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Growth, fatty acid composition and Na⁺/K⁺-ATPase isoform physiology of juvenile chinook salmon (Oncorhynchus… Grant, Amelia Anne May 2006

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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. by AMELIA ANNE MAY GRANT B.Sc. (Biology), Malaspina University-College, 2000 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR T H E DEGREE OF MASTER OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA November 2006 © Amelia Anne May Grant, 2006 A B S T R A C T This research was designed to assess the effects of alternate lipid diets on chinook salmon, Oncorhynchus tshawytscha, where canola oil (CO) was used as a vegetable lipid alternative to marine lipid. Diets supplemented with anchovy oil (AO) or CO blended with A O so that CO composed to a maximum of 41% dietary lipid content were fed to juvenile salmon twice daily. The salmon grew to a maximum of 86 grams over the study with no significant effect of diet treatment on fish growth. Similar results were obtained for an array of whole body physiological parameters including feed efficiency, hematocrit, percent muscle water and plasma Na + and CI" concentrations. Examination of whole body fatty acid composition reflected the respective dietary lipid composition. Salmon were kept in freshwater during the majority of the study and-were periodically challenged to 24 hour seawater transfers. Gill physiology was assessed in response to the transfer and in relation to dietary treatment. Gill Na +/K +-ATPase, a dominant ATP-consuming enzyme present in chloride cells of the fish gill, was used as an indicator of salinity tolerance. Na +/K +-ATPase activity was measured and found not to be significantly different across dietary groups kept in freshwater or between salinity challenged groups. In the gill, two differentially expressed Na +/K +-ATPase isoforms, a la and a lb were determined to differ in response to increased environmental salinity. Na +/K +-ATPase a la mRNA expression demonstrated sensitivity to seawater exposure and was down-regulated, while a lb mRNA expression increased in response to salinity transfer. At the end of the freshwater phase of the study, salmon were transferred to full strength seawater. At this time, Na +/K +-ATPase physiology was assessed for dietary effects. N a + / K + - A T P a s e activity did not differ between salmon fed diets based on A O or the maximum amount o f C O blended with A O . In response to increasing levels of C O in the diet, N a + / K + - A T P a s e a l a m R N A levels did not change in fish exposed to freshwater or seawater. However, as C O content increased in the diet, N a + / K + - A T P a s e a l b m R N A decreased in a linear manner. Fish fed a diet containing only marine l ipid had higher N a + / K + - A T P a s e a l b m R N A levels than those of fish ingesting the diet with the higher C O content. These data suggest that cellular fatty acids may play a role in N a + / K + -ATPase a l b expression in salmon held in freshwater. To determine the effect of time and diet on the osmoregulatory capacity o f these fish, N a + / K + - A T P a s e activity and isoform expression were analyzed using the fish fed with the two most extreme differences in fatty acid composition over 97 days. N a + / K + - A T P a s e a l b m R N A was found to be more highly expressed in fish fed a marine oil-based diet relative to those fish fed the diet with higher C O content during specific times during development. However, these molecular differences were not translated into detectable physiological differences among juvenile salmon. It is concluded that C O is a viable alternative to marine oils for juvenile chinook salmon provided essential fatty acid requirements of the salmon are met through inclusion of some marine oi l . T A B L E O F C O N T E N T S ABSTRACT » T A B L E OF CONTENTS v LIST OF TABLES • »vi LIST OF FIGURES • viii ACKNOWLEDGEMENTS » x 1.0 INTRODUCTION 1 1.1 Aquaculture and alternate lipid diets 1 1.2 Chinook salmon 4 1.3 Lipids and lipid metabolism 5 1.4 Lipids as energy 7 1.5 Lipids in membranes 8 1.6 Lipid and fatty acid requirements of fish 8 1.7 Physiology of gill Na+/K+-ATPase 12 1.8 Lipids and enzymes - protein: lipid interactions 14 1.9 Chloride cells: freshwater vs. seawater and relevance to smolting 16 1.10 Na7K+-ATPase isoforms 17 1.11 Regulation of Na+/K+-ATPase 18 1.12 Hypothesis and predictions 20 2.0 MATERIALS AND METHODS 22 2.1 Fish husbandry and feeding 22 2.2 Diet formulation 23 2.3 Sample collection and biometric analysis 24 2.4 Proximate composition analysis 25 2.5 Hematocrit 26 2.6 Muscle water content 26 2.7 RNA extraction and cDNA preparation 26 2.8 Partial cloning of chinook alb isoform 27 2.9 Na +/K +- ATPase ala and alb qPCR 28 2.10 Na +K +- ATPase activity 29 2.11 Protein quantification ; : 30 2.12 Blood plasma ion concentrations 30 2.13 Lipid extraction : 30 2.14 Fatty acid methyl esters :. 31 iv 2.15 Statistical analysis 32 3.0 RESULTS 35 3.1 EFFECT OF DIET 35 3.1.1 Diet formulation 35 3.1.2 Fish whole body fatty acid composition 36 3.1.3 Proximate analysis 37 3.1.4 Growth 37 3.1.5 Feed intake and feed utilization 38 3.1.6 Hematocrit 38 3.1.7 Muscle water content 39 3.1.8 Plasma Na+ and CI" 39 3.1.9 Mortality 40 3.1.10 Na +/K + ATPase physiology 40 3.2 EFFECT OF AGE AND ACCLIMATION (DIETS 0CO AND 40CO) 43 3.2.1 Days 0 to 97: Freshwater changes with age and acute seawater challenges 43 3.2.2 Day 97 (freshwater-reared) vs. Day 129 (seawater-acclimated) 44 4.0 DISCUSSION 45 4.1 EFFECT OF DIET 45 4.1.1 General growth and whole body physiological parameters 45 4.1.2 Gill Na +/K + physiology 52 4.2 EFFECT OF AGE AND ACCLIMATION ON Na + /K + ATPase PHYSIOLOGY 58 4.2.1 Days 0-97: Freshwater changes with age and acute seawater challenges 58 4.2.2 Day 97 (freshwater reared) vs. Day 129 (seawater acclimated) ; 59 5.0 REFERENCES 75 V ( LIST OF TABLES Section 2.0 Table 2.1 Amounts of supplemental anchovy oil (AO) and canola oil (CO) added to base pellets in the diets fed to juvenile chinook salmon 24 Table 2.2 1 Experimental set up of study. Assays listed are in addition to general growth parameters 34 Section 3.1 Table 3.1.1 Percent fatty acid compositions of dietary lipids and percentages of saturated, monounsaturated (monoenes), total n-6, total n-3 and selected n-3 highly unsaturated fatty acids (EPA and DHA) in the test diets fed to juvenile fall chinook salmon. The diets contained various supplemental amounts of canola oil (CO) by replacement of anchovy oil 62 Table 3.1.2 Percent fatty acid composition of whole body lipids of juvenile fall chinook salmon on day 129 in relation to diet treatment. Refer to Table 3.1.1 for additional information 63 Table 3.1.3 Concentrations of proximate constituents in the test diets (dry weight basis) and whole fish bodies (WB; as is basis) on day 129 in relation to diet treatment 64 Table 3.1.4 Growth performance data for fish fed diets 0CO through 40CO for 129 days 65 Table 3.1.5 Dry feed intake and feed and protein utilization of juvenile chinook salmon over 129 days in relation to diet treatment 66 Table 3.1.6 Percentages of red blood cells (hematocrit) and muscle water in juvenile fall chinook salmon during the 129-day experimental period in relation to diet treatment and water source (i.e. freshwater, FW or seawater, SW) 67 vi Table 3.1.7 Plasma ion concentrations of juvenile fall chinook salmon during the 129 day study in relation to diet treatment and water source 68 LIST OF FIGURES Section 3.1 Figure 3.1.1 Diet effects on gill Na + /K + ATPase activity (A), a l a mRNA expression (B), and alb mRNA expression (C) of fish held in freshwater (FW) and fish challenged to 24 hrs in seawater (SW) at day 97 of the feed trial. Activity data are all normalized to total protein, and mRNA expression data are normalized to a control gene, F l a . Asterisks indicate significance between freshwater and seawater conditions 69 Figure 3.1.2 Diet effects on gill Na + /K + ATPase activity (A), and a la and alb mRNA expression (B) offish challenged to 32 days in seawater. Activity data are all normalized to total protein and mRNA expression data are normalized to a control gene, E F l a . Asterisks indicate significant differences between a la and alb expression levels 70 Figure 3.1.3 Dietary effects on gill Na + /K + ATPase a la (A) and alb (B) mRNA expression offish transferred from freshwater (FW) to seawater at day 97. Data for seawater are 32 days post-transfer. Activity data are all normalized to total protein and mRNA expression data are normalized to a control gene, E F l a . Asterisks indicate significance between freshwater and seawater conditions 71 Section 3.2 Figure 3.2.1 Freshwater gill physiology of salmon fed the 0CO or 40CO diets. (A) Na + /K + ATPase activity (B) gill a la mRNA expression (C) a lb mRNA expression in fish fed the preceding diets over 97 days. Activity data are all normalized to total protein and mRNA expression data are normalized to a control gene, E F l a . Asterisks indicate significance between freshwater and seawater values 72 Figure 3.2.2Salt water gill physiology of salmon fed the 0CO or 40CO diets. (A) Na + /K + ATPase activity (B) a l a mRNA expression and (C) a lb mRNA expression. Activity data are all normalized to total protein and mRNA expression data are normalized to a control gene, E F l a 73 viii Figure 3.2.3 Effects of seawater acclimation on gill (A) Na + /K + ATPase activity (B) a la mRNA (B) alb mRNA expression in fish fed diets OCO and 40CO. Fish were held in freshwater (FW) for 97 days and then held in seawater (SW) for an additional 32 days. Activity data are all normalized to total protein and mRNA expression data are normalized to a control gene, EFlcc. Asterisks indicate significance between diet groups 74 ix A C K N O W L E D G E M E N T S I sure have a lot of people to thank for helping me through the last two and a half years of university! I entered this degree program with very little knowledge of what a Master's degree would entail, and I am not sure how I would have made it through the first year without the following people. Firstly, many thanks to my committee members, Dr. Patricia Schulte, Dr. Colin Brauner and Dr. Dave Higgs for guidance, encouragement and support. Thank you also to Dr. Jeff Richards for advice and for teaching me to use a GC. Secondly, I want to thank my many fish feeders, Shawna Vickerman, Amy Lin, Dan Baker, Susie Huang, Kim Suvajdzic and Mahsa Amirabbasi who put in countless hours meticulously feeding, siphoning and counting mushy, miniscule and unrecognizable feed pellets. A special thanks to Dan Baker for assisting me in setting up my tanks, conducting sample analysis and suggesting ways to make my chaotic sampling weeks go more smoothly. Thirdly, many individuals helped me during those busy sampling weeks. They included the fish feeders mentioned above as well as Anne Todgham, Nann Fangue, Myriam Hofmeister, Anne Dalziel, Erika Eliason, Graham Scott and Rosalind Leggat. Employees at the Department of Fisheries and Oceans also provided valuable advice, assistance with fish feed preparation or just plain exercise and entertainment. They are Janice Oakes, Nancy Richardson, and Mahmoud Rowshandeli and members of the noon-hour volleyball team. Also, thanks to Erin Friesen for great discussions, fun-filled trips to Aquanet conferences and technical advice about diets and GC analysis. Thank you also to Angela Stevenson who volunteered her time to help me with my ion analysis. And, of course, I must mention the 10,000+ Chinook salmon smolts that I had to sacrifice in order for this research to be completed. The last few years have been a lot of fun because of many friendships (new and old) and family members. I can't thank my friends and family enough for everything, from going for dinners or braving many camping and hiking trips to listening to my ramblings during countless coffee breaks. Last, but definitely not least, I would like to especially thank Daniel Mahony, my partner in life. He provided not only financial support during my degree, but unlimited amounts of emotional support. Daniel assisted me with everything from tank scrubbing to driving me back and forth from DFO and UBC to making me a cup of tea when I looked like I needed one. He also provided an invaluable amount of humor and entertainment that always grounded me and brought me back to reality when I was about to forget who I was and what my research was all about. x 1.0 INTRODUCTION 1.1 Aquaculture and alternate lipid diets Marine aquaculture contributes 15.9 million tonnes of biomass to the global fisheries market each year (FAO, 2002). Canada contributes approximately 145 thousand tonnes and its production is increasing at approximately 16.2% per year (DFO, 2004; FAO, 2002). Salmonid aquaculture constitutes 97 thousand tonnes or two-thirds of Canada's production and is becoming an increasingly important contributor to the growing fisheries industry despite uncertain culturing practices and negative public scrutiny (DFO, 2004; Lovell, 1998). Public disapproval towards salmon farming includes foreign fish escapes, competition with and impact on established wild fisheries. Salmon aquaculturists also contend with the public's perception of harm to wild fish stocks used to manufacture salmonid feeds and increased contaminant levels associated with farmed fish (Lovell, 1998). In order for sustainable aquaculture to increase its contribution to the global market, environmental and public concerns need to be addressed. My thesis specifically addresses two of the public's concerns regarding aquaculture: the impact of over-fishing on the fish species used for salmonid feeds, and the potential for increased levels of PCB's (polychlorinated biphenyls) in farmed fish. To address these issues, I examined the effects of replacing marine-derived lipids with vegetable-derived lipids in feed for juvenile fall chinook salmon. In the wild, PCBs are taken up by primary producers such as zooplankton and then are concentrated up the food web (Burreau et al, 2006). The age and size of a fish 1 has been positively correlated to PCB levels such that mature and/or larger fish tend to have increased levels of PCBs relative to immature and/or smaller fish (Thomann, 1989). In an aquaculture setting, fish may consume PCBs through the consumption of commercially-prepared feed based primarily from fishmeal and fish oil derived from the processing of wild adult fish. As a result, some aquacultured salmon have been reported to contain higher levels of PCBs than some wild salmon stocks (Carlson and Hites, 2005). Higher PCB levels can interfere with endocrine systems in wild or cultured salmon (Quabius et al, 2005). Moreover, PCBs can act as receptor agonists that increase intracellular calcium levels and they may reduce stress-related gene expression (Reynaud and Deschaux, 2006; Vaccaro et al, 2005). These effects can be harmful to the fish themselves and potentially to human consumers (Reynaud and Deschaux, 2006; Vaccaro etal, 2005). Alternate diets involving the substitution of plant and/or animal protein and lipid sources for fishmeal and fish oil can de-emphasize our present reliance on protein and lipid from wild fish stocks and address concerns regarding bioaccumulation of contaminants in farmed fish. (Bell et al, 2005). Vegetable sourced protein and oils contain very low levels of contaminants such as PCBs because plants have mechanisms to avoid PCB bioaccumulation (Donnelly and Fletcher, 1995; Satoh et al, 1998). Terrestrial plants contain cell walls for increased toxin selectivity, or they can have symbiotic relationships with PCB-degrading