Nutritional Strategies to Cope with Mildly Elevated Water Temperature in Fish

Nutritional Strategies to Cope with Mildly

Elevated Water Temperature in Fish

Fikremariam Geda Ararame

Fikremariam Geda Ararame

Thesis submitted in fulfillment of the requirements for the academic degree of

Doctor of Philosophy (PhD) in Veterinary Sciences

2016

Promotor:

Prof. dr. Geert P.J. Janssens

Laboratory of Animal Nutrition

Department of Nutrition, Genetics and Ethology

Faculty of Veterinary Medicine

Ghent University

Heidestraat 19, B-9820 Merelbeke, Belgium

Promotor:

Prof. dr. Geert P.J. Janssens, Faculty of Veterinary Medicine, Ghent University

Members of the examination committee:

Prof. dr. Luc Duchateau, Chair, Faculty of Veterinary Medicine, Ghent University

Dr. Gunther Antonissen, Secretary, Faculty of Veterinary Medicine, Ghent University

Prof. dr. Patrick Sorgeloos, Faculty of Bioscience Engineering, Ghent University

Prof. dr. Rune Waagbø, National Institute of Nutrition and Seafood Research, Norway

Prof. dr. Wim Derave, Faculty of Medicine and Health Sciences, Ghent University

Dr. Johan Schrama, Aquaculture and Fisheries, Wageningen University, The Netherlands

Fikremariam Geda Ararame

Geda, F., 2016. Nutritional strategies to cope with mildly elevated water temperature in fish. PhD thesis, Ghent University, Belgium.

Cover design: Fikremariam Geda Ararame

Printed: University Press, Leegstraat 15, B-9060 Zelzate, Belgium.

The author and the promotor give the authorization to consult and to copy parts of this PhD thesis for personal use only. Every other use is subjected to the copyright laws. Prior permission to reproduce any material contained in this PhD thesis in any form or in any way, by print, photo print, microfilm, or any other means should be obtained from the author.

Life is like riding a bicycle.

To keep your balance, you must keep moving.

Albert Einstein

Dedication

To my parents: Geda Ararame and Nedi Beyecha; my brothers and sisters: Ajema, Sebsebe, Gadise, Jalene, Haile, Mekonnen, Megerssa, Bekele, Gebisa and Tolossa; my father in law: Tsegaye Debele; my wife: Emebet Tsegaye, and to my first child: Nobel Fikremariam.

Table of contents

General introduction ...... 1

Scientific aims and objectives ...... 23

Nutrient metabolism in fish at mild changes in water temperature ...... 27

Potential of dietary β-alanine in fish at mild changes in water temperature: Part I ...... 45

Potential of dietary β-alanine in fish at mild changes in water temperature: Part II ...... 56

Effects of dietary protein:lipid ratio in fish at mild changes in water temperature ...... 69

Effects of dietary mannanoligosaccharides in fish at mild changes in water temperature ...... 91

General discussion ...... 107

Summary ...... 119

Samenvatting ...... 125

References ...... 131

Curriculum vitae ...... 149

Bibliography ...... 153

Acknowledgements ...... 157

List of abbreviations

AA Amino acids AB-PAS Alcian blue-periodic acid-Schiff ATP Adenosine triphosphate BA β-Alanine BCAA Branched-chain amino acids CoA Co-enzyme A CRA Cold resistance area CTA Cold tolerance area CTMax Critical thermal maximum CTMin Critical thermal minimum DHA Docosahexaenoic acid DIT Diet-induced thermogenesis DO Dissolved oxygen EPA Eicosapentaenoic acid FAA Free amino acids FCR Feed conversion ratio GABA γ-Aminobutyric acid HAT Higher avoided temperature HRC Histidine related compounds HE Hematoxylin and eosin HPLC High performance liquid chromatography HRA Heat resistance area HTA Heat tolerance area HUFA Highly unsaturated fatty acid LAT Lower avoided temperature LCFA Long-chain fatty acids LILT Lower incipient lethal temperature

List of abbreviations

MCWT Mild changes in water temperature MOS Mannanoligosaccharides NAH N-α-Acetylhistidine NEFA Non-esterified fatty acids PT Preferred temperature PUFA Polyunsaturated fatty acid SE Standard error SEM Standard error of means TAC Total acylcarnitine Tb Body temperature TFAA Total free amino acids TPA Thermal preference area UILT Upper incipient lethal temperature ƩFA Sum of fatty acids Ʃ3OHFA Sum of 3-hydroxy fatty acids Ʃ3OHLCFA Sum of 3-hydroxy long-chain fatty acids ƩLCFA Sum of long-chain fatty acids

List of abbreviations

Biochemical names of acylcarnitines C0 Free-carnitine C2 Acetyl-carnitine C3 Propionyl-carnitine C3-DC Malonyl-carnitine C4 Butyryl-carnitine C4-DC Methylmalonyl-carnitine 3OH-C4 3-Hydroxybutyryl-carnitine C5 (Iso)valeryl-carnitine C5:1 Tiglyl-carnitine C5-DC Glutaryl-carnitine 3OH-C5 3-Hydroxy(iso)valeryl-carnitine C6 Hexanoyl-carnitine C8 Octanoyl-carnitine C10 Decanoyl-carnitine C12 Dodecanoyl-carnitine 3OH-C12 3-Hydroxydodecanoyl-carnitine C14 Tetradecanoyl-carnitine 3OH-C14 3-Hydroxytetradecanoyl-carnitine

C14:1 Tetradecenoyl-carnitine 3OH-C14:1 3-Hydroxytetradecenoyl-carnitine C16 Hexadecanoyl-carnitine 3OH-C16 3-Hydroxyhexadecanoyl-carnitine C16:1 Hexadecenoyl-carnitine 3OH-C16:1 3-Hydroxyhexadecenoyl-carnitine C18 Octadecanoyl-carnitine 3OH-C18 3-Hydroxyoctadecanoyl-carnitine C18:1 Octadecenoyl-carnitine 3OH-C18:1 3-Hydroxyoctadecenoyl-carnitine

Chapter 1 General introduction

General introduction

Global aquaculture, global warming, metabolic changes and nutritional coping strategies in fish

Geda, F., Janssens, G.P.J.

Review in preparation The general introduction presents a brief review of global aquaculture, global warming and nutritional coping strategies in fish. World total fish production was increasing and reached 167.2 million tonnes in 2014 with 93.4 million tonnes from capture fisheries and 73.8 million tonnes from aquaculture. World capture fisheries production has almost stabilized, because there are few opportunities to develop new sustainable fisheries or to increase catch rates in existing fisheries. However, global aquaculture is rising and remaining one of the fastest animal food-producing sectors with its growth rising from 18.5% in 1994, 32.4% in 2004 to 44.1% in 2014. World inland aquaculture grew at average annual rate of 8.5% in the period 1994-2004, 8.4% in the period 2004-2014, compared with 7.8 and 6.7%, respectively for world marine aquaculture. World aquaculture has shown strong growth in developing countries in Asia and the Pacific, sub-Saharan Africa and South America. Although global aquaculture is substantially contributing to world total fish production, its productivity has been affected by global warming. Global warming causes changes in thermal zones and increases vertical stratification, which reduces nutrient availability, primary and secondary production, plankton production, physiology of fish, fish populations (ecology, abundance and distributions) and aquaculture in a warmed world. In short, global warming causes a mild elevation in water temperature, which affects overall physiology and performance of fish, thereby the global total fish production. The metabolic responses of individual fish to the mild changes in water temperature are either not well investigated or poorly described. This is because most physiological studies apply strong-short-term heat stress instead of the constant mild changes in water temperature that is happening due to global warming. More research efforts are needed to know the effects of the mild changes in water temperature in fish and the metabolic responses of individual fish, which should be supported with nutrition. Therefore, the demonstration of effects of the mild changes in water temperature in fish, individual metabolic reactions, and evaluation of model nutritional strategies (e.g., dietary protein:lipid ratio, dietary β-alanine, dietary mannanoligosaccharides) warrant further research.

General introduction

1.1 Fisheries and aquaculture Fisheries or capture fisheries refer to the exploitation of aquatic organisms by the public from populations in extensive water bodies as a common property resource, with or without appropriate licences (Food and Agricultural Organization of the United Nations [FAO], 1997). Aquaculture, which does not include fisheries, is the farming of aquatic (freshwater, brackish, or marine) organisms including fish, shellfish (mollusks or crustaceans), and aquatic plants (seaweed), under controlled or semi-controlled conditions, with some sort of intervention in the rearing process to enhance production, such as regular stocking, feeding, and protection from predators (FAO, 1997). In aquaculture, cultivated stocks of aquatic organisms are harvested by an individual or corporate body which has owned them throughout their rearing period. Aquaculture does not include the culture of terrestrial plants (e.g. hydroponics) or animals and according to Pillay and Kutty (2005), the term can be used in restrictive sense to denote (i) the type of culture techniques (e.g. pond, raceway, cage, pen, raft), (ii) the type of organism cultured (e.g. fish, oyster, mussel, shrimp or seaweed), (iii) the environment in which the culture is done (e.g. freshwater, brackish, salt water or marine aquaculture or mariculture) or (iv) a specific character of the environment used for culture (e.g. cold-water or warm-water aquaculture; upland, low land, inland, coastal, estuarine).

1.2 World fish production and utilization World total fish production and utilization has been summarized and presented for two decades in the period 1994-2014 with some information in the period 1950-1990 (FAO, 2000, 2004, 2008, 2012, 2016) (Table 1). World total fish production was 112.3 million tonnes in 1994, 140.5 million tonnes 2004 and 167.2 million tonnes 2014 with an increase in the growth by 25% in the period 1994-2004 and by 19% in the period 2004-2014. World capture fisheries production (in million tonnes) increased from 18 in 1950 (1.2 inland and 16.8 marine), 56 in 1969, 91.4 in 1994, 95.0 in 2004 to 93.4 in 2014. World inland capture fisheries production has increased by 37% in the period 1994-2004 and by 29% in the period 2004-2014. World marine capture fisheries production increased markedly from 16.8 million tonnes in 1950 to 86.0 million tonnes in 1996 with an increase in the growth by 1.3% in the period 1994-2004 and reduced by 5% in the period 2004-2014.

General introduction

World aquaculture production continues to be the fastest growing animal food-producing sector (Table 1, Figure 1). Its contribution to world total fish production continued to grow, rising from 18.5% in 1994, 32.4% in 2004 to 44.1% in 2014. World aquaculture production grew at annual rate of 5% in the period 1950-1969, 8% in the period 1970-1980, 10.8% in the period 1980-1990, 8.2% in the period 1994–2004 and 5.9% in the period 2004–2014. World inland aquaculture production grew at average annual rate of 8.5% in the period 1994-2004, 8.4% in the period 2004-2014, compared with 7.8 and 6.7%, respectively for world marine aquaculture production. World aquaculture production has shown strong growth in developing countries in Asia and the Pacific (Myanmar and Papua New Guinea), sub-Saharan Africa (Nigeria, Uganda, Kenya, Zambia and Ghana) and South America (Ecuador, Peru and Brazil). Annual growth rates in developed industrialized countries averaged only 2.1% in the 1990s and 1.5% in the 2000s.

Figure 1. World fish production. The data of 2014 are provisional estimates (source: FAO, 2000, 2004, 2008, 2012, 2016).

General introduction

World food fish supply for human consumption was 79.8 million tonnes in 1994, 105.6 million tonnes in 2004 and 146.3 million tonnes in 2014 with an average annual growth rate of 3.3% in the period 1994-2014 and 3.2% in the period 1961-2013, outpacing the increase of 1.5% per year in the world’s population (Table 1, Figure 2). The contribution of world aquaculture production to world food fish supply for human consumption was 4.8% in 1970, 9% in 1980, 13.4% in 1990, 26% in 1994, 43% in 2004 and 50% in 2014. Average per capita world food fish supply steadily increased from 9.9 kg in the 1960s (live weight equivalent), 11.5 kg in the 1970s, 12.5 kg in the 1980s, 14.4 kg in the 1990s, 14.3 kg in 1994, 16.6 kg in 2004 and reached 20.1 kg in 2014. Average per capita world aquaculture food fish supply was 0.7 kg in 1970, 3.7 kg in 1994, 7.1 kg in 2004 and 10.1 kg in 2014.

The percentage contribution of fish proteins to world animal protein supplies for human consumption was 13.7 in 1961, 14.9 in 1992, 16.0 in 1996, 15.9 in 2001, 15.5 in 2003, 15.3 in 2005, 15.7 in 2007, 16.6 in 2009, 16.7 in 2010, and 17.0 in 2013. Worldwide, fish provided about 3.0 billion people in 2009, 2.9 billion people in 2010, 3.1 billion people in 2013 with almost 20% of their average per capita intake of animal protein.

World food fish supply and consumption increased due to such factors as increase in fish production, reductions in wastage, better utilization, improved distribution channels, and growing demand linked to population growth, rising income, urbanization and international trade in providing wider choices to consumers. Fish is predominantly utilized for human consumption with continued reduction into non-food uses such as for fishmeal, fish oil, aquaculture purposes (fingerlings, fry, etc.), ornamental purposes, bait, pharmaceutical purposes and as direct feed for aquaculture, livestock and other animals.

General introduction

Figure 2. World fish utilization and supply. The data of 2014 are provisional estimates (source: FAO, 2000, 2004, 2008, 2012, 2016).

1.3 The role of fish in human nutrition Fish and fishery products are available and distributed as live, fresh, chilled, frozen, heat-treated, fermented, dried, smoked, salted, pickled, boiled, fried, freeze-dried, minced, powdered or canned, or as a combination of two or more of these forms (FAO, 2012). They represent a very valuable source of protein and essential micronutrients for balanced nutrition and good health. Fish provides not only easily digested high-value proteins containing all amino acids (AA), but essential fats (e.g. long-chain omega-3 fatty acids), essential micronutrients, including vitamins (D, A and B), minerals (calcium, iodine, zinc, iron and selenium) and polyunsaturated omega-3 fatty acids [docosahexaenoic acid, DHA (22:6n-3) and eicosapentaenoic acid, EPA (20:5n-3)]. Fish is usually low in saturated fats, carbohydrates and cholesterol. Fish consumption is beneficial in protection against cardiovascular disease (Mozaffarian and Rimm, 2006), stroke, age-related macular degeneration and mental health (Peet and Stokes, 2005; Young and Conquer, 2005). It is also valuable in foetal and infant development of the brain and nervous system, in correcting unbalanced diets, and through substitution, in countering obesity.

General introduction

The dietary contribution of fish is more significant in terms of animal proteins, as a portion of 150 g of fish provides about 50–60% of an adult’s daily protein requirements. Fish proteins can represent a crucial nutritional component in some densely populated countries where total protein intake levels may be low because small quantities of fish can have a significant positive nutritional impact by providing essential AA, fats and micronutrients that are scarce in vegetable-based diets. In fact, many populations, more those in developing countries than developed ones, depend on fish as part of their daily diet. For them, fish and fishery products often represent an affordable source of animal protein that may not only be cheaper than other animal protein sources, but preferred and part of local and traditional recipes.

1.4 Challenges of fisheries and aquaculture Global aquaculture production is vulnerable to adverse impacts of natural, socioeconomic, disease, environmental and technological conditions. Fish are recognized as an integrated part of an aquatic ecosystem such that modifications in one area have the potential to affect other areas (FAO, 2000).

Climate change is bringing substantial changes to the world’s capture fisheries, which are already under stress from overfishing and other anthropogenic influences (FAO, 2010). Inland fisheries – most of which are in developing African and Asian countries – are at particularly high risk, threatening the food supply and livelihoods of some of the world’s poorest populations. There are also consequences for aquaculture, which is especially significant for populations in Asia.

Global aquaculture is concentrated in the world’s tropical and subtropical regions, with Asia’s inland freshwaters accounting for 65% of total production (FAO, 2010). Significant aquaculture activities occur in the delta areas of major rivers. Sea-level rise in the coming decades will increase salinity intrusion further upstream, affecting brackish water and freshwater culture practices. Adaptation would involve moving aquaculture practices further upstream or shifting to more salinity-tolerant strains of cultured species. Such measures are costly, with significant effects on the socio-economic status of the communities involved. On the other hand, aquaculture in temperate zones can be more affected by water warming to levels that exceed the limit for many farmed species and require changes in farmed species.

1.4.1 Growth in human population World population is growing and is expected to grow by another 2 billion to reach 9.6 billion people by 2050 with a concentration in coastal urban areas (FAO, 2014). This conveys huge challenge on global fisheries and aquaculture since global demands for food fish are expected to rise even faster 7

General introduction owing to the emergence of a larger proportion of middle class people who have greater spending power and typically consume more animal protein than people with lower income (Kharas, 2010). Fisheries and aquaculture assure the livelihoods of 10–12% of the world’s population and global fish production continues to outpace world population growth (FAO, 2014). While aquaculture production is rising and remaining one of the fastest food producing sectors, capture fisheries production has almost stabilized, because there are few opportunities to develop new sustainable fisheries or to increase catch rates in existing fisheries. For instance, in 2011, 61% of fished stocks assessed by the FAO globally were estimated to be fully but sustainably fished, 29% were overfished and only 10% under fished (FAO, 2014).

There are distinct global and local sets of environmental challenges related to aquaculture production (Asche, 2015). The main global challenge is that increased demands for fish meal in feed for aquaculture production increased fishing pressure on wild stocks and threaten the sustainability of associated capture fisheries because marine proteins are important ingredients in the diet for cultured fish. The local challenges include discharges from aquaculture sites, destruction of local habitat, and escapees and spreading of potential pathogens. Furthermore, inland capture fisheries are affected by destruction and fragmentation of aquatic habitats, aquatic pollution, impoundment and channelization of water bodies, soil erosion and manipulation of hydrological characteristics of rivers, lakes and flood plains (Hongskul, 1999). Therefore, because of the increasing population and anthropogenic influences on capture fisheries, any future increase of the yield from capture fisheries could be derived only from fishery enhancement activities, i.e., the effects of direct human intervention in the production processes of aquatic environments.

1.4.2 Nutritional challenges Feed is a major constraint to aquaculture development to significantly and sustainably contribute to the declining percentage of non-fed species in global production, to maintain the relatively faster body-growth rates achieved in the culture of fed species and to respond to the increasing consumer demand for higher trophic-level species of fishes and crustaceans.

Fishmeal and fish oil are still considered the most nutritious and digestible ingredients for farmed fish feeds. To offset their high prices, as feed demand increases, the amount of fishmeal and fish oil used in compound feeds for aquaculture has shown a clear downward trend, with their being more selectively used as strategic ingredients at lower concentrations and for specific stages of production, particularly hatchery, broodstock and finishing diets. Fishmeal is the crude flour obtained after milling and drying fish or fish parts, and it is produced from whole fish, fish remains or other fish by- 8

General introduction products resulting from processing (FAO, 2012). Many different species are used for fishmeal and fish-oil production: small pelagics, in particular anchoveta are the main groups; the processing waste from commercial fish species used for human consumption is important source of raw material for the production of fishmeal.

The long-term ability of capture fisheries production to produce the fishmeal and fish-oil supplies used as feed components in aquaculture is of considerable concern (FAO, 2010). Alternatives, such as soybean, corn meal, rice bran and others, have not been perfected according to fish requirements, and the increased demand for these agricultural products created by expanding aquaculture could also have consequences.

1.4.3 Global warming Global warming of oceans causes changes in temperature and salinity, which reduce the density of surface waters and thus increase vertical stratification (FAO, 2010). These changes are likely to reduce nutrient availability in the surface layer and, therefore, primary and secondary production in a warmed world. The consequences of climate change can affect plankton production, physiology of fish, fish populations (ecology, abundance and distributions) and aquaculture production.

As temperatures warm, marine fish populations at the poleward extents of their ranges will increase in abundance whereas populations in more equatorward parts of their range will decline in abundance (FAO, 2010). In general, climate change is expected to drive the ranges of most terrestrial and marine species towards the poles, expanding the range of warmer-water species and contracting that of colder-water species. The most rapid changes in fish communities will occur with pelagic species that are expected to shift to deeper waters to counteract rising surface temperatures. Moreover, the timing of many animal migrations will be affected. Ocean warming will also alter the predator–prey matches because of the differential responses between plankton components (some responding to temperature change and others to light intensity).

Inland waters are also warming but there are differential impacts of climate change on the river runoff that feeds these waters (FAO, 2010). In general terms, high-latitude and high-altitude lakes will experience reduced ice cover, warmer water temperatures, a longer growing season and, as a consequence, increased algal abundance and productivity. In contrast, some deep tropical lakes will experience reduced algal abundance and declines in productivity, probably owing to reduced supply of nutrients. Regarding freshwater systems in general, there are also specific concerns over changes in

General introduction timing, intensity and duration of floods, to which many fish species are adapted in terms of migration, spawning and transport of spawning products, as a result of climate change.

1.5 Changes in temperature and thermal zones 1.5.1 Mild changes in temperature and related terminology The definitions and basic concepts of mild changes in temperature and related terminology have been described in this thesis in context of fish and aquatic environment. Heat stress can be defined as the total temperature or heat related load on the fish from both the water environment and metabolic activities within the fish body. The effect of heat stress can be mild or severe depending on the degree of changes in temperature and the exposure time. Mild heat stress refers to the moderate level of heat stress experienced by the fish under natural conditions or in laboratory, which can contribute to heat tolerance, adaptation and survival of the fish without prominent damage to their physiological activities. Heat strain refers to the response of the fish to the heat stress, mainly in terms of a function of the biochemical and physiological adjustments under the exposure to the changes in temperature. The mild heat strain of the fish depends on the biological requirement factors, adaptation during acclimation period, degree of changes in temperature, and exposure time. Hence, the heat strain of fish can be variable for the same level of heat stress caused by exposure to changes in water temperature. The “mild changes in water temperature” was used throughout this thesis and refers to mild increase in water temperature from the acclimation temperature or reference temperature at a certain rate to a higher temperature. The higher or the changes in water temperature model was tested mainly within the temperature preference area of the experimental fish in this thesis. The changes in water temperature does not refer to the usual heat stress model tested at changes in temperature that ranges outside the temperature preference area for a short time.

1.5.2 Thermal preference area and preferred temperature All fish have ranges of environmental temperature most suitable and comfortable for their survival referred as thermal preference area or thermoneutral zone, which varies from one species of fish to another (Figure 3). The lower and upper ends of the thermoneutral zone are restricted and bounded by low and high avoided temperatures, respectively. At low acclimation temperatures, the organisms have the largest thermal preference interval that diminishes upon increasing that of acclimation as reported in longarm river prawn (Macrobrachium tenellum) (Hernández-Rodríguez and Bückle- Ramirez, 1997), Procambarus clarkii (Espina et al., 1993), allegheny crayfish (Orconectes obscurus) (Mathur et al., 1982), and in some fish (Cherry et al., 1975).

General introduction

The preferred temperature of a species, located within the thermal preference area, is the average optimum temperature most preferred by the species as a function of their thermal acclimation history. This is the most ideal temperature for the performance of the species in terms of growth, feed conversion efficiency, biochemical, and physiological potentials among others. The preferred temperature, also the final preferendum of the species, is the temperature at which the species are stable, free of stress and maximize their physiological processes (Buckle et al., 1996).

1.5.3 Heat tolerance and resistance The heat tolerance area (area of low and high avoided temperatures) is the area limited by the LILT and UILT, bounds the thermal preference area (Figure 3). The heat resistance area is the area limited by the CTMin and CTMax, that is, the thermal area between UILT and CTMax or LILT and CTMin. Like most animals, fish do not have a fixed heat tolerance (Bilyk and DeVries, 2011) or heat resistance (Brett, 1958) and the response and sensitivity to high and low lethal temperatures can be modified by prior thermal history of an individual.

It is well documented that heat tolerance varies apparently between species in different climatic regions and also known to exist between species within biomes (Cossins and Bowler, 1987; Bilyk and DeVries, 2011). The range of heat tolerance of Antarctic fishes is narrower when compared to temperate and tropical fishes (Somero and DeVries, 1967; Elliot, 1981). In heat tolerance studies, the use of survival time of fish at different temperatures as a measure of heat tolerance is an important parameter for comparison between fish from Antarctic, temperate and tropics. Fish exposed to changes in regular daily temperature are characterized by higher heat tolerances than are those acclimated to constant daily mean temperatures (Heath, 1963; Feldmeth et al., 1974; Otto, 1974).

1.5.4 Incipient lethal temperature and critical thermal maximum Fish can experience, in the water environment, temperatures outside of their thermal preference area, which can be lethal for an extended period (upper incipient lethal temperature, UILT) or immediately lethal (critical thermal maximum, CTMax) (Brett, 1956) (Figure 3). The CTMax is the highest temperature that would induce strong stress in the fish at which 50% of the fish lose the ability to escape the stress conditions and ultimately die in short time (Cox, 1974). Fry et al. (1942) defined the incipient lethal level of temperature as the temperature beyond which 50% of the population can no longer survive for an indefinite period of time. The incipient lethal level of temperature in the cold end of the thermal tolerance is the lower incipient lethal temperature (LILT) and that in the hot end is the upper incipient lethal temperature (UILT) (Rajaguru and Ramachandran, 2001).

General introduction

Figure 3. Complete scheme of thermal zones and temperatures (Fry, 1947; Brett, 1956; Giattina and Garton, 1982; Hernández-Rodríguez and Bückle-Ramirez, 1997; Rajaguru and Ramachandran, 2001; Rajaguru, 2002). Preferred temperature (PT), thermal preference area (TPA), higher and lower avoided temperature (HAT, LAT), heat and cold tolerance area (HTA, CTA), upper and lower incipient lethal temperature (UILT, LILT), heat and cold resistance area (HRA, CRA), and Critical thermal maximum and minimum (CTMax, CTMin).

1.5.5 Preferred temperatures of experimental animals In the experimental studies of this thesis, presented in the Chapters three to seven, common carp (Cyprinus carpio), Nile tilapia (Oreochromis niloticus), and European sturgeon (Acipenser sturio L. 1758) were used as experimental animals. Background information has been presented here to introduce the preference or optimum temperatures of these experimental animals.

The common carp, Cyprinus carpio L., is native to Eastern Europe and Central Asia and is distributed in freshwater habitats around the world (Barus et al., 2002). Common carp belongs to cyprinids such that they can tolerate a very wide temperature range; an adult common carp can survive temperatures from 1 to 35oC (Billard, 1999). In common carp, spawning occurs at temperatures of 20°C and in European common carp when water temperature reaches 17-18°C (Flajšhans and Hulata, 2007). The best growth is obtained in common carp at optimum water temperature of 23-30°C. Common carp is 12

General introduction also less sensitive to changes in dissolved oxygen (DO) level, and can be efficiently cultured even at DO level of 3-4 mg/L and can survive low oxygen concentration (0.3-0.5 mg/L, death may occur) as well as supersaturation (Horváth et al., 2002).

