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The effect of dietary lipid and vitamin E on the reproduction of Arctic charr, Salvelinus alpinus (L.) 1992

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THE EFFECT OF DIETARY LIPID AND VITAMIN E ON THE REPRODUCTION OF ARCTIC CHARR, Salvelinus alpinus (L.) by Jo-Anne L e s l i e Tabachek B.Sc. (Hons.)/ U n i v e r s i t y of Manitoba, 1968 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 September 1992 ® Jo-Anne L e s l i e Tabachek, 1992 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 -6 (2/88) ABSTRACT This research was conducted i n an attempt t o improve embryonic s u r v i v a l of Labrador A r c t i c c h a r r by a l t e r i n g the conc e n t r a t i o n s of l i p i d and v i t a m i n E i n the d i e t s fed t o broodstock. Four-year o l d Labrador A r c t i c c h a r r broodstock were fed d i e t s c o n t a i n i n g two l e v e l s of d i e t a r y l i p i d a t two l e v e l s of v i t a m i n E acetate ( d l - a - t o c o p h e r y l acetate) supplementation. The d i e t s were designated LLLE, LLHE, HLLE and HLHE t o denote low l i p i d ( 1 2 % ) ( L L ) , high l i p i d (19%)(HL), low v i t a m i n E (30 mg/kg)(LE) and high v i t a m i n E (600 mg/kg)(HE). For comparison, a f i f t h group was fed t h e i r normal d i e t - a commercial grower d i e t (COMM) c o n t a i n i n g 17% l i p i d and 100 mg v i t a m i n E acetate/kg. Since A r c t i c charr broodstock used i n t h i s research were v a l u a b l e , d i e t s t h a t might have a d e l e t e r i o u s e f f e c t on the s u r v i v a l or f e c u n d i t y of the broodstock c o u l d not be used. D i e t a r y l i p i d concentrations were s e l e c t e d based on the requirements f o r j u v e n i l e A r c t i c c h a r r . D i e t a r y v i t a m i n E co n c e n t r a t i o n s were s e l e c t e d based on requirements s t a t e d i n the l i t e r a t u r e f o r other salmonids. F i s h were fed f o r 71 days before withdrawal of feed p r i o r t o spawning i n Year 1 and f o r 252 days i n Year 2. Since f i s h f e d the LL d i e t s i n Year 1 spawned 5 weeks l a t e r than those fed the HL d i e t s , a crossover i n d i e t s was conducted t o see i f t h i s would a l s o occur i n Year 2. Keeping the v i t a m i n E l e v e l the i i i same, those fed LL d i e t s i n Year 1 were fed HL d i e t s i n Year 2 and v i c e v e r s a . No e f f e c t of d i e t a r y l i p i d on spawning time was observed i n Year 2. The p r o p o r t i o n of females f a i l i n g t o spawn was not r e l a t e d t o the concentrations of d i e t a r y l i p i d or v i t a m i n E. A s i g n i f i c a n t l y higher p r o p o r t i o n of males fed the higher c o n c e n t r a t i o n of v i t a m i n E produced m i l t i n two s u c c e s s i v e spawning seasons. Fecundity of 3894 and 4532 eggs i n Year 1 and 4154 and 8305 eggs i n Year 2 f o r the four experimental and COMM d i e t s , r e s p e c t i v e l y , was d i r e c t l y c o r r e l a t e d w i t h female weight and was not a f f e c t e d by the l e v e l of d i e t a r y l i p i d or v i t a m i n E. The f a t t y a c i d composition of the eggs r e f l e c t e d t h a t of the d i e t and was not i n d i c a t i v e of e s s e n t i a l f a t t y a c i d d e f i c i e n c y . In Year 2, concentrations of t o t a l (22.3%), n e u t r a l (10.6%) and p o l a r l i p i d s (11.7%) i n the eggs were not s i g n i f i c a n t l y a f f e c t e d by the concentrations of d i e t a r y l i p i d or v i t a m i n E. The v i t a m i n E concentrations of the eggs were 52, 202, 54 and 156 u,g/g i n Year 1 and 38, 208, 51 and 140 ixg/g i n Year 2 f o r the LLLE, LLHE, HLLE and HLHE d i e t s r e s p e c t i v e l y . The i n c r e a s e i n d i e t a r y v i t a m i n E r e s u l t e d i n a g r e a t e r increase i n v i t a m i n E c o n c e n t r a t i o n i n eggs from f i s h fed the LL compared to the HL d i e t s . i v D i e t s c o n t a i n i n g 30 or 600 mg v i t a m i n E acetate/kg w i t h 12 or 19% l i p i d met the v i t a m i n E requirements of 4- and 5-year o l d broodstock t o the extent t h a t f e r t i l i z a t i o n and embryonic s u r v i v a l was not a f f e c t e d s i g n i f i c a n t l y . F e r t i l i z a t i o n was 89% i n Year 2 and 80% i n Year 2. There was a high degree of w i t h i n treatment v a r i a t i o n i n embryonic s u r v i v a l . F e r t i l i z a t i o n and embryonic s u r v i v a l were not c o r r e l a t e d w i t h v i t a m i n E c o n c e n t r a t i o n of the eggs. S u r v i v a l t o swimup of f e r t i l i z e d eggs was 27% f o r the LE d i e t s and 55% f o r the HE d i e t s i n Year 2. Embryonic s u r v i v a l t o the eyed, hatch and swimup stages were n e g a t i v e l y c o r r e l a t e d w i t h the percent n e u t r a l l i p i d and w i t h 16:ln7 i n the n e u t r a l l i p i d of the eggs. V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS V LIST OF TABLES v i i i LIST OF FIGURES X LIST OF APPENDICES x i i ACKNOWLEDGEMENTS x i i i 1 INTRODUCTION 1 2 LITERATURE REVIEW 4 2.1 N a t u r a l h i s t o r y of A r c t i c charr 4 2.2 A r c t i c c h a r r c u l t u r e i n Canada 6 2.3 Physiology of reproduction 10 2.4 Feed i n t a k e d u r i n g reproduction 12 2.4.1 Feed i n t a k e 12 2.4.2 S t a r v a t i o n p r i o r t o spawning 14 2.5 P r o t e i n and energy 14 2.6 L i p i d s , f a t t y acids and v i t a m i n E 16 2.6.1 Chemistry of l i p i d s and f a t t y a c i d s 16 2.6.2 D i g e s t i o n and absorption of l i p i d s and v i t a m i n E 18 2.6.3 E s s e n t i a l f a t t y a c i d requirements f o r r eproduction 22 2.6.4 Vitamin E 25 2.6.4.1 Chemistry of tocopherols 25 2.6.4.2 Function of v i t a m i n E 27 2.6.4.3 M o b i l i z a t i o n and requirement of v i t a m i n E f o r reproduction 30 3 MATERIALS AND METHODS 38 3.1 L o c a t i o n and time 38 3.2 D i e t s 38 3.2.1 Formulation 38 3.2.2 Manufacturing 41 3.3 P a r e n t a l f i s h 43 3.3.1 H i s t o r y of the stock 43 3.3.2 S e l e c t i o n of f i s h f o r the experiment 44 3.4 Environmental c o n d i t i o n s f o r broodstock 45 3.4.1 Tanks 45 v i 3.4.2 L i g h t i n g and photoperiod 45 3.4.3 Temperature 4 6 3.4.4 Water chemistry 47 3.5 Feeding regime 47 3.6 Growth of broodstock 50 3.7 Spawning 50 3.8 F e r t i l i z a t i o n r a t e and measurement of water-hardened eggs 54 3.9 Incubation of eggs 54 3.10 Growth and s u r v i v a l of f r y 56 3.11 C o l l e c t i o n of f r y and u n f e r t i l i z e d eggs f o r measurement and/or a n a l y s i s 58 3.12 Analyses of eggs, f r y and/or d i e t s 59 3.12.1 Vitamin E analyses 59 3.12.2 Dry matter and selenium 60 3.12.3 L i p i d and f a t t y a c i d analyses 60 3.12.4 A n a l y s i s of d i e t s 63 3.13 E v a l u a t i o n of methods 63 3.13.1 E v a l u a t i o n of egg r i n s e s and extenders 63 3.13.2 E v a l u a t i o n of number of f r y t o r a i s e i n each r e a r i n g u n i t 64 3.14 S t a t i s t i c a l a n a l y s i s 64 4 RESULTS 68 4.1 Feed composition 68 4.2 Water temperature 71 4.3 Water chemistry 73 4.4 Feed fed and feed e f f i c i e n c y 74 4.5 Growth of broodstock 81 4.6 Spawning 83 4.6.1 Time of spawning 83 4.6.2 Number of r i p e males and females 92 4.6.3 A t y p i c a l spawning 92 4.7 Fecundity and egg s i z e (diameter and weight) 94 4.8 Embryonic s u r v i v a l 102 4.9 Egg and f r y composition 109 4.9.1 Vitamin E 109 4.9.2 L i p i d and dry matter 115 4.9.3 F a t t y a c i d composition 122 4.9.4 Selenium 133 4.9.5 Composition of w i l d A r c t i c c h a r r eggs 133 4.10 Fry s u r v i v a l and growth 136 5 DISCUSSION 141 5.1 Growth of broodstock and feeding a c t i v i t y 141 5.2 R e l a t i o n of f e c u n d i t y t o r a t i o n and weight of female 147 5.3 Spawning time and p r o p o r t i o n of f i s h t h a t spawned 150 5.4 Egg s i z e 153 5.5 V a r i a t i o n i n embryonic s u r v i v a l 154 5.6 L i p i d and f a t t y a c i d compositions of eggs 155 5.7 V i t a m i n E content of eggs 163 5.8 Fry s u r v i v a l and growth 6 CONCLUSIONS 7 LITERATURE CITED 8 APPENDICES v i i i LIST OF TABLES Page Table 1. Formulation of experimental d i e t s . 39 Table 2. Composition of the d i e t s . 69 Table 3. Water chemistry data f o r each tank. 75 Table 4. Water chemistry data f o r each sampling day. 76 Table 5. Feed f e d , weight gain and feed e f f i c i e n c y f o r each tank of f i s h fed f o r 71 days i n Year 1 and 239 days i n Year 2. 77 Table 6. I n i t i a l weight d i s t r i b u t i o n s of f i s h i n each tank. 82 Table 7. I n i t i a l and f i n a l mean and range i n weight of males and females fed each d i e t during the feeding p e r i o d i n Year 1. 84 Table 8. I n i t i a l and f i n a l mean and range i n weight of males and females fed each d i e t d u r i n g the feeding p e r i o d i n Year 2. 85 Table 9. S p e c i f i c growth r a t e of female and male f i s h fed the d i e t s i n Year 1 and 2. 86 Table 10. Number of male and female c h a r r which l o s t or gained weight throughout Year 1 or 2. 87 Table 11. Number of females which spawned duri n g the 14-week spawning p e r i o d i n Year 1 and the 11- week spawning p e r i o d i n Year 1. 88 Table 12. Number of females and males which produced eggs (spawned) and m i l t ( r i p e ) i n Year 1. 90 Table 13. Number of females and males which produced eggs (spawned) and m i l t ( r i p e ) i n Year 2. 91 Table 14. Number of males which produced m i l t ( r i p e ) i n Year 1 which d i d or d i d not produce m i l t i n Year 2. 93 Table 15. Egg production parameters i n Year 1. 95 Table 16. Egg production parameters i n Year 2. 96 IX Table 17. Egg weight and diameter of water-hardened preserved eggs i n Years 1 and 2. 98 Table 18. F e r t i l i z a t i o n and embryonic s u r v i v a l i n Year 1. 103 Table 19. F e r t i l i z a t i o n and embryonic s u r v i v a l i n Year 2. 105 Table 20. Vitamin E c o n c e n t r a t i o n and content of eggs i n Years 1 and 2 and f r y i n Year 2. I l l Table 21. T o t a l l i p i d and dry matter contents of eggs i n Year 1. 116 Table 22. T o t a l l i p i d and dry matter contents of eggs i n Year 2. 118 Table 23. S e l e c t e d major f a t t y acids i n the t o t a l l i p i d of the d i e t s . 123 Table 24. T o t a l s f o r f a m i l i e s of f a t t y a c i d s i n the t o t a l l i p i d of the d i e t s . 124 Table 25. S e l e c t e d major f a t t y acids i n the n e u t r a l l i p i d s of eggs. 129 Table 26. T o t a l s of f a m i l i e s of 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 s of eggs. 130 Table 27. S e l e c t e d major f a t t y acids i n the p o l a r l i p i d s of eggs. 131 Table 28. T o t a l s of f a m i l i e s of f a t t y acids i n the p o l a r l i p i d s of eggs. 132 Table 29. S e l e c t e d major f a t t y acids i n the p o l a r l i p i d s of eggs from A r c t i c c h a r r from Tree R i v e r , NWT. 134 Table 30. T o t a l s of f a m i l i e s of f a t t y a c i d s i n the p o l a r l i p i d s of eggs from A r c t i c c h a r r from Tree R i v e r , NWT. 135 Table 31. Fry s u r v i v a l and s p e c i f i c growth r a t e when stocked at d i f f e r e n t numbers. 137 Table 32. Fry s u r v i v a l and s p e c i f i c growth r a t e f o r 6 weeks at 10°C i n Year 1. 138 Table 33. Fry s u r v i v a l and s p e c i f i c growth r a t e f o r 6 weeks at 10°C i n Year 2. 139 X LIST OF FIGURES Fig u r e 1. Figur e 2. Figure 3. Figur e 4. Figur e 5. Fig u r e 6. Figur e 7. Fig u r e 8. Fig u r e 9. Fig u r e 10. Fig u r e 11. Fig u r e 12. Fig u r e 13. Fi g u r e 14. Sy n t h e s i s , d e s a t u r a t i o n and e l o n g a t i o n of f a t t y a c i d s i n f i s h . Forms of v i t a m i n E. Experimental plan showing crossover of d i e t s fed i n Year 1 and Year 2. Flow diagram showing analyses conducted on eggs and/or f r y i n Year 1 and/or Year 2. Mean weekly water temperatures during Year 1 and Year 2. Egg weight i n a) Year 1 and b) Year 2. T o t a l number of eggs produced i n a) Year 1 and b) Year 2. R e l a t i o n s h i p of t o t a l number of eggs produced and f i s h weight i n a) Year 1 and b) Year 2. F e r t i l i z a t i o n and s u r v i v a l of eggs t o the eyed, hatch and swimup stages i n Year 1. F e r t i l i z a t i o n and s u r v i v a l of eggs t o the eyed, hatch and swimup stages i n Year 2. Vitamin E concen t r a t i o n of eggs i n a) Year 1 and b) Year 2. Vitami n E content of eggs i n a) Year 1 and b) Year 2. Vitamin E concentration and content of f r y i n Year 2. T o t a l l i p i d i n eggs i n Year 1 (a) and b) t o t a l , c) n e u t r a l and d) p o l a r l i p i d i n Year 2. Fi g u r e 15. R e l a t i o n s h i p of a) n e u t r a l l i p i d s and b) 16:ln7 i n n e u t r a l l i p i d s on s u r v i v a l t o the eyed stage i n Year 2. Fig u r e 16. R e l a t i o n s h i p of a) n e u t r a l l i p i d s and b) 16:ln7 i n n e u t r a l l i p i d s on s u r v i v a l t o hatch i n Year 2. Page 19 21 49 52 72 97 100 101 104 106 112 113 114 117 119 120 x i F i g u r e 17. R e l a t i o n s h i p of a) n e u t r a l l i p i d s and b) 16:ln7 i n n e u t r a l l i p i d s on s u r v i v a l t o swimup i n Year 2. 121 F i g u r e 18. Comparison of sa t u r a t e d , n7, n9, n i l monoenoic a c i d s , PUFA and HUFA of the t o t a l l i p i d s of the d i e t s w i t h the n e u t r a l and p o l a r l i p i d s of the eggs i n Year 2. 125 F i g u r e 19. Comparison of n3 f a t t y acids of the t o t a l l i p i d s of the d i e t s w i t h the n e u t r a l and p o l a r l i p i d s of the eggs i n Year 2. 126 F i g u r e 20. Comparison of n6 f a t t y acids of the t o t a l l i p i d s of the d i e t s w i t h the n e u t r a l and p o l a r l i p i d s of the eggs i n Year 2. 127 x i i LIST OF APPENDICES Page Appendix l a . F a t t y a c i d composition of the t o t a l l i p i d s of the d i e t s . 191 Appendix l b . F a t t y a c i d composition of the n e u t r a l l i p i d s of Labrador A r c t i c c h a r r eggs i n Year 2. 192 Appendix l c . F a t t y a c i d composition of the p o l a r l i p i d s of Labrador A r c t i c c h a r r eggs i n Year 2. 193 Appendix I d . F a t t y a c i d composition of the n e u t r a l and p o l a r l i p i d s of Tree R i v e r , NWT A r c t i c c h a r r eggs. 194 Appendix 2a. Mean weekly water temperature i n Year 1 and Year 2. 195 Appendix 2b. T o t a l g a i n , feed fed and feed e f f i c i e n c y of each tank of f i s h f o r each p e r i o d i n Year 1. 196 Appendix 2c. T o t a l g a i n , feed fed and feed e f f i c i e n c y of each tank of f i s h f o r each p e r i o d i n Year 2. 197 Appendix 3a. Method of e x t r a c t i o n and a n a l y s i s of v i t a m i n E i n eggs and f r y . 198 Appendix 3b. Method of e x t r a c t i o n and a n a l y s i s of t o t a l l i p i d i n eggs and d i e t s . 201 Appendix 4. 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 v a r i o u s parameters on f e c u n d i t y , f e r t i l i z a t i o n and s u r v i v a l of eggs i n Year 1 and Year 2. 203 x i i i ACKNOWLEDGEMENTS Many people c o n t r i b u t e d t o the completion of the research f o r t h i s t h e s i s . I would l i k e t o extend s p e c i a l thanks t o Randy Olson and C o l l e e n Engel of Rockwood Aquaculture Research Centre (RARC) f o r t h e i r on-going a s s i s t a n c e i n spawning f i s h and c a r i n g f o r broodstock, eggs and f r y . Thanks are a l s o extended t o other RARC s t a f f , Murray Foster and Danny Chaput. Thanks t o Jim Gibson who a s s i s t e d w i t h chemical analyses and hatchery work and t o Ron Lypka and Bob Jes t a d t i n the computer department who always helped me out when I had problems w i t h graphics and computer programming. Thanks a l s o t o Bob Hunt f o r doing min e r a l analyses and t o Bob Evans f o r doing h i s t o l o g i c a l examination of f r y . I wish t o thank Dr. B e r y l March who always took the time t o di s c u s s my work and o f f e r advice. I thank f r i e n d s and colleagues who provided advice, a s s i s t a n c e and moral support, e s p e c i a l l y Roberta York and Michael Papst. Although I do not normally have the op p o r t u n i t y t o work w i t h other f i s h n u t r i t i o n i s t s , I enjoyed having f r i e n d s i n Vancouver l i k e Heather Ashton and Rob Roy, who shared a common i n t e r e s t i n broodstock n u t r i t i o n . I am indebted t o Management at the Freshwater I n s t i t u t e , Department of F i s h e r i e s and Oceans, who supported my nine-month leave t o attend the U n i v e r s i t y of B r i t i s h Columbia and complete the course work towards my degree. This research was supported by the Department of F i s h e r i e s and Oceans. I thank Hoffmann- LaRoche f o r conducting v i t a m i n analyses on the feed i n g r e d i e n t s . L a s t but c e r t a i n l y not l e a s t , I am t h a n k f u l f o r the c o n t i n u i n g support and encouragement from my f a m i l y , e s p e c i a l l y my husband, Edward. 1 INTRODUCTION As the aquaculture i n d u s t r y i n Canada matures, there i s the need t o develop the technology t o r a i s e d i f f e r e n t species of f i s h and thus a l l o w producers t o r a i s e d i f f e r e n t products f o r which they can develop markets. Beginning i n 1978, stocks of A r c t i c c h a r r [Salvelinus alpinus (L.)] were c o l l e c t e d as eggs from w i l d f i s h i n the Northwest T e r r i t o r i e s (NWT) and Labrador and t r a n s f e r r e d t o the Rockwood Aquaculture Research Centre (RARC), Gunton, Manitoba. This f a c i l i t y became the major source of A r c t i c c h a r r f o r producers across Canada. T r a v e l c o s t s t o o b t a i n eggs from w i l d stocks of A r c t i c c h a r r , u n c e r t a i n spawning times and l a c k of d i s e a s e - f r e e w i l d stocks make i t i m p r a c t i c a l or i m p o s s i b l e t o continue o b t a i n i n g eggs from w i l d s t o c k s . This makes i t necessary f o r producers t o maintain t h e i r own broodstocks and they w i l l r e q u i r e i n f o r m a t i o n on t h e i r d i e t a r y and environmental requirements. H a t c h a b i l i t y of A r c t i c charr from broodstock which have been r a i s e d i n c a p t i v i t y has v a r i e d w i t h the s p e c i f i c stock. H a t c h a b i l i t y of the stocks o r i g i n a t i n g from Nauyuk Lake i n the NWT and from Norway has been high (Tabachek and de March 1990). Since these stocks are r a i s e d i n a n o n - c e r t i f i e d b u i l d i n g at RARC, most producers cannot o b t a i n permits t o t r a n s p o r t eggs from those stocks across p r o v i n c i a l boundaries. One stock from the F r a s e r R i v e r , Labrador i s r a i s e d i n a c e r t i f i e d b u i l d i n g at 2 RARC and s i n c e these eggs can be t r a n s p o r t e d across p r o v i n c i a l boundaries, they have been d i s t r i b u t e d widely t o producers throughout Canada. Unfortunately, embryonic s u r v i v a l of t h i s stock has been v a r i a b l e and o f t e n low when fed the same d i e t and r a i s e d under the same environmental c o n d i t i o n s as the Nauyuk Lake and Norway stocks at RARC. S u r v i v a l at f i r s t - f e e d i n g i s a l s o stock-dependent and some producers have experienced high m o r t a l i t y at f i r s t f eeding w i t h Labrador and Norway A r c t i c charr (Tabachek and de March 1991). Poor or u n c e r t a i n h a t c h a b i l i t y and s u r v i v a l of f r y f o r c e s producers t o maintain a l a r g e inventory of broodstock and makes plann i n g of production d i f f i c u l t . Therefore, there i s a need t o improve both the l e v e l and p r e d i c t a b i l i t y of embryonic s u r v i v a l and s u r v i v a l at f i r s t - f e e d i n g of the Labrador stock of A r c t i c c h a r r . D i f f e r e n c e s a l s o e x i s t e d i n the d u r a t i o n of the spawning p e r i o d w i t h the Labrador stock of A r c t i c c h a r r spawning over 4 t o 5 months w h i l e the other stocks spawned w i t h i n 6 weeks (Tabachek 1990). There i s a s c a r c i t y of i n f o r m a t i o n on the n u t r i t i o n a l requirements of f i s h of reproductive age and on A r c t i c c h a r r broodstock i n p a r t i c u l a r . Therefore, i t was not known i f some n u t r i e n t ( s ) were r e l a t e d t o the poor h a t c h a b i l i t y of Labrador A r c t i c c h a r r broodstock. 3 Maternal n u t r i t i o n a f f e c t s embryonic and f r y s u r v i v a l and can be i n f l u e n c e d by l e v e l s of d i e t a r y p r o t e i n and energy, carotenoids and s p e c i f i c minerals and vitamins (Luquet and Watanabe 1986). The 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 and v i t a m i n E a f f e c t h a t c h a b i l i t y of both f i s h (Watanabe 1985) and p o u l t r y (Jensen 1968). The l e v e l of unsaturated f a t t y a c i d s i n f l u e n c e d the v i t a m i n E requirement of rainbow t r o u t (Oncorhynchus mykiss) f r y (Watanabe et al. 1981a). J u v e n i l e A r c t i c c h a r r r e q u i r e a high l e v e l of d i e t a r y p r o t e i n (44-54%) and l i p i d (15-20%) (Tabachek 1986). J u v e n i l e A r c t i c charr may have a requirement f o r a high l e v e l of e s s e n t i a l f a t t y acids (Yang and Dick 1992) but the requirements f o r A r c t i c charr broodstock are unknown. I t was hypothesized t h a t i f A r c t i c c h a r r broodstock r e q u i r e d a high l e v e l of d i e t a r y l i p i d and/or unsaturated f a t t y a c i d s , they might a l s o have a requirement f o r a high l e v e l of d i e t a r y v i t a m i n E. The o b j e c t i v e of t h i s research was t o examine the e f f e c t s and i n t e r a c t i v e e f f e c t s of both d i e t a r y l i p i d and v i t a m i n E on the reproduction of A r c t i c c h a r r . Reproductive parameters of i n t e r e s t i n c l u d e d embryonic s u r v i v a l , growth and s u r v i v a l of f r y , f e c u n d i t y , p r o p o r t i o n of the p o p u l a t i o n t h a t matured and d u r a t i o n of the spawning p e r i o d . 4 LITERATURE REVIEW 2.1 N a t u r a l h i s t o r y of A r c t i c c harr A r c t i c c h a r r have a circumpolar d i s t r i b u t i o n throughout A r c t i c waters. In Canada, they are found i n Labrador, Newfoundland, throughout the Northwest and Yukon T e r r i t o r i e s (Johnson 1980). Populations a l s o occur i n New Brunswick, northern Quebec and northern Manitoba. There are many d i f f e r e n c e s i n the n a t u r a l l i f e h i s t o r y between some of these pop u l a t i o n s i n c l u d i n g d i f f e r e n c e s i n growth r a t e , l o n g e v i t y , f e c u n d i t y and age at ma t u r i t y . A r c t i c c h a r r used i n the research d e s c r i b e d i n t h i s t h e s i s o r i g i n a t e d from the Fr a s e r R i v e r , Labrador. Charr may make s e v e r a l migrations t o sea before they mature. Dempson and Green (1985) found t h a t Labrador A r c t i c c harr migrated t o sea f o r the f i r s t time when they were 78-191 mm i n length at 3 t o 7 years of age. Charr migrated t o sea i n May and e a r l y June, spent 2-3 months at sea and returned t o f r e s h water from the l a t t e r h a l f of J u l y through August. Larger f i s h returned f i r s t w i t h a higher p r o p o r t i o n of females r e t u r n i n g e a r l y i n the run. Annual l e n g t h increments were 68 mm between 4-5 years, 103 mm between 5-6 years and 71 mm between 6-7 years of age. While 50% of males matured at 5 years of age when they were 245 mm i n length, females matured at an o l d e r age w i t h 50% mature at 7 years when they were 381 mm i n l e n g t h and 75% mature at 8 years. They estimated t h a t 60% of the females r e t u r n i n g t o f r e s h water would spawn t h a t year. 5 This i s i n c o n t r a s t t o ch a r r i n Nauyuk and Willow Lakes, NWT which spawn one year a f t e r r e t u r n i n g from the sea and may spend up t o two years i n f r e s h water before r e t u r n i n g t o the sea (Johnson 1980). Anadromous A r c t i c c h a r r are i t e r o p a r o u s , spawning i n a l t e r n a t e years. However, i n nonanadromous populations they may spawn every year with only one anadromous stock i n Norway spawning an n u a l l y . While some tagged c h a r r i n an anadromous p o p u l a t i o n were found t o spawn every year, others i n the same po p u l a t i o n were not recaptured at the same spawning s i t e f o r two t o f i v e years. I t i s not known i f the l a t t e r f i s h d i d not spawn durin g t h a t i n t e r v a l or i f they spawned at a d i f f e r e n t s i t e but the former seems more probable. A r c t i c c h a r r may spawn s e v e r a l times d u r i n g t h e i r l i f e t i m e but frequency of spawning may decrease w i t h i n c r e a s i n g age. In Labrador, water temperature during l a t e J u l y and August, when c h a r r were r e t u r n i n g t o f r e s h water, v a r i e d between years but ranged from 8.7 t o 12.9°C (Dempson and Greene 1985). Spawning occurred during a three week p e r i o d i n October at a water temperature of 1-3 °C. I t occurred i n areas of f i n e and coarse sand and 40-50 mm g r a v e l i n 1.5-2.0 m of water. Fecundity of 7-13 year olds ranged from 2316-9245 eggs w i t h a mean of 4665 ± 434 ( s t d . dev.). Egg diameter was 4.5 mm. 6 The type of food consumed changes s e a s o n a l l y and an n u a l l y according t o a v a i l a b i l i t y . Small charr are benthic feeders and i n f r e s h water they feed on i n v e r t e b r a t e s such as chironomids, amphipods, copepods and molluscs (Johnson 1980). When c h a r r migrate t o sea, they remain along the c o a s t l i n e and are p e l a g i c feeders. When Labrador c h a r r migrate t o the sea, p r i n c i p a l food items i n c l u d e amphipods (Parathemisto l i b e l l u l a ) , euphasid shrimp (Meganyctiphanes norvegica) and f i s h such as c a p e l i n (Mallotus v i l l o s u s ) , sand lance (Ammodytes americanus) and young mailed s c u l p i n (Triglops p i n g e l i ) . During spawning, eggs are consumed by c h a r r t h a t may or may not have been i n v o l v e d i n spawning. Charr can a l s o be c a n n i b a l i s t i c . Postspawning c h a r r consume l i t t l e , i f any, food i n f r e s h water and they can be very emaciated when they r e t u r n t o sea the f o l l o w i n g year. Dempson and Green (1985) found few A r c t i c c h a r r i n Labrador t h a t were o l d e r than 12 years w i t h the o l d e s t f i s h being 18 yea r s . In other l o c a t i o n s throughout the world, charr may l i v e t o be 30-33 years of age. 2.2 A r c t i c c h a r r c u l t u r e i n Canada Beginning i n 1978, stocks of A r c t i c c h a r r eggs were c o l l e c t e d i n the w i l d during s e v e r a l c o l l e c t i o n s and t r a n s f e r r e d t o the Rockwood Aquaculture Research Centre (RARC), Gunton, Manitoba. They were c o l l e c t e d i n the w i l d from Nauyuk Lake, N.W.T. i n 1978 (Papst and Hopky 1984), Fraser R i v e r , Labrador i n 7 1980 (Baker 1981, 1983), 1981 and 1984 (de March 1991) and Tree R i v e r , NWT i n 1988. They were a l s o c o l l e c t e d from a hatchery stock from Sunndalsora, Norway i n 1980 (Baker 1983). P r i m a r i l y , i t has been the progeny from the Fraser R i v e r , Labrador stock c o l l e c t e d i n 1984 t h a t have been d i s t r i b u t e d from a sm a l l c e r t i f i e d f a c i l i t y a t RARC. They have been t r a n s f e r r e d from RARC t o producers, researchers and government agencies i n every province and t e r r i t o r y i n Canada (Olson, p e r s o n a l communication). When incubated at 6°C, s u r v i v a l of eggs c o l l e c t e d from w i l d stocks was 88% t o the eyed stage f o r the Nauyuk Lake, NWT stock (Papst and Hopky 1984), 93% t o swimup f o r the Fraser R i v e r , Labrador stock c o l l e c t e d i n 1980 (Baker 1981) and 94% t o eye and 87% t o swimup f o r the Tree R i v e r , NWT stock (Olson, personal communication). While the h a t c h a b i l i t y of eggs c o l l e c t e d i n the w i l d from the Nauyuk Lake, NWT stock was hig h , the f i r s t g eneration domestic stock produced eggs which had only 17% s u r v i v a l t o the eyed stage w i t h 65% of eggs being u n f e r t i l i z e d (Papst and Hopky 1984). Changes t o the r e a r i n g and spawning techniques have increased the h a t c h a b i l i t y of t h i s stock t o 73- 98% ( K r i e g e r et al. 1988) wit h c o n s i s t e n t h a t c h a b i l i t y between f i s h and between spawning years. The l a t t e r authors showed t h a t the eggs of A r c t i c charr over-ripened q u i c k l y and the chance of f e r t i l i z a t i o n decreased s i g n i f i c a n t l y between the periods of 4-7 days and 11-14 days a f t e r o v u l a t i o n . They a l s o showed t h a t 8 f e r t i l i z a t i o n improved i f the males had been r a i s e d i n c o l d water (6.5°C) compared t o warm water (8-17°C) p r i o r t o the f i r s t year of spawning. As w i t h the Nauyuk Lake stock, s u r v i v a l of eggs from the Norway stock i s high (> 80% eyed, Olson, personal communication). While h a t c h a b i l i t y of the Nauyuk Lake stock has improved, s u r v i v a l of stocks of A r c t i c c h a r r o r i g i n a t i n g from the F r a s e r R i v e r , Labrador have remained h i g h l y v a r i a b l e between f i s h and between spawning years at RARC. For example, s u r v i v a l t o the eyed stage f o r one stock of Labrador c h a r r was 50%, 72% and 35% i n three successive years (Tabachek and de March 1990) and 68% the f o l l o w i n g year (Olson, personal communication). There has been v a r y i n g success among producers who have r a i s e d Labrador c h a r r t o mat u r i t y (Tabachek and de March 1991). H a t c h a b i l i t y of the Labrador stock of A r c t i c c h a r r has been shown t o be under maternal r a t h e r than p a t e r n a l i n f l u e n c e . When de March (1992) separated the eggs from i n d i v i d u a l Labrador females i n t o groups and f e r t i l i z e d each group w i t h the m i l t from a d i f f e r e n t male, the p a t t e r n of embryonic s u r v i v a l always i n d i c a t e d t h a t s u r v i v a l was i n f l u e n c e d only by the female. This o b s e r v a t i o n suggests t h a t the n u t r i t i o n a l s t a t u s and/or environmental c o n d i t i o n s of the female should be i n v e s t i g a t e d . When S r i v a s t a v a (1991) c o l l e c t e d eggs from Labrador A r c t i c c h a r r which had spawned f o r the f i r s t time, he found t h a t those t h a t spawned i n the middle of the spawning season produced eggs t h a t weighed s i g n i f i c a n t l y more, and had higher p r o t e i n , l i p i d , 9 carbohydrate, ash and amino acids than those t h a t spawned e i t h e r 4 weeks e a r l i e r or 4 weeks l a t e r . In a d d i t i o n , eggs from t h i s middle group a l s o reached the eyed stage, hatched e a r l i e r and had fewer deformed f r y than those spawned e a r l i e r or l a t e r . Age of m a t u r i t y and f e c u n d i t y of stocks r a i s e d f o r one or two generations i n c a p t i v i t y vary from one stock t o another. The Nauyuk Lake stock produced 1200-2000 eggs w h i l e the Labrador stocks produced 3000-4000 eggs and the Norway stock produced 3000 eggs (Tabachek and de March 1990). The Labrador and Norway stocks s t a r t t o spawn at 3 years of age, w h i l e the Nauyuk Lake stock does not s t a r t t o spawn u n t i l at l e a s t 4 years of age w i t h most f i s h spawning f o r the f i r s t time at 5-6 years of age. A l l stocks spawn f o r 4-5 years (Olson, personal communication). The m o r t a l i t y r a t e begins t o increase at 6 years of age and f i s h are g e n e r a l l y euthanized at 7-8 years of age. Nauyuk Lake A r c t i c c harr f i n g e r l i n g s r a i s e d at 12°C gained s i g n i f i c a n t l y more when fed a d i e t c o n t a i n i n g 54% p r o t e i n and 20% l i p i d compared t o those fed a d i e t c o n t a i n i n g 44% p r o t e i n and 15% l i p i d (Tabachek 1986). Feeding d i e t s c o n t a i n i n g 34% p r o t e i n and/or 10% l i p i d r e s u l t e d i n s i g n i f i c a n t l y lower weight g a i n compared t o feeding d i e t s c o n t a i n i n g 44% p r o t e i n and/or 15% l i p i d . The requirements f o r rainbow t r o u t have been reported as 34% p r o t e i n i n d i e t s with 15-20% l i p i d when r a i s e d at 15-18°C (Takeuchi et al. 1978) and as 40% p r o t e i n i n d i e t s w i t h 10% 10 l i p i d when r a i s e d at 16-27°C ( S a t i a 1974). The e s s e n t i a l f a t t y a c i d requirements of A r c t i c charr are discussed i n S e c t i o n 2.6.3. There has been l i t t l e other research reported on the n u t r i t i o n a l requirements of j u v e n i l e or broodstock A r c t i c c h a r r . S u r v i v a l of swimup f r y v a r i e s w i t h the stock ( K r i e g e r 1987). S u r v i v a l and growth of the Labrador and Norway stocks improved when r e a r i n g temperature at f i r s t feeding was i n c r e a s e d from 7 t o 10°C (Tabachek 1992) w h i l e there was only a s l i g h t improvement i n s u r v i v a l and growth when fed a semimoist d i e t from one manufacturer compared t o a dry d i e t from another manufacturer. 2.3 Physiology of reproduction The physiology of reproduction i n t e l e o s t f i s h e s has been reviewed i n d e t a i l (Nagahama 1983, Ng and I d l e r 1983). In summary, environmental f a c t o r s such as photoperiod and temperature act upon the hypothalamus which i s s t i m u l a t e d t o s e c r e t e gonadotropin-releasing hormone (GRH). This hormone act s upon the p i t u i t a r y causing i t t o s e c r e t e the gonadotropins a) l u t e n i z i n g hormone (LH) and b) f o l l i c l e - s t i m u l a t i n g hormone (FSH). Thecal c e l l s are s t i m u l a t e d t o s y n t h e s i z e t e s t o s t e r o n e from c h o l e s t e r o l . FSH s t i m u l a t e s the development of the f o l l i c l e s and s t i m u l a t e s granulosa c e l l s surrounding each oocyte t o produce the enzyme t h a t converts t e s t o s t e r o n e t o estrogen. Estrogen i s t r a n s p o r t e d t o the l i v e r where i t a c t i v a t e s 11 hepatocytes t o s y n t h e s i z e v i t e l l o g e n i n . V i t e l l o g e n i n i s t r a n s p o r t e d t o the ovary where i t i s s e l e c t i v e l y taken up by oocytes through m i c r o p i n o c y t o s i s . V i t e l l o g e n i n i s taken up by the f o l l i c l e c e l l s , t r ansported across the m i c r o v i l l i of the zona p e l l u c i d a i n t o the m i c r o v i l l i i n the zona r a d i a t a of the oocyte. N u t r i e n t s such as vitamins and minerals are a l s o taken up by the oocyte. Most v i t e l l o g e n i n , a g l y c o l i p o p h o s p h o p r o t e i n , i s s y n t h e s i z e d i n the l i v e r by exogenous v i t e l l o g e n e s i s but there i s evidence t h a t a p o r t i o n i s synthesized i n the ovary by endogenous v i t e l l o g e n e s i s . I t i s hydrolyzed i n the oocyte t o form p h o s v i t i n (phosphoprotein) and l i p o v i t e l l i n ( p r o t e o l i p i d which i s 80% p r o t e i n and 20% l i p i d ) . The l i p i d of l i p o v i t e l l i n c o n s i s t s of both p o l a r and n e u t r a l l i p i d s which Léger et a l . (1981) found were 35% t r i a c y l g l y c e r i d e s . Yolk granules, c o n t a i n i n g l i p o v i t e l l i n and p h o s v i t i n , form i n the periphery of the oocyte dur i n g the primary stage and fuse t o form y o l k spheres d u r i n g the secondary stage. They coalesce t o form a s i n g l e y o l k d u r i n g l a t e v i t e l l o g e n e s i s . O i l d r o p l e t s appear at the nucleus, migrate t o the periphery and merge t o form l a r g e r o i l d r o p l e t s . The o i l d r o p l e t s c o n s i s t of t r i a c y l g l y c e r i d e s w i t h t r a c e s of c h o l e s t e r o l and c h o l e s t e r y l e s t e r s (Luquet and Watanabe 1986). When oocytes have matured and f i r s t meiosis ends, the m i c r o v i l l i of the f o l l i c l e c e l l s withdraw and the oocytes are r e l e a s e d . Second meiosis then continues t o metaphase and eggs are ready to be released from the body and f e r t i l i z e d . 