bacteria and fungi capable of catabolising PCBs (Donnelly and Fletcher, 1995; Donnelly et al, 1994). A potential challenge to using vegetable oils in the diets of salmon is that in general, vegetable oils do not contain high levels of highly unsaturated fatty acids of the 2 n-3 or n-6 families of fatty acids that fish require for normal growth, health and physiological functions and which are not synthesized de novo by vertebrates (Lands, 1992). Fatty acid requirements of a given fish species must be known and met before alternative diets can be developed and utilized with optimal success (Halver, 1996; Higgs et al, 2006). A vegetable oil that best meets the overall fatty acid requirements for growth of a salmonid species will be most suitable as a feed alternative. Canola oil is a potential alternative for fish-based lipids in aquaculture feeds. Canola refers to genetically-selected varieties of rapeseed Brassica napus or B: campestris (Satoh et al, 1998). Canola oil contains higher levels of fatty acids such as linoleic and linolenic acid (parent acids of the n-3 and n-6 series of fatty acids that are essential for fish species and other vertebrates) relative to other vegetable oils such as coconut and palm oils, but lower levels of linolenic acids relative to linseed oil (also known as flaxseed oil) (NRC, 1993). Coin, cottonseed, peanut, safflower, soybean and sunflower oils contain high levels of linoleic acid, but with the exception of soybean oil contain very low levels of linolenic acid (NRC, 1993). Moderate levels of both linoleic and linolenic acid make canola oil a potential alternative to marine oil in salmonid feeds, addressing both salmonid and human health concerns. Moreover, potentially harmful substances including erucic acid and glucosinolates are present in low quantities (Higgs et al, 2006). Canola oil contains low levels of erucic acid content (less than 2%) in the oil, and glucosinolate levels or anti-thyroid compounds (<30 /rniol/g of air-dry, oil-free meal) (Higgs et al., 2006). Marine fish oil supplies are predicted to become less available than canola oil by 2010 (Higgs et al, 2006). Research has suggested that global vegetable oil production is 100 times greater than that of fish oil (Caballero et al, 2002). Also, 3 vegetable oil supplementation has been predicted to be more economical than marine-sourced diets in the near future (Higgs, 2006; Menoyo et al, 2002). Numerous studies on Atlantic salmon have indicated that partial substitution of canola oil for fish oil may be viable; however, there has been comparatively less work conducted on Pacific salmonid species such as chinook salmon in this regard (Dosanjh et al, 1988; Koshio et al, 1994; Higgs et al, 1995; Dosanjh et al, 1998; Higgs et al, 2006). It is for the aforementioned reasons that canola oil was chosen as the experimental oil in the present study. 1.2 Chinook salmon Chinook salmon, Oncorhynchus tshawytscha, is an anadromous and semelparous fish species native to the Pacific coast of North America (Healey, 1991). Two small introduced runs are also present off the coast of Korea and the east coast of New Zealand (Healey, 1991). There are two races of chinook, spring and fall. Fall chinook salmon have a very variable life history (Healey, 1991). Juveniles can enter the ocean at 30 days or spend up to 14 months as a fresh water resident (FJeckman, 2003). Fall chinook salmon spend an average of 3-4 years at sea before returning to their natal rivers to spawn (Healey, 1991). While at sea, most runs migrate up the coast to the southern reaches of Alaska where they feed on marine fish such as herring (Healey, 1991). Chinook have variable ages of maturity and times of return to freshwater (Healey, 1991). The majority of fall run chinook return to the rivers in the fall (Healey, 1991). When at the spawning grounds, female chinook spawn in redds, depositing their eggs which are then fertilized 4 by a male (Healey, 1991). The role of dietary fatty acids in all aspects of chinook development and life history are not well understood. 1.3 Lipids and lipid metabolism Lipids play many diverse roles in the daily life of a cell. For instance, lipids are the primary components of cellular membranes and are a source of digestible energy and of fat-soluble vitamins (NRC, 1993). Lipids are insoluble in water and thus are involved in the selection of material entering or exiting the cell (Higgs and Dong, 2000). Lipids are composed of fatty acids and in nature there are more than 40 known fatty acids. (Higgs and Dong, 2000). The individual properties of the fatty acids are critical in determining the contribution of the lipid to local and whole cellular function. Fatty acyl chains are categorized into groups depending on the location and orientation of their double bonds (Lee, 2003). Saturated fatty acids do not contain any double bonds whereas monoenes contain only one double bond. Independent of chain length, fatty acyl chains with the first carbon double bond located 6 carbons from the terminal methyl group carbon are termed omega-6 (n-6) fatty acids while those located 3 carbons from the terminal methyl group carbon are named omega-3 (n-3) fatty acids (Lee, 2003). Fatty acid nomenclature is organized such that 18:2n-6 consists of 18 carbons with 2 double bonds and the first is located 6 carbons counting from the terminal methyl group carbon. Numerous studies have shown that n-3 fatty acids especially the highly unsaturated fatty acids (HUFA), eicosapentanoic (EPA) and docoshexanoic acid (DHA), 5 are beneficial to humans by mitigating against diseases such as Alzheimer's disease, coronary heart disease, rheumatoid arthritis and developmental disorders (La Guardia et al, 2005; Noseda, 2005). Eicosanoid derivatives of n-3 fatty acids are anti-inflammatory and thus may play cardioprotecive roles in human health (DeFilippis and Sperling, 2006). Fish remain the dominant source of n-3 FfUFA for human consumption (DeFilippis and Sperling, 2006; Morrissey, 2006). Fatty acids undergo a series of metabolic processes following ingestion. Lipid metabolism begins in the intestine. Lipases are active in the lumen of the intestine and hydrolyze fatty acid complexes into individual fatty acids which are absorbed by intestinal enterocytes (Higgs and Dong, 2000). Efficiency of absorption depends on the chain length and degree of saturation of the fatty acid (Higgs and Dong, 2000). Saturates and long chain fatty acids having decreased intestinal absorption relative to unsaturated and shorter chain fatty acids (Higgs and Dong, 2000). In the intestinal mucosa enterocytes, the fatty acids are re-esterified and modified (Higgs and Dong, 2000). Chylomicrons (complexes of lipids, proteins and cholesterol) transport lipids to the liver where fatty acid synthesis and modification by desaturases and elongases takes place (Higgs and Dong, 2000). Endogenous and exogenous fatty acids are combined to form very low density lipoproteins (VLDLs) which transport lipids to other tissues where they are hydrolyzed and taken up by cells (Higgs and Dong, 2000). Further desaturation can occur in cardiac and gonad tissue or leukocytes (Higgs and Dong, 2000). Fatty acids can then be stored by red or white muscle, used for energy in the mitochondria ((3-oxidation), or processed through golgi and endoplasmic reticulum (ER) organelles for exchange to the membrane surface (Higgs and Dong, 2000). 6 1.4 Lipids as energy Active animals such as fish use high energy-yielding lipids as a primary source of energy (Kiessling et al, 2005). Both red and white muscle contain the enzymes necessary for mitochondrial and peroxisomal jS-oxidation (Kiessling et al, 2005; Stubhaug et al, 2005). 8 -oxidation is the mechanism whereby acetyl Co-A is sequentially liberated from fatty acyl chains to enter the tricarboxylic acid (TCA) cycle producing reduced co-factors that enter the electron transport chain (ETC) which fuels ATP production (Hochachka and Somero, 2002). On a per-gram basis, red muscle has 10 times the capacity for B -oxidation. However, considering the white muscle comprises approximately 60% of the fish whole body, total oxidative contribution is dominated by the white muscle (Kiessling et al, 2005; Stubhaug et al, 2005). |3-oxidation capacity in the white muscle has been shown to increase over time in post-smolt Atlantic salmon while the capacity of the liver decreases and the activity in red muscle does not change (Stubhaug et al, 2005). j8-oxidation of fatty acids in fish tissues appears to be selective. For instance, saturated fatty acids may be oxidized at a higher rate than either monounsaturated or polyunsaturated fatty acids in the muscle offish (Stubhaug et al, 2005). Moreover, studies on /3-oxidation rates in rainbow trout red muscle have suggested that slower oxidation rates of 18:2n-6 and 18:3n-3 fatty acids may allow sparing of these essential fatty acids (Stubhaug et al, 2005; Higgs et al., 1995). On the other hand, highly unsaturated fatty acids have demonstrated increased tissue retention (and thus decreased oxidation) in Atlantic salmon white muscle (Stubhaug et al, 2005). 7 1.5 Lipids in membranes Cellular membrane properties can be affected by fatty acid composition, interactions with cholesterol, interactions with neighboring fatty acids and imbedded resident proteins/enzymes (Higgs and Dong, 2000). The number and type of double bonds present in the fatty acids allow the cell to regulate the order or phase (liquid or solid) of the membrane (Lee, 2003; Zajchowski and Robbins, 2002). For example, increased unsaturation and decreased chain length can serve to make the membrane more fluid at a given temperature. Two fatty acyl chains, in the sn-1 and sn-2 positions, are required to make up a phospholipid, the primary constituent of the plasma membrane (Hulbert et al, 2005). The sn-2 position always contains an unsaturated fatty acid and the sn-1 position varies in its composition (Hulbert et al, 2005). Cellular functions that can be affected by an unstable and dynamic membrane include the activity of membrane-bound enzymes, hormone binding and signaling, control and expression of nucleus activity and modulations of cyclooxygenase and lipoxygenase eicosanoid products (Higgs and Dong, 2000). Through these means, physiological processes can be directly regulated by lipid composition and indirectly regulated by dietary intake. 1.6 Lipid and fatty acid requirements of fish Lipids are a primary source of energy for fish (Higgs and Dong, 2000). On average, salmon require about 150 to 330 g of lipids per kilogram of feed to fuel their high-energy 8 activities (Higgs and Dong, 2000). With limited capacity for metabolic utilization of digestible carbohydrates and gluconeogenisis in salmon, greater importance is placed on lipid metabolism to meet the daily energetic requirements needed to maintain optimal physiological performance (NRC, 1993). In most fish, the parent acids of the n-6 and n-3 families, namely linoleic (18:2n--6) and linolenic (18:3n-3), a combination of 18:2n-6 and 18:3n-3 or highly unsaturated members of the n-3 and n-6 families of fatty acids are needed for optimal growth and health (Higgs and Dong, 2000). The specific dietary fatty acid needs are influenced by fish species and habitat (freshwater or marine environment), dietary lipid level and other factors (Higgs and Dong, 2006). Dietary fatty acid requirements of many fish, including salmonids, change throughout development (Sargent et al, 1999). Juvenile salmon in a freshwater environment require a diet that is higher in 18:3n-3 and possibly 18:2n-6 whereas an adult salmon living in saltwater requires a diet higher in n-3 H U F A (Sargent et al, 1999; Higgs and Dong, 2000). Regardless of developmental state, all salmonid species require the essential fatty acids linoleic (LA; 18:2n6) and linolenic (LNA; 18:3n3), and fatty acids of nutritional significance, docohexanoic acid (DHA; 22:6n3), eicosapentanoic acid (EPA; 20:5n3) and arachidonic acid (AA; 20:4n6) (Higgs and Dong, 2000). Individual levels of fatty acids required vary by species (Sargent et al, 1999). Fish cannot endogenously make L A or L N A which require delta 12 and 15 desaturases which are not present in vertebrates including fishes (Higgs et al, 1995). L N A is the precursor to EPA and D H A while L A is the precursor to A A . Consequently, L A and L N A must be obtained through dietary means in order to synthesize other nutritionally important fatty acids 9 (NRC, 1993). Salmon in freshwater have a good capacity to convert L N A to D H A and EPA whereas salmon in saltwater have a reduced capacity for bioconversion and rely more extensively on n-3 HUFAs obtained in the diet (Sargent et al, 1999; Higgs and Dong, 2000). The preceding differences in fatty acid needs of salmon in relation to their life history stage largely reflect the lipid composition of their respective prey. Parr in freshwater naturally feed on crustaceans and insects that contain increased concentrations of L A and L N A and lower levels of EPA and D H A relative to a diet obtained by adult salmon in the ocean (Higgs et al, 1995; Higgs et al., 1992; Sargent et al., 1999; Higgs and Dong, 2000). Juvenile salmonids have lower EPA and D H A requirements than adult salmonids and therefore, metabolic production of EPA and D H A is less necessary. (Sargent et al, 1999). Salmon in saltwater feed on prey items that are higher in n-3 H U F A and lower in L N A and thus the fatty acid requirements of these salmon match their prey lipid composition (NRC, 1993; Higgs and Dong, 2000). Prey abundance and prey choice will be reflected in the fatty acid make up of the predator (Menoyo et al, 2002). During the transition of chinook salmon from freshwater to saltwater, an increased amount of n-3 HUFA are required (Higgs et al, 1992). Optimum dietary ratios of n-3/n-6 fatty acids have also been suggested to produce chinook smolts of higher quality (Higgs et al, 1992). To satisfy essential fatty acid requirements, chinook salmon juveniles need about 1% L N A and 1% L A in both freshwater and seawater conditions (Higgs and Dong, 2000). Alternatively, their essential fatty acid needs can be met by n-3 10 HUFAs when they comprise 10% of the dietary lipid content (Higgs and Dong, 2000). This is considered to be true for all Oncorhynchus species (Higgs and Dong, 2000). Fatty acids not obtained through the diet may, in some cases, be obtained through metabolic means. Enzymatic modifications to fatty acids can affect the fatty acid makeup of the fish and subsequently alter dietary fatty acid requirements. Enzymes such as delta-6 and delta-5 desaturases (named for the modified carbon number) and elongases are responsible for the majority of lipid modifications (Zheng, 2005a). The genes for these enzymes have been cloned and characterized in Atlantic salmon (Hastings, 2005). Elongases are responsible for adding 2 carbons to the length of the fatty acyl chain whereas desaturases increase the degree of unsaturation by adding a carbon double bond (Hastings, 2005; Zheng, 2005a). In fish, it has been suggested that the delta 6 enzyme is rate limiting in most vertebrates (Zheng, 2005a; Williams and Burdge, 2006). N-3 and n-6 fatty acids can only be made from parent acids that are of the same series (Sargent et al, 1999). Thus, n-3 fatty acids cannot be synthesized from parent n-6 fatty acids and vice versa. To satisfy the diverse fatty acid needs of an animal, the combination of desaturases and elongases yields the synthesis of many different fatty acyl chain lengths and saturations (Higgs and Dong, 2000). For example, alternating desaturase and elongase activity is required for the conversion of L N A to EPA and D H A (Dosanjh et al, 1998). The activity of desaturases and elongases has been shown to change depending on the diet of the fish and the abiotic factors (Zheng, 2005b). In Atlantic salmon, desaturase and elongase enzymatic activity increases with increasing vegetable oil content in the diet indicating a need for fatty acid classes not provided in the diet (Zheng, 2005b). If dietary 11 n-3 fatty acid requirements are met, n-3 polyunsaturated fatty acids (PUFAs) have been shown to inhibit desaturase and elongase enzyme activities (Zheng, 2005b). Photoperiod may also influence the activity of these enzymes suggesting both a dietary and environmental regulation of fatty acid metabolism (Zheng, 2005b). An imbalance in the n-6/n-3 ratio can have a negative effect on fish health (Higgs et al, 1992; Higgs and Dong, 2000). Balancing saturated and unsaturated fatty acids is critical for maintenance of membrane fluidity and the proper function of membrane-associated proteins (Lee, 2003). For example, elevated dietary n-6 fatty acids (with adequate n-3 fatty acids) fed to Atlantic salmon can result in cardiomyopathy and a compromised immune system (Higgs and Dong, 2000). Moreover, deficient quantities of essential fatty acids such as DHA have been shown to be detrimental to retinal acuity in herring in low light and to brain function (Higgs and Dong, 2000). D H A is a very fluid fatty acid and is not condensable by cholesterol which suggests an important physiological niche for this essential fatty acid (Radhakrishnan et al, 2000; Stillwell and Wassail, 2003; Turner et al, 2003). Dynamic rearrangement of individual fatty acids such as D H A within the membrane can have diverse physiological consequences on overall cellular physiology (Stillwell and Wassail, 2003). 