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Lipid composition and fatty acid profiles of eggs from wild and cultured chinook salmon (oncorhynchus… Ashton, Heather 1991

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L I P I D COMPOSITION AND FATTY ACID PROFILES OF EGGS FROM WILD AND CULTURED CHINOOK SALMON (ONCORHYNCHUS TSHAWYTSCHA) BROODSTOCK by Heather A s h t o n B.Sc. U n i v e r s i t y o f V i c t o r i a , 1976. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n 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 t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Oct o b e r 1991 © Heather A s h t o n , 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of M / A / M L SCl£MC£ The University of British Columbia Vancouver, Canada Date / / o c r o 6 6 / e is 91 DE-6 (2/88) ABSTRACT In Experiment 1, eggs of B i g Qualicum (BQ) and R o b e r t s o n Creek (RC) w i l d and c u l t u r e d Chinook salmon (Oncorhynchus tschawytcha) were a n a l y z e d f o r l i p i d c o m p o s i t i o n and f a t t y a c i d p r o f i l e s . Each of t h e c u l t u r e d b r o o d s t o c k s had been f e d two f o r m u l a t e d d i e t s d e s i g n a t e d as COMM and WV33. S i g n i f i c a n t l y h i g h e r c o n c e n t r a t i o n s of s a t u r a t e d and n3 f a t t y a c i d s and lower c o n c e n t r a t i o n s o f n6 and n9 f a t t y a c i d s were found i n b o t h t h e t o t a l and p o l a r l i p i d s o f t h e eggs from t h e w i l d f i s h t h a n i n t h o s e from e i t h e r group o f c u l t u r e d f i s h . H i g h l y u n s a t u r a t e d f a t t y a c i d s (HUFAs), p r i m a r i l y 20:5n3 and 22:6n3, were t h e major c o n t r i b u t o r s t o t h e n3 s e r i e s i n both t h e w i l d and c u l t u r e d eggs. The n3:n6 r a t i o s o f b o t h t h e t o t a l and p o l a r l i p i d s were up t o 5.3 t i m e s g r e a t e r i n t h e w i l d t h a n i n t h e c u l t u r e d eggs. The monounsaturate c o n c e n t r a t i o n was s i g n i f i c a n t l y g r e a t e r i n t h e c u l t u r e d eggs t h a n i n t h e w i l d eggs. 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 g e n e r a l l y r e f l e c t e d 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 d i e t s f e d t o t h e b r o o d s t o c k . C o n d i t i o n f a c t o r s f o r t h e w i l d f i s h were s i g n i f i c a n t l y lower than f o r e i t h e r group o f c u l t u r e d f i s h , r e f l e c t i n g t h e s i g n i f i c a n t l y g r e a t e r body l e n g t h o f t h e w i l d f i s h t h a n t h e c u l t u r e d f i s h . 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 body weights. S u r v i v a l t o eyeing among the c u l t u r e d eggs was lower than t h a t r e p o r t e d by the r e s p e c t i v e f e d e r a l government h a t c h e r i e s f o r the w i l d eggs. The q u a l i t y of eggs was poor i n both stocks f e d the WV33 d i e t , with high i n c i d e n c e s of abnormal and r e t a i n e d eggs and low f e r t i l i t y , p a r t i c u l a r l y i n the RC f i s h . S u r v i v a l t o eyeing was poorest i n the RC-WV33 eggs. Some f a c t o r other than l i p i d and f a t t y a c i d composition ( p o s s i b i l y Vitamin C) appeared to be r e s p o n s i b l e , as the f a t t y a c i d p r o f i l e s i n the WV33 eggs were g e n e r a l l y c l o s e r to those of the w i l d f i s h than t o those of the COMM f i s h . There was no e x p l a n a t i o n f o r the i n f e r i o r performance of the RC-WV33 f i s h compared t o the BQ-WV33 f i s h . In experiment 2 , feed was withdrawn from a group of Chinook broodstock f o r 7 days beyond the usual 7 day s t a r v a t i o n p e r i o d p r i o r t o t r a n s p o r t t o freshwater f o r f i n a l maturation. There were no d i f f e r e n c e s i n l i p i d composition and few d i f f e r e n c e s i n f a t t y a c i d p r o f i l e s . F ecundity and s u r v i v a l to eyeing were not compromised by t h i s treatment. Experiment 3 was c a r r i e d out to determine whether a formulated d i e t (COMM) augmented with k r i l l (designated as the BROOD d i e t ) f o r s i x weeks p r i o r to the p r e - t r a n s p o r t s t a r v a t i o n p e r i o d i v would a f f e c t l i p i d c o m p o s i t i o n , f a t t y a c i d p r o f i l e s and i n c u b a t i o n s u c c e s s . The c o n t r o l d i e t was the COMM f o r m u l a t i o n . The BROOD eggs were s i g n i f i c a n t l y lower i n t o t a l l i p i d c o n c e n t r a t i o n due t o the presence of s i g n i f i c a n t l y l e s s n e u t r a l l i p i d t h a n i n t h e COMM eggs. S m a l l s i g n i f i c a n t d i f f e r e n c e s i n i n d i v i d u a l f a t t y a c i d s were found between t h e two groups. W h i l e some o f t h e s e d i f f e r e n c e s r e f l e c t e d t h e d i e t a r y f a t t y a c i d s (eg. 2 2 : l n l l ) , o t h e r s appeared t o be due t o r e t e n t i o n ( i e . n3 f a t t y a c i d s ) o r t o e l o n g a t i o n and d e s a t u r a t i o n of f a t t y a c i d s (eg. 1 8 : l n 9 ) . No d i f f e r e n c e s i n i n c u b a t i o n s u c c e s s were found between t h e groups. V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF FIGURES v i i i LIST OF TABLES i x ACKNOWLEDGEMENTS x i v INTRODUCTION 1 LITERATURE REVIEW 7 1 L i p i d s of p h y s i o l o g i c a l importance f o r f i s h 7 2 D i e t a r y f a t t y a c i d s and requirements f o r growth 9 3 D i g e s t i o n of d i e t a r y l i p i d s 13 4 Role of l i p i d s i n f i s h 15 5 F a t t y a c i d s y n t h e s i s and m o b i l i z a t i o n 23 6 V i t e l l o g e n e s i s 28 7 Embryonic development 32 8 Feeding regime f o r c u l t u r e d maturing salmonids 34 SECTION 1 - A comparison of the l i p i d composition and f a t t y a c i d p r o f i l e s of the eggs of two stocks of w i l d and c u l t u r e d Chinook salmon. 1.1 M a t e r i a l s and Methods 37 1.1.1 Experimental design and c o n d i t i o n s 37 1.1.2 Sample c o l l e c t i o n 41 1.1.3 Egg composition 42 1.1.4 D i e t composition 49 1.1.5 Egg s i z e 50 1.1.6 F e r t i l i z a t i o n and eyeing success 51 1.1.7 S t a t i s t i c a l procedures 51 1.2 R e s u l t s and D i s c u s s i o n 53 1.2.1 Morphometric measurements 53 1.2.2 Spawning and i n c u b a t i o n success 55 1.2.3 Composition of the eggs 63 1.2.4 F a t t y a c i d composition of the t o t a l l i p i d s 68 1.2.5 F a t t y a c i d composition of the p o l a r l i p i d s 81 1.2.6 D i e t s 93 1.3 Conclusions 101 v i SECTION 2 - Feed withdrawal from c u l t u r e d Chinook broodstock p r i o r to t r a n s f e r to freshwater f o r maturation. 2.1 M a t e r i a l s and Methods 1 0 9 2.2 R e s u l t s and D i s c u s s i o n 1 1 1 2 . 2 . 1 Morphometric measurements 1 1 1 2.2 . 2 Spawning and i n c u b a t i o n success 1 1 1 2. 2 . 3 Composition of the eggs 1 1 5 2.2.4 F a t t y a c i d composition of the t o t a l l i p i d s 1 1 8 2. 2 . 5 F a t t y a c i d composition of the p o l a r l i p i d s 1 2 3 2.3 Conclusions 1 3 0 SECTION 3 - A l t e r a t i o n of l i p i d composition and f a t t y a c i d p r o f i l e s i n eggs from Chinook broodstock. 3.1 M a t e r i a l s and Methods 1 3 1 3.2 R e s u l t s and D i s c u s s i o n 1 3 3 3. 2 . 1 Morphometric measurements 1 3 3 3.2.2 Spawning and i n c u b a t i o n success 1 3 3 3.2 . 3 Composition of the eggs 1 3 6 3.2.4 F a t t y a c i d composition of the t o t a l l i p i d s 1 3 8 3.2 . 5 F a t t y a c i d composition of the p o l a r l i p i d s 1 4 0 3.2.6 D i e t s 1 4 7 3.3 Conclusions 1 5 2 CONCLUDING REMARKS 1 5 3 REFERENCES 1 5 5 APPENDIX 1 - P r e l i m i n a r y analyses of s e l e c t e d n u t r i e n t parameters i n the eggs of w i l d and c u l t u r e d Chinook salmon - 1 9 8 5 & 1 9 8 6 . 1 6 4 APPENDIX 2 - D i e t Data. Table A 2 . 1 : Manufacturer's feed l a b e l data f o r commercial (COMM) and brood (BROOD) d i e t s . 1 6 5 Table A 2 . 2 : Formulation of the West Vancouver 3 3 ( W V 3 3 ) d i e t . 1 6 6 Table A 2 . 3 : Dry matter and l i p i d composition of the experimental d i e t s used i n S e c t i o n s 1, 2 and 3. 1 6 7 Table A 2 . 4 : F a t t y a c i d p r o f i l e s of d i e t s . 1 6 8 Table A2.5: Dry matter and l i p i d composition and f a t t y a c i d p r o f i l e of a eu p h a u s i i d ( k r i l l ) sample. 1 6 9 v i i APPENDIX 3 - Formulae f o r c a l c u l a t i n g composition parameters. APPENDIX 4 - F a t t y a c i d p r o f i l e s of eggs. 1 7 0 Table A 4 . 1 : Table A 4 . 2 : Table A 4 . 3 : Table A 4 . 4 : Table A 4 . 5 : Table A4.6 Table A 4 . 7 Table A 4 . 8 : F a t t y a c i d p r o f i l e s of TOTAL LIPIDS i n the eggs of BIG QUALICUM broodstock on three d i e t s . 1 7 1 F a t t y a c i d p r o f i l e s of TOTAL LIPIDS i n the eggs of ROBERTSON CREEK broodstock on three d i e t s . 1 7 2 F a t t y a c i d p r o f i l e s of POLAR LIPIDS i n the eggs of BIG QUALICUM broodstock on three d i e t s . 1 7 3 F a t t y a c i d p r o f i l e s of POLAR LIPIDS i n the eggs of ROBERTSON CREEK broodstock on three d i e t s . 1 7 4 F a t t y a c i d p r o f i l e s of TOTAL LIPIDS i n the eggs of 4-year o l d BIG QUALICUM broodstock. Feed was withdrawn from these f i s h f o r 7 days or 1 4 days p r i o r to t r a n s f e r t o freshwater f o r maturation. 1 7 5 F a t t y a c i d p r o f i l e s of POLAR LIPIDS i n the eggs of 4-year o l d BIG QUALICUM broodstock. Feed was withdrawn from these f i s h f o r 7 days or 1 4 days p r i o r to t r a n s f e r to freshwater f o r maturation. 1 7 6 F a t t y a c i d p r o f i l e s of TOTAL LIPIDS i n the eggs of 4-year o l d broodstock on a commercial grower d i e t (COMM) or on a brood (BROOD) d i e t p r i o r to t r a n s f e r to freshwater f o r maturation. 1 7 7 F a t t y a c i d p r o f i l e s of POLAR LIPIDS i n the eggs of 4-year o l d broodstock on a commercial grower d i e t (COMM) or on a brood (BROOD) d i e t p r i o r to t r a n s f e r to freshwater f o r maturation. 1 7 8 v i i i LIST OF FIGURES Fi g u r e Page 1 Conversion of a r a c h i d o n i c a c i d (20:4n6) t o ei c o s a n o i d s . 20 2 F a t t y a c i d s y n t h e s i s i n f i s h . 25 3 Flowchart of procedures f o r l i p i d a n a l y s i s and f a t t y a c i d d e t e r m i n a t i o n s . 43 4 Egg a b n o r m a l i t i e s i n Chinook broodstock on the WV33 d i e t . 60 5 A comparison of s a t u r a t e d , n3, n6 and n9 f a t t y a c i d s and n3:n6 r a t i o s i n t o t a l and p o l a r l i p i d s of eggs from c u l t u r e d and w i l d B i g Qualicum and Robertson Creek broodstock with the COMM and WV33 d i e t s . 95 6 A comparison of s e l e c t e d n3 f a t t y a c i d s , n3 PUFAs and HUFAs i n t o t a l and p o l a r l i p i d s of eggs from c u l t u r e d and w i l d B i g Qualicum and Robertson Creek broodstock with the COMM and WV33 d i e t s . 97 7 A comparison of s e l e c t e d f a t t y a c i d s and n6 PUFAs i n t o t a l and p o l a r l i p i d s of eggs from c u l t u r e d and w i l d B i g Qualicum and Robertson Creek broodstock with the COMM and WV33 d i e t s . 98 8 A comparison of s e l e c t e d f a t t y a c i d s i n the t o t a l and p o l a r l i p i d s of eggs of 4-year o l d broodstock with those i n t h e i r d i e t s (COMM and BROOD). 150 \ i x LIST OF TABLES Table 1 5a 5b 6a 6b 7a 7b 8a 8b 9a Page Morphometric data f o r w i l d and c u l t u r e d B i g Qualicum and Robertson Creek female broodstock. 54 Spawning data f o r w i l d and c u l t u r e d B i g Qualicum and Robertson Creek broodstock. 56 Egg r e t e n t i o n and other r e p r o d u c t i v e a b n o r m a l i t i e s observed i n B i g Qualicum and Robertson Creek c u l t u r e d females on two d i e t s . 58 Incubation data f o r w i l d and c u l t u r e d B i g Qualicum and Robertson Creek broodstock. 62 Composition of the eggs of BIG QUALICUM broodstock on three d i e t s . 64 Composition of the eggs of ROBERTSON CREEK broodstock on three d i e t s . 65 Saturated, n 3 , n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s i n TOTAL LIPIDS of eggs from BIG QUALICUM broodstock on three d i e t s . 70 Saturated, n 3 , n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s i n TOTAL LIPIDS of eggs from ROBERTSON CREEK broodstock on th r e e d i e t s . 71 S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs and t o t a l n3 HUFAs i n TOTAL LIPIDS of eggs from BIG QUALICUM broodstock on th r e e d i e t s . 73 S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs and t o t a l n3 HUFAs i n TOTAL LIPIDS of eggs from ROBERTSON CREEK broodstock on three d i e t s . 74 S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs i n TOTAL LIPIDS of eggs from BIG QUALICUM broodstock on three d i e t s . 76 S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs i n TOTAL LIPIDS of eggs from ROBERTSON CREEK broodstock on three d i e t s . 77 Monounsaturates, r e p o r t e d as n 5 , n7 , n9 , n i l and t o t a l monounsaturated f a t t y a c i d s i n TOTAL LIPIDS of eggs from BIG QUALICUM broodstock on th r e e d i e t s . 79 X 9b Monounsaturates, r e p o r t e d as n5, n7, n9, r i l l and t o t a l monounsaturated f a t t y a c i d s i n TOTAL LIPIDS of eggs from ROBERTSON CREEK broodstock on three d i e t s . 80 10a Saturated, n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s i n POLAR LIPIDS of eggs from BIG QUALICUM broodstock on three d i e t s . 82 10b Saturated, n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s i n POLAR LIPIDS of eggs from ROBERTSON CREEK broodstock on th r e e d i e t s . 83 11a S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs and t o t a l n3 HUFAs i n POLAR LIPIDS of eggs from BIG QUALICUM broodstock on th r e e d i e t s . 85 l i b S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs and t o t a l n3 HUFAs i n POLAR LIPIDS of eggs from ROBERTSON CREEK broodstock on three d i e t s . 86 12a S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs i n POLAR LIPIDS of eggs from BIG QUALICUM broodstock on three d i e t s . 88 12b S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs i n POLAR LIPIDS of eggs from ROBERTSON CREEK broodstock on three d i e t s . 89 13a Monounsaturates, r e p o r t e d as n5, n7, n9, n i l and t o t a l monounsaturated f a t t y a c i d s i n POLAR LIPIDS of eggs from BIG QUALICUM broodstock on th r e e d i e t s . 91 13b Monounsaturates, r e p o r t e d as n5, n7, n9, n i l and t o t a l monounsaturated f a t t y a c i d s i n POLAR LIPIDS of eggs from ROBERTSON CREEK broodstock on three d i e t s . 92 14 Morphometric data f o r Big Qualicum 4-year o l d broodstock. Feed was withdrawn from these f i s h f o r 7 or 14 days p r i o r to freshwater t r a n s f e r . 112 15 Spawning and i n c u b a t i o n data f o r Big Qualicum 4-year o l d broodstock. Feed was withdrawn from these f i s h f o r 7 or 14 days p r i o r t o freshwater t r a n s f e r . 113 16 Composition of the eggs of 4-year o l d Big Qualicum broodstock. Feed was withdrawn from these f i s h f o r 7 or 14 days p r i o r to freshwater t r a n s f e r . 116 x i 17 Absolute volumes of t o t a l and p o l a r l i p i d per egg c a l c u l a t e d from mean egg volumes and percentages of these parameters. 117 18 Saturated, n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s i n TOTAL LIPIDS of eggs from 4-year o l d Big Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r to freshwater f o r maturation. 119 19 S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs and t o t a l n3 HUFAs i n TOTAL LIPIDS of eggs from 4-year o l d Big Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r to freshwater f o r maturation. 120 20 S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs i n TOTAL LIPIDS of eggs from 4-year o l d B i g Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r to freshwater f o r maturation. 121 21 Monounsaturates, r e p o r t e d as n5, n7, n9, n i l and t o t a l monounsaturated f a t t y a c i d s i n TOTAL LIPIDS of eggs from 4-year B ig Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r to freshwater f o r m aturation. 122 22 Absolute volumes of t o t a l s a t u r a t e s , n3 f a t t y a c i d s , n3 HUFAs and 22:6n3 i n the t o t a l and p o l a r l i p i d s per egg c a l c u l a t e d from the ab s o l u t e volumes of t o t a l and p o l a r l i p i d per egg and the percentage of these parameters, as r e p o r t e d i n pr e v i o u s t a b l e s . 124 23 Saturated, n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s i n POLAR LIPIDS of eggs from 4-year o l d Big Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r to freshwater f o r maturation. 126 24 S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs and t o t a l n3 HUFAs i n POLAR LIPIDS of eggs from 4-year o l d Big Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r t o t r a n s f e r t o freshwater f o r maturation. 127 25 S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs i n POLAR LIPIDS of eggs from 4-year o l d Big Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r to freshwater f o r maturation. 128 x i i 26 Monounsaturates, r e p o r t e d as n 5 , n7 , n9 , n i l and t o t a l monounsaturated f a t t y a c i d s i n POLAR LIPIDS of eggs from 4-year B i g Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r to freshwater f o r maturation. 129 27 Morphometric data f o r 4-year o l d B ig Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r t o freshwater f o r maturation. 134 28 Spawning and i n c u b a t i o n data f o r B ig Qualicum 4-year o l d broodstock on a commercial d i e t (COMM) or a brood d i e t (BROOD) p r i o r to t r a n s f e r to freshwater f o r maturation. 135 29 Composition of the eggs of 4-year o l d B ig Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial (COMM) d i e t p r i o r to t r a n s f e r to freshwater f o r maturation. 137 30 Saturated, n 3 , n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s i n TOTAL LIPIDS of eggs from 4-year o l d Big Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. 139 31 S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs and t o t a l n3 HUFAs i n TOTAL LIPIDS of eggs from 4-year o l d Big Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. 141 32 S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs i n TOTAL LIPIDS of eggs from 4-year o l d B i g Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r t o freshwater f o r maturation. 142 33 Monounsaturates, r e p o r t e d as n 5 , n7 , n 9 , n i l and t o t a l monounsaturated f a t t y a c i d s i n TOTAL LIPIDS on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. 143 34 Saturated, n 3 , n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s i n POLAR LIPIDS of eggs from 4-year o l d B i g Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. 144 x i i i 35 S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs and t o t a l n3 HUFAs i n POLAR LIPIDS of eggs from 4-year o l d Big Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r t o t r a n s f e r to freshwater f o r maturation. 145 36 S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs i n POLAR LIPIDS of eggs from 4-year o l d Big Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r t o freshwater f o r maturation. 146 37 Monounsaturates, r e p o r t e d as n5, n7, n9, n i l and t o t a l monounsaturated f a t t y a c i d s i n POLAR LIPIDS of eggs from 4-year B i g Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. 148 x i v ACKNOWLEDGEMENTS My s i n c e r e a p p r e c i a t i o n goes to a l l who p r o v i d e d support and encouragement to me throughout my s t u d i e s . In p a r t i c u l a r I wish to thank Dr. David Groves f o r so w i l l i n g l y a c c e p t i n g me as a student a t Sea Spring Salmon Farm and f o r h i s generous s h a r i n g of knowledge and p r a c t i c a l experience i n the c u l t u r e and n u t r i t i o n of salmon. I w i l l never f o r g e t h i s u n f a i l i n g good humour and ready t a l e s of h i s years as a student and u n i v e r s i t y p r o f e s s o r . I wish a l s o t o thank Dr. B e r y l March f o r her guidance and knowledge and f o r b r i n g i n g t o my a t t e n t i o n the grammatical faux pas of the s p l i t i n f i n i t i v e . Her q u e s t i o n i n g nature, f a s c i n a t i o n with the b i o l o g y of a l l animals and i n s i g h t s i n t o n u t r i t i o n were an i n s p i r a t i o n . Thank you to Dr. David Higgs who p r o v i d e d u s e f u l suggestions and encouragement throughout. His p o s i t i v e a t t i t u d e toward r e s e a r c h problems and l i f e i n general and h i s sunny d i s p o s i t i o n were g r e a t l y a p p r e c i a t e d . Thanks a l s o t o Doug Harpham and the s t a f f ( e s p e c i a l l y Peter and Cathy) of Sea Spring Salmon Farm L t d ; G i l l e s Galzy, Andy Hickman and L e s l i e Hart of the Department of Animal Science, UBC; Gordon M i l l e r , Susan K e l l e r , Henrik K r i e b e r g and I a i n Whyt of the P a c i f i c B i o l o g i c a l S t a t i o n i n Nanaimo and West Vancouver Yvonne Yole of the S c a l e Ageing Lab at DFO, Vancouver; and Don Lawseth and Grant Ladocoeur, managers of Robertson Creek and B i Qualicum H a t c h e r i e s . Thanks a l s o t o my f a m i l y - E r i c , M e r r e l l , Mum and Dad, L a u r i e , B r i a n , John, Elma and Margaret f o r t h e i r l o v e , p a t i e n c e and encouragement throughout my years as a student. T h i s work was supported by Sea Spring Salmon Farm L t d . , Chemainus, BC and by the BC Science C o u n c i l through a GREAT award. F i n a l l y I would l i k e to d e d i c a t e t h i s t h e s i s to my 17 month o l d daughter, M e r r e l l , who w i l l ' l e a r n much more than I ' l l ever know.' 1 "I hear babies c r y ; I watch them grow, T h e y ' l l l e a r n much more than,I'11 ever know, And I t h i n k to myself: What a Wonderful World." Lo u i s Armstrong, "What a Wonderful World." 1 INTRODUCTION The s u r v i v a l of c u l t u r e d Chinook salmon (Oncorhynchus tschawytcha) from f e r t i l i z a t i o n t o hatch and through to ponding has, u n t i l r e c e n t l y , been much lower than t h a t of w i l d Chinook taken from t h e i r n a t a l streams and spawned and incubated using the same hatchery procedures. U n t i l 1987, the best s u r v i v a l to ponding r e p o r t e d by a p r i v a t e hatchery was approximately 70%. T h i s f i g u r e was u n u s u a l l y high f o r the i n d u s t r y and s u r v i v a l to the eyed stage a t some p r i v a t e h a t c h e r i e s had been i n the range of 13 to 35%. S u r v i v a l to ponding had been even lower. Wild Chinook, spawned i n the f e d e r a l government hatchery system and r e a r e d to r e l e a s e as smolts, r o u t i n e l y have s u r v i v a l r a t e s to eyeing of 90% or more and t o ponding of approximately 85% (D. Lawseth, Department of F i s h e r i e s and Oceans, B i g Qualicum Hatchery, p e r s . comm.). Sea S p r i n g Salmon Farm L t d . , a company t h a t r e a r s smolts f o r the salmon farming i n d u s t r y , has h e l d a f e d e r a l government c o n t r a c t with the Department of F i s h e r i e s and Oceans f o r twelve years to take and spawn maturing Chinook from the Chemainus R i v e r on Vancouver I s l a n d and to r e a r the smolts f o r r e l e a s e back to the w i l d . S u r v i v a l r a t e s to eyeing f o r eggs from the w i l d Chemainus f i s h have been 20 to 60% b e t t e r than the s u r v i v a l r a t e s of eggs from c u l t u r e d B i g Qualicum broodstock, spawned and r e a r e d under i d e n t i c a l c o n d i t i o n s . In 1986, the i n a b i l i t y of the p r i v a t e h a t c h e r i e s to produce enough eggs 2 c o n s i s t e n t l y f o r t h e growing salmon f a r m i n g i n d u s t r y i n B r i t i s h Columbia c a l l e d i n t o q u e s t i o n t h e v i a b i l i t y o f salmon f a r m i n g as a whole. Poor s u r v i v a l r a t e s a l s o hampered th e development o f domestic s t r a i n s o f f i s h s e l e c t e d f o r t o l e r a n c e t o s t r e s s , d i s e a s e r e s i s t a n c e , enhanced growth r a t e s , improved r e p r o d u c t i v e performance, e t c . w h i l e a v o i d i n g t h e n e g a t i v e i m p a c t s of i n b r e e d i n g . W i t h o u t adequate numbers of progeny, h a t c h e r i e s c o u l d i l l a f f o r d t o u n d e r t a k e g e n e t i c e x p e r i m e n t s . I n 1985 and 1986, p r e l i m i n a r y i n v e s t i g a t i o n s i n t o t h e problem of poor egg s u r v i v a l i n c u l t u r e d Chinook salmon were i n i t i a t e d by Dr. D a v i d Groves (Sea S p r i n g Salmon Farm L t d . ) i n c o n j u n c t i o n w i t h t h e B r i t i s h Columbia Salmon Farmers A s s o c i a t i o n and t h e Government of Canada, Department o f F i s h e r i e s and Oceans (IRAP (PILP) #CA910-5-0042/866). B r o o d s t o c k h a n d l i n g and spawning t e c h n i q u e s were examined but t h e major emphasis o f t h e i n v e s t i g a t i o n was d i r e c t e d t o b r o o d s t o c k n u t r i t i o n . With t h e p a r t i c i p a t i o n o f s e v e r a l p r i v a t e h a t c h e r i e s , a s u r v e y o f s e l e c t e d n u t r i e n t parameters i n c u l t u r e d eggs and i n t h e eggs from t h r e e w i l d s t o c k s was c a r r i e d o u t . R e s u l t s from t h e 1985 and 1986 a n a l y s e s , p r e s e n t e d i n Appendix 1, r e v e a l e d i m p o r t a n t d i f f e r e n c e s i n f a t t y a c i d c o m p o s i t i o n between c u l t u r e d and w i l d eggs. The o b j e c t i v e s o f t h i s t h e s i s were s e t out i n view o f t h e s e 3 r e s u l t s . The f i r s t g o a l was t h e a c c u r a t e d e t e r m i n a t i o n o f t h e l i p i d p r o f i l e s i n t h e eggs of w i l d and c u l t u r e d f i s h . For t h i s p u rpose, t h e eggs of two s t o c k s ( B i g Qualicum and R o b e r t s o n Creek) of Chinook salmon were sampled. Two groups of c u l t u r e d f i s h o f each s t o c k were f e d e i t h e r a commercial d i e t i n wide use i n t h e i n d u s t r y or t h e open f o r m u l a t i o n , West Vancouver 33 d i e t (D.A. H i g g s , Department of F i s h e r i e s and Oceans, West Vancouver L a b o r a t o r y , unpub. d a t a ) . These d i e t s were manufactured by two independent commercial f e e d companies i n B r i t i s h Columbia. W i l d Chinook eggs were sampled from t h e r e t u r n s t o t h e B i g Qualicum and R o b e r t s o n Creek H a t c h e r i e s , two Government of Canada f a c i l i t i e s . The r e s u l t s o f t h i s s t u d y , documenting l i p i d c o n c e n t r a t i o n s and f a t t y a c i d p r o f i l e s o f t h e t o t a l and p o l a r l i p i d s i n t h e eggs of Chinook salmon f o r t h e f i r s t t i m e , a r e p r e s e n t e d i n S e c t i o n 1. In t h e e a r l y s t u d i e s (D. Groves, 1987 unpub. r e p o r t ) , i t was found t h a t t h e muscle of c u l t u r e d f i s h had h i g h e r l e v e l s of l i p i d t h a n w i l d f i s h and t h a t c u l t u r e d f i s h produced eggs w i t h h i g h e r l i p i d c o n c e n t r a t i o n s (Appendix 1 ) . A g e n e r a l o b s e r v a t i o n was a l s o made t h a t c o n d i t i o n f a c t o r s were lower i n t h e w i l d t h a n i n t h e c u l t u r e d b r o o d s t o c k . I t was p o s t u l a t e d t h a t ' f a t f i s h do not t e n d t o be p r o d u c t i v e b r o o d s t o c k ' and t h a t i n g e s t e d energy i n c u l t u r e d f i s h was b e i n g d e p o s i t e d as f a t r a t h e r t h a n as p r o t e i n . Reasons proposed f o r t h i s were t h a t c u l t u r e d f i s h expend l e s s energy t h a n t h e i r w i l d c o u n t e r p a r t s i n t h e i r d a i l y l i v e s , t h a t they a r e on a h i g h e r 4 plane of n u t r i t i o n than w i l d f i s h or t h a t some n u t r i t i o n a l imbalance may e x i s t which promotes the d e p o s i t i o n of f a t r a t h e r than p r o t e i n . Wild Chinook r e t u r n i n g t o t h e i r n a t a l streams from the ocean cease f e e d i n g upon or p r i o r to en t r y to freshwater. Many stocks then undergo long arduous m i g r a t i o n s p r i o r t o reaching the spawning grounds. Greene (1919) and others have r e p o r t e d the e x t e n s i v e d e p l e t i o n of body r e s e r v e s i n m i g r a t i n g Chinook durin g the p e r i o d when gonad t i s s u e s are p r o l i f e r a t i n g and maturing. T h i s lends credence t o the suggestion t h a t c u l t u r e d f i s h are on a higher plane of n u t r i t i o n than i s necessary and t h a t t h i s higher f e e d i n g regime may be d e t r i m e n t a l t o the p r o d u c t i o n of high q u a l i t y eggs. To t e s t the hypothesis t h a t the plane of n u t r i t i o n i n c u l t u r e d f i s h towards the end of v i t e l l o g e n e s i s i s e x c e s s i v e , feed was withdrawn from a group of c u l t u r e d f i s h f o r 14 days p r i o r to t r a n s f e r t o freshwater f o r maturation and spawning. A second group, the c o n t r o l , was s t a r v e d f o r one week p r i o r t o t r a n s f e r , f o l l o w i n g the usual p r o t o c o l f o r broodstock t r a n s p o r t . L i p i d composition and s u r v i v a l r a t e s of the eggs of both groups were monitored. The t h i r d o b j e c t i v e was aimed a t modifying the f a t t y a c i d p r o f i l e of the eggs of c u l t u r e d b r o o d f i s h by f e e d i n g a commercially manufactured brood d i e t c o n t a i n i n g k r i l l f o r s i x 5 weeks p r i o r t o t r a n s p o r t to the hatchery. I t was thought t h a t enrichment with k r i l l would p r o v i d e a more f a v o u r a b l e balance of f a t t y a c i d s , i e . c l o s e r to what may be found i n eggs from f i s h f e e d i n g i n the w i l d . T h i s work i s presented i n S e c t i o n 3. Hatchery f a c i l i t i e s and a l l c u l t u r e d f i s h f o r t h i s work were pr o v i d e d by Sea Spring Salmon Farm L t d . , Chemainus, BC. The B i g Qualicum and Robertson Creek f i s h f e d the West Vancouver 33 d i e t were p a r t of a l a r g e r study conducted by the Government of Canada, Department of F i s h e r i e s and Oceans; the Government of B r i t i s h Columbia, M i n i s t r y of A g r i c u l t u r e and F i s h ; BC Research; and the BC Salmon Farmers' A s s o c i a t i o n . T h i s study was undertaken to examine the p o t e n t i a l f o r r e a r i n g these and f o u r other stocks at e n v i r o n m e n t a l l y d i v e r s e s i t e s along the P a c i f i c c o a s t . The net pen f a c i l i t y of Sea S p r i n g Salmon Farm L t d . a t Genoa Bay near Duncan on Vancouver I s l a n d was one of these s i t e s . F i s h from each stock, reared a t each s i t e , were from the same g e n e t i c mix and a l l were f e d the West Vancouver 33 d i e t to e l i m i n a t e g e n e t i c and d i e t f a c t o r s from the study ( W i t h l e r et al., 1986). Some f i s h matured i n 1987 a t t h r e e years of age when the sampling f o r the p r e s e n t study was undertaken. y Since the p r e l i m i n a r y s t u d i e s i n 1985 and 1986 (D. Groves, 1987 unpub. r e p o r t ) , the commercial feed companies have made m o d i f i c a t i o n s t o t h e i r f o r m u l a t i o n s i n c l u d i n g the a d d i t i o n of higher l e v e l s of Vitamins C and E and selenium, demonstrated as 6 e s s e n t i a l f o r t h e r e p r o d u c t i v e s u c c e s s o f many s p e c i e s (Watanabe, 1985). Because t h e d i e t s a r e c l o s e d f o r m u l a t i o n s and because no c o n t r o l l e d e x p e r i m e n t s were performed t o t e s t t h e i r e f f e c t s , i t i s i m p o s s i b l e t o a s c e r t a i n p r e c i s e l y which m o d i f i c a t i o n s have been r e s p o n s i b l e f o r the s i g n i f i c a n t i n c r e a s e i n s u r v i v a l o f c u l t u r e d eggs seen s i n c e 1986. W h i l e perhaps not as c o n s i s t e n t l y s u c c e s s f u l as t h e i r w i l d c o u n t e r p a r t s , c u l t u r e d Chinook salmon t y p i c a l l y now have s u r v i v a l r a t e s t o ponding o f 75-85%. 