UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effect of oxidized dietary lipid and vitamin E on growth and immunocompetence of coho salmon (Oncorhynchus… Forster, Ian 1987

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1987_A6_7 F67_7.pdf [ 4.22MB ]
Metadata
JSON: 831-1.0096904.json
JSON-LD: 831-1.0096904-ld.json
RDF/XML (Pretty): 831-1.0096904-rdf.xml
RDF/JSON: 831-1.0096904-rdf.json
Turtle: 831-1.0096904-turtle.txt
N-Triples: 831-1.0096904-rdf-ntriples.txt
Original Record: 831-1.0096904-source.json
Full Text
831-1.0096904-fulltext.txt
Citation
831-1.0096904.ris

Full Text

THE EFFECT OF OXIDIZED DIETARY LIPID AND VITAMIN E ON GROWTH AND IMMUNOCOMPETENCE OF COHO SALMON (Oncorhynchus kisutch) by Ian F o r s t e r B.Sc. The U n i v e r s i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1987 © Ian F o r s t e r , 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada DE-6(3/81) i i A b s t r a c t H i g h l y unsaturated marine l i p i d s are common i n g r e d i e n t s i n salmon d i e t s , and they are prone to o x i d a t i v e change. The present study was undertaken to examine the growth and h e a l t h of coho salmon (Oncorhynchus kisutch) fed d i e t s c o n t a i n i n g h e r r i n g o i l a u t o x i d i z e d to d i f f e r e n t degrees. The e f f i c a c y of d i e t a r y v i t a m i n E i n a m e l i o r a t i n g any adverse e f f e c t on performance was i n v e s t i g a t e d . H e r r i n g o i l was o x i d i z e d to one of two l e v e l s ( r e l a t i v e to a c o n t r o l ) by a e r a t i o n and m i l d h e a t i n g (40 ° C ) . Peroxide values and i o d i n e numbers were recorded to monitor the extent of a u t o x i d a t i o n . D e p l e t i o n of d i e t a r y l i n o l e n i c a c i d s e r i e s f a t t y a c i d s (n3FA), and the l a b i l e v i t a m i n s A, C, and E, pro v i d e d f u r t h e r evidence of the progress of l i p i d o x i d a t i o n . The mean i n i t i a l body weight was 5.1 g / f i s h , and growth (weight and length) was measured at 3 or 6 week i n t e r v a l s f o r 28 weeks. Experimental d i e t s c o n t a i n e d 16.8% l i p i d , p r i m a r i l y as h e r r i n g o i l . One d i e t was made with c o r n o i l r e p l a c i n g h e r r i n g o i l , and another c o n t a i n e d a combination of low and h i g h l y o x i d i z e d o i l . V i t a m i n E (as d l - a - t o c o p h e r y l a c e t a t e ) was added at e i t h e r 30 IU/kg dry d i e t or 1000 IU/kg dry d i e t . At 23 weeks, 1/3 of the f i s h were v a c c i n a t e d a g a i n s t v i b r i o s i s . At 28 weeks the f i s h were twice c h a l l e n g e d with l i v e Vibrio sp. Immunocompetence was e s t i m a t e d by m o r t a l i t y and by plasma a g g l u t i n a t i o n . The i n c l u s i o n of a u t o x i d i z e d h e r r i n g o i l reduced the n u t r i t i v e value of the d i e t s . The poorer growth and feed e f f i c i e n c y of f i s h fed d i e t s c o n t a i n i n g o x i d i z e d o i l s appears to have r e s u l t e d from a combination of a p p e t i t e s u p p r e s s i o n and n u t r i e n t d e f i c i e n c y . The r e l a t i v e importance of these f a c t o r s i n i n f l u e n c i n g growth and feed e f f i c i e n c y depended upon the extent of the o x i d a t i o n , with a p p e t i t e suppression being most apparent in f i s h fed d i e t s c o n t a i n i n g moderately o x i d i z e d o i l . D i e t a r y supplementation with a h i g h l e v e l of v i t a m i n E had no a m e l i o r a t i n g e f f e c t on growth or feed e f f i c i e n c y . H e a l t h and immunocompetence were not impaired by the presence of o x i d i z e d d i e t a r y l i p i d , or improved by the a d d i t i o n of v i t a m i n E. TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS ix 1 INTRODUCTION 2 2 LITERATURE REVIEW 3 2. 1 Requirement and f u n c t i o n of l i p i d i n f i s h 3 2. 1 . 1 Energy 4 2. 1 . 2 Requirement f o r s p e c i f i c f a t t y ac ids 6 2. 1 . 3 E f f e c t of d i e t on body composition 9 2. 2 Processes of l i p i d a u t o x i d a t i o n 10 2. 3 The r o l e of v i t a m i n E i n salmonid d i e t s 1 5 2. 4 L i p i d o x i d a t i o n and v i t a m i n E i n salmonid d i e t s 21 3 MATERIALS AND METHODS 25 3. 1 F i s h h i s t o r y and experimental c o n d i t i o n s 25 3. 2 H e r r i n g o i l h i s t o r y and treatment 26 3. 3 Di e t p r e p a r a t i o n and composition 27 3. 4 F i s h d i s t r i b u t i o n 31 3. 5 S t a r t of Experiment 35 3. 6 Feeding 35 3. 7 Immunocompetence 37 3. 7. 1 Vacc i n a t ion 37 3. 7. 2 Hematocrit and plasma sampling 38 3. 7. 3 L i v e V i b r i o c h a l l e n g e s 39 3. 7. 4 Antibody t e s t 40 3. 7. 5 Agglut i n a t ion 41 3. 8 Body composition 42 3. 8. 1 Dry matter, % l i p i d , f a t t y a c i d composition 43 3. 8. 2 P h o s p h o l i p i d s 44 3. 9 S t a t i s t i c a l procedures 46 V 4 RESULTS 48 4.1 D i e t s and o i l s 48 4.1.1 D i e t composition and proximate a n a l y s i s 48 4.1.2 O x i d a t i o n and f a t t y a c i d p r o f i l e of o i l s 48 4.1.3 P h y s i c a l c h a r a c t e r i s t i c s of o i l and p e l l e t s 58 4.2 Growth and feed e f f i c i e n c y 59 4.3 F i s h m o r t a l i t y d u r i n g "growth phase" 67 4.4 Hematocrit Values 68 4.5 F i s h body composition 68 4.5.1 Dry matter, l i p i d , and p h o s p h o l i p i d 68 4.5.2 F a t t y a c i d p r o f i l e s of l i p i d and p h o s p h o l i p i d 71 4.6 Immunocompetence 71 5 DISCUSSION 76 5.1 O x i d a t i v e s t a t e of the d i e t s 76 5.2 Growth and feed e f f i c i e n c y 79 5.3 F a t t y a c i d composition of f i s h 85 5.4 Disease and Immune Response 86 5.5 Comparison of r e s u l t s to those of other i n v e s t i g a t i o n s 88 6 CONCLUSIONS 91 7 REFERENCES 92 v i LIST OF TABLES Table 1 - Composition of test diet as fed. 29 Table 2 - Types of o i l added to basal d i e t . 30 Table 3a - Vitamin E content of diets sampled at d i f f e r e n t times. 32 Table 3b - Vitamin A content of diets sampled at d i f f e r e n t times. 33 Table 3c - Vitamin C content of diets sampled at d i f f e r e n t times. 34 Table 4 - Proximate analyeses of the d i e t s . 49 Table 5 - Proximate analyeses of freeze-dried pollock and freeze-dried euphausids. 50 Table 6 - Temporal changes in peroxide values of the herring o i l s r e l a t i v e to treatment. 51 Table 7 - n3 Fatty acid p r o f i l e s of dietary l i p i d . 53 Table 8 - Iodine numbers of the herring o i l s used in this study r e l a t i v e to treatment. 54 Table 9 - Iodine number of dietary l i p i d after storage for eight months. 55 Table 9b - Iodine number of dietary l i p i d after storage for eight months. Mean values of diets i n i t i a l l y containing o i l of the same oxidative state. 56 Table 10 - Mean body weights of the groups at di f f e r e n t times during the experiment. 63 Table 11 - Body weight gain, feed intake/fish, BWG/feed intake, s p e c i f i c growth rate • (0 d - 205d). 64 Table 12 - Body weight gain/feed intake (combining values for low and high vitamin E supplementation). 66 v i i Table 13 - Hematocrit v a l u e 69 Table 14 - F i s h body a n a l y s i s 70 Table 15 - % n 3 - f a t t y a c i d composition of t o t a l l i p i d and p h o s p h o l i p i d r e l a t i v e to d i e t a r y treatment. 72 Table 16 - n 3 - f a t t y a c i d content in the t r i g l y c e r i d e nad p h o s p h o l i p i d f r a c t i o n s in the f i s h body r e l a t i v e to d i e t a r y treatment. 73 Table 17 - M o r t a l i t y of v i b r i o c h a l l e n g e d f i s h ( non-vaccinated f i s h ) . 74 v i i i LIST OF FIGURES F i g u r e 1 - The o v e r a l l mechanism of l i p i d o x i d a t i o n . 12 Fi g u r e 2 - The chemical s t r u c t u r e of the t o c o p h e r o l s . 16 Fi g u r e 3a - E f f e c t of treatments 1, 2, 3, 4 on mean body weight over time. 60 Fi g u r e 3b - E f f e c t of treatments 6, 7, 8, 9 on mean body weight over time. 61 Fi g u r e 4 - E f f e c t of d i e t a r y l i p i d o x i d a t i o n on mean body weight over time. 62 ix Acknowledgements I would l i k e t o express my thanks to a l l those who helped me d u r i n g my time as a master's c a n d i d a t e . In p a r t i c u l a r , P r o f . B e r y l March, who not only helped me with s p e c i f i c p r o c e d u r a l problems, but who a l s o , by her u n f l a g g i n g i n t e r e s t i n the nature of t h i n g s , reminded me of why I am p e r s u i n g a c a r e e r i n the s c i e n c e s . I would a l s o l i k e to e s p e c i a l l y thank Dr. David Higgs of F i s h e r i e s and Oceans, whose w i l l i n g n e s s to share h i s knowledge and e x p e r t i s e was e s s e n t i a l to the s u c c e s s f u l completion of my s t u d i e s . Dr. Gordon B e l l , of the P a c i f i c B i o l o g i c a l S t a t i o n , p r o v i d e d me with i n v a l u a b l e a s s i s t a n c e i n the immunocompetence s e c t i o n of my study. Among those who helped me d u r i n g my experimental work, Mark Newcombe deserves s p e c i a l mention f o r c a t c h i n g the f i s h d u r i n g most of the weigh p e r i o d s , and f o r being a good d e b a t i n g opponent while I was honing the i n t e r p r e t a t i o n s of my experimental r e s u l t s . I t i s impossible to convey my a p p r e c i a t i o n to those o t h e r s who helped i n many ways througout my graduate work. In p a r t i c u l a r these a r e : C a r o l M c M i l l a n , Ray Soong, Mark Walsh, and C h r i s t i n a Pozza. In a d d i t i o n to a l l the expert h e l p and support I have r e c e i v e d as a student, my parents have, more than I can say, supported me i n every way throughout my academic c a r e e r ; emmotionally, s p i r i t u a l l y , and f i n a n c i a l l y . For them I have the g r e a t e s t a d m i r a t i o n , and to them I owe the g r e a t e s t a p p r e c i a t i o n . F i n a n c i a l a i d f o r t h i s work was a l s o p r o v i d e d from F i s h e r i e s and Oceans, Canada. 1 INTRODUCTION Salmonid c u l t u r e i s not new i n Canada. Government f i s h h a t c h e r i e s have been i n o p e r a t i o n f o r decades. Recently, however, c u l t u r i n g these f i s h has i n t e n s i f i e d as entrepreneurs seek to grow them p r o f i t a b l y to s i z e s w e l l beyond the scope of a hatchery s i t u a t i o n . The r e l a t i v e "newness" of t h i s i n d u s t r y has made the d i v i d i n g l i n e between a p r o f i t a b l e venture and a l o s i n g concern d i f f i c u l t to surpass. Consequently, i n f o r m a t i o n r e g a r d i n g f a c t o r s i n f l u e n c i n g f i s h h e a l t h and growth r a t e s has become important. Salmonid g e n e t i c s , n u t r i t i o n , d i s e a s e c o n t r o l and r e p r o d u c t i o n , are four main areas of r e s e a r c h that are e s s e n t i a l f o r the c o n t i n u i n g growth and w e l l - b e i n g of the aquaculture i n d u s t r y . The presence of o x i d i z e d f i s h o i l i n salmonid d i e t s has been thought to be r e s p o n s i b l e f o r a number of c o n d i t i o n s r e l a t i n g to h e a l t h . These i n c l u d e the r e d u c t i o n of growth r a t e , feed consumption, feed e f f i c i e n c y and d i s e a s e r e s i s t a n c e . The presumed causes of these problems r e l a t e to some combination of d e s t r u c t i o n of e s s e n t i a l n u t r i e n t s ( f o r i n s t a n c e , e s s e n t i a l f a t t y a c i d s , e s s e n t i a l amino a c i d s and some 1 2 v i t a m i n s ) , lowered p a l a t a b i l i t y , accumulation of t o x i c compounds and reduced energy content of d i e t s c o n t a i n i n g o x i d i z e d f i s h o i l . Reduction i n e i t h e r d i e t a r y v i t a m i n E or p o l y u n s a t u r a t e d f a t t y a c i d s (PUFA) below t h e i r r e s p e c t i v e requirement l e v e l s i s thought to impair the i n t e g r i t y of c e r t a i n c e l l u l a r membranes, such as those of c e l l s r e s p o n s i b l e f o r immune response. T h i s study was conducted to assess the e f f e c t of d i e t a r y l i p i d o x i d a t i o n on the growth, feed e f f i c i e n c y and immunocompetence of j u v e n i l e coho salmon (Oncorhynchus k i s u t c h ) . The a b i l i t y of supplemental v i t a m i n E to counter the e f f e c t of o x i d i z e d d i e t a r y l i p i d on the f o r e g o i n g performance parameters was a l s o e v a l u a t e d . The e v a l u a t i o n of immunocompetence was undertaken subsequent to a s s e s s i n g the e f f e c t of d i e t a r y treatment on growth and i n v o l v e d (1) measuring the antibody t i t r e i n f i s h a f t e r v a c c i n a t i o n a g a i n s t n o n - v i r u l e n t v i b r i o b a c t e r i a and (2) monitoring the r e l a t i v e m o r t a l i t y of the f i s h a f t e r a c h a l l e n g e with v i r u l e n t V i b r i o sp. 3 2 LITERATURE REVIEW 2.1 Requirement and f u n c t i o n of l i p i d i n f i s h D i e t a r y l i p i d s are comprised p r i m a r i l y of f a t t y a c i d s . These are hydrocarbon molecules comprised of 1 2 - 2 4 tandem carbon u n i t s and are u s u a l l y p a r t of other molecules, t y p i c a l l y t r i - a c y l - g l y c e r i d e s , or phospho-l i p i d s . The amount and l o c a t i o n of u n s a t u r a t i o n s i t e s (carbon-carbon double-bonds) are important i n determining the n u t r i t i o n a l q u a l i t i e s of a f a t t y a c i d . I t i s customary i n n u t r i t i o n s t u d i e s , when d e s c r i b i n g f a t t y a c i d s , to s t i p u l a t e the number of carbons l i n k e d , the number of carbon-carbon double-bonds, and the number of carbon atoms between the t e r m i n a l methyl group and the f i r s t carbon that i s part of a double-bond. Thus, the d e s i g n a t i o n Cl8:2n6 r e f e r s to a f a t t y a c i d that c o n t a i n s a carbon c h a i n of 18 u n i t s with two double-bonds, the one c l o s e s t to the t e r m i n a l methyl group being s i x carbons away. F a t t y a c i d s that have a double-bond three carbons from the t e r m i n a l methyl group are designated as n3FA, whatever t h e i r l e n g t h . F a t t y a c i d s i n animal d i e t s serve two main f u n c t i o n s : 1) they are a source of e a s i l y a v a i l a b l e b i o c h e m i c a l energy, and 2) they supply some e s s e n t i a l 4 components of c e l l membranes that the f i s h cannot s y n t h e s i z e (other f u n c t i o n s i n c l u d e : a c t i n g as c a r r i e r s of f a t - s o l u b l e v i t a m i n s , and as components of p a l a t a b i l i t y ) . In salmonids, as i n other animals, the f i r s t f u n c t i o n can be s a t i s f i e d by a wide range of f a t t y a c i d types (Brown and Tappel, 1959; Takeuchi et al. , 1978a,b,c; C a s t l e d i n e and Buckley, 1980) but only a few s p e c i f i c types can f u l f i l l the second f u n c t i o n . The most important f a t t y a c i d s to salmonids f o r c e l l membrane i n t e g r i t y are those that are long-chained and h i g h l y unsaturated; namely, docosahexaenoic a c i d (C22:6n3) and e i c o s a p e n t a e n o i c a c i d (C20:5n3). D i e t a r y l i n o l e n i c a c i d (Cl8:3n3) serves as the p r e c u r s o r f o r C22:6n3 and C20:5n3 (Dosanjh et al . , 1 984). 2.1.1 Energy Salmonids u t i l i z e d i e t a r y p r o t e i n as a source of b i o c h e m i c a l energy to a l a r g e r extent than other animals (Atherton and A i t k e n , 1970; Cowey and Sargent, 1972). T h i s i s probably due to the c a r n i v o u r o u s nature of these f i s h and the low a v a i l a b i l i t y of complex carbohydrates as an energy source, which leads to the u t i l i z a t i o n of some of the p r o t e i n for energy p r o d u c t i o n (Watanabe, 1982). 5 E x t e n s i v e r e l i a n c e on p r o t e i n as a source of energy may be reduced by i n c l u s i o n of l i p i d i n the d i e t (Ringrose, 1971; Lee and Putnam, 1973; R e i n i t z et al. , 1978; Takeuchi et al. , 1978a; Cowey et al. , 1981). The amount and type of l i p i d r e q u i r e d i n salmonid d i e t s i s not adequately understood (NRC, 1981). D i e t a r y i n c l u s i o n l e v e l s of 10 - 20% of dry matter g i v e e x c e l l e n t r e s u l t s f o r a v a r i e t y of s p e c i e s ( S t i c k n e y and Andrews, 1972; Lee and Putnam, 1973; Adron et al . , 1976; Takeuchi et al . , 1978b; NRC, 1981). L e v e l s of l i p i d higher than 20% i n the d i e t s may i n c r e a s e body l i p i d content ( P h i l l i p s et al . , 1 952; P h i l l i p s et al . , 1957; H i g a s h i et al. , 1964; Takeuchi et al . , 1 978b), and decrease body p r o t e i n content ( R e i n i t z et al. , 1978). Boggio et al. (1985) found t h a t f i s h fed d i e t s c o n t a i n i n g h e r r i n g o i l as the primary l i p i d source had a higher p o l y u n s a t u r a t e d f a t t y a c i d content than those fed d i e t s with swine l a r d as the p r i n c i p a l l i p i d . D i e t s c o n t a i n i n g a mixture of animal f a t s with marine or vegetable o i l s c o n t a i n i n g s u f f i c i e n t n3FA are energy r i c h , and produce none of the adverse e f f e c t s a s s o c i a t e d with d i e t s c o n t a i n i n g no n3FA (Cho et al . , 1974; Atherton, 1975; Yu et al. , 1977a,b; Dosanjh et al. , 1984). 