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

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THE EFFECT OF DIETARY L I P I D 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 o f M a n i t o b a , 1968  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department o f A n i m a l S c i e n c e We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1992 ® Jo-Anne L e s l i e Tabachek, 1992  In  presenting  degree freely  this  at the  University  available for  copying  of  department publication  this or of  thesis  partial  of  British Columbia,  reference  thesis by  in  for  his  this thesis  and  for  her  of  The University of British Columbia Vancouver, Canada  -6  (2/88)  of  the  requirements  I agree  I further  purposes  representatives.  that  agree  may  be  It  is  financial gain shall not  permission.  Department  study.  scholarly  or  fulfilment  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall make for  the that  without  head  it  extensive of  my  copying  or  my  written  ABSTRACT  This  research  embryonic  was  conducted  i n an attempt  s u r v i v a l o f Labrador A r c t i c  concentrations broodstock.  of l i p i d  and v i t a m i n  charr E  to  improve  by a l t e r i n g t h e  i n the d i e t s  fed t o  F o u r - y e a r o l d L a b r a d o r A r c t i c c h a r r b r o o d s t o c k were  f e d d i e t s c o n t a i n i n g two l e v e l s o f 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  (dl-a-tocopheryl acetate)  The  d i e t s were d e s i g n a t e d  low  lipid  (12%)(LL), high  supplementation.  LLLE, LLHE, HLLE and HLHE t o denote lipid  (19%)(HL),  low v i t a m i n  mg/kg)(LE) and h i g h v i t a m i n E (600 mg/kg)(HE). a fifth diet  E (30  For comparison,  group was f e d t h e i r normal d i e t - a c o m m e r c i a l grower  (COMM)  acetate/kg.  containing  17%  lipid  and  100  mg  vitamin  S i n c e A r c t i c c h a r r b r o o d s t o c k used i n t h i s  E  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 t h e survival  or fecundity  Dietary  lipid  requirements  of the broodstock  concentrations  were  f o r juvenile Arctic  could  selected  charr.  n o t be based  Dietary  used.  on t h e  vitamin  E  c o n c e n t r a t i o n s were s e l e c t e d based on r e q u i r e m e n t s s t a t e d i n t h e l i t e r a t u r e f o r other  salmonids.  F i s h were f e d f o r 71 days b e f o r e w i t h d r a w a l  o f feed  t o spawning i n Year 1 and f o r 252 days i n Year 2.  Since  prior fish  f e d t h e L L d i e t s i n Year 1 spawned 5 weeks l a t e r t h a n t h o s e f e d t h e HL d i e t s , a c r o s s o v e r i n d i e t s was c o n d u c t e d t o see i f t h i s w o u l d a l s o o c c u r i n Year 2.  Keeping t h e v i t a m i n E l e v e l t h e  iii same, t h o s e f e d LL d i e t s i n Year 1 were f e d 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 o f d i e t a r y l i p i d on spawning t i m e was  o b s e r v e d i n Year 2.  The p r o p o r t i o n o f females f a i l i n g t o spawn was n o t r e l a t e d to  the  concentrations  significantly concentration  higher of  of  dietary  proportion  vitamin  E  lipid of  or  males  produced  milt  vitamin fed  E.  the  i n two  A  higher  successive  spawning seasons.  F e c u n d i t y o f 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  diets,  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 w e i g h t and was not a f f e c t e d by t h e l e v e l o f d i e t a r y l i p i d o r v i t a m i n E.  The f a t t y a c i d c o m p o s i t i o n o f t h e eggs r e f l e c t e d t h a t o f the  diet  and  deficiency. (10.6%)  and  was  not  indicative  of  essential fatty  acid  I n Year 2, c o n c e n t r a t i o n s o f t o t a l ( 2 2 . 3 % ) , n e u t r a l polar  lipids  (11.7%)  in  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 t h e c o n c e n t r a t i o n s o f d i e t a r y l i p i d o r v i t a m i n E.  The v i t a m i n E c o n c e n t r a t i o n s o f t h e 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 t h e 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 i n c r e a s e 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 f e d t h e LL compared to  t h e HL  diets.  iv D i e t s c o n t a i n i n g 30 o r 600 mg v i t a m i n E a c e t a t e / k g w i t h 12 o r 19% l i p i d met t h e v i t a m i n E r e q u i r e m e n t s o f 4- and 5-year o l d broodstock  to  the  extent  that  fertilization  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 . i n Year 2 and 80% i n Year 2.  and  embryonic  F e r t i l i z a t i o n was  There was a h i g h degree o f w i t h i n  t r e a t m e n t v a r i a t i o n i n embryonic s u r v i v a l .  Fertilization  embryonic  with  survival  were  c o n c e n t r a t i o n o f t h e eggs.  89%  not  correlated  and  vitamin  S u r v i v a l t o swimup o f  E  fertilized  eggs was 27% f o r t h e LE d i e t s and 55% f o r t h e HE d i e t s i n Y e a r 2.  Embryonic s u r v i v a l t o t h e eyed, h a t c h and swimup s t a g e s were  negatively c o r r e l a t e d with the percent n e u t r a l l i p i d 16:ln7 i n t h e n e u t r a l l i p i d o f t h e eggs.  and  with  V  TABLE OF CONTENTS ABSTRACT  Page i i  TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES ACKNOWLEDGEMENTS  V viii X x i i xiii  1  INTRODUCTION  1  2  LITERATURE REVIEW  4  2.1 N a t u r a l h i s t o r y o f A r c t i c c h a r r  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 P h y s i o l o g y o f r e p r o d u c t i o n  10  2.4 Feed i n t a k e d u r i n g r e p r o d u c t i o n 2.4.1 Feed i n t a k e  12 12  2.5  2.4.2 S t a r v a t i o n p r i o r t o spawning  14  P r o t e i n and energy  14  2.6 L i p i d s , f a t t y a c i d s and v i t a m i n E 2.6.1 C h e m i s t r y o f l i p i d s and f a t t y a c i d s 2.6.2 D i g e s t i o n and a b s o r p t i o n o f l i p i d s and vitamin E 2.6.3 E s s e n t i a l f a t t y a c i d r e q u i r e m e n t s for reproduction 2.6.4 V i t a m i n E 2.6.4.1 C h e m i s t r y o f t o c o p h e r o l s 2.6.4.2 F u n c t i o n o f v i t a m i n E 2.6.4.3 M o b i l i z a t i o n and r e q u i r e m e n t of v i t a m i n E f o r reproduction 3 MATERIALS AND METHODS 3.1 L o c a t i o n and t i m e 3.2 D i e t s 3.2.1 F o r m u l a t i o n 3.2.2 M a n u f a c t u r i n g 3.3 P a r e n t a l f i s h 3.3.1 H i s t o r y o f t h e s t o c k 3.3.2 S e l e c t i o n o f f i s h f o r t h e e x p e r i m e n t 3.4 E n v i r o n m e n t a l c o n d i t i o n s f o r b r o o d s t o c k 3.4.1 Tanks  16 16 18 22 25 25 27 30 38 38 38 38 41 43 43 44 45 45  vi  3.5 3.6 3.7 3.8 3.9 3.10 3.11 3.12  3.13  3.14  3.4.2 L i g h t i n g and p h o t o p e r i o d 3.4.3 Temperature 3.4.4 Water c h e m i s t r y F e e d i n g regime Growth o f b r o o d s t o c k Spawning F e r t i l i z a t i o n r a t e and measurement o f water-hardened eggs I n c u b a t i o n o f eggs Growth and s u r v i v a l o f f r y C o l l e c t i o n o f 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 A n a l y s e s o f eggs, f r y and/or d i e t s 3.12.1 V i t a m i n E a n a l y s e s 3.12.2 Dry m a t t e r and s e l e n i u m 3.12.3 L i p i d and f a t t y a c i d a n a l y s e s 3.12.4 A n a l y s i s o f d i e t s E v a l u a t i o n o f methods 3.13.1 E v a l u a t i o n o f egg r i n s e s and e x t e n d e r s 3.13.2 E v a l u a t i o n o f number o f f r y t o r a i s e i n each r e a r i n g u n i t S t a t i s t i c a l analysis  45 46 47 47 50 50 54 54 56 58 59 59 60 60 63 63 63 64 64  4 RESULTS 4.1 Feed c o m p o s i t i o n 4.2 Water temperature 4.3 Water c h e m i s t r y 4.4 Feed f e d and f e e d e f f i c i e n c y 4.5 Growth o f b r o o d s t o c k 4.6 Spawning 4.6.1 Time o f spawning 4.6.2 Number o f r i p e males and females 4.6.3 A t y p i c a l spawning 4.7 F e c u n d i t y and egg s i z e (diameter and w e i g h t ) 4.8 Embryonic s u r v i v a l 4.9 Egg and f r y c o m p o s i t i o n 4.9.1 V i t a m i n E 4.9.2 L i p i d and d r y m a t t e r 4.9.3 F a t t y a c i d c o m p o s i t i o n 4.9.4 S e l e n i u m 4.9.5 C o m p o s i t i o n o f w i l d A r c t i c c h a r r eggs 4.10 F r y s u r v i v a l and growth  68 68 71 73 74 81 83 83 92 92 94 102 109 109 115 122 133 133 136  5 DISCUSSION 5.1 Growth o f b r o o d s t o c k and f e e d i n g a c t i v i t y 5.2 R e l a t i o n o f f e c u n d i t y t o r a t i o n and w e i g h t o f female 5.3 Spawning t i m e and p r o p o r t i o n o f f i s h t h a t spawned 5.4 Egg s i z e 5.5 V a r i a t i o n i n embryonic s u r v i v a l 5.6 L i p i d and f a t t y a c i d c o m p o s i t i o n s o f eggs 5.7 V i t a m i n E c o n t e n t o f eggs  141 141 147 150 153 154 155 163  5.8  F r y s u r v i v a l and growth  6 CONCLUSIONS 7 LITERATURE CITED 8 APPENDICES  viii LIST OF TABLES Page T a b l e 1.  Formulation of experimental d i e t s .  39  T a b l e 2.  Composition of the d i e t s .  69  T a b l e 3.  Water c h e m i s t r y d a t a f o r each t a n k .  75  T a b l e 4.  Water c h e m i s t r y d a t a f o r each s a m p l i n g day.  76  T a b l e 5.  Feed f e d , w e i g h t g a i n and f e e d e f f i c i e n c y f o r each t a n k o f f i s h f e d f o r 71 days i n Year 1 and 239 days i n Year 2.  77  I n i t i a l weight d i s t r i b u t i o n s o f f i s h i n each t a n k .  82  I n i t i a l and f i n a l mean and range i n w e i g h t o f males and females f e d each d i e t d u r i n g t h e f e e d i n g p e r i o d i n Year 1.  84  I n i t i a l and f i n a l mean and range i n w e i g h t o f males and females f e d each d i e t d u r i n g t h e f e e d i n g p e r i o d i n Year 2.  85  S p e c i f i c growth r a t e o f female and male f e d t h e d i e t s i n Year 1 and 2.  86  T a b l e 6. T a b l e 7.  T a b l e 8.  T a b l e 9.  fish  T a b l e 10. Number o f male and female c h a r r w h i c h l o s t o r g a i n e d w e i g h t t h r o u g h o u t Year 1 o r 2.  87  T a b l e 11. Number o f females which spawned d u r i n g t h e 14-week spawning p e r i o d i n Year 1 and t h e 11week spawning p e r i o d i n Year 1.  88  T a b l e 12. Number o f females and males which produced eggs (spawned) and m i l t ( r i p e ) i n Year 1.  90  T a b l e 13. Number o f females and males which produced eggs (spawned) and m i l t ( r i p e ) i n Year 2.  91  T a b l e 14. Number o f males w h i c h produced m i l t ( r i p e ) i n Year 1 which d i d o r d i d n o t produce m i l t i n Year 2.  93  T a b l e 15. Egg p r o d u c t i o n parameters i n Year 1.  95  T a b l e 16. Egg p r o d u c t i o n parameters i n Year 2.  96  IX  T a b l e 17. Egg w e i g h t and d i a m e t e r o f water-hardened p r e s e r v e d eggs i n Years 1 and 2. T a b l e 18. F e r t i l i z a t i o n and embryonic Year 1.  survival i n  T a b l e 19. F e r t i l i z a t i o n and embryonic Year 2.  survival i n  98 103  T a b l e 20. V i t a m i n E c o n c e n t r a t i o n and c o n t e n t o f eggs i n Years 1 and 2 and f r y i n Year 2.  105 I l l  T a b l e 21. T o t a l l i p i d and d r y m a t t e r c o n t e n t s o f eggs i n Year 1.  116  T a b l e 22. T o t a l l i p i d and d r y m a t t e r c o n t e n t s o f eggs i n Year 2.  118  T a b l e 23. S e l e c t e d major f a t t y a c i d s i n t h e t o t a l l i p i d of the d i e t s .  123  T a b l e 24. T o t a l s f o r f a m i l i e s o f f a t t y a c i d s i n t h e t o t a l l i p i d of the d i e t s .  124  T a b l e 25. S e l e c t e d major f a t t y a c i d s i n t h e n e u t r a l l i p i d s o f eggs.  129  T a b l e 26. T o t a l s o f f a m i l i e s o f f a t t y a c i d s i n t h e n e u t r a l l i p i d s o f eggs.  130  T a b l e 27. S e l e c t e d major f a t t y a c i d s i n t h e p o l a r l i p i d s o f eggs.  131  T a b l e 28. T o t a l s o f f a m i l i e s o f f a t t y a c i d s i n t h e p o l a r l i p i d s o f eggs.  132  T a b l e 29. S e l e c t e d major f a t t y a c i d s i n t h e p o l a r l i p i d s o f eggs from A r c t i c c h a r r from Tree R i v e r , NWT.  134  T a b l e 30. T o t a l s o f f a m i l i e s o f f a t t y a c i d s i n t h e p o l a r l i p i d s o f eggs from A r c t i c c h a r r from Tree R i v e r , NWT.  135  T a b l e 31. F r y s u r v i v a l and s p e c i f i c growth r a t e when s t o c k e d a t d i f f e r e n t numbers.  137  T a b l e 32. F r y 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 a t 10°C i n Year 1.  138  T a b l e 33. F r y 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 a t 10°C i n Year 2.  139  X  LIST OF FIGURES Page F i 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 o f fatty acids i n f i s h .  19  F i g u r e 2.  Forms o f v i t a m i n E.  21  F i g u r e 3.  E x p e r i m e n t a l p l a n showing c r o s s o v e r o f d i e t s f e d i n Year 1 and Year 2.  49  F i g u r e 4.  Flow d i a g r a m showing a n a l y s e s c o n d u c t e d on eggs and/or f r y i n Year 1 and/or Year 2.  52  F i g u r e 5.  Mean weekly water t e m p e r a t u r e s d u r i n g Year 1 and Year 2.  72  F i g u r e 6.  Egg w e i g h t i n a) Year 1 and b) Year 2.  97  F i g u r e 7.  T o t a l number o f eggs produced i n a) Year 1 and b) Year 2.  100  F i g u r e 8.  R e l a t i o n s h i p o f t o t a l number o f eggs produced and f i s h w e i g h t i n a) Year 1 and b) Year 2.  101  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 o f eggs t o t h e eyed, h a t c h and swimup s t a g e s i n Year 1.  104  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 o f eggs t o t h e eyed, h a t c h and swimup s t a g e s i n Year 2.  106  F i g u r e 11. V i t a m i n E c o n c e n t r a t i o n o f eggs i n a) Year 1 and b) Year 2.  112  F i g u r e 12. V i t a m i n E c o n t e n t o f eggs i n a) Year 1 and b) Year 2.  113  F i g u r e 13. V i t a m i n E c o n c e n t r a t i o n and c o n t e n t o f f r y i n Year 2.  114  F i g u r e 14. 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.  117  F i g u r e 15. R e l a t i o n s h i p o f 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 t h e eyed s t a g e i n Year 2.  119  F i g u r e 16. R e l a t i o n s h i p o f 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 h a t c h i n Year 2.  120  xi F i g u r e 17. R e l a t i o n s h i p o f 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 o f s a t u r a t e d , n7, n9, n i l monoenoic a c i d s , PUFA and HUFA o f t h e 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 and p o l a r l i p i d s o f t h e eggs i n Year 2.  125  F i g u r e 19. Comparison o f n3 f a t t y a c i d s o f t h e t o t a l l i p i d s o f t h e d i e t s w i t h t h e n e u t r a l and p o l a r l i p i d s o f t h e eggs i n Year 2.  126  F i g u r e 20. Comparison o f n6 f a t t y a c i d s o f t h e t o t a l l i p i d s o f t h e d i e t s w i t h t h e n e u t r a l and p o l a r l i p i d s o f t h e eggs i n Year 2.  127  xii LIST OF APPENDICES Page Appendix l a . F a t t y a c i d c o m p o s i t i o n o f t h e 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 c o m p o s i t i o n o f t h e n e u t r a l l i p i d s o f L a b r a d o r 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 c o m p o s i t i o n o f t h e p o l a r l i p i d s o f L a b r a d o r 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 c o m p o s i t i o n o f t h e n e u t r a l and p o l a r l i p i d s o f 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 t e m p e r a t u r e i n Year 1 and Year 2.  195  Appendix 2b. T o t a l g a i n , f e e d f e d and f e e d e f f i c i e n c y o f each t a n k o f f i s h f o r each p e r i o d i n Year 1.  196  Appendix 2 c . T o t a l g a i n , f e e d f e d and f e e d e f f i c i e n c y o f each tank o f f i s h f o r each p e r i o d i n Year 2.  197  Appendix 3a. Method o f e x t r a c t i o n and a n a l y s i s o f v i t a m i n E i n eggs and f r y .  198  A p p e n d i x 3b. Method o f e x t r a c t i o n and a n a l y s i s o f t o t a l l i p i d i n eggs and d i e t s .  201  A p p e n d i x 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 regressions o f 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 o f eggs i n Year 1 and Year 2.  203  xiii ACKNOWLEDGEMENTS Many p e o p l e c o n t r i b u t e d t o t h e c o m p l e t i o n o f t h e r e s e a r c h f o r t h i s t h e s i s . I would l i k e t o e x t e n d s p e c i a l t h a n k s t o Randy O l s o n and C o l l e e n E n g e l o f Rockwood A q u a c u l t u r e R e s e a r c h C e n t r e (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 b r o o d s t o c k , eggs and f r y . Thanks a r e a l s o extended t o o t h e r RARC s t a f f , Murray F o s t e r and Danny Chaput. Thanks t o J i m G i b s o n who a s s i s t e d w i t h c h e m i c a l a n a l y s e s and h a t c h e r y work and t o Ron Lypka and Bob J e s t a d t i n t h e computer department who always h e l p e d me o u t when I had problems w i t h g r a p h i c s and computer programming. Thanks a l s o t o Bob Hunt f o r d o i n g m i n e r a l a n a l y s e s and t o Bob Evans f o r d o i n g h i s t o l o g i c a l e x a m i n a t i o n o f fry. I w i s h t o thank Dr. B e r y l March who always t o o k t h e t i m e t o d i s c u s s my work and o f f e r a d v i c e . I thank f r i e n d s and c o l l e a g u e s who p r o v i d e d a d v i c e , a s s i s t a n c e and m o r a l s u p p o r t , e s p e c i a l l y R o b e r t a Y o r k and M i c h a e l P a p s t . A l t h o u g h I do n o t n o r m a l l y have t h e o p p o r t u n i t y t o work w i t h o t h e r f i s h n u t r i t i o n i s t s , I e n j o y e d h a v i n g f r i e n d s i n Vancouver l i k e Heather Ashton and Rob Roy, who s h a r e d a common i n t e r e s t i n b r o o d s t o c k n u t r i t i o n . I am i n d e b t e d t o Management a t t h e F r e s h w a t e r I n s t i t u t e , Department o f F i s h e r i e s and Oceans, who s u p p o r t e d my nine-month l e a v e t o a t t e n d t h e U n i v e r s i t y o f B r i t i s h Columbia and complete t h e c o u r s e work towards my degree. T h i s r e s e a r c h was s u p p o r t e d by t h e Department o f F i s h e r i e s and Oceans. I thank HoffmannLaRoche f o r c o n d u c t i n g v i t a m i n a n a l y s e s on t h e f e e d i n g r e d i e n t s . L a s t b u t c e r t a i n l y n o t l e a s t , I am t h a n k f u l f o r t h e c o n t i n u i n g s u p p o r t 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 t h e a q u a c u l t u r e i n d u s t r y i n Canada matures, t h e r e i s t h e need t o d e v e l o p t h e t e c h n o l o g y t o r a i s e d i f f e r e n t fish  species of  and thus a l l o w p r o d u c e r s t o r a i s e d i f f e r e n t p r o d u c t s f o r  w h i c h t h e y can d e v e l o p markets.  B e g i n n i n g i n 1978,  A r c t i c c h a r r [Salvelinus  ( L . ) ] were c o l l e c t e d as eggs  from w i l d f i s h and  alpinus  i n t h e Northwest  transferred  t o the  (NWT)  Rockwood A q u a c u l t u r e  (RARC), Gunton, M a n i t o b a . of  Territories  stocks of  and  Labrador  Research  Centre  T h i s f a c i l i t y became t h e major s o u r c e  A r c t i c c h a r r f o r p r o d u c e r s a c r o s s Canada.  Travel costs to  o b t a i n eggs from w i l d s t o c k s o f A r c t i c c h a r r , u n c e r t a i n spawning t i m e s and l a c k o f d i s e a s e - f r e e w i l d s t o c k s make i t i m p r a c t i c a l o r i m p o s s i b l e t o c o n t i n u e o b t a i n i n g eggs from w i l d s t o c k s . makes  i t  necessary  for  producers  to  maintain  This  their  b r o o d s t o c k s and t h e y 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  own  dietary  and e n v i r o n m e n t a l r e q u i r e m e n t s .  Hatchability been r a i s e d  of A r c t i c  i n captivity  c h a r r from b r o o d s t o c k w h i c h  has  varied w i t h the s p e c i f i c  have  stock.  H a t c h a b i l i t y o f t h e s t o c k s o r i g i n a t i n g from Nauyuk Lake i n t h e NWT  and from Norway has been h i g h (Tabachek and de March  Since these  s t o c k s are r a i s e d  RARC, most p r o d u c e r s  cannot  i n a non-certified  1990).  building  at  o b t a i n p e r m i t s t o t r a n s p o r t eggs  from t h o s e s t o c k s a c r o s s p r o v i n c i a l b o u n d a r i e s .  One  s t o c k 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 a t  2 RARC and  s i n c e t h e s e eggs can be t r a n s p o r t e d a c r o s s  boundaries,  they  have  t h r o u g h o u t Canada.  under t h e  distributed  widely  to  producers  U n f o r t u n a t e l y , embryonic s u r v i v a l  s t o c k has been v a r i a b l e raised  been  provincial  of  this  and o f t e n low when f e d t h e same d i e t  same e n v i r o n m e n t a l  c o n d i t i o n s as  the  and  Nauyuk  Lake and Norway s t o c k s a t RARC.  Survival  a t f i r s t - f e e d i n g i s a l s o stock-dependent and some  p r o d u c e r s have e x p e r i e n c e d  high m o r t a l i t y  at f i r s t feeding with  L a b r a d o r and Norway A r c t i c c h a r r (Tabachek and de March 1991). Poor  or  uncertain  hatchability  and  survival  of  fry  producers t o maintain a large inventory of broodstock planning of production d i f f i c u l t .  forces  and makes  T h e r e f o r e , t h e r e i s a need t o  improve b o t h t h e l e v e l and p r e d i c t a b i l i t y o f embryonic and  survival  charr.  survival  a t f i r s t - f e e d i n g of t h e L a b r a d o r s t o c k o f  Arctic  D i f f e r e n c e s a l s o e x i s t e d i n t h e d u r a t i o n o f t h e spawning  p e r i o d w i t h t h e L a b r a d o r s t o c k o f A r c t i c c h a r r spawning o v e r 4 to  5  months w h i l e  the  other  stocks  spawned  within  6  weeks  (Tabachek 1990).  There  is a  requirements of broodstock  scarcity  information  f i s h of reproductive  in particular.  n u t r i e n t ( s ) were r e l a t e d A r c t i c charr  of  broodstock.  Therefore,  age  on and  i t was  the on  nutritional Arctic  charr  not known i f some  t o the poor h a t c h a b i l i t y  of  Labrador  3 Maternal can  be  n u t r i t i o n a f f e c t s embryonic and  influenced  carotenoids  and  by  l e v e l s of  specific  Watanabe 1986).  dietary protein  minerals  and  and  vitamins  The  (Watanabe 1985)  l e v e l of u n s a t u r a t e d  energy,  (Luquet  The l e v e l s o f e s s e n t i a l f a t t y a c i d s and  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 (Jensen 1968).  f r y s u r v i v a l and  and  vitamin poultry  fatty acids influenced  t h e v i t a m i n E r e q u i r e m e n t o f rainbow t r o u t (Oncorhynchus fry  (Watanabe e t al.  high  level  of  1981a).  dietary  (Tabachek 1986).  protein  (44-54%)  and  J u v e n i l e A r c t i c c h a r r may  lipid  a  (15-20%)  have a r e q u i r e m e n t  a high l e v e l of e s s e n t i a l f a t t y acids  but  the requirements f o r A r c t i c charr broodstock are hypothesized  mykiss)  Juvenile A r c t i c charr require  for  I t was  and  (Yang and D i c k  1992)  unknown.  that i f A r c t i c charr broodstock required  a  h i g h l e v e l o f d i e t a r y l i p i d and/or u n s a t u r a t e d  f a t t y a c i d s , they  might  level  also  v i t a m i n E. effects  and  vitamin  E on  have The  a  requirement  for  high  o b j e c t i v e o f t h i s r e s e a r c h was  i n t e r a c t i v e e f f e c t s of the  a  reproduction  both  of  t o examine t h e  dietary  of A r c t i c c h a r r .  dietary  lipid  Reproductive  p a r a m e t e r s o f 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 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 matured and d u r a t i o n o f t h e spawning p e r i o d .  and  and that  4 LITERATURE REVIEW  2.1 N a t u r a l h i s t o r y o f A r c t i c c h a r r Arctic Arctic  c h a r r have a c i r c u m p o l a r d i s t r i b u t i o n  waters.  Newfoundland,  I n Canada,  throughout  (Johnson  1980).  northern  Quebec  Populations also and  fecundity  including  northern  Labrador.  before  they  Manitoba.  There  i n growth Arctic  rate,  Brunswick, are  many  several  and Green  longevity,  c h a r r used  thesis originated  Dempson  Territories  h i s t o r y between some o f t h e s e  C h a r r may make  mature.  and Yukon i n New  differences  research described i n this  i n Labrador,  occur  and age a t m a t u r i t y .  River,  a r e found  t h e Northwest  differences i n the natural l i f e populations  they  throughout  i n the  from t h e F r a s e r  migrations t o sea (1985)  found  that  L a b r a d o r A r c t i c c h a r r m i g r a t e d t o s e a f o r t h e f i r s t t i m e when t h e y were 78-191 mm i n l e n g t h a t 3 t o 7 y e a r s o f age.  Charr  m i g r a t e d t o s e a i n May and e a r l y June, spent 2-3 months a t s e a and r e t u r n e d t o f r e s h water from t h e l a t t e r h a l f o f J u l y t h r o u g h August.  Larger f i s h returned f i r s t with a higher proportion of  f e m a l e s r e t u r n i n g e a r l y i n t h e r u n . Annual l e n g t h  increments  were 68 mm between 4-5 y e a r s , 103 mm between 5-6 y e a r s and 71 mm between 6-7 y e a r s o f age. W h i l e 50% o f males matured a t 5 y e a r s of  age when t h e y were 245 mm i n l e n g t h , females matured  a t an  o l d e r age w i t h 50% mature a t 7 y e a r s when t h e y were 381 mm i n l e n g t h and 75% mature a t 8 y e a r s . the  females  returning  They e s t i m a t e d t h a t 60% o f  t o f r e s h water would  spawn t h a t  year.  5 T h i s i s i n c o n t r a s t t o c h a r r i n Nauyuk and W i l l o w L a k e s ,  NWT  w h i c h spawn one y e a r a f t e r r e t u r n i n g from t h e sea and may spend up t o two y e a r s i n f r e s h w a t e r  before returning  t o the sea  (Johnson 1980).  Anadromous  Arctic  charr  are iteroparous,  spawning  in  a l t e r n a t e y e a r s . However, i n nonanadromous p o p u l a t i o n s t h e y may spawn  every  year  with  spawning a n n u a l l y .  only  one  anadromous  s t o c k i n Norway  W h i l e 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 e v e r y y e a r , o t h e r s i n t h e same p o p u l a t i o n were n o t r e c a p t u r e d a t t h e same spawning s i t e f o r two to f i v e years.  I t i s not known i f t h e l a t t e r f i s h d i d n o t spawn  d u r i n g t h a t i n t e r v a l o r i f t h e y spawned a t a d i f f e r e n t s i t e b u t t h e former seems more p r o b a b l e . times  during  their  lifetime  A r c t i c c h a r r may spawn s e v e r a l  but  frequency  of  spawning  may  d e c r e a s e w i t h i n c r e a s i n g age.  I n L a b r a d o r , water t e m p e r a t u r e d u r i n g l a t e J u l y and A u g u s t , when c h a r r were r e t u r n i n g t o f r e s h w a t e r , v a r i e d between y e a r s but  ranged  from  8.7  t o 12.9°C  (Dempson  and  Greene  1985).  Spawning o c c u r r e d d u r i n g a t h r e e week p e r i o d i n O c t o b e r a t a w a t e r t e m p e r a t u r e o f 1-3 °C. coarse  sand  and  40-50  mm  I t o c c u r r e d i n a r e a s o f f i n e and gravel  i n 1.5-2.0  m  of  water.  F e c u n d i t y o f 7-13 y e a r o l d s ranged from 2316-9245 eggs w i t h a mean o f 4665 ± 434 ( s t d . d e v . ) .  Egg d i a m e t e r was 4.5  mm.  6 The t y p e o f f o o d consumed changes s e a s o n a l l y and a n n u a l l y according t o a v a i l a b i l i t y .  S m a l l c h a r r a r e b e n t h i c f e e d e r s and  i n f r e s h w a t e r t h e y f e e d on i n v e r t e b r a t e s such as c h i r o n o m i d s , amphipods, copepods and m o l l u s c s  (Johnson  1980).  When c h a r r  m i g r a t e t o s e a , t h e y remain a l o n g t h e c o a s t l i n e and a r e p e l a g i c feeders. items  When L a b r a d o r c h a r r m i g r a t e t o t h e s e a , p r i n c i p a l f o o d  include  shrimp  amphipods  (Meganyctiphanes  (Mallotus  (Parathemisto norvegica)  libellula),  and f i s h  such  euphasid  as c a p e l i n  v i l l o s u s ) , sand l a n c e (Ammodytes americanus)  mailed s c u l p i n  (Triglops  pingeli).  and young  D u r i n g spawning,  eggs a r e  consumed by c h a r r t h a t may o r may n o t have been i n v o l v e d i n spawning.  C h a r r c a n a l s o be c a n n i b a l i s t i c .  Postspawning  charr  consume l i t t l e , i f any, food i n f r e s h water and t h e y c a n be v e r y e m a c i a t e d when t h e y r e t u r n t o s e a t h e f o l l o w i n g y e a r .  Dempson and Green (1985) found few A r c t i c c h a r r i n L a b r a d o r t h a t were o l d e r than years.  12 y e a r s w i t h t h e o l d e s t f i s h  b e i n g 18  I n o t h e r l o c a t i o n s throughout t h e w o r l d , c h a r r may l i v e  t o be 30-33 y e a r s o f 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, s t o c k s  of Arctic  charr  eggs  were  c o l l e c t e d i n t h e w i l d d u r i n g 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 to  t h e Rockwood A q u a c u l t u r e  Manitoba.  Research  They were c o l l e c t e d  Centre  i n the wild  (RARC),  Gunton,  from Nauyuk  Lake,  N.W.T. i n 1978 (Papst and Hopky 1984), F r a s e r R i v e r , L a b r a d o r 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 h a t c h e r y s t o c k from S u n n d a l s o r a , Norway i n 1980 (Baker 1983). it  Primarily,  has been t h e progeny from t h e F r a s e r R i v e r , L a b r a d o r  collected  i n 1984 t h a t  have  certified  f a c i l i t y a t RARC.  been  distributed  from  stock  a  small  They have been t r a n s f e r r e d from  RARC t o p r o d u c e r s , r e s e a r c h e r s and government a g e n c i e s i n e v e r y province  and  territory  in  Canada  (Olson,  personal  communication).  When i n c u b a t e d a t 6°C, s u r v i v a l o f eggs c o l l e c t e d f r o m w i l d s t o c k s was 88% t o t h e eyed stage f o r t h e Nauyuk L a k e , NWT s t o c k (Papst  and Hopky 1984), 93% t o swimup f o r t h e F r a s e r  Labrador 87%  s t o c k c o l l e c t e d i n 1980 (Baker 1981)  t o swimup f o r t h e Tree R i v e r , NWT s t o c k  communication). wild  from  generation  River,  and 94% t o eye and (Olson,  personal  W h i l e t h e h a t c h a b i l i t y o f eggs c o l l e c t e d i n t h e  t h e Nauyuk domestic  Lake,  stock  NWT  stock  produced  was  high,  eggs w h i c h  s u r v i v a l t o t h e eyed stage w i t h 65% o f eggs b e i n g (Papst and Hopky 1984).  the f i r s t  had o n l y 17% unfertilized  Changes t o t h e r e a r i n g and spawning  t e c h n i q u e s have i n c r e a s e d t h e h a t c h a b i l i t y o f t h i s s t o c k t o 7398%  ( K r i e g e r e t al. 1988) w i t 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 y e a r s .  The l a t t e r a u t h o r s showed t h a t  t h e eggs o f A r c t i c c h a r r o v e r - r i p e n e d q u i c k l y and t h e chance o f 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 t h e p e r i o d s o f 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  that  8 fertilization  improved  i f t h e males  had been r a i s e d  i n cold  w a t e r (6.5°C) compared t o warm water (8-17°C) p r i o r t o t h e f i r s t y e a r o f spawning.  As w i t h t h e Nauyuk Lake s t o c k , s u r v i v a l o f  eggs from t h e Norway s t o c k i s h i g h (> 80% eyed, O l s o n , p e r s o n a l communication).  W h i l e h a t c h a b i l i t y o f t h e Nauyuk Lake s t o c k has  improved, s u r v i v a l o f s t o c k s o f A r c t i c c h a r r o r i g i n a t i n g the  from  F r a s e r R i v e r , L a b r a d o r have remained h i g h l y v a r i a b l e between  f i s h and between spawning y e a r s a t RARC.  F o r example,  survival  t o t h e eyed s t a g e f o r one s t o c k o f L a b r a d o r c h a r r was 50%, 72% and 35% i n t h r e e s u c c e s s i v e y e a r s (Tabachek and de March 1990) and  68% t h e f o l l o w i n g  year  (Olson, p e r s o n a l communication).  There has been v a r y i n g s u c c e s s among p r o d u c e r s who have r a i s e d Labrador  charr  Hatchability  t o maturity  (Tabachek  and de March  o f t h e Labrador stock of A r c t i c  charr  1991).  has been  shown t o be under m a t e r n a l r a t h e r t h a n p a t e r n a l i n f l u e n c e . de  March  (1992)  s e p a r a t e d t h e eggs  from i n d i v i d u a l  When  Labrador  females i n t o groups and f e r t i l i z e d each group w i t h t h e m i l t from a  different  male,  the pattern  o f embryonic  survival  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 o n l y by t h e f e m a l e . observation  suggests  that  the  nutritional  status  always This and/or  e n v i r o n m e n t a l c o n d i t i o n s o f t h e female s h o u l d 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 L a b r a d o r A r c t i c c h a r r w h i c h had spawned f o r t h e f i r s t t i m e , he found t h a t t h o s e t h a t spawned i n t h e m i d d l e o f t h e 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 h i g h e r p r o t e i n ,  lipid,  9 c a r b o h y d r a t e , ash and amino a c i d s t h a n t h o s e t h a t spawned e i t h e r 4 weeks e a r l i e r o r 4 weeks l a t e r .  I n a d d i t i o n , eggs from t h i s  m i d d l e group a l s o reached t h e eyed s t a g e , hatched e a r l i e r  and  had fewer deformed f r y t h a n t h o s e spawned e a r l i e r o r l a t e r .  Age o f m a t u r i t y and f e c u n d i t y o f s t o c k s r a i s e d f o r one two  g e n e r a t i o n s i n c a p t i v i t y v a r y from one  or  stock t o another.  The Nauyuk Lake s t o c k produced 1200-2000 eggs w h i l e t h e L a b r a d o r s t o c k s produced  3000-4000 eggs and  t h e Norway s t o c k  3000 eggs (Tabachek and de March 1990).  produced  The L a b r a d o r and Norway  s t o c k s s t a r t t o spawn a t 3 y e a r s o f age, w h i l e t h e Nauyuk Lake s t o c k does not s t a r t t o spawn u n t i l a t l e a s t 4 y e a r s o f age w i t h most f i s h spawning  f o r t h e f i r s t time a t 5-6 y e a r s o f age.  All  s t o c k s spawn f o r 4-5 y e a r s ( O l s o n , p e r s o n a l c o m m u n i c a t i o n ) .  The  m o r t a l i t y r a t e b e g i n s t o i n c r e a s e a t 6 y e a r s o f age and f i s h a r e g e n e r a l l y e u t h a n i z e d a t 7-8 y e a r s o f age.  Nauyuk Lake A r c t i c c h a r r f i n g e r l i n g s r a i s e d a t 12°C  gained  s i g n i f i c a n t l y more when f e d a d i e t c o n t a i n i n g 54% p r o t e i n 20% l i p i d and  15%  compared t o t h o s e f e d a d i e t c o n t a i n i n g 44% lipid  (Tabachek  1986).  and  protein  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 l o w e r w e i g h t g a i n compared t o f e e d i n g 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 lipid.  15%  The r e q u i r e m e n t s f o r rainbow t r o u t have been r e p o r t e d as  34% p r o t e i n i n d i e t s w i t h 15-20% l i p i d when r a i s e d a t 15-18°C ( T a k e u c h i e t 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 a t 16-27°C ( S a t i a 1974). acid  requirements  2.6.3.  of  There has  Arctic  charr  been l i t t l e  are  The e s s e n t i a l f a t t y  discussed  other research  in  Section  r e p o r t e d 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 .  Survival 1987).  of  swimup  S u r v i v a l and  f r y varies with  the  stock  growth o f the L a b r a d o r and  Norway  improved when r e a r i n g temperature a t f i r s t f e e d i n g was from 7 t o 10°C  (Tabachek 1992)  improvement i n s u r v i v a l and from  one  manufacturer  w h i l e t h e r e was  (Krieger stocks  increased  only a  slight  growth when f e d a semimoist  compared  to  a  dry  diet  from  diet  another  manufacturer.  2.3  Physiology of The  reviewed summary,  reproduction  p h y s i o l o g y o f r e p r o d u c t i o n i n t e l e o s t f i s h e s has in detail  (Nagahama 1983,  environmental  t e m p e r a t u r e a c t upon t h e  factors  Ng  and  such  as  pituitary  1983).  photoperiod  hypothalamus w h i c h i s s t i m u l a t e d  s e c r e t e g o n a d o t r o p i n - r e l e a s i n g hormone (GRH). upon t h e  Idler  been  causing  i t to secrete  In and to  T h i s hormone a c t s 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). from  T h e c a l 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  cholesterol.  FSH  stimulates  the  testosterone  development  of  the  f o l l i c l e s and s t i m u l a t e s g r a n u l o s a c e l l s s u r r o u n d i n g each o o c y t e to  produce t h e enzyme t h a t c o n v e r t s  Estrogen  is  transported  to  the  testosterone to liver  where  estrogen.  