Science, Faculty of
Zoology, Department of
DSpace
UBCV
Holeton, George F.
Master of Science - MSc
University of British Columbia
A study of factors affecting gas exchange at the gills of rainbow trout during hypoxia was made. Techniques for implanting cannulae in the buccal and opercular chambers, the dorsal aorta, and the ventral aorta are described. The responses of afferent and efferent blood and water pressures, Po₂, PCo₂, pH, and the hematocrit of efferent blood were made while the unanaes thetized free swimming trout was in a sealed respirometer. The fish were allowed to consume the oxygen in the respirometer to produce conditions of hypoxia. Determination of oxygen uptake and a knowledge of the solubility of oxygen in blood and water allowed indirect estimation of ventilation volume, cardiac output and stroke volume of the heart. With this knowledge, inferences on the function of circulatory and respiratory mechanisms during hypoxia were drawn. The rainbow trout possesses a number of homeostatic mechanisms which augment oxygen uptake during hypoxia. It was found that the trout could maintain a fairly uniform oxygen uptake in environmental oxygen tensions as low as 30 to 50 mm Hg„ The major homeostatic responses to hypoxia affecting oxygen uptake were: an increase in ventilation volume, an increase in the functional capacity of the blood, and an apparent vascular shunting of blood closer to the surface of the respiratory epithelium. No significant increase in cardiac output was observed. Since there was a pronounced bradycardia with hypoxia the stroke volume was apparently increasing. The role of the bradycardia, which has usually been associated with a reduction in cardiac output, is not clear. Possible reasons and functions of the bradycardia are discussed.
FACTORS INFLUENCING GAS EXCHANGE IN THE RAINBOW TROUT (5ALIY1D GAIRDNERI) by GEORGE Fo H D L ETON S c , , U n i v e r s i t y o f A l b e r t a , C a l g a r y , 1964 t h e s i s s u b m i t t e d 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 t h e d e g r e e o f MASTER OF SCIENCE i n t h e D e p a r t m e n t o f Z o o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRIT I S H COLUMBIA J u n e , 1966 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 of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the 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 reference and study, I f u r t h e r agree t h a t p e r m i s s i o n - f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s , Tt i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a in s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . ABSTRACT A s t u d y o f f a c t o r s a f f e c t i n g gas e x c h a n g e a t t h e g i l l s o f r a i n b o w t r o u t d u r i n g h y p o x i a was made. T e c h n i q u e s f o r i m p l a n t -i n g c a n n u l a e i n t h e b u c c a l and o p e r c u l a r c h a m b e r s , t h e d o r s a l a o r t a , and t h e v e n t r a l a o r t a a r e d e s c r i b e d . The r e s p o n s e s o f a f f e r e n t and e f f e r e n t b l o o d and w a t e r p r e s s u r e s , P o j , P C 0 2 , pH, and t h e h e m a t o c r i t o f e f f e r e n t b l o o d were made w h i l e t h e u n a n a e s t h e t i z e d f r e e swimming t r o u t was i n a s e a l e d r e s p i r o m e t e r . The f i s h were a l l o w e d t o consume t h e o x y g e n i n t h e r e s p i r o m e t e r t o p r o d u c e c o n d i t i o n s o f h y p o x i a . D e t e r m i n a t i o n o f o x y g e n u p t a k e and a k n o w l e d g e o f t h e s o l u b i l i t y o f o x y g e n i n b l o o d and w a t e r a l l o w e d i n d i r e c t e s t i m a t i o n o f v e n t i l a t i o n v o l u m e , c a r d i a c o u t -p u t ' and s t r o k e volume o f t h e h e a r t . W i t h t h i s k n o w l e d g e , i n f e r -e n c e s on t h e f u n c t i o n o f c i r c u l a t o r y and r e s p i r a t o r y mechanisms d u r i n g h y p o x i a were drawn. The r a i n b o w t r o u t p o s s e s s e s a num-b e r o f h o m e o s t a t i c mechanisms w h i c h augment o x y g e n u p t a k e d u r i n g h y p o x i a . I t was f o u n d t h a t t h e t r o u t c o u l d m a i n t a i n a f a i r l y u n i f o r m o x y g e n u p t a k e i n e n v i r o n m e n t a l o x y g e n t e n s i o n s as l o w a s 30 t o 50 mm Hg„ The m a j o r h o m e o s t a t i c r e s p o n s e s t o h y p o x i a a f f e c t i n g o x y g e n u p t a k e were: an i n c r e a s e i n v e n t i l a t i o n v o l u m e , an i n c r e a s e i n t h e f u n c t i o n a l c a p a c i t y o f t h e b l o o d , and an a p p a r e n t v a s c u l a r s h u n t i n g o f b l o o d c l o s e r t o t h e s u r -f a c e o f t h e r e s p i r a t o r y e p i t h e l i u m . No s i g n i f i c a n t i n c r e a s e i n c a r d i a c o u t p u t was o b s e r v e d . S i n c e t h e r e was a p r o n o u n c e d b r a d y c a r d i a w i t h h y p o x i a t h e s t r o k e volume was a p p a r e n t l y i n c r e a s i n g . The r o l e o f t h e b r a d y c a r d i a , w h i c h has u s u a l l y been a s s o c i a t e d w i t h a r e d u c t i o n i n c a r d i a c o u t p u t , i s n o t c l e a r . P o s s i b l e r e a s o n s and f u n t i o n s o f t h e b r a d y c a r d i a a r e d i s c u s s e d . TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS i v L I S T OF TABLES v L I S T OF FIGURES v i ACKNOWLEDGEMENT i x INTRODUCTION 1 MATERIALS AND METHODS 5 PART ONE - PRESSURES AND RATES I - R e s u l t s and D i s c u s s i o n 17 PART TWO - GAS TENSIONS AND pH I - R e s u l t s and D i s c u s s i o n 33 PART THREE - BLOOD AND WATER FLOWS AND EXCHANGE RELATIONSHIPS I - R e s u l t s and D i s c u s s i o n 44 I I - Summary o f p a r t t h r e e 67 GENERAL DISCUSSION 69 REFERENCES 77 0 V L I S T OF T A B L E S F a c i n g page Io The l e v e l o f b l o o d l a c t a t e b e f o r e and a f t e r 47 an e x p e r i m e n t . D a t a f r o m f o u r l a r g e f i s h . I I . A summary o f some o f t h e a v a i l a b l e i n f o r m a - 55 t i o n on c a r d i a c o u t p u t and s t r o k e v olume o f v a r i o u s v e r t e b r a t e s . v i L I S T OF FIGURES F a c i n g page 1. D e t a i l s o f t h e head o f a t r o u t s h o w i n g r e l a t i v e 7 p o s i t i o n s o f t h e c a n n u l a e . 2. The r e s p i r o m e t e r u s e d i n t h e e x p e r i m e n t s . 11 3. E f f e c t o f h y p o x i a upon b r e a t h i n g and h e a r t r a t e . 18 4. P r e s s u r e r e c o r d i n g s f r o m t h e v e n t r a l a o r t a u n d e r 20 n o r m a l , p a r t i a l l y h y p o x i c , and h y p o x i c e n v i r o n -m e n t a l c o n d i t i o n s . 5. P r e s s u r e r e c o r d i n g s f r o m t h e d o r s a l a o r t a u n d e r 22 n o r m a l , p a r t i a l l y h y p o x i c , and h y p o x i c e n v i r o n -m e n t a l o x y g e n t e n s i o n s . 6. P r e s s u r e r e c o r d i n g s o f b l o o d p r e s s u r e d i f f e r e n - 24 t i a l ( v e n t r a l a o r t i c - d o r s a l a o r t i c ) o v e r t h e g i l l s u n d e r n o r m a l , p a r t i a l l y h y p o x i c , and h y p o x i c e n v i r o n m e n t a l o x y g e n t e n s i o n s . 7. E f f e c t o f h y p o x i a upon v e n t r a l a o r t i c s y s t o l i c 25 and p u l s e p r e s s u r e s . 8. E f f e c t o f h y p o x i a upon d o r s a l a o r t i c s y s t o l i c 26 and p u l s e p r e s s u r e s . 9. E f f e c t o f h y p o x i a upon t h e b l o o d p r e s s u r e d i f - 27 f e r e n t i a l o v e r t h e g i l l s . 10. The r a t i o o f d o r s a l a o r t i c s y s t o l i c p r e s s u r e t o 29 v e n t r a l a o r t i c s y s t o l i c p r e s s u r e , and t h e r a t i o o f d o r s a l a o r t i c p u l s e p r e s s u r e t o v e n t r a l a o r t i c p u l s e p r e s s u r e , a t v a r i o u s o x y g e n t e n s i o n s o f t h e e n v i r o n m e n t a l w a t e r . V 11 The method o f a n a l y s i s and r e s u l t s o f r a t e o f d f e r e n t i a l b l o o d p r e s s u r e d r o p o v e r t h e g i l l s a t v a r i o u s e n v i r o n m e n t a l o x y g e n t e n s i o n s . , The e f f e c t o f f l u s h i n g f r e s h o x y g e n a t e d w a t e r i n t o t h e r e s p i r o m e t e r f i l l e d w i t h w a t e r o f low o x y g e n c o n t e n t , upon t h e h e a r t r a t e and b l o o d p r e s s u r e o f t h e r a i n b o w t r o u t . The e f f e c t o f h y p o x i a on t h e o x y g e n t e n s i o n o f o p e r c u l a r w a t e r . The e f f e c t o f h y p o x i a on t h e o x y g e n t e n s i o n o f v e n t r a l a o r t i c a nd d o r s a l a o r t i c b l o o d . The e f f e c t o f h y p o x i a upon t h e pH o f v e n t r a l a o r t i c b l o o d . The e f f e c t o f h y p o x i a upon t h e pH o f d o r s a l a o r t i c b l o o d . The pH o f e n v i r o n m e n t a l and o p e r c u l a r w a t e r d u r i n g t h e c o u r s e o f t h e p r o g r e s s i v e h y p o x i a . The h e m a t o c r i t o f t h e f i s h and t h e e n v i r o n m e n t a l o x y g e n t e n s i o n a t w h i c h t h e e x p e r i m e n t w i t h h y p o x i a was t e r m i n a t e d due t o s i g n s o f r e s p i r a -t o r y o r c a r d i a c f a i l u r e . The e f f e c t o f h y p o x i a on t h e i a t e o f o x y g e n up-t a k e . -The e f f e c t o f h y p o x i a on v e n t i l a t i o n v o l u m e . The r e l a t i o n s h i p b e t ween h e m a t o c r i t and b l o o d o x y g e n c a p a c i t y . The e f f e c t o f h y p o x i a upon c a r d i a c o u t p u t . The e f f e c t o f h y p o x i a upon s t r o k e v o l u m e . v i i i 24. Changes i n t h e e f f e c t i v e n e s s o f b l o o d o x y g e n a - 59 t i o n and t h e e f f e c t i v e n e s s o f o x y g e n r e m o v a l f r o m w a t e r w i t h i n c r e a s i n g h y p o x i a . 2 5 . Changes i n t h e c a p a c i t y r a t e r a t i o o f b l o o d and 63 w a t e r o c c u r r i n g w i t h i n c r e a s i n g h y p o x i a . 26. Changes i n t r a n s f e r f a c t o r o f t h e g i l l s i n 65 r e s p o n s e t o h y p o x i a . ACKNOWLEDGMENT -I. w i s h t o thank Dr. D. J . R a n d a l l f o r s u g g e s t i n g t h i s t h i s r e s e a r c h problem and f o r h i s g uidance and encouragement, i n t h e . p r e s e n t study. - -I am g r a t e f u l t o Drs. W. S. Hoar, J McFadden, and J . E. P h i l l i p s f o r t h e i r a d v i c e and s u g g e s t i o n s i n the p r e p a r a t i o n o f t h i s m a n u s c r i p t . Thanks are a l s o due' t o Dr. J . R. B r e t t f o r use, of-- ' • f a c i l i t i e s and equipment a t the B i o l o g i c a l S t a t i o n a t "Nanaimo, B. C., and' t o the t e c h n i c a l s t a f f a t the s t a t i o n f o r t h e i r r eady a s s i s t a n c e , a d v i c e , and f o r p r o c u r i n g and c a r i n g f o r some of the f i s h used i n t h i s work. I am i n d e b t e d t o Mrs. C. Beaumont f o r t e c h n i c a l a s s i s t a n c e i n a n a l y s i n g b l o o d samples and f o r a s s i s t a n c e i n p r e p a r i n g i l l u s t r a t i o n s f o r t h i s m a n u s c r i p t . T h i s r e s e a r c h was a s s i s t e d by the N a t i o n a l R e s e a r c h • C o u n c i l o f Canada through, a g r a n t . t o Dr. D. J . R a n d a l l and i s g r a t e f u l l y acknowledged. •I w i s h t o e x p r e s s my a p p r e c i a t i o n f o r the t e c h n i c a l ; . a s s i s t a n c e of Miss G e o r g i n a D o c h e r t y I n a n a l y s i n g b l o o d samples f o r l a c t a t e . - 1 -INTRODUCTION M o t t ( 1 9 5 7 ) r e v i e w e d t h e c a r d i o v a s c u l a r s y s t e m o f f i s h e s and s t r e s s e d t h e l i m i t e d n a t u r e o f t h e p r e s e n t k n o w l e d g e , and t h e need f o r more i n f o r m a t i o n . Much o f t h e p r e s e n t i n f o r m a -t i o n on c i r c u l a t o r y and r e s p i r a t o r y s y s t e m s o f f i s h e s i s o f a f r a g m e n t a r y n a t u r e e n c o m p a s s i n g f i s h o f w i d e l y d i f f e r i n g h a b i t a t , h a b i t , and s p e c i e s . T h e r e i s a d e f i n i t e need f o r an i n t e g r a t e d and c o m p r e h e n s i v e s t u d y o f c i r c u l a t o r y and r e s p i r a -t o r y c h a r a c t e r i s t i c s on a s i n g l e s p e c i e s o f f i s h . T h e r e has been no s t u d y w h i c h has m e a s u r e d a l l t h e m a j o r f a c t o r s gov-e r n i n g gas e x c h a n g e a t t h e g i l l s o f f i s