6 2.1.2 Requirement f o r s p e c i f i c f a t t y a c i d s That salmonids r e q u i r e a d i e t a r y source of f a t t y a c i d s of the l i n o l e n i c a c i d f a m i l y (n3FA) has been w e l l e s t a b l i s h e d (Higashi et al. , 1966; C a s t e l l et al. , 1972a; Watanabe et al. , 1974). Because salmonids have a l i m i t e d a b i l i t y to s y n t h e s i z e f a t t y a c i d s of the l i n o l e n i c a c i d s e r i e s , these are c o n s i d e r e d e s s e n t i a l components of the d i e t (Lee et al. , 1967; C a s t e l l et al . , 1972a,b,c; Sinnhuber et al. , 1972; Yu and Sinnhuber, 1972; Watanabe et al. , 1974). Yu and Sinnhuber (1972) found that d i e t a r y l e v e l s of 1% of e i t h e r Cl8n3:3 or C22:6n3 were s u f f i c i e n t to meet the e s s e n t i a l f a t t y a c i d requirement of rainbow t r o u t . D i e t a r y i n c l u s i o n of Cl8:2n6 at l e v e l s of 1.5%, or g r e a t e r , a d v e r s e l y a f f e c t e d growth, feed c o n v e r s i o n , and m o r t a l i t y i n rainbow t r o u t , even when there was otherwise s u f f i c i e n t l e v e l s of Cl8:3n3 i n the d i e t (Yu and Sinnhuber, 1975). I t was a l s o noted that d i e t a r y Cl8:3n3 was l a r g e l y elongated i n t o C22:6n3 in the body. Improved growth r a t e and feed e f f i c i e n c y has been observed i n coho salmon fed d i e t s c o n t a i n i n g 1 - 2.5% Cl8:3n3 (Yu and Sinnhuber, 1979). N6FA d i d not improve growth or feed e f f i c i e n c y at any l e v e l of d i e t a r y i n c l u s i o n . In f a c t , Cl8:2n6, when present above 1% of 7 d i e t s , r e s u l t e d i n poorer growth and feed e f f i c i e n c y i n these animals, a l s o . Takeuchi and Watanabe (1976, and 1977b), and Watanabe and Takeuchi (1976) demonstrated that d i e t a r y C20:5n3 and C22:6n3 have the same growth enhancing e f f e c t on rainbow t r o u t , and that both have a g r e a t e r b i o l o g i c a l e s s e n t i a l f a t t y a c i d e f f i c i e n c y than Cl8:3n3. V a r i o u s f a c t o r s may a f f e c t the n3FA requirement of salmonids. These i n c l u d e : water temperature (Leger et al. , 1977), and t o t a l d i e t a r y l i p i d content (Watanabe, 1982). Takeuchi and Watanabe (1977a) found t h a t , while rainbow t r o u t grew w e l l with d i e t s c o n t a i n i n g 1% Cl8:3n3 and 4% C12:0, the same d i e t c o n t a i n i n g 9% or 14% C12:0 r e q u i r e d 2% Cl8:3n3 f o r .maximal growth. On the b a s i s of these r e s u l t s , they recommended that the d i e t a r y requirement of n3FA be expressed as a percentage of the t o t a l l i p i d content, with the p o s t u l a t e d l i p i d f r a c t i o n as n3FA at about 20% of t o t a l l i p i d s , f o r rainbow t r o u t . Dosanjh et al. (1984) agreed with t h i s type of recommendat i o n . Takeuchi and Watanabe (1979) found that d i e t a r y n3FA at l e v e l s c o n s i d e r a b l y higher than requirement r e s u l t e d i n poor growth of rainbow t r o u t . They found that f e e d i n g d i e t s c o n t a i n i n g 4% (80% of d i e t a r y l i p i d ) as Cl8:3n3 or 8 2% (40% of d i e t a r y l i p i d ) as a n3 HUFA mixture (C20:5n3, C22:6n3; 1:1) r e s u l t e d i n poor growth and feed c o n v e r s i o n , compared to f i s h f e d d i e t s c o n t a i n i n g 1% (20% of d i e t a r y l i p i d ) as Cl8:3n3, or 1% (20% of d i e t a r y l i p i d ) as n3 HUFA mixture. Yu and Sinnhuber (1979) working with coho salmon found that d i e t a r y Cl8:3n3 l e v e l s of 5% (50% of d i e t a r y l i p i d ) r e s u l t e d i n poorer growth and feed e f f i c i e n c y compared to f i s h f e d d i e t s c o n t a i n i n g 1% - 2.5% of Cl8:3n3 (10% - 25% of d i e t a r y l i p i d ) . Watanabe (1982) notes that care must be taken "about the type and amount of EFA [ e s s e n t i a l f a t t y a c i d s ] in l i p i d s used to supplement d i e t s , s i n c e the EFA requirement changes a c c o r d i n g to the d i e t a r y l i p i d l e v e l s and type of [n3] f a t t y a c i d s i n the l i p i d s . " D e f i c i e n c y s i g n s of n3FA i n salmonids i n c l u d e : poor growth, high n9FA l e v e l s i n t i s s u e s ( e s p e c i a l l y C20:3n9), n e c r o s i s of the caudal f i n , f a t t y p a l e l i v e r , dermal depigmentation, high muscle water content, i n c r e a s e d m i t o c h o n d r i a l s w e l l i n g , i n c r e a s e d r e s p i r a t i o n rate of l i v e r homogenates, heart myopathy, and lowered haemoglobin l e v e l ( N i c o l a i d e s and Woodall, 1962; C a s t e l l et al. , 1972b,c; Sinnhuber et al. , 1972). 9 2.1.3 E f f e c t of d i e t on body composition The d i e t a r y n3FA content i n f l u e n c e s the n3FA content i n the t o t a l body l i p i d . T h i s i s e s p e c i a l l y true of the n e u t r a l l i p i d s , but i t i s a l s o a p p l i e s to p h o s p h o l i p i d s ( C a s t e l l et al. , 1972c; Yu and Sinnhuber, 1975; C a s t l e d i n e and Buckley, 1980). P h o s p h o l i p i d s comprise g e n e r a l l y 60 - 90% (by weight) of the l i p i d p o r t i o n of s u b c e l l u l a r membranes (H a r r i s o n and Lunt, 1980). C a s t l e d i n e and Buckley (1980), found that f a s t i n g j u v e n i l e rainbow t r o u t f o r 4 weeks induced no s i g n i f i c a n t change in the f a t t y a c i d p r o f i l e of t o t a l l i p i d , but l e d to a higher c o n c e n t r a t i o n of n3FA i n the p h o s p h o l i p i d s . T h i s i n d i c a t e s that d u r i n g s t a r v a t i o n a l l f a t t y a c i d s are used f o r the production of energy, but that there i s s e l e c t i v e r e t e n t i o n of n3FA i n the p h o s p h o l i p i d s . Mugrditchian et al. (1981) a l s o found that n e u t r a l body l i p i d i s more i n f l u e n c e d by d i e t than i s p o l a r l i p i d , which s e l e c t i v e l y r e t a i n e d n3FA. C a s t e l l et al. (1972c), however, found that d i e t a r y l i p i d s a f f e c t e d the f a t t y a c i d composition of phospho-l i p i d s to a g r e a t e r degree than those in n e u t r a l l i p i d s . I t i s evident from these o b s e r v a t i o n s that l o n g - c h a i n n3FA are important i n the d i e t of salmonids p r i m a r i l y 10 because of t h e i r i n c o r p o r a c i o n i n t o c e l l u l a r membrane s t r u c t u r e as a pa r t of p h o s p h o l i p i d . There are , however, other important f u n c t i o n s of n3FA (e.g., as p r e c u r s o r s of s p e c i f i c p r o s t a g l a n d i n s (Lands, 1982). I n c r e a s i n g the degree of u n s a t u r a t i o n of membrane f a t t y a c i d s i s thought to i n c r e a s e membrane f l u i d i t y , which i s presumed important i n f i s h that are a c t i v e at low p h y s i o l o g i c a l temperatures ( r e l a t i v e to homeotherms). Popp-Snijders et al. (1986) found that a l t e r i n g the f a t t y a c i d composition of human e r y t h r o c y t e s by d i e t a r y supplementation of n3FA i n a way that b r i n g s about a s i g n i f i c a n t i n c r e a s e i n the number of double-bonds does not i n f l u e n c e membrane f l u i d i t y . They propose that other f a c t o r s , namely a l t e r a t i o n s i n the p r o p o r t i o n s of p h o s p h o l i p i d c l a s s e s , can counter the e f f e c t s of i n c r e a s i n g double-bond d e n s i t y . 2.2 Processes of l i p i d a u t o x i d a t i o n A u t o x i d a t i o n i s the spontaneous ( i . e . , non-enzymatically induced) combination of l i p i d s with molecular oxygen. T h i s process i s a u t o - c a t a l y t i c and once i n i t i a t e d may proceed with no f u r t h e r inducement. The range of pathways and products p o s s i b l e i s l a r g e and much i s not 11 known. The general process has been i l l u m i n a t e d , however, and the b a s i c steps are o u t l i n e d as fo l l o w s : I n i t i a t i o n : RH + 0 2 2R00H Propa g a t i o n : R * + CU > R* + 0 2H' or > RO, + RO* + H 20 > ROO* ROO* + RH > ROOH + R* Ter m i n a t i o n : R* + R* R* + ROO* > > RR ROOR ROO' + ROO* > ROOR + 0 2 (adapted from Sevanian and Hochstein, 1985) where 'R' r e f e r s to a carbon c h a i n and '*' r e f e r s to an unpaired e l e c t r o n (hence a f r e e - r a d i c a l ) . F i g u r e 1 d e p i c t s the o v e r a l l mechanisms of l i p i d o x i d a t i o n . As can be seen, a myriad of compounds, many of which are l a b i l e to f u r t h e r o x i d a t i o n , are formed d u r i n g t h i s process (e.g., p e r o x i d e s ) . Many f a c t o r s may enhance the i n i t i a t i o n p r o c e s s . These i n c l u d e : (1) i n c i d e n t e l e c t r o - m a g n e t i c r a d i a t i o n , (2) the presence of pro - o x i d a n t s and, (3) e l e v a t e d 1 2 F i g u r e 1 - The o v e r a l l mechanisms o f l i p i d o x i d a t i o n , (from Labuza and Dugan, 1971) U n s a t u r a t e d f a t t y a c i d o r t r i g l y c e r i d e Breakdown p r o d u c t s ( i n c l u d i n g r a n c i d o f f - f l a v o u r compounds) such a s , ketones a l d e h y d e s a l c o h o l s hydrocarbons a c i d s e p o x i d e s Free r a d i c a l s I + Oxygen H y d r o p e r o x i d e s P o l y m e r i z a t i o n ( p o s s i b l y t o x i c ) * O x i d a t i o n o f p i g m e n t s , f l a v o u r s , and v i t a m i n s I n s o l u b i l i z a t i o n o f p r o t e i n s 1 3 temperature. Examples of pro-oxidants i n c l u d e the t r a n s i t i o n metals, p a r t i c u l a r l y i r o n ( Ingold, 1962; German and K i n s e l l a , 1986; McCay, 1985; Fong et al. , 1976), and hydrogen peroxide, although the l a t t e r may be transformed i n t o superoxide or hydroxyl r a d i c a l by the a c t i o n of a pro-oxidant ( K e l l o g g and F r i d o v i t c h , 1975). The degree of u n s a t u r a t i o n of a f a t t y a c i d determines i t s p r o c l i v i t y f o r l o s i n g a hydrogen atom (the i n i t i a t i o n s t e p ) . The observed r a t e constant of f r e e r a d i c a l i n i t i a t i o n of unsaturated f a t t y a c i d s c o n t a i n i n g from 1 to 6 double-bonds i n c r e a s e s by the order o f : 0.025: 1: 2: 4: 6: 8 ( W i t t i n g and Horwitt, 1964; W i t t i n g , 1965). Removal of hydrogen from the pentadiene system ([A] - see below) allows the formation of a resonance h y b r i d of three valence-bond s t r u c t u r e s which are d e p i c t e d below. 14 R' [CH==CH--CH0--CH==CH]--R" * H* -CH==CH--CH*--CH==CH-T 3 » -CH*--CH==CH--CH==CH-t -CH==CH—CH==CH—CH * -(adapted from Labuza and Dugan, 1971) Th i s resonance r e p r e s e n t a t i o n i s u s u a l l y a b b r e v i a t e d as: R'-[-CH--CH--CH--CH—CH-]*-R" where R' and R" r e f e r to carbon c h a i n s , ' r e f e r s to an unpaired e l e c t r o n (hence a f r e e - r a d i c a l ) , and CH—CH symbolizes a p a r t i a l double-bond. It i s the s t a b i l i t y of the resonance s t r u c t u r e s formed that r e s u l t s i n the higher r e a c t i v i t y of unsaturated f a t t y a c i d s with oxygen. A n t i o x i d a n t s can prevent the formation of f r e e -r a d i c a l s , or they can scavenge a l r e a d y formed r a d i c a l s , and thereby render them l e s s r e a c t i v e . These r e a c t i o n s have been summarized by Burton and Ingold (1981) and are represented as f o l l o w s : 1 5 ROO* + ArOH > ROOH + ArO* and ROO* + ArO* > u n r e a c t i v e products where 'R' i s a carbon c h a i n , 'Ar' i s an aromatic r i n g and '*' r e f e r s to an unpaired e l e c t r o n (hence a f r e e -r a d i c a l . Vitamin E i s p r i m a r i l y a f r e e - r a d i c a l scavenger (Burton and Ingold, 1981). 2.3 The r o l e of vit a m i n E i n salmonid d i e t s The primary n u t r i t i o n a l a c t i o n of vi t a m i n E i s as an a n t i o x i d a n t . It p r o t e c t s other n u t r i e n t s (e.g., other v i t a m i n s and l i p i d s ) from o x i d i z i n g . T h i s a c t i o n i s important f o r the maintenance of b i o l o g i c a l membranes, which a r e , to a l a r g e degree, made of unsaturated f a t t y a c i d s . C o n s i d e r i n g the importance of b i o l o g i c a l membranes i t i s expected that the e f f e c t s of vi t a m i n E d e f i c i e n c y are numerous and wide-ranging (Kay et al., 1986). There are many forms of vit a m i n E. A l l are toc o p h e r o l s ( F i g u r e 2), and many can be s y n t h e t i c a l l y produced i n racemic mixtures. The b i o l o g i c a l a c t i v i t y of 1 6 F i g u r e 2 - The c h e m i c a l s t r u c t u r e o f t h e t o c o p h e r o l s . Chroman s t r u c t u r e —Phytyl s i d e c h a i n -C H , 1 3 C H 2 ( C H 2 C H 2 C H C H 2 ) 3 H a - T o c o p h e r o l : R l = R2 = R3 = CH, fl-Tocopherol: R l = R3 = CH, ; R2 = H Y - T o c o p h e r o l : R l = R2 = C H 3 ; R3 = H 6 - T o c o p h e r o l : R l = CH,; R2 = R3 = H (from B u r t o n and I n g o l d , 1981) 17 1 mg d l - a - t o c o p h e r y l a c e t a t e i s d e f i n e d as 1 IU ( i n t e r -n a t i o n a l u n i t ) , whereas 1 mg of n a t u r a l l y o c c u r r i n g d-a-tocopherol (a-T) i s e q u i v a l e n t to 1.49 IU. The b i o l o g i c a l a c t i v i t y of the t o c o p h e r o l s i n descending order i s : a-T, B-T, V-T, o-T (Johnson and Peterson, 1974). U n t i l r e c e n t l y , however, i t was thought that the in vitro a n t i - o x i d a n t c a p a c i t y of the t o c o p h e r o l s was i n the reverse order. Recent s t u d i e s using e l e c t r o n s p i n resonance techniques, have shown that the true a n t i -oxidant a b i l i t y of the t o c o p h e r o l s f o l l o w s the same p a t t e r n as the b i o l o g i c a l a c t i v i t y (Burton et al. , 1980; Burton and Ingold, 1981). I t i s the fused chroman r i n g that donates a hydrogen atom, thus reducing f r e e r a d i c a l s to l e s s r e a c t i v e substances. The absence of the p h y t y l s i d e -c h a i n does not a p p r e c i a b l y a f f e c t the in vitro a n t i -oxidant c a p a c i t y of the s t r u c t u r e , but d r a s t i c a l l y d i m i n i s h e s i t s b i o l o g i c a l a c t i v i t y , presumably by reducing i t s a f f i n i t y f o r the hydrophobic environment of the membrane (McCay, 1985). Along with the requirement of salmonids f o r p o l y -unsaturated f a t t y a c i d s , i s a requirement f o r v i t a m i n E, f o r t h e i r p r o t e c t i o n . T h i s requirement depends upon 18 environmental and n u t r i t i o n a l f a c t o r s . For standard salmonid d i e t s the requirement i s 30 IU/kg d i e t (NRC, 1981), although l e v e l s of 20 - 30 IU/kg d i e t meet rainbow t r o u t needs (Brown and Tappel, 1959; N i c o l a i d e s and Woodall, 1962). The requirements of v i t a m i n E i n c r e a s e d i n d i e t s c o n t a i n i n g e l e v a t e d l e v e l s of h i g h l y unsaturated f a t t y a c i d s . By comparison, Wilson et al. (1984) found that channel c a t f i s h (Ictalurus punctatus) r e q u i r e d g r e a t e r than 50 IU/kg d i e t (requirements i n c r e a s e d f o r t h i s s p e c i e s when o x i d i z e d menhaden o i l r e p l a c e d " f r e s h " o i l (Murai and Andrews, 1974)). W i t t i n g and Horwitt (1964), studying c r e a t i n u r i a i n r a t s estimated that the r e l a t i v e l e v e l s of v i t a m i n E r e q u i r e d to p r o t e c t one mole of monoenoic, d i e n o i c , t r i e n o i c , t e t r a e n o i c , pentaenoic, and hexaenoic a c i d s , r e s p e c t i v e l y , a r e : 0.3:2:3:4:5:6. Some d e f i c i e n c y symptoms f o r v i t a m i n E i n salmonids i n c l u d e : e r y t h r o p o i e s i s , extreme anemia, s u s c e p t i b i l i t y to s t r e s s , m o r t a l i t y , reduced d i s e a s e r e s i s t a n c e , muscular dystrophy (Poston et al. , 1976; B e l l and Cowey, 1985). Cowey et al. (1984) sp e c u l a t e t h a t s i n c e lower water temperatures i n c r e a s e p h o s p h o l i p i d p o l y u n s a t u r a t e d f a t t y a c i d content i n salmonids, the need f o r the p r o t e c t i o n a f f o r d e d by vit a m i n E a l s o i n c r e a s e s . They 19 c i t e as evidence f o r t h i s , Hazel's (1980) demonstration that m a i n t a i n i n g biomembrane f l u i d i t y i s more demanding i n terms of p o l y u n s a t u r a t e d f a t t y a c i d s at low temperatures. I t would seem, however, that the lower temperature would, i t s e l f , reduce the s u s c e p t i b i l i t y of p o l y u n s a t u r a t e d f a t t y a c i d s to a u t o x i d a t i o n . V i t a m i n E i s important to the d i s e a s e r e s i s t a n c e and immune response of a v a r i e t y of animals. H e i n z e r l i n g et al. (1974), found that c h i c k m o r t a l i t y to Escherichia coli i n f e c t i o n was s i g n i f i c a n t l y reduced by d i e t a r y v i t a m i n E supplementation. Sheep had i n c r e a s e d r e s i s t a n c e to Chi amydi a when fed d i e t s supplemented with v i t a m i n E (Nockels, 1979). Mice hemagglutinin t i t r e s and plaque-forming c e l l s i n c r e a s e d i n response to d i e t a r y supplementation with v i t a m i n E at a c o n c e n t r a t i o n of 2035 IU/kg d i e t (Tengerdy et al. , 1973). In t h i s l a s t experiment supplemental N,N-diphenyl-p-phenylene diamine (DPPD) without v i t a m i n E i n c r e a s e d plaque-forming c e l l s but not h e m a g l u t i n i n . The authors i n t e r p r e t e d these r e s u l t s as i n d i c a t i o n of the n e c e s s i t y of v i t a m i n E f o r antibody p r o t e c t i o n . Recent work (reviewed by Boxer, 1986) has i l l u m i n a t e d some of the r o l e s v i t a m i n E p l a y s i n r e g u l a t i n g phagocyte f u n c t i o n . The a b i l i t y of the human 20 polymorphonuclear leukocyte to phagocytose was i n c r e a s e d when 1600 IU/day fo r 2 weeks was administered to a d u l t s (Baehner et al. , 1977). There was, however, a m i l d r e d u c t i o n i n a b i l i t y to k i l l b a c t e r i a in the a-toco-p h e r o l - r e p l e t e polymorphonuclear l e u k o c y t e s . These r e s e a r c h e r s found t h a t , whereas a normal amount of superoxide was r e l e a s e d e x t r a c e l l u l a r l y by the a-toco-p h e r o l r e p l e t e polymorphonuclear leu k o c y t e s , only 45% of the normal amount of HjC^ was r e l e a s e d . They concluded that "the depressant e f f e c t on polymorphonuclear l e u k o c y t e s k i l l i n g in vitro must be weighed a g a i n s t reduced polymorphonuclear l e u k o c y t e s a u t o t o x i c i t y l e a d i n g to improved p h a g o c y t o s i s " . B l a z e r and Wolke (1984) found that v i t a m i n E i n rainbow t r o u t d i e t s i n c r e a s e d immune response and non-s p e c i f i c r e s i s t a n c e f a c t o r s . These i n c l u d e d : i n c r e a s e d T - c e l l ( m i g r a t i o n i n h i b i t i o n f a c t o r ) and plaque-forming c e l l response to sheep red blood c e l l a ntigen exposure f o r f i s h fed d i e t s supplemented with a-tocopherol; i n c r e a s e d plaque-forming c e l l response and a g g l u t i n a t i o n t i t r e to Yersina ruckeri exposure. The phagocytic index of p e r i t o n e a l macrophages (measured using l a t e x beads) was 10 times higher i n the f i s h fed the c o n t r o l d i e t (400 IU v i t a m i n E/kg dry d i e t ) , than i n those i n g e s t i n g 21 the d e f i c i e n t d i e t (0 IU v i t a m i n E/kg dry d i e t ) . Selenium a l s o a c t s i n d i r e c t l y as an a n t i - o x i d a n t . It i s i n c o r p o r a t e d i n t o Se-mediated g l u t a t h i o n e peroxidase, which scavenges peroxides (German and K i n s e l l a , 1986). Se and v i t a m i n E may operate independently or s y n e r g i s t i c a l l y , depending on the environmental c o n d i t i o n s . They are both r e q u i r e d n u t r i e n t s , but each can reduce the s e v e r i t y of a d e f i c i e n c y of the o t h e r . 