Vol 6 Issue 2 2018 Hatcheryfeed Advances in feeding early life stage and broodstock aquatic species

Do you know your microalgae?

Trends in micro diets for fish larvae

Essential nutrients and their crucial effects on reproductive performance of fish broodstock

Effects of enriched and non-enriched Artemia on jaw malformation in golden pompano larvae

Enrichment strategies in the early larval rearing of marine fish become a long-term investment for farmers in grow-out 2

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VOL 6 ISSUE 2 2018

Contents

• News Review Replacing live feeds with formulated feed or minced fish improves survival of mud crab megalopae - New salmon hatchery for Marine Harvest in Scotland - BioMar increases fry and RAS feed capacity at mill 5 in Denmark - Scientists make major breakthrough in omega-3 12 production - Breeding for faster-growing bluegills and yellow perch - INVE launches Natura diet range for early-stage marine fish larvae

• Trends in micro diets for fish larvae 12

• Do you know your microalgae? 17

• Essential nutrients and their crucial effects on 20 17 reproductive performance of fish broodstock

• Enrichment strategies in the early larval rearing of marine fish become a long-term investment 26 for farmers in grow-out

• Effects of enriched and non-enriched Artemia on 30 20 jaw malformation in golden pompano larvae • New research developments in aquatic animal 36 larval feeding and nutrition—recent literature

• Calendar of events 38

26 * Cover : Valentin Thépot, James Cook University, Australia. 4

VOL 6 ISSUE 2 2018

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Aliga 21

Aquaculture without Frontiers 4

Aquafeed.com 40

Aquafeed Workshop Mexico 29

Frozen Ocean 16

INVE Aquaculture 2

Lallemand Animal Nutrition 27

Megasupply 11

Reed Mariculture 7

Skretting 10 CONTACT US SUBSCRIBE Sparos 9 Editorial: Digital editions are free to industry. [email protected] You may also purchase print copies. Advertising: [email protected] SUBSCRIBE Technical feed consulting: [email protected] Hatcheryfeed magazine DOWNLOAD YOUR COPY is published by Hatcheryfeed, a division of Aquafeed.com LLC. Aquafeed.com, LLC., Kailua, Hawaii 96734, USA. www.aquafeed.com. ©2018 all rights reserved. Copyright & Disclaimer. www.hatcheryfeed.com. 5

NEWS REVIEW

Replacing live feeds with formulated feed or minced fish improves survival of mud crab megalopae

Mud crab farming has long been established in the Philippines. Scylla serrata, S. tranquebarica and S. olivacea are the common species found in the Philippines; S. serrata is the preferred species for farming by crab growers.

According to Southeast Asian Fisheries Development Center (SEAFDEC), in 2015 the total production of mud crab from aquaculture was estimated at 13,720 tons, valued at US$77,025,000 and 14,437 tons valued at US$86,511,000 in 2009 and 2010, respectively.

Some 13 mud crab hatcheries have been established in the Philippines, nine of which are in the private The use of antibiotics as treatment percent. sector, two are government owned for Vibrio spp. has been shown to The improvement was realized and a further two are at state improve larval survival. However, under a project entitled universities and colleges. A the use of antibiotics can lead to “Improvement of Larval Rearing prototype commercial-scale the development of resistant Protocol,” which is implemented by hatchery produces about 40,000 strains of bacteria and abnormali- the Aquaculture Department, crab instars per run. However, ties that become apparent at the Southeast Asian Fisheries Develop- more hatcheries are needed since juvenile stage. ment Center (SEAFDEC/AQD). farms still depend on diminishing By replacing rotifers and artemia in wild seed stock and are experienc- The project is part of the National the diet with formulated feed or ing low survival rate during the Mud Crab Science and Technology minced fish or mussel, the survival megalopae stage due to Molt (S&T) Program, which is being rate of mud crab from zoea to Death Syndrome (MDS), a disease monitored by the Philippine megalopae has significantly due to luminescent bacteria (Vibrio Council for Agriculture, Aquatic and improved from 3.75 percent to spp.) in which zoea 5 has difficulty Natural Resources Research and 22.5 percent, while the survival molting to megalopa and diseases Development of DOST (DOST- rate from zoea to crab instar has due to bacterial and fungal PCAARRD). improved from 1.0-3.5 percent to 6 infection in eggs and larvae. 6

BioMar increases fry and RAS feed capacity at mill in Denmark

BioMar Group is increasing capacity future demands from the custom- recognized for its products as well at its feedmill in Denmark with the ers.” as expertise within this segment. addition of a new production line. BioMar states that the new line The new line is expected to be The new line will be highly enables a significant step forward ready in Q2 2019 with a budget of specialized and dedicated to fry in developing RAS feed with even DKK 90 million (approx. and RAS feed production, enabling higher growth performance and US$14,420,927). The feedmill in the factory to increase flexibility, physical as well as nutritional Denmark will, after the capacity quality and production perfor- quality. expansion, be able to deliver mance. 150.000 tons of feed per year. It is “We are already recognized in the “BioMar has experienced a solid BioMar’s largest production unit in market for the high quality and growth in market share on our core Europe outside the salmon performance of our fry and RAS markets in Europe and the factory markets. The factory is designed feed from the factory in Denmark in Brande has through the last for agility with a flexible production and the factory is often delivering years been expanding capacity by set-up. It delivers mainly feed for feed to support other business removing bottle necks and species such trout, eel, sturgeon units,” added Diaz. “This is simply a optimizing operational processes,” and salmon in RAS; however, in step making sure we stay in front, explained Carlos Diaz, CEO BioMar total the factory produces feed for delivering a market leading Group. “We foresee that the more than 40 species. customer value proposition in growth will continue and we need terms of agility, quality and The factory has been a pioneer to take a significant leap forward to performance.” within RAS feed and is highly make sure we can deliver on the 7

New salmon hatchery for Marine Harvest in Scotland

Marine Harvest opened a new Ben Hadfield, Managing hatchery June 1, 2018, which Director of Marine Harvest will grow up to 12 million fish (Scotland) Ltd, said: “This new a year and help meet the hatchery is a much needed global demand for Scottish facility which will allow us to farmed salmon. The new supply our expanding network £26.5 million hatchery at of sea farms across the Inchmore in Glenmoriston western Highlands and Islands replaces a much smaller and meet the increasing hatchery which had been on demand for Scottish salmon the site more than 38 years, technically advanced water from across the world”. expanding the workforce from 5 to recirculation systems in the world, The new hatchery is the latest 18. which cuts down the amount of stage in Marine Harvest’s water required while ensuring the The hatchery team will grow expansion plans which includes the water quality, the new facility is salmon from eyed eggs into smolts, creation of new open sea fish farms almost identical to the Lochailort fry and parr which will then be in various locations in the Minch. hatchery opened by Marine transferred to the company’s 49 Farms have already opened off Harvest in 2013. It will allow seawater fish farms and 5 Barra, Uist and Muck with a new Marine Harvest to stock all their freshwater loch sites to be grown site off the Isle of Rum receiving Scottish farms with fish they have into mature fish. planning permission earlier this grown themselves from eggs. year. Featuring one of the most 8

Scientists make major breakthrough in omega-3 production

A major discovery that could after receiving a fellowship from surprising to us to see just how revolutionize the understanding of the Marine Alliance for Science and widespread these genes were, omega-3 production in the ocean Technology Scotland (MASTS). particularly in animals that are so has been made by an international common and abundant in the sea. Dr. Kabeya said: “Since inverte- team of scientists. brates represent a major compo- “It is also intriguing that these Led by the University of Stirling, nent of the biomass in aquatic genes seem to be jumping between Scotland, research has found for ecosystems such as coral reefs, very different organisms, such as the first time that omega-3 fatty abyssal plains and hydrothermal from plants or fungi into an insect acids can be created by many vents, their contribution to the and a spring-tail, by a process of invertebrates inhabiting marine overall omega-3 production is likely horizontal gene transfer. This has ecosystems, including corals, to be remarkable.” been a controversial idea, that worms and molluscs. genes can move around in this way, The research also involved Stirling’s but our data looks rather convinc- The breakthrough challenges the Professor Douglas Tocher, and ing that these genes have done this generally held principle that marine members of an international in at least some of these species." microbes, such as microalgae and consortium of scientists, including bacteria, are responsible for Dr. David Ferrier, of the Scottish “These findings can revolutionize virtually all primary production of Oceans Institute at the University our understanding of omega-3 long omega-3. of St Andrews; Dr. Filipe Castro, of -chain polyunsaturated fatty acids the Interdisciplinary Centre of production on a global scale,” Dr. Lead scientist Dr. Oscar Monroig, of Marine and Environmental Monroig added. the Institute of Aquaculture, said Research (CIIMAR) - University of that the findings strongly suggest The paper, Genes for de novo Porto; the Spanish National that aquatic invertebrates may biosynthesis of omega-3 polyun- Research Council; the Australian make “a very significant contribu- saturated fatty acids are wide- Institute of Marine Science; and tion to global omega-3 produc- spread in animals, was funded by Deakin University. tion”. MASTS and the European Union’s Dr. Ferrier said: “It was very FP7 funding program. “Our study provides a significant paradigm shift, as it demonstrates that a large variety of invertebrate animals, including corals, rotifers, molluscs, polychaetes and crustaceans, possess enzymes called ‘desaturases’ of a type that enable them to produce omega-3, an ability thought to exist almost exclusively in marine microbes,” Dr. Monroig explained.

