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Effects of dietary canola oil level on growth, fatty acid metabolism and physiology of red sea bream… Huang, Shih-Yin Susie 2007

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EFFECTS OF DIETARY CANOLA OIL LEVEL ON GROWTH, FATTY ACID METABOLISM AND PHYSIOLOGY OF RED SEA BREAM FINGERLNGS AND SPRING CHINOOK SALMON PARR by SHIH-YIN SUSIE HUANG B.Sc, The University of British Columbia, 2005 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA July 2007 © Shih-Yin Susie Huang, 2007 ABSTRACT Lipids are an invaluable dietary component for fish because they furnish the indispensable essential fatty acids (EFA) that are required for normal growth and development of the animaf EFA generally refers to 18:3n-3, 18:2n-6 and their metabolic derivatives, 20:5n-3, 22:6n-3 and 20:4n-6. Marine and freshwater species have distinct EFA requirements largely due to differences in the lipid compositions of their naturally available diets as well as differences in their metabolism of lipids and fatty acids (FA). Nonetheless, marine fish oils (FO) are the traditional lipid sources for finfish aquafeeds in both the marine and freshwater environments. However, recent environmental and economical constraints have generated interest in non-marine sources of lipid in finfish aquafeeds. This thesis examined the physiological effects of partially substituting canola oil (CO) for FO in practical commercial diets of two high-value finfish species during their early development. In the first experiment, triplicate groups of red sea bream fingerlings (Pagrus major) were fed 4 isoenergetic, isonitrogenous and isolipid commercial diets with varying levels of refined CO up to 70% of total dietary lipid content over a 12-week period. In the second experiment, triplicate groups of spring chinook salmon parr (Oncorhynchus tshawytscha) were fed practical dry diets in which CO comprised up to 72% of total lipid (mostly FO) over a 30-week period. No adverse effects of diet on growth and whole body proximate constituents were found in either study. Whole body FA composition correlated strongly to the diet FA compositions. Signs of specific FA retention were observed in both species, but to a much greater degree in the spring chinook salmon. Nonetheless, specific FA were strongly retained in the red sea bream liver polar lipids. Ionoregulatory development in the spring chinook salmon was uncompromised by diet treatment. However, whole body [Cl] was influenced by diet at week 10 and 15, where body [Cl"] was -negatively correlated to dietary CO content. Overall, results demonstrated excellent potential for CO to be the main source of supplemental lipid in commercial aquafeeds for juvenile Japanese red sea bream and spring chinook salmon parr. i i T A B L E O F C O N T E N T S Abstract i i Table of Contents i i i List of Tables iv List of Figures v Acknowledgements : vi Co-Authorship Statement vi i C h a p t e r O n e : I n t r o d u c t i o n 1.1 State of World Fisheries and Aquaculture 2 1.2 Lipids and Fatty Acids ......4 1.3 Alternative Lipids 11 1.4 Model Species 15 1.5 Research Purpose and Hypothesis 19 Figures 21 References 22 C h a p t e r T w o : R e d Sea B r e a m Study 2.1 Introduction 29 2.2 Materials and Methods 33 2.3 Results 37 2.4 Discussion 39 Summary 45 Tables 46 Figures 53 References 54 C h a p t e r T h r e e : S p r i n g C h i n o o k S a l m o n Study 3.1 Introduction 58 3.2 Materials and Methods 62 3.3 Results 67 3.4 Discussion 70 Summary 76 Tables 77 Figures : 84 References 87 C h a p t e r F o u r : G e n e r a l Discuss ion a n d Conclusions 4.1 General Discussion and Conclusions 91 References 95 iii LIST OF TABLES Table 2.1: Ingredient compositions of the experimental diets 46 Table 2.2: Mean concentrations of proximate constituents and gross energy in the experimental diets 47 Table 2.3: Percent fatty acid contents in pollock liver oil and canola oil and the experimental diets 48 Table 2.4: Mean initial and final body weight, weight gain, specific growth rate, dry feed intake, feed efficiency, protein efficiency ratio, percent protein deposited, percent lipid deposited, gross energy utilization, and survival of red sea bream in relation to diet treatment 49 Table 2.5: Initial and final mean concentrations of proximate constituents and gross energy contents in the whole bodies of red sea bream in relation to diet treatment ...--50 Table 2.6: Final mean fatty acid contents in the whole bodies of red sea bream in relation to diet treatment 51 Table 2.7: Final mean fatty acid contents in the liver polar lipids of red sea bream in relation to diet treatment 52 Table 3.1: Ingredient compositions of the experimental diets 77 Table 3.2: Mean percentages of proximate constituents, gross energy contents and chloride content in the experimental diets 78 Table 3.3: Percentages of fatty acids in anchovy oil/poultry fat, canola oil, basal mixture, and the experimental diets 79 Table 3.4: Mean initial and final body weight, weight gain, specific growth rate, dry feed intake, feed efficiency, protein efficiency ratio, percent protein deposited, gross energy utilization, and survival of chinook salmon in relation to diet treatment over the 30-week feeding trial .' 80 Table 3.5: Whole body initial and final mean concentrations of proximate constituents and gross energy contents in the whole bodies of spring chinook salmon in relation to diet treatment 81 Table 3.6: Final mean percent fatty acids in the whole bodies of spring chinook salmon in relation to diet treatment 82 Table 3.7: Mean plasma chloride and sodium ion content of spring chinook salmon in freshwater and following a 24-h SW challenge test at week 25 and 30 of the experiment in relation to diet treatment 83 LIST OF FIGURES Figure 1.1: Pathway of biosynthesis of C20 and C22 HUFA from n-3, n-6 and n-9 families of C l 8 PUFA precursors 21 Figure 2.1: Relationship between whole body and liver polar lipid fatty acid concentrations and dietary concentrations of 18:2n-6, 18:3n-3, AA, EPA and DHA in red sea bream juveniles fed either FO, C025, C048 or C O 7 0 . . 53 Figure 3.1: Relationship between dietary fatty acid concentrations and whole body fatty acid concentrations of 18:2n-6, 18:3n-3, EPA(20:5n-3) and DHA (22:6n-3) in total lipids of spring chinook salmon parr fed either dAPF, C025, C049 or C072... 84 Figure 3.2: Post 24-hour seawater challenge survival in spring chinook salmon in relation to diet treatment at 5, 10, 15, 20, 25, and 30 weeks of feeding 85 Figure 3.3: Mean whole body chloride ion content of spring chinook salmon in freshwater and following a 24-hour seawater challenge at week 5, 10, and 15 of the experiment in relation to diet treatment 86 ACKNOWLEDGEMENTS I would like to take this opportunity to thank the many people who have offered their continual assistance, support and guidance throughout the duration of my research. I would like to thank Dr. Colin Brauner and Dr. Dave Higgs for their encouragement, persistence, and patience and for spending valuable time editing and improving papers. I am extremely lucky to have not just one, but two, amazingly supportive supervisors. I would also like to extend my gratitude to Dr. Shannon Balfry and Dr. Patricia Schulte for their support and suggestions. Dr. Shuichi Satoh, who welcomed me to his lab during my 6-month stay in Tokyo and generously covered all experimental expenses, contributed immensely to this project. Without his collaboration, the second chapter of this thesis would not have come into existence. A number of co-op, directed studies and graduate students have supported me during the unbearable feeding trials. My appreciation goes to Sarah Henderson and other co-op students for fish husbandry; Amelia Grant and Dr. Rosalind Leggatt for sampling and the staffs at CAER: Janice Oakes, Mahmoud Rowshandeli and Jill Sutton for their helpful advice and technical assistance. Special thanks go to Erin Friesen for the many great debates, technical advice, and her ever entertaining personality and friendship and also to Miki Nomura, a great friend who showed up at every sampling without my request and kindly showered me with encouragement when the project was not going smoothly. Furthermore, I would like to extend my appreciation to Dan Baker, Clarice Fu, Matt Regan, Jodie Rummer and Dr. Louise Kuchel at the University of British Columbia for their co-authorship, support and covering my lab duties when I was either abroad or at CAER. The same goes to my Japanese colleagues for their research support and great discussions. Finally, I would like to thank my parents, for their unconditional love and support, and for their honest attempts at trying to incorporate my research topic into our daily discussions over dinner. I would not have had the courage to be where I am without their encouragements. I would also like to offer a token of appreciation to my sister, Annie, who still does not have the faintest idea about lipids and fatty acids, for her lawyerly and tireless efforts at editing my works and correspondences, academic or personal, all done in non-billable hours; her sense of humour and the ability to come up with a double meaning for any scientific term have made the thesis writing a much more bearable, albeit longer, process. Had I written the last sentence myself, I could not have said it better. vi CO-AUTHORSHIP STATEMENT The majority of the experiments and analyses for this thesis were completed by myself, S.S.YHuang, and the following co-authors are listed on the manuscripts: AungNang Oo, Dave A. Higgs, Colin J. Brauner; Clarice H L . Fu., Shannon. K. Balfry, and Patricia M. Schulte. A.N Oo assisted with sampling and the fatty acid composition analyses in the red sea bream study. Clarice Fu analyzed the whole body and plasma ion samples for assessment of ionoregulatory development in the spring chinook salmon study as well as commenting on editions of the manuscript. D.A. Higgs and C.J. Brauner assisted in the analysis of the data and commented on editions of the manuscripts for both the red sea bream and the spring chinook salmon studies. S.B. Balfry and P.M. Schulte contributed to the formation of the spring chinook salmon study as well as the editing of the manuscript. vii CHAPTER ONE: Introduction Piscivorous fish have high dietary requirements for lipids and in commercial pellets, marine fish oil is the main source of dietary lipids. However, due to environmental and economical constraints, there is considerable interest in finding alternative lipids (primarily those of terrestrial plant in origin) as sources of lipids in aquafeeds. This thesis will focus on the potential for refined canola oil (the dominant oilseed produced in Canada) in diets of two important Asia-Pacific finfish species - Japanese red sea bream (Pagrus major) and spring chinook salmon (Oncorhynchus tshawytscha). Specifically, the thesis investigates the effect of dietary canola oil on the growth, fatty acid metabolism and ionoregulatory development of the animals during their rapid growth phase. The remainder of the introduction will provide the background information on: 1) the state of world fisheries and aquaculture, 2) lipids and fatty acids, 3) alternative lipids and then leads to the overall objectives of the thesis. The introduction consists of five major sections: 1. The State of World Fisheries and Aquaculture - an overview on the current status of capture fisheries and aquaculture as well as the research initiatives of the thesis. 2. Lipids and Fatty Acids - a comprehensive overview of the properties, metabolism and requirements of fish for dietary lipids and fatty acids. 3. Alternative Lipids - a review on past findings and current research status of the use of alternative lipids in finfish aquaculture. 4. Model Species - a brief description of the life history and commercial significance of the model species used in this thesis as well as the physiology of salmonid smoltification and performance indicators. 5. Research Purpose and Hypothesis - a summary of the objectives and research predictions 1 of the experiments conducted in this thesis. 1.1 The State of World Fisheries and Aquaculture Accompanying the rapid global growth of human populations and the subsequent rising needs for high-quality nutritional sources, the amount of food fish alone has grown to 106 million tons in 2004 (FAO-FD, 2006). Fish provide 2.6 billion people world-wide with at least 20% of their per capita intake of .animal protein. However, reported global capture fisheries have plateaued since the early 1990s, despite the fact that demand for fish products continues to rise, particularly in developing countries such as China (FAO-FD, 2006). Presently, aquaculture output compensates for the short fall in capture fisheries and there has been a one million ton increase in production in 2005 relative to 2004 (FAO-FD, 2006). Aquaculture contributes more than 33.7 percent by weight of global supplies offish, crustaceans and molluscs. This is a significant increase compared to the 27 percent contribution in 2000 (FAO-FD, 2006). Within the sector, finfish represented over half of the total production by weight in 2004 (54%), and the rapid expansion of the major species groups shows no apparent sign of slowing down in the near future. Thus, while the natural fisheries are struggling to meet the rising global consumer demands for food fish, it appears that finfish aquaculture can provide a viable alternative for consumers. However, the rapid growth in aquaculture production is not without limitations. In particular, the cultivation of high-value piscivorous marine finfish may put considerable strain on small pelagic fish stocks worldwide (Naylor et al., 2000), as these populations are exploited to a considerable extent for commercial aquafeed production. In a recent report (FAO-FD, 2006), it was stated that 24% of edible global fish production (excluding China) was reduced to non-food products such as meal and oil. Fish meals and fish oils (FO) derived from small pelagic fish have been used traditionally in aquafeed production for practical, 2 scientific and economic reasons. However, the'present global supply of FO has plateaued and is possibly declining due to over exploitation and natural phenomena such as "El Nino" (Barlow, 2000). At the same time, demand for aquafeed production continues to increase, with several conservative projections of production to increase from 13.6 million tons to 32.6 million tons by 2010, and to 37.5million tons by the year 2015 (IFOMA, 2000). Not only is the utilization of FO environmentally restrictive (due to depleting small pelagic fish stocks), it also imposes a considerable economic burden as prices for FO continue to increase, reflecting a corresponding decrease of supply. Furthermore, as evident by the formulation of the so-called "high energy (fat) diets" (lipid as high as 40% in salmon feeds) that are used to promote rapid salmon growth, there is little doubt that lipid is a significant component in feed ingredients. It is important to note that feed alone can account for 35%-60% of the total production cost of commercially valuable finfish such as salmonids. Moreover, the cost of the dietary oil alone constitutes 25% of the feed expenditures (Forster, 1995; Hardy et al., 2001). Clearly, the acquisition of FO is predicted to become increasingly more difficult and expensive in the near future. Consequently, there is a need to find alternatives that will still allow the aquaculture industry to expand and satisfy the increasing global demand for fish in an economically and environmentally sound manner. While in the short term, non-food grade fisheries by-products (i.e., fishery by-catch and discards from processing plants) can be considered as an alternative ingredient in fish feed, a more long-term effort is needed to scrutinize the possible utilization of the much larger and faster growing terrestrial agricultural production sector, which has the potential to provide alternative sources of non-food proteins and oil for aquafeed production (Hardy et al., 2001; Tacon, 2004; Pike, 2005). However, potential nutritional and physiological effects on the fish due to the differences in fatty acid compositions between marine fish oils and plant oils must be taken into careful 3 ) consideration. 1.2 Lipids and Fatty Acids 1.2.1 Properties of lipids and fatty acids in fish In general, "lipids" refer to the relatively water insoluble organic compounds which can be classified into nonpolar (i.e., triacylglycerols, wax esters, alkyl diacylglycerols, and sterol esters) and polar lipids (i.e., phosphoglycerides). The characteristic of each lipid group depends on the constituent fatty acids that are attached to the glycerol backbone. The fatty acids are carboxylic acids with long-chain hydrocarbon side groups and differ in their degree and position of double bonds and the number of carbon atoms. The nomenclatures of fatty acids are in accordance with their carbon chain lengths, degree of unsaturation (number of ethylenic or double bonds) and the position of their double bonds. Thus, 16:0 designates a fatty acid with 16 carbon atoms with no ethylenic bonds. Whereas 18:2n-6 designates a fatty acid with 18 carbon atoms and two ethylenic bonds, the first of which is located at the 6 , h carbon atom from the methyl end of the molecule. Lipogenesis refers to the biosynthesis of new endogenous lipids. Cytosolic fatty acid synthetase is a multi-enzyme complex in the liver that catalyzes the key pathway of lipogenesis in fish as well as the degradation of pyruvate (from carbohydrate) or amino acids (from protein) which provides the predominant carbon source for lipid biosynthesis (Sargent et al., 1989). Fish that naturally consume a high amount of dietary lipid, such as top piscivorous marine species, are generally less likely to have any significant activity of de novo lipid or fatty acid biosynthesis (Sargent et al., 2002). However, the extent of lipogenesis in freshwater species is quite different, as lipid-rich prey are less common in the environment (Tocher, 2003). The rate of lipogenesis is determined by the absolute amount of dietary lipids and their relative composition. For example, increased consumption of dietary lipid relative to protein 4 ratio depresses lipogenesis in both marine and freshwater species; however, increased intake of lipid relative to carbohydrate stimulates lipogenesis (Shimeno et al., 1995, 1996). Furthermore, elevated levels of dietary omega 3 (n-3) highly unsaturated fatty acids (HUFA) and polyunsaturated fatty acids (PUFA) are also known to depress lipid biosynthesis in fish (Shikata and Shimeno, 1994; Alvarez et al., 2000). Because HUFA and PUFA levels are higher in the diets of marine species, this may be why lipogenesis is generally less prevalent in many marine species than in freshwater species. However, this does not mean that marine fish are not capable of modifying dietary lipid and their constituent fatty acids. Although there are differences between the fatty acid compositions of the northern hemispheric FO (capelin, herrings and menhaden) and the southern hemispheric FO (anchovy, sardine and pilchard), fish lipids are typically high in unsaturated fatty acids. The PUFA can either belong to the oleic (n-9), the linoleic (n-6) or the linolenic (n-3) series and are characterised by the number of double carbon bonds they contain (Sargent et al., 2002). Like other vertebrates, finfish do not possess the A12 and A15 desaturase enzymes necessary for the synthesis of 18:2n-6 (linoleic acid; LA) and 18:3n-3 (linolenic acid; LNA) that are, respectively, the precursors for the n-6 and n-3 family or series of fatty acids (Henderson and Tocher, 1987). Subsequently, those fatty acids must be dietary in origin. The absolute levels and proportions of the parent acids of the n-6 and n-3 series and their members of HUFA are considered to be essential for many physiological processes, under which the specific levels and proportions are species specific (Higgs and Dong, 2000). In general, the constituent members of all series of fatty acids are synthesized through a common enzyme system where desaturases and elongases alter the number of double bonds and the carbon chain length (Figure 1.1; Tocher, 2003). However, the members of each series are not interconvertible and must be derived from distinctive precursors. Within the n-9, n-6 and n-3 series, eicosatrienoic acid (20:3n-9), dihomo-y-linolenic acid (20:3n-6), arachidonic 5 acid (20:4n-6; AA), eicosapentaenoic acid (20:5n-3; EPA), and docosahexaenoic acid (22:6n-3; DHA) are of prominent biological significance. The latter four are precursors of eicosanoids that are essential for the regulation of many physiological processes (Higgs and Dong, 2000; Sargent el al., 2002). EPA and DHA are commonly referred to as n-3 HUFA, a term that can also include other C20 members of the n-3 series. The ability of fish to synthesis these compounds is dependent on the relative activities of the A5 andA6 desaturase enzymes and their access to these fatty acids. Triacylglycerols (TAG) are the major dietary lipid class in both marine and freshwater fish (Tocher, 2003). They are also the main form of lipid storage in fish. TAG are made up of three fatty acids esterified to aL-glycerol backbone in the sn-1, sn-2, and sn-3 positions. The nature of the attached fatty acids and the positions in which they are esterified are highly variable. However, while saturated fatty acids (SFA) and monounsaturated fatty acids (MFA) tend to occupy the sn-1 and sn-3 positions, respectively, PUFA are preferentially esterified in the sn-2 position (Sargent et al., 2002). The polar lipids are another major lipid group found in fish. They typically consist of a glycerol or sphingosine (an amino alcohol) moiety affixed with one or two fatty acids and a polar head group. With the exception of glycolipids, all polar lipids contain a phosphate group and, in general, a nitrogenous base. Thus, they are commonly known as phospholipids. Phospholipids are amphiphilic and along with protein and cholesterol, are the basic components of cellular and subcellular membranes. Palmitic acid (16:0), 18:ln-9, 20:5n-3, and 22:6n-3 are the principal fatty acids of the phospholipids that make up the biological cell membrane bilayers in fish (Tocher, 2003). Furthermore, 22:6n-3 is generally present at twice the level of 20:5n-3 in fish phospholipids (Sargent et al., 2002). However, this ratio can differ between phosphoglyceride classes and the particular tissue. The high abundance of long-chain n-3 HUFA (EPA and DHA) is thought to help establish suitable membrane fluidity 6 in response to changing environmental temperatures, which is particularly important in the aquatic environment (Wodtke and Crossins, 1991). Furthermore, the unique structure of 22:6n-3 facilitates the formation of hexagonal phases of the lipid phospho-bilayer, which in turn, enables rapid conformational changes in membrane proteins and optimization of membrane function (Brown, 1994). Unlike body neutral lipids, polar lipids are less influenced by dietary lipids. Selective retention and catabolism of fatty acids maintain the specific fatty acid profile that is characteristic of this lipid group, and their fatty acid compositions reflect their physiological importance as the major constituents of the biological membrane. 