2.4 L i p i d o x i d a t i o n and v i t a m i n E i n salmonid d i e t s Owing to the high l e v e l of i n c l u s i o n of p o l y u n s a t u r a t e d f a t t y a c i d s of the l i n o l e n i c a c i d f a m i l y i n salmonid d i e t s there i s i n c r e a s e d l i k e l i h o o d of d i e t a r y l i p i d o x i d a t i o n . Since v i t a m i n E i s a strong a n t i - o x i d a n t i t seems l o g i c a l to study the i n t e r r e l a t i o n s h i p s between extent of d i e t a r y l i p i d o x i d a t i o n and d i e t a r y l e v e l of vitamin E on salmonid performance. The a u t o x i d a t i o n of f a t t y a c i d s can a f f e c t the n u t r i t i o n a l q u a l i t y of a d i e t i n a v a r i e t y of ways. O x i d a t i o n of e s s e n t i a l f a t t y a c i d s may r e s u l t i n a low a v a i l a b i l i t y of these n u t r i e n t s . In a d d i t i o n , the products of l i p i d o x i d a t i o n may lower the a v a i l a b i l i t y of 22 other n u t r i e n t s . For example, bovine t r y p s i n has been shown to be s t r o n g l y i n h i b i t e d by polymerized methyl l i n o l e n a t e (Hatate and Toyomizu, 1985). T h i s i n h i b i t i o n was found to be a s s o c i a t e d p r i m a r i l y with the production of i n a c t i v e complexes, and s e c o n d a r i l y by a c c e l e r a t e d a u t o l y s i s of t r y p s i n by the polymer. Hung et al. (1980) o x i d i z e d h e r r i n g o i l by a e r a t i o n at room temperature f o r 0, 100, and 265 h p r i o r to i n c l u s i o n (at 7.5%) i n a n o n - p u r i f i e d d i e t . They found minimal i n c r e a s e s i n o x i d a t i v e i n d i c e s ( i . e . , TBA, peroxide valu e , a n i s i d i n e v a l u e ) , and l i t t l e , but, never-t h e l e s s , s t a t i s t i c a l l y s i g n i f i c a n t , e f f e c t on v i t a m i n E and p o l y u n s a t u r a t e d f a t t y a c i d c o n c e n t r a t i o n s . Rainbow t r o u t fed d i e t s c o n t a i n i n g these o i l s had no s i g n i f i c a n t d i f f e r e n c e s i n body weight g a i n , feed c o n v e r s i o n , or m o r t a l i t y . The v i t a m i n E content of the body was n e g a t i v e l y c o r r e l a t e d to l i p i d o x i d a t i o n . In another experiment (Hung et al. , 1981), the same authors o x i d i z e d o i l by a e r a t i o n at room temperature for 0, 1348, and 1540 h. In t h i s case, there were l a r g e d i f f e r e n c e s i n peroxide valu e , TBA, and AV, as w e l l as a c o n s i d e r a b l e r e d u c t i o n of n3FA. Rainbow t r o u t fed p r a c t i c a l d i e t s c o n t a i n i n g 7.5% of these o i l s showed no d i f f e r e n c e i n growth, or feed e f f i c i e n c y over 24 weeks. 23 The i n c l u s i o n of a s y n t h e t i c a n t i o x i d a n t , ethoxyquin ( 6 - e t h o x y - 1 , 2 - d i h y d r o - 2 , 2 , 4 - t r i m e t h y l q u i n o l i n e ) , at 125 ppm had no i n f l u e n c e . M o r t a l i t y from 9 to 24 weeks was higher i n treatment groups which i n g e s t e d d i e t s c o n t a i n i n g i n c r e a s i n g l y o x i d i z e d o i l . T h i s m o r t a l i t y was, to some extent, reduced by the a d d i t i o n of ethoxyquin at 125 ppm. a-Tocopheryl a c e t a t e , added to -the d i e t c o n t a i n i n g moderately o x i d i z e d HO (aerated f o r 1348 h o u r s ) , completely e l i m i n a t e d m o r t a l i t y between 9 and 24 weeks, even with no ethoxyquin. E r y t h r o c y t e f r a g i l i t y was a f f e c t e d s i g n i f i c a n t l y by d i e t a r y l i p i d o x i d a t i o n . E r y t h r o c y t e f r a g i l i t y i n f i s h r e c e i v i n g the d i e t with moderately o x i d i z e d o i l , was completely a m e l i o r a t e d by supplementing the d i e t with 33 IU/kg of a-t o c o p h e r y l acetate.. Supplemental ethoxyquin (125 ppm) had no e f f e c t , by i t s e l f , on groups i n g e s t i n g d i e t s c o n t a i n i n g moderately or h i g h l y o x i d i z e d o i l . Cowey et al . (1984) examined the growth and h e a l t h of rainbow t r o u t i n response to d i e t a r y l i p i d o x i d a t i o n and v i t a m i n E l e v e l at v a r y i n g water temperatures. These r e s e a r c h e r s aerated marine o i l at 100 °C f o r 7 h, fo l l o w e d by a e r a t i o n f o r a f u r t h e r 14 h without heat. The r e s u l t i n g o i l had v i r t u a l l y the same i o d i n e number as the " f r e s h o i l " (97 vs. 99). N3FA content was unchanged 24 at 11.6% of t o t a l f a t t y a c i d . The peroxide value i n c r e a s e d from 2 to 47 mmol/kg. Heating the o i l to such temperatures seems to have promoted p e r o x i d a t i o n pathways that proceed other than by the d e s t r u c t i o n of n3FA (the primary pathway at lower temperatures). The measured d i e t a r y v i t a m i n E content of unsupplemented d i e t s was 18.4 ppm f o r the f r e s h o i l d i e t , and 7.4 ppm i n the d i e t with o x i d i z e d o i l . T h i s d i f f e r e n c e was a t t r i b u t e d to the l o s s of n a t u r a l l y o c c u r r i n g v i t a m i n E d u r i n g the o x i d a t i o n process. The growth r a t e , hematocrit and feed e f f i c i e n c y were lowest f o r those f i s h fed d i e t s c o n t a i n i n g no supplemental v i t a m i n E. O x i d i z e d o i l had l i t t l e or no e f f e c t . The d e f i c i e n c y of v i t a m i n E was exacerbated by the lower temperature ( 1 2 - 6 °C) of t h i s study, r e l a t i v e to other s t u d i e s ( R e i n i t z et al . , 1978; Takeuchi et al. , 1978a). 25 3 MATERIALS AND METHODS 3.1 F i s h h i s t o r y and experimental c o n d i t i o n s On 05/06/1985 coho salmon f r y (Oncorhynchus kisutch) were obtained from C a p i l a n o Hatchery, North Vancouver, B.C. They were 2 - 3 months of age with a mean weight of about 1.30 g / f i s h . No attempt was made to sex the animals. The f i s h were t r a n s f e r r e d to 9 X 150 L o v a l f i b e r g l a s s tanks with c e n t e r stand-pipe d r a i n s . The water temperature was 9.5 - 15.0 °C and the flow r a t e was 1.8 -2.0 L/min./tank, c r e a t i n g a s m a l l , steady c u r r e n t . The water throughout the experiment was obtained from the M u n i c i p a l i t y of Vancouver Water Works and was not r e c i r c u l a t e d . Sodium t h i o s u l p h a t e (Na2S20^) was added to the water supply at a dosage of about 50 ppm, to remove any c h l o r i n e . Only p o l y v i n a l c h l o r i d e (PVC) p i p i n g was used i n t r a n s p o r t i n g the water. The water passed through an a s p i r a t o r immediately p r i o r to c a s c a d i n g i n t o the tank. F i s h were fed 2 - 3 times d a i l y to s a t i a t i o n with Oregon Moist P e l l e t s . From 27/08/1985 u n t i l 08/10/1985 (the beginning of the experiment) the f i s h were fed once a day to keep growth below the maximum r a t e , but to ensure h e a l t h of the animals. Screens p e r m i t t i n g l i g h t passage were i n s t a l l e d to prevent jumping. Oxygen 26 t e n s i o n was measured p e r i o d i c a l l y and the water was always found to be s a t u r a t e d over the d u r a t i o n of the experiment. L i g h t i n g was p r o v i d e d by 6 X 60 watt incandescent bulbs and was constant throughout the experiment (15.75 hours l i g h t , 8.25 hours d a r k ) . 3.2 H e r r i n g o i l h i s t o r y and treatment Approximately 75 L of u n s t a b i l i z e d h e r r i n g o i l (HO) was obtained from B.C. Packers Imperial P l a n t , Steveston, B.C. The o i l was taken from the top of a storage tank on 19/06/1985. The HO was s t o r e d i n the l a b o r a t o r y i n f i v e almost-f u l l p l a s t i c c o n t a i n e r s with s e a l e d l i d s . I n i t i a l peroxide v a l u e s were 2.2 - 2.7 meq/kg (AOCS method, 1973). O i l s 1 and 2 were s t o r e d at ambient temperature (approximately 1 5 - 2 5 °C) i n the dark. O i l 1 was s t a b i l i z e d with 500 ppm ethoxyquin and bubbled with N 2 f o r 40 minutes to mix (26/06/1985). Oxygen was bubbled through o i l s 3 and 4 f o r 4 hours, a f t e r which time a i r ( f i l t e r e d and bubbled through 5 cm water) was passed through o i l 4 c o n t i n u o u s l y . O i l 4 was heated to 35 °C u n t i l 04/07/1985, when i t was allowed to c o o l to room temperature and a e r a t i o n ceased. A e r a t i o n of o i l s 3 and 27 4 was rebumed on 19/07/1985, while a temperature of approximately 70 °C was reached f o r < 2 hours before being maintained at 50 °C. O i l 3 was removed from heat and a e r a t i o n on 02/08/1985 and s t a b i l i z e d (500 ppm ethoxyquin and m i x i n g ) . O i l 4 was removed from a e r a t i o n and heat on 11/09/1985 and s t a b i l i z e d (500 ppm ethoxyquin, and N 2 m i x i n g ) . The i o d i n e numbers of the v a r i o u s h e r r i n g o i l s were determined using Wij's method (AOAC, 1980) on 18/11/1985. O i l s 3 and 4 were aer a t e d at e l e v a t e d temperatures f o r approximately 360 and 1536 hours, r e s p e c t i v e l y . The 2 - t h i o b a r b i t u r i c a c i d number measures the occurrence of malondialdehyde, a secondary product of l i p i d o x i d a t i o n , and i s used to measure the r e l a t i v e s t a t e of o x i d a t i o n . The malondialdehyde content of the h e r r i n g o i l s were determined by the method of Yu and Sinnhuber (1967). The malondialdehyde content of the d i e t a r y o i l s was determined by the method of T a r l a d g i s et al . (1960). 3.3 Diet p r e p a r a t i o n and composition The compositions of the 10 t e s t d i e t s are given i n Table 1 (proximate a n a l y s i s of d i e t s was performed by 28 Cantest L t d . , Vancouver, B.C.). Except f o r d i e t 5, d i e t s d i f f e r e d from one another only by the o x i d a t i v e s t a t e of HO added and by the presence or absence of a d d i t i o n a l v i t a m i n E supplementation ( a l l d i e t s had a minimum of 30 IU of v i t a m i n E added per kg, which i s the NRC (1981) requirement l e v e l f o r salmonids, and i n s e v e r a l d i e t s supplemental v i t a m i n E was r a i s e d an a d d i t i o n a l 1000 IU/kg). Corn o i l (Mazola, General Foods Canada, Inc., E t o b i c o k e , Ont.) r e p l a c e d HO i n d i e t 5. H e r r i n g o i l was r e p l a c e d by corn o i l (which i s very low i n n3FA) in order to p r o v i d e a n3FA d e f i c i e n c y c o n d i t i o n f o r comparison with f i s h fed d i e t s c o n t a i n i n g h i g h l y o x i d i z e d o i l s . A summary of d i e t a r y d i f f e r e n c e s i s given i n Table 2. D i e t s were c o l d - p e l l e t e d by e x t r u s i o n using a C a l i f o r n i a model CL-type 2 l a b o r a t o r y p e l l e t m i l l . Two p e l l e t s i z e s were made: 3/32" and 5/64". Immediately f o l l o w i n g p e l l e t i n g , d i e t s were a i r - c o o l e d and f i n e s screened out. P e l l e t s were sprayed with a p p r o p r i a t e remaining HO or corn o i l and s t o r e d i n t h i c k p l a s t i c bags at 0 - 6 °C i n the dark throughout the experiment. The bags were t i e d t i g h t l y so that a minimum of a i r remained. The v i t a m i n E, C and A contents of the d i e t s were measured at v a r i o u s times d u r i n g the experiment (data summarized i n Tables 3a,b,c). L i p i d e x t r a c t i o n was 29 T a b l e 1 - C o m p o s i t i o n o f t e s t d i e t s a s f e d . (g/kg) F r e e z e - d r i e d p o l l o c k m u s c l e F r e e z e - d r i e d e u p h a u s i d s D e x t r i n G l u c o s e G r o u n d c e l l u l o s e V i t a m i n s u p p l e m e n t 1 M i n e r a l s u p p l e m e n t 3 O i l s o u r c e 3 C a r b o x y - m e t h y l c e l l u l o s e A s c o r b i c a c i d C h o l i n e c h l o r i d e (60*/.) E t h o x y q u i n * Water 0. 07 75. 27 1000.00 425.34 46. 53 31. 73 31. 73 138.93 36. 39 45490 134.82 27. 30 4. 55 1. 82 ' The v i t a m i n s u p p l e m e n t s u p p l i e d t h e f o l l o w i n g l e v e l s o f n u t r i e n t s p e r kg o f d r y d i e t : D - c a l c i u m p a n t o t h e n a t e , 165.7 mg; p y r i d o x i n e - HC1, 36.5 mg; r i b o f l a v i n , 60. O mg; n i a c i n , 300 mg; f o l i c a c i d , 19.3 mg; t h i a m i n e m o n o n i t r a t e , 36.3 mg; b i o t i n , 3.0 mg; c y a n o c o b a l a m i n e 60 pg; menadione ( a s h e t r a z e e n ) , 30. 3 mg; c h o l e c a l c i f e r o l , 2,400 IU; r e t i n o l a c e t a t e , 10,000 IU; i n o s i t o l , 400 mg; d l - c c - t o c o p h e r y l a c e t a t e , ( d i e t s 1, 2, 3, 4, 5, 10) 30 IU, ( d i e t s 6, 7, 8, 9) 1000 IU. a The m i n e r a l s u p p l e m e n t s u p p l i e d t h e f o l l o w i n g n u t r i e n t s (mg/kg o f d r y d i e t ) : c a l c i u m ( a s CaHPO,, 3014; and a s CaCOa, 5858), 8872; magnesium ( a s M g S 0 « • 7 H 3 0 ) , 663; manganese ( a s MnSO* • H 2 O) , 74.6; z i n c ( a s ZnSO, • 7 H 8 0 ) , 67; c o b a l t ( a s C o C l a - 6 H s 0 ) , 4.00; c o p p e r ( a s CuSO*-5H aO>, 5.64; i r o n ( a s FeSO*•7H eO>, 191.1; f l u o r i n e ( a s NaF), 4.50; i o d i n e ( a s KI0 3>, 4.50; s e l e n i u m ( a s N a 2 S e 0 3 ), 0.097; aluminum ( a s A l C l 3 - 6 H e O ) , 34.1; p h o s p h o r u s < a s CaHPO* ), 2328. 3 O i l u s e d d e p e n d s on t r e a t m e n t : s e e T a b l e 2. * E t h o x y q u i n was n o t added t o d i e t s 1 and 6. 30 Table 2 - Types of o i l added to b a s a l d i e t D i e t s O i l type 1 ,6 U n s t a b i l i z e d 1 2.7 Low o x i d i z e d , s t a b i l i z e d (LOX) 3.8 Medium o x i d i z e d , s t a b i l i z e d (MOX) 4.9 High o x i d i z e d , s t a b i l i z e d (HOX) 5 Corn o i l , s t a b i l i z e d 10 LOX + HOX 1:1, s t a b i l i z e d . U n s t a b i l i z e d o i l had no ethoxyquin added. A l l other o i l s were s t a b i l i z e d with 500 ppm ethoxyquin. 31 conducted by the method of B l i g h and Dyer (1959), and b a s e - c a t a l y s e d methyl e s t e r i f i c a t i o n was c a r r i e d out as d e s c r i b e d by C h r i s t i e (1982). The r e l a t i v e amounts of f a t t y a c i d methyl e s t e r s (by weight) were determined using a V a r i a n 3700 g a s - l i q u i d chromatograph with a CDS 401 i n t e g r a t o r (Varian Instrument Group, Palo A l t o , Ca. USA), equipped with a Supelco SP-2330 c a p i l l a r y column (30 m X .25 mm i d , Supelco Canada L t d , O a k v i l l e , Ont). Temperature program was set at 180 °C f o r 10 min, up 5 °C/min to 195 °C. Detector and i n j e c t o r port temperatures were 230 °C. Flow r a t e was 40 mL/min. Peaks were i d e n t i f i e d r e l a t i v e to f a t t y a c i d methyl e s t e r standards ( p r i m a r i l y PUFA-1, Supelco Canada L t d . ) . The i o d i n e number of d i e t s was determined on e x t r a c t e d l i p i d (09/04/1986) u s i n g Wij's method (AOAC, 1980). The 2-t h i o b a r b i t u r i c a c i d number of d i e t s 1, 2, 3, 4 was determined by the method of T a r l a d g i s et al (1960). 3.4 F i s h d i s t r i b u t i o n On 01/10/1985 a l l f i s h were a n e s t h e t i z e d using 2 -phenoxyethanol (0.5 mL/L). Each f i s h was then weighed to the nearest 0.01 g wet weight (data were not r e c o r d e d ) . Those f i s h weighing between 3.60 g and 6.35 g 32 Table 3a - Vitamin E content of d i e t s sampled at d i f f e r e n t t i m e s 1 . Vitamin E (IU/kg) Date D i e t 13/11/1985 03/02/1986 30/06/1986 1 86.7 18.2 27. 1 2 1 38.9 34.7 18.0 3 71.8 2.8 36. 1 4 26.5 2.8 54.2 5 70.2 18.9 12.8 6 745. 1 329. 1 433.4 7 .688.1 605.5 307.8 8 810.6 542.8 1 70.9 9 674.4 508. 1 252.9 10 32.8 29.2 61 .4 Analyses c a r r i e d out at Cantest L a b o r a t o r i e s L t d . , using the method of Soderhjelm and Andersson, 1978. 33 Table 3b - Vitamin A content of d i e t s sampled at d i f f e r e n t t i m e s 1 . Vitamin A (IU/g) Date D i e t 13/11/1985 03/02/1986 30/06/1986 1 50.9 41 .3 48.3 2 67.6 43.6 50.3 3 33.6 10.0 33.0 4 0.0 6.7 27.6 5 16.0 11.0 8.0 6 52.9 44.0 11.3 7 63.9 52.9 28.6 8 13.7 12.7 24.6 9 0.0 4.7 16.3 1 0 0.0 12.3 20.6 Analyses c a r r i e d out at Cantest L a b o r a t o r i e s L t d . , us i n g the method of Soderhjelm and Andersson, 1978. 34 Table 3c - Vitamin C content of d i e t s sampled at d i f f e r e n t t i m e s 1 . Vitamin C (mg/lOOg) Date Di e t 25/09/1985 07/01/1986 16/05/1986 1 1 32.3 51 .8 28.0 2 1 40.8 46.2 22.0 3 1 32.5 46.7 24.0 4 1 33.6 67.5 30.0 5 135.7 75.7 39.0 6 1 35.5 73.4 24.0 7 131.9 77. 1 35.0 8 1 35.8 56.3 24.0 9 1 25.3 49. 1 33.0 10 1 38.2 56.2 35.0 Analyses c a r r i e d out at Cantest L a b o r a t o r i e s Inc. u s i n g the indophenol method o u t l i n e d i n AOAC, 1980. 