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The effect of dietary fatty acids on body composition, growth, mortality and saltwater tolerance in juvenile… Bernatsky, Ivor Paul 1990

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THE EFFECT OF DIETARY FATTY ACIDS ON BODY COMPOSITION, GROWTH, MORTALITY AND SALTWATER TOLERANCE IN JUVENILE COHO SALMON (Oncorhynchus k i s u t c h ) by IVOR PAUL BERNATSKY B.S.A., The U n i v e r s i t y of Saskatchewan, 1986 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 t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1990 © Ivo r Paul Bernatsky, 1990 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 of The University of British Columbia Vancouver, Canada •ate Zl ; | 9 9 Q DE-6 (2/88) ABSTRACT An e x p e r i m e n t was d e s i g n e d t o d e t e r m i n e whether t h e r e s p o n s e o f f i s h t o g r aded d i e t a r y l e v e l s o f e s s e n t i a l f a t t y a c i d s (n3) was a f f e c t e d by t h e t o t a l d i e t a r y l i p i d c o n c e n t r a t i o n . J u v e n i l e coho salmon were f e d p r a c t i c a l d i e t s v a r y i n g i n l i p i d s o u r c e and t o t a l l i p i d c o n t e n t . The d i e t s were f e d i n d u p l i c a t e d u r i n g a 27-week and a 12-week p e r i o d r a n i n s u c c e s s i o n . The e f f e c t s o f t h e d i f f e r e n t d i e t a r y f a t t y a c i d c o n c e n t r a t i o n s on body f a t t y a c i d s c o m p o s i t i o n were d e t e r m i n e d a f t e r each p e r i o d . The d i e t a r y f a t t y a c i d c l a s s e s were e x p r e s s e d e i t h e r as p e r c e n t o f t h e d r y d i e t o r p e r c e n t o f t h e d i e t a r y l i p i d . A n a l y s i s o f t h e body l i p i d f a t t y a c i d c o m p o s i t i o n was p e r f o r m e d f o r n e u t r a l and p o l a r l i p i d f r a c t i o n s . The e f f e c t o f d i e t a r y f a t t y a c i d c o n c e n t r a t i o n on g r o w t h and m o r t a l i t y was d e t e r m i n e d o v e r a 12-week growth s t u d y ( p e r i o d 2 ) . A 24-hour s a l t w a t e r c h a l l e n g e was p e r f o r m e d a t t h e end o f p e r i o d 2. I t was used t o examine t h e e f f e c t o f d i e t a r y f a t t y a c i d c o n c e n t r a t i o n on s a l t w a t e r t o l e r a n c e . D i e t a r y n6 and n3 f a t t y a c i d s appeared t o be s e l e c t i v e l y i n c o r p o r a t e d i n t o t h e body p o l a r l i p i d p o o l . L i n o l e a t e and l i n o l e n a t e underwent e l o n g a t i o n and d e s a t u r a t i o n w h i c h r e s u l t e d i n t h e i n h i b i t i o n o f t h e e l o n g a t i o n and d e s a t u r a t i o n o f 1 8 : l n 9 . The n e u t r a l l i p i d p o o l s e r v e d as a s o u r c e o f n3 f a t t y a c i d s f o r t h e p o l a r l i p i d when d i e t a r y i n t a k e was l i m i t e d by l o w t e m p e r a t u r e s d u r i n g p e r i o d 1. The body n e u t r a l m o n o u n s a t u r a t e d and 18:1 monounsaturated f a t t y a c i d s c o n s i s t e n t l y r e f l e c t e d t h e c o m p o s i t i o n o f t h e d i e t . The n3 f a t t y a c i d c o n c e n t r a t i o n i n t h e n e u t r a l l i p i d was a l s o d i r e c t l y r e l a t e d t o t h e d i e t a r y f a t t y a c i d c o m p o s i t i o n d u r i n g p e r i o d 2. The e f f e c t o f d i e t a r y f a t t y a c i d s on t h e body n e u t r a l o r p o l a r f a t t y a c i d c o m p o s i t i o n d i d n o t depend on t h e manner i n w h i c h t h e d i e t a r y f a t t y a c i d s were e x p r e s s e d . There was a l s o no s i g n i f i c a n t e f f e c t o f d i e t a r y t o t a l l i p i d c o n c e n t r a t i o n on t h e r e l a t i o n s h i p between d i e t a r y f a t t y a c i d s and t h e i r i n c o r p o r a t i o n i n t o t h e body l i p i d s . The g r o w t h r e s p o n s e was d i f f i c u l t t o i n t e r p r e t because o f t h e h i g h m o r t a l i t y . There was a s i g n i f i c a n t d i f f e r e n c e i n m o r t a l i t y among t r e a t m e n t s . A p o s i t i v e r e l a t i o n s h i p between d i e t a r y c o n c e n t r a t i o n s o f t o t a l n3 f a t t y a c i d s o r n3 h i g h l y u n s a t u r a t e d f a t t y a c i d s and m o r t a l i t y became e v i d e n t f o l l o w i n g a n a l y s i s o f t h e r e g r e s s i o n o f m o r t a l i t y as a f u n c t i o n o f d i e t a r y f a t t y a c i d c o m p o s i t i o n . The d i e t a r y f a t t y a c i d c o m p o s i t i o n d i d not appear t o a l t e r t h e s a l t w a t e r t o l e r a n c e o f t h e 1+ coho salmon. Page TABLE OF CONTENTS Abstract i i Table of Contents i v L i s t of Tables v i i i L i s t of Figures x Acknowledgements xvi Introduction 1 The p r a c t i c a l importance of studying the e f f e c t of dietr a r y f a t t y acids i n coho salmon 3 Studying the e s s e n t i a l f a t t y acid requirement i n f i s h . . . . 7 The salmonids 11 The e s s e n t i a l i t y of n3 fa t t y acids i n salmonids.... 12 The r o l e of n3 f a t t y acids E s s e n t i a l f a t t y acids as prostaglandin precursors..15 E s s e n t i a l f a t t y acids i n b i o l o g i c a l membranes 20 Es s e n t i a l f a t t y acids i n neurological function 21 The e f f e c t of dietary f a t t y acids on body f a t t y acid composition 1 The polar l i p i d 21 Body t o t a l l i p i d and neutral l i p i d 26 The e f f e c t of energy balance on body f a t t y acid composition 28 The e f f e c t of dietary f a t t y acids on growth ...29 The e f f e c t of dietary f a t t y acids on mortality 32 The e f f e c t of feeding dietary n3 f a t t y acids i n excess of the requirement 32 The e f f e c t of dietary protein to l i p i d r a t i o on growth, feed e f f i c i e n c y and body composition 33 iv Page The e f f e c t of dietary l i p i d composition on growth and body f a t t y acid composition 35 The method by which dietary f a t t y acids are quantified..36 The e f f e c t of dietary f a t t y acid concentration on saltwater tolerance 37 The objective 40 Materials and methods E x p e r i m e n t a l d i e t s 42 E x p e r i m e n t a l a n i m a l s and t h e i r e n v i r o n m e n t 48 The t i m e p e r i o d ( P e r i o d s 1 and 2) 52 F e e d i n g and t h e m o n i t o r i n g o f growth and m o r t a l i t y . 5 2 S a m p l i n g p r o c e d u r e 54 A n a l y s i s o f body l i p i d s 55 S a l t w a t e r c h a l l e n g e ..57 S t a t i s t i c a l a n a l y s i s 58 Results Body f a t t y acid composition 61 A. The f a t t y acid composition of f i s h sampled i n period 1 62 I . S a t u r a t e d f a t t y a c i d s 67 I I . M o nounsaturated f a t t y a c i d s 76 I I I . C18 Monounsaturated f a t t y a c i d s 84 IV. T o t a l n3 f a t t y a c i d s 92 V. n3 H i g h l y u n s a t u r a t e d f a t t y a c i d s 92 V I . T o t a l n6 f a t t y a c i d s 93 v Page Summary of dietary e f f e c t s on body composition during period 1 95 B. The f a t t y acid composition of f i s h sampled i n period 2 98 I. Saturated f a t t y acids . 103 I I . Monounsaturated f a t t y acids 103 I I I . C18 Monounsaturated f a t t y acids 108 IV. Total n3 f a t t y acids 114 V. n3 Highly unsaturated f a t t y acids 118 VI. Total n6 f a t t y acids 126 VII. n6 Highly unsaturated f a t t y acids 129 VIII. Body l i p i d 131 IX. Body moisture 131 Summary of dietary e f f e c t s on body composition during period 2 131 Growth 135 Mor t a l i t y 139 Saltwater challenge A. Body composition with respect to saltwater challenge I. Muscle moisture 143 I I . Plasma sodium 145 B. M o r t a l i t y 145 v i Page Discussion Method of quantifying f a t t y acids i n the d i e t (% DMB vs % l i p i d ) 146 The e f f e c t of dietary l i p i d concentration on the re l a t i o n s h i p between dietary f a t t y acids and body f a t t y acid composition 147 The e f f e c t of dietary f a t t y acids on body f a t t y acid composition 149 The e f f e c t of dietary n3 f a t t y acid concentration on growth 158 The e f f e c t of dietary n3 f a t t y acid concentration on mortality 159 The e f f e c t of dietary n3 f a t t y acid concentration on saltwater tolerance 160 Conclusion 163 Li t e r a t u r e c i t e d 167 v i i Page LIST OF TABLES Table 1. Table 2. Table 3. Table 4, Table 5, Table 6, Table 7 Table 8. Table 9. Table 10 Table 11 . Composition of experimental d i e t s 43 L i p i d content of the experimental d i e t s 44 Determined f a t t y acid composition of the d i e t . Concentrations are expressed as percentages of the d i e t on a dry matter basis 45 Determined f a t t y acid composition of the d i e t . Concentrations are expressed as percentages of the dietary l i p i d 46 Determined t o t a l l i p i d and dry matter composition i n the d i e t 47 Dry matter and l i p i d composition of f i s h at the end of period 1. Concentrations of polar and neutral body l i p i d are expressed as percentages of the t o t a l l i p i d or of the no n - l i p i d dry s t r u c t u r a l body weight 62 Fatty acid composition of polar l i p i d extracted from f i s h at the end of period 1. Concentrations of f a t t y acids are expressed as percentages of the polar l i p i d 63 Fatty acid composition of polar l i p i d extracted from f i s h at the end of period 1. Concentrations of f a t t y acids are expressed as percentages of the non-lipid dry body weight..64 Fatty acid composition of neutral l i p i d extracted from f i s h at the end of period 1. Concentrations of f a t t y acids are expressed as percentages of the neutral l i p i d 65 Fatty acid composition of neutral l i p i d extracted from f i s h at the end of period 1. Concentrations of f a t t y acids are expressed as percentages of the no n - l i p i d dry s t r u c t u r a l body weight 66 S t a t i s t i c a l l y s i g n i f i c a n t regressions of body f a t t y acid composition as a function of dietary f a t t y acids from period 1 97 vi i i Page Table 12. Body dry matter and l i p i d composition at the end of period 2. Concentrations of polar and neutral body l i p i d are expressed as percentages of the t o t a l l i p i d or of the n o n - l i p i d dry s t r u c t u r a l body weight 98 Table 13. Fatty acid composition of polar l i p i d extracted from f i s h at the end of period 2. Concentrations of f a t t y acids are expressed as percentages of the polar l i p i d . . . 9 9 Table 14. Fatty acid composition of polar l i p i d extracted from f i s h at the end of period 2. Concentrations of f a t t y acids are expressed as percentages of the n o n - l i p i d dry body weight 100 Table 15. Fatty acid composition of neutral l i p i d extracted from f i s h at the end of period 2. Concentrations of f a t t y acids are expressed as percentages of the neutral l i p i d 101 Table 16. Fatty acid composition of neutral l i p i d extracted from f i s h at the end of period 2. Concentrations of f a t t y acids are expressed as percentages of the n o n - l i p i d dry s t r u c t u r a l body weight 102 Table 17. S t a t i s t i c a l l y s i g n i f i c a n t regressions of body f a t t y acid composition as a function of dietary f a t t y acids from period 2 134 Table 18. The mean body weight of f i s h at the beginning of period 1, between periods 1 and 2, and at the end of period 2 137 Table 19. The number of fish/tank at the beginning of period 1, between periods 1 and 2, and at the end of period 2 as well as the mortality, r e l a t i v e growth rate and s p e c i f i c growth rate observed during period 2 138 Table 20. Percentage dry matter i n muscle of coho salmon before and a f t e r a 24 hour saltwater challenge 144 Table 21. Mean Plasma Sodium Concentration (meq/1) following a 24 hour saltwater challenge 145 i x Page LIST OF FIGURES Figure 1. Water temperature for periods 1 and 2, i n c l u s i v e 50 Figure 2. Water temperature during period 2 51 The e f f e c t of dietary f a t t y acids on body composition Period 1 Figure 3. Saturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary saturated f a t t y acid concentration (% DMB) in period 1. The regression includes the low l i p i d d i e t s (I-V) only 69 Figure 4. Saturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary saturated f a t t y acid concentration (% l i p i d ) i n period 1. The regression involves a l l d i e t s (I-X) 70 Figure 5. Saturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% l i p i d ) i n period 1. The regression involves a l l d i e t s (I-X) 71 Figure 6. Saturated f a t t y acid concentration i n the body neuttal l i p i d as a function of dietary 18:1 f a t t y acid concentration (% DMB) i n period 1. The regression involves a l l diets (I-X) 72 Figure 7. Saturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary 18:1 f a t t y acid concentration (% l i p i d ) i n period 1. The regression involves a l l d i e t s (I-X) 73 Figure 8. Saturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary monounsaturated f a t t y acid concentration (% DMB) i n period 1. The regression involves a l l diets (I-X) 74 x Page Figure 9. Saturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary monounsaturated f a t t y acid concentration (% l i p i d ) i n period 1. The regression involves a l l d i e t s (I-X) 75 Figure 10. Monounsaturated f a t t y acid concentration i n body neutral l i p i d as a function of dietary monounsaturated f a t t y acid concentration (% DMB) i n period 1. The regression involves a l l d i e t s (I-X) the Figure 11. Monounsaturated f a t t y acid concentration i n body neutral l i p i d as a function of dietary monounsaturated f a t t y acid concentration (% l i p i d ) i n period 1. The regression involves a l l diets (I-X) .78 the Figure 12. Monounsaturated f a t t y acid concentration i n body polar l i p i d as a function of dietary monounsaturated f a t t y acid concentration (% DMB) i n period 1. The regression involves a l l diets (I-X) .79 the Figure 13. Monounsaturated f a t t y acid concentration i n body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% l i p i d ) i n period 1. The regression involves a l l d i e t s (I-X) .80 the Figure 14. Monounsaturated f a t t y acid concentration i n body polar l i p i d as a function of dietary n3 f a t t y acid concentration (% DMB) i n period 1. The regression involves the high l i p i d d i e ts (VI-X) only .81 the Figure 15. Monounsaturated f a t t y acid concentration i n body polar l i p i d as a function of dietary n3 f a t t y acid concentration (% l i p i d ) i n period 1. The regression involves the high l i p i d d i e ts (VI-X) only .82 the ,83 Figure 16. 18:1 f a t t y acid concentration i n the body neutral l i p i d as a function of dietary 18:1 f a t t y acid concentration (% DMB) i n period 1. The regression involves a l l d i e t s (I-X) 86 xi Page Figure 17. 18:1 f a t t y acid concentration i n the body neutral l i p i d as a function of dietary 18:1 f a t t y acid concentration (% l i p i d ) i n period 1. The regression involves a l l d i e t s (I-X)...87 Figure 18. 18:1 f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% DMB) i n period 1. The regression involves the low l i p i d d i e t s (I-V) only 88 Figure 19. 18:1 f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% l i p i d ) i n period 1. The regression involves a l l d i e t s (I-X)...89 Figure 20. 18:1 f a t t y acid concentration i n the body polar l i p i d as a function of dietary n3 f a t t y acid concentration (% DMB) i n period 1. The regression involves the high l i p i d d i e t s (VI-X) only 90 Figure 21. 18:1 f a t t y acid concentration i n the body polar l i p i d as a function of dietary n3 f a t t y acid concentration (% l i p i d ) i n period 1. The regression involves the high l i p i d d i e ts (VI-X) only .91 Figure 22. n6 f a t t y acid concentration i n the body polar l i p i d as a function of dietary n6 f a t t y acid concentration (% DMB) i n period 1. The regression involves a l l d i e t s (I-X) 94 The e f f e c t of dietary f a t t y acids on body composition Period 2 Figure 23. Monounsaturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary - monounsaturated f a t t y acid concentration (% DMB) i n period 2. The regression involves a l l d i e t s (I-X) 105 Figure 24. Monounsaturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary monounsaturated f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X) 106 xi i Page Figure 25. Monounsaturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X) 107 Figure 26. 18:1 f a t t y acid concentration i n the body neutral l i p i d as a function of dietary 18:1 f a t t y acid concentration (% DMB) i n period 2. The regression involves a l l d i e t s (I-X) 109 Figure 27. 18:1 f a t t y acid concentration i n the body neutral l i p i d as a function of dietary 18:1 f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X)..110 Figure 28. 18:1 f a t t y acid concentration i n the body polar l i p i d as a function of dietary 18:1 f a t t y acid concentration (% DMB) i n period 2. The regression involves the low l i p i d d i e t s (I-V) only I l l Figure 29. 18:1 f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% DMB) i n period 2. The regression involves the low l i p i d d i e t s (I-V) only 112 Figure 30. 18:1 f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X)..113 Figure 31. n3 f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% DMB) i n period 2. The regression involves a l l d i e t s (I-X) 115 Figure 32. n3 f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X)..116 Figure 33. n3 f a t t y acid concentration i n the body polar l i p i d as a function of dietary n3 f a t t y acid concentration (% lipid), i n period 2. The regression involves a l l d i e t s (I-X) 117 xi i i Page Figure 3 4 . n3 highly unsaturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 highly unsaturated f a t t y acid concentration (% DMB) i n period 2. The regression involves a l l d i e t s (I-X) 120 Figure 3 5 . n3 highly unsaturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 highly unsaturated f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X) 121 Figure 3 6 . n3 highly unsaturated f a t t y acid concentration i n the body polar l i p i d as a function of dietary n3 highly unsaturated f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X) 122 Figure 3 7 . n3 highly unsaturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% DMB) i n period 2. The regression involves a l l d i e t s (I-X) 123 Figure 3 8 . n3 highly unsaturated f a t t y acid concentration i n the body neutral l i p i d as a function of dietary n3 f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X) 124 Figure 3 9 . n3 highly unsaturated f a t t y acid concentration i n the body polar l i p i d as a function of dietary n3 f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X) 125 Figure 4 0 . n6 f a t t y acid concentration i n the body polar l i p i d as a function of dietary n6 f a t t y acid concentration (% DMB) i n period 2. The regression involves the low l i p i d d i e ts (I-V) only 127 Figure 4 1 . n6 f a t t y acid concentration i n the body polar l i p i d as a function of dietary n6 f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X) 128 x i v Page Figure 42. n6 highly unsaturated f a t t y acid concentration i n the body polar l i p i d as a function of die t a r y n6 f a t t y acid concentration (% l i p i d ) i n period 2. The regression involves a l l d i e t s (I-X) 130 Figure 43. Number of m o r t a l i t i e s during period 2 as a function of dietary n3 f a t t y acids (% DMB)..141 Figure 44. Number of m o r t a l i t i e s during period 2 as a function of dietary n3 f a t t y acids (% l i p i d ) 142 xv Acknowledgement The author wishes thank Carol MacMillan, E l l e n Teng, James McGeer, David Farkvam, Chutima T a n t i k i t t i , Heather Ashton and Darin Bennett f o r t h e i r e xcellent t e c h n i c a l assistance. I would l i k e to thank Professor B.E. March fo r her persistence i n educating me. My understanding of the realm of research i s based on her example. I cannot give enough thanks to my family and my friends who have always been there to share my joy and mend my wounds. I wish to acknowledge the contribution of herring o i l , herring meal and feather meal by Moore-Clark Co. (Canada) Inc., Vancouver, B.C. This project was funded i n part by a grant from the National Sciences and Engineering Research Council of Canada. 1 Introduction The farming of salmon i s a v i v i d example of h i s t o r y repeating i t s e l f . The salmon being consumed by the world i s increasingly becoming the r e s u l t of a s h i f t i n man's r o l e from that of a hunter to one of a keeper of domesticated creatures. In t h i s modern day move towards c i v i l i z a t i o n aquatic animals and plants are being removed from t h e i r natural habitat and raised with some degree of in t e n s i t y . Mankind has often prospered as a r e s u l t of i t s a b i l i t y to perform t h i s t r a n s i t i o n e f f e c t i v e l y . I t i s t h i s a r t of manipulating natural systems that allows man to t r u l y e x p l o i t h i s environment. The act of e f f e c t i v e l y t r a n s f e r r i n g an organism from i t s natural environment to an a r t i f i c i a l environment i s referred to as "domestication". The effectiveness of t h i s t r a n s f e r r e f e r s to the a b i l i t y of the organism to survive, reproduce and grow i n i t s a r t i f i c i a l environment. Understanding the creature i s es s e n t i a l to the act of domestication. I n i t i a l l y , the understanding i s e s s e n t i a l only to propagate the species. However, refined knowledge eventually leads to greater e f f i c i e n c y i n manipulating the creature. Behaviour, locomotion, reproduction, disease resistance and n u t r i t i o n are general areas of study. 2 W i t h i n each of these areas are numerous f i e l d s of study. The study of salmon n u t r i t i o n has been l o g i c a l l y separated i n t o the f i e l d s of p r o t e i n , l i p i d , carbohydrate, moisture, v i t a m i n and mineral n u t r i t i o n . W i t h i n each of these f i e l d s l i e s p e c i f i c areas of i n q u i r y which e i t h e r ask a question of immediate relevance or are aimed at f u r t h e r understanding the animal i n question. L i p i d n u t r i t i o n i n salmon i s s t u d i e d f o r both of the above reasons. Questions which are p e r t i n e n t t o the present f i s h farming i n d u s t r y i n v o l v e growth maximization, l i m i t i n g m o r t a l i t y , and the maintenance of o r g a n o l e p t i c p r o p e r t i e s i n the f i s h . Broader areas of i n t e r e s t w i t h i n t h i s f i e l d i n v o l v e the understanding of the metabolism of l i p i d s and other n u t r i e n t s i n the body. The study of f a t t y a c i d s w i t h i n the d i e t a r y l i p i d a l s o address s p e c i f i c questions and general knowledge. P e r t i n e n t questions i n v o l v e the r e l a t i o n s h i p of growth and m o r t a l i t y t o d i e t a r y f a t t y a c i d c o n c e n t r a t i o n s . Are there any c o n s t i t u e n t s of d i e t a r y l i p i d which maximize growth or minimize m o r t a l i t y ? I s d i e t p a l a t a b i l i t y a f f e c t e d by d i e t a r y l i p i d composition or t o t a l content? Do d i f f e r e n t s pecies of salmon r e a c t d i f f e r e n t l y t o d i e t a r y l i p i d s ? Are the d i f f e r e n t stages of the salmon's l i f e c y c l e a f f e c t e d s i m i l a r l y by d i e t a r y f a t t y a c i d composition? 3 The p r a c t i c a l importance of studying the e f f e c t of dietary f a t t y acids on coho salmon Present e s t i m a t i o n s of world marine f i s h o i l s t o r e s would be s u f f i c i e n t t o meet B r i t i s h Columbia's a q u a c u l t u r a l needs i n feed f o r m u l a t i o n . However, increased demand of high q u a l i t y marine f i s h o i l s may be seen i n the human food and medicine s e c t o r as t h e i r a m e l i o r a t i n g e f f e c t s of n3 h i g h l y unsaturated f a t t y a c i d s i s f u r t h e r documented. The r e s i d u a l o i l i n f i s h meal i s a s i g n i f i c a n t source of n3 f a t t y a c i d s . Mugrditchian et a l . (1981) c a l c u l a t e d the n3 content i n h e r r i n g meal t o be approximately 6.7 % of the r e s i d u a l o i l . The reported l e v e l equals approximately 0.6% of the meal i t s e l f on a dry matter b a s i s . Changes i n f i s h meal q u a l i t y and a v a i l a b i l i t y may a f f e c t the n3 content i n f i s h d i e t s . I t i s c r u c i a l t h a t the q u a l i t y of primary i n g r e d i e n t s i n f i s h feeds i s c o n s i s t e n t l y maintained. U n f o r t u n a t e l y , f i s h o i l s can vary d r a m a t i c a l l y i n q u a l i t y . Q u a l i t y of raw i n g r e d i e n t s , processing c o n d i t i o n s and procedures, the use of s t a b i l i z i n g agents, storage and h a n d l i n g are f a c t o r s which d i c t a t e the q u a l i t y of the end product. The q u a l i t y of the f i s h o i l w i l l a f f e c t i t s f a t t y a c i d content. 