7 LITERATURE REVIEW 1 L i p i d s o f p h y s i o l o g i c a l i m p o r t a n c e f o r f i s h L i p i d s i n c l u d e f a t s , o i l s , waxes and r e l a t e d compounds. They a r e r e l a t i v e l y i n s o l u b l e i n water but s o l u b l e i n n o n p o l a r s o l v e n t s such as c h l o r o f o r m , e t h e r and benzene. F a t t y a c i d s a r e c o n s t i t u e n t s o f a l l l i p i d s . They c o n s i s t o f a h y d r o c a r b o n c h a i n w i t h a t e r m i n a l c a r b o x y l a t e group. F a t t y a c i d s can be d i v i d e d i n t o two broad c a t e g o r i e s , t h e s a t u r a t e s and t h e u n s a t u r a t e s . S a t u r a t e d f a t t y a c i d s have no do u b l e bonds. U n s a t u r a t e d f a t t y a c i d s have double bonds between some of t h e c a r b o n atoms i n t h e h y d r o c a r b o n c h a i n and may be c a l l e d monoenoic, d i e n o i c , t r i e n o i c , ... h e x a e n o i c i f one, two, t h r e e , ... s i x d o u b l e bonds a r e p r e s e n t . C o n s e c u t i v e double bonds a r e methylene i n t e r r u p t e d so t h a t a -CH 2- group o c c u r s between two carbons c a r r y i n g double bonds. The p o s i t i o n o f t h e f i r s t d o u b l e bond from t h e methyl end o f t h e m o l e c u l e i n u n s a t u r a t e s i s o f p h y s i o l o g i c a l s i g n i f i c a n c e and d e t e r m i n e s t h e omega (n) number of t h e f a t t y a c i d [11. The f a t s and o i l s a r e u s u a l l y c o n s i d e r e d i n s o l u b l e i n water due [1] F a t t y a c i d nomenclature f o l l o w s t h e n o t a t i o n Crxnp, where C = t h e number o f ca r b o n atoms i n t h e hy d r o c a r b o n c h a i n , x = t h e number of double bonds, n i s an a b b r e v i a t i o n f o r omega and p = t h e p o s i t i o n o f t h e f i r s t d o u b l e bond from t h e methyl end of t h e m o l e c u l e . T h e r e f o r e 16:ln7 i s a monoenoic u n s a t u r a t e d f a t t y a c i d w i t h 16 c a r b o n atoms, 1 double bond and t h e p o s i t i o n o f t h e do u b l e bond i s on ca r b o n 7 c o u n t i n g from t h e methyl end. 8 t o t h e p r e s e n c e o f n o n p o l a r h y d r o c a r b o n groups. However p o l a r groups i n a s s o c i a t i o n w i t h f a t t y a c i d s , f o r m i n g p h o s p h o l i p i d s , s p h i n g o l i p i d s and o t h e r s , i m p a r t v a r i o u s degrees o f water s o l u b i l i t y t o t h e m o l e c u l e . L i p i d s can t h e r e f o r e be c l a s s i f i e d on t h e b a s i s o f t h e i r p o l a r i t y , i e t h e n e u t r a l and t h e p o l a r l i p i d s . N e u t r a l l i p i d s i n c l u d e t h e t r i a c y l g l y c e r i d e s (TAGs), which form t h e major l i p i d energy depot. TAGs c o n s i s t o f t h r e e l o n g - c h a i n f a t t y a c i d s e s t e r i f i e d t o t h e a l c o h o l , g l y c e r o l . S a t u r a t e s , p a r t i c u l a r l y 14:0 and 16:0, and t h e monounsaturates, 16:ln7 and 1 8 : l n 9 , t y p i c a l l y occupy p o s i t i o n s on t h e f i r s t (a) and t h i r d ( T ) c a r b o n o f g l y c e r o l i n TAG m o l e c u l e s . The second (0) car b o n of g l y c e r o l may be a t t a c h e d t o a n o t h e r s a t u r a t e o r t o a monounsaturate o r , as i s common i n f i s h , t o a PUFA o r HUFA ( C h r i s t i e , 1986). I n t h e second p o s i t i o n , t h e s e l o n g c h a i n f a t t y a c i d s a r e l e s s s u s c e p t i b l e t o o x i d a t i o n f o r energy (Murray, 1988). Other common n e u t r a l l i p i d s a r e t h e mono and d i a c y l g l y c e r i d e s ( w i t h one o r two f a t t y a c i d s e s t e r i f i e d t o g l y c e r o l ) ; c h o l e s t e r o l and c h o l e s t e r y l e s t e r s ( c h o l e s t e r o l e s t e r i f i e d t o a f a t t y a c i d ) , and f r e e f a t t y a c i d s . The p o l a r l i p i d s i n c l u d e t h e p h o s p h o g l y c e r i d e s , g l y c o l i p i d s , s p h i n g o m y e l i n s and p l a s m o l a g e n s . These compounds have e i t h e r a g l y c e r o l o r s p h i n g o s i n e (an amino a l c o h o l ) backbone, one o r two f a t t y a c i d r e s i d u e s and a p o l a r head group. With t h e e x c e p t i o n o f t h e g l y c o l i p i d s a l l o f t h e s e p o l a r l i p i d s c o n t a i n a 9 phosphate group and a r e o f t e n r e f e r r e d t o as p h o s p h o l i p i d s . G l y c o l i p i d s c o n t a i n s p h i n g o s i n e , a f a t t y a c i d and one o r more sugar groups. P h o s p h o g l y c e r i d e s and g l y c o l i p i d s a r e i m p o r t a n t c o n s t i t u e n t s o f biomembranes. P h o s p h a t i d y l c h o l i n e (PC), p h o s p h a t i d y l e t h a n o l a m i n e <PE), p h o s p h a t i d y l s e r i n e ( P S ) , p h o s p h a t i d y l i n o s i t o l (PI) and d i p h o s p h a t i d y l g l y c e r o l ( c a r d i o l i p i n ) a r e a l l p h o s p h o g l y c e r i d e s . C e r e b r o s i d e s and g a n g l i o s i d e s a r e g l y c o l i p i d s , i m p o r t a n t t h r o u g h o u t t h e body but e s p e c i a l l y i n n e u r a l t i s s u e and t h e b r a i n . S p h i n g o m y e l i n s a r e a l s o found i n b r a i n and nerve t i s s u e w h i l e p l a s m o l a g e n s c o m p r i s e as much as 10% o f t h e p h o s p h o l i p i d s i n b r a i n and muscle t i s s u e (Murray et al. , 1988). S a t u r a t e d and monounsaturated f a t t y a c i d s a r e f r e q u e n t l y p r e s e n t i n t h e a - p o s i t i o n o f t h e g l y c e r o l backbone i n p o l a r l i p i d s w i t h an n3 PUFA o r HUFA i n t h e P - p o s i t i o n . I n t h i s p o s i t i o n , t h e u n s a t u r a t e i s p r o t e c t e d i f t h e s a t u r a t e o r monoene i s o x i d i z e d (Greene and S e l i v o n c h i c k , 1987). 2 D i e t a r y f a t t y a c i d s and r e q u i r e m e n t s f o r growth S a t u r a t e d f a t t y a c i d s w i t h even numbers of c a r b o n atoms can 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). Some d i e t a r y f a t t y a c i d s can be e l o n g a t e d and d e s a t u r a t e d and i n c o r p o r a t e d i n t o compounds as r e q u i r e d . Other f a t t y a c i d s cannot be s y n t h e s i z e d , have s p e c i f i c m e t a b o l i c f u n c t i o n s and must be p r o v i d e d i n t h e d i e t . These a r e t h e e s s e n t i a l f a t t y a c i d s . 1 10 F i s h cannot s y n t h e s i z e any member o f t h e l i n o l e i c (n6) o r t h e l i n o l e n i c (n3) s e r i e s u n l e s s a p r e c u r s o r w i t h t h e same omega s t r u c t u r e i s p r e s e n t . I n a d d i t i o n , t h e r e i s c o m p e t i t i v e i n h i b i t i o n between t h e n3, n6 and n9 s e r i e s such t h a t e l o n g a t i o n and d e s a t u r a t i o n o f one s e r i e s i s i n h i b i t e d by t h e members o f a n o t h e r s e r i e s . The n3 s e r i e s i s t h e most p o t e n t i n h i b i t o r , f o l l o w e d by t h e n6 and n9 s e r i e s ( H a l v e r , 1980). D i e t a r y s t u d i e s have demonstrated t h e e s s e n t i a l i t y o f t h e n3 ( l i n o l e n i c s e r i e s ) f a t t y a c i d s f o r rainbow t r o u t . C a s t e l l et al. (1972 a,b,c) e s t a b l i s h e d t h e re q u i r e m e n t f o r 18:3n3 a t 1% o f t h e d i e t by weight o r a p p r o x i m a t e l y 2.7% o f t h e d i e t a r y c a l o r i e s . T h i s was based on a c h i e v i n g t h e b e s t growth r a t e s and f e e d c o n v e r s i o n s and on a l l e v i a t i n g s e v e r a l s i g n s 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 , eg. f i n e r o s i o n , h e a r t myopathy and syncope t h a t o c c u r r e d d u r i n g t h e i r s t u d i e s . T a k e u c h i and Watanabe (1977b) found t h a t as t h e t o t a l l e v e l of l i p i d i n c r e a s e d i n t h e d i e t o f rainbow t r o u t , t h e r e q u i r e m e n t f o r 18:3n3 a l s o i n c r e a s e d . For example 1% 18:3n3 was \ s u f f i c i e n t i n d i e t s c o n t a i n i n g r e l a t i v e l y low l i p i d l e v e l s (up t o 5%) but i n d i e t s c o n t a i n i n g 14% l i p i d , t h e r e q u i r e m e n t f o r 18:3n3 was more th a n 2% o f t h e d i e t f o r maximum growth. Hence, i t i s suggested t h a t t h e 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 h o u l d be e x p r e s s e d as a p e r c e n t a g e of t h e d i e t a r y l i p i d and f o r rainbow t r o u t t h e r e q u i s i t e l e v e l was s e t a t a p p r o x i m a t e l y 20% as 18,:3n3. Some workers b e l i e v e t h a t n6 f a t t y a c i d s may a l s o be e s s e n t i a l f o r s a l m o n i d s . N i c o l a i d e s and Woodall (1962) r e p o r t e d 70-80% of j u v e n i l e Chinook salmon e x h i b i t e d dermal d e p i g m e n t a t i o n when t h e y were f e d d i e t s c o n t a i n i n g e i t h e r no l i p i d , 18:0 as t r i o l e i n a t 1% o f t h e d i e t , o r 0.1% o f t h e d i e t as 18:3n3. When t h e f i s h were f e d 1% o f t h e d i e t as 18:2n6 (as t r i l i n o l e i n ) , 86% d i s p l a y e d normal p i g m e n t a t i o n . When 0.1% 18:3n3 and 1% t r i l i n o l e i n were i n c l u d e d i n t h e d i e t , 69% o f t h e f i s h were normal. Growth r a t e s were a l s o s t u d i e d and found t o be h i g h e s t i n t h o s e f i s h r e c e i v i n g 18:3n3 a l o n e a t 0.1% o f t h e d i e t and t h e s e were not improved even when 3% 18:2n6 (as t r i l i n o l e i n ) was added t o t h e d i e t . Ackman and Ta k e u c h i (1986) r e p o r t e d l e v e l s o f 18:3n3 and 20:4n6 t h a t were t h r e e and t e n t i m e s h i g h e r , r e s p e c t i v e l y , i n w i l d t h a n i n h a t c h e r y - r e a r e d A t l a n t i c p r e - s m o l t s o v e r w i n t e r i n g a t 0-0.5° C. The h a t c h e r y s m o l t s e x p e r i e n c e d s e v e r e f i n e r o s i o n which o f t e n l e d t o m o r t a l i t y . T h i s prompted an i n v e s t i g a t i o n i n t o t h e p o s s i b i l i t y o f 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 . A l t h o u g h t h e h a t c h e r y r e a r e d f i s h had h i g h e r l e v e l s o f 18:2n6 t h a n t h e w i l d f i s h , t h e y d i d not e l o n g a t e and d e s a t u r a t e 18:2n6 t o 20:4n6. I t was suggested t h a t h i g h l e v e l s o f n3 f a t t y a c i d s may have i n h i b i t e d t h e e l o n g a t i o n / d e s a t u r a t i o n o f 18:2n6 i n t h e h a t c h e r y f i s h . Moreover, i t was noted t h a t t h e a q u a t i c 12 i n s e c t d i e t o f t h e w i l d f i s h i n c l u d e d h i g h l e v e l s o f pre-formed 20:4n6. There was no c o n c l u s i v e p r o o f t h a t h i g h l e v e l s o f 20:4n6 were p r e v e n t i n g f i n e r o s i o n i n t h e w i l d f i s h . However Ackman and T a k e u c h i suggested t h a t 20:4n6, b e i n g a p r e c u r s o r t o p r o s t a g l a n d i n s (see b e l o w ) , may have been i m p o r t a n t i n a d e f e n s e mechanism a g a i n s t t h e l e s i o n s . I t has a l s o been shown t h a t d i e t a r y i n c l u s i o n o f 18:2n6 p r e v e n t s t h e a c c u m u l a t i o n of 20:3n9, 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 (Greene and S e l i v o n c h i c k , 1987). I t i s g e n e r a l l y b e l i e v e d t h a t t h e n6 f a t t y a c i d s ( l i n o l e i c s e r i e s ) a r e not e s s e n t i a l f o r s a l m o n i d s a l t h o u g h t h e y a r e f o r o t h e r s p e c i e s , p a r t i c u l a r l y t h e homeothermic l a n d - d w e l l i n g a n i m a l s . C a s t e l l (1972a) demonstrated t h a t no c o m b i n a t i o n of 18:2n6 and 18:3n3 r e s u l t e d i n as good a growth r a t e o r f e e d c o n v e r s i o n as 18:3n3 a l o n e a t 1% o f t h e d i e t . Yu e t al. (1979) a l s o showed t h a t t h e b e s t growth r a t e s o c c u r r e d when t r o u t were f e d 1% 18:3n3 as t h e s o l e d i e t a r y e s s e n t i a l f a t t y a c i d . Yu and Sinnhuber (1979b) found t h a t coho salmon (Oncorhynchus kisutch) f r y r e q u i r e d 1 t o 1.5% o f a d i e t c o n t a i n i n g 10% l i p i d as n3 f a t t y a c i d s . The p r e s e n c e of more th a n 1% n6 f a t t y a c i d s o r e x t r e m e l y low or h i g h d i e t a r y l e v e l s o f n3 f a t t y a c i d s d e p r e s s e d growth. The optimum l e v e l o f p o l y u n s a t u r a t e d f a t t y a c i d s (n3+n6) i n t h e d i e t was a p p r o x i m a t e l y 2.5% o r l e s s . 13 T a k e u c h i et al. (1979c) i n v e s t i g a t e d f a t t y a c i d r e q u i r e m e n t s i n chum salmon (Oncorhynchus keta) f r y h e l d i n b o t h f r e s h and s a l t w a t e r . The b e s t weight g a i n and f e e d c o n v e r s i o n was o b t a i n e d when both 1% 18:3n3 and 1% 18:2n6 were p r e s e n t . F a t t y a c i d r e q u i r e m e n t s f o r Chinook salmon (Oncorhynchus tshawytscha) have not been r e p o r t e d f o r any s t a g e o f t h e i r l i f e c y c l e . The p r e s e n c e of l o n g c h a i n h i g h l y u n s a t u r a t e d n3 f a t t y f a c i d s (n3 HUFAs) a r e a n o t a b l e f e a t u r e o f t h e body c o m p o s i t i o n of a l l P a c i f i c salmon ( P l o t n i k o f f e t al., 1984), and j u v e n i l e Chinook salmon a r e a b l e t o c o n v e r t 18:3n3 t o n3 HUFAs r e a d i l y ( M u g r d i t c h i a n e t al., 1982 and Dosanjh e t al., 1988). T h i s s u g g e s t s t h e importance o f t h e l i n o l e n i c (n3) s e r i e s f o r t h i s s p e c i e s . S i g n s 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 i n s a l m o n i d s i n c l u d e : poor growth; e l e v a t e d l e v e l s o f 20:3n9; n e c r o s i s o f t h e c a u d a l f i n ; f a t t y , p a l e l i v e r ; dermal d e p i g m e n t a t i o n ; i n c r e a s e d water . c o n t e n t o f t h e muscle; syncope i n response t o shock; i n c r e a s e d m i t o c h o n d r i a l s w e l l i n g ; h e a r t myopathy; i n c r e a s e d r e s p i r a t i o n o f l i v e r homogenates; and low hemoglobin l e v e l s ( S i n n h u b e r , 1969). 3 D i g e s t i o n o f d i e t a r y l i p i d s H y d r o l y s i s o f t r i a c y l g l y c e r i d e s , t h e major component o f d i e t a r y l i p i d s , i s c a t a l y s e d by p a n c r e a t i c l i p a s e . T h i s r e s u l t s i n t h e 14 l i b e r a t i o n of f a t t y a c i d s and m o n o a c y l g l y c e r i d e s . I f t h e f a t t y a c i d s e s t e r i f i e d t o t h e TAG a r e a l l s a t u r a t e d , t h e n t h e r a t e o f h y d r o l y s i s f o r a l l t h r e e p o s i t i o n s w i l l be t h e same. I f p o s i t i o n two i s o c c u p i e d by an u n s a t u r a t e t h e n i t may not be h y d r o l y s e d , l e a v i n g a m o n o a c y l g l y c e r i d e . I f t h e u n s a t u r a t e i s i n p o s i t i o n one o r t h r e e , i t w i l l be h y d r o l y s e d a t t h e same r a t e as a s a t u r a t e d f a t t y a c i d ( L e g e r , 1985). H y d r o l y s i s o f d i e t a r y p h o s p h o l i p i d s i s thought t o r e s u l t i n t h e f o r m a t i o n o f f r e e f a t t y a c i d s and l y s o p h o s p h o l i p i d s as i n mammals (Henderson and Tocher, 1987). T y p i c a l l y p o l a r l i p i d s have an e s s e n t i a l f a t t y a c i d e s t e r i f i e d t o t h e 3 p o s i t i o n and a s a t u r a t e o r monoene a t t h e a p o s i t i o n . T h i s arrangement p r o b a b l y makes t h e e s s e n t i a l f a t t y a c i d l e s s s u s c e p t i b l e t o h y d r o l y s i s and removal t h a n f a t t y a c i d s i n t h e a p o s i t i o n (Dave et al., 1976, c i t e d i n C a s t l e d i n e and B u c k l e y , 1982). L i b e r a t e d f a t t y a c i d s , m o n o a c y l g l y c e r i d e s and l y s o p h o s p h o l i p i d s a r e absorbed by t h e p r o x i m a l i n t e s t i n e and t h e p y l o r i c caecae. The r a t e o f a b s o r p t i o n i s much s l o w e r t h a n i n mammals and appears t o i n c r e a s e w i t h t h e degree o f u n s a t u r a t i o n and t h e water t e m p e r a t u r e (Henderson and Tocher, 1987). A l l d i e t a r y f a t t y a c i d s , e x c e p t t h o s e t h a t a r e r a p i d l y i n c o r p o r a t e d i n t o p h o s p h o l i p i d s , a r e e i t h e r c a t a b o l i z e d f o r energy o r s t o r e d (Yu et al., 1977). S i r e et al. (1981) has demonstrated t h a t f a t t y a c i d s a r e r e - e s t e r i f i e d t o TAGs w i t h i n t h e i n t e s t i n a l e p i t h e l i u m and i t i s thought t h a t l y s o p h o s p h o l i p i d s a r e r e -15 e s t e r i f i e d w i t h f a t t y a c i d s t o p h o s p h o l i p i d s as o c c u r s i n mammals (Henderson and Tocher, 1987). A f t e r c r o s s i n g t h e i n t e s t i n a l w a l l , most d i e t a r y l i p i d s a r e packaged i n t o c h y l o m i c r o n s and some i n t o l i p o p r o t e i n s (VLDL) f o r t r a n s p o r t by t h e lymph ( L e g e r , 1985). Most l i p o p r o t e i n (eg. VLDL, LDL, HDL) s y n t h e s i s t a k e s p l a c e i n t h e l i v e r i n s a l m o n i d s , i n c l u d i n g t h e l i p o p r o t e i n , v i t e l l o g e n i n , which i s produced by m a t u r i n g female f i s h (Henderson and Tocher, 1987). Uptake and s t o r a g e o f TAGs o c c u r s i n a d i p o s e t i s s u e (Henderson and S a r g e n t , 1985) and i n muscle, p a r t i c u l a r l y i n t h e r e d muscle i n s a l m o n i d s (Hepher, 1988). 4 R o l e o f l i p i d s i n f i s h Energy i s r e q u i r e d f o r maintenance, growth and r e p r o d u c t i o n i n a l l a n i m a l s . W h i l e f i s h p r e f e r e n t i a l l y c a t a b o l i z e p r o t e i n t o meet t h e i r energy r e q u i r e m e n t s , d i e t s f o r farmed f i s h a r e f o r m u l a t e d t o m i n i m i z e p r o t e i n consumption f o r energy. T h i s s p a r e s c o s t l y p r o t e i n f o r growth and t i s s u e r e p a i r . Salmonids have a l i m i t e d a b i l i t y t o u t i l i z e c a r b o h y d r a t e as an energy s o u r c e . Excess (>20%) d i e t a r y c a r b o h y d r a t e f e d t o rainbow t r o u t r e s u l t e d i n e n l a r g e d l i v e r s w i t h g l y c o g e n - f i l l e d v a c u o l e s i n t h e h e p a t o c y t e s , d e p r e s s e d growth r a t e s and i n c r e a s e d m o r t a l i t y ( H i l t o n and S l i n g e r , 1981). The n u t r i t i o n i s t t h e r e f o r e i n c o r p o r a t e s l i p i d s i n t o t h e d i e t as t h e major energy s o u r c e . The m e t a b o l i z a b l e energy v a l u e of l i p i d s i s 9.45 16 k c a l / g l i p i d , more th a n double t h a t a v a i l a b l e from c a r b o h y d r a t e (4.0 k c a l / g f o r d e x t r i n and g l u c o s e ) o r p r o t e i n (4.5 k c a l / g f o r c r u d e p r o t e i n ) (McCallum and H i g g s , 1989). A s i d e from t h e p r o v i s i o n o f energy, l i p i d s f u n c t i o n i n t h e t r a n s p o r t o f t h e f a t s o l u b l e v i t a m i n s , A, D, E and K, t o a l l t i s s u e s . They improve t h e p a l a t a b i l i t y of f o r m u l a t e d d i e t s and p r o v i d e e s s e n t i a l f a t t y a c i d s as d i s c u s s e d p r e v i o u s l y . F a t t y a c i d s a r e i n t e g r a l p a r t s o f many v i t a l components of t h e body of a l l a n i m a l s . The a m p h i p a t h i c n a t u r e ( h a v i n g b o t h p o l a r and n o n p o l a r components) of p o l a r l i p i d m o l e c u l e s i s c e n t r a l t o t h e i r f u n c t i o n i n c e l l membranes. O r i e n t a t i o n o f t h e s e l i p i d s a t w a t e r - o i l i n t e r f a c e s i s such t h a t t h e p o l a r group i s i n t h e water phase and t h e n o n p o l a r p o r t i o n i s i n t h e o i l phase. A b i l a y e r o f t h e s e p o l a r l i p i d s i s t h e b a s i c s t r u c t u r e o f b i o l o g i c a l membranes, b o t h c e l l u l a r and s u b c e l l u l a r . N a t u r a l l y o c c u r r i n g u n s a t u r a t e d f a t t y a c i d s a r e a l m o s t e n t i r e l y o f t h e c i s c o n f i g u r a t i o n and t h e m o l e c u l e i s bent 120° a t a d o u b l e bond. As t h e l e v e l o f u n s a t u r a t i o n i n c r e a s e s i n membranes, the m o l e c u l e s pack t o g e t h e r l e s s t i g h t l y due t o g r e a t e r numbers of t h e s e bonds and t h e c o n c o m i t a n t r e d u c t i o n i n van der Waal a t t r a c t i o n s . High l e v e l s of u n s a t u r a t i o n promote membrane f l u i d i t y and p e r m e a b i l i t y . T h i s i s e s p e c i a l l y i m p o r t a n t i n c o l d a c c l i m a t e d s p e c i e s such as t h e s a l m o n i d s where i t has been shown t h a t as t e m p e r a t u r e d e c r e a s e s a 17 r e s t r u c t u r i n g of membranes with g r e a t e r u n s a t u r a t i o n occurs (Hazel, 1979; L i e et a l . , 1989). Without a s h i f t t o a more unsaturated s t a t e , membrane a s s o c i a t e d f u n c t i o n s such as carbohydrate t r a n s p o r t , r a t e s of m i t o c h o n d r i a l o x i d a t i o n , ATP p r o d u c t i o n , c a t a l y t i c a c t i v i t y of membrane-bound enzymes, n u c l e a r to c y t o p l a s m i c t r a n s p o r t of RNA, and n o n e l e c t r o l y t e p e r m e a b i l i t y would be compromised. Conversely, as temperatures i n c r e a s e , a s h i f t toward l e s s u n s a t u r a t i o n o c c u r s . Without t h i s s h i f t an i n c r e a s e i n membrane p e r m e a b i l i t y would occur with a d i s s i p a t i o n of i o n g r a d i e n t s , i n c r e a s e d n e u r a l e x c i t a b i l i t y and u l t i m a t e l y heat death (Hazel, 1979). The dynamic composition of membranes a l s o r e f l e c t s s a l i n i t y changes. Leray et al. (1984) r e p o r t e d a t r a n s f o r m a t i o n i n the f a t t y a c i d composition of PC i n the i n t e s t i n a l mucosa of rainbow t r o u t , i e from 22.4% to 11.9% f o r 18:0 and from 10.6% to 28.0% f o r 22:6n3 w i t h i n one day of t r a n s f e r from f r e s h to s a l t water. S i m i l a r changes were found i n PE and t o a l e s s e r extent i n PS and PI. These changes were r e l a t e d to a measured i n c r e a s e i n membrane f l u i d i t y and i t was suggested t h a t important i o n t r a n s p o r t mechanisms may r e q u i r e an i n c r e a s e i n f l u i d i t y t o f u n c t i o n i n the more s a l i n e environment. Sheridan et al. (1985) found an i n c r e a s e i n 20:5n3, 22:5n3 and 22:6n3 i n both the t r i a c y l g l y c e r i d e s and p h o s p h o l i p i d s i n 18 h a t c h e r y r e a r e d s t e e l h e a d t r o u t d u r i n g s m o l t i f i c a t i o n . A t t h e same t i m e t h e y noted t h e r e d u c t i o n i n t o t a l body l i p i d s t y p i c a l o f t h e p a r r - s m o l t t r a n s f o r m a t i o n . T h i s l i p i d d e p l e t i o n was due / t o a d e c r e a s e i n monoenes and s a t u r a t e d f a t t y a c i d s r a t h e r t h a n PUFA and i l l u s t r a t e s s e l e c t i v e r e t e n t i o n o f PUFA over o t h e r f a t t y a c i d s by s a l m o n i d s . B e l l et al. (1985) de t e r m i n e d t h a t 22:6n3 was e s s e n t i a l f o r t h e maintenance o f h e a l t h y g i l l e p i t h e l i u m i n t u r b o t , Scophthalmus maximus. Four d i e t s were f e d t h a t c o n t a i n e d n6 f a t t y a c i d s o n l y o r t h a t had 20:5n3 t o 22:6n3 r a t i o s o f 1.8, 2.2 o r 13.8. J u v e n i l e t u r b o t were a b l e t o s u r v i v e w i t h 20:5n3 t o 22:6n3 r a t i o s of 1.8 o r 2.2 but c o u l d not s u r v i v e on t h e d i e t c o n t a i n i n g o n l y n6 f a t t y a c i d s . Those on t h e h i g h 20:5n3 d i e t (13.8 r a t i o ) a l s o e x p e r i e n c e d h i g h m o r t a l i t y . These f i s h were a p p a r e n t l y u n a b l e t o c o n v e r t 20:5n3 t o 22:6n3. When d i e t a r y 22:6n3 was low o r a b s e n t , c h l o r i d e c e l l s d i s a p p e a r e d and t h e e p i t h e l i u m o f t h e p r i m a r y and secondary l a m e l l a e s l o u g h e d o f f l e a v i n g a s k e l e t o n of c o n n e c t i v e t i s s u e w i t h masses o f d e b r i s i n t h e i n t e r l a m e l l a r s p a c e s . The r a p i d t u r n o v e r of g i l l e p i t h e l i u m , and p a r t i c u l a r l y t h e c h l o r i d e c e l l s w i t h t h e i r c o n v o l u t e d plasma membranes and numerous m i t o c h o n d r i a , makes t h e g i l l s a s e n s i t i v e i n d i c a t o r o f t h e e s s e n t i a l f a t t y a c i d s t a t u s o f f i s h and i l l u s t r a t e s t h e a b s o l u t e r e q u i r e m e n t f o r 22:6n3 as a s t r u c t u r a l component o f biomembranes. Langdon and Thorpe (1984) demonstrated a 19 p r o l i f e r a t i o n and enlargement o f c h l o r i d e c e l l s on t h e g i l l f i l a m e n t s o f A t l a n t i c salmon (Salmo salar) s m o l t s on t r a n s f e r t o s a l t w a t e r . These c e l l s produce Na^-IC" ATPase and g i l l s u c c i n i c dehydrogenase, key enzymes i n o s m o r e g u l a t i o n and i o n t r a n s p o r t . F a t t y a c i d s have an i m p o r t a n t r o l e as s u b s t r a t e s f o r e i c o s a n o i d s y n t h e s i s . E i c o s a n o i d s c o m p r i s e t h e p r o s t a g l a n d i n s (PG), p r o s t a c y c l i n s ( P G I ) , thromboxanes (TX) and l e u k o t r i e n e s ( L T ) . PG, PGI and TX t o g e t h e r a r e o f t e n r e f e r r e d t o as p r o s t a n o i d s . A l l a r e p h y s i o l o g i c a l l y a c t i v e compounds d e r i v e d from 20 c a r b o n ( e i c o s a - ) f a t t y a c i d s and c o n t a i n i n g a 5-carbon r i n g . E i c o s a n o i d s a r e e x t r e m e l y p o t e n t and a v e r y s m a l l amount has a pronounced e f f e c t . As l i t t l e as 1 ng PG/ml causes t h e c o n t r a c t i o n o f smooth muscle i n a n i m a l s (Murray e t al., 1988). They a r e s y n t h e s i z e d w i t h i n membranes, r a p i d l y t a k e n up, used and r a p i d l y d e - a c t i v a t e d . I n mammals, t h e predominant pathway i s from a r a c h i d o n i c a c i d , 20:4n6, as shown i n F i g u r e 1 (adapted from Murray et al., 1988) g i v i n g r i s e t o t h e group 2 e i c o s a n o i d s . Groups 1 and 3 a r e d e r i v e d from 20:3n6 and 20:5n3, r e s p e c t i v e l y , i n analogous pathways ( F i s c h e r and Weber, 1984). The s u b s c r i p t o f t h e e i c o s a n o i d i n d i c a t e s t h e number of double bonds i n t h e m o l e c u l e and t h e group t o which i t b e l o n g s . The f u n c t i o n s o f e i c o s a n o i d s a r e many and v a r i e d . TXA 2 (a group 2 thromboxane s y n t h e s i z e d i n p l a t e l e t s from 20:4n6) i s a 20 MEMBRANE PHOSPHOLIPID p h o s p h o l i p a s e 20:4n6 l i p o x y g e n a s e c y c l o o x y g e n a s e LEUKOTRIENES PROSTANOIDS (eg. P G I 2 ) & THROMBOXANES (eg. TXA 2) f F i g u r e 1: C o n v e r s i o n o f a r a c h i d o n i c a c i d (20:4n6) t o group 2 e i c o s a n o i d s . By analogous pathways, e i c o s a t r i e n o i c a c i d (20:3n6) and e i c o s a p e n t a e n o i c a c i d (20:5n3) a r e s u b s t r a t e s f o r t h e s y n t h e s i s of groups 1 and 3 e i c o s a n o i d s , r e s p e c t i v e l y , ( a f t e r Murray et al., 1988) 21 p o t e n t s t i m u l a t o r of p l a t e l e t a g g r e g a t i o n and v a s o c o n s t r i c t i o n . P G I 2 i s produced i n t h e w a l l s o f b l o o d v e s s e l s and i s a n t a g o n i s t i c t o TXA 2. TXA 3 (a group 3 thromboxane, a l s o s y n t h e s i z e d i n p l a t e l e t s but from 20:5n3) a l s o s t i m u l a t e s p l a t e l e t a g g r e g a t i o n but i t has much weaker c l o t t i n g e f f e c t s t h a n TXA 2. P G I 3 e q u a l s P G I 2 i n a n t i - c l o t t i n g p o t e n cy but because TXA 3 i s weaker, t h e b a l a n c e s h i f t s toward a n t i -a g g r e g a t i o n . I n a d d i t i o n , group 3 p r o s t a n o i d s b l o c k t h e s y n t h e s i s o f group 2 p r o s t a n o i d s by i n h i b i t i n g t h e r e l e a s e o f 20:4n6 from membrane p h o s p h o l i p i d s . T h i s i s thought t o be t h e b a s i s o f t h e low i n c i d e n c e o f i s c h e m i c h e a r t d i s e a s e , reduced p l a t e l e t a g g r e g a t i o n and p r o l o n g e d c l o t t i n g t i m e s i n Eskimos whose d i e t i s h i g h i n f i s h o i l s w i t h h i g h c o n c e n t r a t i o n s o f 20:5n3 and low i n c h o l e s t e r o l , t r i a c y l g l y c e r i d e s and v e r y low d e n s i t y l i p o p r o t e i n s (Murray e t al., 1988). L e u k o t r i e n e s a r e s y n t h e s i z e d i n l e u k o c y t e s , p l a t e l e t s and macrophages. They a c t t o promote t h e i n f l a m m a t i o n response i n i n f e c t i o n s and t h e h y p e r s e n s i t i v i t y r e a c t i o n i n a l l e r g i e s . I n mammals e i c o s a n o i d s a l s o modulate t h e a c t i o n o f hormones, r e g u l a t e b l o o d f l o w t o p a r t i c u l a r o r g a n s , c o n t r o l i o n t r a n s p o r t a c r o s s some membranes, and modulate s y n a p t i c t r a n s m i s s i o n a l o n g nerve c e l l s (Murray e t al., 1988). A l t h o u g h 20:5n3 appears t o be t h e t h e main p r o s t a g l a n d i n p r e c u r s o r i n f i s h , t h e r e i s l i t t l e e v i d e n c e f o r t h e p r o d u c t i o n o f t h e PG 3 s e r i e s o r t h e i r e f f e c t s i n f i s h t i s s u e (Greene and 22 S e l i v o n c h i c k , 1987). PGF 2« i s a p o t e n t in v i t r o s t i m u l a t o r o f o v u l a t i o n i n brook t r o u t ( S t a c e y and G o e t z , 1982); however i t i s not c l e a r what n a t u r a l r o l e t h e PGF p r o s t a g l a n d i n s may have a t o v u l a t i o n (Goetz, 1983). Kayama et al. (1986) demonstrated t h e p r o d u c t i o n o f TXA 3, from 20:5n3 i n rainbow t r o u t . S t u d i e s on p l a i c e s k i n i n d i c a t e d t h a t 20:5n3 (or i t s p r o d u c t s ) may i n h i b i t t h e t r a n s f o r m a t i o n o f 20:4n6 t o a muscle and v a s o -c o n t r a c t i n g PG (presumably TXA 2) (Anderson et al., 1979, 1981) and t h a t 20:5n3 may be a p r e c u r s o r t o P G I 3 , t h e p o t e n t vaso-d i l a t i n g compound, as i n man ( F i s c h e r and Weber, 1984). Greene and S e l i v o n c h i c k (1987) suggest t h a t P G I 3 may be i m p o r t a n t i n t e m p e r a t u r e a c c l i m a t i o n i n f i s h . There i s some s p e c u l a t i o n t h a t 22:6n3, o f t e n t h e predominant HUFA i n f i s h , i n c l u d i n g s a l m o n i d s , may be r e t r o c o n v e r t e d t o 20:5n3 f o r PG s y n t h e s i s ( T i n o c o , 1982; Yu and S i n n h u b e r , 1972; Greene and S e v i l o n c h i c k , 1987). The presence o f 22:6n3 i n membrane p h o s p h o l i p i d s may be more th a n a mechanism t o promote f l u i d i t y a t low t e m p e r a t u r e s and may, i n f a c t , be a l o c a l s t o r a g e depot f o r PG p r e c u r s o r s (Greene and S e l i v o n c h i c k , 1987). Other workers have demonstrated t h e s y n t h e s i s o f l e u k o t r i e n e s d i r e c t l y from 22:6n3 (German et al., 1986a, 1986b). The l i p i d s i n t h e r e t i n a l membrane of t h e human eye c o n t a i n 30% o r more of t h e f a t t y a c i d , 22:6n3. Rhodopsin m o l e c u l e s i n t h e eye change shape i n response t o l i g h t , e n a b l i n g images t o be 23 p e r c e i v e d . I t i s thought t h a t t h e f l e x i b i l i t y of t h e r e t i n a l membrane, due t o t h e p r e s e n c e o f 22:6n3, f a c i l i t a t e s t h e response o f r h o d o p s i n ( C a s t e l l , 1988). Tocher and H a r v i e (1988) found h i g h l e v e l s o f 22:6n3 i n t h e p h o s p h o g l y c e r i d e s (PC, PE, PS, and PI) of t h e r e t i n a s o f rainbow t r o u t and cod (Gadus morhua). They p o s t u l a t e d t h a t 22:6n3 may be r e t r o c o n v e r t e d t o 20:5n3 b e f o r e p r o s t a g l a n d i n s y n t h e s i s . No p o t e n t i a l f u n c t i o n f o r p r o s t a g l a n d i n s i n t h e eye was s u g g e s t e d . PI was found t o have h i g h e r l e v e l s o f 20:5n3 tha n t h e o t h e r p h o s p h o g l y c e r i d e s i n b o t h r e t i n a s and b r a i n s o f t h e f i s h i n t h i s s t u d y l e a d i n g t o s p e c u l a t i o n t h a t PI may s e r v e as a r e s e r v o i r f o r p r o s t a g l a n d i n p r e c u r s o r s . 5 F a t t y a c i d s y n t h e s i s and m o b i l i z a t i o n The l i v e r i s t h e p r i n c i p a l s i t e o f de novo f a t t y a c i d s y n t h e s i s i n f i s h . L i t t l e s y n t h e s i s t a k e s p l a c e i n t h e a d i p o s e t i s s u e i n c o n t r a s t t o t h e s i t u a t i o n i n mammals. F a t t y a c i d s y n t h e s i s has a l s o been r e p o r t e d in v i t r o i n t h e o v a r y o f rainbow t r o u t (Weigand and I d l e r , 1982). De novo s y n t h e s i s o f f a t t y a c i d s from p r e c u r s o r a c e t a t e r e s u l t s i n t h e p r o d u c t i o n o f s a t u r a t e d and monoenoic f a t t y a c i d s w i t h even numbers o f carbon atoms. F i s h cannot s y n t h e s i z e any members of t h e n3 o r n6 f a t t y a c i d s e r i e s b u t , when p r o v i d e d i n t h e d i e t , s a l m o n i d s can e l o n g a t e and d e s a t u r a t e n3 and n6 f a t t y 24 a c i d s t o PUFAs and HUFAs as shown i n F i g u r e 2 ( C a s t e l l , 1979). Rahm and Holman (1964) found t h a t by i n c r e a s i n g t h e amounts o f d i e t a r y 18:2n6, t h e e l o n g a t i o n and d e s a t u r a t i o n of 18:3n3 t o 20:5n3, 22:5n3 and 22:6n3 was s u p p r e s s e d i n w e a n l i n g r a t s . T h i s s u p p r e s s i o n c o u l d be d i s p l a c e d i n t h e o t h e r d i r e c t i o n when a h i g h l e v e l o f 18:3n3 was f e d , i e t h e e l o n g a t i o n and d e s a t u r a t i o n o f 18:2n6 t o 20:3n6, 20:4n6 and 22:5n6 was i n h i b i t e d . T h i s i n h i b i t o r y e f f e c t i s a p p a r e n t l y c o n c e n t r a t i o n dependent and, i n s a l m o n i d s , Yu and Sinnhuber (1976, 1979a) d e t e r m i n e d t h a t n3 f a t t y a c i d s a r e more p o t e n t i n h i b i t o r s o f n6 metabolism t h a n t h e r e v e r s e . The mechanism o f c o m p e t i t i v e i n h i b i t i o n i s r e l a t e d t o s u b s t r a t e s p e c i f i c i t y o f t h e enzyme, 6 6 - d e s a t u r a s e , w hich d e s a t u r a t e s 18:2n6 t o 18:3n6 and 18:3n3 t o 18:4n3 (Leger et al., 1981). I t i s g e n e r a l l y a c c e p t e d t h a t t h e p r e f e r e n t i a l a f f i n i t y o f t h i s enzyme i s f o r t h e more u n s a t u r a t e d f a t t y a c i d , i e d e s a t u r a t i o n o c c u r s i n t h e o r d e r n3 > n6 > n9 e x c e p t when t h e r e i s a l a r g e preponderance o f n6 or n9 f a t t y a c i d s (Henderson and Tocher, 1987). I n h i b i t i o n of n3 e l o n g a t i o n and d e s a t u r a t i o n may a l s o o c c u r when t h e l e v e l o f s a t u r a t e s i n t h e d i e t i s r a i s e d . T a k e u c h i and Watanabe (1977b) found t h a t an i n c r e a s e i n d i e t a r y l a u r a t e from 4 t o 14% i n c r e a s e d t h e r e q u i r e m e n t f o r 18:3n3 from 1 t o 2% i n rainbow t r o u t . 