1.2.2 Lipid and fatty acid catabolism in fish Lipids, specifically fatty acids, are the favoured sources of metabolic energy in fish and more so in marine fish, as reflected by the high body lipid content in species such as capelin and herring (Sargent et al., 2002). Consequently, marine fish are especially dependent on dietary lipid due to the scarcity of carbohydrate in their natural diet (Tocher, 2003). Fatty acids are not only the preferred energy source for growth and swimming (Tocher et al., 1985), but are also crucial for reproductive success. The quality and quantity of lipids can have profound effects on the fecundity of the spawners and subsequent egg survival (Henderson et al., 1984; Sargent et al., 1989). In this regard, marine FO is the traditional choice of lipid for inclusion in piscivorous fish feeds because of their rich content of EFA, particularly the highly unsaturated members of the n-3 family (Hardy et al., 2001). Lipids store and provide metabolic energy in the form of ATP generated by the catabolism of fatty acids (Sargent et al., 1989; Froyland et al., 2000). The catabolism of fatty acids, also termed P-oxidation, occurs in cellular mitochondria and is carried out by distinctive sets of enzymes rather than a multi-enzyme complex as in the case of biosynthesis. In coldwater species, monounsaturated and polyunsaturated fatty acids, rather than saturated 7 fatty acids, are preferentially oxidized for energy. This is because the combination of the low-melting point and molecular structure of these fatty acids increases their oxidation rate at low temperatures (Sidell et al., 1995; Henderson and Sargent, 1985; Bendiksen et al., 2003). Thus, the fatty acids used to derive metabolic energy in commercial salmon feeds include mostly 18:ln-9; 20:1 n-9 and 22:In-11 (Sargent et al., 1989). The fatty acid 22: In-11, in particular, has been established to play a single role in fish, as a source of metabolic energy (Tocher, 2003). Fatty acid oxidation also occurs in other tissues in fish besides the liver. Tissues such as the heart, red and white muscles can contribute a significant amount to the overall fatty acid oxidation in Atlantic salmon (Froyland et al., 1998, 2000). P-oxidation of polyunsaturated fatty acids is more complex and can vary between different classes of PUFA molecules. The rate of P-oxidation can also be influenced by environmental signals and the various developmental stages of the fish, such that the rate of oxidation of 18:3n-3 and 18:2n-6 is reduced during the freshwater phase and increased during smoltification in salmonids (Fonseca-Madrigal et al., 2006). However, PUFA are usually not the preferred substrates for catabolism, because many are selectively retained in tissues as metabolic precursors for other C20 and C22 HUFA. Although 20:5n-3 can be oxidized for energy if necessary, 22:6n-3, by contrast, is a relatively poor substrate for catabolism, and is thus, fairly resistant to metabolic degradation. This is because catabolism of 22:6n-3 requires a special mechanism due to the insertion of the A4 ethylenic bond (Madsen et al., 1999). 1.2.3 Lipid and fatty acid requirements in fish High energy diets are commonly used in finfish aquaculture to promote fish growth. By providing an abundance of dietary lipid for metabolic energy, the "protein sparing effect" allows the accumulation of protein for growth and thus shortens market time for growers (Higgs and Dong, 2000). A strong positive correlation exists between dietary lipid levels and levels of lipid in the fish body of many freshwater, marine and salmonid species (Takeuchi et 8 al., 1991; Bell et al., 1998; Caballero et al., 2002). However,, unwanted deposition of excess lipids in flesh and organs has health consequences to the fish and is less desired by the consumers. 'Tree oil," caused by increasing flesh lipid in Atlantic salmon, reduces visualization of pigments, interferes with processing and smoking, and increases the chances of lipid oxidation (Bell et al., 1998). Furthermore, high dietary lipid depresses de novo fatty acid synthesis by inhibiting enzymes involved in hepatic lipogenesis and hepatic activities in general, and can thus affect liver histology and may lead to development of pathology (Sargent et al., 1989). Also, dietary lipid concentrations can influence optimal levels of protein and other nutrients (Takeuchi et al., 1991, 1992b). Thus, the absolute level of dietary lipid is an important parameter for finfish feed due to potential negative impact on fish health, performance and flesh quality. In general, diets having a lipid content of ^ 10% can be considered high energy diets, however, the optimal dietary lipid level is often species specific (Sargent et al., 2002). Salmonids, especially post-smolt salmonids, are typically fed high lipid diets compared to freshwater species. This is necessary because of their dietary history, where high lipid content prey would normally be consumed (Sargent et al., 2002). As previously noted, the amount of dietary lipid in feed can affect the efficacy of other dietary components. The quantitative requirements of essential fatty acids (EFA) have been shown to vary with the amount of total lipid in feed (Izquierdo, 1996). Furthermore, increasing the dietary lipid levels apparently increases the requirement for n-3 HUFA in both red sea bream (Pagrus major) and yellowtail fingerlings (Seriola quinqueradiata) (Takeuchi et al., 1992a, c). Nonetheless, the optimal dietary lipid level can vary between different developmental stages in fish. The term, EFA generally refers to L A and LNA and their metabolic derivatives, collectively referred to as the physiologically important AA, EPA, and DHA. The physiological functions of EFA broadly fall into two roles in fish: the maintenance of the 9 structural and functional integrity of the biological membrane and as precursors of the biologically active paracrine hormones known as eicosanoids (Sargent et al., 1999). As previously noted, fish are incapable of synthesizing LA and LNA, and thus, they must be dietary in origin. However, major differences exit between freshwater and stenohaline marine fish. Freshwater fish, including salmon parr, typically have a requirement for 18:3n-3 and 18:2n-6, as n-3 HUFA are not readily acquired through diets. Consequently, the dietary requirements of AA, EPA and DHA are also lower compared to that of the marine species. Thus, salmon parr, in general, require about 1% in diet of each of LA and LNA or 10% of dietary lipid as n-3 HUFA(Castell et al., 1972; Takeuchi and Watanabe, 1977b, 1982). An equivalent blend of LA and LNA at ~1 -2% diet fulfills the EFA requirement in other freshwater species such as ayu (Plecoglossus altivelis), common carp (Cyprinus carpio), channel catfish (Jctalurus punctatus), Japanese eel (Anguilla japonica), Nile tilapia (Tilapia zillii)and rainbow trout (Oncorhynchus mykiss) (Yu and Sinnhuber, 1979; Takeuchi and Watanabe, 1977a; Takeuchi et al., 1980; Kanazawa et al., 1980,1981; Satoh et al., 1989). Thus, these freshwater species are capable of further desaturating and chain elongating 18:2n-6 and 18:3n-3, which then must be adequately provided in the diets. Unlike freshwater fish, the EFA requirements of marine species, in general, are met specifically by EPA and DHA. It is thought that the abundance of these fatty acids in their natural diets render the fish incapable of further modifications of LNA (Sargent et al., 2002). The Japanese red sea bream has a 1% dietary requirement for a combination of EPA and DHA(Yone et al., 1971); however, this requirement can fluctuate with levels of dietary lipid. Takeuchi et al. (1990; 1992a) stipulated a need for 1% dietary EPA and 0.5% DHA at a level of 10%> total lipid and 3.7% of EPA and DHA combined at 20% dietary lipid for this species. Other common cultured marine species such as the giant sea perch (Lates calcarifer), striped jack (Caranx vinctus), turbot (Psetta maxima), and yellowtail have a 1-2% dietary 10 requirement for EPA and DHA (Gatesoupe et al., 1977; Deshimaru and Kuroki, 1983; Buranapanidgit et al., 1989; Watanabe et al., 1989a). Nonetheless, DHA alone can satisfy the EFA requirement in some species, demonstrating its higher efficacy as an EFA in marine species (Watanabe et al., 1989b). In conclusion, lipids are an invaluable dietary component for finfish because they furnish the indispensable EFA that are crucial to all aspects of general physiology. Marine and freshwater fish species have distinct lipid and fatty acid requirements due to the differences in their metabolism of these compounds. Thus, this must be taken into great consideration when formulating their diets. 1.3 Alternative Lipids 1.3.1 Effects on fish The fatty acid composition of lipids of terrestrial plants arid/or animals is considerably different from marine FO. Plant oils are typically abundant in the unsaturated and highly digestible C18 and n-6 fatty acids, but lack the longer n-3 HUFA characteristic of marine FO, and thus, have a higher n-6/n-3 ratio (Opsahl-Ferstad et al., 2003). On the other hand, animal lipids like beef tallow have increased saturated fatty acid content whereas, other like poultry fat maybe have reduced SFA content but elevated 18:ln-9 (Mugrditchian et al., 1981; Dosanjh et al., 1984; 1988; Higgs et al., 2006). Therefore, the shift in fatty acid compositions associated with incorporating these alternative lipids into finfish diets may have complications, since dietary alteration of body fatty acid compositions may indirectly alter supplies of metabolic energy for growth and compositions of the cellular membranes that are crucial to physiological processes (McKenzie, 2001; Welker and Congleton, 2003). It has been demonstrated that the relative proportions of SFA and the ratio between n-3 and n-6 11 PUFA in diets can affect swim performance and oxygen consumption in Atlantic salmon (McKenzie et al., 1998; Wagner et al., 2004). Activities of membrane-bound proteins, including key enzymes in ionoregulation such as Na'/K.'- ATPase, may also be affected (Bell et al., 1997; Tocher et al., 2000; Stubhaug et al., 2006). Furthermore, EFA deficiency in fish has been found to lead to a reduction in ion permeability and morphological changes in the gills (Bell et al., 1985). Given such findings, incorporation of terrestrial alternative lipids into the diets of piscivorous finfish driven largely by environmental and economical concerns, must be approached with some caution. Extensive studies on alternative lipids have been conducted on commercially valuable species of salmonids, such as Atlantic salmon (Salmo salar, Dosanjh et al., 1998; Rosenlund et al., 2001; Bell et al., 2001, 2002, 2003 ), fall chinook salmon (Oncorhynchus tshawytscha, Dosanjh et al., 1988; Grant, 2006), rainbow trout (O. mykiss; Caballero et al., 2002), coho salmon (O. kisutch, Dosanjh et al., 1984), and brook charr (Salvelinus fontinalis, Guillou et al., 1995). The majority of these studies have focused primarily on post-smolts or adults held in seawater. Some freshwater species have also been examined (Carassius auratus, Pozernick and Wiegand, 1997; Sander lucioperca, Schulz et al., 2005; Maccullochellapeeliipeelii, Turchini, et al., 2006). In contrast, the use of alternative lipids has only been investigated in a few species of stenohaline fish such as the gilthead sea bream (Spams aurata; Montero etal, 2003; Izquierdo et al;, 2005), European sea bass (Dicentrarchus labrax; Izquierdo et al., 2003; Mourente et al., 2005), Japanese sea bass (Lateolabrax japonicus;Xue et al., 2006) and turbot (Psetta maxima; Regost et al., 2003). To dale, Atlantic salmon is, by far, the most extensive species examined, as it is widely regarded as the single most cultured finfish species in America and Europe. While the effects of dietary changes on fish growth are still of great interest, many of these recent studies have also incorporated other physiological measurements, including 12 hepatic lipid/fatty acid metabolism/oxidation, eicosanoid production, gene expression, immunocompetence as well as traits defining product quality such as flesh taste, composition and pigment incorporation (Bransden et al., 2003; Regost et al., 2003; Izquierdo et al., 2005; Jordal et al., 2005; Stubhaug et al., 2006). As evident in these studies, in comparison to marine species, freshwater species, including salmonid parr, lend to exhibit a higher tolerance with respect to the extent to which plant oil can replace dietary FO. Moreover, studies using low erucic acid rapeseed oil or canola oil have yielded very promising results in salmonids. Similar results were achieved when these plant oils were used either alone or blended with other plant oils (flaxseed, linseed, and soybean oil), or with animal lipid (pork lard or poultry fat) (Caballero et al, 2002; Dosanjh et al., 1984, 1988, 1998; Grant, 2006; Higgs et al, 2006; Bendiksen and Jobling, 2003; Rosenlund et al., 2001). These studies conclusively demonstrate that the growth and development of these fish remained uncompromised even when plant oils replaced up tol00% of the supplemental marine fish oil and ^50% of the total lipid in the feed. However, other aspects of the physiology of these fish fed these diets have not been investigated in these studies. Canola oil (CO) has a fatty acid profile that resembles more closely the natural prey of freshwater fish than FO. For this reason, CO appears to be a more appropriate source of lipid in feeds designated for salmon parr reared in fresh water (Bell et al., 1997; Tocher et al., 2000). In fact, Bendiksen et al. (2003) have demonstrated that the inclusion of rapeseed and linseed oil in diets improves parr-smolt transformation and promotes seawater growth in Atlantic salmon. Other studies have also produced similar findings (Tocher et al., 2000; Bendiksen and Jobling, 2003). A recent long-term study (5 months) indicates excellent potential for the use of refined CO in the diets of fall chinook salmon juveniles. Growth and ionoregulatory development of these fish were found to be unaffected, despite the replacement of dietary CO up to 41% of the total dietary lipid in some feeds (Grant, 2006). . 1 3 Similar results have also been demonstrated in gilthead sea bream (Spams aurata; Montero et al., 2003; Menoyo et al., 2004; Izquierdo et al., 2003, 2005), Australian pink snapper (Pagrus auratus: Glencross et al., 2003a, b), European sea bass (Izquierdo et al., 2003; Mourente et al., 2005) and turbot (Regost et al., 2003), all of which are stenohaline species. Short and long-term studies have already confirmed that rapeseed oil can replace up to 60% of the supplemental FO in diets for gilthead sea bream and European sea bass without adversely affecting fish growth, feed efficiency and survival (Izquierdo et al., 2003, 2005; Mourente et al., 2005). As such, alternative lipids, particularly canola oil, appear to be suitable replacements of marine fish oil in finfish aquafeed. 1.3.2 Canola oil Canola is the trademark name given to rapeseed cultivars of genetically selected variant, that contain <2% erucic acid in the oil and glucosinolates or antithyroid compounds in the meal (Canola Council of Canada, 2005). Canola is Canada's major oilseed as well as one of the most important oilseed crops in the world. Canola oil has important economic significance and potential in the Canadian industry and the industry is currently projected to continue to grow due to a steady demand for plant oil for both food consumption and fuel. Furthermore, seed yield and oil extraction efficiency continue to improve with better hybridization and processing techniques (Canola Council of Canada, 2005). A complex process of oil extraction and processing produces a variety of canola oil products for human consumption as well as for animal feeds. To prepare for oil extraction, canola seeds are cleaned, pre-heated, flaked, and then cooked to intensify oil release. The heat-conditioned seeds are then subsequently screw-pressed and are subjected to solvent extraction using hexane. The extracted oils are then desolventized using a multiple stage . solvent evaporation. Crude oils are degummed to remove phosphatides, leaving the oil product glistening and translucent. Finally, the oils are further processed and refined 14 according to specific product needs. In terms of its nutritional properties, canola oil is characterized by having a very low-level of SFA, a high level of MFAand intermediate amount of PUFA. Specifically, it contains about 7% SFA, 62% MFA and 31 % PUFA. Oleic acid is the most prominent MFA and the ratio of linoleic and linolenic acid is about 2:1 (Canola Council of Canada, 2005). In conclusion, whole body fatty acid composition can be influenced by dietary lipid ' composition which has growth and physiological consequences on the animal. Thus, it is crucial to have comprehensive assessments of the potential effects of changes in dietary lipids (inclusion of plant oil) on the animal. Previous studies have demonstrated excellent potential of some plant oils in diets of certain freshwater, salmonid and marine species. However, specific studies focusing on canola oil are necessary to assess the merits of its application for commercial aquafeed production. 