i t activates  11 hepatocytes  to  transported  synthesize  vitellogenin.  Vitellogenin  is  t o t h e o v a r y where i t i s s e l e c t i v e l y t a k e n up  by  oocytes through m i c r o p i n o c y t o s i s . the  follicle cells,  transported  V i t e l l o g e n i n i s t a k e n up across  zona p e l l u c i d a i n t o t h e m i c r o v i l l i oocyte.  of  the  i n t h e zona r a d i a t a o f  the  N u t r i e n t s such as v i t a m i n s and m i n e r a l s a r e a l s o t a k e n  up by t h e o o c y t e . is  the m i c r o v i l l i  by  synthesized  Most v i t e l l o g e n i n , a i n the  liver  by  glycolipophosphoprotein,  exogenous v i t e l l o g e n e s i s but  t h e r e i s e v i d e n c e t h a t a p o r t i o n i s s y n t h e s i z e d i n t h e o v a r y 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 ( p h o s p h o p r o t e i n ) and  lipovitellin  (proteolipid  w h i c h i s 80% p r o t e i n and 20% l i p i d ) .  The  lipovitellin  c o n s i s t s o f b o t h p o l a r and (1981)  found  were  35%  l i p i d of  neutral lipids  w h i c h Léger e t a l .  triacylglycerides.  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 t h e p e r i p h e r y the  oocyte  during  spheres d u r i n g  the  the  primary  stage  secondary s t a g e .  single yolk during late v i t e l l o g e n e s i s . the nucleus, migrate t o the periphery oil  droplets.  The  ends, t h e oocytes  metaphase and fertilized.  of  released.  the  to  They c o a l e s c e  yolk  t o form a  and merge t o form l a r g e r triacylglycerides  cholesteryl esters  f o l l i c l e cells  Second  form  O i l d r o p l e t s appear a t  When o o c y t e s have matured and  microvilli  are  fuse  o i l droplets c o n s i s t of  w i t h t r a c e s o f c h o l e s t e r o l and Watanabe 1986).  and  of  meiosis  eggs are ready t o be r e l e a s e d  (Luquet  first  meiosis  w i t h d r a w and then  and  the  continues  to  from t h e body  and  12 2.4  Feed i n t a k e and  2.4.1  reproduction  Feed i n t a k e The e f f e c t o f l e v e l o f f e e d i n t a k e ( r a t i o n ) on  fecundity, reviewed  egg  size  (Roley  and  1983,  survival  Springate  of  eggs  and  e t a l . 1985,  maturation,  f r y has Hardy  been  1985  and  Luquet and Watanabe 1986).  O r r e t a l . (1982) found t h a t a d u l t r a i n b o w t r o u t consumed more  feed  feeders  and  had  higher  compared t o t h o s e  weight f e d by  r a t e s produced l a r g e r eggs and relative  There was  stage.  ration  when  hand.  had  fed  with  demand  F i s h feeding at  high  f e c u n d i t y but  lower  higher  f e c u n d i t y (eggs p e r body w e i g h t ) t h a n t h o s e who  hand-fed. eyed  gain  were  no d i f f e r e n c e i n s u r v i v a l o f eggs t o  Knox e t  al.  (1988) found t h a t  feeding  (0.7% body w e i g h t ) r e s u l t e d i n r a i n b o w t r o u t  at  swimup f r y stages  body w e i g h t ) .  than f i s h fed at h a l f - r a t i o n  Swimup f r y from p a r e n t s  s i g n i f i c a n t l y h i g h e r p r o t e i n and half-satiation fatty  acid  Springate  group.  However, r a t i o n  composition et  al.  (1985)  e x p e r i m e n t o f Knox e t al. fed  in  of  yolk (0.35%  ration  had  l e v e l s compared t o  the  had  no  effect  polar  or  neutral  fatty  reported  on  another  part  on  the  acids. of  the  (1988) and s t a t e d t h a t r a i n b o w t r o u t  a t f u l l r a t i o n spawned 2-3  half ration.  lipid  fed the f u l l  full  producing  progeny w h i c h were s i g n i f i c a n t l y l a r g e r a t t h e eyed egg, sac and  the  weeks e a r l i e r t h a n t h o s e  The c o n c e n t r a t i o n o f v i t e l l o g e n i n and  fed at  testosterone  t h e serum were s i g n i f i c a n t l y h i g h e r a t 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 f e d a t f u l l r a t i o n spawned w h i l e 11%  of t h o s e f e d a t h a l f r a t i o n d i d not spawn.  In addition,  fish  f e d a t 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 t h e s e eggs were s i g n i f i c a n t l y l a r g e r fed at h a l f r a t i o n . were  t h a n t h o s e from  H i s t o l o g i c a l e x a m i n a t i o n showed t h a t t h e r e  s i g n i f i c a n t l y more a t r e t i c o o c y t e s  ration.  fish  in fish  fed at  half  R a t i o n 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 o r s u r v i v a l t o  t h e eyed s t a g e .  Roley satiation  (1983) f e d rainbow f o r one  year  prior  trout  to satiation  t o spawning.  At  and  half-  cool  water  t e m p e r a t u r e , b o t h egg s i z e and s u r v i v a l i n c r e a s e d f o r t h e h a l f s a t i a t i o n group satiation contrast  compared t o t h e s a t i a t i o n group.  increased the v a r i a t i o n t o Orr  et  a l . (1982),  i n embryonic the high  Feeding t o  survival.  level  In  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 a l t h o u g h s u r v i v a l was independent spawning  o f egg s i z e .  I n c o n t r a s t t o Knox e t a l . ( 1 9 8 8 ) ,  was d e l a y e d b u t o c c u r r e d w i t h i n  a shorter interval i n  f i s h w h i c h had been f e d t o s a t i a t i o n b u t f e e d i n g l e v e l had no e f f e c t on t h e number o f f i s h t h a t spawned.  Jones and Bromage (1987) f e d 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% o f body weight and found t h a t w h i l e r a t i o n had  a  significant  effect  on  fecundity  beyond  the e f f e c t  r a t i o n on body w e i g h t , i t had no e f f e c t on egg d i a m e t e r .  of This  14 is  i n contrast  t o t h e f i n d i n g s o f b o t h O r r e t 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 t h e spawning  period  A s h t o n (1991) found no d i f f e r e n c e i n f a t t y a c i d c o m p o s i t i o n o r s u r v i v a l o f Chinook salmon {Oncorhynchus  tshawytscha)  to the  eyed s t a g e when b r o o d s t o c k were s t a r v e d f o r 7 and 14 days b e f o r e spawning.  Starvation  of  rainbow  trout  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 w e i g h t , egg d i a m e t e r o r p r o x i m a t e c o m p o s i t i o n (Ridelman e t al.  1984).  2.5 P r o t e i n and energy The  influence  reproduction  energy  dietary  protein  has been r e v i e w e d by Hardy  Watanabe (1986). and  of  energy  levels  (1985) and Luquet  on and  I t was b e l i e v e d t h a t r e q u i r e m e n t s f o r p r o t e i n  might  development.  and  increase  However, Hardy  during  the  time  (1985) p o i n t e d  of  out t h a t  ovarian somatic  growth c e a s e s d u r i n g o v a r i a n development and t h a t t h e r e may  not  be  and  an  o v e r a l l increase  i n the requirements f o r p r o t e i n  energy.  Roley  (1983)  f e d rainbow t r o u t d i e t s c o n t a i n i n g  l e v e l s o f 27 t o 57% a t 3.8 k c a l m e t a b o l i z a b l e  protein  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 y e a r s o f age. There  were  diameter,  no  significant  fecundity,  differences  duration  of  i n h a t c h a b i l i t y , egg  spawning  or  prespawning  15 mortality. protein  He  c o n c l u d e d t h a t t h e r e q u i r e m e n t was  a t 3.8  effects  of  k c a l ME/g.  dietary  Washburn  protein  and  (1989)  energy  on  37  to  47%  investigated  the  rainbow  trout  b r o o d s t o c k and found t h a t f e e d i n g h i g h d i e t a r y p r o t e i n (58%) and energy  (3.23 k c a l / g ) c o n c e n t r a t i o n s r e s u l t e d  in significantly  l o w e r h a t c h a b i l i t y compared t o f e e d i n g a d i e t t h a t was p r o t e i n (30%) and energy (2.64 k c a l / g ) .  low i n  This l a t t e r combination  was below t h e l e v e l o f energy a s s e s s e d by R o l e y (1983).  S m i t h e t a l . (1979) f e d rainbow t r o u t f o r 6 months b e f o r e t h e i r f i r s t spawning season and t h e n t h r o u g h 2 more c o n s e c u t i v e spawning  seasons.  levels of protein 3.44  kcal/g).  T e s t d i e t s c o n t a i n e d low, medium and (36, 42 and 48%)  and energy  (2.52, 2.85  high and  F e e d i n g r a t e was 0.8% body weight/day u n t i l f e e d  i n t a k e d e c r e a s e d as spawning approached.  The h i g h 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 w e i g h t g a i n , f e c u n d i t y and egg  size  but  no  significant  effect  on  survival  t o the  eyed  stage.  Long-term  f e e d i n g o f rainbow t r o u t  from t h e  fingerling  s t a g e t h r o u g h t h e second spawning season on d i e t s c o n t a i n i n g 35% protein  with  16-18%  lipid  or  45%  protein  with  15%  lipid  ( T a k e u c h i e t 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 d i a m e t e r o r s u r v i v a l t o t h e eyed (90%) and h a t c h (87%) s t a g e s . al.  T h i s c o n f l i c t s w i t h t h e f i n d i n g s o f Watanabe e t  (1984b) who observed poor h a t c h a b i l i t y when b r o o d s t o c k were  16 f e d 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%) o r 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 a c i d s and v i t a m i n E  2.6.1  C h e m i s t r y o f l i p i d s and f a t t y Neutral  lipids  acids  i n c l u d e mono-, d i - and  triacylglycerides  (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 t h e major s o u r c e o f energy.  P o l a r l i p i d s , which  include  sphingomyelins  phosphoglycerides,  plasmolagens, are required subcellular  Fatty  glycolipids,  f o r the formation  and  of c e l l u l a r  and  membranes.  acids  a r e an i n t e g r a l  part  of these  lipids.  The  p o s i t i o n o f t h e f i r s t double bond from t h e m e t h y l end o f a f a t t y acid  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 d o u b l e bond a f t e r t h e t h i r d c a r b o n from t h e t e r m i n a l end.  A d d i t i o n a l d o u b l e bonds a r e i n t e r r u p t e d by one  carbon  atom.  Double bonds  increase  saturated  the f l e x i b i l i t y  m o l e c u l e and a l s o r e s u l t i n a d e c r e a s e i n t h e m e l t i n g Both these f a c t o r s are important and  f u n c t i o n o f membranes,  l i v e i n c o l d environments. levels  of  unsaturated  of the point.  i n maintaining the f l e x i b i l i t y  especially i n poikilotherms  which  This i s the reason there are high  fatty  b i o l o g i c a l membranes i n f i s h . bonds a r e c a l l e d p o l y u n s a t u r a t e d  acids  i n the p o l a r  lipids  of  F a t t y a c i d s w i t h 2 t o 4 double f a t t y a c i d s (PUFA) w h i l e t h o s e  17 w i t h g r e a t e r t h a n o r e q u a l t o 5 double bonds a r e r e f e r r e d t o as h i g h l y unsaturated mainly  fatty acids  (HUFA).  I n f i s h , PUFA c o n s i s t  o f n6 f a t t y a c i d s w h i l e HUFA c o n s i s t m a i n l y o f n3 f a t t y  acids.  I n TAG, s a t u r a t e d  positions  and monoenoic f a t t y  on t h e 1- (a) and 3- (y) c a r b o n  acids  occupy  of the glycerol  m o l e c u l e , w h i l e PUFA o r HUFA occupy t h e 2- (P) c a r b o n p o s i t i o n . I n p o l a r l i p i d s , monoenoic and s a t u r a t e d f a t t y a c i d s occupy t h e a p o s i t i o n and HUFA o r PUFA occupy t h e p p o s i t i o n .  Saturated  fatty  acids  c a n be s y n t h e s i z e d  de novo  from  a c e t a t e ( C a s t e l l 1979).  F a t t y a c i d s t h a t cannot be s y n t h e s i z e d  but w h i c h a r e n e c e s s a r y  f o r t h e h e a l t h and r e p r o d u c t i o n o f t h e  a n i m a l a r e c o n s i d e r e d t o be e s s e n t i a l f a t t y a c i d s w h i c h must be supplied 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, t h e d i e t must s u p p l y t h e s e two f a t t y a c i d s as p r e c u r s o r s for must  d e s a t u r a t i o n and e l o n g a t i o n i n t o PUFA and HUFA o r t h e d i e t supply  PUFA  o r HUFA  (Figure  1).  Desaturation  e l o n g a t i o n o f t h e n9, n6 and n3 s e r i e s o f f a t t y a c i d s t h r o u g h a common enzyme pathway o f d e s a t u r a s e s  and  occurs  and e l o n g a s e s b u t  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 ( F i g u r e 1 ) . I n mammals, the  desaturase  unsaturated  enzymes have a g r e a t e r  fatty  a c i d a v a i l a b l e (Leray  affinity  and P e l l e t i e r  That i s , A 5 - d e s a t u r a s e has a g r e a t e r a f f i n i t y 20:3n6 w h i c h has g r e a t e r a f f i n i t y t h a n 20:2n9. the a b i l i t y t o desaturate but  f o r t h e most 1985).  f o r 20:4n3 t h a n Thus, t h e r e i s  20:2n9 t o 20:3n9 o r 20:3n6 t o 20:4n6  i n t h e p r e s e n c e o f 20:4n3, A 5 - d e s a t u r a s e  preferentially  18 converts  20:4n3 t o 20:5n3.  I t i s speculated  that a  similar  mechanism o c c u r s i n f i s h and t h e p r e s e n c e o f 20:3n9 i s used as an i n d i c a t o r o f 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 e t al. 1972).  2.6.2 D i g e s t i o n and a b s o r p t i o n o f l i p i d s and v i t a m i n E I n t h e g u t lumen, an emulsion compounds  occurs  phospholipids  on  contact  produced  by  of l i p i d  with  bile  and l i p i d - s o l u b l e salts  the l i v e r .  In  and  rainbow  bile trout,  t r i a c y l g l y c e r i d e s a t the surface o f the emulsion are hydrolyzed through  the action  of pancreatic  lipase  (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 a r e h y d r o l y z e d i n t h e 1and  3 - p o s i t i o n i n t o f r e e f a t t y a c i d s and  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  if  2-monoacylglycerides.  t h e a c y l c h a i n c o n t a i n s a t l e a s t one d o u b l e bond.  d i f f e r e n t from mammals and b i r d s where t h i s does n o t o c c u r . the  liver,  solubilize  latter hydrolysis  M i c e l l e s , c o n s i s t i n g o f b i l e s a l t s produced by  a r e amphipathic  neutral interior  This i s  providing  ( S c o t t e t al. 1982).  monoacylglycerides,  a c i d s and g l y c e r o l .  a polar  exterior  and  This allows m i c e l l e s t o  long-chain  unsaturated  fatty  The mixed m i c e l l e c a n t h e n 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 a c i d s 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 intestine  and/or  pyloric  cecae  cells  along  f o r absorption  the proximal (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 t h e lumen g i v i n g them a h i g h absorptive area.  Acetate  F i 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 o f 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 S a r g e n t 1985).  20 T o c o p h e r o l s and t h e i r e s t e r s ( F i g u r e 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 o f o n l y 20-40% i n mammals ( G a l l o - T o r r e s 1980a) and t h i s may be due t o t h e l o n g p h y t y l s i d e c h a i n .  While absorption  e f f i c i e n c i e s have n o t 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 t h a n t h e p~, y- o r ôforms o f t h e v i t a m i n (Watanabe e t a l . 1981b).  Tocopheryl e s t e r s  a r e h y d r o l y z e d by p a n c r e a t i c e s t e r a s e s t o form t o c o p h e r o l w h i c h 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). A l m o s t no t o c o p h e r y l e s t e r s a r e absorbed. found  that  absorption  o f d-a-tocopherol  t o c o p h e r y l a c e t a t e was s l o w e r and  they speculated  Hung e t a l . (1982) from  dietary dl-a-  t h a n from d i e t a r y  d-a-tocopherol  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 absorption.  Re-esterification  of  glycerol,  monoacylglyceride  l y s o p h o s p h o l i p i d s w i t h f a t t y a c i d s t o form TAG and p o l a r (Léger 1985) o c c u r s i n t h e i n t e s t i n a l mucosa. through  the  chylomicrons transported 1989). to  and  very  by t h e b l o o d  wall, low  become  density  End p r o d u c t s pass  incorporated  lipoproteins  o r lymphatic  lipids  system  into  and  (Sargent  are  et a l .  Lipoproteins transported v i a the blood are transported  the l i v e r  tissues.  intestinal  and  by t h e h e p a t i c  portal  vein  TAG i s t a k e n up by a d i p o s e  S a r g e n t 1985) where i t i s s t o r e d u n t i l  and t h e n  tissue  to  other  (Henderson and  r e q u i r e d as an energy  source while polar 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 o f v i t a m i n E  22 Tocopherol lipoproteins  is  transported  (Hung e t  al.  primarily  1982)  and  in  low  i s taken  up  density  rapidly  plasma and l i v e r but more s l o w l y by w h i t e m u s c l e , h e a r t , and  erythrocytes  (Cowey e t al.  1983,  Hung e t al.  spleen  1982).  When  r a i n b o w t r o u t were f e d a v i t a m i n E-supplemented d i e t and g i v e n an o r a l dose of d - a - t o c o p h e r o l , high  concentration  declined  as  i n the  i t was  taken  (Cowey e t a l . 1981).  i n i t i a l l y i t was  cytosol up  by  of  liver  mitochondria  and  I n c o n t r a s t , when f i s h  had  v i t a m i n E - d e f i c i e n t d i e t , t o c o p h e r o l was by m i t o c h o n d r i a fraction  and  cells  implying  that  then  found i n but  this  microsomes been f e d  a  t a k e n up most r a p i d l y  i n t h e l i v e r f o l l o w e d by microsomes, t h e cytosol  by  it  is  needed  nuclear 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 r e p r o d u c t i o n The e s s e n t i a l f a t t y a c i d r e q u i r e m e n t s o f immature f i s h have  been r e v i e w e d by D i c k 1992)  Kanazawa (1981).  A recent  report  (Yang  and  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  Yang and  0.5%  Dick  desaturating  n6 f a t t y a c i d s .  Both O l s e n e t al.  (1991) and  (1992) showed t h a t A r c t i c c h a r r were c a p a b l e o f and  elongating  18:3n3 and  18:2n6.  The  former  a u t h o r s o b s e r v e d t h a t n3 f a t t y a c i d s were used i n p r e f e r e n c e 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  elongation.  to  23 The  r e q u i r e m e n t s o f b r o o d s t o c k f o r f a t t y a c i d s have been  r e v i e w e d by Watanabe (1985) and Ashton (1991) b u t no work w i t h A r c t i c c h a r r b r o o d s t o c k has been r e p o r t e d .  Yu et al. (1979) f e d  rainbow t r o u t experimental d i e t s throughout t h e i r e n t i r e  life  h i s t o r y and a s s e s s e d t h e h a t c h a b i l i t y o f t h e eggs and t h e growth of the f r y .  They found t h a t  18:2n6 was n o t r e q u i r e d  i n the  p r e s e n c e o f 1.0% 18:3n3 f a t t y a c i d s and b o t h groups had e q u a l hatchability  (60%, n=4).  L e r a y e t al. (1985) f e d a c o m m e r c i a l  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 b r o o d s t o c k f o r one y e a r b e f o r e spawning w i t h t h e two d i e t s d i f f e r i n g i n many  respects  recalculation contained  including of  fatty  the data  acid  presented,  2.5% n6 and 1.3% n3 w h i l e  c o n t a i n e d 5.4% n6 and no n3.  composition.  By  t h e commercial  diet  the semipurified  diet  The h a t c h a b i l i t y from t h e group  f e d t h e c o m m e r c i a l d i e t averaged 79% (n=3) w h i l e t h a t o f t h e n3 d e f i c i e n t group was 20% (n=4).  Those f e d t h e n 3 - d e f i c i e n t d i e t  produced eggs w h i c h had d e v e l o p m e n t a l d i s o r d e r s t h a t c o u l d be seen a t t h e 16-32 c e l l s t a g e and hatched f r y e x h i b i t e d numerous t y p e s o f s p i n a l d e f o r m i t i e s and a s h o r t e r t i m e t o r e s o r b t h e y o l k s a c (50 v s . 65 d a y s ) . that  Watanabe e t al. (1984b)  f e c u n d i t y and embryonic  s u r v i v a l d e c r e a s e d when  observed rainbow  t r o u t were f e d a d i e t w i t h no n3 o r n6 (54% eyed and 36% h a t c h ) compared t o a d i e t supplemented w i t h 5% 18:2n6 (88% eyed and 82% hatch) .  Red s e a bream (Pagrus  major)  fed n3-deficient  diets  a l s o produced eggs w i t h v e r y 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 o b s e r v e d (Watanabe 1984a).  24 As d i s c u s s e d i n S e c t i o n 2.3, v i t e l l o g e n i n i s a v e r y h i g h density  lipoprotein containing  80% p r o t e i n  and 20% l i p i d  which  i s h i g h i n e s s e n t i a l f a t t y a c i d s (Léger e t 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  diet  for6  months p r i o r t o spawning d e c r e a s e d t h e amount o f v i t e l l o g e n i n (Fremont e t a l . 1984) as w e l l as t h e l e v e l s o f 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 i n c r e a s e d t h e l e v e l o f 20:4n6.  These  changes  i n other  serum  during  early  were  similar  but  lipoproteins  and  were  vitellogenesis  indicating  less  pronounced  most that  pronounced feeding  a diet  sufficient i n  e s s e n t i a l f a t t y acids 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 c o m p o s i t i o n o f t h e eggs r e f l e c t e d t h a t o f the  diet.  I n b o t h L e r a y e t al. (1985) and Watanabe e t al.'s  (1984b) e x p e r i m e n t s , f e e d i n g 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 o f n3 f a t t y a c i d s (20:5, 22:5 and 22:6) and h i g h e r l e v e l s o f n6 (18:2 and 20:4) i n b o t h t h e p o l a r and n e u t r a l  l i p i d fractions.  Watanabe e t al. (1984b)  a l s o found t h a t t h e f a t t y a c i d c o m p o s i t i o n o f t h e m i l t r e f l e c t e d t h a t o f t h e d i e t i n t h e same way as f o r t h e eggs and s u g g e s t e d that  the effect  investigated. fed d i e t s al.  of this  change on f e r t i l i z a t i o n  should  be  W h i l e 20:3n9 was not found i n t h e eggs o f p a r e n t s  l a c k i n g i n n3 f a t t y a c i d s f o r 3 months (Watanabe e t  1984a) o r one y e a r ( L e r a y e t al. 1985) p r i o r t o spawning, i t  was found i n t h e m i l t .  When t r o u t  were f e d d i e t s  containing  18:3n3 a l o n e o r i n c o m b i n a t i o n w i t h 18:2n6 f o r t h e i r e n t i r e  life  25 h i s t o r y , 20:3n9 was o b s e r v e d i n t h e eggs from b o t h groups b u t was l o w e r i n t h e group f e d t h e d i e t c o n t a i n i n g b o t h f a t t y  acids  (Yu e t al. 1979).  The t r a n s f e r o f 16:0 and 22:6n3 from t h e y o l k s a c t o t h e body o f t h e d e v e l o p i n g body and c o n c u r r e n t (Salvelinus  f r y was shown by t h e i r i n c r e a s e i n t h e  decrease i n the y o l k  fontinalis)  e t al. 1985).  s a c o f brook  trout  ( A t c h i s o n 1975) and r a i n b o w t r o u t (Hayes  Hayes e t 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 fatty  (74%) i n r a i n b o w  trout  a t t h e y o l k sac stage while  acids  18:1,  20:4 and 20:5 showed o n l y 41% r e t e n t i o n .  such as 16:1, They p o s t u l a t e d  t h a t t h o s e f a t t y a c i d s w h i c h had h i g h r e t e n t i o n were r e q u i r e d for  specific  related  to  unsaturated  purposes. inclusion 22:6n3  Moreover, into  polar  incorporated  these lipids  purposes with  might  the  be  highly  i n t o t h e p* p o s i t i o n and t h e  s a t u r a t e d 16:0 and 18:0 i n c o r p o r a t e d i n t o t h e a p o s i t i o n on t h e g l y c e r o l molecule.  2.6.4 V i t a m i n E 2.6.4.1 C h e m i s t r y o f t o c o p h e r o l s The c h e m i s t r y by K a s p a r e k (1980).  of the tocopherols  has been w e l l documented  Isomers o f t o c o p h e r o l e x i s t which d i f f e r i n  t h e p r e s e n c e and l o c a t i o n o f m e t h y l groups on t h e chromanol r i n g g i v i n g r i s e t o a-,  y- and ô-tocopherols ( F i g u r e 2 ) .  Stereo-  i s o m e r s a l s o e x i s t w i t h d i f f e r e n t c e n t e r s o f symmetry around t h e  26 C-2 carbon o f t h e r i n g and t h e C-4' and C-8' o f t h e i s o p r e n o i d chain.  The d- s t e r e o i s o m e r s a r e found  manufactured  synthetically  tocopherols.  a r e racemic  i n nature while mixtures  o f d- and 1-  Watanabe e t al. (1981b) found t h a t a - t o c o p h e r o l s  accumulated  i n tissues  l i v e r , while  o f rainbow  trout,  y- and Ô- forms d i d n o t .  especially  Vitamin E i s a  g e n e r i c term r e f e r r i n g t o a l l t o c o l o r t o c o t r i e n o l  1980).  the b i o l o g i c a l  activity  i n the  No i n t e r c o n v e r s i o n o f  t h e s e isomers o c c u r r e d i n t h e muscle o r l i v e r .  exhibiting  those  of a-tocopherol  derivatives (Kasparek  U n l e s s o t h e r w i s e s p e c i f i e d , t h e 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 o f t h e major f u n c t i o n s o f v i t a m i n E i s i t s a c t i o n as an antioxidant functional  and t h e p h e n o l i c group.  Based  hydroxyl  on  group  provides the  effectiveness i n preventing  r e s o r p t i o n o f embryos i n t h e r a t , t h e isomers and t h e i r e s t e r s differ i nbiological activity  (NRC 1987):  d-a-tocopherol  1.49 I.U./mg  d-a-tocopheryl acetate  1.36 I.U./mg  dl-a-tocopherol  1.1  I.U./mg  dl-a-tocopheryl acetate  1.0  I.U./mg  d-p-tocopherol  0.12 I.U./mg  d-y-tocopherol  0.05 I.U./mg  O x i d a t i o n o f t o c o p h e r o l s i s enhanced by exposure t o l i g h t , heat,  alkali,  iron  and copper  ions.  Since  a-tocopherol i s  27 e a s i l y o x i d i z e d ( B a u e r n f e i n d 1980), i t i s added t o a n i m a l in  i t s esterified  form  of  the  properties hydroxyl  group  esterases.  form as a - t o c o p h e r y l a c e t a t e , a more s t a b l e  vitamin.  until  feed  the  The  ester  acetate  during  form  has  has  been  group  hydrolysis  no  antioxidant  replaced  initiated  by  by  a  pancreatic  S i n c e t o c o p h e r o l s a r e manufactured by p l a n t s , t h e y  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 a r e a l s o  present  animals  in  i n g r e d i e n t s of  animal  origin  where  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 F u n c t i o n o f v i t a m i n E Vitamin  E  functions  i n preventing  lipid  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  electrons.  In the p e r o x i d a t i o n of l i p i d ,  oxidation  o r more  and  unpaired  a hydrogen atom i s  a b s t r a c t e d from t h e methylene carbon o f a m e t h y l e n e - i n t e r r u p t e d u n s a t u r a t e d 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  abstraction i s initiated  only  by  radicals  degree o f r e a c t i v i t y , such as a h y d r o x y l r a d i c a l radical  (ROO.) o r p o s s i b l y a hydroperoxy  radical  with  a  high  (.OH), peroxy (HOO.).  The  d o u b l e 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 w h i c h can r e a c t with  oxygen  to  form a peroxy  radical  (ROO.) w h i c h  can  a b s t r a c t hydrogen atoms from o t h e r u n s a t u r a t e d f a t t y a c i d s . process  i s autocatalytic  keeps t h e c y c l e g o i n g .  because g e n e r a t i o n o f more The  process  then The  radicals  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 o t h e r compounds.  I t also results  i n t h e f o r m a t i o n o f ( h y d r o ) p e r o x i d e s w h i c h 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). donor, d o n a t i n g  V i t a m i n E a c t s as a hydrogen o r e l e c t r o n  t h e hydrogen from i t s 6-hydroxy1 group t o t h e  p e r o x y r a d i c a l c o n v e r t i n g i t t o t h e l i p i d h y d r o p e r o x i d e (ROOH). A v i t a m i n E r a d i c a l i s formed b u t t h i s i s n o t a c t i v e enough t o a b s t r a c t hydrogen from PUFA as R00. c a n . ROOH i s c o n v e r t e d ROH t h r o u g h t h e a c t i o n o f g l u t a t h i o n e p e r o x i d a s e of  glutathione  (GSH).  The hydroxy  radical  to  i n the presence (OH.), w h i c h i s  a n o t h e r r a d i c a l t h a t can i n i t i a t e t h e o x i d a t i o n p r o c e s s (Cowey 1986), i s formed when t h e s u p e r o x i d e  r a d i c a l (0 _.) and H 0 2  t o form OH. and OH" i n t h e presence o f F e . +3  prevented  by  the  action  catalyzes  the conversion  glutathione peroxidase oxidation.  of of  glutathione H0 2  2  t o H 0. 2  2  react  2  This r e a c t i o n i s peroxidase Vitamin  which E  and  f u n c t i o n i n d i f f e r e n t ways i n p r e v e n t i n g  V i t a m i n E f u n c t i o n s w i t h i n t h e membrane t o p r o t e c t  polyunsaturated  fatty  acids with electron transport  against free r a d i c a l attack.  Glutathione peroxidase  functions  acts w i t h i n  t h e c y t o s o l t o p r e v e n t t h e f o r m a t i o n o f hydroxy r a d i c a l s and i t destroys  h y d r o p e r o x i d e s which form.  V i t a m i n E i s m a i n l y a s s o c i a t e d w i t h t h e m i t o c h o n d r i a l and m i c r o s o m a l membranes and t o a l e s s e r degree w i t h t h e plasma and n u c l e a r membranes (Cowey e t al. 1981). contain lipids The  35-40%  lipid  and 90% o f t h i s  M i t o c h o n d r i a l membranes i s comprised  of polar  ( P i k e and Brown 1967) which a r e h i g h i n PUFA and HUFA.  inner mitochondrial  membrane c o n t a i n s  proteins that  allow  29 t h e passage o f f a t t y a c i d s , p y r u v a t e , oxygen and as C a  +2  and  P0 ~  from the  3  4  space ( A l b e r t s  intermembrane space i n t o t h e  e t al. 1983)  o f f a t t y a c i d and  some i o n s s u c h matrix  where enzymes i n v o l v e d i n o x i d a t i o n  p y r u v a t e t o a c e t y l CoA  are  i s metabolized  located.  v i a the  In  citric  the  matrix  space, acetyl-CoA  acid  cycle.  Enzymes i n t h e i n n e r membrane i n c l u d e t h o s e i n v o l v e d i n  t h e 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 p h o s p h o r y l a t i o n synthetase complex). membrane  and  membrane.  Vitamin  influences  E i s associated  the  degree  of  with  (ATP  the  unsaturation  inner of  F o r example, PUFA have been found t o be were l o w e r i n  h e p a t i c m i t o c h o n d r i a l membranes from d u c k l i n g s f e d a v i t a m i n deficient 1980).  d i e t compared  The  transport rigidity  to  a  control  diet  (Molenaar  et  Eal.  f a t t y a c i d c o m p o s i t i o n o f t h e membrane a f f e c t s t h e  o f compounds a c r o s s or f l u i d i t y  transport proteins.  t h e membrane by  o f t h e membrane and  is  being  reactions.  i n f l u e n c i n g the  the t r a n s l o c a t i o n of  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 a r e a because m i t o c h o n d r i a l oxygen  the  supplied  membranes a r e h i g h i n PUFA  to  mitochondria  and  for  oxidative  V i t a m i n E d e f i c i e n c y has been shown t o be  associated  w i t h i n c r e a s e d membrane f r a g i l i t y o f b o t h c e l l membranes such as erythrocytes  in  fish,  birds  and  mammals  and  mitochondrial  membranes i n b i r d s .  In  mammals,  tocopherone, 1980b) and  vitamin  hydrolyzed  to  E  radicals  tocopheryl  are  converted  quinone  reduced t o t o c o p h e r y l - p - h y d r o q u i n o n e .  to  (Gallo-Torres The  latter  30 compound i s c o n j u g a t e d  w i t h g l u c u r o n i c a c i d i n t h e l i v e r and  excreted i n the b i l e .  I n some c a s e s , t h e need f o r v i t a m i n E i n t h e membrane c a n be r e d u c e d by t h e p r e s e n c e o f s e l e n i u m w h i c h , t h r o u g h i t s a c t i o n i n glutathione peroxidase, the  products  i n the cytosol  peroxidative carotenoids  can l i m i t t h e f o r m a t i o n  process  going.  which  keep  There  i s also  o f some o f  the a u t o c a t a l y t i c evidence  that  a c t as a n t i o x i d a n t s (Tacon 1981) and t h e r e may be  interactions  between  vitamin  E  and c a r o t e n o i d s .  I f this  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 charr, addition of carotenoids to  the diet  Carotenoids  might  reduce  the requirement  f o r vitamin  E.  were n o t added t o t h e d i e t s i n t h i s e x p e r i m e n t 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 vitamin E alone.  2.6.4.3  Mobilization  and  requirement  of  vitamin  E  for  reproduction K i n g (1985) m o n i t o r e d t h e t e m p o r a l changes i n d i s t r i b u t i o n o f v i t a m i n E i n mature female rainbow t r o u t .  D u r i n g t h e month  o f June, f i s h were f e d a d i e t not supplemented w i t h v i t a m i n E. At  the beginning  storage  o f J u l y , she found  there  was  considerable  o f v i t a m i n E i n t h e l i v e r and t h i s c o n s t i t u t e d 45% o f  t h e t o t a l body s t o r a g e o f v i t a m i n E (25 mg).  Distribution of  v i t a m i n E was m o n i t o r e d i n f i s h f e d d i e t s supplemented o r n o t supplemented w i t h t h e v i t a m i n .  I n J u l y and A u g u s t , v i t a m i n E  was t r a n s p o r t e d from t h e l i v e r m a i n l y  t o t h e muscle w i t h some  31 transported  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 t h e muscle t o t h e o v a r y and a d i p o s e t i s s u e , and t h e n from t h e a d i p o s e t i s s u e t o t h e o v a r y between O c t o b e r and December. deposited marked  D u r i n g 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  i n t h e eggs from t h e o v a r i a n t i s s u e and t h e r e was  increase  i n the v i t a m i n  December, t h e r e was  E  a significant difference  c o n t e n t o f t h e eggs from f i s h  diet.  Fish  By  i n the v i t a m i n E  fed the v i t a m i n  d i e t compared t h e unsupplemented J a n u a r y 6 and 27.  c o n t e n t o f t h e eggs.  a  E-supplemented spawned between  A t spawning, f i s h f e d t h e unsupplemented  diet  and supplemented d i e t s , c o n t a i n e d a t o t a l body burden o f 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 t h e o v a r i a n t i s s u e 54.0%  respectively.  After  spawning, t h e body burden was reduced t o 11.5 and 18 mg  vitamin  E  and  49.0%  f o r those  of  this  fed the  respectively.  body  burden,  contained  unsupplemented  At t h i s r a t e ,  one  and  o r two  supplemented additional  diets, spawning  seasons would be r e q u i r e d t o reduce t h e body s t o r e s o f v i t a m i n E drastically.  Kinumaki  et  concentrations  al.  (1972)  found  that  the  vitamin  E  o f o v a r i a n t i s s u e i n c r e a s e d 4- t o 1 2 - f o l d when  r a i n b o w t r o u t were f e d d i e t s supplemented w i t h l a r g e amounts o f vitamins spawning.  A,  D  and  E  f o r 10-17  weeks p r i o r  A t spawning, v i t a m i n E was  t o the  start  of  found t o be d i s t r i b u t e d  m a i n l y i n t h e 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 o f v i t a m i n E p r e s e n t i n t h e o v a r i e s o f supplemented  fish  (57-  32 59%) compared t o t h e unsupplemented  fish  (39%).  In the  latter  group, 17% o f t h e v i t a m i n was s t o r e d i n t h e l i v e r compared t o 37% i n t h e supplemented  The  fish.  requirement of  factors.  fish  for vitamin  These i n c l u d e t h e s p e c i e s  depends on  and s t a g e o f  form o f t h e v i t a m i n , water t e m p e r a t u r e level  E  many  development,  (Cowey e t a l . 1984),  o f PUFA i n t h e d i e t (Watanabe e t a l . 1981a), p r e s e n c e o f  s e l e n i u m ( S c o t t e t al. such  as  1982)  ethoxyquin  trimethylquinoline).  This  and t h e p r e s e n c e o f a n t i o x i d a n t s (6-ethoxy-1,2-di-hydro-2,2,4 makes  i t difficult  to  establish  r e q u i r e m e n t s and compare t h e r e s u l t s o f e x p e r i m e n t s .  V i t a m i n E has been a s s o c i a t e d w i t h r e p r o d u c t i o n s i n c e Evans and B i s h o p (1922) d i s c o v e r e d t h a t v i t a m i n E p r e v e n t e d r e s o r p t i o n o f f e t u s e s i n pregnant r a t s . from t h e Greek " t o b e a r " and 1982). female  "tokos"  The word " t o c o p h e r o l " was  meaning " o f f s p r i n g " ,  "pherein"  " o l " t o denote t h e a l c o h o l group  The v i t a m i n p r o t e c t s t h e i n t e g r i t y reproductive  system  derived meaning  (Scott et  o f membranes i n t h e  o f some a n i m a l s and  the  vascular  s y s t e m o f t h e embryo i n t h e hen, t u r k e y , r a t , cow and ewe. also  prevents  seminiferous and  chicken.  male  sterility  through  al.  i t s effect  on  t u b u l e s o f t h e male r a t , g u i n e a p i g , hamster,  It the dog  33 Several  researchers  have  investigated  the  effects  d i e t a r y v i t a m i n E on t h e r e p r o d u c t i v e performance o f species  of f i s h .  For example, K i n g  different  (1985) f e d r a i n b o w 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 o r 90 mg v i t a m i n E a c e t a t e / k g 8%  lipid  followed spawning,  for by  different  periods  starvation for  there  concentration  was  of  a  eggs  unsupplemented d i e t (5.39  of  time  of  1 month p r i o r  significant only  up  to  those  6  months  spawning.  