UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A genetic basis for swim stamina differences between strains of the rainbow trout, Salmo gairdneri Winz, Robert Alan 1985

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1986_A6_7 W57.pdf [ 4.09MB ]
Metadata
JSON: 831-1.0096880.json
JSON-LD: 831-1.0096880-ld.json
RDF/XML (Pretty): 831-1.0096880-rdf.xml
RDF/JSON: 831-1.0096880-rdf.json
Turtle: 831-1.0096880-turtle.txt
N-Triples: 831-1.0096880-rdf-ntriples.txt
Original Record: 831-1.0096880-source.json
Full Text
831-1.0096880-fulltext.txt
Citation
831-1.0096880.ris

Full Text

A GENETIC BASIS FOR SWIM STAMINA DIFFERENCES BETW EEN STRAINS OF THE RAINBOW TROUT, SALMO GAIRDNERI By ROBERT ALAN WINZ B.Sc. (Highest Honors), Rutgers University, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept thi3 thesis aa conforming to~t.he rejquired standard THE UNIVERSITY OF BRITISH COLUMBIA December 1985 0 Robert Alan Winz, 1985 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 a n d 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 ^ O O i- O G Y 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 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 Dat e 1 O. <+', £6 •7Q \ ABSTRACT Two wild s t r a i n s of B r i t i s h Columbia rainbow trout (Salmo gairdneri) (Deadman Creek s t e e l h e a d and Pennask Lake ) and 2 d o m e s t i c a t e d s t r a i n s (Domestic-McLeary and Duncan River) were compared f o r evidence of genetic changes i n swim s t a m i n a due to n a t u r a l and a r t i f i c i a l s e l e c t i o n i n hatcheries. The w i l d s t r a i n s had approximately twice the swim stamina of the h a t c h e r y s t r a i n s , w i t h c r i t i c a l swim v e l o c i t i e s (Ucrj_-fc) 0 f 1.98 + 0.08 (Pennask) and 1.91 _+ 0.1 0 (Deadman) body l e n g t h s . s e c - 1 vs. 1.36 _+ 0.09 (Domestic) and 1.10 _+ 0.06 (Duncan) body lengths.sec-1. These two categories of s t r a i n s also d i f f e r e d i n c e r t a i n g e n e t i c / b i o c h e m i c a l and m o r p h o l o g i c a l c h a r a c t e r i s t i c s which have been li n k e d to swimming a b i l i t y . The r e s u l t s o f t h i s study reveal that: (1) In the exercised state, the Pennask and Deadman s t r a i n s convert l a c t a t e to pyruvate i n the l i v e r at the f o l l o w i n g maximum rates: 151.92 _+ 5.25 and 150.64 + 5«74 I.U.g-1 wet weight, w h i l e the Domestic and Duncan c o n v e r t at s l o w e r r a t e s : 86.48 + 1.78 and 109.20 _+ 3«11 I.U.g-''. L a c t a t e o x i d a t i o n i n whole blood shows a s i m i l a r t r e n d . (2) No c o r r e l a t i o n was found between swim stamina and LDH B2 genotype. However the two h i g h stamina " w i l d " s t r a i n s were the o n l y heterozygous groups. (3) Lactate l e v e l s i n the brain showed no change with exercise i n the " w i l d " s t r a i n s , while a 2.5 f o l d increase was observed for the "hatchery" s t r a i n s . (4) Hemoglobin c o n c e n t r a t i o n d u r i n g e x e r c i s e was h i g h e r f o r the Pennask and Deadman s t r a i n s , 1.73_+_0.04 and 1.75+^0.05 mM, vs. 1.61 _+ 0.03 and 1.54 +_ 0.03 mM f o r the Domestic and Duncan s t r a i n s . (5) The percentage o f non-Bohr/Root hemoglobin was approximately 30$ f o r a l l 4 s t r a i n s . (6) The Duncan s t r a i n was the o n l y one to show a n u c l e o s i d e triphosphate/hemoglobin (NTP/Hb) r a t i o o f l e s s than u n i t y d u r i n g e x e r c i s e (0.89 0.04)« (7) T h i s s t r a i n i a a l s o monomorphic f o r the r a r e LDH B2" a l l e l e . (8) The Deadman and Duncan s t r a i n s had greater percentages of GTP i n t h e i r e r y t h r o c y t i c NTP pools i n both the exercised and baseline states. Poor s u r v i v a l a f t e r release i n t o the wild i s a negative c h a r a c t e r i s t i c of domesticated stocks of hatchery trout. A major component of the a b i l i t y to survive i n the w i l d i s swim stamina. Domestication s e l e c t i o n (a form of natural selection) i n hatcheries against unnecessary swimming capacity i s a p o t e n t i a l reason why t r o u t r a i s e d from h a t c h e r y brood s t o c k s perform so poorly. High swim stamina i s not aa necessary f o r s u r v i v a l i n the hatchery environment as i t i s i n a t r o u t ' s n a t u r a l environment. A l s o , a d d i t i o n a l a r t i f i c i a l s e l e c t i o n f o r r a p i d growth c h a r a c t e r i s t i c s may be m u t u a l l y exclusive with any type of s e l e c t i o n f o r high swim stamina. i i i TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v L I S T OF FIGURES v i L I S T OF T A B L E S v i i AC KNOW LEDGEMENTS v i i i INTRODUCTION -1 Management I m p l i c a t i o n s 8 MATERIALS AND METHODS 9 E x p e r i m e n t a l A n i m a l s 9 L a t e D o m e s t i c M c L e a r y Rainbow T r o u t 9 Duncan R i v e r Rainbow T r o u t 9 Deadman C r e e k S t e e l h e a d T r o u t 10 Pennask Lake/Spahomin Creek Rainbow Trout 13 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 R e a r i n g C o n d i t i o n s 13 B a s e l i n e S a m p l i n g 16 E x e r c i s e S a m p l i n g 16 B l o o d and T i s s u e S a m p l i n g P r o t o c o l 27 B i o c h e m i c a l A s s a y s 29 S t a t i s t i c a l A n a l y s i s 33 RESULTS... 34 C r i t i c a l Swim V e l o c i t y 34 L a c t a t e A c c u m u l a t i o n 34 LDH B2 G e n o t y p e s • 37 LDH B2 A c t i v i t i e s 39 Hemoglobin 40 E r y t h r o c y t i c N u c l e o s i d e T r i p h o s p h a t e s 40 i v DISCUSSION 4 6 Swim Stamina o f "Hatchery" vs. "Wild" S t r a i n s 46 S e l e c t i o n i n H a t c h e r i e s f o r Rapid Growth 47 S e l e c t i o n i n H a t c h e r i e s f o r Body Depth 50 S e l e c t i o n i n H a t c h e r i e s f o r Reduced H e t e r o z y g o s i t y 50 B r a i n " L a c t i c A c i d o s i s " 52 L a c t a t e T o l e r a n c e i n Deadman S t e e l h e a d 54 L a c t a t e A c c u m u l a t i o n i n " H a t c h e r y " S t r a i n s 55 Hemoglobin, H e m o c o n c e n t r a t i o n and RBC S w e l l i n g 56 LDH B2 i n E r y t h r o c y t e s 59 O n t o g e n e t i c and S e a s o n a l V a r i a t i o n 61 LDH B2 i n H e p a t o c y t e s .61 Decreased NTP:Hb R a t i o s i n the Duncan S t r a i n 62 Linkage between LDH B2 Genotype and NTP:Hb R a t i o .62 GTP i n Rainbow T r o u t E r y t h r o c y t e s 63 Summary and C o n c l u s i o n s 64 REFERENCES 66 v LIST OF FIGURES Figure 1a. General l o c a t i o n s of the Duncan River, Deadman Creek, and Pennask Lake i n B r i t i s h Columbia... 11 Figure 1b. The Duncan River: O r i g i n a l source area of the Duncan s t r a i n o f r a i n b o w t r o u t 11 Figure 2a. Deadman Creek: O r i g i n a l source area of the Deadman steelhead t r o u t s t r a i n . . . . . 14 Figure 2b. Pennask Lake: O r i g i n a l source' area of the Pennask s t r a i n of r a i n b o w t r o u t 14 F i g u r e 3« Schematic diagram of swim; t u n n e l , top view ....19 Figure 4. Schematic diagram of swim tunnel, side view of f i s h chamber 21 Figure 5. F i s h chamber water flow p r o f i l e at transmission s e t t i n g 1 (.slow current) 23 Figure 6. F i s h chamber water flow p r o f i l e at transmission s e t t i n g 4 (.fast current) 25 v i LIST OF TABLES Table 1. Rainbow trout s t r a i n information f o r baseline and exercise sampling 17 Table 2. Stock s o l u t i o n s f o r 7.5% p o l y a c r y l a m i d e g e l s 31 Table 3. Comparison of c r i t i c a l swimming speeds {UCT±t) and cond i t i o n f a c t o r s (,CFj between f o u r s t r a i n s o f rainbow t r o u t 35 Table 4- Comparison of l a c t a t e accumulation i n plasma, brain, l i v e r and white muscle between f o u r s t r a i n s o f rainbow t r o u t 36 Table 5. Comparison of LDH B2 a l l e l e frequencies, l i v e r and erythrocyte (RBC) LDH B2 l a c t a t e o x i d a t i o n a c t i v i t i e s between four s t r a i n s o f r a i n b o w t r o u t 38 Table 6. Comparison of Hemoglobin concentration (Hb), percent non-Bohr/ Root type Hb (% Non-B/R), mean c e l l u l a r Hb concentration (.MCHCJ and hematocrit (.HOT) between four s t r a i n s o f rainbow t r o u t 41 Table -7. Comparison of whole blood (WBj and e r y t h r o c y t i c (RBC) NTP c o n c e n t r a t i o n s , and NTP/Hbratiosbetween f o u r s t r a i n s of rainbow trout, as determined enzymatically (ENZ) and by TLC 42 Table 8. Comparison of whole blood (WBJ and e r y t h r o c y t i c (RBCJ ATP and GTP concentrations, and percent GTP of t o t a l NTP between f o u r s t r a i n s o f rainbow t r o u t 44 v i i ACKNOWLEDGEMENTS Many thanks go to E r i c Parkinson of the B.C. P r o v i n c i a l Pish & W i l d l i f e Branch f o r the o r i g i n a l impetus to do a study comparing s t r a i n s of rainbow trout. I also thank E r i c f o r being a very a c t i v e and int e r e s t e d co-worker, and f o r h i s support, p a r t i c u l a r l y during the more uncertain times during the s t a r t of t h i s study. Drs. A l Burton, Roger Brownsey, R i c h a r d Barton, and the r e s t of the U.B.C. Dept. of Biochemistry provided me with a lab to work i n and access to t h e i r equipment. I am ve r y t h a n k f u l to them f o r p u t t i n g up w i t h me f o r so long. W i t h o u t t h e i r g e n e r o s i t y t h i s study would have been much more d i f f i c u l t to carry out. I am v e r y g r a t e f u l to Dr. Henry T s u y u k i o f F i s h e r i e s & Oceans Canada f o r the time he took to discuss with me several aspects of f i s h metabolism. I also thank him f o r allowing me to use h i s lab equipment and chemicals. The d i s c u s s i o n s I had with Dr. Dennis Powers of Johns Hopkins U n i v e r s i t y were also extremely h e l p f u l and g r e a t l y appreciated. My s u p e r v i s o r , Dr. T.G. N o r t h c o t e , f i n a n c i a l l y supported the bulk o f th i s study on his NSERC grant. The B.C. F i s h & W i l d l i f e Branch also provided funds, equipment, hatchery f a c i l i t i e s , and the f i s h stocks. The s t a f f of the Fraser V a l l e y Trout Hatchery (Research Section): Bob Land, Morley Rem pel and Larry M i t c h e l l , maintained the f i s h stocks throughout the years and provided e x p e r i m e n t a l a s s i s t a n c e on o c c a s i o n . Ian W i l l i a m s of the I n t e r n a t i o n a l P a c i f i c Salmon Commission allowed me to use and modify t h e i r swim tunnel. I a l s o thank Drs. Ha r o l d K a s i n s k y and Hugh Brock of the U.B.C. Dept. of Zoology f o r t o l e r a t i n g long term loans of t h e i r l i q u i d nitrogen equipment. I am v e r y g r a t e f u l to my f r i e n d , Bruce Semple, eh, f o r u s i n g h i s expertise i n computers to b u i l d those invaluable p i a n o - l i k e shelters-from-the-elements f o r the swim t u n n e l . I would a l s o l i k e to thank the c a s t o f thousands, who I had d e c e i v e d i n t o h e l p i n g me i n Abbot s f o r d by p r o m i s i n g them a good time: Joyce Andrew, Alex Brett, John F r y x e l l , Chris Foote, Tom Johnston, Helen Loughery, Howard M u e l l e r , E r i c P a r k i n s o n , Grant Pogson, Kathy Quayle, Ric Taylor, Arlene Tompkins, Rick Ward, Jean-Michel Weber, and Simon Wong. Most i m p o r t a n t l y I'd l i k e to thank my b r o t h e r , S t e f a n , f o r the use o f h i s c o l l e c t i o n of science f i c t i o n books. I t served as an invaluable a id to the development of most of the t h e o r e t i c a l aspects of my discussion. L a s t l y , I'd l i k e to express my g r a t i t u d e to a l l those hundreds o f g a l l a n t f i s h out t h e r e , who gave t h e i r l i v e s so b r a v e l y i n the name of truth, j u s t i c e and s c i e n t i f i c research. ix INTRODUCTION S e l e c t i o n by man f o r d e s i r a b l e t r a i t s i n h a t c h e r y f i s h s t o c k s , both f o r f i s h f a r m i n g and s t o c k i n g , i s not new. T h i s i s e s p e c i a l l y the case f o r salmonids, which have been s e l e c t e d f o r such q u a n t i t a t i v e t r a i t s as fecundity, growth rate, time of maturity, time of s m o l t i f i c a t i o n , disease resistance, a c i d i c pH tolerance, etc. ( f o r reviews and references see Ryman 1980, Donaldson 1968, Kirpichnikov 1981, Stock Concept Symposium 1981, Holm & Naevdal 1977, Webster & F l i c k 1978). However, s e l e c t i o n f o r these types of t r a i t s , f o r example growth, i s always i n response to a hatchery environment, and not the n a t u r a l environment o f the f i s h . T h e r e f o r e , i t f o l l o w s that c e r t a i n a d d i t i o n a l a r t i f i c i a l ( i . e . manmade) s e l e c t i v e pressures are being e x e r t e d , but i t i s a l s o v e r y p r o b a b l e t h a t c e r t a i n n a t u r a l s e l e c t i v e pressures are absent or reduced (Doyle 1983)« I t i s therefore not s u r p r i s i n g to f i n d a l a r g e body o f l i t e r a t u r e d e m o n s t r a t i n g t h a t h a t c h e r y - b r e d f i s h have poorer s u r v i v a l a f t e r r e l e a s e i n the w i l d than c o r r e s p o n d i n g w i l d s t o c k s ( V i n c e n t 1960, F l i c k & Webster 1962, 1964, & 1976, F l i c k 1971, Webster & F l i c k 1978 & 1981, Cordona & Ni c o l a 1970, Smith 1957, Fraser 1981, Maclean et a l . 1981, R i t t e r 1975, Greene 1952, Shuck 1948, M i l l e r 1953). For rainbow t r o u t , Salmo g a i r d n e r i , i n B r i t i s h Columbia, i t i s known that d i f f e r e n c e s e x i s t between d i f f e r e n t s t r a i n s ( w i l d and hatchery) with respect to growth, maturation, etc., and e s p e c i a l l y s u r v i v a l . Bams (1967), working with sockeye migrant f r y , and Burrows (1969), working with chinook salmon, have shown that swim stamina t e s t s can serve as adequate i n d i c a t o r s f o r s u r v i v a l i n the wild. Poorer s u r v i v a l of hatchery-bred stocks could then be p a r t i a l l y explained by i n f e r i o r swimming c a p a b i l i t y , which brings with i t such consequences as i n c r e a s e d v u l n e r a b i l i t y to predation.(Bams 1967), decreased a b i l i t y to maintain f i s h stocks i n the f a s t e r flowing currents of 1 headwater r e g i o n s (Bachman 1984), and o t h e r c o m p e t i t i v e d i s a d v a n t a g e s ( M i l l e r 1958, Moyle 1969, Fenderson et a l . 1968, Bachman 1984). The p u r p o s e o f t h i s s t u d y i s to i n v e s t i g a t e t h e g e n e t i c and p h y s i o l o g i c a l basis of swim stamina i n s t r a i n s of rainbow trout that have been r a i s e d i n h a t c h e r y environments f o r d i f f e r e n t l e n g t h s o f time (and t h e r e f o r e g e n e r a t i o n s ) , f o r up to 40 y e a r s . I t i s b e l i e v e d that t h e r e i s l i t t l e p o s i t i v e s e l e c t i o n o c c u r r i n g i n h a t c h e r i e s f o r stamina (Beamish 1978), i n contrast to wild trout i n t h e i r n atural environment. That changes occur i n the gene p o o l o f t r o u t as a r e s u l t o f h a t c h e r y procedures i s i l l u s t r a t e d i n the study by Ryman and S t a h l (1980) on c o r r e s p o n d i n g p o p u l a t i o n s of w i l d and h a t c h e r y - b r e d brown t r o u t , Salmo t r u t t a ,and i n s i m i l a r studies on cutthroat trout, Salmo c l a r k i (Allendorf & Phelps 1980, Leary et a l . 1985) and A t l a n t i c salmon, Salmo s a l a r ( C r o s s & K i n g 1983, S t a h l 1983). However, these gene p o o l changes are i n randomly s e l e c t e d c h a r a c t e r i s t i c s . There have been many studies done on f i s h where d i f f e r e n t b i o c h e m i c a l / g e n e t i c forms o f t h a t s p e c i e s show d i f f e r e n t swim st a m i n a a b i l i t i e s ( T s u y u k i & W i l l i s c r o f t 1977, K l a r et a l . 1979a & b, Powers 1972, DiMichele & Powers 1982a). I t would therefore be of i n t e r e s t to determine i f s t r a i n s o f rainbow t r o u t which d i f f e r i n swim stamina, a l s o d i f f e r i n a s e r i e s of b i o c h e m i c a l / g e n e t i c c h a r a c t e r i s t i c s which have previously been i n d i v i d u a l l y linked to swim stamina. Several studies have been c a r r i e d out on the allozymes of the l a c t a t e dehydrogenase (LDH) ( L - l a c t a t e : NAD oxidoreductase, EC 1.1.1.27) l i v e r locus ( i . e . B2; a l s o found i n e r y t h r o c y t e s , b r a i n and red muscle) of the rainbow trout, Salmo g a i r d n e r i , to determine the adaptive s i g n i f i c a n c e of t h i s two a l l e l e system (Tsuyuki & W i l l i s c r o f t 1977, Klar et a l . 1979a & b, Redding & Schreck 1 9 7 9 , Northcote & Kelso 1981). I t has been suggested that the rarer 2 LDH "A" a l l e l e (i.e. B2") i s advantageous to populations which inhabit fast flowing and headwater currents (Northcote 1981, Northcote & Kelso 1981). The more common LDH "B" a l l e l e (i.e. B2') i s , however, the t y p i c a l form of most rainbow trout stocks throughout the northwestern regions of North America (Northcote et a l . 1970, Huzyk & Tsuyuki 1974, H. Tsuyuki, personal communication). Due to this general c o r r e l a t i o n of high B2" a l l e l e frequencies with fast currents, i t would be reasonable to assume that evolved adaptations might be in operation, with swim stamina being one of them (Tsuyuki & W i l l i s c r o f t 1977, Klar et a l . 1 9 7 9 a ) . Support f o r such an assumption has not only come from experiments on whole organisms, but also from kinetic studies on the homotetrameric LDH allozymes themselves (Tsuyuki & W i l l i s c r o f t 1973, Kao & Farley 1978a & b). Additional maintenance for the theory that LDH B genotype influences swim performance comes from similar studies that have been performed on the cyprinodontid, Fundulus heteroclitus (DiMichele & Powers 1982b, Place & Powers 1984a & b). In order to f u l l y comprehend th i s s e l e c t i v e advantage given LDH B2" individuals in headwater populations, an understanding of the physiological mechanisms in metabolic terms, and the differences between the two existing alleles must be achieved. In this branch of research, two semi-conflicting views are present. Tsuyuki & W i l l i s c r o f t (1973 & 1977) maintain that rainbow 2' trout homozygous for LDH B2" exhibit a swimming stamina superior to the B homozygotes because in the l i v e r and erythrocytes (where lactate is chiefly oxidized back to pyruvate) the lactate to pyruvate conversion rate for the LDH B2" homotetramer i s 3.6 to 4-0 times higher (Tsuyuki & W i l l i s c r o f t 1973, Kao & Farley 1978a & b) than that of the B2' homotetramer. This in v i t r o difference could very well influence, in vivo, the postponement of the onset of fatigue in B2" homozygotes. Results of this nature would lend support to the stocking of LDH B2" homozygous f i n g e r l i n g s i n headwater regions. 3 However, the c o n c l u s i o n s drawn from t h i s s e t o f r e s u l t s come m a i n l y from experiments performed on j u v e n i l e Salmo g a i r d n e r i (SL: 5 to 6 cm). Some experiments have been done on o l d e r specimens (26 to 27 months), g i v i n g r e s u l t s which indicated the LDH B2' homozygotes to have better swim stamina (Tsuyuki & W i l l i s c r o f t 1977). However i t should be noted that the decrease i n the stamina of LDH B2" genotype c o u l d be r e l a t e d to t h i s homozygote's e a r l i e r apparent onset of secondary c h a r a c t e r i s t i c s of sexual maturity (i.e. darker pigmentation). The major studies done with adult rainbow trout are those of K l a r et a l . (1979a & b). Here i t was shown t h a t at s a t u r a t i n g dissolved oxygen l e v e l s (e.g. 11 to 12 ppm) no d i f f e r e n c e was found between the three possible genotypes. However the LDH B2" homozygote does perform more poorly, but only at low environmental oxygen tensions (2 ppm). This i s thought to be due to the f a c t that t h i s locus i s also the main one expressed i n b r a i n t i s s u e ( a l o n g w i t h the LDH B1 l o c u s ) . T h e r e f o r e because of the a t y p i c a l k i n e t i c s o f the B2" a l l o z y m e and i t s apparent e v o l u t i o n towards muscle-type LDH b e h a v i o u r ( B a i l e y et a l . 