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Effects of rearing density on stress response in tank-reared juvenile steelhead trout, (Salmo Gairdneri) Loftus, Kevin Kenneth 1983

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EFFECTS OF REARING DENSITY ON STRESS RESPONSE IN TANK-REARED JUVENILE STEELHEAD TROUT, (SALMO GAIRDNERI) by KEVIN KENNETH LOFTUS B . S c , G u e l p h U n i v . , G u e l p h , 1978 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 Z o o l o g y ) We a c c e p t t h i s t h e s i s a s 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 J a n u a r y 1983 @ K e v i n K e n n e t h L o f t u s , 1983 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 DE-6 (3/81) i i ABSTRACT The e f f e c t s o f r e a r i n g d e n s i t y on s t r e s s r e s p o n s e i n j u v e n i l e s t e e l h e a d t r o u t (2-60 grams) were examined i n a s e r i e s of e x p e r i m e n t s . F i s h r e a r e d a t h i g h d e n s i t i e s showed e v i d e n c e o f s t r e s s , however, b o t h t h e m a g n i t u d e and t h e n a t u r e o f t h e r e s p o n s e v a r i e d w i t h s i z e . In t h e f i r s t s t u d y , f r y ( i n i t i a l w e i g h t 2.35 gm) were r e a r e d a t 2 t e m p e r a t u r e s (12.5 and 15.0 °C) a n d 3 d e n s i t i e s (0.6 t o 3.0 t i m e s c o n v e n t i o n a l d e n s i t i e s ) . F i s h r e a r e d a t t h e h i g h t e m p e r a t u r e showed a g r e a t e r r e s p o n s e t o d e n s i t y t h a n t h o s e r e a r e d a t t h e low t e m p e r a t u r e . A t 15 °C, g r o w t h r a t e s were d e p r e s s e d by h i g h d e n s i t i e s , however t h e e f f e c t was o f s h o r t d u r a t i o n and e v i d e n t o n l y d u r i n g t h e f i r s t 2 o f 8 weeks. Whole- body p r o x i m a t e c o m p o s i t i o n was a f f e c t e d . At h i g h d e n s i t i e s , m o i s t u r e and p r o t e i n l e v e l s were e l e v a t e d , and l i p i d l e v e l s were l o w e r e d . C o n d i t i o n f a c t o r s were a l s o low i n f i s h r e a r e d a t h i g h d e n s i t i e s . B o t h c o n d i t i o n f a c t o r s and g r o w t h r a t e s f o l l o w e d a c u r v i l i n e a r p a t t e r n w i t h t i m e . A t 12.5 °C, g r o w t h r a t e s , p r o x i m a t e c o m p o s i t i o n and c o n d i t i o n f a c t o r s f o l l o w e d t r e n d s c l o s e t o t h o s e a t 15 °C, however, t e s t s were r a r e l y s i g n i f i c a n t . Plasma C o r t i s o l c o n c e n t r a t i o n s were u n a f f e c t e d by e i t h e r d e n s i t y o r t e m p e r a t u r e . A c t i v i t y was a f f e c t e d by d e n s i t y , b u t n o t by t e m p e r a t u r e . F i n g e r l i n g t r o u t ( i n i t i a l w e i g h t 15 gm), were d i f f e r e n t l y a f f e c t e d o v e r an 8 - f o l d r a n g e i n d e n s i t y (0.6 t o 4.8 t i m e s c o n v e n t i o n a l l e v e l s ) . At h i g h d e n s i t i e s , g r o w t h r a t e s were s u p p r e s s e d , and t h e e f f e c t was of l o n g e r d u r a t i o n t h a n o b s e r v e d i n t h e f i r s t e x p e r i m e n t . Whole body c o m p o s i t i o n f o l l o w e d t h e same p a t t e r n w i t h d e n s i t y as i n t h e f i r s t e x p e r i m e n t , but c o n d i t i o n f a c t o r s r e s p o n d e d i n t h e o p p o s i t e d i r e c t i o n , i n c r e a s i n g a t h i g h d e n s i t i e s . Plasma C o r t i s o l c o n c e n t r a t i o n s were u n a f f e c t e d by d e n s i t y . R a p i d i n c r e a s e s i n d e n s i t y i n d u c e d a r e s p o n s e i n g r o w t h r a t e s o v e r and above t h a t due t o d e n s i t y a l o n e , w h i c h s u g g e s t e d t h a t f i s h become c o n d i t i o n e d t o r e a r i n g d e n s i t i e s . R a p i d r e d u c t i o n s i n d e n s i t y d i d n o t a f f e c t g r o w t h . Growth r a t e s and p l a s m a C o r t i s o l c o n c e n t r a t i o n s o f p r e -s m o l t s and s m o l t s were u n a f f e c t e d by an 8 - f o l d d e n s i t y r a n g e (0.4 t o 3.2 t i m e s c o n v e n t i o n a l l e v e l s ) . However, a f t e r an a c c l i m a t i o n p e r i o d , sudden i n c r e a s e s i n d e n s i t y c a u s e d s i g n i f i c a n t r e d u c t i o n s i n g r o w t h ( g r e a t e r t h a n t h a t e x p e c t e d on t h e b a s i s o f d e n s i t y a l o n e ) and e l e v a t i o n s i n p l a s m a C o r t i s o l c o n c e n t r a t i o n s . Whole body c o m p o s i t i o n f o l l o w e d a s i m i l a r p a t t e r n w i t h d e n s i t y as o b s e r v e d w i t h s m a l l e r f i s h s u g g e s t i n g t h a t t h e l a c k of g r o w t h r e s p o n s e s t o d e n s i t y , d o e s not n e c e s s i t a t e a l a c k of s t r e s s r e s p o n s e . C o n d i t i o n f a c t o r d a t a were i n c o n c l u s i v e . A c t i v i t y l e v e l s were u n a f f e c t e d by d e n s i t y , but d i d v a r y w i t h t i m e . A f t e r e x s p o s u r e t o a s a l t water c h a l l e n g e t e s t , f i s h r e a r e d a t h i g h d e n s i t i e s r e g u l a t e d p l a s m a sodium l e v e l s l e s s e f f i c i e n t l y t h a n f i s h r e a r e d a t low d e n s i t i e s . Flow r a t e s and c o n t a i n e r volumes d i d not s i g n i f i c a n t l y a f f e c t s t r e s s r e s p o n s e . T h e s e r e s u l t s i n d i c a t e t h a t h i g h d e n s i t y r e a r i n g i n d u c e s p h y s i o l o g i c a l , and p o s s i b l y b e h a v i o r a l c h a n g e s , i n s t e e l h e a d t r o u t . T h i s s u g g e s t s t h e f i s h a r e showing a s t r e s s r e s p o n s e . T h e r e i b e v i d e n c e t h a t , i n some c a s e s , f i s h may a d j u s t t o d e n s i t i e s , and t h a t c h a n g e s i n d e n s i t y , n ot d e n s i t y p e r s e , may i n f l u e n c e t h e s t r e s s r e s p o n s e . Growth r a t e s , when u s e d a l o n e , a r e i n a d e q u a t e i n d i c a t o r s of s t r e s s . i v TABLE OF CONTENTS ABSTRACT i i L I S T OF TABLES v i L I S T CF FIGURES v i i ACKNOWLEDGEMENTS v i i i G e n e r a l I n t r o d u c t i o n 1 S t u d y s i t e and r e a r i n g f a c i l i t i e s 11 Water s u p p l y and q u a l i t y 11 E n v i r o n m e n t a l c o n t r o l f a c i l i t i e s 12 G e n e r a l methods 13 C u l t u r e p r a c t i c e s 13 B r o o d s t o c k c o l l e c t i o n and i n i t i a l r e a r i n g 13 Food and f e e d i n g 14 E n v i r o n m e n t a l c o n t r o l 16 T e m p e r a t u r e 16 P h o t o p e r i o d 16 D a i l y m a i n t e n a n c e 17 Water q u a l i t y 18 S e l e c t i o n of r e a r i n g d e n s i t i e s 19 Sample c o l l e c t i o n 21 A n a e s t h e t i c 21 L e n g t h s and w e i g h t s 21 T o t a l w e i g h t s 21 P r o x i m a t e a n a l y s i s 22 B l o o d c o l l e c t i o n 22 B e h a v i o r a l O b s e r v a t i o n s 24 E x p e r i m e n t 1 : S t r e s s R e s p o n s e s i n f r y 26 I n t r o d u c t i o n 26 M a t e r i a l s and Methods 27 R e s u l t s 29 Growth 29 P r o x i m a t e a n a l y s i s 38 M o i s t u r e 38 P r o t e i n 41 L i p i d s 41 Ash 42 C o r t i s o l 43 Weight l e n g t h r e l a t i o n s 43 B e h a v i o r 49 Net a c t i v i t y 50 T o t a l a c t i v i t y 51 Water q u a l i t y 54 C o n c l u s i o n s 55 E x p e r i m e n t 2: S t r e s s r e s p o n s e s i n f i n g e r l i n g s 60 I n t r o d u c t i o n 60 M a t e r i a l s and Methods 61 R e s u l t s 63 Growth 63 P r o x i m a t e a n a l y s i s 72 M o i s t u r e 73 P r o t e i n 73 L i p i d s 76 V Ash 77 C o r t i s o l 77 Weight l e n g t h r e l a t i o n s 78 Water q u a l i t y 80 C o n c l u s i o n s 82 E x p e r i m e n t 3: s t r e s s r e s p o n s e s i n s m o l t s and p r e - s m o l t s ... 84 I n t r o d u c t i o n 84 M a t e r i a l s and methods 86 R e s u l t s 93 Growth 93 E x p e r i m e n t 3a 93 E x p e r i m e n t 3b 96 E x p e r i m e n t 3c 101 P r o x i m a t e A n a l y s i s 102 E x p e r i m e n t 3a 102 E x p e r i m e n t 3b 105 E x p e r i m e n t 3c 107 C o r t i s o l 107 E x p e r i m e n t 3a 108 E x p e r iment 3b 111 E x p e r i m e n t 3c 112 Weight L e n g t h R e l a t i o n s - 113 E x p e r i m e n t 3a 113 E x p e r i m e n t 3b 115 E x p e r i m e n t 3c : 115 B e h a v i o r 116 E x p e r i m e n t 3a 116 E x p e r i m e n t 3b 117 S a l t Water C h a l l e n g e T e s t 123 C o n c l u s i o n s 127 E x p e r i m e n t 3a 127 E x p e r i m e n t 3b 128 E x p e r i m e n t 3c 130 G e n e r a l D i s c u s s i o n 132 S t r e s s r e s p o n s e s i n f r y 133 S t r e s s r e s p o n s e s i n f i n g e r l i n g s .139 S t r e s s r e s p o n s e s i n s m o l t s and p r e s m o l t s 142 R e f e r e n c e s C i t e d 150 A p p e n d i x I . A n a l y t i c a l c o m p o s i t i o n o f a r t i f i c a l s e a s a l t . .161 L I S T OF TABLES T a b l e 1. P r i m a r y , s e c o n d a r y and t e r t i a r y l e v e l r e s p o n s e s t o s t r e s s 9 T a b l e 2. Summary o f t e m p e r a t u r e f l u c t u a t i o n s between t a n k s w i t h i n e x p e r i m e n t s 17 T a b l e 3. D e s i g n o f e x p e r i m e n t 1 28 T a b l e 4. A n a l y s e s of v a r i a n c e o f g r o w t h s l o p e d a t a f o r e x p e r i m e n t 1 33 T a b l e 5. E x p e r i m e n t 1 . C o r t i s o l d a t a 44 T a b l e 6. E x p e r i m e n t 1. C o n d i t i o n f a c t o r d a t a summary. 46 T a b l e 7. Water q u a l i t y d a t a f o r e x p e r i m e n t 1 55 T a b l e 8. D e s i g n o f e x p e r i m e n t 2 62 T a b l e 9. A n a l y s i s of v a r i a n c e o f g r o w t h s l o p e d a t a f o r e x p e r i m e n t 2 70 T a b l e 10. E x p e r i m e n t 2. C o r t i s o l d a t a summary 78 T a b l e 11. E x p e r i m e n t 2. Summary o f c o n d i t i o n f a c t o r d a t a . . 79 T a b l e 12. Water q u a l i t y d a t a f o r e x p e r i m e n t 2. 81 T a b l e 13. P h y s i o l o g i c a l c h a n g e s w h i c h o c c u r d u r i n g 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 i n s a l m o n i d s 85 T a b l e 14. D e s i g n o f e x p e r i m e n t 3 87 T a b l e 15. Summary o f d i s s o l v e d o xygen d a t a f o r e x p e r i m e n t 3 90 T a b l e 16. A n a l y s i s of v a r i a n c e o f g r o w t h s l o p e d a t a f o r e x p e r i m e n t 3, p a r t A 96 T a b l e 17. A n a l y s i s of v a r i a n c e o f g r o w t h s l o p e d a t a f o r e x p e r i m e n t 3, p a r t B 99 T a b l e 18. A n a l y s i s o f v a r i a n c e of g r o w t h s l o p e d a t a f o r e x p e r i m e n t 3, p a r t C ...102 T a b l e 19. E x p e r i m e n t 3, p a r t s 2 and 3. Whole body p r o x i m a t e c o m p o s i t i o n 1 06 T a b l e 20. E x p e r i m e n t 3. C o n d i t i o n f a c t o r d a t a summary 114 T a b l e 21. E x p e r i m e n t 3, p a r t 2. Summary of n e t a c t i v i t y d a t a by t r e a t m e n t and t i m e 121 T a b l e 22. E x p e r i m e n t 3, p a r t 2. Summary of t o t a l a c t i v i t y d a t a 122 T a b l e 23. D e s i g n o f s a l t w a t e r c h a l l e n g e t e s t 126 v i i L I S T OF FIGURES F i g u r e 1. Change i n w e i g h t o v e r t i m e by t e m p e r a t u r e and d e n s i t y f o r e x p e r i m e n t 1 30 F i g u r e 2. Growth s l o p e s a g a i n s t t i m e f o r e x p e r i m e n t 1 35 F i g u r e 3. H i s t o g r a m s o f p r o x i m a t e c o m p o s i t i o n d a t a f o r e x p e r i m e n t 1 39 F i g u r e 4. C o n d i t i o n f a c t o r s a g a i n s t t i m e f o r e x p e r i m e n t 1. 47 F i g u r e 5. E f f e c t o f f i s h d e n s i t y on a c t i v i t y l e v e l s i n e x p e r i m e n t 1 52 F i g u r e 6. Change i n w e i g h t w i t h t i m e f o r e x p e r i m e n t 2 65 F i g u r e 7. Growth s l o p e s a g a i n s t t i m e f o r e x p e r i m e n t 2 68 F i g u r e 8. H i s t o g r a m s of p r o x i m a t e c o m p o s i t i o n d a t a f o r e x p e r i m e n t 2 74 F i g u r e 9. Changes i n w e i g h t o v e r t i m e f o r e x p e r i m e n t s 3a, 3b, 3c 94 F i g u r e 10. Growth s l o p e s a g a i n s t t i m e f o r e x p e r i m e n t s 3a, 3b, 3c 97 F i g u r e 11. H i s t o g r a m s of p r o x i m a t e c o m p o s i t i o n d a t a f o r e x p e r i m e n t 3a 103 F i g u r e 12. H i s t o g r a m s of p l a s m a C o r t i s o l c o n c e n t r a t i o n s f o r e x p e r i m e n t s 3a, 3b, 3c 109 F i g u r e 13. A c t i v i t y a g a i n s t t i m e f o r e x p e r i e m t 3 118 F i g u r e 14. Plasma sodium c o n c e n t r a t i o n s f o l l o w i n g s a l t w a t er c h a l l e n g e t e s t 124 v i i i ACKNOWLEDGEMENTS I would l i k e to express g r a t i t u d e to my s u p e r v i s o r , Dr. T.G. Northcote f o r h i s encouragement, and f i n a n c i a l support. Dr. J.D. McPhail deserves c o n s i d e r a b l e thanks f o r assuming s u p e r v i s o r y d u t i e s d u r i n g Dr. Northcote's s a b b a t i c a l . H i s pati e n c e and thorough e f f o r t s i n reviewing the d r a f t s were a p p r e c i a t e d . S p e c i a l thanks t o Dr. A.F. Tautz of the F i s h and W i l d l i f e Branch f o r h i s encouragement, guidance and support, without which, t h i s study would not have been completed. He and I spent many hours p l a n n i n g and d i s c u s s i n g t h i s r e s e a r c h . Dr. Tautz arranged access to the F r a s e r V a l l e y Trout Hatchery and p r o v i d e d f i n a n c i a l a s s i s t a n c e . He a l s o reviewed s e v e r a l d r a f t s . Dr. P.A. L a r k i n p r o v i d e d encouragement and ad v i c e at s e v e r a l stages d u r i n g the study. H is f i n a n c i a l c o n t r i b u t i o n , which covered a major p o r t i o n of the m a t e r i a l c o s t s , was both i n d i s p e n s i b l e , and a p p r e c i a t e d . H is comments on the f i n a l d r a f t were most u s e f u l . Mr. U.H.M. Fagerlund and Dr. D.A. Higgs of the Department of F i s h e r i e s and Oceans provided advice whenever requested, and were i n s t r u m e n t a l i n d e f i n i n g the problem. Mr. Fagerlund a l s o reviewed a number of d r a f t s . T h e i r support and encouragement was g r e a t l y a p p r e c i a t e d . Drs. G. Greer, J.R. McBride, and D.J. Randa l l p r o v i d e d advice on s e v e r a l o c c a s s i o n s . Dr. Greer k i n d l y arranged f o r the use of the video equipment. S p e c i a l thanks to Bakhshish Dosanjh, Helen Dye and Glen van der Kraak f o r p r o v i d i n g t e c h n i c a l a d v i c e with sample a n a l y s e s . The f o l l o w i n g people deserve c r e d i t and thanks f o r p r o v i d i n g t e c h n i c a l a s s i s t a n c e with sample c o l l e c t i o n : Paul Bentzen, Linda Berg, Gary B i r c h , Michael F a t t o r i , Ron G r i f f i t h s , Mike Henderson, Michele Skwarok, A l S t o c k w e l l , Leonardo T i r o , and John Werring. S i n c e r e s t thanks are extended to the re s e a r c h s t a f f at the hatchery; Bob Land, L a r r y M i t c h e l l , and Morley Rempel, who cared f o r my f i s h when I was not at the hatchery. These gentlemen provided t e c h n i c a l a s s i s t a n c e , encouragement and advice whenever requested. T h e i r s k i l l f u l e f f o r t s ensured the h e a l t h of my f i s h , and success of the p r o j e c t . T h e i r a b i l i t i e s to suppress what must have been n e a r - h y s t e r i c a l laughter d u r i n g some of my more i n v e n t i v e moments, w i l l be remembered. Dr. Dave Z i t t i n , of the B i o s c i e n c e Data Centre p r o v i d e d e s s e n t i a l h e l p with programming and data a n a l y s i s . H is r a t h e r remarkable p a t i e n c e and good humour was a p p r e c i a t e d . i x F i n a l l y , a s p e c i a l t h a n k s t o my f a m i l y f o r m o r a l and m a t e r i a l s u p p o r t t h r o u g h o u t t h i s e f f o r t . P e r s o n a l s u p p o r t d u r i n g t h i s s t u d y was p r o v i d e d by a G r a d u a t e R e s e a r c h and E n g i n e e r i n g T e c h n o l o g y Award from t h e B.C. S c i e n c e C o u n c i l , and f r o m Dr. N o r t h c o t e ' s NSERC g r a n t . 1 GENERAL INTRODUCTION F i s h c u l t u r i s t s i n N o r t h A m e r i c a have been r a i s i n g s a l m o n i d s i n h a t c h e r i e s s i n c e t h e l a t e 1850's (MacCrimmon e t a l . 1974) but w i t h l i t t l e s c i e n t i f i c d a t a g u i d i n g t h e i r s e l e c t i o n o f r e a r i n g d e n s i t i e s . In t h i s s e n s e , f i s h c u l t u r e h as r e m a i n e d a s much an a r t as a s c i e n c e , and e s t a b l i s h i n g a d e q u a t e c r i t e r i a f o r s e l e c t i n g r e a r i n g d e n s i t i e s r e m a i n s an i m p o r t a n t p r o b l e m i n f i s h c u l t u r e . Over t h e p a s t 50 y e a r s , c o m m e r c i a l c a t c h e s o f some P a c i f i c s a l m o n , ( O n c o r h y n c h u s s p p . ) , have d e c l i n e d (MacLeod 1977; L a r k i n 1975) . S i m i l a r l y , c a t c h e s o f t h e most i m p o r t a n t s p o r t s f i s h i n t h e p r o v i n c e , s t e e l h e a d t r o u t , (Salmo g a i r d n e r i ) , have d e c l i n e d ( F o r d 1982; H a r t 1973). C o n c o m i t a n t w i t h t h e s e d e c l i n e s , has been an i n c r e a s e i n a r t i f i c i a l p r o p a g a t i o n as a method t o r e p l e n i s h and s u p p l e m e n t n a t u r a l s t o c k s . A s s o c i a t e d w i t h t h i s h as been an e f f o r t t o i n c r e a s e t h e e f f i c i e n c y o f h a t c h e r i e s t h r o u g h improvements i n d e s i g n , o p e r a t i o n , and management. In n a t u r e , s t r e a m d w e l l i n g s a l m o n i d s r e g u l a t e d e n s i t i e s p r i m a r i l y t h r o u g h b e h a v i o r a l mechanisms ( D i l l 1981; A l l e n 1969; F r a s e r 1969; Chapman 1966, 1962). In h a t c h e r i e s , most of t h e n a t u r a l d e t e r m i n a n t s of d e n s i t y no l o n g e r o p e r a t e , and i t i s t h e f i s h c u l t u r i s t who d e t e r m i n e s o p t i m a l d e n s i t y . U s u a l l y t h i s i s a b a l a n c e between economic e f f i c i e n c y and f i s h s u r v i v a l ( B i r k s e t a l . 1981). Under h a t c h e r y c o n d i t i o n s i t i s p r o b a b l e t h a t f i s h e x p e r i e n c e some d e g r e e of s t r e s s . I f s u c h s t r e s s r e d u c e s t h e i r a b i l i t y t o s u r v i v e i n t h e h a t c h e r y and when r e l e a s e d , t h e n u n d e r s t a n d i n g t h i s s t r e s s r e s p o n s e i s e s s e n t i a l t o f i s h 2 c u l t u r i s t s (Donaldson 1981; Schreck 1981a). Salmonid c u l t u r e p r e s e n t l y i n v o l v e s a r t i f i c i a l spawning, and the subsequent r e a r i n g to a development stage where the f i s h can be s u c c e s s f u l l y r e l e a s e d i n t o the environment. Economic v i a b i l i t y depends on adequate s u r v i v a l of r e l e a s e d f i s h , and t h e i r c o n t r i b u t i o n s to commercial and s p o r t s f i s h e r i e s , as w e l l as to the r e d u c t i o n of p r e s s u r e s on n a t u r a l s t o c k s . To achieve success, r e l e a s e d f i s h must be p h y s i o l o g i c a l l y and b e h a v i o r a l l y capable of s u r v i v i n g and growing in the n a t u r a l environment. L a r k i n (1981) suggested that the high d e n s i t i e s used i n h a t c h e r i e s may impose b e h a v i o r a l and p h y s i o l o g i c a l changes on the reared f i s h , and these changes may a f f e c t s u r v i v a l when r e l e a s e d . He a l s o noted that l a r g e - s c a l e g e n e t i c i n t e r f e r e n c e with w i l d salmonid stocks may be an i n e v i t a b l e consequence of hatchery s t o c k i n g programs, even with c a r e f u l management. Sev e r a l - a u t h o r s have d i s c u s s e d the p o s s i b l e consequences of hatchery r e a r i n g experience on subsequent behavior and s u r v i v a l i n the w i l d . Chapman (1966) suggested that a r t i f i c i a l c o n d i t i o n s i n h a t c h e r i e s i n f l u e n c e behavior upon r e l e a s e . Fenderson and Carpenter (1971) noted d i f f e r e n c e s i n the behavior of n a t u r a l and h a t c h e r y - r e a r e d A t l a n t i c salmon (Salmo s a l a r ) smolts. K a l l e b e r g ( 1 9 5 8 ) observed a suppression of "normal" behavior p a t t e r n s ( a g g r e s s i o n , t e r r i t o r i a l i t y ) i n f i s h reared i n h a t c h e r i e s . In a d d i t i o n , behavior on r e l e a s e was d i f f e r e n t from that of w i l d f i s h , and the over-crowding i n f l u e n c e was found to be long l a s t i n g . These s t u d i e s suggest that hatchery d e n s i t i e s , o f t e n s e v e r a l orders of magnitude above d e n s i t i e s i n the w i l d , 3 may c a u s e c h a n g e s i n b e h a v i o r , and i n t u r n t h e s e may a f f e c t s u r v i v a l . H i g h d e n s i t y r e a r i n g o f s a l m o n i d s i s known t o i n f l u e n c e a number of p h y s i o l o g i c a l p r o c e s s e s . S i n c e most s p e c i e s a r e r e l e a s e d as e i t h e r s m o l t s o r p r e - s m o l t s , e n v i r o n m e n t a l c o n d i t i o n s a f f e c t i n g 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 a r e p o t e n t i a l l y i m p o r t a n t . E x t e n s i v e r e s e a r c h has been d i r e c t e d a t i d e n t i f y i n g and u n d e r s t a n d i n g t h e p h y s i o l o g i c a l and b e h a v i o r a l c h a n g e s a s s o c i a t e d w i t h s m o l t i f i c a t i o n . Many a u t h o r s (Wedemeyer e t a l . 1981; S c h r e c k 1981b; F o l m a r and D i c k h o f f 1980) n o t e t h a t t h e r e q u i r e m e n t s of s m o l t s a r e e x a c t i n g , and i t i s d u r i n g t h e p r o c e s s o f s m o l t i f i c a t i o n t h a t f i s h a r e most s e n s i t i v e t o h a t c h e r y r e a r i n g p r a c t i c e s . A f u n d a m e n t a l p r o b l e m i n f i s h c u l t u r e i s t h e e s t a b l i s h m e n t o f a d e q u a t e c r i t e r i a f o r s e l e c t i n g r e a r i n g d e n s i t i e s . I n c r e a s i n g r e a r i n g d e n s i t i e s does not n e c e s s a r i l y i n c r e a s e p r o d u c t i o n . In f a c t S a n d e r c o c k and S t o n e ( c i t e d i n F a g e r l u n d e t a l . 1981) o b s e r v e d r e d u c t i o n s i n r e t u r n s o f f i s h r e a r e d under h i g h d e n s i t i e s . In most h a t c h e r i e s d e n s i t y c r i t e r i a a r e e s t a b l i s h e d p r i m a r i l y by t r i a l and e r r o r (McLean 1979) and t h e p h y s i o l o g i c a l b a s i s u n d e r l y i n g d e n s i t y e f f e c t s i s n o t u n d e r s t o o d (McLean 1979; S c h r e c k 1981a). A number of s t u d i e s examine t h e e f f e c t s of d e n s i t y on g r o w t h , s u r v i v a l , and c o m p e t i t i o n i n n a t u r a l or s i m u l a t e d s t r e a m s . K a l l e b e r g (1958) n o t e d t h a t t e r r i t o r i e s d i s i n t e g r a t e d when d e n s i t i e s of S. s a l a r and brown t r o u t , Salmo t r u t t a , were h i g h and t h a t under s u c h c o n d i t i o n s s u b o r d i n a t e s were d i s p l a c e d 4 to areas where growth r a t e s were reduced. Chapman (1966) suggested that the minimal space requirements of t e r r i t o r i a l salmonids are f i x e d but that they can be moderated by p h y s i o l o g i c a l and p s y c h o l o g i c a l f a c t o r s a s s o c i a t e d with food. A l l e n (1969) observed that t e r r i t o r y s i z e s i n some species would not s h r i n k below a c e r t a i n minimum s i z e , even under c o n d i t i o n s of h i g h d e n s i t i e s . Mason and Chapman (1965) observed that i n c r e a s e d food abundance allowed i n c r e a s e d d e n s i t i e s of j u v e n i l e coho salmon, Oncorhynchus k i s u t c h , without a f f e c t i n g growth. Slaney and Northcote (1974) made s i m i l a r o b s e r v a t i o n s on rainbow t r o u t , Salmo g a i r d n e r i , i n a r t i f i c i a l streams. They noted in c r e a s e d l e v e l s of ag g r e s s i o n when prey l e v e l s were decreased. Kawanabe (1969) d e s c r i b e d an unusual b e h a v i o r a l response to d e n s i t y i n the ayu, P l e c o g l o s s u s a l t i v e l i s • At hig h d e n s i t i e s and under surplu s food c o n d i t i o n s , t e r r i t o r i a l behavior i n t h i s f i s h was i n h i b i t e d and a l l i n d i v i d u a l s grew w e l l . At middle d e n s i t i e s , some f i s h were t e r r i t o r i a l and grew w e l l , while others were d i s p l a c e d from p r e f e r r e d areas and grew p o o r l y . At low d e n s i t i e s , a l l f i s h occupied t e r r i t o r i e s and grew w e l l . These s t u d i e s made under n a t u r a l and semi-natural c o n d i t i o n s , suggest s e v e r a l g e n e r a l i z a t i o n s . In many salmonids as d e n s i t i e s i n c r e a s e under c o n d i t i o n s of l i m i t e d food, a g g r e s s i v e behavior i n c r e a s e s . T e r r i t o r y s i z e s a l s o may decrease (but r a r e l y d i s a p p e a r ) , and growth ra t e s of some i n d i v i d u a l s w i l l be suppressed. In some sp e c i e s under surplus food c o n d i t i o n s , a s d e n s i t i e s become h i g h , t e r r i t o r i e s d i s i n t e g r a t e . In o t h e r s , however, when minimum t e r r i t o r y s i z e s are reached 5 s u r p l u s f i s h a r e d i s p l a c e d and r e g a r d l e s s o f f o o d s u p p l y , grow p o o r l y . S e v e r a l l a b o r a t o r y s t u d i e s have examined t h e c o n s e q u e n c e s of d e n s i t y on g r o w t h and a g g r e s s i o n . In t h e medaka, ( O r y z i a s  l a t i p e s , Magnuson (1962) f o u n d t h a t r e g a r d l e s s o f f o o d a v a i l a b i l i t y , a t h i g h d e n s i t i e s t h e a d v a n t a g e s o f t e r r i t o r i e s d i m i n i s h e d and e v e n t u a l l y t h e y were abandoned. M i n c h e n (1972) made s i m i l a r f i n d i n g s on a c i c h l i d A q u i d e n s p u l c h e r . Brown (1946a,b) grew S. t r u t t a a t d i f f e r e n t d e n s i t i e s and d e t e c t e d an optimum d e g r e e o f c r o w d i n g i n f r y a n d 2 y e a r o l d s . The h i g h d e n s i t i e s f o u n d i n h a t c h e r i e s make t h e e x t r a p o l a t i o n t o f i s h p r o d u c t i o n f a c i l i t i e s of t h e r e s u l t s from e c o l o g i c a l and some l a b o r a t o r y s t u d i e s of l i m i t e d v a l u e . More m e a n i n g f u l a r e s t u d i e s c o n d u c t e d i n h a t c h e r i e s w i t h h i g h e r ( b ut e c o n o m i c a l l y v i a b l e ) d e n s i t i e s . Under h a t c h e r y c o n d i t i o n s r e d u c e d g r o w t h r a t e s a s s o c i a t e d w i t h h i g h d e n s i t y r e a r i n g have been o b s e r v e d i n c a t f i s h , I c t a l u r u s p u n c t a t u s (Andrews e t a l . 1971), and v a r i o u s s a l m o n i d s ( F a g e r l u n d e t a l . 1981; T r z e b i a t o w s k i e t a l . 1981; R e f s t i e 1977; R e f s t i e and K i t t l e s o n 1976; B r a u h n e t a l . 1976). O t h e r s t u d i e s a l s o have d e s c r i b e d d e n s i t y - r e l a t e d g r o w t h d e p r e s s i o n ; however t h e s e g r o w th d i f f e r e n c e s c o u l d be a t t r i b u t e d t o e i t h e r w a t e r q u a l i t y o r t h e r e a r i n g methods were t o o p o o r l y d e s c r i b e d t o a l l o w c r i t i c a l e v a l u a t i o n . In most s t u d i e s however, d e n s i t y e f f e c t s were e v a l u a t e d on t h e b a s i s o f f i n a l s i z e d i f f e r e n c e s a l o n e , and no e f f o r t was made t o d e t e c t c h a n g e s i n g r o w t h r a t e s o v e r t i m e w i t h i n e a c h e x p e r i m e n t . Such a n a l y s e s may be e s s e n t i a l t o 6 u n d e r s t a n d i n g d e n s i t y - r e l a t e d g r o w t h phenomena. Growth a l o n e i s p r o b a b l y not an a d e q u a t e i n d i c a t o r f o r a s s e s s i n g d e n s i t y e f f e c t s ( B i r k s e t a l . 1981). F a g e r l u n d e t a l . (1981) examined t h e c o n s e q u e n c e s o f d e n s i t y - i n d u c e d s t r e s s on a number o f v a r i a b l e s and f o u n d them u s e f u l i n a s s e s s i n g f i s h h e a l t h . As m e n t i o n e d e a r l i e r , S a n d e r c o c k and S t o n e ( c i t e d i n F a g e r l u n d e t a l . 1981) r e p o r t e d r e d u c e d r e t u r n s o f c oho r e a r e d a t h i g h d e n s i t i e s . A l t h o u g h d e n s i t y / r e t u r n t r e n d s a r e u s e f u l , t h e y must be i n t e r p r e t e d c a u t i o u s l y . S i n c e b o t h s i z e and t i m e o f r e l e a s e c a n a f f e c t s a l t water t o l e r a n c e and r a t e o f r e t u r n ( C o n t e and Wagner 1965; Wedemeyer e t a l . 1980), t h e r e s u l t s of s t u d i e s d e s c r i b i n g r e d u c e d r e t u r n s by f i s h r e a r e d u nder h i g h d e n s i t i e s may be c o n f o u n d e d e i t h e r by s i z e o r t i m e - r e l a t e d e f f e c t s . T h e r e f o r e s u c h s t u d i e s may n o t be m e a s u r i n g d e n s i t y -r e l a t e d s u r v i v a l e f f e c t s . B i l t o n (1978) and B i l t o n e t a l . (1980) d e m o n s t r a t e d t h a t b o t h t i m e and s i z e a t r e l e a s e , when v a r i e d i n d e p e n d a n t l y , a f f e c t e d a d u l t r e t u r n s . They f o u n d t h a t optimum v a l u e s e x i s t f o r e a c h . B e c a u s e i t i s u n c l e a r i f s i z e d i f f e r e n c e s have a f f e c t e d r e t u r n s f r o m j u v e n i l e s r e a r e d u n d e r d i f f e r e n t d e n s i t i e s , t h e e f f e c t s o f r e a r i n g d e n s i t i e s on o cean s u r v i v a l a r e not w e l l e s t a b l i s h e d . To summarize, t h e e f f e c t s of r e a r i n g d e n s i t y on t h e g r o w t h and s u r v i v a l of h a t c h e r y - r e a r e d s a l m o n i d s a r e u n c l e a r . T h e r e i s e v i d e n c e t h a t h i g h r e a r i n g d e n s i t y may r e d u c e g r o w t h r a t e s i n some s p e c i e s . However, i n some s t u d i e s , i n a p p r o p r i a t e f e e d i n g r e g i m e s , water q u a l i t y v a r i a t i o n s , and d i f f i c u l t y i n c o m p a r i n g 7 g r o w t h r a t e s among g r o u p s of d i f f e r e n t w e i g h t s have d i s t o r t e d f i n d i n g s . B e c a u s e o f t h i s , s u i t a b l e d e n s i t y g u i d e l i n e s have n o t been e s t a b l i s h e d . F u r t h e r m o r e , t h e s i g n i f i c a n c e t o s u r v i v a l o f gr o w t h r a t e s p e r se , not a b s o l u t e s i z e , i s f a r from c l e a r . One a p p r o a c h t o g a i n i n g an u n d e r s t a n d i n g of t h e c o n s e q u e n c e s of d i f f e r e n t r e a r i n g d e n s i t i e s on f i s h h e a l t h , and u l t i m a t e l y , s u r v i v a l i n t h e n a t u r a l e n v i r o n m e n t , may l i e i n d e v e l o p i n g an u n d e r s t a n d i n g o f t h e l e v e l s o f s t r e s s p e r c e i v e d by th e f i s h , t h e i r r e s p o n s e t o s t r e s s , and c u l t u r e s t r a t e g i e s f o r c o n t r o l l i n g s t r e s s . I f g r o w t h r a t e s o f f i s h r e a r e d a t h i g h d e n s i t i e s a r e s u p p r e s s e d , t h e n t h e f i s h must have e x p e r i e n c e d some s t r e s s . E v i d e n c e of t h i s may be d e m o n s t r a b l e t h r o u g h e x a m i n a t i o n o f p h y s i o l o g i c a l and b e h a v i o r a l p a r a m e t e r s . B e f o r e p r o c e e d i n g f u r t h e r , a d e f i n i t i o n o f " s t r e s s " i s r e q u i r e d . The c o n c e p t o f s t r e s s as a p p l i e d t o b i o l o g i c a l s y s t e m s has r e c e i v e d c o n s i d e r a b l e a t t e n t i o n . S e l y e (1950) p r o p o s e d one of t h e f i r s t d e f i n i t i o n s : " t h e sum of a l l t h e p h y s i o l o g i c a l r e s p o n s e s by w h i c h an a n i m a l t r i e s t o m a i n t a i n o r r e - e s t a b l i s h a n o r m a l m e t a b o l i s m i n t h e f a c e of a p h y s i c a l o r c h e m i c a l f o r c e . " S i n c e t h e n , s t r e s s has been r e - d e f i n e d many t i m e s . One of t h e most u s e f u l d e f i n i t i o n s i s t h a t of B r e t t ( 1 9 5 8 ) : " s t r e s s i s a s t a t e p r o d u c e d by any e n v i r o n m e n t a l or o t h e r f a c t o r w h i c h e x t e n d s t h e a d a p t i v e r e s p o n s e s of an a n i m a l b e y o n d t h e n o r m a l r a n g e , o r w h i c h d i s t u r b s t h e n o rmal f u n c t i o n i n g t o s u c h an e x t e n t t h a t t h e c h a n c e s of s u r v i v a l a r e s i g n i f i c a n t l y r e d u c e d . " S e y l e ' s (1950) a c c o u n t of s t r e s s i n t r o d u c e d t h e c o n c e p t of 8 t h e g e n e r a l a d a p t a t i o n syndrome ( G . A . S . ) . In s i m p l e terms t h i s i s a common r e s p o n s e t o a wide v a r i e t y o f s t r e s s s t i m u l i ( i n c l u d i n g d i s e a s e , f r i g h t , p a i n , e t c . ) . The t o t a l i t y o f r e s p o n s e s t o a s t r e s s o r i s composed of some r e a c t i o n s s p e c i f i c t o t h a t s t i m u l u s , and o t h e r s common t o a l l s t i m u l i r e g a r d l e s s o f t h e i r n a t u r e . The G.A.S. c a n be c o n v e n i e n t l y d i v i d e d i n t o 3 s t a g e s : an a l a r m r e a c t i o n , a s t a g e of r e s i s t a n c e , and a s t a g e o f e x h a u s t i o n (Wedemeyer 1980; S e l y e 1973, 1950). The i n i t i a l o r a l a r m r e a c t i o n , i s u s u a l l y a c c o m p a n i e d by t h e r e l e a s e o f v a r i o u s s t r e s s hormones. I f t h e s t r e s s i s n o t l e t h a l , a d a p t a t i o n o c c u r s d u r i n g t h e r e s i s t a n c e s t a g e , i n w h i c h b i o c h e m i c a l , p h y s i o l o g i c a l , m o r p h o l o g i c a l and b e h a v i o r a l c h a n g e s t a k e p l a c e . Even i f a d a p t a t i o n o c c u r s , an a n i m a l s ' c a p a c i t y t o a b s o r b o t h e r s t r e s s e s may be r e d u c e d . I f t h e s t r e s s i s s e v e r e enough, a d a p t a t i o n w i l l not o c c u r , o r i f i t i s o f l o n g enough d u r a t i o n , t h e s t a g e o f e x h a u s t i o n may be r e a c h e d . T h i s i s u s u a l l y l e t h a l . T h r e e l e v e l s o f r e s p o n s e t o s t r e s s have been d e f i n e d i n f i s h ( S c h r e c k 1981a; Wedemeyer and McLeay 1981; Wedemeyer 1980; Mazeaud e t a l . 1977). These a r e p r i m a r y , s e c o n d a r y and t e r t i a r y e f f e c t s ( T a b l e 1 ) . Over t h e p a s t 15 y e a r s , a c o n s i d e r a b l e i n t e r e s t i n t h e e f f e c t s of s t r e s s on f i s h has d e v e l o p e d , and many s t r e s s -i n d u c i n g s t i m u l i have been examined ( f o r a r e v i e w , see P i c k e r i n g 1981). S t i m u l i i n v e s t i g a t e d i n c l u d e t r a n s p o r t a t i o n ( B a r t o n e t a l . 1980), a n a e s t h e s i a (Mazeaud e t a l . 1977; S t r a n g e and S c h r e c k 1978; Wedemeyer and Y a s u t a k e 1977), l o a d i n g and d e n s i t y ( F a g e r l u n d e t a l . 