25 SATURATED AND MONOENOIC FATTY ACIDS ACETATE 14: 0 — > 1 4 : l n 5 --> 16:ln5 16 1 0 --> 16:ln7 --> 18:ln7 18: 1 0 --> 18:ln9 --> 20:ln9 20: 1 0 --> 2 0 : l n l l -- > 2 2 : l n l l 22: 0 --> 2 2 : l n l 3 POLYUNSATURATED FATTY ACIDS 1 8 : l n 9 / \ 20:ln9 18:2n9 \ / 20:2n9 \ 20:3n9 18:2n6 / \ 20:2n6 18:3n6 \ / 20:3n6 / \ 22:3n6 20:4n6 \ / 22:4n6 \ 22:5n6 18:3n3 / \ 20:3n3 18:4n3 \ / 20:4n3 / \ 22:4n3 20:5n3 \ / 22:5n3 \ 22:6n3 F i g u r e 2: F a t t y a c i d s y n t h e s i s i n f i s h . (from C a s t e l l , 1979) V 26 C o m p e t i t i o n between s u b s t r a t e s f o r 66 - d e s a t u r a s e e x p l a i n s t h e r e s u l t s o f many of t h e e a r l y f e e d i n g s t u d i e s . Growth r a t e s i n t r o u t f e d d i e t s d e f i c i e n t i n n3 f a t t y a c i d s were improved by the a d d i t i o n o f 18:2n6. However, i f t h e d i e t c o n t a i n e d adequate l e v e l s of n3 f a t t y a c i d s , t h e a d d i t i o n o f 18:2n6 a t 0.5 o r 1% d e p r e s s e d t h e growth r a t e . F u r t h e r m o r e , when 18:3n3 was h e l d a t 0.5 o r 1% and 18:2n6 was i n c r e a s e d from 0 t o 5%, th e 22:6n3 and t o t a l n3 f a t t y a c i d s i n t h e p h o s p h o l i p i d f r a c t i o n d e c r e a s e d w h i l e t h e 18:2n6 l e v e l i n c r e a s e d (Yu and ' Si n n h u b e r , 1981). T h i s i s an example of c o n c e n t r a t i o n dependent i n h i b i t i o n o f n3 e l o n g a t i o n and d e s a t u r a t i o n by h i g h l e v e l s o f n6 f a t t y a c i d s . Leger et al. (1981) showed t h a t a h i g h l e v e l o f n3 HUFAs such as 22:6n3 c o u l d e x e r t a n e g a t i v e feedback on t h e d e s a t u r a t i o n o f b o t h 18:3n3 and 18:2n6. M o b i l i z a t i o n and c a t a b o l i s m o f l i p i d s o c c u r d u r i n g s t a r v a t i o n , when t h e f o o d s u p p l y i s s c a r c e and d u r i n g t h e spawning m i g r a t i o n , when w i l d P a c i f i c salmon cease f e e d i n g . When an energy d e f i c i t i s pending TAGs a r e p r e f e r e n t i a l l y c a t a b o l i z e d o v e r p h o s p h o l i p i d s (Yu et al., 1977; C a s t l e d i n e and B u c k l e y , 1980, 1982). When p h o s p h o l i p i d s a r e c a t a b o l i z e d , i t i s l i k e l y t h a t t h e PUFA or HUFA i n t h e fl p o s i t i o n a r e p r o t e c t e d from o x i d a t i o n and i t i s t h e monoenoic f a t t y a c i d i n t h e a p o s i t i o n t h a t i s broken down. R e c y c l i n g o f l o n g c h a i n u n s a t u r a t e s between p h o s p h o g l y c e r i d e s t o m a i n t a i n v i t a l p h y s i o l o g i c a l 27 f u n c t i o n has been p o s t u l a t e d by C a s t l e d i n e and B u c k l e y (1982). C a s t l e d i n e and B u c k l e y (1980) s t a r v e d j u v e n i l e rainbow t r o u t t o i n d u c e m o b i l i z a t i o n o f f a t t y a c i d s and t o d e t e r m i n e whether t h e r e was any c r o s s o v e r o f PUFAs and HUFAs from t h e n e u t r a l l i p i d p o o l t o p h o s p h o l i p i d s . Changes i n f a t t y a c i d c o m p o s i t i o n i n t h e n e u t r a l l i p i d f r a c t i o n were s l i g h t and no p r e f e r e n t i a l c a t a b o l i s m o f f a t t y a c i d s was e v i d e n t . However i n t h e p h o s p h o l i p i d component, t h e r e was a g e n e r a l d e c r e a s e i n t h e s a t u r a t e s and monoenes but no c a t a b o l i s m o f 18:2n6 o r 22:6n3 was e v i d e n t . No t r a n s f e r o f t h e PUFAs and HUFAs from n e u t r a l t o p h o s p h o l i p i d s o c c u r r e d . M o b i l i z a t i o n a l s o o c c u r s when n u t r i t i o n a l l y u nbalanced o r d e f i c i e n t d i e t s a r e f e d as i s t h e i n t e n t i o n i n some e x p e r i m e n t a l d e s i g n s . The f a t t y a c i d c o n t e n t o f t h e n e u t r a l l i p i d f r a c t i o n o f t h e body l a r g e l y r e f l e c t s t h e c o m p o s i t i o n o f d i e t a r y l i p i d . P h o s p h o l i p i d s a r e b u f f e r e d t o some e x t e n t from d i e t a r y changes and f a t t y a c i d s w i t h s p e c i f i c m e t a b o l i c f u n c t i o n s a r e c o n s e r v e d . Yu e t al. (1977) found t h a t t r o u t r e t a i n e d d i s p r o p o r t i o n a t e l y h i g h l e v e l s o f PUFAs and HUFAs i n t h e p h o s p h o l i p i d f r a c t i o n i n comparison w i t h t h e l e v e l s employed i n t h e i r e x p e r i m e n t a l d i e t s . I n p a r t i c u l a r t h e c o n c e n t r a t i o n o f 22:6n3 was h i g h , i n d i c a t i n g t h e importance of t h i s f a t t y a c i d f o r t r o u t . , S i m i l a r l y when C a s t l e d i n e and B u c k l e y (1980, 1982) f e d d i e t s d e f i c i e n t i n e s s e n t i a l f a t t y a c i d s , n e a r l y complete r e t e n t i o n o f n3 f a t t y a c i d s i n t h e 28 p h o s p h o l i p i d s was found. An i n c r e a s e i n n9 f a t t y a c i d s , p a r t i c u l a r l y 1 8 : l n 9 and 20:3n9, was seen as t h e experiment p r o g r e s s e d . Mechanisms may a l s o e x i s t t o modulate o t h e r l i p i d s . Yu et al. (1977) f e d i s o c a l o r i c d i e t s c o n t a i n i n g v a r i o u s l e v e l s o f s a t u r a t e d f a t t y a c i d s t o t h r e e groups of rainbow t r o u t . R e g a r d l e s s o f t h e s a t u r a t e l e v e l s used i n t h e i r d i e t s , t h e y found t h a t t h e c o n c e n t r a t i o n i n t h e t o t a l body l i p i d s remained f a i r l y c o n s t a n t ( a p p r o x i m a t e l y 2 4 % ) . T h i s s u g g e s t s r e g u l a t i o n and m a i n t a i n a n c e o f a s p e c i f i c degree o f s a t u r a t i o n . As t h e l e v e l o f s a t u r a t e s i n c r e a s e d i n t h e d i e t , t h e c o n c e n t r a t i o n of monoenes, p a r t i c u l a r l y 1 8 :ln9 i n c r e a s e d , i n d i c a t i n g e l o n g a t i o n and d e s a t u r a t i o n o f t h e s a t u r a t e s . M o b i l i z a t i o n of l i p i d s a l s o o c c u r s d u r i n g m a t u r a t i o n and i n t h e p r o c e s s o f v i t e l l o g e n e s i s . y 6 V i t e l l o g e n e s i s V i t e l l o g e n e s i s i s t h e s y n t h e s i s o f y o l k m a t e r i a l s , p a r t i c u l a r l y v i t e l l o g e n i n , and t h e i r a c c u m u l a t i o n by t h e d e v e l o p i n g o o c y t e s . Weigand and I d l e r (1982) demonstrated t h e a b i l i t y o f o o c y t e s t o s y n t h e s i z e l i p i d s e a r l y i n t h e i r development, i n t h e p e r i o d termed 'endogenous v i t e l l o g e n e s i s ' . T h i s t e r m i n o l o g y may be c o n s i d e r e d a misnomer as i t i s t h e l i v e r where v i t e l l o g e n i n i s s y n t h e s i z e d , as d i s c u s s e d below, not t h e o v a r y . Endogenous 29 s y n t h e s i s o f y o l k l i p i d s p r e cedes and o v e r l a p s exogenous v i t e l l o g e n e s i s i n time but i t s c o n t r i b u t i o n i n q u a n t i t a t i v e terms i s r e l a t i v e l y minor (Wiegand and I d l e r , 1982; Mommsen and Walsh, 1988). The importance of endogenous s y n t h e s i s seems t o be r e l a t e d t o t h e p r o v i s i o n o f a s t r u c t u r a l framework f o r t h e subsequent a c c u m u l a t i o n o f y o l k m a t e r i a l s (Fremont et al., 1984). I t i s d u r i n g exogenous v i t e l l o g e n e s i s t h a t t h e o v a r y g a i n s most of i t s mass. The o n s e t o f v i t e l l o g e n e s i s i s under hormonal c o n t r o l . I n response t o t h e p r o d u c t i o n o f a g o n a d o t r o p i n by t h e p i t u i t a r y , t h e o v a r i a n f o l l i c l e s s y n t h e s i z e e s t r o g e n hormones which a r e s e c r e t e d i n t o t h e s y s t e m i c c i r c u l a t i o n . The l i v e r i s t h e p r i m a r y t a r g e t f o r 17 8 - e s t r a d i o l , and i t responds w i t h t h e s y n t h e s i s (exogenous v i t e l l o g e n e s i s ) and e x p o r t o f v i t e l l o g e n i n (Mommsen and Walsh, 1988). V i t e l l o g e n i n i s a v e r y h i g h d e n s i t y , female s p e c i f i c l i p o p h o s p h o p r o t e i n complex which c o n t a i n s c a . 80% p r o t e i n and i s r i c h i n p h o s p h o l i p i d s and e s s e n t i a l f a t t y a c i d s ( L e g e r , 1985.). I t a l s o c a r r i e s c a r b o h y d r a t e s , phosphate groups and m i n e r a l s a l t s (Mommsen and Walsh, 1988). V i t e l l o g e n i n i s s e l e c t i v e l y t a k e n up from t h e c i r c u l a t i o n by t h e d e v e l o p i n g o o c y t e s by m i c r o p i n o c y t o s i s and i s c l e a v e d i n t o i t s components, i n c l u d i n g p h o s v i t i n and l i p o v i t e l l i n . A c c o r d i n g t o Wiegand (1982), v i t e l l o g e n i n i s t h e major s o u r c e o f o v a r i a n p o l a r l i p i d s . Up t o 87% of t h e l i p i d i n t h e v i t e l l o g e n i n o f t h e 30 g o l d f i s h , Carassius auratus, i s p o l a r l i p i d and 58% of t h e l i p o v i t e l l i n l i p i d i n coho salmon, Oncorhynchus kisutch, i s p h o s p h o l i p i d . E a r l y i n v i t e l l o g e n e s i s , p o l a r l i p i d s p redominate i n t h e o v a r y . L a t e r , d u r i n g t h e exogenous s t a g e , TAGs s t a r t t o i n c r e a s e and f i n a l l y predominate (Wiegand, 1982). C i r c u l a t i n g l i p o p r o t e i n s , t h e VLDL, LDL and HDL a r e macromolecular components of t h e b l o o d plasma o f a l l v e r t e b r a t e s . They t r a n s p o r t l i p i d s from t h e s i t e o f a b s o r p t i o n i n t h e i n t e s t i n a l mucosa and from t h e s i t e o f b i o s y n t h e s i s i n th e l i v e r i n t o t h e c i r c u l a t i o n and t o t h e s i t e s o f c o n v e r s i o n , s t o r a g e o r u t i l i z a t i o n . L i p o p r o t e i n s c o n s i s t o f a hyd r o p h o b i c c o r e o f TAGs and c h o l e s t e r o l surrounded by a h y d r o p h i l i c e n v elope o f p o l a r c o n s t i t u e n t s such as p h o s p h o l i p i d s and a p o p r o t e i n s (Murray et al., 1988) and p r o v i d e a n o t h e r s o u r c e o f m a t e r i a l s f o r o o g e n e s i s . D i e t a r y l i p i d s a r e thought t o be d e p o s i t e d d i r e c t l y i n t o t h e eggs d u r i n g t h i s p e r i o d , t r a n s p o r t e d by l i p o p r o t e i n s and b y p a s s i n g t h e a d i p o s e t i s s u e (Fremont et al., 1984; Luquet and Watanabe, 1986). The e x a c t p a r t i t i o n i n g o f v i t e l l o g e n i n and l i p o p r o t e i n , f o r t h e movement o f y o l k p r e c u r s o r s i n t o t h e o o c y t e i s not w e l l u n d e r s t o o d . I t i s thought t h a t b o t h a r e i n v o l v e d perhaps a t d i f f e r e n t t i m e s (Wiegand, 1982). Leger et al. (1981) r e p o r t t h a t egg l i p i d s i n t r o u t a r e found i n two s e p a r a t e f r a c t i o n s o f the egg and t h a t t h e y can be s e p a r a t e d by c e n t r i f u g a t i o n . The o i l g l o b u l e was found t o c o n t a i n p r i m a r i l y TAGs w i t h t r a c e s o f 31 c h o l e s t e r o l and c h o l e s t e r y l e s t e r s ; t h e y o l k g l o b u l e c o n t a i n e d l i p o v i t e l l i n and p h o s v i t i n . The n3 f a t t y a c i d c o n c e n t r a t i o n , and p a r t i c u l a r l y 22:6n3, was h i g h e s t i n t h e l i p o v i t e l l i n and i t was suggested t h a t t h e major s o u r c e o f s t r u c t u r a l components, both l i p i d and p r o t e i n , f o r t h e d e v e l o p i n g embryo i s t h e y o l k g l o b u l e . The o i l g l o b u l e i s t h e major energy r e s e r v o i r t h r o u g h i n c u b a t i o n . A v a r i e t y o f o t h e r compounds i s a l s o accumulated by t h e growing o o c y t e s , i n c l u d i n g g l y c o g e n , c a r o t e n o i d s , l e c t i n s , wax and s t e r o l e s t e r s , and s i a l o g l y c o p r o t e i n s . W h i l e some o f t h e s e a r e energy s o u r c e s , t h e f u n c t i o n s of o t h e r s i s not c l e a r (Mommsen and Walsh, 1988). Maximum growth and t h e a l l e v i a t i o n 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 s i g n s a r e g e n e r a l l y used as t h e c r i t e r i a f o r e s t a b l i s h i n g d i e t a r y r e q u i r e m e n t s f o r l i p i d l e v e l s and e s s e n t i a l f a t t y a c i d s . L i t t l e a t t e n t i o n has been p a i d i n s e t t i n g t h e s e r e q u i r e m e n t s t o p h y s i o l o g i c a l changes such as v i t e l l o g e n e s i s and m a t u r a t i o n . However, r e c e n t i n f o r m a t i o n s u g g e s t s t h a t i n g e n e r a l n u t r i t i o n a l r e q u i r e m e n t s o f b r o o d s t o c k may be q u i t e d i f f e r e n t from f i s h i n t h e so m a t i c s t a g e s o f t h e i r l i f e c y c l e s . For example Luquet and Watanabe (1986) r e p o r t work showing t h a t m a t u r i n g s a l m o n i d s may be b e t t e r a b l e t o u t i l i z e c a r b o h y d r a t e t h a n younger f i s h due t o t h e presence o f an amylase i n t h e i r p y l o r i c caecae. T a k e u c h i et al. (1981) demonstrated t h a t d i e t s r e l a t i v e l y low i n p r o t e i n (33-35%) had 32 no a d v e r s e e f f e c t s on r e p r o d u c t i o n compared w i t h a commercial d i e t h i g h i n p r o t e i n (43-47%) when f e d t o rainbow t r o u t f o r 3 y e a r s as l o n g as t h e d i e t was a l s o h i g h i n energy (390 k c a l / 1 0 0 g., i n c l u d i n g 5-7% beef t a l l o w ) . A s i m i l a r r e s u l t was r e p o r t e d by R o l e y (1983) who found t h a t maximum growth r a t e was a c h i e v e d w i t h a d i e t c o n t a i n i n g between 37 and 47% p r o t e i n and 3.8 k c a l / g m e t a b o l i z a b l e energy but t h a t f e c u n d i t y , egg s i z e and embryo s u r v i v a l were u n a f f e c t e d by a p r o t e i n l e v e l as low as 27% a t t h e same energy l e v e l . Fremont et al. (1984) demonstrated t h a t rainbow t r o u t were a b l e t o m a i n t a i n h i g h l e v e l s o f 22:6n3 i n v i t e l l o g e n i n and l i p o p r o t e i n when f e d an n3 d e f i c i e n t d i e t f o r 6 m o n t h s , p r i o r t o spawning. L e v e l s o f a l l o t h e r n3 f a t t y a c i d s , i n c l u d i n g 20:5n3, were reduced d r a s t i c a l l y d u r i n g t h i s p e r i o d . The t e n a c i o u s r e t e n t i o n o f 22:6n3 over a l l o t h e r n3 f a t t y a c i d s i n d i c a t e s i t s p r o b a b l e importance f o r r e p r o d u c t i v e s u c c e s s and a p o s s i b l e d i f f e r e n c e i n r e q u i r e m e n t s f o r growth v e r s u s r e p r o d u c t i o n i n t h i s s p e c i e s . 7 Embryonic development From t h e f o r e g o i n g , i t i s c l e a r t h a t salmon embryos r e q u i r e a s u p p l y o f f a t t y a c i d s and preformed compound l i p i d s < t r i a c y l g l y c e r i d e s , p h o s p h o l i p i d s , s p h i n g o l i p i d s , c h o l e s t e r o l e t c . ) t o d e v e l o p s u c c e s s f u l l y t h r o u g h t h e i n c u b a t i o n and a l e v i n s t a g e s . These l i p i d s w i l l form t h e major energy sou r c e as w e l l 33 as s t r u c t u r a l components f o r growth and p r e c u r s o r s f o r compounds of p h y s i o l o g i c a l i m p o r t a n c e such as s t e r o i d s and p r o s t a g l a n d i n s . D e f i c i e n c y o f e s s e n t i a l f a t t y a c i d s i n t h e y o l k s u p p l y has d i r e consequences f o r t h e .health and s u r v i v a l of t h e d e v e l o p i n g f i s h . When EFA d e f i c i e n t d i e t s were f e d t o b r o o d s t o c k o f rainbow t r o u t , c a r p and r e d sea bream, low f e c u n d i t y and f e r t i l i z a t i o n r a t e s and poor h a t c h a b i l i t y r e s u l t e d (Watanabe, 1982). I n t h e c a s e of r e d sea bream, EFA d e f i c i e n t d i e t s r e s u l t e d i n egg a b n o r m a l i t i e s and embryo d e f o r m i t i e s . L e r a y et al. (1985) o b s e r v e d d e f o r m i t i e s i n t r o u t embryos when b r o o d s t o c k were f e d n 3 - d e f i c i e n t d i e t s w i t h c o n c o m i t a n t h i g h l e v e l s o f n6 f a t t y a c i d s . Anomalies o c c u r r e d as e a r l y as t h e 16- and 3 2 - c e l l s t a g e , when t h e arrangement of c e l l s was d i s t u r b e d . L a t e r , t h e b o d i e s of t h e a l e v i n s were c u r v e d o r c u r l e d i n t o h e l i c a l shapes. L e r a y et al. (1985) s p e c u l a t e d t h a t t h e s e d e f o r m i t i e s were r e l a t e d t o t h e absence of n3 HUFAs, t h e p r e c u r s o r s o f hydroxy f a t t y a c i d s , t hought t o a c t as m o d u l a t o r s o r m e d i a t o r s i n p r o c e s s e s of c e l l u l a r r e c o g n i t i o n o c c u r r i n g i n embryonic development (Aveldano and S p r e c h e r , 1983; Boukhchache and Lagarde, 1982, c i t e d i n L e r a y e t al., 1985). The r o l e of 20:5n3 as a p r o s t a g l a n d i n p r e c u r s o r was a l s o i m p l i c a t e d . 34 L e r a y e t al. (1985) a l s o r e p o r t e d p r o l o n g e d embryo development t i m e s and more r a p i d (premature) y o l k r e s o r p t i o n t h a n i n c o n t r o l f i s h when n 3 - d e f i c i e n t d i e t s were f e d t o b r o o d s t o c k . 8 Feeding regime f o r c u l t u r e d m a t u r i n g s a l m o n i d s D e p l e t i o n o f a major p o r t i o n o f t h e somati c r e s e r v e s o f w i l d s a l m o n i d s o c c u r s d u r i n g v i t e l l o g e n e s i s , p a r t i c u l a r l y i n t h e l a t t e r s t a g e s when f e e d i n g c e a s e s and t h e f i s h m i g r a t e t o t h e spawning grounds. T h i s j o u r n e y i s l o n g and arduous f o r s t o c k s t h a t spawn hundreds o f m i l e s from t h e ocean. I n Chinook salmon, a l o s s o f c a . 85% o f t h e n e u t r a l l i p i d s , 60-70% o f t h e p h o s p h o l i p i d s and 30% o f t h e p r o t e i n from muscle, t h e major s t o r a g e depot i n t h i s s p e c i e s , was documented a f t e r a 4-5 month j o u r n e y o f more th a n 800 m i l e s from t h e mouth o f t h e Columbia R i v e r t o t h e i r spawning grounds (Greene, 1919). Kato (1975) o b s e r v e d t h a t c u l t u r e d m a t u r i n g t r o u t n a t u r a l l y reduce t h e i r f e e d i n t a k e s e v e r a l months p r i o r t o spawning, but t o a l e s s e r e x t e n t t h a n t h e w i l d f i s h do. C u l t u r e d Chinook salmon a l s o reduce t h e i r f e e d i n t a k e over s e v e r a l months p r i o r t o spawning but t h e y w i l l c o n t i n u e t o t a k e some f e e d f i g h t up u n t i l t h e y a r e moved t o f r e s h w a t e r ( p e r s o n a l o b s e r v a t i o n ) . G u t s e l l (1940) f e d t h r e e l o t s o f 3-year o l d rainbow t r o u t e i t h e r t o s a t i a t i o n , a p p r o x i m a t e l y 50% o f s a t i a t i o n , o r about 25% o f s a t i a t i o n f o r e i g h t months b e f o r e spawning. No d e f i n i t i o n was g i v e n f o r s a t i a t i o n but r e f e r e n c e was made t o th e f i s h f e d t o s a t i a t i o n as b e i n g " o v e r f e d " . Group 1 ^ ( s a t i a t i o n ) had t h e b e s t f e c u n d i t y on an i n d i v i d u a l b a s i s f o l l o w e d by group 2 ( 5 0 % s a t i a t i o n ) and 3 ( 2 5 % s a t i a t i o n ) . However 10% of group 1 females f a i l e d t o spawn. Egg s i z e d e c r e a s e d i n t h e o r d e r : group 1 > group 2 > group 3. P e r c e n t s u r v i v a l t o e y e i n g was g r e a t e s t i n group 3 (80.7%) f o l l o w e d by group 2 (78.7%) and t h e n by group 1 ( 7 0 . 4 % ) . I t was c o n c l u d e d t h a t egg q u a l i t y s u f f e r e d from o v e r f e e d i n g t h e b r o o d s t o c k . Ridelman e t al. (1984) s t a r v e d a group o f rainbow t r o u t b r o o d s t o c k f o r c a . 40 days p r i o r t o spawning. No d i f f e r e n c e s i n egg q u a l i t y , q u a n t i t y o r v i a b i l i t y were fo u n d . V i t e l l o g e n e s i s was a p p a r e n t l y c o m p l e t e , o r n e a r l y so, by 40 days p r i o r t o spawning and no n e g a t i v e e f f e c t s o f t h e t r e a t m e n t were e v i d e n t . S p r i n g a t e e t al. (1985) f e d rainbow t r o u t e i t h e r f u l l o r h a l f r a t i o n s ( 0 . 7 % o r 0.35% o f body weight per day) f o r a year p r i o r t o spawning. The f i r s t f i s h t o be s t r i p p e d were t h o s e f e d f u l l r a t i o n . T h i s o c c u r r e d a p p r o x i m a t e l y 2-3 weeks b e f o r e t h e h a l f r a t i o n f i s h s t a r t e d t o spawn. W h i l e a l l of t h e f u l l - f e d f i s h spawned, c a . 1 1 % of t h e r e s t r i c t e d f i s h f a i l e d t o mature. F e c u n d i t y was g r e a t e r (by 22%) i n t h e former group but when f i s h o f s i m i l a r s i z e s from b o t h groups were compared, f e c u n d i t i e s were s i m i l a r . The eggs of t h e f u l l - f e d f i s h were l a r g e r i n b o t h d i a m e t e r and w e i g h t and produced l a r g e r f r y . 36 A t r e s i a (egg r e s o r p t i o n ) l e v e l s were c a . 7% i n t h e f u l l r a t i o n group v e r s u s c a . 22% i n t h e h a l f r a t i o n group. There were no d i f f e r e n c e s i n t h e t o t a l l i p i d o r p r o t e i n l e v e l s and t h e amino a c i d and m i n e r a l p r o f i l e s were s i m i l a r i n t h e two groups. F e r t i l i z a t i o n s u c c e s s and s u r v i v a l t o e y e i n g and t o 6 months o f age were not d i f f e r e n t . I n i t i a l d i f f e r e n c e s i n f r y s i z e were o b s c u r e d by v a r i a n c e s i n growth i n t h e two groups a f t e r 4 months. S c o t t (1962) r e p o r t e d t h a t t h e degree of f o l l i c u l a r a t r e s i a i n c r e a s e d w i t h t h e l e v e l o f s t a r v a t i o n i n c u l t u r e d t r o u t . Egg s i z e was not a f f e c t e d by a reduced f e e d i n g regime, but t h e p r o p o r t i o n o f f i s h w i t h i n a p a r t i c u l a r age c l a s s i n a p o p u l a t i o n t h a t matured was redu c e d . S i m i l a r r e s u l t s were o b t a i n e d by Bagenal (1969) w o r k i n g w i t h brown t r o u t . The r e s u l t s o f many o f t h e p r e c e d i n g s t u d i e s a r e c o n f l i c t i n g . A t p r e s e n t t h e r e appears t o be no c l e a r c u t b e n e f i t t o f a s t i n g on t h e r e p r o d u c t i v e p r o c e s s e s o f P a c i f i c salmon. D i f f e r e n c e s between s t u d i e s i n e x p e r i m e n t a l c o n d i t i o n s , p a r t i c u l a r l y i n t h e n u t r i t i o n a l h i s t o r y o f t h e f i s h and i n t h e l e v e l o f f o o d d e p r i v a t i o n , may account f o r t h e d i s s i m i l a r f i n d i n g s . However i f b r o o d f i s h can be m a i n t a i n e d w i t h o u t f e e d i n g f o r some p e r i o d p r i o r t o spawning w i t h no d e t r i m e n t , a s i g n i f i c a n t s a v i n g i n f e e d and l a b o u r c o s t s would a c c r u e t o t h e f a r m e r . I f f e c u n d i t y o r s u r v i v a l can be improved by o p t i m i z i n g f e e d i n g p r o t o c o l b e f o r e spawning, an a d d i t i o n a l advantage i s o b t a i n e d . 37 SECTION 1 - A comparison of the l i p i d composition and f a t t y a c i d p r o f i l e s of the eggs of two stocks of w i l d and c u l t u r e d Chinook salmon. 1.1 MATERIALS AND METHODS 1.1.1 Experimental design and c o n d i t i o n s C u l t u r e d 3-year o l d Chinook salmon were h e l d i n s a l t w a t e r net pens at the broodstock r e a r i n g s i t e of Sea Spring Salmon Farm L t d . i n Genoa Bay, near Duncan on Vancouver I s l a n d . Two sto c k s , B i g Qualicum (BQ) and Robertson Creek (RC), were each f e d two formulated d i e t s (designated as COMM or WV33 and de s c r i b e d l a t e r ) . The experimental design c o n s i s t e d of fou r pens of f i s h , each c o n t a i n i n g one stock/ d i e t combination. T h i s f a c i l i t y i s a commercial o p e r a t i o n and i t was not p o s s i b l e to a s s i g n f i s h randomly t o treatment groups because the broodstock were a l r e a d y i n t h e i r t h i r d year a t the s t a r t of t h i s p r o j e c t . However, subsampled f i s h from each pen were sampled randomly, as d e s c r i b e d l a t e r . No r e p l i c a t i o n of pens was p o s s i b l e . The numbers of f i s h i n the pens v a r i e d w i d e l y . In the two pens f e d the COMM d i e t there were 149 (RC) and 811 (BQ) and i n the pens f e d the WV33 d i e t t h e r e were 189 (BQ) and 283 (RC). Two d i f f e r e n t s i z e s of pens were a l s o used, 680 m3 and 340 m3, the 38 s m a l l e r pens h o l d i n g the f i s h f e d the WV33 d i e t . Pen d e n s i t i e s were. A l l were below 6 kg/ m3. Not a l l of the f i s h i n the pens matured i n 1987. During September of t h e i r t h i r d summer, those f i s h maturing as 3-year o l d s (1987 broodstock) were i d e n t i f i e d and t r a n s p o r t e d t o the hatchery where they were h e l d f o r s e v e r a l weeks f o r f i n a l m aturation, p r i o r t o spawning. F i s h not maturing i n 1987 were h e l d f o r spawning i n 1988. When the f i s h were brought to the hatchery they were tagged with c o n s e c u t i v e l y numbered p l a s t i c s p a g h e t t i tags (Floy Tag & Manufacturing, Inc., S e a t t l e , Wash., USA), i n s e r t e d i n the d o r s a l musculature a n t e r i o r t o the d o r s a l f i n . A l l numbers were recorded and used t o generate the random s e l e c t i o n of f i s h to be sampled f o r eggs. Ten f i s h from each group were randomly sampled. Subsequently f i v e f i s h were randomly s e l e c t e d from these t en f o r complete l i p i d a n a l y s i s of t h e i r eggs. A d e v i a t i o n from t h i s procedure r e s u l t e d when only t h r e e of the Robertson Creek females on the COMM d i e t matured. In t h i s case a l l t h ree were sampled. The f i s h were f e d to s a t i a t i o n twice a day d u r i n g the t h i r d year of t h e i r r e a r i n g p e r i o d a t Genoa Bay and r e c e i v e d c a . 1% of t h e i r body weight per day. During t h e i r f i n a l summer, they cut back n a t u r a l l y t o ca. 0.5% of t h e i r body weight per day. Towards the end of the summer they were t a k i n g even l e s s f e e d . 39 Because t h e pens c o n t a i n e d some f i s h t h a t d i d not mature, i t i s i m p o s s i b l e t o s t a t e more p r e c i s e l y t h e l e v e l o f f e e d i n g i n t h e 1987 b r o o d s t o c k . They were not f e d a t a l l d u r i n g t h e f r e s h w a t e r h o l d i n g p e r i o d ( c a . 2-4 weeks) p r i o r t o spawning. One pen o f each s t o c k was f e d a commercial d i e t (COMM); the o t h e r pen of each s t o c k was f e d t h e open f o r m u l a t i o n West Vancouver 33 d i e t (WV33). The f o r m u l a t i o n f o r t h e commercial d i e t was p r o p r i e t a r y and not a v a i l a b l e , o t h e r t h a n th e d a t a p r o v i d e d on t h e f e e d l a b e l . Feed l a b e l i n f o r m a t i o n f o r t h e commercial d i e t i s g i v e n i n Appendix 2 T a b l e A2.1. The f o r m u l a t i o n o f t h e West Vancouver 33 d i e t i s p r e s e n t e d i n Appendix 2 T a b l e A2.2. Males and f e m a l e s were h e l d s e p a r a t e l y and checked r e g u l a r l y f o r o v u l a t i o n o r t h e s t a r t o f s p e r m i a t i o n . When o v u l a t i o n was e v i d e n t , a female was k i l l e d w i t h a sharp blow t o t h e head and t h e eggs were s t r i p p e d i n t o a c o l l e c t i n g b u c k e t . M i l t was c o l l e c t e d and d i v i d e d among t h e egg b u c k e t s so t h a t each bucket of eggs r e c e i v e d t h e m i l t from two d i f f e r e n t males of t h e same s t o c k . M i l t was t a k e n w i t h o u t k i l l i n g t h e f i s h and an i n d i v i d u a l male c o u l d p o t e n t i a l l y be used t o f e r t i l i z e many f e m a l e s . The eggs of each female were i n c u b a t e d i n s e p a r a t e Heath t r a y s ( F l e x - a - L i t e C o n s o l i d a t e d , Tacoma, Wash., USA). A f t e r e y e i n g and t h e f i r s t egg p i c k , s m a l l l o t s o f eggs were o f t e n combined. 40 Consequently data on the i n c u b a t i o n success of many i n d i v i d u a l s were not a v a i l a b l e beyond the eyed stage. Wild f i s h from each stock r e t u r n i n g t o t h e i r n a t a l streams t o spawn were sampled a t B i g Qualicum Hatchery and Robertson Creek Hatchery, by arrangement with the Department of F i s h e r i e s and Oceans, Government of Canada. When the r e t u r n i n g w i l d f i s h reached the hatchery, they were h e l d i n freshwater raceways and checked r e g u l a r l y f o r m a t u r i t y before spawning. O v u l a t i n g females and spermiating males were both k i l l e d and s t r i p p e d of t h e i r eggs or m i l t . Eggs from s e v e r a l females were combined i n an egg bucket p r i o r t o f e r t i l i z a t i o n by s e v e r a l males. A f t e r f e r t i l i z a t i o n , the egg buckets were emptied i n t o Heath t r a y s . No attempt was made to keep eggs from d i f f e r e n t females separate at these h a t c h e r i e s . I t was t h e r e f o r e i m p o s s i b l e to t r a c k the i n c u b a t i o n success of i n d i v i d u a l w i l d f i s h . Data on the success of the e n t i r e run ( a l l age groups together) were, however, a v a i l a b l e . Female Chinook salmon r e t u r n i n g t o the h a t c h e r i e s are 3 , 4, and 5-years o l d . In order to o b t a i n 3-year o l d f i s h f o r comparison w i t h the c u l t u r e d 3-year o l d s , sampling was c a r r i e d out d u r i n g t h a t p o r t i o n of the run when the peak of 3-year o l d f i s h i s known t o r e t u r n . T h i s was determined i n c o n s u l t a t i o n with the hatchery managers, Mr. Grant LaDocoeur at Big Qualicum and Mr. Don Lawseth at Robertson Creek. In a d d i t i o n o n l y those f i s h f a l l i n g w i t h i n the s i z e range of 3-year o l d f i s h were sampled. 41 S c a l e s were taken c o i n c i d e n t a l l y with egg samples f o r subsequent c o n f i r m a t i o n of age. S c a l e s were aged by Ms Yvonne Yole of the Ageing U n i t of the Department of F i s h e r i e s and Oceans. A f t e r the f i s h were aged, f i v e 3-year o l d f i s h of each stock were randomly s e l e c t e d from a l l 3-year o l d f i s h sampled f o r complete l i p i d a n a l y s i s . 1.1.2 Sample c o l l e c t i o n Approximately 50-60 grams of u n f e r t i l i z e d eggs were taken w i t h i n 10 minutes of s t r i p p i n g . They were d r a i n e d of most of the o v a r i a n f l u i d , p l a c e d i n t o p l a s t i c bags and immediately f r o z e n . A l l samples were re-packaged, without thawing, i n t o oxygen b a r r i e r bags (W. Grace & Co. Canada L t d . , M i s s i s a u g a , Ontario) w i t h i n a week of c o l l e c t i o n f o r storage a t -35°C. Samples were analysed w i t h i n 10 months of c o l l e c t i o n . Fork l e n g t h and weight data were taken from a l l females, c u l t u r e d and w i l d , t h a t were spawned. Approximately 12 hours a f t e r f e r t i l i z a t i o n , t e n eggs were taken from the Heath t r a y of each c u l t u r e d female t h a t had been sampled. These eggs were put i n t o Stockard's c l e a r i n g s o l u t i o n (Velsen, 1980) and l a t e r examined under a d i s s e c t i n g microscope to determine whether f e r t i l i z a t i o n had taken p l a c e . Because 42 t h e eggs of s e v e r a l f e males a t B i g Qualicum and R o b e r t s o n Creek h a t c h e r i e s a r e p o o l e d a t spawning, f e r t i l i z a t i o n s u c c e s s c o u l d not be a s s e s s e d f o r t h e w i l d f i s h . 1.1.3 Egg c o m p o s i t i o n A f l o w c h a r t o u t l i n i n g a l l a n a l y t i c a l p r o c e d u r e s i s p r e s e n t e d i n F i g u r e 3. Dry weight determination Samples of c a . 