Nile tilapia water temperature requirement was summarized by Balarin and Haller (1982), Chervinski (1982), Philippart and Ruwet (1982), and Wolfarth and Hulata (1983). The preferred water temperature ranges between 28oC and 35oC with optimal growth between 29oC and 31oC; no reproduction occurs below 20oC and growth is poor between 10oC and 15oC. The optimum spawning temperature for Nile tilapia is between 25oC and 30oC (Mironova, 1977); reproduction slows between 21oC and 24oC, starts at 22oC, stops below 20oC, and growth is poor between 10oC and 15oC. Tilapia can tolerate temperatures up to 40oC, but experience stress-induced disease and mortality when temperatures exceed 37oC or 38oC (Allanson and Noble, 1964; Wohlfarth and Hulata, 1983) or at temperatures lower than 17oC or 18oC. Temperatures ranging from 18oC to 35oC can be tolerated without any apparent ill effects. The upper lethal limits are near 42oC, and lower lethal limits are approximately 8oC to 12oC. Nile tilapia can survive routine dawn DO levels of less than 0.5 mg/L, for instance, to the level of 0.1 mg/L (Stickney, 1986a). Survival in water with low DO (<1 mg/L) is due to either their ability to use oxygen at the air-water interface or their physiological adaptation to optimize oxygen utilization at low environmental levels. Generally DO should be maintained above 2 mg/L at dawn because exposure for prolonged periods depresses metabolism and growth, and frequent exposure to low oxygen is stressful and lowers disease resistance (Teichert-Coddington and Green, 1993). Nile tilapia are amongst the most popular and promising fish for warm water aquaculture (Harpaz et al., 1999).

The spawning temperatures for European (Common or Atlantic) sturgeon range from 7.7 to 22°C (Chebanov and Galich, 2013) and the optimum spawning water temperature is 20°C (Williot et al., 2011). European sturgeon were reportrd to be reared at very low light intensities, a natural photoperiod and an annual temperature variation between 25 and 10°C and at constant temperatures around 20°C at high light intensities, and with a natural photoperiod (Williot et al., 2011). The preferred water temperature for best growth of European sturgeon is 20°C.

1.6 Effects of changes in temperature on thermal zones and fish Global thermal change might result in an increase of average 0.3-5°C in the next century (Thia-Eng and Paw, 1989), which will markedly impact the habitats of the aquatic poikilotherms. The long term increase in the global temperature will modify the thermal preference area of the animals whereby the most sensitive and immediate avoided temperatures will be altered first and the preferred temperature 13

General introduction maintained until next modification occurs (Hernández-Rodríguez and Bückle-Ramirez, 1997). This shifts the thermal preference area of the ectothermic organisms in the direction of the effect of the acclimation temperature to respond positively or negatively and extend or reduce their distribution pattern as reported in Macrobrachium tenellum (Hernández-Rodríguez and Bückle-Ramirez, 1997; Thia-Eng and Paw, 1989) and frogs, salamander, and caecilians (Wyman, 1991). Rajaguru and Ramachandran (2001) reported from the study in three species, Etroplus suratensis, Therapon jarbua and Ambassis commersoni that fishes acclimated at high temperatures had higher low lethal temperatures than those acclimated to low temperatures.

Water temperature plays the most important role in the development and growth of fish (Piper et al., 1982), but exposure of fish to temperature above an optimal range for growth, disease tolerance and reproduction induces a stress response leading to declined feed utilization and performance (Cossins et al., 1995; Ficke et al., 2007; Katersky and Carter, 2005). The occurrence of the thermal stress affects the metabolic and physiological functions in maintaining adaptive metabolic responses (Beitinger et al., 2000; Elliott, 1981; Goldspink, 1995) resulting in increased demand for energy production, changes in metabolic rate and decreased in individual endurance (Wendelaar-Bonga, 1997; Portz et al., 2006). The decline in performance of fish exposed to changes in temperature is mainly due to an increase in turnover of endogeneous protein although their synthesis is declined (Carter et al., 2006).

One of the important metabolic changes in fish due to rise in water temperature is the metabolism of AA. The response of two main transaminases (glutamate oxaloacetate and glutamate pyruvate transaminases) to temperature change in fish (Jürss, 1979) cannot be denied. The rise in water temperature can be a seasonal function. The observation of Squires et al. (1979) for plasma AA in the winter flounder (Pseudopleuronectes americanus) indicated that the total amino acid concentrations decreased in winter and increased during summer. Woo (1990) also reported an increase in total plasma amino acid concentrations of warm acclimated red seabream (Pagrus major), while total amino acid concentrations in gills and kidney of gilthead seabream (Sparus aurata) were significantly higher during cold acclimation (Vargas-Chacoff et al., 2009b). Metabolic changes associated with low temperature (e.g. elevated lipogenesis) may also influence amino acid metabolism in fish (Ballantyne, 2001).

The biochemical effects of temperature show chemical reactions, and especially enzymatic reactions, are temperature dependent (Willmer et al., 2005). With increasing temperature, all chemical reactions run at a higher velocity. A standard temperature quotient (Q10) is a rise in 10°C, which determines

General introduction the temperature dependence of a biological process. The rise of 10°C can increase the velocity of biochemical reactions catalysed by enzymes because of the thermal effects on the reaction of substrates with enzyme catalysts and the consequent faster release of products. In all biological systems, from enzymatic reactions up to whole body processes, Q10 is usually between 2 and 3, which means a 10°C increase of ambient temperature produces a 2-3 fold increase in metabolic rate. Although a rise in water temperature by 10°C was not investigated in this thesis, the effect of the mild changes in water temperature on nutrient metabolism can be related to the concept of Q10 effect.

Temperatures often fluctuate widely in natural environments (Yamanaka et al., 2010); fish often experience seasonal and daily changes in temperature, which can be rapid and large changes with a potential stressor to many fish species (Takahara et al., 2011). Therefore, fish use behavioral responses to select optimal temperatures and simultaneously avoid areas that are suboptimal (Beitinger, 1990). Water temperature has a significant effect on growth, metabolic, and immune function in teleosts (Engelsma et al., 2003; Ruyet et al., 2004).

High water temperatures reduce oxygen solubility such that with increasing water temperature, the dissolved oxygen concentration of water declines, producing hypoxic condition (Tiffany et al., 2010). The high water temperature induced stress produces reactive oxygen species (ROS), the chemically reactive molecules containing oxygen that form as a natural by-product of the normal metabolism of oxygen and have important roles in cell signalling and homeostasis (Cadenas, 1989; Vinegar et al., 2012). The production of ROS during changes in environmental temperature can increase dramatically (Gerschman et al., 1954; Cadenas, 1989). When the production and accumulation of ROS is beyond the organism’s capacity to deal with these reactive species there is oxidative stress, which can damage lipids, proteins and deoxyribonucleic acid (DNA). Some ROS can initiate lipid peroxidation, a self-propagating process in which a peroxyl radical is formed when a ROS has sufficient reactivity to abstract a hydrogen atom from an intact lipid (Halliwell and Gutteridge, 1999). The reaction of ROS with lipids is considered one of the most prevalent mechanisms of cell damage (Halliwell and Gutteridge, 1999). Under most physiological states, ROS production is closely matched by antioxidant responses. Enzymatic antioxidants, such as superoxide dismutases, catalase and peroxidases, form an important part of the antioxidant response (Lesser, 2006).

1.7 Thermoregulation and metabolism in endotherms and ectotherms The response of animals to thermal variation in their environment can be broadly categorized into thermoregulation and regulation of cellular rate functions. Thermoregulation is a neural process that matches information about the external environment with the appropriate animal response to maintain 15

General introduction a more or less stable internal environment relative to external variation (Nakamura and Morrison, 2008). Many vertebrates regulate their body temperature in response to thermal variability of the environment (Seebacher, 2009). Endotherms maintain relatively stable body temperatures through adaptive thermogenesis, a mechanism of heat production by metabolic processes in response to environmental temperature for protection from cold exposure and buffering body temperature from environmental temperature fluctuations (Lowell and Spiegelman, 2000; Morrison et al., 2008). Most ectotherms do not display adaptive thermogenesis, but they do acclimate cellular metabolism to compensate for variation of environmental temperature, which can determine their body temperature and thereby affect metabolism thermodynamically. Like endotherms, however, most ectotherms regulate the metabolic capacity of their tissues in response to longer-term (days to weeks) thermal variation (Guderley, 2004; Seebacher, 2005). Variation in body temperature of ectotherms may result either from environmental variation when body temperature passively follows environmental temperatures or from regulation to different set-points in those animals that do thermoregulate (Seebacher, 2009).

Ectotherms regulate body temperature behaviourally and by cardiovascular modulation of heating and cooling rates (Seebacher, 2000; Seebacher and Grigg, 2001). At the same time, metabolism and other essential rate functions can be regulated so that reaction rates remain relatively constant even when body temperatures vary [a process referred to as temperature compensation, acclimation or acclimatisation (Fry, 1958)]. For example, many fish adjust metabolic capacities to compensate for seasonal variation in water temperature with the result that metabolic performance remains relatively stable throughout the year (Pierre et al., 1998; Wakeling et al., 2000). Reptiles often regulate their body temperature to different levels in different seasons to minimise the behavioural cost of thermoregulation (Seebacher et al., 2003a). At the same time, tissue metabolic capacities are adjusted to counteract thermodynamically-induced changes in rate functions (Seebacher et al., 2003b).

The endothermic adaptive thermogenesis may result from the same regulatory pathways evolutionarily conserved as ectothermic metabolic acclimation, and both could be considered as adaptive metabolic responses to temperature variation. Although metabolic heat production in ectotherms is negligible, the components of the thermoregulatory system are conserved at least between mammals and reptiles. Both groups rely on transient receptor potential ion channels to sense environmental temperatures. The thermosensory proteins [transient receptor potential ion channels (TRPs)] send afferent signals via the dorsal horn to the hypothalamus, resulting in efferent sympathetic outflow to stimulate transcriptional regulators of metabolism (Figure 4).

General introduction

Figure 4. Scheme of thermoregulatory pathways linking cellular metabolism to environmental thermal conditions in endotherms and ectotherms (Seebacher, 2009). Afferent pathways (red) consist of transient receptor potential ion channels (TRP) that sense environmental conditions (heat) and convey thermosensory information to the hypothalamus (red and green in brain) via sensory neurons. The hypothalamus initiates an efferent response (green) mediated by the sympathetic nervous system (NS). Efferent responses include cardiovascular adjustments as well as modulation of cellular metabolism. Adaptive thermogenesis and metabolic acclimation can be mediated by transcriptional controllers such as peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α). Resting metabolic rate of endotherms can also acclimate to changing thermal conditions thereby affecting thermogenesis. The evolutionary conservatism of each component of this scheme suggests that the regulatory pathways are the same in endotherms and ectotherms.

1.8 Nutritional strategies Aquaculture requires comprehensive knowledge and skill of nutrition, feedstuffs, feeds and feeding practices (Hertrampf and Piedad-Pascual, 2000) because aquaculture feed accounts for 30–60% of the total operating budget of aquaculture such that any saving on feed may greatly reduce the total cost and increase returns (Lucas and Southgate, 2012). Fishes are aquatic animals with remarkable taxonomic diversity and wide nutritional requirements, but like terrestrial farm animals, require proteins, lipids, carbohydrates, vitamins and minerals for maintenance, growth, and reproduction. Functional aquafeed additives are specific (micro) dietary ingredients that are supplemented in small amounts (alone or in combination) for such a specific purpose as: (1) to promote the feed intake, 17

General introduction digestion of ingredients and assimilation of nutrients, growth performance, and to improve quality of fish product, (2) to eliminate pathogens, stimulate the immune system, and increase the health status of animals, (3) to maintain the physical and chemical quality of the diet, and (4) to improve the quality of water in the culture system (Hertrampf and Piedad-Pascual, 2000; Bai et al., 2015).

Aquafeed additives are used in fish feed to preserve the nutritional characteristics of a diet or feed ingredients prior to feeding (e.g., antioxidant, mold inhibitors), enhance ingredient dispersion or feed pelleting (e.g., emulsifiers, stabilizers, binders), facilitate feed ingestion and consumer acceptance of the product (e.g., feed stimulants, food colorants), and promote growth (e.g., antibiotics and hormones) (Ajiboye et al., 2012). Consumer interest in the quality and safety of seafood is growing in raising fish in antibiotic-free environments, leading to increased research and use of alternative dietary additives such as natural immunostimulants, immunonutrients, probiotics, prebiotics, and synbiotics (Bai et al., 2015). Although fish species in aquaculture have specific nutrient requirements, the nutrient requirements are affected by the growth rate, growing conditions, and environmental factors. One of the environmental factors that affect the nutrient requirements thereby the performance of fish is changes in water temperature.

Water temperature plays the most important role in the development and growth of fish (Piper et al., 1982), but its rise due to global warming affects the performance of fish (Ficke et al., 2007). The feed utilization and performance of the fish exposed to an changes in water temperature above an optimum level begins to decline (Katersky and Carter, 2005) because their metabolic and physiological functions are affected in maintaining adaptive metabolic responses (Beitinger et al., 2000; Elliott, 1981; Goldspink, 1995). The decline in performance of fish exposed to changes in temperature is mainly due to an increase in turnover of endogeneous protein although their synthesis is declined (Carter et al., 2006).

The responses of fish to the effects of mild changes in water temperature can be observed through feed intake, gut efficiency and metabolic responses. This warrants further research to investigate the effect of the mild changes in water temperature on nutrient metabolism of fish and improve the performance of fish using certain rewarding nutritional strategies such as optimum level of the macro- and micronutrients and potential functional dietary supplements. In this thesis, metabolic responses and gut efficiency of fish exposed to mild changes in water temperature were investigated in Nile tilapia, common carp and European sturgeon. The nutritional strategies studied in this thesis were dietary β-alanine, dietary protein:lipid ratio, and dietary mannanoligosaccharides.

General introduction

1.8.1 Dietary β-alanine β-Alanine (beta-alanine) (β-aminopropionic acid, also known as 3-aminopropanoic acid) was first synthesized by W. Heintz in 1870, only 20 years after the chemical synthesis of α-alanine (Wu, 2013). In 1911, the Russian biochemist W. Gulewitsch discovered that β-alanine is a component of carnosine

(β-alanyl-l-histidine) in beef muscle. β-Alanine (molecular formula: C3H7NO2, molar mass: 89.09 g/mol, melting point: 207oC, density: 1.44 g/cm3) is a naturally occurring beta amino acid in which the amino group is bonded to the β-carbon rather than the α-carbon (Figure 5).

Figure 5. β-Alanine: (A) product image and (B) structural formula.

β-Alanine is formed from six sources (Griffith, 1986): (1) aspartate decarboxylation (primarily in bacteria), (2) hydrolysis of coenzyme A by a combination of several enzymes (phosphomonoesterase, phosphodiesterase, CoA pyrophosphatase, and a type of peptidase), (3) degradation of carnosine by carnosinase, (4) catabolism of pyrimidines through a series of reactions, (5) transamination of malonic acid semialdehyde with glutamate, and (6) polyamine degradation. Concentrations of β-alanine in the plasma are much higher in ruminants than in nonruminants (Kwon et al., 2003; Wu et al., 1995), suggesting an important contribution of bacteria to β-alanine synthesis in the body.

β-Alanine (BA, β-aminopropionic acid, also known as 3-aminopropanoic acid) is a product of aspartate decarboxylation and a constituent of carnosine (β-alanyl-L-histidine), anserine (β-alanyl-1- methyl-L-histidine), carcinine (β-alanyl-histamine), and balenine (β-alanyl-3-methyl-L-histidine) with antioxidative function (Pi et al., 2009; Wu, 2009). β-Alanine is combined with L-histidine to form carnosine; in the synthesis of carnosine, BA is known as a rate-limiting substrate (Derave et al. 2010; Harris et al. 2006; Ng and Marshall 1978) meaning that carnosine levels are limited by the amount of available BA, not histidine. The BA is also a component of CoA, which is important for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle (Wu, 19

General introduction

2009). Supplementation with dietary BA has been shown to increase the concentration of carnosine in muscles (e.g., in yellowtail) (Ogata, 2002). Studies show that carnosine is a muscular dipeptide with physiological roles such as buffering and antioxidant capacity (e.g. Ito et al. 2001; Ogata 2002; Snyder et al. 2008). In poikilothermic animals, imidazole buffers can maintain a certain degree of protonation state over the whole range of environmental temperatures (Van Waarde, 1988), which is important for proper function of many proteins (Somero, 1981). Supplementation of BA can in particular be important in fish at changes in water temperature, the situation in which the fish face challenges of the oxidative stress. This is because dietary BA can affect nutrient metabolism in fish at the changes in water temperature either directly (component of CoA) or indirectly (component of histidine-related compounds). Thereore, BA can be a potential functional dietary supplement that can be added to fish diet and act inside the metabolism of fish.

1.8.2 Dietary protein:lipid ratio Protein levels in aquaculture feed range from 25–60% crude protein because proteins are the most important components of the body of animals, representing 65–85% of the body composition of fish (Jauncey, 1982). As a nutritional strategy, optimal levels of protein and protein:lipid ratio, using nonprotein digestible energy sources such as lipids, are necessary to produce maximum growth and reduce feed costs (Bautista, 1986). The optimum protein:lipid ratio is an environmentally friendly nutritional strategy, which avoids excess protein in the diet thereby decreases the amount of excreted ammonium (Shiau and Chou, 1991). The content of protein in the diets can be reduced through a suitable balance between protein and lipid. Lipids are nutrients with more concentrated energy source and have 2.25 times more energy per unit weight of proteins and carbohydrates. Fish are aquatic animals evolved and adapted to nutrition of aquatic environment, which is rich in proteins and lipids, but scarce in carbohydrates; proteins are the preferred energy source, followed by lipids, while carbohydrates are poorly stored and utilised by fish (Lucas and Southgate, 2012).

Variations in protein requirements of fish can be associated with whether the fish are carnivores and omnivores such that carnivorous fish require 40–50% protein while omnivorous fish require 25–35% (Wilson, 2002). Fish nutrient requirements variations can also be associated with whether the fish are of coldwater or warmwater species because fish species living in warm tropical and subtropical climates require less protein and carbon (Molina-Poveda, 2016). Thus, most warmwater fish require diets with protein levels of 30–36% while most fish species require diets with high protein levels of 23–55% in successful fish feed.

General introduction

Fish species typically require omega-3 and 6 (ω-3 and ω-6) polyunsaturated FAs (PUFAs), but the requirements for the ω-3 are greater than the ω-6 (Molina-Poveda, 2016). Freshwater fish can synthesize C20 or C22 highly unsaturated FA (HUFA) from C18 PUFA by a series of chain elongation and desaturation reactions; thus, their HUFA requirements are met by PUFAs, α-linolenic acid (ALA, C18:3ω-3), and linoleic acid (LA, C18:2ω-6). However, marine fish lack enzymes to elongate and desaturase C18 FA or present a reduced capability (Mourente and Tocher, 1993; Ghioni et al., 1999), consequently they require eicosapentaenoic acid (EPA, C20:5ω-3) and docosahexaenoic acid (DHA, C22:6ω-3) (Kanazawa and Teshima, 1979; Sargent et al., 2002). The higher requirements for ω-3 FAs in fish is necessary to maintain membrane fluidity for the proper functioning of the membranes, for instance at lower environmental temperatures.

The requirements of less protein in warm climates and higher ω-3 FAs at lower environmental temperatures in fish species indicate variations in requirements for protein:lipid ratio at changes in water temperatures. This warrants the need to evaluate the optimum dietary protein:lipid ratio required for fish at changes in water temperature.

1.8.3 Dietary mannanoligosaccharides Dietary mannanoligosaccharides (MOS) is one of the most common prebiotics used as fish functional dietary supplements such as inulin, fructooligosaccharides (FOS), short-chain fructooligosaccharides (scFOS), oligofructose, galactooligosaccharides (GOS), trans-galactooligosaccharides (TOS), xylooligosaccharides (XOS), arabinoxylooligosaccharides (AXOS), isomaltooligosaccharides (IMO), and various commercial products containing multiple prebiotic combinations (Ringø et al., 2010, 2014). Prebiotics are non-digestible feed ingredients that are not metabolized in small intestine. Most prebiotics can be fermented in large intestine by specific health-promoting bacteria, but MOS, the component of yeast cell wall is not fermentable and works through competition with the binding sites for pathogenic bacteria (Ewing, 2008; McDonald et al., 2010) (Figure 6). Prebiotics are known to enhance stress tolerance and disease resistance (Gatlin III, 2002), change gut morphology and oxidative stress (De Los Santos et al., 2005), decrease amino acid catabolism and affect voluntary feed intake (Verbrugghe et al., 2009). Furthermore, prebiotics used in aquaculture to improve growth performance, survival, feed conversion, digestibility, gastrointestinal enzyme activities, immune functions and the presence of beneficial gut bacteria as well as the suppression of potentially pathogenic bacteria (Ringø et al., 2014). Therefore, the use of dietary MOS as a functional dietary supplement in fish can enable the fish cope with the challenges at changes in water temperature, for instance, to enhance the nutrient metabolism.

General introduction

Figure 6. Mode of attachment of bacteria to the gut wall and mode of action of mannanoligosaccharides (Ewing, 2008; McDonald et al., 2010). (A) A mixed population of bacteria with substantial attachment of pathogenic bacteria. (B) Competitive exclusion of pathogens due to preferential attachment of non-pathogens. It should be noted that the recognition of receptor sites (carbohydrates) by the bacterial fimbriae (lectins) is very specific to different types of organism.

1.9 Conclusion Global aquaculture is the fastest growing animal food-producing sector, but its productivity has been affected by global warming. Global warming causes a mild elevation in water temperature, which affects overall physiology and performance of fish, thereby the global fish production. The metabolic responses of individual fish to the mild changes in water temperature are either not well investigated or poorly described. This is because most physiological studies apply strong-short-term heat stress instead of the constant mild changes in water temperature that is happening due to global warming. More research efforts are needed to know the effects of the mild changes in water temperature in fish and the metabolic responses of individual fish, which should be supported with nutrition. Therefore, the demonstration of effects of the mild changes in water temperature in fish, individual metabolic reactions, and evaluation of model nutritional strategies warrant further research.

Chapter 2 Scientific aims and objectives

Scientific aims and objectives

Global aquaculture continues to be the fastest growing animal food-producing sector. Its contribution to global food fish supply for human consumption reached 50 % in 2014. Although the food fish supply from aquaculture is increasing, there is growing demand linked to population growth and continued reduction into non-food uses. Global aquaculture is also vulnerable to adverse impacts of natural, socioeconomic, disease, environmental and technological conditions. Furthermore, there are increased anthropogenic effects on the terrestrial and aquatic environments, which led to global warming.

Global warming causes a mild increase in water temperature, especially in the tropical belt and affects the overall physiology and reduces the feed utilization and performance of fish. To maintain the performance of fish under the changes in water temperature needs understanding of the effects in the physiology of the animal and responses to nutritional strategies. The impact of this mild temperature elevation on aquatic organisms is well-documented on an ecological scale. The individual metabolic reactions to such event are however poorly described, because most physiological studies apply heat stress (short-term, strong temperature increase) as a model instead of the mild but constant elevation of water temperature that is happening due to global warming.

The major scientific aim of this thesis was to investigate the effect of mild changes in water temperature on nutrient metabolism in fish, with an evaluation of nutritional strategies to improve the nutrient metabolism of fish at mild changes in water temperature. The specific objectives of the thesis to address the major scientific aim were:

1. To investigate the effect of mild changes in water temperature in fish with focus on nutrient metabolism in skeletal muscle.

2. To evaluate β-alanine as a dietary metabolic modulator in fish at mild changes in water temperature.

3. To evaluate the effect of dietary macronutrient profile on nutrient metabolism in fish at mild changes in water temperature.

4. To evaluate the effect of mannanoligosaccharides as a dietary intestinal modulator in fish at mild changes in water temperature.

Chapter 3 Nutrient metabolism in fish at mild changes in water temperature

Chapter 3

The metabolic response in fish to mild changes in water temperature relates to species- dependent muscular storage of free amino acids and imidazole compounds

Geda, F., Declercq, A.M., Remø, S.C., Waagbø, R., Lourenço, M., Janssens, G.P.J.

Submitted manuscript

Fish species differ substantially in their muscular concentrations of imidazoles and free amino acids (FAA). This study was conducted to investigate whether metabolic response to mild changes in water temperature (MCWT) relates to species-dependent muscular concentrations of imidazoles and FAA. Thirteen carp and 17 Nile tilapia, housed one per aquarium, were randomly assigned to either acclimation (25°C) or MCWT (30°C) for 14 days. Main muscular concentrations were histidine (HIS; P<0.001) in carp versus N-α-acetylhistidine (NAH; P<0.001) and taurine (TAU; P=0.001) in tilapia. Although the sum of imidazole (HIS+NAH) and TAU in muscle remained constant over species and temperatures (P>0.05), (NAH+HIS)/TAU ratio was markedly higher in carp versus tilapia, and decreased with MCWT only in carp (P<0.05). Many of the muscular FAA concentrations were higher in carp than in tilapia (P<0.05). Plasma acylcarnitine profile suggested a higher use of AA and fatty acids in carp metabolism (P<0.05). On the contrary, the concentration of 3- hydroxyisovalerylcarnitine, a sink of leucine catabolism, (P=0.009) pointed to avoidance of leucine use in tilapia metabolism. Despite a further increase of plasma longer-chain acylcarnitines in tilapia at MCWT (P=0.009), their corresponding beta-oxidation products (3-hydroxy-longer-chain acylcarnitines) remained constant. Together with higher plasma non-esterified fatty acids (NEFA) in carp (P=0.001), the latter shows that carp, being a fatter fish, more readily mobilises fat than tilapia at MCWT, which coincides with more intensive muscular mobilisation of imidazoles. In conclusion, there are differences in metabolism between species, and even interaction between species and temperature, which was demonstrated by changes in muscle imidazole to taurine ratio.

Chapter 3

3.1 Introduction

Exposure of fish to elevations in water temperature can occur due to global warming (Ficke et al., 2007) or because aquaculture is increasingly established in warm countries (FAO, 2014). Fish are poikilothermic ectotherms, whose metabolism and growth is influenced by the water temperature and also farming conditions such as food availability, uptake and utilization (Jobling, 1994). The elevations in water temperature increase basal metabolism that results in a higher energy demand for maintenance and a less energy available for growth (Jobling, 1994). Previous studies have shown that changes in water temperature can lead to changes in nutrient metabolism; for example, it can result in an increased breakdown of amino acids (AA) in fish (Geda et al., 2012). However, there is uncertainty about whether these AA are derived from the diet or from free AA (FAA) concentrated in muscle.

Fish tissues, like that of other animals, contain FAA and non-protein nitrogenous compounds (imidazole compounds, taurine, trimethylamine oxide and other methylamine compounds) (Van Waarde, 1988). The imidazole compounds are the major non-protein nitrogenous constituents, which occur in skeletal muscles of vertebrates (Crush, 1970; Abe, 1983a, 1983b). There are five imidazole compounds in skeletal muscle of fish: histidine (HIS), carnosine (β-alanyl-L-histidine), anserine (β- alanyl-L-methyl-L-histidine), ophidine (balenine; β-alanyl-3-methyl-L-histidine), and N-α- acetylhistidine (NAH) (Van Waarde, 1988; Yamada et al., 2009). The imidazole compounds have a number of biological roles such as H+ buffer (Sewell et al., 1992; Abe, 2000), neurotransmitter (Petroff et al., 2001), non-enzymatic free-radical scavenger (Guiotto et al., 2005), antioxidant (Boldyrev et al., 2004) and a blood glucose regulator (Sauerhofer et al., 2007).