35 comprised the experimental f i s h and were separated from the r e s t . A l l experimental f i s h were pooled i n t o 3 c o n t a i n e r s ( >160 L each) with a e r a t i o n . T h e r e a f t e r , 112 f i s h were randomly assigned, i n groups of 5, to each of 26 experimental tanks. 3.5 S t a r t of Experiment F i s h were s t a r t e d on experimental treatments on 08-09/10/1985. At t h i s time a l l f i s h from a tank were captured and p l a c e d i n t o a p l a s t i c bucket c o n t a i n i n g a e r a t e d water. S i x t y of these f i s h were then randomly s e l e c t e d and subsequently a n a e s t h e t i z e d (0.5 ml 2-phen-oxyethanol/1 water) and i n d i v i d u a l l y weighed and measured (weight +0.01 g and l e n g t h +0.1 mm). A f t e r the f i s h recovered, they were returned to t h e i r r e s p e c t i v e tanks. Weighing and measuring were c a r r i e d out u s i n g these methods throughout the experiment. F i s h were not fed 24 hours before and a f t e r weighing. 3.6 Feeding F i s h were weaned from a d i e t of Oregon Moist P e l l e t s to the c o n t r o l d i e t ( d i e t 2) over a two day p e r i o d . 36 The f e e d i n g response was g e n e r a l l y e n t h u s i a s t i c . At the s t a r t of the experiment, the day a f t e r weighing on 08-09/10/1985, f i s h were fed the t e s t d i e t s . Two treatment r e p l i c a t e s were assig n e d i n completely random f a s h i o n w i t h i n one room. A l l f i s h were fed t h e i r p r e s c r i b e d d i e t s by hand 3 times d a i l y to s a t i a t i o n (about 2 - 3 min./feeding time/tank), except f o r those on treatments 11, 12, and 13, which were p a i r - f e d d i e t 2 at the same l e v e l of consumption as noted f o r f i s h on treatments 1, 3 and 4, r e s p e c t f u l l y . P a i r - f e e d i n g was i n c o r p o r a t e d i n t o the experimental design to examine the p o s s i b l e e f f e c t s of l i p i d o x i d a t i o n on growth and immunocompetence from reduced a p p e t i t e . Feed i n t a k e was recorded f o r each group at each of the three d a i l y f e e d i n g times. When f i s h on treatments 11, 12, and 13 d i d not consume the same amount of food as those on treatments 1, 3 or 4, the a c t u a l amount of food eaten was recorded. The p o i n t of s a t i a t i o n at each f e e d i n g time was determined when p e l l e t s began h i t t i n g the bottom of the tank. R e p l i c a t e groups on treatments 1, 3 and 4 were p a i r - f e d with c o r r e s p o n d i n g r e p l i c a t e groups on treatments 11, 12, and 13. Throughout the study the d i e t s were s t o r e d i n the dark at <5.0 °C. Tanks were c l e a n e d as needed by 37 siphoning out uneaten food and f e c a l matter without removing the f i s h . Water temperature and f i s h deaths were recorded d a i l y (dead f i s h were removed). Weights and lengths were measured every 3 or 6 weeks. Tanks were cleaned thoroughly at t h i s time. 3.7 Immunocompetence 3.7.1 Vacc i n a t ion On 27/03/1986 37 f i s h from each tank were immersed fo r 3 minutes i n an aerated v a c c i n e s o l u t i o n c o n s i s t i n g of 1 L V i b r o g e n 1 : 9 L 1% s a l i n e ( p l u s 1 mL Baker A n t i -Foam B/10 L ) . Each group of f i s h was t r e a t e d i n random order; i . e . , the order of placement i n the room. F i s h recovered i n a separate tank f o r each d i e t a r y treatment. V a c c i n a t e d f i s h from the d u p l i c a t e groups of f i s h per d i e t a r y treatment were mixed without marking and were kept i n tanks i n another room, to prevent c r o s s -v a c c i n a t i o n of non-vaccinated f i s h . Non-vaccinated f i s h were returned to t h e i r o r i g i n a l tanks. Treatments were continued as be f o r e . L i g h t i n g and water c o n d i t i o n s of the two rooms were s i m i l a r . On 1 Vibrio angui 11 arum b a c t e r i a serotypes I + I I . Connaught L a b o r a t o r i e s L t d . Animal H e a l t h D i v . Willowdale, Ont., Canada. 38 03/04/1986 v a c c i n a t e d f i s h were a n a e s t h e t i z e d and each had i t ' s adipose f i n removed, for i d e n t i f i c a t i o n . I t was assumed that there would be no r e s i d u a l a c t i v e v a c c i n e remaining by t h i s time. Since v a c c i n a t e d f i s h from both tanks from each treatment had been mixed together, they were randomly, and e q u a l l y , r e - a s s i g n e d to the two o r i g i n a l tanks f o r that treatment. 3.7.2 Hematocrit and plasma sampling On 01-02/05/1986 a l l f i s h were measured as before and a r e c o r d made of those that were v a c c i n a t e d . While a n a e s t h e t i z e d , blood was taken from 9 non-vaccinated and 15 v a c c i n a t e d f i s h from each treatment r e p l i c a t e (except treatments 11, 12, and 13) by s e v e r i n g the caudal peduncle. Two blood samples were taken from each f i s h ( h e p a r i n i z e d micro-hematocrit tubes, 75 mm, 1.1 -1.2 mm ID) and c e n t r i f u g e d (Clay-Adams A u t o c r i t C e n t r i f u g e ) f o r 3 minutes. Hematocrit was measured and the c e l l s separated from the plasma by breaking the tube. The tube ends were s e a l e d with p l a s t o c i n e and the plasma samples s t o r e d f r o z e n . Carcasses of v a c c i n a t e d f i s h were s t o r e d whole in s e a l e d p l a s t i c f r e e z e r - b a g s by r e p l i c a t e and kept frozen (-18 ° C ) . 39 3.7.3 L i v e V i b r i o c h a l l e n g e s On 09/05/1986 a l l f i s h were removed from each tank by c o l l e c t i o n i n a p e r f o r a t e d bucket that was then 2 immersed f o r 15 minutes i n a s a l i n e s o l u t i o n c o n t a i n i n g l i v e Vibrio anguillarum b a c t e r i a . At the end of t h i s p e r i o d the f i s h were r e t u r n e d as a group to t h e i r a p p r o r i a t e tank. New suspensions were mixed f o r each tank. Tanks were t r e a t e d i n the same order that they o c c u r r e d i n the room. Water temperature was 8.0 °C. Subsequent deaths were recorded d a i l y . I t was decided to c h a l l e n g e the f i s h on 03/06/1986 with V. ordalii because of the f a i l u r e of the pr e v i o u s V. anguillarum c h a l l e n g e . A stock c u l t u r e of a l o c a l l y i s o l a t e d V. ordalii was obtained from the P a c i f i c B i o l o g i c a l S t a t i o n (Nanaimo, B.C.) and i n o c u l a t e d i n t o 2 - 1 0 1 f l a s k s of t r y p t i c soy broth to which 0.5% NaCl was added. In a d d i t i o n , each f l a s k r e c e i v e d 2 ml of Baker Antifoam B. The f l a s k s were v i g o r o u s l y a e r a t e d and incubated at 20 °C f o r 56 hours. At the end of the i n c u b a t i o n p e r i o d the contents of the f l a s k s were mixed and the numbers of v i a b l e organisms determined by 9.9 L g S a l i n e (1% NaCl):0.1 L b a c t e r i a l s o l u t i o n (10 colony forming u n i t s / m l i n growth medium). 40 standard drop p l a c i n g technique. The same c h a l l e n g e procedure as before was used, except that four tanks (which c o n t a i n e d f i s h from treatments 5, 9, 11, and 12) were c h a l l e n g e d f o r 25 minutes while a l l other tanks were c h a l l e n g e d f o r 14 minutes ( i t was decided a f t e r the 25 minute c h a l l e n g e that exposure times longer than 14 minutes s t r e s s e d the f i s h too s e v e r e l y ) . M o r t a l i t y was recorded as b e f o r e . 3.7.4 Antibody t e s t F i s h blood plasma was analyzed f o r a n t i b o d i e s a g a i n s t V. anguillarum, one of the antigens i n the commercial v a c c i n e , using a micro - t i t r a t i o n technique (06 - 08/08/1986). T h i s technique i n v o l v e d p l a c i n g 25 uL of s a l i n e i n t o a l l the w e l l s of U-bottom p l a s t i c micro-t i t r e p l a t e s . Recently thawed f i s h blood plasma was mixed i n t o a w e l l at the beginning of a row, one sample to a row. Using m i c r o - c a p i l l a r y tube (Drummond Microcaps, Drummond S c i e n t i f i c Co., Broomall, Pa.) 25 uL of t e s t plasma and s a l i n e was mixed i n t o the next w e l l c o n t a i n i n g s a l i n e . The f i n a l column co n t a i n e d 25 uL s a l i n e o n l y , as a negative c o n t r o l . Each w e l l then r e c e i v e d 25 uL a n t i g e n (V. anguillarum i n s a l i n e and 41 d i l u t e f o r m a l i n ) . The r e s u l t i n g s e r i a l d i l u t i o n s c o n t a i n e d t e s t plasma:antigen i n the f o l l o w i n g p r o p o r t i o n s : 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128. P o s i t i v e c o n t r o l s c o n t a i n e d r a b b i t anti-serum (Connaught L a b o r a t o r i e s , Willowdale, O n t a r i o ) i n replacement of t e s t plasma. The p l a t e s were incubated o v e r - n i g h t ( >16 hrs.) at 15 °C. The presence of a f l o c u l e n t p r e c i p i t a t e on the bottom of the w e l l i n d i c a t e d a p o s i t i v e r e s u l t . The presence of a w e l l - d e f i n e d " p e l l e t " i n the c e n t r e i n d i c a t e d a negative response. Plasma from f i s h fed d i e t s 2, 3, 4, 5, 7, 10, 11, and 12 were t e s t e d i n t h i s manner. Between 20 and 24 samples from each d i e t a r y treatment were used i n each t e s t ; approximately 9 v a c c i n a t e d and 15 non-vaccinated. 3.7.5 A g g l u t i n a t i o n The a b i l i t y of the f i s h blood plasma to a g g l u t i n a t e and/or immobilize l i v e Vibrio ordalii was estimated r e l a t i v e to a standard obtained from a coho salmon s u r v i v o r of a v i b r i o c h a l l e n g e . T h i s t e s t was made at ambient temperature ( i . e., the temperature on a microscope stage at moderate v i s i b l e l i g h t i n t e n s i t y ) . A c u l t u r e of h i g h l y m o t i l e V. ordalii organisms was mixed 42 wit Ii approximately the same volume of r e c e n t l y thawed blood plasma from t e s t f i s h . The mixture, having a volume of 5 - 10 uL, was observed under o i l - i m m e r s i o n with a l i g h t - m i c r o s c o p e . The m o t i l i t y and a g g l u t i n a t i n g tendencies of the organisms were s u b j e c t i v e l y estimated r e l a t i v e to a p o s i t i v e standard (which c o n t a i n e d m o t i l e organisms and commercially prepared r a b b i t a n tiserum), and a negative c o n t r o l ( c o n t a i n i n g m o t i l e organisms and s a l i n e ) . Observation began approximately 3 minutes post-mixing and l a s t e d s e v e r a l minutes. S e v e r a l f i e l d s were observed. At l e a s t 5 samples from v a c c i n a t e d f i s h and at l e a s t 2 non-vaccinated f i s h were used from each group of f i s h t e s t e d . 3.8 Body composition 3.8.1 Dry matter, % l i p i d , f a t t y a c i d composition The 9 non-vaccinated f i s h from each treatment were froze n whole at -18 °C and s t o r e d without a i r i n p l a s t i c f r e e z e r bags f o r about 2 months. A f t e r t h i s time, one set of f i s h from each treatment was thawed and ground whole i n a Waring blendor u n t i l homogeneous. Dry matter was determined using t r i p l i c a t e samples. The l i p i d content of the f i s h was determined using a m o d i f i c a t i o n 43 of the e x t r a c t i o n method of B l i g h and Dyer (1959). About 50 g of f i s h homogenate was p l a c e d i n a Waring blender and mixed f o r 4 minutes with 50 ml c h l o r o f o r m ( s t a b i l i z e d with 75 ppm w/w b u t y l hydroxy-toluene (BHT)) and 100 ml of methanol. The s l u r r y was then passed through a s i n t e r e d g l a s s f unnel with l i g h t s u c t i o n . The remaining dry matter was mixed with 50 ml c h l o r o f o r m and r e f i l t e r e d . The r e s u l t i n g s o l v e n t s o l u t i o n was t r a n s f e r r e d to a 250 ml g l a s s graduated c y l i n d e r and 50 ml water (0.88% w/w KC1) was added. Chloroform and methanol (1:1 v/v) were added to make the volume up to 250 ml (some c h l o r o f o r m and methanol were l o s t d u r i n g mixing and f i l t e r i n g , presumably mostly from e v a p o r a t i o n ) . The s o l v e n t s were allowed to separate over s e v e r a l hours, a f t e r which time the methanol/aqueous l a y e r was drawn o f f . The weight of l i p i d c o n t ained i n t r i p l i c a t e 4 ml samples of the c h l o r o f o r m / l i p i d l a y e r was used to determine the % l i p i d content of the whole f i s h . The l i p i d was s t o r e d under n i t r o g e n at below 0 °C. E x t r a c t e d d i e t a r y l i p i d was methyl e s t e r i f i e d using the b a s e - c a t a l y z a t i o n procedure d e s c r i b e d by C h r i s t i e (1982). 44 3.8.2 P h o s p h o l i p i d s The p h o s p h o l i p i d s of each l i p i d e x t r a c t were i s o l a t e d with a s i l i c i c a c i d column, a f t e r the manner d e s c r i b e d by C h r i s t i e (1982). The column volume was 21 ml, and the s i l i c i c a c i d was hydrated to about 5% (w/w). S i l i c a Gel 60, 70-230 mesh was o b t a i n e d from E. Merck L t d . (Darmstadt, W. Germany). About 500 mg l i p i d was f i l t e r e d through the column. The column was f l u s h e d with 210 ml c h l o r o f o r m ( c o n t a i n i n g about 1% non-polar s t a b i l i z e r ) , and the p h o s p h o l i p i d s were then f l u s h e d out of the system with 210 ml methanol. The s o l v e n t i n the r e s u l t i n g s o l u t i o n was r a p i d l y removed under reduced pressure and g e n t l e h e a t i n g . The r e s i d u e , assumed to be almost e n t i r e l y p h o s p h o l i p i d , was methyl e s t e r i f i e d u s i n g the b a s e - c a t a l y z e d procedure d e s c r i b e d by C h r i s t i e (1982). The ester/hexane s o l u t i o n s thus prepared were s t o r e d under n i t r o g e n below 0 °C u n t i l i n j e c t i o n i n t o the g a s - l i q u i d chromatograph. The c o n d i t i o n s of the g a s - l i q u i d chromatograph were as d e s c r i b e d e a r l i e r . The n e u t r a l l i p i d s e l u t e d from the s i l i c i c a c i d column were p e r i o d i c a l l y t e s t e d f o r the presence of p h o s p h o l i p i d using the method d e s c r i b e d below and were determined to be p h o s p h o l i p i d f r e e . 45 The p h o s p h o l i p i d content of the f i s h l i p i d s from treatments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 was determined using a m o d i f i c a t i o n of the c o l o u r i m e t r i c procedure of Raheja et al (1973). T h i s technique used a chromogenic s o l u t i o n prepared as f o l l o w s : "16 g of ammonium molybdate i s d i s s o l v e d i n 120 ml H 20 to gi v e s o l u t i o n I. 40 ml of cone. HCI and 10 ml of Hg are shaken with 80 ml of s o l u t i o n I f o r 30 min. to g i v e , a f t e r f i l t r a t i o n , s o l u t i o n I I . 200 ml of cone. H^SO. i s added c a r e f u l l y to the remainder of s o l u t i o n I. To the r e s u l t a n t s o l u t i o n i s added s o l u t i o n II to gi v e s o l u t i o n I I I . 45 ml of methanol, 5 ml of chloroform, and 20 ml of H 20 are added to 25 ml of s o l u t i o n III to gi v e the chromo-genic s o l u t i o n . " About 9 mg of o i l from each treatment was a c c u r a t e l y measured i n t o a g l a s s - s t o p p e r e d t e s t tube and 0.4 ml of chl o r o f o r m and 0.2 ml of chromogenic s o l u t i o n . The r e s u l t i n g s o l u t i o n was heated i n a b o i l i n g water bath f o r 80 s e c , then c o o l e d t o room temperature. A f t e r 5 minutes, 5.0 ml of ch l o r o f o r m was added and the s o l u t i o n was mixed and the absorbency measured on a Beckman DB spectrophotometer (Beckman Instruments, Inc., F u l e r t o n , Ca. USA) at 710 nm. The readings were c a l i b r a t e d using beef b r a i n p h o s p h a t i d y l c h o l i n e as a standard (10 mg/ml from Serdary Research Inc., London, Ont, Canada), and the r e s u l t s were c a l c u l a t e d r e l a t i v e 46 to d i p a l m i t o y l - p h o s p h a t i d y l c h o l i n e . 3.9 S t a t i s t i c a l procedures 4 Mean weights, s p e c i f i c growth r a t e , body weight 5 g a i n s , feed e f f i c i e n c y , hematocrit v a l u e s , and m o r t a l i t y data were su b j e c t e d to a n a l y s i s of v a r i a n c e procedures (Snedecor and Cochran, 1980). Analyses were done on tank means. Treatment means were separated using REGWF^ (maximum experimentwise e r r o r r a t e = 0.05). Analyses were performed using data from a l l treatments, as w e l l as f a c t o r i a l l y (2 l e v e l s of vit a m i n E supplementation by 3 l e v e l s of l i p i d o x i d a t i o n ) , using treatments 2, 3, 4, 6, 7, 8, and 9. T - t e s t s were performed on the hematocrit v a l u e s and average body weights at 205 days to t e s t the e f f e c t of v a c c i n a t i o n on weight and hemato c r i t . In a d d i t i o n , the mean body weights of f i s h i n treatments 2, 3, 4, 7, 8, and 9 were Beef b r a i n , l e c i t h i n i s p r i m a r i l y d i p a l m o t y l . Merck Index. 8 e d i t i o n , 1968. S p e c i f i c growth rate was c a l c u l a t e d as f o l l o w s : 100 ln(body w e i g h t ) t l - ln(body w e i g h t ) t Q / ( t l - t 0 ) . Feed e f f i c i e n c y i s expressed as body weight gain ( g ) / f e e d intake ( g ) . REGWF r e f e r s to a m u l t i p l e range t e s t that holds over-a l l e r r o r r a t e constant (SAS, 1985). 47 analysed f a c t o r i a l l y at a l l weigh times s i m u l t a n e o u s l y 7 . D i f f e r e n c e s between treatment means were c o n s i d e r e d s i g n i f i c a n t at the 5% l e v e l of p r o b a b i l i t y . A c u b i c f u n c t i o n of time was d e r i v e d using a gen e r a l l i n e a r means model (SAS, 1985). 