most efficient Gill-cutting can be regarded as an important method of transforming kinetic energy into a method of ensuring death, irrespective of the shock wave. If the fish is to be killed with a method of anaesthetisation or slaughter sharp-pointed “Iki jime” hammer intended to employed. For salmonids (and possibly cod) penetrate the neurocranium and destroy the cutting the gills while the fish is unconscious is brain, the region around the cerebellum needs to synonymous with an unconscious death, but for be destroyed in order to obtain permanent other species such as turbot and eels, and possi- insensibility. The problem with percussion bly halibut, the fish may regain consciousness. machines is that at present they usually require For this reason, it will be important in the future operation by human beings, and in order to to be able to evaluate the effectiveness of meth- improve the process of automation the fish ods of anaesthetisation combined with slaugh- should preferably be sedated or calm before ter. Bleeding is also regarded as essential for treatment. complete blood loss, but as soon as the fish is quite dead, it can be further processed without Electric anaesthetisation and slaughter negative effects on the loss of blood (29, 30), as Electric anaesthetisation has often been long as bleeding takes place immediately, or at employed on fish. With salmonids, the principal least within 30 minutes of death (31). challenge is to anaesthetise the fish instantaneously without injuring it (24). Injuries occur when the dorsal aorta bursts, causing a haem- Types of processing, transport atoma. However, the injury problem can be and logistics – an industry in the reduced by employing new frequencies in the process of changing 500–1000 Hz range (25, 26). In salmonids, Norway has largely been a supplier of fish raw injury can be completely avoided by using very materials, where much of the value-adding pro- low frequencies and voltages for longer periods cess has been transferred to the receiving coun-

54 Aquaculture Research: From Cage to Consumption tries. This, in conjunction with growing compe- The post-processing of pre-rigor raw materials tition on the production side from countries that has also been focused on, particularly salting often have lower cost levels, has led to more and smoking. Early processing affects both the attention being focused on how to exploit Nor- uptake and distribution of salt in salmon (36) way’s particular advantages to increase value and cod (37). Generally speaking, it is more dif- adding and competitive advantage. The spot- ficult to obtain good salt uptake in pre-rigor fil- light has been turned particularly on how to leted raw material than in post-rigor filleted exploit advantages in the production of salmon, fillets, due to muscle structure and physical trout and cod pre-rigor in an integrated slaugh- conditions during the rigor stage. The results of ter and filleting process. Such a process could a number of studies are not in complete agree- not only lower production costs, but also open ment, but the most promising method of salting up the prospect of supplying the market with so far is injection salting, while methods of super-fresh, high-quality filleted products – at treating raw materials need to be further devel- higher prices than for conventional post-rigor oped. filleted fillets. In the course of the past few years, a number of Pre-rigor filleting industry-oriented projects have aimed at devel- The development and adoption of new methods oping salmon and trout slaughter process lines of rapid, humane slaughter at the end of the that will allow the fish to be filleted and post- 1990s has made pre-rigor filleting a practical processed in their pre-rigor state. In order to in- possibility. Pre-rigor filleting is carried out im- crease value-adding even more, greater atten- mediately after slaughter, which means that the tion has been paid to fillet portions in individual Norwegian fish-processing industry can exploit packages and to the main challenges presented its unique advantage of lying physically close to by greater automation of portion packaging of production sites to supply the European market salmon. Pre-rigor filleting also means that other with super-fresh products. Apart from the po- parts of the fish than the fillets themselves be- tential of even greater freshness on delivery to come available, and both research and industry the customer, it has been shown that pre-rigor have begun to regard it as a matter of impor- filleting of salmon gives fillets better colour, tance to study the optimal utilisation of the rest firmer texture and less gaping early in the of the fish, whether fresh or frozen. course of storage, compared with fillets that have been processed post-rigor (32). A firmer The difficulty of removing bones from pre-rigor texture and less gaping have also been found in raw material has acted as a limiting factor in the pre-rigor than in post-rigor filleted cod (33). To utilisation of this process. With new technology, guarantee good, stable pre-rigor filleting, the however, the fish-processing industry is now ca- time aspect from slaughter until the onset of pable of producing virtually boneless fillets that rigor is critical. In the course of the past few cost less and fetch higher prices. The techno- years, a number of studies have looked at fac- logy is based on pinching out the bones from tors that affect the window of rigor, particularly the fillet and then x-raying it in order to locate the effects of different types of stress, tempera- any remaining bones. Projects that have studied tures and handling during the slaughtering pro- strategies for increasing value-adding in the cess (34, 35). Norwegian seafood industry have pointed out

Thematic area: Quality, Slaughter, Transport 55 that non-material capital, in the shape of com- fresh versus frozen products. As an example of petence and knowledge systems, will be of this trend, industry-oriented projects have been growing importance in attempts to increase carried out on cod super-chilling. These have value-adding in Norway. It is therefore recom- focused on the entire value chain from produc- mended that companies should more than ever tion, via refrigeration during transport, to mar- be creating powerful knowledge systems an- ket acceptance. Most people regard the distance chored in the industry itself, and be developing to the market as long, and it takes four days cooperative models that are capable of out- from the date on which fish are packed in weighing the disadvantages of small-scale Northern Norway until they are delivered for operation. onward distribution in Europe. The most common method of transport is in insulated boxes, Transport and logistics each containing ice-chilled fish or fillets. In or- Process chain traceability and verification of der to keep fillets in good condition for another origin of foodstuffs have been priority areas of couple of days and thus retain their high quality interest for the European food industry in recent for longer, studies of super-chilling have been years. Particularly within the EU, a rising level carried out. This process involves chilling the of interest in these topics has contributed to en- fillets to a temperature at which some of the wa- suring that the final consumer is able to put to ter in their tissue freezes. After it has evened the test the systems that have been developed. out, the temperature will stabilise at around As well as the international standardisation and -1.5 oC, and the ice in the product helps to implementation of traceability systems, the re- maintain a low temperature throughout the search and industrial sectors have also been transport chain. The ice in the product during working on methods of mapping traceability, transport will have thawed by the time the fish new marking technologies, and data-capture are presented for sale in shops, so it is important and IT technologies that will streamline the that they should not be chilled more than neces- transfer of traceability information between sary to maintain the good qualities of the fresh companies in the value chain. fish. Trials have shown that after four days in transport, the temperature of super-chilled fil- More pre-rigor filleting and processing in Nor- lets was still as low as -1–0 oC, and their quality way in general will be of great importance for was judged by both customers and chefs to be the efficient transport of farmed fish. At present just as good as or better than iced fillets, sug- very large quantities of ice, as well as unmarket- gesting that this method has the potential to im- able parts of the fish, are transported. Pre-rigor prove the efficient transport of processed fish processing opens up the logistical prospect of products. bringing fresher fish to the market. The demand for high-quality fresh-fish products is growing, and exports of fresh fillets have increased in re- Packaging – new products – cent years. The use of super-chilling to prolong different markets shelf life could expand the market for fresh fillet The seafood industry is a rapidly growing sector products. Prolonging quality retention will also in Norway. Its range of products is expanding, bring greater flexibility to production and en- and new markets are being developed. Exports able Norwegians to increase the proportion of of whole round fish in traditional expanded

56 Aquaculture Research: From Cage to Consumption polystyrene cartons still make up an over- inate sales, nowadays there is a much greater whelming proportion of turnover of fresh range of packaging and presentation. The need wares, but the degree of processing is on the infor rapid food preparation at home is growing, crease, and with it, the need for packaging ma- and the number of small households is also in- terials. creasing; the keyword here is flexibility.

Packaging makes it possible to transport and Product exposure in the shops is a decisive fac- store fish, maintain product quality and ease the tor when shoppers are deciding what to buy. handling of goods in the transport chain. It also Customers want to see the products on offer. tells customers something about the product, Many people wish to be able to choose products profiling it and thus increasing sales. Packaging on the basis of price and to compare seafood also has the important function of protecting the with meat and chicken. Some shops have there- product from contamination and ensuring its fore chosen to sell seafood in the form of pre- safety in terms of health. This in turn allows priced consumer packs. A steadily rising pro- seafoods to be sold in self-service counters. portion of customers also wish to see their food Suitable packaging reduces transport costs, packed in environmentally friendly packaging. damage and losses. Optimal packaging solu- For the companies offering these products, the tions throughout the transport chain involve cost-effectiveness of the production process is good utilisation of the environment and re- the most important aspect, but the economics of sources, thus benefiting both society and the in- packaging is also important. Companies need to dividual consumer. balance economics against customer satisfaction and to take both national and EU require- Purchasing patterns and trends ments into account. In recent years, the EU has There has been a trend in the direction of offer- issued directives that aim to reduce or eliminate ing consumer packs of seafood in self-service environmental impacts. These directives al- counters. This trend is particularly evident in ready are, and will continue to be, of impor- the European market. There are ongoing re- tance for the evolution of product packaging. search efforts to improve the efficiency of packaging methods. Advertising expert Leo Burnett Trends in consumer packaging once said, “Packaging is the primary display There are three main types of consumer pack- opportunity.” Good packaging sells. It should ages of seafood products: be safe and capable of maintaining quality. It is the combination of appropriate quality, graphic Vacuum packs design and technical design that turns pack- These are used for both fresh and frozen prod- aging into a solid, serious and not least, effec- ucts, and they allow products to be attractively tive sales tool for seafood. No matter whether it profiled. When the air is sucked out and the is fresh, salted or frozen, seafood needs to be plastic tightly surrounds the product, both sold in watertight packaging that can tolerate colour and consistency can be particularly well being turned upside down. profiled. It is also easy to handle products in the store, as they can be presented either lying flat Buying patterns are changing. While traditional or hanging vertically. Studies have shown that fish counters featuring whole fish used to dom- vertical presentation tends to increase sales.

Thematic area: Quality, Slaughter, Transport 57 is chilled is also critical to the quality of the end-product.

This type of packaging enjoys important advantages in that it can be distributed or delivered to stores together with other product groups.

MAP (Modified Atmosphere Packaging) This is winning a growing share of the market in important export markets for Norwegian seafood. Though still relatively small in Norway, MAP is much more important abroad. How- ever, there are good reasons for supposing that this type of packaging will take a larger share of the market in the future.

MAP products are sold in self-service counters and have become a common sight in most food stores (see Figure 6). The gas used in these Figure 6: MAP-packed cod. (Photo: Norwegian Seafood packs slows bacterial growth and thus prolongs Centre) the shelf life of the product. A relatively large 145x100//Kap03-fig01.eps volume of gas is required to obtain the desired shelf-life, which means that such packs take up This packaging method is not particularly a good deal of space on the shelves. Recent re- advanced, and it requires little investment. search suggests that seafood can be pre-satur- Environmental taxes may be imposed in certain ated with gas, thus reducing the large volumes markets, but these focus little on food stores. required. Traditionally, it appears that those This type of packaging largely complies with types of fish that release most liquid are least the wish of customers to see the products on suitable for MAP production. In the interna- offer before they actually decide what to buy. tional market, there is a clear trend in the direction of MAP and vacuum-packing. In recent years, chilled prepared foods have grown in popularity. “Sous vide” food products, Seafood packed in trays which are vacuum-packed and heat-treated, can These products are portion-packed either in the give products a shelf-life of 10–12 weeks when store itself or in a packing plant. This type of they are kept at 0–2 oC. This means that con- presentation is labour-intensive, but it satisfies sumers do not need to freeze their purchases, certain customer requirements. For the cus- and that they stay fresh for a long time. To tomer, pre-packaged portion packs are easy to maintain their quality, products need to be relate to in the sense that they are clearly priced handled in an unbroken cold chain from the and the price can be easily compared with the production stage until they reach the consumer. quantity in the pack. This is thus similar to how The way in which seafood is handled before it meat is presented, and products are cleanly cut

58 Aquaculture Research: From Cage to Consumption and boned. The range of pre-packed portion Cod products is rapidly growing in Norway, and they Commercial activity based on farming cod is are a good supplement to manned fish counters. growing rapidly, largely thanks to goal-oriented Studies show that these products also appeal to research and practical trials. Cod farming is customers who would not normally ask for based on the production of fry or on raising cap- fresh fish. This type of packaging also allows tured cod. Commercial up-scaling in the future fish-counter staff to be more efficiently will require farming of cod from the fry stage, employed in pre-packaging their products. while raising captured cod will be a relatively minor part of the industry. Market orientation and good co-existence between the value chains New aquaculture species – are for farmed and captured cod will also play cen- there alternatives to salmon? tral roles in the development of this sector. The Sustainable growth in the marine sector de- production of fry and juvenile fish is progress- pends largely on an expanding and diversified ing, and work on feeds and cod breeding is un- aquaculture. Not all species can be developed derway. Research financed privately and by the simultaneously, and environmental conditions public sector has improved operational aspects. determine the suitability for growing particular species. Goal-oriented marketing and sales ef- The quality of farmed cod differs to a certain forts, and production of fish with “the right extent from that of wild cod. Farmed cod usual- quality” are also important success factors. ly has a firmer texture, its flesh is whiter in Norway’s government is currently taking steps colour and it produces thicker fillets. Most mar- to ensure that new species can be introduced to kets regard these characteristics in a positive aquaculture by encouraging research and devel- light. On the other hand, farmed cod is more opment efforts within this area. likely to lose moisture during storage, particularly when stored frozen. The past few years Norway lies at the forefront of research on ma- have seen a certain amount of research on this rine coldwater species, but scaling up produc- topic. Appropriate handling before, during and tion and commercialising new aquaculture after slaughter, as well as correct storage, are species are resource-intensive and time-con- important aspects of maintaining quality. Re- suming processes. This was also the case with cent research results also suggest that specially salmon farming, where it took a long time to designed feeds given before slaughter can help reach profitable large-scale commercial pro- to improve the ability to bind water. duction. In the past few years, the development of cod as a new aquaculture species has been The greatest challenge facing fish production prioritised, but halibut and mussels are also at today is that of postponing sexual maturation so the scaling-up stage. Expectations are high for that growth to slaughter size is not held back the economic potential of these species. Other and flesh quality does not deteriorate. Cod species also have potential in terms of volume make up part of the global whitefish market, in and niche production. which farmed cod can complement wild-caught fish. During the first phase, the European fresh- fish market will most likely be the primary market for farmed cod.

Thematic area: Quality, Slaughter, Transport 59 Halibut In Norway, the greatest efforts have been put into farming halibut as a new marine species. Since the mid-1980s, the public and private sectors have invested large sums into making commercial halibut farming possible. To some extent, these efforts have been successful. Pro- duction of fish for human consumption was expected to pass 1,000 tonnes in 2005. Improved control of fry production and greater attention to the sea cage environment has created confidence that we are approaching the stage of being able to implement an efficient halibut production line.

Halibut quality is generally good. Fillets are usually firm and white, but there is a need to standardise methods of slaughter. We also need to ensure that both male and female halibut maintain good growth and good quality year- Cod is considered the aquaculture species with the greatest growth potential today. (Photo: Per Eide, round. Norwegian Seafood Export Council) 145x100//Kap03-fig01.eps Shellfish Shellfish are a important item in terms of global In 2004, production of farmed cod reached consumption, and Norway enjoys good natural 4,500 tonnes. Current prognoses suggest that conditions that could enable it to become a ma- production will surpass 50,000 tonnes as early jor player in shellfish farming. Mussel farming as 2010. is growing in scale and is the shellfish industry

Table 1: Candidate species for aquaculture in Norway, apart from salmon and rainbow trout. (source: KPMG)

Species on which work has Species on which little work has Non-fish species Other marine animals been done in Norway been done in Norway

Cod Hake Shellfish Sea urchins Halibut Angler fish Mussels Sea cucumbers Catfish Atlantic bluefin tuna King scallops Charr Haddock Flat oysters Turbot Redfish Crustaceans Eel Lumpfish Lobster Plaice King crab Sole Edible crab Lemon sole

60 Aquaculture Research: From Cage to Consumption sector that has progressed furthest. The mussel mercialisation; it should be up to the industry it- farming industry has gone through a phase of self to decide whether it is economically consolidation, and now has better control of its interesting to scale up farming of these species. production. Intensive lobster farming is in transition between the pilot phase and up-scaling, while flat Important quality characteristics of shellfish in- oysters, king scallops and catfish have been at clude meat content, colour, texture and shell the pilot stage for some years. Flat oysters and strength. These properties display considerable catfish could be niche products for the Norwe- variation from one site to another and at differ- gian aquaculture industry, but their commercial ent times of the year. Mussel farming has con- potential is considered limited. siderable potential for growth, provided that greater efforts are made in the areas of control- Scallops are believed to have greater potential. ling stocks, ensuring reliable supplies and pro- Ballan wrasse and sea urchins are not yet far viding quality based on market demands. A enough along to be ready for up-scaling; their national harvesting plan based on geographical commercial potential is regarded as medium. and seasonal variations in quality would con- Other species at a very early stage of farming tribute to meeting specific market preferences include angler fish, haddock, king crabs, lemon and ensuring constant quality, and thus better sole, hake, Norway lobster and edible crabs. prices. These species may have potential in the long term, and there are concrete plans for commer- Other species cial development of some of them. Table 1 of- Charr, turbot, plaice and sole are considered so fers an overview of species on which work has well developed that they should be ripe for com- been done in Norway (38–40).

Thematic area: Quality, Slaughter, Transport 61 References Quality (S. Kestin and P. Wariss, Eds.), pp 202–219, 1. Thomassen, M.S. and Røsjø, C., 1989. Different Fats Blackwell Science, Oxford. in Feed for Salmon: Influence on Sensory Parame- 11. Erikson, U., Beyer, A.R. and Sigholt, T., 1997. Ini- ters, Growth Rate and Fatty Acids in Muscle and tial distribution of white muscle high-energy phos- Heart. Aquaculture, 79, 129–135. phates related to handling stress affects K-values 2. Einen, O., Tomter, R. and Fjæra, S.O., 2002. Contrac- during ice storage of Atlantic salmon (Salmo salar). tion and quality changes of pre-rigor fillets of At- J. Food Sci., 62,43–47. lantic salmon as affected by dietary oil and live- 12. Sigholt, T., Erikson, U., Rustad, T., Johansen, S., chilling. Poster. Int. Symp. on Nutrition and Feed- Nordtvedt, T.S. and Seland, A., 1997. Handling ing of Fish. Rhodes, 2–7 June. stress and storage temperature affect meat quality of 3. Johnston, I.A., Manthri, S., Alderson, R., Campbell, farmed-raised Atlantic salmon (Salmo salar). J. P., Mitchell, D., Dingwall, A., Nickell, D., Selkirk, Food Sci., 62, 898–905. C. and Robertson, B., 2002. Effects of dietary pro- 13. Berg, T., Erikson, U. and Nordtvedt, T.S., 1997. tein level on muscle cellularity and flesh quality in Rigor mortis assessment of Atlantic salmon (Salmo Atlantic salmon with particular reference to gaping. salar) and effects of stress. J. Food Sci., 62, 439– Aquaculture, 210, 259–283. 446. 4. Bjørnevik, M., Karlsen, Ø., Johnston, I.A. and 14. Roth, B., Moeller, D., Veland, J.O., Imsland, A. and Kiessling, A., 2003. Effect of sustained exercise on Slinde, E., 2002. The effect of stunning methods on white muscle structure and flesh quality in farmed rigor mortis and texture properties of Atlantic salm- cod. Aqua. Res., 34, 55–64. on (Salmo salar). J. Food Sci., 67, 1462–1466. 5. Espe, M., Ruohonen, K., Bjørnevik, M., Frøyland, L., 15. Roth, B., Slinde, E. and Arildsen, J., 2006b. Pre or Nortvedt, R. and Kiessling, A., 2004. Interaction post mortem muscle activity in Atlantic salmon between ice storage time, collagen composition, (Salmo salar). The effect on rigor mortis and the gaping and textural properties in farmed salmon physical properties of flesh. Aquaculture, in press. muscle harvested at different times of the year. 16. Stien, L.H., Hirmas, E., Bjørnevik, M., Karlsen, Ø., Aquaculture, 240, 489–504. Nortvedt, R., Rørå, A.M.B., Sunde, J., Kiessling, 6. Hagenes, T., 2002. The amount and solubility chang- A., 2005a. The effects of stress and storage temper- es for collagen in Atlantic salmon (Samo salar L.) ature on the colour and texture of pre-rigor filleted muscle with emphasis on smolt type, annual varia- farmed cod (Gadus morhua L.). Aquaculture tions and ice storage. Thesis for the Degree of Can- Research, 36, 1197–1206. didatus Scientiarum in Nutrition. University of 17. Kiessling, A., Espe, M., Ruohonen, K. and Bergen, Institute of Fisheries and Marine Biology Mørkøre, T., 2004. Texture, gaping and colour of Institute of Nutrition, Bergen, Norway. 71 pp. Feb fresh and frozen Atlantic salmon flesh as affected 2002. by pre-slaughter iso-eugenol or CO2 anaesthesia. 7. Johnston, I.A., Alderson, R., Sandham, C., Dingwall, Aquaculture, 236/1–4, 645–657. A., Mitchell, D., Selkirk, C., Nickell, D., Baker, R., 18. Erikson, U., Hultmann, L. and Steen, J.E., 2006. Robertson, B., Whyte, D. and Springate, J., 2000. Live chilling of Atlantic salmon (Salmo salar) com- Muscle fibre density in relation to the colour and bined with mild carbon dioxide anaesthesia. I. − texture of smoked Atlantic salmon (Salmo salar L.) Establishing a method for large-scale processing of Aquaculture, 189, 335–349. farmed fish. Aquaculture, 252, 183–198. 8. Bjørnevik, M., Espe, M., Beattie, C., Nortvedt, R. and 19. Saunders, R.L., 1986. The thermal biology of Atlan- Kiessling, A., 2004. Temporal variation in muscle tic salmon: influence of temperature on salmon cul- fibre area, gaping, texture colour and collagen in ture with particular reference to constraints triploid and diploid Atlantic salmon (Salmo salar imposed by low temperature. Rep.-Inst. Freshw. L.) ranging in body size from 2 to 10 kg. J. Sci. Res., Drottningholm, 63, 77–90. Food Agric., 84, 530–540. 20. Skuladottir, G.V., Schiöth, H.B., Gudmundsdottir, 9. Erikson, U., Sigholt, T. and Seland, A., 1997. Hand- E., Richards, B., Gardarsson, F. and Jonsson, L., ling stress and water quality during live transpor- 1990. Fatty acid composition of muscle, heart and tation and slaughter of Atlantic salmon (Salmo liver lipids in Atlantic salmon, Salmo salar, at ex- salar). Aquaculture, 149, 243–252. tremely low environmental temperature. Aquacul- 10. Erikson, U., 2001. Potential effects of pre-slaughter ture, 84, 71–80. fasting, handling and transport. In: Farmed Fish 21. Roth, B., Imsland, A. K., Gunnarsson, S. and Foss, A., 2006. Short communication. Thermal shocking

62 Aquaculture Research: From Cage to Consumption of turbot using ice water; effect on behavior and trout (Oncorhyncus mykiss) and Atlantic salmon blood chemistry. (In prep.) (Salmo salar). Aquaculture, 250, 796–803. 22. Robb, D.H.F., Wotton, S.B., McKinstry, J.L., 31. Akse, L., Joensen, S., Tobiassen, T., Midling, K.Ø. Sorensen, N.K. and Kestin, S.C., 2000b. Commer- and Eilertsen, G., 2005. Fangsthåndtering på store cial slaughter methods used on Atlantic salmon, snurrevadfartøy. Del 1: Blodtømming av torsk. determination of the onset of brain failure by Fiskeriforskning, Tromsø. Report 9/2005, 26 pp. electroencephalography. Vet. Rec. 147, 298–303. 32. Skjervold, P.O., Rørå, A.M.B., Fjæra, S.O., Synstad, 23. Roth B., Slinde, E. and Robb, D., 2006b. Percussive A.V., Vorre, A. and Einen, O., 2001. Effects of stunning of Atlantic salmon (Salmo salar). (In pre-, in- or post-rigor filleting of live chilled Atlan- prep.) tic salmon. Aquaculture, 194, 315–326. 24. Roth, B., Slinde, E., Imsland, A. and Moeller, D., 33. Tobiassen, T., Akse, L., Midling, K., Aas, K., Dahl, 2003. Effect of electric field strength and current R. and Eilertsen, G., 2005. Pre-rigor processing of duration on stunning and injuries in market-sized wild caught farmed raised cod (Gadus morhua L.): Atlantic salmon held in seawater. N. Am. J. Aqua- Effects on quality and shelf life. WEFTA, Antwerp, cult., 65, 8–13. Belgium, 19–22 September. 25. Lines, J.A., Robb, D.H., Kestin, S.C., Crook, S.C. 34. Mørkøre, T., Mazo, P., Lilleholt, R., Tahirovic, V. and Benson, T., 2003. Electric stunning, a humane and Einen, O., 2004. Kontraksjon av pre-rigor lak- slaughter method for trout. Aquacult. Eng., 28, sefilet. Effekt av fôring og stress. Programme con- 141–154. ference Havbruk [Aquaculture] 2004, Gardermoen, 26. Roth. B., Moeller, D. and Slinde, E., 2004. Ability 23–24 March. of electric field strength, frequency and current du- 35. Stien, L.H., Bjørnevik, M., Hirmas, E., Karlsen, Ø., ration to stun farmed Atlantic salmon (Salmo salar) Nortvedt, R., Rørå, A.M.B., Sunde, J and Kiessling, and pollock (Pollachius virens) and relations to A., 2005. The effects of stress and storage tempera- observed injuries using sinusoidal and squarewave ture on the colour and texture of pre-rigor filleted AC. N. Am. J. Aquacult., 65, 208–216. farmed cod (Gadus morhua L.). Aqua. Res., 36, 27. Morzel, M., Sohier, D. and van de Vis, H., 2003. 1197–1206. Evaluation of slaughtering methods for turbot with 36. Rørå, A.M.B., Furuhaug, R., Fjæra, S.O. and Skjer- respect to animal welfare and flesh quality. J. Sci. vold, P.O., 2004. Salt diffusion in pre-rigor filleted Food Agric., 83, 19–28. Atlantic salmon. Aquaculture, 232, 255–263. 28. Lines, J. and Kestin, S., 2005. Electric stunning of 37. Mørkøre, T. and Berner, M. 2006. Saltet filet av op- trout, power reduction using a two-stage stun. pdrettstorsk. Torskenettverksmøte, Tromsø, 16–17 Aquacult. Eng., 32, 483–491. February. 29. Robb, D.H.F., Phillips, A.J. and Kestin, S.C., 2003. 38. http://odin.dep.no/filarkiv/181 729/ Evaluation of methods for determining the preva- Nye_arter_KPMG.pdf lence of blood spots in smoked Atlantic salmon and 39. http://odin.dep.no/fkd/norsk/dok/regpubl/stmeld/ the effect of ex-sanguination method on prevalence 047 001–040 003/hov011-bn.html of blood spots, Aquaculture, 217, 125–138. 40. http://www.seafoodnorway.no/ 30. Roth, B., Torrissen, O.J. and Slinde, E., 2005b. The effect of slaughtering on blood spotting in Rainbow

Thematic area: Quality, Slaughter, Transport 63 64 Aquaculture Research: From Cage to Consumption Production

■ Repoductive Physiology in Cultured Cold-water Marine Fish ■ From Extensive to Intensive Production of Marine Fish ■ Water Quality – Salmonids ■ Effects of Intensive Production with Emphasis on On-growing Production: Fast Growth, Deformities and Production-related Diseases ■ A New Boost for the Norwegian Shellfish Sector Birgitta Norberg1), Geir Lasse Taranger1) and Helge Tveiten2) 1) Institute of Marine Research 2) Fiskeriforskning (Norwegian Institute of Fisheries and Aquaculture Research)

Reproductive Physiology in Cultured Cold-water Marine Fish

Insight into the mechanisms that control reproduction in cultured fish is important for several reasons. Improved routines in broodstock management are imperative in order to obtain good, stable gamete quality, to ensure fish welfare and for seasonally independent production of viable offspring. Early sexual maturation is a major bottleneck in aquaculture of most fish species. Maturation leads to great weight loss, impaired flesh quality and often increased mortality. Sexual maturation is regulated through the brain-pituitary-gonad (BPG) axis, and many central components of this axis have been recently identified and characterised in salmon, cod and halibut. Effects of the environment, especially photoperiod and temperature, on sexual maturation and spawning have been given special attention. In salmon, gonadal development was accelerated after exposure to long days from March and short days from May, but only partial ovulation was registered at the relatively high (10–12 ˚C) ambient temperatures, while cooling the water to 6–7 ˚C resulted in rapid and efficient ovulation and increased egg survival. In cod, high temperature accelerated spawning, but egg quality was reduced compared to eggs from spawners held at low temperature. While continuous (24-hour) light effectively arrests maturation in salmon, results from cod and halibut are more complex. The wolffish has a reproductive biology that is somewhat different from e.g. salmon or cod. It appears to have internal fertilisation where the male deposits sperm in the female via a small copulatory organ during a “mating act”. Such mating occurs only rarely in captivity, and methods for artificial fertilisation of wolffish eggs have been developed.

66 Aquaculture Research: From Cage to Consumption Photoperiod manipulation can delay sexual maturation by at least one year in Atlantic cod held in tanks. Use of additional light on sea cages has also had good results. (Photo: Geir Lasse Taranger, Institute of Marine Research)

Kap04-bilde01.eps

Introduction incidence of maturation may also increase the Early, or precocious, sexual maturation is a ma- genetic impact of farming on wild stocks. Ma- jor problem in aquaculture of Atlantic salmon turing salmon that escape are likely to return to and rainbow trout, as well as Arctic char, Atlan- the rivers in higher numbers than immature tic halibut and Atlantic cod. Maturation leads to salmon, and may be a large genetic and ecolo- substantial weight loss, impaired flesh quality gical threat to the wild salmon. Cultured cod and often increased mortality. In salmonids, also escape easily, and in addition they will maturation in seawater is a large welfare prob- spawn spontaneously in the sea cages, having a lem, since mature salmon and trout lose their potential genetic influence on local cod stocks. hyperosmoregulatory ability, and hence most of Increased knowledge in broodstock manage- the fish will die of dehydration following matu- ment is crucial in order to secure good gamete ration if kept in the sea. In cod, a weight loss of quality, to ensure fish welfare and for season- up to 30 per cent is often observed through the ally independent egg production by photoperi- spawning season. Increased mortality is also od and temperature control of spawning. To a observed in mature cod held in sea cages, espe- great extent, a lack of knowledge has led to a cially females that appear to have problems re- “trial-and error” strategy for control of matura- leasing their eggs (irregular spawners). High tion in aquaculture, both when it comes to pho-

Thematic area: Production 67 Figure 1. The brain-pituitary-gonad (BPG) axis controls sexual maturation in fish. A number of environmental factors, such as photoperiod and temperature, interact with internal factors such as energy status and biological clocks. Growth, energy depots and onset of maturation (puberty) appear to be connected – but the underlying physiological mechanisms are not clear. The figure shows some of the components that have been identified in salmon. Analytical methods have been established to quantify expression of relevant genes, identify localisation of hormone-producing cells and measure blood plasma concentrations of hormones.

Kap04-fig01.eps

toperiod manipulation for inhibiting early itary-gonad axis in fish as in other vertebrates maturation, and out-of-season spawning. (Figure 1), and a number of central components Therefore, there is a need to increase the basic have recently been characterised in salmon, cod understanding of the morphological, molecular and halibut. In salmon, quantitative methods and physiological changes that take place have been established to measure gene expres- through the process of sexual maturation, as sion of subunits of Follicle Stimulating Hor- well as to understand the impact of environmen- mone (FSH) and Luteinising Hormone (LH) tal factors and handling. Sexual maturation is and their receptors (FSH-R and LH-R), as well under endocrine regulation by the brain-pitu- as two forms of androgen receptors. Changes in

68 Aquaculture Research: From Cage to Consumption expression have been monitored through puber- vation of key components in the brain-pituitary- ty under different photoperiod regimes. In cod gonad (BPG) axis were studied. The objective and halibut, a number of genes have been iden- was to find out how temperature influences the tified that are important in the maturation pro- BPG axis during the spawning season, and to cess. The distribution of hormone producing test if treatment with gonadotropin releasing cells in the pituitary has been described by im- hormone analogue (GnRHa) could counteract munocytochemistry and in situ hybridisation in the negative effects of high temperature on ovu- salmon and halibut. Different techniques for lation that had been observed in previous stud- studying cell division and apoptosis (pro- ies. The study revealed clear effects of both grammed cell death) have been adapted and water temperature and GnRHa on plasma sex tested. The development of germ cells has been steroid hormones, ovulation and spermiation. studied in detail, especially in male salmon, hal- Exposure to high temperature inhibited ovula- ibut and cod. In cod testes, an increase in apop- tion and spermiation in all individuals, while tosis was apparent in fish exposed to continuous treatment with GnRHa enabled ovulation and light treatment, while no such change was spermiation in the high temperature exposed found in salmon where maturation was arrested fish. Cold water advanced ovulation slightly by photoperiod treatment. and improved egg viability, as compared to fish kept on ambient temperature. The most pro- found effects on plasma sex steroid profiles Control of spawning in salmon were seen on the levels of 17α,20β-dihydroxy- The timing of egg and milt production is mainly progesterone (17,20P), the maturation-inducing controlled by seasonal changes in photoperiod, hormone in salmon. High temperature inhibited in salmon as in other teleosts living in temperate the normal increase in this hormone in females waters. Spawning time can be advanced by a just prior to ovulation, while GnRHa treatment combination of long days early in the year and resulted in an apparently normal increase in short days from midsummer, while spawning 17,20P even in those fish that were held at high can be delayed by exposure to long days from temperature. In male fish, GnRHa yielded a dra- midsummer onwards. However, the shift in matic increase in 17,20P at all temperatures, spawning time may often be less than expected, and this was accompanied by a large increase in and in addition egg quality may be variable. sperm volume, compared to males held at ambi- High temperatures (12–14 °C) during spawning ent temperature. However, in untreated males, inhibit ovulation in salmon. When spawning there was only a tendency of lower 17,20P in was advanced by exposure to long days from males kept at high temperatures, and a moderate March and short days from May, only partial increase in males held in cold water compared ovulation was noted in females held at ambient to control males kept at ambient temperature. In temperature (10–12 °C), while exposure to cold addition, there was no marked peak in 17,20P in water (6–7 °C) resulted in 100 per cent ovula- males at ambient temperature, but only a gradu- tion and improved egg quality. An experiment al increase from around the time when running was carried out in which changes in tempera- milt could be expressed. The function of 17,20P ture to either high (14 °C) or low (6–8 °C) were in male fish is not established, but possible combined with hormone therapy during the functions may be stimulation of milt hydration spawning season. Timing of spawning and acti- and sperm activation. Interestingly, the increase

Thematic area: Production 69 in 17,20P after GnRHa treatment was rapid, and content were apparent in June. All experimental occurred one week before the fish had running groups had a very high incidence of maturation, milt. This may indicate that high temperature in particular male salmon where almost 100 per also acts to disturb the function of the 17,20P- cent matured after 1.5 years in the sea as grilse. receptors. A slight reduction in mature males, but not females, was seen in the “low-fat” group. Clear differences existed between different families, Early maturation in salmon both in weight, fat deposition and incidence of A positive correlation has long been assumed maturation – but no obvious correlation be- between growth, energy deposition and age at tween growth and maturation was seen between first maturation (puberty) in fish. This assump- families. However, it was clear that the largest tion is based on the observation that cultured individuals in mid-winter within each family fish mature at a significantly lower age and had a higher probability to mature the next au- smaller size than wild fish, and is also based on tumn than smaller fish. The experiment showed other observations in wild stocks. Cultured cod a connection between somatic growth and sex- normally mature at two years of age – some al- ual maturation within families, but the differ- ready at one year – while wild cod usually ma- ence between the two diets was much smaller ture at 4–8 years of age in Norwegian waters. In than expected. The latter may be due to the salmon, similar observations are made but the compensatory growth of the “low-fat” group situation is complicated by the natural occur- during spring, which in turn may be due to rath- rence of precocious males that mature at a very er high general fat levels in both diets during small size before they migrate to sea. However, spring and summer. If compensatory growth there is a lack of accurate documentation on the was the cause of the very high incidence of connections between energy availability, energy maturation in all groups – also within the “low- deposition and maturation in fish, as well as fat” group – this may indicate that salmon are how these may vary genetically between differ- able to enter maturation late in spring. Analyses ent families. of sex hormones in the blood and histology of gonads are currently being performed to find Fish from different family groups of salmon out when “onset” of puberty occurs in different were individually tagged and fed diets contain- individuals under various environmental condi- ing different levels of fat through their first au- tions. One possible cause of the high male tumn, winter and spring in the sea. From the maturation in the fish in all groups may be that onset of the experiment in September and until the experiment was carried out in small January, fish that were given a relatively high (5x5x8 m) sea cages. High incidence of early fat content in the diet (28 vs. 18 per cent) had male maturation has been observed in small sea significantly higher growth, development in cages in several previous trials, but the reason condition and fat content. However, the fish giv- for this is not known. One possible hypothesis en a “low-fat” diet showed compensatory is that swimming activity is lower in small cag- growth in the period from January to June, es, and that this in turn may affect maturation, when the fat content was increased in both e.g. by affecting energy balance and its endo- groups, to 38 and 28 per cent, respectively, so crine regulation. that no differences in growth, condition or fat

70 Aquaculture Research: From Cage to Consumption Cod oocytes with central (a) and migrating (b) cell nucleus. A migrating cell nucleus indicates that final maturation of the oocyte has commenced. (Photo: Helge Tveiten, Fiskeriforskning)

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Temperature and spawning in sponding numbers at 4 °C and 8 °C were four cod and 15 per cent. Fertilisation rates decreased Temperature affects the timing of spawning and with increasing temperature, and were 43, 34 the length of the spawning period in cod, as well and 23 per cent in the groups held at 4 °C, 8 °C as egg viability and embryonic development. and 12 °C. Almost no embryos originating from Cod held at high temperatures (8 °C and 12 °C) fish held at 12 °C survived until hatching. The started spawning 2–4 weeks earlier than cod amount of eggs produced appeared not to be held at 4 °C. At the same time, there was a ten- affected by temperature. dency for a shorter spawning period in cod held at high temperatures (approx. six weeks at 8 °C and 12 °C, compared to eight weeks at 4 °C). Early maturation in cod There was a clear effect of temperature on egg Photoperiod manipulation can delay or block quality, where the percentage of dead eggs and sexual maturation in cod kept in indoor tanks, embryos with erratic cell division increased and delayed maturation and increased growth with temperature: At collection, dead eggs con- has been seen in cod exposed to continuous stituted 27, 36 and 65 per cent of the total egg light (LL) in sea cages, similar to what is seen volume at 4 °C, 8 °C and 12 °C, respectively. in salmon. However, results from initial experi- The number of dead eggs also increased with ments suggested that maturation could only be length of exposure of the brood fish to high tem- delayed by 4–6 months when exposing cod to perature. Temperature also had a pronounced LL sea cages, while maturation was delayed at effect on the percentage of normally developed least one year in most cod exposed to LL in in- eggs and embryos. At 12C, 88 per cent of ferti- door tanks. LL of high intensities appeared to lised eggs had abnormal early cell division (4– be more effective than low intensities in delay- 64 cells). Abnormal division at this stage is of- ing or arresting maturation in cod in outdoor ten followed by the death of the embryo. Corre- tanks. Therefore, we have studied more closely

Thematic area: Production 71 abc

Normal (a) and abnormal (b and c) cell division in 16-cell stage cod embryo. Loss of cell-to-cell contact (c) was common in eggs originating from broodstock held at high (12 ˚C) temperature. (Photo: Helge Tveiten, Fiskeriforskning)

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the effects of LL of different light intensities in what less efficient. Still, the 300-lux treatment outdoor tanks. The effects of different light in- arrested gonadal growth in most individuals, tensities and colours on plasma levels of mela- and appears to be sufficient in commercial cod tonin were also studied. Melatonin is mainly farming. It was found, however, that male cod produced in the pineal organ during the dark pe- could develop a high degree of maturation, seen riod of the diel cycle, and is believed to transmit as production of gametes, even in very small information of photoperiod signals to give testes. This will most likely not be important for physiologic responses in the individual. Thus, it commercial production as it will not impair so- was hypothesised that if nighttime plasma lev- matic growth, but is very interesting from a els of melatonin could be brought below a cer- more fundamental, physiological perspective. tain threshold with sufficient light intensity, The degree of maturation also varied with local- there would also be good effects on incidence of isation of gametes in the testis, where the most maturation. Experiments in tanks showed that immature stages were found in the periphery. cod are very sensitive to light in terms of mela- This finding may be very important for exami- tonin plasma levels – more so than salmon – and nation of degree of maturation in the fish, and also that a strong biological clock controls the also for understanding the molecular and circadian cycle of melatonin. The biological physiological control of spermatogenesis. clock appears to regulate nighttime melatonin levels even after six months of continuous pho- However, the amount of light that needs to be toperiod in indoor tanks. There were also indi- used to arrest maturation in cod in sea cages re- cations that blue and green light had a greater mains to be established. It was expected initial- suppressive effect on melatonin secretion than ly that cod would be less sensitive to light than yellow and red light given in the same amounts salmon, partly from early measurements of at night. This finding may be important for de- melatonin levels in cod from cages held under velopment of effective photoperiod regimes and continuous, additional light. Practical experi- new lamps. In the outdoor tank experiment with ence with cod in sea cages also indicates that different light intensities, 900 lux of additional cod need more light than salmon to arrest matu- light was approximately as effective in reducing ration. These apparently contrasting results maturation as true continuous light in indoor may be a consequence of different behaviour in tanks, while 300 lux in outdoor tanks was some- the two species: salmon are attracted to light,

72 Aquaculture Research: From Cage to Consumption fore or after Christmas. There appears to be a 100 critical window of determination in October- November (6–7 months post-hatch) for male 80 maturation at one year, as no mature males were 60 found in the groups that received reduced feed ration during this period. Incidence of matura- 40 tion at two years was also affected in the groups % mature females that received reduced feeding, independent of 20 period (Figure 2). However, reduced feeding 0 also results in reduced growth, and the effect on low medium high final size of the fish needs to be investigated fur- feed ration ther. Kap04-fig02.eps Figure 2. Proportion of mature female cod at two years of age, in groups that were given either low (30 %), medium (60 %) or high (100 %) feed ration between six and 10 Reproductive physiology in months post-hatching. Fish fed the high feed ration were fed to satiation every day, Monday to Friday. halibut Since the 1980s, a substantial research effort has been made in order to provide a foundation especially in winter, while cod do not seem to for aquaculture of halibut. After a period with have such behaviour. This may explain why cod highly variable results, intensive production – which appear to be biologically more sensi- methods are now established that seem to give a tive to light – still need more light on their cages stable supply of fry. Some bottlenecks remain, than salmon. A possible practical solution to the however, such as problems connected to brood- problems with photoperiod control of cod in sea stock management and gamete quality, espe- cages may be to construct an even field of light cially in seasonally independent fry production, within the whole cage, and in this way stop the as well as poor growth and early maturation in fish from swimming out of the light. males. Knowledge of basic processes in reproductive physiology is necessary both to ensure Photoperiod manipulation of maturation has good spawning results and to develop methods been relatively well studied in cod, with respect for controlled maturation. Growth patterns and to both seasonally independent production of changes in plasma levels of central hormones in eggs and larvae, and to blocking or delaying male and female halibut from juvenile (two- maturation. Less well known is the connection year-old) fish until first (female) or second between growth and energy acquisition during (male) maturation were therefore documented. the first year of life, and maturation as one- or Female halibut increased their weight more two-year-old fish. So experiments have been than threefold during an 18-month period that carried out to study this connection. The results started two years prior to their first maturation. show that incidence of maturation can be affect- Male halibut matured three years before feed in both one- and two-year old cod, by limit- males (five and eight years of age, respectively) ing the growth rate of cod 6–10 months old. and this was reflected in their growth pattern, This was achieved by giving the fish reduced such that male average weight was 80 per cent feed rations in periods of up to four weeks, be- of female average weight at four years, 65 per

Thematic area: Production 73 cent at five years, and 50 per cent at six years of was to ensure optimal content of nutrients in age. Plasma sex steroid levels varied according halibut eggs, and thus contribute to production to maturity status. One year (in males) and two of high-quality, viable larvae. The composition years (in females) before first maturation, of the feed was determined after analysis of a “dummy runs” were apparent in both sexes, large number of egg batches from halibut that with elevated levels of androgens and estrogens had been held in captivity for varying time peri- during the time of year when gonadal growth is ods. The significance of vitamins A, B and C as usually high, Follicle stimulating hormone well as fatty acids in the feed was investigated. (FSH) and luteinising hormone (LH) and their The results showed a general improvement in two subunits α and β were purified from halibut egg quality at high dietary levels of vitamin C, pituitaries and characterised chemically and specific B vitamins and specific fatty acids. An biologically. This was the first time FSH and increase of fat content in the feed from 20 to 30 LH were characterised in a flatfish. In addition, per cent dry matter resulted in higher fecundity the genes encoding the subunits were cloned in the female fish. Broodfish with a high indi- and sequenced. Eight hormone-producing cell vidual feed intake produced eggs with higher types are present in the halibut adenohypophys- fertilisation and survival, and thus higher poten- is, and these were identified and localised. FSH tial for producing more halibut larvae of good and LH are produced in different cell types; pre- quality. liminary results indicate that both types are active in immature male halibut, although the cell number is lower than in mature fish. Similar re- Early maturation in halibut sults have previously been obtained in tuna, but Even though the timing of spawning is relat- are in contrast to the situation in salmon, where ively easy to control in halibut by photoperiod only FSH appears to be produced before puber- manipulation, and continuous light promotes ty. The receptors for FSH and LH have been growth, there is still no effective protocol to cloned and characterised from halibut ovaries, block or delay puberty and promote growth in and this will enable further studies of the gona- male halibut. Available information suggests dotropins and their function at different stages that a short day period may be important, but we of the maturation process. Synthetic pathways still need more knowledge on the mechanisms for steroid hormones have been characterised in controlling light perception and transmission of the ovaries, in order to identify the maturation- photoperiod signals in halibut. Sustainable inducing hormone (MIH). The halibut testis commercial production requires that halibut was characterised with regard to cell develop- grow from 100 g to 5 kg in 30 months. Effects ment and organisation. Each step in cell of different photoperiod regimes on gonad de- development was related to general testis velopment in halibut as well as the impact of growth and to plasma levels of the androgens age/size of the fish at the start of manipulation testosterone and 11-ketotestosterone. have been investigated, in order to develop protocols to reduce incidence of early maturation in males. Transfer to continuous light or simu- Broodstock nutrition lated natural photoperiod at either summer or A broodstock feed for halibut was developed, winter solstice showed that continuous light did based on microwave technology. The objective not delay or block maturation, and mature, two-

74 Aquaculture Research: From Cage to Consumption year-old males were first recorded in September gan during what can be characterised as a mat- in the group that was held under continuous ing act. Such mating happens only very rarely in light – three months before the first mature captivity, so methods for artificial fertilisation males were found in the group held at a simu- of wolffish eggs have been developed. Sperm lated natural photoperiod. Gonad development cells from wolffish also have some characteris- in halibut starts in summer and continues tics that suggest internal fertilisation. Unlike through autumn and early winter. Males nor- many other fish species, wolffish sperm is ac- mally have running milt from December tive in the seminal fluid (as with mammals), the through May. A strong summer signal in mid- cells can move actively for up to 48 hours after winter may have advanced maturation, and this stripping, and the sperm is inactivated in sea can partly explain that fish that were transferred water. from a simulated natural photoperiod to continuous light in December had the highest inci- In teleost fish, final maturation of the oocyte dence of mature males in September the comprises important processes such as migra- following year. The higher growth rate at con- tion and breakdown of the germinal vesicle tinuous photoperiod may also have enabled the (GVBD). In wolffish and other marine species fish to mature earlier than normal. A desynchro- that are candidates for aquaculture in Norway, nisation of maturation between individuals was endocrine regulation of final maturation has not observed at continuous light. This was supported by GSI and by highly variable plasma testosterone levels in this group.

Results from the project have yielded valuable information on growth and maturation in hali- but, and have been used as a basis for current protocols in halibut farming. However, the results were based on experimental fish that had been held in very good conditions as juveniles, during the six months that preceded the start of the experiment, with respect to both water temperature and size grading. The importance of the juvenile (<500 g) stages for later growth and age at maturation are poorly understood and need further investigation.

Wolffish Wolffish (Anarhicas sp.) have a reproductive biology that is somewhat different from salmon and cod, for example, as they have a form of in-

Kap04-bilde04.eps ternal fertilisation in which the male transfers Female spotted wolffish just prior to spawning. sperm to the female via a small copulatory or- (Photo: Helge Tveiten, Fiskeriforskning)

Thematic area: Production 75 been studied extensively. Hence, an important vealed a high Na+: Mg2+ ratio in seminal fluid goal was to establish a method for investigation from individuals with active sperm, compared of how different steroid hormones can induce to individuals with inactive sperm. A high Na+: final maturation in vitro. The experiments Mg2+ ratio may therefore be a good indicator of showed that three steroids were particularly ef- sperm quality in wolffish. The concentrations of fective in inducing GVBD in wolffish (Tveiten Na+ and Mg2+ in seminal fluid were positively and Scott, 2003). The three steroids are all and negatively correlated to plasma levels of progesterone derivatives, but only one, 17,20P, 11-ketotestosterone, respectively (Figure 3). seems to be synthesised naturally in the wolff- Such correlations may indicate that 11-KT is in- ish ovary. We therefore assume that 17,20P is volved in regulation of the ionic composition of the maturation-inducing hormone in wolffish. the seminal fluid. Further experiments showed Experiments showed, however, that 17,20P is that milt production, and hence fertilisation po- rapidly metabolised by wolffish ovaries to other tential, in wolffish can be increased through compounds, e.g. 17,20P-sulphate, that occur in treatment with sex steroids. Both androgens high concentrations in plasma. Further studies (11-KT) and progestins (17,20P) influence the showed that sulphation of 17,20P, which leads volume of milt produced. Further, it is shown to a complete loss of maturation-inducing ca- that treatment with the stress hormone cortisol pacity, is likely a mechanism that is necessary in reduces milt production as well as the number order to avoid “overexposure” of the oocyte to of individuals with active sperm. Increased maturation-inducing hormone, since a negative plasma cortisol levels also result in a reduction effect on egg viability was apparent if females in motile sperm cells within an individual. were treated with high doses of 17,20P in the These findings indicate that environmental fac- period just prior to final maturation. tors that are perceived as “stressful” to the fish may affect the reproductive potential in wolff- Poor milt production, with low volume and den- ish. sity as well as low motility of sperm cells in male wolffish, is a limitation for efficient hatchery management. The causes of low milt quality Conclusions and future are not known. In order to study how different perspectives environmental conditions affect sperm quality During the programme period, research has in wolffish, it is vital to know which factors and been focussed on understanding how sexual mechanisms are central in this process. Com- maturation and spawning are affected, and can puter Assisted Sperm Analysis (CASA) is now be controlled, by environmental factors such as in use to obtain an objective measure of sperm photoperiod and temperature. A number of new quality. CASA is based on video recordings of molecular tools have been established for study sperm cells through a microscope, where infor- of reproduction in salmon, cod and halibut. mation is generated on percentage of motile These tools will be very important in functional cells, swimming speed and swimming pattern, genomic research in the coming years. Some for example. In fish, the ionic composition of specific research challenges are outlined below, the seminal fluid may have great importance for in which a combination of molecular and physio- the function of sperm cells. Analyses of the ion- logical research has the potential to yield in- ic composition of wolffish seminal fluid re- teresting results of high scientific impact.

76 Aquaculture Research: From Cage to Consumption 150 Circles - Mg2+, central factors and molecular mechanisms in Squares- Na+ Open symbols - Inactive sperm embryonic development, a comprehensive Filled symbols - Active sperm approach is needed, where the different phases

100 in oocyte and embryonic development are seen as parts of a continuous process rather than separate events.

Na+50 Mg2+ (mM) All female production – sex differentiation

0 In halibut, a large sex dimorphism in growth is 0123present, both before and especially after sexual 11-ketotestosterone (ng/ml) maturation, so that females grow to be many Kap04-fig03.eps Figure 3. Interaction between concentrations of Na+ and times larger than males. Finding a solution to Mg2+ in seminal fluid, plasma 11-KT, and sperm motility in the problems connected with grading/elimina- spotted wolffish. tion of male halibut is thus very important. Since sex differences are small and individual Maternal effects on developmental variations are large in juvenile halibut, growth biology rate is not a practical means to separate males In all animals, there is a period between fertili- from females. Continuous photoperiod increas- sation and activation of the genome of the zy- es growth, but as described above, it is not ef- gote. During this period, embryonic develop- fective in blocking or delaying male maturation. ment is completely dependent upon regulatory An alternative solution to the problem is to pro- factors deposited in the egg (maternal factors), duce all female populations of halibut. This is before it is capable of synthesising these factors possible, since sex differentiation in fish can be itself (zygotic factors). In addition, maternal influenced during larval development – and factors will also exert important functions long phenotypic males that are genetic females can after the genome is activated, either by them- be produced. In the next generation, milt from selves or together with zygotic factors. Deposi- these males can be used to fertilise eggs, and the tion of maternal factors may occur through all offspring will thus consist of 100 per cent fe- stages of oogenesis and to a high degree appears males. to be in the form of mRNA. Data available in other oviparous animals show that maternal Production of all-female, all-male or sterile mRNA may code for factors important in cell populations of cod will become increasingly division, cell contact (adhesion) and body axis important since commercial cod production is formation, where the dorsoventral axis specifi- expected to grow rapidly. Production of “mono- cation appears particularly influenced by mater- sex” populations is a means to circumvent cod nal determinants. Deformities that occur late in spawning in sea cages, which will reduce the embryonic or larval development may have possible genetic impact of cultured cod on wild their origin in incorrect processing of maternal populations. However, escaped cod may still be factors as early as during oogenesis, final matu- able to reproduce, so research aimed at produc- ration or early cell division. In order to study the ing sterile cod with the same or improved

Thematic area: Production 77 growth potential as fertile fish will be a pivotal terised in several fish species, and this opens up challenge in coming years. new possibilities to study the connection between growth, lipid depots and puberty in fish. A number of other factors important for the on- Connections between feed intake, set of puberty have been identified in mammals, growth, energy deposition and puberty and with the high (and increasing) availability A number of studies on salmon show a correla- of gene sequence data in fish, it is probable that tion between somatic growth and age at first such factors will also be identified in relevant maturation; results from experiments on cod in- aquaculture species in the near future. However, dicate that this may be true for other species as studies of the mechanisms regulating puberty well. The physiological and endocrine connec- are complicated in species such as salmon, cod tion is not well known and there is a need for and halibut because of their high phenotypic more research on molecular and endocrine reg- plasticity regarding the age at which puberty de- ulation of growth, puberty and recruitment of velops – due to environmental influences and germ cells. In mammals, a clear correlation has possibly also social interactions. Good experi- been found between body size, fat level and age mental design should therefore be combined at puberty: a hormone produced in fat cells has with in-depth genetic knowledge as well as new been identified. Called leptin, it is important for molecular and endocrinological techniques, in both regulation of appetite and puberty. A cor- order to improve the understanding of how pu- responding hormone has recently been charac- berty is regulated in fish.

References Bromage, N.R., Porter, M.J.R. and Randall, C.F. 2001. Hyllner, S.J., Norberg, B. and Haux, C. 1994. Isolation, The environmental regulation of maturation in partial characterization, induction, and the occur- farmed finfish with special reference to the role of rence in plasma of the major vitelline envelope pro- photoperiod and melatonin. Aquaculture, 197: 63– teins in the Atlantic halibut (Hippoglossus 98. hippoglossus) during sexual maturation. Can. J. Dahle, R., Taranger, G.L., Porter, M. and Norberg, B. Fish. Aquat. Sci. 51(8), 1700–1707. 2000. Growth and sexual maturation of Atlantic cod Kagawa, H., Kawazoe, I., Tanaka, H., and Okuzawa, K. (Gadus morhua L.) under different light intensities. 1998. Immunocytochemical identification of two In: Norberg, B., Kjesbu, O. S., Taranger, G.L., distinct gonadotropic cells (GTH I and GTH II) in Andersson, E. and Stefansson, S.O. (Eds.): Repro- the pituitary of bluefin tuna, Thunnus thynnus. Gen. ductive Physiology of Fish. John Grieg Forlag A/S, Comp. Endocrinol. 110, 11–18. p 336. Karlsen, Ø., Norberg, B., Kjesbu, O.S. and Taranger, Dahle, R., Taranger, G.L., Karlsen, Ø., Kjesbu, O.S. and G.L. 2006. Effects of photoperiod and exercise on Norberg, B. 2003. Gonadal development and asso- growth, liver size, and age at puberty in farmed At- ciated changes in liver size and sexual steroids dur- lantic cod (Gadus morhua) at two years of age at ing the reproductive cycle of captive male and different locations along the Norwegian coast. ICES female Atlantic cod (Gadus morhua L.). Comp. J. Mar. Sci. (63)2: 355–364. Biochem. Physiol. A 136: 641–653. Kime, D.E. and Tveiten, H. 2002. Unusual motility Hansen, T., Karlsen, Ø., Taranger, G.L., Hemre, G.I., characteristics of sperm of the spotted wolffish. Holm, J.C. and Kjesbu, O.S. 2001. Growth, gonadal Journal of Fish Biology, 61: 1549–1559. development and spawning time of Atlantic cod Kobayashi, T., and Andersen, Ø. 2003. Molecular study (Gadus morhua) reared under different photoperiod of gonadotropin receptors in Atlantic halibut. 7th regimes. Aquaculture, 203: 51–67. International Symposium on Reproductive Physiol- ogy of Fish, Mie, Japan, May 18–23, 2003.

78 Aquaculture Research: From Cage to Consumption Kobayashi, T., and Andersen, Ø. 2005. Molecular diver- Y., and Andersson, E. 2004. Effects of photoperiod, sity of the gonadotropin receptors in Atlantic hali- temperature and GnRHa treatment on the reproduc- but (H.hippoglossus). International Symposium on tive physiology of Atlantic salmon (Salmo salar L.) Gonadal and Nongonadal actions of LH/hCG, broodstock. Fish Physiol Biochem 28:403–406. Turku, Finland, June 3–4 2005. Tveiten, H. and Scott A.P. 2003. Steroid synthesis in Methven, D.A., Crim, L.W., Norberg, B., Brown, J.A., follicular cells and induction of germinal vesicle Goff, G.P. and Huse, I. 1992. Seasonal reproduction breakdown in spotted wolffish (Anarhichas minor) and plasma sex steroid and vitellogenin levels in At- oocytes. Fish Physiology and Biochemistry, 28: lantic halibut (Hippoglossus hippoglossus). Can. J. 373–374. Fish. Aquat. Sci. 49 (4), 754–759. Tveiten, H., Jobling, M. and Andreassen, I. 2004. Influ- Norberg, B. and Weltzien, F.-A. 2001. Fysiologisk reg- ence of egg lipids and fatty acids on egg viability, ulering av pubertet og kjønnsmodning i kveite. and their utilization during embryonic development Concluding report to Research Council of Norway. of spotted wolffish, Anarhichas minor Olafsen. Norberg, B. and Weltzien, F.-A. 2003. Vekst og kjønns- Aquaculture Research, 35: 152–161. modning hos kveite: Interaksjoner og koblinger til Vermeirssen, E.L.M., Mazorra de Quero, C., Shields, lysperiode. Concluding report, Research Council of R.J., Norberg, B., Kime, D. E. and Scott, A.P. 2004. Norway project 128 754/122 Fertility and motility of sperm from Atlantic halibut Norberg, B., Brown, C.L., Halldorsson, O., Stensland, (Hippoglossus hippoglossus) in relation to dose and K., and Bjørnsson, B. Th. 2004. Photoperiod regu- timing of of gonadotrophin-releasing hormone ago- lates the timing of sexual maturation, spawning, sex nist implant. Aquaculture 230: 547–567. steroid and thyroid hormone profiles in the Atlantic Weltzien, F.-A., Karlsen, Ø. and Norberg, B. 2003. cod (Gadus morhua). Aquaculture 229: 451–467. Growth patterns and plasma levels of 11-ketotest- Nortvedt, R., Halldorson, O., Mangor-Jensen, A., Sma- osterone, testosterone and IGF-1 in male Atlantic radottir, H., Waagbø, R. and Norberg, B. 2001. Vari- halibut (Hippoglossus hippoglossus). Fish Physi- ability in egg composition in captive broodstock of ol.Biochem. 28: 227–228. Atlantic halibut (Hippoglossus hippoglossus) from Weltzien, F.-A., Norberg, B., Helvik, J.V., Andersen, Iceland and Norway. Short communication, Larvi Ø., Swanson, P. and Andersson, E. 2003. Identifica- 2001, Gent, Belgia. tion and localization of eight distinct pituitary cell Nortvedt, R., Mangor-Jensen, A., Waagbø, R. and types in male Atlantic halibut (Hippoglossus hippo- Norberg, B. 2003. Ernæringsbetinget eggkvalitet glossus L.). Comp Biochem Physiol, vol 134/2 pp hos kveite, Hippoglossus hippoglossus. Sluttrapport 315–327. til Nordisk Ministerråd, NMR prosjektnr 661 045– Weltzien, F.-A., Norberg, B. and Swanson, P. 2003. Iso- 90 929. lation and characterization of FSH and LH from Taranger, G.L., Haux, C., Stefansson, S.O., Björnsson, pituitary glands of Atlantic halibut. Gen. Comp. B. Th., Walther, B.T. and Hansen, T. 1998. Abrupt Endocrinol 131(2): 97–105. changes in photoperiod affect age at maturity, tim- Weltzien, F.-A., Kobayashi, T., Andersson, E., Norberg, ing of ovulation and plasma testosterone and oestra- B.and Andersen, Ø. 2003. Molecular characterisa- diol-17b profiles in Atlantic salmon, Salmo salar. tion of the FSHß, LHß, and -subunt of Atlantic hal- Aquaculture 162: 85–98. ibut (Hippoglossus hippoglossus L.). Gen. Comp. Taranger G.L., Stefansson, S.O., Oppedal, F., Anders- Endocrinol.131, 87–96. son, E., Hansen, T. And Norberg, B. 2000. Photope- Weltzien, F.-A., Andersson, E., Andersen Ø., Shal- riod and temperature affect spawning time in chian-Tabrizi, K., and Norberg, B 2004. The brain- Atlantic salmon, Salmo salar. In: Norberg, B., Kjes- pituitary-gonad axis in male teleosts, with emphasis bu, O.S., Taranger, G.L., Andersson, E. and Stefans- on the flatfish (Pleuronectiformes). Comp.Bio- son, S.O. (Eds.): Reproductive Physiology of Fish. chem. Physiol. A 137: 447–477. John Grieg Forlag A/S, p 345. Taranger, G.L., Vikingstad, E., Klenke, U., Mayer, I., Stefansson, S.O., Norberg, B., Hansen, T., Zohar,

Thematic area: Production 79 Anders Mangor-Jensen1), Victor Øiestad2), Terje van der Meeren1), Ingrid Lein3), Torstein Harboe1), Ingegjerd Opstad1), Gunvor Øie4), José Rainuzzo4), Jan Ove Evjemo5), Kjell Inge Reitan4) and Bendik Fyhn Terjesen3)

1) Institute of Marine Research, 2) Akvaplan-niva (Consultancy and Research Services in Aquaculture and Marine and Freshwater Environments), 3) Akvaforsk – The Institute of Aquaculture Research, 4) The SINTEF Group, 5) Norwegian University of Science and Technology (NTNU)

From Extensive to Intensive Production of Marine Fish

Sea ranching of marine fish in Norway has only been experimental and small- scale thus far. Therefore the terms “intensive” and “extensive” refer to the two main methods of juvenile production. Sea ranching of three species (cod, halibut and turbot) has been carried out, although cod is the only candidate that to some extent has been produced in ponds with copepods as the only food source. Cur- rently, some 100,000 cod juveniles are produced extensively each year, and their quality is still regarded as superior. However, the demand for cost efficiency has forced production towards more intensive methods that also permit year-round operation. The following section describes the beginnings of this transition and how the experience from these early trials has formed today’s intensive industry.

80 Aquaculture Research: From Cage to Consumption The beginning... tory studies on the other, thus shedding further The inclusion of the experimental ecosystem as light on those processes in focus in each con- an auxiliary instrument in the study of early life text. history of fish (ELHF) was initiated in 1975 by a team of scientists at the Institute of Marine This new three-pronged method was initially Research (IMR) in Bergen. At face value this applied to Atlantic cod with field studies by on appeared to be a revolutionary and innovative research vessel in Lofoten, detailed studies of move, but in fact it continued a nearly 100-year- cod larvae at the IMR laboratory in Bergen and old Norwegian tradition started by Captain experimental ecosystem studies with cod larvae Gunnar M. Dannevig. In 1886 he began a study in the two saltwater basins at the old Flødevigen on cod larvae in a saltwater basin on land, an Hatchery – a facility that is now part of the activity continued in the 1910s by his son, Alf IMR. The working hypothesis in focus was the Dannevig. Both were directors of the renowned one advanced by Dr. Hjort and re-advanced by Udklekningsanstalten for Saltvannsfisker Dr. Lasker: Starvation as the main reason for (“Hatchery for Saltwater Fish”) in Arendal, larval mass mortality. All activity was designed which was established in 1883 on the recom- to substantiate this hypothesis. Laboratory stud- mendation of Professor Georg Ossian Sars and ies indicated that the required density of prey inspired by similar facility in Woods Hole, animals was close to 1,000 nauplii per litre – in USA. The use of experimental ecosystems was stark contrast to densities observed in Lofoten, continued in the 1930s by the former director at typically in the range of 1–20 nauplii per litre. the IMR Gunnar Rollefsen at the other Norwe- Thus, these results supported the hypothesis gian Hatchery for Saltwater Fish, located in from the very beginning. However, the results Trondheim. He conducted restocking of flatfish from the transitional studies did not fit in, as larvae in a land-locked fjord, while carrying out high larval survival and growth rates were ob- larval studies in an experimental ecosystem or served at prey animal densities below 10 per basin at the Hatchery, which was known as litre in the 4,400-m3 basin. Trondhjem Biologiske Stasjon, and which is now part of the University of Trondheim. The observations from the experimental ecosystem indicated that the nauplii densities observed Nevertheless, the timing of the renewed use of in Lofoten in the range of 1–20 organisms per experimental ecosystem, after a pause of 40 litre should be sufficient for high larval survival years, was somewhat intriguing since it coincid- and growth rates. This observation coincided ed exactly with an expert ELHF meeting at the with another surprising observation from Lofo- Southwest Fisheries Center in La Jolla, Califor- ten, namely that it was impossible to detect cod nia. The conclusions from the meeting strongly larvae in a clear state of starvation. If starvation supported the use of the experimental ecosys- were to blame for high mortality, a large frac- tem as a forceful instrument to better under- tion of the larvae should have been in a state of stand the dynamic processes that take place starvation at any given time. The main reason during the early life history of fish. The method for mortality in the experimental ecosystem was called “transitional studies” as it was meant seemed to be predation, either from other or- to function as a critical link between field obser- ganisms or in the form of cannibalism. Gunnar vations on the one hand and traditional labora- M. Dannevig, carefully observing events in his

Thematic area: Production 81 ecosystem in 1886, reported from a morning success with cod juvenile production thus gave observation on 10 June 1886: “The cod larvae substance and support to a future propagation are attacking and eating each other to a large ex- or restocking programme – as well as to farm- tent”. This observation from the first experi- ing of cod. A few years later a breakthrough was mental ecosystem was confirmed during similar also achieved with juvenile production of At- studies in the 1970s, and as well as in the large- lantic halibut, Dover sole and turbot in pilot scale (1.6 million m3) commercial ecosystem scale. This took place in small-scale experimen- constructed in the 1980s and in two smaller tal ecosystems called mesocosms, and these pilot-scale basins adapted for ecosystem studies events led a number of Norwegian companies to in Austevoll as part of the IMR Aquaculture start commercial juvenile production of three Station facilities. As a result the starvation species – cod, turbot and halibut – in the late hypothesis was weakened, and the current inter- 1980s. national research trends concur with the following observations from the experimental The surprisingly high survival rates observed in ecosystem: predation is the overwhelming rea- experimental ecosystems where no predators son for mortality among the early life stages of were present opened an avenue for exploitation fish and starvation is more of a fine-tuning within aquaculture. However, from the very be- mechanism. ginning, there were calls for juveniles to be produced intensively rather than extensively. This The encouraging experiences from the first six divergence of opinion still exists. Nevertheless, years with experimental ecosystems in Fløde- the reality that halibut, turbot and cod juveniles vigen – resulting in high survival rate for almost could be produced extensively gave Norwegian all of the 10 marine species included in the ex- marine aquaculture a jumpstart. A positive con- periments – triggered similar activities at the sequence of the focus placed on turbot is that a new Aquaculture Station in Austevoll in the Norwegian company is still leading European 1980s, first with cod and subsequently with tur- producer of turbot. All in all, the early push bot, sole and halibut in two ponds, the Hyltro gave Norway a head start in cold-water marine Pond (60,000 m3) and the Svartatjønn (9,000 fish farming – and it still holds the lead. m3). At both locations the same, nearly unchanged survival rate of cod of one to two juve- Naturally produced zooplankton is the nutri- niles per m3 was observed, then repeatedly tional basis for the first-feeding fish larvae. To confirmed in the 250,000-m3 Paris Pond, which maintain the energy flow exclusively for that is still operated by IMR after 20 years of pro- purpose, the pond or basin is treated with rote- duction. The real breakthrough in large-scale none every autumn to exterminate organisms production of juvenile cod came in 1983, just in that otherwise would compete for the feed time for the 100-year anniversary of the resources. Species surviving the treatment are Flødevigen Hatchery, which was celebrated those with a production of bottom-resting eggs with an international conference on the propa- in the autumn, as is the case for numerous cal- gation of Atlantic cod – a rather bold topic since anoid copepod species. Those eggs will typical- large-scale farming of cod juveniles had not yet ly hatch in early spring and due to initial minor been successful when the main theme of the predation pressure, new generations will cas- conference was announced! The coinciding cade, thus supplying the fish larvae with ample

82 Aquaculture Research: From Cage to Consumption food – with each life stage suited to different- have thus been trained to be more aggressive sized fish larvae. Normally the ecosystem is hunters. This might partly explain why these ju- dominated by a single copepod species through- veniles are a faster-growing fish during the on- out the season – sometimes in combination with growing phase and are also better suited to sea succession of one dominant species. Rotifers ranching than the intensively produced juve- might multiply quickly in ponds or basins, func- niles, which are generally accustomed to a tioning as an additional food resource for a slow-moving or inert diet. while. This very simplified ecosystem, where fish larvae often make up the only significant Both production systems have been used in predator of zooplankton, might explain the high Norway for 20 years. The trend is toward inten- survival rate of fish larvae beyond metamorpho- sive production; indeed, all halibut juveniles sis. A characteristic of juveniles produced ac- have been intensively produced since 2003 – a cording to the natural method has been their sharp rise from 2000, when 50 per cent came very high quality without any type of deforma- from extensive systems. A fraction of the cod tion and with perfect pigmentation, both of juveniles are still from extensive systems, a which have until recently been tremendously situation that could last, although their propor- difficult to achieve with juveniles produced in tion percentage will decline from 25 per cent in intensive systems based on rotifers and 2005 to less than 10 per cent by 2010. The rea- Artemia. Still, the production method for inten- son why the intensive method is winning out is sive juveniles will need further improvement to obvious: a company can produce all year and obtain the same stable high quality of juveniles easily scale up production in pace with market as those from simplified ecosystems. needs, as demonstrated with sea bass and sea bream, where a typical hatchery will produce at Extensive juveniles seem to have more appetite least 20 million juveniles per year. and be more voracious then intensively produced fish – probably because the extensive ju- Is the conclusion, then, that the extensive juve- veniles have had to hunt fast-moving prey and nile production method has been made obsolete

Figure 1. A) Svartatjern – former freshwater lake, now seawater pond. B) Wheel-filter for collection and fractionation of zooplankton. (Photo: Institute of Marine Research)

Thematic area: Production 83 forever? Perhaps, but the EU has just funded an- of natural zooplankton of defined sizes. Howev- other R&D project to look into this production er, availability of the plankton from the sea was method. Still, the industry does not control the variable and dependent on weather and wind. juvenile production of a number of valuable The need for controlled production of zoop- niche fish species, and for some of these a first lankton therefore became urgent. step might be to produce them in an extensive system with naturally produced organisms as The experiments in Svartatjern showed that their only food resource – where they may find plankton production in lagoons could be stimu- those tiny nauplii or rotifers needed for their lated and to some extent controlled (2, 4). Use start-feeding. Subsequent progress in rearing of fertilisation to increase primary production methods might bring them into intensive pro- (phytoplankton), together with gentle mixing of duction – one –by –one, as seen with cod and the water masses to prevent stratification and halibut. This two-step strategy may thus still be oxygen depletion close to the lagoon bottom, suited to, for example, grouper species, blue fin were key factors for sustainable and sufficient tuna and numerous other “difficult” fish species production of zooplankton (copepods) that remaining on a list of desirable species to be could survive harsh winter conditions in the farmed in the future. lagoon sediments as dormant eggs (1). Never- theless, large lagoon systems have been difficult to manage. The ideal size for maintaining con- From the extensive to “semi- trol of plankton production is probably below intensive” production in large 100,000 m3. In addition, the lagoon should not plastic bags and tanks be too deep (maximum 8–9 m). The knowledge gleaned from using plastic bags for studies of feeding ecology and growth dy- During the early 1990s, producers of marine namics in different larval fish species during the fish fry intensified their efforts to develop the 1970s was applied by commercial farmers of semi-intensive plastic bag method (5). To attain turbot and halibut juveniles in 1987. The a commercial scale, bag size was increased to Austevoll experiments in Svartatjern (Figure 50–100 m3, or the bags were moved on land in 1a) and Hyltropollen showed that larvae of flat- the form of large tanks (Rørvik, Nordland fishes had survival problems when released di- County). In 1994, almost 400,000 halibut juve- rectly into the lagoon systems, but that the use niles were produced by the semi-intensive of plastic bags with a volume of 5–12 m3 yield- method, but recurring outbreaks of viral diseased considerably better results. In 1988, the bag es such as VER and IPN stopped further devel- method was also tried on a large scale for cod opment of this rearing method for turbot and (Selvågspollen in Os, Hordaland County). halibut in Norway. In the late 1990s, success Water exchange rates and feed supply were easy with intensive production of halibut juveniles in to control in the plastic bags. Similarly, harvest- Iceland encouraged a change of production ing of juveniles was much easier compared to methods for halibut in Norway. Development of the task of collection when the larvae were intensive production of turbot fry at Øye reared directly in the lagoon. Development of Havbruk in Kvinesdal showed that this rearing the wheel-filter by Unik Filtersystem AS method could be an alternative. Although there (Figure 1b) allowed collection of large amounts are success stories for semi-intensive produc-

84 Aquaculture Research: From Cage to Consumption tion of turbot juveniles abroad (Maximus AS in Variation in nutritional composition of copep- Denmark and Stolt Seafarm AS in Spain), ods has also been a subject for discussion. This modification of the semi-intensive method was investigated in 2000 and 2001 in the abroad is primarily related to internal produc- Svartatjern, with support from the Research tion within the rearing units of copepods or Council of Norway. The copepods were collec- rotifers. Additional live feed (e.g. copepods, ro- ted weekly for biochemical analyses from tifers or Artemia) has to be supplied in a manner spring to early winter and during confined peri- similar to intensive production. In this way, the ods where three different copepod species alter- stability characterising large systems is pre- nately dominated the system. The results served while food availability can be secured showed surprising stability with respect to lipid and regulated according to larval needs. While classes, fatty acids, amino acids, protein, pig- plastic bag systems for cod were only occasion- ments, and in part also vitamins (3). In general, ally explored during the 1990s, a full-scale pro- the copepods were rich in phospholipids, ome- duction system for cod juveniles has now been ga-3 fatty acids (mainly DHA and EPA), free established at Lofilab AS in Lofoten. In recent amino acids, astaxanthin, and vitamin C and E. years, Lofilab has been producing 700,000 to The digestive system in fish larvae is probably 900,000 cod juveniles annually, and uses roti- evolutionarily adapted to copepods as their pri- fers and Artemia as larval feed in addition to mary food, leading to specific nutritional re- zooplankton collected from the lagoon or the quirements in fish larvae. Thus, development of sea (6). alternative feed in intensive rearing will be very important for successful production of larvae Relatively little research has been carried out on and juveniles. The results from the copepod in- the combination of using a lagoon for zooplank- vestigation are now used by the different feed ton production and large plastic bags or tanks as producers to develop improved feed formula- larval rearing units in juvenile production of tions or better methods of enriching live feed marine fish. Studies have mainly focused on (such as rotifers and Artemia). growth and survival mechanisms of fish larvae in relation to availability of prey. Efforts to develop intensive rearing methods have shown Intensive production of marine that nutritional deficiencies easily occur in lar- juveniles – the most important val and juvenile fish. With this in mind, the use challenges of copepods from lagoons as supplementary Using intensive production methods, juveniles feed has been investigated. The problem with may be produced year-round, which increases using copepods this way is the possible transfer the utilisation of hatchery installations consid- of disease agents and parasites. In addition, erably. Control of the photoperiod and temp- copepods have a slow generation time from erature for the brood stock is required in order eggs to reproductive adults, which limits pro- to spawn high-quality eggs outside the natural duction and involves a variation that makes it spawning period. Intensive production of juve- difficult to predict the amount of available cope- niles usually means higher larval density, which pods at any given time in a commercial produc- requires better control of different environmen- tion situation. tal parameters compared to extensive production methods. Access to sufficient numbers of

Thematic area: Production 85 high-quality juveniles has always been a major a common problem that the number and size of bottleneck in the development efficient produc- egg batches per female as well as the egg qual- tion of marine cold-water species such as Atlan- ity decrease over the years. This problem might tic cod and Atlantic halibut. The biological be related to nutritional status of the fish, caused bottlenecks in the development of intensive pro- either by insufficient food quality or low feed duction of marine cold-water species, particu- intake in individual fish. It is quite common that larly cod and halibut, are explored below. only a few females produce most of the eggs at one fish farm. Knowledge about nutritional re- Due to high investment and production costs, quirements of brood fish is limited. Experi- year-round access to high-quality eggs is a pre- ments involving brood fish are expensive, both requisite for profitable production. Shifting the because the fish themselves are very valuable spawning from the normal spawning season by and because the experiments often require large photo manipulation is relatively simple, but the experimental facilities. There is, however, a egg quality has been unsatisfactory. Both need for more knowledge about nutritional experimental results and commercial practice requirements, effects of light and temperature have demonstrated that it is necessary to control manipulation, and final maturation of eggs. both the photoperiod and the temperature. The optimal temperature for the brood stock varies Systematic genetic improvement of fish stocks between species, and so far there is limited results in continual improvement of the traits in- knowledge about the optimal temperature for cluded in the breeding goal. However, for seve- each species of commercial interest. Ongoing ral marine fish species, use of wild-caught experiments on cod indicate that the tempera- brood fish is still common. The use of wild- ture of the rearing water for the brood stock is caught brood fish does not provide genetic important for egg quality. Details are scarce as improvement from one generation to the next. to how the rearing temperature should vary dur- The first genetic breeding programme for cod ing the year in order to obtain maximal growth was initiated in 2001, and eggs from selected and optimal egg development. Most marine fish brood stock are now available. A selective species are batch spawners, i.e. one fish spawns breeding programme has not yet been estab- several egg batches during the spawning period. lished for Atlantic halibut. Selective breeding is Some species such as cod spawn spontaneously an important factor for achieving continual in the brood stock tanks, and fertilised eggs can improvement of production. be collected by the use of surface skimmers. For Atlantic halibut, however, stripping of eggs is Compared to other marine fish species, Atlantic still necessary, and knowledge about correct halibut larvae undergo a long yolk-sac stage, timing of stripping is still limited. Experiments and both the survival and the larval quality dur- have demonstrated that stripping of eggs two to ing this stage vary considerably. Results during three hours before the fish is ready causes re- this stage have improved during the last few duced fertilisation and hatching rates in Atlantic years thanks to modifications of production halibut and turbot. equipment and improvement of the water quality by the use of vacuum degassers and protein In contrast to salmon production, marine brood skimmers. However, the variation in larval fish produce eggs for several years. It has been quality and survival is still too high, and larval

86 Aquaculture Research: From Cage to Consumption density in the large silos used for halibut is very a problem during slaughtering and processing low compared to common practices for other and pose an ethical problem with regard to ani- marine fish species. The same type of produc- mal welfare. tion unit has been used for almost 20 years, with only minor modifications. This indicates a need Disease problems are important in all aquacul- for smaller and more efficient units for this de- ture production, particularly in intensive cul- velopmental stage in halibut. For cod and other tures where the risk of infection can be high. In cold-water marine fish, the yolk-sac stage last halibut, the VNN virus became a serious prob- only a few days. For these species, only too high lem in the mid-1990s. The virus killed large temperature has been proven to cause negative numbers of juveniles during the metamorphosis effects such as reduced survival. stage. The problem has been reduced by improved routines for disinfection of eggs and sea- Most marine fish larvae still depend on live water used for the larval stages. Most hatcheries feed. Both rotifers and Artemia are used for cod. have also introduced better routines against the Today several hatcheries wean the larvae to dry spread of infection between different units and feed at an earlier stage, and then rotifers are compartments. Much of the work on developing used as the only live feed. Halibut larvae are better disinfection methods was done at com- larger than most other marine fish larvae, and mercial hatcheries, causing major losses during due to their size they can feed directly on the process. There is still a need for more Artemia. Skeletal deformities are a problem in knowledge about the use of ozone and other dis- cod juveniles; the most common deformity so infectants, and in the future this kind of work far has been a “bent neck”, i.e. the head is bent should be carried out in experimental facilities upwards. The cause of this deformity has not in order to avoid expensive losses of fish at been identified, but one theory is that a hyper- commercial hatcheries. inflated swim bladder causes pressure on the notochord before the vertebrae are calcified. Other disease problems such as IPN (infectious The extent of this problem has been reduced in pancreas necrosis) often appear during meta- the last couple of years, probably because most morphosis, which is a very sensitive stage. IPN hatcheries have installed vacuum degassers and is commonly found in seawater, and outbreaks thereby reduced the problem of gas supersatura- occur happen when the fish are weak. Flexi bac- tion of the water. However, a 2005 survey of teria have been found in both halibut larvae and malformations in cod showed that skeletal de- juveniles, and cause skin wounds. In larvae this formities are still a major problem in the pro- bacteria can cause massive mortality, while in duction of cod juveniles. The survey also larger fish reduced vitality and growth are the demonstrated different types of skeletal defor- most common effects. This kind of outbreak mities, and that the frequencies and types of de- can best be prevented by good water quality and formities vary between different hatcheries. generally good hygiene at the farm. There is These results show the need to identify the still great potential for improvement of the hy- causes of deformities in cod in order to avoid giene routines at Norwegian marine hatcheries. these problems in the future. Deformed fish are

Thematic area: Production 87 50 - Microalgae 600 l

Semi-continuous Harvest Feeding Green water production technique

Rotifers 200 - 5 000 l 100 l

Cultivation Rinsing Short-term Rinsing Long-termenrichment Concentration Enrichment Concentration 50 – 9 000 l (~24 hours)

Larvae tank 50 - Artemia 50 l 1000 l

Hatching Rinsing Short-term Rinsing (~24 hours) Enrichment Concentration (~24 hours) Figure 3.The rotifer Brachionus plicatilis (Nevada) Figure 2. Schematic drawing for production and use of microalgae, rotifers and Artemia in marine fish fry Currently, the intensive production method for production. (Figure from Katja Hoehne Reitan, Norwegian marine fish juveniles primarily uses live feed University of Science and Technology (NTNU)) such as rotifers and Artemia. These organisms 145x100//Kap05-fig02.eps are not the natural food for the fish larvae and From copepods to cultivated live have a lower nutritional quality compared with feed natural zooplankton. In order to improve the Extensive nature-like systems for production of nutritional quality of these organisms, specific marine fish juveniles are based on the use of enrichment methods are being developed to natural zooplankton as feed. In the coastal la- improve the nutritional value of live feed. The goons and large natural enclosed systems which methods for nutritional enrichment and produc- have been used for this purpose, the natural tion technology of live feed are vital to success- plankton communities have been manipulated ful first-feeding of marine fish larvae. Today, in order to increase the number of planktonic automation is increasingly streamlining the cul- organisms that comprise the natural food for the tivation methods for both rotifers and Artemia. fish larvae. This has been done by removing the Much progress has been made, but the produc- natural predators and stimulating the primary tion and quality of live feed remain a bottleneck production of the systems. The natural prey of in the aquaculture of marine fish. the fish larvae satisfy the nutritional requirements of fish larvae and are shown to be of high Microalgae are used as feed for both Artemia nutritional quality. However, this production and rotifers, added directly to the fish larvae method is unpredictable and completely depen- tanks (known as the greenwater technique). A dent seasons of the year. In addition, there is a schematic drawing for production and use of great danger of infection by viruses and para- live feed is shown in Figure 2. sites. This method is also very labour-intensive. Today, mainly due to the risk of infection and There are more than 2,000 different species of the need for year-round production, most hatch- rotifers, but only biotypes from the Brachionus- eries use intensively cultivated live food as feed complex are used in aquaculture. The culture of for the fish larvae. rotifers (Figure 3) was first started in Japan in the early 1960s, and today hatcheries world-

88 Aquaculture Research: From Cage to Consumption but it has now been determined that three different species are used in aquaculture, with different biotypes within each species (Figure 4).

Rotifers can be produced in all kinds of tanks, from small buckets to huge basins. The production methods can be batch, semi-continuous and continuous cultivation. The most common Figure 4. Drawing of the lorica of three Brachionus species. method at European hatcheries is batch cultiva- 145x100//Kap05-fig04.eps tion, where the entire culture is harvested after wide are producing rotifers as feed for fish and a three-to-five-day growth period (Figure 5). crustacean larvae. In the literature rotifers are The rotifers are grown in tanks with a conical often described as S-type and L-type rotifers, bottom (1–3 m3); air and oxygen are added. The

145x100//Kap05-fig05.eps Figure 5. Rotifer production and handling before feeding to the fish larvae.

145x100//Kap05-fig06.eps Figure 6. Recirculation production system for rotifers.

Thematic area: Production 89 temperature and salinity have to be stable, and (Figure 6). This production system is based on the rotifer densities are counted daily to calcu- recirculation and automation, enabling rotifers late the correct amount of feed. Rotifers can be to be produced in a continuous culture. cultivated on different types of feed. After harvesting, the rotifer will be washed and enriched Artemia are distributed commercially as dried with a special diet, and thereafter the rotifer will cysts, and the cysts may be hatched as needed, be washed again and feed to the fish larvae. which means that it is not necessary to keep With rotifers, microbial control and high nutri- continuous cultures of Artemia. Artemia are the tional value are essential. right size for most fish larvae, and it is possible to adapt the nutritional value in order to meet More intensive production of rotifers has been the requirements of the fish larvae. The produc- in focus in recent years. Hatcheries are increas- tion process consists of four steps: 1) Decapsu- ing their fish production and more cost-effec- lation, 2) Hatching, 18–24 hours, 3) Enrichment tive rotifer production is important. At the and 4) Feeding to the fish larvae (Figure 7). Ar- University of Gent (Belgium) and at SINTEF temia start feeding already after the first ecdy- Fisheries and Aquaculture, a new production sis, and are non-selective filter feeders that feed system for rotifers has been developed on particles sized 8–35 m.