1976, Kao & F a r l e y 1978a & b), i t has been suggested that at decreased pH and oxygen l e v e l s as seen during low ambient oxygen t e n s i o n s and/or b u r s t e x e r c i s e "the b r a i n i n t r a c e l l u l a r l a c t a t e concentrations (of LDH B2" homozygotes) might r i s e to toxic l e v e l s f a r exceeding the blood l a c t a t e l e v e l s s u p p l y i n g the b r a i n , w i t h a concomitant l o s s of ATP normally provided by oxidative phosphorylation." Both Tsuyuki <$ W i l l i s c r o f t (1977) and K l a r et a l . (1979a <S b) mention the p l a u s i b i l i t y of ontogenetic change i n swim stamina. K l a r goes one step f u r t h e r and attempts to c o r r e l a t e i t w i t h changes i n oxygen s u p p l y due to changes i n hemoglobin type, i.e. from l a r v a l (high oxygen a f f i n i t y ; no Bohr and Root effects) to adult (low oxygen a f f i n i t y ) . However, at 25 days a f t e r h a t c h i n g , l a r v a l hemoglobin i s no l o n g e r apparent i n the a l e v i n blood 4 samples (Iuchi & Yamagami 1969, Iuchi 1973)' The change i n hemoglobin types i s therefore p r i o r to the age. at which any rainbow trout has ever been tested for swim stamina. One could view ontogenetic change in swim stamina and the adaptive significance of the LDH B2 polymorphism by the following hypothesis. The possibly inferior swim performance of adult B 2 i n d i v i d u a l s i s not as c r i t i c a l i n the natural environment as i s the i n f e r i o r swim performance of j u v e n i l e B2' i n d i v i d u a l s because of the greater size of an adult rainbow trout. In other words, the maximum flows experienced i n a stream or lake, as measured i n body lengths per second, decrease as trout size increases. Hence a s e l e c t i v e ontogenetic bottleneck might exist, whereby the LDH B2" genotype has an advantage in swimming stamina at a more crucial stage during the post-alevin/pre-adult period of the l i f e cycle of headwater rainbow trout. This theory i s consistent with the work done by Northcote & Kelso (1981) in which better position maintenance and upstream movement i s associated with LDH B2" homozygous f r y i n an above-waterfall stock of rainbow trout. This could i n d i c a t e that the advantage of the LDH B2 a l l e l e i s expressed early i n l i f e during times of upstream movement, and i s therefore more important i n above-falls and outlet f r y than i n i n l e t spawners. Perhaps t h i s could be an example of a balanced polymorphism, whereby a heterozygote would have the best of both a l l e l e s : f i r s t with LDH as a juvenile, and then B^' as an adult. Such changeable or s h i f t adaptation of a l l e l e s has been supposed for the LDH B2 locus ( l i v e r ) in sockeye salmon, Oncorhynchus nerka (Kirpichnikov & Muske 1980). Here, selection for the two alleles changes i t s direction during the l i f e span of the salmon, specifically during the early stages of development in the lake period. Other metabolic e n t i t i e s , besides the LDH B2 allozymes, have been described as affecting swim stamina in fish. The proportions of juvenile and 5 adult hemoglobin may be ruled out as being such a factor. However, because more than one type of hemoglobin i s present i n the blood of post-alevin rainbow trout, some of which possess a Bohr e f f e c t (i.e. oxygen binding a f f i n i t y decreases with decreasing pH) and a Root e f f e c t (i.e. oxygen binding capacity decreases with decreasing pH), and some which do not (Brunori 1975), i t would be a reasonable hypothesis to expect differences in the r e l a t i v e amounts of these hemoglobin types between s t r a i n s with d i f f e r e n t swim stamina. Such an adaptive mechanism has been described by Powers (1972) for subspecies of catostomid f i s h . In t h i s instance the subspecies which inhabits faster flowing waters had a higher proportion of non-Bohr/Root hemoglobin. This would give these f i s h supplementary oxygen during exercise when blood pH decreases. It has a l s o been demonstrated that i n Fundulus h e t e r o c l i t u s intracellular erythrocytic ATP (the major al l o s t e r i c modifier of hemoglobin oxygen binding affinity) concentrations appear to be genetically determined and correlated with homozygous LDH B genotypes (Powers et a l . 1979, DiMichele & Powers 1982a & b), with both of these factors influencing swim stamina (DiMichele & Powers 1982b). Here the high stamina LDH B genotype has a larger ATP/hemoglobin ratio, which allows i t to deliver between 18 and 40% more oxygen to muscle tissue than the low stamina LDH B genotype at maximum swimming speed. Whether such a s i m i l a r genetic phenomenon also occurs i n more active fish such as rainbow trout has never been examined. ATP i s also the major a l l o s t e r i c modifier of hemoglobin oxygen binding a f f i n i t y i n rainbow trout, with GTP playing a more potent yet secondary role (Weber et a l 1976, Weber 1982). Both these nucleoside triphosphates can bind to the hemoglobin molecule and thereby reduce i t s a f f i n i t y for oxygen. The more NTP present i n the erythrocyte, the less oxygen i s bound to hemoglobin. Low 6 blood oxygen a f f i n i t i e s are g e n e r a l l y associated with the more a c t i v e f i s h s p e c i e s ( P r o s s e r 1973. Hayden et a l . 1975, G r i g g 1967, Graham et a l . 1985, Dobson & Baldwin 1982). On an i n t e r s p e c i e s l e v e l t h i s may be due to di f f e r e n c e s i n hemoglobin structure, whereas wit h i n a species i t w i l l more l i k e l y be due to d i f f e r e n t NTP/Hb rat i o s . It i s the hypothesis of t h i s study that rainbow trout s t r a i n s with high swim stamina w i l l also d i s p l a y the f o l l o w i n g biochemical c h a r a c t e r _ s t i c s : (1) l a c t a t e o x i d a t i o n a c t i v i t y o f e r y t h r o c y t e and l i v e r LDH w i l l be greater to achieve more rapid l a c t a t e removal and delay fatigue; (2a) s i n c e the f i s h w i l l be t e s t e d at s a t u r a t i n g d i s s o l v e d oxygen l e v e l s , the LDH B 2" a l l e l e s h o u l d be more p r e v a l e n t , because a c c o r d i n g to Ts u y u k i & W i l l i s c r o f t (1977) the ho m o t e t r a m e r i c enzyme produced by the homozygote w i l l be able to convert l a c t a t e to pyruvate more r a p i d l y and thus ai d i n the postponement of fatigue, or (2b) a c c o r d i n g to K l a r et a l . (1979a & b), because the f i s h t e s t e d (17 to 18 cm f o r k length) are c o n s i d e r e d to be s u b - a d u l t s to a d u l t s , no d i f f e r e n c e i n swim stamina should be detected between LDH B2 genotypes; (3) there should also be l e s s l a c t a t e build-up i n the b r a i n because of the e x p r e s s i o n of the LDH B2" a l l e l e i n t h i s t i s s u e and/or because of greater l a c t a t e oxidation a c t i v i t y s i m i l a r to the l i v e r and erythrocytes; (4) hemoglobin concentration i n the blood should be greater to provide increased oxygen transport capacity; (5) r e l a t i v e l y more of the non-Bohr/Root hemoglobin types s h o u l d be present to serve as oxygen " r e s e r v o i r s " u n t i l low blood pH i s achieved; (6) e r y t h r o c y t i c NTP/Hb r a t i o w i l l be higher (to decrease blood oxygen a f f i n i t y , and therefore keep t i s s u e oxygen tension high), and (7) perhaps c o r r e l a t e d w i t h the h i g h stamina LDH B2" genotype (at sa t u r a t i n g d i s s o l v e d oxygen l e v e l s ) ; 7 (8) a greater proportion of t h i s NTP w i l l c o n sist of GTP because of i t s greater e f f e c t i v e n e s s at lowering the oxygen a f f i n i t y of trout hemoglobin. Since the rainbow trout s t r a i n s w i l l be raised i n i d e n t i c a l environments, any s t r a i n - r e l a t e d d i f f e r e n c e s observed e i t h e r i n swim s t a m i n a or i n biochemical c h a r a c t e r i s t i c s w i l l be of a genetic o r i g i n . Management Implications The a n a l y s i s of the preceding blood and tissue components, which have a l l been g e n e t i c a l l y l i n k e d w i t h swimming a b i l i t y i n v a r i o u s s p e c i e s o f f i s h , might give hatcheries t r a i t s which they can s e l e c t from to increase the s t a m i n a o f t h e i r rainbow t r o u t s t r a i n s . G e n e t i c i n f o r m a t i o n o f t h i s nature would be useful i n de-emphasizing the hatchery s e l e c t i o n trends f o r f i s h s o l e l y with high growth/low stamina t r a i t s , and thereby a i d i n reducing the r e l e a s e of h a t c h e r y s t r a i n s p o s s e s s i n g h i g h hooking m o r t a l i t y and i n g e n e r a l , low s u r v i v a l . T h i s can be done by t a k i n g and a n a l y z i n g , f o r example, s m a l l blood samples or l i v e r b i o p s i e s from the brood stock. T h i s would be a more p r a c t i c a l test than performing swim stamina runs (i.e. Ucr-j_-t; determinations) on the e n t i r e brood stock. 8 MATERIALS AND METHODS Experimental Animals Four s t r a i n s of rainbow trout were raised under as i d e n t i c a l conditions as possible to be tested f o r genetic d i f f e r e n c e s i n 3wim stamina. Late Domestic McLeary Rainbow T r o u t : T h i s s t r a i n i s used s o l e l y to s t o c k suburban l a k e s , h a v i n g no i n d i g e n o u s t r o u t p o p u l a t i o n s , f o r r e c r e a t i o n a l angling purposes. These are o r i g i n a l l y Kamloops trout (Behnke 1972), which have been subject to nearly 40 years of s e l e c t i v e breeding and d o m e s t i c a t i o n at a commercial f i s h h a t c h e r y ( T r o u t l o d g e , Inc., M c M i l l a n , Wa.). They were s e l e c t e d to spawn i n the f a l l and p r e d o m i n a n t l y at 2 and 3 y e a r s o f age. S e l e c t i o n f o r an i n c r e a s e d growth r a t e i s a l s o v e r y obvious. This brood l i n e has been held i n c a p t i v i t y at the Fraser V a l l e y P r o v i n c i a l Trout Hatchery i n Abbotsford, B.C. since approximately 1968, when they were a c q u i r e d as eyed eggs from T r o u t l o d g e , Inc., M c M i l l a n , Wa. In A b b o t s f o r d , attempts are b e i n g made to s h i f t m a t u r i t y back to 3 and 4 y e a r s of age (H. Sparrow & E. Parkinson, B.C. F i s h & W i l d l i f e , personal communication). The f i s h used i n these experiments came from the. second batch of eggs (hence the term " l a t e " ) taken from 3 and/or 4 y e a r o l d spawners i n l a t e December, 1983' The e s t i m a t e d hatch date f o r these i f i s h was January 22, 1984. They were then raised on Nelson's S t e r l i n g S i l v e r c u p trout feed i n the Fraser V a l l e y Trout Hatchery i n 9 .50C well water u n t i l a mean s i z e of 23.1 g was reached on August 17, 1984, whereupon they were t r a n s f e r r e d to the quarantine room of that hatchery. Duncan River Rainbow Trout: This s t r a i n has the reputation of having a high hooking m o r t a l i t y (Facchin 1983). I t s usefulness f o r stocking purposes w i l l be more e a s i l y evaluated upon a more q u a n t i t a t i v e determination of i t s swim s t a m i n a r e l a t i v e to o t h e r s t r a i n s . l t o r i g i n a t e s from Kootenay Lake, 9 B.C., where i t once migrated a short distance up the Duncan River, north end o f the l a k e , to spawn ( F i g s . 1a and b). I t has the d e s i r a b l e t r a i t s o f g r o w i n g l a r g e and m a t u r i n g l a t e ( I r v i n e 1978, E. P a r k i n s o n , B.C. F i s h & W i l d l i f e , p e r s o n a l communication). The o r i g i n a l s ource o f the Kootenay P r o v i n c i a l Hatchery brood stock was the capture of adults from the Duncan River, B.C., a short distance upstream from i t s confluence with the Lardeau R i v e r , i n 1970. Eggs from these a d u l t s were used to s t a r t a c a p t i v e brood stock i n the Kootenay Hatchery. The o r i g i n a l intent of egg c o l l e c t i o n s was to restore the rainbow population of Trout Lake, many; of which spawn i n an i n l e t c a l l e d W i l k i e Creek. Some r e l e a s e s were a l s o made i n t o the Duncan R i v e r from 1971 to 1974 (H. Sparrow, B.C. F i s h & W i l d l i f e , p e r s o n a l communication). The wild equivalent of t h i s captive brood stock no longer e x i s t s due to the construction of the Duncan Dam i n 1967* The eggs f o r t h i s study's f i s h were c o l l e c t e d between March 22 and A p r i l 25, 1 983 from Kootenay Hatchery brood s t o c k which were 3, 4, and 5 y e a r s o f age as f i r s t time spawners. Some eggs might a l s o have been taken from f i s h held f o r a second spawning. Hatching took place between A p r i l 22 to May 25, 1983- The f i s h were then r a i s e d i n the Kootenay Hatchery on S i l v e r c u p t r o u t feed i n 8oC water u n t i l a mean s i z e of 2.9 g was a t t a i n e d on August 18, 1983- They were then shipped to the q u a r a n t i n e room of the Fraser V a l l e y Hatchery. Deadman Creek Steelhead Trout: This s t r a i n i s c h a r a c t e r i s t i c a l l y known f o r rapid growth, strong t e r r i t o r i a l i t y , and high swimming stamina (Tsuyuki & W i l l i s c r o f t 1977, E. P a r k i n s o n , B.C. F i s h & W i l d l i f e , p e r s o n a l communication). Deadman steelhead migrate from the ocean approximately 230 km up the Fraser River to i t s confluence with the Thompson River, where they continue t h e i r migration approximately 120 km up to Deadman Creek to spawn 1 0 FIGURE 1 a . G e n e r a l l o c a t i o n s of the Duncan R i v e r ( F i g . 1 b ) , Deadman Creek ( F i g . 2 a ) , and Pennask Lake ( F i g . 2 b ) i n B r i t i s h Columbia. A) Vancouver, B) F r a s e r R i v e r , C) Thompson R i v e r , D) Bonaparte R i v e r , E) N i c o l a R i v e r , F) Columbia R i v e r , G) Kamloops, H) Nelson, I) F i g . 1 b , J) F i g . 2 a , K) F i g . 2 b . FIGURE 1 b. The Duncan R i v e r : O r i g i n a l source ar e a of the Duncan s t r a i n o f rainbow t r o u t . A) Duncan R i v e r , B) Duncan Dam, C) Duncan Lake, D) Lardeau River, E) Trout Lake, F) north arm of Kootenay Lake. 1 1 6 0 K M I 1 1 (Pigs. 1a and 2a). This route contains some of the most strenuous rapids i n the F r a s e r system (e.g. H e l l ' s Gate). The f i s h f o r these experiments o r i g i n a t e d from a d u l t s taken from Deadman Creek, B.C. (about 1.5 km upstream from i t s c o n f l u e n c e w i t h C r i s s Creek), which were spawned on May 3, 1983 (H. Sparrow & J. C a r t w r i g h t , B.C. F i s h & W i l d l i f e , p e r s o n a l communication). The eggs hatched at the F r a s e r V a l l e y Hatchery on approximately May 27, 1983- The f i s h were raised there o_ S i l v e r c u p t r o u t feed i n 13oC w e l l water u n t i l a mean s i z e of 0-7 g was reached on July 25, 1983' Thereafter they were tra n s f e r r e d to the quarantine room of the same hatchery. Pennask Lake/Spahomin Creek Rainbow T r o u t : T h i s s t r a i n of t r o u t i s t y p i c a l l y used to stock i n t e r i o r lakes where there already i s a native trout population, but which requires augmentation f o r r e c r e a t i o n a l angling. I t i s a pure s t r a i n , i . e . o r i g i n a t i n g from a l a k e which has had no s t o c k i n g i n t r o d u c t i o n s . I t i s a l s o a v e r y slow growing s t r a i n ( A y l e s & Baker 1983, Kelso et a l . 1981, H. Sparrow <& E. Parkinson, B.C. F i s h & W i l d l i f e , personal communication). The e x p e r i m e n t a l f i s h are progeny of a d u l t s taken from Spahomin Creek, B.C. (the o u t l e t stream of Pennask Lake ( e l e v a t i o n 1420 m); see F i g s . 1a and 2b) and spawned on June 1, 1983. The fence and t r a p s are located at the lake o u t l e t where i t i s c o n s t r i c t e d and becomes a stream (H. Sparrow & L. Lemke, B.C. F i s h & W i l d l i f e , p e r s o n a l communications). These f i s h hatched a p p r o x i m a t e l y on June 30, 1983 at the Summerland P r o v i n c i a l Hatchery, where they were a l s o r a i s e d on S i l v e r c u p t r o u t feed i n IO .50C water, u n t i l a mean s i z e of 0.6 g was achieved on August 18, 1983' They were then shipped to the quarantine room of the Fraser V a l l e y Hatchery. Fraser V a l l e y Trout Hatchery Rearing Conditions Subsequent rearing conditions of a l l four s t r a i n s at the Fraser V a l l e y 1 3 FIGURE 2a. Deadman Creek: O r i g i n a l source a r e a of the Deadman s t e e l h e a d t r o u t s t r a i n . A) Deadman Creek, B) C r i s s Creek, C) Bonaparte R i v e r , D) Thompson River, E) Kamloops Lake. FIGURE 2b. Pennask Lake: O r i g i n a l source a r e a o f the Pennask s t r a i n o f rainbow t r o u t . A) Pennask Lake, B) Spahomin Creek ( o u t l e t ) , C) Pennask R i v e r ( i n l e t ) . 14 Trout H a t c h e r y q u a r a n t i n e room were as f o l l o w s . S e p a r a t e l o t s of 150 f i s h per s t r a i n were kept i n 4 i d e n t i c a l 300 1 oval culture tanks. Well water at 9.50C was d e l i v e r e d v i a a p r e s s u r i z e d v e r t i c a l m u l t i - h o l e d i n f l o w p i p e , thereby a l l o w i n g the f i s h to be t r a i n e d to swim i n f l o w s of 1 to 2 body l e n g t h s / s e c o n d f o r at l e a s t two months p r i o r to s a m p l i n g and t e s t i n g . An a r t i f i c i a l l y constant 7 hour light/17 hour dark photoperiod was maintained during the e n t i r e time of rearing. I l l u m i n a t i o n consisted of 3 banks of ten 35 watt 4 f t "cool white" fluorescent l i g h t tubes. Only one of the end banks was d i r e c t l y overhead the 4 c u l t u r e tanks. A l l banks were on during the 7 hr l i g h t p e r i o d . W i t h the e x c e p t i o n o f 2 tubes on the f a r end bank (which provided f a i n t i n d i r e c t l i g h t i n g ) , a l l tubes were extinguished during the 17 hr dark phase. A l l 4 s t r a i n s were fed to s a t u r a t i o n on a d a i l y b a s i s . T h i s means t h a t at each f e e d i n g event more food was dropped i n t o the tank than c o u l d be eaten. Large q u a n t i t i e s of r e s i d u a l food were always l e f t over a f t e r feeding. Baseline Sampling Sixteen f i s h per s t r a i n , weighing at l e a s t 55 g (to ensure s u f f i c i e n t a v a i l a b l e blood f o r sampling during t h i s very low a c t i v i t y state) were taken from the reari n g tanks and placed i n separate opaque flow-through i s o l a t i o n chambers i n the quarantine room (Table 1). A f t e r 72 hours of s t a r v a t i o n and i s o l a t i o n , the 16 f i s h were removed one by one w i t h minimum a g i t a t i o n , s a c r i f i c e d w i t h a blow to the head, and sampled a c c o r d i n g to the p r o t o c o l described below. Exercise Sampling A 28 cm diameter swim tunnel (Tsuyuki & W i l l i s c r o f t 1977) belonging to the I n t e r n a t i o n a l P a c i f i c Salmon F i s h e r i e s Commission, Cultus Lake, B.C. was 1 6 TABLE 1. Rainbow trout strain information for baseline(B) and exercise(E) sampling. Stock* LGT Range ¥ GT Range Age Range Test Temp Test D.O. # Fish/Test (cm) (g) (months) (oC) (ppm) DEAD B 17-5-21.2 56.7-100.9 11-12 E 16.9-18.5 45.9-74.4 15-24 9-15 8-13 6, 7 & 1 DUNC B 17.7-21.5 63-2-124-7 12-13 E 16.6-18.3 54-2-80.5 16-20 9-14 8-11 8 & 12 DOM B 19-3-22.0 94.0-143-6 10-11 E 16.8-18.5 73-5-106.1 9-10 11-12 10-13 9 & 9 PEN B 19.4-22.5 62.2-117.9 17-18 E 16.7-18.9 41.9-71.9 16-19 7-9 9-12 10 & 8 * DEAD = Deadman Creek Steelhead Trout, DUNC = Duncan River Rainbow Trout, DOM = Late Domestic McLeary Rainbow Trout, PEN = Pennask Lake/ Spahomin Creek Rainbow Trout 17 used i n an outdoor enclosure at the Fraser V a l l e y Hatchery (see Figs. 3 and 4)- M o d i f i c a t i o n s to e n s u r e a r e l a t i v e l y u n i f o r m r e c t i l i n e a r and m i c r o t u r b u l e n t f l o w p r o f i l e were made ( F i g s . 