4 The n3 content of f i s h v a r i e s among sp e c i e s . The f a t t y a c i d composition of menhaden o i l i s not e q u i v a l e n t t o the composition of h e r r i n g o i l . F i s h o i l s from the Norwegian h e r r i n g o i l and meal i n d u s t r y intended f o r use i n aquaculture (NorSalmOil) are marketed w i t h a guaranteed content of l e s s than 4.5% f r e e f a t t y a c i d s . I t i s assumed t h a t o i l s w i t h a high f r e e f a t t y a c i d content have probably been exposed t o c o n d i t i o n s which may r e s u l t i n high l e v e l s of o x i d i z a t i o n . Austreng and Gjefsen (1981) found crude (unrefined) c a p e l i n o i l s c o n t a i n i n g 1.8 - 11.0 % f r e e f a t t y a c i d s . Variances such as these i n a r e l a t i v e l y unregulated commodity cre a t e concerns f o r f i s h o i l based f i s h d i e t s . As increased demand c a l l s on a world market p l a c e t o provide f i s h o i l , f i s h feed manufacturers could see great v a r i a t i o n i n the q u a l i t y of f i s h o i l based d i e t s . As a precautionary step, feed manufacturers would be wise t o decrease t h e i r r e l i a n c e on f i s h o i l s and spread the n u t r i t i o n a l r e s p o n s i b i l i t y of the l i p i d source t o a l t e r n a t e o i l s i n combination w i t h marine f i s h o i l s . The i n c o r p o r a t i o n of m u l t i p l e i n g r e d i e n t s i n t o d i e t s provides the manufacturer w i t h the f l e x i b i l i t y of d i e t manipulation. Subtle changes i n d i e t composition should not r e s u l t i n d r a s t i c changes i n growth, feed e f f i c i e n c y 5 and s u r v i v a l p r o v i d i n g n u t r i e n t requirements have been met. To date only a few a l t e r n a t e l i p i d sources have been used i n salmonid d i e t s . Yu et a l . (1977). used swine f a t as an energy source i n t r o u t r a t i o n s . N e i t h e r i n t a k e nor feed e f f i c i e n c y was a f f e c t e d by the i n c l u s i o n of l a r d t o a maximum of 50% of the t o t a l l i p i d . Takeuchi e t a l . (1978) implemented 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 carp and rainbow t r o u t . They i n d i c a t e d t h a t a l t e r n a t e o i l s can e f f e c t i v e l y serve as energy sources once the e s s e n t i a l f a t t y a c i d requirement has been met. Cowey et a l . (1979) e f f e c t i v e l y r e p l a c e d a p o r t i o n of the marine o i l i n a rainbow t r o u t w i t h hard f a t from p a r t i a l l y rendered hide f l e s h i n g s without a d v e r s e l y a f f e c t i n g growth. Cho e t a l . (1974) compared soybean o i l , rapeseed o i l or marine o i l i n rainbow t r o u t d i e t s . Weight g a i n i n the f i s h was not a f f e c t e d by l i p i d source. However, feed conversion was s i g n i f i c a n t l y b e t t e r f o r f i s h on the marine o i l d i e t . Mugrditchian et a l . (1981) used 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 fOncorhynchus tshawvtscha). From the l a c k of a s i g n i f i c a n t d i e t a r y e f f e c t i n each example we can conclude t h a t once the e s s e n t i a l f a t t y a c i d requirement has been met a l t e r n a t i v e l i p i d sources can 6 e f f e c t i v e l y be u t i l i z e d i n the d i e t provided t h a t d i g e s t i b i l i t y i s c o n s i s t e n t among l i p i d s . D i f f e r e n c e s i n the growth response t o d i f f e r e n t f a t t y a c i d s among the salmonids may be f u r t h e r j u s t i f i c a t i o n f o r the use of m u l t i p l e l i p i d sources. Dosanjh et a l . (1988) evaluated canola o i l , pork l a r d and marine l i p i d , s i n g l y and i n combination, as supplemental d i e t a r y sources f o r j u v e n i l e chinook salmon (Oncorhynchus tshawytscha). This p r e l i m i n a r y r e p o r t found t h a t d i e t s u s i n g a l t e r n a t e l i p i d sources or combinations of sources r e s u l t i n s i g n i f i c a n t l y b e t t e r feed conversion than do d i e t s u s i n g only h e r r i n g o i l . There were no d i f f e r e n c e s i n growth response among the a l t e r n a t e l i p i d s or t h e i r v a r i o u s combinations. Dosanjh e t a l . (1984) reported on the e f f i c a c y of canola 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 (Oncorhynchus k i s u t c h ) . The l a c k of d i e t a r y i n f l u e n c e i n t h i s salmonid species suggests, as i s the case w i t h other salmonids, t h a t a l t e r n a t e l i p i d sources which supplement an adequate content of e s s e n t i a l f a t t y a c i d s do not impede growth or feed conversion. However, remarks were made regarding the p a l a t a b i l t y and t e x t u r e of d i e t s and the e f f e c t t h a t a l t e r n a t e l i p i d s might have on these two important d i e t a r y t r a i t s . 7 The d i g e s t i b i l i t y of a l t e r n a t e l i p i d source must be considered when d i e t a r y manipulations occur. Takeuchi et a l . (1978) reported t h a t the d i g e s t i b i l i t y of h i g h l y hydrogenated o i l s was seen t o be 20% lower than a combination of soybean and c u t t l e f i s h o i l . The d i g e s t i b i l i t y of the hydrogenated o i l s mixed w i t h n3 h i g h l y unsaturated f a t t y a c i d source o i l s was not d r a s t i c a l l y lower than t h a t of the n3 h i g h l y unsaturated f a t t y a c i d o i l alone w h i l e the d i g e s t i b i l t y of hydrogenated f i s h o i l was markedly lower. Studying the e s s e n t i a l f a t t y acid requirement i n f i s h F a t t y a c i d research i n salmon a l s o addresses the comprehensive questions of which f a t t y a c i d s are e s s e n t i a l t o t h a t animal, a t what l e v e l s , and why. Understanding these important p r i n c i p l e s w i l l a l l o w n u t r i t i o n r esearch t o progress e f f e c t i v e l y i n i t s attempt a t r e s o l v i n g the d a i l y dilemma of a q u a c u l t u r a l endeavors. I n i t i a l l y , l i p i d n u t r i t i o n and f a t t y a c i d metabolism i n salmonids were accepted as being s i m i l a r t o those of t e r r e s t r i a l animals. However, t h i s e x t r a p o l a t i o n proved t o be erroneous. T e r r e s t r i a l animals and f i s h do share the i n a b i l i t y t o elongate and desaturate s p e c i f i c f a t t y 8 a c i d s . Polyunsaturated f a t t y a c i d s cannot be sy n t h e s i z e d de novo i n e i t h e r case. The i n a b i l i t y t o sy n t h e s i z e these compounds d e f i n e s the e s s e n t i a l i t y of the f a t t y a c i d s . However, the idea t h a t d i f f e r e n t f a m i l i e s of f a t t y a c i d s ( l i n o l e i c or l i n o l e n i c ) might be r e q u i r e d f o r d i f f e r e n t animals was not embraced u n t i l a f t e r u n t i l e a r l y r e s e a r c h e r s , P h i l l i p s et a l . (1963), unknowingly reported the importance of n3 polyunsaturated f a t t y a c i d s i n salmonids. They found t h a t the a d d i t i o n of corn o i l t o a d i e t was l i m i t e d i n i t s e f f e c t when f i s h meal was d e l e t e d from the d i e t of brook t r o u t ( S a l v e l i n u s f o n t i n a l i s ) growth. F i s h meal was the only s i g n i f i c a n t source of n3 f a t t y a c i d s i n the d i e t s . This i n i t i a l naive o b s e r v a t i o n has r e s u l t e d i n the statement of n3 e s s e n t i a l i t y i n t e l e o s t s . I t has a l s o l e d t o much debate regarding the r o l e s of n3 and n6 f a t t y a c i d s i n t e r r e s t r i a l animals and f i s h r e s p e c t i v e l y . The f i e l d of a f a t t y a c i d research i n f i s h has p r i m a r i l y i n v o l v e d salmonids. I t i s erroneous t o e x t r a p o l a t e these r e s u l t s across s p e c i e s . The l i m i t e d r esearch t h a t has i n v o l v e d other species of f i s h has found d i f f e r e n c e s between sp e c i e s . Yamada et a l . (1980) has demonstrated t h a t 18:3n3 i s e s s e n t i a l t o rainbow t r o u t (Oncorhynchus m y k i s s j , carp (Cyprinus carpio) and the e e l 9 ( A n g u i l l a i a p o n i c a ) . This f a t t y a c i d i s of much l e s s e r importance t o marine f i s h e s such as Red sea bream (Chrysophrys major), Black sea bream (Mvlio  macrocephalus), opaleye ( G i r e l l a n i g r i c a n s ) , and freshwater f i s h e s such as y e l l o w t a i l ( S e r i o l a  q u i n q u e r a d i a t a ) . I t was assumed t h a t the d i f f e r e n c e i n e s s e n t i a l i t y l i e s i n the a b i l i t y t o convert 18:3n3 t o n3 h i g h l y unsaturated f a t t y a c i d s . From these r e p o r t s i t i s assumed t h a t t h e r e i s much v a r i a t i o n i n e s s e n t i a l i t y of f a t t y a c i d s . E l o n g a t i o n and d e s a t u r a t i o n of f a t t y a c i d s i s thought t o occur i n other s p e c i e s . However, the r a t e of t h i s conversion does seem t o vary from species t o spe c i e s . The mechanisms of l i p i d metabolism and b i o s y n t h e s i s seem t o be u n i v e r s a l among t e l e o s t s (Yamada et a l 1980). A l l s pecies which possess the app r o p r i a t e enzymes do convert some sho r t chain n3 f a t t y a c i d s i n t o longer chained polyunsaturates as w e l l as i n t o s h o r t e r , more sa t u r a t e d f a t t y a c i d s . 18:3 n3 can be broken down through B - o x i d a t i o n and new f a t t y a c i d s b i o s y n t h e s i z e d through acetyl-CoA. Some t e l e o s t s are unable t o elongate and desaturate short-chained n3 f a t t y a c i d s . Owen et a l . (1975) dis c o v e r e d t h a t the t u r b o t (Scophthalmus maximus L.) i s 10 one such species. Using radioactive tracers they showed that the turbot require long-chained highly unsaturated f a t t y acids while rainbow trout are able to elongate and desaturate 18:3n3 to n3 highly unsaturated f a t t y acids. Elongation and desaturation of both the l i n o l e n i c and l i n o l e i c family f a t t y acids occurs i n the rainbow trout when the respective d i e t s were fed (C a s t e l l et a l . 1972c). The elongation and desaturation mechanism for producing >C20 f a t t y acids seems to be common to n3, n6 and n9 f a t t y acids. Yu and Sinnhuber (1972) observed that 20:3n9 was biosynthesized i n rainbow trout from n3 d e f i c i e n t d i e t s . The elongation and desaturation of non-essential f a t t y acids was decreased when dietary n3 l e v e l s were increased thereby providing evidence for the idea of substrate dependent elongation and desaturation. I t was evident to Yu and Sinnhuber (1979) that both n6 and n3 f a t t y acids are able to i n h i b i t the incorporation of 20:3n9 into body phospholipids i n f i s h . The competition between e s s e n t i a l and nonessential f a t t y acids for elongation resulted i n the development of the es s e n t i a l f a t t y acid index. The es s e n t i a l f a t t y acid index i s a t o o l used to estimate e s s e n t i a l f a t t y acid deficiency. I t i s simply the r a t i o of 20:3n9 /22:6n3. Takeuchi et a l . (1979) stated that 20:ln9 and 18:ln9 11 concentrations i n the body decrease when l i n o l e i c or l i n o l e n i c acid i s fed. This can be associated with the elongation of n9 f a t t y acids. Takeuchi and Watanabe (1977a) observed that the addition of l i n o l e n a t e to the d i e t s decreased the amount of 16:1, 18:1 and 20:3 n9 i n the body of rainbow trout. However, n3 highly unsaturated f a t t y acids are more e f f e c t i v e i n decreasing abnormal l e v e l s of the n9 f a t t y acids. There was a low e s s e n t i a l f a t t y a c i d index i n rainbow trout fed high l i p i d d i e t s with only 1% linolenate. The acceptable index l e v e l occured even though that l e v e l of supplementation was not s u f f i c i e n t to maximize growth. Therefore, the e s s e n t i a l f a t t y acid index i s not a v a l i d t o o l f o r growth maximization. The r a t i o of 20:3 n9 / 22:6 n3, which has been used to indicate the adequacy of dietary l i p i d s i n rainbow trout, cannot be used i n turbot because of turbot's i n a b i l i t y to elongate o l e i c acid according to Owen et a l . (1975). This would apply to other marine f i s h with s i m i l a r n3 elongation and desaturation c a p a c i t i e s . The salmonids Differences i n l i f e cycle and d i e t are the two most obvious reason why differences i n f a t t y acid n u t r i t i o n i n 12 these f i s h might be expected. I t can be expected t h a t species which have evolved under a s p e c i f i c environment and d i e t a r y regime might metabolize f a t t y a c i d s d i f f e r e n t l y . Rainbow t r o u t are freshwater r e s i d e n t s w h i l e coho (Oncorhynchus k i s u t c h ) , chum (Oncorhynchus k e t a ) , chinook (Oncorhynchus tshawvtscha), sockeye (Oncorhynchus nerka), pink (Oncorhynchus qorbuscha), A t l a n t i c (Salmo s a l a r ) and steelhead salmon (Oncorhynchus mvkiss) a l l are anadromous under d i f f e r e n t l i f e c y c l e s and environments. Die t does d i f f e r w i t h i n and among species due t o these d i f f e r e n c e s . W i t h i n salmonids the n3 f a t t y a c i d s requirements do d i f f e r from species t o speci e s . Takeuchi and Watanabe (1982) found species d i f f e r e n c e s i n the e f f e c t of sh o r t chained and long-chained n3 polyunsaturated f a t t y a c i d s on rainbow t r o u t , coho salmon, and chum salmon. Hardy et a l . (1987) reported t h a t A t l a n t i c salmon respond t o d i e t a r y f a t t y a c i d s i n a manner s i m i l a r t o P a c i f i c salmon and rainbow t r o u t . S p e c i f i c d i f f e r e n c e s w i l l be expanded on l a t e r i n the t e x t . The e s s e n t i a l i t y of n3 f a t t y a c i d s i n salmonids A compound i s deemed t o be n u t r i t i o n a l l y e s s e n t i a l i f i t i s necessary to maintain l i f e and cannot be s u f f i c i e n t l y synthesized de novo i n the body. n3 f a t t y a c i d s were de f i n e d as e s s e n t i a l i n rainbow t r o u t by C a s t e l l et a l . (1972a, 1972b, 1972c). D i e t s d e f i c i e n t i n n3 f a t t y a c i d s r e s u l t e d i n m o r t a l i t y , f i n e r o s i o n , p a l e enlarged l i v e r s , increased m i t o c h o n d r i a l r e s p i r a t i o n , acute m y o c a r d i t i s , m i l d macrocytic hypochromic anemia and " f a i n t i n g " or shock syndrome and poor growth. The l i n o l e n i c f a m i l y of f a t t y a c i d s were e x c l u s i v e l y r e s p o n s i b l e f o r the c o r r e c t i o n and prevention of the d e f i c i e n c y symptoms. The i n c l u s i o n of 1% l i n o l e n a t e i n the d i e t of rainbow t r o u t c o r r e c t e d m i t o c h o n d r i a l s w e l l i n g , prevented the shock syndrome, decreased m o r t a l i t y , improved growth and completely prevented f i n e r o s i o n . I t has been proven t h a t n3 f a t t y a c i d s are e s s e n t i a l i n preventing the maladies described above. However, the a c t u a l mechanism by which these e s s e n t i a l n u t r i e n t s c o r r e c t and prevent these symptoms s t i l l eludes researchers. What i s i t about t h i s group of f a t t y a c i d s t h a t makes them e s s e n t i a l t o t e l e o s t s ? The r o l e s t h a t these compounds might p l a y i n the physiology of the f i s h w i l l be discussed l a t e r . 14 The s i t e of the double bond w i t h respect t o the t e r m i n a l methyl group does not seem t o be the e x c l u s i v e l y r e s p o n s i b l e f o r the d e f i n i t i o n of e s s e n t i a l i t y . Takeuchi and Watanabe (1982) f a i l e d t o meet the n3 requirement of rainbow t r o u t , coho salmon and chum salmon u s i n g *12,19-C22:2. This nonmethylene i n t e r r u p t e d (NMID) p o l y e t h y l e n i c f a t t y a c i d possesses a double bond at the n3 p o s i t i o n but has another near the carbo x y l end. I t i s evident t h a t the d e f i n i t i o n of n3, a carbon-carbon double bond s i t u a t e d between the t h i r d and f o u r t h carbons on a c h a i n , i s not the s o l e reason f o r e s s e n t i a l i t y . The m e l t i n g p o i n t of f a t t y a c i d s has been a s s o c i a t e d w i t h t h e i r e s s e n t i a l i t y i n p o i k i l o t h e r m s . I t would seem p h y s i o l o g i c a l l y unsound f o r the f i s h t o i n c o r p o r a t e f a t t y a c i d s w i t h high m e l t i n g p o i n t s i n t o the body. The m e l t i n g p o i n t of f a t t y a c i d s u s u a l l y decreases as the degree of u n s a t u r a t i o n i n c r e a s e s . The m e l t i n g p o i n t of C18 f a t t y a c i d s decreases from +16.3°C i n o l e i c a c i d (18:1 n9) t o -5.0°C i n l i n o l e i c a c i d (18:2 n6) t o -11.3°C i n l i n o l e n i c a c i d (18:3 n3). P a r i n a r i c a c i d (18:4n3) i s an exception t o the r u l e having a m e l t i n g p o i n t of +84°C. The high m e l t i n g p o i n t i n t h i s case i s due t o the s t r u c t u r e of compound. I t i s u n c e r t a i n whether p o i k l i o t h e r m s i n c o r p o r a t e f a t t y a c i d s d i f f e r e n t l y at 15 d i f f e r e n t ambient temperatures. For t h i s reason i t i s important t o consider water temperature when f a t t y a c i d n u t r i t i o n experiments are conducted. The f a c t t h a t both l i n o l e a t e and l i n o l e n a t e have m e l t i n g p o i n t s below the l e t h a l temperature f o r t e l e o s t s does not answer the question why n3 and not n6 f a t t y a c i d are e s s e n t i a l i n salmonids. The whole i s s u e of whether or not n6 f a t t y a c i d s are a c t u a l l y e s s e n t i a l i n salmonids w i l l not be f u l l y addressed i n t h i s paper. E s s e n t i a l n u t r i e n t s may be r e q u i r e d i n such small amounts t h a t even p u r i f i e d d i e t s may provide adequate amounts (Yu e t a l . 1979). The Roles of n3 f a t t y a c i d s E s s e n t i a l f a t t y a c i d s as p r o s t a g l a n d i n p r e c u r s o r s The most l i k e l y e s s e n t i a l r o l e of n3 f a t t y a c i d s i n t e l e o s t s i n v o l v e s p r o s t a g l a n d i n s y n t h e s i s . The r o l e of e s s e n t i a l f a t t y a c i d s i n p r o s t a g l a n d i n s y n t h e s i s i n t e r r e s t r i a l animal leads n u t r i t i o n i s t s and biochemists t o hypothesize a s i m i l a r r o l e f o r the n3 f a m i l y of f a t t y a c i d s i n f i s h . However, cu r r e n t research has been unable t o i d e n t i f y a b i o s y n t h e s i s system t h a t i s d i f f e r e n t than 16 t h a t of t e r r e s t r i a l animals. The main pr e c u r s o r f a t t y a c i d i n these animals i s a r a c h i d o n i c a c i d (20:4n6). 20:3n6 and 20:5n3 are the next most important p r e c u r s o r s . P r o s t a g l a n d i n s have been detected i n f i n f i s h and lower animals. Nomura and Ogata (1976) i s o l a t e d p r o s t a g l a n d i n s i n carp, s h e a t - f i s h , leopard shark, as w e l l as i n c r a w f i s h , blue crab, mussel, s c a l l o p , s e a - s q u i r t , and sea-anemone. I t appeared t o these researchers t h a t the g a s t r o - i n t e s t i n a l t i s s u e s , a i r bladder, heart and g i l l of f i s h e s are i n c l i n e d t o have a higher l e v e l of p r o s t a g l a n d i n than other t i s s u e s . I t i s assumed, but not confirmed, t h a t p r o s t a g l a n d i n s i n f i s h are important i n modulating numerous important p h y s i o l o g i c a l f u n c t i o n s i n c l u d i n g c a r d i a c , pulmonary, and muscular. I t i s a l s o u n c e r t a i n whether or not the f i s h c o n t a i n s p r o s t a g l a n d i n s i n a d d i t i o n t o those found i n t e r r e s t r i a l animals. P r o s t a g l a n d i n s of the 1, 2, and 3 s e r i e s are s y n t h e s i z e d from the C20 unsaturated f a t t y a c i d s e i c o s a t r i e n o i c a c i d (20:3 n6), e i c o s a t e t r a n o i c a c i d (2 0:4n6) and eicosapentanoic a c i d (20:5n3) r e s p e c t i v e l y . In t e r r e s t r i a l animals the 2 s e r i e s p r o s t a g l a n d i n s are predominant. Anderson et a l . (1981) suggested t h a t the mechanism f o r P G E 2 i n f i s h i s s i m i l a r t o t h a t found i n h i g h e r v e r t a b r a t e s . 17 In an p r e l i m i n a r y r e p o r t Mai et a l . (1981) announced the d i s c o v e r y of a new p r o s t a g l a n d i n C 2 2 - P G F 4 A , s y n t h e s i z e d from docosahexaenoic a c i d (22:6n3) by t r o u t g i l l . The presence of s e v e r a l p r o s t a g l a n d i n s w i t h three double bonds i n the g i l l of rainbow t r o u t was a l s o reported. I n a d d i t i o n s i g n i f i c a n t amounts of an unknown compound w i t h chain length equivalence of 2 2 carbons was observed. The evidence f o r the presence of a P G F 4 T T was dispu t e d by the l a b o r a t o r y which i n i t i a l l y made the r e p o r t . German et a l . (1983) communicated t h a t the predominant oxygenating enzyme i n t r o u t g i l l t i s s u e i s a lipoxygenase enzyme and the compound p r e v i o u s l y d e s c r i b e d by Mai et a l . (1981) as PGF 4 a i s a c t u a l l y t r i h y d r o x y l a t e d compounds of the hydroperoxyfatty a c i d s generated by the a c t i o n of the lipoxygenase. I t was concluded t h a t c u r r e n t evidence suggests t h a t t r i h y d r o x y p o l y e n o i c f a t t y a c i d s are predominant products and n3 p r o s t a g l a n d i n s are apparently not produced i n q u a n t i t y by t r o u t g i l l t i s s u e . The search f o r s e r i e s 3 p r o s t a g l a n d i n s i n marine f i s h once again was u n f r u i t f u l when Anderson e t a l . (1979) found t h a t P G E 2 was the only p r o s t a g l a n d i n i d e n t i f i e d i n v i t r o i n p l a i c e , Pleuronectes p l a t e s s a L., r e g a r d l e s s of the s i g n i f i c a n t c o n c e n t r a t i o n of 20:5n3 i n the t i s s u e c u l t u r e d . 18 Much of the research i n f i s h p r o s t a g l a n d i n s y n t h e s i s has been c a r r i e d out i n v i t r o on rainbow t r o u t . I t was not s t a t e d , but, i t i s assumed t h a t these were freshwater f i s h . T h i s f a c t may be of s i g n i f i c a n c e when the osmoregulatory mechanisms of the f i s h are considered. Anadromous f i s h , of which salmonids are d e f i n i t e l y i n c l u d e d , develop c h l o r i d e c e l l s i n the g i l l t i s s u e a t s m o l t i f i c a t i o n (immediately p r i o r t o e n t e r i n g the s a l t water h a b i t a t ) . Research i n v o l v i n g marine f i s h may demonstrate the presence of p r o s t a g l a n d i n s i n the f i s h g i l l which are e x c l u s i v e or more important i n the f i s h . B e l l e t a l . (1983) described the importance of n3 and n6 polyunsaturated f a t t y a c i d s i n the s a l t s e c r e t i n g e p i t h e l i a of two marine f i s h s p e c i e s . The turnover of p h o s p h a t i d y l i n o s i t o l i n the g i l l s of seawater e e l s (Ancruilla a n q u i l l a ) was e l i c i t e d by an a-adrenergic s t i m u l u s , a c o n d i t i o n known t o i n h i b i t s a l t s e c r e t i o n by the g i l l s . P h o s p h a t i d y l i n o s i t o l i s a l s o i n v o l v e d i n p r o s t a g l a n d i n formation by being the source of the 20:4n6 pre c u r s o r used f o r P G E 2 a s y n t h e s i s . P G E 2 i s known t o i n h i b i t s a l t s e c r e t i o n by marine t e l e o s t g i l l s ( P i c , 1975) . There i s evidence t h a t 20:4n6 r a t h e r than eicosapentaenoic a c i d i s the p r e f e r r e d p r e c u r s o r of 19 p r o s t a g l a n d i n s i n marine f i s h . Anderson et a l . (1981) found arachidonate t o be the only s i g n i f i c a n t p r o s t a g l a n d i n p recursor of the e s s e n t i a l f a t t y a c i d p r e c u r s o r s f o r s e r i e s 1, 2, and 3 p r o s t a g l a n d i n s i n p l a i c e s k i n . The c o n c e n t r a t i o n of the precursor i n the f i s h d i d not seem t o e f f e c t prevalence, of the corresponding p r o s t a g l a n d i n . In the p l a i c e the eicosapentaenoic a c i d c o n c e n t r a t i o n was approximately three times t h a t of a r a c h i d o n a i c a c i d . However, the conversion of eicosapentaenoic a c i d t o P G E 3 was approximately 7 times l e s s than t h a t of 20:4n6 t o P G E 2 . The importance of p h o s p h a t i d y l i n o s i t o l and more s p e c i f i c a l l y 20:4 n6 may r e d e f i n e the e s s e n t i a l f a t t y a c i d s i n f i s h . The f a c t t h a t p h o s p h a t i d y l i n o s i t o l i s present i n a very minute q u a n t i t y i n the f i s h probably makes the requirement l e v e l s of arachidonate i n the d i e t extremely low. While eicosapentaenoic a c i d has not been found t o be important as a p r o s t a g l a n d i n precursor i t i s l i k e l y t h a t t h i s e s s e n t i a l f a t t y a c i d p l a y s a r e g u l a t o r y r o l e i n p r o s t a g l a n d i n s y n t h e s i s i n f i s h . This was observed i n the p l a i c e s k i n by Anderson e t a l . (1981). This e f f e c t i s evident i n human medicine by the a b i l i t y t h a t eicosapentaenoic a c i d has i n i n h i b i t i n g the conversion of 20 20:4n6 t o thromboxane A 2 / which i s an important aggregator of p l a t e l e t s . P l a t e l e t s seem t o have a l i m i t e d a b i l i t y t o metabolize eicosapentaenoic a c i d . E s s e n t i a l f a t t y a c i d s i n b i o l o g i c a l membranes E s s e n t i a l f a t t y a c i d s from the d i e t can be i n c o r p o r a t e d i n t o the p o l a r l i p i d which i s found throughout the body. These p o l a r l i p i d s p l a y an important r o l e i n maintenance of the i n t e g r i t y and f u n c t i o n of membranes according t o the c u r r e n t understanding of b i o l o g i c a l membranes. C a s t e l l et a l . (1972b) observed t h a t the f l e s h of rainbow t r o u t fed a f a t - f r e e d i e t were abnormally s o f t and f l a c c i d i n comparison to f i s h fed l i n o l e n i c a c i d . Decreasing the l e v e l of l i n o l e n i c a c i d i n the d i e t apparently promoted an i ncrease i n the muscle water content. I f the e f f e c t i s r e a l , i t would suggest t h a t the change i n muscle composition may be due t o a l t e r a t i o n s i n membrane p e r m e a b i l i t y as a r e s u l t of the l a c k of n3 f a t t y a c i d s . Takeuchi and Watanabe (1979) observed a h i g h body moisture content i n rainbow t r o u t fed a d i e t c o n t a i n i n g 4% methyl l i n o l e n a t e . Once again, manipulating the d i e t a r y n3 content may r e s u l t i n changes i n membrane p e r m e a b i l i t y . 