The distribution and abundance of imidazole compounds vary between fish species. For instance, skeletal muscle of carp (Cyprinus carpio) contains higher levels of HIS (Van Waarde, 1988) whereas that of Nile tilapia (Oreochromis niloticus) has higher levels of NAH (Yamada et al., 1992). It is not fully elucidated why animals store high concentrations of the FAA and imidazole compounds in their muscles (Shiau et al., 1997). The reason for the difference in the distribution patterns of the imidazole compounds in the skeletal muscle among fish species is also not yet understood. To our knowledge, it has not been investigated whether the different muscular concentrations of FAA and imidazole compounds would determine the use of nutrients in metabolism of fish at changes in temperature.

When fish cannot store high amounts of body fat (e.g., carp store more fat than tilapia: Abdelghany and Ahmad, 2002), as in many terrestrial animals, readily available muscular concentrations of FAA and imidazole compounds might serve as a rapid source of energy. The species difference in muscular

Chapter 3 concentration of imidazole compounds and their metabolic response to changes in water temperature would relate to the difference in body fat concentration between species. Therefore, due to the aforementioned reasons, carp and Nile tilapia were used as model fish in the present study to investigate the metabolic response of fish to a mild changes in water temperature (MCWT).

3.2 Materials and methods

3.2.1 Animals and experimental design

Thirteen carp (Cyprinus carpio) and 17 Nile tilapia (Oreochromis niloticus) were acclimated to laboratory conditions, in two batches (batch 1: 6 carp and 6 tilapia; batch 2: 7 carp and 11 tilapia) for two weeks. The fish were individually and randomly allocated in 63L-glass aquaria of 60x30x36 cm (JUWEL Aquarium, Rotenburg, Germany). The acclimated fish were fasted for 24 h, weighed (average initial body weight: 119±4 g tilapia, 123±9 g carp), distributed into the aquaria, and randomly assigned to two groups, a control ("T25") and treatment ("T30"). This experiment was set up using two species of fish under two levels of temperature, in a 2x2 factorial design. All experimental methods and procedures used in this study were approved by the ethics committee of animal experiments at Faculty of Veterinary Medicine, Ghent University.

3.2.2 Feed, feeding protocol and changes in temperature The fish were hand-fed (feed: Benelux NV, Wielsbeke - Ooigem, Belgium) (Table 2) for 14 days at a feeding rate of 1.5% of average wet body weight per fish per day twice at 10:00 and 15:00. The experimental diet was analysed for proximate chemical analysis of dry matter (DM), moisture (M), crude protein (CP), crude fat (diethyl ether-extract, EE), crude fibre (CF) and ash (Table 2). The DM and M contents were determined by drying feed samples in freeze dryer for 24 h and heating in a forced air oven at 103°C to a constant weight. The ash content was determined by combustion of the feed at 550°C. The EE was analyzed with the Soxhlet method (ISO, 1973). The CF was determined using the Association of Official Analytical methods (Method 962.09 and 985.29, 1995). The Kjeldahl method (ISO 5983–1, 2005) was used to determine CP (6.25×N). The percentage of nitrogen-free extract (NFE) was calculated as: NFE = 100–(M+Ash+CP+EE+CF). The T25 group was managed at an acclimation temperature of 25°C and the T30 group was managed at a constant MCWT of 30°C, set-up at a rate of 5°C per 60 h. All the aquaria were maintained at 12:12 h light- dark photoperiod using fluorescent lights controlled by timers.

Chapter 3

Table 2. Chemical composition of the experimental dieta (on as fed basis) Proximate composition (g/kg) Dry matter 922.00 Moisture 78.00 Crude protein 326.20 Crude ash 54.40 Crude fat 55.80 Crude fibre 31.30 NFEb 454.30 Amino acid composition (g/kg) Alanine 19.98 Arginine 19.90 Aspartic acid + Asparagine 31.62 Cysteine 5.46 Glutamic acid + Glutamine 67.55 Glycine 13.98 Histidine 8.11 Isoleucine 15.44 Leucine 34.21 Lysine 16.85 Methionine 6.28 Phenylalanine 18.46 Proline 21.18 Serine 16.51 Threonine 13.23 Tryptophan 3.56 Tyrosine 12.52 Valine 17.06 a Aqua-KI feed, manufactured by Benelux NV, Wielsbeke - Ooigem, Belgium. Vitamin and mineral premix added per kg feed (as given in the commercial feed technical sheet): retinol, 3 mg; vitamin C, 100 mg; cholecalciferol, 0.025 mg; vitamin E, 33 mg; calcium, 6 g; phosphorus, 6 g; sodium, 0.8 g; iron, 100 mg; copper, 2.5 mg; manganese, 15 mg; zinc, 50 mg; selenium, 0.25 mg. List of ingredients reported in Aqua-KI in decreasing order of inclusion: Fish products, products and by- products of oil seeds, vegetal products and by-products, algae, AA, vitamins, minerals, calcium propionate, antioxidants, oils and fats. b NFE = 100–(M+Ash+CP+EE+CF).

Chapter 3

Daily feed intake was determined by removing any uneaten feed after 60 min from each aquarium, drying and weighing as previously described (Geda et al., 2015). All fish were fasted for 24 h during measurements of their initial and final body weights. Daily measured water quality parameters were pH (7.7-8.6 at 25°C; 7.8-8.6 at 30°C) (Merck KGaA, Darmstadt, Germany), dissolved oxygen (4.2- 7.5 mg/L at 25°C; 3.8-6.8 mg/L at 30°C) (Hanna Instruments Srl, Nufalau, Romania), ammonium (<0.05 mg/L at 25°C; <0.05 mg/L at 30°C) and nitrite (0.05-0.40 mg/L at 25°C; 0.05-0.20 mg/L at 30°C) (JBL GmbH and Co KG, Neuhofen/Pfalz, Germany).

3.2.3 Blood sampling, plasma collection and analysis

All fish were fasted for 24 h, euthanized using an overdose of a benzocaine (ethyl 4-aminobenzoate) solution (0.1 g/mL acetone) and subjected to blood sampling. Blood samples were collected from the heart (cardiac puncture) using a 1 mL syringe (Becton Dickinson S.A., Madrid, Spain) and a 26 G needle (Becton Dickinson, Drogheda, Ireland) rinsed with heparin (LEO Pharma, Ballerup, Denmark). The blood in the heparinized plasma tubes was centrifuged at 1200 × g and 5oC for 10 min. The plasma samples were collected and stored at –20°C until analyzed for acylcarnitines and non-esterified fatty acids (NEFA). Acylcarnitine profile of the plasma samples was determined using quantitative electrospray tandem mass spectrometry (Zytkovicz et al., 2001). For determination of NEFA, the plasma samples were analyzed spectrophotometrically (EZ Read 400 Microplate Reader, Biochrom Ltd., Cambridge, United Kingdom) by adding acyl-CoA synthetase, acyl-CoA oxidase and peroxidase (Randox NEFA, Randox Laboratories Ltd., Crumlin, United Kingdom). According to manufacturer’s guidelines, using a commercial analysis kit (Randox FA115), series concentrations (CONC) of standard dilutions were made. These were CONC 0 (1.5 mL eppendorf full of demineralized water), CONC 1 (NEFA calibrator), CONC 1/2 (100 μL CONC 0 + 100 μL CONC 1), CONC 1/4 (100 μL CONC 1/2 + 100 μL CONC 0), and CONC 1/8 (100 μL CONC 1/4 + 100 μL CONC 0). In step two, 10 μL standards (except, 2 × CONC 1 = 20 μL of standard) and 10μL samples were pipetted into microtiter plate. In step three, 50 μL of REAGENT 1 (10 mL R1a added to vial R1b) was added to each well of the microtiter plate and incubated for 10 min at 37°C. Then 100 μL of REAGENT 2 (complete bottle of R2a added to vial R2b) was added to each well of the microtiter plate and incubated for 10 min at 37°C. Finally, absorbance in each well of the microtiter plate was measured at 570 nm using the ADAP Software (Biochrom Ltd., Cambridge, United Kingdom).

3.2.4 Skeletal muscle sampling and analysis After the blood sampling, a sample of the dorsal muscle of about 4.5 cm2 was taken on the left side of the body, 1 cm ventral to the base of the dorsal fin along the lateral line (Geda et al., 2015). The

Chapter 3 muscle samples were stored at –80°C until analyzed. Determination of muscle tissue NAH and HIS concentrations was based on the reversed phase HPLC (Waters Corporation) method (O’Dowd et al., 1990) with slight modification (Breck et al., 2005). Muscle tissues were homogenized in 80% (v/v) ethanol and centrifuged at 2000 g for 20 min. The supernatants were dried in a Termaks incubator (40°C, normal atmosphere) (Termaks, Bergen, Norway), dissolved in phosphate buffer (pH 2.0) and filtered through a membrane filter (0.45 µm). An isocratic reverse phase HPLC was performed, using a 4.6 mm ID ×250 mm column with a silica-based packing (ZORBAX SB-C18, Agilent Technologies AS, Kolboth, Norway) and a Waters 600 E pump (Waters Corporation, Milford, MA, USA). A 0.1 M phosphate buffer (pH 2.0) was used as eluting solvent, with a flow rate of 0.6 mL/min. The concentrations of NAH and His were detected by UV absorbance (Waters 486–Tuneable Absorbance Detector, Waters Corporation) at 210 nm, using external standards.

For determination of free basic AA and total FAA (TFAA) concentrations in the muscle, tissue samples were prepared as described above. After complete drying, samples were dissolved in running buffer (Lithium Citrate Loading Buffer, 80-2038-10, Biochrom Ltd, Cambridge, UK) and AA content was determined by ninhydrin detection with Biochrom 20 Plus Amino Acid Analyser (Biochrom Ltd., Cambridge, United Kingdom) based on low pressure ion-exchange chromatography. Different gradient elution systems were used for identification of either TFAA profiles or, in a shortened version, the profile of free basic AA only. After post-column ninhydrin derivatization, colorimetric detection was made at 570 and 440 nm (Waters 486, Waters Corporation).

3.2.5 Statistical analysis Statistical analysis was conducted using SPSS version 20 (IBM Corporation, Armonk, NY, USA). All data were evaluated for normality using boxplot graphs, analyzed with Linear Mixed Model (LMM), and are expressed as means and standard error of means (SEM). Because of inflated Type I error rate due to multiple comparisons of the main effects, a Bonferroni correction was applied as confidence interval adjustment. Statistical significance was accepted at P<0.05. The effects of temperature, species and their interactions were managed as fixed factors; random effects of batch and aquarium nested within the batch were analyzed in the LMM:

Yijklm = μ + Ti + Sj + (TS)ij + Bk + (AB)l(k) + εijklm where μ is the overall mean; Ti is the ith effect of temperature; Sj is the jth effect of species; (TS)ij is the ijth interaction effect between temperature and species; Bk is the kth effect of batch; (AB)l(k) is the klth interaction effect between batch and aquarium in which the effect of the aquarium is nested within that of the batch; and εijklm is the random error.

Chapter 3

3.3 Results

In the present study, the results indicated that the main muscular FAA concentrations were HIS (P<0.001) in carp; NAH (P<0.001) and taurine (TAU) (P=0.001) in Nile tilapia, but temperature had no effect (P>0.05) (Table 3; Figure 7). The sum of imidazole HIS+NAH and TAU in the muscle remained constant over species and temperature zones (P>0.05), whereas the NAH+HIS:TAU ratio was affected by the temperature × species interaction effects (P<0.05) (Figure 7). The concentrations of muscle FAA (Table 3) and plasma acylcarnitines (Table 4) were different for species with some temperature × species interaction effects (P<0.05). The level of muscular TFAA was significantly reduced (P=0.002) at MCWT. The concentration of 3-hydroxyisovalerylcarnitine was significantly affected by temperature × species interaction effects (P=0.009) (Figure 8). The sum of the concentrations of 3-hydroxy-longer-chain acylcarnitines was constant while that of longer-chain acylcarnitines was affected by both species (P=0.009) and temperature × species interaction effects (P=0.009) (Figure 9). The plasma NEFA concentration was higher in carp than in Nile tilapia (P=0.001) (Figure 10). The feed intake was higher in carp than in Nile tilapia (P=0.005) (Table 5).

Chapter 3

Table 3. Selected skeletal muscle FAA in carp (n=13) and tilapia (n=17) at MCWT (mean, SEM)

Amino acids T25 T30 Pooled P (µmol/g) Carp Tilapia Carp Tilapia SEM T S T×S Alanine 2.62 2.28 2.15 2.46 0.12 0.573 0.963 0.203 Aspartate 0.21 0.97 0.16 0.70 0.08 0.178 <0.001 0.362 Asparagine 0.31 2.91 0.17 1.85 0.28 0.119 <0.001 0.228 Arginine 0.85 0.05 0.50 0.04 0.09 0.217 <0.001 0.238 Ornithine 0.09 0.06 0.08 0.05 0.01 0.555 0.139 0.902 Leucine 0.17 0.09 0.16 0.12 0.01 0.180 <0.001 0.097 Isoleucine 0.075 0.046 0.079 0.064 0.004 0.131 0.003 0.315 Valine 0.104 0.063 0.099 0.091 0.005 0.209 0.010 0.079 Glutamate (Glu) 0.78 0.61 0.79 0.62 0.04 0.918 0.032 0.919 Glutamine (Gln) 1.08 1.18 0.77 1.16 0.07 0.224 0.067 0.272 Glu:Gln 0.79 0.54 1.01 0.57 0.06 0.168 0.001 0.311 Glycine 4.40 8.33 2.82 7.88 0.48 0.047 <0.001 0.257 Serine 0.69 0.18 0.47 0.16 0.05 0.086 <0.001 0.143 Histidine (HIS) 9.15 1.15 7.42 0.78 0.73 0.065 <0.001 0.223 NAH 0.01 1.26 0.06 1.32 0.15 0.784 <0.001 0.982 Lysine 1.69 0.10 1.35 0.14 0.15 0.283 <0.001 0.196 Phenylalanine 0.07 0.03 0.07 0.03 0.01 0.508 <0.001 0.694 Tyrosine 0.07 0.03 0.06 0.04 0.01 0.865 0.001 0.418 Proline 1.84 1.82 0.64 0.64 0.17 <0.001 0.972 0.966 Hydroxyproline 0.18 0.75 0.07 0.49 0.06 0.032 0.000 0.360 Taurine (TAU) 8.96 16.54 11.91 16.76 0.97 0.337 0.001 0.409 Threonine 0.93 0.67 0.55 0.58 0.04 0.001 0.074 0.029 Tryptophan 0.021 0.009 0.013 0.011 0.002 0.275 0.007 0.077 Ammonium 1.24 1.39 1.27 1.40 0.07 0.892 0.366 0.963 Urea 9.34 3.48 7.40 2.40 0.81 0.268 <0.001 0.749 TFAA 44.884 43.977 39.077 39.816 0.831 0.002 0.955 0.583 NAH+HIS:TFAA 0.21 0.05 0.19 0.05 0.02 0.618 <0.001 0.678 TAU:TFAA 0.20 0.38 0.31 0.42 0.02 0.021 <0.001 0.327 T, temperature; S, species; T×S, T and S interaction; NAH, N-α-acetylhistidine; TFAA, total FAA.

Chapter 3

Table 4. Selected plasma acylcarnitines in carp (n=13) and tilapia (n=17) at MCWT (mean, SEM)

Carnitine ester T25 T30 Pooled P (µmol/L) Carp Tilapia Carp Tilapia SEM T S T×S Free 6.68 4.77 6.91 4.88 0.39 0.818 0.012 0.940 Acetyl 3.62 1.31 3.58 1.17 0.28 0.816 <0.001 0.901 Propionyl 0.26 0.04 0.26 0.06 0.25 0.660 <0.001 0.834 Butyryl 0.21 0.13 0.17 0.09 0.02 0.242 0.008 0.976 3OH-butyryl 0.047 0.016 0.047 0.029 0.004 0.368 0.001 0.334 Hexanoyl 0.037 0.031 0.037 0.024 0.003 0.510 0.118 0.562 Octanoyl 0.457 0.033 0.032 0.024 0.003 0.039 0.073 0.685 Decanoyl 0.026 0.046 0.022 0.035 0.003 0.222 0.009 0.581 Dodecanoyl 0.037 0.034 0.035 0.040 0.003 0.817 0.876 0.602 3OH-dodecanoyl 0.013 0.009 0.010 0.006 0.001 0.323 0.169 0.968 Tetradecanoyl 0.026 0.036 0.018 0.048 0.003 0.654 0.001 0.066 3OH-tetradecanoyl 0.006 0.008 0.008 0.006 0.001 0.813 0.997 0.373 Tetradecenoyl 0.037 0.033 0.022 0.039 0.003 0.418 0.287 0.099 3OH-tetradecenoyl 0.011 0.010 0.010 0.008 0.001 0.205 0.205 0.726 Hexadecanoyl 0.05 0.07 0.04 0.10 0.01 0.404 0.001 0.021 3OH-hexadecanoyl 0.010 0.008 0.007 0.009 0.001 0.606 0.976 0.350 Hexadecenoyl 0.037 0.032 0.023 0.036 0.003 0.493 0.574 0.216 3OH-hexadecenoyl 0.011 0.007 0.007 0.008 0.001 0.337 0.337 0.175 Octadecanoyl 0.019 0.033 0.013 0.049 0.003 0.271 <0.001 0.031 3OH-octadecanoyl 0.004 0.004 0.005 0.003 0.001 0.750 0.545 0.492 Octadecenoyl 0.11 0.08 0.06 0.13 0.01 0.895 0.253 0.011 3OH-octadecenoyl 0.006 0.006 0.005 0.006 0.001 0.996 0.783 0.722 T, temperature; S, species; T×S, T and S interaction.

Table 5. Growth performance and feed utilization in carp (n=13) and tilapia (n=17) at MCWT (mean, SEM)

T25 T30 Pooled P Carp Tilapia Carp Tilapia SEM T S T×S Initial weight (g/fish) 117 116 130 121 4 0.350 0.600 0.644 Final weight (g/fish) 127 123 134 127 4 0.545 0.526 0.879 Weight Gain (g/fish) 10 7 4 6 1 0.193 0.603 0.252 Feed intake (g/fish) 17 12 18 13 1 0.462 0.005 0.783 FCR (feed/gain) 1.7 1.7 4.5 2.2 0.8 0.847 0.779 0.099 T, temperature; S, species; T×S, T and S interaction.

Chapter 3

Figure 7. Mean skeletal muscle imidazole (NAH and HIS) and TAU in carp (n=13) and tilapia (n=17) at acclimation (25°C) and mild changes in water temperature (30°C): (A) sum of imidazole and taurine: temperature, species, and interaction effects, P>0.05; (B) imidazole to taurine ratio: temperature effect, P=0.012; species effect, P<0.001; interaction effect, P=0.049. a,b,cMean values with different letters were significantly different (P<0.05).

Chapter 3

Figure 8. Mean plasma 3-hydroxyisovalerylcarnitine, valerylcarnitine and their ratios in carp (n=13) and tilapia (n=17) at acclimation (25°C) and mild changes in water temperature (30°C): (A) 3- hydroxyvalerylcarnitine: temperature effect, P=0.070; species effect, P<0.001; interaction effect, P=0.009; (B) valerylcarnitine: temperature effect, P=0.717; species effect, P=0.007; interaction effect, P=0.724; (C) ratio of 3-hydroxyvalerylcarnitine to valerylcarnitine: temperature effect, P=0.209; species effect, P<0.001; interaction effect, P=0.090. a,bMean values with different letters were significantly different (P<0.05).

Chapter 3

Figure 9. Mean plasma 3-hydroxy-long-chain acylcarnitine and long-chain acylcarnitine and their ratios in carp (n=13) and tilapia (n=17) at acclimation (25°C) and mild changes in water temperature (30°C): (A) sum of 3-hydroxy-long-chain acylcarnitines (3OH-tetradecanoyl, 3OH-tetradecenoyl, 3OH-hexadecanoyl, 3OH- hexadecenoyl, 3OH-octadecanoyl, 3OH-octadecenoyl): temperature, species, and interaction effects, P>0.05); (B) sum of long-chain acylcarnitines (tetradecanoyl, tetradecenoyl, hexadecanoyl, hexadecenoyl, octadecanoyl, octadecenoyl): temperature effect, P=0.946; species effect, P=0.009; interaction effect, P=0.009; (C) ratio of (A) to (B): temperature effect, P=0.407; species effect, P=0.001; interaction effect, P=0.036. a,bMean values with different letters were significantly different (P<0.05).

Chapter 3

Figure 10. Mean plasma non-esterified fatty acid (NEFA) in carp (n=13) and tilapia (n=17) at acclimation (25°C) and mild changes in water temperature (30°C): temperature effect, P=0.765; species effect, P=0.009; interaction effect, P=0.536. a,bMean values with different letters were significantly different (P<0.05).

3.4 Discussion

The results of the present study confirm earlier findings that mild elevation of water temperature affects nutrient metabolism in fish (Geda et al., 2012, 2015). The study reported how the different muscular concentrations of FAA and imidazole compounds determine the use of nutrients in metabolism of fish at MCWT, a situation where changes in AA catabolism were triggered.

In Atlantic salmon exposed to normal (13°C) or changes in temperature (19°C), both muscle and plasma TFAA concentrations were lower in the changes in temperature groups, measured at 4–24 h postprandially (Vikeså et al., 2015). This agrees with the MCWT-induced reduction of muscular TFAA in our study. Yet, the concentrations of muscle FAA profile were quite different between both species, with some increasing and some decreasing from tilapia to carp, and, importantly, with some temperature-species interactions.

The most abundant amino acid in both carp and tilapia muscle was taurine with significant difference between the species, the higher concentration being in the tilapia. Taurine was also reported as the most abundant AA among the FAA profiles in marine animals (Park et al., 2002); its abundance in fishmeal was also documented (El-Sayed, 2013). Taurine can be synthesized from methionine and cysteine in fish (Goto et al., 2003). Taurine plays important roles in numerous biological processes such as antioxidation (free radical scavenge to reduce intracellular oxidation), osmoregulation (reduction of membrane permeability) and lipid metabolism (reduction of lipid peroxidation), thereby

Chapter 3 protecting tissue from oxidative injury (Lin and Xiao, 2006; Kim et al., 2008; Cheng et al., 2011; Zhu et al., 2011).

This study demonstrated that temperature-induced mobilization of muscular HIS and NAH pools seems to be compensated by TAU, possibly to safeguard the osmolytic capacity of these three compounds. The concentration of muscle free HIS was more than 8 fold higher in carp than tilapia, while muscle NAH concentration was higher in tilapia compared to carp. The NAH concentration in Nile tilapia muscle was lower than previously reported (Yamada et al., 1992, 1994, 2009), and only low concentrations were found in Cyprinidae (carps) as previously reported (Yamada et al., 2009). However, water temperature did not affect the concentration of NAH in the muscle, whereas the concentration of HIS tended to decrease in both species at the MCWT.

Histidine is an important buffer component in muscle (Abe and Okuma, 1991; Abe, 2000). It has been demonstrated that histidine and other imidazole compounds are released from muscle tissue during starvation (Shiau et al., 2001), yet the effect size is considerably different between the two species in the present study, with carp showing a greater extent of muscular free imidazole decrease than tilapia. The present data also suggest that any change in muscular concentration of imidazole compounds is compensated by taurine. Such compensations can be explained by the necessity of the fish to maintain osmolytic capacity in its muscles. Taurine is indeed known as an osmolyte (Zhang et al., 2006). Imidazole compounds have also been proposed to play a role in for instance osmotic balance, but also as antioxidant, as antiglycation agent or as precursor of urocanic acid as natural sunscreen for the skin (Ezzat et al., 2015).

The changes in NAH+HIS:TAU ratio are thus not necessarily related to nutrient use, nevertheless the fatty acid metabolism – as measured via the plasma acylcarnitine profile – showed specific species- temperature interactions. In particular, the longer-chain acylcarnitines, representing the use of fatty acids increased more in tilapia than in carp. This seems paradoxal at first instance with the lower fat stores in tilapia (about 3%) versus carp (about 6%) (Abdelghany and Ahmad, 2002), and the higher plasma NEFA concentration in carp compared to tilapia. However, the 3-hydroxy acylcarnitines of the corresponding fatty acids were unaltered, suggesting that fatty acids might accumulate as they are not pushed into fatty acid combustion to generate acetyl CoA. Therefore, this metabolic profile can be interpreted as a reduced use of fatty acids, especially given the distinctly higher concentrations of short-chain acylcarnitines in carp versus tilapia.

Tilapia seems to display a higher resistance to use AA in metabolism, with lower concentrations of the AA catabolites valeryl and isovaleryl carnitine in plasma, and a further increase of 3-

Chapter 3 hydroxyisovalerylcarnitine concentration in the plasma at MCWT. The corresponding 3- hydroxyisovaleryl CoA is a dead-end sink in the leucine catabolism, hence avoiding the use of leucine as substrate for acetyl CoA in the citric acid cycle. Although carp showed the highest levels of muscular free leucine, the MCWT tended to trigger relatively more leucine release in tilapia, yet without further promoting its use as an energy substrate.

The hampered metabolisation of both the longer-chain fatty acids and leucine would suggest that tilapia derives its acetyl CoA from another source. This study hypothesized that tilapia stores NAH because the hydrolyzed product would not only provide HIS, but also an acetyl moiety that could serve as source for acetyl CoA, but the lower concentrations of plasma acetylcarnitine do not support this.

In general, carp seems to have a more active nutrient metabolism than tilapia, seen the higher concentrations of many acylcarnitines in the plasma. This can be explained by the higher thyroid metabolism in carp compared with tilapia (Geven et al., 2007). This might again relate to the higher HIS turnover in carp: HIS and in particular its metabolite histamine are known as stimulatory compounds (Abe, 1995; Li et al., 2009; Wu, 2009), hence might serve to increase metabolic rate.

The evolutionary reason for the observed differences between carp and tilapia remain uncovered, but do not seem to be associated with for instance muscle fibre type, since the typical difference found in FAA between fibre types in rats (Turinsky and Long, 1990) do not correspond with the difference found between the fish species in this study. This might be an interesting topic for further study. Muscle HIS concentration increases whereas muscle taurine decreases with growth in milkfish (Shiau et al., 1997) but the inverse occurs during starvation (Shiau et al., 2001), which supports the hypothesis that the higher imidazole to taurine ratio (Figure 7) observed in carp compared with tilapia in this study might be explained by metabolic rate.

In conclusion, this first comparative study demonstrated the species-dependent metabolic response to a mild elevation of water temperature of the muscular concentrations of imidazoles and FAA in aquatic organisms. The study compared two fish species (carp versus tilapia) that clearly differed in muscular concentration profile and demonstrated that muscular concentrations of imidazoles and TAU in fish respond to water temperature in a species-dependent manner. The present results point to a species-dependent relationship between nutrient use and muscular concentrations of imidazoles and FAA as a response to water temperature. The results are also expected to give rise to a range of follow-up studies by research groups active in the domains of imidazole-related compounds and global warming.