5 and 6). At c o n t r o l l e d v e l o c i t i e s 6400 l i t e r s of water were recycled through the tunnel. The tunnel t e s t section, 160 cm i n length, was enclosed i n a black p l a s t i c cover with a v i e w i n g booth to prevent e x c i t i n g the f i s h . A downstream e l e c t r i f i e d (3 v o l t s AC) screened b u t t e r f l y valve was used to capture fatigued f i s h . A f i s h was c o n s i d e r e d f a t i g u e d when i t was unable to remove i t s e l f from t h i s s c r e e n . To d i s c o u r a g e f i s h from r e s t i n g on the bottom, an e l e c t r i f i e d (3 v o l t s AC) f l o o r was i n s t a l l e d a l o n g the tunnel's h o r i z o n t a l d i a m e t e r . The f l o o r was constructed out of s t a i n l e s s s t e e l bars s i t u a t e d p a r a l l e l to the water f l o w ( i . e . l e n g t h w i s e i n the tube) and r e s t i n g on a minimum o f P l e x i g l a s cross-bars. This arrangement minimized drag due to the presence of the f l o o r (Figs. 5 and 6). This f l o o r was turned on only when necessary, as were two e l e c t r i c f i e l d s (6 v o l t s DC) p l a c e d at the f r o n t and back o f the t e s t section. These f i e l d s prevented f i s h from r e s t i n g i n "slow spots" which formed near the walls i n these two areas at the higher flow v e l o c i t i e s . The water used f o r t e s t s was Fraser V a l l e y Hatchery w e l l water. However since t h i s water was taken from a l i n e preceding the aeration tower, water had to be oxygenated by b u b b l i n g compressed oxygen through the system u n t i l the d i s s o l v e d oxygen reached at l e a s t 8 ppm. D i s s o l v e d oxygen was m a i n t a i n e d between 8 ppm and s a t u r a t i o n during tests. Temperature f o r he duration of any one t e s t never v a r i e d more than 1oC. Temperature between t e s t s v a r i e d with the time of year, despite the w e l l water source, ranging from 7 to 15oC (see Table 1). Water v e l o c i t y was monitored i n the t e s t tunnel area with a s m a l l S t r e a m f l o Probe (Nixon I n s t r u m e n t a t i o n , Ltd.). A more thorough d e s c r i p t i o n o f the swim t u n n e l may be o b t a i n e d from the I n t e r n a t i o n a l P a c i f i c Salmon Commission, New Westminster, B.C. Canada. 1 8 FIGURE 3. Schematic diagram of swim t u n n e l , top view. S c a l e : 3 mm = 1 dm. Arrows i n d i c a t e c o u n t e r c l o c k w i s e d i r e c t i o n o f water flow. A) upstream r e s e r v o i r , B) downstream r e s e r v o i r , C) base p l a t f o r m , D) 3.2 mm mesh sc r e e n s , E) 5.0 cm diameter P l e x i g l a s t u b i n g , F) 1.6 mm mesh s c r e e n , G) 6 v o l t s DC e l e c t r i c f i e l d , I) 3 v o l t s AC e l e c t r i f i e d s c reened b u t t e r f l y v a l v e , J) f i s h chamber, K) thermometer, L) fl o w meter, M) b a f f l e , N) 6.4 mm mesh s c r e e n , 0) 1.6 mm mesh s c r e e n , P) 3.2 mm mesh screen, QJ water pump, R) b e l t s , S) 5 hp e l e c t r i c motor and transmission unit. 19 FIGURE 4» Schematic diagram of swim t u n n e l , s i d e view of f i s h chamber. Scale: 3 mm = 1 dm. Arrows i n d i c a t e d i r e c t i o n of water flow. A) upstream r e s e r v o i r , B) downstream r e s e r v o i r , C) base p l a t f o r m , D) 3.2 mm mesh sc r e e n s , E) 5'0 cm d i a m e t e r P l e x i g l a s t u b i n g , F) 1.6 mm mesh s c r e e n , G) 6 v o l t s DC e l e c t r i c f i e l d , H) 3 v o l t s AC e l e c t r i f i e d f l o o r , I) 3 v o l t s AC e l e c t r i f i e d screened b u t t e r f l y valve, J) f i s h chamber, K) thermometer, L) flow meter. 21 22 FIGURE 5. Fish chamber water flow profile at transmission setting 1 (slow current). The range of flow values i s plotted for each point measured in the vertical profile within the fis h chamber, starting with the flow at the l e v e l of the e l e c t r i c f l o o r , and ending with the flow at the top of the fish chamber tube. 23 CM / SEC MINIMUM VALUES MAXIMUM VALUES 24 F I G U R E 6. F i s h c h a m b e r w a t e r f l o w p r o f i l e a t t r a n s m i s s i o n s e t t i n g 4 ( f a s t c u r r e n t ) . T h e r a n g e o f f l o w v a l u e s i s p l o t t e d f o r e a c h p o i n t m e a s u r e d i n t h e v e r t i c a l p r o f i l e w i t h i n t h e f i s h c h a m b e r , s t a r t i n g w i t h t h e f l o w a t t h e l e v e l o f t h e e l e c t r i c f l o o r , a n d e n d i n g w i t h t h e f l o w a t t h e t o p o f t h e f i s h c h a m b e r t u b e . 25 MINIMUM VALUES MAXIMUM VALUES 26 The t e s t i n g procedure used i s a m u l t i - f i s h v e r s i o n of t h a t used by-B r e t t (1964). B e f o r e the s t a r t o f each t e s t , 6 to 1 2 f i s h of a p p r o x i m a t e l y 17 to 18 cm were p l a c e d i n the t u n n e l f o r a 12 to 14 hour ( o v e r n i g h t ) a c c l i m a t i o n p e r i o d , at a t r a i n i n g speed of 10 cm/s. In f a c t , 10 to 12 f i s h were a c t u a l l y placed into the tunnel at the s t a r t of the a c c l i m a t i o n period. However the a c t u a l number of t e s t f i s h v a r i e d because not a l l o f the f i s h would s u r v i v e the 12 to 14 hrs. T h i s was e s p e c i a l l y the case w i t h the steelhead s t r a i n , which was by f a r the most aggressive and t e r r i t o r i a l group (T a b l e 1). The e l e c t r i c s c r e e n was not turned on u n t i l the s t a r t o f a t e s t . As the t e s t f i s h had not been fed f o r 24 hrs p r i o r to the a c c l i m a t i o n period, the t o t a l f a s t i n g period before t e s t i n g ranged from 36 to 38 hrs. The t r i a l was s t a r t e d by elevating the flow to 20 cm/s. Hereafter the flow was increased 10 cm/s every 75 minute period. When a f i s h fatigued, the time i n t o the p e r i o d was noted so t h a t swim st a m i n a c o u l d be determined u s i n g the c r i t i c a l swimming speed ( U o r i t ) f o r m u l a . U c r i t ( b o d y 1 engths/second) = [A + B (Tout/75)J/LGT, where A = cm/s of the l a s t f u l l 75 minute p e r i o d or the 10 cm/s of the a c c l i m a t i o n p e r i o d ; B = the 10 cm/s i n t e r v a l increase; Tout = time into the period when the f i s h fatigued; LGT = the f o r k l e n g t h o f the f a t i g u e d f i s h . F a t i g u e d f i s h were p r o m p t l y removed and sampled f o r blood and tissues according to the f o l l o w i n g protocol. Blood and Tissue Sampling Protocol The s a c r i f i c e d (baseline sampling) or fatigued (exercise sampling) f i s h was b l o t t e d dry, and then measured (FL) and weighed. A c o n d i t i o n f a c t o r was c a l c u l a t e d according to: CF = (WGT x 100)/LGT3 ( L e i t r i t z & Lewis 1980). A 1 cc s y r i n g e w i t h a 26 or 27 gauge needle was then used to sample blood v e n t r a l l y from the c a u d a l blood v e s s e l s . The body of the f i s h was then sampled f o r t i s s u e s (see below) simultaneously. The blood was transferred to 27 a h e p a r i n i z e d m i c r o t e s t tube, from which the sample was d i v i d e d up, as foll o w s , using a micropipettor: (1) 60 u l was put i n t o a cryotube c o n t a i n i n g 120 u l of i c e c o l d t r i c h l o r o a c e t i c a c i d (TCA) f o r enzymatic NTP determination. (2) 100 u l was added to a cryotube c o n t a i n i n g 100 u l of i c e c o l d TCA f o r separation of ATP and GTP by t h i n l a y e r chromatography (TLC). (3) 70 u l was put i n t o a micro-hematocrit tube, centrifuged f o r 5 min, and hematocrit determined. The tube was broken at the buffy coat, separating the plasma and erythrocytes. (3a) The plasma was put i n t o a sample cup, from which 20 to 40 u l was pipetted into a cryotube containing 60 to 80 u l of i c e cold p e r c h l o r i c acid (PCA), f o r plasma l a c t a t e assays. (3b) For " e x e r c i s e sampling" o n l y : the e r y t h r o c y t e s were added to a micro test tube containing i c e cold Alsever's s o l u t i o n (Kolmer 1949)- These c e l l s were subsequently washed twice with i c e cold Alsever's s o l u t i o n , and were then f r o z e n i n a c r y o t u b e c o n t a i n i n g 200 u l of i c e c o l d p r e s e r v i n g s o l u t i o n (Sharp 1969) f o r hemoglobin (Hb) electrophoresis. (4) 20 u l were p i p e t t e d i n t o an i c e c o l d c r y o t u b e f o r subsequent Hb concentration determination. (5) 70 u l was put into a micro test tube containing i c e cold Alsever's sol u t i o n . These c e l l s were l a t e r washed and stored l i k e those i n step (3b), but f o r LDH a c t i v i t y assays. Tissues were sampled at the same time as the blood i n the f o l l o w i n g way: (6) B r a i n , (7) l i v e r , and (8) epaxial white muscle (removed from above the midline equidistant between the dorsal and adipose fins) was sampled and then promptly freeze-28 clamped i n l i q u i d nitrogen (W ollenberger et a l . 1960) f o r subsequent l a c t a t e i assays. (9) Another l i v e r sample from the same f i s h was then taken and frozen i n l i q u i d nitrogen for LDH assays. A l l blood cryotubes and t i s s u e samples were stored i n l i q u i d nitrogen f o r transport back to U.B.C. Lactate t i s s u e samples were pro m p t l y homogenized w i t h a P o l y t r o n t i s s u e horaogenizer i n 5 volumes o f PCA, c e n t r i f u g e d at 30,000g and 40C f o r 30 min, and the supernatant s t o r e d i n an u l t r a l o w f r e e z e r at -70oC. A l l blood cryotubes and the r e m a i n i n g l i v e r LDH samples were a l s o s t o r e d at U.B.C. i n a -70oC f r e e z e r . T i s s u e s s t o r e d f o r enzyme a c t i v i t y a ssays i n t h i s manner show no s t o r a g e time dependent changes (Childress & Somero 1979)« Biochemical Assays (numbers correspond to those of previous section) (1) Erythrocyte NTP was determined enzymatically according to Jaworek et a l . (1974) u s i n g a PGK/GAPDH system. Samples were s t o r e d and thawed according to Brewer and Knudsen (1966). Instead of n e u t r a l i z i n g the TCA, i t was removed with three 1 ml d i e t h y l ether extractions. Residual ether was removed by a 5 min immersion i n a 7O0C water bath (Gilman & Murad 1 974)- The v a l u e f o r whole b l o o d NTP was d i v i d e d by the h e m a t o c r i t to g i v e the erythrocyte NTP concentration. (2) The r e l a t i v e c o n c e n t r a t i o n of ATP and GTP i n e r y t h r o c y t e s was determined by the methods of Johansen et a l . (1976) using P E I - c e l l u l o s e TLC plates. Samples were stored, thawed, and TCA removed as above. One hundred u l samples were applied to the TLC plates. Erythrocyte concentration of ATP, GTP, and NTP were again c a l c u l a t e d as above. (3a), (6), (7) and (8). The f r o z e n PCA e x t r a c t s o f plasma (from s t e p 3a), b r a i n (6), l i v e r (7), and white muscle t i s s u e (8) were assayed 29 a c c o r d i n g to Sigma B u l l e t i n 826-UV (1976) u s i n g Sigma reagents. F i r s t the samples were thawed f o r 5 to 10 min i n a 37oC waterbath. Plasma samples were then c e n t r i f u g e d at 30,000g and 40C f o r 30 min to remove the denatured plasma pr o t e i n residue. The PCA i n a l l sample supernatants was ne u t r a l i z e d with i c e cold 3.0 M K2C03 and 0-5 M triethanolamine-HCl to pH 7»0 (Driedzic & Hochachka 1975). A f t e r 10 min on i c e , the samples were c e n t r i f u g e d at 30,000g and 40C f o r 30 min. The neutralized supernatants were then assayed by the Sigma method modified to include 0.2% EDTA i n the assay medium. (3b) Hb e l e c t r o p h o r e s i s was done i n the f o l l o w i n g manner. The s t o r e d samples were lysed with 1 ml i c e cold deionized water f or 1 hr. I n d i v i d u a l samples were c e n t r i f u g e d at 15»600g and 40C f o r 30 min, the 1 ml Hb s u p e r n a t a n t s removed, and each converted to the cyanomethemoglobin d e r i v a t i v e by the a d d i t i o n of 200 u l of 2.0% K3Fe(CN)6' °* 5^ K C N ' NaHCOj s o l u t i o n (Moss & Ingram 1968). The samples were l e f t standing on i c e f o r 15 min, and then d i a l y s e d with 4 ml o f d i l u t e d i a l y s i s b u f f e r : 10 mM K H 2 P 0 4 , 0.1 mM EDTA, 0.2 mM KCN, pH 8.0 (Giovenco et a l . 1970, H j e r t e n e t a l . 1965) i n C e n t r i f l o membrane cones, type CF 25 (Amicon Corp.). The samples were spun i n the cones f o r 30 min at 1000g and 40C. The dialysed Hb was then combined w i t h an equal volume (approx. 300 u l ) o f i c e c o l d g l y c e r o l . The system of P o u l i k (1 957) was adapted to a 7.3% p o l y a c r y l a m i d e g e l employing the v e r t i c a l m i n i - g e l apparatus made by the Idea S c i e n t i f i c Co. (T a b l e 2). The mixed g e l components were degassed b r i e f l y (< 5 min), added to the g e l apparatus, and l e f t to p o l y m e r i z e at 40 to 50oC. Samples were a p p l i e d to the g e l i n 5 u l volumes v i a a 1 0 u l H a m i l t o n s y r i n g e . Gels were run at 4 mA (40 to 60 V) and 40C f o r 90 min. B e n z i d i n e d i h y d r o c h l o r i d e (Sigma Chem i c a l Co.) and the methods of B r o y l e s et a l . (1979) were used to s t a i n the Hb bands so that densitometric q u a n t i f i c a t i o n could be c a r r i e d out 30 TABLE 2. Stock solutions f o r 7.5$ polyacrylamide gels. Volume Solution Ratio Components 7.5% g e l , pH 9.0 A 2 30 g acrylamide 0.8 g bis-acrylamide to 100 ml w/ deionized H2° B 1 0.48 M TRIS, 5-82 g 2.4 mM c i t r i c acid, 52 mg 0.17 ml TEMED to 100 ml w/ deionized H^Q C 5 82 mg ammonium persulfate 100 ml deionized E^Q electrode b u f f e r , pH 10.0 D 20x 0.06 M boric acid, 3.71 g d i l u t i o n NaOH to pH 10.0 before to 1 L w/ deionized H2^ use 31 using a Beckman DU-8 spectrophotometer with g e l scan compuset. The scanned peaks were then cut out and weighed to determined the percentage of non-Bohr/Root and Bohr/Root hemoglobins. (4) Hb concentration was determined spectrophotometrically according to Riggs (1981) by c o n v e r s i o n to cyanomethemoglobin. Mean c e l l u l a r Hb c o n c e n t r a t i o n was c a l c u l a t e d by d i v i d i n g by the h e m a t o c r i t (Turner et a l . 1983). (5) and (9)« For d e t e r m i n a t i o n o f LDH B2 l a c t a t e o x i d a t i o n a c t i v i t i e s and genotypes, the f r o z e n l i v e r (from step 9) was homogenized i n a known volume of i c e c o l d g r i n d i n g s o l u t i o n (50 mM t r i e t h a n o l a m i n e - K O H , 2.0 mM MgC12» 1-0 mM EDTA, 2-0 mM DTT, to pH 7.5 with KOH ( C r a b t r e e et a l . 1979)) using "Duall type 22" c o n i c a l glass homogenizers with ground-glass contact s u r f a c e s . The homogenates were c e n t r i f u g e d at 2500g and 40C f o r 10 min. Twenty-five u l of supernatant was d i l u t e d 16-fold, and the rest was saved f o r e l e c t r o p h o r e s i s . The f r o z e n e r y t h r o c y t e s o l u t i o n (from step 5) was l y s e d by thawing and the a d d i t i o n o f 250 u l o f i c e c o l d g r i n d i n g s o l u t i o n . I t was then c e n t r i f u g e d at 2500g and 40G f o r 10 min. The s u pernatant r e q u i r e d no f u r t h e r d i l u t i o n . Both l i v e r and e r y t h r o c y t e samples were d i l u t e d to ensure l i n e a r r e a c t i o n r a t e s f o r at l e a s t 5 min. The r e a c t i o n medium consisted of 50 mM immidazole buffer, pH 7-5, 2.0 mM NAD, and 5 u l of l i v e r extract or 40 u l of erythrocyte extract. The r e a c t i o n was s t a r t e d by the a d d i t i o n of n e u t r a l i z e d (KOH) L - l a c t i c a c i d (50 mM i n c u v e t t e ) (T. Mommsen, personal communication). T o t a l assay volume was 2.0 ml. A l l assays were done at room temperature (22oC) i n a Beckman DU-8 spectrophotometer. L i v e r LDH a c t i v i t y was d e t e r m i n e d as i n t e r n a t i o n a l u n i t s (umoles l a c t a t e converted to pyruvate per minute) per gram wet weight of tissue. Erythrocyte LDH a c t i v i t y was c a l c u l a t e d by d i v i d i n g whole blood LDH a c t i v i t y by the hematocrit, with both q u a n t i t i e s being expressed as i n t e r n a t i o n a l units per ml of whole blood or erythrocytes. The r e m a i n i n g l i v e r homogenate supernatants from above were furt h e r centrifuged at 30,000g and 40C f o r 15 min, y i e l d i n g c l e a r supernatants f o r electrophoresis. Starch g e l electrophoresis was performed by the techniques of W i l l i s c r o f t & Tsuyuki (1970), modified by destaining the gels i n a 5:5:1 s o l u t i o n of deionized water: methanol : g l a c i a l a c e t i c acid. S t a t i s t i c a l Analysis A s i n g l e f a c t o r f o u r sample Model I a n a l y s i s o f v a r i a n c e f o r uneven sample s i z e s was used to d e t e c t s i g n i f i c a n t d i f f e r e n c e s between the four s t r a i n s i n e i t h e r the b a s e l i n e or e x e r c i s e d s t a t e f o r each o f the f a c t o r s studied. The c r i t i c a l l i m i t f o r s i g n i f i c a n c e (alpha) was set at P = 0.05- I f the s t r a i n s were found to be s i g n i f i c a n t l y d i f f e r e n t , the Dunn-Sidak method o f p a i r w i s e c o m p a r i s o n was used to d e t e r m i n e w h i c h s t r a i n s were s i g n i f i c a n t l y d i f f e r e n t . This method employs an experimentwise error rate, whereby alpha' f o r an a l p h a = 0.05 w i t h s i x p a i r comparisons i s e q u a l to 0.00851. For a g i v e n f a c t o r and s t r a i n , b a s e l i n e v a l u e s were compared w i t h e x e r c i s e d v a l u e s ( f o l d change) u s i n g a s i n g l e f a c t o r two sample Model I a n a l y s i s of v a r i a n c e f o r uneven sample s i z e s . An a l p h a o f 0.05 was used to determine the l i m i t of s i g n i f i c a n c e . LDH B2 genotype frequencies were compared to expected frequencies f o r Hardy-Weinberg e q u i l i b r i u m u s i n g a c h i - s q u a r e a n a l y s i s w i t h d.f. = 1 and alpha = 0.05. A l l e l e frequencies were tested f o r independence of s t r a i n type by a 4 x 2 c o n t i n g e n c y t e s t w i t h d.f. = 3 and a l p h a = 0.05 ( S o k a l & R o h l f 1981, Kincaid 1982). A l l data are given as means +_ standard errors, except f o r f o l d changes which are c a l c u l a t e d by d i v i d i n g the exercised value by the baseline value. RESULTS C r i t i c a l Swim V e l o c i t y Rainbow t r o u t s t r a i n s o f h a t c h e r y - k e p t b r o o d s t o c k s have a s i g n i f i c a n t l y lower c r i t i c a l swim speed than s t r a i n s r a i s e d from wild brood s t o c k s ( T able 3 ) . The Pennask and Deadman s t r a i n s have s i m i l a r U c r ; j _ ^ g Qf about 2.0 BL/S (i.e. body lengths/sec), which are s i g n i f i c a n t l y greater than the a p p r o x i m a t e l y 1.2 BL/S U c r i t s o f t h e D o m e s t i c and Duncan s t r a i n s . As expected the Domestic s t r a i n had a s i g n i f i c a n t l y g r e a t e r c o n d i t i o n f a c t o r ( i . e . CP) than the Duncan s t r a i n , which i n t u r n was s i g n i f i c a n t l y g r e a t e r than the : Pennask and Deadman s t r a i n s (Table 3)« Many; s i g n i f i c a n t d i f f e r e n c e s were found on the metabolic l e v e l between the four s t r a i n s (Tables 4 through 8 ) . These d i f f e r e n c e s between s t r a i n s are a l l of genetic o r i g i n because of the constant environment, but they are not a l l c o r r e l a t e d with the observed swim stamina differences. Lactate Accumulation Some s i g n i f i c a n t d i f f e r e n c e s between s t r a i n s i n l a c t a t e accumulation f o r the baseline and exercised states were found (Table 4 ) . For the baseline state, no s i g n i f i c a n t d i f f e r e n c e s were observed i n plasma l a c t a t e . For brain l a c t a t e , there was no d i f f e r e n c e between the Pennask and Deadman s t r a i n s , but both these s t r a i n s had s i g n i f i c a n t l y h i g h e r l e v e l s than the Domestic s t r a i n , which was s i g n i f i c a n t l y h i g h e r than the Duncan v a l u e . "Wild" s t r a i n s had approximately two times the baseline l e v e l of br a i n l a c t a t e that "hatchery" s t r a i n s did. The Deadman s t r a i n stood out i n l i v e r l a c t a t e with a s i g n i f i c a n t l y h i g h e r v a l u e than the o t h e r three s t r a i n s . The same can be s a i d f o r white muscle l a c t a t e , except t h a t the Deadman s t r a i n was not s i g n i f i c a n t l y d i f f e r e n t from the Duncan. 