21 E s s e n t i a l f a t t y acids neurological function The " f a i n t i n g " or shock syndrome observed i n n3 f a t t y acid d e f i c i e n t f i s h suggests that these f a t t y acids play a neurological r o l e i n the f i s h . Singh and Chandra (1989) reviewed l i t e r a t u r e which showed substantial amounts of 22:6n3 i n gray matter, white matter, myelin, synaptosomal plasma membrane and synaptic v e s i c l e s i n the brains of humans, the mouse and the rat . The highly unsaturated f a t t y acids seem to be maintained i n the polar l i p i d i n the salmonid brain t i s s u e even a f t e r spawning (Phleger et a l . , 1989). The above factors strongly suggest that n3 highly unsaturated f a t t y acids are es s e n t i a l components of brain l i p i d i n salmonids. The e f f e c t of dietary f a t t y acids on body f a t t y acid composition The body polar l i p i d The l i p i d composition of d i f f e r e n t body organs seem to be affected d i f f e r e n t l y by dietary f a t t y acid composition. Observations regarding t o t a l l i p i d f a t t y a c i d concentration are not as ill u m i n a t i n g as are observations based on the f a t t y acid content of the 22 n e u t r a l or p o l a r f r a c t i o n s . P o l a r l i p i d s , p h o s p h o l i p i d s , are considered as s t r u c t u r a l w h i l e n e u t r a l l i p i d s are considered p r i m a r i l y as body l i p i d r e serves. Body organs d i f f e r i n t h e i r r e l a t i o n s h i p t o d i e t a r y f a t t y a c i d s . Much of these d i f f e r e n c e s r e l a t e t o the r a t i o of p o l a r l i p i d t o n e u t r a l l i p i d i n t h a t organ. The l i v e r i s a source of l i p i d s y n t h e s i s and m o d i f i c a t i o n . The c o n c e n t r a t i o n s of n e u t r a l and p o l a r l i p i d depends on the s t a t e of l e v e l of s y n t h e s i s . The b r a i n , having more p o l a r l i p i d than n e u t r a l , r e t a i n s a r e l a t i v e l y s t a b l e f a t t y a c i d p r o f i l e which i s i n t o l e r a n t t o changes i n d i e t a r y f a t t y a c i d composition. The muscle f a t t y a c i d composition, because of i t s higher n e u t r a l l i p i d content, u s u a l l y r e f l e c t s the d i e t . The b r a i n p o l a r l i p i d does not seems t o be e q u a l l y responsive t o l i n o l e a t e and l i n o l e n a t e ( C a s t e l l e t a l . 1972c). The p o l a r l i p i d does remain c o n s i s t a n t l y high i n h i g h l y unsaturated n3 f a t t y a c i d s r e g a r d l e s s of d i e t a r y f a t t y a c i d content. S i m i l a r f i n d i n g s were reported by Takeuchi and Watanabe (1977b). The l i p o g e n i c nature of the l i v e r does not prevent i t from being a f f e c t e d by d i e t a r y f a t t y a c i d s . The f a t t y a c i d content of t h i s organ i s a f f e c t e d by d i e t a r y l i p i d . C a s t e l l et a l . (1972c) found the h i g h l y unsaturated f a t t y a c i d s i n the p o l a r l i p i d t o be more responsive t o d i e t a r y f a t t y a c i d s than was the n e u t r a l l i p i d . Takeuchi and Watanabe (1982) reported t h a t i n rainbow t r o u t and coho salmon the l i v e r t o t a l l i p i d and nonpolar l i p i d s were i n f l u e n c e d by d i e t . However, l i v e r p o l a r l i p i d i n chum salmon was not a f f e c t e d by d i e t a r y l i p i d content. Takeuchi and Watanabe (1982) observed the s u b s t a n t i a l c o n c e n t r a t i o n s of 20:3n9, 16:1 and 18:1 i n the p o l a r l i p i d of l i v e r s i n rainbow t r o u t fed n3 d e f i c i e n t d i e t s . These f a t t y a c i d s are products of the n o n - e s s e n t i a l f a t t y a c i d s provided i n the d i e t . D i e t a r y f a t t y a c i d c o n c e n t r a t i o n a l s o a f f e c t e d the f a t t y a c i d p r o f i l e s of heart and kidney l i p i d f r a c t i o n s ( C a s t e l l et a l . 1972c). Although the p h o s p h o l i p i d f r a c t i o n f a t t y a c i d content i s l e s s r e f l e c t i v e of d i e t a r y content than the n e u t r a l f r a c t i o n i t i s s t i l l a f f e c t e d by d i e t a r y changes. C a s t e l l et a l . (1972c) found t h a t the n3 content i n the p h o s p h o l i p i d f r a c t i o n of the rainbow t r o u t body l i p i d was inc r e a s e d when the d i e t a r y n3 l e v e l increased t o 1% on a dry matter b a s i s (DMB). n3 f a t t y a c i d s are s e l e c t i v e l y i n c o r p o r a t e d i n t o the p h o s p h o l i p i d as h i g h l y unsaturated f a t t y a c i d s when suboptimal c o n c e n t r a t i o n s of e s s e n t i a l f a t t y a c i d s are 24 fed. D e f i c i e n t d i e t s r e s u l t i n the i n c o r p o r a t i o n of h i g h l y unsaturated n o n - e s s e n t i a l f a t t y a c i d s i n the p o l a r l i p i d . I t i s b e l i e v e d t h a t the mechanism f o r e l o n g a t i o n and d e s a t u r a t i o n of f a t t y a c i d s i n f i s h i s the same f o r n3, n6 and n9 f a t t y a c i d s . The r a t e s of e l o n g a t i o n and d e s a t u r a t i o n are most l i k e l y s u b s t r a t e dependent. I t has been suggested by Brockerhoff et a l . (1966) t h a t 22:6n3 i n h i b i t s the i n c o r p o r a t i o n of 20:3n9 i n t o the B p o s i t i o n of the g l y c e r o p h o s p h o l i p i d s . As a r e s u l t formation of 20:3n9 was l i m i t e d . Therefore i t i s b e l i e v e d t h a t n3 h i g h l y unsaturated f a t t y a c i d s are s e l e c t i v e l y i n corporated i n t o the p o l a r l i p i d without being metabolized. Short-chained n3 f a t t y a c i d s from the d i e t are desaturated and elongated on a su b s t r a t e c o n c e n t r a t i o n b a s i s . They, i n t u r n , are inc o r p o r a t e d i n t o the p o l a r l i p i d s e l e c t i v e l y when they are i n the form of long-chained h i g h l y unsaturates. F u r t h e r evidence of the s e l e c t i v e i n c o r p o r a t i o n of n3 f a t t y a c i d s was reported by Yu and Sinnhuber (1979) and C a s t l e d i n e and Buckley (1980). They found t h a t the p r o p o r t i o n of 22:6n3 incorporated i n t o body p h o s p h o l i p i d from the d i e t in c r e a s e s as the co n c e n t r a t i o n i n the d i e t decreased. N o n - d e f i c i e n t f i s h i n c o r p o r a t e n3 f a t t y a c i d s i n t o the p o l a r l i p i d a t r a t e s which r e f l e c t the d i e t a r y c o n c e n t r a t i o n (Yu e t a l . 1979). This r e l a t i o n s h i p i s not e x c l u s i v e t o the l i n o l e n a t e f a m i l y of f a t t y a c i d s . Lee e t a l . (1967) noted t h a t the body p h o s p h o l i p i d f r a c t i o n of f i s h contained low l e v e l s of 22:6n3 and h i g h l e v e l s of 22:5n6 when the d i e t was composed p r i m a r i l y of l i n o l e i c f a m i l y of f a t t y a c i d s . C a s t l e d i n e and Buckley (1980) concurred w i t h t h i s o b s e r v a t i o n . However, Yu and Sinnhuber (1979) discovered t h a t high c o n c e n t r a t i o n s of 18:3n3 i n the d i e t decrease the i n c o r p o r a t i o n of n6 f a t t y a c i d s i n t o body ph o s p h o l i p i d s . High d i e t a r y n6 co n c e n t r a t i o n s d i d not a f f e c t the i n c o r p o r a t i o n of n3 i n t o body p h o s p h o l i p i d s . Long-chained polyunsaturated f a t t y a c i d s are p r e f e r e n t i a l l y i n corporated i n t o the p h o s p h o l i p i d f r a c t i o n . Lee et a l . (1967) found t h a t d i e t s c o n t a i n i n g n3 h i g h l y unsaturated f a t t y a c i d s r e s u l t e d i n a higher c o n c e n t r a t i o n of 22:6n3 i n the body p o l a r f r a c t i o n than d i e t s c o n t a i n i n g 18:3n3 as the only n3 source. The preference f o r long-chained h i g h l y unsaturated f a t t y a c i d s i n v o l v e d n6 as w e l l as n3 f a t t y a c i d s (Takeuchi and Watanabe 1982). Body p o l a r f a t t y a c i d s other than the n3 and n6 1s do not seem t o be a f f e c t e d by d i e t a r y content. C a s t l e d i n e 26 and Buckley (1980), found t h a t the percentage s a t u r a t e d f a t t y a c i d s remained constant w i t h i n the p h o s p h o l i p i d pool r e g a r d l e s s of the d i e t l i p i d source. I t i s u n c e r t a i n whether or not d i e t a r y f a t t y a c i d composition a f f e c t s the amounts of p o l a r l i p i d i n the body. C a s t l e d i n e and Buckley (1980) s t a t e d t h a t the s i z e of the p h o s p h o l i p i d pool r e l a t i v e t o body weight i n rainbow t r o u t does not vary w i t h changes i n n3 f a t t y a c i d content of the d i e t . I t remained r e l a t i v e l y constant a t 0.97% wet body weight. However, C a s t e l l et a l . (1972c) observed t h a t the amount of p h o s p h o l i p i d i n the body was a s s o c i a t e d w i t h d i e t a r y l e v e l s of l i n o l e n i c a c i d . Body t o t a l l i p i d and n e u t r a l l i p i d F a t t y a c i d composition based on t o t a l body l i p i d i s d i f f i c u l t t o i n t e r p r e t because of the l a c k of homogeneity i n the l i p i d f r a c t i o n s . The p o l a r and n e u t r a l l i p i d f r a c t i o n s are not found i n the body i n equal amounts. These two f r a c t i o n s a l s o r e a c t d i f f e r e n t l y t o d i e t a r y f a t t y a c i d c o n c e n t r a t i o n s . The p o l a r l i p i d i s somewhat s e l e c t i v e w h i l e the n e u t r a l l i p i d masks the d i e t composition. The d i e t w i l l tend t o a f f e c t the t o t a l body l i p i d i n a manner s i m i l a r t o the n e u t r a l l i p i d simply 27 because of the r e l a t i v e abundance of t h a t f r a c t i o n . I n c r e a s i n g d i e t a r y l i n o l e n a t e does change body f a t t y a c i d content (Takeuchi and Watanabe, 1977a). The a d d i t i o n of l i n o l e n a t e t o the d i e t s decreased the amount of 16:1, 18:1 and 20:3 n9 i n the body of rainbow t r o u t . C a s t l e d i n e and Buckley, (1980) found t h a t i n rainbow t r o u t the percentage s a t u r a t e d f a t t y a c i d s i n c r e a s e d i n the body n e u t r a l l i p i d f r a c t i o n w i t h increases i n d i e t a r y s a t u r a t e content. The n6 c o n c e n t r a t i o n i n both the p o l a r and the n e u t r a l l i p i d f r a c t i o n s were a l s o a f f e c t e d by changes i n the d i e t a r y f a t t y a c i d p r o f i l e . C a s t l e d i n e and Buckley (1980) observed what appeared t o be an i n v e r s e r e l a t i o n s h i p between d i e t a r y n3 content and body 22:6n3. The form i n which ingested d i e t a r y f a t t y a c i d s , e s p e c i a l l y n3 f a t t y a c i d s , are in c o r p o r a t e d i n t o the n e u t r a l l i p i d depends on the a b i l i t y of the animal t o desaturate and elongate f a t t y a c i d s . F i s h t h a t are unable t o desaturate and elongate f a t t y a c i d s w i l l possess n e u t r a l l i p i d f r a c t i o n s which c l o s e l y match the p r o f i l e of the d i e t a r y l i p i d (Yamada et a l . 1980). This d i f f e r e n c e i n f a t t y a c i d metabolism i s the primary reason f o r d i f f e r e n c e s i n the n e u t r a l l i p i d f a t t y a c i d p r o f i l e among f i s h e s . Increases i n the n e u t r a l l i p i d pool are seen duri n g 28 pe r i o d s of p o s i t i v e energy balance. These in c r e a s e s are enhanced by d i e t a r y l i p i d , m odified d i e t a r y l i p i d or endogenously synthesized l i p i d . Synthesis and m o d i f i c a t i o n would l i k e l y occur i n the l i v e r because mesenteric adipose t i s s u e has a much l e s s e r l i p o g e n i c a b i l i t y than h e p a t i c t i s s u e . There i s no turnover of the n e u t r a l l i p i d pool d u r i n g t h i s time. The e f f e c t of energy balance on body f a t t y a c i d composition During periods of negative energy balance, f a t t y a c i d s i n the n e u t r a l l i p i d are c a t a b o l i z e d and are probably u n a v a i l a b l e t o the p h o s p h o l i p i d p o o l . C a s t l e d i n e and Buckley (198 0) observed t h a t s t a r v a t i o n caused a r e d u c t i o n i n the 14:0, 16:ln7, 18:ln9 c o n c e n t r a t i o n s and an i n c r e a s e i n the 18:2n6 and 22:6n3 i n the ph o s p h o l i p i d s of rainbow t r o u t except i n f i s h which had r e c e i v e d these two f a t t y a c i d s i n l a r g e amounts. C a s t l e d i n e and Buckley (1980) hypothesized f a t t y a c i d s might be c a t a b o l i z e d on a molar b a s i s i n the n e u t r a l l i p i d . They suggested t h a t 22:6n3 would be c a t a b o l i z e d before s h o r t e r chain f a t t y a c i d s . The p o l a r h i g h l y unsaturated n3 f a t t y a c i d s are maintained i n times of l i p i d catabolism. I t was u n c l e a r whether h i g h l y unsaturated n3 f a t t y a c i d s from the n e u t r a l l i p i d pool are t r a n s f e r r e d t o the p h o s p h o l i p i d pool d u r i n g p e r i o d s of cat a b o l i s m i n the body. D i e t a r y l i p i d s help c r e a t e a p o s i t i v e energy balance which u s u a l l y r e s u l t s i n growth. The f a t t y a c i d themselves and the t o t a l f a t t y a c i d p r o f i l e of d i e t a r y l i p i d seems t o a f f e c t the growth r a t e and feed e f f i c i e n c y . The e f f e c t s of d i e t a r y f a t t y a c i d s on growth In a d d i t i o n t o c o r r e c t i n g the c l a s s s i c a l e s s e n t i a l f a t t y a c i d s i g n s , the a d d i t i o n of 18:3n3 s t i m u l a t e s growth and improves feed e f f i c i e n c y ( C a s t e l l et a l . 1972a). L i n o l e i c a c i d was a l s o found t o improve growth and feed e f f i c i e n c y although l i n o l e n i c a c i d was s u p e r i o r i n t h i s regard ( C a s t e l l e t a l . 1977a) and (Takeuchi and Watanabe 1982) . The con c e n t r a t i o n s and combinations of d i e t a r y f a t t y a c i d s r e q u i r e d t o enhance growth and feed e f f i c i e n c y seem to vary from species t o sp e c i e s . Takeuchi et a l . (1979) observed t h a t the a d d i t i o n of e i t h e r l i n o l e i c or l i n o l e n i c a c i d improved growth i n chum salmon. The best weight g a i n and feed e f f i c i e n c y was seen when the f i s h were fed a d i e t c o n s i s t i n g of both 1% 18:3n3 and 1% 18:2n6. Yu e t a l . (1979) observed weight ga i n and feed e f f i c i e n c y t o be 30 g r e a t e s t f o r rainbow t r o u t fed a 1% 18:3n3 d i e t . Yu and Sinnhuber (1972) observed t h a t growth and feed e f f i c i e n c y were maximized i n rainbow t r o u t when e i t h e r 1% 18:3n3 or 1% 22:6n3 was fed. There seems t o be no s i g n i f i c a n t d i f f e r e n c e i n growth r a t e and feed e f f i c i e n c y between d i e t s c o n t a i n i n g equal amounts of e i t h e r 18:3n3 or h i g h l y unsaturated n3 f a t t y a c i d s i n rainbow t r o u t (Lee et a l . 1967). However, Takeuchi and Watanabe, (1977b, 1982) found l i n o l e n a t e t o be i n f e r i o r t o eicosapentaenoic a c i d and decosahexaenoic a c i d when fed at 0.5% of the d i e t regarding growth enhancement. A 1:1 mixture of these two h i g h l y unsaturated f a t t y a c i d s proved t o be more e f f e c t i v e i n s t i m u l a t i n g growth than they were when fed i n d i v i d u a l l y . Takeuchi and Watanabe (1982) d i d not f i n d much d i f f e r e n c e i n the growth r a t e of rainbow t r o u t fed 0.5% n3 h i g h l y unsaturated f a t t y a c i d o i l or 1% l i n o l e n a t e . Yu and Sinnhuber (1972) s t a t e d t h a t rainbow t r o u t fed d i e t s c o n t a i n i n g 22:6n3 d i d not produce s i g n i f i c a n t l y b e t t e r r e s u l t s than d i d d i e t s c o n t a i n i n g 18:3n3. In coho salmon, growth and feed e f f i c i e n c y are maximized when the d i e t contains 1 - 2.5% 18:3n3 (Yu and Sinnhuber, 1979). Coho respond p o o r l y t o d i e t s l a c k i n g n3 f a t t y a c i d s r e s u l t i n g i n poor growth (Takeuchi and Watanabe 1982). I t has a l s o been s t a t e d t h a t a mixture of n3 h i g h l y unsaturated f a t t y a c i d s fed at 1% of the d i e t i s not e f f e c t i v e i n improving the growth e f f e c t i n coho beyond the r a t e of the f i s h fed a d i e t c o n t a i n i n g l i n o l e a t e as the only l i p i d . In t h a t r e p o r t the researchers concluded t h a t n3 h i g h l y unsaturated f a t t y a c i d s had an adverse e f f e c t on growth. I t was a l s o observed t h a t 1% 18:2n6 could improve growth caused by an n3 d e f i c i e n c y w h i l e 1% arachidonate could not. Takeuchi e t a l . (1979) found t h a t the a d d i t i o n of 20:5n3 and n3 h i g h l y unsaturated f a t t y a c i d s i n chum salmon d i e t s increased growth beyond the l e v e l produced by the a d d i t i o n of the same amount of 18:3n3. Takeuchi and Watanabe (1982) s t a t e d t h a t the best growth was seen i n chum salmon r e c e i v i n g a combination of 1% l i n o l e n a t e and 1% l i n o l e a t e . The l i n o l e i c f a m i l y of f a t t y a c i d s are b e n e f i c i a l t o growth and feed e f f i c i e n c y i n salmonids. There does not seem t o be a d i f f e r e n c e between the response t o 18:2n6 or 20:4n6 (Takeuchi and Watanabe 1982). However, there does seem t o be some d i f f e r e n c e between speci e s . In many cases growth i s maximized when n3 and n6 f a t t y a c i d s are combined i n the d i e t . Takeuchi and Watanabe (1982) addressed the i s s u e of 32 what e f f e c t age and l i f e cycle have on f a t t y acid requirement. They did not observe any s i g n i f i c a n t d i f f e r e n c e i n the requirement of freshwater and sea water chum. This observation suggests that there may not be a d i f f e r e n c e i n requirements of anadromous f i s h i n t h e i r d i f f e r e n t l i f e stages. The e f f e c t of dietary f a t t y acids on mortality High mortaltiy i n f i s h fed n3 d e f i c i e n t d i e t s can be arrested by feeding d i e t s containing the e s s e n t i a l n3 f a t t y acids. Lee et a l . (1967) used rainbow trout to show the e f f e c t of n-3 f a t t y acids on mortality. The high mortality found throughout the n3 d e f i c i e n t f i s h was reduced a f t e r 2 weeks of feeding the n3 supplemented d i e t . E s s e n t i a l f a t t y acid d e f i c i e n t diets resulted i n high mortality i n chum salmon (Takeuchi et a l . 1979). Mo r t a l i t y was not affected by the addition of 0.5% 18:3n3. In coho, an n3 d e f i c i e n t d i e t resulted high mortality (Takeuchi and Watanabe, 1982) . The e f f e c t of feeding dietary n3 i n excess of the requirement Current research suggests that increasing the dietary n3 f a t t y a c i d content above a minimal c o n c e n t r a t i o n improves growth. However, the l i t e r a t u r e r a r e l y pursues t h i s p r i n c i p l e t o examine the e f f e c t of d i e t a r y n3 c o n c e n t r a t i o n s which are two or three times the l e v e l s used f o r growth enhancement. P r a c t i c a l salmonid d i e t s which use marine f i s h o i l s e x c l u s i v e l y w i l l c o n t a i n such c o n c e n t r a t i o n s . Yu and Sinnhuber (1979) found t h a t the a d d i t i o n of 2.5% l i n o l e n i n c o n s i s t e n t l y r e s u l t e d i n decreased average f i n a l weight and s p e c i f i c weight ga i n i n coho salmon. Feed e f f i c i e n c y was not a f f e c t e d . Takeuchi and Watanabe (1979) d i s c o v e r e d t h a t the a d d i t i o n of 4% n-3 f a t t y a c i d t o rainbow t r o u t d i e t s r e s u l t e d i n poor growth and low feed conversion when compared t o d i e t s c o n t a i n i n g 1% 18:3n3. The e f f e c t of d i e t a r y p r o t e i n t o l i p i d r a t i o on growth, feed e f f i c i e n c y and body composition The r a t i o of p r o t e i n t o l i p i d i n salmonid d i e t s i s one area of d i e t manipulation which should be considered. Being the sources of metabolizable energy these two n u t r i e n t s become intermeshed. While, l i k e other areas of salmonid n u t r i t i o n , there i s no d e f i n i t i v e r a t i o , some trends have been reported. U n f o r t u n a t e l y , these r e p o r t s 34 are few and deal p r i m a r i l y w i t h rainbow t r o u t . Takeuchi e t a l . (1978a) reported t h a t i n the rainbow t r o u t weight g a i n and feed e f f i c i e n c y improved as l i p i d i n c r e a s e d and was maximum when the d i e t contained 35% p r o t e i n and 15 or 20% l i p i d . Lee and Wales (1973) found improvement i n feed e f f i c i e n c y w i t h e i t h e r an in c r e a s e i n l i p i d or i n p r o t e i n . Kellems and Sinnhuber (1982) s t a t e d t h a t e f f i c i e n c y of feed conversion increased s i g n i f i c a n t l y w i t h i n c r e a s e s i n d i e t a r y l i p i d content. R e i n i t z e t a l . (197 8) attempted t o e s t a b l i s h n u t r i e n t l e v e l s of d i e t s based on a r a t i o of d i g e s t i b l e energy t o d i e t a r y crude p r o t e i n . The d i e t w i t h the g r e a t e s t energy content provided the best feed e f f i c i e n c y . U n f o r t u n a t e l y , the d i g e s t i b l e energy (DE) values were based on proximate a n a l y s i s value which were the same f o r a l l i n g r e d i e n t s . The authors themselves s t a t e t h a t d i f f e r e n c e s such as d i f f e r e n t amino a c i d l e v e l s could a f f e c t d i g e s t i b i l i t y and negate any c o r r e l a t i o n between DE/CP r a t i o and p r o t e i n r e t e n t i o n . The r a t i o of d i e t a r y l i p i d t o d i e t a r y p r o t e i n d i d a f f e c t body composition as was seen by Takeuchi et a l . (1978b) and R e i n i t z et a l . (1978). I t was suggested t h a t d i e t a r y l i p i d i s able t o increase p r o t e i n u t i l i z a t i o n e f f i c i e n c y . Lee and Wales (1973) documented increa s e s i n 35 l i v e r glycogen l e v e l s i n rainbow t r o u t on a h i g h p r o t e i n low l i p i d d i e t . These r e s u l t s suggest t h a t when p r o t e i n r a t h e r than l i p i d i s metabolized f o r energy there i s g r e a t e r b i o s y n t h e s i s of glycogen i n the body a t l i v e r . The e f f e c t of d i e t a r y l i p i d c o n c e n t r a t i o n on growth and body f a t t y a c i d composition A minimum of 10% l i p i d was deemed necessary t o i n c r e a s e the e f f i c i e n c y of p r o t e i n u t i l i z a t i o n by Takeuchi et a l . (1978a). Takeuchi et a l . (1978b) suggested t h a t 10% d i e t a r y l i p i d was s u f f i c i e n t t o maintain normal growth i n rainbow t r o u t . Takeuchi e t a l . (1978b) reported a decreased growth response t o l i p i d l e v e l s g r e a t e r than 20%. Large q u a n t i t i e s of l i p i d such as t h i s may be p r o v i d i n g excesses of n6 or n3 f a t t y a c i d s . On the other hand, increases i n d i e t a r y l i p i d might cause an increase i n e s s e n t i a l f a t t y a c i d requirement as a percentage of the dry d i e t . Takeuchi and Watanabe (1977a) noted t h a t i n c r e a s i n g d i e t a r y l a u r a t e from 4% t o 9% or 14% d i d not a l t e r t o t a l body f a t t y a c i d composition when 1% 18:3n3 was i n the d i e t . Simply s t a t i n g a crude f a t requirement e i t h e r as a minimum l e v e l or as a r a t i o w i t h p r o t e i n i s not s u f f i c i e n t . A f t e r d i g e s t i b i l i t y has been considered the f a t t y a c i d p r o f i l e of the l i p i d source should be considered. Excesses and d e f i c i e n c i e s of both n3 and n6 f a t t y a c i d s a f f e c t performance. Here again i s a b e n e f i t of m u l t i p l e l i p i d sources. D i f f e r e n t l i p i d sources can be s e l e c t e d and inco r p o r a t e d i n t o d i e t s f o r the purpose of sup p l y i n g n3, n6 or satu r a t e d and monounsaturated f a t t y a c i d s . The method by which dietary f a t t y acid concentrations are expressed D i e t a r y n3 f a t t y a c i d c o ncentrations are not commonly expressed r e l a t i v e t o the t o t a l l i p i d content. Concentrations of n3 f a t t y a c i d s are q u a n t i f i e d as a percent of the d i e t , u s u a l l y on a dry matter b a s i s . Takeuchi and Watanabe, (1977a) observed t h a t at 5% t o t a l l i p i d , 1% n3 l e v e l i n the d i e t produce the best growth response and feed e f f i c i e n c y i n rainbow t r o u t . However, when the l i p i d content was increased t o 9 or 14%, usi n g l a u r a t e , more than 2% l i n o l e n a t e was r e q u i r e d t o produce