1981; Wedemeyer 1976), s o c i a l h e i r a r c h i e s 9 T a b l e 1. Summary o f p r i m a r y , s e c o n d a r y and t e r t i a r y l e v e l r e s p o n s e s t o s t r e s s ( f r o m Wedemeyer and McLeay, 1981) P r i m a r y E f f e c t s ( i ) r e l e a s e of a d r e n o c o r t i c o t r o p h i c hormone (ACTH) from t h e a d e n o h y p o p h y s i s ; ( i i ) r e l e a s e of " s t r e s s hormones" ( c a t e c h o l a m i n e s and c o r t i c o s t e r o i d s ) from t h e i n t e r r e n a l . S e c o n d a r y E f f e c t s ( i ) b l o o d c h e m i s t r y and h e m a t o l o g i c a l c h a n g e s ( h y p e r -g l y c e m i a , h y p e r l a c t i c e m i a , h y p o c h l o r e m i a , l e u c o -p e n i a , r e d u c e d b l o o d c l o t t i n g t i m e ) ; ( i i ) t i s s u e c h a n g e s ( d e p l e t i o n o f l i v e r g l y c o g e n , i n t e r r e n a l v i t a m i n C d e p l e t i o n ) ; ( i i i ) m e t a b o l i c c h a n g e s s u c h as n e g a t i v e n i t r o g e n b a l a n c e and oxygen d e b t ; ( i v ) d i u r e s i s w i t h r e s u l t a n t o s m o r e g u l a t o r y d y s -f u n c t i o n due t o e l e c t r o l y t e l o s s . T e r t i a r y E f f e c t s ( i ) i m p a i r e d g r o w th r a t e and f o o d c o n v e r s i o n ; ( i i ) d e l a y e d m o r t a l i t y ; ( i i i ) i m p a i r e d p a r r - s m o l t t r a n s f o r m a t i o n ; ( i v ) i m p a i r e d normal m i g r a t o r y b e h a v i o r ; (v) r e d u c e d spawning s u c c e s s ; ( v i ) a l t e r e d body c o m p o s i t i o n ; ( v i i ) i n c r e a s e d s u s c e p t i b i l i t y t o d i s e a s e . ( E j i k e and S c h r e c k 1981; Noakes and L e a t h e r l a n d 1977; E r i c k s o n 1967), h i g h d e n s i t y c o n f i n e m e n t ( S p e c k e r and S c h r e c k 1980; B a r t o n e t a l . 1980), f r i g h t i n d u c e d by c h a s e , c a p t u r e o r h a n d l i n g ( B a r t o n e t a l . 1980; S t r a n g e and S c h r e c k 1980; Bouck e t a l . 1978; Wedemeyer 1976; Wedemeyer 1972), d i s e a s e (Mazeaud e t a l . 1977; Wedemeyer 1976; Wedemeyer 1970), c o n t a m i n a n t s ( S c h r e c k and L o r z 1978; McLeay and Gordon 1977; D o n a l d s o n and Dye 1975; H i l l and Fromm 1968), t e m p e r a t u r e ( S t r a n g e e t a l . 1977), s m o l t m i g r a t i o n and b e h a v i o r ( S c h r e c k 1981b; S p e c k e r and S c h r e c k 1980) a n d e x e r c i s e ( Z a l n i k and G o l d s p i n k 1980). Most o f t h e s e s t u d i e s d e m o n s t r a t e d m e a s u r a b l e s t r e s s r e s p o n s e s , and a l l have 10 c o n t r i b u t e d t o an improved understanding o f the requirements of c u l t u r e d f i s h . However, only F a g e r l u n d et a l . (1981) have attempted t o measure the l e v e l s of s t r e s s experienced by f i s h r e ared f o r extended p e r i o d s under d i f f e r e n t d e n s i t i e s , and i t s consequences on p h y s i o l o g i c a l v a r i a b l e s . T h i s study attempted t o examine the e f f e c t s o f d e n s i t y , and changes i n d e n s i t y , on growth and on a number of other p h y s i o l o g i c a l and b e h a v i o r a l v a r i a b l e s that might be u s e f u l i n a s s e s s i n g s t r e s s i n j u v e n i l e s t e e l h e a d t r o u t . A primary c o n s i d e r a t i o n was that the r e s u l t s be of immediate use to s t e e l h e a d production f a c i l i t i e s . To a t t a i n a thorough understanding of t h e e f f e c t s o f s t r e s s , one primary i n d i c a t o r o f s t r e s s (plasma C o r t i s o l ) , and s e v e r a l t e r t i a r y i n d i c a t o r s (growth r a t e , proximate compostion, body c o n d i t i o n , a c t i v i t y , and s a l t - w a t e r t o l e r a n c e ) were monitored. C o r t i s o l was s e l e c t e d as a primary i n d i c a t o r because i t responds q u i c k l y to s t r e s s ( D o n a l d s o n 1981) and i t has important e f f e c t s on metabolic and osmotic p r o c e s s e s . E f f e c t s of excess C o r t i s o l l e v e l s as observed i n mammals are d e s c r i b e d i n Lee and L a y c o c k ( 1 9 7 8 ) . The other measures were s e l e c t e d e i t h e r because o f t h e i r s e n s i t i v i t y , or because changes in them would s i g n i f y metabolic or b e h a v i o r a l changes. In a l l experiments the n u l l hypothesis was that d e n s i t y had no e f f e c t on any of the measured v a r i a b l e s . 11 STUDY S I T E AND REARING F A C I L I T I E S A l l e x p e r i m e n t s were c o n d u c t e d a t t h e F r a s e r V a l l e y T r o u t H a t c h e r y i n A b b o t s f o r d , B r i t i s h C o l u m b i a . T h i s p r o v i n c i a l f a c i l i t y i s o p e r a t e d by t h e M i n i s t r y o f t h e E n v i r o n m e n t . H a t c h e r y o u t p u t i n c l u d e s p r o d u c t i o n of p a r r and s m o l t s o f anadromous s t o c k s of c o a s t a l c u t t h r o a t t r o u t , Salmo c l a r k i , and c s t e e l h e a d t r o u t . Water s u p p l y and q u a l i t y G r o u n d w a t e r , o b t a i n e d f r o m a l o c a l a q u i f e r , i s a v a i l a b l e i n s u f f i c i e n t q u a n t i t i e s f o r a l l p h a s e s o f p r o d u c t i o n . B e f o r e u s e , w a ter i s p a s s e d t h r o u g h an a e r a t i o n tower t o remove e x c e s s n i t r o g e n , and t o b r i n g oxygen l e v e l s t o s a t u r a t i o n . T e m p e r a t u r e i s c o n s t a n t t h r o u g h o u t t h e y e a r a t 9.5° C.(± . 2 ) , and pH i s n e a r l y c o n s t a n t a t 7.5 - 7.8 u n i t s . S p e c i f i c c o n d u c t a n c e r a n g e s from 200 t o 230 umho/cm. A l k a l i n i t y and t o t a l h a r d n e s s a r e 71.0 and 84.0 mg/1 r e s p e c t i v e l y , and f o r a c o a s t a l s o u r c e t h e water i s m o d e r a t e l y b u f f e r e d . O v e r a l l , w a t e r q u a l i t y i s i d e a l f o r f i s h c u l t u r e . 1 2 E n v i r o n m e n t a l c o n t r o l f a c i l i t i e s Most e x p e r i m e n t s were c o n d u c t e d i n t h e q u a r a n t i n e room i n t h e r e s e a r c h s e c t i o n of t h e h a t c h e r y . T w e l v e o v a l , 500 l i t r e , f i b r e g l a s s t a n k s (137 cm by 77 cm) were a v a i l a b l e . T h e s e were f i t t e d w i t h c e n t r a l b ottom d r a i n s , and were l a r g e l y s e l f -c l e a n i n g . Header b o x e s , e a c h w i t h a s i n g l e o v e r f l o w and two v a l v e - r e g u l a t e d o u t f l o w s e n a b l i n g a c c u r a t e f l o w c o n t r o l , were c o n s t r u c t e d f o r e a c h p a i r of t a n k s . E a c h u n i t r e c e i v e d w ater from t h r e e i n f l o w s : one h e a t e d , one c h i l l e d , and one a t a m b i e n t g r o u n d w a t e r t e m p e r a t u r e . By m i x i n g t e m p e r a t u r e c o u l d be c o n t r o l l e d a c c u r a t e l y . E a c h t a n k was e q u i p p e d w i t h an a u t o m a t i c Ewos f e e d e r , e i t h e r model 504 o r 708, b o t h o f w h i c h o p e r a t e s i l e n t l y and v i b r a t i o n f r e e . F r e q u e n c y of f e e d i n g , d u r a t i o n of e a c h f e e d i n g , and q u a n t i t y of f o o d f e d a t e a c h f e e d i n g were c o n t r o l l a b l e . A u t o m a t i c t i m e r s p r o v i d e d p h o t o p e r i o d c o n t r o l . I l l u m i n a t i o n was p r o v i d e d by f l u o r e s c e n t l i g h t i n g . 1 3 GENERAL METHODS C u l t u r e p r a c t i c e s B r o o d s t o c k c o l l e c t i o n and i n i t i a l r e a r i n g The c o l l e c t i o n of b r o o d s t o c k , a r t i f i c i a l s p a w n i n g, and i n i t i a l r e a r i n g o f t h e f i s h u s e d i n t h e s e e x p e r i m e n t s were c a r r i e d o u t by t h e r e s e a r c h s t a f f a t t h e h a t c h e r y . A d u l t e a r l y r u n , C h i l l i w a c k - V e d d e r s y s t e m s t e e l h e a d were c a p t u r e d by a n g l i n g i n t h e h e a d w a t e r s o f t h e s y s t e m . The c o l l e c t i o n p e r i o d e x t e n d e d from l a t e December, 1979, u n t i l m i d - A p r i l , 1980. D u r i n g egg t a k e , f i s h were a n a e s t h e t i z e d w i t h 2-p h e n o x y e t h a h o l . R i p e f e m a l e s were b l e d by c a r d i a c p u n c t u r e , w i p ed c l e a n , and t h e n s l i t a n t e r i o r l y from t h e v e n t a l l o w i n g t h e eggs t o f a l l i n t o a c l e a n , d r y b u c k e t . Sperm from a t l e a s t two males was mixed i n and the n t h e eggs were r i n s e d s e v e r a l t i m e s . D u r i n g water h a r d e n i n g , e r y t h r o m y c i n p h o s p h a t e (40 ppm) was u s e d as an a n t i b i o t i c . F e r t i l i z e d eggs from i n d i v i d u a l f e m a l e s were p l a c e d i n s e p a r a t e H e a t h t r a y s . To s y n c h r o n i z e t i m e t o swim-up, d i f f e r e n t egg b a t c h e s were r e a r e d a t t e m p e r a t u r e s r a n g i n g from 7 t o 12 °C. When a p p r o x i m a t e l y 10% of t h e a l e v i n s r e a c h e d t h e swim-up s t a g e , t h e y were t r a n s f e r r e d t o 170 l i t r e (2.4 m) t r o u g h s a t d e n s i t i e s up t o 8000 f i s h p e r t r o u g h . They were f e d S i l v e r Cup s t a r t e r u n t i l t h e y r e a c h e d a mean w e i g h t of 1.2 gm a t w h i c h t i m e t h e y were t r a n s f e r r e d t o 1 .8 m c i r c u l a r t a n k s . R e a r i n g c o n t i n u e d i n t h e s e t a n k s u n t i l t h e i r w e i g h t s a v e r a g e d 2.3' gm. At t h i s 1 4 w e i g h t t h e e x p e r i m e n t was s t a r t e d . F o o d and f e e d i n g F e e d i n g s c h e d u l e s i n d e n s i t y e x p e r i m e n t s s h o u l d be d e s i g n e d t o e n s u r e t h a t a l l f i s h a r e e x p o s e d t o e x c e s s r a t i o n s , t h e r e b y m i n i m i z i n g t h e l i k e l i h o o d o f a g g r e s s i v e i n t e r a c t i o n s and t h e i r c o n s e q u e n c e s . F u r t h e r m o r e , f r e q u e n t f e e d i n g s s h o u l d on a v e r a g e r e d u c e a p p e t i t e and d e c r e a s e h u n g e r - i n d u c e d a g g r e s s i v e i n t e r a c t i o n s . S l a n e y and N o r t h c o t e ( 1 9 7 4 ) , and Magnuson (1962) p r o v i d e d e v i d e n c e o f t h e i m p o r t a n c e o f p r e y s u p p l y and a p p e t i t e on b o t h f r e q u e n c y of a g g r e s s i o n i n t e r a c t i o n s and t h e outcome o f t h o s e i n t e r a c t i o n s i n t e r r i t o r i a l f i s h . G r o v e e t a l . (1978) d e m o n s t r a t e d t h a t a p p e t i t e s o f r a i n b o w t r o u t c o r r e l a t e d c l o s e l y w i t h t h e r a t e o f g a s t r i c e v a c u a t i o n and t h a t e v a c u a t i o n r a t e i n c r e a s e d w i t h t e m p e r a t u r e . B r e t t and H i g g s (1970) f o u n d t h a t 30-40 gram s o c k e y e , O n c o r h y n c h u s n e r k a , r e a r e d a t 15°C o r a bove, c o u l d f u l l y d i g e s t t h e i r maximum d a i l y r a t i o n i n l e s s t h a n 24 h o u r s . S h e l b o u r n e e t a l . (1973) o b s e r v e d t h a t s o c k e y e f r y f e d c o n t i n u o u s l y a t 20°C e x h i b i t e d g r e a t e r g r o w t h t h a n f i s h f e d t o s a t i a t i o n 3 t i m e s d a i l y . G i v e n t h i s i n f o r m a t i o n , i t was d e c i d e d t h a t f i s h s h o u l d be f e d f r e q u e n t l y , a p p r o x i m a t e l y e v e r y 20 m i n u t e s f o r s m a l l , 2-20 gram f i s h , and e v e r y 50 m i n u t e s f o r l a r g e r f i s h . F e e d e r s were s e t t o t u r n on 15 m i n u t e s a f t e r t h e l i g h t s were t u r n e d on, and o f f 15 m i n u t e s b e f o r e l i g h t s o f f . The d u r a t i o n of t h e f e e d i n g v a r i e d between 30 and 70 s e c o n d s . At any t i m e , however, a l l t r e a t m e n t s were on t h e same f r e q u e n c y and d u r a t i o n s c h e d u l e . The 15 q u a n t i t y of f o o d f e d d u r i n g e a c h f e e d i n g c o u l d be c o n t r o l l e d by a d j u s t i n g t h e f e e d e r p l a t e s . To e s t i m a t e f o o d r e q u i r e m e n t s , p r o j e c t e d g r o w t h r a t e s were c a l c u l a t e d u s i n g a model d e v e l o p e d by Iwama and T a u t z ( 1 9 8 1 ) . T h e i r model, W t ' 3 3 3 = Wo' 3 3 3 + ( T / l 0 0 0 ) * t : where Wt = f i n a l w e i g h t i n grams, Wo = i n i t i a l w e i g h t i n gm, T = t e m p e r a t u r e i n d e g r e e s C e l c i u s , and t = t i m e i n d a y s ) was m o d i f i e d by i n c o r p o r a t i n g a s e a s o n a l a d j u s t m e n t f a c t o r c a l c u l a t e d f r o m h a t c h e r y d a t a . T h i s model e n a b l e d r e a s o n a b l y a c c u r a t e p r e d i c t i o n s o f i n c r e a s e i n w e i g h t of i n d i v i d u a l f i s h . C a l c u l a t i n g t h e minimum amount t o f e e d was a s i m p l e m a t t e r o f m u l t i p l y i n g p r o j e c t e d w e i g h t i n c r e a s e s by a c o n v e r s i o n f a c t o r and t h e number o f f i s h p e r t a n k . S i n c e some f o o d i s a l w a y s w a s t e d , and t o e n s u r e t h a t c o m p e t i t i o n f o r f o o d would be m i n i m a l , I m u l t i p l i e d t h i s amount by a f a c t o r of 3. F i s h were t h e r e f o r e p r o v i d e d w i t h s u f f i c i e n t f o o d t o a l l o w c o n v e r s i o n e f f i c i e n c i e s as p o o r as 9:1 on a d r y w e i g h t f o o d t o d r y w e i g h t f i s h b a s i s ( a s s u m i n g an a v e r a g e f i s h m o i s t u r e c o n t e n t o f 70% and an a v e r a g e f e e d m o i s t u r e c o n t e n t o f 1 0 % ) . G i v e n t h a t c o n v e r s i o n e f f i c i e n c i e s o f 3:1 ( d r y w e i g h t b a s i s ) a r e n o t uncommon i n h a t c h e r i e s , i t i s c l e a r t h a t , even a l l o w i n g f o r w a s t e , e x c e s s f o o d was a v a i l a b l e . I d e a l l y , f i s h s h o u l d have been f e d t o s a t i a t i o n e a r l y e a c h m o r n i n g , and t h e n f r e q u e n t l y t h e r e a f t e r . L o g i s t i c a l l y , t h i s was n o t p o s s i b l e . To c o n f i r m t h a t r a t i o n s were a d e q u a t e , p e r i o d i c v i s u a l i n s p e c t i o n s o f f o o d waste were made. S u r p l u s f e e d was a l w a y s f o u n d on t h e t a n k b o t t o m s i m m e d i a t e l y a f t e r f i r s t f e e d i n g 1 6 i n t h e m o r n i n g s , and t h e amount w a s t e d a t s u c c e s s i v e f e e d i n g s i n c r e a s e d t h r o u g h o u t t h e d a y . T h i s s u g g e s t s a v o l u n t a r y r e d u c t i o n o f f o o d i n t a k e . The f o o d u s e d i n t h e s e e x p e r i m e n t s was t h e A b e r n a t h y d i e t o b t a i n e d f r o m t h e M o o r e - C l a r k e Company i n L a C o n n e r , W a s h i n g t o n . T h i s b r a n d was u s e d s i n c e i t s f o r m u l a t i o n s p e c i f i c a t i o n s a r e p u b l i s h e d and i t i s known t o be a s u i t a b l e f e e d . F e e d s were s t o r e d a t -20°C u n t i l r e q u i r e d i n o r d e r t o r e d u c e o x i d a t i o n . E n v i r o n m e n t a l c o n t r o l T e m p e r a t u r e The a v a i l a b l e t e m p e r a t u r e r a n g e e x t e n d e d f r o m 7 t o 18°C When a d j u s t e d , i t c o u l d be m a i n t a i n e d w i t h i n ± 0.5°C of t h e d e s i r e d l e v e l and f l u c t u a t i o n s f r o m t h o s e l e v e l s a r e summarized by e x p e r i m e n t i n T a b l e 2. P h o t o p e r i o d As d e s c r i b e d e a r l i e r , t h e q u a r a n t i n e room was e q u i p p e d w i t h f l u o r e s c e n t l i g h t s . Dimmers were n o t a v a i l a b l e . B r e t t (1979) n o t e d t h a t c h a n g i n g p h o t o p e r i o d s c a n a f f e c t g r o w t h r a t e s i n s a l m o n i d s . To e l i m i n a t e t h i s i n f l u e n c e , s i m p l e a v e r a g e s o f t h e number o f h o u r s of d a y l i g h t a t t h e s t a r t and f i n i s h o f t h e e x p e r i m e n t s were u s e d t o e s t a b l i s h p h o t o p e r i o d s . 1 7 T a b l e 2. Summary o f t e m p e r a t u r e f l u c t u a t i o n s between t a n k s w i t h i n e x p e r i m e n t s . F l u c t u a t i o n s were not c a l c u l a t e d f o r t a n k s h e l d a t 9.5 C , s i n c e most were r e c e i v i n g w e l l w a t e r , and w e l l w a t e r t e m p e r a t u r e s f l u c t u a t e d o n l y s l i g h t l y . In column 4, t h e g r e a t e s t d i f f e r e n c e between mean t a n k t e m p e r a t u r e s o v e r t h e d u r a t i o n o f e a c h e x p e r i m e n t i s g i v e n . EXPERIMENT DESIRED TEMPERATURE c e l c i u s MAX DIFFERENCE BETWEEN TANKS AT ANY TIME c e l c i u s MAX DIFFERENCE BETWEEN TIME AVERAGES c e l c i u s 1a 1b 2 3 15.0 12.5 12.5 9.5 0.17 0.21 0.66 0 0.13 0.15 0.21 0 D a i l y m a i n t e n a n c e E a c h m o r n i n g f e e d e r s were m a n u a l l y s w i t c h e d on and i n d i v i d u a l l y e xamined t o e n s u r e p r o p e r f u n c t i o n i n g . T h i s r o u t i n e a l s o a l l o w e d f e e d i n g a c t i v i t y t o be o b s e r v e d . A t ' t h e same t i m e , h e a d e r box o u t f l o w s were c h e c k e d . T e m p e r a t u r e s were c h e c k e d f r e q u e n t l y . To m i n i m i z e s t r e s s , a l l - t a n k s were s c r u b b e d t h o r o u g h l y o n l y once p e r week. The t i m e s p e n t on e a c h tank r a r e l y e x c e e d e d 30 s e c o n d s ( e x c l u d i n g p a r t i a l d r a i n i n g t i m e ) . F i s h were n o t removed d u r i n g c l e a n i n g . More f r e q u e n t l y , t a n k s were p a r t i a l l y d r a i n e d w i t h o u t s c r u b b i n g and e x c e s s f o o d and wastes were removed. T h i s p r o c e d u r e was q u i c k and a p p a r e n t l y not s t r e s s f u l s i n c e f i s h u s u a l l y f e d v i g o r o u s l y a t t h e n e x t f e e d i n g . 18 Water q u a l i t y G e n e r a l l y , two a p p r o a c h e s c a n be t a k e n t o e n s u r e a d e q u a t e water q u a l i t y i n d e n s i t y e x p e r i m e n t s . In one, f l o w s c a n be p r o p o r t i o n a l l y i n c r e a s e d i n h i g h d e n s i t y t a n k s t o e n s u r e t h a t a l l t r e a t m e n t s r e c e i v e t h e same amount of water p e r u n i t o f t i m e and p e r u n i t of f i s h w e i g h t . T h i s a p p r o a c h , w h i l e i d e a l f r o m a water q u a l i t y v i e w p o i n t , c a n have s e r i o u s c o n s e q u e n c e s . I n c r e a s e d f l o w s i n t h e h i g h d e n s i t y t a n k s , i f h i g h enough, would n e c e s s i t a t e h i g h e r e n e r g y e x p e n d i t u r e s f o r p o s i t i o n m a i n t e n a n c e . F r y (1957) showed t h a t t h e m e t a b o l i c c o s t o f swimming v a r i e s a p p r o x i m a t e l y as t h e s q u a r e o f t h e s u s t a i n e d swimming s p e e d . B r e t t (1964) r e c o r d e d t h a t i n s o c k e y e (30-50 gm), oxygen c o n s u m p t i o n i n c r e a s e s l o g a r i t h m i c a l l y w i t h v e l o c i t y . T h e s e f i n d i n g s s u g g e s t t h a t s c o p e f o r g r o w t h , as d e f i n e d by Warren and D a v i s ( 1 9 6 7 ) , c o u l d be r e d u c e d a t h i g h f l o w s . An a l t e r n a t e method, and t h e one u s e d i n t h e s e e x p e r i m e n t s , was t o e s t i m a t e t h e maximum water r e q u i r e m e n t s ( n e x t s e c t i o n ) and p r o v i d e a l l t a n k s w i t h t h e same water f l o w . D i f f e r e n c e s i n w a t e r q u a l i t y between t r e a t m e n t s a r e e x p e c t e d . S e v e r a l o t h e r v a r i a b l e s were m o n i t o r e d . T h e s e were d i s s o l v e d oxygen and n i t r o g e n o u s compounds. D i s s o l v e d oxygen was u s u a l l y measured by t h e a z i d e m o d i f i c a t i o n , i o d o m e t r i c ( W i n k l e r ) method ( S t a n d a r d Methods 1976). Samples were a n a l y z e d f o r n i t r i t e , n i t r a t e , t o t a l n i t r i t e and n i t r a t e , and ammonia n i t r o g e n a t t h e E n v i r o n m e n t a l L a b o r a t o r y of t h e M i n i s t r y o f t h e E n v i r o n m e n t . In a d d i t i o n pH, t u r b i d i t y and s p e c i f i c c o n d u c t a n c e were d e t e r m i n e d . Samples were a l w a y s c o l l e c t e d n e a r t h e c e n t r a l 19 b o t t o m d r a i n s . C o l l e c t i o n t i m e v a r i e d , but was a l w a y s i n t h e a f t e r n o o n . S e l e c t i o n o f r e a r i n g d e n s i t i e s The s e l e c t i o n o f d e n s i t i e s was b a s e d upon: ( i ) t h e maximum amount of wa t e r a v a i l a b l e , and ( i i ) a knowledge o f c o n v e n t i o n a l s t e e l h e a d r e a r i n g d e n s i t i e s . In t h e r e s e a r c h s e c t i o n of t h e h a t c h e r y , r e a r i n g d e n s i t i e s i n c i r c u l a r t a n k s r a r e l y e x c e e d 20 gm/1 f o r f i s h 2 t o 60 gm i n w e i g h t . A t C a p i l a n o h a t c h e r y , s t e e l h e a d r e a r i n g d e n s i t i e s a r e o n l y h a l f t h a t h i g h . McLean (1979) examined r e a r i n g d e n s i t i e s of c h i n o o k , O n c o r h y n c h u s t s h a w y t s c h a and c o h o , 0. k i s u t c h a t Quinsam, R o b e r t s o n C r e e k , and B i g Q u a l i c u m h a t c h e r i e s , and f o u n d t h a t f o r f i s h w e i g h t s g r e a t e r t h a n 2 gm, 20 gm/1 was a common upper d e n s i t y . T h e r e f o r e , 20 gm/1 was used as an e s t i m a t e of c o n v e n t i o n a l maximum r e a r i n g d e n s i t i e s f o r s t e e l h e a d . To d e t e r m i n e t h e number o f f i s h t h a t c o u l d be r e a r e d i n a g i v e n f l o w o f w a t e r , minimum a c c e p t a b l e oxygen l e v e l s were u s e d i n s t e a d o f maximum s a f e m e t a b o l i t e l e v e l s . The minimum p e r m i s s i b l e oxygen l e v e l was s e t a t 6.0 mg/1. T h i s c l o s e l y a p p r o x i m a t e d D a v i s ' (1975) "B" s a f e t y l e v e l . T h i s l e v e l i s c o n s e r v a t i v e f o r t h r e e r e a s o n s : ( i ) oxygen l e v e l s s h o u l d n o t a p p r o a c h t h i s l e v e l u n t i l t h e f i n a l s t a g e s o f t h e e x p e r i m e n t s , ( i i ) t h e f r e q u e n t b u t s h o r t d u r a t i o n f e e d i n g s c h e d u l e s h o u l d m i n i m i z e f e e d i n g - i n d u c e d oxygen l e v e l r e d u c t i o n s , and ( i i i ) B r e t t and B l a c k b u r n (1981) f o u n d t h a t i n young coho and s o c k e y e r e a r e d a t 15°C, as l o n g as oxygen l e v e l s were m a i n t a i n e d above a 20 c r i t i c a l minimum o f 4.0 - 4.5 mg/1, n e i t h e r g r o w t h nor c o n v e r s i o n e f f i c i e n c y were l i m i t e d d u r i n g 6 t o 8 week e x p e r i m e n t s . A l t h o u g h m e t a b o l i t e p r o d u c t i o n r a t e s were no t c a l c u l a t e d , r o u g h e s t i m a t e s were made. I t became c l e a r t h a t ammonia c o n c e n t r a t i o n s w o u l d remain low even a t h i g h d e n s i t i e s . Un-i o n i z e d ammonia l e v e l s ( t h e t o x i c form) were e s t i m a t e d by r e f e r r i n g t o a c h a r t ( L i a o e t a l . 1974) w h i c h e x p r e s s e s t h e q u a n t i t y of t h e t o x i c form a t d i f f e r e n t pH and t e m p e r a t u r e l e v e l s . The EPA (1973) handbook recommends t h a t u n - i o n i z e d l e v e l s s h o u l d r e m a i n below 0.010 mg/1. Burrows (1964) recommended 0.003 mg/1 as a s a f e maximum l e v e l , however W e s t e r s and P r a t t (1977) f o u n d e r r o r s i n Burrows method and recommended 0.0125 mg/1. The p r o c e d u r e f o r c a l c u l a t i n g maximum d e n s i t i e s i n v o l v e d f i v e s t e p s . F i r s t , t h e amount of a v a i l a b l e oxygen (above 6 ppm) b a s e d on t h e q u a n t i t y of w a t e r a v a i l a b l e p e r t a n k , and t h e r e a r i n g t e m p e r a t u r e , was c a l c u l a t e d . S e cond, p r o b a b l e f i n a l f i s h s i z e s were e s t i m a t e d u s i n g t h e g r o w t h model d e s c r i b e d e a r l i e r . T h i r d , knowing t h e p r o b a b l e f i n a l s i z e , a p p r o x i m a t e oxygen c o n s u m p t i o n r a t e s were c a l c u l a t e d u s i n g t h e f o l l o w i n g f o r m u l a ( T a u t z , p e r s . comm.): mg o x y g e n / f i s h / h o u r = wt' e * temp (°C) * 0.048 F o u r t h , t o d e t e r m i n e t h e maximum number of f i s h p e r t a n k , t h e t o t a l a v a i l a b l e o xygen was d i v i d e d by t h e e x p e c t e d maximum 21 c o n s u m p t i o n r a t e . F i n a l l y , t o a c h i e v e t h e d e s i r e d w e i g h t / v o l u m e d e n s i t y , t a n k volumes c o u l d be a d j u s t e d once t h e number o f f i s h p e r t a n k were known. Sample c o l l e c t i o n A n a e s t h e t i c When r e q u i r e d , t h e a n a e s t h e t i c 2 - p h e n o x y e t h a n o l was u s e d . The c o n c e n t r a t i o n was v a r i e d a c c o r d i n g t o t h e s p e e d r e q u i r e d by th e s a m p l i n g p r o c e e d u r e . M o r t a l i t i e s were r a r e , and r e c o v e r y was u s u a l l y r a p i d . L e n g t h s and w e i g h t s T h e s e measurements were u s u a l l y t a k e n e v e r y two weeks. F i s h were q u i c k l y n e t t e d , a n a e s t h e t i z e d , and 50 were randomly s e l e c t e d f o r measurement. F o r k l e n g t h s were t a k e n t o t h e n e a r e s t m i l l i m e t e r , and w e i g h t s t o t h e n e a r e s t 0.1 gm, a l t h o u g h d u r i n g th e f i r s t weeks o f t h e f i r s t e x p e r i e n t , t h e y were w e i g h e d t o t h e n e a r e s t 0.01 gm. T o t a l w e i g h t s To o b t a i n an e x a c t measure of mean w e i g h t by t a n k , t o t a l w e i g h t s were d e t e r m i n e d a p p r o x i m a t e l y e v e r y two weeks by a n a e s t h e t i z i n g and s a m p l i n g a l l f i s h i n a t a n k . The e n t i r e p r o c e d u r e r a r e l y l a s t e d more t h a n 5 t o 7 m i n u t e s . The e x p e r i e n c e d i d n ot a p p e a r t r a u m a t i c s i n c e f i s h u s u a l l y f e d a c t i v e l y a t the 22 n e x t o p p o r t u n i t y . P r o x i m a t e a n a l y s i s To d e t e r m i n e t h e e f f e c t s of r e a r i n g c o n d i t i o n s on whole body c o m p o s i t i o n , f i s h were k i l l e d and s u b s e q u e n t l y a n a l y s e d f o r m o i s t u r e , p r o t e i n , l i p i d a nd a s h c o n t e n t . In t h e f i r s t two e x p e r i m e n t s , f i s h were k i l l e d a t t h e end of t h e g r o w t h s t u d i e s f o l l o w i n g 24 h o u r s o f s t a r v a t i o n . Samples f o r t h e t h i r d e x p e r i m e n t were c o l l e c t e d mid-way t h r o u g h t h e e x p e r i m e n t and f i s h were n o t s t a r v e d , s i n c e t h i s w ould have a f f e c t e d g r o w t h . When k i l l e d , f i s h were w e i g h e d a n d me a s u r e d , and t h e n f r o z e n u n t i l r e q u i r e d . D u r i n g p r o c e s s i n g , f i s h f r o m e a c h t a n k were d i v i d e d i n t o two g r o u p s of 2-5 f i s h on t h e b a s i s of s i z e . T h i s e n a b l e d t e s t i n g f o r s i z e d - r e l a t e d d i f f e r e n c e s i n p r o x i m a t e c o m p o s i t i o n w i t h i n t a n k s . F o l l o w i n g H i g g s e t a l . (1975) f i s h were h o m o g e n i z e d b e f o r e a n a l y s i s . The a u t o m a t e d K j e l d a h l p r o c e d u r e was u s e d f o r p r o t e i n d e t e r m i n a t i o n s on a T e c h n i c o n A u t o A n a l y s e r I I . B l o o d c o l l e c t i o n B l o o d s a m p l e s were c o l l e c t e d f o r C o r t i s o l d e t e r m i n a t i o n . C o n s i d e r a b l e c a r e was r e q u i r e d t o m i n i m i z e t h e e f f e c t s of s t r e s s d u r i n g s a m p l i n g . F a g e r l u n d ( p e r s . comm.; 1967) s u g g e s t e d t h a t 5 m i n u t e s between n e t t i n g and t a k i n g o f t h e l a s t sample was an a c c e p t a b l e maximum t i m e . Wedemeyer and Y a s u t a k e (1977) 23 recommended s i m i l a r g u i d e l i n e s . F o r 24 h o u r s p r i o r t o s a m p l i n g , f i s h were n o t d i s t u r b e d . When f i s h were removed from a t a n k , c a r e was t a k e n t o a v o i d d i s t u r b i n g f i s h i n a d j a c e n t t a n k s . F i s h were q u i c k l y n e t t e d and i m m e d i a t e l y p l a c e d i n a b a t h c o n t a i n i n g s t r o n g a n a e s t h e t i c . The c o n c e n t r a t i o n was s u c h t h a t most f i s h " r o l l e d o v e r " w i t h i n 20-30 s e c o n d s . S e v e r a l a u t h o r s have d e m o n s t r a t e d t h a t a n a e s t h e t i z a t i o n p r i o r t o s a m p l i n g r e d u c e d e l e v a t i o n s i n s t r e s s hormones a s s o c i a t e d w i t h s a m p l i n g (Mazeaud e t a l . 1977; S t r a n g e and S c h r e c k 1978; Wedemeyer and Y a s u t a k e 1977). As soon as f i s h began t o r o l l , t h e y were removed i n d i v i d u a l l y , measured and w e i g h e d , and handed t o one of two a s s i s t a n t s . They s e v e r e d t h e c a u d a l p e d u n c l e and c o l l e c t e d b l o o d i n h e p a r i n i z e d 500 lamda m i c r o p i p e t t e s . B l o o d was q u i c k l y e m p t i e d i n t o 2ml c e n t r i f u g e v i a l s and i c e d u n t i l a l l f i s h were p r o c e s s e d . I f i n s u f f i c i e n t b l o o d volume was c o l l e c t e d a s e c o n d f i s h of s i m i l a r s i z e was s e l e c t e d , and i t s b l o o d added t o t h e same v i a l . V i a l s were c e n t r i f u g e d f o r t h r e e m i n u t e s a t h i g h s p e e d and t h e n t h e serum was removed w i t h i n d i v i d u a l P a s t e u r p i p e t t e s and p l a c e d i n l a b e l l e d v i a l s . A l l samples were f r o z e n on d r y i c e , and r e m a i n e d f r o z e n u n t i l a n a l y s e d . C o r t i s o l samples were a n a l y s e d by r a d i o - i m m u n o a s s a y , u s i n g a k i t a v a i l a b l e from C l i n i c a l A s s a y of Canada. P r o c e e d u r e s a r e o u t l i n e d i n t h e manual ( s e e R e f . ) . 24 B e h a v i o r a l O b s e r v a t i o n s The i n i t i a l o b j e c t i v e of t h e b e h a v i o r a l o b s e r v a t i o n s was t o e n a b l e a q u a n t i t a t i v e a s s e s s m e n t o f b o t h t h e l e v e l s o f a g g r e s s i o n and a c t i v i t y w i t h i n d i f f e r e n t t r e a t m e n t s . B e c a u s e r e a r i n g d e n s i t i e s were o f t e n h i g h , i t was d i f f i c u l t t o f o l l o w and o b s e r v e i n d i v i d u a l f i s h . Thus a v i d e o r e c o r d e r , w h i c h e n a b l e d r e p e t i t i v e v i e w i n g , was u s e d . T h i s s y s t e m c o u l d be u s e d w i t h l i t t l e d i s t u r b a n c e t o t h e f i s h . A d d i t i o n a l l y , b e c a u s e a l l o b s e r v a t i o n s c o u l d be made w i t h i n a 2-3 hour p e r i o d , the. p o s s i b l e e f f e c t s of d i u r n a l c h a n g e s i n b e h a v i o r were r e d u c e d . P r i o r t o f i l m i n g , f i s h were l e f t u n d i s t u r b e d f o r a t l e a s t 12 h o u r s . W i t h t h e t e l e v i s i o n m o n i t o r o p e r a t i n g , t h e camera c o u l d be p o s i t i o n e d o v e r t h e tank edge w i t h l i t t l e d i s t u r b a n c e t o f i s h . A c t i v i t y was t h e n f o l l o w e d on t h e m o n i t o r f o r a t l e a s t f i v e m i n u t e s t o e n s u r e t h a t f i s h were b e h a v i n g " n o r m a l l y " . In most c a s e s , a c t i v i t y a p p e a r e d " n o r m a l " w i t h i n two t o t h r e e m i n u t e s . F i s h were t h e n f i l m e d f o r f i v e m i n u t e s . A n a l y s i s o f t h e f i l m s showed t h a t b o t h q u a l i t a t i v e and q u a n t i t a t i v e e s t i m a t e s o f a g g r e s s i o n were d i f f i c u l t t o o b t a i n a n d s u b j e c t t o o b s e r v a t i o n a l b i a s . I n t h e low d e n s i t y t a n k s , i n d i v i d u a l f i s h c o u l d be f o l l o w e d and a g o n i s t i c e n c o u n t e r s , t h o u g h uncommon, c o u l d be measured. However, a t h i g h d e n s i t i e s , i n d i v i d u a l f i s h c o u l d be f o l l o w e d f o r o n l y s h o r t t i m e s . A d d i t i o n a l l y , d i s t r i b u t i o n i n t h e s e t a n k s t e n d e d t o be non-u n i f o r m , and t h e ea s e of making o b s e r v a t i o n s v a r i e d c o n s i d e r a b l y w i t h i n t a n k s . T h e r e f o r e o n l y g r o s s measures o f a c t i v i t y were r e c o r d e d . 25 The two v a r i a b l e s measured were t o t a l a c t i v i t y ( t h e t o t a l d i s t a n c e t h a t a g i v e n f i s h moved i n 15 s e c o n d s ) and n e t a c t i v i t y ( t h e s h o r t e s t d i s t a n c e between a f i s h ' s p o s i t i o n a t t h e s t a r t and end o f a 15 s e c o n d i n t e r v a l ) . A t o t a l o f 100 o b s e r v a t i o n s was r e c o r d e d f o r e a c h t a n k (50 t o t a l and 50 n e t ) on e a c h day t h a t a c t i v i t y was f i l m e d . To r e d u c e o b s e r v a t i o n a l b i a s , and t o d e c r e a s e t h e i n f l u e n c e o f p e r i o d s o f e r r a t i c b e h a v i o r , f i v e s e p a r a t e t i m e i n t e r v a l s , e a c h 15 s e c o n d s i n d u r a t i o n , e v e n l y s p a c e d t h r o u g h o u t t h e r e c o r d i n g p e r i o d , were a n a l y z e d . A t t h e s t a r t o f e a c h i n t e r v a l , t e n f i s h were s e l e c t e d , f i v e e a c h on t h e r i g h t and l e f t h a l f o f t h e f i e l d o f v i e w , and b o t h measures o f a c t i v i t y were made on e a c h f i s h . S e l e c t i o n c r i t e r i a r e q u i r e d o n l y t h a t e a c h f i s h be d i s t i n g u i s h a b l e i n terms of p o s i t i o n f r o m i t s n e i g h b o u r s . I m m e d i a t e l y a d j a c e n t f i s h were n o t measured, s i n c e t h e b e h a v i o r o f one c o u l d a f f e c t t h e b e h a v i o r o f t h e o t h e r . D u r i n g o b s e r v a t i o n a c t i v e f i s h would o c c a s s i o n a l l y swim ou t o f t h e f i e l d o f v i e w . To a v o i d s e l e c t i n g l e s s a c t i v e f i s h , t h e t o t a l d i s t a n c e moved by t h o s e f i s h , was c o r r e c t e d by a f a c t o r p r o p o r t i o n a l t o t h e f r a c t i o n o f t h e r e q u i r e d v i e w i n g t i m e c o m p l e t e d . M e a s u r e s of n e t a c t i v i t y were not p o s s i b l e f o r t h e s e f i s h . A n o t h e r p r o b l e m a s s o c i a t e d w i t h h i g h d e n s i t i e s , was t h a t some f i s h c o u l d not be f o l l o w e d f o r t h e r e q u i r e d l e n g t h of t i m e . In t h e s e c a s e s , a l t e r n a t e s were s e l e c t e d . 26 EXPERIMENT 1: STRESS RESPONSES IN FRY I n t r o d u c t i o n I t i s known t h a t r e l a t i v e g r o w t h r a t e s o f f i s h t e n d s t o d e c r e a s e as body s i z e i n c r e a s e s . T h e r e f o r e , when a n i m a l s a r e s m a l l , t h e i r p o t e n t i a l f o r g r o w t h i s g r e a t e s t . When g r o w t h i s r a p i d , s m a l l v a r i a t i o n s i n r a t e s , r e g a r d l e s s o f c a u s e , a r e more e a s i l y d e t e c t e d t h a n i n s l o w e r g r o w i n g i n d i v i d u a l s . One m i g h t e x p e c t t h e n , t h a t t h e c o n s e q u e n c e s o f d e n s i t y - i n d u c e d s t r e s s s h o u l d , i f p r e s e n t , be r e a d i l y d e m o n s t r a b l e i n s m a l l f i s h . In t h i s e x p e r i m e n t , s t e e l h e a d f r y were r e a r e d f o r e i g h t weeks a t t h r e e d e n s i t i e s and two t e m p e r a t u r e s . Maximum d e n s i t i e s a t t a i n e d were s e v e r a l o r d e r s of m a g n i t u d e above n a t u r a l s t r e a m l e v e l s , and t h r e e t i m e s c o n v e n t i o n a l h a t c h e r y d e n s i t i e s . S i n c e i n a l l g r o u p s , mean w e i g h t s were e x p e c t e d t o i n c r e a s e 400-600%, t h e p h y s i o l o g i c a l / e n d o c r i n o l o g i c a l c o n s e q u e n c e s o f d e n s i t y -i n d u c e d s t r e s s , i f p r e s e n t , s h o u l d be r e a d i l y d e t e c t a b l e . Under n a t u r a l c o n d i t i o n s , j u v e n i l e s t e e l h e a d of t h i s s i z e a r e t e r r i t o r i a l ( C o l e and Noakes 1980), and under t h e s e h i g h l y a r t i f i c a l c o n d i t i o n s , c o n s i d e r a b l e d i f f e r e n c e s i n a c t i v i t y l e v e l s between d e n s i t i e s m i g h t r e s u l t i f . t h e f i s h a t t e m p t t o e s t a b l i s h t e r r i t o r i e s . 