12 2.4 Feed i n t a k e and reproduction 2.4.1 Feed i n t a k e The e f f e c t of l e v e l of feed i n t a k e ( r a t i o n ) on maturation, f e c u n d i t y , egg s i z e and s u r v i v a l of eggs and f r y has been reviewed (Roley 1983, Springate et a l . 1985, Hardy 1985 and Luquet and Watanabe 1986). Orr et a l . (1982) found that a d u l t rainbow t r o u t consumed more feed and had higher weight gain when fed w i t h demand feeders compared t o those fed by hand. F i s h feeding at high r a t e s produced l a r g e r eggs and had higher f e c u n d i t y but lower r e l a t i v e f e c u n d i t y (eggs per body weight) than those who were hand-fed. There was no d i f f e r e n c e i n s u r v i v a l of eggs t o the eyed stage. Knox et al. (1988) found t h a t feeding at f u l l r a t i o n (0.7% body weight) r e s u l t e d i n rainbow t r o u t producing progeny which were s i g n i f i c a n t l y l a r g e r at the eyed egg, y o l k sac and swimup f r y stages than f i s h fed at h a l f - r a t i o n (0.35% body we i g h t ) . Swimup f r y from parents fed the f u l l r a t i o n had s i g n i f i c a n t l y higher p r o t e i n and l i p i d l e v e l s compared t o the h a l f - s a t i a t i o n group. However, r a t i o n had no e f f e c t on the f a t t y a c i d composition of p o l a r or n e u t r a l f a t t y a c i d s . Springate et al. (1985) reported on another p a r t of the experiment of Knox et al. (1988) and s t a t e d t h a t rainbow t r o u t fed at f u l l r a t i o n spawned 2-3 weeks e a r l i e r than those fed at h a l f r a t i o n . The c o n c e n t r a t i o n of v i t e l l o g e n i n and t e s t o s t e r o n e i n the serum were s i g n i f i c a n t l y higher at spawning i n f i s h fed 13 at f u l l r a t i o n . A l l f i s h fed at f u l l r a t i o n spawned w h i l e 11% of those fed at h a l f r a t i o n d i d not spawn. In a d d i t i o n , f i s h fed at f u l l r a t i o n produced s i g n i f i c a n t l y more eggs (22% more) and these eggs were s i g n i f i c a n t l y l a r g e r than those from f i s h fed at h a l f r a t i o n . H i s t o l o g i c a l examination showed t h a t there were s i g n i f i c a n t l y more a t r e t i c oocytes i n f i s h fed at h a l f r a t i o n . R a tion d i d not a f f e c t f e r t i l i z a t i o n r a t e or s u r v i v a l t o the eyed stage. Roley (1983) fed rainbow t r o u t t o s a t i a t i o n and h a l f - s a t i a t i o n f o r one year p r i o r t o spawning. At c o o l water temperature, both egg s i z e and s u r v i v a l i ncreased f o r the h a l f - s a t i a t i o n group compared t o the s a t i a t i o n group. Feeding t o s a t i a t i o n i n c r e a s e d the v a r i a t i o n i n embryonic s u r v i v a l . In c o n t r a s t t o Orr et a l . (1982), the high l e v e l of feeding r e s u l t e d i n s m a l l e r eggs w i t h low s u r v i v a l although s u r v i v a l was independent of egg s i z e . In c o n t r a s t t o Knox et a l . (1988), spawning was delayed but occurred w i t h i n a s h o r t e r i n t e r v a l i n f i s h which had been fed t o s a t i a t i o n but feeding l e v e l had no e f f e c t on the number of f i s h t h a t spawned. Jones and Bromage (1987) fed rainbow t r o u t r a t i o n s v a r y i n g between 0.4 and 1.5% of body weight and found t h a t w h i l e r a t i o n had a s i g n i f i c a n t e f f e c t on fe c u n d i t y beyond the e f f e c t of r a t i o n on body weight, i t had no e f f e c t on egg diameter. This 14 i s i n c o n t r a s t t o the f i n d i n g s of both Orr et a l . (1982) and Roley (1983). 2.4.2 S t a r v a t i o n p r i o r t o the spawning p e r i o d Ashton (1991) found no d i f f e r e n c e i n f a t t y a c i d composition or s u r v i v a l of Chinook salmon {Oncorhynchus tshawytscha) t o the eyed stage when broodstock were starved f o r 7 and 14 days before spawning. S t a r v a t i o n of rainbow t r o u t f o r 40 days before spawning d i d not a f f e c t f e c u n d i t y , h a t c h a b i l i t y , egg weight, egg diameter or proximate composition (Ridelman et al. 1984). 2.5 P r o t e i n and energy The i n f l u e n c e of d i e t a r y p r o t e i n and energy l e v e l s on re p r o d u c t i o n has been reviewed by Hardy (1985) and Luquet and Watanabe (1986). I t was b e l i e v e d t h a t requirements f o r p r o t e i n and energy might increase during the time of o v a r i a n development. However, Hardy (1985) pointed out t h a t somatic growth ceases duri n g ovarian development and t h a t there may not be an o v e r a l l increase i n the requirements f o r p r o t e i n and energy. Roley (1983) fed rainbow t r o u t d i e t s c o n t a i n i n g p r o t e i n l e v e l s of 27 t o 57% at 3.8 k c a l metabolizable energy (ME ) /g d i e t f o r 8 months p r i o r t o t h e i r f i r s t spawning a t 2 years of age. There were no s i g n i f i c a n t d i f f e r e n c e s i n h a t c h a b i l i t y , egg diameter, f e c u n d i t y , d u r a t i o n of spawning or prespawning 15 m o r t a l i t y . He concluded t h a t the requirement was 37 t o 47% p r o t e i n at 3.8 k c a l ME/g. Washburn (1989) i n v e s t i g a t e d the e f f e c t s of d i e t a r y p r o t e i n and energy on rainbow t r o u t broodstock and found t h a t feeding high d i e t a r y p r o t e i n (58%) and energy (3.23 kcal/g) concentrations r e s u l t e d i n s i g n i f i c a n t l y lower h a t c h a b i l i t y compared t o feeding a d i e t t h a t was low i n p r o t e i n (30%) and energy (2.64 k c a l / g ) . This l a t t e r combination was below the l e v e l of energy assessed by Roley (1983). Smith et a l . (1979) fed rainbow t r o u t f o r 6 months before t h e i r f i r s t spawning season and then through 2 more consecutive spawning seasons. Test d i e t s contained low, medium and high l e v e l s of p r o t e i n (36, 42 and 48%) and energy (2.52, 2.85 and 3.44 k c a l / g ) . Feeding r a t e was 0.8% body weight/day u n t i l feed i n t a k e decreased as spawning approached. The high p r o t e i n - h i g h energy d i e t s had a p o s i t i v e e f f e c t on weight g a i n , f e c u n d i t y and egg s i z e but no s i g n i f i c a n t e f f e c t on s u r v i v a l t o the eyed stage. Long-term feeding of rainbow t r o u t from the f i n g e r l i n g stage through the second spawning season on d i e t s c o n t a i n i n g 35% p r o t e i n w i t h 16-18% l i p i d or 45% p r o t e i n w i t h 15% l i p i d (Takeuchi et al. 1981b) r e s u l t e d i n no s i g n i f i c a n t d i f f e r e n c e i n f e c u n d i t y , egg diameter or s u r v i v a l t o the eyed (90%) and hatch (87%) stages. This c o n f l i c t s w i t h the f i n d i n g s of Watanabe et al. (1984b) who observed poor h a t c h a b i l i t y when broodstock were 16 fed a d i e t c o n t a i n i n g 46% p r o t e i n and 15% l i p i d (57%) compared t o 36% p r o t e i n and 18% l i p i d (86%) or 28% p r o t e i n w i t h 21% l i p i d (70%). 2.6 L i p i d s , f a t t y acids and v i t a m i n E 2.6.1 Chemistry of l i p i d s and f a t t y a c i d s N e u t r a l l i p i d s i n c l u d e mono-, d i - and t r i a c y l g l y c e r i d e s (TAG), f r e e f a t t y a c i d s , c h o l e s t e r o l and c h o l e s t e r o l e s t e r s w i t h TAG p r o v i d i n g the major source of energy. P o l a r l i p i d s , which i n c l u d e phosphoglycerides, g l y c o l i p i d s , sphingomyelins and plasmolagens, are r e q u i r e d f o r the formation of c e l l u l a r and s u b c e l l u l a r membranes. F a t t y a c i d s are an i n t e g r a l p a r t of these l i p i d s . The p o s i t i o n of the f i r s t double bond from the methyl end of a f a t t y a c i d i s denoted by i t s "n" number w i t h n3 f a t t y a c i d s having t h e i r f i r s t double bond a f t e r the t h i r d carbon from the t e r m i n a l end. A d d i t i o n a l double bonds are i n t e r r u p t e d by one s a t u r a t e d carbon atom. Double bonds increase the f l e x i b i l i t y of the molecule and a l s o r e s u l t i n a decrease i n the m e l t i n g p o i n t . Both these f a c t o r s are important i n maintaining the f l e x i b i l i t y and f u n c t i o n of membranes, e s p e c i a l l y i n p o i k i l o t h e r m s which l i v e i n c o l d environments. This i s the reason there are high l e v e l s of unsaturated f a t t y acids i n the p o l a r l i p i d s of b i o l o g i c a l membranes i n f i s h . F a t t y a c i d s w i t h 2 t o 4 double bonds are c a l l e d polyunsaturated f a t t y acids (PUFA) w h i l e those 17 w i t h g r e a t e r than or equal t o 5 double bonds are r e f e r r e d t o as h i g h l y unsaturated f a t t y a c i d s (HUFA). In f i s h , PUFA c o n s i s t mainly of n6 f a t t y acids w h i l e HUFA c o n s i s t mainly of n3 f a t t y a c i d s . In TAG, saturated and monoenoic f a t t y a c i d s occupy p o s i t i o n s on the 1- (a) and 3- (y) carbon of the g l y c e r o l molecule, w h i l e PUFA or HUFA occupy the 2- (P) carbon p o s i t i o n . In p o l a r l i p i d s , monoenoic and saturated f a t t y a c i d s occupy the a p o s i t i o n and HUFA or PUFA occupy the p p o s i t i o n . Saturated f a t t y acids can be synthesized de novo from acetate ( C a s t e l l 1979). F a t t y acids t h a t cannot be synthesized but which are necessary f o r the he a l t h and re p r o d u c t i o n of the animal are considered t o be e s s e n t i a l f a t t y a c i d s which must be s u p p l i e d i n the d i e t . Since f i s h cannot s y n t h e s i z e 18:2n6 o r 18:3n3, the d i e t must supply these two f a t t y a c i d s as precursors f o r d e s a t u r a t i o n and el o n g a t i o n i n t o PUFA and HUFA or the d i e t must supply PUFA or HUFA (Figure 1). Desat u r a t i o n and e l o n g a t i o n of the n9, n6 and n3 s e r i e s of f a t t y a c i d s occurs through a common enzyme pathway of desaturases and elongases but w i t h s p e c i f i c enzymes f o r each p o s i t i o n (Figure 1). In mammals, the desaturase enzymes have a greater a f f i n i t y f o r the most unsaturated f a t t y a c i d a v a i l a b l e (Leray and P e l l e t i e r 1985). That i s , A5-desaturase has a greater a f f i n i t y f o r 20:4n3 than 20:3n6 which has greater a f f i n i t y than 20:2n9. Thus, there i s the a b i l i t y t o desaturate 20:2n9 t o 20:3n9 or 20:3n6 t o 20:4n6 but i n the presence of 20:4n3, A5-desaturase p r e f e r e n t i a l l y 18 converts 20:4n3 t o 20:5n3. I t i s speculated t h a t a s i m i l a r mechanism occurs i n f i s h and the presence of 20:3n9 i s used as an i n d i c a t o r of e s s e n t i a l f a t t y a c i d d e f i c i e n c y ( C a s t e l l et al. 1972). 2.6.2 D i g e s t i o n and absorption of l i p i d s and v i t a m i n E In the gut lumen, an emulsion of l i p i d and l i p i d - s o l u b l e compounds occurs on contact w i t h b i l e s a l t s and b i l e p h o spholipids produced by the l i v e r . In rainbow t r o u t , t r i a c y l g l y c e r i d e s at the surface of the emulsion are hydrolyzed through the a c t i o n of pancreatic l i p a s e (Léger 1985) and p o s s i b l y a c o l i p a s e . T r i a c y l g l y c e r i d e s are hydrolyzed i n the 1- and 3 - p o s i t i o n i n t o f r e e f a t t y acids and 2-monoacylglycerides. The h y d r o l y s i s of the f a t t y a c i d i n the 2 - p o s i t i o n a l s o occurs i f the a c y l c h a i n contains at l e a s t one double bond. This i s d i f f e r e n t from mammals and b i r d s where t h i s l a t t e r h y d r o l y s i s does not occur. M i c e l l e s , c o n s i s t i n g of b i l e s a l t s produced by the l i v e r , are amphipathic p r o v i d i n g a p o l a r e x t e r i o r and n e u t r a l i n t e r i o r (Scott et al. 1982). This allows m i c e l l e s t o s o l u b i l i z e monoacylglycerides, long-chain unsaturated f a t t y a c i d s and g l y c e r o l . The mixed m i c e l l e can then s o l u b i l i z e l o n g - c h a i n s a t u r a t e d f a t t y acids and f a t s o l u b l e v i t a m i n s A, D, E and K and t r a n s p o r t them t o e p i t h e l i a l c e l l s along the proximal i n t e s t i n e and/or p y l o r i c cecae f o r abso r p t i o n (Léger 1985). These c e l l s p r o j e c t m i c r o v i l l i i n t o the lumen g i v i n g them a high a b s o r p t i v e area. Acetate Fi g u r e 1. S y n t h e s i s , d e s a t u r a t i o n and e l o n g a t i o n of f a t t y a c i d s i n f i s h ( C a s t e l l 1979, Henderson and Sargent 1985). 20 Tocopherols and t h e i r e s t e r s (Figure 2) have low a b s o r p t i o n e f f i c i e n c i e s of only 20-40% i n mammals (Gallo-Torres 1980a) and t h i s may be due t o the long p h y t y l s i d e c h a i n . While a b s o r p t i o n e f f i c i e n c i e s have not been e s t a b l i s h e d f o r f i s h , a-tocopherol was absorbed more r e a d i l y i n rainbow t r o u t than the p~, y- or ô- forms of the v i t a m i n (Watanabe et a l . 1981b). Tocopheryl e s t e r s are hydrolyzed by pa n c r e a t i c esterases t o form tocopherol which can be s o l u b i l i z e d i n mixed m i c e l l e s and absorbed (Léger 1985). Almost no tocopher y l e s t e r s are absorbed. Hung et a l . (1982) found t h a t absorption of d-a-tocopherol from d i e t a r y d l - a - t o c o p h e r y l acetate was slower than from d i e t a r y d-a-tocopherol and they speculated t h a t t h i s might be due t o l i m i t a t i o n s i n d e e s t e r i f i c a t i o n p r i o r t o absorpti o n . R e - e s t e r i f i c a t i o n of g l y c e r o l , monoacylglyceride and ly s o p h o s p h o l i p i d s w i t h f a t t y acids t o form TAG and p o l a r l i p i d s (Léger 1985) occurs i n the i n t e s t i n a l mucosa. End products pass through the i n t e s t i n a l w a l l , become i n c o r p o r a t e d i n t o chylomicrons and very low de n s i t y l i p o p r o t e i n s and are t r a n s p o r t e d by the blood or lymphatic system (Sargent et a l . 1989). L i p o p r o t e i n s t r a n s p o r t e d v i a the blood are tr a n s p o r t e d t o the l i v e r by the hepatic p o r t a l v e i n and then t o other t i s s u e s . TAG i s taken up by adipose t i s s u e (Henderson and Sargent 1985) where i t i s sto r e d u n t i l r e q u i r e d as an energy source w h i l e p o l a r l i p i d s are transported t o other t i s s u e s f o r i n c o r p o r a t i o n i n t o membranes. F i g u r e 2. Forms of v i t a m i n E 22 Tocopherol i s transported p r i m a r i l y i n low d e n s i t y l i p o p r o t e i n s (Hung et al. 1982) and i s taken up r a p i d l y by plasma and l i v e r but more sl o w l y by white muscle, heart, spleen and e r y t h r o c y t e s (Cowey et al. 1983, Hung et al. 1982). When rainbow t r o u t were fed a v i t a m i n E-supplemented d i e t and then given an o r a l dose of d-a-tocopherol, i n i t i a l l y i t was found i n high c o n c e n t r a t i o n i n the c y t o s o l of l i v e r c e l l s but t h i s d e c l i n e d as i t was taken up by mitochondria and microsomes (Cowey et a l . 1981). In c o n t r a s t , when f i s h had been fed a v i t a m i n E - d e f i c i e n t d i e t , tocopherol was taken up most r a p i d l y by mitochondria i n the l i v e r followed by microsomes, the nuclear f r a c t i o n and c y t o s o l implying t h a t i t i s needed most by mitochondria. 2.6.3 E s s e n t i a l f a t t y a c i d requirements of f i s h f o r reproduction The e s s e n t i a l f a t t y a c i d requirements of immature f i s h have been reviewed by Kanazawa (1981). A recent r e p o r t (Yang and Dick 1992) s t a t e s t h a t the e s s e n t i a l f a t t y a c i d requirements of A r c t i c c h a r r f i n g e r l i n g s were met w i t h a d i e t supplemented w i t h 1.7% n3 and 0.5% n6 f a t t y a c i d s . Both Olsen et al. (1991) and Yang and Dick (1992) showed th a t A r c t i c c h a r r were capable of d e s a t u r a t i n g and e l o n g a t i n g 18:3n3 and 18:2n6. The former authors observed t h a t n3 f a t t y acids were used i n preference t o n6 f a t t y a c i d s f o r d e s a t u r a t i o n and e l o n g a t i o n . 23 The requirements of broodstock f o r f a t t y a c i d s have been reviewed by Watanabe (1985) and Ashton (1991) but no work w i t h A r c t i c c h a r r broodstock has been reported. Yu et al. (1979) fed rainbow t r o u t experimental d i e t s throughout t h e i r e n t i r e l i f e h i s t o r y and assessed the h a t c h a b i l i t y of the eggs and the growth of the f r y . They found t h a t 18:2n6 was not r e q u i r e d i n the presence of 1.0% 18:3n3 f a t t y a c i d s and both groups had equal h a t c h a b i l i t y (60%, n=4). Leray et al. (1985) fed a commercial d i e t and a s e m i p u r i f i e d d i e t t o rainbow t r o u t female broodstock f o r one year before spawning w i t h the two d i e t s d i f f e r i n g i n many respects i n c l u d i n g f a t t y a c i d composition. By r e c a l c u l a t i o n of the data presented, the commercial d i e t contained 2.5% n6 and 1.3% n3 while the s e m i p u r i f i e d d i e t contained 5.4% n6 and no n3. The h a t c h a b i l i t y from the group fed the commercial d i e t averaged 79% (n=3) w h i l e t h a t of the n3 d e f i c i e n t group was 20% (n=4). Those fed the n 3 - d e f i c i e n t d i e t produced eggs which had developmental d i s o r d e r s t h a t c o u l d be seen at the 16-32 c e l l stage and hatched f r y e x h i b i t e d numerous types of s p i n a l d e f o r m i t i e s and a shor t e r time t o resorb the y o l k sac (50 vs. 65 days). Watanabe et al. (1984b) observed t h a t f e c u n d i t y and embryonic s u r v i v a l decreased when rainbow t r o u t were fed a d i e t w i t h no n3 or n6 (54% eyed and 36% hatch) compared t o a d i e t supplemented w i t h 5% 18:2n6 (88% eyed and 82% hatch) . Red sea bream (Pagrus major) fed n 3 - d e f i c i e n t d i e t s a l s o produced eggs w i t h very low h a t c h a b i l i t y and egg and f r y a b n o r m a l i t i e s were observed (Watanabe 1984a). 24 As discussed i n Se c t i o n 2.3, v i t e l l o g e n i n i s a very high d e n s i t y l i p o p r o t e i n c o n t a i n i n g 80% p r o t e i n and 20% l i p i d which i s high i n e s s e n t i a l f a t t y acids (Léger et al. 1981), e s p e c i a l l y 20:5n3 and 22:6n3. Feeding t r o u t an n 3 - d e f i c i e n t d i e t f o r 6 months p r i o r t o spawning decreased the amount of v i t e l l o g e n i n (Fremont et a l . 1984) as w e l l as the l e v e l s of 20:5n3 and 22:6n3 i n v i t e l l o g e n i n w h i l e i t increased the l e v e l of 20:4n6. These changes were s i m i l a r but l e s s pronounced i n other serum l i p o p r o t e i n s and were most pronounced d u r i n g e a r l y v i t e l l o g e n e s i s i n d i c a t i n g t h a t feeding a d i e t s u f f i c i e n t i n e s s e n t i a l f a t t y a c i d s i s important f o r v i t e l l o g e n e s i s . The f a t t y a c i d composition of the eggs r e f l e c t e d t h a t of the d i e t . In both Leray et al. (1985) and Watanabe et al.'s (1984b) experiments, feeding t r o u t d i e t s w i t h no n3 f a t t y a c i d s r e s u l t e d i n eggs which had lower l e v e l s of n3 f a t t y a c i d s (20:5, 22:5 and 22:6) and higher l e v e l s of n6 (18:2 and 20:4) i n both 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 . Watanabe et al. (1984b) a l s o found t h a t the f a t t y a c i d composition of the m i l t r e f l e c t e d t h a t of the d i e t i n the same way as f o r the eggs and suggested t h a t the e f f e c t of t h i s change on f e r t i l i z a t i o n should be i n v e s t i g a t e d . While 20:3n9 was not found i n the eggs of parents fed d i e t s l a c k i n g i n n3 f a t t y acids f o r 3 months (Watanabe et al. 1984a) or one year (Leray et al. 1985) p r i o r t o spawning, i t was found i n the m i l t . When t r o u t were fed d i e t s c o n t a i n i n g 18:3n3 alone or i n combination with 18:2n6 f o r t h e i r e n t i r e l i f e 25 h i s t o r y , 20:3n9 was observed i n the eggs from both groups but was lower i n the group fed the d i e t c o n t a i n i n g both f a t t y a c i d s (Yu et al. 1979). The t r a n s f e r of 16:0 and 22:6n3 from the y o l k sac t o the body of the developing f r y was shown by t h e i r i n c r e a s e i n the body and concurrent decrease i n the y o l k sac of brook t r o u t (Salvelinus fontinalis) (Atchison 1975) and rainbow t r o u t (Hayes et al. 1985). Hayes et al. (1985) found t h a t 18:0 as w e l l as 16:0 and 22:6n3 were p r e f e r e n t i a l l y r e t a i n e d (74%) i n rainbow t r o u t a t the y o l k sac stage while f a t t y a c i d s such as 16:1, 18:1, 20:4 and 20:5 showed only 41% r e t e n t i o n . They p o s t u l a t e d t h a t those f a t t y a c i d s which had high r e t e n t i o n were r e q u i r e d f o r s p e c i f i c purposes. Moreover, these purposes might be r e l a t e d t o i n c l u s i o n i n t o p o l a r l i p i d s w i t h the h i g h l y unsaturated 22:6n3 incorporated i n t o the p* p o s i t i o n and the sa t u r a t e d 16:0 and 18:0 incorporated i n t o the a p o s i t i o n on the g l y c e r o l molecule. 2.6.4 Vitam i n E 2.6.4.1 Chemistry of tocopherols The chemistry of the tocopherols has been w e l l documented by Kasparek (1980). Isomers of tocopherol e x i s t which d i f f e r i n the presence and l o c a t i o n of methyl groups on the chromanol r i n g g i v i n g r i s e t o a-, y- and ô-tocopherols (Figure 2 ) . Stereo- isomers a l s o e x i s t w i t h d i f f e r e n t centers of symmetry around the 26 C-2 carbon of the r i n g and the C-4' and C-8' of the i s o p r e n o i d c h a i n . The d- stereoisomers are found i n nature w h i l e those manufactured s y n t h e t i c a l l y are racemic mixtures of d- and 1- tocop h e r o l s . Watanabe et al. (1981b) found t h a t a-tocopherols accumulated i n t i s s u e s of rainbow t r o u t , e s p e c i a l l y i n the l i v e r , w h i l e y- and Ô- forms d i d not. No i n t e r c o n v e r s i o n of these isomers occurred i n the muscle or l i v e r . V itamin E i s a generic term r e f e r r i n g t o a l l t o c o l or t o c o t r i e n o l d e r i v a t i v e s e x h i b i t i n g the b i o l o g i c a l a c t i v i t y of a-tocopherol (Kasparek 1980). Unless otherwise s p e c i f i e d , the term v i t a m i n E i n t h i s t h e s i s r e f e r s t o a-tocopherol or i t s acetate d e r i v a t i v e . One of the major f u n c t i o n s of v i t a m i n E i s i t s a c t i o n as an a n t i o x i d a n t and the phenolic hydroxyl group provides the f u n c t i o n a l group. Based on e f f e c t i v e n e s s i n pre v e n t i n g r e s o r p t i o n of embryos i n the r a t , the isomers and t h e i r e s t e r s d i f f e r i n b i o l o g i c a l a c t i v i t y (NRC 1987): d-a-tocopherol 1.49 I.U./mg d-a-tocopheryl acetate 1.36 I.U./mg dl - a - t o c o p h e r o l 1.1 I.U./mg d l - a - t o c o p h e r y l acetate 1.0 I.U./mg d-p-tocopherol 0.12 I.U./mg d-y-tocopherol 0.05 I.U./mg Ox i d a t i o n of tocopherols i s enhanced by exposure t o l i g h t , heat, a l k a l i , i r o n and copper i o n s . Since a-tocopherol i s 27 e a s i l y o x i d i z e d (Bauernfeind 1980), i t i s added t o animal feed i n i t s e s t e r i f i e d form as a-tocopheryl a c e t a t e , a more s t a b l e form of the v i t a m i n . The e s t e r form has no a n t i o x i d a n t p r o p e r t i e s u n t i l the acetate group has been replaced by a hydroxyl group during h y d r o l y s i s i n i t i a t e d by p a n c r e a t i c e s t e r a s e s . Since tocopherols are manufactured by p l a n t s , they are present i n feed i n g r e d i e n t s of p l a n t o r i g i n . They are a l s o present i n i n g r e d i e n t s of animal o r i g i n where animals have consumed p l a n t m a t e r i a l c o n t a i n i n g t o c o p h e r o l . 2.6.4.2 Function of v i t a m i n E V i t a m i n E f u n c t i o n s i n preventing l i p i d o x i d a t i o n and a t t a c k by f r e e r a d i c a l s , compounds w i t h one or more unpaired e l e c t r o n s . In the p e r o x i d a t i o n of l i p i d , a hydrogen atom i s a b s t r a c t e d from the methylene carbon of a methylene-interrupted unsaturated f a t t y a c i d t o form a l i p i d r a d i c a l (R.)(Cowey 1986). This a b s t r a c t i o n i s i n i t i a t e d only by r a d i c a l s w i t h a high degree of r e a c t i v i t y , such as a hydroxyl r a d i c a l (.OH), peroxy r a d i c a l (ROO.) or p o s s i b l y a hydroperoxy r a d i c a l (HOO.). The double bond s h i f t s r e s u l t i n g i n a l i p i d r a d i c a l which can r e a c t w i t h oxygen t o form a peroxy r a d i c a l (ROO.) which can then a b s t r a c t hydrogen atoms from other unsaturated f a t t y a c i d s . The process i s a u t o c a t a l y t i c because generation of more r a d i c a l s keeps the c y c l e going. The process destroys the f u n c t i o n of PUFA by c o n v e r t i n g them i n t o other compounds. I t a l s o r e s u l t s i n the formation of (hydro)peroxides which move i n t o a l l p a r t s 28 of the c e l l r e a c t i n g w i t h membranes and s u l f h y d r y l - c o n t a i n i n g enzymes (Tappel 1980). Vitamin E acts as a hydrogen or e l e c t r o n donor, donating the hydrogen from i t s 6-hydroxy1 group t o the peroxy r a d i c a l c o n v e r t i n g i t t o the l i p i d hydroperoxide (ROOH). A v i t a m i n E r a d i c a l i s formed but t h i s i s not a c t i v e enough t o a b s t r a c t hydrogen from PUFA as R00. can. ROOH i s converted t o ROH through the a c t i o n of g l u t a t h i o n e peroxidase i n the presence of g l u t a t h i o n e (GSH). The hydroxy r a d i c a l (OH.), which i s another r a d i c a l t h a t can i n i t i a t e the o x i d a t i o n process (Cowey 1986), i s formed when the superoxide r a d i c a l (0 2_.) and H202 r e a c t t o form OH. and OH" i n the presence of Fe + 3. This r e a c t i o n i s prevented by the a c t i o n of g l u t a t h i o n e peroxidase which c a t a l y z e s the conversion of H202 t o H20. Vitamin E and g l u t a t h i o n e peroxidase f u n c t i o n i n d i f f e r e n t ways i n preventing o x i d a t i o n . Vitamin E f u n c t i o n s w i t h i n the membrane t o p r o t e c t polyunsaturated f a t t y acids w i t h e l e c t r o n t r a n s p o r t f u n c t i o n s a g a i n s t f r e e r a d i c a l a t t a c k . Glutathione peroxidase acts w i t h i n the c y t o s o l t o prevent the formation of hydroxy r a d i c a l s and i t destroys hydroperoxides which form. Vitamin E i s mainly a s s o c i a t e d w i t h the m i t o c h o n d r i a l and microsomal membranes and t o a l e s s e r degree w i t h the plasma and nu c l e a r membranes (Cowey et al. 1981). M i t o c h o n d r i a l membranes c o n t a i n 35-40% l i p i d and 90% of t h i s i s comprised of p o l a r l i p i d s ( P i k e and Brown 1967) which are high i n PUFA and HUFA. The i n n e r m i t o c h o n d r i a l membrane contains p r o t e i n s t h a t a l l o w 29 the passage of f a t t y a c i d s , pyruvate, oxygen and some ions such as Ca + 2 and P0 4~ 3 from the intermembrane space i n t o the m a t r i x space ( A l b e r t s et al. 1983) where enzymes i n v o l v e d i n o x i d a t i o n of f a t t y a c i d and pyruvate t o a c e t y l CoA are l o c a t e d . In the matrix space, acetyl-CoA i s metabolized v i a the c i t r i c a c i d c y c l e . Enzymes i n the inner membrane i n c l u d e those i n v o l v e d i n the e l e c t r o n t r a n s p o r t system and o x i d a t i v e phosphorylation (ATP synthetase complex). Vitamin E i s a s s o c i a t e d w i t h the i n n e r membrane and i n f l u e n c e s the degree of u n s a t u r a t i o n of the membrane. For example, PUFA have been found t o be were lower i n hepatic m i t o c h o n d r i a l membranes from d u c k l i n g s fed a v i t a m i n E- d e f i c i e n t d i e t compared t o a c o n t r o l d i e t (Molenaar et al. 1980). The f a t t y a c i d composition of the membrane a f f e c t s the t r a n s p o r t of compounds across the membrane by i n f l u e n c i n g the r i g i d i t y or f l u i d i t y of the membrane and the t r a n s l o c a t i o n of t r a n s p o r t p r o t e i n s . I t i s p o s s i b l e t h a t f r e e r a d i c a l s form i n t h i s area because m i t o c h o n d r i a l membranes are high i n PUFA and oxygen i s being s u p p l i e d t o mitochondria f o r o x i d a t i v e r e a c t i o n s . Vitamin E d e f i c i e n c y has been shown t o be a s s o c i a t e d w i t h i n c r e a s e d membrane f r a g i l i t y of both c e l l membranes such as e r y t h r o c y t e s i n f i s h , b i r d s and mammals and m i t o c h o n d r i a l membranes i n b i r d s . In mammals, v i t a m i n E r a d i c a l s are converted to tocopherone, hydrolyzed t o tocopheryl quinone (Gallo-Torres 1980b) and reduced t o tocopheryl-p-hydroquinone. The l a t t e r 30 compound i s conjugated w i t h g l u c u r o n i c a c i d i n the l i v e r and excreted i n the b i l e . In some cases, the need f o r v i t a m i n E i n the membrane can be reduced by the presence of selenium which, through i t s a c t i o n i n g l u t a t h i o n e peroxidase, can l i m i t the formation of some of the products i n the c y t o s o l which keep the a u t o c a t a l y t i c p e r o x i d a t i v e process going. There i s a l s o evidence t h a t carotenoids a c t as a n t i o x i d a n t s (Tacon 1981) and there may be i n t e r a c t i o n s between v i t a m i n E and ca r o t e n o i d s . I f t h i s i n t e r a c t i o n d i d e x i s t f o r A r c t i c c h a r r , a d d i t i o n of carotenoids t o the d i e t might reduce the requirement f o r v i t a m i n E. Carotenoids were not added t o the d i e t s i n t h i s experiment i n order t o i n v e s t i g a t e the e f f e c t s of v i t a m i n E alone. 2.6.4.3 M o b i l i z a t i o n and requirement of v i t a m i n E f o r re p r o d u c t i o n King (1985) monitored the temporal changes i n d i s t r i b u t i o n of v i t a m i n E i n mature female rainbow t r o u t . During the month of June, f i s h were fed a d i e t not supplemented w i t h v i t a m i n E. At the beginning of J u l y , she found there was co n s i d e r a b l e storage of v i t a m i n E i n the l i v e r and t h i s c o n s t i t u t e d 45% of the t o t a l body storage of v i t a m i n E (25 mg). D i s t r i b u t i o n of v i t a m i n E was monitored i n f i s h fed d i e t s supplemented or not supplemented w i t h the v i t a m i n . In J u l y and August, v i t a m i n E was t r a n s p o r t e d from the l i v e r mainly t o the muscle w i t h some 31 t r a n s p o r t e d t o the ovary. Between August and October, the v i t a m i n was t r a n s p o r t e d from the muscle t o the ovary and adipose t i s s u e , and then from the adipose t i s s u e t o the ovary between October and December. During t h i s l a t t e r p e r i o d , v i t a m i n E was deposited i n the eggs from the o v a r i a n t i s s u e and there was a marked i n c r e a s e i n the v i t a m i n E content of the eggs. By December, 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 v i t a m i n E content of the eggs from f i s h fed the v i t a m i n E-supplemented d i e t compared the unsupplemented d i e t . F i s h spawned between January 6 and 27. At spawning, f i s h fed the unsupplemented d i e t and supplemented d i e t s , contained a t o t a l body burden of 25 and 35 mg v i t a m i n E, r e s p e c t i v e l y and the o v a r i a n t i s s u e contained 54.0% and 49.0% of t h i s body burden, r e s p e c t i v e l y . A f t e r spawning, the body burden was reduced t o 11.5 and 18 mg v i t a m i n E f o r those fed the unsupplemented and supplemented d i e t s , r e s p e c t i v e l y . At t h i s r a t e , one or two a d d i t i o n a l spawning seasons would be r e q u i r e d t o reduce the body s t o r e s of v i t a m i n E d r a s t i c a l l y . Kinumaki et al. (1972) found t h a t the v i t a m i n E c o n c e n t r a t i o n s of ovarian t i s s u e increased 4- t o 1 2 - f o l d when rainbow t r o u t were fed d i e t s supplemented w i t h l a r g e amounts of vi t a m i n s A, D and E f o r 10-17 weeks p r i o r t o the s t a r t of spawning. At spawning, v i t a m i n E was found t o be d i s t r i b u t e d mainly i n the muscle, s k i n and o v a r i e s w i t h a g r e a t e r p r o p o r t i o n of v i t a m i n E present i n the ovaries of supplemented f i s h (57- 32 59%) compared t o the unsupplemented f i s h (39%). In the l a t t e r group, 17% of the v i t a m i n was stored i n the l i v e r compared t o 3- 7% i n the supplemented f i s h . The requirement of f i s h f o r v i t a m i n E depends on many f a c t o r s . These i n c l u d e the species and stage of development, form of the v i t a m i n , water temperature (Cowey et a l . 1984), l e v e l of PUFA i n the d i e t (Watanabe et a l . 