3 4 TABLE 3. Comparison of c r i t i c a l swimming speeds ( U c r i t ) a n d c o n d i t i o n factors (CF) between four s t r a i n s of rainbow trout. A l l values are means +_ S.E.M. ( U c r i t = b Q d y i e n g t h s / s e c , CF = g/cm3). For S t r a i n Comparison: * = s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) from Pennask s t r a i n x = s i g n i f i c a n t l y d i f f e r e n t from Deadman s t r a i n + = s i g n i f i c a n t l y d i f f e r e n t from Domestic s t r a i n # = s i g n i f i c a n t l y d i f f e r e n t from Duncan s t r a i n N.S. = not s i g n i f i c a n t l y d i f f e r e n t U c r i t CF BASELINE PENNASK 0-99+0.02 (N=16) +#"• DEADMAN 1.06+0.01 +r DOMESTIC 1.32_+0.02 *x# DUNCAN 1.18+0.02 *x+ EXERCISED PENNASK 1-98+0.08 1.02+0.03 (18<N<20) +? +? DEADMAN 1-91+0.10 1-09+0.03 +? DOMESTIC 1 . 3 6 + 0 . 0 9 1-60+0-03 *x *x# DUNCAN 1.10+0.06 1.1 9+0.01 *x *x+ 35 TABLE 4« Comparison of l a c t a t e a c c u m u l a t i o n i n plasma, b r a i n , l i v e r and white muscle between four s t r a i n s of rainbow trout. A l l values are means _+ S.E.M. Fold change from baseline to exercised state also indicated f o r each st r a i n , (plasma l a c t a t e = mM, tissu e l a c t a t e = umole/g). For S t r a i n Comparisons: * = s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) from Pennask s t r a i n x = s i g n i f i c a n t l y d i f f e r e n t from Deadman s t r a i n + = s i g n i f i c a n t l y d i f f e r e n t from Domestic s t r a i n * = s i g n i f i c a n t l y d i f f e r e n t from Duncan s t r a i n N.S. = not s i g n i f i c a n t l y d i f f e r e n t For Fold Change: * = exercised state s i g n i f i c a n t l y d i f f e r e n t from baseline state (P < 0.05) N.S. = not s i g n i f i c a n t l y d i f f e r e n t Plasma Brain L i v e r White Muscle BASELINE PENNASK 0.85+0.14 7.69+0.43 1.06+0.16 14-17+_0.49 (N=16) N.S. +#" X X DEADMAN 0.70+0.17 7.39+0.42 2.91+0.23 21.13+1.41 N.S. +# *+# . * + DOMESTIC 0.60+0.04 4-40+0.26 0.88+0.07 15.86+0.86 N.S. *x# x# X DUNCAN 0.78+0.12 3- 25+0.27 1.42+0.16 16.85+1.18 N.S. *x+ x+ N.S. EXERCISED PENNASK 6.07+_0.96 8.28+0.67 3.88+0.46 29.90+1.49 (17<N<20) + + + +# DEADMAN 6.30+_0.40 6.92+0.49 4.46+0.32 27.35+1.52 + + + +#~ DOMESTIC 11.54+0.96 11.10+0-49 6.65+0-54 37-06+0.96 *x *x# *x# *x DUNCAN 8.55+0.86 7.86+0.52 4.48+0.35 36.01+1.22 N.S. + + *x FOLD PENNASK 7.14 * 1.08 N.S. 3.66 * 2.11 * CHANGE DEADMAN 9-00 * 0-94 N.S. 1.53 * 1.29 * DOMESTIC 19.23 * 2.52 * 7-56 * 2.34 * DUNCAN 10.96 * 2.42 * 3-15 * 2.14 * 36 F o r the e x e r c i s e d s t a t e , i t appears t h a t the Domestic and Duncan s t r a i n s develop higher plasma l a c t a t e l e v e l s than do the two " w i l d " s t r a i n s , even though the Duncan s t r a i n ' s i s not s i g n i f i c a n t l y h i g h e r . The Domestic s t r a i n i s , however, s i g n i f i c a n t l y higher. For b r a i n l a c t a t e , the Domestic s t r a i n i s much higher than the other three s t r a i n s . More i n t e r e s t i n g i s the maintenance of b r a i n l a c t a t e at baseline l e v e l s f o r the two " w i l d " s t r a i n s . When the f o l d change v a l u e s are compared, there i s no s i g n i f i c a n t change between baseline and exercised f o r the Pennask and Deadman, but a 2.5 f o l d change f o r the Domestic and Duncan. The l i v e r l a c t a t e v a l u e s f o r the Domestic s t r a i n are a l s o s i g n i f i c a n t l y h i g h e r than f o r the r e s t o f the s t r a i n s . The Deadman s t r a i n ' s f o l d change value f o r l i v e r l a c t a t e does i n d i c a t e a s i g n i f i c a n t change from baseline to exercised, however i t i s much lower than the o t h e r t h r e e s t r a i n s ' . The same can be s a i d f o r the Deadman s t r a i n ' s white muscle l a c t a t e f o l d change. T h i s i s due to h i g h b a s e l i n e values i n both cases. Exercised white muscle l a c t a t e i s s i g n i f i c a n t l y higher i n the two "hatchery" s t r a i n s than i n the two "wild" s t r a i n s . LDH B2" Genotypes Table 5 shows the amount o f LDH B2 a c t i v i t y p r e s e n t , as w e l l as the a l l e l e f r e q u e n c y at the LDH B2 l o c u s . The two "hatchery" s t r a i n s show no polymorphism at the LDH B2 locus. The Domestic s t r a i n s o l e l y possesses the B2' a l l e l e , and the Duncan s t r a i n the B2" a l l e l e . The two " w i l d " s t r a i n s are p o l y m o r p h i c , w i t h the B2" a l l e l e d o m i n a t i n g i n the Pennask and the B2* a l l e l e dominating i n the Deadman s t r a i n . The a l l e l e frequency values are i n agreement w i t h those from Huzyk & Tsuyuki ( 1 9 7 4 ) , as w e l l as w i t h the concept that those populations o r i g i n a t i n g from i n t e r i o r headwater areas tend to have more of the LDH B2" a l l e l e . The Duncan s t r a i n o r i g i n a t e s from the most i n t e r i o r area. A l l four s t r a i n s are i n Hardy-Weinberg e q u i l i b r i u m 3 7 TABLE 5« Comparison of LDH B2 a l l e l e f r e q u e n c i e s , l i v e r and e r y t h r o c y t e RBC) LDH B2 l a c t a t e oxidation a c t i v i t i e s between four s t r a i n s of rainbow rout. A l l values are means +_ S.E.M. Fold change from baseline to exercised state also i n d i c a t e d f o r each s t r a i n , ( l i v e r LDH = I.U./g wet wgt, blood LDH = I.U./ml whole blood (WB) or RBCs, hematocrit (HCT) = % ) . For S t r a i n Comparisons: * = s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) from Pennask s t r a i n x = s i g n i f i c a n t l y d i f f e r e n t from Deadman s t r a i n + = s i g n i f i c a n t l y d i f f e r e n t from Domestic s t r a i n * = s i g n i f i c a n t l y d i f f e r e n t from Duncan s t r a i n N.S. = not s i g n i f i c a n t l y d i f f e r e n t For Fold Change: * = exercised state s i g n i f i c a n t l y d i f f e r e n t from baseline state (P < 0.05) N.S. = not s i g n i f i c a n t l y d i f f e r e n t For LDH B2 A l l e l e Frequencies: - A l l 4 s t r a i n s ' observed g e n o t y p i c f r e q u e n c i e s not s i g n i f i c a n t l y d i f f e r e n t (P > 0.05) from expected Hardy-Weinberg d i s t r i b u t i o n . - A l l e l e frequency i s s i g n i f i c a n t l y dependent (P < 0.05) on s t r a i n type. - Sample s i z e s range from 3 4 to 4 3 f i s h per st r a i n . L i v e r A l l e l e Freq RBC WB LDH B2' B2" LDH LDH HCT BASELINE PENNASK 108.20+3-13 -324 -676 2.52jK).05 1.00+0.02 39.6411.06 (N=16) x+#~ +0.057 + x+# x+ DEADMAN 1 70.18 + 5*00 .616 -384 2.2510.09 0.79+0.04 35» 31>1 • 12 *+# +0.052 + *+ *+ DOMESTIC 61-31 +2.1 1 1.00 .000 1.7310.06 0.50_+0.02 28.74^0.84 *x# +0.000 *x# *x# *x# DUNCAN 136.61+3.14 -000 1,00 2.44^0.05 0.89 + 0.03 36.46^1.32 *x+ +0.000 + "*+ . + EXERCISED PENNASK 151.92i5.25 1-91 +0.05 0-9910.02 52.3311-35 (17<H<20) +# x+? x+# # DEADMAN 1 50.64+5-74 1.56+0.06 0.78 + 0.03 50.4011.47 +# - ~* ~* N.S. DOMESTIC 86.48+1.78 1.3910.05 0.6810-03 47-4411-61 *xf * * N.S. DUNCAN 109.20+3.1 1 1-50+0-05 0.69+0.03 46.3111-01 FOLD PENNASK 1.40 * 0-76 * 0-99 N.S. 1-32 * CHANGE DEADMAN 0.89 * 0.69 * 0.99 N.S. 1.43 * DOMESTIC 1.41 * 0.80 * 1.36 * 1.65 * DUNCAN 0.80 * 0.61 * 0-78 * 1.27 * 38 f o r the LDH B2 l o c u s . T h i s would i n d i c a t e t h a t the s t r a i n p o p u l a t i o n s ' LDH B2 genotypic frequencies are the product of random mating, and that both LDH B2 a l l e l e s are e q u a l l y competent i n making c o p i e s o f themselves (Futuyma 1979)* T h e r e f o r e , based on each s t r a i n ' s LDH B2 a l l e l i c f r e q u e n c i e s , the observed genotypic frequencies comply with the expected. A l l e l e frequency i s s i g n i f i c a n t l y dependent on the s t r a i n type, meaning t h a t each s t r a i n i s a unique gene pool with respect to the LDH B2 locus. LDH B2" A c t i v i t i e s In the b a s e l i n e s t a t e , each s t r a i n has a l i v e r LDH a c t i v i t y t h a t i s s i g n i f i c a n t l y d i f f e r e n t from the other three s t r a i n s , but not c o r r e l a t e d to swim stamina d i f f e r e n c e s . The Domestic s t r a i n , however, does have a much lower a c t i v i t y than any of the others. Erythrocyte LDH i s also s i g n i f i c a n t l y lower f o r the Domestic s t r a i n than any o t h e r s t r a i n . Whole blood LDH r e f l e c t s the h e m a t o c r i t , as w e l l as the RBC LDH v a l u e . T h i s v a l u e does not i n c l u d e plasma LDH, which has i t s o r i g i n s from c e l l r u p t u r i n g i n v a r i o u s organs and tissues. Hence the Domestic s t r a i n has s i g n i f i c a n t l y the lowest value, and the Pennask the highest. In the e x e r c i s e d s t a t e , the two " w i l d " s t r a i n s have s i m i l a r LDH a c t i v i t i e s which are s i g n i f i c a n t l y higher than the Duncan's, which i n turn i s s i g n i f i c a n t l y h i g h e r than the Domestic's. The " w i l d " s t r a i n s have at : l e a s t 50% more l i v e r LDH a c t i v i t y than the "hatchery" s t r a i n s . A l l four: s t r a i n s show a s i g n i f i c a n t change i n l i v e r LDH a c t i v i t y from b a s e l i n e to exercised. The Pennask and the Domestic increase by a f a c t o r of 1.4, and the Deadman and the Duncan decrease by a f a c t o r of 0.a RBC and whole blood LDH i s s i g n i f i c a n t l y d i f f e r e n t and higher f o r the Pennask s t r a i n . For these two categories there i s a p a r t i a l l y s i g n i f i c a n t c o r r e l a t i o n with swim stamina, 39 meaning the two h i g h e s t v a l u e s are always the two " w i l d " s t r a i n s , but the Deadman are not s i g n i f i c a n t l y h i g h e r than the two "hatchery" s t r a i n s . The two " w i l d " s t r a i n s showed no change i n whole blood LDH a c t i v i t y from baseline to exercised, while the Domestic increased and the Duncan decreased ; s i g n i f i c a n t l y . Hemoglobin There are no s i g n i f i c a n t d i f f e r e n c e s between s t r a i n s with respect to% non-Bohr/Root hemoglobin types, baseline and exercised MCHC (Table 6). These r e s u l t s would i n d i c a t e t h a t the s i g n i f i c a n t d i f f e r e n c e s observed between • s t r a i n s i n hemoglobin c o n c e n t r a t i o n are m a i n l y due to d i f f e r e n c e s i n hematocrit. Baseline Hb f o r the Domestic s t r a i n i s s i g n i f i c a n t l y d i f f e r e n t and lower than the other three s t r a i n s . The same i s true f o r i t s hematocrit. In the e x e r c i s e d s t a t e , a p a r t i a l l y s i g n i f i c a n t c o r r e l a t i o n to swim stamina i s observed for Hb and HCT, as follows. Hemoglobin concentration i s the same f o r the two "wild" s t r a i n s , which are s i g n i f i c a n t l y higher than the Duncan s t r a i n . The Domestic s t r a i n a l s o has a lower v a l u e , but i t i s not s i g n i f i c a n t l y d i f f e r e n t from any of the other three s t r a i n s . For hematocrit, the rank order of values p a r a l l e l s that of U c r i t w i t h Pennask s i g n i f i c a n t l y d i f f e r e n t from Duncan. However Deadman and Domestic have intermediate HCT v a l u e s , which are not s i g n i f i c a n t l y d i f f e r e n t from any o t h e r s t r a i n ' s . Pennask and Duncan showed no change i n whole blood Hb values from baseline to exercised, while Deadman and Domestic increased s i g n i f i c a n t l y . E r y t h r o c y t i c Nucleoside Triphosphates Because o f the l a c k o f agreement between the b a s e l i n e n u c l e o s i d e t r i p h o s p h a t e v a l u e s (Table 7 ) d e t e r m i n e d by the two methods, none of the baseline and f o l d change comparisons w i l l be considered. Also the baseline 40 TABLE 6. Comparison of hemoglobin concentration (Hb), percent non-Bohr/Root type Hb {% Non-B/R), mean c e l l u l a r Hb c o n c e n t r a t i o n (MCHC) and h e m a t o c r i t (HCT) between four s t r a i n s of rainbow trout. A l l values are means +_ S.E.M. Fold change from baseline to exercised s t a t e also indicated f o r each s t r a i n . (Hb = mM whole blood, MCHC = mM RBCs, HCT = % ) . For S t r a i n Comparisons: * = s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) from Pennask s t r a i n x = s i g n i f i c a n t l y d i f f e r e n t from Deadman s t r a i n + = s i g n i f i c a n t l y d i f f e r e n t from Domestic s t r a i n * = s i g n i f i c a n t l y d i f f e r e n t from Duncan s t r a i n N.S. = not s i g n i f i c a n t l y d i f f e r e n t For Fold Change: * = exercised state s i g n i f i c a n t l y d i f f e r e n t from baseline state (P < 0.05) N.S. = not s i g n i f i c a n t l y d i f f e r e n t Hb % Non-B/R MCHC HCT BASELINE PENNASK 1.67+0.06 4-19+0.06 39-64+J.06 (N=16) + N.S. x+ DEADMAN 1-52+0.04 4-35+0.14 35-31+1.12 + N.S. *+ DOMESTIC 1.13+0.04 3.92+0.07 28.74+0.84 *x# N.S. *xF DUNCAN 1.54j^0.06 4.27+0.18 36.46+J.32 + N.S. + EXERCISED PENNASK 1.73+0.04 32.37+0.77 3.32+0.07 52-33+1-35 (17<N<20) # ~ N.S. N. S. # " DEADMAN 1-75+0.05 30.34+1.12 3-48+0.05 50.40+1.47 # N.S. N.S. N.S. DOMESTIC 1.61+0.03 30-94+0.78 3-33+0-06 47.44+L61 N. S. N.S. N. S. N.S. DUNCAN 1-54+0.03 31.02+1.03 3.32+0.04 46.31+1-01 *x N.S. N.S. * — FOLD PENNASK 1.04 N.S. 0.79 * 1.32 * CHANGE DEADMAN 1.15 * 0.80 * 1-43 * DOMESTIC 1.42 * 0.85 * 1-65 * DUNCAN 1.00 N.S. 0.78 * 1.27 * 41 TABLE 1. Comparison of whole blood (WB) and e r y t h r o c y t i c (RBC) NTP concentrations, and NTP/Hb r a t i o s between four s t r a i n s of rainbow trout, as determined e n z y r a a t i c a l l y (ENZ) and by TLC. A l l v a l u e s are means +_ S.E.M. F o l d change from b a s e l i n e to e x e r c i s e d s t a t e a l s o i n d i c a t e d f o r each s t r a i n . (NTP = mM whole blood or RBCs). For S t r a i n Comparison: * = s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) from Pennask s t r a i n x = s i g n i f i c a n t l y d i f f e r e n t from Deadman s t r a i n + = s i g n i f i c a n t l y d i f f e r e n t from Domestic s t r a i n * = ' s i g n i f i c a n t l y d i f f e r e n t from Duncan s t r a i n N.S. = not s i g n i f i c a n t l y d i f f e r e n t For Fold Change: * = exercised state s i g n i f i c a n t l y d i f f e r e n t from baseline state (P < 0.05) N.S. = not s i g n i f i c a n t l y d i f f e r e n t 42 ENZ TLC WB NTP RBC NTP NTP/Hb WB NTP RBC NTP NTP/Hb BASELINE PENNASK 3-2710-09 8.2510.13 1-9810.05 2-75+0.08 6.95 + 0.15 1.67+0.05 (N=16) + N.S. + + xf x DEADMAN 3- 06+^ 0.12 8.68+0.22 2.01+0.05 + *+ *+ DOMESTIC 2.42+0.09 8.41+0.12 2.15+0.04 1.99+0.10 6.88+0.17 1.76j»_0.04 * ~ N.S. * *xj x# x DUNCAN — — ----- 2.92j_0.16 8.CXD+0.27 1.92_+0.09 + *+ N.S. EXERCISED PENNASK 1.88+0.08 3.61+0.16 1.10+0.05 1.85+0.06 3.52+0.15 1.08+0.05 (17<N<20) x+W x+# x+I x#~ x+# x+# DEADMAN 2.55+0.14 5-07+0.24 1-45+0.06 2.27+0.11 4-51+0.19 1.29+0.05 •r *r *r *r *r *r DOMESTIC 2.39+0.12 5-05+0.21 I.5O+O.O6 2.16+0.12 4-57+0.20 1.36+0.06 *#- *r *r #- *r *r DUNCAN I.32+0.06 2.89_+0.15 0.8910.04 1-2910-06 2.83l0.15 0.8710-04 *x+ *x+ *x+ *x+ *x+ *x+ FOLD PENNASK 0.57 * O.44 * 0.56 * 0.67 * 0-51 * 0.65 * CHANGE DEADMAN 0.74 * 0.52 * 0.64 * DOMESTIC 0-99 N.S. 0.60 * 0.70 * 1-09 N.S. 0.66 * 0. 39 * DUNCAN O.44 * 0.35 * 0.68 * Deadman and Duncan samples f o r enzymatic NTP determination were l o s t . The e x e r c i s e d v a l u e s , however, concur. F o r the NTP/Hb r a t i o , as w e l l as whole blood NTP and ery t h r o c y t i c NTP, s i g n i f i c a n t d i f f e r e n c e s are present but not c o r r e l a t e d to U c r i t . Domestic and Deadman are s i m i l a r and s i g n i f i c a n t l y greater than Pennask, which i s s i g n i f i c a n t l y greater than Duncan. The Duncan s t r a i n i s the o n l y s t r a i n to have a NTP/Hb r a t i o t h a t i s l e s s than one. The % GTP (T a b l e 8) o f t h i s NTP a l s o y i e l d s 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 no c o r r e l a t i o n to swim stamina. In both the baseline and exercised states, the Deadman and Duncan are s i m i l a r and s i g n i f i c a n t l y higher than the Pennask and Domestic, which are s i m i l a r . 44 TABLE 8. Comparison of whole blood (WB) and e r y t h r o c y t i c (RBC) ATP and GTP concentrations, and percent GTP of t o t a l NTP between four s t r a i n s of rainbow trout. A l l values are means + S.E.M. Fold change from baseline to exercised s t a t e a l s o i n d i c a t e d f o r each s t r a i n . (ATP and GTP = mM whole blood or RBCs). For S t r a i n Comparisons: * = s i g n i f i c a n t l y d i f f e r e n t (P < 0.05) from Pennask s t r a i n x = s i g n i f i c a n t l y d i f f e r e n t from Deadman s t r a i n + = s i g n i f i c a n t l y d i f f e r e n t from Domestic s t r a i n * = s i g n i f i c a n t l y d i f f e r e n t from Duncan s t r a i n N.S. = not s i g n i f i c a n t l y d i f f e r e n t For Fold Change: * = exercised state s i g n i f i c a n t l y d i f f e r e n t from baseline state (P < 0.05) N.S. = not s i g n i f i c a n t l y d i f f e r e n t WB ATP RBC ATP WB GTP RBC GTP % GTP BASELINE PENNASK 2.55+0.07 6.45+0.1 1 0.20+0.02 0.50+0.05 7-04+0.67 (N=16) + X x r x? x? DEADMAN 2.72 + 0.11 7-73 + 0.1 9 0.34 + 0.01 0.96+0.04 1 1.03 + 0.31 + ~*+ "*+ «+ DOMESTIC 1.83 + 0.08 6.33+.0.1 4 0.1 6 + 0.02 0.56+0.05 7-97 + 0.60 *x# X x# x# x#" DUNCAN 2.57+0.16 7.01+0.26 0.35+0.01 0. 98 + 0.04 1 2.38 + 0.49 + N.S. *+ *+ *+ EXERCISED PENNASK 1.75 + 0.06 3.36 + 0.13 0.08 + 0.01 0.1 4 + 0.02 4.00 + 0.56 (17<N<20) x# - . x+#~ X xF x? DEADMAN 2.1 1 +0.1 0 4-1 9+0.1 8 0.1 6 + 0.02 0.32 + 0.03 . 