➡

➡ ➡

Figure 7. Artemia cysts, hatching of cysts, nauplii and enriched Artemia. (Photo: Tora Bardal, Norwegian University of Science and Technology (NTNU))

145x100//Kap05-fig06.eps

90 Aquaculture Research: From Cage to Consumption Artemia have a basically low nutritional quality dia. High bacterial content in the culture will for larvae of marine cold-water fish, due in par- also yield low-quality Artemia. Predictable pro- ticular to the fatty acid composition of Artemia. duction of live feed of high quality is critical to Artemia contain no DHA, very little EPA, and the success and profitability of marine fish fry have a relatively high total lipid content (12). production. Much research has been done to develop enrichment methods to improve the nutritional quality of these animals. Enrichment protocols and Tank dynamics and technology – several enrichment emulsions have been devel- how to successfully produce fry oped that should ensure sufficient lipid quality From the beginnings of fry production using an of Artemia. The enrichment procedure takes intensive, year-round production method, it has place in tanks of 20–5,000 litres (Figure 8) at frequently been observed that tanks with high densities of 100–250 ind/ml. Short-term enrich- larval densities perform better than larval tanks ment also results in high and disadvantageous with relatively low larval densities with regard bacterial content, and it is necessary to rinse the to both survival and growth. Several theories animals before they are offered to the fish have been suggested to explain this pheno- larvae. menon, but so far none has proven especially reliable. This section discusses factors and The production and enrichment of Artemia empirical models that describe the dynamics in must be conducted with careful attention to a larval rearing tank. Larval growth and survival proper routines. The enrichment emulsions depend on proper, high-quality environmental must be of high quality and the Artemia need conditions. This means that physical factors good culture conditions. Low enrichment re- such as illumination, water movement (turbu- sults may be due to low temperature during the lence), water temperature and water turbidity enrichment period, low concentration of feed must be close to optimal. Years of experience particles, unstable enrichment emulsion and have led to several adjustments in technology low pH and O2 concentration of the culture me- and daily working routines to improve larval survival during the first-feeding stage. Examp- les are automatic cleaning systems for larval tanks and degassing of the water. The average survival rate through the first-feeding and weaning stages for halibut now exceeds 50 per cent with high regularity. Survival rates through similar stages for cod are somewhat lower (on average 20 per cent), but are a significant improvement over a few years ago.

The quality of not only the physical factors but also the biological factors have must be optimal to secure fry production. Obvious biological Figure 8. Production equipment for decapsulation, hatching and short-term enrichment of Artemia. factors are larval and live prey quality. Phyto- (Photo: Jan Ove Evjemo, The SINTEF Group) plankton is commonly used as greenwater tech-

145x100//Kap05-fig08.eps

Thematic area: Production 91 nique, which strongly influences the quantity of bacteria in the rearing water. The flora of the larval gut is also greatly affected by larval feed intake (16). Feed consumption by the larvae has also proven to be influenced by prey density. Several experiments have been conducted dealing with different prey species and different enrichment diets for these species. Experiments dealing with feeding regimes and food amount have also been conducted. Feeding Artemia to halibut larvae in meals yielded significantly better results compared to continuous feeding (17). One explanation for this is probably that the larvae digest the prey better between meals due to Figure 9. Schematic set-up of a first-feeding system for longer retention time in the larval gut. The qual- marine fish larvae. Seawater is added through a vacuum ity of the prey is also dependent on the time be- aerator (A-B). A centrally placed aerator provides sufficient tween its enrichment and its consumption by turbulence to avoid prey patching. At the bottom, a sweeper collects dead material that is subsequently flushed out the larvae. This is the retention time of the prey through the bottom outlet screen (C). Turbulence and in the larval rearing tank. illumination combined with the peripheral shade (D-E) ensure adequate larval distribution and thereby optimal It is important to have a reliable estimate for the feed intake. amount of prey needed in the larval rearing tank 145x100//Kap05-fig09.eps in order to avoid “old” prey and also to be able let. However, experiments have shown that the to estimate the production of prey animals. proportion of Artemia leaving the tank uneaten When the physical and biological factors are is low when fed in meals. The model is also close to optimal, we are able to model the dy- based on the feed requirements of the larvae namics in a larval rearing tank based on the re- (with tables based on larval growth, metabolic tention time of the prey in the tank. This activity and empirical data). The number of lar- retention time is basically dependent on three vae in a tank can be estimated based on “prey factors: clearance” time. These estimates use the fol- 1. Water flow rate lowing equations: 2. Feeding intake of the larvae 3. “Natural” death rate of the prey Remaining Art. (measured) = Art. added – (Art. eaten. + waste Art.) (1) As noted above, the current patterns (turbu- Requirement per tank = Eaten + loss + security lence) must ensure an even distribution of the margin (2) prey in the tank. The loss of prey due to water flowing through can then be estimated based on Eaten = Number of larvae* Artemia demand per the half-life of water exchange in the tank, pro- larva (table) (3) vided that the prey cannot avoid the water out- Loss = mortality (Artemia) + loss through outlet (4)

92 Aquaculture Research: From Cage to Consumption Calculations like these indicate strongly that the larvae number (density) has to be high in order to achieve sufficient clearance of the prey. This is especially important in the beginning of the first-feeding stage when live prey is added in high numbers to secure high feed intake (density dependent).

In the case of halibut, first-feeding in a regular Ø=2.5 m tank with a water flow rate of 10–20 litres per minute, minimum number of larvae is approximately four individuals per litre. At 45x100//Kap05-fig10.eps lower larval stocking densities the prey clear- Figure 10. Comparison of a cod larva (4 mm long at ance will be too slow and “old” prey may accu- hatching), and a salmon larva at the time of first-feeding. The salmon larva is 22 mm long, and 140 times heavier mulate. than the cod larva.

The transition from live prey to a clude in a formulated diet, mainly due to leak- formulated diet age. A key factor for profitable production of marine fish juveniles is the development of formulated In aquaculture cod larvae are weaned to a for- diets for replacement of live prey during the lar- mulated diet at 30–35 days post hatching, but as val stage. The requirements for these diets are the quality of the formulated diets improves, the complex and difficult to achieve, both with re- weaning time is shortened. This principle is also gard to nutritional composition and physical true for halibut larvae, although this species is characteristics. In addition, the feed also has to not weaned until the larvae have begun demer- be accepted by the larva and ensure growth and sal life at about 70 days post hatching (approx- survival. Some marine fish species may readily imately 0.2 gram). In the last few years several be first fed on formulated diets, primarily spe- feed manufacturers have claimed to have devel- cies that have large, well-developed larvae at oped a formulated diet that may be used as ini- hatching. The challenge may be visualised by tial feed. This has yet to be confirmed in feeding comparing the size of a larval salmon to an trials. equivalent cod larva (Figure 10). Results from the hatchery industry show that At hatching, pelagic marine fish larvae have there may be large mortality rates during the poorly developed digestion compared to salm- weaning process. Not only food quality, but also on. In the wild, marine fish larvae prey on cope- environmental factors may affect mortality. It pods rich in low-molecular nutrients such as has been demonstrated that the aged water in re- PUFA (polyunsaturated fatty acids), FFA (free circulated systems suppresses cannibalism, and amino acids) and vitamins (18). Some of these that reduced salinity during the first days on a water-soluble components are difficult to in- formulated diet also reduces mortality. Further, it has been demonstrated that moist feed (70 per

Thematic area: Production 93 cent water content) increases growth compared iour, as do probably the appearance and colour to a dry diet (19). For comparison, live prey of the feed. (Artemia, copepods) contain approximately 80 per cent water. Inadequate tending routines may Formulated larval diets may be classified de- also lead to unhealthy tank conditions, low pending on their types of binder and coating. growth rates at weaning, size differences and The different binders are added to the feed dur- aggression. The halibut larvae live pelagically ing the production process to make the particles during the first 50–70 days after first-feeding, fixed. Gelatine is a binder that forms a stable, until they metamorphose and seek the tank floor durable and soft pellet that allow dry matter at approximately 0.2 gram wet weight. During content down to two per cent without disinte- this stage it is of special importance that the gration of the particle. However, if uncoated, feed have the right characteristics with regard to the gelatine-stabilised pellet has far too high a buoyancy for optimal availability. After meta- rate of leakage of small water-soluble particles. morphosis, halibut juveniles feed at the bottom Leakage from a food particle leads to reduced so buoyancy properties are less important. After nutritional value and to reduced tank hygiene metamorphosis, the gastrointestinal system is and bacterial growth due to increased amounts fully developed, and therefore able to handle of dissolved organic matter (DOM). The first larger and heavier nutrients. Thus the nutrition- particles to disappear are small water-soluble al composition and the physical characteristics molecules such as amino acids, peptides and of an optimal feed may change dramatically vitamins. Up to 50 per cent of these molecules through the different stages of growth. will leak out during the first minutes in water. However, the leakage rate depends on particle In the process of producing a feed for pelagic size since bigger particles leak less than smaller marine fish larvae, it is of special importance to ones (20). consider: • Buoyancy properties A key issue when using formulated diets is pol- • Larval acceptance lution/hygiene in the tanks. Using the optimal • Leakage of water-soluble nutrients combination of feed type, feeding regimes, • Nutritional quality water exchange rates and the employment of a mechanical cleaning viper may minimise pollu- The sinking velocity determines the availability tion. Tank shape may also contribute to solving of the feed to the larvae. If the sinking velocity the problems related to contamination by POM is too high, the larvae may fail to catch it, and if and DOM. Raceways with low water levels the feed is lighter than water, it will float on the have proved to be superior during the weaning surface and thereby be unavailable. The size of period, due to the high food application and low the particles should preferably be as big as the food ingestion by the fish. fish can eat. Larger particles have a more favourable surface-volume ratio than smaller In a weaning experiment, halibut larvae were ones, thus lesser leakage. The fish larva is a transferred directly from live prey (Artemia) to visual predator, irrespective of its adult feeding a formulated diet at different ages (7, 14 and 21 behaviour. Smell may also affect capture behav- days with Artemia). In this trial, growth was observed in the group that had been fed Artemia

94 Aquaculture Research: From Cage to Consumption for 21 days, although there was less growth than by a factor of 10 during the first 23 days of in the control group still feeding on Artemia. At exogenous feeding. the time of first-feeding, the halibut larvae showed lower activity of the digestive enzymes compared to other flatfish species such as turbot Water quality in intensive and Japanese flounder (21). This apparent re- cultures of marine juvenile fish duced ability to digest food is probably the main In early extensive aquaculture methods, there cause for age restrictions in weaning rather than were few options with regard to water treat- ingestion rates. Several studies report onthoge- ment. In these low-density cultures, the fish netic changes, as well as changes in digestive were part of a larger ecosystem that was largely enzyme activity as response to food composi- self-sustainable. As culture intensity has pro- tion, presentation and size. Although a certain gressed, the need for water quality improve- amount of digestive enzymes may originate ment has evolved, as have the possibilities. from the live prey, direct measurements of key enzyme activity have revealed that less than The two prevailing methods for intensive fish 10 per cent originate from Artemia. In the larval cultures also have different demands for water halibut gut the activity of the key enzyme chy- treatment. In flow-through systems, still pre- motrypsine has been demonstrated to increase dominant in Norway, water is sampled from a clean source at sea and adjusted according to

Figure 11. The feeding incidence of larvae after a 24-hour feeding experiment including nine different feeds. Feeds 1–3 have different sinking velocities and Feeds 4–9 have different taste admixture. Before the experiment the larvae had been first-fed on Artemia for 28 days. The highest ingestion incidence was seen in Feeds 2 and 3, where a floating agent was admixed (46 and 44 per cent, respectively). Only four per cent of the larvae had ingested the control feed without the floating agent (Feed 1).

Thematic area: Production 95 the specific water quality required. The water is Gas supersaturation is a phenomenon normally used just once before being discharged, but in found in flow-through systems in connection most cases energy is conserved in heat exchang- with heating and pumping (pressurising). To- ers. In some cases effluent water must also be day, there are several commercial systems avail- treated due to local environmental regulations. able for vacuum stripping of excess gas from In recirculated systems a major part of the efflu- the intake water. These degassers will typically ent water is returned to the fish tanks after sys- decrease total saturation by five per cent, which tematic and comprehensive treatment. Both for most situations is sufficient to bring TGP be- principles have their advantages and disadvan- low 100 per cent. However, traditional vacuum- tages, but the recirculation system now seems to degassers require space drop-height and may be gaining popularity. This section will discuss therefore be difficult to install in existing facili- the most important water quality parameters to ties. be monitored in intensive marine juvenile production, along with the prevailing standards for Oxygen is the most important of the dissolved water treatment. gases and is the prerequisite for aerobic metabolism where organic molecules are enzymati- Gas supersaturation is a common occurrence cally converted to chemical energy, water and that has proved to be difficult to avoid, particu- metabolites. The growth of fish larvae is dy- larly in open flow-through systems. Gas super- namic, and in some species daily growth rates saturation may lead to a condition called gas up to 30 per cent have been observed under con- bubble disease (GBD), characterised by the for- trolled conditions. Although high growth rates mation of small gas bubbles in the blood circu- may be advantageous in aquaculture, the high lation and tissue that may impede oxygen tran- oxygen demand makes the larvae more vulner- sport and nerve signals. The hazard of super- able to fluctuations in the environment. Under saturation is a subject of discussion and its natural conditions cod is relatively sedate and effect on early stages is yet to be scientifically has not developed a high proportion of red aer- documented, at least for the species in question. obic muscles, reflected in its comparatively low Due to uncertainties in assessment methods, it oxygen consumption. In land-based tank opera- has been difficult to establish the connection be- tions, oxygen saturation levels above 85 per tween observed mortalities and gas supersatura- cent in the effluent water are recommended. In tion. In spite of this, gas supersaturation and gas contrast to recirculated systems, the content of bubble disease have in several cases been sus- oxygen in the intake water in flow-through sys- pected as the prime cause for acute mortalities tems may fluctuate considerably through the and high frequencies of deformities in juvenile year due to changes in primary production and cultures. In recent years several trials aimed at vertical mass transport of water. It is considered quantifying negative effects of gas supersatura- unproblematic to measure oxygen content in the tion have shown that cod juveniles cope with actual ranges, and several manufacturers offer TGP of 105 per cent (22). For another species low-priced, reliable instruments for this pur- harmful effects were already observed at 102 pose. The latest development in this field is ox per cent TGP during pre-metamorphic stages meters based on light fluorescence from oxygen (colt). This has also been found for cod, al- molecules dissolved in a matrix membrane. though the results are not conclusive.

96 Aquaculture Research: From Cage to Consumption These have proved to be both accurate and tides either in the fish themselves or from bac- stable. terial breakdown of faeces and feed spill. Depending on pH, ammonia exists as the gas In aerobic metabolism, CO2 is produced at a NH3, or the cation NH4+at low pH, where the rate approximately similar to oxygen consump- ionised form is far less toxic than the ammonia tion (when the respiration coefficient, RQ, is gas. The NH3 fraction will increase with rising one). CO2combines with water molecules in the pH and temperature, and decrease with increas- blood to form the less toxic carbonic acid and ing salinity. Ammonia has been shown to affect bicarbonate that are subsequently transported to neurotransmission, production of neurotrans- the gills and discharged. CO2 reduces the pH of mitters, general cell metabolism and intracellu- the blood, and thereby decreases the oxygen af- lar pH. The symptoms in the fish may include finity of the haemoglobin. This is a finely tuned reduced growth and swimming ability, hyper- mechanism that under normal conditions ventilation, coma, spasms, and mortality in the secures efficient transport and distribution of most severe cases. oxygen to the organism and to the pump that inflates the swim bladder. In the case of cod, observations indicate that low levels of ammonia (0.06 mg/l NH3-N) High partial pressure of CO2 in the ambient wa- cause reduced growth at fish sizes of 17 grams. ter will decrease the discharge effectiveness and The growth suppression gradually decreased as disturb the important acid-base balance. Al- fish size increased probably due to activation of though captive fish in intensive cultures experi- compensatory mechanisms (24). In smaller fish ence elevated levels of CO2 in the blood it has been shown that acute ammonia sensitiv- (hypercapnia) compared to natural situations, ity varies with age, although the tolerance limits the long-term effects of this are not fully were generally higher than expected compared known. In both rainbow trout and wolffish, ele- to other species. Metabolically produced am- vated levels of CO2 have been shown to elicit monia will normally be detoxified within the problems such as nephrocalcinosis, reduced fish by enzymatic transformation to the amino growth and reduced muscle pH (23). It is there- acid glutamine, or directly excreted approach- fore not unlikely that spinal deformations as ing a concentration gradient (25). When fish are found in cod juveniles are also affected by subjected to elevated levels of ammonia, in- hypercapnia through insufficient calcium depo- creased levels of detoxifying enzymes and sitions. Acceptable ambient CO2 values for cod transport enzymes may therefore be detected. have not been mapped, but for other species it Similar to carbonic dioxide, ammonia may ac- has been found that oxygen deficiency occurs cumulate in recirculated systems and have to be prior to CO2 toxicity in flow-through systems. removed to avoid toxic conditions. This is nor- Toxic values of CO2 may therefore only occur mally accomplished by use of biofilters where in systems where flow is restricted and oxygen ammonia is oxidised by nitrifying bacteria to has to be added to avoid hypoxia. the far less toxic compounds nitrite and nitrate. To ensure adequate function of the biofilter, a Ammonia is another important metabolite in relatively long period of priming has to be car- aquatic systems. This molecule is formed in the ried out to establish the proper community of deamination process of amino acids and nucleo- bacteria. However, in temperate fish cultures

Thematic area: Production 97 this process may be accelerated with bacteria time of residence of the water in the skimmer is cultures that are now commercially available. imperative for proper function. Enriching the There is little information about the efficiency bubbles with oxygen and ozone greatly increas- of biofilters in the low temperature systems es the efficiency of the skimmer by increasing used for cod and halibut; clearly more studies surface charge. are needed to fully adapt recirculation technology in cold-water juvenile cultures. In biofilters A large part of the organic waste produced in a nitrate is the end product from the aerobic am- juvenile culture will precipitate and form a lay- monia catabolism. Nitrate has low toxicity, but er of sediment on the tank floor. This is often the should nevertheless be kept within the limits of case during the live feed period, when low flow tolerance for the fish species and stage in ques- rates make self-cleaning impossible. The intro- tion. Nitrate may be removed from the system duction of automated mechanical floor sweep- by a certain water exchange, or be reduced to ers has revolutionised this critical part of the N2 gas by denitrifying bacteria in anaerobic process, and has probably been an important biofilters. All biofilters may be regarded as liv- factor in the very positive trend experienced in ing organisms, and in order to reduce the total the industry during the last decade. number of species to be cultured, alternative methods for ammonia removal have been con- The addition of ozone to the protein skimmers sidered. At the Institute of Marine Research, serves a dual purpose. In addition to enhancing use of precipitating agents for ammonia in re- foam formation, ozone also increases the redox circulated rotifer cultures has been shown to be potential of the water by oxidation of nega- effective, and may therefore in due time replace tively-charged halogen ions (Br-, Cl-, I-) to their the traditional biofilter. respective oxides. These exercise an inhibitory effect on both bacteria and viruses, and have, Organic waste such as faeces and feed remains also at medium levels (up to approximately 350 are found in the form of particulate organic mat- mV), an apparently positive effect on fish wel- ter (POM) or dissolved organic matter (DOM). fare. Over time the redox potential of the water In recirculated systems these compounds need in recirculated systems will decrease due to the to be regularly removed to maintain the neces- oxidation of organic particles. Addition of sary standards of hygiene. The bulk of the waste ozone will therefore within certain limits act to may easily be removed in suitable mechanical recondition the water. In marine juvenile pro- filters that do not fragment the particles. There- duction it is therefore recommended to keep the after the water passes through a skimmer, where redox potential within 250–350 mV. However, cascade aeration removes both POM and DOM in addition to the low-toxic oxidised forms of by the formation of foam. The working prin- halogens, ozone will also to a certain extent pro- ciple is that particles and large molecules are, duce the far more toxic bromate (bromic acid). due to their electrical charge, associated to the This oxidised brome compound is primarily oppositely-charged surface of small gas bubbles formed under high redox potentials, and is very and removed at the surface as foam. The size poisonous to the fish. In successive water circu- and volume of the skimmer has to be adapted to lation cycles, as in recirculation, bromate may the actual water flow through the system and to also be formed at lower redox potentials. A cer- the maximum load of organic matter, since the

98 Aquaculture Research: From Cage to Consumption tain renewal of the water is therefore needed for fish juveniles have progressed, thus enhancing this reason as well. the development of new production industry of both cod and halibut. New species, such as Today, an increasing number of hatcheries pre- wolffish and plaice, will most certainly become fer to recirculate the water during at least parts profitable species of the aquaculture menu. of production. When such systems are working, Knowledge of the specific requirements for the overall experience is that they are more sta- each species with regard to nutrition, water ble and reliable than their counterpart, although quality and physical environment is a prerequi- the water quality measured in total load of both site for successfully moving from extensive/ bacteria and organic matter is less favourable. semi-extensive production to intensive produc- An unexpected and unexplained side effect of tion. So far such knowledge has made it possi- recirculation was that cannibalism decreased ble to create intensive environments that allow drastically. Flow-through systems are still for high fish densities and which to a certain ex- easier to establish not only due to lower cost, tent may be called profitable. Still, this branch but also because current technology is insuffi- of aquaculture needs to be innovative with re- cient for building and running a full-scale high- gard to issues concerning water treatment, dis- tech recirculation system for cold-water ease prophylaxis and formulated replacements species. Nevertheless, increased legislative for live feed organisms. Further achievements demands for health documentation and effluent in the automation of production procedures will control will probably force future aquaculture be an increasing challenge to maintaining high towards water reuse. quality and cost efficiency. Animal welfare in juvenile fish cultures will probably be a focus area issue in the future, as evidenced by average Summary and perspectives mortality rates of more than 70 per cent in the During the last programme period the efforts toperiod from egg to juvenile, as well as high wards fully automating production of marine rates of various different malformations.

Thematic area: Production 99 References 14) Grotmol, S., Totland, G.K., Kvellestad, A., Fjell, K. 1) Næss, T., 1996. Benthic resting eggs of calanoid & Olsen, A.B., 1995. Mass mortality of larval and copepods in Norwegian enclosures used in maricul- juvenile hatchery-reared halibut (Hippoglossus ture: abundance, species composition and hatching. hippoglossus) associated with the presence of virus- Hydrobiologia 320: 161–168. like particles in various lesions of the central ner- 2) Naas, K.E., van der Meeren, T. & Aksnes, D.L., vous system and retina. Bull. Eur. Fish Pathol. 1991. Plankton succession and responses to mani- 15:175–180. pulations in a marine basin for larval fish rearing. 15) Poppe, T., 1999. Fiskehelse og fiskesykdommer. Mar. Ecol. Prog. Ser. 74: 161–173. Universitetsforlaget AS. 411 pp. (ISBN 82-00- 3) Van der Meeren, T., 2003. Kartlegging av biokjemisk 12718-4). innhold i copepoder som basis for kvalitetsvurder- 16) Bergh, Ø., Naas, K.E. & Harboe, T., 1994. Shift in ing av fôr i oppdrett av marin fiskeyngel. Fisken og the intestinal microflora of Atlantic halibut Havet 5: 39. (Hippoglossus hippoglossus) larvae during first 4) Van der Meeren, T., 2004. Yngelproduksjon av kveite feeding.Can. J. Fish. Aquat. Sci. 51:1899–1903. i poll- og bassengsystemer. In: Mangor-Jensen A. & 17) Harboe, T. & K. I. Reitan, 2005. Halibut fry produc- Holm, J.C. (eds.). Håndbok i kveiteoppdrett. Insti- tion. In: Larvi’05 Fish and Shellfish Larviculture tute of Marine Research, Bergen. pp 112–133. Symposium. Hendry, C.I., Van Stappen, G., Wille, (ISBN 82–7461–060–1) M. & Sorgeloos, P. (eds.). European Aquaculture 5) Van der Meeren, T. & Naas, K.E., 1997. Develop- Society, Special Publication No. 36, Oostende, Bel- ment of rearing techniques using large enclosed gium. ecosystems in the mass production of marine fish 18) Van der Meeren, T. 2003. Kartlegging av fry. Reviews in Fisheries Science 5 (4): 367–390. biokjemisk innhold i copepoder som basis for 6) Van der Meeren, T., Pedersen, J.P. & Kolbeinshavn, kvalitetsvurdering av fôr i oppdrett av marin fiske- A.-G., 2005. Yngelproduksjon - larvefase. In: yngel. Fisken og havet, nr.5 Otterå, H., Taranger, G.L. & Borthen, J. (eds). 19) Otterå, H., Hemre, G.I. & Lie, Ø. 1994. Influence of Oppdrett av torsk. Norsk Fiskeoppdrett AS, Bergen, dietary water content on food intake, growth and Norge. pp. 85–110. (ISBN 82–7595–024–4). survival of juvenile Atlantic cod (Gadus morhua 7) Bjørnsson, B.T., Halldórsson, O., Haux, C., Norberg, L.). Aquacult. Fish. Manage. 25:915–926. B. & Brown, C.L., 1998. Photoperiod control of sex- 20) Langdon, C., 2003. Microparticle types for deliver- ual maturation of the Atlantic halibut (Hippoglossus ing Nutrients to marine fish larvae. Aquaculture hippoglossus): plasma thyroid hormone and calcium 227:259–275. levels. Aquaculture, 166:117–140. 21) Gawlicka, A. Parent, B. Horn, M.H. Ross, N. Opstad, 8) Norberg, B., Valkner, V., Huse, J., Karlsen, I. & I. & Torrissen, O.J., 2000. Activity of digestive Grung, G.L., 1991. Ovulatory rhythms and egg via- enzymes in yolk-sac larvae of Atlantic halibut bility in the Atlantic halibut (Hippoglossus hippo- (Hippoglossus hippoglossus): indication of readi- glossus). Aquaculture, 97:365–371. ness for first feeding. Aquaculture 184:303–314. 9) Izquierdo, M.S., Fernández-Palacios, H. & Tacon, 22) Skajaa K., Mortensen A., Toften H., 2004. Abstract, A.G.J. 2001. Effect of broodstock nutrition on re- Program Conferance, Research Council of Norway, productive performance of fish. Aquaculture, March 2004. Clarion Airport Hotel, Gardermoen. 197:25–42. 23) Foss, A., Rosnes, B.A. & Øiestad, V., 2003. Graded 10) Law, R., 2000. Fishing, selection, and phenotypic environmental hypercapnia in juvenile spotted evolution. ICES Journal of Marine Science, wolffish (Anarhichas minor Olafsen): effects on 57:659–668. growth, food conversion efficiency and nephrocal- 11) Mangor-Jensen, A. & Holm, J.C., 2005. Håndbok i cinosis. Aquaculture 220:607–617. kveiteoppdrett. www.imr.no/dokumenter/kveite- 24) Foss, A., Siikavuopio, S., Sæther, B.-S. & Evensen, haandbok T., 2004. Effect of chronic ammonia exposure on 12) Bell, J.G., McEvoy, L.A., Estevez, A., Shields, R.J. growth in juvenile Atlantic cod. Aquaculture & Sargent, J.R., 2003. Optimising lipid nutrition in 237:179–189. first-feeding flatfish larvae. Aquaculture, 227:211– 25) Wright, P.A. & Fyhn, H.J., 2001. Ontogeny of nitro- 220. gen metabolism and excretion. In: Wright, P.A., 13) Lein, I., Bæverfjord, G., Hjelde, K. & Helland, S., Anderson, P.M. (eds.), Fish Physiology vol. 20., Ni- 2005. Kartlegging av deformiteter hos torsk. Foreløpig trogen Excretion. Academic Press, New York, pp. rapport, november 2005. Akvaforsk. 14 pp. 149–200.

100 Aquaculture Research: From Cage to Consumption Sigurd Stefansson1), Grete Bæverfjord2), R. Nigel Finn1), Sigurd Handeland1), Bengt Finstad3), Sveinung Fivelstad4), Torstein Kristensen5), Frode Kroglund5), Trond Rosten5), Bjørn Olav Rosseland6), Brit Salbu6), Hilde Toften7), Olav Breck8), Ellen Bjerkås9) and Rune Waagbø10) 1) University of Bergen, 2) Akvaforsk – The Institute of Aquaculture Research, 3) NINA (Norwegian Institute for Nature Research), 4) Bergen University College, 5) NIVA (Norwegian Institute for Water Research, 6)Norwegian University of Life Sciences, 7) Fiskeriforskning (Norwegian Institute of Fisheries and Aquaculture Research), 8) Marine Harvest ASA, 9) Norwegian School of Veterinary Science, 10) NIFES (National Institute of Nutrition and Seafood Research) Water Quality – Salmonids

Modern production of salmonids can be considered an industrial production of fish. Salmonid aquaculture is characterised by high fish densities, low specific water consumption, high-energy diets, fast growth, reduced generation time and control of key environmental factors in season-independent production. Rearing intensity is particularly high in production of juveniles and smolt, where water consumption per kg fish has been significantly reduced over a short period and production is sustained by increased addition of oxygen. Smolt production takes place along the entire coast in water sources differing greatly in water quality; for example, acidity and metal content show wide variations among sites and regions. Reports from the aquaculture industry describe problems related to deformities, diseases and mortality of smolt after seawater transfer; these problems are further related to increased intensity of production, specifically the deterioration of water quality. Research on these topics over the last few years has revealed critical connections between water quality and the quality of fish produced. High density, low specific water consumption and addition of oxygen cause a build-up of carbon dioxide in the tanks, reducing pH and increasing the toxicity of metals in the water. Density in itself does not seem to be the most critical factor. We have shown that a very low concentration of aluminium can negatively influence smolt quality, and that exposure to acid water/aluminium can increase sensitivity to salmon lice. Poor water quality can further increase sensitivity to IPN, and observations have been made of increased frequency of skeletal deformities in situations of poor and variable water quality. Current research has further contributed to defining critical limits for key water quality parameters.

Thematic area: Production 101 + Developmental stages – critical during which NH4 is released. This may form periods substrate for bacterial growth and cause toxic Salmonids go through critical developmental effects at low water exchange. PVS creates a stages, from fertilisation through embryogene- space for continued growth of the embryo with- sis and hatching to yolk sac fry and free-living in the chorion; hence the size of the PVS ulti- parr. Following a period of growth lasting sev- mately determines the size of the embryo before eral months or years, the parr go through a the egg must hatch. It is therefore important to transformation to become smolt, pre-adapted to note that both low pH and certain ions/salts (in 3+ 2+ 2+ life in seawater. These transitions between life- particular Al , but also Zn , Mg and 2- stages are characterised by fundamental chang- SO4 ) at low concentrations (1–5 mM) reduce es in critical organ systems and functions, mak- the volume of PVS (5). Addition of saltwater to ing the juvenile salmon especially vulnerable to a final salinity of 1 ppt reduces PVS by 50 per environmental disruptions and poor environ- cent (4). The salt effect on PVS acts through an mental quality. This section focuses on specific inhibition of the enzymes which split protein challenges related to water quality during egg into small peptides during the cortical reaction development and smoltification. (6), while the effect of pH is presumed to be related to the net charge of the colloids, and there- The egg fore their ability to pull other ions into the PVS During oogenesis the plasma membrane is per- (7, 8). meable to low-molecular substances so that water and ions can be transported into the Smolt – regulation of hydro-mineral oocyte. After fertilisation and activation, balance permeability to water and ions is drastically re- The dramatic increase in seawater tolerance duced, becoming extremely low (1, 2). The which takes place during smoltification is more outer protein shell (chorion) protects the salmon fundamental than the gradual increase in ability egg from external mechanical disturbances. to regulate hydro-mineral balance during the fry Chorion has high permeability to water ions, and parr stage. In contrast to most other euryha- NH3 and gasses, but is impermeable to large line species this increase in seawater tolerance molecules (3). At fertilisation and contact with takes place while the juvenile salmon is still in fresh water the salmon egg becomes activated, fresh water. The smolt develop several charac- the cortical vesicles empty their contents into teristic features of marine fish, and are consid- the periviteline space (PVS) between the plas- ered to be pre-adapted to seawater. ma membrane and chorion (cortical reaction). Mitochondria-rich ‘chloride cells’ in the gills Peptides from the cortical vesicles are too large differentiate to new forms and increase in num- to pass through pores in the chorion, and their ber concurrent with a redistribution of cells to negative charge at normal pH values causes an the inter-lamellar spaces on the primary fila- equilibrium resulting in influx of water and pos- ments. Leaky junctions are formed between the itive ions into the PVS. During the first hour fol- chloride cells and their accessory cells, provid- lowing spawning and fertilisation, the PVS ing a route for the net transport of Na+ out of the increases in volume until it reaches about 25 per fish, energised by the increase in activity of the + + + + ÷ cent of the total egg mass (4). After swelling the enzyme Na ,K -ATPase. Na ,K ,2Cl -cotrans- ÷ chorion goes through the hardening process, porter (NKCC) drives the transport of Cl into

102 Aquaculture Research: From Cage to Consumption the chloride cell; the Cl÷ then exits the cell studies with salmon, dry fertilised eggs were in- through ion selective channels (Cystic fibrosis cubated in water with different O2 and CO2 transmembrane conductance regulator, CFTR). pressure and metal concentrations, from fertili- K+ enters the cell through active co-transport sation to first-feeding. A significantly higher with Na+ and Cl÷, and exits through specialised mortality was observed in most exposed groups basolateral K+-channels. During the first few after about 100 degree-days (dC). Eggs from hours following seawater transfer, drinking rate AquaGen strain were further incubated with increases to compensate osmotic water loss. different doses of iron (FeII and FeIII) until first- 2+ 2- Excess divalent ions such as Mg and SO4 feeding, and additional groups were exposed to are excreted via more-concentrated urine. The high concentrations of CO2 (20–25 mg CO2/L) fundamental changes in mechanisms for hydro- and O2 (120 and 150 per cent). Fertilisation rate mineral balance make the smolt especially vul- was 100 per cent in controls. After about 100 nerable to poor water quality. One critical con- dC, mortality increased dramatically in most of sequence is that the cellular adaptations to ion the exposed groups, while controls developed regulation in seawater are affected, despite the normally. Preliminary data suggest clear nega- fact that the smolt appear normal and capable of tive effects of iron and CO2, with little addition- maintaining hydro-mineral balance in fresh wa- al stress from high O2 at high CO2. Studies of ter. Accordingly, there will be no early warning uptake of isotopes of Fe, Mn, Cd and Zn from that smolt quality may be seriously compro- the water into the egg showed that metals enter mised. the egg with water during swelling, but continue to enter the egg even after swelling is com- Water quality – effects, water pleted. Our results emphasise the importance of treatments, actions good water quality during the entire period The egg stage from fertilisation through to first-feeding. Studies show that as little as one minute of inflow of water with high Al and low pH may cause reduced fertilisation, increased mortality and deformities in whitefish (9). In preliminary

Figure 1. To the left, a yolk sac fry of salmon in control water. To the right, a yolk sac fry of salmon exposed to iron. Despite equal thermal sum, the iron-exposed fry is significantly less developed than the control fry. (Photo: Bjørn Olav Rosseland)

Thematic area: Production 103 Fry, parr and smolt stages cells and change mucus composition and prop- Aluminium (Al) erties (16), concurrent with a natural reduction Al is found naturally in rock and soil and is dis- in the number of mucus cells associated with solved into the water through erosion. Acid pre- smoltification (17). Changes in permeability in- cipitation causes more Al to become dissolved; crease demands on ion regulatory mechanisms hence acid water will always have a higher con- in the chloride cells. Al can further cause a di- centration of Al (11). Acidity is in itself not the rect inhibition of transport proteins, thereby re- major water-quality problem for salmonids, ducing the capacity for ion absorption in fresh however. Trout experience osmoregulatory water (18, 19, 20, 21) and excretion of ions in problems only below pH 4.6, and salmon can seawater (22, 23). withstand a pH of 5.4 without showing population responses or osmoregulatory problems, There is a close relationship between Al in the provided the water does not contain inorganic water and accumulation of Al on the gills (21, Al. Al is generally found in several different 24ab, 25, 26). At high concentrations the fish forms: either as free ions (Al3+), organically die from failure in respiration and ion regulation bound (Al-org.), inorganically bound to fluo- (27). Low, non-lethal concentrations of Al may rine (AlF2+), or as hydroxides (AlOH 2+, affect the establishment of seawater tolerance. + Al(OH)2 ) (15). Toxicity mainly appears to be In salmonids, the period prior to (winter) and related to the inorganic compounds (“labile Al” during smoltification (spring) is critically im- (LAl) or inorganic monomeric Al, Ali), which portant, since episodes of acid runoff often co- mainly consist of the Al hydroxides, while for- incide with smoltification. The water quality mation of organic complexes reduces Al tox- during spring runoff is largely determined by icity (15). Concentration of organic matter is the winter climate, including sea salt episodes, therefore important for the toxicity of Al, and in snow cover and snow melt (28). Acid water and the presence of high concentrations of humic Al can also have indirect effects by disrupting acids (high total organic carbon, TOC) a larger cell functions (29), cellular differentiation (30), fraction of Al is found as organically bound, the integrity of tissue (29, 31) and through the and therefore less toxic. Further, calcium con- formation of free radicals which react with lip- centration in the water has a major influence on ids in cell membranes and so change the envir- the toxicity of Al and other metals, mainly be- onment and conditions for transport proteins in cause calcium is an important factor in deter- membranes (32, 33, see also discussion on gas- mining the permeability of cell membranes. ses below).

The negative effects of Al in fish are in the short A concentration of 15–20 µg LAl/L is generally term caused by deposition of positively charged considered a limit for damage to salmon smolt metal species to the negatively charged layer of in fresh water (34), while values approaching mucus on the gill. These depositions can cause 10 µg LAl/L have been known to affect sea- problems by eroding gill surface, causing in- water tolerance in smolt (35). In typical ion- creased mucus production and interfering with poor water qualities with low TOC, <10 µg respiration. In a longer perspective, even low Al/g (dry weight gill) are considered back- concentrations of Al at moderately low pH, ground levels (36, 37). Physiological changes around 6.0, may reduce the number of mucus are observed in smolt with gill Al >100–200 µg

104 Aquaculture Research: From Cage to Consumption Al/g and mortalities in freshwater are observed 44). Divalent iron may also be directly toxic by >350 µg Al/g. Seawater tolerance is negatively inhibiting calcium uptake. Fe and Al can also influenced by levels >40 µg Al/g, while mortal- act synergistically, increasing the toxicity of ei- ity in seawater is observed >150 µg/g (36). Re- ther metal alone (45, 46). cent studies, however, have shown a 50 per cent reduction in marine survival of salmon smolt In order to determine the toxicity of iron, it is with gill-Al levels as low as 40 µg Al/g, (35, 21, important to determine the fraction of iron 38, see further below). which is organically bound. Several studies have shown reduced toxicity when the level of Acid water and Al can also influence salmon in humic acids is high (47). Based on studies in sea cages in fjord sites. During periods of mild smolt sites during 1999–2002, problems have weather with snowmelt and heavy rainfall, sur- been observed when the total iron concentration face waters down to 5–6 m may show Al con- exceeds 70 µg/L and the ratio Fe/TOC exceeds centrations >100 mg/L, and gill Al may show 40 (48). In iron-poor water sources, gill iron increase from <10 µg/g under normal condi- levels of 100–150 µg Fe/g have been observed tions to >200 µg/g coinciding with massive (48). Recent studies have shown that oxidation mortalities. A main cause for the increased tox- of <200 µg FeII to FeIII caused considerable ac- icity is that seawater mobilises Al that is bound cumulation of Fe on fish gills (>1000 µg/g) in to humic acids or colloids from the freshwater addition to negative effects, including mortali- source (39, 40, 26). These effects will likely ties (49). Considerable efforts are therefore also be observed in marine fish species which underway to evaluate effects of various actions are exposed to similar mixing zones (41, 42, that can reduce the problem of oxidation, in- 43). cluding the use of neo-silicate (50, see further below). Iron (Fe) In water, Fe may be present in different forms, Carbon dioxide, CO2 ranging from simple divalent and trivalent ions Studies have shown that background levels in to products from hydrolysis, complexes or water sources for more than 100 smolt sites bound to organic (humic) or inorganic (clay) range from 1–2.5 mg CO2/L. During dry colloids and particles. When iron-rich anoxic periods, however, CO2-rich groundwater may groundwater mixes with oxygen-rich surface predominate in a water source, and at low tem- water ‘redox-mixing zones’ are formed in peratures this may cause CO2 super-saturation, which Fe2+ is oxidised to Fe3+-ions (FeIII-com- which cannot easily be removed using traditio- pounds) which hydrolyse, polymerise and nal aeration methods. CO2 from the water will deposit (known in Norway as okerutfelling). come in addition to CO2 produced by the fish in II High concentrations of Fe have been observed the rearing unit. Free CO2 may therefore be- in smolt sites which use groundwater. In such come a problem in land-based production using water sources, aeration or oxygenation may addition of oxygen and in closed transport of cause deposit of iron oxide directly in the fish fish. In situations with sufficient water ex- II tanks. High levels of Fe forms may cause the change and without addition of oxygen, O2 be- formation of free radicals with subsequent oxi- comes limiting long before CO2 even dative stress and lipid peroxidation (‘stale’ fat, approaches critical levels. In intensive produc-

Thematic area: Production 105 tion with addition of oxygen and reduced spe- sure enough O2 in the tanks. Oxygen is often cific water consumption, CO2 from metabolism supplied by super-saturating the inlet water and/ may accumulate in the water. CO2 reacts with or tank with O2, exposing the fish chronically or + water to form H2CO3, which dissociates to H acutely to high O2 levels. By adding pure oxy- ÷, and HCO3 , causing a reduction in pH. CO2 gen to super-saturation, water consumption can concentration may therefore have both direct be reduced to a minimum. However, this strat- and indirect effects on the physiology of fish; it egy can create serious problems. Firstly, these interacts with the important bicarbonate systems must be backed up by advanced secur- ÷ (HCO3 ) buffer system and may affect blood ity and control systems, as failure in oxygen pH, acid-base balance and so also hydro- supply will quickly cause anoxia and suffoca- mineral balance (51). Recent studies have fur- tion. Furthermore, there are serious concerns re- ther pointed to negative effects of high CO2 lev- lated to toxic effects of oxygen itself. els on bone mineralisation (51). Elevated levels of CO2 in intensive culture (30–40 mg CO2/L) Oxygenation using pure O2 gas will create a are a consequence of limited water supply, high toxic O2 environment for the fish, which has fish density and extensive use of oxygen sup- nothing to do with gas bubble disease or high plied at super-saturation. As outlined above, gas pressure in itself. Rather, the toxic effects CO2 reduces pH and thereby affects any sub- are related to the formation of the free oxygen - stances which show different forms depending radical superoxide (O2 ). on pH. Accordingly, a reduction in pH caused - by elevated levels of CO2 may re-mobilise met- O2 → •O2 (1) al ions, e.g. Al (52). The consequences of a - CO2-dependent reduction in pH would be the •O2 → H2O2(2) same as discussed above. High CO2 can further cause deposition of calcium carbonate in kidney H2O2 → HO•+H2O(3) tissue, known as nephrocalcinosis. This condition is characterised by visible, white, cheese- HO• → H2O(4) like, calcium-rich depositions in kidney tissue. Nephrocalcinosis has been observed at concen- Under normal conditions this is a radical which trations as low as 10 mg CO2/L following long- is formed as part of the aerobic respiration - term exposure. Increased capacity for binding chain. The mechanism of action for O2 in water metabolic CO2 can be achieved by increasing will be partly through direct effects on mem- bicarbonate contents of the water using addition brane transport proteins, partly through changes of seawater or liming (53); a reduction from in protein synthesis and partly through oxida- 2–4 mg CO2 has been observed when small tion of membrane lipids (32, 33, see also discus- doses of seawater have been added to smolt sion on metals above). To counteract such rearing tanks. consequences, animals have evolved defence systems which include e.g. the antioxidants vi- Oxygen, O2 tamins A, C and E, and Glutathione. Recent Intensive smolt production generally involves data show that the partial pressure in arterial high biomass, low specific water consumption blood of many fish species contain oxygen lev- and the use of oxygen supplementation to en- els corresponding to no more than 30 per cent

106 Aquaculture Research: From Cage to Consumption saturation (70), most likely because the formation of polymers as pH increases (56, 57, 26, tion of free radicals increases dramatically 58). FeIII species which hydrolyse and polymer- above this level. At oxygen pressures far above ise are extremely reactive and are quickly de- normal, e.g. in the case of super-saturation, posited on gill, causing ion-regulatory and oxygen partial pressure in blood increases, in- respiratory stress and mortality. Following an creasing the formation of free radicals. increase in pH from 6.3 to 7.0, oxidation of FeII Hydrogen peroxide (H2O2) may also negatively and the formation of the extremely reactive Fe influence production of erythropoetin (EPO), species increased through hydrolysis and poly- i.e. reduce production of haemoglobin and merisation of FeIII. Oxidation of FeII increased erythrocytes. At transfer to an environment con- Fe deposition on the gills, resulting in high mor- taining less O2 (e.g. seawater) this may cause tality within 24 hours. The results further sug- further problems. Recent findings suggest that gest that the use of neo-silicate or seawater in Atlantic salmon may be equally or more vulner- water sources rich in FeII reduces Fe deposition able to the formation of free radicals caused by on the gills. hyperoxia compared to other species, unless the salmon has more efficient de-toxification sys- Fish density – water chemistry and smolt tems than other fish species. Ongoing research quality is likely to resolve these questions in more de- The availability of fresh water is considered a tail. bottleneck in several Norwegian smolt production sites. As a consequence, several sites expe- Several smolt producers have installed equip- rience periods of too high biomass-water ment for CO2 removal using within-tank recir- consumption ratio, which requires oxygenation culation. It remains to be confirmed, however, of the production water, with subsequent in- whether this equipment contributes to reducing creases in CO2 and TAN. In order to avoid this the oxidative stress caused by super-oxygen- situation, regulations have been discussed ation, i.e. gradients/zones of oxygen super-satu- which would limit fish density in rearing tanks ration in the tanks. to maximum 20–30 kg/m3. Average density in Norwegian sites is estimated to range between De-toxification of water rich in Al and Fe 40 and 50 kg/m3. Regulations which would When acid, aluminium-rich fresh water changes limit the fish density in production of salmon pH, e.g. by liming, addition of sodium hydrox- juveniles and smolt would therefore represent a ide or by mixing with seawater, changes in pH significant limitation for the Norwegian salmon will cause polymerisation of Al, meaning that aquaculture industry. In a study motivated by low-molecular Al compounds form larger com- the need to resolve potential consequences of plexes (19, 54, 55, 49, 26). During this phase, fish density and water quality on growth and before a new equilibrium is formed, toxicity smoltification in salmon, potential 1+ smolt of may increase significantly as positively charged AquaGen strain were reared at 10C and con- Al complexes bind to the negatively charged trolled photoperiod (6 weeks on LD 12:12 fol- mucus of gill tissue. This is the main reason for lowed by 6 weeks on 24 L). Initial density was recommending a certain retention time after 20, 40, 60 and 80 kg/m3 and specific water con- treatment of aluminium-rich water, and/or the sumption was 0.3–0.4 L/kg fish per min. Oxy- use of neo-silicate, which prevents the forma- gen was added to maintain levels in effluent

Thematic area: Production 107 >80 per cent. Densities were maintained con- Bergen. This network carried out several series stant by regularly removing biomass from the of experiments during the period 1996–2003, tanks. Fish from all groups were transferred to among other things to determine limits for CO2 seawater and growth was monitored. in smolt rearing. The present CO2 limit of 15 mg/L in current rearing regulations is based In freshwater, average growth rates were ap- on this research. Studies during this period in- proximately 0.4 per cent per day in all groups, cluded water consumption during first-feeding with no differences among groups. With the ex- (59) and smoltification (60), studies of pH ef- ception of slightly elevated glucose levels in the fects alone (14), effects of CO2 (52, 53), effects 3 80 kg/m group, no differences were observed of CO2 and pH and effects of CO2, pH and alu- among groups in condition, fin erosion or gill minium (51). When salmon smolt are exposed + + ÷ Na ,K- ATPase, nor in plasma, Cl , CO2, hct, to elevated CO2 levels, respiration frequency pH, HCO3 or Hb during smoltification. These and haematocrit are temporarily increased (52, results are further confirmed by results from the 53). Plasma Cl÷ is always lower in smolt ex- seawater phase, where all groups showed an in- posed to CO2. Long-term effects of CO2 are increase in growth rate from 0.4 to 0.7 per cent/ creased frequency of nephrocalcinosis, reduced day, with no differences among groups. Further, condition factor, a moderate reduction in no differences in fin status or physiology were growth and in some cases increased mortality. observed after 12 weeks in seawater. Briefly Recent studies have further demonstrated that summarised, rearing densities as high as 80 kg/ an increase in CO2 and reduction in pH even at m3 do not appear to be limiting for growth or low Al concentrations may interfere with smol- smolt quality provided that supply of water and tification. The experiment was terminated after feed are sufficient to sustain a good rearing en- 25 days, when mortality had reached 42 per vironment. cent in the group exposed to 19mg/L CO2, pH 5.7, with the rest of the fish hyperventilating. In Water quality limits and long- another study it was shown that CO2 is most term effects of water quality on toxic at low temperatures (62), suggesting that smolt – water consumption, the aquaculture industry needs temperature-ad- CO2, oxygenation, pH, Al justed limits for CO2. Studies have also sug- At Bergen University College, long-term stud- gested that moderate oxygen super-saturation is ies have been performed since 1991 to deter- not harmful as long as total gas pressure is low mine limits for water use and oxygenation in (63). Salmon in intensive aquaculture spend smolt production. As early as 1991, several neg- large parts of their life in an environment with ative effects were observed on smolt from expo- elevated CO2 and further research on this pa- sure to reduced water consumption combined rameter is critical for the industry. with oxygenation. The recommendation of 0.3 L/kg fish/min was later defined based on Water quality – fish health and this work. Based on an initiative from the Re- production disorders search Council of Norway, a cooperative net- Water quality – smolt quality – sensitivity work was established including Bergen to IPN University College, Sævareid fish farm, the Na- Infectious pancreas necrosis (IPN) typically at- tional Veterinary Institute and the University of tacks early fry stages; in recent years, however,

108 Aquaculture Research: From Cage to Consumption Figure 2. Relative mortality (% of mortality of controls) in salmon under different rearing conditions (acid water, increased

CO2 and O2, reduced specific water consumption, increased density) exposed to IPN in seawater.

145x100//Kap06-fig02.eps increasing numbers of outbreaks around the bination of several factors and high variability time of smoltification and up to a year after creating a negative synergy, although stable transfer to seawater have been reported. Previ- high O2 levels seemed to counteract negative ef- ous studies have shown that highly intensive fects of some other parameters. The highest risk rearing conditions, including extensive use of of IPN outbreak was related to high fish dens- oxygen supplementation and low specific water ities, low specific water consumption and high consumption, may increase susceptibility to but variable O2 levels. IPN. These preliminary results suggested poor water quality as a probable cause for the in- Acid water and CO2 reduce growth in crease in IPN. On this basis a series of studies freshwater and can increase susceptibility have been conducted in which groups of salmon to IPN in salmon smolt after transfer to smolt have been reared in freshwater at differ- seawater ent densities and concentrations of CO2 and O2, Groups of smolting salmon were exposed to pH and under different pH conditions. Following 5.7, 5.9 and 6.5 combined with three different transfer to seawater, groups were exposed to the levels of CO2 (9, 13 and 20 mg/L) for 5–6 IPN virus. Mortality increased in all groups weeks in fresh water at 25–30 kg/m3, specific which had been exposed to sub-optimal rearing water consumption 1.3 L/kg/min and 10 °C. conditions in fresh water. CO2, O2 and density Smolt were transferred to seawater and infected were identified as critical factors, with the com- with IPN. Groups exposed to the highest CO2

Thematic area: Production 109 concentrations (13–20 mg/L) and lowest pH Taken together, these results suggest that oxy- (5.7–5.9) grew less than controls, while fish ex- gen concentrations above 134 per cent, specific posed to low pH only did not differ from con- water consumption below 0.14 L/kg/min and trols. After transfer to seawater and IPN CO2 concentrations above 12 mg/L increase the infection there was a tendency towards in- risk of disease and reduce welfare in salmon creased mortality in groups from the highest smolt, both in freshwater and after transfer to CO2 concentrations and lowest pH levels; seawater. again, previous exposure to low pH alone did not cause increased mortality from IPN. Elev- High O2 and CO2 levels can inhibit growth ated CO2 concentrations and low pH further re- and increase risk of IPN duced post-smolt growth in seawater, Groups of smolting salmon were exposed to suggesting that CO2 concentrations above 13 three O2 levels (85, 130, 155 per cent; pH 6.5) mg/L and pH below 5.9 should be avoided. combined with background or elevated levels of CO2 (30–35 mg/L; pH 5.6) for 5–6 weeks in Intensive production can inhibit growth, fresh water at 25–30 kg/m3, specific water con- seawater performance and increase risk sumption 1.4 L/kg/min and 10°C. Smolt were of IPN transferred to seawater and infected with IPN. Groups of smolting salmon were exposed to All exposed groups showed reduced growth high densities, low specific water consumption, during the freshwater phase, with the poorest high levels of CO2 and low pH. Compared with growth recorded in the three groups on elevated 3 controls (density 25 kg/m , specific water con- CO2. Slightly elevated O2 levels seemed to sumption 1.3 L/kg/min) densities in the range counteract some of the negative effects of high 3 80–87 kg/m inhibited growth and reduced re- CO2. Although there were no clear indications sistance to IPN. The combination of high densi- of differences in seawater performance, mortal- ty and low specific water consumption (0.14 L/ ity following IPN infection suggested a reduced kg/min) further reduced growth in freshwater resistance in all groups, with highest mortality and seawater performance. Resistance to IPN in fish exposed to high O2 and CO2 levels. Elev- was further significantly reduced. These prob- ated levels of CO2 and O2 do impose reduced lems were observed irrespective of oxygen lev- health and welfare in salmon smolt. els (103, 134 or 161 per cent), although there was a tendency of more severe problems with Freshwater quality and deformities in increasing oxygen levels. At the highest O2 sea – is there a connection? concentration in combination with high density Ever since deformities were recognised as a and low specific water consumption, observa- problem in salmon aquaculture in the mid- tions were made of mucus accumulating in the 1990s, studies have shown clear connections water, fungal infections and gas bladder disease between temperature and development of defor- in fresh water. This group also had the highest mities, at both egg and fry stages, and similarly mortality after transfer to seawater and IPN in- clear effects of long-term mineral deficiencies. fection, as well as the poorest post-smolt perfor- In modern, intensive smolt production, focus mance during the first three weeks in seawater. has been on changes in water quality, i.e. situa- Clear signs of stress were observed in groups tions where high density and low specific water exposed to low specific water consumption. consumption reduce water quality in fish tanks.

110 Aquaculture Research: From Cage to Consumption Figure 3. Relative growth (% of growth rate of controls) in salmon under different rearing conditions (acid water, increased

CO2 and O2, reduced specific water consumption, increased density).

145x100//Kap06-fig03.eps

Further, uncontrolled use of oxygen may cause Exposure to high levels of CO2 in fresh water additional problems. can be a contributing factor to developmental disorders of vertebrae. No documented effects of high levels of CO2 Excessive doses of O2 cause reduced In situations where CO2 levels increased with growth and irregular vertebrae in increasing biomass in fish tanks, but where con- seawater ditions where otherwise stable, no disorders Effects of excessive doses (125–130 per cent were seen in developing vertebrae, even when saturation) of oxygen in freshwater were exposed to 20–25 mg/L CO2 for 4–6 weeks. studied in an experiment where all other envir- However, in groups for which the tank environ- onmental factors were controlled. Fish were ex- ment was more unstable, a higher frequency of posed during two periods, first during a growth compressed caudal vertebrae was observed in period under constant light, and subsequently harvest-sized fish. Feeding was variable in during a period with photoperiod control to- these tanks and consequently the regulation of wards smoltification. Clear effects on growth in oxygen addition was difficult. CO2 levels fresh water were observed, and differences reached 30–35 mg/L for several weeks, and were still very evident after four months in sea- oxygen levels were unstable, with periods of water, with a 20 per cent reduction in growth of super-saturation in the tanks (>100 per cent). the most exposed groups. We also observed in-

Thematic area: Production 111 creased frequency of irregular vertebrae in the Small problems with open transport in most exposed groups, of a category which sug- well-boats gests subsequent development of deformities. Challenges with water quality during open There was a particularly strong effect of O2 lev- transport are related to the quality of the water el in the weeks prior to smolt transfer. entering the well, and the water consumption relative to the biomass. This is usually not a Water quality limits for gas bladder problem, as water consumption is typically 3–4 disease under hyperoxic conditions times the levels normally observed in juvenile We have previously observed that salmon ex- production (e.g. >100 L/m3 well volume/min). posed to high O2 levels develop gas bladders In most cases the water flow alone is sufficient beneath the skin, and observations have also to provide oxygen and remove waste, with the been made of a film of biological material on possible exception of when docking, during the surface. To resolve which levels of super- which water is pumped through the well. In saturation cause these changes, a short-term some cases additional oxygen must be supplied. study was done where oxygen level in the tank Oxygen saturation should not fall below 50 per was raised gradually. The surface film was ob- cent, nor should it exceed 120–130 per cent sat- served after 8–10 days exposure at 150–170 per uration (see section on oxygen). Densities typi- cent saturation. At about the same time, gas cally range between 115 and 180 kg/m3 for bladders were observed on the fish. Analyses of open transport of large salmon. Problems occur the surface film showed traces of bacteria, when valves have to be closed due to disease amoeba, fungi, scales and leucocytes, suggest- regulations, or smolt are transported for longer ing that skin damage in exposed fish causes the periods with closed valves. These challenges release of organic matter which provides sub- are discussed below for closed transport. strate for microbes. In smolt transport a density of 35–50 kg/m3 is Taken together, these results suggest that re- recommended when transport is mainly with duced water quality may contribute to the de- open valves. CO2 and NH3 do not normally ac- velopment of deformed vertebrae. The results cumulate to dangerous levels under conditions are particularly clear-cut for O2, and it is worth outlined above; CO2 levels in wells with open noticing that growth may also be influenced valves will normally be around 2 mg/L. One im- negatively by O2 levels >100 per cent. The un- portant consideration is the risk of introducing derlying mechanisms are, however, still unre- algae or chemicals. Further, critical mixing solved. We still lack knowledge of early signs zone chemistry may occur if fish are transported of developmental disorders of vertebrae, and we through an acid layer of freshwater in a fjord, or hope that future research will provide better an- acid water is loaded along with smolt into a well swers to these questions. filled with seawater. Problems related to acid fresh water being mixed with seawater and the Water quality during transport mobilisation, bioavailability and deposition of We distinguish between three types of trans- Al on fish gills are discussed above. port: open, intermittently open and closed, and closed transport.

112 Aquaculture Research: From Cage to Consumption Closed transport is challenging welling of bottom water can be observed in the Water quality in closed transport is determined autumn as cold, saline water enters in the deep- by the metabolism of the fish. Of particular con- er layers. In deep fjords with a shallow thresh- + cern is O2, CO2, NH4 + NH3 (TAN) and TOC. old and poor water exchange, the bottom water When CO2 accumulates, pH will drop, faster in can be anoxic and may contain H2S. fresh water than in seawater. For closed transport of large salmon, CO2 should not exceed 60 Large variations in temperature can be observed mg/L when TAN and TOC are high. A prelim- in a cage, directly influencing the distribution of inary study of transport of harvested salmon in the fish. At high seawater temperatures the closed and chilled systems showed that fish can salmon will seek out layers of the water column be transported in closed well for 8–10 hours at with the lowest temperature. Cod show the densities around 120 kg/m3 without mortality same response, seeking out lower temperatures or deterioration of harvest quality. Even if CO2 as oxygen saturation falls. The water quality ex- can exceed 50 mg/L it is NH3 which represents perienced by the fish when congregating in the greatest danger during closed transport. dense layers within the cage is still poorly re- This may become critical when new seawater at solved. higher pH has to be added to the wells. Acute NH3 poisoning may occur due to a rapid in- Seawater will normally be fully saturated with crease in pH, driving the equilibrium from O2 as it enters the sea cages, but blooms of algae + NH4 to NH3. To maintain low concentrations and zooplankton may cause periodic fluctua- of NH3, pH must be kept below 6.5; this will tions in CO2 and O2 concentrations. O2 levels normally occur after 1–2 hours during closed within the cage primarily depend on the con- transport in seawater due to accumulation of sumption of the biomass in the cage, and oxy- CO2. gen is the factor which will first become limiting. Oxygen deficiency in a sea cage can be Water quality in sea cage compensated by addition of O2 and/or use of production large propellers to increase circulation. Water In some fjord systems oxygen conditions may quality in cages is, however, best insured by vary considerably. The most critical conditions regulating fish density and the water flow are generally observed in the autumn, when sur- through (clean) cages, and by maintaining cage face temperature is high, concurrent with high geometry. Measurements of oxygen inside and biomass in the pens. In many fjords, an up-

Figure 4. Smolt exposed to Al are more sensitive to diseases and parasite attacks.

145x100//Kap06-fig06.eps

Thematic area: Production 113 outside the cage and on the side downstream of of these experiments, which were performed at the cage indicate water quality in the cage. the research station at Ims, was to study the relationship between water quality and seawater Water quality – smolt quality survival, and the importance of water quality in and interactions with salmon fresh water for the susceptibility and sensitivity lice of salmon lice. Acidification is an important cause of the reduction or extermination of salmon populations In the years 1999, 2000 and 2002–2004, groups from several rivers in Norway (64, 65). At high of 1200–1500 pre-smolt of Imsa stock were ex- concentrations of Al, mortality is caused by posed to good water quality (average pH>6,5; failure of respiration and ion-regulation (27). At <5 µg LAl/L) or one of three reduced water low concentrations, the physiology may be af- qualities (episodic high Al, long-term low Al, or fected. The ecological consequences may be long-term high Al). Long-term exposure lasted significant if the Al exposure hits the salmon at for a minimum of 30 days prior to smolt migra- critical stages, e.g. shortly before smolt migration, episodes for three days prior to release. tion. Low Al concentrations may affect growth Average pH ranged from 5.9 to 6.4 in low Al and the establishment of seawater tolerance. and from 5.5 to 6.0 in high Al. LAl ranged from While there is a general agreement that high 6 to 11 µg/L in low Al and from 12 to 21 µg/L concentrations of Al in fresh water will affect in high Al. On termination of exposure, the ma- populations, there is less documentation of the jority of smolt released into the Imsa River 150 ecological consequences of sub-lethal expo- m upstream of the river mouth. Migration out of sure. Several salmon populations in Western the river was voluntary. Additional groups were Norway are exposed to moderate acidification released 800 m upstream of a trap in the river to (65), and are also exposed to salmon lice after study downstream migration. Sensitivity to migration into seawater (66, 67). The objective salmon lice was studied by first acclimating two

Control Sea lice treated 120 Episode 100 High Al Untreated 100 Low Al 75 80

60 50 40

20 y = -0,96x + 105 25 R2 = 0,86

Total catch rates, (% of Control) 0 Accum. mortality (%); 42 days 0255075 0 Control Episode Low Al High Al -1 Terminal sample; gill-Al, µg g Freshwater treatment

Figure 5. An increase in gill Al from 25 to 50 • g Al/g reduced Figure 6. Exposure to Al increases sensitivity to further recaptures of adult salmon by 20–50% compared with stress. Gill Al levels of 50 • g Al/g cause a threefold increase controls. (Photo: Bengt Finstad) in mortality from exposure to salmon lice.

145x100//Kap06-fig04.eps 145x100//Kap06-fig05.eps

114 Aquaculture Research: From Cage to Consumption replicate groups of 150 smolt from each of the negative effects than either factor alone, and exposed groups and from controls to seawater, may be the main reason for declining stocks of and after 24 hrs in 33 ‰, exposing one group to salmon in many rivers. salmon lice, the other serving as control (68). Water quality – smolt quality The levels of gill Al were closely correlated and the development of with Al in the water when LAl exceeded 5 µg cataract Al/L. Prior to river release, exposed groups Additional studies were designed to cover the showed gill Al levels from 25 to 60 µg Al/g, topic of cataract and to study how rearing con- while control fish had gill Al in the range 5.9 ± ditions in freshwater prior to and during smolt- 3.3 µg Al/g. The physiology of the fish at re- ification affect development of cataract in lease and recapture rates of adult salmon were salmon, prior to and after transfer to seawater. closely related to gill Al. Recapture rates of Concurrent studies of wild migrating smolt groups released into the Imsa River showed a were performed to resolve possible causal rela- dose-dependent reduction in all groups with gill tionships. Tank studies were combined with Al exceeding 25 µg Al/g at the time of release. studies of wild smolt from Hardangerfjord and The relationship between recaptures, physiolo- Sognefjord. gy, gill Al and LAl suggest that even LAl concentrations approaching current detection limits Gases and cataract may represent critical conditions. Smolting salmon in freshwater were exposed to different levels of super-oxygenation and car- As expected, the salmon lice developed from bon dioxide (six weeks) and transferred to sea- copepodite to adult based on temperature. After water (seven weeks). At termination of the 28 days chalimus stages three and four, pre- study in seawater, increased prevalence of cata- adult males and females were detected. After 48 ract (range 4–32 per cent) was observed, with days stages from chalimus three to adult fe- the highest frequency in the group exposed to males were observed, independent of treatment. the highest level of super-oxygenation. How- Salmon smolt which had been exposed to Al ever, this group also showed the highest growth were more sensitive to salmon lice, and mortal- rate in seawater. Cataracts were low-level, with- ity was dose-dependent. Mortality increased out consequence for vision. Measurements of dramatically when lice reached the pre-adult mRNA levels of antioxidant enzymes SOD and stage after 28 days. This is in accordance with GST, as well as HSP70, showed reduced levels earlier reports showing the increase in mortality with increasing levels of super-oxygenation. is delayed in relation to the time of infestation (68, 69). The levels of acidification defined by Taken together, it cannot be concluded whether the concentrations of LAl (5–15 µg Al/L) were cataract is related to fast growth, which has at a level not normally associated with major been observed previously or directly to effects population effects in salmon. The combined ef- of high oxygen levels. Reduced levels of anti- fect of moderate acidification in freshwater and oxidants however, suggest that super-oxygen- salmon lice in seawater may have more severely ation may represent a stressor for the lens.

Thematic area: Production 115 Smolt quality and cataract Perspectives Osmotic cataract was observed in 111/178 fish Our understanding of the relationships between (Sognefjord 2002) and 16/73 (Sognefjord water quality and production of salmonids, in 2004). None of the 33 examined fish from Har- both culture and nature, is changing constantly. dangerfjord (2005) showed cataract. Osmola- New results from research challenge old con- lity in the chamber fluid was higher in fish from ceptions, limits have to be redefined, production Sognefjord (2002, 2004) than from Hardanger- strategies revised and new technologies devel- fjord (2005), with highest levels in fish showing oped and used. Further progress in this area re- osmotic cataract. Gill Na+,K+-ATPase was quires research activity spanning from basic lower in fish with cataract than in normal fish studies of single physical/chemical and biolog- (statistically significant in 2002), yet lower than ical factors to large-scale multidisciplinary in Imsa controls after three days of seawater ex- projects, where synergies between factors and posure. Histidine levels were lower in fish from the importance of scale can be resolved. Re- Ims than in wild migrating smolt. No differ- search must have the long-term objective of ences in N-acetylhistidine in lenses from the contributing to economically viable and sus- two fjords were observed, and no major salmon tainable production through all life stages, lice infestations were observed. where the environmental requirements are central and where novel findings contribute to ensuring normal development, good health and increased welfare in intensive culture.

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118 Aquaculture Research: From Cage to Consumption 2003b. Long-term sub-lethal effects of carbon diox- 61) Fivelstad, S., Bergheim, A., Hølland, P.M. & Fjer- ide on Atlantic salmon smolts: ion regulation, hae- medal, A.B., 2004a. Water flow requirements in the matology, element composition, nephrocalcinosis intensive production of Atlantic salmon (Salmo and growth parameters. Aquaculture 215: 301–319. salar L.). Aquaculture 231, 263–277. 53) Fivelstad, S., Olsen, A.B., Kløften, H., Ski, H.W. & 62) Fivelstad, S., Wågbø, R. & Olsen, A.B. Sub-lethal Stefansson, S., 1999a. Effects of carbon dioxide for effects of carbon dioxide at two temperatures on Atlantic salmon (Salmo salar L.) smolts at constant Atlantic salmon (Salmo salar L.) parr: physiology pH in bicarbonate rich freshwater. Aquaculture 178, and growth, in prep. 1–20. 171–187. 63) Hosfeld, C.D., Fivelstad, S., Waagbø, R., Olsen, 54) Lydersen, E., Poleo, A.B.S., Nandrup Pettersen, M., A.B., Breck, O., Engevik, A., Mollan, T., Lunde, T. Riise, G., Salbu, B., Kroglund, F. & Rosseland, & Stefansson, S. Long-term separate and combined B.O., 1994. The importance of "in situ" measure- effects of environmental hypercapnia and hyperoxia ments to relate toxicity and chemistry in dynamic in Atlantic salmon (Salmo salar L.) smolts. (in aluminium freshwater systems. - J. Ecol. Chem. 3: prep.) 357–365. 64) Hesthagen, T. & Hansen, L.P., 1991. Estimates of 55) Poleo, A.B.S., Lydersen, E., Rosseland, B.O., Kro- the annual loss of Atlantic salmon, Salmo salar L., glund, F., Salbu, B. Vogt, R. & Kvellestad, A., 1994. in Norway due to acidification. Aquacult.Fish.Man- Increased mortality of fish due to changing Al- age. 22: 85–91. chemistry of mixing zones between limed streams 65) Kroglund, F., Wright, R.F. & Burchart, C., 2002. and acidic tributaries. - Water, Air, and Soil Pollu- Acidification and Atlantic salmon: critical limits for tion 75: 339–351. Norwegian rivers. Oslo, Norwegian Institute for 56) Staurnes, M., Nordtvedt, R. & Rosseland, B.O., Water Research; 61 pp. 1998. Vannkvalitet. (Water quality). Pp 87–113 in: 66) Holst, J.C. Jakobsen, P.J., Nilsen, F., Holm, M. & Hansen, T. (ed.) Oppdrett av laksesmolt. (Produc- Asplin, L., 2001. Lakselusen dreper villaksen. Kan tion of Atlantic salmon smolt). Landbruksforlaget, vi spore effekter av tiltakene så langt? Institute of ISBN 82–529–1722–4. Marine Research, Bergen, Norway, Havbruksrap- 57) Åtland, Å., Teien, H.C., Bjerknes, V. & Rosseland, porten 2001. B.O., 2001. Erfaringer med bruk av silikat i sette- 67) Heuch, P.A., Bjørn, P.A., Finstad, B., Holst, J.C., fiskproduksjon. Norsk Fiskeoppdrett 2 /2001. Asplin, L. & Nilsen, F., 2005. A review of the Nor- 58) Teien, H.-C., Kroglund, F., Salbu, B., Åtland, Å. & wegian ‘National Action Plan Against Salmon Lice Rosseland, B.O., 2006. Sodium silicate as alterna- on Salmonids’: The effect on wild salmonids. tive to liming - reduced aluminium toxicity for At- Aquaculture 246, 79–92. lantic salmon in unstable mixing zones. Sci. Total 68) Bjørn, P.A. & Finstad, B., 1997. The physiological Environ. (In press) effects of salmon lice infection on sea trout post 59) Fivelstad, S., Bergheim, Kløften, H., Haugen, R., smolts. Nordic J. Freshw. Res. 73, 60–72. Lohne, T. & Olsen, A., 1999b. Water flow require- 69) Finstad, B., Bjørn, P.A., Grimnes, A. & Hvidsten, ments in the intensive production of Atlantic salm- N.A., 2000. Laboratory and field investigations of on (Salmo salar L.): Growth and oxygen salmon lice [Lepeophtheirus salmonis (Krøyer)] in- consumption. Aquacultural Engineering, 20, 1–15. festation on Atlantic salmon (Salmo salar L.) post- 60) Fivelstad, S. & Binde, M., 1994. Effects of reduced smolts. Aquacult. Res. 31, 795–803. waterflow in softwater on Atlantic salmon smolts 70) Massabuau, J.C., 2003. Primitive, and protective, (Salmo salar L.) while maintaining oxygen at con- our cellular oxygenation status. Mechanisms of stant level by oxygenation of the inlet water. Aqua- Ageing and Development 124, 857–863. cultural Engineering, 13, 211–238.

Thematic area: Production 119 Trygve T. Poppe1), Grete Bæverfjord2) and Tom Hansen3) 1) Norwegian School of Veterinary Science, 2) Akvaforsk – The Institute of Aquaculture Research AS, 3) Institute of Marine Research

Effects of Intensive Production with Emphasis on On-growing Production: Fast Growth, Deformities and Production-related diseases

Modern fish farming is highly efficient production based on large-scale farming, fast growth, automated feeding and extensive use of modern technology. Such efficient production offers many advantages in a highly competitive international market, but it is also a biological challenge. Most bacterial infections no longer pose a serious threat thanks to effective vaccination programmes and better biological knowledge. Nevertheless, diseases are causing severe losses for the industry. These diseases are primarily viral diseases and multifactorial diseases associated with intensive production. The common denominator for production-related diseases is that they are the result of intensive production, and their etiologies are often complex and diffuse. Examples of production-related diseases in modern fish farming are skeletal and soft-tissue malformation, cataract, side effects following vaccination and malfunction of organs. Some people would also argue that certain viral diseases should be classified as production-related diseases. These diseases cause severe losses for the industry, but also raise important ethical and animal welfare issues. Through different approaches scientists at different universities have tried to address some of these conditions. Improved diagnostic techniques and better knowledge of the mechanisms behind them will hopefully make important contributions to reducing the problems.

120 Aquaculture Research: From Cage to Consumption Figure 1. Production-related diseases in farmed salmon: – Emaciation following IPN or PD – Spinal deformity – Winter ulcer (Photo: T. Poppe)

In the course of a few years modern fish farm- growth has also been improved through deter- ing has developed from small-scale operations mined breeding over many generations. Growth into large-scale, information-sourced, high-tech rate, particularly during the freshwater phase, is industrialised production. Most farming com- therefore far better than for wild fish. Losses panies run an integrated operation, i.e. they pro- caused by diseases such as furunculosis, vibrio- duce both the smolts and run the on-growing sis and cold-water vibriosis have been reduced farms, and often the slaughterhouses as well. to a minimum thanks to effective vaccination The general trend is towards larger units, both programmes and improved environments. This, in freshwater and saltwater. Furthermore, pro- together with highly automated production, duction time has been reduced and production makes productivity very high. There is hardly itself made more effective. The result is bigger any other type of animal production that is more smolts in a shorter time compared to only a few industrialised and automated than the produc- years ago. This has been achieved by combining tion of salmon and rainbow trout. environmental factors influencing growth and development (e.g. light and temperature) with These developments, which are key elements in high-energy feed. The genetic potential for fast economically sustainable production, also pose

Thematic area: Production 121 challenges for the industry, particularly within side effects following vaccination. Today, dif- the fields of ethics and animal welfare. Exam- ferent skeletal abnormalities, soft tissue defor- ples of such challenges include several produc- mities (of the heart in particular), ulcers, fin and tion-related diseases and biological deviations gill diseases, cataract and side effects following that are presented below. vaccination are the most common production- related diseases. It is also a matter of discussion whether severe infectious diseases like IPN Production–related diseases should be considered a production-related Production-related diseases are those directly or disease (Figure 1). indirectly associated with modern, intensive farming procedures. In this context, it is important to appreciate that diseases are not caused by Animal welfare virus, bacteria, fungi and parasites alone. Any Animal welfare issues are both challenging and malfunction and abnormality should also be re- multidisciplinary, encompassing the humani- garded as a disease and treated and handled just ties, biological sciences and veterinary medi- like more traditional diseases. Production- cine. Report No. 12 (2002) to the Storting, related diseases are well known in other types Animal husbandry and animal welfare, invited of animal production, and their occurrence in to a broad debate on animal welfare. One result farmed fish should therefore not come as a sur- was the report Research Needs in Animal Welfa- prise. Such problems can manifest themselves re in Norway published by the Research Coun- in increased susceptibility to infectious diseas- cil of Norway in 2004. Both publications es, different ulcerative diseases, “wear and emphasise the importance of treating fish on tear”, deformities and malfunction of organs equal terms with traditional farm animals and (e.g. digestion, circulation, reproduction) and wild animals. This is a new way of thinking, as

Figure 2 A. Absence of septum transversum in farmed salmon. B: Heart from salmon with absence of septum transversum (top), compared to a normal heart (bottom). (Photo: T. Poppe)

122 Aquaculture Research: From Cage to Consumption fish have traditionally been ranked lower than deformities were identified as a problem in domesticated farm animals and pets. Compared modern salmon farming, and since then there to mammals, fish have few advantages in their has been extensive research to find causes and relationship to humans. They live in a complete- develop prophylactic measures. Problems simi- ly different environment, they have no means of lar to those in farmed salmon have also been ob- communication (sounds, facial expressions), served in farmed rainbow trout and cod. Such and they have body language and behaviour that problems are also found in other fish-farming are unfamiliar to humans. countries, and in other species, e.g. sea bass and sea bream. Current knowledge of pain perception in fish is disputed and insufficient, but recent research in- The prevalence of salmon with deformities is dicates that fish have the ability to perceive pain highly variable; from groups with close to zero according to their environment and needs. Until to groups where more than one-third of the fish there is adequate knowledge, fish should be have grossly visible malformations. The present treated as if they have the ability to perceive prevalence of fish with grossly visible spinal pain and discomfort. deformities at slaughter is 1 to 5 per cent. Fish with malformations have a slower growth rate One set of criteria frequently used for good an- than normal fish and have reduced market imal welfare is the “five freedoms” for domestic value. The financial losses associated with animals published by the Brambell Committee deformities are therefore formidable, even in 1965. With some modifications, these may when there is a relatively moderate prevalence. also be applied to fish, although it is obvious Increased public awareness about fish welfare that these criteria also pose great challenges also signals that such problems should be when it comes to fish. addressed seriously. • Freedom from hunger, thirst or malnutrition • Freedom from abnormal cold and heat Malformations during embryogenesis • Freedom from fear and stress Early egg stages have a lot in common with ear- • Freedom from injury and disease ly embryonic stages in mammals. Some of the • Freedom to behave and act naturally deformities observed in salmon in the 1990s were characteristic for teratogenic developmen- Today’s highly effective, industrialised and tal abnormalities, i.e. abnormalities developed market-oriented production, to which the finan- during early embryogenesis in the early egg cial aspect is crucial, represents huge challeng- stage. es with regard to ethics and animal welfare. High incubation temperature at the egg stage cause abnormalities in salmon and Deformities rainbow trout The first reports on deformities in fish were Preliminary studies in salmon showed that tem- published in the 1930s, and there are scattered peratures above 8 ºC during egg incubation reports of deformities both on the individual ba- were an important factor for initiating these sis and on a larger scale in the scientific litera- types of malformations. Other environmental ture. From the mid-1990s, malformations and factors were of relatively less significance. Lat-

Thematic area: Production 123 the occurrence of situs inversus will be reduced to a considerable extent. Other abnormalities following the same pattern are torsion of the swimbladder, incomplete operculae, pigment abnormalities and incomplete development of pyloric caecae. These abnormalities were observed as individual cases, or in combination in fish groups exposed to high temperature at the Figure 3. Platyspondyly in farmed salmon. Preparation: E. Sterud. (Photo: T. Poppe) egg stage. Vertebral abnormalities such as fusion of vertebrae will also occur when the eggs are exposed to high temperature (Figure 3).

Similar correlations between organ abnormalities, skeletal malformations and incubation temperatures were found in rainbow trout. The optimum temperature for rainbow trout eggs, Figure 4. Shortened vertebra from the mid-section of the however, was approximately 10 ºC, with a toler- vertebral column in a salmon with platyspondyly. (Fjelldal ance window from 8 to 12 ºC. & Grotmol 2005) Mechanisms for temperature-induced er studies showed that temperature sensitivity malformations was at its maximum in the period before eyed Research on mechanisms for induction of mal- eggs, i.e. during organogenesis. However the formations during embryogenesis in salmon formation of vertebrae was also affected by shows that expression of different genes of sig- high temperature in the period following eyed nificance for normal embryonal development is eggs, i.e. towards hatching and during the yolk- affected by temperature stress. Similar mecha- sac phase. Several different malformations nisms are known from other species, both fish show a correlation to high temperature during and mammals. Further research is important in embryogenesis. In fish with an absence of the order to understand what happens when such septum transversum, the tissue wall between deviations occur, not only during embryogene- the pericardial and abdominal cavity is not de- sis, but also during later developmental stages. veloped and the heart ends up in the abdominal Over time this will provide a better basis for cavity, often under or cranially to the liver. The both early diagnosis and prevention. heart thereby attains a divergent and sub-optimal shape. In situs inversus of the heart, the sep- Malformations induced during the tum transversum is normally developed, but the salmon parr stage heart is located more or less upside-down with- The parr stage is the most intensive period in the in the pericardial cavity, with resultant aberrant life of farmed salmon. The pressure on the fish shape and compromised function. When incu- is at its maximum during this stage of produc- bation temperatures are controlled and kept tion, and the environmental factors are stretched lower than 8 ºC, the problems with absent sep- to the limit. Approximately one-third of the tum transversum will be more or less absent and smolts are transferred to seawater as “autumn

124 Aquaculture Research: From Cage to Consumption smolts” (0+), i.e. the period from startfeeding to water/kg fish/minute) and by adding oxygen. seawater transfer is six to seven months. The These measures will result in an accumulation general trend is towards bigger smolts, which of metabolic CO2 in the fish tanks, and pH will again increases the pressure on limited water re- be reduced. The effects on fish have been inves- sources. tigated in several projects. In an aquatic environment, exposure of the fish to increasing Temperature during freshwater levels of CO2 and falling pH as the biomass in- production creases will cause pronounced physiological ef- Based on knowledge about temperature sensi- fects on the fish. However, specific effects on tivity in salmon eggs and fry, it was found nec- the development of deformities have not been essary to investigate whether there are similar registered as long as other environmental fac- mechanisms involved in the post-startfeeding tors have been stable and adequate. period as well. The results showed a clear correlation between high temperature and develop- In an experiment where gaseous CO2 was added, mental abnormalities in the vertebrae, also in internal structural changes of the vertebrae were the period following startfeeding, and this sen- observed in fish exposed to high CO2 levels in sitivity was present until the fish reached 60 freshwater, but there were no visible effects like grams. There was a greater occurrence of fish deformed vertebrae at slaughter. Nevertheless, a with deformed vertebrae when the fish had been high frequency of fish with spinal deformities at kept at water temperatures of 14, 16 and 18 ºC slaughter, primarily compressed and shortened during part of or the entire period from start- vertebrae (platyspondyly), has been observed in feeding to 60 grams than when the fish had been groups where poor water quality was combined kept at 12 ºC. Both the severity of lesions and with unstable conditions in the tank (Figure 4). the number of affected fish increased with tem- Poor water quality is characteristic for high fish perature. These results were confirmed by a density with high CO2 levels, high temperature corresponding higher percentage of fish down- and low pH, where the tank environment is graded from “superior” to “ordinary” or “pro- unstable due to fluctuating CO2 and oxygen duction” at slaughter. A continuous observation levels. Unfortunately, this scenario is highly of 200 individual fish from smolt to slaughter relevant for many farms. showed that temperature-induced malformations increased in severity over time: seemingly Controlled experiments where the specific ef- negligible deviations in the vertebral column at fect of oxygen overdose (superoxygenation) smolt size could result in severe fusions and during the last part of the freshwater phase have focal changes as the fish grew bigger. also been undertaken. This is a relevant scenario for many farms where oxygenation of the water Water quality is not properly controlled. The results after 16 Sufficient water of good quality is a limiting weeks in seawater showed a high percentage of factor in many smolt farms, and this issue be- fish with aberrant, narrow vertebrae in groups comes more pressing as smolt production is exposed to oxygen supersaturation during concentrated in fewer and bigger units. The in- smoltification. These abnormalities were not dividual farms solve this problem by reducing visible prior to seawater transfer and were not their specific water consumption (litres of

Thematic area: Production 125 typical for fully developed platyspondyly, but maximum. The longer the period with feed low nevertheless indicated a possible correlation. in P, the greater the number and the more serious the deformities. Studies of phosphorus In summary, the results of these experiments availability in fishmeal have shown that P di- show that there is a correlation between poor gestibility in fishmeal may be highly variable, freshwater quality and development of spinal and that P from e.g. fishmeal from blue whiting deformities as fish grow, but the correlation can be almost non-available. It is also a well- appears to be more complex than that between known problem that P digestibility can be re- spinal deformities and water temperature. duced in feeds containing vegetable components such as soybeans. It is therefore important Autumn smolts – Spring smolts that the availability of P in the feed be meas- Experience from practical fish farming shows ured, not only the feed’s quantitative chemical that autumn smolts (0+) are more susceptible to contents. developing deformities than spring smolts (1+). On average, the frequency of deformities in 0+ It has been a matter of discussion whether high is two times higher than in 1+, and the percent- CO2 levels in water may have a negative impact age of fish that perform poorly is also higher in on the mineralisation of bony structures and 0+. The production of 0+ and 1+ is basically contribute to the development of deformities by similar. In the production of 0+, however, time creating a conditional deficit of minerals of im- is short and these fish probably experience more portance for normal bone formation. Experi- stress and strain in several fields simultaneously ments combining these two factors showed that than 1+ do. feed had a great impact, while poor water quality had little or no extra impact. Mineralisation of the skeleton Several nutritional factors, primarily those af- Abnormal development of the skeleton is a fecting the mineralisation of bony structures, well-known problem in all species of fast-grow- are relevant as possible explanations for devel- ing animals. When the growth rate is at its max- opment of spinal deformities. Earlier studies imum, inadequate mineralisation of bone may have shown that available phosphorus (P) in the be a limiting factor independent of feed, and feed is the most critical single factor for the de- growth-related bone problems have been diffi- velopment of a normal skeleton. In a recently cult to solve, e.g. in poultry and swine produc- completed research project, salmon were given tion. There is therefore good reason to expect a feed containing 1.0–1.2 per cent P through that a fast growth rate in itself may be a risk fac- parts of or the entire lifecycle, compared to a tor for the development of deformities, and it is feed containing 1.6–1.7 per cent P. The feed therefore important to avoid the most extreme with the lowest P content was in accordance growth rates. with the published needs for salmon. The results from this project demonstrated a clear ef- Several other nutritional factors can also be as- fect of mineral supplementation on the sociated with deformities, both in salmon and in development of spinal and jaw abnormalities. other species. The effect of hypervitaminosis A The effects were at their maximum in early fry has been investigated, and it has been shown phase, when the relative growth rate was at its that bone formation in early fry stages is affec-

126 Aquaculture Research: From Cage to Consumption these parameters than spring smolts. This may indicate that smoltification and the early saltwater phase are a critical period for the maintenance of normal mineral metabolism and vertebral growth in salmon. Mineralisation of vertebrae can explain up to 70 per cent of the variation in the mechanical properties of the vertebrae in the central part of the vertebral col- Figure 5. Radiograph showing the vertebral column of a umn. Based on studies from human medicine it salmon with platyspondyly (a), and a salmon with normal may be surmised that microstructure-related vertebrae (b). (Photo: T. Hansen) factors and protein structure account for the rest of the variation. ted at high levels. Vitamin C deficiency is a classical and well-known condition in several Repeated radiology of individually tagged species. Under practical conditions most of spring smolts has shown that vertebral deformi- these deficiency conditions are irrelevant, ex- ties may develop both in the freshwater and in cept in a very few exceptional cases. the saltwater phase and that lesions initiated during the freshwater phase may get worse dur- Spinal deformities ing the saltwater phase (Figure 5). In experi- Radiology of salmon during the smoltification ments where the smolts have been vaccinated at period has shown that the smoltification process 18 or 20 ºC, significant vertebral fusions were initiates increased growth in the vertebrae in the observed, particularly in small fish. In these tail region and that deformities in this part of the cases growth has also been reduced to a consid- spine may develop at this life stage. Further- erable degree. more, these studies have shown that domestication and age at smoltification are of marginal In several experiments the results have shown significance for regional growth in the vertebral that vaccination interferes with mineralisation column during smoltification, but of great sig- in such a way that vaccinated fish have a higher nificance for the morphology, strength and min- mineral rate and stronger vertebrae than unvac- eralisation. Autumn smolts had weaker and less cinated fish. This shows that vaccination affects mineralised vertebrae than spring smolts, and vertebral growth. Whether this is a result of re- the vertebrae of autumn smolts were more com- duced growth following vaccination or of an pressed along the longitudinal axis. Wild salm- immune reaction to mineralisation is not known on had more rigid vertebrae than farmed spring at the present time. Furthermore, it is not clear and autumn smolts in the early saltwater phase. whether this difference in mineralisation be- At seawater transfer or sea migration, wild tween vaccinated and unvaccinated fish is al- salmon had better vertebrae mineralisation than ways long-lasting and whether it can be linked farmed spring smolts. These studies have also to later development of deformities. shown that the blood plasma levels of minerals (Ca, Mg, P) and stress indicators (lactate) are Genetic variation affected by the smolt production regime. One of the earliest theories launched concern- Autumn smolts had higher plasma levels of ing the etiology of malformations in salmon

Thematic area: Production 127 Figure 6. Vertebral deformity in a cod larva. (Photo: S. Helland, L.T. Poppe)

was a possible inbreeding in the farmed stocks. tion are known, although several different A genetic study of the correlation between procedures are used. There is still a tremendous growth and deformities in four year-classes variation in survival and larvae quality, both be- from the most commonly used strain in Norway tween farms and between different groups with- showed that there was no such correlation. Fur- in the same farm. thermore, there was no correlation between deformities and genetic growth potential, i.e. the Dorsal deviation of neck vertebrae is the most fastest-growing families were not those most commonly observed deformity in farmed cod. susceptible to deformities. A hereditary vari- This condition has been associated with abnor- ation in the occurrence of deformities was mal development of the swimbladder in early found, with a hereditability of 0.2–0.3 for three stages, and some farms have been able to reduce of the four year-classes. the problem through adjustments of their water treatment procedures. Deformities in farmed cod Cod farming is new and relatively small-scale A large-scale mapping of deformities in larvae compared to farming of salmon and rainbow and small cod from all the commercial fry pro- trout, but the outlook for further development of ducers showed many different deformities in this industry is promising. Production of marine the head, neck, backbone and tail areas (Figure larvae is more complicated and complex than in 6). Dorsal deviation of neck vertebrae was only the production of salmonids. The yolk-sac lar- one of several different deviations that were ob- vae of cod are very small and little developed, served, although it was not a major problem. and it is necessary to use live feed for startfeed- There were different types of vertebral fusions, ing. The basic techniques for cod larvae produc- different snout and jaw abnormalities plus axis

128 Aquaculture Research: From Cage to Consumption deviations of the vertebral column. In some ing handling, treatment and transport, or in fish, axis deviations caudal to the vent area were periods with low ambient oxygen levels. In wild observed. These lesions progressed to lordosis, salmonids the cardiac ventricle has a marked the most common skeletal abnormality in triangular or pyramidal shape which is impor- farmed sea bass and sea bream. Other skeletal tant for normal function. However, the shape is abnormalities were more or less comparable to flexible within certain limits as fish living in those observed in salmon. All producers had a lakes have a smaller and more rounded ventricle high percentage of fish with considerable ab- than anadromous fish that ascend rivers during normalities, but there was a wide variation in their spawning migration. It has recently been the severity and the type of abnormalities be- shown that farmed fish have a smaller and more tween farms. However, abnormalities in differ- rounded ventricle than wild fish of similar size ent groups from the same farm were quite (Figure 7). Experiments with rainbow trout in similar in this material, indicating that there is France have shown that fish with small, round much to be gained from standardising and opti- hearts have poorer stamina in swimming tests mising production routines for cod larvae. and a higher condition factor than fish with sharp, triangular ventricles. It is currently not known whether the small rounded hearts in Heart farmed fish are the result of a docile life in cages Recently there has been increased focus on car- or if they represent a gradual development over diac diseases in farmed fish, and there appears several generations. Whatever the causes, fish to be a general consensus in several countries with such hearts are unwanted, as they are far that farmed fish suffer from a wide range of less robust and tolerate less stress and handling more or less well-defined diseases associated than fish with sharp, triangular hearts. with the heart. Knowledge about the different conditions is important, as deviations in heart In addition to aberrant shape, several abnormal morphology and function reduce the tolerance conditions associated with the heart have been for normal physical challenges in the fish farm observed in farmed fish. Absence of septum environment. This may result in “unexplain- transversum can still be found in individual fish, able” mortality of apparently healthy fish dur- but these days its occurrence is close to zero as a result of better control of the incubation temperature during the egg stage. The occurrence of situs inversus has also been reduced, but not to the same extent as absent septum transversum. There are indications that temperature, both at the egg stage and during the fry period, is of significance for relative heart size.