First author of the study, Dr. Naoki Kabeya, of Tokyo University of Dr. Oscar Monroig, of the University of Stirling, said the research suggests that Marine Science and Technology, aquatic invertebrates may make a "very significant contribution to global omega visited the Institute of Aquaculture -3 production." 9

Breeding for faster-growing bluegills and yellow perch

Hanping Wang, who manages The It seems there might be a downside Ohio State University’s Ohio Center to unnaturally mating fish, but for Aquaculture Research and Wang says that’s not the case. The Development, has succeeded in practice of mating females raising faster-growing fish by together or males together might artificially mating them in a not so be unusual but does not produce typical way. Inside cool water-filled problem fish – that is, assuming no tanks in southern Ohio, the laws of relatives are mated with each nature are being defied: female other, Wang said. That allowed the females to mate yellow perch mate with other with other female yellow perch. It Among yellow perch, the females female yellow perch; male bluegills might sound odd, perhaps, but it grow 60 to 70 percent faster than with other male bluegills. On worked. The results were “neo- the males, and they grow larger average, the resulting offspring males,” or “pseudo-males,” as than the males. As a result, it reach market size six months faster Wang calls them. makes sense that a breeder would than bluegills or yellow perch bred want to produce the fastest- The offspring produced by the out of standard male-female growing female yellow perch. So mating of a neo-male with a mating. That’s because among Wang did exactly that. He mated standard female yellow perch were yellow perch, females grow quicker females to females with the help of all females, since there was no Y than males; among bluegills, males grants from the U.S. Department of chromosome in the mix. And the faster than females. Agriculture and the state-based female offspring grew as expected, “We’re using the animals’ Ohio Sea Grant program, which 60 to 70 percent faster than any maximum potential to make them funds research in the Great Lakes female offspring born out of the grow faster for human benefit,” and aquaculture. standard arrangement of a male Wang said. “We have to do it this and female mating with each other. While they remained females at way to meet the growing need for the chromosome level — pos- On average, it takes a farmer 16 food, specifically protein. You need sessing the XX chromosome pair as months to raise a yellow perch to to have a process to produce more opposed to the XY chromosome reach market size. Now it can take animals – more chickens, cows, pair that typical males have — they as little as 10 months if neo-males pigs and fish.” still were able to produce sperm. are mated with typical female 10

yellow perch, Wang said. the three sites, and on average, the breeding program is a key improved fish grew 35 percent accomplishment of Wang’s “The farmer saves on labor, saves faster than the unimproved fish, research team: Completing on feed and saves on space,” he meaning the ones whose parents genome sequencing of both yellow said. came together naturally without perch and bluegills. By changing With bluegills, the males grow any special mating arrangements. the genetic makeup of the fish, faster and bigger than the females. Not only do they grow 35 percent researchers can select for high So, Wang took males and mated faster, they have a higher survival disease resistance and larger, them with males through a process rate, 20 percent higher. Whether faster-growing fish, Wang said. similar to what was done with the bluegills and yellow perch can be “We know long term it will have a yellow perch, so they became what made to grow even faster is huge impact.” Wang calls “neo-females.” The uncertain. “We don’t know,” Wang Wang has also authored a book on offspring of a neo-female bluegill said. “We’re working on that.” selective fish breeding that will be and a male bluegill were all male Critical to the selective fish available this summer. fish that could grow to 1 pound, the size needed to sell them, in about a year, cutting three to five months off the typical time needed for them to mature.

Along with mating females with females and males with males, Wang and his colleagues have conducted standard mating with yellow perch to generate the fastest-growing males and the fastest-growing females. They began with 800 yellow perch, 100 taken from eight states in the Midwest and Northeast, including Ohio. The DNA of the fish was analyzed, then the fish that were related were put in different tanks to prevent the possibility of them mating. Each fish was placed in one of a series of tanks with males and females, and they were allowed to mate as usual, males with females.

From the first round of fry, the scientists selected the 200 fastest- growing male and female fish from each cohort – then those pairs mated, and the same process occured over and over to get genetically improved fish. Across 11

NEW PRODUCT NEWS INVE launches Natura formulated to obtain satisfactory obtain feed acquisition and feed uptake. the most diet range for early - “Feed digestibility is of great optimal stage marine fish larvae importance, especially at the initial uptake of amino acids and peptides stages of larval development when in the initial stages of larval the digestive tract is under INVE Aquaculture has launched development. This together with development,” stated Alessandro ‘Natura’, a new diet range for early high quality n-3 HUFA inclusion Moretti, Product Manager Fish -stage marine fish larvae. The new promotes larval development, Hatcheries. “Larvae fed with formulation has been developed to juvenile growth, survival and Natura show superior acceptance simplify the weaning process and quality. of formulated feed.” provides key nutritional compo- For optimal ease of use, Natura nents for the initial stages of larval The Natura diet range has been diets can be fed daily over multiple development. developed using carefully selected rations according to the larval age, protein sources for the early stages The Natura diets are available in 4 fish density and water tempera- of the fish larvae. Not only proteins different sizes used from co- ture. They are ideally supplement- of sustainable marine origin are feeding to post-weaning. Natura is ed with enriched rotifers and/or used, but also adequate protein a floating, slowly sinking feed Artemia. sources in hydrolyzed form to 12

Trends in micro diets for fish larvae

By Wilson Pinto1*, Sofia Engrola2, Michael Viegas1, Jorge Dias1 and Luís Conceição1 1 Sparos Lda, Área Empresarial de Marim, 8700-221 Olhão, Portugal 2 Centre of Marine Sciences of Algarve (CCMAR), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal

Major research efforts conducted early stages. formulations that are attractive to by the industry and academia ensure a high ingestion; and highly Even if we know that fish larvae during the last decade have led to a digestible for an easy digestion, require diets with high levels of significant evolution in the quality that combine will avoid excessive protein, essential fatty acids and of commercial microdiets. This nutrient losses to the surrounding micronutrients, and that these evolution reflects not only in larval water by leaching and disaggrega- should be provided by highly growth performance and survival tion; 2) physical properties such as digestible ingredients, the exact during weaning, but also later in floatability, sinking speed, nutritional requirement are yet the production of high quality dispersion both in tank surface and poorly understood. Microdiet juveniles. Nevertheless, there is water column; and 3) tank design production technologies are also still room for substantial improve- and operation procedures that evolving day-by-day. The success of ments in microdiets for marine fish allow an easy cleaning of uneaten future microdiets depends on a set larvae, in particular for the very microdiets. It is also clear that early of technological factors like: 1) 13

nutrition has consequences during (e.g., Vibrio sp.) vectored by live- the end of the trial (58 DAH), larvae the larval, but also later in the feeds. Even if a complete replace- from the co-feeding treatment had juvenile stage, in terms of health ment of live-feeds by microdiets is a significantly higher weight than status, survival, skeletal deformities still considered an utopia for the larvae from the Control treatment. and growth performance. majority of cultured species, this is However, although not significantly work in progress. A recent trial different at 20 DAH (65- 70 %),

with Senegalese sole evaluated the survival in larvae from the co- Early weaning may bring effect of an early introduction of feeding treatment was lower (34 benefits microdiets in larval growth and %) than in larvae from the Control survival. Newly-hatched larvae treatment (46 %) at 58 DAH. These Marine fish larvae rearing live-feed were fed at mouth opening (2 days findings indicate that an early dependency and the age to start after hatching, DAH) with Artemia weaning with high quality weaning have also been gradually (Control) or through a combination microdiets may promote an earlier decreasing in the last years. of Artemia and microdiet (high maturation of the digestive system Beyond the benefits of off-the-shelf replacement co-feeding strategy). and unravel the growth potential of availability and nutritional Results showed that larvae from fish larvae, although currently this consistency, an early introduction the co-feeding treatment had a may occur at the expense of larval of microdiets may increase lower growth performance at early survival. Larvae failing to cope with production predictability, avoiding stage (20 DAH) than larvae fed on an early microdiet introduction will live-feed nutritional and availability Artemia alone (Figure 1). Following lead to mortalities, whereas fluctuations and will decrease the larval settling, larvae from both survival of the fittest larvae will introduction of pathogenic agents treatments were weaned and at prevail with large growth benefits.