h i n a s i m u l t a n e o u s and i n t e g r a t e d manner. The o b j e c t o f t h e p r e s e n t s t u d y was t o a t t e m p t t o m e a s u r e , and t o i n t e r p r e t t h e s i g n i f i c a n c e o f , t h e f a c t o r s a f f e c t i n g gas e x c h a n g e a t t h e g i l l s o f a f r e s h w a t e r t e l e o s t , t h e r a i n b o w t r o u t (Salmo q a i r d n e r i R . ) . A m a j o r d i f f i c u l t y i n m e a s u r i n g t h e c i r c u l a t o r y and r e s p i r a t o r y p a r a m e t e r s o f f i s h i s t h a t t h e f i s h must be a b l e t o b r e a t h e , a p r o b l e m w h i c h l i m i t s t h e t i m e o r t h e c o n -d i t i o n s u n d e r w h i c h t h e v a r i o u s o p e r a t i o n s and m e a s u r e m e n t s c a n be made. T h i s , t o a g r e a t e x t e n t , e x p l a i n s t h e l i m i t e d and f r a g m e n t a r y n a t u r e o f t h e c u r r e n t i n f o r m a t i o n . Rushmer and S m i t h ( 1 9 5 9 ) e m p h a s i z e d t h e need f o r , and t h e i m p o r t a n c e o f , m a k i n g s t u d i e s on t h e c a r d i o v a s c u l a r s y s t e m s o f a n i m a l s w h i c h a r e i n t a c t , u n r e s t r a i n e d , and u n a n a e s t h e t i z e d . T h i s w o u l d s u g g e s t t h a t much o f t h e c u r r e n t k n o w l e d g e o f f i s h c i r -c u l a t o r y p a r a m e t e r s may n o t be o f p h y s i o l o g i c a l s i g n i f i c a n c e . Much o f t h e i n f o r m a t i o n has been o b t a i n e d f r o m f i s h w h i c h a i e - 2 -n o t i n t a c t , ( B e r g & S t e e n , 1965; G o l d s t e i n , F o r e s t e r , & ^ a n e l l i , 1964; G r e e n e , 1 904; I t a z a w a , 1957; J o h a n s e n , 1962; S a t c h e l l , 1960, 1962; S t e e n & K r u y s s e , 1964) o r From f i s h w h i c h a r e e i t h e r -r e s t r a i n e d o r a n a e s t h e t i z e d ( B u r g e r & B r a d l e y , 1951; I t a z a w a , 1957; M o t t , 1950; R a n d a l l & S h e l t o n , 1963; S a t c h e l l , 1960, 1 9 6 1 , 1 9 6 2 ) . The e f f e c t o f h y p o x i a upon c i r c u l a t o r y and r e s p i r a t o r y p a r a m e t e r s o f f i s h i s n o t w e l l d o c u m e n t e d . The i n f l u e n c e o f h y p o x i a upon t h e h e a r t r a t e , b r e a t h i n g r a t e , and b r e a t h i n g a m p l i t u d e o f t e n c h ( T i n e a t i n e a L 0 ) has been r e p o r t e d by R a n d a l l and S h e l t o n ( 1 9 6 3 ) . H o w e v e r , l i t t l e i s known a b o u t c h a n g e s i n b l o o d p r e s s u r e and v a s c u l a r r e s i s t a n c e d u r i n g h y p o -x i a . S a t c h e l l ( 1 9 6 2 ) d e m o n s t r a t e d t h a t t h e d o g f i s h ( S q u a l u s a c a n t h i a s ) r e s p o n d e d t o a n o x i a by a t r a n s i e n t d e c r e a s e i n h e a r t r a t e and d o r s a l a o r t i c b l o o d p r e s s u r e a s s o c i a t e d w i t h an i n c r e a s e i n t h e mean and p u l s e p r e s s u r e s i n t h e v e n t r a l a o r t a . I t was shown t h a t an i n c r e a s e o f p e r i p h e r a l r e s i s t a n c e , an i n c r e a s e i n p r e s s u r e d r o p o v e r t h e g i l l s , and an i n c r e a s e i n s t r o k e v olume o f t h e h e a r t were a l l i m p l i c a t e d i n t h e r e s p o n s e o f t h e d o g f i s h t o a n o x i a . An i n s i g h t can be o b t a i n e d i n t o t h e mechanism o f r e g u l a -t i o n o f gas e x c h a n g e i n f i s h by r e c o r d i n g c h a n g e s i n gas t e n -s i o n s i n t h e b l o o d and w a t e r , a f f e r e n t and e f f e r e n t t o t h e g i l l s d u r i n g h y p o x i a . L i t t l e i n f o r m a t i o n o f t h i s k i n d i s a v a i l -a b l e i n t h e l i t e r a t u r e . Van Dam ( 1 9 3 8 ) , I t a z a w a ( 1 9 57 ) , and S a u n d e r s ( 1 9 6 2 ) have l i s t e d some i n f o r m a t i o n on t h e e f f e c t s o f h y p o x i a on b l o o d and w a t e r gas t e n s i o n s , b u t t h e r e has been no i n t e g r a t e d s t u d y o f s e v e r a l o f t h e s e p a r a m e t e r s s i m u l t a n -e o u s l y . - 3 -The measurement o f t h e f l o w o f b l o o d and w a t e r p a s t t h e e x c h a n g e s u r f a c e s , and c h a n g e s i n t h e s e f l o w s i n r e s p o n s e t o h y p o x i a w o u l d a l s o a l l o w an i n s i g h t t o be o b t a i n e d i n t o t h e gas e x c h a n g e m e c h a n i s m s . Changes i n t h e f l o w o f w a t e r p a s t t h e g i l l s i n r e s p o n s e t o h y p o x i a have been d e t e r m i n e d p r e v i o u s l y by Van Dam ( 1 9 3 8 ) and S a u n d e r s ( 1 9 6 2 ) b u t t h e i n f o r m a t i o n i s n o t s u f f i c i e n t f o r t h e p r e s e n t s t u d y . A l t h o u g h B u r g e r and B r a d l e y ( 1 9 5 1 ) , G o l d s t e i n , F o r s t e r , and F a n e l l i ( 1 9 6 4 ) , H a r t ( 1 9 4 3 ) , J o h a n s e n ( 1 9 6 2 ) , and M o t t ( 1 9 5 7 ) have p r e s e n t e d some i n f o r m a -t i o n and e s t i m a t e s o f g i l l b l o o d f l o w o f v a r i o u s f i s h e s , t h e r e i s , t o my k n o w l e d g e , no s a t i s f a c t o r y i n f o r m a t i o n on c h a n g e s i n g i l l b l o o d f l o w i n r e s p o n s e t o h y p o x i a . Hughes and S h e l t o n ( 1 9 6 2 ) and Hughes ( 1 9 6 4 ) have s u g g e s -t e d t h a t p e r h a p s t h e f u n d a m e n t a l r e l a t i o n s h i p s d e r i v e d f r o m h e a t e x c h a n g e t e c h n o l o g y w o u l d p r o v e u s e f u l i n o b t a i n i n g i n f o r -m a t i o n a b o u t t h e f u n c t i o n a l a s p e c t s o f gas e x c h a n g e and r e s p i r a -t o r y h o m e o s t a t i c m echanisms a s s o c i a t e d w i t h t h e g i l l s o f f i s h e s . O n l y f r a g m e n t a r y e x p e r i m e n t a l i n f o r m a t i o n f r o m w i d e l y v a r y i n g s o u r c e s was a v a i l a b l e t o t e s t t h e s e s u g g e s t e d r e l a t i o n s h i p s . A k n o w l e d g e o f t h e q u a l i t a t i v e and q u a n t i t a t i v e a s p e c t s o f t h e f a c t o r s a f f e c t i n g gas e x c h a n g e f r o m a s i n g l e s p e c i e s u n d e r a u n i f o r m c o n d i t i o n w o u l d a l l o w an e x a m i n a t i o n o f t h e a p p l i c a -b i l i t y o f t h e h e a t e x c h a n g e r a n a l o g y r e l a t i o n s h i p s and p o s s i b l y d i s c l o s e some f u n c t i o n a l a s p e c t s o f h o m e o s t a t i c mechanisms o p e r a t i n g d u r i n g h y p o x i a . I t i s t h e o b j e c t o f t h i s s t u d y t o r e c o r d t h e e f f e c t s o f h y p o x i a upon t h e r a t e o f o x y g e n u p t a k e and upon t h e p r e s s u r e s and gas t e n s i o n s o f t h e b l o o d and w a t e r a f f e r e n t and e f f e r e n t _ 4 ~ t o t h e g i l l s o f u n r e s t r a i n e d , u n a n a e s t h e t i z e d , and i n t a c t r a i n -bow t r o u t . T h i s k n o w l e d g e w i l l t h e n be u t i l i z e d t o e s t i m a t e c h a n g e s i n t h e f l o w o f b l o o d and w a t e r p a s t t h e e x c h a n g e s u r -f a c e , w h i c h i n t u r n w i l l a l l o w an e x a m i n a t i o n o f t h e t h e o r e t i -c a l a s p e c t s o f t h e e x c h a n g e p r o c e s s and p o s s i b l e h o m e o s t a t i c m e c h a n i s m s . T e c h n i q u e s have been w o r k e d o u t f o r c a n n u l a t i n g t h e v e n t r a l a o r t a , t h e d o r s a l a o r t a , t h e b u c c a l c h a m b e r , and t h e o p e r c u l a r chamber o f r a i n b o w t r o u t w i t h v e r y l i t t l e t i s s u e damage. The e x p e r i m e n t s were c a r r i e d o u t on t r o u t w h i c h were i n t a c t s a v e f o r t h e i n s e r t i o n o f h y p o d e r m i c t i p p e d c a n n u l a e i n t o t h e d o r s a l and v e n t r a l a o r t a e f r o m t h e e x t e r i o r o f t h e f i s h , and t h e s m a l l h o l e s p u n c h e d i n t h e c a r t i l a g i n o u s p o r t i o n s o f t h e s n o u t and one o f t h e o p e r c u l a e . Thus i t has been p o s s i b l e , by means o f d i r e c t c o n n e c t i o n s , t o s t u d y c o n t i n u o u s l y , r e s p o n s e s t o h y p o x i a o f t h e c i r c u l a t o r y and r e s p i r a t o r y s y s t e m s , e f f e r e n t and a f f e r -e n t t o t h e g i l l s o f u n a n a e s t h e t i z e d , f r e e s w i m m i n g , b u t c o n f i n e d t r o u t . - 5 -MATERIALS AND METHODS The e x p e r i m e n t s were c a r r i e d o u t on 86 r a i n b o w t r o u t ob-t a i n e d f r o m t h e f o l l o w i n g s o u r c e s : A. A s m a l l p r i v a t e h a t c h e r y n e a r Nanoose Bay, V a n c o u v e r I s l a n d , B r i t i s h C o l u m b i a . Age 3 y e a r s * W e i g h t s r a n g e d f r o m 300 t o 450 grams. B. Summerland T r o u t H a t c h e r y a t S u m m e r l a n d , B.C. Ages were v a r i e d and n o t d e t e r m i n e d . A l l were i n e x c e s s o f one y e a r o l d . W e i g h t s r a n g e d f r o m 206 t o 700 grams. C. Sun V a l l e y T r o u t Farm, P o r t C o q u i t l a m , B.C. These f i s h had a h i s t o r y o f f u r o n c u l o s i s . They were q u a r a n t i n e d and t r e a t e d w i t h s u l p h a m e r a z i n e u n t i l s e v e r a l weeks a f t e r a l l m a n i f e s t a t i o n s o f t h e d i s e a s e were no l o n g e r o b s e r v a b l e . Ages were 2 and 3 y e a r s . W e i g h t s r a n g e d f r o m 300 t o 400 grams. D. S a l t s p r i n g I s l a n d , B.C. These f i s h were r a i s e d i n l a r g e o u t d o o r ponds and c o n s e q u e n t l y were l a r g e r t h a n f i s h f r o m t h e o t h e r s o u r c e s . Ages were 2 and 3 y e a r s . \ W e i g h t s r a n g e d f r o m 350 t o 1050 grams. The f i s h were h e l d i n l a r g e t a n k s o f a t l e a s t 500 g a l l o n s c a p a c i t y . A l l e x p e r i m e n t s were c a r r i e d o u t a t t h e same t e m p e r a -t u r e as t h a t o f t h e w a t e r i n t h e h o l d i n g t a n k s . The f i s h were a c c l i m a t e d i n t h e h o l d i n g t a n k s t o t h e e x p e r i m e n t a l t e m p e r a t u r e f o r a t l e a s t one week p r i o r t o e x p e r i m e n t a t i o n . The e x p e r i m e n t s were c a r r i e d o u t a t t h e B i o l o g i c a l S t a t i o n , N a n aimo, B.C. d u r i n g May t o S e p t e m b e r 1965, and 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 f r o m S e p t e m b e r 1965 t o F e b r u a r y 1966. A f i s h was s e l e c t e d f r o m t h e h o l d i n g t a n k and a n a e s t h e t i z e d by i m m e r s i o n i n w a t e r c o n t a i n i n g 1:10,000 t r i c a i n e m e t h a n e s u l p h o -n a t e ( M 5 - 2 2 2 ) . The a n a e s t h e t i z e d f i s h was g e n t l y p l a c e d i n a - 6 -m o i s t c l o t h hammock, v e n t r a l s i d e up. C a r e was t a k e n t o a v o i d i n j u r y t o t h e f i s h . Water c o n t a i n i n g 1:10,000 t o 1:15,000 mS-222 was p e r f u s e d o v e r t h e g i l l s by means o f a r e c i r c u l a t i n g pump. P r o v i s i o n s were made f o r p e r f u s i o n i n t o e i t h e r t h e b u c c a l c a v i t y o r i n t o t h e o p e r c u l a r c a v i t i e s . W i t h h i g h e r w a t e r tem-p e r a t u r e s i t was f o u n d t h a t c o o l i n g and o x y g e n a t i n g t h e r e c i r -c u l a t i n g w a t e r g r e a t l y e x t e n d e d t h e p e r i o d o f t i m e t h a t t h e f i s h c o u l d be k e p t u n d e r a n a e s t h e s i a i n good c o n d i t i o n . W h i l e a n a e s t h e t i z e d t h e f i s h were c a n n u l a t e d a t f o u r s i t e s as f o l l o w s : 1 . B u c c a l C a v i t y C a n n u l a t i o n The t e c h n i q u e s was e s s e n t i a l l y t h e same as t h a t d e s c r i b e d by S a u n d e r s (1961 ) . C a r e was e x e r c i s e d t o a v o i d damage t o t h e b u c c a l v a l v e s and t h e o l f a c -t o r y l o b e s . 80 cm o f P.E. 200 o r P.E. 160 p o l y -e t h y l e n e t u b i n g was u s e d ( F i g . 1 ) . 2. The O p e r c u l a r C a v i t y C a n n u l a t i o n The c a v i t y was c a n n u l a t e d ( F i g . 