1981a), presence of selenium (Scott et al. 1982) and the presence of a n t i o x i d a n t s s u c h as e t h o x y q u i n ( 6 - e t h o x y - 1 , 2 - d i - h y d r o - 2 , 2 , 4 - t r i m e t h y l q u i n o l i n e ) . This makes i t d i f f i c u l t t o e s t a b l i s h requirements and compare the r e s u l t s of experiments. Vi t a m i n E has been a s s o c i a t e d w i t h reproduction s i n c e Evans and Bishop (1922) discovered t h a t v i t a m i n E prevented r e s o r p t i o n of f e t u s e s i n pregnant r a t s . The word "tocopherol" was d e r i v e d from the Greek "tokos" meaning " o f f s p r i n g " , "pherein" meaning "to bear" and " o l " t o denote the a l c o h o l group (Scott et al. 1982). The v i t a m i n p r o t e c t s the i n t e g r i t y of membranes i n the female r e p r o d u c t i v e system of some animals and the v a s c u l a r system of the embryo i n the hen, turkey, r a t , cow and ewe. I t a l s o prevents male s t e r i l i t y through i t s e f f e c t on the seminiferous tubules of the male r a t , guinea p i g , hamster, dog and c h i c k e n . 33 S e v e r a l researchers have i n v e s t i g a t e d the e f f e c t s of d i e t a r y v i t a m i n E on the r e p r o d u c t i v e performance of d i f f e r e n t species of f i s h . For example, King (1985) fed rainbow t r o u t p u r i f i e d d i e t s c o n t a i n i n g 0 or 90 mg v i t a m i n E acetate/kg w i t h 8% l i p i d f o r d i f f e r e n t periods of time of up t o 6 months f o l l o w e d by s t a r v a t i o n f o r 1 month p r i o r t o spawning. At spawning, 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 v i t a m i n E c o n c e n t r a t i o n of eggs only between those always fed the unsupplemented d i e t (5.39 ng/egg) compared t o those always fed the supplemented d i e t (7.37 ng/egg). There was s i g n i f i c a n t l y g r e a t e r s u r v i v a l of eyed eggs a f t e r shocking i f the females had been fe d the d i e t supplemented w i t h 90 mg v i t a m i n E acetate/kg f o r 6 months. O v e r a l l , h a t c h a b i l i t y was e x c e l l e n t w i t h a l l treatments w i t h only 11% m o r t a l i t y i n eggs from females fed the d e f i c i e n t d i e t f o r 6 months and 2% m o r t a l i t y w i t h the supplemented d i e t . S u r v i v a l was poorest at l e v e l s of < 5 \iq v i t a m i n E/egg and best when eggs contained > 7 ng v i t a m i n E/egg. S u r v i v a l was poorest when v i t a m i n E c o n c e n t r a t i o n was < 54 fxg/g and best at > 82 ng/g. A c r i t i c a l p e r i o d e x i s t e d f o r the maximum e f f e c t of v i t a m i n E d e p r i v a t i o n on the rainbow t r o u t egg. F i s h fed the unsupplemented d i e t between August and October produced eggs w i t h lower v i t a m i n E l e v e l s than those from f i s h fed t h a t d i e t l a t e r than October. Vita m i n E was t r a n s f e r r e d e f f i c i e n t l y from the y o l k t o the f r y w i t h swimup f r y having s i m i l a r v i t a m i n E content t o the eggs. King b e l i e v e d t h a t the broodstock had s u f f i c i e n t body s t o r e s of v i t a m i n E at 34 the beginning of the experiment t o supply the o v a r i e s without any supplementation of the d i e t . F u r ther, she s t a t e d t h a t the d i e t s would need t o be fed f o r a longer p e r i o d of time before the body reserves of vi t a m i n E would be depleted s u f f i c i e n t l y f o r t h e r e t o be a s i g n i f i c a n t e f f e c t of d i e t a r y v i t a m i n E co n c e n t r a t i o n on h a t c h a b i l i t y . Spawning time was delayed one week and f i s h d i d not a l l spawn s u c c e s s f u l l y i f they had been fed the unsupplemented d i e t f o r 6 months. Of the 8 f i s h i n t h i s treatment group, 3 spawned s u c c e s s f u l l y , 3 d i d not r i p e n , 1 produced abnormal eggs and 1 di e d p r i o r t o spawning. The abnormal eggs had disp e r s e d l i p i d g l o b u l e s and convoluted membranes. A l l the f i s h i n the supplemented group spawned normally. There were no s i g n i f i c a n t d i f f e r e n c e s i n egg weight or diameter. Kinumaki et al. (1972) fed p r a c t i c a l d i e t s t o rainbow t r o u t broodstock. They fed d i e t s c o n t a i n i n g 66, 4600 and 8200 mg v i t a m i n E/kg f o r 19 weeks. The c o n t r o l d i e t (66 mg v i t a m i n E/kg d i e t ) , which contained only 10% of the l e v e l s of vitamins A and D compared t o the other two d i e t s , r e s u l t e d i n s u r v i v a l t o the eyed stage of 51% compared t o 46 and 37% f o r the other d i e t s r e s p e c t i v e l y . Vitamin E i n the eggs was 4.4 ng/egg (46 u,g/g) f o r the c o n t r o l group compared t o 23 and 32 ixg (240 and 330 M-g/g) f o r the groups fed d i e t s supplemented w i t h 4600 and 8200 mg v i t a m i n E/kg. When the y o l k sac had been absorbed, the f r y 35 contained 3.5, 13 and 30 ng v i t a m i n E / f r y , r e s p e c t i v e l y or 27, 114 and 166 ^g/g. Thus, v i t a m i n E had been t r a n s f e r r e d from the d i e t t o the egg and absorbed from the y o l k sac by the f r y . The m u l t i p l e d i f f e r e n c e s between the l e v e l s of f a t - s o l u b l e v i t a m i n s i n the d i e t s prevent making any f i r m conclusions about t h i s r esearch. Takeuchi et a l . (1981a) fed ayu (Plecoglossus a l t i v e l i s ) f o r 3 months p r i o r t o spawning w i t h d i e t s c o n t a i n i n g graded l e v e l s of v i t a m i n E acetate plus endogenous v i t a m i n E ( t o t a l 10- 2390 mg/kg). Feeding unsupplemented d i e t s (10 mg endogenous v i t a m i n E/kg) decreased s u r v i v a l t o the eyed stage, h a t c h a b i l i t y and s u r v i v a l of f r y immediately a f t e r hatching. Spawning was delayed or f a i l e d t o occur i n o n e - t h i r d of the female p o p u l a t i o n fed the unsupplemented d i e t while a l l f i s h spawned when they were fed d i e t s c o n t a i n i n g more than 34 mg t o t a l v i t a m i n E/kg. Watanabe and Takashima (1977) fed carp (Cyprinus carpio) broodstock s e m i p u r i f i e d d i e t s c o n t a i n i n g e i t h e r 0 or 700 mg v i t a m i n E acetate/kg f o r 17 months. They found t h a t the f i s h fed the unsupplemented d i e t had a very low gonadosomatic index (ovary weight/body weight). Oocyte development was delayed w i t h oocytes l a c k i n g l a r g e y o l k granules and y o l k v e s i c l e s even though the oocytes had reached a s i z e at which these granules and v e s i c l e s should have been deposited. I t appeared t h a t oocytes had begun t o develop and then became a t r e t i c . 36 H i s t o l o g i c a l examination of the p i t u i t a r y gland i n d i c a t e d decreased s e c r e t o r y a c t i v i t y of the gonadotrophs, which produce FSH and LH, and t h y r o t r o p h s , which produce t h y r o i d - s t i m u l a t i n g hormone, and the e p i t h e l i a l c e l l s i n the t h y r o i d t i s s u e were a l s o reduced i n height. Spawning was probably delayed or prevented i n a l a r g e r p o r t i o n of the p o p u l a t i o n as i t was f o r the ayu (Takeuchi et al. 1981a) and rainbow t r o u t (King 1985). In a d d i t i o n t o endocrine gland d y s f u n c t i o n , the f a t t y a c i d composition of the carp ovarian t i s s u e resembled t h a t of f i s h w i t h e s s e n t i a l f a t t y a c i d d e f i c i e n c y s i n c e the t i s s u e contained i n c r e a s e d l e v e l s of 18:ln9 and 20:3n9 and decreased l e v e l s of 20:4n6, 20:5n3 and 22:6n3. I f the eggs had been analyzed f o r f a t t y a c i d composition, they probably would have r e f l e c t e d t h i s e s s e n t i a l f a t t y a c i d d e f i c i e n c y as w e l l . Watanabe and Takashima (1977) p o s t u l a t e d t h a t the ct-tocopherol d e f i c i e n c y caused a r e d u c t i o n of 18:2n6, one of the e s s e n t i a l f a t t y a c i d s f o r carp, and t h a t t h i s created a secondary d e f i c i e n c y i n n3 f a t t y a c i d s . Watanabe et al. (1991) evaluated the i n f l u e n c e of v i t a m i n E acetate c o n c e n t r a t i o n on reproduction i n red seabream. F i s h were fed a c o n t r o l d i e t c o n t a i n i n g 500 mg v i t a m i n E acetate/kg f o r 90 days and subsequently they were fed e i t h e r the c o n t r o l d i e t or a d i e t c o n t a i n i n g 2000 mg v i t a m i n E acetate/kg f o r 10 days p r i o r t o and 30 days a f t e r spawning. The r a t e of buoyant eggs, hatching success and normal l a r v a e were d i r e c t l y i n f l u e n c e d by d i e t a r y treatment. Production of normal l a r v a e 37 increa s e d from 35% t o 76% f o r seabream fed the d i e t c o n t a i n i n g 2000 mg v i t a m i n E acetate/kg. At the time t h a t the current experiment began, most commercial broodstock d i e t s a v a i l a b l e i n Canada were supplemented w i t h 300-500 mg (IU) dl-cc-tocopheryl acetate/kg d i e t , w h i l e two were supplemented w i t h 200 and 800 mg/kg. Although there are no re p o r t s on hypervitaminosis E i n f i s h , March et a l . (1973) showed th a t c h i c k s had decreased growth r a t e s and s k e l e t a l m i t o c h o n d r i a l r e s p i r a t i o n r a t e s when they were fed d i e t s c o n t a i n i n g 2200 compared t o 1000 mg v i t a m i n E/kg. This l e d NRC (1987) t o suggest a " t e n t a t i v e presumed safe use l e v e l of 75 IU/kg of BW/day". 38 MATERIALS AND METHODS 3.1 L o c a t i o n and time The f i s h r e a r i n g and egg i n c u b a t i o n p o r t i o n s of t h i s research were conducted at Rockwood Aquaculture Research Centre (RARC) l o c a t e d 30 km north of Winnipeg (49°54' N, 97°8' W), Manitoba. Biochemical analyses and a l l other r e l a t e d a c t i v i t i e s (except as noted) were conducted at the Freshwater I n s t i t u t e Science Laboratory (FWISL), Winnipeg, Manitoba. Throughout t h i s t h e s i s , Year 1 r e f e r s t o the p e r i o d from June 27, 1988 when the f i s h were weighed and d i s t r i b u t e d t o the tanks u n t i l January 11, 1989 one week a f t e r the f i r s t spawning p e r i o d was completed. Year 2 r e f e r s t o the p e r i o d from January 12, 1989 u n t i l January 3, 1990 when the second spawning p e r i o d was completed. 3.2 D i e t s 3.2.1 Formulation Four d i e t s were formulated t o c o n t a i n two l e v e l s of l i p i d (12 and 19%) at each of two l e v e l s of dl-ct-tocopheryl acetate ( v i t a m i n E acetate) (30 and 600 mg/kg). The formulations are presented i n Table 1. Since broodstock used i n t h i s research were v a l u a b l e , d i e t s were formulated t h a t were not l i k e l y t o have a d e l e t e r i o u s e f f e c t on the s u r v i v a l of the broodstock o r t h e i r f e c u n d i t y . The lower l e v e l of 30 mg v i t a m i n E acetate/kg 39 Table 1. Formulation of experimental d i e t s (g/kg dry d i e t , except as noted). D i e t LLLE LLHE HLLE HLHE COMM1 H e r r i n g meal, steam-dried 2' 3 395 395 395 395 (CP=78%, TL=12%) 4 Soybean meal 2 195 195 195 195 (CP=47%, TL=2.5%) Corn g l u t e n meal 2 86 86 86 86 (CP=68%, TL=8%) Whole wheat, ground 2 183 183 183 183 (CP=16%, TL=4%) Herring o i l 5 44 44 114 114 A l p h a - c e l l u l o s e 70 70 - - Vitamin premix 6 10 10 10 10 M i n e r a l premix 7 10 10 10 10 Choline c h l o r i d e (60%) 6 6 6 6 Asc o r b i c a c i d ( c r y s t a l l i n e ) 1 1 1 1 dl-ct-Tocopheryl acetate 8(mg/kg) 30 600 30 600 Ethoxyquin (mg/kg) 35 35 - - 1 Commercial rainbow t r o u t grower d i e t contained a l l the i n g r e d i e n t s i n the high l i p i d d i e t s plus blood meal, p o u l t r y meal, whey, brewers yeast, limestone, s a l t and methionine. COMM contained wheat shorts and middlings but no whole wheat or i n o s i t o l . 2 Four i n g r e d i e n t s combined t o form a ba s a l mixture. 3 S t a b i l i z e d w i t h ethoxyquin at 250 mg/kg. 4 CP=Crude p r o t e i n , TL= T o t a l l i p i d 5 S t a b i l i z e d w i t h ethoxyquin at 500 mg/kg. 6 Vitamin premix s u p p l i e d the f o l l o w i n g i n mg or I.U./kg dry d i e t : v i t a m i n A acetate ( r e t i n y l a c e t a t e ) , 10,000 I.U.; c h o l e c a l c i f e r o l , 2,400 I.U.; menadione sodium b i s u l f i t e , 25; thiam i n mononitrate, 30; r i b o f l a v i n , 60; c a l c i u m pantothenate, 120; niacinamide, 200; f o l i c a c i d , 15; b i o t i n , 3; py r i d o x i n e h y d r o c h l o r i d e , 30; cyanocobalamin, 0.06; i n o s i t o l , 400. 7 M i n e r a l premix s u p p l i e d the f o l l o w i n g i n mg element/kg dry d i e t : Mn (as MnS0 4.H 20), 86; Fe (as FeS0 4.7H 20), 63; Cu (as CuS0 4.5H 20), 25; Zn (as ZnS0 4.7H 20), 144; I (as K I 0 3 ) , 8. 8 Supplied courtesy of Hoffmann-La Roche, Etobicoke, Ontario. 40 1981). The 600 mg vi t a m i n E acetate/kg c o n c e n t r a t i o n was chosen because i t was above the range added t o most salmonid broodstock d i e t s (300-500 mg/kg). Tabachek (1984) showed t h a t a 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 of 10% was below the growth requirements f o r Nauyuk Lake A r c t i c charr f i n g e r l i n g s w h i l e 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 s of 15-20% r e s u l t e d i n s i g n i f i c a n t i n c r e a s e s i n weight g a i n . L i p i d concentrations of 10-11% and 17-18% were chosen but upon a n a l y s i s the d i e t s contained 12% and 19% l i p i d . Throughout t h i s t h e s i s , the four d i e t s have been denoted as LLLE (Low L i p i d Low v i t a m i n E ) , LLHE (Low L i p i d High v i t a m i n E ) , HLLE (High L i p i d Low v i t a m i n E ) , HLHE (High L i p i d High v i t a m i n E ) . The higher l i p i d l e v e l was achieved by r e p l a c i n g a l p h a - c e l l u l o s e i n the low l i p i d d i e t s w i t h a d d i t i o n a l h e r r i n g o i l s t a b i l i z e d w i t h 0.05% ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4 t r i m e t h y l - q u i n o l i n e ) . To e q u a l i z e the co n c e n t r a t i o n of ethoxyquin i n the d i e t s , a d d i t i o n a l ethoxyquin was added t o the low l i p i d d i e t s . In a d d i t i o n t o the above d i e t s , a commercial rainbow t r o u t grower d i e t (denoted as COMM) was fed t o one tank of f i s h as a refer e n c e d i e t . The broodstock d i e t manufactured by the s u p p l i e r (Martin Feed M i l l s , E l m i r a , Ontario) was a minimum of 6 mm i n diameter. The wide v a r i a t i o n i n the s i z e of c h a r r i n the p o p u l a t i o n r e s u l t e d i n the 6 mm d i e t being too l a r g e f o r a p o r t i o n of the population t o consume. Therefore, the 5 mm grower d i e t had been used r o u t i n e l y at RARC u n t i l a l l the f i s h were l a r g e enough t o consume the 6 mm broodstock d i e t . 41 3.2.2 Manufacturing Soybean meal and whole wheat were ground i n a F i t z m i l l and were ground f i n e l y enough t o pass through an 18 U.S. mesh screen (1.00 mm) and f o r 60% of i t t o pass through a 30 U.S. mesh screen (0.60 mm). A b a s a l mix of h e r r i n g meal, corn g l u t e n meal, ground soybean meal and whole wheat was prepared i n a v e r t i c a l mixer and was placed i n double p l a s t i c bags w i t h an outer brown paper bag. M i n e r a l s were ground t o a f i n e powder i n a c o f f e e g r i n d e r p r i o r t o weighing. Both the m i n e r a l and v i t a m i n premixes were prepared i n p e a r l i z e d c o r n s t a r c h c a r r i e r s , mixed i n a Hobart mixer and stored i n double p l a s t i c bags. H e r r i n g o i l was placed i n three 20 L p l a s t i c c o n t a i n e r s . A l l the above i n g r e d i e n t s and mixes were prepared at UBC and shipped from Vancouver t o Winnipeg under frozen c o n d i t i o n s . Upon a r r i v a l i n Winnipeg, the basal mix, a s c o r b i c a c i d , c h o l i n e c h l o r i d e mixture, v i t a m i n and mineral premixes were f r o z e n at 35°C u n t i l r e q u i r e d f o r manufacturing the d i e t s d u r i n g the two year p e r i o d of the experiment. The d l - a - t o c o p h e r y l acetate was s t o r e d i n a r e f r i g e r a t o r while ethoxyquin was kept at room temperature. Herring o i l was s t a b i l i z e d w i t h the a d d i t i o n of ethoxyquin at 500 mg/kg (NRC 1981), f l u s h e d w i t h n i t r o g e n and s t o r e d a t -35°C u n t i l r e q u i r e d . The d i e t s were manufactured f i v e times d u r i n g the course of the experiment at 9-13 wk i n t e r v a l s (mean = 11 wk). Ingredients were weighed and mixed each time the d i e t s were manufactured. 42 The commercial d i e t (COMM) was from one shipment i n Year 1 and two shipments i n Year 2. Since the h e r r i n g o i l d i d not reach a uniform l i q u i d s t a t e when thawed at room temperature, a warm water bath (37°C) was used t o l i q u e f y the o i l . A f t e r use, n i t r o g e n was bubbled i n t o the o i l i n the c o n t a i n e r before r e t u r n i n g i t t o the f r e e z e r . Each time the four d i e t s were made, a l l the o i l used was only from one c o n t a i n e r . Both d l - a - t o c o p h e r y l acetate and ethoxyquin (when required) were mixed w i t h the p r e s c r i b e d weight of h e r r i n g o i l before adding the o i l mixture t o the other dry i n g r e d i e n t s . D i e t s were c o l d p e l l e t e d through a 5 mm d i e i n a Superior-Templewood p e l l e t m i l l l o c a t e d at the Department of Animal Science, U n i v e r s i t y of Manitoba. D i s t i l l e d water was added at 6-7% t o a i d i n p e l l e t i n g . P e l l e t s were spread out i n a t h i n l a y e r on waxed paper and allowed t o a i r dry f o r 4-5 hours at which time the moisture content was l e s s than 9%. The d i e t s were sieved using a Kason separator t o pass over a No. 12 U.S. sie v e (1.7 mm). D i e t s were placed i n double p l a s t i c bags and an outer brown paper bag, t r a n s p o r t e d t o Rockwood Aquaculture Research Centre and s t o r e d at -10°C. H e r r i n g meal and o i l was purchased from B.C. Packers, Steveston, B.C. Approximately one month p r i o r t o t h i s purchase, DFO's West Vancouver Laboratory purchased h e r r i n g o i l from the same s u p p l i e r . A sample of o i l from t h i s f i r s t shipment was s t a b i l i z e d w i t h 0.05% ethoxyquin and placed i n two c o n t a i n e r s which were f l u s h e d w i t h n i t r o g e n . One c o n t a i n e r was placed i n 43 the r e f r i g e r a t o r and the other c o n t a i n e r was placed i n a f r e e z e r at -15°C. The f o l l o w i n g day, the o i l and a sample of b a s a l mix were shipped on i c e t o Hoffmann-La Roche, Etobicoke, Ontario f o r a n a l y s i s of a-tocopherol. Samples of h e r r i n g o i l used i n t h i s experiment were a l s o shipped t o Hoffmann-LaRoche but they were not analyzed. The h e r r i n g o i l used i n t h i s experiment was analyzed f o r peroxide value each time the d i e t s were made (Woyewoda et a l . 1986). 3.3 P a r e n t a l f i s h 3.3.1 H i s t o r y of the stock A r c t i c c h a r r used i n t h i s experiment o r i g i n a t e d from the F r a s e r R i v e r i n Labrador (56°39'N, 63°10'W). Charr were spawned at the s i t e i n October 1984 and f e r t i l i z e d eggs were t r a n s p o r t e d t o Winnipeg, Manitoba and incubated i n a quarantine area at the FWISL. Once p a r e n t a l f i s h had been t e s t e d and found t o be f r e e of c e r t i f i a b l e d i s eases, 1070 eyed eggs were t r a n s f e r r e d t o the Rockwood Aquaculture Research Centre's (RARC) broodstock b u i l d i n g which had a c e r t i f i e d d i s e a s e - f r e e s t a t u s . The number of f i s h was reduced over the years through c u l l i n g , n a t u r a l m o r t a l i t y and sampling of f i s h f o r c e r t i f i c a t i o n t e s t i n g once a year. F i s h spawned f o r the f i r s t time as 3 year olds i n the f a l l of 1987 w i t h 143 females producing eggs out of the 476 male and female f i s h present at t h a t time. An unknown number of f i s h t h a t had not matured were maintained s e p a r a t e l y because they looked l i k e " p o t e n t i a l spawners" (Olson, personal communication) 44 w i t h 48 females and 95 males i n t h a t group remaining the f o l l o w i n g year. By the time t h i s experiment began i n June 1988, 353 of the o r i g i n a l p o p u l a t i o n remained w i t h an estimated 165 females, 134 males and 54 emaciated f i s h of unknown sex. 3.3.2 S e l e c t i o n of f i s h f o r the experiment The m a j o r i t y of c h a r r used i n t h i s experiment were s e l e c t e d from those t h a t had spawned the previous year w i t h 15 females and 10 males used f o r each of the f i v e treatments. Emaciated f i s h or those w i t h gross a b n o r m a l i t i e s were not used i n t h i s experiment. F i s h were anaesthetized using 0.03% 2- phenoxyethanol and tagged w i t h numbered F l o y tags. Length was measured t o the nearest m i l l i m e t e r and weight was taken t o the nearest gram using a M e t t l e r PE12 zero-tare balance. The wide range i n the weight of the f i s h (345-2190 g) made i t i m p o s s i b l e t o d i s t r i b u t e the f i s h t o the 5 tanks i n a t o t a l l y random manner. The t o t a l and mean weight of f i s h i n each tank was a f f e c t e d d r a m a t i c a l l y i f j u s t 1 or 2 small f i s h were rep l a c e d by 1 or 2 l a r g e f i s h . Therefore, the tag number, sex, l e n g t h and weight of a l l the f i s h considered f o r the experiment were recorded. F i s h were d i s t r i b u t e d t o each tank i n a manner t h a t provided each tank w i t h the same weight d i s t r i b u t i o n of male and of female f i s h . 45 3.4 Environmental c o n d i t i o n s f o r broodstock 3.4.1 Tanks Each of the f i v e d i e t s was assigned t o one tank of f i s h . F i v e independent r e a r i n g u n i t s were used each c o n s i s t i n g of a 1500 L l i g h t blue f i b e r g l a s s tank (1.8 m x 1.8 m) (6 f t x 6 f t ) mounted over a 1100 L b i o l o g i c a l f i l t e r [1.8 m (6 f t ) diameter] c o n t a i n i n g 0.73 m3 g r a n i t e g r a v e l (Papst and Hopky 1982). Flow r a t e s t o each tank averaged 105 L/min (range = 101-112). R e c i r c u l a t e d water entered the tank through a spray bar t o provide a e r a t i o n . Fresh w e l l water (6-7°C) was a l s o added as makeup t o r e p l a c e a p o r t i o n of the water being d i s c a r d e d . The f l o w r a t e of makeup water was approximately 6.5-7 L/min and was adjusted t o achieve the d e s i r e d temperature and t o e q u a l i z e the temperature between tanks. While f i l t e r s were backwashed every 2 weeks duri n g the feeding p e r i o d , i t was not necessary t o backwash the f i l t e r s during the spawning p e r i o d when f i s h were not f e d . 3.4.2 L i g h t i n g and photoperiod L i g h t i n g f o r each tank was provided by 2 c o o l white f l u o r e s c e n t bulbs placed 1.2 m above the water. L i g h t i n t e n s i t y was 165 l u x when measured w i t h a M i n o l t a Autometer I I I and converted from e l e c t r o n v o l t s i n t o l u x . I t was 2.88 microeinsteins/m 2/sec when measured w i t h a Lambda quantum photometer. The photoperiod was adjusted every 10 days t o conform t o the Winnipeg (49°54' N, 97°8' W) photoperiod. This 46 was s i m i l a r t o the photoperiod i n Goose Bay, Labrador (Olson, personal communication) which i s c l o s e t o the spawning grounds of the p a r e n t a l charr (56°39'N, 63°10'W). 3.4.3 Temperature On the n i n t h day of feeding i n Year 1 ( J u l y 6, 1988), i t was found t h a t the water temperatures d i f f e r e d i n the f i v e tanks. At t h a t time, the temperatures were 7.2°C (LLLE), 9.7°C (LLHE), 10.2°C (HLLE), 7.2°C (HLHE), 12.2°C (COMM). On J u l y 7, temperature d i f f e r e n c e s between tanks had been reduced t o 1.5°C or l e s s . V a r i a t i o n i n temperature was due t o d i f f e r e n c e s i n the amount of 7°C makeup water being added t o each tank. The makeup water entered the tank below the water l i n e and could not be adjusted because the flow valves were f a u l t y . Changes t o the makeup l i n e s were made so t h a t the water entered the tank above the s u r f a c e of the water and a new c o n t r o l v a l v e was i n s t a l l e d f o r each tank. When t h i s was c o r r e c t e d , lengths and weights of a l l f i s h were taken again on J u l y 11, 1988. T h e r e a f t e r , the temperature of water i n each tank was measured each morning us i n g a F i s h e r temperature meter. Adjustments t o the amount of makeup water were made t o e q u a l i z e water temperatures i n the f i v e tanks. The amount of makeup water was adjusted t o t r y and match the water temperature i n Year 2 t o t h a t of Year 1. 47 3.4.4 Water chemistry In Year 1, water samples were taken from each tank on J u l y 13 and September 22. A f t e r the spawning season ended and feeding resumed, samples were taken on February 6, March 21, May 29, J u l y 12, August 29, October 4 and December 6 i n Year 2. Water samples were measured f o r pH and analyzed f o r ammonium-N, n i t r i t e - N and n i t r a t e - N according t o the methods of S t a i n t o n et a l . (1974). Temperature and d i s s o l v e d oxygen were measured at the same time using a YSI-temperature oxygen meter. Un-ionized ammonia was c a l c u l a t e d using the t a b l e s of P i p e r (1982) and the f o l l o w i n g equation: Un-ionized ammonia (u,g/L) = un-ionized ammonia (%) x NH„-N (uq/L) 100 3.5 Feeding regime There appeared t o be an e f f e c t of l i p i d l e v e l on the length of the spawning p e r i o d i n Year 1. To evaluate t h i s f u r t h e r , a crossover i n d i e t s was c a r r i e d out i n Year 2 (Figure 3 ) . The d i e t a r y v i t a m i n E content of the d i e t fed t o a given tank of f i s h was the same i n both Year 1 and Year 2. However, f i s h fed a low l i p i d d i e t i n Year 1 were fed a high l i p i d d i e t i n Year 2 and those fed a high l i p i d d i e t i n Year 1 were fed a low l i p i d d i e t i n Year 2. O r i g i n a l l y , i t was expected t h a t the d a i l y s a t i a t i o n r a t i o n c o u l d be e s t a b l i s h e d by feeding f i s h t o s a t i a t i o n f o r 1 or 2 48 days. However, t h i s d i d not prove t o be s a t i s f a c t o r y because f i s h d i d not consume the same amount each day. F i s h consumed much more on the f i r s t day than they d i d on the second or t h i r d day of feeding t o s a t i a t i o n . A regime was e s t a b l i s h e d of feeding t o s a t i a t i o n twice a day f o r 3 consecutive days and using the mean as the d a i l y r a t i o n t o feed f o r the next 18 or 25 days. Feed was weighed f o r an 11 day and one or two 7 day periods w i t h the l a t t e r p o r t i o n s stored i n the f r e e z e r at -10°C u n t i l r e q u i r e d . The weight of feed t o be fed each day was converted t o a volume per day f o r the convenience of hatchery s t a f f . Hatchery s t a f f o f f e r e d t h i s amount of feed over 3-4 feedings throughout the day. I f i t was not p o s s i b l e t o feed the p r e s c r i b e d amount of feed, the feed remaining at the end of each p e r i o d was weighed and net feed fed was c a l c u l a t e d . In Year 2, there seemed t o be 10% of the feed remaining at the end of each p e r i o d . Therefore, the d a i l y amount o f f e r e d was set at 90% of the mean of the 3 day s a t i a t i o n amount. In Year 1, f i s h were fed between June 28, 1988 and September 11, 1988. Feed consumption decreased as spawning time approached and feed was not o f f e r e d from September 12, 1988 t o January 11, 1989. When spawning was complete, feeding was resumed on January 12, 1989 and was o f f e r e d u n t i l the end of the experiment. F i s h were not fed on the day p r i o r t o , or on the day of weighing. Feed was o f f e r e d f o r a t o t a l of 71 days i n Year 1 of the experiment and 252 days p r i o r t o the f i r s t f i s h Year 1 Year 2 4 9 50 spawning i n Year 2. In Year 2, feed was o f f e r e d throughout the spawning p e r i o d on the 2 days f o l l o w i n g spawning. 3.6 Growth of broodstock F i s h were anaesthetized w i t h 2-phenoxyethanol p r i o r t o t a k i n g l e n g t h and weight measurements of i n d i v i d u a l l y tagged f i s h . Data were c o l l e c t e d on the f o l l o w i n g dates: June 27, 1988, J u l y 8, 1988, August 10, 1988, September 14, 1988, January 11, 1989, A p r i l 5, 1989, June 28, 1989, September 13, 1989 and January 3, 1990. S p e c i f i c growth r a t e and c o n d i t i o n f a c t o r were c a l c u l a t e d . S p e c i f i c growth r a t e = l o g Q f i n a l weight (q) - l o q Q i n i t i a l wt (g) x 100 time (days) C o n d i t i o n f a c t o r = weight (g) x 100 l e n g t h 3 (cm) 3.7 Spawning K r i e g e r et al. (1988) found t h a t A r c t i c c h a r r eggs over- ripened q u i c k l y a f t e r o v u l a t i o n and t h a t the f e r t i l i z a t i o n r a t e d e c l i n e d . Therefore once spawning commenced, a l l f i s h were checked a t weekly i n t e r v a l s . Charr were anaesthetized using 0.03% 2-phenoxyethanol. The t a g number and stage of development of each f i s h was noted. Males were judged as Not Ripe or Ripe w h i l e females were judged as No Development, Some Development, Well Developed, Ripe or Spawned. As f i s h were checked, female 51 f i s h t h a t would r e l e a s e t h e i r eggs (Ripe) and males which would r e l e a s e m i l t (Ripe) were separated from the r e s t . A f t e r a l l the f i s h i n a tank had been checked, the f o l l o w i n g spawning procedure was c a r r i e d out. Each r i p e female f i s h was anaesthetized and r i n s e d i n f r e s h water t o remove any a n a e s t h e t i c . The tag number was noted and the f i s h was weighed t o the nearest gram and f o r k length measured t o the nearest mm. A f t e r d r y i n g the v e n t r a l s i d e w i t h a s o f t c l o t h , the eggs were r e l e a s e d manually, c o l l e c t e d and drained of o v a r i a n f l u i d i n 1 or 2 p l a s t i c p re-tared c o n t a i n e r s w i t h aluminum screen bottoms. Ovarian f l u i d was c o l l e c t e d i n a p l a s t i c pan. The t o t a l weight of drained eggs was measured t o the nearest gram. A 50-60 g sample of eggs was taken and processed as described i n S e c t i o n 3.11 (Figure 4). A r i n s e of 1.37% NaHC03 (250 ml) was added t o the o v a r i a n f l u i d and remaining eggs and then decanted. This r i n s e was evaluated as described i n S e c t i o n 3.13.1. Any a b n o r m a l i t i e s ( d i s c o l o u r e d eggs or blood i n the o v a r i a n f l u i d ) were noted. Two male f i s h were anaesthetized and r i n s e d i n f r e s h water before noting t h e i r tag numbers and measuring t h e i r lengths and weights. The v e n t r a l area of each f i s h was d r i e d before c o l l e c t i n g i t s m i l t i n a dry p l a s t i c beaker. A 125 ml a l i q u o t of r i n s e was d i v i d e d between the two beakers, the contents of the beakers combined and added t o the eggs. A f t e r mixing the sperm through the eggs g e n t l y f o r 2 minutes, the f l u i d was poured o f f and 6-7°C w e l l water was used t o r i n s e the eggs u n t i l the f l u i d was c l e a r . A sample of 50 eggs was taken 52 ANALYSES OF EGGS 50-60 g sample of eggs 1 10-15 g sto r e d at -115°C 1 Yr 1 and Yr 1 Vitamin E e x t r a c t e d 1 assay 1 10-15 g stored at -35°( Yr 2 1 TL e x t r a c t e d 1 30 eggs weighed i n d i v i d . 30 g 1 -35°C sto r e d at 1 f r e e z e d r i e d f o r dry matter determination and ground up Yr 1 1 NL and PL separated by column chromatography 1 1 NL _ i _ 1 1 1 1 assay TLC FAMES 1 GLC 1 PL _1_ 1 I TL e x t r a c t i o n 1 assay I and Yr 2 _ i 1 Selenium assay 1 1 1 assay TLC FAMES 1 GLC ANALYSIS OF FRY f r y (Yr 2 only) 1 s t o r e d at -115°C 1 Vitamin E e x t r a c t e d 1 assay TL = t o t a l l i p i d NL = n e u t r a l l i p i d PL = p o l a r l i p i d TLC = t h i n - l a y e r chromatography FAMES = f a t t y a c i d methyl e s t e r s GLC = g a s - l i q u i d chromatography A l l samples of eggs and f r y were flushed w i t h n i t r o g e n p r i o r t o being f r o z e n . F i g u r e 4. Flow diagram showing analyses conducted on eggs and f r y i n Year 1 and/or Year 2. 53 to assess f e r t i l i z a t i o n r a t e as described i n S e c t i o n 3.8. Approximately 1000 eggs i n Year 1 and 500 eggs i n Year 2 were measured v o l u m e t r i c a l l y and placed i n the j a r s e c t i o n of an incubator (described i n Se c t i o n 3.9). Ja r s were placed i n a pan of 6-7°C w e l l water and covered w i t h black p l a s t i c . A f t e r 2 hours the j a r s c o n t a i n i n g the water-hardened eggs were t r a n s f e r r e d from the broodstock b u i l d i n g t o the main b u i l d i n g and placed i n t o the lower s e c t i o n s of the i n c u b a t o r s . I f f e c u n d i t y was low, h a l f the numbers of eggs were sampled and h a l f the amount of r i n s e and extender was used. In Years 1 and 2, r e p l i c a t e groups of eggs were incubated from two and three f i s h , r e s p e c t i v e l y . Excess eggs not r e q u i r e d f o r the experiment were incubated i n Heath t r a y s w i t h i n the broodstock b u i l d i n g . In Year 2, Heath t r a y s were compartmentalized and a comparison between s u r v i v a l t o the eyed stage when incubated i n Heath t r a y s versus j a r incubators was made. Eggs were not a l l r e l e a s e d on the day the f i s h spawned s i n c e some eggs may not have ovulated and/or they remained i n the ovarian c a v i t y . To assess the t o t a l number of eggs produced, f i s h which had spawned were inspected d u r i n g subsequent spawning checks and eggs were c o l l e c t e d and counted. On three occasions i n Year 1, l a r g e numbers of eggs were r e l e a s e d during a subsequent check. These eggs were f e r t i l i z e d and incubated and data was used f o r the time when the highest number of eggs were re l e a s e d . 