5-10 grams of f r o z e n eggs were a c c u r a t e l y weighed i n t o each o f t h r e e d r i e d and t a r e d aluminum pans. The pans were d r i e d o v e r n i g h t t o c o n s t a n t weight a t 70° C. The d r y w e i g h t o f t h e eggs was d e t e r m i n e d by s u b t r a c t i n g t h e pan w e i g h t . Dry weight was c a l c u l a t e d as a p e r c e n t o f t h e o r i g i n a l wet w eight of t h e eggs. P e r c e n t m o i s t u r e was c a l c u l a t e d as t h e d i f f e r e n c e between 100% and t h e p e r c e n t d r y w e i g h t . The means of t h e wet and d r y w e i g h t s were used i n c a l c u l a t i n g p e r c e n t l i p i d i n t h e samples. A l l f o r m u l a e used f o r c a l c u l a t i n g c o m p o s i t i o n parameters a r e p r e s e n t e d i n Appendix 3. Lipid extraction L i p i d was e x t r a c t e d from t h e eggs a c c o r d i n g t o t h e p r o c e d u r e o f B l i g h and Dyer (1959) w i t h m o d i f i c a t i o n s by C h r i s t i e (1982). A sample of c a . 20 grams of f r o z e n eggs was a c c u r a t e l y weighed 4 3 f r o z e n egg sample •> d r y m a t t e r & m o i s t u r e d e t e r m i n a t i o n •> l i p i d e x t r a c t i o n ( B l i g h and Dyer, 1959) > TL FAMES — > t o t a l l i p i d d e t e r m i n a t i o n C H C l 3 / n e u t r a l l i p i d s TLC chromatography ( s i l i c a c a r t r i d g e s ! CH 30H7polar 1 i p i d s > PL FAMES — > GLC > TLC p o l a r l i p i d d e t e r m i n a t i o n F i g u r e 3: F l o w c h a r t of p r o c e d u r e s f o r l i p i d a n a l y s i s and f a t t y a c i d d e t e r m i n a t i o n s (see t e x t f o r f u l l d e s c r i p t i o n ) . TL = t o t a l l i p i d s PL = p o l a r l i p i d s FAMES = f a t t y a c i d methyl e s t e r s TLC = t h i n l a y e r chromatography GLC = gas l i q u i d chromatography 4 4 and homogenized f o r 2 minutes with 50 ml of c h l o r o f o r m and 100 ml of methanol. An a d d i t i o n a l 50 ml of c h l o r o f o r m were added and homogenization continued f o r a f u r t h e r 30 seconds. The homogenate was f i l t e r e d under p r e s s u r e through a Buchner apparatus f i t t e d with a Whatman No.1 f i l t e r paper. The contents of the f u n n e l were ret u r n e d to the homogenizer and a second e x t r a c t i o n was c a r r i e d out u s i n g the same procedure. The two f i l t r a t e s were combined and 100 ml of 2.6% KCl s o l u t i o n was added. The s o l u t i o n was poured i n t o a 500 ml graduated c y l i n d e r . The a d d i t i o n of the KCl caused the s e p a r a t i o n of the s o l u t i o n i n t o an aqueous phase and a c h l o r o f o r m phase, the l a t t e r c o n t a i n i n g the e x t r a c t e d l i p i d . A f t e r s e p a r a t i o n was complete, the volume of the c h l o r o f o r m l a y e r was measured and the aqueous l a y e r removed by a s p i r a t i o n . T h i s procedure was repeated on a second 20 gram sample of eggs from each f i s h a n alysed. The amount of l i p i d i n t r i p l i c a t e subsamples of the c h l o r o f o r m l a y e r of each e x t r a c t i o n was determined by p i p e t t i n g 10 ml a l i q u o t s i n t o d r i e d and weighed aluminum pans. The c h l o r o f o r m was evaporated by g e n t l e warming on a hot p l a t e . The pans were then d r i e d o v e r n i g h t a t 70° C. and weighed to a constant weight. The mean amount of d r i e d l i p i d i n 10 ml was determined f o r each e x t r a c t i o n and used to c a l c u l a t e the t o t a l amount of l i p i d i n each sample, based on the volume of the c h l o r o f o r m l a y e r s (Appendix 3). L i p i d i n the o r i g i n a l egg sample was c a l c u l a t e d as a percent of wet and dry weight f o r each 45 e x t r a c t i o n and the means were determined. Separation of polar l i p i d s An a l i q u o t of the c h l o r o f o r m phase, c o n t a i n i n g ca. 200-250 mg of l i p i d was evaporated under reduced pressure on a Rotovapor (Buchi Rotovapor-R, S w i t z e r l a n d ) . The remaining o i l was a p p l i e d t o two Sep Pak s i l i c a c a r t r i d g e s (Waters A s s o c i a t e s , M i l f o r d , Massachusetts) j o i n e d by p l a s t i c t u b i n g and attached to the Luer l o c k of the b a r r e l of a g l a s s s y r i n g e which was mounted v e r t i c a l l y on a r e t o r t stand. A small stream of n i t r o g e n gas was a p p l i e d through a p l a s t i c t u b i n g and rubber cork assembly i n the open end of the s y r i n g e b a r r e l to f o r c e the l i p i d i n t o the s i l i c a c a r t r i d g e s . A f t e r the l i p i d was loaded onto the top of the f i r s t s i l i c a c a r t r i d g e , 60 ml of a s o l u t i o n of 15% hexane i n c h l o r o f o r m was poured i n t o the top of the s y r i n g e , and the cork and t u b i n g was a t t a c h e d a l l o w i n g a s l i g h t flow of n i t r o g e n to f o r c e the s o l v e n t slowly through the c a r t r i d g e s . The e l u e n t contained the non-polar or n e u t r a l l i p i d f r a c t i o n of the sample. A f t e r the n e u t r a l l i p i d was e l u t e d , 60 ml of methanol was poured i n t o the s y r i n g e and the procedure repeated, e l u t i n g the p o l a r l i p i d s from the sample. The s e p a r a t i o n of n e u t r a l and p o l a r l i p i d s was checked 4 6 f r e q u e n t l y by t h i n l a y e r chromatography (TLC) us i n g S i l i c a G P l a t e s (A. G. Merck, Darmstad, W. Germany) and a s o l v e n t system c o n s i s t i n g of hexane-diethy1 e t h e r - f o r m i c a c i d (80:20:2 by volume), ac c o r d i n g t o the methods of C h r i s t i e ( 1 9 8 2 ) . Bands were v i s u a l i z e d with i o d i n e vapor and s e p a r a t i o n was found to be very s a t i s f a c t o r y . The p o l a r l i p i d e l u e n t was made up to 80 ml with methanol and thre e 10 ml a l i q u o t s were p i p e t e d i n t o dry, pre-weighed aluminum pans; evaporated on a warm hot p l a t e ; d r i e d o v e r n i g h t (70° C); and weighed t o a constant weight. The weight of p o l a r l i p i d i n each a l i q u o t was c a l c u l a t e d by s u b t r a c t i n g the weight of the dry aluminum pan. The mean was used to c a l c u l a t e the t o t a l amount of p o l a r l i p i d i n the sample (Appendix 3 ) . The remaining p o l a r l i p i d was used f o r the p r e p a r a t i o n of p o l a r l i p i d f a t t y a c i d methyl e s t e r s . The e n t i r e f i r s t e l u e n t , c o n t a i n i n g the n e u t r a l l i p i d s , was evaporated and d r i e d o v e r n i g h t t o a constant weight. The n e u t r a l p l u s p o l a r l i p i d weight was used as a check on the c a l c u l a t e d amount of l i p i d o r i g i n a l l y a p p l i e d t o the s i l i c a c a r t r i d g e s . N e u t r a l and p o l a r l i p i d c o n c e n t r a t i o n s were c a l c u l a t e d as percentages of the dry weight of the eggs. P o l a r l i p i d c o n c e n t r a t i o n was a l s o c a l c u l a t e d as a percentage of the t o t a l l i p i d and as a percentage of the f a t f r e e dry weight of the 47 eggs. These c a l c u l a t i o n s are presented i n Appendix 3. P o l a r l i p i d d e t erminations were done i n d u p l i c a t e i n i t i a l l y but t h i s became expensive. Since the p r e c i s i o n of these d e t e r m i n a t i o n s was s a t i s f a c t o r y , i t was decided t h a t one d e t e r m i n a t i o n per sample was s u f f i c i e n t . Fatty acid profiles F a t t y a c i d methyl e s t e r s (FAMEs) were prepared f o l l o w i n g the base c a t a l y z e d methyl e s t e r i f i c a t i o n procedure of C h r i s t i e (1982), u s i n g sodium methoxide. An a l i q u o t of the c h l o r o f o r m phase (of the o r i g i n a l e x t r a c t i o n ) , c o n t a i n i n g c a . 50 mg of l i p i d was used to produce t o t a l l i p i d FAMEs. The balance of the p o l a r l i p i d e l u e n t , a f t e r d e t e r m i n a t i o n of percent p o l a r l i p i d , was used to make p o l a r l i p i d FAMEs. FAMEs were s t o r e d i n hexane i n 2 ml septum v i a l s under n i t r o g e n below 0 ° C. u n t i l they c o u l d be analysed by gas l i q u i d chromatography. FAME s o l u t i o n s (1 u l ) were separated i n t o t h e i r component f a t t y a c i d methyl e s t e r s on a V a r i a n 3700 gas l i q u i d chromatograph (GLC) equipped with a flame i o n i z a t i o n d e t e c t o r . The column was a Supelcowax 10 (Supelco Canada L t d . , O a k v i l l e , O ntario) f u s e d s i l i c a c a p i l l a r y column (30 m x 0.32 mm ID, 0.25 ym f i l m ) . Peak areas were i n t e g r a t e d by a CDS microprocessor (V a r i a n Instrument Group, Palo A l t o , CA, USA). 48 The GLC was temperature programmed at 170° C. f o r 10 minutes then i n c r e a s e d 2° C. per minute to 230° C. and h e l d a t 230° C. f o r 20 minutes. The i n j e c t i o n and d e t e c t i o n temperatures were 220° C. The s p l i t r a t i o was 50:1. Helium c a r r i e r gas was p u r i f i e d through a heated furnace (Supelco Canada Ltd.) to remove a l l t r a c e s of oxygen and water. The l i n e a r v e l o c i t y was 22 cm/sec. F a t t y a c i d peaks were compared with those obtained by running PUFA-1 and Rapeseed standards (Supelco Canada Ltd.) and i d e n t i f i e d a c c o r d i n g to Ackman (1982). Peaks r e p r e s e n t i n g l e s s than 0.20 % of the t o t a l area of the chromatogram were not i n c l u d e d i n the p r o f i l e s . U n i d e n t i f i a b l e peaks were assigned a number or number and l e t t e r code. These codes were used c o n s i s t e n t l y f o r any peak o c c u r r i n g i n the same p o s i t i o n i n the chromatograms of a l l samples and are i n c l u d e d i n Appendix 2, Tables A2.4 and A2.5 and Appendix 4, Tables A4.1-A4.8 i n the order t h a t they were e l u t e d . The u n i d e n t i f i a b l e f a t t y a c i d s were g e n e r a l l y present at l e v e l s l e s s than 0.50 %. F a t t y a c i d p r o f i l e s i n the t o t a l and p o l a r l i p i d s were i n i t i a l l y c a r r i e d out i n d u p l i c a t e . However, s i n c e l i t t l e was gained by the d u p l i c a t i o n of e f f o r t , a s i n g l e d e t e r m i n a t i o n of the f a t t y a c i d p r o f i l e was deemed s u f f i c i e n t as the analyses proceeded. / 49 To a v o i d f a t t y a c i d o x i d a t i o n d u r i n g a n a l y s i s , t h e f o l l o w i n g p r e c a u t i o n s were t a k e n : \ > Egg samples were kept i n t h e f r e e z e r u n t i l i m m e d i a t e l y p r i o r t o a n a l y s i s . F r o z e n eggs were used i n t h e e x t r a c t i o n p r o c e d u r e and t h e b ucket of t h e homogenizer was p l a c e d i n an i c e b a t h d u r i n g t h e g r i n d i n g s t e p . > BHT, b u t y l a t e d h y d r o x y - t o l u e n e (50-100 m g / l i t r e ) , was added t o a l l s o l v e n t s used i n t h e e x t r a c t i o n and s e p a r a t i o n p r o c e d u r e s and i n t h e p r e p a r a t i o n o f methyl e s t e r s . S o l v e n t s 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 use. > A f t e r e v a p o r a t i o n o f s o l v e n t s under reduced p r e s s u r e , t h e vacuum was broken w i t h n i t r o g e n e n s u r i n g t h a t exposure of l i p i d t o a i r was m i n i m a l . > N i t r o g e n was used t o f o r c e l i p i d and s o l v e n t s t h r o u g h t h e s i l i c a c a r t r i d g e s . As t h e e l u e n t l e f t t h e c a r t r i d g e s , t h e s o l v e n t c a r r y i n g t h e l i p i d was run down the s i d e o f t h e c o l l e c t i o n f l a s k t o a v o i d s p l a s h i n g w i t h t h e p o s s i b l e i n c o r p o r a t i o n of a i r i n t o t h e sample. 1.1.4 D i e t c o m p o s i t i o n A sample of each of t h e two f o r m u l a t e d d i e t s was t a k e n and s t o r e d i n oxygen b a r r i e r bags, as f o r t h e egg samples. 50 To determine dry weight c a . 5-10 grams of f r o z e n p e l l e t s were a c c u r a t e l y weighed i n t o each of three d r i e d and t a r e d aluminum pans. Because the p e l l e t s were r e l a t i v e l y hard, 5-10 ml of d i s t i l l e d water were added to the pans to break them down. The s l u r r y i n the pans was then d r i e d to a constant weight at 70° C and mean dry weight and moisture i n the p e l l e t s was c a l c u l a t e d . L i p i d was e x t r a c t e d from the d i e t s f o l l o w i n g the same procedure a p p l i e d t o the eggs, with the e x c e p t i o n t h a t i t was necessary to soak the s t i l l f r o z e n p e l l e t s i n the f i r s t s o l v e n t system f o r s e v e r a l minutes to break them down p r i o r t o g r i n d i n g . T o t a l l i p i d and f a t t y a c i d p r o f i l e s i n the t o t a l l i p i d were determined f o l l o w i n g the procedures f o r the eggs. A l l analyses on the d i e t s were c a r r i e d out i n d u p l i c a t e and the means were c a l c u l a t e d . 1.1.5 Egg s i z e Egg s i z e was measured i n two ways. A minimum of ten eggs from the samples preserved i n Stockard's s o l u t i o n (Velsen, 1980), taken to monitor f e r t i l i z a t i o n success, were p l a c e d i n a trough c a l i b r a t e d i n m i l l i m e t e r s . Mean egg diameter was determined from the t o t a l l e n g t h d i v i d e d by the number of eggs measured. Egg volume was measured by displacement of water i n a b u r e t . Again a minimum of ten eggs was measured and the mean volume determined by d i v i d i n g t o t a l volume d i s p l a c e d by the number of eggs. Egg diameter was then c a l c u l a t e d from t h i s mean. Egg d i a m e t e r s d e t e r m i n e d by each method were compared f o r c o n s i s t e n c y but o n l y d i a m e t e r s d e t e r m i n e d by d i r e c t measurement ( f i r s t method) were r e p o r t e d . 1.1.6 F e r t i l i z a t i o n and e y e i n g s u c c e s s F e r t i l i z a t i o n s u c c e s s was m o n i t o r e d by t a k i n g a minimum of t e n eggs from a l l f i s h sampled 12-24 hours a f t e r f e r t i l i z a t i o n . These eggs were p l a c e d i n S t o c k a r d ' s s o l u t i o n ( V e l s e n , 1980) f o r c l e a r i n g . Egg c l e a v a g e , i n d i c a t i n g t h a t f e r t i l i z a t i o n had o c c u r r e d , was e a s i l y o b s e r v e d by eye or under t h e low power, o b j e c t i v e o f a d i s s e c t i n g m i c r o s c o p e . F e c u n d i t y was appr o x i m a t e d a t spawning by e s t i m a t i n g t h e volume of eggs spawned by each f e m a l e . Eggs a r e t o o f r a g i l e a t t h i s t i m e t o count and i t i s not a d v i s a b l e t o d r a i n t h e o v a r i a n f l u i d so t h a t an a c c u r a t e weight can be o b t a i n e d . At e y e i n g t h e eggs a r e much more r o b u s t and a count of t h e l i v e and t h e dead eggs was made f o r each f i s h . The t o t a l o f l i v e p l u s dead eggs e q u a l s t h e number of eggs spawned or t h e f e c u n d i t y . E y e i n g s u c c e s s was c a l c u l a t e d as t h e p e r c e n t a g e o f v i a b l e eggs a t t h e eyed s t a g e d i v i d e d by t h e f e c u n d i t y . 1.1.7 S t a t i s t i c a l p r o c e d u r e s A l l d a t a r e p o r t e d as p e r c e n t a g e s were t r a n s f o r m e d by the a r c s i n e t r a n s f o r m a t i o n b e f o r e a n a l y s e s . Morphometric d a t a 52 ( c o n d i t i o n f a c t o r s , egg d i a m e t e r s , e t c . ) ; i n c u b a t i o n r e s u l t s ( f e r t i l i z a t i o n r a t e s , e y e i n g s u c c e s s , e t c . ) ; c o m p o s i t i o n parameters ( d r y m a t t e r ; m o i s t u r e ; t o t a l l i p i d ; p o l a r l i p i d ; p o l a r l i p i d , f a t f r e e d r y w e i g h t b a s i s , e t c . ) , i n d i v i d u a l f a t t y a c i d s , and f a t t y a c i d s e r i e s ( s a t u r a t e s , n3, n6, n9, e t c . ) were a l l a n a l y s e d f o r d i f f e r e n c e s among t h e t h r e e d i e t s f o r each s t o c k and f o r d i f f e r e n c e s between s t o c k s f o r each d i e t u s i n g t h e g e n e r a l l i n e a r model p r o c e d u r e (PROC GLM) f o r t h e a n a l y s i s o f v a r i a n c e (1x6 ANOVA), recommended f o r unbalanced d a t a s e t s (SAS, 1985). D i f f e r e n c e s between means were de t e r m i n e d by l e a s t square means (LSMEANS) w i t h t h e PDIFF o p t i o n (SAS, 1985) t o i d e n t i f y which means were d i f f e r e n t . The s i g n i f i c a n c e l e v e l was s e t a t 95% f o r a l l t e s t s . 53 1.2 RESULTS AND DISCUSSION 1.2.1 Morphometric measurements Fork l e n g t h and weight data were recorded by the hatchery s t a f f from a l l c u l t u r e d females spawned at Sea Spring Salmon Farm. T h i s i n f o r m a t i o n i s not r o u t i n e l y taken at B i g Qualicum or Robertson Creek H a t c h e r i e s but a l l f i s h sampled f o r eggs at these h a t c h e r i e s d u r i n g t h i s study were weighed and measured. C o n d i t i o n f a c t o r (CF) was c a l c u l a t e d f o r each f i s h a c c o r d i n g t o the formula of Vanstone and Markert (1968): CF = (W x 1000)/ F L 3 * 2 5 . where W - weight (g) and FL = f o r k l e n g t h (cm) A l l morphometric data are presented i n Table 1. U n f o r t u n a t e l y o n l y 5 of the 10 f i s h sampled a t B i g Qualicum were 3 years o l d as i n d i c a t e d by s c a l e a n a l y s i s and onl y t h r e e females i n the RC-COMM group matured. An ANOVA was c a r r i e d out on a l l three d i e t groups of the BQ stock, i n c l u d i n g BQ-WILD. Because the sample s i z e was so low, the r e s u l t s of t h i s t«|st r i s k e d a Type 1 e r r o r i n the case of the BQ-WILD f i s h . For the RC groups an ANOVA was performed e x c l u d i n g the RC-COMM f i s h as the power of t h i s t e s t would have been very low ( i e the p o s s i b i l i t y of a Type 2 e r r o r would have been h i g h ) . The w i l d f i s h of both stocks were s i g n i f i c a n t l y longer (and 54 i Table 1. Morphometric data (mean and standard e r r o r of the means) f o r w i l d and c u l t u r e d B i g Qualicum (BQ) and Robertson Creek (RC) female Chinook broodstock. D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t BQ BQ BQ WILD WV33 COMM mean sem (n) mean sem (n) mean sem (n) Fork l e n g t h , cm 77. 8- 0.903 (5) 70.1fa«0.678 (22) 6 9 . 8 b 0.447 (46) Weight, kg 5.1" 0.258 (5) 5.1B 0.179 (22) 5.0 0.124 (46) C o n d i t i o n f a c t o r 3.7- 0.126 (5) 5.1 b 0.097 (22) 5.1 b 0.134 (46) RC WILD RC WV33 RC" COMM mean sem (n) mean sem (n) mean sem (n) Fork l e n g t h , cm 8 0 . 3 a 0.547 (12) 67.2 b"0.838 (17) 70.7" 1.764 (3) Weight, kg 6.3- •0.124 (12) 4.1 b«0.166 (17) 4 . 8 " 0.252 (3) C o n d i t i o n f a c t o r 4.1- 0.067 (12) 4.8 b 0.167 (17) 4 . 7 " 0.152 (3) * = The RC-COMM f i s h were excluded from the ANOVA as the sample s i z e was inadequate. See t e x t f o r d i s c u s s i o n . ab = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between stocks f o r the parameter i n d i c a t e d . 55 h e a v i e r i n the case of the RC f i s h ) than the BQ c u l t u r e d groups or the RC-WV33 group. However the mean c o n d i t i o n f a c t o r was s i g n i f i c a n t l y lower f o r the BQ-WILD f i s h than f o r the BQ c u l t u r e d f i s h . C o n d i t i o n f a c t o r was a l s o s i g n i f i c a n t l y lower i n the RC-WILD f i s h than i n the RC-WV33 f i s h . For s i m p l i c i t 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 a parameter between  the stocks f o r each d i e t group have been i n d i c a t e d by '•' i n Table 1 and i n any of the t a b l e s t h a t f o l l o w . The RC-WILD f i s h were s i g n i f i c a n t l y h e a v i e r than the BQ-WILD f i s h but the r e was no d i f f e r e n c e i n len g t h or c o n d i t i o n f a c t o r . By c o n t r a s t , the RC-WV33 f i s h were s m a l l e r i n both length and weight than the BQ-WV33 f i s h . 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 length or weight between stocks on the COMM d i e t . 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 c o n d i t i o n f a c t o r s between the stocks f o r each d i e t group. 1.2.2 Spawning and i n c u b a t i o n success The volume of eggs s t r i p p e d from each female was measured a t spawning but f e c u n d i t y c o u l d not be determined u n t i l the eyed stage as eggs are too f r a g i l e to count u n t i l then (Table 2). Eggs from one or more females were combined at spawning i f th e r e were too few eggs t o f i l l an i n c u b a t i o n t r a y or a t eyeing i f the number of s u r v i v i n g eggs was low. As a r e s u l t , i n d i v i d u a l f e c u n d i t y data were not a v a i l a b l e f o r a l l the f i s h 56 Table 2. Spawning data (mean and standard e r r o r of the means) f o r w i l d and c u l t u r e d B i g Qualicum (BQ) and Robertson Creek (RC) Chinook broodstock. D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD - n a t u r a l d i e t BQ WILD BQ WV33 BQ COMM mean sem (n) mean sem (n) mean sem (n) Volume o f eggs spawned, ml no d a t a " 597.6" 44.8 (21) 605.4 23.9 (46) F e c u n d i t y (# eggs spawned) no d a t a " 3019.9" 249.2 (12) 3204.0 92.9 (40) Egg d i a m e t e r , mm 7.1-«0.271 (5) 7.7* 0.044 (9) 7.9 b 0.038 (17) ab = a s i g n i f i c a n t w i t h d i f f e r e n t d i f f e r e n c e (a : s u p e r s c r i p t s =0.05) was found w i t h i n t h e same between means row. RC WILD RC WV33 RC COMM mean sem (n) mean sem (n) mean sem (n) Volume o f eggs spawned, ml** no d a t a " 311. 5" 39.7 (13) 666.7 44.1 (3) F e c u n d i t y " * (# eggs spawned) no d a t a " 1595.6" 413.6 (5) 2527.5 294.5 (2) Egg d i a m e t e r , mm*" 7.7" 0.061 (12) 7.7 0.125 (15) 8.3 0.133 (3) * = These data are not recorded at the government h a t c h e r i e s ** = ANOVA was not conducted f o r the RC f i s h as t h e r e was no data f o r the WILD f i s h and sample s i z e was inadequate f o r the COMM f i s h . " = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between stocks f o r the parameter i n d i c a t e d . 57 t h a t were spawned. T h i s e x p l a i n s why the number of ob s e r v a t i o n s (n) was lower f o r f e c u n d i t y compared with the n values r e p o r t e d f o r the volume of eggs spawned (Table 2 ) . Combined egg l o t s , r e p r e s e n t i n g the f i s h with the lowest f e c u n d i t i e s , were excluded from the c a l c u l a t i o n of the means. Consequently the f i g u r e s given f o r mean f e c u n d i t y i n Table 2 are o v e r - e s t i m a t e s , p a r t i c u l a r l y i n the case of the BQ and RC f i s h f e d the WV33 d i e t , where combining eggs from more than one female was most p r e v a l e n t (see a l s o Table 3 ) . No f e c u n d i t y or i n c u b a t i o n data f o r i n d i v i d u a l f i s h are r o u t i n e l y c o l l e c t e d at Big Qualicum or Robertson Creek H a t c h e r i e s . And because there were i n s u f f i c i e n t data, ANOVA co u l d not be conducted on the RC f i s h . No s i g n i f i c a n t d i f f e r e n c e was found i n f e c u n d i t y , whether determined by volume or by count, between the BQ-COMM and the BQ-WV33 f i s h (Table 2 ) . An ANOVA c o u l d not be performed on the RC f i s h because the sample s i z e f o r the RC-COMM group was too small and there were no f e c u n d i t y data f o r the w i l d f i s h . However an ANOVA between stocks f o r the WV33 d i e t r e v e a l e d t h a t the BQ-WV33 f i s h were s i g n i f i c a n t l y more fecund than the RC-WV33 group. These f i g u r e s i n Table 2 do not i n c l u d e the eggs from the combined egg l o t s . As shown i n Table 3, the i n c i d e n c e of 58 Table 3. Egg r e t e n t i o n and other r e p r o d u c t i v e a b n o r m a l i t i e s observed i n Big Qualicum (BQ) and Robertson Creek (RC) c u l t u r e d females on two d i e t s , expressed as a percentage of a l l spawners i n each stock and d i e t group. A c t u a l numbers are re p o r t e d i n parentheses. D i e t codes: COMM = commercial; WV33 = West Vancouver 33 S t o c k / D i e t BQ-WV33 RC-WV33 BQ-COMM RC-COMM Number of spawners 22 17 46 3 % (#) % (#) % (#) % (#) L e v e l of retentions-low moderate high 31.8 4.5 4.5 (7) (1) (1) 17 29 29 .6 .4 .4 (3) (5) (5) 6.5 0.0 0.0 (3) (0) (0) 0. 0. 0. 0 0 0 (0) (0) (0) Spawners not r i p e 9.1 (2) 23 .5 (4) 0.0 (0) 0. 0 (0) Spawners with only one r i p e ovary 4.5 (1) 11 .8 (2) 0.0 (0) 0. 0 (0) Spawners with g l a s s y eggs 0.0 (0) 47 .1 (8) 0.0 (0) 0. 0 (0) Spawners with no s u r v i v o r s a t eyeing 18.2 (4) 41 .2 (7) 2.2 (1) 33. 3 (1) Spawners with eyed s u r v i v o r s 68.2 (15) 35 .3 (6) 97.3 (45) 66. 7 (2) 1 L e v e l of egg r e t e n t i o n (unripe eggs; not o v u l a t e d ) : low = < a count of 400 eggs; moderate = > a count of 400 eggs or < a weight of 400 grams; high = > a weight of 400 grams. 59 r e p r o d u c t i v e a b n o r m a l i t i e s was high, p a r t i c u l a r l y among the WV33 groups of both s t o c k s . Many females f e d t h i s d i e t had unripe eggs r e t a i n e d on the skei n s at spawning. U n f o r t u n a t e l y only rough estimates were made of t h i s egg r e t e n t i o n but i t was c l e a r t h a t f e c u n d i t i e s were reduced and t h a t the RC-WV33 f i s h were the most s e v e r e l y a f f e c t e d . In s e v e r a l f i s h only one ovary (the l e f t ) o v u l a t e d , the second remained i n t a c t or n e a r l y so and not r i p e . Some f i s h d i d not mature a t a l l . G lassy, a p p a r e n t l y water-hardened, eggs were frequent i n the RC-WV33 group. Some r e t a i n e d eggs were enlarged to double or t r i p l e normal s i z e and were s o f t and f l a c c i d , o f t e n p a l e i n c o l o u r ( F i g u r e 4, photographs). Egg diameter data are a l s o given i n Table 2. The eggs of the BQ c u l t u r e d f i s h were s i g n i f i c a n t l y l a r g e r than those of the BQ-WILD f i s h . I n t e r e s t i n g l y the RC-WILD f i s h had s i g n i f i c a n t l y l a r g e r eggs than the BQ-WILD f i s h . T h i s may r e f l e c t the lower f e c u n d i t y found i n the RC f i s h as an i n v e r s e r e l a t i o n s h i p between egg s i z e and f e c u n d i t y has been r e p o r t e d ( B l a x t e r , 1969; Roy and Higgs, 1987). T h i s c o u l d not be t e s t e d f o r the w i l d f i s h as no f e c u n d i t y data were a v a i l a b l e but c o r r e l a t i o n c o e f f i c i e n t s between egg diameter and f e c u n d i t y were c a l c u l a t e d f o r BQ-COMM and BQ-WV33. They were -0.1335 (n=15) and -0.2550 (n = 6) r e s p e c t i v e l y but were not s i g n i f i c a n t a t <x = 0.05 (Zar, 1984). C o r r e l a t i o n c o e f f i c i e n t s were not determined f o r the RC-COMM and RC-WV33 groups because f e c u n d i t y data were a v a i l a b l e f o r only 2 and 4 f i s h , r e s p e c t i v e l y . 60 Figu r e 4: Egg a b n o r m a l i t i e s i n Robertson Creek females on the WV33 d i e t . Top: Female with 660 grams of r e t a i n e d eggs. Bottom: Abnormal eggs are pale i n c o l o u r , f l a c c i d and enlarged 2-3 times normal. 61 No s i g n i f i c a n t c o r r e l a t i o n s (a=0.05) between f e c u n d i t y and f o r k l e n g t h , weight or c o n d i t i o n f a c t o r were found i n the BQ-COMM and BQ-WV33 groups as has been r e p o r t e d by B l a x t e r (1969). T h i s may have been because these f i s h e x h i b i t e d v a r y i n g degrees of egg r e t e n t i o n as d i s c u s s e d above. No s i g n i f i c a n t d i f f e r e n c e was found i n f e r t i l i t y between the BQ-COMM and BQ-WV33 groups (Table 4 ) . In the RC-WV33 f i s h , o n l y 40% of the eggs from ten f i s h were f e r t i l e . T h i s together with low f e c u n d i t y (Table 2) and o b s e r v a t i o n s of spawning a b n o r m a l i t i e s (Table 3) suggest t h a t poor performance was due to poor egg q u a l i t y i n the WV33 d i e t groups and p a r t i c u l a r l y i n the RC-WV33 f i s h . F e r t i l i t y was a l s o low i n the RC-COMM f i s h (69% of the eggs of 3 f i s h were f e r t i l e ) but the sample s i z e was too low to assess any d i f f e r e n c e between the RC-COMM and the RC-WV33 f i s h . F e r t i l i t y was s i g n i f i c a n t l y g r e a t e r i n the BQ-WV33 than the RC-WV33 eggs and appeared (not t e s t e d ) t o be g r e a t e r i n the BQ-COMM than i n the RC-COMM eggs. 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 the number of eyed eggs or i n eyeing success (Table 4) between the BQ-COMM and BQ-WV33 eggs. Poor s u r v i v a l r a t e s t o the eyed stage occ u r r e d i n both of the WV33 groups (Table 3 ) , eg. 7 out of 17 RC-WV33 spawners and 4 out of 18 BQ-WV33 spawners had no s u r v i v i n g eggs a t e y e ing. As expected, the RC-WV33 group produced 62 Table 4. Incubation data (mean and standard e r r o r of the means) f o r w i l d and c u l t u r e d B i g Qualicum (BQ) and Robertson Creek (RC) Chinook broodstock. D i e t codes: COMM = -Vancouver 33; WILD commercial; WV33 = n a t u r a l d i e t = West BQ WILD BQ WV33 BQ COMM mean sem (n) mean sem (n) mean sem (n) F e r t i l i t y , % no data" 83.6 s" 7.20 (8) 97.8- 2.20 (7) Eyed eggs, # no data" 1940.2-" 353.4 (16) 2406.3 - 168.5 (44) Eyed, % 90.87" 62.4-"" 9.88 (16) 73.9-" " 4.46 (44) ab = a s i g n i f i c a n t d i f f e r e n c e (a with d i f f e r e n t s u p e r s c r i p t s =0.05) was found w i t h i n the same between means row. RC WILD RC WV33 RC COMM mean sem (n) mean sem (n) mean sem (n) F e r t i l i t y , %""" no data" 40.1" 10.7 (10) 68.9 26.3 (3) Eyed eggs, #""" no data" 706.9" 272.1 2392.5 (8) 280.5 (2) Eyed, % 90.26" 36.3"" 14.39 94.7"" (8) 0.070 (2) * = F e r t i l i t y data not a v a i l a b l e as the eggs of s e v e r a l f i s h are combined at spawning. Eyed f i g u r e s f o r these f i s h are from the r e s p e c t i v e hatchery records and were not i n c l u d e d i n the ANOVA. ** = Data f o r i n d i v i d u a l f i s h were not a v a i l a b l e a f t e r eyeing as small egg l o t s were combined. *** = ANOVA was not conducted f o r the RC f i s h as the r e was no data f o r the WILD f i s h and sample s i z e was inadequate f o r the COMM f i s h . • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  stocks f o r the parameter i n d i c a t e d . 63 s i g n i f i c a n t l y fewer eyed eggs than the BQ-WV33 f i s h although there was no s i g n i f i c a n t difference i n the eyed percentage between these two groups. The figures for the RC-COMM f i s h i n Table 4 are for 3 f i s h only (only 3 females matured in th i s group) and should not be regarded as an accurate assessment of the success of these f i s h . At the time of eyeing, dead eggs were removed from the trays and more groups of surviving eggs were combined to keep the egg trays f u l l . Data for individual f i s h were therefore l o s t beyond the eyed stage. This meant that survival to ponding could not be estimated accurately, even on a group basis. Beyond eyeing, however, mortality among a l l groups was low (D. Groves, pers. comm.). r The values for survival to eyeing for the BQ-WILD and RC-WILD f i s h i n Table 4 are for a l l age groups spawned throughout the entire run. Three year old f i s h are generally somewhat less successful than four year olds and the majority of spawners at Big Qualicum and Robertson Creek Hatcheries are four year olds (G. Ladocoeur and D. Lawseth, pers. comm. and Blaxter, 1969). 1.2.3 Composition of the eggs Composition data for the eggs of BQ and RC cultured and wild broodstock are presented i n Tables 5a & 5b. The eggs of a l l groups of both stocks, except RC-WV33, contained ca. 40% dry 64 Table 5a. Composition of the eggs (mean and standard e r r o r of the means) of BIG QUALICUM (BQ) broodstock on three d i e t s (n=5 f o r each group). D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t BQ BQ BQ WILD WV3 3 COMM mean sem mean sem mean sem Dry matter % 39 .47 0 .75 39 .64" 0 .24 39. 32 0 .49 Moisture % 60 .54 0 .75 60 .38* 0 .25 60. 68 0 .49 L i p i d , % ww" 12 .19 0 .33 11 .82 0 .17 12. 33 0 .20 L i p i d , % dw" 30 .89 0 .62 29 .81 0 .44 31. 37 0 .35 P o l a r l i p i d , % dw 12 .78 0 .17 12 .84 0 .27 13. 24 0 .30 Ne u t r a l l i p i d , % dw 18 .83 0 .56 17 .51 0 .56 18. 95 0 .43 P o l a r l i p i d , % of t o t a l 1 i p i d 41 .33 0 .40 43 .14 0 .41 42. 18 0 .59 Ne u t r a l l i p i d , % of t o t a l 1 i p i d 61 .41" 0 .66 58 .79 0 .58 60. 38« 0 .85 P o l a r l i p i d , % of f a t f r e e dw 19 .35 0 .55 18 .58 0 .45 19. 75 0 .51 P r o t e i n + ash, % WW 27 .28 0 .59 27 .80" 0 .27 26. 99 0 .37 *ww = wet weight; dw = dry weight ab = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 5b. 65 Table 5b: Composition of the eggs (mean and standard e r r o r of the means) of ROBERTSON CREEK (RC) broodstock on t h r e e d i e t s (n=5 f o r each group except RC-COMM where n=3). D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t RC RC RC WILD WV33 COMM mean sem mean sem mean sem Dry m a t t e r % 40 .27- 0. 26 35 .89° • 1 .70 39 .59- 0 .97 M o i s t u r e % 59 .74- 0. 26 64 .11° • 1 .70 60 .41- 0 .97 L i p i d , % ww* 11 .98 0. 13 11 .54 0 .32 12 .47 0 .11 L i p i d , % dw" 29 .75- 0. 30 32 .32° 0 .83 31 .53- 0 .69 P o l a r l i p i d , % dw 12 .75 0. 22 14 .16 0 .55 13 .91 0 .34 N e u t r a l l i p i d , % dw 17 .11 0. 35 19 .05 0 .57 18 .00 0 .78 P o l a r l i p i d , % of t o t a l 1 i p i d 42 .88 0. 86 43 .76 0 .77 44 .11 0 .70 N e u t r a l l i p i d , % of t o t a l l i p i d 57 .50" 0. 93 58 .19 0 .63 57 • 06« 1 .73 P o l a r l i p i d , % of f a t f r e e dw 18 .37 0. 42 20 .86 1 .08 20 .06 0 .81 P r o t e i n + ash. % WW 28 .01 0. 