7.1 1 +0.58 *# *# * * *+ DOMESTIC 2.05 + 0.10 4-35 + 0.18 0.10 + 0.02 0.22 + 0.04 4-54+0.59 # *# N.S. N.S. x? DUNCAN 1.17 + 0.05 2.56+_0.1 2 0.1 2+0.01 0.26 + 0.03 8.88 + 0.63 *x+ *x+ N.S. * #+ FOLD PENNASK 0.69 * 0.52 * 0.40 * 0.28 * 0.57 * CHANGE DEADMAN 0-78 * 0.54 * 0.47 * 0.33 * 0.64 * DOMESTIC 1.12 N.S. 0.69 * 0.62 * 0.39 * 0.57 * DUNCAN 0.46 * 0.37 * 0.34 * 0.27 * 0.72 * 45 DISCUSSION Swim Stamina of "Hatchery" vs. "Wild" S t r a i n s S t r a i n s of rainbow trout tested i n t h i s study that have been reared and bred s o l e l y i n the h a t c h e r y environment f o r s e v e r a l g e n e r a t i o n s possess s u b s t a n t i a l l y lower swim stamina than rainbow trout that o r i g i n a t e from wild brood s t o c k s but were h a t c h e r y r a i s e d . T h i s statement would be more d e f i n i t i v e i f , f o r example, i t had been possible to test Deadman steelhead and Pennask rainbow t h a t had been r e a r e d i n h a t c h e r i e s and bred from h a t c h e r y - k e p t brood s t o c k f o r s e v e r a l g e n e r a t i o n s . T h i s would have e l i m i n a t e d any i n t e r - s t o c k v a r i a t i o n t h a t may have e x i s t e d p r i o r to a r t i f i c i a l s e l e c t i o n by hatcheries. However, such s t r a i n s o r t h e i r converse, i.e. " w i l d " Domestic and Duncan s t r a i n s , were not availa b l e . S t i l l , between my study and the ones done by Thomas & Donahoo (1977) and Green (1964) i t would aeem that s t r a i n s which go through hatchery s e l e c t i o n procedures tend to d i s p l a y p o o r e r swim stamina. Thomas & Donahoo r e p o r t t h a t swimming performance o f New Zealand and Sand Creek s t r a i n s (both o f which a r e streamlined, slow growing and semi-wild) did not d i f f e r , but were superior to the M a n c h e s t e r s t r a i n ( d e e p - b o d i e d , f a s t g r o w i n g and " v e r y domesticated"). Two wild s t r a i n s of brook trout (Salvelinus f o n t i n a l i s ) from Honnedaga Lake and Long Pond O u t l e t ( b o t h i n the Adirondack Mountains, N.Y.), and one domestic ( B e r l i n ) s t r a i n were found to possess g e n e t i c d i f f e r e n c e s i n swimming s t a m i n a by Green (1 964)- At a l l body l e n g t h s the B e r l i n s t r a i n showed s i g n i f i c a n t l y lower stamina. I t was concluded that t h i s "could be the r e s u l t of i n t e n t i o n a l or inadvertent f i s h c u l t u r a l attempts at selection." The two wild s t r a i n s exhibited s i m i l a r high swim stamina values. Reduced f i g h t i n g capacity has also been noted f o r domesticated carp, when compared to equally sized wild carp (Beukema 1969). S e l e c t i o n i n Hatcheries f o r Hapid Growth Possible genetic reasons why the two "hatchery" s t r a i n s should perform so poorly are many. F i r s t l y , there i s much l e s s , i f any, s e l e c t i v e pressure for swim stamina i n the hatchery environment compared to the wild (Beamish 1978). A second p o s s i b l e reason i s t h a t t h e r e i s h i g h s e l e c t i o n f o r r a p i d growth r a t e , and t h i s : c a n be a c h i e v e d i n as l i t t l e as three g e n e r a t i o n s ( K i n c a i d et a l . 1977). I t i s t h e r e f o r e p o s s i b l e t h a t a r t i f i c i a l s e l e c t i o n f o r h i g h growth r a t e s might, from an enzymatic s t a n d p o i n t , c o n c u r r e n t l y preclude s e l e c t i o n f o r the b i o c h e m i c a l machinery n e c e s s a r y f o r h i g h swim s t a m i n a . F o r e x a m p l e , the o p t i m a l e n z y m a t i c p r o p e r t i e s f o r , and concentrations of, metabolic enzymes functioning i n a system geared f o r high growth (such as a Domestic rainbow t r o u t ) may not be the same as f o r a system geared f o r h i g h stamina and " n a t u r a l " growth, e.g. a Deadman s t e e l h e a d t r o u t , (Somero 1978, Z u c k e r k a n d l 1976a & b). In a sense, the Deadman s t r a i n c o u l d be a compromise i n p r o t e i n e v o l u t i o n to a c h i e v e the o p t i m a l balance between enzymatic p r o p e r t i e s f o r growth and stamina, y i e l d i n g maximum f i t n e s s f o r the w i l d organism (Somero 1978). Hence a r t i f i c i a l s e l e c t i o n f or only high growth could involve protein a l t e r a t i o n s which are good f o r growth, but at the same time not good f o r stamina. For example, i t has been shown that l i n e s of T r i b o l i u m castaneum selected f o r high growth have also undergone major changes i n c e l l u l a r response to growth (Medrano & G a l l 1976a & b). These l i n e s have l a r g e r c e l l s , more c e l l s , higher RM content, higher RNA:DNA r a t i o s , higher feeding e f f i c i e n c i e s , and a le s s a c t i v e turnover of metabolites. S i m i l a r changes, as well as decreaseu protein turnover rates, were also seen f o r l i v e r s and kidneys i n high growth s t r a i n s o f mice ( P r i e s t l e y & Robertson 1973). Whether t h i s change i n 47 m e t a b o l i c machinery a l s o r e s u l t s i n decreased s t a m i n a was not examined. However, the s e l e c t e d l i n e s o f T r i b o l i u m a l s o had l o w e r . i s o c i t r a t e dehydrogenase and LDH enzyme a c t i v i t i e s . This observation i s s i m i l a r to the Duncan's and e s p e c i a l l y the Domestic's tendency to have low LDH a c t i v i t i e s and low Hb l e v e l s . Consequently these s t r a i n s would have l e s s c a p a c i t y to o x i d i z e l a c t a t e and to t r a n s p o r t oxygen, e s p e c i a l l y d u r i n g e x e r c i s e . I t would seem that l i n e s and/or s t r a i n s of animals selected f o r high growth put more of t h e i r ingested energy i n t o anabolic s t r u c t u r a l growth and l e s s into the maintenance o f the b i o c h e m i c a l machinery a s s o c i a t e d w i t h e x e r c i s e t r a i n i n g (Hochachka 1961, Johnston & Moon 1980a & b). When increased growth rate i s a r t i f i c i a l l y selected, higher growth rates have been accompanied by a decrease i n energetic costs of maintenance metabolism (Lohn et a l . 1946, S t a n i e r & Mount 1972, Medrano & G a l l 1976a &b). B e i n g capable o f high swimming performance stems from an increased amount of energy put i n t o t h i s m e t a b o l i c maintenance s t a t e (Hochachka 1961, Johnston & Moon 1980a & b, Graham et a l . 1985, S i e b e n a l l e r & Somero 1982, Swezey & Somero 1982, Sie b e n a l l e r et a l . 1982, S u l l i v a n & Somero 1980, Hammond & Hickman 1966). It would be i n t e r e s t i n g to compare, f o r example, the a c t i v i t i e s o f o t h e r enzymes involved i n intermediary metabolism and t i s s u e b u f f e r i n g c a p a c i t i e s between these f o u r s t r a i n s . S i n c e ATP h y d r o l y s i s i s the major source o f hydrogen ions accumulated during exercise, and not l a c t a t e , white and red muscle ATPase and muscle b u f f e r i n g capacity should be looked into (Hochachka & Mommsen 1983)- In salmonids, h i g h speed b u r s t swimming i s performed a n a e r o b i c a l l y w h i l e s u s t a i n e d performance i s , f o r the most p a r t , a e r o b i c (Jones 1982). Because of t h i s the a c t i v i t i e s of c e r t a i n key enzymes involved i n aerobic energy production are worth examining i n white and/or red muscle. These would be, f o r example, c i t r a t e s y n t h e t a s e (an index o f c i t r i c a c i d cycle a c t i v i t y p o t e n t i a l ; Somero & Childress 1980), hexokinase (an index of 48 glucose uptake and oxidation; Newsholme 1980), glutamate dehydrogenase and a s p a r t a t e amino t r a n s f e r a s e ( i n d i c e s o f amino a c i d o x i d a t i v e p o t e n t i a l ; Mommsen et a l . 1980) and 3-hydroxybutyrate dehydrogenase (an index of f a t t y a c i d o x i d a t i o n ; Mommsen et a l . 1980). A n a e r o b i c energy p r o d u c t i o n does contribute to a fish's swim stamina capacity, e s p e c i a l l y at the s t a r t of an increment and towards the end o f an i n c r e a s i n g v e l o c i t y t e s t (Jones & Randall 1978). Therefore baseline values of c e r t a i n stored energy substrates such as white muscle and l i v e r glycogen, and white and red muscle creatine p o o l s (an index o f phosphogen energy s t o r e s ) c o u l d be determined. The a c t i v i t i e s of enzymes involved i n anaerobic energy production i n white and red muscle could also be investigated, f o r example: l a c t a t e dehydrogenase ( l a c t a t e production di r e c t i o n ) and pyruvate kinase (both serve as i n d i c e s of g l y c o l y t i c p o t e n t i a l ; Somero & Childress 1980), creatine phosphokinase (an index of phosphogenic potential) and 5-AMP deaminase (proton consumption and ATP synthesis enhancement v i a adenylate kinase; Hochachka & Mommsen 1983, Driedzic & Hochachka 1978). Thomas and Donahoo (1977) concluded t h a t d i f f e r e n c e s i n swimming a b i l i t y between s t r a i n s was not related to growth rate. They tested t h i s by comparing the swimming performance of the f a s t e s t and slowest growing f i s h w i t h i n one o f the s e m i - w i l d s t r a i n s . T h i s may not be a s e n s i t i v e enough t e s t , i.e. t h i s i n t r a - s t r a i n v a r i a t i o n i n growth r a t e and swimming performance may not be as great as the i n t e r - s t r a i n v a r i a t i o n one wants to test. Also, they may have found that growth rate did a f f e c t swimming a b i l i t y i f they had used length, instead of weight, as t h e i r independent va r i a b l e . Length i s a much more important determinant of swimming a b i l i t y (Beamish 1978), i.e. another reason why t h e i r deep-bodied Manchester s t r a i n performed more p o o r l y was because, at the same t e s t weight as the o t h e r s t r a i n s , i t 49 would always be shorter i n length. S e l e c t i o n i n Hatcheries f o r Body Depth A t h i r d p o s s i b l e reason why the two "hatchery" s t r a i n s should perform so poorly i s that s e l e c t i o n f o r high growth not only involves biochemical changes, but, as can be seen from the condition f a c t o r s (WGT x 100/LGT3) i n T a b l e 3, i t i n v o l v e s m o r p h o l o g i c a l changes as w e l l . The l a r g e r c o n d i t i o n f a c t o r s , and t h e r e f o r e more robust and deeper b o d i e s o f the "hatchery" s t r a i n s are i n d i c a t i v e of high growth f i s h . Taylor (1984) has shown that f o r coho salmon, Oncorhynchus k i s u t c h , c o a s t a l f i s h p o p u l a t i o n s that possess deep, robust bodies have l e s s swim stamina, as w e l l as h i g h e r f r i c t i o n a l drag c o e f f i c i e n t s , than i n t e r i o r populations with slender, fusiform-shaped b o d i e s ( s e e a l s o T a y l o r & M c P h a i l 1985)- The same ' i n t r a s p e c i f i c morphological e f f e c t s on swim stamina are probably i n a c t i o n f o r the robust "hatchery" s t r a i n s and the fusiform "wild" s t r a i n s . S e l e c t i o n i n Hatcheries f o r Reduced Heterozygosity L a s t l y , a p o s s i b l e c o r r e l a t i o n may be e x t r a p o l a t e d from the reduced stamina of the "hatchery" stocks and t h e i r lack of polymorphism at the LDH B2 l o c u s . S i n c e the Duncan and Domestic are v e r y i n b r e d s t r a i n s , l a c k of h e t e r o g e n e i t y i s p r o b a b l y not o n l y l i m i t e d to the LDH B2 l o c u s ( I h s s e n 1976). Heterosis or heterozygote s u p e r i o r i t y f o r swim stamina could be i n e f f e c t f o r the two "wild" s t r a i n s , which are probably more heterogeneous at other l o c i besides the LDH B2 locus. The advantage of greater heterozygosity i s a reduction i n phenotypic v a r i a b i l i t y , thereby a c t i n g as a b u f f e r against genetic or environmental m o d i f i c a t i o n (Berger 1976, Fincham 1972, Johnson 1976). For example, h e t e r o z y g o t e s h a v i n g a l l o z y m e s t h a t vary i n such enzymatic properties as temperature or pH optima, w i l l be able to function b e t t e r over l a r g e r ranges of temperature or pH than w i l l t h e i r homozygous counterparts. In t h i s study, s i n g l e locus LDH B2 h e t e r o s i s f o r swim stamina was not found because wi t h i n s t r a i n comparisons f o r the polymorphic Pennask and Deadman s t r a i n s ( e i t h e r when considered separately or combined) y i e l d e d no s i g n i f i c a n t d i f f e r e n c e s i n c r i t i c a l swim v e l o c i t y between the t h r e e genotypes. When the two s t r a i n s were combined, LDH B2' homozygotes had a mean U c r i t (H - 8) of 2.17 1 0.06 BL/S, w i t h h e t e r o z y g o t e s (N = 18) at 1.84 +_ 0.09 BL/S, and LDH B2" homozygotes (N = 12) at 1-94 +_ 0.12 BL/S. What i s t h e r e f o r e suggested above f o r the two " w i l d " s t r a i n s i s m u l t i l o c u s heterosis, and to determine t h i s more p r e c i s e l y the four s t r a i n s should be screened f o r heterozygosity at many other l o c i . This view i s more sensible i n any case because of the interconnected, coordinated nature of metabolic f u n c t i o n i n g . A l s o u n t i l now, o n l y h e t e r o s i s f o r swim st a m i n a has been discussed. I f t h i s hypothetical heterozygote advantage i s to be relevant to stocking these s t r a i n s i n the wild, i t must be i n the form of euheterosis, or heterosis f o r f i t n e s s . That increased heterozygosity i n f i s h favors the s u r v i v a l components o f f i t n e s s has been shown by Beardmore & Ward (1976). " W i l d " f i s h s u r v i v e to reproduce b e t t e r when r e l e a s e d than do "hatchery" ones ( R e i s e n b a c h l e r & M c l n t y r e 1977). T h i s c o u l d be because o f the previously discussed compromise i n protein evolution to achieve the optimal balance between enzymatic properties f o r growth, reproduction and stamina. H e t e r o z y g o t e s w i t h unique n o n - i n t e r m e d i a t e and i n t e r m e d i a t e enzymatic p r o p e r t i e s ( i . e . between the two homozygotes) have been observed i n many forms (Berger 1 976), and t h e r e f o r e may p r e s e n t a m o l e c u l a r b a s i s f o r t h i s balancing s e l e c t i o n (Johnson 1976, Beardmore & Shami 1979), as w e l l as swim stamina h e t e r o s i s and e u h e t e r o s i s . Inbred "hatchery" f i s h d i r e c t i o n a l l y selected f o r high growth have been skewed g e n e t i c a l l y towards high growth 51 m e t a b o l i s m ( R e i n i t z et a l . 1978), and c o n s e q u e n t l y away from the more balanced metabolism and higher wild f i t n e s s which they had possessed while under natural s e l e c t i o n . Brain " L a c t i c A c i d o s i s " Lactate build-up has always been considered a s i g n of fatigue, so i n some f i s h large build-ups a f t e r exercise are considered l e t h a l and possibly the cause of hooking m o r t a l i t y ( M a r n e l l & Hunsaker 1970, Beamish 1968, Gronlund et a l . 1968, C a i l l o u e t 1968, Black 1958, K l a r et a l . 1979a & b). K l a r et a l . (1979a & b) speculated that blood l a c t a t e build-up i n the brain might r e s u l t i n swim stamina reduction f o r some s t r a i n s of rainbow trout. There i s evidence f o r p r o t o n t o x i c i t y i n r a t b r a i n s , but none yet f o r l a c t a t e alone (Rehncrona et a l . 1981, P a l j a r v i et a l . 1982). Both " w i l d " s t r a i n s i n t h i s study maintain much l a r g e r baseline l e v e l s of l a c t a t e i n the b r a i n than do the " h atchery" s t r a i n s . L a c t a t e i s an i m p o r t a n t source o f energy f o r the b r a i n , a t l e a s t i n W e ddell s e a l s , and e s p e c i a l l y when most o t h e r t i s s u e s are e x p e r i e n c i n g a n a e r o b i c c o n d i t i o n s (Murphy et a l . 1980). Therefore these large maintained pools of l a c t a t e are a v a i l a b l e as a ready source of energy f o r the w e l l oxygenated b r a i n during exercise when oxygen i s i n h i g h demand by most o t h e r t i s s u e s . T h e LDH B2 l o c u s i s a l s o the major one expressed i n rainbow t r o u t b r a i n t i s s u e , a l o n g w i t h the LDH B1 l o c u s (Kao & F a r l e y 1978b, B a i l e y et a l . 1976). The l i v e r LDH a c t i v i t i e s seen i n these f o u r s t r a i n s may p o s s i b l y r e f l e c t the r e l a t i v e magnitude o f LDH a c t i v i t i e s i n the brain. During exercise the "wild" s t r a i n l i v e r s had about 50$ more l a c t a t e o x i d i z i n g a c t i v i t y than the "hatchery" s t r a i n s . This might explain why no changes i n brain l a c t a t e l e v e l s were seen f o r the Deadman and Pennask. The i n d i c a t i o n s are that either, or a combination of the f o l l o w i n g are i n e f f e c t here: more r a p i d metabolism of b l o o d and b r a i n l a c t a t e , o r 52 l e s s production of l a c t a t e , or d i f f e r e n t i a l permeability of the blood/brain b a r r i e r to l a c t a t e , and c o n s e q u e n t l y l e s s a l t e r a t i o n o f b r a i n l a c t a t e l e v e l s . I t would appear tha t the b r a i n t i s s u e o f " w i l d " s t r a i n s i s more s u i t e d to s w i t c h to l a c t a t e from, f o r example, g l u c o s e f o r f u e l d u r i n g e x e r c i s e than t h a t o f the "hatchery" s t r a i n s . In any case, b r a i n l a c t a t e accumulation and p o s s i b l y brain a c i d o s i s were not f a c t o r s i n f a t i g u i n g the two " w i l d " s t r a i n s . However, f o r the two " h atchery" s t r a i n s i t c o u l d have been, due to the 2.5 f o l d i n c r e a s e s i n b r a i n l a c t a t e , p l u s the p o s s i b l y reduced b r a i n LDH a c t i v i t i e s d u r i n g e x e r c i s e . S i n c e b u f f e r i n g c a p a c i t y i n c r e a s e s w i t h a c t i v i t y ( C a s t e l l i n i & Somero 1981), i t would be of g r e a t i n t e r e s t to examine br a i n b u f f e r i n g capacity and t i s s u e pH, before and a f t e r e x e r c i s e , i n these s t r a i n s . The a c t u a l a c t i v i t i e s of b r a i n LDH i n both d i r e c t i o n s , as w e l l as the r e l a t i v e c o n t r i b u t i o n s by the isozymes and allozymes involved, would also be worth examining. The theory of K l a r and colleagues (1979a & b) concerning b r a i n " l a c t i c a c i d o s i s " was however derived from the observation that the common LDH B2' a l l e l e was a s s o c i a t e d w i t h h i g h e r swim stamina d u r i n g c o n d i t i o n s o f low e n v i r o n m e n t a l oxygen. At s a t u r a t i n g oxygen t e n s i o n s , as used d u r i n g t h i s study, a l l three LDH B2 genotypes had equal swim stamina c a p a b i l i t i e s . This also seems to be the case among the two "wild" s t r a i n s , and more noticeably among the two homozygous "hatchery" s t r a i n s . The Duncan s t r a i n , homozygous f o r the r a r e LDH B2" a l l e l e , does not have a s i g n i f i c a n t l y d i f f e r e n t c r i t i c a l swim speed from that of the Domestic s t r a i n , which i s homozygous f o r the common LDH B2' a l l e l e . However when one compares b r a i n and l i v e r l a c t a t e accumulation, and maximum LDH a c t i v i t i e s f o r these two homozygous s t r a i n s , the greater l a c t a t e o x i d i z i n g capacity of