54 3.8 F e r t i l i z a t i o n r a t e and measurement of water-hardened eggs A sample of 50 eggs (25 eggs i f the f e c u n d i t y was very low) were placed i n a compartment of a p e r f o r a t e d styrofoam egg car t o n (Ashton, personal communication). The egg c a r t o n was weighted down and placed i n the top t r a y of a Heath t r a y ( f l o w r a t e 8 L/min) and was covered w i t h b lack p l a s t i c . A f t e r 26 hours (Year 1) or 48 hours (Year 2 ) , the eggs were removed from the egg c a r t o n , drained and t r a n s f e r r e d t o a v i a l c o n t a i n i n g 10 ml Stockard's s o l u t i o n (Velsen 1980). Eggs were measured a f t e r being i n Stockard's s o l u t i o n f o r 68-70 days and 82-86 days i n Year 1 and Year 2, r e s p e c t i v e l y . To assess f e r t i l i z a t i o n , the eggs were viewed under a Zeiss low power microscope. I f no c e l l d i v i s i o n had occurred, the egg was considered not f e r t i l i z e d . Eggs were drained on a paper towel and the weight and diameter of 25 i n d i v i d u a l eggs were determined. The weight was taken t o 0.0001 g on a M e t t l e r AE160 and the diameter was determined by measuring under a Bausch and Lomb microscope f i t t e d w i t h a micrometer. % F e r t i l i z a t i o n = no. f e r t i l i z e d eggs x 100 no. f e r t i l i z e d + no. u n f e r t i l i z e d eggs 3.9 Incubation of eggs Eggs from each mating were incubated s e p a r a t e l y i n up w e l l i n g 1 L j a r incubators as described by Tabachek et a l . (1993). Each l o t of eggs was placed i n an upper s e c t i o n made of ABS pipe w i t h an aluminum screen bottom. The lower s e c t i o n of 55 each in c u b a t o r contained aquarium g r a v e l t o d i s p e r s e any a i r bubbles. Each trough of incubators r e c e i v e d aerated w e l l water at 6-7°C which passed through a household d i r t and r u s t f i l t e r . The f i l t e r s were cleaned every 5-10 days as r e q u i r e d and r e p l a c e d a f t e r 5-10 c l e a n i n g s . In Year 1, eggs were incubated i n f o u r troughs each c o n t a i n i n g 24 incubators w i t h a t o t a l flow r a t e of 14.6 L/min (608 ml/min per i n c u b a t o r ) . In Year 2, eggs were incubated i n two troughs each c o n t a i n i n g 32 i n c u b a t o r s w i t h a t o t a l flow r a t e of 15.1 L/min (470 ml/min per i n c u b a t o r ) . Between 10 days a f t e r spawning and the eyed stage, eggs were t r e a t e d w i t h f o r m a l i n (1/600 or 1667 ppm) f o r 15 min t h r e e times a week t o c o n t r o l fungus growth (Hynes 1984, Fuze et a l . 1985). Incubators were covered w i t h black p l a s t i c and were exposed t o y e l l o w f l u o r e s c e n t and incandescent l i g h t only d u r i n g removal of dead eggs and counting. Yellow f l u o r e s c e n t l i g h t was 350 l u x or 8.2 and 10.1 microeinsteins/mVsec i n Year 1 and Year 2, r e s p e c t i v e l y . A f t e r reaching the eyed stage, the eggs were shocked and incubated one more day before removing the dead eggs. Dead and l i v e eggs were counted at t h i s time. Dead eggs and f r y were removed and counted throughout the r e s t of the i n c u b a t i o n p e r i o d . Dead eggs were preserved i n Stockard's s o l u t i o n i n Year 1 but were i n a d v e r t e n t l y d i s c a r d e d i n Year 2. In Year 2, records were a l s o kept on the numbers of deformed f r y and f r y w i t h blue sac, a c o n d i t i o n i n which a b l u i s h l i q u i d accumulates between the y o l k and the membrane of the y o l k sac (van D u i j n 1973, Balon 1980). In Year 2, samples of f r y w i t h 56 blue sac and other abnormalities were preserved i n Davidson's s o l u t i o n ( f o r m a l i n , 20 ml; ethanol, 30 ml; g l y c e r i n e , 10 ml; g l a c i a l a c e t i c a c i d , 10 ml; d i s t i l l e d water, 30 ml) f o r h i s t o l o g i c a l examination. The equations used t o c a l c u l a t e s u r v i v a l t o the eyed, hatch and swimup stages are as f o l l o w s : % Eyed = no. a l i v e a f t e r shocking eyed eggs x 100 t o t a l no. eggs at beginning of i n c u b a t i o n % Hatched = no. a l i v e a f t e r hatching complete x 100 t o t a l no. eggs at beginning of i n c u b a t i o n % Swimup = no. l i v e f r y at swimup x 100 t o t a l no. eggs at beginning of i n c u b a t i o n % Eyed of f e r t i l i z e d = % eyed x 100 % f e r t i l i z e d % Hatched of eyed = % hatched x 100 % eyed % Swimup of hatched = % swimup x 100 % hatched 3.10 Growth and s u r v i v a l of f r y Fry were r a i s e d i n the same type of system as described f o r the i n c u b a t i o n of eggs i n S e c t i o n 3.9 except t h a t no g r a v e l was pla c e d i n t o the lower s e c t i o n s . Jars were placed i n t o a trough w i t h each j a r connected t o a header pipe running the length of the trough. Water was t r e a t e d by a b i o l o g i c a l g r a v e l f i l t e r 57 l o c a t e d beneath the trough. The f i l t e r (2.46 m x 93 cm x 88 cm), which contained 0.85 m3 g r a v e l , r e s u l t e d i n the water temperature i n c r e a s i n g t o 10°C w i t h no a d d i t i o n a l h e a t i n g . The system r e c e i v e d approximately 15% f r e s h w e l l water as makeup. The temperature was checked d a i l y and was c o n t r o l l e d by a d j u s t i n g the amount of makeup water. The f i l t e r was backwashed once duri n g the time t h a t the c u l t u r e system was used i n Year 1 and i n Year 2. The flow r a t e t o each j a r was 770-820 ml/min. For the f i r s t two days a f t e r p l a c i n g f r y i n a j a r , t h a t j a r was covered w i t h b lack p l a s t i c . I l l u m i n a t i o n was from y e l l o w f l u o r e s c e n t l i g h t s w i t h an i n t e n s i t y of 127 l u x and 1.94 microeinsteins/mVsec w i t h a photoperiod of 10 hr of l i g h t and 14 hr of darkness. The s p e c i f i c growth r a t e and m o r t a l i t y of f r y from each mating were evaluated over a p e r i o d of 41-42 days. Since f i s h spawned over a 3 month p e r i o d , the e v a l u a t i o n of groups of swimup f r y was i n i t i a t e d over a 3 month p e r i o d . At swimup (123 and 125 d a f t e r spawning i n Year 1 and Year 2 r e s p e c t i v e l y ) , groups of f r y were drained of water, batch weighed t o 0.01 g and placed i n t o the r e a r i n g u n i t described above. At t h i s time, the y o l k sac had not been f u l l y absorbed. In Year 1, 50 f r y were placed i n each u n i t while 25 f r y were used i n Year 2 s i n c e the e v a l u a t i o n of the number of f r y t o r a i s e i n each r e a r i n g u n i t (see S e c t i o n 3.13.2) showed th a t the growth r a t e of f r y was s i g n i f i c a n t l y g r eater when f r y number was 25 per u n i t compared 58 to 50. I f adequate numbers of f r y s u r v i v e d , two r e p l i c a t e groups from each female were r a i s e d ; otherwise only one group was r a i s e d . Semimoist s t a r t e r d i e t ( B i o d i e t #1 manufactured by Bioproducts, Warrenton, Oregon) was used. Fresh feed was weighed weekly and i t was placed i n the r e f r i g e r a t o r each n i g h t . The r e s t of the feed was kept frozen (-10°C) i n a p l a s t i c bucket which was f l u s h e d w i t h n i t r o g e n before c l o s i n g . Fry were not fed the day they were placed i n t o the j a r s and they were f e d l i g h t l y 3 times the next day and 6 times a day (between 0800 and 1600) t o excess t h e r e a f t e r . Dead f r y were removed and counted d a i l y and the j a r s were cleaned weekly. At the end of the p e r i o d , f r y were counted and batch weighed t o assess s u r v i v a l and s p e c i f i c growth r a t e . S u r v i v a l (%) = f i n a l no. f r y x 100 i n i t i a l no. f r y S p e c i f i c growth r a t e (%/day) = l o q 0 f i n a l mean wt (mq)-loq Q i n i t i a l mean wt (mg) x 100 no. of days 3.11 C o l l e c t i o n of u n f e r t i l i z e d eggs and f r y f o r measurement and/or a n a l y s i s At spawning, a 50-60 g sample of eggs, which had been drained o f ov a r i a n f l u i d , was placed i n t o a p r e - t a r e d brown g l a s s j a r and tra n s p o r t e d on i c e back t o Winnipeg. From t h i s sample, two 10-15 g samples of eggs were t r a n s f e r r e d i n t o c l e a r 59 g l a s s s c i n t i l l a t i o n v i a l s (Figure 4 ) . Each v i a l was f l u s h e d w i t h n i t r o g e n and wrapped i n a paper towel t o p r o t e c t the sample from exposure t o l i g h t . One v i a l was s t o r e d at -35°C and the other was s t o r e d at -115°C. A f t e r o b t a i n i n g the weights of 30 i n d i v i d u a l eggs t o 0.0001 g on a M e t t l e r AE160 balance, the t o t a l weight of eggs i n the pre-tared j a r was noted, the sample was f l u s h e d w i t h n i t r o g e n and stored at -35°C. In Year 2, samples of swimup f r y were taken f o r a n a l y s i s on the days the f r y e v a l u a t i o n s s t a r t e d . Fry were drained of excess moisture on paper towels, placed i n p l a s t i c tubes w i t h snap tops, f l u s h e d w i t h n i t r o g e n and immediately f r o z e n on dry i c e . They were st o r e d at -115°C u n t i l analyzed f o r v i t a m i n E (Figure 4 ) . 3.12 Analyses of eggs, f r y and/or d i e t s 3.12.1 Vitamin E a n a l y s i s of eggs and f r y Eggs and f r y , stored at -115°C, were analyzed i n d u p l i c a t e f o r v i t a m i n E (a-tocopherol) content (Figure 4) by u s i n g the e x t r a c t i o n method of King (1983) and the adapted spectrophotometric method of the AOAC (1984) us i n g bathophenathroline (Appendix 3a). Precautions were taken t o minimize l o s s of v i t a m i n E through exposure t o heat, l i g h t and oxygen. Subdued f l u o r e s c e n t l i g h t i n g was used (475 l u x ) , v e s s e l s were f l u s h e d w i t h n i t r o g e n and the temperature was kept below 45°C during evaporation of s o l v e n t s . 60 For comparison, samples of eggs which were c o l l e c t e d from w i l d A r c t i c c h a r r a t Tree R i v e r , NWT were analyzed. These eggs were c l o s e t o hatching when sampled. 3.12.2 Dry matter and selenium The 30 g samples of eggs stored at -35°C were f r e e z e - d r i e d (Figure 4) w i t h the d i f f e r e n c e i n i n i t i a l and f i n a l weight used t o c a l c u l a t e percent dry matter. Three samples of f i n e l y ground eggs per treatment per sampling year were analyzed f o r selenium i n the same way as the d i e t s (S e c t i o n 3.12.4). 3.12.3 L i p i d and f a t t y a c i d analyses The 10-15 g samples of eggs taken i n Year 2 and st o r e d a t -35°C (Figure 4) were used f o r e x t r a c t i o n of t o t a l l i p i d s by a modi f i e d B l i g h and Dyer (1959) method (Appendix 3a). Column chromatography w i t h s i l i c a Sepak c a r t r i d g e s was used t o separate t o t a l l i p i d s i n t o n e u t r a l l i p i d and p o l a r l i p i d f r a c t i o n s by m o d i f i c a t i o n of the method of Ashton (1991). T o t a l l i p i d was reduced t o 60-70 mg and only one Sepak was used per sample. N e u t r a l l i p i d s were e l u t e d w i t h 30 ml 15% hexane i n chloroform and p o l a r l i p i d s were e l u t e d w i t h 30 ml methanol f o l l o w e d by 30 ml chloroform: methanol (1:1). D u p l i c a t e a l i q u o t s of the f r a c t i o n s were assayed f o r l i p i d by the dichromate o x i d a t i o n spectrophotometric assay (Fales 1971). Standard curves were conducted using n e u t r a l and p o l a r l i p i d s c o l l e c t e d from A r c t i c 61 c h a r r eggs i n order to have a comparable l e v e l of o x i d a t i o n . R e s u l t s from t h i s assay compared w e l l w i t h r e s u l t s obtained by weighing a l i q u o t s d r i e d at 70°C. The r e l a t i v e percent of n e u t r a l and p o l a r l i p i d s i n each sample of t o t a l l i p i d was e s t a b l i s h e d . To o b t a i n percent t o t a l l i p i d on a dry matter b a s i s , t o t a l l i p i d was a l s o e x t r a c t e d from dry samples of eggs taken i n Year 1 and 2 (Figure 4) and d u p l i c a t e a l i q u o t s of l i p i d e x t r a c t s were d r i e d at 70°C and weighed. N e u t r a l and p o l a r l i p i d s were converted t o percent dry matter: N e u t r a l l i p i d (% dry matter) = n e u t r a l l i p i d ( r e l a t i v e %) x t o t a l l i p i d (% dry matter) 100 P o l a r l i p i d (% dry matter) = p o l a r l i p i d ( r e l a t i v e %) x t o t a l l i p i d (% dry matter) 100 Since the p r e c i s i o n of the n e u t r a l l i p i d and p o l a r l i p i d f r a c t i o n a t i o n s was s a t i s f a c t o r y , only one determination per sample was conducted. The p u r i t y of the 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 was checked by s p o t t i n g a l i q u o t s of the f r a c t i o n s on S i l i c a Gel G (0.25 mm) t h i n - l a y e r p l a t e s and p l a c i n g the p l a t e s i n an ascending s o l v e n t system of hexane:diethyl ether : g l a c i a l a c e t i c a c i d = 80:20:2 ( C h r i s t i e 1981). L i p i d standards used f o r i d e n t i f i c a t i o n of the bands inc l u d e d p h o s p h a t i d y l c h o l i n e , c h o l e s t e r o l , c h o l e s t e r y l e s t e r , o l e i c a c i d , t r i o l e i n , 1,2- and 62 1,3- d i g l y c e r i d e s and monoglyceride. L o c a t i o n of the bands was v i s u a l i z e d by spraying the a i r - d r i e d p l a t e s w i t h s u l p h u r i c a c i d and heating them at 110°C. To determine the f a t t y a c i d composition of the n e u t r a l and p o l a r l i p i d s of the eggs c o l l e c t e d i n Year 2 and of the d i e t s used i n Year 1 and 2, 5 mg a l i q u o t s of t o t a l l i p i d were t r a n s e s t e r i f i e d using the a c i d - c a t a l y z e d method w i t h toluene and methanolic HC1 and e x t r a c t e d i n hexane (Yurkowski 1989). The e x t r a c t s were p u r i f i e d by app l y i n g them on S i l i c a Gel H p l a t e s and p l a c i n g them i n an ascending TLC tank w i t h toluene. The methyl e s t e r band was v i s u a l i z e d by spraying w i t h 0.02% Rhodamine 6G and viewing i n u l t r a v i o l e t l i g h t . The methyl e s t e r band was removed from the p l a t e , e l u t e d w i t h chloroform and concentrated i n hexane. F a t t y a c i d p r o f i l e s were obtained by i n j e c t i n g the samples i n t o a Va r i a n Aerograph Model 3400 Chromatograph f i t t e d w i t h a Supelcowax s i l i c a c a p i l l a r y column (30 m, 0.32 mm I.D., 0.25 jim f i l m t h i c k n e s s ) . I n j e c t o r and d e t e c t o r temperatures were 235 and 250°C, r e s p e c t i v e l y and the column temperature was held at 175°C f o r 25 min, incre a s e d 1°C per min f o r 35 min and held at 210°C f o r 30 min. I d e n t i t y of each peak had been determined (Yurkowski, personal communication) by the methods described by Yurkowski (1989). Peak areas were measured by an e l e c t r o n i c i n t e g r a t o r (Varian S t a r I n t e g r a t o r , R e v i s i o n A, Varian A s s o c i a t e s , Walnut Creek, CA) and converted i n t o area percent of each f a t t y a c i d . One 63 f a t t y a c i d determination per sample was conducted a f t e r determining t h a t the p r e c i s i o n of the t e s t was s a t i s f a c t o r y . 3.12.4 A n a l y s i s of d i e t s Major i n g r e d i e n t s and d i e t s were analyzed f o r crude p r o t e i n by the macro-Kjeldahl method (AOCS 1989). Ash and moisture con c e n t r a t i o n s were a l s o determined (AOAC 1984). The d i e t s were analyzed f o r t o t a l Ca, Na, K, Mg, Mn, Zn, Cu, Fe by n i t r i c a c i d - p e r c h l o r i c a c i d d i g e s t i o n followed by atomic a b s o r p t i o n spectrophotometry. Selenium determination was by the method of V i j a n and Wood (1976). Soluble r e a c t i v e phosphorus was analyzed by the molybdenum blue method of St a i n t o n et a l . (1974). 3.13 E v a l u a t i o n of methods 3.13.1 E v a l u a t i o n of egg r i n s e s and extenders In Year 1, a t o t a l of 5 f i s h from tanks 2 (HLHE) and 5 (COMM) spawned between September 29, 1988 and October 12, 1988. The f e r t i l i z a t i o n r a t e was low (mean = 59 ± 36) and ranged from 0-88%. Egg r i n s e s and extenders were t e s t e d October 26, November 2, 1989 and December 27, 1989 on 2 f i s h which spawned each day t o determine i f the method being used was a f f e c t i n g the f e r t i l i z a t i o n r a t e . The f o l l o w i n g were t e s t e d l a ) w e l l water as r i n s e and extender on October 26, lb) no r i n s e and w e l l water as extender on November 2 (normal procedure at RARC), 2) l a c t o s e - CaCl 2-NaCl (Hamor 1987), 3) Tris-glycine.HC1 ( B i l l a r d 1977) and 4) NaHC03 (Wilcox et a l . 1984). An equal weight (20 or 50 g) of 64 dra i n e d eggs was d i v i d e d i n t o 4 l o t s and 50 ml of r i n s e s o l u t i o n was poured i n t o each pan, s w i r l e d and poured o f f . An equal volume (0.5 ml) of m i l t from each of the same two males was added t o 25 ml of the s o l u t i o n and i t was added t o the eggs. Eggs were mixed gently f o r two minutes and then r i n s e d w i t h w e l l water u n t i l the water was c l e a r . Samples of eggs were incubated as d e s c r i b e d t o assess f e r t i l i z a t i o n r a t e and s u r v i v a l t o swimup. Based on the f e r t i l i z a t i o n r a t e data, the d e c i s i o n was made t o use the NaHC03 r i n s e . In Year 2, s o l u t i o n s l b , 2 and 4 were re- e v a l u a t e d on November 15, 1989 using 30-40 g eggs, 35 ml r i n s e , 15 ml s o l u t i o n t o 0.4-0.7 ml m i l t from each of two males and f e r t i l i z e d and incubated as above. 3.13.2 E v a l u a t i o n of number of f r y t o r a i s e i n each r e a r i n g u n i t During Year 1, the number of f r y t h a t c o u l d be r a i s e d i n each j a r was evaluated. Swimup f r y , a l l o f f s p r i n g of one female, were counted out i n t o two groups of 10, 25, 50, 100 and 300 f r y and batch weighed. The groups of 10 t o 100 f r y were placed i n t o j a r s using a randomized design. Two groups of 300 f r y were placed i n 60 L f i b e r g l a s s tanks as de s c r i b e d by Tabachek (1983) w i t h a flow r a t e of 1.8 L/min. Fry were r a i s e d i n the manner described i n Se c t i o n 3.10. 3.14 S t a t i s t i c a l a n a l y s i s In order t o s a t i s f y the c o n d i t i o n s of the a n a l y s i s of v a r i a n c e , the data must f i t a normal d i s t r i b u t i o n and have 65 va r i a n c e s which are equal and not r e l a t e d t o the means ( L i 1964). A r c s i n transformation was c a r r i e d out on a l l data from b i n o m i a l d i s t r i b u t i o n s ( f e r t i l i z a t i o n and embryonic and f r y s u r v i v a l ) . Log transformation was c a r r i e d out on v i t a m i n E c o n c e n t r a t i o n and content of eggs and f r y as w e l l as egg weights because variances were p r o p o r t i o n a l t o the means. B a r t l e t t ' s t e s t was used t o t e s t f o r homogeneity of variances p r i o r t o conducting a n a l y s i s of va r i a n c e . Since most data sets were unbalanced, the general l i n e a r model (PROC GLM) was used t o detect s i g n i f i c a n t d i f f e r e n c e s between means by a n a l y s i s of variance (SAS I n s t i t u t e 1985). One-way a n a l y s i s of variance was conducted on data f o r a l l f i v e d i e t s u s i n g the model: Y = \i + a + e where Y = measurement, \i = mean of p o p u l a t i o n s , a = e f f e c t of d i e t and E = experimental e r r o r . A n a l y s i s of va r i a n c e was used on data f o r the four d i e t s comprising the 2 x 2 f a c t o r i a l design by u s i n g the model: Y = (x + a + p + ap + e where Y = measurement, \i = mean of p o p u l a t i o n s , a = e f f e c t of l i p i d , p = e f f e c t of v i t a m i n E, ap" = i n t e r a c t i o n of l i p i d and v i t a m i n E and E = experimental e r r o r . Since no r e p l i c a t e tanks of broodstock were subjected t o the r e s p e c t i v e treatments, v a r i a t i o n s among f i s h w i t h i n each treatment served as the t e s t i n g e r r o r . Duncan's m u l t i p l e range t e s t was used t o detect 66 s i g n i f i c a n t d i f f e r e n c e s between means. The l e v e l of s i g n i f i c a n c e was set at 95% i n a l l s t a t i s t i c a l analyses. Regression a n a l y s i s was conducted t o det e c t c o r r e l a t i o n between s e l e c t e d parameters such as the e f f e c t of f i s h weight on f e c u n d i t y or the e f f e c t of s e l e c t e d d i e t a r y f a t t y a c i d s on the f a t t y a c i d composition of eggs (SAS I n s t i t u t e 1985). The l e v e l of s i g n i f i c a n c e was set at 95%. Regression a n a l y s i s detected a c o r r e l a t i o n between embryonic s u r v i v a l and % n e u t r a l l i p i d s i n the eggs and i t appeared th a t t h i s r e l a t i o n s h i p might a l s o be a f f e c t e d by d i e t . A n a l y s i s of covariance was used t o p a r t i t i o n d i f f e r e n c e s due t o d i e t and n e u t r a l l i p i d s on the i n t e r c e p t s and slopes of the r e g r e s s i o n . C h i square a n a l y s i s ( L i 1964) was conducted t o compare the e f f e c t s of s e l e c t e d treatments ( e f f e c t s of v i t a m i n E or l i p i d ) on the p r o p o r t i o n of females t h a t spawned or males t h a t produced m i l t . Data from the same d i e t a r y l i p i d or f o r the same d i e t a r y v i t a m i n E treatments were pooled t o increase the sample s i z e f o r each group t o o b t a i n expected values of 5 ( L i 1964). Data f o r the COMM d i e t s were excluded from t h i s a n a l y s i s . When the expected number was too low f o r Chi square t e s t t o be v a l i d (as i n t e s t i n g f o r the e f f e c t of d i e t on spawning t i m e ) , F i s h e r ' s exact t e s t was used (SAS I n s t i t u t e 1990). 67 Power a n a l y s i s was conducted (Dixon and Massey 1969) as recommended by Peterman (1990) t o determine the power of the a n a l y s i s of va r i a n c e given the variance t h a t was observed i n embryonic s u r v i v a l . 68 RESULTS 4.1 Feed composition Experimental d i e t s contained 46.9-47.8% crude p r o t e i n and 7.5-7.8% ash, on a dry weight b a s i s (Table 2 ) . T o t a l l i p i d contents of the low l i p i d d i e t s were 11.8-12.6% w h i l e those of the high l i p i d d i e t s were 18.8-19.0%. M e t a b o l i z a b l e energy was estimated as 3.23 and 3.90 k c a l / g f o r the LL and HL d i e t s , r e s p e c t i v e l y . COMM contained 42.4% p r o t e i n , 16.8% l i p i d and 7.8% ash and was estimated t o co n t a i n 120 mg t o t a l a-tocopherol /kg (Martin Feed M i l l s , personal communication), 223 mg ethoxyquin/kg and 3.47 k c a l metabolizable energy/g. The fo u r experimental d i e t s contained t o t a l mineral c o n c e n t r a t i o n s of 16 mg Ca/g, 2.4 mg Na/g, 1.4 mg K/g, 1.5 mg Mg/g, 1.3 mg P/g, 91 u,g Mn/g, 228 \ig Fe/g, 26 ng Cu/g, 189 \ig Zn/g and 1.02 \ig Se/g on a dry weight b a s i s . COMM had concentrations of Ca and K s i m i l a r t o the above, lower P of 1.1 mg/g and higher c o n c e n t r a t i o n s of the o t h e r s : 2.8 mg Na/g, 1.9 mg Mg/g, 116 \xg Mn/g, 42 fxg Cu/g, 210 [ig Zn/g and twice as much i r o n at 456 ng Fe/g. COMM used i n Year 1 contained 0.75 jig Se/g w h i l e the two l o t s used i n Year 2 contained 0.88 and 1.96 jig Se/g. In a d d i t i o n t o the supplemented amount of d l - a - t o c o p h e r y l a c e t a t e , endogenous a-tocopherol was present i n the other i n g r e d i e n t s . The basa l mix contained 7.7 ± 0.2 mg a-tocopherol /kg. H e r r i n g o i l , purchased one month p r i o r t o t h a t purchased 69 Table 2. Composition of the d i e t s (dry matter b a s i s ) . D i e t LLLE LLHE HLLE HLHE COMM2 Moisture (%) 8.71 0.80 8.8 0.60 8.6 0.56 8.7 0.72 7.1 1.16 Crude p r o t e i n (%) 46.9 0.53 47.2 0.38 47.4 0.58 47.8 0.51 42.4 1.27 T o t a l l i p i d (%) 11.8 0.34 12.6 0.59 19.0 1.11 18.8 1.20 16.8 1.25 Ash (%) 7.6 0.09 7.6 0.06 7.6 0.23 7.5 0.14 7.8 1.49 F i b e r 3 (%) 9.5 9.5 2.5 2.5 2.5 N i t r o g e n - f r e e e x t r a c t 4 (%) 23.7 22.6 23.5 23.4 30.5 Ethoxyquin 5 (mg/kg) 160 160 160 160 223 [dl]-a-Tocopheryl a c e t a t e 6 (mg/kg) 30 600 30 600 - [ d l ] - a - T o c o p h e r o l 7 (mg/kg) 14 14 24 24 - T o t a l [ d l ] - a - t o c o p h e r o l + to c o p h e r y l acetate (mg/kg) 44 614 54 624 120 M e t a b o l i z a b l e energy 8 (kcal/g) 3.28 3.34 3.86 3.86 3.59 1 Mean (upper l i n e ) ± standard d e v i a t i o n (lower l i n e ) f o r the 5 batches of experimental d i e t s and 3 l o t s of COMM d i e t used throughout t h i s study. 2 Estimated values f o r the commercial d i e t on p r o p r i e t a r y i n f o r m a t i o n s u p p l i e d t o the author by the manufacturer. 3 Estimated using NRC (1981) t a b l e s on composition of i n g r e d i e n t s . 4 Estimated u s i n g 1 0 0 - ( t o t a l l i p i d + crude p r o t e i n + ash + f i b e r ) . 5 T o t a l ethoxyquin i n f i s h meal and o i l . 6 Taken from formulations i n Table 1. 7 Estimated from a n a l y s i s of basa l mix and h e r r i n g o i l as de s c r i b e d i n S e c t i o n 3.3.3. 8 Estimated using values of 4.2 k c a l / g p r o t e i n , 8.0 k c a l / g l i p i d and 1.6 k c a l / g raw s t a r c h ( B r e t t and Groves 1979). 70 f o r t h i s experiment, which was not frozen contained 184 ± 5.7 mg a-tocopherol/kg o i l w h i l e the sample which was frozen contained 144 ± 0.7 mg a-tocopherol/kg o i l . Samples of h e r r i n g o i l used i n t h i s experiment were a l s o shipped t o Hoffmann-LaRoche but they were not analyzed. Since the o i l used i n t h i s experiment had been f r o z e n before use, the l e v e l of endogenous a-tocopherol was estimated t o be 144 mg/kg o i l . H e r r i n g o i l was estimated as c o n t r i b u t i n g 14 mg a-tocopherol/kg t o the low l i p i d d i e t s and 24 mg/kg a-tocopherol t o the high l i p i d d i e t s . I t i s expected t h a t much of the endogenous tocopherol would o x i d i z e d u r i n g the manufacturing process and on storage s i n c e a-tocopherol i s e a s i l y o x i d i z e d (Bauernfeind 1980). Vitamin E acetate content of the d i e t s was not analyzed. When v i t a m i n E a c e t a t e , donated by Hoffmann-LaRoche f o r t h i s research, was compared t o v i t a m i n E acetate obtained from Sigma Chemical Co., S t . L o u i s , MO and ICN Biomedicals, Costa Mesa, CA, the s p e c t r a were i d e n t i c a l and the absorbances were w i t h i n 1.4% when e t h a n o l i c s o l u t i o n s of the same co n c e n t r a t i o n s were compared i n a spectrophotometer. The peroxide value of the h e r r i n g o i l used i n making the four experimental d i e t s increased from 1.44 ± 0.38 meq/kg i n J u l y 1988 t o 3.33 ± 0.11 meq/kg at the f i n a l a n a l y s i s i n September 1989. These values were w i t h i n the g u i d e l i n e s f o r q u a l i t y c o n t r o l of f i s h o i l s of < 5-10 meq/kg (NRC 1981, Canadian Working Group f o r F i n f i s h N u t r i t i o n , personal communication). 71 4.2 Water temperature As d e s c r i b e d i n Se c t i o n 3.5.3, v a r i a t i o n i n the temperatures between tanks was observed on day 9 of the experiment. At t h a t time, water temperatures were 7.2°C (LLLE), 7.2°C (HLHE), 9.7°C (LLHE), 10.2°C (HLLE) and 12.2°C (COMM). That i s , temperatures were low (7.2°C) f o r groups r e c e i v i n g one LL and one HL d i e t and f o r one LE and one HE d i e t . S i m i l a r l y , temperatures were higher (9.7-10.2°C) f o r groups r e c e i v i n g one LL and one HL d i e t and f o r one LE and one HE d i e t . No data on water temperature was c o l l e c t e d during the f i r s t 8 days of the experiment. F o l l o w i n g changes t o the water system t o c o r r e c t the problem, there was l i t t l e d i f f e r e n c e i n water temperature between tanks. D i f f e r e n c e s i n temperatures between tanks a f t e r day 9 ranged from 0 t o 1.5°C (0.27°C ± 0.11) (mean ± standard d e v i a t i o n ) w i t h d i f f e r e n c e s of over 1.0°C o c c u r r i n g on 8 days d u r i n g the 544 day pe r i o d measured. Mean weekly water temperatures d u r i n g Year 1 and 2 are shown i n F i g u r e 5 and Appendix 2a. During Year 1, mean weekly water temperature i n c r e a s e d from 8.3 t o 8.8°C during J u l y and decreased s t e a d i l y down t o 7.1°C from August t o mid-November as outdoor and room temperatures decreased. A f t e r room heaters were turned on i n mid-November, water temperature increased t o 7.7-7.8°C. In Year 1, spawning occurred between September 29 and December 27, 1989 when mean weekly temperatures were 7.1 t o 7.9°C. 7 2 73 In Year 2, mean weekly temperature v a r i e d between 7.7 and 8.4°C from January t o May 1988, between 8.0 and 8.6°C from June t o J u l y (Figure 5) and d e c l i n e d from 8.6 t o 7.2°C from August t o November. As i n Year 1, water temperature i n c r e a s e d t o 8.5°C from December 1989 t o January 1990 when room heaters were used. Mean weekly water temperature was 7.1 t o 8.5°C du r i n g spawning i n Year 2. S i m u l a t i o n of water temperature i n Year 2 t o match t h a t of Year 1 was good except between November 28 and December 25, 1989 when i t averaged 0.6-0.9°C higher than corresponding dates i n Year 1. Flow r a t e of the 6.5°C make-up water was i n c r e a s e d as much as p o s s i b l e during t h i s time but water temperature could not be decreased t o match t h a t of Year 1. 4.2 Water chemistry Chemical composition of the incoming w e l l water has been measured (Province of Manitoba 1987). Measurements i n d i c a t e d the f o l l o w i n g : pH, 7.75; c o n d u c t i v i t y , 918 jAmho/cm; hardness, 424 mg/L; t o t a l d i s s o l v e d s o l i d s , 560 mg/L (1982); a l k a l i n i t y (HC0 3), 486 mg/L; t o t a l a l k a l i n i t y (CaC0 3), 398 mg/L; Ca ( e x t r a c t a b l e ) , 66.1 mg/L, Mg ( e x t . ) , 63.0 mg/L, Na ( e x t . ) , 40.8 mg/L; Fe ( e x t . ) , 0.114 mg/L (1982); Mn ( e x t . ) , <0.02 mg/L; S0 4 ( s o l u b l e ) 68 mg/L; CI ( s o l . ) , 43 mg/L and F, 0.28 mg/L. These parameters are a l l w i t h i n the suggested chemical values f o r hatchery water s u p p l i e s used t o r a i s e t r o u t ( P i p e r et al. 1982) except f o r hardness. The hardness was 424 mg/L which exceeds the suggested range of 10-400 mg/L. 74 Mean values f o r water chemistry parameters f o r each tank over the 8 sampling times are shown i n Table 3 and f o r the 5 tanks f o r each sampling time are shown i n Table 4. Over the repeated samplings, there were no s i g n i f i c a n t d i f f e r e n c e s between tanks i n any water q u a l i t y parameters. Un-ionized ammonia was always l e s s than t h a t l i s t e d i n the water q u a l i t y c r i t e r i a f o r optimum h e a l t h of salmonids s t a t e d by P i p e r et al. (1982) as 0.0125 ppm (12.5 \iq/L). The highest observed un- i o n i z e d ammonia l e v e l (8.96 ng/L) occurred on February 6, 1989. This occurred at a time when the e f f i c i e n c y of the b i o l o g i c a l f i l t e r s may have been reduced when feeding resumed on January 12, 1989 a f t e r feed was w i t h h e l d d u r i n g spawning. The f i r s t backwashing of the f i l t e r s occurred 4 days a f t e r t a k i n g the water samples. N i t r i t e - N was w i t h i n the suggested c r i t e r i a of 0.06 ppm (60 ng/L). While the suggested pH range f o r t r o u t i s 6.5-8.0, the pH of the r e c i r c u l a t e d water on most sampling days was 7.9-8.2 and 8.8-9.0 on two sampling days. 4.4 Feed fed and feed e f f i c i e n c y Data were c o l l e c t e d on the weight of feed o f f e r e d t o each tank of f i s h d u r i n g each feeding p e r i o d and these values were converted t o dry weight of feed. Feed fed and weight gain between each weighing p e r i o d i n Years 1 and 2 are shown i n Appendices 2b and 2c and are provided i n Table 5. I t i s expected t h a t d i f f e r e n c e s i n water temperature d u r i n g the f i r s t 9 days of the experiment a f f e c t e d feed i n t a k e and u l t i m a t e l y 75 Table 3. Water chemistry data f o r each tank (n=8). Tank Temp. D.O.1 pH NH4-N N02-N N03-N °C mg/L JAg/L ng/L Hg/L 2 8.3 11.4 8.2 28.8 2.25 455.3 3 8.5 11.4 8.4 26.3 1.00 366.0 6 8.6 11.4 8.3 35.8 0.88 418.5 7 8.4 11.4 8.3 28.4 0.88 404.5 5 8.2 11.2 8.3 38.5 1.13 364.8 Mean 8.4 11.4 8.3 33.8 1.23 401.8 SD2 0.14 0.15 0.05 4.84 0.58 38.1 1 D i s s o l v e d oxygen 2 Standard d e v i a t i o n Table 4. Water chemistry data f o r each sampling day (n=5). 77 Table 5. Feed f e d, weight gain and feed e f f i c i e n c y f o r each tank of f i s h fed f o r 71 days i n Year 1 and 239 days i n Year 2. Di e t LLLE LLHE HLLE HLHE COMM LL 1 HL2 T o t a l feed fed (kg, d r y ) 3 Year 1 15.2 14.9 16.4 17.1 20.1 15.0 16.7 Year 2 24.2 28.9 34.3 34.2 40.4 26.6 34.3 T o t a l weight gain (kg) Year 1 9.75 8.12 12.24 12.28 15.45 8.94 13.87 Year 2 15.42 13.78 6.73 9.84 24.81 14.60 8.29 Feed e f f i c i e n c y (%) 4 Year 1 64.0 54.6 74.7 71.9 76.8 59.3 73.3 Year 2 63.7 47.8 19.6 29.4 61.4 55.8 24.2 Mean no. f i s h 5 Year 1 25 25 25 25 25 25 25 Year 2 21 23 22 24 24 22 24 Estimated dry weight feed fed per f i s h (g) Year 1 610 595 655 683 805 603 669 Year 2 1152 1254 1561 1425 1683 1203 1493 1 LL = mean of the two LL d i e t s 2 HL = mean of the two HL d i e t s 3 Dry feed= feed, g as fed x (dry matter,% /100) 4 Feed e f f i c i e n c y = ( t o t a l weight g a i n , g x 100)/dry feed f e d , g 5 Average number of f i s h present during the feeding p e r i o d 78 weight g a i n d u r i n g t h i s p e r i o d . During these f i r s t 9 days, tanks of f i s h h e l d at the higher temperatures had higher feed in t a k e s and weight gains than t h e i r counterparts fed d i e t s w i t h the same l i p i d l e v e l . However, at the end of 71 days of feeding i n Year 1, t o t a l weight gains were almost i d e n t i c a l i n both HL groups. In c o n t r a s t , the LLLE group, which had been held at the lower temperature, had gained a t o t a l of 1600 g more than i t s LLHE counterpart held at the higher temperature. The e f f e c t t h a t d i f f e r e n c e s i n t h i s 9-day p e r i o d had on weight gain d u r i n g the remainder of the experimentcannot be estimated. In Year 1, the weight of dry feed fed i n 71 days was s l i g h t l y lower f o r the f i s h fed the LL d i e t s (15.0 kg) compared t o the HL d i e t s (16.7 kg). Feed fed i n Year 2 was a l s o lower f o r f i s h fed the LL d i e t s (26.6 kg) compared t o the HL d i e t s (34.3 kg). More COMM d i e t was fed i n both Year 1 (20.1 kg) and Year 2 (40.4 kg) than a l l other d i e t s . Having equal numbers of f i s h i n each tank i n Year 1 makes comparison of feed fed t o each tank of f i s h simple. However, i n Year 2 the numbers of f i s h v a r i e d between tanks and w i t h i n a tank over the year. To compare feed fed on the b a s i s of equal numbers of f i s h , a gross e s t i m a t i o n of the feed i n t a k e per f i s h has been c a l c u l a t e d and i n d i c a t e s t h a t feed fed per f i s h was not markedly lower w i t h the LL d i e t s compared t o the HL d i e t s . In a d d i t i o n t o the unequal numbers of f i s h i n each tank i n Year 2, a p o r t i o n of the p o p u l a t i o n d i d not feed and t h i s was e s p e c i a l l y n o t i c e a b l e during Year 2. 