33 24 .36" 1 .42 27 .12 0 .92 *ww = wet we i g h t ; dw = d r y weight ab = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e same row. • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  s t o c k s f o r t h e parameter i n d i c a t e d . See Ta b l e 5a. 66 matter and (by s u b t r a c t i o n ) 60% moisture. The c o n c e n t r a t i o n of dry matter was s i g n i f i c a n t l y lower i n the eggs of the RC-WV33 broodstock than i n e i t h e r of the other RC groups or i n the eggs of the BQ-WV33 broodstock. Sinnhuber (1969) has r e p o r t e d an i n c r e a s e i n water content i n muscle of salmonids f e d 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 t d i e t s . However EFA d e f i c i e n c y seems not to have been a f a c t o r i n the highe r water l e v e l s i n the RC-WV33 eggs. There was no s i g n i f i c a n t d i f f e r e n c e i n n3 f a t t y a c i d s of the t o t a l l i p i d s between the RC-WV33 and RC-COMM eggs (Table 6b) and the n3 f a t t y a c i d c o n c e n t r a t i o n of the p o l a r l i p i d s was s i g n i f i c a n t l y g r e a t e r i n the RC-WV33 eggs than i n the RC-COMM eggs. As shown i n Table 3, the RC-WV33 group had a high l e v e l of g l a s s y , a p p a r e n t l y water hard eggs. Fecundity (Table 2 ) , f e r t i l i t y and s u r v i v a l r a t e to eyeing (Table 4) were a l l lower i n t h i s group than i n RC-COMM, although the sample s i z e s i n v o l v e d i n these groups were too low to assess s t a t i s t i c a l s i g n i f i c a n c e . The moisture v a l u e s f o r the groups (other than RC-WV33) are int e r m e d i a t e between those given f o r c u l t u r e d eggs (62.0 -66.1%) and w i l d eggs (57.7%) by Groves (1987, unpub. rep o r t ) and presented i n Appendix 1. The d i f f e r e n c e i n moisture content i n the eggs of the w i l d f i s h between the pre v i o u s study and t h i s study may have been due to stock e f f e c t s . The 67 p r e v i o u s study i n v o l v e d w i l d f i s h from the Chemainus R i v e r . The dry matter i n a l l groups was ca. 30% l i p i d of which c a. 41-44% was p o l a r l i p i d and ca. 57-61% was n e u t r a l l i p i d . On a f a t f r e e dry weight b a s i s , c a . 18-21% of the dry matter was p o l a r l i p i d . 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 d i e t groups of e i t h e r stock i n these parameters. However, there were s i g n i f i c a n t d i f f e r e n c e s i n n e u t r a l l i p i d c o n c e n t r a t i o n between stocks on the same d i e t , i e . the BQ-WILD and BQ-COMM eggs had s i g n i f i c a n t l y g r e a t e r n e u t r a l l i p i d l e v e l s and, although not s t a t i s t i c a l l y g r e a t e r , BQ-WV33 eggs a l s o c o n t a i n e d more n e u t r a l l i p i d than the RC-WV33 eggs. Groves (1987, unpub. re p o r t ) found t h a t l i p i d v a l u e s (wet weight b a s i s ) ranged from 13.5-19.0% i n c u l t u r e d eggs and 6.0-13.2% i n w i l d eggs (Appendix 1) but i n t h i s study they ranged from 11.5-12.5% a c r o s s a l l groups, c u l t u r e d and w i l d (Tables 5a & 5b). The higher l i p i d l e v e l s found i n both the muscle and the eggs of c u l t u r e d f i s h compared to the w i l d f i s h as rep o r t e d by Groves (1987, unpub. r e p o r t ) coupled with the b e t t e r x r e p r o d u c t i v e performance of the w i l d f i s h l e a d to s p e c u l a t i o n t h a t ' f a t f i s h do not tend to be p r o d u c t i v e broodstock'. The lower c o n d i t i o n f a c t o r s i n the w i l d f i s h and g r e a t e r egg diameters i n the c u l t u r e d f i s h , r e p o r t e d here, lend some support to t h i s s uggestion. However i n t h i s study, 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 among any of the BQ groups, c u l t u r e d ( or w i l d , i n t o t a l egg l i p i d l e v e l s (Table 5a). The 68 s i g n i f i c a n t l y higher l i p i d l e v e l (dw) determined f o r the RC-WV33 eggs (Table 5b) appeared t o be a r e f l e c t i o n of the high moisture l e v e l s found i n these eggs. The RC-WILD and RC-COMM l i p i d l e v e l s were s t a t i s t i c a l l y no d i f f e r e n t than those of the BQ eggs. The data of Tables 5a and 5b are presented as percentages. Since the l e v e l of carbohydrate i s l i k e l y to be very low i n these eggs, % p r o t e i n + % ash (composite value) can be c a l c u l a t e d as: 100 % - [% moisture + % l i p i d , wet wt] C o i n c i d e n t with the s i g n i f i c a n t l y higher moisture l e v e l i n the RC-WV33 eggs was a s i g n i f i c a n t l y lower % p r o t e i n + % ash v a l u e . There were no other d i f f e r e n c e s i n the p r o t e i n + ash c o n c e n t r a t i o n between d i e t groups of e i t h e r stock nor between the two stocks f o r each d i e t with the ex c e p t i o n t h a t RC-WV33 was s i g n i f i c a n t l y lower than BQ-WV33. 1.2.4 F a t t y a c i d composition of the t o t a l l i p i d s The complete f a t t y a c i d p r o f i l e s of the t o t a l l i p i d s i n the eggs f o r each stock by d i e t are presented i n Appendix 4, Tables A4.1 and A4.2. Data f o r s e l e c t e d f a t t y a c i d s and assemblages of f a t t y a c i d s are i n c l u d e d here. 69 Because the b i o s y n t h e s i s of po l y u n s a t u r a t e d f a t t y a c i d s i n f i s h proceeds w i t h i n the n 3 , n6 and n9 omega s e r i e s ( F i g u r e 2 ) , the f a t t y a c i d s were grouped a c c o r d i n g l y and s t a t i s t i c a l l y analysed (Tables 6 - 8 ) . As shown i n Tables 6a & 6b , the w i l d f i s h of both stocks had s i g n i f i c a n t l y higher l e v e l s of n3 f a t t y a c i d s i n the t o t a l l i p i d s of t h e i r eggs than the c u l t u r e d f i s h , the d i f f e r e n c e between the w i l d and c u l t u r e d groups being c a. 7-13% i n the BQ eggs and 11-13% i n the RC eggs. The w i l d eggs a l s o had s i g n i f i c a n t l y higher l e v e l s of s a t u r a t e s . The n6 f a t t y a c i d s were s i g n i f i c a n t l y lower (by 4-6%) i n the w i l d eggs than i n the c u l t u r e d eggs. The n9 f a t t y a c i d s were a l s o s i g n i f i c a n t l y lower (by 6-9%) i n the w i l d than i n the c u l t u r e d eggs. The BQ eggs from the c u l t u r e d f i s h on two d i e t s were s i g n i f i c a n t l y d i f f e r e n t i n n3, n6 and n9 s e r i e s of f a t t y a c i d s , with more n6 and n9 f a t t y a c i d s i n the BQ-COMM group and more n3 i n the BQ-WV33 group. There were no s i g n i f i c a n t d i f f e r e n c e s i n s a t u r a t e s , n3, n6, or n9 f a t t y a c i d s e r i e s between the two d i e t s i n the c u l t u r e d RC eggs. The BQ-COMM eggs had a s t a t i s t i c a l l y higher c o n c e n t r a t i o n of n6 f a t t y a c i d s than the RC-COMM eggs. There were no other s i g n i f i c a n t d i f f e r e n c e s i n these f a t t y a c i d s e r i e s between s t o c k s . The r a t i o of n3 to n6 f a t t y a c i d s i n muscle l i p i d s has f r e q u e n t l y been used as an i n d i c a t i o n of the e s s e n t i a l f a t t y 70 Table 6a. Saturated, n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from BIG QUALICUM (BQ) broodstock on three d i e t s (n = 5 f i s h f o r each group). D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . BQ BQ BQ WILD WV33 COMM mean sem mean sem mean sem S- 9- 8-^ -6 -6 S a t u r a t e s 19.40- 0.38 18.00° 0.23 18.66° 0.21 n3 39.95- 0.87 32.30° 0.59 27.22° 0.35 n6 2.34- 0.07 6.61° 0.06 8.47°«0.22 n9 21.71- 0.84 28.01° 0.75 30.33° 0.36 abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (<x = 0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 6b. BQ BQ BQ WILD WV33 COMM mean sem mean sem mean sem n3:n6 r a t i o " 17.07 0.58 4.89 0.07 3.21 0.12 * ANOVA was not conducted on the n3:n6 r a t i o s . 71 Table 6b. Saturated, n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from ROBERTSON CREEK (RC) broodstock on thr e e d i e t s (n = 5 f i s h f o r each group except COMM where n=3). D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . RC RC RC WILD WV33 COMM mean sem mean sem mean sem % % % S a t u r a t e s 19.95- 0.32 18.69° 0.24 18.21° 0.01 n3 41.56- 0.59 30.38° 0.47 28.82° 0.18 n6 2.19- 0.13 6.97° 0.07 8.12°«0.14 n9 21.88- 0.77 29.04° 0.43 29.87° 0.28 ab = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 6a. RC WILD n3:n6 r a t i o ' mean sem 18.98 0.95 RC WV33 RC COMM mean sem 4.36 0.07 mean sem 3.55 0.07 * ANOVA was not conducted on the n3:n6 r a t i o s . 72 a c i d s t a t u s of salmonids (eg. C a s t e l l , 1979). Gruger et al. (1964, c i t e d i n C a s t e l l , 1979) r e p o r t e d an n3:n6 r a t i o of 6.69 i n the muscle of w i l d Chinook salmon i n seawater. The d i f f e r e n c e i n the n3:n6 r a t i o s between Chinook muscle and Chinook eggs i s s t r i k i n g . The n3:n6 r a t i o i n the t o t a l l i p i d s of the eggs of the w i l d f i s h was found t o be ca. 2.6 to 2.8 times g r e a t e r than Gruger's muscle r a t i o , i e . 17.07 i n BQ eggs and 18.98 i n RC eggs (Table 6a & 6 b ) . In c o n t r a s t , the n3:n6 r a t i o i n the t o t a l l i p i d s of the eggs of a l l c u l t u r e d f i s h (both stocks) was ca. 27 - 52% lower than the l e v e l i n the muscle of w i l d f i s h . The n3:n6 r a t i o i n the BQ eggs was ca. 3 to 5 times g r e a t e r i n the w i l d f i s h than i n the c u l t u r e d f i s h (Table 6 a ) , eg. 17.07 f o r BQ-WILD eggs but only 3.21 f o r BQ-COMM and 4.89 f o r BQ-WV33 eggs. S i m i l a r l y , i t was 18.98 f o r the RC-WILD eggs but onl y 3.55 and 4.36 f o r RC-COMM and RC-WV33 eggs r e s p e c t i v e l y (Table 6b) . The n3 s e r i e s of f a t t y a c i d s i n the t o t a l l i p i d s has been f u r t h e r broken down i n t o n3 PUFAs (n3 f a t t y a c i d s with 2 to 4 double bonds), HUFAs (n3 f a t t y a c i d s with 5 or more double bonds) and the thr e e f a t t y a c i d s , 20:5n3, 22:5n3 and 22:6n3 i n Tables 7a & 7b. Over a l l groups i n both s t o c k s , the n3 PUFA present i n the eggs was r e l a t i v e l y low at 2-3.5% of a l l f a t t y a c i d s i n the t o t a l 73 Table 7a. S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs (poly u n s a t u r a t e d f a t t y a c i d s ) and t o t a l n3 HUFAs ( h i g h l y unsaturated f a t t y a c i d s ) (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from BIG QUALICUM (BQ) broodstock on th r e e d i e t s (n = 5 f i s h f o r each group). D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . BQ BQ BQ WILD WV33 COMM mean sem mean sem mean sem % % % 20:5n3 13.70-"0.36 8.88° 0.16 7.45° 0.14 22:5n3 5.49--0.23 3.47° 0.08 2.74°»0.15 22:6n3 17.09- 0.74 17.73- 0.39 14.87° 0.45 T o t a l n3 PUFAs 1 2.92- 0.11 1.91° 0.05 1.91° 0.07 T o t a l n3 HUFAs 2 36.28- 0.73 30.08°«0.56 25.06°"0.38 1 n3 PUFAs = unsaturated n3 f a t t y a c i d s with 2 t o 4 double bonds 2 n3 HUFAs = unsaturated n3 f a t t y a c i d s with 5 or more double bonds abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 7b. 74 Table 7b. S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs (poly u n s a t u r a t e d f a t t y a c i d s ) and t o t a l n3 HUFAs ( h i g h l y unsaturated f a t t y a c i d s ) (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from ROBERTSON CREEK (RC) broodstock on thr e e d i e t s (n = 5 f i s h f o r each group except COMM where n = 3) . D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . RC RC RC WILD WV33 COMM mean sem mean sem mean sem % % % 20:5n3 15.06-B0.46 8.36 b 0.24 7.88 b 0.09 22:5n3 4.81-B0.20 3.10 b 0.07 3.28 b"0.25 22:6n3 17.81- 0.51 16.70- b0.25 15.63 b 0.12 T o t a l n3 PUFAs3- 3.50- 0.42 1.89 b 0.09 1.81 b 0.07 T o t a l n3 HUFAs 2 37.68- 0.30 28.17 bn0.38 26.79 b«0.26 1 n3 PUFAs = unsaturated n3 f a t t y a c i d s with 2 to 4 double bonds 2 n3 HUFAs = unsaturated n3 f a t t y a c i d s with 5 or more double bonds abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. B = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 7a. r 75 l i p i d , compared with the t o t a l HUFA which represented 25-38% of the f a t t y a c i d s i n the t o t a l l i p i d . The l e v e l s of n3 PUFAs and HUFAs were s i g n i f i c a n t l y higher i n the w i l d eggs than i n the c u l t u r e d eggs, r e g a r d l e s s of stock. The BQ-WV33 eggs had a s i g n i f i c a n t l y higher c o n c e n t r a t i o n of HUFA than the BQ-COMM. There were no other s i g n i f i c a n t d i f f e r e n c e s i n n3 PUFAs or HUFAs between the formulated d i e t groups of each stock. T e s t i n g between the stocks r e v e a l e d s i g n i f i c a n t l y more HUFAs i n the BQ-WV33 eggs than i n the RC-WV33 eggs and s i g n i f i c a n t l y more HUFAs i n the RC-COMM eggs than i n the BQ-COMM eggs. The 20:5n3 content of the t o t a l l i p i d s was s i g n i f i c a n t l y higher (almost double) i n the w i l d groups than i n the c u l t u r e d groups of e i t h e r stock and the RC-WILD eggs co n t a i n e d s i g n i f i c a n t l y more 20:5n3 than the BQ-WILD eggs. The w i l d eggs of both stocks a l s o c o n t a i n e d s i g n i f i c a n t l y more 22:5n3 than the c u l t u r e d eggs. The 22:6n3 c o n c e n t r a t i o n i n the WILD and WV33 eggs was not s i g n i f i c a n t l y d i f f e r e n t r e g a r d l e s s of stock but the COMM eggs contained s i g n i f i c a n t l y l e s s 22:6n3 than the WILD eggs, again r e g a r d l e s s of stock. S i g n i f i c a n t d i f f e r e n c e s between stocks f o r the d i e t s were found f o r 20:5n3 i n the WILD groups and f o r 22:5n3 i n the COMM and WILD groups. L e v e l s of the n6 f a t t y a c i d s and the n6 PUFAs as shown i n Tables 8a & 8b, were a l l s i g n i f i c a n t l y lower i n the eggs of w i l d f i s h than i n e i t h e r group of c u l t u r e d f i s h , r e g a r d l e s s of stoc k . In both s t o c k s , the eggs from the COMM groups had 76 Table 8a. S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs (polyunsaturated f a t t y a c i d s ) (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from BIG QUALICUM (BQ) broodstock on thr e e d i e t s (n - 5 f i s h f o r each group). D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . BQ WILD BQ WV33 BQ COMM mean sem mean sem mean sem 18:2n6 20:2n6 20:4n6 T o t a l n6 PUFAs 1 0.79- 0.05 0.00- 0.00 1.15-"0.03 2.34- 0.07 3.43to«0.07 0.37 b 0.02 1.60 b 0.04 6.61 b 0.06 4.80° 0.14 0.39 b 0.04 1.69 b 0.04 8.47°«0.22 1 n6 PUFAs = unsaturated n6 f a t t y a c i d s with 2 to 4 double bonds abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 8b. 77 Table 8b. S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs (polyunsaturated f a t t y a c i d s ) (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from ROBERTSON CREEK (RC) broodstock on thr e e d i e t s (n = 5 f i s h f o r each group except COMM where n = 3) . D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . RC RC RC WILD WV33 COMM mean sem mean sem mean sem % % % 18:2n6 0.85- 0.08 3.85°"0.06 4.59° 0.18 20:2n6 0.00- 0.00 0.32° 0.02 0.46° 0.04 20:4n6 1.02--0.03 1.63° 0.03 1.73° 0.04 T o t a l n6 PUFAs 1 2.19- 0.13 6.97° 0.07 8.12°«0.14 1 n6 PUFAs = unsaturated n6 f a t t y a c i d s with 2 to 4 double bonds abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 8a. 78 s i g n i f i c a n t l y higher l e v e l s of 18:2n6, the predominant n6 f a t t y a c i d , and higher t o t a l n6 PUFAs than the eggs from the WV33 groups. There were no d i f f e r e n c e s i n 20:4n6 i n the eggs of e i t h e r stock of c u l t u r e d f i s h . Small but s i g n i f i c a n t d i f f e r e n c e s were found between the two stocks f o r 18:2n6 (RC-WV33 > BQ-WV33), 20:4n6 (BQ-WILD > RC-WILD) and t o t a l n6 PUFAs (BQ-COMM > RC-COMM). Leray et al. (1985) found d e f o r m i t i e s i n e a r l y rainbow t r o u t embryos when the broodstock was f e d an n3 d e f i c i e n t d i e t with a high n6 content. In t h i s study most of the m o r t a l i t y i n the c u l t u r e d f i s h o c c u r r e d before eyeing, d u r i n g the p e r i o d of h i g h e s t m o r t a l i t y r e p o r t e d by Leray. However n3:n6 r a t i o was not d i r e c t l y r e l a t e d to the observed m o r t a l i t y . Fecundity (Table 2), f e r t i l i t y and s u r v i v a l to the eyed stage (Table 4) were b e t t e r i n the COMM than the WV33 groups i n both stocks although the n3:n6 was lower i n the former (Tables 6a & 6b). The n9, n i l and t o t a l monounsaturated f a t t y a c i d s (Tables 9a & 9b) were a l l s i g n i f i c a n t l y lower i n the eggs of the w i l d f i s h than i n the c u l t u r e d f i s h , r e g a r d l e s s of stock. The l e v e l of n7 monounsaturates i n the eggs of the BQ-WILD f i s h was s i g n i f i c a n t l y higher than i n the BQ c u l t u r e d f i s h . The n7 monounsaturates were lower i n the RC-WILD f i s h than i n the RC c u l t u r e d f i s h , though not s i g n i f i c a n t l y so i n the case of the RC-WV33 eggs. There were no d i f f e r e n c e s i n n5 l e v e l s among the d i e t groups of e i t h e r stock. S i g n i f i c a n t stock d i f f e r e n c e s 79 Table 9a. Monounsaturates, r e p o r t e d as n5, n7, n9, n i l and t o t a l monounsaturated f a t t y a c i d s (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from BIG QUALICUM (BQ) broodstock on thr e e d i e t s (n = 5 f i s h f o r each group). D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . BQ WILD BQ WV33 BQ COMM mean sem % mean % sem mean % sem n5 0.42 0.03 0.45 0.00 0.43 0.00 n7 11.65~"0.31 10.02 b 0.11 10.30 b 0.10 n9 21.71- 0.84 28.01 b 0.75 30.33° 0.36 n i l 0.53- 0.27 1.12 b 0.05 1.19° 0.08 T o t a l monounsaturates 34.31-«0.98 39.60° 0.77 42.25 b 0.33 abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 9b. 80 Table 9b . Monounsaturates, r e p o r t e d as n 5 , n7 , n 9 , n i l and t o t a l monounsaturated f a t t y a c i d s (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from ROBERTSON CREEK (RC) broodstock on thr e e d i e t s (n = 5 f i s h f o r each group except COMM where n = 3) . D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . RC RC RC WILD WV33 COMM mean sem mean sem mean sem % % % n5 0.47 0.05 0.44 0.01 0.43 0.02 n7 9.97-"0.22 10.05- b0.16 10.62 b 0.11 n9 21.88- 0.77 29.04 b 0.43 29.87 b 0.28 n i l 0.52- 0.26 1.03 b 0.41 0.81- b0.19 T o t a l monounsaturates 32.83-"0.56 40.56 b 0.49 41.73 b 0.19 abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e same row. • = a s i g n i f i c a n t d i f f e r e n c e (<x = 0.05) was found between  s t o c k s f o r t h e parameter i n d i c a t e d . See T a b l e 9a. 81 were found on l y i n the w i l d eggs f o r n7 f a t t y a c i d s and t o t a l monounsaturates (BQ-WILD > RC-WILD i n both c a s e s ) . D i f f e r e n c e s i n f a t t y a c i d s between the eggs of the w i l d BQ and RC f i s h are probably a r e f l e c t i o n of the d i f f e r e n c e s i n the d i e t s t h a t these f i s h consumed throughout t h e i r l i v e s . An example of t h i s i s the n7 c o n c e n t r a t i o n i n the t o t a l l i p i d s which was 11.65 i n the BQ-WILD eggs and 9.97 i n the RC-WILD eggs. D i f f e r e n c e s i n temperature (Hazel, 1979) and s a l i n i t y (Leray et a i . , 1984) i n the marine environments of the two stocks may a l s o c o n t r i b u t e t o d i f f e r e n c e s between the stocks by a f f e c t i n g the l e v e l of u n s a t u r a t i o n u l t i m a t e l y a t t a i n e d by the eggs. 1.2.5 F a t t y ,acid composition of the p o l a r l i p i d s The complete f a t t y a c i d p r o f i l e s of the p o l a r l i p i d f r a c t i o n of t o t a l l i p i d are l i s t e d i n Appendix 4, Tables A4.3 and A4.4, f o r each stock by d i e t . Data f o r s e l e c t e d f a t t y a c i d s and f o r assemblages of f a t t y a c i d s are presented here. As f o r the t o t a l l i p i d , the s a t u r a t e d and n3 f a t t y a c i d s were s i g n i f i c a n t l y higher and the n6 and n9 f a t t y a c i d s were s i g n i f i c a n t l y lower i n the p o l a r l i p i d s of the w i l d eggs than i n those from the c u l t u r e d f i s h (Tables 10a & 10b) i n both s t o c k s . T h i s r e s u l t e d i n higher n3:n6 r a t i o s i n the w i l d than i n the c u l t u r e d eggs of both s t o c k s . There were no s i g n i f i c a n t 82 Table 10a. Saturated, n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from BIG QUALICUM (BQ) Chinook broodstock on three d i e t s (n = 5 f i s h f o r each group). D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . BQ BQ BQ WILD WV33 COMM mean sem mean sem mean sem % % % S a t u r a t e s 27.80- 0.52 23.78 b 0.53 •24.95 b 0.30 n3 45.57- 0.86 39.66 b 0.40 37.93 b 0.51 n6 2.41- 0.15 • 5.49 b 0.08 7.04° 0.10 n9 13.73- 0.74 •20.68 b 0.72 19.95 b 0.26 abc = a s i g n i f i c a n t d i f f e r e n c e (ot = 0.05) was found between means w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e same row. • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  s t o c k s f o r t h e parameter i n d i c a t e d . See Ta b l e 10b. BQ BQ BQ WILD WV33 COMM mean sem mean sem mean sem n3:n6 r a t i o " 1 18.91 1.51 7.22 0.07 5.39 0.14 * ANOVA was not conducted on the n3:n6 r a t i o s . 83 Table 10b. Saturated, n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from ROBERTSON CREEK (RC) Chinook broodstock on three d i e t s (n = 5 f i s h f o r each group except COMM where n=3) Di e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t • RC RC RC WILD WV33 COMM mean % sem mean sem % mean sem % Saturates 27.61- 0.67 24.80° 0.14 B25.86° 0.55 n3 46.81- 0.71 41.22° 0.87 37.99° 0.35 n6 2.54- 0.10 • 6.11° 0.12 6.76° 0.21 n9 13.75- 0.55 •18.37° 1.62 18.61° 0.34 ab = a s i g n i f i c a n t difference (a=0.05) was found between means with d i f f e r e n t superscripts within the same row. • = a s i g n i f i c a n t difference (a=0.05) was found between  stocks for the parameter indicated. See Table 10a. RC RC RC WILD WV33 COMM mean sem mean sem mean sem n3:n6 r a t i o " 18.43 0.89 6.76 0.08 5.62 0.15 * ANOVA was not conducted on the n3:n6 r a t i o s . 84 d i f f e r e n c e s between the w i l d stocks i n s a t u r a t e d , n3, n6 or n9 f a t t y a c i d s but the RC-COMM eggs had s i g n i f i c a n t l y more s a t u r a t e s than the BQ-COMM eggs and the RC-WV33 eggs had s i g n i f i c a n t l y more n6 f a t t y a c i d s and l e s s n9 f a t t y a c i d s than the BQ-WV33 eggs. As shown i n Tables 11a & l i b , s i g n i f i c a n t l y higher l e v e l s of 20:5n3, 22:5n3, n3 PUFAs and HUFAs were present i n the p o l a r l i p i d s of the eggs of the w i l d f i s h than i n the c u l t u r e d f i s h of both s t o c k s . The ex c e p t i o n to t h i s p a t t e r n was the f a t t y a c i d , 22:6n3. 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 the l e v e l s of 22:6n3 among any of the d i e t groups, i n e i t h e r s tock. C a s t l e d i n e and Buckley (1980 and 1982) rep o r t e d the presence of u n f l u c t u a t i n g l e v e l s of 22:6n3 i n the p o l a r l i p i d of rainbow t r o u t muscle d u r i n g s t a r v a t i o n and e s s e n t i a l f a t t y a c i d d e p r i v a t i o n . The p h y s i o l o g i c a l importance of t h i s f a t t y a c i d as a membrane component and p o s s i b l e e i c o s a n o i d p r e c u r s o r i s r e f l e c t e d by t h i s t e n a c i o u s r e t e n t i o n . Fremont et al. (1984) found t h a t rainbow t r o u t f e d an n3 d e f i c i e n t d i e t f o r 6 months before spawning maintained high l e v e l s of 22:6n3 i n the v i t e l l o g e n i n and l i p o p r o t e i n while l e v e l s of other n3 f a t t y a c i d s , i n c l u d i n g 20:5n3, d e c l i n e d d r a s t i c a l l y . T h i s f a t t y a c i d may t h e r e f o r e be e s s e n t i a l f o r the r e p r o d u c t i v e success of salmonids. A s i m i l a r p a t t e r n of n6 f a t t y a c i d s was seen i n the p o l a r 85 Table 11a. S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs (poly u n s a t u r a t e d f a t t y a c i d s ) and t o t a l n3 HUFAs ( h i g h l y unsaturated f a t t y a c i d s ) (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from BIG QUALICUM (BQ) broodstock on thr e e d i e t s (n = 5 f i s h f o r each group). D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . BQ BQ BQ WILD WV33 COMM mean sem mean sem mean sem % % % 20:5n3 14.97- 0.45 10.70° 0.25 10.59° 0.16 22:5n3 5.22--0.37 3.48° 0.04 3.19° 0.19 22:6n3 24.14 0.96 24.69 0.33 23.59 0.73 T o t a l n3 PUFAs 1 1.10- 0.24 0.44° 0.11 0.57° 0.09 T o t a l n3 HUFAs 2 44.47- 0.76 39.23°"0.41 37.36° 0.50 1 n3 PUFAs = unsaturated n3 f a t t y a c i d s with 2 or 4 double bonds 2 n3 HUFAs = unsaturated n3 f a t t y a c i d s with 5 or more double bonds abc = a s i g n i f i c a n t d i f f e r e n c e (ot = 0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (<x = 0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table l i b . 86 Table l i b . S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 PUFAs (poly u n s a t u r a t e d f a t t y a c i d s ) and t o t a l n3 HUFAs ( h i g h l y unsaturated f a t t y a c i d s ) (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from ROBERTSON CREEK (RC) broodstock on thr e e d i e t s (n = 5 f i s h f o r each group except COMM where n = 3) . D i e t codes: COMM = commercial; WV33 = West Vancouver 3 3 ; WILD = n a t u r a l d i e t . RC RC RC WILD WV33 COMM mean sem mean sem mean sem % % % 20:5n3 15.59- 0.76 11.05 b 0.48 10.39 b 0.03 22:5n3 4.42-«0.38 3.35 b 0.13 3.33 b 0.36 22:6n3 25.16 0.42 25.56 0.25 23.93 0.41 T o t a l n3 PUFAs 1 1.41- 0.21 0.41 b 0.14 0.34 b 0.18 T o t a l n3 HUFAs 2 45.40- 0.82 40.81 b"0.85 37.65° 0.27 1 n3 PUFAs = unsaturated n3 f a t t y a c i d s with 2 or 3 double bonds 2 n3 HUFAs = unsaturated n3 f a t t y a c i d s with 4 or more double bonds abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 11a. 87 l i p i d s as was seen i n the t o t a l l i p i d s , namely, s i g n i f i c a n t l y lower l e v e l s of a l l n6 f a t t y a c i d s i n the w i l d than i n the c u l t u r e d eggs (Tables 12a & 12b). Within each stock, i t was the COMM eggs t h a t c o n s i s t e n t l y had the h i g h e s t , and u s u a l l y s i g n i f i c a n t l y h i g h e r , l e v e l s of n6 f a t t y a c i d s . Between sto c k s , the RC-COMM eggs contained s i g n i f i c a n t l y higher l e v e l s of 20:4n6 and t o t a l n6 PUFAs and the RC-WV33 eggs contained a s i g n i f i c a n t l y higher l e v e l of 20:2n6. T h i s p a t t e r n was r e f l e c t e d i n the p o l a r l i p i d n3:n6 r a t i o s (Tables 10a and 10b). The absence of 20:2n6 (and 20:3n6, see Appendix 4) from both the t o t a l and the p o l a r l i p i d s of the w i l d eggs (both stocks) suggests t h a t e l o n g a t i o n and d e s a t u r a t i o n of 18:2n6 to 20:4n6 was not o c c u r r i n g . I t i s known t h a t high l e v e l s of n3 f a t t y a c i d s i n the d i e t i n h i b i t e l o n g a t i o n and d e s a t u r a t i o n of n6 and n9 f a t t y a c i d s (Halver, 1980). I t was the w i l d eggs, with the hi g h e s t n3 c o n c e n t r a t i o n , t h a t had the lowest l e v e l of 20:4n6 i n both the t o t a l and p o l a r l i p i d s . The source of the 20:4n6 i n a l l groups may have been d i e t a r y . Both 18:2n6 and 20:4n6 were present i n the COMM and WV33 d i e t s and a small amount (0.33%) of 18:3n6 was present i n the WV33 d i e t . A d i e t a r y source c o u l d t h e r e f o r e account f o r the appearance of these f a t t y a c i d s i n the eggs of the c u l t u r e d f i s h . I f t h i s was the case, i t seems u n l i k e l y t h a t 20:4n6, the primary p r e c u r s o r f o r e i c o s a n o i d s y n t h e s i s i n mammals, has much p h y s i o l o g i c a l s i g n i f i c a n c e f o r Chinook, p a r t i c u l a r l y s i n c e the 88 Table 12a. S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs (poly u n s a t u r a t e d f a t t y a c i d s ) (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from BIG QUALICUM (BQ) broodstock on thr e e d i e t s (n = 5 f i s h f o r each group). D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . BQ BQ BQ WILD WV3 3 COMM mean sem % mean sem % mean sem % 18:2n6 0.28- 0.04 1.40° 0 . 0 7 2.03° 0.04 2 0 :2n6 0.00- 0.00 0.42° 0 . 0 2 0.45°B0.03 20:4n6 1.68- 0.04 2.43°«0.07 2.88° 0.12 T o t a l n6 PUFAs 1 2.41- 0.15 5.49 ° n 0.08 7.04° 0.10 1 n6 PUFAs = unsaturated n6 f a t t y a c i d s with 2 to 4 double bonds abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 12b. 89 Table 12b. S e l e c t e d n6 f a t t y a c i d s and t o t a l n6 PUFAs (polyunsaturated f a t t y a c i d s ) (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from ROBERTSON CREEK (RC) broodstock on three d i e t s (n = 5 f i s h f o r each group except COMM where n = 3) . D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . RC RC RC WILD WV33 COMM mean sem mean sem mean sem % % % 18:2n6 0.34- 0.02 1.57 b 0.05 1.83° 0.02 20:2n6 0.00- 0.00 0.40 b 0.03 0.59°«0.05 20:4n6 1.56- 0.04 2.68 bn0.05 2.84 b 0.02 T o t a l n6 PUFAs 1 2.54- 0.10 6.11 bn0.12 6.76° 0.21 1 n6 PUFAs = unsaturated n6 f a t t y a c i d s with 2 t o 4 double bonds abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (ot = 0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 12a. 90 l e v e l o f 20:4n6 was s i g n i f i c a n t l y l o w e r i n t h e w i l d t h a n i n t h e c u l t u r e d e g g s . A n o t h e r e x p l a n a t i o n f o r t h e absence o f 20:2n6 i n t h e w i l d eggs and i t s p r e s e n c e i n t h e c u l t u r e d eggs may l i e i n t h e use o f t h e Supe lcowax 10 ( S u p e l c o Canada L t d . , O a k v i l l e , O n t a r i o ) co lumn employed on t h e GLC i n t h e s e a n a l y s e s . A c c o r d i n g t o Ackman ( 1 9 8 6 ) , t h i s co lumn does no t s e p a r a t e 20:3n9 and 20:2n6 v e r y s a t i s f a c t o r i l y . Ackman (1986) s u g g e s t s t h a t n o r m a l l y t h i s i s no t a p r o b l e m i n n u t r i t i o n a l b i o c h e m i s t r y u n l e s s t h e r e i s a d e f i c i e n c y o f e s s e n t i a l f a t t y a c i d s w h i c h l e a d s t o t h e f o r m a t i o n o f 2 0 : 3 n 9 . I n v i e w o f t h e measures w h i c h r e s e a r c h e r s must t a k e i n o r d e r t o i n d u c e an EFA d e f i c i e n c y i n f i s h ( e g . C a s t l e d i n e and B u c k l e y , 1980 and 1 9 8 2 ) , i t i s u n l i k e l y t h a t t h e f o r m a t i o n o f 20 :3n9 was o c c u r r i n g i n t h e c u l t u r e d f i s h i n t h i s s t u d y . R a t h e r t h e f a t t y a c i d i d e n t i f i e d as 2 0 : 2 n 6 , r e a l l y was 2 0 : 2 n 6 . M o n o u n s a t u r a t e s , p a r t i c u l a r l y 1 6 : l n 7 and 1 8 : l n 9 , a r e i m p o r t a n t components o f TAG and p o l a r l i p i d m o l e c u l e s , as men t ioned p r e v i o u s l y . These two f a t t y a c i d s were t h e p r edominan t monoenoic f a t t y a c i d s i n b o t h t h e p o l a r and t h e t o t a l l i p i d s . As shown i n T a b l e s 13a & 13b , n9 f a t t y a c i d s were s i g n i f i c a n t l y l o w e r i n t h e p o l a r l i p i d s o f w i l d eggs t h a n c u l t u r e d e g g s . The n7 f a t t y a c i d s were no t s i g n i f i c a n t l y d i f f e r e n t among t h e d i e t g roups o f t h e c u l t u r e d BQ o r RC e g g s . T h i s may i n d i c a t e a r e q u i r e m e n t f o r a s p e c i f i c l e v e l o f n7 f a t t y a c i d s . 