Dry weight pelagic Dry weight end of phase (mg larva-1) trial (mg larva-1)

Live-Feed Co-Feeding Live-Feed Co-Feeding

Fig. 1. Dry weight of Solea senegalensis fed on live-feed only or using a co-feeding strategy with a high replacement of live- feeds by microdiets from mouth opening onwards. On the left, larval dry weight at the end of the pelagic phase (18 DAH). On the right, larval dry weight at the end of the trial (58 DAH). 14

Microdiet formulations high quality plant-based proteins or maturation and development of a mixture of all these ingredients the digestive tract, which may only should be species and (Figure 2). Making it even more be able to process complex stage of development- challenging for fish larval nutrition- proteins following a few weeks of specific ists, it is also very likely that a given sole development. species may require different

One of the factors contributing to a microdiet formulations at the higher microdiet success within last various developmental stages. Diets formulations need years was the increasing percep- Although initially surprising, a to be optimized tion that ingredient formulations closer look on human dietary should be adapted according to habits reminds us that a baby´s diet Even if several commercial inert requirements and preferences of changes several times during the microdiets have been shown to target species. Current knowledge first year of life. A study conducted provide good results in terms of on larval nutrition leave no choice by Canada et al. (2017) showed growth and survival for species but using an “educated guess” that Senegalese sole larvae (3-16 such as European seabass, gilthead strategy when developing a DAH) grow better when feeding on seabream, red seabream and olive formulation for a given species. a diet including high levels of a flounder, there is certainly still Namely, trials using different moderately hydrolysed protein room for improvement. Dietary ingredients as main protein source source, whereas after larval settling macro and micro-nutrient contents revealed that gilthead seabream a higher growth performance is may be optimized, as shown shows preference to a squid meal- obtained when larvae feed on a recently in a study were microdiets based diet, compared to a fishmeal diet composed by low levels of with two different protein/lipid or a mixture of high quality plant- hydrolysed proteins and mostly ratios were tested in both meagre based proteins, whereas Senega- with complex proteins (19-60 and gilthead seabream larvae lese sole is more eclectic, dealing DAH). The most likely explanation (Candeias-Mendes et al. 2016; better with fishmeal, a mixture of for these findings relates to the Castanho et al. 2016). This study

Relative Growth Rate Relative Growth Rate (% day-1) (% day-1)

7 15 6 12 5

4 9

3 6 2 3 1

0 0 Fishmeal Vegetable mix Squid meal Fishmeal Vegetable mix Squid meal

Fig. 2. Growth rates of Senegalese sole (left) and gilthead seabream (right) reared using microdiets based on ingredients from marine or plant origin. 15

As knowledge of larval Low level Medium level

nutrition progresses and High level newer technologies become available, better performing microdiets for fish will

become available. Frequency (%) Frequency

shows that both gilthead seabream and meagre larvae require a high protein/high lipid diet for an Grey Level optimal performance. Diets with lower lipid contents (i.e. 15-17% crude fat), which are currently used in commercial hatcheries, may X-raymicrosadiograph 16-color conversion for result in a lower growth in meagre. analysis In gilthead seabream, this scenario may also occur unless a very high Fig. 3. Analysis of vertebral bone density by X-ray microradiograph. A higher pixel protein diet is used. Moreover, frequency, indicating a higher density of bone, is observed in sole fed a diet diets containing high lipid contents containing an optimal phosphorus levels. did not affect liver histology or skeletal development in both species.

Also revealing how microdiet formulations still show room for improvement, a trial conducted with Senegalese sole larvae showed that dietary phosphorous levels can have a strong impact on larval development. In this scenario, high phosphorous levels led to a depression of larval growth, whereas optimal levels increased larval growth (ranging from 4.4 to 6.4 % day -1) and survival (from 60 to 81 %). In addition, optimal phosphorous levels also led to an increased Fig. 4. Analysis of vertebral growth by double staining with Alizarin Red S and vertebral bone density (Figure 3) Calcein. Vertebral growth is assessed by analysing the bone formation between and vertebral growth (Figure 4). first (Calcein) and second (Alizarin) stainings. 16

Future microdiets will (Argyrosomus regius). Aquaculture Senegalese sole larvae. Aquacul- Europe 16 - Food for Thought. ture 479, 273-284. bring improvements Edinburgh (Scotland), 20-23rd

As knowledge of larval nutrition September. progresses and newer technologies Castanho, S., Viegas, S., Candeias- ΩHF become available, better perform- Mendes, A., Pinto, W., Conceição, ing microdiets for fish will become L.E.C., Pousão-Ferreira, P. (2016). A available. This will mean faster high protein/high energy micodiet growing larvae, higher survival improves growth and survival in More information rates and better quality juveniles, larval gilthead seabream (Sparus with an early live-feed replacement aurata). Aquaculture Europe 16 - increasingly being a reality. Food for Thought. Edinburgh (Scotland), 20-23rd September. References Canada, P., Conceição, L.E.C., Mira, S., Teodósio, R., Fernandes, J.M.O., Candeias-Mendes, A., Viegas, S., Barrios, C., Millán, F., Pedroche, J., Castanho, S., Pinto, W., Pousão- Valente, L.M.P., Engrola, S., 2017. Ferreira, P., Conceição, L.E.C. Dietary protein complexity Wilson Pinto, Ph.D., Sparos Ltd., (2016). A microdiet designed for modulates growth, protein Portugal fast-growing fish larvae improves utilisation and the expression of E: [email protected] performance in meagre protein digestion-related genes in 17

Do you know your microalgae?

By Emil Rue, MSc Agro and Plant Sciences, Aliga Microalgae

Breakfast is the most important meal of the day, or at least so the saying goes. Simplified logic and whether true or not, the underlying message is quite clear: make sure that you get off to a good start. Rearing robust and high performing marine finfish larvae cultures with low The use of rotifers B. plicatilis and essential fatty acid profile and B. rotondiformis as well as brine nutritional value regardless of mortality rates requires shrimp Artemia as live prey for whether you cultivate your own skills and is a multi- marine fish larvae is today microalgae or purchase concentrat- commonplace since they are of ed paste from a third party. disciplinary field on its major importance in the larvae’s The most frequent microalgae own. Effects of early nutrition. However, these genera used by hatcheries as zooplankton naturally lack mistakes can amplify rotifers and Artemia feed are sufficient amounts of the essential Nannochloropsis, Isochrysis, throughout the fish’s n-3 HUFA’s, yet they are rich in Pavlova, Tetraselmis, Dunaliella, growth stages and linolenic and linoleic acid n-6 PUFA. Spirulina and Chlorella. Some of With the larvae’s inability to therefore it is important these are rich in DHA, some in EPA synthesize the linoleic acid into and others in proteins. Due to their that larvae are grown essential fatty acids 1, it is required different nutritional profile, a that the live prey is correctly and under good hatchery mixture of genera is normally fed sufficiently enriched with eicosa- practises and feed to give the best balance between pentaenoic (EPA), docosahexaenoic growth and fatty acid enrichment nutritiously during its first (DHA) and arachidonic acid (ARA)2. of the zooplankton. A role of Thus, it is pivotal that the microal- few weeks. thumb for live prey to be used as gae fed to zooplankton, either marine larvae feed is that the directly in its tanks or through DHA:EPA ratio be above 1, with a Green Water, has the necessary tad of ARA on top of it. It must 18

the essential EPA, DHA and ARA 3 4. fairly straightforward, but to get Our findings have shown that constant pH levels, temperature … fatty acid composition can results obtained under small scale levels and desired water quality, as change, not only when going laboratory conditions might not well as induce shifts and stress on easily be transferrable to a larger the culture for it to produce the from lab to larger scale scale production of the very same fatty acid levels described in the production, but also when strains. Many scientific studies scientific papers is often more parameters such as light, regarding induction of fatty acid challenging. production in different microalgae temperature and composition At our production facility in strains revolves around the northern Denmark we have within the growth media application and adjustments of managed to get these parameters changes. light, temperature, pH value, water under control by using LED light quality, salinity, growth media, and a software-controlled stresses and sudden shifts in temperature and pH sensor system however be remembered that live culture conditions. Under in our proprietary photobioreactor prey’s ability to absorb essential laboratory conditions it is possible systems. Additionally, we formu- fatty acids from different microal- to adjust and control these late our own artificial sea water gae genera varies widely between parameters to achieve the desired mainly in order to eliminate the prey types, therefore the feeding fatty acid composition owning to risk of waterborne pathogens and rates of microalgae for the the relatively small volumes that contaminants being brought in to zooplankton must be analysed and are used in experimental setups. our cultures via collected seawater. adjusted accordingly 2 . Applying these parameters to a However, in doing so we can also larger on-site production is often Several microalgae species are control exactly what is in our more challenging. To control the capable of producing high amounts growth media, as we know from strain selection and the nutrient of fatty acids (see table 1) including experience that the same strain of composition of the growth media is

Table 1: Effects on EPA and DHA content as percentage of total fatty acids in algae- fed live prey. Taken and modified from 1.