Abnormal architecture of the ventricle has been shown in both rainbow trout and salmon. In these cases, the outer compact muscle layer is

Figure 7. Heart of a farmed salmon (left), compared to the poorly developed or not developed at all. Thus, heart of a wild salmon of equal size (right). (Photo: T. Poppe) the normal function of the heart is severely

Thematic area: Production 129 compromised and the fish typically die before they reach the smolt stage. The causes are unknown.

Vaccines All farmed salmon are immunised with oil- adjuvanted vaccines. The vaccine is given in the peritoneal cavity by injection (intraperitoneally) prior to smoltification and gives good protection Figure 8. Black pigment (melanin) on the peritoneal surface against certain bacterial diseases, while protec- of a salmon with severe vaccination side effects. tion against IPN and winter ulcers is somewhat (Photo: T. Poppe) variable. All oil-adjuvanted vaccines cause a local inflammation around the injection site. gical status of the fish. Furthermore, the approv- Such minor side effects cause hardly any prob- al of fish vaccines should be re-evaluated, and lems for the fish and will normally not reduce control and reporting improved. the quality at slaughter. In some cases, however, the vaccine causes severe side-effects with An epidemiological survey shows significant adhesions between visceral organs and the body differences in the occurrence of malformations wall, melanin patches in the muscle, reduced in autumn smolts depending on the time of vac- growth, increased number of fish with spinal de- cination. Smolts that were vaccinated during formities and some other diffuse side effects. In the photomanipulation period (the dark period) addition to the financial losses, which may be had less serious malformations than fish vacci- considerable, there are obvious animal welfare nated during the following period with continu- issues. Huge batches of fish have been culled af- ous light. It has also been shown that vaccina- ter six months in seawater because of serious tion strategy may affect the development of side effects. The occurrence of these side effects deformities. Two experiments revealed that has been highly unpredictable. Even if some groups with severe side effects (adhesions) have vaccine batches have caused more severe prob- developed more deformities than groups with lems than others, this has not explained the huge less severe side effects and/or unvaccinated fish. variation in occurrence (Figure 8). In most vaccination experiments, however, deformities do not develop. The mechanisms Vaccine side-effects and deformities and interrelations between adhesions and mal- During the project period it has been shown that formations are at the present time unknown, but early vaccination (small fish) and high temper- will hopefully be clarified by future research. ature contribute to serious side effects and a high percentage of fish with spinal deformities. Furthermore, it has been shown that other dis- Prenatal stress eases and stress during and after vaccination Prenatal stress is defined as stress experienced will contribute to more serious problems. Thus, by the gravid mother animal that can affect the the potential improvement lies in optimising the development of the offspring. Stress may result time of vaccination in relation to the physiolo- in increased production of glucocorticoids that

130 Aquaculture Research: From Cage to Consumption can pass from the mother to egg or embryo. If result of osmotic changes in the environment. It the levels of steroids in the mother remain ele- is usually reversible unless the swelling of the vated over time, it may negatively affect the off- fibres is extensive or the fibres are torn apart. spring. Prenatal stress may result in reduced Cataract may occur throughout the entire life growth, increased mortality, depressed immune cycle of the salmon, but salmon appear to be system, altered behaviour and increased occur- most susceptible during the smoltification rence of deformities (morphological devia- period and the first summer in seawater. Cata- tions). Such mechanisms are well known in ract results in impaired vision, and therefore mammals and birds, and have now been experi- reduced feed uptake, growth and welfare. The mentally demonstrated in salmon by injecting degree of vision impairment depends on the broodfish with cortisol. Relevant stressors in severity of the lens lesions. broodfish salmon include high temperature, low oxygen levels, pollution, different pathogens In addition to osmoregulatory and nutritional and parasites, high stocking density and hand- factors, several other conditions may cause ling prior to spawning. Improved routines for cataract in fish. Fast growth, water temperature broodfish handling and optimising the environ- fluctuations, fluke infestations (Diplostomum ment are therefore important in preventing and Tylodelphus spp.), exposure to UV light, these types of problems. side effects following medication, gas supersaturation, salinity fluctuations and genetic predis- position have all been shown to be involved in Cataract the development of cataract. Cataract is opacity of the lens leading to impaired vision (Figure 9). Cataract is considered Cataract in modern salmon farming is primarily to be a multifactorial production disease where associated with nutrition and fast growth. The several nutritional and environmental factors problem became very evident when the use of may lead to the same result. Cataract is the result of structural changes of the lens leading to light diffraction instead of normal refraction, and may include destruction of lens fibres and/ or growth of abnormal lens epithelium cells on the surface of the lens. Osmotic cataract is the

Figure 10. Diagnosis of cataract in salmon using a slit lamp Figure 9. Cataract in farmed salmon. (Photo: T. Poppe) biomicroscope. (Photo: E. Bjerkås)

Thematic area: Production 131 Figure 11. Winter ulcers. (Photo: T. Poppe)

bloodmeal in the feed was prohibited, and it ap- cause growth loss, mortality and downgrading/ pears that the amino acid histidine is of great cassation at slaughter (Figure 11). importance for nutritional cataract. Histidine- supplemented feed is therefore used during critical periods of production (Figure 10). Challenges, the road ahead Thanks to the effectivity and skill of fish farmers, as well as intensive research in several Ulcers fields, the health status of farmed fish is gener- Different types of ulcers are common in farmed ally good. Most bacterial diseases are under fish and may have different causes (physical control, while viral diseases still cause mortal- damage, infections). One of the best-known ity and problems for the industry. In addition to conditions is “winter ulcers”, in which the bac- well-known viral diseases, “new” viral diseases terium Moritella viscosa plays a key, but not de- have appeared on the scene, creating challenges cisive, role. The fish develop skin ulcers with for both farmers and scientists. Modern fish exposure of the underlying muscle, the osmotic farming is extremely rationalised and stream- barrier is broken, and bacteria from the environ- lined, resulting in highly efficient production. ment may gain access. This is typically a low- Large-scale units, integrated production and in- temperature problem, and healing of the ulcers tensification throughout the production cycle will not take place until the temperature in- also represent formidable challenges. Under creases in the spring. Modern multivalent vac- such circumstances, it is easy to forget that fish cines provide some protection, but the problem farming comprises biological production based is nevertheless extensive. In addition to the eth- on organisms with very specific needs and re- ical and animal welfare aspects, winter ulcers quirements with regard to environment, feed, care and biological framework if they are to

132 Aquaculture Research: From Cage to Consumption In the white paper Animal welfare and animal husbandry, Norway signalled that it intends to be at the forefront in animal welfare and that healthy, well-functioning animals should be a prerequisite in all farming. Norway is heavily involved in international fish farming and therefore has a particular responsibility to guide developments in the right direction.

Even though the primary aim of fish farming is to provide consumers with safe food of first- class quality, documentation of farming under Figure 12. Production-related diseases may cause severe losses in fish farming. (Photo: T. Poppe) ethically acceptable conditions must also be provided. This requires the survey and documentation of the environmental parameters and function optimally. In fish farming, as in swine health status throughout the production cycle as and poultry production, “production diseases” well as sustained focus on animal welfare and are playing an increasingly important role. Un- production diseases. This will enable the im- less society makes accommodations for ethical provement of production systems, resulting in production, ethics will always be on the losing better health and a better environment for the end when profit and ethics are opposing forces. fish. It is of great importance that this work has A major challenge is therefore to establish con- a long-term perspective and that it remains in- ditions that provide a balance between the two dependent of periods of growth and recession in in the context of both national and international the industry. competition.

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Thematic area: Production 133 of adherences, growth and spinal deformities in At- Fjelldal, P.G., Grotmol, S., Kryvi, H., Gjerdet, N.R., lantic salmon (Salmo salar L.). Dis. Aquat. Org. (in Taranger, G.L., Hansen, T., Porter, M.J.R., Totland, press). G.K., 2004. Pinealectomy induces malformation of Bjerkås, E., Bjørnestad, E., Breck, O. & Waagbø, R., the spine and reduces the mechanical strength of 2001. Water temperature regimes affect cataract de- vertebrae in Atlantic salmon, Salmo salar. J. Pineal velopment in smolting Atlantic salmon, Salmo sa- Res. 36,1–8. lar L. J.Fish.Dis. 24, 281–291. Fjelldal, P.G., Nordgarden, U., Berg, A., Grotmol, S., Bjerkås, E., Sveier, H., 2004. The influence of environ- Totland, G.K., Wargelius, A., Hansen, T., 2005. mental and nutritional factors on osmoregulation Vertebrae of the trunk and tail display different and cataract in Atlantic salmon (Salmo salar L). growth rates in response to photoperiod in Atlantic Aquaculture 235, 101–122. salmon, Salmo salar L., post-smolts. Aquaculture Bjerkås, E., Waagbø, R., Sveier, H., Breck, O., Bjerkås, (in press). I., Bjørnestad, E., Maage, A., 1996. Cataract devel- Fjelldal, P.G., 2005. Growth and mineralisation of the opment in Atlantic salmon (Salmo salar L.) in fresh vertebral column in cultured Atlantic salmon water. Acta Vet Scand 37, 351–360. (Salmo salar, L.). Ph.D. Thesis, University of Ber- Breck, O., 2004. Histidine nutrition and cataract devel- gen. opment in Atlantic salmon. Dissertation for the de- Fjelldal, P.G., Berg, A., Hansen, T., 2005. The develop- gree of Doctor Scientiarum, University of Bergen, ment of vertebral column deformations in Atlantic Bergen, Norway. salmon Salmo salar yearling smolts. Eur. Aquacult. Breck, O., Bjerkås, E., Campbell, P., Arnesen, P., Hal- Soc. Spec. Pub. No. 35, 199–200. dorsen, P., Waagbø, R., 2003. Cataract preventative Fjelldal, P.G., Hansen, T., 2004. Hurtig smoltvekst kan role of mammalian blood meal, histidine, iron and gi skjelettdeformasjoner. Norsk Fiskeoppdrett nr. zinc in diets for Atlantic salmon (Salmo salar L) of 10. different strains. Aquacult. Nutr. 9, 341–350. Fjelldal, P.G., Wargelius, A., Nordgarden, U., Hansen, Breck, O., Bjerkås, E., Campbell, P., Rhodes, J.D., T., 2004. Ryggvirvler hos laks i saltvann: Styrke og Sanderson, J., Waagbø, R., 2005. Histidine nutrition mineralinnhold påvirkes av lysregime og vekst- and genotype affect cataract development in Atlan- hastighet. Norsk Fiskeoppdrett nr. 11. tic salmon, Salmo salar L. J. Fish Dis. 28, 357–371. Forskningsbehov innen dyrevelferd i Norge, 2005. Rap- Breck, O., Bjerkås E., Sanderson J., Waagbø R., Camp- port fra styringsgruppen. Norges forskningsråd. bell P., 2005. Dietary histidine affects lens protein Gjerde, B., Pante, M.J.R., Baeverfjord, G., 2005. Genet- turnover and synthesis of N-acetylhistidine in At- ic variation for a vertebral deformity in Atlantic lantic salmon (Salmo salar L.) undergoing parr- salmon. Aquaculture, 244, 77–87. smolt transformation. Aquacult. Nutr. 11, 321–332. Grotmol, S., Kryvi., H., Totland, G.K., 2005. Deforma- Breck, O., Sveier, H., 2001. Growth and cataract devel- tion of the notochord by pressure from the swim opment in two groups of Atlantic salmon (Salmo sa- bladder may cause malformation of the vertebral lar L) post smolts transferred to sea with a four- column in cultured Atlantic cod Gadus morhua lar- week interval. Bull. Eur. Ass. Fish Pathol. 21, 91– vae: a case study. Dis. Aquat Org. 65, 115–120. 103. Helland, S., Refstie, S. Espmark, Å.M., Hjelde, K, Bae- Brønstad, I., Bjerkås I, Waagbø R., 2002. The need for verfjord, G., 2005. Mineral balance and bone for- riboflavin supplementation in high and low energy mation in fast-growing Atlantic salmon parr (Salmo diets for Atlantic salmon (Salmo salar L.) parr. salar) in response to dissolved metabolic carbon di- Aquacult. Nutr. 8, 209–220. oxide and restricted dietary phosphorus supply. Ersdal, C., Midtlyng, P., Jarp, J., 2001. An epidemiolog- Aquaculture, 250, 364–376. ical study of cataracts in seawater farmed Atlantic Kvellestad, A., Høie, S., Thorud, K., Tørud, B., Lyngøy, salmon Salmo salar. Dis. Aquat. Org. 45, 229–236. A., 2000. Platyspondyly and shortness of vertebral Fivelstad, S., Olsen, A.B., Åsgård, T., Baeverfjord, G., column in farmed Atlantic salmon Salmo salar in Rasmussen, T., Vindheim, T. and Stefansson, S. Norway – description and interpretation of patho- 2003. Long-term sublethal effects of carbon dioxide logical changes, Dis. Aquat. Org. 39, 97–108. on Atlantic salmon smolts (Salmo salar L.): Ion Kjønniksen, I., Bjerkås, E., Jellum, E., Ringvold, A., regulation, haematology, element composition, 2003. A comparative study of free amino acids in nephrocalcinosis and growth parameters. Aquacul- aqueous humour in various animal species. Ophth. ture, 215, 301–319. Res. 35, 208–216.

134 Aquaculture Research: From Cage to Consumption Kjønniksen, I., Bjerkås, E., Jellum, E., Ringvold, A.; Stortingsmelding nr. 12. Om dyrehold og dyrevelferd. 2002. Comparison of free amino acids in aqueous 2003. Landbruksdepartement. humor of farmed and wild salmon and of six species Takle, H., Baeverfjord, G. and Andersen, O. Identifica- additionally. Ophth. Res. 34, 366–370. tion of stress sensitive genes in hyperthermic Atlan- Menzies, F.D., Crockford, T., Breck, O., Midtlyng, P.J., tic salmon (Salmo salar) embryos by RAP-PCR. 2002. Estimation of direct costs associated with cat- Fish Physiol Biochem, 30: 275. 281. aracts in farmed Atlantic salmon (Salmo salar). Takle, H., Baeverfjord, G., Helland, S., Kjørsvik, E. and Bull. Eur. Ass. Fish Pathol. 22, 27–32. Andersen, O. Hyperthermia induced atrial natriuret- Midtlyng, P.J., Ahrend, M., Bjerkås, E., Waagbø, R., ic peptide (ANP) expression and deviant heart de- Wall, T., 1999. Current research on cataracts in fish. velopment in Atlantic salmon Salmo salar embryos. Bull. Eur. Ass. Fish Pathol. 19, 299–301. Gen. Comp. Endocrinol. (in press). Nordgarden, U., Hansen, T., Hemre, G.I., Sundby, A., Takle, H., Baeverfjord, G., Lunde, M., Andersen, Ø., Björnsson, B.Th., 2005. Endocrine growth regula- 2005. The effect of heat and cold exposure on tion of adult Atlantic salmon in seawater: The ef- HSP70 expression and development of deformities fects of light regime on plasma growth hormone, during embryogenesis of Atlantic salmon (Salmo insulin-like growth factor-I, and insulin levels. salar). Aquaculture, 249, 515–524. Aquaculture (in press) Waagbø, R., Bjerkås, E., Hamre, K., Berge, R., Wathne, Olsvik, P.A., Kristensen, T., Waagbø, R., Rossseland, E., Lie, Ø., Torstensen, B., 2003. Cataract formation B.O., Tollefsen K.E., Baeverfjord, G., Berntssen, in Atlantic salmon, Salmo salar L. smolt, relative to M.H.G., 2005. SOD, CAT and GSH-Px mRNA ex- dietary pro- and antioxidants and lipid level. J. Fish pression and lipid peroxidative stress in liver of At- Dis. 26, 213–229. lantic salmon Salmo salar exposed to hyperoxic Waagbø, R., Breck, O., Torstensen, B., 2004. Dietary conditions during smoltification. Comp. Biochem. lipid regimes can affect cataract development in At- Physiol. C, 141, 314–323. lantic salmon (Salmo salar L.) growers. Proceed- Oppedal, F., Hansen T., Taranger, G.L., Berg, A., Olsen, ings 11th International Symposium on Nutrition R.E., 2005. Photoperiod in seawater influenced sea- and Feeding in Fish (ISNFF), 2–7 May 2004, sonal growth and chemical composition in autumn Phuket, Thailand. Abstract O59. sea-transferred Atlantic salmon (Salmo salar L.). Waagbø, R., Sveier, H., Breck, O., Bjørnestad, E., Aquaculture (in press). Maage, A.., Bjerkås, E., 1998. Cataract formation in Poppe, T.T., Helberg, H., Griffiths,D., Meldal, H., 1997. smolting Atlantic salmon, Salmo salar, fed low and Swimbladder abnormality in farmed Atlantic salm- high energy diets. Bull. Eur. Ass. Fish Pathol. 18, on Salmo salar. Dis. Aquat. Org. 30, 73–76. 201–205. Poppe, T.T., Midtlyng, P.J., Sande, R.D., 1998. Exami- Wall, T., Bjerkås, E., 1999. A simplified method of nation of abdominal organs and diagnosis of defi- scoring cataracts in fish. Bull. Eur. Ass. Fish Pathol. cient septum transversum in Atlantic salmon, Salmo 19, 162–165. salar L., using diagnostic ultrasound imaging. J. Wargelius, A., Fjelldal, P.G., Benedet, S., Hansen, T., Fish Dis. 21, 67–72. Björnsson, B. Th., Nordgarden, U., 2005. A peak in Poppe, T.T., Taksdal, T., 2000. Ventricular hypoplasia in gh-receptor expression is associated with growth farmed Atlantic salmon Salmo salar. Dis. Aquat. activation in Atlantic salmon vertebrae, while up- Org. 42, 35–40. regulation of igf-I receptor expression is related to Poppe, T.T., Johansen, R., Tørud, B., 2002. Cardiac ab- increased bone density. Gen. Comp. Endocrin. 142, normality with associated hernia in farmed rainbow 163–168. trout Oncorhynchus mykiss. Dis. Aquat. Org. 50, Wargelius, A.., Fjelldal, P.G., Hansen, T., 2005. Heat 153–155. shock during early somitogenesis induces caudal Poppe, T.T., Johansen, R., Gunnes, G., Tørud, B., 2003. vertebral column defects in Atlantic salmon (Salmo Heart morphology in wild and farmed Atlantic salar). Devel. Genes Evol. 215, 350–357. salmon Salmo salar and rainbow trout Oncor- Wargelius, A., Nordgarden, U., Fjelldal, P.G., Berg, A., hynchus mykiss. Dis. Aquat. Org. 57, 103–108. Hansen, T., 2004. Spring growth in the vertebrae of Skilbrei, O.T., Hansen, T., 2004. Effects of pre-smolt Atlantic salmon (Salmo salar) is characterized by a photoperiod regimes on post-smolt growth rates of gradual increase in ihh expression, while a certain different genetic groups of Atlantic salmon (Salmo photoperiod triggers shh induction. J. Bone Min. salar). Aquaculture 242, 671–688. Res. 19, 217–217.

Thematic area: Production 135 Stein Mortensen1), Øivind Strand1), Arne Duinker2), Sissel Andersen1) and Tore Aune3) 1) Institute of Marine Research, 2) NIFES (National Institute of Nutrition and Seafood Research), 3) Norwegian School of Veterinary Science

A New Boost for the Norwegian Shellfish Sector

The shellfish sector has made a great deal of progress in the course of the past few years. Some of the airy visions that have been offered still lie far in the future, but we have experienced slow but sure growth based on laboriously acquired knowledge. However, improvements still need to be made. There is still a great deal of potential for improving methods of cultivation, identifying optimal sites and operating routines for farms, if we are to meet the challenges of marketing high- quality toxin-free shellfish for as much of the year as possible.

136 Aquaculture Research: From Cage to Consumption The Norwegian shellfish industry was given a adapt our cultivation practices to them, there boost in the 1990s by the “Scallop Programme”. will always be farms somewhere on the coast An important component of the progress was that are capable of supplying high-quality shell- the coordinated, goal-oriented R&D that was fish. initiated by this programme, which accelerated the build-up of knowledge and transformed the The coast of Norway stretches relatively far to Scallop Programme into a professional forum the north, and many coastal and fjord areas have that brought together representatives of indus- a relatively low range of temperatures and lim- try, research and the management authorities. ited availability of light and nutrients for much However, the “scallops” concept soon became of the year. It has been generally assumed that too narrow in scope. The project was turned into these conditions were synonymous with slower the “Shellfish Programme”, and a professional growth and low cultivation profitability, and it platform for the industry gradually emerged. A has been a challenging task to obtain good doc- number of R&D projects were generated under umentation regarding growth conditions and to the organisational umbrella of this programme. explain the wide range in growth rates displayed by different sites and cultivation systems. Mussels on long stretches of Norway’s Norway’s natural advantages elongated coastline grow well and have high for shellfish cultivation meat content. The pattern of growth of Norwe- Norway’s long coastline offers great potential gian scallops shows good growth over a longer for shellfish cultivation. On the basis of the total period of years than scallops from farther south area of sheltered productive areas on the coast, in Europe, so that there is a greater availability we could theoretically produce millions of of large scallops here. Norwegian oysters have tonnes of shellfish a year. The fjords and the been shown to grow to harvesting size in three archipelago offer a wealth of sites with a wide years, when suitable sites and cultivation prac- range of different characteristics. In an initial tices are employed. phase, such a wide range of variation may actually be a disadvantage, since a production strat- All experience confirms the fact that appropri- egy that is suitable for one site may not be ate siting and cultivation practices are often transferable to another. It has become more and more important than differences between differ- more clear that cultivation practices need to be ent parts of the coastline. In the course of the adapted to the site, and that we will need to ob- past ten years, we have gained better knowledge tain essential knowledge of how the environ- of the natural conditions that are most appropri- ment affects production conditions. In the ate for shellfish cultivation, and the shellfish longer term, differences in site conditions are farming industry will gradually implement the likely to be a major advantage, since they will best solutions. Thanks to good R&D projects, provide an opportunity to draw up a range of the knowledge-based development of the shell- strategies for dealing with toxic algae, preda- fish sector will play a key role in the optimal tors, fouling, etc. If we have good knowledge of utilisation of this country’s good natural condi- the characteristics of different areas and can tions for shellfish farming.

Thematic area: Production 137 Shellfish production perience, realising that knowledge, practical ex- Shellfish cultivation in Norway is concentrated perience and a long-term view are all essential on scallops, mussels and oysters. As this chap- components of success. A lack of profit has led ter makes clear, the methods of production em- to bankruptcies and a weeding out of this sector, ployed in raising these three species are quite leaving us with players with more experience different. Projects related directly to shellfish and better chances of obtaining good results. On production, therefore, have naturally had quite the negative side, however, it is difficult to at- different points of focus. However, there are a tract investors, and many companies are strug- number of scientific problems common to the gling with a lack of equipment due to a shortage three species. The most obvious of these is the of capital, even though most of them are now problem of algae that produce toxins. There are prepared to cultivate and care for their shellfish also problems of health and disease, the spread properly. However, it is important to bring out of infection, the effects of joint production fa- the fact that the initial phases of the shellfish cilities, the carrying capacity of sites, sea ranch- farming sectors in such countries as Canada and ing and questions related to management. We New Zealand had several features in common will discuss these questions in the course of this with what we are currently seeing in Norway. chapter. The production figures that make up the statistics only indicate harvested volumes, while the Mussels volumes of mussels growing in the sea have Mussel cultivation is an extensive form of been much larger. The limitations have not been aquaculture, based on the collection of wild those of production, but rather of harvesting, spat. The mussel cultivation industry has passed logistics, product quality and sales. In order to through a process marked by optimism and rap- solve these problems, more advanced strategies id build-up, followed by an almost equally rapid have gradually been drawn up. Many cultivators return to an existence marked by clear limita- have implemented suitable production routines, tions on what is possible and strategically ap- involving socking, building efficient harvesting propriate. The aims that parts of this sector set vessels and upgrading their equipment. We for itself about 10 years ago were unrealistic. have witnessed a promising development of Companies have gradually begun to learn by ex- strategies for a two-phase production system. In the first phase, seed are produced in separate spat systems in the inner fjords, where they are readily available. Such areas suffer serious problems of toxic algae, but this production model harvests and moves the seed out of the problem areas. Specialist seed producers then supply pre-sorted seed to ongrowing sites, which produce mussels for consumption in areas that suffer fewer problems of toxicity. Specialisation at both stages improves both efficiency and quality.

145x100//Kap08-fig01.eps Appetising mussels. (Photo: Arne Duinker)

138 Aquaculture Research: From Cage to Consumption The development of the mussel industry has ing food availability. This is expected to be an been held back by uneven quality (meat yield) important factor in efforts to improve our and the toxic algae problem. In Norwegian knowledge of how farms should be sited and waters, most of the annual production of algae how their biomass should be distributed in order takes place during the spring bloom, while a to cultivate the best-quality mussels. lack of nutrient salts can hamper production during the summer. Toxic algae have caused the On the processing side, we find that experi- greatest problems in the autumn, particularly in ments are being carried out on different prod- the fjords. A supply of nutrient-rich deepwater ucts that could give producers more legs to is capable of increasing the production of algae stand on. Shellfish packed in plastic modified- again during nutrient-poor periods, and the atmosphere packs (MAP) are becoming more Maricult programme recommended continuing and more common. Growing quantities of such efforts to test out methods of increasing shell- products are being supplied to the supermarket fish production via upwelling of nutrient-rich chains. Some producers are going in for frozen deepwater in fjords. In the course of the past products as a solution to logistics problems. few years, a number of large-scale trials have Cooking shellfish in processing plants and can- been carried out, involving various methods of ning them in a range of sauces is an alternative bring deepwater up to the productive zone of the to sending shellfish with unacceptable levels of water column. In the Lysefjord, it was shown growth organisms to overseas boiling compa- for the first time that brackish water-driven up- nies. Efforts are also being made to develop al- welling of nutrient-rich deepwater can increase ternative areas of use for mussels in Norway the production of algae in fjord areas. and Sweden. Mussels have turned out to be a good replacement for fish meal in salmon and In the inner part of the Lysefjord, upwelling re- cod feeds, although they do not offer improve- sulted in about a trebling of the biomass of al- ments in tastiness or feed uptake compared to gae within an area of about 10 km2 in 2004 and modern fish meals. It has also been shown that 2005. There were obvious changes in the algal salmon react physiologically to high concentra- picture within the area of influence of the artifi- tions of algal toxins in mussels that result in cial upwelling. A stable increase in the produc- diarrhoea, and similar trials are currently under tion of algae – predominantly non-toxic algae – way in cod. Mussels in poultry feeds have been could form the basis of more predictable mussel shown to have a highly positive effect on yolk farming in the fjords. colour. The hens did not appear to react to the toxic algae, but more sensitive responses have In order to ensure that all the shellfish in a farm yet to be investigated. Consideration is also be- obtain sufficient food, cultivators need to take ing given to the possibility of fractioning the such factors as location, water flow-through and mussel raw material via processes which would shell density into account. Studies of these fac- also produce saleable products from toxic al- tors, in conjunction with the meat content of gae. In the course of time, it seems likely that mussels from long-line farms, have formed the other uses will be found for mussels than only basis of a numerical model that has demonstra- as food for human beings or animals. ted the importance of farm design for optimis-

Thematic area: Production 139 145x100//Kap08-fig02.eps Live scallops ready for the market. (Photo: Arne Duinker)

Scallops ulating temperature, length of day, amount of It has proved to be difficult to base scallop farm- feed and the duration of the conditioning pro- ing on the collection of wild spat, so these are cess. It has also been shown that the yield of lar- produced under controlled conditions in hatch- vae can be improved by modifying the mixture eries. In Norway, there is one full-scale scallop of algae in the diet of the broodstock, so that hatchery: Scalpro AS in Øygarden near Bergen. they receive both summer and winter diets. The Since the company was launched in 1995, the results of the larval production phase, however, production of spat ready for release into the sea are still variable. Particularly in April, after the has steadily increased, thanks to a series of spring bloom in the sea has broken up, it is dif- R&D projects, most of which have been per- ficult to obtain optimal results in the hatchery. formed by research groups in Bergen. During the past few years, however, the hatchery has succeeded in producing groups totalling The first step in the hatching and spat phase is more than 100 million newly hatched larvae. to condition the broodstock shellfish, in order to ready them for spawning. Broodstock are The larval phase is critical. The high mortality brought from the counties of Trøndelag and of larvae and spat is well known in all areas Hordaland. The hatchery has obtained accept- where shellfish are cultivated, and both bacteria able results from spawnings of stocks from both and viruses that are fatal to shellfish larvae have counties at different times of the year by manip- been described. In many places, therefore, pro-

140 Aquaculture Research: From Cage to Consumption duction is supported by the preventive use of After the larval phase, the larvae are transferred antibiotics (Chloramphenicol). Norwegian to cylindrical sieves partly submerged in tanks, scallop production has also used Chloram- where they attach themselves by their byssus phenicol, and the results of several years of pro- threads to the netting at the bottom of the sieves duction have demonstrated that this improves and metamorphose into post-larvae and spat. production in larvae systems. However, The spat attach themselves to the substrate if Chloramphenicol is not permitted for use in conditions are good, and their “attachment be- food production. Research has therefore con- haviour” is much weaker if the salinity is low. centrated on microbiological studies and the de- The spat display better rates of growth and sur- velopment of cultivation systems for larvae. vival at temperatures above 15 oC, while a salinity below 30 ppm has negative effects. The knowledge we already possessed regarding Following metamorphosis, growth rates in- the production of other marine larvae was also crease, as does the need for food and a constant used in scallop production. Flow-through tanks renewal of water. At an early stage, studies were with volumes of several thousand litres, which carried out in order to determine how great the were originally developed for halibut larvae, flow-through of water needed to be as the spat have also been very successfully utilised for grew. These studies resulted in much improved scallop larvae. Scalpro AS currently uses a sim- survival rates in the post-larval and spat phases. ilar system for its larvae. This has led to major Heating the seawater and cultivating feed algae improvements as far as both working practices are significant cost elements in spat production. and the environment are concerned, since anti- Costs can be reduced by raising recirculation biotics are no longer used. However, flow- rates of water and algae in the cultivation sys- through systems, which are in operation for tem. The result was that 80 per cent of the water three weeks at a time without the need for drain- that runs through the sieves is now recirculated ing or washing, are more liable to experience by means of an airlift, while only 20 per cent is growth of organic material than the old static fresh seawater. Studies of the cultivation envi- systems. This has made it essential to optimise ronment in the larval phase and survival in the food availability in order to match it to the post-larval phase are important, since only lar- growth and requirements of the larvae. vae in good condition manage to survive metamorphosis and turn into post-larvae with good Feed uptake studies have determined the opti- rates of growth. mal amounts of feed required by different sizes of larvae. Studies of swimming patterns have Both larvae and spat are fed several species of identified the factors that are capable of influ- microscopic algae, which are cultivated in con- encing larvae, and showed for the first time that centrated intensive cultures. A project that char- larvae alter their patterns of swimming under acterised the capacity to digest lipids and the influence of the quantity of feed, their own carbohydrates in scallop spat (2–4 mm) showed density and light conditions. These results, in that feed algae should be harvested during their addition to others from Europe and Canada, led late growth phase, when they have a high carbo- to production-scale studies of optimal growth hydrate content and high rates of activity of car- conditions for larvae being carried out. bohydrate breakdown enzymes. These results

Thematic area: Production 141 could be important in the development of for- environment and are very liable to display poor mulated feeds in the future. growth and mortality rates.

At a size of around 2 mm, the spat are trans- It is not regarded as profitable to keep scallops ferred from controlled indoor cultivation condi- in cultivation systems until they are of harvest- tions to more natural conditions in the sea or able size for consumption. Cost-effective culti- onshore growth facilities. During this phase, the vation from the spat stage until harvesting shellfish change from being fed on cultivated therefore requires the shellfish to be kept on the algae to living on natural food. It has been seabed under sea-ranching conditions. shown that controlling conditions in the aqueous environment (temperature, salinity and In northern Europe – particularly in Norway – food availability), transfer methods, choice of predation by the edible crab Cancer pagurus site and time of transfer are decisive factors in has been the main problem facing scallop- ensuring good growth rates and predictable sur- ranching. Starfish (Asterias rubens and vival in scallop spat. Sea temperature is impor- Marthasterias glacialis) also eat scallops, but tant for growth. Transferring small spat to are much less of a threat to the sea ranching than seawater at temperatures below 7 oC has given the crabs. Studies of survival in sea-ranching poor results, but survival rates can be improved cultures have shown that cultivated scallops by acclimating the spat to colder water before need to be at least 6–7 cm in size before survival they are released, or by releasing them when rates increase. Shell strength is of decisive im- they are larger. Ongrowing farms that utilise portance in determining how well scallops man- sun-warmed semi-enclosed bays as their age vis-à-vis crabs. Shell strength increases sources of water can accelerate growth in the with age and size. However, it turns out that in spring. Much better results and cost reductions cultivated scallops, the strength of the shells of have been obtained by means of shore-based individuals of the same age and size varies ongrowing facilities in which the water is fil- widely with cultivation site. Although the shells tered in order to prevent the entry of fouling at certain sites are just as strong as those of wild organisms and predators. shellfish, at other sites they may be only half as strong. We believe that this can be explained in During the mid-culture phase, the shellfish are terms of the difference between the natural and grown from a size of 15–20 mm in nets or trays farming environment conditions. Research has suspended from long-lines, or in trays standing shown that we need a better understanding of in ramps on the seabed. The idea here is to pro- how shell formation, and thus shell strength, are tect the spat from predators and keep them in a affected by environmental conditions. beneficial environment in which they can be well cared for. Farms with good siting, equip- It is essential to identify effective measures to ment and cultivation procedures should be cap- keep the crabs away. Passive measures involve able of producing scallops with good results all using our knowledge of animal biology to re- along the coast of Western Norway and Trøn- duce the chances of crabs being able to prey on delag, but here too, cultivation has shown that scallops. What largely determines this likeli- scallops are particularly vulnerable in this hood is the relative size of the two species. Set- ting out shellfish of a single size that are at low

142 Aquaculture Research: From Cage to Consumption 145x100//Kap08-fig03.eps In seabed scallop cultivation it is vital to prevent crabs from getting at the shellfish. Fenced enclosure place directly on the sand are an effective measure, but they still need to be regularly inspected. (Photo: Esben Helland) risk of being eaten is a passive measure to re- predators on these shellfish. Ballan wrasse duce this threat. Shell size is the usual measure (Labrus bergylta) eat scallops, but scallops are for deciding when the scallops can be placed on at relatively little risk once they have reached a the seabed. As well as shell size, it is also ne- size of 25 mm. The production of spat in hatch- cessary to know something about differences in eries early in the season makes it possible to re- the strength of shells that are to be released. lease large spat (more than 30 mm) to the Fences that can be placed on the seabed have seabed in their first autumn, which thus offers been developed, and these prevent the crabs the opportunity of cultivating scallops without from getting at the scallops. We can take Hel- the expensive mid-culture phase. Studies have land Skjell AS in Nordhordland as an example, recently started on how fenced scallop sea- as this company has developed an effective ranching affects the seabed fauna. fence that has enabled them to cultivate scallops with an 80 per cent survival rate. Fencing allows Cooperative efforts involving industrial compa- small scallops to be set out, which in turn re- nies, the University of Bergen and the Institute duces or even eliminates the costly mid-culture of Marine Research have developed financial phase and increases the potential for profitable models for scallop ranching. The standard operation. Work has also been done on methods model is based on income from the fifth year of of releasing smaller spat, and on studies of operation onwards. The values used by these

Thematic area: Production 143 145x100//Kap08-fig04.eps A dozen oysters from Sunnhordland, ready for measurement of meat content and taste. (Photo: Arne Duinker)

models are based on experiments and informa- food availability resulting from high density tion provided by the industry. The models show will affect production. that there is significant economic potential once a company has come into regular production, but building up biomass is an expensive pro- Oysters cess. Oyster cultivation can rightly be called a niche production sector of Norwegian aquaculture. Scallops are usually released to the seabed at a Production has been extremely modest for sev- density of 10 shells per square metre, which is eral decades. The only naturally occurring oys- no higher than the densities that we find in nat- ter in our waters is the common oyster (Ostrea ural stocks. Relative to scallop ranching in other edulis), which is regarded as the best edible oys- countries, which is extensive in nature, Norwe- ter. In the 1980s and early 1990s a few spat of gian sea ranching will be rather more intensive. the imported Pacific oyster (Crassostrea gigas) It has been shown that the density of scallops on were also produced. Oysters from this produc- the seabed has a major influence on profitabil- tion were sold for a few seasons. However, they ity. Apart from our experience of extensive in- were gradually mixed together with imported dustrial sea-ranching, at a density of 10 shells oysters for consumption, which were released per square metre, we still lack knowledge of into Norwegian farms. This type of production maximum permissible densities inside fenced is both illegal and risky because of the risk of areas. We also lack knowledge of how limited spreading disease. We can only hope that this

144 Aquaculture Research: From Cage to Consumption 145x100//Kap08-fig05.eps Agapollen in Sunnhordland is an old, traditional oyster production site, at which Bømlo Skjell AS has collaborated with the Institute of Marine Research in the development of a semi-intensive production method for oyster spat. (Photo: Eivind Bergtun) practice has come to an end, and that the indus- in Sunnhordland. For the time being, it looks as try will focus on producing locally adapted oys- though these producers have managed to make ters. themselves regionally self-sufficient in oyster spat, but a build-up of oyster production will re- The current situation is one of limited availabil- quire further development of stable and cost- ity of oyster spat. Most of our traditional oyster effective spat production. polls have been abandoned, or have only produced oyster spat every few years. The excep- The network of cultivators in Sunnhordland has tion is Bømlo Skjell AS in Aga, where carried out a pilot cultivation project with the traditional semi-enclosed bay production has support of Hordaland County Council, the been extended by means of a “floating” hatch- Sunnhordland Cooperative Council, and in co- ery and spat farm (see photo), at which studies operation with the Institute of Marine Research of environmental and feeding factors in various and NIFES. The results show that we can iden- larval production systems have been carried tify a number of suitable cultivation sites on the out. Scalpro AS in Øygarden also produces coast of Western Norway. They also confirm the spat. Most of this company’s oyster spat are importance of adequate current conditions, supplied to an organised network of producers optimal shell density and a good flow of water

Thematic area: Production 145 The Norwegian Seafood Export Council is now profiling Norwegian mussels under the logo “Norwegian Fjord Mussels”.

145x100//Kap08-fig07.eps

145x100//Kap08-fig06.eps A well-filled steamed mussel. (Photo: Arne Duinker) out more or less at random into the big wide through the cultivation systems. Under optimal world. We must either customise batches of conditions, high-quality oysters ready for con- shellfish for the markets that they are intended sumption can be harvested after 2.5 and for, or Norwegian shellfish must develop a 3.5 years. These results are very promising, and brand identity with well-defined quality, so that offer a good basis for establishing a regional the market learns about, and becomes familiar model of oyster cultivation. However, future de- with, just what the product represents. velopments are still uncertain. The market situation will be the main determinant of how the As companies have gained experience of their situation develops. More groups of cultivators markets, the mussel farming industry has grad- are likely to be formed in the future, and if these ually shifted from focusing solely on volume at choose to adopt the strategy that has been laid the end of the 1990s to a sharper focus on qual- out during the past few years, there is every ity. In 2003, this trend resulted in a resolution to reason to be optimistic about the future. the effect that the industry wished to have a brand name for mussels with a defined standard of quality. The Norwegian Export Council for The market Fish followed the matter up, and in spring 2005 In the 1980s and 1990s, there was little focus on was ready to pilot the brand name “Norwegian quality and brand development for Norwegian Fjord Mussels”, with defined quality require- shellfish. A few limited projects carried out by ments that had been drawn up in collaboration the shellfish industry itself had little or no long- with the industry itself. This process has since term effect, and it gradually became clear that been adopted as a template for quality efforts what was needed was a better coordinated and concerning other shellfish species. more general effort. Most Norwegian mussels are sold on the central The market is a challenge. Of course, shellfish European markets of Belgium and France, but are not a single item, but a whole range of prod- efforts are also being put into marketing in other ucts. Different markets have different prefer- regions, such as Eastern Europe and Russia. ences, and the right choice of strategy is Mussels are classified as either industrial or therefore essential for what we wish to produce fresh shellfish, a terminology that offers a good and sell. There is no point in sending shellfish picture of production. Many producers are

146 Aquaculture Research: From Cage to Consumption forced to sell their shellfish to the canning in- quality assurance of oysters have been started dustry because of their low meat content and by shellfish cultivators in Sunnhordland, growth rates, factors that in turn are due to inad- NIFES, the Institute of Marine Research, the equate husbandry, poor location or to the shell- Norwegian Export Council for Fish and Horda- fish not having been harvested in time. We can land County Council. also see a tendency for suitable production strategies and quality sorting to produce clear The situation is somewhat different for scallops, benefits in the form of shellfish that can be sold which have been harvested from wild stocks, so in the best paying market segments. In the that their quality varies according to the season course of the past few years, shellfish have risen of the year. There is neither a harvesting season in price from less than NOK 1 to up to nearly nor a minimum size for scallops, so that quality NOK 40 per kilo in a few cases. Prices tend to assurance has so far been left to the industry it- lie between NOK 5 and NOK 10 per kilo – a self. One important element in this respect is price level that shows that it is of decisive im- that the size of scallops is an important quality portance to employ cost-effective systems in criterion, for which consumers are prepared to mussel cultivation. pay. So far, the ability of Norwegian suppliers to deliver large scallops (more than 12 cm) has Long transportation times after harvesting and been an advantage in the market. A higher rate packing can be a problem. One solution is to of harvesting of scallop stocks using current send the product in bulk to the importing coun- methods would probably initially result in the tries for packing there, possibly after relaying in oldest part of the stock disappearing. An initia- seawater for a while. One Norwegian company tive has recently been taken to propose regulat- has bought a processing plant abroad in order to ing scallop harvesting levels in such a way as to maintain control of this part of the process. An- ensure the constant availability of large shell- other possibility is to transport the shellfish by fish. boat in specialised systems in seawater at low temperature, and to pack them in the importing country. Principal challenges Although significant progress in developing the Norwegian oyster production remains at a mod- shellfish farming industry has been made, we est level, but where quality and brand develop- can still see important challenges facing the fu- ment are concerned, this may be an advantage. ture development of this sector. Toxic shellfish If the sector is developed in a serious and well are still a problem, sufficient supplies of spat thought out way, it should be possible to estab- and good health are absolute success criteria, lish a standard of quality and develop a profile the utilisation of areas of the coast is bringing for Norwegian oysters right from the beginning up new problems and the industry will have to – i.e. before large quantities of Norwegian oys- learn to master new markets as production in- ters enter the market. We would prefer to see creases. The availability of mussels without al- imported Pacific oysters either leaving the mar- gal toxins, stable access to large quantities of ket or being sold at a low price. Internationally, high-quality scallop and oyster spat, cost-effec- these fetch at best half the price of the native flat tive sea-ranching methods and knowledge oysters. Collaborative efforts in marketing and about the health status of individual shellfish

Thematic area: Production 147 145x100//Kap08-fig08.eps Efforts are being put into developing methods of chemical analysis as a replacement for tests using mice. (Photo: Thomas Bjørnflaten) Availability of spat stocks are important success criteria that will determine the development of the Norwegian Large-scale production of high-quality spat for aquaculture industry. sea ranching will require more knowledge of the relationship of broodstock from different local stocks and the quality of the offspring produced at different times of the year. An emphasis on producing spat early in the season can significantly reduce production times for scallops and greatly improve profitability. A better understanding of disease and of genetic differences between stocks will improve the chances of developing a reliable industry without contaminating genetic material. As in all areas of aquaculture, shellfish farming is dependent on stable supplies of spat. Both scallop and oyster production are vulnerable, since spat production is restricted to only a few facilities.

145x100//Kap08-fig09.eps An increase in production could make this stage Dinophysis cells that produce a toxin that causes diarrhoea. a bottleneck, which means that it is extremely (Photo: Linda G. Møland).

148 Aquaculture Research: From Cage to Consumption important to keep hatcheries and nurseries in of such symptoms. Fatal cases of PSP poisoning operation. Importing spat involves a high risk of have not been reported in Norway for several introducing diseases, and this must not be al- years, thanks to steady improvements in moni- lowed to happen. toring and control programmes.

Problems of toxic algae Diarrhoea-producing toxins (DSP toxins/OA International research projects have identified group) were reported for the first time in Nor- eight different groups of marine algal toxins in way in the mid-1980s. These toxins are prima- shellfish. In Norwegian waters, most toxins de- rily produced by dinoflagellate algae of the rive from four groups: paralysing toxins (PSP genus Dinophysis. Recent research indicates toxins/STX group), diarrhoea-producing toxins that D. acuta is the most common source of (DSP toxins/OA group), the azaspiracids (AZA diarrhoea toxins. High densities of D. acuta oc- group) and yessotoxins (YTX group). To date, cur most frequently in the autumn, so that DSP the last of these groups has not been linked to problems tend to arise after problems caused by cases of poisoning of human beings. In the PSP have already passed. Here too, it appears to course of the past few years, traces of spirolides be the case that mussels have the greatest ability have also been found in mussels at some Nor- to accumulate these toxins. Symptoms in hu- wegian sites. These are toxins that belong to the mans include diarrhoea, vomiting, nausea, group of cyclic imines, together with exotic tox- stomachaches and headaches. These effects are ins such as gymnodimine and other toxins that noticed after a few hours, and normally last for have not been found in Norwegian water. Pec- a couple of days. Medical treatment is usually tenotoxins (PTX group) have occasionally also unnecessary, although sufferers may feel ex- been identified, but these have not been report- tremely unwell. Relatively few cases have been ed to be a problem for people who eat shellfish. reported in Norway, and there is good reason to believe that this is due to the monitoring and Paralysing toxins (PSP toxins/STX group) have control apparatus that has been set up. been recognised for several hundred years. The most important source of these is the dinofla- Azaspiracid (AZP) poisoning has not been re- gellate Alexandrium tamarense, which occurs ported in Norway so far, but these toxins have relatively often in Norwegian waters, particu- been identified in Norwegian mussels in recent larly in late winter and spring. Shellfish species years, which has led to the closure of certain that filter these algae can accumulate high lev- harvesting areas. Irish shellfish have caused els of their toxins, and mussels in particular can episodes of AZP poisoning in a number of become highly poisonous. The symptoms that European countries in the course of the past 10 appear within a short time of eating shellfish years. Symptoms are similar to those of DSP, containing PSP toxins include numbness of the but the mechanisms of action are quite differ- mouth and lips, spreading to the rest of the face, ent, and the toxins are not produced by a prickly feeling in fingers and toes, headaches, Dinophysis species of algae. An important chal- dizziness and difficulty in speaking. If the level lenge at present is to map the producers of AZA of toxins is high, there may be problems of res- toxins, in order to improve control mechanisms. piration, and in the most severe cases, death This is one of the most important areas of a new may follow. Rapid treatment is vital in the event Norwegian research project that started in 2006,

Thematic area: Production 149 and which is financed by the Research Council mechanisms of action. It is thus obvious that the of Norway. mouse-test method for the fat-soluble toxins is highly unsuitable, as far as sensitivity and spec- Neither yessotoxins, pectenotoxins nor spiro- ificity are concerned, not to mention on ethical lides – all of which have been found in Nor- grounds. Several research groups have been wegian shellfish – have been linked to food working hard to develop methods capable of re- poisoning. It is essential that research on their placing the biological methods, and a number of toxic properties should continue, so that per- alternatives are now available. The international missible levels of these toxins in shellfish can organisations FAO, IOC and WHO set up an ex- be related to their risk potential for human be- pert consultation process in 2004, which was in- ings. To date, the regulation of YTX and PTX tended to evaluate all aspects of the problems of toxins has been based on their toxicity to mice algal toxins. This expert group suggested a following injection into the abdominal cavity. number of changes in the permitted limits of The spirolides are so recent a find that special different groups of toxins and in the choice of regulation of permitted levels has yet to be in- analytical methods. Instrumental methods were troduced, but this is needed, since they provoke proposed as reference methods for all groups of a response in the traditional method of analysis toxins. The EU’s experts endorsed the propos- using mice. als made by the expert group and submitted a set of recommendations regarding revisions of All the available data suggest that mussels are both methods and permitted limits to the EU’s the most important problem where accumula- decision-making bodies. As soon as chemical tion of algal toxins is concerned. Although we standards become commercially available for have much less experiental data for oysters and all the relevant groups of toxins – as was expect- scallops (because far fewer analyses have been ed to happen in 2006 – we will be in a position carried out), there is good reason to believe that to carry out international ring tests on the ana- oysters and scallops, in that order, do accumu- lytical methods and to establish routine meth- late marine algal toxins. However, we should be ods that do not involve the use of experimental aware that if more toxin-producing strains of animals. At the same time, research in progress Pseudonitzschia enter Norwegian waters, scal- will help to establish more scientifically justifi- lops could come to be the main problem. Inter- able upper limits for the different groups of tox- national research suggests that scallops have the ins. greatest ability to accumulate “memory-loss toxins” (ASP toxins/DA group). Detoxification As the mussel farming industry has developed, Norway is obliged to follow EU regulations methods of detoxification of shellfish have been concerning analyses of algal toxins. EU direc- a recurring topic. Proposals have been put for- tives require shellfish extracts to be analysed us- ward for intensive methods involving feeding ing biological methods before they can be sold. mussels in land-based facilities with artificial or There are specific mouse tests for the water-sol- cultivated feeds, and strategies have been sug- uble PSP toxins and for fat-soluble toxins, gested for moving shellfish to areas that are free which comprise several different groups of tox- of toxic algae or where attempts have been ins (DSP, AZA, YTX and PTX) with different made to alter the algal community by upwelling

150 Aquaculture Research: From Cage to Consumption of nutrient-rich seawater. Calculations have ties, given that the EU has a relatively liberal been made that show that the costs of intensive system for trade in live animals, and the Norwe- cultivation can be anything from 10 to 100 gian authorities have adopted a defensive atti- times as high as those of extensive methods. tude to the introduction of protective measures However, recent results of studies of how envi- that might deviate from EU regulations. In this ronmental factors and availability of food affect respect, there is a definite need for self-disci- changes in the content of diarrhoea-producing pline within this industry – for example by toxins have suggested that the basis of all solu- avoiding illegal releases of imported Pacific tions that depend on feeding needs to be com- oysters. We have seen cases of this happening pletely reassessed. The results show that the without sanctions being imposed by the veteri- excretion of toxins by shellfish is not affected nary authorities. The second challenge involves by environmental factors, and pilot-scale stud- the establishment of models capable of rapidly ies in Sweden have demonstrated that toxins are identifying outbreaks of disease and limiting excreted just as rapidly in filtered water or deep- their spread. This is also difficult under the water as in algae-rich surface water. This points present system. With the exception of regular to the possibility that mussels can be detoxified health checks of oysters, there is no systematic at reasonable cost by keeping them in systems monitoring of shellfish diseases in Norway, and in which they are not fed, if the levels of toxins the coast has not been divided up into delimited are low enough to fall below limiting values autonomous geographical zones that could limit within a reasonable period of time. the spread of disease in the event of an outbreak.

Health status User conflicts and use of area No diseases have been identified in Norwegian Modern shellfish cultivation is a relatively new shellfish stocks. As we described in the section activity in the coastal zone. When mussel farms about scallops, most problems have been at the started to appear along the coastline, the ques- larval and spat stages. So far, we have been tion of their visual impact was raised, and the spared outbreaks of disease in adult shellfish. appearance of the floating elements in particu- The situation is different in most of the rest of lar has been the subject of debate. Today, these Europe, and oyster stocks in particular have farms are better adapted to local natural condi- been hit hard by diseases caused by parasites. tions than they were a few decades ago, and it is Studies of Norwegian oysters have shown no accepted by and large that shellfish farms repre- signs of disease. Oysters are a valuable re- sent relatively modest incursions in the land- source, which ought to be carefully managed. scape. Nor has disease been registered in large scallops, probably because the industry is still lim- Scallop sea-ranching gives the licence-holder ited in size, and few shellfish have been exclusive rights to recapture released animals imported from other regions. It is naturally a within a defined area. In the development of the clear goal to keep Norwegian stocks free of dis- scallop-farming sector it has been a clear aim to ease. There are two challenges at present. The reduce conflicts with other users. In a sea- first is that it is of quite decisive importance to ranching area, therefore, marine traffic is unre- avoid importing live shell spat that are carriers stricted and the use of fishing gear is permitted, of disease. This is a challenge for the authori- as long as the ranched organisms are not fished.

Thematic area: Production 151 The siting of shellfish farms is always evaluated means that it is essential to obtain a good know- with respect to other aquaculture activities. One ledge of how this environment can best be uti- aspect of disease management is the require- lised for shellfish production, or how shellfish ment that there should be a certain distance be- cultivation can be carried out in such a way as to tween fish farms. The aim of this requirement is maximise the exploitation of carrying capacity. to minimise the possibilities of carrying disease from one species or farm to another. This is an Logistics and marketing in the mussel important measure, but the requirements are cultivation sector largely based on the preventive principle, which In the mussel cultivation industry, we are begin- does little to take into account local variations ning to see the contours of a change in scenario in oceanographic conditions, or specific risk from that of a large number of small players who evaluations. It is a general problem that we have sell mussels to one in which large contracts are too little concrete knowledge about the spread signed, by which the seller agrees to make stable of disease that can be transferred directly to a deliveries throughout the year. This requires a specific on-site situation. During the past few quite different network of producers than we years, a number of studies have looked at the have had until now. The experience of the past role played by shellfish in spreading diseases, few years suggests that there will be a growing and of the host-specificity of the pathogens in- need to buy and sell shellfish across regions all volved. The results suggest that in almost all along the long coastline. An important natural cases, shellfish do not present an increased risk advantage for Norway is its long coastline with of spreading infections to other species. its wide variations in climate, on which shellfish can maintain an acceptable level of quality at Carrying capacity different times of the year in different parts of The carrying capacity for shellfish production in the country. Growing inter-regional trade will usually based on an evaluation of the availability also mean that we need better knowledge of of food, and the loads imposed by the shellfish on quality aspects of transport and handling at re- the environment. However, other environmental ception facilities, as well as more unified sys- factors may also affect carrying capacity. Put in tems for logistics and for estimating biomass. extreme terms, a locality with a source of pollution that leads to the production of shellfish that are detrimental to health or are unacceptable in Vision within reach other ways naturally has no carrying capacity for We can hope that our vision of a viable Norwe- shellfish. However, areas with toxic algae will gian shellfish cultivation industry is within often have a limited carrying capacity. The load reach. One of the greatest challenges facing us that a shellfish farm imposes on the environment is that of how the industry can adopt the knowl- will set certain limits on what is generally accep- edge that has been generated in the course of the table. On a Norwegian scale, shellfish farms are past few years, and how it can find sufficient unlikely to cause effects that would be regarded capital to make use of this knowledge in long- as environmentally irresponsible. We have a term, sustainable and cost-effective production. wide range of environmental conditions, but The sector needs cooperation, organisation and there are also fjord and coastal areas with rela- a sharp focus on quality. It still needs both en- tively low concentrations of food particles. This thusiasts and optimists!

152 Aquaculture Research: From Cage to Consumption References Grefsrud, E.S. & Strand, Ø., 2006. Comparison of shell Andersen, S., Burnell, G. & Bergh, Ø., 2000. Flow- strength in wild and cultured scallops (Pecten max- through systems for culturing great scallop larvae. imus). Aquaculture, 251:306–313. Aquaculture International, 8:249–257. Hovgaard, P., Mortensen, S. & Strand, Ø., 2001. Skjell, Asp, T., Larsen, S. & Aune, T., 2004. Analysis of PSP biologi og dyrking, Kystnæringen forlag og bokk- toxins in Norwegian mussels by post-column de- lubb, Bergen, 255 pp. rivatization HPLC method. Toxicon, 43(3):319– Magnesen, T., Bergh, Ø. & Christophersen, G., 2005. 327. Scallop larval yields in a commercial flow-through Aune, T., Torgersen, T., Arff, J. & Tangen, K., 2004. De- rearing system with continuous feeding. Aquacul- tection of pectenotoxin in Norwegian blue mussels ture International (in press). (Mytilus edulis). Proc. Xth International Confer- Mortensen, S.H., Bachere, E., LeGall, G. & Mialhe, E., ence on Harmful Algae, Florida, USA, 21–25 Octo- 1992. Persistence of infectious pancreatic necrosis ber, 2002 (red: K.A. Steidinger, J.H. Landsberg, virus (IPNV) in scallops (Pecten maximus). Diseas- C.R. Tomas, G.A. Vargo), s. 306–308. es of Aquatic Organisms, 12: 221–227. Bergh, Ø. & Strand, Ø., 2001. Great Scallop, Pecten Mortensen, S.H., 1993. A health survey of selected maximus, research and cultivation strategies in Nor- stocks of commercially exploited Norwegian bi- way: a review. Aquaculture International, 9(4): valve molluscs. Diseases of Aquatic Organisms, 305–317. 16:149–156. Cristophersen, G., 2005. Effects of environmental con- Mortensen, S.H., 1993. Passage of infectious pancreatic ditions on culturing scallop spat (Pecten maximus). necrosis virus (IPNV) through invertebrates in an PhD thesis, University of Bergen, 58 pp. aquatic food chain. Diseases of Aquatic Organisms, Cristophersen, G. & Magnesen, T., 2001. Effects of de- 16:41–45. ployment time and acclimation on survival and Mortensen, S.H., 2000. Introductions and transfers of growth of hatchery-reared scallop (Pecten maxi- scallops, from an animal health point of view. mus) spat transferred to sea. Journal of Shellfish Re- Aquaculture International, 8:123–138. search, 20:1043–1050. Mortensen, S.H., van der Meeren, T., Fosshagen, A., Duinker, A., Bergslien, M., Strand, Ø., Ohlseng, C.D. & Hernar, I., Harkestad, L. & Bergh, Ø., 2000. Mortal- Svardal, A. The effect of size and age on depuration ity of scallop spat in cultivation, infested with tube rates of diarrhetic shellfish toxins (DST) in mussels dwelling bristle worms, Polydora sp. Short commu- (Mytilus edulis L.). Submitted to Harmful Algae. nication, Aquaculture international, 8:267–271. Duinker, A. & Nylund, A., 2002. Seasonal variations in Sandlund, N., Torkildsen L., Magnesen, T., Mortensen, the ovaries of the great scallop (Pecten maximus) S. & Bergh, Ø., 2006. Immunohistochemistry of from western Norway. Journal of the Marine Biolo- Great Scallop, Pecten maximus larvae experimen- gy Association of the United Kingdom, 82(3):477– tally challenged with pathogenic bacteria. Diseases 482. of Aquatic Organisms (in press). Duinker, A., Torsteinsen, B. E. & Lie, Ø., 2004. Lipid Sandaa, R.A., Magnesen, T., Torkildsen, L., & Bergh, classes and fatty acid composition in female gonads Ø., 2003. Characterisation of bacterial communities of great scallops – a selective field study. Journal of associated with early life stages of cultured great Shellfish Research, 23(2): 507–514. scallop (Pecten maximus), using denaturing gra- Duinker, A., Saout, C. & Paulet, Y. M., 1999. Effect of dient gel electrophoresis (DGGE) Systematic and photoperiod on conditioning of the great scallop. Applied Microbiology, 26:302–311. Aquaculture International, 7(6):449–457. Strand Ø, Grefsrud, E.S., Haugum, G.A., Bakke, G., Espenes, A., Aasen, J., Hetland, D., Satake, M., Smith, Helland, E., & Helland, T., 2004. Release strategies A., Eraker, N, & Aune, T., 200_ Toxicity of YTX in in scallop (Pecten maximus) sea ranching vulnera- mice after repeated oral exposure. ICMSS Galway ble to crab predation I: Stock Enhancement and Sea 2004. Proceedings (in press). Ranching (Ed: K. Leber, S. Kitada, H.L. Blanken- FAO/IOC/WHO: Report of the Joint FAO/IOC/WHO ship & T. Svåsand) Blackwell Science Ltd, Oxford, ad hoc Expert Consultation on Biotoxins in Bivalve pp 544 – 555 molluscs. FAO 2005. Strohmeier, T., Duinker, A. & Lie, Ø., 2000. Seasonal Grefsrud, E.S., 2006. Predation on cultured and wild variations in chemical composition of the female scallops Pecten maximus L. by the crab Cancer pa- gonad and storage organs in Pecten maximus (L.) gurus L. Phd thesis, University of Bergen, 44 pp. suggesting that somatic and reproductive growth are

Thematic area: Production 153 separated in time. Journal of Shellfish Research, fadiazine and flumequine in seawater of bacteria as- 19(2):741–747. sociated with scallops (Pecten maximus) larvae. Strohmeier, T., Aure, J., Duinker, A., Castberg, T., Aquaculture, 185:1–12 Svardal, A., & Strand, Ø., 2005. Flow reduction, Aasen, J., MacKinnon, S.L., LeBlanc, P., Walter, J.A., seston depletion, meat content and distribution of Hovgaard, P., Aune, T. & Quilliam, M.A., 2005. diarrhea shellfish toxins in a long-line blue mussel Detection and identification of spirolides in Norwe- (Mytilus edulis) farm. Journal of Shellfish Re- gian shellfish and plankton. Chem. Res. Toxicol., search, 24:15–23. 18:509–515. Strohmeier, T., Oppegård, GG & Strand, Ø. Predation Aasen, J., Torgersen, T. & Aune, T., 2003. Application of hatchery-reared scallop spat (Pecten maximus L.) of an improved method for detection of lipophili by the Ballan wrasse (Labrus bergylta)-conse- marine algal toxins (OA/DTXs, PTXs, YTXs and quences for sea ranching. Aquaculture. AZAs) with LC/MS. I: Molluscan Shellfish Safety Torkildsen, L., Coyne, R., Samuelsen, O.B., Magnesen, (red. A. Villalba, B. Reguera, J.L. Romalde, R. Bei- T. & Bergh, Ø., 2002. Treatment of early life stages ras), IOC of UNESCO, pp. 49–55. of scallop (Pecten maximus) with antimicrobial Aasen, J., Samdal, I.A., Miles, C.O., Dahl, E., Briggs, agents; searching for an alternative to chloram- L.R., & Aune, T., 2005. Yessotoxins in Norwegian phenicol. Aquaculture International, 1:399–409 blue mussels (Mytilus edulis); Uptake from Torkildsen, L., Lambert, C., Nylund, A., Magnesen, T. Protoceratium reticulatum, metabolism and depu- & Bergh, Ø., 2005. Bacteria associated with early ration. Toxicon, 45:265–272. life stages of the great scallop, Pecten maximus: im- Aasen, J., Torgersen, T., Dahl, E., Naustvoll, L.-J. & pact on larval survival. Aquaculture International, Aune, T., 200_. Confirmation of azaspiracids in 13:575–592 mussels in Norwegian coastal areas, and full profile Torkildsen, L., Samuelsen, O.B., Lunestad, B.T. & at one location. ICMSS Galway 2004. Proceedings Bergh, Ø., 2000. Minimum inhibitor concentrations (in press). of chloramphenicol, florfenicol, trimethoprim/sul-

154 Aquaculture Research: From Cage to Consumption Health

■ New Diseases – Phenomena Developing into Problems ■ Virulece Mechanisms ■ The Fish Immune System Atle Lillehaug1) and Aud Skrudland2) 1) National Veterinary Institute, 2) Norwegian Food Safety Authority

New Diseases – Phenomena Developing into Problems

Overall losses in fish farming include mortality, escape, predation and sorting out of fish at slaughter, in addition to other unregistered demise of fish. These losses represent substantial reductions in profit. The overall proportion of fish lost has been quite stable during the last 15 years. In salmonids, it is assumed that “new” diseases are a major cause of losses, including pancreas disease (PD), heart and skeletal muscle inflammation (HSMI), cardiomyopathy syndrome (CMS), epiteliocystis / proliferative gill inflammation (PGI), and disease caused by Parvicapsula pseudobranchiola. In marine fish aquaculture, bacteria such as Vibrio anguillarum and Aeromonas salmonicida create problems, as does infection with nodavirus. Parasites such as “the Scottish louse” (Caligus elongatus) and other lice (Caligus curtus, Argulus sp.), as well as gill worms, flagellates, ciliates, microsporidiae, nematodes, and different gyrodactylus species, can turn problematic for marine fish production.

156 Aquaculture Research: From Cage to Consumption Measures for fish health to more secrecy relating to the causes of losses, The state of fish health in Norwegian aquaculture and fear of negative reactions from the market is considered substantially improved during re- may contribute to reduced openness in health cent years, compared to the situation in the late problems. Some diseases must be reported in ac- 1980s and the early 1990s. One yardstick for cordance with Norway’s Food Safety Act, and all demonstrating an improved health situation is the outbreaks should be registered. consumption of antibacterial drugs in fish. The number of outbreaks of ISA is another health Phenomena developing into problems parameter used. One can assume that a predominant cause of losses is “new” diseases such as CMS, HSMI, Using losses of fish as a measure, statistics from PD, etc. These disease conditions seem to have Kontali Analyse AS illustrate that after a reduc- gained increased importance in recent years, de- tion in overall losses during the early 1990s, the veloping from phenomena into problems, and percentages of lost fish have been quite constant they will be described in more detail in this chap- or slightly increasing, as can be seen in Figure 1. ter. In general, most infections causing disease outbreaks under aquaculture conditions develop A great proportion of both governmental and pri- from rare phenomena in wild fish. This has been vate research funding for aquaculture purposes the case for major bacterial infections, such as has been applied in the area of fish health and vibriosis and furunculosis. But “new” diseases closely related topics. However, this effort has have also been discovered in farmed fish, such as not resulted in a reduction of losses. coldwater vibriosis, IPN and ISA, which have never been described in wild fish. These infec- There are no statistics describing the different tions do, in all probability, also have reservoirs in causes of overall losses. The organisation of the wild stocks of fish or other feral organisms. In aquaculture industry in big companies may lead aquaculture, a great number of individuals are

Overall losses in Norwegian Atlantic salmon production 1986–2005, Loss % expressed as per cent of total number of fish per generation 40 % 35 % 30 % 25 % 20 % 15 % 10 % 5% 0% 86G 87G 88G 89G 90G 91G 92G 93G 94G 95G 96G 97G 98G 99G 00G 01G 02G 03G

145x100//Kap09-fig01.eps Figure 1. The graph shows the progression of losses calculated per generation of Atlantic salmon from 86G to 03G in the period 1986–2005. The figures represent percentages lost from the total number of smolt put to sea. 92G designates fish put to sea in the calendar year 1992, minus the number of fish of that generation slaughtered. Loss includes mortality, escape, predation, culled fish at slaughter, and any unrecorded losses.

Thematic area: Health 157 concentrated in a limited volume of water, and nid fish farming industry in the western parts of the conditions may be optimal for infectious Norway. In 2003, the first outbreaks were report- agents to flourish and spread. The examples al- ed in Troms and Finnmark, probably as a result ready mentioned have been thoroughly described of spreading from the western parts of the coun- earlier, and will not be dealt with in this chapter. try with infected smolt. From 2002 to 2005, the number of farms experiencing outbreaks per year Vibrio infections and furunculosis in salmonids has increased from 12 to 35. In 2004, 44 out- are effectively controlled by use of vaccines, re- breaks were reported, and the same year the dis- sulting in limited use of antibiotics in Norwegian ease has spread to the counties of Nordland and fish farming for many years. However, the num- Rogaland. ber of prescriptions for antibacterials now seems to be increasing; these are mainly associated with Pancreas disease affects both Atlantic salmon the cultivation of cod larvae. Hence, in this con- and rainbow trout in the seawater phase; usually text, we can observe that new infections, and, outbreaks occur half a year or more after the fish moreover, new variants of well-known disease have been transferred to sea. A typical disease agents, may develop from phenomena into prob- outbreak has chronic characteristics with symp- lems. toms including loss of appetite and low mortalities, but a long duration of an outbreak may result in high overall mortality numbers. Losses of sev- Pancreas disease (PD) eral hundreds of tonnes of fish have been report- Pancreas disease is a viral disease first described ed in one single farm location. Moreover, in Scotland by Munro et al. (1984). Later, PD has surviving fish may have reduced growth rate, and been reported in North America, and the disease fish slaughtered after an outbreak may have re- is particularly significant in Ireland. In Norway, duced meat quality due to muscle damage. PD has been present as a disease problem since the second half of the 1980s (Poppe et al. 1989), The development of the disease first results in with a geographic localisation in the county of necroses in pancreas, caused by the virus, which Hordaland and the southern parts of Sogn og has given rise to the name of the condition. Next, Fjordane. Today, PD is probably one of the dis- damage develops in the heart muscle, and later eases inflicting the greatest losses to the salmo- even in skeletal muscle. In Norway, pathological changes have even been seen in the kidney and spleen late in the course of the disease.

Diagnostic examinations for PD should be done on fish showing clinical signs of disease (Taksdal et al., manuscript). Post mortem may demonstrate mucous intestinal contents, caused by anorexia, and signs of circulatory disturbances may be seen, such as oedema in scale pockets, pro-

145x100//Kap09-fig02.eps Figure 2. Atlantic salmon surviving an outbreak of truding eyes and liquid contents in the abdominal pancreas disease may suffer from reduced growth and cavity. Petechial haemorrhages in the adipose tis- have poor quality at slaughter. The two fishes are from the sue surrounding pancreas may be found. Early in same population. (Photo: T.T. Poppe)

158 Aquaculture Research: From Cage to Consumption the course of the disease, typical acute changes in in Europe (Welsh et al. 2000). It has been sug- pancreas may be found in histological sections, gested to unite the three types in a species named and positive immunohistochemical staining for “Salmonid alphavirus”. the PD virus may be demonstrated. Next, inflammatory cells and tissue damage can be found in Experience indicates that PD contamination is the heart muscle, and later in skeletal muscle. connected to specific locations, such that outbreaks often reappear when fish are transferred It has been difficult to isolate the PD virus in cell to sea at a site where the disease has been seen culture. However, the methods have been im- earlier. Spread of the infection to new areas proved. Moreover, the virus can be detected by seems to be connected to movements of infected use of PCR. Demonstration of specific anti-PD fish. A vaccine against pancreas disease is being virus antibodies in fish blood is crucial in the dia- tested. gnosis of pancreas disease, but such tests can only be employed some time after the infection Current topics for further research has been established. Antibodies may be detec- Studies which have contributed to create knowl- ted for a long time after infection, and may be edge on PD, including the development of diag- used to find out if a fish population has been in- nostic methods, have been carried out in fected with the PD virus. cooperation between research institutions in Norway, Ireland and Scotland (Taksdal et al. Causality manuscript). The cooperation is being continued Three different but closely related PD viruses in new research projects aimed at elucidating the have been described. Salmon pancreas disease geographical distribution of the pertinent virus virus (SPDV) is the cause of PD in Atlantic salm- types, and identifying risk factors for acquiring on in Scotland and Ireland (Nelson et al. 1995). infection and for disease outbreaks, as well as Hodneland et al. (2005) describe a Norwegian further characterisation of virus isolates and val- strain called salmonid alphavirus (NSAV) caus- idation of diagnostic methods. ing disease both in Atlantic salmon and rainbow trout. A third variant of the virus gives rise to “sleeping disease” in rainbow trout in fresh water

145x100//Kap09-fig03.eps Figure 3. Fish suffering from heart and skeletal muscle inflammation. The heart is visibly paler than normal. (Photo: T. Taksdal)

Thematic area: Health 159 145x100//Kap09-fig04.eps Figure 4. The histological section to the left shows normal, red skeletal muscle, and to the right is a section of muscle from an Atlantic salmon with heart and skeletal muscle inflammation. The muscle is infiltrated with inflammatory cells, and many of the muscle cells shrink and degenerate. (Photo: R.T. Kongtorp)

20 per cent in some pens. Stressing of the fish Heart and skeletal muscle seems to result in increased mortality (Kongtorp inflammation (HSMI) et al. 2004a). Results of a study of the develop- HSMI was found for the first time during routine ment of a disease outbreak indicate that HSMI diagnostic investigations of farmed Atlantic can act as a sub-clinical inflammation in the heart salmon in 1999; the disease has since created in- several months prior to the onset of increased creasing problems for the Norwegian aquacul- mortalities in the farm, and heart injuries may be ture industry. Most of the outbreaks are occurring observed for a long period after mortality num- in Central Norway, but HSMI is reported in most bers have normalised (Kongtorp et al. 2006). fish farming coastal areas (Kongtorp et al. 2004a). No cases have been confirmed outside The diagnosis is based on clinical signs, includ- Norway, but outbreaks have been suspected in ing increased mortality and increasing numbers Scotland (Ferguson et al. 2005). of lethargic individuals, together with observations during autopsy and histopathology. Typical HSMI affects Atlantic salmon in seawater. Out- findings are coagulated blood in the pericardium, breaks usually occur between five and nine pale and flabby heart, liquid contents in the ab- months after sea transfer of the smolt, and body dominal cavity, yellowish or spotted liver with a weights are usually below 1.5 kg (Kongtorp et al. fibrinous veil covering the capsule, tiny haemor- 2004a). Results from field studies and challenge rhages in the liver parenchyma, and a swollen trials indicate that close to 100 per cent of the in- spleen (Kongtorp et al. 2004a). dividuals in a farm can be affected, although not all fish show obvious symptoms (Kongtorp et al. Histological characteristics of HSMI include se- 2004b; Kongtorp et al. 2006). During an HSMI vere inflammation and necroses in the heart and outbreak, lethargic fish and increased mortality in the red skeletal muscle. In the heart ventricle, may be observed for one to six months. Mortality all layers are involved. Usually, an extensive in- numbers vary from almost negligible to nearly filtration of inflammatory cells is observed in the

160 Aquaculture Research: From Cage to Consumption epicardium, together with inflammation and necroses in both the compact and spongy layers of the heart muscle. The atrium is usually less affected, and in bulbus arteriosus (a pear-shaped expansion of the first part of the aorta, made up of elastic tissue) no inflammation has been observed in connection with HSMI. There are inflammatory reactions and necroses in red skeletal muscle, but the white muscle is rarely affected. Necroses may be found in the liver, probably as a result of impaired blood circulation. Figure 5. Atlantic salmon dead from cardiomyopathy Some individuals even have oedematous stretch- syndrome. Coagulated blood can be observed in the es in the spleen, and other organs may show dif- pericardium, causing a so-called heart tamponade. (Photo: T.T. Poppe) ferent levels of circulation disturbances 145x100//Kap09-fig05.eps (Kongtorp et al. 2004a). Important aims for future research will be to Causality identify the etiological agent and to develop dia- Challenge trials have demonstrated that the dis- gnostic tools in order to improve the sensitivity ease can be transmitted both by intraperitoneal and specificity of the diagnosis. It is also crucial injection of heart tissue homogenate from sick to develop further competence regarding patho- fish, as well as by cohabitation with injected fish genesis, applying pathological and microbiolo- (Kongtorp et al. 2004b). A viral cause of HSMI gical studies. An epidemiological project is is suspected. However, isolation and characteri- necessary in order to map geographic distribu- sation of an etiological agent has not been pub- tion and spread of disease, together with risk fac- lished yet. tors for acquiring the disease. Such knowledge is basic for the implementation of control measures Due to the contagious characteristics of HSMI, it and eradication strategies for HSMI. is important to prevent further spread of the disease by measures such as disinfection of equipment and fallowing of contaminated sites Cardiomyopathy syndrome between fish generations. Due to the high infec- (CMS) tion rate in affected populations, together with a Cardiomyopathy syndrome is a serious heart prolonged course of the disease, any transfer of condition, mainly causing disease in farmed At- fish during the seawater phase will involve a risk lantic salmon late in the seawater phase, when of spread of the disease. This is also the case for the fish is approaching slaughter 14–18 months the introduction of new populations to sites al- after sea transfer. At this time, the monetary ready inhabited by salmon. value of the fish is greatest. CMS was first observed in Norway in 1985 (Amin & Trasti Current topics for further research 1988). Later, it has been diagnosed in Scotland A study of the inflammatory reactions in fish (Rodger & Turnbull 2000) and on the Faeroe with HSMI is in progress, aiming at an improved Islands, and there have been suspected cases in understanding of the development of the disease. Canada (Brocklebank & Raverty 2002). Similar

Thematic area: Health 161 disease conditions have been observed in wild to deaths. Due to the high value of each fish dy- salmon as well (Poppe & Seierstad 2003). ing from CMS, the economic losses may be significant (Brun et al. 2003; Østvik & Kjerstad In Norway, CMS occurs along the entire coast- 2003). line, but is most prevalent in Central Norway. The disease is rare in the counties of Troms and Losses may be limited by avoiding stresses such Finnmark (Østvik & Kjerstad 2003). There has as sorting and moving of fish, delousing or other been an increase in the annual number of diag- handling, when CMS is suspected or diagnosed nosed outbreaks, from 25 in 1998 to 101 in 2002 in the farm. Emergency slaughtering may con- (Brun et al. 2003), and statistics from diagnostic tribute to the limitation of losses (Skrudland et al. work at the National Veterinary Institute demon- 2002). strate a lasting high prevalence in recent years. Autopsy of fish dying from CMS will typically A clinical outbreak can last from one to six reveal haemorrhages in the skin, and protruding months, even longer. Typically, an outbreak ari- scales and eyeballs. Fluid contents in the abdom- ses more than one year after sea transfer (mean: inal cavity and fibrinous coverings on the liver 400 days), at a fish body weight of 2–3 kg. Ac- surface can be seen. In the heart, the atrium and cording to a study by Østvik & Kjerstad (2003), the adjacent great veins are usually dilated and mean total mortality during an outbreak was six enlarged, and the pericardium may contain blood per cent. In most cases, only some of the net pens coagula (Bruno & Poppe 1996). Sometimes, the in a farm were affected by the disease outbreak. wall of the atrium may have ruptured (Amin & Still, up to 80 per cent of the total losses in affect- Trasti 1988). The pathological changes de- ed farms were caused by CMS. The course of the scribed may also be studied in live, anaesthetised disease can go in one of two directions: either fish by use of ultrasound examinations. In histo- sudden onset of mortalities in a population of ap- logical sections, CMS may be characterised by parently healthy fish (Skrudland et al. 2002), or inflammatory reactions and degenerations in the moderately increased mortalities over several spongy layers of the heart muscle (Ferguson et months (Brun et al. 2003). During a prolonged al. 1990). Studies have documented that the course of the disease, abnormal swimming pathological changes develop over several movements and anorexia may be observed prior months, and in early stages the changes are de-

145x100//Kap09-fig06.eps Figure 6. The histological section to the left shows a normal gill filament, and to the right is a filament with proliferative gill inflammation. Thickened lamellae with infiltrations of inflammatory cells can be seen. (Photo: A. Kvellestad)

162 Aquaculture Research: From Cage to Consumption marcated. However, gradually the changes be- viral aetiology. In an epidemiological study, come generally distributed, and the inflammat- Brun et al. (2003) found a connection between ory reactions and necroses in the muscular cells outbreaks of CMS and earlier cases of infectious may weaken the wall of the atrium and lead to a pancreas necrosis (IPN) in the same farm. In- rupture. Necroses may be observed even in the flammation of the heart muscle is also found in liver (Ferguson et al. 1990). In other organs, e.g. connection with “erythrocytic inclusion body the gills and spleen, congestion of blood can be syndrome” (EIBS) (Rodger & Richards 1998). seen, probably as a consequence of a general cir- Recently, it was reported that a variant of the PD culatory failure (Amin & Trasti 1988). virus, Norwegian salmonid alphavirus, had been isolated from CMS fish (Hodneland et al. 2005). Causality It is not known whether this was merely a coinci- An infection hypothesis of CMS is based on the dental finding. findings of inflammatory reactions and necroses in the heart muscle. Other theories have been Current topics for further research launched, relating to malnutrition, auto immune There are many unanswered questions related to reactions or environmental influences. However, CMS, particularly connected to the aetiology, the alternative causal relations (other than an infec- elucidation of the pathogenesis, and the mapping tious agent) have received little attention in re- of risk factors. An approach to these problems search projects. can include observing groups of fish during the entire seawater phase – preferably even during As early as in the first description of the condi- outbreaks of CMS – and making clinical records tion, Amin & Trasti (1988) suggested a viral aeti- and taking samples in order to study pathological ology, based on the detection of inclusion bodies changes and examine for microorganisms. De- in muscle cells adjacent to damaged cells. Later tection of microorganisms should be followed by studies, however, have not succeeded in cultivat- characterisation studies and challenge trials, in ing any virus or in demonstrating viral particles order to try to reproduce the disease condition. by use of electron microscopy (Rogder & Turn- Possible connections between CMS and other bull 2000; Ferguson et al. 1990). Still, Grotmol et diseases, including IPN, should be studied fur- al. (1997) reported the detection of nodavirus- ther, and other risk factors should be identified in like particles in heart muscle of fish suffering epidemiological studies. Based on the results of from CMS, both by use of immunohistochemis- pathological and microbiological studies, better try against nodavirus, as well as by demonstrat- diagnostic tools should be developed. ing virus-like particles 25 nm in size with electron microscopy. It could not be concluded, however, that the virus particles were the cause Epitheliocystis / proliferative gill of the disease, and similar findings have not been inflammation (PGI) reported from other outbreaks of CMS. Comprehensive gill problems in Atlantic salmon, resulting in impaired growth and significant Transmission of the disease by injection of cell- mortalities, have been observed to varying de- free material based on heart, liver and kidney tis- grees along the entire coast since the 1980s. sue from sick fish has been reported (Watanabe Some fish health services do report increasing et al. 1995; Nylund 2001), supporting a theory of

Thematic area: Health 163 problems in recent years (Myklebust & Holm No effective treatment has been identified for 2005). PGI. Bathing in different solutions has been tested, as well as antibiotics added to feed, but no Outbreaks typically occur the first autumn in sea- evident effect has been demonstrated (Myklebust water, a few months after sea transfer. Mortalities & Holm 2005). may vary from negligible to more than 20 per cent. Even higher mortality numbers have been The Research Council of Norway is supporting reported in some outbreaks (Myklebust & Holm two ongoing projects aimed at identifying risk 2005). In addition, there are losses due to re- factors for outbreaks of PGI and developing duced growth in survivors. Fish suffering from diagnostics for the disease condition. such gill problems may also be more vulnerable to other disorders. Hence, the overall economic impact of the disease is substantial. Parvicapsula pseudobranchiola During the spring and early summer of 2002, dis- So far, diagnosis of PGI has mainly been based ease outbreaks in Atlantic salmon were recorded on clinical symptoms and histological findings, in five sea farms in Northern Norway (Karlsbakk the latter revealing intracellular accumulations of et al. 2002; Sterud et al. 2003). Mortality num- rickettsia- or clamydia-like organisms, termed bers in the farms varied from three to 50 per cent. “epitheliocysts” in the gills. These formations A single-celled parasite, Parvicapsula pseudo- give rise to the term “epitheliocystis” for this branchiola, was found to be the cause of deaths. condition. Typical findings in histology from The diagnosis was based on histology. The para- gills are inflammation and increased numbers of site is a myxosporidium, and the life cycle prob- surface cells; in recent years the disease has been ably includes a switch between Atlantic salmon called “proliferative gill inflammation” (PGI). (intermediate host) and an invertebrate (end host). There is no known treatment for the dis- So far, it has not been possible to cultivate the mi- ease. croorganisms in question, so challenge experiments have not been possible. Based on molecular Disease caused by Parvicapsula has previously characterisations of the organism, a new species been reported in salmon in North America (Jones has been suggested: Candidatus pisciclamydia et al. 2004). The discovery of a new parasite in salmonis (Draghi et al. 2004). A paramyxovirus Norway attracted considerable attention, particu- has been isolated from the gills of sick post-smolt, larly in relation to a possible risk for important Atlantic salmon paramyxovirus (ASPV) (Kvelle- wild stocks of Atlantic salmon. During 2003, the stad et al. 2003), and the virus has been detected parasite was found in more than 30 fish farming in fish suffering from clinical outbreaks in the sites from the county of Møre og Romsdal in field (Kvellestad et al. 2005). However, it has not Central Norway, and northwards. In 2004, the been possible to reproduce the disease in chal- parasite was even found in Sunnhordaland. The lenge trials (Fridell et al. 2004). Therefore, the eti- findings in 2002 resulted in the employment of ology for PGI is not fully elucidated, but the new routines for histological investigations of pathogenesis probably includes an interaction be- diagnostic material. The increased numbers of tween clamydia, ASPV and unfavourable envi- identifications of Parvicapsula in recent years ronmental conditions (Holm et al. 2005). should be interpreted in this context.