Chapter 3

This comparative investigation of the effect of mild changes in water temperature, which focused on nutrient metabolism in skeletal muscle in carp and tilapia, was followed by evaluation of dietary β- alanine in chapters 4 and 5, as a dietary metabolic modulator in fish at the mild changes in water temperature.

Acknowledgements

The authors acknowledge Herman De Rycke (Laboratory of Animal Nutrition, Faculty of Veterinary Medicine, Ghent University) for the proximate analysis and Ansynth Service B.V., The Netherlands for AA analysis of the experimental diet. The authors do also acknowledge the late Prof. Brigitte Wuyts (Department of Clinical Chemistry, Microbiology and Immunology, Ghent University) for analysis of the selected plasma acylcarnitines. This study was financially supported by the Flemish Interuniversity Council (VLIR) scholarship of Institutional University Cooperation (IUC) programme between Ghent University (Belgium) and Jimma University (Ethiopia). None of the authors has any conflicts of interest to declare.

Chapter 4 Potential of dietary β-alanine in fish at mild changes in water temperature: Part I

β-Alanine does not act through branched-chain amino acid catabolism in carp, a species with low muscular carnosine storage

Geda, F., Declercq, A., Decostere, A., Lauwaerts, A., Wuyts, B., Derave, W., Janssens, G.P.J., 2015.

Fish Physiol Biochem 41:281–287

This study was executed to investigate the effect of dietary β-alanine (BA) on amino acid (AA) metabolism and voluntary feed intake in carp (Cyprinus carpio) at mild changes in temperature to exert AA catabolism. Twenty-four fish in 12 aquaria were randomly assigned to either a control diet or the same diet with 500 mg BA/kg. A 14-day period at an acclimation temperature (23°C) was followed by 15 days at constant mild changes in temperature (27°C). After the 15 days, all fish were euthanized for muscle analysis on histidine-related compounds (HRC), whole blood on free AA (FAA) and carnitine esters. The carnosine and anserine analysis indicated that all analyses were below the detection limit of 5 µmol/L, confirming that carp belongs to a species that does not store HRC. The increases in FAA concentrations due to BA supplementation failed to reach the level of significance. The effects of dietary BA on selected whole blood carnitine esters and their ratios were also not significant. The supplementation of BA tended to increase body weight gain (P=0.092) and feed intake (P=0.081). The lack of differences in the selected nutrient metabolites in combination with tendencies of improved growth performance warrants further investigation to unravel the mechanism of BA affecting feed intake. This first trial on the effect of BA supplementation on AA catabolism showed that its metabolic effect in carp at constant mild changes in temperature was very limited. Further studies need to evaluate which conditions are able to exert an effect of BA on AA metabolism.

4.1 Introduction An optimal temperature range plays a fundamental role in the development and growth of fish (Cossins et al., 1995; Piper et al., 1982). When fish are exposed to changes in water temperature above the optimum level, thermal stress occurs resulting in increased metabolic rate and energy production, but decreased individual endurance (Portz et al., 2006; Wendelaar-Bonga, 1997). The feed utilization and performance of the fish also begins to decline (Katersky and Carter, 2005) because their metabolic and physiological functions are affected in maintaining thermal equilibrium (Beitinger et al., 2000; Elliott, 1981; Goldspink, 1995), and due to an increase in turnover but a decline in synthesis of endogenous protein (Carter et al., 2006).

An changes in temperature, even within the optimum range, affects amino acid (AA) metabolism in fish; a former study has demonstrated increased AA catabolism in carp at mild changes in temperature (Geda et al., 2012). This induction of AA catabolism, especially under constant conditions in open aquaculture systems, would have economic importance at harvest unless managed in time. Most fish have a metabolism that preferentially oxidizes AA, but AA are inefficient sources of energy; fats generate twice the ATP of AA. The classical technique to increase the dietary protein:lipid ratio might improve the response of the fish to the increased rate of protein turnover (Barnes et al., 2006), but would not be the best option because protein shares the highest cost in the formulation of fish diets.

Therefore, the use of functional supplements that limit AA breakdown and support the fish might be a rewarding strategy to minimize the inefficient energy use at changes in temperature, e.g. in warmer climate countries. A candidate supplement for such purposes might be dietary β-alanine (3- aminopropanoic acid). β-Alanine (BA) is mainly known as the often rate-limiting substrate (Derave et al., 2010; Harris et al., 2006; Ng and Marshall, 1978) that combines with L-histidine in the synthesis of carnosine (β-alanyl-L-histidine). Carnosine is a muscular dipeptide with physiological roles such as buffering and antioxidant capacity (e.g., Ito et al., 2001; Ogata, 2002; Snyder et al., 2008). It is not stored in all animals; hence the BA limited carnosine synthesis pathway would not work in all animals.

Yet, BA is linked to the metabolism of branched-chain AA (BCAA) (Figure 11) (Michal and Schomburg, 2012). These particular AA are preferentially used as substrates for the citric acid cycle, hence crucial in energy metabolism (Costas et al., 2012; Storey, 2004). Through, for instance, the interaction with γ-aminobutyric acid (GABA), BA can be linked to voluntary feed intake (Kim et al.,

2003), which makes it attractive to be used in conditions of changes in water temperature. Therefore, the objective of this study was to investigate the effect of dietary BA on AA metabolism and voluntary feed intake in fish at mild changes in temperature, independent of its role in carnosine.

Figure 11. Overview of metabolic pathways linking BA, BCAA, and GABA. Note that BA and GABA compete for the same enzyme: GABA-T; based on Michal and Schomburg (2012).

Therefore, we executed this study on carp because it was described that this fish species does not accumulate carnosine (Boldyrev et al., 2013). The investigation of the BA metabolic pathway involving BCAA in a species that does not store carnosine or histidine-related compounds (HRC) provides insights in the direct value of dietary BA in relieving the negative effects of changes in temperature on fish metabolism. Any meaningful change in metabolism due to BA supplementation warrants evaluation of its effect on growth performance at highly changes in temperature in future larger scale studies.

4.2 Materials and methods 4.2.1 Experimental animals and design

Twenty-four carp (Cyprinus carpio) were obtained from a local producer in Belgium. The fish were transported in a double polyethylene bag with sufficient oxygen to the Laboratory of Animal Nutrition, Faculty of Veterinary Medicine, Ghent University, Belgium. The fish were randomly placed pairwise in twelve 63L-glass aquaria of 60x30x36 cm (JUWEL Aquarium, Rotenburg, Germany) and fed a commercial carp diet (Benelux NV, Wielsbeke - Ooigem, Belgium) (Table 6) for 14 days of acclimatisation to laboratory conditions. At the end of the 14 days, the fish were weighed (average initial body weight: 206 ± 8 g), randomly distributed pairwise to the 12 aquaria and assigned

to either a control ("CON") or BA ("BA") treatment group, each containing six aquaria. The experimentation, housing and ethical procedures carried out in this experiment were approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University.

Table 6. Nutrient composition of the control dieta

Proximate analysis (g/kg)b Dry matter 922.0 Moisture 78.0 Crude protein 326.2 Crude fat 55.8 Crude fibre 31.3 Crude ash 54.4 NFEc 454.3 a Aqua-KI feed, manufactured by Benelux NV, Wielsbeke - Ooigem, Belgium. Vitamin and mineral premix added per kg feed (as given in the commercial feed technical sheet): vitamin A,

10000 IU; vitamin C, 100 mg; vitamin D3, 1000 IU; vitamin E, 33 mg; calcium, 6 g; phosphorus, 6 g; sodium, 0.8 g; iron, 100 mg; copper, 2.5 mg; manganese, 15 mg; zinc, 50 mg; selenium, 0.25 mg. List of ingredients reported in Aqua-KI in decreasing order of inclusion: Fish products, products and by- products of oil seeds, vegetal products and by-products, algae, amino acids, vitamins, minerals, calcium propionate, antioxidants, oils and fats. b The result of laboratory analysis. c Nitrogen-free extract: %NFE = 100% – (%moisture + %crude protein + %crude fat + %crude fiber + %crude ash).

4.2.2 Experimental diets and changes in temperature The CON group was fed the commercial carp diet (the same diet during acclimatisation) (Table 6) and the BA group was given the same CON group diet supplemented with 500 mg BA/kg (Sigma-Aldrich BVBA, Diegem, Belgium) using Comfort Pressure Sprayer (AVEVE Group, Leuven, Belgium). The carps were hand-fed at a feeding rate of 1.5% of body weight per day. Feeding occurred at 09:00 and 15:00 and the feeding regime remained the same for 15 days. To determine the daily feed intake, any uneaten feed was removed after 60 min from each aquarium, dried and weighed as described elsewhere (Helland et al., 1996; Jimoh et al., 2010). At the end of the acclimatisation period, an acclimation temperature of 23°C was gradually (1°C/12 h) increased to a constant mild changes in temperature of 27°C. All aquaria were maintained at 12:12 h light-dark photoperiod with fluorescent lights controlled by timers.

4.2.3 Water quality parameters Water quality parameters were monitored daily: pH (7.0) (Merck KGaA, Darmstadt, Germany), temperature (27°C) and dissolved oxygen (2.0-4.0 mg/L) (Hanna Instruments Srl, Nufalau, Romania), ammonium (<0.05-0.15 mg/L) and nitrite (0.10-0.20 mg/L) (JBL GmbH and Co KG, Neuhofen/Pfalz, Germany).

4.2.4 Blood sampling and analysis At the end of the 15 days of feeding trial, all fish were fasted for 24 h and the final body weights determined. From each aquarium, two fish were euthanized using an overdose of a benzocaine solution (10 g benzocaine/ 100 mL acetone) and subjected to blood sampling. Blood samples were collected from the heart (cardiac puncture) using a 1 mL syringe (Becton Dickinson S.A., Madrid, Spain) and a 26 G needle (Becton Dickinson, Drogheda, Ireland) rinsed with heparin (LEO Pharma, Ballerup, Denmark). Whole blood was sampled using Whatman 903TM dried blood spot card (Westborough, MA, USA) and the samples were stored airtight at –20°C until analysis for indicators of BA and nutrient metabolism. Acylcarnitine profile and selected free amino acids (FAA) of the dried blood spot samples were determined using quantitative electrospray tandem mass spectrometry (Zytkovicz et al., 2001).

4.2.5 Muscle sampling and analysis After the blood sampling, muscle of about 2.25 cm2 was sampled on the left side of the body about 1 cm ventral to the base of the dorsal fin across the lateral line. The muscle samples were stored at – 80°C until sent for HPLC analysis of HRC – carnosine, anserine and taurine metabolites. Muscle samples from various species are generally analysed for the following metabolites with the standard curves determined prior to analysis: taurine (~12.5 min), because taurine is another abundant β-AA, which competes with BA for incorporation into the muscle; carnosine (~17 min); anserine (~19 min), methylated carnosine because some species possess mainly carnosine, others mainly anserine (Boldyrev et al., 2013).

4.2.6 Statistical analysis Statistical analysis was conducted with One-Way ANOVA using SPSS version 20. The normality of the data was assessed using the Kolmogorov-Smirnov test. Fixed effect of BA treatment was analysed for the dependent variables: selected whole blood FAA, whole blood acylcarnitines, body weight, weight gain, feed intake, and feed conversion ratio. All results were expressed as means and standard

error of means (SEM). Statistical significance was accepted at P<0.05 and P-values between 0.05 and 0.10 were considered tendency.

4.3 Results The carnosine and anserine analysis indicated that all analyses were below the detection limit of 5 µmol/L, confirming that carp belongs to a species that does not store HRC. The increases in FAA concentrations due to BA supplementation failed to reach the level of significance (Table 7). The effects of dietary BA on selected whole blood carnitine esters and their ratios were also not significant (Table 8). The supplementation of BA tended to increase body weight gain (P=0.092) and feed intake (P=0.081) (Table 9).

Table 7. Effect of dietary BA on selected whole blood FAA in carp (mean, SEM, n=6)

Dietary treatment Pooled Amino acids (µmol/L) CON BA SEM P Alanine 528 559 32 0.654 Citrulline (CIT) 24 33 3 0.180 Ornithine (ORN) 41 73 15 0.310 CIT:ORN 0.6 0.5 0.1 0.662 Leucine (LEU) 322 341 18 0.618 Valine (VAL) 278 287 17 0.812 VAL:LEU 0.86 0.84 0.03 0.787 CON, control; BA, β-alanine.

Table 8. Effect of dietary BA on selected whole blood acylcarnitine profile in carp (mean, SEM, n=6)

Dietary treatment Pooled Carnitine ester (µmol/L) CON BA SEM P Free 13.74 17.16 1.07 0.114 Acetyl 11.6 12.9 1.2 0.588 Methylmalonyl 0.04 0.05 0.01 0.332 3-hydroxybutyryl 0.12 0.13 0.01 0.353 Ratios Acetyl:Free 0.9 0.8 0.1 0.515 Methylmalonyl:Valine 0.0002 0.0002 0.000 0.551 3-hydroxybutyryl:Acetyl 0.010 0.011 0.001 0.697 Ʃ3OH-FA:ƩFA* 0.47 0.41 0.04 0.477 TAC:Free 0.99 0.87 0.09 0.512 CON, control; BA, β-alanine; TAC, total acylcarnitine. * Ʃ3OH-FA: [3OHC4+3OHC5+3OHC12+3OHC14+3OHC14:1+3OHC16+3OHC16:1+3OHC18+ 3OHC18:1]; ƩFA: [C4+C5+C12+C14+C14:1+C16+C16:1+C18+C18:1].

Table 9. Effect of dietary BA on growth performance and feed utilization in carp (mean, SEM, n=6)

Dietary treatment Pooled CON BA SEM P Initial weight (g/fish) 202 211 11 – Final weight (g/fish) 213 230 11 0.468 Weight gain (g/fish) 11 18 2 0.092 Feed intake (g/fish) 38 44 2 0.081 FCR (feed/gain) 3.5 2.4 0.5 0.255 CON, control; BA, β-alanine. Note: this table has been revised and updated from its original publication.

4.4 Discussion Our previous study demonstrated that even within the normal temperature range reported for carp, an elevation of water temperature increased AA catabolism (Geda et al., 2012). Although the present study applied the same water temperature regime, with the same fish species and the same diet, the metabolic response of the fish appeared to be different. For instance, as a measure of metabolic rate, the ratio of acetylcarnitine to free carnitine can be considered: this ratio was 1.4 in our previous study, whereas only 0.8 in the present study. We cannot identify the cause of this difference, but the heavier fish in the present experiment had a lower relative growth rate than in the previous study, hence a lower metabolic rate.

Anyhow, the hypothesised effect of BA on AA metabolism was not observed under the present conditions, hence the question remains whether this lack of effect is due to the lower metabolic activity of the fish. The experiment was set up as a small scale in-depth study, hence not powered for performance parameters and balance studies. Yet, the observed tendencies for increased growth rate and feed intake are intriguing. The question arises why these tendencies in increased performance were not reflected in the studied metabolites.

The acylcarnitine and AA profile has been proven sensitive to metabolic changes in several other studies (e.g. sows: Cools, 2013; carp: Geda et al., 2012; cats: Verbrugghe et al., 2009), and the choice of the presented parameters here was based on available biochemical pathways, mainly about the citric acid cycle and BCAA metabolism. The ratios of acetylcarnitine to free carnitine and total acylcarnitines to free carnitine have been described as indicators of metabolic activity (Adams et al., 2009; Bremer, 1983; Ramsay and Arduini, 1993), hence suggesting that BA supplementation had not induced changes in metabolic activity under the present conditions.

Our hypothesis was that BA would interact with the energy generation in the citric acid cycle through its potential effect on pyruvate synthesis and the catabolism of BCAA as displayed in Figure 11, but the similar ratios of 3-hydroxybutyrylcarnitine to acetylcarnitine suggest that the deviation of acetyl coenzyme A to the energetically less efficient ketone synthesis did not differ. The mobilised amount of BCAA such as valine and leucine was also not different, neither was valine catabolised at a different rate (methylmalonylcarnitine:valine). Any effect on puryvate metabolism could have induced a difference in fatty acid combustion to generate acetyl coenzyme A, but the ratio of 3-OH fatty acids to the respective fatty acids cannot support that hypothesis.

We therefore propose an alternative hypothesis that could explain the observed tendencies in feed intake stimulation: BA can promote glutamine (Figure 11), which enhances feed intake (Lin and Zhou, 2006). With this mechanism, BA can antagonise GABA, a known inhibitor of voluntary feed intake (Jonaidi et al., 2002; Kim et al., 2003; Lin et al., 2000). We did unfortunately not measure this compound or its direct neural effects, so this is left as an interesting topic for further study, especially to evaluate if the glutamine pathway can exert differences in feed intake at a larger scale. Furthermore, we could suggest that there might be an evolutionary reason why certain species have not developed muscular storage of dipeptides containing BA (such as carnosine). As a consideration on the present data, species storing carnosine (or eventually anserine) might be better study objects for evaluating the effect of BA supplementation. This might especially be the case under catabolic conditions, e.g. lactation in mammals, moulting in crustaceans, natural phases of feed deprivation encountered in for instance salmon, or particular pathogenic challenges.

In conclusion, this first trial on the effect of BA supplementation on AA catabolism showed that its metabolic effect in carp subjected to a constant mild change in water temperature was very limited. The lack of differences in the selected nutrient metabolites in combination with tendencies of improved growth performance in the present trial warrants further investigation at a production scale to unravel the mechanism of BA affecting feed intake. Further studies are also needed to evaluate which conditions are able to exert an effect of BA on AA metabolism.

The present results confirm that β-alanine does not act through branched-chain amino acid catabolism in carp, a species with low muscular carnosine storage. In chapter 5, sturgeon, a fish species known for muscular storage of carnosine was used to investigate the effect of graded levels of BA on nutrient metabolism at mild changes in water temperature.

Acknowledgements The authors acknowledge Herman De Rycke for proximate analysis of the control diet. The authors are also grateful to the anonymous reviewers for suggesting valuable comments to improve this paper. The authors declare that they have no conflict of interest.

Chapter 5 Potential of dietary β-alanine in fish at mild changes in water temperature: Part II

Chapter 5

Dietary β-alanine does not support muscular carnosine storage in sturgeon but alters nutrient metabolism

Geda, F., Remø, S.C., Waagbø, R., Kalmar, I.D., Cools, A., Vanhauteghem, D., Janssens, G.P.J.

Manuscript in preparation

A study was conducted to investigate the effect of graded levels of dietary β-alanine (BA) on nutrient metabolism in sturgeon at mild changes in water temperature. Fourteen sturgeons were randomly distributed, individually or pairwise in eight aquaria, and assigned to eight feeding groups on diets prepared without (control) or with graded levels of BA for a week. Dietary BA had no effect on serum FAA, but linearly decreased acetylcarnitine (P=0.038) and propionylcarnitine (P=0.010). Remarkably, BA supplementation increased neither BA, nor carnosine in sturgeon muscle, but induced a distinct increase in glycine (P=0.034), glycine to total FAA (P=0.019), citrulline (P=0.031), cystathionine 2 (P=0.031), hence reducing the concentrations of many other amino acids. Also muscle ammonium (P=0.001) was reduced by BA. The changes observed in acylcarnitines point to a down regulation of the citric acid cycle. The marked BA-induced increase in muscle glycine and citrulline suggests a shift in amino acid metabolism that might be beneficial to counteract the increase in amino acid breakdown at changes in water temperature, but a performance trial should evaluate what the reduction of the citric acid cycle means for the overall metabolism in these fish. Sturgeon, as a carnosine-storing fish species, did not show a dose-response in muscular carnosine storage due to BA supplementation, but instead developed a considerable increase in glycine, citrulline and cystathionine, suggesting an important metabolic modulator function of BA.

Chapter 5

5.1 Introduction An optimal temperature plays a fundamental role in the development and growth of fish (Piper et al., 1982; Cossins et al., 1995). Exposure of fish to changes in water temperature results in increased metabolic rate and respiration, ATP breakdown, amino acid (AA) breakdown, production of reactive oxygen species, and decreased endurance (Wendelaar-Bonga, 1997; Portz et al., 2006; Geda et al., 2012). Thermal stress thus affects metabolic and physiological functions in maintaining thermal equilibrium (Elliott, 1981; Goldspink, 1995; Beitinger et al., 2000), and is associated with an increase in turnover but a decline in synthesis of endogenous protein (Carter et al., 2006).

Many functional AA are known to regulate key metabolic pathways, which are important to maintenance, growth, reproduction, and immune responses (Li et al., 2009). A former study was conducted in carp using dietary β-alanine (BA) as a functional AA, because of its biochemical role in for instance valine metabolism (Geda et al., 2015). β-Alanine is often known as the rate-limiting substrate (Derave et al., 2010; Harris et al., 2006; Ng and Marshall, 1978) that combines with L- histidine in the synthesis of carnosine (β-alanyl-L-histidine). Carnosine is a muscular dipeptide with physiological roles such as buffering and antioxidant capacity (e.g., Ito et al., 2001; Ogata, 2002; Snyder et al., 2008). Since carp does not accumulate carnosine in its muscle, BA supplementation could only affect metabolism through other mechanisms than carnosine synthesis.

In the carp study, the dietary BA was thus used as a functional supplement to limit AA breakdown and minimize the inefficient energy use at changes in temperature, but the metabolic effect of the BA on AA catabolism was very limited. To investigate whether the impact of BA would be greater in fish with reported carnosine storage, such as sturgeon (Boldyrev et al., 2013), the present study was conducted in sturgeon to investigate the effect of graded levels of dietary BA on nutrient metabolism at mild changes in water temperature.

Chapter 5

5.2 Materials and methods 5.2.1 Experimental animals and design Fourteen European Sturgeon (Acipenser sturio L. 1758) were acclimated to laboratory conditions for two weeks and were allocated either pairwise or individually in 63L-glass aquaria of 60x30x36 cm (JUWEL Aquarium, Rotenburg, Germany). The acclimated fish were fasted for 24 h, weighed (average initial body weight: 81±19 g). The animals were distributed into the aquaria, and randomly assigned to eight feeding groups on diets prepared with graded levels of BA except for the control where they were managed for a week. The experimental animal protocol was approved by the local ethics committee of animal experiments at Faculty of Veterinary Medicine, Ghent University.

5.2.2 Feed, feeding protocol and changes in temperature The sturgeons were fed on a commercial diet (basal) for the species during the acclimatization period (Table 10). At the onset of the dietary treatments, the fish were assigned to eight feeding groups on diets prepared with graded levels of BA except for the control. The diets were: control (basal, BA0), BA1 (basal + 0.025% BA), BA2 (basal + 0.05% BA), BA3 (basal + 0.075% BA), BA4 (basal + 0.1% BA), BA5 (basal + 0.125% BA), BA6 (basal + 0.15% BA), BA7 (basal + 0.175% BA) (Sigma- Aldrich BVBA, Diegem, Belgium). The BA was sprayed on the commercial pellets using Comfort Pressure Sprayer (Gardena GmbH, Ulm, Germany). The basal diet was analysed for proximate chemical analysis of dry matter (DM), moisture (M), crude protein (CP), crude fat (diethyl ether- extract, EE), crude fibre (CF) and ash (Table 10). The DM and M contents were determined by drying feed samples in freeze dryer for 24 h and heating in a forced air oven at 103°C to a constant weight. The ash content was determined by combustion of the feed at 550°C. The EE was analyzed with the Soxhlet method (ISO, 1973). The CF was determined using the Association of Official Analytical methods (Method 962.09 and 985.29, 1995). The Kjeldahl method (ISO 5983–1, 2005) was used to determine CP (6.25×N). The percentage of nitrogen-free extract (NFE) was calculated as: NFE = 100–(M+Ash+CP+EE+CF). The sturgeons were hand-fed at a feeding rate of 2% (based on FAO recommendation for the species) of average initial wet body weight per day. Feeding occurred at 10:00 and 15:00 and the feeding regime remained the same for a week. To determine daily feed intake, any uneaten feed was removed after 60 min from each aquarium, dried and weighed as described elsewhere (Helland et al., 1996; Jimoh et al., 2010). At the end of the acclimatisation period, an acclimation temperature of 20°C was gradually (1°C/12 h) increased to a constant mild changes in temperature of 24°C. All aquaria were maintained at 12:12 h light-dark photoperiod with fluorescent lights controlled by timers.

Chapter 5

Table 10. Proximate chemical analysis and nutrient composition of the control dieta

Proximate analysis (g/kg) Dry matter 924.50 Crude protein 416.10 Crude fat 99.20 Crude fibre 6.10 Crude ash 73.50 NFEb 329.60 Amino acid composition (g/kg) Alanine 29.30 Arginine 24.89 Aspartic acid + Asparagine 42.07 Cysteine 6.24 Glutamic acid + Glutamine 76.90 Glycine 20.45 Histidine 13.09 Isoleucine 17.59 Leucine 48.18 Lysine 25.26 Methionine 8.84 Phenylalanine 24.93 Proline 25.84 Serine 20.69 Threonine 16.74 Tryptophan 4.74 Tyrosine 16.09 Valine 25.39 a Aqua-KI feed, manufactured by Benelux NV, Wielsbeke - Ooigem, Belgium. Vitamin and mineral premix added per kg feed (as given in the commercial feed technical sheet): Vitamin A,

15000 IU; vitamin C, 200 mg; vitamin D3, 200 IU; vitamin E, 200 mg; calcium, 6.5 g; phosphorus, 7 g; sodium, 2.5 g; iron, 100 mg; copper, 2.5 mg; manganese, 15 mg; zinc, 50 mg; selenium, 0.25 mg. List of ingredients reported in Aqua-KI in decreasing order of inclusion: Fish products, products and by- products of oil seeds, vegetal products and by-products, algae, amino acids, vitamins, minerals, calcium propionate, antioxidants, oils and fats. b Nitrogen-free extract.

Chapter 5

5.2.3 Water quality parameters Water quality parameters were measured daily: pH (7.6-8.9) (Merck KGaA, Darmstadt, Germany), temperature (24°C) and dissolved oxygen (4.9-7.8 mg/L) (Hanna Instruments Srl, Nufalau, Romania), ammonium (<0.05 mg/L) and nitrite (0.02 mg/L) (JBL GmbH and Co KG, Neuhofen/Pfalz, Germany).