1) by p u n c h i n g a h o l e i n t h e c e n t e r o f t h e o p e r c u l u m w i t h an 18 gauge h y p o d e r m i c n e e d l e , and a n c h o r i n g a h e a t f l a r e d 80 cm l e n g t h o f P.E. 60 p o l y e t h y l e n e t u b i n g i n t h e h o l e ( S a u n d e r s , 1 9 6 1 ) . A one cm l e n g t h o f h e a t f l a r e d P.E. 90 t u b i n g was p a s s e d o v e r t h e o u t s i d e o f t h e o p e r c u l a r c a n n u l a . T h i s s h o r t p i e c e o f t u b i n g was b r o u g h t up s n u g l y a g a i n s t t h e o p e r c u l u m and s e c u r e d w i t h t h r e a d , t h u s a n c h o r i n g t h e c a n n u l a i n p l a c e . P.E. 60 t u b i n g was u s e d i n t h i s a p p l i c a t i o n i n o r d e r t o m a i n t a i n a h i g h l e v e l o f f l e x i b i l i t y so as n o t t o i m p a i r t h e n o r m a l f u n c t i o n i n g o f t h e o p e r c u l u m . OPERCULUM _ 7 -L e o e n d f o r F i g u r e 1 D e t a i l s o f t h e head o f a t r o u t s h o w i n g r e l a t i v e p o s i t i o n s o f t h e c a n n u l a e . The D o r s a l A o r t i c C a n n u l a t i o n C a n n u l a t i o n o f t h e d o r s a l a o r t a was c a r r i e d o u t as d e s c r i b e d by S m i t h and B e l l ( 1 9 6 4 ) . P.E. 60 p o l y e t h y l e n e t u b i n g , BO cm l o n g t i p p e d w i t h t h e end o f a 21 gauge h u b e r p o i n t h y p o d e r m i c n e e d l e , was u s e d . The p o i n t o f c a n n u l a t i o n was a t t h e j u n c t i o n o f t h e r i g h t and l e f t s u p r a - b r a n c h i a l a r t e r i e s ( F i g . 1 ) . The V e n t r a l A o r t i c C a n n u l a t i o n C a n n u l a t i o n was c a r r i e d o u t u s i n g an 80 cm l e n g t h o f P.E. 60 p o l y e t h y l e n e t u b i n g , t i p p e d w i t h a 21 gauge s h o r t b e v e l h y p o d e r m i c n e e d l e b e n t a t an a n g l e o f a p p r o x i m a t e l y 60 d e g r e e s a b o u t 1-2 cm back f r o m t h e p o i n t . The v e n t r a l a o r t a was u s u a l l y v i s i b l e f r o m t h e l a t e r a l v i e w as i t p a s s e d t h r o u g h t h e i s t h m u s . I t was p o s s i b l e t o p i e r c e t h e v e n t r a l a o r t a f r o m t h e v e n t r a l m e d i a n s u r f a c e w h i l e o b s e r v i n g t h e p r o g r e s s o f t h e c a n n u l a t i p f r o m a l a t e r a l v i e w . The c o r o n a r y a r t e r y w h i c h a l s o p a s s e s i n t h i s r e g i o n i s s u f f i c i e n t -l y s m a l l and s l i p p e r y t h a t i t i s n o t c u t by t h e c a n -n u l a t i p . I t was f o u n d c o n v e n i e n t t o have a s e l e c t i o n o f c a n n u l a t i p s o f v a r i o u s l e n g t h s and a n g l e s on hand b e f o r e a t t e m p t i n g a c a n n u l a t i o n i n o r d e r t o accommo-d a t e t h e v a r i a b i l i t y o f p o s i t i o n and a n g l e o f t h e v e n t r a l a o r t a t h a t was f o u n d f r o m f i s h t o f i s h . I t was f o u n d t h a t t h e v e n t r a l a o r t a s h o u l d be c a n n u l a t e d as f a r a n t e r i o r l y as i s p r a c t i c a b l e . C a n n u l a e p l a c e d t o o n e a r t h e b u l b u s a r e p r o n e t o l e a k a g e due t o t h e d i s t e n s i b i l i t y o f t h e v e s s e l i n t h i s r e g i o n . Such l e a k age c a n f i l l t h e p e r i c a r d i a l c a v i t y w i t h b l o o d and d e a t h may e n s u e . Once t h e v e n t r a l a o r t a was p i e r c e d t h e c a n n u l a was a n c h o r e d w i t h two s t i t c h e s , one and two c e n t i m e t e r s p o s t e r i o r t o t h e p o i n t o f emergence o f t h e c a n n u l a f r o m t h e f i s h ' s i s t h m u s . When t h e p r o p e r c h o i c e o f t i p l e n g t h and a n g l e were made, t h e c a n n u l a w o u l d l i e f l a t on t h e v e n t r a l body s u r f a c e . I f t h e t i p u s e d was t o o l o n g and t h e bend i n t h e c a n n u l a t i p e x t e n ded b e y o n d t h e p o i n t o f e m e r g e n c e , a pad was p l a c e d u n d e r t h e bend t o p r e v e n t t h e c a n n u l a f r o m b e i n g p u s h e d c o m p l e t e l y t h r o u g h t h e v e n t r a l a o r t a . T h i s p r e p a r a t i o n was f o u n d t o be f l e x i b l e and r e s i s t a n t t o s u d d e n s t r a i n s ( F i g . 1 ) . T h e r e was no a p p a r e n t e f f e c t o f t h e v e n t r a l a o r t i c c a n n u l a t i o n on t h e a c t i o n o f t h e s t e r n o h y o i d e u s m u s c l e s The c a n n u l a p a s s e d b e tween t h e p a i r e d s t e r n o h y o i d e u s m u s c l e s and b e c a u s e o f t h e p r o x i m i t y o f t h e c a n n u l a t o t h e p o i n t o f i n s e r t i o n o f t h e s e m u s c l e s o n t o t h e c l e i t h r u m , movement was m i n i m a l and had no e f f e c t on t h e p o s i t i o n o f t h e c a n n u l a . The d o r s a l and v e n t r a l a o r t i c c a n n u l a e were f i l l e d w i t h h e p a r i n i z e d (10 i . u . p e r ml) C o u r t l a n d s a l i n e ( W o l f 1 9 6 3 ) , and t h e ends p l u g g e d w i t h t a p e r e d s t a i n l e s s s t e e l p i n s . I n many i n s t a n c e s a l l f o u r c a n n u l a e were n o t p l a c e d i n a s i n g l e f i s h , b u t w i t h e x p e r i e n c e i t became p o s s i b l e t o p l a c e a l l f o u r c a n n u l a e i n p o s i t i o n i n 15 - 25 m i n u t e s . A f t e r c a n n u l a t i o n t h e f i s h were p a r t i a l l y r e v i v e d by - 10 -c i r c u l a t i n g f r e s h w a t e r o v e r t h e g i l l s . When r e g u l a r b r e a t h i n g movements were o b s e r v e d t h e f i s h was q u i c k l y and g e n t l y t r a n s -p o r t e d t o a r e s p i r o m e t e r ( F i g . 2 ) . The r e s p i r o m e t e r was f l u s h e d w i t h f r e s h w a t e r and t h e f i s h was a l l o w e d t o r e c o v e r f r o m t h e o p e r a t i o n f o r a t l e a s t e i g h t h o u r s . The r e s p i r o m e t e r was d a r k e n e d by a b l a c k p o l y e t h y l e n e c o v e r d u r i n g t h i s p e r i o d . The r e s p i r o m e t e r c o n s i s t e d o f an a c r y l i c p l a s t i c t u b e ( w a l l t h i c k n e s s 1.27 cm) w i t h c o n i c a l ends ( F i g . 2 ) . I t s c a p a -c i t y was 12.23 l i t e r s . W a ter c o u l d be f l u s h e d o r c i r c u l a t e d t h r o u g h i t f r o m a t a p s u p p l y o r r e c i r c u l a t e d by means o f a s m a l l e l e c t r i c pump. The c a p a c i t y o f t h e pump was a p p r o x i -m a t e l y 13 l i t e r s p e r m i n u t e . I n c l u d e d i n t h e r e c i r c u l a t i n g w a t e r c i r c u i t was a c o i l o f m e t a l t u b i n g i m m e r s e d i n a c o n s t a n t t e m p e r a t u r e b a t h , s e r v i n g a s a h e a t e x c h a n g e r . T h i s a r r a n g e -ment a l l o w e d c o n t r o l o f t h e t e m p e r a t u r e o f t h e r e s p i r o m e t e r . The u p s t r e a m end o f t h e r e s p i r o m e t e r chamber h e l d a s e r i e s o f g r i d s w h i c h p r o d u c e d s m a l l t u r b u l e n c e e d d i e s i n t h e i n c o m i n g w a t e r ( B r e t t , 1 9 6 4 ) . These r e d u c e d t h e l a m i n a r f l o w c o n d i -t i o n s w h i c h , i f w e l l d e v e l o p e d , w o u l d r e s u l t i n u n e q u a l r e s i -d e n c e t i m e and p o o r m i x i n g o f w a t e r i n t h e r e s p i r o m e t e r . The c a n n u l a e p a s s e d o u t t h r o u g h one o f a p a i r o f e x i t t u b e s i n t h e t o p o f t h e r e s p i r o m e t e r . The r e s p i r o m e t e r was s u f f i c i e n t l y l a r g e t h a t a l l b u t t h e l a r g e s t f i s h were a b l e t o t u r n a r o u n d and swim f r o m end t o end o f t h e chamber. The f i s h u s u a l l y a ssumed a p o s i t i o n n e a r t h e i n l e t o f t h e chamber f a c i n g up-s t r e a m , and r e m a i n e d q u i e t . To measure o x y g e n c o n s u m p t i o n , t h e e x i t t u b e s were s e a l e d w i t h a f l o a t i n g l a y e r o f l i q u i d p e t r o l a t u m and t h e CANNULAE TO PRESSURE AND GAS MEASURING EQUIPMENT ^ — HEAT EXCHANGER - 11 L e g e n d f o r F i g u r e 2 The r e s p i r o m e t e r u s e d i n t h e e x p e r i m e n t s . The f i s h i l l u s t r a t e d i s c o n s i d e r a b l y l a r g e r t h a n t h e a v e r a g e s i z e o f f i s h u s e d i n t h e e x p e r i m e n t s . - 12 -w a t e r movement - i n s i d e s w i t c h e d f r o m f l u s h i n g t o pump r e c i r c u l a -t i o n . P e r i o d i c o x y g e n d e t e r m i n a t i o n s o f t h e d e c l i n i n g o x y g e n c o n t e n t o f t h e r e c i r c u l a t i n g w a t e r a l l o w e d c o m p u t a t i o n o f o x y -gen c o n s u m p t i o n ( B r e t t , 1964; B r e t t & S u t h e r l a n d , 1965; R a n d a l l , S m i t h , and B r e t t , 1 9 6 5 ) . D u r i n g t h e p e r i o d May 1965 t o J u l y 1965 o x y g e n was measured by means o f t h e u n m o d i f i e d W i n k l e r method as a p p l i e d by B r e t t ( 1 9 6 4 ) . Th i s method had t h e d i s a d v a n t a g e t h a t a b o u t 130 ml o f w a t e r was w i t h d r a w n f o r ea c h d e t e r m i n a t i o n r e -s u l t i n g i n c h a n g e s i n t h e t o t a l volume o f w a t e r i n t h e r e s p i r o -m e t e r . A f t e r A u g u s t 1965 a l l o x y g e n d e t e r m i n a t i o n s were made by means o f a Beckman o x y g e n e l e c t r o d e . T h i s method gave de-t e r m i n a t i o n s o f P0^ w h i c h c o u l d be c o n v e r t e d t o o x y g e n c o n t e n t by a p p l i c a t i o n o f s o l u b i l i t y c o n s t a n t s . The l a t t e r t e c h n i q u e r e q u i r e d o n l y a b o u t 1 ml o f w a t e r f o r a d e t e r m i n a t i o n . The t e m p e r a t u r e o f t h e w a t e r v a r i e d f r o m 9°C t o 19°C d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t s . I n any one e x p e r i m e n t t e m p e r a -t u r e c h a n g e s n e v e r e x c e e d e d 1.2°C and u s u a l l y were l e s s t h a n 0.6°C. In a l l c a s e s t h e t e m p e r a t u r e t e n d e d t o i n c r e a s e d u r i n g an e x p e r i m e n t . E x p e r i m e n t s were c a r r i e d o u t between 9:00 A.M. and 3:00 A.m. A l l e x p e r i m e n t s c o n s i s t e d o f s e a l i n g t h e r e s p i r o m e t e r and a l l o w i n g t h e f i s h t o consume t h e a v a i l a b l e o x y g e n . The r a t e o f o x y g e n c o n s u m p t i o n was m o n i t o r e d c o n t i n u o u s l y and a v a r i e t y o f p a r a m e t e r s o f t h e c i r c u l a t o r y and b r e a t h i n g s y s t e m s r e c o r d e d . In t h i s way t h e r e s p o n s e o f t h e c i r c u l a t o r y and b r e a t h i n g s y s t e m s t o h y p o x i a was d e t e r m i n e d . When t h e f i s h showed s i g n s o f c a r -d i a c o r r e s p i r a t o r y f a i l u r e due t o h y p o x i a , f l u s h i n g o f t h e - 13 -r e s p i r o m e t e r w i t h f r e s h w a t e r was resumed and t h e e x p e r i m e n t t e r m i n a t e d . W h i l e t h i s method i s a c o n v e n i e n t way o f v a r y i n g e n v i r o n -m e n t a l o x y g e n , i t has t h e d i s a d v a n t a g e o f a c o n c o m i t a n t CO^ b u i l d u p . However t h e s o l u b i l i t y o f CO^ i n w a t e r i s more t h a n 33 t i m e s t h a t o f o x y g e n and c h a n g e s i n t h e p a r t i a l p r e s s u r e o f CO^ were o f t h e o r d e r o f 1 - 2 mm Hg. The r e c o r d i n g o f p r e s s u r e s f r o m t h e c i r c u l a t o r y and b r e a t h -i n g s y s t e m s was c a r r i e d o u t i n t h e f o l l o w i n g manner. B u c c a l and o p e r c u l a r c a n n u l a e were c o n n e c t e d t o S t a t ham P 23 - AA p r e s s u r e t r a n s d u c e r s . D o r s a l and v e n t r a l a o r t i c c a n n u l a e were c o n n e c t e d t o S t a t h a m P 23 - BB p r e s s u r e t r a n s d u c e r s . A l t e r -n a t i v e l y t h e d o r s a l a nd v e n t r a l a o r t i c c a n n u l a e were c o n n e c t -ed t o a S a n b o r n model 26BB d i f f e r e n t i a l p r e s s u r e t r a n s d u c e r . The o u t p u t f r o m t h e t r a n s d u c e r s was r e c o r d e d c o n t i n u o u s l y w i t h a Beckman Q f f n e r Type R D y n o g r a p h r e c o r d e r . C a r e was t a k e n t o e n s u r e t h a t a l l c a n n u l a e were f r e e o f c o n s t r i c t i o n s and t h a t a l l were t h e same l e n g t h . A l l c a n n u l a e were o f PE 60 p o l y e t h y l e n e t u b i n g e x c e p t t h e b u c c a l c a n n u l a w h i c h was PE 200 o r PE 160. A l l d e t e r m i n a t i o n s o f h e a r t and b r e a t h i n g r a t e s o b t a i n e d f r o m t h e p r e s s u r e r e c o r d i n g s were b a s e d on a t l e a s t t w e n t y s e c o n d s r e c o r d i n g . E x p e