79 F i s h behaviour a f f e c t e d feed i n t a k e i n two cases. On the t w e l f t h day of the experiment, i t was observed t h a t f i s h i n tank 6 stayed crowded together i n the area under the water i n l e t . This behaviour appeared t o i n h i b i t feeding a c t i v i t y and water turbulence i n t h i s area made i t impossible t o determine i f s a t i e t y had been reached. D i s s o l v e d oxygen was 10.4 mg/L and temperature was 8.5°C i n both tanks. When the d i r e c t i o n of water f l o w was a l t e r e d , f i s h would d i s p e r s e throughout the tank, but w i t h i n a short time they resumed t h e i r p o s i t i o n s under the water i n l e t . On day 43 of the experiment, f i s h from tank 6 were placed i n tank 7 and those i n tank 7 were placed i n tank 6. Both groups acted normally and dispersed throughout the tank. Therefore, i t appeared t o be n e i t h e r the e f f e c t of the tank nor the f i s h themselves but a t a n k - f i s h i n t e r a c t i o n t h a t caused t h i s behaviour t o be e x h i b i t e d . A f t e r t h i s change was made, cha r r were l e f t i n these tanks and the d i e t s each group had been fed were maintained. A l l records were changed t o show t h a t tank 7's f i s h and t h e i r r e s p e c t i v e d i e t were o r i g i n a l l y assigned t o tank 6 and v i c e v e r s a (Figure 3 ) . I t i s expected t h a t feed i n t a k e i n tank 6 was reduced p r i o r t o t h e i r being moved t o tank 7. When feeding was resumed a f t e r spawning at the beginning of Year 2, one emaciated male f i s h i n tank 3 (fed LLLE i n Year 1 and HLLE i n Year 2) became very aggressive. This f i s h , which was one of the sma l l e s t i n the tank, became very t e r r i t o r i a l and fo r c e d a l l the other f i s h i n t o the area under the water i n l e t 80 where water turbulence d i d not a l l o w them t o feed normally. I f a f i s h ventured out of t h i s area, the aggressive f i s h would challe n g e i t . When the aggressive f i s h was removed from the tank on March 16, 1989, the remaining f i s h a t t a i n e d a more normal d i s t r i b u t i o n i n the tank and feed i n t a k e i n c r e a s e d . The aggressive f i s h was not replaced. Small numbers of f i s h were considered t o be " l o n e r s " - f i s h t h a t stayed o u t s i d e of the main group of f i s h and always remained at the periphery of the tank. These f i s h g e n e r a l l y d i d not feed. Even when a feed p e l l e t was thrown d i r e c t l y i n f r o n t of these f i s h , the p e l l e t was e i t h e r ignored completely or taken i n t o the mouth and then s p i t out. In Year 1, feed e f f i c i e n c y was higher f o r f i s h fed the HL d i e t s (73%) and COMM d i e t (77%) than observed f o r those fed the LL d i e t s (59%). The reverse occurred i n Year 2 when feed e f f i c i e n c y was lower f o r the HL d i e t s than the LL d i e t s (56%) and COMM d i e t (61%). Feed e f f i c i e n c y was 29% f o r the HLHE d i e t and 20% f o r the HLLE d i e t . P a r t of the reason f o r the extremely poor feed e f f i c i e n c y of the l a t t e r group was the e f f e c t t h a t the aggressive male f i s h had on the feeder's assessment of s a t i e t y . The feed o f f e r e d was probably not a l l consumed. A r c t i c c h a r r took feed from the bottom of the tank as w e l l as throughout the water column. One f i s h i n each tank fed LLLE 81 and COMM i n Year 1 and HLLE i n Year 2 r e l e a s e d almost a l l of i t s eggs i n t o the tank. The small screen l o c a t e d i n the middle of the tanks had p e r f o r a t i o n s which d i d not a l l o w e i t h e r feed p e l l e t s or f i s h eggs t o pass through i t . When s e v e r a l eggs were placed i n a tank t o observe the response of the f i s h , the eggs were r e a d i l y consumed. 4.5 Growth of broodstock F i s h had not been tagged during the previous spawning season and had no spawning c o l o u r a t i o n at the time t h i s experiment was i n i t i a t e d . An experienced hatchery manager found t h a t i t was d i f f i c u l t t o determine the sex of some f i s h . In Year 1, 11 out of 75 f i s h and an a d d i t i o n a l 3 f i s h i n Year 2 ( t o t a l of 2-5 f i s h i n each t a n k ) , i n i t i a l l y b e l i e v e d t o be female, developed primary and secondary sexual c h a r a c t e r i s t i c s of males (mean i n i t i a l weight = 993 g, range = 690-1455 g ) . E i g h t of them gained weight throughout Year 2, w h i l e 6 l o s t weight. A l l those which gained weight produced m i l t w h i l e only 2 of the f i s h t h a t l o s t weight produced m i l t . F i s h which d i d not mature u n t i l Year 2 were l a r g e f i s h (2-3 kg at spawning time) and they had a very s i l v e r y appearance u n t i l they began t o mature. The a c t u a l number of male and female f i s h i n each tank and u l t i m a t e l y the length and weight d i s t r i b u t i o n of males and females i n each tank was d i f f e r e n t than expected. I n i t i a l weight d i s t r i b u t i o n s of male and female f i s h i n each tank are presented i n Table 6. 8 2 83 I n i t i a l and f i n a l weights of f i s h i n Year 1 and 2 are presented i n Tables 7 and 8. High d i e t a r y l i p i d content s i g n i f i c a n t l y i ncreased the s p e c i f i c growth r a t e of females i n Year 1 but not i n Year 2 wh i l e d i e t a r y v i t a m i n E had no i n f l u e n c e (Table 9). There were no e f f e c t s of l i p i d o r v i t a m i n E on the s p e c i f i c growth r a t e s of males i n e i t h e r year. In Year 1, feeding the COMM d i e t r e s u l t e d i n s i g n i f i c a n t l y higher growth r a t e s i n females than f o r those fed other d i e t s except HLLE and i n s i g n i f i c a n t l y higher growth r a t e s i n males compared t o those fed LLHE. There was a low incidence of f i s h l o s i n g weight i n Year 1, but t h i s incidence increased i n Year 2 (Table 10). There was no s i g n i f i c a n t d i f f e r e n c e between the p r o p o r t i o n of males or females which l o s t weight and no s i g n i f i c a n t e f f e c t of d i e t a r y l i p i d or v i t a m i n E on the p r o p o r t i o n of males or females l o s i n g weight. F i s h which l o s t weight c o n t i n u a l l y throughout Year 1 and/or Year 2 became emaciated but only 2 of these f i s h d i e d p r i o r t o spawning i n Year 2 (one fed LE and 1 fed COMM). 4.6 Spawning 4.6.1 Time of spawning In Year 1, there appeared t o be an e f f e c t of d i e t a r y l i p i d l e v e l on the i n i t i a l spawning date and the le n g t h of the spawning p e r i o d (Table 11). F i s h fed the HL d i e t s began t o spawn on September 29, 1988 w i t h s i x out of 26 f i s h spawning d u r i n g the f i r s t 5 weeks. Those fed the LL d i e t s d i d not begin t o spawn u n t i l November 2, 1988. F i s h e r ' s exact t e s t was used 8 4 8 5 8 6 87 Table 10. Number of male and female c h a r r which l o s t o r gained weight throughout Year 1 or 2. Tank 3 6 2 7 5 T o t a l D i e t a r y v i t a m i n E LE LE HE HE COMM LE HE Lo s t weight only i n Year 1 Male 1 0 0 2 0 1 2 Female 0 0 0 0 0 0 0 Lo s t weight onl y i n Year 2 Male 6 6 2 4 1 12 6 Female 5 3 5 4 1 8 9 L o s t weight i n both Year 1 and 2 Male 0 0 2 0 1 0 2 Female 0 0 0 0 0 0 0 Lo s t weight i n e i t h e r Year 1 or 2 Male 7 6 4 6 2 13 10 Female 5 3 5 4 1 8 9 Gained weight i n Year 1 and 2 Male 4 6 8 9 11 10 17 Female 6 10 7 6 9 16 13 88 Table 11. Number of females which spawned durin g the 14-week spawning p e r i o d i n Year 1 and the 11-week spawning p e r i o d i n Year 2. Week of Die t spawning Date LLLE LLHE HLLE HLHE COMM LL HL YEAR It 1- 2 Sep. 29-Oct. 5/88 - - - 1 1 — 1 3- 4 Oct. 12-Oct. 19/88 — — 1 2 1 — 3 5- 6 Oct. 26-Nov. 2/88 - 1 2 1 2 1 3 7- 8 Nov. 9-Nov 17/88 6 4 3 2 6 10 5 9-10 Nov. 23-Nov. 30/88 2 1 4 3 - 3 7 11-12 Dec. 7-Dec. 14/88 3 - 1 3 - 3 4 13-14 Dec. 21-Dec. 27/88 2 1 2 1 - 3 3 T o t a l 13 7 13 13 10 20 1 26 No. weeks of spawning 8 8 11 14 8 9 14 YEAR 2: 1- 2 Oct. 11-Oct. 18/89 _ 1 _ 3- 4 Oct. 25-Nov. 2/89 3 - — 1 2 3 1 5- 6 Nov. 8-Nov. 15/89 2 - 1 2 6 2 3 7- 8 Nov. 22-Nov. 29/89 3 4 3 3 — 7 6 9-10 Dec. 6-Dec. 13/89 1 2 2 — - 3 2 11 Dec. 20/89 1 1 - - - 2 - T o t a l 10 72 62 6 9 17 2 12: No. weeks of spawning 8 5 6 6 6 8 8 1 I n c l u d i n g one f i s h which had ovulated eggs when found dead on f l o o r . 2 P l u s 2 f i s h fed LLHE and 1 f i s h fed HLLE which contained maturing eggs when they die d as a r e s u l t of human e r r o r at 8 weeks. 89 and showed t h a t 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 pr o p o r t i o n of f i s h fed the HL d i e t s spawning e a r l i e r than those fed the LL d i e t s . In Year 1, f i s h fed both the LL and the HL d i e t s f i n i s h e d spawning on December 27, 1988 w i t h spawning ending 12 weeks a f t e r i t had begun. The spawning p e r i o d l a s t e d 9 weeks f o r f i s h fed the LL d i e t s and 14 weeks f o r f i s h fed the HL d i e t s . F i s h fed the COMM d i e t spawned over a 9-week p e r i o d which began during the t h i r d week of spawning. To observe i f the e f f e c t of d i e t a r y l i p i d on spawning day was repea t a b l e , f i s h fed the LL d i e t s i n Year 1 were fed the HL d i e t s i n Year 2 and v i c e versa w h i l e keeping the v i t a m i n E l e v e l s the same (Figure 3). Spawning began a minimum of 12 days l a t e r i n Year 2 compared t o Year 1 beginning on October 11, 1989 f o r f i s h fed the COMM d i e t and not u n t i l on or a f t e r October 22 f o r those fed the other d i e t s (Table 11). D i e t a r y l i p i d had no e f f e c t on the spawning p e r i o d i n Year 2. Spawning day was p o s i t i v e l y c o r r e l a t e d w i t h egg f e r t i l i z a t i o n i n Year 1 (r 2=0.143, Appendix 4) and n e g a t i v e l y c o r r e l a t e d w i t h egg weight i n Year 1 (r 2=0.143). There was no s i g n i f i c a n t e f f e c t of spawning day on egg weight, egg f e r t i l i z a t i o n or embryonic s u r v i v a l but f e c u n d i t y was n e g a t i v e l y c o r r e l a t e d w i t h spawning day (r 2=0.249). 9 0 9 1 92 4.6.2 Number of r i p e males and females Chi square a n a l y s i s showed t h a t d i e t a r y l i p i d or v i t a m i n E c o n c n t r a t i o n had no s i g n i f i c a n t e f f e c t on the p o r p o r t i o n of males which produced m i l t i n Year 1 or Year 2 (Tables 12 and 13). F i s h e r ' s exact t e x t showed there was no e f f e c t of d i e t a r y v i t a m i n E c o n c e n t r a t i o n on the p r o p o r t i o n of females which spawned i n Year 1 or 2. A t o t a l of 9 males d i d not mature i n e i t h e r spawning season w i t h 5 fed the LE d i e t s and 4 fed the HE d i e t s (Table 14). A p o r t i o n of the po p u l a t i o n which produced m i l t i n Year 1 d i d not produce i t i n Year 2 and v i c e versa. F i s h fed HE d i e t s had a s i g n i f i c a n t l y higher p r o p o r t i o n of males which produced m i l t i n both years compared t o the LE d i e t s (x 2=4.030). The low sample s i z e and the closeness of the P value t o s i g n i f i c a n c e suggests t h a t t h i s area should be i n v e s t i g a t e d f u r t h e r . D i e t had no s i g n i f i c a n t e f f e c t on changing the p r o p o r t i o n of males becoming r i p e i n Year 2 which had not been not r i p e i n Year 1 (x 2 =0.202). 4.6.3 A t y p i c a l spawning In Year 1, one f i s h fed LLLE and one fed COMM rel e a s e d t h e i r eggs i n the tank and both f i s h d i e d w i t h i n 2-3 weeks. Embryonic s u r v i v a l was poor being 14% eyed f o r the former and 0% eyed f o r the l a t t e r . One f i s h fed HLHE produced eggs which were spotted and appeared o v e r r i p e (38% eyed) and there was blood i n the o v a r i a n f l u i d . This f i s h died w i t h i n 2 weeks. 93 Table 14. Number of males which produced m i l t ( r i p e ) i n Year 1 which d i d or d i d not produce m i l t i n Year 2. Diet fed i n Year 1 HLLE HLHE LLLE LLHE COMM Diet fed i n Year 2 LLLE LLHE HLLE HLHE COMM LE HE Tank 6 2 3 7 5 Year 1 Year 2 Ripe Ripe 2 4 2 8 8 4 12 Ripe Not r i p e 2 2 5 2 0 7 4 T o t a l 4 6 7 10 8 11 16 X 2 f o r v i t a m i n E 4.030 P<0 .05 Year 1 Year 2 Not r i p e Ripe 3 4 2 2 4 5 6 Not r i p e Not r i p e 4 2 1 2 0 5 4 T o t a l 7 6 3 4 4 10 10 X 2 f o r v i t a m i n E 0.202 P>0.10 94 Problems occurred w i t h 3 f i s h fed HLLE i n Year 2, w i t h one f i s h r e l e a s i n g most of i t s eggs i n the tank and blood o c c u r r i n g i n the o v a r i a n f l u i d of one f i s h which a l s o r e l e a s e d 50-60 browncoloured eggs. Brown de b r i s was present i n the o v a r i a n f l u i d of a t h i r d f i s h . One f i s h fed LLLE had eggs which c o u l d not be r e l e a s e d and they appeared t o be i n a s o l i d mass. One f i s h fed LLHE produced eggs which i n c l u d e d about 12 dark orange eggs and t h i s f i s h died w i t h i n 4 weeks of spawning. In Year 2, m o r t a l i t y , p r i m a r i l y of females, increased at spawning i n a l l groups except those fed LLLE and COMM and t h i s was due t o fun g a l i n f e c t i o n which spread r a p i d l y over the s k i n . 4.7 Fecundity and egg s i z e (diameter and weight) D i e t had no s i g n i f i c a n t e f f e c t on u n f e r t i l i z e d egg weight i n e i t h e r Year 1 or Year 2 (Tables 15 and 16, Fi g u r e 6a and 6b). There was no e f f e c t of d i e t on the weight and diameter of water- hardened preserved eggs except f o r a s i g n i f i c a n t e f f e c t of v i t a m i n E on egg diameter i n Year 1 (Table 17). The c o e f f i c i e n t of v a r i a t i o n (CV) f o r eggs produced by each f i s h ranged from 4.5 t o 24.1% (mean=8.5) f o r egg weight and from 1.2 t o 6.3% (mean=3.7) f o r diameter i n Year 1. There was l i t t l e change i n Year 2 w i t h CV ranging from 3.0 t o 16.6% (mean=7.5) f o r egg weight and 1.9 t o 8.2% (mean=3.7) f o r diameter. No s i g n i f i c a n t e f f e c t s of d i e t on the t o t a l weight of eggs or number of eggs produced per kg body weight (Table 15) were 9 5 9 6 9 7 Figure 6. Egg weight i n a) Year 1 and b) Year 2 showing minimum, 25, 50, 75 p e r c e n t i l e s and maximum values. 9 8 99 observed. Some eggs were rele a s e d a f t e r spawning and 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 number of eggs r e l e a s e d at spawning or the t o t a l number of eggs produced. InYear 2, f i s h f ed COMM weighed s i g n i f i c a n t l y more than those fed other d i e t s and they produced s i g n i f i c a n t l y more eggs and a g r e a t e r t o t a l weight of eggs than those fed a l l d i e t s except LLLE (Table 16). 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 number of eggs produced per kg body weight. The numbers of eggs produced were h i g h l y v a r i a b l e w i t h i n each treatment i n both years as shown i n Figures 7a and 7b. The t o t a l number of eggs produced was d i r e c t l y c o r r e l a t e d w i t h f i s h weight i n both Year 1 (r 2=0.334) and Year 2 (r 2=0.853) (Figures 8a and 8b, Appendix 4 but was not s i g n i f i c a n t l y c o r r e l a t e d w i t h egg weight. Some f i s h l o s t weight throughout Year 2 and were not expected t o produce any eggs. However, some of these f i s h produced s m a l l numbers of eggs. For example, 2 f i s h fed LLHE r e l e a s e d l e s s than 350 eggs w i t h s u r v i v a l of 61 and 94% t o the eyed stage and 1 f i s h fed HLHE relea s e d 525 eggs w i t h 95% s u r v i v a l t o the eyed stage. Fecundity was not c o r r e l a t e d w i t h f e r t i l i z a t i o n , s u r v i v a l t o the eyed, hatch or swimup stage or w i t h s u r v i v a l or s p e c i f i c growth r a t e of f r y . F e r t i l i z a t i o n and s u r v i v a l t o hatch and swimup stages was d i r e c t l y r e l a t e d t o egg weight i n Year 2 (r 2=0.11-0.12) (Appendix 4) but was not c o r r e l a t e d i n Year 1. 1 0 0 F i g u r e 7. T o t a l number of eggs produced i n a) Year 1 and b) Year 2 showing minimum, 25, 50, 75 p e r c e n t i l e s and maximum values. 1 0 1 F i g u r e 6. R e l a t i o n s h i p of t o t a l number of eggs produced and f i s h weight i n : a) Year 1 (Y= 2.71X + 351.88, r 2=0.334) and b) Year 2 (Y= 3.88X + 2202.25, r 2=0.853). 102 4.8 Embryonic s u r v i v a l More eggs were g e n e r a l l y c o l l e c t e d and f e r t i l i z e d than r e q u i r e d f o r i n c u b a t i o n f o r t h i s experiment. Excess eggs were incubated i n Heath t r a y s u n t i l they reached the eyed stage and then they were d i s t r i b u t e d t o producers. In Year 2, Heath t r a y s were compartmentalized and eggs from each female were maintained s e p a r a t e l y . S u r v i v a l t o the eyed stage was comparable f o r eggs incubated i n Heath t r a y s compared t o the j a r system. When f e r t i l i z e d eggs were viewed under a low power micro- scope, d i v i d e d c e l l s could be seen which v a r i e d from the 4 - c e l l t o 16-32 c e l l stage (Tables 18 and 19, Figures 9 and 10). In Year 2, 2 f i s h fed LLHE produced eggs i n which abnormal c e l l d i v i s i o n and they had v i t a m i n E concentrations of 236 and 242 fxg/g. Abnornal c e l l d i v i s i o n was a l s o observed i n eggs from 3 f i s h fed HLLE and the v i t a m i n E concentrations were 37, 54 and 59 M-g/g. C e l l s appeared t o have d i v i d e d unevenly and/or were some d i s t a n c e apart. As the incidence of abnormal eggs i n c r e a s e d , the percentage which reached the eyed stage decreased. Abnormal c e l l s were s i m i l a r i n appearance t o those i l l u s t r a t e d i n Leray et a l . (1985) which were apparently due t o an e s s e n t i a l f a t t y a c i d d e f i c i e n c y . There were no s i g n i f i c a n t d i f f e r e n c e s between the 5 d i e t a r y treatments i n f e r t i l i z a t i o n or s u r v i v a l t o the eyed, hatch or swimup stage i n Year 1 or 2 (Table 18 and 19, Figures 9 and 10). 103 Table 18. F e r t i l i z a t i o n 1 and embryonic s u r v i v a l 1 (mean ± standard d e v i a t i o n ) i n Year 1. Die t fed n F e r t i l i z e d Eyed Hatch Swimup i n Year 1 % + SD % + SD % + SD % + SD LLLE 12 92 + 5 67 + 28 48 + 32 42 + 32 LLHE 7 90 + 13 81 + 14 76 + 15 65 + 31 HLLE 13 92 + 8 77 + 19 63 + 25 58 + 25 HLHE 12 86 + 27 60 + 34 46 + 37 38 + 36 COMM 10 87 + 18 68 + 37 63 + 34 57 + 31 S i g n i f i c a n c e l e v e l from a n a l y s i s of var i a n c e Source of v a r i a t i o n A l l d i e t s ns ns ns ns E x c l u d i n g COMM L i p i d ns ns ns ns V i t a m i n E ns ns * * L i p i d x V i t E ns ns * * Eved/Fert Hatch/Eved Swimup/Hatch % + SD % t : SD % + SD LLLE - 71 + 29 64 b + 27 84 + 19 LLHE - 90 + 7 94 a + 6 82 + 32 HLLE - 83 + 18 7gab + 17 91 + 7 HLHE - 70 + 31 66 b + 34 82 + 24 COMM _ 74 + 39 93 a + 6 91 + 6 S i g n i f i c a n c e l e v e l from a n a l y s i s of vari a n c e Source of v a r i a t i o n A l l d i e t s ns ** ns Ex c l u d i n g COMM L i p i d ns ns ns Vit a m i n E ns ** ns L i p i d x V i t E * ** ns 1 A r c s i n t r a n s f o r m a t i o n was used on a l l data p r i o r t o a n a l y s i s of v a r i a n c e . ns = not s i g n i f i c a n t , P>0.05, * = P < 0 , 0 5 , * * = P < 0 . 0 1 1 0 4 F i g u r e 9. F e r t i l i z a t i o n and s u r v i v a l of eggs to the eyed, hatch and swimup stages i n Year 1 showing minimum, 25, 50, 75 p e r c e n t i l e s and maximum values. 105 Table 19. F e r t i l i z a t i o n and embryonic s u r v i v a l 1 (mean ± standard d e v i a t i o n ) i n Year 2. Die t fed n F e r t i l i z e d Eyed Hatch Swimup i n Year 2 % ± SD % + SD % + SD % + SD LLLE 10 87 ± 13 40 + 35 31 + 31 23 + 30 LLHE 7 83 ± 11 53 + 42 45 + 39 42 + 39 HLLE 6 69 ± 37 28 + 40 24 + 35 19 + 32 HLHE 6 88 ± 11 65 + 33 59 + 34 51 + 38 COMM 9 73 ± 28 37 + 29 29 + 25 24 + 20 S i g n i f i c a n c e l e v e l from a n a l y s i s of vari a n c e Source of v a r i a t i o n A l l d i e t s ns ns ns ns E x c l u d i n g COMM L i p i d ns ns ns ns Vitam i n E ns ns ns ns L i p i d x V i t E ns ns ns ns Eved/Fert. Hatch/Eved Swimup/Hatct % + SD % + SD % + SD LLLE 44 + 38 67 + 21 74 + 25 LLHE 56 + 42 81 + 24 82 + 21 HLLE 37 + 47 75 + 34 45 + 44 HLHE 75 + 36 84 + 15 77 + 27 COMM 46 + 32 76 + 13 83 + 9 S i g n i f i c a n c e l e v e l from a n a l y s i s of variance Source of v a r i a t i o n A l l d i e t s ns ns ns ns E x c l u d i n g COMM L i p i d ns ns ns ns Vi t a m i n E ns ns ns ns L i p i d x V i t E ns ns ns ns 1 Arcsin transformation was used on a l l data prior to analysis of variance, ns = not s i g n i f i c a n t , P>0.05 F i g u r e 10. F e r t i l i z a t i o n and s u r v i v a l of eggs to the eyed, hatch and swimup stages i n Year 2 showing minimum, 25, 50, 75 p e r c e n t i l e s and maximum values. 107 In Year 1, there was a s i g n i f i c a n t e f f e c t of d i e t a r y v i t a m i n E. An i n t e r a c t i o n between l i p i d and v i t a m i n E on s u r v i v a l t o hatch and swimup r e s u l t e d i n an increase i n s u r v i v a l as d i e t a r y v i t a m i n E increased i n the LL d i e t s and a decrease i n s u r v i v a l as v i t a m i n E increased i n the HL d i e t s . To a s c e r t a i n i f d i e t r e s u l t e d i n a d i f f e r e n c e i n the stage at which embryonic development ceased, the f o l l o w i n g values were c a l c u l a t e d and compared: F e r t i l i z e d , E y e d / F e r t i l i z e d , Hatch/Eyed and Swimup/Hatch (Table 18 and 19). A value was not i n c l u d e d i n the a n a l y s i s i f both the denominator and numerator were zero. In Year 2, most m o r t a l i t y occurred p r i o r t o reaching the eyed stage. Only 37-56% of eggs t h a t were f e r t i l i z e d reached the eyed stage i n a l l groups except the HLHE group, where 75% of those f e r t i l i z e d reached the eyed stage. Of the eggs t h a t reached the eyed stage i n Year 2, 67 t o 84% hatched. S u r v i v a l was low (45%) between hatch and swimup f o r f r y from the HLLE group compared t o other groups (74-83%). When t h i s experiment was i n i t i a t e d , approximately one-half of the stock of A r c t i c charr was used. The remaining f i s h were maintained as usual and fed the COMM d i e t (although i t may have been from d i f f e r e n t shipments as the d i e t used i n the present experiment). Although records were not kept on the s u r v i v a l of eggs from i n d i v i d u a l f i s h , the o v e r a l l s u r v i v a l t o the eyed stage was 72% i n Year 1 and f i s h r eleased 3300 eggs at spawning. 108 In Year 2, records were kept f o r i n d i v i d u a l f i s h and s u r v i v a l t o the eyed stage was 35% ± 33 ( s t d . dev.)(n=30) and f i s h r e l e a s e d 4270 ± 2456 eggs at spawning. This was s i m i l a r t o s u r v i v a l t o the eyed stage of 68% ± 37 i n Year 1 and 37% ± 29 i n Year 2 f o r f i s h fed the COMM d i e t i n t h i s experiment. Fecundity was higher f o r the f i s h fed COMM i n the experimental group w i t h them r e l e a s i n g 4532 ± 1596 eggs i n Year 1 and 8305 ± 3313 eggs i n Year 2. Approximately 4-5% of f r y had blue-sac and 4-5% had d e f o r m i t i e s i n Year 2 but t h i s d i d not appear t o be c o r r e l a t e d w i t h d i e t . Most d e f o r m i t i e s were s p i n a l d e f o r m i t i e s and conjoined twins. There was a l s o evidence of "coagulated y o l k " i n a t l e a s t 8 cases w i t h proportions of over 20% of the f r y a f f e c t e d . A hemorrhagic area was v i s i b l e i n the coagulated y o l k i n some cases. Fry from the HLHE group had the lowest i n c i d e n c e of coagulated y o l k . Premature hatch was observed i n 5 cases i n Year 2 w i t h 10-50% m o r t a l i t y o c c u r r i n g . A c o n d i t i o n s i m i l a r t o blue-sac has been reported i n sunapee ( S a l v e l i n u s a l p i n u s oquassa) and lake c h a r r ( S a l v e l i n u s namaycush) (Balon 1980) when they were incubated at a high temperature (9.5°C). Blue-sac has a l s o been a s s o c i a t e d w i t h low d i s s o l v e d oxygen and t o x i c a n t s . As r e p o r t e d by Balon (1980), h i s t o l o g i c a l examination showed the y o l k sac separated from the y o l k and became i n f l a t e d as f l u i d accumulated. 109 4.9 Egg and f r y composition 4.9.1 V i t a m i n E R e p r o d u c i b i l i t y of the assay f o r v i t a m i n E c o n c e n t r a t i o n i n d i c a t e d a mean c o e f f i c i e n t of v a r i a t i o n of 3.7% f o r f r y (n=29), and 6.1 (n=57) and 4.1% (n=38) f o r eggs i n Year 1 and Year 2, r e s p e c t i v e l y . When eggs were weighed out f o r v i t a m i n E e x t r a c t i o n and assay, they d i s i n t e g r a t e d as soon as the eggs began t o thaw. This made v i t a m i n E a v a i l a b l e f o r o x i d a t i o n and r e s u l t e d i n a r e d u c t i o n of the r e p r o d u c i b i l i t y of the assay. R e p r o d u c i b i l i t y improved when the a n t i o x i d a n t , b u t y l a t e d hydroxytoluene (BHT), was added t o hexane, the e x t r a c t i o n s o l v e n t , at 50 mg/L. A d d i t i o n of BHT was not necessary during e x t r a c t i o n of v i t a m i n E from f r y . The e x t r a c t i o n blank i n d i - c ated a need f o r a c o r r e c t i o n f a c t o r of 9.92 \ag v i t a m i n E per assay f o r the egg samples and 5.63 jig f o r the f r y samples, w i t h the d i f f e r e n c e being due t o the a d d i t i o n of BHT f o r the a n a l y s i s of the eggs. Recovery was measured by s p i k i n g a sample of f r y w i t h a known amount of 95% pure d l - a - t o c o p h e r o l (Sigma Chemical Company, S t . L o u i s , MO). Recovery was 100.8 ± 0.54% (SD). A sample of the f i n a l e t h a n o l i c e x t r a c t from f r y from the HLHE treatment was subjected t o a spectrophotometric scan i n the u l t r a v i o l e t range. The major peak occurred at a wavelength of 288-292 m\i which corresponded t o a-tocopherol at 292 m\i (Budvari 110 et al. 1989). A small peak was evident at 280 mu, but no peaks occurred above 310 m\i i n d i c a t i n g t h a t r e t i n o l , w i t h an abso r p t i o n peak of 325 m\i was not present i n the e x t r a c t . I n c r e a s i n g d i e t a r y v i t a m i n E co n c e n t r a t i o n r e s u l t e d i n s i g n i f i c a n t increases i n the v i t a m i n E co n c e n t r a t i o n and content of eggs and f r y i n both years (Table 20, Figur e 11-13). In a d d i t i o n , i n Year 2 there was a l s o a s i g n i f i c a n t e f f e c t of d i e t a r y l i p i d and an i n t e r a c t i o n between the e f f e c t of l i p i d and v i t a m i n E on the eggs but not the f r y . The rank of v i t a m i n E co n c e n t r a t i o n and content a s s o c i a t e d w i t h the 5 d i e t a r y treatments remained the same i n both years f o r eggs and f r y , i n c r e a s i n g i n the order of LLLE, HLLE, COMM, HLHE, LLHE. In Year 2, 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 v i t a m i n E co n c e n t r a t i o n and content of eggs from f i s h fed each d i e t w h i l e i n Year 1 there were no s i g n i f i c a n t d i f f e r e n c e s i n v i t a m i n E of eggs from f i s h fed LLLE, HLLE or COMM. At high d i e t a r y l i p i d , i n c r e a s i n g d i e t a r y v i t a m i n E had l e s s impact on egg vi t a m i n E con c e n t r a t i o n ( 2 - 3 - f o l d increase) than at the low l i p i d c o n c e n t r a t i o n ( 3 - 5 - f o l d i n c r e a s e ) . Except f o r the LLHE d i e t , swimup f r y g e n e r a l l y had a higher v i t a m i n E co n c e n t r a t i o n and content than the eggs. 1] F i g u r e 11. Vitamin E concentration of eggs i n a) Year 1 and b) Year 2 showing minimum, 25, 50, 75 p e r c e n t i l e s and maximum values. F i g u r e 12. Vitamin E content of eggs i n a) Year 1 and b) Year 2 showing minimum, 25, 50, 75 p e r c e n t i l e s and maximum values. Figure 13. Vitamin E concentration and content of f r y i n Year 2 showing minimum, 25, 50, 75 p e r c e n t i l e s and maximum values 115 There was no overlap i n the range of v i t a m i n E conce n t r a t i o n s of the LE eggs (30-74 \ig/g) compared t o the HE eggs (109-275 ng/g). The COMM eggs were intermediate between the LE and HE eggs (42-94 \ig/g). There was much gre a t e r v a r i a b i l i t y i n v i t a m i n E conc e n t r a t i o n f o r the HE eggs compared t o the LE eggs. Within the HE eggs, LLHE had gr e a t e r v a r i a b i l i t y than HLHE w i t h eggs from f i s h fed LLHE ranging from 126-275 \xg/g w h i l e HLHE ranged from 109-187 yig/g. 4.9.2 L i p i d and dry matter There were no s i g n i f i c a n t d i f f e r e n c e s i n percentages of dry matter o r t o t a l l i p i d i n eggs from f i s h f ed any of the d i e t s i n Year 1 (Table 21, Figure 14a). There was no c o r r e l a t i o n between t o t a l l i p i d or dry matter and s u r v i v a l of embryos or f r y or s p e c i f i c growth r a t e of f r y . In Year 2, eggs from f i s h fed COMM had g r e a t e r percent dry matter than those of HLLE and s i g n i f i c a n t l y g r eater percent t o t a l and n e u t r a l l i p i d than noted f o r f i s h fed a l l other d i e t s except LLLE (Table 22, Figure 14b and 14c). No s i g n i f i c a n t d i f f e r e n c e s e x i s t e d i n percent p o l a r l i p i d (Table 22, Figure 14d). Ne u t r a l l i p i d was n e g a t i v e l y c o r r e l a t e d t o s u r v i v a l t o the eyed, hatch and swimup stages (r 2=0.26-0.34, P<0.003) (Figures 15a, 16a, 17a and Appendix 4 ) . A n a l y s i s of covariance showed th a t there was no d i f f e r e n c e between d i e t s i n t h i s r e l a t i o n s h i p . 116 Table 21. T o t a l l i p i d and dry matter contents (mean 1 standard d e v i a t i o n ) of eggs i n Year 1. Di e t Dry T o t a l matter l i p i d % ± SD % ± SD LLLE 37.0±2.6 2 2 . 5 1 1 . 1 LLHE 38.1 ± 1.5 22.2 ± 1.3 HLLE 3 8 . 4 1 1 . 1 2 3 . 2 1 1 . 0 HLHE 3 8 . 6 1 0 . 9 2 2 . 3 1 1 . 0 COMM 3 8 . 3 1 0 . 8 2 2 . 2 1 0 . 9 S i g n i f i c a n c e l e v e l from a n a l y s i s of vari a n c e Source of v a r i a t i o n A l l d i e t s ns ns Excl u d i n g COMM L i p i d ns ns Vita m i n E ns ns L i p i d x V i t E ns ns ns = not s i g n i f i c a n t , P>0.05 F i g u r e 14. T o t a l l i p i d of eggs i n Year 1 ( a ) , b) t o t a l , c) n e u t r a l and d) p o l a r l i p i d i n Year 2 showing minimum, 25, 50, 75 p e r c e n t i l e s and maximum values. 1 1 8 F i g u r e 15. R e l a t i o n s h i p of : a) n e u t r a l l i p i d s (Y= -17.29X + 233.36, r 2=0.263) and b) 16:ln7 i n n e u t r a l l i p i d s (Y= -13.68X + 202.52, r 2=0.238) on s u r v i v a l to the eyed stage i n Year 2. Figure 16. R e l a t i o n s h i p of : a) n e u t r a l l i p i d s (Y= -18.24X + 236.20, r 2=0.321) and b) 16:ln7 i n n e u t r a l l i p i d s (Y= -12.92X + 186.58, r 2=0.234) on s u r v i v a l to hatch i n Year 2. F i g u r e 17. R e l a t i o n s h i p of : a) n e u t r a l l i p i d s (Y= -18.44X + 232.38, r 2=0.338) and b) 16:ln7 i n n e u t r a l l i p i d s (Y= -12.57X + 176.66, r 2=0.228) on s u r v i v a l to swimup i n Year 2. 122 4.9.3 F a t t y a c i d composition The f a t t y a c i d compositions of the d i e t s are shown i n Tables 23 and 24 and Appendix l a . Comparison of the f a t t y a c i d composition of the d i e t s and the n e u t r a l and p o l a r l i p i d s of eggs are shown i n Figures 18-20. The LL and HL d i e t s were s i m i l a r i n most sat u r a t e d , monoenoic, n6 and n3 f a t t y a c i d s . T o t a l l i p i d s of the HL d i e t s had s i g n i f i c a n t l y more t o t a l monoenoic and n i l f a t t y a c i d s ( s p e c i f i c a l l y , 16:ln7, 2 0 : l n l l , 22:ln9, 2 2 : l n l l ) , 18:4n3, 20:5n3 and 22:5n3 compared t o the LL d i e t s . The HL d i e t s had s i g n i f i c a n t l y l e s s 18:2n6, 18:3n3, t o t a l n6 and t o t a l PUFA than the LL d i e t s . The COMM d i e t fed i n Year 1 d i f f e r e d d r a m a t i c a l l y i n f a t t y a c i d composition from t h a t fed i n Year 2. Fa t t y acids i n the COMM d i e t which d i f f e r e d the most between Year 1 vs Year 2 incl u d e d 16:0 (15.8 vs 12.2%), 20:ln9 (4.5 vs 11.3%), 2 2 : l n l l (5.4 vs 15.5%), 20:5n3 (10.5 vs 5.1%), t o t a l saturated (24 vs 18%) and monoenoic f a t t y a c i d s (35 vs 52%). F a t t y a c i d composition of n e u t r a l and p o l a r l i p i d s of the eggs, at l e v e l s above 0.1% of l i p i d , appear i n Appendices l b and l c . S e l e c t e d f a t t y acids and t o t a l values f o r f a m i l i e s of f a t t y a c i d s present i n the 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 are shown i n Tables 25-28 w i t h i n d i c a t i o n s of the s i g n i f i c a n c e of d i f f e r - ences among them. 123 Table 23. Selected major f a t t y acids i n the t o t a l l i p i d of the d i e t s . Values are % of t o t a l l i p i d . F a t t y a c i d D i e t LLLE LLHE HLLE HLHE COMM COMM LL 2 Year 1 Year 2 HL2 % % 14:0 4 .16 4 .11 16:0 16 .40 16 .43 18:0 2 .72 2 .69 16:ln7 5 .06 5 .05 18:ln7 3 .70 3 .72 18:ln9 18 .71 18 .60 20:ln9 3 .30 3 .29 22:ln9 1 .01 1 .00 2 0 : l n l l 3 .84 3 .77 2 2 : l n l l 6 .60 6 .50 18:2n6 7 .46 7 .40 20:4n6 0 .62 0 .62 18:3n3 1 .32 1 .32 18:4n3 0 .88 0 .88 20:5n3 5 .97 5 .92 22:5n3 0 .92 0 .90 22:6n3 6 .77 6 .75 % % % 4.44 4.09 5.14 15.97 15.61 15.77 2.68 2.69 2.53 5.51 5.36 7.76 3.81 3.62 2.64 18.48 18.83 12.44 3.52 3.32 4.49 1.17 1.16 0.61 4.63 5.04 0.43 7.49 7.83 5.39 4.90 4.97 10.68 0.63 0.64 0.39 1.00 1.01 1.32 1.00 1.00 1.77 6.20 6.36 10.45 1.03 1.08 1.13 6.33 6.58 5.89 % % % 3 .86 4 .14 4 .27 12 .16 16 .42 15 .79 1 .63 2 .71 2 .69 6 .97 5 .06a 5 .44b 2 .35 3 .71 3 .72 10 .93 18 .66 18 .66 11 .32 3 .30 3 .42 1 .68 1 .01 a 1 .17 b 0 .68 3 .81 a 4 .84b 15 .52 6 .55 a 7 .66b 10 .52 7 .43 a 4 .94b 0 .25 0 .62 0 .64 1 .01 1 .20 a 1 .07 b 1 .03 0 .88 a 1 .00 b 5 .05 5 .95 a 6 .28 b 0 .61 0 .91 a 1 .06b 4 .90 6 .76 6 .46 1 Complete f a t t y a c i d p r o f i l e appears i n Appendix l a . 2 Values w i t h i n a row which have d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (P<0.05). 124 Table 24. T o t a l s f o r f a m i l i e s of f a t t y a c i d s i n the t o t a l l i p i d of the d i e t s . Values are % of t o t a l l i p i d . F a t t y D i e t a c i d LLLE LLHE HLLE HLHE COMM COMM LL 1 HL1 type Year 1 Year 2 % % % % % % % % Saturated 24, .87 24 .88 24, .80 24, .06 24, .26 18 .49 24, .88 24, .43 Monoenoic 44, .60 44 .27 47, .15 47, .72 35, .35 51, .97 44, .44a 47, .43* n5 0, .50 0. 52 0, .55 0, .53 0, .48 0, .63 0, .51 0, .54 n7 9, .54 9 .54 10, .19 9, .86 10, .86 10, .49 9, .54 10, .03 n9 24, .12 23 .94 24, .29 24, .46 18, .19 24, .66 24, .03 24, .38 n i l 10, .44 10 .27 12. .12 12, .87 5. .82 16, .19 10. .36a 12. .50b n6 9. .04 9 .02 6. .62 6. ,69 12. .46 11. .57 9. ,03a 6. .66b n3 16. .87 16 .81 16. .70 17. .16 23. .04 13. .80 16. .84 16. .93 n3/n6 1. .87 1 .86 2. ,52 2. .56 1. ,85 1. .19 1. .86a 2. ,54b PUFA 11. .70 11 .70 9. ,15 9. .21 16. .33 14. .06 11. .70a 9. .18b HUFA 14. .36 14 .30 14. ,34 14, .80 19. ,30 11. ,41 14. ,33 14. ,57 1 Values w i t h i n a row which have d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (P<0.05). LLLE 1 2 5 SAT n7 n9 SAMPLE D I E T n i l PUFA HUFA Figure 18. Comparison of saturated, n7, n9, n i l monoenoic a c i d s , PUFA and HUFA of the t o t a l l i p i d s of the d i e t s w i t h the neutral(NL) and polar l i p i d s ( P L ) of the eggs i n Year 2 LLLE "V" JStwm 1 2 6 LLHE 50 - HLLE 40 - 30 - 20 - 10 n u - 50 - HLHE 18:3n3 20:5n3 22:5n3 22:6n3 SAMPLE DIET NL n3 PL Figure 19. Comparison of n3 f a t t y a c i d s of the t o t a l l i p i d s of the d i e t s with the n e u t r a l (NL) and polar l i p i d s (PL) of the eggs i n Year 2. 