27 M a t e r i a l s and Methods R e p l i c a t e g r o u p s o f s t e e l h e a d f r y were r e a r e d a t e i t h e r 12.5 o r 15°C, a t one o f t h r e e d e n s i t i e s . In t o t a l , t h e r e weie 6 t r e a t m e n t s and 12 t a n k s . I n i t i a l f r y w e i g h t a v e r a g e d 2.35 gm. The e x p e r i m e n t began on A u g u s t 26, 1980 and was t e r m i n a t e d i n l a t e O c t o b e r , 1980. Maximum p e r m i s s i b l e d e n s i t i e s were c a l c u l a t e d a s d e s c r i b e d e a r l i e r ( G e n e r a l M e t h o d s ) . Water f l o w s were s e t a t 25 1/min. The t h r e e d e n s i t i e s were 200, 600, and 1000 f i s h p e r t a n k . On a w e i g h t p e r volume b a s i s , p r e d i c t e d f i n a l d e n s i t i e s e x t e n d e d from 0.6 t o 3.0 t i m e s c o n v e n t i o n a l d e n s i t i e s a t t h e h a t c h e r y . The d e s i g n i s summarized i n T a b l e 3. P h o t o p e r i o d was f i x e d on a 12L:12D c y c l e . L i g h t s were a u t o m a t i c a l l y s w i t c h e d on a t 0700 h o u r s and o f f a t 1900 h o u r s . I n i t i a l l y , f i s h were f e d e v e r y 18-19 m i n u t e s f o r 60 s e c o n d s e a c h t i m e . On day 39, f e e d e r s were a d j u s t e d - t o f e e d e v e r y 23-24 m i n u t e s f o r 68 s e c o n d s . F e e d e r p l a t e s were a d j u s t e d f r e q u e n t l y t o f u r t h e r c o n t r o l f o o d q u a n t i t y r e l e a s e d . A l s o on day 39, f e e d s i z e was s w i t c h e d from 3/64ths t o 4/64ths i n c h e s . T o t a l w e i g h t s , and i n d i v i d u a l l e n g t h s and w e i g h t s , were t a k e n on d a y s 1, 14, 32, 46, and 61. Plasma s a m p l e s f o r C o r t i s o l a n a l y s i s were c o l l e c t e d on day 58, and on day 60 samples were c o l l e c t e d f o r p r o x i m a t e a n a l y s i s . On day 36, w a t e r samples were c o l l e c t e d f r o m t h e f o u r h i g h d e n s i t y t a n k s , and from t h e i n f l o w . On day 58, a f t e r C o r t i s o l s a m p l i n g was c o m p l e t e , water samples were a g a i n c o l l e c t e d , t h i s t i m e f r om a l l t a n k s . A l l samples were r e f r i g e r a t e d and kept i n 28 T a b l e 3. D e s i g n o f e x p e r i m e n t 1. I n i t i a l d e n s i t i e s (gm/1) b a s e d on i n i t i a l mean w e i g h t o f 2.35 grams. F i n a l d e n s i t i e s a r e p r o j e c t e d v a l u e s b a s e d on g r o w t h s l o p e model (Iwama and T a u t z 1981) u s i n g t h e s e a s o n a l a d j u s t m e n t f a c t o r of T/1000 (see t e x t , page 1 5 ) . TANK TEMP C e l c i u s DENSITY LOADING NUMBER VOLUME FLOW I N I T I A L I N I T I A L FINAL gm/l/min l i t r e s 1/m f/1 gm/1 gm/1 i n f l o w 1 15.0 1000 250 25 4.0 9.4 60 600 2 15.0 600 250 25 2.4 5.6 36 360 3 15.0 200 250 25 0.8 1.9 1 2 1 20 4 15.0 1000 250 25 4.0 9.4 60 600 5 15.0 600 250 25 2.4 5.6 36 360 6 15.0 200 250 25 0.8 1.9 1 2 1 20 7 12.5 1000 250 25 4.0 9.4 48 480 8 12.5 600 250 25 2.4 5.6 29 288 9 12.5 200 250 25 0.8 1 .9 10 96 1 0 12.5 1000 250 25 4.0 9.4 48 480 1 1 12.5 600 250 25 2.4 5.6 29 288 1 2 12.5 200 250 25 0.8 1 .9 10 96 d a r k n e s s u n t i l d e l i v e r e d t o t h e e n v i r o n m e n t a l l a b o r a t o r y w i t h i n 24 h o u r s . D i s s o l v e d oxygen c o n c e n t r a t i o n s were d e t e r m i n e d on a l l t a n k s on days 46 and 61. A YSI model 54 oxygen meter was u s e d . V i d e o r e c o r d i n g s of f i s h b e h a v i o r were made on d a y s 25, 39, and 53. M o r t a l i t i e s were r e c o r d e d d a i l y , b u t were r a r e , and were u s u a l l y c a u s e d by e i t h e r p h y s i c a l damage w h i l e b e i n g n e t t e d , o r by g i l l i n g i n t h e p e r f o r a t i o n s o f t h e i n f l o w p i p e . 29 Results Growth During the course of the experiments, technica l d i f f i c u l t i e s resul ted in the loss of some data. In tank 4, feeder problems l as t ing four days, between days 14 and 32, resul ted in the loss of growth slope data for that i n t e r va l . S im i l a r l y , growth slope data was los t between days 32 and 46 for tank 12. A plot of adjusted f i s h weights against time is given (Figure 1) in which weights were corrected to a common i n i t i a l s i z e . This was accomplished using the grand mean s ta r t ing weight as a common i n i t i a l weight and reca lcu la t ing s izes at spec i f i ed times using the growth slopes from successive i n t e r va l s . This f igure shows the re la t i ve changes in weight over time. As expected, at 15°C, f i sh grew faster than at 12.5°C. At 15°C, as density increased, mean f i n a l weight decreased. At 12.5°C, the trend was s im i l a r , but less c l e a r . Linear regressions of mean f i sh weights to the one-third power against time were p lot ted for each treatment. The regression equations f i t the data c lose ly ( a l l co r re la t ion coe f f i c i en t s were greater than 0.99). However, c lose inspect ion revealed a consistent pattern in the res iduals between a l l treatments. It is un l ike ly that the s l i gh t c u r v i l i n e a r i t y in the data f i t was of su f f i c i en t magnitude to e i ther inva l idate the use of the growth model (Iwama and Tautz 1981), or to a l t e r the conclusions of any of the analyses. 30 F i g u r e 1. Change i n w e i g h t o v e r t i m e by t e m p e r a t u r e and d e n s i t y f o r e x p e r i m e n t 1. A l l w e i g h t s have been a d j u s t e d t o a common i n i t i a l w e i g h t u s i n g t h e g r a n d mean s t a r t i n g w e i g h t (2.35 gm) and t h e i n d i v i d u a l g r o w t h s l o p e s by t i m e i n t e r v a l . -16 T 0 -I 1 1 1 1 1 1 1 0 10 20 30 HO 50 60 70 TIME (DAYS) 32 Growth s l o p e s ( b o t h t e m p e r a t u r e s ) Growth d a t a were f i r s t a n a l y z e d i n a 3-way ANOVA ( t e m p e r a t u r e , d e n s i t y , t i m e ) u s i n g g r o w t h s l o p e s c a l c u l a t e d a c c o r d i n g t o Iwama and T a u t z (1981) ( T a b l e 4 ) . The t e m p e r a t u r e e f f e c t was, a s e x p e c t e d , s i g n i f i c a n t . At b o t h t e m p e r a t u r e s t h e c a l c u l a t e d g r o w t h s l o p e s p o o l e d by d e n s i t y were h i g h e r t h a n model p r e d i c t i o n s . T h a t i s a t 15°C g r o w th s l o p e s a v e r a g e d 0.0182 (model p r e d i c t i o n i s 15/1000, o r 0.0150). S i m i l a r i l y , a t 12.5°C, t h e o b s e r v e d v a l u e was 0.0143 (model p r e d i c t i o n i s 0.0125). D e n s i t y e f f e c t s were s i g n i f i c a n t . The two e x t r e m e d e n s i t i e s were d i f f e r e n t f r o m e a c h o t h e r , b u t n o t f r o m t h e m i d - d e n s i t y (Duncan's m u l t i p l e range t e s t ( 5 % l e v e l ) ) . On a p e r c e n t a g e b a s i s , t h e low d e n s i t y t a n k s were g r o w i n g a b o u t 10% f a s t e r t h a n t h e h i g h d e n s i t y t a n k s . Time e f f e c t s were s i g n i f i c a n t . E x a m i n a t i o n of t h e d a t a by t i m e i n t e r v a l r e v e a l e d a c l e a r p a t t e r n ( F i g u r e 2 ) ; low s l o p e s i n i t i a l l y , r i s i n g t o a peak by t h e s e c o n d i n t e r v a l , and t h e n d e c l i n i n g t h e r e a f t e r . F u r t h e r d i s c u s s i o n o f t h e p a t t e r n w i l l f o l l o w . The d e n s i t y by t e m p e r a t u r e i n t e r a c t i o n was n o t s i g n i f i c a n t i n d i c a t i n g t h a t d i f f e r e n c e s w i t h t e m p e r a t u r e were s i m i l a r a t e a c h d e n s i t y . The o t h e r i n t e r a c t i o n s , t e m p e r a t u r e by t i m e , and d e n s i t y by t i m e were s i g n i f i c a n t . T hese s u g g e s t t h a t t h e i n f l u e n c e on growth o f d e n s i t y and t e m p e r a t u r e c h a n g e d o v e r t i m e . The 3-way i n t e r a c t i o n was a l s o s i g n i f i c a n t . Of a l l i n t e r a c t i o n s , t h e most i n t e r e s t i n g was d e n s i t y by t i m e . S i n c e b o t h f a c t o r s t a k e n - a l o n e were s i g n i f i c a n t , t h i s s u g g e s t e d t h a t one o r more i n t e r v a l s w i t h i n t h e g r o w th p e r i o d 33 may be c o n t r i b u t i n g t o most o f t h e o b s e r v e d d e n s i t y e f f e c t s . T a b l e 4. A n a l y s i s o f v a r i a n c e t a b l e s o f growth s l o p e a g a i n s t t r e a t m e n t f o r e x p e r i m e n t 1. P a r t A i n c l u d e s b o t h d e n s i t y and t e m p e r a t u r e e f f e c t s . In p a r t s B and C, a n a l y s e s of t h e 15 and 12.5 C d a t a a r e g i v e n . The d e g r e s s of fr e e d o m v a l u e s f o r b o t h t h e r e s i d u a l and t o t a l terms i n e a c h a n a l y s i s a r e lo w e r t h a n e x p e c t e d s i n c e a t e a c h t e m p e r a t u r e , d a t a from one t i m e was l o s t f o r one t a n k . MEAN SOURCE DF SQUARE PROBABILITY A BOTH TEMPERATURES T e m p e r a t u r e 1 0. 00017606 0. 0 D e n s i t y 2 0. 00000468 0. 01 52 Time 3 0. 00018975 0. 0 T e m p * D e n s i t y 2 0. 00000227 0. 1 072 Temp*Time 3 0. 00000700 0. 001 1 D e n s i t y * T i m e 6 0. 00000383 0. 0060 Temp*Dens*Time 6 0. 00000337 0. 0112 R e s i d u a l 22 0. 00000092 -T o t a l 45 - -D e n s i t y 2 0. Time 3 0. D e n s i t y * T i m e 6 0. R e s i d u a l •1 1 0. T o t a l 22 00000444 0.0292 00011205 0.0 00000393 0.0167 00000090 12.5 C D e n s i t y Time D e n s i t y * T i m e R e s i d u a l T o t a l 2 0.00000225 3 0.00008380 6 0.00000327 11 0.00000094 22 0.1367 0.0 0.0352 To i n v e s t i g a t e t h i s , i t was d e c i d e d t o a n a l y z e t h e d a t a o v e r s e l e c t e d t i m e i n t e r v a l s . A c u r s o r y i n s p e c t i o n i n d i c a t e d t h a t t h e f i r s t i n t e r v a l may have been t h e most c r u c i a l . C o n s e q u e n t l y , two more ANOVAs were p e r f o r m e d , one f o r t h e f i r s t i n t e r v a l , and a n o t h e r f o r t h e r e m a i n i n g t h r e e . Data f o r t h e f i r s t t i m e i n t e r v a l were a n a l y z e d i n a 2-way 34 ANOVA ( t e m p e r a t u r e , d e n s i t y ) . As e x p e c t e d , t e m p e r a t u r e p r o d u c e d s i g n i f i c a n t e f f e c t s (df=1,11 p<.01). D e n s i t y a l s o p r o v e d s i g n i f i c a n t ( d f = 2,11 p<.C)1). T h i s c l e a r l y d e m o n s t r a t e d t h a t d e n s i t y a f f e c t e d g r o w t h d u r i n g t h e f i r s t t i m e p e r i o d . Duncan's t e s t showed t h a t a l l t h r e e d e n s i t i e s were s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r . Mean g r o w t h s l o p e s i n c r e a s e d as d e n s i t y d e c r e a s e d . U n e x p e c t e d l y , t h e i n t e r a c t i o n o f t e m p e r a t u r e and d e n s i t y was f o u n d t o be s i g n i f i c a n t (df=2,11 p<.05), i n d i c a t i n g t h a t t h e e f f e c t of d e n s i t y on g r o w t h v a r i e d w i t h t e m p e r a t u r e . The r e m a i n i n g 3 i n t e r v a l s were a n a l y s e d i n a 3-way ANOVA ( t e m p e r a t u r e , d e n s i t y , t i m e ) . As b e f o r e , t h e e f f e c t of t e m p e r a t u r e (df=1,33 p < . 0 0 l ) and t i m e (df=2,33 p<.00l) were s i g n i f i c a n t . Growth s l o p e s d e c l i n e d s t e a d i l y w i t h t i m e . S u r p r i s i n g l y , d e n s i t y was n o t s i g n i f i c a n t (df=2,33 p=.74) and t h e r e f o r e , d i d n o t a f f e c t growth a f t e r t h e f i r s t t i m e i n t e r v a l . T h i s means t h a t most of t h e o b s e r v e d d i f f e r e n c e s i n growth r a t e , and d e n s i t y e f f e c t s on f i n a l s i z e s , o c c u r r e d d u r i n g t h e f i r s t 14 d a y s . N e i t h e r i n t e r a c t i o n o f d e n s i t y by t i m e , o r d e n s i t y by t e m p e r a t u r e , were s i g n i f i c a n t . However, b o t h were n e a r l y s i g n i f i c a n t (df=4,33 .05<p<.1 and df=2,33 . 0 5 < p < . 1 , r e s p e c t i v e l y ) s u g g e s t i n g t h a t t h e e f f e c t s of t e m p e r a t u r e and t i m e may have been d i f f e r e n t a t d i f f e r e n t d e n s i t i e s . To f u r t h e r c l a r i f y t h e d a t a , i t was d e s i r a b l e t o e l i m i n a t e t e m p e r a t u r e e f f e c t s . T h i s was j u s t i f i e d s i n c e t h e i n t e r a c t i o n t erms s u g g e s t d i f f e r e n c e s . 35 F i g u r e 2. Growth s l o p e s a g a i n s t t i m e ( m i d - p o i n t o f s a m p l i n g i n t e r v a l ) f o r e x p e r i m e n t 1. Mean v a l u e s a r e g i v e n . Growth s l o p e s were c a l c u l a t e d a c c o r d i n g t o Iwama and T a u t z ( 1 9 8 1 ) . V e r t i c l e b a r s a r e s t a n d a r d e r r o r s . 0.005 0 10 20 30 40 50 60 MID-POINT OF TIME INTERVAL 37 Growth s l o p e s (15°C) The h i g h t e m p e r a t u r e d a t a were f i r s t a n a l y s e d i n a 2-way ANOVA ( d e n s i t y , t i m e ) ( T a b l e 4 ) . The r e s u l t s , computed o v e r a l l f o u r t i m e p e r i o d s , i n d i c a t e d t h a t b o t h d e n s i t y and t i m e had s i g n i f i c a n t e f f e c t s on g r o w t h . C l e a r l y , a t 15°C, d e n s i t y a f f e c t e d g r o w t h . However, as b e f o r e , t h e d e n s i t y by t i m e i n t e r a c t i o n was s i g n i f i c a n t i n d i c a t i n g t h a t t h e e f f e c t of d e n s i t y c h a n g e d o v e r t i m e . To d e t e r m i n e w h i c h t i m e s were most a f f e c t e d by d e n s i t y , t h e d a t a were s u b d i v i d e d by t i m e . D e n s i t y e f f e c t s on g r o w t h were s i g n i f i c a n t (df=2,5 p < . 0 l ) d u r i n g t h e f i r s t t i m e i n t e r v a l , c o n f i r m i n g t h e f i n d i n g s o f t h e p r e v i o u s i n t e r a c t i o n e f f e c t . When a n a l y s e d o v e r t h e l a s t t h r e e t i m e s * d e n s i t y had no e f f e c t (df=2,16 p=.87), c o n f i r m i n g t h a t t h e e f f e c t o f d e n s i t y was o f s h o r t d u r a t i o n . Time e f f e c t s were s i g n i f i c a n t (df=2,16 p<.001), but t h e i n t e r a c t i o n was n o t . Growth s l o p e s (12.5°C) When g r o w t h d a t a from a l l t i m e s were a n a l y s e d f o r t h e low t e m p e r a t u r e t r e a t m e n t s , d e n s i t y had no e f f e c t ( T a b l e 4 ) . However b o t h t i m e and t h e i n t e r a c t i o n t e r m were s i g n i f i c a n t . The l a c k of a d e n s i t y e f f e c t was u n e x p e c t e d i n view of t h e r e s u l t s a t 15°C and was i n i t i a l l y t h o u g h t t o be a f u n c t i o n of s m a l l sample s i z e s . However, r e p l i c a t e t a n k s were c l o s e , s u g g e s t i n g t h a t t h e l a c k of e f f e c t was r e a l . F u r t h e r m o r e , t h e t r e n d w i t h d e n s i t y was s i m i l a r , t h o u g h l e s s c l e a r t h a n a t 1 5 ° C , and t h e p r o b a b i l i t y l e v e l a t t a i n e d was c l o s e . In a d d i t i o n , t h e s i g n i f i c a n t 38 i n t e r a c t i o n t e r m s u g g e s t e d t h a t t h e same p r o c e s s w h i c h o c c u r r e d a t t h e 1 5 ° C was o c c u r r i n g h e r e . T h e s e d a t a ( 1 2 . 5 ° C ) were t h e n r e a n a l y z e d by ti m e i n t e r v a l . A g a i n , d e n s i t y e f f e c t s were n ot s i g n i f i c a n t d u r i n g t h e f i r s t t i m e i n t e r v a l (df= 2 , 5 p = . l 8 ) , a l t h o u g h a s i m i l a r , b u t l e s s c l e a r t r e n d t o t h a t o b s e r v e d a t 1 5 ° C , was f o u n d . Over t h e r e m a i n i n g 3 i n t e r v a l s , d e n s i t y had no e f f e c t (df= 2 , 1 6 p= . 5 9 ) . P r o x i m a t e a n a l y s i s M o i s t u r e A 2-way, n e s t e d ANOVA ( t e m p e r a t u r e , d e n s i t y , r e p l i c a t e s ) was p e r f o r m e d f o r t h e m o i s t u r e d a t a . R e p l i c a t e s were n ot d i f f e r e n t . A s u b s e q u e n t a n a l y s i s on p o o l e d d a t a r e v e a l e d t h a t b o t h t e m p e r a t u r e (df= 1 , 2 9 p<.0CM) and d e n s i t y (df= 2 , 2 9 p<.01) had s i g n i f i c a n t , e f f e c t s . M o i s t u r e c o n t e n t d e c r e a s e d a s t e m p e r a t u r e i n c r e a s e d . As d e n s i t y i n c r e a s e d , so d i d m o i s t u r e . The i n t e r a c t i o n was n o t s i g n i f i c a n t . In v i e w o f t h e u n u s u a l t e m p e r a t u r e e f f e c t on gr o w t h s l o p e s i t was d e c i d e d t o a n a l y s e t h e d a t a f o r e a c h t e m p e r a t u r e s e p a r a t e l y . A t 15°C t h e e f f e c t of d e n s i t y was s i g n i f i c a n t (df= 2 , 1 7 p< . 0 5 ) . A t 1 2 . 5 ° C i t was n o t , however a s i m i l a r t r e n d was a p p a r e n t . These r e s u l t s a r e summarized i n F i g u r e 3a. To d e t e r m i n e whether w e i g h t a f f e c t e d m o i s t u r e , l i n e a r r e g r e s s i o n s o f mean f i s h w e i g h t by sample a g a i n s t p e r c e n t a g e m o i s t u r e c o n t e n t were c a l c u l a t e d f o r d i f f e r e n t t r e a t m e n t s . In a l l c a s e s , s l o p e s were n ot s i g n i f i c a n t l y d i f f e r e n t from z e r o . 39 F i g u r e 3. H i s t o g r a m s o f p r o x i m a t e c o m p o s i t i o n d a t a f o r e x p e r i m e n t 1. A - m o i s t u r e , B - p r o t e i n , C - l i p i d , D-ash. S t i p p l e d b a r s a r e 15 °C d a t a , and open b a r s a r e 12.5 °C d a t a . E a c h f i g u r e i s d i v i d e d i n t o 3 s e c t i o n s . The f i r s t 2 b a r s a r e mean l e v e l s a t e a c h t e m p e r a t u r e ( d e n s i t i e s p o o l e d ) . B a r s 3-5 a r e 15 °C d a t a a t ea c h o f t h e t h r e e d e n s i t y t r e a t m e n t s . B a r s 6-8 a r e 12.5 °C d a t a a t e a c h of t h e t h r e e d e n s i t y t r e a t m e n t s . V e r t i c a l b a r s a r e s t a n d a r d e r r o r s . W i t h i n e a c h s e c t i o n , common l e t t e r s r e f e r t o t r e a t m e n t s n o t s t a t i s t i c a l l y d i f f e r e n t (Duncan's o r S c h e f f e ' s m u l t i p l e r a n g e t e s t s - 5% l e v e l ) . 4-0 B 75 L U Dd i 73 i GO o 71 • * 69 y— ••i 68 z 1 C D i i i 66 L U f— o >- 64 cc: f—i 62 60 /•—^ 32 C D 30 L U i 28 . . . J 26 24 10 C D GO 8 + b a a b a b 15.0 12.5 (°C) MEANS a b b 1 1 a a i i i i 1000 600 200 1000 600 200 DENSITY (FISH PER TANK) 41 P r o t e i n P r o t e i n v a l u e s were e x p r e s s e d as p e r c e n t a g e d r y w e i g h t ( F i g u r e 3 b ) . W i t h r e p l i c a t e s p o o l e d , a 2-way ANOVA was c a l c u l a t e d . D e n s i t y (df=2,29 p<.00l) and t e m p e r a t u r e (df=1,29 p<.05) e f f e c t s were s i g n i f i c a n t . As w i t h t h e m o i s t u r e d a t a , p r o t e i n l e v e l s i n c r e a s e d w i t h a d e c r e a s e i n t e m p e r a t u r e , and d e c r e a s e d w i t h d e c r e a s i n g d e n s i t i e s . The i n t e r a c t i o n t e r m was not s i g n i f i c a n t . The d a t a f o r e a c h t e m p e r a t u r e were a n a l y s e d s e p a r a t e l y t o d e t e r m i n e i f d e n s i t y e f f e c t s were i n f l u e n c e d by t e m p e r a t u r e . A t 15°C",- t h e e f f e c t of d e n s i t y was s i g n i f i c a n t ( d f = 2,17 p < . 0 0 l ) . P r o t e i n l e v e l s i n c r e a s e d w i t h d e n s i t y . At 12.5°C, t h e e f f e c t , a s w i t h t h e m o i s t u r e d a t a , was n o t s i g n f i c a n t (df=2,11 p=.24). A g a i n however, a d e c r e a s i n g t r e n d i n p r o t e i n l e v e l s w i t h d e c r e a s i n g d e n s i t y was o b s e r v e d . L i n e a r r e g r e s s i o n s of sub-sample mean f i s h w e i g h t s a g a i n s t p e r c e n t a g e p r o t e i n were c a l c u l a t e d t o t e s t f o r s i z e - d e p e n d a n t e f f e c t s . O n l y a t t h e m i d d l e d e n s i t y a t 15°C was a s l o p e s i g n i f i c a n t l y d i f f e r e n t f r o m z e r o o b t a i n e d . C o n s e q u e n t l y , i t a p p e a r s u n l i k e l y t h a t s i z e a f f e c t e d p r o t e i n c o n t e n t . L i p i d s L i p i d v a l u e s were e x p r e s s e d as p e r c e n t of d r y w e i g h t s as were t h e p r o t e i n d a t a . W i t h o u t p o o l i n g of r e p l i c a t e s , d e n s i t y e f f e c t s were s i g n i f i c a n t , and t e m p e r a t u r e e f f e c t s were n e a r l y s o . 42 When r e p l i c a t e s were p o o l e d , t h e e f f e c t s o f d e n s i t y (df=2,29 p<.00l) and t e m p e r a t u r e (df=1,29 p<.05) were s i g n i f i c a n t . The i n t e r a c t i o n was n o t . L i p i d c o n t e n t d e c r e a s e d a s t e m p e r a t u r e d e c r e a s e d and i n c r e a s e d as d e n s i t y d e c r e a s e d . B o t h p a t t e r n s were o p p o s i t e i n d i r e c t i o n t o t h o s e o b s e r v e d f o r m o i s t u r e and p r o t e i n . To i n v e s t i g a t e t e m p e r a t u r e - r e l a t e d d i f f e r e n c e s i n d e n s i t y r e s p o n s e , t e m p e r a t u r e s were a n a l y s e d s e p a r a t e l y . A t 15°C, d e n s i t y e f f e c t s were s i g n i f i c a n t (df=2,17 p<.005), l i p i d l e v e l s d e c r e a s i n g as d e n s i t y i n c r e a s e d . At 12.5°C, t h e e f f e c t was n o t s i g n i f i c a n t (df=1,11 p=.29). However, a s w i t h m o i s t u r e and p r o t e i n l e v e l s , t h e t r e n d a t t h i s t e m p e r a t u r e was i n t h e same d i r e c t i o n a s a t 15°C. The r e s u l t s of t h e s e a n a l y s e s a r e p r e s e n t e d i n F i g u r e 3c. L i n e a r r e g r e s s i o n s were c a l c u l a t e d t o i n v e s t i g a t e s i z e -r e l a t e d e f f e c t s . Most r e g r e s s i o n s p r o d u c e d s l o p e s n o t d i f f e r e n t f r o m z e r o . As b e f o r e , t h e r e was one e x c e p t i o n - t h e m i d d l e d e n s i t y a t t h e h i g h t e m p e r a t u r e . T h i s o c c u r r e n c e was c o n s i d e r e d u n i m p o r t a n t f o r t h e r e a s o n s d e s c r i b e d e a r l i e r . A s h T h e r e were no d e n s i t y e f f e c t s on a s h c o n t e n t , e i t h e r when t e m p e r a t u r e s were a n a l y s e d t o g e t h e r , or s e p a r a t e l y . T h e r e were no t r e n d s i n t h e d a t a . I t t h e r e f o r e a p p e a r e d t h a t whole-body a s h c o n t e n t was r e l a t i v e l y i n s e n s i t i v e t o t h e e f f e c t s o f d e n s i t y and t e m p e r a t u r e . L i n e a r r e g r e s s i o n s were not c a l c u l a t e d . 4 3 C o r t i s o l To i n c r e a s e a s s a y p r e c i s i o n , e a c h p l a s m a s a m p l e was a n a l y s e d i n d u p l i c a t e . H owever, t h e r e were c a s e s i n w h i c h one o f t h e d u p l i c a t e s p r o d u c e d q u e s t i o n a b l e r e s u l t s , o r p l a s m a v o l u m e s were i n s u f f i c i e n t t o e n a b l e d u p l i c a t i o n . To a v o i d i n a p p r o p r i a t e w e i g h t i n g o f t h e r e s u l t s , d u p l i c a t e s were p o o l e d s o t h a t o n l y one C o r t i s o l v a l u e p e r s a m p l e was e n t e r e d i n t h e a n a l y s i s . T h e r e were no e f f e c t s o f e i t h e r d e n s i t y o r t e m p e r a t u r e when d a t a were a n a l y s e d i n a n e s t e d ANOVA. The r e p l i c a t e s t e r m was s i g n i f i c a n t . When r e a n a l y s e d by t e m p e r a t u r e , a g a i n no d e n s i t y e f f e c t s were o b s e r v e d , a n d i m p o r t a n t l y no t r e n d s were d i s t i n g u i s h a b l e . T h e r e was a t r e n d h o w e v e r , t o h i g h e r v a l u e s a t t h e low t e m p e r a t u r e . As m e n t i o n e d , t h i s t r e n d was n o t s i g n i f i c a n t . T h e s e d a t a a r e s u m m a r i z e d i n T a b l e 5. . B e c a u s e t h e v a r i a n c e s were h e t e r o g e n e o u s f o r t e m p e r a t u r e , a n d t h i s was n o t c o r r e c t e d by any o f t h e t r a n s f o r m a t i o n s t e s t e d , a number o f n o n - p a r a m e t r i c M a n n - W h i t n e y U - t e s t c o m p a r i s o n s were made. None o f t h e t e s t s p r o d u c e d s i g n i f i c a n t d i f f e r e n c e s w i t h t h e 2 - t a i l e d t e s t . Some were s i g n i f i c a n t w i t h 1 - t a i l e d t e s t s , h o w e v e r t h e r e was l i t t l e a p r i o r i j u s t i f i c a t i o n f o r i t s u s e . W e i g h t l e n g t h r e l a t i o n s W e i g h t - l e n g t h r e l a t i o n s were e x a m i n e d i n a two s t e p p r o c e d u r e . F i r s t , a f u n c t i o n a l , g e o m e t r i c mean (G.M.) r e g r e s s i o n was c a l c u l a t e d f o r t h e n a t u r a l l o g ( I n ) l e n g t h , n a t u r a l l o g ( l n ) w e i g h t r e l a t i o n s h i p . S e c o n d l y , u s i n g t h i s r e g r e s s i o n , a 44 T a b l e 5. Summary of C o r t i s o l d a t a r e s u l t s . T h e r e were no s i g n i f i c a n t e f f e c t s due t r e a t m e n t . V a l u e s a r e g i v e n i n ng/ml. f i s h / t a n k 1000 600 200 r e p l i c a t e ' 1 2 1 2 1 n 12 13 12 14 16 15 15.0 C. mean 10.8 19.8 14.4 16.0 12.9 22.9 S.E. 2.88 2.77 2.88 2.67 2.50 2.58 n 15 16 15 15 15 16 12.5 C. mean 19.6 27.6 22.7 12.3 16.1 24.7 S.E. 2.58 2.50 2.58 2.58 2.58 2.50 c o n d i t i o n f a c t o r e q u a t i o n " c u s t o m i z e d " t o t h e d a t a , w i t h a v a l u e of one, was c r e a t e d . T h i s t e c h n i q u e i s p r e f e r r e d by R i c k e r (1973, 1975). I t was i m p o r t a n t t o have a c u s t o m i z e d e q u a t i o n s i n c e d i f f e r e n t p o p u l a t i o n s o f f i s h have v a r i a b l e w e i g h t l e n g t h r e l a t i o n s . S m a l l e r r o r s i n c e r t a i n p a r t s o f a c o n d i t i o n e q u a t i o n , e s p e c i a l l y t h e l e n g t h e x p o n e n t , b, can p r o d u c e a r t i f i c i a l c u r v i 1 i n e a r i t i e s i n c o n d i t i o n t r e n d s w i t h s i z e . Such t r e n d s can l e a d t o e r r o r s i n i n t e r p r e t a t i o n . As d e s c r i b e d e a r l i e r , w e i g h t and l e n g t h d a t a (50 p a i r s p e r t a n k ) , were c o l l e c t e d a t 5 t i m e s t h r o u g h o u t t h e c o u r s e o f t h e e x p e r i m e n t . A p r e l i m i n a r y o v e r v i e w o f t h e d a t a i n d i c a t e d t h a t i n t h o s e t a n k s where f e e d e r p r o b l e m s had e x i s t e d , w e i ght l e n g t h r e l a t i o n s were not s t r o n g l y a f f e c t e d . However, t o m a i n t a i n c o n s i s t e n c y w i t h o t h e r a n a l y s e s , t h e a f f e c t e d t a n k s , 4 and 12, were e x c l u d e d . The G.M. f u n c t i o n a l r e g r e s s i o n o f l n l e n g t h a g a i n s t l n w e i g h t p r o d u c e d t h e f o l l o w i n g e q u a t i o n : l n Wt. = -5.017 + 3.269 In L. 45 When a n t i l o g g e d , t h e e q u a t i o n becomes: c o n d i t i o n f a c t o r = w e i g h t / ( . 0 0 6 6 2 4 3 7 * l e n g t h 3 ' 2 6 9 ) , w h i c h i s t h e c o n d i t i o n e q u a t i o n . U s i n g t h i s e q u a t i o n , 50 c o n d i t i o n v a l u e s were c a l c u l a t e d f r o m t h e l e n g t h w e i g h t d a t a from e a c h t a n k . The s t a t i s t i c a l a n a l y s e s p r e s e n t e d were p e r f o r m e d on d a t a w i t h r e p l i c a t e s p o o l e d a l t h o u g h i n some o f t h e c o m p a r i s o n s t h e y were s i g n i f i c a n t l y d i f f e r e n t . C o n d i t i o n f a c t o r s ( b o t h t e m p e r a t u r e s ) C o n d i t i o n f a c t o r d a t a were f i r s t a n a l y s e d i n a 3-way, n e s t e d ANOVA ( t e m p e r a t u r e , d e n s i t y , t i m e , r e p l i c a t e s ) . R e p l i c a t e s were s i g n i f i c a n t l y d i f f e r e n t . However, on c l o s e i n s p e c t i o n , i t a p p e a r e d t h a t o n l y one o r two c a s e s c a u s e d t h e s i g n i f i c a n c e . To i d e n t i f y o v e r a l l t r e n d s , r e p l i c a t e s were p o o l e d . The p a t t e r n s o b s e r v e d i n main e f f e c t s a nd i n t e r a c t i o n s were s i m i l a r t o t h o s e o b s e r v e d i n g r o w t h s l o p e d a t a . T e m p e r a t u r e e f f e c t s were not s i g n i f i c a n t . However, b o t h d e n s i t y (df=2,2499 p<.005) and t i m e e f f e c t s (df=4,2499 p<.00l) were s i g n i f i c a n t . C o n d i t i o n t e n d e d t o i n c r e a s e w i t h d e c r e a s i n g d e n s i t i e s , a l t h o u g h t h e p a t t e r n was not a l w a y s c o n s i s t e n t . S c h e f f e ' s t e s t p r o d u c e d two homogeneous g r o u p s w h i c h were t h e h i g h e s t and l o w e s t d e n s i t i e s , and the low and m i d d l e d e n s i t i e s . Duncan's t e s t i n d i c a t e d t h a t c o n d i t i o n f a c t o r s a t e a c h t i m e were d i f f e r e n t t h a n a t a l l o t h e r t i m e s . The p a t t e r n i n c o n d i t i o n o v e r t i m e was i d e n t i c a l t o t h a t f o r growth s l o p e . The i n t e r a c t i o n of t e m p e r a t u r e by d e n s i t y was not s i g n i f i c a n t , b u t was c l o s e 46 (p=.09). However, a l l o t h e r s , t e m p e r a t u r e by t i m e , d e n s i t y by t i m e , and t e m p e r a t u r e by d e n s i t y by t i m e were s i g n i f i c a n t (df=4/2/2,2499 p < . 0 0 l ) . Most i m p o r t a n t l y , t h e e f f e c t s o f d e n s i t y on c o n d i t i o n c h a n g e d o v e r t i m e . P l o t s o f c o n d i t i o n f a c t o r s o v e r t i m e , by d e n s i t y , a r e p r e s e n t e d i n F i g u r e 4. T a b l e 6. C o n d i t i o n f a c t o r d a t a summary by t e m p e r a t u r e and t i m e f o r e x p e r i m e n t 1. C o n d i t i o n s were c a l c u l a t e d f r o m t h e GM f u n c t i o n a l r e g r e s s i o n of n a t u r a l l o g o f l e n g t h a g a i n s t n a t u r a l l o g of w e i g h t from samples o f 50 l e n g t h s and w e i g h t s t a k e n f r om e a c h t a n k . I n a l l c e l l s , n=50 and S.E. o f t h e mean = 0.00923. TEMP DENSITY 1 14 32 46 61 1 000 0.919 1 .009 1 .030 1 .009 0.985 15.0 C. 600 0.938 1 .036 1.0-61 1 .025 0.981 0.915 1 .071 1 .007 1 .030 1 .005 200 0.930 1 .057 1 .026 1.014 0.938 0.948 1 . 1 00 1 .071 1.014 0.974 1 000 0.941 1 .046 1 .039 1 .005 0.949 0.945 1 .045 1 .036 1.017 0.955 12.5 C. 600 0.954 1 .055 1 .023 0.986 0.961 0.959 1 . 1 27 1 .023 1.013 0.959 200 0.949 1 .064 1.010 1.015 0.952 C o n d i t i o n f a c t o r s (15°C) To s i m p l i f y i n t e r p r e t a t i o n , t e m p e r a t u r e s were a n a l y z e d s e p a r a t e l y ( T a b l e 6 ) . At 15°C, w i t h r e p l i c a t e s p o o l e d , d e n s i t y e f f e c t s were s i g n i f i c a n t (df=2,1249 p<.005), c o n d i t i o n 47 F i g u r e 4. C o n d i t i o n f a c t o r s a g a i n s t t i m e f o r e x p e r i m e n t 1. V e r t i c a l b a r s a r e s t a n d a r d e r r o r s . S i n c e t e m p e r a t u r e e f f e c t s were not s i g n i f i c a n t , t e m p e r a t u r e t r e a t m e n t s were p o o l e d . E a c h p o i n t r e p r e s e n t s 200 l e n g t h w e i g h t o b s e r v a t i o n s . I.IOT DAY NUMBER 49 i n c r e a s i n g as d e n s i t y d e c r e a s e d . S c h e f f e ' s t e s t i n d i c a t e d t h a t t h e h i g h d e n s i t y t r e a t m e n t s had c o n d i t i o n s s i g n i f i c a n t l y l o w e r t h a n t h e o t h e r s . The e f f e c t o f t i m e was s i g n i f i c a n t (df=4,1249 p<.001), w i t h t h e p r e v i o u s l y d e s c r i b e d p a t t e r n o c c u r r i n g . The i n t e r a c t i o n o f d e n s i t y by t i m e was s i g n i f i c a n t (df=8,1249 P<.001). As d e n s i t y i n c r e a s e d , t h e t i m e r e q u i r e d t o peak i n c r e a s e d , and t h e m a g n i t u d e of t h e peak d e c r e a s e d . C o n d i t i o n f a c t o r s (12.5°C) At 12.5°C, t h e d e n s i t y e f f e c t was not s i g n i f i c a n t , and t h e t r e n d f o l l o w e d e x a c t l y t h a t f o r g r o w t h s l o p e . The e f f e c t o f t i m e was s i g n i f i c a n t (df=4,1249 p < . 0 0 l ) , and f o l l o w e d t h e e x p e c t e d p a t t e r n . The i n t e r a c t i o n t e r m was a l s o s i g n i f i c a n t (df=8,1249 P<.001), but t h e p a t t e r n was l e s s c l e a r t h a n a t 15°C as had been t h e c a s e f o r growth s l o p e d a t a . B e h a v i o r B e h a v i o r d a t a i s o f t e n n o m i n a l o r o r d i n a l , o r i f on a measurement s c a l e may f r e q u e n t l y n o t meet t h e h o m o g e n e i t y o f v a r i a n c e a s s u m p t i o n r e q u i r e d f o r s t a t i s t i c a l a n a l y s i s . However, w i t h l a r g e sample s i z e s , F - v a l u e s a r e r e l a t i v e l y i n s e n s i t i v e t o h e t e r o g e n e i t y p r o b l e m s (Dr. T. Kozak, F a c u l t y of F o r e s t r y , UBC, p e r s . comm.). T h e r e f o r e , i t was d e c i d e d t o use p a r a m e t r i c methods i n t h e s e a n a l y s e s s i n c e t h e y a r e more p o w e r f u l . As a c h e c k , some c o m p a r i s o n s w h i c h were m a r k e d l y h e t e r o g e n e o u s were r e a n a l y s e d u s i n g n o n - p a r a m e t r i c t e s t s . A l l o b s e r v a t i o n s were t r a n s f o r m e d as f o l l o w s : 50 n a t u r a l l o g ( a c t i v i t y + 1 . 1 ) . I t should be kept i n mind that the u n i t s of measurement although a r b i t r a r y , were c o n s i s t e n t . U n f o r t u n a t e l y , t e c h n i c a l problems with the recorder r e s u l t e d i n poor f i l m q u a l i t y and only one of three f i l m s c o u l d be a n a l y s e d . In a d d i t i o n , data from tank 4 was d e l e t e d s i n c e at the time of f i l m i n g feeder problems had caused the f i s h to become h y p e r a c t i v e . Net a c t i v i t y When the pooled data were analysed i n a 2-way ANOVA (temperature, d e n s i t y ) d e n s i t y e f f e c t s were s i g n i f i c a n t (df=2,514 p < . 0 l ) . S c h e f f e ' s m u l t i p l e range t e s t i n d i c a t e d two homogeneous groups as f o l l o w s : ( i ) the lowest and hi g h e s t d e n s i t i e s , and ( i i ) the highest and middle d e n s i t i e s . The middle d e n s i t y was s i g n i f i c a n t l y d i f f e r e n t from only the lowest d e n s i t y . S t a t i o n maintenance was l e a s t p r e c i s e at the middle d e n s i t y , and most p r e c i s e at the low d e n s i t y . N e i t h e r temperature nor the i n t e r a c t i o n terms were s i g n i f i c a n t . B a r t l e t t ' s t e s t i n d i c a t e d that the v a r i a n c e s were homogeneous when grouped by d e n s i t y , but not when grouped by temperature. T h i s l a c k of homogeneity was not c o n s i d e r e d important because of the robustness of the t e s t procedure. Next, i t was decided to analyse the data f o r each temperature s e p a r a t e l y in view of the d i f f e r e n t e f f e c t s of the two temperatures on the other v a r i a b l e s measured. At n e i t h e r temperature were s i g n i f i c a n t d e n s i t y e f f e c t s found. However, 51 b o t h were c l o s e (15°C df=2,233 p=.055; 12.5°C df=2,280 p=.08). I t was i n t e r e s t i n g t o n o t e t h a t a t b o t h t e m p e r a t u r e s , t h e p a t t e r n i n net a c t i v i t y w i t h d e n s i t y was s i m i l a r ( F i g u r e 5 a ) . T o t a l a c t i v i t y A n a l y s i s o f t o t a l a c t i v i t y d a t a r e v e a l e d a d i f f e r e n t p a t t e r n ( F i g u r e 5b) f r o m t h a t o b s e r v e d f o r n e t a c t i v i t y . W i t h o u t p o o l i n g , a 2-way, n e s t e d ANOVA ( t e m p e r a t u r e , d e n s i t y , r e p l i c a t e s ) d i d n o t p r o d u c e s i g n i f i c a n t e f f e c t s . C o n s e q u e n t l y , r e p l i c a t e s were p o o l e d and t h e d a t a were r e a n a l y z e d . As w i t h t h e n e t a c t i v i t y d a t a , t h e e f f e c t s of t e m p e r a t u r e were n o t s i g n i f i c a n t . However, b o t h d e n s i t y (df=2,549 p<.005) and t h e i n t e r a c t i o n (df=2,549 p<.05) were s i g n i f i c a n t . T o t a l a c t i v i t y i n c r e a s e d as d e n s i t y d e c r e a s e d . To i n v e s t i g a t e i n t e r a c t i o n e f f e c t s , d a t a . f o r e a c h t e m p e r a t u r e was s u b s e q u e n t l y a n a l y z e d s e p a r a t e l y . At 15°C, t h e e f f e c t o f d e n s i t y was s i g n i f i c a n t (df = 2,2-49 p<.005). When t h e d a t a were u n t r a n s f o r m e d , t o t a l a c t i v i t y d o u b l e d from 4.2 u n i t s p e r 15 s e c o n d i n t e r v a l a t t h e h i g h d e n s i t y t o 8.5 a t t h e low d e n s i t y . S c h e f f e ' s t e s t i n d i c a t e d t h a t t h e h i g h d e n s i t y t a n k had s i g n i f i c a n t l y l o w e r a c t i v i t y l e v e l s t h a n t h e o t h e r two w h i c h were not d i f f e r e n t from e a c h o t h e r . At 12.5°C, t h e e f f e c t of d e n s i t y was not s i g n i f i c a n t (df=2,299 p=.59), a l t h o u g h a s i m i l a r t r e n d was a p p a r e n t . To examine v a r i a n c e h e t e r o g e n e i t y e f f e c t s , and t o c o n f i r m t h a t p a r a m e t r i c t e s t s were a c c e p t a b l e , 2 - t a i l e d , Mann-Whitney l i -t e s t c o m p a r i s o n s were c a l c u l a t e d f o r a l l d e n s i t y c o m b i n a t i o n s a t 52 F i g u r e 5. E f f e c t o f f i s h d e n s i t y on a c t i v i t y l e v e l s . P a r t A i s n e t a c t i v i t y , t h e t e n d a n c y t o m a i n t a i n a f i x e d p o s i t i o n . H i g h v a l u e s i n d i c a t e l e s s p r e c i s e s t a t i o n m a i n t e n a n c e . P a r t B, t o t a l a c t i v i t y , i s t h e t o t a l d i s t a n c e moved i n a g i v e n t i m e . A l l v a l u e s a r e g i v e n i n a r b i t r a r y u n i t s per 15 s e c o n d i n t e r v a l . S2> 1.6T 1.1 + CO 1.0 + 0.8 -12.5 ° C •temperatures p o o l e d (± 95% C L . ) -+• -+-B •— LU > to H— LH I—> i—I «=C \ oo —I t -<£ ~ I— z O Z> 10 T 8+ 6 + 4+ 1 5 . 