difference  between  to  in  vitamin  always  fed  ng/egg).  There was  At E the  significantly  g r e a t e r s u r v i v a l o f eyed eggs a f t e r s h o c k i n g i f t h e females been f e d t h e d i e t supplemented w i t h 90 mg v i t a m i n E 6 months.  with  ng/egg) compared t o t h o s e always f e d  t h e supplemented d i e t (7.37  for  of  O v e r a l l , h a t c h a b i l i t y was  had  acetate/kg  excellent with a l l  treatments  w i t h o n l y 11% m o r t a l i t y i n eggs from females f e d t h e  deficient  diet  for  supplemented d i e t .  6  months  S u r v i v a l was  and  2%  poorest  mortality  with  the  a t l e v e l s o f < 5 \iq  v i t a m i n E/egg and b e s t when eggs c o n t a i n e d > 7 ng v i t a m i n E/egg. S u r v i v a l was  p o o r e s t when v i t a m i n E c o n c e n t r a t i o n was  and  >  best  at  82  ng/g.  A  critical  period  maximum e f f e c t o f v i t a m i n E d e p r i v a t i o n on egg.  Fish  fed  the  unsupplemented  O c t o b e r produced eggs w i t h from  fish  fed  existed the  d i e t between  than October.  for  the  rainbow t r o u t  lower v i t a m i n E l e v e l s  that diet later  < 54 fxg/g  August  and  than those  Vitamin  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 t h e 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 vitamin E content  t o t h e eggs.  King  believed  t h a t t h e b r o o d s t o c k had s u f f i c i e n t body s t o r e s o f v i t a m i n E a t  34 the beginning any  o f t h e experiment t o s u p p l y  supplementation  of the diet.  the ovaries  without  F u r t h e r , she s t a t e d t h a t t h e  d i e t s would need t o be f e d f o r a l o n g e r p e r i o d o f t i m e t h e body r e s e r v e s for  there  o f v i t a m i n E would be d e p l e t e d  t o be a s i g n i f i c a n t  before  sufficiently  effect of dietary vitamin  E  c o 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 t i m e was d e l a y e d  one week and f i s h d i d n o t a l l  spawn s u c c e s s f u l l y i f they had been f e d t h e unsupplemented d i e t f o r 6 months.  Of t h e 8 f i s h i n t h i s t r e a t m e n t group, 3 spawned  s u c c e s s f u l l y , 3 d i d n o t r i p e n , 1 produced abnormal eggs and 1 d i e d p r i o r t o spawning. globules  The abnormal eggs had d i s p e r s e d  and c o n v o l u t e d  membranes.  supplemented group spawned n o r m a l l y .  A l lthe f i s h  lipid  i n the  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 w e i g h t o r diameter.  K i n u m a k i e t al. (1972) f e d p r a c t i c a l d i e t s t o r a i n b o w t r o u t broodstock.  They f e d d i e t s c o n t a i n i n g  v i t a m i n E/kg f o r 19 weeks.  66, 4600 and 8200 mg  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 ) , w h i c h c o n t a i n e d o n l y 10% o f t h e l e v e l s o f v i t a m i n s A and D compared t o t h e o t h e r 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 t h e eyed s t a g e  o f 51% compared t o 46 and 37% f o r t h e o t h e r d i e t s  respectively.  V i t a m i n E i n t h e eggs was 4.4 ng/egg (46 u,g/g)  f o r t h e c o n t r o l group compared t o 23 and 32 ixg (240 and 330 M-g/g) f o r t h e groups f e d d i e t s supplemented w i t h 4600 and 8200 mg v i t a m i n E/kg.  When t h e y o l k s a c had been a b s o r b e d , t h e f r y  35 contained  3.5,  13 and  114 and 166 ^g/g.  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 o r  27,  Thus, v i t a m i n E had been t r a n s f e r r e d from t h e  d i e t t o t h e egg and absorbed from t h e y o l k sac by t h e f r y .  The  m u l t i p l e d i f f e r e n c e s between t h e l e v e l s o f f a t - s o l u b l e v i t a m i n s in  the  d i e t s p r e v e n t making any  firm conclusions  about  this  research.  T a k e u c h i e t a l . (1981a) f e d ayu for  3 months p r i o r  t o spawning w i t h  (Plecoglossus  altivelis)  diets containing  graded  l e v e l s o f v i t a m i n E a c e t a t e p l u s endogenous v i t a m i n E ( t o t a l 2390 mg/kg). v i t a m i n E/kg) and  F e e d i n g unsupplemented d i e t s  (10  mg  10-  endogenous  d e c r e a s e d s u r v i v a l t o t h e eyed s t a g e , h a t c h a b i l i t y  s u r v i v a l of f r y immediately a f t e r hatching.  Spawning  was  d e l a y e d o r f a i l e d t o o c c u r i n o n e - t h i r d o f t h e female p o p u l a t i o n fed the  unsupplemented d i e t w h i l e  a l l fish  were f e d d i e t s c o n t a i n i n g more t h a n 34 mg  Watanabe and broodstock  t o t a l vitamin  Takashima (1977) f e d c a r p  semipurified diets containing  vitamin E acetate/kg  f o r 17 months.  spawned when t h e y E/kg.  (Cyprinus  e i t h e r 0 or  carpio) 700  They found t h a t t h e  f e d t h e unsupplemented d i e t had a v e r y low gonadosomatic (ovary weight/body weight). oocytes  lacking  large  though t h e o o c y t e s had and  v e s i c l e s should  yolk  Oocyte development was d e l a y e d granules  and  yolk  fish index with  v e s i c l e s even  reached a s i z e a t which t h e s e  have been d e p o s i t e d .  mg  granules  I t appeared  o o c y t e s had begun t o d e v e l o p and t h e n became a t r e t i c .  that  36 H i s t o l o g i c a l e x a m i n a t i o n of the p i t u i t a r y g l a n d  indicated  d e c r e a s e d s e c r e t o r y a c t i v i t y o f t h e gonadotrophs, w h i c h produce FSH  and LH,  and  hormone, and also  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  the  reduced  epithelial  in  height.  cells  i n the  Spawning  was  t h y r o i d t i s s u e were probably  delayed  or  p r e v e n t e d i n a l a r g e r p o r t i o n o f t h e p o p u l a t i o n as i t was t h e ayu In  (Takeuchi  addition  composition  to  e t al.  for  1981a) and r a i n b o w t r o u t ( K i n g 1985).  endocrine  gland  dysfunction,  the  fatty  acid  o f t h e c a r p o v a r i a n t i s s u e resembled t h a t o f  fish  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 increased  l e v e l s o f 18:ln9 and  20:4n6, 20:5n3 and  22:6n3.  f a t t y a c i d composition,  20:3n9 and  I f the eggs had  been a n a l y z e d  for  t h e y p r o b a b l y 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 . (1977) p o s t u l a t e d  decreased l e v e l s of  that  the  Watanabe and Takashima  ct-tocopherol  d e f i c i e n c y caused  a  r e d u c t i o n o f 18:2n6, one o f t h e e s s e n t i a l f a t t y a c i d s f o r c a r p , and t h a t t h i s c r e a t e d 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 e t al.  (1991) e v a l u a t e d  the i n f l u e n c e of  E a c e t a t e c o n c e n t r a t i o n on r e p r o d u c t i o n  i n r e d seabream.  were f e d a c o n t r o l d i e t c o n t a i n i n g 500 mg f o r 90 days and  eggs,  hatching  vitamin E  Fish  acetate/kg  s u b s e q u e n t l y t h e y were f e d e i t h e r t h e c o n t r o l  d i e t o r a d i e t c o n t a i n i n g 2000 mg days p r i o r t o and  vitamin  vitamin E acetate/kg  30 days a f t e r spawning. success  and  i n f l u e n c e d by d i e t a r y t r e a t m e n t .  normal  The  larvae  Production  for  10  r a t e o f buoyant were  directly  o f normal  larvae  37 i n c r e a s e d from 35% t o 76% f o r seabream f e d t h e d i e t c o n t a i n i n g 2000 mg v i t a m i n E a c e t a t e / k g .  At  the time  commercial  that  broodstock  supplemented  with  diet,  two were  while  Although there  the current diets  300-500 mg  experiment  available  in  began, Canada  (IU) d l - c c - t o c o p h e r y l  supplemented  with  e t a l . (1973) showed t h a t c h i c k s  rates  and s k e l e t a l  mitochondrial  were  acetate/kg  200 and 800 mg/kg.  a r e no r e p o r t s on h y p e r v i t a m i n o s i s  March  most  E i n fish,  had d e c r e a s e d  respiration  growth  r a t e s when t h e y  were f e d 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. T h i s l e d NRC (1987) t o suggest a " t e n t a t i v e presumed s a f e use l e v e l o f 75 IU/kg o f BW/day".  38 MATERIALS AND METHODS  3.1 L o c a t i o n and t i m e The  fish  rearing  and  egg  incubation  portions  of  this  r e s e a r c h were conducted a t Rockwood A q u a c u l t u r e R e s e a r c h C e n t r e (RARC) l o c a t e d Manitoba.  30 km n o r t h o f Winnipeg  (49°54' N, 97°8'  B i o c h e m i c a l a n a l y s e s and a l l o t h e r r e l a t e d  ( e x c e p t as noted) were conducted  a t the Freshwater  W),  activities Institute  S c i e n c e L a b o r a t o r y (FWISL), Winnipeg, M a n i t o b a .  Throughout t h i s t h e s i s , Year 1 r e f e r s t o t h e p e r i o d  from  June 27, 1988 when t h e f i s h were weighed and d i s t r i b u t e d t o t h e t a n k s u n t i l J a n u a r y 11, 1989 one week a f t e r t h e f i r s t p e r i o d was completed.  spawning  Year 2 r e f e r s t o t h e p e r i o d from J a n u a r y  12, 1989 u n t i l J a n u a r y 3, 1990 when t h e second spawning  period  was c o m p l e t e d .  3.2 D i e t s 3.2.1 F o r m u l a t i o n Four d i e t s were f o r m u l a t e d t o c o n t a i n two l e v e l s o f l i p i d (12 and 19%) a t each o f two l e v e l s o f d l - c t - t o c o p h e r y l a c e t a t e ( v i t a m i n E a c e t a t e ) (30 and 600 mg/kg). p r e s e n t e d i n T a b l e 1.  The f o r m u l a t i o n s a r e  S i n c e b r o o d s t o c k used i n t h i s r e s e a r c h  were v a l u a b l e , d i e t s were f o r m u l a t e d t h a t were n o t 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 t h e s u r v i v a l o f t h e b r o o d s t o c k o r t h e i r fecundity.  The lower l e v e l o f 30 mg v i t a m i n E a c e t a t e / k g  39 T a b l e 1. F o r m u l a t i o n e x c e p t as n o t e d ) .  of experimental  diets  (g/kg  dry diet,  Diet LLLE  LLHE  HLLE  HLHE  395  395  395  395  195  195  195  195  86  86  86  86  183  183  183  183  44  44  114  114  Alpha-cellulose  70  70  -  -  Vitamin  premix  6  10  10  10  10  Mineral premix  7  10  10  10  10  H e r r i n g m e a l , steam-dried ' (CP=78%, TL=12%) Soybean m e a l (CP=47%, TL=2.5%) Corn g l u t e n m e a l (CP=68%, TL=8%) Whole wheat, ground (CP=16%, TL=4%) Herring o i l 2  3  COMM  1  4  2  2  2  5  Choline c h l o r i d e Ascorbic acid  (60%)  (crystalline)  6  6  6  6  1  1  1  1  d l - c t - T o c o p h e r y l acetate (mg/kg)  30  600  30  600  Ethoxyquin  35  35  -  -  8  1  2 3 4 5 6  7  (mg/kg)  Commercial r a i n b o w t r o u t grower d i e t c o n t a i n e d a l l t h e 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 p l u s blood meal, p o u l t r y meal, whey, brewers y e a s t , l i m e s t o n e , s a l t and m e t h i o n i n e . COMM c o n t a i n e d wheat s h o r t s and m i d d l i n g s b u t no whole wheat or i n o s i t o l . F o u r i n g r e d i e n t s combined t o form a b a s a l m i x t u r e . S t a b i l i z e d w i t h e t h o x y q u i n a t 250 mg/kg. CP=Crude p r o t e i n , TL= T o t a l l i p i d S t a b i l i z e d w i t h e t h o x y q u i n a t 500 mg/kg. V i t a m i n premix s u p p l i e d t h e f o l l o w i n g i n mg o r I.U./kg d r y d i e t : v i t a m i n A a c e t a t e ( 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; t h i a m i n m o n o n i t r a t e , 30; r i b o f l a v i n , 60; c a l c i u m p a n t o t h e n a t e , 120; n i a c i n a m i d e , 200; f o l i c a c i d , 15; b i o t i n , 3; p y r i d o x i n e h y d r o c h l o r i d e , 30; c y a n o c o b a l a m i n , 0.06; i n o s i t o l , 400. M i n e r a l premix s u p p l i e d t h e f o l l o w i n g i n mg element/kg d r y d i e t : Mn (as MnS0 .H 0), 86; Fe (as FeS0 .7H 0), 63; Cu (as CuS0 .5H 0), 25; Zn (as ZnS0 .7H 0), 144; I (as K I 0 ) , 8. S u p p l i e d c o u r t e s y o f Hoffmann-La Roche, E t o b i c o k e , O n t a r i o . 4  4  8  2  2  4  4  2  2  3  40 1981).  The 600 mg v i t a m i n E a c e t a t e / k g c o n c e n t r a t i o n was chosen  because i t was above t h e range added t o most s a l m o n i d diets  (300-500 mg/kg).  broodstock  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 o f 10% was below t h e growth r e q u i r e m e n t s f o r Nauyuk  Lake  Arctic  charr  fingerlings  while  dietary  concentrations  o f 15-20% r e s u l t e d i n s i g n i f i c a n t  weight g a i n .  Lipid  concentrations  lipid  increases i n  o f 10-11% and 17-18% were  chosen b u t upon a n a l y s i s t h e d i e t s c o n t a i n e d 12% and 19% l i p i d . Throughout t h i s t h e s i s , t h e f o u r 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 H i g h 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 H i g h v i t a m i n E ) . The h i g h e r l i p i d l e v e l was a c h i e v e d by r e p l a c i n g a l p h a - c e l l u l o s e in  t h e low l i p i d  with  diets with additional herring o i l s t a b i l i z e d  0.05% e t h o x y q u i n  quinoline).  (1,2-dihydro-6-ethoxy-2,2,4 t r i m e t h y l -  To e q u a l i z e t h e c o n c e n t r a t i o n o f e t h o x y q u i n  d i e t s , a d d i t i o n a l e t h o x y q u i n was added t o t h e l o w l i p i d  i n the diets.  I n a d d i t i o n t o t h e above d i e t s , a c o m m e r c i a l r a i n b o w t r o u t grower d i e t reference supplier  (denoted as COMM) was f e d t o one t a n k o f f i s h as a diet.  The  broodstock  diet  manufactured  by t h e  ( M a r t i n Feed M i l l s , E l m i r a , O n t a r i o ) was a minimum o f  6 mm i n d i a m e t e r .  The wide v a r i a t i o n i n t h e s i z e o f c h a r r i n  t h e p o p u l a t i o n r e s u l t e d i n t h e 6 mm d i e t b e i n g t o o l a r g e f o r a portion  of the population  t o consume.  Therefore,  t h e 5 mm  grower d i e t had been used r o u t i n e l y a t RARC u n t i l a l l t h e f i s h were l a r g e enough t o consume t h e 6 mm b r o o d s t o c k  diet.  41 3.2.2  Manufacturing Soybean meal and whole wheat were ground i n a F i t z m i l l  were ground f i n e l y enough t o pass t h r o u g h an 18 U.S. (1.00  mm)  screen  and  (0.60  m e a l , ground  f o r 60% mm).  A b a s a l mix  was  o u t e r brown paper bag. coffee  grinder  mesh s c r e e n  i t t o pass t h r o u g h a 30  soybean meal and  v e r t i c a l m i x e r and  a  of  placed  U.S.  o f h e r r i n g meal, c o r n whole wheat was  mesh gluten  prepared  i n double p l a s t i c  and  in a  bags w i t h  an  M i n e r a l s were ground t o a f i n e powder i n  prior  to  weighing.  Both  the  mineral  and  v i t a m i n premixes were p r e p a r e d 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  in a  Hobart m i x e r  H e r r i n g o i l was  and  stored  i n double p l a s t i c  p l a c e d i n t h r e e 20 L p l a s t i c c o n t a i n e r s .  t h e above i n g r e d i e n t s and mixes were p r e p a r e d from  Vancouver  arrival  bags.  to  Winnipeg  i n Winnipeg,  the  under  basal  frozen  mix,  a t UBC  and  shipped  conditions.  ascorbic  acid,  All  Upon choline  c h l o r i d e m i x t u r e , v i t a m i n and m i n e r a l premixes were f r o z e n a t 35°C u n t i l r e q u i r e d f o r m a n u f a c t u r i n g year p e r i o d of the experiment. stored  in a  temperature. ethoxyquin  refrigerator  while  H e r r i n g o i l was  a t 500  mg/kg (NRC  the d i e t s d u r i n g the  two  The d l - a - t o c o p h e r y l a c e t a t e  was  ethoxyquin  was  kept  at  room  s t a b i l i z e d w i t h the a d d i t i o n of 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 t h e c o u r s e t h e e x p e r i m e n t a t 9-13  wk i n t e r v a l s (mean = 11 wk).  of  Ingredients  were weighed and mixed each t i m e the d i e t s were m a n u f a c t u r e d .  42 The c o m m e r c i a l d i e t (COMM) was two shipments i n Year 2. uniform l i q u i d water  bath  nitrogen returning  Since the h e r r i n g o i l d i d not reach a  s t a t e when thawed a t room t e m p e r a t u r e , a warm  (37°C) was  was  from one shipment i n Y e a r 1 and  used  bubbled  into  to liquefy the  i t t o the f r e e z e r .  the o i l .  o i l i n the  After  container  a c e t a t e and  before  Each t i m e t h e f o u r d i e t s  made, a l l t h e o i l used was o n l y from one c o n t a i n e r . tocopheryl  use,  were  Both d l - a -  e t h o x y q u i n (when r e q u i r e d ) 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 ingredients.  D i e t s were c o l d p e l l e t e d  t h r o u g h a 5 mm d i e i n a Superior-Templewood at  t h e Department  pellet mill  of Animal Science, U n i v e r s i t y  located  of Manitoba.  D i s t i l l e d w a t e r was added a t 6-7% t o a i d i n p e l l e t i n g .  Pellets  were s p r e a d o u t i n a t h i n l a y e r on waxed paper and a l l o w e d t o a i r d r y f o r 4-5 l e s s t h a n 9%.  hours a t which t i m e t h e m o i s t u r e c o n t e n t  was  The d i e t s were s i e v e d u s i n g a Kason s e p a r a t o r t o  pass o v e r a No.  12 U.S.  s i e v e (1.7 mm).  D i e t s were p l a c e d i n  d o u b l e p l a s t i c bags and an o u t e r brown paper bag, t r a n s p o r t e d t o Rockwood A q u a c u l t u r e Research C e n t r e and s t o r e d a t -10°C.  Herring  meal  S t e v e s t o n , B.C.  and  o i l was  purchased  from  B.C.  Packers,  A p p r o x i m a t e l y one month p r i o r t o t h i s p u r c h a s e ,  DFO's West Vancouver L a b o r a t o r y purchased h e r r i n g o i l from t h e same s u p p l i e r .  A sample  s t a b i l i z e d w i t h 0.05%  o f o i l from t h i s  first  shipment  e t h o x y q u i n and p l a c e d i n two  w h i c h were f l u s h e d w i t h n i t r o g e n .  was  containers  One c o n t a i n e r was p l a c e d i n  43 t h e r e f r i g e r a t o r and t h e o t h e r c o n t a i n e r was p l a c e d i n a f r e e z e r a t -15°C.  The f o l l o w i n g day, t h e o i l and a sample o f b a s a l mix  were s h i p p e d on i c e t o Hoffmann-La Roche, E t o b i c o k e , O n t a r i o f o r a n a l y s i s of a-tocopherol.  Samples o f h e r r i n g o i l used i n t h i s  e x p e r i m e n t were a l s o s h i p p e d t o Hoffmann-LaRoche but t h e y were not  analyzed.  analyzed  The  herring  f o r peroxide  value  o i l used each  i n this  time  the  experiment diets  was  were made  (Woyewoda e t a l . 1986).  3.3  Parental fish  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  F r a s e r R i v e r i n L a b r a d o r (56°39'N, 63°10'W).  the  C h a r r were spawned  a t t h e 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 W i n n i p e g , Manitoba and i n c u b a t e d i n a q u a r a n t i n e a r e a a t t h e 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  of c e r t i f i a b l e Rockwood  free  d i s e a s e s , 1070 eyed eggs were t r a n s f e r r e d t o t h e (RARC)  broodstock  b u i l d i n g w h i c h 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  fish  Aquaculture  was  reduced  Research  Centre's  over t h e y e a r s  through  culling,  m o r t a l i t y and s a m p l i n g o f f i s h f o r c e r t i f i c a t i o n year.  natural  t e s t i n g once a  F i s h spawned f o r t h e f i r s t time as 3 y e a r o l d s i n t h e  f a l l o f 1987 w i t h 143 females p r o d u c i n g eggs out o f t h e 476 male and female f i s h p r e s e n t a t t h a t t i m e . that  had  not matured were m a i n t a i n e d  An unknown number o f f i s h s e p a r a t e l y because  they  l o o k e d l i k e " p o t e n t i a l spawners" ( O l s o n , p e r s o n a l communication)  44 with  48  females  f o l l o w i n g year.  and  95  males  i n that  group  remaining  By t h e t i m e t h i s experiment began i n June  the 1988,  353 o f t h e o r i g i n a l p o p u l a t i o n remained w i t h an e s t i m a t e d 165 f e m a l e s , 134 males and 54 emaciated f i s h o f unknown s e x .  3.3.2  S e l e c t i o n o f f i s h f o r t h e experiment The  majority  of  charr  used  in  this  experiment  were  s e l e c t e d from t h o s e t h a t had spawned t h e p r e v i o u s y e a r w i t h females  and  10 males  used  f o r each  of the  15  f i v e treatments.  E m a c i a t e d f i s h o r t h o s e w i t h g r o s s a b n o r m a l i t i e s were n o t used in  this  experiment.  F i s h were a n a e s t h e t i z e d  using  p h e n o x y e t h a n o l and tagged w i t h numbered F l o y t a g s .  0.03%  2-  Length  was  measured t o t h e n e a r e s t m i l l i m e t e r and w e i g h t was t a k e n t o t h e n e a r e s t gram u s i n g a M e t t l e r PE12  zero-tare balance.  The  wide  range i n t h e w e i g h t o f t h e f i s h (345-2190 g) made i t i m p o s s i b l e to  distribute  manner.  The  the total  fish  t o the  5 tanks  in a  and mean w e i g h t o f f i s h  totally  random  i n each t a n k  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 o r 2 s m a l l f i s h were r e p l a c e d by 1 or 2 large f i s h .  T h e r e f o r e , t h e t a g number, s e x , l e n g t h and  weight  fish  of  recorded.  a l l the  considered f o r the  experiment  were  F i s h were d i s t r i b u t e d t o each t a n k i n a manner t h a t  p r o v i d e d each t a n k w i t h t h e same weight d i s t r i b u t i o n o f 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 o f t h e f i v e d i e t s was  F i v e independent  a s s i g n e d t o one  tank of  fish.  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 o f a  1500 L l i g h t b l u e 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 o v e r 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 ) d i a m e t e r ] c o n t a i n i n g 0.73  m  rates  tank  to  each  Recirculated  g r a n i t e g r a v e l (Papst and Hopky 1982).  3  water  averaged entered  provide aeration.  105  the  L/min  tank  F r e s h w e l l water  (range  through  =  a  (6-7°C) was  Flow  101-112).  spray  bar  to  a l s o added as  makeup t o r e p l a c e a p o r t i o n o f t h e water b e i n g d i s c a r d e d .  The  f l o w r a t e o f makeup water was  was  a p p r o x i m a t e l y 6.5-7  a d j u s t e d t o a c h i e v e t h e d e s i r e d temperature temperature  between t a n k s .  2 weeks d u r i n g t h e  L/min and  and t o e q u a l i z e t h e  W h i l e f i l t e r s were backwashed e v e r y  f e e d i n g p e r i o d , i t was  not  necessary  to  backwash t h e f i l t e r s d u r i n g t h e 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 Lighting  photoperiod  f o r each  tank  was  provided  by  f l u o r e s c e n t b u l b s p l a c e d 1.2 m above t h e water. was  165  2  cool  Light intensity  l u x when measured w i t h a M i n o l t a Autometer  converted  from  electron  microeinsteins/m /sec 2  photometer.  The  when  volts  into  measured  photoperiod  was  lux. with  adjusted  It  a  white  was  Lambda  every  I I I and  10  2.88  quantum days  c o n f o r m t o t h e Winnipeg (49°54' N, 97°8' W) p h o t o p e r i o d .  to  This  46 was  s i m i l a r t o t h e p h o t o p e r i o d i n Goose Bay, L a b r a d o r ( O l s o n ,  p e r s o n a l communication) w h i c h i s c l o s e t o t h e spawning  grounds  o f t h e p a r e n t a l c h a r r (56°39'N, 63°10'W).  3.4.3  Temperature On t h e n i n t h day o f f e e d i n g i n Year 1 ( J u l y 6, 1988), i t  was  found  tanks.  that  t h e water  temperatures  differed  i n the  A t t h a t t i m e , t h e t e m p e r a t u r e s were 7.2°C ( L L L E ) , 9.7°C  (LLHE), 10.2°C (HLLE), 7.2°C (HLHE), 12.2°C (COMM).  On J u l y  t e m p e r a t u r e d i f f e r e n c e s between t a n k s had been reduced t o or l e s s .  five  7,  1.5°C  V a r i a t i o n i n t e m p e r a t u r e was due t o d i f f e r e n c e s i n t h e  amount o f 7°C makeup water b e i n g added t o each t a n k . w a t e r e n t e r e d t h e t a n k below t h e w a t e r l i n e a d j u s t e d because t h e f l o w v a l v e s were f a u l t y .  The makeup  and c o u l d not  be  Changes t o t h e  makeup l i n e s were made so t h a t t h e water e n t e r e d t h e t a n k above t h e s u r f a c e o f t h e water and a new c o n t r o l v a l v e was f o r each t a n k . all  installed  When t h i s was c o r r e c t e d , l e n g t h s and w e i g h t s o f  f i s h were t a k e n a g a i n on J u l y 11, 1988.  t e m p e r a t u r e o f water  i n each t a n k was  u s i n g a F i s h e r t e m p e r a t u r e meter.  Thereafter,  measured each  the  morning  A d j u s t m e n t s t o t h e amount o f  makeup w a t e r were made t o e q u a l i z e w a t e r t e m p e r a t u r e s i n t h e f i v e tanks.  The amount o f makeup water was a d j u s t e d t o t r y and  match t h e w a t e r t e m p e r a t u r e i n Year 2 t o t h a t o f Year 1.  47 3.4.4 Water c h e m i s t r y In Year 1, w a t e r samples were t a k e n from each t a n k on J u l y 13  and September  22.  After  t h e spawning  season  ended  and  f e e d i n g resumed, samples were t a k e n on F e b r u a r y 6, March 2 1 , 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 a n a l y z e d f o r ammonium-N,  n i t r i t e - N and n i t r a t e - N a c c o r d i n g t o t h e methods o f S t a i n t o n e t al.  (1974).  Temperature and d i s s o l v e d oxygen were measured a t  the  same t i m e u s i n g a Y S I - t e m p e r a t u r e oxygen meter.  Un-ionized  ammonia was c a l c u l a t e d u s i n g t h e t a b l e s o f P i p e r (1982) and t h e following equation: U n - i o n i z e d ammonia (u,g/L) = u n - i o n i z e d ammonia (%) x NH„-N (uq/L) 100 3.5 F e e d i n g regime There appeared t o be an e f f e c t o f l i p i d l e v e l on t h e l e n g t h of  t h e spawning p e r i o d i n Year 1.  To e v a l u a t e t h i s f u r t h e r , a  c r o s s o v e r i n d i e t s was c a r r i e d o u t i n Year 2 ( F i g u r e 3 ) . The d i e t a r y v i t a m i n E content of the d i e t f e d t o a given tank of f i s h was t h e same i n b o t h Year 1 and Year 2.  However, f i s h f e d  a l o w l i p i d d i e t i n Year 1 were f e d a h i g h l i p i d d i e t i n Year 2 and t h o s e f e d a h i g h l i p i d d i e t i n Year 1 were f e d a l o w 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 e x p e c t e d t h a t t h e 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 f e e d i n g 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  f i s h d i d not  consume t h e same amount each day.  because  F i s h consumed  much more on t h e f i r s t day t h a n t h e y d i d on t h e second o r t h i r d day  of  feeding  to satiation.  feeding t o s a t i a t i o n  A  t w i c e a day  regime  was  established  f o r 3 c o n s e c u t i v e days  of and  u s i n g t h e mean as t h e d a i l y r a t i o n t o f e e d f o r t h e n e x t 18 o r 25 days.  Feed was  weighed  f o r an 11 day  and one  o r two  7  periods w i t h the l a t t e r portions stored i n the f r e e z e r at until  required.  The  weight of feed  t o be  f e d each  day -10°C  day  was  c o n v e r t e d t o a volume p e r day f o r t h e c o n v e n i e n c e o f h a t c h e r y staff.  Hatchery s t a f f  offered this  f e e d i n g s t h r o u g h o u t t h e day.  amount o f f e e d o v e r  3-4  I f i t was not p o s s i b l e t o f e e d t h e  p r e s c r i b e d amount o f f e e d , t h e f e e d r e m a i n i n g a t t h e end o f each p e r i o d was weighed and net f e e d f e d was c a l c u l a t e d .  I n Year 2,  t h e r e seemed t o be 10% o f t h e f e e d r e m a i n i n g a t t h e end o f each period. the  T h e r e f o r e , t h e d a i l y amount o f f e r e d was  s e t a t 90% o f  mean o f t h e 3 day s a t i a t i o n amount.  In  Year  1,  September 11, 1988.  fish  were  fed  between  June  28,  1988  and  Feed consumption d e c r e a s e d as spawning t i m e  approached and f e e d was not o f f e r e d from September  12, 1988 t o  January  feeding  11,  1989.  When spawning  was  complete,  was  resumed on J a n u a r y 12, 1989 and was o f f e r e d u n t i l t h e end o f t h e experiment.  F i s h were not f e d on t h e day p r i o r t o , o r on t h e  day o f w e i g h i n g .  Feed was  o f f e r e d f o r a t o t a l o f 71 days i n  Year 1 o f t h e e x p e r i m e n t and 252 days p r i o r t o t h e f i r s t  fish  49  Year 1  Year 2  50 spawning i n Year 2.  I n Year 2, f e e d was o f f e r e d t h r o u g h o u t t h e  spawning p e r i o d on t h e 2 days f o l l o w i n g  spawning.  3.6 Growth o f b r o o d s t o c k Fish  were  anaesthetized  with  2-phenoxyethanol  prior  taking  l e n g t h and weight measurements o f i n d i v i d u a l l y  fish.  Data were c o l l e c t e d  on t h e f o l l o w i n g  to  tagged  d a t e s : June  27,  1988, J u l y 8, 1988, August 10, 1988, September 14, 1988, J a n u a r y 11, 1989, A p r i l 5, 1989, June 28, 1989, September 13, 1989 J a n u a r y 3, 1990.  and  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  calculated. S p e c i f i c growth r a t e = log  Q  f i n a l w e i g h t (q) - l o q i n i t i a l wt (g) x 100 time (days)  Condition factor =  Q  w e i g h t (g) length (cm)  x 100  3  3.7  Spawning K r i e g e r e t al.  (1988) found t h a t A r c t i c c h a r r eggs o v e r -  r i p e n e d 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 t h e f e r t i l i z a t i o n  rate  declined.  were  checked  T h e r e f o r e once spawning  a t weekly  intervals.  0.03% 2-phenoxyethanol. of  each f i s h was noted.  commenced, a l l f i s h  C h a r r were a n a e s t h e t i z e d  using  The t a g number and s t a g e o f development Males were judged as Not R i p e o r R i p e  w h i l e f e m a l e s were judged as No Development, W e l l Developed, R i p e o r Spawned.  Some  Development,  As f i s h were c h e c k e d , 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 w h i c h w o u l d r e l e a s e m i l t (Ripe) were s e p a r a t e d fish  in  a  procedure  tank was  anaesthetized anaesthetic.  had  been  carried and The  checked,  out.  rinsed  from t h e r e s t .  in  t a g number was  the  A f t e r a l l the  following  Each  ripe  fresh  water  female to  spawning fish  was  remove  any  noted and t h e f i s h was  weighed  t o t h e n e a r e s t gram and f o r k l e n g t h measured t o t h e n e a r e s t  mm.  A f t e r d r y i n g t h e 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 , t h e eggs were r e l e a s e d m a n u a l l y , c o l l e c t e d and d r a i n e d o f o v a r i a n f l u i d i n 1 o r 2 p l a s t i c p r e - t a r e d c o n t a i n e r s w i t h aluminum s c r e e n bottoms. O v a r i a n f l u i d was of drained  eggs was  sample o f eggs was 3.11  (Figure 4).  the ovarian rinse  was  c o l l e c t e d i n a p l a s t i c pan. measured t o t h e n e a r e s t  The t o t a l w e i g h t gram.  A  50-60 g  t a k e n and p r o c e s s e d as d e s c r i b e d i n S e c t i o n A r i n s e o f 1.37%  f l u i d and evaluated  remaining as  NaHC0  eggs and  described  in  abnormalities  ( d i s c o l o u r e d eggs o r b l o o d  were n o t e d .  Two  male f i s h  (250 ml) was  3  added t o  then decanted. Section  3.13.1.  i n the o v a r i a n  were a n a e s t h e t i z e d  This  and  Any fluid)  rinsed i n  f r e s h w a t e r b e f o r e n o t i n g t h e i r t a g numbers and m e a s u r i n g t h e i r lengths before  and  weights.  The  v e n t r a l a r e a o f each f i s h was  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. of  contents  o f t h e beakers combined and added t o t h e eggs. the  f l u i d was  divided  sperm t h r o u g h t h e  between  the  eggs g e n t l y  two  A 125  aliquot  mixing  r i n s e was  clear.  beakers,  ml the  After  f o r 2 minutes,  poured o f f and 6-7°C w e l l water was  eggs u n t i l t h e f l u i d was  dried  the  used t o r i n s e the  A sample o f 50 eggs was  taken  52 ANALYSES OF EGGS 50-60 g sample o f eggs  1  1  1  10-15 g stored a t -35°(  10-15 g stored a t -115°C  30 eggs weighed i n d i v i d . 1 30 g s t o r e d a t -35°C  1  1  freeze d r i e d f o r d r y matter d e t e r m i n a t i o n and ground up  Y r 1 and Y r  1  Yr 2  Vitamin E extracted  I  Y r 1 and Y r 2 _ i  1  TL e x t r a c t e d  1  1  assay  I  NL and PL s e p a r a t e d by column chromatography  1  1  1  TL e x t r a c t i o n  1  1  Selenium assay  assay  1  PL  NL _ i _  1 11  a s s a y TLC FAMES  1  1  _1_ 1 11  a s s a y TLC FAMES  1  GLC  GLC  ANALYSIS OF FRY fry  (Yr 2 only)  1  s t o r e d a t -115°C  1  Vitamin E extracted  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 m e t h y l e s t e r s GLC = g a s - l i q u i d chromatography  A l l samples o f eggs and f r y were f l u s h e d w i t h n i t r o g e n p r i o r t o being frozen. F i g u r e 4. Flow diagram showing a n a l y s e s c o n d u c t e d on eggs and f r y i n Y e a r 1 and/or Year 2.  53 to  assess  fertilization  Approximately  rate  as  described  i n Section  3.8.  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 p l a c e d  i n t h e j a r s e c t i o n o f an  i n c u b a t o r ( d e s c r i b e d i n S e c t i o n 3.9). J a r s were p l a c e d i n a pan o f 6-7°C w e l l water and covered hours  the  jars  containing  the  t r a n s f e r r e d from t h e b r o o d s t o c k and  placed  into  f e c u n d i t y was  t h e lower  with black p l a s t i c . water-hardened  After 2 eggs  were  b u i l d i n g t o t h e main b u i l d i n g  sections  of the incubators.  If  low, h a l f t h e numbers o f eggs were sampled and  h a l f t h e amount o f r i n s e and e x t e n d e r was u s e d . 2, r e p l i c a t e groups o f eggs were i n c u b a t e d  I n Y e a r s 1 and  from two and t h r e e  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 t h e e x p e r i m e n t were i n c u b a t e d  i n Heath t r a y s w i t h i n t h e b r o o d s t o c k  building.  I n Year 2, Heath t r a y s were c o m p a r t m e n t a l i z e d and a c o m p a r i s o n between s u r v i v a l t o t h e eyed stage when i n c u b a t e d i n Heath t r a y s versus  j a r i n c u b a t o r s was made.  Eggs were n o t a l l r e l e a s e d on  t h e day t h e f i s h spawned s i n c e some eggs may n o t have o v u l a t e d and/or t h e y remained i n t h e o v a r i a n c a v i t y . To a s s e s s t h e t o t a l number o f eggs produced, f i s h which had spawned were i n s p e c t e d d u r i n g subsequent spawning checks and eggs were c o l l e c t e d and counted. were  On t h r e e o c c a s i o n s  released  during  a  i n Year 1, l a r g e numbers o f eggs  subsequent  check.  These  eggs  were  f e r t i l i z e d and i n c u b a t e d and d a t a was used f o r t h e t i m e when t h e h i g h e s t number o f eggs were r e 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 o f w a t e r - h a r d e n e d eggs A sample o f 50 eggs (25 eggs i f t h e f e c u n d i t y was v e r y low) were  placed  carton  i n a compartment  of a perforated  ( A s h t o n , p e r s o n a l communication).  s t y r o f o a m egg  The egg c a r t o n was  w e i g h t e d down and p l a c e d i n t h e t o p t r a y o f a Heath t r a y rate  8 L/min) and was c o v e r e d w i t h b l a c k p l a s t i c .  (flow  A f t e r 26  hours (Year 1) o r 48 hours (Year 2 ) , t h e eggs were removed from the  egg c a r t o n , d r a i n e d 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 S t o c k a r d ' s s o l u t i o n ( V e l s e n 1980).  Eggs were measured a f t e r  b e i n g i n S t o c k a r d ' 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 a s s e s s f e r t i l i z a t i o n , t h e  eggs were v i e w e d under a Z e i s s low power m i c r o s c o p e . division  had o c c u r r e d , t h e egg was c o n s i d e r e d n o t f e r t i l i z e d .  Eggs were  drained  on a paper t o w e l  and t h e w e i g h t and  d i a m e t e r o f 25 i n d i v i d u a l eggs were d e t e r m i n e d . taken  I f no c e l l  t o 0.0001 g on a M e t t l e r  determined  The w e i g h t was  AE160 and t h e d i a m e t e r was  by measuring under a Bausch and Lomb  microscope  f i t t e d w i t h a micrometer. % Fertilization  = no. f e r t i l i z e d eggs no. f e r t i l i z e d + no. u n f e r t i l i z e d  x 100 eggs  3.9 I n c u b a t i o n o f eggs Eggs upwelling (1993).  from  each  mating  were  incubated  separately  in  1 L j a r i n c u b a t o r s as d e s c r i b e d by Tabachek e t a l . Each l o t o f eggs was p l a c e d i n an upper s e c t i o n made o f  ABS p i p e w i t h an aluminum s c r e e n bottom.  The l o w e r s e c t i o n o f  55 each i n c u b a t o r bubbles.  