91 T a b l e 13a. Monounsaturates, r e p o r t e d as n5, n7, n9, n i l and t o t a l monounsaturated f a t t y a c i d s (mean and s t a n d a r d e r r o r o f t h e means) i n POLAR LIPIDS of eggs from BIG QUALICUM (BQ) b r o o d s t o c k on t h r e e d i e t s (n = 5 f i s h D i e t codes: COMM = Vancouver 33; WILD f o r each g r o u p ) . c o m m e r c i a l ; WV33 : = n a t u r a l d i e t . = West BQ WILD BQ WV33 BQ COMM mean sem % mean sem % mean sem % n5 0.50 0.05 0.52 0.02 0.48 0.03 n7 6.59" 0.21 6.30" 0.27 5.98 0.17 n9 13.73- 0.74 20.68 b»0.72 19.95° 0.26 n i l 0.37 0.34 0.71 0.36 0.37 0.04 T o t a l monounsaturates 21.19- 0.91 28.21 f anQ.63 26.78 b"0.14 abc = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e same row. s = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between  s t o c k s f o r t h e parameter i n d i c a t e d . See T a b l e 13b. T a b l e 1 3 b . M o n o u n s a t u r a t e s , r e p o r t e d a s n 5 , n 7 , n 9 , n i l a n d t o t a l m o n o u n s a t u r a t e d f a t t y a c i d s ( m e a n a n d s t a n d a r d e r r o r o f t h e m e a n s ) i n P O L A R L I P I D S o f e g g s f r o m R O B E R T S O N C R E E K ( R C ) b r o o d s t o c k o n t h r e e d i e t s ( n = 5 f i s h f o r e a c h g r o u p e x c e p t COMM w h e r e n = 3 ) . D i e t c o d e s : COMM = c o m m e r c i a l ; WV33 = W e s t V a n c o u v e r 3 3 ; W I L D = n a t u r a l d i e t . R C R C R C W I L D WV33 COMM mean % sem mean sem % mean % sem n5 0.59 0.07 0.48 0.01 0.50 0.04 n7 •5.21" 0.21 5.75" 0.07 5.99 0.17 n9 13.75- 0.55 18.37 bnl.62 18.61° 0.34 n i l 0.17 0.11 0.23 0.06 0.00 0.00 T o t a l monounsaturates 19.72- 0.56 24.82~H1.58 25.10° • 0.24 a b c = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) w a s f o u n d b e t w e e n m e a n s w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e s a m e r o w . • = a s i g n i f i c a n t d i f f e r e n c e (ct = 0.05) was found between  stocks f o r the parameter i n d i c a t e d . See Table 13a. 93 Between the two st o c k s , where th e r e were s i g n i f i c a n t d i f f e r e n c e s i n monounsaturates, the BQ eggs c o n s i s t e n t l y had the g r e a t e r c o n c e n t r a t i o n , i e . f o r the n7 s e r i e s , BQ-WILD > RC-WILD and BQ-WV33 > RC-WV33; f o r the n9 s e r i e s , BQ-WV33 > RC-WV33; and f o r t o t a l monounsaturates, BQ-WV33 > RC-WV33 and BQ-COMM > RC-COMM. 1.2.6 D i e t s A l l d i e t data are presented i n Apppendix 2. T h i s i n c l u d e s feed tag data f o r the COMM d i e t (Table A2.1) and the f o r m u l a t i o n of the WV33 d i e t (Table A2.2). Both d i e t s were analysed f o r dry matter, moisture, l i p i d on a wet and dry weight b a s i s (Table A2.3) and f o r t o t a l l i p i d f a t t y a c i d s (Table A2.4). Dry matter, moisture and l i p i d l e v e l s were found to match s p e c i f i c a t i o n s s t a t e d on the feed tags of both d i e t s (Table A2.1). For c l a r i t y , comparisons of i n d i v i d u a l f a t t y a c i d s and f a t t y a c i d s e r i e s i n the t o t a l and p o l a r l i p i d s of the eggs are presented together with the same parameters i n the d i e t s i n Fig u r e s 5, 6 and 7. P a r a l l e l egg data f o r the w i l d f i s h are shown a t the bottom of each f i g u r e . D i e t data were not a v a i l a b l e f o r the w i l d f i s h . i The s a t u r a t e d , n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s i n the t o t a l and p o l a r l i p i d s of the eggs of both stocks on a l l 94 d i e t s are d i s p l a y e d as histograms i n Fi g u r e 5. Data f o r the d i e t s (dark bars) are presented a l s o . Except f o r the s a t u r a t e s , there was a strong tendency f o r f a t t y a c i d s p r e s e n t i n the d i e t t o accumulate i n the t o t a l l i p i d s . Both the COMM and the WV33 d i e t s were high i n s a t u r a t e d f a t t y a c i d s . I t appears t h a t some s a t u r a t e s were converted to monounsaturates (see F i g u r e 7) or t h a t they were o x i d i z e d . The f a t t y a c i d 16:0 was the major s a t u r a t e d f a t t y a c i d i n the t o t a l and p o l a r l i p i d s of the eggs of both the c u l t u r e d and w i l d f i s h . I t i s o f t e n present i n TAGs and to a l e s s e r extent i n p o l a r l i p i d s . The n9 f a t t y a c i d s , predominant i n the COMM d i e t , were the major s e r i e s present i n the t o t a l l i p i d s of the COMM eggs. However i t was the n3 s e r i e s , the t h i r d l a r g e s t component of the COMM d i e t and the second l a r g e s t component i n the WV33 d i e t , t h a t formed the l a r g e s t p r o p o r t i o n of f a t t y a c i d s i n the p o l a r l i p i d s of the COMM eggs and i n both the t o t a l and p o l a r l i p i d s of the WV33. The n3 s e r i e s was a l s o dominant i n the t o t a l and p o l a r l i p i d s of the w i l d eggs of both s t o c k s . The l e v e l s of s a t u r a t e d and n3 f a t t y a c i d s were w i t h i n 2% of each other i n the WV33 d i e t , f o l l o w e d by the n9 s e r i e s . However, i n the eggs, the n3 f a t t y a c i d s were the major s e r i e s i n both the t o t a l and p o l a r l i p i d s , f o l l o w e d by the n9 f a t t y a c i d s i n the t o t a l l i p i d s and the s a t u r a t e s i n the p o l a r l i p i d s . In t h i s study, d i e t was a g r e a t e r f a c t o r than stock a f f e c t i n g 95 Figure 5: A compar ison of saturated, n3, n6 and n9 fatty acids (%) andn3.n6 ratios in total (TL) and polar (PL) lipids of eggs from cultured and wild (WILD) Big Qual icum (BQ) and Robertson Creek (RC) broodstock with the commercial (COMM) and West Vancouver (WV33) diets. BQ Eggs - COMM Diet SATURATES n3 L i R A T I O n9 n3:n6 RATIO RC Eggs - COMM Diet R A T I O ] TL L_S PL SATURATES n3 n6 nS n3:n6 RATIO • I COMM R88 TL [Ml PL BQ Eggs - WV33 Diet R A T I O SATURATES r>3 n6 n9 n3:n6 RATIO • • WV33 ^ TL C'_! PL RC Eggs - WV33 Diet R A T I O SATURATES n3 n6 n9 n3:n6 RATIO •D WV33 KS> TL EES PL BQ Eggs - WILD Diet I 1 r R A T I O SATURATES n3 n6 n9 n3:tl6 RATIO TL MB PL RC Eggs - WILD Diet l 1 R A T I O i SATURATES n3 n6 n9 r3:n6 RATIO JSSiTL E U P L 96 f a t t y a c i d p r o f i l e s i n the eggs of the c u l t u r e d f i s h . D i f f e r e n c e s i n f a t t y a c i d composition were g r e a t e r between the COMM and WV33 d i e t groups than between the two stocks on the same d i e t , ( F i g u r e 5). T h i s was a l s o t r u e f o r the other f a t t y a c i d s , as d e s c r i b e d below and i l l u s t r a t e d i n Fi g u r e s 6 and 7. The i n f l u e n c e of temperature on the f a t t y a c i d composition of membranes and the a b i l i t y of f i s h to a l t e r l e v e l s of u n s a t u r a t i o n i n membranes i n response to temperature changes has been documented (eg. Hazel, 1979). A l l c u l t u r e d f i s h were rea r e d at the same s i t e and were exposed to the same temperature regime. Temperature was t h e r e f o r e not a f a c t o r . Although a w i l d d i e t would c o n t a i n high l e v e l s of n3 f a t t y a c i d s and p a r t i c u l a r l y high l e v e l s of HUFAs, the e f f e c t of temperature cannot be r u l e d out as a major i n f l u e n c e on the high l e v e l s of u n s a t u r a t i o n i n the w i l d eggs. During t h e i r ocean m i g r a t i o n , the w i l d f i s h would have been exposed to lower water temperatures than the c u l t u r e d f i s h i n t h e i r near shore net pens. Since the Robertson Creek stock matures o f f the west c o a s t of Vancouver I s l a n d while the B i g Qualicum stock comes i n t o the warmer S t r a i t of Georgia, i t may be expected t h a t t h e i r temperature regimes and d i e t s would both d i f f e r . The most apparent d i f f e r e n c e was the presence of higher l e v e l s of n3 and s a t u r a t e d f a t t y a c i d s and lower l e v e l s of n9 f a t t y a c i d s i n the p o l a r l i p i d f r a c t i o n of the eggs of the RC-WILD f i s h than i n the eggs of the BQ-WILD f i s h ( F i gure 5). These d i f f e r e n c e s are c o n s i s t e n t with t h e i r r e s p e c t i v e temperature 97 Figure 6: A compar ison of se lected n3 fatty acids, n3 PUFAs and HUFAs in total (TL) and polar (PL) l ipids of eggs from cultured and wild (WILD) Big Qualicum (BQ) and Robertson Creek (RC) broodstock with the commercial (COMM) and West Vancouver 33 (WV33) diets. BQ Eggs - COMM Diet RC Eggs - COMM Diet i 22:6n3 22:6n3 HUFAs • i COMM XKK TL H U P L n3 PUFAs 1 5 1 _••=> • ! 22:6n3 22:6n3 HUFAs n3 PUFAs • • C O M M E53TL 151 PL BQ Eggs - WV33 Diet RC Eggs - WV33 Diet 20:6n3 22:6n3 22:6n3 HUFAs n3 PUFAs • WV33 KSSTL EUDPL 22:6n3 22:6n3 HUFAs n3 PUFAs ^ TL EES PL BQ Eggs - WILD Diet RC Eggs - WILD Diet 20:6n3 22:6n3 22:6n3 HUFAs n3 PUFAs EfflTL EEDPL ^ TL ,' J PL 98 Figure 7: A compar ison of selected fatty acids and n6 PUFAs in total (TL) and polar (PL) l ipids of eggs from cultured and wild (WILD) Big Qualicum (BQ) and Rober tson Creek (RC) broodstock with the commercial (COMM) and West Vancouver (WV33) diets. BQ Eggs - COMM Diet RC Eggs - COMM Diet 18:1n9 16:1n7 22:1n11 18:2n6 20:4n6 n6 PUFAs } TL E l PL 30 26 20 % 16 10 6 0 • 16:107 22;1n11 18:2n6 20:4n8 n6 PUFAs U COMM t\V»3 TL EH PL BQ Eggs - WV33 Diet RC Eggs - WV33 Diet 16:1n7 22:1n11 18:2n6 20:4n6 n6 PUFAs • • WV33 ESS! TL [MO PL % 16 16:1n7 22:1n11 18:2n6 20:4n6 n8 PUFAs H WV33 E33 TL EMI PL BQ Eggs - WILD Diet RC Eggs - WILD Diet H I 18:1n9 16:1n7 22:1n11 18:2n6 20:4n6 n6 PUFAs EMI TL EM PL 30 26 20 % 16 10 6 I 1 18:1n9 16:1n7 22:1n11 18:2n6 20:4n6 n6 PUFAs ESS TL EMI PL 9 9 regimes. The two stocks were remarkably similar i n composition otherwise (see also Figures 6 and 7 ) . The major f a t t y acids in the n3 series of both the cultured and wild eggs were 22:6n3 and 20:5n3. These f a t t y acids were present i n the eggs i n concentrations that were notably higher than in the diets and i t i s evident that they had been accumulated and conserved i n the eggs (Figure 6). While the fa t t y acid, 20:5n3 was higher in both the t o t a l and polar l i p i d s of the wild eggs than i n the cultured eggs, 22:6n3 level s were remarkably similar, p a r t i c u l a r l y in the polar f r a c t i o n , i n the cultured and wild eggs (Figure 6). In view of th e i r physiological significance as membrane components and eicosanoid precursors and because n3 fa t t y acids have essential status for salmonids, i t i s reasonable to expect consistent high levels of these HUFAs i n the egg where they would be required by the growing embryo. As shown i n Figure 7 both the COMM and WV33 diets contained high levels of monounsaturates, p a r t i c u l a r l y 18:ln9, 16:ln7 and 2 2 : l n l l . The f i r s t two are the primary monoenes i n f i s h egg l i p i d s ( C h r i s t i e , 1986) and were re f l e c t e d accordingly i n the egg composition but 2 2 : l n l l was apparently not deposited i n the eggs or i t was oxidized as i t was present in only very small amounts. As previously mentioned, 18:ln9 and 16:ln7 are important constituents of both TAGs and of many polar l i p i d s . Both were present in the wild eggs but not i n as large amounts 100 as was found i n the cultured eggs. The major n6 f a t t y acid present i n the COMM and WV33 diets was 18:2n6 followed by 20:4n6. The eggs of the cultured f i s h contained small amounts of these f a t t y acids and the COMM eggs of both stocks contained s l i g h t l y more 18:2n6 than the WV33 eggs consistent with s l i g h t l y higher levels of t h i s f a t t y acid i n the COMM d i e t . The low levels of 20:4n6, an important prostaglandin precursor for mammals, in the eggs of both cultured and wild f i s h leads to speculation that t h i s f a t t y acid has l i t t l e physiological significance for Chinook. Total n6 PUFAs were considerably higher i n the cultured eggs than i n the wild eggs. Dietary sources of these f a t t y acids are few in the marine environment (Ackman, 1986). 101 1.3 CONCLUSIONS The i n t e n t i o n of S e c t i o n 1 was to compare l i p i d composition and f a t t y a c i d p r o f i l e s i n w i l d and c u l t u r e d Chinook salmon. While not an o b j e c t i v e of t h i s experiment, i t i s tempting to s p e c u l a t e on p o s s i b l e reasons f o r the s u p e r i o r r e p r o d u c t i v e performance of the w i l d over the c u l t u r e d broodstock (Table 4). The most obvious d i f f e r e n c e s found i n the l i p i d s between the c u l t u r e d and w i l d f i s h were t h a t the w i l d f i s h had much higher l e v e l s of n3 f a t t y a c i d s , p a r t i c u l a r l y the HUFAs, and lower l e v e l s of n6 and n9 f a t t y a c i d s . The p h y s i o l o g i c a l importance of long c h a i n f a t t y a c i d s as membrane components and e i c o s a n o i d p r e c u r s o r s has been d i s c u s s e d . F u r t h e r , Leray et al. (1985) re p o r t e d embryonic d e f o r m i t i e s l e a d i n g to m o r t a l i t y as e a r l y as the 16-and 3 2 - c e l l stages when rainbow t r o u t broodstock were fe d low l e v e l s of n3 and high l e v e l s of n6 f a t t y a c i d s . I t was suggested t h a t the HUFAs (20:5n3 and 22:6n3) are the p r e c u r s o r s f o r hydroxy f a t t y a c i d s which are thought to be c r i t i c a l to the processes of c e l l u l a r r e c o g n i t i o n . Most of the m o r t a l i t y i n the c u l t u r e d eggs i n t h i s study occ u r r e d before eyeing and, as shown i n Table 6, the n3:n6 r a t i o s i n both t o t a l and p o l a r l i p i d s of the c u l t u r e d eggs (both d i e t s ) were c o n s i d e r a b l y lower than i n the w i l d eggs. The q u a n t i t a t i v e requirements of Chinook eggs f o r HUFAs du r i n g i n c u b a t i o n are unknown. The e i c o s a n o i d s are e f f e c t i v e 102 i n very low c o n c e n t r a t i o n s which suggests t h a t a small pool of pr e c u r s o r f a t t y a c i d s should s a t i s f y the requirement f o r t h e i r s y n t h e s i s . However as work proceeds i n t h i s area, more e i c o s a n o i d s are being d e t e c t e d and more f u n c t i o n s are being a t t r i b u t e d t o them. E i c o s a n o i d s are r a p i d l y taken up and de-a c t i v a t e d and i n order to maintain a response f o r a continuous p e r i o d , as may occur i n a c h r o n i c i n f e c t i o n , f o r example, a l a r g e pool of p r e c u r s o r s may be necessary. E i c o s a n o i d s are sy n t h e s i z e d from the membrane HUFAs and whether t h i s r e s e r v o i r has p r i o r i t i e s f o r other f u n c t i o n s i s not c l e a r . A l s o , l i t t l e i s known about the k i n e t i c s of the s y n t h e t i c p r o c e s s . Future work i n t h i s area i s bound to be very i n t e r e s t i n g . The eggs of the c u l t u r e d f i s h on the two formulated d i e t s were more s i m i l a r to each other than to the w i l d eggs, r e g a r d l e s s of stock ( F i g u r e s 5, 6 & 7). Too few f i s h were analysed f o r f e c u n d i t y , f e r t i l i t y and eyeing success (Tables 2 & 4) r e l a t i v e to the standard e r r o r of the means, f o r r e l i a b l e s t a t i s t i c a l s i g n i f i c a n c e to be assessed. However the COMM f i s h g e n e r a l l y out-performed the WV33 f i s h (see a l s o Table 3 ) . I t i s u n l i k e l y t h a t the success of the COMM eggs r e l a t i v e to the WV33 eggs was due to f a t t y a c i d composition. The n3:n6 r a t i o s i n the COMM and WV33 d i e t s were 2.73 and 5.68 (Appendix 2, Table A2.4), r e s p e c t i v e l y , and were lower i n the eggs of the COMM group than i n the WV33 group i n both the t o t a l and p o l a r l i p i d s of both stocks (Tables 6a & 6b and 10a & 10b). 103 The WV33 d i e t was manufactured by a commercial feed company and, d u r i n g the year p r i o r to t h i s study, i t was manufactured only t w i c e . Product storage t h e r e f o r e exceeded the usual recommended s h e l f l i f e of 3 months (NRC, 1981). While degradation t o v a r y i n g degrees of many of the n u t r i e n t s i n the d i e t probably o c c u r r e d and nothing d e f i n i t i v e can be concluded from t h i s study, i t i s l i k e l y t h a t l o s s e s of v i t a m i n C were p a r t i c u l a r l y c r i t i c a l t o r e p r o d u c t i v e success. The d i e t a r y requirement f o r v i t a m i n C i n coldwater f i s h i s 100 mg/kg dry d i e t (NRC, 1981). The COMM d i e t was supplemented with a minimum of 700 mg/kg; the WV33 d i e t was supplemented with 1900 mg/kg (Appendix 2, Tables A2.1 and A2.2). Vitamin C i s g e n e r a l l y added because t h i s v i t a m i n i s h i g h l y s u s c e p t i b l e to d e gradation by heat and moisture d u r i n g p r o c e s s i n g and storage. Despite high supplementation of the WV33 d i e t , prolonged storage times probably r e s u l t e d i n very l i t t l e r e s i d u a l v i t a m i n C. S l i n g e r et al. (1979, c i t e d i n Poston, 1986) r e p o r t l o s s e s of 67 to 83% due to storage at about 20°C f o r 6 months. Storage c o n d i t i o n s a t the net pen s i t e may have f u r t h e r i n c r e a s e d the r a t e of deg r a d a t i o n . The feed f o r the week was s t o r e d i n black p l a s t i c b a r r e l s i n the sun. In the study by S l i n g e r et al. (1979, c i t e d i n Poston, 1986), l e e c h i n g d u r i n g exposure of feed to water f o r up to 10 seconds caused f u r t h e r l o s s e s of 15 to 67% of the remaining v i t a m i n C a c t i v i t y . New forms of v i t a m i n C (eg. a s c o r b a t e - 2 - s u l f a t e ( C 2 ) ) are now commonly used i n formulated d i e t s . They are heat 104 and water s t a b l e at pH 4-13 (Tucker and Halver, 1986). Vitamin C has many important biochemical f u n c t i o n s i n c l u d i n g r o l e s i n the p r e v e n t i o n of s c o l i o s i s and l o r d o s i s (a s c o r b u t i c m a n i f e s t a t i o n due to impaired s y n t h e s i s of c o l l a g e n and c a r t i l a g e ) ; l i p i d and amino a c i d c a t a b o l i s m ; catecholamine s y n t h e s i s ; d e - t o x i f i c a t i o n of p o l l u t a n t s (eg. cadmium) and f r e e r a d i c a l s v i a the cytochrome P 4 5 0 system; i n the immune system and i n s t e r o i d o g e n e s i s (Sandnes, 1988). Based on t h e i r poor spawning performance, i t i s p o s s i b l e t h a t s t e r o i d o g e n e s i s was compromised i n the f i s h f e d the WV33 d i e t . Other f u n c t i o n s may a l s o have been impaired. S e v e r a l s t u d i e s i n v a r i o u s s p e c i e s ( H i l t o n et ai.,1979; Seymour, 1981 a,b; T o l b e r t , 1979; Lutwak-Mann, 1958; and Sandnes and Braekkan, 1981 - a l l c i t e d i n Sandnes, 1988) p o i n t to a r e l a t i o n s h i p between the high l e v e l s of v i t a m i n C found i n f i s h o v a r i e s with the s y n t h e s i s of sex s t e r o i d s . A r i s e i n v i t a m i n C c o n c e n t r a t i o n i n the ovary d u r i n g v i t e l l o g e n e s i s f o l l o w e d by a decrease i n c o n c e n t r a t i o n p r i o r t o o v u l a t i o n p a r a l l e l s the s y n t h e s i s of sex s t e r o i d s by the o v a r i a n f o l l i c l e c e l l s and suggests t h a t v i t a m i n C may be r e q u i r e d i n the h y d r o x y l a t i n g r e a c t i o n s necessary to convert c h o l e s t e r o l t o sex s t e r o i d s such as the estrogens (Sandnes, 1988). Waagbo et al. (1989) demonstrated a s i g n i f i c a n t l y higher l e v e l , of 1 7 - P - e s t r a d i o l i n f i s h f e d adequate l e v e l s of v i t a m i n C than 105 i n those f e d d e f i c i e n t d i e t s . A higher l e v e l of c i r c u l a t i n g v i t e l l o g e n i n i n a supplemented group was a l s o found. Sandnes et al. (1984) found t h a t rainbow t r o u t broodstock f e d a d i e t d e f i c i e n t i n v i t a m i n C f o r 3 months p r i o r to spawning tended to produce fewer eggs of s m a l l e r diameter than d i d supplemented broodstock fed a d i e t c o n t a i n i n g 1000 mg/kg. T h e i r f i g u r e s f o r f e c u n d i t y and egg s i z e were not s i g n i f i c a n t and there was a high l e v e l of v a r i a b i l i t y a s s o c i a t e d with the data as was the case f o r the f e c u n d i t y data r e p o r t e d i n t h i s study (Table 2 ) . A l s o r e p o r t e d was a s i g n i f i c a n t l y (a=0.05) g r e a t e r s u r v i v a l r a t e to eyeing and h a t c h i n g i n the supplemented progeny than i n the d e f i c i e n t group. A t h i r d group, used as a c o n t r o l a g a i n s t the experimental d i e t s , was f e d a commercial d i e t and produced eggs of s i m i l a r q u a l i t y to those of the supplemented group. A n a l y s i s of the eggs r e v e a l 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 C l e v e l s between the d e f i c i e n t (15 ug/g wet weight ± 7, s d ) , supplemented (31 ug/g wet weight ± 9, sd) and commercial (20 yg/g wet weight ± 10, sd) groups. In a study undertaken to determine the e f f e c t s of broodstock d i e t s v a r y i n g i n moisture, amount and composition of l i p i d s and content of a - t o c o p h e r o l and a s c o r b i c a c i d f o r A t l a n t i c salmon, E s k e l i n e n (1989) r e p o r t e d t h a t the h i g h e s t t o t a l s u r v i v a l from f e r t i l i z a t i o n t o s t a r t f e e d i n g (92.7%) was achieved with a semi-moist feed which had a low f a t and moderate energy content 106 t h a t had been supplemented with a s c o r b i c a c i d at 1050 mg/kg. The second h i g h e s t s u r v i v a l r a t e (90.3%) was obtained with a dry d i e t which had higher f a t and p r o t e i n l e v e l s and a s c o r b i c a c i d at the second h i g h e s t l e v e l i n the study - 230 mg/kg. No e f f e c t on s u r v i v a l was a t t r i b u t a b l e to a-tocopherol l e v e l s i n the d i e t s . Soliman et al (1986) demonstrated t h a t a d i e t supplemented to 1250 mg/kg s i g n i f i c a n t l y improved h a t c h a b i l i t y i n t i l a p i a (Oreochromis mossambicus). They a l s o showed t h a t v i t a m i n C d e f i c i e n t t i l a p i a reached m a t u r i t y two weeks l a t e r than the supplemented group. Waagbo et al. (1989) i n t e r p r e t e d a s i m i l a r f i n d i n g i n rainbow t r o u t , f e d a v i t a m i n C d e f i c i e n t d i e t , t o impaired s t e r o i d o g e n e s i s , a f f e c t i n g o v a r i a n growth and maturation d u r i n g v i t e l l o g e n e s i s . In the present study, f i s h f e d the WV33 d i e t e x h i b i t e d high l e v e l s of egg r e t e n t i o n and impaired maturation (one or both o v a r i e s d i d not mature) (Table 3) . Rainbow t r o u t f e d a d i e t c o n t a i n i n g 1400 ppm v i t a m i n C produced fewer 'blank' ( u n f e r t i l i z e d ) eggs than t r o u t f e d a d i e t c o n t a i n i n g 800 ppm (Ridelman, 1981, c i t e d by Hardy, 1985). In t h i s study, the r a t e of f e r t i l i z a t i o n was lower among the WV33 eggs than i n the COMM eggs, although the d i f f e r e n c e was not s i g n i f i c a n t i n the case of the BQ group and there were too few f i s h to assess s i g n i f i c a n c e i n the RC group (Table 4 ) . 107 V i t a m i n C i s a l s o r e q u i r e d f o r c o l l a g e n s y n t h e s i s by the embryo. The f u n c t i o n of v i t a m i n C i n c o l l a g e n s y n t h e s i s i s a g a i n as a reducing agent i n h y d r o x y l a t i o n r e a c t i o n s . The amino a c i d s , p r o l i n e and l y s i n e , are h y d r o x y l a t e d promoting the formation of hydrogen bonds between p r o t e i n c h a i n s . L i t t l e i s known about the metabolism of v i t a m i n C a f t e r f e r t i l i z a t i o n i n f i s h eggs but i n sea u r c h i n eggs c o l l a g e n s y n t h e s i s s t a r t e d at g a s t r u l a t i o n and i n c r e a s e d many times before h a t c h i n g (Golub et al., 1974). Sandnes et al. (1988) demonstrated an i n c r e a s e "in the percentage of h y d r o x y p r o l i n e i n p r o t e i n i n the eggs of rainbow t r o u t through i n c u b a t i o n which was probably c o i n c i d e n t with i n c r e a s i n g l e v e l s of c o l l a g e n i n the embryo. Impaired c o l l a g e n s y n t h e s i s may lead to l a r v a l d e f o r m i t i e s , p a r t i c u l a r l y t o severe damage of the s p i n a l column, and to poor hat c h i n g performance (Soliman et al., 1986). T i l a p i a f r y , hatched from broodstock f e d a low l e v e l of v i t a m i n C, grew sl o w l y , had low feed c o n v e r s i o n r a t e s and poor s u r v i v a l . When the broodstock were f e d supplemented d i e t s , f r y performance was improved (Soliman et al., 1986). From the f o r e g o i n g , the i n f l u e n c e of v i t a m i n C i n broodstock n u t r i t i o n on o v a r i a n growth and maturation and i n the s u r v i v a l and growth of the progeny appears c r i t i c a l . F u r t h e r work on e s t a b l i s h i n g optimum l e v e l s f o r t h i s v i t a m i n i n d i e t s f o r 108 Chinook broodstock should be c a r r i e d out. Reasons f o r the apparent success of the BQ-WV33 f i s h over the RC-WV33 f i s h (Tables 2, 3 & 4 ) can only be guessed a t . There i s always some 'wild feed' i n the net pens a t Genoa Bay, the marine broodstock f a c i l i t y f o r Sea Spring Salmon Farm. However, the c u l t u r e d f i s h g e n e r a l l y do not u t i l i z e t h i s f e e d . Perhaps some of the BQ-WV33 broodstock were more i n c l i n e d to take advantage of t h i s source than were the RC-WV33 f i s h . Otherwise, t h e r e may have been some stock e f f e c t i n response t o the low l e v e l s of v i t a m i n C or to other f a c t o r s i n the d i e t or environment of these f i s h . As i n d i c a t e d i n M a t e r i a l s and Methods, there were 283 f i s h i n the RC-WV33 pen and 189 i n the BQ-WV33 pen f o r d e n s i t i e s of ca. 3 . 41 kg/m3 and 2 . 8 4 kg/m3, r e s p e c t i v e l y . There were 811 BQ f i s h and 149 RC f i s h reared on the COMM d i e t f o r d e n s i t i e s of ca. 5.96 kg/m3 and 1.05 kg/m 3, r e s p e c t i v e l y . Pen d e n s i t i e s below 6 kg/m 3 are co n s i d e r e d a p p r o p r i a t e i n the i n d u s t r y (D. Groves, p e r s . comm.) and as the morphometric data presented i n Table 1 i n d i c a t e s , growth was not compromised i n the c u l t u r e d f i s h at the h i g h e s t pen d e n s i t i e s . I t i s t h e r e f o r e u n l i k e l y t h a t d e n s i t y was a s i g n i f i c a n t f a c t o r i n the poor performance of the RC-WV33 broodstock. 109 SECTION 2 - Feed withdrawal from c u l t u r e d Chinook broodstock p r i o r t o t r a n s f e r t o f r e s h water f o r maturation. 2.1 MATERIALS AND METHODS Two groups of c u l t u r e d 4-year o l d Chinook salmon were h e l d i n 680 m3 marine net pens at Genoa Bay. Pen d e n s i t i e s were l e s s than 2.5 kg/ m3 i n both pens. Both groups were f e d the same commercial d i e t (COMM) at the same r a t e as was f e d to the f i s h i n S e c t i o n 1. Again, not a l l of the f i s h i n these pens matured i n 1987 which made i t impossible t o a c c u r a t e l y determine feed consumption by the broodstock. Feeding l e v e l s were approximately 1% of body weight per day at the s t a r t of t h e i r f i n a l summer but they cut back n a t u r a l l y to ca. 0.5% or l e s s by the end of the summer. The o r i g i n a l i n t e n t i o n of t h i s experiment was to withdraw a l l fe e d from the f i s h i n Pen 16 (the r e s t r i c t e d group) f o r one f u l l month bef o r e t r a n s f e r t o f r e s h water f o r maturation and spawning. The f i s h i n Pen 4 ( f u l l r a t i o n group) were to a c t as c o n t r o l s and were to be s t a r v e d f o r one week p r i o r t o t r a n s f e r , the usual p r o t o c o l f o r broodstock t r a n s p o r t . The design was s i n g l e f a c t o r with no r e p l i c a t i o n . The f i s h were progeny of Big Qualicum females and Chemainus males. The pure Chemainus stock t y p i c a l l y spawns before the 110 pure B i g Qualicum s t o c k . Consequently, the f i s h matured e a r l i e r than expected. Feeding of the r e s t r i c t e d f i s h was h a l t e d on September 2, 1987 but they were darkening r a p i d l y ten days l a t e r and had to be t r a n s p o r t e d on September 16 with o n l y 14 days of s a l t w a t e r s t a r v a t i o n . Feeding of the c o n t r o l f i s h was stopped on September 28 and they were t r a n s p o r t e d on October 5 f o r a t o t a l of 7 days of s a l t w a t e r s t a r v a t i o n . Maturing f i s h were brought to the hatchery, h e l d u n t i l f u l l y mature (ca. 2 - 4 weeks) and spawned a c c o r d i n g to the methods o u t l i n e d i n S e c t i o n 1. Sample c o l l e c t i o n , techniques f o r l i p i d and f a t t y a c i d a n a l y s e s , c o l l e c t i o n of morphometric data, and procedures f o r measuring f e r t i l i z a t i o n and eyeing success were i d e n t i c a l t o those employed i n S e c t i o n 1. A l l data r e p o r t e d as percentages were transformed by the a r c s i n e t r a n s f o r m a t i o n before s t a t i s t i c a l a n a l y s e s . One-way ANOVA (Proc GLM, SAS 1985) were c a r r i e d out on the morphometric and i n c u b a t i o n data, composition parameters, i n d i v i d u a l f a t t y a c i d s and f a t t y a c i d s e r i e s to d e t e c t any s i g n i f i c a n t d i f f e r e n c e s due to p e r i o d of s t a r v a t i o n . The s i g n i f i c a n c e l e v e l was set a t 95%. I l l 2.2 RESULTS AND DISCUSSION 2.2.1 Morphometric measurements Morphometric measurements are presented i n Table 14. 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 f o r k l e n g t h , weight or c o n d i t i o n f a c t o r between the two groups of f i s h . 2.2.2 Spawning and i n c u b a t i o n success The r e s t r i c t e d f i s h produced s i g n i f i c a n t l y s m a l l e r eggs than c o n t r o l f i s h (Table 14). I f v i t e l l o g e n e s i s was s t i l l o c c u r r i n g d u r i n g the p e r i o d of s t a r v a t i o n , i t i s reasonable f o r the eggs of the f i s h s t a r v e d f o r the longer p e r i o d to be s m a l l e r . Fecundity, whether determined by volume or number of eggs spawned, was not a d v e r s e l y a f f e c t e d by s t a r v a t i o n as no s i g n i f i c a n t d i f f e r e n c e was found between the two groups (Table 15). I t i s u n l i k e l y t h a t the s h o r t p e r i o d of s t a r v a t i o n would have an e f f e c t on f e c u n d i t y because egg r e c r u i t m e n t would have been determined much e a r l i e r i n v i t e l l o g e n e s i s (personal o b s e r v a t i o n ) . T h e r e f o r e the major e f f e c t of s t a r v a t i o n was on egg s i z e r a t h e r than on egg number. There was no apparent e f f e c t of s m a l l e r egg s i z e on s u r v i v a l to eyeing i n t h i s study. No s i g n i f i c a n t d i f f e r e n c e due to p e r i o d of s t a r v a t i o n was found between groups i n f e r t i l i z a t i o n r a t e , number of eyed eggs produced, or r a t e of s u r v i v a l to eyeing (Table 15). 112 Table 14. Morphometric data (mean and standard e r r o r of the means) f o r Big Qualicum 4-year o l d broodstock. Feed was withdrawn from these f i s h f o r 7 or 14 days p r i o r to freshwater t r a n s f e r . P e r i o d of S t a r v a t i o n , days 14 Fork l e n g t h , cm sem (n) 84.16 0.69 (56) 83.58 0.74 (57) Weight, kg sem (n) 8.44 0.19 (56) 8.41 0.21 (57) C o n d i t i o n f a c t o r sem (n) 4.69 0.10 (56) 4.78 0.12 (57) Egg diameter, mm sem (n) 8.6-0.06 (19) 8.3 t o 0.11 (16) ab = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. 113 Table 15. Spawning and i n c u b a t i o n data (mean and standard e r r o r of the means) f o r Big Qualicum 4-year o l d broodstock. Feed was withdrawn from these f i s h f o r 7 or 14 days p r i o r t o freshwater t r a n s f e r . P e r i o d of S t a r v a t i o n , days '7 14 Volume of eggs spawned, ml sem (n) 1266 52.7 (55) 1259 61.8 (57) Fecundity (# eggs spawned) sem (n) 4702 155.3 (54) 4831 149.6 (53) Eyed eggs, # sem (n) 4201 164.8 (53) 4474 166.5 (53) F e r t i l i t y , % sem (n) 92.8 2.90 (18) 93.8 2.39 (16) Eyed, % sem (n) 89.05 1.91 (53) 91.96 1.41 (53) A one-way ANOVA conducted on p e r i o d of s t a r v a t i o n i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e (ot = 0.05) f o r any of the spawning and i n c u b a t i o n parameters, above. 114 Ridelman et al. (1984) compared the r e p r o d u c t i v e performance of rainbow t r o u t f e d ad libitum or s t a r v e d f o r 45 days p r i o r t o spawning. 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 h y s i c a l or chemical c h a r a c t e r i s t i c s , i n c l u d i n g egg s i z e , proximate composition, f e c u n d i t y or egg v i a b i l i t y , were found i n the groups. I t was suggested t h a t v i t e l l o g e n e s i s was complete or n e a r l y so by 45 days p r i o r t o spawning. The p e r i o d of s t a r v a t i o n i n t h i s study was i n i t i a t e d 42 - 49 days p r i o r to the peak of spawning. B l a x t e r (1970) has re p o r t e d t h a t f e c u n d i t y i n c r e a s e s with l e n g t h and weight i n many s p e c i e s . S i g n i f i c a n t (ot = 0.05) p o s i t i v e c o r r e l a t i o n s ( C r i t i c a l v alues of the c o r r e l a t i o n c o e f f i c i e n t , r . Zar, 1984) f o r both groups were found between f e c u n d i t y and f o r k l e n g t h (r=0.465, n=60 i n the r e s t r i c t e d group and r=0.280, n=66 i n the c o n t r o l group) and between f e c u n d i t y and weight (r=0.371, n=60 i n the r e s t r i c t e d group; r=0.583, n=66 i n the c o n t r o l group). C o r r e l a t i o n a nalyses of f e c u n d i t y and c o n d i t i o n f a c t o r r e v e a l e d a small s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n i n the c o n t r o l f i s h (r=0.276, n=66) but a small n e gative c o r r e l a t i o n (not s i g n i f i c a n t ) i n the r e s t r i c t e d f i s h (r=-0.213, n=60). B l a x t e r (1970) and Roy and Higgs (1987) r e p o r t e d t h a t t h e r e i s a strong tendency f o r egg s i z e and f e c u n d i t y to be i n v e r s e l y r e l a t e d . In the r e s t r i c t e d f i s h t h e r e was a s i g n i f i c a n t n e g a tive c o r r e l a t i o n (r=-0.628, n=14) between egg diameter and f e c u n d i t y but there was no s i g n i f i c a n t c o r r e l a t i o n between 115 these parameters i n the c o n t r o l f i s h (r=-0.210, n=18). S u r v i v a l t o eyeing i n both groups was approximately 90%. The BQ f i s h of S e c t i o n 1 on the COMM d i e t (Table 4) produced progeny with s u r v i v a l to eyeing of only 74%. Although the two stocks were d i f f e r e n t , i e . BQ i n S e c t i o n 1 and BQ c r o s s e d with Chemainus males i n S e c t i o n 2, the success of the S e c t i o n 2 f i s h was probably due to t h e i r age at m a t u r i t y . Incubation success i n progeny from f o u r year o l d broodstock i s g e n e r a l l y g r e a t e r than t h a t from three year o l d s (Groves, p e r s . comm. and Lawseth, p e r s . comm.). The high l e v e l s of egg r e t e n t i o n and other a b n o r m a l i t i e s found i n the three year o l d f i s h f e d the WV33 d i e t were not present i n the f o u r year o l d f i s h r e g a r d l e s s of treatment. 