0 U C 1 2 . 5 ° C •temperatures p o o l e d (± 95% C L . ) "0 +-200 400 600 800 1000 1200 DENSITY (FISH P E R T A N K ) 54 e a c h t e m p e r a t u r e . The r e s u l t s c o n f i r m e d t h o s e l i s t e d a b o v e . Water q u a l i t y Oxygen l e v e l s were d e t e r m i n e d on d a y s 46 and 61 ( T a b l e 7 ) . The p a t t e r n i n d i s s o l v e d oxygen c o n c e n t r a t i o n s w i t h d e n s i t y was c o n s i s t e n t w i t h t h a t e x p e c t e d . The o b s e r v e d l e v e l s were c l o s e t o e x p e c t e d l e v e l s when t e m p e r a t u r e , f i s h s i z e , a n d f i s h number were a c c o u n t e d f o r . T h e r e f o r e t h e models u s e d i n d e t e r m i n i n g f l o w s and d e n s i t i e s were c o n s i d e r e d a d e q u a t e . On day 46 t h e l o w e s t oxygen o b s e r v e d was 8.0 mg/1. By day 61 t h i s had d e c l i n e d t o 7.1 mg/1. In most o f t h e c a l c u l a t i o n s , t h e a c t u a l oxygen l e v e l s were s l i g h t l y h i g h e r t h a n e x p e c t e d . A p p a r e n t l y , t h e model p r e d i c t e d oxygen c o n s u m p t i o n r a t e s on t h e h i g h ( c o n s e r v a t i v e ) s i d e . E r r o r s i n t h i s d i r e c t i o n were a c c e p t a b l e . As e x p e c t e d , oxygen l e v e l s were l o w e s t i n t h e h i g h d e n s i t y t a n k s . W h i l e t h i s may be s u s p e c t e d o f c o n t r i b u t i n g t o r e d u c e d g r o w t h r a t e s i n t h e s e t a n k s , t h e r e were no d i f f e r e n c e s i n g r o w t h r a t e s d u r i n g t h e l a s t 70% o f t h e e x p e r i m e n t when oxygen demand was g r e a t e s t . In a d d i t i o n , t h e r e was a n o n - s i g n i f i c a n t t e n d e n c y f o r f i s h f r o m h i g h d e n s i t y t a n k s a t 15°C t o grow f a s t e r d u r i n g t h e f i n a l weeks. Water samples were c o l l e c t e d on d a y s 36 and 58 f o r a n a l y s i s . The r e s u l t s a r e p r e s e n t e d i n T a b l e 7 a l o n g w i t h c a l c u l a t e d u n - i o n i z e d ammonia l e v e l s . I t i s c l e a r t h a t m e t a b o l i t e l e v e l s were w e l l w i t h i n s a f e l e v e l s . 55 T a b l e 7. Water q u a l i t y d a t a f o r e x p e r i m e n t 1. U n - i o n i z e d ammonia v a l u e s were d e t e r m i n e d from L i a o (1974) u s i n g pH and t e m p e r a t u r e d a t a . N i t r i t e l e v e l s were below d e t e c t a b l e l e v e l s (<0.005 mg/1). TOTAL DAY UN-1ONI ZED TOTAL TANK AMMONIA AMMONIA NITRATE pH mg/1 mg/1 mg/1 1 0. 1 05 0.0018 5.60 7.8 4 0.069 0.0012 5.80 7.8 7 0. 1 25 0.0022 5.80 7.8 . 10 0.161 0.0028 5.90 7.8 INFLOW 0.030 0.0005 5.90 7.8 _ DAY 58 DISSOLVED OXYGEN TOTAL UN-IONIZED TOTAL S P E C I F I C mg/1 TANK AMMONIA AMMONIA NITRATE pH CONDUCTANCE DAY No. mg/1 mg/1 mg/1 umho/cm 46 61 1 0.201 0.0024 4.90 7.6 211 8.8 7.1 2 0. 1 32 0.0017 4.80 7.7 210 9.3 8.5 3 0.045 0.0006 4.90 7.7 209 10.0 9.8 4 0. 1 53 0.0020 5.00 7.7 209 8.8 7.8 5 0. 1 39 0.0017 5.00 7.6 209 9.1 8.2 6 0.044 0.0006 5.00 7.7 208 10.0 9.4 7 0.110 0.0010 5.00 7.6 210 8.5 8.6 8 0. 154 0.0014 5.10 7.6 209 8.7 8.0 9 0.064 0.0009 5.10 7.8 209 9.0 8.9 1 0 0. 194 0.0023 5.00 7.6 21 1 8.0 7.6 1 1 0. 183 0.0022 5.00 7.7 210 9.0 9.0 1 2 0.041 0.0006 5.20 7.8 210 9.7 10.0 C o n c l u s i o n s R e s u l t s o f t h i s e x p e r i m e n t d e m o n s t r a t e d c l e a r t r e a t m e n t -i n d u c e d d i f f e r e n c e s i n g r o w t h , p r o x i m a t e c o m p o s i t i o n , and c o n d i t i o n f a c t o r s ( t e r t i a r y r e s p o n s e s t o s t r e s s ) . A d d i t i o n a l l y , d e n s i t y r e l a t e d d i f f e r e n c e s i n a c t i v i t y were d e t e c t e d . P l asma C o r t i s o l l e v e l s (a p r i m a r y r e s p o n s e t o s t r e s s ) showed no e v i d e n c e o f t r e a t m e n t e f f e c t s . The e f f e c t o f d e n s i t y v a r i e d 56 between t e m p e r a t u r e s . A t t h e h i g h t e m p e r a t u r e , 15°C, e f f e c t s were u s u a l l y s i g n i f i c a n t . At t h e low t e m p e r a t u r e , t h e t r e n d s o b s e r v e d were u s u a l l y i n t h e same d i r e c t i o n a s a t t h e h i g h t e m p e r a t u r e . E v a l u a t i o n o f t h e most i m p o r t a n t water q u a l i t y d a t a , d i s s o l v e d oxygen and u n - i o n i z e d ammonia l e v e l s , d e m o n s t r a t e d t h a t w ater q u a l i t y d i d not a t any t i m e p r o v e e i t h e r l i m i t i n g , o r a l t e r g r o w t h r e s p o n s e s . As e x p e c t e d , t e m p e r a t u r e e f f e c t s were s i g n i f i c a n t when a l l t r e a t m e n t s were compared w i t h r e s p e c t t o g r o w t h s l o p e s . A d d i t i o n a l l y , d e n s i t y and t i m e were s i g n i f i c a n t . S e v e r a l i n t e r a c t i o n s were s i g n i f i c a n t , t h e two most i m p o r t a n t b e i n g d e n s i t y by t i m e and t e m p e r a t u r e by t i m e . T o g e t h e r t h e s e s u g g e s t e d t h a t t h e e f f e c t s of d e n s i t y on g r o w t h were d i f f e r e n t a t d i f f e r e n t t i m e s and t h a t t h e e f f e c t s o f t e m p e r a t u r e v a r i e d o v e r t i m e . C o n s e q u e n t l y , g r o w t h was a n a l y s e d s e p a r a t e l y f o r e a c h t e m p e r a t u r e . In t h e s e a n a l y s e s , f u r t h e r s i g n i f i c a n t d e n s i t y by t i m e i n t e r a c t i o n t e r m s s u g g e s t e d t h a t d e n s i t y e f f e c t s v a r i e d o v e r t i m e . T h e r e f o r e , d a t a were a n a l y s e d by tim e i n t e r v a l g r o u p i n g . At t h e h i g h t e m p e r a t u r e , d e n s i t y e f f e c t s on growth were s i g n i f i c a n t o n l y d u r i n g t h e f i r s t of f o u r t i m e i n t e r v a l s . The s h o r t d u r a t i o n of t h e d e n s i t y e f f e c t was i n d i c a t i v e o f c o n d i t i o n i n g . About 90% of t h e d i f f e r e n c e s i n f i n a l w e i g h t c o u l d be a s c r i b e d t o t h e f i r s t t i m e i n t e r v a l . At t h e l o w e r t e m p e r a t u r e , t r e n d s s i m i l a r t o t h o s e a t t h e h i g h e r t e m p e r a t u r e a p p e a r e d d u r i n g t h e f i r s t i n t e r v a l , but were 57 n o t s i g n i f i c a n t d u r i n g t h a t o r t h e r e m a i n i n g i n t e r v a l s . O v e r a l l however, g r o w t h r a t e s were s u p p r e s s e d a t t h e h i g h d e n s i t y . A n o n - s i g n i f i c a n t , but p e r s i s t e n t t r e n d t o w a r d s g r e a t e r g r o w t h s l o p e s i n t h e m i d d l e d e n s i t y o v e r t h e low d e n s i t y was o b s e r v e d f o r most t i m e i n t e r v a l s . In a l l g r o w t h a n a l y s e s , t h e r e was an u n u s u a l p a t t e r n w i t h t i m e . Growth r a t e s i n a l l t r e a t m e n t s were i n i t i a l l y low, i n c r e a s e d t o a peak by t h e s e c o n d i n t e r v a l , and t h e n d e c l i n e d s t e a d i l y t h e r e a f t e r . The d e c l i n e w i t h t i m e was n o t a f i s h s i z e e f f e c t , s i n c e g r o w t h s l o p e a n a l y s i s c o m p e n s a t e s f o r s i z e . T h i s p a t t e r n , as w i t h t h e s h o r t d u r a t i o n d e n s i t y e f f e c t , was s u g g e s t i v e o f a c o n d i t i o n i n g e f f e c t . Whole body c o m p o s i t i o n d a t a r e v e a l e d b o t h d e n s i t y and t e m p e r a t u r e e f f e c t s . W i t h t e m p e r a t u r e e n t e r e d a s a f a c t o r i n t h e a n a l y s i s , s i g n i f i c a n t d e n s i t y e f f e c t s were o b s e r v e d i n m o i s t u r e , p r o t e i n and l i p i d s , b ut not i n a s h c o n t e n t . In s e v e r a l a n a l y s e s , t h e d e n s i t y by t e m p e r a t u r e i n t e r a c t i o n s were s i g n i f i c a n t , n e c e s s i t a t i n g f u r t h e r a n a l y s i s by t e m p e r a t u r e . A t t h e h i g h t e m p e r a t u r e , m o i s t u r e and p r o t e i n l e v e l s were s i g n i f i c a n t l y l o w e r , and l i p i d l e v e l s s i g n i f i c a n t l y g r e a t e r , t h a n a t t h e low t e m p e r a t u r e . Ash was u n a f f e c t e d . A t 15°C, m o i s t u r e and p r o t e i n l e v e l s i n c r e a s e d s i g n i f i c a n t l y , and l i p i d l e v e l s d e c r e a s e d s i g n i f i c a n t l y , as d e n s i t y i n c r e a s e d . As b e f o r e , a s h c o n t e n t was n o t a f f e c t e d . A t 12.5°C, s i g n i f i c a n t d e n s i t y e f f e c t s on p r o x i m a t e c o m p o s i t i o n were not f o u n d . A l t h o u g h few of t h e c o m p a r i s o n s were s i g n i f i c a n t , some were c l o s e , and t r e n d s i n m o i s t u r e , p r o t e i n 58 and l i p i d data suggested s i m i l a r r e l a t i o n s h i p s to those at the higher temperature. With temperatures pooled, 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 net a c t i v i t y (the tendency to maintain p o s i t i o n s ) a t t r i b u t a b l e to d e n s i t y e f f e c t s . However, at each temperature, p r o b a b i l i t i e s while not s i g n i f i c a n t , were c l o s e , and the p a t t e r n s observed were i d e n t i c a l . L e v e l s were g r e a t e s t at the middle d e n s i t y , and lowest at the low d e n s i t y . The c o n s i s t e n c y of t h i s p a t t e r n and the near s i g n i f i c a n c e , suggest t h a t the p a t t e r n was a r e a l consequence of d e n s i t y , and not a chance occurrence. T o t a l a c t i v i t y decreased as d e n s i t y i n c r e a s e d ( s i g n i f i c a n t at 15°C, not so at 12.5°C). The p o s s i b l e s i g n i f i c a n c e of these f i n d i n g s i n c o n j u n c t i o n with the net a c t i v i t y r e s u l t s w i l l be d i s c u s s e d . l a t e r . No trends i n plasma C o r t i s o l c o n c e n t r a t i o n s were pr e s e n t . T h i s would i n d i c a t e that f i s h were not d i f f e r e n t i a l l y s t r e s s e d by d i f f e r e n t d e n s i t i e s , or that i f i n i t i a l l y s t r e s s e d , a compensatory adjustment of i n t e r r e n a l a c t i v i t y as d e s c r i b e d by Schreck (1981) may have oc c u r r e d . The f i r s t h y pothesis, that of no s t r e s s appears u n l i k e l y i n view of a l l other r e s u l t s presented. C o n d i t i o n f a c t o r data showed s i m i l a r trends to those of growth slope data. At both temperatures, c o n d i t i o n s v a r i e d s i g n i f i c a n t l y with time; the p a t t e r n being i d e n t i c a l to that f o r growth s l o p e s . Only at '15°C were d e n s i t y e f f e c t s s i g n i f i c a n t , c o n d i t i o n f a c t o r s d e c r e a s i n g as d e n s i t y i n c r e a s e d . At 12.5°C, no 59 t r e n d was a p p a r e n t . The i n t e r a c t i o n term was s i g n i f i c a n t a t b o t h t e m p e r a t u r e s , and was r e f l e c t e d by a t e n d e n c y t o w a r d s d e l a y e d p e a k i n g a t h i g h e r d e n s i t i e s . From t h e s e d a t a i t i s c l e a r t h a t a t any t i m e , a s t r o n g c o r r e l a t i o n between g r o w t h s l o p e and c o n d i t i o n e x i s t e d . In summary, t h e r e s p o n s e t o r e a r i n g d e n s i t y i s d i f f e r e n t a t t h e two t e m p e r a t u r e s . A t 15°C, t h e r e a r e c l e a r and c o n s i s t e n t i n d i c a t i o n s o f s t r e s s - i n d u c e d m e t a b o l i c and b e h a v i o r a l d i f f e r e n c e s a s s o c i a t e d w i t h d e n s i t y . At 12.5°C, th e d i f f e r e n c e s n o t e d , a l t h o u g h n o t s t a t i s t i c a l l y s i g n i f i c a n t , f o l l o w e d t r e n d s s i m i l a r t o t h o s e a t 15°C, s u g g e s t i n g t h a t e i t h e r t h e m a g n i t u d e of t h e r e s p o n s e i s r e d u c e d a t t h e l o w e r t e m p e r a t u r e , o r t h a t i t s m a n i f e s t a t i o n i s d e l a y e d . 60 EXPERIMENT 2: STRESS RESPONSES IN FINGERLINGS I n t r o d u c t i o n T h i s e x p e r i m e n t e x a m i n e d t h e e f f e c t s o f d e n s i t y on g r o w t h a n d s t r e s s i n j u v e n i l e s t e e l h e a d t r o u t (15 gm i n i t i a l w e i g h t ) . W e i g h t s were e x p e c t e d t o d o u b l e d u r i n g t h e e x p e r i m e n t a n d t h u s e n a b l e d e t e c t i o n o f g r o w t h d i f f e r e n c e s and o t h e r p h y s i o l o g i c a l c o n s e q u e n c e s o f d e n s i t y - i n d u c e d s t r e s s . S i n c e t h e r e s u l t s o f t h e f i r s t e x p e r i m e n t s u g g e s t e d t h a t t h e f i r s t few d a y s ( o r weeks) a t a new d e n s i t y ( t h e p e r i o d i m m e d i a t e l y a f t e r t r a n s f e r t o a t e s t d e n s i t y f r o m a common d e n s i t y ) may be most s e n s i t i v e , t h e e f f e c t s o f s u d d e n c h a n g e s i n d e n s i t y were e x a m i n e d . I f i t i s i s t h e r e l a t i v e c h a n g e i n d e n s i t y f i r s t e x p e r i e n c e d upon t r a n s f e r , and n o t c t h e a c t u a l d e n s i t y , . t h a t d e t e r m i n e s t h e m a g n i t u d e o f t h e s t r e s s r e s p o n s e t h i s c o u l d h ave i m p o r t a n t i m p l i c a t i o n s i n b o t h h a t c h e r y management an d i n t h e i n t e r p r e t a t i o n o f r e s u l t s o f d e n s i t y e x p e r i m e n t s . L a s t l y , t h i s e x p e r i m e n t e x a m i n e s c h a n g e s i n g r o w t h o v e r t i m e t o d e t e r m i n e i f t h e p a t t e r n o b s e r v e d i n t h e f i r s t e x p e r i m e n t i s r e p e a t e d w i t h l a r g e r f i s h . S i n c e t h e o b s e r v e d p a t t e r n s u g g e s t s a c o n d i t i o n i n g e f f e c t , s u dden d e n s i t y c h a n g e s s h o u l d e i t h e r a c c e l e r a t e o r d e c e l e r a t e c h a n g e s i n g r o w t h o v e r t i m e . 61 M a t e r i a l s and Methods The e x p e r i m e n t was s t a r t e d on December 6, 1980. R e p l i c a t e d g r o u p s o f s t e e l h e a d j u v e n i l e s were r e a r e d a t one of two n u m e r i c a l d e n s i t i e s - - (200 o r 800 f i s h p e r t a n k ) . I n i t i a l mean w e i g h t o v e r a l l t a n k s was 15.1 ± 0.61 gm, ( r a n g e 13.9-16.1). Tank volumes were a d j u s t e d t o e i t h e r 250 o r 500 l i t r e s . T h i s r e s u l t e d i n f o u r d e n s i t y g r o u p s and r e p r e s e n t e d an e i g h t - f o l d r a n g e . On a w e i g h t / v o l u m e b a s i s t h i s was 6 t o 48 gm/1 (1/3 .- 3 t i m e s s t a n d a r d d e n s i t i e s ) . The e x p e r i m e n t a l d e s i g n i s p r e s e n t e d i n T a b l e 8. A l l f i s h u s e d i n t h i s e x p e r i m e n t had s i m i l a r r e a r i n g h i s t o r i e s . On November 13, 1980, a b o u t 4 weeks b e f o r e t h e s t a r t o f t h e s t u d y , t h e f i s h were t r a n s f e r r e d from 2 m c i r c u l a r t a n k s t o t h e o v a l t a n k s and h e l d a t 12.5 °C. T h e r e were a p p r o x i m a t e l y 650 f i s h p e r t a n k , c o r r e s p o n d i n g t o a d e n s i t y of a b o u t 40 gm/1 i m m e d i a t e l y p r i o r t o t h e s t a r t o f t h e s t u d y . F i s h were f e d r e d u c e d r a t i o n s u n t i l one week b e f o r e t h e e x p e r i m e n t began, when e x c e s s r a t i o n s were g i v e n . To examine t h e e f f e c t s o f sudden c h a n g e s i n d e n s i t y on a w e i g h t / v o l u m e o r number/volume b a s i s ( n o t a n u m e r i c a l d e n s i t y b a s i s ) t h r e e volume t r e a t m e n t s were u s e d . In one t r e a t m e n t , t a n k s i n i t i a l l y h e l d a t 250 l i t r e s were d o u b l e d i n volume t o 500 l i t r e s . I n a s e c o n d t r e a t m e n t , t h e o p p o s i t e was done. In t h e t h i r d t r e a t m e n t , t h e c o n t r o l , volumes were unchanged. I t was d e c i d e d t h a t t a n k volumes would n o t be a d j u s t e d u n t i l t h e p a t t e r n i n g r o w t h s l o p e s w i t h t i m e showed e v i d e n c e of a d e c l i n e , s i m i l a r t o t h a t o b s e r v e d i n t h e f i r s t e x p e r i m e n t . U n f o r t u n a t e l y , t h e number of t a n k s was l i m i t e d , and t h e e x p e r i m e n t a l d e s i g n 62 c o u l d not be b a l a n c e d . C h a n g i n g t h e a v a i l a b l e s p a c e p e r f i s h ( d e n s i t y ) was e x p e c t e d t o a l t e r t h e r a t e o f change i n g r o w t h s l o p e s . T h e s e c hanges were e x p e c t e d o v e r d e n s i t y r a n g e s n o t sho w i n g e v i d e n c e of growth d i f f e r e n c e s p r i o r t o volume c h a n g e s . T a b l e 8. D e s i g n of e x p e r i m e n t 2. Tank volumes were c h a n g e d on day 32 ( d e n o t e d by t h e s e p a r a t i o n i n t h e t a b l e ) . E x p e c t e d maximum and f i n a l d e n s i t i e s were c a l c u l a t e d a c c o r d i n g t o Iwama and T a u t z (1981) u s i n g T/1000. FIRST FIRST DENSITY TNK NUMBER VOLUME I N I T I A L MAX FISH l i t r e s f / 1 gm/1 gm/1 SECOND SECOND DENSITY VOLUME - I N I T I A L - FINAL AVE l i t r e s f / 1 gm/1 gm/1 f / 1 1 200 250 0.8 1 2 18.8 500 0.4 9.4 1 2 0.6 2 800 250 3.2 48 75.2 500 1 .6 37.6 49 2.4 3 200 250 0.8 1 2 18.8 500 0.4 9.4 12 0.6 4 800 250 3.2 48 75.2 500 ' 1 .6 37.6 49 2.4 5 200 500 0.4 6 9.4 500 0.4 9.4 12 0.4 6 800 500 1 .6 24 37.6 500 1 .6 37.6 49 1 .6 7 200 500 0.4 6 9.4 500 0.4 9.4 1 2 0.4 8 800 500 1 .6 24 .37.6 500 1 .6 37.6 49 1 .6 9 200 500 0.4 6 9.4 250 0.8 18.8 24 0.6 1 0 800 500 1 .6 24 37.6 250 3.2 75.2 98 2.4 1 1 200 500 0.4 6 9.4 250 0.8 18.8 24 0.6 1 2 800 500 1 .6 24 37.6 250 3.2 75.2 98 2.4 A l l f i s h were r e a r e d a t 12.5°C P h o t o p e r i o d was s e t a t 10L:14D. L i g h t s were a u t o m a t i c a l l y s w i t c h e d on a t 0730 h o u r s and o f f a t 1730 h o u r s . A l l t a n k s r e c e i v e d f l o w s o f 25 1/min. S i n c e an o p t i m i s t i c p r o j e c t e d g r o w t h r a t e was u s e d , maximum d e n s i t y e s t i m a t e s were c o n s e r v a t i v e . F e e d i n g r a t e s were c a l c u l a t e d a s f o r t h e p r e v i o u s e x p e r i m e n t . The d e s i g n of the e x p e r i m e n t n e c e s s i t a t e d t h a t s e v e r a l d i f f e r e n t a n a l y s e s be r u n on t h e same d a t a , b u t on d i f f e r e n t 63 g r o u p i n g s . P a r t of t h e e x p e r i m e n t was a n a l y z e d i n t h e same manner a s t h e f i r s t e x p e r i m e n t . D e t a i l s w i l l be g i v e n where a p p r o p r i a t e . T o t a l w e i g h t s were measured on d a y s 1, 10, 17, 31, 46, and 53. L e n g t h and w e i g h t d a t a were t a k e n on days 3, 10, 31, 46, and 53. D u r i n g t h e c o u r s e o f t h e e x p e r i m e n t , g r o w t h s l o p e s were p l o t t e d a g a i n s t t i m e . At t h e f i r s t i n d i c a t i o n s of t h e a n t i c i p a t e d c h a n g e s i n g r o w t h p a t t e r n tank volumes, were a l t e r e d . The t r e n d was d e t e c t e d on day 31, and c h a n g e s were made on day 32. Plasma samples f o r C o r t i s o l a n a l y s i s were c o l l e c t e d on day 56. A f t e r two d a y s o f s t a r v a t i o n , on day 58, sam p l e s were c o l l e c t e d f o r p r o x i m a t e c o m p o s i t i o n . D i s s o l v e d oxygen l e v e l s were m o n i t o r e d on two d a y s , 41 and 49, u s i n g W i n k l e r r e a g e n t s . Water s a m p l e s were c o l l e c t e d f o r a n a l y s i s of pH, m e t a b o l i t e s , e t c . on t h e same d a y s . B e h a v i o r a l o b s e r v a t i o n s were n o t r e c o r d e d . M o r t a l i t i e s were r e c o r d e d d a i l y b u t were r a r e . T e m p e r a t u r e s and f l o w s were c h e c k e d e v e r y 2-3 d a y s . R e s u l t s Growth D u r i n g t h e c o u r s e of t h i s e x p e r i m e n t some d a t a were l o s t due t o t e c h n i c a l d i f f i c u l t i e s . D u r i n g t h e f i r s t t i m e i n t e r v a l , f e e d e r p r o b l e m s i n t a n k 2 r e s u l t e d i n t h e l o s s o f g r o w t h s l o p e d a t a f o r t h a t p e r i o d . More p e r s i s t e n t f e e d e r p r o b l e m s i n t a n k 5 r e s u l t e d i n t h e l o s s of g r o w t h s l o p e i n f o r m a t i o n f o r t h r e e 64 i n t e r v a l s . Changes i n a d j u s t e d w e i g h t s o v e r t i m e a r e g i v e n i n F i g u r e 6. Growth s l o p e s i n c o n s t a n t d e n s i t y t r e a t m e n t s To examine t h e e f f e c t s of c o n s t a n t d e n s i t y on g r o w t h s l o p e s , t h e c o n t r o l t a n k s (5,6,7,8) were a n a l y z e d s e p a r a t e l y . On b o t h a w e i g h t / v o l u m e and number/volume b a s i s , t h e s e t r e a t m e n t s s panned most of t h e r a n g e t e s t e d i n t h e f i r s t e x p e r i m e n t . On a f i s h / l i t r e b a s i s , d e n s i t y r a n g e d from 0.4 t o 1.6 (ab o u t 0.33 t o 1.3 t i m e s c o n v e n t i o n a l maximum h a t c h e r y d e n s i t i e s ) . The d a t a were a n a l y z e d i n a 2-way ANOVA. D e n s i t y e f f e c t s were n o t s i g n i f i c a n t (df=1,16 p=.11). However, f i s h i n t h e h i g h d e n s i t y t r e a t m e n t a p p e a r e d t o grow a t 90% o f t h e r a t e o f t h e low d e n s i t y t r e a t m e n t . A l t h o u g h t h e low d e n s i t y t r e a t m e n t r e a c h e d a h i g h e r peak g r o w t h s l o p e , t h e r e was a t r e n d f o r a more r a p i d d e c l i n e i a gr o w t h s l o p e w i t h t i m e than a t t h e h i g h d e n s i t y . F o r s u c h a t r e n d t o be d e t e c t a b l e s t a t i s t i c a l l y , t h e i n t e r a c t i o n t e r m w o u l d have t o be s i g n i f i c a n t . I t was n o t (df=4,16 p=.11). The e f f e c t of t i m e , however, was ( d f = 4 , l 6 p<.05) and t h e p a t t e r n c l o s e l y f o l l o w e d t h a t e x p e c t e d . Growth s l o p e s ( a v e r a g e d e n s i t i e s ) G rowth r a t e s were a n a l y s e d b a s e d on a v e r a g e d e n s i t y g r o u p s ( T a b l e 8, l a s t c o l . ) . A c c o r d i n g l y t h e r e were s i x t r e a t m e n t s , e a c h d e f i n e d by one of two n u m e r i c a l d e n s i t i e s and one o f t h r e e volume t r e a t m e n t s . I t was not p o s s i b l e t o e n t e r b o t h f i s h number and volume i n t o an ANOVA s i n c e t h e y c o v a r i e d a l m o s t p e r f e c t l y . 65 F i g u r e 6. Change i n w e i g h t w i t h t i m e f o r e x p e r i m e n t 2. A l l w e i g h t s have been a d j u s t e d t o a common i n i t i a l s i z e u s i n g an i n i t i a l w e i g h t of 15 grams and t h e g r o w t h s l o p e s by t i m e i n t e r v a l . 35 T TIME (DAYS) 67 F o l l o w i n g t h e work o f L i and B r o c k s e n (1977) i t was assumed t h a t volume e f f e c t s would be m i n i m a l ( G e n e r a l D i s c u s s i o n ) . T h e r e f o r e , when v o l u m e s were c h a n g e d , i t was assumed t h a t any e f f e c t s were due t o d e n s i t y c h a n g e s . The d a t a were e n t e r e d i n a 2-way ANOVA ( a v e r a g e d e n s i t y , t i m e ) ( T a b l e 9 ) . A v e r a g e d e n s i t y , t i m e and t h e i n t e r a c t i o n terms were a l l s i g n i f i c a n t . As a v e r a g e d e n s i t y i n c r e a s e d o v e r a l l g r o w t h s l o p e d e c r e a s e d , a l t h o u g h t h e p a t t e r n was n o t c o n s i s t e n t . S c h e f f e ' s t e s t i n d i c a t e d t h a t o n l y t h e t a n k s c o n t a i n i n g 200 f i s h ( c o n s t a n t d e n s i t y ) and t h o s e c o n t a i n i n g 800 f i s h i n w h i c h d e n s i t i e s were d o u b l e d , were d i f f e r e n t . The i n t e r a c t i o n s u g g e s t e d t h a t t h e e f f e c t o f d e n s i t y c h a n g e d o v e r t i m e , however t h i s c o u l d have been a f u n c t i o n o f sudden d e n s i t y c h a n g e s . The o v e r a l l p a t t e r n w i t h t i m e was a s e x p e c t e d -- low i n i t i a l l y , r i s i n g t o a peak, and t h e n d e c l i n i n g . D i f f e r e n c e s between t i m e s were o f s u f f i c i e n t m a g n i t u d e t h a t S c h e f f e ' s t e s t p r o d u c e d 3 homogeneous g r o u p s . When i n d i v i d u a l t r e a t m e n t s were examined, p a t t e r n s o v e r t i m e d i d n o t a l w a y s f o l l o w t h i s t r e n d ( F i g u r e 7 ) . T h i s p r o b a b l y r e f l e c t e d t h e sudden c h a n g e s i n d e n s i t y . Growth b e f o r e d e n s i t y c h a n g e s To examine g r o w t h d i f f e n c e s b e f o r e t a n k v olumes, and t h e r e f o r e w e i g h t d e n s i t i e s were c h a n g e d , t h e f i r s t t h r e e i n t e r v a l s were a n a l y z e d s e p a r a t e l y . T h e r e were f o u r g r o u p s c o v e r i n g an 8 - f o l d d e n s i t y r a n g e ( T a b l e 8, c o l . 4 ) , e i t h e r 200 of 800 f i s h , and e i t h e r h i g h o r low v o l u m e s . The d a t a were e n t e r e d i n a 2-way ANOVA. D e n s i t y e f f e c t s were s i g n i f i c a n t 68 F i g u r e 7. Growth s l o p e s a g a i n s t t i m e ( m i d - p o i n t o f s a m p l i n g i n t e r v a l ) f o r e x p e r i m e n t 2. Growth s l o p e s were c a l c u l a t e d a c c o r d i n g t o Iwama and T a u t z ( 1 9 8 1 ) . V e r t i c a l b a r s a r e s t a n d a r d e r r o r s . LI 0.020T 200 fish 200 fish 200 fish 0.015 + D _ O I O O CD CD 0.010+ constant low density high to low density low to high density 800 fish 800 fish 800 fish constant low density high to low density low to high density 0.005-0 10 20 30 40 50 60 MID-POINT OF TIME INTERVAL 70 T a b l e 9. A n a l y s i s o f v a r i a n c e o f g r o w t h s l o p e d a t a f o r e x p e r i m e n t 2. A v e r a g e d e n s i t y t r e a t m e n t s were e n t e r e d i n t o t h e a n a l y s i s ( T a b l e 8, l a s t c o l u m n ) . T e s t s a r e c o n s i d e r e d s i g n i f i c a n t i f p < 0.05. SOURCE DF MEAN SQUARE PROBABILITY D e n s i t y 5 Time 4 D e n s i t y * T i m e 20 R e s i d u a l 26 T o t a l 55 0.00000637 0.00005694 0.00000731 0.00000144 0.00483 0.0 0.00080 (df=3,33 p<.05). W i t h e a c h i n c r e a s e i n d e n s i t y , w i t h i n t h e l o w e s t t h r e e g r o u p s , t h e r e was a s l i g h t r e d u c t i o n i n mean g r o w t h s l o p e . However, between t h i r d h i g h e s t (1.6 f/1) and t h e h i g h e s t (3.2 f / 1 ) , t h e d e c l i n e was marked. T h i s s u g g e s t s a t h r e s h o l d e f f e c t . The e f f e c t s o f t i m e were s i g n i f i c a n t (df=2,33 p < . 0 0 l ) , and t h e means of e a c h o f t h e t h r e e t i m e s e n t e r e d were d i f f e r e n t . The i n t e r a c t i o n t e r m , t h o u g h n o t s i g n i f i c a n t was c l o s e (df=6,33 p=. 10), s u g g e s t i n g t h a t i t may have been t h e change i n d e n s i t y i n h e r e n t i n t h e p r e v i o u s a n a l y s i s on a l l 6 t r e a t m e n t s t h a t c a u s e d t h e s i g n i f i c a n t i n t e r a c t i o n e f f e c t . Growth a f t e r d e n s i t y c h a n g e s To examine g r o w t h c h a n g e s a f t e r sudden d e n s i t y c h a n g e s , t h e l a s t two t i m e s were examined s e p a r a t e l y . F o u r new d e n s i t y g r o u p s ( T a b l e 8, c o l . 8) were compared. D e n s i t y e f f e c t s were s i g n i f i c a n t (df=3,21 p<.05), as were t h e e f f e c t s o f t i m e Xdf =1,21 p<.005). As b e f o r e , t h e p a t t e r n showed a r e d u c t i o n i n g rowth s l o p e w i t h i n c r e a s e s i n d e n s i t y . The more r a p i d d e c l i n e i n g r o w t h r a t e s a t t h e l o w e r n u m e r i c a l d e n s i t i e s was e s p e c i a l l y 71 a p p a r e n t . The i n t e r a c t i o n t e r m was n o t s i g n i f i c a n t (p=.54). D i d t h e c h a n g e s i n d e n s i t y a l t e r g r o w t h s l o p e s t o a g r e a t e r e x t e n t t h a n e x p e c t e d on t h e b a s i s o f d e n s i t y a l o n e ? R e c a l l i n g t h a t t h e i n t e r a c t i o n e f f e c t s were s i g n i f i c a n t when a v e r a g e t r e a t m e n t s were compared o v e r a l l t i m e s , b u t not when d e n s i t i e s were a n a l y z e d b e f o r e and a f t e r volume c h a n g e s , i t i s a p p a r e n t t h a t t h e d e n s i t y c h a n g e s had a l t e r e d g r o w t h . To t e s t whether g r o w t h c h a n g e s were l e s s t h a n o r g r e a t e r t h a n t h a t e x p e c t e d from d e n s i t y e f f e c t s a l o n e , t h o s e t a n k s i n w h i c h volumes were n o t ch a n g e d were u s e d as c o n t r o l s . Changes i n g r o w t h s l o p e o v e r t i m e c o u l d t h e r e f o r e be compared w i t h e x p e c t e d c h a n g e s . L i n e a r r e g r e s s i o n s f o r t h e l a s t t h r e e i n t e r v a l s were c a l c u l a t e d f o r v a r i o u s t r e a t m e n t c o m b i n a t i o n s , and compared u s i n g e q u a l i t y o f s l o p e t e s t s . ' F i r s t , t h e c o n t r o l s were compared, and as e x p e c t e d from t h e e a r l i e r a n a l y s i s , t h e i r s l o p e s were not d i f f e r e n t (p=.09). However, i t a p p e a r e d t h a t t h e r a t e of d e c l i n e i n g r o w t h s l o p e s was a c c e l e r a t e d a t t h e low e r d e n s i t y . U n f o r t u n a t e l y , d a t a f r o m one of t h e c o n t r o l s were m i s s i n g , r e d u c i n g t h e s e n s i t i v i t y o f s u b s e q u e n t c o m p a r i s o n s . When t h e low d e n s i t y c o n t r o l s (200 f i s h , c o n s t a n t volume) were compared w i t h t h e low d e n s i t y t r e a t m e n t i n w h i c h volumes were i n c r e a s e d ( d e n s i t i e s d e c r e a s e d , i . e . b o t h had e q u a l d e n s i t i e s a t t i m e o f c o m p a r i s o n ) , s l o p e s were not s i g n i f i c a n t l y d i f f e r e n t . However, t h e r e was a t r e n d t o w a r d s a l e s s s t e e p d e c l i n e i n g r o w t h s l o p e i n t h e g r o u p w h i c h had undergone t h e sudden d e n s i t y r e d u c t i o n , and i n f a c t , i n t h e f i r s t i n t e r v a l 72 f o l l o w i n g d e n s i t y c h a n g e s , g r o w t h r a t e s a c t u a l l y i n c r e a s e d . When t h e same c o n t r o l s were compared w i t h t h e low d e n s i t y t r e a t m e n t i n w h i c h volumes were h a l v e d , s l o p e d i f f e r e n c e s were s i g n i f i c a n t (p<.05). The p a t t e r n was c o n s i s t e n t w i t h t h e p r e d i c t i o n , i . e . an i n c r e a s e ( 2 - f o l d ) i n t h e r a t e o f d e c l i n e o f g r o w t h s l o p e s w i t h t i m e . A l t h o u g h t h i s c o m p a r i s o n was between two d i f f e r e n t d e n s i t i e s , i t i s u n l i k e l y t h a t t h e a b s o l u t e d e n s i t y d i f f e r e n c e was i m p o r t a n t , s i n c e i n none of t h e e a r l i e r a n a l y s e s were t h e r e s i g n i f i c a n t d i f f e r e n c e i n g r o w t h o v e r t h i s d e n s i t y r a n g e . D e c r e a s i n g t h e s p a c e p e r f i s h ( i n c r e a s i n g t h e d e n s i t y ) a f t e r t h e f i s h had been a c c u s t o m e d t o one d e n s i t y , a p p e a r e d t o a f f e c t growth more t h a n one would e x p e c t on t h e b a s i s o f d e n s i t y a l o n e . In n e i t h e r c o m p a r i s o n a t t h e h i g h n u m e r i c a l d e n s i t y , were s l o p e s s i g n i f i c a n t l y d i f f e r e n t . In b o t h , however, t r e n d s were i n t h e e x p e c t e d d i r e c t i o n . I f c o n d i t i o n i n g had no e f f e c t , i t would seem i m p r o b a b l e t h a t when d e n s i t i e s were d e c r e a s e d ( i . e . v o l u m e s i n c r e a s e d ) , t h e r e would be a c o n s i s t e n t ( a l t h o u g h n o n - s i g n i f i c a n t ) t r e n d f o r f a s t e r g r o w t h t h a n t h e c o n t r o l s t h a t had c o n t i n u o u s l y e x p e r i e n c e d t h e same d e n s i t y . P r o x i m a t e a n a l y s i s S t a t i s t i c a l a n a l y s e s were p e r f o r m e d on a v e r a g e d e n s i t y g r o u p i n g s . In a l l a n a l y s e s , w e i g h t was u s e d as a c o v a r i a t e . 73 M o i s t u r e The r e s u l t s of a 1-way n e s t e d ANOVA w i t h w e i g h t a s a c o v a r i a t e f o r t h e 6 a v e r a g e d e n s i t y t r e a t m e n t s a r e p r e s e n t e d i n F i g u r e 8a. The e f f e c t s o f d e n s i t y were s i g n i f i c a n t (df=5,49 p < . 