contained  aquarium g r a v e l t o d i s p e r s e  any  Each t r o u g h o f i n c u b a t o r s r e c e i v e d a e r a t e d w e l l w a t e r  a t 6-7°C w h i c h passed t h r o u g h a household d i r t and r u s t The  air  filters  were  r e p l a c e d a f t e r 5-10  cleaned  every  cleanings.  5-10  days  as  filter.  required  I n Year 1, eggs were  and  incubated  i n f o u r t r o u g h s each c o n t a i n i n g 24 i n c u b a t o r s w i t h a t o t a l r a t e o f 14.6  L/min (608 ml/min per i n c u b a t o r ) .  flow  I n Year 2, eggs  were i n c u b a t e d 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 total  flow r a t e of  Between 10  15.1  L/min  (470  days a f t e r spawning and  ml/min p e r  t h e eyed s t a g e ,  t r e a t e d w i t h f o r m a l i n (1/600 o r 1667 ppm)  were covered  8.2  and  10.1  shocked eggs. and  and  Y e l l o w f l u o r e s c e n t l i g h t was  A f t e r reaching incubated  Fuze e t a l . 1985).  l i g h t o n l y d u r i n g removal o f  microeinsteins/mVsec  respectively.  one  the  in eyed  more day  Year stage,  before  1  f r y were removed and period.  Dead  the  Year  preserved  2,  eggs were  removing t h e  counted t h r o u g h o u t t h e eggs were  350 l u x o r  and  Dead and l i v e eggs were counted a t t h i s t i m e .  incubation  times  w i t h b l a c k 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 i n c a n d e s c e n t dead eggs and c o u n t i n g .  eggs were  f o r 15 min t h r e e  a week t o c o n t r o l fungus growth (Hynes 1984, Incubators  incubator).  dead  Dead eggs  r e s t of  in  the  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.  I n Year 2, r e c o r d s were a l s o kept on the numbers o f deformed f r y and  f r y with blue  sac,  a c o n d i t i o n i n which a b l u i s h  a c c u m u l a t e s between the y o l k and (van D u i j n 1973,  Balon  1980).  liquid  t h e membrane o f t h e y o l k  sac  I n Year 2, samples o f f r y w i t h  56 b l u e sac and o t h e r a b n o r m a l i t i e s were p r e s e r v e d i n solution glacial  (formalin, acetic  histological  20 ml; e t h a n o l , 30 ml;  acid,  10  ml;  distilled  Davidson's  glycerine,  water,  30  10 ml)  ml; for  examination.  The e q u a t i o n s used t o c a l c u l a t e s u r v i v a l t o t h e e y e d , h a t c h and swimup s t a g e s a r e as f o l l o w s : % Eyed  =  no. a l i v e a f t e r s h o c k i n g eyed eggs x t o t a l no. eggs a t b e g i n n i n g o f i n c u b a t i o n  100  % Hatched =  no. a l i v e a f t e r h a t c h i n g complete x t o t a l no. eggs a t b e g i n n i n g o f i n c u b a t i o n  100  % Swimup  no. l i v e f r y a t swimup x t o t a l no. eggs a t b e g i n n i n g o f i n c u b a t i o n  100  =  % Eyed o f f e r t i l i z e d  = %  % Hatched o f eyed  =  x  % hatched % eyed  % Swimup o f hatched =  3.10  % eyed fertilized  % swimup % hatched  100  x  100  x  100  Growth and s u r v i v a l o f f r y Fry  were r a i s e d i n t h e same t y p e o f system as d e s c r i b e d f o r  t h e i n c u b a t i o n o f eggs i n S e c t i o n 3.9 e x c e p t t h a t no g r a v e l was p l a c e d i n t o t h e lower s e c t i o n s .  J a r s were p l a c e d i n t o a t r o u g h  w i t h each j a r connected  t o a header p i p e r u n n i n g t h e l e n g t h o f  the trough.  t r e a t e d by a b i o l o g i c a l  Water was  gravel  filter  57 l o c a t e d beneath t h e t r o u g h . cm),  which  contained  0.85  The m  3  filter  gravel,  (2.46 m x 93 cm x resulted  i n the  88  water  t e m p e r a t u r e 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 a p p r o x i m a t e l y 15% f r e s h w e l l w a t e r as makeup. The  temperature  was  checked  daily  a d j u s t i n g t h e amount o f makeup w a t e r .  and  was  controlled  by  The f i l t e r was backwashed  once d u r i n g t h e t i m e t h a t t h e c u l t u r e system was used i n Year 1 and i n Year 2. For  The f l o w r a t e t o each j a r was  770-820 ml/min.  t h e 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 ,  covered  with  fluorescent  black lights  plastic. with  an  Illumination intensity  of  t h a t j a r was  was 127  from  yellow  l u x and  1.94  m i c r o e i n s t e i n s / m V s e c w i t h a p h o t o p e r i o d o f 10 h r o f l i g h t  and  14 h r o f d a r k n e s s .  The  specific  growth r a t e and m o r t a l i t y  m a t i n g were e v a l u a t e d o v e r a p e r i o d o f 41-42 spawned o v e r a 3 month p e r i o d ,  o f f r y from each days.  the e v a l u a t i o n  swimup f r y was i n i t i a t e d o v e r a 3 month p e r i o d . and  Since f i s h  o f groups  of  A t swimup (123  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 o f f r y were d r a i n e d o f w a t e r , b a t c h weighed t o 0.01 g and p l a c e d i n t o t h e r e a r i n g u n i t d e s c r i b e d above. y o l k sac had not been f u l l y absorbed.  At t h i s time, the  I n Year 1, 50 f r y were  p l a c e d i n each u n i t w h i l e 25 f r y were used i n Year 2 s i n c e t h e e v a l u a t i o n o f t h e number o f f r y t o r a i s e i n each r e a r i n g (see  Section  3.13.2) showed t h a t t h e growth r a t e  s i g n i f i c a n t l y g r e a t e r when f r y number was  unit  o f f r y was  25 p e r u n i t compared  58 to  50.  I f adequate  numbers o f 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 ; o t h e r w i s e o n l y one group was r a i s e d . Bioproducts,  Semimoist s t a r t e r d i e t ( B i o d i e t #1 m a n u f a c t u r e d by Warrenton,  Oregon) was  used.  Fresh  feed  was  weighed weekly and i t was p l a c e d i n t h e r e f r i g e r a t o r each n i g h t . The r e s t o f t h e f e e d was kept f r o z e n (-10°C) i n a p l a s t i c b u c k e t which was f l u s h e d w i t h n i t r o g e n b e f o r e c l o s i n g . fed  F r y were n o t  t h e day t h e y were p l a c e d i n t o t h e j a r s and t h e y were f e d  l i g h t l y 3 t i m e s t h e n e x t day and 6 t i m e s a day (between 0800 and 1600) t o e x c e s s t h e r e a f t e r . daily  Dead f r y were removed and c o u n t e d  and t h e j a r s were c l e a n e d weekly.  A t t h e end o f t h e  p e r i o d , f r y were c o u n t e d and b a t c h weighed t o a s s e s s 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) = loq  0  f i n a l mean wt ( m q ) - l o q i n i t i a l mean wt (mg) x 100 no. o f days Q  3.11 C o l l e c t i o n  of unfertilized  eggs and f r y f o r measurement  and/or a n a l y s i s At drained  spawning, o f ovarian  a 50-60 g sample fluid,  was p l a c e d  o f eggs, into  which  had been  a pre-tared  g l a s s j a r and t r a n s p o r t e d on i c e back t o W i n n i p e g .  brown  From t h i s  sample, two 10-15 g samples o f eggs were t r a n s f e r r e d i n t o c l e a r  59 glass s c i n t i l l a t i o n  vials  (Figure 4).  Each v i a l  was  flushed  w i t h n i t r o g e n and wrapped i n a paper t o w e l t o p r o t e c t t h e sample from exposure t o l i g h t . o t h e r was  One  s t o r e d a t -115°C.  individual  eggs t o  v i a l was  s t o r e d a t -35°C and  A f t e r o b t a i n i n g the weights of  0.0001 g on  30  a M e t t l e r AE160 b a l a n c e ,  t o t a l w e i g h t o f eggs i n the p r e - t a r e d j a r was was  the  the  n o t e d , t h e sample  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.  I n Year 2, samples o f swimup f r y were t a k e n f o r a n a l y s i s on the  days  the  f r y evaluations  started.  e x c e s s m o i s t u r e on paper t o w e l s ,  placed  snap t o p s , f l u s h e d w i t h n i t r o g e n and ice.  Fry  were d r a i n e d  i n p l a s t i c tubes  with  i m m e d i a t e l y f r o z e n on  They were s t o r e d a t -115°C u n t i l  analyzed  of  dry  for vitamin  E  (Figure 4).  3.12  A n a l y s e s of eggs, f r y and/or d i e t s  3.12.1 V i t a m i n E a n a l y s i s o f eggs and f r y Eggs and f r y , s t o r e d a t -115°C, were a n a l y z e d for  vitamin  extraction  E  (a-tocopherol)  method  spectrophotometric bathophenathroline  of  content  King  method  ( F i g u r e 4)  (1983)  of  (Appendix 3 a ) .  i n duplicate  the  and  by  using  the  AOAC  adapted  (1984)  Precautions  using  were t a k e n  m i n i m i z e l o s s o f v i t a m i n E t h r o u g h exposure t o h e a t , l i g h t oxygen.  Subdued  fluorescent  lighting  was  used  (475  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 t e m p e r a t u r e was b e l o w 45°C d u r i n g e v a p o r a t i o n  of s o l v e n t s .  the  to and  lux), kept  60  F o r c o m p a r i s o n , samples o f eggs w h i c h were c o l l e c t e d f r o m 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 a n a l y z e d .  These eggs  were c l o s e t o h a t c h i n g when sampled.  3.12.2 Dry m a t t e r and s e l e n i u m The 30 g samples o f eggs s t o r e d a t -35°C were f r e e z e - d r i e d ( F i g u r e 4) w i t h t h e 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 w e i g h t used t o c a l c u l a t e percent dry matter.  Three samples o f f i n e l y ground  eggs p e r t r e a t m e n t p e r s a m p l i n g y e a r were a n a l y z e d  f o r selenium  i n t h e same way as t h e 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 o f eggs t a k e n i n Year 2 and s t o r e d a t -35°C ( F i g u r e 4) were used f o r e x t r a c t i o n o f t o t a l l i p i d s by a modified  B l i g h and Dyer (1959) method  (Appendix 3 a ) .  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 s e p a r a t e total  lipids  into neutral l i p i d  and p o l a r  lipid  m o d i f i c a t i o n o f t h e method o f A s h t o n (1991). r e d u c e d t o 60-70 mg and o n l y Neutral  f r a c t i o n s by  Total l i p i d  was  one Sepak was used p e r sample.  l i p i d s were e l u t e d w i t h 30 ml 15% hexane i n c h l o r o f o r m  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  f r a c t i o n s were assayed spectrophotometric  (1:1).  for lipid  assay  (Fales  Duplicate  aliquots  by t h e d i c h r o m a t e 1971).  of the  oxidation  Standard curves  were  c o n d u c t e d u s i n g 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 charr  eggs i n o r d e r t o have a comparable l e v e l o f  Results  oxidation.  from t h i s assay compared w e l l w i t h r e s u l t s o b t a i n e d  weighing  aliquots  neutral  and  dried  polar  established.  To  at  lipids obtain  b a s i s , t o t a l l i p i d was  70°C.  The  i n each  r e l a t i v e percent  sample o f  percent t o t a l  also extracted  by  total  lipid  on  of  lipid  a dry  was  matter  from d r y samples o f eggs  t a k e n i n Year 1 and 2 ( F i g u r e 4) and d u p l i c a t e a l i q u o t s o f  lipid  extracts  polar  were d r i e d a t  70°C and  weighed.  Neutral  and  l i p i d s were c o n v e r t e d t o p e r c e n t d r y m a t t e r : Neutral  lipid  (% d r y m a t t e r ) =  n e u t r a l l i p i d ( r e l a t i v e %) 100 Polar l i p i d  the  fractionations  The  ( r e l a t i v e %) 100  precision  sample was  total lipid  was  of  the  x  total lipid  neutral  lipid  s a t i s f a c t o r y , only  one  the  neutral  and  c h e c k e d by  s p o t t i n g a l i q u o t s o f the  (0.25  thin-layer  plates  and  polar  =  and  polar  lipid  determination  80:20:2  identification  ( C h r i s t i e 1981). of  the  bands  lipid  f r a c t i o n s on placing  a s c e n d i n g s o l v e n t system o f h e x a n e : d i e t h y l acid  (% d r y m a t t e r )  per  conducted.  p u r i t y of  mm)  (% d r y m a t t e r )  (% d r y m a t t e r ) =  polar l i p i d  Since  x  Lipid  included  the  fraction  was  S i l i c a Gel plates  in  G an  ether : g l a c i a l a c e t i c standards  used  for  phosphatidylcholine,  cholesterol, cholesteryl ester, oleic acid, 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 m o n o g l y c e r i d e .  L o c a t i o n o f t h e bands was  v i s u a l i z e d by s p r a y i n g t h e 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 h e a t i n g them a t 110°C.  To d e t e r m i n e t h e f a t t y a c i d c o m p o s i t i o n  o f t h e n e u t r a l and  p o l a r l i p i d s o f t h e eggs c o l l e c t e d i n Year 2 and o f t h e d i e t s used  i n Year  1 and 2, 5 mg  aliquots of t o t a l  lipid  were  t r a n s e s t e r i f i e d u s i n g t h e a c i d - c a t a l y z e d method w i t h t o l u e n e 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 a p p l y i n g them on S i l i c a G e l H p l a t e s and  p l a c i n g them i n an a s c e n d i n g  methyl  ester  band  was  TLC t a n k w i t h t o l u e n e .  visualized  by  spraying  Rhodamine 6G and v i e w i n g i n u l t r a v i o l e t l i g h t . band was removed concentrated injecting  t h e samples  0.02%  The m e t h y l e s t e r  from t h e p l a t e , e l u t e d w i t h  i n hexane.  with  The  chloroform  and  F a t t y a c i d p r o f i l e s were o b t a i n e d into  a  Varian  Aerograph  Model  by  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 t e m p e r a t u r e s were 235 and 250°C, r e s p e c t i v e l y and t h e column t e m p e r a t u r e was h e l d a t 175°C f o r 25 m i n , i n c r e a s e d 1°C p e r min f o r 35 min and h e l d a t 210°C f o r 30 min. each  peak  had  been  determined  Identity of  (Yurkowski,  communication) by t h e methods d e s c r i b e d  by Y u r k o w s k i  personal (1989).  Peak a r e a s were measured by an e l e c t r o n i c i n t e g r a t o r ( V a r i a n S t a r I n t e g r a t o r , R e v i s i o n A, V a r i a n A s s o c i a t e s , Walnut Creek, CA) and c o n v e r t e d  i n t o area percent  o f each f a t t y a c i d .  One  63 fatty  acid  d e t e r m i n a t i o n p e r sample  was  conducted  after  d e t e r m i n i n g t h a t t h e p r e c i s i o n o f t h e 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 diets  Major ingredients by  and d i e t s were a n a l y z e d f o r c r u d e p r o t e i n  t h e m a c r o - K j e l d a h l method  (AOCS 1989).  A s h and m o i s t u r e  c o n 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  a n a l y z e d f o r t o t a l Ca, Na, K, Mg, Mn, Zn, Cu, Fe by n i t r i c perchloric  acid  digestion  spectrophotometry.  followed  by  absorption  S e l e n i u m d e t e r m i n a t i o n was by t h e method o f  V i j a n and Wood (1976).  S o l u b l e r e a c t i v e phosphorus was a n a l y z e d  by t h e molybdenum b l u e method o f S t a i n t o n  3.13 E v a l u a t i o n  atomic  acid-  e t a l . (1974).  o f methods  3.13.1 E v a l u a t i o n  o f egg r i n s e s and e x t e n d e r s  I n Year 1, a t o t a l  of 5 fish  from t a n k s 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 e x t e n d e r s  were  tested  October  26,  November 2, 1989 and December 27, 1989 on 2 f i s h w h i c h spawned each day t o d e t e r m i n e i f t h e method b e i n g used was a f f e c t i n g t h e f e r t i l i z a t i o n rate.  The f o l l o w i n g were t e s t e d l a ) w e l l w a t e r as  r i n s e and e x t e n d e r on October 26, l b ) no r i n s e and w e l l water as e x t e n d e r on November 2 (normal p r o c e d u r e a t RARC), 2) l a c t o s e C a C l - N a C l (Hamor 1987), 3) T r i s - g l y c i n e . H C 1 ( B i l l a r d 2  4) NaHC0  3  ( W i l c o x e t a l . 1984).  1977) and  An e q u a l w e i g h t (20 o r 50 g) o f  64 d r a 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 o f 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  volume  (0.5 ml) o f m i l t  and poured o f f .  An  equal  from each o f t h e same two males  added t o 25 ml o f t h e s o l u t i o n and i t was  was  added t o t h e eggs.  Eggs were mixed g e n t l y f o r two minutes and t h e n r i n s e d w i t h w a t e r u n t i l t h e water was c l e a r . as  described  swimup.  to  assess  Samples o f eggs were i n c u b a t e d  fertilization  rate  and  survival  Based on t h e f e r t i l i z a t i o n r a t e d a t a , t h e d e c i s i o n  made t o use t h e NaHC0 r i n s e . 3  well  I n Year 2, s o l u t i o n s  to was  l b , 2 and 4  were r e - e v a l u a t e d on November 15, 1989 u s i n g 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 o f two males and f e r t i l i z e d and i n c u b a t e d as above.  3.13.2 E v a l u a t i o n  o f number o f f r y t o r a i s e i n each r e a r i n g  D u r i n g Year 1, t h e number o f f r y t h a t c o u l d each  j a r was  evaluated.  unit  be r a i s e d i n  Swimup f r y , a l l o f f s p r i n g  of  one  f e m a l e , were counted out i n t o two groups o f 10, 25, 50, 100 and 300  f r y and b a t c h weighed.  The groups o f 10 t o 100 f r y were  p l a c e d i n t o j a r s u s i n g a randomized d e s i g n . fry  were  Tabachek  placed  i n 60  L  fiberglass  (1983) w i t h a f l o w r a t e o f 1.8 L/min.  i n t h e manner d e s c r i b e d i n S e c t i o n  3.14  Statistical In  variance,  tanks  order  Two groups o f 300 as  described  by  F r y were r a i s e d  3.10.  analysis  to satisfy  the data  must  the conditions f i t a normal  of the analysis  d i s t r i b u t i o n and  of have  65 variances 1964).  which  are equal  and not r e l a t e d  t o t h e means ( L i  A r c s i n t r a n s f o r m a t i o n was c a r r i e d o u t on a l l d a t a  binomial  distributions  survival).  (fertilization  and embryonic  Log t r a n s f o r m a t i o n was c a r r i e d  from  and f r y  o u t on v i t a m i n E  c o n c e n t r a t i o n and c o n t e n t o f eggs and f r y as w e l l as egg w e i g h t s because v a r i a n c e s were p r o p o r t i o n a l t o t h e means. t e s t was used t o t e s t  Bartlett's  f o r homogeneity o f v a r i a n c e s  prior to  conducting a n a l y s i s of variance.  S i n c e most d a t a s e t s were unbalanced, t h e g e n e r a l model  (PROC GLM) was used  t o detect  linear  significant differences  between means by a n a l y s i s o f v a r i a n c e  (SAS I n s t i t u t e  1985).  One-way a n a l y s i s o f v a r i a n c e was conducted on d a t a f o r a l l f i v e d i e t s u s i n g t h e model: Y = \i + a + e where Y = measurement, \i = mean o f p o p u l a t i o n s , a = e f f e c t o f d i e t and E = e x p e r i m e n t a l e r r o r .  A n a l y s i s o f v a r i a n c e was used  on d a t a f o r t h e f o u r d i e t s c o m p r i s i n g t h e 2 x 2 f a c t o r i a l d e s i g n by u s i n g t h e model: Y = (x + a + p + ap + e where Y = measurement, \i = mean o f p o p u l a t i o n s , a = e f f e c t o f lipid,  p = e f f e c t o f v i t a m i n E, ap" = i n t e r a c t i o n o f l i p i d and  v i t a m i n E and E = e x p e r i m e n t a l e r r o r . of  broodstock  variations  were  among  testing error.  subjected  fish  S i n c e no r e p l i c a t e  t o the respective  w i t h i n each  treatment  tanks  treatments,  served  as t h e  Duncan's m u l t i p l e range t e s t was used t o d e t e c t  66 significant  differences  between  means.  The  level  of  s i g n i f i c a n c e was s e t a t 95% i n a l l s t a t i s t i c a l a n a l y s e s .  Regression  analysis  was conducted  t o detect  correlation  between s e l e c t e d parameters such as t h e e f f e c t o f f i s h w e i g h t on fecundity  or the e f f e c t of selected  dietary  f a t t y a c i d s on t h e  f a t t y a c i d c o m p o s i t i o n o f eggs (SAS I n s t i t u t e 1985).  The l e v e l  o f s i g n i f i c a n c e was s e t a t 95%. R e g r e s s i o n a n a l y s i s d e t e c t e d 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 the  eggs and i t appeared t h a t  a f f e c t e d by d i e t . differences  Analysis  lipids i n  t h i s r e l a t i o n s h i p might a l s o  be  o f c o v a r i a n c e was used t o p a r t i t i o n  due t o d i e t and n e u t r a l l i p i d s on t h e i n t e r c e p t s and  slopes of the regression.  C h i square a n a l y s i s effects of selected  ( L i 1964) was c o n d u c t e d t o compare t h e  treatments ( e f f e c t s of v i t a m i n E or l i p i d )  on t h e p r o p o r t i o n o f females t h a t spawned o r males t h a t milt.  produced  Data from t h e same d i e t a r y l i p i d o r f o r t h e same d i e t a r y  v i t a m i n E t r e a t m e n t s were p o o l e d t o i n c r e a s e t h e sample s i z e f o r e a c h group t o o b t a i n e x p e c t e d v a l u e s o f 5 ( L i 1964). the  COMM d i e t s  were e x c l u d e d from t h i s  analysis.  Data f o r When t h e  e x p e c t e d number was t o o low f o r C h i 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 t h e e f f e c t o f d i e t on spawning t i m e ) , e x a c t t e s t was used (SAS I n s t i t u t e 1990).  Fisher's  67 Power  a n a l y s i s was  recommended by Peterman  conducted  (Dixon  and Massey  (1990) t o d e t e r m i n e t h e power  a n a l y s i s o f v a r i a n c e g i v e n t h e v a r i a n c e t h a t was embryonic  survival.  1969)  as  of the  observed i n  68 RESULTS  4.1  Feed  composition  Experimental 7.5-7.8% a s h ,  d i e t s c o n t a i n e d 46.9-47.8% c r u d e p r o t e i n and  on a d r y w e i g h t b a s i s  (Table  2).  Total  lipid  c o n t e n t s o f t h e low l i p i d d i e t s were 11.8-12.6% w h i l e t h o s e o f t h e h i g h l i p i d d i e t s were 18.8-19.0%. estimated  M e t a b o l i z a b l e energy was  as 3.23 and 3.90 k c a l / g f o r t h e L L and HL d i e t s ,  respectively.  COMM c o n t a i n e d  42.4%  p r o t e i n , 16.8%  7.8%  ash and was e s t i m a t e d t o c o n t a i n 120 mg t o t a l  /kg  (Martin  ethoxyquin/kg  Feed  Mills,  personal  l i p i d and  a-tocopherol  communication),  and 3.47 k c a l m e t a b o l i z a b l e e n e r g y / g .  223 mg The f o u r  e x p e r i m e n t a l d i e t s c o n t a i n e d t o t a l m i n e r a l c o n c e n t r a t i o n s o f 16 mg Ca/g, Mn/g,  2.4 mg Na/g,  228 \ig Fe/g,  26 ng Cu/g,  a dry weight b a s i s . to  1.4 mg K/g,  1.5 mg Mg/g,  189 \ig Zn/g  1.3 mg P/g,  91 u,g  and 1.02 \ig Se/g on  COMM had c o n c e n t r a t i o n s o f Ca and K s i m i l a r  t h e above, lower P o f 1.1 mg/g and h i g h e r c o n c e n t r a t i o n s o f  t h e o t h e r s : 2.8 mg Na/g,  1.9 mg Mg/g,  116 \xg Mn/g,  210 [ig Zn/g and t w i c e as much i r o n a t 456 ng Fe/g.  42 fxg  Cu/g,  COMM used i n  Year 1 c o n t a i n e d 0.75 jig Se/g w h i l e t h e two l o t s used i n Year 2 c o n t a i n e d 0.88 and 1.96 jig Se/g.  In acetate,  a d d i t i o n t o t h e supplemented amount o f d l - a - t o c o p h e r y l endogenous  ingredients. /kg.  a-tocopherol  was p r e s e n t  i n t h e other  The b a s a l mix c o n t a i n e d 7.7 ± 0.2 mg a - t o c o p h e r o l  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 T a b l e 2. C o m p o s i t i o n o f t h e d i e t s ( d r y m a t t e r b a s i s ) . Diet LLLE Moisture  (%)  LLHE  HLLE  HLHE  COMM  2  8.7 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  9.5  9.5  2.5  2.5  2.5  22.6  23.5  23.4  30.5  1  (%)  Fiber  3  (%)  N i t r o g e n - f r e e e x t r a c t (%) 23.7 4  160  160  160  160  30  600  30  600  (mg/kg)  14  14  24  24  -  Total [dl]-a-tocopherol + t o c o p h e r y l a c e t a t e (mg/kg)  44  614  54  624  120  3.28  3.34  3.86  3.86  3.59  Ethoxyquin  5  (mg/kg)  [dl]-a-Tocopheryl (mg/kg) [dl]-a-Tocopherol  acetate 7  M e t a b o l i z a b l e energy (kcal/g) 1  2  3  4  5 6 7  8  6  223  8  Mean (upper l i n e ) ± s t a n d a r d d e v i a t i o n ( l o w e r l i n e ) f o r t h e 5 b a t c h e s o f e x p e r i m e n t a l d i e t s and 3 l o t s o f COMM d i e t used throughout t h i s study. E s t i m a t e d v a l u e s f o r t h e 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 t h e a u t h o r by t h e m a n u f a c t u r e r . E s t i m a t e d u s i n g NRC (1981) t a b l e s on c o m p o s i t i o n o f ingredients. E s t i m a t e d 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 + a s h + fiber). T o t a l e t h o x y q u i n i n f i s h meal and o i l . Taken f r o m f o r m u l a t i o n s i n T a b l e 1. E s t i m a t e d from a n a l y s i s o f b a s a l mix and h e r r i n g o i l as d e s c r i b e d i n S e c t i o n 3.3.3. E s t i m a t e d u s i n g v a l u e s o f 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 e x p e r i m e n t , which was n o t f r o z e n c o n t a i n e d 184 ± 5.7 mg a - t o c o p h e r o l / k g o i l w h i l e t h e sample which was f r o z e n c o n t a i n e d 144 ± 0.7 mg a - t o c o p h e r o l / k g o i l . in  this  experiment  Samples o f h e r r i n g o i l u s e d  were a l s o s h i p p e d  t h e y were n o t a n a l y z e d .  t o Hoffmann-LaRoche b u t  S i n c e t h e o i l used i n t h i s  experiment  had been f r o z e n b e f o r e u s e , t h e l e v e l o f endogenous a - t o c o p h e r o l was e s t i m a t e d t o be 144 mg/kg o i l .  H e r r i n g o i l was e s t i m a t e d as  c o n t r i b u t i n g 14 mg a - t o c o p h e r o l / k g t o t h e l o w l i p i d d i e t s and 24 mg/kg a - t o c o p h e r o l t o t h e h i g h l i p i d d i e t s . much  o f t h e endogenous  manufacturing  process  easily oxidized  I t i s expected t h a t  t o c o p h e r o l would o x i d i z e  and on s t o r a g e  ( B a u e r n f e i n d 1980).  of t h e d i e t s was n o t a n a l y z e d .  since  during the  a-tocopherol i s  Vitamin E acetate content  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 r e s e a r c h , was compared t o v i t a m i n E a c e t a t e o b t a i n e d from Sigma Chemical  Co., S t . L o u i s , MO and  ICN B i o m e d i c a l s , C o s t a Mesa, CA, t h e s p e c t r a were i d e n t i c a l and t h e 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 o f t h e  same c o n c e n t r a t i o n s were compared i n a  spectrophotometer.  The p e r o x i d e v a l u e o f t h e h e r r i n g o i l used i n making t h e four experimental diets July  1988 t o 3.33  September 1989. quality Canadian  control  ± 0.11 meq/kg  1.44 ± 0.38 meq/kg i n  at the f i n a l  analysis i n  These v a l u e s were w i t h i n t h e g u i d e l i n e s f o r of fish  Working  communication).  i n c r e a s e d from  Group  oils for  of <  5-10  Finfish  meq/kg  Nutrition,  (NRC 1981, personal  71 4.2 Water t e m p e r a t u r e As  described  temperatures experiment.  between  in  Section  tanks  was  3.5.3, observed  variation on  i n the  day  9  of the  A t t h a t t i m e , water t e m p e r a t u r e s were 7.2°C ( L L L E ) ,  7.2°C (HLHE), 9.7°C (LLHE), 10.2°C  (HLLE) and 12.2°C  (COMM).  That i s , t e m p e r a t u r e s 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 .  Similarly,  t e m p e r a t u r e s were h i g h e r (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 d a t a on  w a t e r t e m p e r a t u r e was c o l l e c t e d d u r i n g t h e f i r s t 8 days o f t h e experiment.  F o l l o w i n g changes t o t h e w a t e r system t o c o r r e c t  t h e p r o b l e m , t h e r e was l i t t l e between t a n k s .  d i f f e r e n c e i n water  temperature  D i f f e r e n c e s i n t e m p e r a t u r e s between t a n k s a f t e r  day 9 ranged from 0 t o 1.5°C (0.27°C ± 0.11) (mean ± s t a n d a r d d e v i a t i o n ) w i t h d i f f e r e n c e s o f o v e r 1.0°C o c c u r r i n g on 8 days during  t h e 544  temperatures Appendix  2a.  day  period  d u r i n g Year  measured.  Mean  weekly  water  1 and 2 a r e shown i n F i g u r e 5 and  D u r i n g Year  1, mean weekly  water  temperature  i n c r e a s e d from 8.3 t o 8.8°C d u r i n g J u l y and d e c r e a s e d  steadily  down t o 7.1°C from August t o mid-November as o u t d o o r and room temperatures decreased.  A f t e r room h e a t e r s were t u r n e d on i n  mid-November, w a t e r temperature i n c r e a s e d t o 7.7-7.8°C.  I n Year  1, spawning o c c u r r e d between September 29 and December 27, 1989 when mean weekly t e m p e r a t u r e s were 7.1 t o 7.9°C.  72  73 In Year 2, mean weekly t e m p e r a t u r e v a r i e d between 7.7 8.4°C from J a n u a r y t o May  and  1988, between 8.0 and 8.6°C from June  t o J u l y ( F i g u r e 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 t e m p e r a t u r e i n c r e a s e d t o 8.5°C  from December 1989 t o J a n u a r y 1990 when room h e a t e r s were used. Mean w e e k l y w a t e r t e m p e r a t u r e was i n Year 2.  7.1 t o 8.5°C d u r i n g  spawning  S i m u l a t i o n o f water t e m p e r a t u r e i n Year 2 t o match  t h a t o f Year 1 was good e x c e p t between November 28 and December 25, 1989 when i t averaged 0.6-0.9°C h i g h e r t h a n c o r r e s p o n d i n g dates  i n Year  increased  as  1.  Flow r a t e  much  as  o f t h e 6.5°C make-up w a t e r  possible  during  this  time  but  was  water  t e m p e r a t u r e c o u l d not be d e c r e a s e d t o match t h a t o f Year 1.  4.2 Water c h e m i s t r y C h e m i c a l c o m p o s i t i o n o f t h e i n c o m i n g w e l l w a t e r has been measured the  ( P r o v i n c e o f Manitoba 1987).  f o l l o w i n g : pH,  424 mg/L; (HC0 ), 3  jAmho/cm; h a r d n e s s ,  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  mg/L;  total  ( e x t r a c t a b l e ) , 66.1 mg/L, mg/L;  Fe ( e x t . ) , 0.114  ( s o l u b l e ) 68 mg/L; parameters  indicated  c o n d u c t i v i t y , 918  486  7.75;  Measurements  alkalinity  (CaC0 ),  Mg ( e x t . ) , 63.0 mg/L,  mg/L  (1982); Mn  CI ( s o l . ) , 43 mg/L  are a l l w i t h i n  398  mg/L;  Ca  Na ( e x t . ) ,  40.8  3  ( e x t . ) , <0.02 mg/L; and F, 0.28  mg/L.  the  4  These  t h e suggested c h e m i c a l v a l u e s f o r  h a t c h e r y w a t e r 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 e t al. except f o r hardness.  S0  The hardness was  s u g g e s t e d range o f 10-400  mg/L.  424 mg/L  which  1982)  exceeds  74 Mean v a l u e s f o r w a t e r c h e m i s t r y parameters  f o r each t a n k  o v e r t h e 8 s a m p l i n g t i m e s a r e shown i n T a b l e 3 and f o r t h e 5 t a n k s f o r each s a m p l i n g t i m e a r e shown i n T a b l e 4. repeated between  samplings, tanks  ammonia was  there  i n any  were  water  Over t h e  no  significant  differences  quality  parameters.  Un-ionized  always l e s s t h a n t h a t l i s t e d i n t h e w a t e r  quality  c r i t e r i a f o r optimum h e a l t h o f s a l m o n i d s s t a t e d by P i p e r e t al. (1982) as  0.0125 ppm  (12.5  \iq/L).  The  h i g h e s t observed  i o n i z e d ammonia l e v e l (8.96 ng/L) o c c u r r e d on F e b r u a r y 6,  un1989.  T h i s o c c u r r e d a t a t i m e when t h e e f f i c i e n c y o f t h e b i o l o g i c a l f i l t e r s may 12, 1989  have been reduced when f e e d i n g resumed on J a n u a r y  a f t e r f e e d was  backwashing  of the f i l t e r s  w a t e r samples. 0.06  ppm  w i t h h e l d d u r i n g spawning. o c c u r r e d 4 days  after  The  first  taking  the  N i t r i t e - N was w i t h i n t h e s u g g e s t e d c r i t e r i a o f  (60 ng/L).  W h i l e t h e suggested pH range f o r t r o u t i s  6.5-8.0, t h e pH o f t h e r e c i r c u l a t e d w a t e r on most s a m p l i n g days was  4.4  7.9-8.2 and 8.8-9.0 on two s a m p l i n g days.  Feed f e d and f e e d e f f i c i e n c y Data were c o l l e c t e d on t h e w e i g h t o f f e e d o f f e r e d t o each  t a n k o f f i s h d u r i n g each f e e d i n g p e r i o d and t h e s e v a l u e s were converted  t o dry weight of  feed.  between each w e i g h i n g p e r i o d Appendices  2b  and  2c  and  Feed  i n Years  f e d and 1 and  weight  gain  2 a r e shown i n  are provided i n Table  5.  It is  e x p e c t e d t h a t d i f f e r e n c e s i n water t e m p e r a t u r e d u r i n g t h e f i r s t 9 days o f t h e experiment a f f e c t e d f e e d i n t a k e and u l t i m a t e l y  75 T a b l e 3.  N0 -N  N0 -N  JAg/L  ng/L  Hg/L  8.2  28.8  2.25  455.3  11.4  8.4  26.3  1.00  366.0  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  SD  0.14  D.O.  °C  mg/L  2  8.3  11.4  3  8.5  6  1  2  1  D i s s o l v e d oxygen Standard d e v i a t i o n  0.15  pH  (n=8).  NH -N  Temp.  Tank  2  Water c h e m i s t r y d a t a f o r each t a n k  0.05  4  4.84  2  0.58  3  38.1  T a b l e 4.  Water c h e m i s t r y d a t a f o r each s a m p l i n g day  (n=5).  77 T a b l e 5. Feed f e d , w e i g h t g a i n and f e e d e f f i c i e n c y f o r each t a n k o f f i s h f e d f o r 71 days i n Year 1 and 239 days i n Year 2.  Diet  LLLE  LLHE  T o t a l feed f e d (kg, d r y )  HLLE  HLHE  COMM  LL  1  HL  2  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 w e i g h t g a i n (kg) Year 1  9.75  8.12  Year 2  15.42  13.78  Feed e f f i c i e n c y ( % )  12.24 12.28 15.45 6.73  9.84  8.94 13.87  24.81 14.60  8.29  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  Year 1  25  25  25  25  25  25  25  Year 2  21  23  22  24  24  22  24  Mean no. f i s h  5  E s t i m a t e d d r y w e i g h t f e e d f e d p e r f i s h (g) Year 1  610  595  655  683  805  603  669  Year 2  1152  1254  1561  1425  1683  1203  1493  1 2 3 4 5  LL = mean o f t h e two LL d i e t s HL = mean o f t h e two HL d i e t s Dry feed= f e e d , g as f e d x ( d r y m a t t e r , % /100) Feed e f f i c i e n c y = ( t o t a l w e i g h t g a i n , g x 1 0 0 ) / d r y f e e d f e d , g Average number o f f i s h p r e s e n t d u r i n g t h e f e e d i n g p e r i o d  78 weight  gain during this  period.  During these f i r s t  9  days,  t a n k s o f f i s h h e l d a t t h e h i g h e r t e m p e r a t u r e s had h i g h e r f e e d i n t a k e s and w e i g h t g a i n s t h a n t h e i r c o u n t e r p a r t s f e d d i e t s w i t h the  same l i p i d l e v e l .  However, a t t h e end o f 71 days o f f e e d i n g  i n Year 1, t o t a l w e i g h t g a i n s were a l m o s t i d e n t i c a l i n b o t h HL groups.  I n c o n t r a s t , t h e LLLE group, w h i c h had been h e l d a t t h e  l o w e r t e m p e r a t u r e , had g a i n e d a t o t a l  o f 1600  g more t h a n i t s  LLHE c o u n t e r p a r t h e l d a t t h e h i g h e r t e m p e r a t u r e .  The  effect  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 w e i g h t g a i n d u r i n g the  r e m a i n d e r o f t h e experimentcannot be e s t i m a t e d .  I n Year 1,  the  w e i g h t o f d r y f e e d f e d i n 71 days was s l i g h t l y l o w e r f o r t h e  f i s h f e d t h e LL d i e t s (15.0 kg) compared t o t h e HL d i e t s kg).  Feed  diets  (26.6 kg) compared t o t h e HL d i e t s  d i e t was  f e d i n Year 2 was  a l s o lower f o r f i s h (34.3 k g ) .  (16.7  f e d t h e LL More COMM  f e d i n b o t h Year 1 (20.1 kg) and Year 2 (40.4 kg) t h a n  a l l other d i e t s .  Having e q u a l numbers o f f i s h i n each t a n k i n  Y e a r 1 makes comparison o f f e e d f e d t o each t a n k o f f i s h s i m p l e . However, i n Year 2 t h e numbers o f f i s h v a r i e d between t a n k s and w i t h i n a tank over the year.  To compare f e e d f e d on t h e b a s i s  of e q u a l numbers o f f i s h , a g r o s s e s t i m a t i o n o f t h e f e e d i n t a k e per f i s h  has been c a l c u l a t e d and  indicates that  feed fed per  f i s h was not markedly lower w i t h t h e LL d i e t s compared t o t h e HL diets.  I n a d d i t i o n t o t h e unequal numbers o f f i s h i n each t a n k  i n Year 2, a p o r t i o n o f t h e p o p u l a t i o n d i d not f e e d and t h i s e s p e c i a l l y n o t i c e a b l e d u r i n g Year 2.  was  79 F i s h b e h a v i o u r a f f e c t e d f e e d i n t a k e i n two c a s e s .  On t h e  t w e l f t h day o f t h e e x p e r i m e n t , i t was o b s e r v e d t h a t f i s h i n t a n k 6 s t a y e d crowded t o g e t h e r i n t h e a r e a under T h i s b e h a v i o u r appeared turbulence  i n this  to inhibit  t h e water  feeding a c t i v i t y  inlet.  and w a t e r  a r e a made i t i m p o s s i b l e t o d e t e r m i n e i f  s a t i e t y had been r e a c h e d .  D i s s o l v e d oxygen was 10.4 mg/L and  t e m p e r a t u r e was 8.5°C i n b o t h t a n k s .  When t h e d i r e c t i o n o f  w a t e r 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 t h r o u g h o u t t h e t a n k , but w i t h i n a s h o r t t i m e t h e y resumed t h e i r p o s i t i o n s under t h e water i n l e t . placed  On day 43 o f t h e e x p e r i m e n t , f i s h from t a n k 6 were  i n t a n k 7 and t h o s e i n t a n k 7 were p l a c e d i n t a n k 6.  