the LDH B2" allozyme i s s t i l l e v i d e n t ( T s u y u k i & W i l l i s c r o f t 1973, Kao & F a r l e y 1978a & b). The 53 Duncan s t r a i n was probably able to maintain i t s much lower l e v e l s of br a i n and l i v e r l a c t a t e because o f t h i s h i g h e r LDH a c t i v i t y , e s p e c i a l l y d u r i n g e x e r c i s e , i n the l i v e r , and p o s s i b l y the b r a i n . The Duncan s t r a i n had the l o w e s t hemoglobin l e v e l s , and by f a r the lo w e s t NTP/Hb r a t i o s . T h i s would r e s u l t i n a r e d u c t i o n i n blood oxygen c a p a c i t y and i n t i s s u e oxygen t e n s i o n s . Perhaps t h i s d i s a d vantage n u l l i f i e s any advantage that would be a c h i e v e d by the LDH B2" a l l o z y m e i n the l i v e r , and hence the Duncan s t r a i n had the lowest U c r i t > T h e lower l i v e r l a c t a t e to pyruvate conversion rate of the LDH B2' a l l o z y m e i n the Domestic s t r a i n c o u l d be compensated f o r by p o t e n t i a l l y h i g h e r t i s s u e oxygen t e n s i o n s (due to i t s h i g h NTP/Hb r a t i o ) , which would a l l o w i t to u t i l i z e pyruvate a e r o b i c a l l y and d r i v e the LDH re a c t i o n from l a c t a t e to pyruvate. In contrast, the LDH B2" allozyme of the Duncan s t r a i n would convert l a c t a t e to pyruvate more r a p i d l y , independent of the oxygen supply, and therefore could develope a build-up of pyruvate which would require oxygen f o r furth e r catabolism. The lower l e v e l of l a c t a t e i n the Duncan s t r a i n c o u l d a l s o be because i t d i d not swim as l o n g as the Domestic s t r a i n . However, as mentioned p r e v i o u s l y , U c r i t 3 w e r e n o ^ s i g n i f i c a n t l y d i f f e r e n t f o r these two s t r a i n s . An a d d i t i o n a l consideration should also be kept i n mind: The LDH l a c t a t e oxidation assays were done at pH 7-5, but the LDH B2' homotetramer has a narrow pH optimum at 7.430 and the LDH B2" homotetramer one at 7-302 (Ts u y u k i & W i l l i s c r o f t 1973)-Therefore to a t t a i n a truer i n v i t r o estimate of what i s occurring i n vivo, b a s e l i n e and exercised t i s s u e pH should be determined f o r each s t r a i n and/or genotype. LDH assays can then be performed at the appropriate t i s s u e pH, or at the corresponding pH optimum i f maximum a c t i v i t i e s are desired. Lactate Tolerance i n Deadman Steelhead According to Hammond & Hickman (1966) baseline ti s s u e (muscle) l a c t a t e 54 i n rainbow t r o u t t r a i n e d at 2 to 3 BL/S was g r e a t e r than t h a t o f t r o u t t r a i n e d at 1 to 2 BL/S, which was g r e a t e r than t r o u t kept i n s t i l l water. Swim stamina also increased with the degree of t r a i n i n g . A l l s t r a i n s i n t h i s study were trained at 1 to 2 BL/S. The Deadman steelhead have s i g n i f i c a n t l y g r e a t e r b a s e l i n e l i v e r and white muscle l a c t a t e l e v e l s , and c o n s e q u e n t l y l ower f o l d changes than the o t h e r t h r e e s t r a i n s . They a l s o have by f a r the l a r g e s t l i v e r LDH a c t i v i t y , which i s t y p i c a l o f more a c t i v e f i s h , but more so f o r muscle LDH ( S u l l i v a n & Somero 1980). Most l i k e l y these f i s h also have higher muscle LDH a c t i v i t y , as w e l l as a higher t i s s u e b u f f e r i n g capacity, a l l o w i n g them to " s t o r e " and make b e t t e r use of i n i t i a l " a c i d i c " energy s u p p l i e s such as l a c t a t e (Hochachka 1961). However the Deadman s t e e l h e a d , which m i g r a t e up and down the f a s t - f l o w i n g Thompson R i v e r o f the F r a s e r R i v e r system, i s the o n l y s t r a i n to show such an a d a p t a t i o n a l s t r a t e g y . Therefore, e i t h e r t h i s s t r a i n i s more responsive than the other s t r a i n s to the same degree o f t r a i n i n g , or the Deadman s t r a i n e x i s t s i n a b e t t e r " g e n e t i c a l l y conditioned " state than the others previous to t r a i n i n g , and upon t r a i n i n g a l l respond e q u a l l y . T h i s might be an a d a p t a t i o n t h a t steelhead derive, because t h i s " l a c t a t e response" or "genetic conditioning" i s not seen i n the Pennask, the other high stamina s t r a i n . Lactate Accumulation i n "Hatchery" S t r a i n s The s t r a i n t h a t stands out f o r l a c t a t e a c c u m u l a t i o n i n the e x e r c i s e d state i s the Domestic. In a l l four exercised l a c t a t e categories i t had the h i g h e s t l e v e l s . These h i g h l e v e l s , as w e l l as the Duncan's h i g h plasma and white muscle l e v e l s , seem to be c h a r a c t e r i s t i c of "hatchery" s t r a i n s . Higher plasma l a c t a t e l e v e l s have been determined to e x i s t i n " h a t c h e r y " f i s h , w h i l s t being a c t i v e i n a stream with "wild" f i s h ( M i l l e r 1958). In the case of the Domestic and Duncan s t r a i n s , t h i s could be due to greater anaerobic ATP generation, e s p e c i a l l y i n white muscle, and/or l e s s e f f i c i e n t l a c t a t e removal, e s p e c i a l l y i n the l i v e r ( B i l i n s k i & Jonas 1972). That at l e a s t the l a t t e r occurs i s indicated by the greater l a c t a t e o x i d i z i n g a c t i v i t i e s i n l i v e r and b l o o d f o r e x e r c i s e d " w i l d " s t r a i n s . The e f f e c t that these h i g h plasma l a c t a t e l e v e l s p o t e n t i a l l y e x e r t on b r a i n m e t a b o l i s m have a l r e a d y been p o i n t e d out. The f a c t t h a t they are h i g h e r i n "hatchery" s t r a i n s j u s t a m p l i f i e s the problem. The high exercised l e v e l s of l a c t a t e i n the Domestic s t r a i n ' s l i v e r and b r a i n , are p r o b a b l y not s o l e l y due to the low LDH a c t i v i t i e s , but a l s o due to the n ature of the LDH B2' a l l o z y m e , i . e . l e s s e f f i c i e n t l a c t a t e oxidation. The Domestic s t r a i n , w i t h i t s over 40 y e a r s of h a t c h e r y s e l e c t i o n behind i t , i s a f i s h i d e a l l y suited to grow large fast. Competition f o r food i s such that whichever f i s h c o n s i s t e n t l y a r r i v e s at the p e l l e t feeder f i r s t w i l l get the most food, grow l a r g e r , and chase o t h e r f i s h away from the feeder. This e s s e n t i a l l y requires s o l e l y burst swimming a c t i v i t y , which i s powered a n a e r o b i c a l l y (Jones 1982). S i n c e the l a r g e s t f i s h are u s u a l l y selected f o r brood stock, one would expect to f i n d an inadvertant s e l e c t i o n f o r b u r s t swimming to o c cur as w e l l . T h i s c o u l d e x p l a i n the Domestic strain's increased dependence on l a c t a t e anaerobiosis during exercise. Hemoglobin, Hemoconcentration and Erythrocyte Swelling During exercise, the Duncan s t r a i n has s i g n i f i c a n t l y l e s s hemoglobin i n i t s blood, i n d i c a t i n g l e s s blood oxygen capacity and l e s s scope f o r a c t i v i t y than any of the other s t r a i n s . The Domestic s t r a i n has the next lowest whole blood hemoglobin content i n the exercised state. When hemoglobin l e v e l s are reduced i n rainbow t r o u t by removing blood and c r e a t i n g an ^nemic s t a t e , c r i t i c a l swim v e l o c i t i e s decrease a l s o (Jones 1971)- A decrease i n blood 56 oxygen capacity La compensated f o r by an increase i n cardiac output (Cameron & Davis 1970). T h e r e f o r e the two low hemoglobin "hatchery" s t r a i n s would r e s t r i c t oxygen supplied to the tissues much sooner during exercise because o f the i n c r e a s e d oxygen demand o f the b r a n c h i a l and c a r d i a c pumps. T h i s brings about prematurely low tissue oxygen tensions and po s s i b l y low tiss u e pH; both o f which are l i k e l y causes o f f a t i g u e i n f i s h (Jones & R a n d a l l 1978). Both o f the h i g h stamina " w i l d " s t r a i n s have h i g h e r whole blood hemoglobin concentrations during exercise than the two "hatchery" s t r a i n s . The Domestic s t r a i n maintains a much lower whole blood hemoglobin l e v e l d u r i n g i n a c t i v i t y than any of the o t h e r s t r a i n s . T h i s may be due to i t s p r e v i o u s l y discussed s e l e c t i o n f o r a n a e r o b i c b u r s t swimming (see " L a c t a t e A c c u m u l a t i o n i n "Hatchery" S t r a i n s " ) and r a p i d growth (see " S e l e c t i o n i n H a t c h e r i e s f o r Rapid Growth"). I f there i s no s e l e c t i o n f o r a e r o b i c swimming, the u n d e r l y i n g b i o c h e m i c a l machinery i s l e s s l i k e l y to be maintained and might diminish due to genetic d r i f t ; whereas the other three s t r a i n s have a l l been i n the w i l d , and s u b j e c t to i t s n a t u r a l s e l e c t i o n , much more recently. The f o l l o w i n g table shows the e f f e c t of exercise on approximate percent changes i n h e m a t o c r i t (HCT), whole blood hemoglobin (Hb), e r y t h r o c y t e hemoglobin (MCHC), whole blood LDH a c t i v i t y (WB LDH), and e r y t h r o c y t e LDH a c t i v i t y (RBC LDH). The increase due to exercise i n the erythrocyte f r a c t i o n of whole blood i s caused by a combination of hemoconcentration and RBC swe l l i n g , i.e. $ CH HCT = $ CH Hb minus $ CH MCHC, r e s p e c t i v e l y . Hemoconcentration i s due to a number of processes, but mainly water los s from the plasma v i a d i u r e s i s and PENNASK DEADMAN DOMESTIC DUNCAN $ CH HCT +25$ +40$ +60$ +20$ $ CH Hb $ CH MCHC $ CH WB LDH $ CH RBC LDH +40$ -20$ +40$ -20$ 0$ -20$ -20$ -40$ osmotic s h i f t i n t o muscle due to l a c t a t e load. RBC release from the spleen i s a l s o a f a c t o r (Jones & R a n d a l l 1978, Yamamoto et a l . 1980). A l s o , synthesis of hemoglobin protein i n the nucleated f i s h erythrocytes can't be' excluded (Weber 1982). RBC s w e l l i n g i s due to bicarbonate formation w i t h i n the red blood c e l l , as w e l l as s t i m u l a t i o n by catecholamines released during exercise (Jones & Randall 1978, Nikinmaa 1982 & 1983, Primmett 1984). The Duncan s t r a i n shows no heraoconcentration, thereby l e a v i n g the 20% i n c r e a s e i n RBC volume s o l e l y due to RBC s w e l l i n g . As a consequence, t h i s s t r a i n has s i g n i f i c a n t l y l e s s hemoglobin i n i t s blood during exercise, than any o f the o t h e r s t r a i n s . Of the Domestic s t r a i n ' s 60% i n c r e a s e i n the red c e l l f r a c t i o n of whole blood, 2/3 i s due to heraoconcentration and 1/3 due to RBC swe l l i n g . Therefore, t h i s strain's U c r i t a n d ^ c o n t e n t a t e x e r c i s e raay not be much g r e a t e r than the Duncan's, but i t s h e m a t o l o g i c a l response to exercise i s quite d i f f e r e n t . A s i m i l a r d i f f e r e n c e i n response can be seen i n the two high stamina "wild" s t r a i n s . Of the 40$ hematocrit increase i n the Deadman s t r a i n , 1/2 i s from hemoconcentration and the other h a l f from RBC swelling. In contrast, the Pennask has a 25$ RBC volume increase, e n t i r e l y due to RBC s w e l l i n g without hemoconcentration. Thus the Duncan and Pennask s t r a i n s seem to both lack hemoconcentration as a response to exercise. They j u s t maintain t h e i r baseline Hb concentration. This i s quite d i f f e r e n t from the expected norm (Primmett 1984, Jones & Randall 1978). Hemoconcentration i 3 presumed to o c c u r i n salmonids d u r i n g e x e r c i s e to augment oxygen t r a n s p o r t and f a c i l i t a t e a c i d / b a s e r e g u l a t i o n . Furthermore, i t has been p o s t u l a t e d t h a t b l o o d oxygen c a p a c i t y e v o l v e d so t h a t the h e a r t o p e r a t e s over a favorable e f f i c i e n c y range. E f f i c i e n c y i s c a l c u l a t e d as the r a t i o of c a r d i a c output to energy (oxygen) used by the he a r t . In s a l m o n i d s p e c i e s , hemoglobin l e v e l s may have been selected so that the maximum e f f i c i e n c y of 58 the heart occurs during prolonged exercise, as observed during the migratory-periods. Therefore hemoconcentration elevates hemoglobin l e v e l s to allow the heart to function at peak e f f i c i e n c y (Cameron & Davis 1970). I t i s therefore s u r p r i s i n g to see the Domestic s t r a i n w i t h a 40$ h e m o c o n c e n t r a t i o n , the Deadman with 20$, and both the Duncan and Pennask with 0$. One would expect the Deadman, but a l s o the Pennask, to show i n c r e a s e d hemoglobin l e v e l s . A c t u a l l y since these are a l l salmonids. a l l four s t r a i n s should have shown hemoc o n c e n t r a t i o n . However i f any o f the s t r a i n s were not to e x h i b i t o r e x h i b i t d i m i n i s h e d h e m o c o n c e n t r a t i o n w i t h e x e r c i s e , one would at l e a s t expect i t to be the Duncan (which did) and the Domestic (which didn't) because f i s h w i t h l o w e r swimming stamina (e.g. the t o a d f i s h , Opsanus tau) may have maximum cardiac e f f i c i e n c i e s that span lower l e v e l s of a c t i v i t y (Cameron & Davis 1970). N e v e r t h e l e s s , the bottom l i n e i s , whether they hemoconcentrated or not, the two "hatchery" s t r a i n s have lower blood oxygen c a p a c i t i e s d u r i n g e x e r c i s e , p o s s i b l y i m p l i c a t i n g t h e i r maximum c a r d i a c e f f i c i e n c i e s to e x i s t at lower l e v e l s of a c t i v i t y . Determination of cardiac e f f i c i e n c i e s f o r the f o u r s t r a i n s would be another w o r t h w h i l e avenue o f i n v e s t i g a t i o n . LDH B2 _in Erythrocytes One would expect the blood LDH percent change values to be i d e n t i c a l to those f o r Hb, and t h a t $ CH HCT = $ CH WB LDH minus $ CH RBC LDH. O b v i o u s l y the f o r m e r i s not always t r u e , w h i l e the l a t t e r does hol d . The hemoglobin values i n d i c a t e changes i n hemoglobin pr o t e i n concentration, whereas the LDH v a l u e s are enzyme a c t i v i t i e s . T h i s means that f o r the Duncan and Deadman s t r a i n s , $ CH WB LDH i s e q u a l to the a c t i v i t y change due to hemoconcentration ($ CH Hb) plus a 20$ decrease due to some form of enzyme r e g u l a t i o n b rought on by e x e r c i s e . L i k e w i s e f o r $ CH RBC LDH, i t i s determined by the decrease i n a c t i v i t y due to RBC s w e l l i n g (% CH MCHC) plus that same 20% decrease from some type of enzyme regulation. However, under the present assay conditions (i.e. not the c e l l u l a r m i l i e u of the exercised state, e s p e c i a l l y with regard to catecholamines, i n h i b i t o r s , etc.), the only type of a d d i t i o n a l a c t i v i t y change that could be detected involves e i t h e r a change i n mean enzyme p r o t e i n concentration or i n mean r e l a t i v e amounts of a l l o z y m e s . The l a t t e r would o n l y be p o s s i b l e f o r the Deadman, s i n c e the Duncan s t r a i n i s monomorphic. S u b s t r a t e and/or product suppression d/or a c t i v a t i o n o f gene a c t i v i t y f o r s y n t h e s i s of a p a r t i c u l a r enzyme was mentioned i n B e r g e r (1976) as a v a l i d mechanism f o r c o n t r o l l i n g enzyme a c t i v i t y , even over r e l a t i v e l y short periods. That p r o t e i n synthesis i n f i s h i s a r e l a t i v e l y r a p i d p r o c e s s has been shown by i n j e c t i n g Fundulus h e t e r o c l i t u s w i t h 1 4 C - l e u c i n e . A f t e r two hours muscle t i s s u e showed 30% i n c o r p o r a t i o n and l i v e r 10% i n c o r p o r a t i o n i n t o p r o t e i n . These r a t e s were a l s o shown to be d e c r e a s e d by, among o t h e r t h i n g s , low oxygen l e v e l s and s t r e s s (Jackim & LaRoche 1973)* P r o t e i n d e g r a d a t i o n a l s o i n c r e a s e s d u r i n g exercise i n rat l i v e r and muscle (Dohm et a l . 1985)' So temporally, changes i n p r o t e i n l e v e l s are quite possible during the average 1 to 4 hour stamina t r i a l s used i n t h i s study. T h i s i s mentioned o n l y as a p o s s i b l e mechanism f o r the r e d u c t i o n i n LDH a c t i v i t y , s i n c e LDH s u b s t r a t e l e v e l s would be expected to d i f f e r markedly between the r e s t e d and e x e r c i s e d s t a t e s , and since f i s h erythrocytes are nucleated. I t i s i n t e r e s t i n g to note th a t the Duncan i s the o n l y s t r a i n to decrease i t s l a c t a t e o x i d i z i n g a c t i v i t y i n whole blood, and the Domestic the o n l y one to r a i s e i t . However, d u r i n g e x e r c i s e both a l t e r e d t h e i r whole blood LDH a c t i v i t i e s to a l m o s t the same low l e v e l . The two " w i l d " s t r a i n s maintained t h e i r whole blood LDH at the same high l e v e l s . 60 Ontogenetic and Seasonal V a r i a t i o n Another p o s s i b l e e x p l a n a t i o n f o r these u n c o r r e l a t e d d i f f e r e n c e s i n hemoglobin and blood LDH p e r c e n t change v a l u e s i s t h a t d i f f e r e n t s t r a i n -related seasonal rhythms or ontogenetic changes could be i n operation. Since the sample dates between baseline and exercised, and between s t r a i n s u s u a l l y e n t a i l e d months, samples had to be taken at d i f f e r e n t times of the year and at d i f f e r e n t f i s h ages (Table 1). Size was the main determinant o f whether or not a f i s h s t r a i n could be sampled. Since s t r a i n - r e l a t e d d i f f e r e n c e s i n the d a i l y rhythm of glyceraldehyde-3-phosphate -dehydrogenase a c t i v i t y have been d e t e c t e d i n the mouse ( P e l e g et a l . 1982), i t i s q u i t e p o s s i b l e t h a t enzyme a c t i v i t i e s may a l s o v a r y s e a s o n a l l y (e.g., Gabbott & Head 1 980) and o n t o g e n e t i c a l l y . S e a s o n a l changes i n rainbow t r o u t and European e e l (Anguilla a n g u i l l a ) hematology have already been observed (Denton & Yousef 1975, Anderson et a l . 