1.4 Model Species 1.4.1 Red sea bream (Pagrus major) Pagrus major, commonly known as red sea bream, is one of the most important species for commercial coastal fisheries in Japan. The fish, known as ~? ^4 (madai) in Japanese, is usually served as high-priced sashimi or presented in traditional ceremonies such as weddings, where it is seasoned with salt and grilled (Watanabe and Vassallo-Agius, 2003). In recent decades, consumer demand for fish in Japan has progressively shifted towards the more expensive marine fish. The traditional cultural significance attached to red sea bream has increased the demand for the fish in the domestic market has increased steadily over time and the current production level is only second to that of yellowtail (Watanabe and Vassallo-Agius, 2003). Red sea bream were initially cultivation for stock enhancement in response to a decline in the fishery in shallow coastal regions of Japan, as annual catch dropped from 136 metric tons in the 1960s to 20 metric tons in the late 1970s. The decrease in wild fisheries of this species was in part caused by rapid urbanization and industrialization of the coastal regions as such fast-paced economic development reduced suitable nursery areas for red sea bream larvae and juveniles which tend, unlike adults, to stay near shallow waters (Imai, 2005). Thanks to a comprehensive knowledge of the biology and behaviour of red sea bream in the wild, as well as a thorough understanding of the nutritional requirements for the species' various life history stages, red sea bream culture has been fairly successful (Foscarini, 1988). Furthermore, its relatively fast growth (2 years to market size), tolerance to a wide range of temperatures (10-22°C) and ability to naturally spawn in captivity has made this species particularly attractive for Japanese fish farmers. 1.4.2 Spring chinook salmon (Oncorhynchus tshawytscha) Chinook salmon are anadromous and semelparous fish indigenous to the North American Pacific coast (Healey, 1991) and are the largest of the 7 species of Pacific salmon (Netboy, 1958). Spring chinook salmon are a population of chinook salmon, known as King salmon. Spring chinook salmon are the largest of the chinook salmon and are integral to salmon fisheries and sport fishing (Healey, 1991). Spring chinook salmon emigrate to sea during their second or, in more rare instances, their third spring post-hatching and thus are also termed stream-type. They are unable to smolt as subyearlings under natural conditions (Myer et al., 1998). A combination of environmental and genetic factors has been observed to influence the divergence of the runs and time of smoltification in chinook salmon (Randall et al., 1987). Stream-type chinook salmon exhibit a slower growth rate relative to other runs; such slow growth has been found to be heritable but influenced by,photoperiod (Clarke et al., 1992). 16 Spring chinook salmon parr are opportunistic feeders. While residing in streams, they feed extensively on drift insects, but zooplankton become their main food source when they move into main river systems and estuaries (Allen and Hassler, 1986; Healey, 1991). Thus, their dietary lipid composition varies with changes in habitat throughout their freshwater residency. The aquatic insects and zooplankton they feast on typically contain higher levels of the C18 n-6 and n-3 series of fatty acids relative to n-3 HUFA. Thus, spring chinook parr naturally consume a large quantity of LNA, LA and other C18 PUFA. Compared to other chinook populations, juvenile spring chinook salmon are more aggressive, and display morphometric differences such as larger and more colourful fins which may relate to social display and territoriality (Myers et al., 1998). These traits which are absent in other populations are likely to acuminate with their extended freshwater residency and the limited resources in these freshwater systems (Carl and Healey, 1984; Taylor and Larkin, 1986). Prior to emigration, juveniles exhibit downstream dispersal near the time of migration and they utilize various habitats. Shifts in habitat occur during overwintering as spring chinook salmon juveniles hide under large rocks and debris to avoid low water temperature (Allen and Hassler, 1986). At the time of seawater entry (April-June), stream-type smolts are much larger than other runs (-73-134 mm) and they tend to have a high tolerance to elevated serum chloride and are able to rapidly acclimate to high salinities (Allen and Hassler, 1986). They are known to enter estuaries without first developing the morphological characteristics ofasmolt (Hoar, 1976). 1.4.3 Salmonid smoltification and the 24-h seawater challenge test Anadromous salmonids undergo profound changes in morphology, behaviour and physiology in preparation for their seaward migration. The changes are known collectively as parr-smolt transformation or smoltification (Folmar and Dickhoff, 1980). In general, 17 seawater (SW) entry in salmonids appears to be a period of high mortality due to the dramatic physiological changes that accompany this change in environment (Parker, 1962; Healy, 1982). Smoltification processes involve the reorganization of biochemical components within the tissues, which prepare the fish for the hyper-osmotic marine environment while still residing in freshwater (Hoar, 1976). Studies on. salmon of the genus Oncorhynchus have shown that a pre-adaptive change in tissue fatty acid composition occurs during smoltification, during which there is a switch between the typical freshwater fatty acid pattern (low in n-3 HUFA) to a typical marine pattern (high in n-3 HUFA) (Sheridan et al., 1985; Li and Yamada, 1992). This change in tissue fatty acid composition is also known to affect the activity of a number of ion pumps and membrane-bound enzymes such as Na +, K+-ATPase (Spector and Yorek, 1985; Bell et al., 1997). The 24-h SW challenge test is used to evaluate the hypo-osmoregulatory status of smolting salmonids prior to their release or transfer into seawater. The seawater adaptability of juvenile salmonids is assessed by comparing plasma ion levels (Cl" and/or Na+) offish exposed to SW for 24-h relative to freshwater control values (Blackburn and Clarke, 1987). A large increase in plasma ions following the 24-h SW challenge has been found to be associated with high mortality or subsequent stunted growth in SW of survivors (Clarke, 1982). Changes in osmolarity and body weight (water content) are also measurements of seawater adaptability (Blackburn and Clarke, 1987). Thus, successful smolts are able to maintain physiological plasma ion concentrations and body water content following SW exposure. In conclusion, the red sea bream is a relatively well studied species in which its basic husbandry and nutritional requirements are well-established. However, the use of alternative 18 lipid has not been addressed in. this species. Similarly, the same topic has not yet been addressed in the spring chinook salmon, whose nutritional requirement is not as well understood compared to other members of the genus Oncorhynchus and even to other chinook salmon populations. 1.5 Research Purpose and Hypothesis This thesis was undertaken to investigate the merits of using refined canola oil as an alternative supplemental lipid source in commercial practical finfish diets. The studies were conducted to determine the growth and physiological effects of replacing all of the supplemental marine fish oil in the diets of two commercially significant Asia-Pacific species - the stenohaline red sea bream (Pagrus major) and the anadromous spring chinook salmon (Oncorhynchus tshawylscha). The experiments conducted from the fry to the juvenile stage allowed for a comprehensive and chronic study on the effects of feeding non-traditional dietary lipid during the early development of the preceding fish species as well as a comparative study of the responses of a marine and freshwater species to the inclusions of dietary canola oil. Four practical diets containing graded amounts of refined canola oil up to a maximum of 70% and 72% of the total dietary lipid were used to rear triplicate groups of red sea bream and spring chinook salmon fry respectively over periods of 12 and 30 weeks, in a randomized block design for assessment of diet treatment in each case. Growth parameters, survival, and terminal whole body proximate and fatty acid compositions were examined in both trials. Terminal liver polar fatty acid compositions were also examined in the red sea bream study, while effects on ionoregulatory development, as determined by periodical 24-h SW challenge tests, were studied in the spring chinook salmon trial. Based on previous short and long-term studies on gilthead sea bream (Spams aurata) 19 fed diets with rapeseed oil (Izquierdo et al., 2003, 2005; Montero et al., 2003, 2004), we postulated that the red sea bream in^our study would have a lower tolerance to increasing dietary canola oil due to their limited ability to modify the metabolic precursors into essential fatty acids. By contrast, previous studies on fall chinook salmon have demonstrated that they exhibit a high tolerance to reduction of dietary marine fish oil (Mugrditchian et al., 1981; Dosanjh et al., 1988; Grant, 2006). Hence, we postulated that spring chinook salmon parr in our study (which possess a prolonged freshwater residency period) would display an even higher tolerance to dietary plant oil inclusion than fall chinook salmon. Also, we postulated that this life history type of chinook would exhibit increased physiological capacities by selectively retaining and utilizing specific fatty acids as well as increasing their capability to modify precursor fatty acids to supplement their essential fatty acid needs. 20 Figures Figure 1.1 Pathway of biosynthesis of C20 and C22 HUFA from n-3, n-6 and n-9 families of C18 PUFA precursors. Fatty acyl desaturases (A5, A6, A6*, A9, A12, A15) and elongases (elong) work in alternation to increase carbon chain length and double bonds of fatty acids. A9 desaturase is found in all animals, whereas A12 and A15 desaturases are only found in plants. The A6* desaturase acting on C24 fatty acids may or may not be the same desaturase (A6) that acts on C18 fatty acids. 18:0 A9 A6 elong AS 22:5n-6 4 A12 A15 A6 elong AS elong elong A6* 18.3n-3 —18:4n-3 — • 20:4n-3 — • 20:5n-3—• 22:5n-3—•24:5n-3—• 24:fin-3 short 22,-tin-I Tocher, 2003 •i 21 References Allen, M.A., Hassler, T.J., 1986. Species profiles: life histories and environmental requirements of coastal fishes and invertebrates (Pacific Southwest) - chinook salmon. U.S. Fish Wild. Serv. Biol. Rep. 82(11.49). U.S. Army Corps of Engineers, TR EL-82-4,26 p. Alvarez, M.J., Diez, A., Lopez-Bote, C , Gallego, M., Bautistia, J M , 2000. Short-term modulation of lipogenesis by macronutrients in rainbow trout (Oncorhynchus mykiss) hepatocytes. Br. J. Nutr. 84, 619-628. Barlow, S., 2000. Fishmeal and fish oil: sustainable feed ingredients for aquafeeds. Glob. Aquacult. Advocate 4, 85-88. Bell, J.G, Tocher, D.R, Farndale, B M , Cox, D.I, McKinney, R W , Sargent, J.R, 1997. 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Effect of dietary ©3 and co6 fatty acids on growth and feed conversion efficiency of coho salmon (Oncorhynchus kisutch). Aquaculture 16, 31-38. 27 Xue, M , Lin, K , We, X , Ren, Z , Gao, R, Yu. Y , Pearl, G, 2006. Effects of six alternative lipid sources on growth and tissue fatty acid composition in Japanese sea bass (Lateolabrax japonicus). Aquaculture 260, 206-214. 28 CHAPTER TWO: Effect of dietary canola oil level on the growth performance and fatty acid composition of juvenile red sea bream, Pagrus major1 2.1 Introduction Fish oil (FO) is an invaluable dietary component for fish because it furnishes the essential fatty acids (EFA) such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) which are needed for optimal growth and development. The constituent fatty acids in FO also influence biological membrane structure and function. Moreover, FO is an important source of lipid soluble vitamins and highly digestible energy (Higgs and Dong, 2000). Indeed, marine fish ingest little carbohydrate as part of their natural diet. Consequently, they are incapable of metabolically utilizing high dietary levels of digestible carbohydrate. Thus, lipids are the favoured form of non-protein metabolic energy (Sargent et a l , 2002). Fish species, especially those of marine origin, provide excellent dietary sources of high quality protein, vitamins and minerals. Fish, in particular, contain a relatively high proportion of n-3 highly unsaturated fatty acids (HUFA), which have a wide range of health benefits in humans such as prevention of cardiovascular disease, Alzheimer's disease and abnormal neurological and ocular development (Mozzaffarian and Rimm, 2006; Schaefer et a l , 2006; Ikonomou et a l , 2007). Consequently, global consumer demands for food fish have grown tremendously and aquaculture is playing an increasingly important role to maintain the percent per capita consumption of fish (FAO-FD, 2004; Tacon, 2004). At the same time, global demand for FO for aquafeeds has grown in direct proportion to the increase in aquaculture output. This is particularly true for carnivorous species such as salmon which require high energy (lipid) content diets (lipid <40% in grower diets) for optimal growth, 1 A version of this chapter has been accepted for publication and the reference is as follows: Huang, S.S.Y., Oo, A.N., Higgs, D.A., Brauner, C.J., Satoh, S., 2007. Effect of dietary canola oil level on the growth performance and fatty acid composition of juvenile red sea bream, Pagrus major. Aquaculture, in press. 29 feed efficiency and minimization of seawater culture time (Sleffens, 1993; Hardy et al., 2001). However, the global supply of FO is limited and the prices of FO have been forecasted to rise (Hardy et al., 2001; New and Wijkstrom, 2002). Plant oil prices today, are generally less than those of FO and for reasons related to sustainability, it is not possible to further increase the annual global harvest of pelagic fish stocks and consequently the supply of FO (Barlow, 2000). Plant oils are rich in C18 fatty acids but unlike FO, the n-3 HUFA (EPA and DHA) are absent. Nevertheless, plant oils are rich in unsaturated fatty acids and therefore are highly digestible (Opsahl-Ferstad et al., 2003). Fish, like other vertebrates, lack the enzymes necessary to synthesize the parent acids of the n-3 and n-6 families of fatty acids namely, linolenic acid (18:3n-3; LNA) and linoleic acid (18:2n-6; LA), respectively (Bell et al., 1986; Tocher et al., 1989; Mourente and Tocher, 1993a, b). These parent acids are abundant in plant oils. However, marine fish species have dietary requirements for both EPA and DHA since they have limited capacity to synthesize these compounds from LNA (Ghioni et al., 1999; Tocher and Ghioni, 1999). Some marine species appear to have a dietary requirement for AA and there may be a need for an optimal balance between EPA, DHA and A A for good growth and normal development (Higgs and Dong, 2000). In Atlantic salmon (Salmo salar), alterations between the dietary proportions of saturated fatty acids (SFA), n-3 and n-6 polyunsaturated fatty acids (PUFA) can affect aerobic swimming performance and gill function (McKenzie et al., 1998; Wagner et al., 2004). A study on the European sea bass (Dicentrarchus labrax), a marine species, has also shown that dietary inclusion of plant oil (canola oil or palm oil) can positively influence cardio-respiratory and swimming performance (Chatelier et al., 2006). Recent studies aimed at assessing the merits of using plant oils as partial replacements for FO in diets for marine fish have demonstrated promising results with respect to gilthead 30 sea bream (Spams aurata; Montero et al., 2003; Izquierdo et al., 2003, 2005; Menoyo et al., 2004), European sea bass (Izquierdo et al., 2003; Mourente et al., 2005) and turbot (Psetta maxima, Regost et al., 2003). Indeed, short and long-term studies have shown that rapeseed oil can replace up to 60% of the supplemental FO in diets for gilthead sea bream and European sea bass without adverse effects on fish growth, feed efficiency and survival (Izquierdo et al., 2003, 2005; Mourente et al., 2005). More recently, Glencross et al. (2003a) demonstrated that refined canola oil could comprise 40% of the total dietary lipid of juvenile Australian pink snapper (Pagrus auralus) without compromising their growth over a 54-day period. Also, in a second study, Glencross et al. (2003b) found that canola oil could comprise up to 68%) of the dietary lipid for this species without any adverse effects on fish growth over a 32-day period. Nonetheless, plant oils referred to as canola oil (CO), apart from the aforementioned studies of Glencross et al. (2003a, b) and Chatelier et al. (2006) on European sea bass, have received little attention in marine fish diets. Canola is the trademark name given to rapeseed cultivars that have been selected genetically to contain low erucic acid content in the oil (<2%) and glucosinolates or antithyroid compounds in the meal (now <30 umoles/g of air dry oil-free meal; Canola Council of Canada, 2004). Like rapeseed oil, CO has an excellent balance between 18:ln-9 (oleic acid), 18:2n-6 and 18:3n-3. Canola is the dominant oilseed crop of Canada, and the oil, along with low erucic acid rapeseed oil, has been shown to be an excellent source of supplemental dietary lipid for finfish, provided that their respective EFA needs have been met (Dosanjh et al., 1984, 1988, 1998; Bell et al., 2001, 2003; Rosenlund et al., 2001; Grant, 2006; Higgs e ta l , 2006). The red sea bream (Pagrus major), a strictly carnivorous marine fish, is a major finfish species cultured in Japan. In fact, the demand for red sea bream has grown tremendously within the last decade primarily because it is a high-quality sashimi grade fish with high 31 market value (Watanabe and Vassallo-Agius, 2003). While extensive research has been done to define the basic nutritional and husbandry requirements for this species, the merits of including alternative protein and lipid sources to fish meal and FO have only been explored recently (Watanabe, 2002). Since Japan is the largest importer of Canadian canola seed (Canola Council of Canada, 2005), there is a strong incentive to consider this source of CO as a potentially suitable alternative lipid source for this species, especially given the success of using low erucic acid type rapeseed oil in the diets of other marine species and of canola oil on P. auratus, as previously mentioned. Based on taxonomical analyses, Paulin (1990) proposed that P. major and P. auratus are independent and reproductively isolated populations of the same species found in Japan and Australiasia. However, P. major has been traditionally treated as an independent species in Japan despite Paulin's recommendations (Nakabo, 1993). Tabata and Taniguchi (2000) partly support Paulin's finding in that P. major and P. auratus may not differ enough to be classified as different species. However, significant differences at the mitochondrial DNA control region and morphology of the head bump suggest that the relationship of the two populations is at the subspecies level (Tabata and Taniguchi, 2000). Furthermore, the same researchers did not find any data that showed interbreeding between the populations. Therefore, the present study was undertaken to evaluate the efficacy of using refined CO as a partial or total substitute for the supplemental FO in a practical diet for juvenile Japanese red sea bream fingerlings that were in a rapid growth phase over a 12-week (84-day) period. The assessment criteria included not only the possible dietary CO concentration effects on fish growth performance and survival, but also on whole body proximate and lipid composition as well as liver polar lipid composition. 