maximum growth. This f i n d i n g suggests t h a t the d i e t a r y requirement f o r n3 f a t t y a c i d s might be b e t t e r s t a t e d as a percentage of the t o t a l l i p i d r a t h e r than as a percentage of the dry d i e t . D i f f e r e n c e s i n d i e t a r y l i p i d l e v e l s 37 confound the i s s u e by a f f e c t i n g consumption, energy balance and body l i p i d storage. The e f f e c t of dietary f a t t y acid concentration on saltwater tolerance The e f f e c t of d i e t a r y f a t t y a c i d composition on s a l t w a t e r t o l e r a n c e has been addressed by researchers u s i n g a 24 hour s a l t w a t e r challenge. The method f o r the s a l t w a t e r challenge has been standardized by Blackburn and Clarke (1987). M o r t a l i t y and plasma sodium concentrations have been the primary parameters of study. Clarke and Blackburn (1977) have s t a t e d t h a t plasma Na conc e n t r a t i o n s below 170 meq/1 i n d i c a t e s a l t w a t e r t o l e r a n c e . Blackburn and Clarke (1987) have s t a t e d t h a t m o r t a l i t y tends t o be more pronounced when water s a l i n i t y i s g r e a t e r than 26 ppt. Uno (1989) found m o r t a l i t y t o d i f f e r between salmonid spec i e s . Markert et a l . (1984) noted t h a t plasma Na conc e n t r a t i o n s i n coho salmon (Oncorhynchus k i s u t c h ) were not a f f e c t e d by d i f f e r e n c e s i n d i e t a r y crude l i p i d content or i n dry matter content f o l l o w i n g a 24 hour s a l t w a t e r c hallenge. A s i m i l a r response was observed by P l o t n i k o f f et a l . (1983) i n Chinook salmon (Oncorhynchus tshawytscha) fed d i e t s of d i f f e r e n t crude l i p i d content. D i e t a r y 38 treatments of 8.16, 12.86, 16.70 and 17.27% crude l i p i d (DMB) d i d not produce s i g n i f i c a n t d i f f e r e n c e s i n plasma Na c o n c e n t r a t i o n s f o l l o w i n g a 24 hour challenge. In t h i s study the f a t t y a c i d the d i e t a r y l i p i d contained 15.4, 21.3, 22.9, or 13.6% n3 f a t t y a c i d s . These values could be t r a n s c r i b e d t o n3 % of d i e t concentrations of 1.26, 2.74, 3.82, and 2.35, r e s p e c t i v e l y . The n6 f a t t y a c i d c o n c e n t r a t i o n s of the l i p i d were 10.3, 7.1, 5.5, and 7.1 r e s p e c t i v e l y (0.84, 0.91, 0.92, 1.23 % of d i e t ) . The l a c k of s i g n i f i c a n t d i f f e r e n c e i n plasma Na content suggests t h a t d i f f e r e n c e s i n d i e t a r y f a t t y a c i d c o n c e n t r a t i o n does not lead t o d i f f e r e n c e s i n osmoregulatory a b i l i t y . However, the mean plasma Na concentrations were above the l e v e l i n d i c a t i v e of s a l t w a t e r t o l e r a n c e . A second study i n v o l v i n g j u v e n i l e Chinook salmon fed d i f f e r e n t d i e t s r e s u l t e d i n lower plasma Na c o n c e n t r a t i o n s . P l o t n i k o f f (1984) suggested t h a t increased body s i z e could be r e s p o n s i b l e f o r the improved osmoregualtion. Unfortunately, s t a t i s t i c a l a n a l y s i s was 39 not used t o evaluate the e f f e c t of d i e t a r y treatments. Osmoregulation at s m o l t i f i c a t i o n seems t o be a p e r f e c t f i e l d of study i n the p u r s u i t of the reason f o r " e s s e n t i a l i t y " i n salmonids. The p h y s i o l o g i c a l f a c t o r s r e q u i r e d t o overcome the systemic s t r e s s of d r a s t i c s a l i n i t y change i n v o l v e two areas t h a t may i n v o l v e e s s e n t i a l f a t t y a c i d s , namely p r o s t a g l a n d i n s y n t h e s i s and b i o l o g i c a l membrane p e r m e a b i l i t y due t o composition. The o b j e c t i v e 40 The study was designed t o add t o the pool of knowledge r e q u i r e d f o r the e f f e c t i v e c u l t u r e of t h i s a q u a t i c s p e c i e s . However, the p r a c t i c a l importance of t h i s research does not d e t r a c t from i t s importance i n the understanding of l i p i d metabolism i n the f i s h . The s p e c i f i c o b j e c t i v e of the experimet was t o determine whether the response of f i s h t o graded l e v e l s of e s s e n t i a l f a t t y a c i d s (n3) was a f f e c t e d by the t o t a l d i e t a r y l i p i d c o n c e n t r a t i o n . The e f f e c t of d i e t a r y f a t t y a c i d s on body composition was s t u d i e d over two p e r i o d s . The primary d i f f e r e n c e between experimental c o n d i t i o n s during the two periods was water temperature. The two p e r i o d scheme was implemented i n an attempt t o g a i n a b e t t e r understanding of t h i s r e l a t i o n s h i p under d i f f e r e n t p h y s i o l o g i c a l s t a t e s and environmental c o n d i t i o n s . The metabolism of e s s e n t i a l and n o n - e s s e n t i a l f a t t y a c i d s were s t u d i e d by observing the d e s a t u r a t i o n and e l o n g a t i o n products of o l e i c (18:ln9), l i n o l e i c (18:2n6) and l i n o l e n i c (18:3n3) f a m i l i e s of d i e t a r y f a t t y a c i d s w i t h i n the body. A l a r g e p a r t of the body composition study examined the r e l a t i o n s h i p s between body f a t t y a c i d composition and the method by which the d i e t a r y f a t t y a c i d c o n c e n t r a t i o n was expressed. The three methods c a t e g o r i z e d the d i e t i n terms of the d i e t a r y t o t a l n3 f a t t y a c i d s content, the d i e t a r y c o n c e n t r a t i o n of the f a t t y a c i d c l a s s being examined i n the body l i p i d ( t o t a l s a t u r a t e d f a t t y a c i d s , t o t a l monounsaturated f a t t y a c i d s , t o t a l 18:1 f a t t y a c i d s , t o t a l n3 f a t t y a c i d s , n3 f a t t y a c i d s longer than C18, t o t a l n6 f a t t y a c i d s , and n6 f a t t y a c i d s longer than C18), or the method by which the f a t t y a c i d was q u a n t i f i e d (percent of the dry d i e t or percent of the d i e t a r y l i p i d ) . The r e l a t i o n s h i p between d i e t a r y l i p i d composition and growth or m o r t a l i t y was s t u d i e d d u r i n g p e r i o d 2 of the experiment. This p e r i o d was d e f i n e d as the p e r i o d i n which the growth t r i a l occurred. P e r i o d 2 began when water temperature began t o r i s e i n the s p r i n g . The r i s e i n water temperature provided a f a v o r a b l e environment f o r growth. A s a l t w a t e r t o l e r a n c e study was performed f o l l o w i n g p e r i o d 2. The challenge was used t o judge the e f f e c t of d i e t a r y l i p i d composition on a b i l i t y of 1(+) year o l d coho salmon t o e f f e c t i v e l y withstand d i r e c t t r a n s f e r t o s a l t w a t e r . The t i m i n g of the study i s q u i t e l o g i c a l as coho salmon commonly undergo s m o l t i f i c a t i o n a f t e r one year i n freshwater. 42 Materials and methods Experimental d i e t s Ten isonitrogenous d i e t s were formulated from p r a c t i c a l and p u r i f i e d ingredients (table 1). The d i e t s were formulated to contain 35.0 % crude protein on a dry matter basis (DMB). The crude f i b e r was c a l c u l a t e d as 12.7 % or 6.7 % (DMB) i n the low and high l i p i d d i e t s r e s p e c t i v e l y . The l i p i d s used i n each of the d i e t s are l i s t e d i n table 2. The actual f a t t y a c i d composition i s summarized on the basis of dry d i e t (% DMB) i n tabl e 4 and on the basis of dietary l i p i d (% l i p i d ) i n table 5. The t o t a l l i p i d l e v e l i n the d i e t was 18% or 25% of the t o t a l d i e t (DMB) as i s shown i n table 3. The higher l i p i d concentration was achieved by s u b s t i t u t i n g t r i - o l e i n f o r a- c e l l u l o s e . The di e t s were isonitrogenous and i s o c a l o r i c within a l i p i d l e v e l . The herring o i l had been s t a b i l i z e d with the antioxidant ethoxyquin (1,2-dihydro-6 ethoxy-2,2,4-tri-methylquinoline) at i t s source. Additional ethoxyquin was added to the d i e t s through the corn o i l . The d i e t s were formulated to maintain n6 f a t t y acids at the same concentration. Increases i n the n3 l i p i d source, herring o i l , were matched by corresponding decreases i n t r i o l e i n . Table 1. Composition of experimental d i e t s D i e t s I - V % V I - X % Soy p r o t e i n Wheat 14.0 15.0 10.0 23.6 5.0 1.0 5.0 0.6 0.2 2.0 12.1 11.4 14.0 15.0 10.0 23.6 5.0 1.0 5.0 0.6 0.2 2.0 6.1 17.5 Dextrose H e r r i n g meal Feather meal Vitamin m i x 1 M i n e r a l m ix 2 60% Choline c h l o r i d e A s c o r b i c a c i d L i g n o s u l f o n a t e a - C e l l u l o s e L i p i d 3 1 The v i t a m i n premix s u p p l i e d the f o l l o w i n g l e v e l s of n u t r i e n t per kg of dry d i e t : v i t a m i n A acetate 7900 IU, c h o l e c a l c i f e r o l 790 IU, DL-alpha-tocopheryl acetate 790 IU, menadione 25 mg, D-calcium pantothenate 160 mg, py r i d o x i n e HC1 40 mg, r i b o f l a v i n 80 mg, n i a c i n 315 mg, f o l i c a c i d 20 mg, thiamin HC1 50 mg/kg, b i o t i n 4 mg, cyanocobalamin 0.1 mg, i n o s i t o l 1580. 2 The mineral premix s u p p l i e d the f o l l o w i n g l e v e l s of n u t r i e n t per kg of dry d i e t : calcium {as Ca(H2PO4)2H2O, CaHPC>4, Ca^o (°H) 2 ( p o4) 6) 3370 mg, phosphorus {as Ca(H 2P0 4)2H 20, CaHP0 4, C a 1 9 ( O H ) 2 ( P 0 4 ) 6 , Na 2HP0 4) 18283 mg, magnesium {as MgS04*7H20) 300 mg, manganese {as MnSO/*5H20) 15 mg, z i n c {as ZnS04*7H20} 40 mg, i r o n {as FeS04*7H20} 70 mg, copper {as Cu S04*5H20) 1.5 mg, c o b a l t {as CoCl*6H20) 2.5 meg, potassium {as K2SO4 and KI0 3} 710 mg, sodium {as NaCl} 79 0 mg, f l u o r i n e {as NaF} 4 mg, selenium {as (Na2Se03*5H20) 0.1 mg, i o d i n e ( as KIO3) 4 mg. 3 See t a b l e 2 f o r type of l i p i d used. 44 Table 2 L i p i d content of the experimental d i e t s L i p i d Source H e r r i n g o i l 1 T r i o l e i n 2 Corn o i l I 1.0 8.9 1.5 I I 2.4 7.5 1.5 I I I 3.8 6.1 1.5 IV 5.2 4.7 1.5 V 6.6 3.3 1.5 VI 1.0 15.0 1.5 V I I 2.4 13.6 1.5 V I I I 3.8 12.2 1.5 IX 5.2 10.8 1.5 X 6.6 9.4 1.5 1 The h e r r i n g o i l was generously s u p p l i e d by Moore-Clark Co.(Canada) Inc., Vancouver, B r i t i s h Columbia, Canada 2 Obtained from United States Biochemical Corporation, Cleveland, Ohio, USA 45 Table 3 Determined t o t a l l i p i d and dry matter composition i n the d i e t s .  Dry Crude Matter L i p i d D i e t % % DMB I 77.88 17.33 I I 74.02 17.51 I I I 78.04 17.85 IV 71.19 17.91 V 81.04 18.39 VI 81.66 24.15 VI I 83.26 25.12 V I I I 79.24 25.06 IX 82.95 24.51 X 81. 33 25.23 46 Table 4 Determined f a t t y a c i d composition of the d i e t s . Concentrations are expressed as percentages of the d i e t on a dry matter b a s i s . (% DMB) Classes of f a t t y a c i d s D i e t s a t * monounsat* 18:1 n3 n3 HUFA* n6 % % % % % % I 2.48 10.21 7.60 1.11 0.93 2.04 I I 2.74 9.61 6.94 1.64 1.34 1.95 I I I 3.18 9.27 7.19 2.07 1.76 2.03 IV 3 . 38 7.93 5.70 2.72 2.34 1.89 V 3.59 7.43 4.90 2.89 2.47 1.84 VI 2.99 15.06 12.15 1.38 1.10 2.39 V I I 3.49 15.47 12.52 1.94 1.70 2.52 V I I I 3.93 14.10 11.31 2.21 1.89 2.33 IX 4.00 13.55 10.65 2.74 2.35 2.33 X 4.24 13 . 05 10.07 3.29 2.88 2.28 * sat = t o t a l of a l l saturated f a t t y a c i d s monounsat = t o t a l of a l l monounsaturated f a t t y a c i d s n3 HUFA = t o t a l of a l l n3 f a t t y a c i d s > C18 47 Table 5 Determined f a t t y a c i d composition of the d i e t s . Concentrations are expressed as percentages of the d i e t a r y l i p i d . (% of l i p i d ) Classes of f a t t y a c i d s D i e t s a t * monounsat* 18:1 n3 n3 HUFA* n6 % % % % % % I 14.29 58.90 43.88 6.41 5 .38 11.78 I I 15. 63 54.86 39.61 9.38 7 .68 11.16 I I I 17.80 51.91 40.29 11.57 9 .86 11.38 IV 18.88 44.28 31.85 15.19 13 .06 10.58 V 19.51 40.41 26. 62 15.71 13 .43 10.02 VI 12.37 62.36 50.30 5.71 4 .54 9.91 V I I 13.90 61.58 49.85 7.73 6 .76 10.04 V I I I 15.67 56.27 45.15 8.83 7 .53 9.30 IX 16.30 55.29 43.45 11.17 9 .60 9.51 X 16.80 51.73 39.91 13.03 11 .40 9.04 * s a t = t o t a l of a l l s a t u r a t e d f a t t y a c i d s monounsat = t o t a l of a l l monounsaturated f a t t y a c i d s n3 HUFA = t o t a l of a l l n3 f a t t y a c i d s > C18 48 The d i e t s were mixed and c o l d - p e l l e t e d i n the Animal Science f a c i l i t i e s i n the MacMillan b u i l d i n g and South campus, UBC. The p e l l e t e d d i e t possessed the complete l i p i d content p r i o r t o p e l l e t i n g (high l i p i d d i e t s o f t e n r e q u i r e p a r t of the l i p i d t o be added a f t e r p e l l e t i n g ) . Water was added t o the dry mixture before being processed through the p e l l e t m i l l . The d i e t s were s t o r e d i n a chest f r e e z e r at -19° C. Experimental animals and t h e i r environment J u v e n i l e coho salmon (Oncorhynchus k i s u t c h ) from three B.C. s t r a i n s were u n s e l e c t i v e l y d i s t r i b u t e d i n t o 30 150 1. tanks i n the Aquaculture centre at South campus, UBC u s i n g a completely random block design. S t r a i n i d e n t i t y was not r e t a i n e d . Twenty tanks were s i t u a t e d i n one room and the remaining ten were i n an s i m i l a r room across the h a l l . Each tank contained 60 x 9.5 g. f i s h . Each tank was s u p p l i e d w i t h d e c h l o r i n a t e d a s p i r a t e d Vancouver c i t y water at the r a t e of 2 l./min. Figures l and 2 p o r t r a y the water temperature changes over the t o t a l experimental p e r i o d and the growth t r i a l , r e s p e c t i v e l y . The water temperature at the beginning of the experiment was 11.5° C. and f e l l t o 3.5° C. during the w i n t e r months. 49 In May a heat exchanger was i n s t a l l e d which provided heated water which remained at a s t a b l e temperature ± 1° C. The water temperature was monitored d a i l y . The d i s s o l v e d O2 i n the water ranged from 9.2 t o 11.8 ppm duri n g the experiment. Throughout the study a constant 12 hr. photoperiod was provided by incandescent l i g h t i n g . The experiment began i n October, 1988 and ended i n mid J u l y , 1989. The growth t r i a l was postponed u n t i l the s p r i n g of 1989 because of the low water temperatures duri n g the w i n t e r months. 50 F i g u r e 1. Water temperature f o r periods 1 and 2, i n c l u s i v e . 51 F i g u r e 2. Water temperature during p e r i o d 2. 52 The time period (Periods 1 and 2) The complete term of the experiment was separated into two periods. Period 1 began i n October, 1988 and lasted 27 weeks u n t i l the water warmed up and the growth t r i a l (period 2) began i n A p r i l , 1989. Period 2 was 12 weeks long. Feeding and the monitoring of growth and mo r t a l i t y The f i s h were hand-fed i n excess of s a t i a t i o n twice d a i l y at the same times each day (0900 and 1500 h r s ) . The l i b e r a l feeding p r a c t i c e was implemented i n an attempt to compensate f o r the poor response to the feed. The amount fed was recorded at each feeding. M o r t a l i t y was recorded d a i l y . Each f i s h that died was examined and weighed. The i n i t i a l response to the d i e t was poor. At the beginning of the experiment most of the f i s h were unresponsive to the d i e t and the method i n which i t was d i s t r i b u t e d . The f i s h began to respond to the d i e t a f t e r 6 weeks of feeding. However, water temperature began to severely drop at that time. The i n i t i a l unexpected appetite f a i l u r e caused the f i s h to lose weight as they catabolized body stores f o r energy. At the lower 5 3 temperature the f i s h maintained that lower body weight and d i d not grow over the winter months. When the water temperature f i n a l l y began to r i s e during period 2 many of the f i s h d i d not survive. These f i s h were anorexic and l e t h a r g i c . The increased water temperature led to an increase i n metabolic rate. Growth would have occurred had the f i s h been eating. However, i n the absence of nutrient intake these animals were forced to r e l y on body stores which were already depleted. Feed intake data from both experimental periods were deemed as u n r e l i a b l e because of the large number of unresponsive f i s h . This i s associated with the f a c t that the amount fed was based on a mean f i s h weight b a s i s . The large number of f i s h that were not eating r e s u l t e d i n substantial overfeeding. I t can be said that those f i s h which were eating d i d so to s a t i a t i o n . This, however, can not be quan t i f i e d . For the i n d i v i d u a l weighings the f i s h were anaesthesized i n a 100 ppm methane tricainesulphonate (MS-222), 100 ppm sodium bicarbonate s o l u t i o n . The f i s h were i n d i v i d u a l l y weighed. This procedure was used at the beginning and end of period 1 and 2. Throughout the complete term of the experiment each tank of f i s h were weighed as a group every three weeks. For the group 54 weighings the f i s h were mildly sedated with 2-phenoxyethanol (50 ppm), counted and group-weighed. The group weights were used to monitor the progress of the experiment. They were not, however, used f o r an a l y s i s . The experiment followed a complete randomized block design. The environments i n aquarium rooms 4 and 5 were i n i t i a l l y thought to be very s i m i l a r . However, room 5 was shared with another group of researchers. The block of tanks i n room 5 had to be removed from the experiment when the s u b s t a n t i a l l y higher t r a f f i c i n that room was deemed responsible f o r the poor response from the f i s h . The mean f i s h body weight was used i n comparisons of growth because the f i s h were not i d e n t i f i e d i n d i v i d u a l l y . Sampling procedures Period 1 An unselected sample of f i v e f i s h per tank was c o l l e c t e d near the end of period 1 while the water temperature was low (5° C). These f i s h are r e f e r r e d to as the "period 1" specimens. These f i s h had been on experimental d i e t s f o r a period of 27 weeks. The f i s h were anaesthesized, k i l l e d and then stored at -20° C. f o r four months before they were analyzed f o r l i p i d content and f a t t y acid composition. L i p i d and f a t t y a c i d analyses 55 were performed on pooled samples c o n s i s t i n g of one f i s h from each of the three blocks per treatment. These f i s h were selected f o r analysis on the basis of body condition and evidence of ingestive a c t i v i t y . Period 2 At the termination of the 12-week feeding t r i a l the f i s h were i n d i v i d u a l l y weighed. Those f i s h not being used for a saltwater challenge described below were k i l l e d . These f i s h were stored for two weeks at -20° C. before being analyzed for dry matter and l i p i d . L i p i d and f a t t y acid analyses were performed on four f i s h per tank. F i s h from the f i r s t two blocks were analyzed. The blocks were not pooled i n t h i s case (see above). Analysis of body l i p i d s T o tal l i p i d was extracted from ground whole f i s h according to Bligh and Dyer (1959) with modifications by C h r i s t i e (1973) and the r e s u l t i n g l i p i d separated i n t o neutral and polar fract i o n s with the use of two Waters-M i l l i p o r e Sep-Paks (Waters Associates, M i l f o r d , Massachussetts) connected i n s e r i e s . Fatty a c i d methyl esters were produced by base-catalyzed f a t t y a c i d methyl e s t e r i f i c a t i o n ( C h r i s t i e , 1982 ). Fatty a c i d methyl 56 esters were analyzed using a Varian 3700 gas chromatograph equipped with a Supelcowax-10 column (30m,.32mm ID) (Supelco Canada Ltd., O a k v i l l e , Ont.). The i n j e c t i o n and detector port temperatures were both 220°C. Oven temperatures ranged from 170° to 230°C, increasing at a rate of 2°C/min a f t e r an i n i t i a l 6 minutes at 170°C. The f a t t y acids were i d e n t i f i e d by reference to Supelco PUFA-1 and Rapeseed o i l standards (Supelco Canada Ltd., O a k v i l l e , Ont.). The f a t t y acid concentrations were calcula t e d as e i t h e r a percentage of t h e i r respective l i p i d f r a c t i o n or as a percentage of the n o n - l i p i d dry body weight of the f i s h . The l a t t e r method of c a l c u l a t i o n was used to remove v a r i a t i o n i n body l i p i d content within and between treatments. In the neutral f r a c t i o n , the percent of each c l a s s of f a t t y a c i d ( NFA) was c a l c u l a t e d as follows: NFA = ( F n X N)/(D + P) X 100 where NFA = percent of that f a t t y acid c l a s s i n the neutral l i p i d as a function of the dry n o n l i p i d s t r u c t u r a l body weight F n = concentration of a f a t t y a c i d c l a s s i n the neutral l i p i d N = quantity of neutral l i p i d i n the body (g) D = dry n o n l i p i d body weight (g) P = quantity of polar l i p i d i n the body (g) The polar l i p i d was added to the denominator i n the standardization of the neutral l i p i d because the polar 57 l i p i d i s believed to be s t r u c t u r a l . In the case of body polar l i p i d , the percent of each cla s s of f a t t y acid ( PFA) was calcul a t e d as follows: PFA = ( F p x P)/D x 100 where PFA = percent of that f a t t y a c i d c l a s s i n the polar l i p i d as a function of the dry n o n l i p i d body weight Fp = concentration of a f a t t y a c i d c l a s s i n the polar l i p i d P = quantity of polar l i p i d i n the body (g) D = dry n o n l i p i d body weight Both NFA and PFA are expressed as percent (%). Saltwater challenge Five f i s h from each tank, using a l l three blocks, were revived a f t e r anaesthesia and used f o r a saltwater challenge. The saltwater challenge was based on the procedure of Blackburn and Clark (1987). The f i s h were placed i n t o one of ten chambers used f o r the challenge. The f i s h from a l l three blocks of a treatment were placed into the same chamber. Caudal f i n c l i p s were used to d i f f e r e n t i a t e the d i f f e r e n t blocks. The f i s h remained i n the blackened chambers f o r 24 hours. The chambers were provided with f u l l strength a r t i f i c i a l saltwater (28 parts per thousand). The water was r e c i r c u l a t e d , aerated (9 ppm O2) and cooled (16° C ) . At the end of the 24 hour saltwater challenge the 58 f i s h were anaesthesized f o r the c o l l e c t i o n of blood samples. Blood was c o l l e c t e d i n heparinized c a p i l l a r y tubes from two f i s h f o r each treatment and each block. The blood was centrifuged and the plasma was r e f r i g e r a t e d f o r 24 hours before being analyzed f o r plasma sodium l e v e l s using a Corning 410 flame photometer. Muscle samples were taken from two u n s e l e c t i v e l y chosen f i s h i n freshwater and from two f i s h immediately following the 24 hour saltwater challenge. A section of ski n l e s s , boneless muscle was extracted from k i l l e d f i s h from an area p o s t e r i o r to the dorsal f i n , a n t e r i o r to the adipose f i n on the dorsal side of the l a t e r a l l i n e on one side of the sp i n a l column. Muscle samples were stored at -19° C. f o r 48 hours before determining dry matter concentration. S t a t i s t i c a l analysis A comparison of regressions was used f o r the s t a t i s t i c a l analysis of the body f a t t y a c i d composition. The dependent v a r i a b l e i n the regressions was d i e t f a t t y acid content. I t was expressed as ei t h e r percent of the dry d i e t (% DMB) or percent of the dietary l i p i d (% l i p i d ) . Seven classes of f a t t y acids were used as 59 independent varia b l e s . They were t o t a l saturated, t o t a l monounsaturated, t o t a l 18:1, t o t a l n3, n3 highly unsaturated, t o t a l n6, and n6 highly unsaturated f a t t y acids. The dependent v a r i a b l e was one of the seven classes of f a t t y acids found i n the polar or neutral f r a c t i o n of the body l i p i d . I t was c a l c u l a t e d as grams of f a t t y a c i d (in e i t h e r polar or neutral l i p i d ) divided by the dry n o n l i p i d body weight. The data on f a t t y acid composition were analysed according to Snedecor and Cochran (1967). F i r s t , the r e s i d u a l mean squares of the i n d i v i d u a l regressions f o r the body l i p i d f a t t y acid composition of e i t h e r the high or low dietary l i p i d group as a function of a d i e t a r y f a t t y a c i d c l a s s was compared by a two-tailed F-test to determine whether there was homogeneity of r e s i d u a l variances. I f there were homogeneous r e s i d u a l variances then the two slopes were compared using another F-test. I f the slopes were not s i g n i f i c a n t l y d i f f e r e n t then an F-t e s t of the difference between adjusted means was performed to determine whether the elevations of two regression d i f f e r e d s i g n i f i c a n t l y . I f the elevations were not found to be s i g n i f i c a n t l y d i f f e r e n t then pooling of the data from the two dietary groups (diets I-V and VI-X) was j u s t i f i e d . T-tests were performed on a l l regressions 60 to determine whether they were s i g n i f i c a n t l y d i f f e r e n t from a slope of zero. The regressions with s i g n i f i c a n t slope were then compared on the basis of t h e i r r 2 value. Analysis of variance was performed on the growth rate data. The m o r t a l i t y data were analyzed using a Chi squared t e s t and by the regression analysis mentioned above. Analysis of variance was performed on the muscle dry matter data from the saltwater challenge. Results Body Fatty acid composition The r e s u l t s from t h e f a t t y a c i d a n a l y s i s a r e r e p o r t e d s e p a r a t e l y f o r t h e two s a m p l i n g p e r i o d s . These p e r i o d s , " p e r i o d 1" and " p e r i o d 2", have been d e f i n e d i n t h e m a t e r i a l s and methods s e c t i o n o f t h i s document. The major f a t t y a c i d groups i n t h e p o l a r l i p i d o f t h e p e r i o d 1 f i s h i s r e p o r t e d i n t a b l e 7 as a p e r c e n t o f t h e p o l a r l i p i d . I n t a b l e 8 t h e p o l a r f a t t y a c i d groups a r e r e p o r t e d as a p e r c e n t a g e o f t h e d r y n o n - l i p i d body w e i g h t . The l a t t e r method o f q u a n t i f y i n g t h e f a t t y a c i d s was used i n an a t t e m p t t o compensate f o r v a r i a t i o n i n t h e r a t i o o f p o l a r l i p i d t o t o t a l l i p i d as w e l l as t h e amounts o f t o t a l l i p i d among and w i t h i n t r e a t m e n t s t h e r e b y g i v i n g an a c c u r a t e assessment o f t h e r e l a t i o n s h i p between t h a t l i p i d f r a c t i o n and s t r u c t u r a l body mass. T a b l e 6 l i s t s t h e p o l a r , n e u t r a l and t o t a l l i p i d c o n t e n t o f t h e f i s h . The n e u t r a l l i p i d f a t t y a c i d c o m p o s i t i o n i s e x p r e s s e d as a p e r c e n t o f t h e n e u t r a l l i p i d i n t a b l e 9. T a b l e 10 r e p o r t s t h e major f a t t y a c i d groups i n t h e n e u t r a l l i p i d as p e r c e n t a g e s o f t h e d r y n o n l i p i d body w e i g h t p l u s t h e amount o f p o l a r l i p i d . The d a t a f o r t h e p e r i o d 2 f i s h a r e l i s t e d i n 62 tables 12 through 16. These tables correspond with tables 6 - 1 0 f o r the period 1 f i s h . A. The f a t t y acid composition of f i s h sampled i n Period l Table 6 Dry matter and l i p i d composition of f i s h at the end of period 1. Concentrations of polar and neutral body l i p i d are expressed as percentages of the t o t a l l i p i d or of the non - l i p i d dry s t r u c t u r a l body weight. polar/ neutral/ polar/ neutral/ % t o t a l t o t a l n o n l i p i d n o n l i p i d Diet % l i p i d l i p i d l i p i d dry b.wt dry b.wt+ DM* DMB m m m oolar (%) I 21.91 25.35 23 .79 76.21 8.81 25.93 II 23.16 24.35 16. 