48 4 R e s u l t s 4.1 D i e t s and o i l s 4.1.1 D i e t composition and proximate a n a l y s i s The r e s u l t s of proximate a n a l y s e s of the d i e t s and f r e e z e - d r i e d p o l l o c k muscle and euphausids are given i n Tables 4, and 5, r e s p e c t i v e l y 1 . The proximate compositions of the d i e t s were s i m i l a r , as would be expected, s i n c e a l l were made using the same b a s a l mix. Most d i e t a r y l i p i d was s u p p l i e d by h e r r i n g o i l , with the r e s i d u a l l i p i d i n p o l l o c k muscle and euphausids c o n t r i b u t i n g very l i t t l e . 4.1.2 O x i d a t i o n and f a t t y a c i d p r o f i l e of o i l s Temporal changes i n the peroxide values of the h e r r i n g o i l s used i n r e l a t i o n to the treatments i n t h i s study are recorded i n Table 6. As expected, net peroxide formation was highest i n the o i l s which were s u b j e c t e d to the most heat and a e r a t i o n . Those o i l s which were not s u b j e c t e d to a e r a t i o n and h e a t i n g d i d not achieve high net l e v e l s of peroxide c o n c e n t r a t i o n . The o i l that was heated and aerated f o r a moderate amount of time ( o i l 3) 1 Proximate analyses were c a r r i e d out at Cantest L t d . 49 Table 4 - Proximate analyses of the d i e t s ( m o i s t u r e - f r e e b a s i s ; 25/09/1985). Di e t Treatment H„0 Ash CL CP 1 Unstab. 10 .71 6. 56 16.80 48. 49 2 LOX 10 .49 6. 57 16.49 48. 49 3 MOX 10 .73 6. 41 16.93 48. 95 4 HOX 10 .64 6. 35 1 6.26 48. 34 5 Corn 10 .78 6. 43 17.07 47. 97 6 Unstab. +E 10 .61 6. 37 16.47 48. 55 7 LOX + E 10 .70 6. 44 16.53 48. 60 8 MOX + E 10 .66 6. 31 16.12 48. 69 9 HOX + E 10 .56 6. 47 16.04 48. 64 10 LOX+HOX 10 .33 6. 45 1 6.57 48. 51 See t e x t f o r f u l l d e s c r i p t i o n of treatments. Unstab. = u n s t a b i l i z e d o i l ; LOX = unoxidized, s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l ; +E = e x t r a supplemental vi t a m i n E (1000 IU/kg dry d i e t ) . Crude l i p i d content was determined by the B l i g h and Dyer e x t r a c t i o n procedure d i s c u s s e d i n M a t e r i a l s and Methods. Crude p r o t e i n was estimated from n i t r o g e n (N) d e t e r m i n a t i o n , using K j e l d a h l ' s technique (CP = %N X 6.25) . 50 Table 5 - Proximate a n a l y s i s of f r e e z e - d r i e d p o l l o c k and f r e e z e - d r i e d euphausids ( a i r - d r y ) . %H 2° %Ash %CL1 %CP 2 P o l l o c k muscle 2. 82 6. 30 2.92 89. 05 Euphausids 5. 88 12. 89 16.06 66. 0 Crude l i p i d content was determined by the B l i g h and Dyer (1959) e x t r a c t i o n procedure. Crude p r o t e i n was e s timated from n i t r o g e n (N) d e t e r m i n a t i o n , u s i n g K j e l d a h l ' s technique (CP = %N X 6.25). 51 Table 6 - Temporal changes in peroxide values of the h e r r i n g o i l s r e l a t i v e to treatment (AOCS, 1973). O i l Number 1 2 3 4 DATE DIETS [2,7,10] [1,6] [3,8] [4,9,10] Peroxide value (meq/kg) 20/06/85 2.3 21 2.5 23 25 26 2.8 28 01/07 2.2 02 2.8 05 07 08 09 10 1 1 17 5.0 22 24 6.5 31 6.6 02/08 10/09 10.1 2.2 2.5 2.6 2.7 3.2 4.3 3.4 20.3 5.3 13.7 33.1 81.0 82.2 89.7 22.3 89.5 89.0 5.0 29.2 67. 1 50.9 89.5 6.4 53.8 81.1 6.1 55.8 57. 1 59.5 53.9 10.2 44.7 59.0 O i l 1 = s t a b i l i z e d , low o x i d i z e d o i l ( c o n t r o l ) . O i l 2 = u n s t a b i l i z e d o i l ; no ethoxyquin added. O i l 3 = moderately o x i d i z e d o i l (MOX). O i l 4 = h i g h l y o x i d i z e d o i l (HOX). See t e x t f o r more complete d e s c r i p t i o n . 52 had a moderate p r o d u c t i o n of peroxides as measured. The t o t a l p r o d u c t i o n of peroxides i s hard to q u a n t i f y , s i n c e r a t e s of formation and breakdown are not known. No attempt has been made i n t h i s study to do so. Knowledge of the peroxide c o n c e n t r a t i o n present over time can provide u s e f u l data, which, when coupled with other i n f o r m a t i o n , may a i d i n determining the r e l a t i v e o x i d a t i v e s t a t e of l i p i d s . The n 3 - f a t t y a c i d (n3FA) content ( r e l a t i v e to the t o t a l f a t t y a c i d content) of the d i e t s , the i o d i n e numbers of the o i l s used to make the d i e t s , and the i o d i n e numbers of the o i l s e x t r a c t e d from the d i e t s are pr o v i d e d i n Tables 7, 8, and 9. The d i e t a r y n3FA contents were hi g h e s t i n d i e t 2 ( c o n t r o l ) and d i e t 7 ( c o n t r o l with e x t r a v i t a m i n E ) . Diet 5 (corn o i l ) had the lowest n3FA content of the d i e t s . D i e t 4 ( c o n t a i n i n g h i g h l y o x i d i z e d o i l , (HOX)) and d i e t 9 ( c o n t a i n i n g HOX with e x t r a v i t a m i n E) had the lowest amount of n3FA of any of the d i e t s c o n t a i n i n g h e r r i n g o i l . D i e t s 3 and 8 ( c o n t a i n i n g moderately o x i d i z e d o i l (MOX), and MOX + e x t r a v i t a m i n E, r e s p e c t i v e l y ) had n3FA present in somewhat lower c o n c e n t r a t i o n s than d i e t s 2 or 7. D i e t s 1 and 6 ( c o n t a i n i n g u n s t a b i l i z e d o i l , and u n s t a b i l i z e d o i l + v i t a m i n E, r e s p e c t i v e l y ) had l e s s n3FA than d i d 53 Table 7 - n3 F a t t y a c i d (n3FA) p r o f i l e s of d i e t a r y l i p i d D i e t 2 Treatment %n3FA 3 %n3FA 4 1 Unstab. o i l 7.69 1 .29 2 LOX 12.31 2.03 3 MOX 10.71 1.81 4 HOX 5.79 0.94 5 Corn 3.03 0.52 6 Unstab. + E 10.08 1 .66 7 LOX + E 1 2.02 1 .99 8 MOX + E 1 0.40 1 .68 9 HOX + E 5.53 0.89 1 0 LOX + HOX(1:1) 7.23 1 .20 Date e s t e r i f i e d : 10/04/1986. Date run on g a s - l i q u i d chromatograph: 11/04/1986. See t e x t f o r f u l l d e s c r i p t i o n of treatments. Unstab. = u n s t a b i l i z e d o i l ; LOX = unox i d i z e d , s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l ; +E = e x t r a supplemental v i t a m i n E (1000 IU/kg dry d i e t ) . Sum of n3 f a t t y a c i d s (18:3, 20:5, 22:5, and 22:6) as percent weight of t o t a l f a t t y a c i d s . Sum of n3 f a t t y a c i d s (18:3, 20:5, 22:5, and 22:6) as percent of d i e t (assuming weight of crude l i p i d i s due to f a t t y a c i d s ) . 54 Table 8 - Iodine nurnbers of the h e r r i n g o i l s used i n t h i s study r e l a t i v e to treatment. 1 O i l number D i e t s 2 Treatment 3 Iodine number 1 2,7,10 LOX 1 12.7 a 2 1,6 Unstab. 111.0 3 3,8 MOX 102.4 b 4 4,9,10 HOX 80.6° +SEM4 0.472 Wij's method (AOAC, 1973). A n a l y s i s performed on 18/11/1985. Values are means of 2 samples. Value of o i l number 2 i s from one sample. See t e x t f o r f u l l d e s c r i p t i o n of treatments. Unstab. = u n s t a b i l i z e d o i l ; LOX = un o x i d i z e d , s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l . Means with d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p < 0.05) . Standard e r r o r of the means. 55 Table 9 - Iodine number 1 (IN) of d i e t a r y l i p i d a f t e r storage f o r e i g h t months. D i e t Treatment IN* 1 Unstab. 2 LOX 3 MOX 4 HOX 5 Corn o i l 6 Unstab. + E 7 LOX + E 8 MOX + E 9 HOX + E 10 LOX+HOX (1:1) +SEM4 114.5! 112.0' 97.7 89.8 J 113.6 108, 1 06, 108, 90, 96, a, b ,a,b Ja,b 3.36 Wij's method AOAC (1980). Date of d e t e r m i n a t i o n : 08/04/1986. Values are means of 2 samples. Value f o r d i e t 5 i s from one sample. See t e x t f o r f u l l d e s c r i p t i o n of treatments. Unstab. = u n s t a b i l i z e d o i l ; LOX = un o x i d i z e d , s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX h i g h l y o x i d i z e d o i l ; +E = e x t r a supplemental v i t a m i n E (1000 IU/kg dry d i e t ) . Means with d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p < 0.05). Standard e r r o r of the means. 56 Table 9b - Iodine number (IN) of d i e t a r y l i p i d a f t e r storage f o r e i g h t months. Mean values of d i e t s i n i t i a l l y c o n t a i n i n g o i l of the same o x i d a t i v e s t a t e . O i l D i e t s IN LOX 2 + 7 109.3 a MOX 3 + 8 103.4 a HOX 4 + 9 90.0 b +SEM4 2.15 LOX = un o x i d i z e d , s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l . D i e t s combined to c a l c u l a t e treatment means. Means with d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p < 0.05) Standard e r r o r of the means. 57 d i e t 2, although d i e t 6 had c o n s i d e r a b l y more than d i d d i e t 1. D i e t 10 ( c o n t a i n i n g _low o x i d i z e d o i l (LOX) + HOX) co n t a i n e d l e s s n3FA than d i d d i e t 2. D i e t s 1, 4, 5, 9, and 10 had l e s s than the NRC (1981) requirement f o r n3FA, as a percent of d i e t . The i o d i n e number measures the degree of u n s a t u r a t i o n of l i p i d s and as such can be used as an in v e r s e measure of the r e l a t i v e extent of l i p i d o x i d a t i o n . The iodine' number of the o i l used f o r d i e t s 2, 7, and 10 ( o i l 1) was the h i g h e s t , i n d i c a t i n g the hig h e s t c o n c e n t r a t i o n of u n s a t u r a t i o n s i t e s on the f a t t y a c i d s . O i l 4 had a c o n s i d e r a b l y lower i o d i n e number than d i d o i l 1. The o i l used f o r d i e t s 3 and 8 ( o i l 3) had a lower i o d i n e number than o i l 1, but a higher i o d i n e number than o i l 4. The p a t t e r n f o r i o d i n e numbers of o i l s of d i e t s c o n t a i n i n g LOX, MOX and HOX (near the end of the "growth" pa r t of the experiment) was s i m i l a r to that observed f o r n3FA con t e n t . Those d i e t s c o n t a i n i n g HOX ( o i l 4) had c o n s i d e r a b l y lower i o d i n e number than the other h e r r i n g o i l d i e t s . D i e t 3, c o n t a i n i n g MOX with no e x t r a v i t a m i n E, had a lower i o d i n e number than the c o n t r o l , but one that was higher than those c o n t a i n i n g HOX. D i e t 8, c o n t a i n i n g MOX with e x t r a v i t a m i n E, had as high an 58 i o d i n e number as those d i e t s c o n t a i n i n g LOX. O i l s 1, 3, and 4 had 2 - t h i o b a r b i t u r i c a c i d numbers of 41, 272, and 252 mmols/kg r e s p e c t i v e l y , whereas the t o t a l o i l i n d i e t s 1, 2, 3, and 4 had v a l u e s of 97, 10, 71, and 66 mmols/kg o i l , r e s p e c t i v e l y . Some of the t e s t s on the d i e t s r e s u l t e d i n the formation of a yellow pigment. The extent of the i n t e r f e r e n c e of t h i s pigment on the normal pink pigment i s not known, and those r e s u l t s were not recorded. 4.1.3 P h y s i c a l c h a r a c t e r i s t i c s of o i l and p e l l e t s The p e l l e t s were very l i g h t brown in c o l o u r , with those c o n t a i n i n g more h i g h l y o x i d i z e d HO being somewhat darker. The p e l l e t s were denser than water and, in s t i l l water, sank at about 10 cm/sec. Those d i e t s c o n t a i n i n g more h i g h l y o x i d i z e d f i s h o i l had more f i n e s and had a d i f f e r e n t odour than those c o n t a i n i n g HO at the lowest l e v e l of o x i d a t i o n . A l l d i e t s maintained t h e i r shape in water f o r many hours. 59 4.2 Growth and feed e f f i c i e n c y F i g u r e s 3a-b show the p a t t e r n of growth of the f i s h i n r e l a t i o n to each treatment (see a l s o Table 10) d u r i n g stage one of the experiment (the f i n a l body weight i s of non-vaccinated f i s h o n l y ) . The o v e r a l l body weight ga i n s , feed e f f i c i e n c i e s , and s p e c i f i c growth r a t e s of the groups are given i n Table 11. No s i g n i f i c a n t e f f e c t of treatment was found when the mean body weight gains of the groups were compared between 0 weeks and 29 weeks, 12 weeks and 29 weeks, or 0 weeks and 6 weeks (p > 0.05). There was no s i g n i f i c a n t e f f e c t of l i p i d o x i d a t i o n or v i t a m i n E l e v e l on growth ra t e when treatments 2, 3, 4, 7, 8, and 9 were compared f a c t o r i a l l y (p > 0.05) u s i n g data from the same times. Using data from a l l times s i m u l t a n e o u s l y i n a 2X3 f a c t o r i a l f a s h i o n , however, demonstrated a s i g n i f i c a n t c u b i c i n t e r a c t i o n of time with l i p i d o x i d a t i o n (p < 0.05), but no e f f e c t of v i t a m i n E supplementation. The c u b i c r e l a t i o n of time to mean body weight i s apparent from the p a t t e r n of body weights d e p i c t e d i n F i g u r e s 3a-b, and i s a t t r i b u t a b l e to the change of water temperature. F i g u r e 4 shows the mean body weights of the f i s h f ed d i e t s c o n t a i n i n g o i l o x i d i z e d to d i f f e r e n t Figure 3a - Effect of treatments 12,3,4 on mean body weight over time 2 5 n 20 H «3 >» T J O DD C o (D 15 10 H Diet used A 1, unstab. oil + Water temp. y • 2, Low 0^id_ized_Oil__ _ O 3, Moderately Oxidized Oil O 4, Highly Oxidized Oil 5 - A - 2 0 a o 15 D 10 CD CL E .CD 0 5 10 15 20 Time (weeks) 25 30 CTl O Figure 3b - Effect of treatments 6,7,8,9 on mean body weight over time Diets used. A 6, unstab. oil+vit.E 5-A .A* • 7, Low Ox id jzedOi l+v i t ^ _ _ O 8, Moderately Oxidized.Oil+vit.E O 9, Highly Oxidized Oil+vit.E 0 10 15 20 Time (weeks) 25 30 Fioure 4 - Effect of dietary lipid oxidation on mean body weight over time 25 1 20 H CT) x : CJ) x> o CQ C o CD 15 10 H Diets used • 2 and 7 - Low 0xidi_zed_0i]_ _ O 3 and 8 - Moderately Oxjdjzed^Oil O 4 and_9 - Highly Oxidized Oil • ..cr 5-0 4 5 >-...-o* CTl to 0 10 -T~~ 15 20 25 30 Time (weeks) 63 Table 10 - Mean body weights (g) of the groups at d i f f e r e n t times d u r i n g the experiment. Time on treatment (days) D i e t Treatment 2 0 d 42 d 86 d 205 d ( T ) 3 205 d(NV) 4 1 unstab. 4. 99 8.79 10. 70 19. 42 19. 66 2 c o n t r o l 5. 16 10.08 1 1 . 04 20. 86 21 . 19 3 MOX 5. 10 8.37 9. 46 18. 47 18. 41 4 HOX 5. 38 8.83 10. 70 18. 47 18. 89 5 corn o i l 5. 49 8.71 9. 90 18. 40 18. 73 6 unstab.+E 5. 01 8.42 9. 57 17. 93 18. 03 7 control+E 5. 1 3 9.50 10. 93 20. 40 20. 91 8 MOX+E 5. 06 9.32 10. 47 18. 92 19. 61 9 HOX+E 5. 1 1 9.24 10. 89 19. 42 20. 26 10 control+HOX 5. 25 9.72 1 1 . 75 19. 62 20. 24 1 1 p-f unstab. 5. 1 5 9.43 10. 79 20. 30 20. 77 12 p-f MOX 5. 21 8.89 10. 44 18. 94 19. 61 13 p-f HOX 5. 21 8.97 10. 1 3 19. 36 19. 77 + SEM 5 0. 083 0.346 0. 527 1 . 063 1 . 052 Means w i t h i n columns were not s i g n i f i c a n t l y d i f f e r e n t (p > 0.05) . See tex t f o r f u l l d e s c r i p t i o n of treatments. Unstab. = u n s t a b i l i z e d o i l ; c o n t r o l = u n o x i d i z e d , s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l ; +E = e x t r a supplemental v i t a m i n E; p-f = p a i r - f e d with c o n t r o l d i e t to the intake of s p e c i f i e d treatment. Mean body weight of a l l f i s h i n treatment. Mean body weight of non-vaccinated f i s h . Standard e r r o r of the means. 6 4 Table 11 - Body weight gain (BWG), feed i n t a k e / f i s h ( F I ) , BWG/feed intake ( F I ) , s p e c i f i c growth r a t e (SGR) ( 0 d - 2 0 5 d ) . Treatment 1 BWG(g) 2 F l ( g ) BWG/FI(g/g) 3 SGR 2 1 unstab. 1 4 . 6 7 2 c o n t r o l 1 6 . 0 3 3 MOX 1 3 . 3 1 4 4 HOX 1 3 . 5 1 5 4 corn o i l 1 3 . 5 4 6 unstab.+E 1 3 . 0 2 7 control+E 1 5 . 7 8 8 MOX+E 1 4 . 5 5 9 HOX+E 1 5 . 1 5 1 0 control+HOX 1 4 . 9 9 1 1 p-f unstab. 1 5 . 6 2 1 2 p-f MOX 1 4 . 4 0 1 3 p-f HOX 1 4 . 5 6 +SEM 1 . 0 8 0 1 6 . 6 . 8 6 8 £ . 6 6 7 1 7 . 3 . 9 1 6 : ! L - 6 8 9 1 5 . 2 . 8 7 4 a D C . 6 2 6 1 7 . 1 . 7 6 4 e . 6 1 3 1 7 . 3 - 7 9 5 , , . 6 2 6 1 5 . 6 . 8 2 6 D r . 6 2 5 1 7 . 8 • 9 0 5 ? ^ , . 6 8 6 1 6 . 4 . 8 4 6 ^ C d e . 6 6 0 1 8 . 3 - 7 7 4 ° ^ o . 6 7 2 1 8 . 2 . 8 0 5 c a e . 6 5 9 1 6 . 1 . 9 3 3 a . 6 7 7 1 4 . 6 . 9 4 1 . 6 4 6 1 6 . 0 . 8 8 8 a D C . 6 5 1 0 . 0 1 8 9 0 . 0 2 2 7 See t e x t f o r f u l l d e s c r i p t i o n of treatments. Unstab. = u n s t a b i l i z e d o i l ; c o n t r o l = un o x i d i z e d , s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l ; +E = e x t r a supplemental v i t a m i n E; p-f = p a i r - f e d with c o n t r o l d i e t to the intake of s p e c i f i e d treatment. Body weight gai n , and s p e c i f i c growth r a t e were not s i g n i f i c a n t l y a f f e c t e d by o x i d a t i o n or v i t a m i n E (p > 0 . 0 5 ) . Means w i t h i n columns with d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p < 0 . 0 5 ) . C a l c u l a t i o n s used non-vaccinated f i s h , except body weight g a i n / f e e d i n t a k e , which used a l l f i s h . Body weight g a i n / f e e d intake was not used i n ANOVA because fewer f i s h i n one tank made feed intake data u n r e l i a b l e . Standard e r r o r of the means. 65 degrees (combining both l e v e l s of v i t a m i n E ) . F i s h f e d the c o n t r o l d i e t c o n s i s t e n t l y had the f a s t e s t growth r a t e , and those fed d i e t s c o n t a i n i n g moderately o x i d i z e d o i l had the slowest growth r a t e . V a c c i n a t e d f i s h had a s i g n i f i c a n t l y lower mean body weight than non-vaccinated f i s h (p < 0.01; data not shown). The average weight d i f f e r e n c e was 1.28 g. The reduced weight gain of the v a c c i n a t e d f i s h can be a t t r i b u t e d to the s t r e s s of ha n d l i n g and v a c c i n a t i o n . Body weight g a i n / f e e d intake was s i g n i f i c a n t l y i n f l u e n c e d by treatment (p < 0.01). When c a l c u l a t i n g o v e r a l l body weight g a i n / f e e d i n t a k e , the weighted average of a l l f i s h i n each tank was used, and the feed intake was ad j u s t e d f o r f i s h m o r t a l i t y . A 2-way f a c t o r i a l ANOVA of body weight g a i n / f e e d intake (using treatments 2, 3, 4, 7, 8, and 9; Table 12) showed that o x i d a t i o n s i g n i f i c a n t l y i n f l u e n c e d body weight g a i n / f e e d intake (p < 0.01) and that d i e t a r y v i t a m i n E had no s i g n i f i c a n t e f f e c t (p > 0.05). The f i s h fed d i e t s c o n t a i n i n g o i l with the lowest amount of o x i d a t i o n had the h i g h e s t body weight g a i n / f e e d i n t a k e , those fed d i e t s with the hig h e s t l e v e l of o x i d a t i o n had the lowest body weight g a i n / f e e d i n t a k e . The f i s h i n the " p a i r - f e d " treatments o f t e n would 66 Table 12 - Body Weight Gain/Feed Intake (combining values f o r low and high v i t a m i n E supplementation). O i l 1 D i e t s 2 BWG/FI(g/g) 3 LOX 2 + 7 0.910 a MOX 3 + 8 0.860 b HOX 4 + 9 0.769° + SEM 4 0.0108 LOX = low o x i d i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l . D i e t s combined to c a l c u l a t e treatment means. Means with d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (p < 0.05) Standard e r r o r of the means. 