1.7 Physiology of gill Na +/K +-ATPase The sodium pump, or Na +/K +-ATPase, is a highly conserved membrane-associated enzyme that is rarely mutated in nature (Mobasheri, 2000). Its presence in almost all cell types across kingdoms lends support for its role in basic physiological functions 12 (Mobasheri, 2000). Maintenance of high intracellular K + and lower Na + concentrations is paramount to fundamental cellular activities such as proper protein function, cell volume regulation and solvent capacity (Hochachka and Somero, 2002). Na +/K +-ATPase, a p-type ATPase, plays an active role in ion transport (Kaplan, 2002). Na +/K +-ATPase is a large, multi-subunit enzyme present in the baso-lateral membrane of chloride cells that are found in the gill epithelium of fish (Evans et al, 2005; Kaplan, 2002). Na +/K +-ATPase plays a central role in osmoregulation by exporting 3 Na + ions out of the cell and importing 2 K + ions into the cell (Marshall, 2002; Mobasheri, 2000). In doing so, Na +/K +-ATPase establishes a local membrane potential across the gill epithelium (Kaplan, 2002). Other proteins such as Na+:K+:2C1" co-transporter (NKCC), K + and CI" channels can then operate using the established electrochemical gradient as an energy source and work in concert with Na +/K +-ATPase to maintain ion homeostasis in the fish gill (Kaplan, 2002; Mobasheri, 2000). Other functions of the Na +/K +-ATPase include pH regulation, generation of action potentials, and secondary transport of other molecules (Kaplan, 2002). The largest and most active subunit of the Na +/K +-ATPase enzyme is the a-subunit (McCormick et al, 2003). ATP binding and hydrolysis occur on the oc-subunit (Evans et al, 2005). It has been hypothesized that this enzyme uses 19-28% of a cell's energy budget (Hochachka and Somero, 2002; Hulbert et al, 2005). This subunit is found in most tissues with different alpha isoforms combining with other subunit (8 and y, discussed below) isoforms to create an enzyme with a more specialized function and with different sensitivities to ouabain, a natural inhibitor (Hirose et al, 2003; Mobasheri, 13 2000). On the other hand, other alpha subunit isoforms have been deemed housekeeping isoforms due to their ubiquitous tissue distribution (Mobasheri, 2000). Other subunits of the Na +/K +-ATPase complex include the beta and gamma subunits. The beta subunit of ATPase complex is thought to anchor the enzyme to the membrane (Hirose et al., 2003). It crosses the membrane only once and is heavily glycosylated on its extracellular domain (Kaplan, 2002). The function of the gamma subunit is largely unknown; however, it is thought to play a regulatory role, as it is not always present (Hirose et al, 2003). Na +/K +-ATPase subunits are synthesized as independent units in the ER followed by assembly into the functional enzyme (Kaplan, 2002). The beta subunit is required for normal assembly of the alpha subunit (Kaplan, 2002). The enzyme is assembled on vesicle membranes where it is then shuttled to the plasma membrane by an unknown mechanism that is thought to involve actin (Kaplan, 2002). Na +/K +-ATPase is restricted to the basolateral membrane in the fish gill and remains polarized due to the actin cytoskeleton (Mobasheri, 2000). 1.8 Lipids and enzymes - protein: lipid interactions Membrane-imbedded proteins interact with several distinct environments including the extra and intracellular environments along with the lipid environment of the membrane. The lipid environment can have dramatic effects on the function of a membrane-bound protein (Lee, 2003). The membrane can restrict or stimulate protein activity by altering the surrounding lipid composition (Lee, 2003; Zajchowski and Robbins, 2002). Evidence 1 4 suggests DHA, an essential fatty acid to all cells, can alter the function of Na +/K +-ATPase (Turner et al, 2003). The methyl end of the fatty acid has been implicated in altering Na +/K +-ATPase function (Esmann and Marsh, 2006; Turner et al, 2003). Moreover, D H A is present in high concentrations in tissues associated with high Na +/K +-ATPase activity such as retinal and muscle cells; however, the mechanism mediating this effect remains unclear (Hulbert et al, 2005). Incorporation of D H A into the membrane also has effects on rhodopsin, protein kinase C, voltage-sensitive Na + channels and Ca 2 + /Mg 2 + -ATPase (Stillwell and Wassail, 2003). Phase changes in the membrane can also affect the function of proteins. A more liquid phase, characterized by higher unsaturated fatty acid incorporation, has been suggested to increase enzymatic activity (Esmann and Marsh, 2006; Lee, 2003). In addition, fatty acyl carbon chain length can be a factor that can affect enzyme performance (Lee, 2003). A mismatch between lipid and protein transmembrane domain lengths could lead to a decreased enzyme activity, altered rate of protein domain rotation, increased energy consumption and increased strain between lipid and protein bonds (Lee, 2003). Therefore, the function of membrane-imbedded protein may be particularly susceptible to changes in the membrane environment. For example, it has been shown that ectotherms have Na +/K +-ATPase pumps that increase in activity when surrounding lipids are rich in polyunsaturated fatty acids (Turner, 2005). Moreover, reconstituted shark NaVK - ATPase enzymes have also been shown to increase in activity with surrounding phospholipids increasing in chain length (Cornelius, 2001). 15 1.9 Chloride cells: freshwater vs. seawater and relevance to smolting Salinity exerts great selective pressure on aquatic organisms such that the mechanisms involved in salinity tolerance are highly integrated (Varsamos et al, 2005). Chloride cells first appear in the integument of the yolk sac of a developing fry (Varsamos et al, 2005). In an adult, chloride cells are located on the afferent region of the gill filament and are the dominant site of osmoregulation in an anadromous fish species; however, the intestine and kidneys are also involved in whole body ion balance (Hirose et al, 2003; Perry, 1997; Wilson, 2002). Freshwater-type chloride cells facilitate gill ion absorption while the primary function of seawater chloride cells is to secrete chloride ions and co-ordinate paracellular movement of sodium ions (Hirose et al, 2003). Significant morphological and molecular differences exist between gill chloride cells when a fish is in freshwater or saltwater. In freshwater, chloride cells must compensate for ion loss through the active uptake of Ca 2 + , Na + and CI" (Hirose et al, 2003). In freshwater, ion uptake is mediated by H +-V-ATPase, C17HC03" exchanger, Na +/K +-ATPase, C a 2 + channel, and Ca 2 +-ATPase (Hirose et al, 2003; Marshall, 2002; Varsamos et al, 2005). H +-V-ATPase and Na + /K + -ATPase work to establish electrochemical gradients utilized by the other transporters (Marshall, 2002). Two types of chloride cells appear to be present in the freshwater epithelium of fish, each with subtle differences in transporter complements (Wilson, 2002). The cc-cell is thought to have higher Na +/K +-ATPase immunoreactivity while the P-cell appears to have less (Wilson, 2002). Coordinated function between these two cell types may help to explain some of the observed differences in ion transporter behavior 16 and cell morphology following fish acclimation to either freshwater or saltwater (Richards et al, 2003; Wilson, 2002). In seawater, chloride cells have different capabilities relative to freshwater chloride cells. Acclimation to seawater results in the deterioration of the [3-cell type and proliferation of the a-cell type chloride cell (Wilson, 2002). Seawater chloride cells are generally larger in size than freshwater chloride cells and are closely associated with assessory cells to facilitate ion excretion (Wilson, 2002). Current hypotheses include Na +/K +-ATPase, N K C C , and K+ and CI- channels as participants in the saltwater gill chloride cell function (Evans et al, 2005; Varsamos et al, 2005). Na +/K +-ATPase activity also increases in fish acclimated to saltwater (Kaplan, 2002). Cell membrane permeability also decreases in response to seawater acclimation (Marshall, 2002). Using these and other morphological and biochemical changes of the chloride cells, the smoltification status (seawater readiness) in anadromous fish can be identified (Wilson et al, 2002). Taken together, these data demonstrate that the Na +/K +-ATPase pump plays an important role in the gill in both freshwater and seawater environments. 1.10 Na +/K +-ATPase isoforms The Na +/K +-ATPase alpha subunit has several distinct isoforms (Bystriansky et al, 2006; Richards et al, 2003). Thus far, five a l subunits have been cloned and identified in trout: ala, alb, ale, a2, a3; however, of the five, ala, a lb have been identified as highly expressed in the gill (Richards et al, 2003). Richards et al (2003) discovered these two Na +/K +-ATPase isoforms are differentially expressed in trout gills exposed to FW, 40% 17 seawater or 80% seawater. These researchers suggested the a l a isoform was predominantly expressed in fish exposed to freshwater while, a lb was more highly expressed in fish exposed to seawater (Richards et al., 2003). In these experiments, it was found that trout exposed to freshwater had high levels of a la mRNA while a lb mRNA levels were low and remained low. After abrupt transfer to 80% seawater, a lb expression levels transiently increased for 10-15 days (Richards et al., 2003). Differences between a la are a lb isoforms may also include location of expression, regulation, kinetics or response to environmental salinity (Bystriansky et al., 2006; Richards et al., 2003) and many questions remain surrounding the differentiated function of these isoforms. 1.11 Regulation of Na +/K +-ATPase Na +/K +-ATPase is capable of being regulated on a short-term or long-term basis (Evans et al., 2005; Wagner, 2005). Research on brown trout has suggested that cAMP has an immediate inhibitory effect on Na +/K +-ATPase activity (Tipsmark and Madsen, 2001; Wagner, 2005). On the other hand, Cortisol, prolactin, growth hormone (GH) and insulin-like growth factor-1 (IGF-1) appear to exert a diverse range of effects relating to seawater adaptation in a more constant manner (Evans et al., 2005). Current models suggest Cortisol works in conjunction with G H and IGF-1 to stimulate ion secretion and increase salinity tolerance while Cortisol and prolactin work together to promote ion uptake in a freshwater environments (Evans et al, 2005; Hirose et al, 2003). Further regulation of Na +/K +-ATPase can be provided by the gamma subunit, prolactin, dopamine, norepinephrine, protein kinase A, protein kinase C, elements of the cytoskeleton, free 18 radicals and membrane-associated lipids or proteins (Evans et al, 2005; Bogdanova, 2005; Kaplan, 2002). Na +/K +-ATPase is also sensitive to ion regulation (Kaplan, 2002). An increased intracellular Na + concentration increases the pump's activity to lower Na + levels back to physiological levels (Kaplan, 2002). Cortisol is perhaps the hormone that most directly influences Na +/K +-ATPase activity. It has been previously shown that Cortisol stimulates Na +/K +-ATPase activity and is required for increased salinity tolerance in fish (Evans, 2002). Cortisol also appears to increase expression of N K C C co-transporter and contributes to increasing cell size in fish acclimated to seawater (Evans, 2002). Fish in a hypoosmotic environment may also require cortisol-mediated osmoregulation, as plasma Na + levels appear to increase in cortisol-treated killifish in freshwater (McCormick et al, 2003). Cortisol-activated processes may also require other biotic (hormones or molecules such as prolactin) or abiotic (photoperiod) factors and these work in a combined synergism to produce multiple or species-specific effects (Evans, 2002). GH is another dominant regulator of Na +/K +-ATPase activity that is thought to increase salinity tolerance in fishes (Evans, 2002; McCormick, 2006). G H effects on salinity tolerance appear to be independent of growth and not restricted to salmonids (McCormick, 2006). GH has been implicated in stimulating Na +/K +-ATPase activity as well as N K C C expression (Evans, 2002). The effects of G H and Cortisol may be synergistic, as seen in killifish where their combined effects produced greater Na + /K + -ATPase activities than either treatment alone (Evans, 2002). Na +/K +-ATPase also functions indirectly as a signaling molecule (Kaplan, 2002). Pump inhibition can alter intracellular Na + levels that can have physiological 19 consequences on the cell. Increased Na + can lead to a change in intracellular pH via the Na + /H + exchanger as well as changes in intracellular C a 2 + concentrations via theNa :Ca' exchange system (Xie and Cai, 2003). Changes in Ca can initiate other signaling cascades such as ERK and Src pathways (Kaplan, 2002; Xie and Cai, 2003). Alterations in intracellular pH can rapidly alter cellular physiology mediated by changes in protein kinetics (Kaplan, 2002). In addition, it has been suggested that a Na +/K +-ATPase signalosome composed of Na +/K +-ATPase, Src, PLC (phospholipase-C), IP3 and many other players can spatially and temporally transmit cellular signals (Xie and Cai, 2003). Xie et al, (2003) suggest Na +/K +-ATPase complexes induce effects on gene expression, cell attachment, formation of tight junctions, modification of immune responses and protein trafficking. Clearly, regulation of Na +/K +-ATPase is complex and more research needs to be conducted in order to ascertain the roles of each component in this complex system. 