67 83.33 5.37 25.46 III 23.31 20.27 24.56 75.44 6.24 18.03 IV 21.77 21.87 20.35 79.65 5.69 21. 09 V 23.84 24.20 31.78 68.22 10.15 19.78 VI 21.28 29.98 24.22 75.78 10.37 29.40 VII 21.18 33.52 23.73 76.27 11.97 34.35 VIII 24.88 30.71 22.61 77.39 10.02 31.17 IX 20.16 23.11 31.53 68.47 9.48 18.80 X 26.97 26.40 22.22 77.78 7.97 25.84 *DM = dry matter 63 Table 7 F a t t y a c i d composition of p o l a r l i p i d e x t r a c t e d from f i s h at the end of p e r i o d 1. Concentrations of f a t t y a c i d s are expressed as percentages of the p o l a r l i p i d . Classes of f a t t y a c i d s D i e t s a t * monounsat* 18:1 n3 n3 HUFA* n6 n6 HUFA' % % % % % % % I 21.93 34.67 18.30 34.84 30.88 3.60 ND** I I 17.26 43.94 16.42 28.23 28.23 2.81 ND I I I 17.59 28.57 15.21 25.78 25.78 3 .87 ND IV 22.91 25.97 13 .95 26.70 26.70 3.06 ND V 22.12 36.13 15.74 30.62 29.12 3.01 ND VI 22.85 47.23 27.04 24.96 23.74 4.95 ND V I I 20.68 44.91 25.90 29.53 28.14 4.33 ND V I I I 23.25 47.43 26.16 24.08 22.70 5.25 ND IX 28.13 37.77 22.03 30.28 30.28 3.60 ND X 22.22 38.12 20.65 30.68 29.33 4.43 ND * s a t = t o t a l of a l l s a t u r a t e d f a t t y a c i d s monounsat = t o t a l of a l l monounsaturated f a t t y a c i d s n3 HUFA = t o t a l of a l l n3 f a t t y a c i d s > C18 n6 HUFA = t o t a l of a l l n6 f a t t y a c i d s > C18 ** ND = not detected 64 Table 8 F a t t y a c i d composition of p o l a r l i p i d e x t r a c t e d from f i s h at the end of p e r i o d 1. Concentrations of f a t t y a c i d s are expressed as percentages of the n o n - l i p i d dry body weight. Classes of f a t t y a c i d s D i e t s a t * monounsat* 18;1 n3 n3 HUFA* n6 n6 HUFA* % % % % % % % I 1.93 3.05 1.61 3.07 2.72 0.32 ND I I 0.93 2.36 0.88 1.51 1.51 0.15 ND I I I 1.10 1.78 0.95 1.61 1.61 0.24 ND IV 1.30 1.48 0.79 1.52 1.52 0.17 ND V 2.24 3.67 1.60 3.11 2.96 0.31 ND VI 2.37 4.90 2.80 2.59 2.46 0. 51 ND V I I 2.47 5.37 3.10 3.53 3.37 0. 52 ND V I I I 2.33 4.75 2.62 2.41 2.27 0. 53 ND IX 2.67 3.58 2.09 2.87 2.87 0. 34 ND X 1.77 3.04 1.65 2.45 2.34 0. 35 ND * s a t = t o t a l of a l l s a t u r a t e d f a t t y a c i d s monounsat = t o t a l of a l l monounsaturated f a t t y a c i d s n3 HUFA = t o t a l of a l l n3 f a t t y a c i d s > C18 n6 HUFA = t o t a l of a l l n6 f a t t y a c i d s > C18 ** ND = not detected 65 Table 9 F a t t y a c i d composition of n e u t r a l l i p i d e x t r a c t e d from f i s h a t the end of p e r i o d l . Concentrations of f a t t y a c i d s are expressed as percentages of the n e u t r a l l i p i d . C lasses of f a t t y a c i d s D i e t s a t * monounsat* 18:1 n3 n3 HUFA* n6 n6 HUFA* % % % % % % % I 17.14 53.03 28.02 14.52 12.05 7. 25 0.78 I I 16.69 54.42 23.85 16.09 13 .71 5. 31 1.10 I I I 16.76 55.00 24.23 14.30 11.73 5. 86 0.74 IV 15.34 46.61 18.90 15.29 12.75 5. 80 2.17 V 15.98 53.15 22.07 16.26 14.04 6. 35 1.11 VI 15.49 56. 67 30.75 11.25 9.46 6. 65 1.11 V I I 16.10 57.65 32.21 13.67 11.47 6. 94 0.66 V I I I 16.76 58.53 34.19 13.31 11.35 7. 25 0.33 IX 17.10 59.70 24.99 15.10 12.91 4. 89 0.36 X 17.20 56.28 28.77 15.80 13.33 6. 96 0.61 * s a t = t o t a l of a l l saturated . f a t t y a c i d s monounsat = t o t a l of a l l monounsaturated f a t t y a c i d s n3 HUFA = t o t a l of a l l n3 f a t t y a c i d s > C18 n6 HUFA = t o t a l of a l l n6 f a t t y a c i d s > C18 ** ND = not detected 66 Table 10 F a t t y a c i d composition of n e u t r a l l i p i d e x t r a c t e d from f i s h a t the end of p e r i o d 1. Concentrations of f a t t y a c i d s are expressed as percentages of the n o n - l i p i d dry s t r u c t u r a l body weight. Classes of f a t t y a c i d s D i e t s a t * monounsat* 18:1 n3 n3 HUFA* n6 n6 HUFA* % % % % % % % I 4.44 13.75 7.27 3.77 3.12 1.88 0.20 I I 4.25 13.85 6.10 4.10 3.49 1.35 0.28 I I I 3.02 9.92 4.37 2.58 2.11 1.06 0.13 IV 3.23 9.83 3.99 3.22 2.69 1.22 0.46 V 3.16 10.51 4.36 3.22 2.78 1.26 0.22 VI 4. 55 16. 66 9. 04 3 .31 2 .78 1. 96 0. 32 V I I 5. 53 19. 80 11. 06 4 .70 3 .94 2. 38 0. 23 V I I I 5. 22 18. 25 10. 66 4 . 15 3 .54 2. 26 0. 10 IX 3 . 22 11. 23 4 . 70 2 .84 2 .43 0. 92 0. 07 X 4 . 44 14. 54 7. 43 4 . 08 3 .44 1. 80 0. 16 * s a t = t o t a l of a l l s a t u r a t e d f a t t y a c i d s monounsat = t o t a l of a l l monounsaturated f a t t y a c i d s n3 HUFA = t o t a l of a l l n3 f a t t y a c i d s > C18 n6 HUFA = t o t a l of a l l n6 f a t t y a c i d s > C18 ** ND = not detected 67 I . Saturated f a t t y a c i d s Pooled r e g r e s s i o n s were not obtained from the r e g r e s s i o n s of s a t u r a t e d f a t t y a c i d s i n the body n e u t r a l l i p i d f r a c t i o n as a f u n c t i o n of d i e t a r y s a t u r a t e s (% DMB) and of d i e t a r y n3 (% DMB) because of s i g n i f i c a n t d i f f e r e n c e s i n the e l e v a t i o n s of the high and low l i p i d groups. The r e g r e s s i o n s of s a t u r a t e d f a t t y a c i d s i n the body p o l a r l i p i d as f u n c t i o n s of n3 i n the dry d i e t (% DMB) and i n the d i e t a r y l i p i d (% l i p i d ) a l s o produced s i g n i f i c a n t d i f f e r e n c e s i n the e l e v a t i o n s of high and low l i p i d groups. There were s i g n i f i c a n t slopes produced i n the r e g r e s s i o n s of sa t u r a t e d f a t t y a c i d s i n the body n e u t r a l l i p i d as a f u n c t i o n of sa t u r a t e d f a t t y a c i d s (% l i p i d ) or d i e t a r y n3 (% l i p i d ) . These re g r e s s i o n s had r 2 values of 0.54 and 0.47 r e s p e c t i v e l y . The r e g r e s s i o n s are shown i n f i g u r e s 4 and 5, r e s p e c t i v e l y . The only other s i g n i f i c a n t slope was produced by a r e g r e s s i o n of s a t u r a t e d f a t t y a c i d s i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y s a t u r a t e s (% DMB) i n the low l i p i d group. This r e g r e s s i o n produced an r 2 value of 0.83. The r e g r e s s i o n i s shown i n f i g u r e 3. 68 There also were s i g n i f i c a n t slopes produced by the regressions of neutral body saturated f a t t y acids as functions of dietary monounsaturates (% DMB and % l i p i d ) and dietary 18:1 (% DMB and % l i p i d ) . The r 2 values of these regressions were 0.50, 0.49, 0.51 and 0.50, respectively. These regressions are shown i n figures 6 -9, respectively. 69 Figure 3 . Saturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y 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 (% DMB) i n p e r i o d 1. The r e g r e s s i o n i n c l u d e s the low l i p i d d i e t s (I-V) only. *body f a t t y a c i d s expressed as % NFA (see page 56) y = - 1 . 3 4 X + 7.74 r 2 = 0.83 70 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 2 0 . 0 0 Diet saturated fatty ac ids (% lipid) F i g u r e 4. Saturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y 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 (% l i p i d ) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = -0.30x + 8.88 r 2 = 0.54 71 4.00 6.00 8.00 10.00 12.00 14.00 16.00 Diet n3 fatty acids (% lipid) F i g u r e 5 . Saturated f a t t y a c i d concentration i n the body neutral l i p i d as a function of d i e t a r y n3 f a t t y acid concentration (% l i p i d ) i n 'period 1. The regression involves a l l d i e t s (I-X). *body f a t t y acids expressed as % NFA (see page 56) y = 0.18X + 5.98 r 2 = 0.47 72 Figure 6. Saturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y 18:1 f a t t y a c i d c o n c e n t r a t i o n (%. DMB) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.23x + 2.05 r 2 = 0.50 73 25 .00 30 .00 35 .00 40 .00 45 .00 50 .00 55 .00 Diet 18:1 fatty ac ids (% lipid) F i g u r e 7. Saturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y 18:1 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) • *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.09X + 0.59 r 2 = 0.49 74 F i g u r e 8. Saturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y monounsaturated f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d l . The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.22x + 1.61 r 2 = 0.51 7 5 F i g u r e 9. Saturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y monounsaturated f a t t y a c i d c o n c e n t r a t i o n U l i p i d ) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.09X - 0.77 r2 = 0.50 76 I I . Monounsaturated f a t t y a c i d s S i g n i f i c a n t d i f f e r e n c e s i n e l e v a t i o n between the high and low d i e t a r y l i p i d groups were observed f o r the re g r e s s i o n s of monounsaturated f a t t y a c i d s as a f u n c t i o n of n3 i n the dry d i e t (% DMB). S i m i l a r d i f f e r e n c e s i n e l e v a t i o n s occurred w i t h i n the r e g r e s s i o n s of monounsaturated f a t t y a c i d s i n the body p o l a r l i p i d as f u n c t i o n s of d i e t a r y n3 (% DMB and % l i p i d ) . There were s i g n i f i c a n t slopes produced i n the re g r e s s i o n s of monounsaturated f a t t y a c i d s i n the body n e u t r a l l i p i d as f u n c t i o n s d i e t a r y monounsaturates (% DMB and % l i p i d ) shown i n f i g u r e s 10 and 11, r e s p e c t i v e l y . The slope of the r e g r e s s i o n of monounsaturates as a f u n c t i o n of d i e t a r y n3 (% l i p i d ) was a l s o s i g n i f i c a n t ( f i g u r e 13). The r 2 values of these three r e g r e s s i o n s were 0.66, 0.54 and 0.49 r e s p e c t i v e l y . The r e g r e s s i o n of monounsaturated f a t t y a c i d content i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y monounsaturates (% DMB) produced a s i g n i f i c a n t slope. This r e g r e s s i o n produced an r 2 value of 0.63. The r e g r e s s i o n i s shown i n f i g u r e 12. The r e g r e s s i o n s of monounsaturated f a t t y a c i d content i n the body p o l a r l i p i d as f u n c t i o n s of d i e t a r y n3 (% DMB) 77 and (% l i p i d ) f o r the high l i p i d group produced s i g n i f i c a n t slopes. The r 2 values of these two r e g r e s s i o n s were 0.79 and 0.81. The r e g r e s s i o n s are shown i n f i g u r e s 14 and 15, r e s p e c t i v e l y . 78 6.00 8.00 10.00 12.00 • 14.00 16.00 Diet monounsaturated fatty ac ids (%DMB) F i g u r e 10. Monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y monounsaturated f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.95X + 2.79 r 2 = 0.66 79 Fi g u r e 11. Monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y monounsaturated f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.37x - 6.10 r 2 = 0.54 80 Fi g u r e 12. Monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y monounsaturated f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % PFA (see page 57) y = 0.35x - 0.63 r 2 = 0.63 81 4.00 6.00 8.00 10.00 12.00 14.00 16.00 Diet n3 fatty ac ids (% lipid) F i g u r e 13. Monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = -0.71x + 21.30 r 2 = 0.49 82 1.00 1.50 2.00 2.50 3.00 3.50 Diet n3 fatty ac ids (% DMB) F i g u r e 14. Monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s the high l i p i d d i e t s (VI-X) only. *body f a t t y a c i d s expressed as % PFA (see page 57) y = -1.18x + 7.06 r 2 = 0.79 83 F i g u r e 15. Monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d l . The r e g r e s s i o n i n v o l v e s the high l i p i d d i e t s (VI-X) only. *body f a t t y a c i d s expressed as % PFA (see page 57) y = -0.31X +5.57 r 2 = 0.81 84 I I I . C18 Monounsaturated f a t t y a c i d s In the body n e u t r a l l i p i d f r a c t i o n a s i g n i f i c a n t d i f f e r e n c e i n e l e v a t i o n s between high and low d i e t a r y l i p i d groups occurred when d i e t a r y n3 (% DMB) was used as the independent v a r i a b l e . S i g n i f i c a n t slopes r e s u l t e d from r e g r e s s i o n s of body n e u t r a l 18:1 f a t t y a c i d content as a f u n c t i o n of d i e t a r y 18:1 (% DMB or % l i p i d ) and d i e t a r y n3 ( % l i p i d ) . the r 2 values of these r e g r e s s i o n s were 0.64, 0.57 and 0.50. These r e g r e s s i o n s are shown i n f i g u r e s 16, 17 and 19, r e s p e c t i v e l y . These values i n d i c a t e d t h a t c a t e g o r i z i n g the independent v a r i a b l e i n terms of the amount i n the dry d i e t was only s l i g h t l y b e t t e r than using the percent i n the l i p i d . D i e t a r y 18:1 (% l i p i d ) proved t o be only s l i g h t l y more r e s p o n s i b l e f o r v a r i a t i o n s i n the n e u t r a l body l i p i d than was d i e t a r y n3 (% l i p i d ) . A s i g n i f i c a n t slope was produced by the r e g r e s s i o n of 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 (% DMB) i n the low l i p i d group. The r 2 value f o r t h a t r e g r e s s i o n was 0.85. This r e g r e s s i o n i s shown i n f i g u r e 18. The r e g r e s s i o n s i n v o l v i n g 18:1 f a t t y a c i d s i n the body p o l a r l i p i d were d i f f i c u l t t o i n t e r p r e t . The 85 r e g r e s s i o n s u s i n g d i e t a r y n3 (% DMB and % l i p i d ) both produced s i g n i f i c a n t d i f f e r e n c e s i n the e l e v a t i o n s between hig h and low d i e t a r y l i p i d groups. Of these, the r e g r e s s i o n of 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 (% DMB or % l i p i d ) i n the high d i e t a r y l i p i d group produced a s i g n i f i c a n t s l o p e s . The r 2 values of t h i s r e g r e s s i o n were 0.8 0 and 0.82. The re g r e s s i o n s are shown i n f i g u r e s 2 0 and 21, r e s p e c t i v e l y . A s t a t i s t i c a l d i f f e r e n c e i n the r e s i d u a l v a r i a n c e between the high and low d i e t a r y l i p i d groups occurred when the 18:1 f a t t y a c i d content i n the body p o l a r l i p i d was r e l a t e d t o d i e t a r y 18:1 (% DMB) suggesting l a c k of homogeneity. There was a s i g n i f i c a n t d i f f e r e n c e between the slopes of high and low d i e t a r y l i p i d groups when a r e g r e s s i o n u s i n g d i e t a r y 18:1 (% l i p i d ) was created. 86 F i g u r e 16. 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y 18:1 f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.77X + 0.05 r 2 = 0.64 87 25 .00 30 .00 35 .00 40 .00 45 .00 50 .00 55 .00 Diet 18:1 fatty ac ids (% lipid) F i g u r e 17. 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y 18:1 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0 . 2 7 X - 4.29 r 2 = 0.57 88 1.00 1.50 2.00 2 .50 3.00 3.50 Diet n3 fatty ac ids (% DMB) F i g u r e 18. 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s the low l i p i d d i e t s (I-V) only. *body f a t t y a c i d s expressed as % NFA (see page 56) y = -1.75x + 8.86 r 2 = 0.85 89 4.00 6.00 8.00 10.00 12.00 14.00 16.00 Diet n3 fatty ac ids (% lipid) F i g u r e 19. 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = - 0 . 5 4 X + 12.53 r 2 = 0.50 90 Fi g u r e 20. 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (%. DMB) i n p e r i o d 1. (V?-xroSlJ 1 0 n i n V o l v e s t h e h i < ? h l i p i d d i e t s *body f - t t y ^ ^ ^ . ^ a s % P , f ^ 5 7 ) 91 Fi g u r e 21. 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s the high l i p i d d i e t s (VI-X) only. *body f a t t y a c i d s expressed as % PFA (see page 57) y = -0.18x + 4.16 r 2 = 0.82 92 IV. T o t a l n3 f a t t y a c i d s There were s i g n i f i c a n t d i f f e r e n c e s between e l e v a t i o n s of h i g h and low l i p i d groups f o r the re g r e s s i o n s of body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 (% DMB and % l i p i d ) . There were no s i g n i f i c a n t slopes produced by the reg r e s s i o n s of body n3 content, e i t h e r n e u t r a l or p o l a r , as a f u n c t i o n of d i e t a r y n3 (% DMB or % l i p i d ) . V. n3 h i g h l y unsaturated f a t t y a c i d s (n3 HUFA's) There were no s i g n i f i c a n t slopes produced by any of the r e g r e s s i o n s f o r body n3 HUFA i n e i t h e r the body n e u t r a l or p o l a r l i p i d f r a c t i o n s . This was t r u e f o r both independent v a r i a b l e s , d i e t n3 and d i e t n3 HUFA re g a r d l e s s of the method i n which they were q u a n t i f i e d (% DMB or % l i p i d ) . 93 VI. T o t a l n6 f a t t y a c i d s There were no s i g n i f i c a n t slopes produced f o r r e g r e s s i o n s of the n6 c o n c n e t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of e i t h e r d i e t a r y n3 or d i e t a r y n6. This was t r u e r e g a r d l e s s of the method i n which the independent v a r i a b l e s were q u a n t i f i e d . S i g n i f i c a n t d i f f e r e n c e s between e l e v a t i o n s of high and low l i p i d groups f o r the r e g r e s s i o n s of n6 c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 (% DMB and % l i p i d ) or d i e t a r y n6 (% l i p i d ) . The r e g r e s s i o n of n6 f a t t y a c i d content i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n6 (% DMB) produced a s i g n i f i c a n t slope. This r e g r e s s i o n i s shown i n f i g u r e 22. The r 2 value f o r t h i s r e g r e s s i o n i s 0.71. 94 1.80 1.90 2.00 2.10 2.20 2.30 2 .40 2.50 2.60 diet n6 fatty ac ids (% DMB) F i g u r e 22. n6 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n6 f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 1. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % PFA (see page 57) y = 0.49x - 0.72 r 2 = 0.71 95 Summary of d i e t a r y e f f e c t s on body composition d u r i n g p e r i o d 1 Saturated f a t t y a c i d content i n the body n e u t r a l l i p i d decreased w i t h increased d i e t a r y s a t u r a t e d (% l i p i d ) or n3 f a t t y a c i d s (% l i p i d ) . Increases i n d i e t a r y monounsaturates, e i t h e r as percent of the d i e t or of d i e t a r y l i p i d i ncreased the monounsaturate content i n the body n e u t r a l l i p i d . An incr e a s e i n d i e t a r y n3 (% l i p i d ) decreased the monounsaturate c o n c e n t r a t i o n i n the body n e u t r a l l i p i d . Increases i n the d i e t a r y monounsaturate content (% DMB) r e s u l t e d i n increased monounsaturates i n the p o l a r l i p i d f r a c t i o n . Increases i n the d i e t a r y n3 (% DMB or % l i p i d ) r e s u l t e d i n decreases i n the monounsaturated f a t t y a c i d c o n c n e t r a t i o n i n the body p o l a r l i p i d of the high d i e t a r y l i p i d groups. Increases i n the c l 8 monounsaturates, both as a percent of the d i e t or of the d i e t a r y l i p i d , increased the 18:1 f a t t y a c i d content i n the body n e u t r a l l i p i d . Increases i n d i e t a r y n3 (% l i p i d ) caused a decrease i n the 18:1 content i n the body n e u t r a l l i p i d . The 18:1 co n c e n t r a t i o n i n the body n e u t r a l l i p i d decreased i n the low l i p i d group when d i e t a r y n3 (% DMB) increased. Increased d i e t a r y n3 (% l i p i d ) decreased the c o n c e n t r a t i o n of 18:1 f a t t y a c i d s i n the body p o l a r l i p i d of the high 96 d i e t a r y l i p i d treatment group. D i e t a r y n3 content had no s i g n i f i c a n t e f f e c t on n3 content i n e i t h e r the n e u t r a l or p o l a r l i p i d f r a c t i o n s i n the body. Body n3 HUFA co n c e n t r a t i o n was not s i g n i f i c a n t l y i n f l u e n c e d by changes i n d i e t a r y n3 HUFA or t o t a l n3 co n c e n t r a t i o n s . n6 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d i n c r e a s e d as d i e t a r y n6 co n c e n t r a t i o n increased (% DMB). 97 Table 11 S t a t i s t i c a l l y s i g n i f i c a n t r e g r e s s i o n s of body f a t t y a c i d composition as a f u n c t i o n of d i e t a r y f a t t y a c i d s from p e r i o d l . V a r i a b l e s  Dependent Independent c l a s s l i p i d crroup ** c l a s s r e a r e s s i o n r 2 s a t s * N* low s a t s ( % DMB) y = -1. 34X+7.74 0. 83 s a t s N high s a t s ( % DMB)*** y = -0. 53X+6.57 0. 09 s a t s N pool s a t s ( % l i p i d ) y = -0. 30X+8.88 0. 54 sa t s N pool n3 ( % l i p i d ) y = 0. 18X+5.98 0. 47 sa t s N pool 18:1(% DMB) y 0. 2 3 X + 2 . 0 5 0. 50 s a t s N pool 1 8 : l ( % l i p i d ) y = 0. 09X+0.59 0. 49 sa t s N pool mono(% DMB) y = 0. 22X+1.61 0. 51 sa t s N pool mono(%lipid) y = 0. 0 9 X - 0 . 7 7 0. 50 mono N pool mono(% DMB) y 0. 95X+2.79 0. 66 mono N pool mono(%lipid) y = 0. 37X-6.10 0. 54 mono P pool mono(% DMB) y = 0. 35X-0.63 0. 63 mono N pool n3 ( % l i p i d ) y = -0. 7 1 X+21.30 0. 49 mono P high n3 (% DMB) y = -1. 1 8 X + 7 . 0 6 0. 79 mono P low n3 (% DMB)*** y — -0. 0 8 X + 2 . 6 3 0. 00 mono P high n3 ( % l i p i d ) y = -0. 31X+5.57 0 . 81 mono P low n3 ( % l i p i d ) * * * y = -0. 02X+2.73 0. 01 18:1 N pool 1 8 : l ( % DMB) y = 0. 7 7 X + 0 . 0 5 0. 64 18:1 N pool 1 8 : l ( % l i p i d ) y = 0. 27X-4.29 0. 57 18:1 N low n3 (% DMB) y = -1. 7 5 X + 8 . 8 6 0. 85 18:1 N high n3 (% DMB)*** y = -2. OlX+13.24 0. 32 18:1 N pool n3 ( % l i p i d ) y = -0. 54X+12.53 0. 50 18:1 P high n3 (% DMB) y = -0. 71X+4.09 0. 80 18:1 P low n3 (% DMB)*** y = -0. 09X+1.35 0. 03 18:1 P high n3 ( % l i p i d ) y = -0. 1 8 X+4.16 0. 82 18:1 P low n3 ( % l i p i d ) * * * y -0. 02X+1.40 0. 04 n6 P pool n6 (% DMB) y = 0. 4 9 X - 0 . 7 2 0. 71 *sat = t o t a l s a t u r a t e d f a t t y a c i d s mono = t o t a l monounsaturated f a t t y a c i d s 18:1 = C18 monounsaturated f a t t y a c i d s N = body n e u t r a l l i p i d f r a c t i o n P = body p o l a r l i p i d f r a c t i o n **group = treatment group i n v o l v e d i n the r e g r e s s i o n low = a r e g r e s s i o n u s i n g the low l i p i d d i e t s ( I - V ) only h i g h = a r e g r e s s i o n u s i n g the high l i p i d d i e t s ( V I - X) only pool = a r e g r e s s i o n using a l l d i e t s (I - X) ***This r e g r e s s i o n d i d not have a slope t h a t was s i g n i f i c a n t l y d i f f e r e n t from zero (P>0.05). 98 B. The f a t t y acid composition of f i s h sampled i n Period 2. Table 12 Body dry matter and l i p i d composition a t the end of p e r i o d 2. Concentrations of p o l a r and n e u t r a l body l i p i d are expressed as percentages of the t o t a l l i p i d or of the n o n - l i p i d dry s t r u c t u r a l body weight. p o l a r / n e u t r a l / % l i p i d l i p i d n o n l i p i d n o n l i p i d % l i p i d % % dry b.wt dry b.wt+ Di e t DM* DMB p o l a r n e u t r a l (%) o o l a r m I a 27.41 38. 60 17.62 82.38 11. 08 46. 62 b 28.94 34.10 17.49 82 .51 9. 05 39.16 I I a 23.64 34.43 22.93 77.07 12.04 36.12 b 33.14 30.89 22.28 77.72 9.19 29.37 I I I a 29 .23 34.11 20.81 79.19 10.37 35.75 b 19.89 37.12 30.30 69 .70 17.87 34 . 88 IV a 24.55 30.83 23.94 76.06 10. 67 30.64 b 30. 03 39.03 14 . 49 85.51 9 . 27 50. 09 V a 27.43 34 . 05 19.49 80.51 10. 06 37.77 b 26.99 29.94 18.75 81.25 8 . 01 32.14 VI a 33.76 31. 34 13.76 86.24 6.28 37 . 04 b 30. 05 46.46 10.67 89.33 9.26 70.94 V I I a 30.32 35.36 18.98 81. 02 10.38 40.15 b 27.44 38 . 67 14 . 23 85.77 8.97 49. 62 V I I I a 36.36 35.51 22 .22 77.78 12 .23 38 .15 b 29.88 48. 06 13 .16 86.84 12 .18 71. 63 IX a 29 . 03 40.27 9.05 90.95 6.10 57.79 b 31.50 39.78 13.24 86.76 8.75 52.70 X a 21.15 24.73 27.60 72.40 9.07 21.81 b 26.22 40.24 20. 