r i m e n t s were c a r r i e d o u t t o t e s t i n t e r a c t i o n e f f e c t s t h a t t h e d o r s a l and v e n t r a l a o r t i c c a n n u l a e m i g h t have upon b l o o d p r e s s u r e . F i s h w i t h o n l y one o f t h e two c a n n u l a e i n them were s u b j e c t e d t o t h e same t e s t s as were f i s h w i t h b o t h c a n n u l a e i n p l a c e . No s i g n i f i c a n t i n t e r a c t i o n e f f e c t o r - 14 -b l o c k a g e was d e t e c t e d as a r e s u l t o f t h e c a n n u l a t i o n s „ The PO^, PCo^, and pH o f b l o o d and w a t e r a f f e r e n t and e f f e r e n t t o t h e g i l l s was me a s u r e d u s i n g a Beckman model 160 p h y s i o l o g i c a l b l o o d gas a n a l y s e r . The m e a s u r e m e n t s were r e -c o r d e d s i m u l t a n e o u s l y w i t h m e a s u r e m e n t s o f b l o o d and b r e a t h i n g p r e s s u r e s . A l s o a t t h e same t i m e s e r i a l d e t e r m i n a t i o n s o f t h e h e m a t o c r i t and r e d b l o o d c e l l c o u n t o f t h e f i s h e s b l o o d were made. The s a m p l e s f o r gas t e n s i o n and pN a n a l y s i s were c o n -d u c t e d i n t o m o d u l a r c u v e t t e s m a i n t a i n e d a t t h e same t e m p e r a -t u r e as t h a t o f t h e r e s p i r o m e t e r . Oxygen d e t e r m i n a t i o n s were made u s i n g a Beckman p l a t i n u m - s i l v e r p o l a r i z i n g e l e c t r o d e c o v e r e d w i t h p o l y p r o p y l o n membrane. The PC02 d e t e r m i n a t i o n s were made u s i n g a Beckman S e v e r i n h a u s t y p e e l e c t r o d e c o v e r e d w i t h a t e f l o n membrane. The pH o f t h e s a m p l e s was d e t e r -m i n e d by means o f a g l a s s pH e l e c t r o d e . O n l y two o f t h e s e t h r e e p a r a m e t e r s were m e a s u r e d on any s i n g l e e x p e r i m e n t , b u t o x y g e n t e n s i o n was m e a s u r e d i n a l l c a s e s . The e l e c t r o d e s u s e d were mounted i n m o d u l a r c u v e t t e s m a n u f a c t u r e d by t h e Beckman i n s t r u m e n t company, i n w h i c h t h e s a m p l e volume r e q u i r e d t o c o v e r t h e e l e c t r o d e t i p s was l e s s t h a n 1.0 m l . S a m p l e s o f b l o o d o r w a t e r were a d m i t t e d d i r e c t l y i n t o t h e c u v e t t e s f r o m t h e f o u r c a n n u l a e w h i c h have a l r e a d y been d e s c r i b e d . The b l o o d s a m p l e s were a d m i t t e d i n t o t h e c u v e t t e s by means o f t h e f i s h e s ' b l o o d p r e s s u r e . The w a t e r s a m p l e s were a s s i s t e d i n t o t h e c u v e t t e s by a p p l y i n g a s l i g h t s u c t i o n on t h e o u t l e t o f t h e c u v e t t e s . The a n a l y s i s t i m e f o r P0^ was f r o m 30 t o 120 s e c o n d s , - 15 -f o r pH a n a l y s i s t i m e was 1 t o 3 m i n u t e s , and f o r PC02 t h e t i m e f o r a n a l y s i s was 5 t o 11 m i n u t e s . The b l o o d s a m p l e s were r e t u r n e d t o t h e f i s h i m m e d i a t e l y a f t e r e a c h a n a l y s i s . A l l t h e e l e c t r o d e s were c a l i b r a t e d i m m e d i a t e l y p r i o r t o e a c h e x p e r i m e n t and c h e c k e d a t t h e end o f e a c h e x p e r i m e n t f o r d r i f t . I n a d d i t i o n t h e pH e l e c t r o d e was c h e c k e d and r e c a l i -b r a t e d a f t e r e v e r y 2 o r 3 d e t e r m i n a t i o n s u s i n g a f r e s h l y p r e -p a r e d b u f f e r s o l u t i o n . B o t h t h e PQ^ and P C ^ e l e c t r o d e s were c a l i b r a t e d w i t h m o i s t gas m i x t u r e s , n i t r o g e n and a t m o s p h e r i c a i r i n t h e c a s e o f t h e PO^ e l e c t r o d e and s t a n d a r d i z e d gas m i x t u r e s i n t h e c a s e o f t h e PC02 e l e c t r o d e . H e m o l y s i s and c l o t t i n g o f t h e b l o o d s a m p l e s was p r e v e n t e d by f l u s h i n g t h e c u v e t t e chambers t h o r o u g h l y w i t h C o u r t l a n d s a l i n e ( W o l f 1963) c o n t a i n i n g a w e t t i n g a g e n t (Tween - 80, one d r o p p e r l i t e r ) and 10 i . u . / m l o f s o d i u m h e p a r i n . The c u v e t t e s were f l u s h e d w i t h t h i s s o l u t i o n b e f o r e a d m i s s i o n o f e v e r y b l o o d s a m p l e . S m a l l q u a n t i t i e s o f b l o o d were t a k e n f r o m t h e d o r s a l a o r -t i c c a n n u l a a t t h e b e g i n n i n g o f e v e r y e x p e r i m e n t i n o r d e r t o d e t e r m i n e t h e h e m a t o c r i t v a l u e o f t h e f i s h ' s b l o o d . In some c a s e s t h e h e m a t o c r i t s a m p l e s were t a k e n p e r i o d i c a l l y t h r o u g h -o u t t h e c o u r s e o f an e x p e r i m e n t . B l o o d s a m p l e s were drawn f r o m t h e d o r s a l a o r t i c c a n n u l a p e r i o d i c a l l y i n o r d e r t o d e t e r m i n e t h e r e d b l o o d c e l l c o u n t as w e l l a s t h e h e m a t o c r i t v a l u e s o f t h e b l o o d i n o r d e r t o d e t e r -mine w h e t h e r o r n o t t h e r e was any marked c h a n g e i n t h e c h a r a c -t e r i s t i c s o f t h e b l o o d d u r i n g h y p o x i a . As many mea s u r e m e n t s as p o s s i b l e were made d u r i n g t h e c o u r s e o f an e x p e r i m e n t , A c h e c k was made on t h e l a c t a t e l e v e l s o f t h e b l o o d o f t h e f i s h b e f o r e and a f t e r e x p e r i m e n t a t i o n . F o u r f i s h were e x a m i n e d . B l o o d s a m p l e s o b t a i n e d f r o m t h e d o r s a l a o r t i c c a n n u l a e (0„5 ml) were p l a c e d i n a l i q u o t s o f 10% t r i c h l o r o -a c e t i c a c i d i m m e d i a t e l y a f t e r w i t h d r a w a l . The s a m p l e s were t h e n f i l t e r e d and t h e f i l t r a t e s were s t o r e d i n a r e f r i g e r a t o r ( n o t f r o z e n ) u n t i l t h e y were a n a l y s e d f o r l a c t a t e by t h e method o f B a r k e r and Summerson (1941 ) . - 17 -RESULTS AND DISCUSSION - I The t r o u t r e s p o n d e d t o h y p o x i a by i n c r e a s i n g t h e r a t e and a m p l i t u d e o f b r e a t h i n g movements ( F i g . 3 ) . B r e a t h i n g r a t e i n -c r e a s e d f r o m 83 movements p e r m i n u t e t o 123 p e r m i n u t e . B r e a t h i n g r a t e was v a r i a b l e between f i s h o w i n g l a r g e l y t o t h e d i v e r s e s i z e o f t h e f i s h e x a m i n e d . L a r g e f i s h t e n d e d t o r e s p i r e a t l o w e r r a t e s . A m p l i t u d e o f b r e a t h i n g movements, as i n d i c a t e d by v i s u a l o b s e r v a t i o n s and r e c o r d e d p r e s s u r e s , i n c r e a s e d w i t h l o w e r e n v i r o n m e n t a l o x y g e n l e v e l s . Changes i n e n v i r o n m e n t a l P02 b e t w e e n 150 and 90 mm Hg p r o -d u c e d no s i g n i f i c a n t c h a n g e s i n h e a r t r a t e . Between 90 and 30 mm Hg Po^ t h e h e a r t r a t e d e c r e a s e d f r o m n o r m a l l e v e l s o f 75 b e a t s p e r m i n u t e t o v a l u e s o f 25 p e r m i n u t e ( F i g . 3 ) . The o n s e t o f t h e b r a d y c a r d i a d i d n o t a l w a y s o c c u r g r a d u a l l y . I n some c a s e s t h e h e a r t r a t e w o u l d d r o p a b r u p t l y . The o b s e r v e d r e s p o n s e s o f h e a r t r a t e , b r e a t h i n g r a t e and b r e a t h i n g a m p l i t u d e ( F i g . 3) a r e s i m i l a r t o t h o s e o b s e r v e d by R a n d a l l and S h e l t o n ( 1 9 6 4 ) i n t e n c h ( T i n e a t i n e a L.) b u t i n t e n c h t h e o n s e t o f b r a d y c a r d i a o c c u r r e d a t l o w e r o x y g e n l e v e l s t h a n i n r a i n b o w t r o u t . V e n t r a l a o r t i c s y s t o l i c b l o o d p r e s s u r e was f o u n d t o be a b o u t 54 mm Hg and t h e p u l s e p r e s s u r e was f o u n d t o be a b o u t 16 mm Hg, i n t h e r e s t i n g f i s h i n w e l l o x y g e n a t e d w a t e r ( F i g s . 7 and 8 ) . T h i s v a l u e i s c o n s i d e r a b l y h i g h e r t h a n t h o s e r e -p o r t e d f o r e l a s m o b r a n c h s , 39/28 ( B u r g e r and B r a d l e y 1 9 5 1 ) , and t h e e e l , 37.5 mm Hg ( M o t t 1 9 5 0 ) , b u t l o w e r t h a n t h a t r e p o r t e d f o r c h i n o o k s a l m o n ( 7 4 . 6 mm Hg Greene 1 9 0 4 ) . T h e r e a r e two r e a s o n s w h i c h a c c o u n t , i n p a r t , f o r t h e d i f f e r e n c e i n - 18 -L e g e n d f o r F i g u r e 3 The e f f e c t o f h y p o x i a upon b r e a t h i n g and h e a r t r a t e . E a c h c i r c l e r e p r e s e n t s an a v e r a g e d v a l u e f o r a l l d e t e r -m i n a t i o n s w i t h i n a 10 mm Hg e n v i r o n m e n t a l o x y g e n t e n s i o n i n t e r v a l . The v e r t i c a l b a r s r e p r e s e n t p l u s o r minus 2 s t a n d a r d e r r o r s . - 19 -v e n t r a l a o r t i c b l o o d p r e s s u r e s r e p o r t e d h e r e f o r r a i n b o w t r o u t and t h o s e p r e s s u r e s r e c o r d e d i n c h i n o o k s a l m o n by Greene ( 1 9 0 4 ) . B o t h Greene ( 1 9 0 4 ) and B u r g e r and B r a d l e y ( 1 9 5 1 ) who worked w i t h S q u a l u s a c a n t h i a s o b s e r v e d t h a t l a r g e r f i s h had h i g h e r b l o o d p r e s s u r e s . The s a l m o n t h a t Greene ( 1 9 0 4 ) worked w i t h were two t o f o u r t i m e s as l o n g as t h e r a i n b o w t r o u t u s e d i n t h e p r e s e n t s t u d y . S e c o n d l y t h e s a l m o n u s e d by Greene ( 1 9 0 4 ) were h e l d r e s t r a i n e d o u t o f w a t e r w i t h an a r t i f i c i a l means o f i r r i g a t i n g t h e i r g i l l s . I t i s q u i t e p o s s i b l e t h a t t h e s e s a l -mon were i n an h y p o x i c s t a t e i n w h i c h c a s e t h e b l o o d p r e s s u r e s d e v e l o p e d w o u l d be c o m p a r a b l e t o t h e b l o o d p r e s s u r e s o b t a i n e d i n t h i s s t u d y when t h e r a i n b o w t r o u t were i n an h y p o x i c c o n d i -t i o n . The e f f e c t o f i n c r e a s i n g h y p o x i a upon t h e v e n t r a l a o r t i c b l o o d p r e s s u r e o f r a i n b o w t r o u t was a g e n t l e i n c r e a s e i n s y s -t o l i c p r e s s u r e s b e c o m i n g i n c r e a s i n g l y p r o n o u n c e d a t l o w e r e n v i r o n m e n t a l o x y g e n l e v e l s ( F i g s . 4 and 7 ) . The h i g h e s t s y s t o l i c p r e s s u r e s o f 81 mm Hg were r e c o r d e d ,at 40 mm Hg e n v i r o n m e n t a l Po£. The r e s u l t s i n d i c a t e d t h a t a t e n v i r o n -m e n t a l o x y g e n l e v e l s b e l o w 40 mm Hg Po2> t h e v e n t r a l a o r t i c s y s t o l i c p r e s s u r e d e c r e a s e d . T h i s was s y m p t o m a t i c o f t h e o n s e t o f r e s p i r a t o r y and c a r d i a c c o l l a p s e . Few e x p e r i m e n t s were c o n t i n u e d b e l o w e n v i r o n m e n t a l o x y g e n l e v e l s o f 30 mm Hg Pct2° P u l s e p r e s s u r e i n c r e a s e d a s h y p o x i a d e v e l o p e d , t h e i n c r e a s e s b e c o m i n g more i n t e n s i f i e d a t t h e l o w e r e d e n v i r o n -m e n t a l Po^ l e v e l s . The h i g h e s t p u l s e p r e s s u r e s o f 40 mm Hg were r e c o r d e d a t an e n v i r o n m e n t a l o x y g e n l e v e l o f 30 mm Hg P o ? . U n l i k e t h e s y s t o l i c p r e s s u r e s , t h e v e n t r a l a o r t i c p u l s e V E N T R A L AORT IC B L O O D P R E S S U R E R E C O R D I N G S f- IOO.MM.HG IO.SEC. I. S E C . - 20 -Legend for Figure 4 Pressure recordings from the v e n t r a l aorta under normal, p a r t i a l l y hypoxic, and hypoxic environmental oxygen tensions. There i s a s l i g h t d i s t o r t i o n of the t r a c i n g s due to curve-l i n e a r writeout from the recorder. p r e s s u r e s d i d n o t show any marked t e n d e n c y t o d e c r e a s e a t v e r y l o w e n v i r o n m e n t a l o x y g e n l e v e l s ( F i g , 7 ) . D u r i n g h y p o x i a t h e r e were i n c r e a s e s i n d o r s a l a o r t i c s y s t o l i c and p u l s e p r e s s u r e s ( F i g , 5 and 8 ) . S y s t o l i c p r e s -s u r e s i n c r e a s e d f r o m a mean o f 34 mm Hg when t h e r e s t i n g f i s h was i n w e l l o x y g e n a t e d w a t e r t o 47 mm Hg a t e n v i r o n m e n t a l P02 o f 3D mm Hg. P u l s e p r e s s u r e i n t h e d o r s a l a o r t a s i m i l a r l y i n c r e a s e d f r o m 6 