1.12 Hypothesis and predictions The research performed for this thesis attempts to determine the effects of partially replacing marine oil in diets for juvenile fall chinook salmon (Oncorhynchus tshawytscha) with canola oil. Six diets were formulated to contain graded amounts of canola oil. Of total dietary lipid, a maximum of 41% canola oil was supplemented with the remainder composed of anchovy oil. Reared on one of six diets, growth, feed efficiency, plasma ion concentrations, blood parameters, whole body proximate composition, gill Na +/K +-ATPase activity and gill Na +/K +-ATPase isoform expression 20 was assessed when the salmon were held in freshwater and seawater. Previous studies on fall chinook salmon have shown an increased rate of return to their natal hatchery when they were fed diets containing a blend of corn oil with herring oil rather than a diet based on supplemental herring oil or corn oil alone (Higgs et al, 1992). It was postulated that juvenile fall salmon in the current study would display increased physiological capacities that might explain the increased rate of return of the fall chinook salmon seen in the previous study. It was predicted that the increased monounsaturated content in CO-containing diets would increase digestibility and thus increase the amount of fatty acids available to the chinook salmon for growth and/or energy. Moreover, it was also predicted that osmoregulatory processes would be impacted favorably by one or more of the test blends of canola oil and anchovy oil because of the likely effects of changes in membrane lipids associated with the Na +/K +-ATPase pump. 21 2.0 M A T E R I A L S AND M E T H O D S 2.1 Fish husbandry and feeding Chinook salmon (fall run Oncorhynchus tshawytscha) were obtained from Big Tree Creek hatchery (Campbell River, BC) and kept at the Department of Fisheries and Oceans/ University of British Columbia, Center for Aquaculture and Environmental Research located at West Vancouver, British Columbia, Canada (49° 15'N, 123° 10'W). The salmon had an initial weight 11.0 grams +/- 0.2 grams. They were randomly distributed into 18 1,100-litre tanks at 400 fish per tank. Fish were kept in flow through well water at 10 liters per minute from July 28, 2004 until November 9, 2004. At the end of this period, freshwater was gradually replaced with full-strength seawater (averaged 28-30 ppt) over 4 days. Fish were exposed to saltwater until December 16, 2004. Average temperatures for fresh and saltwater phases ranged from approximately 9.7 to 12.1°C and 9.5 to 10.4 °C , respectively. Oxygen saturation ranged from 6.8 to 9.6 milligrams per liter. A randomized block design of "6 X 3", representing 6 diets (described below) and 3 replicate groups per diet, was used to assign diet type to a tank. Fish were fed to satiation twice daily at 900 and 1300 hours for 129 days (Day 0 was the first day of feeding). Satiation was determined to be the time in which salmon swum lower in the water column and surface feeding was discontinued. Uneaten pellets were siphoned from the bottom of each tank into a bucket lined with mesh (1.5mm2) and counted. Pellets consumed per tank were calculated by subtracting the number of uneaten pellets from the number of pellets given to each tank. Dry feed intake as well as mortality 22 and general health were recorded daily throughout the feeding trial. Fish were cared for according to the prescribed procedures of the Canadian Council for Animal Care (certificate #A04-0185-R003). 2.2 Diet formulation Low lipid-containing extruded pellets without any supplemental lipids were obtained from Skretting Canada (Vancouver, Canada). Base pellets were pre-made with fish meal supplying residual marine oil. Base pellets were isonitrogenous and composed of 81.53% Peruvian prime fish meal, 17.77% wheat flour, 0.47% vitamin premix and 0.23% mineral premix. Base pellets were determined to contain an average initial marine-sourced lipid content of 12.6% on a dry weight basis (DWB) and digestibility was assumed to be 95%. Lipid supplementation was performed by coating the base pellets with either canola oil (CO), anchovy oil (AO) or a combination of both oils (see Table 2.1) bringing the total lipid (DWB) to a measured amount of 19.4% among the diets. A total of 104.5 grams supplemental lipid per kilogram pellets was added. For simplicity, diets were named according to the approximate final %CO of total lipid in diet. 23 Table 2.1. Amounts of supplemental anchovy oil (AO) and canola oil (CO) added to base pellets in the diets fed to juvenile chinook salmon. Diet Intended Measured Grams lipid /kg Grams lipid/kg name ratios of ratios of feed of feed of AO:CO in AO:CO in supplemental supplemental total dietary total dietary A O CO lipid lipid OCO 100:0 100:0 104.50 0 6CO 90:10 94:6 83.60 20.90 12CO 80:20 88:12 62.70 41.80 20CO 70:30 80:20 41.80 62.70 30CO ,- 60:40 70:30 20.90 83.60 40CO 50:50 60:40 0 104.50 2.3 Sample collection and biometric analysis Three days during each interval (see Table 2.2) were required to sample all salmon. Sampling days were staggered to accommodate the workload and to standardize sampling procedures. Each tank was sampled on the last day of each interval. Salmon were deprived of food for 24 hours prior to all sampling. During each sampling period (see Table 2.2 for days), individual live body weights and fork lengths were taken from samples of fish from each tank. To accomplish this, a sample of 60 fish from each tank (n=180 for each diet treatment) was removed and placed into an aerated 30-litre basin containing 80 ppm tricainemethanesulfonate (MS-222; Syndel Laboratories Ltd., Vancouver, Canada). Salmon anesthetized under freshwater conditions were buffered with sodium bicarbonate (Sigma). One day prior to each sampling period, 10 fish were randomly removed from each tank and placed into a saltwater tank for a 24 hour seawater challenge and sampled as described below (with the exception of the final sampling period in which the fish were already in full strength seawater). On each sampling day, an 24 additional 10 fish were removed from each freshwater tank and killed by a blow to the head. Blood was taken for hematocrit analyses (see section 2.5). Blood was spun down and plasma was kept for plasma sodium and chloride analysis (see section 2.12). Gill arches 2 and 3 were removed from the left side of the body for Na + /K + - ATPase activity and mRNA expression analysis (sections 2.9 and 2.10). Gill samples were wrapped in aluminum foil and placed immediately in LN2. Finally, dorsal lateral muscle was removed for percent water analysis (see section 2.6). During final sampling (day 129), 10 fish were randomly sampled for determination of whole body proximate composition. Fish were killed using an overdose of MS-222 (approximately 150 ppm) and were immediately placed in vacuum-sealed bags and stored at -40°C pending analysis (see section 2.4). 2.4 Proximate composition analysis Proximate composition was performed on homogenized whole fish and ground experimental diets according to the procedures of Higgs et al. (2006). Briefly, percent moisture was determined by drying samples at 100°C for 16 hours in a drying oven. To determine percent ash, dried samples were subsequently burned at 600°C for 2 hours in a muffle furnace. Lipids were extracted using the chloroform/methanol (1:1 v/v) procedure according to Bligh and Dyer (1959). Protein was determined according to the Technicon industrial method no. 334-74 W7B, revised March 1977 (Technicon Industrial Systems, Tarrytown, NY, USA). Percent nitrogen was multiplied by 6.25 to obtain percentage of protein. 25 2.5 Hematocrit Fish were bled from the caudal vein using a 23-gauge needle, and blood was transferred to a heparinized hematocrit tube (VWR, Canada) and centrifuged at 3000 x g for 3 minutes. Percent red blood cell (RBC) was read using a ruler. 2.6 Muscle water content A rectangular sample of muscle was taken from below the dorsal fin and above the lateral line so as to isolate a single muscle type. The tissue was weighed and placed in a pre-weighed aluminum boat, heated for 24 hours at 100°C and then weighed again. Percent water content was calculated from these data. 2.7 RNA extraction and cDNA preparation Gill arches 2 and 3 were removed from the left side of the salmon and immediately frozen in liquid nitrogen. Samples were homogenized using 500 fil Trizol (Invitrogen, Ontario, Canada) and 1 /^l of 10 iLgJpX glycogen stock/sample. One hundred pi of chloroform were added to each homogenized sample and centrifuged at 10 000 x g for 15 minutes at 4°C. The clear phase was removed, placed in a new tube, and 250 fil isopropanol were then added. Samples were vortexed and centrifuged at 10 000 x g for 10 minutes at 4°C. Five hundred pX of fresh 75% EtOH were added to each sample. Samples were vortexed vigorously and centrifuged at 10 000 x g for 5 minutes. Pellets 26 were left to air dry and were resuspended in 50 uA RNase-free water. Three fig of RNA were used per reverse transcription reaction. RNA, Oligo (dTis) primer and water were incubated at 70°C for 10 minutes to allow primers to anneal. First strand cDNA was synthesized from 5X reaction buffer, 1 mM dNTPs, 0.5 /il RnaseGuard ™, 0.1 Revert Aid™ H Minus M-MuLV-RTase according to manufacturer's instructions (MBI Fermentas Inc., Burlington, ON, Canada). The reaction was incubated at 37°C for 1 hour and stopped by heating at 70°C for 10 minutes. Samples were stored at -30°C until further use. 2.8 Partial cloning of chinook alb isoform Chinook 3' UTR sequences were desired in order to make a comparison to the known 3' UTR rainbow trout (Oncorhynchus mykiss) sequences. Rainbow trout Na + /K + ATPase alb primers have been used previously to discriminate between ala and alb isoforms (Richards et al., 2003). To obtain chinook Na + /K + ATPase alb 3'UTR sequences, partial gill alb sequences located approximately 1000 bp upstream from the 3' UTR were obtained (Dalziel, unpublished). Alignment of chinook and rainbow trout sequences (accession number AY319390; 3087 base pairs in length) revealed conserved regions in the 1000 bp upstream area. A primer was designed using GeneTool Lite software ( targeting the 1000 bp upstream area. The forward primer (NaK) was 5' CGC C C T C A G A A T G T A C C C CCT C A 3' and the reverse primer (3' adaptor) were provided by 3' Race kit (Ambion FirstChoice, Texas, USA). Sequences were amplified with a PTC-200 MJ Research thermocycler using Tag polymerase (MBI Fermentas) and 27 cDNA isolated from the aforementioned tissues. Transcripts were amplified with a polymerase chain reaction (PCR) program consisting of: 3 minutes at 94 °C; 35 cycles at 94 °C for 30 seconds; 1 cycle at 60 °C for 30 seconds and 1 cycle of 72 °C for 1 minute and 1 cycle of 72 °C for 7 minutes. PCR products were run on a 2% gel for 1 hour and appropriate bands were extracted using a QIAquick gel extraction kit (Qiagen Inc., Mississauga, ON, Canada). Sequencing reactions consisted of 4 [i\ Big Dye 3.1 terminator mix (Applied Biosystems, California, USA), 0.3 itl NaK primer, 5 id purified PCR product and 11.7 /xl of ddFI^ O. Final products were run using an Applied Biosystems PRISM 337 automated sequencer (California, USA). Resulting chinook salmon and rainbow trout 3' UTR sequences were aligned and determined to be 100% identical in the 3' UTR regions targeted by primers used by Richards et al, (2003) for Na + /K + ATPase alb isoform amplification. Thus, primers designed for rainbow trout Na + /K + ATPase alb isoform expression analysis were also used in the current study. 2.9 Na + /K + - ATPase al a and alb qPCR The mRNA levels for Na + /K + - ATPase isoforms ala and alb were determined by real-time PCR amplification from gill cDNA and standardized to mRNA levels for EFla , a standard control gene (as seen in Richards et al., 2003). Two /xl of cDNA were used per well of each qPCR reaction. All genes were amplified with SYBR green PCR mix (Applied Biosystems, California, USA) using an ABI Prism 7000 (Applied Biosystems) Primers used were as follows: E F l a forward 5' TGC T C A C A T CGC C T G C A A 3'; E F l a reverse 5' C G G A A C G A C GGT C A A TCT TC 3'; ala forward 5' GGC C G G 28 C G A GTC C A A T 3'; ala reverse 5' G A G C A G C T G T C C A G G A T C CT 3'; alb forward 5' C T G C T A C A T CTC A A C C A A C A A C A T T 3'; alb reverse 5' C A C C A T C A C A G T GTT C A T T G G A T 3' (Richards et al, 2003). Transcripts were amplified by 1 cycle at 50 °C for 2 min; 1 cycle at 95 °C for 10 minutes; 40 cycles of 95 °C for 15 seconds and 1 cycle at 60 °C for 1 minute. Expected products were 66 and 81 bps in length, respectively. PCR results were analyzed using ABI Prism 7000 SDS Software (California, USA). 2.10 Na + K + - ATPase activity Na + /K + - ATPase activity was assessed according to McCormick (1993). Briefly, gill samples were homogenized for 15 seconds in homogenization buffer [Na-deoxycholic acid (final concentration of 0.1%) added to 250mM sucrose; lOmM EDTA-Na2; 50mM imidazole, pH 7.3]. Assay buffer was prepared in multiple steps. Firstly, a stock assay solution was made with imidazol (50 mM) buffer, pH 7.5 supplemented with 0.16 mM NADH, 2 mM phosphoenolpyruvate, 0.5 mM ATP, 3.3 U/ml lactate dehydrogenase and 3.6 U/ml pyruvate kinase. Secondly, the total volume of the stock solution was divided in half and supplemented (1:15) either with imidazole buffer or imidazole buffer containing 10.5 mM ouabain. Thirdly, to obtain a working solution, the stock solution was further diluted at a ratio of 3:1 with salt solution (50 mM imidazole; 189 mM NaCl; 10.5 mM MgCb; 45 mM KC1). To assess enzyme activity, 10 id of gill homogenate were added to a 96 well plate with 200 /il of working assay buffer with and without ouabain. Ten til of ADP standards were used to compare unknowns. N A D H depletion was observed using a 29 spectrophotometer (SpectraMAX 190; Molecular Devices, CA, USA) at 340 nm. Maximum activity was determined by choosing a time period corresponding to the steepest part of the graph. The specific maximum activity of each sample was calculated as the difference between samples incubated with and without ouabain. 2.11 Protein quantification Na + /K + - ATPase activity was standardized to total protein present in each sample. Total protein was determined using a 1:40 dilution of copper(II) sulfate and bicinchoninic acid solutions (Sigma). Each sample was developed for Vi hour at 37°C. Protein concentrations were quantified spectrophometrically at 540nm (Molecular Devices, CA, USA) 2.12 Blood plasma ion concentrations Plasma samples were diluted 1:1000 in ddHbO, and sodium ion concentration was determined using a Corning Flame Photometer 410 (New York, USA). Plasma chloride concentration was directly determined using an HB1 digital chloridometer (Haake Buehler Instruments, Inc., Saddlebrook, NJ). 2.13 Lipid extraction Total lipid was extracted from approximately 2 grams of ground feed pellets or a whole body fish sample according to Bligh and Dyer (1959). Briefly, samples were 30 homogenized in 8 ml ddFkO, 20 ml chloroform and 20 ml methanol. Each sample was vacuum filtered, and the filtrate was poured into a graduated cylinder and left to sit for a minimum of 1 hr at room temperature (RT). The methanol and water layers were aspirated and the chloroform layer was stored at -80°C in a glass vial until methyl esterification could be completed (refer to section 2.14). 