164 Aquaculture Research: From Cage to Consumption 145x100//Kap09-fig07.eps Figure 7. Vibrio anguillarum-isolates from Atlantic cod examined for antibiotic resistance. The plates from left to right show a resistant, an intermediate and a sensitive isolate, respectively. (Photo: D.J. Colquhoun)

The Research Council of Norway has funded a 2006), and the disease is already of concern for project to build up knowledge regarding the para- the cod farming industry. site, its spread, life cycle and ability to cause disease. Such knowledge will be important in Vibriosis order to evaluate different preventive measures. Globally, vibriosis is probably the most common systemic bacterial infection in farmed fish. The disease is caused by the Gram-negative bacteri- Bacterial infections in marine um usually known as Vibrio anguillarum. The fish species has been reclassified in the genus Great effort goes into developing the cultivation Listonella (MacDonald & Colwell 1985), but its of marine fish species as an aquaculture industry. taxonomic transfer from genus Vibrio has not Production is increasing, particularly for Atlantic been fully recognised (Austin et al. 1995) and is cod, and even for halibut. The range of diseases still a subject of discussion (Thompson et al. is reminiscent of what was seen in salmonids 2004). during the 1980s and early 1990s: bacterial infections are dominating these relatively early Vibriosis has caused sporadic mortalities in feral phases of developing production of marine fish- fish, but the disease is primarily a problem under es. In 2005, a new bacterial species was found marine aquaculture conditions in temperate and isolated from cod with granulomatous for- zones, including fish farming in brackish water. mations in different organs. The bacterium be- Outbreaks are most common during relatively longs to the genus Francisella (Olsen et al. high water temperatures in summer. Sørensen &

Thematic area: Health 165 Larsen (1986) originally suggested 10 different of genetic differences between isolates of V. an- serovariants of the bacterium. In addition, a num- guillarum O2α and O2β from different Euro- ber of sub-groups have been identified (Rasmus- pean countries. Their results will hopefully sen 1987; Bolinches et al. 1990; Tiainen et al. provide important knowledge of the population 1997). Vibriosis in cod in Norway is caused structure of V. anguillarum. Further research solely by V. anguillarum serovars O2α and O2β, should aim at improved vaccines on the one the latter being dominating. hand, and at the optimisation of fry cultivation systems on the other, in order to reduce the infec- Vibriosis is the most important bacterial infection pressure. tion in the production of cod fry in Norway, and the majority of prescriptions of antibiotics for Aeromonas salmonicida cod fry are against this disease. Vibriosis has also A. salmonicida can cause serious systemic infec- been seen in coalfish. The pathological findings tions in many fish species around the world. The usually include ulcers and haemorrhages in the bacterial species can be divided into five subspe- skin, particularly in the head region, and at the cies, but in connection with fish diseases, isolates bases of the fins. With a more chronic disease are categorised into two main groups; subspecies course, bloodshot, protruding eyeballs may be salmonicida, and “atypical” isolates. Isolates seen. causing disease outbreaks in farmed marine species usually belong to the atypical group (Bergh Losses due to vibriosis can be limited by vacci- et al. 2001; Lund et al. 2002; Gudmundsdóttir et nation. While the vaccines used today are with- al. 2003), but infections caused by subsp. salmo- out adjuvants, and do not offer full protection nicida do occur as well. against disease, experiments with adjuvanted injection vaccines have shown promising results In Norwegian aquaculture, A. salmonicida caus- (Mikkelsen et al. 2004). These vaccines cannot, es problems primarily in halibut, Atlantic cod, however, be applied to small fishes. Results from wolffish and turbot. Typical external symptoms experiments with fry have demonstrated better are seen as skin lesions, including ulcers (Wik- protection of fish of two grams weight, compared lund & Dalsgaard 1998). Acute mortality can be to those vaccinated at one gram weight. Fish vac- observed, but more common is a moderately in- cinated at five grams are protected for at least six creased level of mortalities over time. Fish may months (Schrøder et al. 2006). Vibrio bacteria be covertly infected with the bacterium, showing are usually sensitive to antibiotics, and oxolonic no signs of disease. One may expect that an in- acid is usually the drug of choice. However, re- crease in the farming of marine fish species in the sistant isolates have been identified (Colquhoun North Atlantic region can be followed by prob- et al. 2005). lems with different variants of A. salmonicida. The reservoir for infection is supposed to be in- Ongoing studies (Colquhoun, underway) indi- fected fish, wild or in farms. Outbreaks are prob- cate that Norwegian V. anguillarum O2β are ably activated by stressors such as high fish quite homogenous, while there are greater varia- densities, handling of fish, or poor water quality. tions between O2α isolates. These differences may be of importance for the design of vibriosis Losses during an outbreak may be reduced by vaccines intended for cod. Studies are underway keeping proper routines. Treatment with antibi-

166 Aquaculture Research: From Cage to Consumption otics is usually effective, but the disease may reappear after some time, and A. salmonicida has the ability to quite frequently develop strains showing multi-resistance against several antibiotics (L’abée-Lund & Sørum 2001).

Vaccination against atypical furunculosis in marine fish species has not offered protection comparable to that achieved against furunculosis in salmonids. Levels of protection seem to vary with the bacterial strain causing disease (Lund et al. 2003), and differences in the bacterial surface A-layer protein seem to be of importance. Varia- tion in the A-layer reflected in DNA of the iso-

145x100//Kap09-fig08.eps lates is a promising epidemiological marker for Figure 8. Cod louse (Caligus curtus) situated on the skin of virulence (Colquhoun, underway). Ongoing re- an Atlantic cod. (Photo: T.T. Poppe) search activities are to a great extent focusing on selection of isolates for vaccine development as VER (Johansen et al. 2004b). However, the virus well as epidemiological typing systems based on was somewhat different from the one causing molecular biological characteristics. disease in halibut. Disease is also reported in cod, the virus being quite similar to the halibut virus (Starkey et al. 2001). Nodavirus Nodavirus is the cause of viral encephalopathy Methods for cultivating the virus in cell culture and retinopathy (VER) in halibut fry (Grotmol et have been established (Dannevig et al. 2000) and al. 1997; Grotmol et al. 1999). The disease antibodies have been produced and are being uti- caused substantial losses throughout the 1990s, lised in immunohistochemistry (Johansen et al. but in more recent years, reported outbreaks have 2002). An ELISA to monitor the immune re- been few. It can be assumed that general hygienic sponse in fish has been established (Grove et al. management procedures implemented in the 2003), and PCR methods have been developed farming of halibut have had a positive effect. for the detection of virus (Grotmol et al. 2000; Grove et al. 2003). Experimental vaccines have Infection of larger fish does not seem to cause been designed and tested in turbot, and a vaccine disease (Grove et al. 2002); however, they can based on a recombinant capsid protein gave pro- become carriers of the virus. Although they show tection (Sommerset et al. 2005). Further studies no obvious symptoms, growth rate may be af- concerning the pathogenesis of VER are needed, fected (Johansen et al. 2004a). The virus may be as is the development of better diagnostic tools. transmitted to spotted wolffish, resulting in a disease condition resembling that seen in halibut (Johansen et al. 2003; Sommer et al. 2004). A Parasites in marine fish nodavirus has been isolated from turbot, as well, When a fish species is kept in monoculture for in connection with a disease outbreak similar to farming purposes, the conditions are ideal for the

Thematic area: Health 167 multiplication of parasites, particularly for spe- cod, and the “fish louse”, genus Argulus (Schram cies which do not need intermediate hosts. The et al. 2005). All these species are parasitic crus- parasites get access to a great number of hosts taceans. living in high population densities, compared to wild populations. Therefore, it is of great impor- Other parasites in cod include the gill worm tance to know which parasite species may be (Lernaeocera branchialis, copepod) (Lysne & found in wild stocks of the same fish species, and Skorping 2002), the flagellate Spironucleus to follow the development of parasites burdening torosa in the intestine, and the microsporidium farmed fish. The multiplication of parasites in Pleistophora gadi in the muscle. Several ciliates aquaculture may also affect wild fish, due to in- (e.g. Trichodina), flagellates (e.g. Ichthyobodo) creased infection pressure. and myxosporidia (e.g. Pleistophora) can infest different marine fish species, such as cod, halibut The “Scottish louse”, Caligus elongatus, consti- and wolffish. The nematodes include species tutes a particular challenge; this louse can be a such as Anisakis and Pseudoterranova, giving parasite on many different fish species, including rise to intermediate stages of the worm in muscle Atlantic salmon, Atlantic cod (Karlsbakk et al. and other organ systems (Hemmingsen & 2001), and halibut. In a research project, lice MacKenzie 2001), and they may cause health sampled from different fish species are compared problems in humans (zoonoses). Monogenean in order to reveal any differences in morphology, ectoparasites also need investigating, including genetic material or specific preferences for dif- various species of gyrodactylus, which may ferent fish species. There are even other “lice” in cause problems in farmed cod. cod, such as Caligus curtus, which attack only

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Thematic area: Health 171 Øystein Evensen1), Nina Santi1), Ann-Inger Sommer2) and Siri Mjaaland1) 1) Norwegian School of Veterinary Science, 2) Fiskeriforskning (Norwegian Institute of Fisheries and Aquaculture Research)

Virulence Mechanisms

Virulence is the ability of infectious agents to cause disease in the infected animal. This is a trait that has developed over millions of years in close interaction between the host and the agent. The relationship that has developed over millions of years of co-existence has resulted in a situation where there is on one side a balance between the agent’s ability to spread within a susceptible population and on the other side the outcome of an infection both at the level of the individual and the host. Rapid viral replication and spread within the host will result in severe damage to internal organs and rapid death, which will limit the ability of the agent to spread and survive within the population. It is anticipated that agents will trade off virulence for persistence and less-efficient spreading mechanisms in lower vertebrates as well as mammals. This chapter will describe characteristics of two fish viruses and present results from recent years’ research into infectious pancreatic necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV). For the IPN virus, a good understanding has been gained of the association between a strain’s amino acid residues at defined positions in the VP2 protein and the virulence of the strain, defining a virulence signature. Similarly, defined mutations in the same amino acid residues can easily be associated with loss of virulence. For the ISA virus it has been shown there are large variations in some of the surface molecules between strains, although it has not been possible to associate these differences with virulence traits. It has also been shown that viral proteins can counteract the defence mechanisms of the host.

172 Aquaculture Research: From Cage to Consumption Definitions Viral replication Virulence is used as a term to describe the rela- The speed at which replication takes place plays tive pathogenicity of the relative ability (capa- a key role in determining virulence. Fast repli- city) of the agent to cause tissue damage/ cation will frequently be associated with dis- disease. While virulence traits in bacteria are ease symptoms and clinical disease. The ability often associated with different toxins and the of the virus to replicate in vivo will depend on level at which they are produced, virulence in the ability of the virus to replicate under the viruses is often less well defined. This chapter conditions “provided” by the infected cell plus will focus on virulence traits of viruses for the the extent to which the virus can resist the de- reason that it has been the main topic of the fence mechanisms of the cell aimed at prevent- Aquaculture programme for this reporting ing replication and eliminating the virus. A period. In no way does this mean that the viru- fine-tuned balance has developed over years of lence traits of fish pathogenic bacteria are all co-existence and relates to different stages of an known and well described. Many factors con- infection: cell adherence, penetration, release of tribute to the overall pathogenicity of a virus, the virus particle to the inner compartment of such as the host cell entry mechanisms, replica- the cell, access to energy, synthesis of compo- tion capacity, ability to counteract or resist the nents of importance during early stage of repli- defence mechanisms of the cell, and the ability cation, synthesis of virus proteins and genome to cause temporary or permanent damage to the replication, and assembly and release from the host through cell lysis, production of toxic sub- cell. stances, cell transformation, and production of antiviral substances by the infected cell in re- Tissue damage sponse to an infection. In addition, induction of A successful infection will often result in cell structural changes in the cell nucleus and the lysis, typical of cytolytic viruses. Typically, cytoplasm are observed. A virus can infect the membrane integrity is lost, followed by leakage host through damaged skin or an insect bite (in of enzymes and cell damage. Apoptosis is an- fish it has been proposed that the salmon louse other classical cellular response to a virus infec- – Lepeophteirus salmonis – can act as a carrier tion and because of this many viruses have and vector of infectious agents). However, in developed strategies whereby they counteract both lower and higher vertebrates the mucosal the induction of apoptosis. Virus proteins inter- surfaces are considered a prime port of entry of act at different levels of the apoptosis “ma- pathogens; because of this, pathogenic viruses chinery” and the details are largely unknown for have developed the ability to exist in company many viruses; the consequence to the host cells with the normal flora, both viruses and bacteria. is a delay in the onset of apoptosis, which al- Replication of a virus can occur in the mucosal lows the virus to produce more progeny before lining, i.e. this site is the primary port of infec- the cell machinery shuts down. Non-cytolytic tion (as seen for koi herpes virus) but more viruses, on the other hand, will often cause cell often the mucosal lining is the port of entry of damage through immunological defence mech- systemic infection. anisms, typically through an expression of capsid or membrane components of the virus being expressed in the cell membrane of the infected cell. Antibodies in combination with

Thematic area: Health 173 complement can result in cytolysis while cyto- toxic T cells can induce cell lysis through direct interaction.

An important virulence trait of many viruses is the ability to circumvent the immune responses of the host. Different viruses have developed different strategies; inhibition of presentation of virus proteins via MHC molecules is one, yet another is the ability of the virus to mutate single amino acids in immunodominant virus proteins.

145x100//Kap10-fig01.eps Cellular effects Figure 1. Electron micrograph of IPN virus (negative A virus infection can have toxic effects on the staining; Trygve Eliassen). infected cells. Examples are formation of syn- cytia, fusion of chromosomes (polykaryocyto- sis) and so on. Virus infections can also result in Methods relevant to studies of viral cell transformation (pre-cancerous stages) and virulence mechanisms suppression of immune responses as inhibition Both biotechnological methods and fish chal- of interferon responses. Cytoplasmic and nuc- lenge studies are important tools when investi- lear inclusion bodies are structural cell changes gating the virulence mechanisms of IPNV. It observed in association with virus infections. has been known for a while that some strains cause higher mortality, and that passage in cell In the ensuing paragraphs you will find a pre- culture can alter the virulence characteristics of sentation of virulence mechanisms of infectious the virus. pancreatic necrosis virus and infectious salmon anaemia virus. IPNV is a small virus, 60 nm in Virulence differences among virus stains can be diameter with an icosahedral symmetry in the revealed through experimental challenge of capsid (Figure 1). The genome consists of two salmonid fish. A number of factors influence double-stranded RNA molecules which encode the mortality caused by IPN, both in field out- five virus proteins (VP1–5). VP2 and possibly breaks and under experimental conditions. Ac- VP5 carry virulence traits. VP2 is exposed on cordingly, virulence comparison of IPNV the virus surface and is important for the bind- strains must be coordinated, and performed in a ing to and uptake in target cells. The VP2 pro- controlled environment to attain credible re- tein also carries serotype specific and sults. All conditions, such as the number of fish, neutralising epitopes. VP5 can promote the virus dose, challenge time, temperature, tanks, ability of the virus to replicate and it has been and water flow, must be equal for all experi- shown that certain strains of IPN virus have an mental groups. By challenging susceptible rain- anti-apoptotic effect. bow trout fry, early investigators detected virulence differences among IPNV strains of

174 Aquaculture Research: From Cage to Consumption 145x100//Kap10-fig01.eps Figure 2. The reverse genetics technique for production of recombinant IPN virus. First, a CDNA copy of the viral genome is cloned into a plasmid vector. In front of the genomic sequences is a short sequence recognised by a RNA polymerase (T7). This enables in vitro synthesis of RNA copies of viral genomic segments A and B. When this copy RNA is transfected into a susceptible cell culture, new viral proteins and genomic segments are made, and later assembled into new viral particles. Mutations introduced in the cDNA copy of the viral genome will be expressed as alteration in the proteins of the new, recombinant virus isolate.

the same serotype, and mapped virulence traits sible to alter the viral proteins by introducing to RNA-segment A. specific mutations of the viral genome. It is, for instance, possible to change one amino acid res- Sequencing and subsequent comparisons of the idue of a highly virulent strain to match the cor- viral genomes of different IPNV strains can re- responding amino acid of a low virulent strain. veal differences and lead to identification of mo- The resulting recombinant viral strain can be lecular motifs involved in virulence. However, further tested to confirm the role of this residue the strains may differ in more than one way, mak- as a virulence marker. The reverse genetics ing it hard to decide which motif is most signifi- technique provides a unique tool for studies of cant for the virulence traits of the virus. the viral life cycle, viral assembly, the role of viral proteins in pathogenicity, and the interplay The reverse genetics technique can be applied of viral protein with components of the host’s to gain a more detailed map of the virulence immune response. A reverse genetics technique markers (Figure. 2). By this technique it is pos- has been developed for the IPN virus as well.

Thematic area: Health 175 Identified virulence mechanisms of the The significance of the individual VP2 amino IPN virus acids was also investigated using the reverse When Atlantic salmon fry are challenged with genetics technique. These studies pinpoint VP2 different field strains of IPNV, great variation in residues 217 and 221 as the major virulence de- the pathogenicity of the isolates is displayed. terminants of Norwegian IPNV isolates. Highly Some of the strains cause high mortality and se- virulent strains encode Thr217 and Ala221, vere lesions in the pancreas and liver of infected while moderate to low virulent strains have fish, while other strains cause low mortality and Pro217 and Ala221. Strains with Thr at residue no detectable tissue damage. The same strains 221 are almost avirulent, irrespective of the cause varying mortality in experimental chal- amino acid in position 217. lenge of Atlantic salmon post-smolt as well. The virulence was also tested after 10 passages The molecular basis for cell culture attenuation in cell culture. was realised by detection of an Ala-Thr substitution at residue 221 after a few passages of the From these studies, several molecular motifs as- virus in cell culture. The cell culture adapted sociated with IPNV virulence were identified. strains are strongly attenuated and almost aviru- Four amino acid residues of the capsid protein lent in experimental challenge of Atlantic VP2, as well as the size of the non-structural salmon fry. A single mutation at VP2 residue protein VP5, appeared to be important. These 221 can be detected after 10 passages in cell motifs have since been subject to further studies culture. The resulting mutated strain has signi- to attain detailed knowledge of their individual ficantly reduced virulence, causing very low influence on the pathogenicity of the virus. mortality as compared to the original strain.

The reverse genetics technique has been used to The three-dimensional structure of VP2 of study the effect of VP5 on IPNV virulence. Re- IBDV was recently determined. IBDV is a combinant viruses coding for different size birnavirus causing infectious bursitis in chick- variants of the VP5 protein have been made, as en. From the IBDV VP2 crystal structure it can well as a VP5 knock-out mutant. Challenge be predicted that the amino acids 217 and 221 studies of both Atlantic salmon fry and post- of IPNV are exposed at the viral surface. Their smolt did not reveal any differences in the viru- exposed orientation combined with the marked lence of the VP5 variant strains. Additional effect on virulence may suggest that residues studies have demonstrated that VP5 is not es- 217 and 221 are engaged directly with receptors sential for viral persistence. A cell protective of the host cell. Further investigations are function (anti-apoptotic) has been recorded for necessary to decide if this is the case. the VP5 protein of an Asian IPNV strain. This anti-apoptotic effect was not found in cell cul- Field studies ture or in fish infected with the recombinant In a field study of IPN including 20 different strains studied. Taken together, these results op- fish farms with post-smolt vaccinated against pose a role of VP5 as a virulence factor of Nor- IPN, an accumulated mortality varying from wegian IPNV strains. one to 45 per cent was registered three to nine weeks after transfer to seawater. There might be different reasons for the large variation in mor-

176 Aquaculture Research: From Cage to Consumption tality. One possibility was that salmon from dif- Whether this applies to other fish species sus- ferent localities was infected with IPN viruses ceptible to IPN is still to be investigated. of varying virulence, and IPN virus isolates from these outbreaks were therefore sequenced. Carrier conditions, reactivation of the IPN Most of them showed the motif of the four virus and virulence amino acids in VP2 consistent with a highly Because the IPN virus is very common in Nor- virulent strain, while only a few of the field wegian fish farms, primary infections may strains were equal to the variants of moderate occur after transfer of the salmon to the sea. virulence. Nevertheless, there is reason to believe that reactivation of a persistent IPN virus infection One field strain was interesting because it con- could be responsible for a great part of the out- tained only two of the four “markers of high vir- breaks. Increased resistance to IPN in new ulence”, Thr217 and Ala221, but nevertheless generations of farmed Atlantic salmon may also was characterised as highly virulent in a chal- represent a genetic pressure allowing more lenge test. Again this indicated that the two virulent IPN viruses to be carried asymptoma- amino acids in position 217 and 221 are the tically in the fish. For instance, the N1 strain of most important ones for virulence in related IPNV previously isolated from most of the IPN virus strains. All genetic characterisations outbreaks of IPN in salmon fry and from virus of the field isolates as highly or moderately carriers is now characterised as a low virulent virulent were confirmed when the strains were strain. tested in challenge experiments under equal conditions. Several trials have shown that non- Experimental challenge and knowledge about vaccinated post-smolt challenged with strains the virulence of the IPN virus strains are also of high virulence usually results in 40–70 per used to ascertain the significance of virulence in cent accumulated mortality and zero to 35 per the establishment of a carrier condition, and in cent using strains of moderate to low virulence. reactivation of IPN as well. Experimental carri- The mortality also seems to vary with the ers of the IPN virus were established under susceptibility to IPN in the families of Atlantic equal conditions by bath challenge in fresh- salmon used. water, using both a highly virulent and a low virulent strain. The highly virulent strain was There is obviously a lack of correspondence be- reactivated after smoltification and transfer to tween mortality registered out in the field and in seawater, which resulted in an IPN outbreak. controlled challenge experiments. This reflects The accumulated mortality was comparable to that many factors, besides the quality of the IPN that observed after challenge of post-smolt virus itself, have an impact on the result of an salmon with the same strain in seawater. This infection. It should be mentioned, though, that occurred even if the virus at some time points in no low virulent virus strain was isolated from the freshwater phase was not detectable in cell any of the IPN outbreaks. Results from all the culture using a traditional carrier test. This type studies together show that the same virulence of IPN virus was isolated from more than ¾ of factors are important for development of IPN all collected field strains, but it is not known both in Atlantic salmon fry and post-smolt. whether the salmon was infected in seawater or

Thematic area: Health 177 already carried the virus in the early freshwater of segment 8, while the larger ORF2 encodes an phase. RNA-binding structural protein with putative interferon antagonistic activity. The mechanisms behind an IPN virus carrier condition are not known. Studies have shown The ISA disease picture is complex, as ob- that the mutations that happen during passages served by large differences in disease develop- in cell cultures and result in a change from Ala- ment and clinical signs. Typically, an ISA nine to Threonine in position 221 in VP2 may outbreak may initially appear as diffuse, unspe- explain a reduced virulence, which seems to co- cific disease problems and variable mortality incide with the establishment of a persistent in- over long time periods, before finally cumulat- fection. ing into an outbreak, while acute disease outbreaks occur more seldom. Virulence is Virulence mechanisms for the influenced by a) the genetic constitution of the ISA virus fish, b) the genetic constitution of the virus iso- ISA virus late, and c) environmental circumstances. The ISA virus is an aquatic orthomyxovirus (genus: Isavirus), belonging to the same family Possible virulence mechanisms as the influenza A virus causing disease in birds The study of ISA virus genes and gene product and mammals. The virus is enveloped, 80–120 is still in its infancy, and there are at present ma- nm in size (Figure 3), with surface projections jor gaps in our knowledge around the mecha- performing hemagglutination (receptor-bind- nisms behind ISA virus virulence. It was not ing), receptor-destroying (viral release) and until 2002, for instance, that all the genes had fusion (virus entry into the cell) activity. The been sequenced, and not until 2005 that the total genome consists of eight single-stranded RNA sequence of one isolate (Glesvaer 2/90), includ- molecules with negative polarity, encoding at ing the non-coding 3’- and 5’-terminal viral least 10 proteins, where nine are known to be RNA sequences, was completed. This last work present in the mature virus particle. The largest was initiated as the first step in the establish- genomic segments (segments 1, 2 and 4) encode ment of a reverse genetics system on ISAV (see the viral polymerases, while segment 3 encodes below). Current knowledge of virulence is the viral nucleoprotein. Segments 5 and 6 en- based on results from experimental trials, se- code the two surface glycoproteins, i.e. the quence analyses of viral genes, and functional fusion protein (F) and the hemagglutinin- studies on cloned recombinant viral proteins. esterase (HE). The HE is responsible for receptor- binding and -release, while the F protein In orthomyxoviruses, reassortment of gene seg- enables the entrance of viral nucleic acids into ments occurs frequently and is a major contrib- the cell. The two smallest genomic segments utor to the evolution of these viruses and the encode two proteins each. The largest open emergence of new virulent strains. Extensive reading frame (ORF1) of segment 7 encodes a molecular and phylogenetic sequence analyses non-structural protein (NS), a putative inter- of 14 full-length sequences of Norwegian ISA feron antagonist, while the function of the struc- virus isolates together with a parallel study on tural ORF2 protein remains to be characterised. the two surface glycoproteins provide strong The matrix (M) protein is encoded by the ORF1

178 Aquaculture Research: From Cage to Consumption evidence for the occurrence of genetic reassort- length precursor gene (HPR0) as a result of ment involving several ISAV gene segments. strong functional selection pressure, possibly,due to a newly or ongoing crossing of spe- Moreover, properties like receptor-binding and cies barrier, or as a results of changes in -release, fusion and interferon antagonism, ecological conditions related to fish farming. So which are all important factors of virulence for far, the number of different HPR groups is 25 in influenza virus, have also been identified for the Europe and four in North America. The pattern ISA virus. However, it is still not known to what of variation is constrained to the 35 amino acids extent variations in these activities influence the defined as the HPR. outcome of an ISA virus infection. The presence of a long HPR0 gene has been ISA virus HE protein confirmed by RT-PCR of tissue samples from Cloning and early characterisation of the he- healthy wild and farmed Atlantic salmon. magglutinin-esterase (HE) gene was published HPR0 is therefore assumed to represent a low- in 2001. It was soon realised that HE is the ISA pathogenic /avirulent virus. The main reservoir virus protein with the overall highest sequence for the ISA virus is today believed to be wild variation, concentrated in a small highly poly- salmonids (brown trout and Atlantic salmon) morphic region (HPR) near the transmembrane and the farmed salmon itself. region. This region is characterised by the presence of gaps rather than single-nucleotide sub- HPR/HPR0 stitutions. Alignments and analyses of a large All viruses isolated from ISA-diseased fish so number of HE gene sequences suggested that far contain deletions in the HPR region. It is the polymorphism in this region could arise therefore assumed that deletions in the HPR are from differential deletions of a theoretical full- a prerequisite for disease development, and that the HPR most likely represents an important virulence marker. In fact, this is so far the only genetic marker associated more or less directly with virulence in the ISA virus. The virulence of the ISA virus cannot, however, be attributed to the HE-HPR alone, as isolates with identical HPR vary in virulence in experimental infections using standardised experimental fish.

Virus-host interactions Experimental trials have been designed to study the interactions between the ISA virus and the salmon host in detail, with emphasis on disease susceptibility and immune responses. To reduce the effect of host genes, half-siblings identical in MHC class I and II genes were used as experimental fish. The ISA virus isolates were

145x100//Kap10-fig01.eps Figure 3. Electron micrograph of ISA virus particles (Ellen selected according to a) variation in their HE- Namork).

Thematic area: Health 179 145x100//Kap10-fig01.eps Figure 4. Reverse genetics on the ISA virus. A. The bidirectional transcription system. Viral cDNA is oriented in a positive direction to the RNA polymerase II promoter (pII) and poly adenylation site (aII), and a negative orientation to the RNA polymerase I promoter (pI) and termination sequence (tI). In this way both viral mRNA (positive-stranded) and viral vRNA (negative-stranded) may be produced from the same construct following transfection into susceptible cells. B. Total viral RNA sequence of each of the eight ISA virus genomic segments is converted to cDNA and placed in separate plasmid vectors. The eight plasmids are co-transfected into fish cells (ASK or TO) and recombinant ISA virus synthesised. C. Detailed description of plasmid-based replication of recombinant ISA virus.

180 Aquaculture Research: From Cage to Consumption HPR region and b) the isolates’ differences in tative cleavage site. More detailed analyses virulence in field outbreaks. The mortality revealed extensive sequence homology in this induced after cohabitant challenge ranged from region. This, together with the fact that exactly zero to 47 per cent in the test-group fish, and the same recombination occurred in two unre- three to 75 per cent in the injected cohabitants. lated ISA virus isolates, suggests the presence of a recombinational hot spot. Hypothetically, The use of MHC compatible experimental fish this could lead to alterations in the cleavage made it possible – for the first time – to estimate specificity of the fusion protein, with potential the relative importance of humoral versus cellu- changes in tissue or organ tropism, analogous to lar responses in protection against the ISA avian influenza A virus HA of the subtypes H5 virus. Most interestingly, the ability to induce a and H7, where sequential insertion of several strong proliferative response correlated with basic amino acid residues at the HA1/HA2 pro- survival, while induction of a humoral response teolytic cleavage site leads to systemic infection in itself was less protective. The differences in and multi-organ failure in poultry. induced immune responses could not be as- cribed to the variation in the HE-HPR alone, as ISA virus interferon antagonists two isolates with identical HE-HPR induced Induction of type I interferon responses repre- opposite immune responses and hence survival. sents an important first-line defence against viral infections. Many viruses have developed ISA virus biobank material strategies to counteract this response. For the Due to the lack of a reverse genetics system for influenza viruses, this is accomplished by a this virus at the time, detailed sequence- and non-structural protein with RNA-binding prop- phylogenetic analyses were the methods of erties. The ISA virus, on the other hand, pro- choice in the search for additional virulence duces two proteins potentially interfering with traits. The 14 ISA virus isolates described the cells’ interferon response: a non-structural above were therefore cloned and full-length protein without RNA-binding properties, and a sequenced. Following extended amino acid small RNA-binding structural protein. The two sequence analyses coupled to data on virulence, related virus groups may therefore seem to use candidate genes and areas of genes of impor- different strategies to achieve the same goal: to tance in ISAV virulence have been suggested inhibit the cells’ defence against viral infection. (see below). For final proof, however, a reverse The detailed mechanisms behind the ISA virus genetics system is needed. interferon antagonism have not yet been characterised. ISA virus F protein The fusion activity is associated with uptake of Reverse genetics on the ISA virus a virus into the cell during infection, and chang- Reverse genetics is the optimal tool for studying es in this protein may thus have important im- virulence mechanisms and virus-host interac- plications for the virulence of an isolate. Most tions. The method also has great potential in interestingly, when aligning all available fusion future productions of vaccines against the ISA gene sequences (segment 5), a 30-nucleotide- virus. For several positive-stranded RNA virus- long insertion was found in several virus iso- es (such as the IPN virus) this technology has lates, immediately upstream of the protein’s pu- already been established and been in use for

Thematic area: Health 181 several years. However, in contrast to positive- tive-stranded mRNA from each cDNA template stranded RNA molecules, negative-stranded (Figure 4). RNA molecules (vRNA; as the ISA virus) are not infectious. By including expression of func- The total vRNA sequences from all eight of the tional viral RNA polymerases and ribonucle- ISA virus genomic segments have been mapped oprotein complexes, synthetic vRNA segments and cDNA cloned into the pol I/II vector can be converted to an infectious virus from pHW2000, and the work towards producing full-length cDNA. A plasmid-based expression viable artificial viral particles is promising. system is used, similar to the one developed for After successful isolation of recombinant viral the influenza A virus, where viral cDNA from particles, mutations will be introduced into can- each of the genomic segments are placed be- didate genes. The molecular analyses of the tween a RNA polymerase I promoter and termi- above mentioned biobank material will help to nator sequence, flanked by a RNA polymerase define relevant genes and areas of genes. The II promoter and polyadenylation site. The ori- effects of the mutations will be tested in vitro entation of the two transcription units allows (infection of cell cultures) and in vivo (experi- synthesis of negative-stranded vRNA and posi- mental trials).

182 Aquaculture Research: From Cage to Consumption References Melby, H.P., Christie, K.E., 1994. Antigenic analysis of reference strains and Norwegian field strains of Brun, E., 2003. Epidemiology. In: Evensen Ø, Rimstad aquatic birnaviruses by the use of 6 monoclonal E, Stagg R, Brun E, Midtlyng P, Skjerstad B, Jo- antibodies produced against the infectious hansen LH, Jensen I (eds): IPN in salmonids; a re- pancreatic necrosis virus N1 strain. J. Fish Dis. view. Trondheim, FHL and VESO, pp 51−67. 17(4), 409−415. Coulibaly, F., Chevalier, C., Gutsche, I., Pous, J., Nava- Mjaaland, S., Hungnes, O., Teig, A., Dannevig, B.H., za, J., Bressanelli, S., Delmas, B., Rey, F.A., 2005. Thorud, K., Rimstad, E., 2002. Polymorphism in The birnavirus crystal structure reveals structural the infectious salmon anemia virus hemagglutinin relationships among icosahedral viruses. Cell 120, gene: importance and possible implications for evo- 761−772. lution and ecology of infectious salmon anemia dis- Cunningham, C.O., Gregory, A., Black, J., Simpson, I., ease. Virology 304, 379−391 Raynard, R.S., 2002. A novel variant of infectious Mjaaland, S., Markussen, T., Sindre, H., Kjoglum, S., salmon anaemia virus (ISAV) haemagglutinin gene Dannevig, B.H., Larsen, S., Grimholt, U., 2005. suggests mechanisms for virus diversity Bull. Eur. Susceptibility and immune responses following ex- Ass. Fish Pathol. 22, 366−374 perimental infection of MHC compatible Atlantic Devold, M., Karlsen, M., Nylund, A., 2006. Sequence salmon (Salmo salar L.) with different infectious analysis of the fusion protein gene from infectious salmon anaemia virus isolates. Arch Virol. 150, salmon anemia virus isolates: evidence of recombi- 2195−2216. nation and reassortment. J.Gen.Virol. 87, 2031– Nylund, A., Devold, M., Plarre, H.. Isdal, E., Aarseth, 2040 M., 2003. Emergence and maintenance of infec- Dorson, M., Castric, J., Torchy, C., 1978. Infectious tious salmon anaemia virus (ISAV) in Europe: a pancreatic necrosis virus of salmonids: biological new hypothesis Dis. Aquat. Organ. 56, 11−24. and antigenic features of a pathogenic and of a non- Plarre,H., Devold, M., Snow, M., Nylund, A., 2005. pathogenic variant selected in RTG-2 cells. J. Fish Prevalence of infectious salmon anaemia virus Dis. 1, 309−320. (ISAV) in wild salmonids in western Norway. Dis. Garcia-Rosado, E., Markussen, T., Mjaaland, S., Aquat. Org. 66, 71−79. Bækkevold,E., Rimstad, E., 2007. The un-spliced Raynard, R.S., Murray, A.G., Gregory, A., 2001. Infec- mRNA of infectious salmon anemia virus genomic tious salmon anaemia virus in wild fish from Scot- segment 7 encodes a non-structural interferon an- land. Dis. Aquat. Organ. 46(2), 93−100. tagonist (submitted) Rimstad, E., Mjaaland, S., Snow, M., Mikalsen, A.B., Hoffmann, E., Webster, R.G., 2000. Unidirectional Cunningham, C., 2001. Characterization of the in- RNA polymerase I-polymerase II transcription sys- fectious salmon anemia virus genomic segment that tem for the generation of influenza A virus from encodes the putative hemagglutinin. J. Virol. 75 eight plasmids. J. Gen. Virol. 81, 2843−2847 (11), 5352−5356. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G., Roberts, R.J., McKnight, I.J., 1976. The pathology of Webster, R.G., 2000. A DNA transfection system infectious pancreatic necrosis. II. Stress-mediated for generation of influenza A virus from eight plas- recurrence. Br. Vet. J. 132, 209−214. mids. Proc. Natl. Acad. Sci. USA, 97(11), 6108− Sano, T., 1971. Studies on viral diseases of Japanese 6113 fishes. II. Infectious pancreatic necrosis of rainbow Krossoy, B., Devold, M., Sanders, L., Knappskog, P.M., trout: pathogenicity of isolates. Bull. Jap. Soc. Sci. Aspehaug, V., Falk, K., Nylund, A., Koumans, S., Fish 37, 499−503. Endresen, C., Biering, E., 2001. Cloning and identi- Sano, M., Okamoto, N., Fukuda, H., Saneyoshi, M., fication of the infectious salmon anaemiavirus Sano, T., 1992. Virulence of infectious pancreatic haemagglutinin. J. Gen. Virol. 82, 1757−1765. necrosis virus is associated with the larger RNA Lyngstad, T.M., Brun, E., Jansen, P.A., Heier, B.T., segment (RNA segment A). J. Fish Dis. 15, 283− 2005. Sammendrag av epidemiologiske rapporter 293. om utbrudd av ILA i Norge 2004. Rapport til Mat- Santi, N., Sandtrø, A., Sindre, H., Song, H., Hong, J.R., tilsynet fra Veterinærinstituttet. pp 1−36. Thu, B., Wu, J.L., Vakharia, V.N., Evensen, Ø., Markussen, T.,Jonassen, C.M., Numanovic, S., Braaen, 2005. Infectious pancreatic necrosis virus induces S. & Mjaaland, S. 2007. Evolutionary mechanisms apoptosis in vitro and in vivo independent of VP5. of infectious salmon anaemia virus (ISAV): full- Virology 342, 13−25 length sequence analyses of 14 isolates diverging in virulence (submitted)

Thematic area: Health 183 Santi, N., Song, H., Vakharia, V.N., Evensen, Ø., 2005b. Song, H., Santi, N., Evensen, Ø., Vakharia, V.N., 2005. Infectious pancreatic necrosis virus VP5 is dispens- Molecular determinants of infectious pancreatic able for virulence and persistence. J. Virol. 79, necrosis virus virulence and cell culture adaptation. 9206−9216. J. Virol. 79, 10 289−10 299. Santi, N., Vakharia, V.N., Evensen, Ø., 2004. Identifica- Stangeland K. et al. 1996. Experimental induction of in- tion of putative motifs involved in the virulence of fectious pancreatic necrosis in Atlantic salmon, infectious pancreatic necrosis virus. Virology 322, Salmo salar L., post-smolts. J. Fish Dis. 19, 323− 31−40. 327. Sommer, A.-I., Knappskog, D., Rødseth, O.-M., 1998. Taksdal et al. 1998. Induction of infectious oancreatic Ny smittemodell for IPN – et viktig bidrag i kampen necrosis (IPN) in covertly infected Atlantic salmon, mot sykdommen. Norsk Fiskeoppdrett nr. 18, 30− Salmo salar L., post-smolts by stress exposure, by 32. injection of IPN virus (IPNV) and by cohabitation. Sommer, A-I., 2004. Studier av virulensfaktorer hos J. Fish Dis. 21, 193−204. norske feltisolater av IPN-virus. Fact sheet, Final Vestergård Jørgensen, P.E., Kehlet, N.P., 1971. Infec- Report, Project 1 418 877/120. Research Council of tious pancreatic necrosis (IPN) viruses in Danish Norway’s website: www.forskningsradet.no/ rainbow trout. Nord. Vet.-Med. 23, 568−575. havbruk Yao, K., Vakharia, V.N., 1998. Generation of infectious pancreatic necrosis virus from cloned cDNA. J. Virol. 72, 8913−8920.

184 Aquaculture Research: From Cage to Consumption Børre Robertsen1), Ivar Hordvik2) and Trond Jørgensen1) 1) Norwegian College of Fishery Science, University of Tromsø 2) University of Bergen

The Fish Immune System

Why do Atlantic salmon smolts develop infectious pancreatic necrosis (IPN) when released into the sea? Why are viral vaccines less efficient than bacterial vaccines in salmon? Why does infectious salmon anaemia virus (ISA-virus) kill Atlantic salmon, but not trout? These are some of the questions that occupy fish immunology researchers. It is the immune status of the fish, which determines whether the fish develops disease after contact with an infectious microorganism. Mo- lecular and functional studies of the fish immune system are thus very important to understand why fish get sick and how fish vaccines can be developed to work optimally. The defence against microorganisms of vertebrates consists of the innate and the adaptive immune system. The innate immune system is comprised of cells and mechanisms that defend the host from infection by microorganisms, in a non-specific manner. This means that the cells of the innate system recognize and respond to molecules that are structurally conserved in viruses, bacteria or fungi. The innate immune defence is active only for a short period of time and may also be deceived by many pathogens. In contrast, the adaptive immune system is activated later during the infection and is directed specifically against the invader. Moreover, the adaptive immune defence is more effective, possesses memory and thus has a longer duration than the innate immune defence. The main executing cells of the adaptive immune system are B and T lymphocytes. B cells are the origin of antibodies whereas T cells kill virus infected cells and stimulate macrophages to kill intracellular bacteria. The goal of vaccination is to acti- vate the adaptive immune system. Increased knowledge about the fish immune systems may be used to improve fish vaccines and to develop better methods for measuring the immune/health status of the fish. Such methods may be used to reveal conditions that predispose the fish for infections.

Thematic area: Health 185 Atlantic salmon is one of the most important The interferon system model species for obtaining new knowledge The interferon system plays a major role in the about the fish immune system and many of the innate defence against viruses and recent re- key genes involved have now been cloned and search has shown that teleost fish have an inter- sequenced. Research on the cod immune sys- feron system, which is very similar to that of tem has also made good progress. The Aquacul- mammals [1]. Interferons (IFNs) are defined as ture Program of the Research Council of proteins that induce an antiviral state in host Norway has contributed with significant fund- cells. Virus infection activates the IFN system ing in this field, which has resulted in increased of the host cell because the cell recognizes viral knowledge and competence in fish immuno- nucleic acids [1, 2]. Henceforth, the infected logy. During the Program period several cell produces IFN, which is an alarm protein projects have been devoted to studies of the that circulates in the body and tells other cells to antibody repertoire of salmon and cod and on start production of antiviral proteins (Figure 1). the innate immunity against viruses of Atlantic As a result the IFN system stops further inva- salmon. These topics are thus the main focus of sion of the virus. The importance of the IFN this chapter. system in the innate immunity against viruses in mammals is well documented. Mice that have defective genes in the IFN system are for exam- Innate immunity against virus ple very susceptible to viral infection. The IFN in fish system is likely to be equally important in the Although viral diseases cause major losses of antiviral innate immunity of fish. The mecha- farmed Atlantic salmon, we tend to forget that nisms used by host cells for recognizing viruses fish like mammals, are normally quite resistant have recently been uncovered. This occurs by to viral infections. The fact is that vertebrates binding of viral single-stranded or double- possess a powerful innate immune defence, stranded RNA to intracellular receptor proteins which stops viral infections in an early phase. (RIG-I, MDA5, TLR3, TLR7, TLR8 or PKR). The reason why fish or humans sometimes de- The synthetic dsRNA poly I:C is in fact a pow- velop disease due to virus infection may be that erful inducer of the IFN system because it imi- the innate defence is weakened by stress, poor tates a virus infection in vertebrate cells. nutrition, poor environmental conditions or because some very aggressive viruses are able to Whereas the first IFN-genes from humans were avoid or inhibit the defence mechanisms of the cloned in 1980, the first IFN-genes from fish in- animal. Atlantic salmon is for example quite re- cluding Atlantic salmon were cloned in 2003 [1, sistant to IPN except for the early stages after 3]. The reason why it has taken such a long time hatching and after release of the smolt into the to clone the first fish IFNs is that the sequence sea. Different salmon strains also display a identity between IFNs from fish and mammals large variation in susceptibility to IPN-virus. is very low. Similar to mammals, Atlantic salm- Atlantic salmon thus has an innate immunity on was shown to possess two types of IFN, type against IPN-virus that varies with inherited I IFN, which is involved in innate immunity and traits, ontogeny/age and living conditions. As type II IFN, which is mainly involved in adap- described below Atlantic salmon appears to tive immunity. Type II IFN is identical to IFN-γ have poor innate immunity against ISA-virus. and will not be further discussed here. Humans

186 Aquaculture Research: From Cage to Consumption Virus IFN IFN-receptor

dsRNA Antiviral receptor proteins

IFN

NF-kB IFN mRNA mRNA IRE ISRE IFN gene IFN stimulated genes

Virus-infected cell IFN-stimulated cell

145x100//Kap11-fig01.eps Figure 1. Role of interferon (IFN) in the defence against viruses. Upon infection by a virus, cytoplasmic receptor proteins recognize double-stranded RNA (dsRNA) produced during viral replication.. Binding of dsRNA to receptor protein produces signals that tell the nucleus to produce IFN mRNA. Accordingly the cell starts production of the alarm protein IFN, which is secreted and circulates in the blood stream. IFN binds to a receptor protein present on the surface of most cells and this starts a signalling process, which tells the cells to produce Mx, ISG15 and other antiviral proteins. As a result IFN protects cells against virus infection. The arrows indicate signals that are produced in the cell as a response to dsRNA and IFN. have many different subtypes of type I IFN IFN gene into cultured human cells [3]. The re- (IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFNτ, combinant IFN was demonstrated to induce IFN-ω and IFN-λ). Only three type I IFN sub- strong antiviral activity against IPNV in cul- types have as yet been discovered in salmonids tured Atlantic salmon cells (Figure 2). The test [3, and unpubl. results]. These are most similar was performed by incubating salmon cells with to IFN-α from mammals. serial dilutions of IFN for 24 hours after which the cells were infected with IPN-virus. When the control cells were killed by the virus after Salmon interferon protects host cells about 72 h, the cells were fixed and stained with from IPN-virus infection crystal violet. Surviving cells become strongly Recombinant IFN-α1 from Atlantic salmon stained by crystal violet whereas dead cells re- was produced by insertion of the cloned salmon main colourless.

Thematic area: Health 187 Antiviral proteins are produced in Mx protein of Atlantic salmon inhibits salmon cells in response to dsRNA and replication of IPN-virus IFN Genetic engineering methods have enabled In human and mouse, IFN has been shown to in- studies of the antiviral activity of Atlantic salm- duce transcription of several hundred genes on Mx protein [8]. The gene encoding salmon some of which are encoding antiviral proteins Mx protein was inserted into a plasmid, which [2]. Several IFN-induced genes have recently allows continuous expression of the protein in been identified in fish [2]. Mx protein is one of animal cells. The plasmid was introduced into the most studied antiviral proteins and is known salmon cells some of which incorporated the to inhibit replication of influenza virus and sev- Mx gene in their chromosomes. Cells produc- eral other virus types. ISG15 is one of the pro- ing Mx protein continuously were subsequently teins that is induced earliest and in the largest selected and grown (Figure 3). amounts in cells by IFN. It has the ability to conjugate to other cellular proteins and appears These Mx producing salmon cells were infected to be involved in antiviral mechanisms. Both with IPN-virus. Salmon cells, which had been Mx and ISG15 have been cloned from Atlantic gene manipulated/engineered to produce green salmon and were shown to be induced by IFN fluorescent protein (GFP) were used as a nega- and dsRNA in live fish and/or cultured cells tive controls. Three days after infection surviv- [4–7]. ing cells in the different cultures were estimated by crystal violet staining. Figure 4 illustrates the

145x100//Kap11-fig02.eps Figure 2. Antiviral assay, which illustrates that recombinant Atlantic salmon IFN protects salmon TO cells against IPN virus infection. The picture shows wells with TO cells in a 96 well culture plate after staining with crystal violet. Non-treated cells or cells treated with different dilutions of recombinant salmon IFN were first incubated for 24 hours. Cells in wells labelled IPNV or IFN+IPNV were next infected with IPNV whereas wells labelled Uinf or IFN were not infected. Three days later the cells were fixed and stained with crystal violet. The colour intensity correlates with the number of living cells in the wells.

188 Aquaculture Research: From Cage to Consumption Figure 3. Expression of Mx protein in Chinook salmon embryo cells after staining with a specific antibody. ASMx1: Cells genetically manipulated to produce Atlantic salmon Mx protein continuously. IFN: Cells stimulated for 48 hours with IFN. Control: Non-treated cells.

145x100//Kap11-fig03.eps protection against IPN-virus obtained in the dif- ISA-virus is not inhibited by the IFN ferent cell types. Similar to IFN-treated cells, system of salmon almost 100 % of the Mx-producing cells sur- ISA-virus kills Atlantic salmon very efficiently vived the virus infection whereas the control in infection experiments. The aggressive nature cells died. This demonstrates that Atlantic of this virus may in part be explained by its abil- salmon Mx protein protects the cells from the ity to trick the IFN system of the fish. Neither damaging effects of IPN-virus and suggests that IFN nor dsRNA was able to protect salmon cells Mx protein is very important for the ability of against ISA-virus in spite of the fact that the salmon to resist IPN-virus infections. cells contained large amounts of Mx protein [5, 9]. Moreover, the virus itself stimulates production of both Mx protein and ISG15 during infection of live salmon and salmon cells in vitro. These results indicate that ISA-virus either avoids or inhibits key antiviral proteins of the IFN system in salmon.

The adaptive immune system in fish Cells and central molecules The most important cells in the adaptive immune system are T cells and B cells (with their derived plasma cells responsible for antibody production). Antibodies recognise non-self structures on for example bacterial or viral sur- 145x100//Kap11-fig04.eps Figure 4. Effect of IPN virus infection on survival of salmon faces while T cells recognise foreign structures cells, which produce Mx protein continuously (ASMx1). IFN on the surface of other host cells. These foreign treated cells (IFN) were included as a positive control and structures might be parts of proteins that are di- non-treated cells (Control) were included as a negative control. The assay was carried out as described in Figure 2. gested by multi-protease complexes in a speci- Percent surviving cells three days after infection were fic manner and bound to a family of molecules calculated by measuring the light absorption at 550 nm known as major histocompatibility complex after staining of cells with crystal violet.

Thematic area: Health 189 T cell T cell CD4+ CD8+

T cell receptor- CD8 T cell receptor- complex complex

CD4 MHC II MHC I

Antigen presenting cell

145x100//Kap11-fig05.eps Figure 5. T cells recognize foreign non-self peptides displayed on other cells. The T cell receptor complex consists of a group of molecules indicated in blue. Nearly all cells in the body are capable of displaying internally derived peptides on their surfaces bound to MHC class I antigen (dark green). MHC class II antigens are expressed by many cells of the immune system (light green) and bind proteins endocytosed from their surroundings. Interactions between MHC class I and II antigens and T cells are supported by CD8 and CD4 co-receptors (violet and brown respectively)

(MHC) antigens. All cells constantly digest mune response in order to eradicate the foreign proteins, which may include non-self proteins if material. While MHC class I peptides are de- the cell is infected or has taken up foreign mo- rived from intracellular proteins, MHC class II lecules. The peptides are loaded onto MHC antigens display peptides that have been en- antigens and presented on the cell surface. docytosed from the extra-cellular environment.

MHC antigens are extremely polymorphic, Genes that encode antibodies, T cell receptors which means that the corresponding genes and MHC antigens represent some of the most show higher individual variation than any other complex genes studied. In fish there is much to gene families. This polymorphism accounts for be done before these genes are characterised in different capabilities displayed by individuals detail, but already we can say there are molecu- for the presentation of certain proteins. MHC lar and functional similarities between teleosts antigens loaded with peptide are transported to and higher vertebrates [10–13]. The enzymes the cell membrane where they are constantly and mechanisms responsible for the generation monitored by circulating T cells. Detection of of the almost infinite number of different anti- non-self proteins causes activation of the im- bodies and T cell receptors are also present in

190 Aquaculture Research: From Cage to Consumption fish together with the machinery surrounding Identification of immune genes in fish: a the MHC antigens. Thus many of the estab- rapidly developing field. lished characteristics of the mammalian im- In addition to salmonids other species such as mune system can be found in fish. fugu, carp, Japanese flounder and zebrafish have been prominent in the study of fish immunology during recent years. Several large-scale Fish have three classes of sequencing projects have been initiated, provid- immunoglobulin ing a better basis for identification of immune- Antibody molecules are immunoglobulins com- related genes. Mapping of gene positions has posed of light and heavy glycoprotein chains. A also made it possible to find many previously typical monomer consists of two identical light unidentified genes by their comparative loca- chains and two identical heavy chains, the latter tions in the genome (so-called synteny analy- of which determines the class and characteris- sis). tics of the immunoglobulin. The different classes can form monomers, dimers, trimers, tetramers, pentamers and even, hexamers. Salmonids: a duplicate set of genes. Due to a tetraploid ancestry, many genes in For a long time fish were considered to have a salmonid fish have two very similar copies. In relatively simple immune system with only one Atlantic salmon, for example, there are A and B class of immunoglobulin, IgM, whereas mam- sub-variants of IgM, IgD and (probably) IgT mals have IgG, IgD, IgA and IgE in addition. [18, 19]. The pseudo-tetraploid state of the ge- However, a new immunoglobulin was cloned nome in salmonid fish has been known for a from channel catfish and named IgD in 1997. long time, but the biologically consequences of The name IgD was proposed due to its sequence this phenomenon are not, so far, known. It has similarity with mammalian IgD and to its gene been speculated that salmonid fish with their localization i.e. adjacent to IgM. In addition anadromous life cycle have benefited from their IgD and IgM can be expressed simultaneously pseudo-tetraploidy to adapt more quickly to in the same cell, a unique property of IgD in various environmental conditions. mammals. Circulating IgD in the blood has only been found in channel catfish who express this soluble form of IgD from a gene remote from The cod immune system that coding for the membrane form albeit in the Atlantic cod has a unique position within the same gene complex. In other teleost fish only Norwegian fishing industry and the efforts in the membrane form of IgD transcripts has been establishing cod as an aquaculture species will reported [14, 15]. The function of IgD is still hopefully give this industry an extra dimension. unknown in fish. Interestingly, a third class of Many see great potential in cod farming, but the immunoglobulin has recently been cloned from road ahead is still long and several multi disci- bony fish, designated IgT since it is found only plinary research programs are needed before the in teleosts. IgT can be both membrane bound domestication can be fulfilled in a scale compa- and soluble and is most similar to IgM [16, 17]. rable to Atlantic salmon. The role of IgT is also unknown at the present.

Thematic area: Health 191 Variable“domain”/sites IgM & IgD Constant domains, IgM & IgD

IgD, IgD, cod salmon L-chain Active site H-chain

IgM, cod and salmon

Fc part

B cell B cell B cell

145x100//Kap11-fig06.eps Figure 6. Comparison of IgM and IgD in salmon and cod.

A research field that has received attention for that such studies could explain the species dif- some years are studies of the immune system in ference in antibody production [22]. The anti- cod, or more precisely, the antibody dependant body genes are relatively complex, but have the or humoral part of it. These studies started al- same organisation “pattern” in vertebrates as di- most 20 years ago, and a finding that surprised vergent as fish and mammals. In short, the anti- the researchers was the relatively low amount of body gene cluster contains several gene antibodies produced after immunisation of cod segments (V, D, J and C) with individual genes with various antigens. Compared to salmon, that are combined in a complete transcript that cod was clearly a non- or low-responder against is translated into the H- and L-chain of the anti- both bacterial and model antigens, although se- body molecule. What makes these molecules rum concentrations of immunoglobulins in non- special is that they possess so-called active sites immunised cod was approximately 10 times (see figure 6) which bind to particular antigen higher than in the Atlantic salmon [20, 21]. The structures / epitopes on the pathogens. In addi- reason for this “discrepancy” is not known in tion, the antibody “tail” or Fc part determines any detail but both regulatory and genetic issues the functions or immune mechanisms involved have been discussed. during infection and eventually humoral immune protection. A main issue has been to compare the antibody repertoire in cod with the one in salmon, hoping

192 Aquaculture Research: From Cage to Consumption Immunoglobulin classes

mammals IgM IgD IgG IgE IgA

birds IgM IgG IgA

amphibians IgM IgY IgX Class shift bony fish IgM IgD, IgT

cartilaginous fish IgM NAR, NARC

145x100//Kap11-fig07.eps Figure 7. IgM is the predominant antibody class found in fish, amphibians, birds and mammals. In teleosts IgM is found mainly as a tetramer whereas in higher vertebrates it is a pentamer. Previously it was thought that IgD occurred late in evolution due to a duplication of IgM but it is now known that IgD was present more than 400 million years ago. Several lines of evidence suggest that the modern Ig classes found in mammals are present as a result of the duplication of the IgD gene. The advent of “class switching” has been an important development of the immune system of higher vertebrates. This involves the switching of a specific IgM antibody production to IgG, IgA or IgE production with the same specificity in progeny cells. Recently a new class of immunoglobulin has been identified in teleosts, called IgT, which most closely resembles IgM.

The gene segments encoding the variable (V) Humans produce nine different antibody class- part of an antibody H and L chains contain es / subclasses, bony fish produce only two or about 100–150 genes (V, D and J genes), but the three (isotypes) characterised as IgM, IgD and ultimate diversity is composed of random re- IgT / IgZ (see above). combination of these genes (combinatorial diversity), in addition to various mutation The studies of IgD genes in cod demonstrated a mechanisms normally giving a functional diver- peculiar organisation of the IgD constant (Fc) sity of at least 109 antibody specificities. genes compared to the ones in salmon and other fish species [26]. It was further shown that this The low antibody responses in cod might reflect antibody class does not exist in a secreted/se- a limited number of functional V-genes encod- rum form, but only as membrane bound anti- ing antibody sites compared to the repertoire body receptors on B lymphocytes. This possessed by i.e. the salmon. However, scien- function of IgD is probably the same in cod and tists have now characterised and compared the salmon. The peculiar organisation of the IgD genetic variation of the antibody genes in cod constant genes in cod does not fully explain and salmon [23–25] and although differences why cod B-lymphocytes do not express a proper were shown, these variations cannot fully ex- IgD receptor diversity, because membrane plain the low responses in cod. bound IgM and IgD receptor molecules share the same antibody V-gene repertoire. Altogeth-

Thematic area: Health 193 er, the antibody gene repertoire in cod and salm- memory, which means that antigens / vaccines on B-lymphocytes seem similar and is probably “educate” the immune system to remember the not responsible for the diverse antibody re- antigen structure and mount stronger reactions sponses in these two species. when the antigen reappears i.e. during a secondary infection. Stimulation of B-lymphocytes As already mentioned, cod express much higher with protein antigens also includes stimulation natural immunoglobulin concentrations in of T-lymphocytes providing helper molecules blood than salmon [20, 21]. These immunoglo- (cytokines) important for B-cell proliferation bulins or antibodies do not, however, reflect a and differentiation into plasma cells. The T- response towards known antigens or pathogens cells express specific antigen receptors (TCR) from natural infection or immunisation. It is not encoded by genes related to the antibody genes known whether antigens such as bacterial com- and with a similar number of V-genes express- ponents taken up through the intestines may ing the TCR repertoire (see above). As the B- stimulate such production as is probably the and T- lymphocytes cooperate during an anti- case in mammals. The immunoglobulin produc- body response, it could be that a limited anti- tion happens either through a massive stimula- body response is caused by inefficient tion of the repertoire of specific (BCR) stimulation of T cells and a subsequent lack of receptors, or more likely, by the pan-B-cell or helper molecules important for B-cell stimula- mitogen receptors present on these cells. An in- tion and proliferation into antibody producing teresting question is, whether these “unspecif- cells. Although the mechanisms for T and B cell ic” antibodies protect against pathogenic cooperation in cod and other fish species have infections and represent a “natural” immune not been studied in sufficient detail, the genes strategy in cod? As haddock and coalfish are encoding the cod TCR are well characterised. also low antibody responders, this may reflect a Neither the diversity nor the organisation of the common “strategy” of the gadoid species. Fur- cod TCR genes differ substantially from the ther investigations are needed to verify this “hy- ones in other fish species, including salmon. As pothesis”. also mentioned above, a third cell type, the antigen presenting cell (APC; macrophages, den- It should also be mentioned that the low respon- dritic cells etc.) plays an important role in siveness in cod is not absolute, as recent data immune stimulation by presenting antigen pep- have shown that the bacterial fish pathogen tide fragments to the T–cell receptor. The APC Aeromonas salmonicida expresses antigens that phagocytose (“eat”) antigens / pathogens and to some degree stimulate antibody production process protein antigens into peptides which are [27]. Since the antigen “nature” is unknown, it then bound to the MHC molecules, and present is too early to speculate about tentative mecha- this peptide-MHC complex to the TCR on T- nisms of this stimulation. lymphocytes. Although MHC molecules exist as two classes (MHC I and MHC II), only the Stimulation of B cells and antibody production class II is involved in stimulation of the T-lym- is only one of several immune reactions against phocytes and helping the B-cell in antibody pathogens and belongs to the so-called “adap- production. Thus, if cod lack or possess crip- tive” arm of the immune system. These mecha- pled MHC II molecules, stimulation of cells in- nisms include immunological education and volved in antibody responses will be subopti-

194 Aquaculture Research: From Cage to Consumption mal and result in low levels of specific anti- Prospects and challenges bodies [28]. In support of this idea, several The molecular characterisation of the fish im- research groups searching for MHC genes in mune system is important for future applica- cod have so far discovered only the MHC I tions not only for improvement of vaccination, class, but a final conclusion in this issue awaits but also for description of various types of im- further gene screening, eventually genomic se- mune responses of fish to different stimuli. The quencing. regulation of immune genes is both dependent on the type of pathogen that the animal encoun- Over the years many vaccine experiments have ters and on the physiological and immunologi- been performed against bacterial pathogens cal state of the animal. In human medicine causing cold-water vibriosis (Vibrio salmonici- blood tests have long been used to trace im- da), classical vibriosis (Vibrio anguillarum) and mune responses that might indicate early devel- atypical furunculosis (atypical Aeromonas sal- opment of disease. One example is the test for monicida). The overall conclusion is that vacci- C-reactive protein, which is an indicator of bac- nated cod is protected against disease to the terial infection. So-called “immune profiling” same degree as vaccinated salmon [29]. The may also be applied within aquamedicine in the mechanisms involved in this protection are not near future. Mx, IFN and other molecules may known, but commercial vaccines are now under be important as molecular markers for the way and will hopefully be as valuable as vac- health status of the fish. Such markers may be cines developed for the salmon farming indus- used to, reveal conditions that down-regulate try. the immune system of the host and indicate how to stimulate the defence against viruses during The lack of specific antibodies after immuniza- critical stages of production. Information about tion of cod has challenged scientists for almost immune genes may also be useful for breeding two decades, and has also led to studies of the programs. innate arm of the fish/cod immune apparatus. The best studied molecule so far is the so-called The research on the IFN system of Atlantic “Bacterial Permeability Increasing Protein”, salmon has revealed strengths and weaknesses BPI [30] and later also the lysozyme C mole- in the innate immune defence against IPN- and cule. These molecules are well known from ISA-virus, two of the most important viral other species to protect against bacterial infec- pathogens in the Norwegian aquaculture indus- tions, and presumably also in cod. The search try. Future experiments will study how these for “new” molecules and potential immune viruses counteract the IFN-system of salmon. mechanisms against bacterial infections in cod continue, and hopefully, the future cod farming From mammalian research it is well known that industry will benefit from this research to the the IFN-system collaborates with the adaptive same extent as for established aquaculture spe- immune system to combat infection. IFN and cies. IFN-stimulating compounds can function as adjuvants by augmenting the protective effect of vaccines. This area of research also deserves more attention in aquamedicine. So-called CpG-oligonucleotides represent one of the most

Thematic area: Health 195 promising vaccine adjuvants and may also be mammals. Although methods for measuring beneficial in fish vaccines since recently they antibody responses have been available for a have been shown to induce Mx protein and prolong time, methods for measuring T cell medi- tection against IPN-virus infection in salmon ated immune responses in fish are still lacking. [30]. The importance of continued research on the adaptive immune system of salmon and cod is The research on fish antibodies has made good underlined by the fact that protection obtained progress during the last 30 years. Research has by vaccination does not always correlate with also established that fish have a T cell mediated the concentration of circulating IgM antibodies. adaptive immune system similar to that of

References

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196 Aquaculture Research: From Cage to Consumption [16] Hansen JD, Landis ED, Phillips RB. Discovery of [24] Hordvik I, De Vries Lindstrom C, Voie AM, Lily- a unique Ig heavy-chain isotype (IgT) in rainbow bert A, Jacob J, Endresen C. Structure and organi- trout: Implications for a distinctive B cell develop- zation of the immunoglobulin M heavy chain genes mental pathway in teleost fish. Proc Natl Acad Sci in Atlantic salmon, Salmo salar. Mol Immunol USA 2005; 102, 6919–6924. 1997; 34, 631–639. [17] Danilova N, Bussmann J, Jekosch K, Steiner LA. [25] Solem ST, Hordvik I, Killie JA, Warr GW, Jørgen- The immunoglobulin heavy-chain locus in ze- sen TØ. Diversity of the immunoglobulin heavy brafish: identification and expression of a previ- chain in the Atlantic salmon (Salmo salar L.) is ously unknown isotype, immunoglobulin Z. Nat contributed by genes from two parallel IgH isoloci. Immunol 2005; 6, 295–302. Dev Comp Immunol 2001; 25:403–417. [18] Hordvik I. The impact of ancestral tetraploidy on [26] Stenvik J, Jørgensen TØ. Immunoglobulin D (IgD) antibody heterogeneity in salmonid fishes. Immu- of Atlantic cod has a unique structure. Immunoge- nol Rev 1998; 166, 153–157. netics 2000; 51, 452–461. [19] Hordvik I, Berven FS, Solem ST, Hatten F, En- [27] Lund V, Bordal S, Kjellsen O, Mikkelsen H, Schro- dresen C. Analysis of two IgM isotypes in Atlantic der MB. Comparison of antibody responses in At- salmon and brown trout. Mol Immunol 2002; 39, lantic cod (Gadus morhua L.) to Aeromonas 313–321. salmonicida and Vibrio anguillarum. Dev Comp [20] Espelid S, Rødseth OM, Jørgensen TØ. Vaccina- Immunol. 2006;30:1145–55. tion experiments and studies of the humoral im- [28] Pilstrøm L, Warr GW, Strømberg S. Why is anti- mune responses in cod, Gadus morhua L., to four body response of Atlantic cod so poor? Fisheries strains of monoclonal-defined Vibrio anguillarum. Science 2005;30: 961–971. Journal of Fish Diseases 1991; 14, 185–191. [29] Schrøder MB, Espelid S, Jørgensen TØ. Two sero- [21] Israelsson O, Petersson A, Bengtén E, Wiersma EJ, types of Vibrio salmonicida isolated from diseased Andersson J, Gezelius G, Pilström L. Immunoglo- cod (Gadus morhua L.); virulence, immunological bulin concentration in Atlantic cod, Gadus morhua studies and vaccination experiments. Fish and L., serum and cross-reactivity between anti-cod- Shellfish Immunology 1992;2: 211–221. antibodies and immunoglobulins from other spe- [30] Stenvik J, Solstad T, Strand C, Leiros I, Jørgensen cies. Journal of Fish Biology 1991; 39, 291–302. TT. Cloning and analyses of a BPI/LBP cDNA of [22] Solem ST, Stenvik J. Antibody repertoire develop- the Atlantic cod (Gadus morhua L.). Dev Comp ment in teleosts-a review with emphasis on salmo- Immunol 2004;28: 307–323. nids and Gadus morhua L. Dev Comp Immunol [31] Jørgensen JB, Johansen LH, Steiro K, Johansen A. 2006; 30, 57–76. CpG DNA induces protective antiviral immune re- [23] Stenvik J, Schrøder MB, Olsen K, Zapata A, Jør- sponses in Atlantic salmon (Salmo salar L.). J Vi- gensen TØ. Expression of immunoglobulin heavy rol 2003;77: 11 471–11 479. chain transcripts (VH-families, IgM, and IgD) in head kidney and spleen of the Atlantic cod (Gadus morhua L.). Dev Comp Immunol 2001; 25, 291– 302.

Thematic area: Health 197 198 Aquaculture Research: From Cage to Consumption Feed, Nutrition, Feeding

■ Nutritional Biology in Farmed Fish

■ Nutritional Aspects – Marine Fish Larvae

■ Feed Resources – Feed Technology

■ Metabolism and Uptake of Carotenoids in Farmed Fish Olai Einen1), Henriette Alne1), Barbara Grisdale-Helland1),2), Ståle J. Helland1),2), Gro-Ingunn Hemre3), Bente Ruyter1), Ståle Refstie1),2) and Rune Waagbø3) 1) Akvaforsk –The Institute for Aquaculture Research AS, 2) Aquaculture Protein Centre, 3) NIFES (Norwegian Institute of Fisheries and Aquaculture Research)

Nutritional Biology in Farmed Fish

Correct nutrition of farmed fish is vital to cost-effective production of healthy, high-quality food. Feed is the greatest single cost in fish farming, and as efficiency increases, feed costs become even more important. With the quantities produced by the aquaculture industry today, relatively small changes in feed composition and feeding regimens can generate substantial benefits. Many aspects of the appetite of fish and their ability to utilise feed for growth are still unknown. The demand for development of more cost-efficient feed means that we have to gain a fundamental understanding of the nutritional needs and tolerance limits of farmed fish. There are increasingly new requirements for feed and raw materials for feed, creating a great need for research in nutritional biology. As more species are becoming relevant for aquaculture, it is important to build specialised expertise, as the needs and preferences of each species can vary greatly. For this reason comparative nutritional studies have been done in recent years. The Norwegian Research Council’s Aquaculture programme, which has now been concluded, has placed particular focus on salmon and cod;, this is reflected in this chapter.

200 Aquaculture Research: From Cage to Consumption This chapter presents new knowledge in nutri- over the significance of the variation parameters tional biology relating to feed composition, in experiments and their effects on feed utilisa- feeding regimens, absorption, metabolism and tion. fish health. Atlantic salmon and cod are discussed in particular, as these species have been Differences between species? the focus of projects on nutrition carried out Are there differences in the feed intake between under the Aquaculture programme during the species under the same conditions? This ques- programme period. Results from these projects tion was studied in an experiment that com- have been included, as have results from strate- pared feed intake and growth in salmon, gic institute programmes and projects funded rainbow trout and cod; the groups had the same by the Fishery and Aquaculture Industry Re- starting weight (450 g) and the same environ- search Fund (FHF) for which the Aquaculture mental conditions. The feed intake was highest programme has been responsible, or in which it in the salmon and somewhat lower in the trout, has taken part. Subjects such as gene-modified while the cod only consumed half of the aver- raw materials, contaminants, pigmentation and age intake for salmon and trout. When compar- larval nutrition are discussed in separate chap- ing the three species, we observed that the cod ters in this book. partly compensated for its lower feed intake with greater nutrient utilisation. However, the level of nutrient utilisation was not high enough Feeding and feed intake to account for the differences in growth related The growth potential of fish differs from spe- to the appetite of the fish. It was expected that cies to species, but is highly dependent on tem- nutrient digestibility would be somewhat re- perature, size and genetic origin. The degree to duced in the cod when the feed intake was in- which this growth potential is realised is highly creased. This was demonstrated in salmon, but dependent on feed intake and on how well the the effect on cod was marginal. A comparison feed has been adjusted to the nutritional needs of feed utilisation results from several experi- of the fish. Maximum growth is usually ob- ments with Atlantic halibut, cod, salmon and tained when the fish are fed until the point of trout indicates that this parameter is relatively satiation. In earlier years, it was difficult to similar in small fish (Figure 1). It appears, how- accurately measure feed intake in nutritional ever, that there are differences between the spe- experiments in tanks and net pens, and it was cies when fish of different species, but the same even more difficult in practical fish farming. In weight, are compared in the same experiment. recent years, feed intake measurement has be- In the comparison of trout, salmon and cod come a standard routine in many nutritional ex- mentioned above, the feed utilisation was 30– periments, and has proven to be a useful tool. 40 per cent high in the cod than in the salmon Feed intake is quantified by measuring the and trout (Figure1; centre ellipse). There were amount of feed given, minus the accumulated no differences in feed utilisation between the feed waste after the fish have been fed in surplus salmon and the trout in this experiment, but (10–20 per cent overfeeding). In practical fish other experiments indicate that salmon utilise farming, feeding surveillance systems based on feed 20–30 per cent better per kg growth than video, sensors or collection of feed waste have trout (Figure 1; left and right ellipse). been implemented. This has given better control

Theme: Feed, Nutrition, Feeding 201 1.7 Trout Salmon 1.5 Halibut Cod 1.3

1.1

0.9

Feed conversion ratio

0.7

0.5 0 200 400 600 800 1000 1200 Mean weight in experiment, g

145x100//Kap12-fig01.eps Figure 1. Feed conversion ratio in farmed fish of different sizes; ellipses represent values from the same experiment.

How does energy content in the feed Vegetable protein ingredients contain a high affect the feed intake? percentage of indigestible carbohydrates (30– To compare the effect on feed intake caused by 50 per cent) in the form of oligosaccharides and the energy concentration in the feed, rainbow different fibre fractions, giving a lower concen- trout, salmon and cod were given two different tration of digestible energy in the feed. In experi- feeds – one feed with a medium energy level ments with rainbow trout and cod it has been and high protein level (22.6 MJ and 60 per cent shown that the fish can compensate for the protein) and one feed with a high energy level lower energy level in the feed by increasing the and medium protein level (24.9 MJ and 53 per feed intake. In some experiments with cod, the cent protein). The feed intake in the salmon and proportion of gastrointestinal tract to body trout was higher with high-energy feed than weight has increased as a consequence of long- with medium-energy feed, but the cod con- term use of soybean meal in the feed, whereas sumed about the same amount of each of the other experiments have not revealed the same two feeds. It seems the salmon and the trout tendency. It has also been shown that cod digest regulated the feed intake so that the protein in- vegetable protein better than salmon. The take per kg fish for the two feeds was the same. growth rate in both cod and salmon is sustained The cod, however, did not show any obvious re- because the fish compensate for poorer diges- sponse to the energy or protein levels in the tion with higher feed intake. This leads to re- feeds, consuming about the same amount of duced feed utilisation when the percentage of both feeds. vegetable protein in raw materials increases, i.e. a higher feed conversion ratio and lower reten-

202 Aquaculture Research: From Cage to Consumption tion of protein and energy. Cod seem to be more Do high energy feeds give the same flexible than salmon and trout in adjusting feed results in salmon and cod? intake and sustaining good digestion of low- As mentioned above, differences in feed intake cost vegetable raw materials in the feed. were found in salmon, rainbow trout and cod that were given feeds with a medium or high How often should the cod be fed? energy content. In this comparison of the three Feeding regimens are important in fish farming species, no difference was found in the salmon in order to optimise the feed intake of the fish in and the trout’s retention of amino acids (per- relation to feed utilisation and fish growth. centage of the intake of amino acids) when There has been much discussion on how often given a medium energy and a high energy feed. cod should be fed, and whether feeding fre- However, the cod, the fish fed a high energy/ quency affects feed intake and fish growth. In medium protein diet retained 45 per cent more an experiment where young cod were held at a essential amino acids than the fish fed a me- water temperature of 8°C, no significant differ- dium energy/high protein diet with. In this ex- ences in growth were found between groups periment, the salmon retained 17 per cent more that were fed to satiation every day and groups of the digested energy than the trout, while the that were fed until satiation every other day. retention of digestible energy in the cod was no Moreover, the feed conversion ratio and liver different than in the other two species. The index were not affected by the various feeding comparison between the three species showed regimens used in this study. that the cod retained on average 14 per cent more of the digested protein than the salmon, and 34 per cent more than the trout. Feed composition In recent years, there has been increased aware- What is the optimal feed composition for ness of the scarcity of marine raw materials cod? such as fish meal and fish oil. There is no longer An experiment with Atlantic cod (from 200 to a feed quota regime in Norway for salmon approx. 850 g) showed that growth was nega- farming, which gives greater opportunities for tively affected by low protein and high starch using new raw materials in feed. This, in turn, concentrations in the feed. The fat content, gives rise to a new demands for knowledge however, had no significant effect on growth. about the nutritional needs of fish and their util- Feed utilisation was improved by increasing the isation and conversion of each nutrient. Good amount of fat and protein in the feed, while it feed utilisation is a prerequisite for good was reduced by increasing the concentration of growth, and is also related to a high retention of starch in the feed. One challenge with farmed protein and fat in salmon. It is important to note cod is unwanted deposits of fat in the liver, that feed manufacturing companies have carried which raises the liver index (liver weight to out a considerable amount of unpublished re- body weight ratio). The liver index rose with a search on feed composition in order to develop higher content of fat in the feed, and fell with a new feeds and feed concepts; this is not dis- higher content of protein. The level of starch in cussed here. the feed, however, had an insignificant effect on the liver index. The conclusion is that in order to obtain good growth and protein retention,

Theme: Feed, Nutrition, Feeding 203 avoid a high liver index and achieve low feed the feed can, without problem, contain >200 g conversion ratios in young cod, the feed should fat/kg feed and <120 g starch/ kg feed. There contain – according to the results in this experi- were no advantages gained by increasing the ment – 500–600 g raw protein/kg feed, 130–200 protein level to over 510 g/kg feed. g fat/kg feed and <150 g starch/kg feed. Should the feed be adjusted to the In another experiment in which cod were al- season? lowed to grow from 125 to 250 g, growth was Large seawater salmon have a seasonally deter- not greatly affected by feed composition. Four- mined rhythm of energy use from feed in rela- teen different feeds, containing 45–60 per cent tion to their own energy depots. These natural protein, 10–27 per cent fat and 5–21 per cent seasonal rhythms are also found in farmed starch, were compared. Feed utilisation was, as salmon which are fed year-round according to in the experiment above, reduced when the con- appetite with a well-balanced feed, even when centration of starch in the feed was increased. continuously exposed to simulated daylight. Feed utilisation was improved when the amount The salmon become leaner in spring and grow of protein in the feed was increased; however, extensively in the summer and autumn, accu- the results indicated that this improvement only mulating large lipid deposits. Due to the annual continued until a certain level of protein in the variation in salmon body energy stores, we feed was reached. The liver index rose from 7 to aimed to discover if, by manipulating the 13 per cent when protein was replaced by fat in energy density of the feed contrary to the the feed. The liver index fell when there was a salmons’ natural annual rhythm (i.e. by feeding higher content of ash in the feed. The conclu- the fish a lipid-rich “spring” feed and a lean sion in this experiment was somewhat different “autumn” feed), we could achieve more uni- from the experiment above; in order to obtain form fillet quality year-round.. If such feed good growth and feed utilisation in young cod, could guarantee invariable fillet quality, it

Growth 9000 Feed Conversion Ratio (FCR) 1,41.4 8000 1,21.2 7000 1,01.0 6000 0,80.8 5000 0,60.6 4000 0,40.4 3000 0,20.2 2000 ÅrstidDirect KontrollControl 0 1000 SesongSeason SpringVår Sommer Summer HøstAutumn VinterWinter start spring vår sommersummer høstautumn vinterwinter

145x100//Kap12-fig02.eps Figure 2. The Direct group was given feed that varies with the marine raw materials’ protein-energy composition (P/E). (The raw material was filleting waste from herring.) The control group was given feed with a constant P/E ratio. The Season group was given an energy-dense spring feed and a lean autumn feed. The figure to the left shows growth development with samples taken in March (start), May (spring), August (summer), November (autumn) and February (winter). The Season group was almost half a kilo heavier than the Direct group after one year in the sea. The figure to the right shows FCR values (g dry feed eaten/g growth) for the three feeding groups: Direct, Control and Season, measured in the period March-May (spring) June-August (summer), September-November (autumn) and December-February (winter). Red=Direct, yellow=Control, blue=Season.