32 2.2 Materials and methods 2.2.1 Experimental diets Four diets of equivalent crude protein (-46%), energy (-21.9 MJ/kg) and lipid (-15%) concentration on a dry weight basis were formulated. The diets had identical ingredient compositions, except CO replaced either 0%, 33%, 67%, or 100% of the supplemental dietary lipid content (100 g/kg diet) with the remainder originating from FO (pollock liver oil). Thus, CO comprised either 0% (diet FO), 25% (C025), 48% (C048), or 70% (CO70) of total dietary lipid content (Table 2.1). The diets were cold-pelleted with a laboratory pellet mill (AEZ12M, Hiraga-Seikakusho, Kobe, Japan). Thereafter, the pellets (2.5mm) were dried using a vacuum freeze-drier (RLE-206, Kyowa Vacuum Engineering, Tokyo, Japan), and stored at 4°C until use. 2.2.2 Fish maintenance and experimental design Red sea bream (Pagrus major) fingerlings were obtained from Seiho Suisan Co. Ltd. (Mie, Japan) and were fed commercial larval feed (Amblose, Nippon Feed Mfg. Co. Ltd, Tokyo, Japan) for 3 weeks while they were acclimated to the culture conditions at the Laboratory of Fish Nutrition located at the Tokyo University of Marine Science and Technology, Tokyo, Japan. Following this, 25 fish (average weight 3.61±0."12g (1 SD)) were distributed randomly into 60-L glass tanks that were each supplied with 700-800 ml/min of aerated, re-circulated, 25±1.2°C artificial seawater (Sea Life®, Tokyo, Japan; salinity, 30g/L). Moreover, a 12-h light/12-h dark photoperiod regime was in effect during the study. Half of the water was renewed weekly to maintain the acceptable water quality limits for the preceding parameters and water quality was monitored daily. Triplicate groups of fish were each fed one of the four aforementioned diets by hand to apparent satiation three times daily (0900h, 1200h and 1600h) for 84 days and the diet treatments were assigned using a 33 randomized block design. 2.2.3 Fish weighing and sampling All fish in each group were anaesthetized (2-phenoxyethanol at 0.5 ml/L) and then weighed individually, after removal of excess surface moisture, to the nearest 0.0 lg at 21-day intervals during the study. On day 0, 15 fish from a common pool of fish were sampled randomly and stored at -30°C for subsequent determinations of their initial proximate and lipid compositions (the analyses were conducted on 3 composite samples of 5 fish each; n =3). On day 84, 3 fish were sampled randomly from each replicate group (tank) per diet treatment for subsequent determinations of whole body proximate and lipid compositions and 5 fish were sampled randomly from each group for assessment of liver polar lipid composition. Whole body samples were ground to a homogeneous consistency using a centrifugal mill (Retsch Z M 100, Haan, Germany) fitted with a 0.25mm screen. The homogenate from each replicate tank was pooled (n = 3/diet treatment) and stored at -30°C under nitrogen pending analysis. Liver samples were ground by hand and analyzed immediately following terminal sampling. 2.2.4 Chemical analyses 2.2.4.1 Moisture, ash, protein and gross energy determinations Determinations of moisture, ash, protein, and gross energy concentrations in the diets and fish samples were conducted as described below. Percent moisture was measured by oven drying each sample at 110°C for 4-h and then weighing each sample at hourly intervals until constant weight was obtained. Ash content was determined by ashing each dried sample in a porcelain crucible using a muffle furnace at 600°C overnight (Woyewoda et al., 1986). Crude protein concentration was determined by the Kjeldahl procedure using a Kjeltec Auto Sampler System 1030 Analyzer (Foss Ltd., Tokyo, Japan). Percent nitrogen was multiplied 34 by 6.25 to obtain an estimate of percent protein. Dietary gross energy concentrations were determined by bomb calorimetry ( IKA Calorimeter System C5001 duo control, IKA® Werke GmbH & Co. K G , Staufen, Germany) whereas whole body energy concentrations were estimated by ascribing 23.64 kJ/g and 39.54 kJ/g for protein and lipid, respectively. 2.2.4.2 Lipid extraction and fatty acid analysis Total lipids were extracted from homogenized (Nissei 250 Ace Homogenizer, Nihonseiki Kaisha L t d , Tokyo, Japan) whole bodies and livers using chloroform/methanol (2:1, v/v) according to the methods of Folch et al. (1957) with minor revisions. Total lipids extracted from the livers were separated into neutral and polar lipid fractions via silica gel cartridges (Sep-Pack, Waters C o , Millford, U.S.A.), as described by Juaneda and Rocquelin (1985). The total lipids from the diets and whole bodies and the polar lipids from the livers were trans-methylated (Christie, 1973, modified), and then the fatty acid methyl esters (FAME) were separated and quantified by a gas chromatograph (GC14B; Shimadzu C o , Tokyo, Japan) equipped with a Supercowax-10 fused silica wall coated 30m x 0.32mm x 0.22pm capillary column (Supleco Inc., Pennsylvania, U.S.A.) and a hydrogen flame ionization detector. F A M E s were eluted from the column using helium as the carrier gas. The initial and final temperatures of the column were 170°C and 250°C, respectively. The gradient increment was set at 2°C per minute. The injector and detector temperatures were set at 250°C. The individual fatty acids were identified with known standards. Subsequently, individual F A M E concentrations were expressed as a percentage of the total of the identifiable fatty acids (>95 %). Area percentage normalized values for the fatty acids were considered to be equivalent to weight percentage values since there were insignificant amounts of fatty acids with less than 12 carbon atoms ( A O A C , 2000). 7 35 2.2.5 Data calculation and statistical analyses The effect of diet treatment on the growth performance of the fish was assessed by the following: (1) Wet weight gain (WG) (g) = (final mean wet weight (FW) (g) - initial mean wet weight (IW) (g)) (2) Specific growth rate (SGR) (% body weight/day) = [(In FW (g) - In IW (g))/time (days)] x 100 (3) Dry feed intake (DFI) (g/fish) = total daily dry feed intake/fish over 84 days (4) Feed efficiency (FE) (g/g) = WG (g)/DFI (g/fish) (5) Protein efficiency ratio (PER) (g/g) = WG (g)/protein intake (g) (6) Percent protein deposited (PPD) (%) = protein gain (g) x 100/protein intake (g) (7) Percent lipid deposited (PLD) (%) = lipid gain (g) x 100/lipid intake (g) (8) Gross energy utilization (GEU) (%) = gross energy gain (MJ/fish) x 100/gross energy intake (MJ/fish) (9) Survival (S) (%) = (number of fish in each group remaining on day 84/initial number of fish)x 100 Al l data were subjected to randomized block Analyses of Variance (ANOVA; SigmaStat 3.0, SPSS, Chicago, U.S.A.) to test for possible diet and block effects. Arcsine square root transformations of percentage data were conducted to achieve homogeneity of variance before statistical analysis. Tukey's test with P = 0.05 was used to detect significant differences among means where appropriate. Graphical relationships between selected dietary fatty acid concentrations and their respective fatty acid concentrations in whole body and liver lipids were examined according to Bell et al. (2002) to gain further insights into the metabolic fates of these fatty acids. 36 2.3 Results All test diets contained similar concentrations of the proximate constituents that were in each case similar to the expected values (Table 2.1 and 2.2). Dietary fatty acid compositions reflected the concentrations of FO and CO in the supplemental lipids and their respective fatty acid compositions (Table 2.3). Thus, dietary CO concentrations were directly related to percentages of 20:0, 18:1 n-(9+7), Smonounsaturated fatty acids (MFA), 18:2n-6, Sn-6 PUFA, and 18:3n-3, and inversely related to 14:0, 16:0, 18:0, ^ saturated fatty acids (SFA), 16:ln-7, 20:ln-(ll+9), 22:ln-(ll+13+9), 20:2n-6, 20:4n-6, 22:4n-6, 22:5n-6, 18:4n-3, 20:4n-3, 20:5n-3, 22:5n-3, 22:6n-3, Zn-3 HUFA, Sn-3 PUFA and ratio of n-3 to n-6 fatty acids (Table 2.3). Diet treatment did not influence growth performance of the red sea bream in this study. For instance, all groups, irrespective of diet fed, had similar initial mean weight and values for FW, WG, SGR, DFI, FE, PER, PPD, PLD, and GEU and %S at the end of the 84-day feeding period. Furthermore, it is noteworthy that the final mean weights of all groups were about 13-fold higher than their respective mean initial weights (Table 2.4). In addition, except for percentages of whole body moisture, which were higher in FO and C025-fed fish relative to those fed diets C048 and CO70, diet treatment had no influence on the terminal concentrations of ash, crude protein, lipid, and gross energy in the fish (Table 2.5). Total body lipid compositions of the juvenile red sea bream following the 12-week feeding trial mirrored the trends described above for dietary fatty acid compositions. Dietary CO level was positively correlated with fish 20:0, 18:ln-(9+7), SMFA, 18:2n-6, 20:2n-6, Zn-6 PUFA (mainly due to step-wise elevations in 18:2n-6), 18:3n-3, and negatively correlated with 14:0, 16:0, SSFA, 16:ln-7, 20:ln-(ll+9), 22:ln-(ll+13+9), 20:4n-6, 22:4n-6, 22:5n-6, 18:4n-3, 20:4n-3, 20:5n-3, 22:5n-3, 22:6n-3, Sn-3 HUFA, En-3 PUFA (primarily due to progressive reductions in n-3 HUFA) and the ratio of n-3 to n-6 fatty acids. Key fatty 37 acids of physiological importance in body lipids, namely, 20:4n-6 (AA), 20:5n-3 (EPA) and 22:6n-3 (DHA) were reduced respectively by 60%, 83%, and 54% in fish ingesting diet CO70 versus those consuming diet FO. Total levels of PUFA in body lipids were not influenced significantly by diet treatment largely because the progressive declines in concentrations of n-3 PUFA were offset by the incremental rises in n-6 PUFA (Table 2.6). Fatty acid compositions of liver polar lipids of the fish on day 84 (Table 2.7) generally exhibited different profiles relative to respective dietary and whole body lipid fatty acid compositions (Table 2.3 and 2.6). Concentrations of SFA (mainly due to elevations of 16:0 and 18:0), n-3 HUFA (especially 22:6n-3), n-3 PUFA, and total PUFA were markedly higher than noted in the dietary and body lipids; however, those for SMFA were lower (Tables 2.3, 2.6 and 2.7). Further, SSFA did not follow a consistent trend in relation to the dietary CO concentration and were highest for C025-fed fish followed by those fed diets FO, C048 and CO70. Moreover, within MFA, key fatty acids such as 16: ln-7 and 20: ln-(l 1+9) did not show a consistent relationship with dietary CO concentration. Thus, incremental changes in SMFA among treatments were not found, unlike in body lipids. Within n-6 PUFA, levels of 20:4n-6 and 22:4n-6 in fish fed diets C025, C048 and CO70 were relatively maintained whereas 22:5n-6 was found to be higher in fish fed diets C048 and CO70 relative to fish fed diet FO, which differed from what was seen in the body lipids. With respect to n-3 PUFA, levels of 22:6n-3 were maintained and in all cases exceeded 29% of total fatty acids and did not differ among the fish fed diets with varying levels of CO. Moreover, liver polar lipid concentrations of EPA and 22:5n-3, although inversely related to dietary CO concentration, showed less of a stepwise decline across treatments than observed in the body lipids (Tables 2.6 and 2.7). Also, ratios of n-3 to n-6 fatty acids and of 18:1 to Sn-3 HUFA in liver polar lipids were found to be inversely and directly related to dietary CO concentration, respectively. 38 To better illustrate the findings for fatty acid utilization and retention, whole body and liver polar lipid concentrations of 18:2n-6, 18:3n-3, AA, EPA, and DHA were graphed as functions of their respective dietary levels (Figure 2.1). The retentions of these fatty acids were found to differ between the body and liver polar lipids. In body lipids, 18:2n-6, 18:3n-3 and EPA were relatively well-conserved, and their rates of utilization increased slightly as their respective dietary levels were raised. However, in liver polar lipids, these fatty acids were noted to be preferentially utilized, particularly at high dietary levels. Nonetheless, levels of A A and DHA were maintained in both body and liver polar lipids regardless of dietary level. These fatty acids also appeared to be produced in the fish as indicated by the slopes of 0.73 and 1.24 for A A and 0.98 and 1.12 for DHA obtained for whole body and liver polar lipids, respectively (Figure 2.1c, d). 2.4 Discussion The present study indicates that all of the supplemental pollock liver oil (10% of diet) in a premium quality practical diet for red sea bream can be replaced by refined canola oil without any adverse effects on their growth, general health (survival) or whole body proximate composition. Indeed, our results showed that CO could comprise up to 70% of total dietary lipid content provided that some fish oil (residual oil from fish meal) was present concurrently to furnish adequate quantities of EFA, specifically EPA, DHA and likely AA, for good growth and health. In this regard, diet CO70 contained about 0.75% n-3 HUFA and 0.03%> A A and it is conceivable that these dietary concentrations were just adequate or marginal. This is because the growth rate, feed intake and feed and protein utilization of the red sea bream fed diet CO70 tended to be numerically lower than respective values noted for fish fed the other diets; however, the differences were not statistically significant. Interestingly, the trends described above were not seen for fish fed diet C048 which 39 contained about 1.28% n-3 HUFA and 0.045% AA. Takeuchi et al. (1990, 1992) proposed that the EFA needs of juvenile red sea bream are satisfied when the diet contains 1% EPA, 0.5% DHA or 2.8% n-3 HUFA, when dietary lipid is at the optimal level of 15% (Takeuchi et al., 1991). Al l of the test diets in this study met the 0.5% DHA but not the 1% EPA requirement. Hence, our results support the notion that DHA has higher efficacy than EPA in red sea bream (Takeuchi et al., 1990). Nevertheless, chronic assessments should be conducted to confirm the acceptable dietary CO concentration found in this study in view of the trends noted above. While our results did not clearly indicate that there was an optimal balance between dietary levels of EPA, DHA and AA, it is interesting to note that the red sea bream fed diet C025 exhibited a trend toward improved growth, feed intake and feed utilization relative to those consuming other test diets. However, again, the differences between the groups for each of the foregoing performance parameters were not significant. A study of longer duration may provide more definitive evidence with respect to this point. In any case, the present findings agree with and extend those obtained in short and long-term studies on gilthead sea bream, European sea bass, and pink snapper in which growth performance measures were found to be uncompromised when rapeseed oil or refined canola oil was used to replace 60% (former two species) to 100% (latter species) of the supplemental fish oil (Glencross et al., 2003a; Montero et al., 2003; Izquierdo et al., 2005; Mourente et al., 2005). Furthermore, the present study exceeded the -40% total dietary lipid substitution with refined CO achieved by Glencross et al., (2003a) who worked with older juveniles (IBW = 28g) and over a shorter time period (54 days). Although the second study by Glencross et al. (2003b) did observe that CO could comprise 68% of the dietary lipid of pink snapper, it is noteworthy that the fish were in a relatively slow growth phase (final body weights were ~1.5-fold higher than the initial body weights) and the study duration was short 40 i.e., only 32 days. By contrast, the red sea bream in this study exhibited a 13-fold increase over their initial body weight over an 84-day period, irrespective of diet treatment. Moreover, all groups in the present study showed excellent values for feed and protein utilization that were generally markedly improved relative to the values noted for the same parameters in the studies by Glencross et al. (2003a, b). Our results also agree with the findings of studies on Pacific and Atlantic salmon which have shown that canola oil is an excellent source of supplemental dietary lipid provided that the diets contain sufficient concentrations of EFA for good growth and health (Dosanjh et a l , 1984, 1988, 1998; Rosenlund et a l , 2001). Whole body proximate compositions, with the exception of percent moisture, were not affected by diet treatment. Although differences in whole body moisture percentages are often accompanied by reciprocal changes in whole body lipid content (Bendiksen et a l , 2003), the latter effect was not found in this study. Since feed intake, feed efficiency, and the ratio of dietary protein to lipid or energy also did not vary among treatments and these variables are known to influence whole body and fillet proximate constituents in other fish species (Higgs et a l , 1995; Rasmussen, 2001), it is likely that the dissimilar moisture contents found between groups in this study did not represent an effect of biological significance. Whole body fatty acid compositions of the fish generally reflected the trends that were observed in the fatty acid concentrations of the diet treatments. In this regard, CO is a richer source of unsaturated fatty acids, 18:ln-9, 18:2n-6, and 18:3n-3 relative to pollock liver oil. Also, unlike the latter lipid source, CO is devoid of n-3 HUFA. Furthermore, marine finfish species exhibit limited or no ability to desaturate and elongate 18:3n-3 to n-3 HUFA and sometimes EPA to DHA as well as 18:2n-6 to 20:4n-6 due to low or absent A6 and A5 desaturase activities (Bell et a l , 1994; Montero et a l , 2004; Izquierdo et a l , 2005). Thus, alterations in the whole body fatty acid compositions of the red sea bream were strongly 41 influenced by the dissimilar fatty acid compositions of pollock liver oil and canola oil and their respective dietary levels as well as the inherent abilities of the red sea bream to metabolize fatty acids. In regard to the latter, our findings revealed that the red sea bream is similar to other marine finfish species investigated since they exhibited little ability to utilize saturated fatty acids for energy purposes. Lipolytic activities and fatty acid adsorption in red sea bream may be inversely related to the melting-point of the fatty acids as has been observed in some other studies on fish (Lie et al., 1987; Johnsen et al., 2000). Thus, whole body concentrations of SFA in red sea bream were strongly negatively related to the dietary CO concentration. Values of SMFA, on the other hand, were positively influenced by dietary canola oil concentration. Other researchers have also observed similar results when studying the effects of rapeseed or canola oil inclusion in diets of salmonids and marine species (Bell et al., 2001, 2003; Glencross et al., 2003a; Montero et al., 2003). Further, our results suggest that one of the dietary MFA, namely, 22:ln-(ll+13+9) was used preferentially as a source of energy by the red sea bream. The others that may have been utilized similarly as sources of energy included L A and LNA and possibly EPA, although the latter fatty acid appeared to be metabolically converted to 22:6n-3 as well. This conclusion is based on our observation of lower concentrations of EPA in body lipids than in respective dietary lipids of fish given the different treatments and the lower decline of DHA relative to EPA in the body lipids of fish fed diet CO70. Collectively, the preceding findings explain some of the departures that we observed in whole body fatty acid compositions of the red sea bream relative to their dietary fatty acid compositions. This is further illustrated in Figure 2.1, in which at higher dietary concentration, EPA is preferentially oxidized or bioconverted in body lipid. Furthermore, the percent decline of 22:6n-3 in the body lipids was not as great in fish fed diet CO70 as that seen for 20:5n-3 (Tables 2.3 and 2.6). Moreover, these findings are in accord with the fatty 42 acid metabolism abilities of marine finfish species in general as described above. Our observation of limited bioconversion of 18:2n-6 in the red sea bream of this study, based upon the relationship between the respective diet and terminal whole body lipid levels of LA and 20:4n-6 (Figure 2.1a, c), also agrees with the findings of others who have shown some degree of A6 desaturation in marine species such as turbot (Linares and Henderson, 1991), gilthead sea bream, and golden grey mullet (Liza aurata; Mourente and Tocher, 1993a, b). This is further supported by terminal levels of body 18:3n-6 which were not present in dietary lipids. As mentioned previously, liver polar lipid compositions generally exhibited different trends to those observed in whole body and dietary lipids. Signs of selective utilization and retention of specific fatty acids were much more profound in liver polar lipids. In this regard, LSFA were highest and lowest in C025 and CO70-fed fish, respectively. However, those for SMFA did not differ among fish fed diets with varying CO levels and they were all significantly higher than found in fish fed diet FO (control diet). However, SFA were accumulated to a greater extent and MFA to a lesser extent in liver polar lipids relative to body lipids. Likely, this reflected the preferential retention of saturated as opposed to monounsaturated fatty acids on position 1 of the phospholipids. Moreover, the red sea bream were also noted to accumulate and retain far more 22:6n-3 (all treatments) and to a lesser extent 20:5n-3 (fish fed diets with CO) and 22:5n-3 (all treatments) in polar lipids versus body lipids. The preceding findings agree with those of Yone and Fujii (1975), who observed similar increases in liver phospholipid DHA levels as more LNA was added to corn oil-based diets for juvenile red sea bream. Further, the accumulation of increased quantities of n-3 HUFA in polar lipids versus body lipids of the red sea bream explains why we noted much higher levels of PUFA in liver polar lipids of all groups relative to body lipids, which are mostly comprised of triacylglycerols in fish (Higgs et a l , 1995). The elevated levels of PUFA 43 in polar lipids versus body lipids were not due to elevations in n-6 PUFA, as these showed a narrower range in the former lipids than in the latter, even though the predominant n-6 fatty acid, 18:2n-6, was positively correlated.with dietary CO concentration in each case. However, we noted marked increases in all of the 20 carbon fatty acids of the n-6 family in liver polar lipids relative to levels in dietary and body lipids, particularly 20:3n-6, which correlated to dietary CO level. Thus, there may be bioconversion of 18:2n-6 to 20:4n-6 in liver polar lipids of the red sea bream and/or selective incorporation of the 20 carbon n-6 fatty acids into the membranes of hepatocytes (Seliez et al., 2003). Undesirable elevations in liver lipid content have been seen in red sea bream when the diets have contained less than 0.5% of EPA and/or DHA (Takeuchi et al., 1990). Ratios of 18:ln-9/n-3 HUFA in excess of 1 have also been associated with poor growth performance and feed efficiency in fingerlings (Fujii et al., 1976; Yone, 1978). The latter effects were not observed in this study and it is noteworthy that all of our test diets contained adequate concentrations of DHA for red sea bream and the ratios of 18:1 to n-3 HUFA were not greater than 0.51 in the liver polar lipids. Hence the red sea bream in this study did not exhibit any signs of EFA deficiency even when dietary CO concentration was as high as 70% of the total lipid content. In conclusion, our findings indicate that refined canola oil is a potentially suitable dietary lipid source for juvenile red sea bream under our test conditions. However, the long-term effects of using canola oil as the main source of supplemental dietary lipid still need to be assessed before definitive recommendations can be made. Future work should also include evaluation of the haematological and immunological responses of the fish in relation to the dietary canola oil level and the concomitant reductions in the levels of the essential highly unsaturated fatty acids of the n-3 and n-6 families. 