2.2.3 Composition of the eggs There were no s i g n i f i c a n t e f f e c t s due to the treatment on dry matter; l i p i d c o n c e n t r a t i o n on a wet or dry weight b a s i s ; p o l a r l i p i d c o n c e n t r a t i o n , as a percent of the t o t a l l i p i d or on a f a t f r e e dry weight b a s i s ; l e v e l of n e u t r a l l i p i d s ; or p r o t e i n p l u s ash content i n the two groups (Table 16). Absolute volumes of t o t a l , p o l a r and n e u t r a l l i p i d per egg were c a l c u l a t e d (Table 17). C o n s i s t e n t l y , the r e s t r i c t e d group had l e s s of each of these l i p i d c l a s s e s . T h i s i n d i c a t e s t h a t had the s t a r v a t i o n p e r i o d been longer, g r e a t e r e f f e c t s on 116 Table 16. Composition of the eggs (mean and standard e r r o r of the means) of 4-year o l d Big Qualicum broodstock. Feed was withdrawn from these f i s h f o r 7 or 14 days p r i o r t o freshwater t r a n s f e r . (n=7 f i s h f o r those s t a r v e d 7 days; n=5 f i s h f o r those s t a r v e d 14 days) P e r i o d of S t a r v a t i o n , days 7 14 mean sem mean sem Dry matter 40.11 0.86 39.21 0.55 Moisture 59.89 0.86 60.77 0.55 L i p i d , ww* 12.84 0.35 12.85 0.19 L i p i d , dw" 32.01 0.40 32.78 0.48 P o l a r l i p i d , dw 12.41 0.03 12.70 0.22 N e u t r a l l i p i d , dw 19.76 0.35 20.15 0.62 P o l a r l i p i d , of t o t a l l i p i d 38.79 0.46 38.75 0.39 N e u t r a l l i p i d , of t o t a l l i p i d 61.73 0.42 61.42 1.29 P o l a r l i p i d , of f a t f r e e dw 18.25 0.13 18.93 0.48 P r o t e i n + ash, ww 27.27 0.56 26.39 0.49 * ww = wet weight; dw = dry weightA one-way ANOVA conducted on p e r i o d of s t a r v a t i o n i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e f o r any of the composition parameters, above (a=0.05). 117 Table 17. Absolute volumes of t o t a l and p o l a r l i p i d per egg c a l c u l a t e d from mean egg volumes and percentages of these parameters. Eggs are from 4-year o l d Big Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r t o freshwater f o r maturation. (n=5 f i s h f o r each group) P e r i o d of S t a r v a t i o n , days 7 14 Mean egg r a d i u s , mm 4.3 - 4.15 b Mean egg volume, mm3 333.0 299.4 (4/3 IT r 3 ) L i p i d c o n c e n t r a t i o n (ww"), % 12.84 12.85 Absolute volume l i p i d / e g g , mm3 42.8 38.5 P o l a r l i p i d of t o t a l l i p i d , % 38.79 38.75 Absolute volume p o l a r l i p i d , mm3 16.6 14.9 N e u t r a l l i p i d of t o t a l l i p i d , % 61.73 61.42 Absolute volume n e u t r a l l i p i d , mm3 26.4 23.6 ab = o r i g i n a l ANOVA r e s u l t s , see Table 14 *ww = wet weight 118 composition may have been found and t h a t even with t h i s s h o r t p e r i o d of r e s t r i c t i o n , a d e c l i n e i n l i p i d c o n c e n t r a t i o n was o c c u r r i n g . 2.2.4 F a t t y a c i d composition of the t o t a l l i p i d s The complete f a t t y a c i d p r o f i l e s of the t o t a l l i p i d s i n the eggs of both groups are presented i n Appendix 4, Table A4.5. The eggs from the r e s t r i c t e d broodstock had s i g n i f i c a n t l y higher l e v e l s of the f a t t y a c i d , 14:0, i n the t o t a l l i p i d s than the c o n t r o l eggs. No other s i g n i f i c a n t d i f f e r e n c e s were present i n the t o t a l l i p i d f a t t y a c i d p r o f i l e (Table A4.5). As shown i n Table 18, 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 t o t a l s a t u r a t e d , n3, n6 or n9 f a t t y a c i d s i n the t o t a l l i p i d s between treatment groups. The n3:n6 r a t i o s were not s u b j e c t e d to ANOVA but they were very c l o s e at 3.98 f o r the r e s t r i c t e d group and 4.01 f o r the c o n t r o l group. 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 the long c h a i n n3 (Table 19) or n6 (Table 20) f a t t y a c i d s or i n the n5, n7, n9 or n i l monounsaturates (Table 21) i n the t o t a l l i p i d s of the two groups of eggs. ' The eggs from the r e s t r i c t e d f i s h had lower a b s o l u t e volumes of t o t a l s a t u r a t e d f a t t y a c i d s , n3 f a t t y a c i d s , n3 HUFAs and 119 Table 18. Saturated, n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from 4-year o l d Big Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) P e r i o d of S t a r v a t i o n , days 7 14 mean sem mean sem Satu r a t e s 16. ,83 0, .23 17. .54 0. ,36 n3 31. ,91 0. .57 31. ,50 0. .78 n6 8. .02 0, .35 7. .93 0. .19 n9 29, .19 0, .52 28, .92 0, .19 A one-way ANOVA conducted on p e r i o d of s t a r v a t i o n i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (ct = 0 . 0 5 ) f o r any of the s e r i e s of f a t t y a c i d s above. P e r i o d of S t a r v a t i o n , days 7 14 mean sem mean sem % % n3:n6 r a t i o " 4.01 0.21 3.98 0.18 * ANOVA was not conducted on the n3:n6 r a t i o s . 120 Table 1 9 . S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 p o l y u n s a t u r a t e d f a t t y a c i d s (n3 PUFAs) and t o t a l h i g h l y unsaturated f a t t y a c i d s (HUFAs) (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from 4-year o l d Big Qualicum broodstock p r i o r t o t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) P e r i o d of S t a r v a t i o n , days 7 14 mean % sem mean % sem 20:5n3 8.76 0 .24 * 8 .41 0 .26 22 :5n3 3 .89 0 .13 3 .73 0 .14 22 :6n3 16 .64 0 .43 16 .88 0 .48 n3 PUFAs 1 2 .23 0 .05 2 .17 0 .06 n3 HUFAs 2 29 .68 0 .62 29 .33 0 .69 1 n3 PUFAs = = unsaturated n3 f a t t y a c i d s with 2 to 4 double bonds 2 n3 HUFAs = unsaturated n3 f a t t y a c i d s with 5 or more double bonds A one-way ANOVA conducted on p e r i o d of s t a r v a t i o n i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (a=0.05) f o r any of the f a t t y a c i d s or s e r i e s of f a t t y a c i d s above. 121 Table 20. S e l e c t e d n6 f a t t y a c i d s , t o t a l n6 p o l y u n s a t u r a t e d f a t t y a c i d s (n6 PUFAs) (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from 4-year o l d B i g Qualicum broodstock p r i o r to t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) P e r i o d of S t a r v a t i o n , days 7 ,14 mean % sem mean % sem 18:2n6 4.56 0.16 4.51 0.18 20:2n6 0.42 0.02 0.40 0.02 20:4n6 1.62 0.06 1.64 0.07 n6 PUFAs 1 8.02 0.29 7.93 0.32 1 n6 PUFAs = unsaturated n6 f a t t y a c i d s with 2 to 4 double bonds A one-way ANOVA conducted on p e r i o d of s t a r v a t i o n i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (a=0.05) f o r any of the f a t t y a c i d s or s e r i e s of f a t t y a c i d s above. 122 Table 21. Monounsaturates, r e p o r t e d as n5, n7, n9, n i l and t o t a l monounsaturated f a t t y a c i d s (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from 4-year o l d Big Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) P e r i o d of S t a r v a t i o n , days 7 14 mean sem mean sem n5 0. .46 .02 0, .44 0. .01 n7 9. .66 0, .12 9, .82 0. .13 n9 29, .19 0, .41 28, .92 0, .46 n i l 0, .77 0, .07 0, .95 0. .08 T o t a l monounsaturates 40, .08 0, .47 40, .13 0. .52 A one-way ANOVA conducted on p e r i o d of s t a r v a t i o n i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (a=0.05) f o r any of the f a t t y a c i d s or s e r i e s of f a t t y a c i d s above. 123 22:6n3 per egg (Table 22) than the c o n t r o l group, probably i n d i c a t i n g t h a t a d e c l i n e i n these f a t t y a c i d s had begun. 2.2.5 F a t t y a c i d composition of the p o l a r l i p i d s The complete f a t t y a c i d p r o f i l e s of the p o l a r l i p i d s i n the eggs of each group are presented i n Appendix 4, Table A4.6. As f o r the t o t a l l i p i d f a t t y a c i d p r o f i l e , the eggs 7of the r e s t r i c t e d f i s h a l s o had s i g n i f i c a n t l y higher l e v e l s of the f a t t y a c i d 14:0 i n the p o l a r l i p i d f r a c t i o n than was present i n the eggs of the c o n t r o l f i s h (Table A4.6). Another s a t u r a t e , 16:0, was a l s o s i g n i f i c a n t l y higher i n the p o l a r f r a c t i o n of these eggs. The only other s i g n i f i c a n t l y d i f f e r e n t f a t t y a c i d was 21:5n3, which was lower i n the eggs of the r e s t r i c t e d f i s h than i n the eggs of the c o n t r o l f i s h . The higher c o n c e n t r a t i o n of s a t u r a t e s i n the eggs from the r e s t r i c t e d broodstock was onl y apparent, an o f f s e t t o the lower c o n c e n t r a t i o n of the n3 f a t t y a c i d s ( p a r t i c u l a r l y 22:6n3) found i n these eggs (Tables 23 and 2 4 ) . T h i s was f u r t h e r demonstrated when the ab s o l u t e volumes of s a t u r a t e d and n3 f a t t y a c i d s per egg were c a l c u l a t e d (Table 2 2 ) . In a b s o l u t e terms, t o t a l s a t u r a t e d f a t t y a c i d s , n3 f a t t y a c i d s , n3 HUFAs and 22:6n3 were a l l lower i n the r e s t r i c t e d eggs than i n the c o n t r o l group. 124 Table 2 2 . Absolute volumes of t o t a l s a t u r a t e s , n3 f a t t y a c i d s , n3 HUFAs and 22 :6n3 i n the t o t a l and p o l a r l i p i d s per egg c a l c u l a t e d from the ab s o l u t e volumes of t o t a l and p o l a r l i p i d per egg and the percentages of these parameters, as re p o r t e d i n prev i o u s t a b l e s . Eggs were from 4-year o l d B i g Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r t o t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) P e r i o d of S t a r v a t i o n , days 7 14 Egg diameter, mm3 8. .6- 8. 3 b Absolute volume t o t a l l i p i d , mm3 42. ,8 38 . 5 Absolute volume p o l a r l i p i d , mm3 16. ,6 1 4 . 9 Composition of T o t a l L i p i d : S a t u r a t e s , % 16. ,83 1 7 . 54 n3 f a t t y a c i d s , % 31. ,91 3 1 . 50 n3 HUFAs, % 29. .68 2 9 . 33 2 2 : 6 n 3 , % 16. .64 1 6 . 88 Composition of Po l a r L i p i d : S a t u r a t e s , % 24. .69 2 5 . 26 n3 f a t t y a c i d s , % 41, .33 - 39 . 20 b n3 HUFAs, % 40, .94 - 38 . 81 b 2 2 : 6 n 3 , % 25, .10 2 3 . 92 Absolute volumes i n T o t a l L i p i d o f : T o t a l s a t u r a t e s , mm3 7. ,2 6 . 8 T o t a l n3 f a t t y a c i d s , mm3 13, .7 1 2 . 1 T o t a l n3 HUFAs, mm3 12, .7 1 1 . 3 T o t a l 2 2 : 6 n 3 , mm3 7, .1 6 . 5 Absolute volumes i n Po l a r L i p i d o f : T o t a l s a t u r a t e s , mm3 4, .1 3 . 8 T o t a l n3 f a t t y a c i d s , mm3 6, .9 5 . 8 T o t a l n3 HUFAs, mm3 6, .8 5 . 8 T o t a l 2 2 : 6 n 3 , mm3 4, .2 3 . 6 ab = o r i g i n a l ANOVA r e s u l t s , see Tables 23 and 24 125 The n3:n6 rat i o s were not tested by ANOVA but there was only a V small difference in the n3:n6 f a t t y acid r a t i o s , 6.90 versus 6.62 for the control and r e s t r i c t e d groups, respectively. There were no other s i g n i f i c a n t differences between the two groups i n t o t a l saturates, n6 or n9 f a t t y acids (Table 23) or in individual long chain f a t t y acids or the n3 PUFAs (Table 24) . There were no s i g n i f i c a n t differences in individual n6 f a t t y acids, i n t o t a l n6 PUFAs (Table 25), or in any of the series (n5, n7, n9, n i l ) of monounsaturates (Table 26) i n the two treatment groups. A loss of n3 f a t t y acids i s contrary to the l i t e r a t u r e which suggests that n3 f a t t y acids are tenaciously retained p a r t i c u l a r l y in polar l i p i d s during starvation while saturates and others are oxidized (Castledine and Buckley, 1980; Castledine and Buckley, 1982; Fremont et al., 1984; B e l l et al. 1985a and B e l l et al.r 1985b). However i f v i t e l l o g e n e s i s was s t i l l occurring, the lower level of n3 f a t t y acids i n the eggs of the r e s t r i c t e d f i s h may indicate that transfer of these f a t t y acids to the eggs was incomplete when feed was withdrawn. Again, had the period of starvation been longer, more pronounced differences in f a t t y acid composition of both the t o t a l and polar l i p i d s may have been seen. 126 Table 23. Saturated, n.3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from 4-year o l d Big Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r to t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) P e r i o d o f S t a r v a t i o n , d a y s 7 14 mean % sem mean % sem S a t u r a t e s 24 .69 0 .24 25. 26 0 .30 n3 41 .33- 0 .29 39. 2 0 b 0 .68 n6 6 .05 0 .27 5. 95 0 .21 n9 18 .64 0 .57 20. 23 0 .79 ab = a s i g n i f i c a n t d i f f e r e n c e (a = 0. 05) was f o u n d between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. P e r i o d o f S t a r v a t i o n , d a y s 7 14 mean sem mean sem % % n3:n6 r a t i o " 6.90 0.37 6.62 0.26 * ANOVA was not conducted on the n3:n6 r a t i o s . 127 Table 24. S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 p o l y u n s a t u r a t e d f a t t y a c i d s (n3 PUFAs) and t o t a l h i g h l y unsaturated f a t t y a c i d s (HUFAs) (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from 4-year o l d Big Qualicum broodstock s t a r v e d f o r 7 or 14 days p r i o r t o t r a n s f e r to freshwater f o r maturation • <n = 5 f i s h f o r each < group) P e r i o d of S t a r v a t i o n , days 7 14 mean % sem mean % sem 20:5n3 11.55 0.26 11.08 0.29 22:5n3 3.94 0.14 3.72 0.15 22:6n3 25.10 0.53 23.92 0.59 n3 PUFAs 1 0.39 0.03 0.39 0.01 n3 HUFAs 2 40.94 - 0. 44 3 8 . 8 1 b 0 .50 1 n3 PUFAs = unsaturated n3 f a t t y a c i d s with 2 to 4 double bonds 2 n3 HUFAs = unsaturated n3 f a t t y a c i d s with 5 or more double bonds ab = a s i g n i f i c a n t d i f f e r e n c e (ot = 0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. 128 Table 25. S e l e c t e d n6 f a t t y a c i d s , t o t a l n6 p o l y u n s a t u r a t e d f a t t y a c i d s (n6 PUFAs) (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from 4-year o l d B i g Qualicum broodstock s t a r v e d 7 or 14 days p r i o r to t r a n s f e r t o freshwater f o r maturation. (n=5 f i s h f o r each group) P e r i o d of S t a r v a t i o n , days 7 14 mean % sem mean % sem 18:2n6 1.74 0.08 1.68 0.10 20:2n6 0.43 0.03 0.50 0.03 20:4n6 2.65 0.12 2.71 0.14 n6 PUFAs 1 6.05 0.24 5.95 0.27 1 n6 PUFAs = unsaturated n6 f a t t y a c i d s with 2 to 4 double bonds A one-way ANOVA conducted on p e r i o d of s t a r v a t i o n i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (a=0.05) f o r any of the f a t t y a c i d s or s e r i e s of f a t t y a c i d s above. 129 T a b l e 26. Monounsaturates, r e p o r t e d as n5, n7, n9, n i l and t o t a l monounsaturated f a t t y a c i d s (mean and s t a n d a r d e r r o r of t h e means) i n POLAR LIPIDS of eggs from 4-year o l d B i g Qualicum b r o o d s t o c k s t a r v e d f o r 7 or 14 days p r i o r t o t r a n s f e r t o f r e s h w a t e r f o r m a t u r a t i o n . (n=5 f i s h f o r each group) P e r i o d o f S t a r v a t i o n , days 7 14 mean sem mean sem % % n5 0.58 0.02 0.52 0.03 n7 5.47 0.13 5.62 0.15 n9 18.64 0.63 20.22 0.71 n i l - - -T o t a l monounsaturates 24.70 0.57 26.37 0.64 A one-way ANOVA conducted on p e r i o d o f s t a r v a t i o n i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (a=0.05) f o r any o f t h e f a t t y a c i d s o r s e r i e s o f f a t t y a c i d s above. 130 2.3 CONCLUSIONS T h i s study was confounded by the e a r l i e r than expected maturation of the broodstock. Had i t been c a r r i e d out as planned, g r e a t e r d i f f e r e n c e s i n egg composition, f a t t y a c i d p r o f i l e s and s u r v i v a l r a t e s may have o c c u r r e d . The s h o r t term s t a r v a t i o n treatment had no e f f e c t on the l i p i d content of the eggs or on the c o n d i t i o n f a c t o r of the broodstock. However l i p i d c o n c e n t r a t i o n (as percent l i p i d , wet weight b a s i s ) was c o n s i d e r a b l y lower i n the eggs of both groups (12.84%; 12.85%) of f i s h compared with the l e v e l s r e p o r t e d by Groves (1987) f o r c u l t u r e d eggs i n 1985 (13.5-19.0%) and i n 1986 (13.5-15.5%) (see Appendix 1). I t appears from t h i s work t h a t the e f f e c t s of s h o r t term s t a r v a t i o n of broodstock on f e c u n d i t y and s u r v i v a l of Chinook salmon eggs are n e g l i g i b l e . Not examined i n t h i s study was the p o t e n t i a l e f f e c t of broodstock s t a r v a t i o n on the growth of a l e v i n s and e a r l y f r y . An experiment of longer d u r a t i o n , i n c o r p o r a t i n g a l e v i n and e a r l y f r y growth, i s needed t o determine at what stage feed withdrawal has a negative e f f e c t . P r i o r t o t h i s a farmer may w e l l cease f e e d i n g broodstock i n order to save labour and feed c o s t s . 131 SECTION 3 - A l t e r a t i o n of l i p i d composition and f a t t y a c i d p r o f i l e s i n eggs from Chinook broodstock. 3.1 MATERIALS AND METHODS Two groups of c u l t u r e d 4-year o l d Chinook salmon were h e l d i n 680 m3 marine net pens at Genoa Bay. There were 245 f i s h i n Pen 3 and 262 f i s h i n Pen 11. Pen d e n s i t i e s were l e s s than 3 kg/ m3 i n both pens. A l l f i s h were f e d a commercial d i e t (COMM) durin g the year b e f o r e t h i s experiment. For s i x weeks p r i o r to the pr e -t r a n s p o r t s t a r v a t i o n p e r i o d , Pen 11 was f e d a brood d i e t (BROOD), commercially a v a i l a b l e t o the i n d u s t r y . The f o r m u l a t i o n of the BROOD d i e t was p r o p r i e t a r y but feed l a b e l i n f o r m a t i o n i s presented i n Appendix 2 Table A2.1. More v i t a m i n E (200 mg), more v i t a m i n C (500 mg) and l e s s crude f a t (2%) were present i n the BROOD d i e t than i n the COMM d i e t . Another d i f f e r e n c e between the two d i e t s , not shown on the feed t a g , was the a d d i t i o n of 4-5% of low temperature d r i e d k r i l l meal t o the BROOD d i e t . Dry matter, l i p i d c o n c e n t r a t i o n and f a t t y a c i d p r o f i l e s of both d i e t s were determined. These data are shown i n Tables A2.3 and A2.4 of Appendix 2. The f i s h were f e d at approximately the same r a t e as the f i s h d e s c r i b e d i n S e c t i o n s 1 and 2. Again, because not a l l the f i s h matured i n 1987, i t was im p o s s i b l e t o c a l c u l a t e feed 132 consumption of the broodstock a c c u r a t e l y . However f e e d i n g l e v e l was approximately 1% of body weight per day at the s t a r t of the summer before spawning and d e c l i n e d to ca. 0.5%, or l e s s , p r i o r t o t r a n s p o r t to the hatchery. Maturing f i s h were t r a n s f e r r e d from the net pens and h e l d i n freshwater u n t i l f u l l y mature and spawned a c c o r d i n g t o the methods o u t l i n e d i n S e c t i o n 1. Sample c o l l e c t i o n procedures, morphometric measurements, a n a l y t i c a l techniques and c a l c u l a t i o n s , d e t e r m i n a t i o n of egg s i z e and procedures f o r measuring f e r t i l i z a t i o n and eyeing success were the same as those d e s c r i b e d i n S e c t i o n 1. A one-way ANOVA (Proc GLM, SAS 1985) was c a r r i e d out on the morphometric and i n c u b a t i o n data; composition parameters; and i n d i v i d u a l f a t t y a c i d s and f a t t y a c i d s e r i e s t o determine s i g n i f i c a n t d i f f e r e n c e s due to the d i e t s . A l l data r e p o r t e d as percentages were transformed by the a r c s i n e t r a n s f o r m a t i o n before a n a l y s e s . The s i g n i f i c a n c e l e v e l was set at 95%. 133 3.2 RESULTS AND DISCUSSION 3.2.1 Morphometric measurements Fork length and weight were measured f o r each female spawned and c o n d i t i o n f a c t o r s were c a l c u l a t e d . These data are presented i n Table 27. 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 f o r k l e n g t h or weight between the two d i e t groups but the c o n d i t i o n f a c t o r of the f i s h on the BROOD d i e t was s i g n i f i c a n t l y lower than those on the COMM d i e t . Egg diameters were a l s o measured and no s i g n i f i c a n t d i f f e r e n c e was found between the two groups (Table 27). 3.2.2 Spawning and i n c u b a t i o n success As shown i n Table 28 the f i s h on the BROOD d i e t produced s i g n i f i c a n t l y g r e a t e r volumes of eggs than those on the COMM d i e t . However, there was no s i g n i f i c a n t d i f f e r e n c e i n f e c u n d i t y or egg diameters between the two groups. The method of measuring the volume of eggs spawned i s crude. The eggs are f r a g i l e at the time of spawning and handl i n g must be minimized. Egg volumes were measured by comparison with l i n e s marked o f f at 100ml i n t e r v a l s on the spawning bucket. Therefore the d i f f e r e n c e i n egg volumes between the two groups was probably due to the in a c c u r a c y of the method of measurement. 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 f e r t i l i z a t i o n success, 134 Table 27. Morphometric data (mean and standard e r r o r of the means) f o r 4-year o l d B i g Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. D i e t COMM BROOD Fork l e n g t h , cm sem (n) 81.5 0.36 (136) 81.8 0.69 (140) Weight, kg sem (n) 7.6 0.07 (136) 7.5 0.10 (140) C o n d i t i o n f a c t o r sem (n) 4.74 -0.05 (136) 4.49 b 0.05 (140) Egg diameter, mm sem (n) 8.41 0.10 (16) 8.30 0.06 (29) ab•= a s i g n i f i c a n t d i f f e r e n c e (a = 0.05) was found between means with a d i f f e r e n t s u p e r s c r i p t w i t h i n the same row. 135 Table 28. Spawning and i n c u b a t i o n data (mean and standard e r r o r of the means) f o r B i g Qualicum 4-year o l d broodstock on a commercial d i e t (COMM) or a brood d i e t (BROOD) p r i o r to t r a n s f e r to freshwater f o r maturation. D i e t COMM BROOD Volume of eggs spawned, ml sem (n) 972 -21.2 (136) 1033 b 22.9 (140) Fecundity (# eggs spawned) sem (n) 4220 130.6 (51) 4413 114.7 (67) Eyed eggs, # sem (n) 3866 137.4 (51) 3979 118.3 (67) F e r t i l i t y , % sem (n) 94.8 2.08 (13) 93.9 3.96 (18) Eyed, % sem (n) 91.79 1.75 (51) 91.89 1.42 (67) ab = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with a d i f f e r e n t s u p e r s c r i p t w i t h i n the same row. 136 number of eyed eggs produced or i n the s u r v i v a l to eyeing between the two groups (Table 2 8 ) . C o r r e l a t i o n analyses of both groups r e v e a l e d s i g n i f i c a n t c o r r e l a t i o n s ( a=0 .05; Z a r , 1984) between f o r k length and f e c u n d i t y (COMM d i e t : r= 0 . 4 0 3 , n=51; BROOD d i e t : r= 0 . 3 2 0 , n=67) and between body weight and f e c u n d i t y (COMM d i e t : r= 0 . 2 9 0 , n=51; BROOD d i e t : r= 0 . 4 3 8 , n=67) as p r e d i c t e d by B l a x t e r (1970) but no s i g n i f i c a n t c o r r e l a t i o n s were found between c o n d i t i o n f a c t o r and f e c u n d i t y (COMM d i e t : r= - 0 . 2 0 7 , n=51; BROOD d i e t : r= 0 . 0 9 9 , n=67) i n e i t h e r group. Roy and Higgs (1987) r e p o r t e d a strong tendency f o r egg s i z e and f e c u n d i t y t o be i n v e r s e l y r e l a t e d but no s i g n i f i c a n t c o r r e l a t i o n between egg diameter and f e c u n d i t y (COMM d i e t : r= 0 . 2 5 0 , n=13; BROOD d i e t : r= - 0 . 2 1 2 , n=18) was found i n e i t h e r group i n t h i s study. 3 . 2 . 3 Composition of the eggs As shown i n Table 29 , no s i g n i f i c a n t d i f f e r e n c e i n dry matter c o n c e n t r a t i o n was found between the two d i e t groups. S i g n i f i c a n t l y g r e a t e r l i p i d l e v e l s (on a wet and dry weight b a s i s ) were evident i n the eggs of f i s h f e d the COMM d i e t than i n those f e d the BROOD d i e t . The p o l a r l i p i d c o n c e n t r a t i o n (as a percent of t o t a l l i p i d ) was s i g n i f i c a n t l y lower i n the COMM group. However, on a f a t - f r e e dry weight b a s i s , the p o l a r l i p i d c o n c e n t r a t i o n was not s i g n i f i c a n t l y d i f f e r e n t between the 137 Table 29. Composition of the eggs (mean and standard e r r o r of the means) of 4-year o l d B ig Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial (COMM) d i e t p r i o r to freshwater t r a n s f e r . (n=5 f i s h f o r each group) D i e t COMM BROOD mean sem mean sem Dry matter Moisture 41.36 58.64 0.40 0.40 40.75 59.25 0.26 0 . 26 L i p i d , ww" L i p i d , dw" 13.58 32.89 0.14 0.65 12.39 fa 0.15 30.29 b 0.42 P o l a r l i p i d , dw Neu t r a l l i p i d , dw 12.04 21.20 0.11 0.19 12.16 0.19 18.38 b 0.16 P o l a r l i p i d , of t o t a l l i p i d 36.66 0.85 40.12 b 0.99 N e u t r a l l i p i d , of t o t a l l i p i d 64.35 0.97 60.67 0.45 P o l a r l i p i d , of f a t f r e e dw P r o t e i n + ash, ww 17.99 27.76 0.43 0.54 18.35 28.36 0.40 0.33 ww wet weight; dw = dry weight ab = a s i g n i f i c a n t d i f f e r e n c e (ot = 0.05) was found between means with a d i f f e r e n t s u p e r s c r i p t w i t h i n the same row. 138 two groups. The lower p o l a r l i p i d l e v e l found i n the eggs from the COMM d i e t group was t h e r e f o r e thought to r e f l e c t the s i g n i f i c a n t l y higher n e u t r a l l i p i d c o n c e n t r a t i o n present i n these eggs. Feed l a b e l i n f o r m a t i o n , presented i n Appendix 2, Table A2.1, shows t h a t the COMM d i e t c o ntained up to 2% more crude f a t than the BROOD d i e t and the same l e v e l s of moisture, crude p r o t e i n , crude f i b r e and ash. The c o n c e n t r a t i o n of n i t r o g e n f r e e e x t r a c t was not l i s t e d . A s i n g l e proximate a n a l y s i s of each d i e t r e v e a l e d l i p i d c o n c e n t r a t i o n s (wet weight b a s i s ) of 16.31% and 15.85% f o r the COMM and BROOD d i e t s , r e s p e c t i v e l y . The higher l i p i d c o n c e n t r a t i o n i n the eggs of f i s h on the COMM d i e t t h e r e f o r e appears t o be d i r e c t l y r e l a t e d to the higher l e v e l of crude l i p i d i n the d i e t . 3.2.4 F a t t y a c i d composition of the t o t a l l i p i d s The complete f a t t y a c i d p r o f i l e s of the t o t a l l i p i d s i n the eggs of both d i e t groups are presented i n Appendix 4, Table A4.7. S i g n i f i c a n t l y higher l e v e l s of 1 6 : l n 9 , 18:4n3 and 2 0 : l n l l and lower l e v e l s of 1 6 : l n 7 and 2 0 : l n 9 were present i n the eggs of f i s h f e d the COMM d i e t than i n those f e d the BROOD d i e t . 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 t o t a l s a t u r a t e s , n3, n6 or n9 f a t t y a c i d s i n the t o t a l l i p i d f a t t y a c i d s were found (Table 30) i n the two d i e t groups. The n3:n6 r a t i o s were not s t a t i s t i c a l l y analyzed but were c l o s e i n value at 3.62 and 139 T a b l e 30. S a t u r a t e d , n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s (mean and s t a n d a r d e r r o r o f t h e means) i n TOTAL LIPIDS o f eggs f r o m 4 - y e a r o l d B i g Q u a l i c u m b r o o d s t o c k on a b r o o d d i e t (BROOD) o r on a r e g u l a r c o m m e r c i a l d i e t (COMM) p r i o r t o t r a n s f e r t o f r e s h w a t e r f o r m a t u r a t i o n . (n=5 f i s h f o r e a c h g r o u p ) D i e t COMM BROOD mean % sem mean % sem S a t u r a t e s 17.03 0.36 18.02 0.36 n3 30.44 0.99 29.59 0.20 n6 7.85 0.17 8.17 0.15 n9 30.13 0.71 29.06 0.24 A one-way ANOVA c o n d u c t e d on d i e t i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (a=0.05) f o r any o f t h e s e r i e s o f f a t t y a c i d s a b o v e . D i e t COMM BROOD mean sem % n3:n6 r a t i o " 3.88 0.07 * ANOVA was not conducted on the n3:n6 r a t i o s . mean sem % 3.62 0.05 140 3.88 f o r the BROOD and COMM eggs r e s p e c t i v e l 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 i n the n3 or n6 PUFAs or i n the n3 HUFAs i n the t o t a l l i p i d s of the two groups as shown i n Tables 31 and 32. The eggs from the f i s h on the COMM d i e t had s i g n i f i c a n t l y g r e a t e r l e v e l s of n i l monounsaturates i n the t o t a l l i p i d s than those from f i s h on the BROOD d i e t (Table 33). The COMM d i e t contained more 2 0 : l n l l and 2 2 : l n l l as shown i n the d i e t a nalyses i n Appendix 2, Table A2.4. Otherwise there were no s i g n i f i c a n t d i f f e r e n c e s between the two d i e t groups i n n5, n7, n9 or t o t a l monounsaturates. 3.2.5 F a t t y a c i d composition of the p o l a r l i p i d s The complete f a t t y a c i d p r o f i l e s of the p o l a r l i p i d s i n the eggs of both groups are presented i n Appendix 4, Table A4.8. As shown i n Table 34, 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 t o t a l s a t u r a t e s , n3, n6 or n9 f a t t y a c i d s i n the p o l a r l i p i d f r a c t i o n of the eggs. The n3:n6 r a t i o s were not subj e c t e d to ANOVA but i t was s l i g h t l y higher f o r the COMM eggs than f o r the BROOD eggs. No s i g n i f i c a n t d i f f e r e n c e s were found i n the i n d i v i d u a l long c h a i n n3 f a t t y a c i d s (Table 35) or i n the long c h a i n n6 f a t t y a c i d s (Table 36). However, the eggs from f i s h on the COMM d i e t contained s i g n i f i c a n t l y more n3 PUFA than d i d those on the BROOD d i e t (Table 35). The f a t t y a c i d analyses of 141 Table 31. S e l e c t e d n3 . f a t t y a c i d s , t o t a l n3 p o l y u n s a t u r a t e d f a t t y a c i d s (n3 PUFAs) and t o t a l h i g h l y unsaturated f a t t y a c i d s (HUFAs) (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from 4-year o l d Big Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) D i e t COMM BROOD mean % sem mean % sem 20:5n3 8.12 0.45 8.29 0.15 22:5n3 3.32 0.22 3.48 0.08 22:6n3 16.38 0.33 15.58 0.14 n3 P U F A s 1 2.27 0.08 2.00 0.10 n3 HUFAs 2 28.16 0.92 27.58 0.25 1 n3 PUFAs = unsaturated n3 f a t t y a c i d s with 2 to 4 double bonds 2 n3 HUFAs = unsaturated n3 f a t t y a c i d s with 5 or more double bonds A one-way ANOVA conducted on d i e t i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (a=0.05) f o r any of the s e r i e s of f a t t y a c i d s r above. 142 Table 32. S e l e c t e d n6 f a t t y a c i d s , t o t a l n.6 p o l y u n s a t u r a t e d f a t t y a c i d s (n6 PUFAs) (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from 4-year o l d B i g Qualicum broodstock on a brood d i e t (BROOD) or on a commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) D i e t COMM BROOD mean sem mean sem Q. 0, 18:2n6 4.65 0.19 4.80 0.20 20:2n6 0.40 0.04 0.47 0.02 20:4n6 1.55 0.02 1.64 0.07 n6 PUFAs 1 7.85 0.17 8.17 0.15 1 n6 PUFAs = unsaturated n6 f a t t y a c i d s with 2 to 4 double bonds A one-way ANOVA conducted on d i e t i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (<x = 0.05) f o r any of the f a t t y a c i d s or s e r i e s of f a t t y a c i d s above. 143 Table 33. Monounsaturates, r e p o r t e d as n5, n7, n9, n i l and t o t a l monounsaturated f a t t y a c i d s (mean and standard e r r o r of the means) i n TOTAL LIPIDS of eggs from 4-year o l d B i g Qualicum broodstock on a brood d i e t (BROOD) or a commercial d i e t (COMM) p r i o r t o t r a n s f e r t o freshwater f o r maturation. (n=5 f i s h f o r each group) D i e t COMM BROOD mean % sem mean % sem n5 0.45 0.01 0.44 0.01 n7 10.09 0.13 10.69 0.18 n9 30.13 0.71 29.06 0.24 n i l 1.02- 0.07 0.68° 0.06 Total monounsaturates 41.69 0.63 40.88 0.26 ab = a s i g n i f i c a n t d i f f e r e n c e (a=0.05) was found between means with a d i f f e r e n t s u p e r s c r i p t w i t h i n the same row. 144 T a b l e 34. S a t u r a t e d , n3, n6 and n9 f a t t y a c i d s e r i e s and n3:n6 r a t i o s (mean and s t a n d a r d e r r o r of t h e means) i n POLAR LIPIDS of eggs from 4-year o l d B i g Qualicum b r o o d s t o c k on a brood d i e t (BROOD) or a commercial d i e t (COMM) p r i o r t o t r a n s f e r t o f r e s h w a t e r f o r m a t u r a t i o n . (n=5 f i s h f o r each group) D i e t COMM BROOD mean sem mean sem -6 -6 S a t u r a t e s 24. .68 0 . 41 25. .85 0, .54 n3 39, .87 0, .52 38, .88 0, .53 n6 6, .20 0, .14 6, .54 0, .17 n9 18, . 31 0, .35 17, .93 0, .79 A one-way ANOVA conducted on d i e t i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (<x = 0.05) f o r any of t h e s e r i e s o f f a t t y a c i d s above. D i e t COMM BROOD mean sem mean sem % % n3:n6 r a t i o " 6.45 0.21 5.95 0.12 * ANOVA was not conducted on t h e n3:n6 r a t i o s . 