B o t h groups a c t e d n o r m a l l y and d i s p e r s e d t h r o u g h o u t t h e t a n k . T h e r e f o r e , i t appeared t o be n e i t h e r t h e e f f e c t o f t h e t a n k n o r the  f i s h t h e m s e l v e s b u t 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  b e h a v i o u r t o be e x h i b i t e d .  A f t e r t h i s change was made, c h a r r  were l e f t i n t h e s e t a n k s and t h e d i e t s each group had been f e d were m a i n t a i n e d .  A l l r e c o r d s were changed t o show t h a t t a n k 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 6 and v i c e v e r s a ( F i g u r e 3 ) .  assigned t o tank  I t i s expected t h a t feed i n t a k e i n  t a n k 6 was reduced p r i o r t o t h e i r b e i n g moved t o t a n k 7.  When f e e d i n g was resumed a f t e r spawning a t t h e b e g i n n i n g o f Y e a r 2, one emaciated male f i s h i n t a n k 3 ( f e d LLLE i n Year 1 and HLLE i n Year 2) became v e r y a g g r e s s i v e .  This f i s h ,  which  was one o f t h e s m a l l e s t i n t h e t a n k , became v e r y t e r r i t o r i a l and f o r c e d a l l t h e o t h e r f i s h i n t o t h e a r e a under t h e w a t e r  inlet  80 where w a t e r t u r b u l e n c e d i d not a l l o w them t o f e e d n o r m a l l y . I f a  fish  ventured  out of t h i s  area, the aggressive  fish  would  challenge i t .  When t h e a g g r e s s i v e f i s h was removed f r o m t h e  tank  16, 1989,  on March  t h e remaining  fish  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 t h e t a n k and f e e d i n t a k e i n c r e a s e d . The a g g r e s s i v e f i s h was not r e p l a c e d .  S m a l l numbers o f f i s h were c o n s i d e r e d t o be " l o n e r s " - f i s h that  stayed  outside  o f t h e main  group  remained a t t h e p e r i p h e r y o f t h e t a n k . not f e e d .  of fish  and always  These f i s h g e n e r a l l y d i d  Even when a f e e d p e l l e t was thrown d i r e c t l y i n f r o n t  o f t h e s e f i s h , t h e p e l l e t was e i t h e r i g n o r e d c o m p l e t e l y o r t a k e n i n t o t h e mouth and t h e n s p i t o u t .  In Y e a r 1, f e e d e f f i c i e n c y was h i g h e r f o r f i s h f e d t h e HL diets LL  (73%) and COMM d i e t (77%) than observed  diets  (59%).  The r e v e r s e  occurred  e f f i c i e n c y was lower f o r t h e HL d i e t s and COMM d i e t  (61%).  f o r those fed the  i n Year  2 when  than the LL d i e t s  feed (56%)  Feed e f f i c i e n c y was 29% f o r t h e HLHE d i e t  and 20% f o r t h e HLLE d i e t .  P a r t o f the reason f o r the  extremely  p o o r f e e d e f f i c i e n c y o f t h e l a t t e r group was t h e e f f e c t t h a t t h e a g g r e s s i v e male f i s h had on the f e e d e r ' s assessment o f s a t i e t y . The  f e e d o f f e r e d was p r o b a b l y not a l l consumed.  A r c t i c c h a r r t o o k f e e d from the bottom o f t h e t a n k as w e l l as t h r o u g h o u t  t h e water column.  One f i s h i n each t a n k f e d LLLE  81 and COMM i n Year 1 and HLLE i n Year 2 r e l e a s e d a l m o s t a l l o f i t s eggs i n t o t h e t a n k . the  tanks  had  The s m a l l s c r e e n l o c a t e d i n t h e m i d d l e o f  perforations  which  d i d not  allow  either  feed  p e l l e t s o r f i s h eggs t o pass t h r o u g h i t . When s e v e r a l eggs were p l a c e d i n a t a n k t o o b s e r v e t h e response o f t h e f i s h , t h e eggs were r e a d i l y consumed.  4.5 Growth o f b r o o d s t o c k Fish season  had  and  not  had  been tagged  no  spawning  e x p e r i m e n t was i n i t i a t e d . t h a t i t was  during the colouration  previous at  the  spawning  time  this  An e x p e r i e n c e d h a t c h e r y manager found  d i f f i c u l t t o d e t e r m i n e t h e sex o f some f i s h .  In  Year 1, 11 out o f 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 (total  of  2-5  fish  i n each  tank),  initially  believed  to  be  f e m a l e , d e v e l o p e d p r i m a r y and secondary s e x u a l 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  w e i g h t = 993  g, range  E i g h t o f them g a i n e d w e i g h t t h r o u g h o u t weight.  Year  = 690-1455 g ) . 2, w h i l e 6  lost  A l l t h o s e which g a i n e d weight produced m i l t w h i l e o n l y  2 o f t h e f i s h t h a t l o s t w e i g h t produced m i l t . n o t mature u n t i l  Year  2 were l a r g e  fish  F i s h which d i d  (2-3 kg a t  spawning  t i m e ) and t h e y had a v e r y s i l v e r y appearance u n t i l t h e y began t o mature.  The a c t u a l number o f male and female f i s h i n each t a n k  and u l t i m a t e l y t h e l e n g t h and weight d i s t r i b u t i o n o f males and females  i n each  t a n k was  different  than  expected.  Initial  w e i g h t d i s t r i b u t i o n s o f male and female f i s h i n each t a n k a r e p r e s e n t e d i n T a b l e 6.  82  83 Initial presented  and  final  i n Tables  weights of f i s h  7  and  8.  High  i n Year dietary  1 and lipid  2 are content  s i g n i f i c a n t l y i n c r e a s e d t h e s p e c i f i c growth r a t e o f females i n Year  1  but  not  i n Year  i n f l u e n c e (Table 9 ) .  2 while  dietary  vitamin  E  had  no  There were no e f f e c t s o f l i p i d o r v i t a m i n  E on t h e s p e c i f i c growth r a t e s o f males i n e i t h e r y e a r .  I n Year  1, f e e d i n g t h e 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 h i g h e r growth r a t e s i n females t h a n f o r t h o s e f e d o t h e r d i e t s e x c e p t HLLE and i n s i g n i f i c a n t l y h i g h e r growth r a t e s i n males compared t o t h o s e fed  LLHE.  Year  1,  There was  There was  but t h i s  a low i n c i d e n c e o f f i s h l o s i n g w e i g h t i n  incidence  increased  i n Year  no s i g n i f i c a n t d i f f e r e n c e between  2  (Table 10).  the proportion of  males o r females which l o s t w e i g h t and no s i g n i f i c a n t e f f e c t o f d i e t a r y l i p i d o r v i t a m i n E on t h e p r o p o r t i o n o f males o r females l o s i n g weight.  F i s h which l o s t w e i g h t c o n t i n u a l l y t h r o u g h o u t  Y e a r 1 and/or Year 2 became e m a c i a t e d but o n l y 2 o f t h e s e f i s h d i e d p r i o r t o spawning i n Year 2 (one f e d LE and 1 f e d COMM).  4.6  Spawning  4.6.1  Time o f spawning In Y e a r 1, t h e r e appeared t o be an e f f e c t o f d i e t a r y  level  on  spawning  the period  initial  spawning  (Table 11).  spawn on September  Fish  and  the  f e d t h e HL  length  of  the  d i e t s began t o  29, 1988 w i t h s i x o u t o f 26 f i s h  d u r i n g t h e f i r s t 5 weeks. to  date  lipid  spawning  Those f e d t h e LL d i e t s d i d not b e g i n  spawn u n t i l November 2, 1988.  F i s h e r ' s e x a c t t e s t was  used  84  85  86  87 T a b l e 10. Number o f male and female c h a r r w h i c h l o s t o r g a i n e d w e i g h t t h r o u g h o u t Year 1 o r 2.  Tank Dietary vitamin E  3 6 2 7 5 Total LE LE HE HE COMM LE HE  L o s t w e i g h t o n l y i n Year 1 Male Female  1 0  0 0  0 0  2 0  0 0  1 0  2 0  L o s t w e i g h t o n l y i n Year 2 Male Female  6 5  6 3  2 5  4 4  1 1  12 8  6 9  L o s t w e i g h t i n b o t h Year 1 and 2 Male Female  0 0  0 0  2 0  0 0  1 0  0 0  2 0  L o s t w e i g h t i n e i t h e r Year 1 o r 2 Male Female  7 5  6 3  4 5  6 4  2 1  13 8  10 9  G a i n e d w e i g h t i n Year 1 and 2 Male Female  4 6  6 10  8 7  9 6  11 9  10 16  17 13  88 T a b l e 11. Number o f females w h i c h spawned d u r i n g t h e 14-week spawning p e r i o d i n Year 1 and t h e 11-week spawning p e r i o d i n Year 2.  Week o f spawning  Diet LLLE LLHE HLLE HLHE COMM  Date  LL  HL  YEAR I t 1- 2 3- 4 5- 6 7- 8 9-10 11-12 13-14  Sep. Oct. Oct. Nov. Nov. Dec. Dec.  29-Oct. 12-Oct. 26-Nov. 9-Nov 23-Nov. 7-Dec. 21-Dec.  5/88 19/88 2/88 17/88 30/88 14/88 27/88  Total No. weeks o f spawning  -— -6  -—  -1  1 4 1  -1  2 3 4 1 2  1 2 1 2 3 3 1  13 8  7 8  13 11  13 14  3 2 3 1 1  4  —  10 8  7 5  2 3 2  1 1 2 6  -  1 10 3 3 3  1 3 3 5 7 4 3  10 8  20 9  26 14  1 2 6  _  -  — —  1  YEAR 2: 1- 2 3- 4 5- 6 7- 8 9-10 11  Oct. Oct. Nov. Nov. Dec. Dec.  11-Oct. 25-Nov. 8-Nov. 22-Nov. 6-Dec. 20/89  Total No. weeks o f spawning  18/89 2/89 15/89 29/89 13/89  _  1 2 3  1 3 2  2 1  2  6 6  2  —  -  -  6 6  9 6  —  3 2 7 3 2  1 3 6 2  17 8  12 8  2  -  :  I n c l u d i n g one f i s h which had o v u l a t e d eggs when found dead on floor. P l u s 2 f i s h f e d LLHE and 1 f i s h f e d HLLE w h i c h c o n t a i n e d m a t u r i n g eggs when t h e y d i e d as a r e s u l t o f human e r r o r a t 8 weeks. 1  2  89 and  showed  that  there  was a s i g n i f i c a n t  difference  in  the  p r o p o r t i o n o f f i s h f e d t h e HL d i e t s spawning e a r l i e r t h a n t h o s e f e d t h e LL d i e t s . diets  I n Year 1, f i s h f e d b o t h t h e L L and t h e HL  f i n i s h e d spawning  on December  e n d i n g 12 weeks a f t e r i t had begun.  27, 1988 w i t h  spawning  The spawning p e r i o d l a s t e d  9 weeks f o r f i s h f e d t h e LL d i e t s and 14 weeks f o r f i s h f e d t h e HL d i e t s .  F i s h f e d t h e COMM d i e t spawned o v e r a 9-week p e r i o d  w h i c h began d u r i n g t h e t h i r d week o f spawning.  To o b s e r v e i f t h e e f f e c t o f d i e t a r y l i p i d  on spawning  day was r e p e a t a b l e , f i s h f e d t h e LL d i e t s i n Year 1 were f e d t h e HL d i e t s i n Year 2 and v i c e v e r s a w h i l e k e e p i n g t h e v i t a m i n E l e v e l s t h e same ( F i g u r e 3 ) .  Spawning began a minimum o f 12 days  l a t e r i n Year 2 compared t o Year 1 b e g i n n i n g on O c t o b e r 11, 1989 for  f i s h f e d t h e COMM d i e t and not u n t i l on o r a f t e r October 22  f o r t h o s e f e d t h e o t h e r d i e t s (Table 1 1 ) .  D i e t a r y l i p i d had no  e f f e c t on t h e spawning p e r i o d i n Year 2.  Spawning fertilization  day was  positively  correlated  i n Year 1 (r =0.143, Appendix 2  significant  effect  of  spawning  day on  egg  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 w e i g h t i n Year 1 (r =0.143). 2  with  There was no  egg w e i g h t , egg  f e r t i l i z a t i o n o r 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 =0.249). 2  90  91  92 4.6.2 Number o f r i p e males and females Chi  s q u a r e 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 o r v i t a m i n E  concntration  had no s i g n i f i c a n t  effect  on t h e p o r p o r t i o n o f  males w h i c h produced m i l t i n Year 1 o r Year 2 ( T a b l e s 12 and 13).  F i s h e r ' s e x a c t t e x t showed t h e r e was no e f f e c t o f d i e t a r y  vitamin  E concentration  on t h e p r o p o r t i o n  o f females  which  spawned i n Year 1 o r 2.  A t o t a l o f 9 males d i d n o t mature i n e i t h e r spawning season w i t h 5 f e d t h e LE d i e t s and 4 f e d t h e HE d i e t s  (Table 14). A  p o r t i o n o f t h e p o p u l a t i o n which produced m i l t i n Year 1 d i d n o t produce i t i n Year 2 and v i c e v e r s a . significantly  F i s h f e d HE d i e t s had a  h i g h e r p r o p o r t i o n o f males w h i c h produced m i l t i n  b o t h y e a r s compared t o t h e LE d i e t s (x =4.030). 2  The l o w sample  s i z e and t h e c l o s e n e s s o f t h e P v a l u e t o s i g n i f i c a n c e s u g g e s t s that  this  a r e a s h o u l d be i n v e s t i g a t e d  further.  Diet  s i g n i f i c a n t e f f e c t on c h a n g i n g t h e p r o p o r t i o n o f males  had no becoming  r i p e i n Year 2 w h i c h had n o t been n o t r i p e i n Year 1 ( x 0 . 2 0 2 ) . 2 =  4.6.3 A t y p i c a l In their  Year  spawning 1, one f i s h  f e d LLLE  and one f e d COMM r e l e a s e d  eggs i n t h e t a n k and b o t h f i s h  died within  2-3 weeks.  Embryonic s u r v i v a l was poor b e i n g 14% eyed f o r t h e former and 0% eyed f o r t h e l a t t e r .  One f i s h f e d HLHE produced eggs which were  s p o t t e d and appeared o v e r r i p e (38% eyed) and t h e r e was b l o o d i n the  ovarian f l u i d .  T h i s f i s h d i e d w i t h i n 2 weeks.  93 T a b l e 14. Number o f males w h i c h produced m i l t ( r i p e ) i n Year 1 w h i c h d i d o r d i d n o t produce m i l t i n Year 2.  D i e t f e d i n Year 1 D i e t f e d i n Year 2 Tank  Year 1  Year 2  Ripe Ripe Total  Ripe Not r i p e  X  2  f o rvitamin E  Year 1  Year 2  Not r i p e Not r i p e Total  Ripe Not r i p e  X  2  f o rvitamin E  HLLE LLLE 6  2 2 4 4.030  3 4 7 0.202  HLHE LLHE 2  LLLE HLLE 3  4 2 6  2 5 7  LLHE HLHE 7  COMM COMM 5  LE  HE  8 2 10  8 0 8  4 7 11  12 4 16  2 2 4  4 0 4  5 5 10  6 4 10  P<0 .05  4 2 6  2 1 3  P>0.10  94 Problems o c c u r r e d w i t h 3 f i s h f e d HLLE i n Year 2, w i t h f i s h releasing in  the ovarian  one  most o f i t s eggs i n t h e t a n k and b l o o d o c c u r r i n g fluid  o f one  b r o w n c o l o u r e d eggs.  f i s h which  Brown d e b r i s was  f l u i d of a t h i r d f i s h .  One  also  released  present i n the  50-60 ovarian  f i s h f e d LLLE had eggs w h i c h c o u l d  not be r e l e a s e d and t h e y appeared t o be i n a s o l i d mass.  One  f i s h f e d LLHE produced eggs w h i c h i n c l u d e d about 12 d a r k orange eggs and t h i s f i s h d i e d w i t h i n mortality,  primarily  4 weeks o f spawning.  2,  I n Year  o f f e m a l e s , i n c r e a s e d a t spawning  in a l l  groups e x c e p t t h o s e f e d LLLE and COMM and t h i s was due t o f u n 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  F e c u n d i t y and egg s i z e  ( d i a m e t e r and w e i g h t )  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 w e i g h t i n e i t h e r Year 1 o r Year 2 ( T a b l e s 15 and 16, F i g u r e 6a and 6 b ) . There was no e f f e c t o f d i e t on t h e w e i g h t and d i a m e t e r o f w a t e r hardened  p r e s e r v e d eggs  except  for a  significant  v i t a m i n E on egg d i a m e t e r i n Year 1 ( T a b l e 1 7 ) . of v a r i a t i o n to  24.1%  of  coefficient  (CV) f o r eggs produced by each f i s h ranged from 4.5  (mean=8.5)  f o r egg  weight  (mean=3.7) f o r d i a m e t e r i n Year 1. Year  The  effect  2 w i t h CV  r a n g i n g from 3.0  w e i g h t and 1.9 t o 8.2%  and  from  There was t o 16.6%  1.2  to  6.3%  l i t t l e change i n  (mean=7.5) f o r egg  (mean=3.7) f o r d i a m e t e r .  No s i g n i f i c a n t e f f e c t s o f d i e t on t h e t o t a l w e i g h t o f eggs o r number o f eggs produced p e r kg body w e i g h t ( T a b l e 15) were  95  96  97  F i g u r e 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 v a l u e s .  98  99 observed. no  Some eggs were r e l e a s e d a f t e r spawning and t h e r e was  significant  spawning  difference  i n t h e number o f eggs r e l e a s e d a t  o r t h e t o t a l number o f eggs produced.  InYear 2,  fish  f e d COMM weighed s i g n i f i c a n t l y more t h a n t h o s e f e d o t h e r d i e t s and t h e y 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  w e i g h t o f eggs t h a n t h o s e f e d a l l d i e t s e x c e p t LLLE ( T a b l e 1 6 ) . There  was  no  significant  difference  produced p e r kg body w e i g h t .  i n the  number  of  eggs  The numbers o f eggs produced were  h i g h l y v a r i a b l e w i t h i n each t r e a t m e n t i n b o t h y e a r s as shown i n Figures  7a  and  7b.  The  total  number o f  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 b o t h Year 1 (r =0.334) 2  and Year 2 (r =0.853) ( F i g u r e s 8a and 8b, Appendix 4 but was 2  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 w e i g h t .  Some  fish  lost  weight any  throughout  expected  t o produce  eggs.  produced  s m a l l numbers o f eggs.  Year  2  and  However, some o f  were these  not fish  F o r example, 2 f i s h f e d LLHE  r e l e a s e d l e s s t h a n 350 eggs w i t h s u r v i v a l o f 61 and 94% t o t h e eyed  stage  and  1  fish  f e d HLHE r e l e a s e d 525  s u r v i v a l t o t h e eyed s t a g e . fertilization,  F e c u n d i t y was  eggs w i t h  95%  not c o r r e l a t e d w i t h  s u r v i v a l t o t h e eyed, h a t c h o r swimup s t a g e o r  w i t h s u r v i v a l o r s p e c i f i c growth r a t e o f 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 h a t c h and swimup s t a g e s  was  d i r e c t l y r e l a t e d t o egg w e i g h t i n Year 2 (r =0.11-0.12) (Appendix 2  4) but was  not c o r r e l a t e d i n Year 1.  100  F i g u r e 7. T o t a l number o f 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 v a l u e s .  101  F i g u r e 6. R e l a t i o n s h i p o f t o t a l number o f eggs produced and f i s h weight i n : a) Year 1 (Y= 2.71X + 351.88, r =0.334) and b) Year 2 (Y= 3.88X + 2202.25, r = 0 . 8 5 3 ) . 2  2  102 4.8 Embryonic  survival  More eggs were g e n e r a l l y  collected  and  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.  fertilized  than  Excess eggs were  i n c u b a t e d i n Heath t r a y s u n t i l t h e y r e a c h e d t h e eyed s t a g e and t h e n t h e y were d i s t r i b u t e d t o p r o d u c e r s . I n Year 2, Heath t r a y s were c o m p a r t m e n t a l i z e d and eggs from each female were m a i n t a i n e d separately.  S u r v i v a l t o t h e eyed s t a g e was comparable f o r eggs  i n c u b a t e d i n Heath t r a y s compared t o t h e j a r system.  When f e r t i l i z e d eggs were viewed under a low power m i c r o s c o p e , d i v i d e d c e l l s c o u l d be seen w h i c h v a r i e d from t h e 4 - c e l l to  16-32  c e l l s t a g e ( T a b l e s 18 and 19, F i g u r e s 9 and 1 0 ) .  Year 2, 2 f i s h  f e d LLHE produced eggs i n w h i c h abnormal  cell  d i v i s i o n and t h e y had v i t a m i n E c o n c e n t r a t i o n s o f 236 and fxg/g. fish  242  A b n o r n a l c e l l d i v i s i o n was a l s o o b s e r v e d i n eggs from 3 f e d HLLE and t h e v i t a m i n E c o n c e n t r a t i o n s were 37, 54 and  59 M-g/g. some  In  C e l l s appeared t o have d i v i d e d u n e v e n l y and/or were  distance  apart.  As  the  percentage  incidence  which  reached  of  abnormal  the  eyed  eggs  increased,  the  stage  decreased.  Abnormal c e l l s  illustrated  i n L e r a y e t a l . (1985) which were a p p a r e n t l y due t o  were s i m i l a r i n appearance t o t h o s e  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 t h e 5 d i e t a r y treatments i n f e r t i l i z a t i o n  o r s u r v i v a l t o t h e eyed, h a t c h o r  swimup s t a g e i n Year 1 o r 2 ( T a b l e 18 and 19, F i g u r e s 9 and 1 0 ) .  103 T a b l e 18. F e r t i l i z a t i o n and embryonic s u r v i v a l (mean ± s t a n d a r d d e v i a t i o n ) i n Year 1. 1  Diet fed i n Year 1  n  1  Fertilized %  +  SD  Eyed  Hatch  Swimup  %  +  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 o f v a r i a n c e Source o f v a r i a t i o n A l l diets E x c l u d i n g COMM Lipid Vitamin E Lipid x V i t E  LLLE LLHE HLLE HLHE COMM  ns  ns  ns  ns  ns ns ns  ns ns ns  ns * *  ns * *  Eved/Fert % + SD 71 + 29  Hatch/Eved % t: SD + 27 64  Swimup/Hatch % + SD 84 + 19  90  +  7  94  82  +  32  83  +  18  7  17  91  +  7  70  +  31  66  b  +  34  82  +  24  74  +  39  93  a  +  6  91  +  6  _  b  a  gab  + +  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 o f v a r i a n c e Source o f v a r i a t i o n A l l diets ns ** ns E x c l u d i n g COMM Lipid ns ns ns Vitamin E ns ** ns Lipid x V i tE * ** ns 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 d a t a p r i o r t o a n a l y s i s of variance. ns = n o t s i g n i f i c a n t , P>0.05, * = P < 0 , 0 5 , * * = P < 0 . 0 1 1  104  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 o f eggs t o t h e eyed, h a t c h and swimup s t a g e s i n Year 1 showing minimum, 25, 50, 75 p e r c e n t i l e s and maximum v a l u e s .  105 T a b l e 19. F e r t i l i z a t i o n and embryonic s u r v i v a l (mean ± s t a n d a r d d e v i a t i o n ) i n Year 2. 1  Diet fed i n Year 2  n  Fertilized %  ± SD  Eyed  Hatch  Swimup  %  +  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 o f Source o f v a r i a t i o n A l l diets ns ns ns E x c l u d i n g COMM Lipid ns ns ns Vitamin E ns ns ns L i p i d x V i t E ns ns ns  variance ns ns ns ns  Eved/Fert. % + SD  Hatch/Eved % + SD  Swimup/Hatct % + 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 o f v a r i a n c e Source o f v a r i a t i o n A l l diets E x c l u d i n g COMM Lipid Vitamin E Lipid x V i tE  ns  ns  ns  ns  ns ns ns  ns ns ns  ns ns ns  ns ns ns  Arcsin transformation was used on a l l data p r i o r t o analysis of variance, ns = n o t s i g n i f i c a n t , P>0.05 1  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 o f eggs t o t h e eyed, h a t c h and swimup s t a g e s i n Year 2 showing minimum, 25, 50, 75 p e r c e n t i l e s and maximum v a l u e s .  107 In Year 1, t h e r e was a s i g n i f i c a n t e f f e c t o f d i e t a r y 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 h a t c h and  swimup  resulted  i n an  increase  i n survival  as  dietary  v i t a m i n E i n c r e a s e d i n t h e LL d i e t s and a d e c r e a s e i n s u r v i v a l as v i t a m i n E i n c r e a s e d i n t h e 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 t h e s t a g e a t w h i c h embryonic development c e a s e d , t h e f o l l o w i n g v a l u e s were calculated  and  compared:  Fertilized,  Hatch/Eyed and Swimup/Hatch ( T a b l e 18 and 1 9 ) .  Eyed/Fertilized, A v a l u e was not  i n c l u d e d i n t h e a n a l y s i s i f b o t h t h e denominator and were z e r o . the  eyed  numerator  I n Year 2, most m o r t a l i t y o c c u r r e d p r i o r t o r e a c h i n g stage.  r e a c h e d t h e eyed  Only  37-56%  o f eggs  that  were  fertilized  s t a g e i n a l l groups e x c e p t t h e HLHE group,  where 75% o f t h o s e f e r t i l i z e d reached t h e eyed s t a g e .  Of t h e  eggs t h a t r e a c h e d t h e eyed s t a g e i n Year 2, 67 t o 84% h a t c h e d . S u r v i v a l was low (45%) between h a t c h and swimup f o r f r y from t h e HLLE group compared t o o t h e r groups ( 7 4 - 8 3 % ) .  When t h i s e x p e r i m e n t was i n i t i a t e d , a p p r o x i m a t e l y o n e - h a l f of t h e s t o c k o f A r c t i c c h a r r was used.  The r e m a i n i n g f i s h were  m a i n t a i n e d as u s u a l and f e d t h e COMM d i e t ( a l t h o u g h i t may  have  been from d i f f e r e n t shipments as t h e d i e t used i n t h e p r e s e n t experiment).  A l t h o u g h r e c o r d s were not k e p t on t h e s u r v i v a l o f  eggs from i n d i v i d u a l  fish,  the o v e r a l l  survival  t o the  eyed  s t a g e was 72% i n Year 1 and f i s h r e l e a s e d 3300 eggs a t spawning.  108 I n Year 2, r e c o r d s 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 t h e eyed s t a g e 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 a t spawning.  T h i s was s i m i l a r t o s u r v i v a l t o  t h e eyed s t a g e o f 68% ± 37 i n Year 1 and 37% ± 29 i n Year 2 f o r f i s h f e d t h e COMM d i e t i n t h i s e x p e r i m e n t . for  the fish  F e c u n d i t y was h i g h e r  f e d COMM i n t h e e x p e r i m e n t a l  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% o f f r y had b l u e - s a c  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 with diet.  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. in  at least  affected.  There was a l s o e v i d e n c e o f " c o a g u l a t e d y o l k "  8 cases  w i t h p r o p o r t i o n s o f over  A hemorrhagic a r e a was v i s i b l e i n t h e c o a g u l a t e d y o l k  i n some c a s e s .  F r y from t h e HLHE group had t h e l o w e s t i n c i d e n c e  of coagulated yolk.  Premature h a t c h was observed  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 . blue-sac  20% o f t h e f r y  has been  reported  i n 5 cases i n  A condition similar t o  i n sunapee  (Salvelinus alpinus  oquassa) and l a k e c h a r r ( S a l v e l i n u s namaycush) ( B a l o n 1980) when t h e y were i n c u b a t e d a t a h i g h temperature  (9.5°C).  B l u e - s a c 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 B a l o n  the y o l k sac separated fluid  accumulated.  (1980), h i s t o l o g i c a l e x a m i n a t i o n  showed  from the y o l k and became i n f l a t e d as  109 4.9 Egg and f r y 4.9.1  composition  Vitamin E R e p r o d u c i b i l i t y o f 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  indicated  a mean  coefficient  of variation  o f 3.7% f o r  fry  (n=29), and 6.1 (n=57) and 4.1% (n=38) f o r eggs i n Y e a r 1 and Year 2, r e s p e c t i v e l y . When eggs were weighed o u t f o r v i t a m i n E e x t r a c t i o n and assay, began t o thaw.  they  d i s i n t e g r a t e d as soon as t h e eggs  T h i s 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 o f the r e p r o d u c i b i l i t y o f t h e assay. Reproducibility hydroxytoluene  improved (BHT),  s o l v e n t , a t 50 mg/L.  when  was  added  the  antioxidant,  t o hexane,  butylated  the extraction  A d d i t i o n o f BHT was n o t n e c e s s a r y  e x t r a c t i o n o f v i t a m i n E from f r y .  The e x t r a c t i o n b l a n k  during indi-  c a t e d a need f o r a c o r r e c t i o n f a c t o r o f 9.92 \ag v i t a m i n E p e r a s s a y f o r t h e egg samples and 5.63 jig f o r t h e f r y samples, w i t h t h e d i f f e r e n c e b e i n g due t o t h e a d d i t i o n o f BHT f o r t h e a n a l y s i s o f t h e eggs.  R e c o v e r y was measured by s p i k i n g a sample o f f r y w i t h a known  amount  of  95% pure  Company, S t . L o u i s , MO). sample o f t h e f i n a l treatment  (Sigma  Chemical  Recovery was 100.8 ± 0.54% (SD). A  e t h a n o l i c e x t r a c t from f r y from t h e HLHE  was s u b j e c t e d  u l t r a v i o l e t range.  dl-a-tocopherol  t o a spectrophotometric  scan  i n the  The major peak o c c u r r e d a t a w a v e l e n g t h o f  288-292 m\i w h i c h c o r r e s p o n d e d t o a - t o c o p h e r o l a t 292 m\i ( B u d v a r i  110 et al. 1989).  A s m a l l peak was e v i d e n t a t 280 mu, b u t no peaks  o c c u r r e d 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 a b s o r p t i o n peak o f 325 m\i was not p r e s e n t i n t h e e x t r a c t .  Increasing  dietary vitamin  E concentration  resulted i n  s i g n i f i c a n t i n c r e a s e s i n t h e v i t a m i n E c o n c e n t r a t i o n and c o n t e n t o f eggs and f r y i n b o t h y e a r s addition,  i n Year  2 there  (Table  20, F i g u r e  11-13).  was a l s o a s i g n i f i c a n t  In  effect 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 t h e e f f e c t o f l i p i d and v i t a m i n E on t h e eggs b u t not t h e f r y . concentration treatments  and c o n t e n t  associated  The rank o f v i t a m i n E with  remained t h e same i n b o t h y e a r s  the 5  dietary  f o r eggs and f r y ,  i n c r e a s i n g i n t h e o r d e r o f LLLE, HLLE, COMM, HLHE, LLHE. Year  2,  there  were  significant  differencesin  vitamin  In E  c o n c e n t r a t i o n and c o n t e n t o f eggs from f i s h f e d each d i e t w h i l e i n Year 1 t h e r e 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 o f eggs from f i s h f e d LLLE, HLLE o r 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 v i t a m i n E c o n c e n t r a t i o n  (2-3-fold increase)  t h a n a t t h e 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 t h e LLHE d i e t , swimup f r y g e n e r a l l y had a h i g h e r v i t a m i n E c o n c e n t r a t i o n and c o n t e n t t h a n t h e eggs.  1]  F i g u r e 11. V i t a m i n E c o n c e n t r a t i o n 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 v a l u e s .  F i g u r e 12. V i t a m i n E c o n t e n t o f 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 v a l u e s .  F i g u r e 13. V i t a m i n E c o n c e n t r a t i o n and c o n t e n t 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 v a l u e s  115 There  was  concentrations  no  overlap  i n t h e range  of  vitamin  E  o f t h e LE eggs (30-74 \ig/g) compared t o t h e HE  eggs (109-275 ng/g).  The COMM eggs were i n t e r m e d i a t e  between  the  (42-94  greater  LE and HE eggs  \ig/g).  There  was much  v a r i a b i l i t y i n v i t a m i n E c o n c e n t r a t i o n f o r t h e HE eggs compared to  t h e LE eggs.  Within  t h e HE eggs,  LLHE  had g r e a t e r  v a r i a b i l i t y t h a n HLHE w i t h eggs from f i s h f e d LLHE r a n g i n g  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 d r y m a t t e r 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 p e r c e n t a g e s o f d r y m a t t e r o r t o t a l l i p i d i n eggs from f i s h f e d any o f t h e d i e t s i n Y e a r 1 ( T a b l e 2 1 , F i g u r e 14a). total  lipid  o r d r y m a t t e r and s u r v i v a l  s p e c i f i c growth r a t e o f f r y . had  There was no c o r r e l a t i o n between  greater  percent  o f embryos o r f r y o r  I n Year 2, eggs from f i s h f e d COMM  d r y matter  than  those  o f HLLE and  s i g n i f i c a n t l y g r e a t e r p e r c e n t t o t a l and n e u t r a l l i p i d t h a n noted for  f i s h f e d a l l o t h e r d i e t s e x c e p t LLLE ( T a b l e 22, F i g u r e 14b  and  14c).  lipid  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 p e r c e n t  (Table  correlated  22, F i g u r e  t o survival  14d).  Neutral  lipid  t o t h e eyed, h a t c h  polar  was n e g a t i v e l y  and swimup  stages  (r =0.26-0.34, P<0.003) ( F i g u r e s 15a, 16a, 17a and Appendix 4 ) . 2  Analysis  o f covariance  between d i e t s i n t h i s  showed  that  relationship.  there  was no d i f f e r e n c e  116 T a b l e 21. T o t a l l i p i d and d r y m a t t e r c o n t e n t s (mean 1 s t a n d a r d d e v i a t i o n ) o f eggs i n Year 1.  Diet  Dry matter % ± SD  Total lipid %  ± SD  LLLE  37.0±2.6  22.511.1  LLHE  38.1 ± 1.5  HLLE  38.411.1  23.211.0  HLHE  38.610.9  22.311.0  COMM  38.310.8  22.210.9  22.2 ± 1.3  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 o f v a r i a n c e Source o f v a r i a t i o n A l l diets E x c l u d i n g COMM Lipid Vitamin E Lipid x V i tE  ns  ns  ns ns ns  ns ns ns  ns = n o t 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 v a l u e s .  118  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 =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 =0.238) on s u r v i v a l t o t h e eyed s t a g e i n Year 2. 2  2  F i g u r e 16. R e l a t i o n s h i p o f : a) n e u t r a l l i p i d s (Y= -18.24X + 236.20, r =0.321) and 2  b) 16:ln7 i n n e u t r a l l i p i d s (Y= -12.92X + 186.58, r =0.234) on s u r v i v a l t o h a t c h i n Year 2. 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 =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 =0.228) on s u r v i v a l t o swimup i n Year 2. 2  2  122 4.9.3  Fatty acid composition The  fatty  acid  compositions  of the diets  T a b l e s 23 and 24 and Appendix l a . composition o f the diets  similar Total  i n most s a t u r a t e d , lipids  Comparison o f t h e f a t t y a c i d  and t h e n e u t r a l  eggs a r e shown i n F i g u r e s  a r e shown i n  18-20.  and p o l a r  lipids of  The LL and HL d i e t s  monoenoic, n6 and n3 f a t t y  o f t h e HL d i e t s  monoenoic and n i l f a t t y a c i d s  had s i g n i f i c a n t l y more (specifically,  16:ln7,  were acids. total  20:lnll,  2 2 : l n 9 , 2 2 : l n l l ) , 18:4n3, 20:5n3 and 22:5n3 compared t o t h e LL diets.  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  t o t a l n6 and t o t a l PUFA t h a n the LL d i e t s . Year 1 d i f f e r e d d r a m a t i c a l l y  18:2n6,  18:3n3,  The COMM d i e t f e d i n  i n f a t t y a c i d c o m p o s i t i o n from t h a t  f e d i n Y e a r 2. F a t t y a c i d s i n the COMM d i e t w h i c h d i f f e r e d most between Year 1 v s Year 2 i n c l u d e d 20:ln9 (4.5  v s 11.3%), 2 2 : l n l l  5.1%), t o t a l s a t u r a t e d vs  (5.4  (24 v s 18%)  16:0 (15.8  vs 12.2%),  v s 15.5%), 20:5n3 (10.5 v s  and monoenoic f a t t y a c i d s (35  52%).  F a t t y a c i d c o m p o s i t i o n o f n e u t r a l and p o l a r eggs, a t l e v e l s above 0.1% lc.  the  Selected  l i p i d s of the  o f l i p i d , appear i n A p p e n d i c e s l b  and  f a t t y a c i d s and t o t a l v a l u e s f o r f a m i l i e s o f f a t t y  a c i d s p r e s e n t 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 a r e shown i n T a b l e s 25-28 w i t h i n d i c a t i o n s o f the s i g n i f i c a n c e o f d i f f e r ences among them.  123 T a b l e 23. S e l e c t e d major f a t t y a c i d s i n t h e t o t a l l i p i d o f t h e diets. Values are % o f t o t a l l i p i d .  Fatty acid  LLLE  %  Diet HLLE  LLHE  %  HLHE  COMM COMM L L Year 1 Year 2  %  %  %  %  2  %  HL  2  %  14:0  4 .16  4 .11  4.44  4.09  5.14  3 .86  4 .14  4 .27  16:0  16 .40  16 .43  15.97  15.61  15.77  12 .16  16 .42  15 .79  18:0  2 .72  2 .69  2.68  2.69  2.53  1 .63  2 .71  2 .69  16:ln7  5 .06  5 .05  5.51  5.36  7.76  6.97  5 .06  5 .44  18:ln7  3 .70  3 .72  3.81  3.62  2.64  2 .35  3 .71  3 .72  18:ln9  18 .71  18 .60  18.48  18.83  12.44  10 .93  18 .66  18 .66  20:ln9  3 .30  3 .29  3.52  3.32  4.49  11 .32  3 .30  3 .42  22:ln9  1 .01  1 .00  1.17  1.16  0.61  1 .68  1 .01  a  1 .17  b  20:lnll  3 .84  3 .77  4.63  5.04  0.43  0.68  3 .81  a  4 .84  b  22:lnll  6.60  6.50  7.49  7.83  5.39  15 .52  6 .55  a  7.66  b  18:2n6  7.46  7.40  4.90  4.97  10.68  10 .52  7 .43  a  4 .94  b  20:4n6  0.62  0.62  0.63  0.64  0.39  0 .25  0.62  0.64  18:3n3  1 .32  1 .32  1.00  1.01  1.32  1 .01  1 .20  a  1 .07  b  18:4n3  0.88  0.88  1.00  1.00  1.77  1 .03  0.88  a  1 .00  b  20:5n3  5 .97  5 .92  6.20  6.36  10.45  5 .05  5 .95  a  6.28  b  22:5n3  0.92  0.90  1.03  1.08  1.13  0 .61  0.91  a  1 .06  b  22:6n3  6 .77  6 .75  6.33  6.58  5.89  4 .90  6 .76  a  b  6 .46  Complete f a t t y a c i d p r o f i l e appears i n Appendix l a . V a l u e s 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 a r e 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). 1  2  124 T a b l e 24. T o t a l s f o r f a m i l i e s o f f a t t y a c i d s i n t h e t o t a l of t h e d i e t s . Values are % o f t o t a l l i p i d .  Fatty acid type  LLLE  LLHE  %  Diet HLLE HLHE  %  %  COMM COMM LL Year 1 Year 2 %  %  %  18 .49  S a t u r a t e d 24,.87  24 .88 24,.80 24,.06  24,.26  Monoenoic 44,.60  44 .27 47,.15 47,.72  35,.35 51,.97  n5  0,.50  n7  9,.54  n9  24,.12  23 .94 24,.29 24,.46  nil  10,.44  10 .27 12..12  n6  9..04  n3  16..87  0.52  lipid  HL  1  %  1  %  24,.88  24,.43  44,.44 47,.43* a  0,.55  0,.53  0,.48  0,.63  0,.51  0,.54  9.54 10,.19  9,.86  10,.86  10,.49  9,.54  10,.03  18,.19 24,.66  24,.03  24,.38  9.02  6..62  16 .81 16..70  12,.87  5..82  16,.19  10..36  a  12..50  b  6.,69  12..46  11. .57  9.,03  a  6..66  b  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..86  a  2.,54  b  PUFA  11. .70  11 .70  9.,15  9..21  16..33  14..06  11. .70  a  9..18  b  HUFA  14..36  14 .30 14.,34  14,.80 19.,30 11. ,41  14.,33  V a l u e s w i t h i n a row which have 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). 1  different  14.,57  superscripts are  125  LLLE  SAT  n7 SAMPLE  n9 D  I  E  nil  PUFA  HUFA  T  F i g u r e 18. Comparison o f s a t u r a t e d , n7, n9, n i l monoenoic a c i d s , PUFA and HUFA o f the t o t a l l i p i d s o f t h e d i e t s w i t h the n e u t r a l ( N L ) and p o l a r l i p i d s ( P L ) o f t h e eggs i n Year 2  126  LLLE  "V"  JStwm  LLHE  50 -  HLLE  40 30 20 10 n u50 -  HLHE  18:3n3 SAMPLE  20:5n3  22:5n3 DIET  22:6n3 NL  n3 PL  F i g u r e 19. Comparison o f n3 f a t t y a c i d s o f t h e t o t a l l i p i d s of t h e d i e t s w i t h t h e n e u t r a l (NL) and p o l a r l i p i d s (PL) o f the eggs i n Year 2.  12 10 8 % 6 4 2 0  127  LLLE  12 10 8 % 6 4 2 0 12 10 8 % 6 4 2 0  HLLE  12 10 8 % 6 4 2 0 12 10 8 % 6 4 2 0  ure . Comparison o f n6 f a t t y a c i d s o f t h e t o t a l l i p i d s the « e t s w i t h the n e u t r a l (NL) and p o l a r l i p i d s (PL) o f egg; i n Year 2.  128 The  fatty  acid profiles of theneutral  lipids  show t h a t  eggs from f i s h f e d 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  f e d LL and COMM and more 22:5n3 t h a n t h o s e o f  f i s h f e d LLLE.  Eggs from f i s h f e d COMM had s i g n i f i c a n t l y h i g h e r  18:2n6, 2 0 : l n 9 , t o t a l n6 and PUFA and l o w e r 18:ln9 and n3/n6 t h a n eggs from f i s h  fed other d i e t s .  