12 10 8 % 6 4 2 0 12 10 8 % 6 4 2 0 12 10 8 % 6 4 2 0 12 10 8 % 6 4 2 0 12 10 8 % 6 4 2 0 ure the « egg; LLLE 1 2 7 HLLE . Comparison of n6 f a t t y a c i d s of the t o t a l l i p i d s e t s with the n e u t r a l (NL) and p o l a r l i p i d s (PL) of i n Year 2. 128 The f a t t y a c i d p r o f i l e s of the n e u t r a l l i p i d s show t h a t eggs from f i s h fed HL d i e t s had s i g n i f i c a n t l y more 20:5n3 than eggs from f i s h fed LL and COMM and more 22:5n3 than those of f i s h fed LLLE. Eggs from f i s h fed COMM had s i g n i f i c a n t l y higher 18:2n6, 20:ln9, t o t a l n6 and PUFA and lower 18:ln9 and n3/n6 than eggs from f i s h fed other d i e t s . Negative c o r r e l a t i o n s e x i s t e d between 16:ln7 and t o t a l n7 i n the n e u t r a l l i p i d s and s u r v i v a l t o eyed, hatch and swimup stages (r 2=0.23-0.24, P<0.0008) (Figures 15b, 16b, 17b, Appendix 4 ) . The s i g n i f i c a n t p r o b a b i l i t y i n d i c a t e s t h a t the slope 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. A n a l y s i s of covariance showed there were no d i f f e r e n c e s between d i e t s . The f a t t y a c i d p r o f i l e s show t h a t the p o l a r l i p i d s of the HL eggs had s i g n i f i c a n t l y l e s s 20:2n6, 20:3n6, t o t a l n6 and PUFA than i n the LL and COMM eggs. P o l a r l i p i d s of HLHE eggs had more 20:5n3 than found i n LL eggs. There was an e f f e c t of d i e t a r y v i t a m i n E concent r a t i o n and an i n t e r a c t i o n between d i e t a r y l i p i d and vit a m i n E concentrations i n 20:ln9 w i t h LLHE eggs having s i g n i f i c a n t l y more 20:ln9 i n the p o l a r l i p i d s than LLLE and HLHE eggs. COMM eggs were higher i n 18:2n6 and 20:ln9 and lower i n t o t a l saturated f a t t y acids i n the p o l a r l i p i d s compared t o a l l other treatments. Means w i t h i n a row which share a common s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t . 1 Complete f a t t y a c i d p r o f i l e i s given i n Appendix l b . Table 25. S e l e c t e d major f a t t y a c i d s 1 i n the n e u t r a l l i p i d s eggs. Values are % of n e u t r a l l i p i d . 130 Table 26. T o t a l s of f a m i l i e s of 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 s of eggs. Values are % of n e u t r a l l i p i d . F a t t y D i e t a c i d LLLE LLHE HLLE HLHE COMM % % % % % Saturated 16.64 15.64 15.31 15.39 15.46 Monoenoic 54.79 55.47 54.89 54.96 53.71 n5 0.62 0.58 0.62 0.58 0.77 n7 18.38 17.60 17.54 17.48 18.81 n9 34.39 36.19 35.37 35.64 32.62 n i l 1.07 1.10 1.35 1.26 1.52 n6 8.16a 8.26a 7.85a 8.17a 10.65 b n3 16.02 16.32 17.91 17.71 15.68 n3/n6 1.98a 2.03a 2.40a 2.21 a 1.48b PUFA 10.10 a 9.81a 9.86a 10.34 a 12.88 b HUFA 14.63 15.28 16.43 16.08 14.17 Means w i t h i n a row which share a common s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t . Table 27. Selected major f a t t y acids 1 i n the p o l a r l i p i d s eggs. Values are % of p o l a r l i p i d . Means w i t h i n a row which share a common s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t . 1 Complete f a t t y a c i d p r o f i l e i s given i n Appendix l c . Table 28. T o t a l s of f a m i l i e s of f a t t y a c i d s i n the p o l a r l i p i d s of eggs. Values are % of p o l a r l i p i d . Means w i t h i n a row which share a common s u p e r s c r i p t are not s i g n i f i c a n t l y d i f f e r e n t . 133 4.9.4 Selenium Selenium concentrations ranged from 1.38-3.09 \ig/g i n the eggs i n Year 1 and 1.70-3.58 \ig/g i n Year 2 on a dry ma t t e r b a s i s . Selenium concentrations were not i n f l u e n c e d by d i e t a r y treatment and had no s i g n i f i c a n t e f f e c t on embryonic s u r v i v a l . 4.9.5 Composition of w i l d A r c t i c charr eggs Eggs which had been c o l l e c t e d from w i l d stocks at Tree R i v e r , NWT were analyzed when they were c l o s e t o hatching. Eggs weighed 122 mg and contained 73.8 \ig v i t a m i n E/g (9.0 jAg/egg) and 3.97 ng selenium/g. S u r v i v a l r a t e s were e x c e l l e n t w i t h 94% eyed and 87% swimup. Tree Ri v e r eggs contained 67.3% dry matter, 22.7% t o t a l l i p i d , 11.9% n e u t r a l and 10.8% p o l a r l i p i d s on a dry matter b a s i s . Compared t o the Labrador eggs i n t h i s experiment, n e u t r a l l i p i d from the Tree R i v e r eggs contained 22:5n3 and l e s s 18:ln9 and 18:2n6 (Tables 29 and 30). O v e r a l l , n e u t r a l l i p i d s contained l e s s HUFA and more PUFA, l e s s n6 and more n3 r e s u l t i n g i n a much higher n3/n6 r a t i o . The monoenoic f r a c t i o n contained more n7 and l e s s n9 f a t t y a c i d s . D i f f e r e n c e s i n the f a t t y a c i d p r o f i l e of the p o l a r l i p i d f r a c t i o n between the Tree R i v e r andexperimental eggs were much l e s s pronounced compared t o the n e u t r a l f r a c t i o n . Tree R i v e r eggs had more 18:0, 20:5n3, 22:5n3, t o t a l n3, n7 and an incre a s e d n3/n6 r a t i o , but l e s s 18:ln9, 22:6n3, t o t a l n6 and n9. 134 Table 29. Se l e c t e d major f a t t y a c i d s 1 of the n e u t r a l and p o l a r l i p i d of eggs of A r c t i c charr from Tree R i v e r , NWT. F a t t y N e u t r a l P o l a r a c i d l i p i d l i p i d % % 14:0 2.42 0.88 16:0 9.66 15.23 18:0 1.88 4.95 16:ln7 17.71 3.46 18:ln7 7.92 8.53 18:ln9 21.28 10.62 20:ln9 1.61 3.07 18:2n6 2.15 0.55 20:4n6 0.48 0.82 20:5n3 11.74 12.66 22:5n3 4.27 6.21 22:6n3 9.59 25.50 1 Complete f a t t y a c i d p r o f i l e i s given i n Appendix Id. 135 Table 30. T o t a l s of f a m i l i e s of f a t t y a c i d s of the n e u t r a l and p o l a r l i p i d of eggs of A r c t i c c h a r r from Tree R i v e r , NWT. F a t t y N e u t r a l P o l a r a c i d l i p i d l i p i d % % Saturated 14.57 21.53 Branch 0.24 0.27 Monoenoic 50.56 27.94 n5 0.84 0.79 n7 26.02 12.63 n9 23.35 13.92 n i l 0.35 0.60 n3 28.17 45.07 n6 3.71 2.41 n3/n6 7.59 18.70 PUFA 6.36 2.97 HUFA 25.94 44.61 136 4.10 Fry s u r v i v a l and growth When d i f f e r e n t numbers of f r y were r a i s e d i n the 1 L j a r s d e s c r i b e d i n S e c t i o n 3.9.2, the s p e c i f i c growth r a t e decreased as the number of f r y increased. I t was s i g n i f i c a n t l y lower f o r 100 f r y compared t o 10 and 25 f r y per j a r (Table 31). In a d d i t i o n , the s p e c i f i c growth r a t e was s i g n i f i c a n t l y g r e a t e r (2.68% body weight/day) when 300 f r y were reared i n 60 L tanks compared t o any number reared i n the j a r s . Although there were no s i g n i f i c a n t d i f f e r e n c e s i n s u r v i v a l , c a n n i b a l i s m was observed i n the j a r s c o n t a i n i n g 100 f r y . Based on these r e s u l t s , the number of f r y r a i s e d i n the j a r system was decreased from 50 per j a r i n Year 1 t o 25 per j a r i n Year 2. There were no e f f e c t s of d i e t on s p e c i f i c growth r a t e o r s u r v i v a l of f r y i n Year 1 (Table 32). Data f o r HLLE was excluded from a l l analyses of variance (Table 33) because only 2 l o t s of eggs i n the HLLE group produced s u f f i c i e n t f r y f o r use i n the Year 2 growth t r i a l . The s p e c i f i c growth r a t e was s i g n i f i c a n t l y lower f o r f r y from the LLHE group than a l l other treatments. O v e r a l l s u r v i v a l of f r y decreased from 82% i n Year 1 t o 76% i n Year 2 which was s i m i l a r t o the decrease from 88% t o 79% t h a t was p r e d i c t e d from the decrease i n the number of f r y r a i s e d i n the j a r s . With the exception of the LLHE group, the s p e c i f i c growth r a t e increased i n Year 2 from 1.7% i n Year 1 t o 2.4% i n Year 2 which was greater than the p r e d i c t e d 0.4% in c r e a s e r e s u l t i n g from the decrease i n number of f r y i n the 1 3 7 1 3 8 1 3 9 140 j a r s . Environmental c o n d i t i o n s were as s i m i l a r as p o s s i b l e i n both Year 1 and 2. Feed was purchased from the same company and a n a l y s i s showed the proximate composition was 39.7% crude p r o t e i n , 25.5% t o t a l l i p i d , 11.8% ash and 82.6% dry matter i n Year 1 and 49.7% crude p r o t e i n , 20.0% t o t a l l i p i d , 11.8% ash and 80.6% dry matter i n Year 2. The d i f f e r e n c e s i n p r o t e i n and l i p i d and/or changes i n the i n g r e d i e n t s used by the manufacturer i n Year 2 may a l s o have c o n t r i b u t e d t o the d i f f e r e n c e s i n s p e c i f i c growth r a t e . 141 DISCUSSION 5.1 Growth of broodstock and feeding a c t i v i t y In the w i l d , A r c t i c c h a r r have the p o t e n t i a l f o r a high r a t e of growth i n a short p e r i o d . They migrate t o sea i n May and e a r l y June, feed h e a v i l y i n t h e i r short time at sea r e t u r n i n g t o f r e s h water i n the l a t t e r h a l f of J u l y and August (Dempson and Greene 1985). They feed h e a v i l y w h i l e at sea and f i s h of a l l s i z e s have high s p e c i f i c growth r a t e s d u r i n g t h i s short p e r i o d (Johnson 1980). This i n d i c a t e s t h a t they have the p o t e n t i a l f o r a high r a t e of growth at l e a s t f o r t h i s short p e r i o d of time at sea. There i s l i t t l e documented i n f o r m a t i o n a g a i n s t which t o compare the growth r a t e s achieved i n t h i s experiment. At t h i s stage of l i f e , growth r a t e s are seldom monitored i n h a t c h e r i e s . Growth data c o l l e c t e d on w i l d stocks are g e n e r a l l y c o l l e c t e d as len g t h r a t h e r than weight data. Dempson (1982) showed t h a t mark-recaptured 8-year o l d Labrador A r c t i c c h a r r had increased i n l e n g t h by 52 mm (455 t o 507 mm) i n one year. In the cu r r e n t experiment, 4-year olds increased 23 t o 38 mm (414 t o 445 mm) i n the 71 day feeding p e r i o d i n Year 1. In Year 2, c h a r r fed the experimental d i e t s increased from 42 mm (443 t o 485 mm) which i s s l i g h t l y l e s s than the increase found by Dempson (1982) w h i l e those fed COMM increased 76 mm (453 t o 529 mm). 142 In Year 1, female f i s h fed the LL d i e t s had s i g n i f i c a n t l y lower s p e c i f i c growth r a t e s than those fed the HL and COMM d i e t s . This e f f e c t of d i e t a r y l i p i d l e v e l was a l s o observed when Nauyuk Lake A r c t i c charr f i n g e r l i n g s were fed d i e t s c o n t a i n i n g 10% l i p i d compared to 15 and 20% l i p i d at d i e t a r y p r o t e i n l e v e l s of e i t h e r 34, 44 or 54% (Tabachek 1986). D i e t a r y l e v e l s of 10% l i p i d and/or 34% p r o t e i n not considered adequate t o meet the growth requirements of A r c t i c c h a r r f i n g e r l i n g s . There was a crossover i n d i e t s at the beginning of Year 2 and f i s h fed LL d i e t s i n Year 1 were fed HL d i e t s i n Year 2 and v i c e v e r s a . Since mean weight of f i s h fed each d i e t d i f f e r e d (although not s i g n i f i c a n t l y ) at the time of the crossover i n d i e t s , comparisons of the growth r a t e s i n Year 2 should be viewed w i t h t h i s i n mind. S p e c i f i c growth r a t e s d e c l i n e d 20-51% from Year 1 f o r females and 24-39% f o r males fed 4 of the 5 d i e t s and 4% f o r males fed HLLE. Labrador A r c t i c charr have been shown t o d i s p l a y high v a r i a b i l i t y i n growth r a t e during j u v e n i l e stages of l i f e (Papst and Hopky 1983, 1989). This wide v a r i a t i o n i n growth r a t e and the s m a l l p o p u l a t i o n s i z e r e s u l t e d i n a wide v a r i a t i o n i n the s i z e of f i s h at the beginning of the experiment. A p o r t i o n of the male and female populations i n each tank l o s t weight dur i n g some p e r i o d or throughout the e n t i r e experiment. Although a p a r t i c u l a r l y high p r o p o r t i o n of males fed HLLE i n Year 2 l o s t weight, other males i n that tank had s p e c i f i c growth r a t e s 143 e q u i v a l e n t t o those of f i s h fed other d i e t s . The high p r o p o r t i o n of males which l o s t weight i n t h a t tank may have been a f f e c t e d by the presence of one aggressive male. S i x out of 10 males and 5 out of 11 females continued t o l o s e weight a f t e r removal of the aggressive f i s h . I t i s not known i f t h i s was caused by the a f t e r - e f f e c t s of the aggressive f i s h or by the d i e t o r some other unknown f a c t o r . Since aggressive f i s h were not observed i n other tanks, some a d d i t i o n a l f a c t o r ( s ) must be considered. The tanks used t o r a i s e the broodstock had been i n use f o r many years. The p o s i t i o n of the four tanks used f o r the f o u r experimental d i e t s were s i m i l a r , a l l being along one w a l l and along the same catwalk. The b u i l d i n g had l i m i t e d access and s t a f f were near these tanks only when they were feeding or spawning f i s h or maintaining the tanks. The p o s i t i o n of the tank used f o r the COMM d i e t was i n the opposite row and was not along the same catwalk. This tank may have had more disturbance s i n c e i t was not along a w a l l and there were other tanks opposite from i t where feeding and/or spawning occurred at d i f f e r e n t times from the other four tanks. Other than the problems w i t h temperature d i f f e r e n c e s between tanks d u r i n g the f i r s t 9 days of the experiment, there were few d i f f e r e n c e s i n temperature, water chemistry and l i g h t i n g between the tanks. F i s h g r a d u a l l y reduced t h e i r feed consumption as spawning 144 approached i n Year 1. Feed was w i t h h e l d from 14 days p r i o r t o spawning u n t i l a l l spawning was complete which r e s u l t e d i n feed being w i t h h e l d f o r a t o t a l of 120 days. Whether t h i s long p e r i o d of feed withdrawal i n f l u e n c e d t h e i r resumption of feeding i n Year 2 i s not known. I t i s a common p r a c t i c e t o withdraw feed d u r i n g spawning or f o r s e v e r a l days p r i o r t o spawning d u r i n g each week of the spawning season. This i s done t o reduce the chance of f e c a l m a t e r i a l contaminating the eggs and m i l t when males and females are s t r i p p e d . At RARC, feed i s w i t h h e l d f o r 2 weeks p r i o r t o and during the e n t i r e spawning p e r i o d . However, there are g e n e r a l l y s u f f i c i e n t tanks a v a i l a b l e so t h a t spawned f i s h can be placed i n t o a tank and feed can be o f f e r e d when f i s h are ready t o resume feeding. A d d i t i o n a l tanks were not a v a i l a b l e f o r t h i s experiment. Records of the number of emaciated f i s h i n the nonexperimental group ( f i s h from the same stock as the experimental groups) were not kept but there was evidence t h a t emaciated f i s h e x i s t e d and t h a t they were r o u t i n e l y c u l l e d during the annual sampling f o r c e r t i f i c a t i o n purposes (Olson, personal communication). In the w i l d , post-spawning charr consume l i t t l e i f any food i n f r e s h water and hence they l o s e weight although they continue t o i n c r e a s e i n length (Johnson 1980). C o n d i t i o n f a c t o r , which i s a measure of the plumpness of the f i s h , decreased from 0.97- 1.00 i n the prespawning run t o 0.67-0.74 during the same f i s h ' s post-spawning m i g r a t i o n the f o l l o w i n g summer. In the present 145 experiment, male and female charr t h a t had l o s t weight had c o n d i t i o n f a c t o r s of 0.81-0.96 and 0.71-0.96, r e s p e c t i v e l y , p r i o r t o spawning i n Year 2, compared t o c o n d i t i o n f a c t o r s of 1.08-1.48 and 1.35-1.73 f o r males and females t h a t had gained weight. F i s h t h a t had l o s t weight g e n e r a l l y produced few, i f any, gametes and e v e n t u a l l y these emaciated f i s h , r e f e r r e d t o as " s l i n k s " , were expected t o d i e of m a l n u t r i t i o n . Regarding the weight g a i n of post-spawning " s l i n k s " when they r e t u r n t o sea, Johnson (1980) s t a t e s : "The recovery of emaciated c h a r r i s phenomenal when food i s a v a i l a b l e . " Therefore, one expects t h a t they should be able t o resume feeding and recover a f t e r t h e i r p e r i o d of spawning and s t a r v a t i o n . The f a i l u r e of Labrador A r c t i c c h a r r t o resume feeding a f t e r spawning has been reported by other producers (Van Toever, personal communication). Broodstock s u r v i v a l and p r o d u c t i v i t y are of importance t o producers and techniques and/or d i e t s which enhance the resumption of feeding a f t e r spawning need t o be i n v e s t i g a t e d . Labrador c h a r r broodstock consumed more COMM d i e t than the experimental d i e t s and more HL compared t o LL d i e t s but the e f f e c t s of d i e t a r y l i p i d on feed i n t a k e c o u l d not be analyzed s t a t i s t i c a l l y . In research w i t h Nauyuk Lake A r c t i c c harr f i n g e r l i n g s , d i e t a r y l i p i d d i d not i n f l u e n c e feed i n t a k e (Tabachek 1986). One explanation regarding the higher i n t a k e of the COMM d i e t i s t h a t f i s h were more accustomed t o t h i s d i e t and t h e r e f o r e consumed i t more r e a d i l y . Since other Labrador A r c t i c 146 c h a r r broodstock at RARC have adjusted w e l l t o a change i n d i e t from the dry COMM d i e t t o a semi-moist product manufactured by Bioproducts, i t i s u n l i k e l y t h a t broodstock would not accept a "new" feed. COMM may have had a d i f f e r e n t f l a v o u r than the other d i e t s and COMM d i d have a harder t e x t u r e than the experimental d i e t s , w i t h the HL d i e t s having the s o f t e s t c o n s i s t e n c y . Orr et al, (1982) and Ridelman et a l . (1984) reported t h a t s t a r v a t i o n of rainbow t r o u t f o r 40 days p r i o r t o spawning had no e f f e c t on f e c u n d i t y , egg weight, diameter, proximate composition or h a t c h a b i l i t y . The e f f e c t t h a t s t a r v a t i o n p r i o r t o spawning had on resumption of feeding was not i n v e s t i g a t e d . Ashton (1991) found t h a t s t a r v a t i o n f o r 7-14 days p r i o r t o spawning had no e f f e c t on the l i p i d content of eggs, c o n d i t i o n f a c t o r o r s u r v i v a l t o the eyed stage i n chinook salmon. The general d e c l i n e i n feeding a c t i v i t y i n Year 2 compared t o Year 1 was observed i n f i s h fed a l l d i e t s i n c l u d i n g COMM. The experimental d i e t s were manufactured at about 11 week i n t e r v a l s . Ingredients and feeds were kept f r o z e n , as described i n S e c t i o n 3.2.2 and 3.5, and feeds were s t o r e d at room temperature only during the week they were fed. COMM used i n Year 2 was from a l a t e r shipment than t h a t used i n Year 1. Hung et a l . (1980) recommended using peroxide value t o measure the o x i d a t i o n of f i s h o i l s . Peroxide value showed t h a t h e r r i n g o i l , 147 s t o r e d i n a f r o z e n s t a t e and f l u s h e d w i t h n i t r o g e n , had not d e t e r i o r a t e d d u r i n g storage over the le n g t h of the experiment. O f f - f l a v o u r s , t h a t might have reduced feed consumption i n a l l d i e t s i n Year 2, are not expected t o have developed. 5.2 R e l a t i o n of f e c u n d i t y t o r a t i o n and s i z e of the female The f e c u n d i t y of stocks of A r c t i c c h a r r from d i f f e r e n t l o c a t i o n s d i f f e r widely both i n the w i l d and i n c a p t i v i t y . Johnson (1980) presents data on s e v e r a l stocks of w i l d c h a r r of d i f f e r e n t ages which vary i n mean f e c u n d i t y from 2000-4954. At RARC, stocks of c h a r r from Norway, Nauyuk Lake i n NWT and F r a s e r R i v e r i n Labrador produce 3000, 1200-2200 and over 3000-4000 eggs, r e s p e c t i v e l y (Tabachek and de March 1990). Therefore, the data provided by Dempson (1982) and Dempson and Green (1985) i n t h e i r sampling of A r c t i c charr from the Fraser R i v e r i n Labrador provide very u s e f u l comparisons of w i l d and c a p t i v e stocks which o r i g i n a t e d from the same l o c a t i o n . Fecundity of f i s h spawned i n Labrador, which were 7-13 years o l d (weight = 2.14 kg, le n g t h = 55.3 cm) ranged from 2316-9245 eggs (mean = 5242) (Dempson 1982). In the present experiment, the number of eggs r e l e a s e d at spawning ranged from 763-7455 i n Year 1 and from 327-13429 i n Year 2 w i t h the l a t t e r extending the range of Dempson (1982). F i s h fed COMM re l e a s e d the highest number of eggs i n both years, w i t h ranges of 2183-6525 (4532 ± 1596) [mean ± standard d e v i a t i o n ] ) i n Year 1 and 4047-13429 (8305 ± 3313) i n Year 2. While the va r i a n c e i s high, 34 and 85% of t h i s v a r i a n c e was 148 accounted f o r by the strong c o r r e l a t i o n between f e c u n d i t y and f i s h weight i n Year 1 and 2, r e s p e c t i v e l y . Although females f e d COMM had s i g n i f i c a n t l y higher f e c u n d i t y than f i s h fed a l l o t her d i e t s except LLLE i n Year 2, they weighed more and d i d not have a s i g n i f i c a n t l y higher r e l a t i v e f e c u n d i t y . Jones and Bromage (1987) showed t h a t f e c u n d i t y depended both on r a t i o n and f i s h weight. I t i s expected t h a t the higher feed i n t a k e of f i s h fed COMM l e d t o t h e i r higher weight which was r e l a t e d t o t h e i r higher f e c u n d i t y than those fed other d i e t s . I n i t i a l l y , the pop u l a t i o n of cha r r at RARC was d i v i d e d i n h a l f w i t h one group a l l o c a t e d f o r the experiment and the remainder f o r the non- experimental group which were maintained as u s u a l . Although the non-experimental group was fed COMM (although i t may have been from d i f f e r e n t shipments f o r part of the t i m e ) , f e c u n d i t y d i d not reach the same high numbers as observed i n the experimental group fed COMM. The non-experimental group had a mean f e c u n d i t y of 3300 i n Year 1 and 3900 i n Year 2. The higher f e c u n d i t y of the experimental group fed COMM was probably due t o t h e i r being fed t o s a t i a t i o n . Jones and Bromage (1987) suggested an optimum r a t i o n of 0.75-1.0% body weight/day at 11.7°C f o r rainbow t r o u t broodstock. In the c u r r e n t experiment, A r c t i c c h a r r were fed t o s a t i a t i o n but r a t i o n could be estimated from feed fed (dry feed f e d , g x 100)/(no. days fed x (average body weight, g ) . When fed a t water temperatures of 7.5-8.5°C, r a t i o n i n Year 1 was 149 approximately 0.77-0.88% f o r the LL and HL d i e t s and 0.98% body weight/day f o r COMM. In Year 2, t h i s decreased t o 0.31% f o r f i s h fed the LL d i e t s and 0.50% and 0.42% f o r those fed the HL and COMM d i e t s , r e s p e c t i v e l y , which were much lower than the optimum r a t i o n suggested f o r rainbow t r o u t of 0.75-1.0%. Dempson and Green (1985) showed t h a t there was a r e l a t i o n s h i p between f e c u n d i t y and f o r k length of w i l d Labrador c h a r r . These authors a l s o estimated f e c u n d i t y t o be 2450 eggs/kg body weight at 7-13 years of age. McGeachy and Delabbio (1989) showed t h a t f i r s t - t i m e spawning 4-year o l d s (mean weight=968 g) r a i s e d i n c a p t i v i t y had a r e l a t i v e f e c u n d i t y of 3850 eggs/kg. In the present experiment, f i s h r e l e a s e d an average of 2842-3200 eggs/kg body weight i n Year 1 (4-year old s ) and 2112-3098 i n Year 2 (5-year o l d s ) . Both ranges were lower than expected based on the f i n d i n g s of McGeachy and Delabbio (1989). The improvement i n the c o r r e l a t i o n of f e c u n d i t y and f i s h weight from 34% i n Year 1 t o 85% i n Year 2 may be r e l a t e d t o the f a c t t h a t f i s h were fed t o s a t i a t i o n f o r the e n t i r e season i n Year 2 whereas they were fed t o s a t i a t i o n f o r only 71 days p r i o r t o spawning i n Year 1. Scott (1962) showed t h a t r e s t r i c t e d feed i n t a k e r e s u l t e d i n an increa s e d p r o p o r t i o n of a t r e t i c f o l l i c l e s . Springate et a l . (1985) reported t h a t f e e d i n g rainbow t r o u t at f u l l r a t i o n (0.7% body weight/day) r e s u l t e d i n s i g n i f i c a n t l y higher l e v e l s of v i t e l l o g e n i n i n females and te s t o s t e r o n e i n males at spawning compared t o those 150 fed at h a l f - r a t i o n (0.35%). S i g n i f i c a n t l y more a t r e s i a occurred i n f i s h fed at h a l f - r a t i o n . Females fed at f u l l r a t i o n produced s i g n i f i c a n t l y more and l a r g e r eggs but had lower r e l a t i v e f e c u n d i t y (eggs per body weight) and these f i n d i n g s were confirmed by Orr et a l . (1982). During the p e r i o d p r i o r t o the c u r r e n t experiment, i t i s p o s s i b l e t h a t some f i s h may have r e c e i v e d a s m a l l e r p r o p o r t i o n of the r a t i o n than o t h e r s . I f r a t i o n was i n s u f f i c i e n t during the p e r i o d p r i o r t o the experiment, eggs may have s t a r t e d t o become a t r e t i c p r i o r t o the experiment. This may p a r t i a l l y account f o r the f a c t t h a t the data were more s c a t t e r e d about the r e g r e s s i o n l i n e i n Year 1. 5.3 Spawning time and p r o p o r t i o n of f i s h t h a t spawned A r c t i c c h a r r i n Labrador (Dempson 1982) spawned i n a 3-week p e r i o d i n October which peaked i n the middle of October. In the c u r r e n t experiment, charr spawned over a much longer p e r i o d of time. For example, they spawned over 14 weeks i n Year 1 and 11 weeks i n Year 2 peaking i n the middle of November. This long spawning p e r i o d i s c h a r a c t e r i s t i c of A r c t i c c h a r r r a i s e d at RARC from three c o l l e c t i o n s i n Labrador. In c o n t r a s t , stocks of A r c t i c c h a r r from Nauyuk Lake, NWT and Norway spawn e a r l i e r than the Labrador stocks and spawn w i t h i n 4-6 weeks (Olson, personal communication). While d i e t a r y l i p i d had an e f f e c t on spawning time i n Year 1, w i t h f i s h fed LL d i e t s spawning 5 weeks l a t e r than those fed HL d i e t s , t h i s p a t t e r n was not repeated i n Year 2. I t i s assumed th a t t h i s e f f e c t was not c o n s i s t e n t or th a t 151 there was no d i r e c t e f f e c t of l i p i d on spawning time. Delayed spawning was not ass o c i a t e d w i t h d i e t s t h a t had low conc e n t r a t i o n s of v i t a m i n E as i t was w i t h rainbow t r o u t (Takeuchi et a l . 1981b). F i s h fed COMM spawned i n 8 weeks i n Year 1 and 6 weeks i n Year 2. Roley (1983) found t h a t spawning was delayed but occurred w i t h i n a s h o r t e r p e r i o d of time when rainbow t r o u t were fed t o s a t i a t i o n . In c o n t r a s t , A r c t i c c h a r r i n the non-experimental group, fed at a lower r a t i o n , spawned 3- 4 weeks l a t e r but over the same length of time compared t o the p o p u l a t i o n i n the experiment i n both years. In Year 1, there was a small p o s i t i v e e f f e c t of spawning day on f e r t i l i z a t i o n . Since t h i s r e g r e s s i o n accounted f o r only 14% of the v a r i a n c e , i t i s expected t h a t a d d i t i o n a l f a c t o r s are r e s p o n s i b l e f o r the v a r i a t i o n i n f e r t i l i z a t i o n . In Year 2, there was an negative e f f e c t of spawning time on f e c u n d i t y , w i t h those spawning l a t e i n the season producing the s m a l l e s t numbers of eggs. A r c t i c c h a r r are iteropareous and may spawn s e v e r a l times d u r i n g t h e i r l i f e t i m e i n the w i l d although the frequency of spawning may decrease w i t h i n c r e a s i n g age (Johnson 1980). Charr i n t h i s experiment had spawned the year p r i o r t o the experiment. This iteropareous tendency makes i t d i f f i c u l t t o evaluate the e f f e c t of d i e t on the p r o p o r t i o n of the p o p u l a t i o n which spawned. The variance i n t h i s parameter i s n a t u r a l l y high and 152 r e s u l t s i n the need f o r l a r g e sample s i z e s t o i n c r e a s e the power of the t e s t (Dixon and Massey 1969). While a l l f i s h spawned i n Year 2 t h a t had not spawned i n Year 1, a p o r t i o n of the population t h a t spawned i n Year 1 f a i l e d t o spawn i n Year 2. D i e t a r y v i t a m i n E had no s i g n i f i c a n t e f f e c t on the p r o p o r t i o n of females t h a t spawned. This i s i n c o n t r a s t t o rainbow t r o u t (King 1985) and ayu (Takeuchi et a l . 1981a) where a p o r t i o n of the p o p u l a t i o n f a i l e d t o spawn when fed d i e t s w i t h no v i t a m i n E supplementation. The f a c t t h a t s i m i l a r p r o p o r t i o n s of the population fed the experimental d i e t s f a i l e d t o spawn i n Year 2 supports the f a c t t h a t c h a r r do not a l l spawn every year. Continued s t u d i e s w i t h l a r g e populations of tagged A r c t i c c h a r r and good record-keeping can a s c e r t a i n i f there are patterns i n t h e i r spawning. Roley (1983) showed t h a t feeding l e v e l had no e f f e c t on the number of rainbow t r o u t t h a t spawned w h i l e S c o t t (1962) showed t h a t i n c r e a s e d feed i n t a k e increased the number of females t h a t spawned. The p r o p o r t i o n of A r c t i c c h a r r t h a t spawned i n Year 2 was lower than i n Year 1 even though they had been fed t o s a t i a t i o n f o r a l l of Year 2. Not a l l males matured and produced m i l t ( i e . ripened) each year and 9 out of 59 males f a i l e d t o mature i n e i t h e r year. S i m i l a r p r o p o r t i o n s of males became r i p e when fed the LE versus 153 HE d i e t s i n Year 1 and Year 2. However, a s i g n i f i c a n t l y higher p r o p o r t i o n (12/16) of males fed HE d i e t s produced m i l t i n two consecutive years compared t o those fed LE d i e t s (4/11). This area warrants f u r t h e r i n v e s t i g a t i o n w i t h l a r g e r numbers of f i s h and w i t h r e p l i c a t e tanks of f i s h . There are r e p o r t s of v i t a m i n E d e f i c i e n c y r e s u l t i n g i n s t e r i l i t y i n male r a t s , guinea p i g s , hamsters, dogs and chickens (Scott et al. 1982) through i t s e f f e c t on the c e l l membranes of the seminiferous t u b u l e s . Male p o u l t r y were not a f f e c t e d by v i t a m i n E d e f i c i e n c y unless they were fed the d e f i c i e n t d i e t f o r a long p e r i o d of time or they were fed d i e t s c o n t a i n i n g o x i d i z e d unsaturated o i l s (Jensen 1968). There are no reports of male i n f e r t i l i t y i n f i s h r e s u l t i n g from a v i t a m i n E d e f i c i e n c y . Watanabe et al. (1984b) found t h a t the f a t t y a c i d composition of m i l t was a f f e c t e d by e s s e n t i a l f a t t y a c i d d e f i c i e n c y but suggested f u r t h e r research was r e q u i r e d t o determine i f t h i s would have an e f f e c t on f e r t i l i z a t i o n or h a t c h a b i l i t y . 5.4 Egg s i z e No s i g n i f i c a n t r e l a t i o n s h i p was found between f e c u n d i t y and egg diameter i n chinook salmon (Ashton 1991) or between f e c u n d i t y and egg weight or diameter w i t h A r c t i c c h a r r i n the present experiment. In c o n t r a s t , Roy and Higgs (1987) reported an i n v e r s e r e l a t i o n s h i p between egg weight and f e c u n d i t y i n chinook salmon (Roy and Higgs 1987). 154 There was a d i r e c t r e l a t i o n s h i p of egg weight on s u r v i v a l i n Year 2 but the low r 2 shows t h a t the r e g r e s s i o n accounts f o r only 11-12% of the variance i n s u r v i v a l . In a d d i t i o n , the slope of the l i n e i s so low t h a t a 1 mg increase i n egg weight would r e s u l t i n a 0.75% increase i n f e r t i l i z a t i o n and l e s s than 1.5% i n c r e a s e i n s u r v i v a l t o a l l stages. Although no s i g n i f i c a n t d i f f e r e n c e s between d i e t s were found, the g r e a t e s t d i f f e r e n c e between means was 7 mg which t r a n s l a t e s i n t o an i n c r e a s e of l e s s than 10.5% i n s u r v i v a l . The diameter of eggs from charr sampled i n Labrador was 4.5 mm (Dempson and Green 1985) which was s m a l l e r than those i n Year 1 (4.94-5.15 mm) or Year 2 (4.90-5.13 mm) i n t h i s experiment. 5.5 V a r i a t i o n i n embryonic s u r v i v a l Labrador A r c t i c c h a r r commonly produce eggs which e x h i b i t wide v a r i a t i o n i n h a t c h a b i l i t y . de March (1992) showed t h a t h a t c h a b i l i t y of Labrador A r c t i c c h a r r was under maternal i n f l u e n c e and was not a f f e c t e d by the source of the sperm. Therefore, i t s seems u n l i k e l y that s u r v i v a l was i n f l u e n c e d by the male. The m i l t from two males was used t o f e r t i l i z e the eggs of each female t o reduce the chance of a male e f f e c t . Records were maintained regarding the number of times each male was used and an attempt was made to e q u a l i z e the number of times each male was used over the course of the long spawning seasons. 155 V a r i a t i o n i n embryonic s u r v i v a l i s not expected t o be r e l a t e d t o the method of i n c u b a t i o n . A few r e p l i c a t e s groups of eggs were incubated each year and no d i f f e r e n c e was found i n s u r v i v a l between r e p l i c a t e s . In a d d i t i o n , i n Year 2, i f more eggs were spawned than were re q u i r e d f o r the experiment, the excess eggs were incubated i n compartmentalized Heath t r a y s . S u r v i v a l t o the eyed stage was almost i d e n t i c a l t o t h a t of the eggs incubated i n i n d i v i d u a l j a r i n c u b a t o r s . Springate et a l . (1985) found no s i g n i f i c a n t d i f f e r e n c e i n h a t c h a b i l i t y or v i a b i l i t y of f r y from rainbow t r o u t fed at h a l f - r a t i o n compared t o f u l l - r a t i o n . However, Roley (1983) found t h a t feeding t o s a t i a t i o n increased the v a r i a t i o n i n h a t c h a b i l i t y and decreased s u r v i v a l at c o o l water temperature. F i s h i n the non-experimental groups, fed at a lower r a t e , produced eggs w i t h the same mean s u r v i v a l t o the eyed stage and the same var i a n c e as those fed COMM i n t h i s experiment. Since there was wide v a r i a t i o n i n the h a t c h a b i l i t y of Labrador A r c t i c c h a r r , the e f f e c t of r a t i o n on h a t c h a b i l i t y i s an area t h a t warrants f u r t h e r i n v e s t i g a t i o n . 