19

-229

3. Catelina Adarme-Vega, David K. Y. Lim, Matthew Timmins, Felicitas Vernen, Yan Li and Peer M. Schenk, 2012: Microalgal biofactories, a promising approach towards sustainable omega-3 fatty acid production. Microbial Cell Factories 11:96

4. Yubin Ma, Zhiyao Wang, Changjiang Yu, Yehu Yin and Gongke Zhou, 2014: Evaluation of the potential of 9 Nannochloropsis strains for biodiesel production. Microalgae production at Aliga Microalgae’s facilities in northern Denmark. Bioresource Technology 167, pp.503-509. microalgae can behave significantly larger scale production, but also 5. Elina Peltomaa, Matthew D. differently, even under slight when parameters such as light, Johnson and Sami J. Taipale, 2018: changes in the mineral composition temperature and composition Marine chryptophytes are great of the growth media. An important within the growth media changes. sources of EPA and DHA. Marine aspect also lies in choosing the Therefore, to ensure your Drugs, 16:3 right microalgae strains, as some microalgae cultures have the fatty strains, even though they are from acid profile you think they do, the same species, will produce profiling of your cultures on a ΩHF higher levels of n-3 HUFA’s than regularly basis is advisable as others 4. Finally, it is important to without it, it is difficult to know the remember that inducing fatty acid nutritional value your rotifer and More information formation in microalgae via stress Artemia provide your larvae. will most often be at the cost of growth, since the metabolic pathways in the cells will be References redirected into energy storage. It 1. Josianne G. Støttrup and Lesley must therefore be a balance A. McEvoy, 2003: Live feeds in between achieving the right fatty marine aquaculture, Blackwell acid composition and at the same Science Ltd, Oxford, UK. time maintaining acceptable culture growth of the microalgae. 2. John Sargent, Lesley McEvoy, Alicia Estevez, Gordon Bell, Michael Most hatcheries are today Emil Rue, MSc Agro. and Plant Bell, James Henderson and Douglas cultivating their own microalgae, to Sciences, Aliga Microalgae Tocher, 1999: Lipid nutrition of a greater or lesser extent. From our marine fish during early develop- E: [email protected] experience we have seen that the ment: current status and future fatty acid composition can change, www.aliga.dk directions. Aquaculture 179, pp.217 not only when going from lab to 20

Essential nutrients and their crucial effects on reproductive performance of fish broodstock

By Chandan Haldar, ICAR - Central Institute of Fisheries Education, Mumbai, India

Broodstock nutrition is indisputably reduction in feeding rate has been fecundity, has been used to one of the most poorly understood reported to cause an inhibition of determine egg quality, which is and researched areas of finfish gonadal maturation in several fish also affected by a nutritional nutrition. To a large extent, this has species, including goldfish, deficiency in broodstock diets. been due to the necessity of European seabass (Dicentrachus Fecundity is the total number of suitable indoor or outdoor culture labrax) and male Atlantic salmon eggs produced by each fish facilities for maintaining large (Salmo salar). In seabass, after six expressed either regarding eggs/ groups of adult fish and the months of feeding broodstock with spawn or eggs/body weight. consequent higher cost of running a half food ration, growth rates Reduced fecundity, reported in and conducting extended decreased and spawning time was several marine fish species, could broodstock feeding trials. However, delayed and eggs, as well as newly be caused either by the influence as in human and livestock hatched larvae, were smaller than of a nutrient imbalance on the nutrition, it is clear that the dietary those obtained from fish fed full brain-pituitary-gonad endocrine nutrient requirements of brood- rations. In female seabass, the system or by the restriction in the stock will be different from those detrimental effects of food availability of a biochemical of rapidly growing juvenile animals. restriction were associated with component for egg formation. Moreover, as in other animals, it is reduced plasma estradiol levels. The elevation of dietary lipid levels also clear that many of the However, the expression of the from 12% to 18% in broodstock deficiencies and problems GtH genes was not affected by diets for rabbitfish (Siganus encountered during the early food restriction in mature female guttatus) increased fecundity and rearing phases of newly hatched goldfish. hatching, although this effect could finfish larvae are directly related to also be related to a gradual the feeding regime (including increase in the dietary essential nutrient level and duration) of the Effects of nutrition on fatty acid content. Indeed, one of broodstock. fecundity of broodstock the major nutritional factors that fish has been found to significantly affect reproductive performance in Effects of food restriction Several methods have been fish is the dietary essential fatty Food restriction itself can seriously developed to assess the egg quality acid content. Fecundity in gilthead affect spawning success. A of fish. One of the parameters, seabream (Sparus aurata) was 21

found to significantly increase with This fact has been demonstrated in an increase in dietary n−3 HUFA mammals, where some leukotri- (polyunsaturated fatty acids with enes (LTB4) enhance the steroido- 20 or more carbon atoms, essential genic action of LH. for marine fish) levels up to 1.6 %, Since reduced fecundity was and similar results have been associated with high n−3 HUFA egg reported in other sparids. content, increased EFA content However, studies on the reproduc- alone should not be used as a tive performance of Nile tilapia criterion to assess the egg quality (Oreochromis niloticus), as of gilthead seabream broodstock. indicated by the number of females High dietary n−3 HUFA levels could that spawn, spawning frequency, affect the brain-pituitary-gonad number of fry per spawning and endocrine axis since both EPA and total fry production over a 24-week DHA have been found to reduce in period, show that the performance vitro the steroidogenic action of was much higher in fish fed a basal gonadotropin in the ovary of diet supplemented with soybean teleost fish. This is similar to oil (high in n−6 fatty acids, essential mammals where an increased for this fish species; and relatively dietary level of n−3 fatty acids low in fish fed a 5% cod liver oil delays the onset of puberty. supplemented diet (high in n−3 fatty acids). Fish fed the diet Other nutrients which have been containing cod liver oil showed the shown to affect fecundity include highest weight gain. vitamin E and ascorbic acid. An increase in dietary α-tocopherol Polyunsaturated fatty acids can levels up to 125 mg/kg resulted in also regulate eicosanoid produc- an improvement in fecundity of tion, particularly prostaglandins, gilthead seabream as expressed by which are involved in several the total number of eggs pro- reproductive processes, including duced/female and egg viability. the production of steroid hor- However, the reduced fecundity mones and gonadal development observed in broodstock fed a diet such as ovulation. Fish ovaries have deficient in α-tocopherol was not a high capacity to generate associated with reduced vitamin E eicosanoids, among them content of eggs, and only very high prostaglandin E (PGE) derived from dietary vitamin E levels (2020 mg/ cycloxygenase action and leukotri- kg) were found to increase egg α- enes LTB4 and LTB5 derived from tocopherol content. In other lipoxygenase action. Inhibitors of species such as turbot or Atlantic the latter enzyme reduced the salmon, vitamin E was mobilized gonadotropin-induced maturation from peripheral tissues during of European seabass oocytes, vitellogenesis although the plasma suggesting that products derived vitellogenin content was not from lipoxygenase action could also affected, suggesting that lipopro- be involved in oocyte maturation. teins may be involved in the 22

integrity after freeze-thawing. However, research did not find any effect of dietary fatty acids (n−3 and n−6 polyunsaturated fatty acids) on sperm freeze-thaw fertilizing ability, whereas low membrane cholesterol– phospholipid ratios were correlat- ed with a better sperm freezing resistance.

Another hypothesis to explain the beneficial effect of EPA and AA on fertilization rates has been proposed by several investigators. Rohu fish brooder on a farm in India Both EPA and AA are involved in cell-mediated functions and are transport of vitamin E during this significant increase in the serum precursors of eicosanoids. EPA is period. Vitamin C content of testosterone levels thus advancing known to be a precursor of rainbow trout eggs reflected the the time of spermiation in males prostaglandins (PG) from series III, content of this nutrient in the diet and induced maturation of whereas AA is a precursor of PG and was associated with improved females. from series II. In vitro AA, but not egg quality. Changes in the vitamin EPA or DHA, stimulates testicular C content of cod ovaries did not testosterone in goldfish testis significantly affect hatching rates. Effects of broodstock through its conversion to prosta- Dietary antioxidant requirements nutrition on fertilization glandin PGE. On the contrary, EPA increase during reproduction. This or DHA blocked the steroid genic may be related to the formation of Certain dietary nutrients also exert action of both arachidonic acid and oxygen radicals during steroid a marked effect on fertilization. PGE2. Both AA and EPA modulate hormone biosynthesis as observed Dietary eicosapentaenoic (EPA) and steroid genesis in the goldfish in higher vertebrates. For example, arachidonic acid (AA) levels show a testis. Thus, the timing of spermia- levels of antioxidant compounds correlation with fertilization rates tion may be delayed and subse- were correlated with progesterone in gilthead seabream broodstock. quently fertilization rates reduced levels in bovine corpus luteum Since sperm fatty acid composition by depressed steroid genesis suggesting the activation of depends upon the essential fatty caused by a broodstock EFA antioxidative mechanisms to cope acid content of broodstock diet in deficiency or imbalance. Moreover, with steroidogenesis dependent species such as rainbow trout and prostaglandins are also recognized oxyradical formation. European seabass, it is possible as important pheromones in some that sperm motility and in turn Dietary tryptophan, a precursor of teleost fish. Some PGs produced by fertilization would be affected. the neurotransmitter serotonin, female goldfish, such as PGFs, have Particularly in salmonids, where may positively affect gonad been shown to stimulate male cryopreservation of sperm is maturation in both males and sexual behavior and synchronize currently utilized, sperm fatty acid females. Supplementation of 0.1% male and female spawnings, thus composition could be a factor that tryptophan in the diets of ayu directly affecting them. The determines the membrane (Plecoglossus altivelis) resulted in a antioxidant function of vitamins C 23

and E can provide an important diet enriched with high-quality fish protective role for the sperm cells oil. Whereas the comparison during spermatogenesis and until between brackish water and “Several nutrients are fertilization by reducing the risk of seawater cod eggs showed that AA lipid peroxidation, which is and DHA/EPA contents in the PL essential for the normal detrimental for sperm motility. The fraction of eggs are positively development of the embryo, ascorbic acid concentration in the correlated with egg symmetry and and their optimum level in seminal fluid reflected the viability. These fatty acids play an concentration of this vitamin in the important structural role as broodstock diets improves egg broodstock diet, and it did not components of phospholipids in morphology and hatching affect semen quality at the fish bio membranes and are beginning of the spawning season.. associated with the membrane rates” However, a deficiency of ascorbic fluidity and correct physiological acid reduced sperm concentration functions for bound membrane and motility during the later part of enzymes and cell functions in the spawning period. marine fish. In some species, such formation and differentiation of as halibut (Hippoglossus hippoglos- immune cells. An increased retinol