Negative  correlations  e x i s t e d between 16:ln7 and t o t a l n7 i n t h e n e u t r a l l i p i d s and survival  t o eyed,  hatch  and swimup  stages  (r =0.23-0.24, 2  P<0.0008) ( F i g u r e s 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 t h e s l o p e 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.  Analysis  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 t h e p o l a r l i p i d s o f t h e 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 t h a n i n t h e LL and COMM eggs. more 20:5n3 dietary  than  found  P o l a r l i p i d s o f HLHE eggs had  i n LL eggs.  There was an e f f e c t o f  v i t a m i n E c o n c e n t 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 v i t a m i n E c o n c e n t r a t i o n s i n 2 0 : l n 9 w i t h LLHE eggs h a v i n g s i g n i f i c a n t l y more 20:ln9 i n t h e p o l a r l i p i d s LLLE and HLHE eggs. and  lower i n t o t a l  than  COMM eggs were h i g h e r i n 18:2n6 and 20:ln9 saturated fatty  compared t o a l l o t h e r t r e a t m e n t s .  acids i n thepolar  lipids  T a b l e 25. S e l e c t e d major f a t t y a c i d s i n t h e n e u t r a l l i p i d s eggs. V a l u e s a r e % o f n e u t r a l l i p i d . 1  Means w i t h i n a row w h i c h s h a r e a common s u p e r s c r i p t a r e not significantly different. Complete f a t t y a c i d p r o f i l e i s g i v e n i n Appendix l b . 1  130 T a b l e 26. T o t a l s o f f a m i l i e s o f f a t t y a c i d s i n t h e n e u t r a l l i p i d s o f eggs. V a l u e s a r e % o f n e u t r a l l i p i d .  Fatty acid  LLLE  Diet HLLE  LLHE  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  1.07  1.10  1.35  1.26  1.52  nil n6  8.16  n3  16.02  a  8.26  a  16.32  7.85  a  17.91  a  2.03  a  2.40  PUFA  10.10  a  9.81  a  9.86  HUFA  14.63  15.28  16.43  a  17.71  1.98  n3/n6  8.17  a  a  2.21  b  15.68  a  10.34  10.65  a  16.08  Means w i t h i n a row which share a common s u p e r s c r i p t significantly different.  1.48  b  12.88  b  14.17  are not  T a b l e 27. S e l e c t e d major f a t t y a c i d s eggs. V a l u e s a r e % o f p o l a r l i p i d .  1  i n the polar l i p i d s  Means w i t h i n a row w h i c h s h a r e a common s u p e r s c r i p t a r e not significantly different. Complete f a t t y a c i d p r o f i l e i s g i v e n i n Appendix l c . 1  T a b l e 28. T o t a l s o f f a m i l i e s o f f a t t y a c i d s i n t h e p o l a r l i p i d s o f eggs. V a l u e s a r e % o f p o l a r l i p i d .  Means w i t h i n a row which significantly different.  s h a r e a common s u p e r s c r i p t  are  not  133 4.9.4  Selenium S e l e n i u m c o n c e n t r a t i o n s ranged from 1.38-3.09 \ig/g i n t h e  eggs  i n Year  matterbasis. dietary  1  and  1.70-3.58  \ig/g i n Year  Selenium concentrations  treatment  2  on  a  dry  were n o t i n f l u e n c e d by  and had no s i g n i f i c a n t  effect  on embryonic  survival.  4.9.5 C o m p o s i t i o n o f w i l d A r c t i c c h a r r eggs Eggs w h i c h had been c o l l e c t e d from w i l d s t o c k s  a t Tree  R i v e r , NWT were a n a l y z e d when they were c l o s e t o h a t c h i n g . weighed 122 mg and c o n t a i n e d  Eggs  (9.0 jAg/egg)  73.8 \ig v i t a m i n E/g  and 3.97 ng s e l e n i u m / 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  Tree  and  matter,  87%  swimup.  River  eggs  contained  67.3% d r y  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 d r y m a t t e r b a s i s .  Compared t o t h e L a b r a d o r eggs i n t h i s  experiment, n e u t r a l l i p i d  from t h e Tree R i v e r eggs  contained  22:5n3 and l e s s 18:ln9 and 18:2n6 (Tables 29 and 3 0 ) . 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 h i g h e r n3/n6 r a t i o .  The monoenoic  f r a c t i o n c o n t a i n e d more n7 and l e s s n9 f a t t y a c i d s . in  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  Differences  fraction  between  t h e Tree R i v e r a n d e x p e r i m e n t a l eggs were much l e s s pronounced compared t o t h e 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 i n c r e a s e d n3/n6 r a t i o , but l e s s 1 8 : l n 9 , 22:6n3, t o t a l n6 and n9.  134 T a b l e 29. S e l e c t e d major f a t t y a c i d s o f t h e n e u t r a l and p o l a r l i p i d o f eggs o f A r c t i c c h a r r from Tree R i v e r , NWT. 1  Fatty acid  Neutral lipid  Polar lipid  %  %  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 g i v e n i n Appendix I d .  135 T a b l e 30. T o t a l s o f f a m i l i e s o f f a t t y a c i d s o f t h e n e u t r a l and p o l a r l i p i d o f eggs o f A r c t i c c h a r r from Tree R i v e r , NWT.  Fatty acid  Neutral lipid  Polar lipid  %  %  14.57  21.53  0.24  0.27  50.56  27.94  n5  0.84  0.79  n7  26.02  12.63  n9  23.35  13.92  nil  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  Saturated Branch Monoenoic  136 4.10 F r y s u r v i v a l  and growth  When d i f f e r e n t  numbers o f 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, t h e s p e c i f i c growth r a t e d e c r e a s e d as t h e number o f f r y i n c r e a s e d .  I t was s i g n i f i c a n t l y l o w e r f o r  100 f r y compared t o 10 and 25 f r y p e r j a r ( T a b l e 3 1 ) . addition,  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  In  greater  (2.68% body weight/day) when 300 f r y were r e a r e d i n 60 L t a n k s compared t o any number r e a r e d i n t h e j a r s .  A l t h o u g h t h e r e 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 o b s e r v e d i n the jars  c o n t a i n i n g 100 f r y .  number o f f r y r a i s e d  Based on t h e s e r e s u l t s , t h e  i n t h e j a r system was d e c r e a s e d from 50 p e r  j a r i n Y e a r 1 t o 25 p e r j a r i n Year 2.  There were no e f f e c t s survival  o f f r y i n Year  of diet 1  on s p e c i f i c  (Table 32).  Data  growth r a t e o r f o r HLLE  was  e x c l u d e d from a l l a n a l y s e s o f v a r i a n c e ( T a b l e 33) because o n l y 2 l o t s o f eggs i n t h e HLLE group produced s u f f i c i e n t f r y f o r use in  t h e Year  2 growth  trial.  The s p e c i f i c  growth  rate  was  s i g n i f i c a n t l y l o w e r f o r f r y from t h e LLHE group t h a n a l l o t h e r treatments.  O v e r a l l s u r v i v a l o f f r y d e c r e a s e d from 82% i n Year  1 t o 76% i n Year 2 which was s i m i l a r t o t h e d e c r e a s e from 88% t o 79% t h a t was p r e d i c t e d from t h e d e c r e a s e i n t h e number o f f r y raised  i n the jars.  W i t h t h e e x c e p t i o n o f t h e LLHE group, t h e  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  increase resulting  was  greater  than  the predicted  0.4%  from t h e d e c r e a s e i n number o f f r y i n t h e  137  138  139  140 jars.  E n v i r o n m e n t a l 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  b o t h Year 1 and 2. analysis  showed  p r o t e i n , 25.5%  Feed was purchased from t h e same company and the  total  proximate lipid,  composition  11.8%  was  ash and 82.6%  39.7%  crude  dry matter i n  Year 1 and 49.7% c r u d e 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%  d r y m a t t e r i n Year  2.  The  differences  i n protein  and  l i p i d and/or changes i n t h e i n g r e d i e n t s used by t h e m a n u f a c t u r e r in  Year  specific  2 may  also  growth r a t e .  have  contributed  t o the  differences  in  141 DISCUSSION  5.1 Growth o f b r o o d s t o c k  and f e e d i n g  activity  I n t h e w i l d , A r c t i c c h a r r have t h e p o t e n t i a l f o r a h i g h r a t e o f growth i n a s h o r t p e r i o d . and  early  June,  feed  heavily  They m i g r a t e  i n their  t o s e a i n May  short  time  a t sea  r e t u r n i n g t o f r e s h w a t e r i n t h e l a t t e r h a l f o f J u l y and August (Dempson and Greene 1985).  They f e e d h e a v i l y w h i l e a t s e a and  f i s h o f a l l s i z e s have h i g h s p e c i f i c growth r a t e s d u r i n g s h o r t p e r i o d (Johnson 1980). potential  f o r a high  this  T h i s i n d i c a t e s t h a t t h e y have t h e  r a t e o f growth a t l e a s t  for this  short  p e r i o d o f time a t 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  compare t h e growth r a t e s a c h i e v e d stage o f l i f e ,  i n t h i s experiment.  which t o At this  growth r a t e s a r e seldom m o n i t o r e d i n h a t c h e r i e s .  Growth d a t a c o l l e c t e d on w i l d s t o c k s a r e g e n e r a l l y c o l l e c t e d as length  r a t h e r than weight data.  Dempson  (1982) showed  that  m a r k - r e c a p t u r e d 8-year o l d L a b r a d o r A r c t i c c h a r r had i n c r e a s e d in  l e n g t h by 52 mm (455 t o 507 mm) i n one y e a r .  In the current  e x p e r i m e n t , 4-year o l d s i n c r e a s e d 23 t o 38 mm (414 t o 445 mm) i n t h e 71 day f e e d i n g p e r i o d i n Year 1.  I n Year 2, c h a r r f e d t h e  e x p e r i m e n t a l d i e t s i n c r e a s e d from 42 mm (443 t o 485 mm) which i s slightly  l e s s t h a n t h e i n c r e a s e found by Dempson (1982) w h i l e  t h o s e f e d COMM i n c r e a s e d 76 mm (453 t o 529 mm).  142 In Year 1, female f i s h f e d t h e LL d i e t s had lower  specific  diets. when  growth  rates  t h a n t h o s e f e d t h e HL  This e f f e c t of d i e t a r y Nauyuk  containing protein  Lake  Arctic  10% l i p i d  levels  of  charr  to  meet  fingerlings.  lipid  l e v e l was  fingerlings  either  the  34,  44  or  54%  and COMM  a l s o observed  were  compared t o 15 and 20% l i p i d  D i e t a r y l e v e l s o f 10% l i p i d adequate  significantly  fed  diets  at dietary  (Tabachek  1986).  and/or 34% p r o t e i n not c o n s i d e r e d  growth  requirements  of  Arctic  charr  There was a c r o s s o v e r i n d i e t s a t t h e b e g i n n i n g o f  Year 2 and f i s h f e d LL d i e t s i n Year 1 were f e d HL d i e t s i n Year 2 and v i c e differed  versa.  (although  S i n c e mean w e i g h t o f f i s h not  significantly)  at  f e d each  the  time  diet  of  the  c r o s s o v e r i n d i e t s , comparisons o f t h e growth r a t e s i n Year 2 should  be  viewed w i t h  this  i n mind.  Specific  growth  rates  d e c l i n e d 20-51% from Year 1 f o r females and 24-39% f o r males f e d 4 o f t h e 5 d i e t s and 4% f o r males f e d HLLE.  Labrador  Arctic  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 d u r i n g j u v e n i l e s t a g e s o f l i f e ( P a p s t and Hopky 1983, 1989). the  T h i s wide v a r i a t i o n i n growth r a t e and  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 t h e  s i z e of f i s h a t the beginning of the experiment. the  A portion of  male and female p o p u l a t i o n s i n each t a n k l o s t w e i g h t d u r i n g  some p e r i o d o r t h r o u g h o u t t h e e n t i r e e x p e r i m e n t .  Although a  p a r t i c u l a r l y h i g h p r o p o r t i o n o f males f e d HLLE i n Year 2 weight,  o t h e r males  i n that  tank had  specific  growth  lost rates  143 equivalent  to  those  of  fish  fed  other  diets.  The  p r o p o r t i o n o f males which l o s t weight i n t h a t t a n k may a f f e c t e d by t h e presence o f one a g g r e s s i v e male. males and  5 out o f 11 females  S i x out o f  10  after  I t i s not known i f t h i s  the a f t e r - e f f e c t s of the aggressive f i s h  d i e t o r some o t h e r unknown f a c t o r . not o b s e r v e d  have been  continued t o l o s e weight  removal o f t h e a g g r e s s i v e f i s h . caused by  high  was  o r by  the  S i n c e a g g r e s s i v e f i s h were  i n o t h e r t a n k s , some a d d i t i o n a l f a c t o r ( s ) must be  considered.  The t a n k s used t o r a i s e the b r o o d s t o c k had been i n use f o r many y e a r s .  The  p o s i t i o n o f the f o u r t a n k s used f o r t h e  e x p e r i m e n t a l d i e t s were s i m i l a r , a l l b e i n g a l o n g one along the staff  same c a t w a l k .  were near t h e s e  spawning f i s h  The tanks  b u i l d i n g had  since  i t was  opposite different  not  from times  i t where  the  i n t h e o p p o s i t e row and was  not  wall  other  and  The  have had more d i s t u r b a n c e there  and/or  were  spawning  four tanks.  other  tanks  occurred  Other t h a n  at the  d i f f e r e n c e s between t a n k s d u r i n g t h e  9 days o f t h e experiment,  temperature,  and  p o s i t i o n of  feeding  from t h e  problems w i t h temperature first  a  l i m i t e d access  or  T h i s t a n k may  along  and  were f e e d i n g  or m a i n t a i n i n g the tanks.  a l o n g t h e same c a t w a l k .  wall  o n l y when t h e y  t a n k used f o r t h e COMM d i e t was  four  t h e r e were few d i f f e r e n c e s i n  water c h e m i s t r y and l i g h t i n g between t h e t a n k s .  F i s h g r a d u a l l y reduced  t h e i r f e e d 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 f e e d being withheld  for a total  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 w i t h d r a w  feed  f o r several  during  spawning  or  days  d u r i n g each week o f t h e spawning season. t h e chance  to  spawning  T h i s i s done t o reduce  o f f e c a l m a t e r i a l c o n t a m i n a t i n g t h e eggs and  when males and females a r e s t r i p p e d . for  prior  2 weeks p r i o r  t o and  milt  A t RARC, f e e d i s w i t h h e l d  during the e n t i r e  spawning  period.  However, t h e r e a r e g e n e r a l l y s u f f i c i e n t t a n k s a v a i l a b l e so t h a t spawned f i s h can be p l a c e d i n t o a t a n k and f e e d can be o f f e r e d when f i s h a r e ready t o resume f e e d i n g . not a v a i l a b l e  f o r t h i s experiment.  A d d i t i o n a l t a n k s were  Records o f t h e number o f  e m a c i a t e d f i s h i n t h e n o n e x p e r i m e n t a l group ( f i s h from t h e same s t o c k as t h e e x p e r i m e n t a l groups) were not kept but t h e r e was evidence  that  emaciated  fish  existed  and  that  they  were  r o u t i n e l y c u l l e d d u r i n g t h e annual s a m p l i n g f o r c e r t i f i c a t i o n purposes  ( O l s o n , p e r s o n a l communication).  I n t h e w i l d , post-spawning c h a r r consume l i t t l e i f any f o o d i n f r e s h w a t e r and hence t h e y l o s e w e i g h t a l t h o u g h t h e y c o n t i n u e t o i n c r e a s e i n l e n g t h (Johnson 1980).  C o n d i t i o n f a c t o r , which  i s a measure o f t h e plumpness o f t h e f i s h , d e c r e a s e d from 1.00  0.97-  i n t h e prespawning r u n t o 0.67-0.74 d u r i n g t h e same f i s h ' s  p o s t - s p a w n i n g m i g r a t i o n t h e f o l l o w i n g summer.  In the present  145 experiment, condition  male  and female  factors  charr  o f 0.81-0.96  that  had l o s t  and 0.71-0.96,  w e i g h t had  respectively,  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 o f 1.08-1.48 and 1.35-1.73 f o r males and females 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 t h e s e emaciated " s l i n k s " , were expected weight  (1980)  f i s h , r e f e r r e d t o as  t o d i e o f m a l n u t r i t i o n . Regarding  g a i n o f post-spawning  Johnson  t h a t had g a i n e d  states:  " s l i n k s " when t h e y r e t u r n t o s e a ,  "The r e c o v e r y  phenomenal when f o o d i s a v a i l a b l e . "  o f emaciated  charr i s  T h e r e f o r e , one e x p e c t s t h a t  t h e y s h o u l d be a b l e t o resume f e e d i n g and r e c o v e r a f t e r period  the  o f spawning and s t a r v a t i o n .  The f a i l u r e  their  o f Labrador  A r c t i c c h a r r t o resume f e e d i n g a f t e r spawning has been r e p o r t e d by  other  producers  Broodstock  (Van Toever,  survival  personal  and p r o d u c t i v i t y and/or  communication).  a r e o f importance  diets  which  to  producers  and t e c h n i q u e s  enhance t h e  resumption  o f f e e d i n g a f t e r spawning need t o be i n v e s t i g a t e d .  L a b r a d o r c h a r r b r o o d s t o c k consumed more COMM d i e t t h a n t h e experimental  diets  and more HL compared t o L L d i e t s  effects of dietary l i p i d statistically. fingerlings,  on f e e d i n t a k e c o u l d n o t be a n a l y z e d  In research dietary  (Tabachek 1986).  but the  lipid  with  Nauyuk  Lake  d i d not influence  Arctic feed  charr intake  One e x p l a n a t i o n r e g a r d i n g t h e h i g h e r i n t a k e o f  t h e 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  Arctic  146 c h a r r b r o o d s t o c k a t RARC have a d j u s t e d w e l l t o a change i n d i e t from t h e d r y COMM d i e t t o a semi-moist p r o d u c t m a n u f a c t u r e d by B i o p r o d u c t s , i t i s u n l i k e l y t h a t b r o o d s t o c k would not a c c e p t a "new"  feed.  COMM may have had a d i f f e r e n t  other  diets  and COMM  experimental  diets,  d i d have  with  f l a v o u r than t h e  a harder  t h e HL d i e t s  texture  having  than t h e  the softest  consistency.  O r r e t al, (1982) and Ridelman e t a l . (1984) r e p o r t e d t h a t s t a r v a t i o n o f 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 w e i g h t , d i a m e t e r , p r o x i m a t e c o m p o s i t i o n or had  hatchability. on r e s u m p t i o n  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 o f f e e d i n g was n o t 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 no  effect  on t h e l i p i d  c o n t e n t o f eggs,  s u r v i v a l t o t h e eyed s t a g e i n c h i n o o k  condition  had  factor or  salmon.  The g e n e r a l d e c l i n e i n f e e d i n g a c t i v i t y i n Year 2 compared to The  Year  1 was o b s e r v e d  experimental  intervals. in  diets  i nfish were  f e d a l l d i e t s i n c l u d i n g COMM.  manufactured  a t about  11 week  I n g r e d i e n t s and feeds were k e p t f r o z e n , as d e s c r i b e d  S e c t i o n 3.2.2 and 3.5, and feeds  were  stored  t e m p e r a t u r e o n l y d u r i n g t h e week t h e y were f e d .  a t room  COMM used i n  Y e a r 2 was from a l a t e r shipment t h a n t h a t used i n Year 1. Hung et  a l . (1980) recommended u s i n g p e r o x i d e v a l u e t o measure t h e  oxidation of fish o i l s .  P e r o x i d e v a l u e showed t h a t h e r r i n g o i l ,  147 stored  i n a frozen  s t a t e and  flushed with  nitrogen,  had  not  d e t e r i o r a t e d d u r i n g storage over the l e 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 f e e d consumption i n a l l d i e t s i n Year 2, a r e not e x p e c t e d t o have d e v e l o p e d .  5.2 R e l a t i o n o f f e c u n d i t y t o r a t i o n and s i z e o f t h e female The  fecundity  locations  differ  of stocks  of A r c t i c  w i d e l y both  charr  i n the w i l d  from  and  different  in  captivity.  Johnson (1980) p r e s e n t s d a t a on s e v e r a l s t o c k s o f w i l d c h a r r o f d i f f e r e n t ages w h i c h v a r y i n mean f e c u n d i t y from 2000-4954. RARC, s t o c k s o f c h a r r from Norway, Nauyuk Lake i n NWT R i v e r i n L a b r a d o r produce  At  and F r a s e r  3000, 1200-2200 and  o v e r 3000-4000  eggs, r e s p e c t i v e l y (Tabachek and de March 1990).  Therefore, the  d a t a p r o v i d e d by Dempson (1982) and Dempson and Green (1985) i n t h e i r s a m p l i n g o f A r c t i c c h a r r from t h e F r a s e r R i v e r i n L a b r a d o r p r o v i d e v e r y u s e f u l comparisons o f w i l d and c a p t i v e s t o c k s w h i c h o r i g i n a t e d from t h e same l o c a t i o n .  F e c u n d i t y o f f i s h spawned i n  L a b r a d o r , which were 7-13 y e a r s o l d (weight = 2.14 55.3  cm)  1982).  ranged  from  2316-9245 eggs  (mean =  kg, l e n g t h =  5242)  (Dempson  I n t h e p r e s e n t e x p e r i m e n t , t h e number o f eggs r e l e a s e d  a t spawning ranged from 763-7455 i n Year 1 and from 327-13429 i n Year 2 w i t h t h e l a t t e r e x t e n d i n g t h e range o f Dempson ( 1 9 8 2 ) . F i s h f e d COMM r e l e a s e d t h e h i g h e s t number o f eggs i n b o t h y e a r s , with  ranges  of  2183-6525  (4532  ±  1596)  [mean  ±  d e v i a t i o n ] ) i n Year 1 and 4047-13429 (8305 ± 3313) While the variance  i s high,  34 and  85% o f t h i s  standard  i n Year  variance  2. was  148 accounted f o r by t h e s t r o n g 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 w e i g h t i n Year 1 and 2, r e s p e c t i v e l y .  A l t h o u g h females f e d  COMM had s i g n i f i c a n t l y h i g h e r f e c u n d i t y t h a n f i s h f e d a l l o t h e r d i e t s e x c e p t LLLE i n Year 2, t h e y 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 fecundity. (1987)  Jones and Bromage  showed t h a t f e c u n d i t y depended b o t h 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 o f f i s h f e d  COMM l e d t o t h e i r  h i g h e r weight  which  was r e l a t e d  higher f e c u n d i t y than those fed other d i e t s .  to their  I n i t i a l l y , the  p o p u l a t i o n o f c h a r r a t RARC was d i v i d e d i n h a l f w i t h one group allocated  f o r t h e experiment  and t h e remainder  e x p e r i m e n t a l group which were m a i n t a i n e d as u s u a l .  f o r t h e nonAlthough the  n o n - e x p e r i m e n t a l group was f e d COMM ( a l t h o u g h i t may have been from d i f f e r e n t shipments  f o r part o f the time), fecundity d i d  n o t r e a c h t h e same h i g h numbers as o b s e r v e d i n t h e e x p e r i m e n t a l group f e d COMM. The n o n - e x p e r i m e n t a l group had a mean f e c u n d i t y o f 3300 i n Year 1 and 3900 i n Year 2.  The h i g h e r f e c u n d i t y o f  t h e e x p e r i m e n t a l group f e d COMM was p r o b a b l y due t o t h e i r b e i n g fed t o s a t i a t i o n .  Jones 0.75-1.0% broodstock.  and Bromage (1987) body  weight/day  suggested an optimum r a t i o n o f at  11.7°C  f o r rainbow  trout  I n t h e c u r r e n t e x p e r i m e n t , A r c t i c c h a r r were f e d t o  s a t i a t i o n but r a t i o n c o u l d be e s t i m a t e d from f e e d f e d ( d r y f e e d fed, fed  g x 100)/(no. days f e d x (average body w e i g h t , g ) . a t w a t e r t e m p e r a t u r e s o f 7.5-8.5°C, r a t i o n  i n Year  When 1 was  149 a p p r o x i m a t e l y 0.77-0.88% f o r t h e LL and HL d i e t s and 0.98% weight/day  f o r COMM.  I n Year 2, t h i s d e c r e a s e d t o 0.31%  f i s h f e d t h e LL d i e t s and 0.50% and COMM d i e t s ,  body  and 0.42%  for  f o r t h o s e f e d t h e HL  r e s p e c t i v e l y , which were much l o w e r t h a n t h e  optimum r a t i o n suggested f o r rainbow t r o u t o f 0.75-1.0%.  Dempson  and Green  (1985)  showed  that  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 l e n g t h o f w i l d L a b r a d o r charr.  These  authors  also  estimated fecundity  eggs/kg body w e i g h t a t 7-13 y e a r s o f age. (1989)  showed  that  first-time  t o be 2450  McGeachy and D e l a b b i o  spawning  4-year  olds  (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 o f 3850 eggs/kg.  I n t h e present experiment,  fish  r e l e a s e d an  average o f 2842-3200 eggs/kg body w e i g h t i n Year 1 ( 4 - y e a r o l d s ) and 2112-3098 i n Year 2 (5-year o l d s ) .  Both ranges were l o w e r  t h a n e x p e c t e d based on t h e f i n d i n g s o f McGeachy and D e l a b b i o (1989).  The improvement i n t h e c o r r e l a t i o n o f f e c u n d i t y and  f i s h w e i g h t from 34% i n Year 1 t o 85% i n Year 2 may be r e l a t e d to  the fact  that  fish  were f e d t o s a t i a t i o n  for the entire  season i n Year 2 whereas t h e y were f e d t o s a t i a t i o n f o r o n l y 71 days  prior  restricted  t o spawning  Scott  (1962)  showed  that  f e e d i n t a k e r e s u l t e d i n an i n c r e a s e d p r o p o r t i o n o f  atretic  follicles.  feeding  rainbow t r o u t  resulted  i n Year 1.  S p r i n g a t e e t a l . (1985) at f u l l  i n significantly  ration  reported  (0.7% body  higher levels  that  weight/day)  of vitellogenin i n  f e m a l e s and t e s t o s t e r o n e i n males a t spawning compared t o t h o s e  150 f e d a t h a l f - r a t i o n ( 0 . 3 5 % ) . S i g n i f i c a n t l y more a t r e s i a o c c u r r e d i n f i s h fed at half-ration. significantly fecundity  more  (eggs  and  per  Females f e d a t f u l l r a t i o n  larger  body  weight)  c o n f i r m e d by O r r e t a l . (1982). current  experiment,  eggs  but and  had these  was  i t i s possible  insufficient  lower  relative  findings  were  During the period p r i o r t o the that  some f i s h  received a smaller proportion of the r a t i o n ration  produced  during  the  may  have  than others.  period  prior  to  If the  e x p e r i m e n t , 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 t h e experiment.  T h i s may p a r t i a l l y account f o r t h e f a c t t h a t t h e  d a t a were more s c a t t e r e d about t h e r e g r e s s i o n l i n e i n Year 1.  5.3 Spawning t i m e and p r o p o r t i o n o f f i s h t h a t spawned A r c t i c c h a r r i n L a b r a d o r (Dempson 1982) spawned i n a 3-week p e r i o d i n O c t o b e r which peaked i n t h e m i d d l e o f O c t o b e r .  In the  c u r r e n t e x p e r i m e n t , c h a r r spawned o v e r a much l o n g e r p e r i o d o f time.  F o r example, t h e y spawned o v e r 14 weeks i n Year 1 and 11  weeks i n Year 2 p e a k i n g i n t h e m i d d l e o f 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 o f A r c t i c c h a r r r a i s e d a t RARC from t h r e e c o l l e c t i o n s  i n Labrador.  In contrast,  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 t h a n t h e L a b r a d o r s t o c k s and spawn w i t h i n 4-6 weeks ( O l s o n , p e r s o n a l communication).  While d i e t a r y  l i p i d had an e f f e c t on  t i m e i n Year 1, w i t h f i s h f e d LL d i e t s  spawning  spawning  5 weeks l a t e r  t h a n t h o s e f e d HL d i e t s , t h i s p a t t e r n was not r e p e a t e d i n Year 2.  I t i s assumed t h a t t h i s e f f e c t was not c o n s i s t e n t o r t h a t  151 t h e r e was no d i r e c t e f f e c t o f l i p i d on spawning t i m e . spawning  was  not  concentrations (Takeuchi  associated  of vitamin  e t a l . 1981b).  E  as  diets  i t was  that  with  had  rainbow  low trout  F i s h f e d COMM spawned i n 8 weeks i n  Year 1 and 6 weeks i n Year 2. was d e l a y e d  with  Delayed  Roley  (1983) found t h a t spawning  b u t o c c u r r e d w i t h i n a s h o r t e r p e r i o d o f t i m e when  r a i n b o w t r o u t were f e d t o s a t i a t i o n .  In contrast, A r c t i c  charr  i n t h e n o n - e x p e r i m e n t a l group, f e d a t a lower r a t i o n , spawned 34 weeks l a t e r b u t o v e r t h e same l e n g t h o f t i m e compared t o t h e p o p u l a t i o n i n t h e experiment i n b o t h y e a r s .  I n Year 1, t h e r e was a s m a l l p o s i t i v e e f f e c t o f 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 o n l y  14% o f t h e v a r i a n c e , i t i s e x p e c t e d t h a t a d d i t i o n a l f a c t o r s a r e responsible  f o r the v a r i a t i o n  in fertilization.  I n Year  2,  t h e r e was an n e g a t i v e e f f e c t o f spawning t i m e on f e c u n d i t y , w i t h t h o s e spawning l a t e i n t h e season p r o d u c i n g t h e s m a l l e s t numbers o f eggs.  A r c t i c c h a r r a r e i t e r o p a r e o u s and may spawn s e v e r a l during  their  lifetime  i n the w i l d although  times  the frequency  spawning may d e c r e a s e w i t h i n c r e a s i n g age (Johnson 1980).  of  Charr  i n t h i s e x p e r i m e n t had spawned t h e y e a r p r i o r t o t h e e x p e r i m e n t . This  iteropareous  effect  of  spawned.  diet  tendency makes i t d i f f i c u l t t o e v a l u a t e t h e on  the proportion  of  the population  which  The v a r i a n c e i n t h i s parameter i s n a t u r a l l y h i g h and  152 r e s u l t s i n t h e 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 t h e power o f t h e t e s t (Dixon and Massey 1969).  W h i l e a l l f i s h spawned i n Year 2 t h a t had Year  1,  a  p o r t i o n of  the  f a i l e d t o spawn i n Year 2.  population  that  not spawned i n  spawned i n Year  1  D i e t a r y v i t a m i n E had no  significant  e f f e c t on t h e p r o p o r t i o n o f females t h a t spawned.  This i s i n  c o n t r a s t t o r a i n b o w t r o u t ( K i n g 1985)  and ayu  (Takeuchi  et a l .  1981a) where a p o r t i o n o f the p o p u l a t i o n f a i l e d t o spawn when fed  diets  with  no  vitamin E  supplementation.  The  fact  s i m i l a r p r o p o r t i o n s of the p o p u l a t i o n fed the experimental f a i l e d t o spawn i n Year 2 s u p p o r t s a l l spawn e v e r y y e a r .  that diets  t h e f a c t t h a t c h a r r do  not  Continued s t u d i e s w i t h l a r g e populations  o f t a g g e d A r c t i c c h a r r and good r e c o r d - k e e p i n g  can a s c e r t a i n i f  t h e r e a r e p a t t e r n s i n t h e i r spawning.  Roley  (1983) showed t h a t f e e d i n g l e v e l had no e f f e c t on t h e  number o f r a i n b o w 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 f e e d i n t a k e i n c r e a s e d t h e number o f females t h a t spawned. was  lower  The p r o p o r t i o n o f A r c t i c c h a r r t h a t spawned i n Year 2 than  i n Year  1 even though  they  had  been  fed  to  s a t i a t i o n f o r a l l o f Year 2.  Not a l l males matured and produced m i l t ( i e . r i p e n e d ) year  and  9 out  of  59 males f a i l e d  t o mature i n e i t h e r  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 f e d t h e LE  each year. versus  153 HE d i e t s i n Year 1 and proportion  Year 2.  However, a s i g n i f i c a n t l y h i g h e r  (12/16) o f males f e d HE  d i e t s produced m i l t i n  c o n s e c u t i v e y e a r s compared t o t h o s e f e d LE d i e t s  two  (4/11).  This  a r e a w a r r a n t s 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 o f and w i t h r e p l i c a t e t a n k s of f i s h . E deficiency  There are r e p o r t s  of  vitamin  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 , g u i n e a  h a m s t e r s , dogs and  chickens  (Scott  et  al.  1982)  fish  pigs,  through i t s  e f f e c t on t h e c e l l membranes of the s e m i n i f e r o u s t u b u l e s .  Male  poultry  unless  they  of time or  they  were not  were f e d the were  fed  1968).  affected  containing  There  are  no  r e s u l t i n g from a v i t a m i n the  oxidized  reports  unsaturated  of  male  E deficiency.  required  oils  infertility  Watanabe e t al.  f a t t y a c i d c o m p o s i t i o n o f m i l t was  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 was  E deficiency  d e f i c i e n t d i e t f o r a long period  diets  found t h a t  by v i t a m i n  to  determine  would  have  in  an  fish  (1984b)  affected  suggested f u r t h e r  i f this  (Jensen  by  research  effect  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  Egg  size  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  diameter  (Ashton  fecundity  and  in egg  chinook  inverse  In c o n t r a s t , Roy  relationship  c h i n o o k salmon (Roy  1991)  or  between  weight or diameter w i t h A r c t i c c h a r r i n  present experiment. an  salmon  and  between egg Higgs 1987).  and  and  the  Higgs (1987) r e p o r t e d  weight  and  fecundity  in  154 There was a d i r e c t r e l a t i o n s h i p o f egg w e i g h t on i n Year 2 but t h e low r  2  survival  shows t h a t t h e r e g r e s s i o n a c c o u n t s f o r  o n l y 11-12% o f t h e v a r i a n c e i n s u r v i v a l .  In a d d i t i o n , the slope  o f t h e l i n e i s so low t h a t a 1 mg i n c r e a s e i n egg w e i g h t would r e s u l t i n a 0.75%  i n c r e a s e i n f e r t i l i z a t i o n and l e s s t h a n  increase i n survival d i f f e r e n c e s between  t o a l l stages.  A l t h o u g h no  significant  d i e t s were found, t h e g r e a t e s t  between means was 7 mg which t r a n s l a t e s  1.5%  difference  i n t o an i n c r e a s e o f l e s s  t h a n 10.5% i n s u r v i v a l .  The d i a m e t e r o f eggs from c h a r r sampled i n L a b r a d o r was mm  (Dempson and Green 1985) which was s m a l l e r t h a n t h o s e i n Year  1 (4.94-5.15 mm)  5.5 V a r i a t i o n  o r Year 2 (4.90-5.13 mm)  i n embryonic  i n t h i s experiment.  survival  L a b r a d o r A r c t i c c h a r r commonly produce eggs w h i c h wide v a r i a t i o n hatchability influence  i n hatchability. of  and  Labrador  was  de March  Arctic  not a f f e c t e d  by  charr  male.  The m i l t  eggs o f each  exhibit  (1992) showed was  under  from two males was  that  maternal  the source of the  T h e r e f o r e , i t s seems u n l i k e l y t h a t s u r v i v a l was the  4.5  sperm.  influenced  by  used t o f e r t i l i z e t h e  female t o reduce the chance  o f a male  effect.  Records were m a i n t a i n e d r e g a r d i n g t h e number o f t i m e s each male was used and an attempt was made t o e q u a l i z e t h e number o f t i m e s each male was used o v e r t h e c o u r s e o f t h e l o n g spawning s e a s o n s .  155 Variation  i n embryonic  survival  r e l a t e d t o t h e method o f i n c u b a t i o n .  i s not expected  t o be  A few r e p l i c a t e s groups o f  eggs were i n c u b a t e d each y e a r 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 addition,  eggs were spawned t h a n were r e q u i r e d excess  eggs were  Survival  i n Year 2, i f more  f o r t h e experiment, t h e  incubated i n compartmentalized  Heath  trays.  t o t h e eyed s t a g e was almost i d e n t i c a l t o t h a t o f t h e  eggs i n c u b a t e d 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 .  S p r i n g a t e e t 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 o r v i a b i l i t y o f f r y from rainbow t r o u t f e d a t h a l f ration that  compared t o f u l l - r a t i o n . feeding  to  satiation  However, R o l e y increased  the  (1983)  found  variation  in  h a t c h a b i l i t y and d e c r e a s e d s u r v i v a l a t c o o l water  temperature.  Fish  lower  i n t h e non-experimental  groups,  fed at a  rate,  produced eggs w i t h t h e same mean s u r v i v a l t o t h e eyed s t a g e and t h e same v a r i a n c e as t h o s e f e d COMM i n t h i s e x p e r i m e n t .  Since  t h e r e was wide v a r i a t i o n i n t h e h a t c h a b i l i t y o f L a b r a d o r A r c t i c charr,  the effect  warrants f u r t h e r  of ration  on h a t c h a b i l i t y  i s an a r e a  that  investigation.  5.6 L i p i d and f a t t y a c i d c o m p o s i t i o n o f eggs The  essential  fatty  b r o o d s t o c k a r e n o t known.  acid  requirements  of A r c t i c  The r e q u i r e m e n t s o f f i n g e r l i n g s a r e  met when d i e t s  c o n t a i n 1.7% 18:3n3 and 0.5% 18:2n6  Dick  The  1992).  diets  charr  used  here  contained  (Yang and  exceeded  the  156 r e q u i r e m e n t s o f 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.  T a k e u c h i and Watanabe (1979) found t h a t t h e growth o f j u v e n i l e r a i n b o w t r o u t d e c r e a s e d when f e d d i e t s c o n t a i n i n g 2-4% acids. not  The e f f e c t o f 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  known.  2.5%  n3 f a t t y  COMM c o n t a i n e d  n3 and 2.1%  3.6% n3 and 2.0% n6 i n Year 1 and  n6 i n Year 2.  COMM used i n Year 2 was h i g h e r  i n 2 0 : l n 9 and 2 2 : l n l l and l o w e r i n 20:5n3 t h a n t h a t used i n Year 1.  Atlantic  herring  o i l contains  P a c i f i c h e r r i n g o i l (10%) 20:1  and 22:1 w h i l e  1989).  more  (Ackman 1982).  22:1 (15-30%)  than  Capelin o i l i s high i n  low i n 20:5n3 and 22:6n3 (Sargent  eta l .  P a c i f i c h e r r i n g o i l may have been used i n m a n u f a c t u r i n g  COMM i n Year 1 and A t l a n t i c  h e r r i n g o i l o r a mixed f i s h o i l  c o n t a i n i n g a h i g h p r o p o r t i o n o f c a p e l i n o i l was used i n Year 2.  L a b r a d o r and Tree R i v e r A r c t i c c h a r r eggs c o n t a i n e d 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 . two  10-12%  Eggs from w i l d as w e l l as  s t r a i n s o f c u l t u r e d c h i n o o k salmon had a l m o s t t w i c e t h e  c o n c e n t r a t i o n (18-19%) o f 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) River Arctic  charr.  