mm Hg t o 14 mm Hg a t 30 mm Hg P02 i n t h e w a t e r . No d r o p i n e i t h e r s y s t o l i c o r p u l s e p r e s s u r e was ob-s e r v e d i n t h e d o r s a l a o r t a a t t h e low e n v i r o n m e n t a l o x y g e n l e v e l s as was p r e v i o u s l y o b s e r v e d w i t h v e n t r a l a o r t i c s y s t o -l i c p r e s s u r e . The d o r s a l a o r t i c b l o o d p r e s s u r e o f 34 mm Hg a g r e e s c l o s e -l y w i t h t h e v a l u e o f 38.7 - 2.9 mm Hg i n f r e e swimming r a i n -bow t r o u t a t a p p r o x i m a t e l y t h e same t e m p e r a t u r e r e p o r t e d by R a n d a l l , S m i t h , and B r e t t ( 1 9 6 5 ) . The d o r s a l a o r t i c b l o o d p r e s s u r e f o r t h e r a i n b o w t r o u t i s c o n s i d e r a b l y l o w e r t h a n t h e v a l u e o f 53.3 mm Hg i n t h e d o r s a l a o r t a o f c h i n o o k s a l m o n r e -p o r t e d by Greene ( 1 9 0 4 ) . The r e a s o n s f o r t h e d i f f e r e n c e a r e p r o b a b l y t h e same a s t h o s e s u g g e s t e d f o r t h e c o r r e s p o n d i n g d i s c r e p a n c y i n v e n t r a l a o r t i c p r e s s u r e s . R a n d a l l , S m i t h , and B r e t t ( 1 9 6 5 ) n o t e d t h a t t h e b l o o d p r e s s u r e s o f r a i n b o w t r o u t ( u s i n g t h e same t e c h n i q u e as was u s e d i n t h i s w o r k ) w e r e , i n g e n e r a l , h i g h e r t h a n o t h e r t e l e o s t b l o o d p r e s s u r e s r e p o r t e d i n t h e l i t e r a t u r e . They s u g g e s t e d t h a t t h e d i f f e r e n c e s were a r e s u l t o f r e c o r d i n g f r o m a f r e e swimming i n t a c t f i s h as o p p o s e d t o r e s t r a i n e d and i n some c a s e s a n a e s t h e t i z e d a n i m a l s . T h e r e were s l i g h t i n c r e a s e s i n t h e d i f f e r e n t i a l p r e s s u r e D O R S A L A O R T I C B L O O D P R E S S U R E R E C O R D I N G S E N - P 0 2 " l 4 0 , M M - H G f»( f(M^ 7 0 M M . H G r- 30 -MM.HG E N . Pn • 9 0 . M M . H G u 2 - 7 0 . M M . H G - 3 0 M M . H G E N . P Q 2 = 4 0 . M M . H G - 70 .MM.HG - 3 0 . M M . H G IO. S E C . I. S E C . - 2 2 -L e g e n d f o r F i g u r e 5 P r e s s u r e r e c o r d i n g s f r g m t h e d o r s a l a o r t a u n d e r n o r m a l , p a r t i a l l y h y p o x i c , and h y p o x i c e n v i r o n m e n t a l o x y g e n t e n s i o n s . T h e r e i s a s l i g h t d i s t o r t i o n o f t h e t r a c i n g s due t o c u r v e -l i n e a r w r i t e - o u t f r o m t h e r e c o r d e r . _ 23 -b e t w e e n t h e d o r s a l and v e n t r a l a o r t a e i n r e s p o n s e t o i n c r e a s -i n g l y h y p o x i c c o n d i t i o n s ( F i g s . 6 and 9 ) . T h e r e was, as w e l l , an i n c r e a s e i n t h e p u l s a t i l i t y o f t h e d i f f e r e n t i a l p r e s s u r e . The g e n e r a l f o r m and p a t t e r n o f r e s p o n s e o f d i f f e r e n t i a l p r e s -s u r e was v e r y s i m i l a r t o t h a t o f t h e d o r s a l a o r t i c p r e s s u r e s . The p r e s s u r e d i f f e r e n t i a l o v e r t h e g i l l s r o s e f r o m a maximum o f 17 mm Hg and a minimum o f 6 mm Hg a t an e n v i r o n m e n t a l o x y g e n P02 o f 140 mm Hg, t o a maximum o f 35 mm Hg and a minimum o f 9.5 mm Hg a t an e n v i r o n m e n t a l o x y g e n l e v e l o f 40 mm Hg P02 ( F i g . 9 ) . The g e n e r a l i n c r e a s e i n b l o o d p r e s s u r e s c o u l d be c a u s e d by two f a c t o r s ; an i n c r e a s e i n c a r d i a c o u t p u t , and by an i n -c r e a s e i n p e r i p h e r a l r e s i s t a n c e t o b l o o d f l o w . I f i t i s assumed t h a t t h e p r e s s u r e r i s e i s due t o i n c r e a s e d c a r d i a c o u t p u t a l o n e , t h e n t h e c a r d i a c o u t p u t w o u l d have t o v i r t u a l l y d o u b l e . The b a s i s o f t h i s h y p o t h e s i s i s t h a t a p a s s i v e b l o o d v e s s e l w i l l d i s t e n d somewhat under i n c r e a s e d p r e s s u r e , and t h u s an i n c r e a s e i n p r e s s u r e w o u l d r e s u l t i n a c o n s i d e r a b l y g r e a t e r f l o w . F u r t h e r m o r e , i f c a r d i a c o u t p u t a l o n e were r e s p o n s i b l e f o r t h e i n c r e a s e i n b l o o d p r e s s u r e s d u r i n g h y p o x i a , t h e r e w o u l d be a t l e a s t a s i x - f o l d i n c r e a s e i n s t r o k e volume s i n c e h e a r t r a t e d e c r e a s e s w i t h i n c r e a s i n g h y p o -x i a . A s i x - f o l d i n c r e a s e i n s t r o k e volume o f t h e h e a r t seems r a t h e r l a r g e so i t i s l i k e l y t h a t t h e i n c r e a s e i n b l o o d p r e s -s u r e s w i t h h y p o x i a a r e due, t o some e x t e n t , t o an i n c r e a s e i n p e r i p h e r a l v a s c u l a r r e s i s t a n c e . I f an i n c r e a s e i n v a s c u l a r r e s i s t a n c e i s t o t a l l y o r i n p a r t r e s p o n s i b l e f o r t h e i n c r e a s e i n b l o o d p r e s s u r e d u r i n g DIFFERENTIAL B L O O D PRESSURE RECORDINGS EN. P n = 140. MM.HG ° 2 4QMM.HG - O.MM.HG EN. P^ = 9 0 . MM.HG ° 2 - 4QMM.HG - QMM.HG EN. P 0 = 4 0 . MM.HG ° 2 - 4QMM.HG 1 QMM.HG 8-SEC. I.SEC - 24 -Legend for Figure 6 Pressure recordings of the blood pressure d i f f e r e n t i a l over the g i l l s ( v e n t r a l a o r t i c - dorsal a o r t i c ) under normal, p a r t i a l l y hypoxic, and hypoxic environmental oxygen te n s i o n s . C u r v e l i n e a r write-out has again caused some very s l i g h t d i s -t o r t i o n of the t r a c i n g s . 120 S Y S T O L I C V E N T R A L A O R T I C S Y S T O L I C & P U L S E P R E S S U R E S M M HG 8 0 4 0 + 4 0 8 0 I20 E N V I R O N M E N T A L PQ^ M M HG - 25 -L e g e n d f o r F i g u r e 7 The e f f e c t o f h y p o x i a upon v e n t r a l a o r t i c s y s t o l i c and p u l s e p r e s s u r e s . E a c h p o i n t p l o t t e d i s an a v e r a g e v a l u e f o r a l l d e t e r m i n a t i o n s w i t h i n a t e n mm Hg e n v i r o n m e n t a l o x y g e n t e n s i o n i n t e r v a l . The v e r t i c a l b a r s r e p r e s e n t p l u s and minus two s t a n d a r d e r r o r s . 6 0 D O R S A L A O R T I C 4 0 S Y S T O L I C & P U L S E P R E S S U R E S M M H G 2 0 O 4 0 8 0 I 2 0 E N V I R O N M E N T A L P Q M M H G 160 - 26 -L e g e n d f o r F i g u r e 8 The e f f e c t o f h y p o x i a upon d o r s a l a o r t i c s y s t o l i c and p u l s e p r e s s u r e s . The v e r t i c a l b a r s r e p r e s e n t p l u s o r minus 2 s t a n d a r d e r r o r s . Each p o i n t p l o t t e d i s an a v e r a g e d v a l u e f o r a l l d e t e r m i n a t i o n s w i t h i n a 10 mm Hg e n v i r o n m e n t a l o x y g e n t e n s i o n i n t e r v a l . 6 0 E N V I R O N M E N T A L P Q 2 M M H G - 27 -L e g e n d f o r F i g u r e 9 The e f f e c t s o f h y p o x i a upon b l o o d p r e s s u r e d i f f e r e n t i a l o v e r t h e g i l l s ( v e n t r a l a o r t i c - d o r s a l a o r t i c ) . E a c h p o i n t p l o t t e d i s an a v e r a g e d v a l u e f o r a l l d e t e r m i n a t i o n s w i t h i n a 10 mm Hg e n v i r o n m e n t a l o x y g e n t e n s i o n i n t e r v a l . - 28 -h y p o x i a , i t s h o u l d be p o s s i b l e t o d e t e c t where t h e c h a n g e s i n r e s i s t a n c e a r e o c c u r r i n g by e x a m i n a t i o n o f t h e p r e s s u r e r e c o r d -i n g s . The r a t i o o f d o r s a l a o r t i c t o v e n t r a l a o r t i c p r e s s u r e i s f a i r l y u n i f o r m a t a l l e n v i r o n m e n t a l P02 l e v e l s ( F i g , 1 0 ) , T h i s s u g g e s t s t h a t any r e s i s t a n c e c h a n g e s a r e o c c u r r i n g i n g i l l and p e r i p h e r a l v e s s e l s a t a l m o s t t h e same t i m e . A s t u d y was made o f t h e r a t e o f p r e s s u r e d r o p i n d i f f e r -e n t i a l p r e s s u r e a c r o s s t h e g i l l s , a t a c h o s e n p r e s s u r e o f 10 mm Hg„ R e c o r d s f r o m e x p e r i m e n t s on s i x f i s h were u s e d . F o u r of t h e s i x f i s h showed a d e f i n i t e d e c r e a s e i n t h e r a t e o f p r e s -s u r e d r o p a c r o s s t h e g i l l s i n r e s p o n s e t o h y p o x i a ( F i g 0 1 1 ) . The o t h e r two f i s h e x a m i n e d showed no s i g n i f i c a n t c hange i n t h e r a t e o f p r e s s u r e d r o p . T h i s i n f o r m a t i o n s u g g e s t e d t h a t t h e r e i s an i n c r e a s e i n g i l l r e s i s t a n c e w i t h i n c r e a s i n g h y p o x i a I f i t i s a c c e p t e d t h a t g i l l r e s i s t a n c e i n c r e a s e s t h e n t h e r e must, when we c o n s i d e r t h e p r e v i o u s a r g u m e n t , be an i n c r e a s e i n p e r i p h e r a l r e s i s t a n c e as w e l l . In some i n s t a n c e s when f l u s h i n g t h e r e s p i r o m e t e r a t t h e end o f an e x p e r i m e n t Evans b l u e dye was i n j e c t e d i n t o t h e o x y g e n a t e d w a t e r . The f r o n t o f t h e dye was o b s e r v e d c a r e -f u l l y , C a r d i o a c c e l e r a t i o n t o o k p l a c e i n l e s s t h a n t h r e e s e c o n d s f r o m t h e t i m e t h a t t h e dyed o x y g e n a t e d w a t e r was o b s e r v e d t o r e a c h t h e h y p o x i c f i s h e s mouth ( F i g , 1 2 ) . I n -t a k e o f o x y g e n a t e d w a t e r by t h e h y p o x i c f i s h was f o l l o w e d by a r i s e i n v e n t r a l and d o r s a l a o r t i c s y s t o l i c b l o o d p r e s -s u r e and a l a r g e d e c r e a s e i n p u l s e p r e s s u r e ( F i g , 1 2 ) . The r i s e i n d o r s a l a o r t i c b l o o d p r e s s u r e was n o t as p r o n o u n c e d - 29 -L e g e n d f o r F i g u r e 10 The r a t i o o f d o r s a l a o r t i c s y s t o l i c p r e s s u r e t o v e n t r a l a o r t i c s y s t o l i c p r e s s u r e , and t h e r a t i o o f d o r s a l a o r t i c p u l s e p r e s s u r e t o v e n t r a l a o r t i c p u l s e p r e s s u r e a t v a r y i n g e n v i r o n m e n t a l o x y g e n t e n s i o n s . - 30 -L e g e n d f o r F i g u r e 11 The u p p e r i l l u s t r a t i g n shows t h e method o f d e t e r m i n i n g t h e r a t e o f d i f f e r e n t i a l b l o o d p r e s s u r e ( v e n t r a l a o r t i c minus d o r s a l a o r t i c ) d e c r e a s e o v e r t h e g i l l s . The l e n g t h o f t i m e f o r t h e p r e s s u r e d i f f e r e n t i a l t o d e c r e a s e 10 mm Hg was u s e d as a measure o f t h e r e s i s t a n c e t o f l o w o f b l o o d t h r o u g h t h e g i l l s . The r a t e was d e r i v e d f r o m an e x t r a p o l a t i o n o f t h e r a t e o f p r e s s u r e d r o p w h i c h was f o u n d a t a p r e s s u r e d i f f e r e n t i a l o f 10 mm Hg. O n l y r e c o r d i n g s i n w h i c h t h e f l a t t e r p o r t i o n s o f t h e p r e s s u r e p u l s e t r a c i n g s i n c l u d e d t h e 10 mm Hg l e v e l were u s e d . The l o w e r f i g u r e i l l u s t r a t e s t h e e f f e c t s o f h y p o x i a upon t h e r a t e o f d i f f e r e n t i a l b l o o d p r e s s u r e d r o p o b t a i n e d f r o m two e x p e r i m e n t s . A t o t a l o f s i x e x p e r i m e n t s were e x a m i n e d ; f o u r o f w h i c h showed i n c r e a s e s i n t i m e f o r p r e s s u r e t o d r o p 10 mm Hg i n d i c a t i n g f l o w r e s i s t a n c e i n c r e a s e s w i t h h y p o x i a s u c h as shown i n t h e u p p e r l i n e ( o p e n c i r c l e s ) , and two o f w h i c h showed no c h a n g e s u c h as t h e l o w e r l i n e ( c l o s e d c i r c l e s ) . The a b s o l u t e v a l u e s o f t i m e shown a r e n o t i m p o r t a n t b u t t h e r e l a t i v e c h a n g e s w i t h i n an e x p e r i m e n t a r e u s e f u l a s e v i d e n c e o f f l o w r e s i s t a n c e c h a n g e s w i t h i n t h e g i l l s . E F F E C T O F F R E S H WATER U P O N B L O O D PRESSURE IN HYPOXIC RAINBOW TROUT - 5 0 . M M . HG. • O. M M . HG. F W - 8. SEC. V E N T R A L AORTA AUG. 23 ,1965 1-70. M M . HG. J- 3 0 . M M . HG. FW. a S E C DORSAL AORTA AUG. 26, 1965 + M O . M M . H G h O. MM.HG. I. S E C . F.W. l o . SEC. D IFFERENTIAL SEPT. 2 , 1965 I. SEC . - 31 -L e g e n d f o r F i g u r e 12 The e f f e c t o f f l u s h i n g f r e s h o x y g e n a t e d w a t e r i n t o t h e r e s p i r o m e t e r a t t h e end o f an e x p e r i m e n t when t h e e n v i r o n -m