sus), the n−3 PUFA concentration in the liver of turbot Effects of nutrition on (polyunsaturated fatty acids) are has been observed during the embryo development also regarded as major energy gonad maturation as the length of sources during early embryonic the day increased, whereas retinol Several nutrients are essential for development. Nevertheless, fatty content in gonads was reduced the normal development of the acid composition of fish egg lipids is during maturation. embryo, and their optimum level in not only determined by the Other dietary nutrients which have broodstock diets improves egg broodstock diet but is also related been found to affect the reproduc- morphology and hatching rates. to species and stock differences. tive performance of marine fish The percentage of morphologically Essential fatty acid requirements include dietary protein intake. For normal eggs (as a parameter to for sparids broodstock range example, a low protein– high- determine egg viability) has been between 1.5% and 2% n−3 HUFA in calorie diet caused a reduction in found to increase with an increase diet ., being higher than those red seabream reproduction. In in the n−3 HUFA levels in brood- determined for juveniles which another sparid, gilthead seabream, stock diets and incorporation of range between 0.5% and 0.8% n−3 a broodstock diet well balanced in these fatty acids into the eggs, thus HUFA in diet. These values are essential amino acids, improved indicating the importance of EFAs higher than the optimum essential vitellogenin synthesis. Moreover, for normal development of gilthead fatty acid levels determined for reduction of dietary protein levels seabream eggs and embryo. salmonids which are approximately from 51% to 34% together with an Gilthead seabream fed EFA around 1% n−3 HUFA. increase in dietary carbohydrate deficient diets also showed an Despite the fact that little is known levels from 10% to 32% reportedly increased number of lipid droplets about the vitamin A requirement reduced egg viability in seabass. in the egg as also reported in red during gonadal maturation and These diets have been shown to seabream. Improved egg quality spawning, it is considered cause alterations in GnRH release has been associated with higher important for embryo and larval in seabass broodstock during total n−3 fatty acids content in development due to its important spawning and plasma hormonal European seabass fed a pelleted role in bone development, retina levels of the gonadotropin GtH II, 24

Effects of broodstock nutrition on reproduction

Reproduction of animals can be affected by several influences at different stages of development, which are mediated by changes in the hypothalamic-pituitary-gonadal (HPG) axis. Gonadotropins constitute an important group of pituitary regulatory hormones which comprise luteinizing hormone (LH) and follicle- Catla brood fish before breeding stimulating hormone (FSH). The secretion of Gonadotropin the latter known to play an increase in survival 14 days after hormones is controlled by the essential role in oocyte maturation hatching. Increased n−3 HUFA hypothalamic gonadotropin- and ovulation. (particularly docosahexaenoic acid) releasing hormone (GnRH) pulse levels in broodstock diets were Research should also be directed to generator that is an integrator of reported to significantly enhance establishing the requirement of hormonal, metabolic and neural the weight of fish larvae and their pyridoxine (vitamin B6) in signals. Experimental data in resistance to osmotic shock. broodstock diets. Vitamin B6 is mammals (and observations in Similarly, increasing n−3 HUFA known to be important in the humans) revealed that early life levels in broodstock diets for synthesis of steroid hormones and exposures influence the develop- gilthead seabream significantly folic acid since its deficiency may ment and functioning of the HPG improved the percentage of live result in reduced cell division due from a programming perspective. larvae after yolk reabsorption. to impaired synthesis of DNA and Effects of nutrition of the reproduc- Moreover, growth, survival and RNA and it has a role in hatchability tive health of fish have not been swim bladder inflation in gilthead of eggs. Unfortunately, there is no studied until now. However, seabream larvae were improved information available on the effect studies conducted in other when fish oil was used instead of of other B vitamins on fish vertebrates could be very useful for soybean oil in broodstock diets. reproduction. further focus. They have shown However, excessive levels of n−3 that, at earlier stages of fetal

HUFA in broodstock diets (over 2%) development, the normal ontogeny Effects of broodstock caused yolk sac hypertrophy in of gonadal development and gilthead seabream larvae and a function can be disrupted by under nutrition on larval quality decrease in larval survival rates. nutrition or the influence of This is probably associated with an Few studies have been able to endocrine-disrupting compounds. increase in antioxidant nutrient show the improvement of seed Any effect of under nutrition on the requirement since an increase in quality through implementation of process of tissue differentiation, dietary α tocopherol levels from broodstock nutrition. Increasing gonad formation and the establish- 125 to 190 mg/kg prevented the lipid levels from 12% to 18% in ment of associated enzyme appearance of yolk sac hypertro- broodstock rabbitfish produced systems is likely to have a phy and larval mortality. large newly hatched larvae and an fundamental effect on the 25

subsequent function of these these larvae digestive features the global effect of nutrients on organs. Specifically, in female allowed to develop a compound developmental processes. fetuses, the onset of meiosis is diet that can be used from the Important information on genome delayed, whereas, in male fetuses, earliest larval stages. It is now and gene expression in now testosterone synthesis is increased possible to refine the nutritional available for several fish species as a result of enhanced testicular requirements of the fish larvae and and this information allows steroid genic enzyme activity. As identify the potential physiological undertaking genomic studies on evoked above, most of the effects of some particular fish development. Fish could then nutritional studies undertaken in nutrients. Indeed, it has been constitute an interesting model for this area have compared fasting to shown in upper vertebrates that studying the effect of nutrition on feeding conditions. It is then some nutrients, even at low dietary development processes in upper necessary to investigate the effects concentrations, may influence vertebrates. of different dietary components as morphogenesis particularly by proteins and other specific acting on bone differentiation. References nutrients, like HUFA and vitamins. These nutrients, such as vitamins Halver, J. and Hardy, R. (1989).The It is then well established in and HUFA, recognize nuclear vitamins J.E. Halver (Ed.), Fish mammals that the reproductive receptors which are transcription Nutrition, Academic Press, San Diego, performance can be influenced by factors for several genes involved USA, 32–111. factors acting during the earliest in tissue differentiation and Lee, C.S. and Donaldson, E.M. (2001). developmental periods; however, organogenesis. These nuclear Reproductive Biotechnology in Finfish much further work is required to receptors can link together and Aquaculture. Aquaculture 197:25–42. identify these critical developmen- form heterodimers, particularly Mercure, F. and Van Der Kraak, G. tal periods and the relationships with RXR. In consequence, HUFA (1995). Inhibition of gonadotropin‐ between developmental abnormal- and the oxidative balance of lipids, stimulated ovarian steroid production ities and adult reproductive itself regulated by vitamin C and E, by polyunsaturated fatty acids in performance. may act on retinoid pathways and, teleost fish. Lipids, 30:547-554. in the same way, molecular Stacey, N.E. and Goetz, F.W. (1982). pathways of vitamin A and D can Role of prostaglandins in fish Conclusion interact together. The formation of reproduction. Canadian Journal of nuclear receptor heterodimers Fisheries and Aquatic Sciences, 39:92- These last 20 years, most of the leads to cross-talks between 98. studies on fish larvae nutrition metabolic processes and then, may ΩHF aimed to develop a compound diet affect the quality of the future able to efficiently replace live prey juvenile fish. It is then possible to More information in the larvae feeding sequence. It orientate the metabolic pathways was hypothesized that fish larvae of the juvenile fish by appropriate lacked a functional digestive nutritional conditioning during the system. Consequently, the larval period. This conditioning functioning of the digestive tract offers the possibility to have fish during the larval period was mainly with an enhanced immunological examined and, unexpectedly, system and fish able to use revealed that larvae have a primary efficiently some vegetable diets. but efficient mode of digestion that Chandan Haldar For this, nutritional studies on fish progressively matures during larvae need to take into account E: [email protected] development. The knowledge of 26 PRODUCT PROFILE Enrichment strategies in the early larval rearing of marine fish become a long- term investment for farmers in grow-out

Why hatcheries should focus even more on early-stage nutrition

By Alessandro Moretti, Product Manager Fish Hatchery, Inve Aquaculture

Marine fish farming need to look respect, specialists at INVE Division - has always dedicated for continuous improvements to Aquaculture see a clear and direct special attention to fry quality and keep profitability and sustainability relation between optimized robustness as a key success factor high. Given the pressure for an hatchery protocols and the in aquaculture. From our vast intensifying production and a animals’ performance, growth and technical experience in the growing attention to cost- survival at the farm gate. industry, we can see clear effectiveness, fry quality and correlations between nutrition in As a recognized pioneer, reference performance measured during the the early larval rearing and and innovator in hatchery products whole cycle have proven to be one performance and robustness in the and protocols INVE Aquaculture – of the keys to success. In that grow-out. Benchmark’s Advanced Nutrition 27

More specifically, the use of optimized live feed and enrichment strategies and the feeding at specific moments play a crucial role. Hatcheries who want to meet the demands of today’s stringent requirements in grow-out, will need to rely on long-term solutions that are especially developed to support superior juvenile quality.