44 Summary 1) Refined canola oil comprised up to 70% of the total dietary lipids in the diets of juvenile red sea bream over a period of 12 weeks and did not adversely affect growth performance when the remainder of the dietary lipids stemmed from marine fish oil. 2) Whole body fatty acid compositions were influenced by dietary fatty acid compositions, however, liver polar lipids were more resistant to dietary influence. 3) Specific fatty acid byconversion (AA) and retention (DHA) were evident in the liver polar lipids. 45 Tables Table 2.1: Ingredient compositions of the experimental diets. Numerical values after CO refer to the percentage of CO expressed in relation to the total dietary lipid content. Ingredients Diet (g/kg air-dry basis) FO C025 C048 CO70 Jack mackerel meal; 500 500 500 500 steam-dried Soybean meal; defatted 50 50 50 50 Corn gluten meal 50 50 50 50 Wheat flour 134 134 134 134 Pre-gelatinized potato 100 100 100 100 starch Pollock liver oil (FO); 100 67 33 0 stabilizeda Canola oil (CO); 0 33 67 100 stabilizedb P-free mineral 10 10 10 10 supplementc NaH 2 PO„ 10 10 10 10 Vitamin supplementd 30 30 30 30 Choline chloride (100%) 5 5 5 5 . Vitamin E (500 IU/g) 1 1 1 1 Cr 2O 3(50%) 10 10 10 10 a Supplemented with 200 mg/kg BHT. b Supplied by Hayashichemical Co. Ltd, Tokyo, Japan with no anti-oxidant added. cMineral supplement supplied (mg/kg diet): Na (as NaCI) 197; Mg (as MgS0 4 .7H 2 0) 735; Fe (as FeC 6 H 5 0 7 .5H 2 0) 258; Zn (as ZnS0 4 .7H 2 0) 40; Mn (as MnS0 4 .5H 2 0) 18; Cu (as CuS0 4 .5H 2 0) 3.9; Al (as A1C13.6H20) 0.56; Co (as CoCl 2 .6H 2 0) 0.15; I (as KI0 3) 0.89; a-cellulose carrier. d Vitamin supplement supplied (amount/kg diet): thiamin hydrochloride, 60 mg; riboflavin, 100 mg; pyridoxine hydrochloride, 40 mg; cyanocobalamin, 0.1 mg; ascorbic acid, 5000 mg; niacin, 400 mg; calcium pantothenate, 100 mg; inositol, 2000 mg; biotin, 6 mg; folic acid, 15 mg; p-aminobenzoic acid, 50 mg; vitamin K3,50 mg; vitamin A acetate, 9,000 IU; vitamin D3, 9,000 IU. 46 Table 2.2: Mean concentrations (% dry weight basis except for % moisture ± 1SD) of proximate constituents and gross energy (MJ/kg dry weight) in the experimental diets. The supplemental lipid stemmed from either pollock liver oil (FO), different blends of canola oil (CO) with FO, or CO. Numerical values after CO refer to the percentage of CO expressed in relation to the total dietary lipid content. Proximate constituent Diet FO C 0 2 5 C 0 4 8 CO70 Moisture 7.73 ± 0.42 7.01 ± 0.56 6.94 ±0.17. 7.18 ±0.71 Ash 10.2 ±0.08 10.4 ±0.08 10.1 ±0.25 9.98±0:05 Crude protein 46.510.18 45.6± 1.14 45.7 ± 0.45 47.2 ± 0.05 Crude lipid 15.1 ± 1.12 14.2 ± 0.42 15.0 ±0.15 15.4 ±0.79 Gross energy (MJ/kg) 21.9 21.6 21.9 22.1 47 Table 2.3: Percent fatty acid contents (g/lOOg fatty acids) in pollock liver oil (FO) and canola oil (CO) and the experimental diets. Numerical values after CO refer to the percentage of CO expressed in relation to the total dietary lipid content. Fat ty acid Pol lock l iver oil Diet C a n o l a oil F O C 0 2 5 C 0 4 8 C O 7 0 14:0 5.36 0.00 4.78 3.63 1.75 0.85 16:0 11.3 2.77 14.6 13.0 9.79 8.10 18:0 1.39 1.04 2.58 2.52 2.46 2.22 20:0 0.07 0.37 0.13 0.23 0.33 0.35 E S F A 18.1 4.18 22.1 19.4 14.3 11.5 16:ln-7 7.85 0.11 6.55 4.72 2.21 0.82 18:ln-(9+7) 15.9 64.6 17.7 31.7 44.7 52.8 20: ln- ( l l+9) 12.2 0.89 9.44 6.08 3.78 1.37 22: l n - ( l 1+13+9) 18.1 0.20 11.1 6.29 2.85 0.60 L M F A 54.0 65.8 44.8 48.8 53.6 55.6 18:2n-6 1.27 21.1 4.65 9.52 14.5 19.0 20:2n-6 0.18 0.05 0.20 0.15 0.12 0.08 20:3n-6 0.04 0.00 0.05 0.00 0.00 0.00 20:4n-6 0.51 0.00 0.61 0.46 0.33 0.21 22:4n-6 0.08 0.00 0.07 0.05 0.04 0.03 22:5n-6 0.08 0.00 0.19 0.14 • 0.12 0.09 L n - 6 P U F A 2.16 21.2 5.78 10.3 15.1 19.4 18:3n-3 0.80 7.60 0.84 2.33 3.40 5.69 18:4n-3 2.40 0.82 1.74 1.24 0.50 0.12 20:4n-3 0.60 0.00 0.51 0.35 0.22 0.10 20:5n-3 10.8 0.00 8.34 5.53 3.04 1.16 22:5n-3 0.90 0.06 1.04 0.77 0.59 0.35 22:6n-3 7.74 0.00 9.95 7.51 6.48 4.25 L n - 3 H U F A 18.5 0.00 18.3 13.0 9.52 5.41 2n -3 P U F A 23.2 8.48 22.4 17.7 14.2 11.7 22:4n-9 0.40 0.00 0.32 0.21 0.11 0.03 Others 2:40 0.00 4.96 3.80 2.73 1.90 Total P U F A 25.8 29.7 28.5 28.2 29.4 31.1 n-3/n-6 10.8 0.40 3.88 1.72 0.94 0.60 48 Table 2.4: Mean (± 1SD) initial (IBW, g) and final (FBW, g) body weight, weight gain (WQ g), specific growth rate (SGR, %/day), dry feed intake (DFI, g/fish), feed efficiency (FE, g/g), protein efficiency ratio (PER, g/g), percent protein deposited (PPD, %), percent lipid deposited (PLD, %), gross energy utilization (GEU, %), and survival (S, %) of red sea bream in relation to diet treatment. The supplemental dietary lipid stemmed from either pollock liver oil (FO), different blends of canola oil (CO) with FO, or CO. Numerical values after CO refer to the percentage of CO expressed in relation to the total dietary lipid content.3 Performance parameters Diet F O C 0 2 5 C 0 4 8 C O 7 0 IBW 3.61 ± 0 . 2 9 3.67 ± 0 . 2 6 3.66 ± 0 . 3 1 3.47 ± 0 . 1 5 FBW 46.6 ± 5 . 5 7 52.3 ± 5 . 8 8 46.4 ± 1.69 44.1 ± 2 . 6 4 W G 43.0 ± 5 . 4 1 48.7 ± 5 . 6 0 42.7 ± 1 . 5 4 40.6 ± 2 . 7 6 SGR 3 . 0 4 ± 0 . 1 3 3.16 ± 0 . 0 6 3.03 ± 0 . 0 9 3.02 ± 0 . 1 2 DFI 39.9 ± 3 . 9 9 43.2 ± 3 . 1 7 39.2 ± 1 . 3 8 37.7 ± 2 . 5 1 FE 1.08 ± 0 . 0 5 1.13 ± 0 . 0 6 1.09 ± 0 . 0 2 1.08 ± 0 . 0 3 PER 2.32 ± 0 . 1 0 2.48 ± 0 . 1 2 2.38 ± 0 . 0 4 2.29 ± 0 . 0 6 PPD , 46.3 ± 1 . 8 2 46.6 ± 2 . 1 6 45.4 ± 0 . 6 4 44.8 ± 1.00 PLD 85.0 ± 3 . 2 8 88.9 ± 3 . 9 8 82.1 ± 1.77 82.7 ± 1 . 7 5 GEU 44.5 ± 1 . 7 3 44.6 ± 2 . 0 3 42.8 ± 0 . 5 4 43.9 ± 0 . 9 4 S 98 97 98 98 a Data for each parameter (n=3) were analyzed by randomized block ANOVA. No significant differences were found for any of the performance parameters due to diet treatment. 49 T a b l e 2.5: Initial (n = 3) and final mean (n = 3) concentrations (% of wet weight ± 1 S D ) of proximate constituents and gross energy contents (kJ/g) in the whole bodies of red sea bream in relation to diet treatment. The supplemental dietary lipid stemmed from either pollock liver oil (FO), different blends of canola oil (CO) with FO, or CO. Numerical values after CO refer to the percentage of CO expressed in relation to the total dietary lipid content.3 Diet D a y Mois ture A s h C r u d e protein C r u d e l ip id Gross energy 0 77.2 ± 0 . 7 1 4.00 ± 0 . 0 1 14.6 ± 0 . 4 3 3.26 ± 0 . 1 2 4.73 ± 0 . 2 1 F O 84 69.4 ± 0.72 a 3.97 ± 0 . 5 0 18.2 ± 0 . 7 5 9.57 ± 0.34 8.08 ± 0 . 2 1 C025 84 69.5 ± 0.17 a 3.60 ± 0 . 1 7 17.4 ± 0 . 5 7 9.22 ± 0.68 7.84 ± 0.44 C048 84 67.8 ± 0.83 b 4.42 ± 0 . 1 6 17.4 ± 0 . 3 2 9.13 ± 1.06 7.73 ± 0.28 C O 7 0 84 67.6 ± 0.59 b 4.54 ± 1.13 18.0 ± 0 . 8 8 9.66 ± 0 . 5 1 7.95 ± 0.26 a Percentages for each proximate constituent were arcsine square root transformed before being subjected to randomized block ANOVA. Where appropriate, differences among treatment means were detected using Tukey's test with P = 0.05. Different superscripts within a column denote significant differences among the means (P<0.05). 50 Table 2.6: Final mean (± 1 SD) fatty acid contents (g/1 OOg total fatty acids) in the whole bodies of red sea bream in relation to diet treatment. The supplemental dietary lipid stemmed from either pollock liver oil (FO), different blends of canola oil (CO) with FO, or CO. Numerical values after CO refer to the percentage of CO expressed in relation to the total dietary lipid content.3 Diet Fatty acid Initial FO C025 C048 CO70 14:0 4.17 3.04 ±0.83 a 2.51 ±0.30 b 1.63 ± 0.06 b 0.60 + 0.03 c 16:0 23.4 17.5 ± 2.45 a 15.9 ±0.71b 14.0 ± 0.49 b 9.7 ± 0.54c 18:0 5.60 3.81 ±0.10 3.65 ±0.25 3.60 ±0.18 3.43 + 0.36 20:0 0.14 0.14±0.02d 0.20 ±0.01 0 0.24 ±0.01 b 0.32 ± 0.02 a LSFA 33.3 24.5 ± 3.32 a 22.2 ± 0.87ab 19.5 ±0.67b 14.1 ±0.85° 16:ln-7 6.00 6.00 ± 0.96 a 4.52 ± 0.06 b 2.95 ±0.17c 1.26 ± 0.09d 18:ln-(9+7) 23.5 22.3 ± 1.06 d 35.1 ±0.52° 44.6 ± 0.27 b 52.2 ± 0.27a 20:ln-(ll+9) 2.45 8.88 + 1.14 a 5.91 ±0.24 b 3.58±0.15c 2.11 ±0.11d 22: ln-(l 1+13+9) 0.96 8.59 ± 1.92' 4.33 ± 0.66 b 1.83 ±0.13° 0.41 ±0.05d EMFA 32.9 45.8 ± 1.17d 49.8 ± 0.86 c 53.0 ± 0.29 b 56.0 ± 0.27a 18:2n-6 3.83 4.24 ± 0.08 d 8.66 ±0.23 0 12.5 ± 0.43 b 16.2 ± 0.40a 20:2n-6 0.18 0.20 ±0.03 b 0.21 ±0.01 b 0.21 ±0.01 b 0.28 ± 0.02 a 20:3n-6 0.07 0.06 ± 0.00 0.06 ±0.00 0.06 ±0.01 0.07 ±0.01 20:4n-6 0.80 0.50 ± 0.04 a 0.37 + 0.01 b 0.27 ± 0.00 c 0.20 ± 0.02 d 22:4n-6 0.24 0.19 ± 0.05 a 0.14 ±0.01 * 0.10 ±0.01 b 0.10±0.02b 22:5n-6 0.29 . 0.18 + 0.02" 0.14 + 0.01 a b 0.11 ±0.00 b 0.10 +0.03 b£n-6 PUFA 5.41 5.36 ± 0.21 d 9.58 ±0.210 13.3 ± 0.43 b 17.0 ± 0.48 a 18:3n-3 0.56 0.62 ± 0.02 d 1.71 ±0.14° 2.66 ± 0.25 b 3.97 ± 0.12 a 18:4n-3 0.49 0.89 ±0.03 a 0.61 ± 0.05 b 0.32 ± 0.02 c 0.09 ± 0.01 d 20:4n-3 0.04 0.62 ±0.05 a 0.44 ± 0.02 b 0.29 ±0.01 c 0.17±0.01d 20:5n-3 5.49 5.56 ±0.53 a 3.61 ± 0.19 b 2.01 ± 0.05 c 0.94 +0.09 d22:5n-3 1.28 1.71+0.21 a 1.19 + 0.09 a b 0.76 ± 0.04 b 0.55 ± 0.11 b 22:6n-3 .15.8 11.4 ± 1.42 a 8.21 ± 0.59 * 5.89 ±0.23° 5.26 ±0.40° Ln-3 HUFA 21.3 17.0 ±2.01 a 11.8 ± 0.72 b 7.90 ± 0.32 c 6.20 ± 0.50d Sn-3 PUFA 23.7 20.8 ± 2.24 a 15.8 ± 0.75 b 11.9 ± 0.47c 11.0 ± 0.38c 22:4n-9 0.16 0.25 ± 0.02 a 0.16 ±0.01 b 0.09 ±0.00° 0.04 ±0.01 d Others 8.55 3.65 ±0.05 a 2.59 ± 0.42 b 2.36 ± 0.07 b 1.85 ± 0.25 c Total PUFA 29.3 26.4 ± 2.43 25.5 ±0.61 25.3 ±0.88 28.1 ±0.86 n-3/n-6 4.38 3.87 ± 0.29 a 1.65 ± 0.11 b 0.90 ±0.01 c 0.66 ±0.01c a Percentage data of each fatty acid (n = 3/diet treatment with each mean based on the analysis of 3 fish) were arcsine square root transformed and then analyzed by randomized block ANOVA and, where appropriate, Tukey's test with P = 0.05. Different superscripts within a row denote significant differences among the means (P<0.05). 51 T a b l e 2.7: Final mean (± 1SD) fatty acid contents (g/lOOg total fatty acids) in the liver polar lipids of red sea bream in relation to diet treatment. The supplemental dietary lipid stemmed from either pollock liver oil (FO), different blends of canola oil (CO) with FO, or CO. Numerical values after CO refer to the percentage of CO expressed in relation to the total dietary lipid content.'1 Fat ty acid D i c t F O C 0 2 5 C 0 4 8 C O 7 0 14:0 0.64 +0.02 a 0.52 ± 0.09 b 0.41 ± 0 . 0 3 c 0.26 ± 0.04 d 16:0 25.6 ± 1.28 a 25.8 ± 1.88 a 23.5 ± 2.24 a b 20.9 ± 1.18 b 18:0 8.38 ± 0.45 10.3 ± 0.90 10.2 ± 0.66 9.74 ± 0.75 20:0 0.09 ± 0 . 0 4 0 J 0 ± 0 . 0 3 0.12 ± 0 . 0 2 ' 0.14 ± 0 . 0 4 S S F A 34.7 ± 1.76 b 36.6 ± 2 . 7 7 a 34.3 ± 2.55 b 31.0 ± 0 . 7 8 ° 16:ln-7 1.33 ± 0.09 a 1.46 ± 0 . 3 1 a 1.16 ± 0.02 a 0.61 ± 0 . 1 4 b 18:ln-(9+7) 8.06 ± 0.89 c 12.9 ± 2.25 b 13.2 ± 1.29 b 16.2 ± 0.68 3 20: ln- ( l l+9) 0.83 ± 0 . 1 0 1.18 ± 0 , 3 6 0.81 ± 0 . 1 1 0.97 ± 0 . 1 7 22: l n - ( l 1+13+9) 0.79 ± 0 . 1 7 a 0.51 ± 0 . 1 0 b 0.32 ± 0.04 b c 0.14 ± 0.06 c S M F A 1 1 . 0 ± 0 . 8 7 b 16.0 ± 2.84 a 15.5 ± 1.39 a 17.9 ± 0.60 a 18:2n-6 2 . 1 9 ± 0 . 1 4 d 4.57 ± 1 . 7 1 0 7.01 ± 1.26 b 10.6 ± 1.00 a 20:2n-6 0.30 +0.05 b 0.40 ± 0.14 b 0.54 ± 0 . 0 7 a b 0.69 ± 0 . 1 5 a 20:3n-6 0.12 ± 0 . 0 1 c 0:15 ± 0.04 c 0.22 ± 0.02 b 0.32 ± 0.03 a 20:4n-6 2.70 ± 0.25 a 2.22 ± 0.32 b 2.25 ± 0.17 b 2 . 1 5 ± 0 . 3 4b 22:4n-6 1.16 ± 0 . 1 7 a 0.37 ± 0.06 b 0.45 ± 0.13 b 0.50 ± 0.03 b 22:5n-6 0.32 ± 0 . 0 1 b 0.32 ± 0.08 a b 0.39 ± 0 . 0 5 a b 0.44 ± 0.07 a Ln-6 P U F A 6.78 ± 0.48 c 8.03 ± 1.79 c 10.9 ± 1.37 b 14.7 ± 0.90 a 18:3n-3 0 . 1 7 ± 0 . 0 2 c 0.43 +0.12 c b 0.61 ± 0 . 0 7 b 1.19 ± 0.19 a 18:4n-3 0.03 ± 0.00 b 0.09 ± 0 . 0 1 b 0.10 ± 0.02 a b 0.16 ± 0 . 0 5 3 20:4n-3 . 0.51 ± 0.06 a 0.32 ± 0.02 b 0.30 ± 0.04 b 0.22 ± 0.02 c 20:5n-3 5.26 ± 0 . 1 1 a 3.99 ± 0.39 b 3.99 ± 0 . 2 7 b 2.63 ± 0.35 c 22:5n-3 2.58 ± 0.29 a 1.99 ± 0.04 b 1.74 ± 0 . 1 5 ° 1.26 ± 0.12 d 22:6n-3 34.8 ± 1.06 a 30.0 ± 1.73 b 30.3 ± 1.24 b 29.0 ± 1.07 b En-3 H U F A 40,1 ± 1.08 a 34.0 ± 2.02 b 34.3 + 1.52 b 31.6 ± 1.42 c E n - 3 P U F A 43.3 ± 1.05 a 36.8 ± 1 .96 b c 37.0 ± 1.57 b 34.4 ± 0.89 0 22:4n-9 0.07 ± 0 . 0 1 a 0.05 ± 0.00 b 0.04 ± 0.00 b 0.03 ± 0.00 0 Others 4.14 ± 1.69 a 2.36 ± 0.19 b 2.32 ± 0.33 b 1.97 ± 0 . 5 1 b Tota l P U F A 50.2 ± 1 . 0 1 44.9 ± 3 . 6 0 47.9 ± 1.92 49.1 ± 0 . 6 4 n-3/n-6 6.41 ± 0.53 a 4.59 ± 0.72 b 3.39 ± 0.45 b 2.35 ± 0.19 c 18:1/ Ln-3 H U F A 0.20 ± 0.02 c 0.38 ± 0.08 b 0.38 ± 0.05 b 0.51 ± 0 . 0 3 a a Percentage data of each fatty acid (n = 3/diet treatment with each mean based on the analysis of 5 fish) were arcsine square root transformed and then analyzed by randomized block ANOVA and, where appropriate, Tukey's test with P = 0.05. Different superscripts within a row denote significant differences among the means (P<0.05). 52 Figures Figure 2.1: Relationship (±1 SD) between whole body (WB) and liver polar lipid (LP) fatty acid concentrations and dietary concentrations of (a).18:2n-6, (Slopewu = 0.8305, RWB = 0.9989; SlopeLP = 0.5752, R 2 L P= 0.9835), (b). 18:3n-3 (SlopewB = 0.6954, R2WB= 0.9928; SlopeLp = 0.2099, R \ P = 0.9884), (c). AA(SlopewB = 0.7333, R2WB= 0.9942; SlopeLP = 1.2362, R 2 L P= 0.7568), (d). E P A (SlopewB = 0.6464, R 2 W B = 0.9984; Slopes = 0.3237, R 2 L P= 0.8827) and (e). D H A (SlopewB = 0.9834, R2WB= 0.8168; SlopeLP= 1.1128, R 2 L P= 0.9114) in red sea bream juveniles fed either FO, C025, C048 or CO70 (n = 3/diet treatment based on 3 (WB) or 5 (LP) fish per replicate). The grey line indicates the line of linearity. Standard deviations are plotted but are within the boundaries of the data points. 2D.1 I 2 1: i2 (b) 13:3»3-/ 25: •ss->. ,._..-0 id) EPA Ml! II', 10 I f >»:• ?5 X< EH* .:*:;:;£'!4J'#ii :-.. •c.: :';:Lxr.^.^li%!J4 Diet (• fart)- arid/MJOg total fatty acufc J 53 References AOAC, 2000. Horwitz, W. (Ed.), Official Methods of Analysis of AOAC International. AOAC International, Gaithersburg, MD. Barlow, S, 2000. Fishmeal and fish oil: sustainable feed ingredients for aquafeeds. Glob. Aquacult. Advocate 4, 85-88. Bell, J.G, Tocher, D.R, MacDonald, F . M , Sargent, J.R, 1994. Effects of diets rich in linoleic (18:2n-6) and alpha-linolenic (18:3n-3) acids on the growth, lipid class and fatty acid compositions and eicosanoid production in juvenile turbot (Scophthalmus maximus L.). Fish Physiol. Biochem. 13, 105-118. Bell, J.G, McEvoy, J , Tocher, D.R, McGhee, F , Campbell, P.J, Sargent, J.R, 2001. Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism. J. Nutr. 131, 1535-1543. 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Koseisha-kosei-kaku, Tokyo, pp. 43-49. 57 CHAPTER THREE: Chronic effects of dietary canola oil level on growth performance, fatty acid composition and ionoregulatory development of spring chinook salmon parr, Oncorhynchus tshawytscha 3.1 Introduction Pacific salmon (Oncorhynchus spp.\ like other vertebrates, are incapable of synthesizing the parent acid of the omega-3 (n-3) series of fatty acids, 18:3n-3 (linolenic acid; LNA), which is one of several precursors of the n-3 long chain C20 and C22 highly unsaturated fatty acids (n-3 HUFA) which are required for normal growth and development (Tocher et al , 1989; Tocher, 2003). Thus, the acquisition of LNA and possibly its metabolic derivatives, notably eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA), must be dietary in origin (Sargent et al, 2002; Tocher, 2003). Juvenile Pacific salmon, e.g., chum salmon, may also have a dietary need for linoleic acid (18:2n-6; LA), the parent acid of the n-6 family of fatty acids, and a small amount of its highly unsaturated derivative, arachidonic acid (20:4n-6; AA), for normal physiological functions (Higgs et al , 1995). The foregoing essential fatty acids (EFA) have critical physiological roles in fish. These include the maintenance of cellular functions and integrity, lipid homeostasis, the production of eicosanoids, and the regulation of lipid mediators (Tocher, 2003). Since Pacific salmon species generally have a higher dietary requirement for the n-3 family of fatty acids than for the n-6 family of fatty acids (Higgs and Dong, 2000), marine fish oils (FO), which are rich in n-3 HUFA, have been used as the predominant sources of dietary lipids for these species (Higgs and Dong, 2000). Increasing global demands for food fish and the recent plateau in global capture fisheries have led to the progressive growth in global finfish aquaculture output and in aquafeed production (FAO-FD, 2006). The latter has been accompanied by escalating 2 Aversion of this chapter has been submitted for publication and the reference is as follows: Huang, S.S.Y., Fu, C.H.L, Higgs, D A , Balfry. S.K, Schulte, P.M., Brauner, C.J., 2007. Chronic effects of dietary canola oil level on growth performance, fatty acid composition and ionoregulatory development of spring chinook salmon parr, Oncorhynchus tshawytscha. Aquaculture. 