2.14 Fatty acid methyl esters For fish and diet samples, 100 /xl of the chloroform layer (from lipid extraction; section 2.14) were pipetted into a test tube. Chloroform was allowed to evaporate under medical grade N2 gas (Praxair, 99%) at 30°C. To methylate samples, 1 ml of benzene (Sigma), and 200 [i\ of sodium methoxide (methylating agent; Sigma) were added to each tube. Samples were heated at 50°C for 10 minutes. Non-polar fatty acid methy esters (FAMEs) were separated out by adding 50 fi\ of glacial acetic acid (>99%, Sigma), 2.5 ml distilled water and 2.5 ml hexane (Sigma) to each tube. Samples were shaken vigorously and incubated at RT for 20 minutes to allow for phase separation. Samples were centrifuged at 4000 x g to pellet debris precipitate and the hexane layer was transferred to a new tube. Samples were dried over anhydrous sodium sulfate (Fluka, USA) and incubated for 10 minutes at RT. All hexane was transferred to a new tube and samples were completely dried under N2 gas. Lipids were resuspended in 500 (i\ of hexane and placed in a GC vial (Supelco, USA) and stored at 4°C until analysis. Fatty acid detection was carried out using a Varion model 3900 gas chromatograph (California, USA). FAMEs were separated on a Varian WCOT fused Silica column (100M x 0.25 id) and flame ionization 31 detector. An initial temperature of 140 °C was held for 5 minutes and gradually raised at a rate of 4 °C per minute to 216 °C. To further separate out C18-C20 FAMEs, the heating rate was slowed to 2 °C per minute until the column temperature was 240 °C where it was held for an additional 10 minutes. Injector and detector temperatures were set at 260 °C, and helium was used as the carrier gas. FAMEs were identified by comparison to known standards (RM-1, Marine PUFA and Supelco 37; Supelco). F A M E analysis was carried out using Galaxie Workstation software (Varion) and data are reported as a percent of total identifiable fatty acids. 2.15 Statistical analysis Random sampling of salmon was ensured by using large catch nets. Nets were lowered to the bottom of each tank and quickly raised back to the surface. These salmon were then placed in large aerated containers for biometric or tissue sampling. Growth and physiological performance variables examined were specific growth rate (SGR=[ln final weight (g)-ln initial weight (g) • number of experimental day"1]* 100), dry feed intake (DFI=total dry feed intake (g) •• fish"1), feed efficiency ratios (FER=[wet weight gained (g) • dry feed consumption (g)"1]), protein efficiency ratios (PER, [wet weight gain (g) • protein consumption (g)"']), whole body proximate composition (percent ash, moisture, protein and lipid), whole body fatty acid analysis, plasma Na+(mmol/L), plasma Cr(mmol/L), percent hematocrit and percent muscle water. Data were analyzed using an one-way A N O V A with Tukey's post hoc test (of=0.05). Where 32 appropriate, a Dunn's analysis on ranks was performed if data did not fit normality assumptions. For molecular data (NaV K +-ATPase activity, Na + / K +-ATPase a l a and alb mRNA expression), a two-way A N O V A was used with a Holm-Sidak post-hoc test or Tukey's test (a=0.05). If the data did not fit the normal assumptions of distribution, data were log transformed. 2.16 Experimental setup Due to the complex design associated with this project, the following table is shown to help clarify the experimental setup (Table 2.2). 33 Table 2.2 Experimental set up of the study. Assays listed are in addition to general growth parameters.1 Diet treatment OCO 6CO 12CO 20CO 30CO 40CO Time (primary water source) Day 0 (FW) [Na+]/[CT]* Hcrit* [Na^/fCn* Hcrit* [Na /^fCT]* Hcrit* [Na+]/[Cl-]* Hcrit* [Na+]/[Cl-]* Hcrit* [Na+]/[Cl-]* Hcrit* %MW* %MW* %MW* %MW* %MW* 1 %MW* 24 hr seawater challenge? Yes Yes Yes Yes Yes Yes Interval 1 Day 0 to 19 [Na+]/[Cr]* Hcrit* %MW* [Natter]* Hcrit* [Na^ /CCr]* Hcrit* [Na+]/[ClT* Hcrit* [Na /^fClT* Hcrit* [Na+]/[Cr]* Hcrit* %MW* (FW) alb or alaf %MW* %MW* %MW* %MW* alb or alaj 24 hr seawater challenge? ATPasef Yes Yes Yes Yes Yes ATPasef Yes Interval 2 Day 20-43 (FW) [Na /^fCT]* Hcrit* %MW* alb or alaf [Na /^fCrj* Hcrit* %MW* [Na^Cl"]* Hcrit* %MW* [Na^ /tCl"]* Hcrit* %MW* [Na^ /fCl"]* Hcrit* %MW* [Na+]/[Cl-]* Hcrit* %MW* alb or ala | 24 hr seawater challenge? ATPasef Yes Yes Yes Yes Yes ATPasef Yes Interval 3 Day 44-70 (FW) [Na+]/[Cl]* Hcrit* %MW* alb or alaf [Na^Cl"]* . Hcrit* %MW* [Na+]/[Cr]* Hcrit* %MW* [Na+]/[Cl-]* Hcrit* %MW* [Na+]/[Cl"]* Hcrit* %MW* [Na+]/[ClT* Hcrit* %MW* alb or alaf 24 hr seawater challenge? ATPasef Yes Yes Yes Yes Yes ATPasej Yes Interval 4 Day 71-97 (FW) [Na+]/[Cr]* Hcrit* %MW* alb or ala*f [Na /^fCT]* Hcrit* %MW* alb or ala* [Na+MCl]* Hcrit* %MW* alb or ala* [Na /^fCT]* Hcrit* %MW* alb or ala* [Na+]/[Cl']* Hcrit* %MW* alb or ala* [Na+]/[Cl]* Hcrit* %MW* alb or ala*| ATPase*f ATPase* ATPase* ATPase* ATPase* ATPase*f 24 hr seawater challenge? Yes Yes Yes Yes Yes Yes Interval 5 [Na+]/[Cl-]* Hcrit* %MW* [Na+]/[Cr]* Hcrit* %MW* [Na+]/[Cl-]* Hcrit* %MW* [Na+]/[Cl-]* Hcrit* %MW* [Na^/tCl-]* Hcrit* %MW* [Na+]/[Cr]* Hcrit* %MW* Day 98-129 alb or ala*f alb or ala* alb or ala* alb or ala* alb or ala* alb or ala*f (SW) ATPase*f ATPase* ATPase* ATPase* ATPase* ATPase*f PA*6 PA* PA* PA* PA* PA* FAME* 6 FAME* FAME* FAME* FAME* FAME* 24 hr seawater challenge? No No No No No No weight, length, feed intake, specific growth rate, feed efficiency ratio and protein efficiency ratio data were collected at all intervals and treatments. FW = freshwater; SW = seawater. [Na+]/[Cr]= plasma Na+and CI' levels. ATPase = gill Na+/ K+-ATPase activity, alb or ala = qPCR of gill Na7 K+-ATPase alb or ala isoform mRNA. Hcrit = blood hematocrit; %MW = % muscle water. FAME = fatty acid methyl ester analysis; PA = proximate analysis. * = data discussed in section 3.1, f = data discussed in section 3.2. 34 3.0 RESULTS 3.1 EFFECT OF DIET This section contains results pertaining to dietary effects on juvenile chinook salmon with an emphasis on feed, growth and blood parameters measured over 129 days. During the latter part of the study (day 97 and day 129), molecular indicators of salinity tolerance such as Na +/K +-ATPase activity and isoform expression are also presented. 3.1.1 Diet formulation Triplicate groups of juvenile chinook salmon were fed one of six diets for 129 days. These alternate lipid diets were formulated to meet minimum essential fatty acid requirements as well as other needs of Pacific salmon for dietary nutrients and energy as defined by the National Research Council (NRC, 1993; Higgs and Dong, 2000). Essential fatty acids such as EPA (20:5n-3) and D H A (22:6n-3) were in sufficient quantities to satisfy normal physiological functions for Pacific salmon. The dietary fatty acid compositions reflected the different supplemental amounts of CO and A O that were included in the diet and their respective fatty acid compositions (Table 3.1.1). The percentages of monounsaturated fatty acids (mainly 18:ln-9) and n-6 fatty acids (mainly 18:2n-6), were noted to increase progressively with each step-wise increment in dietary CO concentration whereas the reverse trend was seen for percentages of saturated fatty acids, total n-3 fatty acids, n-3 HUFAs and ratios of n-3:n-6 fatty acids (Table 3.1.1). The results therefore indicate that the salmon did ingest different levels of fatty acids in their 35 diets across treatment groups. Therefore, since the other factors (water quality, temperature and photoperiod) were kept constant across treatment groups, the effects of dietary change can likely be attributed to the changes in dietary fatty acid composition. In this study CO composed a maximum of 41% of the total dietary lipid content. 3.1.2 Fish whole body fatty acid composition Results for the fatty acid compositions of whole body fish lipids (sampled on day 129) are shown in relation to diet treatment in Table 3.1.2. The ingestion of diets with different concentrations of CO and A O resulted in significant differences in the fatty acid composition whole body lipid. The trends for monounsaturated and polyunsaturated (n-6 and n-3) fatty acids mirrored those seen in the dietary lipids. These percentages of CI 6:0 (palmitic acid), CI8:0 (stearic acid), C20:4n-6 (arachidonic acid), C22:5n-3 (docohexanoic acid), and n-3 HUFA were inversely related and C18:ln-9, C18:2n-6c and C18:3n-3 were directly related to dietary CO concentration (see Table 3.1.2). Indeed, diet 40CO contained 72.8% less EPA than diet OCO, and fish whole body lipids of the same 40CO treatment group contained 72.2% less EPA than those fed diet OCO. Fish on diet OCO ingested 89.8% more D H A than their counterparts fed diet 40CO; however, fish whole body lipids of those fed diet OCO contained only 51.4% more D H A as compared to fish on diet 40CO. Although present at low quantities, saturated fatty acids CI5:0, C20:0 and C22:0 as well as longer chain monoenes C22:ln-9 and C24.T did not differ significantly in content across fish fed the different diets. Likewise, C18:3n-6 content did not differ among treatment groups. These data suggest that juvenile chinook salmon 36 incorporated the majority of their dietary fatty acids into their body lipids mainly in accordance with their respective dietary concentrations. 3.1.3 Proximate analysis Concentration of dietary and whole body proximate constituents of the groups in relation to diet treatment on day 129 are shown in Table 3.1.3. Total lipid, protein, ash and moisture were analyzed. All diets contained similar levels of protein, lipid, moisture and ash, as expected. Terminal whole body moisture and ash percentages did not show significant differences due to diet treatment. Moreover, this was generally true for whole body lipid concentrations except fish fed diets 6CO and 30CO had more body lipid than those consuming diet 20CO. The protein content offish fed low CO diets (OCO, 6CO, and 12CO) generally was lower than noted for those fed higher CO diets (20CO, 30CO and 40CO). 3.1.4 Growth Data for fish wet weight, fork length, and specific growth rate (SGR) are shown in Table 3.1.4. There were no statistical differences between dietary treatments for weight or length of the chinook salmon during the 129-day feeding trial. Juvenile salmon gained approximately 70-75 grams during the trial and increased in fork length by an average of 9.0 cm. Values for SGR over the 129-day period ranged from 1.16 to 2.10 percent per day and did not differ significantly across dietary treatments. SGR peaked during interval 37 2 from day 20 to 43 of the study, and minimum SGR values were observed during the last interval of the study (days 98 to 129) when the fish were kept in saltwater. The condition factors of the fish did not differ between treatments at any point during the study (data not shown). 3.1.5 Feed intake and feed utilization Results for dry feed intake (DFI), protein efficiency ratio (PER) and feed efficiency (FE) are shown in Table 3.1.5. As expected, DFI increased during each interval of the study from approximately 3.2 grams per fish at the outset to 23 grams per fish at the end of the study. In general, dietary treatment did not significantly affect feed intake of the salmon. Overall, values for FE and PER were unaffected by diet treatment. PER, during days 71 to 97 were higher for fish fed diet 30CO than noted for fish fed diet 12CO but there was no consistent trend due to dietary CO content. 3.1.6 Hematocrit Percent RBC or hematocrit was measured immediately after each fish was removed from its respective tank (see Table 3.1.6). Overall, there was no significant difference across dietary treatments. However, during interval 3 (day 44 to 70), fish fed diet 30CO had significantly lower hematocrit than those fed diets 12CO and 20CO. Also, during days 71 to 97, differences in hematocrit were detected in seawater-challenged fish fed diets OCO, 6CO, 30CO and 40CO. However, the results did not bear a consistent relationship 38 with dietary CO content, and hence the biological significance of these differences remains unclear. Differences in hematocrit were observed between salmon held in freshwater and seawater with seawater-challenged salmon generally exhibiting consistently lower mean hematocrit values. Hematocrit values increased over time in all treatment groups whether the fish were in freshwater or seawater and this developmental trend did not appear to be related to diet treatment. 3.1.7 Muscle water content Muscle water content is a general indicator of a fish's ability to osmoregulate and maintain water balance. Overall, there were no statistical difference between the dietary treatments at each time point of measurement during the study (Table 3.1.6). Some significant differences were detected among treatments during the later intervals. However, the data were difficult to interpret due to the random nature of the differences (i.e. not linear with diet treatment). Overall, muscle water content of salmon in freshwater was greater than those of salmon challenged for 24 hours in seawater; however, data were not consistent for each treatment. Over the course of the study, muscle water content was observed to decrease. 3.1.8 Plasma Na +and CI" Plasma Na + and CI" ions were measured for each treatment group over the course of the study (Table 3.1.7). No significant differences were detected between any of the 39 treatment groups in the experiment. Over time and within each diet, plasma Na + and CI" also did not differ significantly. Plasma from the fish challenged in seawater had higher chloride values than those observed for their freshwater counterparts. On the other hand, sodium content did not differ between fish held in freshwater or challenged to seawater. 3.1.9 Mortality There were no significant differences in the fish mortality rate associated with dietary treatments at any time during the feed trial. Mortality possibly due to treatment did not exceed 0.003% (data not shown). 3.1.10 Na + /K + ATPase physiology Two time points were chosen to assess whether or not diet treatment affected the osmoregulatory performance of the fish before and after transition to seawater, a critical stage in the life cycle of an anadromous species. These time points coincided with the natural cycles of movement of the fish to seawater of local populations of fall chinook salmon . At day 97, prior to the transition to seawater, freshwater-reared and seawater-challenged fish were compared while at day 129, seawater-acclimated fish (32 days post-transfer) were assessed. All groups were analyzed for gill Na + /K + ATPase activity, N a / K ATPase a la mRNA expression and Na+/K+ ATPase a lb mRNA expression. The results for each situation are presented below. 40 3.1.10a Day 97: Salmon reared in freshwater and challenged to an acute salinity transfer • No significant difference in gill arch Na + /K + ATPase a l a and alb mRNA expression activity or isoform expression was detected and therefore, the data were pooled (data not shown). At day 97, gill Na + /K + ATPase activity did not differ across dietary treatments; between fish held in freshwater or fish challenged to seawater for 24 hours (Figure 3.1.1 A). NaTKT" ATPase mRNA levels were analyzed for both isoforms for each diet in fish reared in freshwater and challenged to seawater for 24 hours. At day 97, a la mRNA levels in freshwater gills were highest for fish fed diets OCO, 30CO and 40CO and lowest in fish fed diets 6CO, 12CO and 20CO (see Figure 3.LIB). When fish were challenged to seawater for 24 hours, a l a expression did not decline significantly as anticipated. However, on average, a l a mRNA levels in fish fed diets 30CO and 40CO (highest CO concentrations) were found to be less in fish challenged to seawater than in fish kept in freshwater, but these differences were not found to be statistically significant. Fish consuming diets 12CO and 20CO had lower alb mRNA expression levels when challenged to seawater than those fish fed all other diets (Figure 3.1.1C). There was a statistically significant negative trend between alb expression in freshwater and dietary CO. Na7K + ATPase a la mRNA expression levels displayed no statistical significance between freshwater or seawater groups across diets. However, for fish ingesting diet 40CO only, Na + /K + ATPase a lb transcript levels were significantly different between freshwater and seawater conditions. 