e n t a l w a t e r i n t h e r e s p i r o m e t e r was o f low o x y g e n c o n t e n t , upon t h e h e a r t r a t e and b l o o d p r e s s u r e s o f t h e r a i n b o w t r o u t . - 32 -as the r i s e i n v e n t r a l a o r t i c s y s t o l i c b l o o d p r e s s u r e . S i x of the f i s h examined had p o s t - h y p o x i c r e c o v e r y s y s t o l i c b l o o d p r e s s u r e s over 110 mm Hg i n t h e i r v e n t r a l a o r t a e . In one c a s e , a 308 gram female developed a s y s t o l i c b l o o d p r e s s u r e of over 128 mm Hg i n her v e n t r a l a o r t a . S i n c e the d o r s a l a o r t i c p r e s s u r e s d i d not i n c r e a s e as much as the v e n t r a l a o r t i c p r e s s u r e s d u r i n g t h i s p e r i o d vas-c u l a r changes, e i t h e r i n c r e a s e d r e s i s t a n c e of the g i l l s or r e -duced p e r i p h e r a l r e s i s t a n c e i s i m p l i c a t e d . I t was not p o s s i b l e to compare the r a t e of decrease of d i f f e r e n t i a l p r e s s u r e as done i n the p r e v i o u s s t u d i e s because the post h y p o x i c d i f f e r -e n t i a l p r e s s u r e s were too h i g h to compare on an e q u i t a b l e b a s i s w i t h h y p o x i c d i f f e r e n t i a l p r e s s u r e s . V e n t r a l a o r t i c and d o r s a l a o r t i c p r e s s u r e s d e c l i n e d slow-l y a f t e r t h e i r i n i t i a l a b rupt r i s e and r e t u r n e d to normal or o r i g i n a l r e s t i n g l e v e l s i n about 1 to 1 j hours. Some id e a of the e f f e c t s of v a s c u l a r changes on p r e s s u r e s was o b t a i n e d by i n j e c t i n g 0.25 mg of e p i n e p h r i n e i n t o a f i s h w e i g h i n g 497 grams. Blood p r e s s u r e i n c r e a s e d but the h e a r t r a t e remained almost c o n s t a n t . The r a t i o of d o r s a l a o r t i c : v e n t r a l a o r t i c s y s t o l i c p r e s s u r e s i n c r e a s e d to 0.82 and the r a t i o of p u l s e p r e s s u r e s i n c r e a s e d to 0.56 (compare w i t h F i g . 8 ) . In t h i s case e i t h e r the g i l l v e s s e l s decreased t h e i r r e s i s t a n c e or p e r i p h e r a l r e s i s t a n c e i n c r e a s e d , or a combina-t i o n of both f a c t o r s o c c u r r e d . The i n t r o d u c t i o n of f r e s h water r e s u l t e d i n a g r a d u a l (30 to 120 minute) d e c l i n e i n b r e a t h i n g r a t e and a m p l i t u d e , back to normal r e s t i n g l e v e l s . - 33 -RESULTS AND DISCUSSION I I S i n c e a s a m p l e f r o m o n l y one o f t h e f o u r c a n n u l a e c o u l d be a n a l y s e d a t one t i m e i t was n e c e s s a r y t o compare a l l o t h e r d e t e r m i n a t i o n s o f b l o o d and w a t e r Pp^, PC02 > and pH t o i n t e r -p o l a t i o n s b e t ween t h e s e r i a l d e t e r m i n a t i o n s o f e n v i r o n m e n t a l o x y g e n . The i n t e r p o l a t i o n s between t h e e n v i r o n m e n t a l o x y g e n l e v e l s w i t h r e s p e c t t o t i m e d u r i n g t h e e x p e r i m e n t s a r e r e a s o n -a b l y r e l i a b l e as t h e l a r g e volume o f w a t e r i n t h e r e s p i r o m e t e r ( a p p r o x i m a t e l y 12 l i t e r s ) was r e s i s t a n t t o s u d d e n c h a n g e s i n o x y g e n l e v e l . The e f f e c t o f i n c r e a s i n g h y p o x i a i n t h e medium was t o p r o d u c e a r a p i d d e c r e a s e i n t h e p e r c e n t a g e o f t h e o x y g e n i n i n s p i r e d w a t e r t h a t was u t i l i z e d by t h e f i s h ( F i g . 1 3 ) . T h i s e f f e c t was v e r y p r o n o u n c e d b e t w e e n e n v i r o n m e n t a l P02 l e v e l s o f 160 mm t o 140 mm Hg. Below e n v i r o n m e n t a l P02 o f 140 mm Hg t h e r e was no s i g n i f i c a n t c hange i n t h e p e r c e n t a g e u t i l i -z a t i o n o f o x y g e n f r o m i n s p i r e d w a t e r . B o t h Van Dam ( 1 9 3 8 ) and S a u n d e r s ( 1 9 6 2 ) d e s c r i b e a d e c r e a s e i n p e r c e n t a g e u t i l i -z a t i o n o f o x y g e n i n i n s p i r e d w a t e r w i t h f i s h u n d e r h y p o x i c c o n d i t i o n s . The d o r s a l a o r t i c b l o o d o f q u i e t f i s h was f o u n d t o have an a v e r a g e P02 o f 122 mm Hg a t an e n v i r o n m e n t a l P02 l e v e l o f 150 mm Hg. The d o r s a l a o r t i c b l o o d P02 d e c r e a s e d t o 19 mm Hg a t an h y p o x i c e n v i r o n m e n t a l o x y g e n l e v e l o f 30 mm Hg P02 ( F i g . 1 4 ) . I t i s i n t e r e s t i n g t o n o t e t h a t t h e d o r s a l a o r t i c b l o o d P02 a t t h e h i g h e s t l e v e l s o f e n v i r o n m e n t a l o x y g e n i s s i g n i f i c a n t l y h i g h e r t h a n t h e P02 o f e x p i r e d w a t e r a t t h e same e n v i r o n m e n t a l o x y g e n l e v e l . T h i s v e r i f i e s a g a i n t h e - 34 -L e g e n d f o r F i g u r e 13 The e f f e c t o f h y p o x i a upon o x y g e n t e n s i o n o f o p e r c u l w a t e r . V e r t i c a l b a r s r e p r e s e n t p l u s o r minus 2 s t a n d a r d e r r o r s o f t h e a v e r a g e o f e a c h o f t h e s u b s a m p l e s p l o t t e d . - 35 -L e g e n d f o r F i g u r e 14 The e f f e c t o f h y p o x i a on t h e o x y g e n t e n s i o n o f v e n t r a l a o r t i c and d o r s a l a o r t i c b l o o d . V e r t i c a l b a r s r e p r e s e n t p l u s o r minus 2 s t a n d a r d e r r o r s o f t h e s u b s a m p l e a v e r a g e s p l o t t e d . - 36 -e x i s t e n c e o f a f u n c t i o n a l c o u n t e r c u r r e n t f l o w a r r a n g e m e n t o f b l o o d and w a t e r p a s t t h e r e s p i r a t o r y e x c h a n g e e p i t h e l i u m o f t h e g i l l s p r e v i o u s l y d ocumented by K r o g h ( 1941) . The Po^ o f v e n o u s b l o o d o f r a i n b o w t r o u t i s s u r p r i s i n g l y h i g h e r t h a n v a l u e s e s t i m a t e d by M o t t ( 1957) , and S a u n d e r s ( 1 9 6 2 ) , o r t h e l e v e l s r e p o r t e d by I t a z a w a ( 1 9 5 7 ) f o r c a r p . The r e s t i n g r a i n b o w t r o u t were f o u n d t o have an a v e r a g e v e n -t r a l a o r t i c P02 o f 30 - 35 mm Hg a t e n v i r o n m e n t a l o x y g e n l e v e o f 140 t o 110 mm Hg P02. V e n t r a l a o r t i c P02 l e v e l s d r o p p e d g r a d u a l l y t o an a v e r a g e 6 mm hg a t e n v i r o n m e n t a l o x y g e n t e n -s i o n s o f 30 mm Hg P02 ( F i g . 1 4 ) . The p a r t i a l p r e s s u r e s o f CO2 i n t h e b l o o d o f q u i e s c e n t r a i n b o w t r o u t r e s t i n g i n w e l l o x y g e n a t e d w a t e r were f o u n d t o be v e r y l o w . The CO2 e l e c t r o d e e m p l o y e d was c a l i b r a t e d t o measure a r a n g e o f CO2 p a r t i a l p r e s s u r e s f r o m 3 mm t o 30 mm Hg. In a l l c a s e s t h e d o r s a l and v e n t r a l a o r t i c b l o o d , and t h e b u c c a l and o p e r c u l a r w a t e r PC02 l e v e l s were l e s s t h a n 3 mm Hg PC02. I t was p o s s i b l e t o g a i n an e s t i m a t e o f some o f t h e PC02 l e v e l s i n t h e s a m p l e s by e m p i r i c a l means f r o m o f f -s e t t i n g t h e m e a s u r i n g r a n g e o f t h e i n s t r u m e n t . S i n c e t h e i n s t r u m e n t s c a l e was l o g a r i t h m i c , t h e e s t i m a t e s o f PC02 a r e n o t as a c c u r a t e as t h e y w o u l d be i f t h e y were t a k e n i n t h e m i d d l e o f t h e i n s t r u m e n t r a n g e . The v e n t r a l a o r t i c PC02 l e v e l s were t h e c l o s e s t t o r e g i s t e r i n g on t h e c a l i b r a t e d r a n g e o f t h e i n s t r u m e n t and were e s t mated t o be 2.5 mm Hg PC02, w i t h r e s t i n g f i s h i n a w e l l o x y g e n a t e d e n v i r o n m e n t . W i t h t h e o n s e t o f h y p o x i c c o n d i t i o n s t h e v e n t r a l a o r t i c PC02 l e v e l s r o s e t o l e v e l s o f 4.5 t o 5 mm Hg. - 37 -The PCo^ o f d o r s a l a o r t i c b l o o d o f t h e q u i e t t r o u t i n w e l l o x y g e n a t e d w a t e r was l o w e r t h a n t h e PCo^ o f t h e v e n t r a l a o r t i c b l o o d . I t was e s t i m a t e d t o be 1 - 1.5 mm Hg. D u r i n g a d e c l i n e i n e n v i r o n m e n t a l o x y g e n t e n s i o n s f r o m 80 mm Hg t o 30 mm Hg t h e d o r s a l a o r t i c b l o o d P C ^ i n c r e a s e d t o 3.5 t o 4 mm Hg. H a n i n g and Thompson ( 1 9 6 5 ) have s t r e s s e d t h a t t h e b l o o d P C 0 2 l e v e l s i n f i s h a r e much l o w e r t h a n i n l a n d v e r t e b r a t e s o r a m p h i b i a n s . They r e p o r t e d a v a l u e o f 9.2 mm Hg P C 0 2 f o r a r t e r -i a l b l o o d o f t h e c a t f i s h , I c t a l u r u s p u n c t a t u s . I r v i n g , B l a c k , and S a f f o r d (1941 ) e s t i m a t e d a r t e r i a l b l o o d P C 0 2 l e v e l s i n t r o u t t o be n e a r 2 mm Hg. B l a c k , K i r k p a t r i c k , and T u c k e r ( 1 9 6 6 ) r e p o r t e d v e n o u s b l o o d s a m p l e s f r o m B r o o k t r o u t had P C 0 2 l e v e l s o f 5.6 t o 6.5 mm Hg. F e r g u s o n and B l a c k ( 1 9 4 1 ) l i s t e d v e n o u s P C 0 2 l e v e l s i n r a i n b o w t r o u t o f 8 t o 10 mm Hg b u t t h e s a m p l e s were o f v e r y low o x y g e n c o n t e n t , a d i f f e r e n t c o n d i t i o n t h a n o b s e r v e d i n t h e p r e s e n t s t u d y . The p r e s e n t f i n d i n g s a r e c l o s e t o t h e v a l u e s r e p o r t e d by t h e p r e v i o u s w o r k e r s and a f f i r m t h a t t h e P C 0 2 l e v e l s i n t r o u t b l o o d a r e c o n s i d e r a b l y l o w e r t h a n t h e P C 0 2 l e v e l s o f t h e b l o o d o f t e r r e s t r i a l v e r t e b r a t e s . T h i s d i f f e r e n c e i s p r o b a b l y due t o t h e l o w CO2 c o n t e n t and h i g h c a p a c i t y o f t h e e n v i r o n m e n t a l w a t e r w h i c h most f i s h b r e a t h . The P C 0 2 l e v e l s o f b u c c a l and o p e r c u l a r w a t e r were c o n -s i d e r a b l y l o w e r t h a n t h o s e o f t h e b l o o d . The w a t e r P C 0 2 l e v e l s were n o t v e r y a c c u r a t e l y d e t e r m i n e d b u t were e s t i m a t e d t o be l e s s t h a n 2 mm Hg P C ^ . The pH o f v e n t r a l and d o r s a l a o r t i c b l o o d was f o u n d t o be v e r y c l o s e t o 7.7 i n t h e q u i e t t r o u t i n w e l l o x y g e n a t e d e n v i r o n m e n t a l c o n d i t i o n s . The pH o f t h e b l o o d o f t h e t r o u t - 38 -c h a n g e d v e r y l i t t l e w i t h t h e o n s e t o f h y p o x i c c o n d i t i o n s and o n l y u n d e r t h e mast e x t r e m e h y p o x i a (30 mm Hg e n v i r o n m e n t a l P 0 2 ) d i d t h e b l o o d pH d r o p t o a l e v e l o f 7,4 ( F i g s . 15 and 16) . The pH o f t h e w a t e r s a m p l e d f r o m t h e b u c c a l and o p e r c u l a r c h a m b e r s d r o p p e d d u r i n g t h e c o u r s e o f e a c h e x p e r i m e n t . T h i s was p r o b a b l y due t o t h e f o r m a t i o n o f c a r b o n i c a c i d f r o m CO^ e x p i r e d by t h e f i s h . T h i s h y p o t h e s i s i s f u r t h e r s t r e n g t h e n e d by t h e o b s e r v a t i o n t h a t t h e pH o f w a t e r s a m p l e d f r o m t h e o p e r -c u l a r chamber o f t h e f i s h was a b o u t . 2 pH u n i t s l o w e r t h a n t h a t o f w a t e r s a m p l e s t a k e n f r o m t h e b u c c a l chamber a t s i m i -l a r e n v i r o n m e n t a l o x y g e n t e n s i o n s . T h i s d i f f e r e n c e a g a i n was p r e s u m a b l y a c o n s e q u e n c e o f e x p i r e d CO2 f r o m t h e g i l l s f o r m i n g c a r b o n i c a c i d ( F i g . 