58 demands for FO which has increased FO prices and decreased global supplies of this valuable commodity for other purposes. Furthermore, the high lipid content in some commercial salmonid diet formulations (high-energy (fat) diets) further exacerbates the problem of meeting aquafeed FO needs from a finite annual global supply (Barlow, 2000; Hardy et al. 2001; Higgs and Dong, 2000; Tacon, 2004). The FO crisis is further intensified during El Nino events off coastal South America, as annual global fish oil supplies have declined even further due to the reduced harvest of pelagic fish species that are used for much of the global fish meal and oil production (Hardy et al , 2001). Clearly, there is strong incentive and need to diversify protein and lipid sources for aquafeed from both sustainability and economic standpoints. Alternative lipids of terrestrial plant and/or animal origin have been considered in feeds for cultured salmonids, such as Atlantic salmon (Salmo salar, Dosanjh et al , 1998; Rosenlund et al , 2001; Bell et al , 2001, 2002), fall chinook salmon (O. tshawytscha, Dosanjh et al, 1988), rainbow trout (O. mykiss, Geurden et al , 2005), coho salmon (O. kisutch, Dosanjh et al , 1984) and brook charr (Salvelinus fontirtalis; Guillou et a l , 1995). However, these lipid sources differ greatly from FO in their fatty acid profiles. Plant oils are typically rich in unsaturated and highly digestible C18 fatty acids but lack the n-3 HUFA that are characteristic of marine FO (Opsahl-Ferstad et al , 2003). On the other hand, animal fats such as lard or poultry fat are generally richer in saturated fatty acids (SFA) and in the case of poultry fat, may have an appreciable content of the monounsaturated fatty acid (MUFA), oleic acid (18:ln-9; OLA) (Mugrditchian et a l , 1981; Dosanjh et a l , 1984, 1988; Higgs et al , 2006). Thus, shifts in dietary fatty acid.profiles will have profound effects on the fatty acid profiles in whole body and tissue lipids of fish which, in turn, may affect their physiological processes by altering supplies of metabolic energy, membrane structure and function, and the types and concentrations of the eicosanoids (Higgs and Dong, 2000; McKenzie, 2001; 59 Welker and Congleton, 2003). Indeed, the relative proportions of SFA and n-3 and n-6 polyunsaturated fatty acids (PUFA) in the diet of post-smolt Atlantic salmon can affect their aerobic swimming and oxygen consumption rate (McKenzie et al , 1998; Wagner et al , 2004). Activities of membrane-bound proteins, including key enzymes in ionoregulation such as Na +/K +- ATPase, may also be affected (Bell et al , 1997; Tocher et al. 2000; Stubhaug et al, 2006). Furthermore, EFA deficiency in fish has led to reduced ion permeability and morphological changes in the gills (Bell et al, 1985). However, studies using low erucic acid rapeseed oil or canola oil (the trademark name of rapeseed cultivars that contain <2% erucic acid in the oil and < 30 umoles of glucosinolates or antithyroid compounds in the oil-free meal; Canola Council of Canada, 2005), as sources of supplemental dietary lipid have yielded promising results in salmonids when used either as partial replacements for FO either alone or blended .with other plant oils (flaxseed and soybean oil), or with animal lipid (pork lard or poultry fat) (Dosanjh et a l , 1984,1988, 1998; Caballero et al , 2002; Rosenlund et al , 2001; Bendiksen and Jobling, 2003; Grant, 2006; Higgs et al , 2006). Furthermore, the fatty acid profile of canola oil (CO) is more similar to the profiles in the freshwater (FW) prey of juvenile salmon, like the chinook salmon, than those of FO and other plant oils (Higgs et a l , 1995). Thus, dietary lipids that contain an appropriate mixture of n-6 and n-3 fatty acids, such as those found in low-erucic acid rapeseed oil and CO, may be more appropriate for inclusion in feeds destined for salmon parr reared in freshwater (Bell et al , 1997; Tocher et a l , 2000). Bendiksen et al. (2003) demonstrated that the dietary inclusion of rapeseed and linseed oil improved parr-smolt transformation and the growth of Atlantic salmon in seawater. Other studies have also observed similar findings (Tocher et al , 2000; Bendiksen and Jobling, 2003). A recently conducted long-term study (5 months) on fall chinook salmon parr also noted that refined canola oil is an excellent source of supplemental dietary lipid (Grant, 2006). Growth and 60 ionoregulatory development in fish were unaffected when CO comprised as much as 41% of the total dietary lipid content (Grant, 2006). However, the lack of dietary effects on the preceding parameters in this study may have stemmed from the life history type of chinook salmon that was used (i.e., fall instead of spring chinook salmon), insufficient replacement of FO with CO, or the possibility that a critical stage in ionoregulatory development was overlooked. These aspects were addressed in the current study. The Canadian salmonid aquaculture industry has been actively incorporating alternative lipids into their formulated grower diets to reduce dependence on imported marine FO for major cultured species such as Atlantic salmon and rainbow trout (Higgs and Dong, 2000; Higgs et a l , 2006). Some emphasis in this regard has also been placed on diets for fall chinook salmon and coho salmon in both the pre- and post-smolt phases of their life history. This study was conducted to assess the potential for long-term inclusion of CO in a practical commercial diet for a strain of chinook salmon (O. tshawytscha), the spring chinook salmon, which is an important indigenous species of the Pacific coast that has a prolonged freshwater stage before seawater entry (Healey, 1991). The extended freshwater residency time of this anadromous species was considered to be ideal for examining possible dietary lipid composition effects on the early growth and ionoregulatory development in salmon. In this regard, the supplemental dietary lipid of spring chinook salmon was varied to contain either a 1:1 blend of anchovy oil and poultry fat (APF) or CO alone or different mixtures of these two sources to determine their effects on the growth and ionoregulation development of this life history type of chinook salmon during a significant portion of the freshwater residency period. In the extreme case, the dietary canola oil concentration comprised 72% of the total dietary lipid content with the remainder being mostly from fish oil. 61 3.2 Material and Methods 3.2.1 Experimental diets Four diets of equivalent protein (-51.3% protein), gross energy (-24.3 MJ/kg) and lipid (~21.6%o) content on a dry weight basis were prepared using a commercially-produced mash (Skretting Canada, Vancouver, BC) which after pelleting (see below) was supplemented with lipid (14.1%) from either a 1:1 blend of anchovy oil and poultry fat (APF), refined canola oil (CO) or various combinations of these sources (Table 3.1). Thus, the diet ingredient compositions were identical except for the proportions of APF and CO in the supplemental lipid. Canola oil comprised either 0%, 33%, 67% or 100% of the supplemental lipid content or 0% (dAPF), 25 % (C025), 49% (C049), or 72% (C072) of the total determined dietary lipid content (Tables 3.1 and 3.2). The diets were steam-pelleted using a California model CL type 2 laboratory pellet mill equipped with a 4 mm die. Subsequently, the diets were cooled and dried rapidly in a custom-made vertical cooler. Diet crumbles were generated using a crumbier (model.7.06S roller mill; W.W. Grinder Corporation, Wichita Kansas) and these were screened into appropriate sizes in relation to the fish size using a model K24-1-SS Kason vibroscreen separator (Separator Engineering Ltd, Montreal, Canada). Thereafter, the aforementioned supplemental lipid sources were sprayed onto the diets of different sizes using an electrically operated sprayer and a cement mixer. Al l finished feed was stored at 4 °C in air-tight bags until use. 3.2.2 Fish maintenance and experimental design Five-month post-hatched spring chinook salmon fry (O. tshawytscha) were obtained from Spius Creek Hatchery (Merritt, Canada) and fed a commercially-prepared starter diet (Nutra Plus, Skrettings Canada, Vancouver, Canada) for a 2-week acclimation period. Subsequently, 320 fry (average weight, 0.80 ± 0.03g (1SD)) were distributed randomly into 62 each of 12, 200-L oval fibreglass tanks that were supplied with aerated, flow-through well water at a flow rate of 10-12 L/min, and a natural photoperiod regime was provided. At week 15 of the feeding trial, the fish were transferred to 1100-L tanks to maintain fish density below 1 kg/m3. Water quality parameters, temperature (8.0-11.5°C) and dissolved oxygen (9.25-11.2 mg/L) were monitored daily. Triplicate groups of fish were fed each of the four test diets by hand to apparent satiation. Fish were fed six times a day for the first 10 weeks (0800h, 0900h, lOOOh, 1200h, 1400h, and 1600h), four times a day for the next 10 weeks (0800h, lOOOh, 1200h and 1600h) and three times daily for the last 10-week period (0800h, HOOh, and 1500h). Estimates of the actual rations ingested by each group were determined between weeks 20 to 30 only by subtracting the weight of waste feed (number of waste feed pellets x air-dry mean pellet weight in each case) from the total weight of feed dispensed each day. Actual rations were not estimated before week 20 due to the difficulty in obtaining accurate estimates of waste feed when the crumbled diets were fed. 3.2.3 Fish performance and sampling 3.2.3.1 Growth trial sampling Prior to the start of the experiment, 200 fish from an initial pool were randomly sampled, vacuum packed, and stored at -80°C for determination of initial concentrations of whole body proximate constituents (ash, moisture, crude lipid and protein) and fatty acid (FA) composition. Thereafter, all individuals were starved for 24-h and then sampled from each group for wet weight (nearest 0.01 g) after being anaesthetized in MS 222 (100 mg/L, buffered with 100 mg/L sodium bicarbonate). Upon termination of the experiment (end of week 30), 15 randomly selected individuals from each replicate tank were euthanized by an overdose of MS 222 to determine whole body proximate constituents and FA composition. Samples were vacuum-packed in oxygen impermeable bags and stored at -80°C until 63 analysis. 3.2.3.2 24-h seawater challenge test Twenty-four-hour seawater (SW) challenge tests (Blackburn and Clarke, 1987) were conducted concurrently with growth assessments every 5 weeks by measuring survival, whole body or muscle water content and whole body or plasma chloride and sodium values following SW exposure. In this regard, 8 randomly selected individuals per tank (24 per diet) were placed into aerated floating buckets submerged in seawater (salinity, 33g/L). At the end of 24-h, survivors were euthanized and sampled along with 8 additional individuals from the fresh water tanks which served as controls. For the first 15 weeks, whole bodies were frozen for later analyses of whole body chloride (fish were too small to provide sufficient blood plasma samples for ion analyses). During the last 10 weeks, blood was withdrawn from the caudal vein of each fish into heparanized hematocrit tubes that were then centrifuged immediately to obtain plasma. The plasma was frozen at -80°C for later sodium and chloride analyses. Whole bodies and skinless portions of the left dorsal epaxial muscle above the lateral line were removed for muscle water content determination immediately following sampling (refer to method below). 3.2.4 Chemical analysis 3.2.4.1 Moisture, ash, protein, lipid and gross energy determinations Samples of ground basal diet ingredients, experimental diets, and homogenized whole body samples (3 pools of 5 fish per tank) were analyzed in duplicate for moisture (16h at 100°C in a drying oven), ash (2h at 600°C in a muffle furnace), lipid (1:1 chloroform/methanol procedure of Bligh and Dyer, 1959), and protein (Technicon industrial method no.334-74W/B, revised March, 1997, Technicon Industrial System, Tarrytown, NY). Percent nitrogen was multiplied by 6.25 to obtain an estimate of percent protein. Dietary 64 gross energy concentrations were determined by adiabatic bomb calorimetry (IKA Calorimeter System C5000 duo control, IKA® Werke GmbH & Co. KG, Staufen, Germany), whereas whole body energy concentrations were calculated based on estimates of 23.64 kJ/g protein and 39.54 kJ/g for lipid (NRC, 1993). 3.2.4.2 Fatty acid analysis The fatty acid compositions of the supplemental lipid sources, as well as dietary and whole body lipids were determined using gas chromatography (GC; Varian 3900, Varian, Mississauga, Canada) of fatty acid methyl esters (FAME) that had been prepared according to Christie (1973). The GC was equipped with a CP sil-88, 50m x 0.25mm x 0.20um, capillary column (Varian, Mississauga, Canada), an automatic sample injector and a flame ionization detector. FAME were eluted from the column with helium as the carrier gas with column temperature set initially at 80°C for lmin. Thereafter, the column temperature was increased by 20°C/min until 170°C was attained, then by l°C/min until 190°C was reached and finally by 5°C until a final temperature of 220°C was achieved. The injector and detector temperatures were set at 250°C. Individual fatty acids were identified by comparing their retention times to those in a known standard (GLC-566, Nu-Check Inc., Elysian, USA). Individual FAME concentrations were calculated as a percentage area of the total of the identifiable fatty acids (>98%). Area percentage normalized values for the fatty acids were considered to be equivalent to weight percentage values since there were insignificant amounts of fatty acids with less than 12 carbon atoms (AOAC, 2000). 3.2.4.3 Diet, whole body and plasma ion concentrations Dried whole bodies and diets were weighed and digested in 5mL of acid (50% IN nitric acid, 50% deionized water) for a minimum of seven days to allow separation of the liquid and solid layers. One mL of the liquid phase was transferred to a 2.5mL tube, vortexed and centrifuged for 90s at 10 000 rpm. A total of 200uL was extracted from the solution and 65 stored at 5°C pending analysis. Whole body concentrations were determined by spectrocolorimetry (SpeetraMAX 190, Molecular Devices, Sunnyvale, USA) and compared to known standards. Plasma and chloride concentrations were determined using a HB 1 digital chloridometer (Haake Buchler Instruments Inc., Saddlebrook, USA). Plasma sodium concentrations were measured using a flame photometer (Corning 410, Corning Inc., New York, USA) with samples diluted 1000-fold in deionized water. Due to time constraints, only three (randomly selected) of the eight fish initially sampled from each replicate tank were analysed for whole body and/or plasma chloride and sodium ion content. 3.2.5 Data calculation and statistical analysis The effect of diet treatment on the growth performance of the fish was assessed by the following: (1) Wet weight gain (WG) (g) = [final mean wet weight (FW) (g) - initial mean wet weight (IW)(g)] (2) Specific growth rate (SGR) (% body weight/day) = [(In FW (g) - In IW (g))/time (days)] xlOO (3) Dry feed intake (DFI) (g/fish) = total daily dry feed intake/fish over 210 days (4) Feed efficiency (FE) (g/g) = WG (g)/DFI (g/fish) (5) Protein efficiency ratio (PER) (g/g) = WG (g)/protein intake (g) (6) Percent protein deposited (PPD) (%) = protein gain (g) x 100/protein intake (g) (7) Gross energy utilization (GEU) (%) = gross energy gain (MJ/fish) x 100/gross energy intake (MJ/fish) (8) Survival (%) = (number of fish in each group remaining on day 210/initial number of fish) x 100 Al l data were subjected to randomized block Analyses of Variance (ANOVA; SigmaStat 66 3.0, SPSS, Chicago, USA) to test for possible diet and block effects. Arcsine square root transformations were conducted on percentage data to achieve homogeneity of variance before statistical analysis. Tukey's test with P = 0.05 was used to detect significant differences among means where appropriate. Graphical relationships between selected dietary fatty acid concentrations and their respective fatty acid concentrations in whole body lipids were examined according to Bell et al. (2002) to gain further insights into the metabolic fates of these fatty acids. 3.3 Results The determined concentrations of proximate constituents and gross energy in the test diets were observed to be similar and close to the expected values (Table 3.2). Further, the dietary lipid compositions reflected the proportions of APF and CO that furnished the supplemental lipid and their respective fatty acid compositions (Table 3.3). Thus, increasing dietary CO concentration directly increased percentages of 20:0, 22:0, 18:ln-(9+ll), IMFA, 18:2n-6, 2n-6 PUFA, and 18:3n-3. Alternatively, progressive increases in dietary CO reduced levels of SSFA, 16:ln-(9+7), 24:ln-9, 20:2n-6, 20:4n-6, Zn-3 PUFA', n-3 HUFA, and ratios of n-3/n-6. The known requirements of chinook salmon for essential fatty acids were clearly achieved in the dAPF diet since the IEPA + DHA exceeded 10% of the dietary lipid content (Higgs et al., 1995; Higgs and Dong, 2000). Partial and total replacement of APF with CO in the supplemental lipid did not influence the growth performance of spring chinook salmon parr during the 30-week study. Fish in all groups exhibited about a 20-fold gain in weight over their respective initial mean weights (~0.80g) and attained final mean weights close to 14.2g (Table 3.4). Indeed, values for FW, WG, and SGR did not differ among diet treatments. This was also true for the values estimated for DFI, FE, PER, PPD, and GEU as well as the percent survivals of the fish over 67 the course of the study (exceeded 92%; Table 3.4). In addition, terminal concentrations of whole body proximate constituents and gross energy remained similar despite the dissimilarity in diet treatment (Table 3.5). There were however, significant differences in the final body lipid compositions of the spring chinook salmon, as a result of the different dietary treatments. Following the 30-week feeding trial, the fatty acid concentrations in the fish body lipids closely mirrored the aforementioned trends described for dietary fatty acids as CO was substituted progressively for APF in the supplemental lipid (Tables 3.3 and 3.6). For instance, the whole body lipids from fish fed C072 contained the lowest and highest percentages of ESFA and SMFA, respectively. In the body SFA, levels of 16:0 and 18:0 were most influenced by diet treatment and both of these fatty acids, as well as ESFA, were inversely related to dietary CO concentration. With respect to MUFA, levels of 16:ln-7 and 18:ln-9 correlated negatively and positively, respectively to increasing dietary CO. The latter effect resulted in increased levels of SMUFA in fish fed the diets with more CO. The totals for n-6 PUFA in fish whole body lipids were very similar to the observed values and trends in dietary lipids. However, concentrations of LA in fish body lipids were consistently lower than in respective dietary lipids containing supplemental CO. Furthermore, A A concentrations were maintained within a narrow range in fish fed diets with dissimilar levels of CO. Thus, the terminal levels of this fatty acid in fish lipids were unaffected by diet treatment. In relation to n-3 PUFA, the concentrations of LNA in the fish increased with increasing dietary CO level. Levels of other n-3 PUFA, including EEPA and DHA, were reduced by CO treatment. However, levels of 18:4n-3 remained unaffected. Concentrations of LNA in fish were observed to be consistently below those noted in dietary lipids. Nonetheless, there was no evidence of bioconversion of LNA to EPA, irrespective of dietary CO concentration. Interesting trends were observed in fish lipid concentrations of EPA, 22:5n-3, and DHA with increasing dietary CO content. 68 Relative to dietary values, we observed a decline in fish EPA but found increases in levels of 22:5n-3 and DHA as the supplemental CO level was increased in the diet. These trends -suggested that all fish groups were striving to maintain DHA concentrations within a narrow range. This appeared to be achieved in fish fed dAPF, C025 and C049. However, DHA in fish fed diet C072 was significantly reduced. The same trend was also observed in Sn-3 PUFA in fish lipids. Although ratios of n-3 to n-6 fatty acids were found to be inversely related to dietary CO concentration, SPUFA remained unaffected. This occurred because the increases in Sn-6 PUFA that were associated with increasing dietary CO level were offset by the decreases in Sn-3 PUFA.' To better illustrate the findings for fatty acid utilization and retention, whole body lipid concentrations of 18:2n-6, 18:3n-3, AA, EPA, and DHA were graphed as functions of their respective dietary levels (Figure 3.2). Slopes less than 1 suggested that the fatty acid under consideration was preferentially utilized for energy and/or bioconverted to a more unsaturated fatty acid derivative when provided at an increased dietary concentration. Thus, at low dietary concentration, 18:2n-6 was relatively well conserved in fish (e.g., fish fed the dAPF diet). However, the rate of 18:2n-6 utilization increased as its dietary levels were raised. This was indicated by the reduced steepness of the 18:2n-6 slope relative to the 1:1 relationship. This same trend was also seen for 18:3n-3 and EPA levels. DHA, however, appeared to be produced in the fish as indicated by the slope of 1.25. Furthermore, no decrease in DHA retention was seen at higher dietary content unlike the situation noted for the aforementioned fatty acids. Diet treatment had little effect on the overall ionoregulatory development of spring chinook salmon fry. Survival rates following 24-h SW challenge tests were found to be similar at all sample times and no significant differences in whole body or muscle water content (71.5- 72.4%) were observed among fish given the different diets (Figure 3.2). 