41 3.1.10b Day 129: Seawater acclimation At day 129 (end of trial; fish acclimated to seawater only), there were no significant differences found between the maximum gill Na + /K + ATPase activities of the fish across dietary treatments (Figure 3.1.2A). Na + /K + ATPase a l a mRNA expression was lowest in fish fed diet 30CO and highest in fish fed diets 6CO and 12CO (Figure 3.1.2B). There were no significant difference detected in Na + /K + ATPase a lb expression across dietary treatments (Figure 3.1.2B). Statistical significance was found between a lb and a l a mRNA levels in fish fed diets 0CO, 30CO and 40CO, and in each case, the former isoform was higher than the latter. 3.1.10c Comparison of Na + /K + ATPase isoform expression of freshwater (day 97) and seawater- acclimated (day 129) salmon To assess whether diet treatment could affect the expression of gill Na + /K + ATPase isoforms in salmon reared in freshwater or seawater, a comparison of isoform levels was made between day 97 freshwater fish and day 129 seawater fish. In this regard, gill Na7K7 ATPase a la mRNA expression was observed to vary between treatment groups reared in freshwater (Figure 3.1.3A). However, 5 weeks after seawater transfer, it was noted that all treatment groups had down regulated a la mRNA levels to a very similar ratio of 1 (relative to EFla) . By contrast, the a lb mRNA levels were not only variable across treatment groups kept in freshwater but also they varied considerably in fish given the test diets after seawater acclimation for 5 weeks (Figure 3.1.3B). 42 3.2 EFFECT OF AGE AND ACCLIMATION (DIETS OCO AND 40CO) A more in-depth look at the physiological responses of juvenile fall chinook salmon fed diets based on fish oil or the highest concentration of CO and some fish oil was performed by assessing molecular indicators of seawater tolerance over the course of the study. In addition to growth (see section 3.1.4), gill Na + /K + ATPase activity and Na + /K + ATPase a la and a lb mRNA levels were monitored over 4 months in salmon fed the OCO and 40CO diets only. 3.2.1 Days 0 to 97: Freshwater changes with age and acute seawater challenges Over the first 97 days of the study, all groups offish were kept in freshwater. At each sampling interval, a sub sample of salmon fed diets OCO and 40CO was removed from their respective tanks and challenged to seawater for 24 hours. Gill Na + /K + ATPase activity was analyzed and no statistically significant differences were detected between the dietary treatments (Figures 3.2.1 A and 3.2.2A). Na + /K + ATPase a l a mRNA levels also did not differ significantly between diet groups in either freshwater or seawater conditions (Figures 3.2.1B and 3.2.2B). On the other hand, at days 19 and 97 (intervals 1 and 4, respectively), fish reared in freshwater and fed diet OCO had significantly higher alb mRNA levels as compared to fish fed diet 40CO (Figure 3.2.IC). Fish challenged to seawater for 24 hours did not show significant differences in gill Na + /K + ATPase a lb mRNA levels (Figure 3.2.2C). 43 3.2.2 Day 97 (freshwater-reared) vs. Day 129 (seawater-acclimated) To examine the effect of acclimation on Na + /K + ATPase physiology, a comparison was made between fish fed diets OCO and 40CO kept only in freshwater for 97 days verses those fish fed the same diets and who were acclimated to seawater at day 97 for 32 days (sampled on day 129). Significant differences were not detected in gill Na + /K + ATPase activity between the two groups for either the OCO or 40CO diet groups (Figure 3.2.3A). Gill NaVK ATPase a la mRNA expression declined in fish gills acclimated to seawater. However, there were no differences in expression patterns of Na + /K + ATPase a l a between dietary treatment groups (Figure 3.2.3B). Na + /K + ATPase a lb expression, however, was significantly different between dietary treatments in freshwater but not seawater (Figure 3.2.3C). In this regard, Na + /K + ATPase a lb mRNA levels were inversely related to dietary CO content as seen in an earlier graph (Figure 3.1.1C). 44 4.0 DISCUSSION 4.1 EFFECT OF DIET 4.1.1 General growth and whole body physiological parameters The findings of this study indicate that canola oil can comprise 41% of the dietary lipid of juvenile fall chinook salmon without any adverse consequences on fish growth, feed intake, and feed and protein utilization for 129 days provided that their essential fatty acid needs are met through the inclusion of some fish oil. No differences in growth were detected between dietary treatment groups. These findings agree with those of previous studies on Atlantic salmon (Salamo salar) (Bell et al., 2003; Bendiksen et al, 2003; Bransden et al, 2003; Dosanjh et al, 1998; Higgs et al, 2006; Menoyo, 2005) and coho salmon (Oncorhynchus kisutch) (Dosanjh et al, 1984). These studies examined the growth performance of both juvenile and adult salmon reared on a range of diets containing marine and vegetable oils including canola, sunflower, linseed and rapeseed oils. Relatively little work has been conducted on chinook salmon fed diets containing plant and/or animal lipid sources as partial replacement for fish oil. A short-term study using fall chinook salmon in which the fish were fed diets supplemented with pork lard, canola oil or herring oil singly or in various combinations of 1:1 combinations did not find any effect of diet treatment on fish growth or feed and protein utilization relative to fish fed the herring oil-supplemented control diet (Dosanjh et al, 1988). Salmon were reared in freshwater and no differences in growth were found (Dosanjh et al, 1988). 45 Moreover, in another study, chinook salmon fed combinations of salmon oil, linseed oil and beef suet did not exhibit differences in growth (Mugrditchian et al., 1981). Further, Atlantic salmon smolts fed diets with up to 100% of total lipid replacement of fish oil (unspecified type) with rapeseed oil had no differences in growth (Bell et al, 2001). Taken together, these data suggest that supplemental vegetable oils in diets for salmonids do not inhibit growth over a prolonged period (up to 6 months) provided that the diets contain adequate concentrations of essential fatty acids. Initially, it was hypothesized that a combination of canola oil with anchovy oil would improve the digestibility of the diet and possibly this would increase the growth and feed efficiency of juvenile fall chinook salmon. Anchovy oil is characterized by a greater concentrations of saturated fatty acids, lower concentration of monounsaturated fatty acids and a higher level of polyunsaturated fatty acids than present in canola oil (Higgs et al, 2006). The more unsaturated nature of canola oil including the high levels of C18:ln-9 conceivably could have improved the overall digestibility of dietary lipids (Higgs and Dong, 2000) and possibly led to improved feed efficiency. However, feed utilization was not improved in fall chinook salmon fed diets in which CO provided up to 41% of the lipid content over a prolonged period. Hence, this potentially beneficial effect of including canola oil as a replacement for anchovy oil was not observed. Diet composition can affect lipid biosynthesis in salmon (Higgs et al, 1992; Higgs and Dong, 2000). Researchers have found that dietary marine oil (unspecified) replacement by rapeseed oil resulted in compensatory desaturase and elongase activities in post-smolt Atlantic salmon (Bell et al, 2001). In an additional study, desaturase and elongase activities were found to be under nutritional regulation in Atlantic salmon livers 46 such that fish fed diets with vegetable oil (containing rapeseed, palm and linseed oils in a 3.7:2:1 ratio) had higher levels of desaturase and elongase enzymes than fish fed a diet based on fish oil (Zheng, 2005b). Sargent et al, (1999) reported that Atlantic salmon parr r fed vegetable oils (combinations of rapeseed and linseed oils) had an enhanced ability to further desaturate and chain elongate C18:2n-6 and especially C18:3n-3 to their respective C20 and C22 end products as compared to fish fed a diet based on fish oil. Vegetable oils, including canola oil can be rich in C-18 PUFA's such as C18:3n-3 and C18:2n-6 as well as the monoene C18:ln-9; however, these oils are lacking the highly unsaturated fatty acids EPA and DHA that are required for normal fish growth, feed efficiency, health and development (Zheng, 2005b). Although not addressed in the present study, research thus far suggests that salmon in freshwater are capable of tolerating vegetable-oil containing diets by utilizing compensatory mechanisms that involve desaturase and elongase enzymes which work to match the salmon's essential fatty acid requirements. The individual species capacity for desaturation and elongation will determine whether a vegetable oil-based diet would be suitable for that species or alternatively, inclusion of some n-3 H U F A would be required in the diet (Zheng, 2005b). The data from the current study suggest that fall chinook salmon pre-smolts were capable of tolerating high canola oil content in their diet but it is also noteworthy that n-3 H U F A also comprised at least 10% of the dietary lipid content to ensure that the essential fatty acid needs of the fish were being met (Higgs and Dong, 2000). Caution should still be taken when using canola oil as a major substitute for marine oils in chinook salmon diets until additional comprehensive studies are completed. While our data suggest no negative effects were associated with canola oil 47 replacement of anchovy oil up to 41% of the dietary lipid, care should be taken to ensure that over-production of arachidonic acid-derived eicosanoid compounds do not occur (Dosanjh et al, 1998; Mesa Garcia et al, 2006). Canola oil contains greater levels of linoleic acid (C18:2n-6) which can be metabolically converted to arachidonic acid which in a non-esterified form is a precursor of the pro-inflammatory eicosanoids (Dosanjh et al, 1998; Balfry and Higgs, 2001). An abundance of n-6 derived eicosanoid compounds can lead to chronic inflammation in a variety of tissues (Mesa Garcia et al, 2006). Moreover, the present study did not address effects of dietary canola oil content on adult chinook salmon. Additionally, the effect of fatty acid absorptive efficiencies has not been assessed in this study. Digestive and absorptive efficiencies of fatty acids have been shown to decrease with increasing meal size (Kinne, 1960). Other physiological parameters considered in this study including Na + , CI" and hematocrit values seem to be in general agreement with other studies on juvenile chinook salmon (Tables 3.1.6 and 3.1.7) (Alcorn et al, 2002). To my knowledge, the percent muscle water values measured in this study are a first for juvenile chinook salmon (Table 3.1.6). The values obtained for chinook salmon are higher than those reported for Atlantic salmon smolts by Bendiksen et al. (2003). When exposed to freshwater, Atlantic smolts were found to have percent muscle water values of approximately 72-74%, while values for chinook smolts ranged between 74-79% (Bendiksen et al, 2003). The reason for the discrepancy in these findings remains unclear; however these values could be species-specific. The whole body proximate composition data revealed some differences between treatment groups for lipid and protein percentages. For example, fish fed diet 6CO and 48 3 OCO had higher lipid content than those fish fed diet 20CO. Also, fish fed diet 12CO had reduced protein content relative to fish fed diets 6CO, 20CO, 3 OCO and 40CO. Consistent relationships were not found between the preceding whole body proximate constituent concentrations and dietary CO content. Hence, the biological significance of the findings remain unclear. The findings in the present study agree with previous studies which suggest that whole body lipid profiles mirror those seen in the diet (Dosanjh et al, 1998; Wagner et al, 2004). Fish fed diet OCO contained higher levels of saturated fatty acids and lower levels of monounsaturated and n-6 fatty acids, as seen in the diet. The fish also contained higher levels of n-3 HUFA, especially C20:5n-3 and C22:6n-3, which was also true for the OCO diet. Fish fed diet 40CO contained the lowest percentages of saturated and n-3 fatty acids (including EPA and DHA) while monounsaturated (eg. C18:ln-9) and n-6 fatty acids (particularly C18:2n-6) were highest; trends also observed for the dietary lipids. However, it is notable that percentages of EPA in the dietary lipids were higher than noted for D H A whereas the opposite was true for body lipids. This suggests that these may have been some bioconversion of EPA and DHA in the salmon lipids. Also, there may have been some oxidation of EPA for energy. Studies in rats have shown EPA is preferentially oxidized over D H A in purified mitochondria (Madsen, 1999). The reason for close agreement of percentages of many fatty acids between dietary and body lipids probably relates to the fact that salmon are not able to synthesize C18:2n-6 or C18:3n-3 or probably not able to rapidly convert these parent acids into highly unsaturated derivatives relative to the large daily influx of fatty acids from their respective diets. As a result, triacylglycerols, the primary form of fatty acids in fish, are composed primarily of 49 fatty acids obtained in the diet (Higgs et al, 1995). Fatty acids such as C18:3n-6, C24:l, C22:ln-9, C20:0 and C22:0 did not appear to change with dietary intake. C20:0, C22:0 and C24:l were undetectable in diet samples, but were present in fish whole body samples in low quantities (<1%). These phenomenon have been documented previously, as chinook salmon consuming a variety of supplemental dietary lipid sources (eg. canola, pork, lard and marine oil) have shown body lipid profiles similar to the fatty acid components of their dietary lipids; however, DHA percentages were higher in the body lipids than in the dietary lipids and the ranges of saturated fatty acids in body lipids was always observed to be narrower than for other unsaturated fatty acids in the diet (Dosanjh et al, 1988). It is possible that these saturated fatty acids are highly regulated within certain tissues, as they are capable of being synthesized de novo by most fish (Henderson, 1996). The present data also suggest that juvenile fall chinook salmon have a large range for utilization of most fatty acids and yet they still maintain normal physiological functions. With respect to lipid profiles, our fish were reared on diets where the extreme treatments could be considered representative of different salmon life situations. Diet OCO contained supplemental lipid that was entirely marine-sourced and thus this diet was more typical of that encountered by adult chinook salmon in the ocean, feeding on fish such as herring and sandlance (Higgs et al, 1995). Diet OCO was also similar in content to those fed to commercial farmed fish, as the protein and oil sources were marine-derived (Higgs et al, 1995; Sargent et al, 1999). On the other hand, diet 40CO contained approximately 41% of total dietary lipid as CO and accordingly this diet was higher in n-6 fatty acids and lower in n-3 fatty acids (especially EPA and DHA). In nature, juvenile 50 FW-phase salmon would normally feed on aquatic insects and crustaceans which have higher levels of C18:3n-3 and C18:2n-6 and lower levels of C20:5n-3 and C22:6n-3 than seen in anchovy or herring oils (Higgs et al, 1995; Sargent et al, 1999). Thus, diet 40CO or diets with less content of CO, had a more similar lipid profile in comparison to those insects available to juvenile fish in a stream environment than fish preyed on by adult salmon in the ocean. Stream-dwelling juvenile salmon have a different dietary lipid requirement than adult salmon residing in the ocean, possibly facilitated by the availability of different dietary fatty acids provided by their respective prey species at either the juvenile or adult life stages (Higgs et al, 1995; Lovell, 1998). As a result, possible mismatches between dietary lipid and fatty acid levels and the dietary requirements of the fish for lipids and fatty acids may result in suboptimal performance measures for juvenile salmon raised in captivity while fed diets based on fish oils that are normally encountered by adults. By this logic, results obtained from salmon fed the 40CO diet or diets containing intermediate levels of CO may be more physiologically relevant to juvenile chinook salmon when cultured. This reasoning is offered with some reservation however, since CO also contains other components (antioxidants and isoflavones) that a high n-6 diet provided by insects would not contain (Jenkins et al., 2002). However, regardless of these issues, the data presented here suggest that juvenile fall chinook salmon can tolerate a diet containing either 100% AO or up to 41% CO content (and thus 59% AO) as a lipid source. 