compared t o L a b r a d o r and w i l d Tree  Kaitaranta  (1982) o b s e r v e d t h a t  lipids  made up o v e r 50% o f t h e t o t a l  eggs.  I t i s u n f o r t u n a t e t h a t many a u t h o r s who r e p o r t f a t t y a c i d  analyses  o f rainbow t r o u t  on t h e 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 o f eggs  to provide observed.  lipid  neutral  t h e percent There  o f n e u t r a l and p o l a r  i s little  information  lipids  that  reported  in  fail they the  157 l i t e r a t u r e f o r comparison o f n e u t r a l and p o l a r l i p i d c o n t e n t s o f eggs i n o t h e r s a l m o n i d s .  C r a i k and Harvey  (1984)  found  that  rainbow t r o u t eggs w i t h g r e a t e r t h a n 50% h a t c h had s i g n i f i c a n t l y more  total  lipid  (24.5%).  (25.7%)  Neutral  lipids  t h r o u g h o u t embryogenesis, prior  compared are  to  those  utilized  with  as  0%  energy  hatch sources  e s p e c i a l l y i n the l a t e r stages  t o h a t c h i n g ( S a r g e n t e t a l . 1989).  W h i l e most  just polar  l i p i d s a r e i n c o r p o r a t e d i n t o membranes, b o t h 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 A t l a n t i c salmon  Polar  t h r o u g h o u t embryogenesis  (Henderson and Tocher  lipids  by  1987).  contained higher l e v e l s  of saturated  fatty  a c i d s and HUFA and lower l e v e l s o f monoenoic f a t t y a c i d s t h a n neutral  lipids.  Monoenoic a c i d s ,  1 6 : l n 7 , 1 8 : l n 7 , 18:ln9  and  2 0 : l n 9 , were i n c o r p o r a t e d i n t o both t h e n e u t r a l and p o l a r l i p i d s of  the  eggs,  with  high  levels  of  16:ln7  and  n e u t r a l l i p i d s and 18:ln9 i n t h e p o l a r l i p i d s . acids  can  be  (Castell  1979,  was  incorporation  i n the  These two  s y n t h e s i z e d de novo o r o b t a i n e d from  ( F i g u r e 1) little  18:ln9  fatty  the  diet  Henderson and S a r g e n t 1985). of 22:ln9, 2 0 : l n l l  or  There  22:lnll  into  e i t h e r t h e n e u t r a l o r p o l a r l i p i d s o f eggs even though t h e 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% o f t h e t o t a l l i p i d i n t h e LL and HL  diets.  (Ashton 1991). when  22:lnll  incorporated  T h i s was  a l s o the case f o r chinook  salmon  A c c o r d i n g t o Henderson and S a r g e n t (1985), even is  a  major  dietary  i n t o the p o l a r l i p i d s  fatty  acid,  i t is  not  o f f i s h membranes and  may  158 f u n c t i o n mainly  as an energy s o u r c e t h r o u g h m i t o c h o n d r i a l  R-  oxidation.  O l s e n e t a l . (1991) f e d j u v e n i l e A r c t i c c h a r r a d i e t  containing  15.5% l i p i d  with  12% o f t h i s  lipid  as 2 2 : l n l l  and  found i t made up o n l y 1.2 and 1.8% o f p o l a r l i p i d s o f t h e l i v e r and m u s c l e , r e s p e c t i v e l y and 6.4 and 12.3% o f 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 t h e n e u t r a l l i p i d s o f eggs was 18:2n6 (28-32%) w h i l e t h e r e were low l e v e l s o f 18:2n6 and 20:4n6 (1.2-2.3%) i n t h e p o l a r 20:3n6.  lipids  and s l i g h t l y  l e s s 20:2n6 and  A s h t o n (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  o f c h i n o o k salmon eggs a l t h o u g h 20:4n6 t h a n  she o b s e r v e d h i g h e r  18:2n6 i n t h e p o l a r l i p i d s  20:2n6 and 20:3n6.  levels of  and minor amounts o f  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 t h e major  n6 f a t t y a c i d i n t h e n e u t r a l l i p i d s and 20:4n6 was t h e major n6 f a t t y a c i d i n the polar l i p i d s  (Cowey e t a l . 1985).  Egg p o l a r l i p i d s from t h e LL groups c o n t a i n e d more 20:2n6, 20:3n6, t o t a l t h e HL g r o u p s .  significantly  n6 and PUFA compared t o t h o s e from  T h i s i n c r e a s e 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 e t a l . (1979) and Watanabe e t a l . (1984b).  S i n c e t h e 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 o n l y 0.6% 20:4n6, i t appears t h a t d i e t a r y 18:2n6 had undergone c h a i n 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  d e s a t u r a t i o n o f 20:3n6.  have been from t h e d i e t o r from  D e s a t u r a t i o n and e l o n g a t i o n o f t h e n9,  159 n6 and n3 s e r i e s o f f a t t y a c i d s o c c u r s t h r o u g h a common enzyme pathway b u t w i t h s p e c i f i c enzymes f o r t h e d e s a t u r a t i o n o f each p o s i t i o n ( F i g u r e 1 ) . I n mammals, t h e d e s a t u r a s e enzymes have a g r e a t e r a f f i n i t y f o r t h e most u n s a t u r a t e d f a t t y a c i d (Leray greater  and P e l l e t i e r affinity  affinity  than  That  f o r 18:3n3 than  18:ln9.  chain molecules.  1985).  Similar  Both O l s e n  available  i s , A6-desaturase  18:2n6 which  desaturases  has a  has a g r e a t e r  exist  f o r longer  e t al. (1991) and Yang and D i c k  (1992) found t h a t A r c t i c c h a r r f i n g e r l i n g s c o u l d d e s a t u r a t e and elongate  dietary  n6PUFA.  The former a u t h o r s observed  elongated  18:3n3 t o n3HUFA and 18:2n6  and d e s a t u r a t e d  and 20:4n6 t o  t h a t n3 f a t t y a c i d s were  i n preference  t o n6  fatty  acids.  W h i l e HUFA, e s s e n t i a l t o m a i n t a i n i n g membrane f l u i d i t y , made up 40-42% o f t h e f a t t y a c i d s i n t h e p o l a r l i p i d s , t h e y were p r e s e n t a t much lower l e v e l s (14-17%) i n t h e n e u t r a l l i p i d s o f t h e eggs. T h i s i s i n agreement w i t h d a t a on rainbow t r o u t (Watanabe e t al. 1984b, Léger e t al. 1985) and c h i n o o k salmon (Ashton 1991) eggs. T o t a l l i p i d s o f t h e LL and HL d i e t s c o n t a i n e d a p p r o x i m a t e l y 6-7% 20:5n3 o r 22:6n3 and t h e r e were minor amounts o f 18:3n3, 18:4n3, 20:3n3, 20:4n3 and 22:5n3. While egg l i p i d s c o n t a i n e d l e s s t h a n 1% o f t h e 18 c a r b o n 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 a t l e v e l s t h a t were 2-5 t i m e s h i g h e r t h a n p r e s e n t i n the d i e t s . came  Some o f t h i s h i g h l y u n s a t u r a t e d f a t t y a c i d p r o b a b l y  directly  from  dietary  22:6n3  while  t h e remainder  s y n t h e s i z e d by d e s a t u r a t i o n and e l o n g a t i o n o f t h e l e s s u n s a t u r a t e d n3 f a t t y a c i d s .  was  highly  160 Dramatic d i f f e r e n c e s between t h e f a t t y a c i d c o m p o s i t i o n o f COMM compared t o t h e LL and HL d i e t s composition consisted 16:0,  o f t h e eggs.  The most  striking  i n the  differences  o f COMM's b e i n g l o w e r t h a n t h e LL and HL d i e t s i n  18:ln9  22:lnll.  were r e f l e c t e d  and 2 0 : l n l l  and h i g h e r  i n 18:2n6,  2 0 : l n 9 and  T h i s was r e f l e c t e d i n eggs from f i s h f e d COMM h a v i n g  significantly  less  18:ln9  in  the  neutral  lipids  and  s i g n i f i c a n t l y more 20:ln9 and 18:2n6 i n b o t h t h e n e u t r a l and polar l i p i d s .  As was found w i t h eggs from f i s h f e d L L and HL  d i e t s , t h e r e was l i t t l e t h e eggs o f f i s h  incorporation of n i l fatty acids  f e d COMM even though  total  lipid  into  contained  15.5% as 2 2 : l n l l .  Yang and D i c k  (1992) showed  that  Arctic  charr  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 . 20:3n9 was n o t found i n t h e eggs o f rainbow  trout  While  fed diets  l a c k i n g i n n3 f a t t y a c i d s f o r 3 months (Watanabe e t al. 1984b) o r one y e a r ( L e r a y e t al. 1985) p r i o r t o spawning, i t was found i n t h e m i l t a f t e r 3 months (Watanabe e t al. 1984b) o f f e e d i n g . When  trout  were  fed diets  containing  18:3n3  alone  ori n  c o m b i n a t i o n w i t h 18:2n6 f o r t h e e n t i r e l i f e t i m e o f t h e f i s h (Yu e t al. 1979), 20:3n9 was observed i n t h e eggs from b o t h b u t was l o w e r from t h e group f e d b o t h f a t t y a c i d s . reduced  i n the m i l t o f trout  supplemented takes  a  with  long  18:2n6  I t was a l s o  i f t h e n 3 , n 6 - d e f i c i e n t d i e t was  (Watanabe e t al. 1984b).  feeding period  groups  before  20:3n9  Since i t  i s observed i n  161 broodstock, ratio  as  Leray  an  e t a l . (1985) s u g g e s t e d t h e  i n d i c a t o r of  n3  deficiency  use  [20:3n9  of +  another 20:4n6  +  22:5n6]/[20:5n3 + 22:5n3 + 22:6n3] w i t h a h i g h r a t i o (6-12) f o r d e f i c i e n t eggs and This  ratio  20:3n9  was  from  inhibited Dietary  a low  low  18:ln9  by  the  lipids  ratio  (0.5)  f o r n o n - d e f i c i e n t eggs.  i n the  present  experiment.  (18%)  and/or  20:ln9  presence  of  also contained  18:2n6 6%  (3%)  (7%)  and  20:5n3 and  6%  Synthesis was  probably  18:3n3  (1%).  22:6n3 w h i c h  c o u l d have reduced t h e r e q u i r e m e n t f o r d e s a t u r a t i o n o f chain  shorter  molecules.  (Leray broodstock  et  a l . 1985)  found  that  feeding  rainbow  trout  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 a t the 16-32  cell  incidence  fry.  of  d e f o r m i t i e s i n the  stage  as w e l l as a  Abnormalities  d i v i s i o n were o b s e r v e d i n eggs from 2-3  in  o t h e r eggs i n any c o n s i s t e n t way. these  high cell  f i s h f e d LLHE o r HLLE  but t h e f a t t y a c i d p r o f i l e s o f t h e s e eggs d i d not d i f f e r  in  of  from  I n a d d i t i o n , t o t a l n3 and  eggs ranged from 15.2-17.6% of t h e n e u t r a l l i p i d  n6 and  40.3-44.8% of t h e 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 fatty acid deficiency.  L e r a y e t a l . (1985) s p e c u l a t e d t h a t t h e  i n c i d e n c e o f egg a b n o r m a l i t i e s i n t h e i r r e s e a r c h might have been due  t o t h e h i g h l e v e l of d i e t a r y 18:2n6 but l e v e l s o f 18:2n6 i n  diets 0.9%  i n the present i n HL and  (5.4%).  1.6%  experiment were much l o w e r i n COMM) t h a n those o f L e r a y  Some o t h e r  f a c t o r ( s ) must  be  (0.6%  in  LL,  e t a l . (1985)  responsible  for  the  162 a b n o r m a l i t i e s observed i n t h i s experiment.  W h i l e s i g n s o f f a t t y a c i d d e f i c i e n c y were o b s e r v e d i n c a r p broodstock Takashima  fed  diets  deficient  i n vitamin  E  (Watanabe  and  1977), t h e s e were not observed i n A r c t i c c h a r r f e d any  of the d i e t s .  No changes i n t h e f a t t y a c i d c o m p o s i t i o n o f eggs  t h a t would be i n d i c a t i v e o f an e s s e n t i a l f a t t y a c i d  deficiency  were found i n A r c t i c c h a r r and i t i s e x p e c t e d t h a t  sufficient  e s s e n t i a l f a t t y a c i d s were a v a i l a b l e from t h e d i e t and/or body s t o r e s  t o meet t h e r e q u i r e m e n t s .  There was and  16:ln7  survival difference and  a s i g n i f i c a n t negative e f f e c t of neutral  and  to  total  each  n7  stage  i n the of  between d i e t s .  neutral  development The  respectively.  The  lipids with  no  of  fact  t h e r e i s a low p r o b a b i l i t y  b e i n g due  t o chance.  indicates  that  there  that  the  correlation  indicates  relationship  However, t h e low r e g r e s s i o n  correlation  other  covariate(s)  that  are  not  No r e a s o n f o r t h e  l i p i d o r 16:ln7 on h a t c h a b i l i t y i s known but When L e r a y  (1985) f e d rainbow t r o u t b r o o d s t o c k a s e m i p u r i f i e d  containing  was  linear  are  of t h i s  t h i s i s an a r e a t h a t w a r r a n t s f u r t h e r i n v e s t i g a t i o n . e t al.  lipid  19-21% o f t h e v a r i a n c e i n  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 . e f f e c t of neutral  on  significant  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 that  lipid  eggs  correlation with neutral  16:ln7 a c c o u n t e d f o r 26-34% and  survival,  from  diet  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 d e c r e a s e d  163 s i g n i f i c a n t l y compared t o t h o s e f e d a c o m m e r c i a l 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 o f t h e d a t a i n t h e p a p e r ) .  I n t h e n 3 - d e f i c i e n t group, t h e n e u t r a l and p o l a r l i p i d s o f t h e eggs and m i l t c o n t a i n e d levels  very  low l e v e l s o f n3 b u t i n c r e a s e d  o f n6 g i v i n g them a s i g n i f i c a n t l y  lower  ( L e r a y and P e l l e t i e r 1985, L e r a y e t a l . 1985).  n3/n6  There was a l s o  a d e c r e a s e i n n7 (15% v s 5%) f a t t y a c i d s i n t h e n e u t r a l o f eggs o f t h e n 3 - d e f i c i e n t group.  ratio  lipids  T h i s l e a d s one t o s p e c u l a t e  t h a t i f n7 f a t t y a c i d s d e c r e a s e d i n t h e n 3 - d e f i c i e n t group w h i c h had  low s u r v i v a l ,  then  n7  fatty  acids  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 t h e y were w i t h A r c t i c c h a r r .  When  c h i n o o k salmon (Ashton 1991) were f e d d i e t s c o n t a i n i n g s i m i l a r amounts  o f 16:ln7  to this  experiment,  the concentration  16:ln7 i n t h e n e u t r a l l i p i d s o f t h e eggs c o u l d be e s t i m a t e d the data provided experiment  of from  a t 4-5% which i s l o w e r t h a n o b s e r v e d i n t h i s  (9-14%).  5.7 V i t a m i n E i n eggs King  (1985) found t h a t t h e t i m e p r i o r t o spawning  during  w h i c h d i e t a r y v i t a m i n E was e i t h e r p r e s e n t o r absent a f f e c t e d the v i t a m i n E concentration  o f t h e eggs.  F o r example, when she  sampled r a i n b o w t r o u t one month p r i o r t o spawning, she o b s e r v e d 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 c o n t e n t and c o n c e n t r a t i o n of  eggs  from  broodstock  fed vitamin  E - s u f f i c i e n t d i e t s (90  mg/kg) f r o m J u l y t o December compared t o t h o s e f e d d e f i c i e n t d i e t s (0 mg/kg) from August o r October t o December.  However, a t  164 spawning t i m e t h e o n l y s i g n i f i c a n t d i f f e r e n c e was content  between t h o s e f e d e i t h e r  the  s u f f i c i e n t or  d i e t from J u l y u n t i l spawning i n J a n u a r y . the  greatest  occurred  dietary  effects  p r i o r t o October  on  egg  July).  spawning  However,  she  vitamin  E  ( i e . p r i o r t o the  observed  and December - i e . i n the 3-4  that  the  32  days  respectively.  before  spawning  3 months  the  first  HE  LE  withdrawn spawned,  f i r s t o f f e r e d 13 and  15 weeks  close to  t i m e t h a t t h e maximum r a t e o f d e p o s i t i o n o f v i t a m i n E  was  of  a  crossover  must be c o g n i z a n t diets  I n Year  fish  p r i o r t o t h e commencement o f spawning w h i c h was  One  of  between O c t o b e r  f e e d was  and  the from  rate  months p r i o r t o spawning.  That i s , f e e d was  i n K i n g ' s work.  before  p r i o r to  greatest  occurred  1, A r c t i c c h a r r were f e d f o r 71 days b e f o r e and  at  ( i e . those fed d e f i c i e n t d i e t s  d e p o s i t i o n o f v i t a m i n E i n t o the egg  18  deficient  W h i l e she s t a t e d t h a t  s p a w n i n g ) , her d a t a seems t o i n d i c a t e t h a t i t was 6 months b e f o r e  i n vitamin E  i n Year  2.  occurred  of the f a c t t h a t That  the  there  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 t h e same, f i s h f e d LL d i e t s i n Year 1 were f e d HL  d i e t s i n Year 2 and  t h o s e f e d HL  were f e d LL d i e t s i n Year 2 ( F i g u r e 3 ) .  d i e t s i n Year 1  I t i s i n t e r e s t i n g that  t h e v i t a m i n E l e v e l s o b s e r v e d i n the HE eggs i n Year 1 were v e r y s i m i l a r t o t h o s e o b s e r v e d i n Year 2 even though t h e d i e t s were f e d f o r a much l o n g e r p e r i o d o f 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 t h e v i t a m i n E c o n t e n t concentrations d i e t s and  of  eggs from b r o o d s t o c k  between those f e d the two  fed  lipid  the  LE  versus  l e v e l s at the  and HE high  165 l e v e l o f v i t a m i n E, t h e t i m e t h a t t h e d i e t s were f e d 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 o f v i t a m i n E i n t o t h e eggs. I n 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 o c c u r r e d between t h o s e f e d LLLE, HLLE and COMM which were not a p p a r e n t i n Year 1 w i t h LLLE <  HLLE  <  COMM.  concentration  Initially,  the reduction  i n vitamin  E  i n t h e LLLE eggs between Year 1 and 2 s u g g e s t s  t h a t t h e a d d i t i o n a l y e a r o f b e i n g f e d a t t h e low l e v e l o f l i p i d and v i t a m i n E (LLLE) reduced t h e body s t o r e s o f v i t a m i n E and r e s u l t e d i n l o w e r d e p o s i t i o n i n t h e eggs.  W h i l e f i s h f e d LLLE  i n Year 2 produced more eggs t h a n t h o s e f e d HLLE, t h e r e 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 vitamin E t r a n s f e r r e d t o the eggs i n t h e LLLE and HLLE groups i n Year 1 (12.6 mg) o r Year 2 (13.6 mg v s 15.2 mg).  F i s h f e d 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 o f v i t a m i n E p e r kg f i s h w e i g h t t o t h e eggs (6.4 mg/kg) as t h o s e f e d HLLE (7.0 mg/kg). Year 1 d e p o s i t e d  I n c o n t r a s t , f i s h f e d LLHE i n  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 t h e eggs  compared t o t h o s e f e d HLHE (48.9 mg v s 35.0 mg v i t a m i n E o r 35.1 vs 24.6 mg v i t a m i n E/kg f i s h w e i g h t ) b u t t h e 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 v s 41.9 mg v i t a m i n E o r 37.4 vs 24.4 mg v i t a m i n E/kg f i s h w e i g h t ) . for  a longer  W h i l e t h e d i e t s were f e d  p e r i o d o f t i m e i n Year 2, t h e r e  were no marked  i n c r e a s e s i n t h e v i t a m i n E c o n c e n t r a t i o n o r c o n t e n t o f t h e eggs. This c o n f i r m s t h a t the p e r i o d o f time f o r t r a n s f e r o f d i e t a r y vitamin  E t o t h e eggs was met d u r i n g  feeding  i n Year 1.  vitamin  E from s t o r a g e s i t e s  the shorter  period of  W h i l e some f i s h had more t i m e t o m o b i l i z e t o t h e eggs p r i o r  t o spawning,  166 there  was  no  significant  relation  between  spawning  day  and  vitamin E content.  K i n g (1985) showed t h e r e was 25 mg o f v i t a m i n E i n t h e body of r a i n b o w t r o u t a t t h e b e g i n n i n g o f h e r e x p e r i m e n t i n J u l y w i t h 45% o f t h i s s t o r e d i n t h e l i v e r .  V i t a m i n E was t r a n s p o r t e d from  the  i n July  l i v e r m a i n l y t o t h e muscle  August and O c t o b e r , i t was  and August.  Between  t r a n s p o r t e d from t h e muscle t o t h e  o v a r y and a d i p o s e t i s s u e and t h e n from t h e a d i p o s e t i s s u e t o t h e o v a r y between October and December.  V i t a m i n E was  transferred  i n t o t h e eggs between October and December w i t h f i s h throughout January. the  spawning  A t spawning, t h e body burden o f f i s h f e d  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 c o n t a i n e d 25 mg  vitamin  E w h i l e t h o s e f e d t h e d i e t supplemented a t 90 mg/kg c o n t a i n e d 35 mg v i t a m i n E.  A f t e r spawning, t h e 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 h a v i n g 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 t h e eggs.  I f t h e unsupplemented  f i s h had been  m a i n t a i n e d on t h a t d i e t f o r a n o t h e r y e a r , one can o n l y s p e c u l a t e t h a t t h e v i t a m i n E c o n t e n t o f t h e eggs and/or t h e number o f eggs might have d e c r e a s e d .  K i n g ' s experiment shows t h a t i t t o o k a t  l e a s t one spawning season b e f o r e t h e body s t o r e s were reduced by half.  T h i s was  a l s o observed i n p o u l t r y where  hatchability  d e c l i n e d o v e r t i m e when t h e y were f e d 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 b e f o r e 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  current  e x p e r i m e n t , d i e t s c o n t a i n e d 30 and 600 mg s u p p l e m e n t a l 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 g r e a t e r r e d u c t i o n i n t h e c o n c e n t r a t i o n o f v i t a m i n E i n t h e LE eggs i n Year 2.  Watanabe  et  a l . (1981a)  found  that  the  vitamin  E  r e q u i r e m e n t o f r a i n b o w t r o u t f r y i n c r e a s e d as e i t h e r t h e l i p i d l e v e l o r degree o f u n s a t u r a t i o n o f t h e d i e t a r y l i p i d  increased.  The v i t a m i n E c o n c e n t r a t i o n o f t h e l i v e r d e c r e a s e d 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 t h e g r e a t e s t 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 e d low l i p i d When f r y were  (3 and 5%) and h i g h l i p i d  fed diets  containing  (10 and 15%) d i e t s .  50 mg v i t a m i n  E/kg, t h e  v i t a m i n E c o n c e n t r a t i o n i n t h e l i v e r was l o w e s t when f r y were f e d t h e d i e t w i t h t h e h i g h e s t l e v e l o f u n s a t u r a t i o n (5.7% PUFA) and i t was h i g h e s t when t h e y were f e d t h e d i e t w i t h t h e l o w e s t level of unsaturation decrease i n l i v e r  (1.6% PUFA).  I t was s p e c u l a t e d t h a t t h e  v i t a m i n E was t h e r e s u l t  of i t s increased  r e q u i r e m e n t i n p r e v e n t i n g o x i d a t i o n o f PUFA and HUFA w i t h i n t h e tissues.  T h i s i s i n agreement w i t h r e s e a r c h 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 r e q u i r e m e n t s i n c r e a s e d w i t h d i e t a r y PUFA o n l y when d i e t a r y l i p i d was h i g h (Fukaba 1980 c i t e d by Watanabe e t 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 c o n t e n t o f t h e  eggs were l o w e r from A r c t i c c h a r r f e d t h e 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  stores o f v i t a m i n E i n broodstock f e d the high l i p i d occurred  with  f r y i n Watanabe  e t al.  (1981a)'s  W h i l e t h i s was o b s e r v e d when d i e t a r y l i p i d  body  d i e t s as  experiment.  i n c r e a s e d i n t h e 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 LE d i e t s .  increased i n the  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 a t 600 mg/kg i n t h e h i g h l i p i d d i e t , b u t t h i s l e v e l o f supplementation  was  excessive  at  the  lower  lipid  r e s u l t i n g i n i n c r e a s e d body s t o r e s o f v i t a m i n E and amounts  of  vitamin  E  to  transfer  to  the  level, increased  eggs.  A  final  p o s s i b i l i t y i s t h a t of decreased absorption o f v i t a m i n E i n the h i g h l i p i d d i e t s which may have r e s u l t e d i n d e c r e a s e d t r a n s f e r o f v i t a m i n E t o t h e eggs.  Decreased a b s o r p t i o n o f v i t a m i n E was  o b s e r v e d i n r a t s f e d f i s h o i l compared t o c o r n o i l (Leka e t a l . 1989).  King and  (1985) o b s e r v e d a d e c r e a s e i n t h e v i t a m i n E  concentration  o f rainbow t r o u t eggs  content  (5.39 uq/egg o r 58.3  fj,g/g) when b r o o d s t o c k were f e d 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 t h o s e f e d 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 o r 85.8 \ig/g) f o r 6 months p r i o r t o spawning. et  a l . (1972) a l s o o b s e r v e d i n c r e a s e s  i n vitamin  Kinumaki  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 vitamin  E increased  advisable  t o examine  from 66 t o 4600 the trends  and 8200 mg/kg.  that  occur  rather  compare t h e a c t u a l v i t a m i n E c o n t e n t s i n c e c o n t e n t on egg w e i g h t .  It is than  to  i s dependent  Rainbow t r o u t eggs i n Kinumaki e t a l . (1972)'s  e x p e r i m e n t were a l m o s t t w i c e t h e weight o f A r c t i c c h a r r eggs i n the present  experiment.  169 Vitamin generally  content  higher  experiment. salmon  E  concentration of  K i n u m a k i e t a l . 1972).  King  rainbow  in  was  Atlantic  and  eggs  fry  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 l . 1985)  the  the  present  et  that  in  the  (Cowey  than  and  trout  (King  1985,  (1985) found t h a t t h e v i t a m i n  c o n t e n t o f r a i n b o w t r o u t eggs was  almost c o n s t a n t  E  from t h e t i m e  o f spawning u n t i l h a t c h w h i l e 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 . K i n u m a k i e t a l . (1972) found s l i g h t r e d u c t i o n s i n v i t a m i n E i n the  egg  between spawning and  absorbed.  the  time the  yolk  sac  had  been  Cowey e t a l . (1985) showed t h a t t h e v i t a m i n E  content  o f A t l a n t i c salmon f e r t i l i z e d eggs d e c r e a s e d from 3.10  \iq/eqq  (39.7  \iq/q) t o 2.29  \iq per  f r y (32.7  The  higher v i t a m i n E content  fry  compared t o eggs may  \iq/q) a t t h e  swimup s t a g e .  and c o n c e n t r a t i o n i n A r c t i c  have been due  t o the  charr  fact that  less  t h a n o n l y 2 months e l a p s e d p r i o r t o a n a l y z i n g t h e f r y compared to  8-9  months f o r t h e eggs, a l t h o u g h  nitrogen  and  immediately egg  and  laboratory. fry  at  -115°C.  Tubes  f l u s h e d w i t h n i t r o g e n and  samples  nitrogen  frozen  samples were f l u s h e d w i t h  were  packed  frozen  on  until  ice  the  but  containing  f r y were  f r o z e n on d r y i c e w h i l e were  not  samples were  flushed  returned  to  with the  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 o r a n a l y s i s and/or use  i n a growth t r i a l  and  values  for  swimup f r y are r e p r e s e n t a t i v e o n l y of s u r v i v o r s from each l o t o f eggs.  I t i s not  different  from t h e  (1985) a n a l y z e d  known i f the non-survivors  composition from t h e  t h e dead eyed eggs and  of  survivors  same p a r e n t .  l i v e eyed eggs from  was King one  170  parent  and  although  found  no  d i f f e r e n c e i n the  content  of  the  eggs  t h e dead eggs had a h i g h e r c o n c e n t r a t i o n because  they  were s m a l l e r t h a n t h e l i v e eggs.  K i n g (1985) recommended a minimum o f 7 ng/egg (82 \ig/g) f o r b e s t s u r v i v a l o f r a i n b o w t r o u t and found s u r v i v a l was p o o r e s t 5 ng/egg (54  (xg/g).  Eggs from the w i l d s t o c k o f A r c t i c  from Tree R i v e r c o n t a i n e d v i t a m i n E a t 73.8  »ug/g o r 9.0  at  charr  \ig/egq.  T h i s c o n c e n t r a t i o n i s 1.3-1.9 times h i g h e r t h a n t h a t o f L a b r a d o r c h a r r eggs i n e i t h e r LE  group, y e t t h e c o n t e n t  i s similar  to  t h a t o f t h e HLHE group because t h e Tree R i v e r c h a r r eggs weighed twice  as  much as  the  Labrador c h a r r  eggs.  W h i l e Tree  River  c h a r r eggs were c l o s e t o h a t c h when sampled, b o t h Cowey e t a l . (1985) and  K i n u m a k i e t al.  (1981) showed t h a t t h e r e was  d e c l i n e i n v i t a m i n E between t h e u n f e r t i l i z e d egg Atlantic fish  salmon and  i n the present  rainbow t r o u t ,  respectively.  experiment produced eggs w i t h  and  little  hatch  in  I n Year  2,  1.5-2.8  and  2.4-4.9 \ig v i t a m i n E/egg when f e d t h e 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 . criteria,  eggs i n t h e  LE  Using King  group were a l l below t h e  (1985)'s 5  (xg/egg  t h r e s h o l d w h i l e t h o s e i n the HE group were a l m o s t a l l above t h e 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  t h a n r a i n b o w t r o u t eggs and i t i s e x p e c t e d t h a t a s m a l l e r embryo w o u l d r e q u i r e l e s s v i t a m i n E. less trout.  for Arctic  charr  than  Therefore, recommended  the t h r e s h o l d may by  King  for  be  rainbow  171 The f e e d i n g o f 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 in  decreased  hatchability  i n turkeys  (Jensen  et  a l . 1955,  A t k i n s o n e t a l . 1955) and c h i c k e n s (Tengerdy and N o c k e l s The  involvement  of  vitamin  E  i n survival  of  the  s u b s t a n t i a t e d by showing t h a t i n j e c t i o n o f v i t a m i n E  1973).  egg  was  directly  i n t o v i t a m i n E - d e f i c i e n t eggs i n c r e a s e d h a t c h a b i l i t y (Jensen and McGinnis content  1957).  As has been found w i t h  of the chicken  egg was  d i e t a r y vitamin E content  directly  fish,  the vitamin  r e l a t e d t o maternal  ( B a r t o v e t a l . 1965).  King  (1985)  found eggs, from rainbow t r o u t f e d d i e t s c o n t a i n i n g 8% with  0 mg  vitamin  E/kg f o r 6 months  E  prior  t o spawning,  lipid had  s i g n i f i c a n t l y l o w e r s u r v i v a l (89%) t h a n t h o s e f e d 90 mg v i t a m i n E/kg  (98%).  Takeuchi  e t a l . (1981a) a l s o found  hatchability  d e c r e a s e d s i g n i f i c a n t l y when ayu were f e d 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 t h a n 34 mg v i t a m i n E/kg.  I n t h e p r e s e n t e x p e r i m e n t , s i g n i f i c a n t d i f f e r e n c e s were n o t observed  i n t h e embryonic  survival  o f c h a r r when f e d t h e LE  compared t o HE d i e t s i n e i t h e r y e a r .  I n Year 2, t h e medians f o r  s u r v i v a l t o swimup were 45 and 57% f o r t h e HE d i e t s i n d i c a t i n g t h a t h a l f o f t h e s e females produced eggs which had s u r v i v a l t o swimup o f g r e a t e r t h a n 45-57%.  I n c o n t r a s t , 50% o f t h e f i s h f e d  t h e LE d i e t s had s u r v i v a l t o swimup o f g r e a t e r t h a n 9 and 1% and t h o s e f e d COMM had a median o f 17%.  172 The w i t h i n t r e a t m e n t v a r i a n c e f o r s u r v i v a l o f eggs t o t h e eyed, h a t c h and swimup s t a g e s was v e r y h i g h and t h e sample s i z e was  low w i t h 7-12  f i s h and 6-10  Year 1 and 2, r e s p e c t i v e l y .  f i s h spawning p e r t r e a t m e n t  in  The low sample s i z e was due t o m i s -  sexing of f i s h at the beginning of the experiment,  failure  of  some females t o spawn each y e a r , premature r e l e a s e o f eggs i n t o the  tank  and  v a r i a n c e s and  a  low  number o f  fish  mortalities.  The  high  low sample number r e s u l t e d i n t h e need f o r v e r y  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 d e t e c t e d w i t h an a c c e p t a b l e degree o f c e r t a i n t y .  Therefore,  w h i l e t h e d i f f e r e n c e s i n t h e mean s u r v i v a l s i n Year 2 l e a d t o s u s p e c t t h a t t h e r e was  a p o s i t i v e e f f e c t o f v i t a m i n E,  d i f f e r e n c e s a r e not s i g n i f i c a n t .  one  these  For example, f e e d i n g t h e  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 t h e two HE r e s u l t e d i n 42 and 51% s u r v i v a l t o swimup.  two  diets  U s i n g 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 d a t a f o r s u r v i v a l t o t h e eyed s t a g e , t h e r e i s a 40% rejecting  ( i e . accepting)  the  null  hypothesis  chance o f when t h e  not null  h y p o t h e s i s i s f a l s e (^=45, o =1089, s =953, n=7.5, a=0.05). 2  In  2  o t h e r words, g i v e n the v a r i a n c e 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 t h e r e 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 , t h e chance o f d e t e c t i n g i t would be 40%.  Sample s i z e s o f 15 and  would  power t o  be  required to  i n c r e a s e the  respectively.  The  planned  sample s i z e was  sample s i z e was  as low as 6 i n Year 2.  0.75  and  25  0.95,  15 but t h e a c t u a l  173 The  c a s e o f low  sample s i z e o f t e n w i t h a h i g h degree  v a r i a t i o n i s common i n b r o o d s t o c k 3-6  f i s h per treatment  Yu e t al.  n u t r i t i o n w i t h sample s i z e s o f  reported  ( K i n g 1985,  1979, Watanabe e t a l . 1984b).  a l l broodstock Knox e t al. per treatment  Leray  e t al.  1985,  That i s not t o say t h a t  n u t r i t i o n i s conducted on s m a l l groups o f  (1988) used 40  of  fish.  females p e r t a n k w i t h 2 r e p l i c a t e s  i n h i s work on the e f f e c t o f r a t i o n , but g e n e r a l l y  h i g h numbers o f b r o o d s t o c k  are not  available.  In f u t u r e , i t  w i l l be n e c e s s a r y t o reduce the number o f t r e a t m e n t s  i n order to  have r e p l i c a t e t a n k s o f f i s h and t o i n c r e a s e t h e number o f  fish  per tank.  year  I n a d d i t i o n , not a l l males produced m i l t each  and i t i s s u g g e s t e d t h a t the number o f males a l s o be  The  fact  that  v i t a m i n E content any  stage  in this  there  was  no  direct  increased.  correlation  between  o r c o n c e n t r a t i o n o f t h e eggs and s u r v i v a l  to  experiment i n d i c a t e s e i t h e r t h a t t h e r e  was  adequate v i t a m i n E even a t the lower l e v e l o f d i e t a r y v i t a m i n E for  adequate d e p o s i t i o n i n t h e  i n d i r e c t e f f e c t ( s ) o f v i t a m i n E.  egg  o r t h a t t h e r e were  other  Other i n d i r e c t e f f e c t s such as  p r o m o t i n g 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 o f eggs.  King  (1985) r e p o r t e d  one  c a s e o f eggs w i t h  the  dispersed  l i p i d g l o b u l e s and c o n v o l u t e d membranes i n r a i n b o w t r o u t groups fed  the  unsupplemented d i e t  f o r 6 months p r i o r  to  spawning.  Spawning a b n o r m a l i t i e s o c c u r r e d i n b o t h y e a r s i n t h i s s t u d y  but  i n Year 2, 4 o f t h e 5 cases o c c u r r e d i n f i s h f e d LE d i e t s and w i t h t h e HE d i e t s .  1  These a b n o r m a l i t i e s i n c l u d e d eggs w h i c h were  174 r e l e a s e d i n t o t h e t a n k , eggs which formed a s o l i d mass and c o u l d not be  released,  t h e presence o f b l o o d i n t h e o v a r i a n  brown p a r t i c u l a t e m a t t e r  i n the f l u i d ,  and  small  fluid,  numbers  of  u n u s u a l brown and d a r k o r a n g e - c o l o u r e d eggs w i t h t h e orange eggs o c c u r r i n g i n a f i s h f e d a HE d i e t . in  the  ovarian  fluid  might  While the presence of b l o o d  be  i n d i c a t i v e of  a  vitamin  E  d e f i c i e n c y i n w h i c h t h e r e was d e t e r i o r a t i o n o f t h e w a l l s o f t h e capillaries,  the incidence  dietary vitamin  of t h i s  s i g n was  very small.  If  E had some o t h e r 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 e x p e r i m e n t .  5.8 F r y growth and s u r v i v a l S u r v i v a l o f f r y d e c r e a s e d from 82% i n Year 1 t o 76% i n Year 2 w h i c h was  s i m i l a r t o t h e d e c r e a s e from 88% t o 79% t h a t  was  p r e d i c t e d from t h e d e c r e a s e i n number o f f r y r a i s e d i n t h e j a r s . W i t h t h e e x c e p t i o n o f t h e LLHE group, t h e s p e c i f i c growth increased  i n Year 2 from 1.7%  i n Year 1 t o 2.4% i n Year 2 w h i c h  was g r e a t e r t h a n t h e p r e d i c t e d 0.4%  i n c r e a s e r e s u l t i n g from t h e  d e c r e a s e i n number o f f r y i n t h e j a r s .  Environmental conditions  were as s i m i l a r as p o s s i b l e i n both Year purchased  from  the  same  proximate  c o m p o s i t i o n was  company 39.7%  1 and  2.  and  analysis  crude  protein,  Feed  was  showed  the  25.5%  l i p i d , 11.8% ash and 82.6% d r y m a t t e r i n Year 1 and 49.7% p r o t e i n , 20.0% Year 2.  total lipid,  rate  11.8% ash and 80.6%  total crude  dry matter i n  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  t h e i n g r e d i e n t s used by t h e m a n u f a c t u r e r i n Year 2 may a l s o have  175 c o n t r i b u t e d t o t h e 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 o f v i t a m i n E c o n t e n t  or  concentration  o f t h e f r y on s u r v i v a l o f f r y i n e i t h e r y e a r o r on growth r a t e i n Year 1.  specific  However, i n Year 2 f r y from t h e 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 t h a n t h o s e f e d t h e o t h e r LL o r HE d i e t s . LLHE group had t h e l o w e s t  I n a d d i t i o n , f r y from t h e  s u r v i v a l during  t h e 6 week  t r i a l a l t h o u g h 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 .  