145 Table 35. S e l e c t e d n3 f a t t y a c i d s , t o t a l n3 p o l y u n s a t u r a t e d f a t t y a c i d s (n3 PUFAs) and t o t a l h i g h l y unsaturated f a t t y a c i d s (HUFAs) (mean and standard e r r o r of the mean) i n POLAR LIPIDS of eggs from 4-year o l d B i g Qualicum broodstock on a brood d i e t (BROOD) or on a r e g u l a r commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) D i e t COMM BROOD mean % sem mean % sem 20:5n3 10.77 0.28 11.00 0.33 22:5n3 3.50 0.18 3.64 0.64 22:6n3 24.27 0.40 23.89 0.38 n3 PUFAs 1 1.33- 0.31 0.35 b 0.04 n3 HUFAs 2 38.54 0.31 38.53 0.53 1 n3 PUFAs = unsaturated n3 f a t t y a c i d s with 2 to 4 double bonds 2 n3 HUFAs = unsaturated n3 f a t t y a c i d s with 5 or more double bonds ab = a s i g n i f i c a n t d i f f e r e n c e (ot = 0.05) was found between means with a d i f f e r e n t s u p e r s c r i p t w i t h i n the same row. 146 Table 36. Selected n6 f a t t y a c i d s , t o t a l n6 polyunsaturated f a t t y acids (n6 PUFAs) (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from 4-year o l d Big Qualicum broodstock on a brood d i e t (BROOD) or on a commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) Diet COMM BROOD mean % sem mean % sem 18:2n6 2.02 0.09 1.90 0 . 05 20:2n6 0.47 0.06 0.56 0.04 20:4n6 2.57 0.05 2.67 0.09 n6 PUFAs 1 6.20 0.14 6.54 0.17 1 n6 PUFAs = u n s a t u r a t e d n6 f a t t y a c i d s w i t h 2 t o 4 double bonds A one-way ANOVA conducted on d i e t i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s (<x = 0.05) f o r any o f t h e f a t t y a c i d s o r s e r i e s of f a t t y a c i d s above. 147 the d i e t s (Appendix 2, Table A2.4) d i d not r e v e a l any l a r g e d i f f e r e n c e s i n i n d i v i d u a l n3 PUFAs. No s i g n i f i c a n t d i f f e r e n c e s were found i n n3 HUFAs (Table 35) or i n n6 PUFAs (Table 36) between eggs of the two groups. As found i n the t o t a l l i p i d s , the eggs of the f i s h on the COMM d i e t c o ntained s i g n i f i c a n t l y more n i l f a t t y a c i d s i n the p o l a r l i p i d s than d i d those on the BROOD d i e t (Table 37). There was ca. 1% more 2 2 : l n l l i n the COMM d i e t than i n the BROOD d i e t t h a t probably accounted f o r t h i s d i f f e r e n c e i n the eggs. 3.2.6 D i e t s Feed tag data f o r the COMM and BROOD d i e t s are provided i n Appendix 2, Table A2.1. The l e v e l of crude f a t i n the BROOD d i e t was reported to be 14% (minimum) versus 16% (minimum) i n the COMM d i e t . The BROOD d i e t c o n tained 5% low temperature d r i e d k r i l l meal. T h i s was expected t o a f f e c t the f a t t y a c i d composition i n the eggs of the f i s h on t h i s d i e t . There were d i f f e r e n c e s i n the supplemented l e v e l s of Vitamin E and C i n the two d i e t s a l s o . Otherwise the tag data was i d e n t i c a l f o r the two d i e t s . P a r t i a l proximate analyses of the d i e t s are presented i n Table A2.3. Contrary to the feed tag data, the moisture l e v e l i n the COMM d i e t was 8.50% and the l i p i d l e v e l s were c a . 16% f o r both d i e t s . Therefore the major d i f f e r e n c e s between the d i e t s were 148 Table 37. Monounsaturates, r e p o r t e d as n5, n7, n9, n i l and t o t a l monounsaturated f a t t y a c i d s (mean and standard e r r o r of the means) i n POLAR LIPIDS of eggs from 4-year o l d Big Qualicum broodstock on a brood d i e t (BROOD) or a commercial d i e t (COMM) p r i o r to t r a n s f e r to freshwater f o r maturation. (n=5 f i s h f o r each group) D i e t COMM BROOD mean sem mean sem -6 n5 0. .52 0. .03 0. ,52 0. .05 n7 5, .73 0. .14 6. ,07 0, .13 n9 18, .31 0, .35 17, .93 0, .79 n i l 0, .45- 0, .07 0, .19° 0, .05 To t a l monounsaturates 25, .18 0, .41 24, .79 0, .80 ab = a s i g n i f i c a n t d i f f e r e n c e ( a = 0 . 0 5 ) was found between means with a d i f f e r e n t s u p e r s c r i p t w i t h i n the same row. 149 t h e V i t a m i n C a n d E l e v e l s a n d t h e p r e s e n c e o f k r i l l m e a l i n t h e BROOD d i e t . 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 d i e t s a r e l i s t e d i n T a b l e A 2 . 4 a n d , f o r c o m p a r i s o n w i t h t h e BROOD d i e t , a e u p h a u s i i d ( k r i l l ) s a m p l e i s p r e s e n t e d i n T a b l e A 2 . 5 . Some o f t h e d i f f e r e n c e s i n f a t t y a c i d c o n c e n t r a t i o n b e t w e e n t h e two d i e t s seem t o r e f l e c t t h e p r e s e n c e o f k r i l l i n t h e BROOD d i e t ; o t h e r s do n o t . F o r e x a m p l e t h e k r i l l a n d t h e BROOD d i e t h a d h i g h e r l e v e l s o f 1 4 : 0 a n d 1 6 : 0 t h a n t h e COMM d i e t b u t , b a s e d on t h e c o n c e n t r a t i o n o f 2 0 : 5 n 3 a n d 22 :6n3 i n t h e k r i l l s a m p l e , h i g h e r l e v e l s o f t h e s e f a t t y a c i d s m i g h t h a v e b e e n e x p e c t e d i n t h e BROOD d i e t t h a n was f o u n d i n t h e COMM d i e t . T h i s r a i s e s t h e p o s s i b i l i t y t h a t t h e BROOD d i e t o r t h e c o n s t i t u e n t k r i l l m e a l h a d o x i d i z e d w i t h t h e l o s s o f some o f t h e l o n g c h a i n f a t t y a c i d s . When c o m p a r e d w i t h t h e f a t t y a c i d p r o f i l e o f t h e WV33 d i e t ( f e d t o t h e b r o o d s t o c k i n S e c t i o n 1 ) , t h e COMM a n d BROOD d i e t s were r e l a t i v e l y s i m i l a r t o e a c h o t h e r ( T a b l e A 2 . 4 ) . F i g u r e 8 d i s p l a y s s e l e c t e d f a t t y a c i d s a n d s e r 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 a n d p o l a r l i p i d s o f t h e COMM a n d t h e BROOD g r o u p s o f eggs t o g e t h e r w i t h t h e same p a r a m e t e r s f o r t h e r e s p e c t i v e d i e t s . T h e i n d i v i d u a l g r a p h s o f t h i s f i g u r e c l e a r l y show t h e s i m i l a r i t y i n f a t t y a c i d p r o f i l e s b e t w e e n t h e d i e t s t h e m s e l v e s a n d b e t w e e n t h e e g g s o f t h e two d i e t g r o u p s i n b o t h t h e t o t a l a n d p o l a r l i p i d s . 150 Figure 8: A comparison of selected fatty acids in the total (TL) and polar (PL) lipids of eggs of 4-year old broodstock with those in their diets (COMM and BROOD) COMM Diet R A T I O SATURATES n3 nS nS n3:n6 RATIO HH COMM E33TL EES PI-B R O O D Diet R A T I O SATURATES n3 n6 n9 n3m6 RATIO • • BROOD ESS TL HZiPL COMM Diet BROOD Diet 2ftSn3 22:5n3 22:6n3 n3 PUFAs n3 HUFAs •JB COMM !§33 TL E2Z1 PL 20:6n3 22:6n3 22:6n3 n3 PUFAs n3 HUFAs •JB BROOD M TL EM PL SO COMM Diet j 1 1 1 1 ^ i l l 16:1n7 18:109 22:1n11 18:2n6 20:4(|8 n6 PUFAs • • COMM SS! TL [11 PL 60 BROOD Diet 4 1 1 1 BSU B l i 18:1n7 18:1n9 22:1n11 18:2nS 20:4n6 nS PUFAs • • BROOD ^ TL EM PL 151 Egg composition was not c o n s i s t e n t l y a r e f l e c t i o n of the d i e t . Saturates and n9 f a t t y a c i d s were the predominant s e r i e s i n the d i e t s but the n3 f a t t y a c i d s were found i n the g r e a t e s t c o n c e n t r a t i o n i n the t o t a l l i p i d s of both groups of eggs with the n9 f a t t y a c i d s ( p r i m a r i l y 18:ln9) a c l o s e second. The p o l a r l i p i d s of both groups of eggs contained n3 f a t t y a c i d s i n the h i g h e s t c o n c e n t r a t i o n . The n3 HUFAs were the primary c o n t r i b u t o r s to the high n3 l e v e l s i n both the t o t a l and p o l a r f a t t y a c i d s . The d i e t s were not the s o l e source of f a t t y a c i d s . R e t e n t i o n of e s s e n t i a l f a t t y a c i d s , p a r t i c u l a r l y the n3 HUFAs, and e l o n g a t i o n and d e s a t u r a t i o n of f a t t y a c i d s a l s o c o n t r i b u t e d to the f i n a l egg p r o f i l e s . The source of the high 18:ln9 c o n c e n t r a t i o n i n the egg t o t a l l i p i d s appeared to be p a r t i a l l y due to e l o n g a t i o n and d e s a t u r a t i o n of s a t u r a t e d f a t t y a c i d s as w e l l as to the presence of high l e v e l s i n the d i e t s . 152 3.3 CONCLUSIONS The BROOD and COMM d i e t s were not s u f f i c i e n t l y d i f f e r e n t i n f a t t y a c i d composition to produce major d i f f e r e n c e s i n egg composition. Had the d i e t s been f e d over the e n t i r e p e r i o d of v i t e l l o g e n e s i s , g r e a t e r d i f f e r e n c e s may have r e s u l t e d . The higher l i p i d l e v e l i n the COMM d i e t r e s u l t e d i n a g r e a t e r p r o p o r t i o n of n e u t r a l to p o l a r l i p i d s i n the eggs of the broodstock f e d t h i s d i e t . No advantage or disadvantage c o u l d be d i r e c t l y a t t r i b u t e d to higher l i p i d l e v e l s during the e a r l y i n c u b a t i o n p e r i o d . The egg m o r t a l i t y which has caused concern i n the past t y p i c a l l y o c c u r r e d between f e r t i l i z a t i o n and eyeing. 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 eyeing r a t e between the two groups i n t h i s study. Beyond eyeing and up to f i r s t f e e d i n g , an e x t r a s t o r e of l i p i d may be u s e f u l i f l i p i d c o n c e n t r a t i o n became l i m i t i n g t o growth and maintenance. The BROOD d i e t t h e o r e t i c a l l y c o n tained more (200 I.U./kg) v i t a m i n E and more (500 mg/kg) v i t a m i n C than the COMM d i e t . No e f f e c t of these d i f f e r e n c e s was evi d e n t i n t h i s study. No s i g n i f i c a n t advantage i n f e c u n d i t y , f e r t i l i z a t i o n r a t e or i n c u b a t i o n success was c o n f e r r e d on the eggs of the f i s h f e d the BROOD d i e t . Unless f e e d i n g t h i s a d i e t over a longer p e r i o d can be shown to r e s u l t i n some s i g n i f i c a n t b e n e f i t , the a d d i t i o n a l c o s t of t h i s d i e t i s not warranted. 153 CONCLUDING REMARKS r Since 1986, the s u r v i v a l of eggs from c u l t u r e d Chinook broodstock has improved markedly. Since 1988 an annual s u r p l u s of smolts has been produced by the i n d u s t r y and at present a high i n c i d e n c e of pre-eyeing egg m o r t a l i t y appears to be i n the p a s t . The year 1987 was p i v o t a l f o r hatchery p r o d u c t i o n of smolts. The eyed s u r v i v a l r a t e s observed i n these s t u d i e s r e f l e c t e d those obtained throughout the i n d u s t r y . Eyed s u r v i v a l s from broodstock f e d the COMM d i e t r e p o r t e d here, ranged from c a . 74% i n the progeny of the three year o l d broodstock to 92% i n the progeny of fou r year o l d broodstock - s i g n i f i c a n t l y b e t t e r than i n 1986 and p r i o r y e a r s . Changes i n d i e t f o r m u l a t i o n made by manufacturers appear to be the main reason f o r t h i s success at Sea Spring Salmon Farm L t d . Improved broodstock management and spawning and i n c u b a t i o n techniques may have c o n t r i b u t e d to the success at some f a c i l i t i e s . Commercial d i e t f o r m u l a t i o n s are c l o s e d . I t i s t h e r e f o r e impossible to determine which m o d i f i c a t i o n s promoted b e t t e r s u r v i v a l . 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Reproduction and s u r v i v a l of rainbow t r o u t (Salmo gairdneri) f e d l i n o l e n i c a c i d as the only source of e s s e n t i a l f a t t y a c i d s . L i p i d s 14(6):572-575. Yu, T . C , R.O. Sinnhuber and G.B. Putnam. 1977. E f f e c t of d i e t a r y l i p i d s on f a t t y a c i d composition of body l i p i d i n rainbow t r o u t (Salmo gairdneri). L i p i d s 12(6):495-499. Zar, J.H. 1984. " B i o s t a t i s t i c a l A n a l y s i s . " P r e n t i c e - H a l l , Inc. Englewood C l i f f s , N.J. 718 pp. 164 APPENDIX 1 P r e l i m i n a r y analyses of s e l e c t e d n u t r i e n t parameters i n the eggs of w i l d and c u l t u r e d Chinook salmon - 1985 & 1986. (Mean or range of values g i v e n ) 1 1985: C u l t u r e d Eggs Wild Eggs % Moisture 66.1 57.7 % L i p i d , wet weight b a s i s 13.5-19.0 8.7-13.2 F a t t y a c i d s %18:1 31.4 21.9 %18:2 5.0 0.9 %20:5 20.5 32.0 Selenium, ug/g dry weight 1.03 1.46 Vitamin A, ug/g dry weight 4.1 7.7 1986: % Moisture 62 .0 57 .7 % L i p i d (eggs), wet weight b a s i s 13 .5-15.5 6 .0 % L i p i d (muscle), wet weight b a s i s 2 .4-8.0 1 .0 F a t t y a c i d s %18:1 26 .4 23 .7 %18:2 3 .8 1 .0 %20: 5 10 .1 17 .4 Selenium, ug/g dry weight 2 .16 3 .24 Vitamin A, ug/g dry weight 3 .2 3 .9 Vitamin E, IU/kg dry weight 106 .7 73 .4 1 from Groves, 1987. 165 APPENDIX 2 D i e t Data Table A2.1: Manufacturer's feed l a b e l data f o r commercial (COMM) and brood (BROOD) d i e t s . Commercial (COMM) Brood (BROOl Moisture 10.0% 10.0% Min. Crude P r o t e i n 47.0% 47.0% Min. Crude Fat 16.0% 14.0% Max. Crude F i b r e 3.0% 3.0% Maximum Ash 10.0% 10.0% A c t u a l Phosphorus 1.5% 1.5% A c t u a l Calcium 2.2% 2.2% A c t u a l Sodium 0.7% 0.7% Min. I.U. Vitamin "A" per kg 10,000 10,000 Min. I.U. Vitamin "D" per kg 2,400 2,400 Min. I.U. Vitamin "E" per kg 300 500 Min. I.U. Vitamin "C" per kg 700 1,200 Included i n BROOD d i e t but not s t a t e d on the feed l a b e l was 5% low temperature d r i e d k r i l l meal. 166 APPENDIX 2 D i e t Data Table A2.2: Formulation of the West Vancouver 33 (WV33) d i e t . I n g r e d i e n t : g/kg steam d r i e d h e r r i n g meal 550 d r i e d whey 80 corn d i s t . d r i e d s o l u b l e s 45 blood f l o u r 50 whole f r o z e n euphausiids 20 f r e e z e - d r i e d euphausiids 10 wheat middlings 81 v i t a m i n supplement 1 43 mineral supplement 2 20.4 h e r r i n g or salmon o i l 75 (with a n t i o x i d a n t ) permapel ( l i g n i n sulphonate binder) 18.9 a s c o r b i c a c i d 1.9 c h o l i n e c h l o r i d e (60%) 4.7 canthaxanthin (10% potency) 0.4 1000.3 1 The v i t a m i n supplement s u p p l i e d the f o l l o w i n g l e v e l s of n u t r i e n t s / kg d i e t : v i t a m i n A a c e t a t e 15000 I.U.; c h o l e c a l c i -f e r o l 3000 I.U.; DL-alpha-tocopherol a c e t a t e 567 I.U.; menadione (as Hetrazeen) 24.8 mg; D-calcium pantothenate 182.9 mg; p y r i d o x i n e HCl 42.2 mg; r i b o f l a v i n 56.7 mg; n i a c i n 284 mg; f o l i c a c i d 18.9 mg; thiamine mononitrate 38.4 mg; b i o t i n 2.84 mg; cyanocobalamin 0.057 mg; i n o s i t o l 378 mg 2 The mineral supplement s u p p l i e d the f o l l o w i n g l e v e l s of m i n e r a l s / kg d i e t : Mn (as MnS0 4 * H 20) 69.2 mg; Zn (as ZnS0 4 * 7H 20) 45.0 mg; Co (as C o C l 2 * 6H 20) 0.94 mg; Cu (as CuS0 4 * 5H 20) 4.50 mg; Fe (as FeS0 4 * 7H 20) 47.3 mg; I (as KI0 3) 5.1 mg / 167 APPENDIX 2 D i e t Data Table A2.3: Dry matter and l i p i d composition of the experimental d i e t s used i n S e c t i o n s 1, 2 and 3. COMM WV3 3 BROOD Dry matter % Moisture % L i p i d , % wet weight L i p i d , % dry weight 91.50 8.50 16.31 17.83 90.33 9.67 14.38 15.92 89.91 10.09 15.85 17.63 COMM = commercial d i e t ( S e c t i o n s 1 & 2) WV33 = West Vancouver 33 d i e t ( S e c t i o n 1) BROOD = brood d i e t ( S e c t i o n 3) 168 APPENDIX 2 Diet Data Table A2.4 Fatty a c i d p r o f i l e s of d i e t s . Diet codes : COMM = commercial, WV33 = Wes Vancouver 33, BROOD = commercial brood Fatty COMM WV33 BROOD Acid % % % 14:0 5.37 5.59 6.15 0 ** 0.87 1.75 0.55 14:ln9 0.34 0.51 0.39 l a ** 0.26 0.00 0.28 16:0 16.75 17.00 18.36 16:ln9 0.00 0.24 0.21 16:ln7 5.75 5 . 07 6.70 16:ln5 0.00 0.24 0.21 4 * * 1.36 1.27 1.46 4a ** 0.22 0.34 0.23 5 ** 0.41 0.65 0.70 6 ** 0.50 0.33 0.59 18:0 2.76 3.14 2.76 18:ln9 19.40 17.08 19.60 18:ln7 3.09 2.77 3.25 18:ln5 0.35 0.54 0.34 18:2n6 5.03 3.42 5.66 18:3n6 0.00 0.33 0.00 18:3n3 0.99 0.79 0.95 18:4n3 1.03 1.52 1.08 20: 0? 0.26 0.00 0.00 2 0 : l n l l 2.70 4.28 2.39 20:ln9 5.78 2.76 5.79 20:ln7 0.28 0.36 0.28 20:4n6 0.44 0.48 0.40 20:4n3 0.29 0.83 0.22 20:5n3 5.32 6.96 5.11 2 2 : l n l l 9.58 5.11 8.47 22:ln9 0.65 0.79 0.66 21:5n3 1.56 4.59 1.46 22:4n6? 0.57 1.04 0.00 22:5n3 0.62 1.28 0.56 22:6n3 5.15 8.07 4.30 t o t a l 97.68 99.13 99.11 t o t a l n3 14.96 24.04 13.68 t o t a l n6 5.47 4.23 6.06 n3:n6 r a t i o 2.73 5.68 2.26 ** = unknown/ u n i d e n t i f i e d f a t t y a c i d s 169 APPENDIX 2 D i e t Data Table A2.5: Dry matter, l i p i d composition and f a t t y a c i d p r o f i l e of a eupha u s i i d ( k r i l l ) sample. Fatty K r i l l A c i d % 14: 0 7.11 0 ** 0.43 14:ln9 1.71 l a ** 0.89 16: 0 21.25 16:ln9 8.05 16:ln7 0.60 2a ** 0.46 3 ** 0.28 4 * * 1.47 4a ** 2.94 5 ** 1.55 ? * * 0.46 6 ** 1.16 18:0 1.24 18:ln9 7.66 18:ln7 5.09 18:2n6 1.44 18:3n6 0.24 18:3n3 2.04 18:4n3 4.89 20:0? 1.47 20:ln7 0.25 -? * * 1.07 20:4n6 0.71 20:4n3 0.31 20:5n3 10.45 21:5n3 0.44 22:4n6 • 4.49 22:5n6 1.59 22:5n3 0.26 22:6n3 6.00 t o t a l 98.00 t o t a l n3 24.39 t o t a l n6 8.47 n3:n6 r a t i o 2.88 Dry matter % 21.54 Moisture % 78.47 L i p i d , % wet weight 5.06 L i p i d , % dry weight 23.47 ** = unknown/ u n i d e n t i f i e d f a t t y a c i d s 170 APPENDIX 3 Formulae f o r C a l c u l a t i n g Composition Parameters. Percent L i p i d (wet weight b a s i s ) : [mean dry weight of l i p i d i n 10 ml a l i q u o t of l i p i d e x t r a c t * (volume of l i p i d e x t r a c t / 10 ml)] * 100 % = t o t a l l i p i d i n sample ( t o t a l l i p i d i n sample/ wet weight of sample) * 100 % = Percent L i p i d (wet weight b a s i s ) Percent L i p i d (dry weight b a s i s ) : ( t o t a l l i p i d i n sample/ dry weight of sample) * 100 % = Percent L i p i d (dry weight b a s i s ) Percent P o l a r L i p i d of T o t a l L i p i d : ( t o t a l amount l i p i d i n sample/ volume of chl o r o f o r m l a y e r ) * volume of a l i q u o t a p p l i e d to s i l i c a c a r t r i d g e s = amount l i p i d i n the a l i q u o t a p p l i e d to s i l i c a c a r t r i d g e s . ( p o l a r l i p i d i n a l i q u o t a p p l i e d to s i l i c a c a r t r i d g e s / amount l i p i d i n a l i q u o t ) * 100% = Percent P o l a r L i p i d of T o t a l L i p i d Percent Neutral L i p i d of T o t a l L i p i d : ( n e u t r a l l i p i d i n a l i q u o t a p p l i e d to s i l i c a c a r t r i d g e s / amount l i p i d i n a l i q u o t ) * 100% = Percent N e u t r a l L i p i d of T o t a l L i p i d Percent P o l a r L i p i d (dry weight b a s i s ) : percent p o l a r l i p i d of t o t a l l i p i d * percent l i p i d (dry weight) = Percent P o l a r L i p i d (dry weight b a s i s ) Percent Neutral L i p i d (dry weight b a s i s ) : percent n e u t r a l l i p i d of t o t a l l i p i d * percent l i p i d (dry weight) = percent n e u t r a l l i p i d (dry weight b a s i s ) Percent P o l a r L i p i d of Fat Free Dry Weight: ( p o l a r l i p i d / volume a l i q u o t a p p l i e d to s i l i c a c a r t r i d g e s ) * volume of chloroform l a y e r = t o t a l p o l a r l i p i d i n whole sample t o t a l p o l a r l i p i d / (sample dry weight - t o t a l amount l i p i d i n sample) * 100% = Percent P o l a r L i p i d of Fat Free Dry Weight Percent P r o t e i n + Percent Ash (wet weight b a s i s ) : 100 % - [% moisture + % l i p i d ] = (wet weight b a s i s ) Percent P r o t e i n + Percent Ash 171 APPENDIX 4 F a t t y A c i d P r o f i l e s o f Eggs T a b l e A4.1: F a t t y a c i d p r o f i l e s (mean and s t a n d a r d e r r o r of t h e means) of TOTAL LIPID i n the eggs o f BIG QUALICUM b r o o d s t o c k on t h r e e d i e t s . D i e t codes: COMM = com m e r c i a l ; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . (n = 5 f o r a l l groups) WV33 COMM WILD F a t t y A c i d mean sem mean 2-sem mean sem 14: 0 O 2.08 0.04 'o 2.39 0.12 -6 2.43 0.23 14: l n 9 1.73 0.59 1.11 0.19 1.71 0.46 ^ * * 0.25 0.01 0.25 0.01 0.26 0.03 16: 0 11.71 0.22 11.90 0.16 11.85 0.41 16:ln9 0.60 0.06 0.57 0.08 0.42 0.10 16:ln7 . 5.72 0.13 6 .42 0.07 7.76 ° • 0 .17 4 ** 0.21 0.00 0.10 t> 0.06 0.56 ° B 0.02 5 ** 0.41 0.03 0.42 0.02 0.42 0.03 18:0 4.21 Ek 0.04 4.36 A B 0.09 5.12 to 0.04 18:ln9 23.73 Ek 0.26 26.55 t> 0.25 18.71 c= 0.53 18:ln7 4.30 EL 0.05 3.87 to 0.08 3.89 B B 0.15 18:l n 5 0.45 0.01 0.43 0.01 0.42 0.03 1 * * 0.29 EL 0.06 0.36 2 L 0.06 0.00 to 0.00 18:2n6 3.43 ~B 0.07 4.79 to 0.14 0.79 0.05 18:2n4 0.00 Ek 0.00 0.00 0.00 0.32 B B 0.02 18:3n6 0.25 EL 0.07 0.44 k> 0.05 0.00 0.00 18:3n4 0.11 Ek 0.05 0.17 0.04 0.47 to 0.02 18:3n3 0.54 0.02 0.63 0.03 0.72 0 . 05 18:4n3 0.68 0.03 0.71 0.04 0.85 0.05 1 8 : 4 n l 0.23 EL 0.00 0.21 0.03 0.50 to 0.05 2 0 : l n l l 0.72 0.03 0.73 0.03 0.40 0.19 20:ln9 1.94 Ek 0.06 2.10 .a. 0.11 0.86 to 0.05 £ * * 0.32 0.03 0.34 £ L 0.04 0.00 to 0.00 20:2n6 0.36 a. 0.01 0.39 0.04 0.00 to 0.00 20:3n6 0.70 Ek 0.03 0.91 to 0.05 0.00 e= 0.00 20:4n6 1.60 EL 0.04 1.68 SL 0.07 1.15 B B 0.02 20:4n3 0.69 Ek 0.04 0.56 £ L 0.04 1.34 B B 0.04 20:5n3 8.88 ^ 0.16 7.45 fa 0.14 13.70 ° B 0.36 2 2 : l n l l 0.39 EL 0.02 0.45 21 0.05 0.13 to 0.08 21:5n3 0.31 0.04 0.24 0.00 0.75 0.36 y * * 0.11 0.05 0.11 0.08 0.18 0.07 22:4n6 0.26 • 0.02 0.23 D 0.03 0.40 0.12 22:5n3 3.47 Ek 0.08 2.74 FAB 0.15 5.49 c- m 0.23 22:6n3 17.73 Ek 0.39 14.87 t> 0.45 17.09 SL 0.74 t o t a l 98.38 98.49 98.66 ** = unknown/ u n i d e n t i f i e d f a t t y a c i d s abc = a s i g n i f i c a n t d i f f e r e n c e (a = 0.05) was found between means w i t h d i f f e r e n t s u p e r s c r i p t s w i t h i n t h e same row. B = a s i g n i f i c a n t d i f f e r e n c e (<x = 0.05) was found between  s t o c k s f o r t h e f a t t y a c i d i n d i c a t e d . See T a b l e A4.2. 172 APPENDIX 4 Fa t t y A c i d P r o f i l e s of Eggs Table A4.2: v Fa t t y a c i d p r o f i l e s (mean and standard e r r o r of the means) of TOTAL LIPID i n the eggs of ROBERTSON CREEK broodstock on three d i e t s . D i e t codes: COMM = commercial; WV33 = West Vancouver 33; WILD = n a t u r a l d i e t . (n = 5 f o r WV33 and WILD; n = 3 f o r COMM) WV33 COMM WILD Fa t t y A c i d mean & sem mean ?. sem mean sem 14:0 o 2.20 0.06 *o 2.29 0.07 'o 2.54 0.26 14:ln9 2.01 0.53 1.04 0.13 0.91 0.17 1 * * 0.25 0.01 0.24 0.00 0.20 0.05 16: 0 12.15 0.22 11.15 0.10 12.17 0.32 16:ln9 0.36 0 .15 0.54 0.04 0.54 0.08 16:ln7 5.99 A B 0.13 6.47 0.10 7.01 0.14 4 * * 0.06 0.04 0.04 0.03 0.16 • 0.05 5 ** 0.40 0.02 0.33 0.02 0.35 0.01 18: 0 4.34 SL 0.12 4.77 0.14 5.24 0.07 18:ln9 24.96 i - 0.64 26.16 0.20 19.56 i b 0.66 18:ln7 4.06 a 0.04 4.15 a 0.06 2.95 B B 0.09 18:ln5 0.44 0.01 0.42 0.01 0.47 0.05 7 * * 0.32 a 0.02 0.31 a. 0.01 0.00 fa 0.00 18:2n6 3.85 A B 0.06 4.59 t> 0.14 0.84 o 0.07 18:2n4 0.00 0.00 0.00 a 0.00 0.27 B B 0.03 18:3n6 0.30 a 0.03 0.29 a 0.02 0.00 fa 0.00 18:3n4 0.17 •zafa 0.02 0.11 a. 0.05 0.29 fa 0.04 18:3n3 0.60 0.01 0.63 0.03 0.71 0.09 18:4n3 0.63 a. 0.06 0.56 a 0.02 1.08 fa 0.13 18:4nl 0.17 a 0.05 0.25 a 0.01 0.45 fa 0.06 2 0 : l n l l 0.66 0.04 0.54 0.04 0.41 0.19 20:ln9 1.70 0.09 2.13 fa 0.11 0.87 cz: 0.06 f * * 0.31 0.05 0.37 a 0.04 0.00 fa 0.00 20:2n6 0.32 0.01 0.46 fa 0.03 0.00 cr 0.00 20:3n6 0.78 a 0.05 0.98 a 0.03 0.03 fa 0.03 20:4n6 1.62 a 0.03 1.73 a 0.03 1.02 B B 0.03 20:4n3 0.66 a 0.04 0.62 a 0.04 1.71 *>• 0.21 20:5n3 8.36 a. 0.24 7.88 a 0.07 15.06 FAB 0.46 2 2 : l n l l 0.37 3L 0.01 0.27 a f a 0.10 0.11 fa 0.08 21:5n3 0.33 0.03 0.21 0.03 0.37 0.05 Y * * 0.27 0.06 0.20 0.15 0.25 0.11 22:4n6 0.10 A B 0.05 0.07 a H 0.05 0.30 fa 0.02 22:5n3 3.10 a. 0 .07 3.28 a H 0.19 4.81 B B 0.20 22:6n3 16 .70 a. t> 0.25 15.63 0.09 17.81 fa 0.51 t o t a l 98.51 98.67 98.47 ** = unknown/ u n i d e n t i f i e d f a t t y a c i d s abc = a s i g n i f i c a n t d i f f e r e n c e (a = 0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row, H = a s i g n i f i c a n t d i f f e r e n c e (a = 0.05) was found between  stocks f o r the f a t t y a c i d i n d i c a t e d . See Table A4.1. 173 APPENDIX 4 Fatty A c i d P r o f i l e s of Eggs Table A 4 . 3 : F a t t y a c i d p r o f i l e s (mean and standard e r r o r of the means) of POLAR LIPID i n the eggs of BIG QUALICUM broodstock on three d i e t s . Diet codes: COMM = commercial; WV33 = West Vancouver 33; WILD = natu r a l d i e t . (n = 5 f i s h f o r a l l groups) WV33 COMM WILD F a t t y A c i d mean 9-sem mean 9-sem mean 9-sem 14: 0 o 1.09 0.04 '0 0.92 EL- 0.03 "6 1.32 to 0.10 1 4 : l n 9 5.64 0 .79 3 40 0.42 2 .44 0 .69 2 * * 0 .21 0 .01 0 .13 0 .05 0 .23 0 .06 16 :0 15 .22 0 .37 15 .55 0.27 16 .41 0 .38 1 6 : l n 9 0 .45 0.06 0 . 35 m 0 .09 0 .39 0 .06 1 6 : l n 7 2 .05 0 .15 2 .08 &L 0.10 2 .87 bB 0.19 4 * * 0.44 0 .19 1.05 b B 0.39 0 .22 EL 0.17 4a ** 0.28 0 .03 0 .36 0 .02 0 .24 0 .06 18 :0 7.46 St 0.25 8.48 b B 0.29 10 .07 C3 0.31 1 8 : l n 9 12 .27 EL 0.39 13 .73 t> 0.23 9 .93 O 0.38 1 8 : l n 7 4 .04 0 .13 3.76 0.08 3 .72 • 0.19 1 8 : l n 5 0 .51 0 .02 0.48 0 .03 0 .49 0 .05 18:2n6 1.39 EL 0.07 2 .03 to 0.04 0.28 0 .04 18 :3n3 0.05 0 .05 0.18 0 .05 0 .25 0.07 18:4n3 0 .09 0 .04 0 .14 0 .06 0 .19 0 .10 1 8 : 4 n l 0 .00 EL 0.00 0 .00 0 .00 0 .20 b B 0.05 2 0 : l n l l .0 .31 £ L 0.02 0 .35 a B 0.02 0 .12 to 0.09 2 0 : l n 9 2 .31 EL 0.10 2 .46 EL 0.14 0.96 to 0.15 2 0 : l n 7 0 .20 ~ B 0.05 0 .14 EL 0.06 0 .00 to 0.00 20:2n6 0 .42 0 .02 0 .45 0 .03 0 .00 to 0.00 20:3n6 0.77 a B 0.03 1.12 to 0.03 0 .00 c= 0.00 20:4n6 2 .43 a II 0.07 2 .88 to 0.12 1.68 C3 0.04 20:4n3 0 .29 0 .04 0 .25 ZL 0.06 0 .66 to 0.09 20:5n3 10 .69 0 .25 10 .59 0 .16 14 .97 to 0.45 2 2 : l n l l 0 .40 0 .37 0 .02 0 .03 0 .25 0 .25 21:5n3 0 .35 0 .14 0 .00 to 0.00 0.14 0 .07 Y * * 0 .11 0 .08 0 .25 0 .12 0 .29 0 .13 22:4n6 0.47 0 .01 0.56 0 .02 0 .45 0 .09 22:5n3 3.48 EL 0.04 3 .19 0 .19 5 .22 faB 0.37 22:6n3 24 .69 0 .33 23 .59 0 .73 24 .14 0.96 t o t a l 98 .13 98 .59 98 .11 ** = unknown/ u n i d e n t i f i e d f a t t y acids abc = a s i g n i f i c a n t d i f f e r e n c e (a = 0 .05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (a = 0 .05) was found between  stocks f o r the f a t t y a c i d i n d i c a t e d . See Table A4.4. 174 APPENDIX 4 Fa t t y A c i d P r o f i l e s of Eggs Table A4.4: Fa t t y a c i d p r o f i l e s (mean and standard e r r o r of the means) of POLAR LIPID i n the eggs of ROBERTSON CREEK broodstock on thr e e d i e t s . D i e t codes: COMM = commercial; WV33 = West Vancouver 3 3 ; WILD = n a t u r a l d i e t . (n = 5 f o r WV33 and WILD; n = 3 f o r COMM) WV33 COMM WILD F a t t y A c i d mean 3-sem mean 9-sem mean Q . sem 14: 0 ^> 0.99 0.06 "o 1.09 0 .06 -6 1.33 to 0 .09 1 4 : l n 9 3.83 1.66 2 .30 0 .35 2 .23 0 .50 ^ * * 0.18 0 .06 0 .24 0 .01 0 .24 0 . 01 16 :0 15 .73 0 .25 15 .37 0 .49 16 .28 0 .64 1 6 : l n 9 0 . 32 S L 0.12 0 .00 *=>• 0.00 0 .21 - 0 . 07 1 6 : l n 7 1.94 0.08 2 .18 iS . t> 0.12 2 .43 b B 0.12 4 * * 0 .21 0 .13 0 .22 • 0.22 0 .05 0 .03 4a ** 0.17 0 .07 0 .08 0 .08 0.26 0 .04 1 8 : 0 8.08 0 .27 9 .39 0.27 9 .99 to 0.13 1 8 : l n 9 12 .26 a . 0.22 13 .48 fa 0.48 10 .25 C2 0.32 1 8 : l n 7 3.76 Sk 0.07 3.73 0 .28 2 .77 B H 0.12 1 8 : l n 5 0.47 0 .01 0 .50 0 .04 0 .59 0 .07 18 :2n6 1.57 0.05 1.83 fa 0 .02 0 .34 c= 0.02 18 :3n3 0.02 0 .02 0.07 0 .07 0.17 0.06 18 :4n3 0.09 0 .05 0 .00 0 .00 0 .31 0 .05 1 8 : 4 n l 0 .00 0 .00 0 .00 0 .00 0 .12 b B 0.06 2 0 : l n l l 0 .23 SL 0.06 0 .00 0 .00 0 .17 0 .11 2 0 : l n 9 1.96 0.12 2 .83 0 .21 1.06 <Z5 0.12 2 0 : l n 7 0 .04 • 0.04 0 .08 0.08 0 .00 0 .00 20:2n6 0 .40 0 .03 0 .59 0 .04 0 .00 C2 0.00 20:3n6 0.90 * * • 0.05 1.10 to 0 .07 0 .00 C J 0.00 20:4n6 2.68 » • 0.05 2 .84 0 .02 1.55 to 0. 04 20:4n3 0.30 A . 0.08 0 .27 Si 0.14 0 .92 to 0 .11 20:5n3 11 .05 0.48 10 .39 0 .03 15 .59 to 0.76 2 2 : l n l l 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 21:5n3 0.85 a B 0.20 0 .00 fa 0 .00 0 .23 c : 0.03 Y * * 0 .00 0 .00 0 .33 to 0.07 0 .38 to 0.09 22:4n6 0.55 0 .09 0 .41 0 .09 0 .64 0 .05 22 :5n3 3.35 0 .13 3.33 0 .36 4 .41 B B 0.38 22:6n3 25.56 0 .25 23 .93 0 .41 25 .16 0 .42 t o t a l 97 .49 96 .77 97 .67 ** = unknown/ u n i d e n t i f i e d f a t t y a c i d s abc = a s i g n i f i c a n t d i f f e r e n c e (a = 0 .05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. • = a s i g n i f i c a n t d i f f e r e n c e (a = 0 .05) was found between  stocks f o r the f a t t y a c i d i n d i c a t e d . See Table A 4 . 3 . 175 APPENDIX 4 F a t t y A c i d P r o f i l e s of Eggs Table A4.5: F a t t y a c i d p r o f i l e s (mean and standard e r r o r of the means) of TOTAL LIPIDS i n the eggs of 4-year o l d Big Qualicum broodstock. Feed was withdrawn from these f i s h f o r 7 days or 14 days p r i o r to t r a n s f e r to freshwater f o r maturation. (n=5 f o r those s t a r v e d 7 days; n=7 f o r those s t a r v e d 14 days) Period of S t a r v a t i o n , days 7 14 Fatty A c i d mean % sem mean % sem 14: 0 1.70 - 0 . 04 1.95 b 0.06 14:ln9 0.37 0.05 0.53 0.08 ^ * * 0.19 0.05 0.26 0.00 16: 0 10.65 0.13 11.27 0.31 16:ln9 0.77 0.05 0.71 0.03 16:ln7 6.00 0.20 6.10 0.04 5 ** 0.38 0.10 0.44 0.02 18:0 4.48 0.10 4.33 0.04 18:ln9 26.67 0.55 26.08 0.18 18:ln7 3.66 0.11 3.72 0.10 18:ln5 0.46 0.02 0.44 0.01 7 * * 0.34 0.03 0.35 0.02 18:2n6 4.56 0.16 4.51 0.18 18:3n6 0.36 0.03 0.40 0.03 18:3n3 0.68 0.02 0.66 0.01 18:4n3 0.77 0.04 0.78 0.03 18:4nl 0.20 0.02 0.23 0.01 2 0 : l n l l 0.53 0.03 0.61 0.04 20:ln9 1.38 0.07 1.60 0.08 20:2n6 0.42 0.02 0.40 0.02 20:3n6 0.84 0.06 0.78 0.05 20:4n6 1.62 0.06 1.64 0.07 20:4n3 0.78 0.05 0.73 0.05 20:5n3 8.76 0.24 8.41 0.26 2 2 : l n l l 0.24 0.06 0.34 0.03 21:5n3 0.38 0.16 0.30 0.05 22:4n6 0.23 0.11 0.19 0.04 22:5n3 3.89 0.13 3.73 0.14 22:6n3 16.64 0.43 16.88 0.48 Total 98.04 98.37 ** = unknown/ u n i d e n t i f i e d f a t t y acids ab = a s i g n i f i c a n t d i f f e r e n c e (a = 0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. 176 APPENDIX 4 F a t t y A c i d P r o f i l e s of Eggs Table A4.6: F a t t y a c i d p r o f i l e s (mean and standard e r r o r of the means) of POLAR LIPIDS i n the eggs of 4-year o l d Big Qualicum broodstock. Feed was withdrawn from these f i s h f o r 7 or 14 days p r i o r t o t r a n s f e r to freshwater f o r maturation. (n=5 f o r those s t a r v e d 7 days; n=7 f o r those s t a r v e d 14 days) P e r i o d of S t a r v a t i o n , days 7 14 F a t t y A c i d mean sem mean % sem 14: 0 0.88 - 0 .01 1.06 b 0.03 1 4 : l n 9 3.08 0 .64 4 .51 0 .92 ^ * * 0.29 0 .01 0 .34 0.04 16: 0 14 .79 - 0 .15 15 .72 fa 0.28 1 6 : l n 9 0 .50 0 .04 0 .35 0 .14 1 6 : l n 7 1.80 0.08 1.93 0 .10 4 * * 0 .31 0 .11 0 .41 0 .14 4a ** 0 .24 0.06 0 .29 0 .03 18: 0 9 .02 0.18 8.48 0 .25 1 8 : l n 9 13 .38 0.14 13 .27 0.05 1 8 : l n 7 3.67 0.08 3 .69 0.14 1 8 : l n 5 0 .58 0 .02 0 .52 0 .03 18:2n6 1.74 0 .08 1.68 0 .10 2 0 : l n 9 1.68 0 .09 2 .09 0.27 20:2n6 0 .43 0 .03 0 .50 0 .03 20:3n6 0 .85 0 .06 0 .82 0 .04 20:4n6 2 .65 0 .12 2 .71 0 .14 20:4n3 0 .39 0 .03 0 .39 0 .01 20:5n3 . 11 .55 0 .26 11 .08 0 .29 21:5n3 0.34 - 0 .05 0 .08 b 0.04 22:4n6 0.38 0 .06 0 .25 0 .12 22:5n3 3.94 0 .14 3 .72 0 .15 22:6n3 25 .10 0 .53 23 .92 0 .59 T o t a l 97 .57 97 .80 ** = unknown/ u n i d e n t i f i e d f a t t y a c i d s ab = a s i g n i f i c a n t d i f f e r e n c e (a = 0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. 177 APPENDIX 4 Fa t t y A c i d P r o f i l e s of Eggs Table A4.7: Fa t t y a c i d p r o f i l e s (mean and standard e r r o r of the means) of TOTAL LIPIDS i n the eggs of 4-year o l d broodstock on a commercial grower d i e t (COMM) or on a brood (BROOD) d i e t p r i o r to t r a n s f e r to freshwater f o r spawning. (n=5 f i s h f o r both groups) D i e t COMM BROOD F a t t y A c i d mean % sem mean % sem 14: 0 1.95 0.10 2.12 0.08 14:ln9 0.38 0.07 0.64 0.12 1 * * 0.25 0.01 0.25 0.01 16: 0 11.11 0.22 11.72 0.32 16:ln9 0.75 0.08 0.39 to 0.12 16:ln7 6.35 a. 0.06 7.02 to 0.18 5 ** 0.50 0.0 3 0.49. 0.03 18:0 3.96 0.07 4.17 0.11 18:ln9 27.42 0.74 26.17 0.21 18:ln7 3.73 0.08 3.66 0.18 18:ln5 0.45 0.01 0.44 0.01 ~j * * 0.38 0.10 0.23 0.04 18:2n6 4.65 0.19 4.80 0.20 18:3n6 0.43 0.09 0.29 0.02 18:3n4 0.16 0.05 0.13 0.05 18:3n3 0.70 0.05 0.70 0.04 18:4n3 0.84 «5L 0.08 0.60 to 0.05 18:4nl 0.20 0.05 0.20 0.05 2 0 : l n l l 0.68 0.05 0.40 to 0.07 20:ln9 1.58 0.09 1.85 to 0.06 20:2n6 0.40 0.04 0.47 0.02 20:3n6 0.69 0.02 0.78 0.04 20:4n6 1.54 0.02 1.64 0.07 20:4n3 0.73 0.06 0.70 0.03 20:5n3 8.11 0.45 8.29 0.15 2 2 : l n l l 0.34 0.03 0.28 0.03 21:5n3 0.35 0.04 0.24 0.03 22:4n6 0.13 0.06 0.19 0.04 22:5n3 3.32 0.22 3.47 0.08 22:6n3 16.38 0.33 15.58 0.14 Total 98.43 97.90 ** = unknown/ u n i d e n t i f i e d f a t t y a c i d s ab = a s i g n i f i c a n t d i f f e r e n c e (a = 0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. 178 APPENDIX 4 F a t t y A c i d P r o f i l e s of Eggs Table A4.8: Fa t t y a c i d p r o f i l e s (mean and standard e r r o r of the means) of POLAR LIPIDS i n the eggs of 4-year o l d broodstock on a commercial grower d i e t (COMM) or on a brood (BROOD) d i e t p r i o r to t r a n s f e r to freshwater f o r spawning. (n=5 f i s h f o r both groups) D i e t COMM BROOD F a t t y A c i d mean % sem mean % sem 14: 0 1.02 0 .04 1. 01 0 .05 1 4 : l n 9 2 .14 0.46 2 .39 0 .79 j_ * * 0 .26 0 .04 0.27 0 .01 16 :0 15 .62 0.28 16 .33 0 .54 1 6 : l n 9 0.54 0 .04 0 .10 to 0.10 1 6 : l n 7 2 .06 0 .08 2 .19 0 .10 4 * * 1.74 a. 0.62 0.16 to 0.10 4a ** 0 .30 0 .01 0 .25 0 .07 18 :0 8 .03 0 .22 8.51 0 .21 1 8 : l n 9 13 .67 0 .38 13 .15 0.08 1 8 : l n 7 3 .67 0 .12 3 .89 0 .09 1 8 : l n 5 0 .52 0 .03 0 .52 0 .05 18:2n6 2 .02 0 .09 1.90 0 .05 2 0 : l n l l 0 .28 0 .04 0 .14 0.06 2 0 : l n 9 1.95 0 .15 2 .29 0 .03 2 0 : l n 7 0.16 0 .05 0.08 0 .05 20:2n6 0 .47 0.06 0.56 0.04 20:3n6 0 .77 0 .06 0.88 0 .04 20:4n6 2 .56 0 .05 2 .67 0 .09 20:3n3 0 .91 EK 0.29 0 .00 to 0.00 20:4n3 0 .42 0 .04 0 .35 0 .04 20:5n3 10 .77 0.28 11 .00 0 .33 2 2 : l n l l 0 .16 0.08 0 .05 0 .05 Y * * 0 .10 a. 0.05 0 .35 to 0.07 22:4n6 0.36 a. 0.05 0 .54 to 0.04 22:5n3 3 .49 0.18 3.64 0.06 22:6n3 24 .27 0 .40 23 .89 0 .38 T o t a l 98 .27 97 .10 ** = unknown/ u n i d e n t i f i e d f a t t y a c i d s ab = a s i g n i f i c a n t d i f f e r e n c e (a = 0.05) was found between means with d i f f e r e n t s u p e r s c r i p t s w i t h i n the same row. 

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