5.2.4 Blood sampling, serum collection and analysis At the end of the 7 days of feeding trial, all fish were fasted for 24 h and the final body weights determined. The fish were euthanized using an overdose of a benzocaine (ethyl 4-aminobenzoate) solution (1 g benzocaine/ 10 mL aceton) and subjected to blood sampling. Blood samples were collected from the heart (cardiac puncture) using a 2 mL syringe (Becton Dickinson S.A., Madrid, Spain) and a 23 G needle (BD Microlance, Fraga, Spain) rinsed with heparin (LEO Pharma, Ballerup, Denmark). The blood was sampled in the heparinized serum tubes (Sigma, Bornem, Belgium) and centrifuged at 1200 × g and 5oC for 10 min. The serum samples were collected in 0.5 mL Eppendorf (Netheler-Hinz-GmbH, Hamburg, Germany) and stored at –80°C until analyzed for acylcarnitines or non-esterified fatty acids (NEFA). Acylcarnitine profile of the serum samples was determined using quantitative electrospray tandem mass spectrometry (Zytkovicz et al., 2001). For determination of NEFA, the serum samples were analyzed spectrophotometrically (EZ Read 400 Microplate Reader, Biochrom Ltd., Cambridge, United Kingdom) by adding acyl-CoA synthetase, acyl-CoA oxidase and peroxidase (Randox NEFA, Randox Laboratories Ltd., Crumlin, United Kingdom). According to manufacturer’s guidelines, using a commercial analysis kit (Randox FA 115), series concentrations (CONC) of standard dilutions were made. These were CONC 0 (1.5 mL eppendorf full of demineralized water), CONC 1/4 (100 μL CONC 1/2 + 100 μL CONC 0), CONC 1/2 (100 μL CONC 0 + 100 μL CONC 1), CONC 1 (NEFA calibrator: 5 μL), and and CONC 2 (2 x CONC 1: 10 μL). In step two, 5 μL standards (except, 2 × CONC 1 = 10 μL of standard) and 5 μL samples were pipetted into microtiter plate. In step three, 50 μL of REAGENT 1 (10 mL R1a added to vial R1b) was added to each well of the microtiter plate and incubated for 10 min at 37°C. Then 100 μL of REAGENT 2 (complete bottle of R2a added to vial R2b) was added to each well of the microtiter plate and incubated for 10 min at 37°C. Finally, absorbance in each well of the microtiter plate was measured at 570 nm using the ADAP Software (Biochrom Ltd., Cambridge, United Kingdom).

Chapter 5

5.2.5 Skeletal muscle sampling and analysis After the blood sampling, a sample of the dorsal muscle of about 4.5 cm2 was taken on the left side of the body, 1 cm ventral to the base of the dorsal fin along the lateral line (Geda et al., 2015). The muscle samples were stored at –80°C until analyzed. Determination of muscle tissue HIS concentrations was based on the reversed phase HPLC (Waters Corporation) method (O’Dowd et al., 1990) with slight modification (Breck et al., 2005). Muscle tissues were homogenized in 80% (v/v) ethanol and centrifuged at 2000 g for 20 min. The supernatants were dried in a Termaks incubator (40°C, normal atmosphere) (Termaks, Bergen, Norway), dissolved in phosphate buffer (pH 2.0) and filtered through a membrane filter (0.45 µm). An isocratic reverse phase HPLC was performed, using a 4.6 mm ID ×250 mm column with a silica-based packing (ZORBAX SB-C18, Agilent Technologies AS, Kolboth, Norway) and a Waters 600 E pump (Waters Corporation, Milford, MA, USA). A 0.1 M phosphate buffer (pH 2.0) was used as eluting solvent, with a flow rate of 0.6 mL/min. The concentrations of HIS were detected by UV absorbance (Waters 486–Tuneable Absorbance Detector, Waters Corporation) at 210 nm, using external standards.

5.2.6 Statistical analysis Statistical analysis was conducted using SPSS version 20 (IBM Corporation, Armonk, NY, USA). Individual fish values were used in the statistical analysis. All data were evaluated for normality using boxplot graphs and presented as means and standard error (SE). The correlation between graded levels of dietary β-alanine and parameters of serum and skeletal muscle nutrient metabolism were presented using the Pearson correlation coefficient (r).

Chapter 5

5.3 Results Dietary BA had no significant correlation with serum FAA (Table 11), and within the acylcarnitine profile, only a negative correlation with acetylcarnitine (P=0.038) and propionylcarnitine (P=0.010) (Table 12) was observed.

Table 11. Correlation between dietary β-alanine and selected serum amino acids in sturgeon

Amino acids Serum (µmol/L)a r P Alanine 19021 -0.051 0.863 Citrulline 213 0.058 0.843 Ornithine 164 -0.122 0.677 Leucine 23329 -0.028 0.924 Valine 17022 -0.044 0.882 Glycine 364 -0.271 0.348 Phenylalanine 807 -0.170 0.560 Tyrosine 434 0.044 0.881 Methionine 799 0.202 0.488 a MeanSE (n=8)

Table 12. Correlation between dietary β-alanine and selected serum acylcarnitines in sturgeon

Carnitine ester Serum (µmol/L)a r P Free (C0) 3.20.3 -0.138 0.639 Acetyl (C2) 1.50.2 -0.558 0.038 Propionyl (C3) 0.120.02 -0.664 0.010 Butyryl (C4) 0.190.01 -0.248 0.393 3OH-butyryl (3OHC4) 0.050.01 -0.153 0.600 Malonyl (C3DC) 0.0160.003 0.367 0.196 Methylmalonyl (C4DC) 0.0390.004 -0.403 0.153 Ʃ3OHFA 0.170.02 -0.221 0.448 Ʃ3OHLCFA 0.0200.004 -0.159 0.587 a MeanSE (n=8); Ʃ3OHFA: sum of 3-OH fatty acids; Ʃ3OHLCFA: sum of 3-OH long-chain fatty acids.

In the skeletal muscle, dietary BA positively correlated with the concentrations of glycine (P=0.034), glycine to total FAA (P=0.019), citrulline (P=0.031), cystathionine (P=0.031), and showed tendency for positive correlation of glutamine (P=0.065) (Table 13). The dietary BA negatively correlated with the concentrations of branched-chain AA (valine, P=0.004; leucine, P=0.005; isoleucine, P=0.008), methionine (P=0.009), phenylalanine (P=0.002), tyrosine (P=0.018), tryptophan (P=0.010), and ammonium (P=0.001).

Chapter 5

Table 13. Correlation between dietary β-alanine and selected muscle free amino acids in sturgeon

Amino acids Muscle (µmol/g)a r P Alanine 0.980.10 -0.037 0.900 β-Alanine (BA) 1.40.2 0.015 0.959 Carnosine (CAR) 7.00.8 -0.167 0.568 BA:CAR 0.230.04 0.220 0.450 Anserine 0.0010.001 -0.045 0.880 Aspartate 0.130.01 0.285 0.323 Asparagine 0.050.03 0.373 0.189 Arginine 0.30.1 -0.099 0.737 Citrulline 0.0010.001 0.577 0.031 Ornithine 0.060.02 0.236 0.416 Leucine 0.180.03 -0.705 0.005 Isoleucine 0.110.02 -0.677 0.008 Valine 0.160.02 -0.720 0.004 Glutamate 0.220.04 -0.064 0.828 Glutamine 0.170.03 0.506 0.065 Glycine 2.50.5 0.568 0.034 Serine 0.340.03 -0.056 0.850 Histidine 0.260.07 -0.413 0.143 Lysine 0.290.02 -0.038 0.897 Phenylalanine 0.070.01 -0.757 0.002 Tyrosine 0.080.01 -0.621 0.018 Proline 0.430.14 0.127 0.665 Hydroxyproline 0.190.05 0.231 0.428 Methionine 0.070.01 -0.672 0.009 Taurine 1.870.51 0.082 0.780 Threonine 0.510.06 0.217 0.456 Tryptophan 0.0040.002 -0.662 0.010 Cystathionine 0.050.01 0.577 0.031 Ammonium 3.80.2 -0.770 0.001 Urea 3.40.5 -0.427 0.128 TFAAb 24.60.8 -0.229 0.432 a MeanSE (n=8) b TFAA: total free amino acids

Table 14. Correlation between dietary β-alanine and growth performance and feed utilization in sturgeon

Amino acids MeanSE (n=8) r P Initial weight (g/fish) 815 -0.519 0.057 Final weight (g/fish) 845 -0.417 0.138 Weight Gain (g/fish) 3.31.8 0.317 0.270 Feed intake (g/fish) 8.60.5 -0.386 0.173 FCR (feed/gain) -0.050.70 0.296 0.305

Chapter 5

5.4 Discussion Although change in temperature is known to increase basal metabolic rate in fish (Johnston and Dunn, 1987), dietary BA showed no change in the serum free amino acid profile. Despite the lack of change in peripheral amino acid concentrations, the clear BA-induced decrease in the two major carnitine esters, acetylcarnitine and propionylcarnitine, points to a substantial interference with nutrient metabolism. The corresponding coenzyme A (CoA) esters, acetyl CoA and propionyl CoA, are crucial in the citric acid cycle, possibly indicating that the citric acid cycle was slowed down. The lack of effect on ketones such as beta-hydroxy-butyrate (measured as 3-OH-butyrylcarnitine) yet indicates that there is no specific blockage of the citric acid cycle that would urge for other routes of energy production, and the lack of changes in long-chain fatty acids (LCFA) use also suggest that lipid metabolism remained unaltered. Additional measures such as mRNA expression of selected genes or measurements of energy use in respiration chambers might reveal better insights in the net effect of BA supplementation.

The lack of peripheral changes in amino acid profile contrasted with the profound AA changes seen in skeletal muscle. The marked increase in muscular glycine is likely related to one of its central non- protein functions, including antioxidant defense (Li et al., 2009). The muscle serine concentrations do not support an increased glycine to serine conversion, but is indirectly confirmed by the increased muscular cystathionine. The increased concentration of citrulline and the increased tendency of glutamine concentration showed enhanced ammonia detoxification (Li et al., 2009), both having common routes in biochemistry with glycine. This ammonia detoxification action agrees with the observed reduction in muscular ammonia and urea.

The reduction of concentrations of branched-chain AA (leucine, isoleucine, valine), methionine, phenylalanine, tyrosine, and tryptophan might just be a consequence of the increase of the above mentioned major components, but might still refer to changes in their biochemistry. The reduced concentration of BCAA with increasing dietary BA at mild changes in temperature shows not only reduced balance between the BCAA, but also reduced synthesis of alanine and glutamine, which are important in gluconeogenesis and ammonia detoxification (Wu, 2009). Skeletal muscle concentration of methionine decreased probably because of its increased utilization as antioxidant, energy metabolism in muscle, and osmoregulation (Li et al., 2009; Wu, 2009). The reduced methionine concentration might also be an indication of reduced methionine catabolism, which might be an

Chapter 5 indirect indication of reduced amino acid catabolism in the muscle. Moreover, the reduced concentration of ammonium suggests less protein catabolism.

In conclusion, in the present study, the changes observed in serum AA and acylcarnitines showed the down regulation of the citric acid cycle. β-Alanine surprisingly does not increase carnosine in muscle but instead has profound effect on other muscle amino acids, such as glycine, potentially leading to increased antioxidant activity and reduced amino acid catabolism at changes in water temperature.

The last two chapters, chapter 4 and 5, revealed basic information on the evaluation of β-alanine as a dietary metabolic modulator in carp and sturgeon independently at mild changes in water temperature. In chapter 6, evaluation of the effect of dietary protein:lipid ratio on nutrient metabolism was conducted in Nile tilapia at mild changes in water temperature.

Acknowledgements The authors acknowledge Herman De Rycke (Laboratory of Animal Nutrition, Faculty of Veterinary Medicine, Ghent University) for the proximate analysis and Ansynth Service B.V., The Netherlands for AA analysis of the experimental diet. The authors do also acknowledge Hedwig Stepman (Department of Clinical Chemistry, Microbiology and Immunology, Ghent University) for analysis of the selected serum acylcarnitines.

Chapter 6 Effects of dietary protein:lipid ratio in fish at mild changes in water temperature

Chapter 6

Dietary protein:lipid ratio interacts with the effect of water temperature in Nile tilapia

Geda, F., Quintelier, K., Remø, S.C., Waagbø, R., Kalmar, I.D., Cools, A., Janssens, G.P.J.

Manuscript in preparation

A study was conducted to investigate the effect of changes in dietary protein:lipid ratio on nutrient metabolism of Nile tilapia fed at acclimation or mild changes in water temperature (MCWT). Twenty six Nile tilapia were randomly distributed in 12 aquaria and assigned to either acclimation (22°C) or MCWT (28°C) for 14 days. The protein:lipid ratio interacted with MCWT for the serum concentrations of glycine (P=0.012), phenylalanine (P=0.004), tyrosine (P=0.006), and sum of the serum free amino acids (P=0.037). The protein:lipid ratio linearly affected serum concentrations of freecarnitine (P=0.005), dodecanoylcarnitine (P=0.041), 3OH-hexadecanoylcarnitine (P=0.029), octadecenoylcarnitine (P=0.029), 3OH-octadecenoylcarnitine (P=0.018), sum of 3OH-fatty acids (P=0.042), sum of 3OH-longer-chain fatty acids (P=0.024), and ratio of sum of 3OH-longer-chain fatty acids to sum of longer-chain fatty acids (P=0.036). The temperature-squared protein:lipid ratio interaction quadratically affected serum concentrations of tetradecanoylcarnitine (P=0.003), octadecanoylcarnitine (P=0.001), and sum of longer-chain fatty acids (P<0.001). There was also a quadratic relationship between squared protein:lipid ratio and the serum concentration of hexadecenoylcarnitine (P=0.012). In the skeletal muscle there was a linear relationship between the protein:lipid ratio and the skeletal muscle FAA concentrations of glutamate (P=0.008) and glutamine (P=0.007). The temperature-diet interaction linearly affected the concentrations of muscle ornithine (P=0.046), tyrosine (P=0.007), proline (P=0.012), taurine (P=0.018), and urea (P=0.008). The squared protein:lipid ratio quadratically affected the skeletal muscle β-alanine (P=0.009), asparagine (P=0.031), and threonine (P=0.038). The temperature-squared protein:lipid ratio interaction quadratically affected muscle phenylalanine (P=0.025). In conclusion, this study demonstrated that changes in fish nutrient metabolism occur depending on changes in water temperature and the dietary protein:lipid ratio can affect this metabolism.

Chapter 5

6.1 Introduction Dietary proteins are functional in fish for maintenance, growth and reproduction. The formulation of balanced diets and their adequate feeding are important for successful aquaculture (Pillay, 1990). Protein is one of the major dietary macronutrients that affect the weight gain of fish and feed cost (Lovell, 1989). Dietary energy has a major impact on the utilization of dietary protein in fish because energy affects the quantitative requirements for protein (Wilson, 2002). Too little energy in the diet results in the utilization of dietary protein for energy rather than for protein synthesis, which is wasteful.

On the contrary, excess energy in the diets may result in a lower nutrient intake by the fish, due to lower feed consumption (Metailler et al., 1981; Alsted and Jokumsen, 1989). The fish do not consume enough diet to meet their protein and other nutrient requirements, leading to a reduced growth rate on the one hand, or on the other, in fatty fish (Webster and Lim, 2002). The dietary inclusion of energy yielding nutrients such as lipids and carbohydrates can also reduce the oxidation of protein to lipid, which improves the utilization of dietary protein for growth. Therefore, providing the appropriate energy levels in feeds for fish is essential.

Nowadays, considerable attention has been paid to fish farms releasing high concentrations of nitrogen, phosphorous and other organic compounds into waters. Nutrients that cause water contamination can come either directly from feeds or from fish excrement (Watanabe et al., 1999). One of the possibilities to reduce the input of such substances into waters is to prepare feeds that contain optimally balanced nutritional components that fish are able to assimilate to the maximum (McGoogan and Gatlin, 2000; Cho and Bureau, 2001). As Jahan et al. (2003) reported, reduction of the nitrogen load in post-production waters is possible by applying components with highly digestible protein and a diet with balanced energy content to protein quantity.

In fish, dietary lipids are an important source of essential fatty acids (EFA) for regular growth, health, reproduction, and bodily functions. All vertebrate species, including fish, have an absolute dietary requirement for both n-6 and n-3 polyunsaturated fatty acids (PUFAs) (Tocher, 2010). Studies showed that tilapia appears to have a requirement for n-6 series, linoleic acid (LA, 18:2n-6), or arachidonic acid (ARA, 20:4n-6), and to a lesser extent, a requirement for n-3 fatty acids (ALA, α- linolenic acid) for normal growth and reproduction (Lim et al., 2011), whereas tilapia has high levels

Chapter 6 of ARA, an n-6 PUFA, and DHA, an n-3 PUFA, in muscle, even when these fatty acids are not present in the diet (Huang et al., 1998).

With the rapid development in aquaculture, effective feed targeting the nutritional needs of individual species seems highly demanded under changes in water temperature, probably in the tropical and subtropical regions. Fish fed on high protein diet are usually characterized by fast growth rate, effective feed utilization and high survival. This study was conducted to investigate the effect of changes in dietary protein:lipid ratio on nutrient metabolism of Nile tilapia at acclimation or mild changes in water temperature.

6.2 Materials and methods 6.2.1 Experimental fish and design

Twenty-six Nile tilapia (Oreochromis niloticus) were obtained from a local producer and transported to the Laboratory of Animal Nutrition, Faculty of Veterinary Medicine, Ghent University, Belgium. The fish were randomly placed pairwise or triplicate in twelve 63L-glass aquaria of 60x30x36 cm (JUWEL Aquarium, Rotenburg, Germany) and fed a commercial diet (Benelux NV, Wielsbeke - Ooigem, Belgium) (Table 15) for 14 days of acclimatisation to laboratory conditions. At the end of the 14 days, the fish were weighed (average initial body weight: 81±10 g), randomly redistributed pairwise or triplicate to the 12 aquaria and assigned to either an acclimation or changes in temperature group, each containing six aquaria. A 2x6 factorial experiment with two replicates was used. The experimentation, housing and ethical procedures carried out in this experiment were approved by the Ethical Committee of the Faculty of Veterinary Medicine, Ghent University.

6.2.2 Experimental diets: formulation, preparation and feeding Six experimental diets ranging in protein:lipid ratio (Diet1: 2.94, Diet2: 3.48, Diet3: 4.33, Diet4: 5.12, Diet5: 5.77, Diet6: 7.51) were formulated (Table 15). Aquaria were used as experimental units; the tilapia in the acclimated and changes in temperature groups were hand-fed the six dietary treatments at a feeding rate of 3% of body weight per day. Feeding occurred at 09:00 and 15:00 and the feeding regime remained the same for 14 days. To determine the daily feed intake, any uneaten feed was removed after 60 min from each aquarium, dried and weighed (Geda et al., 2012, 2015).

Chapter 5

Table 15. Proximate chemical analysis and nutrient composition of the experimental dietsa

Proximate analysis (g/kg) Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6

Dry matter 866.6 902.3 905.1 912.1 923.3 895.5 Crude protein 422.0 392.0 365.9 343.7 326.0 309.2 Crude fat 56.2 67.9 71.5 79.3 93.7 105.0 Crude fibre 10.5 18.8 27.3 36.2 41.6 48.5 Crude ash 42.4 51.4 59.8 69.9 82.2 85.7

NFEb 335.5 363.8 380.6 383.0 379.8 347.1 a Aqua-KI feed, manufactured by Benelux NV, Wielsbeke - Ooigem, Belgium. Amino acid composition (g/kg: as analyzed in the commercial feed): Alanine, 29.30; arginine, 24.89; aspartic acid + asparagine, 42.07; cysteine, 6.24; glutamic acid + glutamine, 76.90; glycine, 20.45; histidine, 13.09; isoleucine, 17.59; leucine, 48.18; lysine, 25.26; methionine, 8.84; phenylalanine, 24.93; proline, 25.84; serine, 20.69; threonine, 16.74; tryptophan, 4.74; tyrosine, 16.09; valine, 25.39. Proximate analysis (g/kg: as analysed in the commercial feed): Dry matter, 924.50; crude protein, 416.10; crude fat, 99.20; crude fibre, 6.10; crude ash, 73.50; NFE, 329.60. Vitamin and mineral premix added per kg feed (as given in the commercial feed technical sheet): Vitamin A,

15000 IU; vitamin C, 200 mg; vitamin D3, 200 IU; vitamin E, 200 mg; iron, 100 mg; copper, 2.5 mg; manganese, 15 mg; zinc, 50 mg; selenium, 0.25 mg. List of ingredients reported in Aqua-KI in decreasing order of inclusion: Fish products, products and by- products of oil seeds, vegetal products and by-products, algae, amino acids, vitamins, minerals, calcium propionate, antioxidants, preservatives, colorants, oils and fats. b Nitrogen-free extract.

6.2.3 Changes in temperature and water quality parameters At the end of the acclimatisation period at an acclimation temperature of 22°C, the fish were managed in aquaria either at the acclimation temperature of 22°C or those in which the acclimation temperature was gradually increased (1°C/12 h) to a constant mild change in temperature of 28°C. All aquaria were maintained at 12:12 h light-dark photoperiod with fluorescent lights controlled by timers. Daily monitored water quality parameters were: pH (6.0-7.3 mg/L at 22°C; 6.5-7.4 mg/L at 28°C) (Merck KGaA, Darmstadt, Germany), temperature (28°C) and dissolved oxygen (3.2-6.1 mg/L at 22°C; 2.7- 5.7 mg/L at 28°C) (Hanna Instruments Srl, Nufalau, Romania), ammonium (<0.05 mg/L) and nitrite (0.02 mg/L) (JBL GmbH and Co KG, Neuhofen/Pfalz, Germany).

Chapter 6

6.2.4 Blood sampling and analysis At the end of the 14 days of feeding trial, all fish were fasted for 24 h and the final body weights were measured. The fish were euthanized using an overdose of a benzocaine solution (1 g benzocaine/ 10 mL acetone) and subjected to blood sampling. Blood samples were collected from the heart (cardiac puncture) using a 2 mL syringe (Becton Dickinson S.A., Madrid, Spain) and a 26 G needle (Becton Dickinson, Drogheda, Ireland) rinsed with heparin (LEO Pharma, Ballerup, Denmark). Blood serum samples were stored airtight at –80°C until analysed for acylcarnitine profile and selected free amino acids. Acylcarnitine profile and selected free amino acids of the serum samples was determined using quantitative electrospray tandem mass spectrometry (Zytkovicz et al., 2001).

6.2.5 Skeletal muscle sampling and analysis After the blood sampling, a sample of the dorsal muscle of about 4.5 cm2 was taken on the left side of the body, 1 cm ventral to the base of the dorsal fin along the lateral line (Geda et al., 2015). The muscle samples were stored at –80°C until analyzed. Determination of muscle tissue NAH and HIS concentrations was based on the reversed phase HPLC (Waters Corporation) method (O’Dowd et al., 1990) with slight modification (Breck et al., 2005). Muscle tissues were homogenized in 80% (v/v) ethanol and centrifuged at 2000 g for 20 min. The supernatants were dried in a Termaks incubator (40°C, normal atmosphere) (Termaks, Bergen, Norway), dissolved in phosphate buffer (pH 2.0) and filtered through a membrane filter (0.45 µm). An isocratic reverse phase HPLC was performed, using a 4.6 mm ID ×250 mm column with a silica-based packing (ZORBAX SB-C18, Agilent Technologies AS, Kolboth, Norway) and a Waters 600 E pump (Waters Corporation, Milford, MA, USA). A 0.1 M phosphate buffer (pH 2.0) was used as eluting solvent, with a flow rate of 0.6 mL/min. The concentrations of NAH and His were detected by UV absorbance (Waters 486–Tuneable Absorbance Detector, Waters Corporation) at 210 nm, using external standards.

Chapter 5

6.2.6 Statistical analysis Statistical analysis was conducted using SPSS version 20 (IBM Corporation, Armonk, NY, USA). All data were evaluated for normality using boxplot graphs and analyzed with General Linear Model Univariate Analysis of Covariance (GLM Univariate ANCOVA) procedure in the SPSS. Aquaria were used as experimental units. ANCOVA analysis was performed with temperature as fixed factor and protein:lipid ratio as covariate. Per temperature, linear and quadratic regression analysis was first performed. If the quadratic term explained the variation substantially better than the linear term, a quadratic term was added to the linear term when subjecting the data to ANCOVA. All the data are expressed as means and standard error of the means (SEM). Statistical significance was accepted at P<0.05 and P-values between 0.05 and 0.10 were considered tendency. The full ANCOVA model used is indicated below:

Yij = μ + Ti + D(xij – x̄ ) + (TD)ij + εij

Where: Yij is the value of the response variable for jth level of protein:lipid ratio in the ith level of temperature; μ is the overall (constant) mean value of the response variable; Ti is effect of ith level of temperature: (i=22, 28); D is a combined regression coefficient representing the pooling of the regression slopes relating response variable to the jth effect of protein:lipid ratio covariate within each temperature group (j=2.94, 3.48, 4.33, 5.12, 5.77, 7.51); (TD)ij is ijth interaction effect between the fixed factor and the covariates; εij is the random or unexplained error associated with the jth replicate observation from the ith level of temperature representing the component of the response variable not explained by the effects of the factor or the relationship with the covariate.

Chapter 6

6.3 Results In the present study, the protein:lipid ratio affected serum alanine (P=0.034) and ornithine (P=0.016) and temperature-diet interaction affected the concentrations of serum glycine (P=0.012; increased at 22oC, R2=0.81; decreased at 28oC, R2=0.03), phenylalanine (P=0.004; increased at 22oC, R2=0.67; increased at 28oC, R2=0.21), tyrosine (P=0.006; increased at 22oC, R2=0.64; increased at 28oC, R2=0.16), and sum of FAA (P=0.037; increased at 22oC, R2=0.72; increased at 28oC, R2=0.05) and (Table 16; Figure 12). There was a linear relationship between protein:lipid ratio and the concentrations of serum alanine and ornithine (Figure 12). Serum alanine increased as protein:lipid ratio increased at 22oC with stronger explaination of the relationship (R2=0.60), but serum alanine decreased at 28oC. The concentration of serum ornithine increased linearly with increased protein:lipid ratio both at 22oC (R2=0.78) and 28oC (R2=0.42), which indicated stronger relationship at 22oC.

The GLM Univariate ANCOVA regression revealed that there was a linear relationship between the protein:lipid ratio and the serum concentrations of freecarnitine (P=0.005; increased both at 22oC (R2=0.50) and at 28oC (R2=0.86)), dodecanoylcarnitine (P=0.041; increased at 22oC (R2=0.41) and decreased at 28oC (R2=0.28)), 3OH-hexadecanoylcarnitine (P=0.029; decreased at 22oC (R2=0.81) and increased at 28oC (R2=0.04)), octadecenoylcarnitine (P=0.029; increased both at 22oC (R2=0.03) and at 28oC (R2=0.69)), 3OH-octadecenoylcarnitine (P=0.018; increased both at 22oC (R2=0.02) and at 28oC (R2=0.17)), sum of 3OH-fatty acids (P=0.042; increased both at 22oC (R2=0.60) and at 28oC (R2=0.03)), sum of 3OH-longer-chain fatty acids (P=0.024; increased both at 22oC (R2=0.09) and at 28oC (R2=0.58)), and ratio of sum of 3OH-longer-chain fatty acids to sum of longer-chain fatty acids (P=0.036; increased both at 22oC (R2=0.50) and at 28oC (R2=0.52)) (Table 17; Figure 13).

The quadratic ANCOVA regression revealed that there was a curvilinear relationship between the temperature (T) squared protein:lipid ratio (D2) interaction (TD2) and the serum concentrations of tetradecanoylcarnitine (TD2: P=0.003; increased and decreased quadratically both at 22oC (R2=0.38) and 28oC (R2=0.34)), octadecanoylcarnitine (TD2: P=0.001; increased and decreased quadratically both at 22oC (R2=0.49) and 28oC (R2=0.78)), and sum of longer-chain fatty acids (TD2: P<0.001; increased and decreased quadratically at 22oC (R2=0.97), but decreased and increased quadratically at 28oC (R2=0.93)) (Table 17; Figure 13). There was also a quadratic relationship between protein:lipid ratio (D2) and the serum concentration of hexadecenoylcarnitine (P=0.012; increased and decreased quadratically at 22oC (R2=0.18), but decreased and increased quadratically at 28oC (R2=0.95)).