67 not eat as much c o n t r o l d i e t as t h e i r c o h o r t s i n the " o x i d i z e d o i l " treatments. T h i s i s evident from the data on o v e r a l l feed consumption (Table 11). D e s p i t e the lower feed consumption in treatments 11, 12, and 13, growth was g r e a t e r , r e l a t i v e to that noted f o r f i s h on treatments 1, 3, and 4, r e s p e c t i v e l y . T h i s i s f u r t h e r evidence that the c o n t r o l d i e t was b e t t e r a b l e to supply the n u t r i e n t s r e q u i r e d f o r growth than were the d i e t s c o n t a i n i n g o x i d i z e d o i l . 4.3 F i s h m o r t a l i t y d u r i n g "growth phase" On 07/10/1985 66 f i s h from treatment 5 (one tank) were i n a d v e r t e n t l y l o s t due to oxygen d e p r i v a t i o n . As the r e l i a b i l i t y of the estimate of feed i n t a k e was b e l i e v e d to be impaired, data from t h i s treatment (both tanks) were not used in a n a l y s i s of body weight g a i n / f e e d i n t a k e . On 13/02/1986 18 f i s h f a i l e d to recover from a n a e s t h e t i c i n one group on treatment 7. I t was not f e l t t h a t feed intake e s t i m a t i o n was impaired, i n t h i s i n s t a n c e , and so data from t h i s treatment were used ( a d j u s t e d f o r the fewer f i s h ) f o r body weight g a i n / f e e d i n t a k e a n a l y s i s . There were four other deaths d u r i n g stage one of the experiment. Body weight g a i n / f e e d 68 i n t a k e e s t i m a t e s were a d j u s t e d a c c o r d i n g l y . 4.4 H e m a t o c r i t V a l u e s H e m a t o c r i t v a l u e s of n o n - v a c c i n a t e d f i s h from each tr e a t m e n t a r e g i v e n i n T a b l e 13. The average h e m a t o c r i t v a l u e of v a c c i n a t e d f i s h was 45.3%, which was s i g n i f i c a n t l y (p < 0.01) lower than the 47.2% average h e m a t o c r i t v a l u e of n o n - v a c c i n a t e d f i s h . There was no s i g n i f i c a n t e f f e c t of d i e t a r y t r e a t m e n t on h e m a t o c r i t v a l u e (p > .10) f o r n o n - v a c c i n a t e d f i s h . 4.5 F i s h body c o m p o s i t i o n 4.5.1 Dry m a t t e r , l i p i d , and p h o s p h o l i p i d L i p i d c o n t e n t , dry m a t t e r c o n t e n t , and p h o s p h o l i p i d c o n t e n t (as p e r c e n t of t o t a l body l i p i d ) of the whole body a r e g i v e n i n T a b l e 14. The m o i s t u r e c o n t e n t of f i s h may v a r y d u r i n g p r o l o n g e d f r o z e n s t o r a g e . A l t h o u g h t h e r e a r e s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s between t r e a t m e n t s , t h e r e i s no o b v i o u s p a t t e r n f o r l i p i d or p h o s p h o l i p i d c o n t e n t among the t r e a t m e n t s . N e i t h e r o x i d a t i v e s t a t e of d i e t a r y l i p i d , nor v i t a m i n E c o n t e n t , by themselves seemed t o have had any c o n s i s t e n t i n f l u e n c e 69 Table 13 - Hematocrit value D i e t Treatment % hematocrit 1 Unstab. 47.3 2 LOX 46.6 3 MOX 47.0 4 HOX 44.8 5 Corn o i l 46.6 6 Unstab. +E 47.4 7 LOX + E 47.9 8 MOX + E 47.7 9 HOX + E 45.6 10 LOX + HOX (1:) 51.4 +SEM4 1 .52 Means of 2 r e p l i c a t e s of 9 non-vaccinated f i s h . See t e x t f o r f u l l d e s c r i p t i o n of treatments. Unstab. = u n s t a b i l i z e d o i l ; c o n t r o l = u n o x i d i z e d , s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l ; +E = e x t r a supplemental v i t a m i n E (1000 IU/kg dry d i e t ) . There was no s i g n i g i c a n t d i f f e r e n c e between means (p > 0.05). Standard e r r o r of the means. 70 Table 14 - F i s h Body A n a l y s i s D i e t Treatment %dry matter % l i p i d % p h o s p h o l i p i d 1 Unstab. 27.60 23.90 6.07 a 2 LOX 28.30 28.50 4.38 c d 3 MOX 27.32 28. 15 3.76 c d 4 HOX 27.54 24.39 4 . 5 8 b c d 5 Corn O i l 29.83 25.56 4 . 4 9 a b c 6 Unstab. +E 27.41 28.06 3.24 d 7 LOX + E 27.39 27.67 5.68 a b 8 MOX + E 27.53 24.89 3.29 d 9 HOX + E 27.31 24.92 3.97 c d 10 LOX+HOX (1:1) 28.45 26.89 4.07 c d A n a l y s i s of 9 whole f i s h from one group on each treatment. Values f o r %dry matter and % l i p i d are means of 3 o b s e r v a t i o n s . % P h o s p h o l i p i d values are means of 4 o b s e r v a t i o n s (value f o r d i e t 6 i s the mean of 2 o b s e r v a t i o n s ) . See t e x t f o r f u l l d e s c r i p t i o n of treatments. Unstab. = u n s t a b i l i z e d o i l ; LOX = un o x i d i z e d , s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l ; +E = e x t r a supplemental v i t a m i n E. %Dry matter b a s i s . P h o s p h o l i p i d as % l i p i d . D i f f e r e n t s u p e r s c r i p t s denote s t a t i s t i c a l l y d i f f e r e n t means (p < 0.05). Standard e r r o r of the means = 0.260. 71 on these c h a r a c t e r i s t i c s . 4.5.2 F a t t y a c i d p r o f i l e s of l i p i d and p h o s p h o l i p i d Table 15 g i v e s the l e v e l of f a t t y a c i d s of the n3 ( l i n o l e n i c ) s e r i e s as percent of body l i p i d and phospho-l i p i d . The n3FA content of t o t a l body l i p i d v a r i e d i n accordance with d i e t a r y content. The n3FA content of p h o s p h o l i p i d v a r i e d i n r e l a t i o n to d i e t a r y content to a l e s s e r degree suggesting that there may be some mechanism that p r e f e r e n t i a l l y i n c o r p o r a t e s n3FA i n t o p h o s p h o l i p i d s . T h i s became even more evident when the body n3FA content was c o n s i d e r e d (Table 16). Most n3FA e x i s t e d as docosa-hexaenoic a c i d (C22:6n3) i n both the t o t a l l i p i d , and p h o s p h o l i p i d f r a c t i o n s . 4.6 Immunocompetence The m o r t a l i t i e s of the f i s h a f t e r exposure to l i v e Vibrio ordalii are given i n Table 17. Very few v a c c i n a t e d f i s h d i e d as a consequence of exposure to the pathogen and n e a r l y a l l dead f i s h were p o s i t i v e f o r the organism. V a r i a t i o n between r e p l i c a t e groups w i t h i n a treatment was very l a r g e , and any d i f f e r e n c e s i n 72 Table 15 - % n 3 - f a t t y a c i d composition of t o t a l body l i p i d . and p h o s p h o l i p i d r e l a t i v e to d i e t a r y treatment . %TOTAL BODY FATTY ACIDS %PHOSPHOLIPID FATTY ACIDS D i e t 3 22:6 22:5 20:5 18:3 sum 22:6 22:5 20:5 18:3 sum 1 8.8 1 .3 3.2 0.6 13.9 36.3 2.0 4.0 2 ND 42.0 2 9.2 1 .3 3.1 0.5 14.2 35.5 1 .9 4.7 ND 42. 1 3 8.2 1 . 1 3.6 0.4 13.3 30.6 1 .4 4.3 ND 36.2 4 6.5 0.6 1 .6 0.4 9.1 35. 1 1 .2 4.3 ND 40.6 5 5.6 0.6 1 . 1 ND 7.3 22.2 0.8 1 .4 ND 24.4 6 8.4 1 .3 3.0 0.6 13.9 23.8 1 .6 3.2 ND 28.6 7 7.6 1 .4 3.3 0.6 12.9 25.6 1 .8 4.3 0.3 32.0 8 7.4 1 .1 2.7 0.5 11.6 31 .6 1 .8 3.6 ND 37.1 9 6.2 0.7 1 .8 0.4 9.0 24.2 1 .4 3.5 ND 29. 1 10 6.1 0.7 1 .9 0.5 9.1 19.8 1 .4 2.7 2.9 26.8 % weight b a s i s . Not d e t e c t e d . (< 0.5%). See t e x t f o r f u l l d e s c r i p t i o n . D i e t 1 - u n s t a b i l i z e d o i l ; D i e t 2 - s t a b i l i z e d , low o x i d i z e d o i l ; D i e t 3 -s t a b i l i z e d , moderately o x i d i z e d o i l ; D i e t 4 - s t a b i l i z e d , h i g h l y o x i d i z e d o i l ; D i e t 5 - corn o i l ; D i e t 6 -u n s t a b i l i z e d , o i l ; D i e t 7 - s t a b i l i z e d , low o x i d i z e d o i l , + v i t a m i n E; D i e t 8 - s t a b i l i z e d , moderately o x i d i z e d o i l + v i t a m i n E; D i e t 9 - s t a b i l i z e d , h i g h l y o x i d i z e d o i l + v i t a m i n E; D i e t 10 - LOX + HOX (1:1). 73 Table 16 - n 3 - f a t t y a c i d content i n the t r i g l y c e r i d e (TG) and p h o s p h o l i p i d (PL) f r a c t i o n s i n the f i s h body r e l a t i v e to d i e t a r y treatment . TG PL D i e t 2 Treatment %n3FA %n3FA 2 LOX 4.05 1 .84 3 MOX 3.74 1 .36 4 HOX 2.22 1 .86 5 Corn o i l 1 .87 1.10 7 LOX + v i t . E 3.57 1 .82 8 MOX + v i t . E 2.89 1 .22 9 HOX + v i t . E 2.24 1.16 10 LOX + HOX (1: 1) 2.45 1 .09 % weight b a s i s See t e x t f o r f u l l d e s c r i p t i o n of treatments. Unstab. = u n s t a b i l i z e d o i l ; LOX = u n o x i d i z e d , s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l ; +E = e x t r a supplemental v i t a m i n E (1000 IU/kg dry d i e t ) . 74 Table 17 - M o r t a l i t y of v i b r i o c h a l l e n g e d f i s h . (%non-vaccinated f i s h ) D i e t Treatment % M o r t a l i t y 1 Unstab. o i l -2 LOX 25. 0 3 MOX 33. 3 4 HOX 32. 6 5 Corn o i l -6 Unstab.+vit.E 37. 6 7 LOX + v i t . E 21 . 3 8 MOX + v i t . E 32. 6 9 HOX + v i t . E -10 LOX + HOX (1:1) -+ SEM 3 9. 31 See t e x t f o r f u l l d e s c r i p t i o n of treatments. Unstab. = u n s t a b i l i z e d o i l ; LOX = unoxidized, s t a b i l i z e d o i l ; MOX = moderately o x i d i z e d o i l ; HOX = h i g h l y o x i d i z e d o i l ; +E = e x t r a supplemental v i t a m i n E. % M o r t a l i t y of non-vaccinated f i s h . Standard e r r o r of the means. No s i g n i f i c a n t d i f f e r e n c e s between treatment means (p < 0.05). 75 m o r t a l i t y due to treatment were completely masked. There were no s i g n i f i c a n t d i f f e r e n c e s (p > 0.05) between groups i n antibody t i t r e s or a g g l u t i n a t i o n c h a r a c t e r i s t i c s . Antibody t i t r e s averaged g r e a t e r than 1:3 ( i . e . , antibody l e v e l was below d e t e c t i o n i n most in s t a n c e s ) f o r a l l treatment groups, and there was no c o n s i s t e n t t r e n d by treatment; although no non-vaccinated f i s h had any sign of antibody. There was a l s o no obvious d i f f e r e n c e by treatment i n a g g l u t i n a t i o n or immobil-i z a t i o n of V. anguillarum b a c i l l u s i n response to plasma from v a c c i n a t e d f i s h . 76 5 DISCUSSION 5.1 O x i d a t i v e s t a t e of the d i e t s The r e s u l t s of the a n a l y s e s of the o i l s and d i e t s used i n t h i s study confirm that there was a g r a d a t i o n i n the o x i d a t i v e s t a t e of d i e t a r y l i p i d s . The r e l a t i v e order observed was c o n s i s t e n t with the treatment of the h e r r i n g o i l . Those o i l s s u b j e c t e d to the g r e a t e s t h e a t i n g and a e r a t i o n had the h i g h e s t i n d i c e s of a u t o x i d a t i o n . The order of i n c r e a s i n g r e l a t i v e d i e t a r y o x i d a t i o n , as determined from f a t t y a c i d a n a l y s e s , peroxide g e n e r a t i o n , and i o d i n e numbers, were as f o l l o w s : 2,7 < 3,8 < 4,9. Peroxides and f r e e r a d i c a l s are examples of p o t e n t i a l l y t o x i c substances produced by a u t o x i d a t i o n of o i l . These products are t r a n s i t o r y and t h e i r turnover r a t e i s very s e n s i t i v e to environmental c o n d i t i o n s , making p r e d i c t i o n of the course of o x i d a t i o n d i f f i c u l t . In t h i s experiment the peroxide c o n c e n t r a t i o n of the o i l s was p o s i t i v e l y c o r r e l a t e d to the extent of o x i d a t i o n , as measured by a combination of i n d i c e s . - I t i s d i f f i c u l t to p r e d i c t f u t u r e c o n c e n t r a t i o n s of peroxides i n a d i e t , even i f the d i e t c o n t a i n s p r o - o x i d a n t s , s i n c e the peroxides may decay even more r a p i d l y than they are formed. For example, the h i g h e s t content of 77 peroxides, as measured by peroxide v a l u e , i n the h e r r i n g o i l s used i n these d i e t s was 90 meq/kg, whereas Hung et al. (1981) r e p o r t e d peroxide values i n excess of 300 meq/kg f o r h e r r i n g o i l . T h i s does not, i n i t s e l f , i n d i c a t e a g r e a t e r degree of a u t o x i d a t i o n i n the o i l s used by Hung et al. (1981), because the higher temperatures to which the o i l s were su b j e c t e d i n t h i s experiment c o u l d have reduced the h a l f - l i f e of any peroxides that were formed. Peroxides break down i n t o a myriad of aldehydes, and ketones, among other c l a s s e s of compounds, any of which may be t o x i c ( F igure 1). Iodine number and r e l a t i v e f a t t y a c i d composition are u s e f u l e s t i m a t o r s of r e l a t i v e a u t o x i d a t i o n s i n c e they i n d i c a t e the amount of u n o x i d i z e d o i l (the i n i t i a l r e a c t a n t ) remaining, and not the presence of a t r a n s i t o r y i n t e r m ediate product. Iodine number v a r i e s with o i l composition (the g r e a t e r the degree of u n s a t u r a t i o n of the o i l s , the higher the i o d i n e number), and consequently i s l e s s u s e f u l when comparing the r e l a t i v e extent of a u t o x i d a t i o n of o i l s with widely d i f f e r e n t f a t t y a c i d p r o f i l e s . The s u s c e p t i b i l i t y of f a t t y a c i d s to r e a c t i o n with molecular oxygen i n c r e a s e s with u n s a t u r a t i o n . A d e c l i n e i n n3FA present i n an o i l i s , a c c o r d i n g l y , an i n d i c a t i o n of the occurrence of a u t o x i d a t i o n . In the 78 present study, the i o d i n e numbers of the o i l s and d i e t s , and the d i e t a r y n3FA contents f o l l o w e d the order expected due to i n c r e a s i n g a u t o x i d a t i o n . The observed decrease i n r e l a t i v e n3FA content accords with that r e p o r t e d by o t h e r s . Hung et a l . (1981) a l s o found a decrease i n d i e t a r y n3FA content of about the same magnitude, although the d i e t s i n the present experiment had a somewhat higher range: 20.3 - 8.9 mg/kg dry d i e t ( e x c l u d i n g corn o i l ) , vs 15.2 - 7.6 mg/kg dry d i e t (Hung et al. , 1981) . Other i n d i c e s which measure breakdown products of peroxides seem l e s s u s e f u l as i n d i c a t o r s of a u t o x i d a t i o n l e v e l s (Gray, 1978). T h i s i s p a r t l y a r e s u l t of the p a u c i t y of s t a n d a r d i z e d procedures of measurement, and p a r t l y a r e s u l t of the u n p r e d i c t a b i l i t y of peroxide breakdown pathways and the t r a n s i t o r y nature of the pro d u c t s . An example i s the 2 - t h i o b a r b i t u r i c a c i d t e s t , which measures malondialdehyde l e v e l s . In t h i s experiment, the TBA value s obtained f o r the o i l s g e n e r a l l y agreed with the p a t t e r n of o x i d a t i o n e s t a b l i s h e d by the methods p r e v i o u s l y mentioned. The t r a n s i t o r y nature of t h i s product and the u n p r e d i c t a b l e p r o d u c t i o n of other pigment-forming substances reduce the a t t r a c t i v e n e s s of t h i s measure as an i n d i c a t o r of 79 r e l a t i v e o x i d a t i o n . Some v i t a m i n s , notably A, E, and C, are s u s c e p t i b l e to degradation i n the presence of l i p i d that i s under-going o x i d a t i o n (Johnson and Peterson, 1974). T h i s i s probably due to the r e a c t i v i t y of peroxides and subsequent f r e e r a d i c a l s . The observed p a t t e r n of d i e t a r y v i t a m i n E and A content accords with the gr a d a t i o n of l i p i d a u t o x i d a t i o n i n the d i e t s . Both v i t a m i n s E and A are commonly found i n marine o i l s , so that t h e i r r e l a t i v e l y lower c o n c e n t r a t i o n s i n the d i e t c o n t a i n i n g corn o i l does not n e c e s s a r i l y i n d i c a t e a high l e v e l of o x i d a t i o n i n t h i s l a t t e r d i e t . In the case of vi t a m i n E, t h i s r e l a t i o n s h i p holds only f o r those d i e t s that were formulated to c o n t a i n low v i t a m i n E l e v e l s . The complexity of the r e a c t i o n s i n v o l v e d , and the i n f l u e n c e that vitamin E (or any a n t i o x i d a n t ) has on these r e a c t i o n s make the r e l a t i v e l y small d i f f e r e n c e s i n vi t a m i n c o n c e n t r a t i o n s among the v a r i o u s d i e t s d i f f i c u l t to e x p l a i n . 