204 Aquaculture Research: From Cage to Consumption would have a competitive advantage on the retention was achieved in summer by feeding a market. This group was given the acronym Sea- lipid-rich (44 per cent) feed with a medium son A second group of triplicate sea cages were amount of protein (44 per cent). There were fed a diet that was the opposite of the Season also differences in growth rate between dietary diet. This diet was designed to be lean in spring treatments during the various seasons. Total and lipid rich in autumn, following the natural growth throughout one year of feeding was variation in energy deposits in herring catches. from 1.7 to 7.6 kg for the fish in group Season. This group was given the acronym Direct. Her- ring trimmings were used as a major source of lipids and protein in the diets. The control diet Digestion and absorption did not vary in composition throughout the four Knowledge about how the digestive system seasons, corresponding to a high-energy salmon works and how different nutrients behave in the feed with a composition similar to that of a gastrointestinal tract is critical to optimising the commercial feed. The control diet was included utilisation of new raw materials in feed. There in order to compare data from the experimental are also significant differences between species; situations against data from a normal situation. for example, Atlantic salmon and rainbow trout The fish were fed according to appetite, two have short gastrointestinal tracts, whereas cod meals a day until satiation. There were slight have a considerably longer one. In recent years, differences in growth in the course of a year; several studies have generated new knowledge however, the fish fed the Season diet were 0.5 in this area, and are a result of, among other kg larger on average compared to the fish fed things, newly developed methods which makes the Direct diet. Feed utilisation also varied such studies possible. throughout the year (Figure 2). The results illustrate that when salmon are fed according to ap- Digestion of proteins petite, they adjust their intake of energy and In fish the majority of amino acids are absorbed protein themselves. For fish farmers, the mea- from digested protein as di- and tri-peptides ac- surable differences related to production data tively transported through the membranes of in- first and foremost concern the amount of feed testinal cells. Oligo-peptides are furthermore necessary to achieve the same growth. It is pos- substrates for enzymes – aminopeptidases – in sible to use less feed by giving fish energy- the intestinal mucosa. It appears that young fish dense feeds in the spring and summer, when the can grow almost normally when amino acids diet richest in energy gives the best feed utilisa- are provided in the form of dipeptides, contrary tion compared to the two other diets. In spring to when they are provided as free amino acids. the feed conversion ratio (FCR) varied from Hence, the need for amino acids in young fish 0.58 to 1.18, and in summer it varied from 0.83 can be determined by using feeds in which lim- to 1.12. The control diet was utilised better than ited amino acids are provided in the form of di- the Season diet during spring, but worse during and tripeptides as substrate for specific peptide summer. The salmon showed the highest transporters. This also has consequences for op- growth rate in the fall; this was also the season timisation of the amino acid profile in feed for during which dietary energy levels did not af- fish larvae with incompletely developed diges- fect feed utilisation, within a variation in fat tion channels (see separate chapter). levels of 27–44 per cent. The highest protein

Theme: Feed, Nutrition, Feeding 205 Figure 3. Methodology for studies of uptake of fat in the intestine and liver. In recent years, a method for quantifying fat in the intestine and liver has been established. Thin cuts of intestine and liver tissue are stained with toluidine blue and alcaline fuchsine. Afterwards the drops of fat appear with a green colour against cytosol, which appears violet or pink under light microscopy. The fat can thus be quantified by digital image analysis. The method has proven to be useful for studying how water temperature and fat content in feed affect deposition of fat in the intestine and liver. (Photo: A. Vegusdal/Akvaforsk)

145x100//Kap12-fig03.eps

Anti-nutrients from vegetable protein raw mate- tion in the mucous membrane of the hindgut of rials present challenges with regard to distur- Atlantic salmon. At the same time, the activity bances in the digestive processes. In plants of enzymes in the intestinal mucous membranes these substances are a heterogeneous group of of the brush border is reduced, depending on the structural components and/or chemical de- amount of soybean meal in the feed. Active fences against herbivores. Anti-nutrients are transport of amino acids into the enterocytes de- therefore often found in low-refined vegetable creases, and the activity of cytosolic enzymes in ingredients, for example soybean meal, an inex- the intestinal mucous membrane is reduced, pensive and easily accessible protein source. which indicates reduced super nuclear forma- Soybeans and other legumes contain proteins tion of vacuoles and therefore reduced absorp- which bind themselves covalently to the active tion of intact macro molecules (proteins) by sites in pancreas proteases such as trypsine and endocytosis. All in all, this will reduce the fish’s chymotrypsine. In vitro experiments have ability to digest and absorb proteins. This seems shown that phytic acid (myo-inositol-6-phos- to be due to an increased exchange of cells in phate) also inhibits the activity of both cationic the intestinal mucous membranes surface as a and anionic salmon trypsine. Moreover, these response to the intestinal inflammation. The in- anti-nutrients inhibit the proteolytic activity in testinal mucous membrane of the fish is strong- the intestinal contents, which in turn leads to in- ly folded, but has no villi. creased loss of proteases through the faeces. This shows that high levels of soybean meal in With soya in the feed, the number of proliferat- the feed leads to depletion of enzymes containing cells on the surface of the folds increases. ing granules in exocrine pancreas cells in rain- These immature cells have reduced function be- bow trout. Soybean meal also contains cause of a lower number of functional proteins unidentified anti-nutrients that cause inflamma- (enzymes, transporters etc.) in the apical cell

206 Aquaculture Research: From Cage to Consumption membrane and/or other glucosylation profile in tine and transport to different tissues. The pyloric membrane-bound receptors. caeca area appeared to be the most important area for absorption of both short and long Corresponding intestinal changes were not chained fatty acids, but longer fatty acids were found in cod in an experiment in which the fish absorbed in more parts of the intestine, including were given feed with up to 44 per cent soya and large parts of the middle intestine. Both short and maize gluten. Plant protein mixtures up to 44 long chained fatty acids were principally drained per cent did not affect the capacity for enzym- from the intestine via portal blood, and trans- atic hydrolysis of protein and absorption of ported directly to the liver. In the liver, the fatty amino acids in any part of the cod intestine, but acids were incorporated into lipoproteins before did reduce the digestibility of fat and led to in- further transport. Significant amounts of the long creased microbial activity and changes in chained fatty acids from the feed were directly microflora in the intestine. Reduced digestibili- deposited in visceral fat depots. ty of fat with vegetable proteins in the feed was also observed in salmon. When partly digested fat is incorporated into the enterocytes, it has to be reassembled into Digestion, intake and transport of fat triglycerides before it can be transported to the Despite the fact that today’s commercial high- organs. A concrete question studied during the energy feeds can contain up to 40 percent fat, programme period was whether fish have a high little is known about how the various fatty acids activity of monoacylglycerol acyltrasferase from different sources affect the absorption pro- (MGAT), the pathway of re-synthesis of triglyc- cess along the digestive tract. Furthermore, erides after the fat has been digested in the in- little is known about how different fatty acids testine and absorbed. Initially, it was assumed are converted in intestinal cells (enterocytes) that the fish could have two mechanisms for and thereafter transported in lipoproteins to dif- synthesis: the MGAT pathway and the glycerol ferent tissues, to be used for combustion, meta- 3-phosphate (GPAT) pathway. The MGAT bolic conversion and/or storage. Increased pathway is very fast, demands little energy and knowledge in this area is especially important is dominant among mammals. The alternative because new dietary fat sources will be used to GPAT pathway is energy-intensive and rela- a greater extent in commercial feeds. tively slow. The problem with the MGAT pathway is that it requires a partly digested There is little knowledge about how lipids are triglyceride, 2-monoacylglycerol (2-MAG), as transported from the fish’s intestine to the tissues the first building block. Earlier studies have where they are to be utilised. Therefore, one ob- shown that fish possibly do not have the diges- jective has been to obtain a fundamental under- tive enzyme that creates 2-MAG, but probably standing of absorption of fat in different parts of have a less specific enzyme which creates all the intestine and further transport of fatty acids kinds of products. Thus, it was also considered with different chain lengths to different tissues in a possibility that the MGAT pathway was not salmon. By feeding salmon with isotope-marked active in fish. A partial characterisation of the fatty acids of short (C10) and long (18:1) chain enzyme MGAT (+DGAT) from salmon micro- lengths, it has been possible to follow the fatty somes did, however, show that the enzyme was acids’ absorption into various parts of the intes- very active in the intestine. A further compari-

Theme: Feed, Nutrition, Feeding 207 son of the two alternative pathways of synthe- tinal lumen and fixed bacteria associated with sis, using entire intestinal segments, also the intestinal epithelium in a number of farmed indicated that the MGAT pathway is far more species, among them salmon and cod. The fixed active than the alternative GPAT pathway. It is bacteria are associated with the viscous “car- also possible that these two pathways of synthe- pet” of hydrated mucines which are excreted sis have different functions in the cells. Further- from the intestinal epithelium to protect the sur- more, it was shown that the fat from the diet can face of the enterocytes against the intestinal be used directly for creating phospholipids in lumen. For the time being, it is unknown to the enterocytes (CPT), probably for direct use what degree intestinal flora contribute to micro- in the lipoprotein synthesis, but also for direct bial digestion in fish. incorporation into membranes. Therefore, this makes the enterocytes very vulnerable to The intestinal flora in fish are sensitive to stress, changes in the composition of the diet, and antibiotics and changes in feed composition. It symptoms of badly balanced fat mixtures will has been shown that different types of oils and often first manifest themselves here. There was, protein meals affect and change the intestinal however, little that indicated that the composi- flora in fish. Low stability in the microflora tion of the diet had any effect on the activity of might have consequences for the fish’s resis- these enzymes (MGAT, DGAT, CPT). tance to disease, as it can result in the growth of pathogen bacteria which damage the intestinal It is known that the fat from the liver is princi- epithelium at the expense of neutral and/or pally transported in lipoproteins in the blood of favourable bacteria (for example, lactic acid the fish, but there is not much knowledge about bacteria). Pathogens may also be absorbed synthesis, secretion, transport, reception and re- through the intestinal mucosal membrane by moval of the different lipoproteins. To provide endocytosis, as shown in salmon with electron improved analytical tools for identification and microscopy. This indicates that the intestine is quantification of lipoproteins at the apo-lipo- one of the main routes of infection in fish. protein and lipoprotein levels, NIFES has developed polyclonal antibodies against HDL and The use of molecular methods for characterisa- VLDL from salmon. A method for quantification of the micro biota in fish and how it is af- tion of vitellogenine in salmon plasma has also fected by feed and water environment will been developed. therefore be important. This will pave the way for a more correct use of raw materials in feed, Intestinal flora as well as for promoting the growth of favour- Like other animals, fish have bacterial flora in able microflora through feed or water (probiot- the intestinal channel. This micro biota consists ics) as a prophylactic measure. of aerobe, facultative anaerobe and obligate anaerobe bacteria, but the composition varies with age, nutrition and water environment. The From intestine to muscle and fat amount of bacteria increases along the intestine tissue: conversion of nutrients and most is found in the distal part of the intes- When the feed is digested and absorbed, the nu- tine. Using electron microscopy this has been trients are distributed via the blood to the liver, shown with both transient bacteria in the intes- muscles and other tissues. Here the nutrients are

208 Aquaculture Research: From Cage to Consumption deposited, combusted or metabolised into other high-quality protein source grow more rapidly substances, which in turn can be excreted and show both increased synthesis and in- through the gills or urine, or transported back to creased degradation. In other words, they have the intestine. The way in which and the efficacy a higher total metabolism of protein than slower with which the different nutrients are converted growing individuals. Synthesis of protein costs are important to feed utilisation, growth, health the same for the two types of fish, but the fish and the quality of the final product. The access to that retain more of the synthesised protein have nutrients often controls the expression of genes a net gain which is higher than the fish that has which are essential to the metabolism of nutri- a higher decomposition rate. ents, such as enzymes, growth factors and factors that are important for the fish immune system. Previously, there has been little research done on how the amino acid balance affects the growth Protein metabolism process in fish. Protein metabolism, and with it In the body of fish, synthesis and degradation of growth, is modulated by a series of nutrients protein is ongoing. Most studies show that pro- which behave as individuals or through interac- tein synthesis increases together with increases tions with other nutrients or hormones. Interac- in protein intake, up to a maximum level (de- tions occur when one nutrient directly affects the pendent on genetic, environmental and nutri- utilisation of another nutrient, either on an ab- tional conditions). The linear proportion which sorptive level or on a cellular level. This can have is found between protein intake and synthesis is a positive or negative influence on fish growth. explained by the fact that the feed’s amino acids stimulate protein synthesis to different degrees. Results from experiments have shown a correla- By measuring feed intake and protein synthesis, tion between the concentration of polyamines in it has been found that approximately 70–80 per muscle and growth. The polyamines spermidine cent of a measured growth variation can be ex- and spermine are growth-regulating compounds plained by variation in feed intake. The degra- that are related to cell division. These are syn- dation rate of protein will also strongly affect thesised from the amino acid arginine, but the the growth rate; it is uncertain whether this is process is also dependent on a form of the dependent on feed intake. Some individuals amino acid methionine. The amino acid lysine grow rapidly, while others grow slowly with the can give reduced availability of arginine. An same amount and type of feed. Thus, in the imbalance in content and availability of argin- aquaculture industry there is the potential for ine, lysine and methionine in the feed can there- selective breeding of individuals with both a by affect the growth process in fish. Inclusion of high growth rate and good feed utilisation. Rap- more than 16 g lysine per kg feed in an experi- idly growing individuals seem to store a larger mental feed based on wheat gluten gave re- share of the synthesised protein than individuals duced growth, most likely because of the with a lower growth rate. When protein meta- lysine’s effect on arginine metabolism. Taurine bolism is measured in order to investigate the in salmon feed has to some extent resulted in quality of a protein source, it is necessary to improved growth and feed utilisation as long as give standardised amounts of feed and energy the methionine level in the feed is low. The because both these factors affect the synthesis branched amino acids leucine, iso-leucine and and degradation rates. Fish that are given a valine have appeared to have a modulating

Theme: Feed, Nutrition, Feeding 209 effect on protein metabolism in fish. An imbal- need for arginine for maintenance and growth. anced intake may possibly affect the meta- This finding also strengthens the hypothesis bolism of branched amino acids as well as the that the urea cycle is expressed in fish larvae protein synthesis capacity in muscle tissue, and fry, and is significant in order to remove which in turn can affect the growth process. In ammonia, which is a toxic end-product created an experiment with salmon, it was shown that through the degradation of amino acids. The ex- these three branched amino acids had an inhib- periment shows the importance of developing itive effect on each other’s reception in the in- experimental dry feeds for larvae in order to de- testine, measured in vitro. termine the quantitative need for arginine and other amino acids in the feed. Such experiments There is little known about the need for arginine can contribute to reduced mortality and in- and other amino acids in the feed in the early creased growth in larvae and fry in culture. life stages of fish larvae and fry. Contrary to adult fish, the enzymes in the urea cycle of lar- Degradation of nucleic acids is also associated vae and fry are often measurable. This cycle with urea production in fish. In humans, overly can, in theory, take part in producing the essen- high levels of nucleic acids in the diet can lead tial amino acid arginine. It is therefore possible to gout. BioProtein (based on bacteria cultiv- that the need for arginine in fry can be covered ated on natural gas) is a product with a high by endogene synthesis. In an experiment with content of nucleic acids (approx. 10 per cent), trout under initial feeding, feed in which the and it was therefore of interest to investigate protein had been replaced with di-peptides (two how salmon handles such relatively large amino acids chained together) was used. One amounts. A central enzyme in the metabolism feed contained di-peptide arginine, while an- of these substances is urate oxidase; mRNA for other contained no arginine. The results were this enzyme was first isolated and sequenced in clear: trout do need arginine in the feed during several fish species. The sequence was then initial feeding, and any synthesis of arginine in used to make a probe in order to measure the the fry themselves is not sufficient to cover the regulation of this enzyme. These results, to-

Figure 4. A method for cultivating fat cells from fish in culture has been established to improve the understanding of development of fat tissue and deposition of fat in fish. The method makes it possible to follow the development of fat tissue step by step, from pre-adipocytes, which are the precursors of the fat cells, to mature fat cells (adipocytes). The method is used in studies to see how high-energy feeds affect the deposition of fat in Atlantic salmon. Using this method, the presence of PPAR (left), C/EBP (centre), leptin and cPLA2 is shown. The accumulated amount of intracellular lipids is a good indicator of the degree of cell differentiation: oil red-O colours drops of lipid red (right). Results achieved using the method indicate that the development of fat tissue in salmon is regulated in the same way as it is in mammals. (Photo: A. Vegusdal/Akvaforsk)

145x100//Kap12-fig04.eps

210 Aquaculture Research: From Cage to Consumption gether with traditional enzyme measurements decyl-thio-acetic acid (TTA) in the salmon feed and measurement of several metabolites, gave resulted in increased mitochondrial oxidation of no indication that the high content of nucleic fatty acids in both the liver and red muscle as acids was problematic for Atlantic salmon. The well as a moderate reduction of fat in the mus- salmon responded well to the BioProtein feed, cle. By replacing up to 70 per cent of the fish growing better than on fishmeal-based control (capelin) oil with rapeseed oil in a salmon feed, feeds. It is possible that the nucleic acids had a the ß-oxidation capacity in white muscle was positive effect on the salmon’s growth. increased compared to fish whose only oil source was fish oil. Metabolism of fat In recent years, substantial research has been It has been proven that water temperature can af- carried out on the consequences of and possibil- fect both the fat deposition level and the fatty ities for using an increased amount of vegetable acid ß-oxidation capacity in fish. In an experi- fat and protein sources in fish feed. The fish’s ment in which the oil source was either fish oil or ability to metabolise and, in turn, deposit fat de- soybean oil, it was found, among other things, pends on the complex interaction of many fac- that low water temperature resulted in increased tors, among them life span, water temperature, deposition of fat in both the intestine and the liv- and the types of fat and protein sources used in er. Low water temperature also seems to induce the feed. In most cases, fat is a less expensive the peroxysomal metabolism of fat in fish. energy source than protein, and the goal is to optimise fat metabolism in fish, so that protein Composition of fatty acids in the fish, can be saved for muscle growth. An unwanted and product quality effect of a high fat level in the feed is increased The composition of fatty acids in the fish large- deposition of fat. Until now, little has been ly mirrors the composition of fatty acids in the known about the function of fat tissue in fish, feed, which is significant for the evaluation of even though it was proven relatively recently product quality. When fish oil is replaced with that fat tissue has great significance for the reg- vegetable oil in salmon, the level of long-chain, ulation of energy metabolism in mammals. Fat healthy, favourable n-3 fatty acids is reduced in tissue produces several important regulatory the fish muscle. To avoid too high a loss of these factors such as leptine, PPARy, adiponection, healthy fatty acids, much research has been etc. To increase the understanding of the devel- done on stimulating the capacity of Atlantic opment of fat tissue in fish and how it is affected salmon to produce the healthy n-3fatty acids, by different fatty acids in the feed, Akvaforsk EPA and DHA from 18:3n-3 from plant oils. has established a method for cultivating fat cells Among other things, it has been shown that the from fish in culture (Figure 4). addition of both soybean oil and rapeseed oil to salmon feed gives increased 5-desaturase activ- Several experiments on the addition of both nat- ity, which in turn leads to increased formation ural and synthesised bioactive fatty acids to of DHA. Even though it is possible to stimulate salmon feed have been done to see whether the capacity of Atlantic salmon to produce EPA these additives will lead to increased oxidation and DHA by adding plant oils to the feed, the of fatty acids, thus the desired effect of salmon increase is not sufficient to maintain the level of with a lower fat content. The addition of tetra- healthy n-3 fatty acids in the fish muscle. Partial

Theme: Feed, Nutrition, Feeding 211 reestablishment of EPA and DHA levels in the questions regarding the health and welfare of fish muscle can be achieved by implementing a the fish. Several diseases and production- washout period before slaughter, during which related disorders have been proven to be pre- plant oil in the feed is replaced by an EPA/ ventable by correct nutrition. DHA-rich fish oil. Like the addition of fatty acids to the feed, water temperature may also Alternative feed ingredients – How do affect the capacity of Atlantic salmon to meta- they affect health? bolise fatty acids. A large number of experiments have shown that a relatively high intake of vegetable oil under It has been tested whether the flesh of Atlantic normal production conditions does not affect salmon that had been fed a diet rich in vegetable the growth and feed utilisation of salmon to any fatty acids had less liquid-binding properties notable degree. However, much research re- than the flesh of Atlantic salmon fed a diet rich mains to be done on the amounts and length of in fish oil. In studies with both small (approx. 1 time fish can be fed various types of vegetable kg) and large (approx. 3 kg) Atlantic salmon, no oil without negative consequences on fish substantial loss of liquid from the muscle after health, especially under suboptimal conditions ice storage or smoking was found. However, such as fluctuating farming conditions, stress feeding fish with vegetable oil did seem to af- and poor health. Experiments with Atlantic fect fillet colour and lipid oxidation. Fish fed salmon have shown that substantial changes in with fish oil have shown somewhat better the composition of fatty acids can affect the im- colouration than fish fed with vegetable oil. On mune system by affecting phagocytotic capa- the other hand, feeds containing vegetable oil city of macrophages and production of signal did seem to have a positive effect on lipid substances (eicosanoids). These effects were oxidation, which can occur during frozen stor- especially clear at low water temperatures age, because the fish fed with vegetable oil have (5 °C). It has also been proven that rapidly a reduced content of n-3 fatty acids that easily growing salmon that were only given vegetable oxidise during storage. Quality parameters such oil in the feed (with fish meal as the protein as fillet gaping, texture and consumer prefer- source) had a higher frequency of cataract. ence were not affected. A high fat level in the blood has been proven to be a serious risk factor for the development of Nutrition and health in fish lifestyle-related diseases in humans, for exam- In the growing aquaculture industry there are al- ple cardiovascular diseases and Type 2 diabetes. ways new challenges concerning fish health and It is uncertain whether this is a risk factor in fish fish diseases. In Norway, effective vaccines as well. Experiments have shown that the lipo- have been developed for many diseases affect- protein secretion from liver cells is significantly ing Atlantic salmon, thus limiting the spread of higher in salmon fed with vegetable oil than in these diseases. Nevertheless, disease and health salmon fed with fish oil. Little is known about problems lead to substantial losses every year. whether the composition of fatty acids in the Aquaculture has been undergoing rapid devel- feed can affect the development of cardiovascu- opment, and fish growth has increased signifi- lar diseases in salmon. One experiment investi- cantly in the past decade. This gives rise to new gated the short-term effects of replacing marine

212 Aquaculture Research: From Cage to Consumption oils with vegetable oils on atherosclerosis in At- Cataract lantic salmon. Looking at the results of this ex- Cataract is an eye disorder which appears peri- periment, the possibility that changes in the odically in and has a high impact on Atlantic composition of fatty acids in fish feed can affect salmon. Several nutritional factors seem to af- the development of such disorders cannot be fect the development of cataract. Both high fat ruled out, but more research is needed before levels in the feed and high concentrations of any conclusions can be drawn. It is still impor- pro-oxidants such as iron, zinc and manganese tant to keep this danger in mind when replacing have been proven to increase the frequency of increasing amounts of the fat in salmon feed irreversible cataract in salmon. Meanwhile, with vegetable oil, which have a lower content antioxidants (astaxanthin and vitamin C) have of the healthy n-3 fatty acids. been proven to reduce the frequency. This indicates that a balanced diet with pro- and anti- Bone deformities oxidants will have a preventive effect against Deformities are a problem in aquaculture, fin- cataract. The content of fat in the feed also af- ancially and ethically. Nutrition, especially vi- fects the growth rate, and it is possible that the tamins and minerals, seems to have significance effect of fat content on frequency of cataract for the development of deformities. Results may be related to fast growth. There is also an from experiments indicate that a deficiency of increased potential for oxidation in high-fat phosphorus and zinc in periods of rapid growth feeds because of a higher content of polyunsat- can affect bone mineralisation in Atlantic salm- urated fatty acids. on, and thus give a higher frequency of deformities. If this deficiency is present already at start- A deficiency of the amino acids methionine and feeding, and becomes chronic, deformities will tryptophan as a factor in the development of cata- occur even if there is only a moderate defi- ract has long been a topic of discussion in aquac- ciency of these minerals. Once needs for phos- ulture research. However, in recent years, phorus and zinc are met, the deformities will be particular focus has been placed on histidine. mineralised and become permanent. Vitamin A Several studies have shown that the optimal level is probably a contributing factor to the develop- of histidine in the feed is often far higher than the ment of spinal deformities in several species. assumed requirement in order to prevent cataract. However, in an experiment with salmon eggs no relationship was found between vitamin A sta- In an experiment, Atlantic salmon fed a com- tus in eggs and the development of deformities mercial feed to which histidine, iron and zinc in the growing embryo. Too much vitamin A in had been added showed a lower frequency of the feed has, in other experiments, proven to cataract than Atlantic salmon fed a commercial lead to abnormal development of the vertebrae control feed. It has since been proven that histi- in several species, among them salmon. Vita- dine alone can reduce the development of cata- min C deficiency in salmon fry gives classic ract, and very high histidine concentrations in vertebrae deformities in the form of scoliosis the lens of the fish have been identified, which and lordosis. Vitamin C deficiency over a short- might explain the relationship between dietary er period of time in larger fish may also lead to histidine and the development of cataract. The deformities; this has been suggested as the experiments that have concluded that iron can cause of deformities in the jaw area. increase the frequency of cataract were most

Theme: Feed, Nutrition, Feeding 213 likely done with feed that contained a signi- gene technology offer the potential for enhanc- ficantly higher amount of added iron. Blood ing the fundamental understanding of fish nutri- meal in the feed has been proven to reduce the tion, the interaction between nutrients, and the frequency of cataract for pre-smolt, smolt and interaction between nutrients and various con- adult fish in the sea. This is related to the natu- ditions in aquaculture. These new methodolo- rally high content of histidine in the blood meal. gies also offer significant possibilities for developing practical, easy-to-use tests for both IPN raw materials and fish. To ensure that the Infectious pancreatic necrosis (IPN) is a conta- knowledge developed in nutritional biology is gious viral disease that attacks farmed fish and utilised on a broad basis, it is necessary to can result in serious economic losses for fish strengthen the ties between the Research Coun- farmers. Experiments have been done to identi- cil’s research initiatives and research being car- fy additives in the feed that can strengthen the ried out in the aquaculture industry, both immune system. Among other things, it has nationally and internationally. been found that feed with added nucleotide reduced IPN mortality in rainbow trout; however, Acknowledgements there were only a small number of fish used in The authors would like to thank Sjur E. Sævik the experiment. Several experiments indicate for translating this chapter from Norwegian to that increased access to energy for smolt in the English. weeks after transfer to seawater, when the need for energy is high, results in increased survival in the case of natural IPN outbreaks after seawater transfer. References Azevedo, P.A., Leeson, S., Cho, C.Y. and Bureau, D.P., 2004. Growth and feed utilization of large size rainbow trout (Oncorhynchus mykiss) and Atlantic Future challenges salmon (Salmo salar) reared in freshwater: diet and To maintain the growth of the aquaculture in- species effects, and responses over time. Aquacul- dustry in Norway, it is important to continually ture Nutrition 10, 401–411. generate new knowledge that can be utilised to Bakke-McKellep, A-.M. and Refstie, S., 2005. Alterna- tive protein sources and digestive function alter- formulate optimal, cost-effective fish feeds that ations in teleost fishes. In: Cyrino, J.E.P., Roubach, will promote normal growth, good health and R., Bureau, D., Kapoor, B.G. (eds.), Feeding and high quality. There is still a great lack of know- Digestive Functions of Fishes, Science Publishers, Inc., Enfield, NH, USA, in press. ledge in nutritional biology regarding salmon, Bjerkås, E. and Sveier, H., 2004. The influence of nutri- trout, cod and Atlantic halibut. As the aquacul- tional and environmental factors on osmoregulation ture industry develops, feeds will have to be for- and cataract in Atlantic salmon (Salmo salar L.). mulated much more specifically in accordance Aquaculture 235, 101–122. Breck, O., Bjerkås, E., Campbell, P., Arnesen, P., Hal- with the environment, life cycles of the various dorsen, P. and Waagbø, R., 2003a. Cataract preven- species and critical life stages. A continued fo- tive role for mammalian blood meal, histidine, iron cus on food safety, quality and fish health is also and zinc in diets for Atlantic salmon (Salmo salar vital to future economic growth and to main- L.) of different strains. Aquaculture Nutrition 9, 341–350. taining the good reputation of the Norwegian Breck, O., Rhodes, J., Waagbø, R., Bjerkås, E. and aquaculture industry. Molecular biology and Sanderson, J., 2003b. Role of histidine in cataract

214 Aquaculture Research: From Cage to Consumption formation in Atlantic salmon (Salmo salar L.). zation and health of Atlantic salmon Salmo salar L. Investigative Ophthalmology & Visual Science 44, fed genetically modified compared to non-modified U261-U261. commercial hybrid soybeans. Aquaculture Nutri- Breck O., Bjerkås E., Campbell P., Rhodes J.D., Sand- tion 11, 157–167. erson J. and Waagbø R., 2005. Histidine nutrition Hillestad, M., Krogdahl, Å., McKellep, A.M.B., Berge, and genotype affect cataract development in Atlan- G.M., Holm, H. and Ruyter, B., 2005. Fat digestion, tic salmon (Salmo salar L.). Journal of Fish Dis- transport and effect on metabolism in salmonids. eases 28, 357–371. Project 137 476. Breck O., Bjerkås E., Sanderson J., Waagbø R. and Hollung, K., Øverland, M., Hrustic, M., Sekulic, P., Campbell P., 2005. Dietary histidine affects lens Miladinovic, J., Martens, H., Narum, B., Sahlstrøm, protein turnover and synthesis of N-acetylhistidine S., Sørensen, M., Storebakken, T. and Skrede, A., in Atlantic salmon (Salmo salar L.) undergoing 2005. Evaluation of non-starch polysaccharides and parr-smolt transformation. Aquaculture Nutrition oligosaccharide content of different soybean vari- 11, 321–332. eties (Glycine max) by near-infrared spectroscopy Dabrowski, K., Terjesen, B.F., Zhang, Y., Phang, J.M. and proteomics. Journal of Agricultural and Food and Lee, K-.Y., 2005. A concept of dietary dipep- Chemistry, in press. tides: a step to resolve the problem of amino acid Krogdahl, Å., Sundby, A. and Olli, J.J., 2004. Atlantic availability in the early life of vertebrates. Project salmon (Salmo salar) and rainbow trout 159 934/120. (Oncorhynchus mykiss) digest and metabolize nu- Denstadli, V., Vegusdal, A., Krogdahl, Å., McKellep, trients differently. Effects of water salinity and A.M.B., Berge, G.M., Holm, H., Hillestad, M. and dietary starch level. Aquaculture 229, 335–360. Ruyter, B., 2004a. Lipid absorption in different seg- Moya-Falcón, C., Stefansson, S.O., Vegusdal A., Hvat- ments of the gastrointestinal tract of Atlantic salm- tum, E., Thomassen, M.S., Jakobsen, J.V., Berge, on (Salmo salar L). Aquaculture 240, 385–398. R.K. and Ruyter B., 2004. Effect of dietary supple- Denstadli, V., Willassen, N.P., Eijsink, V.G.H. and mentation of sulphur-substituted fatty acid ana- Storebakken, T., 2004b. Effect of phytic acid (IP6) logues on growth, survival, lipid composition and on the activity of bovine trypsin and trypsin from Na+, K+-ATPase activity in Atlantic salmon. Comp. Atlantic salmon (Salmo salar). 11th ISNFF, Phuket, Biochemistry and Physiology Part B. 139, 657–668. Thailand, 2–7 May 2004. Moya-Falcón, C., Thomassen, M.S., Jakobsen, J.V. and Gjøen, T., Obach, A., Røsjø, C., Helland, B.G., Rosen- Ruyter B., 2005. Effects of supplementation of lund, G., Hvattum, E. and Ruyter, B., 2004. Effect rapeseed oil on metabolism of [1–14C] 18:1 n-9, [1– of dietary lipids on macrophage function, stress sus- 14C] 20:3 n-6 and [1–14C] 20:4 n-3 in Atlantic ceptibility and disease resistance in Atlantic salmon salmon hepatocytes. Lipids 40, 709–717. (Salmo salar). Fish Physiology and Biochemistry Nordgarden U. Oppedal F., Taranger G.L., Hemre G.-I. 30, 149–161. and Hansen T., 2003. Seasonally changing metabo- Grisdale-Helland, B. Energy utilization in fish: conse- lism in Atlantic salmon (Salmo salar L.), I – Growth quences for the economy and environment. Project and feed conversion ratio. Aquaculture Nutrition 9, 146 469 (2002–2006). 287–294. Grisdale-Helland, B., Ruyter, B., Rosenlund, G., Olsen, R.E. Krill as feed source for fish. Project Obach, A., Helland, S.J., Sandberg, M.G., Standal, 146 871/120 (2002–2004). H. and Røsjø, C., 2002. Influence of high contents Oxley, A., Torstensen, B.E., Rustan, A.C. and Olsen, of dietary soybean oil on growth, feed utilization, R.E., 2005a. Enzyme activities of intestinal tria- tissue fatty acid composition, heart histology and cylglycerol and phosphatidylcholine biosynthesis standard oxygen consumption of Atlantic salmon in Atlantic salmon (Salmo salar L.) Comparative (Salmo salar) raised at two temperatures. Aquacul- Biochemistry and Physiology Part B 141, 77–87. ture 207, 311–329. Oxley, A., Tocher, D.R., Torstensen, B.E. and Olsen, Grisdale-Helland, B., Shearer, K.D. and Helland, S.J., R.E., 2005b. Fatty acid utilisation and metabolism 2007. Energy and nutrient utilization of Atlantic in caecal enterocytes of rainbow trout cod, Atlantic salmon and rainbow trout fed diets dif- (Oncorhynchus mykiss) fed dietary fish or copepod fering in energy content. Aquaculture Nutrition, in oil. Biochimica et Biophysica Acta, in press. press. Refstie, R. and Glencross, B., 2005. Different lupin Hemre, G-.I, Sanden, M., Bakke-McKellep, A.M, Sag- meals and concentrates to Atlantic salmon at low stad, A. and Krogdahl, Å., 2005. Growth, feed utili- temperature: Digestibility and intestinal physi-

Theme: Feed, Nutrition, Feeding 215 ology. “Seeding a Future for Grains in Aquaculture Rørvik, K.-A. and Thomassen, M.S. Effekt av samspill Feeds Part III”, Fremantle, Western Australia, 14 mellom økt energitilgang og vaksinering på IPN og April 2005. PD hos laks i sjø. Project 152 045 (2003–2005). Refstie, S., Korsøen, Ø.J., Storebakken, T., Baeverfjord, Rørå, A.M.B., Ruyter, B., Skorve, J., Berge, R.K. and G., Lein, I. and Roem, A.J., 2000. Differing nutri- Slinning, K-E., 2005. Influence of high content of tional responses to dietary soybean meal in rainbow dietary soybean oil on quality of market sized raw, trout (Oncorhynchus mykiss) and Atlantic salmon smoked and frozen Atlantic salmon (Salmo salar). (Salmo salar). Aquaculture 190, 49–63. Aquaculture International 13, 217–231. Refstie, S., Førde-Skjærvik, O., Rosenlund, G. and Rør- Sanden, M., Berntssen, M.H.G., Krogdahl, Å., Hemre, vik, K-.A., 2005a. Feed intake, growth, and utilisa- G-.I. and Bakke-McKellep, A-.M., 2004. An exam- tion of macronutrients and amino acids by 1- and 2- ination of the intestinal tract of Atlantic salmon, year old Atlantic cod (Gadus morhua) fed standard Salmo salar L., parr fed different varieties of soy or bioprocessed soybean meal. Aquaculture, in and maize. Journal of Fish Disease 28, 317–330. press. Seierstad, S.L., Poppe, T.T. and Larsen, S., 2005a. Refstie, S., Sahlström, S., Bråthen, E., Baeverfjord, G. Introduction and comparison of two methods of and Krogedal, P., 2005b. Lactic acid fermentation assessment of coronary lesions in Atlantic salmon, eliminates indigestible carbohydrates and antinutri- Salmo salar L. Journal of Fish Diseases 28, tional factors in soybean meal for Atlantic salmon 189–197. (Salmo salar). Aquaculture 246, 331–345. Seierstad, S.L., Poppe, T.T., Koppang, E.O., Svindland, Refstie S., Krogdahl, Å., Førde, O., Ringø, E. and A., Rosenlund G., Frøyland, L. and Larsen, S., Shearer, K.D., 2005c. Nutrient digestion and ab- 2005b. Influence of dietary lipid composition on sorption along the intestinal tract of Atlantic cod cardiac pathology in farmed Atlantic salmon, Salmo (Gadus morhua) fed standard or bioprocessed soy- salar L. Journal of Fish Diseases 28, 677–690. bean meal. Aquaculture Europe 2005 “Optimising Stubhaug, I., Frøyland L. and Torstensen, B. E., 2005. the Future”, Trondheim, 5–8 August 2005. β-oxidation capacity of red and white muscle and Ringø, E., Lødemel, J.B., Myklebust, R., Jensen, L., liver in Atlantic salmon (Salmo salar. L.)-Effects of Lund, V., Mayhew, T.M. and Olsen, R.E., 2002. The increasing dietary rapeseed oil and olive oil to re- effects of soybean, linseed and marine oils on aero- place capelin oil. Lipids 40, 39–47. bic gut microbiota of Artic char Salvelinus alpinus Terjesen, B., Lee, K.-J., Failla, Y.Z.M. and Dabrowski, L. before and after challenge with aeromonas sal- K., 2005. Optimization of dipeptide-protein mix- monicida ssp. salmonicida. Aquaculture Research tures in experimental diet formulations for rainbow 333, 591–606. trout (Oncorhynchus mykiss) alevins. Aquaculture, Ringø, E., Olsen, R.E., Mayhew, T.M. and Myklebust, in press. R., 2003. Electron microscopy of the intestinal Torstensen, B.E., 2003. Kartlegging av lipoproteinme- microflora of fish. Aquaculture 227, 395–415. tabolismen hos laks. Project 134 289/120. Ruyter, B., Røsjø, C., Grisdale-Helland, B. Rosenlund, Vegusdal, A. Sundvold, H., Gjøen, T. and Ruyter, B., G., Obach, A. and Thomassen M.S., 2003. Influ- 2003. An in vitro method for studying the prolifer- ence of temperature and high dietary linoleic acid ation and differentiation of Atlantic salmon preadi- content on the elongation and desaturation of poly- pocytes. Lipids 38, 289–296. unsaturated fatty acids in Atlantic salmon hepato- Vegusdal A., Gjøen T., Berge R. K., Thomassen, M.S. cytes. Lipids 38, 833–839. and Ruyter B., 2005. Effect of 18:1n-9, 20:5n-3 and Ruyter, B., Jacobsen, J.V. 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216 Aquaculture Research: From Cage to Consumption Kristin Hamre1), Ivar Rønnestad2), José Rainuzzo3), Yoav Barr4) and Torstein Harboe5) 1) NIFES (National Institute of Nutrition and Seafood Research), 2) University of Bergen, 3) The SINTEF Group, 4) Akvaforsk – The Institute of Aquaculture Research, 5) Institute of Marine Research

Nutritional Aspects – Marine Fish Larvae

In the course of the most recent programme period we have determined the nutrient content of the live feed organisms, rotifers and Artemia, which are used in the intensive aquaculture of marine fish larvae. These were subsequently compared with the nutrient content of copepods, which are the natural prey of these larvae. Rotifers and Artemia may be particularly low in omega-3 fatty acids and have low levels of specific vitamins and minerals. This may partly explain why fish larvae in intensive farming grow more slowly and suffer a higher proportion of developmental anomalies than larvae that are kept under natural conditions and feed on copepods. We can enrich the prey species with a number of the nutrients that they possess in insufficient amounts, but it is still difficult to obtain a satisfactory combination of fatty acids in Artemia. One of the goals of nutritional research on marine fish larvae is to generate knowledge that will enable us to replace live feed organisms with formulated feed as early as possible in the larval stage. However, such a development is held back by a number of factors, such as the fact that the tiny feed particles have an extremely high rate of leaching of water-soluble nutrients. Fat and protein, in the form in which they are normally given to larger fish, are difficult for the larvae to digest, and thus have poor availability to their absorption processes, since their digestive tract is not fully developed. The mapping of larval development, for example the capacity for digestion and absorption of nutrients, should take place in parallel with the development of feeds and feedstuff raw materials with good technical characteristics and high nutrient availability.

Thematic area: Feed, Nutrition, Feeding 217 Cod and halibut are the main marine fish spe- duced in semi-enclosed bays. The percentage of cies with altricial larvae (i.e. larvae that lack a deformities is currently declining, but there is stomach) that are currently being farmed in still room for improvement. There are no reli- Norway. Halibut have been farmed continu- able figures regarding different rates of growth ously since the mid-1980s, while the most rein halibut, but intensively produced juveniles cent efforts using cod began around 2000. Work often display faulty pigmentation and incom- on larval nutrition has been divided between plete eye migration, conditions that are obvi- studies of live feed aimed at characterising ously related to nutrition. One important goal of optimal nutritional content, the development of research on larval nutrition should be to gener- formulated feeds, and research on larval biolo- ate the knowledge that will enable us to raise gy, nutritional physiology and nutritional re- growth and survival rates and lower deformity quirements. Developments in raising larvae rates in intensive aquaculture to levels equal to have led to techniques of stocking in semi- those found in semi-enclosed bay conditions. enclosed bays being abandoned and being replaced by the adoption of more intensive methods that employ indoor tanks and feeding Results and status with rotifers and Artemia. This has brought a Nutritional content of live feed number of challenges with respect to lower In the course of this and the previous pro- growth rates and higher incidences of develop- gramme period we have gained a fairly com- mental anomalies and deformities in juveniles, plete understanding of the basic nutritional conditions that appear to be partly due to subop- content of several live feed organisms. We still timal nutrition. do not know the nutritional requirements of marine fish larvae, but the nutritional content of Cod larvae raised in intensive aquaculture grow copepods and the requirements of larger fish much more slowly than cod produced in semi- can be used as indicators of whether rotifers and enclosed bays. (Ten per cent per day compared Artemia contain adequate levels of particular with up to 30 per cent per day.) There have also nutrients. been problems of deformities in cod juveniles produced under intensive farming conditions; Data from the literature concerning the protein such problems are extremely rare in cod pro- content of live feeds are highly variable (e.g. 24–61 per cent of dry weight in rotifers), and seem to primarily depend on the analytical methods employed in individual studies. Differ- ent colorimetric methods (e.g. Lowry) have a tendency to underestimate protein because they are intended only for analyses of water-soluble proteins, while live feed organisms also contain proteins that are insoluble in water. Protein can also be measured by first measuring the nitrogen content of the sample and then multiplying it by a particular factor to indicate nitrogen con-

Rotifers.145x100//Kap13-bilde01.eps Photo: The Institute of Marine Research tent. A factor of 6.25 is usually employed, but in

218 Aquaculture Research: From Cage to Consumption fact, each individual organism has its own fac- obtain the same fatty acid composition as that of tor, which needs to be determined by measuring copepods. This is often explained by reference total amino acid content. This has recently been to Artemia’s self-metabolism. done for rotifers, which proved to have a factor of 4.46 and a protein content of about 40 per Artemia have low levels of thiamine, vitamin A, cent of dry weight, irrespective of the protein iodine and zinc. However, halibut are capable of content of their diet. The proportion of water- converting canthaxanthin, of which there are soluble protein was about 50 per cent, and the high levels in Artemia, to vitamin A, and we es- amount of free amino acids was between 5 and timate that feeds need to contain about 500 mg/ 14 per cent of total protein. Changes in diet pro- kg astaxanthin or canthaxanthin if they are to duced only minor changes in the amino acid meet the vitamin A requirements of halibut lar- composition of the rotifers. As was to be ex- vae. Cod larvae, which also feed on copepods pected, there were stable levels of protein and that lack vitamin A, probably have a similar amino acids in rotifers because such factors are mechanism for converting carotenoids. In roti- largely genetically determined. Such work has fers, levels of vitamin A, manganese and sele- not been done on Artemia or copepods, so the nium are lower than in copepods and are also precise levels of protein in these feed organisms lower than the requirements of larger fish. Roti- are still not known. By and large, the amino acid fers contain insufficient carotenoids to enable composition of total protein in Artemia and roti- these to meet larval vitamin A requirements. The fers is satisfactory when we compare it with that following nutrients may be present in lower in copepods, the amino acid requirements of amounts than in copepods, but equal to or higher larger fish and the composition of the halibut than the requirements of larger fish: vitamin C, larva body. thiamine, vitamin E, iodine, copper and phosphorus. The lipid content of the stages of copepods that are eaten by marine fish larvae is 6–16 per cent Nutritional requirements of marine fish of dry weight. In enriched Artemia it can vary larvae from 22 to more than 30 per cent. Rotifers that We know little about the real nutritional require- have not been fat-enriched have a lipid content ments of cod and halibut larvae. These needs may similar to that of copepods, but after enrichment differ from those of larger fish, since fish larvae it often lies between 20 and 30 per cent. The fat- are undergoing metamorphosis at the same time ty acid composition of rotifers reflects that of as they are growing extremely rapidly. Neverthe- their feed, whether it is ingested during cultiva- less, their requirements may not be as high as the tion or in short-term enrichment, and is there- level of nutrients in copepods might suggest. The fore easy to manipulate. Unenriched Artemia do task of determining nutritional requirements will not contain DHA (docosahexaenoic acid, be simplified when we can design classical nutri- 22:6n-3), have a low content of EPA (eicosap- tional studies for marine larvae in order to im- entaenoic acid, 20:5n-3) and a high content of prove our knowledge in this field. ARA (arachidonic acid, 20:4n-6), while copepods contain 20–40 per cent DHA, 15–20 per However, some studies of the fatty acid compo- cent EPA and <1 per cent ARA. Even if we en- sition of larvae have been carried out. It was rich Artemia with marine fats, it is difficult to originally thought that feeds for fish larvae

Thematic area: Feed, Nutrition, Feeding 219 should contain a high level of DHA and have a Enrichment of rotifers and Artemia with high DHA/EPA ratio, on the basis of the fatty n-3 PUFA, free amino acids, lipid classes acid composition of cod roe. Studies of pigmen- and micronutrients tation in flatfish have since shown that the ratio There are two traditional methods of enriching of DHA to EPA is not so important, but that a rotifers. In the first method, which is known as high level of ARA and thus a low EPA/ARA long-term enrichment, we combine growth feed- ratio has a dramatic negative effect. However, it ing with an enrichment of nutrients in which the is important to maintain a high level of DHA in enrichment process takes place throughout the order to ensure that this essential fatty acid is production phase. The other method, known as available for the development of the vision and short-term enrichment, consists of modifying the nervous system. Just what the minimum the nutrient content via a brief final feeding level of content of n-3 fatty acids might be for phase. In this case we usually employ a fat emul- cod and halibut larvae is still unknown, but sion to which other nutrients may be added. >20 per cent DHA, > 10 per cent EPA and < 2 per cent ARA should provide a good feed as There are several commercial sources of feed and far as fatty acids are concerned. This can easily enrichment additives that yield rotifers with an im- be achieved with rotifers, although enriching proved fatty acid profile; e.g. yeast and marine oil Artemia is rather more difficult. or emulsions, algal concentrates and products that consist of dehydrated cells of heterotrophic single- Protein requirements are relatively unknown, celled organisms (Figure 1, Schizochytrium sp., but it has been shown that only a small propor- e.g. Algamac, Rotimac, or Crypthecodinium tion of the protein in rotifers is utilised for cohnii, Aquagrow Advantage). growth in rapidly growing turbot larvae. The larvae presumably compensate for their low To enrich Artemia with polyunsaturated n-3 protein utilisation by eating more. This must fatty acids (PUFA), we usually utilise a 24-hour also be considered in the context of the need of enrichment period, employing more or less the fish larvae for an energy substrate, since fish same products as those used for rotifers. SIN- larvae obtain between 60 and 90 per cent of TEF’s experience is that MAROL E™ is an ef- their energy from amino acid metabolism. Free ficient means of raising the DHA content of amino acids are easily available to fish larvae

because they do not need to be digested before DHA EPA they are absorbed. We also assume that water- soluble protein is more available than insoluble 50 protein, because it is more available for diges- 40

tion than insoluble protein. DW 30 20 mg/g It has been shown that 400–500 mg/kg vitamin 10

C, which is found in enriched Artemia, and also 0 often in rotifers, will be sufficient to meet the START 0.2 0.4 0.8 1.2

145x100//Kap13-fig01.eps needs of marine fish larvae. Figure 1. DHA and EPA in rotifers enriched with different doses (μg/ind.) of Thraustochytride cells after 24 h enrichment at 20 ºC.

220 Aquaculture Research: From Cage to Consumption 145x100//Kap13-bilde03.eps Cod larva fed on rotifers. Photo: The Institute of Marine

145x100//Kap13-bilde02.eps Research Newly hatched Artemia. Photo: NIFES

Artemia (i.e. 42 mg/g, 25 per cent of total fatty The composition of lipid classes in live feeds is acids; total lipids 25.5 per cent and DHA/EPA not directly related to the composition of lipid ratio 2.3). The experience of NIFES and the In- classes in the emulsions used for enrichment. stitute of Marine Research, however, is that tar- Rotifers enriched with an emulsion rich in wax get-oriented Artemia enrichment is extremely esters increased their proportion of triglycer- difficult to reproduce. The Pronova oil TG ides. Artemia also has a tendency to retain its 5010, which has a fatty acid composition simi- original lipid class composition even after 12 lar to that of MAROL E, can give Artemia a hours of enrichment with emulsions of different DHA content that ranges between 10 and 25 per lipid classes. This is probably because phospho- cent, even when identical emulsification and lipids and wax esters are rapidly broken down in enrichment methods are employed. Commer- the organism’s gut, and the fatty acids that are cial fry producers also experience wide varia- liberated are incorporated into triglycerides. tions in the fatty acid composition of their However, phospholipid enrichment may have Artemia, even when they use the same enrich- other positive effects on the nutritional content ment media. In the literature, values of DHA in of feed organisms, for example by increasing Artemia enriched using emulsions tend to be their content of phosphorus and nitrogen bases between 6 and 10 per cent of total fatty acids. such as choline, ethanolamine and inositol. However, the adoption of heterotrophic algae in recent years has improved both the general fatty A recently developed method of enrichment is acid profile and the reproducibility of Artemia based on intensive short-term “boosting”. The quality in both commercial aquaculture and re- method enriches rotifers or Artemia with 1–5 g search. This has made faulty pigmentation less enrichment feed l-1 for 30–120 minutes. When of a problem in halibut fry production than it the feed organisms are enriched at a high feed used to be. concentration, the gut is rapidly filled, while the short enrichment time reduces the amount of nutrient metabolism. The short enrichment time

Thematic area: Feed, Nutrition, Feeding 221 also prevents the tank environment from be- the increase in individual FAA matched the coming unduly contaminated and thus allows concentrations of the same AA in the liposomes for higher live feed density. This method offers very well. The effect in Artemia was less pro- new possibilities for modifying the nutrient nounced but still significant, with an increase of content of live feed organisms. 50 per cent (Figure 3).

This new enrichment method was used in a study A combination of boosting and the use of lipo- aimed at increasing the phospholipids and free somes for enrichment might provide a good tool amino acids content of live feed, using liposome for further studies of nutrition and physiology loaded with free amino acids. A new method has in fish larvae. In this regard, we can envisage a been developed for the mass production of large further development whereby marine phospho- liposomes with diameters of 2–8 m. These lipo- lipids replace soya lecithin. Another aspect that somes are based on soy lecithin (Epikuron™), ought to be investigated concerns how long which forms a phospholipid membrane around a phospholipid and FAA levels are maintained in solution of free amino acids (FAA). The loss of the live feed organism in the fish larvae feeding FAA from the liposomes in seawater was 9.2 per situation, since both of these nutrients undergo cent in the course of two hours. rapid metabolism in the feed organisms.

A large rise in the phospholipid content of roti- A number of studies have also been carried out fers and Artemia that had been enriched with on enrichment of Artemia and rotifers with liposomes by the boosting method has been micronutrients. Both Artemia and rotifers can demonstrated (Figure 2). The rotifers displayed be sufficiently enriched with thiamine by add- an increase of 298 per cent in FAA content and ing 200 mg thiamine HCl per kg emulsion.

120

100

40 74.8 73.2

20 37.2

mg PL/gr dry weight 30.4 0 Standard Boosting with Standard Boosting with enriched liposome 2gr/liter enriched liposome 1gr/liter 30 min 60 min

Artemia Rotifer

145x100//Kap13-fig02.eps Figure 2. Effects of boosting enrichment with liposomes on the content of polar lipids in rotifers previously enriched with Algamac 2000• , and on Artemia nauplii previously enriched with DHA-Selco•

222 Aquaculture Research: From Cage to Consumption 60 Rotifers enriched with Algamac 2000 Rotifers enriched with Algamac 2000 + boosting1g60minepikuren 50 DM) -1 40

30 FAA (nmol mg 20

Individual

0 ile tyr thr ala val leu ser glu gln pro cys aba aba asn asp lys * gly * his * g tau * arg * a met * phe *

45x100//Kap13-fig03.eps Figure 3. Effects of Epikuron-based liposome boosting on FAA content of rotifers. The liposomes were filled with a solution of amino acids.

Iodine has been used both in its available form obtained with vitamin C and E enrichment. It is I- (as sodium iodide) and in an oil containing possible to produce rotifers with graded levels bound iodine, Lipiodol. Both methods offer of certain of these nutrients, opening up the pos- good iodine enrichment of Artemia. We found a sibility of studies of nutrition in cod larvae. linear increase in iodine concentration in rotifers with increasing amounts of Lipiodol added Formulated feeds for larvae to the feed of the rotifers. Using Lipiodol for One important goal of research on nutrition in enrichment is simpler than using water-soluble marine larvae is to generate knowledge on compounds, but there is some uncertainty as to which to base the development of formulated whether Lipiodol is converted to I- in the feed feeds that can be used as early as possible in the organisms and/or larvae, thus becoming avail- larval phase. This will reduce the need for live able for the synthesis of thyroid hormone. feed organisms, which are both labour-intensive Enrichment of Artemia and rotifers with vita- and complicated to culture. min A is difficult to reproduce, probably because vitamin A is unstable when it is exposed In cod farming, current practice is usually to to light, oxygen and high temperature in the move to formulated feed at about 25 to 30 days enrichment tank, while good results have been after hatching, i.e. at the end of the phase of

Thematic area: Feed, Nutrition, Feeding 223 feeding with rotifers. Artemia are thus little liposomes and lipid spray beads can be used for used at present in cod farming. Halibut are this purpose. transferred to formulated feed somewhat later. With financial support from the Research Coun- Most currently available feeds are microbound, cil of Norway, Ewos Innovation and Maripro which is to say that the components of the feed have developed two different formulated feeds are bound together by a polymer that forms a for fish larvae; both production processes have network within the particles. Various types of been patented. Promal, from Ewos, is based on capsules based on alginate, chitosan or cross- marine phospholipids that encapsulate water- linked protein are also available. Experiments soluble nutrients in order to prevent leaching have also been carried out on fat encapsulation. and supply the larvae with what is believed to be an optimal combination of fats. This feed is al- The most serious bottleneck holding back the de- ready available on the market. Minipro, from velopment of formulated feed for larvae is the Maripro, is a feed whose nutrients are encapsu- high rate of leaching during administration, lated with the aid of a polymerising agent. which is due to the tiny particle size, which gives the particles an extremely high surface/volume Digestibility of protein sources used in ratio. A test of four commercial feeds and test formulated feeds for larvae feeds showed that 18–40 per cent of the protein The digestibility of the source of protein is a de- leached out in the course of two minutes in sea- cisive factor in determining how efficiently the water. The leaching rate of small water-soluble dietary protein can be utilised by the fish larvae. molecules such as FAA, water-soluble vitamins Very little is known about protein digestibility and minerals may be as high as 90 per cent in the in fish larvae, and data based on studies of adult course of two minutes. Leaching occurs most fish are not directly relevant because of differ- rapidly in microbound feeds, but encapsulated ences in the digestive systems of larvae and feeds, whose particles are often treated with wa- adult fish. Among other factors, the lack of a ter, may suffer high rates of leaching during feed stomach at the larval stage means that ingested processing, and micronutrients in the end prod- protein is neither exposed to the denaturing uct may be completely leached out. Although conditions imposed by the gastric acids nor pre- commercial feeds also leach, the production of digested by pepsin before entering the mid-gut. cod fry with an early transition to formulated feeds has been successful. Nevertheless, feeds Live feed, which usually contains large with lower leaching rates would offer a better amounts of soluble nitrogen, produces the best guarantee that the nutritional requirements of the growth and survival rates under farming condi- larvae are met, and probably also result in im- tions. Earlier experiments on carp larvae proved growth and survival rates. showed that higher levels of soluble protein in the feed improved growth and survival rates. It The latest development in formulated feeds is is possible that the soluble protein is more the production of “complex particles”, in which readily available to proteases and also that it water-soluble nutrients are encapsulated in or- may be more easily absorbed through pinocyto- der to prevent leaching, after which the capsules sis. The soluble protein content of the source of are incorporated into larger feed particles. Both

224 Aquaculture Research: From Cage to Consumption protein may therefore have an important influ- the form of phospholipids if they are to utilise it ence on the digestibility of the protein. optimally, although the optimal levels of phospholipids for cod and halibut larvae are not The results of an in vitro study of digestion known. Phospholipids must therefore be added showed that live feed and hydrolysed cod fillet to formulated feeds, and it is an open question contained most soluble protein. The study was whether live feeds, which contain phospho- based on fractionating soluble and insoluble lipids in the form of cell membranes, possess components, which were subsequently exposed them in sufficient amount. As mentioned above, to a mixture of alkaline proteases. The protein it is difficult to enrich live feed with phospho- under digestion was later sampled for analysis lipids. at various points in time. There was no anterior gastric component (pepsin under acidic condi- Phospholipids in microdiets have been shown to tions) in the experimental design. These exper- improve growth and survival rates in cod larvae iments showed that the soluble fraction was when the feed is used in a dietary regime to- consistently more readily digested than the in- gether with rotifers until around 26 days after soluble fraction. Overall digestibility was best hatching. Larvae fed microdiets containing 40 for casein, while live feeds were also high (84– per cent phospholipids had better survival rates 86 per cent) and roe meal was lowest at 48 per than those on microdiets that contained either cent. Meal was generally relatively indigestible, 30 or 50 per cent phospholipids. Phospholipids even though its soluble fraction was itself high- from marine sources resulted in better growth ly digestible. and survival rates than soya lecithin.

A preliminary stage of pepsin hydrolysis of cod Marine fish larvae have an incompletely devel- fillet produced an increase in the soluble frac- oped gut. As in the case of protein, it may be tion, but did not result in higher overall digest- supposed that the enzymes that digest fat have a ibility in vitro. If increased solubility also limited capacity, and that partially digested fat increases leaching from particles of feed, this in the form of diacylglycerol (DAG) and could reduce the availability of protein in fish monoacylglycerol (MAG) will be more avail- larvae feeds. able to fish larvae than triacylglycerol (TAG), which is the form of fat usually added to feed. The production process may affect protein di- A tube feeding experiment, in which individual gestibility. One study showed that the protein halibut larvae were fed radioisotope- labelled digestibility of the raw materials was higher TAG, DAG, MAG or phospholipids, showed a than in the final feed, which had been produced rate of uptake of around 90 per cent for MAG using a protein encapsulation technique. and 20 per cent for TAG. DAG and phospholipid uptake rates were around 40–60 per cent. Effects of fat quality of feeds on growth These results show that the utilisation of fat and survival rates of larvae and quality of added to formulated feeds is capable of being juveniles considerably improved. We also attempted to Lipid quality is characterised by its fatty acid enrich Artemia with MAG, DAG and phospho- and lipid class composition. It appears that ma- lipids, but this produced only insignificant rine fish larvae need to ingest part of their fat in changes in lipid class composition in Artemia, a

Thematic area: Feed, Nutrition, Feeding 225 finding that has been confirmed by the results of larvae lack a food reservoir, and that the amount other studies. of food entering into the gut is regulated only by ingestion rates. Nor do fish larvae have the abil- Digestive tract development in marine ity to release pepsinogen and HCl. fish larvae In marine fish, the digestive tract gradually de- Digestive enzymes velops from a short, straight, simple and histo- Most of the proteolytic enzymes are produced logically undifferentiated tube, often closed at in the pancreas and are released into the diges- both the mouth and anal ends in yolk-sac larvae, tive tract. Trypsin levels rise throughout the to a segmented tract in juvenile fish. The first yolk-sac stage, but after the yolk has been ab- rapid change takes place just ahead of onset of sorbed, they fall rapidly before rising again in exogenous feeding, when the digestive tract dif- older larvae. The fall in trypsin after the yolk- ferentiates into three sections: the foregut, mid- sac has been used up can be explained by inad- gut and hindgut, which are histologically and equate rates of new synthesis or possibly hun- functionally different from each other. At this ger-induced intracellular breakdown of the stage, gut length is often less than 50 per cent of enzyme in the pancreas. Absolute levels of body length. The individual segments of the gut trypsin vary widely between different groups of are separated by muscular sphincters or valves. larvae. It is still uncertain whether extracellular With few exceptions, the gut is coiled into a digestion based on pancreatic enzymes is suffi- loop before start-feeding. In the subsequent cient to hydrolyse proteins rapidly enough. phase of development the surface of the gut in- Brush-border membrane (BBM) enzymes are creases in area (thicker gut epithelium, larger located in the gut cell membrane that faces the microvilli). During metamorphosis the foregut gut lumen. These enzymes hydrolyse peptides, differentiates into a functional stomach cavity which are the result of cleaving by pancreatic with acid and pepsin secretion capability. Si- proteases, into free amino acids and peptides multaneously, the pyloric appendices also de- that are small enough to be absorbed. In halibut velop; these increase the absorptive area of the the activity of the BBM enzyme leucine amino- digestive tract and are important for protein and peptidase (LAP) is low and stable until about fat absorption. Other changes in the gut take day 30 after start-feeding, whereupon it rises place after metamorphosis; these changes in- rapidly to its maximum level at 60 days after clude an increase in its relative length. In cod, start-feeding, only to fall somewhat again. Cod metamorphosis is less well-defined than in oth- larvae have a stable level of LAP activity er species and the development of the stomach throughout the start-feeding period, apart from is very slow. a peak in activity around day 35 after start- feeding. The liver, pancreas and gall bladder with their outlets form as early as the egg and yolk-sac Mechanisms of absorption stage, and are assumed to be functional by the Several quantitative studies of the ability of the time that the yolk-sac has been absorbed. As gut to digest and absorb nutrients have been mentioned earlier, fish larvae lack a morpho- performed in recent years. These studies have logically differentiated and functional stomach focused on the absorption kinetics of isotope- at the time of start-feeding. This means that the labelled whole protein and amino acids (see be-

226 Aquaculture Research: From Cage to Consumption 145x100//Kap13-fig04.eps Figure 4. Distribution of CCK-producing cells in the gut of fish larvae (based on Kamisaka, 2005). low). As yet, few details of the absorption pro- have been shown to play a leading role in regu- cess are known, and efforts to characterise the lating gut movement and enzyme secretion. molecular basis of amino acid and peptide Cholecystokinin (CCK) plays a central role in transport in the digestive tract have just begun. regulating the liberation of bile and enzymes The gene for the peptide transporter in the cod from the pancreas, as well as in influencing gut gut has been cloned, and preliminary studies peristalsis and appetite control. The amino acid suggest that this gene is expressed from start- sequence of CCK is extremely conservative feeding onwards. To date, no sequences for throughout the vertebrates. Two types have amino acid transporters have been published, been demonstrated in teleosts: fish CCK1 and but two of the transporters for basic amino acids fish CCK2, and we find variations in the sixth have been partially characterised. position from the C-terminal end.

Hormones The teleosts are a complex group, and we have Digestion is a complex process, which we as- found distinct differences between patterns of sume is closely controlled in larvae in order to distribution and the development of CCK-pro- optimise the uptake of nutrients in the natural ducing cells at the larval stage (Figure 4). In lar- environment. In adult fish, peptide hormones vae that develop from pelagic eggs and which

Thematic area: Feed, Nutrition, Feeding 227 have a coiled gut when they begin to eat (e.g. chain lengths and intraluminal proteolytic ac- halibut and tuna), it is difficult to demonstrate tivity, regulates CCK levels. the presence of CCK-producing cells in the gut until some time after the larvae have started Protein digestion, absorption and feeding. In such species, the CCK-producing metabolism cells initially develop in the area of the appendi- The results of in vivo experiments on fish larvae ces and the forepart of the midgut, and are never based on tube feeding with radio-labelled amino found in the rear two-thirds of the gut. In view acids show that FAA are absorbed two to five of the leading role played by CCK in regulating times as fast as whole protein (Figure 5), depend- gut functions, we may therefore ask how well ing on the type of protein concerned, species and the digestive process is controlled during the developmental stage. Peptides appear to be ab- start-feeding phase. In larvae with a straight gut, sorbed somewhat more slowly than FAA. So far, such as herring and ayu, CCK-producing cells our experiments on halibut, herring and Senegal are present ahead of start-feeding and through- sole suggest that both essential and non-essential out the whole length of the gut. A point of inter- amino acids are absorbed rapidly and efficiently est in this connection is that such larvae are if they are given in free form. Even if FAA are in- easier to start-feed. We also find CCK-produc- troduced into the gut in extremely high concen- ing cells in the brain, and during the early stag- trations, losses of intact amino acids to the es, the amount of CCK in the nervous system is surrounding water seems to be low. predominant. Recent data support earlier assumptions that the Preliminary studies of herring larvae indicate ability of larvae to digest protein improves after that CCK is involved in the secretion of trypsin. metamorphosis (Figure 6). In halibut, about 30 One study showed that the presence of proteins per cent of a protein administered via a tube was in the digestive system led to higher levels of absorbed at the larval stage, while after meta- CCK in larvae, while the presence of FAA did morphosis, absorption efficiency had risen to 60 not have the same effect. Higher levels of CCK per cent. correlated with increased trypsin activity in the larvae. In this study, the gut was also supplied About 60 per cent of the protein was also ab- with saline in such amounts as to overfill it. This sorbed at the larval stage if it had been prehy- did not lead to changes in the level of CCK, al- drolysed with pepsin. It was also demonstrated though trypsin activity increased after a while. that the capacity to digest protein decreases as This indicates that distension of the gut wall via the amount of protein in the gut increases. This stretch receptors is not a triggering factor in the may be related both to digestive capacity and to CCK-based regulation of trypsin release. the evacuation rate of the gut.

A higher percentage of added protein hydroly- Essential amino acids are incorporated into tis- sate in the diet of sea bass larvae lowered sue proteins to a greater extent than non-essen- trypsin secretion and CCK levels. Taken togeth- tial amino acids (80–90 per cent as against 20– er, these data indicate that dietary protein con- 30 per cent). Non-essential amino acids tend to tent, in combination with protein and peptide be used more as a substrate in energy metabolism. This indicates that fish larvae can deal

228 Aquaculture Research: From Cage to Consumption tissue to be followed. In larvae of Senegal sole, about 30 per cent of the amino acids in Artemia were taken up and transferred to tissue within an hour of being consumed. In juveniles that had developed a stomach, around 55 per cent of the amino acids were transferred within the same period of time. This shows that the digestion of live feed is more efficient when it is pre- digested in a functional stomach, even though the difference is less than might have been expected.

Effects of hydrolysed protein in the feed

145x100//Kap13-fig05.eps on cod and halibut larvae Figure 5. Absorption of protein at different degrees of Cod larvae fed on formulated feed have higher hydrolysis by halibut larvae (based on Tonheim et al, survival rates when some of the protein in the 2005). feed is given in the form of hydrolysate. Exper- with and regulate amino acid metabolism to a iments have been done in which as much as 40 greater extent than was previously believed. per cent of the protein was given as pepsin hydrolysate, and survival rates rose in line with the Amino acid retention can be improved if the proportion of hydrolysate. In halibut, on the amino acid profile of the feed is improved by other hand, a gradual decline in survival rates the addition of peptides. Studies have shown was noted as the proportion of pepsin hydroly- that lipid absorption has little influence on ami- sate in the feed protein was raised to 45 per cent. no acid absorption. Methods for tagging amino These results are not in agreement with the ef- acids in Artemia (largely protein) with isotopes fect of hydrolysis on the uptake of tube-admin- have also been developed, allowing the rate of istered protein by halibut larvae (Figures 5 and amino acid uptake, transfer and metabolism in 6), but they can be explained by the leaching of

145x100//Kap13-fig06.eps Figure 6. Comparison of the absorption of intact and hydrolysed protein in halibut larvae and juveniles (Tonheim et al, 2005).

Thematic area: Feed, Nutrition, Feeding 229 the market recently have improved pigmentation in commercially farmed halibut juveniles. We do not know to what extent thyroid hormone affects pigmentation, but we believe that the process of pigment cell development involves interactions among the three above-mentioned factors, and possibly others as well. We are thus now in a position to avoid faulty pigmentation, but we do not know the mechanisms involved in pigmentation. An understanding of this process might prevent the reappearance of faulty pigmentation in the event of changes in fish farm-

145x100//Kap13-bilde04.eps ing procedures. Atlantic halibut juvenile. Photo: Øystein Sæle, University of Bergen Incomplete eye migration is still a recurring problem in halibut farming. The literature on water-soluble protein from feeds that contain a this problem is sparse, even though the problem high proportion of hydrolysate. Halibut are is also found in other flatfish, but some hypo- slow to tackle feed in the water, and the feed theses have been put forward. Thyroid hormone particles remain suspended for a long time be- controls metamorphosis in flatfish, as it does in fore they are consumed. Cod, on the other hand, other vertebrates. Iodine deficiency may result are very rapid feeders, with the result that there in reduced thyroid hormone synthesis. Iodine is less leaching to the water. Furthermore, the levels are 50–700 times as high in copepods as protein requirements of halibut are probably they are in Artemia. Marine organisms usually higher than those of cod (see below), and pro- obtain the iodine they need from seawater, but tein leaching is thus more critical for halibut. iodine is found as I-, its available form, only in Hydrolysed protein had no effect on growth rate the photic zone. Fish farms with deep water in- in either species. lets may therefore find themselves pumping in seawater that is low in available iodine. In con- Effects of nutrition on metamorphosis in junction with a low iodine content in the feed, halibut this may result in iodine deficiency, reduced A survey of the literature shows that vitamin A, thyroid hormone synthesis and incomplete fatty acid composition and thyroid hormone all metamorphosis. There are also certain indica- influence pigmentation in flatfish. Iodine is es- tions that a more intense supply of energy to the sential for the synthesis of thyroid hormone. larvae, for example through feeding with “on- Even though Artemia lack vitamin A, halibut grown” Artemia (i.e. Artemia that have been fed larvae appear to obtain a sufficiency of vitamin on fish meal for four days) may improve eye mi- A through the conversion of canthaxanthin from gration. Better eye migration has also been Artemia. It is generally agreed that fatty acid found in a group of larvae whose level of fat was composition affects pigmentation in halibut, a four times as high as that of the control group. supposition that is supported by the fact that the Juveniles with incomplete eye migration are better enrichment methods that have reached often doubly pigmented.

230 Aquaculture Research: From Cage to Consumption Optimal composition of macronutrients enough to be able to control the fat composition in weaning feeds for cod and halibut of rotifers. We need to continue to study the We carried out a series of experiments on cod question of a reproducible and satisfactory fat and halibut with the aim of identifying the opti- composition in Artemia and to control the con- mal composition of macronutrients in weaning tent of potentially rate-limiting micronutrients feeds and feeds for small juveniles. The protein in both of these feed organisms. When this has requirement of halibut was less than 60 per cent. been done, we will be in a position to design A search of the literature and unpublished data classical nutrition studies aimed at identifying indicated that the requirement was around 58 the needs of larvae for particular nutrients. per cent. Raising fat content from 5 to 25 per- More knowledge in this area will enable us to cent did not increase growth rate. Raising the improve survival and growth rates and reduce proportion of carbohydrates from 0 to 15 per the incidence of deformities in both cod and cent resulted in a strong increase in the liver in- halibut. In both species, it is essential to identify dex and a rise in liver glycogen concentration. the causes of faulty development, since a better At the highest carbohydrate levels we also understanding of this area will prevent such found glycogen and glucose in other tissues anomalies from recurring when farming condi- than the liver. We concluded that feed for hali- tions and possibly also feeds are altered. We but fry ought to contain less than five per cent know that malpigmentation in halibut is a func- carbohydrate. tion of nutrition, but just what causes developmental anomalies in cod and impaired eye- The protein requirement for maximal growth of migration in halibut is not known. Where for- cod is 41 per cent, and the fat content of the feed mulated feeds are concerned, it will be impor- can be varied between 15 and 25 per cent. When tant to identify means of preventing water- the fat content of the feed in one study was re- soluble micronutrients and amino acids, pep- duced to below 15 per cent, cannibalism in- tides and protein from leaching into the water creased. With more than 25 per cent fat in the column. We also need to examine the potential feed, the liver’s fat storage capacity was a limiting for improving the digestibility of protein and fat factor. Cod fry appear to tolerate and even to ben- sources used in formulated feeds for larvae. efit by carbohydrate levels of up to 15 per cent. A high level of protein in the feed (>63 per cent) re- The development of enzymes involved in protein duced growth in 0.2–1.0-g fry. Our hypothesis is digestion has been partly characterised. A Cana- that this effect is due to late development of the dian group has made important progress on cod in stomach, and thus limited protein digestion, a hy- this area. In halibut, we still lack data on both pan- pothesis that is supported by the fact that pepsin creatic enzymes and pepsin. Generally speaking, activity in stomachs of cod larvae and juveniles extremely little is known about mechanisms of up- continues to increase until the fish weigh 5 g. take of whole protein, peptides and amino acids, how these are modified in the course of development, and how they are influenced by the feed Conclusions and perspectives eaten by larvae at different stages of development. for the future We now know the basic nutrient content of We know little about fat digestion and absorption Artemia, rotifers and copepods and we know in cod and halibut, and it would be useful to start

Thematic area: Feed, Nutrition, Feeding 231 characterising digestive enzymes, uptake mecha- ledge of metabolic plasticity in fish with respect nisms and the uptake process itself. We have just to their ability to process fat and protein, and how begun to study how digestion in fish larvae is these processes may limit growth rates. regulated, and we also still lack sufficient know-

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234 Aquaculture Research: From Cage to Consumption Mette Sørensen1,2), Rune Waagbø3) and Rolf Erik Olsen4) 1) Akvaforsk – The Institute of Aquaculture Research, 2) Aquaculture Protein Centre, 3) NIFES (National Institute of Nutrition and Seafood Research), 4) Institute of Marine Research

Feed Resources – Feed Technology

Aquaculture is the fastest growing food-producing sector in the world. The national and international aquaculture industry considers the availability of suitable feed resources to be one of the greatest challenges to future growth. The world's marine fish resources are limited, and although a large proportion of these are used for aquaculture, sustainable alternatives must be sought lower in the food chain, both in the sea and on land. We have already seen a global shortage in the supply of marine oils, and there has therefore been a particular focus on the supply and quality of fat sources in feed for salmonids. Although the fat sources do not affect the growth of the fish to a great extent, aspects such as well- being and health, quality of the end product, and not least safety are highly relevant. Research related to feed resources and technology has largely concerned studies of biological suitability and optimisation of existing production processes.

Thematic area: Feed, Nutrition, Feeding 235 The marine fish resources that have been used per year. Although Norway at present utilises in the production of fishmeal and oil are at up to 75 per cent of its by-products, from a glo- present very limited and to some extent overex- bal perspective there is great room for improve- ploited. Evaluating the use of other available in- ment. Every year large quantities of fish also gredients for feed from the sea and from land disappear as by-catch, especially from the has thus been recognised as essential for further trawler fleet. This resource has great potential growth in the aquaculture industry. An investi- for utilisation. Further, we know that the annual gation into feed resources concluded that there production of biomass at lower trophic levels is were many potential future resources applicable large, while only a fraction of this biomass is in fish farming under certain conditions. In this used by fish and marine mammals. chapter, we will try to summarise the research in feed resources and feed technology carried One of the greatest challenges involved in the out under the Aquaculture programme. On the use of trimmings and by-catch is logistics and basis of great differences in the challenges and intermediate storage of the products before pro- approaches relating to different feed resources, duction of feedstuffs or feed. Fermentation and we divided this chapter into research on marine ensiling are the methods assumed to have the resources, on plant resources, and on micro- greatest potential, even though these processes organisms produced in culture. In evaluating may change the quality of the feedstuffs. Fish the suitability of raw materials, the industry silage has also been used as raw material for must take into account issues other than price further processing into fishmeal and fish oil. and availability. Documentation and research The results show that when the quality of the must be undertaken to investigate the nutritional raw materials is good, it is possible to produce quality of new raw materials, their technical fish silage products of excellent quality. Poor quality, their safety for fish, the environment quality raw materials, however, result in signif- and the consumer, and the logistics involved. It icant deterioration in the silage quality, and in is also vital to confirm that the feed resources meal and oil produced from the silage. In partic- provide seafood in line with ethical standards ular, rancidification of the fat may cause prob- and the consumer’s needs and demands. The lems. Changes in technical properties have also section on research into feed technology prima- been observed in extruded feed based on fish- rily covers alternative production methods for meal mixed with fish silage. The feed becomes feed and processing of feed ingredients. less expanded and more compact. Although this will reduce the fat-holding capacity of high- energy feed, there are advantages in using silage Marine feed resources in fishmeal production. Growth trials on salmon By-products and by-catch have shown that feed with up to 20 per cent sil- One way to increase the availability of marine age in the production process yields the same ingredients for fish feed is to improve the util- growth, colour and feed conversion as feed isation of marine by-products and by-catch. In based on LT (low temperature) fishmeal. New the fishing industry, up to 50 per cent may end methods for production of feed ingredients up as by-products in the form of trimmings, from silage, by-products or by-catch make an heads, viscera, skin, backbones, etc., and na- important contribution to improving the tionally this comprises about 600,000 tonnes exploitation of marine raw materials. Various

236 Aquaculture Research: From Cage to Consumption 145x100//Kap14-fig01.eps Meganyctiphanes norvegica (Photo: Line Anfinsen, Institute of Marine Research) technologies are described in the section on amphipod from Northern waters as well as feed technology. Antarctic krill have confirmed that krill can replace fishmeal as the protein source of at least Krill and plankton 40–60 per cent of the protein in the feed, with- At a lower trophic level, krill and plankton pro- out challenging growth, feed conversion or vide another significant source of marine feed quality. For higher inclusion levels growth may ingredients. Although we still do not have a be comparable with fishmeal, but the feed con- complete overview of biomass and production, version ratio has a tendency to increase, which it is assumed that in Northern waters, primarily means that the fish must eat more feed to the Norwegian Sea and Barents Sea, the volume achieve the same growth. The reason for this is amounts to several hundred million tonnes. still not clear, but there are strong indications Euphasiid and Calanus spp probably have a that the fish get a mild form of diarrhoea, due to production capacity high enough to allow prof- the high levels of chitin in krill meal. itable harvesting. There are also strong indications that only a fraction of this production is Although krill and amphipods contain the used by fish and mammals in the ocean. Utilisa- essential fatty acids EPA (20:5n-3) and DHA tion of these products could provide raw mate- (22:6n-3), they are, with few exceptions, prima- rials for the aquaculture industry far into the rily protein sources. A shortage of marine fat future. for fish in aquaculture is a major concern in the coming years. Here, Calanus finmarchicus is a Feeding trials with salmon, cod and halibut relevant resource. The biomass of Calanus in given feed based on three krill species and an the ocean outside Norway is conservatively es-

Thematic area: Feed, Nutrition, Feeding 237 a) b)

145x100//Kap14-fig01.eps Figure 1a. Levels of dioxins (PCDD/F) and dioxin-like PCBs (DLPCB) in farmed salmon fed with feed based on fish oil (FO) or vegetable oil (VO) during a complete production cycle (Berntssen et al., 2005). Figure 1b.Correlation between observed levels of dioxins and dioxin-like PCB congeners in commercially farmed salmon, and predicted levels based on known levels in feed and a simple first-order kinetics model (Berntssen, 2007. Pers. comm.).

timated to constitute several hundred million in seawater, and very low concentrations are de- tonnes. Depending on the time of year, this posited in the fillet. It therefore remains to as- plankton may contain large quantities of fat. sess the implications of elevated dietary This fat is, however, in the form of wax esters, fluoride for the health of the fish. Results from and not triglycerides as in fish oils. Since these such trials will improve the scientific basis for may be toxic in large quantities, they are not establishing more appropriate limits, making it suitable for human consumption. If fish can possible to use krill meal safely as a future feed convert wax esters to “healthy” fat, use of resource. Preliminary indications show that cer- Calanus in fish feed could make a significant tain krill meals also contain concentrations of contribution of marine fatty acids for human selected undesirable metals above the upper consumption. These issues are the subject of a limits stipulated in feed legislation. Despite project in which fish oil is replaced with wax higher concentrations, feeding trials indicate from Calanus finmarchicus in feed for salmon, that replacing fishmeal with krill meal in fish cod and halibut. Although the project is in the feed indeed reduces the levels of these metals in start-up phase, we already know that salmon cod and salmon fillets. utilise wax esters about as well as fat from fish oil. By-products from mussels The mussel industry in Norway has consider- A challenge with krill as a feedstuff is its high able production potential, and by-products from fluoride content, which in certain species may mussels are a potential feed resource. There is be as high as 6 g/kg dry weight. The EU’s upper significant wastage in today’s production due to limit of 150 mg fluoride/kg feedstuff currently rejection of small sizes, breakage during pro- prevents the aquaculture sector from using duction, attached barnacles, and the content of these meals as a protein source in fish feed. It is, algae toxins, which in certain cases are discov- however, known that most of the fluoride from ered after harvesting. Controlled disposal of by- the krill meal in the feed is not absorbed by fish products involves substantial expenses for the

238 Aquaculture Research: From Cage to Consumption producers. Utilising these products as ingredi- pared with fish fed diets based on marine oils. ents in fish feed would both reduce the costs of Studies have also been conducted in which as- disposal and provide a market for the whole similation and excretion rates for 29 different mollusc. In a research project, the possibility of chemical forms of dioxins, furans, and dioxin- using mussels in feed for salmon and cod was like PCBs have been estimated. The results of investigated, using mussels both with and with- such trials can be used to predict the end-point out detected diarrhoea toxins. Mussels were level of POPs in commercially farmed salmon processed through boiling, cleaning and drying, when the levels in feed are known (Figure 1). and then ground to a fine powder. The protein and fat content, as well as the composition of While fish oil is the principal source of many amino acids in the mussel meal, were compar- organic pollutants, fishmeal is the principal able with fishmeal. Feeding experiments in- source of undesirable elements, such as arsenic cluding up to 8 per cent mussel meal in feed for and mercury. The toxicity of these metals and salmon showed no change in palatability, de- the transfer from feed to fish is determined by spite the fact that mussel meal contains some the chemical form. In marine feed ingredients, flavour-promoting free amino acids in higher both arsenic and mercury occur in organic concentrations than fishmeal. Short-term trials forms. The organic form of mercury (methyl with feed supplemented with pure toxins mercury) is toxic, while the organic forms of ar- showed that salmon tolerated toxin levels which senic (like arsenobetaine) are not toxic. Experi- are regarded as threshold values for human con- ments have been conducted to evaluate the sumption, although this affected certain clinical dietary upper limit for methyl mercury toxicity, blood parameters. At these toxin levels, the using early biomarkers specifically selected for growth of the fish was also reduced. This indi- dietary exposure. Increased intestinal cell pro- cates that mussel meal can be used in fish feed, liferation, indicated by PCNA immunohis- but meal with toxins should be avoided. tochemistry, was observed in salmon fed with 1 mg methyl mercury per kg of feed for two Undesirable compounds in traditional months. After four months, reduced cell prolif- and new marine raw materials eration and histopathological changes (shorter Feed ingredients are unfortunately also sources and thicker villi) were observed in the intestine of a number of undesirable substances, which (Figure 2). have drawn attention relative to the production of safe seafood. Certain marine oils may in- Arsenobetaine is known to be non-toxic to ani- clude substantial quantities of persistent org- mals and humans. While a great deal is known anic pollutants (POPs), such as dioxins, PCBs about the assimilation and excretion of arseno- (polychlorinated biphenyls) and brominated betaine in humans, the mechanism in fish is still flame retardants. The problem with these com- unclear. To study deposition of the compound in pounds is that they are stored in the fat, and bio- fish, salmon were fed with isotope-marked arse- accumulate in the food chain. Thus, oily farmed nobetaine. The results showed that the com- fish may retain the dietary undesirables in the pound is distributed throughout the fish, with muscle. With the use of plant oils, it has been the greatest accumulation in the muscle. This is shown that the level of dioxins and dioxin-like in contrast to mammals and humans, where ar- PCBs can be reduced by eight to 12 times com- senobetaine is not stored in any organs, but is

Thematic area: Feed, Nutrition, Feeding 239 a) b) c)

145x100//Kap14-fig01.eps Figure 2. Pronounced increase in cell proliferation, shown immunohistochemically coloured with PCNA, in intestine of salmon after feeding for two and four months with feed supplemented with methylmercury (Berntssen et al., 2004).

rapidly excreted in the urine. By analysis of to- these affect the commercial value, the nutri- tal quantity of arsenic in feed ingredients, feed tional and technical quality of the feed, and the and fish fillets, potentially high levels of arseno- actual processing of the feed. In addition, betaine found in marine raw materials could be processes which could reduce the content of misinterpreted as hazardous arsenic. It is there- anti-nutrients in plant protein sources are being fore important to analyse and report the exact explored, especially with regard to any negative chemical species of the undesirable substances. effect on the intestine of the fish and potential implications on feed conversion efficiency, Research results within undesirable substances physiology and well-being. are very important to the operating conditions for the aquaculture sector, since the authorities Plant fat sources in salmon feed in the EU and in Norway take such results into Various plant oils have been used as a substitute account when the legislation on feedstuffs is to for fish oil in feeding trials with salmon, either be revised or new safe upper limits are to be de- added as pure oils (for example soybean oil, termined. rapeseed oil or linseed oil) or as oil mixtures. The main conclusion is that salmon tolerates a relatively high content of plant oils in the feed, Feed ingredients from plant without any impact on growth or feed utilisation sources in salmon of different sizes. The concerns on There is currently a general national ban on the the use of plant oils in feed for fish has primar- use of slaughter trimmings from land animals in ily focused on changing the composition of fat- fish feed due to the fear of spreading bovine ty acids in the fish fillet and how this affects spongiform encephalopathy. The search for al- other quality characteristics of the product. Fish ternative protein and fat ingredients for fish is traditionally associated with a high content of feed has therefore resulted in a strong focus on healthy long-chain n-3 fatty acids (the “fishy the use of plant ingredients, as well as of ingre- fatty acids” EPA and DHA). The level of these dients based on micro-organisms. fatty acids is reduced in the fish fillet when the fish is fed with plant oils, and the level of C18 Research on the use of plant and microbial fatty acids increases. It has therefore been con- sources in feed for fish is concentrated on how sidered important to focus the research on

240 Aquaculture Research: From Cage to Consumption health aspects of both the farmed fish and the onment, such as the pesticide endosulfan. The consumer in relation to the replacement of ma- existing upper limit for endosulfan in fish feed rine oils with plant oils in fish feed, with a spe- is considerably lower than for other livestock. It cial focus on effects related to immunology, is based on the naturally low concentrations of lipid oxidation, undesirable substances (diox- the pesticide in marine feed resources and the ins) and cardiovascular diseases (the “from high sensitivity of the fish to water exposure. fjord to table” concept). Research has shown Trials are being conducted with the objective of that changes take place in the cardiovascular determining toxic concentrations for endosul- system of salmon corresponding to the develop- fan in fish feed. ment of atherosclerosis in humans, and the degree of changes is related to the size or age of Plant protein ingredients in feed for the fish. No clear associations have, however, salmon and cod been found between the fatty acid composition Plant protein sources comprise a large group of in the feed and the degree of cardiovascular potential ingredients. Soybeans dominate the changes in the fish. Although results from on- world market because of supply, price and nu- going research projects are still being pro- tritional quality. There are two key challenges cessed, the results so far confirm that plant oil regarding the use of soybean for fish. First, soya can make up a considerable proportion of the contains indigestible components and anti- energy in the feed without affecting the health nutrients that are harmful to the fish. It has been of the salmon. shown that soybean meal in feed for Atlantic salmon causes inflammation-like reactions in A clinical trial on nutrition in humans showed a the posterior intestine, which may be unfavour- therapeutic effect associated with increasing the able because of reduced immunological func- content of marine n-3 fatty acids in the salmon tion of the intestine and reduced feed conver- fillet, so that the daily intake averaged 3 g. De- sion efficiency. The second major challenge is spite a lower content of marine n-3 fatty acids in related to the use of genetically modified (GM) salmon fed with plant oil, patients eating this soya. Globally, the production of GM plants is salmon showed a neutral or positive response in increasing – primarily glyphospate tolerant key markers related to the development of car- soya, maize and cotton. The use of GM ingredi- diovascular diseases. This means that there are ents is regulated by Norwegian and EU feed also factors other than n-3 fatty acids in salmon legislation, with regard to both approved prod- fillets that are beneficial in prevention of these ucts and mandatory labelling of products that diseases. In the same clinical study, the intake include GM ingredients. Because of the gener- of environmental pollutants was also studied, ally negative attitude to GM products in public and the results from this study may provide a opinion, the aquaculture sector wishes to use contribution to the debate about product man- GM-free ingredients (nGM) in fish feed, and agement and risk related to the intake of oily these currently are more expensive than GM fish. products. Results from a project which aimed to study the health risks associated with GM Use of plant oils as feedstuffs may introduce plants in salmon feed showed that DNA from new undesirable substances that usually occur GM soya passes unchanged through the intesti- only in low concentrations in the marine envir- nal tract. The DNA sequences were not found in

Thematic area: Feed, Nutrition, Feeding 241 tissue from the intestine, liver, brain or muscle ive digestive enzymes, as well as the presence of the salmon. Another study investigated of a ‘fermentation chamber’ with high micro- whether incorporation of 12 per cent of GM bial activity in the distal part of the intestine, soya and maize into salmon feed resulted in which may participate in digestion. There have measurable changes in the intestinal mucosa in been some concerns that maize in diets for cod relation to fish given feed based on GM-free could discolour its white flesh. Research so far ingredients and fishmeal. There were no differ- has shown that maize gluten caused a yellow ences between GM and nGM ingredients, but hue in the skin, while the muscle was not dis- feed based on soya induced changes in intest- coloured. inal cells compared with maize and fishmeal- based feed. The main conclusion of a PhD thesis on GM ingredients in salmon feed is that in- Micro-organisms as a feedstuff corporation of low levels of GM plant materials for fish (soya and maize) can be regarded as safe, both Bacteria for fish and for consumers. Changes in spleen Today, substantial volumes of natural gas are size which may be attributable to the use of GM produced on the Norwegian continental shelf. feed have, however, caused concern, prompting In addition to serving as fuel, gas can be used as a cautious attitude to GM feedstuffs for fish un- an energy and carbon source for heterotrophic til more research has been conducted. biomass production. Bioprotein® (BPM) is a promising new feed ingredient produced in Cod is a relatively new species in the aquacul- Norway by Norferm AS; however, production ture context, and several projects have been un- was, at least temporarily, discontinued in 2006. dertaken to study feed efficiency and product The single cell protein was produced using aer- quality in relation to the choice of ingredients obic fermentation of bacteria, primarily and chemical composition of cost-effective Methylococcus capsulatus. Natural gas was the feed. Methodology used to determine the di- energy source, while ammonia, water, phos- gestibility of main nutrients may influence the phate, and minerals were added to the process. result, and attempts have therefore been made The product was spray-dried and had a chemi- to standardise this for cod. The main conclusion cal composition of 70 per cent protein, 10 per from this research is that cod have a greater cent fat and about 7 per cent ash, and an amino ability than salmon to adapt to plant feed ingre- acid profile fairly similar to a high-quality fish- dients. Even the use of relatively high levels of meal. The fat consists primarily of phospho- soybean meal in the diets for cod resulted in lipids in which the fatty acid composition is good growth, health, and product quality. In sin- dominated by the fatty acids C16:0 and C16:1. gle trials, however, minor histological changes BPM is legally approved for inclusion at 33 per in the intestine were observed. The digestibility cent in feed for salmon in seawater and 19 per of the fat was reduced somewhat with the use of cent in feed for salmon in freshwater. The main 25 per cent soybean meal. The cod seem to conclusion of a PhD thesis studying use of BPM compensate for this with a higher intake of feed. in diets for Atlantic salmon, rainbow trout and The finding that cod utilises soya-based feed Atlantic halibut was a slight reduction in appar- better than salmon is probably explained by the ent digestibility coefficients with increasing in- longer gastrointestinal tract in cod, less sensit- clusion level of BPM replacing LT fishmeal in

242 Aquaculture Research: From Cage to Consumption the diet. The growth rates and feed efficiency diets in which BPA and BPM replaced up to ratio, however, were highest in Atlantic salmon 250 g kg-1 of crude protein from high-quality and rainbow trout fed the highest levels of BPM fish meal. in the diet, 36 per cent and 27 per cent respectively (Figure 3). BPM in diets for Atlantic hal- Microalgae ibut can be included up to 9 per cent without Microalgae form the basis for most of the negative effects. Atlantic halibut fed up to 18 aquatic food chains and therefore play a key per cent BPM in the diet showed reduced feed role in the nutrition of fish. For example, micro- intake, growth rates and feed efficiency ratio. algae are currently used as feed for zooplankton, which are in turn used for start-feeding of BPM bacterial protein meal autolysate (BPA) is marine fish fry such as halibut, turbot and cod produced from BPM under high pressure by hy- (see separate chapters). Microalgae may also drolysis catalysed by endogenous enzymes and have a potential for direct use as a raw material is characterised by a high content of free amino in fish feed. The chemical composition of acids and low molecular weight proteins. A microalgae varies depending on the species, but higher digestibility for most indispensable ami- it is foremost as a net producer of long-chain no acids in the BPA compared to BPM has been polyunsaturated n-3 fatty acids EPA (20:5 n-3) reported in feeding experiments with mink. No and DHA (22:6 n-3) that microalgae are inter- such differences in digestibility of nitrogen and esting. The concentration of unsaturated fatty amino acids were observed in rainbow trout fed

Specific growth rate (SGR)

120 a a ab b b 100 a a 80

60 b

40 Salmon

SGR (% of control group) 20 Trout Halibut 0 0 9 18 27 36 Dietary BPM inclusion level (%)

145x100//Kap14-fig01.eps Figure 3. Specific growth rate (% of body weight per day) of salmon, trout and halibut fed diets containing increasing levels of bacterial protein meal (BPM). The values are standardised to per cent of the specific growth rate of the control group of each species. Significant differences within each species are denoted with different letters. No significant differences were found in trout. (Aas, 2006..)