0 0 l ) . As a v e r a g e d e n s i t y i n c r e a s e d , so d i d m o i s t u r e . The e f f e c t of t h e c o v a r i a t e , w e i g h t , was s i g n i f i c a n t (df=1,49 p<.001), i n d i c a t i n g t h a t m o i s t u r e l e v e l s were a f f e c t e d by f i s h w e i g h t . The e q u a l i t y of s l o p e term, d e f i n e d by t h e i n t e r a c t i o n o f t h e c o v a r i a t e w i t h d e n s i t y , was a l s o s i g n i f i c a n t (df=5,49 p<.005). T h i s s u g g e s t e d t h a t t h e e f f e c t o f w e i g h t was n o t t h e same f o r a l l t r e a t m e n t s . C o n s e q u e n t l y , i t i s l i k e l y t h a t s i g n i f i c a n t e f f e c t s between t r e a t m e n t s would d i s a p p e a r as t h e i r c o v a r i a t e - d e n s i t y r e g r e s s i o n l i n e s a p p r o a c h e d and c r o s s e d e a c h o t h e r . However, s i n c e t h e mean sample w e i g h t s were c l o s e , t h e o b s e r v e d d e n s i t y e f f e c t i s l i k e l y r e a l . P r o t e i n The r e s u l t s of t h e 1-way, n e s t e d ANOVA w i t h w e i g h t as a c o v a r i a t e , p r o d u c e d s i g n i f i c a n t d e n s i t y e f f e c t s (df=5,49 p<.05). P r o t e i n l e v e l s t e n d e d t o i n c r e a s e w i t h a v e r a g e d e n s i t y , a l t h o u g h t h e p a t t e r n was i r r e g u l a r ( F i g u r e 8 b ) . At t h e t i m e t h a t samples were c o l l e c t e d , one of t h e high, d e n s i t y t r e a t m e n t s had a d e n s i t y of 3.2 f / 1 , even though i t s a v e r a g e d e n s i t y was 2.4 f / 1 . The o t h e r h i g h d e n s i t y t r e a t m e n t , w h i c h had a d e n s i t y of 1.6 f / 1 a t th e t i m e of s a m p l i n g , had l o w e r p r o t e i n l e v e l s . S i n c e b o t h had s i m i l a r a v e r a g e d e n s i t i e s , t h i s s u g g e s t s t h a t t h e - m e t a b o l i c 74 F i g u r e 8. Histograms of proximate composition data f o r experiment 2. A-moisture, B - p r o t e i n , C - l i p i d , D-ash S t i p p l e d bars are treatments c o n t a i n i n g 200 f i s h . E r r o r bars are i n c l u d e d . Bars b e a r i n g common s m a l l -case l e t t e r s are not s t a t i s t i c a l l y d i f f e r e n t (Duncan' or S c h e f f e ' s m u l t i p l e range t e s t s - 5% l e v e l ) . Uppe case l e t t e r s at the base of the bottom graph i n d i c a t e d e n s i t y treatments (e.g. H-L means i n i t i a l l y h i g h d e n s i t y , then decreased). ASH •<Z DRY WEIGHT) I D -4-00 PROTEIN (% DRY WEIGHT) MOISTURE CD cn cn at tr a 76 c o n s e q u e n c e s of d e n s i t y on body c o m p o s i t i o n a p p e a r q u i c k l y when d e n s i t i e s i n c r e a s e , and a l s o may d i s a p p e a r q u i c k l y when d e n s i t i e s a r e r e d u c e d . As w i t h m o i s t u r e , b o t h t h e c o v a r i a t e and t h e e q u a l i t y o f s l o p e s t e s t t e r m s were s i g n i f i c a n t (df=1,49 p<.05 and df=5,49 p<.005). The p a t t e r n i n c o v a r i a t e s l o p e s s u g g e s t e d t h a t a t h i g h d e n s i t i e s , w i t h i n t a n k s i z e v a r i a t i o n s had a g r e a t e r e f f e c t on p r o t e i n l e v e l s t h a n i n t h e low d e n s i t y t a n k s . T h i s may be i n d i c a t i v e o f g r e a t e r d i f f e r e n c e s i n l e v e l s o f s t r e s s w i t h i n h i g h d e n s i t y t a n k s t h a n w i t h i n low d e n s i t y t a n k s . L i p i d s The r e s u l t s o f t h e l i p i d a n a l y s i s ( F i g u r e 8c) were l e s s c l e a r . T h i s was due t o a h i g h e r d e g r e e o f v a r i a b i l i t y between r e p l i c a t e s . In t h e 1-way n e s t e d ANOVA w i t h w e i g h t a s a c o v a r i a t e , o n l y t h e r e p l i c a t e s t e r m was s i g n i f i c a n t (df=6,49 p<.01), a l t h o u g h t h e e f f e c t s o f b o t h d e n s i t y and t h e c o v a r i a t e were n e a r l y so (df=5,49 p=.09; df=1,49 p<.09). Upon v i s u a l i n s p e c t i o n i t was c l e a r , however, t h a t d i f f e r e n c e s d i d e x i s t e s p e c i a l l y between t h e h i g h d e n s i t y t r e a t m e n t , i n w h i c h volumes were h a l v e d , and a l l o t h e r s . T h e r e was a t r e n d t o w a r d s a r e d u c t i o n i n l i p i d s a t h i g h d e n s i t i e s . 77 A s h As w i t h t h e p r e v i o u s a n a l y s i s f o r l i p i d s , d i f f e r e n c e s between r e p l i c a t e s were g r e a t e r t h a n between t r e a t m e n t s . U n l i k e t h e l i p i d a n a l y s i s , t h e p r o b a b i l i t y v a l u e f o r t h e d e n s i t y e f f e c t was n o t s i g n i f i c a n t . T h e r e were no t r e n d s i n t h e d a t a ( F i g u r e 8 d ) . C o r t i s o l E i g h t p l a s m a s a m p l e s from e a c h tank were a s s a y e d i n d u p l i c a t e . T h i s r e s u l t e d i n 16 C o r t i s o l d e t e r m i n a t i o n s p e r t a n k . In a l l c a s e s , d u p l i c a t e s were s u c c e s s f u l l y a n a l y s e d and t h e r e was, t h e r e f o r e , no need t o p o o l d u p l i c a t e s . B e f o r e t h e d a t a were a n a l y z e d s t a t i s t i c a l y , p l o t s were made of C o r t i s o l v a l u e s . a g a i n s t b o t h sample w e i g h t and s a m p l i n g t i m e . T h e r e was a t r e n d t o w a r d s i n c r e a s e d C o r t i s o l l e v e l s a s sample t i m e p r o g r e s s e d . However, w i t h i n t a n k v a r i a t i o n was l a r g e making i t d i f f i c u l t t o a p p l y a c o r r e c t i o n f a c t o r . S i n c e sample p r o c e s s i n g t i m e s were c l o s e ( w i t h i n 1 m i n ) , t h e e f f e c t was p r o b a b l y c o n s t a n t between t a n k s . No t r e n d s i n C o r t i s o l w i t h f i s h w e i g h t were a p p a r e n t . D a t a were a n a l y s e d i n a 1-way, n e s t e d ANOVA (6 a v e r a g e d e n s i t y t r e a t m e n t s , 2 r e p l i c a t e s ) . The t r e a t m e n t g r o u p s were t h o s e d e s c r i b e d p r e v i o u s l y a n a l y s e s ( T a b l e 8, l a s t c o l . ) . T h e r e were no e f f e c t s o f a v e r a g e d e n s i t y on p l a s m a C o r t i s o l c o n c e n t r a t i o n s . The r e p l i c a t e s t e r m was s i g n i f i c a n t . E a r t l e t t ' s t e s t i n d i c a t e d a marked h e t e r o g e n e i t y o f v a r i a n c e between 78 T a b l e 10. Summary o f C o r t i s o l d a t a . T h e r e was no e v i d e n c e of s i g n i f i c a n t d i f f e r e n c e s between t r e a t m e n t s . V a l u e s a r e g i v e n i n n g / m l . Samples s i z e s i n a l l c e l l s were 16. S t a n d a r d e r r o r s ( o f means) were 0.71. f i s h / t a n k 200 800 r e p l i c a t e ' 1 2 1 2 h i g h t o low d e n s i t y 15.1 11.3 11.4 12.4 c o n s t a n t d e n s i t y 12.3 12.6 14.9 12.1 low t o h i g h d e n s i t y 12.0 12.5 12.3 15.4 t r e a t m e n t s . S e v e r a l t r a n s f o r m a t i o n s were t e s t e d t o e l i m i n a t e t h i s p r o b l e m , but none were s u c c e s s f u l . C o n s e q u e n t l y , non-p a r a m e t r i c c o m p a r i s o n s were p e r f o r m e d on t h e d a t a w i t h r e p l i c a t e s p o o l e d . O n l y 4 o f t h e 15 Mann-Whitney L J - t e s t c o m p a r i s o n s i n d i c a t e d s i g n i f i c a n t d i f f e r e n c e s . However, t h e r e were no p a t t e r n s i n t h e d a t a ( T a b l e 10) s u g g e s t i n g t h e d i f f e r e n c e s were p o s s i b l y c h a n c e o c c u r e n c e s a n d were t h e r e f o r e c o n s i d e r e d u n i m p o r t a n t . Weight l e n g t h r e l a t i o n s S t a t i s t i c a l t r e a t m e n t of t h e l e n g t h - w e i g h t d a t a was t h e same as t h a t o u t l i n e d i n t h e f i r s t e x p e r i m e n t . In c a l c u l a t i n g t h e l i n e a r r e g r e s s i o n r e l a t i o n s h i p between l n w e i g h t and l n l e n g t h , d a t a from t i m e i n t e r v a l s where f e e d e r p r o b l e m s o c c u r r e d were e x c l u d e d . C o n s e q u e n t l y , 2650 of 3000 d a t a p a i r s were a c c o u n t e d f o r i n t h e e q u a t i o n . A f u n c t i o n a l GM r e g r e s s i o n o f n a t u r a l l o g of l e n g t h a g a i n s t n a t u r a l l o g of w e i g h t was c a l c u l a t e d . The d a t a f i t was good ( r 2 = . 9 3 ) . The e q u a t i o n of t h e l i n e was: 79 l n Wt. = -4.317 + 2.956 * l n L. R e a r r a n g e d and a n t i l o g g e d , t h e e q u a t i o n becomes: c o n d i t i o n f a c t o r = w e i g h t / ( 0 . 0 1 3 3 3 9 8 * l e n g t h 2 ' 3 5 6 ) , w h i c h i s t h e w o r k i n g e q u a t i o n . T a b l e 11. C o n d i t i o n f a c t o r d a t a summary. V a l u e s a r e g i v e n f o r t r e a t m e n t g r o u p s on t h e b a s i s of a v e r a g e d e n s i t y ( see T a b l e 8 ) . D e n s i t i e s were c h a n g e d on day 32 as d e s c r i b e d i n t h e methods s e c t i o n . R e p l i c a t e s were n o t s i g n i f i c a n t l y d i f f e r e n t and were t h e r e f o r e p o o l e d . L e t t e r s i n column 2 r e f e r t o r e l a t i v e d e n s i t y c h a n g e s . F o r example, H-L, means i n i t i a l d e n s i t y was h i g h , and t h e n on day 32 i t was halved,. AVE DENSITY DAY f / 1 TREAT. 3 10 31 46 53 n S.E. 0.4 L-L 0 .989 0 .985 1.017 0 .981 1 .016 50 0.0096 0.6 H-L 1 .003 0 .995 1.017 0 .996 1 .030 1 00 0.0068 0.6 L-H 0 .984 1 .010 1.014 0 .997 0 .983 1 00 0.0068 1 .6 L-L 0 .988 0 .980 1.015 1 .009 1 .026 1 00 0.0068 2.4 H-L 1 .009 1 .014 1 .024 1 .010 1 .041 50 0.0096 2.4 L-H 0 .988 0 .991 1 .025 0 .996 1 .024 100 0.0068 C o n d i t i o n f a c t o r d a t a The c o n d i t i o n d a t a ( T a b l e 11) were a n a l y s e d i n a 2-way ANOVA ( a v e r a g e d e n s i t y , t i m e ) , w i t h r e p l i c a t e s p o o l e d . D e n s i t y ( d f = 5 , 2 4 9 9 ) , t i m e (df=4,2499) and t h e i n t e r a c t i o n (df=20,2499) terms were s i g n i f i c a n t ( p < . 0 0 l ) . The range i n mean v a l u e s was s m a l l , and s u r p r i s i n g l y , t h e l o w e s t mean v a l u e was f o u n d i n t h e l o w e s t d e n s i t y t r e a t m e n t (200 f i s h , c o n s t a n t v o l u m e ) , w h i l e t h e h i g h e s t was f o u n d i n one of t h e t r e a t m e n t s c o n t a i n i n g 800 (low t o h i g h v o l u m e ) . O n l y t h e s e t r e a t m e n 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 . N o t a b l y , t h i s p a t t e r n was i n t h e o p p o s i t e d i r e c t i o n 80 t o t h e one o b s e r v e d i n t h e f i r s t e x p e r i m e n t . T h e r e was no c l e a r p a t t e r n w i t h d e n s i t y w i t h i n t h e o t h e r t r e a t m e n t s t e s t e d . The p a t t e r n w i t h t i m e d i d not f o l l o w t h a t i n g r o w t h s l o p e s as had been t h e c a s e i n t h e f i r s t e x p e r i m e n t . However, d u r i n g t h e f i r s t p a r t o f t h e e x p e r i m e n t , b e f o r e volumes were a d j u s t e d , t h e two v a r i a b l e s f o l l o w e d e a c h o t h e r c l o s e l y . The s i g n i f i c a n t i n t e r a c t i o n was a t t r i b u t a b l e t o t h e sudden c h a n g e s i n d e n s i t y . Water q u a l i t y Oxygen l e v e l s i n t h e h i g h d e n s i t y t a n k s were measured t h r e e t i m e s on day 41, between 1000 and 1500 h o u r s . D i f f e r e n c e s between t i m e s were s m a l l ( T a b l e 1 2 ) . S i n c e t h i s t i m e i n t e r v a l c o r r e s p o n d s t o t h e t i m e of day when oxygen l e v e l s s h o u l d be m a x i m a l l y d e p r e s s e d due t o f e e d i n g , a c t i v i t y , and s p e c i f i c dynamic a c t i o n , i t i s u n l i k e l y t h a t g r o w t h was c o n s t r a i n e d by oxygen l e v e l s . F u r t h e r m o r e , t h i s s u p p o r t s t h e v i e w t h a t s i n g l e -p o i n t - i n - t i m e measures o f oxygen l e v e l s were a d e q u a t e f o r t h e p u r p o s e i n t e n d e d . A d d i t i o n a l m e a s u r e s were c o l l e c t e d n e a r t h e end of t h e e x p e r i m e n t when oxygen demand s h o u l d have been g r e a t e s t . I n a l l t a n k s , l e v e l s were above t h e minimum a c c e p t a b l e l e v e l s . The r e s u l t s o f t h e l a b o r a t o r y a n a l y s i s of w a t e r s a m p l e s c o l l e c t e d on day 41 ( T a b l e 12) a l s o i n d i c a t e t h a t w ater q u a l i t y was a c c e p t a b l e and u n l i k e l y t o have a f f e c t e d e i t h e r g r o w t h o r s t r e s s r e s p o n s e . U n - i o n i z e d ammonia l e v e l s were w e l l below t h e g u i d e l i n e maximum d i s c u s s e d i n t h e G e n e r a l M e thods. I t was u n l i k e l y t h a t , i n t h e r e m a i n i n g d a y s of t h e e x p e r i m e n t , T a b l e 12. Water q u a l i t y d a t a summary f o r e x p e r i m e n t 2. To i n v e s t i g a t e v a r i a t i o n s d u r i n g t h e t i m e o f day when oxygen demand i s g r e a t e s t , oxygen l e v e l s were d e t e r m i n e d a t t h r e e t i m e s on day 41. Water samples f o r l a b o r a t o r y a n a l y s i s were c o l l e c t e d t h e same day. Oxygen d a t a were a g a i n c o l l e c t e d on day 49. N i t r i t e l e v e l s were below d e t e c t a b l e l e v e l s (<0.005 mg/1). U n - i o n i z e d ammonia l e v e l s were d e t e r m i n e d from L i a o (1974) . D A Y 4! TANK TIME OXYGEN TIME OXYGEN TIME OXYGEN h r s . mg/1 h r s . mg/1 h r s . mg/1 2 10: 1 4 8.35 13:00 8.50 1 5:22 8.60 4 10. 44 8.48 1 3:00 8.20 15:25 8.50 6 1 0 44 7.75 1 3:00 7.80 1 5:29 7.40 8 1 1 1 6 7.10 1 3:00 7.10 15:20 7.00 1 0 1 1 1 6 7.00 1 3:00 7.30 15:15 7.60 1 2 1 1 16 7.60 13:00 8.30 15:17 7.80 DAY 41 DAY 49 TOTAL UN-IONIZED TOTAL TANK AMMONIA AMMONIA NITRATE pH mg/1 mg/1 mg/1 S P E C I F I C DISSOLVED CONDUCT TURBIDITY OXYGEN umho/cm J.T.U. mg/1 1 - - - - - - 9.75 2 0.260 0.0018 5.65 7.5 219 1 .5 -3 - - - - - - 9.90 4 c 0.306 0.0021 5.70 7.5 220 1 .5 -O 6 0.464 0.0032 5.65 7.5 225 4.1 — 8 0.470 0.0033 5.70 7.5 224 2.7 -1 0 0.414 0.0024 5.70 7.4 221 3.5 -1 2 0.265 0.0019 5.75 7.5 219 1 .4 -m e t a b o l i t e l e v e l s i n c r e a s e d s u f f i c i e n t l y t o a l t e r t h i s c o n c l u s i o n . 82 C o n c l u s i o n s The r e s u l t s of t h i s e x p e r i m e n t were l e s s c l e a r t h a n t h o s e o f t h e p r e v i o u s e x p e r i m e n t . However, t h e r e was e v i d e n c e o f a s t r e s s - r e l a t e d r e s p o n s e t o d e n s i t y . As i n t h e f i r s t e x p e r i m e n t , p l a s m a c o r t i c o s t e r o i d l e v e l s , a p r i m a r y l e v e l s t r e s s i n d i c a t o r , showed no e v i d e n c e o f d e n s i t y e f f e c t s . However, b o t h g r o w t h s l o p e s and p r o x i m a t e c o m p o s i t i o n , t e r t i a r y l e v e l i n d i c a t o r s , p r o d u c e d r e s u l t s t h a t s u g g e s t i n c r e a s e d s t r e s s a t h i g h d e n s i t i e s . C o n d i t i o n f a c t o r r e s u l t s were i n c o n c l u s i v e , and i f d e c r e a s e d body c o n d i t i o n i s t r u l y a s s o c i a t e d w i t h s t r e s s , t h e s e r e s u l t s were somewhat c o n t r a d i c t o r y . The c u r v i l i n e a r g r o w t h p a t t e r n o b s e r v e d i n t h e f i r s t e x p e r i m e n t was r e p e a t e d i n t h e s e d a t a . T h i s s u p p o r t s t h e i d e a t h a t f i s h become c o n d i t i o n e d t o t h e i r r e a r i n g e n v i r o n m e n t . I t a l s o removes doub t t h a t t h e t r e n d w i t h t i m e was an a r t i f a c t o f t h e m o d e l . A d d i t i o n a l l y , a f t e r a p e r i o d of a c c l i m a t i o n t o r e a r i n g d e n s i t i e s , sudden c h a n g e s i n d e n s i t y t e n d e d t o a l t e r t h e g r o w t h r e s p o n s e more t h a n e x p e c t e d on t h e b a s i s o f d e n s i t y a l o n e . T h i s f u r t h e r s u p p o r t s t h e i d e a o f a c o n d i t i o n i n g i n f l u e n c e on g r o w t h r e s p o n s e . I n s p e c t i o n o f t h e w a t e r q u a l i t y d a t a i n d i c a t e t h a t oxygen and m e t a b o l i t e l e v e l s d i d n o t i n f l u e n c e t h e r e s u l t s . D e n s i t y e f f e c t s on g r o w t h were not o b s e r v e d when t r e a t m e n t s • c o n t a i n i n g 0.4 and 1.6 f i s h / l i t r e were compared; however, t h e e x p e c t e d p a t t e r n o v e r t i m e o c c u r r e d . When a l l 6 t r e a t m e n t s were compared, o n l y t h e l o w e s t and one o f t h e h i g h e s t d e n s i t i e s were d i f f e r e n t . In t r e a t m e n t s w i t h s i m i l a r a v e r a g e d e n s i t i e s , i n c r e a s i n g d e n s i t i e s mid-way t h r o u g h t h e e x p e r i m e n t p r o d u c e d 83 l o w e r o v e r a l l g r o w t h r a t e s t h a n t h o s e i n w h i c h d e n s i t i e s were d e c r e a s e d . T h i s s u g g e s t s t h a t d e c r e a s i n g t h e s p a c e p e r f i s h a f t e r a p e r i o d of a c c l i m a t i o n has a g r e a t e r e f f e c t on g r o w t h t h a n i n c r e a s i n g t h e s p a c e a f t e r a s i m i l a r a c c l i m a t i o n p e r i o d . In a l l c o m p a r i s o n s g r o wth r a t e s r e s p o n d e d i n t h e e x p e c t e d d i r e c t i o n t o c h a n g e s i n d e n s i t y ( a l t h o u g h 3 o f 4 o f t h e t e s t s were n o t s i g n i f i c a n t ) . Changes i n whole body c o m p o s i t i o n w i t h a v e r a g e d e n s i t y f o l l o w e d t h e same p a t t e r n as i n t h e p r e v i o u s e x p e r i m e n t s . However, t h e d a t a were l e s s c l e a r . As a v e r a g e d e n s i t y i n c r e a s e d , m o i s t u r e and p r o t e i n l e v e l s t e n d e d t o i n c r e a s e . A l t h o u g h no s i g n i f i c a n t e f f e c t s o f d e n s i t y on l i p i d c o n t e n t were f o u n d , t h e r e was e v i d e n c e of r e d u c e d l e v e l s a t h i g h d e n s i t i e s . No e f f e c t s on a s h c o n t e n t were d e t e c t e d . T h e r e were no t r e n d s i n p l a s m a c o r t i c o s t e r o i d s a s s o c i a t e d w i t h a v e r a g e d e n s 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 c o n d i t i o n d a t a were d e t e c t e d w i t h b o t h d e n s i t y and t i m e . As d i s c u s s e d e a r l i e r i n t h i s s e c t i o n , t h e r e s u l t s were c o n t r a d i c t o r y . I t a p p e a r s t h a t f o r f i s h 15-30 gm i n w e i g h t , o v e r t h e d e n s i t y r a n g e t e s t e d , body c o n d i t i o n may n o t be a u s e f u l i n d i c a t o r of d e n s i t y - i n d u c e d s t r e s s . 84 EXPERIMENT 3: STRESS RESPONSES IN SMOLTS AND PRE-SMOLTS I n t r o d u c t i o n T h i s e x p e r i m e n t was d i v i d e d i n t o 3 s e c t i o n s i n w h i c h t h e r e s p o n s e s t o r e a r i n g d e n s i t y , tank volume, f l o w r a t e and sudden d e n s i t y c h a n g e s were examined. I n i t i a l w e i g h t s a v e r a g e d 30 grams. The p e r i o d o v e r w h i c h t h e s e s t u d i e s were c o n d u c t e d c o r r e s p o n d e d t o t h e l a s t few months o f r e a r i n g b e f o r e r e l e a s e . T h i s i s p r o b a b l y t h e most c r i t i c a l p e r i o d i n t h e r e a r i n g p r o c e s s . From F e b r u a r y u n t i l A p r i l , w i l d s t r e a m d w e l l i n g p o p u l a t i o n s o f j u v e n i l e s t e e l h e a d of a p p r o p r i a t e s i z e u n d e r g o t h e p r o c e s s of s m o l t i f i c a t i o n . T h i s p r o c e s s i n v o l v e s b i o c h e m i c a l , p h y s i o l o g i c a l , m o r p h o l o g i c a l and b e h a v i o r a l c h a n g e s . Hoar ( 1 9 7 6 ) , F o l m a r and D i c k h o f f ( 1 9 8 1 ) , and Wedemeyer (1981) have d e s c r i b e d t h e s e c h a n g e s ( T a b l e 1 3 ) . T h e s e , and o t h e r a u t h o r s have e m p h a s i z e d t h e s e n s i t i v i t y o f t h e s m o l t i f i c a t i o n p r o c e s s t o h a t c h e r y r e a r i n g p r a c t i c e s . The e x p e r i m e n t s d e s c r i b e d h e r e e xamined s t r e s s r e s p o n s e s d u r i n g t h i s p e r i o d . The f i r s t of t h e s e (3a) compared s t r e s s - r e l a t e d r e s p o n s e s a t two w i d e l y d i f f e r e n t and c o n s t a n t d e n s i t i e s . The s e c o n d , ( 3 b ) , was a p a r t i a l r e p e t i t i o n of t h e s e c o n d major e x p e r i m e n t . The t h i r d , ( 3 c ) , examined t h e e f f e c t s of t a n k volume a t e q u a l d e n s i t i e s and l o a d i n g r a t e s , on g r o w t h and s t r e s s r e s p o n s e . A l t h o u g h L i and B r o c k s o n (1977) f o u n d no volume e f f e c t on g r o w t h of r a i n b o w t r o u t , o t h e r a u t h o r s have d e t e c t e d s u c h an e f f e c t ( G e n e r a l D i s c u s s i o n ) . Most of t h e s e s t u d i e s were not c o n d u c t e d under h a t c h e r y c o n d i t i o n s . A u l s t a d and R e f s t i e (1975) n o t e d 85 T a b l e 13. P h y s i o l o g i c a l c h a n g e s a s s o c i a t e d w i t h 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 i n s a l m o n i d s ( f r o m Wedemeyer e t a l . 1980). LEVEL IN SMOLTS PHYSIOLOGICAL CHARACTERISTICS COMPARED WITH PARR - body s i l v e r i n g , f i n m a r g i n b l a c k e n i n g i n c r e a s e s - h y p o o s m o t i c r e g u l a t o r y c a p a b i l i t y i n c r e a s e s - s a l i n i t y t o l e r a n c e and p r e f e r e n c e i n c r e a s e s - w e i g h t p e r u n i t l e n g t h ( c o n d i t i o n f a c t o r ) . d e c r e a s e s - g r o w t h r a t e i n c r e a s e s - body t o t a l l i p i d c o n t e n t d e c r e a s e s - oxygen c o n s u m p t i o n i n c r e a s e s - ammonia p r o d u c t i o n i n c r e a s e s - l i v e r g l y c o g e n d e c r e a s e s - b l o o d g l u c o s e i n c r e a s e s - e n d o c r i n e a c t i v i t y i n c r e a s e s t h y r o i d (T4) i n t e r r e n a l p i t u i t a r y g r o w t h hormone - g i l l microsome Na+ K+ -ATPase enzyme a c t i v i t y , i n c r e a s e s - a b i l i t y t o grow i n f u l l s t r e n g t h s ea water .. . i n c r e a s e s - b u oyancy i n c r e a s e s - m i g r a t o r y b e h a v i o r i n c r e a s e s s i g n i f i c a n t d i f f e r e n c e s i n g r o w t h r a t e s i n coho r e a r e d a t d i f f e r e n t t a n k d e p t h s , b u t t h e y d i d not d e t e c t t h i s e f f e c t i n rai n b o w t r o u t . The n u l l h y p o t h e s e s f o r e a c h of t h e e x p e r i m e n t s a r e t h a t t h e r e w i l l be no d i f f e r e n c e s , a t t r i b u t a b l e t o t r e a t m e n t e f f e c t s , i n any o f t h e v a r i a b l e s m e a s u r e d . 86 M a t e r i a l s and methods T h e s e e x p e r i m e n t s began on 13 F e b r u a r y 1981, and were d e s i g n e d s a c o n t i n u a t i o n s of t h e f i r s t two e x p e r i m e n t s . Maximum p e r m i s s i b l e d e n s i t i e s were c a l c u l a t e d as b e f o r e . A l l f i s h u s e d i n t h e s e e x p e r i m e n t s were h e l d i n c i r c u l a r t a n k s a t 9.5 t o 10 °C f o r a t l e a s t two weeks p r i o r t o t h e s t a r t o f t h i s s t u d y . A l l f i s h were f e d f u l l r a t i o n s d u r i n g t h e f i n a l week. R e a r i n g d e n s i t i e s d u r i n g t h i s p e r i o d were 20-30 gm/1. In t h e f i r s t e x p e r i m e n t , ( 3 a ) , a s i m p l e d e n s i t y s t u d y was c o n d u c t e d i n w h i c h t h e e f f e c t s o f w i d e l y v a r y i n g d e n s i t i e s on g r o w t h and s t r e s s i n p r e s m o l t s were e x a m i n e d . I n i t i a l f i s h w e i g h t a v e r a g e d 30-32 grams. The r e a r i n g t e m p e r a t u r e was 9.5°C Tank v o l u m e s were a d j u s t e d t o 350 1, and t h e f l o w s were s e t a t 24 1/min. T h e s e c o n d i t i o n s a l l o w e d a maximum d e n s i t y o f 400 f i s h p e r t a n k . T h i s c o r r e s p o n d e d t o a maximum e x p e c t e d d e n s i t y a b o u t 3.8 t i m e s t h e maximum d e n s i t y commonly u s e d a t t h e h a t c h e r y . The low d e n s i t y (50 f i s h ) was o n e - e i g h t h t h e h i g h d e n s i t y and had a p r o j e c t e d f i n a l d e n s i t y h a l f o f t r a d i t i o n a l l e v e l s . W h i l e an i n t e r m e d i a t e d e n s i t y was d e s i r a b l e , i t was more i m p o r t a n t t o a s c e r t a i n t h e e f f e c t s o f f l o w r a t e s on g r o w t h s i n c e , due t o water s u p p l y c o n s t r a i n t s , t h e o t h e r s m a l l - s c a l e e x p e r i m e n t s would have d i f f e r e n t f l o w s . T h e r e f o r e , two more t a n k s , e a c h c o n t a i n i n g 50 f i s h , r e c e i v e d a f l o w o f 10 1/min. The d e s i g n of t h i s e x p e r i m e n t i s summarized i n T a b l e 14, t a n k s 1-6. P h o t o p e r i o d was s e t a t 10L:14D. In t h e s e c o n d s t u d y , ( 3 b ) , as i n t h e s e c o n d major e x p e r i m e n t , t h e e f f e c t s of c h a n g e s i n d e n s i t y were examined. A l l 87 T a b l e 14. D e s i g n o f e x p e r i m e n t 3. Tan k s 1-6 (upper t a b l e ) , c o m p r i s e d s t u d y 3a. Tanks 13-16 (upper t a b l e ) , c o m p r i s e d s t u d y 3c. Tanks 7-12 ( l o w e r t a b l e ) c o m p r i s e d s t u d y 3b. A l l t a n k s were h e l d a t 9.5 C. Flow r a t e i n s t u d y 3b was 18 1/min. DENSITY TANK NUMBER VOLUME FLOW — I N I T I A L — FINAL PHOTOPERIOD FISH l i t r e s 1/min f i s h / 1 gm/1 gm/1 '. h o u r s l i g h l 1 400 350 24.0 1.14 34.2 62.7 10 2 50 350 24.0 0.14 4.2 7.8 10 3 50 350 10.0 0.14 4.2 7.8 10 4 400 350 24.0 1.14 34.2 62.7 10 5 50 350 24.0 0.14 4.2 7.8 10 6 50 350 10.0 0.14 4.2 7.8 10 1 3 250 250 12.5 1 .0 30.0 55.0 1 4 1 4 250 250 12.5 1 .0 30.0 55.0 1 4 1 5 500 500 25.0 1 .0 30.0 55.0 1 4 1 6 500 500 25.0 1.0 30.0 55.0 1 4 FIRST — D E N S I T Y - - SECOND DENSITY- PHOTO TNK VOLUME I I N I T I A L FINAL VOLUME I N I T I A L FINAL AVE h r s ' FISH l i t r e s f/1 gm/1 gm/1 l i t r e s f/1 " - gm/1 l i g h t 7 300 500 0.6 18 27 500 0.6 27 33 0.6 10 8 300 500 0.6 18 27 500 0.6 27 33 0.6 10 9 300 500 0.6 18 27 250 1 .2 54 66 0.9 10 1 0 300 500 0.6 18 27 250 1 .2 54 66 0.9 10 1 1 300 250 1 .2 36 54 500 0.6 27 33 0.9 10 12 300 250 1 .2 36 54 500 0.6 27 33 0.9 10 t a n k s c o n t a i n e d 300 f i s h , and r e c e i v e d f l o w s of 18 1/min a t 9.5°C. A g a i n , t h r e e r e p l i c a t e d t r e a t m e n t s o f 2 t a n k s e a c h , were u s e d . As b e f o r e , two t a n k s were h e l d a t a c o n s t a n t volume of 500 1 t h r o u g h o u t t h e s t u d y . Two t a n k s had t h e i r volumes h a l v e d ( i . e . d e n s i t i e s d o u b l e d ) midway t h r o u g h t h e s t u d y , and two o t h e r s were g i v e n t h e r e v e r s e t r e a t m e n t . T h i s e x p e r i m e n t i s summarized i n T a b l e 14, t a n k s 7-12. Based on p r o j e c t e d f i n a l w e i g h t s , d e n s i t i e s would a p p r o a c h 66 gm/1 i n t h e h i g h d e n s i t y (low volume) t a n k s , and h a l f t h a t i n t h e low d e n s i t y t a n k s . 88 P h o t o p e r i o d was s e t a t 10L:14D. In t h e t h i r d s m a l l - s c a l e s t u d y , ( 3 c ) , t h e e f f e c t s o f d i f f e r e n t t a n k volumes on g r o w t h and s t r e s s were i n v e s t i g a t e d . B o t h t a n k s had i d e n t i c a l d e n s i t i e s (on a gm/1 b a s i s ) , and were e q u a l l y l o a d e d (on a gm/l/min o f i n f l o w b a s i s ) . To a c h i e v e s i m i l a r l o a d i n g r a t e s f l o w s d i f f e r e d by a f a c t o r o f 2. Tanks c o n t a i n i n g 500 l i t r e s had 500 f i s h and t a n k s c o n t a i n i n g 250 l i t r e s had 250 f i s h . E x p e c t e d f i n a l d e n s i t y i n a l l t a n k s was a b o u t 55 gm/1. P e r t i n e n t d e t a i l s o f t h e d e s i g n o f t h e e x p e r i m e n t a r e g i v e n i n T a b l e 14, t a n k s 13-16. T h i s s t u d y , u n l i k e a l l t h e o t h e r s , was c o n d u c t e d i n t h e wet l a b o r a t o r y , n o t i n t h e q u a r a n t i n e room. P h o t o p e r i o d h e r e was s e t on a 14L:10D c y c l e . T h i s s t u d y was i m p o r t a n t s i n c e i n t e r p r e t a t i o n o f t h e r e s u l t s o f e x p e r i m e n t 3b assumed no volume e f f e c t s . U n f o r t u n a t e l y , i n s u f f i c i e n t numbers of p r o g e n y of 3-ocean a d u l t s were a v a i l a b l e f o r t h i s s t u d y . T h e r e f o r e , i t was n e c e s s a r y t o i n c l u d e 2-ocean f i s h . T h i s e f f e c t i s c o n s i d e r e d i n t h e a n a l y s e s . S i n c e most t a n k s r e c e i v e d w e l l w a t e r d i r e c t l y , t e m p e r a t u r e s were measured i n f r e q u e n t l y . W e l l w ater t e m p e r a t u r e d e c r e a s e d t h r o u g h o u t t h e e x p e r i m e n t from 9.5 t o 9.2°C. B e c a u s e o f w ater s h o r t a g e s some t a n k s r e c e i v e d w ater from s e v e r a l s o u r c e s . T h e s e t a n k s were m o n i t o r e d more f r e q u e n t l y , and t e m p e r a t u r e s r a r e l y f l u c t u a t e d by more t h a n 0.3°C from t h e w e l l s o u r c e . The q u a n t i t y of f o o d and f r e q u e n c y of f e e d i n g were d e t e r m i n e d as b e f o r e . F o r t h e f i r s t 8 d a y s of t h e e x p e r i m e n t , a l l f i s h r e c e i v e d A b e r n a t h y 6/64th i n c h c r u m b l e s . A f t e r t h i s d a t e 3/32 p e l l e t s were u s e d w h i c h , b e c a u s e of t h e i r d i f f e r e n t 89 shape, were l a r g e r . T o t a l w e i g h t s were c o l l e c t e d on d a y s 1, 11, 18, 32, 39, 48, 54, 61 and 72. Not a l l t a n k s were measured on e a c h of t h e s p e c i f i e d d a y s . L e n g t h - w e i g h t d a t a were c o l l e c t e d on t h e f o l l o w i n g d a y s : 1, 8, 18, 32, 61 and 72. No measurements were c o l l e c t e d between days 32 and 61 s i n c e f i s h were f r e q u e n t l y b e i n g sampled f o r C o r t i s o l and i t was d e s i r a b l e t o m i n i m i z e h a n d l i n g s t r e s s . In e x p e r i m e n t 3b d e n s i t i e s were a d j u s t e d on day 48. To a s s e s s C o r t i s o l c o n c e n t r a t i o n s mid-way t h r o u g h t h e e x p e r i m e n t s , and t o o b t a i n p r e - d e n s i t y a d j u s t m e n t d a t a f o r s t u d y 3b, b l o o d samples were c o l l e c t e d on day 47. To a s s e s s t h e e f f e c t s o f sudden d e n s i t y c h a n g e s , samples were a g a i n c o l l e c t e d f r o m s t u d y 3b t a n k s on day 54. A l l t a n k s were sampled a g a i n on day 74. Samples were c o l l e c t e d on d a y s 47 and 74 f o r p r o x i m a t e a n a l y s i s . S i n c e t h e g r o w t h e x p e r i m e n t s were s t i l l i n p r o g r e s s on day 47, i t was n o t a p p r o p r i a t e t o s t a r v e f i s h b e f o r e s a m p l i n g . However, t h e e f f e c t o f f o o d i n t h e stomach s h o u l d have been b o t h s m a l l and c o n s t a n t between t r e a t m e n t s . F o r t u n a t e l y , t h e s e s a m p l e s e n a b l e d i n s p e c t i o n o f s tomach c o n t e n t f u l l n e s s . A l l f i s h s a mpled had f u l l s t o m a c h s . F o r l o g i s t i c r e a s o n s , o n l y samples c o l l e c t e d on day 47 were p r o c e s s e d . B e h a v i o r a l o b s e r v a t i o n s were r e c o r d e d on v i d e o on d a y s 17, 31, 45, 52, 59, and 73. T e c h n i c a l p r o b l e m s on day 17 r e s u l t e d i n a p o o r f i l m and t h e s e d a t a were e x c l u d e d from t h e a n a l y s i s . Water q u a l i t y was m o n i t o r e d l e s s f r e q u e n t l y i n t h e s e 90 e x p e r i m e n t s t h a n i n t h e p r e v i o u s o n e s . T h i s was a c c e p t a b l e f o r two r e a s o n s . F i r s t , r e s u l t s o f t h e f i r s t two e x p e r i m e n t s s u g g e s t e d t h a t t h e method of c a l c u l a t i n g maximum d e n s i t i e s was c o n s e r v a t i v e . S e c o n d l y , t h e maximum d e n s i t i e s a c t u a l l y u s e d i n t h e s e l a s t e x p e r i m e n t s were o n l y 70-80% as h i g h a s p r e d i c t i o n s a l l o w e d . C o n s e q u e n t l y , samples were c o l l e c t e d a t t h e end of t h e e x p e r i m e n t f r o m t h e h i g h d e n s i t y . t r e a t m e n t s o n l y . In a d d i t i o n however, oxygen l e v e l s were p e r i o d i c a l l y c h e c k e d . A t a l l t i m e s , oxygen l e v e l s were s a t i s f a c t o r y . Oxygen d a t a f o r day 73 a r e summarized i n T a b l e 15. T a b l e 15. Summary of d i s s o l v e d oxygen d a t a f o r e x p e r i m e n t 3. Measurements a r e p r e s e n t e d f o r day 73. TANK TIME OXYGEN TANK TIME OXYGEN hr s. mg/1 h r s . mg/1 1 - 12:45 8.80 9 11:05 8.35 2 13:15 1 0.95 1 0 11:30 9.40 3 13:15 10.75 1 1 11:30 8.50 4 13:55 9.1 5 1 2 12:35 8.90 5 1 3:55 10.90 1 3 1 5:00 8.30 6 1 4:22 10.90 1 4 1 5:00 8.50 7 10:40 9.20 1 5 15:00 8.60 8 10:52 9.00 1 6 - -The g r o w t h phase o f t h e e x p e r i m e n t s ended on day 74 (27 A p r i l 1 9 8 1 ) ; however, f e e d i n g c o n t i n u e d and on May 6 (day 82) g r o u p s of f i s h were g i v e n a s a l t w a t e r c h a l l e n g e t e s t . S a l t w a t e r was n o t a v a i l a b l e a t t h e h a t c h e r y and t r a n s p o r t i n g t e s t f i s h t o V a n c o u v e r may have p r o d u c e d s t r e s s . T r a n s p o r t s t r e s s has been shown t o r e d u c e t h e a b i l i t y of s m o l t s t o r e g u l a t e s a l t s i n a h y p e r o s m o t i c e n v i r o n m e n t (Iwama 1979). 91 C o n s e q u e n t l y , t h e t e s t was c o n d u c t e d a t t h e h a t c h e r y u s i n g a r t i f i c i a l s e a w a t e r . I d e a l l y , a f l o w - t h r o u g h s a l t w a ter s y s t e m s h o u l d have been u s e d , or a c l o s e d s y s t e m e q u i p p e d w i t h b i o - f i l t e r s . N e i t h e r were a v a i l a b l e , and i t was t h e r e f o r e n e c e s s a r y t o use a s t a t i c b a t h . To m i n i m i z e t h e l i k l i h o o d of m e t a b o l i t e s r e a c h i n g s t r e s s f u l l e v e l s , ammonia p r o d u c t i o n r a t e s were a p p r o x i m a t e d u s i n g i n f o r m a t i o n p r e s e n t e d i n B r e t t and Z a l a (1975) and t h e oxygen model ( T a u t z , p e r s . comm.). The e f f e c t s o f f i s h s i z e , t e m p e r a t u r e , pH, c a r b o n d i o x i d e p r o d u c t i o n , and t h e a d d i t i o n a l s t r e s s due t o s a l t water were c o n s i d e r e d . W i t h t h i s i n f o r m a t i o n t h e minimum r e q u i r e d volume o f water f o r t h e t e s t was a p p r o x i m a t e d . A v e x a r mesh h o l d i n g c a g e , t h a t a l l o w e d f r e e c i r c u l a t i o n of water w i t h i n compartments, was c o n s t r u c t e d . A d j a c e n t compartments were s e p a r a t e d w i t h b l a c k p o l y e t h y l e n e s h e e t s . T h i s e n a b l e d f i s h t o be removed f r o m one s e c t i o n w i t h o u t d i s t u r b i n g n e i g h b o u r s . M i n i m i z i n g s t r e s s d u r i n g s a m p l i n g was i m p o r t a n t s i n c e c a t e c h o l a m i n e r e l e a s e a s s o c i a t e d w i t h s t r e s s c a n c a u s e r a p i d s h i f t s i n g i l l p e r m e a b i l i t y and o s m o r e g u l a t o r y p e r f o r m a n c e (Eddy 1981). I n d i v i d u a l c o m p a r t m e n t s were a p p r o x i m a t e l y 30 cm wide by 27 cm a c r o s s by 50 cm deep. T e m p e r a t u r e was s e t a t 9.5°C, and t o p r e v e n t i t f r o m i n c r e a s i n g t o a m bient a i r t e m p e r a t u r e , a h e a t - e x c h a n g i n g c o i l was i m p r o v i s e d t o c i r c u l a t e c h i l l e d w a t e r . The a r t i f i c i a l s a l t u s e d was " F o r t y Fathoms M a r i n e m i x " b r a n d . The c h e m i c a l c o m p o s i t i o n i s p r o v i d e d i n A p p e n d i x I . A 92 h y d r o m e t e r ( F i s h e r S c i e n t i f i c ) , a c c u r a t e t o ± 0.1 °C, was u s e d t o c h e c k s a l i n i t i e s . T a b l e 2.1 i n R i l e y and C h e s t e r (1977) was u s e d t o c o r r e c t f o r t e m p e r a t u r e e f f e c t s on s p e c i f i c volume anomaly. The s a l t s o l u t i o n was a e r a t e d v i g o r o u s l y f o r 24 h o u r s p r i o r t o i n t r o d u c i n g t h e t e s t f i s h and l e s s v i g o r o u s l y a f t e r . The t e s t p r o c e d u r e s c o r r e s p o n d e d c l o s e l y t o t h o s e d e s c r i b e d by Iwama (1979) and C l a r k e and B l a c k b u r n ( 1 9 7 7 ) . F i s h were n o t f e d f o r 24 h o u r s p r i o r t o t e s t i n g . F i s h t o be u s e d i n t h e c h a l l e n g e were not a n a e s t h e t i z e d , but were i n d i v i d u a l l y w e i g h e d i n a p r e - t a r e d c o n t a i n e r of w a t e r . A l l f i s h f r o m one t r e a t m e n t were h e l d and i n t r o d u c e d i n t o t h e s a l t b a t h as a g r o u p . Twenty f i s h f r o m e a c h t r e a t m e n t were u s e d i n t h e t e s t . Time o f i n t r o d u c t i o n was r e c o r d e d . As n e a r t o 24 h o u r s as p o s s i b l e , f i s h were removed f r o m t h e a p p r o p r i a t e compartment, and p l a c e d i n a b u c k e t c o n t a i n i n g s a l t w ater and a s t r o n g s o l u t i o n o f a n a e s t h e t i c . B e f o r e s a m p l i n g , t h e c a u d a l p e d u n c l e was d r i e d t o p r e v e n t d i l u t i o n of t h e p l a s m a . Plasma was c o l l e c t e d w i t h e i t h e r s y r i n g e s o r m i c r o c a p i l l a r y t u b e s . B o t h had been t r e a t e d w i t h c a t i o n - f r e e h e p a r i n . Samples were c e n t r i f u g e d and i c e d . P lasma sodium l e v e l s were d e t e r m i n e d on samples d i l u t e d w i t h a l i t h i u m s t a n d a r d , and measured i n an I n s t u m e n t a l L a b o r a t o r i e s I n c . , model 443 f l a m e p h o t o m e t e r . Z e r o and 140 meq Na + / l s o l u t i o n s were u s e d as s t a n d a r d s . Samples were a s s a y e d i n d u p l i c a t e . I f v a r i a t i o n between d u p l i c a t e s was g r e a t e r t h a n 10 u n i t s , t h e y were r e - r u n , and i f d i f f e r e n c e s were s t i l l l a r g e , t h e y were d i s c a r d e d . 