5.6 L i p i d and f a t t y a c i d composition of eggs The e s s e n t i a l f a t t y a c i d requirements of A r c t i c c harr broodstock are not known. The requirements of f i n g e r l i n g s are met when d i e t s c o n t a i n 1.7% 18:3n3 and 0.5% 18:2n6 (Yang and Dick 1992). The d i e t s used here contained exceeded the 156 requirements of f i n g e r l i n g A r c t i c c h a r r w i t h LL d i e t s c o n t a i n i n g 2.0% n3 and 1.1% n6 and HL d i e t s c o n t a i n i n g 3.2% n3 and 1.2% n6. Takeuchi and Watanabe (1979) found t h a t the growth of j u v e n i l e rainbow t r o u t decreased when fed d i e t s c o n t a i n i n g 2-4% n3 f a t t y a c i d s . The e f f e c t of excess n3 f a t t y a c i d s on A r c t i c c h a r r i s not known. COMM contained 3.6% n3 and 2.0% n6 i n Year 1 and 2.5% n3 and 2.1% n6 i n Year 2. COMM used i n Year 2 was higher i n 20:ln9 and 2 2 : l n l l and lower i n 20:5n3 than t h a t used i n Year 1. A t l a n t i c h e r r i n g o i l contains more 22:1 (15-30%) than P a c i f i c h e r r i n g o i l (10%) (Ackman 1982). C a p e l i n o i l i s high i n 20:1 and 22:1 whil e low i n 20:5n3 and 22:6n3 (Sargent et a l . 1989). P a c i f i c h e r r i n g o i l may have been used i n manufacturing COMM i n Year 1 and A t l a n t i c h e r r i n g o i l or a mixed f i s h o i l c o n t a i n i n g a high p r o p o r t i o n of c a p e l i n o i l was used i n Year 2. Labrador and Tree Ri v e r A r c t i c c h a r r eggs contained 10-12% n e u t r a l l i p i d and 11-12% p o l a r l i p i d . Eggs from w i l d as w e l l as two s t r a i n s of c u l t u r e d chinook salmon had almost t w i c e the co n c e n t r a t i o n (18-19%) of n e u t r a l l i p i d s and s l i g h t l y more p o l a r l i p i d s (13-14%) (Ashton 1991) compared t o Labrador and w i l d Tree R i v e r A r c t i c c h a r r . K a i t a r a n t a (1982) observed t h a t n e u t r a l l i p i d s made up over 50% of the t o t a l l i p i d of rainbow t r o u t eggs. I t i s unfortunate t h a t many authors who re p o r t f a t t y a c i d analyses on the 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 of eggs f a i l t o p r o v i d e the percent of n e u t r a l and p o l a r l i p i d s t h a t they observed. There i s l i t t l e i n f o r m a t i o n reported i n the 157 l i t e r a t u r e f o r comparison of n e u t r a l and p o l a r l i p i d contents of eggs i n other salmonids. C r a i k and Harvey (1984) found t h a t rainbow t r o u t eggs w i t h greater than 50% hatch had s i g n i f i c a n t l y more t o t a l l i p i d (25.7%) compared t o those w i t h 0% hatch (24.5%). N e u t r a l l i p i d s are u t i l i z e d as energy sources throughout embryogenesis, e s p e c i a l l y i n the l a t e r stages j u s t p r i o r t o hatching (Sargent et a l . 1989). While most p o l a r l i p i d s are i n c o r p o r a t e d i n t o membranes, both t r i a c y l g l y c e r o l and p h o s p h a t i d y l c h o l i n e were u t i l i z e d throughout embryogenesis by A t l a n t i c salmon (Henderson and Tocher 1987). P o l a r l i p i d s contained higher l e v e l s of s a t u r a t e d f a t t y a c i d s and HUFA and lower l e v e l s of monoenoic f a t t y a c i d s than n e u t r a l l i p i d s . Monoenoic a c i d s , 16:ln7, 18:ln7, 18:ln9 and 20:ln9, were in c o r p o r a t e d i n t o both the n e u t r a l and p o l a r l i p i d s of the eggs, w i t h high l e v e l s of 16:ln7 and 18:ln9 i n the n e u t r a l l i p i d s and 18:ln9 i n the p o l a r l i p i d s . These two f a t t y a c i d s can be synthesized de novo or obtained from the d i e t (Figure 1) ( C a s t e l l 1979, Henderson and Sargent 1985). There was l i t t l e i n c o r p o r a t i o n of 22:ln9, 2 0 : l n l l or 2 2 : l n l l i n t o 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 s of eggs even though the two n i l f a t t y a c i d s c o n s t i t u t e d 10.4-12.5% of the t o t a l l i p i d i n the LL and HL d i e t s . This was a l s o the case f o r chinook salmon (Ashton 1991). According t o Henderson and Sargent (1985), even when 2 2 : l n l l i s a major d i e t a r y f a t t y a c i d , i t i s not 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 s of f i s h membranes and may 158 f u n c t i o n mainly as an energy source through m i t o c h o n d r i a l R- o x i d a t i o n . Olsen et a l . (1991) fed j u v e n i l e A r c t i c c h a r r a d i e t c o n t a i n i n g 15.5% l i p i d w i t h 12% of t h i s l i p i d as 2 2 : l n l l and found i t made up only 1.2 and 1.8% of p o l a r l i p i d s of the l i v e r and muscle, r e s p e c t i v e l y and 6.4 and 12.3% of n e u t r a l l i p i d s . The major n6 f a t t y a c i d i n the n e u t r a l l i p i d s of eggs was 18:2n6 (28-32%) w h i l e there were low l e v e l s of 18:2n6 and 20:4n6 (1.2-2.3%) i n the p o l a r l i p i d s and s l i g h t l y l e s s 20:2n6 and 20:3n6. Ashton (1991) found s i m i l a r f i n d i n g s f o r n e u t r a l l i p i d s of chinook salmon eggs although she observed higher l e v e l s of 20:4n6 than 18:2n6 i n the p o l a r l i p i d s and minor amounts of 20:2n6 and 20:3n6. The f i n d i n g s w i t h c h a r r were i n c o n t r a s t t o A t l a n t i c salmon (Salmo salar) eggs, where 18:2n6 was the major n6 f a t t y a c i d i n the n e u t r a l l i p i d s and 20:4n6 was the major n6 f a t t y a c i d i n the p o l a r l i p i d s (Cowey et a l . 1985). Egg p o l a r l i p i d s from the LL groups contained s i g n i f i c a n t l y more 20:2n6, 20:3n6, t o t a l n6 and PUFA compared t o those from the HL groups. This increase i n n6 f a t t y a c i d s i n egg l i p i d s w i t h i n c r e a s e d d i e t a r y n6 i s i n agreement w i t h Yu et a l . (1979) and Watanabe et a l . (1984b). Since the major d i e t a r y n6 f a t t y a c i d was 18:2n6 w i t h only 0.6% 20:4n6, i t appears t h a t d i e t a r y 18:2n6 had undergone chain e l o n g a t i o n and d e s a t u r a t i o n t o 20:2n6 and 20:3n6 w h i l e 20:4n6 may have been from the d i e t or from d e s a t u r a t i o n of 20:3n6. Desaturation and e l o n g a t i o n of the n9, 159 n6 and n3 s e r i e s of f a t t y acids occurs through a common enzyme pathway but w i t h s p e c i f i c enzymes f o r the d e s a t u r a t i o n of each p o s i t i o n (Figure 1). In mammals, the desaturase enzymes have a gr e a t e r a f f i n i t y f o r the most unsaturated f a t t y a c i d a v a i l a b l e (Leray and P e l l e t i e r 1985). That i s , A6-desaturase has a grea t e r a f f i n i t y f o r 18:3n3 than 18:2n6 which has a gre a t e r a f f i n i t y than 18:ln9. S i m i l a r desaturases e x i s t f o r longer chain molecules. Both Olsen et al. (1991) and Yang and Dick (1992) found t h a t A r c t i c c harr f i n g e r l i n g s c ould desaturate and elongate d i e t a r y 18:3n3 t o n3HUFA and 18:2n6 and 20:4n6 t o n6PUFA. The former authors observed t h a t n3 f a t t y a c i d s were elongated and desaturated i n preference t o n6 f a t t y a c i d s . While HUFA, e s s e n t i a l t o maintaining membrane f l u i d i t y , made up 40-42% of the f a t t y acids i n the p o l a r l i p i d s , they were present at much lower l e v e l s (14-17%) i n the n e u t r a l l i p i d s of the eggs. This i s i n agreement wi t h data on rainbow t r o u t (Watanabe et al. 1984b, Léger et al. 1985) and chinook salmon (Ashton 1991) eggs. T o t a l l i p i d s of the LL and HL d i e t s contained approximately 6-7% 20:5n3 o r 22:6n3 and there were minor amounts of 18:3n3, 18:4n3, 20:3n3, 20:4n3 and 22:5n3. While egg l i p i d s contained l e s s than 1% of the 18 carbon n3 f a t t y a c i d s , 22:6n3 was i n c o r p o r a t e d i n t o egg l i p i d s at l e v e l s t h a t were 2-5 times higher than present i n the d i e t s . Some of t h i s h i g h l y unsaturated f a t t y a c i d probably came d i r e c t l y from d i e t a r y 22:6n3 w h i l e the remainder was syn t h e s i z e d by d e s a t u r a t i o n and el o n g a t i o n of the l e s s h i g h l y unsaturated n3 f a t t y a c i d s . 160 Dramatic d i f f e r e n c e s between the f a t t y a c i d composition of COMM compared t o the LL and HL d i e t s were r e f l e c t e d i n the composition of the eggs. The most s t r i k i n g d i f f e r e n c e s c o n s i s t e d of COMM's being lower than the LL and HL d i e t s i n 16:0, 18:ln9 and 2 0 : l n l l and higher i n 18:2n6, 20:ln9 and 2 2 : l n l l . This was r e f l e c t e d i n eggs from f i s h fed COMM having s i g n i f i c a n t l y l e s s 18:ln9 i n the n e u t r a l l i p i d s and s i g n i f i c a n t l y more 20:ln9 and 18:2n6 i n both the n e u t r a l and p o l a r l i p i d s . As was found w i t h eggs from f i s h fed LL and HL d i e t s , t h e r e was l i t t l e i n c o r p o r a t i o n of n i l f a t t y a c i d s i n t o the eggs of f i s h fed COMM even though t o t a l l i p i d contained 15.5% as 2 2 : l n l l . Yang and Dick (1992) showed th a t A r c t i c c h a r r produced 20:3n9 when e i t h e r 18:3n3 and/or 18:2n6 were d e f i c i e n t . While 20:3n9 was not found i n the eggs of rainbow t r o u t fed d i e t s l a c k i n g i n n3 f a t t y acids f o r 3 months (Watanabe et al. 1984b) or one year (Leray et al. 1985) p r i o r t o spawning, i t was found i n the m i l t a f t e r 3 months (Watanabe et al. 1984b) of feeding . When t r o u t were fed d i e t s c o n t a i n i n g 18:3n3 alone or i n combination w i t h 18:2n6 f o r the e n t i r e l i f e t i m e of the f i s h (Yu et al. 1979), 20:3n9 was observed i n the eggs from both groups but was lower from the group fed both f a t t y a c i d s . I t was a l s o reduced i n the m i l t of t r o u t i f the n3, n 6 - d e f i c i e n t d i e t was supplemented w i t h 18:2n6 (Watanabe et al. 1984b). Since i t takes a long feeding p e r i o d before 20:3n9 i s observed i n 161 broodstock, Leray et a l . (1985) suggested the use of another r a t i o as an i n d i c a t o r of n3 d e f i c i e n c y [20:3n9 + 20:4n6 + 22:5n6]/[20:5n3 + 22:5n3 + 22:6n3] w i t h a high r a t i o (6-12) f o r d e f i c i e n t eggs and a low r a t i o (0.5) f o r n o n - d e f i c i e n t eggs. This r a t i o was low i n the present experiment. Synthesis of 20:3n9 from 18:ln9 (18%) and/or 20:ln9 (3%) was probably i n h i b i t e d by the presence of 18:2n6 (7%) and 18:3n3 ( 1 % ) . D i e t a r y l i p i d s a l s o contained 6% 20:5n3 and 6% 22:6n3 which c o u l d have reduced the requirement f o r d e s a t u r a t i o n of s h o r t e r c h a i n molecules. (Leray et a l . 1985) found t h a t feeding rainbow t r o u t broodstock an n 3 - d e f i c i e n t d i e t r e s u l t e d i n a b n o r m a l i t i e s i n egg development v i s i b l e at the 16-32 c e l l stage as w e l l as a high i n c i d e n c e of d e f o r m i t i e s i n the f r y . A b n o r m a l i t i e s i n c e l l d i v i s i o n were observed i n eggs from 2-3 f i s h fed LLHE or HLLE but the f a t t y a c i d p r o f i l e s of these eggs d i d not d i f f e r from other eggs i n any c o n s i s t e n t way. In a d d i t i o n , t o t a l n3 and n6 i n these eggs ranged from 15.2-17.6% of the n e u t r a l l i p i d and 40.3-44.8% of the p o l a r l i p i d which i s not c o n s i s t e n t w i t h an n3 f a t t y a c i d d e f i c i e n c y . Leray et a l . (1985) speculated t h a t the i n c i d e n c e of egg abnormalities i n t h e i r research might have been due t o the high l e v e l of d i e t a r y 18:2n6 but l e v e l s of 18:2n6 i n d i e t s i n the present experiment were much lower (0.6% i n LL, 0.9% i n HL and 1.6% i n COMM) than those of Leray et a l . (1985) (5.4%). Some other f a c t o r ( s ) must be r e s p o n s i b l e f o r the 162 a b n o r m a l i t i e s observed i n t h i s experiment. While signs of f a t t y a c i d d e f i c i e n c y were observed i n carp broodstock fed d i e t s d e f i c i e n t i n v i t a m i n E (Watanabe and Takashima 1977), these were not observed i n A r c t i c c h a r r fed any of the d i e t s . No changes i n the f a t t y a c i d composition of eggs t h a t would be i n d i c a t i v e of an e s s e n t i a l f a t t y a c i d d e f i c i e n c y were found i n A r c t i c c harr and i t i s expected t h a t s u f f i c i e n t e s s e n t i a l f a t t y acids were a v a i l a b l e from the d i e t and/or from body s t o r e s t o meet the requirements. There was a s i g n i f i c a n t negative e f f e c t of n e u t r a l l i p i d and 16:ln7 and t o t a l n7 i n the n e u t r a l l i p i d s of eggs on s u r v i v a l t o each stage of development w i t h 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 s . The c o r r e l a t i o n w i t h n e u t r a l l i p i d and 16:ln7 accounted f o r 26-34% and 19-21% of the v a r i a n c e i n s u r v i v a l , r e s p e c t i v e l y . The f a c t t h a t the c o r r e l a t i o n was h i g h l y s i g n i f i c a n t (P=0.0006-0.0032 ) f o r n e u t r a l l i p i d i n d i c a t e s t h a t t h e r e i s a low p r o b a b i l i t y of t h i s l i n e a r r e l a t i o n s h i p being due t o chance. However, the low r e g r e s s i o n c o r r e l a t i o n i n d i c a t e s t h a t there are other c o v a r i a t e ( s ) t h a t are not accounted f o r i n t h i s l i n e a r r e l a t i o n s h i p . No reason f o r the e f f e c t of n e u t r a l l i p i d or 16:ln7 on h a t c h a b i l i t y i s known but t h i s i s an area t h a t warrants f u r t h e r i n v e s t i g a t i o n . When Leray et al. (1985) fed rainbow t r o u t broodstock a s e m i p u r i f i e d d i e t c o n t a i n i n g no n3 and 5.4% n6 f a t t y a c i d s , h a t c h a b i l i t y decreased 163 s i g n i f i c a n t l y compared t o those fed a commercial d i e t c o n t a i n i n g 1.3% n3 and 2.5% n6 (by r e c a l c u l a t i o n of the data i n the paper). In the n 3 - d e f i c i e n t group, the n e u t r a l and p o l a r l i p i d s of the eggs and m i l t contained very low l e v e l s of n3 but inc r e a s e d l e v e l s of n6 g i v i n g them a s i g n i f i c a n t l y lower n3/n6 r a t i o (Leray and P e l l e t i e r 1985, Leray et a l . 1985). There was a l s o a decrease i n n7 (15% vs 5%) 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 s of eggs of the n 3 - d e f i c i e n t group. This leads one t o speculate t h a t i f n7 f a t t y a c i d s decreased i n the n 3 - d e f i c i e n t group which had low s u r v i v a l , then n7 f a t t y a c i d s were not n e g a t i v e l y c o r r e l a t e d w i t h s u r v i v a l as they were w i t h A r c t i c c h a r r . When chinook salmon (Ashton 1991) were fed d i e t s c o n t a i n i n g s i m i l a r amounts of 16:ln7 t o t h i s experiment, the c o n c e n t r a t i o n of 16:ln7 i n the n e u t r a l l i p i d s of the eggs co u l d be estimated from the data provided at 4-5% which i s lower than observed i n t h i s experiment (9-14%). 5.7 Vi t a m i n E i n eggs King (1985) found t h a t the time p r i o r t o spawning duri n g which d i e t a r y v i t a m i n E was e i t h e r present or absent a f f e c t e d the v i t a m i n E con c e n t r a t i o n of the eggs. For example, when she sampled rainbow t r o u t one month p r i o r t o spawning, she observed s i g n i f i c a n t d i f f e r e n c e s i n vi t a m i n E content and c o n c e n t r a t i o n of eggs from broodstock fed vi t a m i n E - s u f f i c i e n t d i e t s (90 mg/kg) from J u l y t o December compared t o those fed d e f i c i e n t d i e t s (0 mg/kg) from August or October t o December. However, at 164 spawning time the only s i g n i f i c a n t d i f f e r e n c e was i n v i t a m i n E content between those fed e i t h e r the s u f f i c i e n t or d e f i c i e n t d i e t from J u l y u n t i l spawning i n January. While she s t a t e d t h a t the g r e a t e s t d i e t a r y e f f e c t s on egg v i t a m i n E at spawning occurred p r i o r t o October ( i e . p r i o r t o the 3 months before spawning), her data seems t o i n d i c a t e t h a t i t was p r i o r t o the 6 months before spawning ( i e . those fed d e f i c i e n t d i e t s from J u l y ) . However, she observed t h a t the g r e a t e s t r a t e of d e p o s i t i o n of v i t a m i n E i n t o the egg occurred between October and December - i e . i n the 3-4 months p r i o r t o spawning. In Year 1, A r c t i c c h a r r were fed f o r 71 days before feed was withdrawn 18 and 32 days before the f i r s t HE and LE f i s h spawned, r e s p e c t i v e l y . That i s , feed was f i r s t o f f e r e d 13 and 15 weeks p r i o r t o the commencement of spawning which was c l o s e t o the time t h a t the maximum r a t e of d e p o s i t i o n of v i t a m i n E occurred i n King's work. One must be cognizant of the f a c t t h a t there was a crossover of d i e t s i n Year 2. That i s , keeping the d i e t a r y v i t a m i n E l e v e l s the same, f i s h fed LL d i e t s i n Year 1 were fed HL d i e t s i n Year 2 and those fed HL d i e t s i n Year 1 were fed LL d i e t s i n Year 2 (Figure 3). I t i s i n t e r e s t i n g t h a t the v i t a m i n E l e v e l s observed i n the HE eggs i n Year 1 were very s i m i l a r t o those observed i n Year 2 even though the d i e t s were fed f o r a much longer p e r i o d of time i n Year 2. Since there were h i g h l y 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 v i t a m i n E content and c o n c e n t r a t i o n s of eggs from broodstock fed the LE versus HE d i e t s and between those fed the two l i p i d l e v e l s at the high 165 l e v e l of v i t a m i n E, the time t h a t the d i e t s were fed i n Year 1 was s u f f i c i e n t f o r i n c o r p o r a t i o n of v i t a m i n E i n t o the eggs. In Year 2, s i g n i f i c a n t d i f f e r e n c e s a l s o occurred between those fed LLLE, HLLE and COMM which were not apparent i n Year 1 w i t h LLLE < HLLE < COMM. I n i t i a l l y , the re d u c t i o n i n v i t a m i n E con c e n t r a t i o n i n the LLLE eggs between Year 1 and 2 suggests t h a t the a d d i t i o n a l year of being fed at the low l e v e l of l i p i d and v i t a m i n E (LLLE) reduced the body s t o r e s of v i t a m i n E and r e s u l t e d i n lower d e p o s i t i o n i n the eggs. While f i s h fed LLLE i n Year 2 produced more eggs than those fed HLLE, 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 t o t a l v i t a m i n E t r a n s f e r r e d t o the eggs i n the LLLE and HLLE groups i n Year 1 (12.6 mg) or Year 2 (13.6 mg vs 15.2 mg). F i s h fed LLLE a l s o t r a n s f e r r e d a s i m i l a r amount of v i t a m i n E per kg f i s h weight t o the eggs (6.4 mg/kg) as those fed HLLE (7.0 mg/kg). In c o n t r a s t , f i s h fed LLHE i n Year 1 deposited s i g n i f i c a n t l y more t o t a l v i t a m i n E i n the eggs compared t o those fed HLHE (48.9 mg vs 35.0 mg v i t a m i n E or 35.1 vs 24.6 mg v i t a m i n E/kg f i s h weight) but the d i f f e r e n c e s were not s i g n i f i c a n t i n Year 2 (54.6 mg vs 41.9 mg v i t a m i n E or 37.4 vs 24.4 mg v i t a m i n E/kg f i s h weight). While the d i e t s were fed f o r a longer p e r i o d of time i n Year 2, there were no marked i n c r e a s e s i n the v i t a m i n E conc e n t r a t i o n or content of the eggs. This confirms t h a t the p e r i o d of time f o r t r a n s f e r of d i e t a r y v i t a m i n E t o the eggs was met during the s h o r t e r p e r i o d of feeding i n Year 1. While some f i s h had more time t o m o b i l i z e v i t a m i n E from storage s i t e s t o the eggs p r i o r t o spawning, 166 there was no s i g n i f i c a n t r e l a t i o n between spawning day and v i t a m i n E content. King (1985) showed there was 25 mg of v i t a m i n E i n the body of rainbow t r o u t at the beginning of her experiment i n J u l y w i t h 45% of t h i s s t o r e d i n the l i v e r . Vitamin E was t r a n s p o r t e d from the l i v e r mainly t o the muscle i n J u l y and August. Between August and October, i t was transported from the muscle t o the ovary and adipose t i s s u e and then from the adipose t i s s u e t o the ovary between October and December. Vitamin E was t r a n s f e r r e d i n t o the eggs between October and December w i t h f i s h spawning throughout January. At spawning, the body burden of f i s h fed the d i e t c o n t a i n i n g no v i t a m i n E s t i l l contained 25 mg v i t a m i n E w h i l e those fed the d i e t supplemented at 90 mg/kg contained 35 mg v i t a m i n E. A f t e r spawning, the body burden was reduced t o 11.5 and 18 mg v i t a m i n E w i t h f i s h having t r a n s f e r r e d h a l f t h e i r body burden i n t o the eggs. I f the unsupplemented f i s h had been maintained on t h a t d i e t f o r another year, one can only speculate t h a t the v i t a m i n E content of the eggs and/or the number of eggs might have decreased. King's experiment shows t h a t i t took at l e a s t one spawning season before the body st o r e s were reduced by h a l f . This was a l s o observed i n p o u l t r y where h a t c h a b i l i t y d e c l i n e d over time when they were fed a v i t a m i n E - d e f i c i e n t d i e t and t h a t 40 eggs had t o be l a i d before a v i t a m i n E d e f i c i e n c y developed (Jensen and McGinnis 1957). In the c u r r e n t experiment, d i e t s contained 30 and 600 mg supplemental v i t a m i n 167 E acetate/kg. I f a d i e t w i t h 0 mg v i t a m i n E/kg had been f e d , i t might have r e s u l t e d i n a greater r e d u c t i o n i n the c o n c e n t r a t i o n of v i t a m i n E i n the LE eggs i n Year 2. Watanabe et a l . (1981a) found t h a t the v i t a m i n E requirement of rainbow t r o u t f r y increased as e i t h e r the l i p i d l e v e l or degree of unsatu r a t i o n of the d i e t a r y l i p i d i n c r e a s e d . The v i t a m i n E co n c e n t r a t i o n of the l i v e r decreased as d i e t a r y l i p i d i n c r e a s e d w i t h the greatest d i f f e r e n c e s o c c u r r i n g between f r y f ed low l i p i d (3 and 5%) and high l i p i d (10 and 15%) d i e t s . When f r y were fed d i e t s c o n t a i n i n g 50 mg v i t a m i n E/kg, the vi t a m i n E c o n c e n t r a t i o n i n the l i v e r was lowest when f r y were fed the d i e t w i t h the highest l e v e l of u n s a t u r a t i o n (5.7% PUFA) and i t was highest when they were fed the d i e t w i t h the lowest l e v e l of u n s a t u r a t i o n (1.6% PUFA). I t was speculated t h a t the decrease i n l i v e r v i t a m i n E was the r e s u l t of i t s incre a s e d requirement i n preventing o x i d a t i o n of PUFA and HUFA w i t h i n the t i s s u e s . This i s i n agreement w i t h research w i t h d u c k l i n g s and r a t s where v i t a m i n E requirements increased w i t h d i e t a r y PUFA only when d i e t a r y l i p i d was high (Fukaba 1980 c i t e d by Watanabe et a l . 1981a). The v i t a m i n E c o n c e n t r a t i o n and content of the eggs were lower from A r c t i c charr fed the HLHE d i e t compared t o the LLHE d i e t . This might be i n d i c a t i v e of decreased body st o r e s of v i t a m i n E i n broodstock fed the high l i p i d d i e t s as occurred w i t h f r y i n Watanabe et al. (1981a)'s experiment. While t h i s was observed when d i e t a r y l i p i d i n c r e a s e d i n the HE 168 d i e t s , i t was not apparent when d i e t a r y l i p i d i n c r e a s e d i n the LE d i e t s . Another p o s s i b i l i t y i s t h a t the v i t a m i n E requirement was met at 600 mg/kg i n the high l i p i d d i e t , but t h i s l e v e l of supplementation was excessive at the lower l i p i d l e v e l , r e s u l t i n g i n increased body stores of v i t a m i n E and in c r e a s e d amounts of v i t a m i n E t o t r a n s f e r t o the eggs. A f i n a l p o s s i b i l i t y i s t h a t of decreased absorption of v i t a m i n E i n the high l i p i d d i e t s which may have r e s u l t e d i n decreased t r a n s f e r of v i t a m i n E t o the eggs. Decreased absorption of v i t a m i n E was observed i n r a t s fed f i s h o i l compared t o corn o i l (Leka e t a l . 1989). King (1985) observed a decrease i n the v i t a m i n E content and c o n c e n t r a t i o n of rainbow t r o u t eggs (5.39 uq/egg or 58.3 fj,g/g) when broodstock were fed d i e t s c o n t a i n i n g no v i t a m i n E compared t o those fed d i e t s c o n t a i n i n g 90 mg v i t a m i n E/kg (7.37 Hg/egg or 85.8 \ig/g) f o r 6 months p r i o r t o spawning. Kinumaki et a l . (1972) a l s o observed increases i n v i t a m i n E from 4.4 jig/egg t o 23 and 32 jig/egg (27 t o 114 and 166 (xg/g) when d i e t a r y v i t a m i n E increased from 66 t o 4600 and 8200 mg/kg. I t i s a d v i s a b l e t o examine the trends t h a t occur r a t h e r than t o compare the a c t u a l v i t a m i n E content s i n c e content i s dependent on egg weight. Rainbow t r o u t eggs i n Kinumaki et a l . (1972)'s experiment were almost twice the weight of A r c t i c c h a r r eggs i n the present experiment. 169 V i t a m i n E content and c o n c e n t r a t i o n i n the f r y was g e n e r a l l y higher than t h a t of the eggs i n the present experiment. This i s i n c o n t r a s t t o the f i n d i n g s w i t h A t l a n t i c salmon (Cowey et a l . 1985) and rainbow t r o u t (King 1985, Kinumaki et a l . 1972). King (1985) found t h a t the v i t a m i n E content of rainbow t r o u t eggs was almost constant from the time of spawning u n t i l hatch while v i t a m i n E c o n c e n t r a t i o n d e c l i n e d . Kinumaki et a l . (1972) found s l i g h t reductions i n v i t a m i n E i n the egg between spawning and the time the y o l k sac had been absorbed. Cowey et a l . (1985) showed t h a t the v i t a m i n E content of A t l a n t i c salmon f e r t i l i z e d eggs decreased from 3.10 \iq/eqq (39.7 \iq/q) t o 2.29 \iq per f r y (32.7 \iq/q) at the swimup stage. The higher v i t a m i n E content and c o n c e n t r a t i o n i n A r c t i c c h a r r f r y compared t o eggs may have been due t o the f a c t t h a t l e s s than o n l y 2 months elapsed p r i o r t o a n a l y z i n g the f r y compared to 8-9 months f o r the eggs, although samples were f l u s h e d w i t h n i t r o g e n and frozen at -115°C. Tubes c o n t a i n i n g f r y were immediately f l u s h e d w i t h nitrogen and f r o z e n on dry i c e w h i l e egg samples were packed on i c e but were not f l u s h e d w i t h n i t r o g e n and frozen u n t i l the samples were returned t o the l a b o r a t o r y . Some groups of eggs d i d not r e s u l t i n s u f f i c i e n t f r y f o r a n a l y s i s and/or use i n a growth t r i a l and values f o r swimup f r y are r e p r e s e n t a t i v e only of s u r v i v o r s from each l o t of eggs. I t i s not known i f the composition of s u r v i v o r s was d i f f e r e n t from the non-survivors from the same parent. King (1985) analyzed the dead eyed eggs and l i v e eyed eggs from one 170 parent and found no d i f f e r e n c e i n the content of the eggs although the dead eggs had a higher c o n c e n t r a t i o n because they were s m a l l e r than the l i v e eggs. King (1985) recommended a minimum of 7 ng/egg (82 \ig/g) f o r best s u r v i v a l of rainbow t r o u t and found s u r v i v a l was poorest at 5 ng/egg (54 (xg/g). Eggs from the w i l d stock of A r c t i c c h a r r from Tree R i v e r contained v i t a m i n E at 73.8 »ug/g or 9.0 \ig/egq. This c o n c e n t r a t i o n i s 1.3-1.9 times higher than t h a t of Labrador c h a r r eggs i n e i t h e r LE group, yet the content i s s i m i l a r t o t h a t of the HLHE group because the Tree R i v e r charr eggs weighed t w i c e as much as the Labrador charr eggs. While Tree R i v e r c h a r r eggs were c l o s e t o hatch when sampled, both Cowey et a l . (1985) and Kinumaki et al. (1981) showed t h a t there was l i t t l e d e c l i n e i n v i t a m i n E between the u n f e r t i l i z e d egg and hatch i n A t l a n t i c salmon and rainbow t r o u t , r e s p e c t i v e l y . In Year 2, f i s h i n the present experiment produced eggs w i t h 1.5-2.8 and 2.4-4.9 \ig v i t a m i n E/egg when fed the LE d i e t s and 7.1-17.2 and 5.9-10.5 |xg v i t a m i n E/egg i n the HE d i e t s . Using King (1985)'s c r i t e r i a , eggs i n the LE group were a l l below the 5 (xg/egg t h r e s h o l d w h i l e those i n the HE group were almost a l l above the 7 |xg/egg t h r e s h o l d . However, A r c t i c c h a r r eggs were smaller than rainbow t r o u t eggs and i t i s expected t h a t a s m a l l e r embryo would r e q u i r e l e s s v i t a m i n E. Therefore, the t h r e s h o l d may be l e s s f o r A r c t i c c h a r r than recommended by King f o r rainbow t r o u t . 171 The feeding of v i t a m i n E - d e f i c i e n t d i e t s t o hens r e s u l t e d i n decreased h a t c h a b i l i t y i n turkeys (Jensen et a l . 1955, Atk i n s o n et a l . 1955) and chickens (Tengerdy and Nockels 1973). The involvement of v i t a m i n E i n s u r v i v a l of the egg was s u b s t a n t i a t e d by showing th a t i n j e c t i o n of v i t a m i n E d i r e c t l y i n t o v i t a m i n E - d e f i c i e n t eggs increased h a t c h a b i l i t y (Jensen and McGinnis 1957). As has been found w i t h f i s h , the v i t a m i n E content of the chicken egg was d i r e c t l y r e l a t e d t o maternal d i e t a r y v i t a m i n E content (Bartov et a l . 1965). King (1985) found eggs, from rainbow t r o u t fed d i e t s c o n t a i n i n g 8% l i p i d w i t h 0 mg v i t a m i n E/kg f o r 6 months p r i o r t o spawning, had s i g n i f i c a n t l y lower s u r v i v a l (89%) than those fed 90 mg v i t a m i n E/kg (98%). Takeuchi et a l . (1981a) a l s o found h a t c h a b i l i t y decreased s i g n i f i c a n t l y when ayu were fed f o r 3 months p r i o r t o spawning on d i e t s c o n t a i n i n g no v i t a m i n E compared t o more than 34 mg v i t a m i n E/kg. In the present experiment, s i g n i f i c a n t d i f f e r e n c e s were not observed i n the embryonic s u r v i v a l of ch a r r when fed the LE compared t o HE d i e t s i n e i t h e r year. In Year 2, the medians f o r s u r v i v a l t o swimup were 45 and 57% f o r the HE d i e t s i n d i c a t i n g t h a t h a l f of these females produced eggs which had s u r v i v a l t o swimup of gr e a t e r than 45-57%. In c o n t r a s t , 50% of the f i s h fed the LE d i e t s had s u r v i v a l t o swimup of greater than 9 and 1% and those fed COMM had a median of 17%. 172 The w i t h i n treatment variance f o r s u r v i v a l of eggs t o the eyed, hatch and swimup stages was very high and the sample s i z e was low w i t h 7-12 f i s h and 6-10 f i s h spawning per treatment i n Year 1 and 2, r e s p e c t i v e l y . The low sample s i z e was due t o mis- sexing of f i s h at the beginning of the experiment, f a i l u r e of some females t o spawn each year, premature r e l e a s e of eggs i n t o the tank and a low number of f i s h m o r t a l i t i e s . The high v a r i a n c e s and low sample number r e s u l t e d i n the need f o r very l a r g e d i f f e r e n c e s between means f o r a s i g n i f i c a n t d i f f e r e n c e t o be detected w i t h an acceptable degree of c e r t a i n t y . Therefore, w h i l e the d i f f e r e n c e s i n the mean s u r v i v a l s i n Year 2 lead one t o suspect t h a t there was a p o s i t i v e e f f e c t of v i t a m i n E, these d i f f e r e n c e s are not s i g n i f i c a n t . For example, feeding the two LE d i e t s r e s u l t e d i n 19 and 23% s u r v i v a l w h i l e the two HE d i e t s r e s u l t e d i n 42 and 51% s u r v i v a l t o swimup. Using power a n a l y s i s d e s c r i b e d by Peterman (1990) and Dixon and Massey (1969) on data f o r s u r v i v a l t o the eyed stage, there i s a 40% chance of not r e j e c t i n g ( i e . accepting) the n u l l hypothesis when the n u l l hypothesis i s f a l s e (^=45, o2=1089, s 2=953, n=7.5, a=0.05). In other words, given the variance i n embryonic s u r v i v a l i n the p o p u l a t i o n , i f there was a s i g n i f i c a n t d i f f e r e n c e t o d e t e c t , the chance of d e t e c t i n g i t would be 40%. Sample s i z e s of 15 and 25 would be r e q u i r e d t o increase the power t o 0.75 and 0.95, r e s p e c t i v e l y . The planned sample s i z e was 15 but the a c t u a l sample s i z e was as low as 6 i n Year 2. 173 The case of low sample s i z e o f t e n w i t h a high degree of v a r i a t i o n i s common i n broodstock n u t r i t i o n w i t h sample s i z e s of 3-6 f i s h per treatment reported (King 1985, Leray et al. 1985, Yu et al. 1979, Watanabe et a l . 1984b). That i s not t o say t h a t a l l broodstock n u t r i t i o n i s conducted on small groups of f i s h . Knox et al. (1988) used 40 females per tank w i t h 2 r e p l i c a t e s per treatment i n h i s work on the e f f e c t of r a t i o n , but g e n e r a l l y high numbers of broodstock are not a v a i l a b l e . In f u t u r e , i t w i l l be necessary t o reduce the number of treatments i n order t o have r e p l i c a t e tanks of f i s h and t o i n c r e a s e the number of f i s h per tank. In a d d i t i o n , not a l l males produced m i l t each year and i t i s suggested t h a t the number of males a l s o be i n c r e a s e d . The f a c t t h a t there was no d i r e c t c o r r e l a t i o n between v i t a m i n E content or c o n c e n t r a t i o n of the eggs and s u r v i v a l t o any stage i n t h i s experiment i n d i c a t e s e i t h e r t h a t there was adequate v i t a m i n E even at the lower l e v e l of d i e t a r y v i t a m i n E f o r adequate d e p o s i t i o n i n the egg or t h a t there were other i n d i r e c t e f f e c t ( s ) of v i t a m i n E. Other i n d i r e c t e f f e c t s such as promoting a f a t t y a c i d d e f i c i e n c y were not found i n any of the eggs. King (1985) reported one case of eggs w i t h d i s p e r s e d l i p i d g l o b u l e s and convoluted membranes i n rainbow t r o u t groups fed the unsupplemented d i e t f o r 6 months p r i o r t o spawning. Spawning ab n o r m a l i t i e s occurred i n both years i n t h i s study but i n Year 2, 4 of the 5 cases occurred i n f i s h fed LE d i e t s and 1 w i t h the HE d i e t s . These abnormalities i n c l u d e d eggs which were 174 r e l e a s e d i n t o the tank, eggs which formed a s o l i d mass and c o u l d not be r e l e a s e d , the presence of blood i n the o v a r i a n f l u i d , brown p a r t i c u l a t e matter i n the f l u i d , and small numbers of unusual brown and dark orange-coloured eggs w i t h the orange eggs o c c u r r i n g i n a f i s h fed a HE d i e t . While the presence of blood i n the o v a r i a n f l u i d might be i n d i c a t i v e of a v i t a m i n E d e f i c i e n c y i n which there was d e t e r i o r a t i o n of the w a l l s of the c a p i l l a r i e s , the incidence of t h i s s i g n was very s m a l l . I f d i e t a r y v i t a m i n E had some other i n d i r e c t e f f e c t , i t was not measured i n t h i s experiment. 