Thematic area: Feed, Nutrition, Feeding 243 acids is affected by incubation factors including Feed technology food supply and temperature. Most fish feed production in Norway today is based on extrusion technology. The process in- Nannochloropsis oceanica is an alga species volves mixing, shearing and heating under ele- that both satisfies the requirement as a high vated pressure, before the extrudate is forced PUFA source, and can be produced efficiently through a die to produce an expanded pelleted in significant quantities. A number of trials have feed. Great effort has been made to optimise the been conducted, aimed at optimising produc- extrusion process in relation to the type of in- tion conditions such as light, temperature, CO2 gredients to produce a high-quality pellet that and food supply. The results have shown that N. readily absorbs oil in a vacuum coater. This is oceanica can be produced at 100 g/m2/day, and will continue to be an ongoing initiative which results in a potential of approximately 30 from the feed producers, especially when new kg dry algae mass/m2/year using additional raw materials with different technical character- light. The content of polyunsaturated fatty acids istics are introduced. Optimisation of feed tech- can be doubled or tripled by reducing the water nology is aimed at improving technical quality, temperature from 25 to 10 oC a few days before increasing the availability of nutrients and re- harvesting. N. oceanica has also proved to grow ducing the content of anti-nutrients in the feed. very well in 200-litre pilot reactors of the Research has shown promising results in reduc- Biofence type. The project indicates that it is ing the heat stable anti-nutritive factor phytate possible to develop the production of micro- under conditions that can be directly applied in algae into a new industry. This is, however, a an extrusion line. More research is, however, new research field and a major effort is required needed to develop a fully integrated online tech- before such production can be initiated. nology for this purpose.

Similarly, biotechnological production of the During the past decade, alternative production lipid-rich marine micro-organisms methods for fish feed have been launched in- Traustochytrids through large-scale fermenta- volving the direct use of by-products and indus- tion may represent valuable contributions as trial fish, without separating the fat and protein feed resources and a source of essential fatty phases into fish oil and meal. The techniques acids for the aquaculture sector. They may be were developed to improve the exploitation of used both in marine start-feeding and as an in- by-products, and also represent energy-saving gredient in growth feed to complement the long production methods. One of the methods is chained n-3 fatty acids EPA and DHA which based on the use of microwave technology to plant oils lack. The microbes contain 47 per heat (and thus disinfect) and bind pellets cent fat on a dry matter basis and can be used di- through protein coagulation. Another technique rectly or fractionated into oil and a meal frac- uses the gelling properties of certain binding tion rich in phospholipids. In the continuation agents, which are mixed with minced fish and of the ongoing chemical characterisation, the activated in an acid bath, known as the Gelly- products will be evaluated as feedstuffs for fish Feed technique. The advantage of both technol- and as an enrichment media for rotifers for use ogies is that feed can be processed locally, with in the production of marine larvae. local resources and players. It is also possible to vary the feed ingredients to a greater extent, and

244 Aquaculture Research: From Cage to Consumption 145x100//Kap14-fig01.eps GellyFeed production plant – from production of preserved paste to finished pellets (Photo: GellyMar) to choose between producing fresh moist feed EU’s new hygiene requirements. Bacteria and and drying the produced feed. On the other viruses are inactivated. The unique aspect of hand, the supply of raw materials for alternative alkaline preservation is that the enzyme activity production of fish feed is unpredictable, an ef- normally present in by-products from fish and fect which is intensified in a large-scale system. in traditional silage is also inactivated. This The concept of feed produced using microwave results in a low level of protein autolysis, con- technology has been refined into the Sea Grain® tributing to good water-binding properties. In process, in which an alternative type of fat-rich addition, an ‘osmosis effect’ is achieved, caus- fishmeal is manufactured from marine by-prod- ing the water to be absorbed into the pellet so ucts and from pelagic species using microwave that the consistency of the paste becomes firm. heating. The second stage of the GellyFeed process en- GellyFeed technology tails making pellets from the preserved paste. GellyFeed is a patented process for the produc- Before pelleting, the paste is processed in a tion of fish feed, developed by Norsk Hydro. mixer into which alginate and other additives The process comprises two stages. The first such as vitamins and antioxidants are incorpo- stage is preservation of by-products from the rated. Finally, the mixture is pelleted and the fish industry by adding an alkaline preservative. final structure is set by immersing the pellet in This results in a fish paste with a firm consist- an acidic bath. The acid bath neutralises the ency and storage stability, which satisfies the alkaline pellet and immediately starts a gelling

Thematic area: Feed, Nutrition, Feeding 245 process that makes the pellet water-stable, non- protein, which has been shown to result in dif- sticky, and durable. This results in a pellet ferences in appetite, growth, and feed conver- which may consist of 97 per cent marine ingre- sion. dients. The finished pellet has a pH of 4–5 and a dry matter content of between 30–40 per cent. The idea behind the Sea Grain® concept is The chemical composition can be adjusted to future on-board production of high-quality fat- satisfy the nutrient requirements by adjusting rich meal with better utilisation of by-products the ingredient composition. For a leaner feed, a and by-catch, lower transport costs for dry mixture of white-fish paste and oily-fish paste is products, and sustained quality of these reused. If only herring trimmings are used, the resources. A challenge related to the fat-rich sult is a salmon feed with a high content of fat products is the lower potential for removal of and protein. The digestibility of the main nutri- contaminants with the use of, for example, by- ents for salmon is good, and often better than products with a high content of organic pollut- commercial dry feed, with a digestibility of 96 ants and other contaminants in production (see per cent for fat, 81 per cent for protein and 87 below). per cent for energy. The salmon also showed improved feed conversion. Arrangements are Technology for modification and refining being made for local production under licence of marine resources with feed factories that produce less than New national and international legislation im- 10,000 tonnes per year, that is, considerably poses restrictions on the use of by-products. smaller units than in the dry-feed industry. Many available raw materials with a low level of utilisation or high content of undesirable sub- Sea Grain• technology stances must be modified or refined if they are The process of manufacturing Sea Grain® prod- to be used as feedstuffs or supplements in fish ucts involves mincing, emulsifying and drying feed. the marine by-products or raw materials on the basis of a patented process. By-products from Marine by-products are valuable raw materials, herring, capelin, sand eel and blue whiting are and efforts are being made to achieve increased used in the production of Sea Grain®, which is utilisation of these in the manufacture of new then used in extruded feed for salmon smolt. consumer products, biochemicals and feed- These Sea Grain® products have low water con- stuffs. In volume terms, however, it is the pro- tent and are therefore easy to store. Depending duction of feedstuffs, in the form of silage on the ingredients, the products varied between concentrates, fish oils and meal products con- 37–41 per cent fat and 50–56 per cent protein on taining protein, for which there is processing a dry matter basis, but it is possible to adjust the capacity and a market large enough to absorb composition further using other combinations the largest quantities of by-products. As men- of ingredients. The results of the trials have tioned before, a knowledge base has been devel- shown that the Sea Grain® products are well oped for commercial application of fish silage suited to salmon feed, without negative effects in the fishmeal industry, and it is estimated that on growth and health of the fish. The composi- some 20 per cent of silage could be used in tration of the products has, however, varied some- ditional fishmeal and fish oil production with no what, primarily in the quantity of soluble deterioration in quality.

246 Aquaculture Research: From Cage to Consumption Hydrolysis of by-products using industrial en- to be able to remove up to 90 per cent of pollut- zymes is another way to improve the efficiency ants in the laboratory, and this is better than or of utilisation of by-products and other industrial equivalent to what can be achieved with solvent fish. This process can be compared with silage. extraction of fishmeal. Solvent extraction is not The protein is hydrolysed into smaller peptides desirable since it may change the nutrient com- and amino acids, and can be used as a readily position of the finished meal. Removing con- available feed supplement for farmed fish. taminants with soybean oil, however, results in Feeding trials with salmon have shown that 18– an undesirable change in the fatty acid compo- 24 per cent of the fishmeal protein can be re- sition of the fishmeal. This may be solved in the placed with fish protein hydrolysate. The ad- extraction process by replacing soybean oil vantage of hydrolysis with industrial enzymes with marine oil, which is itself purified after- is the increased predictability and reproducibil- wards. These are regarded as processes that can ity of the products, and it is a significantly mild- easily be integrated into the existing production er and less time-consuming treatment than the of fishmeal and fish oil. ensiling process. Activated carbon is an effective technique for For certain marine raw materials, the content of removing dioxins from fish oil, either alone or undesirable pollutants will exceed the upper in combination with a deodorisation technique limits specified in the legislation. The focus on (vacuum pump distillation). Dioxin-like PCB organic pollutants such as dioxins and PCBs has (DLPCB) compounds are less efficiently re- been strong, especially for industrial fish caught moved, while brominated flame retardants (PB- in overtaxed areas such as the North Sea, the DE) cannot be removed using this method. Baltic Sea and Skagerak, and for by-products Alternatively, molecular distillation appears to from the white fish industry. Feeding trials with enable highly efficient removal of dioxins, salmon have shown deposition of PCB of 80–90 dioxin-like PCBs and brominated flame retar- per cent and deposition of dioxins of 50 per cent dants in fish oil (more than 90 per cent). The in salmon (Figure 1). The international legisla- process also reduces the content of fat-soluble tion will be revised to include PCBs, and one vitamins, while the fatty acid composition and should be prepared for the prospect of require- oxidation status are not changed. The research ments for removal of contaminants from raw concludes that technology for removing pollut- materials. Research on several cost-effective ants exists, but that it is a challenge to find cost- cleaning techniques for fishmeal and fish oil has effective solutions that take into account the been evaluated and tested. The organic pollut- other qualities of the raw materials and, in turn, ants are fat-soluble, and simply reducing the fat the quality of the seafood products. component of the fishmeal will be an adequate ‘decontamination technique’ for certain raw materials (30–40 per cent efficiency). Other raw Perspectives materials will require more drastic cleaning. Fishmeal and fish oil have traditionally been the One of the initiatives tested entailed a new prin- dominant ingredients in salmon feed. The ciple for removal of pollutants based on the ex- aquaculture industry has already experienced traction of press cake from fishmeal production limitations in the marine resource base, and fur- with soybean oil. The process has been shown ther pressure on these resources is expected

Thematic area: Feed, Nutrition, Feeding 247 with reallocation of a higher proportion directly novel raw materials may, however, influence to human consumption. This has necessitated both the nutrient content and the technical qual- an intensified research effort to find sustainable ity of the feed. These issues require further re- alternatives that can provide a sound basis for search. New species in aquaculture also result further growth and profitability in the aquacul- in new problems and challenges. Cod requires ture sector. It is especially urgent to find alter- feed with a lower fat content, and at the same native fat sources of acceptable quality which time appears to tolerate plant protein sources take into account economic factors, production better than salmon. This will probably reduce capacity, the aquaculture environment, the future competition for feed ingredients between health of the fish, and not least the production of our foremost farmed species. Development of a safe and healthy product. Research has gone a technology and strategies for refining and re- long way towards confirming that there are moving contaminants and undesirable sub- many alternative fat and protein raw materials stances from existing and potential future among marine crustaceans, plant products and feedstuffs must continue. In the same way, single cell organisms. Short-term challenges are knowledge about undesirable substances must related to the influence of the fat sources on the be improved through research to establish real- seafood fatty acid composition and content of istic upper limits that protect the environment, harmful and undesirable substances. For protein the health of the fish, and product safety for the ingredients, the main challenges are related to consumer. For many alternative feed ingredi- anti-nutrients and undesirable substances, ents, such research documentation is absolutely which often depend on the degree of refine- vital to consider even using them. ment. Research related to modelling and the development of feeding strategies shows that with The authors wish to express their deep appreci- the alternative raw materials of today there are ation to the project managers and project colle- opportunities for full or partial replacement of agues who have contributed information to this fishmeal and fish oil in aquaculture. The use of chapter.

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Thematic area: Feed, Nutrition, Feeding 251 Bjørn Bjerkeng1), Trine Ytrestøyl1) and Rolf Erik Olsen2) 1) Akvaforsk – The Institute of Aquaculture Research 2) Institute of Marine Research

Metabolism and Uptake of Carotenoids in Farmed Fish

Considerable progress in the determination of factors that influence carotenoid pigmentation of salmonid fishes has been made in recent years. Comparative trials with red-fleshed Atlantic salmon and white-fleshed Atlantic halibut have revealed that gastrointestinal absorption of dietary carotenoids does not influence the amount of carotenoids deposited in the muscle tissue of the latter species. After intraperitoneal injection of astaxanthin in Atlantic salmon, the concentration was 20 times higher in the blood and 15 times higher in the muscle than was the case after adding astaxanthin to the diet, indicating that the potential for muscle uptake in Atlantic salmon is considerably higher than previously anticipated. Similar indications have come from studies on binding astaxanthin to muscle protein fractions. Comparative studies have shown that fish species with white muscle may absorb substantial amounts in the blood, and that their muscle proteins may associate with astaxanthin. This is evidence for regulation of astaxanthin uptake at the muscle cell level.

252 Aquaculture Research: From Cage to Consumption Appearance is among the most important prop- ucts of salmonid fishes without negative effects erties of food items including fish, and colour is during processing [9]. perhaps the most conspicuous feature of food appearance. Preferences for food items with a Animal colours, including those in seafood, certain colouration are learned and elicit psy- have different characteristics and may be divid- chological expectations based on experience, ed into two main categories. The first category tradition and customs, and also trigger an emo- includes physical or structural colours, and is tional response in the consumer. Muscle colou- not due to true pigments. Such colours are due ration is among the most important quality to light reflections in structural elements of the criteria for salmonid fishes [1]. In a recent real- material. Tyndall blues and diffraction colours choice experiment it was found that consumers in animals are caused by light scattering in col- were willing to pay significantly more for fillets loid systems of solid material in gel. Iridescent of Atlantic salmon (Salmo salar) that had nor- or metallic blue is caused by light interference mal or above normal redness than for paler fil- with thin laminates. Such colours can be found lets [2]. However, there are large differences in in the external ornaments, also those of com- muscle carotenoid concentrations among mercially important salmonid fishes. The sec- salmonid fish species. Muscle concentrations of ond main category consists of true pigments, astaxanthin may range as high as 59 mg kg-1 in and includes carotenoids, lipofuscins, quinones, wild sockeye salmon (Oncorhynchus nerka) flavonoids and anthocyanins, indoles, tetrapyr- [3], whereas the muscle carotenoid concentra- rols, flavins, purins and pterins, and inorganic tion in large rainbow trout (O. mykiss) may pigments. Combinations of the colour catego- reach 25 mg kg-1 [4–5]. ries, for instance Tyndall blue and yellow pigments, may give rise to green colours. In Consumers show preference for pink-coloured addition, the translucency of the material is of products of salmonid fishes. The red colour importance for the appearance. contributes strongly to the pleasure of eating salmon and may have a signalling effect with All aspects of colouration may be altered in fish respect to quality [6–7], and carotenoids act as products post mortem, and certain aspects may an antioxidant in such products [8]. Colour may be manipulated during farming or processing. therefore be used as a direct estimate of quality The main focus in aquaculture has been on mus- since it is required to be within a certain range cle tissue pigmentation of salmonid fishes. The for a given product. Correct colour determines colour of the muscle tissue of the different consumer preference for and prices of salmon salmonid fishes is caused by uptake, accumula- products. It is therefore important to have tion and metabolic transformation of ingested knowledge of variation in colouration among carotenoids, which is the topic of this article. individual fish and between various salmonid Pigmentation of salmonid fishes and fish in fish species, and of the factors that govern this general is a small field internationally. How- variation. Colour may also directly indicate ever, in Norway, where the farming of Atlantic quality by signalling the degree of maturity of salmon in particular is of great economic im- vegetables and fruits. Astaxanthin has a minor portance, it is an important topic of research. influence on taste, and may be added to prod- Much of the research in the field of salmonid fish pigmentation has therefore been conducted

Thematic area: Feed, Nutrition, Feeding 253 145x100//Kap15-fig01.eps Figure 2. Chemical structures of the quantitatively most important geometrical isomers of astaxanthin, all-E- (a), 9Z- (b), 13Z- (c), and 15Z- (d) (3R,3´R)-astaxanthin. Chiral centra are indicated with an asterisk for structure a.

145x100//Kap15-fig01.eps Figure 1. Chemical structures of optical RS isomers of tenoids. Astaxanthin (3,3´-dihydroxy-β,β-caro- astaxanthin, (3R,3´R)- 1a, (3R,3´S)- 2a and (3S,3´S)- tene-4,4´-dione) and canthaxanthin (β,β- astaxanthin 3a. carotene-4,4´-dione) produced by chemical synthesis are, either alone or in combination, in Norway, where continuity and expertise have the most commonly used carotenoids for pig- been accumulated over a period of more than 30 mentation of salmonid fishes. Astaxanthin pos- years. This article focuses on publicly funded sess three optical R/S isomers (Figure 1) and research during the past decade, as well as pro- 272 possible geometrical E/Z isomers, whereof vides references to important literature. the quantitatively most important are the all-E-, 9Z-, 13Z- and 15Z-isomers (Figure 2). The most important alternative sources of astaxanthin are Carotenoids in seafood the yeast Phaffia rhodozyma, the microalgae The conspicuous colour of seafood is often Haematococcus pluvialis or products based on caused by the presence of carotenoids. All ani- crustaceans. mals depend on a dietary supply of carotenoids. Carotenoids are usually tetraterpenes that are Supplementation of fish feed with carotenoids biosynthesised by photosynthesising organisms is expensive, and previously represented up to and certain bacteria and fungi. Carotenoids add 15–20 per cent of total feed costs. Recently, it colour to important aquaculture species, for ex- has become practice to use considerably less ample, the muscle tissue of salmonid fish spe- carotenoids in the feed. Only about 5–15 per cies, the exoskeleton and muscle epithelium of cent of the dietary carotenoids are utilised for crustaceans, the external colour of the integu- muscle pigmentation. The low degree of utilisa- ment of fishes and the gonads of molluscs. It is tion is partly due to a low absorption rate in the highly desirable that fillets of Atlantic salmon gastrointestinal tract, deposition in other organs have a rich pink colour, and for this reason it is and metabolic transformation into colourless necessary to supplement the feed with caro- compounds that may eventually be excreted.

254 Aquaculture Research: From Cage to Consumption Certain fish species, such as salmonid fishes, ed in the faeces. For carotenoids to be absorbed are capable of transforming the 4(4´)-ketocaro- and enter the systemic circulation they must be tenoids astaxanthin and canthaxanthin into vita- released from the matrix that surrounds them, min A1 and A2 (retinol and 3,4-didehydro- be incorporated into mixed micelles, enter retinol) [10–12]. The bioavailability of caro- through the brush border membrane and be tenoids is altered with age and physiological transported through the enterocytes, as well as status in salmonid fishes. Small fish preferen- incorporated into the chylomicrons and lipopro- tially deposit carotenoids in the skin, whereas teins. Typical values for the ADC of astaxanthin deposition in the muscle increases as the fish and canthaxanthin are between 30 and 60 per grow [13]. At the onset of sexual maturation, cent, but higher or lower values may be found sex hormones induce a relocalisation of caro- depending on carotenoid source, dietary dose tenoids from muscle to skin, which contributes and feed composition. Astaxanthin dipalmitate to the characteristic nuptial colouration [14]. and esterified carotenoid are more poorly uti- The colour is of great importance since male lised than unesterified astaxanthin [20]. This sockeye salmon (Oncorhynchus nerka) selec- also appears to be true for natural astaxanthin tively prefer spawning with female models of a esters of the green algae Haematococcus pluvi- deeper red colour [15]. Carotenoids are bioac- alis [21]. Astaxanthin is about 1.4 times more tive compounds. Thus the astaxanthin content efficient as a carotenoid source than canthaxan- of rainbow trout eggs is correlated to fertilisa- thin for muscle pigmentation of rainbow trout tion and hatching rates, which indicates that as- [22,23]. However, in Atlantic salmon canthax- taxanthin is required for optimum reproduction anthin is a better source for muscle pigmenta- [16]. High dietary astaxanthin concentrations tion than astaxanthin [24–26]. The blood may also reduce cataract in Atlantic salmon concentration of carotenoids has been regarded [17]. Astaxanthin and canthaxanthin have been as a good indicator of their bioavailability in regarded as vitamins, and a positive effect on salmonid fishes [27–29]. In Atlantic salmon it growth of salmon larvae has served as a basis was found that more canthaxanthin than astax- for the recommendation that all fish diets anthin was taken up when the dietary inclusion should contain at least 10 mg of these caro- level was similar to but higher than 30 mg/kg. tenoids per kg feed [18]. When the diet was supplemented with both carotenoids the interaction between them caused a lower utilisation of both, but the reduc- Uptake of carotenoids tion was highest for astaxanthin [26]. A study of Uptake of carotenoids from the gastrointestinal the ADC, muscle accumulation and relative tract is critical for their utilisation. A character- concentrations of astaxanthin isomers in rain- istic feature of the uptake process is that maxi- bow trout showed that the E/Z isomers were ab- mum concentrations in the blood are found up sorbed from the gastrointestinal tract to a differ- to 30 hours after force feeding [19]. A common ent degree, while this was not true for the measure of gastrointestinal absorption is the ap- optical R/S isomers [30,31]. The absorption of parent digestibility coefficient (ADC), which the geometrical isomers descended in the fol- estimates the difference in the amount of a nu- lowing order: All-E-astaxanthin > 13Z-astaxan- trient present in the diet and the amount excret- thin > 9Z-astaxanthin.

Thematic area: Feed, Nutrition, Feeding 255 Increasing the dietary lipid content causes an ed ration, respectively. A negative correlation increased deposition of astaxanthin in the mus- was found between feed intake and ADC. The cle of Atlantic salmon [22,23,32]. The compo- ADC was 1.5 times higher in the fish fed to 40 sition of dietary fatty acid may also influence per cent of apparent satiation compared to the uptake and deposition. This is indicated by fish fed the full ration. However, due to the rel- higher astaxanthin concentrations in Atlantic atively low feed intake in the fish fed the re- salmon fed diets supplemented with capelin or stricted diet, the total amount of digested Peruvian oil high in polyunsaturated fatty acids astaxanthin only represented about 50 per cent compared to Atlantic salmon fed diets supple- of that digested by the fish fed the full ration. mented with herring or sandeel oils [33,34]. Estimates based on the results from this trial in- The effect of lipid sources and bile acids have dicate that a low retention of astaxanthin at a been studied in dorsal aorta cannulated Atlantic high feed intake level may cause a reduction in salmon [35]. The salmon were fed diets supple- the muscle concentration of astaxanthin in mented with herring oil, soy lecithin, lard, or rapidly growing salmon [36]. This may further herring oil supplemented with taurocholin. Soy explain observations of poor pigmentation in lecithin caused a poorer uptake of astaxanthin periods of extreme thermal growth. In a trial compared to herring oil, whereas lard had a pos- with small Atlantic salmon (approximately itive effect on the uptake. Dietary supplementa- 500 g), a 10 per cent lower ADC of astaxanthin tion with taurocholin did not significantly affect was found at 8 ºC compared to 12 ºC [37]. the blood concentration of astaxanthin. Thus, it appears that alteration of the mixed micelle Species differences in muscle structure in the intestine does not improve the pigmentation astaxanthin uptake. The positive effect of lard Several different causes may explain the differ- on the blood concentration of astaxanthin may ences in carotenoid pigmentation found in dif- have been affected by the increased concentra- ferent wild fish species. For instance, different tions of 16:0, 18:1n-9 or 18:2n-6 fatty acids or fish species may have different carotenoid di- the lower content of the 20:1n-9 and 22:1n-9 gestibility and gastrointestinal absorption rate fatty acids of lard compared to herring oil, caus- of ingested carotenoids. The catabolic transfor- ing an increased solubility of astaxanthin in the mation (metabolism) of carotenoids may be dif- intestinal micelles. ferent, and there may be differences in ingested doses and foraging habits as well. Most fish Recently, the authors have shown that feed in- species are capable of absorbing a certain take [36] and temperature [37] influence the amount of carotenoids, but most of them depos- ADC of carotenoids. Groups of Atlantic salmon it these carotenoids or their metabolites in the weighing about 2 kg were fed either a full or a integument. At present it is virtually unknown restricted ration that corresponded to about 100 to what extent the gastrointestinal absorption per cent and 40 per cent of apparent satiation, rate, catabolic transformation rate, ingestion of respectively, followed by a switch in rations af- different doses, or other factors are responsible ter 14 days. The control group was fed a full ra- for the differences in muscle carotenoid pig- tion during the entire experiment. The feed mentation between salmonid and other fish spe- intake corresponded to 0.45 per cent and 0.16 cies that normally have white flesh. Thus, the per cent of the biomass for the full and restrict- ADCs of individual astaxanthin E/Z isomers

256 Aquaculture Research: From Cage to Consumption and total astaxanthin were determined in a com- To investigate whether the carotenoid transport parative experiment with Atlantic salmon and capacity of the blood could represent an impor- Atlantic halibut (Hippoglossus hippoglossus) tant limitation to muscle supply of carotenoids, fed the same diet supplemented with 66 mg as- experiments involving intraperitoneal injection taxanthin/kg consisting of 75.3 per cent all-E-, of an astaxanthin preparation were performed to 2.6 per cent 9Z-, and 21.5 per cent 13Z-astaxan- study astaxanthin uptake in the blood and depo- thin, respectively [38]. The results showed that sition in various tissues and organs [41,42]. Dif- the ADC of total astaxanthin was in fact signi- ferent doses of commercial water-dispersible ficantly higher in the Atlantic halibut than in the astaxanthin were suspended in a phosphate Atlantic salmon after 56 days and 112 days of buffer and injected into the intraperitoneum of feeding (77.8 and 45.9 per cent, and 71.7 and Atlantic salmon, rainbow trout and Atlantic cod 59.2 per cent, respectively). A possible explana- (Gadus morhua) to determine uptake and depo- tion for this difference in ADC could be that sition of the individual geometrical E/Z isomers specific growth rate of the latter species is 3.5 of astaxanthin, as well as the idoxanthin con- times higher. Furthermore, the ADC of all-E-as- centration of plasma, muscle, liver, kidney and taxanthin was significantly higher than the skin as an indicator of metabolic transforma- ADC of 9Z-astaxanthin in both species. The ex- tion. In the first trial [41], duplicated groups of periment showed that astaxanthin digestibility rainbow trout (initial weight 550 g) were inject- and gastrointestinal absorption is not a limiting ed with astaxanthin, and were either subse- factor for utilisation of astaxanthin in Atlantic quently fed a diet supplemented with a low level halibut compared to Atlantic salmon, and that of astaxanthin (10 mg kg-1), a diet supplement- other factors must be identified to explain the ed with 60 mg astaxanthin kg-1 (fed control), or absence of astaxanthin deposition in the muscle a diet supplemented with 10 mg astaxanthin/kg- of Atlantic halibut. Despite the higher ADC in 1 (0-group) for eight weeks. In the second trial Atlantic halibut, the plasma concentration of as- [42], duplicated groups of Atlantic salmon (ini- taxanthin was considerably lower than in Atlan- tial weight 370 g) and Atlantic cod (initial tic salmon (the total carotenoid concentration weight 210 g) were injected with 0, 12.5, 25 or was 0.7 and 4.0 mg l-1, respectively). This may 50 mg astaxanthin, respectively. Subsequently, be due to different catabolic transformation all fish were fed a diet supplemented with a low rates, astaxanthin transport in the blood, and up- level of astaxanthin (10 mg kg-1) for four take at the muscle cell level. Blood uptake of weeks. In plasma the astaxanthin concentration geometrical E/Z isomers in humans following curve increased in a dose dependent fashion, ingestion of a meal containing 100 mg astaxan- and was positively linearly correlated with the thin indicates that the absorption process in hu- injected dose in both Atlantic salmon and At- mans is different than that in fish, since a lantic cod. Individual concentrations up to 90, selective accumulation of astaxanthin Z-iso- 50 and 20 mg astaxanthin l-1 were detected in mers takes place [39]. As in fish, the uptake of the plasma of Atlantic salmon, Atlantic cod and astaxanthin was lower, only 25 per cent based rainbow trout, respectively. In comparison, an on the AUC (area under the concentration time in vitro investigation of the saturation level of curve), following administration of astaxanthin canthaxanthin in the lipoproteins of rainbow fatty acyl esters compared to unesterified astax- trout revealed that the binding capacity of the anthin [40]. lipoproteins was about 100 times as high as the

Thematic area: Feed, Nutrition, Feeding 257 concentration obtained by feeding the fish a diet of the total carotenoids of the muscle in Arctic supplemented with 100 mg canthaxanthin per charr (Salvelinus alpinus; 1.1 kg) [45,46], the kg-1 [43]. Thus, the transport capacity of the corresponding figure for Atlantic salmon is blood of salmonids and other fish species does about 10 per cent [38], while idoxanthin is bare- not limit the utilisation of carotenoids. ly detectable in rainbow trout [30]. Recently, the authors isolated and quantified 4´-hydroxy- Metabolism of carotenoids in salmonid echinenone in the muscle of Atlantic salmon fishes 0+ and 1+ smolts fed a diet supplemented with Salmonid fishes possess enzyme systems that canthaxanthin [47]. We found that the (4´S)- can transform ingested astaxanthin and can- isomer (Figure 3b) accumulated selectively (81 thaxanthin or certain other carotenoids into per cent 4´S- and 19 per cent 4´R-isomer, re- vitamin A1 and A2 as mentioned previously. spectively, of total amount of 4´-hydrox- Recent findings show that Atlantic halibut pos- yechinenone), which strongly indicates that sess this capability as well [44], and suggest canthaxanthin is reduced selectively by the that this metabolic conversion may be more enzyme involved. A selective reduction of can- widespread among fish than previously recog- thaxanthin in favour of the (4´S)-hydroxy- nised. The gastrointestinal epithelium and liver echinenone has previously been reported for are presumably the quantitatively most impor- laying hens [48] and skin of rainbow trout [13]. tant organs for catabolic transformation of caro- The concentration of 4´-hydroxyechineone in tenoids, even though reductive metabolites the muscle was relatively low (<3.1 per cent of have been detected in the kidney, spleen, go- total carotenoids) and diminished with increas- nads, skin and retina. The quantitative impor- ing fish size and age. This corresponds to find- tance of the various organs for metabolic ings of idoxanthin in Arctic charr and Atlantic transformation has not yet been precisely deter- salmon, in which the concentration of idoxan- mined, and requires the identification of the thin was higher in small fish than large fish, and genes and enzymes of different metabolic path- indicates that the metabolic capacity to trans- ways and the metabolites that are produced. form absorbed astaxanthin decreases with age However, the authors have found that metabolic and size in salmonid fishes [45,49]. However, products are formed rather rapidly because the the metabolic capacity may increase at the onset reductive astaxanthin metabolite idoxanthin of sexual maturation [50]. Although the factors (3,3´,4´-trihydroxy-β,β-carotene-4-one) was that govern carotenoid metabolism in salmonid detected in the plasma 6 hours after force-feed- fishes have not been identified, it has been ing Atlantic salmon with radioactively labelled shown that hormones may be involved, since astaxanthin [19]. Idoxanthin is formed by re- sex hormones (11-ketotestosterone and 17β- duction of one of the keto-groups of astaxan- estradiol) stimulate metabolic transformation thin, whereas 4´-hydroxyechinenone (4´- and redistribution, which take place during sex- hydroxy-β,β-carotene-4-one) is a correspond- ual maturation [51]. The idoxanthin content of ing reductive metabolite of canthaxanthin the muscle of Atlantic salmon is partly geneti- (Figure 3). Considerable differences between cally determined and has a heritability coeffi- species are found with regard to idoxanthin ac- cient of 0.4 [52]. No effects of temperature and cumulation in the blood and various tissues. individual feed intake were detected on astax- Whereas idoxanthin comprises 40 to 60 per cent anthin metabolism measured as idoxanthin con-

258 Aquaculture Research: From Cage to Consumption Changes in pigmentation pattern during sexual maturation It is well recognised that sexual maturation causes dramatic changes in the pigmentation pattern of salmonid fishes [14]. In addition to a redistribution of the carotenoids from the muscle to the skin and gonads, high metabolic activity causes a 25 per cent drop in the total body burden of carotenoids in female rainbow trout compared to sexually immature fish of the same age and size, while the drop in male rainbow trout is about 80 per cent of the total body burden [5]. In a trial with Arctic charr, it was found that intraperitoneal implants containing hormones influenced pigmentation. Thus, Arctic charr supported with implants containing 11- ketotestosterone or 17β-estradiol had higher carotenoid concentrations in the skin than Arc- tic charr supported with implants containing 3,5,3´-triiodo-L-thyronine or with implants that did not contain hormones; in addition, idoxanthin comprised a higher proportion of the total carotenoids in the blood [51]. In Arctic charr a considerably higher proportion of idoxanthin 145x100//Kap15-fig01.eps Figure 3. Structural formulas of the metabolites a) (15 per cent of total carotenoids) was found in idoxanthin (3,3´,4´-trihydroxy-β,β-carotene-4-one) and b) the muscle of sexually maturing fish compared 4´-hydroxyechinenone (4´-hydroxy-β,β-carotene-4-one). to immature fish of the same age and size [50]. This trial also showed that astaxanthin com- centration in plasma in an experiment with prised only a small part of the total carotenoids small Atlantic salmon (500 g) [37]. Large indi- (<20 per cent of total carotenoids), whereas the vidual variation in the plasma concentration of major carotenoid was idoxanthin (57 per cent of idoxanthin was, however, found (<0.5–70 per total carotenoids). Furthermore, it was shown cent of the total carotenoid content). In groups that crustaxanthin (3,4,3´4´-tetrahydroxy-β,β- of Atlantic salmon that were fed restrictively, carotene) isomers comprised about 20 per cent fish with visible damage to the pectoral fins had of the total carotenoids in the gonads, and that a higher idoxanthin concentration in the plasma the reduction in astaxanthin was selective in than individuals without fin damage, and indi- favour of the sterically most hindered cates that stress may influence carotenoid 3,4(3´,4´)-cis glycolic isomers, whereof the metabolism. Stress has previously been sug- 3,4,3´4´-di-cis; 3,4-trans 3´4´-cis; 3,4,3´4´-digested to cause increased metabolism of caro- trans glycolic isomers comprised 40, 45, and 16 tenoids in salmonid fishes [49]. per cent of the total crustaxanthin, respectively. In the skin, astaxanthin esters represented about

Thematic area: Feed, Nutrition, Feeding 259 85 per cent of the total carotenoids. The bio- Accumulation of carotenoids in chemical basis for selective accumulation of muscle and other tissue various carotenoids in different tissues and or- Considerable progress has recently been made gans still remains to be elucidated. However, se- with respect to the binding of astaxanthin in the lective accumulation should be related to tissue muscle tissue of salmonid fishes. Even though specific metabolism, uptake or excretion. astaxanthin is liposoluble, it has long been recognised that it is primarily associated with the protein, not the lipid, fraction of muscle tissue Metabolism of E/Z and R/S [53]. A newly developed method has enabled isomers the determination of astaxanthin binding pro- Some features regarding the distribution of geo- teins in fish muscle tissue [54]. An ultracentri- metrical E/Z isomers and optical R/S isomers fugation method and the use of sodium cholate have implications for a selective metabolic for dispersion of astaxanthin complexes were transformation. Whether rainbow trout were fed developed to separate unbound astaxanthin by diets high or low in all-E-astaxanthin (97 per filtration over a 30 kDa filter. After fractional cent vs. 64 per cent of total astaxanthin) a selec- extraction of the muscle proteins, the various tive accumulation of 13Z-astaxanthin was fractions that bound astaxanthin could be iden- found in the liver (48–59 per cent of total astax- tified. Fractional extraction of protein-bound anthin), whereas 9Z-astaxanthin only com- astaxanthin using different brine concentrations prised about 2.5 per cent of the total astaxanthin and pH have also indicated that a water soluble [31]. 9Z-Astaxanthin comprised only about 2.5 extract had the highest affinity to astaxanthin, per cent of the total astaxanthin of the blood. i.e. the highest ratio between astaxanthin and This indicates that a selective metabolic trans- protein, and indicated that a sarcoplasmaprotein formation of the various geometrical E/Z iso- was involved in the binding of astaxanthin [55]. mers takes place, presumably in the liver. A By using fractional extraction of muscle pro- selective accumulation of 13Z-astaxanthin in teins from Atlantic salmon, Atlantic halibut and the liver has also been found in Atlantic salmon haddock, it was shown that certain fractions had and Atlantic halibut [38]. A slight selective ac- a greater ability to bind to astaxanthin than oth- cumulation of (3S,3´S)-astaxanthin took place er fractions, but the same protein fractions from in skin and kidney tissue [31]. This may indi- all three species were able to associate with cate that a selective stereochemical conjugation astaxanthin [56]. Recent work has provided takes place during detoxification reactions evidence that the primary binding protein of (Phase II) in the kidney, by potential formation astaxanthin in the muscle of Atlantic salmon is of glucuronides or sulphates. In the skin, the se- a-actinin; a-actinin isolated from Atlantic hali- lective accumulation is probably related to the but also combined with astaxanthin in about the esterification process, similar to the one that same stoichiometric ratio [57]. These experi- takes place during gastrointestinal hydrolysis. ments indicate that the astaxanthin-binding ca- A selective accumulation of (3S,3´S)-zeaxan- pacity of the muscle proteins does not limit the thin, a metabolite of astaxanthin, in the skin of deposition of astaxanthin in the muscle; rather, rainbow trout supports this view [5]. a high metabolic transformation rate may limit the retention of ingested astaxanthin.

260 Aquaculture Research: From Cage to Consumption Smolt production regimes may influence caro- higher than levels that can be achieved by feed- tenoid uptake and deposition of carotenoids. ing Atlantic salmon diets supplemented with This was investigated in an experiment with At- astaxanthin. In both Atlantic salmon and Atlan- lantic salmon transferred to seawater either as tic cod the astaxanthin accumulated in the mus- 0+ or 1+ smolts, and fed a diet supplemented cle in a dose-dependent manner. Individual with 30 mg astaxanthin and 30 mg canthaxan- concentrations up to 30 mg kg-1 were detected thin per kg for 35 weeks [47]. The 0+ smolts in Atlantic salmon, which represent about 15 had a significantly higher carotenoid concentra- times higher concentrations than can be tion in the muscle than the 1+ smolts (approxi- achieved by feeding fish of a similar size diets mately 10 per cent) after correcting for weight supplemented with astaxanthin. A good linear differences. This indicates that the growth pat- correlation was found between plasma and tern may influence carotenoid utilisation, possi- muscle concentrations of astaxanthin, which in- bly due to the reduction in ADC caused by dicates that increased uptake causes increased increased feed intake during more rapid growth muscle deposition and that muscle is not satu- [36]. Similarly, continuous illumination during rated at this level. Only about 1 mg astaxanthin the seawater phase may cause better pigmenta- kg-1 was present in Atlantic cod injected with tion than the natural photoperiod in autumn astaxanthin despite the high plasma concentra- smolts [58]. tions (about 20 mg l-1). A patent has been applied for for a method of injecting astaxanthin Extremely high plasma concentrations of astax- into salmonid fishes. In conclusion, neither the anthin have been achieved in Atlantic salmon, binding capacity of the muscle for astaxanthin, rainbow trout and Atlantic cod by intraperito- nor plasma transport appears to be factors that neal injection of astaxanthin (50–100 mg) limit the efficient utilisation of astaxanthin in [41,42]. The concentrations were up to 20 times Atlantic salmon. Furthermore, it appears obvi-

145x100//Kap15-fig01.eps Figure 4. Atlantic cod intraperitoneally injected with 0 or 100 mg astaxanthin, respectively. (Photo: Arild Linseth, Akvaforsk, Sunndalsøra)

Thematic area: Feed, Nutrition, Feeding 261 ous that receptors related to the uptake of astax- cle carotenoids, carotenoid digestibility and anthin in the muscle have a species-specific retention, and so on, mathematical models can distribution in different tissues, which may ex- be produced to suggest optimal regimes. The re- plain the differences observed in uptake be- sults indicate that the expenditure on astaxan- tween salmonid fish species and white-fleshed thin can be reduced considerably compared to fish species, although the latter may have a few years ago, while still achieving accepted good capability of depositing carotenoids in the final concentrations in the muscle (7 mg kg-1) skin and gonads (Figure 4). of the slaughtered fish (+4 kg). Pigmentation models for farmed Atlantic salmon have recently emerged. Nonlinear regression metaanalysis Concluding remarks of published experimental data has served as a Considerable progress has been made in re- basis for the development of a pigmentation search on carotenoids and salmonid fish spe- model [59]. An excellent relationship was cies. The authors are convinced that a found between modelled astaxanthin concen- breakthrough in determination of factors that tration in the muscle and the explanatory vari- govern gastrointestinal uptake and uptake in ables dietary astaxanthin concentration and fish liver and muscle cells is imminent. Work with size. Based on this model the authors developed muscle cells in culture and proteom studies has a mathematical programming model designed been initiated and, provided projects receive to optimise dietary astaxanthin concentrations financial support, it will contribute to such a throughout the grow-out period that results in breakthrough. This in turn may create tools to well-pigmented salmon at minimal cost [60]. investigate variation in various populations and These models are rather simple, and other para- may contribute to improving selective breeding meters that influence carotenoid utilisation as programmes for commercially important spe- mentioned above have not been taken into ac- cies. count, although they may improve the models. A dynamic ordinary differential equation model Small fish exhibit a relatively poor utilisation of was recently developed to further the under- carotenoids – 0+ smolts utilise carotenoids bet- standing of the fundamental processes that reg- ter than 1+ smolts. Carotenoid utilisation de- ulate the absorption and metabolism of pends on seasonal variations, and carotenoid carotenoids in Atlantic salmon [61]. The model digestibility depends on feed intake and temper- mimics feeding experiments well and indicates ature. Nevertheless, it may be of practical im- that uptake over the muscle membrane is pos- portance to provide the correct dosage for the sibly an important source of variation and that a different stages of production. There may be dynamic model can be instrumental in deter- more optimal dosage regimes than simply feed- mining underlying determinants of complex ing fish a constant dose throughout the produc- traits. tion cycle. Research on pigment strategies has provided models that estimate economically Quality issues comprise an important aspect of optimal pigmentation regimes, or the lowest to- pigmentation of products of salmonid fishes. tal amount of carotenoid required to achieve a Documentation of the effects of process param- given carotenoid level in the muscle at slaugh- eters such as salting technique, temperature and ter. By using data on feed intake, growth, mus- duration of processing on the stability of astax-

262 Aquaculture Research: From Cage to Consumption 145x100//Kap15-fig01.eps 145x100//Kap15-fig01.eps Figure 5. Extraordinary decolouration of an Atlantic Figure 6. Dramatic decolouration of the surface of an salmon fillet (right-hand side) after injection-salting. Atlantic salmon fillet (unsmoked). (Photo: Sveinung (Photo: Sveinung Birkeland, Norconserv, Stavanger) Birkeland, Norconserv, Stavanger) anthin, colouration, texture and more during thin in a red yeast (Phaffia rhodozyma) during processing of cold-smoked salmon has recently extrusion processing [68]. The study revealed been provided [62–66]. Although important in- that enzymatic degradation of the cell wall formation on factors that influence the quality caused poorer stability, but that this was by far of smoked salmon during processing has al- outweighed by the increased availability of the ready been published, work is still needed, astaxanthin for gastrointestinal absorption among other things, to avoid problems with pig- (muscle retention 3.7 and 17.4 per cent, respec- mentation as shown in Figures 5 and 6 [67]. tively) [69]. The research on the identification of genes involved in carotenoid biosynthesis A considerable amount of work has been done gives hope for future development of astaxan- to develop alternative astaxanthin sources. We thin and other carotenoid hyperproducing or- have recently studied the stability of astaxan- ganisms.

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266 Aquaculture Research: From Cage to Consumption Selective Breeding and Genetics

■ Selective Breeding and Genetics – Atlantic Salmon ■ Breeding and Genetics – New Species Bjarne Gjerde1), Anna Sonesson1), Arne Storset2) and Morten Rye3) 1) Akvaforsk – The Institute of Aquaculture Research, 2) Aqua Gen AS, 3) Akvaforsk Genetics Center AS

Selective Breeding and Genetics – Atlantic Salmon

Systematic genetic improvement work in Atlantic salmon has been a cornerstone in developing the Norwegian aquaculture industry. In three decades, this work has produced stocks of farmed salmon that are better adapted to commercial farming environments, grow twice as fast and demonstrate significantly improved feed conversion as compared to the base material collected from wild Norwegian river strains. The family-based Norwegian breeding programmes are among the most advanced in international aquaculture, currently targeting improvements in growth rate, age at first sexual maturation, disease resistance and product quality. The overall effect of breeding work has been continuous reduction in production costs and improved competitive power of the industry. In spite of this success, the leading breeding companies in Norway are facing significant challenges. As similar programmes are rapidly gaining ground in other salmon farming countries, only a marginal fraction of the real added value resulting from Norwegian breeding programmes is returned to the breeding companies operating. This is significantly limiting their capability to conduct R&D necessary to maintain their leading position in the field, which in the longer run constitutes a real, major threat to the entire Norwegian salmon industry as it faces increased competition in its major markets.

268 Aquaculture Research: From Cage to Consumption The main objective of research in selective the breeding candidates for growth, age at sex- breeding and genetics is to produce knowledge ual maturity and carcass quality traits; a dedi- that can be used to develop more cost-effective cated facility for challenge testing of additional and sustainable selective breeding programmes. full- and half sibs for specific disease resis- In Norway, breeding programmes for Atlantic tance; and finally, a few multiplier stations for salmon provide farmers with genetic material commercial production of eyed eggs for sale to that for each generation has a higher genetic po- smolt producers (Figure 1). Currently, full-sib tential for efficient biological production. Since families are reared separately until individuals the mid-1970s this has contributed significantly are large enough to be tagged by physical to reduced production costs, giving Norwegian methods (~10 gram). farmers a competitive advantage. For the last 10–15 years selection has also been done for The generation interval is generally four years, carcass quality and disease-resistant traits for resulting in four parallel breeding populations. increased product quality and improved animal In the breeding nucleus, selection of parents for health and welfare. a new generation is mainly based on data recorded on full- and half sibs of the breeding For several years, Norwegian breeding work candidates. Phenotypic records on the breeding targeted national production, but this has now candidates themselves are only obtained for changed. Applied genetic improvement work growth and sexual maturity. Normally parents and trade of genetic material has become an are not reused across generations, which neces- international business, and currently more than sitates the use of cryopreserved milt. The gener- ten competing companies and organisations are ations are therefore discrete. working with Atlantic salmon. Hence both scientists and breeding companies are facing new A breeding company will maximise the short- challenges and increased competition, leading term annual genetic gain in order to maintain or to more secrecy about the individual improve- increase its market share. At the same time, ment programmes. Paradoxically this trend however, the rate of inbreeding in the breeding appears at a time when demand is dramatically nucleus must be kept at an acceptable level to increasing for documentation of the various ma- attain a high annual genetic gain in the long terials available. Due to stiff competition and possibly to “cultural” reasons as well, breeding companies are facing significant difficulties in Nucleus

Breeding Selection Selected securing operating margins which enable them Multiplier candidates breeders Fert. eggs/ to invest in further R&D for development of fry/fing. Tagged Families fish of fert. Fry/fing. their breeding programmes. eggs Startfeeding Hatchery production

Nucleus breeding design Grow-out A typical Atlantic salmon breeding programme Field test 1 Chall. test 1 Tagged fish consists of: full-sib and paternal half-sib family Field test 2 Chall. test 2 groups; a sea-unit location for rearing of the Field test n Chall. test n breeding candidates; a few sea-unit locations

145x100//Kap16-fig01.eps for performance testing of full- and half sibs of Figure 1. Outline of a typical breeding programme for Atlantic salmon (13).

Thematic area: Selective Breeding and Genetics 269 term. This is a complicated optimisation prob- in net-cages in the sea where they are kept for lem, especially when selection is performed two years before preselected sexually maturing simultaneously for many traits. An optimal fish are transferred to a freshwater facility four breeding nucleus design also depends on the to five months before the mating season and available technology. New technologies that al- finally reproduce at four years of age. The cost low the recording of more traits of live breeding and risk associated with this production cycle is candidates, or cost-effective use of genetic very high, with a total of two – and during part markers for parental assignment, will have a of the year three – year-classes of fish off- and significant effect on the structure of an optimal on-shore. If the generation interval could be re- breeding nucleus design. duced to three years, both the cost and risk of production could be reduced, while at the same Published work on the design of optimal breed- time the annual genetic gain would be 33 per ing programme design for aquaculture species cent higher. In addition most of the 1+ smolt is scarce. Available literature is limited to de- stocked is harvested at 2.5 to 3 years age, while sign of programmes using individual (mass) se- the four-year-old selected breeders are on aver- lection (6, 13, 16) or combined (own, full- and age 15–20 kg. It is therefore of interest to pro- half sib records) selection (48, 57, 58) for a sin- duce the families from younger and smaller fish gle trait. Because Atlantic salmon, like other than is the practice today. This has become even aquaculture species, has a high fecundity and more interesting as an increasing proportion of external fertilisation permits a variety of possi- the salmon is put into the sea as 0+ (8-month- ble mating designs, optimal design for aquacul- old) smolt in September-October. However, the ture species are significantly different from problem with the 0+ smolt is that a relatively optimal design for traditional farm animals. low proportion of these fish become sexually mature at three years of age. This makes it dif- For production of full-sib families, milt from ficult to obtain three-year-old breeders from one male is typically used to fertilise the eggs families with a low proportion of sexually ma- from two or three females. New knowledge ture fish. Hopefully this can be solved using ar- shows that this kind of fixed mating design is tificial light programmes to stimulate the not optimal. The output from optimum contri- development of a higher proportion of mature bution (OC) selection (33) is the optimum num- fish at three years of age. This requires more re- ber of sexually mature offspring to be produced search and should include experiments to obtain from each selected breeder to obtain maximum estimates of the magnitude of possible genotype annual genetic gain at a predefined rate of in- by light regime (natural and artificial) interac- breeding. More research is needed in close co- tion for important traits. operation with the breeding companies on how OC selection can be implemented in aquacul- Fewer breeding populations? ture breeding programmes. Due to the four-year generation interval in At- lantic salmon, breeding companies operate with Shorter generation interval? several parallel nucleus breeding populations, Up to now, the length of the generation interval mainly in order to produce commercial eyed for Atlantic salmon in Norway has been kept at eggs for smolt producers and 1+ smolt to the four years; 1+ (1.5-year-old) smolt are stocked multiplier stations annually from four-year-old

270 Aquaculture Research: From Cage to Consumption breeders. For the quality of the breeding programme per se this is not necessary. The multi- Table 1. The traits presently selected for in the four breeding programmes for Atlantic salmon in plier stations could for example be provided Norway. with smolt from the same breeding population two subsequent years, either by use of 0+, 1+, 2 Trait Breeding programme or 2+ smolt produced from breeders four (alter- A B C D natively three and five) years old. This strategy Growth in freshwater X X X X would require keeping only two parallel breed- Body weight at slaughter X X X X ing populations and could also involve the use Early sexual maturity X X of cryopreserved milt from the breeding nucle- Furunculosis X X us populations. One breeding company in Nor- ISA X X way is implementing the use of cryopreserved IPN X X X milt into its breeding scheme, in combination Dressing percentage X X with merging of the four breeding populations Fillet yield X X into one and reduction of the generation interval Fillet colour X X X X to three years. Fillet fat X X Deformity X X The optimum number of breeding populations Body shape X is a function of effective population size, risk and company strategy. If each population is produced based on a low number of breeders (i.e. feed conversion efficiency, survival); traits re- few families), a larger breeding population can lated to health and functionality (disease resis- be obtained by keeping two or more parallel tance and reproduction); and traits related to sub-populations and systematically exchanging product quality (pigmentation and fat deposi- genetic material between them to obtain in- tion). creased selection intensity and thus increased genetic gain at a predefined rate of inbreeding. Table 1 lists the traits selected for by the main Access to more than one breeding population breeding companies in Norway. The choice of would also facilitate larger product differentia- traits and their relative weights in the pro- tion for serving different market segments, and grammes reflect the judgment and strategic as- production of crossbred animals for on-grow- sessment made by the individual company. In ing, which would provide better protection of its strategic research programme “Future ani- the genetic material against unauthorised repro- mal breeding goals” (1), AKVAFORSK is cur- duction. An alternative strategy could be a long- rently developing objective economic weights term and strategic cooperation between breed- for key traits recorded in various phases of the ing companies with similar breeding objectives production cycle. and markets. Accurate information about heritabilities and Breeding goal genetic correlations among traits under selec- Efficient and sustainable genetic improvement tion is critical for correct ranking of breeding work involves: balanced selection for traits that candidates. In Atlantic salmon as for most primarily reduce production cost (growth rate, aquaculture species, few reliable estimates of

Thematic area: Selective Breeding and Genetics 271 genetic correlations among different traits have been published. Table 2. Growth, feed intake and feed efficiency of the offspring of Atlantic salmon selected for five generations and offspring of wild salmon Traits from the river Namsen (54) Growth rate Trait Selected Wild Difference By improving growth rate of the salmon, the % target numbers of smolt or amount of market- Body weight at start of 814 533 - sized fish can be reached in a shorter time and experiment, g at lower fixed and variable production costs. The genetic correlation between body weight Body weight at end of 1455 743 - recorded at smolt stage and at standard market experiment, g Growth, TGC x 10–3 2.96 1.39 113 size is relatively low (rg~0.5), nor is it very high between body weights recorded several months Feed intake, g feed/g body 0.67 0.48 40 apart during the seawater phase (15). These re- weight, % sults strongly indicate genetic variability in the Feed efficiency, g gain/g 1.16 0.93 25 shape of the growth curve of Atlantic salmon, feed intake which may be utilised in advanced selection programmes to develop a more cost-effective animal. Selection for modified growth curves unknown. An experimental design which will depends on gentle recording technology allow- provide accurate estimates of this genetic corre- ing repeated measurements of individual body lation using currently available technology is weights throughout the production cycle. proposed (30). Scientists at the Norwegian Uni- versity of Science and Technology (NTNU) are Feed efficiency currently developing a “feeding sensitive” te- Feed costs account for 40 to 50 per cent of the lemetry system that can be employed in studies total variable costs in salmon production. of individual feeding behaviour of fish in net Hence high utilisation of the main feed compo- pens (3). This work may facilitate direct selec- nents protein, fat and carbohydrates is therefore tion for feed conversion efficiency, and also pivotal for the economic results of production. provide estimates of genetic correlations be- Direct selection for improved feed conversion tween FCE and the range of other traits current- efficiency (FCE) is currently inhibited by lack ly under selection. of cost-effective technology for accurate recording of feed intake for individual or groups Extensive short-term growth trials of 12 weeks’ of fish. Short-term experiments with pre- and duration showed that Atlantic salmon selected post-smolt have demonstrated genetic variabili- for five generations for improved growth rate ty for FCE (expressed as body weight incre- had a 25 per cent higher FCE as compared to ment/weight of feed eaten) and a positive wild salmon (54, Table 2). This is an example of correlation between family averages for FCE a correlated response, i.e. a genetic change in a and growth rate (r=0.74, 56; r=0.60, 28). given trait (in this case FCE) resulting from se- Whether these estimates reflect the true corre- lection for another, genetically linked trait sponding genetic correlations throughout the (growth rate). Although not yet quantified due main growth phase taking place in seawater is to lack of knowledge about the magnitude of the

272 Aquaculture Research: From Cage to Consumption genetic correlations involved, it is assumed that Both traits demonstrate genetic variability selection for reduced fat deposition also leads to when recorded on fish at the same age (43). As a favourable correlated response in FCE. larger salmon tend to deposit a higher amount of fillet fat, it is still not clear to what extent the Age at sexual maturation genetic variability, in particular for fillet fat Salmon developing secondary sex characteris- content, reflects size differences between indi- tics before harvest size are downgraded at vidual fish at the same age. Further studies on slaughter. The focus in breeding has so far been fish sampled at the same size are needed for to reduce the percentage of fish reaching sexual clarification of this important issue. maturation after one sea winter (grilsing). Grils- ing is heritable, but very few heritability esti- The average fat content in salmon fillet is 15 to mates or estimates of genetic correlations 16 per cent, but may vary from six to 22 per cent involving age at sexual maturation have been among individual fish (45). It appears that con- published (12, 15, 61). A possible emerging sumers’ preference for salmon, within certain problem may be fish becoming sexually mature limits, is not significantly affected by the fat as early as during the first (1+ smolt) and sec- content in the product. If this is the case, selec- ond (0+ smolt) autumn in sea water. In the tion should be made for reduced fat content, as 1970s and the 1980s there was a certain occur- theoretical calculations made by Akvaforsk rence of fish maturing in freshwater (precocious suggest that the production cost of 1 kg salmon males). This is less of a problem today, likely may be lowered by as much as NOK 0.30 per due to the fact that the breeding companies con- %-unit reduction in fat content. These estimates sistently culled these fish and did not use them however need to be verified in designed experi- in reproduction. ments.

Fillet pigmentation and fillet fat It is not known whether observed differences in Fillet pigmentation and fillet fat content is in- pigmentation between individual fish are pri- cluded in the breeding goal to ensure competi- marily due to individual differences in caro- tive product quality and lower production costs. tenoid utilisation or simply reflect differences in individual feed intake and thus amount of

250 pigments consumed. Scientific clarification of

200 this issue requires methodology for recording individual feed uptake. 150

NOK 100 Figure 2 illustrates that consumers are willing 50 to pay a premium price for a stronger pigmented 0 salmon (2, 51), but lack of rapid and cost effec- R21 R22 R23 R24 R25 R26 R27 R28 R29 R30 R31 R32 Colour tive technology for grading the product with re- Without information With information spect to pigmentation level prohibits price

145x100//Kap16-fig02.eps differentiation. Development of an adequate Figure 2. Willingness to pay for filet colour (Rosche-scale) technology for this purpose should be given obtained from a market experiment with Atlantic salmon (51). Before and after information refer to the fact that high priority by the industry. The same applies information was given about the source of the colour in the for technology facilitating grading with respect feed.

Thematic area: Selective Breeding and Genetics 273 to fat content. Accurate information of custom- 1.6 er preferences when offered products of varying pigment and fat levels is essential for determin- 1.5

ing the correct relative weight assigned to these 1.4 traits in selection work. The ongoing research 1.3 project On-line determination of fat and carote- rg = 0.88 ± 0.26 noids in whole salmon (The Research Council 1.2

of Norway, 174 237/S40), involving several re- 1.1 search and industrial partners, is addressing this challenge. 1.0

Field test, number0.9 of lice per fish External pigmentation 0.8 A large proportion of farmed salmon is sold as 45 50 55 60 65 70 75 80 85 90 95 round whole or gutted fish. For marketing pur- Challenge test, number of lice per fish

poses it is important that the skin colour of the 14x100//Kap16-fig03.eps Figure 3. Breeding values for number of sea lice per fish of product is similar to the skin colour of wild 50 Atlantic salmon full-sib families under controlled salmon. External pigmentation is likely to be in- (challenge test with post-smolt) and natural infection fluenced by few genes and therefore expected to (data from 29). change readily as a consequence of direct or indirect selection, or due to random genetic drift or breeding values of the same precision as be- in the population. Skin colour should therefore fore with fewer animals tested. be regularly recorded and monitored in selective breeding programmes in order to prevent For biosecurity reasons, fish surviving the chal- undesirable changes. lenge tests are usually culled and disposed of. These are animals with high levels of genetic re- Disease resistance sistance to the respective pathogen tested for, Designed challenge test experiments carried out and thus of high potential value as breeding with Atlantic salmon pre-smolt (10, 11, 20, 52) candidates. If adequate systems could be devel- and post-smolt (50) have demonstrated substan- oped that facilitate safe introduction and use of tial additive genetic variation in tolerance to spe- these surviving animals in the breeding nucleus, cific viral and bacterial diseases. Breeding it would speed up response to selection for in- companies in Norway now select for improved creased resistance to diseases. resistance to furunculosis, ISA (infectious salmon anaemia) and IPN (infectious pancreatic ne- Currently, challenge tests for bacterial diseases crosis) based on challenge test survival data. involve non-vaccinated animals, while fish used in regular production are usually vaccinated Recent research work has developed improved against the same diseases. No information is statistical models for analysis of this type of available about the genetic correlation between data (9, 60). These models produce more accu- innate resistance and the animals’ response to rate breeding values based on survival data ob- the protective effect of the vaccine. If this corre- tained from a given number of tested animals, lation is low, it may be more efficient to use vaccinated fish in the challenge tests. Likewise,

274 Aquaculture Research: From Cage to Consumption information is lacking regarding the potential heritability estimate based on analysis of data effect of increased levels of innate disease resis- from a controlled challenge test infection was tance on the immune response to vaccination or higher (0.26±0.07; on average 74.6 lice counted possible adverse effects of vaccination (e.g. in- on individual fish (29)). Figure 3 demonstrates flammation, adhesions and pigmentation). a very high correlation between the ranking of families based on the average number of lice per Survival animal under controlled infections and the rank- Survival is of key economic importance in ing of the same families under natural out- salmon farming, in particular during the seawa- breaks. This suggests that results from a ter rearing phase. This trait is thus an obvious challenge test serve as a good basis for ranking candidate for inclusion in the breeding goal, but families with respect to their ability to resist sea surprisingly scarce information is available lice infections. Data recorded during infections about the magnitude of additive genetic vari- under field conditions are less suited for this ability for survival. This essential information purpose, since natural infections are usually can readily be obtained from analysis of surv- less intense due to lower infection pressure. ival data from a population of individually Furthermore, Norwegian regulations require tagged and pedigreed animals, provided that treatment of the affected fish when the average mortality is thoroughly recorded and records of number of female lice observed per fish exceeds animals dying of specific diseases and other 0.5 during spring and summer (27). known causes are omitted from the dataset before analysis. The magnitude of the genetic cor- Selection based on infection experiments is a relation between survival, a measure of viability valuable tool for obtaining improved resistance and general disease resistance, and resistance to to sea lice infections in farmed salmon, and specific diseases measured in controlled chal- should be considered as a supplemental strategy lenge tests should also be investigated. to the current biological and chemical means used to control this problem (8, 27). Resistance to sea lice infestation Economic losses due to sea lice infestation are Malformations substantial in the salmon industry, and affected Studies have documented substantial genetic populations of farmed salmon serve as reser- variability for spinal deformities in Atlantic voirs for infection of vulnerable wild salmon salmon and conclude that this class of defects is populations (27). Results from controlled infec- not caused by inbreeding or as a correlated re- tion experiments have demonstrated statistical- sponse to selection for fast growth (19, 32). ly significant differences between salmon While other developmental problems have been stocks with respect to the number of lice infect- significantly reduced as the industry has imple- ing individual fish (22). Analyses of data ob- mented newer guidelines for water temperature tained during natural sea lice infections under during egg incubation (not above 8ºC), spinal commercial rearing conditions indicate low deformities still represent a serious challenge heritability for this trait (0.07±0.02 (23), (5). It is shown that phosphorus deficiency in 0.14±0.02 (29)). The average number of lice the diet, particularly when combined with zinc counted on individual fish during these ana- deficiency, induces skeletal malformations lyzed outbreaks ranged from 1.1 to 5.2. The (41). This indicates that the genetic variation

Thematic area: Selective Breeding and Genetics 275 expressed for spinal deformity may in part be today. However, since 2005 Debio has approved related to genetic variability in mineral utilisa- feed additives from algae, fungi and bacteria tion (55). Although it is likely that the main cultures for pigmentation. problems related to malformations are most efficiently solved through optimisation of feed Different ways to include and value traits re- composition and other non-genetic measures, lated to animal welfare and environment have breeding companies should avoid using breed- been discussed (39, 40) and one method has ers from families with high incidence of de- been suggested (37). Still, more research is formed fish. needed in this field.

Animal welfare and environmental Non-additive genetic effects concerns A diallel cross experiment including five wild Some groups of consumers heavily emphasise river strains of Atlantic salmon demonstrated animal welfare and environmental issues when low non-additive genetic variability for growth buying food. In Norway, organic salmon certi- rate (14), but a study carried out on extensive fied by Debio (www.debio.no), for example, re- data from a farmed population revealed non- quires that feed additives be of natural origin, additive genetic variation of intermediate mag- implying that synthetic pigmentation additives nitude for the same trait (44). Similar informa- are prohibited. This has made it difficult to ob- tion for other traits is not known. Optimal tain the same degree of pigmentation as conven- implementation of non-additive genetic effects TM tionally produced salmon. Freedom Food is in salmon breeding programmes requires a brand certified by the British animal welfare further studies. It is assumed that its potential organisation Royal Society for the Prevention of benefit is highest at the multiplier level and may Cruelty to Animals (RSCPA; www.rsp- also provide the basis for a strategy to prohibit ca.org.uk), which has particularly high require- non-authorised reproduction of genetically im- ments for secured and documented animal proved material. welfare. Most salmon produced under the Free- dom Food certificate are sold via Internet shops. Genotype by environment None of these products are found in shops in interaction Norway, so the position of Norwegian consum- Growth trials with salmon originating from sev- ers is unknown with regard to organic salmon or eral Norwegian river strains conducted at differ- salmon certified to be produced with special ent sites along the Norwegian west coast emphasis on animal welfare. An experimental indicated low strain by location interaction for market with organic salmon and salmon certi- harvest weight (21). On the other hand, a rela- fied by Freedom FoodTM showed that consum- tively strong family by location interaction ef- ers preferred these salmon to conventionally fect was demonstrated for age at sexual produced salmon, if the price was not too high maturation (61). These are presently the only and if the pigmentation of the fillet was similar published data on genotype by environment in- (40). For the ecological production of salmon, it teractions (GxE) in salmon. is therefore important to find natural sources of pigmentation additives which colour the meat The magnitude of GxE for a given trait may be more efficiently than shrimp peel, which is used assessed from genetic correlations estimated

276 Aquaculture Research: From Cage to Consumption from records obtained in different environ- If Chile is considered a strategic market for ments. Likewise, the same correlations can be Norwegian breeding companies (or vice versa), used to estimate the genetic gain in one particu- reliable data on the magnitude of a possible lar environment when selection is based on re- GxE for key traits between Chile and Norway cordings made in another. In a situation with are critically needed for determining optimal low GxE reflected by a high genetic correlation testing and selection strategies. This informa- of 0.90, the accumulated response after five tion can be calculated from performance data generations is 59 per cent ((0.9)5) as compared obtained from testing the same salmon families with the projected response if testing and selec- in both environments. tion were done in the same environment. If the interaction effect is higher as demonstrated by a Vertical transmission of genetic correlation between environments of infectious diseases 0.80, the corresponding estimate of the accumu- Reliable knowledge about risk for vertical lated response after five generations will only transmission of pathogens via eggs or milt is be 33 per cent ((0.8)5). This clearly demon- scarce (36). This knowledge is very important strates the need to select based on performance for breeding companies in order to avoid dis- data recorded in the target environment. Hence semination of disease agents to the industry a breeding company which decides not to estab- through transfer of genetically improved seed- lish routine local performance testing may rap- stock. This has been a current topic for years, idly lose market share if competing breeding but in Norway brood fish are not routinely test- companies base their selection on test data ob- ed for pathogens. For export of eyed eggs to tained in that particular production environ- Chile, testing of brood fish for the presence of ment. This may be a highly relevant situation IPN virus is mandatory. Testing for the ISA for Norwegian breeding companies targeting virus has been proposed in order to reduce the the egg market in Chile and facing competition risk for transferring the avirulent variant of the from local breeding companies. virus, which can mutate to the virulent variant in the progeny (38). A field trial with ISA- In recent years Norwegian breeding companies infected brood fish demonstrated lack of know- have significantly reduced the number of loca- ledge with respect to potential vertical transmis- tions (in some cases, down to just one or two) sion of this disease in Atlantic salmon (53). In used for regular performance testing during the this trial, 10 of 11 examined brood fish had high seawater phase. This strategy may be risky, con- titres of the ISA virus irrespective of clinical sidering the fact that farming of salmon in Nor- findings, and the correlation between clinical way is done across a very large geographical area symptoms or virus titre in the brood fish and the with significant variations in local light and tem- ISA virus in eggs was not significantly different perature conditions. Some of these companies from zero. The trial showed that RNA frag- also export significant volumes of eggs to Chile, ments from the ISA virus can be transferred representing farming conditions with higher av- from brood fish to eggs, but the trial did not give erage water temperature and day length. an unequivocal answer to what this means for transferring viable viruses and disease to the progeny.

Thematic area: Selective Breeding and Genetics 277 New technologies breeding candidates. Hence, genotyping costs Genetic markers for parentage testing can be greatly reduced, while still maintaining Genetic markers can indicate a close genetic re- high genetic gain. For schemes with 5,000 or lationship between two individuals, and there- 10,000 breeding candidates, little extra genetic fore be a useful tool to control the rate of gain was achieved by genotyping 1,000 instead inbreeding in breeding programmes based on of 100 candidates. For multi-trait selection pro- individual selection without physical tagging of grammes or where the trait cannot be measured the candidates. For species with high survival in on the breeding candidates, a higher number of all life stages, e.g. Atlantic salmon, similar con- candidates must be genotyped. Also note that trol of the rate of inbreeding can be achieved breeding values are somewhat biased in these without the use of marker information, by as- programmes, because only data from a small suring that in each generation, the population and selected sample of the candidates is used to consists of at least 50 parental pairs, each with estimate the breeding values. at least 100 offspring each (6, 16). Today, some breeding companies utilise genetic The advantage of using genetic markers for the markers instead of physical tagging to identify control of relationships and inbreeding in individuals for selection of traits that cannot be breeding programmes, where the information measured on the breeding candidates. However, on full- and half sibs is used to estimate breed- no documentation of the design and profit (mea- ing values, was studied by computer simulation sured as value of the genetic gain minus costs of (48). Selection was for one trait, recorded on running the breeding programme) of these 1,000, 5,000 or 10,000 breeding candidates breeding programmes is available. The poten- each generation. In general, results show that by tial increase in genetic gain makes the optimis- genotyping the 100 individuals with the highest ation of genetic marker use in breeding phenotypic values, genetic gain was 76–92 per programmes an important topic for further stud- cent of that achieved when genotyping all ies, especially for multi-trait selection, where several traits cannot be measured on the breeding candidates.

Father x Mother M1M2 M3M4 Marker-assisted selection Quantitative trait loci (QTL) are loci whose variability underlies variation in expression of a quantitative character. Selection for QTL is called marker-assisted selection (MAS). The Test offspring Breeding M1M3 + Fenotype candidates advantage of MAS compared to conventional M1M3 M1M4 + Fenotype selection is expected to be largest when the trait M1 M2M3 + Fenotype M4 M3 under selection has a low heritability or when M2M4 + Fenotype M2 M2M4 the traits cannot be measured on the breeding candidates. Examples of these traits are fillet 145x100//Kap16-fig04.eps Figure 4. Outline of marker assisted selection (MAS). The quality traits where fish are slaughtered before two marker-alleles M1 and M3 in bold print have a positive the traits are measured or disease traits, where effect on the actual trait. Individuals that possess one or survivors from a challenge tests are not allowed both of these alleles will be selected.

278 Aquaculture Research: From Cage to Consumption to be used in the nucleus because of the high increased the accuracy of the breeding values, risk of infecting fish in the nucleus. In addition, and thus genetic gain. Rate of inbreeding was one single fish can only be tested for one dis- lower for the MAS schemes as compared to the ease trait. For MAS, however, a DNA test for conventional breeding scheme, because the high- the QTL will identify the candidates with the er degree of within-family selection. favourable alleles, making the phenotypic recording of the candidates redundant. The increased gain for MAS makes it important to identify QTL for traits that are not measured Figure 4 shows a model for the design of a MAS on the breeding candidates. QTL for resistance to breeding scheme. Here, sires with marker geno- ISA has been identified (35). Figure 5 shows that type M1M2 and dams with genotype M3M4 and a QTL for resistance to ISA is probably linked to their progenies are genotyped. For the tested markers 4 and 5. Studies to identify QTL for IPN progenies, we also know the phenotype of the (4) and fillet colour (7) are done today. trait, and in this group of progeny the association between the QTL genotype and the phenotype is For Atlantic salmon, thousands of nucleotides estimated. The test progenies are, as explained made up of small pieces of DNA are identified, above, no candidates for selection. Markers M1 but only 13 genes have been identified and reg- and M3 have a positive effect on the trait under istered at NCBI (National Center for Biotech- selection. When using MAS, all M1M3-proge- nology Information, http://www.ncbi.nlm.nih. nies, some M1M4- and M2M3-progenies, but no gov/entrez). Even though the function of these M2M4-progeny, will become selected. genes is known, it is not known if they contain mutations and what effects these mutations Stochastic simulation of the breeding scheme in have on important production traits. Figure 4 has shown that MAS for one trait that cannot be measured on the breeding candidates Genomic selection can yield up to twice as much genetic gain com- Today, a genetic marker map with thousands of pared to conventional breeding schemes where single nucleotide polymorphisms (SNP) spread sib selection is practiced for such a trait, because over the whole genome is under development MAS could differentiate between breeding val- for Atlantic salmon. In the future, it is expected ues of animals within families, whereas the con- that faster and cheaper DNA analysis will allow ventional breeding scheme could not (49). This genotyping of many individuals for a large number of markers. This would make it possi-

20 ble to utilise high density genetic marker maps

15 to select individuals, which is needed for a method called genomic selection (34). It is ex- 10 pected that the high precision of the breeding 5 values – due to genetic values being based on 2LogLikelihoodratio 0 DNA information that also comes from the can- 123456 Marker no. didates themselves – will increase genetic gain.

145x100//Kap16-fig05.eps Figure 5. The figure shows that there is most likely a QTL for In the purest form of genomic selection, breed- ISA which is coupled to marker alleles 4 and 5 on an Atlantic salmon chromosome (35). ing values for one or several traits are calculated

Thematic area: Selective Breeding and Genetics 279 for the selection candidates based on the marker genotypes only. The association between the Definitions phenotypes and the markers has been calcul- Heritability ated beforehand in the population under selec- The proportion of the total phenotypic tion. For each individual, the effects of its variation of a trait that is caused by addi- markers are first calculated, and thereafter tive gene effects. summed to get a whole genome estimated breeding value. The phenotypic data needed to Additive gene effects estimate the association between markers and Gene effects that are transferred from phenotypes can come from e.g. a slaughter parents to offspring. house or a natural disease outbreak, which could make the sib tests redundant. Non-additive genetic variation Genetic variation that is caused by non- A method to calculate breeding values based additive gene effects. on a large number of haplotypes has been proposed (34). However, implementation of ge- Non-additive gene effects nomic selection in practical breeding schemes Gene-combination effects that are not, or must be preceded by theoretical comparisons only to a minor degree, transferred from to existing schemes using existing data. The parents to offspring. new statistical models and methods to estimate the association of the haplotype effects must be Breeding value adapted to practical breeding schemes, making The value of an animal as parent, judged this an exciting but demanding field. by the expected mean value of its progenies. Documentation of genetic changes Genetic correlation For the purposes of internal control and infor- The correlation between the true breed- mation to customers, it is of vital importance ing values of many animals for two traits. for the breeding company to have reliable documentation of the genetic changes for all traits Correlated response selected for and the economic value of these Genetic change in a trait caused by selec- changes. It is also of importance to monitor tion for a genetically correlated trait. possibly correlated genetic responses for traits not directly selected for. en, corresponding to an average realised genetic There is little published documentation on ge- gain of 15 per cent per generation (18) and sim- netic changes for traits selected for in Atlantic ilar to the expected genetic gain of 13 per cent salmon breeding programmes. Offspring of per generation calculated based on realised se- salmon selected for increased growth for five lection differentials (17). For the trait early sex- generations grew better in the freshwater phase ual maturity (grilse) the latter-mentioned study and were significantly heavier at slaughter than documented a 3.1 %-unit expected reduction offspring of wild salmon from the river Nams- per generation in the proportion of early sexual

280 Aquaculture Research: From Cage to Consumption maturing fish over four generations of selection. for and such estimates are only available for For feed efficiency an accumulated genetic gain growth and to some extent for feed efficiency of 25 per cent over five generations of selection and early sexual maturity. has been documented, most likely as a correlated response of the selection performed for in- For feed efficiency we can make a simple calcu- creased growth over the same period of time lation by assuming that the salmon of today use (54). For the other traits selected for, such as fil- 25 per cent less feed per kg gain than the off- let fat content and fillet colour and specific dis- spring of wild salmon (see Table 2; 1.125 kg vs. ease resistance recorded in challenge tests, no 1.5 kg) and a feed cost of NOK 8 per kg dry published documentation of expected or rea- feed. Then the accumulated economic value of lised responses is available. better feed efficiency becomes NOK 3 (0.375 kg feed/kg gain x NOK 8/kg feed) per kg With respect to methods for obtaining unbiased fish produced, or in total NOK 1,500 million for estimates of genetic changes for traits in aqua- 500,000 tons of salmon produced per year. The culture species, no reports are available. A com- economic value of better growth comes from a mon approach in farm animal species is to reduction in the fixed production costs due to estimate the difference between the average the much-reduced time to harvest (from about BLUP (best linear unbiased prediction, 26) 40 to 20 months). The value of lower propor- breeding values of animals of different generations of early sexually maturing fish is difficult tions. However, for completely discrete genera- to calculate because an increasing proportion of tions, no sires and/or dams are reused over the salmon are slaughtered before sexually ma- generations and estimates of genetic trend can ture fish become a problem. In addition comes only be obtained through the parent-offspring the value of reduced costs due to genetic chang- ties. Possible bias in genetic gain estimates thus es in the other traits selected for, i.e. disease re- obtained has not been investigated. The estab- sistance, fillet colour and fillet fat. A rough lishment of some direct ties through the reuse of calculation shows that the total economic value some sires and/or dams or some sire-dam com- of the selective breeding work for Atlantic binations may be required. salmon in Norway over 7–8 generations of selection adds up to at least NOK 15, or about One option is the establishment of a control NOK 2 per generation, or NOK 0.50 per year, population for each generation through the use all expressed per kg of fish produced. of some breeders with average breeding values. However, when selection is practised for more The total value of the Norwegian salmon pro- than one trait this may be difficult. duction for 2004 is estimated at NOK 9,710 million (31) and is distributed among the differ- The economic value of selective ent sectors of the value chain as follows: Nucle- breeding us breeding NOK 40 million; egg production No estimate is available for the economic value NOK 70 million; smolt production NOK 1,200 of the selective breeding work conducted for million; and grow-out production NOK 8,400 Atlantic salmon in Norway since the start in the million. Based on this only 0.41 per cent of the early 1970s. This would require reliable esti- total value (40/9710), or NOK 0.07 per kg of mates of the genetic gain for the trait selected fish produced, is returned to the companies run-

Thematic area: Selective Breeding and Genetics 281 ning the breeding nucleus in which the genetic be (a) continuous upgrading and documentation gain is produced. of the genetic material so that the breeding company is always ahead of its competitors; (b) If the economic value of selective breeding is crossbreeding and sale of hybrids; or (c) sale of NOK 0.50 per kg of fish produced per year, it is sterile production animals. Legal protection can evident that most of the economic value of the be secured through (a) branding and trade- selective breeding work is harvested by the marks; (b) material transfer agreements be- grow-out producers and consumers and not the tween the breeding company and the multiplier breeding companies. A change in this will most or farmer; or (c) patents. Worth mentioning is probably first come as a result of marketing that only single genes can be patented, not pop- forces when the added value of the use of the ulations. best genetic material has been documented, creating a willingness to pay more for the best An alternative approach is the establishment of genetic material. a mandatory certification system based on the known pedigree of farmed fish (42). This could The current imbalance between the added value be an integrated part of a larger traceability sys- of the improved genetic material and the modest tem for farmed fish production (47). The verifi- profit margin the breeding companies earn from cation of such a system could be done using the sale of the improved genetic material makes different types of genetic markers (24, 25, 46, it difficult for the breeding companies to invest 59) and at a relatively low cost because it will in research and development. In view of the in- only require the genotyping of tissue samples of creased international competitiveness in the alleged parent fish. Such a system would allow salmon industry, this is a serious challenge for the breeding companies to check if the actual the whole Norwegian salmon industry. fish come from their material. Combined with branding and trademarks, continuous upgrading Who owns the improved genetic of the genetic material through selective breed- material? ing and material transfer agreement, this may Different biological protection measures related become a balanced strategy. A dialog between to access to and exclusive rights of improved breeding companies and public authorities is re- genetic material have been discussed (42). Al- quired to obtain legal approval of such a system. ternative biological protection mechanisms can

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284 Aquaculture Research: From Cage to Consumption Kjersti Turid Fjalestad1), Svein-Erik Fevolden2), Knut Jørstad3) and Ingrid Olesen4) 1) Fiskeriforskning (Norwegian Institute of Fisheries and Aquaculture Research), 2) Norwegian College of Fishery Science, University of Tromsø, 3) Institute of Marine Research, 4) Akvaforsk – The Institute of Aquaculture Research

Breeding and Genetics – New Species

Today’s methods for population genetics studies of Atlantic cod enable researchers to differentiate between North-east Arctic cod and Norwegian coastal cod, and to a certain extent, between individual coastal cod populations. Cod seem to be less genetically homogeneous than formerly assumed. This has had certain consequences for stock management and may also turn out to have implications for cod farming and breeding. A large-scale cod breeding programme has been launched, based on quantitative genetics methods involving the stocking of individual families. Broodstock fish have been collected, base populations established, and methods have been developed for stripping cod of eggs and milt and for keeping families in small units. Genetic differences between families in growth and resistance to vibriosis have been demonstrated. These characteristics have been used as selection criteria in the breeding programme. The most important result so far is that the first selected year class of cod has been produced. DNA methods are now available for testing family relationships in cod. Further studies of genetic markers and the production of genetic maps may lead to the inclusion of marker-assisted selection in breeding programmes. A wide range of genetic variation has also been demonstrated in halibut growth, and this can be exploited in breeding. Stripping and the production of groups of offspring are functioning satisfactorily, but to date, a halibut breeding programme has not been launched. Finally, it has proved possible to produce sterile cod and halibut, but a great deal of work remains to be done before this can be implemented in practice.

Thematic area: Selective Breeding and Genetics 285 This chapter presents three main areas of re- opment of cod farming has triggered a wide- search in the breeding and genetics of new spe- ranging discussion of the potential environmen- cies. These areas are: tal impact of cod farming, including the possible genetic effects on wild stocks on the one Population genetics studies, mostly of Atlantic hand, and the question of which cod popula- cod. These studies have largely concerned the tions would be most suitable for farming on the characterisation of genetic similarities and dif- other. Knowledge of genetic variation, both ferences between different stocks of cod. This within and between cod populations, is thus im- research will be of benefit both to the manage- portant for the successful launching of a cod ment of our wild cod stocks and the breeding breeding programme. programme. North-east Arctic cod vs. Norwegian Breeding research and breeding efforts on At- coastal cod lantic cod and Atlantic halibut. Interest in farm- For several years now, studies have been carried ing marine species has grown in recent years. A out to test the theory that stationary coastal cod breeding programme ought to be set up for ev- and migratory North-east Arctic cod represent ery farmed species when the life-cycle is con- two genetically distinct stocks. The gene that trolled and long-term production levels are high controls production of the protein pantophysin enough to justify investment in such a pro- (the gene itself is called Pan I) has been most gramme. Based on this, cod and halibut are cur- frequently used to distinguish between the two rently the only species that have been seriously stocks (1, 2, 3). There are wide differences be- evaluated in connection with the establishment tween Norwegian coastal cod and North-east of breeding programmes. Other prospective Arctic cod in the frequencies of two variants of species include turbot and wolffish, as well as the gene (the A and B allele classes) (1, 3). shellfish such as scallops, oysters and mussels. Since the start of these studies in 1993, cod Production of these species is currently at low from North Norwegian fjords and from coastal levels, and to a certain extent insufficient areas further south have displayed consistently knowledge of controlled farming procedures for high frequencies of the A allele variant (80 per these species does not yet allow breeding pro- cent or more), while North-east Arctic cod have grammes to be established. had similarly high frequencies of the B allele variant (90 per cent or higher). Chromosome manipulation and gene technology methods. This section discusses the potential During the past few years, much effort has been role of these methods in cod and halibut farm- put into identifying other markers that could ing and in breeding programmes. support the theory of genetic divergence between coastal cod and North-east Arctic cod, as Population genetics studies well as between different populations of coastal of cod cod. Promising results have been found by the Norway has a long tradition of studies of Atlan- use of microsatellites (variation in number of tic cod genetics, both in connection with the repetitions of very short DNA sequences). identification of wild populations and in efforts These markers support the hypothesis that there related to sea ranching and farming. The devel- is a clear genetic difference between coastal cod

286 Aquaculture Research: From Cage to Consumption and North-east Arctic cod, as suggested by the Specific genetic mapping of individual Pan I gene. Moreover, the microsatellites indi- populations of coastal cod cate that within North-east Arctic cod, and es- For many years, both fishermen and scientists pecially within coastal cod, there are further have acknowledged that individual fjords in divisions into sub-populations (see below). Northern Norway may have their own stationary cod stocks. In the course of determining the Objections have been raised to such a genetic extent of genetic differences between various division of cod populations (4, 5), because some coastal cod populations, the past few years have of the gene variants that display large variations seen the collection of material from 70 different between coastal cod and North-east Arctic cod sites, from Hvaler in the south to Varangerfjord are influenced by the environment in which the in the north. These samples have been gathered fish live. Nevertheless, the differences in the from spawning grounds on the coast and in the frequencies of variants at Pan I and some of the fjords, for the most part during the spawning microsatellites are so great that it would be dif- season. Thanks to cooperation with Russian re- ficult to explain them without recourse to the search institutions, we can now also compare theory of genetically different spawning popu- cod from Russian waters with fish taken on the lations. In other words, there is growing agree- Norwegian coast. Both older (e.g. haemoglo- ment that the genetic differences that can be bin) and more recent (e.g. Pan I, microsatellite) measured between coastal cod and North-east genetic analytical methods are being used, so Arctic cod are due first and foremost to the low that fish sampled at present can be directly com- frequency of genetic exchange between the two pared with analyses of earlier catches. stocks. In spite of the fact that coastal cod and North-east Arctic cod partially overlap on cer- The results of analysing the recently collected tain spawning grounds, there appear to be fish largely confirm those obtained in the 1960s, mechanisms that prevent them from spawning which even then demonstrated genetic differ- together. ences between North-east Arctic cod and coastal cod, and between coastal cod from different Outside the spawning season, coastal cod and regions. As part of this study, a more detailed North-east Arctic cod may inhabit the same comparison has been made on material collect- areas, such as when immature North-east Arctic ed from the Lofoten and Vesterålen region, the cod enter fjords in the course of feeding migra- main spawning site for North-east Arctic cod. tions. The number of such cod in the fjords, This material has been thoroughly tested using whether they are fry or adult fish, will vary from new statistical methods to assign individual fish year to year, depending on the availability of directly to the North-east Arctic or coastal cod prey and on hydrographic conditions. This can population (6). The total collection of fish in explain the observed differences in the fre- this ongoing study is thought to provide a com- quency of certain gene variants from one year to plete database of genetic profiles of all our cod another. populations. This database will be important reference material in order to evaluate any genetic changes that take place in populations of cod in the future.