69 Analyses of whole body chloride ion (Cl") content showed diet effects in both the freshwater control groups and following a 24-h SW challenge test at weeks 10 and 15 of the experiment (Figure 3.3). Freshwater fish fed diets which contained higher CO contents generally exhibited lower whole body Cl" level, with the exception of the C025 group at week 10. The same trend was also seen following the 24-h seawater challenge tests. Differences in whole body Cl" were much greater in magnitude, but correlated with that of the diets (Table 3.2), where Cl" level was inversely related to the dietary CO content. No significant differences were seen in plasma Cl" and sodium (Na+)concentrations in of both freshwater and SW-challenged fish at weeks 25 and 30 (Table 3.7). In general, whole body and plasma [Cl"] or [Na+] in fish subjected to a 24-h seawater challenge were consistently higher than that of their freshwater counterparts, confirming that the fish had not smolted. 3.4 Discussion The present study demonstrates that refined canola oil can be used as the sole source of supplemental lipid in a practical fish meal-based diet without compromising the growth performance of spring chinook salmon parr. Indeed, there were no adverse effects of diet treatment on the growth rate, dry feed intake, feed efficiency, utilization of dietary protein and energy, or whole body proximate composition of the fish after 30 weeks of feeding. Furthermore, varying dietary CO level did not affect % survival. Our findings are in accordance with previous studies on freshwater non-salmonid species (gold fish, Carassius auratus L.; common carp, Cyprinus carpio) as well as salmonids (Atlantic salmon; coho salmon, fall chinook salmon, rainbow trout; brook charr) in which canola oil or low-erucic acid rapeseed oil, alone or blended with other plant oils and/or animal fats, did not affect the growth parameters of these animals over either short or long-term periods (Dosanjh et al., 1984, 1988, 1998; Guillou et al., 1995; Steffens et al., 1995; Pozernick and Wiegand, 1997; 70 Caballero, et a l , 2002; Bell et a l , 2003; Higgs et a l , 2006; Grant, 2006). It is noteworthy, that the level of CO inclusion employed in our present study far exceeded that of all previous studies. This study revealed that growth performance of juvenile spring chinook salmon over 7 months was not compromised by feeding diets in which 72% of the lipid content was comprised of CO. The preceding findings strongly suggest that the quantities of AA, EPA, and DHA as provided by the residual lipid in the fish meal and krill meal in the C072 diet and also by APF in the other test diets, met the needs of the chinook salmon in this study. High percentages of the aforementioned fatty acids in the basal mix lipid (0.87% AA; 7.29% EPA; 11.7% DHA) further indicate the practicality and efficiency of our diet formulations. The 2% dietary 18:3n-3 and 18:2n-6 requirement for various salmonids of genus Oncorhynchus (Yu and Sinnhuber, 1979; Takeuchi and Watanabe, 1982) were met in all treatments. However, only dAPF and C025 met the proposed 2.1% EPA and DHA requirement outlined by Takeuchi and Watanabe (1977) for these salmonids. Unfortunately, the EFA needs of juvenile chinook salmon have not been determined but it is noteworthy that signs of EFA deficiency were lacking in all groups regardless of dietary CO content. This again, supports our conclusion that all test diets had adequate EFA content. Although we did not see conclusive evidence of bioconversion of LNA to EPA we did observe some bioconversion of LA to A A and also bioconversion of EPA to DHA in fish fed diets with supplemental CO. This suggests that an adequate amount of L A and LNA may be sufficient to meet the EFA requirement and that these n-3 HUFA precursors may be more important during the freshwater stage for spring chinook parr. It is noteworthy that the natural foods of this species at this life history stage are rich in C18 PUFA (Higgs et a l , 1995). The whole body lipid compositions observed for the chinook salmon in this study generally reflected the proportions of dietary APF, CO and their respective fatty acid 71 compositions. Specifically, chinook parr fed diets with more CO exhibited reduced percentages of total SFA, n-3 HTJFA and ratios of n-3 to n-6 fatty acids and increased percentages of total MFA, L A and LNA. These trends were seen because CO is a richer source of MFA, LNA and LA in comparison to APF. But unlike FO, CO is devoid of n-3 HUFA and AA. Interestingly, the spring chinook parr may be capable of compensating for dietary fatty acid alterations, as the effects of different proportions of APF and CO were generally less profound in fish lipids than in dietary lipids. It is likely, that the fish preferentially metabolized some of the ingested fatty acids for energetic or physiological purposes. With respect to the latter, the fish maintained A A and generally DHA levels within defined limits in the fish lipids whereas other fatty acids such as 16:0, 18:ln-9, 22:ln-9 and LNA appeared to be utilized for energy depending upon the diet treatment or they were bioconverted e.g., L A and EPA to more unsaturated derivatives. Similar findings have also been seen in the whole body or muscle lipids of Atlantic salmon parr and post-smolts when they have been fed diets with various levels of rapeseed or canola oil (Dosanjh et al., 1998; Rosenlund et al., 2001; Bell et al., 2003). Thus, levels of some SFA and MFA are not as rigidly regulated in salmonids as are levels of DHA and AA, which suggest that the former fatty acids are the more ready sources of energy (Sargent et al, 2002; Tocher, 2003). Alternatively, the reason may be related to the roles of AA, EPA and DHA in biomembrane structure and function and as progenitors of the physiologically active eicosanoid compounds (Higgs and Dong, 2000). Bendiksen et al. (2003) have shown that inclusion of plant oils (1:1 linseed and rapeseed) in diets for Atlantic salmon parr improves their retention of total n-3 fatty acids. The authors suggest that a reduction in dietary n-3 HUFA by the inclusion of plant oils enhances the retention of n-3 fatty acids in fish neutral lipids to ensure a reservoir that can be mobilized when needed. In the present study, however, the regressions obtained between selected ' 72 dietary and fish fatty acid concentrations showed that increased inclusion of dietary CO improved the retention of a specific n-3 fatty acid (i.e., DHA) but not total n-3 fatty acids. In fact, the retention of L N A in the body lipids (mostly neutral lipid) of chinook salmon pan-decreased at high dietary CO. Other studies on salmon have also observed that reductions in dietary DHA due to the inclusion of more plant oil have enhanced DHA production in fish lipids (Bell et a l , 1997, 2001, 2002; Torstensen et a l , 2004). Indeed, the replacement of dietary FO with increased levels of rapeseed oil or other plant oils has been shown to trigger hepatocyte fatty acyl desaturation/elongation activities (Tocher et a l , 2000; Bell et a l , 2001). In this study, enhanced DHA production appeared to be mainly due to bioconversion of EPA to 22:5n-3 and then increased elongation and desaturation of this fatty acid via two intermediates (Higgs and Dong, 2000) to DHA. When supplied in abundance, 18:3n-3 can be readily catabolized via P-oxidation to provide metabolic energy (Henderson and Sargent 1985; Henderson, 1996; Tocher et a l , 2000; Bendiksen et a l , 2003). This was strongly suggested in this study by the progressive departure of the 18:3n-3 regression slope from linearity when the diets contained high concentrations of LNA. An identical trend was also seen with 18:2n-6, however in this case, LA did not appear to be utilized for energy purposes but rather for bioconversion to maintain A A in the body lipids within a narrow range. This conclusion is supported by the elevated concentrations of 18:3n-6. 20:2n-6 and 20:3n-6 that were observed in the body lipids with increasing dietary CO. Dietary FO can inhibit the pre-adaptive surge of desaturase activities in salmon parr prior to smoltification (Bell et a l , 1997). This is because high levels of dietary EPA and DHA exert powerful feedback inhibition on A5 and A6 desaturase enzymes (Brenner, 1981; Tocher et a l , 1997). Thus, inclusion of plant oils may prevent this inhibition and could be beneficial in facilitating the smoltification process (Tocher et a l , 2000). In our study, the overall 73 ionoregulatory development of spring chinook salmon from the fry to parr stage was largely, unaffected by the dietary CO level. Diet also had no effect on the percent survival of the fish following 24-h SW challenges. However, our data indicate that the whole body Cl" content of spring chinook salmon can be influenced by changes in diet composition. For example, spring chinook parr fed diets with greater CO content generally exhibited lower body Cl" levels both in the FW control and SW-challenged groups, i.e. diet Cl" content was inversely related to CO content. Although one study suggests that almost all the dietary salt is bioavaliable in the closely related salmonid species rainbow trout (Salmon, 1987), this alone cannot account for the observed significantly greater Cl" content in the body as compared to the diets themselves. The basis for this difference clearly requires further investigation. Dietary salt (NaCl) supplementation before the onset of smoltification in salmonids may enhance their ionoregulatory ability and/or survival rate following transfer to seawater. This has been observed in coho salmon (Zaugg and McLain, 1969), fall chinook salmon (Zaugg et al., 1983), Atlantic salmon (Basulto, 1976) and brook trout (Pelletier and Besner, 1992). However, under intensive culture conditions, total salt intake (branchial + diet) can easily exceed the animal's requirement since formulated diets often contain a much higher salt content than is present in natural foods (aquatic invertebrates) of parr (Smith et al., 1989). Furthermore, excessive dietary salt may suppress appetite and result in poor feed conversion and growth in salmonids (Salmon and Eddy, 1988; Duston, 1993). Thus, high dietary salt content may influence both the ionoregulatory and growth development in parr. However, no such effects were observed in the current study as the dietary salt content was varied. Previous studies on Atlantic salmon parr have shown that diets with blends of plant oils have improved the ability of the fish to transfer to seawater based upon lower plasma Cl" values in fish following a 24-h SW challenge test (Bell et a l , 1997; Tocher et al., 2000; Bendiksen et al., 2003). Assuming diet has an effect on body and/or plasma ion levels, it is 74 important to look at differences between FW and post 24-h SW values rather than rely solely on the latter to assess salinity tolerance. Bearing this in mind, there were no significant effects of diet on the changes in whole body and muscle water content, or whole body Cl" or plasma.Na+ and Cl" concentrations between FW and 24-h SW values. However, as whole body and plasma ion levels were not measured simultaneously within a given time period, we do not know whether the fish were able to compensate for the differences in dietary salt content in week 25-30, where no difference in plasma Cl" and Na + were observed, or whether parr maintained a continuous tight regulation of plasma [Na+] and [Cl"] despite large variation in whole body ions. Thus, potential changes in dietary salt content in association with levels of CO may need to be taken into consideration to ensure their normal osmoregulatory ability. In conclusion, the present study shows that refined CO can comprise as much as 72% of the lipid content of diets for spring chinook parr without adversely affecting their growth and ionoregulatory development over a prolonged period (210 days). Moreover, other studies conducted on these fish have not found any effects of diet treatment on their health status or swimming performance (Kuchel et a l , unpublished). Our combined findings suggest that the compositions of our test diets adequately fulfilled the essential nutritional requirements of these fish, despite the dietary inclusion of a non-traditional feed ingredient, CO, as a significant proportion of the total lipid content. An abundance of dietary linoleic acid and linolenic acid provided by CO in combination with the n-3 HUFA present in the, residual lipid of the marine protein sources in the diets appeared to be sufficient for spring chinook salmon parr to meet their EFA needs under the conditions of this study. Furthermore, the ability of this species to selectively retain DHA and bioconvert EPA to DHA, and to metabolically convert L A to A A to some extent may explain the apparent high tolerance of the spring chinook salmon to the test diets that differed markedly in fatty acid composition. 75 Summary 1) Refined canola oil can comprise up to 72% of the total dietary lipids in diets of pre-smolt spring chinook salmon without adversely affecting the growth or ionoregulatory development of the fish over a period of 30 weeks. 2) Whole body fatty acid compositions were influenced by dietary lipids, however, pronounced fatty acid bioconversions (EPA to DHA) and retentions (DHA and AA) were observed. 3) Levels of dietary canola oil may affect whole body chloride content of the animal by affecting dietary chloride content. 76 Tables Table 3.1: Ingredient compositions of the experimental diets. Numerical values after CO refer to the percentage of total dietary lipid comprised by canola oil (CO). dAPF served as the control diet. This diet was only supplemented with a 1:1 blend of anchovy oil and poultry fat (APF). Ingredients* Diet (g/kg air-dry) dAPF C025 C049 C072 Fish meaf; steam-dried 419 419 419 419 Corn gluten meal 200 200 200 200 Canola meal 79 79 79 79 Feather meal 56 56 56 56 Wheat flour 82 82 82 82 Krill meal 15 15 15 15 Anchovy oil/Poultry fat 141 94 47 0 (APF) Canola oil (CO) 0 47 94 141 Vitamin (98 ARC) 4 4 4 4 Mineral (99 WC) 2 2 2 2 Ascogen (yeast-based) 2 2 2 2 * Essential vitamin and mineral requirements of the species were met in accordance to NRC (1993). The ethoxyquin content of all diets was adjusted to 125 mg/kg. a Austral (224 g/kg) and Hayduk (195 g/kg) fish meal; 77 Table 3.2: Mean percentages (% dry-weight ± 1SD) of proximate constituents, gross energy content (MJ/kg dry-weight) and chloride content (mmol/kg) in the experimental diets. Numerical values after CO refer to the percentage of total dietary lipid comprised of canola oil. Proximate Diet Constituent dAPF C025 C049 C072 Moisture 7.38±0.05 7.15±0.01 7.05±0.11 7.23±0.11 Crude ash 8.61±0.06 8.63±0.05 8.61±0.14 8.60±0.11 Crude protein 51.2±0.49 51.4±0.54 51.0±0.37 51.6±0.57 Crude lipid 22.0±0.19 21.3±0.17 21.5±0.37 21.6±0.35 Gross energy (MJ/kg) , 24.3 24.2 24.3 24.3 Cl" (mmol/kg)* 47.2i0.193 46.2±0.21 b 45.1±0.12c 44.4±0.10d * Dietary chloride data (n = 2) were analyzed by one-way ANOVA and where appropriate, the Tukey's Test with P = 0.05. Different superscripts denote significant differences among the means (PO.05). 78 Table 3.3: Percentages of fatty acids (g respective fatty acid/lOOg total fatty acids) in anchovy oil/poultry fat (APF), canola oil (CO), basal mixture, and the experimental diets. Numerical values after CO refer to the percentage of total dietary lipid comprised of canola oil. Diet Fatty acid A n c h o v y / Pou l try fat C a n o l a oil Basa l mix d A P F C 0 2 5 C 0 4 9 C 0 7 2 14:0 3.60 0.00 3.30 3.85 2.88 1.91 1.02 15:0 0.24 0.00 0.28 0.30 0.22 0.16 0.00 16:0 18.3 4.12 20.1 20.7 16.4 12.3 8.62 17:0 0.26 0.00 0.24 0.43 0.34 0.25 0.17 18:0 4.02 2.02 . 4.98 4.66 3.98 3.37 2.81 20:0 0.21 0.71 0.00 0.23 0.51 0.64 0.78 22:0 0.00 0.35 0.16 0.07 0.17 0.21 0.22 E S F A 26.6 7.20 29.1 30.2 24.5 18.8 13.6 16:ln-9 0.52 0.00 0.20 0.49 0.31 0.23 0.12 16:ln-7 5.75 0.22 4.21 4.61 3.74 2.49 1.45 18:ln-9 33.6 59.1 21.9 29.0 35.4 42.2 48.1 18:ln-7 4.42 5.34 4.04 3.89 4.26 4.85 5.13 20:ln-9 1.45 1.56 0.97 1.26 1.30 1.25 1.33 22:ln-9 0.24 0.00 0.27 0.35 0.52 0.55 0.41 24:ln-9 0.25 0.12 0.32 0.25 0.21 0.15 0.00 E M F A 46.2 66.3 31.9 39.8 45.7 51.7 56.6 18:2n-6 (LA) 11.5 18.8 14.0 11.7 13.6 15.9 17.6 18:3n-6 0.29 0.58 0.32 0.26 0.16 0.27 0.43 20:2n-6 0.51 0.00 0.15 0.48 0.29 0.18 0.00 20:3n-6 0.53 0.00 ' 0.14 0.39 0.29 0.15 0.34 20:4n-6 (AA) 0.58 0.00 0.87 0.68 0.58 0.52 0.34 22:4n-6 0.00 0.00 0.87 0.18 0.21 0.20 0.19 En -6 P U F A 13.4 19.4 16.3 13.7 15.2 17.2 18.9 18:3n-3 (LNA) 0.98 6.20 1.34 1.19 2.58 3.89 5.04 18:4n-3 1.09 0.00 1.05 1.06 0.80 0.53 0.30 20:4n-3 0.21 0.00 0.29 0.30 0.23 0.13 0.00 20:5n-3 (EPA) 6.73 0.00 7.29 6.52 5.09 3.35 2.03 22:5n-3 0.78 0.00 0.95 0.80 0.68 0.44 0.30 22:6n-3 (DHA) 4.97 0.00 11.7 6.44 5.11 4.02 3.32 L n -3 P U F A 14.8 6.20 22.6 16.3 14.5 12.4 11.0 n-3/n-6 1.10 0.32 1.38 1.19 0.96 0.72 0.58 E P U F A 28.1 . 25.6 39.0 30.0 29.6 29.5 29.9 E E P A + D H A 11.7 0.00 19.0 13.0 10.2 7.36 5.35 79 Table 3.4: Mean (± 1SD) initial (IBW, g) and final (FBW, g) body weight, weight gam (WQ g), specific growth rate (SGR, %/day), dry feed intake (DFI, g/fish), feed efficiency (FE, g/g), protein efficiency ratio (PER, g/g), percent protein deposited (PPD, %), gross energy utilization (GEU, %), and survival (S, %) of chinook salmon in relation to diet treatment over the 30-week feeding trial. Numerical values after CO refer to the percentage of total dietary lipid comprised of canola oil . 3 Growth Diet Performance (1APF C025 C049 C072 IBW 0.77 ±0.04 0.79 ±0.01 0.81 ±0.03 0.81 ±0.01 FBW 15.2 ±0.49 15.1 ±0.48 14.9 ± 1.03 14.9± 0.58 W G 14.4 ±0.51 14.3±0.49 14.1 ± 1.03 14.1 ±0.58 SGR 1.42 ±0.04 1.40 ±0.02 1.39+ 0.01 1.39± 0.02 DFI 23.7 ±0.25 25.8 ±2.77 23.9 ±0.67 23.6 ±0.38 FE 0.61 ±0.02 0.56 ±0.05 0.59 ± 0.05 0.60 ± 0.02 PER .1.18 ± 0.10 1.09 ±0.10 1.16± 0.09 1.16 + 0.04 PPD 18.6 ±0.73 16.7 ± 1.52 17.3 ± 1.32 17.6 ±0.63 GEU 20.1 ± .77 18.5± 1.69 19.9± 1.50 20.1 ±0.71 S 92 92 93 95 a Data for each parameter (n=3) were analyzed by randomized block A N O V A . No significant differences were found between diet treatments for any of the performance parameters. 80 Table 3.5: Whole body initial (n = 3) and final mean (n = 3) concentrations (% of wet weight ± 1SD) of proximate constituents and gross energy contents (kJ/g) of spring chinook salmon in relation to diet treatment. Numerical values after CO refer to the percentage of total dietary lipid comprised of canola oil. 3 Diet Day Moisture Ash Crude protein Crude lipid Gross energy 0 81.3 ±0.27 1.80 ± 0.17 13.3 + 0.16 4.30 + 0.12 4.83 ± 0.02 dAPF 280 71.8 + 0.05 2.03 + 0.03 15.4 + 0.30 10.8 + 0.30 7.89 + 0.05 C025 280 71.8 ±0.30 2.17 + 0.09 15.3 + 0.51 10.7 + 0.30 7.86 + 0.13 C049 280 71.6 + 0.19 2.02 ± 0.05 15.2 + 0.39 11.3+0.35 8.00 + 0.30 C072 280 71.4 ±0.04 2.05 + 0.06 15.1+0.65 11.2 + 0.32 8.00 ± 0.09 a Percentages of each proximate constituent were arcsine square root-transformed before they were subjected to randomized block ANOVA. 81 Table 3.6: Final mean (± 1SD) percentages of fatty acids (g respective fatty acid/1 OOg total fatty acids) in the whole bodies of spring chinook salmon in relation to diet treatment. Numerical values after CO refer to the percentage of total dietarv lipid comprised by canola oil.3 Fatty acid dAPF C 0 2 5 Diets C 0 4 9 C 0 7 2 14:0 2.89 ± 0.14 a 2.39 ± 0.05 b 1.64 ± 0.21 c 1.10 ± 0.04 d 15:0 0.24 ± 0 . 0 6 0.20 ± 0.08 0.21 ± 0 . 1 0 0.11 ± 0 . 0 5 16:0 17.6 ± 0.43 a 15.2 ± 0.57 b 13.1 ± 1.05 0 9.2 ± 0.54 d 17:0 0.24 ± 0.01 3 0.21 ± 0 . 0 1 b 0.17 ± 0.01 c 0.13 ± 0 . 0 1 d 18:0 4.25 ± 0 . 4 3 a 3.73 ± 0.10 a b 3.37 ± 0.19 a b 3.25 ± 0 . 3 1 b 20:0 0.00 ± 0.00 d 0.16 ± 0 . 0 3 0 0.24 ± 0.02 b 0.32 ± 0 . 0 1 a 22:0 0.00 ± 0.00 b 0.00 ± 0.00 b 0.04 ± 0.03 b 0.18 ± 0.02 a DSFA 25.2 ± 0 . 7 2 a 21.9 ± 0.63 b 19.0 ± 0.26 c 14.2 ± 0.30 d 16:ln-9 0.48 ± 0.01 0.45 ± 0.03 0.44 ± 0.03 0.41 ± 0.06 16:ln-7 5.06 ± 0.28 a 3.73 ± 0.10 b 2.61 ± 0.20 c 1.80 ± 0.04 d 18:ln-9 32.4 ± 0.14 d 36.0 ± 1.44 c 40.4 ± 1.48 b 46.3 ± 0.85 a 18:ln-7 4.53 ± 0.29 4.64 ± 0 . 1 1 4.78 ± 0.25 5.12 ± 0.06 20:ln-9 1 . 