93 R e s u l t s G r o w t h E x p e r i m e n t 3a I n t h i s e x p e r i m e n t , t h e e f f e c t s o f d e n s i t y a n d f l o w on s t r e s s r e s p o n s e were e x a m i n e d ( T a b l e 1 4 ) . F e e d e r p r o b l e m s d u r i n g f o u r o f t h e f i r s t e l e v e n d a y s n e c e s s i t a t e d t h e d e l e t i o n o f g r o w t h s l o p e i n f o r m a t i o n f r o m t a n k s 5 a n d 6 f o r t h a t i n t e r v a l . C h a n g e s i n a d j u s t e d w e i g h t s o v e r t i m e a r e g i v e n i n F i g u r e 9. A 2-way ANOVA ( T a b l e 16) was c a l c u l a t e d f o r t h e g r o w t h s l o p e d a t a . T r e a t m e n t e f f e c t s were n o t s i g n i f i c a n t . H o wever, B a r t l e t t ' s t e s t showed t h a t v a r i a n c e s were h e t e r o g e n e o u s , a n d i t was t h e r e f o r e n e c e s s a r y t o a n a l y s e by n o n - p a r a m e t r i c means. M a n n - W h i t n e y LT-test c o m p a r i s o n s ( 2 - t a i l e d , 5% l e v e l ) were c a l c u l a t e d . No s i g n i f i c a n t e f f e c t o f e i t h e r f l o w o r d e n s i t y was d e t e c t e d . O v e r t h e r a n g e t e s t e d , d e n s i t y h a d no e f f e c t on g r o w t h . A l t h o u g h t h e r e was a n o n - s i g n i f i c a n t t r e n d t o w a r d s r e d u c e d g r o w t h a t r e d u c e d f l o w s , t h e e f f e c t was n o t c o n s i d e r e d i m p o r t a n t . R e f e r r i n g b a c k t o t h e ANOVA p r e s e n t e d i n T a b l e 16, B a r t l e t t ' s t e s t i n d i c a t e d t h a t g r o w t h r a t e s c o u l d be a n a l y z e d by t i m e w i t h o u t h e t e r o g e n e i t y p r o b l e m s . The e f f e c t s o f t i m e were s i g n i f i c a n t ( d f = 6 , 3 9 p < . 0 5 ) . I n c o n t r a s t t o t h e r e s u l t s o f t h e p r e v i o u s e x p e r i m e n t s , g r o w t h s l o p e s d i d n o t f o l l o w t h e e x p e c t e d p a t t e r n ( i . e . r i s e t o a peak a f t e r a low i n i t i a l p e r i o d , a n d 94 F i g u r e 9. Change i n w e i g h t o v e r t i m e f o r e x p e r i m e n t s 3a, 3b, and 3c. A l l w e i g h t s have been a d j u s t e d t o a common i n i t i a l w e i g h t of e i t h e r 30 o r 33 grams (33 grams i f f i r s t i n t e r v a l g r o w t h d a t a had t o be d e l e t e d ) . Changes o v e r t i m e were c a l c u l a t e d u s i n g g r o w t h s l o p e i n f o r m a t i o n . ^ 60 t 50 + 400 fish - 24 1/min 50 fish - 24 1/min 50 fish - 10 1/min 40 + 30 60 T 50 + S 40 + 30 TIME (DAYS) constant density low to high density high to low density 0 10 20 30 40 50 60 70 80 - 60 T 50 + 40 + 30 TIME (DAYS) high volume low volume 0 10 20 30 40 50 60 70 TIME (DAYS) 96 T a b l e 16. A n a l y s i s of v a r i a n c e of g r o w t h s l o p e d a t a from e x p e r i m e n t 3, p a r t A ( s e e t a b l e 14, p a r t A ) . T e s t s a r e c o n s i d e r e d s i g n i f i c a n t i f p < 0.05. D a t a from a l l t i m e i n t e r v a l s were a n a l y s e d . SOURCE DF MEAN SQUARE PROBABILITY T r e a t m e n t 2 Time 6 T r e a t m e n t * T i m e 12 R e s i d u a l 19 T o t a l 39 0.00000666 0.00000944 0.00000298 0.00000253 0.09846 0.01284 0.36346 t h e n d e c l i n e t h e r e a f t e r ) . D u r i n g t h e f i r s t 4 t i m e s , g r o w t h was e r r a t i c ( a l t h o u g h c l o s e t o model p r e d i c t i o n s ) . Over t h e l a s t t h r e e i n t e r v a l s , t h e r e was a t r e n d f o r g r o w t h t o i n c r e a s e . A l t h o u g h t h e d i f f e r e n c e s between t h e l a s t t h r e e t i m e s were not s i g n i f i c a n t (Duncan's t e s t ) , a l l 6 t a n k s showed t h i s p a t t e r n . T h i s i n c r e a s e i f r e a l , may be p a r t o f t h e g r o w t h a c c e l e r a t i o n p r o c e s s a s s o c i a t e d w i t h s m o l t i f i c a t i o n . Growth s l o p e s a g a i n s t t i m e a r e p l o t t e d i n F i g u r e 10a. E x p e r iment 3b D u r i n g t h e f i r s t d a y s o f t h i s e x p e r i m e n t , f e e d e r p r o b l e m s i n 4 o f 6 t a n k s , r e s u l t e d i n u n r e l i a b l e d a t a . C o n s e q u e n t l y , g r o w t h s l o p e i n f o r m a t i o n f o r a l l 6 t a n k s was d e l e t e d f o r t h e f i r s t i n t e r v a l . As a r e s u l t of t h e f e e d e r p r o b l e m s , f i s h became h y p e r a c t i v e , and l a r g e numbers jumped out of some t a n k s . T h i s n e c e s s i t a t e d r e c o u n t s a t t h e t i m e o f n e x t w e i g h i n g . A l l f e e d e r p r o b l e m s were c o r r e c t e d w i t h i n a few d a y s of t h e s t a r t o f t h e e x p e r i m e n t , and f i s h a p p e a r e d t o behave " n o r m a l l y " by day 7. B e c a u s e t h e f i r s t t i m e i n t e r v a l u s e d i n t h e a n a l y s i s e x t e n d e d 97 F i g u r e 10. Growth s l o p e s a g a i n s t t i m e ( m i d - p o i n t o f s a m p l i n g i n t e r v a l ) f o r e x p e r i m e n t s 3a, 3b, and 3c. Growth s l o p e s were c a l c u l a t e d a c c o r d i n g t o Iwama ad T a u t z ( 1 9 8 1 ) . V e r t i c a l b a r s a r e s t a n d a r d e r r o r s . D _ O I OO 0.020 0.015 § 0.010 CD 0.005 400 fish 50 fish 50 fish 24 1/min 24 1/min 10 1/min B 0.020 r LU D_ O i 0.015 -OO rn 1— 0.010 to 1 1 • CD 0.005 •• 0.000 +• 0.020 T £ 0.015 + o oo 0.010 + o oc CD ^ 0.005 + 0.000 0 constant density low to high density high to low density high volume low volume 10 H h-20 30 40 50 60 70 MID-POINT OF TIME INTERVAL 99 from day 11 t o 18, i t i s u n l i k e l y t h a t t h e f e e d e r p r o b l e m s a f f e c t e d s u b s e q u e n t r e s u l t s . The d a t a were a n a l y s e d i n a 2-way ANOVA ( T a b l e 1 7 ) . B o t h d e n s i t y and t i m e e f f e c t s were s i g n i f i c a n t . Duncan's t e s t p r o d u c e d two homogeneous g r o u p s . Growth i n t h e t r e a t m e n t i n w h i c h v o l u m e s were r e d u c e d ( d e n s i t y d o u b l e d ) was s i g n i f i c a n t l y l o w e r t h a n i n t h e o t h e r g r o u p s w h i c h were n o t d i f f e r e n t . The two g r o u p s w i t h t h e same a v e r a g e d e n s i t y , b u t d i f f e r e n t volume t r e a t m e n t s (low t o h i g h , o r h i g h t o l o w ) , were not e x p e c t e d t o have s i g n i f i c a n t l y d i f f e r e n t mean growth s l o p e s . Growth s l o p e p a t t e r n s o v e r t i m e a r e g i v e n i n F i g u r e 10b. T a b l e 17. A n a l y s i s o f v a r i a n c e o f g r o w t h s l o p e d a t a from e x p e r i m e n t 3, p a r t B ( s e e t a b l e 14, p a r t B ) . T e s t t e r m s a r e c o n s i d e r e d s i g n i f i c a n t i f p < 0.05. Due t o t e c h n i c a l d i f f i -c u l t i e s , d a t a f r o m t h e f i r s t t i m e i n t e r v a l were d e l e t e d . The s i g n i f i c a n t i n t e r a c t i o n term was t h e b a s i s f o r r e a n a l y s i s by s e l e c t e d t i m e i n t e r v a l s . MEAN SOURCE DF SQUARE PROBABILITY T r e a t m e n t 2 Time 6 T r e a t m e n t * T i m e 12 R e s i d u a l 21 T o t a l 41 0.00002167 0.00001294 0.00000908 0.00000273 0.00270 0.00335 0.00773 Duncan's t e s t p r o d u c e d s e v e r a l g r o u p s on t h e b a s i s o f t i m e , however, none of t h e s e g r o u p s o f f e r e d i n s i g h t i n t o g r o w t h p r o c e s s e s s i n c e no p a t t e r n s were d e t e c t e d . The c u r v i l i n e a r p a t t e r n o b s e r v e d i n t h e p r e v i o u s major e x p e r i m e n t s was a b s e n t . The i n t e r a c t i o n t e r m was s i g n i f i c a n t , and i t was t h e r e f o r e 100 d e c i d e d t o r e a n a l y z e t h e d a t a by t i m e g r o u p i n g s b e f o r e and a f t e r v o l u m e s were a d j u s t e d . When t h e f i r s t f o u r t i m e s were a n a l y z e d i n a 2-way ANOVA d e n s i t y e f f e c t s were n o t s i g n i f i c a n t (df=2,23 p=.42). T h e r e f o r e g r o w t h was u n a f f e c t e d o v e r a 2 - f o l d range i n d e n s i t y d u r i n g t h e f i r s t 47 d a y s o f t h e e x p e r i m e n t . On a w e i g h t / v o l u m e b a s i s , by day 47, t h e h i g h d e n s i t y t a n k s were s l i g h t l y more t h a n t w i c e c o n v e n t i o n a l d e n s i t i e s a t t h e h a t c h e r y . Time e f f e c t s were s i g n i f i c a n t (df=3,23 p<.005). Growth s l o p e s were c o n s t a n t o v e r t i m e w i t h t h e e x c e p t i o n of t h e t h i r d i n t e r v a l w h i c h was s i g n i f i c a n t l y l o w e r i n a l l g r o u p s . The i n t e r a c t i o n t e r m was n o t s i g n i f i c a n t . The r e s u l t s o f a 2-way ANOVA f o r t h e l a s t 3 t i m e s p r o d u c e d t r e a t m e n t - r e l a t e d d i f f e r e n c e s . D e n s i t y e f f e c t s were s g n i f i c a n t (df=2,17 p < . 0 0 l ) . Those g r o u p s a t h i g h d e n s i t y (low volume) grew a b o u t 60% s l o w e r t h a n t h e low d e n s i t y g r o u p s . B o t h low d e n s i t y -t r e a t m e n t s had s t a t i s t i c a l l y e q u i v a l e n t g r o w th s l o p e s , d e s p i t e t h e f a c t t h a t t h e y had d i f f e r e n t d e n s i t y h i s t o r i e s ( i . e . two of t h e t a n k s had been r e a r e d a t t h e h i g h d e n s i t y u n t i l day 4 7 ) . The p e r i o d i m m e d i a t e l y f o l l o w i n g d e n s i t y c h a n g e s e l i c i t e d t h e g r e a t e s t r e s p o n s e . I n t e r e s t i n g l y , mean growth s l o p e s o v e r t h e l a s t 3 i n t e r v a l s were c o n s i d e r a b l y h i g h e r t h a n f o r t h e f i r s t f o u r i n t e r v a l s (0.0125 v e r s u s 0.0097) when t a n k s 7, 9, 10, and 12 were compared. T h i s s u p p o r t s t h e t r e n d o b s e r v e d i n e x p e r i m e n t 3a. 101 E x p e r i m e n t 3c T e c h n i c a l d i f f i c u l t i e s d u r i n g t h e f i r s t few days of t h e e x p e r i m e n t , n e c e s s i t a t e d d e l e t i o n o f g r o w t h d a t a f r o m t h e f i r s t t i m e i n t e r v a l . C o n s e q u e n t l y , o n l y t h e r e m a i n i n g 6 t i m e i n t e r v a l s were a n a l y s e d . The d a t a were a n a l y z e d i n a 2-way ANOVA ( T a b l e 18). Volume e f f e c t s were n o t s i g n i f i c a n t . However, f i s h i n t h e low volume t r e a t m e n t s t e n d e d t o w a r d s l o w e r g r o w t h , s l i g h t l y below model p r e d i c t i o n s ( 0 . 0 0 9 1 ) , t h a n t h o s e f i s h a t h i g h volume w h i c h grew s l i g h t l y a b o v e p r e d i c t i o n s ( 0 . 0 1 0 9 ) . Growth s l o p e s v a r i e d s i g n i f i c a n t l y w i t h time (df=5,23 p<.05), and t h e o v e r a l l p a t t e r n ( F i g u r e 10c) a p p r o x i m a t e d t h o s e i n e x p e r i m e n t s 3a and 3b. E s p e c i a l l y i m p o r t a n t was t h e t r e n d t o w a r d s i n c r e a s e d g r o w t h o v e r t h e l a s t t h r e e i n t e r v a l s , s i n c e i t r e d u c e d t h e p o s s i b i l i t y t h a t t h e p a t t e r n was an a r t i f a c t of t h e room i n w h i c h t h e r e s e a r c h was c o n d u c t e d . S i n c e t h e t r e a t m e n t means were d i f f e r e n t by a l m o s t 15%, i t was s u r p r i s i n g t h a t s i g n i f i c a n c e was n o t o b t a i n e d . O b v i o u s l y v a r i a n c e s were l a r g e and t h e most l i k e l y s o u r c e o f v a r i a t i o n was t h e two d i f f e r e n t s t o c k s u s e d . To examine t h i s p o s s i b i l i t y , s i m i l a r s t o c k s were p o o l e d and a n a l y s e d i n a 1-way ANOVA. The s t o c k e f f e c t was not s i g n i f i c a n t , p o s s i b l y due t o t h e v a r i a n c e a s s o c i a t e d w i t h volume e f f e c t s . However t h e r e was a t r e n d f o r f a s t e r g r o w t h i n t h e f i s h w h i c h t h e were p r o g e n y o f 3-ocean p a r e n t s . T h e r e f o r e some o f t h e v a r i a n c e i n t h e ANOVA c o n d u c t e d on volume d a t a may be due t o s t o c k e f f e c t s . A volume e f f e c t may have been p r e s e n t b u t , i f s o , i t was s m a l l . 1 02 T a b l e 18. A n a l y s i s of v a r i a n c e o f growth s l o p e d a t a f r o m e x p e r i m e n t 3, p a r t C ( s e e t a b l e 14, p a r t C K T e s t s a r e c o n -s i d e r e d s i g n i f i c a n t i f p < 0.05. Due t o t e c h n i c a l d i f f i c u l -t i e s , d a t a f r o m t h e f i r s t t i m e i n t e r v a l were d e l e t e d . MEAN SOURCE DF SQUARE PROBABILITY Volume Time Volume*Time R e s i d u a l T o t a l P r o x i m a t e A n a l y s i s  E x p e r iment 3a R e s u l t s o f t h e s e a n a l y s e s a r e summarized i n F i g u r e 11. M o i s t u r e : The f i r s t ANOVA c a l c u l a t e d d e m o n s t r a t e d t h a t non-p a r a m e t r i c c o m p a r i s o n s were n e c e s s a r y . Mann-Whitney U - t e s t t e s t s were c a l c u l a t e d f o r t r e a t m e n t r e p l i c a t e s . In one c a s e o n l y were r e p l i c a t e s s t a t i s t i c a l l y d i f f e r e n t . S u b s e q u e n t c o m p a r i s o n s were done on d a t a w i t h r e p l i c a t e s p o o l e d . The h i g h d e n s i t y t r e a t m e n t had s i g n i f i c a n t l y h i g h e r m o i s t u r e l e v e l s t h a n t h e low d e n s i t y t a n k s (Us = 57, Uc = 4 9 ) . T h i s t r e n d a g r e e d w i t h t h a t o b s e r v e d i n t h e f i r s t two e x p e r i m e n t s . No d i f f e r e n c e s were f o u n d between t r e a t m e n t s c o n t a i n i n g 50 f i s h w i t h d i f f e r e n t f l o w s . P r o t e i n : P a r a m e t r i c t e s t s were a d e q u a t e f o r t h e s e d a t a w h i c h were e n t e r e d i n a 1-way ANOVA w i t h w e i g h t as a c o v a r i a t e . T r e a t m e n t s had no e f f e c t on p r o t e i n c o n t e n t . N e i t h e r t h e c o v a r i a t e nor t h e e q u a l i t y of s l o p e t e s t were s i g n i f i c a n t . L i p i d : W i t h r e p l i c a t e s p o o l e d , d e n s i t y e f f e c t s were examined i n a 1-way ANOVA. The e f f e c t , a l t h o u g h n o t s i g n i f i c a n t , 1 0.00001175 0.06749 5 0.00001449 0.01063 5 0.00000311 0.42484 12 0.00000291 23 -1 03 F i g u r e 11. H i s t o g r a m o f p r o x i m a t e c o m p o s i t i o n d a t a f o r e x p e r i m e n t 3a. A - m o i s t u r e , B - p r o t e i n , C - l i p i d , D-ash. Means a r e g i v e n w i t h s t a n d a r d e r r o r s . B a r s b e a r i n g common s m a l l - c a s e l e t t e r s a r e n o t s t a t i s t i c a l l y d i f f e r e n t (Duncan's o r S c h e f f e ' s m u l t i p l e r a n g e t e s t -5% l e v e l ) . S t a t i s t i c a l c o m p a r i s o n s were made between t h e f i r s t 2 columns ( d i f f e r e n t d e n s i t y , e q u a l f l o w s ) , and t h e l a s t 2 co l u m n s ( e q u a l d e n s i t y , d i f f e r e n t f l o w s ) . o CD m i 1 i C O » « —1 J=--< C D C D \ -n ro t—• CO X -1 > z v—* o ro -n _t i— o ^ —i vn m C D CO 1—1 \ O z ASH (% DRY WEIGHT) C O O H H LIPID (% DRY WEIGHT) ro ro -Cr r o cn PROTEIN (% DRY WEIGHT) C D - C r cn cn cn oo MOISTURE C D r o J r (7 105 was c l o s e (df=1,15 p=.12), and t h e t r e n d f o l l o w e d t h e e x p e c t e d d i r e c t i o n , i . e . d e c r e a s e d l i p i d c o n t e n t a t t h e h i g h e r d e n s i t y . F u r t h e r m o r e t h e r e were no s i g n i f i c a n t e f f e c t s o f t h e c o v a r i a t e or t h e i n t e r a c t i o n . The e f f e c t s of v a r y i n g f l o w r a t e s were t h e n e x amined i n t a n k s c o n t a i n i n g i d e n t i c a l d e n s i t i e s . At t h e d e c r e a s e d f l o w , l i p i d l e v e l s were s i g n i f i c a n t l y r e d u c e d (df=1,15 p<.005). A s h : W i t h r e p l i c a t e s p o o l e d , t h e d e n s i t y d a t a were a n a l y z e d i n a 1-way ANOVA. T h e r e was a s i g n i f i c a n t e f f e c t o f d e n s i t y (df=1,15 p<.05), a s h c o n t e n t d e c r e a s i n g as d e n s i t y was r e d u c e d . Flow e f f e c t s were a l s o s i g n i f i c a n t (df=1,15 p<.05). A t low f l o w s a s h c o n t e n t was h i g h e r . E f f e c t s o f t h e c o v a r i a t e were n o t s i g n i f i c a n t . E x p e r i m e n t 3b As m e n t i o n e d i n t h e methods, samples f o r p r o x i m a t e a n a l y s i s were c o l l e c t e d j u s t p r i o r t o volume a d j u s t m e n t . U n t i l t h a t t i m e , t h e r e were f o u r t a n k s a t t h e low d e n s i t y and two a t t h e h i g h d e n s i t y . T h e r e were, t h e r e f o r e , two t r e a t m e n t s . R e s u l t s of t h e s e a n a l y s e s a r e summarized i n T a b l e 19. M o i s t u r e : U s i n g w e i g h t as a c o v a r i a t e , a 1-way ANOVA was c a l c u l a t e d . The e f f e c t of d e n s i t y was not s i g n i f i c a n t , a l t h o u g h r e p l i c a t e s were d i f f e r e n t (df=4,23 p < . 0 0 l ) . N e i t h e r t h e c o v a r i a t e t e r m n o r t h e e q u a l i t y o f s l o p e t e s t t e r m were s i g n i f i c a n t . P r o t e i n : A s i m i l a r a n a l y s i s p e r f o r m e d on t h e s e d a t a i n d i c a t e d t h a t r e p l i c a t e s c o u l d be p o o l e d . When p o o l e d t h e 106 T a b l e 19. Whole body p r o x i m a t e c o m p o s i t i o n of j u v e n i l e s t e e l h e a d t r o u t f r o m samples c o l l e c t e d from e x p e r i m e n t s 3b and 3c a f t e r 47 d a y s o f r e a r i n g . F i v e f i s h were c o l l e c t e d from e a c h t a n k , and d i v i d e d i n t o two s i z e g r o u p s . F i s h f r o m e a c h g r o u p were h o m o g e n i z e d and a n a l y s e d i n d u p l i c a t e . The two s t u d i e s were a n a l y s e d s e p a r a t e l y w i t h a n a l y s i s of c o -v a r i a n c e and Duncan's t e s t . Common l e t t e r s r e p r e s e n t no s i g -n i f i c a n t d i f f e r e n c e s . DENSITY VOLUME 0.6 1 .2 250 500 f i s h / 1 f i s h / 1 l i t r e s l i t r e s n 16 8 8 8 MOISTURE mean 72.81a 72.71a 73.78a 73.61a S.E. 0.058 0.082 0. 1 02 0. 102 n 16 8 8 8 PROTEIN mean 66.11a 66.53a 67.96a 68.26a S.E. 0.313 0.446 0.549 0.549 n 16 8 8 8 L I P I D mean 25.16a 25.31a 22.40a 23.48a S.E. 0.284 0.404 0.219 0.219 n 1 6 8 8 8 ASH mean 8.74a 8. 1 6a 9.64a 8.27a S.E. 0.270 0.384 0.562 0.562 ef f e c t s o f d e n s i t y were not s i g n i f i c a n t (df=1,23 p=.45) c o v a r i a t e t e r m was s i g n i f i c a n t l y l e s s t h a n z e r o (df=1,23 p<.001), i n d i c a t i n g t h a t a s f i s h w e i g h t i n c r e a s e d , p r o t e i n l e v e l s d e c r e a s e d . The i n t e r a c t i o n t e r m was n o t s i g n i f i c a n t . L i p i d : As w i t h t h e p r o t e i n a n a l y s i s , o n l y t h e r e p l i c a t e s t e r m was s i g n i f i c a n t (df=4,23 p<.05). No e f f e c t o f d e n s i t y was d e t e c t a b l e . None of t h e o t h e r t e r m s were s i g i f i c a n t . A s h : W i t h r e p l i c a t e s p o o l e d , no d e n s i t y e f f e c t s of d e n s i t y on a s h c o n t e n t was d e t e c t e d . A l l o t h e r t e r m s were non-s i g n i f i c a n t . 107 E x p e r i m e n t 3c As w i t h t h e g r o w t h d a t a a n a l y s i s , s t o c k d i f f e r e n c e s between r e p l i c a t e s p r o b a b l y i n c r e a s e d v a r i a n c e s . A c c o r d i n g l y a l l d a t a were a n a l y z e d b o t h f o r volume and s t o c k e f f e c t s . O n l y t h e r e s u l t s of t h e a n a l y s e s on volume e f f e c t s w i l l be p r e s e n t e d ( T a b l e 19); however, t h e s t o c k a n a l y s e s d i d s u g g e s t t h a t s t o c k e f f e c t s e x i s t e d . M o i s t u r e : A 1-way n e s t e d ANOVA, u s i n g w e i g h t as a c o v a r i a t e , i n d i c a t e d no volume e f f e c t s (df=1,15 p=.92). A l l o t h e r t e r m s were n o n - s i g n i f i c a n t w i t h , t h e e x c e p t i o n o f t h e r e p l i c a t e s t e r m (df=1,15 p < . 0 0 l ) . P r o t e i n : S i m i l a r a n a l y s e s were p e r f o r m e d on p r o t e i n d a t a . As w i t h m o i s t u r e , volume t r e a t m e n t s had no e f f e c t on p r o t e i n c o n t e n t . V a r i a t i o n s between r e p l i c a t e s were l a r g e (df=1,15 p=.051) but not q u i t e s i g n i f i c a n t . T h e r e was a t r e n d t o w a r d s i n c r e a s e d p r o t e i n l e v e l s a t t h e low e r volume. L i p i d : Volume d i d ' not s i g n i f i c a n t l y a f f e c t l i p i d c o n t e n t . R e p l i c a t e s were d i f f e r e n t (df=1,15 p < . 0 0 l ) . A s h : A l t h o u g h , n o t s i g n i f i c a n t , t h e r e was a t r e n d (df=1,15 p=.15) t o w a r d s l o w e r a s h c o n t e n t a t h i g h v o l u m e s . C o r t i s o l As d e s c r i b e d e a r l i e r , a l l t a n k s were s a m p l e d a t l e a s t t w i c e and t h o s e from e x p e r i m i e n t 3b were sampled t h r e e t i m e s . I n some a n a l y s e s v a r i a n c e s were h e t e r o g e n e o u s . S e v e r a l t r a n s f o r m a t i o n s were t r i e d b u t none were s u f f i c i e n t t o c o r r e c t t h e p r o b l e m . In 108 t h e s e c a s e s n o n - p a r a m e t r i c t e s t s were r e q u i r e d . E x p e r i m e n t 3a C o r t i s o l d a t a ( F i g u r e 12a) from t h e f i r s t s a m p l i n g d a t e were i n i t i a l l y a n a l y z e d by ANOVA. B a r t l e t t ' s t e s t i n d i c a t e d t h e need f o r n o n - p a r a m e t r i c t e s t s . Mann-Whitney U - t e s t c o m p a r i s o n s between r e p l i c a t e s i n d i c a t e d t h e y c o u l d be p o o l e d w i t h t h e e x c e p t i o n o f t h o s e from t h e h i g h d e n s i t y t r e a t m e n t . When t h e e f f e c t s of f l o w were examined no d i f f e r e n c e s were d e t e c t e d . C o m p a r i s o n s between t h e two d e n s i t y t r e a t m e n t s (400 v e r s u s 50 f i s h ) , were not s i g n i f i c a n t due t o h i g h v a r i a n c e between t h e h i g h d e n s i t y t a n k s . I t a p p e a r e d u n l i k e l y t h a t d e n s i t y e f f e c t s were p r e s e n t . D a t a from t h e s e c o n d s a m p l i n g d a t e were a n a l y s e d i n a 1-way n e s t e d ANOVA. 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 due t o e i t h e r t r e a t m e n t ( d f = 2 , 9 l p=.93) o r w i t h i n r e p l i c a t e s . When t h e two t i m e s were compared, n o n - p a r a m e t r i c t e s t s were a g a i n r e q u i r e d . W i t h r e p l i c a t e s p o o l e d , c o m p a r i s o n s were made between t i m e s f o r e a c h t r e a t m e n t s e p a r a t e l y . In a l l c a s e s C o r t i s o l l e v e l s were s i g n i f i c a n t l y r e d u c e d by t h e s e c o n d d a t e (400 f i s h , 24 1/min - c a l c t=2.8685; 50 f i s h , 24 1/min - c a l c t = 3.064; 50 f i s h 10 1/min - c a l c t = 4 . 4 0 5 ) ( c r i t i c a l t = 1 . 9 6 ) . On th e s e c o n d d a t e , t e s t s i n d i c a t e d t h a t t h e r e were no d i f f e r e n c e s between r e p l i c a t e s . Due t o t h e h i g h v a r i a n c e , i t i s u n l i k e l y , w i t h o u t p o o l i n g , t h a t a d e c l i n e w i t h t i m e would have been d e t e c t e d a t t h e h i g h d e n s i t y . 109 F i g u r e 12. H i s t o g r a m o f p l a s m a C o r t i s o l c o n c e n t r a t i o n s f o r e a c h of e x p e r i m e n t s 3a, 3b, and 3c, from samples c o l l e c t e d on d a y s 47, 54, and 74. Mean v a l u e s a r e g i v e n w i t h s t a n d a r d e r r o r s . E x p e r i m e n t s 3a and 3c were sampled o n l y on d a y s 47 and 74. T h e s e d a t a d e m o n s t r a t e t h e r e d u c t i o n i n p l a s m a C o r t i s o l c o n c e n t r a t i o n w i t h t i m e . D a t a f r o m p a r t B, e x p e r i m e n t 3b, c o l l e c t e d on d a y s 47 and 54 d e m o n s t r a t e t h e C o r t i s o l r e s p o n s e t o c h a n g i n g d e n s i t i e s . B a r s b e a r i n g common l e t t e r s a r e n o t s t a t i s t i c a l l y d i f f e r e n t (Duncan's o r S c h e f f e ' s m u l t i p l e r a n g e t e s t s - 5% l e v e l ) . o CORTISOL (NG/ML) oo CD — t -ISJ - f --Cr cn oo H 1 oo o —f-CD o o x < O CD VAI CD CD CD CD X o t=3 CO m CD CD o o VAl CD CD I o -Cr CD U l CD CD hH " x < CD CD ONI CD CD CD CD X o 03 CORTISOL ( N G / M L ) CORTISOL ( N G / M L ) hO -Er cn oo —I 1 1 1 oo -Cr -Cr CD CD 01 -Cr CD CD I CD to -Cr -Cr CD CD I-H o ro KJI -Cr CD I—1 u n CD CD CD hO -Cr cn oo H 1 1 1 hH °" hH °-111 E x p e r iment 3b Plasma sa m p l e s were c o l l e c t e d on t h r e e d a t e s , t h e f i r s t and l a s t were t h e same as i n t h e o t h e r two s t u d i e s . The f i r s t d a t e was one day p r i o r t o t h e d e n s i t y (volume) a d j u s t m e n t s . The s e c o n d d a t e was one week l a t e r and was i n t e n d e d t o d e t e c t s t r e s s r e s p o n s e s a s s o c i a t e d w i t h t h e c h a n g e s i n d e n s i t y . When d a t a from t h e f i r s t t i m e were a n a l y s e d i n a 1-way ANOVA ( r e p l i c a t e s p o o l e d ) , t h e e f f e c t s of d i f f e r e n t r e a r i n g d e n s i t i e s were n o t s i g n i f i c a n t (df=2,79 p=.36). D a t a f r o m t h e s e c o n d s a m p l i n g d a t e were a n a l y s e d i n a 1-way ANOVA ( r e p l i c a t e s p o o l e d ) . A l t h o u g h d e n s i t y t r e a t m e n t e f f e c t s were not s i g n i f i c a n t (df=2,95 p=.13), t h e r e were i n t e r e s t i n g t r e n d s i n t h e d a t a ( s e e b e l o w ) . The r e s u l t s o f t h e l a s t s a m p l i n g p e r i o d showed no d i f f e r e n c e s between d e n s i t y t r e a t m e n t s on p o o l e d d a t a (df=2,87 P=.19). I n t e r e s t i n g l y , mean plas m a C o r t i s o l l e v e l s had d e c r e a s e d t o l e v e l s s i m i l a r t o t h o s e of t h e l a s t s a m p l i n g d a t e i n e x p e r i m e n t 3a. To t e s t f o r c h a n g e s w i t h t i m e , t h e d a t a were a n a l y s e d i n two 2-way, n e s t e d ANOVAs. E a c h a n a l y s i s i n c l u d e d two t i m e s . When d a t a from t h e f i r s t two t i m e s were compared, r e p l i c a t e s were n o t s i g n i f i c a n t l y d i f f e r e n t (df=5,170 p=.17) and were p o o l e d . When p o o l e d t h e e f f e c t s of t i m e ( o r more c o r r e c t l y , t h e e f f e c t s o f sudden c h a n g e s i n d e n s i t y ) were s i g n i f i c a n t ( d f = 1 , l 7 0 p<.005). T r e a t m e n t e f f e c t s were n o t s i g n i f i c a n t (df=2,170 p=.95), however, t h e i n t e r a c t i o n t e r m was n e a r l y so (df=2,170 p=.08). T h i s s u g g e s t s t h a t t h e e f f e c t o f ' t i m e ' may not have been t h e 1 1 2 same i n e a c h t r e a t m e n t . T h i s was e x p e c t e d s i n c e e a c h t r e a t m e n t underwent d i f f e r e n t d e n s i t y c h a n g e s . I n s p e c t i o n o f t h e d a t a r e v e a l e d t h a t C o r t i s o l l e v e l s f r o m t h o s e t a n k s i n w h i c h d e n s i t i e s were unchanged d i d not change w i t h t i m e . In t a n k s i n w h i c h volumes were h a l v e d ( i . e . d e n s i t y d o u b l e d ) C o r t i s o l l e v e l s i n c r e a s e d s i g n i f i c a n t l y , i n d i c a t i n g a s t r e s s r e s p o n s e . S u r p r i s i n g l y , i n t a n k s where volumes were d o u b l e d ( i . e . d e n s i t y h a l v e d ) , t h e r e was a s l i g h t , a l t h o u g h not s i g n i f i c a n t (df=1,61 P=.19) i n c r e a s e i n pla s m a C o r t i s o l l e v e l s . In t h e 2-way ANOVA c o m p a r i n g t h e s e c o n d and l a s t s a m p l i n g d a t e s r e p l i c a t e s were p o o l e d . The e f f e c t of t i m e was s i g n i f i c a n t ( d f =1,178 p < . 0 0 l ) , c o n f i r m i n g t h e p a t t e r n o b s e r v e d i n e x p e r i m e n t 3a. A l t h o u g h n o t s p e c i f i c a l l y t e s t e d , i t i s l i k e l y t h a t t h e d e c r e a s e w i t h t i m e was s i g n i f i c a n t i n a l l t r e a t m e n t s . T h e s e d a t a a r e p r e s e n t e d i n F i g u r e 12b. E x p e r i m e n t 3c As w i t h t h e g r o w t h and p r o x i m a t e a n a l y s i s d a t a , i n t e r p r e t a t i o n of t h e s e d a t a was c o n f o u n d e d by s t o c k d i f f e r e n c e s . N o n - p a r a m e t r i c t e s t i n g p r o c e d u r e s were r e q u i r e d . F o r s i m p l i c i t y , r e p l i c a t e s were p o o l e d f o r t h e p a i r w i s e c o m p a r i s o n s . T h i s p r o c e d u r e i s not s t r i c t l y c o r r e c t ; however, much of t h e v a r i a n c e was l i k e l y a t t r i b u t a b l e t o s t o c k d i f f e r e n c e s and so s h o u l d be o f s i m i l a r m a g n i t u d e between volume t r e a t m e n t s . When t h e e f f e c t s of d i f f e r e n t v o l u m e s were compared a t b o t h s a m p l i n g d a t e s ( F i g u r e 1 2 c ) , f i s h r e a r e d i n low volume 1 1 3 c o n t a i n e r s had s i g n i f i c a n t l y l o w e r p l a s m a l e v e l s ( t i m e 1, t=3.417 p < . 0 0 l ; ti m e 2, t=4.231 p < . 0 0 l ) . The m a g n i t u d e o f t h e d i f f e r e n c e s was s i m i l a r i n b o t h c a s e s . I t was s u r p r i s i n g t h a t C o r t i s o l l e v e l s were h i g h e r f o r f i s h i n t h e h i g h volume t a n k s , s i n c e t h e y t e n d e d t o be g r o w i n g somewhat f a s t e r . To i n v e s t i g a t e t i m e e f f e c t s , f u r t h e r c o m p a r i s o n s were computed. C o r t i s o l l e v e l s were s i g n i f i c a n t l y r e d u c e d by t h e s e c o n d s a m p l i n g d a t e i n t h e low volume t a n k s ( t = 2 . 5 9 l p<.01) and were i n f a c t a l m o s t i d e n t i c a l t o t h o s e l e v e l s o b t a i n e d i n t h e q u a r a n t i n e room w i t h t h e o t h e r two e x p e r i m e n t s . C o r t i s o l l e v e l s were t h e r e f o r e u n a f f e c t e d by p h o t o p e r i o d . No d i f f e r e n c e s were f o u n d between t i m e s when t h e h i g h volume t a n k s were compared. T h i s r e s u l t was u n e x p e c t e d i n view o f a l l o t h e r C o r t i s o l t r e n d s . C o n s e q u e n t l y r e p l i c a t e s were a n a l y s e d s e p a r a t e l y . F i s h i n one t a n k d i d show a s i g n i f i c a n t r e d u c t i o n w h i l e t h e o t h e r , s u r p r i s i n g l y , showed an i n c r e a s e . The r e a s o n f o r t h i s anomalous r e s u l t i s u n c l e a r . However, t h i s t a n k was most s u s c e p t i b l e t o d i s t u r b a n c e , as i t was n e a r a walkway, and n e a r o t h e r f r e q u e n t l y v i s i t e d t a n k s . Weight L e n g t h R e l a t i o n s E x p e r i m e n t 3a The GM f u n c t i o n a l r e g r e s s i o n o f l n l e n g t h a g a i n s t l n w e i g h t p r o d u c e d t h e f o l l o w i n g e q u a t i o n ( r 2 = .9731 n = 1383 d a t a p a i r s ) : l n Wt. = (-4.438) + 2.993 * l n L. 1 1 4 T a b l e 20. C o n d i t i o n f a c t o r d a t a summary. V a l u e s were c a l c -u l a t e d f r o m a GM f u n t i o n a l r e g r e s s i o n of n a t u r a l l o g l e n g t h a g a i n s t n a t u r a l l o g w e i g h t . D e t a i l s of t h e d e s i g n a r e i n T a b l e 14. I n p a r t 2, d e n s i t i e s were c h a n g e d on day 48. The 8 i n b r a c k e t s r e f e r s o n l y t o p a r t 2. In b o t h o t h e r g r o u p s t h e f i r s t s a m p l i n g d a t e was on day 1. D A Y TANKS 1 ( 8 ) * 18 32 61 72 n S.E. 1 0 .998 1 .011 0 .998 0 .962 0.991 50 0.00645 4 1 .001 1 .026 1 .019 0 .988 1 .035 50 0.00645 p a r t 1 2 0 .984 1 .016 1 .017 0 .942 1 .001 50 0.00645 5 0 .995 1 .024 1 .032 1 .008 1 .020 50 0.00645 3. 0 .993 0 .999 1 .010 0 .942 0.984 50 0.00645 6 1 .000 1 .010 1 .018 0 .991 0.992 50 0.00645 7 1 .005 1 .002 1 .014 0 .976 1 .001 50 0.00647 * 10 1 .003 1 .006 0 .990 0 .986 1.014 50 0.00647 p a r t 2 8 0 .996 1 .023 1 .014 0 .975 1.012 50 0.00647 1 1 1 .004 1 .014 1 .003 0 .968 1 .019 50 0.00647 9 0 .996 1 .007 0 .976 0 .994 1.017 50 0.00647 1 2 0 .993 1 .010 0 .997 0 .992 1 .024 50 0.00647 1 3 0 .997 1 .004 1 .015 0 .968 0.995 50 0.00702 14 0 .968 1 .002 1 .000 0 .975 0.997 50 0.00702 p a r t 3 1 5 0 .988 1 .015 1 .016 1 .008 1 .043 50 0.00702 1 6 0 .997 1 .002 1 .026 1 .002 1 .033 50 0.00702 When r e o r g a n i z e d and a n t i l o g g e d , t h e w o r k i n g c o n d i t i o n e q u a t i o n was: c o n d i t i o n f a c t o r = w e i g h t / ( 0 . 0 1 1 8 1 9 5 5 * l e n g t h 2 ' 9 9 3 ) . A 2-way n e s t e d ANOVA p r o d u c e d no s i g n i f i c a n t t r e a t m e n t e f f e c t s (df=2,1382 p=.47). R e p l i c a t e s were s i g n i f i c a n t l y d i f f e r e n t (df=15,1382 p < . 0 0 l ) . C o n d i t i o n v a r i e d s i g n i f i c a n t l y o v e r t i m e ( d f = 4 , l 3 8 2 p<.01). The p a t t e r n w i t h t i m e was s i m i l a r t o t h a t f o r growth s l o p e w i t h t i m e and was c o n s i s t e n t i n a l l t a n k s . T h e s e d a t a a r e p r e s e n t e d i n T a b l e 20. 1 15 E x p e r i m e n t 3b The GM f u n c t i o n a l r e g r e s s i o n o f l n l e n g t h a g a i n s t l n w e i g h t p r o d u c e d the- f o l l o w i n g e q u a t i o n ( r 2 = .9779, n= 1 5 0 0 ) : l n Wt. = (-4.549) + 3.035 * l n L., w h i c h when c o n v e r t e d t o t h e w o r k i n g f o r m u l a was: c o n d i t i o n f a c t o r = w e i g h t / ( 0 . 0 1 0 5 7 7 7 8 * l e n g t h 3 ' 0 3 5 ) . The c o n d i t i o n d a t a were s u b s e q u e n t l y a n a l y s e d i n a 2-way ANOVA ( r e p l i c a t e s p o o l e d , d f = l 5 , 1 4 9 9 p=.14). T r e a t m e n t e f f e c t s were n o t s i g n i f i c a n t (df=2,1499 p=.55), however, t h e e f f e c t s of t i m e were (df=4,1499 p < . 0 0 l ) . The p a t t e r n o b s e r v e d f o l l o w e d t h a t o f g r o w t h s l o p e s o v e r t i m e . The same t r e n d o c c u r r e d i n e x p e r i m e n t 3a. The i n t e r a c t i o n t e r m was s i g n i f i c a n t (df=8,1499 P<.001). I n s p e c t i o n o f t h e d a t a r e v e a l e d o n l y minor p a t t e r n d i f f e r e n c e s between t r e a t m e n t s and t h e s o u r c e o f t h e i n t e r a c t i o n was n o t r e a d i l y a p p a r e n t . T h e r e was no e v i d e n c e of a c l e a r r e l a t i o n between t h e e f f e c t s o f c h a n g i n g d e n s i t y and c o n d i t i o n f a c t o r r e s p o n s e . Mean v a l u e s a r e p r e s e n t e d i n T a b l e 20. E x p e r iment 3c The GM f u n c t i o n a l r e g r e s s i o n o f l n w e i g h t on l n l e n g t h ( r 2 = .9707, n = 1000) i s p r e s e n t e d below: l n Wt. = (-4.849) + 3.138 * l n L., w h i c h r e a r r a n g e d becomes: c o n d i t i o n f a c t o r = weight/(0.00783621 * l e n g t h 3 ' 1 3 8 ) , and i s t h e w o r k i n g f o r m u l a . When c o n d i t i o n d a t a were a n a l y s e d i n a 2-way ANOVA 11 6 ( r e p l i c a t e s p o o l e d - d f = l 0 , 9 9 9 p=.55), b o t h volume an d t i m e e f f e c t s were s i g n i f i c a n t (df=1,999 p < . 