1 7 ) . I t was o b s e r v e d t h a t i n most c a s e s t h e h e m a t o c r i t o f t h e b l o o d o f t h e f i s h i n c r e a s e d w i t h t h e o n s e t o f h y p o x i a . T h i s i n c r e a s e was most p r o n o u n c e d i n f i s h w i t h i n i t i a l l y h i g h h e m a t o c r i t v a l u e s ; f i s h w i t h low h e m a t o c r i t s g e n e r a l l y d i d n o t show s u c h a r e s p o n s e . The i n c r e a s e i n h e m a t o c r i t c o u l d have been a r e s u l t o f c e l l u l a r s w e l l i n g o r an i n c r e a s e i n c e l l numbers i n t h e b l o o d . The r e d b l o o d c e l l c o u n t o f t h e b l o o d o f f i v e f i s h was t a k e n s e r i a l l y w i t h t h e o n s e t o f h y p o x i a . In e a c h o f t h e s e e x p e r i m e n t s t h e r e was a marked i n c r e a s e i n hema-t o c r i t w i t h deep h y p o x i a (15% to E>0%> more t h a n t h e i n i t i a l v a l u e s ) , b u t t h e r e was no s i g n i f i c a n t i n c r e a s e o r d e c r e a s e i n t h e number o f r e d b l o o d c e l l s i n t h e b l o o d o f t h e s e f i s h . From t h i s i t w o u l d a p p e a r t h a t t h e o b s e r v e d i n c r e a s e i n hema-t o c r i t c o n c o m i t a n t w i t h d e e p e n i n g h y p o x i a i s p r i m a r i l y a E N V I R O N M E N T A L R~ M M HG - 39 -L e g e n d f o r F i g u r e 15 The e f f e c t o f h y p o x i a upon t h e pH o f v e n t r a l a o r t i c b l o o d . S p l i t c i r c l e s r e p r e s e n t 2 v a l u e s a t t h a t p o i n t . - 40 -L e g e n d f o r F i g u r e 16 The e f f e c t o f h y p o x i a upon t h e pH o f d o r s a l a o r t i c b l o o d . S p l i t c i r c l e s r e p r e s e n t 2 v a l u e s a t t h a t p o i n t . - 41 -L e g e n d F o r F i g u r e 17 The pH o f e n v i r o n m e n t a l and o p e r c u l a r w a t e r d u r i n g t h e c o u r s e o f p r o g r e s s i v e l y s e v e r e h y p o x i a . S p l i t c i r c l r e p r e s e n t two v a l u e s a t t h a t p o i n t . - 42 -r e s u l t o f c e l l u l a r s w e l l i n g . F e r g u s o n and B l a c k ( 1 9 4 1 ) have shown in_ v i t r o t h a t t h e r e d b l o o d c e l l s o f r a i n b o w t r o u t s w e l l c o n s i d e r a b l y w i t h r i s e s i n CO- i n t h e b l o o d . T h e r e was a s u g g e s t i o n o f a l o o s e i n v e r s e c o r r e l a t i o n b e t w e e n t h e h e m a t o c r i t o f t h e f i s h and t h e o x y g e n t e n s i o n a t w h i c h t h e f i s h began t o show s i g n s o f r e s p i r a t o r y f a i l u r e . F i g u r e 18 shows t h e r e l a t i o n s h i p b e t w e e n t h e i n i t i a l hemato-c r i t o f t h e f i s h a n d t h e o x y g e n t e n s i o n a t w h i c h t h e v a r i o u s e x p e r i m e n t s were t e r m i n a t e d . I t a p p e a r s t h a t t h e i n i t i a l hema-t o c r i t and c o r r e s p o n d i n g l y t h e c a p a c i t y o f t h e b l o o d f o r o x y g e n i s o f some i m p o r t a n c e t o t h e f i s h f o r s u r v i v a l o r a t l e a s t r e s i s t a n c e t o h y p o x i c c o n d i t i o n s i n t h e e n v i r o n m e n t . 3 0 2 5 2 0 H E M A T O C R I T 1 5 I O 5 E N V I R O N M E N T A L P Q 2 M M H G - 43 -L e g e n d f o r F i g u r e 18 The h e m a t o c r i t o f t h e f i s h and t h e e n v i r o n m e n t a l o x y g e n t e n s i o n a t w h i c h t h e e x p e r i m e n t w i t h h y p o x i a was t e r m i n a t e d due t o s i g n s o f r e s p i r a t o r y o r c a r d i a c f a i l u r e . S p l i t p o i n t s i n d i c a t e two v a l u e s f o r t h a t p o i n t . 44 RE5LJLT5 AND DISCUSSION I I I The r a t e o f o x y g e n u p t a k e o f t h e f i s h was r e c o r d e d i n a l l e x p e r i m e n t s ( F i g . 1 9 ) . The c h a n g e s i n o x y g e n u p t a k e w i t h h y p o -x i a ( F i g . 19) i n t h e medium a r e n o t s t a t i s t i c a l l y s i g n i f i c a n t and a r e p r o b a b l y r e l a t e d t o s p o n t a n e o u s a c t i v i t y o f t h e f i s h . No e f f o r t was made t o e i t h e r r e s t r a i n t h e u n a n a e s t h e t i z e d f i s h d u r i n g t h e e x p e r i m e n t s o r t o a p p l y any c o r r e c t i o n f o r a c t i v i t y , i n o r d e r t o s t a n d a r d i z e t h e o x y g e n u p t a k e r e s u l t s . However, % t h e a v e r a g e r a t e o f o x y g e n c o n s u m p t i o n (100 - 125 mg/Kg/hr) was o n l y s l i g h t l y h i g h e r t h a n t h e s t a n d a r d l e v e l s o f o x y g e n consump-t i o n f o r b r o o k t r o u t o f s i m i l a r s i z e and a t t h e same t e m p e r a t u r e r e p o r t e d by B e a m i s h (1963 - C ) . T h i s w o u l d s u p p o r t t h e e v i d e n c e o b t a i n e d f r o m t h e s e n s i t i v e b u c c a l and o p e r c u l a r p r e s s u r e r e c o r -d i n g s t h a t t h e a c t i v i t y l e v e l o f f i s h i n t h e r e s p i r o m e t e r was g e n e r a l l y q u i t e l o w . The o x y g e n u p t a k e i s a measure o f t h e e x c h a n g e b e t w e e n t h e w a t e r and t h e f i s h , n o t t h e b l o o d and c e l l s . Oxygen d i s s o l v e d i n t h e b l o o d and m u s c l e a t t h e s t a r t o f t h e e x p e r i m e n t i s n o t t a k e n i n t o c o n s i d e r a t i o n and does n o t r e p r e s e n t a s o u r c e o f e r r o r I t has been assumed t h a t o x y g e n u p t a k e i s t h e q u a n t i t y o f gas p a s s i n g t h r o u g h t h e r e s p i r a t o r y e p i t h e l i u m . O b v i o u s l y t h e m a j o r s o u r c e o f e r r o r i n t h i s a s s u m p t i o n i s gas e x c h a n g e w h i c h does n o t o c c u r t h r o u g h t h e r e s p i r a t o r y e p i t h e l i u m . F r y ( 1 957) s u g g e s t s t h a t a l t h o u g h t h e body s u r f a c e s o f a l l b u t t h e most h e a v i l y a r m o u r e d f i s h a r e p e r m e a b l e t o r e s p i r a t o r y g a s e s , c u t a n e o u s r e s p i r a t i o n i s n o r m a l l y o f no a p p r e c i a b l e s i g n i f i -c a n c e i n a q u a t i c r e s p i r a t i o n o f most f i s h e s . B e r g and S t e e n 140 120 IOO OXYGEN 8 0 CONSUMPTION 6 0 M G / K G / H R 4 0 2 0 h 4 0 8 0 I20 ENVIRONMENTAL P Q MM HG 160 - 45 -L e g e n d f o r F i g u r e 19 The e f f e c t o f h y p o x i a upon t h e r a t e o f o x y g e n u p t a k e . The c i r c l e s r e p r e s e n t t h e a v e r a g e s o f sub-samples f r o m e a c h 10 mm Hg o x y g e n t e n s i o n i n t e r v a l . - 46 -( 1 9 6 5 ) s t a t e t h a t t h e e e l , w h i c h has a c o n s i d e r a b l e c u t a n e o u s r e s -p i r a t o r y c a p a c i t y , r e c e i v e s a b o u t 85 t o 90 p e r c e n t o f i t s o x y g e n u p t a k e i n w a t e r t h r o u g h i t s g i l l s , T hese f i g u r e s w o u l d i n d i c a t e t h a t w i t h some f i s h , s u c h as t h e e e l , t h e r e w o u l d be an a p p r e c i a b l e e r r o r i n a s s u m i n g t h a t o x y g e n u p t a k e was e x c l u s i v e l y v i a t h e g i l l s . However i t i s f e l t t h a t t h i s e r r o r w o u l d be q u i t e s m a l l i n r a i n -bow t r o u t , w h i c h , a s i d e f r o m t h e l a c k o f e x c e p t i o n a l c u t a n e o u s v a s c u l a r i z a t i o n , have a r a t h e r t h i c k l a y e r o f s l i m e o v e r t h e i r body s u r f a c e (Van Dam, 1 9 3 8 ) . To o b t a i n i n d i c a t i o n s o f t h e d e g r e e o f a n a e r o b i c m e t a b o l i s m t h a t t h e f i s h were u s i n g d u r i n g t h e e x p e r i m e n t s , b l o o d s a m p l e s were t a k e n b e f o r e a n d a f t e r t h e e x p e r i m e n t s on f o u r f i s h w h i c h w e i g h e d b e t w e e n 520 and 1015 grams. The b l o o d s a m p l e o f 0.7 ml i f t a k e n f r o m a f i s h w e i g h i n g 500 grams w o u l d r e p r e s e n t a p p r o -x i m a t e l y 5% o f t h e b l o o d volume o f t h e f i s h a s s u m i n g a b l o o d v o l u m e o f 2.8/6 o f t h e t o t a l w e i g h t o f t h e f i s h ( P r o s s e r and Brown 1 9 6 1 ) . The a v e r a g e l e v e l o f l a c t a t e i n t h e f i s h b e f o r e t h e e x p e r i -ments i s q u i t e c l o s e t o t h e v a l u e s o f b l o o d l a c t a t e l e v e l i n r a i n b o w t r o u t r e p o r t e d by B l a c k ( 1 9 5 5 ) , and B l a c k et. a_l ( 1 9 6 2 ) . I n t h e p r e s e n t s t u d y i t was f o u n d t h a t l a c t a t e l e v e l s i n t h e b l o o d i n c r e a s e d by a f a c t o r o f 2 t o 3, a f t e r t h e f i s h had been s u b j e c t e d t o a p e r i o d o f h y p o x i a i n t h e medium ( T a b l e I ) . The i n c r e a s e i s p r e s u m a b l y due t o d i f f u s i o n o f l a c t a t e f r o m m u s c l e where t h e r e c a n be c o n s i d e r a b l e a n a e r o b i c g l y c o l y s i s o f c a r b o -h y d r a t e ( B l a c k 1 9 5 5 ) . The s o u r c e o f t h e l a c t a t e c o u l d be b r a n c h i a l m u s c l e s w h i c h o b v i o u s l y i n c r e a s e t h e i r o u t p u t , o r p o s s i b l y i t c o u l d come f r o m a g e n e r a l i n c r e a s e i n m u s c u l a r a c t i v i t y . TABLE I BLOOD LACTATE CHANGES EXPT. LACTATE BEFORE LACTATE AFTER % CHANGE #1 22.52 mq% 50.66 mg % 225 #2 10.60 30.46 288 #3 9,27 36.75 396 #4 8.69 21,56 248 Av g . 12.77 34,86 273 - 47 L e g e n d f o r T a b l e I L e v e l o f l a c t i c a c i d i n t h e b l o o d (mg/100 ml o f b l o o d ) o f r a i n b o w t r o u t b e f o r e and a f t e r s u b j e c t i o n t o h y p o x i a . - 48 -Changes i n v e n t i l a t i o n volume d u r i n g h y p o x i a were d e t e r -m i n ed by a p p l i c a t i o n o f t h e F i c k p r i n c i p l e , u s i n g t h e f o l l o w i n g e q u a t i o n : R 0„ V e n t i l a t i o n v o l u m e : V w - C x 0 o . - C xO-, , w 2 i n w 2 o u t Where R O2 ~ ®2 u P ^ a ' < e m l / m i n u t e / K g . C 0„ . = Oxyqen c o n t e n t o f w a t e r a f f e r e n t t o t h e w 2 i n ' y g i l l s . C 0 , = Oxyqen c o n t e n t o f w a t e r e f f e r e n t t o t h e w 2 o u t 7 y g i l l s . Oxygen c o n t e n t i n t h e w a t e r was d e t e r m i n e d by m u l t i p l y i n g t h e r e c o r d e d p a r t i a l p r e s s u r e o f o x y g e n i n t h e w a t e r by t h e s o l u b i l i t y c o e f f i c i e n t f o r o x y g e n i n t h e w a t e r a t t h e p r e v a i l i n g e x p e r i m e n t a l t e m p e r a t u r e . The l a r g e i n c r e a s e i n v e n t i l a t i o n volume w h i c h was i n d i -c a t e d f r o m p r e s s u r e and r a t e s t u d i e s i s shown a l s o by t h e F i c k p r i n c i p l e e s t i m a t e s ( F i g . 20) w h i c h showed an i n c r e a s e f r o m 274 m l /min/Kg a t an e n v i r o n m e n t a l l e v e l o f 150 mm Hg P0^ t o a maximum o f 3560 m l / m i n / K g a t 40 mm Hg e n v i r o n m e n t a l PO2. The d r o p i n v e n t i l a t i o n v o lume w i t h e n v i r o n m e n t a l l e v e l s o f o x y g e n b e l o w 40 mm Hg was due p r i m a r i l y t o r e s p i r a t o r y f a i l u r e . From F i g u r e 20 i t i s o b v i o u s t h a t t h e r a i n b o w t r o u t has a w i d e s c o p e f o r i n c r e a s i n g i t s v e n t i l a t i o n v o l u m e ; i n t h i s s t u d y t h e i n c r e a s e i n a v e r a g e v e n t i l a t i o n v o lume was 13 f o l d . The v e n t i l a t i o n v o lume o f t h e q u i e s c e n t d o g f i s h ( M u s t e l u s c a l i f o r n i c u s ) was r e p o r t e d by Ogden ( 1 9 4 5 ) t o be 500 m l /min/Kg w h i c h i s q u i t e s i m i l a r t o t h e t r o u t i n a w e l l o x y g e n a t e d en-v i r o n m e n t i n t h i s s t u d y . Van Dam ( 1 9 3 8 ) r e p o r t e d a f i v e f o l d ENVIRONMENTAL P n M M HG ° 2 _ 49 -L e g e n d F o r F i g u r e 20 The e f f e c t o f h y p o x i a upon v e n t i l a t i o n v o l u m e . Each p o i n t i s t h e a v e r a g e o f s e v e r a l v a l u e s w i t h i n p l u s o r minus 5 mm Hg e n v i r o n m e n t a l o x y g e n t e n s i o n o f t h a t p o i n t . 