Early stimulation from 3 days post hatching onwards as rotifers and Artemia is consid- system can only be affected after ered to be imperative in order to metamorphosis is probably INVE Aquaculture scientists are obtain the best possible fry quality destined to be changed in the near continuously developing new and future fish performances and future. INVE researchers have now solutions, enrichment formulations robustness. proven that lasting positive effects and easy-to-use protocols to of stimulation can already be improve fry quality. Optimizing the The assumption that key factors induced in the first month of the nutritional value of live feed such such as the animals’ immune 28

A new, complete and suitable for fast growing marine fish larvae and tropical species. smart approach to live Completing the nutritional balance This product is a dry feed enrichment of the early-stage fish feed, this enrichment with a new enrichment contributes As a result of continuous research considerably to optimizing fry formulation that boosts the and development, INVE Aquacul- quality and robustness. rotifers and Artemia with key ture has recently expanded its enrichment portfolio with a brand- natural ingredients, turning it new formulation: Easy Dry Selco® Remarkable results into a functional enrichment. (EDS). Extensive tests and trials have This product is a dry enrichment shown that enriching rotifers and with a formulation that boosts the Artemia with EDS can trigger the life cycle. These important effects rotifers and Artemia with key immune system of the fish larvae in of early immune system stimula- natural ingredients, turning it into a very early stages (even before 28 tion linked with other functional functional enrichment. dph) with visible effects at 56 dph. effects of specific nutrition and Because it adds a lot more than just This is the first time ever that this microbiological management of the selected fatty acids to the live feed, unique effect has been scientifically tank system can actually increase the complete and rich formula of reported at such an early stage. production efficiency and quality. EDS is extremely flexible and also

A significant increase of IL-1β, TNF-α and COX-2 levels mean that the fry are better prepared to fight against inflammation and pathogen threats. 29

How to use

Despite the very complex action and the extremely high score in quality of the fry, EDS is simple and effective in use. As a rotifer enrichment product, Easy Dry Selco® can be used in the tradition- al way after harvesting in a separate enrichment tank, or even directly into the culture tank. This means an incredible advantage in More information terms of space and workload. Rotifer enrichment is finalized in 6- Alessandro Moretti, Product Manager Fish 9 hours. For Artemia, INVE Hatchery, INVE Aquaculture Aquaculture recommends harvesting 18 hours after the start E: [email protected] of the enrichment. www.inveaquaculture.com

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Effects of enriched and non- enriched Artemia on jaw malformation in golden pompano larvae

Skeleton malformation is a major 2003). Among different lipid pompano larvae have been well bottleneck continually hindering components, fatty acids are studied (Ma et al. 2014a, 2014b), the production of marine fish indispensable to modulate the high malformation during early fingerlings (Sandel et al. 2010; Ma transcription of genes involved in development stage of this species et al. 2014c). Malformed fish are metabolism (Kliewer et al. 1997). has continually affected its usually sold at a low price or are Although previous studies have production efficiency in hatchery manually removed before sale to demonstrated that feeding with a (Ma et al. 2014d). Furthermore, market (Ma et al. 2014d). high level of dietary lipid can factors causing malformations on Moreover, deformation in fish can improve fish growth performance this fish are still unclear. A previous negatively affect fish growth, and reduce skeletal malformation study has found that enriching survival, food conversion ratio, and (Cahu et al. 2003), excessive formula for Artemia nauplii can susceptibility to stress and dietary lipids and unbalanced fatty affect the expression of retinoid X pathogens (Andrades et al. 1996; acid ratios can also lead to low receptors (RXRs) and jaw malfor- Koumoundourous et al. 1997; survival (Fernández & Gisbert mation in golden pompano, but Boglione et al. 2013). Although 2011) and skeleton malformation there was no clear correlation genetic factors (Ma et al. 2014c), (Izquierdo et al. 2010). Nutrient between retinoid X receptors environmental factors (Hattori et enrichment for Artemia nauplii has (RXRs) expressions and malfor- al. 2004), parasites and pesticides been used in larval fish culture for mation rates when golden (Liang et al. 2012; Liu et al. 2012) decades, but suitable enrichment pompano larvae were subjected to can affect fish bone development, formula is not available to lower nutrient change (Yang et al. 2015). increasingly evidence has showed skeleton malformation in larval fish In order to further explore the that nutritional factors during larval culture. relationships between BMPs gene fish rearing can directly cause expression and jaw malformation Due to high flesh quality, fast skeleton malformation (Villeneuve during the Artemia nauplii feeding growth, and suitability for cage et al. 2005a; Yang et al. 2015). phase, we cloned BMPs genes in culture, golden pompano golden pompano larvae and Lipid is the main source of energy (Trachinotus ovatus) has become a evaluated the correlation between supply for larval fish (Sargent et al. good candidate species for gene expressions and jaw 1999a). The morphogenesis of aquaculture (Guo et al. 2014). malformation. Such information marine fish larvae can be altered by Although several aspects pertaining would contribute to improvement changing dietary lipids (Cahu et al. to hatchery rearing of golden of larval quality and production 31

efficiency in the aquaculture of of 90% by number. For the analysis golden pompano and other related of gene expression, samples were species. collected from each tank. For each dietary treatment, samples in three In the present study, fish fed replicates were collected. Artemia nauplii enriched with Materials and methods Fish growth was determined by Algamac 3080 showed The experimental material in the specific growth rate (SGR) as%/day. present study was collected from At the end of this experiment, 50 twofold lower jaw an earlier study on nutritional trial fish larvae from each tank were malformation than those fed sampled for assessing growth and and fish growth measurement, non-enriched Artemia nauplii fatty acid analysis, and jaw jaw malformation. The remaining malformation reported in Yang et fish in each rearing tank were or Artemia nauplii enriched al. (2015). Fertilized eggs of golden harvested and counted for survival with Nannochloropsis. pompano hatched in 500-L determination. . fiberglass incubators at 26°C with a On 28 DPH, approximately 50 hatch rate of 97.1 ± 1.9%. On two individuals were collected from days post hatch (DPH), larvae were each tank and jaws were collected stocked into four 1000-L larval for total RNA extraction. RNA was Jaw malformation = (malformed rearing tanks at a density of 60 fish reverse-transcribed to cDNA, which larvae/total larvae) × 100%. L−1. Rotifers Brachionus rotundi- was used as a template in The highest SGR was obtained in formis at a density of 10–20 rotifers subsequent PCR. Quantitative real- fish fed Artemia nauplii enriched mL−1 were used to feed fish larvae time PCR (qPCR) was used to with Algamac 3080 and the lowest from 2 to 12 DPH. On the morning analyze the expression levels of SGR was observed in fish fed non- of 11 DPH, fish larvae were BMP genes in golden pompano enriched Artemia nauplii. The restocked into 12 500-L larval larvae. rearing tanks at a density of 20 fish highest survival was achieved in

L−1. fish fed non-enriched Artemia or Nanochloropsis enriched Artemia The nutritional manipulation Fatty acid and jaw malfor- (P < .05, Table 1), and the lowest experiment included three dietary mation analysis survival was observed when fish treatments with three replicates were fed with Algamac 3080 each. Artemia nauplii were treated The nutritional content of Artemia enriched Artemia nauplii (P < .05). in three methods (1) enriched with nauplii was assessed when fish On 28 DPH, the jaw deformity of instant micro-algal paste, (2) larvae were at 18 DPH. Fatty acids fish larvae fed Algamac 3080 enriched with Algamac 3080®, and were analyzed at South China Sea enriched Artemia nauplii was (3) without any enrichment as Fisheries Research Institute, significantly lower than fish fed non control. For each treatment, three Chinese Academy of Fishery -enriched Artemia nauplii or replicate tanks were used, and a Sciences, Guangzhou, China Nannochloropsis enriched Artemia total of nine tanks were used in this following the method described by nauplii (P < .05). However, the jaw study. Artemia nauplii were fed to Ma and Qin (2014). Jaw malfor- deformity was not significantly fish from 11 to 27 DPH. On 11 DPH, mation was examined under a different between fish fed non- Artemia nauplii were first stereo microscope (Olympus SZ, enriched Artemia nauplii and introduced at 200 nauplii L−1, and Tokyo, Japan) using the criteria Nannochloropsis enriched Artemia then added with a daily increment described by Ma et al. (2014d). Jaw nauplii (P > .05). malformation (%) was calculated as 32

Table 1. SGR, coefficients of variation, final survival rate, and jaw deformity rate of golden pompano larvae fed with enriched and non-enriched Artemia nauplii. Different letters represent significant differences at P < 0.05. Non-enriched Nannochloropsis Algamac 3080 SGR (%/day) 5.68 ± 0.22a 6.25 ± 0.08b 6.49 ± 0.05c Final survival rate (%) 31.83 ± 8.60b 29.33 ± 5.32b 10.33 ± 0.90a Jaw deformity rate (%) 15.91 ± 0.68b 15.39 ± 0.69b 8.33 ± 0.74a