Chapter 5

In the skeletal muscle, the GLM Univariate ANCOVA regression revealed that there was a linear relationship between the protein:lipid ratio and the skeletal muscle FAA concentrations of glutamate (P=0.008; increased both at 22oC (R2=0.72) and at 28oC (R2=0.40)) and glutamine (P=0.007; decreased both at 22oC (R2=0.51) and at 28oC (R2=0.39)) (Table 18; Figure 14). The temperature-diet interaction (T×D or TD) also linearly affected the concentrations of some other skeletal muscle FAA: ornithine (P=0.046; increased at 22oC (R2=0.87) and decreased at 28oC (R2=0.09)), tyrosine (P=0.007; increased at 22oC (R2=0.63) and decreased at 28oC (R2=0.03)), proline (P=0.012; increased both at 22oC (R2=0.76) and at 28oC (R2=0.14)), taurine (P=0.018; decreased at 22oC (R2=0.57) and increased at 28oC (R2=0.61)), and urea (P=0.008; increased at 22oC (R2=0.80) and decreased at 28oC (R2=0.31)). The quadratic ANCOVA regression revealed that there was a curvilinear relationship between the squared protein:lipid ratio and the skeletal muscle FAA concentrations of β-alanine (D2: P=0.009; decreased and increased quadratically both at 22oC (R2=0.44) and 28oC (R2=0.92)), asparagine (D2: P=0.031; decreased and increased quadratically both at 22oC (R2=0.85) and 28oC (R2=0.64)), and threonine (D2: P=0.038; decreased and increased quadratically both at 22oC (R2=0.94) and 28oC (R2=0.39)) (Table 18; Figure 14). There was a curvilinear relationship between the temperature and squared protein:lipid ratio interaction (TD2) and the skeletal muscle phenylalanine concentrations (TD2: P=0.025; decreased and increased quadratically at 22oC (R2=0.93), but increased and decreased quadratically at 28oC (R2=0.26).

Temperature, diet or temperature-diet interaction did not affect the growth performance and feed utilization parameters and the proximate percentage of liver fat (P>0.05) (Table 19).

Chapter 6

Chapter 5

Chapter 6

Chapter 5

Chapter 6

Chapter 5

Chapter 6

Chapter 5

Chapter 6

Chapter 5

6.4 Discussion This study investigated the potential of nutritional strategies of dietary protein:lipid ratio to minimize the effect of mild changes in water temperature on nutrient metabolism and performance of Nile tilapia. The study identified the effects of temperature, diet, and temperature-diet interactions. The present observations provide an insight that dietary strategies can be implemented to reduce the effects of changes in water temperature on nutrient metabolism and growth performance and feed utilization in fish. In general, the importance of dietary strategies on the aforementioned parameters was identified both at acclimation and mild changes in water temperatures. In this study there was clear temperature effect on nutrient metabolism in fish. For almost all temperature effects, there was the protein:lipid ratio interaction. This shows that the level of water temperature in which fish are managed determines the optimum level of protein:lipid ratio necessary for the fish.

A lot of serum free amino acids were increased with higher dietary protein:lipid ratio, showing a higher involvement of amino acids in biochemical reactions. This supports that temperature affects the metabolism of amino acids in fish, for instance, in winter flounder (Pseudopleuronectes americanus), total plasma amino acids decreased in winter and increased in summer (Squires et al., 1979). In the present study, the effect of changes in water temperature on serum free amino acids showed that temperature differentially affects the metabolism of specific amino acids as well as the overall importance of amino acids as energy sources (Ballantyne, 2001). The serum citrulline to ornithine ratio was not significantly affected by dietary protein:lipid ratio although the ratio would increase. This shows arginine cycle was capable to manage the detoxification of the products of protein breakdown that increases with increasing the protein:lipid ratio. The amino acids might have increased in the serum as a result of peripheral protein breakdown due to a catabolic effect of the changes in temperature for energy production in liver and other tissues (Costas et al., 2012). This could also be due to the increased dietary protein:lipid ratio, which might have increased the concentration of amino acids in metabolism.

In this study, the concentration of free carnitine increased with increasing dietary protein:lipid ratio without a decrease in acylcarnitines. This suggests increased de novo synthesis of carnitine, which requires methionine and lysine (Li et al., 2009). This showed the overall improved metabolic activity of the fish because protein promoted the combustion of fatty acids in the citric acid cycle. The changes in the sums and ratios of longer-chain fatty acids showed effects of protein:lipid ratio on fat metabolism, which for instance demonstrated by the ratio of the sum of 3-hydroxy longer-chain fatty

Chapter 6 acids to the sum of the respective longer-chain fatty acids. The use of longer-chain fatty acids (3OHLCFA:LCFA) seems to be promoted by increasing the dietary protein:lipid ratio, which might seem contradictory at first sight, but likely indicates that the higher availability of amino acids might have increased the efficiency of using LCFA for energy. However, this is not confirmed, for example, by a decrease in liver fat or a decrease in ketone formation, so remains to be elucidated.

In the skeletal muscle, there was a linear relationship between the protein:lipid ratio and the skeletal muscle FAA concentrations of glutamate increased while that of glutamine decreased both at 22oC and at 28oC. The temperature-diet interaction linearly affected the skeletal muscle FAA concentrations of ornithine (increased at 22oC and decreased at 28oC), tyrosine (increased at 22oC and decreased at 28oC), proline (increased both at 22oC and at 28oC), taurine (decreased at 22oC and increased at 28oC), and urea (increased at 22oC and decreased at 28oC). There was a curvilinear relationship between the squared protein:lipid ratio and the skeletal muscle FAA concentrations of β- alanine (decreased and increased quadratically both at 22oC and 28oC), asparagine (decreased and increased quadratically both at 22oC and 28oC), and threonine (decreased and increased quadratically both at 22oC and 28oC). There was a curvilinear relationship between the temperature and squared protein:lipid ratio interaction and the skeletal muscle phenylalanine concentrations (decreased and increased quadratically at 22oC, but increased and decreased quadratically at 28oC).

Although the concentration of muscle FAA are quite constant over species, the present study demonstrated that changes in water temperature affects the concentrations of FAA, which are regulated by a set of other amino acids due to conversion of one into another. The effects of mild changes in water temperature on skeletal muscle FAA directly or indirectly affect the physiological roles of the FAA such as carnitine synthesis, ATP synthesis, citrulline synthesis, arginine cycle, ammonia detoxification, osmoregulation, antioxidation, and others (Costas et al., 2012; Li et al., 2009; Wu, 2009).

Although the other results indicated that water temperature determines the detary protein:lipid ratio level necessary for the fish, there was no significant effect of temperature, diet or temperature-diet interaction on growth performance and feed utilization parameters. The absence of significant effect on the percent of liver fat shows as there was no real pathology or development of fatty liver due to either the effect of the changes in temperature or the dietary protein:lipid treatment. This is because the development of fatty liver is basically an indication of extreme scenario.

Chapter 5

In conclusion, this study demonstrated the potential of increasing dietary protein:lipid ratio to improve changes in nutrient metabolism of Nile tilapia at mild changes in water temperature. The study demonstrated that changes in fish nutrient metabolism occur depending on changes in water temperature and the dietary protein:lipid ratio can affect this metabolism.

The present demonstration of the potential dietary macronutrient profile to improve the response of fish to mild changes in water temperature was followed by the evaluation of the effect of mannanoligosaccharides as a dietary intestinal modulator in fish at mild changes in water temperature in chapter 7.

Acknowledgements The authors acknowledge Herman De Rycke (Laboratory of Animal Nutrition, Faculty of Veterinary Medicine, Ghent University) for the proximate analysis of the experimental diet and Hedwig Stepman (Department of Clinical Chemistry, Microbiology and Immunology, Ghent University) for analysis of the selected serum acylcarnitines.

Chapter 7 Effects of dietary mannanoligosaccharides in fish at mild changes in water temperature

Chapter 7

Changes in intestinal morphology and amino acid catabolism in common carp at mildly elevated temperature as affected by dietary mannanoligosaccharides

Geda, F., Rekecki A., Decostere A., Bossier, P., Wuyts, B., Kalmar, I.D., Janssens, G.P.J., 2012.

Anim Feed Sci Tech 178:95–102

A study was conducted to evaluate whether dietary mannanoligosaccharides (MOS) modulate the effect of mild changes in water temperature on intestinal morphology and some selected whole blood amino acids and acylcarnitines in common carp (Cyprinus carpio carpio). Twenty fish in 10 aquaria were randomly assigned to either a control diet or the same diet with 4 g/kg MOS. A 35-day period at acclimation temperature (23.2°C) was followed by 14 days at changes in temperature (26.1°C). After both temperature periods, one fish per aquarium was euthanized for the intestinal morphology and whole blood analysis on free amino acids and carnitine esters. The changes in temperature period decreased the number of neutral (P=0.031) and acid (P=0.004) mucin-secreting goblet cells and tended to reduce (P=0.056) the fold height in the midgut, irrespective of MOS supplementation. In both periods, supplementation of MOS increased (P=0.035) the number of goblet cells in the hindgut, but other histomorphometries were unaffected. Free concentrations of whole blood amino acids were increased after the mild changes in temperature period: valine (P=0.002), leucine (P=0.001), methionine (P<0.001), phenylalanine (P=0.001) and tyrosine (P=0.001), but not affected by MOS supplementation. None of the carnitine esters were altered by the changes in temperature except propionyl carnitine that was higher (P=0.003) after the mild changes in temperature of 14 days. Supplementation of MOS only tended to reduce the tiglyl carnitine (P=0.069) and methylmalonyl carnitine (P=0.078) concentrations. The analysis of free amino acids and carnitine esters could not support the hypothesis that MOS counteracts depressing effects of changes in temperature on amino acid catabolism. In conclusion, moderate elevation of water temperature could lead to considerable changes in gut histology and metabolism.

Chapter 7

7.1 Introduction

The consumption of aquatic products keeps increasing because of their importance as dietary protein source and high contents of health-associated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and other nutrients that are considered healthy (Liao, 2009). However, global climate change was suggested to potentially affect aquaculture sector in warmer climate developing countries by lowering productivity in wild fish populations and in intensive aquaculture systems worldwide (Ficke et al., 2007). Rising water temperature up to a certain limit may favor aquaculture production by increasing growth rate and reducing the maturation period of fish (Chatterjee et al., 2004; Debnath et al., 2006). On the other hand, several studies indicated that a change in water temperature beyond optimum limits of a particular fish species influences the body temperature, oxygen demand, health, feed utilization and growth performance (Linthicum and Carey, 1972; Houlihan et al., 1993; Azevedo et al., 1998; Wedemeyer et al., 1999; Chatterjee et al., 2004; Debnath et al., 2006).

Unlimited feed supply under changes in water temperature reduces feed intake in fish and gut absorption capacity due to a decrease in surface area of microvilli, both resulting in depressed growth performance (Jobling, 1994; Garriga et al., 2005; Song et al., 2010). A study in broiler chickens has shown that fermentable oligosaccharides alter gut morphology with some concomitant changes in oxidative stress (De Los Santos et al., 2005). A study by Dimitroglou et al. (2008) under normal temperature reported that MOS increased the absorptive surface area and microvilli density and length in rainbow trout. Yet, a study in pigeons under thermoneutral environment concluded that dietary MOS reduced the intestinal morphology (villus length, crypt depth, muscularis thickness) because of reduced bacterial challenge (Abd El-Khalek et al., 2012). Furthermore, even in species with a relatively undersized large bowel like the cat, prebiotic-induced changes in the intestinal environment can limit amino acid catabolism, thus promoting the use of lipids and carbohydrates as energy source (Verbrugghe et al., 2009). The concomitant drop in heat production per gained adenosine triphosphate (ATP) can especially be helpful under heat stress conditions.

The potential of dietary MOS to support intestinal morphology and reduce amino acid catabolism has so far not been studied in fish under changes in temperature conditions. Therefore, we investigated the effect of dietary MOS supplementation on gut morphology and some selected whole blood amino acids and acylcarnitines during mild changes in temperature in common carp.

Chapter 7

7.2 Materials and methods 7.2.1 Experimental animals and design Twenty common carp (Cyprinus carpio carpio) fingerlings (average body weight of 45.1 ± 1.4 g) were obtained from Wageningen University Animal Sciences, Zodiac, Wageningen, The Netherlands. The fish were transported in a double polyethylene bag with sufficient aeration to the Laboratory of Animal Nutrition, Faculty of Veterinary Medicine, Ghent University, Belgium. All the experimental fish were acclimated to the laboratory conditions on a common diet for 14 days and randomly allocated per two in ten 63L-glass aquaria of 60x30x36 cm (JUWEL Aquarium, Rotenburg, Germany) of recirculating tap water with continuous aeration through a biological filter. Following the acclimation period, the fish were fasted for 24 h, weighed (average initial body weight: 48±2 g) and stratified into the ten aquaria. The aquaria were randomly assigned to two treatment groups, control (“Control”) and MOS (“MOS”), each group with five aquaria with two fish each. The experimentation, housing and ethical procedures carried out in this experiment have been approved by the Ethical Committee of Laboratory Animals, Faculty of Veterinary Medicine, Ghent University, Belgium.

7.2.2 Feed, feeding protocol and changes in temperature The control group was fed a complete carp diet, S9029-S012/-S015 (ssniff Spezialdiäten GmbH, Soest, Germany) (Table 20) and the MOS group was given the same control diet supplemented using Comfort Pressure Sprayer (AVEVE Group, Leuven, Belgium) with 4 g/kg yeast-derived MOS (Alltech, Dunboyne, Ireland) (Figure 15) at a feeding rate of 1.5% of body weight per day. The two experimental diets were provided at normal temperature of 23.2°C for 35 days. At the end of the 35- day feeding period, each fish was fasted for 24 h and the changes in temperature experiment was conducted on individual fish, those not sampled for normal temperature analysis. The water temperature in each aquarium was increased from 23.2°C to 26.1°C at a rate of 3°C/36 h. The changes in temperature was applied for two weeks. Feeding occurred at 09:00 and 17:00 and the feeding regime remained the same throughout the study. All aquaria were maintained at 12:12 h light–dark photoperiod with fluorescent lights controlled by timers.

Chapter 7

Figure 15. Yeast-derived mannanoligosaccharides (MOS) used as a dietary supplement to the carp diet.

7.2.3 Water quality parameters At the temperature of 23.2°C, daily pooled average pH (7.0, 7.0) (Merck KGaA, Darmstadt,

Germany), dissolved oxygen (6.52, 6.54 mg/L) (Hanna Instruments Srl, Nufalau, Romania) and two- week intervals ammonium (<0.05, <0.05 mg/L) and nitrite (0.10, 0.09 mg/L) (JBL GmbH and Co

KG, Neuhofen/Pfalz, Germany) were measured for the control and MOS groups, respectively.

Similarly, at the changes in temperature of 26.1°C, the daily pooled average pH (7.6, 7.6), dissolved oxygen (5.44, 5.30 mg/L) and two-week intervals ammonium (<0.05, <0.05 mg/L) and nitrite (0.06,

0.08 mg/L) were measured for the control and MOS groups, respectively.

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Table 20. Nutrient composition of the experimental diet (on as fed basis)

Diet Control MOS Ingredients (g/kg) Fish meal, high CP, hydrolyseda 300.00 300.00 Poultry protein, hydrolyseda 40.00 40.00 Soybean concentrate 150.00 150.00 Wheat 134.15 134.15 Corn, pre-treated 260.00 260.00 L-Lysine HCl 1.20 1.20 DL-Methionine 3.60 3.60 L-Threonine 2.30 2.30 Vitamin and trace element premixb 10.00 10.00 Mannanoligosaccharide 0 4.00 Calcium carbonate 12.00 12.00 Calciumphosphate, monobasic 9.00 9.00 Magnesium oxide 2.00 2.00 Choline Cl (500 g/kg) 2.00 2.00 Stabilized Vitamin C (350 g/kg) 3.60 3.60 Butylated hydroxytoluene 0.15 0.15 Sodium carboxymethyl cellulose 10.00 10.00 Eicosapentaenoic acid oil 50.00 50.00 Soybean oil 10.00 10.00 Calculated chemical composition (g/kg)c Dry matter 900.00 900.00 Crude ash 70.00 70.00 Crude protein 411.00 411.00 Crude fat 40.00 40.00 Starch 250.00 250.00 Sugar 13.00 13.00 Lysine 30.50 30.50 Methionine 14.70 14.70 Methionine+Cystine 20.00 20.00 Threonine 20.50 20.50 Tryptophan 4.30 4.30 Calcium 9.50 9.50 Phosphorus 7.50 7.50 Sodium 4.20 4.20 Magnesium 2.80 2.80 a Low ash. b Vitamin and trace element premixture provides per kg feed: 25.000 IU Vitamin A, 1.500 Vitamin D3, 120 mg

Vitamin E, 20 mg Vitamin K3, 80 mg Vitamin B1; 30 mg Vitamin B2; 25 mg Vitamin B6; 150 μg Vitamin B12; 50 mg Ca Pantothenate; 9 mg Folic acid; 500 μg Biotin; 100 mg Iron; 50 mg Zinc; 30 mg Manganese; 5 mg Copper; 0.1 mg Selenium; 2 mg Iodine; 2 mg Cobalt. c Concentrations were calculated with Fumi (Hybrimin, Hessisch Oldendorf, Germany) from a feedstuff matrix based on the respective provider's information.

Chapter 7

7.2.4 Blood sampling and analysis At the end of the 35-day feeding period at normal temperature, all fish were fasted for 24 h, final body weights were measured, and one random fish from each aquarium was euthanized using an overdose of a benzocaine solution (300 mg/L) and subjected to blood sampling. The remaining half of the fish underwent the same procedure after the changes in temperature experiment. Blood samples were collected from the heart (cardiac puncture) using a 1 mL syringe (Becton Dickinson S.A., Madrid, Spain) and a 26 G needle (Becton Dickinson, Drogheda, Ireland) rinsed with heparin (LEO Pharma, Ballerup, Denmark). Whole blood was sampled using Whatman 903TM dried blood spot card (Westborough, MA, USA) and the samples were stored airtight at −20°C until analysis of selected amino acid and acylcarnitine profile. Selected free amino acids and acylcarnitine profile of the whole blood samples was determined using quantitative electrospray tandem mass spectrometry (Zytkovicz et al., 2001).

7.2.5 Histological and morphometric analysis

After blood sampling, the fish were dissected to measure the fold heights and widths in the mid- and hindguts at the normal and changes in temperature. The midgut and hindgut were differentiated as described elsewhere (FAO, 1980; Wilson and Castro, 2011). Each intestinal section was cut in 10 portions (Figure 16). Three samples of 5 mm each were taken at the second, fourth and sixth portion from the cranial region of the midgut and at the fifth, seventh and ninth portion in the caudal end of the hindgut. Light microscopy was used to examine and evaluate morphological and histological effects.

For histomorphometrical analysis, hematoxylin and eosin (HE) staining for fold measurements and Alcian blue-periodic acid-Schiff (AB-PAS) staining for histochemistry of goblet cells were performed. The samples were fixed for 24 h in Bouin’s fluid (saturated picric acid, formaldehyde 37%, glacial acetic acid 100%), dehydrated in graded alcohols (50, 70, 80, 94, and 100%) and embedded in paraffin wax using the STP 420 Microm Tissue Processor and the embedding center EC350-1 and 2 (Microm, Prosan, Merelbeke, Belgium), respectively as described by Rekecki et al. (2009). Transverse sections of 8 µm thickness were cut with the HM 360 Microtome using the Section Transfer System (Microm, Prosan, Merelbeke, Belgium). The histological analysis was performed after the sections were stained with HE, and AB-PAS for neutral (magenta) and acid (blue) mucins (Figure 17); using the Olympus BX61 light microscope and Olympus DP50 camera (Olympus Belgium, Aartselaar, Belgium). The variables measured were fold height, fold width, and number of

Chapter 7 goblet cells in the mid- and hindgut. The intestinal fold height was measured from the tip of the fold to basal area and fold width – across the bottom end of fold height in the basal area (Sanden et al., 2005). The fold height and fold width measurements of twenty-seven folds and counts of the number of goblet cells on nine folds in the mid- and hindgut were performed per fish and their mean values were used in statistical analysis.

Figure 16. Photo image and diagrammatic view of sampling of common carp mid- and hindgut for gut morphology. (A) Mid- and hindgut were cut into 10 portions and three samples of 5 mm each were taken at the second, fourth and sixth portion from cranial of midgut and at the fifth, seventh and ninth portion in the caudal of hindgut. (B) The samples were embedded in paraffin wax and (C) transverse sections of 8 μm thickness were cut with the HM 360 Microtome using the Section Transfer System (Microm, Prosan, Merelbeke, Belgium) for light microscopic observations. (Photograph by Geda, F.)

Chapter 7

Figure 17. Light micrograph of mucin-secreting neutral (magenta, indicated by letter N) and acid (blue, indicated by letter A) goblet cells of common carp midgut folds surface epithelial cells (AB-PAS stain: x200, Photograph by Geda, F.)

7.2.6 Statistical analysis Statistical analysis was performed using SPSS version 20. All data were evaluated for normality using boxplot graphs, and were analyzed with the General Linear Model Univariate Analysis of Variance (GLM Univariate ANOVA) procedure in SPSS with MOS treatment and temperature effects as fixed factors and aquarium effect as random factor nested within the MOS treatment. All data are expressed as means and standard error of means (SEM). Statistical significance was accepted at P<0.05. P- values between 0.05 and 0.10 were considered as tendency. The GLM Univariate ANOVA model used is indicated below:

Yij(k) = μ + Mi + Tj + MiTj + MiAk + εij(k) where, μ = mean, Mi = ith effect of MOS: (i=0,1), Tj = jth effect of temperature: (j=1,2), MiTj = ijth interaction effect between MOS and temperature, MiAk = ikth interaction effect between MOS and aquarium in which the effect of aquarium is nested in that of MOS: (k=1,2,3,4,5), and εij(k)= the random error.

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7.3 Results

The pooled final body weights of both groups (control and MOS) were 67 ± 2 and 74 ± 3 g at the end of the normal and changes in temperature, respectively; no differences (P>0.05) were observed between the control and MOS groups at any of the time points (Table 24). The mild changes in temperature decreased the number of neutral (P=0.031) and acid (P=0.004) mucin-secreting goblet cells and tended to reduce (P=0.056) the fold height in the midgut, irrespective of MOS supplementation (Table 21). There were no significant effects of mild changes in temperature on hindgut morphology. In both periods, supplementation of MOS increased (P=0.035) the number of goblet cells in the hindgut.

Table 21. Effect of dietary MOS on gut histomorphometry in common carp (mean, SEM, n=5)

23.2°C 26.1°C Pooled P Histomorphometry Control MOS Control MOS SEM Temp MOS T x M Midgut Fold height (µm) 534.73 548.44 499.31 520.77 9.59 0.056 0.459 0.790 Fold width (µm) 107.19 102.14 102.74 105.24 1.51 0.819 0.724 0.210 Number of neutral goblet cells 49.47 54.40 42.13 41.60 2.17 0.031 0.593 0.499 Number of acid goblet cells 64.33 69.73 52.27 50.53 2.91 0.004 0.757 0.389 Total number of goblet cells 113.80 124.13 94.40 92.13 4.73 0.009 0.638 0.421 Hindgut Fold height (µm) 563.82 555.41 499.75 516.62 15.73 0.166 0.889 0.717 Fold width (µm) 125.17 119.74 116.12 119.05 1.77 0.247 0.691 0.315 Number of neutral goblet cells 40.80 44.07 33.87 41.60 1.96 0.347 0.073 0.648 Number of acid goblet cells 46.60 56.80 53.87 60.87 1.95 0.113 0.039 0.630 Total number of goblet cells 87.40 100.87 87.74 102.47 3.48 0.903 0.035 0.936 MOS, mannanoligosaccharides; Temp, temperature; TxM, interaction of temperature and MOS; the number of goblet cells were counted on nine folds in the mid- and hindgut per fish.

Free concentrations of whole blood amino acids were increased after the mild changes in temperature period: valine (P=0.002), leucine (P=0.001), methionine (P<0.001), phenylalanine (P=0.001) and tyrosine (P=0.001) (Table 22). None of the blood amino acid concentrations were affected by MOS supplementation.

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Table 22. Effect of dietary MOS on selected whole blood amino acids in common carp (mean, SEM, n=5)

23.2°C 26.1°C Pooled P Amino acids (µmol/L) Control MOS Control MOS SEM Temp MOS T x M Alanine 781 292 636 530 113 0.831 0.266 0.390 Citrulline 16 15 17 23 1 0.112 0.386 0.248 Ornithine 25 22 29 29 2 0.180 0.744 0.730 Leucine 428 362 633 563 33 0.001 0.263 0.955 Valine 170 154 290 255 18 0.002 0.443 0.715 Glycine 438 383 472 436 19 0.272 0.283 0.811 Phenylalanine 84 83 185 171 14 0.001 0.714 0.767 Tyrosine 71 68 134 123 9 0.001 0.703 0.768 Methionine 127 103 209 205 14 <0.001 0.571 0.501 MOS, mannanoligosaccharides; Temp, temperature; TxM, interaction of temperature and MOS.

None of the carnitine esters were altered by the changes in temperature except propionyl carnitine that was higher (P=0.003) after the mild changes in temperature of 14 days (Table 23). Supplementation of MOS had no major effect on the acylcarnitine profile, although it tended to reduce the tiglyl carnitine (P=0.069) and methylmalonyl carnitine (P=0.078) concentrations, the latter tending (P=0.086) to an interaction between environmental temperature and MOS supplementation: methylmalonyl carnitine concentration was lower in the MOS group during the normal temperature period only.

Table 23. Effect of dietary MOS on whole blood acylcarnitine profile in common carp (mean, SEM, n=5)

23.2°C 26.1°C Pooled P Carnitine ester (µmol/L) Control MOS Control MOS SEM Temp MOS T x M Free 11.2 8.3 11.2 11.0 0.700 0.256 0.362 0.278 Acetyl 16.9 11.8 14.7 15.2 1.200 0.819 0.393 0.292 Propionyl 2.3 2.1 5.5 3.8 0.500 0.003 0.326 0.226 Butyryl 0.690 0.500 0.420 0.540 0.070 0.477 0.799 0.321 Tiglyl 0.122 0.060 0.078 0.082 0.011 0.665 0.069 0.215 Isovaleryl 0.144 0.138 0.162 0.174 0.012 0.303 0.909 0.723 3-OH-butyryl 0.222 0.108 0.154 0.120 0.024 0.546 0.161 0.394 Malonyl 0.048 0.030 0.034 0.030 0.005 0.398 0.322 0.398 Methylmalonyl 0.058 0.030 0.032 0.030 0.004 0.086 0.078 0.086 Glutaryl 0.040 0.016 0.024 0.022 0.004 0.468 0.132 0.132 MOS, mannanoligosaccharides; Temp, temperature; TxM, interaction of temperature and MOS.