0 0 l ; df=4,999 p<.001, r e s p e c t i v e l y ) . F i s h f r o m t h e low volume t r e a t m e n t s had l o w e r c o n d i t i o n f a c t o r s t h a n t h o s e i n t h e h i g h volume t a n k s . T h i s i s i n g e n e r a l agreement w i t h t h e t r e n d f o r i n c r e a s e d g r o w t h a t h i g h e r v o l u m e s . The p a t t e r n w i t h t i m e c l o s e l y f o l l o w e d t h a t i n e x p e r i m e n t s 3a and 3b, and m i r r o r e d i t s own g r o w t h s l o p e p a t t e r n . T h e s e d a t a a r e summarized i n T a b l e 20. B e h a v i o r B e h a v i o r a l a n a l y s e s w i l l be p r e s e n t e d s e p a r a t e l y f o r e x p e r i m e n t s 3a and 3b. D a t a were f i r s t a n a l y s e d by i n d i v i d u a l t i m e s and t h e n a n a l y s e d o v e r t i m e . D a t a from t a n k 5 (50 f i s h , 24 1/min) were d e l e t e d from t h e a n a l y s i s . A f a i n t shadow, o n l y v i s i b l e on t h e t a p e s , a f f e c t e d a c t i v i t y and f i s h d i s t r i b u t i o n . In a d d i t i o n , d a t a from t h e l a s t t h r e e d a t e s were d e l e t e d f o r t a n k 12 (300 f i s h , low t o h i g h v o l u m e ) , s i n c e s u r f a c e g l a r e o b s c u r e d a c t i v i t y . E x p e r i m e n t 3a Net A c t i v i t y : No d i f f e r e n c e s between t r e a t m e n t s were f o u n d d u r i n g t h e f i r s t t h r e e d a t e s when d a t a were a n a l y s e d i n 1-way n e s t e d ANOVAs. On t h e l a s t day a c t i v i t y i n t h e h i g h d e n s i t y t r e a t m e n t was g r e a t e r t h a n i n t h e o t h e r t r e a t m e n t s (df=2,209 p<.05). To i d e n t i f y p a t t e r n s w i t h t i m e , t h e d a t a were r e a n a l y s e d i n 1 1 7 a 2-way n e s t e d ANOVA. No s i g n i f i c a n t t r e a t m e n t e f f e c t s were f o u n d . T h e r e was a s l i g h t t r e n d f o r i n c r e a s e d a c t i v i t y i n h i g h e r d e n s i t i e s . R e p l i c a t e s were s i g n i f i c a n t l y d i f f e r e n t ( d f = 8 , 8 l 9 p<.005). Net a c t i v i t y d i d n o t v a r y s i g n i f i c a n t l y w i t h t i m e ( d f = 3 , 8 l 9 p=.07), however, t h e r e was a p a t t e r n , r e p e a t e d i n e a c h t a n k , f o r an i n c r e a s e i n a c t i v i t y w i t h t i m e o v e r t h e f i r s t 3 p e r i o d s , and t h e n a marked d e c l i n e on t h e l a s t day ( F i g u r e 1 3 a ) . T o t a l A c t i v i t y : When t i m e s were a n a l y s e d s e p a r a t e l y i n 1-way n e s t e d ANOVAs, o n l y d u r i n g t h e f i r s t d a t e (day 31) were d i f f e r e n c e s f o u n d . On t h i s day, t o t a l a c t i v i t y i n t h e low f l o w t r e a t m e n t was r e d u c e d (df=2,230 p < . 0 0 l ) . As w i t h n e t a c t i v i t y , t h e d a t a were a n a l y s e d t o d e t e c t t r e n d s w i t h t i m e . T r e a t m e n t e f f e c t s were n o t s i g n i f i c a n t . As i n t h e n e t a c t i v i t y d a t a , t h e r e was a t r e n d t o w a r d s h i g h e r a c t i v i t y i n t h e h i g h d e n s i t y t r e a t m e n t . R e p l i c a t e s were d i f f e r e n t (df=8,980 p<.001). Time e f f e c t s were s i g n i f i c a n t (df=3,980 p<.05) and t h e o b s e r v e d p a t t e r n matched t h a t f o r n e t a c t i v i t y ( F i g u r e 13b). The i n t e r a c t i o n t e r m was not s i g n i f i c a n t (df=6,980 p=.84). E x p e r i m e n t 3b Net A c t i v 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 were f o u n d between t r e a t m e n t s i n a l l b ut one s a m p l i n g d a t e . O n l y on day 59 were d i f f e r e n c e s n o t s i g n i f i c a n t . T r e a t m e n t s i n w h i c h t h e s p a c e p e r f i s h was l a r g e ( h i g h volume, low d e n s i t y ) a t t h e t i m e o f s a m p l i n g , e x h i b i t e d g r e a t e r a c t i v i t y l e v e l s t h a n t h o s e i n w h i c h t h e s p a c e p e r f i s h was low. Th e s e d a t a a r e summarized i n 1 18 F i g u r e 13. A c t i v i t y a g a i n s t t i m e f o r e x p e r i m e n t 3a. P a r t A- n e t a c t i v i t y , p a r t B - t o t a l a c t i v i t y . Mean v a l u e s a r e i n d i c a t e d by heavy s o l i d l i n e s w i t h 95% C L . L i n e s a r e o f f s e t by one day e i t h e r s i d e of t h e t r u e s a m p l i n g day f o r c l a r i t y . DAY NUMBER 24 T -| 1 i —I 1 1 \ 20 30 40 50 60 70 80 DAY NUMBER 121 T a b l e 21 . T a b l e 21. Summary of n e t a c t i v i t y d a t a by t i m e and t r e a t m e n t f o r t h e s e c o n d s t u d y , 3b, (sudden d e n s i t y c h a n g e s ) o f t h e t h i r d e x p e r i m e n t . V a l u e s p r e s e n t e d a r e t r a n s f o r m e d ( n a t u r a l l o g + 1.1), and a r e e x p r e s s e d i n a r b i t r a r y u n i t s p e r 15 s e c o n d s . To d e t e r m i n e t h e e f f e c t o f sudden c h a n g e s i n d e n s i t y , f o l l o w t r e a t m e n t s a c r o s s t i m e i n t e r v a l s . The mean v a l u e s o v e r a l l t i m e s ( l a s t column) shows t h e e f f e c t most c l e a r l y . As m e n t i o n e d i n t h e t e x t , t h e o b s e r v e d e f f e c t s may be due t o volume d i f f e r e n c e s . FIRST SECOND --DENSITY-- DENSITY DAY 31 45 52 59 73 ALL n 38 54 32 78 49 267 L--L mean 1 .819 1 .736 1 .877 1 .656 1 .742 1.731 S.E. 0 . 1 174 0 . 1 003 0 . 1 2.03 0 .081 7 0 .0974 0.0441 n 70 83 90 73 83 412 L--H mean 1 .765 1 .410 1 .262 1 .480 1 .279 1 .364 S.E. 0 .0865 0 .0809 0 .071 7 0 . 0844 0 .0748 0.0354 n 86 56 29 30 39 210 H--L mean 1 .246 1 .347 1 .708 1 .659 1 .449 1 .461 S.E. 0 .0781 0 .0985 0 . 1 264 0 .1310 0 . 1 092 0.0496 L-L - c o n s t a n t low d e n s i t y ( h i g h volume) L-H - low d e n s i t y t o h i g h ( h i g h volume t o low) H-L - h i g h d e n s i t y t o low (low volume t o h i g h ) When a n a l y s e d i n a 2-way ANOVA o v e r a l l 5 t i m e s t h e r e s u l t s were c o n s i s t e n t w i t h t h e above a n a l y s e s . A c t i v i t y i n t r e a t m e n t s i n w h i c h volumes were unchanged was g r e a t e r (df=2,888 .p<.00l) t h a n i n t r e a t m e n t s i n w h i c h i t was n o t . A t h i g h volumes (low d e n s i t i e s ) f i s h s p e n t l e s s t i m e h o l d i n g p o s i t i o n s t h a n a t low v o l u m e s . As e x p e c t e d , t h e i n t e r a c t i o n t e r m was c l o s e t o s i g n i f i c a n c e (df=8,888 p=.06) s u g g e s t i n g t h a t c h a n g i n g volumes had i n f l u e n c e d a c t i v i t y . 1 22 T a b l e 22. Summary of t o t a l a c t i v i t y d a t a by t i m e and t r e a t -ment f o r s t u d y 2 (sudden d e n s i t y c h a n g e s ) o f e x p e r i m e n t 3. V a l u e s p r e s e n t e d a r e t r a n s f o r m e d v a r i a b l e s ( n a t u r a l l o g + 1.1), and a r e g i v e n i n a r b i t r a r y u n i t s p e r 15 s e c o n d s . To d e t e r m i n e t h e e f f e c t s of c h a n g e s i n d e n s i t y , f o l l o w mean v a l u e s a c r o s s t i m e i n t e r v a l s . The mean v a l u e s o v e r a l l t i m e i n t e r v a l s ( l a s t column) d e p i c t s t h e e f f e c t most c l e a r l y . As m e n t i o n e d i n t h e t e x t , t h e o b s e r v e d e f f e c t s may be due t o vol u m e . FIRST SECOND DENSITY DENSITY DAY 31 45 52 59 73 ALL n 100 100 100 100 99 499 L--L mean 3 .861 3 .450 3.688 2 .788 3.651 3.487 S.E. 0 .1041 0 . 1 041 0. 1041 0 . 1 041 0.1046 0.0467 n 100 100 1 00 100 1 00 500 L--H mean 3 .079 2 .543 2.470 2 .818 2.630 2.708 S.E. 0 .1041 0 . 1 041 0.1041 0 . 1 041 0.1041 0.0466 n 1 00 100 60 50 50 360 H' -L mean 2 .376 3 .018 3. 104 3 .428 2.611 2.859 S.E. 0 .1041 0 . 1 041 0.1344 0 . 1 472 0.1472 0.0553 L-L - c o n s t a n t low d e n s i t y ( h i g h volume) L-H - low d e n s i t y t o h i g h ( h i g h volume t o low) H-L - h i g h d e n s i t y t o low (low volume t o h i g h ) T o t a l A c t i v i t y : When a n a l y s e d i n 1-way n e s t e d ANOVAs, s i g n i f i c a n t d i f f e r e n c e s were f o u n d a t most t i m e s . However, t h e d i f f e r e n c e s were l e s s c o n s i s t e n t t h a n w i t h n e t a c t i v i t y . T h i s s u g g e s t s t h a t t o t a l a c t i v i t y i s l e s s a f f e c t e d by d e n s i t y ( a n d / o r volume) t h a n n e t a c t i v i t y . The g e n e r a l p a t t e r n was t h e same ( T a b l e 2 0 ) . When a n a l y z e d i n a 2-way n e s t e d ANOVA o v e r a l l 5 t i m e s , t h e p a t t e r n was i d e n t i c a l t o t h a t a b o v e . T r e a t m e n t e f f e c t s were s i g n i f i c a n t (df=2,1358 p<.00l) and r e p l i c a t e s were s i g n i f i c a n t l y d i f f e r e n t (df=13,1358 p < . 0 0 l ) . In a d d i t i o n t h e i n t e r a c t i o n t e r m 1 23 was s i g n i f i c a n t (df=8,1358 p<.005) and t h i s i n d i c a t e s a h e f f e c t o f volume a n d / o r d e n s i t y c h a n g e s . S a l t Water C h a l l e n g e T e s t T e c h n i c a l d i f f i c u l t i e s , i n c l u d i n g i n a d v e r t e n t m i x i n g o f f i s h from d i f f e r e n t t r e a t m e n t s n e c e s s i t a t e d an e a r l y t e r m i n a t i o n o f t h e f i r s t t e s t . The s e c o n d a t t e m p t was s u c c e s s f u l . F o r l o g i s t i c a l r e a s o n s f i s h from r e p l i c a t e t a n k s o f e a c h t r e a t m e n t were p o o l e d . A d d i t i o n a l l y , t h e two t r e a t m e n t s c o n t a i n i n g 50 f i s h w i t h d i f f e r e n t f l o w s were p o o l e d . The t r e a t m e n t c o n t a i n i n g 300 f i s h (low t o h i g h volume) was n o t t e s t e d . A t o t a l o f 20 f i s h f r o m e a c h t r e a t m e n t was randomly s e l e c t e d ( e q u a l numbers f r o m r e p l i c a t e t a n k s ) . D e s i g n d e t a i l s a r e p r e s e n t e d i n T a b l e 23. Da t a were f i r s t a n a l y z e d p a r a m e t r i c a l l y , however, h e t e r o g e n e i t y of v a r i a n c e p r o b l e m s n e c e s s i t a t e d n o n - p a r a m e t r i c t e s t s . E v i d e n t l y sample s i z e s were l a r g e enough t h a t F - v a l u e s were u n a f f e c t e d by t h e h e t e r o g e n e o u s v a r i a n c e s , o r d i f f e r e n c e s were l a r g e , s i n c e t h e r e s u l t s of b o t h methods were i d e n t i c a l . The p a r a m e t r i c a n a l y s e s a r e p r e s e n t e d h e r e . B e c a u s e f i s h f r o m s t u d y 3c were r e a r e d under a d i f f e r e n t p h o t o p e r i o d t h a n t h o s e from 3a and 3b, t h e s e d a t a were i n i t i a l l y a n a l y s e d s e p a r a t e l y . When t h e f o u r t r e a t m e n t s f r o m 3a and 3b were t e s t e d a c l e a r d e n s i t y e f f e c t was a p p a r e n t (df=3,72 p<.001) ( F i g u r e 14)* S c h e f f e ' s t e s t p r o d u c e d two g r o u p s . F i s h f r o m t r e a t m e n t s c o n t a i n i n g h i g h d e n s i t i e s , a p p r o x i m a t e l y 1 f i s h / 1 (50-60 gm/1), r e g u l a t e d plasma sodium l e v e l s p o o r l y . F i s h f r o m t r e a t m e n t s c o n t a i n i n g low d e n s i t i e s , e i t h e r 0.14 f / 1 (9 gm/1) o r 1 24 F i g u r e 14. Plasma sodium c o n c e n t r a t i o n s f o l l o w i n g s a l t w a t e r c h a l l e n g e t e s t . P a r t A- sodium c o n c e n t r a t i o n s a g a i n s t r e a r i n g d e n s i t y , p a r t B- sodium c o n c e n t r a t i o n s a g a i n s t t a n k volume. . V e r t i c a l b a r s a r e s t a n d a r d e r r o r s . The d a s h e d h o r i z o n t a l l i n e i s an a r b i t r a r y c u t - o f f v a l u e . Plasma sodium v a l u e s above t h i s l e v e l a f t e r 24 h o u r s , i n d i c a t e t h a t t e s t f i s h were i n c o m p l e t e l y s m o l t e d , and n o t f u l l y c a p a b l e of s u r v i v i n g i n s a l t w a t e r . 115 190 T 180+ o C l ° ^ 170-r-si UJ o CO oo 160+ 150 0. i 0 i 0.3 0.6 0.9 1.2 DENSITY ( F I S H P E R L I T R E ) B cc c__> CD O OO OO 190 T 180+ S 170-1" 160+ 150 200 f ^ o " 400 i 500 600 VOLUME ( L I T R E S ) 1 26 T a b l e 23. D e s i g n o f s a l t w a ter c h a l l e n g e t e s t . F i s h from r e p l i c a t e t a n k s were p o o l e d . The d e n s i t i e s g i v e n a r e t h e a c t u a l r e a r i n g d e n s i t i e s e x p e r i e n c e d p r i o r t o t e s t i n g ( i . e . m o r t a l i t i e s and sampled f i s h a r e a c c o u n t e d f o r ) . NUMBER REARING DENSITY MEAN STD. OUP TANKS SAMPLED PHOTOPERIOD WEIGHT DEV. pe r t a n k f i s h / 1 gm/1 h o u r s l i g h t gm gm 1 1,4 10 1.01 59.6 10 58.3 15.1 2 2,3,5,6 5 0.10 5.6 1 0 56.5 11.5 3 7,10 10 0.41 21.0 10 66.4 12.7 4 8,11 10 0.96 53.0 1.0 51 .6 9.2 5 13,14 10 0.93 46.5 1 4 56.9 14.7 6 15,16 10 0.96 52.8 14 52.9 10.3 0.45 f/1 (30 gm/1) were s i m i l a r i n t h e i r c a p a c i t y and r e g u l a t e d sodium w e l l . ! The e f f e c t of t a n k volume on s a l t r e g u l a t i o n was examined by c o m p a r i n g t h e two t r e a t m e n t s from e x p e r i m e n t 3c. No d i f f e r e n c e s were f o u n d (df=1,59 p=.21). To d e t e r m i n e i f t h e d i f f e r e n t , b u t c o n s t a n t , p h o t o p e r i o d s had a f f e c t e d t h e d e v e l o p m e n t of h y p e r o s m o r e g u l a t o r y c a p a b i l i t y , t r e a t m e n t s c o n t a i n i n g s i m i l a r d e n s i t i e s f r o m t h e two rooms were compared. No d i f f e r e n c e s due t o p h o t o p e r i o d were d e t e c t e d (df=3,77 p=.12). S i n c e t h e mean s i z e of t h e t e s t f i s h u s e d i n e a c h t r e a t m e n t v a r i e d , i t was d e s i r a b l e t o d e t e r m i n e i f i n d i v i d u a l w e i g h t s were c o r r e l a t e d w i t h p l a s m a sodium l e v e l s . T h i s was not u n l i k e l y s i n c e s m o l t i f i c a t i o n i s m o d i f i e d by f i s h s i z e . L i n e a r r e g r e s s i o n s of mean f i s h w e i g h t a g a i n s t p l a s m a sodium were c a l c u l a t e d f o r e a c h t r e a t m e n t . In most c a s e s , s l o p e s were not s i g n i f i c a n t l y d i f f e r e n t f r o m z e r o . In one c a s e , however, a 127 s i g n i f i c a n t n e g a t i v e s l o p e (b = -0.39) was o b t a i n e d . S i n c e t h i s c a s e was i s o l a t e d , and b e c a u s e t h e mean f i s h w e i g h t i n t h e t r e a t m e n t was a b o u t t h e same as i n o t h e r g r o u p s of c o m p a r a b l e mean sodium l e v e l s , i t s e f f e c t was i g n o r e d . T h e r e was some c o n c e r n t h a t t a n k volumes may have a f f e c t e d p l a s m a s o d i u m l e v e l s . T h i s was c o n s i d e r e d u n l i k e l y i n v i e w o f t h e r e s u l t s of c o m p a r i s o n s of t r e a t m e n t s from s t u d y 3c. P l o t s of plas m a sodium l e v e l s a g a i n s t volume ( F i g u r e 14b) r e i n f o r c e t h i s c o n c l u s i o n . C o n c l u s i o n s E x p e r i m e n t 3a Growth s l o p e d a t a showed no e v i d e n c e of d e n s i t y e f f e c t s o v e r an 8 - f o l d range from 0.5 t o 3.8 t i m e s h a t c h e r y l e v e l s . Flow r a t e d i d n o t a f f e c t g r o w t h . Growth s l o p e s , i n i t i a l l y e r r a t i c , showed e v i d e n c e o f a n o n - s i g n i f i c a n t i n c r e a s e d u r i n g t h e l a s t 3 p e r i o d s . P r o x i m a t e c o m p o s i t i o n d a t a were l e s s c l e a r t h a n i n p r e v i o u s e x p e r i m e n t s . F i s h f r o m t h e h i g h d e n s i t y t r e a t m e n t had e l e v a t e d m o i s t u r e and a s h l e v e l s . P r o t e i n c o n t e n t was u n a f f e c t e d . L i p i d c o n t e n t was n o t a f f e c t e d by t r e a t m e n t s a l t h o u g h t h e p r o b a b i l i t y was near t h e s i g n i f i c a n t l e v e l and c o n t e n t was r e d u c e d a t h i g h d e n s i t y . T h e s e f i n d i n g s a r e s i m i l a r t o t h o s e from t h e f i r s t two s t u d i e s , and t o o t h e r s r e p o r t e d i n t h e l i t e r a t u r e . T h e r e were no t r e a t m e n t - r e l a t e d d i f f e r e n c e s i n p l a s m a C o r t i s o l c o n c e n t r a t i o n s , however, t h e r e was a r e d u c t i o n w i t h 128 t i m e . C o n d i t i o n f a c t o r s showed no e v i d e n c e of t r e a t m e n t e f f e c t s . As w i t h t h e growth d a t a , d i f f e r e n c e s w i t h t i m e were s i g n i f i c a n t and a p a t t e r n m i r r o r i n g growth s l o p e s was o b s e r v e d . N e i t h e r n e t nor t o t a l a c t i v i t y were s i g n i f i c a n t l y a f f e c t e d by e i t h e r d e n s i t y o r f l o w ( e x c e p t f o r some i s o l a t e d c a s e s ) . T h e r e was a p a t t e r n w i t h t i m e , however, t h a t was s i g n i f i c a n t f o r t o t a l a c t i v i t y and n e a r l y so f o r n e t a c t i v i t y . In a l l t a n k s a c t i v i t y i n c r e a s e d s t e a d i l y f r o m day 31 u n t i l i t p e a k e d a t day 59. On t h e l a s t d a t e l e v e l s d e c l i n e d . E x p e r i m e n t 3b D u r i n g t h e f i r s t f o u r s a m p l i n g i n t e r v a l s t h e r e were no d e n s i t y e f f e c t s on any of t h e v a r i a b l e s t e s t e d , w i t h t h e e x c e p t i o n of t h e a c t i v i t y d a t a . The d e n s i t y r a n g e e x t e n d e d f r o m 1.8 t o 3.6 t i m e s s t a n d a r d h a t c h e r y d e n s i t i e s . D e n s i t y e f f e c t s were a p p a r e n t d u r i n g t h e l a s t 3 i n t e r v a l s . U s i n g g r o w th s l o p e i n f o r m a t i o n from t h e t r e a t m e n t i n w h i c h volumes r e m a i n e d unchanged from t h i s s t u d y and t h e h i g h d e n s i t y t r e a t m e n t from s t u d y 3a as c o n t r o l s , t h e p o s s i b i l i t y o f c o n d i t i o n i n g was e x p l o r e d . When t a n k s i n w h i c h v o l u m e s were d o u b l e d ( i . e . d e n s i t i e s h a l v e d - 0.6 f / 1 ) were compared w i t h t a n k s h e l d a t t h e h i g h volume (0.6 f / 1 ) , no d i f f e r e n c e s i n g r o w t h s l o p e were f o u n d . However, when t a n k s i n w h i c h volumes were h a l v e d ( i . e . d e n s i t i e s d o u b l e d - 1.0 f / 1 ) were compared w i t h t a n k s o f s i m i l a r d e n s i t y f r o m s t u d y 3a, t h e r e was e v i d e n c e of s i g n i f i c a n t g r o w t h d e p r e s s i o n . T h i s c l e a r l y s u g g e s t s t h e p o s s i b i l i t y of a 129 c o n d i t i o n i n g e f f e c t . S i n c e t h e t a n k s u s e d a s s t a n d a r d s f o r t h i s s e c o n d c o m p a r i s o n c o n t a i n e d d i f f e r e n t v olumes and f l o w s , i t was n e c e s s a r y t o e s t a b l i s h t h a t t h e s e d i d n o t a f f e c t r e s u l t s . C o n s e q u e n t l y t r e a t m e n t s o f s i m i l a r d e n s i t i e s b u t d i f f e r e n t v o l u m e s were compared f o r t h e p e r i o d when volume s were c h a n g e d . No volume e f f e c t was f o u n d . When t h e e f f e c t o f f l o w was examined i n s t u d y 3a, i t was f o u n d t o be n o n - s i g n i f i c a n t . S i n c e t h e range t e s t e d was t w i c e t h a t of t h e g r o u p s b e i n g compared between s t u d i e s 3a and 3b, t h e e f f e c t o f f l o w was u n i m p o r t a n t . D e n s i t y d i d not a f f e c t p r o x i m a t e c o m p o s i t i o n p r i o r t o t h e t i m e of volume c h a n g e s . When C o r t i s o l d a t a were a n a l y s e d by s e p a r a t e s a m p l i n g d a t e s no d i f f e r e n c e s between t r e a t m e n t s were d e t e c t e d . However, when t h e f i r s t two p e r i o d s were compared, d i f f e r e n c e s were d e t e c t e d . T h i s was e x p e c t e d s i n c e t h e s a m p l i n g . d a t e s were t h o s e i m m e d i a t e l y b e f o r e , and one week a f t e r , v olumes were c h a n g e d . T a n k s i n w h i c h d e n s i t i e s were d o u b l e d had s i g n i f i c a n t l y h i g h e r C o r t i s o l l e v e l s t h a n b e f o r e d e n s i t i e s were d o u b l e d , i n d i c a t i v e of a s t r e s s r e s p o n s e . T a n k s i n w h i c h d e n s i t i e s were r e d u c e d showed e v i d e n c e of a s l i g h t ( n o n - s i g n i f i c a n t ) i n c r e a s e . When t h e f i r s t and l a s t s a m p l i n g d a t e s were compared, a l l e v i d e n c e of t h e s t r e s s e f f e c t p r e s e n t i n t h e s e c o n d p e r i o d was gone. In a l l t r e a t m e n t s C o r t i s o l l e v e l s d e c r e a s e d s i g n i f i c a n t l y by t h e l a s t s a m p l i n g t i m e . C o n d i t i o n d a t a were u n a f f e c t e d by t r e a t m e n t . O v e r a l l p a t t e r n s w i t h t i m e t e n d e d t o f o l l o w t h o s e f o r g r o w t h . A s i g n i f i c a n t i n t e r a c t i o n t e r m (df=8,1499 p<.00l) d e m o n s t r a t e d 1 30 t h a t sudden c h a n g e s i n d e n s i t y c a u s e d m e a s u r e a b l e c h a n g e s i n body form. A l t h o u g h b e h a v i o r a l d a t a , p a r t i c u l a r l y t o t a l a c t i v i t y m e a s u r e s , were not a l w a y s c l e a r a t i n d i v i d u a l t i m e s , when a l l t i m e s were p o o l e d , a c o n s i s t e n t p a t t e r n emerged. Under h i g h d e n s i t y (low volume) c o n d i t i o n s b o t h a c t i v i t y m e a sures were s i g n i f i c a n t l y below l e v e l s o b t a i n e d under low d e n s i t y ( h i g h volume) c o n d i t i o n s . When d e n s i t i e s were c h a n g e d a c t i v i t y l e v e l s a d j u s t e d v e r y q u i c k l y . T h i s s u g g e s t s t h a t p r i o r e x p e r i e n c e d i d n o t have a l a s t i n g e f f e c t . The r e s p o n s e s o b s e r v e d may have been a r t i f a c t s of t a n k v o l u m e s . E x p e r iment 3c The r e s u l t s of t h i s s t u d y were m i x e d . A n e a r l y s i g n i f i c a n t t r e n d f o r r e d u c e d growth a t low volumes was d e t e c t e d . T h e r e were no volume r e l a t e d e f f e c t s on p r o x i m a t e c o m p o s i t i o n . F i s h f r o m low volume t a n k s had r e d u c e d c o n d i t i o n s . C o n d i t i o n f a c t o r s f o l l o w e d a p a t t e r n w i t h t i m e w h i c h a p p r o x i m a t e d t h e p a t t e r n i n g r o w t h s l o p e s . In a p p a r e n t c o n t r a d i c t i o n w i t h g r o w t h and c o n d i t i o n d a t a , f i s h r e a r e d a t h i g h v o l u m e s had c o n s i s t e n t l y h i g h e r p l a s m a C o r t i s o l l e v e l s . As i n e x p e r i m e n t s 3a and 3b C o r t i s o l l e v e l s d e c r e a s e d s i g n i f i c a n t l y w i t h t i m e i n a l l but one t a n k . P e r i o d i c q u a l i t a t i v e o b s e r v a t i o n s i n d i c a t e d t h a t a c t i v i t y was c o n s i s t e n t l y h i g h e r i n t h e h i g h volume t a n k s . S i n c e d e n s i t i e s were s i m i l a r , i t a p p e a r s l i k e l y t h a t t h e r e s p o n s e was a f u n c t i o n o f volume. 131 S a l t Water C h a l l e n g e : The s a l t w a ter c h a l l e n g e t e s t d e t e c t e d s t r e s s - r e l a t e d d i f f e r e n c e s i n s m o l t f i t n e s s a t t r i b u t a b l e t o r e a r i n g c o n d i t i o n s . The r e s u l t s c o u l d n o t be a t t r i b u t e d t o f i s h s i z e , f l o w r a t e , volume, o r p h o t o p e r i o d e f f e c t s . F i s h r e a r e d a t d e n s i t i e s as h i g h as 30 gm/1 r e g u l a t e d s a l t b a l a n c e w e l l (as w e l l as f i s h r e a r e d a t much l o w e r d e n s i t i e s ) . F i s h r e a r e d a t h i g h d e n s i t i e s (60 gm/1.) showed e v i d e n c e of a marked r e d u c t i o n i n o s m o r e g u l a t o r y p e r f o r m a n c e . 1 32 GENERAL DISCUSSION The s t r e s s - r e l a t e d p h y s i o l o g i c a l c o n s e q u e n c e s of e x t e n d e d p e r i o d s o f h i g h d e n s i t y r e a r i n g have n o t been t h o r o u g h l y i n v e s t i g a t e d i n s a l m o n i d s . In c oho salmon F a g e r l u n d e t a l . (1981) examined t h e e f f e c t s o f d e n s i t i e s on g r o w t h , body c o m p o s i t i o n , c o n d i t i o n f a c t o r , and i n t e r r e n a l n u c l e a r d i a m e t e r . They f o u n d t h a t g r o w t h and body c o n d i t i o n was s u p p r e s s e d by h i g h d e n s i t i e s . In a d d i t i o n , f i s h r e a r e d i n h i g h d e n s i t i e s had r e d u c e d whole-body l i p i d l e v e l s and e l e v a t e d m o i s t u r e l e v e l s . F u r t h e r m o r e , s m a l l f i s h s e l e c t e d f r o m t h e h i g h d e n s i t y t r e a t m e n t s had s i g n i f i c a n t l y i n c r e a s e d i n t e r r e n a l n u c l e a r d i a m e t e r s . T h i s i s one o f t h e few t h o r o u g h i n v e s t i g a t i o n s i n t h i s a r e a . However, f o r p a r t o f t h e i r s t u d y , f i s h were f e d r e d u c e d r a t i o n s . A l t h o u g h t h i s f o l l o w e d n o r m a l h a t c h e r y p r a c t i c e s , a d e s i r e d o b j e c t i v e , i t may a l s o have c o n t r i b u t e d t o t h e o b s e r v e d d e n s i t y - i n d u c e d e f f e c t s . Wedemeyer (1976) examined t h e s h o r t - t e r m s t r e s s r e s p o n s e s t o c r o w d i n g i n b o t h c oho and s t e e l h e a d and f o u n d t h a t when d e n s i t i e s were i n c r e a s e d f r o m 8.5 gm/1 t o 17, 34, 68, 102, o r 204 gm/1., a l l g r o u p s e x h i b i t e d h y p e r g l y c e m i a . T h i s r i s e i n b l o o d g l u c o s e was t r a n s i t o r y and i n most g r o u p s l e v e l s r e t u r n e d t o n o r m a l w i t h i n a few weeks. T h i s s u g g e s t s a d a p t a t i o n . Growth r e s p o n s e s were not examined d u r i n g t h a t s t r e s s p e r i o d . F u r t h e r m o r e t h e h i g h e r d e n s i t i e s t e s t e d were more t y p i c a l o f t r a n s p o r t t h a n r e a r i n g d e n s i t i e s . I e x a m i n e d t h e c o n s e q u e n c e s of r e a r i n g j u v e n i l e s t e e l h e a d t r o u t of t h r e e s i z e r a n g e s under v a r i o u s d e n s i t y r e g i m e s . The r e s p o n s e s t o d e n s i t y were not t h e same i n e a c h o f t h e t h r e e 133 e x p e r i m e n t s . T h e r e f o r e g e n e r a l i z a t i o n s r e g a r d i n g r e s p o n s e a r e n o t e a s i l y made, and f o r c l a r i t y , e a c h e x p e r i m e n t i s d i s c u s s e d s e p a r a t e l y . Where a p p r o p r i a t e , g e n e r a l i z a t i o n s a r e a t t e m p t e d . S t r e s s r e s p o n s e s i n f r y R e s u l t s o f t h e f i r s t e x p e r i m e n t d e m o n s t r a t e d t h a t s m a l l s t e e l h e a d t r o u t (2-16 grams) a r e p h y s i o l o g i c a l l y a f f e c t e d by r e a r i n g d e n s i t y . As d e s c r i b e d e a r l i e r , d e n s i t y e f f e c t s a t t h e h i g h t e m p e r a t u r e (15°C) were s t a t i s t i c a l l y c l e a r . At 12.5°C, s i m i l a r e f f e c t s , a l t h o u g h not s t a t i s t i c a l l y s i g n i f i c a n t , were o b s e r v e d . However t h e c o n s i s t e n c y i n t h e o b s e r v e d p a t t e r n s s t r o n g l y s u g g e s t t h a t d e n s i t y d i d have e f f e c t s s i m i l a r t o t h o s e a t 15°C. P e r h a p s a t l o w e r t e m p e r a t u r e s d e n s i t y t o l e r a n c e s may be h i g h e r , or i f r e s p o n s e s t o d e n s i t y a r e r e l a t e d t o m e t a b o l i c r a t e , t h e n more t i m e may be r e q u i r e d f o r c e r t a i n p h y s i o l o g i c a l c o n s e q u e n c e s t o be m a n i f e s t e d . The d i f f e r e n t d e n s i t y r e s p o n s e s c o u l d not be a s c r i b e d t o d i f f e r e n c e s i n swimming a c t i v i t y a t t h e two t e m p e r a t u r e s . Growth r a t e s were a f f e c t e d by d e n s i t y d u r i n g an i n i t i a l s h o r t p e r i o d ( l e s s t h a n 2 w e e k s ) . A f t e r t h e f i r s t few weeks t h e r e were no l o n g e r s t a t i s t i c a l d i f f e r e n c e s between t r e a t m e n t s . Such s h o r t t e r m d e n s i t y e f f e c t s a r e not m e n t i o n e d i n t h e l i t e r a t u r e p r i o r t o 1981. R e c e n t l y however, B i r k s e t a l . (1981) d e s c r i b e d s i m i l a r s h o r t - d u r a t i o n d e n s i t y e f f e c t s on g r o w t h i n e x p e r i m e n t s w i t h c h i n o o k salmon. P e r h a p s t h e s h o r t d u r a t i o n of d e n s i t y e f f e c t s s h o u l d not be s u r p r i s i n g , s i n c e Wedemeyer (1976) f o u n d t h a t b o t h coho and s t e e l h e a d a d a p t (as measured by 2 b l o o d 134 v a r i a b l e s ) t o h i g h d e n s i t i e s w i t h i n 2-3 weeks. F u r t h e r m o r e , S c h r e c k (1981) p o i n t e d o u t t h a t c o m p l e t e a d a p t a t i o n ( i n t e r m s o f C o r t i s o l l e v e l s ) t o some t y p e s o f c h r o n i c s t r e s s (not i n c l u d i n g c r o w d i n g ) c a n o c c u r w i t h i n a few weeks ( 1 - 3 ) . I f r e d u c e d g r o w t h r e s u l t i n g f r o m h i g h d e n s i t y r e a r i n g i s i n d i c a t i v e o f s t r e s s , t h e n my r e s u l t s d e m o n s t r a t e a c l e a r s t r e s s r e s p o n s e ( T a b l e 4, F i g u r e s 1,2). Many a u t h o r s have d e m o n s t r a t e d d e n s i t y - i n d u c e d g r o w t h s u p p r e s s i o n i n h a t c h e r i e s ( F a g e r l u n d e t a l . 1981; T r z e b i a t o w s k i e t a l . 1981; R e f s t i e and K i t t l e s o n 1976; R e f s t i e 1977; Brauhn e t a l . 1976; Andrews e t a l . 1971). None have examined t e m p o r a l c h a n g e s i n g r o w t h . The a b s e n c e o f 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 l a s m a C o r t i s o l l e v e l s between t r e a t m e n t s a p p e a r s t o i n d i c a t e t h a t d e n s i t y e f f e c t s were of s h o r t d u r a t i o n . However, as S c h r e c k (1981) p o i n t s o u t , some i n d i c a t o r s i n c l u d i n g p l a s m a C o r t i s o l show a d a p t a t i o n under c o n d i t i o n s of c h r o n i c s t r e s s . F u r t h e r m o r e H i l l and Fromm (1968) o b s e r v e d t h a t f o r some forms o f s t r e s s e l e v a t i o n s i n p l a s m a c o r t i c o s t e r o i d s a r e t r a n s i t o r y . U n f o r t u n a t e l y few s t u d i e s have f o l l o w e d t h e C o r t i s o l r e s p o n s e t o c r o w d i n g s t r e s s under h a t c h e r y c o n d i t i o n s . A l s o , a d a p t a t i o n t o c h r o n i c s t r e s s c o u l d i n v o l v e b e h a v i o r a l as w e l l a s p h y s i o l o g i c a l mechanisms. E j i k e and S c h r e c k (1980) n o t e d t h a t c o n d i t i o n i n g a l t e r s s t r e s s r e s p o n s e t o h a n d l i n g i n s a l m o n i d s . P o s s i b l y s a l m o n i d s c a n become c o n d i t i o n e d t o o t h e r t y p e s o f s t r e s s , i n c l u d i n g h i g h d e n s i t y r e a r i n g . I f , as t h e C o r t i s o l d a t a s u g g e s t s , c o m p l e t e r e c o v e r y f r o m t h e e f f e c t s of d e n s i t y - i n d u c e d s t r e s s had o c c u r r e d by t h e end of 135 t h e e x p e r i m e n t , and by t h e end o f t h e f i r s t week or so a c c o r d i n g t o g r o w t h s l o p e d a t a , t h e n v a r i o u s o t h e r i n d i c a t o r s of m e t a b o l i c s t a t e ( a n d s t r e s s ) a l s o s h o u l d show no d i f f e r e n c e s . T h i s was n o t t h e c a s e . B o t h p r o x i m a t e c o m p o s i t i o n and c o n d i t i o n f a c t o r d a t a r e f l e c t p e r s i s t e n t d e n s i t y e f f e c t s . F u r t h e r m o r e r e a r i n g d e n s i t y a l s o i n d u c e d c h a n g e s i n b e h a v i o r ( a c t i v i t y ) . The e x a c t c o n s e q u e n c e s of d i f f e r e n c e s i n t h e p h y s i o l o g i c a l i n d i c a t o r s o f s t r e s s ( p r o x i m a t e c o m p o s i t i o n , c o n d i t i o n , e t c . ) , i n t e r m s of s u r v i v a l and u l t i m a t e f i t n e s s , a r e not w e l l u n d e r s t o o d . However i t i s c l e a r t h a t d i f f e r e n t r e a r i n g d e n s i t i e s p r o d u c e d m e a s u r a b l e p h y s i o l o g i c a l c h a n g e s i n t h e f i s h , and t h u s a l t e r e d t i s s u e g r o w t h and e n e r g y m e t a b o l i s m . A s s o c i a t e d w i t h i n c r e a s e s i n d e n s i t y were e l e v a t e d m o i s t u r e and p r o t e i n l e v e l s and r e d u c t i o n s i n l i p i d c o n t e n t . F a g e r l u n d e t a l . (1981) a l s o o b s e r v e d s i g n i f i c a n t e l e v a t i o n s i n m o i s t u r e and r e d u c t i o n s i n l i p i d and a n o n - s i g n i f i c a n t t r e n d t o w a r d s r e d u c t i o n s i n p r o t e i n c o n t e n t a t h i g h d e n s i t i e s . They s u g g e s t t h a t r e d u c e d l i p i d r e s e r v e s c o u l d s i g n i f y r e d u c e d c a p a b i l i t i e s f o r s u s t a i n e d a c t i v i t y and p r e d a t o r a v o i d a n c e . Lee and L a y c o c k (1978) o b s e r v e d t h a t i m p a i r e d l i p i d m e t a b o l i s m i s a s s o c i a t e d w i t h e l e v a t e d c o r t i c o s t e r o i d s i n mammals. Burrows (1969) s u g g e s t e d t h a t l i p i d r e s e r v e s of 8% (32% o f d r y body w e i g h t a s s u m i n g 75% m o i s t u r e ) a r e o p t i m a l f o r s u r v i v a l of r e l e a s e d f i n g e r l i n g s . L i and B r o c k s e n (1977) and L i (1973) o b s e r v e d r e d u c t i o n s i n l i p i d l e v e l s i n s u b o r d i n a t e r a i n b o w t r o u t r e a r e d a t h i g h d e n s i t i e s . Noakes and L e a t h e r l a n d (1977) d e m o n s t r a t e d c l e a r s t r e s s r e s p o n s e s i n r a i n b o w t r o u t a s s o c i a t e d w i t h 136 h e i r a r c h i e s and f o u n d t h i s r e f l e c t e d i n l i p i d r e s e r v e s . T h e s e s t u d i e s s u g g e s t t h a t t h e t i s s u e c h a n g e s w h i c h accompany h i g h d e n s i t y r e a r i n g may r e d u c e c h a n c e s of s u r v i v a l . The s i g n i f i c a n c e o f i n c r e a s e d p r o t e i n l e v e l s a t h i g h d e n s i t i e s i s o b s c u r e . In s e v e r e c a s e s o f s t r e s s i n mammals a n e g a t i v e n i t r o g e n b a l a n c e d e v e l o p s and t h i s can l e a d t o m u s c l e w a s t i n g -(Lee and L a y c o c k 1978). Such a r e s p o n s e may be a d a p t i v e i n s t a r v i n g a n i m a l s but i t i s d i f f i c u l t t o comprehend i n t h i s c a s e . In f i s h , under crowded c o n d i t i o n s but w i t h e x c e s s f o o d , i t a p p e a r s t h a t t h e e f f i c i e n c y of l i p i d e n e r g y s t o r a g e i s r e d u c e d . T h i s may have c o n t r i b u t e d t o t h e o b s e r v e d r e l a t i v e i n c r e a s e i n body p r o t e i n . Even t h o u g h f o o d may be p r o v i d e d i n e x c e s s a t h i g h d e n s i t i e s , f o o d c o n v e r s i o n and g r o w t h e f f i c i e n c i e s may be r e d u c e d ( F a g e r l u n d e t a l . 1981; L i and B r o c k s e n 1977; Andrews e t a l . 