51 4.1.2 Gill Na + /K + physiology To further pursue potential effects of alteration of dietary and fish lipid compositions, we next .turned to the biochemical level. The current study confirmed that a l a mRNA levels are, in general, higher in freshwater and decreased in seawater, while alb levels are increased during seawater exposure and decreased during freshwater exposure in salmonids (Bystriansky et al, 2006; Richards et al, 2003; Shrimpton et al, 2005). Rainbow trout abruptly transferred to 80% seawater showed a transient increase in Na + /K + ATPase crib levels with a concomitant decline in ala levels (Richards et al, 2003). Conversely, fish kept in freshwater had high ala levels and lowered alb levels (Richards et al, 2003). A study examining isoform switching in migrating sockeye salmon (Oncorhynchus nerka) showed that a la levels increase in fish moving to freshwater sources while alb mRNA levels remained the same as those seen in ocean conditions (Shrimpton et al, 2005). Moreover, isoform data obtained from Artie char (Salvelinus alpinus L.), rainbow trout (Oncorhynchus mykiss), and Atlantic salmon (Salmo salar L.) demonstrated isoform switching regardless of ascribed osmoregulatory capacity (Bystriansky et al, 2006). Members of the Oncorhynchus and Salmo genus are generally accepted to have higher osmoregulatory capacities than members of the Salvelinus genus. Rainbow trout was shown to have the highest ala levels in freshwater conditions followed by Artie char and Atlantic salmon (Bystriansky et al, 2006). Similarly, alb levels in seawater were noted to be highest in rainbow trout, followed by Artie char and then Atlantic salmon (Bystriansky et al, 2006). Our values suggest that chinook salmon have isoform levels that are distinct from the aforementioned species. 52 Chinook appear to have a l a levels in freshwater that are higher than any o f the other 3 species mentioned above, but a l b levels that are lower in seawater when compared to all 3 species at 30 days seawater exposure. The studies mentioned here looked at adult populations either from the wi ld or in captivity. Differences in expression levels in the preceding species relative to chinook salmon may be attributed to the age o f the fish, species differences, seasonal differences or rearing conditions including diet. Caution should be exercised when determining physiologically relevant N a + / K + ATPase a l b levels in cultured salmon where the diet source is unknown, as diet may influence its levels. A t day 97 (interval 4) o f the study, it was found that a l a expression did not differ between diet treatment when fish were kept in freshwater or challenged for 24 hours in seawater; however, an effect of declining a l b expression was observed in a step-wise manner with diet treatment. A s dietary C O content increased, a l b m R N A expression declined in fish exposed to F W , but not S W . These data are o f interest, as regulatory mechanisms surrounding differential N a + / K + ATPase isoform expression have not yet been elucidated. In juvenile chinook salmon, changes in the consumed fatty acid profiles may offer a potential role for regulating N a + / K + ATPase a l b isoform expression independent of a l a expression in a freshwater environment. Richards et al. (2003) speculated that amino acid sequence differences in the 5 t h transmembrane domain between a l a and other a l isoforms (including a l b ) may result in different cation binding affinities in freshwater verses a saltwater environment in rainbow trout. Differences such as these in chinook salmon may also have implications on the specific function of the isoforms; however, it remains to be seen i f interactions between surrounding fatty acid 53 chains and amino acids of transmembrane domains would translate into differences at the transcriptional level. Previous isoform research has documented a similar phenomenon whereby differential expression and activation of peroxisome proliferator-activated receptor alpha (PPARct) isoforms in mammals was determined to be facilitated in part by linoleic acid, an essential fatty acid (Kliewer et al, 1994). However, the regulatory mechanism behind this effect remains unknown. As Na + /K + ATPase a lb is the putative seawater isoform, it is unclear why this response was seen only in freshwater conditions, but it may suggest a regulatory difference between the isoforms. A gradual trend of decreased Na + /K + ATPase alb expression in freshwater as a result of increased CO in the diet was evident in groups tested at day 97 (interval 4). Moreover, lipid-mediated changes in Na + /K + ATPase a lb levels may have ecological significance for stream-dwelling juvenile chinook salmon. As previously discussed, in freshwater conditions, salmonid prey tend to be higher in L A and LNA, as reflected in our 40CO diet. It is logical to infer that diets of this nature may act to selectively decrease expression of the seawater isoform, as it is less required under freshwater conditions. This dietary influence could potentially play a role in salinity tolerance acquisition of juvenile salmon. Conversely, as changes in diet occur from insects to small fish (accompanied by an increase in DHA and EPA content) during seaward migrations, suppression of this isoform may be lifted and thereby its activity is increased as environmental salinity increases. The levels of gill Na + /K + ATPase activity observed in this study were higher than previously reported in chinook salmon reared in freshwater (Beckman, 2003). Studies have found gill Na + /K + ATPase values ranging between 4-8 /xmol PO^mg protein/hr in 54 spring chinook salmon cultured in de-chlorinated city water (Beckman, 2003). Other studies have found gill Na + /K + ATPase values ranging from 1-4 ttmol POVmg protein/hr in juvenile fall chinook salmon sampled directly from the Rogue River, Oregon (Ewing et al, 2001). The reason for the higher values seen in the juvenile fall chinook salmon of this study remain unclear, but may be attributed to differences in the techniques used, rearing temperature or seasonal effects. Environmental salinity has shown to change the V m a x of Na + /K + ATPase activity (Amaral et al, 2001). However, our acute seawater transfer Na + /K + ATPase activity values are not significantly different from samples obtained from freshwater salmon. The activity levels observed in our study may represent maximum ATPase capacity for pre-smolt chinook salmon and that maximum enzyme capacity was already attained prior to testing in the fish exposed to freshwater and seawater conditions. A lack of increased Na + /K + ATPase activity in chinook salmon in response to seawater also suggests that the enzymes were already working at maximum activity and that the salmon were pre-prepared for salinity exposure. However, a 24 hour salinity transfer may not represent sufficient time to detect increases in activity. Detectable increases in Na + /K + ATPase activity has previously only been observed after 24 hours post-transfer in Atlantic salmon and up to 11 days post-transfer reported in other salmonids. (Bystriansky et al, 2006; Richards ef .al., 2003; D'Cotta et al, 2000; Madsen et al, 1995). Delays between sodium pump expression and increases in activity have been attributed to de novo synthesis of the sodium pump (Bystriansky et al, 2006; Richards et al., 2003). It is also possible that initial salinity tolerance was achieved by other means including the compensatory activity of other enzymes, suggesting the sodium pump isn't the rate limiting enzyme after first exposure to SW. Moreover, 55 changes in percent contribution by the Na + /K + ATPase isoforms or changes in percent contribution of M R C cell types which are thought to contain different enzyme compliments has yet to be explored. Evidence suggests that several unsaturated fatty acids play a key role in Na + /K + ATPase activity modulation. Bystriansky and Ballantyne (2006) found Na + /K + ATPase activity was positively correlated to the amount of 18:2n-6 (LA) in the basal lateral membrane (BLM) of mitochondrial rich (MCR) cells in the gill of Arctic Char. Na + /K + ATPase isoform mRNA levels were also assessed; however, levels were not correlated to any particular fatty acid component in the B L M (Bystriansky and Ballantyne, 2006). There is evidence that D H A has direct effects on Na + /K + ATPase activity (Hulbert et al, 2005; McKenzie, 2001; Wu et al, 2001). Within the cell, it has been shown that unsaturated fatty acids (primarily from the n-3 series including DHA) have been selected for in the phospholipid fraction in fish (Higgs and Dong, 2000). Experiments using molecular dynamic simulations and proton leak experiments show that the methyl end of the D H A molecule may interact with the Na + /K + ATPase pump and increase its activity (Brookes et al, 1998; Stillwell et al, 1997; Turner et al, 2003). Moreover, interactions between phospholipids and the Na + /K + ATPase pump have been documented in mammals. Na + /K + ATPase in the cellular membranes of brain tissue in weaned and adult rats fed diets containing fish oils (high in D H A and EPA) demonstrated sensitivity to ouabain relative to membranes isolated from rats fed a standard laboratory diet (DHA and EPA) (Gerbi et al, 1994). Our whole body fish samples contained D H A content ranging from 13.73% (diet OCO) to 7.06% (40CO); almost a 2 fold difference in content; however, we were unable to detect differences in Na + /K + ATPase activity. The 56 mechanism linking membrane D H A content and Na + /K + ATPase activity modulation remains unknown. 57 4.2 EFFECT OF AGE AND ACCLIMATION ON Na + /K f ATPase PHYSIOLOGY 4.2.1 Days 0-97: Freshwater changes with age and acute seawater challenges Following the finding that gill Na + /K + ATPase a lb expression levels are diet dependent, a more in-depth analysis of Na + /K + ATPase isoform expression was performed. Given that the two extreme dietary treatments (OCO and 40CO) yielded the greatest differences in a lb expression at week 15 (Figure 3.1.1C), similar time-related trends may be reflected in samples taken at 3, 7,11 and 15 weeks. qPCR was performed on gill samples from freshwater salmon and challenged to 24 hours in SW. These data revealed that at only weeks 3 and 15, gill Na + /K + ATPase a lb mRNA levels were higher in the OCO fed salmon relative to the 40CO fed salmon. This effect was not seen at weeks 7 or 11. Moreover, this effect was seen only in salmon held in freshwater and not in salmon challenged to 24 hours of seawater (Figure 3.2. IC and 3.2.2C). Changes in a lb expression during intervals 1 and 4 could signal crucial developmental stages involving lipid turnover where Na + /K + ATPase performance was most affected. Na + /K + ATPase ala and alb isoforms have been suggested to be differentially regulated in response to seawater exposure in rainbow trout (Richards et al, 2003). Richards et al (2003) found that at 80% seawater, alb mRNA was up regulated relative to freshwater values within 24 hours. Na + /K + ATPase alb expression was transient, with significant expression seen after 24 hours, reaching maximum levels at 3 days and declining to pre-transfer levels at 15 days (Richards et al, 2003). Analysis of these isoforms under these conditions has not been conducted before on juvenile chinook 58 salmon. Therefore, these data are novel. However, a 24 hour seawater challenge may not be a sufficient time to accurately determine expression levels of the isoforms in chinook salmon and a more thorough examination of this effect would be necessary to conclude that this effect is FW-specific. A concomitant decline in Na + /K + ATPase activity was not seen with a decline in Na + /K + ATPase alb mRNA levels; however, current Na + /K + ATPase activity assays can not delineate between isoform contributions. A compensatory change in a l a mRNA levels was also not seen as a means of explaining why a lb may be differentially expressed in fish ingesting the OCO and 40CO diets. 4.2.2 Day 97 (freshwater reared) vs. Day 129 (seawater acclimated) Gill Na + /K + ATPase physiology in salmon held in freshwater for 97 days were compared to salmon acclimated to seawater for an additional 32 days. Physiological responses to long term acclimation can be significantly different from short term challenges. Na + /K + ATPase a la mRNA transcript levels were significantly lowered in salmon given both diet treatments when acclimated to seawater relative to the freshwater groups (Figure 3.2.3). This finding is expected, as a l a is the putative freshwater isoform and was expected to remain depressed during seawater exposure. Na + /K + ATPase a lb mRNA levels were not significantly different between diet or acclimation groups, alb mRNA expression has previously found to be transient, with peak expression occurring at 5 days (Richards et al, 2003). The sampling schedule did not sample at these times, and most likely the peak alb expression was missed. At this time, it can only be concluded 59 that a lb mRNA was not constitutively expressed at high levels in seawater-acclimated fall chinook salmon (Figure 3 .2.3C). 60 4.3 G E N E R A L CONCLUSIONS Taken together, the data from this study indicate that dietary CO content had no adverse effects on the growth, feed intake, feed efficiency, hematocrit, or Na + /K + ATPase activity of juvenile fall chinook salmon and therefore, CO may comprise 41% of the dietary lipid content provided that the essential fatty acid requirements of the fish are met by some fish oil included in the diet. The chinook salmon can utilize a wide range of available fatty acids, as the majority of whole body fatty acid levels mirrored those found in the diet without any apparent ill-effects on the salmon. Gill Na + /K + ATPase a lb mRNA levels were found to be diet-dependent. In salmon reared in FW, Na + /K + ATPase a lb levels declined with increasing CO content (and consequently decreasing A O content) in the diet. These effects were not seen in seawater conditions nor were they seen in gill Na + /K + ATPase a l a levels. Cellular lipid content may influence a lb mRNA expression under these conditions. Further research is recommended to elucidate the mechanisms involved in a lb mRNA modulation. 