Thematic area: Selective Breeding and Genetics 287 Do different populations of coastal cod pattern even after almost three years in aquacul- have different characteristics? ture. This is an indication that genetic compo- One question that needs consideration is nents are involved in determining specific whether locally adapted cod populations with spawning times, which could represent adapta- their own genetic features perform differently tion to particular environmental conditions. under aquaculture conditions. To evaluate the performance of possibly genetically different Genetically marked cod? populations, the various groups need to be com- A discussion concerning genetic interactions pared under identical environmental conditions. between farmed cod and wild cod arose as early This means that the offspring of different as the end of the 1980s, and led to the develop- groups must be placed together as early as the ment of a genetically marked cod (10, 11). This hatching stage and fed together in the same fish carried a gene marker that is rare in nature tank. Microsatellite DNA analyses for the iden- (approximately 1 in 10,000 individuals), and tification of families and strains (7) enable the gene is easily identifiable. Fish from a pop- researchers to do this. ulation that possessed this gene were used in releases in the early 1990s (12). This led to a large The offspring of broodstock from four different increase in the frequency of the marker gene in regions (Porsangerfjord, Tysfjord, Helgeland local populations, but repeated samplings and Øygarden; two year classes of each popula- showed that the frequency fell rapidly in the tion) are currently being studied at the Parisvatn course of the years following the releases (13). Field Station in Øygarden and the Austevoll Aquaculture Station. Fish that were produced in A number of cod carrying this genetic marker 2004 are now also being tested at the breeding were recently found among wild fish that were station in Tromsø. Weight and length of these being collected as broodstock for the produc- fish are regularly recorded. The results of these tion of cod fry at Parisvatnet in Øygarden. efforts will provide useful knowledge about the These fish are probably descendents of the fish aquaculture potential of these cod strains. We released in the early 1990s. Thanks to these will also be able to study any interactions be- fish, this strain is once again available, opening tween genotype and environment, given that the up the possibility of carrying out important same families and strains are being kept in both studies in connection with a growing cod-farm- Bergen and Tromsø. All broodstock have been ing industry. Not of least importance is the pos- classified for a number of genetic markers (8). sibility of generating new knowledge of genetic The broodstock were collected well in advance interactions between escaped farmed cod and of spawning in order to acclimatise them to the wild cod. aquaculture environment. Our expectation was that spawning dates in the different strains Population genetics studies of would become as similar as possible. However, lobster it turned out that the spawning times of fish Studies of lobster genetics were originally car- from different areas were very different. The ried out in connection with a large-scale release broodstock from Helgeland in particular experiment on Kvitsøy that started in 1990. spawned much later than the other strains (9). Marked young lobsters were released and re- We were able to observe an identical spawning captured, and rates of marked lobsters have

288 Aquaculture Research: From Cage to Consumption been registered (14, 15). Details of the genetic Choice of breeding strategy studies carried out in connection with the re- Breeding programmes based on methods that lease trials are discussed in another chapter of depend on quantitative genetics are still the this volume. most relevant, even though it could be useful to supplement them with gene-technology meth- Parallel to these releases, a major study of ge- ods. If we wish to select for higher disease re- netic variation in lobsters was also conducted sistance and better slaughter quality traits, the (16). The sample material covered the whole methods available imply that we need to base area in which lobsters are found, and the results our efforts on a family-based breeding pro- indicated small but significant genetic variation gramme. To obtain a well-balanced family ma- between different geographical areas (17, 18). terial, it is difficult to avoid keeping families in The first lobster sea ranching permits have al- separate tanks during the early stages of the life ready been issued, and plans for commercial cycle, although this is an expensive procedure. lobster farming have been drawn up. In connec- Bringing up all the families together in large tion with the plans for lobster farming on common tanks before the numbers of fish in Kvitsøy, microsatellite DNA could also act as a each family have stabilised would lead to tool for family group identification. smaller effective numbers in the breeding population, because fewer families would be repre- Breeding research in cod and sented and because of big variation in family halibut size (19–22). This can both reduce the selection Experience from salmon farming and domestic response and lead to inbreeding. Problems of animal husbandry tells us that breeding efforts this sort will be particularly acute in species will also be important for the profitable produc- with low and variable survival rates, such as tion of marine species. There is a great deal to halibut and cod. be gained in terms of productivity by starting an efficient breeding programme as soon as pos- Establishing a base population – choice of sible. A prerequisite for launching a breeding strains and populations programme is control of the complete life cycle, When a breeding programme is being started, it and a relatively secure production of families, is essential to start out with the best possible or alternatively of other small groups, in which material, and to use many unrelated fish as the relevant traits are recorded. Reliable infor- broodstock. By using fish from different strains, mation regarding the family background of the the initial material will provide the widest pos- fish is also essential for family-based breeding sible genetic basis for later selection. This is programmes. critical to carrying out efficient breeding over several generations. Important challenges facing breeding research and the development of breeding programmes Breeding goals include the choice of breeding strategy, evalua- Rational breeding programmes must have clear- tions of suitable strains, estimates of genetic ly defined goals. Generally speaking, the goal parameters such as degree of heritability and will be to develop a fish that possesses good genetic correlations, assessments of breeding production characteristics and is of good quality goals and genotype-environment interactions. for human consumption when it is raised under

Thematic area: Selective Breeding and Genetics 289 normal fish-farming conditions. In principle, all amples of slaughter quality traits include dress- important economic characteristics should form ing percentage, liver index, texture, taste and part of the breeding programme, while proper- colour. ties that are of purely social economic value (e.g. ethical values) may also be relevant. It Improved disease resistance. Goal-oriented must be remembered that the greater the num- breeding can prevent disease and raise disease ber of traits included in the breeding goal, the survival rates in a sustainable manner, and thus less pronounced the genetic improvement in reduce losses in fish farming operations. It is breeding will be for each individual characteris- important to include this characteristic in a tic. breeding programme as early as possible, for example by infecting fish from a number of Higher growth rates. Growth rates are one of families with one or more diseases and then us- the most economically important traits in fish ing their survival data as a selection criterion for farming. A fish that grows rapidly has many ad- broodstock. vantages, and the growth rate is of decisive importance for profitability. In a breeding pro- Development of test procedures gramme, this property is usually registered in To operate an efficient family-based breeding terms of weight at the normal time of slaughter. programme, full control of broodstock and reproduction is needed in order to know the fam- Feed conversion efficiency. Feed costs make up ily background of all the fish. The families must an important proportion of production costs. be kept in separate tanks until the fry are big Developing a fish that consumes less feed per enough to be tagged. As an alternative, a system unit growth is therefore of great economic im- is needed for testing the family relationships of portance. Animals that grow rapidly tend to all the fish. consume less feed per unit of growth than those that grow slowly. For this reason, selecting for It is also essential to have a reliable, objective rapid growth will often have a desirable effect and rational methodology for registering the in- on this trait as well. dividual traits being selected for. For certain traits it may be necessary to develop new meth- Age at sexual maturation. The fish should not ods such as how to measure traits related to become sexually mature before reaching an ac- product quality, or challenge tests for bacteria, ceptable slaughter weight, because at maturity viruses and parasites. the fish often grow more slowly or stop growing altogether, or because the flesh quality becomes Genetic parameters poorer. With cod this is an especially serious Genetic improvement by selective breeding re- challenge, since the male fish in particular tend quires genetic variation for the trait in question; to become sexually mature at an extremely ear- the genetic correlations between all traits in- ly age. cluded in the breeding goal must also be known.

Slaughter quality. It is to be expected that the Genotype-environment interactions economic value of slaughter quality traits will An important question in all breeding efforts is grow in step with greater market coverage. Ex- whether families that perform well in one par-

290 Aquaculture Research: From Cage to Consumption ticular environment (e.g. a geographical region) kept in large tanks at least 10 metres in diameter also perform well in another one. Is it the case (25). that families that score highly under certain conditions will also do so under other condi- Incubation, storage and start-feeding in tions? For example, are there families that are small units particularly well adapted for aquaculture in the Standard production systems are not suitable north or the south of the country? It is known for the production of halibut family groups be- from several other fish species that genotype- cause there can be wide variations in survival environment interactions are relatively low rates between families. It is also impossible to within small or moderate environmental ranges incubate different age groups of larvae in the of difference. In such cases, it would not be nec- same tank. Even if the age difference is only a essary to develop separate breeding pro- few days, the older larvae will outcompete their grammes for different production environ- younger brethren. The development of small ments. systems was therefore a breakthrough for breeding programmes, and has allowed con- Atlantic halibut trolled production of family groups without Broodstock and stripping making heavy demands on area or costs. Roe Current methods of stripping halibut provide us have been incubated in small, 25-litre units (26) with roe and milt of reasonably good quality in that require little work and are suitable for the sufficient quantities to run efficient breeding production of family groups. The same prin'ci- programmes. The broodstock are kept under ple may also be suitable for larger units if controlled light conditions, and water tempera- desired (26). ture is kept closely under control (23). This has made it possible to extend the spawning season Several systems for start-feeding halibut larvae until early autumn, which in turn means a great- have been tested and compared, and a method er spread of breeding activities throughout the has been developed for start-feeding family year. Choosing the right time for stripping is groups in small, 20-litre aquaria. Comparisons perhaps the most important factor in determin- of different tanks, water flow rates and lighting ing good egg quality and satisfactory rates of conditions have shown that larval growth from hatching. The methodology is labour-intensive, the start-feeding stage can be improved in such as it requires checking the rhythm of egg release small tanks. These findings were useful when a of each female fish. Successful experiments in- relatively small but efficient system for produc- volving freezing halibut milt have also been car- ing cod families was later being developed. ried out (24). More details regarding the production of halibut larvae can be found elsewhere in this volume. Today’s knowledge is sufficient to allow manipulation of the spawning time, making it possible The number of broodstock is still a bottleneck to produce family groups throughout the year. when breeding programmes are being started, However, this is a resource-intensive process but cooperation between scientists and produc- that requires large numbers of broodstock. Ex- ers of fry who maintain their own broodstocks perience has shown that broodstock should be may be a way of solving this problem. Fry producers with their own broodstock can produce

Thematic area: Selective Breeding and Genetics 291 2.5

1.5

Growth g/day

0.5

E3C J5B Pi7A E3A J5A X2A X2B P5A P3A Ø6A P7B Q3A K5A Ø6B Family

145x100//Kap17-fig01.eps Figure 1: Variation in second-year growth (g/d) in halibut families produced in 1998 (26).

family groups which will subsequently be trans- growth from about one-and-a-half to two-and- ferred to a “breeding station” for testing and se- a-half in the 1998 year class. Although the ma- lection purposes. Cooperation of this sort will terial involved is limited, there are large varia- exploit existing resources better and more effi- tions both within and between families. The ciently. Proposals for developing and operating results of subsequent registrations at the ages of a future family-based halibut breeding pro- four and five showed similarly wide variations gramme have been drawn up, and the prospects in inter-family growth rates (28). This genetic of breeding for better feed utilisation, growth variation can be exploited in a breeding pro- rates, age of sexual maturation, slaughter quali- gramme to improve growth rates in halibut ty and resistance to disease have been evaluated farming. Reliable estimates of genetic parame- (27). An initiative for a halibut breeding pro- ters for use in selective breeding, a more exten- gramme has been launched, but the programme sive material and registrations of more traits are has yet to start. needed. More knowledge is also needed regarding age at first sexual maturation, synchronisa- Genetic variation in survival and growth tion of spawning, optimal re-use of broodstock In the course of 1997 and 1998, 22 full-sib fam- and methods for registering more traits in live ilies were produced. At around two-and-a-half fish. and three-and-a-half years of age there were large differences in growth and survival rates Atlantic cod between these family groups (25). Survival There has been renewed interest in intensive rates ranged from 30 to 85 per cent, and the cod farming in recent years, and large-scale cod mean family live weight ranged from 0.4 to 1.1 breeding programmes have been launched. The kg at two-and-a-half years of age and from 1.0 Norwegian Institute of Fisheries and Aquacul- to 1.6 kg at around three-and-a-half. Figure 1 ture Research in Tromsø has been given respon- shows the differences between families in daily sibility for the national breeding programme,

292 Aquaculture Research: From Cage to Consumption which is financed by the Ministry of Fisheries marker, has been used to distinguish between and Coastal Affairs. Marine Breed, a commer- North-east Arctic cod and coastal cod, to con- cial company, is also involved in cod breeding. trol which fish are being used as broodstock (31). Broodstock and stripping We still know little about the suitability of Stripping eggs and milt from cod instead of al- North-east Arctic cod and different populations lowing pairs of fish to spawn naturally has been of coastal cod for aquaculture. This can be test- successful with good egg quality. Figure 2 ed by keeping different strains under common shows female and male fish ready for stripping. environmental conditions (same sea-cage or tank) throughout the growth phase. All impor- Fry production tant traits such as growth and disease resistance In principle, fry are produced in the same way should be tested for in this way. So far, the re- as in other intensive-production fish farms. The sults suggest that there are minor differences in most important difference is that family groups growth rates between North-east Arctic cod and are held in separate units until the fry are large coastal cod, although certain results suggest enough to be tagged. This means that instead of that North-east Arctic cod grow better. Other having a few large tanks, as we find in commer- studies (29) have found no differences in cial farms, the breeding station has a large num- growth rates, sexual maturation and deformities ber of small units that hold one family each. between coastal cod and North-east Arctic cod. Hence, it is important that all family groups be A number of coastal cod populations are cur- raised under as similar environmental condi- rently being tested for their aquaculture poten- tions as possible. Figure 3 illustrates tanks for tial. start-feeding of families.

Broodstock for starting breeding programmes Tagging have been collected from both North-east Arc- About six months after hatching, when the fry tic cod and several different populations of have attained a minimum weight of 5–10 coastal cod (30, 31). Pantophysin, a genetic grams, a number of fry from each family group

Figure 2: Female fish with swollen anal region, ready for stripping (left) and male fish being stripped (right). (Photo: Kjell Ingebrigtsen)

145x100//Kap17-fig01.eps

Thematic area: Selective Breeding and Genetics 293 are individually tagged with passive integrated of production, the fish are weighed regularly, transponder (PIT) tags. These are tiny electron- and other records are gathered (e.g. date of sex- ic tags containing unique numerical codes that ual maturation, and deformities). can be read by a special instrument and uploaded directly to a computer program. The tag is At the same time as the groups of fry are trans- placed in the abdominal cavity of the fish, en- ferred to the breeding programme’s sea-cages, abling it to be identified for the rest of its life other groups of fry are transferred to testing sta- until it is slaughtered. Once they have been tions elsewhere in the country. There they are tagged, fry from different tanks are placed to- held until slaughter, and records are collected gether in large tanks until they are transferred to just as in the sea-cages of the breeding pro- the breeding station’s sea facility and test sta- gramme. Data from the test stations are of par- tions. ticular importance as a means of revealing whether the performance of different families The marine phase differs under different farming conditions About nine to 10 months after hatching, the fry (genotype-environment interactions). The test from the breeding nucleus are transferred to stations also provide useful data for the selec- sea-cages, where the fish material that includes tion process and provide a degree of backup in representatives of all the families is raised until the event of the fish at the breeding station the broodstock are selected. The best indi- being lost through disease, damage to facilities viduals are brought back to the breeding station or for other reasons. as broodstock, while the remaining ones are slaughtered. The groups are neither sorted nor split up during the marine phase. In the course

145x100//Kap17-fig01.eps Figure 3: Tank for start-feeding of cod families. Part of the facilities of the national breeding programme in Tromsø. Photo Atle Mortensen

294 Aquaculture Research: From Cage to Consumption Table 1: Heritability estimates for body weight at various ages

Trait Heritability (S.E.M.) Reference

Body weight at tagging 0.52 (0.26) 30 Body weight at 16–18 months 0.44 (0.28) unpublished Body weight at 2 years 0.51 (0.10) 29 Body weight at 2.5 years 0.50 32

Production of family groups and genetic Growth variation in traits of interest A number of studies of genetic variation in In 2002, Akvaforsk produced around 50 full-sib growth have been carried out. The results so far families of cod. This material has been taken suggest that there is a wide range of genetic over by Marine Breed, which produced 86 fam- variation in ability to grow, and the heritability ilies of first-generation selected material in is about the same as we have found in other spe- 2005. The Norwegian Institute of Fisheries and cies, or somewhat higher (Table 1). Figure 4 Aquaculture Research started the national cod shows mean family breeding values for weight breeding programme with 47 tagged families in at an age of two-and-a-half years in one year 2003, 77 families in 2004 and 84 families in class. One study concluded that there appear to 2005. The target is to produce 200 full-sib fam- be only slight differences in growth between ilies a year. Since 2006, selected material from North-east Arctic cod and coastal cod (29), the cod breeding programme has been pro- while the cod breeding programme in Tromsø duced. has observed better growth in North-east Arctic cod, at least in one year class (unpublished results).

Coastal cod North-east Arctic cod 140

120 100

alues 80 60

Breeding v Breeding 40 20 0 1 3 5 7 9 11131517192123252729313335373941

Families

145x100//Kap17-fig01.eps Figure 4: Breeding values for growth at 2.5 years of age; 2003 year class (32).

Thematic area: Selective Breeding and Genetics 295 Disease resistance (Figure 5). These results demonstrate that sur- Cod are still a relatively new species in inten- vival after vibriosis infection can be used as a sive aquaculture, and it is not known which dis- criterion for selecting more-resistant cod (35). eases are likely to be most serious in the future, although vibriosis is already a problem in sever- Age at sexual maturation al places. An infection model for vibriosis in Early sexual maturation is a major problem in cod has been developed (33), and the first infec- cod farming. The costs of early sexual maturation challenge trials on a cod genetic material tion in cod are high, involving reduced appetite, were carried out in 2004 in order to study genet- major weight loss, an increased feed factor and ic variation in vibriosis resistance (34). This ex- lower survival rates. Genetic variation between periment demonstrated a clear variation in families in the frequency of early sexual matu- resistance to vibriosis, in spite of the limited ration has been demonstrated with a heritability amount of family material. In winter 2005 a of 0.22 (29), which implies a possibility of new vibriosis challenge test was performed on modifying this trait by selective breeding. 3,576 cod fry from 50 different families of coastal cod and North-east Arctic cod. At the Deformities end of the experiment, overall survival was 13.6 A series of skeletal deformities have been ob- per cent, but the coastal cod had a distinctly served in cod, and these are a serious problem in higher survival rate (17.0 per cent) than the intensive fish farming. Most deformities are due North-east Arctic cod (10.3 per cent). Survival to either spinal malformations or neck bend. It rates of individual families ranged from 1.4 to is difficult to observe such deformities before 37.9 per cent, and calculations so far suggest the fry have reached the tagging size. that the heritability of the trait “days of survival following infection” varies from 0.10 to 0.18

145x100//Kap17-fig01.eps Figure 5: Survival rates of different families following vibriosis challenge (35).

296 Aquaculture Research: From Cage to Consumption We do not know when the deformities occur – Production of female halibut by probably at different points in time. They may gynogenesis and gene technology even be determined during the larval phase. With the aim of producing only female fish, ex- There also appears to be a genetic component in periments on the production of gynogenetic lar- this trait, with a heritability of 0.27 (29). We do vae have been carried out. Milt was exposed to not know whether there is a correlation between gamma radiation in order to enable it to trigger genetic disposition and environmental factors. cell division in the egg without the sperm contributing their genetic material. A cold shock Genotype-environment interactions caused the chromosomes, which are normally A number of studies aimed at testing interac- expelled from the egg, to be retained by it. This tions between genotype and geographical envi- process produces diploid individuals, as if the ronment are currently underway. The results of mother fish had mated with itself. In both stud- these studies are not yet clear, but they may af- ies, the gynogenesis groups had extremely low fect the development of the cod breeding pro- survival rates to hatching and later in the larval gramme. phase (25). This method of producing female fish is not yet fully developed, and more re- Chromosome manipulation and search and development efforts are still re- gene technology methods quired. Sterile fish – triploidy Early sexual maturation in cod has negative ef- Two alternative gene technology strategies for fects such as reduced growth and survival rates solving the problem of farming male halibut are and poorer flesh quality. Producing sterile trip- also being studied. Identifying gender-specific loid fish avoids such problems, as well as elim- DNA sequences would allow male fish to be inating the possibility of genetic interactions sorted out at the larval stage. So far, this has not and impacts on wild populations. Efforts to pro- been done successfully. An alternative strategy duce triploid cod started in 2003, and studies would be to increase the number of hen fish by have already shown that such fish can be pro- influencing sexual differentiation at the larval duced with the aid of cold shock, although only stage. The gene responsible for aromatase, the a small proportion (2–14 per cent) of the larvae enzyme involved in this process, has been became triploid (36). There are now plans to ex- cloned in order to study the effects of water tend these experiments to include heat shock temperature on the activity of the gene (38). It and hydrostatic pressure treatments. has been shown that raising the water temperature down-regulates the aromatase gene, thus Early sexual maturation and growth stagnation raising the proportion of male halibut (39). are also a problem in male halibut. Triploid halibut larvae have been produced by means of Gene technology methods both heat shock and cold shock (37). The latter Developments in the field of gene technology technique produced the high survival rates (50– may open up the possibility of new breeding 82 per cent of control-group rates). strategies. Such methods will probably supplement classical breeding strategies.

Thematic area: Selective Breeding and Genetics 297 DNA fingerprinting, parental assignment Summary Genetic markers can be used to identify strains Population genetics studies suggest that Atlan- (2), families and individual relationships (7, 40) tic cod in Norwegian waters are more genetical- in cod. Such identifications are useful in breed- ly differentiated than formerly believed. This ing programmes, since they allow individual has implied consequences for management of fish to be assigned to the correct family. DNA the wild populations. To what degree this find- tests of this sort enable us to stock different ge- ing should be taken into account in cod farming netic groups together at an early stage of devel- and cod breeding programmes is still a matter of opment – at hatching, for example – thus discussion. Fry producers have already crossed avoiding the problem of keeping families in North-east Arctic cod and Norwegian coastal separate units for long periods of time. The dis- cod, and there are no restrictions on transferring advantage of this procedure is that it could re- fry from one region to another. It seems unreal- sult in a badly balanced material because the istic to design specific breeding programmes survival rates of individual families are very dif- for each individual farming region or every sin- ferent. DNA testing is still relatively expensive gle fjord. On the other hand, many coastal cod and time-consuming, and it cannot replace tag- populations are small and may be vulnerable to ging (41). Physical tagging is sometimes im- being crossed with foreign genetic material. possible, particularly with small fish such as The process of gaining more genetic knowledge early-stage cod larvae used for virus resistance of coastal cod populations, in particular to find testing. In such cases, parental assignment of out whether some of them possess special adap- individuals that have been used for such tests tations, should therefore be continued. can be a very useful tool. Large-scale cod breeding programmes have Marker-assisted selection (MAS) been launched both in the public sector and by One way of making use of genotype informa- private companies. Broodstock have been col- tion in a breeding programme is to utilise mark- lected, base populations established and the ers that are linked to genes that influence first selected year classes of cod have been pro- important traits. Genetic markers can be used to duced. The necessary infrastructure is also in search for the loci that are important for particu- place. Methods of stripping cod and of keeping lar traits; this is known as quantitative trait loci families in small units until tagging function (QTL) mapping. In 2005, a project with the goal reasonably well. However, there is still plenty of finding QTL for disease resistance in cod of room for improvement, particularly in in- was launched. So far, the project has produced creasing survival rates in the start-feeding data from vibriosis challenge experiments on phase. The breeding strategy that has been families (34) and has developed genetic mark- adopted is based on combined family and indi- ers for use as microsatellites and single nucleo- vidual selection and requires families to be held tide polymorphisms (SNPs). If markers linked in separate tanks until the fish are tagged. This to disease resistance can be found, breeding is expensive, but it does ensure that the genetic programmes could probably be made more effi- material is well balanced. A wide range of gen- cient. etic variation in growth and a moderate degree of variation in vibriosis resistance have been demonstrated. More knowledge is needed re-

298 Aquaculture Research: From Cage to Consumption garding genetic variation in a number of impor- improving growth in farmed halibut. Accurate tant traits, and not least, correlations between estimation of genetic parameters for breeding them. For example, genetic studies of age at purposes requires a larger material and the reg- sexual maturation have been performed, but istration of several other traits. More knowledge these need to be supplemented by studies that is also required regarding age at first sexual employ better methods of assessing the status of maturation, synchronisation of spawning, opti- sexual maturation. For certain traits, it may be mal re-use of broodstock and methods for re- necessary to develop new methods such as tech- cording more traits in live fish. nology for measuring slaughter quality traits and developing new challenge tests for different If it turns out to be desirable to produce sterile types of bacteria, viruses and parasites. cod and halibut by inducing triploidy or gynogenesis or by gene technology, these methods Large-scale halibut breeding programmes have will need to be further developed and much im- not yet been started. Methods of stripping and proved. producing groups of offspring function satisfactorily, and currently do not act as a bottleneck in Good DNA-based methods already exist for halibut breeding efforts. Nevertheless, there is distinguishing between North-east Arctic cod still room for improvements that would make and coastal cod and for testing kinship in cod. production more reliable and rational. There is Further studies aimed at identifying genetic a wide range of genetic variation in growth be- markers could help us to complement current tween halibut families, which could be exploit- breeding strategies with new strategies such as ed in a breeding programme aimed at marker-assisted selection.

Thematic area: Selective Breeding and Genetics 299 References 1) Fevolden, S.E., Pogson, G.H., 1997. Genetic diver- Enhancement and Sea Ranching (eds. Howell, B., gence at the synaptophysin (Syp-1) locus among Moksness, E., & Svåsand, T). Fishing News Books, Norwegian coastal and north-east arctic populations Blackwell Science Oxford, UK. of Atlantic cod, Gadus morhua. J. Fish. Biol. 12) Jørstad, K.E., 2004. Genetic studies in marine stock 51:895–908. enhancement in Norway. In: K. Leber, S. Kitada, 2) Pogson, G.H. & Fevolden S.E., 2003. Natural selec- H.L. Blankenship & T. Svåsand (eds.). Proceedings tion and the genetic differentiation of coastal and of Second International Symposium on Stock En- Arctic populations of the Atlantic cod in northern hancement and Sea Ranching. Blackwell Science Norway: a test involving nucleotide sequence vari- Ltd., Oxford. ation at the Pantophysin (Pan I) locus. Molecular 13) Jørstad, K.E., Nævdal, G., Karlsen, Ø., Torkildsen, ecology 12:63–74. S., Paulsen, O.I., Otterå, H., 2004. Long term stud- 3) Sarvas, T.H. & Fevolden, S.E. 2005. Pantophysin ies on genetic interaction between wild and ranched (Pan I) locus divergence between inshore vs. off- cod (Gadus morhua) by use of a genetic marked shore and northern vs. southern populations of At- strain. Fisheries Society of the British Isles Annual lantic cod in the north-east Atlantic. J.Fish. Biol. Symposium, 19–23 July 2004, Imperial, college, 67:444–469. London. 4) Mork, J., and Giaever, M., 1999. Genetic structure 14) Agnalt, A.-L., van der Meeren, G.I., Jørstad, K.E., of cod along the coast of Norway: Results from Næss, H., Farestveit, E., Nøstvold, E., Sfåsand, T., isozyme studies. SARSIA 84(2):157–168 Korsøen, E. and Ydstebø, L., 1999. Stock enhance- 5) Karlsson, S. and Mork, J., 2003.Selection-induced ment of European lobster (Homarus gammarus): A variation at the pantophysin locus (Pan I) in a Nor- large scale experiment off south-western Norway wegian fjord population of cod (Gadus morhua L.) (Kvitsøy). In: Stock Enhancement and Sea Ranch- Molecular Ecology 12 (12):3265–3274 ing (eds. Howell, B., Moksness, E., & Svåsand, T.) 6) Wennevik, V., Jørstad, K.E., Fevolden, S.-E., Dahle, pp. 401–419. Fishing News Books, Blackwell Sci- G., and Nedreaas, K., 2005. Application of new ence Oxford, UK.. DNA techniques to reveal population genetic struc- 15) Agnalt, A.-L., Jørstad, K.E., Nøstvold, E., Fares- ture of Atlantic cod (Gadus morhua.) in the north- tveit, E., Næss, H., Kristiansen, T. and Paulsen, O.I., east Atlantic. International Scientific and Applied 2004. Long term lobster (Homarus gammarus) en- Conference, Moscow, November 2005. hancement at Kvitsøy Islands – perspectives for re- 7) Delghandi, M., Mortensen, A. and Westgaard, J.I., building Norwegian stocks. In: K. Leber, S. Kitada, 2003. Simultanous analysis of six microsatellite H.L. Blankenship & T. Svåsand (eds.). Proceedings markers in Atlantic cod (Gadus morhua L): A novel of Second International Symposium on Stock En- multiplex assay for use in selective breeding stud- hancement and Sea Ranching. Blackwell Science ies. Mar. Biotechnol. 5:141–148 Ltd., Oxford. 8) Dahle, G., Jørstad, K.E., Rusaas, H.E., and Otterå, 16) Jørstad, K.E. and Farestveit, E., 1999. Population H., 2006. Genetic characterization of broodstock of genetic structure of lobster (Homarus gammarus) in Atlantic cod, Gadus morhua, collected from four Norway and implications for enhancement and Norwegian coastal cod populations. ICES Journal ranching operation. Aquaculture, 173:447–457. of Marine Science (in press) 17) Triantafyllidis A., Apostolidis, A. P., Katsares, V., 9) Otterå, H., Agnalt, A-L. and Jørstad, K.E., 2006. Kelly, E., Mercer, J., Hughes, M., Jørstad, K.E., Differences in spawning time in captive Atlantic Tsolou, A., Hynes, R and C. Triantaphyllidis. 2005. cod from four regions of Norway, spawned under Mitochondrial DNA variation in the European lob- identical conditions. ICES Journal of Marine Sci- ster throughout the range. Marine Biology ence (in press) 146(2):223–235. 10) Jørstad, K.E., Ø. Skaala and G. Dahle, 1991. The 18) Jørstad, K.E., Prodohl, P.A., Kristiansen, T.S, development of biochemical and visible genetic Hughes, M., Farestveit, E., Taggart, J.B., Agnalt, markers and their potential use in evaluating inter- A.-L. and Ferguson, A., 2005. Communal larval action between cultured and wild fish populations. rearing of European lobster (Homarus gammarus): ICES mar. Sci. Symp. 192:200–205. family identification by microsatellite DNA profil- 11) Jørstad, K.E., Ø. Skaala and G. Nævdal., 1999. ing and offspring fitness comparison. Aquaculture Genetic diversity and the Norwegian Sea Ranching 247:275–285. Programme: a retrospective perspective. In: Stock

300 Aquaculture Research: From Cage to Consumption 19) Frank-Lawale, A.S., Taggart, J.B., McAndrew, B.J., 30) Gjerde, B., Terjesen, B.F., Barr, Y., Lein I. and Woolliams, J.A., 2005. The use of microsatellite Thorland, I., 2004. Genetic variation for juvenile markers for pedigree analysis in the Atlantic hali- growth and survival in Atlantic cod (Gadus mor- but, Hippoglossus hippoglossus: the first step to- hua). Aquaculture 236:167–177. wards a sustainable breeding programme. 31) Finne, M.Aa., Fjalestad K.T., Delghandi M., Hans- Aquaculture 247:13. en Ø.J., Haug L., Kettunen, A., Skajaa K., Wesma- 20) Jackson, T.R., Martin-Robichaud, D.J., Reith, M.E., jervi M.S. and Mortensen A., 2004. Developing a 2003. Application of DNA markers to the manage- genetic improvement programme in Atlantic cod ment of Atlantic halibut (Hippoglossus hippoglos- (Gadus morhua L.) European Aquaculture Society sus) broodstock. Aquaculture 220:245–259. Special publication No. 34 21) Sekino M, Saitoh K, Yamada T, Kumagai A, Hara 32) Kettunen, A.,and Fjalestad, K.T. 2006 Genetic pa- A, Yamashita Y., 2003. Microsatellite-based pedi- rameters for important traits in the breeding pro- gree tracing in a Japanese flounder Paralichthys oli- gram for Atlantic cod (Gadus morhua L.). Abstract vaceus hatchery strain: implications for hatchery submitted to ISGA VIII, to be presented Montpelli- management related to stock enhancement pro- er, France, 26–30 June 2006 gram. Aquaculture 221:255–263. 33) Mikkelsen, H., Schrøder, M.B. and Lund, V., 2004. 22) Fishback AG, Danzmann RG, Ferguson MM, Gib- Vibriosis and atypical furunculosis vaccines; effica- son JP., 2002. Estimates of genetic parameters and cy, specificity and side-effects in Atlantic cod, genotype by environment interactions for growth Gadus morhua L. Aquaculture 242:81–91. traits of rainbow trout (Oncorhynchus mykiss) as in- 34) Kettunen, A.. and Fjalestad, K.T., 2006. Resistance ferred using molecular pedigrees. Aquaculture against vibriosis in Atlantic cod (Gadus morhua 206:137–150. L.): first challenge test results. Submitted to Aquac- 23) Næss, T., Harboe, T., Magnor-Jensen, A., Naas, ulture K.E., Kristiansen, A., Hennø, J. og Norberg, 35) Kettunen, A., Serenius, T. and Fjalestad, K.T., 2006. B.,1995. Vellykket startfôring av kveitelarver fra Three approaches for genetic analysis of disease re- gytetidsforskjøvet stamfisk. Norsk fiskeoppdrett, sistance against vibriosis in Atlantic cod (Gadus 21 (5):36. morhua L.) submitted to Journal of Animal Science 24) Bolla, S., Holmefjord, I., og Refstie, T., 1987. Cryo- 36) Kettunen, A., Kauric, G. and Peruzzi, S., 2004. In- genic preservation of Atlantic halibut sperm. duction of triploidy in Atlantic cod (Gadus morhua) Aquaculture, 65: 371–374. by cold shocks. European Aquaculture Society Spe- 25) Olesen, I., Barr, Y., Holmefjord, I., Refstie, T., cial Publication No. 34 Pante, M.J.R. og Lein, I., 2001. Avlsforsking på 37) Holmefjord, I. and T. Refstie, 1997. Induction of kveite. Final Report, Akvaforsk, Fagrapport 37/01. triploidy in Atlantic halibut by temperature shocks. 16 p. Aquaculture International 5:169–173. 26) Barr, Y., Holmefjord, I., Pante, J. og Lein, I., 2001. 38) Van Nes, S., Moe, M and Andersen, Ø. 2005. Mo- Towards halibut breeding program – some achieve- lecular characterization and expression of two ments of the SIP «Breeding Research on Halibut». cyp19 (P450 aromatase) genes in embryos, larvae, Poster presentation, Programme Conference and adults of Atlantic halibut (Hippglossus hippo- Havbruk 19–21 March 2001. glossus). Molecular Reproduction and Develop- 27) Gjerde, B., Refstie T., Salte, R., Andersen, Ø., ment 72: 437–449. Holmefjord, I., Lein, I. og Rye, M., 1999. Strategiar 39) Van Nes S. and Andersen Ø. 2006. Temperature ef- for avlsarbeid. Akvaforsk Report, 42 pp. fects on sex determination and ontogenetic gene ex- 28) Erdahl, R., 2003. Studium av familiegrupper hos at- pression of the aromatases cyp19a and cyp19b, and lantisk kveite (Hippoglossus hippoglossus L.) på the estrogen receptors esr1 and esr2 in Atlantic hal- eigenskapane overleving, vekt, tilvekst, lengde, au- ibut (Hippoglossus hippoglossus). Submitted to geplassering og pigmentering. Thesis, Department Mol.Reprod,Dev., of Animal and Aquacultural Sciences, Norwegian 40) Stenvik, J., Wesmajervi, M.S., Damsgård, B.and University of Life Sciences, 59 pp. Delghandi, M. Genotyping of panthophysin I (Pan 29) Kolstad, K., Thorland, I., Refstie, T. and Gjerde, B., I) of Atlantic cod (Gadus morhua L.) by allele –spe- 2006. Body weight, sexual maturity and spinal de- cific PCR, Molecular Ecology Notes, in press formity in strains and families of Atlantic cod 41) Gjerde, B., 2003. Genetic markers for parental as- (Gadus morhua) at two years of age at different lo- signment in fish breeding programs. ISGA VII, Pu- cations along the Norwegian coast. ICES Journal of erto Varas, Chile, 9–15 November 2003 Marine Science (in press)

Thematic area: Selective Breeding and Genetics 301 302 Aquaculture Research: From Cage to Consumption Technology

■ Aquaculture Technology Egil Lien1), Leif Magne Sunde1) and Kjell Midling2) 1) SINTEF Fisheries and Aquaculture, 2) Fiskeriforskning (Norwegian Institute of Fisheries and Aquaculture Research)

Aquaculture Technology

Development of new aquaculture technology has been a major contributing factor to the success of the Norwegian fish farming industry. There has been a mutual interaction between development of technology and its use in production, especially for salmon. The Norwegian aquaculture industry is widely considered an international leader in the cultivation of seafood, including cage technology, well-boats/vessels and utility equipment. In recent years there has been a significant reorganisation of the fish farming industry. At the same time, changes in external regulations mean that producers of aquaculture technology will have to meet different and stricter requirements. The current situation is that the research community together with industrial partners is attempting to establish well functioning and profitable production methods for “new” species such as halibut, cod, mussel and scallop.

304 Aquaculture Research: From Cage to Consumption Challenges, tendency and goes further; fish farms are considered demands for aquaculture processing plants where sea cages, well- technology boats and slaughterhouses will be more The aquaculture industry is strongly influenced integrated. by both international and national frameworks. • Documentation The last five years have clearly shown that the The focus on quality, health, animal welfare, industry faces a broad range of challenges, and traceability and food safety is increasing. As success will require the proper technology. consumers sharpen their focus on the • Production areas environment and food quality, they set Expected increases in marine biomass higher demands on products and production production, combined with increased conditions. The result of this is that the competition in coastal areas, means that not technology and its use must be documented only will existing locations for fish farming very thoroughly. Breeding of cod and need to be exploited better, but also that halibut is becoming ever more important, so aquaculture will have to make use of new rationalised production of these species will and more exposed sea areas. Regardless of require specially adapted technology and how exposed the production areas are, knowledge. applied technology will need to satisfy the • Other factors demands. This technology must also be well With increased production growth, it is only documented. fitting that society raise its expectations with • Health, environment and safety respect to environmentally friendly Working in the aquaculture industry is technology, aesthetics, ethics and more. One considered hazardous and stressful, so the can see that life cycle analysis (LCA) is industry faces the challenge of ensuring a gaining relevance in the fish farming supply of personnel at all competence industry as well. levels. • Improving the efficiency of production In cooperation with both the Research Council With increasing quantity of production, of Norway and other funding sources, the last there will be a continuous need to improve 10 years have seen several strategic institute the competitive characteristics of the final programmes and projects with the goal of products. Both competence and technology producing knowledge which ensures that the will be important contributors, although Norwegian aquaculture industry is ready to face some investment components such as feed these coming challenges. Two of those strategic ingredients may become limiting factors. institute programmes carried out at SINTEF • Centralisation of functions Fisheries and Aquaculture have been chosen to To achieve rationalised production, present work that has been done within the field centralisation – as with meat production is of aquaculture technology, namely, PROMAR one method of improvement, although it (Processes and equipment for farming of means greater distances between production marine species) and SIKTEK (Safe and sites and the processing site. Because of this, environmentally friendly aquaculture the demands for the technology employed constructions). will change. The industrialisation process

Thematic area: Technology 305 Methods for product of the technology and the existing environ- development and mental terms for production make it vital that documentation technology and biology are seen in context with Aquaculture technology encompasses the each other. relatively simple to very complex products. At the same time, increasingly advanced solutions With increasing complexity, it will be more have been developed and presented on the important to test new technology before it is market in recent years. To be able to deliver introduced to the market. Today this can be seen competitive products, focus has to be directed within the mussel farming industry, where on product development. This is also a cause of problems persist in establishing production the increasing requirements for documentation lines with clarified and functional technology of the products used within aquaculture. and operations. Modelling experiments with Through research programmes such as floating net cages and submersible cages have PROMAR and SIKTEK, expertise in product been carried out at MARINTEK in Trondheim design and new methods has been introduced and at the flume tank in Hirsthals, Denmark. for the development of aquaculture technology. However, modelling experiments as a tool for documenting loads at aquaculture installations Up to now the development of products has are not as well established as for the other mainly been in response to equipment produ- maritime products, primarily due to their cers identifying needs or receiving orders for expense. Another method for documentation is new technology from fish farmers. The to use numerical simulations. As in all other technology is then realised and in many cases industries, this is becoming a more and more introduced through prototypes for a pilot cus- powerful tool. The complexity of analysis is tomer. There has been a tradition of putting new very high, even in comparison to the offshore prototypes on the market, in order to achieve industry. Cost-effective tools must be devel- rapid recognition and product sales. This has oped to keep the costs of documentation worked acceptably and has been a development reasonable. strategy with satisfactory solutions. But more expensive technology – and greater economic The increasing complexity of products makes it consequences when the technology is essential to perform testing during product unsatisfactory – are highlighting the need to development. A final documentation of the item thoroughly lay the groundwork for product before introducing it to the market is demanded development. Contractors have expressed a by more and more farmers, so this should be great need for know-how that will allow the focused on. companies to evolve further. Increased emphasis on competence in product It is important that both technology and biology development, combined with a wish to secure are assigned equal weight in connection with transfer of competence from other industries R&D work in the future, since they mutually (oil, fisheries etc.), has led to a clear picture of influence each other greatly. So far projects how this can be done in practice. NTNU have either focused on technology or biology. (Norwegian University of Science and Techno- Industry players now realise that the complexity logy) has recently established a Centre of

306 Aquaculture Research: From Cage to Consumption 145x100//Kap18-fig01.eps 145x100//Kap18-fig02.eps Figure 1. Single-point moored plastic cage (Frøyaringen A/ Figure 2. Anti-skid walkway patented by PolarCirkel AS. S). (Photo: SINTEF) (Photo: Helgeland Plast)

Excellence, the Centre for Ships and Ocean In the last five years, farmers have invested in Structures, with a goal of long-term develop- more and more advanced and expensive ment of expertise. Establishment of practical technology. One example of this technology is facilities for testing equipment will be a plastic cages, which have undergone only minor required tool in the future. modifications to the main concept in 20 years. At the same time, these have mainly been upgraded to larger sizes. Larger scale of Aquaculture farming existing concepts and optimisation of existing There has been a significant upgrade in marine technology have been the overall trends. aquaculture technology since the beginning of Through the SIKTEK programme, methods of the 1990s. The main focus has been on salmon analyses for different cage concepts are evalu- farming; only limited progress has been made ated; this work is carried out according to the for the marine species. NYTEK standard NS4915. The most prevalent cage types are the circular plastic cages, PEH The average production per man-labour year cages and hinged steel cages. By experience, has increased from about 58 tonnes in 1990 to the plastic cages are far less vulnerable to harsh 295 tonnes in 2000 (Norwegian Directorate of environmental conditions than the steel cages. Fisheries). During the same period, annual Much research has been done on the dynamic production has increased from 80,000 to behaviour of these cage types in waves and 450,000 tonnes. The trend at that time was currents. towards larger companies (more licenses) and increased production at each location. This Different types of constructions have now been tendency towards larger production at each introduced. Several cage systems now have location has continued through 2005 and has integrated feed storage and feeding systems (for been the basic challenge with respect to example, Procean AS and Storm Havbruk AS). technological research. Plastic and steel are the commonly used materials and the typical circumference of the cages is 70–100 m (monAqua POL 99). The

Thematic area: Technology 307 Figure 3. Storm Havbruk, a rigid steel farm with two cages of 45x45 m each. Integrated silos and feeding systems. A truss construction designed by Project Services A/S with a total steel weight of 350 tonnes. (Photo: SINTEF)

same study shows that the plastic cages are the moored system, and also introduced a heavy most common type in use north of Stadt, plastic bottom ring to replace individual sinkers Norway, while steel cages are the most preval- (Figure 1). For marine species such as halibut, ent in the southern part of Norway. This is AquaLine AS has developed shelves to be mainly due to harsher weather conditions in the placed inside existing cages. northern region. The sizes of the cage units are still increasing (160 m) and many people in the PolarCirkel AS has developed anti-skid sec- business predict that even units of 200-m tions on its plastic cages in order to improve circumference are to come. working conditions. These are grated sections similar to these for steel cages but made of The trend is towards having feeding stations polypropylene (PP) instead of steel. located directly on fish farms of larger and larger size in more and more exposed locations. Hinged steel cages remain an excellent working Meanwhile, the need for land-based aquacul- platform. They are stable and have high free- ture is decreasing as more of these facilities are moved offshore. Thus more locations are available, but the need to withstand the harsher weather conditions becomes a great challenge. Challenges in operational tasks as well as hydrodynamic loading and proper choice of materials become significant.

Innovations related to plastic cages have been

145x100//Kap18-fig04.eps limited, but some modifications are relevant. Figure 4. A new cage design in steel developed by SINTEF Frøyaringen AS has developed a single point Fisheries and Aquaculture. (Photo: SINTEF)

308 Aquaculture Research: From Cage to Consumption boards and wide walkways. Unfortunately, they • Flexible mounting – single cage units are are not very durable when they are located in easy to move exposed areas. The hinges or the area around • Tolerates waves and currents from all direc- the hinges seem to be troublesome areas where tions well, not only from primary direction fatigue problems often occur. • Working conditions on par with existing hinged systems New designs were proposed (Figure 4). The system as a whole is based on identical Critical review of existing concepts for produc- modules. Four modules make one cage frame, tion of halibut is performed. Two main factors and an unlimited number of cages may be are critical for a successful production. There is assembled. Each module has a float at each end. a demand for a special solution for production A trusswork construction allows sufficient of small halibut (<1 kg). This production bending and torsion stiffness. On each end there requires smaller units easy to inspect. At the are mountings where flexible rubber/neoprene same time a basic requirement for larger bands are mounted. These bands join the production units is that of practical cleaning construction and induce sufficient flexibility. methods. For this purpose, a cage system has The rubber bands are resistant to seawater and been designed which is possible to lift com- sunlight and have a calculated effective lifetime pletely out of the water. The halibut are of 10–15 years. Its advantages include: transferred to a clean unit during this lifting • Hinge systems with sufficient flexibility in operation. While on the surface, high pressure bending and torsion cleaning may be performed (Figure 5). • Modular construction suitable for mass production In this concept hydrodynamic and structural • High redundancy in all components analyses are performed. A large floor area is • Simple maintenance – worn components are achieved by multiple bottom layers. Thus some visible and easy to replace basic requirements have to be fulfilled:

145x100//Kap18-fig05.eps Figure 5. Multi-module cage. (Photo: SINTEF)

Thematic area: Technology 309 • There must be openings in the front to allow for the farming industry, but the overall concern fish to be transferred from one unit to an- is the impact on the wild fish population. This other during the lifting operation. impact may in the form of immediately • Net channels for fish transfer must be pre- increased competition with the wild stock on installed. habitat, food or partners, and/or lead to such • One extra clean cage unit is necessary. long-term effects as spreading of diseases, • The units have vertical floats whose buoy- parasites or genetic impact. ancy is controlled by air pressure for full control of the lifting and lowering There are many causes of fish escapes. The operations. By using the additional hori- prevailing reported cause during the last five zontally positioned floats at the bottom, the years has been structural failure of the farm cage units are lifted completely out of the itself. There are significant variations between water (Figure 5). different species: salmon and trout need relatively large holes in the net pen before they tend to escape, while cod tends to sneak along Escape-proof cages – a vision the net and passes through the smallest holes. Escapees from cages are the largest environ- Cod is also disposed to bite holes in the netting. mental challenge of the farming industry. None The tiniest fleece of thread seems to trigger the of today’s aquaculture cages are sufficiently biting behaviour. A technological development escape-proof. Released farmed salmon threaten towards stronger and safer farms is an ongoing the wild salmon population. Statistically, about process, one that involves the equipment indus- 0.2 per cent of all salmon escapes after being try, the farmers and the research institutes. The put in the cages. With cod the number is perhaps Norwegian authorities have introduced new ten times higher. Farmed fish are genetically standards and stricter regulations for fish farms. more identical to their wild relatives compared Standards on technical equipment, inspection to farmed species in the agricultural industry. and maintenance methods have to be carefully Thus most escapees will survive in the wild. documented. Hopefully, these new standards and regulations will decrease the number of There is insufficient scientific knowledge as to escapees in the future. why or how the fish escape. Recapture of escaped fish has shown to be very unsuccessful. The coming research in this field will be con- The environmental impact on the wild popula- centrated on preventive actions (technological tion is not yet clarified, but there is great improvements and use of sterile fish) and concern surrounding the biological impact on increased knowledge of fish behaviour. the wild species. After some years of reduction Research on the overall impact on the wild of escapees in the 1990s, today’s escapee population must be redoubled. numbers are rising dramatically. In 2005, some 700,000 farmed fish were reported lost. There is no international supervision in this field, so we Net technology have no knowledge whether these numbers are The net pen is normally a nylon bag enclosing relevant for the international aquaculture the fish. Both knotless and knitted net bags are industry. Escapes cause great financial losses used. Most nets are knotless, ladder-proof

310 Aquaculture Research: From Cage to Consumption 145x100//Kap18-fig06.eps Figure 6. Model testing of deformation of the net pen in current. (Photo: SINTEF)

types. Several alternative materials have been significant impact on fish behaviour, prosperity studied. and growth. The problem is demonstrated in Figure 6 from a test performed in the flume tank The available farming locations are often in Hirtshals. limited by current speed. High current speeds cause large deflection of the net bag, reducing Furthermore, fish behaviour in the cages with the available volume inside the bag. Often these high biomass density has been studied in full deflections cause large folds, trapping the fish scale at sites with high current speeds up to 0.8 and causing contact wounds and stress. Docu- m/s. Large mortality and less growth were mentation of drag forces and deflection of net detected. Abnormal swimming patterns were pens has been a central task within the research observed: groups of fish circulated in shoals programme SIKTEK. while other fish were steadily swimming against the current. No wounds due to contact Nowadays, fish farms tend to be located at more were observed as there was no large wave and more exposed locations. Net bags are action during the observation period. getting larger and larger; the enclosed volume has increased by up to 40 times since the cage The development of larger net pens has mainly industry was established. One unit may keep as been an up-scaling of dimensions very little much as 1,000 tonnes of fish enclosed in upgrading of materials has occurred. Recently, extreme cases. Larger units at greater depths there has been an increased import of net and with higher exposure to current create an materials from abroad, mainly from Asia. This increased technological challenge. includes completely mounded net pens as well as net materials. Three full-scale studies have been performed by towing empty cages in the flume tank. The The increased net sizes cause increased stress forces and deflections have been measured; the while in operation and during activities such as data have been compared with theoretical net changing. New construction methods have models and used to improve them. Enormous been evaluated in cooperation with fish farmers, reductions of available volume – up to 80 per net manufactories and SINTEF (Figure 7). cent –have been measured under conditions Intensive research in this field is ongoing. A with moderate current velocities of 0.5 m/s. PhD study is focusing on design and fatigue This increase in biomass density may have a properties of the net pens (Figure 8).

Thematic area: Technology 311 145x100//Kap18-fig08.eps

145x100//Kap18-fig07.eps Figure 8. Numerical analyses of tension in the net during Figure 7. Alternative net pen design: net with twisted the lifting operation. (Photo: SINTEF) bottom panel. (Photo: SINTEF)

Net bags are normally bottom-loaded, either by Classification rules for marine fish farms, such individual clump or by continuous circular as NS 9415, were established in 2004. These are bottom rings, with the purpose of reducing the requirements for design, dimensioning, produc- deflection in current. Several new methods have tion installation and operation. been evaluated. The main issue has been to develop safe systems reducing the risk of dam- There has been an increasing interest in age during net-handling operations. Extremely computational methods in order to improve the large net bags require well-established cage systems: tools for analysing cages and nets operational routines in order to avoid overload. individually as well as methods for analysing Manual labour is no longer sufficient, net- complete systems such as cage structures, net handling operations require large cranes great pens and moorings. care needs to be taken with such heavy loads to avoid tearing a net apart. Numerous programs exist for analysing large- volume constructions such as vessels and offshore installations. Most fish farms are, Analytical tools for cages, nets however, mainly small-volume constructions, and moorings usually very flexible. Many farms consist of Technological developments in the aquaculture complex hinged frame systems; together with industry have come largely through experience extremely flexible net systems, this makes for and the trial and error method. This practical very complex constructions. approach has been successful together with theoretical methods. Larger units are however Fish farms are very large and occupy a great causing significant environmental impact as a amount of surface area, whether large indi- failure consequence. A demand for certification vidual cages or systems with several integrated rules for fish farms has been a demand from cage units. Traditional methods involving farming companies and the equipment industry extreme waves will not be realistic since the alike. wave surface elevations must be regarded as a stochastic process, and the significant

312 Aquaculture Research: From Cage to Consumption 145x100//Kap18-fig09.eps Figure 9. Regular and irregular waves; significant wave height of 1 m. wavelength is small compared to the overall Some basic tools for analysing fish farms are: size of the farm. ConMotion Mooring (Mooring analysis of fish farms moored in frames. It includes calcu- Studies of farms in both regular and irregular lation of forces acting on the net pen from seas are performed. Studies on short-crested waves and current. Developed by SINTEF wave and their impact on structures have been Fisheries and aquaculture) especially focused upon. Use of a regular wave MIMOSA (Mooring analysis tool developed approach shows unrealistically large excitation for vessels and floating rigs. Special ver- forces compared to irregular waves. Regular sions MIMOSA-2F were previously used and irregular waves are shown in figure 9. intensively for rigid fish farms. Developed by MARINTEK, distributed by DNV Analyses of traditional hinged steel cages in Software) irregular seas yields large second-order Aquastructures (This is a FEM-based deflections of the structure. These deformations computation tool for nonlinear analysis of will not be detected by standard regular ana- complete systems. Includes complete time lyses, however they may cause many of the domain analyses of the structure, net pen fatigue problems detected on these types of and the mooring. Developed by Aquastruc- structures. tures A/S) Riflex (Nonlinear FEM program for analysing Analysis in short-crested sea is very time- slender constructions. Originally developed consuming and requires large computational for analysing flexible risers for the offshore resources. The aim is to develop simplified industry. Modified to include net models. methods to be implemented as a standard, Developed by MARINTEK) avoiding the need for costly and time-consuming analyses. This work is closely connected to For the nets in particular, the following analyt- NS4915, Norwegian standard for floating fish ical programs are available: farms. Notdrag (Calculation of current forces on net bags. This program is based on empirical

Thematic area: Technology 313 data from model tests of nets performed at Shellfish farming technology the towing tank at MARINTEK. Includes The development of the Norwegian shellfish net panel deformations and shielding effects industry has been a tedious process with from multiple net bags) piecemeal continuity. At the end of 1970s small 3D-Not (3-dimensional motion analyses of mussel farms were established along the coast. dynamic response of net system in waves Great potential was seen and the number of and current, developed by SINTEF Fisheries farms gradually increased. At the end of the and aquaculture) 1980s shellfish farming ended abruptly as a NetSim (2-dimensional analyses of net bag consequence of problems with poisonous algae response in wave and current, developed by and insufficient technology. The industry was SINTEF Fisheries and aquaculture) revitalised in the mid-1990s, when both scallop and mussel farms were established. The Dynamic analyses of net pens are generally numbers of mussel farms in particular sky- very complicated. Still, there is extensive rocketed all along the coast. research in progress to improve these models. The main challenge for both mussel and scallop Considerable efforts are being directed towards production is establishing a rationalised developing a computational method for ana- production line. Expectations of easy produc- lysing longline systems for the shell farming. tion were crushed; intensive care and handling Their responses to the layout and the influence would be needed. The technology was mainly of environmental stresses have been included. adopted from abroad, developed for enviro- Guidelines were developed for the layout and nments quite different from those in Norway, installation in order to reduce the risk of and the technology was not properly adapted. sinking; results have been transferred to the The main focus was on the investment costs and industry in a series of articles. very little on operational costs. The expected growth speed was low. The quality, especially The mooring layout is essential to the safety of the meat content, was very variable. Production floating fish farms. Especially the overall forces costs were far too high compared with the price on rigid or hinged steel farms are very sensitive fetched on the marked. Poisonous mussels to the mooring layout. An unfavourable moor- became a huge problem as well. Fetching a ing layout may easily cause structure damage. good market price and continuity of deliveries Visual observations of a mooring system are are essentials. practically impossible. Guidelines have been developed for installation of these types of fish An example of a new mussel farming method is farms in order to minimise the tension from the shown in Figure 10. mooring forces. In general, a lengthwise flexibility variation along the long sides of the Norwegian shellfish technology has been farms is preferable. This is achieved by adapted to the characteristic environmental positioning these anchors along as straight a conditions. The Norwegian coast has a very line as possible, achieving linear flexibility. variable topography. The locations are often highly exposed, with large variations in the wind, waves and current conditions. The

314 Aquaculture Research: From Cage to Consumption 145x100//Kap18-fig10.eps Figure 10: Shell culture concept developed by Smart Farm A/S. A unique machine performs the harvesting of a 2-m-deep net. (Photo: SINTEF)

Norwegian coastline is extremely long, but the buoy may have been torn off or punctured. This fjords are normally very deep. Deep waters in loss of buoyancy may cause the adjacent buoys particular are a great challenge, so the need for to sink and collapse from the increasing water redundant and safer floating systems is obvious. pressure. One local loss of buoyancy may then start a chain reaction causing the whole farm to Norwegian mussel farms consist mainly of sink. If this occurs at a deep-water location, the hanging culture systems and longline systems. farm may be lost forever. Bundles of ropes and Mussel collectors are attached to a main line floats may be sink to the sea bottom. rope carried by buoyancy elements. Floats are equally spaced along the main line. The main The weight of the mussels is about 20 per cent line is moored at each end. In many cases many when they are immersed. To keep a system with lines are moored in parallel. The lines are 50 tonnes of mussels afloat, a minimum of 10 attached to tubes made of PEH or aluminum at tonnes of buoyancy is required. To achieve a both ends. One such raft may have more than 10 minimum redundancy the actual buoyancy km of collectors for cultivating mussels. should be at least 30 per cent higher, so it is important to select technology with respect to Several mussel farms using floats have been environmental conditions and topography. destroyed, mainly due to loss of buoyancy. One

Thematic area: Technology 315 A modified Swedish concept with floating end kilometres of these are lost at the bottom. They tubes and several parallel lines was introduced very often break down during harvesting. at the end of the 1990s. This concept, with series of lines linked together, increases the risk Fiskevegn A/S has developed collectors with of a total loss. SINTEF Fishery and Aquacul- integrated weighting. These seem to be very ture has performed intensive analysis of this promising with respect to automation of the concept and found it unsuitable at many installation and harvesting process. locations, especially deep ones. A typical production strategy has been to Today’s concepts are not suitable for mechan- minimise labour costs by minimising work on ical handling. They require lots of manpower the farms. In practice, this means only adjusting with respect to their installation, operation and the buoyancy and otherwise letting the farm go harvesting. This is a main reason for the lack of through its cycles for 2–3 years before harvest- profitability in the industry. ing. This results in uneven size and quality and the price fetched in the demanding European The weakness of the systems discussed above market is very low. Also, tangling of collectors has led to the development of new concepts causes complications during harvesting. Losses based upon the use of long plastic tubes replac- from starfish and eider ducks also point to the ing the main line and buoys. These concepts need for active tending. seem to be more secure at deep-water locations, although some of these systems also have sunk. Equipment for harvesting, sorting and re- Several full-scale prototypes are now being installation is now in use, but the technology is tested, but they have not been verified yet. not sufficiently documented or automated to prove commercially feasible. A development of The collectors are attached to the main line. The automated processes for installation, sorting, mussel spat attach to these collectors and re-installation and harvesting seems to be continue to grow until they reach a market size necessary in order to develop a sustainable of 40–50 mm in 2–3 years. A sinker is attached large-scale industry. to the bottom of the collector to keep it hanging until the weight of the mussels is satisfactory. Cultivation of scallops is done only on a very There are several types of collectors on the small scale in Norway and is still in the experi- market. It is not yet known which if any of these mental phase. There is a focus on bottom culture types is preferable. Different types seem to be production strategy. Use of nets and boxes for working better at different locations. Some on-growing is used to a small extent. In many collectors are developed for collecting spat, countries more intensive production methods are other types for on-growing. Lack of documen- being studied. Hanging solutions, more or less tation of their effectiveness is, however, one of similar to the longline technology, is used in the industry’s biggest challenges in selecting an other parts of the world (Canada, Ireland, Asia). optimal technology. Indistinct results from sporadic tests cause confusion. Several Cultivation of scallops requires large areas. collector types deteriorate quickly, and many Predictable production of large quantities requires effective production methods.

316 Aquaculture Research: From Cage to Consumption Large losses from predators in bottom cultiv- other technical demands. The greatest chal- ation have initiated new concept development, lenge today is to drastically reduce escapes. with the goal of industrial on-growing concepts. Temporary housekeeping in boxes/traps until As floating production units steadily increase in the last on-growing phase using long-tube and size, the consequences of failure are ever hanging culture is one possibility. Different greater. The overall redundancy should be hanging methods are being tried out, such as increased for these enormous production units “ear hanging”, featuring scallops hanging in and should reflect the overall risk. pre-drilled holes in the shell, or attached by gluing methods. The environmental forces acting on these gigantic units are of course also increasing not A large number of the activities were focused only due to their increased size, but also on the basic production method of longline because the best sheltered locations become technology. Good expertise has been developed occupied so the production plants are forced to on this technology, and advisory services for accept more exposed areas. safer systems have been conducted. Safety studies with respect to design and buoyancy Net-handling operations become far more risky distribution for risk analyses are key contribu- with larger nets. Large vessels and cranes cause tions. Preliminary studies of the floats, buoys, heavy point loads on the nets. Comprehensive and their risk of collapsing were performed at manuals now describe operations and handling the ocean laboratory at MARINTEK. General methods; lifting on the wrong rope may cause a information on the system’s safety was complete loss. disseminated through several articles in popular science magazines and outreach activities. Today’s largest uncertainty is the anchors’ Dissemination efforts have led to positive holding power. There is no clear connection feedback from the industry, insurance comp- with their weight and holding power. There is anies and public authorities. insufficient knowledge about the different bottom sediments and preferred anchor types. The sizes of anchors now in use are so large that Conclusion full-scale testing of holding power is unrealistic. The last five years for the salmon industry have The aquaculture industry should collaborate been a down period. No extensive entrepreneur- with the offshore industry in this field. ial activities within the shellfish industry have paid off. There is a demand for new net materials. Large numbers of cod escape by biting holes in the net The importance of the technological challenges mesh. This is a significant threat to the entire in new industry has been underestimated. The industry and it is urgent to find new materials or salmon industry throughout its future growth methods of net treatment to avoid these escapes. will face increased demands for technological This problem may be solved through extensive development and documentation. New cultured collaboration between the fields of biology, species such as cod, shellfish and halibut have ethology and technology.

Thematic area: Technology 317 Referanser Berstad, A. J. Aqua Structures: Utvikling av bereg- Fredheim, A., and Faltinsen, O.M. Current forces on ningsverktøy for havbruksanlegg (2002–2004). and flow throug three dimensional fish net cages. Verktøy for beregning av styrke og sikkerhet av nye Jorunal of Marine Science (in prep.). og eksisterende anlegg. Norges forskningsråd, Hamarøy Skjellfarm AS, 2000–2001. Utprøving og prosjekt 151 649/120. tilpassing av ny produksjonsteknologi for kamskjell Bømlo Skjell AS og Tarovekst AS 2001–2002. Effektiv og haneskjell. NUMARIO v/SND. dyrkningsteknologi for kamskjell og østers. Knudsen, F. R., 2000–2002. Overvåkning av laks i merd NUMARIO v/SND K32–3058/3059/00. med ekkolodd (Simrad AS). Research Council of Digre, H., Hagen, N., and Lader, P., 2001. Norway, project 138 538/120. Pilotproduksjon av laks på strømutsatt lokalitet på Lader, P., 2000–2002. 3-dimensjonal dynamisk Færøyene. SINTEF Fisheries & Aquaculture AS, modellering og simulering av oppdrettsmerd. report STF80 A014 047. SINTEF Fisheries & Aquaculture AS. Research Edulis AS, SINTEF Fiskeri og havbruk AS, Helgeland Council of Norway, project 134 491/120. Plast AS og Mørenot AS 2000. Utvikling av Lien, E., 2000. Oppdrett på strømutsatte lokaliteter. langrørsteknologi for blåskjelldyrkning. SINTEF Fisheries & Aquaculture AS, report STF90 NUMARIO v/SND K32–2038/00. A00 001. Ellingsen, H., 2001. Marine ressurser og teknologi- Lien, E., Fredheim, A. and Sunde, L. M., 2000. utvikling. SINTEF Fiskeri og havbruk rapport Blåskjelldyrking i en tynn tråd? – Krefter i A013 051. langlineanlegg. Norsk Fiskeoppdrett, 6/2000. Fredheim, A., Lien, E. and Sunde, L.M., 2000. Lien, E., Fredheim, A. and Sunde, L. M., 2001. Blåskjelldyrking – produksjon i en tynn tråd? – Blåskjelldyrking – fortsatt produksjon i en tynn Komponenter i et langlineanlegg. Norsk tråd? – Stor havaririsiko i norske skjellanlegg. Fiskeoppdrett, 4/2000, 24–26. Norsk Fiskeoppdrett, 1/2001. Fredheim, A. and Faltinsen, O.M., 2001. A numerical Lien, E. and Fredheim, A., 2001. Development of model for the fluid structure interaction of a three- Longtube Mussel Systems for Cultivation of dimensional net structure. Proceedings of the Fifth Mussels, Open Ocean Aquaculture IV 2001, St. International Workshop on Methods for the Andrews, Canada. ISBN 1–888 807–13-X Development and Evaluation of Maritime Lien, E., Vikestad, K. and Jensen, Ø. Mechanics of technologies. Methods for the Development and hinged steel cages. Aquaculture Engineering, Evaluation of Maritime Technologies, DEMAT Jorunal of Marine Technology (in prep.). 2001, 7–10 November, Rostock, Germany. ISBN 3– Linga Laks AS, Bolstad Fjordbruk AS and Bergen 935 319–88–6 Aqua AS 2000–2001. Driftsoptimalisering og Fredheim, A. and Faltinsen, O.M., 2001 Hydroelastic slakteforsøk ved merdoppdrett av kveite. analysis of a fishing net in steady inflow conditions. NUMARIO v/SND K32–2026/2000. Proceedings of the third international conference on Malm, M. and Boxaspen, K., 2002. Bruk av lys for hydroelasticity in marine technology. Third optimalisering av biologisk effekt i matfiskanlegg. International Conference on Hydroelasticity in Research Council of Norway, project 143 289/120. Marine Technology, Oxford England. ISBN 0–952– Maritime Transportation and Exploitation of Ocean and 62 081–2 Coastal Resources, Volume 2: Exploitation of Fredheim, A. and Lien, E., 2001. General Analysis of Ocean and Coastal Resources. (IMAM 2005), Long-line Constructions used for Cultivation of Lisboa, Portugal. ISBN 0–415–39 036–2 Blue Mussels (mytilis edulis). Open Ocean Moe, H., Fredheim, A. and Heide, M., 2005. New net Aquaculture IV 2001, St. Andrews Canada, ISBN cage designs to prevent tearing during handling. 1–888 807–13-X Maritime Transportation and Exploitation of Ocean Fredheim, A., 2002. Sikre og miljøvennlige havbruks- and Coastal Resources, Volume 2: Exploitation of konstruksjoner (Strategic institute programme). Ocean and Coastal Resources. Research Council of Norway, project 143 223/140. Moe, H., Sunde, L.M., and Dempster, T. Escape of Fredheim, A. and Faltinsen, O.M., 2002. Current forces Atlantic Cod (Gadus morhua) from sea-cage fish on net structures. Fifth International Conference on farms and possible technological solutions: Hydrodynamics, ISBN 957–8845–66–9 preliminary studies. Aquaculture International (in prep.).

318 Aquaculture Research: From Cage to Consumption Mosjøen Kveite AS 1999–2002. Bruk av lys i matfisk- Sunde, L. M., Lien, E., and Fredheim, A., 2000. oppdrett av kveite i merd. NUMARIO v/SND. Blåskjelldyrking – produksjon i en tynn tråd? Norsk Myklebust, H. Fiskevegn AS: Utvikling av påslags- og Fiskeoppdrett: 3/2000, 34–35. vekstmateriale for dyrking av blåskjell (2001– Sunde, L.M., Lien, E., og Fredheim, A., 2000. 2004). Research Council of Norway, project Blåskjelldyrking – produksjon i en tynn tråd? Norsk 145 537/120. Fiskeoppdrett: 3/2000, 34–35. Otterå, H., Sjøtun, K., Meeren, G., Boxaspen, K. and Sunnfjord Kamskjell AS, 2000–2001. Utprøving av Taranger, G.L., 2002. Fullskala utprøving av stor- produksjonsegenskaper for ny burtype i aluminium merd mekanisk notskift. Research Council of til dyrkning av kamskjell og østers i mellomkultur. Norway project. NUMARIO v/SND K32–2042/00. Rømningsutvalget, 2000. Nasjonal tiltaksplan mot Sørensen, A., Lindegaard, K.P.E. and Hansen, E.D.D., rømming (utvalgsleder Håvard Vannebo). 2002. Locally Multiobjective H2and H~ Control of Storm Havbruk. Pilotproduksjon av laks på eksponert Large-Scale Interconnected marine Structures, lokalitet i STORM havbruksmerd. Research IEEE CDC’02, Las vegas. Council of Norway, project 144 244/120. Vikestad, K. and Lien, E. Non-Dimensional Weight- Storøy, J. and Sunde, L. M., 2000. Forprosjekt Velocity Parameter For Estimating Drag Force And AKVATEK 2005. “Teknologiutvikling av utstyr til Net Deformation Of Gravity Fish Cages, 2005, oppdrettsnæringen”. SINTEF Fisheries & Halkidiki, Greece. ISBN: 0–415–39 036–2 Aquaculture AS, report no. 840 026 commissioned Vikestad, K. and Lien, E., 2005. Bending stiffness of by Norwegian Suppliers to the Aquaculture clamps connected 2-rings PE fish cage collar. Industry (NLTH). Vikestad, K., Lien, E., Jensen, Ø. and Leira, B. Sunde, L. M. and Forås, E., 2002. Utredning om de Simulation of floating fish farms exposed to forskningsmessige behov relatert til fiskeoppdrett irregular sea states. Aquaculture Engineering, på eksponerte lokaliteter i Norden. SINTEF Jorunal of Marine Technology (in prep.). Fisheries & Aquaculture AS, report A024 033. Økonomiske analyser, Fiskeoppdrett, 2000. Sunde, L. M. and Fredheim, A., 2001. Prosesser og Fiskeridirektoratets lønnsomhetsundersøkelse for utstyr for marine arter i havbruksnæringen. Faglig matfiskproduksjon av laks og ørret. sluttrapport for PROMAR – Strategic institute programme. SINTEF Fisheries & Aquaculture AS, report A01 015.

Thematic area: Technology 319 320 Aquaculture Research: From Cage to Consumption The Environment

■ Salmon Lice: Importance, Problem and Treatment ■ How Much Aquaculture Can the Norwegian Coast Tolerate? ■ Genetic Interactions Karin Boxaspen1), Peter Andreas Heuch2), Pål Arne Bjørn3), Bengt Finstad4), Petter Frost1) and Kevin Glover1) 1) Institute of Marine Research, 2) National Veterinary Institute, 3) Fiskeriforskning (Norwegian Institute of Fisheries and Aquaculture Research), 4) NINA (Norwegian Institute of Nature Research)

Salmon Lice: Importance, Problem and Treatment

Salmon lice (Lepeophtheirus salmonis) on farmed fish can be kept under control, but they are regarded as the most expensive disease problem the aquaculture industry has to deal with. Outbreaks are controlled by medication, either in the form of bath treatments or as feed additives. One source of concern is that lice have developed resistance to several types of medication. The ultimate strategy for combating lice would be a salmon louse vaccine. Initial trials suggest that it is possible to vaccinate farmed salmon in order to reduce the number of lice that they carry. It has also been shown that different strains of salmon differ in their sensitivity to salmon lice. It is theoretically possible to breed for resistance, with the goal of developing a strain of resistant salmon. The salmon louse is also a problem for wild stocks of salmonids. Monitoring wild salmonids from rivers and fjords has shown that wild and farmed salmon are capable of co-existing in Nor- wegian fjords, as long as the total number of louse larvae is adapted to local hydrographic conditions and the characteristics of wild salmon populations. A combination of methods, including coordinated strategic delousing, biological control, the use of wrasse as cleaner-fish, the possibility of anti-lice vaccines and breeding salmon to reduce their sensitivity to lice will be important means of controlling levels of salmon lice in the future.

322 Aquaculture Research: From Cage to Consumption 145x100//Kap19-fig01.eps A copepodite (left) and a chalimus attached to a salmon Mobile stages of male lice. The smallest individual (left) is scale. (Photo: Karin Boxaspen) a pre-adult I, the two individuals in the centre are pre- adults II, and the one on the right is an adult male. Salmon lice: past and present (Photo: Karin Boxaspen) Salmonid fish and salmon lice have co-existed in coastal areas of Norway since the last ice age. a requirement that they should be deloused if The growth of salmon farming, however, has these levels were exceeded. significantly increased the number of hosts in the sea, which in turn has resulted in higher pro- The lowest level of salmon lice infestation must duction of salmon lice. The presence of many be maintained from December 1 until July 1. salmon in the sea also increases the likelihood This period was selected specifically in order to that salmon lice drifting with the currents will reduce the number of free-living salmon lice in find a host. Today, there are between 100 and the spring, when the wild salmon smolt migrate 300 times as many salmon lice hosts in fish seaward through long or short fjords that are po- farms as in wild salmonid populations on the tentially full of salmon lice. coast. While earlier research had largely concentrated Salmon lice became a problem for aquaculture on chemical treatment of salmon lice, it was be- in the mid-1970s. The problem was solved by coming clear that other types of research were bathing the fish in insecticide products that re- also needed. This realisation resulted in projects moved the lice. Then, in 1989–1990, reports that have studied the behaviour of the salmon came in of epidemic infestations of wild salmon louse and how it finds host fish, studies of levels in Ireland and Norway. These epidemics were of salmon lice and their significance for wild related to high levels of salmon production and stocks, studies of heritable resistance to salmon the associated production of salmon lice by the lice in hosts, molecular biology and projects aquaculture industry. In 1997, Norway that may lead to the development of a vaccine, launched a national action plan that set guide- as well as the development of alternative meth- lines for permitted levels of salmon lice on ods of treatment. In the following paragraphs, farmed fish during particular periods, as well as we discuss some of the results of these lines of research.

Thematic area: The Environment 323 The salmon louse is a copepod crustacean, an The copepodite has a single eye, which is locat- external parasite that is capable of developing ed beneath the rostrum. The eye has two lenses, and surviving on the skin of salmonids, i.e. in in addition to a lensless light-sensitive area. The Norwegian waters, salmon, sea trout, rainbow eye is theoretically capable of registering po- trout and artic charr are all potential hosts. larised light (i.e. light whose rays are perpen- Salmon lice have a direct life cycle, and they dicular to its direction of travel) and UVA light need only a single host to complete the cycle. (wavelengths 320–400 nm). In such light, a Salmon louse larvae are spread by currents in salmon will appear as a clearly defined silhou- three free-living larval stages, remain attached ette against the background, and we might to the host for four stages, and undergo three imagine that the copepodite should be capable mobile stages during which they are capable of of exploiting this situation to see and move in moving around on the host. The adult lice mate the direction of a potential host. However, stud- on the fish, and their fertilised eggs are released ies carried out in large containers have found no in two long egg sacs which are attached to the statistical differences in the number of lice on genital segment of the female louse. A single fish under different light conditions (Figure 1). egg sac may contain as many as 800 eggs. The This leads us to the conclusion that copepodites eggs develop into the first larval stage, nauplius I. The nauplius larvae hatch out and swim freely in the water column, where they rapidly develop into the nauplius 2 stage, and then into copepodites. At this stage, when approximately 1 mm long, they find and attach themselves to a salmonid, where they resume a parasitic lifestyle. The length of their free-living stages varies with temperature (stages are shorter at high temperatures), but under certain physical conditions that provide high current speeds, they are capable of spreading for hundreds of kilometres from where they hatched. The behaviour of the salmon louse: how does it find the salmon? The infestation of a host by a copepodite can be divided schematically into three stages: in the first place, host and parasite must be in the same area. Then, the copepodite needs to be able to react rapidly as soon as the fish is within range, and to settle on it. Finally, the copepodite must be able to recognise the correct host, i.e. that it

is a salmonid. How does a wide range of light 145x100//Kap19-fig01.eps Figure 1: Settling of salmon lice on different parts of the and water conditions affect the copepodite’s body under various types of light. (Figure 3 from Browman ability to orient itself to the host? et al., 2004)

324 Aquaculture Research: From Cage to Consumption do not need light to infest a fish when the dis- saltwater, and the movements of the copep- tance between them is as short as it is in an ex- odites were filmed in two and three dimensions, perimental tank (maximum distance about 1 m). in light and in darkness.

The copepodite is thus capable of infesting a When the head was moved forward, 65 per cent fish in complete darkness. How then does it of the copepodites moved towards it. They nor- know that the fish is approaching it, and how mally swam in wavy tracks when the head ap- should it swim in order to attach itself to the fish proached them, at an average speed of about 95 without being eaten itself? Previous studies mm/s. Copepodites that were swimming in the have shown that copepodites react to low-fre- water directly in front of the head first moved quency pressure waves in the water by a violent aside a little before circling in again, so that increase in their swimming activity. Such pres- they often encountered the head further back as sure waves are created by swimming fish. By it passed them (Figure 2). Their reactions usual- filming the activity of copepodites when a salm- ly started when the head was about 2.5 cm away on head is approaching them, the parasites’ pat- from them. Their behaviour was identical in tern of swimming can be analysed. In one light and in darkness, and the number of attacks experiment, a silicon cast of the head of a salm- was the same under all light conditions. on smolt was moved around in a container of

145x100//Kap19-fig01.eps Figure 2: The swimming track of a salmon louse copepodite (in colour) when a salmon head approaches it. Often, the copepodite first swims away before circling back and settling further back on the head of the salmon.

Thematic area: The Environment 325 145x100//Kap19-fig01.eps Figure 3: Prevalence and median intensity of salmon lice on post-smolt caught in the Sognefjord between 1998 and 2004. (From Holst et al., 2005, Final report, p. 18)

The results of these two behavioural projects a serious population-regulating factor for Nor- show that light is not necessary for a copepodite wegian salmon and trout stocks. It has also been to be able to infest a fish. The copepodites rec- shown that the salmon louse problem takes dif- ognise the agitation of the water produced by a ferent forms in salmon and trout. Salmon en- swimming fish, and swim towards it. In the trials counter lice for the first time as post-smolt on that used the silicon fish head, there was no trace their way to the sea. In Norway, most strains of of chemical signals from the fish in the water, salmon have to migrate through more or less but the copepodites still attacked, often at such long fjords before they reach the sea. Fjords dif- high speeds that they bounced off again like rub- fer in terms of length, the number of fish farms ber balls. The conclusion here is that biological located within or outside them, and in the gen- molecules are not needed to trigger an infesta- eral freshwater runoff that determines their sation. Such molecules are probably most impor- linity. The salmon later return to their fjords as tant during the final phase of the infection, when adults in order to migrate upriver to spawn. the parasite tastes the fish in order to find out Trout also leave their rivers to optimise their ac- whether it has landed on the right species. cess to food, but they often remain within the fjord system and do not migrate as far from their Infestation rates and the own rivers. What has been called “premature re- consequences of salmon lice for turn” has also been registered in sea trout. This wild salmonids phenomenon involves the trout migrating upriv- Research results and reports that appeared dur- er in early summer, thus losing the better avail- ing the 1990s pointed out that salmon lice act as

326 Aquaculture Research: From Cage to Consumption ability of food offered by the sea. Such fish has been downwards. The conclusion to be often have very high infestations of salmon lice. drawn is that in Alta, salmon lice have most probably never been a problem for migrating The salmon louse problem has been studied in wild salmon, while in Sogn the situation has im- more detail in three Norwegian fjords: the Al- proved from being “serious” in the years prior tafjord, the Hardangerfjord and the Sognefjord. to 2002 to “very good” in 2002–2004 A comparative study of interactions between (Figure 3). salmon lice, farmed fish and wild fish was carried out in the Altafjord and the Sognefjord be- In the Hardangerfjord, stocks of wild salmon tween 1998 and 2004. In 2004, another inter- have been dwindling for many years. Only in disciplinary study was launched with the aim of the course of the past three or four years have studying similar interaction in the Hardanger- studies of infestation levels in wild salmon fjord system. This study also included certain smolt been carried out. These levels have been measures implemented by fish farmers. The low, with a somewhat higher rate of infestation Hardangerfjord is an ideal system for studies of around the outlet of the Etnefjord in both 2004 this sort because it is primarily influenced by and 2005. internal factors and is the location of important wild salmon and trout stocks, all of which are Sea trout affected by salmon lice, as well as housing a Where sea trout are concerned, infestation rates large farming industry that cooperates via a fish generally appear to be higher than they are in health network. A central aim of the study was salmon. In the Altafjord, no improvement has to consider whether the measures carried out on been observed in lice infestation rates in sea the farms themselves are appropriate, and above trout. In Sogn, the data suggests a general im- all, whether they are sufficient. provement, in parallel with the efforts made by the fish farming industry to lower the produc- Salmon tion potential of salmon lice in their installa- On average, it is colder the further north we go, tions. More data is needed, however, to be and this also affects the numbers of salmon lice. conclusive on the trend in Sogn. In the Hardan- Farmed fish in the Altafjord tended to have low- gerfjord, sea trout stocks have seriously de- er levels of infestation of salmon lice than those clined since the mid-1990s. Studies of infesta- on the Sognefjord during the period of the tion rates on sea trout revealed extremely high study. The threshold of 0.5 adult female lice per values at the beginning of the 1990s. fish, at which veterinary regulations require measures to be taken, was occasionally exceed- One method of studying the effects of salmon ed in Sogn, with an average of 0.6 female lice lice on trout is to treat a proportion of the mi- per fish in the first week of sampling in 2003. At grating trout population for lice. This has been roughly the same time, the average in Alta was done at the field station in the River Guddal in 0.13 adult female lice per fish. The rate of infes- the Hardangerfjord, where traps have been in- tation on migrating post-smolt show that Alta stalled for both migrating and returning fish. A salmon are either free of lice or have very few total of 711 trout smolt were treated with sub- lice. In Sogn, the level of lice has been high, but stance EX, which protects the fish against louse the records suggest that the trend in recent years attack for up to 16 weeks, while 1,380 fish were

Thematic area: The Environment 327 200 2005

150

32 23

100

50 26 4 11 7 6

Average number of lice per infected sea trout 7 0 0 0 0 22 24 26 29 22 24 26 29 22 24 26 29 Hardangerfjorden Ryfylke Jæren & Dalane

145x100//Kap19-fig01.eps Figure 4: Mean levels of salmon lice on sea trout caught in different regions at four points in time during the summer of 2005. (Data: Rådgivende biologer AS)

used as controls. Return migration rates were Sea trout that inhabit areas closer to the coast extremely low in both the control group (0.5– and, in many cases, within fjords, thus appear to 0.7 per cent) and the EX group (1.7–5.0 per be more exposed to the salmon lice threat, and cent), but taken together, the figures show that they act as carriers of lice within the fjord sys- the re-catch rate of the treated sea trout was tem, as do farmed salmon. More detailed stud- about twice as high as that of the control group. ies are needed to give us a complete overview of This shows that salmon lice are an important the overall situation. mortality factor for sea trout stocks in the basin. The effects of synchronised delousing The sea trout situation can also be evaluated by In 1996, a proposal for a national salmon lice registering salmon lice numbers on trout return- action plan was drawn up, and the plan was giving prematurely in the lower reaches of the riv- en its final shape in 1997. At the same time the er. This will be a measure of minimum levels, “Regulations for combating lice on salmonids”, since the salmon lice begin to fall off their hosts which specified how many lice per fish were when they move upriver in fresh water. Studies permitted in fish farms, were approved. These of this sort have been carried out at several loca- regulations have been regularly modified in re- tions on the coast of Norway in recent years. cent years. These studies have demonstrated that the highest infestation rates were observed in the Har- In the winter of 2004/2005 a synchronised de- dangerfjord basin. In the Jaeren area, where lousing programme was carried out in the Har- there is no fish farming, levels of infestation are dangerfjord, with the strong support of fish lower (Figure 4). farmers. The level of infestation in wild salmon

328 Aquaculture Research: From Cage to Consumption smolt and sea trout was monitored in spring and probability of development of resistance over summer 2005 in order to evaluate the effects of time, as happened with the organophosphates the programme. The results showed that out- that were used until the mid-1990s. Pesticides ward migrating salmon had low levels of infes- similar to those currently being used to deal tation, and that fewer than three per cent would with salmon lice have also been used to combat die as a direct result of infection. However, sea insect pests, and many species have evolved re- trout infestation rates were higher, particularly sistant strains. in the outer areas of the fjord, and the infestation pressure in the Hardangerfjord was still At the moment, there are no serious problems of higher than in adjacent fjords where there was resistance associated with the delousing com- no aquaculture, although it was significantly pounds used by the Norwegian aquaculture in- better than at the end of the 1990s. The results dustry, but problems of this sort are likely to thus indicate that coordinated measures are ca- occur in the future. The development of new de- pable of improving the situation, particularly lousing strategies, vaccination or new types of for salmon smolt, but that rises in infestation medication will therefore be of vital importance levels during the summer can still have a nega- for aquaculture. tive effect on wild sea trout stocks. However, sets of data covering several more years are still Molecular biological studies of salmon needed before safe conclusions can be drawn. lice and the potential development of a vaccine We may thus conclude that wild and farmed Molecular biological studies of salmon lice may salmon can co-exist in Norwegian fjords as long provide knowledge to be used in developing as total releases of lice larvae are adapted to lo- new methods of controlling lice on farmed fish. cal hydrographic conditions and the character- The strategy employed involves gaining an un- istics of the wild salmon populations. In order derstanding of central biological processes (di- to ensure long-term sustainable management, gestion, development and egg production) in the present level of knowledge needs much en- order to identify points in the salmon louse that hancement regarding the relevant conditions in can be blocked, and thus exploited to combat many fjord areas, particularly where the pro- the lice. Vaccination and the identification of duction of farmed fish is likely to be escalated. potential vaccine antigens are important aspects of these efforts. Potential anti-salmon lice measures Vaccines Although wrasse (Labridae) are currently used Developing vaccines against ectoparasites such to a certain extent for biological delousing, the as salmon lice is a difficult process. Ectopara- salmon lice problem is actually kept under con- sites live on the external surfaces of their hosts, trol by the widespread use of a few effective de- whose immune systems react to the parasite lousing compounds: two pyrethroids only slightly, compared with the way they do to (deltamethrin and cypermethrin) and emamec- viruses and bacteria. At present, only one com- tin benzoate (“SLICE”). The limited number of mercially available ectoparasite vaccine exists; types of medication available, combined with a vaccine for cattle ticks (Boophilus microplus). their widespread use, means that there is a high Cattle ticks and salmon lice are very different

Thematic area: The Environment 329 In trials of anti-lice vaccines based on hidden antigens, large numbers of salmon lice were hatched out and female lice were raised to the appropriate stage. A protein complex was extracted and purified and a test vaccine produced. Vaccinating fish with these antigens stimulated the production of specific antibodies. A controlled infection experiment showed a significant decrease in the number of salmon lice on fish that had been vaccinated with the hidden salmon louse antigen, compared with the number on fish that had been vaccinated with serum proteins from cattle (control vaccine). The difference was particularly great in terms of the number of egg-laying female lice per fish (2.8 145x100//Kap19-fig01.eps Figure 5: Number of adult female lice per fish found on in the control group vs. 0.75 in the vaccinated salmon vaccinated with hidden antigen from salmon lice. fish) (Figure 5). The control group was vaccinated with an equivalent dose of cattle serum protein. Irrespective of whether components of our cur- animals, but they do have some features in com- rent test vaccine can be commercialised or not, mon. Among other things, they suck the blood the trials have made it clear that it is possible to of the host organism when they are about to pro- vaccinate specifically in order to reduce the duce eggs. The cattle tick vaccine is based on a number of salmon lice on farmed salmon. How- single protein (Bm86) that is found in the gut of ever, the “right” antigen remains to be identi- the tick. Since this protein exists only in the tick fied. gut, it will not be “perceived” by the immune system of cattle during a tick infestation. How- Digestion in the salmon louse ever, when the protein is extracted from the tick, Studies of the digestive system are important purified and injected into cattle as a vaccine, for several reasons, since this is the organ in antibodies to it are created. (The protein is then called an “antigen”.) When the tick sucks blood, it also ingests these antibodies, which probably bind themselves to Bm86 in its gut and thus inhibit the functions of Bm86 there. Antigens of this sort are known as “hidden antigens”. Studies of ticks have shown that a vaccine based on such hidden antigens should preferably consist of only a few antigens. In order to be able to identify candidates for vaccines

of this sort in salmon lice, we require detailed 145x100//Kap19-fig01.eps Mobile stages of the female louse; an adult louse (top), a knowledge of the life cycle and biological pro- pre-adult II (left) and a pre-adult I (right). (Photo: Karin cesses of the louse. Boxaspen)

330 Aquaculture Research: From Cage to Consumption Immature Mature adult female adult female

18-20 days

145x100//Kap19-fig01.eps Various stages of maturity of the female salmon louse. (Photo: Lars A. Hamre) which the antibody from the salmon is found. A time. This method has been used to identify number of genes and gene products for various which genes are active in various biological enzymes related to digestion and other process- processes such as egg production. es have been identified and characterised. For example, we have found more than 20 different The maturation process that takes place in adult trypsin-like genes. Typical trypsins in salmon female lice ahead of egg production has been lice are produced by cells in the gut and are re- documented in salmon lice of homogeneous an- leased into the gut, which means that they are cestry produced in our laboratory. More than candidates for a vaccine antigen. However, im- 100 adult salmon lice at various stages of matu- munisation trials performed on rabbits suggest rity were collected (see photo) and systematised that the antigenicity of recombinant proteins is by means of digital structure analyses. The re- low, which makes them less likely to be suitable productive process is a potential point of attack as vaccine antigens. for a salmon lice vaccine. The aim is to identify the genes that are involved in egg production Egg production in the salmon louse and in the structural (anatomical) changes that The genome of the salmon louse is virtually un- take place in female lice ahead of egg produc- known, but some 2,500 genes have already been tion. We have identified four genes that code for cloned. Analyses of these genes have revealed the proteins that make up most of the nutrient that a large proportion of them (more than 40 stores in salmon louse eggs, and have demon- per cent) are unknown (i.e. there is no similarity strated just when in the louse’s growth these between them and genetic data on other organ- genes are switched on. In order to perform these isms stored in various databases). This large tasks we have established a range of microarray proportion of unknown genes has led to the de- techniques. Microarrays for salmon louse genes velopment of methods for studying the expres- do not exist anywhere else in the world, so the sion of a large number of genes at the same first phase of the project was to establish the

Thematic area: The Environment 331 technique. Microarray technology has revolu- In order to study the long-term stability of in- tionised certain parts of medical research, but festations of salmon lice on individual fish until now, it has not been available for organ- within a family group, two separate studies isms such as salmon lice. Microarrays permit were carried out. These studies were designed thousands of genes (i.e. information about with the aim of answering the following impor- whether they are switched on or off) to be stud- tant question: do fish that carry higher or lower ied simultaneously. Microarrays have turned numbers of lice at a given time also have a cor- out to be a very successful method for large- respondingly higher or lower number at a later scale searches for candidates for vaccine status. point in time? This is important, because when we search for fish that carry lower numbers of Genetic resistance to salmon lice: a lice (i.e. resistant individuals?) it is essential possible solution? that this characteristic should be stable both A number of studies have been dedicated to over time and in terms of variations in lice finding out whether particular families of salm- infestation pressure. on display characteristics that could be exploited to breed for resistance to salmon lice. The first study showed that we could not predict the number of lice carried by an individual fol- In order to study some of the variables that af- lowing a second round of infestation on the basis fect individual variations in receptivity to salm- of its rank following the first round. In practice, on lice, some 300 salmon from 30 full-sibling this means that individuals cannot be identified groups were kept in sea cages. The fish were as either resistant or receptive in the course of naturally infested with salmon lice in the sea two rounds of infestation. The implications of cages, and the number of lice settling on them this finding led us to carry out a second experi- was recorded. It turned out that two main com- ment, in order to see whether the results could ponents – the size of the individual fish and its be repeated in a sea-cage situation. This time, family background – had a clear influence on the salmon were kept in a sea cage for nine the level of infestation. The genetic component, months, during which they were sampled three denominated “heritability”, was estimated to be times. Once again, there was no correlation be- 0.074 ± 0.022. This value indicates that less tween the individuals classified as resistant in than 10 per cent of the variation in the level of the first round and those in the second. salmon louse infestation in this study was due to genetic variables, while more than 90 per cent Another study looked at three year-classes of was a consequence of environmental parame- salmon from 300 family groups from 2000 and ters, including the size of the individual fish. 2001, as well as 50 family groups from 2002. This value must be regarded as relatively low in This study found a heritability index of 0.14 ± the context of selective breeding, and limits the 0.02 (14 per cent). A follow-up study in which expected effect of a programme of breeding for salmon were infested under controlled condi- resistance to salmon lice. At the same time, tions found a heritability of 0.26 ± 0.07 (26 per however, it does indicate that it is theoretically cent). possible to reduce receptivity to salmon lice by means of selective breeding. One interesting finding of these studies was the high correlation (0.88) between the number of

332 Aquaculture Research: From Cage to Consumption lice per fish under controlled and natural in- are smaller than those of the other species. festation conditions. This shows that controlled Wrasse farming will also provide small ballan infestations can be utilised to distinguish be- wrasse for small salmon, enabling the two spe- tween families with high and low susceptibility cies to grow up together. Ballan wrasse aqua- to lice. This is difficult to achieve in the field, culture is focusing on two main bottlenecks: the where the fish carry few lice, because infesta- supply of good-quality fertilised eggs and satis- tion pressure is lower and because the fish must factory survival rates during the period follow- be treated for lice as soon as the number of lice ing start-feeding until the fry have been weaned exceeds 0.5 adult females per fish. onto dry feed.