00 80. 00 13 .47 47.47 * DM = dry matter 99 Table 13 F a t t y a c i d composition of p o l a r l i p i d e x t r a c t e d from f i s h a t the end of p e r i o d 2. Concentrations of f a t t y a c i d s are expressed as percentages of the p o l a r l i p i d . Classes of f a t t y a c i d s D i e t Rep s a t * monounsat* 18:1 n3 n3 HUFA* n6 n6 HUFA* I a 20 .82 38 .82 28 .92 21 .76 20 .48 12 .56 4.20 b 22 .06 43 .06 32 .98 20 .70 20 .70 11 .49 2.91 I I a 23 .84 36 . 65 28 .76 29 .79 29 .79 9 .72 1.97 b 23 .56 38 . 01 29 .37 26 . 14 25 .48 10 .99 2 .44 I I I a 20 .27 37 . 12 27 .55 28 .83 27 .68 10 .48 2.95 b 17 .79 33 .03 24 .56 26 .63 25 .31 11 .38 4.01 IV a 25 .52 32 .40 24 . 11 32 .52 32 .52 8 .27 1.50 b 27 .37 38 .50 26 .33 26 .41 26 .41 7 .71 ND** V a 24 .77 27 .85 20 .74 34 .82 33 . 69 9 .14 2.29 b 20 .84 29 .55 19 . 15 36 .81 35 .73 8 .82 3.33 VI a 19 . 03 46 . 82 35 .57 20 .97 20 . 62 10 .45 2 .17 b 20 .99 44 .29 30 .77 24 .30 24 . 30 10 .42 3 . 55 VI I a — — — _ _ b 19 .70 43 .38 35 .18 20 .74 20 .74 12 .05 4.57 V I I I a 21 .94 43 .04 34 .92 25 .51 24 .56 8 .01 1.90 b 23 .42 44 . 12 34 .46 19 .95 19 . 08 8 .67 0.90 IX a 25 .91 34 .83 26 .63 31 . 85 31 . 85 7 .41 ND b 28 .97 32 . 17 23 .35 31 .25 31 .25 6 .25 ND X a 18 . 17 36 .30 25 .27 25 .73 24 . 69 8 .44 2 .20 b 23 .95 43 .69 35 . 14 25 .76 25 .76 6 . 60 ND * s a t = t o t a l of a l l s a t u r a t e d f a t t y a c i d s monounsat = t o t a l of a l l monounsaturated f a t t y a c i d s n3 HUFA = t o t a l of a l l n3 f a t t y a c i d s > C18 n6 HUFA = t o t a l of a l l n6 f a t t y a c i d s > C18 ** ND = not detected 100 Table 14 F a t t y a c i d composition of p o l a r l i p i d e x t r a c t e d from f i s h a t the end of p e r i o d 2. ._ Concentrations of f a t t y a c i d s are expressed as percentages of the n o n - l i p i d dry body weight. Classes of f a t t y a c i d s D i e t Rep s a t * monounsat* 18:1 n3 n3 HUFA* n6 n6 i HUFA* % % % % % % % I a 2.31 4.30 3.20 2.41 2.27 1.39 0.47 b 2.00 3.90 2.99 1.87 1.87 1.04 0.26 I I a 2.87 4.41 3.46 3 .59 3 .59 1.17 0.24 b 2.17 3 .49 2.70 2.40 2.34 1. 01 0.22 I I I a 2.10 3.85 2 .86 2.99 2.87 1. 09 0.31 b 3 .18 5.90 4.39 4.76 4.52 2.03 0.72 IV a 2.72 3.46 2.57 3.47 3.47 0. 88 0.16 b 2.54 3.57 2.44 2.45 2.45 0.72 ND** V a 2.49 2.80 2.09 3.50 3.39 0.92 0.23 -b 1.67 2.37 1.53 2.95 2.86 0.71 0. 27 VI a 1. 20 2 . 94 2 .23 1.32 1.30 0. 66 0.14 b 1. 94 4 .10 2 .85 2.25 2.25 0. 96 0.33 V I I a — — _ _ _ _ b 2. 05 4.50 3 . 65 2.15 2.15 1. 25 0.47 V I I I a 1. 97 3 .86 3 . 13 2.29 2.20 0. 72 0.17 b 2. 86 5.40 4 .22 2.44 2.33 1. 06 0.11 IX a 3. 15 4.24 3 .24 3.88 3.88 0. 90 ND b 1. 77 1.96 1 .42 1.91 1.91 0. 38 ND X a 1. 59 3 .17 2 .21 2.25 2 .16 0. 74 0.19 b 2 . 17 3 .96 3 . 19 2 . 34 2 . 34 0. 60 ND * s a t = t o t a l of a l l s a t u r a t e d f a t t y a c i d s monounsat = t o t a l of a l l monounsaturated f a t t y a c i d s n3 HUFA = t o t a l of a l l n3 f a t t y a c i d s > C18 n6 HUFA = t o t a l of a l l n6 f a t t y a c i d s > C18 ** ND = not detected 101 Table 15 F a t t y a c i d composition of n e u t r a l l i p i d e x t r a c t e d from f i s h a t the end of p e r i o d 2. Concentrations of f a t t y a c i d s are expressed as percentages of the n e u t r a l l i p i d . Classes of f a t t y a c i d s D i e t Rep s a t * monounsat* 18:1 n3 n3 HUFA* n6 n6 HUFA* % % % % % % % I a 18 .84 58 .83 47 .61 4 .89 4 .05 11.98 0.63 b 17 .66 55 .39 44 .91 6 .63 5 .05 12.53 1.68 I I a 17 .46 55 .08 41 .69 8 .88 7 .26 12.55 1.43 b 18 .86 56 .04 44 . 17 6 .92 5 .78 12.30 0.59 I I I a 19 . 65 55 .20 43 . 09 10 .19 8 .48 11.86 ND** b 17 . 65 52 .70 40 .83 10 .55 8 .98 12.52 0.84 IV a 18 . 62 51 .40 36 .77 12 .79 10 .80 11.73 0.30 b 20 .94 48 .89 35 .50 11 .31 9 .71 9. 33 ND V a 23 . 11 47 . 63 35 .33 13 .54 11 .40 11.67 0.35 b 20 .84 49 .25 35 . 61 14 .54 12 .50 11.67 0. 60 VI a 14 .70 61 .46 49 . 62 4 .96 4 . 30 10.52 0.96 b 18 .39 66 .50 56 . 47 3 .44 3 . 44 10.34 ND VI I a 15 .44 59 .61 48 . 67 6 . 08 5 .01 11.45 0.82 b 15 .24 61 .27 49 . 16 6 .78 5 . 62 10. 09 0.85 V I I I a 18 .74 59 .49 48 .06 8 .11 6 .69 9.53 ND b 15 . 66 52 .07 40 .53 9 .27 7 .80 11.83 3.21 IX a 17 . 12 58 . 15 45 .38 9 .14 7 .72 9.71 0.32 b 16 .27 56 .42 45 .38 9 .61 8 .17 10.94 0.59 X a 20 . 69 46 .52 33 . 57 15 .59 13 .40 11.88 0.35 b 18 . 57 58 . 51 46 . 92 8 .85 7 .91 10.02 ND * s a t = t o t a l of a l l s a t u r a t e d f a t t y a c i d s monounsat = t o t a l of a l l monounsaturated f a t t y a c i d s n3 HUFA = t o t a l of a l l n3 f a t t y a c i d s > C18 n6 HUFA = t o t a l of a l l n6 f a t t y a c i d s > C18 ** ND = not detected 102 Table 16. F a t t y a c i d composition of n e u t r a l l i p i d e x t r a c t e d from f i s h a t the end of p e r i o d 2. Concentrations of f a t t y a c i d s are expressed as percentages of the n o n - l i p i d dry s t r u c t u r a l body weight. Classes of f a t t y a c i d s D i e t Rep s a t * monounsat* 18:1 n3 3 HUFA* : n6 n6 HUFA* % % % % % % % I a 9.76 30.47 24.66 2.53 2.10 6.20 0.33 b 7.54 23.65 19.18 2.83 2.16 5.35 0.72 I I a 7.07 22.29 16.87 3.59 2.94 5.08 0.58 b 6.05 17.97 14.16 2.22 1.85 3.94 0.19 I I I a 7.75 21.78 17.00 4.02 3.35 4 . 68 ND** b 7.26 21.67 16.79 4.34 3.69 5.15 0.35 IV a 6.31 17.43 12.47 4.34 3.68 3.98 0.10 b 11.46 26.76 19.43 6.19 5.31 5.11 ND V a 9.61 19.80 14 . 69 5.63 4.73 4.85 0.15 b 7.24 17.10 12.36 5. 05 4.35 4.05 0.21 VI a 5. 79 24. 19 19. 53 1. 95 1. 69 4 . 14 0.38 b 14. 25 51. 54 43. 77 2. 67 2. 67 8. 01 ND VI I a 6. 84 26. 41 21. 57 2. 69 2 . 22 5. 07 0.36 b 8. 24 33 . 13 26. 58 3. 67 3 . 04 5. 46 0.46 V I I I a 8. 02 25. 47 ,20. 58 3 . 47 2 . 86 4 . 08 ND b 12. 58 41. 84 32. 57 7. 45 6. 27 9. 51 2.58 IX a 10. 50 35. 65 27. 82 5. 60 4 . 73 5. 95 0.20 b 9. 32 32. 33 26. 01 5. 51 4. 68 6. 27 0.34 X a 4. 92 11. 06 7. 98 3. 71 3 . 19 2 . 83 0.08 b 10. 00 31. 51 25. 27 4 . 77 4 . 26 5. 40 ND * s a t = t o t a l of a l l s a t u r a t e d f a t t y a c i d s monounsat = t o t a l of a l l monounsaturated f a t t y a c i d s n3 HUFA = t o t a l of a l l n3 f a t t y a c i d s > C18 n6 HUFA = t o t a l of a l l n6 f a t t y a c i d s > C18 ** ND = not detected 103 I. Saturated f a t t y a c i d s The content of sa t u r a t e d f a t t y a c i d s i n e i t h e r the p o l a r or the n e u t r a l f r a c t i o n s of body l i p i d d i d not produce a s t a t i s t i c a l l y s i g n i f i c a n t slope when r e l a t e d t o d i e t a r y s a t u r a t e d or d i e t a r y n3 f a t t y a c i d s . T h i s l a c k of s i g n i f i c a n c e was not a l t e r e d by method of q u a n t i f y i n g the d i e t a r y f a t t y a c i d c a l c u l a t e d as % of d i e t a r y l i p i d or as % of the d i e t on a dry matter b a s i s . I I . Monounsaturated f a t t y a c i d s A s i n g l e r e g r e s s i o n f o r the r e l a t i o n of monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d t o d i e t a r y n3 (% DMB) could not be produced because of a s i g n i f i c a n t d i f f e r e n c e i n e l e v a t i o n s of the low and high d i e t a r y l i p i d treatment groups. The i n d i v i d u a l r e g r e s s i o n s f o r each of these groups d i d not produce s i g n i f i c a n t slopes. There were s i g n i f i c a n t slopes produced when the monounsaturated f a t t y a c i d content i n the body n e u t r a l l i p i d was a s s o c i a t e d w i t h d i e t a r y monounsaturates (% DMB and % d i e t a r y l i p i d ) and the d i e t a r y n3 (% of d i e t a r y l i p i d ) . These r e g r e s s i o n s are shown i n f i g u r e s 23, 24 and 25, r e s p e c t i v e l y . 104 A s i g n i f i c a n t slope was not produced by any of the independent v a r i a b l e s when the monounsaturated f a t t y a c i d content i n the body p o l a r l i p i d was analyzed. The r 2 v a r i e d only s l i g h t l y f o r the r e l a t i o n of n e u t r a l body monounsaturate content as a f u n c t i o n of e i t h e r d i e t a r y n3 or monounsaturate (% d i e t a r y l i p i d ) . The r 2 values were 0.24 and 0.27 r e s p e c t i v e l y . There was only a small d i f f e r e n c e i n the r 2 values f o r monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of the monounsaturate i n the d i e t .(% DMB) or i n the l i p i d ( % l i p i d ) . The values were 0.34 and 0.27 r e s p e c t i v e l y . 105 F i g u r e 23. Monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y monounsaturated f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 1.71x + 4.37 r 2 = 0.34 106 s 10 40 .00 45 .00 50 .00 55 .00 60 .00 65 .00 Diet monounsaturates (% lipid) F i g u r e 24. Monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y monounsaturated f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.66x - 11.20 r 2 = 0.27 107 F i g u r e 25. Monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = -1.24x + 37.20 r 2 = 0.24 108 I I I . C18 Monounsaturated f a t t y a c i d s Once again there were s i g n i f i c a n t d i f f e r e n c e s i n the e l e v a t i o n s of the low and high l i p i d groups when the body n e u t r a l 18:1 content was r e l a t e d t o the d i e t n3 (% DMB). The r e g r e s s i o n i n v o l v i n g the low d i e t a r y l i p i d group produced a s i g n i f i c a n t slope w i t h an r 2 value of 0.41. The r e g r e s s i o n i s shown i n f i g u r e 29. S i g n i f i c a n t slope were a l s o produced by the re g r e s s i o n s of 18:1 f a t t y a c i d s i n the body n e u t r a l l i p i d and d i e t a r y 18:1 (% DMB and % l i p i d ) . These r e g r e s s i o n s are shown i n f i g u r e s 26 and 27, r e s p e c t i v e l y . The e l e v a t i o n s between high and low groups were a l s o s i g n i f i c a n t l y d i f f e r e n t when the 18:1 f a t t y a c i d s i n the body p o l a r l i p i d was r e l a t e d t o d i e t a r y 18:1 (% of dry d i e t ) . The r e g r e s s i o n i n v o l v i n g the low d i e t a r y l i p i d group ( f i g u r e 28) produced a s i g n i f i c a n t slope w i t h an r 2 value of 0.58. The r 2 value was s l i g h t l y higher when the n e u t r a l body 18:1 was a s s o c i a t e d w i t h d i e t 18:1 (% l i p i d ) r a t h e r than d i e t n3 (% l i p i d ) . The r 2 values were 0.37 and 0.29, r e s p e c t i v e l y . There was very l i t t l e d i f f e r e n c e between the r 2 values of 18:1 co n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of 18:1 i n the d i e t expressed as % DMB 109 or as % l i p i d . The r 2 values were 0.38 and 0.37 r e s p e c t i v e l y . F i g u r e 26. 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y 18:1 f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 1.71x + 3.80 r 2 = 0.38 110 F i g u r e 27. 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y 18:1 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.63x - 6.81 r ^ = 0.37 I l l ~g o o o o CL o 4.5 4 3.5 3 2.5 2 1.5 1 0.5 JL p a D a a • a 4.00 6.00 8.00 10.00 12.00 Diet 18:1 fatty ac ids (% DMB) 14.00 diets l - V • diets VI—X regression F i g u r e 28. 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y 18:1 f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s the low l i p i d d i e t s (I-V) only. *body f a t t y a c i d s expressed as % PFA (see page 57) y = 0.56x - 0.79 r 2 = 0.58 112 F i g u r e 29. 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s the low l i p i d d i e t s (I-V) only. *body f a t t y a c i d s expressed as % NFA (see page 56) y = -3.06x + 21.51 r 2 = 0.41 113 F i g u r e 3 0 . 18:1 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = -1.19x + 31.53 r 2 = 0.29 114 IV. T o t a l n3 f a t t y a c i d s There was a s i g n i f i c a n t d i f f e r e n c e i n e l e v a t i o n between the low and high l i p i d groups when n3 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d was r e l a t e d t o d i e t a r y n3 f a t t y a c i d s (%DMB). There was only a s l i g h t d i f f e r e n c e i n the r 2 f o r body n e u t r a l n3 as a f u n c t i o n of d i e t n3 as a percent of the d i e t (DMB) or of the d i e t a r y l i p i d . The r 2 values were 0.44 and 0.39, r e s p e c t i v e l y . Figures 31 and 32 show these r e g r e s s i o n s . A s i g n i f i c a n t slope was produced when the n3 f a t t y a c i d content i n the body p o l a r l i p i d was r e l a t e d t o d i e t n3 (% l i p i d ) . This r e g r e s s i o n produced an r 2 value of 0.22. I t i s shown i n f i g u r e 33. 115 Fi g u r e 31. n3 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 1.29X + 0.89 r 2 = 0.44 116 Fi g u r e 32. n3 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.25X + 1.16 r 2 = 0.39 117 F i g u r e 33. n3 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % PFA (see page 57) y = O . l l x + 1.48 r 2 = 0.22 118 V. n3 h i g h l y unsaturated f a t t y a c i d s (n3 HUFA's) There were s i g n i f i c a n t d i f f e r e n c e s i n e l e v a t i o n between the low and high l i p i d groups when n3 HUFA co n c e n t r a t i o n i n the body p o l a r l i p i d was r e l a t e d t o d i e t n3 (%DMB) or n3 HUFA (% DMB). There was only a s l i g h t d i f f e r e n c e i n the r 2 f o r body n e u t r a l n3 HUFA as a f u n c t i o n of d i e t n3 HUFA or d i e t t o t a l n3 when r e f e r r e d t o as e i t h e r percent of the d i e t (DMB) or of the d i e t a r y l i p i d . The r 2 values were 0.46 and 0.47 r e s p e c t i v e l y when the independent v a r i a b l e was c a l c u l a t e d as a percent of the d i e t (DMB). The reg r e s s i o n s produced r 2 values of .41 and .40 r e s p e c t i v e l y when the independent v a r i a b l e c a l c u l a t e d as a percent of the d i e t l i p i d . The r 2 values were c o n s i s t e n t l y higher when the re g r e s s i o n s used d i e t a r y f a t t y a c i d s as a percent of the d i e t r a t h e r than was a percent of the l i p i d . F i g ures 34 and 35 show the reg r e s s i o n s f o r n3 HUFA co n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t n3 HUFA (DMB), n3 HUFA (% l i p i d ) . Figures 37 and 38 show the re g r e s s i o n s f o r n3 HUFA content i n the body n e u t r a l l i p i d as a f u n c t i o n of n3 (DMB) and n3 (% l i p i d ) , r e s p e c t i v e l y . There was only a s l i g h t d i f f e r e n c e i n the r 2 f o r body 119 p o l a r n3 HUFA as a f u n c t i o n of d i e t n3 HUFA or d i e t t o t a l n3 c a l c u l a t e d as a percent of the d i e t a r y l i p i d . The r 2 values were 0.22 and 0.23 r e s p e c t i v e l y . I t should be noted t h a t n3 h i g h l y unsaturated f a t t y a c i d s made up 85% of the t o t a l n3 i n the l i p i d of a l l of the d i e t s r e g a r d l e s s of t h e i r c o n c e n t r a t i o n of n3 f a t t y a c i d s . These r e g r e s s i o n s are shown i n f i g u r e s 36 and 39, r e s p e c t i v e l y . 120 0.50 1.00 1.50 2.00 2.50 3.00 Diet n3 HUFA's (% DMB) F i g u r e 34. n3 h i g h l y unsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 h i g h l y unsaturated f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 1.25x + 0.82 r 2 = 0.46 121 4.0 6.0 8.0 10.0 12.0 14.0 Diet n3 HUFA's (% lipid) F i g u r e 35. n3 h i g h l y unsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 h i g h l y unsaturated f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.24x + 1.00 r 2 = 0.41 122 Figure 36. n3 h i g h l y unsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 h i g h l y unsaturated f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % PFA (see page 57) y = 0.12x + 1.52 r 2 = 0.22 123 1.00 1.50 2.00 2.50 ' 3 .00 3.50 Diet n3 fatty ac ids (% DMB) F i g u r e 37. n3 h i g h l y unsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 1 . 1 3 X + 0.67 r 2 = 0.47 124 Fi g u r e 3 8 . n3 h i g h l y unsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % NFA (see page 56) y = 0.21X + 0.94 r 2 = 0.40 125 F i g u r e 3 9 . n3 h i g h l y unsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l ' d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % PFA (see page 57) y = O . l l x + 1.45 r 2 =0.23 126 VI. T o t a l n6 f a t t y a c i d s There were no s t a t i s t i c a l l y s i g n i f i c a n t slopes produced i n r e g r e s s i o n s i n v o l v i n g the n e u t r a l body n6 content. There were s i g n i f i c a n t d i f f e r e n c e s i n e l e v a t i o n between the high and low d i e t a r y l i p i d groups when re g r e s s i o n s were drawn f o r n6 f a t t y a c i d s i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 (% l i p i d ) and n6 f a t t y a c i d s (% DMB). The r e g r e s s i o n of n6 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of the d i e t a r y n6 (% DMB) produced a s i g n i f i c a n t slope i n the low d i e t a r y l i p i d groups. The r 2 value f o r t h i s r e g r e s s i o n was 0.46. The r e g r e s s i o n i s shown i n f i g u r e 40. There was a s i g n i f i c a n t slope produced i n the r e g r e s s i o n of n6 f a t t y a c i d s i n the body p o l a r l i p i d as a f u n c t i o n of d i e t n6 (% l i p i d ) . The r 2 value f o r t h i s r e g r e s s i o n was 0.39. The r e g r e s s i o n i s shown i n f i g u r e 41. The r e g r e s s i o n of n6 f a t t y a c i d content i n the body p o l a r l i p i d as a f u n c t i o n of d i e t n3 (% DMB) d i d not produce a s i g n i f i c a n t slope. 127 a o o ID c 1 d) C >, o D a a • diets l - V a diets VI—X regression 0| r I I I I r I 1.80 1.90 2.00 2.10 2.20 2.30 2.40 2.50 2.60 Diet n6 fatty ac ids (% DMB) F i g u r e 40. n6 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n6 f a t t y a c i d c o n c e n t r a t i o n (% DMB) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s the low l i p i d d i e t s (I-V) only. *body f a t t y a c i d s expressed as % PFA (see page 57) y = 3.21x - 5.16 r 2 = 0.46 128 Figure 41. n6 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n6 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % PFA (see page 57) y = 0.24X - 1.52 r 2 = 0.39 129 V I I . n6 h i g h l y unsaturated f a t t y a c i d s (n6 HUFA's) No s i g n i f i c a n t slopes were produced as a r e s u l t of re g r e s s i o n s of body n6 HUFA as a f u n c t i o n of d i e t a r y n3. This was the case i n both the n e u t r a l and p o l a r body l i p i d f r a c t i o n . There was a s i g n i f i c a n t d i f f e r e n c e i n the e l e v a t i o n between the low and high d i e t a r y l i p i d groups f o r the re g r e s s i o n s " o f n6 HUFA co n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n3 (% l i p i d ) . n6 HUFA's were not found i n the d i e t a r y l i p i d and could t h e r e f o r e not be used as an independent v a r i a b l e . No s i g n i f i c a n t slopes were produced as a r e s u l t of re g r e s s i o n s of n6 HUFA i n the body n e u t r a l l i p i d as a f u n c t i o n of d i e t a r y n6. There was a s i g n i f i c a n t d i f f e r e n c e i n the e l e v a t i o n between the low and high d i e t a r y l i p i d groups f o r the re g r e s s i o n s of n6 HUFA co n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n6 (% DMB). A s i g n i f i c a n t slope was produced f o r the r e g r e s s i o n of n6 HUFA f a t t y a c i d content i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n6 (% l i p i d ) . The r e g r e s s i o n produced an r 2 value of 0.32. This r e g r e s s i o n i s shown i n f i g u r e 42. 130 Figure 4 2 . n6 h i g h l y unsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d as a f u n c t i o n of d i e t a r y n6 f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) i n p e r i o d 2. The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . *body f a t t y a c i d s expressed as % PFA (see page 57) y = O . l l x - 0.93 r 2 = 0.32 131 V I I I . Body l i p i d The t o t a l body l i p i d ( t a b l e 12) d i d not d i f f e r s i g n i f i c a n t l y among d i e t a r y treatments. The percentage of body p o l a r l i p i d or n e u t r a l l i p i d over n o n - l i p i d dry body weight d i d not d i f f e r s i g n i f i c a n t l y among treatments. IX. Body moisture The percentage dry matter of the body 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 treatment. The data are shown i n t a b l e 12. Summary of d i e t a r y e f f e c t s on body composition d u r i n g p e r i o d 2 From the s i g n i f i c a n t r e g r e s s i o n s only a few trends i n f a t t y a c i d composition can be commented on. These r e g r e s s i o n s are l i s t e d i n t a b l e 17. Increases i n d i e t a r y monounsaturates, e i t h e r as percent of the d i e t or d i e t a r y l i p i d i ncreased the monounsaturate content i n the body n e u t r a l l i p i d . An inc r e a s e i n d i e t a r y n3 as a percent of the l i p i d decreased the content of monounsaturates i n the body n e u t r a l l i p i d . Increases i n the C18 monounsaturates, both as a percent of the d i e t or of the d i e t a r y l i p i d , i n creased the 132 18:1 f a t t y a c i d content i n the body n e u t r a l l i p i d . Increases i n d i e t a r y n3 (% l i p i d ) caused decreases i n 18:1 content i n the n e u t r a l body l i p i d . n3 f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d i s i n c r e a s e d by i n c r e a s i n g the d i e t a r y n3 con c e n t r a t i o n s e i t h e r as a percent of the d i e t (% DMB) or a percent of the l i p i d . Increases i n d i e t a r y n3 HUFA as e i t h e r percent of d i e t (% DMB) or percent of l i p i d r e s u l t i n increa s e d body n e u t r a l n3 HUFA content. Increases i n n3 HUFA co n c e n t r a t i o n i n the body n e u t r a l l i p i d a l s o occur as a r e s u l t of i n c r e a s i n g the t o t a l n3 i n the d i e t (% DMB) or i n the d i e t a r y l i p i d (% l i p i d ) . However, t h i s may depend on the p r o p o r t i o n of n3 HUFA i n the t o t a l d i e t a r y n3. T o t a l n3 i n the body p o l a r l i p i d i ncreased as d i e t a r y t o t a l n3 (% l i p i d ) increased although C18 n3 f a t t y a c i d s o n l y made up 2 % of the t o t a l p o l a r n3. n3 HUFA content i n the body p o l a r l i p i d i n creased w i t h i n c r e a s e s i n e i t h e r d i e t a r y t o t a l n3 (% l i p i d ) or d i e t a r y n3 HUFA (% l i p i d ) . n6 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d i n c r e a s e d as d i e t a r y n6 co n c e n t r a t i o n increased ( % l i p i d ) . n6 HUFA co n c e n t r a t i o n i n the body p o l a r l i p i d a l s o i n c r e a s e d w i t h i n c r e a s i n g l e v e l s of n6 i n the d i e t (% 133 l i p i d ) . No other s i g n i f i c a n t e f f e c t s of d i e t a r y f a t t y a c i d s were observed. 134 Table 17 S t a t i s t i c a l l y s i g n i f i c a n t r e g r e s s i o n s of body f a t t y a c i d composition as a f u n c t i o n of d i e t a r y f a t t y a c i d s from p e r i o d 2. V a r i a b l e s Dependent Independent r e g r e s s i o n r2 c l a s s l i p i d aroup ** c l a s s mono* N* pool mono (% DMB) y = 1. 71X+4.37 0. 34 mono N pool mono (% l i p i d ) y = 0. 66X-11.10 0. 27 mono N pool n3 (% l i p i d ) y -1. 24X+37.20 0. 24 18:1 N pool 18: 1 (% DMB) y = 1. 71X+3.80 0. 38 18:1 N pool 18: 1 (% l i p i d ) y = 0. 63X-6.81 0. 37 18:1 P low 18: 1' (% DMB) y = 0. 5 6 X - 0 . 7 9 0. 58 18:1 P high 18: 1 (% DMB)*** y 0. 2 3 X + 0 . 3 1 0. 06 18:1 N low n3 (% DMB) y = -3. 06X+21.51 0. 41 18:1 N high n3 (% DMB)*** y = -5. 85X+36.47 0. 23 18:1 N pool n3 (% l i p i d ) y -1. 19X+31.53 0. 29 n3 N pool n3 (% DMB) y 1. 29X+0.89 0. 44 n3 N pool n3 (% l i p i d ) ,y = 0. 25X+1.16 0. 39 n3 P pool n3 (% l i p i d ) y — 0. llx+1.48 0. 22 n3 HUFA N pool n3 HUFA (% DMB) y = 1. 25X+0.82 0. 46 n3 HUFA N pool n3 HUFA ( % l i p i d ) y = 0. 2 4 X + 1 . 0 0 0. 41 n3 HUFA P pool n3 HUFA ( % l i p i d ) y = 0. 12X+1.52 0. 22 n3 HUFA N pool n3 (% DMB) y = 1. 13X+0.67 0. 47 n3 HUFA N pool n3 (% l i p i d ) y = 0. 21X+0.94 0. 40 n3 HUFA P pool n3 (% l i p i d ) y 0. llx+1.45 0. 23 n6 P low n6 (% DMB) y 3 . 21X-5.16 0. 46 n6 P high n6 (% DMB)*** y = 2 . 2 7 X - 4 . 5 3 0. 40 n6 P pool n6 (% l i p i d ) y = 0. 2 4 X - 1 . 5 2 0. 39 n6 HUFA P pool n6 (% l i p i d ) y 0. l l x - 0 . 9 3 0. 32 *mono = t o t a l monounsaturated f a t t y a c i d s 18:1 = C18 monounsaturated f a t t y a c i d s n3 HUFA = n3 h i g h l y unsaturated f a t t y a c i d s ( >C18) n6 HUFA = n6 h i g h l y unsaturated f a t t y a c i d s ( >C18) N = body n e u t r a l l i p i d f r a c t i o n P = body p o l a r l i p i d f r a c t i o n **group = treatment group i n v o l v e d i n the r e g r e s s i o n low = a r e g r e s s i o n using the low l i p i d d i e t s (I - V) only h i g h = a r e g r e s s i o n using the high l i p i d d i e t s ( V I - X) only pool = a r e g r e s s i o n using a l l d i e t s (I - X) ***This r e g r e s s i o n d i d not have slope t h a t was s i g n i f i c a n t l y d i f f e r e n t from zero (P>0.05). 135 Growth The weights of the f i s h a t the beginning of the experiment (wtjj , as w e l l as before (wtg) and a f t e r (wtf) the growth t r i a l are shown i n t a b l e 18. The data f o r wt^ corresponds t o the i n i t i a l weight f o r p e r i o d 1. The f i n a l weight f o r p e r i o d 1 i s the same as the i n i t i a l weight f o r p e r i o d 2 (wtg). Wtf corresponds t o the f i n a l weight f o r p e r i o d 2. The r e l a t i v e growth r a t e and s p e c i f i c growth r a t e s were c a l c u l a t e d f o r the growth t r i a l ( p eriod 2) only. Those values are shown i n t a b l e 19. R e l a t i v e growth r a t e i s d e f i n e d as the f i n a l body weight d i v i d e d by the i n i t i a l body weight. S i m i l a r l y , there was no s i g n i f i c a n t d i f f e r e n c e i n the s p e c i f i c growth r a t e among d i e t a r y treatments. S p e c i f i c growth (SGR) r a t e has been d e f i n e d by Yu and Sunnhuber (1979) as f o l l o w s : SGR = (log wtf - l o g wtj j / d a y s where wtf i s f i n a l wet body weight and wtj_ i s i n i t i a l wet body weight. A n a l y s i s of va r i a n c e showed t h a t d i e t a r y treatments had no s i g n i f i c a n t e f f e c t on r e l a t i v e growth r a t e or s p e c i f i c growth r a t e . Regression a n a l y s i s was performed on r e l a t i v e growth r a t e and s p e c i f i c growth r a t e as f u n c t i o n s of d i e t a r y n3 (% DMB and % l i p i d ) . 