61 Table 3.1.1. Percent fatty acid compositions of dietary lipids and percentages of saturated, monounsaturated (monoenes), total n-6, total n-3 and selected n-3 highly unsaturated fatty acids ( E P A and D H A ) in the test diets fed to juvenile fall chinook salmon. The diets contained various supplemental amounts of canola o i l (CO) by replacement of anchovy oi l . Fatty acid OCO 6CO 12CO 20CO 30CO 40CO C14:0 6.74 6.20 5.81 4.78 1.75 1.90 C15:0 0.16 0.03 0.00 0.00 0.00 0.00 . C16:0 22.86 21.64 19.76 18.55 14.57 13.08 C18:0 4.80 4.76 4.31 4.45 3.72 3.64 Total saturated 34.56 32.63 29.88 27.78 20.04 18.62 C16:ln-7c 8.22 7.09 5.90 4.97 3.77 2.75 C18:ln-9 15.36 22.64 27.25 30.98 40.90 47.67 C18:ln-7 2.16 3.22 3.25 3.19 3.20 3.22 C20:ln-9 1.25 1.12 0.09 0.18 1.04 0.16 C22:ln-9 0.39 0.00 0.00 0.00 0.00 0.00 Total monoene 27.38 34.07 36.49 39.32 .48.91 53.8 C18:2n-6t 0.08 0.03 0.05 0.00 0.16 0.09 C18:2n-6c 5.89 8.58 8.75 10.50 14.63 16.81 C18:3n-6 0.11 0.03 0.00 0.05 0.27 0.24 C20:4n-6 0.55 0.00 0.07 0.00 0.00 0.00 Total n-6 6.63 8.64 8.87 10.55 15.06 17.14 C18:3n-3 1.00 0.99 2.29 2.86 3.88 4.67 C20:3n-3c 0.73 0.12 0.07 0.08 0.14 0.02 C20:5n-3 16.23 13.48 . 12.04 10.18 6.29 4.41 C22:5n-3 1.18 0.16 0.11 0.10 0.13 0.00 C22:6n-3 12.06 9.78 10.10 8.96 5.40 1.22 Total n-3 31.2 24.53 24.61 22.18 15.84 10.32 n-3/n-6 4.71 2.84 2.77 2.10 1.05 0.60 Total HUFA 28.29 23.26 22.14 19.14 11.69 5.63 to Table 3.1.2. Percent fatty acid composition o f whole body lipids of juvenile fall chinook salmon on day 129 in relation to diet treatment. Refer to Table 3.1.1 for additional information. 1 Fatty acid OCO 6CO 12CO 20CO 30CO 40CO Significance C14:0 4.69±0.92 a 5.45i0.15a 4.24±0.39 a b 3.48i0.35ab 3.10i0.12ab 1.87i0.20b P<0.05 C15:0 0.27±0.07 0.22i0.07 0.22i0.05 0.10i0.04 0.15i0.04 0.07i0.02 ns C16:0 21.46±0.29 a 20.48±0.29 a b 18.56i0.31c 16.77i0.36d 15.09i0.13e 13.17i0.24f PO.05 C17:0 0.31±0.10 a 0.17i0.09ab 0.25i0.08ab 0.06i0.05b 0.12i0.09ab 0.11i0.04ab P<0.05 C18:0 5.12±0.09 a 4.81i0.06b 4.63i0.10bc 4.31i0.07d 4.07i0.06de 3.91i0.03ef P<0.05 C20:0 0.06±0.03 0.02i0.02 0.02i0.01 O.OliO.OO O.OliO.Ol 0.05i0.02 ns C22:0 0.07±0.02 0.06i0.03 0;55i0.50 0.09i0.03 0.15i0.08 0.19i0.03 ns Total Saturated 31.98il.161 31.21±0.31 a 28.47i0.68a 24.82i0.66ab 22.69i0.07ab 19.37i0.38b PO.05 C16:ln-7c 8.38i0.34a 7.49i0.12b 6.36i0.14c 5.17i0.12d 4.32i0.0.10e 3.27i0.07f PO.05 C18:ln-9 19.43i0.49a 25.00±0.28 b 29.78i0.22c 34.94i0.63d 38.51i0.40e 43.39i0.33f PO.001 C18:ln-7 3.72i0.08a 3.50±0.05 a b 3.54i0.08ab 3.42i0.06b 3.23^.09^ 3.18i0.03c PO.05 C20:ln-9 1.38i0.05ab 1.32i0.03a 1.43i0.04ab 1.37±0.06a b 1.25i0.31ab 1.58i0.05b PO.05 C22:ln-9 0.03i0.02 0.02i0.01 0.04i0.01 0.02i0.01 0.09i0.06 0.06i0.01 ns C24:l 0.04i0.02 O.OliO.Ol 0.02i0.01 O.OliO.Ol O.OliO.Ol 0.00 ns Total Monoene 32.98i0.98a 37.34i0.40b 41.17i0.33c 44.93i0.65de 47.41i0.22e 51.48i0.36f PO.001 C18:2n-6t 0.16±0.03 a 0.15±0.05 a 0.39i0.05ab 0.41i0.08ab 0.58i0.04ab 0.67i0.04b PO.05 C18:2n-6c 5.80i0.14a 7.54i0.14b 9.14±0.17 c 10.79i0.13d 11.91i0.25e 13.26i0.07f PO.001 C18:3n-6 0.64i0.09 0.43i0.09 0.49i0.12 0.42i0.10 0.69i0.09 0.70i0.05 ns C20:4n-6 0.77i0.08a 0.54i0.09ab 0.59i0.03ab 0.33i0.08b 0.28i0.13b 0.29i0.04b PO.01 Total n-6 7.37i0.12a 8.66±0.25b 10.61i0.19c 11.95i0.21d 13.46i0.17e 14.92i0.07f PO.001 C18:3n-3 l'.13i0.03a 1.51i0.13a 2.24i0.04a 2.50i0.22ab 3.06i0.07ab 3.42i0.02b PO.05 C20:5n-3 9.71i0.32a 7.73±0.13 b 6.05i0.20c 5.19i0.10dc 3.30i0.54ef 2.80i0.15f PO.001 C22:5n-3 3.00±0.11 a 2.13i0.25a 1.49i0.30ab 1.36i0.13ab 1.28i0.03ab 0.73i0.08b PO.05 C22:6n-3 13.73i0.53a 11.34i0.32c 9.83i0.53bc 9.13i0.70b 8.59i0.13bd 7.06i0.19d PO.05 Total n-3 27.57i0.93a 22.71i0.65b 19.61±0.76 c d e 18.18i0.76de 16.23i0.43ef 14.01i0.25f PO.05 Total HUFA 23.44i0.81a 19.07i0.41b 15.88±0.66 c 14.32i0.65cd 11.89i0.46de 9.86i0.24ef PO.01 n-3/n-6 3.74i0.09a 2.64i0.12b 1.85i0.09c 1.52i0.05de 1.21i0.05ef 0.94i0.02f PO.05 Values are ± SEM (n values range from 3-11). Significance between diets is indicated with superscripts. O N Table 3.1.3 Concentrations o f proximate constituents in the test diets (dry weight basis) and whole fish bodies ( W B ; as is basis) on 129 in relation to diet treatment.1 Proximate Constituent Diet OCO 6CO 12CO 20CO 30CO 40CO Significance Diet - Lipid (%) 19.10±0.60 18.59±0.93 18.66±0.44 18.53±1.78 18.83±3.27 20.75-1.22 NA Diet - Protein (%) 57.50±0.53 55.98±2.21 57.74±0.14 57.88-1.16 56.39±0.89 57.28±0.16 NA Diet - Ash (%) 12.90±0.00 12.95±0.12 12.80±0.19 12.90±0.06 12.86±0.14 12.82±0.08 NA WB - Lipid (%) 8.60±0.13ab 8.79±0.14b 8.42±0.17ab 8.18±0.15a 9.01±0.17b 8.76±0.14ab PO.05 WB - Protein (%) 16.39±0.17ac 16.85±0.07cd 15.89±0.17a 17.68±0.34b 17.34±0.17bd 18.10±0.31b PO.05 WB - Moisture (%) 72.92±0.14 72.56±0.13 72.78±0.17 73.11_0.16 73.00±0.21 72.57±0.14 ns WB - Ash (%) 2.13±0.04 2.22±0.02 2.26±0.04 2.21±0.04 2.15±0.05 2.28±0.04 ns WB values are ± SEM representing 3 replicate tanks per diet. Proximate constituents for diet (n=2) are represented ± the range between the two replicates. Significance between fish whole bodies is indicated with superscripts. 4^ Table 3.1.4. Growth performance data for fish fed diets OCO through 40CO for 129 days. 1 Diet OCO 6CO 12CO 20CO 30CO 40CO Significance DayO Length (cm) 9.8±0.1 9.9±0.1 9.9±0.1 9.9±0.1 9.8±0.1 9.9±0.1 ns Live weight (g) 10.6±0.2 11.1±0.3 11.1±0.3 11.2±0.3 10.9±0.3 11.1±0.2 ns Interval 1 - Day 0 to 19 Length (cm) 10.7±0.06 10.8±0.05 10.8±0.05 10.9±0.06 10.8±0.06 10.7±0.07 ns Live weight (g) 14.0±0.3 14.9±0.3 14.5±0.3 15.08±0.3 14.7±0.3 14.3±0.3 ns SGR 2 1.5±0.03 1.5±0.07 1.4±0.01 1.6±0.08 1.6±0.10 1.4±0.13 ns Interval 2 - Day 20-43 Length (cm) 12.3±0.08 12.6±0.09 12.5±0.08 12.5±0.09 12.4±0.08 12.4±0.11 ' ns Live weight (g) 23.0±0.4 24.8±0.5 24.2±0.5 24.8±0.5 23.9±0.5 23.2±0.5 ns SGR 2.0±0.05 2.1±0.06 2.1±0.03 2.1±0.07 2.0±0.13 2.0±0.05 ns Interval 3 - Day 44-70 Length (cm) 14.3±0.08 14.8±0.08 14.2±0.08 14.3±0.10 14.3±0.08 14.5±0.08 ns Live weight (g) 39.0±0.73 40.9±0.72 38.8±0.72 39.6±0.76 37.7±0.65 38.4±0.72 ns SGR 1.96±0.03 1.84±0.11 1.72±0.04 1.72±0.06 1.69±0.06 1.9±0.07 ns Interval 4 - Day 71-97 Length (cm) 16.1±0.09 16.3±0.08 16.0±0.08 16.0±0.11 16.0±0.10 16.1±0.10 ns Live weight (g) 55.9±1.03 56.9±1.05 54.4±0.91 55.3±1.15 55.5±1.11 56.1±1.13 ns SGR 1.39±0.02 1.27±0.03 1.31±0.10 1.28±0.02 1.48±0.04 1.46±0.04 ns Interval 5 - Day 98-129 Length (cm) 18.3±0.12 18.1±0.12 18.1±0.11 18.0±0.13 18.0±0.12 18.2±0.13 ns Live weight (g) 85.0±1.81 84.5±1.64 81.2±1.61 80.9±2.01 81.9±1.73 86.2±1.83 ns SGR 1.26±0.04 1.19±0.04 1.22±0.07 1.16±0.06 1.18±0.02 1.29±0.03 ns Entire Trial - Day 0 to 129 SGR 1.61±0.01 1.57*0.00 1.54*0.01 1.53±0.02 1.57±0.02 1.59±0.02 ns ' Values are means ± SEM of 3 replicate tanks for each diet. 2 1 SGR = specific growth rate (% body weight • gain day)" ). 65 Table 3.1.5. Dry feed intake and feed and protein utilization of juvenile chinook salmon over 129 days in relation to diet treatment.1 Feed Parameters Diet OCO 6CO 12CO 20CO 30CO 40CO Significance Interval 1 - Day 0 to 19 F I 2 3.20±0.1 F E 4 1.05±0.02 PER 3 (%) 2.23±0.06 Interval 2 - Day 20 to 43 FI 7.31±0.3 FE 1.22±0.03 PER(%) 2.59±0.12 Interval 3 - Day 44 to 70 FI 10.41±0.5 FE 1.54±0.03 PER(%) 2.68±0.14 Interval 4 - Day 71 to 97 FI 13.72±1.0 FE 1.25±0.05 PER(%) 2.16±0.18 Interval 5 - Day 98 to 129 FI FE PER (%) Entire Trial - Day 0 to 129 FI 57.29±3.10 FE 1.30±0.02 PER (%) 2.25±0.05 ab 3.50±0.03 1.09±0.06 2.24±0.24 7.74±0.11 I. 29±0.04 2.65±0.15 II. 02±0.05 1.45±0.09 2.55±0.28 14.21±0.25 1.13±0.02. 2.06±0.07 a b 22.62±1.2 a b 22.53±0.44 a b 1.28±0.02 1.22±0.05 2.22±0.15 2.23±0.17 59.00±0.54 1.24±0.02 2.27±0.05 3.57±0.16 0.97±0.03 2.00±0.12 8.12±0.38 I. 20±0.03 2.47±0.09 II. 08±0.05 1.3O±0.O3 2.25±0.12 14.09±0.93 1.11±0.07 1.92±0.21a 23.62±1.32 a 1.14±0.07 1.97±0.21 60.47±3.15 1.16±0.02 2.01±0.05 3.30±0.06 1.17±0.06 2.42±0.20 7.46±0.26 1.31±0.03 2.71±0.12 10.73±0.37 1.37±0.04 2.39±0.11 13.26±0.32 1.19±0.03 2.03±0.11 a b 21.59±0.47 a b 1.19±0.08 2.04±0.23 56.64±1.17 1.23±0.03 2.11±0.11 3.23±0.12 1.19±0.09 2.39±0.34 7.37±0.35 1.26±0.07 2.52±0.27 10.21±0.08 1.35±0.05 2.39±0.18 13.01±0.59 1.37±0.03 2.44±0.08 b 20.40±1.22 b 1.30±0.05 2.33±0.16 53.91±1.97 1.32±0.02 2.36±0.07 3.24±0.13 1.00±0.08 2.02±0.31 7.39±0.16 1.20±0.03 2.43±0.11 10.51±0.42 1.44±0.05 2.52±0.17 14.15±0.34 1.25±0.02 2.19±0.04 a b 22.59±0.64 a b 1.32±0.05 2.31±0.15 57.88±2.17 1.29±0.03 2.26±0.09 ns ns ns ns ns ns ns ns ns ns ns PO.01 P<0.01 ns ns ns ns ns 1 Values are ± S E M representing 3 replicate tanks per diet. Significance is denoted with superscripts. 2 F I = dry feed intake (g fish"1) 3 P E R = protein efficiency ratios (wet weight gain (g) • protein consumption (g)"1) 4 F E = feed efficiency (weight gain • total dry feed intake"1) of salmon at each interval. Table 3.1.6. Percentages o f red blood cells (hematocrit) and muscle water in juvenile fall chinook salmon during the 129-day experimental period in relation to diet treatment and water source (i.e. freshwater, F W or seawater, S W ) . 1 Parameter Water Source Diet OCO 6CO 12CO 20CO 30CO 40CO Significance Day 0 Hematocrit (%) FW 38.0±2.1 ND ND ND ND ND SW 33.4±1.0 ND ND ND ND ND % muscle water FW 80.05±0.23 ND ND ND ND ND SW 79.55i0.23 ND ND ND ND ND Interval 1 - Day 0 to 19 Hematocrit (%) FW 36.93-1.77 36.64il.94 36.47il.08 37.77il.68 35.76il.23 38.05il.30 ns SW 32.39-1.08* 32.65il.24* 33.16il.32* 29.73il.52* 33.14il.53 30.76il.48* ns % muscle water FW 79.48±0.24 79.42i0.48 79.46i0.36 79.48i0.42 79.61i0.32 79.28i0.51 ns SW 77.36i0.49* 77.90i0.50* 78.26i0.45 78.24i0.63 77.99i0.42* 78.45i0.36 ns Interval 2 - Day 20-43 Hematocrit (%) FW 36.30-1.45 35.68i0.96 38.10i0.87 37.04il.01 . 36.66il.06 37.96il.15 ns SW 30.88il.19* 33.67il.36 33.94il.22* 34.78il.31 31.43i0.96* 32.93il.12* ns % muscle water FW 79.09±0.98 b 77.16i0.42a 77.79i0.53ab 78.09i0.27ab 78.78i0.45b 77.66il.27ab P<0.05 SW 75.21±1.74 75.25il.29 75.61il.23 75.86il.37* 76.87i0.46* 77.93i0.35 ns Interval 3 - Day 44-70 43.12il.48ab 42.91i4.39ab Hematocrit (%) FW 42.63±1.08 a b 43.88il.41a 45.03il.94a 38.80il.38b PO.05 SW 37.71-1.40* 39.15il.55 36.00il.30* 38.32il.71* 35.23il.15 38.01il.51 ns % muscle water FW 75.70i2.00a 76.90i0.35ab 75.54i0.83a 74.82il.29a 76.64i0.50a 78.84i0.88b PO.05 SW 76.75±0.47 a 75.01i0.80ab 73.77il.20b 72.09i2.17b 76.48i0.44ab 73.64il.40b* PO.05 Interval 4 - Day 71-97 Hematocrit (%) FW 46.28±1.43 49.28il.42 47.91il.50 48.57il.54 45.95il.64 43.89il.42 ns SW 47.68±1.33 a 42.88il.54bc* 46.58il.73abc 45.63il.48abc 47.64i2.76ac 42.04il.47b PO.05 % muscle water FW 77.68±0.40 76.86i0.35 77.39i0.28 77.03i0.31 77.17i0.89 77.26i0.38 ns SW 75.95i0.314* 75.11i0.28ab* 75.38i0.40ab* 73.05i0.63b* 75.04i0.66ab* 75.88i0.43a* PO.05 Interval 5 - Day 98-129 Hematocrit (%) SW 47.04il.00 44.82il.34 46.75il.26 47.51il.28 46.26il.30 46.72il.49 ns % muscle water SW 76.30i0.38 75.88i0.38 75.92i0.48 75.20i0.58 76.74i0.46 75.15i0.71 ns Values are means ± SEM of 3 replicate tanks for each diet. FW = freshwater; SW = fish challenged for 24 hours in full strength saltwater. ND = no data. Asterix indicates significance between freshwater and seawater groups within a physiological measurement (pO.001). f indicates p< 0.01 and % indicates p<0.05. Table 3.1.7. Plasma ion concentrations of juvenile fall chinook salmon during the 129 day study in relation to diet treatment and water source. 1 Diet Water Source OCO 6CO 12CO 20CO 30CO 40CO Signific DayO Na + (mmoI/L) FW 148.54-4.11 ND ND ND ND ND -Na+ (mmol/L) SW 170.12±4.04 t ND ND ND ND ND -CI' (mmol/L) FW 119.60±3.26 ND ND ND ND ND -CI (mmol/L) SW 139.14-1.88* ND ND ND ND ND -Interval 1 - Day 0 to 19 Na+ (mmol/L) FW 172.12±5.67 157.61-11.84 164.99±8.98 163.26±14.26 166.01±5.08 158.84±12.54 ns Na + (mmol/L) SW 189.07±7.55 181.27-5.80 182.45±9.28 148.46±25.62 182.91±6.46 180.43±6.34 ns CI (mmol/L) FW 122.91±1.57 123.67±2.36 119.90±2.79 123.32_1.96 120.44_1.73 124.54-1.32 ns CI" (mmol/L) SW 137.58-1.94* 136.92±2.22 130.59±2.36 133.94±2.09 139.53_2.19* 139.46±2.02* ns Interval 2 - Day 20 to 43 Na + (mmol/L) FW 175.82±5.28 179.04-10.71 180.71±5.00 181.53-25.62 176.50±5.36 178.52_5.11 ns Na + (mmol/L) SW 185.11±6.82 176.21-13.74 171.19-15.48 189.77±6.71 187.53±4.84 176.39-13.44 ns CT (mmol/L) FW 124.14±2.16 126.54_1.24 126.00±1.09 124.54_1.40 125.62-1.27 122.39-1.66 ns CI" (mmol/L) SW 138.29±2.05* 135.92_1.80 133.46±3.13 142.61-1.15* 139.82-2.35* 134:08-2.30* ns Interval 3 - Day 44 to 70 Na + (mmol/L) FW 169.57±4.98 170.42±4.21 179.46±7.38 173.23±6.08 165.05±5.29 178.67±5.46 ns Na + (mmol/L) SW 187.83±6.18 189.87±9.60 195.27±5.04 176.89±4.18 191.02-4.34 181.97±5.06 ns CI" (mmol/L) FW 124.33±1.54 120.57±1.57 116.70±3.70 121.13±1.35 123.00±2.92 121.00 ns CT (mmol/L) SW 135.36±1.20* 142.46-5.19* 149.13±7.03* 140.13±2.00* 138.95±2.07* 135.88-1.68 ns Interval 4 - Day 71 to 97 Na+ (mmol/L) FW 173.84±7.84 176.93±5.95 175.42±6.63 188.81±6.43 173.41±4.64 194.96±14.87 ns Na + (mmol/L) SW 166.98±4.96 193.55-8.11 186.21-12.33 171.18±9.98 185.03±4.60 197.72±5.53 ns CI" (mmol/L) FW 117.50-3.51 118.50-1.83 123.17±2.57 122.00±1.71 120.64_1.76 127.00±4.42 ns CI' (mmol/L) SW 129.50-1.78 149.38±3.06* 138.14±2.82* 136.50±3.46 136.50±2.02* 137.88-3.55 ns Interval 5 - Day 98 to 129 Na + (mmol/L) . SW 177.47±3.88 180.23±3.98 179.48±3.99 181.82-3.00 176.93±2.94 174.28±3.75 ns CI" (mmol/L) SW 129.75-1.22 130.39±2.10 126.29±2.03 129.33-1.65 128.67±1.65 129.58±2.08 ns FW = fish reared i n freshwater; SW = fish challenged to SW; ND = no data; asterisks indicates significance at p O . 0 0 1 ; | indicates significance at p< 0.01; J indicates significance at p< 0.05 between freshwater and seawater groups. O N C O Figure 3.1.1. Diet effects on gi l l N a + / K + ATPase activity (A), a l a m R N A expression (B), and a l b m R N A expression (C) of fish held in freshwater (FW) and fish challenged to 24 hrs in seawater (SW) at day 97 of the feed trial. Activi ty data are all normalized to total protein, and m R N A expression data are normalized to a control gene, F l a . Asterisks indicate significance between freshwater and seawater conditions. A • FW • SW 1 x 1 A T I* I B 12CO 20CO Diet ul iFW • aUSW OCO 6C0 I2CO 20CO 30CO 40CO Diet c 69 Figure 3.1.2. Diet effects on gi l l N a + / K + ATPase activity (A), and a l a and a l b m R N A expression (B) o f fish challenged to 32 days in seawater. Act ivi ty data are all normalized to total protein and m R N A expression data are normalized to a control gene, E F l a . Asterisks indicate significant differences between a l a and a l b expression levels. B OCO 6C0 12C0 20CO 30CO 40CO 70 Figure 3.1.3. Dietary effects on gi l l N a + / K + ATPase a l a (A) and a l b (B) m R N A expression o f fish transferred from freshwater (FW) to seawater at day 97. Data for seawater are 32 days post-transfer. Act ivi ty data are all normalized to total protein and m R N A expression data are normalized to a control gene, E F l a . Asterisks indicate significance between freshwater and seawater conditions. B 71 Figure 3.2.1. Freshwater gi l l physiology of salmon fed the OCO or 40CO diets. (A) N a + / K + ATPase activity (B) gi l l a l a m R N A expression (C) a l b m R N A expression in fish fed the preceding diets over 97 days. Activi ty data are all normalized to total protein and m R N A expression data are normalized to a control gene, E F l a . Asterisks indicate significance between freshwater and seawater values. A < 20 40 60 80 100 Time (days) B C Time (days) Time (days) 72 Figure 3.2.2. Salt water gill physiology of salmon fed the OCO or 40CO diets. (A) Na + /K + ATPase activity (B) a la mRNA expression and (C) alb mRNA expression. Activity data are all normalized to total protein and mRNA expression data are normalized to a control gene, E F l a . A 20 40 60 Time (days) 80 100 O OCO < 40CO A -< I. 1 X X r : B X • B 60 Time (days) • OCO O 40CO Time (days) 73 Figure 3.2.3. Effects of seawater acclimation on gi l l (A) N a + / K + ATPase activity (B) a l a m R N A (B) a l b m R N A expression in fish fed diets OCO and 40CO. 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