1985). T h e r e f o r e the v a l i d i t y of these Hb and LDH percent change observations should be v e r i f i e d on cannulated i n d i v i d u a l f i s h before and a f t e r exercise. LDH B2 in Hepatocytes For l i v e r LDH a c t i v i t i e s , u n c o r r e l a t e d percent changes with exercise, s i m i l a r i n trend to erythrocyte LDH, were observed. The Pennask and Domestic increased t h e i r l a c t a t e o x i d i z i n g a b i l i t i e s with exercise, while the Deadman and the Duncan d i d the o p p o s i t e . The Duncan and Deadman are the same two s t r a i n s that showed the extra reduction i n erythrocyte a c t i v i t y . Whatever mechanism might be at work i n e r y t h r o c y t e s may a l s o be i n o p e r a t i o n i n h e p a t o c y t e s d u r i n g e x e r c i s e . In a d d i t i o n to the enzyme r e g u l a t i o n and the s e a s o n a l / o n t o g e n e t i c change t h e o r i e s , another l i n e of e x p l a n a t i o n i s possible i n the l i v e r because no measure of l i v e r c e l l s w e l l i n g / s h r i n k i n g 61 was made. This means that i f the hepatocyte volume, f o r example increases, fewer c e l l s and hence l e s s enzyme would be found per wet weight of tissue. These volume changes might be brought on by d i f f e r e n t i a l a d r e n e r g i c response, as i n the s w e l l i n g o f e r y t h r o c y t e s (Nikinmaa 1982), or by some other possible s t r a i n r e l a t e d d i f f e r e n c e i n how water i s regulated i n the tissues during exercise. Decreased NTP:Hb Ratios i n the Duncan S t r a i n Of a l l the observed d i f f e r e n c e s regarding organo-phosphate modifiers of hemoglobin oxygen b i n d i n g a f f i n i t y , o n l y one seems to be c o r r e l a t e d with swim stamina. The Duncan s t r a i n during exercise i s the only group to possess an NTP:Hb r a t i o t h a t i s l e s s t h a n u n i t y . ATP and GTP a m p l i f y the d i s s o c i a t i o n o f oxygen from hemoglobin. T h i s a f f e c t s oxygen u n l o a d i n g i n muscle t i s s u e , as w e l l as a l l o t h e r t i s s u e s . The l e s s ATP and/or GTP pr e s e n t , the l o w e r the t i s s u e oxygen t e n s i o n has to be b e f o r e oxygen i s released to i t from the r e l a t i v e l y high oxygen tension erythrocyte. On each hemoglobin molecule there i s one NTP binding s i t e (Weber 1982). This means that i n the Duncan s t r a i n , even i f a l l the NTP were to remain t i g h t l y bound to the hemoglobin, there would always be non-NTP-bound high oxygen a f f i n i t y hemoglobin i n the blood. Linkage between LDH B2 Genotype and NTP:Hb Ratio I n t e r e s t i n g but not c o r r e l a t e d to swim stamina i s the apparent linkage between LDH B2 genotypes and the exercised NTP:Hb r a t i o . DiMichele & Powers (1982b) observed i n k i l l i f i s h t h a t the h i g h stamina LDH B homozygote a l s o had a higher r e s t i n g NTP:Hb r a t i o than did the low stamina LDH B homozygote. He t e r o z y g o t e s , though not t e s t e d f o r swim stamina, had a h i g h but s t i l l 62 i n t e r m e d i a t e r e s t i n g NTP:Hb r a t i o (Powers et a l . 1979). Among the two homozygous t r o u t s t r a i n s , the s l i g h t l y h i g h e r s t a m i n a LDH B2' Domestic s t r a i n a l s o had the h i g h e s t NTP:Hb r a t i o , and the l o w e r stamina LDH Duncan s t r a i n the l o w e s t . T h i s i s c o n t r a r y to what Tsuyuki <S W i l l i s c r o f t (1977) would expect, but i s i n agreement w i t h what K l a r et a l . (1979a & b) predicted but f o r low oxygen environments. Possible reasons why the Duncan s t r a i n was not a b l e to make f u l l use o f the LDH B2" a l l o z y m e have been discussed p r e v i o u s l y (see "Brain "Lactic Acidosis""). The two "heterozygous" s t r a i n s , Deadman and Pennask, had i n t e r m e d i a t e NTP:Hb r a t i o s , though the Deadman were not s i g n i f i c a n t l y lower than the Domestic. One c o u l d even observe that the high "intermediate" NTP:Hb r a t i o of the Deadman i s linked w i t h a h i g h LDH B2' a l l e l e frequency. L i k e w i s e the Pennask have a "low i n t e r m e d i a t e " NTP:Hb r a t i o and a high LDH B2" a l l e l e frequency. The mechanism f o r t h i s linkage i s not known (DiMichele & Powers 1982b), but has been s p e c u l a t e d upon (Hochachka& Somero 1984). U n l i k e the k i l l i f i s h , the "heterozygous" LDH B2 t r o u t s t r a i n s possessed swim stamina superior to both homozygous s t r a i n s . F u r t h e r a n a l y s i s o f the LDH B2 genotypes and NTP:Hb r a t i o s within the two high stamina s t r a i n s gave the f o l l o w i n g r e s u l t s : For the Deadman s t r a i n the three LDH B2 genotypes possessed these NTP:Hb r a t i o s : LDH B2'B2' (N - 7) = 1-53 1 0.08; LDH B2"B2" (N = 3) = 1.51 1 0.26; LDH B2'B2" (N = 10) = 1-38 1 0.08. L i k e w i s e f o r the Pennask s t r a i n : LDH B2'B 2' (N = 1) = 1.24 1 0.00; LDH B2"B2" (N = 9) = 1.13 1 0.07; LDH B2'B2" (N = 8) = 1.04 1 0.07* None o f the t h r e e genotypes are s i g n i f i c a n t l y d i f f e r e n t w i t h i n e i t h e r s t r a i n . However i t i s doubtful that the Deadman LDH B2"B2" and the Pennask LDH B2'B2' sample s i z e s are adequately large. GTP i n Rainbow Trout Erythrocytes Another i n t e r e s t i n g observation that i s not correlated to the observed 63 A * + 4 > a ^ n a nr, TT . - 4 . i s the percentage of NTP which i s made up of GTP. The d i f f e r e n c e i n u c r i t * Deadman and the Duncan s t r a i n s have s i g n i f i c a n t l y higher percentages of GTP i n both the b a s e l i n e and e x e r c i s e d s t a t e s than do the Pennask and the Domestic. The Duncan and the Deadman are the same two s t r a i n s which showed an added reduction i n erythrocyte LDH l e v e l s . Because of GTP's importance i n mammalian, and hence p o s s i b l y p i s c i n e , protein synthesis (Lehninger 1975), perhaps t h e r e i s a r e l a t i o n s h i p between e r y t h r o c y t i c GTP and i n t r a -e r y t h r o c y t i c r e g u l a t i o n o f enzyme p r o t e i n d u r i n g e x e r c i s e . In any case, t h e r e i s a wide s p e c i e s d i v e r g e n c e i n whether ATP or GTP i s the major a l l o s t e r i c m o d i f i e r o f hemoglobin oxygen b i n d i n g a f f i n i t y (Weber 1982). T h e r e f o r e a g e n e t i c d i f f e r e n c e between s t r a i n s i s not s u r p r i s i n g . The q u e s t i o n i s what are the s t r a i n r e l a t e d d i f f e r e n c e s i n e r y t h r o c y t i c metabolism which b r i n g about these d i f f e r e n t r e l a t i v e ATP/GTP l e v e l s . They could be i n p r o t e i n synthesis, the TCA cycle and oxidative phosphorylation (Greaney & Powers 1977), guanosine monophosphokinase and/or n u c l e o s i d e d i p h o s p h o k i n a s e a c t i v i t i e s (Weber 1982), o r g a n i s m i c r e g u l a t i o n of erythrocyte ATP/GTP metabolism (Tetens & Lykkeboe 1981), or something else. Summary and Conclusions The predominant reasons why "hatchery" s t r a i n s of rainbow trout have po o r e r swimming s t a m i n a than t h e i r " w i l d " c o u n t e r p a r t s now can be summarized. T r o u t o f " w i l d " o r i g i n have e x p e r i e n c e d more s e l e c t i o n f o r swimming performance, so that a metabolic balance e x i s t s between growth and stamina. The deep, r o b u s t b o d i e s o f "hatchery" f i s h , which are i n p a r t a consequence o f h i g h growth s e l e c t i o n , are a l s o l e s s adapted to endurance swimming. The p o s s i b l e decrease i n heterozygosity of these two "hatchery" s t r a i n s may be the cause o f an i n b r e e d i n g d e p r e s s i o n i n swim stamina. I t 64 a l s o appears th a t h a t c h e r y s t r a i n s are more s u s c e p t i b l e to b r a i n l a c t a t e build-up, and therefore p o s s i b l y a c i d o s i s , because i n general they create a b i g g e r body oxygen debt v i a i n c r e a s e d l a c t a t e f o r m a t i o n d u r i n g e x e r c i s e . P a r t of the cause o f t h i s a n a e r o b i c b u i l d - u p i s a decreased amount o f hemoglobin i n the b l o o d and a reduced l a c t a t e o x i d i z i n g c a p a c i t y i n l i v e r and blood. An a d d i t i o n a l reason f o r the poor performance of the Duncan s t r a i n can be a t t r i b u t e d to an NTP:Hb r a t i o l e s s than unity, and consequent further reduction i n t i s s u e oxygen tension during exercise. Another reason why the Deadman steelhead had better swimming endurance i s t h e i r apparent superior a b i l i t y to u t i l i z e " a c i d i c " energy sources, i.e. l a c t a t e . 6 5 REFERENCES A l l e n d o r f , F.W. and S.R. Phelps. 1980. Loss o f g e n e t i c v a r i a t i o n i n a hatchery stock of cutthroat trout. Trans. Amer. Fish. Soc. 109:537-543. Anderson, N.A., J.S. Laursen, and G. Lykkeboe. 1985. Seasonal v a r i a t i o n s i n h e m a t o c r i t , r e d c e l l h e m o g l o b i n and n u c l e o s i d e t r i p h o s p h a t e concentrations, i n the European e e l A n g u i l l a a n g u i l l a . Comp. Biochem. P h y s i o l . 81A:87-92. A y l e s , G.B. and R.F.Baker. 1983. G e n e t i c d i f f e r e n c e s i n growth and s u r v i v a l between s t r a i n s and hybrids of rainbow trout (Salmo gairdneri) stocked i n aquaculture lakes i n the Canadian p r a i r i e s . Aquaculture 33:269-280. Bachman, R.A. 1984. F o r a g i n g b e h a v i o r o f f r e e - r a n g i n g w i l d and ha t c h e r y brown trout i n a stream. Trans. Amer. Fish. Soc. 113:1-32. B a i l e y , G.S., H. T s u y u k i , and A.C. W i l s o n . 1976. The number o f genes f o r l a c t a t e dehydrogenase i n sal m o n i d f i s h e s . J. F i s h . . Res. Board Can. 33:760-767-Bams, R.A. 1967- D i f f e r e n c e s i n performance o f n a t u r a l l y and a r t i f i c i a l l y propagated sockeye salmon migrant f r y , as measured with swimming and p r e d a t i o n t e s t s . J. F i s h . Res. Board Can. 24:11 17-11 53-Beamish, F.W.H. 1968- Glycogen and l a c t i c a c i d c o n c e n t r a t i o n s i n A t l a n t i c cod (Gadus morhua) i n r e l a t i o n to e x e r c i s e . J. F i s h . Res. Board Can. 25:837-851-Beamish, F.W.H. 1978. Swimming capacity. In "Fish Physiology". W.S. Hoar and D.J. R a n d a l l , eds. Vol.7, p. 1 01. Academic P r e s s , N.Y. Beardmore, J.A. and S.A. Shami. 1979« H e t e r o z y g o s i t y and the optimum phenotype under s t a b i l i s i n g s e l e c t i o n . Aquilo Ser. Zool. 20:100-110. Beardmore, J.A. and R.D. Ward. 1976. Polymorphism, s e l e c t i o n , and m u l t i -l o c u s h e t e r o z y g o s i t y i n the p l a i c e , P l e u r o n e c t e s p l a t e s s a L. In "Measuring S e l e c t i o n i n N a t u r a l P o p u l a t i o n s " . F.B. C h r i s t i a n s e n and T.M. Fenchal, eds. p. 207- Springer-Verlag, B e r l i n . Behnke, R.J. 1972. The systematics of salmonid f i s h e s of recently g l a c i a t e d l a k e s . J. F i s h . Res. Board Can. 29:639-671. Berger, E. 1976. Heterosis and the maintenance of enzyme polymorphism. Amer. Natur. 110:823-839. Beukema, J.J. 1969. A n g l i n g experiments w i t h c a r p ( C y p r i n u s c a r p i o L.). I. D i f f e r e n c e s between w i l d , d o m e s t i c a t e d , and h y b r i d s t r a i n s . Neth. J. Zool . 19:596-609-B i l i n s k i , E. and R.E.E. Jonas. 1972. O x i d a t i o n o f l a c t a t e to carbon d i o x i d e by rainbow t r o u t (Salmo g a i r d n e r i ) t i s s u e s . J. F i s h . Res. Board Can. 29:1467-1471. 6 6 Black, E.C. 1958. H y p e r a c t i v i t y as a l e t h a l f a c t o r i n f i s h . J. F i s h . Res. Board Can. 15:573-586. B r e t t , J.R. 1964- The r e s p i r a t o r y m etabolism and swimming performance o f young sockeye salmon. J. Fish. Res. Board Can. 21:1183-1226. Brewer, G.J. and CA. Knudsen. 1966. A technique f o r the processing of blood samples f o r subsequent assay of ATP, and an i n v e s t i g a t i o n of the method of s t a n d a r d i z a t i o n of the f i r e f l y - l u c i f e r a s e ATP assay. C l i n . Chim. Acta 14:836-839-B r o y l e s , R.H., B.M. Pack, S. Berger, and A.R. Dorn. 1979. Q u a n t i f i c a t i o n o f s m a l l amounts o f hemoglobin i n p o l y a c r y l a m i d e g e l s w i t h b e n z i d i n e . Anal. Biochem. 94:211 -21 Brunori, M. 1975- Molecular adaptation to p h y s i o l o g i c a l requirements: the hemoglobin system of trout. In "Current Topics i n C e l l u l a r Regulation". B.L. Horecker and E.R. Stadtman, eds. Vol.9, p.1. Academic P r e s s , N.Y. Burrows, R.E. 1969- The i n f u e n c e of f i n g e r l i n g q u a l i t y on a d u l t salmon s u r v i v a l s . Trans. Amer. F i s h . Soc. 4:777-784. C a i l l o u e t , J r . , C.W. 1968. L a c t i c a c i d o s i s i n channel c a t f i s h . J. F i s h . Res. Board Can. 25:15-23. Cameron, J.N. and J.C. Davis. 1970- Gas exchange i n rainbow t r o u t w i t h varying blood oxygen capacity. J. Fish. Res. Board Can. 27:1069-1085-C a s t e l l i n i , M.A. and G.N. Somero. 1981. B u f f e r i n g c a p a c i t y of v e r t e b r a t e muscle: C o r r e l a t i o n s with p o t e n t i a l s f o r anaerobic function. J. Comp. P h y s i o l . 143:191-198. C h i l d r e s s , J.J. and G.N. Somero. 1979- D e p t h - r e l a t e d enzymic a c t i v i t i e s i n muscle, b r a i n and heart of de e p - l i v i n g pelagic marine teleosts. Marine B i o l . 52:273-283. Cordona, A.J. and S.J. N i c o l a . 1970. Harvest of f o u r s t r a i n s of rainbow t r o u t , Salmo g a i r d n e r i , from B e a r d s l e y R e s e r v o i r , C a l i f o r n i a . C a l i f . F i s h & Game 56:271 -287-Crabtree.B., A.R. Leech, and E.A. Newsholme. 1979. Measurement of enzyme a c t i v i t i e s i n crude extracts of tissues. Tech. Metab. Res. B211:1-37-Cross, T.F. and J. King. 1983- Ge n e t i c e f f e c t s o f h a t c h e r y r e a r i n g i n A t l a n t i c salmon. Aquaculture 33:33-40. Denton, J.E. and M.K. Yousef. 1975- Seasonal changes i n hematology o f rainbow trout, Salmo g a i r d n e r i . Comp. Biochem. Physiol. 51A:151-153-DiMichele, L. and D.A. Powers. 1982a. LDH-B genotype-specific hatching times of Fundulus h e t e r o c l i t u s embryos. Nature 296:563-564-D i M i c h e l e , L. and D.A. Powers. 1982b. P h y s i o l o g i c a l b a s i s f o r swimming endurance d i f f e r e n c e s between LDH-B genotypes of Fundulus h e t e r o c l i t u s . S cience 216:1014-1016. 67 Dobson, G.P. and J. Baldwin. 1982. R e g u l a t i o n o f Dlood oxygen a f f i n i t y i n the A u s t r a l i a n b l a c k f i s h Gadopsis marmoratus. I. Correlations between oxygen-binding p r o p e r t i e s , h a b i t a t and swimming behaviour. J. Exp. B i o l . 99:223-243. Dohm, G.L., G.J. Kasperek, E.B. T a p s c o t t , and H.A. Barakat. 1985- P r o t e i n metabolism during endurance exercise. Fed. Proc. 44:348-352. Donaldson, L.R. 1968. S e l e c t i v e b r e e d i n g of s a l m o n i d f i s h e s . In "Marine A q u a c u l t u r e " . W.J. M c N e i l , ed. p.65- Oregon S t a t e U n i v . P r e s s , C o r v a l i s . Doyle, R.W. 1983- An approach to the q u a n t i t a t i v e a n a l y s i s of domestication s e l e c t i o n i n aquaculture. Aquaculture 33:167-185-D r i e d z i c , W.R. and P.A-. Hochachka. 1975. The unanswered q u e s t i o n of h i g h anaerobic c a p a b i l i t i e s of carp white muscle. Can. J. Zool. 53:706-712. Driedzic, W.R. and P.W. Hochachka. 1978. Metabolism i n f i s h during exercise. In " F i s h P h y s i o l o g y " . W.S. Hoar and D.J. R a n d a l l , eds. Vol.7, P-503-Academic Press, N.Y. Facchin, A. 1983. Hooking m o r t a l i t y of fly-caught Duncan River rainbow trout (Salmo g a i r d n e r i ) i n Harper Lake, B r i t i s h Columbia. F i s h . Tech. C i r c . No. 58. 11 p. B.C. F i s h & W i l d l i f e Branch. Fenderson, O.C, W.H. E v e r h a r t , and K.M. Muth. 1968. Comparative a g o n i s t i c and feeding behavior of hatchery-reared and wild salmon i n aquaria. J. F i s h . Res. Board Can. 25:1-14. Fincham, J.R.S. 1972. Heterozygous advantage as a l i k e l y g e n e r a l b a s i s f o r enzyme polymorphism. Heredity 28:387-391* F l i c k , W.A. 1971. New t r o u t f o r o l d waters. N.Y. S t a t e Conserv. 25:18-21. F l i c k , W.A. and D.A. Webster. 1962. Problems i n s a m p l i n g w i l d and domestic s t o c k s o f brook t r o u t , S a l v e l i n u s f o n t i n a l i s . Trans. Amer. F i s h . Soc. 91:140-144-F l i c k , W.A. and D.A. Webster. 1964. Comparative f i r s t y e a r s u r v i v a l and p r o d u c t i o n i n w i l d and domestic s t r a i n s o f brook t r o u t , S a l v e l i n u s  f o n t i n a l i s . Trans. Amer. F i s h . Soc. 93:58-69. F l i c k , W.A. and D.A. Webster. 1976. P r o d u c t i o n o f w i l d , domestic, and i n t e r s t r a i n hybrids of brook trout (Salvelinus f o n t i n a l i s ) i n natural ponds. J. F i s h . Res. Board Can. 33:1525-1539-Fraser, J.M. 1981. Comparative s u r v i v a l and growth of planted wild, hybrid, and domestic s t r a i n s of brook trout (Salvelinus f o n t i n a l i s ) i n Ontario l a k e s . Can. J. F i s h . Aquat. S c i . 38:1672-1684. Futuyma, D.J. 1979- E v o l u t i o n a r y B i o l o g y . 565 P« S i n a u e r Ass., Inc., Sunderland, Mass. 68 Gabbott, P.A. and E.J.H. Head. 1980. Seasonal changes i n the s p e c i f i c a c t i v i t i e s of the pentose phosphate pathway enzymes, G6PDH and 6PGDH and NADP-dependent i s o c i t r a t e dehydrogenase i n the b i v a l v e s M y t i l u s e d u l i s , O s t r e a e d u l i s and C r a s s o s t r e a g i g a s . Comp. Biochem. P h y s i o l . 66B: 21<f^m. Gilman, A.G. and F. Murad. 1974* Assay of c y c l i c n u c l e o t i d e s by r e c e p t o r p r o t e i n binding displacement. In "Methods i n Enzymology". J.G. Hardman and B.W. O'Malley, eds. V o l . 38, p.49« Academic P r e s s , N.Y. Giovenco, S., I. B i n o t t i , M. B r u n o r i , and E. A n t o n i n i . 1970. S t u d i e s on the f u n c t i o n a l p r o p e r t i e s of f i s h hemoglobins, I. The 0 2 e q u i l i b r i u m o f trout hemoglobin. Int. J. Biochem. 1:57-61. Graham, M.S., R.L. Hae d r i c h , and G.L. F l e t c h e r . 1985- Hematology of three deep-sea f i s h e s : A r e f l e c t i o n of low m e t a b o l i c r a t e s . Comp. Biochem. P h y s i o l . 80A:79-84. Greaney, G.S. and D.A. Powers. 1977. C e l l u l a r r e g u l a t i o n of an a l l o s t e r i c m o d i f i e r of f i s h haemoglobin. Nature 270:73-74* Greene, C.W. 1952. R e s u l t s from s t o c k i n g brook t r o u t o f w i l d and h a t c h e r y s t r a i n s at S t i l l w a t e r Pond. Trans. Amer. Fish. Soc. 81:43-52. Green, D.M. Jr. 1964- A comparison of stamina of brook trout from wild and domestic parents. Trans. Amer. Fish. Soc. 93:96-100. G r i g g , G.C. 1967. Some r e s p i r a t o r y p r o p e r t i e s o f the b l o o d of f o u r s p e c i e s o f Antarctic fishes. Comp. Biochem. Physiol. 23:139-148. Gronlund, W.D., H.O. Hodgins, R.C. Simon, and D.D. Weber. 1968. .Blood l a c t a t e c o n c e n t r a t i o n s and m o r t a l i t y i n sockeye and chinook salmon (Oncorhynchus nerka and 0. tshawytscha) a f t e r e x e r c i s e . J. F i s h . Res. Board Can. 25:473-484. Hammond, B.R. and CP. Hickman,Jr. 1966. The e f f e c t of p h y s i c a l conditioning on the metabolism of l a c t a t e , phosphate, and glucose i n rainbow trout, Salmo g a i r d n e r i . J. F i s h . Res. Board Can. 23:65-83. Hayden, J.B., J.J. Cech, and D.W. Bridges. 1975* Blood oxygen d i s s o c i a t i o n c h a r a c t e r i s t i c s of the winter flounder, Pseudopleuronectes americanus. J. F i s h . Res. Board Can. 32:1 539-1544. H j e r t e n , S., S. J e r s t e d t , and A. T i s e l i u s . 1965- Some a s p e c t s o f the use of "continuous" and "discontinuous" b u f f e r systems i n p o l y a c r y l a m i d e g e l electrophoresis. Anal. Biochem. 11:219-223-Hochachka, P.W. 1961- The e f f e c t o f p h y s i c a l t r a i n i n g on oxygen debt and glycogen reserves i n trout. Can. J. Zool. 39:767-776. Hochachka, P.W. and T.P. Mommsen. 1983. Protons and a n a e r o b i o s i s . Science 219:1391-1397-Hochachka, P.W. and G.N. Somero. 1984. B i o c h e m i c a l A d a p t a t i o n . 537 p. Princeton Univ. Press, Princeton, N.J. 69 Holm, M. and G. Naevdal. 1977- Quantitative genetic v a r i a t i o n i n f i s h - i t s s i g n i f i c a n c e f o r s a l m o n i d c u l t u r e . In "Marine Organisms: G e n e t i c s , Ecology, and E v o l u t i o n " . B. B a t t a g l i a and J.A. Beardmore, eds. p.679. Plenum Press, N.Y. Huzyk, L. and H. Tsu y u k i . 