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Table 24. Effect of dietary MOS on growth performance and feed utilization in common carp (mean, SEM, n=5)

23.2°C 26.1°C Pooled P Control MOS Control MOS SEM Temp MOS T x M Initial weight (g/fish) 53.6 59.8 62.4 70.5 1.9 0.001 0.109 0.534 Final weight (g/fish) 62.9 71.3 69.7 78.1 2.0 0.002 0.112 0.992 Weight gain (g/fish) 17.1 19.1 10.8 12.2 0.6 0.003 0.226 0.232 Feed intake (g/fish) 9.3 11.5 7.2 7.6 0.9 <0.001 0.106 0.416 FCR (feed/gain) 1.84 1.66 1.50 1.61 0.1 0.992 0.802 0.395 MOS, mannanoligosaccharides; Temp, temperature; TxM, interaction of temperature and MOS.

7.4 Discussion

Heat production in animals arises from basal metabolism, physical exercise and diet-induced thermogenesis (DIT) (Westerterp-Plantenga et al., 1999; Johnston et al., 2002; Scott and Devore, 2005; Brotherhood, 2008). A strategy to reduce the effect of the heat load involves reducing feed intake that reduces DIT as observed in many species among the animal kingdom, including fish (Garriga et al., 2005; Katersky and Carter, 2005; Song et al., 2010; Mujahid, 2011). Yet, a decrease in DIT can also be accomplished by reducing the absorption of nutrients. Studies in poultry by Marchini et al. (2011) have for instance demonstrated that changes in environmental temperature reduces the absorptive surface of the intestine. In the present study, the tendency to lower fold height in the midgut after the mild changes in temperature period suggests a reduction in absorptive surface. This feature can be part of a strategy to overcome the negative effects of changes in temperature, but will not benefit growth performance of the fish. In case of reduced supply of dietary energy, a higher proportion of the required energy has to come from body reserves. If lipid stores have to be mobilized to provide acetyl CoA for the citric acid cycle, a source of oxaloacetate is needed to form citrate and render ATP (Goldstein and Newsholme, 1976; Owen et al., 2002). Oxaloacetate can only be synthesized from glucose or certain amino acids. Because of reduced absorption, less glucose is available, and the main source for oxaloacetate will be amino acids from body reserves. Nutritional modulation for reduced absorptive capacity of gut during changes in temperature requires a shift in energy substrate to induce less heat stress because the decarboxylation of amino acids will render less ATP per unit of metabolisable energy compared to combustion of fat or carbohydrates (Wu, 2009), and should be restricted to the minimum when animals have to limit heat production.

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In the present study, several of the studied free amino acids in the fasted blood samples were increased after the changes in temperature period, suggesting increased mobilisation of amino acids from body stores. The hypothesis that they serve as energy substrate, is supported by the considerable increase in propionyl carnitine. The latter, for instance, reflects the concentration of propionyl coenzyme A (CoA), a catabolite of methionine and isoleucine (Zhang et al., 2004; Maerker et al., 2005; Ibrahim-Granet et al., 2008). Based on the absence of a MOS effect on intestinal fold height and fold width, MOS supplementation seemed to have no impact on absorptive capacity under the tested conditions. This result is similar to that reported for red drum (Sciaenops ocellatus) in which the fold height in mid- and hindguts was not significantly affected by dietary MOS supplementation (Zhou et al., 2010). The main mode of action of MOS is to avoid bacterial fimbriae to attach to the intestinal mucosa (Newman, 1994; Spring et al., 2000), and the concomitant reduction of the intestinal bacterial load, could result in a higher efficiency of nutrient absorption per unit of intestinal surface (Abd El-Khalek et al., 2012) or in an increased absorptive surface (Dimitroglou et al., 2008). Although more detailed analysis of absorptive capacity might need to confirm this statement, no major improvement in nutrient absorption is to be expected: the trends towards lower tiglyl carnitine and methylmalonyl carnitine were only present before the changes in temperature period. Tiglyl CoA reflects the breakdown of isoleucine, whereas methylmalonyl CoA indicates isoleucine, leucine and methionine breakdown. The reason why this would be most prominent under normal temperature, is yet unclear, but needs confirmation anyhow.

From the decrease in number of goblet cells in the midgut after the changes in temperature period, it can be assumed that mucin production was decreased, thereby lowering the intestinal barrier for pathogens (Blomberg et al., 1993), and reducing the facilitation of movement of digesta along the intestine (Hoerr, 2001). We hypothesize that this feature is an anticipation to the lower need for mucus when heat stress reduces feed intake. Turning down goblet cell activity will likely also limit heat production, and will spare nutrients for other processes in the body. This effect was not seen in the hindgut, which might still be related to the fact that the midgut is the main site of nutrient absorption (Lovett and Felder, 1990; Wallace et al., 2005; Wilson and Castro, 2011). In the hindgut, supplementation of MOS increased the number of goblet cells, which suggests that MOS had more potential to reduce the bacterial load in this part of the intestine because the hindgut typically contains more microbiota than the midgut (Mondal et al., 2010). Studies in avian species earlier identified an influence of dietary MOS on mucin secretion (De Los Santos et al., 2007; Baurhoo et al., 2009), but to our knowledge, no studies have been published in aquatic species so far.

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In conclusion, a two-week period of changes in water temperature resulted in decreased absorptive capacity of the gut and reduced mucin-secreting goblet cells, associated with increased amino acid catabolism. Supplementation of MOS could not counteract this change, but increased the number of goblet cells in the hindgut, irrespective of water temperature.

This thesis investigated the effect of mild changes in water temperature on nutrient metabolism in fish, with an evaluation of nutritional strategies to improve the nutrient metabolism of fish at the mild changes in water temperature. The thesis addressed the effect of mild changes in water temperature in fish with focus on nutrient metabolism in skeletal muscle. Three dietary strategies were evaluated for their effects on the mild changes in water temperature in fish: (1) β-alanine was evaluated as a dietary metabolic modulator, (2) effect of dietary protein:lipid ratio was evaluated on nutrient metabolism, and (3) mannanoligosaccharides was evaluated as a dietary intestinal modulator in fish at the mild changes in water temperature. The general discussions of these dietary strategies were presented in chapter 8.

Acknowledgements The authors acknowledge Prof. Johan Verreth, Head of Aquaculture and Fisheries Group and Sietze Leenstra, Manager of Aquatic Research Facilities of Wageningen University, The Netherlands for kindly providing the carp and its feed and the German ssniff Spezialdiäten GmbH Company for kindly providing the composition and proximate contents of the carp diet. We also thank the staff of Laboratory of Animal Nutrition, for help during blood sampling; and all staff of the Laboratory of Aquaculture and Artemia Reference Center, especially Prof. Patrick Sorgeloos for his material support and valuable comments during the study. We also wish to thank the support of Prof. Wim Van den Broeck and Prof. Paul Simoens at the Department of Morphology for the histological and morphometric analysis. We are also grateful to the anonymous reviewers for suggesting valuable comments to improve this paper.

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Chapter 8 General discussion

General discussion

Helicopter view of the study results

Chapter 3. Nutrient metabolism in fish at mild changes in water temperature

 There are differences in metabolism between fish species, and even interaction between species and temperature.

 The difference in metabolism between fish species and species-temperature interaction was demonstrated by changes in muscle imidazole to taurine ratio.

Chapter 4. Potential of dietary β-alanine in fish at mild changes in water temperature: Part I

 Metabolic effect of dietary β-alanine (BA) supplementation on carp amino acid (AA) catabolism was very limited.

 Limited effects in selected nutrient metabolites and tendencies of improved growth performance warrants further investigation.

 Further studies are needed to evaluate conditions that exert an effect of BA on AA metabolism.

Chapter 5. Potential of dietary β-alanine in fish at mild changes in water temperature: Part II

 Sturgeon, as a carnosine-storing fish species, did not show a dose-response in muscular carnosine storage due to BA supplementation.

 BA supplemented sturgeon developed a considerable increase in glycine, citrulline and cystathionine, suggesting an important metabolic modulator function of BA.

Chapter 6. Effects of dietary protein:lipid ratio in fish at mild changes in water temperature

 Response of fish nutrient metabolism clearly changes with water temperature.

 Dietary protein:lipid ratio to improve fish nutrient metabolism is determined by the specific temperature.

Chapter 7. Effects of dietary mannanoligosaccharides in fish at mild changes in water temperature

 Mild changes in water temperature decreased absorptive capacity of the gut and reduced mucin- secreting goblet cells, associated with increased AA catabolism.

 Supplementation of dietary mannanoligosaccharides could not counteract the effect of mild changes in water temperature, but increased the number of goblet cells in the hindgut, irrespective of water temperature.

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General discussion

8.1 Searching for an evolutionary reason for increased amino acid catabolism at changes in water temperature

The general aim of this thesis was to identify the changes that happen in fish nutrient metabolism when exposed to mild changes in water temperature, and to evaluate different nutritional strategies to support the fish in performing under such conditions. The work in this thesis focused primarily on the changes in nutrient biochemistry, requiring intensive monitoring studies with less emphasis on growth performance and feed utilization. Although some studies still demonstrated significant effect on the performance and feed utilization of the fish, the next step in research should be the evaluation of our findings at larger scale in order to verify if the nutritional strategies can aid fish productivity when exposed to changes in water temperature. The value of the present studies lies in the demonstration of changes in nutrient metabolism in the fish as a response to changes in water temperature that are commonly considered as “within normal range”. The practical importance of these findings is that guidelines for nutrient requirements that were established at optimum temperature might need to be adapted in function of the actual water temperature and its associated oxygen saturation.

In all studies, signs have been observed that point to increased amino acid catabolism. This seems somehow in contrast with many studies indicating that fish perform better with increasing water temperature. This paradox is quickly solved if higher feed intakes at higher temperatures can explain the increased growth, which then is not necessarily linked to improved feed utilization efficiency.

The increase in amino acid catabolism typically implies a higher use of protein as an energy source, which is commonly considered as disadvantageous for energetic efficiency. In homeothermic animals, the increased use of protein as an energy source leads to increased heat production due to the low conversion efficiency of amino acids to energy. Information on the effect of protein use in poikilothermic animals such as fish is scarce, but the principle can be assumed similar across higher organisms. At first sight, it is therefore remarkable that fish tend to increase amino acid catabolism when exposed to changes in water temperature. In terrestrial mammals, the increased use of protein as an energy source can be a rescue route for providing oxaloacetate precursors in case of problems with glucose metabolism (e.g. insulin resistance). In such cases, the biochemical profile would typically show increased levels of ketone bodies because of the relative excess of acetyl coenzyme A that diverts from the citric acid cycle to ketone synthesis.

In the studies of this thesis, no obvious changes were observed in ketone synthesis, as could have been seen through changes in 3-OH-butyrylcarnitine and 3-OH-butyrylcarnitine:acetylcarnitine ratio. Therefore, the increased protein catabolism does not appear to be a way to maintain the citric acid cycle, but serves another goal. In nature, fish – and aquatic organisms in general – have a very low

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General discussion availability of glucose sources in their diet. Assuming that the limited digestive tract of many fish does not allow them to provide considerable amounts of volatile fatty acids as energy source, the main energy sources will be fat and protein. Although protein is a less efficient energy source, the consumption of oxygen when burning protein is lower than when burning fat per unit of gained energy. Given the fact that changes in water temperature inherently reduces the solubility of oxygen in the water, a strategy of shifting from fat to protein use might therefore make sense. In the blood acylcarnitine profile, there are however no indications of reduced fatty acid metabolism. This should be further explored, but evidently the increased availability of amino acids in energy metabolism will also aid the combustion of fatty acids, as was demonstrated in our study with increasing dietary protein:lipid ratio (Chapter 6). The lack of noticeable changes in fatty acid metabolism therefore not necessarily contradict the hypothesis that fish try to save oxygen through a higher proportion of amino acid use as energy source. To verify this hypothesis in future studies, respiration chamber studies could be helpful.

8.2 A comparative view on metabolic responsiveness to changes in water temperature

Nutrient requirements have been established for the main economically important fish species, hence acknowledging the interspecies differences in nutrient metabolism. What is far less studied is the difference in response of this nutrient metabolism across fish species to challenges such as changes in water temperature.

Considering the role of amino acid metabolism in responding to changes in water temperature, it was logical to look into strategic body reserves of amino acids. Histidine-related compounds (HRC) have been intensively studied in a wide range of species across the animal kingdom, and to date, there is no clear explanation for the diversity in amount and type of muscular storage of HRC. In our work, we for instance confirm the difference in HRC storage in skeletal muscle between tilapia (mainly N-α- acetylhistidine), carp (mainly histidine) and sturgeon (mainly carnosine) (Chapters 3 and 5). The initial hypothesis that histidine might serve as energy source was not clearly supported by the findings in this work, and the energetic efficiency of histidine as energy source is one of the lowest of all amino acids. This hypothesis might therefore not be very likely. It is however clear that HRC somehow play a role in the response to changes in water temperature, and that this role is distinctly different between fish species. This was demonstrated by the difference (both absolute and as response to water temperature) in the blood ratio of histidine+N-α-acetylhistidine: taurine in carp versus tilapia (Chapter 3), and further substantiated in by the distinct decrease in muscular histidine and N-α-acetylhistidine concentrations in tilapia when exposed to changes in water temperature.

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This thesis did not elaborate on other hypotheses than the direct impact on nutrient biochemistry, but other hypotheses can be distilled from literature, such as the precursor role of histidine for histamine, a proinflammatory compound that also induces vasodilation, a feature that is beneficial in situations of hypoxia and increased need for heat dissipation. To our knowledge, this has not been studied in fish.

Identification of the evolutionary reasons for the interspecies diversity in muscular HRC storage might reveal the necessary insights in their physiological role, especially when this feature can be associated with species-related lifestyles and diets. A first theme to look into would then be the changes in oxygen availability in the natural living conditions of fish. It is remarkable that for instance carnivorous fish that might need to quickly shift from aerobic to anaerobic conditions during hunt, show the highest muscular HRC storage (Boldyrev et al., 2013). One of the highest muscular storage of HRC found in nature are those in whales, which is interesting because they shift to severe anaerobic conditions during their long dives (Boldyrev et al., 2013).

There are five distribution patterns of HRC in skeletal muscle of fish (Yamada et al., 2009; Van Waarde, 1988): (1) migratory pelagic fish (tuna family) contain extremely large amounts (>50 μmol/g) of histidine and anserine; (2) in the families Anguillidae (eels) and Acipenseridae (sturgeons), carnosine is the major imidazole-related compound; (3) anserine is the major component of the imidazole pool in the families Salmonidae (salmons) and Gadidae (cods), and the subclass Elasmobranchii (sharks and rays); (4) in the families Clupeidae (herrings and sardines) and Cyprinidae (carps), only histidine is present, at high concentrations; and, (5) very low levels (<μmol/g) of imidazolerelated compounds are found in the families Pleuronectidae (flounders) and Percidae (perciform fish).

Until we know the underlying evolutionary drivers for this HRC storage, we must be aware of the interspecies differences in order to anticipate potentially different responses in nutrient requirements to changes in water temperature.

8.3 Metabolic transformations and pathways for synthesis of amino acids in animal cells

Fish tissues such as extracts of skeletal muscle contain FAA, which are not incorporated in proteins (Shiau et al., 2001). The FAAs have been implicated as being responsible for the characteristic taste of seafood (Fuke, 1994). In addition, FAAs play important roles in physiological functions such as osmoregulation and buffer capacity in the tissues of aquatic animals (Van Waarde, 1988; Abe, 1995).

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The synthesis of AA occurs in a tissue- and cell-specific manner, requires energy, and also involves inter-organ and compartmentalized metabolism of AA (Arentson et al., 2012). This shows that the changes in amino acid metabolism in the present study might have occurred depending on changes in temperature, due to dietary factors, differences between species, and the cellular specificity of the metabolism.

Figure 18. Overview of metabolic transformations of nutritionally essential AA into nutritionally nonessential AA in animals. BCAA, branched-chain amino acids; D3GP, d-3-glyceraldehyde phosphate; HYP, hydroxyproline; P5C, pyrroline-5-carboxylate; Tau, taurine; TF, tetrahydrofolate (Wu, 2013).

The overall pathways for synthesis of alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, taurine, serine, and tyrosine in animal cells are illustrated in Figure 19 (Wu, 2013). With the exception of arginine and taurine, pathways for the synthesis of these AA were uniformly conserved in the animal lineage. Notably, except for arginine and cysteine, only one or a few steps are required for the synthesis of these AA. Selective conservation of a metabolic pathway during evolution indicates its essentiality for the survival, growth, and reproduction of the organism. Pyruvate, oxaloacetate, and α-ketoglutarate are the ultimate sources of the carbon skeletons of alanine, arginine, asparagine, aspartate, glutamate, glutamine, glycine, proline, and serine. In most cases, glutamate provides the amino group and ATP supplies the energy for AA synthesis (Brosnan 113

General discussion

2000). Enzyme-catalyzed transamination of AA in the biological system, discovered by A.E. Braunstein in 1937, is crucial for AA syntheses. While AA transaminases have broad substrate specificities for AA and α-ketoacids, not all exogenous α-ketoacids can be used to form the corresponding AA in organisms.

Figure 19. Synthesis of amino acids in animal tissues in a cell- and species-dependent manner. The enzymes catalyzing the indicated reactions are: (1) BCAA transaminase; (2) glutamate dehydrogenase; (3) pyrroline-5- carboxylate (P5C) synthase; (4) P5C reductase; (5) aspartate transaminase; (6) alanine transaminase; (7) asparagine synthetase; (8) enzymes for tryptophan catabolism; (9) enzymes for converting glucose into α-KG; (10) glutamine:fructose-6-phosphate transaminase; (11) phosphate-activated glutaminase; (12) glutamine synthetase; (13) ornithine aminotransferase; (14) arginase; (15) ornithine carbamoyltransferase; (16) enzymes for protein synthesis, hydroxylation of peptide-bound proline, and protein degradation; (17) enzymes for converting hydroxyproline into glycine; (18) enzymes for converting threonine into glycine; (19) enzymes for converting choline into glycine; (20) serine hydroxymethyltransferase; (21) cystathionine β-synthase; (22) cystathionine γ-lyase; (23) enzymes for converting cysteine into taurine; (24) enzymes for converting d-3- phosphoglycerate (D3PG) and glutamate into serine; (25) enzymes for methionine catabolism; (26) enzymes for GTP synthesis; (27) enzymes for tetrahydrobiopterin synthesis; (28) phenylalanine hydroxylase; (29) dihydrobiopterin reductase; (30) enzymes of the pentose cycle; (31) proline oxidase; (32) enzymes for pyrimidine synthesis; (33) enzymes for pyrimidine catabolism; (34) enzymes for coenzyme A (CoA-SH) catabolism; (35) carnosinase; (36) aspartate decarboxylase; (37) β-alanine-α-KG transaminase, with malonic acid semialdehyde being produced from propionyl-CoA and malonyl-CoA semialdehyde. GlcN-6-P, glucosamine-6-phosphate; Hcys, homocysteine; KGM, α-ketoglutaramate; MTH, N5-N10-methylene tetrahydrofolate; OH-Pro, hydroxyproline; OAA, oxaloacetate; THF, tetrahydrofolate (Wu, 2013).

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The catabolic pathways of amino acids are also very important. For example, the breakdown of BCAA (valine, isoleucine, leucine) can be reflected by the corresponding acylcarnitines. The effect of mild changes in water temperature on BCAA catabolism was reported in particular in Chapter 7 in line with their corresponding acylcarnities. Acylcarnitines are esterified derivatives of carnitine, which are products of reactions catalyzed by carnitine acyltransferases that utilize acyl-CoA (Sandor et al., 1987). Free carnitine is the major carnitine pool representative as demonstrated in this thesis in the plasma or serum samples. The proportion of acylcarnitines, with acyl moiety ranging from the short-chain acetyl to the long-chain was demonstrated to vary with mild changes in water temperature.

8.4 Dietary tools to support fish at changes in water temperature

Nutrient metabolism, whether anabolic or catabolic, is of a complex pathway in nature. However, with advances in applied nutritional research, scientists have identified and described a lot. Among the nutritional research areas are found the rapidly growing aquaculture sector, which has become increasingly dependent upon the use of external feed inputs, and in particular upon the use of compound aquafeeds. There are changes in production technology and marketing in this sector, which needs improved and advanced changes in feed ingredients to grow. For nutritional research, it is necessary to know how each component of a diet functions with its specific routes through which it affects nutrient metabolism of the animal. On the other hand, animals in terrestrial or aquatic environment live under frequent chlimate challenges including the effect of changes in water temperature, for instance in the aquatic aspect.

Therefore, from this point of view, this thesis explored examples of three main routes to affect nutrient metabolism in fish: (1) the effect of supplementing a metabolic modulator, i.e. β-alanine (Chapters 4 and 5); (2) the effect of dietary macronutrient profile (Chapter 6); (3) the effect of supplementing a modulator of the gut, i.e. mannanoligosaccharides, as a way to affect nutrient metabolism through changes in the gut (Chapter 7).

The model additives are very likely not be representative for all of their kind, but at least it demonstrates that modulation of nutrient metabolism can be achieved, in particular on amino acid metabolism. The fact that for instance in the protein:lipid study many of the biochemical parameters showed interaction between diet and temperature, indicate that there are ways to interfere with the metabolic response of fish to changes in water temperature (Chapter 6). The linear and curvilinear responses in the protein:lipid ratio study still left unexplained relationships which made clear interpretation difficult, but at least can give rise to further studies that can link such interactions with performance at larger scale.

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8.5 Future research perspectives

Although the contribution of global aquaculture to global food fish supply for human consumption reached 50% in 2014, global warming is reducing growth performance and feed utilization of fish. Given the impacts of the changes in water temperature, the consequence of global warming, the studies in this thesis demonstrated to gain general insight about the nutrient metabolism in fish at mild changes in water temperature using, examples of three model nutritional strategies that have different routes of functionalities. These studies were conducted with intensive monitoring at laboratory conditions. It is necessary to further validate the present hypotheses and the findings at large scale outdoor conditions. Some of the points to take into account in future research are:

 It is worth to investigate other strategies of dietary interventions, for instance, managing under dietary treatments for some period and exposing both the control and treatment groups to the effect of changes in water temperature still for some period.

 Future research is necessary to investigate the effects of specific amino acids, omega-3 and 6 FA, HRC, fructooligosaccharide, and other dietary supplemts on fish nutrient metabolism at changes in water temperature. Further research should target nutrient requirements of fish as a function of water temperature.

 The number of fish species, duration of study and sources of fish (wild or hatchery) should be get attention in future research. The ingredients and composition of experimental diets in particular in commercial diets should be known not only for research but also from animal welfare and customer perspectives.  The consistency of blood (plasma or serum) or muscular parameters should be maintained throughout a package of studies like in doctoral researches for comparison for the results of different experiments. The effect of fasting on fish blood and muscular metabolites must be considered in future research at changes in water temperature. During fasting periods, energy reserves could be allocated away from the normal growth and into support of vital processes (Sumpter et al., 1991). For instance, protein and fat were demonstrated to be important sources of energy during fasting in adult Atlantic salmon (Einen et al., 1998).  To link the comparison and interpretation of free amino acids in the serum or plasm and muscle, currently there was a limitation due to the few amino acids measured using tandem mass spectrometry technique. A number of enzymes are involved in nutrient metabolism in blood and muscle that affect the concentrations of free amino acids, acylcarnitines, and HRC. Gene

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expression techniques might work better in future research for detection and expression of those concentrations.  The take-home message: In conclusion, this thesis demonstrated that there were differences in nutrient metabolism between fish species, and even interaction between species and temperature. The changes in fish nutrient metabolism, including amino acids, acylcarnitines, and muscular histidine-related compounds occur depending on changes in water temperature. The evaluated dietary β-alanine, protein:lipid ratio, and mannanoligosacharides showed that dietary factors can affect the changes in fish nutrient metabolism that occur depending on changes in water temperature.

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Summary

The rise in aquaculture contributes to the world’s increasing demand for protein sources in human consumption. Gradually the proportion of aquaculture to total fish production is on the rise, which should reduce the overfishing pressure on fish populations in the wild. In addition, fish consumption has benefits for human health. Yet, aquaculture faces major challenges, such as the lack of sustainable feed resources and the effects of climate change. Global warming does affect water temperature, especially in subtropical and tropical regions, where aquaculture is growing most intensively (Chapter 1).

Each fish species has a preferred temperature range that is considered safe for the fish and should allow normal development. Nevertheless, a mild but constant increase in water temperature might still affect fish metabolism, therefore interfering with the ways that metabolism is using dietary nutrients. The general introduction of this thesis further describes the mechanisms how fish react to increased water temperature, including the principles of heat tolerance and heat resistance. Although heat stress has been intensively studied in fish, the effects of mild but constant elevation on fish nutrient metabolism are not well documented. Concomitantly, nutritional strategies to help fish to cope with any changes in nutrient metabolism due to mild changes in water temperature are still uncovered. This thesis presents three potential strategies to interfere with nutrient metabolism at mild changes in water temperature: 1) adding a metabolic modulator such as β-alanine, 2) changing the macronutrient ratio, and 3) adding a gut modulator such as mannanoligosaccharides.

The major scientific aim of this thesis thus was to investigate the effect of mild changes in water temperature on nutrient metabolism in fish, with an evaluation of nutritional strategies to improve the nutrient metabolism of fish at mild changes in water temperature (Chapter 2). The specific objectives were 1) to investigate the effect of mild changes in water temperature in fish with focus on nutrient metabolism in skeletal muscle, 2) to evaluate β-alanine as a dietary metabolic modulator in fish at mild changes in water temperature, 3) to evaluate the effect of dietary macronutrient profile on nutrient metabolism in fish at mild changes in water temperature, and 4) to evaluate the effect of mannanoligosaccharides as a dietary intestinal modulator in fish at mild changes in water temperature.

Fish species show distinct differences in their muscular concentrations of free amino acids (FAA) and imidazoles. In Chapter 3, a study was conducted to investigate whether the metabolic response to mild changes in water temperature (MCWT) relates to species-dependent muscular concentrations of FAA and imidazoles. Thirteen carp and 17 Nile tilapia, housed one per aquarium, were randomly assigned to either an acclimation temperature (25°C) or a MCWT (30°C) for 14 days. The main muscular concentrations were histidine (HIS) in carp versus N-α-acetylhistidine (NAH) and taurine (TAU) in tilapia. Although the sum of imidazole (HIS+NAH) and TAU in the muscle, remained

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Summary constant over species and temperature zones, the (NAH+HIS)/TAU ratio was markedly higher in carp versus tilapia, and decreased with the MCWT only in carp. Many of the muscle FAA showed higher concentrations in carp than in tilapia, and plasma acylcarnitine profile suggested a higher use of AA as well as fatty acids in the metabolism of carp. On the contrary, the concentration of 3- hydroxyisovalerylcarnitine, a sink of leucine catabolism, pointed to an avoidance of leucine use in the metabolism of tilapia. Despite a further increase of plasma longer-chain acylcarnitines in tilapia at MCWT, their corresponding beta-oxidation products (3-hydroxy-longer-chain acylcarnitines) remained constant. Together with the higher plasma non-esterified fatty acids (NEFA) in carp, the latter shows that carp, being a fatter fish, more readily mobilises fat than tilapia when exposed to MCWT, which coincides with a more intensive muscular mobilisation of imidazoles. In conclusion, there are differences in metabolism between species, and even interaction between species and temperature, which was demonstrated by changes in muscle imidazole to taurine ratio.