5.8 Fry growth and s u r v i v a l S u r v i v a l of f r y decreased from 82% i n Year 1 t o 76% i n Year 2 which was s i m i l a r t o the decrease from 88% t o 79% t h a t was p r e d i c t e d from the decrease i n number of f r y r a i s e d i n the j a r s . With the exception of the LLHE group, the s p e c i f i c growth r a t e i n c r e a s e d i n Year 2 from 1.7% i n Year 1 t o 2.4% i n Year 2 which was g r e a t e r than the p r e d i c t e d 0.4% increase r e s u l t i n g from the decrease i n number of f r y i n the j a r s . Environmental c o n d i t i o n s were as s i m i l a r as p o s s i b l e i n both Year 1 and 2. Feed was purchased from the same company and a n a l y s i s showed the proximate composition was 39.7% crude p r o t e i n , 25.5% t o t a l l i p i d , 11.8% ash and 82.6% dry matter i n Year 1 and 49.7% crude p r o t e i n , 20.0% t o t a l l i p i d , 11.8% ash and 80.6% dry matter i n Year 2. The d i f f e r e n c e s i n p r o t e i n and l i p i d and/or changes i n the i n g r e d i e n t s used by the manufacturer i n Year 2 may a l s o have 175 c o n t r i b u t e d t o the d i f f e r e n c e s i n s p e c i f i c growth r a t e . There was no e f f e c t of v i t a m i n E content or c o n c e n t r a t i o n of the f r y on s u r v i v a l of f r y i n e i t h e r year or on s p e c i f i c growth r a t e i n Year 1. However, i n Year 2 f r y from the LLHE group had a s i g n i f i c a n t l y lower mean s p e c i f i c growth r a t e than those fed the other LL or HE d i e t s . In a d d i t i o n , f r y from the LLHE group had the lowest s u r v i v a l during the 6 week growth t r i a l although t h i s was not s i g n i f i c a n t l y d i f f e r e n t . Since the f r y i n the LLHE group had the highest l e v e l s of v i t a m i n E, one must con s i d e r the p o s s i b i l i t y of hypervitaminosis but there was no c o r r e l a t i o n between s p e c i f i c growth r a t e or s u r v i v a l and v i t a m i n E content or c o n c e n t r a t i o n . Although s t u d i e s w i t h c h i c k s showed t h a t growth r a t e and r e s p i r a t i o n r a t e of s k e l e t a l mitochondria were decreased when c h i c k s r e c e i v e d d i e t a r y l e v e l s of 2200 mg v i t a m i n E acetate/kg compared to 1000 mg v i t a m i n E acetate/kg (March et a l . 1973), there have been no s t u d i e s reported on hypervitaminosis E i n f i s h . Kinumaki et a l . (1972) fed d i e t s c o n t a i n i n g 4600, 8200 mg and 10,000 mg v i t a m i n E/kg and d i d not r e p o r t increased m o r t a l i t y of eggs or f r y . 176 CONCLUSIONS Feeding 4-5 year o l d Labrador A r c t i c c h a r r broodstock d i e t s c o n t a i n i n g 12 or 19% l i p i d w i t h 30 or 600 mg v i t a m i n E acetate/kg d i e t d i d not r e s u l t i n s i g n i f i c a n t d i f f e r e n c e s i n f e r t i l i z a t i o n of eggs or s u r v i v a l t o the eyed, hatch or swimup stages. In Year 2, f i s h fed the HE d i e t s produced eggs w i t h s u r v i v a l s 1.5-2.2 times higher than those fed the LE d i e t s . The high degree of w i t h i n treatment v a r i a t i o n i n s u r v i v a l t o the eyed, hatch and swimup stages and the small number of females which spawned r e s u l t e d i n the need f o r very l a r g e d i f f e r e n c e s i n means i n order t o detect s i g n i f i c a n t d i f f e r e n c e s . Future research w i t h Labrador A r c t i c charr broodstock r e q u i r e s higher numbers of f i s h per treatment and r e p l i c a t e tanks i n order t o i n c r e a s e the power of the t e s t . Feeding LL d i e t s r e s u l t e d i n delayed spawning i n Year 1 but t h i s was not apparent i n Year 2. The p r o p o r t i o n of females f a i l i n g t o spawn was not r e l a t e d t o concentrations of d i e t a r y l i p i d or v i t a m i n E. A s i g n i f i c a n t l y lower p r o p o r t i o n of males fed the higher c o n c e n t r a t i o n of v i t a m i n E produced m i l t i n two s u c c essive years. Fecundity was h i g h l y v a r i a b l e w i t h i n treatments but v a r i a t i o n was d i r e c t l y c o r r e l a t e d w i t h female weight and was not a f f e c t e d by the c o n c e n t r a t i o n of d i e t a r y l i p i d or v i t a m i n E. F i s h fed COMM consumed more feed, weighed more and produced s i g n i f i c a n t l y more eggs than those fed other 177 d i e t s i n Year 2 except f o r LLLE. S u r v i v a l t o swimup of the COMM group was h a l f t h a t of the HE groups. D i e t a r y v i t a m i n E had a s i g n i f i c a n t e f f e c t on the v i t a m i n E c o n c e n t r a t i o n and content of the eggs even when fed the d i e t s f o r o n l y 71 days i n Year 1. Inc r e a s i n g d i e t a r y v i t a m i n E co n c e n t r a t i o n had a greater impact on egg v i t a m i n E co n c e n t r a t i o n at the low compared t o the high c o n c e n t r a t i o n of d i e t a r y l i p i d . This might be due t o decreased ab s o r p t i o n or inc r e a s e d o x i d a t i o n of vi t a m i n E w i t h i n the t i s s u e s when the high l i p i d d i e t was fed. F e r t i l i z a t i o n and embryonic s u r v i v a l were not c o r r e l a t e d w i t h v i t a m i n E content or c o n c e n t r a t i o n of the eggs. I f the broodstock had been fed a d i e t c o n t a i n i n g 30 mg v i t a m i n E/kg d i e t f o r more of t h e i r l i f e t i m e , they may have had lower s t o r e s of vi t a m i n E at the beginning of the experiment and more pronounced e f f e c t s of feeding a d i e t w i t h a low co n c e n t r a t i o n of vi t a m i n E may have been observed. D i e t a r y l i p i d and vi t a m i n E concentrations d i d not a f f e c t the t o t a l , n e u t r a l or p o l a r l i p i d c oncentrations of the eggs. Feeding COMM r e s u l t e d i n s i g n i f i c a n t l y higher l e v e l s of t o t a l and n e u t r a l l i p i d i n the eggs of f i s h fed a l l d i e t s except LLLE. The f a t t y a c i d composition of eggs d i d not i n d i c a t e a d e f i c i e n c y of e s s e n t i a l f a t t y a c i d s . The f a t t y a c i d a s s o c i a t e d w i t h e s s e n t i a l f a t t y a c i d d e f i c i e n c y , 20:3n9, was not detected 178 i n any eggs analyzed. The con c e n t r a t i o n of 22:6n3 was un i f o r m l y high i n the p o l a r l i p i d s of eggs from f i s h fed a l l d i e t s . The f a t t y a c i d composition of the eggs r e f l e c t e d t h a t of the d i e t . The HL d i e t s had s i g n i f i c a n t l y lower n6 and PUFA and a higher n3/n6 r a t i o than the LL d i e t s and s i g n i f i c a n t d i f f e r e n c e s i n these f a t t y a c i d s and the n3/n6 r a t i o were apparent i n the p o l a r l i p i d s of the eggs. COMM eggs were s i g n i f i c a n t l y higher i n n6 and PUFA i n the n e u t r a l l i p i d s than eggs from a l l other treatments. S u r v i v a l t o the eyed, hatch and swimup stages was n e g a t i v e l y c o r r e l a t e d w i t h the percent of n e u t r a l l i p i d and 16:ln7, one of the major n7 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 f r a c t i o n . The low c o e f f i c i e n t of determination ( r 2 ) f o r the e f f e c t of n e u t r a l l i p i d on s u r v i v a l i n d i c a t e s t h a t a d d i t i o n a l c o v a r i a t e ( s ) must e x i s t which a l s o a f f e c t embryonic s u r v i v a l . Eggs from w i l d A r c t i c charr from the Tree R i v e r , NWT had high s u r v i v a l t o swimup. They had v i t a m i n E c o n c e n t r a t i o n higher than those fed the LE d i e t and t o t a l , n e u t r a l and p o l a r l i p i d s i m i l a r t o Labrador charr i n t h i s experiment. 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L i p i d s 14: 572-575. Yurkowski, M. 1989. L i p i d content and f a t t y a c i d composition of muscle from some freshwater and marine f i s h from c e n t r a l and A r c t i c Canada, pp. 547-557. In Chandra, R.K. (ed.) Health E f f e c t s of F i s h and F i s h O i l s . ARTS P u b l i s h e r s . S t . John's, N f l d . 191 Appendix l a . F a t t y a c i d s i n the t o t a l l i p i d i n the d i e t s . F a t t y LLLE LLHE HLLE HLHE COMM COMM a c i d Year 1 Year 2 % % % % % % 14:0 4.16 4.11 4.44 4.09 5.14 3.86 15:01 0.15 0.08 0.17 0.15 0.16 0.17 15:0 0.34 0.36 0.36 0.33 0.31 0.31 16:01 0.11 0.14 0.13 0.10 0.09 0.09 Unknown 0.25 0.27 0.27 0.26 0.11 0.22 16:0 16.40 16.43 15.97 15.61 15.77 12.16 16:ln7 5.06 5.05 5.51 5.36 7.76 6.97 16:ln5 0.18 0.19 0.20 0.18 0.22 0.24 Unknown 0.29 0.33 0.34 0.31 0.30 0.15 16:2n9 0.15 0.17 0.17 0.16 0.13 0.10 Unknown 0.42 0.44 0.47 0.45 0.79 0.48 17:0 0.83 0.86 0.94 0.92 0.14 0.22 Unknown 0.24 0.26 0.26 0.24 0.22 0.15 17 18 16 18 18 18 18 18 18 19 18 19 18 18 20 20 20 20 20 20 20 20 20 22 22 22 22 21 22 22 22 24 ln9 0.32 0.31 0.32 0.31 0.23 0.15 01 0.22 0.20 0.19 0.18 0.11 0.13 5n3 0.37 0.40 0.42 0.39 1.38 0.60 0 2.72 2.69 2.68 2.69 2.53 1.63 ln9 18.71 18.60 18.48 18.83 12.44 10.93 ln7 3.70 3.72 3.81 3.62 2.64 2.35 ln5 0.32 0.33 0.35 0.35 0.26 0.40 2n6 7.46 7.40 4.90 4.97 10.68 10.52 3n6 0.15 0.16 0.17 0.17 0.23 0.12 0 0.09 0.10 0.10 0.09 0.14 0.08 4n6 0.11 0.12 0.13 0.12 0.17 0.07 ln7 0.10 0.10 0.10 0.10 0.04 0.04 3n3 1.32 1.32 1.00 1.01 1.32 1.01 4n3 0.88 0.88 1.00 1.00 1.77 1.03 0 0.20 0.20 0.20 0.21 0.14 0.17 l n l l 3.84 3.77 4.63 5.04 0.43 0.68 ln9 3.30 3.29 3.52 3.32 4.49 11.32 ln7 0.48 0.48 0.56 0.57 0.29 0.81 2n6 0.18 0.18 0.20 0.20 0.14 0.13 4n6 0.62 0.62 0.63 0.64 0.39 0.25 3n3 0.08 0.08 0.09 0.09 0.06 0.05 4n3 0.29 0.29 0.33 0.34 0.66 0.24 5n3 5.97 5.92 6.20 6.36 10.45 5.05 0 0.13 0.13 0.11 0.12 0.09 0.09 l n l l 6.60 6.50 7.49 7.83 5.39 15.52 ln9 1.01 1.00 1.17 1.16 0.61 1.68 ln7 0.20 0.19 0.21 0.21 0.13 0.33 5n3 0.21 0.21 0.23 0.25 0.38 0.19 5n6 0.12 0.12 0.13 0.14 0.07 0.07 5n3 0.92 0.90 1.03 1.08 1.13 0.61 6n3 6.77 6.75 6.33 6.58 5.89 4.90 ln9 0.78 0.74 0.80 0.84 0.42 0.59 Unknown 0.17 0.16 0.12 0.11 0.07 0.06 192 Appendix l b . F a t t y a c i d composition (mean % and standard d e v i a t i o n ) of the n e u t r a l l i p i d s of the eggs i n Year 2. F a t t y D i e t a c i d LLLE LLHE HLLE HLHE COMM % SD % SD % SD % SD % SD 14:0 2. 20 0. 14 1 .97 0. 24 2 .22 0 .35 2 .07 0. 36 2.52 0.23 14:ln5 0. 11 0. 01 0 .09 0. 02 0 .10 0 .02 0 .07 0. 04 0.11 0.02 15:01 0. 10 0. 02 0 .09 0. 02 0 .11 0 .01 0 .10 0. 01 0.11 0.01 15:0 0. 25 0. 03 0 .21 0. 02 0 .26 0 .02 0 .25 0. 05 0.26 0.03 16:01 0. 11 0. 03 0 .09 0. 01 0 .11 0 .00 0 .09 0. 02 0.08 0.01 16:0 11. 81 0. 87 10 .81 2. 97 10 .46 1 .11 10 .64 1. 29 10.58 0.41 Unkn. 0. 15 0. 03 0 .15 0. 06 0 .17 0 .02 0 .14 0. 02 0.28 0.04 16:ln9 0. 69 0. 14 0 .78 0. 19 0 .65 0 .10 0 .59 0. 11 0.55 0.30 16:ln7 11. 29 1. 09 10 .89 1. 43 10 .98 0 .93 11 .34 0. 78 12.65 0.87 16:ln5 0. 19 0. 02 0 .17 0. 01 0 .20 0 .01 0 .19 0. 03 0.26 0.02 Unkn. 0. 23 0. 04 0 .19 0. 01 0 .21 0 .03 0 .20 0. 04 0.08 0.05 16:2n9 0. 19 0. 04 0 .16 0. 01 0 .22 0 .04 0 .17 0. 01 0.22 0.03 Unkn. 0. 16 0. 03 0 .16 0. 03 0 .19 0 .02 0 .20 0. 03 0.30 0.03 17:0 0. 21 0. 06 0 .16 0. 08 0 .23 0 .03 0 .21 0. 04 0.01 0.01 Unkn. 0. 14 0. 04 0 .11 0. 01 0 .13 0 .00 0 .13 0. 02 0.10 0.01 17:ln9 0. 52 0. 05 0 .43 0. 03 0 .56 0 .05 0 .55 0. 09 0.30 0.11 18:0 1. 53 0. 14 1 .54 0. 45 1 .46 0 .10 1 .43 0. 12 1.26 0.12 18:ln9 31. 06 1. 04 32 .10 2. 65 31 .67 1 .49 32 .40 2. 94 27.67 1.98 18:ln7 6. 55 0. 35 6 .16 1. 25 6 .03 0 .46 5 .58 0. 76 5.66 0.58 18:ln5 0. 32 0. 01 0 .31 0. 04 0 .34 0 .00 0 .31 0. 04 0.39 0.01 Unkn. 0. 25 0. 07 0 .33 0. 17 0 .20 0 .06 0 .24 0. 10 0.16 0.05 18:2n9 0. 17 0. 03 0 .18 0. 02 0 .10 0 .04 0 .13 0. 01 0.15 0.01 18:2n6 5. 52 0. 48 5 .50 1. 37 5 .47 1 .03 5 .84 1. 04 7.88 0.75 18:3n6 0. 22 0. 02 0 .20 0. 02 0 .23 0 .02 0 .23 0. 03 0.18 0.09 19:0 0. 64 0. 21 0 .85 0. 82 0 .57 0 .33 0 .69 0. 38 0.71 0.32 18:4n6 0. 20 0. 01 0 .20 0. 04 0 .22 0 .01 0 .24 0. 02 0.24 0.01 19:ln7 0. 12 0. 01 0 .11 0. 02 0 .11 0 .02 0 .19 0. 20 0.07 0.00 18:3n3 0. 61 0. 07 0 .46 0. 11 0 .62 0 .09 0 .65 0. 12 0.56 0.07 18:4n3 0. 55 0. 11 0 .42 0. 11 0 .55 0 .04 0 .67 0. 10 0.64 0.13 1 8 : 4 n l l 0 .19 0 .03 0 .17 0. 04 0 .21 0 .01 0 .24 0. 02 0.35 0.03 2 0 : l n l l 0 .77 0 .13 0 .70 0. 20 0 .90 0 .18 0 .84 0. 27 0.66 0.09 20:ln9 2. 16 0. 21 2 .65 0. 61 2 .27 0 .38 1 .91 0. 17 3.85 0.40 20:ln7 0. 42 0. 04 0 .45 0. 07 0 .41 0 .03 0 .39 0. 07 0.43 0.05 Unkn. 0. 32 0. 09 0 .42 0. 13 0 .18 0 .07 0 .19 0. 08 0.17 0.09 20:2n6 0. 72 0. 10 0 .71 0. 07 0 .60 0 .06 0 .56 0. 03 0.79 0.08 20:3n6 0. 71 0. 16 0 .80 0. 16 0 .57 0 .19 0 .58 0. 12 0.90 0.02 20:4n6 0. 67 0. 09 0 .76 0. 14 0 .66 0 .05 0 .64 0. 09 0.59 0.08 20:4n3 0. 33 0. 10 0 .26 0. 06 0 .41 0 .06 0 .41 0. 11 0.38 0.06 20:5n3 3. 55 0. 65 3 .46 0. 50 4 .58 0 .22 4 .72 0. 36 3.92 0.47 2 2 : l n l l 0 .32 0 .06 0 .40 0. 23 0 .45 0 .06 0 .42 0. 09 0.86 0.20 22:ln9 0. 17 0. 01 0 .23 0. 08 0 .21 0 .04 0 .19 0. 01 0.26 0.05 21:5n3 0. 22 0. 02 0 .20 0. 02 0 .25 0 .01 0 .27 0. 02 0.24 0.01 22:5n6 0. 11 0. 02 0 .10 0. 01 0 .10 0 .01 0 .09 0. 02 0.06 0.02 22:5n3 1. 25 0. 24 1 .33 0. 14 1 .63 0 .12 1 .51 0. 07 1.38 0.22 22:6n3 9. 50 1. 59 10 .19 1. 24 9 .88 0 .75 9 .49 1. 41 8.58 1.46 193 Appendix l e . F a t t y a c i d composition (mean % ± standard d e v i a t i o n ) of the p o l a r l i p i d s of the eggs i n Year 2, F a t t y D i e t a c i d LLLE LLHE HLLE HLHE COMM % SD % SD % SD % SD % SD 14:0 1. 14 0. 09 0 .94 0 .11 0. 88 0.13 0 .97 0. 15 1.08 0.07 15:0 0. 27 0. 03 0. 22 0. 02 0. 24 0.02 0 .28 0. 02 0.25 0.03 16:0 16. 49 0. 59 15 .69 1 .13 15. 99 0.28 16 .43 0. 51 15.60 0.95 Unkn. 0. 18 0. 06 0 .18 0 .06 0. 10 0.03 0 .12 0. 10 0.27 0.05 16:ln9 0. 58 0. 12 0 .67 0 .18 0. 45 0.15 0 .48 0. 14 0.54 0.09 16:ln7 2. 39 0. 14 2 .57 0 .24 2. 09 0.21 2 .31 0. 31 2.45 0.18 16:ln5 0. 10 0. 01 0 .09 0 .02 0. 09 0.04 0 .10 0. 01 0.13 0.01 Unkn. 0. 23 0. 03 0 .18 0 .01 0. 21 0.01 0 .21 0. 03 0.17 0.02 16:2n9 0. 11 0. 01 0 .09 0 .01 0. 10 0.01 0 .10 0. 01 0.08 0.01 17:0 0. 17 0. 04 0 .14 0 .01 0. 13 0.02 0 .12 0. 03 0.08 0.02 Unkn. 0. 16 0. 01 0 .12 0 .01 0. 17 0.03 0 .15 0. 02 0.12 0.02 17:ln9 0. 24 0. 02 0 .20 0 .03 0. 22 0.02 0 .23 0. 01 0.17 0.02 18:01 0. 22 0. 10 0 .20 0 .03 0. 20 0.13 0 .21 0. 10 0.18 0.02 18:0 3. 34 0. 23 3 .39 0 .60 3. 44 0.43 3 .26 0. 25 2.91 0.30 18:ln9 12. 39 0. 67 13 .55 2 .76 12. 75 1.97 13 .35 1. 55 11.62 0.72 18:ln7 6. 82 0. 35 6 .44 1 .32 6. 59 1.18 6 .23 0. 75 6.07 0.45 18:ln5 0. 39 0. 03 0 .36 0 .06 0. 43 0.03 0 .41 0. 05 0.47 0.03 19: OA 0. 18 0. 09 0 .21 0 .09 0. 12 0.04 0 .16 0. 06 0.10 0.06 Unkn. 0. 11 0. 04 0 .09 0 .02 0. 08 0.00 0 .08 0. 00 0.09 0.00 18:2n6 1. 74 0. 23 1 .80 0 .17 1. 49 0.34 1 .62 0. 16 2.29 0.15 18:3n6 0. 11 0. 01 0 .08 0 .01 0. 11 0.02 0 .11 0. 02 0.10 0.01 19:0 0. 12 0. 04 0 .13 0 .08 0. 06 0.03 0 .08 0. 03 0.11 0.03 19:ln7 0. 26 0. 02 0 .21 0 .05 0. 25 0.08 0 .22 0. 05 0.15 0.02 18:3n3 0. 15 0. 02 0 .11 0 .02 0. 14 0.03 0 .16 0. 04 0.12 0.02 18:4n3 0. 14 0. 03 0 .10 0 .02 0. 09 0.01 0 .13 0. 01 0.13 0.03 2 0 : l n l l 0. .30 0. .06 0 .27 0 .14 0. 34 0.12 0 .35 0. 13 0.31 0.07 20:ln9 3. 80 0. 33 4 .68 0 .96 4. 29 0.88 3 .64 0. 47 6.04 0.49 20:ln7 0. 42 0. 02 0 .49 0 .09 0. 49 0.10 0 .40 0. 06 0.46 0.05 Unkn. 0. 12 0. 06 0 .20 0 .08 0. 04 0.06 0 .08 0. 03 0.09 0.02 20:2n6 1. 01 0. 12 1 .06 0 .13 0. 89 0.14 0 .82 0. 08 1.15 0.14 20:3n6 0. 99 0. 30 1 .15 0 .07 0. 62 0.28 0 .62 0. 11 1.15 0.14 20:4n6 1. 63 0. 27 1 .79 0 .47 1. 24 0.18 1 .37 0. 37 1.48 0.29 20:4n3 0. 13 0. 05 0 .11 0 .03 0. 14 0.03 0 .14 0. 05 0.14 0.03 20:5n3 7. 46 0. 76 7 .05 0 .69 8. 10 1.11 8 .58 0. 60 7.96 0.61 2 2 : l n l l 0. .10 0. .03 0 .14 0 .05 0. 16 0.04 0 .14 0. 03 0.26 0.04 21:5n3 0. 09 0. 06 0 .12 0 .01 0. 12 0.01 0 .12 0. 01 0.09 0.06 22:4n6 0. 10 0. 05 0 .13 0 .04 0. 08 0.01 0 .06 0. 03 0.04 0.03 22:5n6 0. 28 0. 03 0 .24 0 .06 0. 23 0.05 0 .21 0. 04 0.12 0.08 22:5n3 2. 32 0. 30 2 .58 0 .17 2. 76 0.20 2 .57 0. 18 2.82 0.29 22:6n3 30. 51 0. 84 29 .86 1 .42 31. 38 1.45 30 .87 0. 99 29.99 0.88 2 4 : l n l l 0. .24 0. .17 0 .36 0 .07 0. 36 0.07 0 .30 0. 16 0.38 0.06 24:ln9 0. 13 0. 02 0 .12 0 .01 0. 13 0.01 0 .16 0. 08 0.12 0.01 26:0 0. 10 0. 02 0 .07 0 .04 0. 08 0.04 0 .07 0. 03 0.11 0.03 194 Appendix I d . F a t t y a c i d composition of the n e u t r a l and p o l a r l i p i d of eggs of w i l d A r c t i c charr from Tree R i v e r , NWT. F a t t y N e u t r a l P o l a r a c i d l i p i d l i p i d % % 14: 0 2.42 0.88 15: 01 0.11 t r 15: 0 0.22 0.22 16: 01 0.13 t r 16: 0 9.66 15.23 16: ln9 t r 0.29 16:ln7 17.71 3.46 16:ln5 0.30 0.15 Unknown t r 0.16 16:2n9 0.13 0.10 Unknown 0.24 t r 17:0 0.15 0.15 Unknown t r 0.13 17: ln9 0.34 0.16 18! 01 t r 0.23 18: 0 1.88 4.95 18: ln9 21.28 10.62 18: ln7 7.92 8.53 18: ln5 0.45 0.64 18: 2n9 0.10 t r 18: 2n6 2.15 0.55 18: 3n6 0.23 0.12 19: :0 0.16 t r 18: •4n6 0.20 t r 19 :ln7 0.06 0.12 18: :3n3 0.77 0.15 18 s4n3 0.64 t r 18 : 4 n l l 0.19 t r 20 : l n l l 0.18 0.11 20 :ln9 1.61 3.07 20 :ln7 0.33 0.52 20 :2n6 0.29 0.41 20 :3n6 0.30 0.27 20 :4n6 0.48 0.82 20 !4n3 0.88 0.34 20 :5n3 11.74 12.66 22 : l n l l 0.17 0.06 22 :ln9 0.12 t r 21 :5n3 0.28 0.12 22 :4n6 t r 0.12 22 :5n6 t r 0.12 22 :5n3 4.27 6.21 22 :6n3 9.59 25.50 24 s l n l l t r 0.43 195 Appendix 2a. Weekly water temperature (mean and standard deviation) i n Year 1 and Year 2 (n=5). Year 1 Year 2 Week Date Temperature Week Date Temperature °C SD °C SD 28 020189-080189 8. 09 0. 05 29 090189-150189 8. 18 0. 07 30 160189-220189 8. 28 0. 06 31 230189-290189 8. 34 0. 00 32 300189-050289 8. 07 0. 07 33 060189-120289 8. 21 0. 06 34 130289-190289 8. 10 0. 20 35 200289-260289 7. 95 0. 07 36 270289-050389 7. 84 0. 11 37 060389-120389 8. 08 0. 17 38 130389-190389 8. 14 0. 17 39 200389-260389 8. 23 0. 14 40 270389-020489 8. 08 0. 04 41 030489-090489 8. 13 0. 03 42 100489-160489 8. 18 0. 15 43 170489-230489 8. 39 0. 19 44 240489-300489 7. 87 0. 53 45 010589-070589 7. 61 0. 26 46 080589-140589 8. 01 0. 09 47 150589-210589 8. 17 0. 14 48 220589-280589 7. 87 0. 15 49 290589-040689 8. 02 0. 16 50 050689-110689 8. 11 0. 10 51 120689-180689 8. 17 0. 07 52 190689-250689 8. 42 0. 13 1 280688 -040788 53 260689-020789 8. 20 0. 20 2 050788 -110788 8. 57 0. 58 54 030789-090789 8. 00 0. 26 3 120888 -180788 8. 58 0. 12 55 100789-160789 8. 35 0. 14 4 190788 -250788 8. 69 0. 08 56 170789-230789 8. 64 0. 24 5 260788 -010888 8. 76 0. 18 57 240789-300789 8. 56 0. 12 6 020888 -080888 8. 55 0. 16 58 010889-070889 8. 50 0. 22 7 090888 -150888 8. 61 0. 16 59 080889-140889 8. 59 0. 28 8 160888 -220888 8. 50 0. 12 60 150889-210889 8. 63 0. 19 9 230888 -290888 8. 28 0. 10 61 220889-280889 8. 65 0. 18 10 300888 -050988 8. 33 0. 15 62 290889-040989 8. 56 0. 26 11 060988 -120988 8. 15 0. 14 63 050989-110989 8. 35 0. 31 12 130988 -190988 8. 16 0. 13 64 120989-180989 8. 27 0. 25 13 200988 -260988 7. 97 0. 05 65 190989-250989 7. 83 0. 15 14 270988 -031088 7. 89 0. 14 66 260989-021089 7. 83 0. 29 15 041088 -101088 7. 66 0. 21 67 031089-091089 7. 61 0. 32 16 111088 -171088 7. 65 0. 22 68 101089-161089 7. 59 0. 14 17 181088 -241088 7. 55 0. 14 69 171089-231089 7. 55 0. 36 18 251088 -311088 7. 14 0. 22 70 241089-301089 7. 48 0. 40 19 011188. -071188 7. 33 0. 09 71 311089-061189 7. 15 0. 49 20 081188 -141188 7. 15 0. 14 72 071189-131189 7. 28 0. 55 21 151188--211188 7. 84 0. 16 73 141189-201189 8. 02 0. 50 22 221188 -281188 7. 69 0. 13 74 211189-271189 8. 14 0. 10 23 291188--041288 7. 68 0. 06 75 281189-041289 8. 50 0. 20 24 051288--111288 7. 66 0. 09 76 051289-111289 8. 54 0. 07 25 121288--181288 7. 71 0. 05 77 121289-181289 8. 49 0. 10 26 191288--251288 7. 93 0. 16 78 191289-251289 8. 43 0. 12 27 261288--010189 7. 72 0. 03 79 261289-010190 8. 49 0. 07 196 Appendix 2b. T o t a l gain, feed fed and feed e f f i c i e n c y of each tank of f i s h f o r each period i n Year 1. I n i t i a l date 280688 090888 110888 280688 F i n a l date 080788 100888 130988 130988 Period A TO B B TO C C TO D A TO D Days fed 9 30 32 71 Days not fed 2 2 2 6 Diet LLLE Tank 3 I n i t i a l no. f i s h 25 25 25 25 F i n a l no. f i s h 25 25 25 25 T o t a l i n i t i a l f i s h wt (g) 21172 21806 26709 T o t a l f i n a l f i s h wt (g) 21806 26709 30924 T o t a l gain (g) 634 4903 4215 9752 Feed fed (g, dry) 1274 6784 7190 15248 Feed e f f i c i e n c y (%) 49.8 72.3 58.6 64.0 Diet LLHE Tank 7 I n i t i a l no. f i s h 25 25 25 25 F i n a l no. f i s h 25 25 25 25 T o t a l i n i t i a l f i s h wt (g) 21160 21941 27766 T o t a l f i n a l f i s h wt (g) 21941 27766 33440 To t a l gain (g) 813 3253 4050 8116 Feed fed (g, dry) 1452 6370 7043 14864 Feed e f f i c i e n c y (%) 56.0 51.1 57.5 54.6 Diet HLLE Tank 6 I n i t i a l no. f i s h 25 25 25 25 F i n a l no. f i s h 25 25 25 25 T o t a l i n i t i a l f i s h wt (g) 21162 22525 29789 T o t a l f i n a l f i s h wt (g) 21525 29789 36616 T o t a l gain (g) 985 5358 5893 12236 Feed fed (g, dry) 1228 7124 8022 16374 Feed e f f i c i e n c y (%) 80.2 75.2 73.5 74.7 Diet HLHE Tank 2 I n i t i a l no. f i s h 25 25 25 25 F i n a l no. f i s h 25 25 25 25 T o t a l i n i t i a l f i s h wt (g) 21160 21941 27766 T o t a l f i n a l f i s h wt (g) 21941 27766 33440 T o t a l gain (g) 781 5825 5674 12280 Feed fed (g, dry) 1127 7449 8503 18381 Feed e f f i c i e n c y (%) 69.3 78.2 66.7 66.8 Diet COMM Tank 5 I n i t i a l no. f i s h 25 25 25 25 F i n a l no. f i s h 25 25 25 25 T o t a l i n i t i a l f i s h wt (g) 21162 22525 29789 T o t a l f i n a l f i s h wt (g) 22525 29789 36616 T o t a l gain (g) 1363 7264 6827 15454 Feed fed (g, dry) 2054 8758 9322 20135 Feed e f f i c i e n c y (%) 66.4 82.9 73.2 76.8 197 Appendix 2c. T o t a l gain, feed fed and feed e f f i c i e n c y of each tank of f i s h f o r each period i n Year 2. I n i t i a l date 120189 060489 290689 120189 F i n a l date 230189 280689 130989 130989 Period E to F F to G G to H E to H Days fed 82 82 75 239 Days not fed 2 2 2 6 Diet LLLE Tank 6 I n i t i a l no. f i s h 24 23 23 F i n a l no. f i s h 23 23 22 T o t a l i n i t i a l f i s h wt (g) 27709 36616 49114 T o t a l f i n a l f i s h wt (g) 36616 49114 52520 To t a l gain (g) 5081 7239 3096 15416 Feed fed (g, dry) 7401 8627 8156 24184 Feed e f f i c i e n c y (%) 68.7 83.9 38.0 63.7 Diet LLHE Tank 2 I n i t i a l no. f i s h 24 24 24 F i n a l no. f i s h 24 24 24 T o t a l i n i t i a l f i s h wt (g) 25622 29160 34623 To t a l f i n a l f i s h wt (g) 29160 34623 39405 To t a l gain (g) 3538 5463 4782 13783 Feed fed (g, dry) 9497 10891 8470 28859 Feed e f f i c i e n c y (%) 37.3 50.2 56.5 47.8 Diet HLLE Tank 3 I n i t i a l no. f i s h 23 21 21 F i n a l no. f i s h 21 21 20 T o t a l i n i t i a l f i s h wt (g) 22467 24259 27288 To t a l f i n a l f i s h wt (g) 24259 27288 29201 T o t a l gain (g) 1792 3029 1913 6734 Feed fed (g, dry) 10536 13804 10006 34347 Feed e f f i c i e n c y (%) 17.0 21.9 19.1 19.6 Diet HLHE Tank 7 I n i t i a l no. f i s h 24 24 24 F i n a l no. f i s h 24 24 24 T o t a l i n i t i a l f i s h wt (g) 25622 29160 34623 T o t a l f i n a l f i s h wt (g) 29160 34623 39405 T o t a l gain (g) 4587 3992 1258 9837 Feed fed (g, dry) 10009 12675 11523 34207 Feed e f f i c i e n c y (%) 45.9 31.5 10.9 28.8 Diet COMM Tank 5 I n i t i a l no. f i s h 23 23 23 F i n a l no. f i s h 23 23 22 T o t a l i n i t i a l f i s h wt (g) 27709 36616 49114 T o t a l f i n a l f i s h wt (g) 36616 49114 52520 T o t a l gain (g) 8907 12498 3406 24811 Feed fed (g, dry) 15474 15671 9248 40393 Feed e f f i c i e n c y (%) 57.6 79.8 36.8 61.4 198 Appendix 3a. Method of e x t r a c t i o n (adapted from King 1985) and assay (adapted from AOAC 1984) of tocopherol i n eggs and f r y . REAGENTS FOR EXTRACTION; Etha n o l , 99% Hexane, d i s t i l l e d i n glas s (B.P.=68-69°C) - Caledon Isooctane (2,2,4-Trimethylpentane) (B.P.=99-100°C) - Caledon A c e t o n i t r i l e , HPLC grade - Sigma EXTRACTION: Weigh sample d i r e c t l y i n t o a 16 x 125 mm c u l t u r e tube c o n t a i n i n g 1 ml 99% ethanol and 2 ml hexane c o n t a i n i n g 50 mg/1 b u t y l a t e d hydroxy-toluene (BHT). Use 0.4 g eggs and 0.2-0.4 g f r y . Eggs must be kept f r o z e n during the weighing process or they w i l l r upture and immediately expose the v i t a m i n E t o oxygen. Therefore, keep the sample v i a l i n a styrofoam block and keep the weighing u t e n s i l s c o l d throughout the weighing process. F l u s h the sample v i a l w i t h n i t r o g e n and r e t u r n i t t o the f r e e z e r immediately. Weigh out and homogenize one sample at a time. Keep the samples on i c e and flushed w i t h n i t r o g e n throughout a l l the steps i n the e x t r a c t i o n . Homogenize sample 30 sec using a P o l y t r o n homogenizer. Sample must be kept i n i c e throughout the homogenizations. Add another 1 ml 99% ethanol and 2 ml hexane. Homogenize 15 sec w i t h P o l y t r o n . T r a n s f e r homogenate t o a 16 x 125 mm screw-cap tube w i t h a Pasteur p i p e t t e . Add 1 ml ethanol and 2 ml hexane t o the c u l t u r e tube and r i n s e tube and P o l y t r o n generator by homogenizing f o r 15 sec. Tra n s f e r the r i n s e t o the above screw-cap tube and f l u s h tube w i t h n i t r o g e n . Keep samples on i c e or i n r e f r i g e r a t o r u n t i l a l l the samples are e x t r a c t e d . C e n t r i f u g e tubes 2 min i n c l i n i c a l c e n t r i f u g e . T r a n s f e r the upper hexane l a y e r w i t h a Pasteur p i p e t t e t o a c l e a n 16 x 125 mm screw-cap tube i f i t i s t o be evaporated i n a heati n g b l o c k or a 50 ml f l a s k i f i t i s t o be evaporated i n a r o t a r y evaporator. Add 4 ml hexane and rehomogenize p e l l e t by v o r t e x i n g 1 min. C e n t r i f u g e and combine hexane l a y e r w i t h the f i r s t hexane l a y e r . Evaporate hexane under ni t r o g e n i n a heating b l o c k or w i t h a r o t a r y evaporator at l e s s than 50°C. 199 E x t r a c t twice w i t h 2 ml a c e t o n i t r i l e and t r a n s f e r e x t r a c t s t o a c l e a n 16 x 125 mm screw-cap tube. E x t r a c t a c e t o n i t r i l e e x t r a c t w i t h 3 ml isooctane by v o r t e x i n g 1 minute fo l l o w e d by c e n t r i f u g i n g at 6 f o r 1-2 min t o separate the phases. Transfer the upper isooctane phase t o a c l e a n 16 x 125 mm screw-cap tube. E x t r a c t the a c e t o n i t r i l e e x t r a c t a second time w i t h 3 ml isooctane by v o r t e x i n g f o r 0.5-1 min and c e n t r i f u g i n g as before. Combine the upper phase wi t h previous e x t r a c t . Evaporate isooctane under ni t r o g e n i n a heating block o r w i t h a r o t a r y evaporator at l e s s than 50°C. D i s s o l v e e x t r a c t i n 3.0-4.0 ml 99% ethanol (eggs) or 2.0 ml et h a n o l . Adjust volume as r e q u i r e d f o r the assay. Assay the e x t r a c t by the f o l l o w i n g AOAC (1984) spectrophotometric method. Note: Throughout the e x t r a c t i o n , a l l hexane and isooctane contained 50 mg/1 BHT as an a n t i o x i d a n t . A blank should a l s o be e x t r a c t e d t o c o r r e c t f o r i n t e r f e r i n g substances. Use of BHT w i l l r e s u l t i n an increase i n t h i s c o r r e c t i o n v a l u e . ASSAY: REAGENTS FOR ASSAY: 1) Bathophenanthroline (Sigma) 0.003M Weigh 100 mg bathophenanthroline. B r i n g up t o 100 ml w i t h 99% et h a n o l . Store i n amber b o t t l e at 5°C. Prepare f r e s h every 3 weeks. 2) F e r r i c c h l o r i d e (Fisher) 0.002M Weigh 55 mg FeCl 3.6H 20. B r i n g up t o 100 ml w i t h 99% et h a n o l . Store i n amber b o t t l e at 5°C. 3) o-Phosphoric a c i d (Baker) 0.172M B r i n g 1.1 ml concentrated phosphoric a c i d (86%) up t o 100 ml w i t h 99% e t h a n o l . 4) Standard: Weigh 0.1 g a-tocopherol (95% p u r i t y ) (Sigma) t o 4-5 decimals and t r a n s f e r t o a vol u m e t r i c f l a s k w i t h 99% et h a n o l . Make up t o 100 ml wi t h ethanol. D i l u t e the above stock t o 1 ml i n 50 ml w i t h e t h a n o l . Concentration (g/ml) = 0.95 x weight g/ 5000 ml (approx 20 ng/ml). Add volumes of up t o 2.00 ml of the d i l u t e d stock tocopherol s o l u t i o n (0-40 and make up t o 2.0 ml w i t h 99% e t h a n o l . Make up a blank w i t h 2.0 ml ethanol. 200 METHOD ; Add tocopherol e x t r a c t ( i n 99% ethanol) t o a 13 x 100 mm c u l t u r e tube. Add 99% ethanol t o make up t o 2.0 ml. Add 0.5 ml bathophenanthroline reagent and vo r t e x . Add 0.25 ml f e r r i c c h l o r i d e reagent. S t a r t t i m i n g once l a s t of reagent added. Vortex. Add 0.25 ml phosphoric a c i d reagent e x a c t l y 15 seconds a f t e r the a d d i t i o n of the f e r r i c c h l o r i d e reagent. Vortex. A f t e r 3 minutes the col o u r which has developed i s s t a b l e f o r 90 minutes. Read samples at 534 m\i against the blank. NOTE: This method has been scaled down by h a l f the volumes used by AOAC (1984) . CALCULATION: C a l c u l a t e slope of standard curve Slope should be approx 0.0310. = absorbance / ng t o c o p h e r o l . Vitamin E (\ig/g) =absorbance x 2.0 ml x 1 slope v o l assayed (ml) wt sample (g) Vitamin E content of egg (jig/egg) = v i t a m i n E co n c e n t r a t i o n i n eggs (\ig/g) x egg weight (g) Vitamin E content of f r y (\ig/fry) = v i t a m i n E (\ig/g) x f r y weight (g) 201 Appendix 3b. Method of e x t r a c t i o n and a n a l y s i s of t o t a l l i p i d i n eggs and d i e t s . REAGENTS ; Chloroform - d i s t i l l e d i n glas s Methanol - d i s t i l l e d i n glas s Concentrated s u l p h u r i c a c i d Potassium dichromate (0.167M): 49.0 g reagent grade potassium dichromate B r i n g t o 1 l i t e r w i t h d i s t i l l e d water. L i p i d standard: 0.1 g/10 ml i n chloroform. In order t o have a comparable l e v e l of o x i d a t i o n i n the standard and the samples, standard curve should be conducted w i t h the samples being analyzed. In the cu r r e n t research, n e u t r a l and p o l a r l i p i d s e x t r a c t e d from A r c t i c c h a r r eggs were used as standards. EXTRACTION: Weigh sample i n t o l a r g e screw-cap tube (25 mm x 150 mm). Use dry or wet samples - 0.4 g dry feed or eggs or 1.0-1.5 g wet eggs. Add 5.0 ml chloroform 10.0 ml methanol 4.0 ml d i s t i l l e d water f o r a dry sample* or 3.4 ml f o r a wet sample. Homogenize 20 sec. Add 5.0 ml chloroform 5.0 ml d i s t i l l e d water. Homogenize 10 sec. Rinse homogenizer between samples w i t h chloroform:methanol (1:1) and d i s c a r d r i n s e . F i l t e r homogenate through a S t e f i s i n t e r e d g l a s s funnel u s i n g Whatman No. 1 f i l t e r paper (42.5 mm) i n t o a medium screw-cap tube (20 x 150 mm). Rinse funnel between samples w i t h chloroform:methanol (1:1) and d i s c a r d r i n s e . A l l o w l a y e r s t o separate overnight i n r e f r i g e r a t o r . Many samples can be processed t o t h i s step u n t i l ready t o proceed w i t h the assay. The upper l a y e r w i l l p r o t e c t the lower l a y e r from any chance of evaporation. When ready t o conduct the c o l o u r i m e t r i c assay, a s p i r a t e o f f and d i s c a r d upper methanol- water l a y e r . Transfer chloroform l a y e r t o a sm a l l screw-cap tube (16 x 125 mm). 202 Appendix 3b. Continued. ASSAY: Tran s f e r a l i q u o t of the lower chloroform l a y e r ( i e . 100-250 \il or s u f f i c i e n t f o r approx 1 mg l i p i d ) t o a 20 mm x 150 mm tube (not screw-cap). Rinse p i p e t t e between samples. Evaporate s o l v e n t i n a stream on n i t r o g e n or other gas or a l l o w t o dry i n fume hood. While evaporating, place tubes i n a warm water bath or a heating block at 70°C. Tubes must be heated f o r 10 min at 70°C a f t e r s olvent has evaporated i n order t o ensure no t r a c e s of s o l v e n t remain. This i s c r i t i c a l i n order t o o b t a i n good r e p l i c a t e s . Add 2.0 ml of 0.167M potassium dichromate t o each tube. Add 4.0 ml concentrated s u l p h u r i c a c i d . Mix by v o r t e x . Heat i n oven at 115°C f o r 30 min (set our oven at 225 F ) . Leave an empty row between tubes i n the rack and do not overload oven w i t h more than 2 racks. Cool f o r 3 minutes at room temperature. Add 2.0 ml d i s t i l l e d water and mix w i t h v o r t e x . Cool i n water bath or s i n k of c o l d water. Mix w i t h v o r t e x . Read absorbance at 600 nm i n 1 cm c e l l s u sing blank as a r e f e r e n c e . STANDARD CURVE: Standard: 1.0 g/100 ml = 10.0 mg/ml Add 25-200 \il standard i e . 0.25-2.00 mg l i p i d . The standard curve i s not l i n e a r beyond 2 mg l i p i d . CALCULATION: % l i p i d = absorbance x t o t a l volume chloroform used x 100 slope x volume a l i q u o t x sample weight x 1000 Where: volume of chloroform and volume of a l i q u o t i n ml sample weight i n gm 1000 converts mg i n standard t o gm. slope = slope of standard curve = absorbance/mg l i p i d 203 Appendix 4. 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 of v a r i o u s parameters on f e c u n d i t y , f e r t i l i z a t i o n and s u r v i v a l of eggs i n Year 1 and Year 2. Dependent Parameter Regression equation r 2 S i g n i f . v a r i a b l e X Y Year 1; No. eggs F i s h wt. (g) Y = 2.40 X + 501.07 0 .315 0 .0001 re l e a s e d No. eggs F i s h wt. (g) Y = 2.71 X + 351.88 0 .334 0 .0001 produced % P e r t . Spawning day Y = 0.299 X + 73.58 0 .143 0 .004 % F e r t . No. Eggs Y = 0.0038 X + 72.88 0 .147 0 .004 % Eyed No. Eggs Y = 0.0067 X + 40.73 0 .142 0 .005 % Hatch No. Eggs Y 0.0059 X + 32.18 0 .083 0 .033 % Swimup No. Eggs Y 0.0059 X + 24.80 0 .091 0 .025 Year 2: % F e r t . F i s h wt. (g) Y = -0.009 X + 101.25 0 .112 0 .040 No. eggs F i s h wt. (g) Y = 3.47 X + 2061.01 0 .844 0 .0001 r e l e a s e d No. eggs F i s h wt. (g) Y = 3.88 X + 2202.25 0 .853 0 .0001 produced No. eggs Spawning day Y -109.74X + 11056.72 0 .249 0 .002 produced % F e r t . Egg wt (mg) Y = 0.75 X + 41.10 0 .118 0 .037 % Hatch Egg wt (mg) Y = 1.43 X - 44.54 0 .118 0 .037 % Swimup Egg wt (mg) Y = 1.32 X - 44.29 0 .108 0 .047 % Eyed NL (%) Y = -17.29 X + 233.36 0 .263 0. .0032 % Hatch NL (%) Y = -18.24 X + 236.20 0 .321 0. .0009 % Swimup NL (%) Y = -18.44 X + 232.38 0 .338 0. .0006 % Eyed NL 16:ln7 (%) Y = -13.68 X + 202.52 0, .238 0. .006 % Hatch NL 16:ln7 <%) Y = -12.92 X + 186.58 0, .234 0. .007 % Swimup NL 16:ln7 (%) Y = -12.57 X + 176.66 0, .228 0. .008

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