Taken together, these results demonstrate the In Norway, only monocultivation of fish, i.e. existence of a weak-to-moderate genetic com- one species per farm, is practised. When labrids ponent in the susceptibility of salmon to lice at- are introduced, even as a delousing method, tacks. It may be possible to exploit this in a there are two species in the same system, a sit- selective breeding programme. uation which poses challenges in terms of potential infections being carried over from one Wrasse species to the other, as well as general problems The ballan wrasse (Labrus bergylta) is an inex- of combating disease. In studies of the rock pensive, ecological and environmentally friend- cook and the ballan wrasse, in which the fish ly method of combating salmon lice, and its use were injected with infectious salmon anaemia is regarded as an advantage possessed by Nor- (ISA) virus, no replication in the fish could be way vis-à-vis other salmon-producing coun- detected. Atypical aeromonas (a bacterial dis- tries. These fish are found in shallow waters ease), which has often been found in labrid spe- along much of the coast of Norway, as far north cies, has also been studied, but neither trans- as the Counties of Trondelag. The species has mission to salmon nor disease outbreaks have many popular names and is familiar to young been demonstrated. Labrids have also been fishermen who have caught it while fishing screened for other diseases and parasites, but from the pier head. Studies carried out in the nothing that could be transmitted from one early 1990s showed that wrasse could act as species to the other has been found. cleaner-fish for salmon, just like certain species of tropical fish on coral reefs. Not all labrids are The use of chemicals and development of suitable, and today only the rock cook resistance in salmon lice (Centrolabrus exoletus) and the corkwing It is known from the treatment of both animals (Crenilabrus melops) are used on small salmon, and human beings that diseases and parasites and the ballan wrasse on larger fish. Wrasse can develop resistance to particular treatments, species tend to be sedentary and do not spread whether antibiotics or other pharmaceutics. rapidly; the eggs of some species are even at- This is also happening with salmon lice. In or- tached to the substrate and the young are cared der to be able to study this development, bioas- for by their parents. This increases their chances says have been developed to test resistance to of being fished to extinction, and wrasse them- the pyrethroids deltamethrin and cypermethrin, selves are now being farmed. This applies in the organophosphorus compound azame- particular to the ballan wrasse, stocks of which thiphos, the avermectin emamectin and the

Thematic area: The Environment 333 chitin synthesis inhibitor teflubenzuron. These acetylcholinesterase: a “sensitive” type that is methods have been employed in a systematic rapidly inhibited by the organophosphorus study of salmon louse population samples col- compound azamethiphos, and a “resistant” type lected from different parts of Norway. that is not particularly inhibited by such concentrations likely to be used for delousing pur- Reduced sensitivity to pyrethroids has been poses. The study of Norwegian strains has been demonstrated on several occasions. However, compared with similar studies of Canadian salmon lice that settled on fish that had recently strains. The results showed that in the Norwe- been released in the same farms after a period of gian strains, there is a significantly higher pro- fallowing displayed a normal level of sensitiv- portion of heterozygotic individuals than in the ity, suggesting that the strains involved came Canadian strains. However, a few heterozygotic from another location. The results also indicate individuals have also been found among salmon that pyrethroid resistance has not “settled lice from Canada, Ireland and Scotland. These down“ in Norwegian populations of salmon results indicate that the serious problems of re- lice. A rise in the metabolic capacity of the sistance to organophosphates that arose in the salmon louse is assumed to be the main mecha- 1990s were due to the selection of individuals nism underlying the development of reduced with a resistant type of acetylcholinesterase. sensitivity to pyrethroids. It is also possible that This mechanism can still be found, ten years af- mutations in the salmon louse gene that regu- ter these drugs were taken out of use. The relates tension-dependent sodium channels are sults indicate that this mechanism has “settled important in this connection. However, the best- in”. We can expect that any reintroduction of described mutation, the kdr mutation, has not this drug would lead to the reappearance of re- been found in any strains, although another mu- sistance in the course of a relatively short time tation in the gene that codes for tension-depen- (for further information see the publications of dent sodium channels (the site of action of Horsberg and Sevatdal). pyrethroids) has been found. This mutation results in an amino acid that has quite different Challenges and vistas electrochemical properties from the original At the present time, only farmed salmon in sea ones being incorporated in this protein. This cages can be treated for salmon lice, and usually mutation has not been described in any other ar- by means of chemical treatments. The day may thropod species, and studies of its importance come when this will no longer be acceptable to are underway. the market, or when salmon lice develop resistance to the treatments we possess. Even given Reduced sensitivity to emamectin has been the some uncertainty, investing in the development subject of merely anecdotal reports. It has not of a salmon lice vaccine would be appropriate been possible to confirm that such cases are due and might be able to solve the problem on a to reduced sensitivity to this drug by means of more permanent basis. such methods as have been developed to date. Both timing and the lice population itself are These studies have also shown that in Norwe- important factors in this respect. Present regula- gian strains of salmon there is a relatively high tions allow more lice to be attached to farmed proportion of individuals that have two types of fish during the summer and autumn. This is the

334 Aquaculture Research: From Cage to Consumption time of year during when salmon are in the sea, stocks. For this purpose, the models of the while sea trout and artic charr stay in the fjords aquatic environment, water movements and lice to graze. These two species, along with the populations that have been developed in recent farmed fish, thus suffer from a build-up of the years will be important tools for testing the con- lice population. A number of Norwegian salm- sequences of different quantities of fish and lice on lice researchers have therefore advised the and of a range of hydrographic conditions. Such authorities that the strictest limits on the permit- studies of the spread of salmon lice, perhaps es- ted number of lice per fish should be maintained pecially the development of models, may also year-round. These limits are already enforced in provide good tools for strategic assessments of certain fjords where the salmon farmers them- fish farm sites as well as the optimal use of area. selves have gone on the offensive. Highly motivated and well-coordinated fish farmers can Because there are so many farmed fish, even a keep salmon lice populations down, to the ben- slight increase in the number of adult female efit of both themselves and wild salmon stocks. lice per fish will have serious consequences for However, a few “black sheep” do not participate wild stocks of salmonids. A rise from 0.25 to in these efforts. Mathematical modelling shows 0.5 lice per fish, for example, means a doubling that a minority of fish farmers with high levels of the production of lice in the system. How- of infestation in their fish can easily destroy ever, well-coordinated anti-lice measures in the other people’s efforts. This makes it important Sognefjord and the Hardangerfjord have shown for such farms to be identified and controlled, that extremely low rates of infestation can be for example through unannounced visits from achieved in fish farms. This appears to be pos- the Norwegian Food Safety Authority. sible without particularly high consumption of pharmaceuticals, and the effect is that the infec- It is extremely important to monitor wild stocks tion pressure on both wild and domesticated of salmonids so that the results of the struggle hosts is significantly reduced. The situation is against salmon lice can be evaluated. The num- different for wild stocks, particularly those of ber of lice on wild fish is the ultimate measure sea trout. In several places, migrating smolt still of whether control measures have been success- carry so many lice that they have the effect of ful. This is of particular importance in the Na- reducing stocks. The challenge here will be to tional Salmon Fjords, where migrating wild identify a level, i.e. a total population of salmon smolt ought to be monitored. The challenge will lice, that is so low that stocks of neither wild nor be to tailor the acceptable number of lice in farmed fish are weakened. farms to the tolerance thresholds of the wild

Thematic area: The Environment 335 References Lepeophtheirus salmonis. J. Exp. Biol., 203, 1649– 1657. Bjørn, P.A. and Finstad, B., 1997. The physiological ef- Frost, P. and Nilsen, F., 2003. Validation of Reference fects of salmon lice infections on sea trout post Genes for Transcription Profiling in the Salmon smolts. Nordic Journal of Freshwater Research, 73, Louse, Lepeophtheirus Salmonis, by Quantitative 60–72. Real-Time Pcr. Veterinary Parasitology, 118, 169– Bjørn, P.A. and Finstad, B., 2002. Salmon Lice, 174. Lepeophtheirus Salmonis (Kroyer), Infestation in Glover, K.A., Aasmundstad, T., Nilsen, F., Storset, A. Sympatric Populations of Arctic Char, Salvelinus and Skaala, O., 2005. Variation of Atlantic Salmon Alpinus (L.), And Sea Trout, Salmo Trutta (L.), In Families (Salmo Salar L.) In Susceptibility to the Areas Near and Distant From Salmon Farms. Ices Sea Lice Lepeophtheirus Salmonis and Caligus Journal of Marine Science, 59, 131–139. Elongatus. Aquaculture, 245, 19–30. Bjørn, P.A., Finstad, B. and Kristoffersen, R., 2001. Glover, K.A., Hamre, L.A., Skaala, O. and Nilsen, F., Salmon Lice Infection of Wild Sea Trout and Arctic 2004. A Comparison of Sea Louse (Lepeophtheirus Char in Marine and Freshwaters: the Effects of Salmonis) Infection Levels in Farmed and Wild At- Salmon Farms. Aquaculture Research, 32, 947– lantic Salmon (Salmo Salar L.) Stocks. Aquacul- 962. ture, 232, 41–52. Boxaspen, K., 2005. Spredning av lakselus − Hardan- Glover, K.A., Nilsen, F. and Skaala, O., 2004. Individu- gerfjorden som et laboratorium. Fiskehelse, 7, 29– al Variation in Sea Lice (Lepeophtheirus Salmonis) 31. Infection on Atlantic Salmon (Salmo Salar). Bredal, W., Vågen, I. and Reinnel, H., 2000. Terapibe- Aquaculture, 241, 701–709. handling: Behandling mot lakselus i oppdrettsan- Glover, K.A., Nilsen, F. and Skaala, O., 2004. Is inter- legg. Statens Legemiddel Kontroll, Oslo, Norway. individual variation in salmon lice (Lepeophtheirus 43 pp. salmonis) infection random? Aquaculture, 241, Browman, H.I., Boxaspen, K. and Kuhn, P., 2004. The 701–709. Effect of Light on the Settlement of the Salmon Heuch, P.A., Bjørn, P.A., Finstad, B., Holst, J.C., Asp- Louse, Lepeophtheirus Salmonis, on Atlantic Salm- lin, L. and Nilsen, F., 2005. A Review of the Norwe- on, Salmo Salar L. Journal of Fish Diseases, 27, gian 'National Action Plan Against Salmon Lice on 701–708. Salmonids': the Effect on Wild Salmonids. Aqua- Butler, J.R.A., 2002. Wild salmonids and sea louse in- culture, 246, 79–92. festations on the west coast of Scotland: sources of Heuch, P.A. and Mo, T.A., 2001. A Model of Salmon infection and implications for the management of Louse Production in Norway: Effects of Increasing marine salmon farms. Pest Management Science, Salmon Production and Public Management Mea- 58, 595–608. sures. Diseases of Aquatic Organisms, 45, 145–152. Denholm, I., Devine, G.J., Horsberg, T.E., Sevatdal, S., Johnson, S.C., Treasurer, J.W., Bravo, S., Nagasawa, K. Fallang, A., Nolan, D.V. and Powell, R., 2002. and Kabata, Z., 2004. A Review of the Impact of Analysis and management of resistance to chemo- Parasitic Copepods on Marine Aquaculture. Zoo- therapeutants in salmon lice, Lepeophtheirus sal- logical Studies, 43, 229–243. monis (Copepoda: Caligidae). Pest Management Kolstad, K., Heuch, P.A., Gjerde, B., Gjedrem, T. and Science, 58, 528–536. Salte, R., 2005. Genetic Variation in Resistance of Fallang, A., Denholm, I., Horsberg, T. E. and William- Atlantic Salmon (Salmo Salar) to the Salmon Louse son, M. S., 2005. Novel Point Mutation in the Sodi- Lepeophtheirus Salmonis. Aquaculture, 247, 145– um Channel Gene of Pyrethroid-Resistant Sea Lice 151. Lepeophtheirus Salmonis (Crustacea: Copepoda). Kvamme, B.O., Frost, P. and Nilsen, F., 2004. The Diseases of Aquatic Organisms, 65, 129–136. Cloning and Characterisation of Full-Length Finstad, B., Bjørn, P.A., Grimnes, A. and Hvidsten, Trypsins From the Salmon Louse Lepeophtheirus N.A., 2000. Laboratory and field investigations of Salmonis. Molecular and Biochemical Parasitology, salmon lice (Lepeophtheirus salmonis, Krøyer) in- 136, 303–307. festation on Atlantic salmon (Salmo salar L.) post- Kvamme, B.O., Kongshaug, H. and Nilsen, F., 2005. smolts. Aquaculture Research, 31, 795–803. Organisation of Trypsin Genes in the Salmon Louse Flamarique, I.N., Browman, H.I., Belanger, M. and (Lepeophtheirus Salmonis, Crustacea, Copepoda) Boxaspen, K., 2000. Ontogenetic changes in visual Genome. Gene, 352, 63–74. sensitivity of the parasitic salmon louse Kvamme, B.O., Skern, R., Frost, P. and Nilsen, F., 2004. Molecular Characterisation of Five Trypsin-Like

336 Aquaculture Research: From Cage to Consumption Peptidase Transcripts From the Salmon Louse Sevatdal, S., Copley, L., Wallace, C., Jackson, D. and (Lepeophtheirus Salmonis) Intestine. International Horsberg, T.E., 2005. Monitoring of the Sensitivity Journal for Parasitology, 34, 823–832. of Sea Lice (Lepeophtheirus Salmonis) to Pyre- Pike, A.W. and Wadsworth, S.L., 1999. Sea lice on throids in Norway, Ireland and Scotland Using Bio- salmonids: Their biology and control. Advances in assays and Probit Modelling. Aquaculture, 244, 19– Parasitology, 44, 234–337. 27. Revie, C.W., Gettinby, G., Treasurer, J.W. and Wallace, Sevatdal, S. and Horsberg, T. E., 2003. Determination C., 2005. Evaluating the Effect of Clustering When of Reduced Sensitivity in Sea Lice (Lepeophtheirus Monitoring the Abundance of Sea Lice Populations Salmonis Kroyer) Against the Pyrethroid Delta- on Farmed Atlantic Salmon. Journal of Fish Bio- methrin Using Bioassays and Probit Modelling. logy, 66, 773–783. Aquaculture, 218, 21–31. Revie, C.W., Robbins, C., Gettinby, G., Kelly, L. and Tjensvoll, K., Hodneland, K., Nilsen, F. and Nylund, Treasurer, J.W., 2005. A Mathematical Model of the A., 2005. Genetic Characterization of the Mito- Growth of Sea Lice, Lepeophtheirus Salmonis, Pop- chondrial Dna From Lepeophtheirus Salmonis ulations on Farmed Atlantic Salmon, Salmo Salar (Crustacea: Copepoda). A New Gene Organization L., In Scotland and Its Use in the Assessment of Revealed. Gene, 353, 218–230. Treatment Strategies. Journal of Fish Diseases, 28, Tully, O. and Nolan, D.T., 2002. A Review of the Pop- 603–613. ulation Biology and Host-Parasite Interactions of Rikardsen, A.H., 2004. Seasonal Occurrence of Sea the Sea Louse Lepeophtheirus Salmonis (Copepo- Lice Lepeophtheirus Salmonis on Sea Trout in Two da: Caligidae). Parasitology, 124, 165–182. North Norwegian Fjords. Journal of Fish Biology, 65, 711–722.

Thematic area: The Environment 337 Arne Ervik1), Jan Aure1) and Pia Kupka Hansen1) 1) Institute of Marine Research

How Much Aquaculture Can the Norwegian Coast Tolerate?

The coast is the basis of human settlement and production, and it has the potential for further development. We have great expectations of the aquaculture industry, but if they are to be met, we need to exploit the coast rationally and sustainably. The key concepts here are “area use” and “carrying capacity”. The coast of Norway is 21,000 km long, and the sea area within the datum line is 90,000 km2. Both hydrography and topography are extremely varied, alternating between shallow sea and deep fjords, calm bays and areas of rapid currents. Tempera- ture and salinity are stable and suitable for the farming of cold-water species. Water quality is good, and eutrophication and pollution are restricted to a few enclosed areas. The Norwegian coast is thus highly suitable as a host for a large, diversified aquaculture industry. However, the coast is also used for many other purposes, including transport, fishing, leisure activities, tourism, conservation areas, etc. The aquaculture industry is thus forced to compete for area and resources, while it also needs to develop new forms of operation and a wider range of products. At present, the industry is vulnerable to competition and changes in the market, and it does not fully exploit the potential for fish farming offered by the coast. The aquaculture industry of the future will continue to be dominated by the intensive production of salmonids and marine species such as cod and halibut, and future trends are likely to be in the direction of large fish farms concentrated in particularly suitable areas. This will offer efficient use of area and production capacity, and reduce the number of user conflicts. But we can also expect to see a major increase in shellfish culture and niche products such as various species of crustaceans, echinoderms and kelp. Any development in such a direction will make specific demands as far as area, topography, depth, water exchange rates, etc. are concerned. Rational use of aquaculture areas also means that we need to be capable of exploiting production capacity while continuing to take the environment into account, ensuring that product quality is high and providing for the welfare of the species being cultivated.

338 Aquaculture Research: From Cage to Consumption Carrying capacity Figure 1 illustrates the relationship between The term “carrying capacity” refers to how carrying capacity for different feed factors and much we can produce in a given area without environmental standards. The curve for feed the effects on the environment exceeding cer- factor 2.2 (dry weight of feed/weight of salmon) tain upper limits. Such limits are known as en- rises more steeply than the curve for feed factor vironmental standards. The carrying capacity 1.1, which means that the environmental impact depends on natural, operational and political per unit of fish produced increases with the feed conditions, and it is not always an easy matter to factor. The two environmental standards, for ex- find a common denominator for these and to set ample, may indicate different degrees of impact suitable environmental standards. on the seabed, as these are defined by Norwe- gian Standard 9410 “Environmental monitoring In other words, it is difficult to concretise just of marine ongrowing farms”. There are thus which, and how large, environmental effects so- two conditions that determine how much we ciety is prepared to accept from the aquaculture can produce at a given farm-site; the feeding re- industry. A milestone was therefore passed in gime, and how much impact on the seabed we 1993, when the environmental, fisheries, veter- are prepared to accept. The figure shows that inary and health authorities drew up a joint set the carrying capacity (production potential) is of environmental goals for the aquaculture in- four times as high when the producer keeps the dustry (1). The environmental objectives were feed factor low and we are willing to accept a revised in 1999 (2), when problematic areas high seabed impact (environmental standard 2), were described and ranked, and long-term en- as when feed is wasted (feed factor 2.2) and we vironmental objectives and short-term aims for accept only a minor impact on the seabed (envi- each problem were drawn up. These aims form ronmental standard 1). the essential basis for the development of operational and measurable environmental stan- Aquaculture affects the environment in several dards for each environmental impact. For some ways, such as the exploitation of wild fish stocks time, annual reports of the results achieved for feed, genetic interactions between farmed were also produced, and these showed that most and wild species, the spread of infections and of the short-term and many of the long-term ob- parasites, emissions of foreign substances such jectives were being met. These reports were as medicines and chemicals, as well as of organic very useful, and it would be a good idea to take them up again. We have continued to make Impact on seabed progress, and the situation has become much better in several areas (3), but certain problems Feed factor 2.2 Feed factor 1.1 remain, including escapes and their genetic im- Environmental standard 2 pacts on wild populations, salmon lice and the Environmental use of copper as antifouling for sea-cages. New standard 1 environmental problems may also emerge with the introduction of new aquaculture species or Carrying capacity changes in operating practices. 145x100//Kap20-fig01.eps Figure 1: Carrying capacity of a site with two different feeding regimes and two different environmental standards.

Thematic area: The Environment 339 compounds and nutrient salts, changes in com- aquaculture, and that is what we take as an exam- petitive and production conditions in the coastal ple here. The bottom becomes loaded with faeces zone, culling of animal pests, etc. Impacts can and waste food, and a number of studies have also be divided into geographic areas such as shown that the impact follows the classical mod- water quality within farms, effects on water el of organic effects as described in (4). The masses and the seabed on-site and in the reci- chemical milieu within the sediment is modified pient, and various regional effects. as the load increases, and fauna come to be dominated by opportunistic species that both tolerate A sustainable aquaculture industry will require and are competitive under such conditions. If the that all these environmental impacts are kept well load becomes too great, the benthic fauna will within the limits of what nature can tolerate, and disappear completely, a development that is most that the environmental standard that results in likely to occur directly beneath the sea-cages. If least production should determine how much we the organic load is removed, for example by can produce within a given area. It does not help moving the fish farm, conditions will gradually that nature can tolerate major emissions of nutri- return to normal. Significant changes take place ents from sea-cages if we overload the seabed be- in the seabed when the burrowing benthic fauna neath the site, or if so many parasites are released disappear. When burrowing ceases, less oxygen that wild salmon stocks disappear. We also need enters the sediments and waste products accumu- to operationalise the concept of carrying capa- late. Organic material is then converted two or city, which means that we need to understand the three times as slowly, and the waste accumulates connection between emissions – broadly defined more readily (5). When the carrying capacity for – and their effects on the environment, we have seabed impact was being determined, it was to be able to predict the environmental effects of therefore natural to set it lower than the load at a planned activity, we need to set scientifically the point where the benthic fauna disappear. responsible environmental standards, and finally, we must have relevant measurement parameters We have designed a regulation system that is in- and programmes of study for monitoring im- tended to ensure that the impact on the seabed pacts. does not exceed its carrying capacity. The system is called Monitoring – Ongrowing farms – Mod- Environmental monitoring elling (MOM), and it consists of a monitoring “Carrying capacity”, then, is defined as the programme with appropriate limiting values, maximum fish production that an area is cap- plus simulation models that can estimate the car- able of sustaining without the environmental rying capacity (6–8). As mentioned above, the impact exceeding acceptable limits. The carry- limiting value for the impact of a site on the sea- ing capacity of a site is determined on the basis bed is that there should still be a benthic infauna of natural criteria, while the environmental cri- in the sediment. A basic premise is that the farm teria are often flexible, and in the final analysis, should be able to use the same site over a long pe- it is the question of what impact society is pre- riod of time without its environmental quality be- pared to accept that is decisive. coming unacceptable. In the recipient around the farm, other environmental standards are applic- The impact on the seabed beneath a fish farm is able; these are derived from the composition of the best understood environmental impact of the benthic fauna. MOM has been utilised as the

340 Aquaculture Research: From Cage to Consumption Surface current 8,0 O2a - summer (water exchange within sea-cage) O2a - winter 7,0 6,0 Current between sea-cage and seabed 5,0 (diffuses particles) 4,0 Compact farm 3,0 (60x40 m) Oksygen (ml/l) Bottom current 2,0 Ft = 25 kg/m3 (brings oxygen to the seabed) k = 0.5 1,0

145x100//Kap20-fig02.eps 0,0 Figure 2: The functions of the current in a fish farm; it 1234567891011121314151617 exchanges the water inside the sea-cages, it spreads the Background current Vo (cm/s) particles that sink out of the farm, and it carries oxygen down to the bottom, so that waste products are broken 145x100//Kap20-fig03.eps down. Figure 3: Relationship between background current (Vo) and oxygen concentration (O2a) in a standard compact farm (2,400 m2) in summer and winter, for fish density (Ft) = 25 kg/m3and a reduction in current velocity of K = 0.5. basis for a Norwegian Standard for monitoring fish farms (9). stress are also of importance. The oxygen con- Estimating environmental sumption of salmon can range from 120 to 940 impact milligrams per kilogram per hour. As an aver- Local carrying capacity age value for a fish farm in the sea, we can The carrying capacity of a site depends in the reckon on a consumption of around 290 mg first instance on current and depth conditions. oxygen/kg/h in the summer, and about The current can be divided into three parts: the 180 mg/kg/h in the winter. surface current exchanges the water in the farm and is decisive in determining the environmen- The water exchange rate in a fish farm is deter- tal conditions within the sea-cages; the currents mined by the background current flow in the between the sea-cages and the seabed spread vicinity, the design of the farm and the friction waste feed and faeces from the farm, while the produced by the sea-cages. The amount of fric- current close to the bottom brings oxygen to the tion is primarily determined by mesh size, the sediment and is thus important for the break- length of the farm in the main current direction, down of organic matter such as waste food and fish density and net fouling. It has been noted faeces (Figure 2). that current velocity may be reduced by 50–70 per cent in traditional compact farms. Oxygen conditions in a fish farm The oxygen conditions inside sea-cages depend If we assume that the farm’s main axis is normal among other things on water exchange rates and to the main current direction and that there is a the respiration of the fish (oxygen consump- balance between oxygen added by water trans- tion). The oxygen consumption of the fish is in- port and the oxygen consumption of the fish, the fluenced by a number of conditions, of which mean oxygen concentration within the farm the most important are temperature and the size (O2a) can be estimated as follows: of the fish, although activity level, feeding and

Thematic area: The Environment 341 O2a = O2o – B x Ft x Pf/(Vo x k) (ml/l) change rates in the sea-cages will be greater than in compact farms (k < 0.5), and such farms where O2o is the oxygen concentration of the will therefore experience fewer problems inflowing water (ml/l). B is the width of the caused by periodically low oxygen concentra- farm (m), Ft is fish density in the farm (kg/m3), tions. Pf is the specific oxygen consumption of the fish (mg O2/s/kg). Vo is the background current Spread of waste food and faeces (cm/s), and the reduction in current speed is ex- The areas of seabed beneath and around fish pressed by k = Va/Vo, where Va is the mean cur- farms receive waste food and faeces. Calcula- rent inside the farm (cm/s). tions using the MOM 3 program for the PC (10) show that the production of 1 tonne of fish with Figure 3 provides an example of how the oxy- a 20 per cent rate of feed wastage results in the gen conditions in a standard compact 2,400-m2 sedimentation of around 270 kg waste feed and fish farm can vary with current conditions in a faeces. Sedimentation is usually greatest in the summer and a winter situation, at a fish density area of the farm itself and decreases rapidly of 25 kg/m3. The current flow is normal to the over the rest of the recipient. In order to be able long axis of the farm, and the reduction in cur- to assess the environmental effects of organic rent velocity is set at 50 per cent (k = 0.5). particles, we need to know something of how they spread, i.e. how much of them settle dir- If the lower limit for oxygen concentration in a ectly under the farm and how much are trans- fish farm is set at 4.0 ml/l, for example, the fig- ported outwards to the rest of the recipient. ure shows that the background current flow rate Faeces sink more slowly than food pellets, and should be greater than 5–6 cm/s during the sum- are more easily broken up into small particles mer, while this requirement is reduced to that remain suspended longer in the water around 2 cm/s in the winter. However, for vari- column. These particles therefore tend to be dif- ous reasons, including temperature, algae, feed- fused over a much wider area than food parti- ing, etc., the fish will often be unevenly cles. In order to estimate how far particles from distributed throughout the volume of the sea- a fish farm can be transported throughout the re- cage. Observations made in early autumn in a cipient before they fall to the bottom, we need farm with a mean fish density of about 20 kg/m3 to know how fast they sink, the depth of the wa- revealed occasional fish densities of up to 60– ter and the current conditions between the un- 100 kg/m3, with significantly lower oxygen derside of the sea-cage and the seabed. A concentrations inside the farm than outside it. detailed simulation of sedimentation around a At that time of the year, there are often large fish farm thus requires detailed knowledge of numbers of fish in the farms, feeding is intense, bottom topography, current conditions and rate growth rates are high, there is relatively little of sinking, as well as a large amount of calcula- oxygen in the seawater and nets are most heavi- tions. ly fouled. The risk of low oxygen concentrations in fish farms is therefore higher in the We can illustrate the most important features of summer and autumn than during the winter. the diffusion of waste feed and faeces around a Other things remaining equal, in farms in which fish farm by means of some examples using a the sea-cages are widely spaced, water ex- simplified seabed topography and current con-

342 Aquaculture Research: From Cage to Consumption 300 we increase the distance between sea-cage and Vo = 5 cm/s Vo = 10 cm/s seabed to 150 m, the maximum diffusion dis- 250 Vo = 15 cm/s tances for feed and faeces will be 150 m and Vo = 20 cm/s 200 500 m respectively. Since the environmental 150 impact of organic loads on the seabed must not

Diffusion (m) 100 exceed certain maximum values, current conditions and water depth are thus critical factors in 50 determining how much fish can be produced at 0 10 30 50 70 90 110 130 150 any given fish-farm site. Depth under sea-cage (m)

145x100//Kap20-fig04.eps Impact on the seabed Figure 4: Diffusion of waste feed from a sea-cage as a Apart from current and depth, the design of the function of the depth beneath the sea-cage and a range of mean current velocities between the sea-cage and the fish farm and its operating procedures are im- seabed. Vo is the mean current velocity between the sea- portant factors in determining how much waste cage and the seabed. food and faeces will settle on the seabed at the site, and there are great differences in the carry- ditions. The diffusion distance (L) can be ex- ing capacities of fish farms whose sea-cages lie pressed as follows: in closely spaced rows on both sides of a central access channel (compact farms), and those L = Vo x D/v (m) whose sea-cages are widely spaced. The operating procedures of the farm also affect carrying where Vo is the mean current velocity between capacity, and rates of feed wastage and the sink- the sea-cage and the seabed (cm/s), D (m) is the ing rates of waste feed and faeces are important distance between sea-cage and seabed and factors in this respect. v(cm/s) is the rate of sinking (feed and faeces). The rate of sinking of the feed particles is about In order to illustrate how current conditions af- 10 cm/s, while that of faeces is less than 3–4 fect carrying capacity, we used the MOMS 3 PC cm/s, depending on particle size. program (10) to estimate the biomass of fish in a standard compact fish farm with a sea-cage Figure 4 shows how far a particle of feed or area of 2,400 m2, in which the distance between faeces is transported away from its point of the bottom of the sea-cages and the seabed is 90 emission under various seabed depth and cur- metres. The calculations utilise a feed waste rent velocity conditions. At many fish-farm rate of 20 per cent. As we can see from Figure sites, current velocities range from 0 to 5, the permissible biomass rises with current 10 cm/s. The figure shows that at a current ve- speed (expressed as current variance) and the locity of between 0 and 10 cm/s, feed particles biomass can be twice as high during the winter will be transported a maximum distance of as in summer. The highest legal biomass of fish around 50 m from the sea-cages when the depth in a standard fish farm is about 800 tonnes, and is less than 50 m. Other estimates show that un- this example shows that we need a current vari- der the same conditions, faeces will be trans- ance of 7–8 (cm/s)2 to have such a large bio- ported about 200 metres at most from the farm. mass. Under the same current conditions, but when

Thematic area: The Environment 343 900 Ft summer 800 Ft winter 700 600 500 400 300

Ft (tonnes of fish) 200 100 0 2, 3, 4, 5, 6, 7, 8,

V diffusion current, variance (Cm/s)2

Figure 5: Estimated carrying capacity (tonnes of fish) in summer and winter for a compact fish farm (2,400 m2, two rows of sea-cages) with a distance between the sea-cages and the seabed of 90 m. Feed wastage: 20 per cent. V (diffusion current) = 8 (cm/s)2 corresponds to a maximum current velocity of around 20 cm/s and V = 4 (cm/s)2 corresponds to a maximum current velocity of about 10 cm/s.

145x100//Kap20-fig05.eps Regional carrying capacity spread over about 260 km2, i.e. about 40 tonnes/ 2 Algae production km . Estimates show that the percentage reduc- The production of farmed fish adds nutrients to tion in Secchi depth (depth of visibility) in a the water, and these nutrients are converted into fjord area as a result of fish farming is about algae production. The proportion of the nutri- three per cent (10, 11). This shows that even in ents that are converted locally within an area de- a fjord where the density of fish farms is rela- pends on the residence time of the water (Tv) tively high, the increase in algae production will and the rate of division of the algae (Ta). When be insignificant under present conditions. Tv is small and Ta large, the algae will be transported out of the immediate vicinity of the farm The environmental standards that will be set before all the nutrient salts have been converted, will determine how much aquaculture can be and the production will be spread over a larger permitted in a given fjord area in the future. For area. In such cases, we can say that we have a example, permitted increases of 10 per cent or regional effect. For the nutrients to be converted 20 per cent in algae production in the central within the local fjord or sound, Tv must there- parts of the Hardangerfjord would mean that the fore be considerably larger than Ta, i.e. production of fish could be raised from the cur- Ta/Tv<<1. By Norwegian standards, between rent level of 10,000 tonnes to around 30,000 Vikingnes and Ånuglo the Hardangerfjord has a tonnes or 70,000 tonnes respectively. A 10 or 20 relatively high density of fish farms, with a total per cent increase in algae production could typ- annual production of around 10,000 tonnes ically lead to a reduction in mean Secchi depth

344 Aquaculture Research: From Cage to Consumption the oxygen consumption of the basin water and 6.5 Hordaland Møre thus reduce the level of dissolved oxygen. The 6.0 Nordland carrying capacity of threshold basins is highly 5.5 5.0 dependent on the residence time of the water in 4.5 them. The water exchange rate, i.e. the time be- 4.0 tween each new inflow of oxygen-rich water to Oxygen min ml/l 3.5 3.0 the basin, is controlled by vertical mixing with- 2.5 in the basin, which in turn is driven by the tides. 2.0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 We know that the vertical difference between Fish production (1000 tonnes/year) high and low tides increases as we move northwards along the Norwegian coast, from about

145x100//Kap20-fig06.eps Figure 6: Estimated aquaculture carrying capacity (tonnes 0.2 m on the coast of southern Norway to about per year) in the Counties of Hordaland, Møre og Romsdal 2.5 m in Finnmark. and Nordland, for a threshold basin with a mean depth of 70 m, if the oxygen level in the basin water is to be kept This is of great importance for the carrying 2 above 4 ml/l. The fjord has a surface area of 40 km and capacity of a threshold basin for fish farming. If the depth of the threshold is 30 m. we assume that the oxygen concentration of a fjord basin where there is fish farming activity in the summer of 0.5 to 1.0 m. This suggests should be no lower than 4 ml per litre, Figure 6 that, at current levels of production and distribu- can tell us that a given fjord basin in the County tion of fish farms, there is very little likelihood of Hordaland has no aquaculture carrying of identifiable changes taking place in the algae capacity, while a similar basin in Møre og situation in Norwegian fjord and coastal areas. Romsdal will have a carrying capacity of about This is in accordance with observations already 27,000 tonnes per year, while in Nordland it made in a number of aquaculture areas. In the would be around 70,000 tonnes per year. Differ- future, therefore, there will be room for a con- ences in carrying capacity between the north siderable increase in aquaculture activity, at and south of the country are thus related to least as far as alga production is concerned. For higher water exchange rates in fjord basins as example, given a permissible eight per cent in- we move north along the coast, and also to hy- crease in the production of algae, we could at drographic conditions outside the fjords (10). least quadruple Norway’s current production of farmed fish (12). Today, only a small proportion of fish farms are located over fjord basins. In the future, more of Oxygen in fjord basins them may come to be located in sites of this Once it has been decided what changes in the type, as a result of both increased production level of production of algae we can accept, the and greater competition for sea areas close to next question is: how does aquaculture affect the land. Many fjord basins are vulnerable to the deeper parts of the recipient? If the farms lie higher rates of addition of organic matter, par- above a threshold basin in a fjord or a sound, the ticularly in the southern part of the country. In basin will receive organic matter in the form of the future, therefore, it may become necessary waste feed and faeces, and possibly also algae to estimate the carrying capacity and to monitor from increased local production. This will raise oxygen concentrations in fjord basins.

Thematic area: The Environment 345 References Regulating the local environmental impact of extensive marine fish farming. II. The monitoring pro- (1) Anonymous 1993. Miljømål for norsk havbruk. gramme of MOM (Modelling - Ongrowing fish Rapport prosjektgruppe, v/ SFT. 17 pp. farms - Monitoring). Aquaculture. 194: 75–92. (2) Anonymous 1999. Miljømål for norsk opp- (8) Stigebrandt, A., Aure, J., Ervik, A. & Hansen, P.K. drettsnæring. Nye miljømål for perioden 1998– 2004. Regulating the local environmental impact of 2000. DN-rapport 199–1. 34 pp. intensive marine fish farming. III: A model for esti- (3) Holm, M. & M. Dalen. 2005. Miljøstatus for norsk mation of the holding capacity in the MOM system havbruk. Bellona rapport Nr.7–2003. 90 pp. (Modelling - Ongrowing fish farms - Monitoring). (4) Pearson, T.H. & R. Rosenberg. 1978. Mactobenthic Aquaculture, 234:239–261. succession in relation to organic enrichment and (9) NS 9410. 2000. Norsk standard Miljøovervåkning pollution of the marine environment. Oceanography av marine matfiskanlegg. Norges standardiserings- and Marine biology Annual review, 16: 229–311. forbund. 22 pp. (5) Perason, T.H. & Black. 2001. Environmental im- (10) Stigebrandt, A. 2001. FjordEnv. a water quality pacts if marine fish cage culture. In Black (ed) En- model for fjords and inshore waters. Gøteborg Uni- vironmental impacts of aquaculture. Sheffield versity, Earth Science Centre, Report C40. 41 pp. Academic Press. ISBN 0–8493–0501–2. 214 pp. (11) Aure, J. & A. Stigebrandt. 1990. Quantitative esti- (6) Ervik, A., P.K. Hansen, J. Aure, A. Stigebrandt, P. mation of the eutrofication effects of fish farming in Johannessen & T. Jahnsen. 1997. Regulating the lo- fjords. Aquaculture 90: 135–156. cal environmental impact of extensive marine fish (12) Ervik, A., J. Aure, H.R. Skjoldal & J. Alvsvåg. farming. I. The concept of MOM (Modelling - On- 2005. Konsekvensutredning av regionale mil- growing fish farms - Monitoring). Aquaculture 158: jøvirkninger av et framtidig økende fiskeoppdrett i 85–94. Norge. Rapport Havforskningsinstiuttet. 13pp. (7) Hansen, P., A. Ervik, M. Schaanning, P. Johannes- sen, J. Aure, T. Jahnsen & A. Stigebrandt. 2001.

346 Aquaculture Research: From Cage to Consumption Øystein Skaala1), Knut E. Jørstad1) and Reidar Borgstrøm2) 1)Institute of Marine Research, 2)Norwegian University of Life Sciences

Genetic Interactions

It has been well documented that escapes of farmed salmon have negative genetic, ecological and infectious consequences for wild Atlantic salmon stocks. Escaped farmed salmon produce viable offspring, and wild salmon differ genetically from domesticated salmon in terms of gene frequency and allele variation in neutral characteristics, as well as in complex characteristics that have important influences on survival and propagation in nature. Studies have also shown that the genetic influence of escaped farmed salmon reduces both survival rates in wild salmon stocks and the production of wild salmon. Important tasks for research include the development of methods for the identification of the sources of unreported escaped salmon. It is possible that the genetic influences of farmed salmon may also be exerted via other mechanisms than direct crossing, such as changes in the immune genes in wild salmon and other species, caused by infections and outbreaks of disease. These question are currently of great importance, since they concern not only salmon but also other species. A large proportion of the environmental monitoring centring on the aquaculture industry is still based on voluntary inputs, fragmented financing and fragmented efforts. The monitoring programme of national salmon fjords and rivers, whose contours we can now glimpse, will have a great need for a goal-oriented, coordinated research effort on the methodological side before it takes its final shape. The environmental challenges of cod farming are considerable. It is important to learn from salmon farming, where we have always lagged behind with essential environmental studies. Where cod are concerned, spawning in sea-cages, escapes and genetic interactions with wild stocks, disease and the spread of pathological organisms are all potential problems. Thematic area: The Environment 347 The Atlantic salmon, a species Stocks of anadrome species such as Atlantic with genetically differentiated salmon have geographically separated spawn- populations ing grounds, on which their young live for up to Genetic variation within a biological species is several years before they set out on their feeding distributed both within and between popula- migrations in the sea, in the course of which tions, but the pattern of distribution is not the several populations mix before returning to same in all species. In freshwater species, a rel- their own rivers to spawn. Given what we know atively high proportion of the variation is dis- about the influence of evolutionary forces on tributed between populations, because in such populations, and about migration in salmon, the species, stocks are often physically separate fact that we find significant genetic differences from each other, and migration rates between between individual stocks is not surprising. populations are low. Marine species live in more open systems, in which exchanges of indi- The past 35 years have seen the growth of a sig- viduals and genes between populations are nificant scientific literature on Atlantic salmon, more pronounced, which means that in marine a literature that has documented geographical species, a lower proportion of their genetic vari- separation, with important genetic differences ation is distributed among populations (1, 2). between North American and European popula-

145x100//Kap21-fig01.eps Figure 1: Regional genetic structure of European populations of Atlantic salmon, illustrated by plotting the genetic distance between populations, based on gene frequences in protein-coding genes (after 10). The seven regions are: 1: Iceland/ Greenland, 2: Northern Russia/Northern Norway, 3: Southern Norway/Western Sweden, 4: the Baltic Sea, 5: the Northern British Isles, 6: the Southern British Isles/Northern France, 7: Southern France/Spain. Both DNA microsatellite markers and protein-coding genes display sharp differentiations between populations, even within regions.

348 Aquaculture Research: From Cage to Consumption tions, and with regional and local differentia- aggression and the flight response. A number of tions within continental stocks (3–10). Such a studies have examined problems of this sort via pattern of distribution of genetic variation with- a wide range of methods for comparing domes- in a species enables several populations to de- ticated and wild salmon. Such methods com- velop somewhat different characteristics and prise studies of individual protein-coding adaptations via the continuous interplay of evo- genes, DNA mini- and microsatellites and phe- lutionary forces. notypical properties that are under the control of several different genes. Studies of protein- Demonstrating in scientific terms that individu- coding genes (20–23) have revealed genetic dif- al populations have different patterns of genetic ferentiation of domesticated salmon and the variation is no longer a problem. Showing that wild populations of origin, and a reduced level populations whose genetic variation is differ- of genetic variation measured in terms of allele ently distributed have local adaptations (11) that diversity and heterozygosity. A recent study of make them vulnerable to influences of escaped wild and farmed salmon in Norway found less domesticated salmon is a much bigger chal- genetic variation in the farmed salmon, mea- lenge (Figure 1). sured as number of alleles, heterozygosity and the number of variable loci (24). When we In the course of the past few years, the scientific study VNTR (variable-number-of-tandem- findings that have documented genetic differenc- repeats) loci, which usually have much more es between salmon populations have grown variation than protein-coding loci, we find a dramatically, primarily due to developments in larger reduction in the degree of genetic varia- molecular genetics and statistics. It has gradually tion in the farmed stock lines. Comparative also become generally accepted that the genetic studies of farmed and wild salmon in Ireland influence of escaped domesticated salmon (12, (25–27) found that farmed lines had 56 per cent 13) that have been selected for a number of of the alleles and 53 per cent of the mean specific characteristics, such as rapid growth and heterozygosity of three DNA minisatellite loci delayed sexual maturation, is capable of nega- compared with wild salmon. Another study of tively affecting wild salmon populations and variation in 15 microsatellite loci (28) in salmon reducing their survival and fitness (14–19). in Ireland showed that cohorts of farmed salmon had 52 and 80 per cent of the alleles and How different are wild and heterozygosity respectively of wild salmon. farmed salmon in reality? The genetic influence of escaped domesticated In one of our own projects, the five most impor- salmon depends on the number of escapees in tant breeding lines in Norway were compared the wild populations, as well as on the degree of with four wild salmon populations from the genetic differentiation between domesticated Rivers Neiden, Namsen, Vosso and Lone. The and wild salmon. Such genetic differentiation project first tested a large selection of relevant can occur both via founder effects in the domes- microsatellite markers, and then compared the ticated salmon, as a result of active selection in breeding lines and wild stocks in terms of 12 the breeding process, and also through the pas- markers. On average, the breeding lines had 58 sive selection of characteristics that do not form per cent of the allele variation in the wild salm- a direct part of the breeding programme, such as on samples (8). Not unexpectedly, considering

Thematic area: The Environment 349 Table 1: Observed genetic differences between wild and farmed salmon in neutral individual genes and fitness- related characteristics

Characteristic Observation References

Allozyme loci Reduction in genetic variation 20–24 Minisatellite loci Large reduction in genetic variation 25–27 Microsatellite loci Large reduction in genetic variation 8, 28 Growth rate Offspring of farmed fish grow faster than offspring of wild fish 16, 30, 31, 33 Aggression Offspring of farmed fish are more aggressive than offspring of wild fish 31 Predator response Reduced in offspring of farmed fish 31, 40 Growth hormone Higher content of growth hormone in farmed fish than in wild fish 18

founder effects and the high degree of isolation Finnmark and the Kola Peninsula belong to a between the breeding lines, measures of genetic separate sub-group (10). distance were several times as high between the breeding lines as in the wild stocks. In this We have been domesticating salmon in Norway project, the genetic data were also used in com- for 40 years, and the breeding programme was bination with assignment tests, which are statis- designed at an early date (29) to modify such tical tests for the identification of individual characteristics as growth rates, sexual matura- fish. Interestingly enough, the results showed tion, fat distribution and resistance to disease. that the DNA markers identified individuals Many characteristics that are not directly in- from specific breeding lines with a high degree cluded in the breeding programme, such as ag- of accuracy (93–97 per cent), and farmed and gression, stress tolerance and temperature wild salmon were separated by a margin of tolerance (30), can also be modified in farmed error of identification of three to four per cent. salmon via the domestication process. As a re- Naturally enough, since the differences be- sult of the programme of actively breeding for tween the wild stocks were smaller than we improved production characteristics, it is not found between the breeding lines, the degree of surprising that farmed salmon grow faster than accuracy of identification was somewhat lower wild salmon in both the farm environment (18, in the wild stocks. Nevertheless, there were 31, 32) and in the wild (16, 17, 31, 33, 34). great differences between the wild stocks. While individuals from Vosso and Namsen Selection for growth affects not only this mech- could be identified to an accuracy of about 67 anism but also components of hormone regula- per cent, the corresponding figure was no less tion and behaviour. Experimental studies have than 93 per cent for salmon from the River shown that administering growth hormone in- Neiden in Finnmark. This result supports creases appetite (34, 35), as well as aggression claims that salmon from Northern Norway be- and activity (36), i.e. behaviour that is related to long to a different sub-group than those from survival in the wild (35, 37, 38). It is thus not further south. Other studies of a larger body of surprising that farmed salmon differ from their material have since shown that salmon from wild counterparts in several characteristics that

350 Aquaculture Research: From Cage to Consumption affect their ability to survive in the wild, such as it has been shown that escaped Atlantic salmon growth, aggression, dominance and the ability are capable of producing viable offspring (45). to escape predators (18, 31, 39, 40). In order to determine whether Norwegian wild The consequences of genetic in- salmon stocks have changed genetically in the fluence of escaped salmon: what course of time as a result of the immigration of do the empirical data tell us? escaped farmed salmon, we created DNA pro- With the aid of various biochemical and molec- files of seven salmon stocks from the Rivers ular genetic methods, it has been shown that es- Namsen, Etne, Opo, Vosso, Granvin, Eio and caped farmed salmon spawn in rivers and that Hå. We studied old samples of fish scales as wild salmon stocks have been modified. Studies well as recently collected material, following a of a pigment found in salmon roe and fry that long period of immigration by escaped farmed reflects differences between the diets of wild salmon (46). In the River Hå in Jæren in South- and farmed salmon (41) have shown that es- western Norway, where there is almost no salm- capees do in fact produce viable offspring in on farming, and the proportion of escaped rivers. A Scottish study found pigment from salmon in the wild population has been very escaped salmon in 14 of 16 rivers studied, with low, probably less than 5 per cent, no changes in a mean contribution of 5.1 per cent coming genetic profiles were found. Significant chang- from escapees (42). In Norway’s River Vosso es in genetic profiles over time were found in the contribution made by escaped salmon was three other populations in the Rivers Opo, Vos- estimated at almost 80 per cent using this so and Eio in Hordaland. However, it was more method (13). Evidence that escaped salmon surprising that no changes were found in salm- produce viable offspring has also been found in on from the Rivers Etne, Namsen or Granvin, Ireland using genetic markers (26, 43, 44). One all of which have had high proportions of salm- study (26) found that escapees that migrated upon escapees in their spawning stocks, either per- river as juveniles survived and returned to the manently or for some periods of time. river to spawn when they were sexually mature. In British Columbia too, which is well beyond Although there is an extremely large literature the natural distribution area of Atlantic salmon, on population genetics theory and on the basic evolutionary forces (mutation, natural selec-

Table 2. Survival and lifetime success of the groups “wild”, “farmed” and “hybrid” of Atlantic salmon. The results are mean values of three year classes: the survival of the wild group was set at 1.0.

Group Eyed roe Eyed roe/smolt Smolt/sexually mature Lifetime

Wild 1.0 1.0 1.0 1.0 BC:Hybrid x Wild 1.0 0.89 1.0 1.0 Hybrid (wild female x farmed male) 1.0 0.73 0.58 0.58 Hybrid (wild male x farmed female) 0.87 0.50 0.61 0.33 BC: hybrid x farmed 1.0 0.79 0.39 0.62 Farmed 0.79 0.41 0.07 0.04

Thematic area: The Environment 351 tion, genetic drift and migration) that influence classes at the eyed roe stage, upriver of fish- and shape the genetic composition of popula- traps in Burrishoole, while equivalent groups tions, few empirical studies that have actually were set out as smolt. A major effort was put assessed the genetic effects of escaped farmed into collecting and genotyping fish, with the salmon crossing with wild salmon populations aim of identifying the ancestry of individual have been carried out. Until recently, there have fish, ranging from fry to spawners that returned been limitations in the methods available for after two and a half years in the sea. identifying the genetic effects of escapees, but with new molecular genetic methods such as In all three year classes, the farmed salmon DNA mini- and microsatellites for parent- were significantly underrepresented in compar- offspring identification, a quite new situation ison with the wild salmon in samples of 0+ has arisen, and good tools are now available for parr. Not surprisingly, it turned out that the studies of these effects. farmed salmon grew better than the wild salmon, and that the larger farmed parr displaced the An extremely direct and informative approach wild parr downriver by out-competing them. to this problem involves comparing the growth rates, behaviour and survival of well-defined Even though the farmed salmon grew better and family groups of farmed salmon, wild salmon displaced some of the juvenile wild salmon, the and hybrids in what are known as “common farmed salmon’s smolt production success rates garden” studies in a natural environment. This in the three years classes were only 34, 34 and involves either setting out salmon roe from 55 per cent respectively of that of the wild fish. well-defined and DNA-identifiable families of The average re-catch rate after a period in the farmed salmon, wild salmon and hybrids (16, sea was 0.5 per cent for the farmed salmon and 33), or setting out sexually mature individuals 8 per cent for the wild salmon. The hybrids of- with known genetic profiles (17) in a natural ten turned out to occupy an intermediate posi- river environment, where all their offspring at tion between the wild and farmed salmon. different life stages from roe to sexual maturity can subsequently be identified with the aid of A similar project was carried out in the River DNA markers. Imsa in Norway (17). This project set out sexually mature wild and farmed salmon with The most wide-ranging detailed project that has known genetic profiles upriver of the fish traps been carried out in this field was performed in in the Imsa. The two groups showed similar pat- Burrishoole in Ireland as part of a major EU- terns of migration and chose the same spawning financed project (16, 33, 47). The project grounds in the river, but the wild male salmon studied growth and survival rates and popula- were more active in their courtship of the hen tion dynamics in wild and farmed salmon and fish than the farmed salmon, and also had less hybrids through the F1 and F2 generations. A gonadal tissue left after spawning than the large number of individuals from several fami- farmed salmon had. The spawning success of lies of wild salmon, farmed salmon, F1 wild x farmed salmon was much lower in both males farmed hybrids, F2 wild x farmed hybrids, (24 per cent) and females (32 per cent) than back-crosses to wild fish and back-crosses to among the wild salmon. Throughout the fresh- farmed salmon, were planted out in three year water phase, the proportion of farmed salmon

352 Aquaculture Research: From Cage to Consumption genotypes gradually fell, and most of the were set out as eyed roe in the “common gar- farmed salmon genes were represented in the den” study could be identified to family level. form of hybrids, produced by farmed hen fish and wild males. During the project, juvenile salmon from all of the three year classes that had been set out were Dietary studies revealed a significant overlap in collected from their river habitat, and growth the choice of food components, showing that and survival rates could thus be calculated for there was competition between the farmed and each family. Since making representative col- wild salmon. Smolt production was 28 per cent lections of material is often a problem in field lower than expected on the basis of the amount studies, the emigration of smolt through the of roe, given the relationship that has been fish-traps, where DNA samples of all the smolt found in the Imsa between quantity of eggs and were gathered, represented a good measure- number of smolt (48). The farmed salmon smol- ment point that offered the possibility of ex- tified and emigrated earlier and at a lower age tremely high accuracy in making comparisons. than the wild salmon. Unlike the results of the So far, the results indicate a high percentage of Burrishoole project, the Imsa project found no survival of the eyed roe, and by the end of 2006, differences between the groups in survival rates genotyping will also have indicated growth and at the marine stage. survival rates in the individual family groups.

To date, only two “common garden” studies Another question that this study is attempting to have been carried out that have looked at the ef- answer, concerns what mechanisms lead to dif- fects of gene transport from escaped to wild ferences in the survival rates of domesticated salmon by comparing defined, identifiable and wild salmon. Do these include food compe- groups. This is a slim basis for making general- tition, or can the process be a matter of preda- isations, particularly since the two studies pro- tion mortality? (Figure 2) duced different results in some respects. One of our own projects is based on the Burrishoole A number of studies of resistance to the salmon set-up, in that it defined family groups of wild louse (Lepeophtheirus salmonis) in Atlantic and domesticated salmon, and hybrids of these salmon have been carried out in the course of two groups. At the same time as eggs from these the past few years. Large differences (up to 180 family groups were being set out in a natural per cent) in resistance to salmon louse infec- river habitat, upriver of the smolt trap in the tions in trout (Salmo trutta L.) have previously Guddal River in the Hardangerfjord, eggs were been found (49, 50). In salmon, it has already also being set at the Institute of Marine been shown that sensitivity to another parasite, Research’s station in Matre, as controls for Caligus elongatus, a close relative of the salm- hatching and growth rates and comparative on louse, has a heritability of 0.22 (51). Con- studies of resistance to infectious pancreatic trolled trials of infectiousness using salmon necrosis (IPN), furunculosis and salmon lice in from a range of wild populations and farmed wild and domesticated salmon. Since all the lines in the same tank have shown that they be- parent fish had been genotyped with microsatel- come infected by salmon lice to different de- lite DNA markers, all of the individuals that grees (Table 3, 52).

Thematic area: The Environment 353 145x100//Kap21-fig02.eps Figure 2: IPN infection experiments showed that mortality in salmon was related to smoltification rather than to group or size (65, 66). As expected, domesticated salmon grew significantly faster than wild salmon or hybrids. No difference in resistance to furunculosis or IPN was found between domesticated and wild salmon (65, 66).

It was thus a natural next step to try to deter- while to include selection for resistance to lice mine whether the heritability of resistance to in a breeding programme. lice is large enough to make it possible to reduce the salmon lice problem via a breeding Aquaculture of marine species programme. It would be particularly interesting and genetic effects on natural if we could find close relationships between populations MHC class II genes and sensitivity to salmon Escapes of farmed salmon and negative genetic lice, as this might enable us to improve the effi- effects on wild salmon have been a topic of dis- ciency of breeding by using genetic markers. cussion for several decades. It has only recently These questions were addressed in one of our been demonstrated that crossing wild and user-guided projects. Controlled trials of re- farmed salmon reduces the fitness of the wild peated salmon louse infections in tanks and sea- fish. However, problems of this sort are general cages showed that there was little correlation in nature, and this situation probably applies to between which individuals were most or least all species that are being adopted for aquacul- severely infected by the first, second or third in- ture. Marine species spend the whole of their fection (53). This project demonstrated that the life cycle in the sea, a situation that could rein- heritability of salmon lice tolerance is low. It is force the potential environmental impact of in- thus by no means certain that it would be worth- dustrial farming. The species that are most

354 Aquaculture Research: From Cage to Consumption Table 3. Comparison of salmon lice infections between three wild salmon populations and two lines of farmed salmon, 31 days following infection (52). In terms of relative amounts, the level in Dale salmon has been set at zero. There is a significant difference between salmon from the River Dale and the River Vosso (p = 0.019), and the difference between Dale salmon and farmed line 2 is also significant (p 0 0.037).

Population No. of lice Range Prevalence Relative amount

Vosso 23.4 (1.76) 8–59 100 29 Dale 20.6 (0.83) 7–54 100 0 Årdal 27.9 (0.99) 10–91 100 15 Oppdrettslinje-1 29.0 (0.87) 6–59 100 7 Oppdrettslinje-2 35.9 (1.40) 11–63 100 22 affected at this moment are conventionally escapes are likely to become a problem. The farmed cod and sea-ranched lobster, and a num- most vulnerable stocks will be the more local ber of studies have been carried out on both of coastal cod populations. These are currently at these species. a low level, and for this reason are more sensitive to genetic influences. Cod As with salmon, the most serious problems are In Norway, we have a relatively long tradition related to physical escapes from sea-cages, as of genetic studies of cod. Dag Møller was one we have already experienced. Behavioural dif- of the first to adopt genetic methods of studying ferences mean that cod are much more adept cod stocks. As early as the mid-1960s, large- than salmon at finding small holes in the net, scale genetic studies based on antibodies and and escaping through them. There is thus a blood proteins were being carried out. Genetic much greater potential for salmon escapes of differences between the main groups of coastal this type than we are used to with salmon. Fur- and yearling cod were demonstrated, and also thermore, sexually mature cod spawn naturally between coastal cod from different parts of the in sea-cages, and are therefore capable of re- coast. Subsequently, a number of studies based leasing large amounts of fertilised eggs into the on tissue proteins (allozymes) and performed wild. with the aid of a number of DNA methods were carried out. Some of these are described by Unwanted genetic factors resulting from es- Fjalestad et al in this volume. Møller’s earlier capes of farmed cod will also depend on the de- results concerning genetic differences between gree of genetic variation between the various the large oceanic stocks of yearling cod and of wild cod strains involved. If the genetic differ- coastal cod have largely been confirmed by ences between Norwegian cod stocks are small these recent studies. However, there are wide or insignificant, the problems of the genetic ef- variations within the latter group, and there is fects of escapes will not be so serious, at least no doubt that in this area we still lack more ba- not in the short run. As the volume of farmed sic knowledge. Most coastal cod migrate to cod increases, and their genetic make-up is only a limited extent, and fishing pressure has modified as a result of goal-oriented breeding, been heavy for many years. At the same time,

Thematic area: The Environment 355 0,30.3

0,20.2

0,10.1

Frequency of marker gene

0 1980 1985 1990 1995 2000 2005

145x100//Kap21-fig03.eps Figure 3: Frequency of the marker gene in cod sampled in Masfjorden between 1984 and 2004. The arrows show when the genetically marked cod were released.

these stocks are an important basis for much of used, for example, in several of the releases car- the pattern of human settlement on the coast. ried out by the PUSH programme in the early Trends in coastal cod stock development have 1990s (56, 57). These releases were subse- been so negative that the International Council quently evaluated at the end of the programme for the Exploration of the Sea (ICES) has re- (58). At that time, however, it was still too early commended a complete stop on fishing. It is to measure any genetic effects on subsequent these stocks that are likely to be most vulner- generations. We therefore gathered samples able to the negative genetic influences of from three release areas in order to compare the escaped farmed cod in the future. frequency of the genetic marker over a longer period of time. The longest time series was The potential problems of genetic interactions from Masfjorden in Hordaland, where the re- between farmed and wild cod were a matter of leases led, as had been expected, to a large in- discussion as early as the end of the 1980s. At crease in the frequency of the marker gene, that time, the question primarily concerned the although repeated sampling also showed a rapid major cod release programmes. The use of gen- decrease in the years following the releases etic marking was also proposed, and this led to (Figure 3). a programme that had the aim of developing a genetically marked strain of cod (55) for use in Samples obtained 10–12 years after the releases such studies. This fish had a gene marker that is revealed no demonstrable effect (59). A similar very rarely found in nature (approximately 1 in trend was found in Heimarkspollen in 10,000 individuals), and it can easily be identi- Austevoll, near Bergen, while there was a statis- fied by the particular pattern of bands produced tically significant increase in the marker gene by electrophoresis of the enzyme glucose phos- for six to eight years following releases in Øy- phate isomerase. Offspring of this strain were garden (60).

356 Aquaculture Research: From Cage to Consumption 145x100//Kap21-fig04.eps Figure 4: Survival of lobster larvae from wild roe-bearing lobsters (KW) and farmed roe lobsters (KR) raised together in the same environment. The larvae were identified to family level by microsatellite DNA identification. (Modified from Jørstad et al., 2004)

Lobster take undesirable effects on the environment, in- Escapes from farms have often been regarded as cluding genetic effects, into account. This is unintentional releases, but escapes of this sort made explicit, for example, in the Regulations to have also been compared with sea ranching and the Act, which set out requirements that brood- cultivation (salmon, trout) in freshwater. Where stock should be used in their native location. lobsters and scallops are concerned, the recent Sea-ranching Act has opened the way for com- Genetic studies of lobsters were also a product mercial sea ranching. The provisions of the Act of the PUSH sea-ranching programme. These

Thematic area: The Environment 357 studies were carried out on the island of Kvitsøy ed, and it is clear that escapes are capable of in the County of Rogaland, and a number of having negative genetic and ecological effects, studies that aimed to survey the population gen- as well as negative effects on infections. For this etics structure of lobster in both Norway and the reason, it is an explicit goal of both the authori- rest of Europe were also implemented (for a ties and the aquaculture industry that the num- summary, see Fjalestad et al., this volume). In ber of escapes should be reduced. As a follow- connection with the large-scale release trials on up of the Report to the Storting no. 12 (2001– Kvitsøy (61, 62), genetic analyses of both the 2002), Protecting the Riches of the Sea, and local wild strain, the broodstock that had been Recommendation to the Storting no. 134 used, and the fry that had been released were in- (2002–2003) regarding the setting up of nation- corporated. This procedure was repeated in al- al salmon rivers and salmon fjords, the Ministry together three releases. Genetic comparisons of Fisheries and Coastal Affairs took the initia- revealed either no or negligible changes in the tive in 2003 to set up a national committee to course of the production process, nor did they study questions related to marking farmed demonstrate changes in stocks for the releases salmon. According to the Marking Committee (59). However, these studies were based on gen- (Norwegian Directorate of Fisheries, 2004), etic changes in allozymes, and a new series of there is a great deal of uncertainty as to just how comparisons based on more accurate microsat- many salmon actually escape from Norwegian ellite DNA analyses have now been started. fish farms. Where do these escapees come from, and what are the relative proportions of reported In connection with the lobster releases on and unreported escapes? How many escape Kvitsøy, more highly controlled studies were from smolt farms? also performed. The establishment of a local hatchery made it possible to carry out more The fisheries authorities are on the lookout for a studies that included genetic components. In means of identifying unreported escaped salm- connection with this programme, a test set-up on. In the course of the past few years it has was developed that was based on individual been shown that DNA microsatellites and statis- families (offspring of single roe-bearing lob- tical testing can often identify salmon with a sters). This set-up was also utilised in compari- high degree of accuracy (8, 54), but molecular sons of survival rates of the offspring of wild genetics is developing rapidly and the accuracy and farmed lobsters, in which larvae from sev- of established methods could still be improved, eral families were mixed in the same tank, and while new methods are also being introduced. were later identified to family group by means One current task for research is to determine of microsatellite marker DNA analyses. The re- whether escapees can be identified with the aid sults demonstrated that in the larval phase, the of the salmon’s own natural characteristics, offspring of wild lobsters had the highest sur- such as DNA profiles, fatty acid profiles, trace vival rates (Figure 4, 64). elements and isotopes.

Needs for research Another important task for research is the de- Salmon velopment and testing of methods for monitor- The negative environmental consequences of ing the genetic makeup of wild salmon stocks. escapes of farmed fish are now well document- For the most part, the DNA markers that have

358 Aquaculture Research: From Cage to Consumption 145x100//Kap21-fig05.eps Figure 5: The Atlantic salmon is important both for the Norwegian economy and for many local communities. The vitality of wild salmon stocks depends on a good interaction between nature and culture. Important tasks include reducing escapes and establishing a professional national programme for monitoring the environmental effects of aquaculture. Strong wild salmon populations are a positive element in the marketing of Norwegian nature and Norwegian aquaculture. (Photo: Svein I. Opdal).

Thematic area: The Environment 359 been utilised to investigate the genetic impact of Cod domesticated salmon on wild fish are selective- The environmental challenges of cod farming ly neutral. It is possible that the influence makes are considerable. It is important to learn from itself evident faster than the neutral characteris- salmon farming, where we have always been tics can register, and that the genetic impact can behindhand with essential environmental stud- also take place via mechanisms that have been ies. Where cod are concerned, spawning in sea- little discussed so far. Recent studies suggest cages, escapes and genetic interactions with that the genetic impact of farmed salmon is ca- wild stocks, disease and the spread of patholog- pable of taking place via other mechanisms than ical organisms are all potential problems. direct crossing. Immune genes in local stocks of wild salmon can probably be altered through The material from Øygarden was based on wild the introduction of infections and outbreaks of fish that had been collected as broodfish, and in- fish diseases in cultivated fish. These recently cluded a small number of cod with the genetic presented results also suggest that the immune marker (60). These fish were the point of depar- genes in other species such as trout (Salmo ture for the re-establishment of the genetically trutta L.) can be altered through similar mecha- marked strain of cod. A large number of fish nisms. These are potential environmental ef- were produced at IMR’s Aquaculture Research fects, about which we need more knowledge. Station in Austevoll in 2003 and 2004, and these cod are currently being ongrown to be- For easily understandable reasons, there is an- come broodstock. This process will enable us to nual large-scale monitoring of our important carry out a number of important studies of ge- productive ocean regions, and of the major fish netic interactions between escaped farmed cod stocks in these areas, with quality assurance of and wild cod. Both small-scale and large-scale the data obtained. A large proportion of the en- studies of how spawning in sea-cages and es- vironmental monitoring centring on the aqua- capes of farmed cod affect local stocks can be culture industry is still based on voluntary carried out. This is extremely important for as- inputs, fragmented financing and fragmented sessing the risks of undesirable genetic effects efforts. The monitoring programme of national on wild cod, and not least, for generating the salmon fjords and rivers, whose contours we knowledge required for the development of can now glimpse, will have a great need for a commercial cod farming with minimal impact goal-oriented, coordinated research effort on on the environment. An experiment based on the methodological side before it takes its final spawning in sea-cages, which is currently at the shape. planning stage, can be carried out in the course of 2006.

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362 Aquaculture Research: From Cage to Consumption 45) Volpe, J.P., Taylor, E.B., Rimmer, D.W. & Glick- 56) Jørstad, K.E., Paulsen, O.I., Nævdal, G. and man, B.W. (2000) Natural reproduction of aquacul- Thorkildsen, S., 1994. Genetic studies of cod, ture escaped Atlantic salmon (Salmo salar) in a Gadus morhua L., in Masfjord, western Norway: coastal British Columbia river. Conservation comparisons between the local stock and realed, Biology, 14, 899–903. artificially reared cod. Aquaculture and Fisheries 46) Skaala, Ø.; Wennevik, V., Glover, K.A., 2006. Evi- Management, 25 (Supplement 1), 77–91. dence of temporal genetic change in wild Atlantic 57) Jørstad, K.E., G. Nævdal, O.I. Paulsen and S. salmon (Salmo salar L) populations affected by Torkildsen, 1994. Release and recapture of geneti- farmed escapees. ICES J. Marine Science. cally tagged cod fry in a Norwegian fjord system. 47) Ferguson, A., McGinnity, P., Baker, N., Cotter, D., In: Beaumont, A.R. (ed.) Genetics and Evolution of Hynes, R. O’Hara, B., O’Maoileidigh, N., Prodöhl, Aquatic Organisms, pp. 519–528. P, Rogan, G., 2002. A two-generation experiment 58) Jørstad, K.E., Ø. Skaala and G. Nævdal, 1999. comparing the fitness and life-history traits of na- Genetic diversity and the Norwegian Sea Ranching tive, ranched, non-native, farmed, and hybrid Programme: a retrospective perspective. In: Stock Atlantic salmon under natural conditions. ICES CM Enhancement and Sea Ranching (eds. Howell, B., 2002/T:04. Moksness, E., & Svåsand, T). Fishing News Books, 48) Jonsson, N., Jonsson, B. & Hansen, L.P. (1998) The Blackwell Science Oxford, UK. relative role of density-dependent and density- 59) Jørstad, K.E., 2004. Genetic studies in marine stock independent survival in the life cycle of Atlantic enhancement in Norway. In: K. Leber, S. Kitada, salmon Salmo salar. Journal of Animal Ecology, H.L. Blankenship & T. Svåsand (eds.). Proceedings 67, 751–762. of Second International Symposium on Stock En- 49) Glover, K.A.; Nilsen, F.; Skaala, Ø; Taggart, J.B; hancement and Sea Ranching. Blackwell Science Teale, A., 2001. Differences in susceptibility to sea Ltd., Oxford lice infection between a sea run and a freshwater 60) Jørstad, K.E., Nævdal, G., Karlsen, Ø., Torkildsen, resident population of brown trout. J. Fish Biol., 59, S., Paulsen, O.I., Otterå, H., 2004. Long term stud- 1512–1519. ies on genetic interaction between wild and ranched 50) Glover, K.A., Skaala, Ø.; Nilsen, F.; Olsen, R.; cod (Gadus morhua) by use of a genetic marked Teale, A.J.; Taggart, J.B., 2003. Differing suscepti- strain. Fisheries Society of the British Isles Annual bility of anadromous brown trout (Salmo trutta L.) Symposium, 19–23 July 2004, Imperial, college, populations to salmon louse (Lepeophtheirus London. salmonis (Krøyer, 1837)) infection. ICES J. Marine 61) Agnalt, A.-L, van der Meeren, G.I., Jørstad, K.E., Science, 60, 1139–1148. Næss, H., Farestveit, E., Nøstvold, E., Svåsand, T., 51) Mustafa, A., MacKinnon, B.M., 1999. Genetic vari- Korsøen, E. and Ydstebø, L., 1999. Stock enhance- ability in susceptibility of Atlantic salmon to the sea ment of European lobster (Homarus gammarus): A louse Caligus elongatus Nordmann, 1832. Can. J. large scale experiment off south-western Norway Zool., 77, 1332–1335. (Kvitsøy). In: Stock Enhancement and Sea Ranch- 52) Glover, K.A.; Hamre, L.A.; Skaala, Ø.; Nilsen, F., ing (eds. Howell, B., Moksness, E., & Svåsand, T.) 2004. A comparison of sea louse (Lepeophtheirus pp. 401–-419. Fishing News Books, Blackwell Sci- salmonis) infection levels in farmed and wild Atlan- ence Oxford, UK. tic salmon (Salmo salar L.) stocks. Aquaculture, 62) Agnalt, A.-L., Jørstad, K.E., Nøstvold, E., Far- 232, 41–52. estveit, E., Næss, H., Kristiansen, T. and Paulsen, 53) Glover, K. A., Skaala, Ø., 2006. Temporal stability O.I., 2004. Long term lobster (Homarus gammarus) of sea louse (Lepeophtheirus salmonis Krøyer) pop- enhancement at Kvitsøy Islands – perspectives for ulations on Atlantic salmon (Salmo salar L.) of rebuilding Norwegian stocks. In: K. Leber, S. Kita- wild, farm and hybrid parentage. J. Fish Biol. (in da, H.L. Blankenship & T. Svåsand (eds.). Proceed- press). ings of Second International Symposium on Stock 54) Wennevik, V.; Skaala, Ø.; Glover, K., 2006. Tracing Enhancement and Sea Ranching. Blackwell Sci- escaped cultured salmon to farm of origin. Reviews ence Ltd., Oxford. in Fish Biology and Fisheries (in press). 63) Jørstad, K.E., Nøstvold, E., Kristiansen, T. and 55) Jørstad, K.E., Ø. Skaala and G. Dahle. (1991). The Agnalt, A-L., 2001. High survival and growth of development of biochemical and visible genetic European lobster juveniles (Homarus gammarus), markers and their potential use in evaluating inter- reared communally with natural bottom substrate. action between cultured and wild fish popula- Marine and Freshwater Research, 52,,1431–1438. tions.ICES mar. Sci. Symp., 192: 200–205.

Thematic area: The Environment 363 64) Jørstad, K.E., Prodohl, P.A., Kristiansen, T.S, 65) Glover, K.A.; Bergh, Ø.; Rudra, H.; Skaala, Ø., Hughes, M., Farestveit, E., Taggart, J.B., Agnalt, 2006 Juvenile growth and susceptibility to Aeromo- A.-L. and Ferguson, A., 2005. Communal larval nas salmonicida subsp. salmonicida in Atlantic rearing of European lobster (Homarus gammarus): salmon (Salmo salar L.) of farmed, hybrid and wild family identification by microsatellite DNA profil- parentage. Aquaculture (in press). ing and offspring fitness comparison. Aquaculture, 66)Glover, K.A.; Skår, C.; Christie, K.E.; Glette, J.; 247, 275–285. Rudra, H.; Skaala, Ø., 2006. Size-dependent susceptibility to infectious salmon anemia virus (ISAV) in Atlantic salmon (Salmo salar L.) of farm, hybrid and wild parentage. Aquaculture (in press).

364 Aquaculture Research: From Cage to Consumption Appendix

■ Members of the Aquaculture Programme Board

■ Publications

■ Projects Members of the Aquaculture Programme Board 01.01.1999–30.06.2006

Magny Thomassen, Professor, Norwegian Sigurd Stefansson, Professor, University of University of Life Sciences (chair) Bergen

Roar Gudding, Managing Director, National Guri Eggset, Professor Hedmark University Veterinary Institute College; deputy for Birgitta Norberg, 01.08.2000–31.07.2001 Leif Jørgensen, Senior Research Scientist, Nord-Trondelag Research Institute Arne Mikal Arnesen, Director of Research, Fis- keriforskning (Norwegian Institute of Fisheries Oddbjørn Larsen, Sales Director, Isoterm AS and Aquaculture Research); deputy for Sigurd Stefansson, 01.08.2003–31.07.2004 Morten Lund, Managing Director, Åsen Settefisk AS Observers: Kari Morvik, Head of Section, Directorate of Kjell Maroni, Director R&D Aquaculture, Nor- Fisheries, Region West wegian Seafood Federation; observer on behalf of the Norwegian Fishery and Aquaculture Birgitta Norberg, Professor, Institute of Marine Industry Research Fund Research Svein Hallbjørn Steien, Senior Adviser, Børre Robertsen, Professor, Norwegian College Innovation Norway of Fishery Science, University of Tromsø Anne-Marie Voll, Adviser, Ministry of Aud Skrudland, Senior Inspector, Norwegian Fisheries and Costal Affairs, until 31.12.2002 Food Safety Authority Rune Bildeng, Senior Adviser, Ministry of Fisheries and Coastal Affairs, from 01.01.2003

366 Aquaculture Research: From Cage to Consumption Publications

The programme board initiated several Norwegian-language reports, which have formed the basis for development of the programme and for the establishment of the new Aquaculture – An industry in growth programme.

“Farming of cod. Strategy for a coordinated ac- “Possibilities and challenges connected to tion from SND and the Research Council of breeding of marine species in culture” Norway, 2001–2010” Group appointed by the Research Council of Group appointed by the Norwegian Industrial Norway: and Regional Development Fund (SND, now Chair Gunnar Nævdal (University of Bergen), Svein- Innovation Norway) and the Research Council Erik Fevolden (Norwegian College of Fishery Science, University of Tromsø), Michael Møller Hansen (Danish of Norway: Institute For Fisheries Research), Odd Vangen (Norwe- Chair Leif Jørgensen (SINTEF), Karl-Petter Myklebust gian University of Life Sciences). (Nærøy Marin Yngel AS), Magny Thomassen (Norwe- The Research Council of Norway, December gian University of Life Sciences), Birgitta Norberg (In- stitute of Marine Research), Guri Eggset (Tromsø 2002. Research Park), Eva Borch Elvevold (SND), Rolf Sør- land (SND), Elin Erdal (SND), Rolf Giskeødegård (Re- “Status of technology in aquaculture” search Council of Norway). Secretary: Per Gunnar Project assigned to SINTEF: Kvenseth (KPMG/Norwegian Seafood Center). The Research Council of Norway/SND, March Project leader Leif Magne Sunde, Mats Augdal, Nils Hagen, Arne Fredheim, Eskil Forås and Øyvind Prest- 2001. vik (all of SINTEF). SINTEF Fisheries and Aquaculture, December An updated version of the report, June 2003: 2002. “Farming of cod. Strategy for a coordinated action from SND and the Research Council of Norway, 2001–2010” “Action Plan for Research and Development Chair Leif Jørgensen (Nord-Trondelag Research Insti- Activity on Feed Resources for Aquaculture” tute), Eva Borch Elvevoll (SND), Reidulf Juliussen Group appointed by the Research Council of (Fjord Marine AS), Atle Mortensen (Fiskeriforskning), Norway: Karl-Petter Myklebust (Nærøysund Matfisk AS), Birgitta Norberg (Institute of Marine Research), Svein Hallbjørn Chair Magny Thomassen (Norwegian University of Steien (SND). Observers: Rolf Giskeødegård (Research Life Sciences), Morten Lund (FHF/FHL Aquaculture), Council of Norway), Peder Jansen (Ministry of Fisheries Birgitta Norberg (Institute of Marine Research), Aud and Coastal Affairs). Secretary: Monica Stadsøy. Skrudland (Nordvest Fiskehelse AS), Svein Hallbjørn Steien (SND), Hans-Petter Næs (Norwegian Seafood Export Council), Grete Skrede (MATFORSK). “Feed and feed resources are the main chal- The Research Council of Norway, July 2003. lenge for growth in Norwegian aquaculture” Group appointed by the Research Council of Norway: Rune Waagbø (NIFES), Ole J. Torrisen (Institute of Ma- rine Research) and Erland Austreng (Akvaforsk). The Research Council of Norway, June 2001.

367 “Health and disease problems in Norwegian “Aquaculture 2020: Transcending the Bounda- aquaculture species. Review, discussion and ries – as long as…” suggestions of research priorities.” Magny Thomassen (Norwegian University of Group appointed by the Research Council of Life Sciences) participated in the steering com- Norway: mittee for the foresight project “Aquaculture Chair Øystein Evensen (Norwegian School of Veteri- 2020”. nary Science), Olav Breck (Marine Harvest Norway AS), Brit Hjeltnes (National Veterinary Institute), Frank Nilsen (Institute of Marine Research), Merete Bjørgan Schrøder (Norwegian College of Fishery Science, Uni- versity of Tromsø). Secretary: Tore Håstein (National Veterinary Institute). The Research Council of Norway, April 2004.

Projects Three hundred and twenty-nine R&D projects were carried out under the Aquaculture programme during the period 2000-2005. Information on these projects is available (primarily in Norwegian) in the project database. The database is accessible via the Norwegian homepage of the HAVBRUK (AQUACULTURE) programme: http://www.rcn.no/havbruk. Click on the link “Projektoversikt” on the left-hand side.

368 Aquaculture Research: From Cage to Consumption Norway is one of the world’s largest producers This book is part of the final report from the of Atlantic salmon; much effort is going into the research programme Aquaculture – Production production of more species as well. Norway is of aquatic organisms and summarises research also home to a sizeable aquaculture equipment findings from the period 2000 – 2005. industry that supplies goods and services to the domestic and international markets. Norwegian During this period, the Aquaculture programme aquaculture represents tremendous assets in allocated over NOK 470 million to nearly 400 terms of exports, jobs and maintenance of R&D projects at research institutes, universities, coastal settlement patterns. university colleges, companies and industry institutions. Research and development, along with the optimism and entrepreneurial spirit of many companies, have been key elements in the success of Norway’s aquaculture industry. These will also be essential to future efforts to make the most of new opportunities and promote the sustainable growth and advancement of the industry.

P.O.Box 2700 St. Hanshaugen NO–0131 OSLO Telephone: +47 22 03 70 00 Telefax: +47 22 03 70 01 [email protected] ISBN 978-82-12-02408-3 (printed version) www.forskningsradet.no/english ISBN 978-82-12-02409-0 (pdf)

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