Cloning and expressions Discussion variation of fish length in the treatment of Algamac 3080 could of the BMP genes Dietary n-3 highly unsaturated fatty also contribute to the high SGR due acids such as DHA and EPA are Partial sequences of BMP1, BMP2, to the death of small larvae in this essential to growth of fish larvae BMP4, BMP5, and BMP10 genes treatment. (Rezek et al. 2010), and their were obtained after sequencing Although a high content of dietary requirements are species-specific analysis. The BMP1 gene exhibited lipid can improve fish survival, (Dantagnan et al. 2010). Fish high identities with other fish overdosed dietary lipid or growth rates are related to dietary species. Nutrient enhancements unbalanced lipid class composition DHA in many fish. Furthermore, the significantly affected the gene can also lead to poor growth and growth response of fish larvae to expressions of BMP4 and BMP10 (P abnormal development in fish different enrichment products is < .05, Figure 1). The expression of larvae (Salhi et al. 1999). For also varied among species. For BMP4 in fish fed non-enriched instance, Ma and Qin (2014) example, the growth rates of Artemia nauplii or Nannochloropsis reported that a high DHA/EPA ratio striped bass Morone saxatilis and enriched Artemia nauplii was in live feed could lead to low gilthead seabream S. aurata larvae significantly higher than fish fed survival in yellowtail kingfish are not affected by feeding the Algamac 3080 enriched Artemia Seriola lalandi. In the present Artemia nauplii enriched with nauplii (P < .05, Figure 1). The study, a higher DHA/EPA ratio Algamac 2000 or PL-Cr (DHA-rich expression of BMP4 was not (0.36:1) was achieved by enriching phospholipid extract of Crypthe- significantly different between fish Artemia nauplii with Algamac 3080. codinium sp.), but the growth rate fed non-enriched Artemia nauplii The high DHA/EPA ratio in the of halibut Hippoglossus hippoglos- and Nannochloropsis enriched Algamac 3080 treatment led to fast sus larvae fed Artemia nauplii Artemia nauplii (P > .05). The fish growth but low survival. In enriched with DHA Seleco is lower lowest level of BMP10 expression contrast, better survival was than the larvae fed PL-Cr (Harel et was observed when fish were fed obtained in the non-enriched and al. 2002). with non-enriched Artemia nauplii, Nannochloropsis treatments where and the highest BMP10 expression In this study, fish growth was the DHA/EPA ratio was 0.07:1– was found in fish fed Algamac 3080 improved when fish larvae were 0.22:1. Low fish survival in the enriched Artemia nauplii (Figure 1). fed with Artemia nauplii enriched Algamac 3080 treatment supports with Algamac 3080 or Nannochlo- Nutrient enhancements were the claim that a high DHA content ropsis. The best fish SGR was correlated to SGR, jaw deformity, and a high DHA/EPA ratio may achieved in the treatment of and expressions of BMP4 and reduce larval fish survival (Planas & Algamac 3080, which is consistent BMP10. Cunha 1999) as unbalanced lipid with the high dietary DHA levels in classes in the diet affect digestion

Artemia nauplii. Nevertheless, the and absorption of fatty acids in fish low survival and high coefficients of larvae (Salhi et al. 1999). 33

skeletal development of marine fish larvae (Villeneuve et al. 2006). In the present study, the expres- sion levels of BMP4 in fish fed non- enriched Artemia nauplii and Nannochloropsis enriched Artemia nauplii were significantly higher than fish fed Algamac 3080 enriched Artemia nauplii. Further- more, jaw malformation in the treatment of non-enriched and Nannochloropsis was significantly higher than in the treatment of Algamac 3080. These results are consistent with the finding reported by Villeneuve et al. (2006), in which jaw malformation increases when the expression of Fig. 1. Relative expression levels of BMPs in golden pompano larvae fed BMP4 is up-regulated. enriched and non-enriched Artemia nauplii. Unlike other BMPs, BMP10 is expressed predominantly in the Previous studies have indicated fed Artemia enriched with Algamac adult heart and to a lesser extent in that poly unsaturated fatty acids 3080, which is coincident with the the liver and lung (Neuhaus et al. play an important role in bone high DHA content in the feed. This 1999). In the present study, formation of fish (Izquierdo et al. indicates that a dietary DHA level nutrient enhancement altered the 2010), and dietary fatty acids can of 2.56% may be suitable for expression of BMP10 in golden alter the composition of bone and skeletal development in golden pompano larvae, and the expres- cartilage (Liu et al. 2004). Abnormal pompano larvae. sion of BMP10 was corresponding development of fish larvae may be BMP2 and BMP4 are closely related to jaw malformation. caused by insufficient dietary n-3 proteins involved in key embryonic highly unsaturated fatty acids In summary, nutrient enhancement processes. In the present study, the (HUFA) in live food (Hamre et al. can affect jaw malformation in fish expressions of BMP1 and BMP2 2002). A 50% reduction of larvae during the Artemia nauplii were not significantly affected by deformed fish was observed when feeding phase. Feeding golden the nutrient enrichment by 28 DPH, fish larvae were fed with higher pompano larvae with enriched suggesting that the expression of levels of dietary DHA (Izquierdo et Artemia nauplii significantly BMP1 and BMP2 in golden al. 2010). In the present study, fish reduced the jaw malformation rate, pompano may be less sensitive to fed Artemia nauplii enriched with but also decreased survival at the nutrient enrichment after the bone Algamac 3080 showed twofold same time. Reduction of jaw structure is formed and mineral- lower jaw malformation than those malformation may be due to the ized. fed non-enriched Artemia nauplii mortality of fish larvae during test or Artemia nauplii enriched with BMP4 plays diverse roles during time. However, this may need Nannochloropsis. Skeletal vertebrate development. Thus, it further verification. Nutritional malformation was reduced in fish has been used to evaluate the manipulations can significantly effect of micronutrients on the affect the expression levels of 34

BMP4 and BMP10, and the larvae reared under different salinity receptors α and β. Proc Natl Acad Sci U concentration of dietary DHA can regimes. Aquacult Res. 41:e239–e244. S A. 94:4318–4323. significantly affect the expression Fernández I, Gisbert E. 2011. The effect Koumoundourous G, Oran G, Divanach of BMP10. The expressions of of vitamin A on flatfish development P, Stefanakis S, Kentouri M. 1997. The BMP4 and BMP10 varied between and skeletogenesis: a review. opercular complex deformity in different dietary treatments, and Aquaculture. 315:34–48. intensive gilthead sea bream Sparus aurata L. larviculture. Moment of the expressions of BMP4 and Guo H, Ma Z, Jiang S, Zhang D, Zhang N, apparition and description. Aquacul- BMP10 correspond to jaw Li Y. 2014. Length-weight relationship ture. 149:215–226. malformation in golden pompano of oval pompano, Trachinotus ovatus larvae during the Artemia nauplii (Linnaeus 1758) (Pisces; Carangidae) Liang Q, Xie Y, Fang Z. 2012. Individual feeding phase. This study suggests cultured in open sea floating sea cages and joint toxicity of Zn2+ and Cd2+ that measures of BMP4 and BMP10 in South China Sea. Indian J Fish. 61:93 during the early embryonic develop- –95. ment of zebrafish (Danio rerio). J Fish in golden pompano may serve as a Sci China. 19:283–292. suitable indicator for jaw malfor- Hamre K, Opstad I, Espe M, Solbakken mation in the field and aquaculture J, Hemre G-I, Pittman K. 2002. Nutrient Liu Z, Zhang S, Yang J. 2012. Toxic facility, leading to rapid assessment composition and metamorphosis effects of chlorobenzene on embryonic success of Atlantic halibut development and larva of zebrafish. of nutrient status affecting fish jaw (Hippoglossus hippoglossus, L.) larvae Env Sci Technol. 35:25–28. malformation. fed natural zooplankton or Artemia. Liu D, Veit HP, Denbow DM. 2004. Aquac Nutr. 8:139–148. Effects of long-term dietary lipids on Harel M, Koven W, Lein I, Bar Y, mature bone mineral content, collagen, References Behrens P, Stubblefield J, Zohar Y, Place crosslinks, and prostaglandin E2 Andrades JA, Becerra J, Fernández- A. 2002. Advanced DHA, EPA, and ARA production in Japanese quail. Poult Sci. Llebrez P. 1996. Skeletal deformities in enrichment materials for marine 83:1876–1883. aquaculture using single cell hetero- larval, juvenile and adult stages of Ma Z, Guo H, Zhang D, Hu CQ, Jiang S. trophs. Aquaculture. 213:347–362. cultured gilthead sea bream 2014a. Food ingestion, consumption, (Sparusaurata L.). Aquaculture. 141(1– Hattori M, Sawada Y, Kurata M, and selectivity of pompano, Trachino- 2):1–11. Yamamoto S, Kato K, Kumai H. 2004. tus ovatus (Linnaeus 1758) under Boglione C, Gisbert E, Gavaia P, Witten Oxygen deficiency during somitogene- different rotifer densities. Aquacult PE, Moren M, Fontagne S, Koumoun- sis causes centrum defects in red sea Res. bream, Pagrus major (Temminck et douros G. 2013. Skeletal anomalies in Ma Z, Guo H, Zheng P, Wang L, Jiang S, Schlegel). Aquaculture Res. 35:850– reared European fish larvae and Qin JG, Zhang D. 2014b. Ontogenetic 858. juveniles. Part 2: main typologies, development of digestive functionality occurrences and causative factors. Rev Izquierdo MS, Socorro J, Roo J. 2010. in golden pompano Trachinotus ovatus Aquacult. 5:S121–S167. Studies on the appearance of skeletal (Linnaeus 1758). Fish Physiol Biochem. Cahu CL, Infante JLZ, Barbosa V. 2003. anomalies in red porgy: effect of 40:1157–1167. culture intensiveness, feeding habits Effect of dietary phospholipid level and Ma Z, Qin JG. 2014. Replacement of and nutritional quality of live preys. J phospholipid: neutral lipid value on the fresh algae with commercial formulas App Ichth. 26:320–326. development of sea bass to enrich rotifers in larval rearing of (Dicentrarchus labrax) larvae fed a Kliewer SA, Sundseth SS, Jones SA, yellowtail kingfish Seriola lalandi compound diet. British J Nutr. 90 (1):21 Brown PJ, Wisely B, CKoble CS, (Valenciennes, 1833). Aquacult Res. –28. Devchand, P, Wahli W, Willson TM, 45:949–960. Lenhard JM, Lehmann JM. 1997. Fatty Dantagnan P, Borquez A, Hernandez A, Ma Z, Tan DAY, Qin JG. 2014c. Jaw acid and eicosanoids regulated gene Izquierdo M. 2010. Effect of EPA/DHA deformities in the larvae of yellowtail expression through direct interactions ratios on the growth and survival of kingfish (Seriola lalandi Valenciennes, with peroxisome proliferator-activated Galaxias maculatus (Jenyns, 1842) 1833) from two groups of broodstock. 35