In Chapter 4, a study was executed to investigate the effect of dietary β-alanine (BA) on amino acid (AA) metabolism and voluntary feed intake in carp (Cyprinus carpio) at mild changes in temperature to exert AA catabolism. β-Alanine was chosen as a potential metabolic modulator of amino acid metabolism to support the fish metabolism at higher water temperature. Carp was used because we hypothesised that a fish that does not store BA-related dipeptides such as carnosine and anserine, could benefit most from dietary BA. Twenty-four fish in 12 aquaria were randomly assigned to either a control diet or the same diet with 500 mg BA/kg. A 14-day period at an acclimation temperature (23°C) was followed by 15 days at constant mild changes in temperature (27°C). After the 15 days, all fish were euthanised for muscle analysis on histidine-related compounds (HRC), whole blood on FAA and carnitine esters. The carnosine and anserine analysis indicated that all analyses were below the detection limit of 5 µmol/L, confirming that carp belongs to a species that does not store HRC. The increases in FAA concentrations due to BA supplementation failed to reach the level of significance. The effects of dietary BA on selected whole blood carnitine esters and their ratios were also not significant. The supplementation of BA tended to increase body weight gain and feed intake. The lack of differences in the selected nutrient metabolites in combination with tendencies of improved growth performance warrants further investigation to unravel the mechanism of BA affecting feed intake. This first trial on the effect of BA supplementation on AA catabolism showed that its metabolic effect in carp at constant mild changes in temperature was very limited. Further studies need to evaluate which conditions are able to exert an effect of BA on AA metabolism.

Anyhow, seen this limited effect, it was proposed that contrary to our initial hypothesis, a fish species such as sturgeon that does apply muscular storage of BA-related dipeptides, might be more adapted to

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Summary use BA in its metabolism, and therefore might better respond to dietary BA (Chapter 5). Fourteen sturgeons were randomly distributed, individually or pairwise in eight aquaria, and assigned to eight feeding groups on diets prepared without (control) or with graded levels of BA for a week. Dietary BA had no effect on serum FAA, but linearly decreased acetylcarnitine and propionylcarnitine. Remarkably, BA supplementation increased neither β-alanine, nor carnosine in sturgeon muscle, but induced a distinct increase in glycine, glycine to total FAA, citrulline, cystathionine, hence reducing the concentrations of many other amino acids. Also muscle ammonium was reduced by BA. The changes observed in acylcarnitines point to a down regulation of the citric acid cycle. The marked BA-induced increase in muscle glycine and citrulline suggests a shift in amino acid metabolism that might be beneficial to counteract the increase in amino acid breakdown at changes in water temperature, but a performance trial should evaluate what the reduction of the citric acid cycle means for the overall metabolism in these fish. Sturgeon, as a carnosine-storing fish species, did not show a dose-response in muscular carnosine storage due to BA supplementation, but instead developed a considerable increase in glycine, citrulline and cystathione, suggesting an important metabolic modulator function of BA.

Given the changes in amino acid metabolism that occur depending on changes in temperature, a study was conducted to investigate the effect of changes in dietary protein:lipid ratio on nutrient metabolism of Nile tilapia fed at acclimation or mild changes in water temperature (MCWT) (Chapter 6). Twenty six Nile tilapia were randomly distributed in 12 aquaria and assigned to either acclimation (22°C) or MCWT (28°C) for 14 days. The protein:lipid ratio interacted with MCWT on serum free amino acid concentrations of glycine, phenylalanine, tyrosine, and sum of the serum free amino acids. The protein:lipid ratio linearly affected serum concentrations of freecarnitine, dodecanoylcarnitine, 3OH- hexadecanoylcarnitine, octadecenoylcarnitine, 3OH-octadecenoylcarnitine, sum of 3OH-fatty acids, sum of 3OH-longer-chain fatty acids, and ratio of sum of 3OH-longer-chain fatty acids to sum of longer-chain fatty acids. The temperature-squared protein:lipid ratio interaction quadratically affected serum concentrations of tetradecanoylcarnitine, octadecanoylcarnitine, and sum of longer-chain fatty acids. There was also a quadratic relationship between squared protein:lipid ratio and the serum concentration of hexadecenoylcarnitine. In the skeletal muscle there was a linear relationship between the protein:lipid ratio and the skeletal muscle FAA concentrations of glutamate and glutamine. The temperature-diet interaction linearly affected the concentrations of muscle ornithine, tyrosine, proline, taurine, and urea. The squared protein:lipid ratio quadratically affected the skeletal muscle β-alanine, asparagine, and threonine. The temperature-squared protein:lipid ratio interaction quadratically affected muscle phenylalanine. In conclusion, this study demonstrated that changes in fish nutrient

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Summary metabolism occur depending on changes in water temperature and the dietary protein:lipid ratio can affect this metabolism.

The study in Chapter 7 was conducted to evaluate whether dietary mannanoligosaccharides (MOS) modulate the effect of mild changes in water temperature on intestinal morphology and some selected whole blood amino acids and acylcarnitines in common carp (Cyprinus carpio carpio). Twenty fish in 10 aquaria were randomly assigned to either a control diet or the same diet with 4 g/kg MOS. A 35- day period at acclimation temperature (23.2°C) was followed by 14 days at changes in temperature (26.1°C). After both temperature periods, one fish per aquarium was euthanized for the intestinal morphology and whole blood analysis on free amino acids and carnitine esters. The changes in temperature period decreased the number of neutral and acid mucin-secreting goblet cells and tended to reduce the fold height in the midgut, irrespective of MOS supplementation. In both periods, supplementation of MOS increased the number of goblet cells in the hindgut, but other histomorphometries were unaffected. Free concentrations of whole blood amino acids were increased after the mild changes in temperature period: valine, leucine, methionine, phenylalanine and tyrosine, but not affected by MOS supplementation. None of the carnitine esters were altered by the changes in temperature except propionyl carnitine that was higher after the mild changes in temperature of 14 days. Supplementation of MOS only tended to reduce the tiglyl carnitine and methylmalonyl carnitine concentrations. The analysis of free amino acids and carnitine esters could not support the hypothesis that MOS counteracts depressing effects of changes in temperature on amino acid catabolism. In conclusion, moderate elevation of water temperature could lead to considerable changes in gut histology and metabolism.

The general discussion in Chapter 8 explores the reasons for changes in amino acid metabolism that occur depending on changes in temperature, and takes a comparative angle to consider the differences between species in metabolic response to changes in water temperature. The nutritional strategies to support the fish at changes in water temperature are also revisited since dietary factors can affect their metabolism, including the methodological challenges to come to final answers, and how they can be applied to practice.

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Samenvatting

De stijging van de aquacultuur draagt bij tot de wereldwijd stijgende vraag naar eiwitbronnen voor menselijke consumptie. Het aandeel van aquacultuur in de totale visproductie neemt gestaag toe, hetgeen de druk van overbevissing op natuurlijke vispopulaties zou moeten terugdringen. Bovendien heeft visconsumptie voordelen voor humane gezondheid. De aquacultuur staat echter voor uitdagingen, zoals het gebrek aan duurzame voedselbronnen en de invloeden van klimaatverandering. De opwarming van de aarde verhoogt inderdaad de watertemperatuur, vooral in subtropische en tropische gebieden, waar nu vooral de aquacultuur het sterkste groeit.

Elke vissoort heeft een geprefereerd temperatuurbereik dat als veilig wordt verondersteld voor de vis en ook normale ontwikkeling toelaat. Echter, een milde maar chronische verhoging van de watertemperatuur kan het metabolisme van vissen beïnvloeden, en daarom tussenkomen in de manier waarop het metabolisme voedingsstoffen benut.

De inleiding van deze thesis beschrijft de mechanismen waarmee vissen reageren op verhoogde watertemperatuur, inclusief de begrippen hittetolerantie en hitteresistentie. Hoewel hittestress intensief werd bestudeerd bij vissen, zijn de effecten van milde maar chronische verhoging niet goed gedocumenteerd. Als logisch gevolg zijn ook de nutritionele strategieën onbekend om vissen te helpen omgaan met veranderingen ten gevolge van milde verhoging van watertemperatuur. Deze thesis stelt drie mogelijke strategieën voor om in te grijpen op het nutriëntenmetabolisme bij verhoogde watertemperatuur: 1) veranderen van de macronutriëntenverhouding; 2) toevoeging van een metabole modulator zoals β-alanine, en 3) toevoeging van een darmmodulator, zoals mannanoligosacchariden.

De voornaamste wetenschappelijke doelstelling van deze thesis was dus om de invloed te onderzoeken van mild verhoogde watertemperatuur op nutriëntenmetabolisme bij vissen, met daarbij een evaluatie van nutritionele strategieën om het nutriëntenmetabolisme bij mild verhoogde watertemperatuur te verbeteren. De specifieke doelstelling waren als volgt: 1) de invloed van mild verhoogde watertemperatuur bij vissen te onderzoeken, met aandacht voor het nutriëntenmetabolisme in skeletspieren, 2) de invloed van β-alanine als metabole modulator onderzoeken bij vissen bij mild verhoogde watertemperatuur, 3) de invloed van macronutriëntenverhouding in visvoeding onderzoeken, en 4) de invloed bestuderen van mannanoligosacchariden als darmmodulator in visvoeding bij mild verhoogde watertemperatuur.

Vissoorten vertonen beduidende verschillen in hun spierconcentraties van vrije aminozuren (VA) en imidazoles. In hoofdstuk 3 werd een studie uitgevoerd om te onderzoeken of de metabole respons op

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Samenvatting mild verhoogde watertemperatuur (MVWT) verband houdt met de soortspecifieke spierconcentraties va VA en imidazoles. Dertien karpers en 17 Nijltilapia’s, telkens één per aquarium, werden willekeurig toegewezen aan ofwel acclimatisatie temperatuur (25°C) of aan MVWT (30°C) gedurende 14 dagen. De voornaamste VA concentraties in spieren waren histidine bij karper en N-α- acetylhistidine en taurine (TAU) bij tilapia. Hoewel de som van imidazoles (HIS+NAH) en TAU in de spier constant bleef over soorten en temperatuurzones, was de verhouding (NAH+HIS)/TAU opmerkelijk hoger bij karper dan bij tilapie, en werd alleen hoger door MVWT bij de karper. Vele spier VA vertoonden hogere concentraties in karper dan in tilapia, en het plasma acylcarnitineprofiel suggereerde een hoger gebruik van aminozuren en vetzuren in het metabolisme van de karpers. Daarentegen toonde de concentratie van 3-hydroxyisovalerylcarnitine, een doodlopende pathway van leucinecatabolisme, dat leucine werd vermeden in het metabolisme van tilapia. Ondanks een verdere stijging van plasma langeketen acylcarnitines in tilapia bij MVWT, bleven de overeenkomstige beta- oxidatieproducten (3-hydroxy-langeketen acylcarnitines) constant. Tezamen met de hogere non- esterified fatty acids (NEFA), toont het dat de karper – een vettere vis – gemakkelijker vet mobiliseert dan tilapia bij MVWT, hetgeen samenvalt met het intensievere mobiliseren van imidazoles. Deze studie toont aan dat vissoorten verschillen in hun metabole respons om MVWT, en dat dit geassocieerd is met soortspecifieke veranderingen in de imidazole:taurine ratio in de spieren.

Een studie in hoofdstuk 4 werd uitgevoerd om de invloed van BA te onderzoeken op aminozuurmetabolisme en vrijwillige voederopname bij karpers (Cyprinus carpio) bij MVWT (om aminozuurcatabolisme uit te lokken). β-Alanine werd gekozen als mogelijke metabole modulator van aminozuurmetabolisme om het vismetabolisme te ondersteunen bij MVWT. Karpers werden gebruikt vanuit onze hypothese dat vissen die geen BA-gerelateerde dipeptiden stapelen zoals carnosine en anserine, mogelijks het meeste voordeel zouden genieten van BA-supplementatie. Vierentwintig vissen in 12 aquaria werden willekeurig toegewezen aan ofwel een controledieet ofwel dit diet met 500 mg BA/kg. Een 14-daagse priode bij acclimatisatie temperatuur (23°C) werd gevolgd door 15 dagen bij chronische MVWT (27°C). Na die 15 dagen werden alle vissen geëuthanaseerd voor spieranalyse op histidine-bevattende dipeptiden, voor bloedanalyse op vrije aminozuren en carnitine- esters. De analyse van carnosine en anserine toonde dat alle analyses onder de detectielimiet lagen van 5 µmol/l, hetgeen bevestigde dat de karper een soort is die geen histidine-gerelateerde dipeptiden opslaat. De stijgingen in VA concentraties vanwege BA supplementatie behaalden geen significante niveaus. De invloed van BA op bepaalde carnitine-esters in bloed en hun ratios waren eveneens niet significant. De BA supplementatie vertoonde een tendens naar hoger lichaamsgewicht en voederopname. Het gebrek aan verschillen in de geselecteerde nutriëntenmetabolieten in combinatie met de tendens naar verbeterde groei heeft verder onderzoek nodig om het mechanisme van BA op

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Samenvatting voederopname te ontrafelen. Deze eerste studie over het effect van BA op aminozuurmetabolisme toont dat het metabole effect bij karper bij MVWT eerder beperkt was. Verdere studies zouden moeten nagana welke voorwaarden nodig zijn om een BA effect uit te lokken op aminozuurmetabolisme. Hoedanook, gezien het beperkte effect bij karpers, zou het kunnen dat – integenstelling tot onze aanvankelijke hypothese – een vissoort die wel BA-gerelateerde dipeptiden stapelt, meer aangepast is om BA te gebruiken in het metabolisme, en daarom beter reageert op BA supplementatie. Zo’n soort is de steur. Veertien steuren werden willekeurig verdeeld, individueel of paarsgewijs over acht aquaria, en toegewezen aan acht voedergroepen met diëten zonder (controle) of met gradueel wijzigende concentraties van BA, gedurende een week. β-Alanine had geen effect op de serum aminozuren, maar induceerde een lineaire daling van acetyl- en propionylcarnitine. Opmerkelijk was dat BA supplementatie geen stijging veroorzaakte in β-alanine en carnosine in de spier, maar wel in glycine (absoluut en relatief), citrulline, en cystathionine. Ook ammonium was gedaald in de spier. De verandering in de acylcarnitines lijken te duiden op een afremming van de citroenzuurcyclus. De opmerkelijke stijging in glycine en citrulline suggereert een verschuiving in het aminozuurmetabolisme die mogelijks gunstig is om de stijging van het aminozuurcatabolisme door MVWT tegen te gaan, maar een zoötechnische studie zou moeten nagana wat de eventuele terugdringing van de citroenzuurcyclus betekent voor het algemene metabolisme van de vissen. De steur, een carnosine-stapelende vis, vertoonde geen dosis-afhankelijke respons van carnosinestapeling in de spier wegens BA, maar wel een stijging in glycine, citrulline en cystathionine, wat doet vermoeden dat BA een belangrijke metabole modulator kan zijn.

Gezien de impact van MVWT op aminozuurmetabolisme, werd een studie uitgevoerd om de invloed van het macronutriëntenprofiel in de voeding te onderzoeken van Nijltilapia bij acclimatisatie of mild verhoogde watertemperatuur. Zesentwintig Nijltilapia’s werden willekeurig verdeeld over 12 aquaria bij ofwel acclimatisatie temperatuur (22°C) of MVWT (28°C) gedurende 14 dagen. De eiwit:vet verhouding interageerde met MVWT voor de serumconcentraties van verschillende vrije aminozuren: glycine, alanine, fenylalanine, tyrosine en de some van de VA in het serum. De temperatuur-dieet interacties beïnvloedden ook acetylcarnitine, tetradecanoylcarnitine, hexadecanoylcarnitine, octadecanoylcarnitine, de som van de 3-hydroxy vetzuren, de som van de 3-hydroxy langeketen vetzuren, de som van de langeketenvetzuren, en de verhouding van de som van de 3-hydroxy vetzuren tot de som van de vetzuren. In de spieren was er een temperatuur-dieet interactie voor de concentraties van glutamine, aspartaat, fenylalanine en tyrosine. Deze studie demonstreerde dat de metabole reactie van vissen duidelijke verandert naargelang de watertemperatuur, en dat de invloed van diëtaire eiwit:vet verhouding op het metabolisme afhangt van de watertemperatuur.

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Samenvatting

In hoofdstuk 6 werd een studie uitgevoerd om de invloed van mannanoligosacchariden (MOS) bij MVWT na te gaan op darmmorfologie en geselecteerde aminozuren an acylcarnitines in het bloed van de karper (Cyprinus carpio). Twintig vissen werden willekeurig toegewezen aan 10 aquaria met ofwel een controledieet of dit diet met 4 g MOS/kg. Na een periode van 35 dagen bij acclimatisatie temperatuur (23,2°C) volgden 14 dagen bij verhoogde temperatuur (26,1°C). Na beide temperatuurperioden, werd een vis per aquarium geëuthanaseerd voor het bepalen van darmmorfologie en analyse van aminozuren en acylcarnitines in het bloed. De periode met verhoogde watertemperatuur verlaagde het aantal neutrale en zure mucus-secreterend slijmbekercellen en toonde een tendens naar lagere villushoogte in de middendarm, onafhankelijk van MOS supplementatie. In beide perioden verhoogde MOS supplementatie het aantal slijmbekercellen in de einddarm, maar andere histomorfometrische parameters bleven onveranderd. De concentraties aan VA in het blood waren gestegen na de MVWT periode: valine, leucine, methionine, fenylalanine en tyrosine, maar werden niet beïnvloed door MOS supplementatie. Geen van de carnitine-esters waren gewijzigd door de MVWT behalve propionylcarnitine dat hoger was na de 14 dagen MVWT. Supplementatie van MOS vertoonde alleen een tendens naar lagere tiglylcarnitine en methylmalonylcarnitine concentraties. De analyse van VA en carnitine-esters kon de hypothese niet onderbouwen dat MOS de nadelige effecten van verhoogde temperatuur zou kunnen teniet doen. De MVWT kon dus wel aanleiding geven tot opmerkelijke veranderingen in de darmhistologie en metabolisme.

De algemene discussie in hoofdstuk 7 verkent de redenen voor het verhoogde aminozuurcatabolisme bij MVWT, en gaat vanuit een comparatief uitgangspunt de verschillen tussen soorten beschouwen qua reactive op MVWT. De nutritionele strategieën om vissen te ondersteunen bij verhoogde watertemperatuur worden ook herbelicht, inclusief de methodologische uitdagingen om te komen tot finale antwoorden en hoe die in de praktijk kunnen worden aangewend.

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Curriculum vitae

Fikremariam Geda Ararame was born on 31 October 1981 in West Shewa, Oromia, Ethiopia. In 2002, he completed secondary school in natural sciences stream and took Ethiopian school leaving certificate examination at the then Kokebe Tsibah Comprehensive Secondary School in Addis Ababa. In the same year, he started his education at Jimma University (JU) and graduated with the diploma of Bachelor of Science in Animal Production and Health in 2006. After graduation, he was employed by JU and served as Graduate Assistant I to Assistant Lecturer from 2006 to 2009. Fikremariam has lectured of the courses Fisheries and Aquaculture and Fisheries and Fish Diseases at JU since 2012.

In 2009, Fikremariam was awarded a VLIR-UOS International Course Program scholarship and studied his master education at Laboratory of Aquaculture and Artemia Reference Center, Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, Belgium. He conducted his master thesis research with the topic, “Prebiotic effects on energy source preference, gut morphology and feed intake regulation in common carp (Cyprinus carpio carpio) during mild heat stress,” at Laboratory of Animal Nutrition, Faculty of Veterinary Medicine. In 2012, he obtained the diploma of Master of Science in Aquaculture, which belongs to the academics of Applied Biological Sciences.

With his diploma of Master of Science in Aquaculture and the MSc research he conducted, Fikremariam got an opportunity to continue his PhD research with a Ghent University scholarship in 2012. From August 2013 onwards, his PhD scholarship was financed by Ghent University and VLIR- UOS JU-IUC program.

Fikremariam Geda has authored and co-authored several scientific publications in international journals. He was also a speaker at several international conferences.

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Bibliography

Articles in international journals

Abstracts in conference proceedings

Geda, F., Declercq, A., Decostere, A., Remø, S., Waagbø, R., Wuyts, B., Lourenço, M., Janssens, G.P.J., 2015. Effect of mild changes in temperature on nutrient metabolism in carp and Nile tilapia. Proceedings of the 19th Congress of the European Society of Veterinary and Comparative Nutrition, September 17-19, 2015, Toulouse, France, pp.92.

Geda, F., Declercq, A., Decostere, A., Lauwaerts, A., Wuyts, B., Derave, W., Janssens, G.P.J., 2013. Dietary β-alanine does not affect amino acid metabolism under mild constant changes in temperature in carp. Proceedings of the 17th Congress of the European Society of Veterinary and Comparative Nutrition, September 19-21, 2013, Ghent, Belgium, pp.61. (ISBN 978-90-586435-3- 7).

Geda, F., Rekecki, A., Decostere, A., Bossier, P., Wuyts, B., Kalmar, I.D., Janssens, G.P.J., 2012. Changes in intestinal morphology and amino acid catabolism in common carp at changes in temperature as affected by dietary mannanoligosaccharides. Proceedings of the 16th Congress of the European Society of Veterinary and Comparative Nutrition, September 13-15, 2012, Bydgoszcz, Poland, pp.55. (ISBN 978-83-921732-2-9).

Janssens, G.P.J., De Graeve, M., Tagliabue, M., Geda, F., Pauwels, J., Van Hecke, T., Akbarian, A., Michiels, J., 2012. Association between intestinal allometry, nutritient utilization and growth in broiler chickens. Proceedings of the 16th Congress of the European Society of Veterinary and Comparative Nutrition, September 13-15, 2012, Bydgoszcz, Poland, pp.111. (ISBN 978-83- 921732-2-9).

Workshops attended

CTA, 2013. Fish farming: the new drive of the blue economy. Brussels development briefing 32, Technical center for agricultural and rural cooperation (CTA), July, Brussels, Belgium.

VLIR-UOS INCO, 2016. International conference (INCO); Aquaculture and food security: defining potential opportunities for the development of aquaculture in Ethiopia, 14-16 April, Jimma, Ethiopia.

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Acknowledgements

First of all, I would like to thank my promotor Prof. dr. Geert P.J. Janssens for giving me the opportunities to work on fish both my MSc and PhD researches. I wish to express my appreciation for his inspired advice and guidance, constructive comments and suggestions, encouragement and kindness during the entire course of the researches. I strongly believe that all his advice and guidance have contributed to my scientific maturity. The first day I met him at his lab, I remember his words: “It is ‘possible’ to organize such type of a trial in this lab.”

It is with great pleasure that I would like to acknowledge members of the examination committee: Prof. dr. Luc Duchateau, Dr. Gunther Antonissen, Prof. dr. Patrick Sorgeloos, Prof. dr. Rune Waagbø, Prof. dr. Wim Derave, and Dr. Johan Schrama for reading, constructive comments and suggestions to improve the scientific quality of this thesis.

I would also like to acknowledge the cooperative colleagues at the Laboratory of Animal Nutrition where I have conducted my studies and spent a joyful time; thank you everyone, the past and the present: Alireza Khadem, Amy Deluycker, An Cools, Andrea Brenes Soto, Annelies De Cuyper, Arturo Muñoz Saravia, Daniël Temsy, Daisy Liu, Donna Vanhauteghem, Galena Rybachuk, Hannelore Van de Velde, Herman De Rycke, Isabelle Kalmar, Isolde Tack, Jana Pauwels, Jenny Martens, Jia Xu, Kristel Rochus, Lien Bruynsteen, Luk Sobry, Mariëlle Van Zelst, Marta Ribeiro Alves Lourenço, Myriam Hesta, Ruben Decaluwe, Ruben Van Gansbeke, Sandra Debevere, Sarah Depauw, Sofie Dupont, Veerle Vandendriessche, Veronique Dermamaw, Wendy Wambacq, and others.

I would like to extend my grateful thank to the following professors with their laboratories and teams whose collaborations substantially contributed to the success of my researches: Ghent University: Prof. dr. Annemie Decostere (Department of Morphology), the late Prof. dr. Brigitte Wuyts (passed away on 19 March 2015, rest in peace; Department of Clinical Chemistry, Microbiology and Immunology), Prof. dr. Wim Derave (Department of Movement and Sports Sciences); Prof. dr. Johan Verreth (Aquaculture and Fisheries, Wageningen University, The Netherlands), and Prof. dr. Rune Waagbø (National Institute of Nutrition and Seafood Research (NIFES), Norway).

I would like to express my appreciation of the IUC-JU team for their smooth management and care during my study; thank you all: South (Kora Tushune, Kassahun Eba, Esubalew Ayalew) and North (Luc Duchateau, Annick Verheylezoon, Madina Abukaeva), and others. I would not forget the friendship cares, encouragements and motivations of my colleagues and friends during my study, thank you all: Abebew Gashaw, Adugna Eneyew, Akalu Dafisa, Ayele Bacha, Belay Duguma, Benti

159

Acknowledgements

Deressa, Berhanu Belay, Derbew Belew, Dessalegn Obsi, Duguma Adugna, Efrem Wakjira, Fikadu Mitiku, Gezahegn Berecha, Girum Ketema, Lidia Tesfaye, Melkamu Dumessa, Oda Bashir, Osman Rahmeta, Sileshi Belew, Solomon Tullu, Tadele Tolossa, Taye Tolamariam, Tsegaye Girma, Worku Jimma, Zeleke Mekonnen, Zerihun Assefa, Zerihun Kebebew, and others. This is also a great opportunity to thank Leen Christiaens and Johan Six, Micheline Bruwaert and René Morel, and all members of their family for the special cares, supports and motivations during my study; thank you all.

My mother, Nedi Beyecha, “You were the making and everything of me. I would not be at this level of education without your stand and believe in education. You really made the right decision the day you sent me to school with a pen and an exercise book of thirty two pages. You were so sure that I would be at some level of education at some point and live for you, but I missed you a few months before I joined Jimma University, ‘rest in peace my mother’. It is difficult, but I must not be disappointed because you have had special love and memory in me, which remains a blessing to me.” My special thanks do also go to my brothers Megerssa, Bekele and Tolossa who have paid a special care to make me a man of today and tomorrow. I would also like to extend my acknowledgement to all members of my family.

My lovely wife, Emebet Tsegaye has significant contributions for the success of my PhD research. I always remember her patience, advice and cool discussions during my study. She has really played a critical role and backup to motivate and encourage me until the final write up of this thesis. I have a special care, respect and consideration for her as my natural partner and as my mother. I missed my mother, but I am happy that I have Emebet.

I have special place and memory with the special kindness, care and support of my father in law, Mr. Tsegaye Debele who above all, was taking me every time to and from Bole international airport during my flights to Belgium and back to Ethiopia. Really thank you very much my father in law!

Finally, I wish to extend my sincere acknowledgements to all whose name I have not mentioned.

Fikremariam Geda Ararame

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