growth  Since the  f r y i n t h e LLHE group had t h e h i g h e s t l e v e l s o f v i t a m i n E, one must c o n s i d e r t h e p o s s i b i l i t y o f h y p e r v i t a m i n o s i s no  c o r r e l a t i o n between  vitamin  E  content  specific  growth  or concentration.  rate  but t h e r e  was  o r s u r v i v a l and  Although  studies  with  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 o f s k e l e t a l mitochondria  were d e c r e a s e d 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  o f 2200 mg v i t a m i n E a c e t a t e / k g acetate/kg  (March  compared t o 1000 mg v i t a m i n E  e t a l . 1973), t h e r e  r e p o r t e d on h y p e r v i t a m i n o s i s  E in fish.  have been  no  studies  Kinumaki e t a l . (1972)  f e d 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 and d i d n o t r e p o r t i n c r e a s e d m o r t a l i t y o f eggs o r f r y .  E/kg  176 CONCLUSIONS  F e e d i n g 4-5 y e a r o l d Labrador A r c t i c c h a r r b r o o d s t o c k containing  12  acetate/kg  or  diet  19%  lipid  d i d not  with  result  30  or  600  in significant  mg  diets  vitamin  E  differences i n  f e r t i l i z a t i o n o f eggs o r s u r v i v a l t o t h e eyed, h a t c h o r swimup stages.  I n Year 2,  fish  f e d 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 h i g h e r t h a n t h o s e f e d t h e LE d i e t s .  The  h i g h degree o f w i t h i n treatment  the  eyed, h a t c h  and  swimup stages  variation  and  i n survival to  t h e s m a l l number o f  females  w h i c h spawned r e s u l t e d i n the need f o r v e r y l a r g e d i f f e r e n c e s i n means  in  order  to  detect  significant  differences.  research w i t h Labrador A r c t i c charr broodstock numbers o f f i s h p e r t r e a t m e n t  and  Future  requires  r e p l i c a t e tanks  higher  i n order  to  i n c r e a s e t h e power o f t h e t e s t .  F e e d i n g LL d i e t s r e s u l t e d i n d e l a y e d spawning i n Year 1 but this  was  not  apparent i n Year 2.  f a i l i n g t o spawn was l i p i d o r v i t a m i n E. fed  The  p r o p o r t i o n of  not r e l a t e d t o c o n c e n t r a t i o n s  females  of d i e t a r y  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 o f males  t h e h i g h e r c o n c e n t r a t i o n o f v i t a m i n E produced m i l t i n  successive  years.  Fecundity  treatments  but  v a r i a t i o n was  weight  was  not  and  l i p i d o r v i t a m i n E.  was  highly  directly  a f f e c t e d by  the  two  variable  within  correlated with  female  concentration  of d i e t a r y  F i s h f e d COMM consumed more f e e d , weighed  more and produced s i g n i f i c a n t l y more eggs t h a n t h o s e  fed  other  177 d i e t s i n Year 2 e x c e p t f o r LLLE.  S u r v i v a l t o swimup o f t h e COMM  group was h a l f t h a t o f t h e 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 t h e v i t a m i n E c o n c e n t r a t i o n and c o n t e n t o f t h e eggs even when f e d t h e d i e t s for  only  71 days  concentration  i n Year  had  a  1.  greater  Increasing impact  dietary vitamin E  on  egg  vitamin  E  c o n c e n t r a t i o n a t t h e low compared t o t h e h i g h c o n c e n t r a t i o n o f dietary  lipid.  T h i s might be due t o d e c r e a s e d a b s o r p t i o n o r  i n c r e a s e d o x i d a t i o n o f v i t a m i n E w i t h i n t h e t i s s u e s when t h e h i g h l i p i d d i e t was f e d .  F e r t i l i z a t i o n and embryonic  were n o t 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 t e n t t h e eggs.  I f t h e broodstock  survival  or concentration o f  had been f e d 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 o f t h e i r l i f e t i m e , t h e y may have had l o w e r s t o r e s o f v i t a m i n E a t t h e b e g i n n i n g o f t h e e x p e r i m e n t and  more  pronounced  e f f e c t s of feeding  a diet  with  a low  c o n c e n t r a t i o n o f v i t a m i n E may have been o b s e r v e d .  D i e t a r y l i p i d and v i t a m i n E c o n c e n t r a t i o n s d i d n o t a f f e c t the t o t a l ,  neutral or polar l i p i d  concentrations  F e e d i n g COMM r e s u l t e d i n s i g n i f i c a n t l y h i g h e r  o f t h e eggs.  levels of total  and n e u t r a l l i p i d i n t h e eggs o f f i s h f e d a l l d i e t s e x c e p t LLLE.  The  fatty  a c i d composition  o f eggs  deficiency of essential fatty acids.  did  not i n d i c a t e a  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 n o t d e t e c t e d  178 i n any eggs a n a l y z e d .  The c o n c e n t r a t i o n o f 22:6n3 was  uniformly  h i g h i n t h e p o l a r l i p i d s o f eggs from f i s h f e d a l l d i e t s .  The  f a t t y a c i d c o m p o s i t i o n o f t h e eggs r e f l e c t e d t h a t o f t h e 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 t h a n t h e 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 t h e s e f a t t y a c i d s and t h e n3/n6 r a t i o were a p p a r e n t i n t h e p o l a r l i p i d s o f t h e eggs. and  PUFA  i n the  treatments. negatively  COMM eggs were s i g n i f i c a n t l y h i g h e r neutral  lipids  than  from  a l l other  S u r v i v a l t o t h e eyed, h a t c h and swimup s t a g e s was correlated with  the percent  1 6 : l n 7 , one o f t h e major n7 f a t t y a c i d s fraction.  eggs  i n n6  of neutral  and  i n the neutral  The low c o e f f i c i e n t o f d e t e r m i n a t i o n  effect of neutral l i p i d  lipid  lipid  ( r ) f o r the 2  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 c h a r r from t h e Tree R i v e r , NWT high  s u r v i v a l t o swimup.  They had v i t a m i n  E  had  concentration  h i g h e r t h a n t h o s e f e d t h e 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.  Fry  s u r v i v a l and  vitamin E or l i p i d fry  growth  were  not  affected  by  dietary  i n Year 1 o r Year 2 w i t h t h e e x c e p t i o n  from p a r e n t s f e d LLHE i n Year 2 had s i g n i f i c a n t l y  s p e c i f i c growth r a t e s t h a n t h o s e f e d o t h e r d i e t s .  Since  that  poorer these  f r y had s i g n i f i c a n t l y h i g h e r v i t a m i n E c o n c e n t r a t i o n , t h e i r poor growth r a t e may have been r e l a t e d t o  hypervitaminosis.  179 LITERATURE CITED Ackman, R.G. 1982. F a t t y a c i d c o m p o s i t i o n o f f i s h o i l s . 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Academic P r e s s , London, England. Watanabe, T., T. Arakawa, C. K i t a j i m a and S. F u j i t a . 1984a. E f f e c t o f n u t r i t i o n a l q u a l i t y o f b r o o d s t o c k d i e t s on r e p r o d u c t i o n o f r e d sea bream. B u l l . J a p . Soc. S c i . F i s h . 50: 495-501. Watanabe, T., T. F u j i m u r a , M.-J. Lee, K. Fukusho, S. S a t o h and T. T a k e u c h i . 1991. E f f e c t o f p o l a r and n o n p o l a r l i p i d s from k r i l l on q u a l i t y o f eggs o f r e d seabream Pagrus major. Nippon S u i s a n G a k k a i s h i 57: 695-698. Watanabe, T., A. I t o h , S. S a t o h , C. K i t a j i m a and S. F u j i t a . 1985a. E f f e c t o f d i e t a r y p r o t e i n l e v e l s and f e e d i n g p e r i o d b e f o r e spawning on c h e m i c a l components o f eggs produced by r e d s e a bream b r o o d s t o c k . B u l l . J a p . Soc. S c i . F i s h . 51: 1501-1509. Watanabe, T., T. K o i z u m i , H. S u z u k i , S. S a t o h , T. T a k e u c h i , N. Y o s h i d a , T. K i t a d a and Y. Tsukashima. 1985b. Improvement o f q u a l i t y o f r e d sea bream eggs by f e e d i n g b r o o d s t o c k on a d i e t c o n t a i n i n g c u t t l e f i s h meal o r on raw k r i l l s h o r t l y b e f o r e spawning. B u l l . J a p . Soc. S c i . F i s h . 51: 1511-1521. Watanabe, T., C. Ogino, Y. K o s h i s h i and T. Matsunaga. 1974a. Requirement o f r a i n b o w t r o u t f o r e s s e n t i a l f a t t y a c i d s . B u l l . J a p . Soc. S c i . F i s h . 40: 493-499. Watanabe, T. and F. Takashima. 1977. E f f e c t of a-tocopherol d e f i c i e n c y on c a r p - V I . D e f i c i e n c y symptoms and changes o f f a t t y a c i d and t r i g l y c e r i d e d i s t r i b u t i o n s i n a d u l t c a r p . B u l l . J a p . Soc. S c i . F i s h . 43: 819-830. Watanabe, T., F. Takashima and C. Ogino. 1974b. Effect of d i e t a r y m e t h y l l i n o l e n a t e on growth o f r a i n b o w t r o u t . B u l l . J a p . Soc. S c i . F i s h . 40: 181-188. Watanabe, T., T. T a k e u c h i , M. S a i t o and K. N i s h i m u r a . 1984b. E f f e c t o f low p r o t e i n - h i g h c a l o r y o r 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 d i e t on r e p r o d u c t i o n o f r a i n b o w t r o u t . Bull. Jap. Soc. S c i . F i s h . 50: 1207-1215. Watanabe, T., T. T a k e u c h i , M. Wada and R. Uehara. 1981a. The relationship between d i e t a r y lipid and a-tocopherol requirement o f rainbow t r o u t . B u l l . J a p . Soc. S c i . F i s h . 47: 1463-1471.  190 Watanabe, T., M. Wada and T. T a k e u c h i . 1981b. Absence o f i n t e r c o n v e r s i o n o f t o c o p h e r o l s i n rainbow t r o u t . Bull. J a p . Soc. S c i . F i s h . 47: 1455-1462. W i l c o x , K.W., J . S t o s s and E.M. Donaldson. 1984. Broken eggs as a cause o f i n f e r t i l i t y o f coho salmon gametes. A q u a c u l t u r e 40: 77-87. Woyewoda, A.D., S . J . Shaw, P . J . Ke and B.6. Burns. 1986. Recommended l a b o r a t o r y methods f o r assessment o f f i s h q u a l i t y . Can. Tech. Rep. F i s h . Aquat. S c i . 1448: v + 156 p. Yang, X. and T. D i c k . 1992. E s s e n t i a l f a t t y a c i d r e q u i r e m e n t s o f A r c t i c c h a r r , (Salvelinus alpinus L . ) . P r o c . Annual M e e t i n g A q u a c u l t u r e A s s o c . Canada. Vancouver, BC. June 1-3, 1992. Abstract. Yu, T.C., R.O. Sinnhuber and J.D. H e n d r i c k s . 1979. R e p r o d u c t i o n and s u r v i v a l o f rainbow t r o u t (Salmo gairdneri) fed l i n o l e n i c a c i d as t h e o n l y source o f e s s e n t i a l f a t t y a c i d s . L i p i d s 14: 572-575. Y u r k o w s k i , M. 1989. L i p i d c o n t e n t and f a t t y a c i d c o m p o s i t i o n o f m u s c l e from some f r e s h w a t e r 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.) H e a l t h E f f e c t s o f 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 t h e t o t a l l i p i d i n t h e d i e t s . Fatty acid  LLLE  14:0 15:01 15:0 16:01 Unknown 16:0 16:ln7 16:ln5 Unknown 16:2n9 Unknown 17:0 Unknown 17 l n 9 18 01 16 5n3 18 0 18 l n 9 18 l n 7 18 l n 5 18 2n6 18 3n6 19 0 18 4n6 19 l n 7 18 3n3 18 4n3 20 0 20 l n l l 20 l n 9 20 l n 7 20 2n6 20 4n6 20 3n3 20 4n3 20 5n3 22 0 22 l n l l 22 l n 9 22 l n 7 21 5n3 22 5n6 22 5n3 22 6n3 24 l n 9 Unknown  4.16 0.15 0.34 0.11 0.25 16.40 5.06 0.18 0.29 0.15 0.42 0.83 0.24 0.32 0.22 0.37 2.72 18.71 3.70 0.32 7.46 0.15 0.09 0.11 0.10 1.32 0.88 0.20 3.84 3.30 0.48 0.18 0.62 0.08 0.29 5.97 0.13 6.60 1.01 0.20 0.21 0.12 0.92 6.77 0.78 0.17  %  LLHE %  4.11 0.08 0.36 0.14 0.27 16.43 5.05 0.19 0.33 0.17 0.44 0.86 0.26 0.31 0.20 0.40 2.69 18.60 3.72 0.33 7.40 0.16 0.10 0.12 0.10 1.32 0.88 0.20 3.77 3.29 0.48 0.18 0.62 0.08 0.29 5.92 0.13 6.50 1.00 0.19 0.21 0.12 0.90 6.75 0.74 0.16  HLLE %  4.44 0.17 0.36 0.13 0.27 15.97 5.51 0.20 0.34 0.17 0.47 0.94 0.26 0.32 0.19 0.42 2.68 18.48 3.81 0.35 4.90 0.17 0.10 0.13 0.10 1.00 1.00 0.20 4.63 3.52 0.56 0.20 0.63 0.09 0.33 6.20 0.11 7.49 1.17 0.21 0.23 0.13 1.03 6.33 0.80 0.12  HLHE %  4.09 0.15 0.33 0.10 0.26 15.61 5.36 0.18 0.31 0.16 0.45 0.92 0.24 0.31 0.18 0.39 2.69 18.83 3.62 0.35 4.97 0.17 0.09 0.12 0.10 1.01 1.00 0.21 5.04 3.32 0.57 0.20 0.64 0.09 0.34 6.36 0.12 7.83 1.16 0.21 0.25 0.14 1.08 6.58 0.84 0.11  COMM COMM Year 1 Year 2 %  5.14 0.16 0.31 0.09 0.11 15.77 7.76 0.22 0.30 0.13 0.79 0.14 0.22 0.23 0.11 1.38 2.53 12.44 2.64 0.26 10.68 0.23 0.14 0.17 0.04 1.32 1.77 0.14 0.43 4.49 0.29 0.14 0.39 0.06 0.66 10.45 0.09 5.39 0.61 0.13 0.38 0.07 1.13 5.89 0.42 0.07  %  3.86 0.17 0.31 0.09 0.22 12.16 6.97 0.24 0.15 0.10 0.48 0.22 0.15 0.15 0.13 0.60 1.63 10.93 2.35 0.40 10.52 0.12 0.08 0.07 0.04 1.01 1.03 0.17 0.68 11.32 0.81 0.13 0.25 0.05 0.24 5.05 0.09 15.52 1.68 0.33 0.19 0.07 0.61 4.90 0.59 0.06  192 Appendix l b . F a t t y a c i d c o m p o s i t i o n (mean % and s t a n d a r d d e v i a t i o n ) o f t h e n e u t r a l l i p i d s o f t h e eggs i n Year 2. Fatty acid  LLLE %  2. 20 14:0 0. 11 14:ln5 15:01 0. 10 0. 25 15:0 0. 11 16:01 16:0 11. 81 0. 15 Unkn. 0. 69 16:ln9 16:ln7 11. 29 0. 19 16:ln5 0. 23 Unkn. 0. 19 16:2n9 0. 16 Unkn. 0. 21 17:0 0. 14 Unkn. 0. 52 17:ln9 18:0 1. 53 18:ln9 31. 06 18:ln7 6. 55 0. 32 18:ln5 0. 25 Unkn. 0. 17 18:2n9 5. 52 18:2n6 0. 22 18:3n6 0. 64 19:0 18:4n6 0. 20 0. 12 19:ln7 0. 61 18:3n3 0. 55 18:4n3 0 .19 18:4nll 0 .77 20:lnll 2. 16 20:ln9 0. 42 20:ln7 0. 32 Unkn. 0. 72 20:2n6 0. 71 20:3n6 0. 67 20:4n6 0. 33 20:4n3 20:5n3 3. 55 0 .32 22:lnll 0. 17 22:ln9 0. 22 21:5n3 0. 11 22:5n6 22:5n3 1. 25 9. 50 22:6n3  LLHE SD 0. 14 0. 01 0. 02 0. 03 0. 03 0. 87 0. 03 0. 14 1. 09 0. 02 0. 04 0. 04 0. 03 0. 06 0. 04 0. 05 0. 14 1. 04 0. 35 0. 01 0. 07 0. 03 0. 48 0. 02 0. 21 0. 01 0. 01 0. 07 0. 11 0 .03 0 .13 0. 21 0. 04 0. 09 0. 10 0. 16 0. 09 0. 10 0. 65 0 .06 0. 01 0. 02 0. 02 0. 24 1. 59  %  1 .97 0 .09 0 .09 0 .21 0 .09 10 .81 0 .15 0 .78 10 .89 0 .17 0 .19 0 .16 0 .16 0 .16 0 .11 0 .43 1 .54 32 .10 6 .16 0 .31 0 .33 0 .18 5 .50 0 .20 0 .85 0 .20 0 .11 0 .46 0 .42 0 .17 0 .70 2 .65 0 .45 0 .42 0 .71 0 .80 0 .76 0 .26 3 .46 0 .40 0 .23 0 .20 0 .10 1 .33 10 .19  Diet HLLE  HLHE  COMM  % SD SD % % SD SD 2 .22 0 .35 2 .07 0. 36 2.52 0.23 0. 24 0 .10 0 .02 0 .07 0. 04 0.11 0.02 0. 02 0 .11 0 .01 0 .10 0. 01 0.11 0.01 0. 02 0 .26 0 .02 0 .25 0. 05 0.26 0.03 0. 02 0. 01 0 .11 0 .00 0 .09 0. 02 0.08 0.01 2. 97 10 .46 1 .11 10 .64 1. 29 10.58 0.41 0 .17 0 .02 0 .14 0. 02 0.28 0.04 0. 06 0 .65 0 .10 0 .59 0. 11 0.55 0.30 0. 19 1. 43 10 .98 0 .93 11 .34 0. 78 12.65 0.87 0 .20 0 .01 0 .19 0. 03 0.26 0.02 0. 01 0 .21 0 .03 0 .20 0. 04 0.08 0.05 0. 01 0 .22 0 .04 0 .17 0. 01 0.22 0.03 0. 01 0 .19 0 .02 0 .20 0. 03 0.30 0.03 0. 03 0 .23 0 .03 0 .21 0. 04 0.01 0.01 0. 08 0 .13 0 .00 0 .13 0. 02 0.10 0.01 0. 01 0. 03 0 .56 0 .05 0 .55 0. 09 0.30 0.11 1 .46 0 .10 1 .43 0. 12 1.26 0.12 0. 45 2. 65 31 .67 1 .49 32 .40 2. 94 27.67 1.98 6 .03 0 .46 5 .58 0. 76 5.66 0.58 1. 25 0. 04 0 .34 0 .00 0 .31 0. 04 0.39 0.01 0 .20 0 .06 0 .24 0. 10 0.16 0.05 0. 17 0. 02 0 .10 0 .04 0 .13 0. 01 0.15 0.01 5 .47 1 .03 5 .84 1. 04 7.88 0.75 1. 37 0 .23 0 .02 0 .23 0. 03 0.18 0.09 0. 02 0 .57 0 .33 0 .69 0. 38 0.71 0.32 0. 82 0 .22 0 .01 0 .24 0. 02 0.24 0.01 0. 04 0 .11 0 .02 0 .19 0. 20 0.07 0.00 0. 02 0. 11 0 .62 0 .09 0 .65 0. 12 0.56 0.07 0. 11 0 .55 0 .04 0 .67 0. 10 0.64 0.13 0. 04 0 .21 0 .01 0 .24 0. 02 0.35 0.03 0. 20 0 .90 0 .18 0 .84 0. 27 0.66 0.09 0. 61 2 .27 0 .38 1 .91 0. 17 3.85 0.40 0 .41 0 .03 0 .39 0. 07 0.43 0.05 0. 07 0 .18 0 .07 0 .19 0. 08 0.17 0.09 0. 13 0. 07 0 .60 0 .06 0 .56 0. 03 0.79 0.08 0 .57 0 .19 0 .58 0. 12 0.90 0.02 0. 16 0 .66 0 .05 0 .64 0. 09 0.59 0.08 0. 14 0 .41 0 .06 0 .41 0. 11 0.38 0.06 0. 06 4 .58 0 .22 4 .72 0. 36 3.92 0.47 0. 50 0. 23 0 .45 0 .06 0 .42 0. 09 0.86 0.20 0. 08 0 .21 0 .04 0 .19 0. 01 0.26 0.05 0 .25 0 .01 0 .27 0. 02 0.24 0.01 0. 02 0. 01 0 .10 0 .01 0 .09 0. 02 0.06 0.02 1 .63 0 .12 1 .51 0. 07 1.38 0.22 0. 14 9 .88 0 .75 9 .49 1. 41 8.58 1.46 1. 24  193 Appendix l e . F a t t y a c i d c o m p o s i t i o n (mean % ± s t a n d a r d d e v i a t i o n ) o f t h e p o l a r l i p i d s o f t h e eggs i n Year 2, Fatty acid  %  Diet HLLE  LLHE  LLLE SD  %  SD  %  SD  0. 88 0.13 0 .94 0 .11 14:0 1. 14 0. 09 0. 24 0.02 15:0 0. 27 0. 03 0. 22 0. 02 16:0 16. 49 0. 59 15 .69 1 .13 15. 99 0.28 0. 10 0.03 0 .18 0 .06 0. 18 0. 06 Unkn. 0. 45 0.15 0 .67 0 .18 16:ln9 0. 58 0. 12 2. 09 0.21 2 .57 0 .24 16:ln7 2. 39 0. 14 0. 09 0.04 0 .09 0 .02 16:ln5 0. 10 0. 01 0. 21 0.01 0 .18 0 .01 0. 23 0. 03 Unkn. 0. 10 0.01 0 .09 0 .01 16:2n9 0. 11 0. 01 0. 13 0.02 0 .14 0 .01 17:0 0. 17 0. 04 0. 17 0.03 0 .12 0 .01 0. 16 0. 01 Unkn. 0. 22 0.02 0 .20 0 .03 17:ln9 0. 24 0. 02 0. 20 0.13 0 .20 0 .03 18:01 0. 22 0. 10 3. 44 0.43 3 .39 0 .60 18:0 3. 34 0. 23 18:ln9 12. 39 0. 67 13 .55 2 .76 12. 75 1.97 6 .44 1 .32 6. 59 1.18 6. 82 0. 35 18:ln7 0. 43 0.03 0 .36 0 .06 18:ln5 0. 39 0. 03 0. 12 0.04 0 .21 0 .09 19: OA 0. 18 0. 09 0. 08 0.00 0 .09 0 .02 0. 11 0. 04 Unkn. 1. 49 0.34 1 .80 0 .17 18:2n6 1. 74 0. 23 0. 11 0.02 0 .08 0 .01 18:3n6 0. 11 0. 01 0. 06 0.03 0 .13 0 .08 0. 12 0. 04 19:0 0. 25 0.08 0 .21 0 .05 19:ln7 0. 26 0. 02 0. 14 0.03 0 .11 0 .02 0. 15 0. 02 18:3n3 0. 09 0.01 0 .10 0 .02 18:4n3 0. 14 0. 03 0. 34 0.12 20:lnll 0..30 0..06 0 .27 0 .14 4. 29 0.88 4 .68 0 .96 20:ln9 3. 80 0. 33 0. 49 0.10 0 .49 0 .09 20:ln7 0. 42 0. 02 0. 04 0.06 0 .20 0 .08 0. 12 0. 06 Unkn. .13 0. 89 0.14 .06 0 0. 1 01 12 20:2n6 1. 0. 62 0.28 1 .15 0 .07 20:3n6 0. 99 0. 30 1. 24 0.18 1 .79 0 .47 20:4n6 1. 63 0. 27 .03 0. 14 0.03 0 0. 0 .11 0. 13 05 20:4n3 8. 10 1.11 0 .69 7 .05 46 0. 76 20:5n3 7. .05 0. 16 0.04 .14 0 0. .03 0 0..10 22:lnll 12 0.01 0 0. .12 .01 0 0. 09 0. 06 21:5n3 08 0.01 .04 0. 0 .13 0 0. 10 0. 05 22:4n6 .06 0. 23 0.05 0 .24 0 22:5n6 0. 28 0. 03 .17 2. 76 0.20 2 .58 0 22:5n3 2. 32 0. 30 22:6n3 30. 51 0. 84 29 .86 1 .42 31. 38 1.45 0. 36 0.07 24:lnll 0..24 0..17 0 .36 0 .07 0. 13 0.01 0 .12 0 .01 24:ln9 0. 13 0. 02 0. 08 0.04 0 .07 0 .04 0. 10 0. 02 26:0  HLHE %  0 .97 0 .28 16 .43 0 .12 0 .48 2 .31 0 .10 0 .21 0 .10 0 .12 0 .15 0 .23 0 .21 3 .26 13 .35 6 .23 0 .41 0 .16 0 .08 1 .62 0 .11 0 .08 0 .22 0 .16 0 .13 0 .35 3 .64 0 .40 0 .08 0 .82 0 .62 1 .37 0 .14 8 .58 0 .14 0 .12 0 .06 0 .21 2 .57 30 .87 0 .30 0 .16 0 .07  SD  COMM %  SD  1.08 0.07 0. 15 0. 02 0.25 0.03 0. 51 15.60 0.95 0. 10 0.27 0.05 0. 14 0.54 0.09 0. 31 2.45 0.18 0. 01 0.13 0.01 0. 03 0.17 0.02 0. 01 0.08 0.01 0. 03 0.08 0.02 0. 02 0.12 0.02 0. 01 0.17 0.02 0. 10 0.18 0.02 0. 25 2.91 0.30 1. 55 11.62 0.72 0. 75 6.07 0.45 0. 05 0.47 0.03 0. 06 0.10 0.06 0. 00 0.09 0.00 0. 16 2.29 0.15 0. 02 0.10 0.01 0. 03 0.11 0.03 0. 05 0.15 0.02 0. 04 0.12 0.02 0. 01 0.13 0.03 0. 13 0.31 0.07 0. 47 6.04 0.49 0. 06 0.46 0.05 0. 03 0.09 0.02 0. 08 1.15 0.14 0. 11 1.15 0.14 0. 37 1.48 0.29 0. 05 0.14 0.03 0. 60 7.96 0.61 0. 03 0.26 0.04 0. 01 0.09 0.06 0. 03 0.04 0.03 0. 04 0.12 0.08 0. 18 2.82 0.29 0. 99 29.99 0.88 0. 16 0.38 0.06 0. 08 0.12 0.01 0. 03 0.11 0.03  194 Appendix I d . F a t t y a c i d c o m p o s i t i o n o f t h e n e u t r a l and p o l a r l i p i d o f eggs o f w i l d A r c t i c c h a r r from Tree R i v e r , NWT. Fatty acid 14: 0 15: 01 15: 0 16: 01 16: 0 16: l n 9 16:ln7 16:ln5 Unknown 16:2n9 Unknown 17:0 Unknown 17: l n 9 18! 01 18: 0 18: l n 9 18: ln7 18: l n 5 18: 2n9 18: 2n6 18: 3n6 19: :0 18: •4n6 19 :ln7 18: :3n3 18 s4n3 18 : 4 n l l 20 : l n l l 20 :ln9 20 :ln7 20 :2n6 20 :3n6 20 :4n6 20 !4n3 20 :5n3 22 : l n l l 22 :ln9 21 :5n3 22 :4n6 22 :5n6 22 :5n3 22 :6n3 24 s l n l l  Neutral lipid %  2.42 0.11 0.22 0.13 9.66 tr 17.71 0.30 tr 0.13 0.24 0.15 tr 0.34 tr 1.88 21.28 7.92 0.45 0.10 2.15 0.23 0.16 0.20 0.06 0.77 0.64 0.19 0.18 1.61 0.33 0.29 0.30 0.48 0.88 11.74 0.17 0.12 0.28 tr tr 4.27 9.59 tr  Polar lipid %  0.88 tr 0.22 tr 15.23 0.29 3.46 0.15 0.16 0.10 tr 0.15 0.13 0.16 0.23 4.95 10.62 8.53 0.64 tr 0.55 0.12 tr tr 0.12 0.15 tr tr 0.11 3.07 0.52 0.41 0.27 0.82 0.34 12.66 0.06 tr 0.12 0.12 0.12 6.21 25.50 0.43  195 Appendix 2a. Weekly water temperature (mean and s t a n d a r d d e v i a t i o n ) i n Year 1 and Year 2 (n=5). Year 1 Week  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27  Date  280688 -040788 050788 -110788 120888 -180788 190788 -250788 260788 -010888 020888 -080888 090888 -150888 160888 -220888 230888 -290888 300888 -050988 060988 -120988 130988 -190988 200988 -260988 270988 -031088 041088 -101088 111088 -171088 181088 -241088 251088 -311088 011188.-071188 081188 -141188 151188--211188 221188 -281188 291188--041288 051288--111288 121288--181288 191288--251288 261288--010189  Year 2 Temperature °C SD  8. 57 8. 58 8. 69 8. 76 8. 55 8. 61 8. 50 8. 28 8. 33 8. 15 8. 16 7. 97 7. 89 7. 66 7. 65 7. 55 7. 14 7. 33 7. 15 7. 84 7. 69 7. 68 7. 66 7. 71 7. 93 7. 72  0. 58 0. 12 0. 08 0. 18 0. 16 0. 16 0. 12 0. 10 0. 15 0. 14 0. 13 0. 05 0. 14 0. 21 0. 22 0. 14 0. 22 0. 09 0. 14 0. 16 0. 13 0. 06 0. 09 0. 05 0. 16 0. 03  Week  28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79  Date  020189-080189 090189-150189 160189-220189 230189-290189 300189-050289 060189-120289 130289-190289 200289-260289 270289-050389 060389-120389 130389-190389 200389-260389 270389-020489 030489-090489 100489-160489 170489-230489 240489-300489 010589-070589 080589-140589 150589-210589 220589-280589 290589-040689 050689-110689 120689-180689 190689-250689 260689-020789 030789-090789 100789-160789 170789-230789 240789-300789 010889-070889 080889-140889 150889-210889 220889-280889 290889-040989 050989-110989 120989-180989 190989-250989 260989-021089 031089-091089 101089-161089 171089-231089 241089-301089 311089-061189 071189-131189 141189-201189 211189-271189 281189-041289 051289-111289 121289-181289 191289-251289 261289-010190  Temperature °C SD 8. 09 8. 18 8. 28 8. 34 8. 07 8. 21 8. 10 7. 95 7. 84 8. 08 8. 14 8. 23 8. 08 8. 13 8. 18 8. 39 7. 87 7. 61 8. 01 8. 17 7. 87 8. 02 8. 11 8. 17 8. 42 8. 20 8. 00 8. 35 8. 64 8. 56 8. 50 8. 59 8. 63 8. 65 8. 56 8. 35 8. 27 7. 83 7. 83 7. 61 7. 59 7. 55 7. 48 7. 15 7. 28 8. 02 8. 14 8. 50 8. 54 8. 49 8. 43 8. 49  0. 05 0. 07 0. 06 0. 00 0. 07 0. 06 0. 20 0. 07 0. 11 0. 17 0. 17 0. 14 0. 04 0. 03 0. 15 0. 19 0. 53 0. 26 0. 09 0. 14 0. 15 0. 16 0. 10 0. 07 0. 13 0. 20 0. 26 0. 14 0. 24 0. 12 0. 22 0. 28 0. 19 0. 18 0. 26 0. 31 0. 25 0. 15 0. 29 0. 32 0. 14 0. 36 0. 40 0. 49 0. 55 0. 50 0. 10 0. 20 0. 07 0. 10 0. 12 0. 07  196 Appendix 2b. T o t a l g a i n , f e e d f e d and f e e d e f f i c i e n c y o f each tank o f f i s h f o r each p e r i o d i n Year 1.  280688 080788 A TO B  I n i t i a l date F i n a l date Period Days f e d Days not f e d Diet  LLLE  Tank  LLHE  Tank  HLLE  Tank  HLHE  Tank  COMM  Tank  30 2  32 2  71 6  25 25 21172 21806 634 1274 49.8  25 25 21806 26709 4903 6784 72.3  25 25 26709 30924 4215 7190 58.6  25 25  25 25 21160 21941 813 1452 56.0  25 25 21941 27766 3253 6370 51.1  25 25 27766 33440 4050 7043 57.5  25 25 21162 21525 985 1228 80.2  25 25 22525 29789 5358 7124 75.2  25 25 29789 36616 5893 8022 73.5  25 25 21160 21941 781 1127 69.3  25 25 21941 27766 5825 7449 78.2  25 25 27766 33440 5674 8503 66.7  25 25 21162 22525 1363 2054 66.4  25 25 22525 29789 7264 8758 82.9  25 25 29789 36616 6827 9322 73.2  9752 15248 64.0  25 25 8116 14864 54.6  25 25 12236 16374 74.7  2  I n i t i a l no. f i s h F i n a l no. f i s h T o t a l i n i t i a l f i s h wt (g) T o t a l f i n a l f i s h wt (g) T o t a l g a i n (g) Feed f e d (g, d r y ) Feed e f f i c i e n c y (%) Diet  9 2  6  I n i t i a l no. f i s h F i n a l no. f i s h T o t a l i n i t i a l f i s h wt (g) T o t a l f i n a l f i s h wt (g) T o t a l g a i n (g) Feed f e d ( g , d r y ) Feed e f f i c i e n c y (%) Diet  280688 130988 A TO D  7  I n i t i a l no. f i s h F i n a l no. f i s h T o t a l i n i t i a l f i s h wt (g) T o t a l f i n a l f i s h wt (g) T o t a l g a i n (g) Feed f e d (g, d r y ) Feed e f f i c i e n c y (%) Diet  110888 130988 C TO D  3  I n i t i a l no. f i s h F i n a l no. f i s h T o t a l i n i t i a l f i s h wt (g) T o t a l f i n a l f i s h wt (g) T o t a l g a i n (g) Feed f e d (g, d r y ) Feed e f f i c i e n c y (%) Diet  090888 100888 B TO C  25 25 12280 18381 66.8  5  I n i t i a l no. f i s h F i n a l no. f i s h T o t a l i n i t i a l f i s h wt (g) T o t a l f i n a l f i s h wt (g) T o t a l g a i n (g) Feed f e d ( g , d r y ) Feed e f f i c i e n c y (%)  25 25 15454 20135 76.8  197 Appendix 2c. T o t a l g a i n , f e e d f e d and f e e d e f f i c i e n c y o f each tank o f f i s h f o r each p e r i o d i n Year 2.  I n i t i a l date F i n a l date Period  120189 230189 E to F  060489 280689 F to G  Days f e d Days n o t f e d  82 2  82 2  Diet  LLLE  Tank  LLHE  Tank  HLLE  Tank  HLHE  Tank  COMM  Tank  23 23 36616 49114 7239 8627 83.9  23 22 49114 52520 3096 8156 38.0  15416 24184 63.7  24 24 25622 29160 3538 9497 37.3  24 24 29160 34623 5463 10891 50.2  24 24 34623 39405 4782 8470 56.5  13783 28859 47.8  23 21 22467 24259 1792 10536 17.0  21 21 24259 27288 3029 13804 21.9  21 20 27288 29201 1913 10006 19.1  6734 34347 19.6  24 24 25622 29160 4587 10009 45.9  24 24 29160 34623 3992 12675 31.5  24 24 34623 39405 1258 11523 10.9  9837 34207 28.8  23 23 27709 36616 8907 15474 57.6  23 23 36616 49114 12498 15671 79.8  23 22 49114 52520 3406 9248 36.8  24811 40393 61.4  7  I n i t i a l no. f i s h F i n a l no. f i s h T o t a l i n i t i a l f i s h wt (g) T o t a l f i n a l f i s h wt (g) T o t a l g a i n (g) Feed f e d (g, d r y ) Feed e f f i c i e n c y (%) Diet  24 23 27709 36616 5081 7401 68.7  3  I n i t i a l no. f i s h F i n a l no. f i s h T o t a l i n i t i a l f i s h wt (g) T o t a l f i n a l f i s h wt (g) T o t a l g a i n (g) Feed f e d (g, d r y ) Feed e f f i c i e n c y (%) Diet  239 6  2  I n i t i a l no. f i s h F i n a l no. f i s h T o t a l i n i t i a l f i s h wt (g) T o t a l f i n a l f i s h wt (g) T o t a l g a i n (g) Feed f e d (g, d r y ) Feed e f f i c i e n c y (%) Diet  75 2  120189 130989 E to H  6  I n i t i a l no. f i s h F i n a l no. f i s h T o t a l i n i t i a l f i s h wt (g) T o t a l f i n a l f i s h wt (g) T o t a l g a i n (g) Feed f e d (g, d r y ) Feed e f f i c i e n c y (%) Diet  290689 130989 G to H  5  I n i t i a l no. f i s h F i n a l no. f i s h T o t a l i n i t i a l f i s h wt (g) T o t a l f i n a l f i s h wt (g) T o t a l g a i n (g) Feed f e d (g, d r y ) Feed e f f i c i e n c y (%)  198 Appendix 3a. Method o f e x t r a c t i o n (adapted from K i n g 1985) and a s s a y (adapted from AOAC 1984) o f t o c o p h e r o l i n eggs and f r y . REAGENTS FOR EXTRACTION; E t h a n o l , 99% Hexane, d i s t i l l e d i n g l a s s (B.P.=68-69°C) - Caledon I s o o c t a n e ( 2 , 2 , 4 - T r i m e t h y l p e n t a n e ) (B.P.=99-100°C) - C a l e d o n 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% e t h a n o l 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 h y d r o x y - t o l u e n e (BHT). Use 0.4 g eggs and 0.2-0.4 g f r y . Eggs must be k e p t f r o z e n d u r i n g t h e w e i g h i n g p r o c e s s o r t h e y w i l l r u p t u r e and i m m e d i a t e l y expose t h e v i t a m i n E t o oxygen. T h e r e f o r e , keep t h e sample v i a l i n a s t y r o f o a m b l o c k and keep the weighing u t e n s i l s c o l d throughout t h e weighing process. F l u s h t h e 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 t h e f r e e z e r immediately. Weigh o u t and homogenize one sample a t a t i m e . Keep t h e samples on i c e and f l u s h e d w i t h n i t r o g e n t h r o u g h o u t a l l the steps i n t h e e x t r a c t i o n . Homogenize sample 30 s e c u s i n g a P o l y t r o n homogenizer. must be k e p t i n i c e t h r o u g h o u t t h e h o m o g e n i z a t i o n s .  Sample  Add a n o t h e r 1 ml 99% e t h a n o l and 2 ml hexane. Homogenize 15 s e c 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 e t h a n o l and 2 ml hexane t o t h e c u l t u r e tube and r i n s e tube and P o l y t r o n g e n e r a t o r by homogenizing f o r 15 s e c . T r a n s f e r t h e r i n s e t o t h e above screw-cap tube and f l u s h tube with nitrogen. Keep samples on i c e o r i n r e f r i g e r a t o r u n t i l a l l t h e samples a r e extracted. C e n t r i f u g e t u b e s 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 t h e upper hexane l a y e r w i t h a P a s t e u r 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 e v a p o r a t e d i n a h e a t i n g b l o c k o r a 50 ml f l a s k i f i t i s t o be e v a p o r a t e d i n a rotary 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 t h e f i r s t hexane l a y e r . E v a p o r a t e hexane under n i t r o g e n i n a h e a t i n g b l o c k o r w i t h a r o t a r y e v a p o r a t o r a t l e s s t h a n 50°C.  199 E x t r a c t t w i c e 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 t u b e . 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 i s o o c t a n e by v o r t e x i n g 1 m i n u t e f o l l o w e d by c e n t r i f u g i n g a t 6 f o r 1-2 min t o s e p a r a t e t h e p h a s e s . T r a n s f e r t h e upper i s o o c t a n e phase t o a c l e a n 16 x 125 mm screw-cap t u b e . E x t r a c t t h e a c e t o n i t r i l e e x t r a c t a second t i m e w i t h 3 ml i s o o c t a n e 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 b e f o r e . Combine t h e upper phase w i t h p r e v i o u s e x t r a c t . E v a p o r a t e i s o o c t a n e under n i t r o g e n i n a h e a t i n g b l o c k o r w i t h a r o t a r y e v a p o r a t o r a t l e s s than 50°C. Dissolve ethanol.  e x t r a c t i n 3.0-4.0 ml 99% e t h a n o l (eggs) o r 2.0 ml A d j u s t volume as r e q u i r e d f o r t h e a s s a y .  Assay the extract by s p e c t r o p h o t o m e t r i c method.  the  following  AOAC  (1984)  N o t e : Throughout t h e e x t r a c t i o n , a l l hexane and i s o o c t a n e c o n t a i n e d 50 mg/1 BHT as an a n t i o x i d a n t . A b l a n k s h o u l d 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 o f BHT w i l l r e s u l t i n an i n c r e a s e 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) B a t h o p h e n a n t h r o l i n e (Sigma) 0.003M Weigh 100 mg b a t h o p h e n a n t h r o l i n e . B r i n g up t o 100 ml w i t h 99% e t h a n o l . S t o r e i n amber b o t t l e a t 5°C. P r e p a r e f r e s h e v e r y 3 weeks. 2) F e r r i c c h l o r i d e ( F i s h e r ) 0.002M Weigh 55 mg F e C l . 6 H 0 . B r i n g up t o 100 ml w i t h 99% e t h a n o l . S t o r e i n amber b o t t l e a t 5°C. 3  2  3) o - P h o s p h o r i c a c i d (Baker) 0.172M B r i n g 1.1 ml c o n c e n t r a t e d p h o s p h o r i c w i t h 99% e t h a n o l .  acid  (86%) up t o 100 ml  4) S t a n d a r d : Weigh 0.1 g a - t o c o p h e r o l ( 9 5 % p u r i t y ) (Sigma) t o 4-5 d e c i m a l s and t r a n s f e r t o a v o l u m e t r i c f l a s k w i t h 99% e t h a n o l . Make up t o 100 ml w i t h e t h a n o l . D i l u t e t h e above s t o c k t o 1 ml i n 50 ml w i t h e t h a n o l . C o n c e n t r a t i o n (g/ml) = 0.95 x w e i g h t g/ 5000 ml (approx 20 ng/ml). Add volumes o f up t o 2.00 ml o f t h e d i l u t e d s t o c k t o c o p h e r o l 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 b l a n k w i t h 2.0 ml e t h a n o l .  200 METHOD ; Add t o c o p h e r o l e x t r a c t ( i n 99% e t h a n o l ) t o a 13 x 100 mm c u l t u r e tube. Add 99% e t h a n o l t o make up t o 2.0 m l . Add 0.5 m l b a t h o p h e n a n t h r o l i n e r e a g e n t and v o r t e x . Add 0.25 ml f e r r i c c h l o r i d e r e a g e n t . S t a r t t i m i n g once l a s t o f r e a g e n t added. V o r t e x . Add 0.25 ml p h o s p h o r i c a c i d reagent e x a c t l y 15 seconds a f t e r t h e 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 t h e c o l o u r which has d e v e l o p e d i s s t a b l e f o r 90 minutes. Read samples a t 534 m\i a g a i n s t t h e b l a n k . NOTE: T h i s method has been s c a l e d down by h a l f t h e volumes used by AOAC (1984) . CALCULATION: C a l c u l a t e s l o p e o f s t a n d a r d c u r v e = absorbance S l o p e s h o u l d be approx 0.0310. V i t a m i n E (\ig/g) =absorbance x 2.0 ml slope v o l assayed  x (ml)  V i t a m i n E c o n t e n t o f egg (jig/egg) = v i t a m i n E c o n c e n t r a t i o n i n eggs (\ig/g) V i t a m i n E c o n t e n t o f f r y (\ig/fry) = v i t a m i n E (\ig/g) x f r y weight (g)  / ng t o c o p h e r o l .  x  1 wt sample (g)  egg w e i g h t (g)  201 Appendix 3b. Method o f e x t r a c t i o n and a n a l y s i s o f t o t a l i n eggs and d i e t s .  lipid  REAGENTS ; Chloroform - d i s t i l l e d i n glass Methanol - d i s t i l l e d i n glass Concentrated sulphuric acid P o t a s s i u m d i c h r o m a t e (0.167M): 49.0 g r e a g e n t grade p o t a s s i u m d i c h r o m a t e B r i n g t o 1 l i t e r with d i s t i l l e d water. L i p i d s t a n d a r d : 0.1 g/10 ml i n c h l o r o f o r m . In o r d e r t o have a comparable l e v e l o f o x i d a t i o n i n t h e s t a n d a r d and t h e samples, s t a n d a r d c u r v e s h o u l d be conducted w i t h t h e samples b e i n g a n a l y z e d . I n t h e c u r r e n t r e s e a r c h , 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 d r y or wet samples - 0.4 g d r y f e e d o r eggs o r 1.0-1.5 g wet eggs. Add  5.0 ml c h l o r o f o r m 10.0 ml methanol 4.0 ml d i s t i l l e d water f o r a d r y sample* o r 3.4 ml f o r a wet sample. Homogenize 20 s e c . Add 5.0 ml c h l o r o f o r m 5.0 ml d i s t i l l e d w a t e r . Homogenize 10 s e c . R i n s e homogenizer between samples w i t h c h l o r o f o r m : m e t h a n o l (1:1) and d i s c a r d r i n s e . F i l t e r homogenate t h r o u g h a S t e f i s i n t e r e d g l a s s f u n n e l 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 t u b e (20 x 150 mm). R i n s e f u n n e l between samples with c h l o r o f o r m : m e t h a n o l (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 c a n be p r o c e s s e d t o t h i s s t e p u n t i l r e a d y t o p r o c e e d w i t h t h e a s s a y . The upper l a y e r w i l l p r o t e c t t h e l o w e r l a y e r from any chance o f e v a p o r a t i o n . When ready t o c o n d u c t t h e c o l o u r i m e t r i c a s s a y , a s p i r a t e o f f and d i s c a r d upper m e t h a n o l water l a y e r . T r a n s f e r c h l o r o f o r m l a y e r t o a s m a l l screw-cap t u b e (16 x 125 mm).  202 Appendix 3b.  Continued.  ASSAY: T r a n s f e r a l i q u o t o f t h e lower c h l o r o f o r m 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 s c r e w - c a p ) . R i n s e p i p e t t e between samples. E v a p o r a t e s o l v e n t i n a stream on n i t r o g e n o r o t h e r gas o r a l l o w t o d r y i n fume hood. W h i l e e v a p o r a t i n g , p l a c e tubes i n a warm w a t e r b a t h o r a h e a t i n g b l o c k a t 70°C. Tubes must be h e a t e d f o r 10 min a t 70°C a f t e r s o l v e n t has e v a p o r a t e d i n o r d e r t o ensure no t r a c e s o f s o l v e n t remain. This i s c r i t i c a l i n order to o b t a i n good r e p l i c a t e s . Add 2.0 ml o f 0.167M p o t a s s i u m dichromate t o each t u b e . Add 4.0 ml c o n c e n t r a t e d 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 a t 115°C f o r 30 min ( s e t our oven a t 225 F ) . Leave an empty row between tubes i n t h e r a c k and do not o v e r l o a d oven w i t h more t h a n 2 r a c k s . C o o l f o r 3 minutes a t 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 . C o o l i n w a t e r b a t h o r s i n k o f c o l d w a t e r . Mix w i t h v o r t e x . Read absorbance a t 600 nm i n 1 cm c e l l s u s i n g b l a n k as a reference. STANDARD CURVE: S t a n d a r d : 1.0 Add  g/100  ml = 10.0 mg/ml  25-200 \il s t a n d a r d i e . 0.25-2.00 mg  lipid.  The s t a n d a r d c u r v e i s not l i n e a r beyond 2 mg  lipid.  CALCULATION: % l i p i d = absorbance x t o t a l volume c h l o r o f o r m used x 100 s l o p e x volume a l i q u o t x sample w e i g h t x 1000 Where:  volume o f c h l o r o f o r m and volume o f a l i q u o t i n ml sample weight i n gm 1000 c o n v e r t s mg i n s t a n d a r d t o gm. s l o p e = s l o p e o f s t a n d a r d c u r v e = 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 o f o f 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 o f eggs i n Year 1 and Y e a r 2. Dependent variable Y  Parameter X  Regression equation  r  2  Signif.  Year 1; No. eggs F i s h wt. (g) released  Y =  2.40 X + 501.07  0 .315  0 .0001  No. eggs produced  F i s h wt. (g)  Y =  2.71 X + 351.88  0 .334  0 .0001  % Pert.  Spawning day  Y =  0 .143  0 .004  % % % %  No. No. No. No.  Y = 0.0038 X + Y = 0.0067 X + Y 0.0059 X + Y 0.0059 X +  0 .147 0 .142 0 .083 0 .091  0 .004 0 .005 0 .033 0 .025  0 .112  0 .040  Fert. Eyed Hatch Swimup  Year 2: % Fert.  Eggs Eggs Eggs Eggs  0.299 X + 73.58 72.88 40.73 32.18 24.80  F i s h wt. (g)  Y = -0.009 X + 101.25  F i s h wt. (g)  Y =  3.47 X + 2061.01  0 .844  0 .0001  No. eggs F i s h wt. (g) produced  Y =  3.88 X + 2202.25  0 .853  0 .0001  No. eggs produced  Spawning day  Y  % Fert.  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  No. eggs released  -109.74X + 11056.72 0 .249  0 .002  % 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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