5.2 Growth and feed e f f i c i e n c y The l e v e l of o x i d a t i o n of the d i e t a r y o i l i n f l u e n c e d the growth r a t e and feed e f f i c i e n c y ( d e f i n e d as: body 80 w e i g h t g a i n d i v i d e d by f e e d i n t a k e , G/F) o f t h e f i s h . The n a t u r e and s i z e o f t h e s e e f f e c t s were i n f l u e n c e d by f e e d i n t a k e . F e e d i n t a k e i t s e l f was a f f e c t e d by d i e t a r y l i p i d o x i d a t i o n . H i g h d i e t a r y l e v e l s o f v i t a m i n E d i d not a m e l i o r a t e t h e g r o w t h - r e d u c i n g c a p a c i t y o f o x i d i z e d d i e t a r y l i p i d . The a b s e n c e o f e t h o x y q u i n ( d i e t s 1 and 6), w i t h o r w i t h o u t e x t r a v i t a m i n E had no s i g n i f i c a n t e f f e c t on g r o w t h r a t e or G/F, p r o v i d i n g f u r t h e r e v i d e n c e t h a t v i t a m i n E was n o t l i m i t i n g i n t h e d i e t s o f t h i s e x p e r i m e n t . I t i s my c o n t e n t i o n t h a t t h e p a t t e r n of gr o w t h r e s p o n s e and G/F, i n r e l a t i o n t o t h e e x t e n t of d i e t a r y l i p i d o x i d a t i o n , was p r i m a r i l y i n f l u e n c e d by n u t r i t i o n a l v a l u e , b u t was o f f s e t i n some c a s e s by f e e d i n t a k e . D i f f e r e n c e s between t h e r e s u l t s o f t h i s e x p e r i m e n t and t h o s e o f e x p e r i m e n t s c o n d u c t e d by o t h e r i n v e s t i g a t o r s c a n r e a s o n a b l y be e x p l a i n e d w i t h i n t h i s framework. T h e r e a r e two f a c t o r s w h i c h must be c o n s i d e r e d when e x a m i n i n g t h e e f f e c t o f l i p i d o x i d a t i o n on t h e n u t r i t i o n a l q u a l i t y o f d i e t s . F i r s t , a u t o x i d a t i o n of d i e t a r y l i p i d c a n r e d u c e t h e n u t r i t i o n a l v a l u e of a d i e t by s e v e r a l mechanisms w h i c h p r o b a b l y a c t i n c o n c e r t . T h e s e i n c l u d e : (1) r e d u c t i o n i n a v a i l a b i l i t y o f e s s e n t i a l f a t t y a c i d s t h r o u g h t h e i r c o m b i n a t i o n w i t h m o l e c u l a r 81 oxygen; (2) d e s t r u c t i o n of other n u t r i e n t s (e.g., v i t a m i n E) by peroxides and f r e e r a d i c a l s generated by l i p i d a u t o x i d a t i o n r e a c t i o n s ; (3) production of t o x i c substances; and, (4) p r o d u c t i o n of a p p e t i t e depressant agents. A l t e r n a t i v e l y , the o x i d a t i o n of d e l e t e r i o u s substances i n the d i e t may improve i t s n u t r i t i o n a l v a l u e . The r e s u l t s of t h i s experiment provide evidence fo r the a c t i o n of a combination of these f a c t o r s . The problem of d i s c e r n i n g d i e t a r y d e f i c i e n c y or t o x i c i t y becomes more d i f f i c u l t to i n v e s t i g a t e when there i s a p o s s i b i l i t y that the presence of a substance, while not harmful i t s e l f , may i n c r e a s e the requirements f o r some n u t r i e n t . An example of t h i s i s the o b s e r v a t i o n by Takeuchi and Watanabe (1977a) of the i n c r e a s e d requirement f o r n3FA by rainbow t r o u t , when t o t a l d i e t a r y l i p i d i s i n c r e a s e d . I t c o u l d be concluded from t h i s study that the t o x i c compounds of l i p i d o x i d a t i o n d i d not exert any adverse e f f e c t on coho salmon performance. For example, f i s h i n g e s t i n g d i e t s c o n t a i n i n g h i g h l y o x i d i z e d h e r r i n g o i l had g r e a t e r feed i n t a k e s and body weight gains than those consuming d i e t s c o n t a i n i n g the moderately o x i d i z e d o i l . Caution should be e x e r c i s e d , however, in reaching such a c o n c l u s i o n because, as was mentioned p r e v i o u s l y , 82 the products of o x i d a t i o n are o f t e n s u s c e p t i b l e to f u r t h e r o x i d a t i o n , and so may be most co n c e n t r a t e d i n marine l i p i d s that have undergone moderate o x i d a t i o n . The p a t t e r n of G/F f o r f i s h fed d i e t s c o n t a i n i n g h e r r i n g o i l s o x i d i z e d to d i f f e r e n t degrees i n d i c a t e s that observed growth d i f f e r e n c e s of f i s h fed d i e t s c o n t a i n i n g o x i d i z e d o i l were more a f u n c t i o n of feed intake than of the o x i d a t i v e s t a t e of d i e t a r y l i p i d per se. Whatever caused the r e d u c t i o n in feed consumption by f i s h fed d i e t s c o n t a i n i n g the moderately o x i d i z e d o i l , r e l a t i v e to that noted f o r f i s h on the c o n t r o l d i e t , was l e s s evident f o r f i s h f ed d i e t s c o n t a i n i n g the h i g h l y o x i d i z e d o i l . For the purposes of t h i s study, a d i e t a r y n u t r i e n t can be d e f i n e d as being d e f i c i e n t i f i t can be demonstrated that the growth and immunocompetence of the animal i s improved upon i t s a d d i t i o n to the d i e t . In regard to the d i e t s used i n t h i s study, the n3FA were most l i k e l y to become l i m i t i n g as a r e s u l t of extreme l i p i d o x i d a t i o n . T h i s i s because these were the only e s s e n t i a l n u t r i e n t s s u p p l i e d e x c l u s i v e l y by the h e r r i n g o i l , and because they are most h i g h l y s u s c e p t i b l e to l o s s d u r i n g a u t o x i d a t i o n . I t was found, however, that f i s h fed d i e t s 4 and 9 (which c o n t a i n e d h i g h l y o x i d i z e d o i l ) grew f a s t e r than those fed d i e t s 3 and 8 (which contained 8 3 moderately o x i d i z e d o i l ) d e s p i t e the f a c t that these f i s h consumed l e s s n3FA r e l a t i v e to body weight g a i n . Hence, although f a t t y a c i d s of the l i n o l e n i c a c i d s e r i e s may i n f l u e n c e growth at the c o n c e n t r a t i o n s present in moderately and h i g h l y o x i d i z e d o i l s , under the c o n d i t i o n s of t h i s experiment, the order of growth response cannot be e x p l a i n e d e n t i r e l y on t h i s b a s i s . D i e t 5, i n which corn o i l r e p l a c e d h e r r i n g o i l , had the lowest n3FA content and promoted the 'lowest r a t e of growth. E i t h e r the n3FA content i n d i e t 5 was below that r e q u i r e d f o r maximum growth, or a p p e t i t e was suppressed by t h i s d i e t . When p o s s i b l e f i s h , l i k e other animals, eat to s a t i s f y d a i l y energy needs (Lee and Putnam, 1973). That i s , animals fed one of two d i e t s d i f f e r i n g only i n c a l o r i c content w i l l eat more of the d i e t with lower a v a i l a b l e energy. L i p i d o x i d a t i o n has been shown to decrease the m e t a b o l i z a b l e energy of feeds i n p o u l t r y (March, et al., 1965; Opstvedt, 1973). In t h i s way, l i p i d o x i d a t i o n may induce animals to consume more of d i e t s c o n t a i n i n g o x i d i z e d l i p i d , thus p o t e n t i a l l y o f f s e t t i n g the lower a v a i l a b i l i t y of e s s e n t i a l n u t r i e n t s . F i s h fed d i e t s c o n t a i n i n g h i g h l y o x i d i z e d o i l may, t h e r e f o r e , have obtained enough n u t r i e n t s to outgrow f i s h fed moderately 84 o x i d i z e d o i l even though the e s s e n t i a l n u t r i e n t s may have been l e s s a v a i l a b l e as a p r o p o r t i o n of the former d i e t . A l t e r n a t i v e l y , there may have been a higher c o n c e n t r a t i o n of o f f - f l a v o u r components in the d i e t s c o n t a i n i n g moderately o x i d i z e d o i l than in the ones with h i g h l y o x i d i z e d o i l f o r reasons e x p l a i n e d p r e v i o u s l y , and t h i s may have l e d to suppression of a p p e t i t e . D i e t 10, which co n t a i n e d a blend of " f r e s h o i l " and h i g h l y o x i d i z e d o i l , supported a r a t e of growth comparable to that o b t a i n e d with the c o n t r o l d i e t , but with a s i g n i f i c a n t l y lower G/F (p < 0.05). If the d i e t a r y energy content had been a d v e r s e l y a f f e c t e d by l i p i d o x i d a t i o n , then d i e t 10 would have had a higher a v a i l a b l e energy content than d i e t s 4 and 9 (which c o n t a i n e d only h e r r i n g o i l that had been h i g h l y o x i d i z e d ) . The a p p e t i t e of f i s h fed d i e t s 3 and 8 (which c o n t a i n e d moderately o x i d i z e d h e r r i n g o i l ) , however, was l e s s than noted f o r f i s h fed d i e t 10. Assuming that the a v a i l a b l e energy contents of 3 and 10 were s i m i l a r , the low r a t e of consumption of d i e t 3 r e l a t i v e to d i e t 10 i n d i c a t e s t h a t , under the c o n d i t i o n s of t h i s experiment, some product of l i p i d o x i d a t i o n a d v e r s e l y a f f e c t e d a p p e t i t e , but was, i t s e l f , not present i n the h i g h l y o x i d i z e d o i l . 85 5.3 F a t t y a c i d composition of f i s h F a t t y a c i d s of the l i n o l e n i c a c i d s e r i e s p r i m a r i l y f u n c t i o n as components of c e l l membrane p h o s p h o l i p i d s . A lower body n3FA p h o s p h o l i p i d content may t h e r e f o r e impair f i s h growth. The r e s u l t s of t h i s experiment, however, i n d i c a t e that the l e v e l s of n3FAs i n p h o s p h o l i p i d s were g e n e r a l l y not a s s o c i a t e d with growth response. I f the degree to which o i l s were o x i d i z e d n e g a t i v e l y a f f e c t e d the a b i l i t y of the f i s h to i n c o r p o r a t e n3FAs i n t o p h o s p h o l i p i d s so as to reduce f i s h growth r a t e , then n3FA c o n c e n t r a t i o n s would be lowest i n the p h o s p h o l i p i d p o r t i o n of f i s h that grew the l e a s t . T h i s was not always the case, however. For example, the growth of f i s h fed d i e t 10 was not s i g n i f i c a n t l y l e s s than that observed f o r f i s h f e d d i e t s 3, 4, 8, and 9 (p > 0.05), even though f i s h f e d d i e t 10 had c o n s i d e r a b l y l e s s n3FA i n t h e i r p h o s p h o l i p i d s than f i s h i n the other groups. Although the p r o p o r t i o n of n3FAs i n the t o t a l body l i p i d was v a r i a b l e , that i n the p h o s p h o l i p i d f r a c t i o n was l e s s so. T h i s i s c o n s i s t e n t with the o b s e r v a t i o n that p h o s p h o l i p i d s c o n t a i n i n g n3FAs are p r e f e r e n t i a l l y r e t a i n e d i n c e l l membranes of f i s h ( C a s t l e d i n e and Buckley, 1980). 8 6 Hematocrit i s a measure of t o t a l volume of e r y t h r o c y t e c e l l s i n a volume of blood. I f the hematocrit i s lower i n one of two otherwise s i m i l a r groups of f i s h , t h i s c o u l d be evidence that c e l l membrane i n t e g r i t y has been impaired. In t h i s experiment hematocrit was not s i g n i f i c a n t l y a f f e c t e d by d i e t a r y l i p i d o x i d a t i o n . T h i s suggests that o x i d a t i o n of h e r r i n g o i l , or the r e d u c t i o n of n3FA i n t a k e , d i d not l i m i t e r y t h r o c y t e formation, or i n c r e a s e e r y t h r o c y t e f r a g i l i t y s i g n i f i c a n t l y . 5.4 Disease and Immune Response There were few deaths throughout the growth phase of the experiment that were not a t t r i b u t a b l e to e i t h e r a c c i d e n t a l 0 2 d e p r i v a t i o n or excess a n e s t h e t i c . There were no observed gross p a t h o l o g i c s i g n s of e s s e n t i a l f a t t y a c i d or v i t a m i n E d e f i c i e n c y , as d e s c r i b e d p r e v i o u s l y . There was no apparent e f f e c t of d i e t a r y l i p i d o x i d a t i o n or of d i e t a r y v i t a m i n E c o n c e n t r a t i o n on r e s i s t a n c e s p e c i f i c a l l y to v i b r i o s i s . T h i s c o n t r a s t s with r e s u l t s of Cowey et al. (1984), and Hung et al. (1983), which i n d i c a t e d that l i p i d o x i d a t i o n and vitamin 87 E d e f i c i e n c y i n c r e a s e m o r t a l i t y g e n e r a l l y i n f i s h , under t h e i r experimental c o n d i t i o n s . B l a z e r and Wolke (1984) found that o x i d a t i o n and vi t a m i n E can a f f e c t d i s e a s e r e s i s t a n c e and immune response. The r e l a t i v e l y low temperatures at which the f i s h were exposed to the pathogen and vac c i n e i n the present experiment, may have caused p o s t - c h a l l e n g e m o r t a l i t y , and other measures of immunocompetence (antibody t i t r e and a g g l u t i n a t i o n c a p a c i t y of plasma), to be low and v a r i a b l e . Although the power of these t e s t s of immunocompetence was reduced by the v a r i a b i l i t y of r e s u l t s , i t i s evident that the d i s e a s e r e s i s t a n c e of these animals cannot have been extremely d i f f e r e n t among the treatments t e s t e d . As mentioned p r e v i o u s l y i n the c o n s i d e r a t i o n of growth and feed e f f i c i e n c y , a u t o x i d a t i o n of d i e t a r y l i p i d does not seem to have induced a gross d e f i c i e n c y of v i t a m i n E or of n3FA, under the c o n d i t i o n s of t h i s study. I t i s not s u r p r i s i n g , t h e r e f o r e , that there i s no d e t e c t a b l e e f f e c t of l i p i d o x i d a t i o n on immunocompetence of f i s h i n t h i s experiment. Under other c o n d i t i o n s , i t has been found that salmonids r e q u i r e d i e t a r y n3FA l e v e l s of g r e a t e r than 1% of the d i e t , or 10% of d i e t a r y l i p i d ( r e f e r to Watanabe, 1982). In t h i s study, f i s h fed d i e t s c o n t a i n i n g h i g h l y o x i d i z e d h e r r i n g o i l ( d i e t s 4, and 8), 88 or u n s t a b i l i z e d h e r r i n g o i l with 30 IU/kg v i t a m i n E, or corn o i l , or a mixture of c o n t r o l o i l and h i g h l y o x i d i z e d h e r r i n g o i l s , e x h i b i t e d l i t t l e , or u n d e t e c t a b l e , r e d u c t i o n i n t h e i r growth r a t e and immunocompetence, when compared to f i s h fed d i e t s c o n t a i n i n g the c o n t r o l o i l ( d i e t s 2, and 7). 5.5 Comparison of r e s u l t s to those of other i n v e s t i g a t i o n s There are s e v e r a l elements i n experiments of t h i s nature which make meaningful comparison of r e s u l t s with those of other i n v e s t i g a t o r s d i f f i c u l t . These i n c l u d e v a r i a b i l i t y i n the c h a r a c t e r i s t i c s of o i l , f i s h , and experimental c o n d i t i o n s . The f a t t y a c i d p r o f i l e of h e r r i n g o i l can vary c o n s i d e r a b l y s e a s o n a l l y and g e o g r a p h i c a l l y (see, f o r examples, Ackman and Eaton, 1966; Dosanjh et al. , 1984). V a r i a t i o n s i n h e r r i n g o i l composition can be due to d i f f e r e n c e s i n consumption by h e r r i n g of n3FA, and the i n f l u e n c e of temperature on i t s r e t e n t i o n . These d i f f e r e n c e s may have c o n s i d e r a b l e impact on the c h a r a c t e r i s t i c s of l i p i d a u t o x i d a t i o n , both i n products formed and i n r a t e s of o x i d a t i o n , which i n t u r n c o u l d i n f l u e n c e growth and G/F of f i s h . The f i s h i n t h i s experiment were not from a 8 9 c u l t u r e d , d e f i n e d gene p o o l , and there was c o n s i d e r a b l e v a r i a t i o n i n growth response w i t h i n groups (how much t h i s w i t h i n tank v a r i a t i o n i s due to d i f f e r e n c e s i n genotype and how much i s a r e s u l t of s o c i a l i n t e r a c t i o n s i s not known). Rainbow t r o u t (Salmo gairdneri), which have a d i f f e r e n t l i f e h i s t o r y and p h y s i o l o g i c a l makeup from coho salmon (Oncorhynchus kisutch), have been more commonly used i n l i p i d o x i d a t i o n s t u d i e s . D i f f e r e n c e s i n s i z e of f i s h used in experiments may i n f l u e n c e r e s u l t s , and t h i s a l s o must be c o n s i d e r e d when comparing r e s u l t s of d i f f e r e n t experiments. D i f f e r e n c e s i n experimental c o n d i t i o n s , p a r t i c u l a r l y i n water temperature and d i e t composition, make comparisons among experiments d i f f i c u l t . In t h i s experiment water temperature was more v a r i a b l e , and reached lower values than i n other experiments. The d i e t s were designed to c o n t a i n at l e a s t the minimum requirement l e v e l of v i t a m i n E, whereas some d i e t s i n other experiments had lower than r e q u i r e d l e v e l s . F i n a l l y , the use of t i m e - s e r i e s a n a l y s i s ( i . e , using a l l mean weights c o l l e c t e d over time), p r o v i d e s a more powerful determination of s i g n i f i c a n c e of treatment e f f e c t s . T h i s technique estimates the v a r i a t i o n about the mean at each time of measurement and removes the 90 v a r i a t i o n due to time (which was a s i g n i f i c a n t c u b i c f u n c t i o n i n t h i s experiment, due to the f l u c t u a t i o n i n water temperature). Care must be taken when us i n g techniques of t h i s kind i f the treatment e f f e c t v a r i e s over time. T h i s i s e s p e c i a l l y important when examining l i p i d s , s i n c e they tend to be unstable over time, p a r t i c u l a r l y when p a r t i a l l y o x i d i z e d . Keeping these caveats i n mind, i t i s s t i l l u s e f u l to compare and c o n t r a s t s i m i l a r experiments i n order to judge the a p p l i c a b i l i t y of c o n c l u s i o n s to other c o n d i t i o n s , and i n so doing achieve a b e t t e r understanding of the u n d e r l y i n g p r i n c i p l e s . 91 6 CONCLUSIONS The r e s u l t s of t h i s study on j u v e n i l e coho salmon i n d i c a t e that the n u t r i t i o n a l q u a l i t y of d i e t s , as measured by growth and feed e f f i c i e n c y , can be a d v e r s e l y i n f l u e n c e d by o x i d a t i o n of i n c l u d e d o i l . I f there i s adequate vi t a m i n E and e s s e n t i a l f a t t y a c i d s , the r e d u c t i o n i n growth response due to d i e t a r y l i p i d o x i d a t i o n can be moderated by i n c r e a s e d feed i n t a k e . The higher growth of f i s h f i s h fed d i e t s c o n t a i n i n g h i g h l y o x i d i z e d h e r r i n g o i l r e l a t i v e to f i s h fed d i e t s c o n t a i n i n g moderately o x i d i z e d h e r r i n g o i l accords with the higher consumption of the former d i e t . T h i s i n d i c a t e s that perhaps some int e r m e d i a t e compound i s formed by l i p i d a u t o x i d a t i o n , that i s i t s e l f s u b j e c t to o x i d a t i o n , and that can reduce a p p e t i t e i n coho salmon. I n c l u s i o n of l a r g e amounts of v i t a m i n E i n the d i e t had no e f f e c t on growth, feed e f f i c i e n c y , hematocrit, or immunocompetence of j u v e n i l e coho salmon. T h i s suggests that the lower l e v e l of supplemental v i t a m i n E (30 IU/kg) which was added to the d i e t s was adequate f o r growth and di s e a s e r e s i s t a n c e , under the c o n d i t i o n s of t h i s study. D i e t a r y n3FA l e v e l s had l i t t l e e f f e c t on the percentages of n3FAs i n p h o s p h o l i p i d , or on growth or d i s e a s e r e s i s t a n c e . Thus, i t i s concluded t h a t , under the present experimental c o n d i t i o n s , n3FAs were not n u t r i t i o n a l l y l i m i t i n g . 92 7 REFERENCES Ackman, R.G., and C A . Eaton. 