136 The slopes from the r e g r e s s i o n s of r e l a t i v e growth r a t e as f u n c t i o n s of d i e t a r y n3 (% DMB or % l i p i d ) were not s i g n i f i c a n t . The hig h and low l i p i d groups could not be compared i n terms of s p e c i f i c growth r a t e because the d i f f e r e n c e s i n r e s i d u a l v a r i a n c e s meant t h a t the high and low l i p i d groups were heterogeneous. Simply s t a t e d , n e i t h e r the d i e t a r y t o t a l l i p i d content nor the f a t t y a c i d composition of the d i e t s i g n i f i c a n t l y a f f e c t e d growth. 137 Table 18 The mean body weight of f i s h a t the beginning of p e r i o d 1, between per i o d s 1 and 2, and at the end of p e r i o d 2. ^ D i e t Rep W t i * ± : SD** Wt 0 r ± SD Wtf i ± SD I l 9.49 + 1.04 7. 78 + 3.02 21. 52 + 14. 90 2 9.61 + 0.97 7. 94 + 2.87 29. 25 + 19. 39 I I 1 9.37 + 1. 09 7. 01 + 2.32 24. 63 + 18. 78 2 9.54 + 0.93 8. 26 + 3 . 32 29 . 31 + 16. 03 I I I 1 9.59 + 1.14 7. 95 + 2.51 25. 29 + 11. 06 2 9.13 + 0.95 6. 19 + 1.40 14. 63 + 9. 36 IV 1 9.43 + 0.86 6. 77 + 1.66 15. 76 + 9. 82 2 9.54 + 1.13 6. 85 + 1.61 18. 39 ± 10. 67 V 1 9.29 + 0.96 6. 82 + 1.62 19. 45 + 11. 89 2 9.56 + 1.05 6. 85 + 2.43 24. 20 + 16. 61 VI 1 9 .20 + 0.97 7. 90 + 3 . 02 25. 62 + 18. 97 2 9.71 + 1. 06 8. 20 + 3 . 55 24 . 00 + 17 . 92 V I I 1 9.73 + 1.02 6. 90 + 2.25 20. 20 + 11. 84 2 9.27 + 0.98 6. 91 + 2.53 21. 37 + 15. 15 V I I I 1 9.70 + 0.93 8. 27 + 2.93 25. 49 + 14 . 07 2 9.56 + 1.04 7. 53 + 3.05 26. 87 + 15. 65 IX 1 9.45 + 0.91 7. 93 + 3.17 22 . 26 + 14 . 84 2 9.38 + 0.92 6. 90 + 2.35 21. 46 + 11. 72 X 1 9.41 + 1. 10 6. 29 + 2 .31 19. 12 + 12 . 27 2 9.47 1.10 7 . 07 + 2.91 17 . 58 + 14. 31 * Wtj^ = beginning of p e r i o d 1 = October 28/88 Wtg = i n t e r f a c e of p e r i o d 1 and 2 = A p r i l 21/89 Wtf = end of p e r i o d 2 = J u l y 13/89 ** SD = one standard d e v i a t i o n 138 Table 19. The number of f i s h / t a n k at the beginning of p e r i o d 1, between p e r i o d s 1 and 2, and a t the end of p e r i o d 2 as w e l l as the m o r t a l i t y , r e l a t i v e growth r a t e and s p e c i f i c growth r a t e observed d u r i n g p e r i o d 2. R e l a t i v e S p e c i f i c D i e t Rep N i N« N f m o r t a l i t y (Nq-N-fO growth Rate growth Rate I 1 60 5§ 33 ?2 2.77 0.44 2 60 55 26 29 3.68 0.57 I I 1 60 56 22 34 3 .51 0.55 2 60 55 37 18 3.55 0.55 I I I 1 60 55 34 21 3.18 0.50 2 60 55 15 40 2.36 0.37 IV 1 60 56 19 37 2.33 0.38 2 60 55 26 29 2.68 0.43 V 1 60 56 27 29 2.85 0.45 2 60 55 12 43 3 . 53 0. 55 VI 1 60 53 29 24 3.24 0.51 2 60 55 31 24 2.93 0.47 V I I 1 60 55 20 35 2.93 0.47 2 60 54 19 35 3.09 0.49 V I I I 1 60 55 31 24 3.08 0.49 2 60 55 27 28 3.57 0.55 IX 1 60 56 31 25 2.81 0.45 2 60 54 20 34 3.11 0.49 X 1 60 55 13 42 3.04 0.48 2 60 55 22 33 2.48 0.40 M o r t a l i t y 139 The numbers of f i s h per tank at d i f f e r e n t times of the experiment and the r e s p e c t i v e number of m o r t a l i t i e s are l i s t e d i n t a b l e 19. S t a t i s t i c a l a n a l y s i s u s i n g a C h i -square t e s t showed t h a t the number of m o r t a l i t y d i d d i f f e r s i g n i f i c a n t l y (P>0.05) among treatments. There were s i g n i f i c a n t slopes produced i n re g r e s s i o n s of recorded m o r t a l i t y f u n c t i o n s of d i e t a r y n3 (% DMB or % l i p i d ) . The re g r e s s i o n s are shown i n f i g u r e s 43 and 44, r e s p e c t i v e l y . The r 2 values f o r these r e g r e s s i o n s were 0.27 and 0.22 r e s p e c t i v e l y . I t i s evident from the r e g r e s s i o n t h a t increased m o r t a l i t y can be a s s o c i a t e d w i t h increased d i e t a r y n3 f a t t y a c i d content. However, i t should be noted t h a t the changes i n d i e t a r y n3 are only r e s p o n s i b l e f o r approximately one quarter of the d i f f e r e n c e s i n m o r t a l i t y . The only independent varuable which had a higher r 2 value was the n3 h i g h l y unsaturated f a t t y a c i d s . The r 2 values were 0.2 8 and 0.24 f o r n3 HUFA (% DMB) and n3 HUFA (% l i p i d ) r e s p e c t i v e l y . These values might have been expected knowing t h a t the t o t a l d i e t a r y n3 i s comprised of 85 % n3 h i g h l y unsaturated f a t t y a c i d s . The m o r t a l i t y was due t o anorexia or co m p l i c a t i o n s of 140 anorexia. The c o r r e l a t i o n of d i e t a r y f a t t y a c i d content t o m o r t a l i t y might be more a c c u r a t e l y expressed as a c o r r e l a t i o n of i n g e s t i v e response t o d i e t a r y f a t t y a c i d content. U n f o r t u n a t e l y , the l a c k of i n t a k e data f o r c e s s p e c u l a t i o n on t h i s c o r r e l a t i o n r a t h e r than numerical proof. 141 F i g u r e 43. M o r t a l i t y d u r i n g p e r i o d 2 as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d s (% DMB). The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . y = 5.41X + 18.40 r 2 = 0.27 142 F i g u r e 44. M o r t a l i t y d u r i n g p e r i o d 2 as a f u n c t i o n of d i e t a r y n3 f a t t y a c i d s (% l i p i d ) . The r e g r e s s i o n i n v o l v e s a l l d i e t s ( I - X ) . y = l.OOx + 19.78 r 2 = 0.22 143 Saltwater tolerance A. Body composition with respect to saltwater challenge I . Muscle moisture There was no s i g n i f i c a n t d i f f e r e n c e i n the dry matter content of muscle among treatments before or a f t e r a 24 hour s a l t - w a t e r challenge. I t should be noted t h a t the muscle dry matter c o n c e n t r a t i o n was a f f e c t e d by the s a l t w a t e r challenge. The means of the d i f f e r e n c e s (post minus p r e - s a l t w a t e r challenge) d i d not d i f f e r s i g n i f i c a n t l y between treatments. The data are shown i n t a b l e 20. 144 Table 20. Percentage dry matter i n muscle of coho salmon before and a f t e r a 24 hour s a l t w a t e r c hallenge. D i e t Rep Pre-Challenge muscle dry matter % Mean Post-Challenge muscle dry matter Mean % Mean D i f f e r e n c e a b 22.19 22.87 22.53 23.09 23.28 23.18 0.65 I I a b 22.87 22.38 22.62 23.54 22.84 23.19 0.57 I I I a b 21.45 21.49 21.47 22.86 22 .77 22 .81 1.34 IV a b 21.99 22.08 22.03 23.44 22.82 23 .13 1.10 V a b 22.23 20.70 21.46 23.26 22.62 22.94 1.48 VI a b 22.42 23.19 22 .80 24.80 24.48 24.64 1.84 VI I a b 22.28 20.25 21.26 23.44 24.49 23.96 2 .70 V I I I a b 22.76 22.73 22.74 23.12 23 .80 23.46 0.72 IX a b 21.99 22.18 22. 08 24.71 23 .57 24.14 2.06 X a b 21.45 22 . 61 22 . 03 23 . 03 23 .76 23 . 39 1.36 I I . Plasma Sodium 145 The mean plasma Na concentrations f o r the d i e t a r y treatments ranged from 138 - 158 mmol/L. These l e v e l s suggest t h a t a l l d i e t a r y treatments produced s a l t w a t e r -t o l e r a n t f i s h . The data i s shown i n t a b l e 21. Table 21 Mean Plasma Sodium Concentration (meq/l) f o l l o w i n g a 24 hour s a l t w a t e r challenge Plasma Na Die t fmeo/l) 4 SD* I 157.7 + 9.2 I I 158.8 + 8.2 I I I 145.1 + 6.1 IV 138 . 3 + 20.8 V 141.6 + 22 . 0 VI 153 . 0 + 12 . 5 V I I 152.0 + 11.1 V I I I 157.0 + 15.7 IX 158.7 + 12.6 X 149.0 + 18. 6 *SD = standard d e v i a t i o n B. M o r t a l i t y No m o r t a l i t y occurred as a r e s u l t of the s a l t w a t e r c h a l l e n g e . A l l of the f i s h seemed healthy and a c t i v e f o l l o w i n g the 24 hour challenge. 146 D i s c u s s i o n Method of quantifying f a t t y acids i n the d i e t . (% DMB vs % l i p i d ) The standard method of q u a n t i f y i n g d i e t a r y f a t t y a c i d s i s as a percent of the d i e t on a dry matter b a s i s . The e s s e n t i a l f a t t y a c i d s are most o f t e n q u a n t i f i e d i n t h i s manner. Obviously, one f a t t y a c i d group, such as n3 f a t t y a c i d s , only makes up a f r a c t i o n of the t o t a l d i e t a r y l i p i d . T h i s simple statement was the b a s i s f o r studying whether the response of f i s h t o graded d i e t a r y l e v e l s of e s s e n t i a l f a t t y a c i d (n3) i s a f f e c t e d by the d i e t a r y l i p i d c o n c e n t r a t i o n . The experimental d i e t s were formulated t o t o d u p l i c a t e f i v e l e v e l s of n3 f a t t y a c i d , as a percent of the dry d i e t , i n the low and h i g h - d i e t a r y l i p i d d i e t s . The t r i o l e i n , however, was an unexpected source of l i n o l e i c and l i n o l e n i c a c i d s . The higher l i p i d l e v e l i n d i e t s VI - X was due e x c l u s i v e l y t o a d d i t i o n a l t r i o l e i n . I t i s f o r t h i s reason t h a t d i e t s V I - X have higher c o n c e n t r a t i o n s of 18:2n6 and 18:3n3 than d i e t s I - V . The r 2 values f o r re g r e s s i o n s of body f a t t y a c i d composition as a f u n c t i o n of d i e t a r y f a t t y a c i d s , q u a n t i f i e d as percent dry d i e t (% DMB) or percent d i e t a r y l i p i d (% l i p i d ) , d i d not d i f f e r markedly. However, a 147 comparison of r e g r e s s i o n s i n t a b l e s 11 and 17 shows t h a t the r 2 values were c o n s i s t e n t l y higher f o r the pooled r e g r e s s i o n s which q u a n t i f i e d the independent v a r i a b l e u s i n g the percent of dry d i e t (% DMB) r a t h e r than u s i n g the percent of the d i e t a r y l i p i d (% l i p i d ) . T h i s suggests t h a t r e l a t i n g body f a t t y a c i d composition t o dry d i e t f a t t y a c i d c o n c e n t r a t i o n (% DMB) i s only s l i g h t l y more accurate than i s r e l a t i n g i t t o d i e t a r y l i p i d f a t t y a c i d c o n c e n t r a t i o n (% l i p i d ) . The magnitude of the d i f f e r e n c e s does not j u s t i f y emphasizing one method of q u a n t i f y i n g d i e t a r y f a t t y a c i d s over the other. The e f f e c t of dietary l i p i d concentration on the r e l a t i o n s h i p between dietary f a t t y acids and body f a t t y acid composition. Two t o t a l l i p i d c o ncentrations were used i n t h i s experiment t o create the d i f f e r e n t r a t i o s of f a t t y a c i d group t o d i e t (% DMB or % l i p i d ) . A l t e r i n g d i e t a r y l i p i d l e v e l s leads t o compl i c a t i o n s i n understanding the metabolism and d e p o s i t i o n of d i e t a r y f a t t y a c i d s . D i e t a r y i n t a k e and i t s a s s o c i a t i o n t o energy i n the d i e t a f f e c t the experiment. A l t e r i n g the t o t a l d i e t a r y l i p i d c o n c e n t r a t i o n confounds a study profoundly. One problem l i e s i n the e f f e c t of increased l i p i d on d i g e s t i b l e energy l e v e l s i n 148 the d i e t . The low ( d i e t s I-V) and high ( d i e t s VI-X) l i p i d d i e t s i n t h i s study were not i s o c a l o r i c . This d i e t a r y d i f f e r e n c e d i c t a t e s t h a t the p r i n c i p l e of d i e t a r y i n t a k e as a f u n c t i o n of d i e t a r y energy l e v e l be considered. I t i s b e l i e v e d t h a t f i s h base t h e i r food i n t a k e on energy i n t a k e . F i s h fed a low t o t a l l i p i d d i e t consume more, on a dry matter b a s i s , than they would on a high l i p i d d i e t (Lee and Putnam, 1973) and (Page and Andrews, 1973). I f a low l i p i d d i e t and high l i p i d d i e t had the same amount of a c e r t a i n f a t t y a c i d group (% DMB), f o r example n3, then the f i s h consuming the lower l i p i d d i e t should be i n g e s t i n g more grams of n3 f a t t y a c i d s f o r a g i ven body weight. However, i f i n t a k e does not d i f f e r w i t h changes i n the t o t a l d i e t a r y l i p i d c o n c e n t r a t i o n then f i s h consuming the h i g h l i p i d d i e t would consume more d i e t a r y l i p i d . Observations r e l a t i n g d i e t a r y f a t t y a c i d t o body f a t t y a c i d content at d i f f e r e n t d i e t a r y l i p i d l e v e l s r e l y on an understanding of what e f f e c t the d i e t a r y l i p i d has on growth and t o t a l l i p i d composition of the body. I s the d i e t a r y l i p i d being c a t a b o l i z e d f o r energy thereby r e s u l t i n g i n growth or i s i t being s t o r e d i n the body? The f i s h which were fed the high l i p i d d i e t s i n t h i s experiment showed n e i t h e r a s i g n i f i c a n t i n c r e a s e i n body 149 l i p i d nor a s i g n i f i c a n t increase i n growth when compared t o those fed the low l i p i d d i e t . These c o n t r a d i c t o r y r e s u l t s n e c e s s i t a t e e v a l u a t i o n of the a c t u a l i n g e s t i v e response of the f i s h t o determine what i s o c c u r r i n g w i t h i n the f i s h . U n f o r t u n a t e l y , the poor response t o the d i e t and the environment by the experimental animals made such data unobtainable. F o r t u n a t e l y , the f a c t t h a t there was s t a t i s t i c a l j u s t i f i c a t i o n f o r p o o l i n g high and low l i p i d d i e t a r y groups i n t o one r e g r e s s i o n d u r i n g the f a t t y a c i d a n a l y s i s makes the statement t h a t body f a t t y a c i d composition i s a f f e c t e d by d i e t a r y f a t t y a c i d composition i n a c o n s i s t e n t manner r e g a r d l e s s of the t o t a l l i p i d content of the d i e t . This statement holds t r u e f o r both the p o l a r and n e u t r a l body l i p i d pools and was c o n s i s t e n t i n the p e r i o d 1 and p e r i o d 2 samples. The e f f e c t of d i e t a r y f a t t y a c i d s on body f a t t y a c i d composition The i n t e r a c t i o n s between d i e t a r y f a t t y a c i d content and body f a t t y a c i d composition were not c o n s i s t e n t i n the two samples of f i s h (periods 1 and 2). D i f f e r e n c e s i n body f a t t y a c i d composition between the p e r i o d 1 samples and the p e r i o d 2 samples are d i f f i c u l t t o i n t e r p r e t . 150 These samples of f i s h , although from the same p o p u l a t i o n s , were of d i f f e r e n t body weights, body l i p i d content, and n e u t r a l l i p i d content. The d i f f e r e n c e i n age of these two groups was 98 days. I t must be noted t h a t the p e r i o d 1 sample were taken from 5° C. water. They had been r a i s e d at between 3 - 5 ° C. f o r 61 days. The p e r i o d 2 sample was taken from a 15° C. environment. The growth t r i a l ( p e r i o d 2) began at 7.5 ° C. I t took 3 3 days f o r the ambient temperature t o r i s e t o 10° C. F o l l o w i n g t h i s the temperature was heated one degree per day f o r two days and then remained c o n s i s t e n t l y at 12° C. f o r 7 days. The temperature was again el e v a t e d by one degree per day t o 14.5° C. where i t remained f o r the remaining 39 days of the growth t r i a l . Although there was an obvious d i f f e r e n c e i n mean environmental (temperature) between periods 1 and 2 a s s o c i a t i n g the d i f f e r e n c e s i n f a t t y a c i d composition e x c l u s i v e l y w i t h water temperature may l e a d t o erroneous c o n c l u s i o n s . The d i f f e r e n c e s i n age, s i z e , p h y s i c a l s t a t e and body composition were s u b s t a n t i a l . The data i n t a b l e s 6 and 12 show con s i d e r a b l e d i f f e r e n c e s i n t o t a l , p o l a r and n e u t r a l l i p i d c o n c entrations i n the body t i s s u e s . The age d i f f e r e n c e may i n f l u e n c e hormonal a c t i v i t y w h i l e the body s i z e and p h y s i c a l s t a t e may r e l a t e t o the a c t i v i t y of the 151 f i s h . For the r e l a t i o n s h i p of water temperature and d i e t a r y f a t t y a c i d e f f e c t t o be p r o p e r l y addressed the d i e t a r y treatments should be administered simultaneously t o a homogeneous p o p u l a t i o n of f i s h at the d i f f e r e n t water temperatures. I t was the p e r i o d 1 f i s h which d i s p l a y e d an e f f e c t of d i e t on body s a t u r a t e d f a t t y a c i d content. The i n c r e a s e s i n s a t u r a t e content i n the d i e t seemed to be s l i g h t l y more r e s p o n s i b l e f o r the decreases i n n e u t r a l s a t u r a t e s i n the body than were the increases i n d i e t a r y n3 as i s seen i n t a b l e 11. This t r e n d was a l s o noted i n a r e p o r t by C a s t l e d i n e and Buckley (1980). However, the d i e t a r y e f f e c t was not repeated i n the p e r i o d 2 sample i n t h i s experiment. Table 4 shows t h a t the d i e t a r y s a t u r a t e c o n c e n t r a t i o n increased when d i e t a r y n3 f a t t y a c i d s increased. The n3 f a t t y a c i d s i n the d i e t a r y treatments incre a s e d at n e a r l y twice the r a t e i n which s a t u r a t e s i n c r e a s e d . This r e l a t i o n s h i p of s a t u r a t e d and n3 f a t t y a c i d s might have been expected to produce a p o s i t i v e e f f e c t on n e u t r a l body sat u r a t e d f a t t y a c i d s r a t h e r than the negative r e l a t i o n s h i p which occurred. D i e t a r y monounsaturated f a t t y a c i d s , i n c l u d i n g 18:1, a l s o a f f e c t e d the n e u t r a l s a t u r a t e content i n the body. Increased d i e t a r y monounsaturated f a t t y a c i d s caused s a t u r a t e d f a t t y 152 a c i d s i n the body n e u t r a l l i p i d t o i n c r e a s e . Table 4 shows t h a t the monounsaturated f a t t y a c i d s i n the d i e t i n c r e a s e d as d i e t a r y n3 and s a t u r a t e d f a t t y a c i d s decreased. Reviewing the r e g r e s s i o n s and r 2 values f o r these r e l a t i o n s h i p s ( t a b l e 11) shows t h a t the monounsaturated f a t t y a c i d s are only s l i g h t l y more r e s p o n s i b l e f o r the changes i n body n e u t r a l s a t u r a t e d f a t t y a c i d concentrations than are the d i e t a r y n3 f a t t y a c i d s . I t i s u n c e r t a i n what caused the unexpected r e l a t i o n s h i p between d i e t a r y and body f a t t y a c i d s . The e s s e n t i a l f a t t y a c i d requirement i n the f i s h remains r e g a r d l e s s of low growth r a t e or metabolic a c t i v i t y . The maintenance of b i o l o g i c a l membranes w i t h i n the body i s the most probable requirement f o r these f a t t y a c i d s . Perhaps the low water temperature caused a decrease i n i n g e s t i v e a c t i v i t y i n comparison to p e r i o d 2 f i s h . At t h i s low i n t a k e l e v e l the d i e t w i t h the lowest n3 f a t t y a c i d c o n c e n t r a t i o n may not have been s u f f i c i e n t f o r p r o v i d i n g the amount of n3 r e q u i r e d f o r maintenance of the c e l l membranes and other e s s e n t i a l , f u n c t i o n s . I f t h i s was the case, the n e u t r a l l i p i d pool would be s e r v i n g as a source of n3 f a t t y a c i d s . As the n e u t r a l l i p i d was depleted of t h i s c l a s s of f a t t y a c i d s the c o n c e n t r a t i o n s of other c l a s s e s of f a t t y a c i d s i n the n e u t r a l f r a c t i o n 153 would i n c r e a s e . I t seems as though t h i s i s what has happened i n the body w i t h the n e u t r a l s a t u r a t e d f a t t y a c i d f r a c t i o n . T h i s may have been what C a s t l e d i n e and Buckley (1980) observed when they hypothesized t h a t f a t t y a c i d s are c a t a b o l i z e d on a molar b a s i s i n the n e u t r a l l i p i d d u r i n g s t a r v a t i o n . I t i s p o s s i b l e t h a t long-chained h i g h l y unsaturated f a t t y a c i d s i n the n e u t r a l l i p i d were being removed and incorporated i n t o the p o l a r l i p i d as maintenance of the c e l l membranes occurred. The l a c k of a s i g n i f i c a n t r e l a t i o n s h i p between d i e t a r y n3 f a t t y a c i d s and body n3 f a t t y a c i d s a l s o r e l a t e s t o the above s c e n a r i o . I t seems t h a t i n p e r i o d 1 the body n e u t r a l l i p i d pool was a more important source of n3 f a t t y a c i d s f o r the p o l a r l i p i d than was the d i e t a r y l i p i d . Perhaps the e s s e n t i a l f a t t y a c i d requirement of f i s h should be s t a t e d as a f u n c t i o n of body weight d i r e c t l y r a t h e r than i n d i r e c t l y . By s t a t i n g the requirement as a f u n c t i o n of the d i e t n u t r i t i o n i s t s are erroneously i m p l y i n g t h a t d i e t a r y i n t a k e remains as a constant f u n c t i o n of the body weight, as i s the case w i t h homeotherms. The metabolic r a t e and i n g e s t i v e r a t e i n p o i k i l o t h e r m s do change when ambient temperature changes. 154 However, i t i s u n c e r t a i n whether these two r a t e s are i n n a t e l y synchronized t o ensure adequate n u t r i t i o n when the d i e t does not change. I f the lower metabolic r a t e does not compensate f o r the lower i n g e s t i v e r a t e then the n3 f a t t y a c i d requirement, as a f u n c t i o n of the d i e t , may change by a d i f f e r e n t increment than would the n3 f a t t y a c i d requirement as a f u n c t i o n of body weight. Increased d i e t a r y monounsaturate c o n c e n t r a t i o n c o n s i s t e n t l y r e s u l t e d i n increased monounsaturate content i n the body n e u t r a l l i p i d f r a c t i o n . S i m i l a r l y , an i n c r e a s e i n the d i e t a r y n3 content, which corresponded t o a decreased d i e t a r y monounsaturate content, decreased the monounsaturate f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d . This was a l s o observed by C a s t l e d i n e and Buckley (1980). A s i m i l a r t r e n d occurred i n the p o l a r l i p i d of the p e r i o d 1 f i s h . P o l a r monounsaturate increased w i t h increased d i e t a r y monounsaturate and decreased w i t h increased d i e t a r y n3 c o n c e n t r a t i o n s . C18 monounsaturated f a t t y a c i d c o n c e n t r a t i o n i n the body n e u t r a l l i p i d f r a c t i o n was a f f e c t e d p o s i t i v e l y by d i e t a r y C18 c o n c e n t r a t i o n and i n v e r s e l y by changes i n d i e t a r y n3 i n both p e r i o d s . This trend was a l s o observed by Takeuchi and Watanabe (1977a). The monounsaturated f a t t y a c i d content i n the body p o l a r l i p i d was a f f e c t e d by 155 d i e t o n l y i n the p e r i o d 1 sample. Increased d i e t a r y n3 r e s u l t e d i n decreased 18:1 f a t t y a c i d content i n the body p o l a r l i p i d . The e f f e c t of n3 f a t t y a c i d s i n the d i e t on body n3 f a t t y a c i d s was d i f f e r e n t f o r the two sample groups. The p e r i o d 2 sample showed an increase i n n3 f a t t y a c i d s i n the body n e u t r a l l i p i d when d i e t a r y n3 was increa s e d . I t has already been mentioned t h a t the p e r i o d 1 body n3 content was not a f f e c t e d by changes i n d i e t a r y n3 co n c e n t r a t i o n . n3 h i g h l y unsaturated f a t t y a c i d s (HUFA's) i n the body n e u t r a l l i p i d were a f f e c t e d i n a s i m i l a r manner t o the t o t a l n3 i n t h a t l i p i d f r a c t i o n . During p e r i o d 2, f i s h s t o r e d l e v e l s of n3 HUFA's i n the n e u t r a l l i p i d which corresponded t o d i e t a r y n3 HUFA and t o t a l n3 i n c r e a s e s . The n e u t r a l l i p i d i n the p e r i o d 1 f i s h was not a f f e c t e d by increased d i e t a r y n3 HUFA or t o t a l n3 l e v e l s . n3 HUFA content i n the body p o l a r l i p i d a l s o i n c r e a s e s w i t h i n c r e a s e s i n d i e t a r y n3 HUFA or t o t a l n3 i n the p e r i o d 2 f i s h . T h i s r e l a t i o n s h i p was reported by C a s t l e d i n e and Buckley (1980). The p o l a r n3 HUFA c o n c e n t r a t i o n i n f i s h d u r i n g p e r i o d 2 was again not a f f e c t e d by changes i n d i e t a r y n3 HUFA or t o t a l n3 content. Increased d i e t a r y n6 content c o n s i s t e n t l y increased 156 n6 content i n the body p o l a r l i p i d i n both the p e r i o d 1 and p e r i o d 2 f i s h . I n the p e r i o d 2 f i s h , n6 HUFA co n c e n t r a t i o n i n the body p o l a r l i p i d i n c r e a s e d as d i e t a r y n6 l e v e l s rose. The increase i n n6 polyunsaturated f a t t y a c i d s i n the p o l a r f r a c t i o n provides f u r t h e r evidence towards the competition of C18 polyunsaturated f a t t y a c i d s f o r e l o n g a t i o n , d e s a t u r a t i o n and i n c o r p o r a t i o n i n t o p h o s p h o l i p i d s . S i m i l a r f i n d i n g s were reported by Lee et a l . (1967) and C a s t l e d i n e and Buckley (1980). The i s s u e of competition of 18:ln9, 18:2n6 and 18:3n3 f o r the e l o n g a t i o n and d e s a t u r a t i o n mechanism i s not e a s i l y addressed by t h i s study. I t i s d i f f i c u l t t o examine the e l o n g a t i o n and d e s a t u r a t i o n of 18:1 because 16:ln9 and 20:ln9 were a l s o found