1974- D i s t r i b u t i o n o f LDH-B" i n r e s i d e n t and anadromous rainbow t r o u t (Salmo g a i r d n e r i ) from streams i n B r i t i s h Columbia. J. P i s h . Res. Board (Jan. 3 1 : 1 0 b - 1 0 8 -Ihssen, P. 1976. S e l e c t i v e b r e e d i n g and h y b r i d i z a t i o n i n f i s h e r i e s management. J. Fish. Res. Board Can. 33:316-321. I r v i n e , J.R. 1978. The G e r r a r d rainbow t r o u t of Kootenay Lake, B r i t i s h Columbia - A discu s s i o n of t h e i r l i f e h i s t o r y with management, research and enhancement recommendations. F i s h . Man. Rep. No. 72. 58 p. B.C. Fish & W i l d l i f e Branch. I u c h i , I. and K. Yamagami. 1969- E l e c t r o p h o r e t i c p a t t e r n of l a r v a l hemoglobins o f the s a l m o n i d f i s h , Salmo g a i r d n e r i i r i d e u s . Comp. Biochem. Physiol. 28:977-979-Iuchi, I. 1973- Chemical and p h y s i o l o g i c a l properties of the l a r v a l and the a d u l t hemoglobins i n rainbow t r o u t , Salmo g a i r d n e r i i r i d e u s . Comp. Biochem. Physiol. 44B: 1087-1101. Jackim, E. and G. LaRoche. 1973- Protein synthesis i n Fundulus h e t e r o c l i t u s muscle. Comp. Biochem. Physiol. 44A:851-866. Jaworek, D., W. Gruber, and H.U. Bergmeyer. 1974- Adenosine-5'-triphosphate determination with 3-phosphoglycerate kinase. In "Methods of Enzymatic A n a l y s i s " , 2nd ed. H.U. Bergmeyer, ed. Vol.4, p-2097- V e r l a g Chemie, Weinheim. Johansen, K., G. Lykkeboe, and R.E. Weber, G.M.O. Ma l o i y . 1976. R e s p i r a t o r y p r o p e r t i e s o f blood i n awake and e s t i v a t i n g l u n g f i s h , P r o t o p t e r u s  amphibius. Resp. Physiol. 27:335-345. Johnson, G.B. 1976. Genetic polymorphism and enzyme function. In "Molecular E v o l u t i o n " . F.J. A y a l a , ed. p.46. S i n a u e r Ass.,Inc., Sunderland, Mass. Johnston, I.A. and T.W. Moon. 1980a. Endurance exercise braining i n the fast and slow muscles o f a t e l e o s t f i s h ( P o l l a c h i u s v i r e n s ) . J. Comp. Physiol. 135:147-1 56-Johnston, I.A. and T.W. Moon. 1980b. Exercise t r a i n i n g i n s k e l e t a l muscle of brook trout (Salvelinus f o n t i n a l i s ) . J. Exp. B i o l . 87:177-194-Jones, D.R. 1971- The e f f e c t o f hypoxia and anemia on the swimming performance of rainbow t r o u t (Salmo g a i r d n e r i ) . J. Exp. B i o l . 55:541-551-Jones, D.R. 1982. Anaerobic e x e r c i s e i n t e l e o s t f i s h . Can. J. Z o o l . 60:1131-1134-70 Jones, D.R. and D.J. R a n d a l l . 1978. The r e s p i r a t o r y and c i r c u l a t o r y systems d u r i n g e x e r c i s e . In " F i s h P h y s i o l o g y " . W.S. Hoar and D.J. R a n d a l l , eds. Vol.7, p-425« Academic P r e s s , N.Y. Kao, Y.J. and T.M. F a r l e y . 1978a. Thermal m o d u l a t i o n o f py r u v a t e s u b s t r a t e i n h i b i t i o n i n the B2' and B2" l i v e r l a c t a t e dehydrogenases of rainbow trout, Salmo gaird n e r i . Comp. Biochem. Physiol. 60B: 153-155* Kao, Y.J. and T.M. F a r l e y . 1978b. P u r i f i c a t i o n and p r o p e r t i e s o f a l l e l i c l a c t a t e dehydrogenase isozymes at the B2 locus i n rainbow trout, Salmo  ga i r d n e r i . Comp. Biochem. Physiol. 61B: 507-512. K e l s o , B.W., T.G. N o r t h c o t e , and C F . Wehrhahn. 1981. G e n e t i c and environmental aspects of the response to water current by rainbow trout (Salmo g a i r d n e r i ) o r i g i n a t i n g from i n l e t and o u t l e t s t reams of two l a k e s . Can. J. Zool. 59:2177-2185-K i n c a i d , D.T. 1982. B i o s t a t i s t i c s programs f o r t e a c h i n g and r e s e a r c h . C.U.N.Y. Bronx, N.Y. K i n c a i d , H.L., W.R. B r i d g e s , and B. von Limbach. 1977. Three g e n e r a t i o n s o f s e l e c t i o n f o r growth rate i n f a l l spawning rainbow trout. Trans. Amer. F i s h . Soc. 106:621-628. K i r p i c h n i k o v , V.S. 1981. Genet.c Bases of F i s h S e l e c t i o n . 4.10 p. S p r i n g e r -Verlag, B e r l i n . K i r p i c h n i k o v , V.S. and G.A. Muske. 1980- The a d a p t i v e v a l u e of b i o c h e m i c a l Solymorphisms i n animal and plant populations. In "Animal Genetics and v o l u t i o n " . N.N. Vorontsov and J.M. van Brink, eds. -p. 183. Dr. W. Junk B.V. P u b l i s h e r s , The Hague. Klar, G.T., C.B. Stalnaker, and T.M. Farley. 1979a. Comparative p h y s i c a l and p h y s i o l o g i c a l performance o f rainbow t r o u t , Salmo g a i r d n e r i , o f d i s t i n c t l a c t a t e dehydrogenase B2 phenotypes. Comp. Biochem. Physiol. 63A:229-235-K l a r , G.T., C.B. S t a l n a k e r , and T.M. F a r l e y . 1979b. Comparative blood l a c t a t e response to low oxygen concentrations i n rainbow trout, Salmo  g a i r d n e r i , LDH B2 phenotypes. Comp. Biochem. Physiol. 63A:237-240. Kolmer, J.A. 1949- C l i n i c a l D i a g n o s i s by L a b o r a t o r y E x a m i n a t i o n s , 2nd ed. p-483- A p p l e t o n - C e n t u r y Crofts Inc., N.Y. L e a r y , R.F., F.W. A l l e n d o r f , and K.L. Knudsen. 1 985- D e v e l o p m e n t a l i n s t a b i l i t y as an i n d i c a t o r o f reduced genetic v a r i a t i o n i n hatchery t r o u t . Trans. Amer. F i s h . Soc. 1 14:230-235-Lehn i n g e r , A.L. 1975- B i o c h e m i s t r y , 2nd. ed. 1104 P- Worth Publ., Inc., N.Y. L e i t r i t z , E. and R.C. Lewis. 1976. Tro u t and Salmon C u l t u r e (Hatchery Methods). C a l i f . F i 3 h & Game F i s h B u l l . 164. 197 p. Lohn, C, L.S. Palmer, and C. Kennedy. 1946. Differences i n the biochemistry and p h y s i o l o g y i n f l u e n c i n g food u t i l i z a t i o n f o r growth i n r a t s . I I . 71 E f f i c i e n c y of metabolism f o r maintenance of mature animals d i f f e r i n g i n e f f i c i e n c y of food u t i l i z a t i o n during growth. Univ. Minn. Ag. Exp. Sta. Tech. B u l l . 176:29-42. MacLean, J.A., D.O. Evans, N.V. M a r t i n , and R.C. D e s j a r d i n e . 1981. S u r v i v a l , growth, spawning d i s t r i b u t i o n , and movements of introduced and native lake trout (Salvelinus namaycush) i n two inland Ontario lakes. Can. J. F i s h . Aquat. S c i . 38:1685-1700. M a r n e l l , L.F. and D. Hunsaker I I . 1970. Hooking m o r t a l i t y o f l u r e - c a u g h t c u t t h r o a t t r o u t (Salmo c l a r k i ) i n r e l a t i o n to water temperature, fatigue, and reproductive maturity of released f i s h . Trans. Amer. Fish. Soc. 99:684-688. Medrano, J.F. and G.A.E. G a l l . 1976a. Growth r a t e , body c o m p o s i t i o n , c e l l u l a r growth and enzyme a c t i v i t i e s i n l i n e s of Tr i b o l i u m casteneum selected f o r 21-day pupa weight. Genetics 83:379-391. Medrano, J . F . and G.A.E. G a l l . 1976b. Food consumption, feed e f f i c i e n c y , m e t a b o l i c r a t e and u t i l i z a t i o n o f gl u c o s e i n l i n e s of T r i b o l i u m  casteneum selected f o r 21-day pupa weight. Genetics 83:393-407. M i l l e r , R.B. 1953* Comparative s u r v i v a l of w i l d and h a t c h e r y - r e a r e d cutthroat trout i n a stream. Trans. Amer. Fish. Soc. 83:120-130. M i l l e r , R.B. 1958. The r o l e o f c o m p e t i t i o n i n the m o r t a l i t y o f h a t c h e r y t r o u t . J. F i s h . Res. Board Can. 15:27-45* Mommsen, T.P., C.J. French, and P.W. Hochachka. 1980. S i t e s and p a t t e r n s o f p r o t e i n and amino a c i d u t i l i z a t i o n d u r i n g the spawning m i g r a t i o n o f salmon. Can. J. Z o o l . 58:1785-1799. Moss, B. and V.M. Ingram. 1968. Hemoglobin s y n t h e s i s d u r i n g amphibian metamorphosis. I. Chemical studies of the hemoglobins from the l a r v a l •'. and adult stages of Rana catesbeiana. J. Mol. B i o l . 32:481-492. Moyle, P.B. 1969. Comparative behavior of young brook trout of domestic and wild o r i g i n . Prog. F i s h Cult. 31:51-56. Murphy, B., W.M. Za p o l , and P.W. Hochachka. 1980. M e t a b o l i c a c t i v i t i e s o f heart, lung, and brain during d i v i n g and recovery i n the Weddell seal. J. Appl. P h y s i o l . : Resp. Env. Ex. P h y s i o l . 48:596-605-Newsholme, E.A. 1980. Use of enzyme a c t i v i t y measurements i n studies on the biochemistry of exercise. Int J. Sports Medicine 1:100-102. N i k i n m a a , N. 1982. E f f e c t s o f a d r e n a l i n e on r e d c e l l v o l u m e and concentration gradient of protons across the red c e l l membrane i n the rainbow trout, Salmo ga i r d n e r i . Mol. Physiol. 2:287-297-Nikinmaa, N. 1983. Adrenergic regulation of haemoglobin oxygen a f f i n i t y i n rainbow trout red c e l l s . J. Comp. Physiol. 152:67-72. No r t h c o t e , T.G. 1981- J u v e n i l e c u r r e n t response, growth and m a t u r i t y o f above and below w a t e r f a l l stocks of rainbow trout, Salmo gairdneri- J. 72 F i s h B i o l . 18:741 -751 -N o r t h c o t e , T.G., S.N. W i l l i s c r o f t , and H. Tsuyuki. 1970. M e r i s t i c and l a c t a t e dehydrogenase genotype d i f f e r e n c e s i n stream p o p u l a t i o n s o f rainbow t r o u t below and above a w a t e r f a l l . J. F i s h . Res. Board Can. 27:1987-1995-Northcote, T.G. and B.W. Kelso. 1981- D i f f e r e n t i a l response to water current by two homozygous LDH phenotypes of young rainbow t r o u t (Salmo  g a i r d n e r i ) . Can. J. F i s h . Aquat. S c i . 38:348-352. P a l j a r v i , L., B. S o d e r f e l d t , H. Kalimo, Y. Olsson, and B.K. S i e s j o . 1 982. The b r a i n i n extreme r e s p i r a t o r y a c i d o s i s . A l i g h t - and e l e c t r o n -microscopic study i n the rat. Acta Neuropath. 58:87-94-P e l e g , L., M.N. N e s b i t t , and I.E. Ashkenazi. 1982. A s t r a i n d i f f e r e n c e i n the d a i l y rhythm of glyceraldehyde-3-phosphate dehydrogenase a c t i v i t y i n the mouse. J. Comp. Physiol. 148:137-142. Place, A.R. and D.A. Powers. 1984a. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of the l a c t a t e dehydrogenase (LDH-B) alloizymes of Fundulus h e t e r o c l i t u s . J. B i o l . Chem. 259:1299-1308. P l a c e , A.R. and D.A. Powers. 1984b. K i n e t i c c h a r a c t e r i z a t i o n of the l a c t a t e dehydrogenase (LDH-B) a l l o z y m e s of Fundulus h e t e r o c l i t u s . J. B i o l . Chem. 259:1309-1318. Poulik, M.D. 1957- Starch gel electrophoresis i n a discontinuous system of buffers. Nature 180:1477-1479-Powers, D.A. 1972- Hemoglobin adaptation f o r fast and slow water habitats i n sympatric catostomid fishes. Science 177:360-361-Powers, D.A., G.S. Greaney, and A.R. Pl a c e . 1979- P h y s i o l o g i c a l c o r r e l a t i o n between l a c t a t e dehydrogenase genotype and haemoglobin f u n c t i o n i n k i l l i f i s h . Nature 277:240-241. P r i e s t l e y , G.C. and M.S.M. Robertson. 1973- P r o t e i n and n u c l e i c a c i d m e t a b o l i s m i n organ3 from mice s e l e c t e d f o r l a r g e r and s m a l l e r body s i z e . Genet. Res. 22:255-278. Primmett, D.R.N. 1984- The r o l e of c a t e c h o l a m i n e s i n e r y t h r o c y t e pH r e g u l a t i o n and oxygen t r a n s p o r t i n rainbow t r o u t (Salmo g a i r d n e r i ) d u r i n g and f o l l o w i n g e x h a u s t i v e a c t i v i t y . M.Sc. T h e s i s . Univ. B r i t i s h Columbia. P r o s s e r , C.L. 1973- Comparative Animal P h y s i o l o g y , 3rd ed. W.B. Saunders, P h i l a d e l p h i a . Redding, J.M. and C.B. Schreck. 1979- P o s s i b l e a d a p t i v e s i g n i f i c a n c e o f c e r t a i n enzyme polymorphisms i n steelhead trout (Salmo gairdneri). J. F i s h . Res. Board Can. 36:544-551. Rehncrona, S., I. Rosen, and B.K. S i e s j o . 1981. B r a i n l a c t i c a c i d o s i s and i s c h e m i c c e l l damage: 1. B i o c h e m i s t r y and ne u r o p h y s i o l o g y . J. Cer. 73 Blood Flow & Metab. 1:297-311-R e i n i t z , G.L., L.E. Orme, CA. Lemm, and F.N. H i t z e l . 1978. D i f f e r e n t i a l performance of four s t r a i n s of rainbow trout reared under standardized conditions. Prog. F i s h Cult. 40:21-23. R e i s e n b a c h l e r , R.R. and J.D. M c l n t y r e . 1977- G e n e t i c d i f f e r e n c e s i n growth and s u r v i v a l o f j u v e n i l e h a t c h e r y and w i l d s t e e l h e a d t r o u t , Salmo  g a i r d n e r i . J. F i s h . Res. Board Can. 34:123-128. Riggs, A. 1981. Preparation of blood hemoglobins of vertebrates. In "Methods i n Enzymology". E. A n t o n i n i , L. R o s s i - B e r n a r d i , and E. Chiancone, eds. Vol.76, p«5« Academic P r e s s , N.Y. R i t t e r , J.A. 1975- Lower ocean s u r v i v a l rates for hatchery-reared A t l a n t i c salmon (Salmo salar) stocks released i n r i v e r s other than t h e i r native streams. I n t . Connc. E x p l o r . Sea CM. 1975/M:26. 10 p. Ryman, N. (ed.). 1980. F i s h Gene P o o l s - P r e s e r v a t i o n of G e n e t i c Resources i n Relation to Wild Fish Stocks. Ecol. Bull.(Stockholm) 34. 111 p. Ryman, N. and G. S t a h l . 1980. G e n e t i c changes i n h a t c h e r y s t o c k s o f brown t r o u t (Salmo t r u t t a ) . Can. J. F i s h . Aquat. S c i . 37:82-87-Sharp, G.D. 1969- E l e c t r o p h o r e t i c study of tuna hemoglobins. Comp. Biochem. P h y s i o l . 31:749-755-Shuck, H.A. 1948- S u r v i v a l of hatchery trout i n streams and possible methods of improving the q u a l i t y of hatchery trout. Prog. F i s h Cult. 10:3-14« Siebenaller, J.F. and G.N. Somero. 1982. The maintenance of d i f f e r e n t enzyme a c t i v i t y l e v e l s i n c o n g e n e r i c f i s h e s l i v i n g at d i f f e r e n t depths. P h y s i o l . Z o o l . 55:171-179-S i e b e n a l l e r , J.F., G.N. Somero, and R.L. Haedrich. 1982. B i o c h e m i c a l c h a r a c t e r i s t i c s o f m a c r o u r i d f i s h e s d i f f e r i n g i n t h e i r depths o f d i s t r i b u t i o n . B i o l . B u l l . 163:240-249. Sigma T e c h n i c a l B u l l e t i n 826-UV. 1976. The q u a n t i t a t i v e d e t e r m i n a t i o n o f l a c t i c a c i d i n whole blood at 340 nm. Sigma Chemical Co., St. L o u i s , Missouri. Smith, S.B. 1957- S u r v i v a l and growth o f w i l d and h a t c h e r y rainbow t r o u t (Salmo gai r d n e r i ) i n Corbett Lake, B.C. Can. F i s h Cult. 20:7-12. S o k a l , R.R. and F.J. Rohlf. 1981. Biometry, 2nd ed. 859 P« W.H. Freeman & Co., San F r a n c i s c o . Somero, G.N. 1978. T e m p e r a t u r e a d a p t a t i o n o f enzymes: B i o l o g i c a l o p t i m i z a t i o n through s t r u c t u r e - f u n c t i o n compromises. Ann. Rev. E c o l . Syst. 9:1-29. Somero, G.N. and J.J. C h i l d r e s s . 1980. A v i o l a t i o n of the m e t a b o l i s m - s i z e s c a l i n g paradigm: A c t i v i t i e s of g l y c o l y t i c enzymes i n muscle increase i n l a r g e r - s i z e f i s h . Physiol. Zool. 53:322-337. 74 S t a h l , G. 1983- D i f f e r e n c e s i n the amount and d i s t r i b u t i o n o f g e n e t i c v a r i a t i o n between natural populations and hatchery stocks of A t l a n t i c salmon. Aquaculture 33:23-32. S t a n i e r , M.W. and L.E. Mount. 1972. Growth r a t e , food i n t a k e and body composition before and a f t e r weaning i n s t r a i n s of mice selected for mature body-weight. Br. J. Nutr. 28:307-325-Stock Concept I n t e r n a t i o n a l Symposium. 1981- Can. J. P i s h . Aquat. S c i . 38(12). 1923 P-S u l l i v a n , K.M. and G.N. Somero. 1980. Enzyme a c t i v i t i e s of f i s h s k e l e t a l muscle and b r a i n as i n f l u e n c e d by depth o f occurence and h a b i t s o f feeding and locomotion. Marine B i o l . 60:91-99-Swezey, R.R. and G.N. Somero. 1982- S k e l e t a l muscle a c t i n c o n t e n t i s s t r o n g l y c onserved i n f i s h e s having d i f f e r e n t depths of d i s t r i b u t i o n and c a p a c i t i e s of locomotion. Mar. B i o l . Letters 3:307-315-T a y l o r , E.B. 1984- Ev i d e n c e f o r a d a p t i v e r e l a t i o n s h i p s between body morphology, body s i z e and swimming performance i n B r i t i s h Columbia p o p u l a t i o n s of j u v e n i l e coho salmon, Oncorhynchus k i s u t c h . M.Sc. Thesis. Univ. B r i t i s h Columbia. T a y l o r , E.B. and J.D. M c P h a i l . 1985- V a r i a t i o n i n b u r s t and prolonged swimming performance among B r i t i s h Columbia populations of coho salmon, Oncorhynchus kisutch. Can. J. Fish. Aquat. Sci. 42:2029-2033. Tetens, V. and G. Lykkeboe. 1981. Blood r e s p i r a t o r y p r o p e r t i e s o f rainbow t r o u t , Salmo g a i r d n e r i : responses to hypoxia a c c l i m a t i o n and anoxi c incubation o f blood i n v i t r o . J. Comp. Physiol. 145:117-125-Thomas, A.E. and M.J. Donahoo. 1977- D i f f e r e n c e s i n swimming performance among s t r a i n s o f rainbow t r o u t (Salmo g a i r d n e r i ) . J. F i s h . Res. Board Can. 34:304-306. Turner, J.D., CM. Wood, and D. C l a r k . 1983- L a c t a t e and pro t o n dynamics i n the rainbow trout (Salmo gairdneri). J. Exp. B i o l . 104:247-268-Tsuyuki, H. and S.N. W i l l i s c r o f t . 1973. The pH a c t i v i t y r e l a t i o n s of two LDH homotetramers from trout l i v e r and t h e i r p h y s i o l o g i c a l s i g n i f i c a n c e . J. F i s h . Res. Board Can. 30:1023-1026. Tsuyuki, H. and S.N. W i l l i s c r o f t . 1977- Swimming stamina dif f e r e n c e s between g e n o t y p i c a l l y d i s t i n c t forms o f rainbow (Salmo gairdneri) and steelhead t r o u t . J. F i s h . Res. Board Can. 34:996-1003. V i n c e n t , R.E. 1 960- Some i n f l u e n c e s o f d o m e s t i c a t i o n upon t h r e e s t o c k s o f brook t r o u t , S a l v e l i n u s f o n t i n a l i s M i t c h i l l . Trans. Amer. F i s h . Soc. 89:35-50-Weber, R.E. 1982. I n t r a s p e c i f i c adaptation of hemoglobin function i n f i s h to oxygen a v a i l a b i l i t y . In "Exogenous and Endogenous I n f l u e n c e s on M e t a b o l i c and N e u r a l C o n t r o l " . A.D.F. Addink and N. Spronk, eds. Vol.1, 75 p.87« Pergamon Press, Oxford. Weber, R.E., S.O. Wood, and J.P. Lomholt. 1976. Temperature a c c l i m a t i o n and oxygen-binding pr o p e r t i e s of blood and mu l t i p l e haemoglobins of rainbow t r o u t . J. Exp. B i o l . 65:333-345. Webster, D.A. and W.A. F l i c k . 1978. Sp e c i e s Management. W i l d Trout -Catchable Trout Symposium, Eugene, Oregon:40-47. Webster, D.A. and W.A. F l i c k . 1981. Performance of i n d i g i n o u s , e x o t i c , and hybrid s t r a i n s of brook trout (Salvelinus f o n t i n a l i s ) i n waters of the Adirondack Mountains, N.Y. Can. j . jj'isn. Aquat. S c i . 38:1701-1707-W i l l i s c r o f t , S.N. and H. Tsuyuki. 1970. L a c t a t e dehydrogenase systems o f rainbow t r o u t . E v i d e n c e f o r polymorphism i n l i v e r and a d d i t i o n a l s u b u n i t s i n g i l l s . J. F i s h . Res. Board Can. 27:1563-1567-Wollenberger, A., 0. Ristau, and G. Schoffa. 1960. Eine einfache Technik der extrem schnellen Abkuehlung groesserer Gewebestuecke. P f l u e g e r s Arch, ges. Physiol. Mensch., Tiere. 270:399-412. Yamamoto, K.I., Y. Itazawa, and H. Kobayashi. 1980. Supply o f e r y t h r o c y t e s i n t o the c i r c u l a t i n g b l o o d from the s p l e e n o f e x e r c i s e d f i s h . Comp. Biochem. Physiol. 65A:5-11« Zuckerkandl, E. 1976a. Evolutionary processes and evolutionary noise at the m o l e c u l a r l e v e l . I. F u n c t i o n a l d e n s i t y i n p r o t e i n s . J. Mol. E v o l . 7:167-183. Zuckerkandl, E. 1976b. Evolutionary processes and evolutionary noise at the m o l e c u l a r l e v e l . II. A s e l e c t i o n i s t model f o r random f i x a t i o n i h p r o t e i n s . J. Mol. E v o l . 7:269-31 1. 76 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0096880/manifest

Comment

Related Items