1966. Some commercial A t l a n t i c h e r r i n g o i l s ; f a t t y a c i d c omposition. J . F i s h . Res. Bd. Can. 23:991-1006 Adron, J.W., A. B l a i r , C.B. Cowey, and A.M. Shanks. 1976. E f f e c t s of d i e t a r y energy l e v e l and d i e t a r y energy source on growth, feed c o n v e r s i o n , and body composition of t u r b o t (Scophthalmus maximus L . ) . Aquaculture 7:125 - 132 AOAC. 1980. A s s o c i a t i o n of O f i c i a l A n a l y t i c a l Chemists. The methods of a n a l y s i s . 13th e d i t i o n . AOAC, Washinton, USA. p.440, 746. AOCS. 1973. American O i l Chemists' S o c i e t y . O f f i c i a l and T e n t a t i v e Methods. Champaign, 111. Cd 8 - 53. A t h e r t o n , W.D. 1975. The e f f e c t of d i f f e r e n t l e v e l s of d i e t a r y f a t on the growth of rainbow t r o u t . J . F i s h B i o l . 7:565 - 571 A t h e r t o n , W.D., and A. A i t k e n . 1970. Growth, n i t r o g e n metabolism and f a t metabolism in rainbow t r o u t . Comp. Biochem. P h y s i o l . 36:719 -.747 Baehner, R.L., L.A. Boxer, J.M. A l l e n , and J . D a v i s . 1977. A u t o x i d a t i o n as a b a s i s f o r a l t e r e d f u n c t i o n by polymorphonuclear l e u k o c y t e s . Blood 50:327 - 335 B e l l , J.G., and C.B. Cowey. 1985. Roles of v i t a m i n E and selenium in the p r e v e n t i o n of p a t h o l o g i e s r e l a t e d to f a t t y a c i d o x i d a t i o n i n salmonids. I n : N u t r i t i o n and Feeding in F i s h . eds. Cowey, C.B., A.M. Mackie, and J.G. B e l l . Acad. P r e s s . London, pp. 333 - 347. B l a z e r , V.S., and R.E. Wolke. 1984. The e f f e c t s of a-tocopherol on the immune response and n o n - s p e c i f i c r e s i s t a n c e f a c t o r s of rainbow t r o u t . Aquaculture, 37:1-9 B l i g h , E.G., and W.J. Dyer. 1959. A r a p i d method of t o t a l l i p i d e x t r a c t i o n and p u r i f i c a t i o n . Can. J . Biochem. P h y s i o l . 37:911 - 917 93 Boggio, S.T., R.W. Hardy, J.K. B a b b i t t and E.L. Brannon. 1985. The i n f l u e n c e of d i e t a r y l i p i d source and a-tocopherol a c e t a t e l e v e l on product q u a l i t y of rainbow t r o u t . Aquaculture 51:13-24 Boxer, L.A. 1986. R e g u l a t i o n of phagocyte f u n c t i o n by a-t o c o p h e r o l . Proc. Nutr. Soc. 45: 333 - 344 Brown, W.D., and A.L. Tappe l . 1959. F a t t y a c i d o x i d a t i o n by c a r p mitochondria. Arch. Biochem. Biophys. 85:149-158 Burton, G.W., Y. LePage, E . J . Gabe, and K.U.Ingold. 1980. A n t i o x i d a n t a c t i v i t y of v i t a m i n E and r e l a t e d phenols. Importance of s t e r e o e l e c t r o n i c f a c t o r s . J . Am. Chem. Soc. 102:7791 - 7792 Burton, G.W., and K.U. I n g o l d . 1981. A u t o x i d a t i o n of b i o l o g i c a l molecules. 1. The a n t i o x i d a n t a c t i v i t y of vi t a m i n E and r e l a t e d c h a i n - b r e a k i n g p h e n o l i c a n t i o x i d a n t s in vitro. J . Am. Chem. Soc. 103:6472 - 6477 C a s t e l l , J.D., R.O. Sinnhuber, J.H. Wales, and D.J. Lee. 1972a. E s s e n t i a l f a t t y a c i d s i n the d i e t of rainbow t r o u t : growth, feed c o n v e r s i o n , and some gross d e f i c i e n c y symptoms. J . Nutr. 102:77-86 C a s t e l l , J.D., R.O. Sinnhuber, D.J. Lee, and J.H. Wales. 1972b. E s s e n t i a l f a t t y a c i d s i n the d i e t s of rainbow t r o u t : p h y s i o l o g i c a l symptoms of e s s e n t i a l f a t t y a c i d d e f i c i e n c y . J . Nutr. 102:87-92 C a s t e l l , J.D., D.J. Lee, and R.O. Sinnhuber. 1972c. E s s e n t i a l f a t t y a c i d s i n the d i e t of rainbow t r o u t : l i p i d metabolism and f a t t y a c i d composition. J . Nutr. 102:93-100 C a s t l e d i n e , A.J., and J.T. Buckley. 1980. D i s t r i b u t i o n and m o b i l i t y of w3 f a t t y a c i d s i n rainbow t r o u t fed v a r y i n g l e v e l s and types of d i e t a r y l i p i d . J . Nutr. 110:675-685 Cho, C.Y., H.S. Bayley, and S.J. S l i n g e r . 1974. P a r t i a l replacement of h e r r i n g meal with soybean meal and other changes i n a d i e t f o r rainbow t r o u t . J . F i s h . Res. Bd. Can. 31:1523 - 1528 94 C h r i s t i e , W.W. 1982. L i p i d A n a l y s i s . 2 1 ed. Pergammon Pre s s . UK. chap. 4, 6. Cowey, C.B., E. Degener, A.G.J. Tacon, A. Youngson, and J.G. B e l l . 1984. The e f f e c t of v i t a m i n E and o x i d i z e d f i s h o i l on the n u t r i t i o n of rainbow t r o u t grown at n a t u a l , v a r y i n g water temperatures. B r i t . J . Nutr. 51:443-451 Cowey, C.B., J.W. Adron, M.J. Walton, J . Murray, A. Youngson and D. Knox. 1981. T i s s u e d i s t r i b u t i o n , uptake, and requirement f o r a-tocopherol of rainbow t r o u t fed d i e t s with a minimal content of unsaturated f a t t y a c i d s . J . Nutr. 111:1556-1567 Cowey, C.B., J.W. Adron, and A. Youngson. 1983. The v i t a m i n E requirement of rainbow t r o u t given d i e t s c o n t a i n i n g p o l y u n s a t u r a t e d f a t t y a c i d s d e r i v e d from f i s h o i l . Aquaculture 30:85-93 Cowey, C.B., and J.R. Sargent. 1972. F i s h n u t r i t i o n . Adv. Mar. B i o l . 10:383 - 492 Dosanjh, B.S., D.A. Higgs, M.D. P l o t n i k o f f , J.R. McBride, J.R. Markert, and J.T. Buckley. 1984. E f f i c a c y of ca n o l a o i l , pork l a r d and marine o i l s i n g l y and i n combination as supplemental d i e t a r y l i p i d sources f o r j u v e n i l e coho salmon. Aquaculture 36:333-345 Fong, K-L., P.B. McCay, and J.L. P o r t e r . 1976, +Evidence f o r superoxide-dependent r e d u c t i o n of Fe and i t s r o l e i n enzyme-generated hydroxyl r a d i c a l formation. Chem. B i o l . I n t e r a c t . 15:77 - 89 German, J.B., and J.E. K i n s e l l a . 1986. Hydroperoxide metabolism i n t r o u t g i l l - t i s s u e : e f f e c t of g l u t a t h i o n e on lipoxygenase products generated from arachadonic a c i d and docosahexaenoic a c i d . Biochem. Biophs. Acta 879:378 - 387 Gray, M. 1978. Measurement of l i p i d o x i d a t i o n : a review. JAOCS 55:539 - 546 H a r r i s o n , R., and G.G. Lunt. 1980. B i o l o g i c a l Membranes. 2nd ed. B l a c k i e . Glasgow, UK. p. 86. 95 Hatate, H., and M. Toyomizu. 1985. I n h i b i t i o n and a c c e l e r a t e d a u t o l y s i s of t r y p s i n due to i n c o r p o r a t i o n of polymers of o x i d i z e d methyl l i n o l e n a t e . B u l l . Jpn. Soc. S c i . F i s h . 51:627 - 633. K a z e l , J.R. 1979. Infl u e n c e of thermal a c c l i m a t i o n on membrane l i p i d composition of rainbow t r o u t l i v e r . Amer. J . P h y s i o l . 236:R91 - R101 H e i n z e r l i n g , R.H., C F . Nockels, C.L. Quarry, and R.D. Tengerdy. 1974. P r o t e c t i o n of c h i c k s a g a i n s t E. col i . i n f e c t i o n by d i e t a r y supplementation with v i t a m i n E. Proc. Soc. Exp. B i o l . Med. 146:279 - 283 H i g a s h i , H., T. Kaneko, S. I s h i i , I. Masuda, and T. Su g i h a s h i . 1964. E f f e c t of d i e t a r y l i p i d on f i s h under c u l t i v a t i o n . I. E f f e c t of l a r g e amounts of l i p i d on h e a l t h and growth r a t e of rainbow t r o u t . B u l l . Jpn. Soc. S c i . F i s h . 30:778 - 785 Hung, S.S.O., B.L. Walker, and S.J. S l i n g e r . 1983. E f f e c t of o x i d i z e d o i l on the l i v e r f a t t y a c i d s of rainbow t r o u t . Comp. Biochem. P h y s i o l . 72B:349-353 Hung, S.S.O., C Y . Cho, and S.J. S l i n g e r . 1980. Measurement of o x i d a t i o n i n f i s h o i l and i t s e f f e c t on v i t a m i n E n u t r i t i o n of rainbow t r o u t . Can. J . F i s h . Aquatic S c i . 37:1248-1253. Hung, S.S.O., C Y . Cho, and S.J. S l i n g e r . 1981. E f f e c t of o x i d i z e d f i s h o i l , d l - a - t o c o p h e r y l a c e t a t e and ethoxyquin supplementation on the v i t a m i n E n u t r i t i o n of rainbow t r o u t fed p r a c t i c a l d i e t s . J . Nutr. 111:648-657 Ingold, K. 1962. Metal c a t a l y s i s . In: L i p i d s and T h e i r O x i d a t i o n . eds. S c h u l t z , H., E. Day, R. Sinnhuber. A v i , Conn., USA pp. 93 - 121. Johnson, A.H., and M.S. Peterson 1974 E n c y c l o p e d i a of Food Technology v o l . 2. A v i , Conn., USA. pp.896-934. Kay, M.B., G. Bosman, S. Shapiro, A. Bendich, and P. B a s s e l . 1986. O x i d a t i o n as a p o s s i b l e mechanism of c e l l u l a r aging: v i t a m i n E d e f i c i e n c y causes premature aging and IgG b i n d i n g to e r y t h r o c y t e s . Proc. N a t l . Acad. S c i . USA 83:2463 - 2467 96 K e l l o g g , E.W., and I. F r i d o v i t c h . 1975. Superoxide, hydrogen peroxide, and s i n g l e t oxygen i n l i p i d p e r o x i d a t i o n by a xanthine oxidase system. J . B i o l . Chem. 250:8812 - 8817 Labuza, T.P., and L.R. Dugan, j r . 1971 K i n e t i c s of o x i d a t i o n i n foods. C r i t i c a l Reviews of Food Tech. 2:355 - 405. Lands, W.E.M. 1982. Biochemical o b s e r v a t i o n s on d i e t a r y l o n g - c h a i n f a t t y a c i d s from f i s h o i l and t h e i r e f f e c t on p r o s t a g l a n d i n s y n t h e s i s i n animals and humans. I n : N u t r i t i o n a l E v a l u a t i o n of Long-chain F a t t y A c i d s i n F i s h O i l . eds. Barlow, S.M., and M.E.' Stansby. Acad. Press. NY pp. 267 - 282. Lee, D.J., J.N. Roehm, T.C. Yu, and R.O. SinnhuberV' 1967. E f f e c t of w3 FA on the growth rate of rainbow t r o u t . J . Nutr. 92:93-98 Lee, D.J., and G.B. Putnam. 1973. The response of rainbow t r o u t to v a r y i n g p r o t e i n : e n e r g y r a t i o s i n a t e s t d i e t . J . Nutr. 103:916 - 922 Leger, C , P. Bergot, P. Luquet, J . F l a n z y , and J.Meurot. 1977. S p e c i f i c d i s t r i b u t i o n of f a t t y a c i d s i n the t r i g l y c e r i d e s of rainbow t r o u t adipose t i s s u e : I n f l u e n c e of temperature. L i p i d s 12:538 - 543 McCay, P.B. 1985. Vitamin E: i n t e r a c t i o n s with f r e e r a d i c a l s and a s c o r b a t e . Ann. Rev. Nutr. 5:323-340. March, B.E., J . B i e l y , H.L.A. T a r r , and F. C l a g g e t t . 1965. The e f f e c t of a n t i o x i d a n t treatment on the m e t a b o l i z a b l e energy and p r o t e i n value of h e r r i n g meal. Poul. S c i . 44:679 - 685. Merck Index. 1968. E i g h t h e d i t i o n . P.G. Stecher, ed. Merck and Co., Inc., Rahway, N.J. p. 615. Mugr d i t c h i a n , D.S., R.W. Hardy, and W.T. Iwaoka. 1981. L i n s e e d o i l and animal f a t as a l t e r n a t i v e l i p i d sources i n dry d i e t s f o r chinook salmon. Aquaculture 25:161 - 172 97 Murai, T., and J.W. Andrews. 1974. I n t e r a c t i o n s of d i e t a r y a - t o c o p h e r o l , o x i d i z e d menhaden o i l , and ethoxyquin, on channel c a t f i s h (Ictalurus punctatus). J . Nutr. 104:1416 - 1431 NRC. 1981. N u t r i e n t Requirements of Coldwater F i s h e s . Nat. Acad. Press, Washington D.C. p.41 N i c o l a i d e s , N., and A.N. Woodall. 1962. Impaired pigmentation i n chinook salmon fed d i e t s d e f i c i e n t i n e s s e n t i a l f a t t y a c i d s . J . Nutr. 78: 431 - 437. Nockels, C F . 1979. P r o t e c t i v e e f f e c t s of supplemental v i t a m i n E a g a i n s t i n f e c t i o n . Fed. Proc. 38:2134 - 2138 Opstvedt, J . 1973. I n f l u e n c e of r e s i d u a l l i p i d s on the n u t r i t i v e value of f i s h meal. IV. E f f e c t of d r y i n g and storage on the energy value of p r o t e i n and l i p i d f r a c t i o n s of h e r r i n g meal. Acta A g r i c . Scand. 23:200 - 208. P h i l l i p s , A.M., D.R. Brockway, and J.M. M a x e l l . 1952. The e f f e c t s of supplemental f a t s i n the brook t r o u t . Prog. F i s h . C u l t . 14:19 - 22 P h i l l i p s , A.M., and H. P o d o l i a k . 1957. The n u t r i t i o n of t r o u t . I I I . Fats and m i n e r a l s . Prog. F i s h . C u l t . 19:68 - 75 Popp-Snijders, C , J . Schouten, W. van B l i t t e r s w i j k , and E. van der Veen. 1986. Changes i n membranes l i p i d composition of human e r y t h r o c y t e s a f t e r d i e t a r y supplementation of n-3 p o l y - u n s a t u t a t e d f a t t y a c i d s . Maintenance of membrane f l u i d i t y . Biochem. Biophys. Acta 854:31 - 37 Poston, H.A., G.F. Combs, and L. L e i b o v i t z . 1976. Vitamin E and selenium i n t e r r e l a t i o n s i n the A t l a n t i c salmon: gross, h i s t o l o g i c a l , and b i o c h e m i c a l d e f i c i e n c y s i g n s . J . Nutr. 106:892 - 904 Raheja, R.J., C. Kaur, A.A. Singh, and I.S. B h a t i a . 1973. New c o l o r i m e t r i c method f o r the q u a n t i t a t i v e e s t i m a t i o n of p h o s p h o l i p i d s without a c i d d i g e s t i o n . J . L i p i d Res. 14:695 - 697 98 R e i n i t z , G.L., L.E. Orme, C A . Lemm, and F.N. H i t z e l . 1978. I n f l u e n c e of v a r y i n g l i p i d c o n c e n t r a t i o n s with two p r o t e i n c o n c e n t r a t i o n s i n d i e t s f o r rainbow t r o u t . Trans. Am. F i s h . Soc. 107:751 - 754 Ringrose, R.C. 1971. C a l o r i e - t o - p r o t e i n r a t i o f o r brook t r o u t . J . F i s h . Res. Bd. Can. 28:1113 - 1117 SAS Users Guide: S t a t i s t i c s v e r s i o n 5. 1985. SAS I n s t i t u t e Inc., Carey, N.C pp. 433 - 506. Sinnhuber, R.O., J.D. C a s t e l l , and D.J.Lee. 1972. E s s e n t i a l f a t t y a c i d requirement of rainbow t r o u t . Fed. Proc. 31:1436 - 1441 Soderhjelm, P., and B. Andersson. 1978. Simultaneous d e t e r m i n a t i o n of v i t a m i n s A and E i n feeds and foods by reversed phased high-performance l i q u i d chromatography. J . S c i . Fd. A g r i c . 29:697 - 702. Snedecor, G.W., and W.G. Cochran. 1980. S t a t i s t i c a l Methods. 7th ed. Iowa St a t e Univ. Press, p 298 - 333 S t i c k n e y , R.R., and J.W. Andrews. 1972. E f f e c t of d i e t a r y l i p i d s on growth, feed c o n v e r s i o n , l i p i d and f a t t y a c i d composition of channel c a t f i s h (Ictalurus punctatus). J . Nutr. 102:249-258 Takeuchi, T., T. Watanabe. 1976. N u t r i t i v e v alue of w3 h i g h l y unsaturated f a t t y a c i d s i n p o l l o c k l i v e r o i l fo r rainbow t r o u t . B u l l . Jpn. Soc. S c i . F i s h . 42:907 - 919. Takeuchi, T., T. Watanabe. 1977a. D i e t a r y l e v e l s of methyl l a u r a t e and e s s e n t i a l f a t t y a c i d requirement of rainbow t r o u t . B u l l . Jpn. Soc. S c i . F i s h . 43:893 -898. Takeuchi, T. , T. Watanabe. 1977b. E f f e c t of e i c o s a p e n t a -enoic a c i d and docosahexaenoic a c i d i n p o l l o c k l i v e r o i l on growth and f a t t y a c i d composition of rainbow t r o u t . B u l l . Jpn. Soc. S c i . F i s h . 43:947 - 953 Takeuchi, T., T. Watanabe, and C. Ogino. 1978a Supplementary e f f e c t of l i p i d s i n a high p r o t e i n d i e t of rainbow t r o u t . B u l l . Jpn. Soc. S c i . F i s h . 44:677 - 681 99 Takeuchi, T., T. Watanabe, and C. Ogino. 1978b Optimum r a t i o of p r o t e i n to l i p i d s i n d i e t s of rainbow t r o u t . B u l l . Jpn. Soc. S c i . F i s h . 44:683 - 688 Takeuchi, T., T. Watanabe, and C. Ogino. 1978c Use of hydrogenated f i s h o i l and beef t a l l o w as a d i e t a r y energy source f o r ca r p and rainbow t r o u t . B u l l . Jpn. Soc. S c i . F i s h . 44:875 - 881 Takeuchi, T., and T. Watanabe. 1979. E f f e c t of excess amounts of e s s e n t i a l f a t t y a c i d s on growth of rainbow t r o u t . B u l l . Jpn. Soc. S c i . F i s h . 45:1517 - 1529. T a r l a d g i s , B.G., B.M. Watts, H, Younathon, and L. Dugan. 1960. D i s t i l l a t i o n method f o r q u a n t i t a t i v e d e t e r m i n a t i o n of malondialdehyde i n food. JAOCS 37:44 - 48 Tengerdy, R.P., R.H. H e i n z e r l i n g , G.L. Brown, and M.M. Mathias. 1973. Enhancement of the humoral immune response by vit a m i n E. I n t . Arch. A l l e r g y Appl. Immunol. 44:221 - 232 Watanabe, T. 1982. L i p i d n u t r i t i o n i n f i s h . Comp. Biochem. P h y s i o l 73B:3-15 Watanabe, T., C. Ogino, and Y. K o s h i s h i . 1974. Requirement of rainbow t r o u t f o r e s s e n t i a l f a t t y a c i d s . B u l l . Jpn. Soc. S c i . F i s h . 40:493 - 499 Watanabe, T., and T. Takeuchi. 1976. E v a l u a t i o n of p o l l o c k l i v e r o i l as a supplement to d i e t s f o r rainbow t r o u t . B u l l . Jpn. Soc. S c i . F i s h . 42:893 - 906. Wilson, R.P., P.R. Bowser, and W.E. Poe. 1984. D i e t a r y v i t a m i n E requirement of f i n g e r l i n g channel c a t f i s h . J . Nutr. 114:2053 - 2058 W i t t i n g , L.A. 1965. L i p i d p e r o x i d a t i o n in vivo. JAOCS 42:908 - 913 W i t t i n g , L.A., and M.K. H o r w i t t . 1964. E f f e c t of d i e t a r y f a t t y a c i d u n s a t u r a t i o n i n to c o p h e r o l d e f i c i e n c y -induced c r e a t i n u r i a . J . Nutr. 82:19 - 33 100 Yu, T.C. , R.O. Sinnhuber, and G.B. Putnam. 1977. E f f e c t of d i e t a r y l i p i d s on f a t t y a c i d composition of body l i p i d in rainbow t r o u t . L i p i d s 12:495-499 Yu, T . C , R.O. Sinnhuber, and G.B. Putnam. 1977. Use of swine f a t as an energy source i n t r o u t r a t i o n s . Prog. F i s h C u l t . 39:95 - 97 Yu, T . C , and R.O. Sinnhuber. 1 967. An improved 2-t h i o b a r b a r t u r i c a c i d procedure f o r measurement of a u t o x i d a t i o n i n f i s h o i l s . JAOCS 44:256 - 258 Yu, T . C , and R.O. Sinnhuber. 1972. E f f e c t of d i e t a r y l i n o l e n i c a c i d and docosahexaenoic a c i d on growth and f a t t y a c i d composition of rainbow t r o u t . L i p i d s 7:450-454 Yu, T . C , and R.O. Sinnhuber. 1 975. E f f e c t of d i e t a r y l i n o l e n i c and l i n o l e i c a c i d s on growth and l i p i d metabolism of rainbow t r o u t . L i p i d s 10:63 - 66 Yu, T . C , and R.O. Sinnhuber. 1979. E f f e c t of d i e t a r y w3 and w6 f a t t y a c i d s on growth and feed c o n v e r s i o n e f f i c i e n c y of coho salmon. Aquaculture 16:31 - 38 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0096904/manifest

Comment

Related Items