i n the d i e t s i n s u b s t a n t i a l amounts. A l l of these f a t t y a c i d s were observed i n the body l i p i d s . However, the l a c k of 20:3n9 suggests t h a t 18:2n6 and 18:3n3 were e f f e c t i v e i n i n h i b i t i n g the e l o n g a t i o n and d e s a t u r a t i o n of 18:ln9. At f i r s t glance the 18:3n3 seems t o be more e f f e c t i v e than 18:2n6 i n c r e a t i n g long-chained h i g h l y unsaturated f a t t y a c i d s . Table 4 shows d i e t s I I I and V I I I as having equal c o n c e n t r a t i o n s of n3 and n6 f a t t y a c i d s . The con c e n t r a t i o n s of h i g h l y unsaturated f a t t y a c i d s i n t a b l e s 12 - 15 c o n s i s t e n t l y shows n3 t o exceed n6 f a t t y a c i d s i n 157 the body. However, 85% of the t o t a l n3 f a t t y a c i d s i n the d i e t are h i g h l y unsaturated. Knowing t h i s , the r a t i o of n3 HUFA t o n6 HUFA i n the body l i p i d i s l e s s impressive s i n c e 85% of the body n3 HUFA's could have been obtained d i r e c t l y from the d i e t without e l o n g a t i o n or d e s a t u r a t i o n . U n f o r t u n a t e l y , not much can be s a i d about t h i s i s s u e because of the v a r i e t y o f , d i e t a r y c o n s t i t u e n t s i n t h i s experiment. The i s s u e of el o n g a t i o n and d e s a t u r a t i o n should be addressed u s i n g r a d i o a c t i v e l y l a b e l l e d 18:ln9, 18:2n6 and 18:3n3 i n the d i e t or by feeding p u r i f i e d d i e t s which only have short-chained f a t t y a c i d s . The e s s e n t i a l f a t t y a c i d index, 20:3n9/22:6n3, reported by Owen et a l . (1975) and Takeuchi and Watanabe (1979) could not be addressed i n t h i s study because 20:3n9 was not detected i n the body l i p i d . The d i e t a r y c o n c e n t r a t i o n of n3 and n6 f a t t y a c i d s probably i n h i b i t e d the e l o n g a t i o n and d e s a t u r a t i o n of n9 monounsaturated f a t t y a c i d s i n agreement w i t h the f i n d i n g s of Brockerhoff e t a l . (1966). This r e l a t i o n s h i p was a l s o reported by Yu and Sinnhuber (1972), Takeuchi et a l . (1979) and Takeuchi and Watanabe (1977a). Body moisture content d i d not seem t o be a f f e c t e d by d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n . This o b s e r v a t i o n i s co n t r a r y t o one re p o r t of a evidence of a negative 158 r e l a t i o n s h i p (Takeuchi and Watanabe, 1979) and one of a p o s i t i v e r e l a t i o n s h i p ( C a s t e l l e t a l . , 1972b.). The e f f e c t of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n on growth The i n i t i a l poor i n g e s t i v e response t o the d i e t s coupled w i t h the disadvantageous environmental c o n d i t i o n s d u r i n g p e r i o d 1 r e s u l t e d i n high m o r t a l i t y and much v a r i a t i o n i n the s i z e of the f i s h i n p e r i o d 2. The high and v a r i a b l e r a t e s of m o r t a l i t y make the growth r a t e s d i f f i c u l t t o evaluate. The l a c k of a s i g n i f i c a n t d i f f e r e n c e i n growth agrees w i t h the r e s u l t s of Yu and Sinnhuber (1979) f o r the d i e t a r y n3 f a t t y a c i d concentrations between 1.0 and 2.5% (% DMB). Growth i n coho salmon i s not improved by increased amounts of d i e t a r y n3 f a t t y a c i d s as was observed i n rainbow t r o u t (Takeuchi and Watanabe, 1977a). This f i n d i n g may prove t o be important t o the aquaculture i n d u s t r y i f e i t h e r the p r i c e or the a v a i l a b i l i t y of marine f i s h o i l s makes the use of t h i s i n g r e d i e n t i m p r a c t i c a l . However, f u r t h e r research i s r e q u i r e d t o b e t t e r determine the optimum d i e t a r y c o n c e n t r a t i o n of n3 f a t t y a c i d s . A long-term study of the e f f e c t of d i e t a r y f a t t y a c i d s on growth and body composition would be very u s e f u l i n 159 mapping out the dietary e f f e c t over d i f f e r e n t environmental and p h y s i o l o g i c a l conditions. The e f f e c t of dietary n3 f a t t y acids concentration on m o r t a l i t y There were l i t t l e m o r t ality during period 1 of t h i s experiment. During period 2, on the other hand, mo r t a l i t y ranged from 33 - 78 % among tanks. M o r t a l i t y was associated with anorexia and lethargy. F a i l u r e to feed during period 1 meant that the f i s h had to c a t a b o l i z e body stores f o r nutrients. The drop i n water temperature during period 1 would have reduced metabolic rate i n the f i s h . The low metabolic rate allowed the f i s h to survive during period 1. However, the metabolic rate would have increased during period 2 when the water temperature increased. This elevation i n metabolic rate increased the energy requirement of the f i s h . At t h i s point i n the experiment some of the f i s h were so d e b i l i t a t e d that they did not commence eating and subsequently died when they had depleted t h e i r body stores. I t i s very l i k e l y that the anorexic f i s h never ate during the whole experiment. I t i s f o r t h i s reason that a s s o c i a t i n g the mortality to the ingestive response i s more l o g i c a l than associating i t to an e f f e c t of dietary f a t t y acids on body f a t t y acid composition. 160 Chi-square a n a l y s i s showed a s i g n i f i c a n t d i f f e r e n c e (P>0.05) i n the m o r t a l i t y among treatments. This e f f e c t of d i e t a r y f a t t y a c i d c o n c e n t r a t i o n on m o r t a l i t y was examined by r e g r e s s i o n s . Increases i n d i e t a r y n3 and n3 h i g h l y unsaturated f a t t y a c i d s were p a r t i a l l y r e s p o n s i b l e f o r i n c r e a s e s i n m o r t a l i t y . The tre n d of i n c r e a s i n g m o r t a l i t y w i t h i n c r e a s i n g n3 or n3 HUFA c o n c e n t r a t i o n i n the d i e t has not been p r e v i o u s l y reported i n the l i t e r a t u r e . I t i s unc l e a r why such a r e l a t i o n s h i p would occur. There have been r e p o r t s of a negative e f f e c t of high d i e t a r y n3 concentrations i n growth i n coho salmon (Yu and Sinnhuber, 1979) and rainbow t r o u t (Takeuchi and Watanabe, 1979). The small r 2 values f o r the re g r e s s i o n s of m o r t a l i t y as a f u n c t i o n of these d i e t a r y f a t t y a c i d c l a s s e s suggests t h a t some unknown f a c t o r i s i n v o l v e d i n the abnormally hi g h m o r t a l i t y . Perhaps there was a c o n s t i t u e n t i n the h e r r i n g o i l which was d i s l i k e d by the f i s h i n t h i s experiment. The l a c k of accurate i n t a k e data l i m i t s assessment of the cause of the high m o r t a l i t y . The e f f e c t of d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n on sa l t w a t e r t o l e r a n c e There was no m o r t a l i t y as a r e s u l t of a 24 hour 161 challenge i n f u l l s t r e n g t h a r t i f i c i a l sea water. The f i s h were e q u a l l y able t o withstand the d i r e c t immersion i n t o s a l t w a t e r r e g a r d l e s s of d i e t a r y treatment. There was a l s o no s i g n i f i c a n t d i f f e r e n c e i n the dry moisture content of muscle p r i o r t o or f o l l o w i n g the 24 hour challenge as was the case p r i o r t o the challenge. The a n a l y s i s of v a r i a n c e of the mean dry matter content (post s a l t w a t e r challenge minus freshwater) showed no s i g n i f i c a n t d i f f e r e n c e between d i e t a r y treatments. This o b s e r v a t i o n suggests t h a t the d i f f e r e n c e s i n p o l a r f a t t y a c i d c o n c e n t r a t i o n , due t o the d i e t , do not impair the e f f e c t i v e n e s s of the b i o l o g i c a l membranes i n ma i n t a i n i n g osmosis. The mean treatment plasma sodium con c e n t r a t i o n s ( p o s t - s a l t w a t e r challenge) d i d not exceed the 170 meq/l. This c o n c e n t r a t i o n has been s t a t e d by Clarke and Blackburn (1977) as evidence of s a l t w a t e r t o l e r a n c e . I t appears t h a t d i e t a r y n3 f a t t y a c i d c o n c e n t r a t i o n does not a f f e c t s a l t w a t e r t o l e r a n c e i n j u v e n i l e coho salmon. This f i n d i n g i s i n agreement w i t h Markert et a l . (1984) and P l o t n i k o f f et a l . (1983). The coho i n t h i s experiment were 1+ years o l d . Under most c o n d i t i o n s these f i s h would undergo t r a n s f e r t o s a l t w a t e r a t t h i s age. I t i s important t h a t the d i e t a r y treatment d i d not impede the 162 a b i l i t y of the f i s h t o undergo t h i s c r i t i c a l p h y s i o l o g i c a l change i n t h e i r l i f e . Conclusion 163 Body concentrations of t o t a l l i p i d , p o l a r l i p i d and n e u t r a l l i p i d were not a f f e c t e d by d i e t a r y l i p i d content or d i e t a r y f a t t y a c i d composition. The composition of p o l a r and n e u t r a l l i p i d s was s i g n i f i c a n t l y a l t e r e d by d i e t a r y f a t t y a c i d c o n c e n t r a t i o n . P o l a r l i p i d composition was, however, comparatively s t a b l e r e l a t i v e t o changes i n d i e t a r y f a t t y a c i d s . The response of body l i p i d s t o d i e t a r y f a t t y a c i d s appeared t o be modified by water temperature. However, cognizance must be taken of the d i f f e r e n c e between the samples of f i s h . As a r e s u l t of feed consumption i n the f i s h during p e r i o d 2, the f i s h had gained weight and accumulated body l i p i d . The n6 f a t t y a c i d c o n c e n t r a t i o n i n the body p o l a r l i p i d c o n s i s t e n t l y increased w i t h increases i n d i e t a r y n6 content. The p e r i o d 1 f i s h showed t h a t p o l a r monounsaturate co n c e n t r a t i o n corresponded d i r e c t l y w i t h d i e t a r y monounsaturate content and i n v e r s e l y w i t h d i e t a r y n3 content. During p e r i o d 2, n3 l e v e l s i n the p o l a r l i p i d of the f i s h increased w i t h higher d i e t a r y concentrations of n3 f a t t y a c i d s . Higher n3 HUFA concentrations i n the p o l a r l i p i d were l i k e w i s e increased when d i e t a r y n3 or n3 HUFA content increased. During p e r i o d 2, increases i n 164 dietary n6 f a t t y acids also increased n6 HUFA l e v e l s i n the body polar l i p i d when dietary n6 l e v e l s increased. The changes i n the polar l i p i d f a t t y a c i d p r o f i l e may be at t r i b u t e d to the competitive elongation and desaturation between 18:ln9, 18:2n6 and 18:3n3 to t h e i r respective long-chained highly unsaturated f a t t y acids. These three f a m i l i e s of f a t t y acids compete fo r the same enzymatic mechanism f o r elongation and desaturation. This mechanism i s substrate concentration dependent. The f a c t that 20:3n9 was not detected i n body l i p i d strongly suggests that dietary 18:3n3 and 18:2n6 i n h i b i t elongation and desaturation of 18:ln9. The differences i n d i e t a r y concentrations of 18:2n6 and 18:3n3 within the experimental d i e t s make i t d i f f i c u l t to evaluate the i n h i b i t i v e e f f e c t of these f a t t y acids upon each other. Total monounsaturated and 18:1 monounsaturated f a t t y acid concentration i n the body corresponded d i r e c t l y with the respective concentration of dietary t o t a l monounsaturated or 18:1 f a t t y acids. Each of these f a t t y acid groups were decreased i n the neutral l i p i d by increases i n dietary n3. Saturated f a t t y a c i d content i n the body neutral l i p i d decreases with increases i n dietary n3 or saturated f a t t y acids i n the period 1 f i s h . However, t h i s r e l a t i o n s h i p was probably the r e s u l t of 165 inadequate intake of e s s e n t i a l f a t t y acids and the subsequent t r a n s f e r of n3 f a t t y acids from the body neutral l i p i d pool to the polar. n3 HUFA concentrations i n the body neutral l i p i d increased during period 2 when feeding occurred and di e t s containing the higher concentrations of t o t a l n3 or n3 HUFA were fed. The f a t t y a c i d composition of body l i p i d s reacted s i m i l a r l y to changes i n f a t t y acid content regardless of the manner i n which the d i e t content was quantified, percent of the dry d i e t or percent of the diet a r y l i p i d . I t seems that dietary nutrient concentrations i n poikilotherms should be stated as absolute amounts ingested rather than amounts i n the d i e t or i n a di e t a r y component such as l i p i d . This i s because nutrient requirements may be maintained regardless of metabolic rate r e s u l t i n g i n inadequate n u t r i t i o n when a c t i v i t y and food ingestion decreases at low temperatures. The growth response was d i f f i c u l t to i n t e r p r e t because of the high mortality. Mortality during period 2 increased as t o t a l n3 or n3 HUFA content i n the d i e t increased. The poor appetitive response i s probably more responsible f o r the mortality than i s dietary f a t t y a c i d content. Saltwater tolerance was not impeded by changes i n the dietary treatment. A l l dietary treatments showed 166 evidence of the a b i l i t y to e f f e c t i v e l y undergo t r a n s f e r to f u l l strength sea water. 167 L i t e r a t u r e c i t e d Anderson, A.A., Fletcher, T.C., and Smith, G.M., 1981. Prostaglandin biosynthesis i n the skin of the p l a i c e Pleuronectes platessa L. Comp. Biochem. Physiol. 70C:195-199 Anderson, A.A., Fletcher, T.C., and Smith, G.M., 1979. The release of prostaglandin E2 from the ski n of p l a i c e , Pleuronectes platessa L. Br. J . Pharmac. 66:547-552 Austreng, E. and Gfefsen, T., 1981. Fi s h o i l s with d i f f e r e n t contents of free f a t t y acids i n d i e t s f o r rainbow trout f i n g e r l i n g s and salmon parr. Aquaculture, 25:173-183. B e l l , M.V., Simpson, C.M.F., and Sargent, J.R., 1983. (n3) and (n6) polyunsaturated f a t t y acids i n the phosphoglycerides of s a l t - s e c r e t i n g e p i t h e l i a from two marine f i s h species. L i p i d s 18(10):720-726. Blackburn, J . and Clarke, W.C, 1987. Revised procedure for the 24 hour seawater challenge t e s t to measure seawater a d a p t a b i l i t y of ju v e n i l e salmonids. Can. Tech. Rep. Fish . Aquat. S c i . No. 1515 Bligh,E.G. and Dyer, W.J., 1959. A rapid method of t o t a l l i p i d extraction and p u r i f i c a t i o n . Can. J . Biochem. Physiol.37:911-917 Brockerhoff, H., Hoyle, R.J. and Wolmark, N., 1966. P o s i t i o n a l d i s t r i b u t i o n of f a t t y acids i n t r i g l y c e r i d e s of animal depot f a t s . Biochim. Biophys. Acta, 116:67 C a s t e l l , J.D., Sinnhuber, R.O., Wales, J.H. and Lee, D.J. 1972a. E s s e n t i a l f a t t y acids i n the d i e t of rainbow trout (Salmo g a i r d n e r i ) : Growth, feed conversion and some gross deficiency symptoms. J.Nutr. 102:77-86. C a s t e l l , J.D.,Sinnhuber, R.O., Lee, D.J. and Wales, J.H., 1972b. E s s e n t i a l f a t t y acids i n the d i e t of rainbow trout (Salmo g a i r d n e r i ) : Physiological symptoms of EFA def i c i e n c y . J . Nutr. 102:87-92. C a s t e l l , J.D., Lee, D.J. and Sinnhuber, R.O., 1972c. E s s e n t i a l f a t t y acids i n the d i e t of rainbow trout (Salmo gairdneri}: L i p i d metabolism and f a t t y acid composition. J . Nutr. 102:93-100 168 C a s t l e d i n e , A.J. and Buckley, J.T., 1980. D i s t r i b u t i o n and m o b i l i t y of n3 f a t t y a c i d s i n rainbow t r o u t f e d 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 C h r i s t , E.J. and Van Dorp, D.A., 1972. Comparative aspects of p r o s t a g l a n d i n b i o s y n t h e s i s i n animal t i s s u e s Biochim. Biophys. Acta 270:537-545 C h r i s t i e , W.W., 1973. L i p i d A n a l y s i s , Pergamon Press, New York, p. 90 C h r i s t i e , W.W., 1982. L i p i d a n a l y s i s . 2nd e d i t i o n , Pergamon Press, Oxford, p53 C l a r k e , W.C. and Blackburn, J . , 1977. A seawater challenge t e s t t o measure smolting i n j u v e n i l e salmon. Can. F i s h . Mar. Serv. Tech. Rep. 705 C l a r k e , W.C, Higgs, D.A., Markert, J.R., Shelbourn, J.E. and C a s t l e d i n e , A . J . , 1982. E f f e c t of v a r y i n g d i e t a r y p r o t e i n : l i p i d r a t i o s on growth and body composition of coho salmon f r y (Oncorhynchus k i s u t c h ) reared a t d i f f e r e n t temperatures. Can. Data Rep. F i s h . Aquat. S c i . , 373,iv + 18 pp. Cowey, C.B., Andron, J.W., Hardy, R., Smith. J.G.M., and Walton, M.J., 1979. U t i l i z a t i o n by rainbow t r o u t of d i e t s c o n t a i n i n g p a r t i a l l y rendered hide f l e s h i n g s . Aquaculture 16:199-209 Dosanjh, B.S., Higgs, D.A., P l o t n i k o f f , M.D., McBride, J.R., Markert, J.R., and Buckley, J.T., 1984. E f f i c a c y of canola 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 (Oncorhynchus  k i s u t c h ) . Aquaculture, 36:333-345. Dosanjh, B.S., Higgs, D.A., P l o t n i k o f f , M.D., Markert, J.R., and Buckley, J.T., 1988. P r e l i m i n a r y e v a l u a t i o n of canola 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 f a l l Chinook salmon (Oncorhynchus tshawytscha). Aquaculture, 68:325-343 German, B., Bruckner, G., and K i n s e l l a , J . , 1983. Evidence a g a i n s t a PGF4a p r o s t a g l a n d i n s t r u c t u r e i n t r o u t t i s s u e - a c o r r e c t i o n . P r o s t a g l a n d i n s 26(2):207-210 169 German, B., Bruckner, G., and K i n s e l l a , J . , 1986. Lipoxygenase i n t r o u t g i l l t i s s u e a c t i n g on a r a c h i d o n i c , eicosapentanoic and docosahexanoic a c i d s . Biochim. Biophys. Acta 875:12-20 Hardy, R.W., S c o t t , T.M., and H a r r e l l , L.W., 1987. Replacement of h e r r i n g o i l w i t h menhaden o i l , soybean o i l , or t a l l o w i n the d i e t s of A t l a n t i c salmon r a i s e d i n marine net-pens. Aquaculture 65:2 67-277 Kellems, R.O. and Sinnhuber, R.O., 1982. Performance of rainbow t r o u t fed gelatin-bound d i e t s of f i s h p r o t e i n concentrate of c a s e i n c o n t a i n i n g 25 t o 45 percent h e r r i n g o i l . Prog. F i s h C u l t . , 44(3) :131-134. Lee, D.J., Roehm, J.N., Yu, T.C. and Sinnhuber, R.O., 1967. E f f e c t of n3 f a t t y a c i d s on the growth r a t e of rainbow t r o u t , Salmo q a i r d n e r i . J . Nutr., 92:93-98. Lee, D.J. and Putnam, G.B., 1973. The response of rainbow t r o u t t o v a r y i n g protein/energy r a t i o s i n a t e s t d i e t . J . Nutr., 103:916-922 Lee, D.J. and Wales, J.H., 1973. Observed l i v e r changes i n rainbow t r o u t (Salmo q a i r d n e r i ) fed v a r y i n g l e v e l s of a c a s e i n - g e l a t i n mixture and h e r r i n g o i l i n experimental d i e t s . J . F i s h . Res. Board Can., 30:1017-1020. Mai, J . , Goswami, S.K., Bruckner, G., and K i n s e l l a , J.E., 1981. A new p r o s t a g l a n d i n C22-PGF4a, sy n t h e s i z e d from docosahexaenoic a c i d (22:6n3) by t r o u t g i l l . P r o s t a g l a n d i n s 21(5):691-698 Markert, J.R., Higgs, D.A., MacQuarrie, D., McBride, J.R., Dosanjh, B.S., Van Tine, J . and Reinhardt, R., 1984. E v a l u a t i o n of the p o t e n t i a l of u s i n g dry r a t h e r than semi-moist food f o r c u l t u r i n g coho salmon i n B r i t i s h Columbia hatchery f a c i l i t i e s 2. Quinsam hatchery 1977 brood. Can. Tech. Rep. F i s h . Aquat. S c i . 1260. Mugrditchian, D.S., Hardy, R.W. and Iwaoka, W.T., 1981. Linseed o i l and animal f a t as a l t e r n a t e l i p i d sources i n dry d i e t s f o r Chinook salmon (Oncorhynchus  tshawytscha). Aquaculture, 25:161-172. 170 Nomura, T. and Ogata, H. 1976. D i s t r i b u t i o n of P r o s t a g l a n d i n s i n the animal kingdom. Biochim. Biophys. Acta 431:127-131 Oliw, E., Granstrom. E., and Anggard, E. 1983. The p r o s t a g l a n d i n s and e s s e n t i a l f a t t y a c i d s . i n P r o s t a g l a n d i n s and Related Substances. Pace-Asciak and Granstrom ed. E l s e v i e r , pp 1-34 Owen, J.M., Adron, J.W., Middleton, C. and Cowey, C.B. 1975. E l o n g a t i o n and d e s a t u r a t i o n of d i e t a r y f a t t y a c i d s i n t u r b o t Scophthalmus maximus L., and rainbow t r o u t , Salmo g a i r d n e r i R i c h . L i p i d s , 10:528-531. Page, J.W. and Nadrews, J.W., 1973. I n t e r a c t i o n s of d i e t a r y l e v e l s of p r o t e i n and energy on channel c a t f i s h ( I c t a l u r u s punctatus). J . Nutr., 103:1339-1346 P i c , P., 1975. J . P h y s i o l . ( P a r i s ) 71, 146A. c i t e d i n B e l l e t a l . 1983. P h i l l i p s , A.M., L i v i n g s t o n , D.L., Poston, H.A., and Booke, H.A., 1963. The e f f e c t of d i e t mixture and c a l o r i e source on growth, m o r t a l i t y , conversion, and chemical composition of brook t r o u t . Prog. F i s h C u l t . 25:8-14. Phleger, C.F., Laub, R.J., and Benson, A.A., 1989. S k e l e t a l l i p i d d e p l e t i o n i n spawning salmon. L i p i d 24:286-289 P l o t n i k o f f , M.D., Higgs, D.A., Markert, J.R., Dosanjh, B.S., McBride, J.R., and Buckley, J.T., 1983. N u t r i t i o n and marine s u r v i v a l of chinook salmon (Oncorhynchus tshawytscha). I . P o t e n t i a l r o l e of smolt body composition (Robertson creek hatchery 1979 brood). Can. Tech. Rep. F i s h . Aquat. S c i . No. 1206. P l o t n i k o f f , M.D., Higgs, D.A., Markert, J.R., Dosanjh, B.S., McBride, J.R., and Buckley, J.T., 1984. N u t r i t i o n and marine s u r v i v a l of chinook salmon (Oncorhynchus tshawytscha). I I . Further i n v e s t i g a t i o n of the p o t e n t i a l r o l e of smolt body composition (Robertson Creek hatchery 1980 brood). Can. Tech. Rep. F i s h . Aquat. S c i . No. 12 35. 171 R e i n i t z , G.L., Orme, L.E., Lemm, C A . and H i t z e l , F.N., 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 w i t h two p r o t e i n c o n centrations i n d i e t s f o r rainbow t r o u t (Salmo q a i r d n e r i ) . Trans. Am. F i s h . S o c , 107 (5) : 751-754. Singh, G. and Chandra, R.K., 1988. Biochemical and c e l l u l a r e f f e c t s of f i s h o i l s . Progress i n Food and N u t r i t i o n Science 12:371-419 Snedecor, G.W., and Cochran, W.G. 1967. S t a t i s t i c a l Methods 6th e d i t i o n , Iowa State U n i v e r s i t y Press, Ames, Iowa, pp. 428 - 438. Takeuchi, T. and Watanabe, T., 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 . Jap. Soc. S c i . F i s h . 43(7):893-898. Takeuchi, T. and Watanabe, T., 1977b. E f f e c t of eicosapentaenoic 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 . Jap. Soc. S c i . F i s h . , 43(8):947-953. Takeuchi, T. and Watanabe, T., 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 . Jap. Soc. S c i . F i s h . 45:1517-1519. Takeuchi, T. and Watanabe, T., 1982. E f f e c t s of v a r i o u s polyunsaturated f a t t y a c i d s on growth and f a t t y a c i d compositions of rainbow t r o u t Salmo q a i r d n e r i . coho salmon Oncorhynchus k i s u t c h . and chum salmon Oncorhynchus keta. B u l l . Jap. Soc. S c i . F i s h . 48(12) :1745-1752 Takeuchi, T. and Watanabe, T. and Nose, T., 1979. Requirement f o r e s s e n t i a l f a t t y a c i d s of chum salmon (Oncorhynchus keta) i n freshwater, environment. B u l l . Jap. Soc. S c i . F i s h . , 45:1319-1323. Takeuchi, T., Watanabe, T. and Ogino, C , 1978a. Optimum r a t i o of p r o t e i n t o l i p i d i n d i e t s of rainbow t r o u t . B u l l . Jap. Soc. S c i . F i s h . , 44(6):683-688. Takeuchi, T., Watanabe, T. and Ogino, C., 1978b. 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 . Jap. Soc. S c i . F i s h . , 44(6):677-681. 172 Takeuchi, T., Watanabe, T. and Ogino, C., 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 carp and rainbow t r o u t . B u l l . Jap. Soc. S c i . F i s h . , 44 (8):875-881 Uno, M., 1989. Seawater adaptation i n Autumn, and freshwater adaptation i n s p r i n g of j u v e n i l e s of s e v e r a l salmonid spec i e s . Nippon Suissan Gak., 55(2):191-196. Yamada, K., Kobayashi, K. and Yone, Y., 1980. Conversion of l i n o l e n i c a c i d t o n3 h i g h l y unsaturated f a t t y a c i d s i n marine f i s h e s and rainbow t r o u t . B u l l . Jap. SOC. S c i . 46:1231-1233 Yu, T.C., Sinnhuber, R.O. and Putnam, G.B., 1977a. 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 i n rainbow t r o u t (Salmo g a i r d n e r i ) . L i p i d s , 12(6):495-499. Yu, T.C., Sinnhuber, R.O. and Putnam, G.B., 1977b. 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 Sinnhuber, R.O., 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 (Salmo  g a i r d n e r i ) . L i p i d s , 7(7):450-454. Yu, T.C. and Sinnhuber, R.O., 1979. E f f e c t of d i e t a r y n3 and n6 f a t t y a c i d s on growth and feed e f f i c i e n c y of coho salmon (Oncorhynchus k i s u t c h ) . Aquaculture, 16:31-38. Yu, T.C. and Sinnhuber, R.O., 1981. Use of beef t a l l o w as an energy source i n coho salmon (Oncorhynchus  k i s u t c h ) r a t i o n s . Can. J . F i s h . Aquat. S c i . 38:367-370. Yu, T.C. and Sinnhuber, R.O. and Hendricks, J.D., 1979. r e p r o d u c t i o n and s u r v i v a l of rainbow t r o u t (Salmo  g a i r d n e r i ) fed l i n o l e n i c a c i d as the only source of e s s e n t i a l f a t t y a c i d s . L i p i d s , 14(6):572-575. Yu, T.C, Sinnhuber, R.O., and Putnam, G.B., 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 i n rainbow t r o u t (Salmo g a i r d n e r i ) . L i p i d s 12(6):495-499 

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