1971). H i g h c o n d i t i o n f a c t o r s a r e sometimes a s s o c i a t e d w i t h i n c r e a s e d p h y s i c a l f i t n e s s and a r e r o u t i n e l y c a l c u l a t e d i n most h a t c h e r i e s a s a means of a s s e s s i n g t h e g e n e r a l h e a l t h of t h e f i s h . The a s s u m p t i o n i s t h a t f i s h o f a g i v e n l e n g t h w i t h a h i g h c o n d i t i o n f a c t o r have g r e a t e r e n e r g y r e s e r v e s t h a n f i s h w i t h a low c o n d i t i o n f a c t o r . In r a i n b o w t r o u t f e d t o e x c e s s R e f s t i e (1977) n o t e d d e c r e a s e d c o n d i t i o n f a c t o r s a t h i g h d e n s i t i e s . He a t t r i b u t e d t h i s t o r e d u c e d f o o d i n t a k e by s m a l l f i s h ( s u b o r d i n a t e s ) . A f t e r s i x months of r e a r i n g o v e r a 2 - f o l d d e n s i t y r a n g e F a g e r l u n d e t a l . (1981) f o u n d no e v i d e n c e o f c o n d i t i o n d i f f e r e n c e s i n coho s a l m o n . However, f o u r months l a t e r , s i g n i f i c a n t d i f f e r e n c e s were f o u n d . C o n d i t i o n f a c t o r s 137 were l o w e r a t t h e h i g h d e n s i t i e s . S i n c e f i s h were r e c e i v i n g r e d u c e d r a t i o n s f o r much o f t h i s l a s t p e r i o d , c o m p e t i t i o n f o r f o o d may have c o n t r i b u t e d t o t h e d e v e l o p m e n t o f h e i r a r c h i e s a t t h e h i g h e r d e n s i t i e s , and t h e s e i n t u r n r e s u l t e d i n u n f a v o u r a b l e g r o w t h c o n d i t i o n s f o r much o f t h e p o p u l a t i o n . My f i r s t e x p e r i m e n t d e m o n s t r a t e d t h a t c o n d i t i o n f a c t o r s were r e d u c e d a t h i g h d e n s i t i e s , even when f i s h were f e d e x c e s s r a t i o n s . Growth r a t e and body c o n d i t i o n were c l o s e l y c o r r e l a t e d . Under c o n d i t i o n s of r a p i d g r o w t h c o n d i t i o n f a c t o r s were h i g h . T h i s s u g g e s t s t h a t d u r i n g p e r i o d s o f good g r o w t h f i s h i n c r e a s e e n e r g y r e s e r v e s . D e n s i t y r e l a t e d d i f f e r e n c e s i n c o n d i t i o n were marked d u r i n g t h e f i r s t t i m e p e r i o d . I n t e r p r e t a t i o n of t h e b e h a v i o r d a t a was d i f f i c u l t . S i n c e t h e o b s e r v e d p a t t e r n s were c o n s i s t e n t and c l e a r a t b o t h t e m p e r a t u r e s , i t a p p e a r s l i k e l y t h a t t h e y were r e a l c o n s e q u e n c e s of d e n s i t y . A t b o t h t e m p e r a t u r e s , n e t a c t i v i t y l e v e l s were g r e a t e s t a t t h e m i d d l e d e n s i t y and l o w e s t a t t h e l o w e s t d e n s i t y . T h i s i n d i c a t e s t h a t a t t h e l o w e s t d e n s i t y f i s h m a i n t a i n e d p o s i t i o n s most r i g i d l y . T o t a l a c t i v i t y ( t o t a l d i s t a n c e moved i n a g i v e n t i m e ) d e c r e a s e d as d e n s i t i e s i n c r e a s e d . A v a i l a b l e s p a c e p e r f i s h may have l i m i t e d t o t a l movement. T h e r e a r e no s t u d i e s r e p o r t e d i n t h e l i t e r a t u r e o f a c t i v i t y l e v e l s o f s a l m o n i d s r e a r e d a t h i g h d e n s i t i e s u nder h a t c h e r y c o n d i t i o n s . Q u a n t i t a t i v e measures o f a g g r e s s i o n were not c o l l e c t e d , and t h e r e a r e no r e p o r t e d s t u d i e s on a g g r e s s i o n i n f i s h a t h a t c h e r y d e n s i t i e s . F o r t h i s r e a s o n t h e f o l l o w i n g d i s c u s s i o n i s e n t i r e l y s p e c u l a t i v e . H i g h t o t a l a c t i v i t y l e v e l s , c o m b i n e d w i t h p r e c i s e 1 38 s t a t i o n m a i n t e n a n c e a t low d e n s i t i e s , may i n d i c a t e t e r r i t o r i a l b e h a v i o r . H i g h a c t i v i t y l e v e l s may be t h e r e s u l t o f p e r i o d i c , s h o r t - d u r a t i o n c h a s e s e q u e n c e s . Some a c t s o f a g g r e s s i o n were v i s i b l e a t t h e low d e n s i t y ; however, t h e s e were n o t q u a n t i f i e d s i n c e a g g r e s s i v e a c t s c o u l d not be r e l i a b l y e s t i m a t e d a t t h e h i g h e r d e n s i t i e s . A t t h e m i d d l e d e n s i t y , p o s i t i o n m a i n t e n a n c e was l e a s t p r e c i s e , and t o t a l a c t i v i t y l e v e l s were d e c r e a s e d . T h i s c o u l d r e f l e c t c o n t i n u e d a t t e m p t s t o d e f e n d t e r r i t o r i e s u n d e r c o n d i t i o n s where n e a r e s t n e i g h b o u r d i s t a n c e i s s m a l l and, t h e r e f o r e , p e r c e i v e d i n t r u d e r e n c o u n t e r r a t e may be e l e v a t e d . T h i s may r e s u l t i n an i n c r e a s e i n d e f e n c e t i m e , but i n a l o c a l i z e d a r e a . At t h e h i g h e s t d e n s i t i e s , i n t r u d e r p r e s s u r e may have been so h i g h t h a t t e r r i t o r i a l b e h a v i o r was i n h i b i t e d , and f i s h p a s s i v e l y m a i n t a i n e d p o s i t i o n . I f t h e r e l a t i v e l y low n e t and t o t a l a c t i v i t y l e v e l s a t t h e h i g h e s t d e n s i t y i n d i c a t e t h e a b s e n c e o r r e d u c t i o n o f a g o n i s t i c b e h a v i o r t h e n t h e s e r e s u l t s a g r e e w i t h t h o s e of Magnuson (1962) and Kawanabe ( 1 9 6 9 ) . A n o t h e r use o f t h e s e m e a s u r e s , was t o o b t a i n a q u a l i t a t i v e e s t i m a t e o f t h e e n e r g y e xpended i n swimming a c t i v i t y , s i n c e s u c h e x p e n d i t u r e s , i f h i g h enough, c o u l d l i m i t g r o w t h . M i n c h e n (1972) f o u n d t h a t i n A. p u l c h e r , as a b s o l u t e s p a c e i n c r e a s e d a c t i v i t y i n c r e a s e d . E n e r g y e xpended on swimming was g i v e n as a c a u s e o f r e d u c e d g r o w t h i n l a r g e c o n t a i n e r s . L i and B r o c k s e n (1977) f o u n d no s u c h e f f e c t i n r a i n b o w t r o u t . I n my e x p e r i m e n t s g r o wth d i f f e r e n c e s c a n n o t be a t t r i b u t e d t o a c t i v i t y d i f f e r e n c e s , s i n c e t h e most a c t i v e f i s h grew f a s t e s t . 1 39 A l t h o u g h t h e s i g n i f i c a n c e o f t h e o b s e r v e d p a t t e r n s i n b e h a v i o r a r e u n c l e a r , t h e y do d e m o n s t r a t e t h a t r e a r i n g c o n d i t i o n s a f f e c t b e h a v i o r . As n o t e d e a r l i e r , s e v e r a l a u t h o r s have d e s c r i b e d d i f f e r e n c e s i n t h e b e h a v i o r o f w i l d and h a t c h e r y -r e a r e d s a l m o n i d s i n s t r e a m s . P e r h a p s a l t e r e d h a t c h e r y r e a r i n g s t r a t e g i e s c o u l d be u s e d t o b e n e f i c i a l l y i n f l u e n c e t h e b e h a v i o r o f r e l e a s e d f i s h . S t r e s s r e s p o n s e s i n f i n g e r l i n g s T h i s e x p e r i m e n t was d e s i g n e d t o answer two q u e s t i o n s : f i r s t do l a r g e r t r o u t ( t h a n t e s t e d i n t h e f i r s t e x p e r i m e n t ) r e s p o n d s i m i l a r l y t o d e n s i t y s t r e s s ; s e c o n d l y , s i n c e d e n s i t y e f f e c t s on g r o w t h a r e o f s h o r t d u r a t i o n and t h e g r o w t h p a t t e r n i s c u r v i l i n e a r ( b o t h i n d i c a t i v e o f a c o n d i t i o n i n g e f f e c t ) what i s t h e e f f e c t o f sudden c h a n g e s i n d e n s i t y ? McDonald e t a l . (1968) s u g g e s t e d t h a t p r i o r e x p e r i e n c e may be i m p o r t a n t i n d e t e r m i n i n g b e h a v i o r a l r e a c t i o n s i n f i s h . P e r h a p s p r i o r e x p e r i e n c e a l s o i n f l u e n c e s p h y s i o l o g i c a l r e a c t i o n s . The r e s u l t s d e m o n s t r a t e d t h a t j u v e n i l e s t e e l h e a d t r o u t , 15-30 grams i n w e i g h t , were p h y s i o l o g i c a l l y a f f e c t e d by r e a r i n g d e n s i t y . Growth r a t e , p r o x i m a t e c o m p o s i t i o n and c o n d i t i o n f a c t o r d a t a s u p p o r t e d t h i s v i e w . In t h o s e t r e a t m e n t s where volum e s were not a d j u s t e d d e n s i t i e s were no t h i g h enough t o e l i c i t a g r o w t h r e s p o n s e . When a l l t r e a t m e n t s were compared t h e r e was e v i d e n c e o f g r o w t h s u p p r e s s i o n a t h i g h d e n s i t i e s , and t h e d e g r e e o f g r o w t h s u p p r e s s i o n s u g g e s t s a t h r e s h h o l d e f f e c t . In t h e l i t e r a t u r e , 1 40 e v i d e n c e f o r t h r e s h h o l d d e n s i t y e f f e c t s i s r a r e a l t h o u g h Brown (1946a) f o u n d t h a t i n t e r m e d i a t e d e n s i t i e s p r o d u c e d o p t i m a l g r o w t h i n t a n k - r e a r e d brown t r o u t f r y and f i n g e r l i n g s . Growth r a t e s i n t h e s e c o n d s t u d y f o l l o w e d a c u r v i l i n e a r p a t t e r n w i t h t i m e , s i m i l a r t o t h a t i n t h e f i r s t e x p e r i m e n t , and a g a i n s u g g e s t a c o n d i t i o n i n g e f f e c t . As i n t h e r e s p o n s e t o d e n s i t y i n t h e f i r s t e x p e r i m e n t , d e n s i t y e f f e c t s were g r e a t e s t i n t h e f i r s t t i m e i n t e r v a l . However, t h e m a g n i t u d e of t h e r e s p o n s e d u r i n g t h i s i n t e r v a l was s m a l l e r . U n l i k e t h e f i r s t e x p e r i m e n t , t h e e f f e c t of d e n s i t y on g r o w t h p e r s i s t e d o v e r t i m e . No o t h e r s t u d i e s have d e m o n s t r a t e d a m o d e r a t i n g i n f l u e n c e o f f i s h s i z e on d e n s i t y r e s p o n s e i n s a l m o n i d s . To d e t e c t d e n s i t y - c o n d i t i o n i n g or a c c l i m a t i o n e f f e c t s , o b s e r v e d g r o w t h r a t e s were compared w i t h e x p e c t e d r a t e s a f t e r v o l u m e s ( i . e . d e n s i t i e s ) had been c h a n g e d . T h e r e was some c o n c e r n t h a t t a n k volume may have i n f l u e n c e d t h e r e s u l t s , however, L i and B r o c k s e n (1977) and Brown (1946b) f o u n d no e f f e c t s o f volume i n t h e i r s t u d i e s . O n l y one of t h e f o u r c o m p a r i s o n s p r o v e d s i g n i f i c a n t . T h a t i s , o n l y i n one t r e a t m e n t was t h e r e s p o n s e t o c h a n g i n g d e n s i t i e s g r e a t e r t h a n t h a t w h i c h would be e x p e c t e d on t h e b a s i s o f d e n s i t y a l o n e . In t h i s c o m p a r i s o n , a d o u b l i n g o f d e n s i t i e s i n t a n k s c o n t a i n i n g 200 f i s h , r e s u l t e d i n a l a r g e r e d u c t i o n i n g r o w t h r a t e . In f a c t , f i s h i n t a n k s i n w h i c h d e n s i t i e s were d o u b l e d , began g r o w i n g a t a r a t e l e s s t h a n t h a t of some o t h e r t a n k s w i t h h i g h e r f i s h d e n s i t i e s . As i n t h e f i r s t e x p e r i m e n t t h e r e was no e v i d e n c e of s t r e s s 141 f r o m t h e C o r t i s o l d a t a . F o r r e a s o n s d e s c r i b e d e a r l i e r t h i s was n o t s u r p r i s i n g a l t h o u g h i n t h i s s t u d y , a t t h e t i m e o f s a m p l i n g , t h e r e was e v i d e n c e o f d e n s i t y - i n d u c e d , s t r e s s - r e l a t e d d i f f e r e n c e s i n g r o w t h r a t e s . T h i s f i n d i n g p r o v i d e s f u r t h e r e v i d e n c e t h a t t h e use o f p l a s m a C o r t i s o l a s an i n d i c a t o r o f c h r o n i c s t r e s s i s o f l i m i t e d v a l u e . A l t h o u g h p l a s m a C o r t i s o l , a p r i m a r y l e v e l s t r e s s i n d i c a t o r , d i d n o t i n d i c a t e a s t r e s s r e s p o n s e , t h e r e were c h a n g e s i n p r o x i m a t e c o m p o s i t i o n , a t e r t i a r y l e v e l i n d i c a t o r . The e f f e c t s , w h i l e l e s s p r o n o u n c e d t h a n t h o s e o f t h e f i r s t e x p e r i m e n t , d i d show s i m i l a r t r e n d s . The l a c k o f c l e a r r e s u l t s was p r o b a b l y due t o t h e e f f e c t s o f s udden d e n s i t y c h a n g e s , a n d a l s o t h e s m a l l e r r e l a t i v e c h a n g e i n w e i g h t t h a n t h a t w h i c h o c c u r r e d i n t h e f i r s t e x p e r i m e n t . W i t h r e s p e c t t o e n e r g y m e t a b o l i s m , i t a p p e a r s t h a t j u v e n i l e s t e e l h e a d t r o u t (2-30 g r a m s ) , r e s p o n d s i m i l a r l y t o d e n s i t y s t r e s s . Under h i g h d e n s i t i e s l i p i d r e s e r v e s , t h e most i m p o r t a n t e n e r g y s o u r c e f o r s u s t a i n e d e x e r c i s e , a r e c o m p r o m i s e d . D i f f e r e n c e s i n t h e r e s p o n s e o f body c o n d i t i o n t o d e n s i t y were o b s e r v e d b e t w e e n t h e s e c o n d and t h e f i r s t e x p e r i m e n t . A l t h o u g h s i g n i f i c a n t d i f f e r e n c e s b e t w e e n d e n s i t i e s were f o u n d i n t h i s e x p e r i m e n t , t h e t r e n d was i n t h e o p p o s i t e d i r e c t i o n t o t h e f i r s t e x p e r i m e n t . I n a d d i t i o n , t h e r e were no c l e a r p a t t e r n s w i t h t i m e i n " e i t h e r body c o n d i t i o n o r g r o w t h s l o p e s a s had been o b s e r v e d i n t h e f i r s t s t u d y . T h e s e f i n d i n g s s u g g e s t t h a t c o n d i t i o n f a c t o r may n o t a l w a y s p r o v i d e an a c c u r a t e a s s e s s m e n t of t h e w e l l - b e i n g o f a g r o u p o f f i s h . 142 S t r e s s r e s p o n s e s i n s m o l t s and p r e s m o l t s I n t e r p r e t a t i o n o f t h e r e s u l t s o f t h e f i n a l g r o u p o f e x p e r i m e n t s was c o m p l i c a t e d by t h e f a c t t h a t n o r m a l g r o w t h p r o c e s s e s and r e s p o n s e s t o d e n s i t y may have been m o d e r a t e d by s m o l t i f i c a t i o n . T a b l e 13 p r o v i d e s a l i s t o f some c h a n g e s t h a t o c c u r d u r i n g s m o l t i f i c a t i o n . Some o f t h e v a r i a b l e s u s e d t o a s s e s s s t r e s s a l s o change d u r i n g t h e t r a n s f o r m a t i o n from p a r r t o s m o l t . In p a r t i c u l a r , c o n d i t i o n f a c t o r and whole body l i p i d c o n t e n t t e n d t o d e c r e a s e , whereas g r o w t h r a t e s i n c r e a s e ( F o l m a r and D i c k h o f f 1980; Hoar 1976). In a d d i t i o n , t h e c a p a c i t y t o o s m o r e g u l a t e i n a h y p e r t o n i c medium i n c r e a s e s ( K o m o u r j d i a n e t a l . 1976a; Wagner 1974a). R e s u l t s of t h e f i r s t s m a l l - s c a l e e x p e r i m e n t (3a) were complex. Growth r a t e s , c o n d i t i o n f a c t o r s , and p l a s m a C o r t i s o l l e v e l s s u g g e s t e d no d i f f e r e n c e s i n s t r e s s r e s p o n s e between t r e a t m e n t s . However, body c o m p o s i t i o n and h y p e r - o s m o r e g u l a t o r y c a p a b i l i t y s u g g e s t e d o t h e r w i s e . T r o u t (30-60 grams) r e a r e d o v e r an e i g h t - f o l d d e n s i t y r a n g e showed no d i f f e r e n c e s i n g r o w t h r a t e s . T h i s s u g g e s t s t h a t l a r g e r t r o u t ( n e a r s m o l t s i z e ) a r e u n a f f e c t e d by r e a r i n g d e n s i t i e s up t o t h r e e t i m e s c o n v e n t i o n a l h a t c h e r y l e v e l s . T h e r e a r e no s i m i l a r f i n d i n g s i n t h e f i s h c u l t u r e l i t e r a t u r e ; however, i n p r i n c i p l e , t h e s e f i n d i n g s a g r e e w i t h t h o s e of Kawanabe (1969) and Magnuson ( 1 9 6 2 ) . C o r t i s o l l e v e l s were u n a f f e c t e d by r e a r i n g d e n s i t y . -However, t h e r e was a d e c l i n e w i t h t i m e t o common l e v e l s i n a l l t r e a t m e n t s . I f f i s h were s t r e s s e d , a d a p t a t i o n may have o c c u r r e d . 1 43 I f t h i s was t h e c a s e t h e n , d u r i n g t h e p e r i o d o f s m o l t i f i c a t i o n , a t i m e s e n s i t i v e t o r e a r i n g s t r e s s ( S c h r e c k 1981; F o l m a r and D i c k h o f f 1980), C o r t i s o l s h o u l d not be u s e d t o a s s e s s c h r o n i c s t r e s s . As m e n t i o n e d e a r l i e r , p r o x i m a t e c o m p o s i t i o n p a t t e r n s were not c l e a r . However, b o t h a s h and m o i s t u r e c o n t e n t were h i g h e r a t h i g h d e n s i t i e s . P r o t e i n l e v e l s were u n a f f e c t e d , and l i p i d s showed a t e n d e n c y t o d e c l i n e as d e n s i t y i n c r e a s e d . T h e s e f i n d i n g s compare f a v o u r a b l y w i t h t h o s e of F a g e r l u n d e t a l . ( 1 9 8 1 ) , but a r e d i f f e r e n t from t h o s e of my f i r s t two e x p e r i m e n t s i n t h a t p r o t e i n l e v e l s d i d n o t i n c r e a s e w i t h d e n s i t y . I f i n c r e a s e d m o i s t u r e l e v e l s i n d i c a t e r e d u c e d a b i l i t y t o s u r v i v e , t h e n h i g h d e n s i t y r e a r i n g c a u s e d s t r e s s . I n t e r p r e t a t i o n of l i p i d d a t a i s d i f f i c u l t . R e d u c t i o n s i n l i p i d c o n t e n t a r e a s s o c i a t e d w i t h s m o l t i f i c a t i o n (Hoar 1976; K o m o u r d j i a n e t a l . 1976b; V a n s t o n e and M a r k e r t 1968). T h e r e f o r e , r e d u c e d l e v e l s a t h i g h d e n s i t i e s c o u l d be a t t r i b u t e d t o e i t h e r a s t r e s s - r e l a t e d r e d u c t i o n , or° a more a d v a n c e d s t a t e o f s m o l t i f i c a t i o n . Burrows (1969) f o u n d t h a t s m o l t s u r v i v a l i n c r e a s e d as l i p i d l e v e l s i n c r e a s e d . C l e a r l y , t r e n d s i n l i p i d s must be u s e d w i t h c a u t i o n as an i n d i c a t o r of e i t h e r s m o l t r e a d i n e s s or s t r e s s r e s p o n s e i n d e n s i t y e x p e r i m e n t s , e s p e c i a l l y i f t h e d a t a a r e t a k e n n e a r t h e t i m e of s m o l t i f i c a t i o n . A l t h o u g h th e s i g n i f i c a n c e of t h e a l t e r e d body c o m p o s i t i o n i s u n c l e a r , i t does i m p l y c h a n g e s i n m e t a b o l i s m a s s o c i a t e d w i t h r e a r i n g d e n s i t y . T h i s s u g g e s t s t h a t under c e r t a i n c o n d i t i o n s g r o w t h r a t e s a r e i n a d e q u a t e i n d i c a t o r s o f s t r e s s . B i r k s e t a l . ( 1 9 8 1 ) , 1 44 s t u d y i n g g r o w t h o f c h i n o o k s a l m o n , r e a c h e d s i m i l a r c o n c l u s i o n s . In d e n s i t y e x p e r i m e n t s , t h e y f o u n d g r o w t h s u p p r e s s i o n t o be s h o r t - l i v e d . A f t e r s e v e r a l months of a p p a r e n t l y s t r e s s f r e e g r o w t h ( i . e . e q u a l growth) f i s h w h i c h had been r e a r e d a t h i g h d e n s i t i e s had r e d u c e d o s m o r e g u l a t o r y c a p a b i l i t y . C o n d i t i o n f a c t o r s showed no e v i d e n c e o f d e n s i t y e f f e c t s . However, t h e y d i d f o l l o w a p a t t e r n s i m i l a r t o g r o w t h r a t e s . As w i t h t h e p r o x i m a t e a n a l y s i s d a t a , i n t e r p r e t a t i o n o f t h e c h a n g e s i n c o n d i t i o n d u r i n g t h i s p e r i o d i s d i f f i c u l t . Many a u t h o r s d e s c r i b e r e d u c t i o n s i n c o n d i t i o n ( i n c r e a s e d s t r e a m l i n i n g ) d u r i n g s m o l t i f i c a t i o n ( F o l m a r and D i c k h o f f 1980; K o m o u r d j i a n e t a l . 1976a; Wagner 1974b; Hoar 1976). I n d e e d , Wagner c o n s i d e r e d r e d u c t i o n i n c o n d i t i o n f a c t o r s t o be a s a t i s f a c t o r y means of a s s e s s i n g s m o l t s t a t u s . C l e a r l y t h e e f f e c t s of s t r e s s were n o t m a n i f e s t e d i n c h a n g e s i n c o n d i t i o n f a c t o r i n my d a t a , and t h e r e f o r e a p p e a r t o be of l i m i t e d v a l u e as a means of a s s e s s i n g s t r e s s under t h e s e c o n d i t i o n s . F u r t h e r m o r e , t h e i r v a l u e i n a s s e s s i n g s m o l t s t a t u s a p p e a r s l i m i t e d s i n c e t h e y d i d n o t i n d i c a t e d i f f e r e n c e s i n s t r e a m l i n i n g , y e t d i f f e r e n c e s i n s a l t w a t e r r e a d i n e s s were d e t e c t e d . T h e r e were no t r e a t m e n t - r e l a t e d d i f f e r e n c e s i n e i t h e r n e t or t o t a l a c t i v i t y l e v e l s . E n e r g y e x p e n d i t u r e d i f f e r e n c e s c o u l d n o t be a t t r i b u t e d t o a c t i v i t y . However, t h e p a t t e r n w i t h t i m e was i n t e r e s t i n g . As d e s c r i b e d e a r l i e r , b o t h m e asures o f a c t i v i t y i n c r e a s e d e v e n l y f r o m day 31 t o day 59 (3 i n t e r v a l s ) , and t h e n s h a r p l y d e c l i n e d . I t seems p r o b a b l e t h a t t h e i n c r e a s e may have been a s s o c i a t e d w i t h t h e p r o c e s s of s m o l t i f i c a t i o n . S e v e r a l 1 45 r e v i e w s (Wedemeyer e t a l . 1981; S c h r e c k 1981b; F o l m a r and D i c k h o f f 1980), n o t e i n c r e a s e s i n a c t i v i t y o f s m o l t s and an a s s o c i a t e d t e n d a n c y t o s c h o o l . M i g r a t o r y u r g e a l s o i n c r e a s e s . I f t h e i n c r e a s e d a c t i v i t y o b s e r v e d i n my d a t a i s a p a r t o f t h e s m o l t i f i c a t i o n p r o c e s s , t h e n a l l g r o u p s were e q u a l l y p r e p a r e d . The d e c l i n e i n a c t i v i t y on t h e l a s t d a t e may i n d i c a t e t h a t t h e p e r i o d of optimum s m o l t r e a d i n e s s had p a s s e d . C o n t e and Wagner (1965) and Wagner (1974b) n o t e d t h a t t h e s m o l t t r a n s f o r m a t i o n i n s t e e l h e a d i s t r a n s i e n t . The r e s u l t s of t h e s a l t water c h a l l e n g e t e s t s u g g e s t e d d i f f e r e n c e s , a t t r i b u t a b l e t o t r e a t m e n t e f f e c t s , i n t h e a b i l i t y o f s m o l t s t o s u r v i v e . A t low d e n s i t i e s , p l a s m a sodium l e v e l s were r e g u l a t e d t o 165 meq/1 a f t e r 24 h o u r s , s l i g h t l y above t h e optimum of 150-160 ( C l a r k e and B l a c k b u r n 1978) but s t i l l below t h e a r b i t r a r y c u t - o f f v a l u e of 170 meq/1. Above t h i s l e v e l f i s h a r e c o n s i d e r e d l e s s c a p a b l e o f s u r v i v i n g i n s e a w a t e r . F i s h f r o m h i g h d e n s i t y t r e a t m e n t s r e g u l a t e d p l a s m a sodium l e v e l s l e s s e f f i c i e n t l y ( a v e r a g e 180 meq/1), i n d i c a t i n g t h a t h i g h d e n s i t y r e a r i n g imposed some d e g r e e o f s t r e s s . R e s u l t s o f t h e s e c o n d s m a l l - s c a l e e x p e r i m e n t (3b) c o n f i r m e d , i n p a r t , some o f t h e r e s u l t s f r o m s t u d y 3a. U n t i l v o l u m e s were c h a n g e d , d e n s i t y had not a f f e c t e d e i t h e r g r o w t h r a t e , c o n d i t i o n f a c t o r , p r o x i m a t e a n a l y s i s , o r p l a s m a C o r t i s o l . T h i s s u g g e s t s t h a t l a r g e f i s h a r e r e l a t i v e l y i n s e n s i t i v e t o r e a r i n g d e n s i t i e s . However, when volumes were c h a n g e d , f i s h i n t a n k s where d e n s i t i e s were d o u b l e d r e s p o n d e d w i t h a marked d e c r e a s e i n g r o w t h r a t e and a s l i g h t r e d u c t i o n i n body 1 46 c o n d i t i o n . The d e c r e a s e i n g r o w t h r a t e was g r e a t e r t h a n e x p e c t e d on t h e b a s i s of d e n s i t y a l o n e . F i s h i n t a n k s where d e n s i t i e s were h a l v e d d i d not show c h a n g e s i n any of t h e v a r i a b l e s t e s t e d . T h e s e r e s u l t s s u g g e s t t h a t f i s h become " c o n d i t i o n e d " t o t h e i r e n v i r o n m e n t , i n c l u d i n g r e a r i n g d e n s i t y , and t h a t sudden r e d u c t i o n s i n s p a c e p e r f i s h ( i n c r e a s e s i n d e n s i t y ) a r e more s t r e s s f u l t h a n i n c r e a s e s i n s p a c e p e r f i s h . T h a t sudden r e d u c t i o n s i n s p a c e p e r f i s h c a u s e d s t r e s s was e v i d e n t f r o m g r o w t h and c o n d i t i o n d a t a and f r o m plasma C o r t i s o l l e v e l s . Changes i n C o r t i s o l l e v e l s were s m a l l a l t h o u g h s t a t i s t i c a l l y s i g n i f i c a n t . S i n c e p l a s m a samples were c o l l e c t e d one week a f t e r volume c h a n g e s , p e r f e c t a d a p t a t i o n as d e s c r i b e d by S c h r e c k (1981) had n o t o c c u r r e d . T h e r e f o r e p l a s m a C o r t i s o l l e v e l s a p p e a r t o be u s e f u l i n d i c a t o r s o f c h r o n i c s t r e s s f o r a t l e a s t one week a f t e r t h e i m p o s i t i o n o f t h e s t r e s s . T h i s i n t e r p r e t a t i o n i s s u p p o r t e d by t h e f i n d i n g s o f Wedemeyer ( 1 9 7 6 ) . C o r t i s o l measurements c o l l e c t e d on day 74 c o n f i r m e d t h e t e m p o r a l d e c l i n e o b s e r v e d i n e x p e r i m e n t 3a p r o v i d i n g e v i d e n c e of a s e a s o n a l t r e n d . The l a c k of t r e a t m e n t e f f e c t s a t t h e l a s t s a m p l i n g d a t e , d e s p i t e t h e s i g n i f i c a n t d i f f e r e n c e s i n g r o w t h w h i c h p e r s i s t e d a f t e r t h e volume a d j u s t m e n t , i n d i c a t e t h a t C o r t i s o l l e v e l s do a d a p t and a f t e r a few weeks a r e not s e n s i t i v e t o c h r o n i c s t r e s s . In v i e w of t h e a b s e n c e of s i g n i f i c a n t d i f f e r e n c e s i n a c t i v i t y o v e r t h e e i g h t - f o l d d e n s i t y r a n g e i n s t u d y 3a, i t i s l i k e l y t h a t t h e a c t i v i t y d i f f e r e n c e s o b s e r v e d i n s t u d y 3b were a r t i f a c t s of volume d i f f e r e n c e s . At low d e n s i t i e s ( h i g h v o l u m e s ) 147 b o t h net and t o t a l a c t i v i t y l e v e l s were c o n s i s t e n t l y h i g h e r t h a n a t t h e h i g h d e n s i t y (low v o l u m e ) . However, t h e d e n s i t y r a n g e i n e x p e r i m e n t 3a b r a c k e t e d t h e r a n g e t e s t e d h e r e . F u r t h e r m o r e M i n c h e n (1972) f o u n d t h a t a c t i v i t y i n c r e a s e d as a b s o l u t e r e a r i n g volume i n c r e a s e d . In a d d i t i o n , q u a l i t a t i v e o b s e r v a t i o n s on a c t i v i t y l e v e l s i n s t u d y 3c ( d i f f e r e n t v o l umes, e q u a l d e n s i t i e s ) , s u g g e s t s t h a t i n c r e a s e d volumes i n d u c e d i n c r e a s e d a c t i v i t y . O n l y two t r e a t m e n t s were t e s t e d i n t h e s a l t w a ter c h a l l e n g e : t h o s e i n w h i c h volumes were unchanged, and t h o s e i n w h i c h v o l u m e s were h a l v e d ( d e n s i t i e s d o u b l e d ) . T h e r e f o r e , a t t h e t i m e of t h e t e s t , f i s h had r e c e n t l y been e x p o s e d t o d e n s i t i e s o f e i t h e r 0.5-0.6 o r 1.0-1.1 f i s h / 1 . T h o s e f i s h r e a r e d a t t h e l o w e r d e n s i t y had s i g n i f i c a n t l y b e t t e r o s m o r e g u l a t o r y p e r f o r m a n c e . T h e s e f i n d i n g a g r e e w i t h t h o s e of e x p e r i m e n t 3a and t h e r e s u l t s of B i r k s e t a l . ( 1 9 8 1 ) . R e s u l t s of t h e t h i r d s m a l l - s c a l e e x p e r i m e n t (3c) i n d i c a t e d t h a t r e a r i n g volumes had a s l i g h t , t h o u g h n o t s i g n i f i c a n t , e f f e c t on g r o w t h r a t e . T h i s i s i n c o n t r a s t t o t h e f i n d i n g s o f L i and B r o c k s e n (1977) and Brown ( 1 9 4 6 a ) . B e c a use t h e t r e n d was n o t s i g n i f i c a n t , i t i s u n l i k e l y t h a t volume e f f e c t s i n f l u e n c e d t h e r e s u l t s of o t h e r s t u d i e s . R e a r i n g volume had no e f f e c t on s a l t w a t er t o l e r a n c e . When f i s h f r o m e x p e r i m e n t 3c (14L:1OD p h o t o p e r i o d c y c l e ) were compared w i t h f i s h r e a r e d under s i m i l a r d e n s i t i e s f r o m e x p e r i m e n t s 3a and 3b (10L:14D p h o t o p e r i o d c y c l e ) , no d i f f e r e n c e s i n s a l t t o l e r a n c e were d e t e c t e d . T h i s i n d i c a t e s t h a t p h o t o p e r i o d e f f e c t s were u n i m p o r t a n t . F i s h r e a r e d 148 a t low v olumes had r e d u c e d c o n d i t i o n f a c t o r s . A l t h o u g h t h e y t e n d e d t o grow s l o w e r , t h e y had l o w e r p l a s m a C o r t i s o l l e v e l s t h a n f i s h r e a r e d under h i g h v o l u m e s . In summary, t h e s t r e s s - r e l a t e d c o n s e q u e n c e s o f r e a r i n g d e n s i t y on s t e e l h e a d t r o u t v a r y w i t h f i s h s i z e . I n e a c h o f t h e s t u d i e s d e s c r i b e d h e r e , t h e r e was e v i d e n c e f r o m some or most of t h e v a r i a b l e s t e s t e d , o f s t r e s s i n f i s h r e a r e d a t h i g h d e n s i t i e s . B o t h t h e n a t u r e and t h e a p p a r e n t d u r a t i o n o f t h e s t r e s s v a r i e d between e x p e r i m e n t s . D i f f e r e n t v a r i a b l e s o f t e n p r o d u c e d d i f f e r e n t c o n c l u s i o n s r e g a r d i n g e i t h e r t h e i n t e n s i t y o r t h e d u r a t i o n of t h e r e s p o n s e . I t i s c l e a r t h a t none of t h e v a r i a b l e s t e s t e d , when u s e d a l o n e , p r o v i d e an a c c u r a t e a s s e s s m e n t of t h e d e g r e e o f s t r e s s e x p e r i e n c e d by s t e e l h e a d t r o u t as a c o n s e q u e n c e o f h i g h d e n s i t y r e a r i n g , P r a c t i c a l l y , t h e s e r e s u l t s s u g g e s t t h a t t h e r e a r i n g d e n s i t i e s u s e d i n h a t c h e r i e s f o r s t e e l h e a d t r o u t c u l t u r e may be more c o n s e r v a t i v e t h a n n e c e s s a r y . On a c a r e f u l l y managed b a s i s , i t w ould a p p e a r r e a s o n a b l e t o e x p e r i m e n t a l l y i n c r e a s e r e a r i n g d e n s i t i e s i n some h a t c h e r i e s by 50 t o 100%. However, t o f u l l y u n d e r s t a n d t h e mechanisms by w h i c h d e n s i t y i n d u c e d s t r e s s a f f e c t s h a t c h e r y p r o d u c t i o n and s u r v i v a l o f t h e r e l e a s e d f i s h , a d d i t i o n a l , f u n d a m e n t a l r e s e a r c h a t b o t h b e h a v i o r a l and p h y s i o l o g i c a l l e v e l s i s r e q u i r e d . The s i g n i f i c a n c e t o f i s h o f d e n s i t y i n d u c e d c h a n g e s i n body c o m p o s i t i o n , g r o w t h r a t e , and b e h a v i o r , i s f a r f r o m c l e a r . F u r t h e r m o r e , t h e n a t u r e o f t h e c o n d i t i o n i n g e f f e c t i s u n c l e a r . L a s t l y , t h e r a t e a t w h i c h t h e " a d v e r s e " e f f e c t s of h i g h d e n s i t i e s a p p e a r as d e n s i t i e s 1 49 i n c r e a s e , and d i s a p p e a r as t h e y d e c r e a s e i s unknown, y e t t h i s knowledge c o u l d have c o n s i d e r a b l e management v a l u e . 1 50 REFERENCES CITED A l l e n , K.R. 1969. L i m i t a t i o n s on p r o d u c t i o n i n s a l m o n i d p o p u l a t i o n s i n s t r e a m s , pp. 3-18 I_n, T.G. N o r t h c o t e [ e d . ] Symposium on salmon and t r o u t i n s t r e a m s . H.R. M a c M i l l a n L e c t u r e s i n F i s h e r i e s . U.B.C. Andrews, J.W., L.H. K n i g h t , T.W. Page, Y. Ma t s u d a , and E.E. Brown. 1971. I n t e r a c t i o n s o f s t o c k i n g d e n s i t y and w a t e r t u r n o v e r on g r o w t h and f o o d c o n v e r s i o n o f c h a n n e l c a t f i s h r e a r e d i n i n t e n s i v e l y s t o c k e d t a n k s . P r o g . F i s h C u l t . 33: 197-203. A u l s t a d , D. and T. R e f s t i e . 1975. The e f f e c t o f water d e p t h i n r e a r i n g t a n k s on growth and m o r t a l i t y of salmon and ra i n b o w t r o u t f i n g e r l i n g s . P r o g . F i s h C u l t . 37:113-114. B a r t o n , B.A., R.E. P e t e r , and C R . P a u l e n c u . 1980. P l a s m a C o r t i s o l l e v e l s o f f i n g e r l i n g r a i n b o w t r o u t (Salmo  g a i r d n e r i ) a t r e s t , and s u b j e c t e d t o h a n d l i n g , c o n f i n e m e n t , t r a n s p o r t and s t o c k i n g . Can. J . F i s h . A q u a t . S c i . 37:805-811. B i l t o n , H.T. 1978. R e t u r n s of a d u l t c o ho salmon i n r e l a t i o n t o mean s i z e and t i m e a t r e l e a s e o f j u v e n i l e s . F i s h . Mar. S e r v . T e c h . Rep. '832. D e p t . o f t h e E n v i r o n m e n t . 73pp. B i l t o n , H.T., D.F. A l d e r d i c e and J . S c h n u t e . 1980. I n c r e a s i n g a d u l t r e t u r n s of h a t c h e r y - p r o d u c e d coho salmon t h r o u g h o p t i m i z a t i o n o f t i m e and s i z e a t j u v e n i l e r e l e a s e . P r o c . N o r t h P a c i f i c A q u a c u l t u r e Symp. A u g u s t 1980. A n c h o r a g e , A l a s k a , pp 171-181. B i r k s , E.K., A.R. Hemmingsen, J . L . D e n t l e r and R.D. E w i n g . 1981. H a t c h e r y p r a c t i c e s . A n n u a l P r o g r e s s R e p o r t . F i s h R e s e a r c h P r o j e c t . O regon D e p t . of F i s h and W i l d l i f e . P r o j e c t ' F-104-R-2. Bouck, G.R., M.A. C a i r n s and R.R. C h r i s t i a n . 1978. E f f e c t o f c a p t u r e s t r e s s on plasma enzyme a c t i v i t i e s i n r a i n b o w t r o u t (Salmo g a i r d n e r i ) J . F i s h . Res. B o a r d Can. 35:1485-1488. 151 B r a u h n , J . L . , R.C. Simon and W.R. B r i d g e s . 1976. Rainbow t r o u t g r o w t h i n c i r c u l a r t a n k s : c o n s e q u e n c e s o f d i f f e r e n t l o a d i n g d e n s i t i e s . T e c h . Rep. '86. U.S. D e p t . I n t e r i o r . F i s h and W i l d l i f e S e r v i c e , W a s h i n g t o n , D.C. 16 pp. B r e t t , J.R. 1958. I m p l i c a t i o n s and a s s e s s m e n t s of e n v i r o n m e n t a l s t r e s s , pp. 66-97. I_n, The i n v e s t i g a t i o n s of f i s h power p r o b l e m s . H.R. M a c M i l l a n L e c t u r e s i n F i s h e r i e s . U n i v e r s i t y o f B r i t i s h C o l u m b i a . B r e t t , J.R. 1964. The r e s p i r a t o r y m e t a b o l i s m and swimming p e r f o r m a n c e o f young s o c k e y e salmon. J . F i s h . Res. B o a r d Can. 21:1183-1226. B r e t t , J.R. 1971. S a t i a t i o n t i m e , a p p e t i t e , and maximum f o o d i n t a k e of s o c k e y e salmon ( O n c o r h y n c h u s n e r k a ) . J . F i s h . Res. B o a r d Can. 28:409-415. 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W i n d e l l , J . T . , D.O. N o r r i s , J.K. K i t c h e l l , and J . S . N o r r i s . 1969. D i g e s t i v e r e s p o n s e o f r a i n b o w t r o u t , Salmo  g a i r d n e r i , t o p e l l e t d i e t s . J . F i s h . Res. B o a r d Can. 26: 1801-1812. Z e l n i k , P.R. and G. G o l d s p i n k . 1980. E f f e c t s of e x e r c i s e a t d i f f e r e n t swimming s p e e d s on b l o o d C o r t i s o l l e v e l s i n t h e r a i n b o w t r o u t , Salmo g a i r d n e r i R i c h a r d s o n . A b s t r a c t I n , A.D. P i c k e r i n g [ e d . ] S t r e s s and f i s h . A c a demic P r e s s . T o r o n t o . 161 APPENDIX I . ANALYTICAL COMPOSITION OF ARTIFICAL SEA SALT. A v e r a g e s o l u t i o n of F o r t y Fathoms M a r i n e m i x h y d r a t e d t o a d e n s i t y of 1.025 u s i n g d i s t i l l e d w a t e r . F i g u r e s c i t e d f r o m a c t u a l i n d e p e n d a n t l a b o r a t o r y a n a l y s i s . C o n c e n t r a t i o i n s i n p a r t s p e r m i l l i o n (ppm). Upon h y d r a t i o n , pH i s 8.3. aluminum 0.06 a n t i m o n y 0.0005 a r g o n t r a c e arsenum 0.01 b a r i u m 0.12 b i c a r b o n a t e .... 174 b e r y l l i u m 0.0002 b i s m u t h t r a c e b o r o n 2.1 br o m i d e 62 cadmium 0.009 c a l c i u m 410 c a r b o n a t e 10 c e r i u m 0.0007 c e s i u m t r a c e chromium 0.02 c h l o r i d e 18600 c o p p e r 0.007 c o b a l t 0.0025 d y s p r o s i u m t r a c e e r b i u m t r a c e e u r o p i u m t r a c e f l u o r i d e 1.9 g a d o l i n i u m ....... t r a c e g a l l i u m 0.0004 germanium 0.00005 g o l d t r a c e h a f n i u m t r a c e h e l i u m t r a c e holmium t r a c e i n d i u m t r a c e i o d i n e 0.03 i r o n 0.03 k r y p t o n t r a c e l a n t h a n u m t r a c e l e a d . t r a c e l i t h i u m 0.24 l u t e t i u m t r a c e magnesium 1290 manganese 0.008 m e r c u r y 0.0007 molybdenum 0.005 neodymium t r a c e neon t r a c e n i c k e l 0.009 n i o b i u m t r a c e n i t r o g e n 0.85 p a l l a d i u m t r a c e p h o s p h o r u s 0.04 p o t a s s i u m 380 praeseodymium t r a c e p r o t a c t i n i u m t r a c e r a d i u m t r a c e r a d o n t r a c e r u b i d i u m 0.06 r u t h e n i u m t r a c e samarium t r a c e s c a n d i u m t r a c e s e l e n i u m t r a c e s i l i c o n 4.5 sodium 10400 s t r o n t i u m 12.4 s u l f u r ( s u l f a t e ) 2600 t a n t a l i u m t r a c e t e l l u r i u m t r a c e t e r b i u m t r a c e t h a l i u m 0.00007 t h u l i u m t r a c e t i n 0.006 t i t a n i u m 0.004 t u n g s t e n 0.004 u r a n i u m 0.00005 vana d i u m 0.0009 xenon t r a c e y t t e r b i u m t r a c e y t t r i u m t r a c e z i n c 0.24 z i r c o n i u m t r a c e 

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