4 8 ± 0.10 c 1 . 5 2 ± 0 . 1 0 b 1.69 ± 0 . 1 2 a b 1.92 ± 0.07 a 2 2 : l n - l l 0.70 ± 0 . 0 5 a 0.37 ± 0.02 b 0.28 ± 0.03 0 0.22 ± 0.02 c 22:ln-9 0 . 1 9 ± 0 . 0 1 0.23 ± 0.04 0.25 ± 0.02 0.21 ± 0.03 24:ln-9 0.46 ± 0 . 0 8 0.32 ± 0.05 0.26 ± 0.09 0.59 ± 0.20 LMFA 45.1 ± 0.74 c 47.3 ± 1.54 c 50.7 ± 1.90 b 5 6 . 7 ± 1.01 a 18:2n-6 (LA) 11.1 ± 1.24 b 13.0 ± 0.61 a b 13.6 ± 1.05 a b 15.6 ± 0 . 5 4 a 18:3n-6 0.32 ± 0.07 c 0.35 ± 0.08 c 0.50 ± 0.05 b 0.65 ± 0.02 a 20:2n-6 0.56 ± 0 . 0 3 0.47 ± 0.11 0.49 ± 0.01 0.49 ± 0.02 20:3n-6 . 0.52 ± 0 . 0 2 a b 0.48 ± 0.01 b 0.51 ± 0 . 0 3 a b 0.55 ± 0.02 a 20:4n-6 (AA) 0.66 ± 0.04 0.66 ± 0.14 0.66 ± 0.09 0.52 ± 0.05 22:4n-6 0.13 ± 0 . 0 5 b 0.27 ± 0.09 b 0.47 ± 0.01 a 0.16 ± 0.10 b Ln-6 PUFA 13.3 ± 1.39 0 15.3 ± 0.65 b c 16.3 ± 0.97 a b 18.0 ± 0.41 8 18:3n-3 (LNA) 0.92 ± 0.08 d 1.88 ± 0.07 c 2.52 ± 0.19 b 3 . 1 7 ± 0.09 a 18:4n-3 0.55 ± 0.06 0.51 ± 0 . 0 2 0.48 ± 0.05 0.52 ± 0 . 0 3 20:4n-3 0.35 ± 0.02 a 0.32 ± 0.01 a 0.23 ± 0.00 b 0 . 1 9 ± 0 . 0 2 ° 20:5n-3 (EPA) 3.02 ± 0.29 a 2.38 ± 0.15 b 1.77 ± 0.32 c 1.01 ± 0.15 d 22:5n-3 1.34 ± 0.29 a 1.07 ± 0.29 a 0.65 ± 0.09 b 0.45 ± 0.11 b 22:6n-3 (DHA) 10.2 ± 0.98 a 9 . 3 8 ± 1.30 a 8.38 ± 1.55 a 5.75 ± 1.45 b £n-3 PUFA 16.3 ± 1.44 a 15.5 ± 1.50 a 14.0 ± 1.68 a 11.1 ± 1.49 b n-3/n-6 1.24 ± 0.25 a 1.02 ± 0 . 1 4 a b 0.87 ± 0.14 b c 0.62 ± 0 . 0 1 c EPUFA 2 9 . 6 ± 0.17 30.8 ± 0.92 30.3 ± 1.05 29.1 ± 1.08 DEPA+DHA 13.2 ± 1.26 a 11.8 ± 1.26 a b 10.2 ± 1.84 b 6.76 ± 1 . 6 0 ° a Percentage data for each fatty acid (n = 3/diet treatment with each mean based on the analysis of 5 fish) were arcsine square root-transformed and then analyzed by randomized block ANOVA and where appropriate, the Tukey's Test with P = 0.05. Different superscripts within a row denote significant differences among dietary treatment means (PO.05) 82 Table 3.7: Mean (± 1SD) plasma chloride and sodium ion content (mmol/L) of spring chinook salmon in freshwater (left) and following a 24-h SW challenge test (right) at week 25 (top) and 30 (bottom) of the experiment in relation to diet treatment (n = 3/diet treatment based on 3 fish per replicate). Numerical values after CO refer to the percentage of total dietary lipid comprised by canola oil. FW SW Diet Cl Na+ Cl Na+ dAPF 117±3.14 143±4.98 158±7.39 191±4.85 C025 116±5.26 147±5.82 166±24.9 194±24.5 C049 123±10.5 146±6.07 171±18.8 199±19.0 C072 116±4.41 145±3.55 166±12.8 192±11.5 FW SW Diet Cl Na + Cl Na + dAPF 127±17.0 151±2.89 180±21.1 210±24.0 C025 117±2.91 152±3.46 165±11.4 196±9.56 C049 116±3.57 150±3.07 176±14.3 211±15.7 C072 116±2.84 155±4.38 179±17.8 211±18.8 83 Figures Figure 3.1: Relationship (± 1SD) between dietary fatty acid concentrations and whole body fatty acid concentrations of 18:2n-6, 18:3n-3, EPA (20:5n-3) and DHA (22:6n-3) in total lipids of spring chinook salmon parr fed either dAPF, C025, C049 or C072 (n = 3/diet treatment based on 15 fish per replicate). The grey line in each graph indicates the line of linearity. Standard deviations are plotted but are-within the boundaries of the data points. •a. "ST <s o •;3 I "8 so C': :•::»• .if is M / 1; >^ :i-•Cl1 -1 rxu •2- -^ -i . « B- 12 Diet <g fatty acid'IOOg total fatly at ids j 84 Figure 3.2: Post 24-hourseawater challenge survival (%) in spring chinook salmon in relation to diet treatment at 5, 10, 15, 20, 25, and 30 weeks of feeding. Numerical values after CO refer to the percentage of CO expressed in relation to the total dietary lipid content." 120 ! 85 Figure 3.3: Mean (±1 SD) whole body chloride ion content (mmol/kg) of spring chinook salmon in freshwater (top) and following a 24-hourseawater challenge (bottom) at week 5, 10, and 15 of the experiment in relation to diet treatment. Numerical values after CO refer to the percentage of total dietary lipid comprised of canola oil. a 120 100 A 80 A •= 60 A 40 20 a r b c ^ A dAPF —o- C025 — CO50 —V" C075 10 15 20 Weeks of Experiment 100 A | 80 ° 60 A 40 A 20 .1 10 15 Weeks of Experiment 20 a Whole body chloride data (n = 3/diet treatment based on 3 fish per replicate) were analyzed by one-way ANOVA and where appropriate, the Tukey's Test with P = 0.05. Different superscripts within a time interval denote significant differences among the means (P<0.05). 86 References AO AC official method 963.22. 2000. Methyl Esters of Fatly Acids in Oils and Fats. In: Horwitz, W. (Ed.), Official Methods of Analysis of AOAC International. AOAC International, Gaithersburg, MD, 1, pp. 24-26. Barlow, S., 2000. Fish meal and fish oil: sustainable feed ingredients for aquafeeds. Glob. Aquacult. Advocate 4, 85-88. Basulto, S., 1976. 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Aquacult. Res. 28, 75-83. Rosenlund, G, Obach, A., Sandberg, M G , Standal, H., Tveit, K., 2001. Effect of alternative lipid sources on long-term growth performance and quality of Atlantic salmon (Salmo salar L.). Aquacult. Res. 32 (Suppl. 1), 323-328. Salmon, N.A., 1987. Nutritional and physiological effects of NaCl on rainbow trout (Salmon gairdneri Richardson) and its application in fish culture. Ph.D. Thesis, University of Dundee, Dundee, Scotland, 370 p. Salmon, N.A., Eddy, F.B., 1988. Effect of dietary sodium chloride on growth, food intake and conversion in rainbow trout (Salmo gairdneri Richardson). Aquaculture 70, 131-144. Sargent, JR., Tocher, DR., Bell, J.G, 2002. The Lipids. In: Halver, J.E., Hardy, R.W. (Eds), Fish Nutrition. Academic Press, San Diego, pp. 181-257. Smith, N.F., Talbot, C , Eddy, F.B., 1989. Dietary salt intake and its relevance to ionic regulation in freshwater salmonids. J. Fish Biol. 35, 749-753. Steffens, W., Wirth, M. , Rennert, B., 1995. Effects of adding various oils to the diet on growth, feed conversion and chemical composition of carp (Cyprinus carpio) Arch. Anim. Nutr. 47, 381-389. Stubhaug, I., Lie, O., Torstensen, B.E., 2006. P-oxidation capacity in liver increases during parr-smolt transformation of Atlantic salmon fed vegetable oil and fish oil. J. Fish Biol. 69,504-517. Tacon, A.G.J., 2004. Use of fish meal and fish oil in aquaculture: a global perspective. Aquatic Resources, Culture and Development 1, 3-14. Takeuchi, T, Watanabe, T, 1977. Dietary levels of methyl laurate and essential fatty acid requirement of rainbow trout. Bull. Jpn. Soc. Sci. Fish. 43, 893-898. Takeuchi, T, Watanabe, T, 1982. Effects of various polyunsaturated fatty acids on growth and fatty acid compositions of rainbow trout Salmo gairdneri, coho salmon Oncorhynchus kisutch, and chum salmon Oncorhynchus keta. Bull. Jpn. Soc. Sci. Fish. 48,1745-1752. Tocher, D.R, 2003. Metabolism and functions of lipids and fatty acids in teleost fish. Reviews in Fish. Sci. 11, 107-184. Tocher, D.R, Carr, J., Sargent, J.R., 1989. Poly-unsaturated fatty-acid metabolism in fish cells: differential metabolism of (n-3) and (n-6) series acids by cultured-cells originating from a fresh-water teleost fish and from a marine teleost fish, Comp. Biochem. Physiol. 94B, 367-374. Tocher, D.R., Bell, J.G., Dick, R.J., Sargent, J.R, 1997. Fatty acyl desaturation in isolated hepatocytes from Atlantic salmon (Salmo salar): Stimulation by dietary borage oil containing y- linolenic acid. Lipids 32, 1237-1247. Tocher, D.R, Bell, J.G., Dick, R.J., Henderson, R.J., McGhee, F., Michell, D., Morris, P C , 2000. Poly unsaturated fatty acid metabolism in Atlantic salmon (Salmo salar) undergoing parr-smolt transformation and the effects of dietary linseed and rapeseed 89 oils. Fish Physiol. Biochem. 23, 59-73. Torstensen, B . E , Froyland, L , Lie, O , 2004. Replacing dietary fish oil with increasing levels of rapeseed oil and olive oil - effects on Atlantic salmon {Salmo salar L.) tissue and lipoprotein lipid composition and lipogenic enzyme activities. Aquacult. Nutr. 10, 175-192. Wagner, G N , Balfry, S.K, Higgs, D A , Lall, S P , Farrell, A.P., 2004. Dietary fatty acid composition affects the repeat swimming performance of Atlantic salmon in seawater. Comp. Biochem. Physiol. 137A, 567-576. Welker, T.L, Congleton, J.L, 2003. Relationship between dietary lipid source, oxidative stress, and the physiological response to stress in sub-yearling chinook salmon (Oncorhynchus tshawytscha). Fish Phys. Bioc. 29, 225-235. Wiegand, M . D , 1993. A study on the use of canola oil in the feed of larval goldfish, Carassius auratus L. Aquaculture and Fisheries Management. 24, 223-228. Yu, T.C, Sinnhuber, R.O, 1979. Effect of dietary co3 and co6 fatty acids on growth and feed conversion efficiency of coho salmon (Oncorhynchus kisutch). Aquaculture 16, 31-38. Zaugg, W.S, McLain, L .R , 1969. Inorganic salt effects on growth, salt water adaptation, and gill ATPase of Pacific salmon, In: Neuhaus, O.W, Halver, I E , (Eds.), Fish in Research. Academic Press, New York, pp. 293-306. Zaugg, W.S, Roley, D.D, Prentice, E.F, Gores, K X , Waknitz, F W , 1983. Increased seawater survival and contribution to the fishery of chinook salmon (Oncorhynchus tshawytscha) by supplemental dietary salt. Aquaculture 32, 183-188. 90 Chapter Four: General Discussion and Conclusions 4.1 General Discussion and Conclusions The objective of this thesis was to determine the suitability of using a non-marine source of lipid (namely canola oil) as the primary source of supplemental lipid in practical diets for Japanese red sea bream fingerlings and spring chinook salmon parr. This was achieved by the assessment of the effects of different dietary canola oil concentrations on fish growth, fatty acid metabolism and physiological development (spring chinook salmon). The studies provided a comprehensive understanding of the acute (red sea bream) and chronic (spring chinook salmon) responses of the preceding finfish species to dietary refined canola oil during early development. Furthermore, working with different model species (i.e., an anadromous and a marine species) from freshwater and marine environments, it was possible to compare and contrast the differences in their nutritional physiology. Research on alternative lipids, with a focus on those of terrestrial plant origin, has been previous studied in many salmonid species and some marine species (Bell et a l , 2001, 2002; Geurden et a l , 2005; Guillou et a l , 1995; Grant, 2006; Izquierdo et a l , 2003, 2005; Menoyo et a l , 2004; Montero et a l , 2003; Regost et a l , 2003; Rosenlund et a l , 2001). Al l of these studies have shown excellent potential for the inclusion of plant oil in finfish aquafeed production. The research conducted in this thesis, however, demonstrates for the first time, the results of using canola oil in the diets of Japanese red sea bream and spring chinook salmon. In addition, these studies showed for the first time, that it was possible for canola oil to comprise up to 70 to 72% of the total dietary lipid content of the foregoing fish species without any adverse effects on their growth provided that the remaining dietary lipid was marine fish oil in origin. The findings in this thesis partially confirmed the initial predictions, since spring chinook salmon demonstrated very high tolerance to changes in dietary fatty acid 91 composition, particularly with the dramatic increase in dietary C18 n-3 and n-6 PUFA as a result of dietary canola oil inclusion. However, it was unexpected, that we found no adverse effects of dietary canola oil concentration on the growth performance of red sea bream, as it has been well-established that marine species are much less tolerate to changes in dietary fatty acid composition (Sargent et al., 2002). Furthermore, results indicate that there may be an optimal concentration of canola oil in the diets of the red sea bream to achieve maximum growth rate and feed efficiency (i.e., 25% of the dietary lipid content). Further work however, will be necessary to confirm this possibility, as such findings have not been reported for any other marine species. Results from the red sea bream study suggest that levels of some dietary fatty acids may trigger specific fatty acid elongation and desaturation to produce A A , despite the known inactivity of the A5 and A6 desaturases in many marine finfish (Bell et al., 1994; Montero et al., 2004; Izquierdo et al., 2005). The levels of intermediate fatty acids between 18:2n-6 and A A were found consistently higher in the fish lipids than in their respective diets that contained various levels of canola oil (Table 2.3, 2.6). Preferential retention and selective incorporation of fatty acids into the cell membranes were also seen and these observations are in accord with the literature (Seliez et al., 2003). Thus, this study clearly showed that fatty acid metabolism and mobilization did differ in the whole body and polar lipids of the red sea bream (Figure 2.1). The present findings on the red sea bream may also challenge the existing EFA requirements established for this species, since growth indicators and survival of the fish were not compromised even though some of the diet treatments failed to meet the known EFA requirements of this species (Takeuchi et al., 1990, 1992). Thus, the present findings suggest that all the test diets met the required dietary EPA, DHA, and A A requirements despite the extremely high inclusion level of canola oil. Further work, however, is recommended to confirm this viewpoint. 92 The spring chinook salmon study demonstrated that this salmon species can tolerate high dietary levels of C18 n-3 and n-6 PUFA, during their freshwater residency, as a result of plant oil inclusion. Indeed, extremely high dietary inclusion of refined canola oil had no adverse effects on any of the growth indicators or on the ionoregulation during development from the fry to the parr stage of the spring chinook salmon. The present study demonstrated that these fish were capable of selectively retaining specific EFA (i.e., EPA and DHA). In addition, they exhibited the capability to modifying the abundant metabolic precursors (C18 n-3 and n-6 PUFA) into the subsequent n-3 HUFA and A A products (Figure 3.1 and Table 3.6). The high dietary tolerance of the spring chinook salmon to increased C18 n-3 and n-6 PUFA and decreased n-3 HUFA, relative to other members of the genus Oncorhynchus and even among other chinook populations, may be associated with the fatty acid composition of their natural diet (aquatic invertebrates) and their extended freshwater residency prior to ocean entry. The findings suggest that there is a need to carefully establish the EFA requirements of this particular life history of chinook salmon which are presently unknown and assumed to be similar to other Oncorhynchus species. Furthermore, it was also shown in this study that canola oil inclusion may affect other dietary components since alteration in the dietary chloride levels were noted. Dietary chloride levels accompanying the various levels of canola oil had an effect on whole body chloride content during early development in both freshwater and following the 24-h SW challenge tests (Figure 3.3). Dietary chloride did not seem to affect plasma chloride at least in the later developmental stages (Table 3.7) and had no adverse effects on the general ionoregulatory development of the spring chinook salmon in this study. Additional physiological studies on these chinook (Kuchel et a l , unpublished) have also confirmed that dietary canola oil inclusion does not adversely affect the swim performance or hypoxia tolerance. In conclusion, the findings in this thesis st5rongly suggest that refined canola oil can 93 comprise a major portion of the dietary lipid of a marine species- the red sea bream, and an anadromous salmon species- spring chinook salmon. Further chronic studies are needed to confirm the results of the present study; red sea bream that are cultured over a prolonged period, and in spring chinook salmon after seawater transfer. In regards to the latter, possible changes in fatty acid metabolism in the spring chinook salmon after smoltification may reduce the acceptable dietary level of canola oil and it will be interesting to determine whether the biological mechanisms associated with the retention and mobilization of specific fatty acids such as DHA and A A are altered. Also, it would be important to ascertain how diet treatment influences the haematological and immunological responses of the fish (red sea bream). ) 94 References Bell, J.G, Tocher, DR., MacDonald, F.M., Sargent, JR., 1994. Effects of diets rich in linoleic (18:2n - 6) and alpha-linolenic (18:3n - 3) acids on the growth, lipid class and fatty acid compositions and eicosanoid production in juvenile turbot (Scophthalmus maximusL.). Fish Physiol. Biochem. 13, 105-118. Bell, J.G, McEvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J., Sargent, J.R, 2001. Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism. J. Nutr. 131, 1535-1543. Bell, J.G, Henderson, R.J., Tocher, DR., McGhee, F., Dick, J.R, Porter, A., Smullen, R.P., Sargent, J.R, 2002. Substituting fish oil with crude palm oil in the diet of Atlantic salmon (Salmo salar) affects muscle fatty acid composition and hepatic fatty acid metabolism. J. Nutr. 132, 222-230. Geurden, I., Cuvier, A. Gondouin, E., Olsen, R.E., Ruohonen, K., Kaushik, S., Boujard, T, 2005. Rainbow trout can discriminate between feeds with different oil sources. Physiol. Behav. 82, 107-114. Guillou, A., Soucy, P., Khalil, M. , Adambounou, L., 1995. Effects of dietary vegetable and marine lipid on growth, muscle fatty acid composition and organoleptic quality of flesh of brook charr (Salvelinus fontinalis). Aquaculture 136, 351-362. Grant, A.A.M. , 2006. Growth, fatty acid composition and Na+/K+-ATPase isoform physiology of juvenile chinook salmon (Oncorhynchus tshawytscha) fed diets supplemented with anchovy or blends of anchovy and canola oil. MSc. thesis, University of British Columbia, Vancouver, Canada, pp. 35-61. Izquierdo, M.S., Obach, A., Arantzamendi, L., Montero, D., Tobaina, L., Rosenlund, G , 2003. Dietary lipid sources for seabream and seabass: growth performance, tissue composition and flesh quality. Aquacult. Nutr. 9, 397-407. Izquierdo, M.S., Montero, D., Tobaina, L., Caballero, M.J., Rosenlund, G, Gines, R , 2005. Alterations in fillet fatty acid profile and flesh quality in gilthead seabream (Sparus aurata) fed vegetable oils for a long term period. Recovery of fatty acid profiles by fish oil feeding. Aquaculture 250, 431-444. Menoyo, D., Izquierdo, M.S., Robaina, L., Gines, R , Lopez-Bote, C.J., Bautista, C.J., 2004. Adaptation of lipid metabolism, tissue composition and flesh quality in gilthead sea' bream (Sparus aurata) to the fish oil replacement by linseed and soybean oils. Brit. J. Nutr. 92,41-52. Montero, D., Kalinowski, T, Obach, A., Robaina, L., Tort, L., Caballero, M.J., Izquierdo, M.S., 2003. Vegetable lipid sources for gilthead seabream (Sparus aurata): effects on fish health. Aquaculture 225, 353-370. Montero, D., Socorro, J., Tort, L., Caballero, M.J., Robaina, L.E., Vergara, J.M., Izquierdo, M.S., 2004. Glomerulonephritis and immunosuppression associated with dietary essential fatty acid deficiency in gilthead seabream, Sparus aurata L., juveniles. J. Fish Dis. 24, 297-306. National Research Council, 1993. Nutrient Requirements of Fishes. National Academic Press. Washington, D.C., pp. 114. Regost, C , Arzel, J., Robin, J., Rosenlund, G , Kaushik, S.J., 2003. Total replacement of fish oil by soybean or linseed oil with a return to fish oil in turbot (Psetta maxima): 1. Growth performance, flesh fatty acid profile, and lipid metabolism. Aquaculture 217, 465-482. Rosenlund, G, Obach, A., Sandberg, M.G, Standal, H., Tveit, K., 2001. Effect of alternative lipid sources on long-term growth performance and quality of Atlantic salmon (Salmo 95 salar L.). Aquacult. Res. 32 (Suppl. 1), 323-328. Sargent, J.R, Tocher, D.R, Bell, J.G, 2002. The Lipids. In: Halver, J.E., Hardy, R. W. (Eds.), Fish Nutrition. Academic Press, San Diego, pp. 181-257. Seliez, I., Panserat, S., Corraze, G, Kaushik, S., Bergot, P., 2003. Cloning and nutritional regulation of a A6-desaturase-like enzyme in the marine teleost gilthead seabream (Sparus aurata). Comp. Biochem. Physiol. 135B, 449-460. Takeuchi, T., Toyota, M . , Satoh, S., Watanabe, T., 1990. Requirement of juvenile red seabream Pagrus major for eicosapentaenoic and docosahexaenoic acids. Nippon Suisan Gakkaishi 56, 1263-1269. Takeuchi, T., Shiina, Y. , Watanabe, T , 1992. Suitable levels of n-3 highly unsaturated fatty acids in diet for fingerlings of red sea bream. Nippon Suisan Gakkaishi 58, 509-514. 96 

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