Indian J Fish. 61(4):137–140. histological structure of gilthead Villeneuve LAN, Gisbert E, Moriceau J, seabream (Sparus aurata) larvae. Cahu CL, Infante JLZ. 2006. Intake of Ma Z, Zheng P, Guo H, Zhang N, Jiang S, Aquaculture. 179(1–4):253–263. high levels of vitamin A and polyun- Zhang D, Qin JG. 2014d. Jaw malfor- saturated fatty acids during different mation of hatchery reared golden Sandel E, Nixon O, Lutzky S, Ginsbourg developmental periods modifies the pompano Trachinotus ovatus (Linnaeus B, Tandler A, Uni Z, Koven W. 2010. The expression of morphogenesis genes in 1758) larvae. Aquacult Res. effect of dietary phosphatidylcholine/ European sea bass (Dicentrarchus phosphatidylinositol ratio on Planas M, Cunha I. 1999. Larviculture of labrax). Br J Nutr. 95:677–687. malformation in larvae and juvenile marine fish: problems and perspec- gilthead sea bream (Sparus aurata). tives. Aquaculture. 177:171-190. Aquaculture. 304(1–4):42–48. From Zhenhua Ma, Jing Hu, and Gang Rezek TC, Watanabe WO, Harel M, Sargent J, Bell G, McEvoy L, Tocher D, Yu, South China Sea Fisheries Research Seaton PJ. 2010. Effects of dietary Estevez A. 1999a. Recent develop- Institute, and Jian G. Qin, Flinders docosahexaenoic acid (22:6n-3) and ments in the essential fatty acid University Adelaide. Gene expression of arachidonic acid (20:4n-6) on the nutrition of fish. Aquaculture. 177(1– bone morphogenetic proteins and jaw growth, survival, stress resistance and 4):191–199. malformation in golden pompano fatty acid composition in black sea bass Trachinotus ovatus larvae in different Centropristis striata (Linnaeus 1758) Yang Q, Zheng P, Ma Z, Li T, Jiang S, Qin feeding regimes. Journal of Applied larvae. Aquacult Res. 41:1302–1314. JG. 2015. Molecular cloning and Animal Research, 46:1. Under CC expression analysis of the retinoid X Salhi M, Hernández-Cruz CM, Bessonart license 4.0 receptor (RXR) gene in golden M, Izquierdo MS, Fernández-Palacios H. pompano Trachinotus ovatus fed 1999. Effect of different dietary polar Artemia nauplii with different lipid levels and different n-3 HUFA ΩHF enrichments. Fish Physiol Biochem. content in polar lipids on gut and liver

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New research developments in aquatic animal larval feeding and nutrition—recent literature

Abstract excerpts compiled by Meredith Brooks, Hatcheryfeed

Growth performance and gradual reduction of nauplii Researchers developed four diet combined with an increase in dry groups A through D, each contain- intestinal morphology of feed stimulated morphological ing equal levels of total protein African catfish Clarias( development, resulting in content, and tested them against gariepinus, Burchell, improved growth performance. one no-feeding control. Higher amounts of oriental river prawn Onura, C.N., Van den Broeck, W., 1822) larvae fed on live Nevejan, N., Muendo, P., and Van larvae and postlarvae were and dry feeds Stappen, G. Growth performance and achieved from diet C containing intestinal morphology of African catfish yeast and fermented soybean meal Researchers investigated the (Clarias gariepinus, Burchell, 1822) and diet D containing yeast, effects of both live and dry feeds larvae fed on live and dry feeds. fermented soybean meal and a on the growth and intestinal Aquaculture, March 2018; 489: 70-79. microparticulate diet. The trials morphology of African catfish Full paper: https://doi.org/10.1016/ also found that the increase of (Clarias gariepinus, Burchell, 1822) j.aquaculture.2018.01.046 larvae can result in an increase in larvae. They developed five diets, phytoplankton in the water, including two diets containing a helping to improve water quality. combination of live and dry feeds, Combined diet of yeast, Final results demonstrated that and tested them on larvae from 2 diet D was the most effective at days post hatch to 6 and 11 days fermented soybean meal, increasing per unit larval yield and post hatch. After the feeding trial, and microparticulate as has a low cost of production, which both final weight and specific larval feed in extensive can assist in the expansion of growth rate were significantly oriental river prawn aquaculture. higher in larvae fed nauplii rearing systems for seed combined with dry feed. The best production of the oriental Yao, Z.L., Zhao, Y., Ma, H.Y., Liu, H.J., FCR rates were achieved with the Wang, H., and Ji, X.S. Combined diet of diet containing only nauplii and the river prawn Macro- yeast, fermented soybean meal, and microparticulate as larval feed in diet containing both nauplii and dry brachium nipponense extensive rearing systems for seed feed. This study aimed to evaluate the production of the oriental river prawn Results found that different starter effects of different types of larval Macrobrachium nipponense. Aquacult feeds affect intestinal morphologi- feed on seed production of the Int (2018) 26: 757. cal development and growth of oriental river prawn Macro- Full paper: https://doi.org/10.1007/ African catfish larvae. Specifically, a brachium nipponense. s10499-018-0240-8 37

Growth, Survival, and and Whole‐body Proximate and Fatty Liu, X., Sha, Z., Wang, C., Li, D., and Acid Composition of Haddock, Bureau, D.P. A web-based combined Whole‐body Proximate Melanogrammus aeglefinus L., nutritional model to precisely predict and Fatty Acid Composi- Postlarvae Fed a Practical Microparticu- growth, feed requirement and waste late Weaning Diet. J World Aquacult output of gibel carp (Carassius auratus tion of Haddock, Melano- Soc, 49: 83-95. doi:10.1111/jwas.12462 gibelio) in aquaculture operations. Aquaculture, July 2018; 492: 335-348. grammus aeglefinus L., Full paper: https:// onlinelibrary.wiley.com/doi/ Full paper: https://doi.org/10.1016/ Postlarvae Fed a Practical full/10.1111/jwas.12462 j.aquaculture.2018.04.019 Microparticulate Wean- ing Diet ΩHF A web-based combined In an effort to further the develop- ment of high quality hatchery feeds nutritional model to for haddock (Melanogrammus precisely predict growth, aeglefinus L.) larvae, Researchers in Canada developed a practical feed requirement and microparticulate diet (PMD). The waste output of gibel diet was compared with a high- carp (Carassius auratus Next issue quality imported feed through a series of feed and weaning trials. gibelio) in aquaculture September ‘18

The PMD formulation was operations demonstrated to be a highly A new combined nutritional model suitable diet for haddock larvae. In has been constructed to predict Shrimp & addition to a high rate of feed growth, feed requirement, and acceptance and survival, the fish waste output of gibel carp Crustacean fed the PMD had higher final fork (Carassius auratus gibelio). The lengths, wet weights, and weight web-based model was developed feed and gains. This indicates the diet has using data from several fish farms good potential for future use in and published studies, and is able hatcheries and research studies. to predict nutritional needs for fish nutrition The study was the first to demon- during various stages of life. strate that a high rate of survival Researchers classified carp into (>88%) of haddock can be achieved three growth stanzas using the through the weaning stage from thermal-unit growth coefficient Submit editorial to: days 54 to 61 post-hatch; previous (TGC) model; stanza 1 is first [email protected] studies achieved between 35% and feeding from 1 to 13.5g, followed 2-5%. Researchers compared the by 13.3 to 175.5g, and >175.5g. co-feeding periods for all studies An online platform was developed Advertising enquiries: and suggested that earlier co- for farmers and managers to use [email protected] feeding periods may result in lower the system, and the design and survival rates. implementation of the model is Lall, S. P., Lewis‐McCrea, L. M. and described in the published study. Tibbetts, S. M. (2018), Growth, Survival, 38

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