50 -i n c r e a s e i n v e n t i l a t i o n volume i n r e s p o n s e t o l o w e r e d e n v i r o n -m e n t a l o x y g e n i n r a i n b o w t r o u t w h i c h i s l e s s t h a n h a l f t h e i n -c r e a s e o b s e r v e d i n t h e p r e s e n t s t u d y . The d i f f e r e n c e may, i n p a r t , be due t o i n t e r f e r e n c e by Van Dam's a p p a r a t u s w i t h t h e n o r m a l b r e a t h i n g a c t i o n o f t h e f i s h . S a u n d e r s (1961 ) r e p o r t e d i n c r e a s e s as h i g h as 82 f o l d f o r t h e s u c k e r , 70 f o l d f o r t h e b u l l h e a d , and 31 f o l d f o r t h e c a r p . These v a l u e s a r e c e r t a i n l y f a r h i g h e r t h a n t h e r e l a t i v e i n c r e a s e f o u n d i n t h i s s t u d y f o r r a i n b o w t r o u t , b u t r e p r e s e n t e x t r e m e v a l u e s , w h e r e a s t h e v a l u e s f o r i n c r e a s e i n r e s p i r a t o r y volume i n t h e p r e s e n t s t u d y a r e a v e r a g e d . An e s t i m a t e o f c a r d i a c o u t p u t c a n be o b t a i n e d i n t h e same manner as t h e v e n t i l a t i o n v o l u m e : h o w e v e r , a p p l i c a t i o n o f t h e F i c k p r i n c i p l e t o b l o o d f l o w i s c o n s i d e r a b l y more d i f f i c u l t o w i n g t o t h e s o l u b i l i t y c h a r a c t e r i s t i c s o f b l o o d . The c a p a c i t y o f b l o o d v a r i e s i n a n o n - l i n e a r manner w i t h c h a n g e s i n o x y g e n and c a r b o n d i o x i d e t e n s i o n s . The d i s s o c i a t i o n c u r v e s r e q u i r e d t o c o n v e r t t h e r e c o r d e d b l o o d P0^ l e v e l s t o o x y g e n c o n t e n t were c o n s t r u c t e d i n t h i s l a b o r a t o r y ( R a n d a l l , Beaumont, and H o l e t o n ; u n p u b l i s h e d ) . The c u r v e s o b t a i n e d a r e s i m i l a r t o t h o s e r e p o r t e d by I r v i n g , B l a c k , and S a f f o r d (1941 ) and B l a c k , K i r k p a t r i c k , and T u c k e r ( 1 9 6 6 ) f o r b r o o k t r o u t , b u t a r e a l l a t 14-15°C and c o v e r P C 0 2 r a n g e s b e t w e e n 0-1 mm and 10 mm Hg 0 The c a p a c i t y o f t h e b l o o d f o r o x y g e n a l s o v a r i e s w i t h t h e h e m a t o c r i t . I n t h e s e e x p e r i m e n t s t h e h e m a t o c r i t v a r i e d i n t h e r a i n b o w t r o u t f r o m 9 t o 28.5 p e r c e n t . M e a s u r e m e n t s were made o f t h e o x y g e n c a p a c i t y o f t h e b l o o d o f 35 f i s h o f d i f f e r i n g h e m a t o c r i t s ( r a n g e - 51 -0 t o 32^) and f r o m t h i s d a t a i t was p o s s i b l e t o o b t a i n a l i n e a r r e g r e s s i o n e q u a t i o n ( c a p a c i t y = .311 h e m a t o c r i t + 0.7) ( F i g . 21) r e l a t i n g t h e h e m a t o c r i t t o t h e c a p a c i t y o f t h e b l o o d i n v o l u m e s p e r c e n t o f o x y g e n . The p e r c e n t s a t u r a t i o n o f t h e b l o o d a f f e r e n t and e f f e r e n t t o t h e g i l l s o f t h e f i s h was d e t e r m i n e d f r o m t h e r e c o r d e d b l o o d PQ^ and PC02 l e v e l s and t h e o x y g e n d i s s o c i a t i o n c u r v e s . The p e r c e n t s a t u r a t i o n was t h e n c o n v e r t e d i n t o o x y g e n c o n t e n t u s i n g t h e c a l c u l a t e d o x y g e n c a p a c i t y . Thus t h e o x y g e n c o n t e n t o f t h e b l o o d a f f e r e n t and e f f e r e n t t o t h e g i l l s was e s t i m a t e d . The volume o f o x y g e n e n t e r i n g t h e b l o o d was known, and t h e r e f o r e by a p p l i c a t i o n o f t h e F i c k p r i n c i p l e t h e c a r d i a c o u t p u t was c a l c u l a t e d . No s i g n i f i c a n t c h a n g e s i n c a r d i a c o u t p u t were o b s e r v e d when t h e f i s h was s u b j e c t e d t o an h y p o x i c e n v i r o n -ment ( F i g . 2 2 ) . S t r o k e v o l u m e , e s t i m a t e d by d i v i d i n g m i n u t e c a r d i a c o u t -p u t by m i n u t e h e a r t r a t e ? i n c r e a s e d m a r k e d l y w i t h h y p o x i a i n t h e medium ( F i g . 2 3 ) . T h i s w o u l d be e x p e c t e d i f c a r d i a c o u t p u t r e m a i n s c o n s t a n t and h e a r t r a t e f a l l s . The s t r o k e v olume a t e n v i r o n m e n t a l o x y g e n l e v e l s o f 40 mm Hg P0^ i s a b o u t t w i c e t h a t c a l c u l a t e d f o r f i s h i n w a t e r o f o x y g e n l e v e l s n e a r a i r s a t u r a -t i o n . A t o x y g e n p a r t i a l p r e s s u r e s b e l o w 40 mm Hg and a t l o w e r h e a r t r a t e s , h o w e v e r , t h e r e a r e a p p a r e n t l y much l a r g e r c h a n g e s i n s t r o k e v o l u m e . O n l y a s i n g l e d e t e r m i n a t i o n o f s t r o k e v olume a t 30 mm Hg P0^ i s a v a i l a b l e , b u t t h i s v a l u e o f 5.2 ml f o r a one Kg f i s h i s some f i v e t i m e s g r e a t e r t h a n t h e a v e r a g e s t r o k e v o l u m e o f 1 ml/Kg f o r f i s h i n a e r a t e d w a t e r . A n o t h e r e s t i m a -t i o n o f s t r o k e v o l u m e was made by u t i l i z i n g a v e r a g e d d a t a f r o m F i g u r e 14 on b l o o d 0 ? l e v e l s and F i g u r e 19 on o x y g e n u p t a k e HEMATOCRIT L e g e n d f o r F i g u r e 21 The r e l a t i o n s h i p b e t ween h e m a t o c r i t and b l o o d o x y g e n c a p a c i t y i n v o l u m e s p e r c e n t . 140 IOO CARDIAC OUTPUT M L / M I N 6 o 2 0 4 0 8 0 I20 160 ENVIRONMENTAL P Q ^ MM HG - 53 L e g e n d f o r F i g u r e 22 The e f f e c t o f h y p o x i a upon c a r d i a c o u t p u t c a l c u l a t e d by t h e F i c k p r i n c i p l e . V e r t i c a l b a r s a r e p l u s o r minus two s t a n d a r d e r r o r s o f t h e a v e r a g e v a l u e s p l o t t e d . STROKE VOLUME ML /STROKE - 54 -L e g e n d f o r F i g u r e 23 The e f f e c t o f h y p o x i a upon s t r o k e v o l u m e . TABLE I I ANIMAL TEMP. CARDIAC OUTPUT ML.,/KG,/MIN, STROKE VOLUME ML./KG. METHOD USED REFERENCE B o w f i n , C a r p S u c k e r , C a t f i s h 10° .44, .36, .22, .52 L i g a t i o n and s u b s e q u e n t w e i g h i n g o f h e a r t cham-b e r s H a r t 1943 S q u a l u s s p . 11-17° 9.. 0 - 33.0 .25 - .93 L i g a t i o n s , n a t u r a l f l o w f r o m c u t v e n t r a l a o r t a . B u r g e r & B r a d -l e y 1951 • p s a n u s s p . 10.1 F i c k p r i n c i p l e , minimum M o t t 1957 T e t r a o d o n s p . 15.5 t h e o r e t i c a l v a l u e s . S t e n o t o m u s s p . 15.7 Gadus morhua 9.3 .31 c a n d o u b l e E l e c t r o m a g n e t i c f l o w m e t e r J o h a n s e n 1962 Amphiuma S D . 40.0 .96 J o h a n s e n 1963 S c u l p i n 15-18° 21.4 - 34.2 F i c k p r i n c i p l e G o l d s t e i n e t a l 1964 O c t o p u s 7-9° 5,0 - 32.2 .41-2.0 F i c k p r i n c i p l e J o h a n s e n 1964 F r o g 20° 57 1.0 a p p r o x . V e n t r i c u l a r d i s p l a c e -ment S h e l t o n & J o n e s 1964 Human C a t 60 - 100 690 P r o s s e r & Brown 1961 T r o u t 12-18° 65 - 100 .85 - 2.0 F i c k p r i n c i p l e P r e s e n t s t u d y 55 L e g e n d f o r T a b l e I I A summary o f some o f t h e a v a i l a b l e i n f o r m a t i o n on c a r d i a c o u t p u t and s t r o k e v olume o f v a r i o u s v e r t e b r a t e s . - 56 -r a t e s . I n t h i s c a s e t h e s t r o k e volume o f a f i s h , a t p a r t i a l p r e s s u r e s o f o x y g e n o f 30 mm Hg i n t h e w a t e r was 2.47 ml/Kg o f f i s h and t h e c a r d i a c o u t p u t was 69 m l / m i n / K g . The f i n a l e s t i -m a t i o n s o f s t r o k e v o l u m e a t 30 mm Hg P0^ i n t h e medium were de-r i v e d f r o m a number o f s i n g l e d e t e r m i n a t i o n s and n o t f r o m p a i r e d o r m a tched a r t e r i a l - v e n o u s gas t e n s i o n m e a s u r e m e n t s , as were t h e o t h e r c a l c u l a t i o n s o f c a r d i a c o u t p u t and s t r o k e v o l u m e . One m i g h t e x p e c t c o n s i d e r a b l e v a r i a t i o n i n t h e d a t a , e s p e c i a l l y be-twee n t h e two e s t i m a t e s o f s t r o k e volume a t 30 mm Hg PQ^ i n t h e w a t e r . A c o m p a r i s o n b e t w e e n t h e v a l u e s f o r c a r d i a c o u t p u t and s t r o k e v olume o b t a i n e d i n t h e p r e s e n t s t u d y w i t h v a l u e s l i s t e d i n t h e l i t e r a t u r e has been made i n T a b l e I I . The v a l u e s f o r c a r d i a c o u t p u t r e p o r t e d i n t h e l i t e r a t u r e a r e g e n e r a l l y a good d e a l l o w e r t h a n t h o s e o f t h e p r e s e n t s t u d y . The l i t e r a t u r e v a l u e s f o r s t r o k e v olume a r e a l s o l o w e r t h a n t h e e s t i m a t e s o f s t r o k e volume o f t h e p r e s e n t s t u d y . I t i s n o t e w o r t h y t h a t t h e e s t i m a t e s o f H a r t (1943 ), B u r g e r & B r a d l e y (19 51 ) , J o h a n s e n ( 1962 ), and G o l d s t e i n et. a l ( 1 9 6 4 ) a l l were o b t a i n e d f r o m f i s h t h a t were n o t i n t a c t , u n a n a e s t h e t i z e d , and f r e e swimming. I t i s q u i t e l i k e l y t h a t f a i l u r e t o p r o v i d e t h e s e p r e v i o u s l y men-t i o n e d r e q u i r e m e n t s c o u l d have a marked e f f e c t upon b l o o d c i r -c u l a t o r y c h a r a c t e r i s t i c s , i n c l u d i n g c a r d i a c o u t p u t and s t r o k e v o l u m e . The minimum t h e o r e t i c a l c a r d i a c o u t p u t v a l u e s f o r t h r e e m a r i n e f i s h e s o f r a t h e r s l u g g i s h b e h a v i o u r , r e p o r t e d by M o t t ( 1 9 5 7 ) a r e a c t u a l l y q u i t e c l o s e t o what w o u l d be e x p e c -t e d i n r a i n b o w t r o u t i f t h e ve n o u s b l o o d were c o m p l e t e l y de-o x y g e n a t e d . A good d e a l o f c a u t i o n must be e x e r c i s e d h owever - 57 -when c o n s i d e r i n g d i f f e r e n c e s i n p h y s i o l o g i c a l f e a t u r e s o f a n i m a l s o f d i f f e r e n t s p e c i e s , o f d i f f e r i n g s i z e , and w h i c h have been e x a m i n e d u n d e r d i f f e r i n g c o n d i t i o n s . Hughes and S h e l t o n ( 1 9 6 2 ) and Hughes ( 1 9 6 4 ) p r e s e n t e d a c o m p a r i s o n o f gas e x c h a n g e a t t h e g i l l s u r f a c e w i t h known r e l a -t i o n s h i p s d e r i v e d f r o m s t u d i e s upon compact h e a t e x c h a n g e r s ( K a y s & London 1 9 5 8 ) . They show how s e v e r a l h e a t e x c h a n g e r r e l a -t i o n s h i p s a r e p r o b a b l y o f use i n a n a l y s i n g gas e x c h a n g e s y s t e m s . One s u c h r e l a t i o n s h i p i s t h e e f f e c t i v e n e s s o f t r a n s f e r . E f f e c -t i v e n e s s i s t h e r a t i o o f t h e a c t u a l gas t r a n s f e r (RO^) c o m p a r e d w i t h t h e maximum amount o f gas (Rmax) t h a t c o u l d be t r a n s f e r r e d i f t h e e x c h a n g e were c o m p l e t e , a c o n d i t i o n f o r w h i c h Hughes ( 1 9 6 4 ) p r e s e n t e d t h e f o l l o w i n g e x p r e s s i o n s f o r e f f e c t i v e n e s s : RQ 2 E f f e c t i v e n e s s o f t r a n s f e r = —57, x 100 R0^ max Vb S b o 2 ( T b o 2 o u t - T b o 2 i n ) vb S b o 2 ( T w o 2 i~n - T b o 2 i n ) x 100 Amount o f 0 2 t a k e n o u t o f w a t e r ( e n t e r i n g b l o o d ) Amount o f 0 2 t h a t c o u l d p o s s i b l y be t a k e n up by b l o o d R a t e o f 0 2 u p t a k e ml/min/Kg W b S b o 2 ( T w o 2 i n - T b o 2 i n ) Where l/b and Vw a r e t h e v o l u m e s o f t h e f l o w o f b l o o d and w a t e r r e s p e c t i v e l y and Sw and Sb a r e t h e s o l u b i l i t y o r a b s o r p t i o n c o e f f i c i e n t s o f 0 2 i n w a t e r and b l o o d . Sb w i l l v a r y w i t h t h e s l o p e o f t h e d i s s o c i a t i o n c u r v e o f b l o o d . Tw and Tb a r e t h e p a r t i a l p r e s s u r e s o f o x y g e n i n w a t e r and b l o o d r e s p e c t i v e l y . The e f f e c t i v e n e s s o f gas e x c h a n g e a c r o s s t h e g i l l s has been c a l c u l a t e d f o r r a i n b o w t r o u t u s i n g t h e i n f o r m a t i o n o b t a i n e d - 58 -i n t h i s s t u d y . The e f f e c t o f h y p o x i a upon e f f e c t i v e n e s s o f e x c h a n g e i s p r e s e n t e d i n F i g u r e 24 ( o p e n c i r c l e s ) . A l t h o u g h t h e r e i s l i m i t e d a c c u r a c y i n s u c h e s t i m a t e s , t h e r e s u l t s w o u l d i n d i c a t e t h a t u n d e r w e l l o x y g e n a t e d c o n d i t i o n s (90 t o 140 mm Hg P 0 2 ) t h e e f f e c t i v e n e s s o f t h e g i l l s i s v e r y n e a r 100/S. As h y p o x i c c o n d i t i o n s d e v e l o p , t h e e f f e c t i v e n e s s d e c r e a s e s , r e a c h i n g v a l u e s o f 30/£ - 40% a t e n v i r o n m e n t a l o x y g e n t e n s i o n s o f 40 mm Hg P®2' T h i s d e c r e a s e i n e f f e c t i v e n e s s i s a r e s u l t o f t h e RO^ max (maximum p o s s i b l e amount o f 0^ t h e b l o o d c o u l d c a r r y away) i n c r e a s i n g . T h r o u g h o u t t h e e x p e r i m e n t t h e o x y g e n c o n s u m p t i o n has n o t c h a n g e d a p p r e c i a b l y so t h e "RO2 max" has t o i n c r e a s e . From t h i s one c o u l d i n f e r t h a t a t h i g h o x y g e n t e n s i o n s i n t h e e n v i r o n m e n t , t h e a r e a , d i f f u s i o n c h a r a c t e r i s t i c s , and t h e amount o f w a t e r p r e s e n t e d t o t h e g i l l s a l l a r e s u f f i c i e n t t o l o a d a l m o s t c o m p l e t e l y t h e b l o o d p a s s i n g t h r o u g h t h e g i l l s . U n d e r h y p o x i c c o n d i t i o n s t h e r e a p p e a r s t o be a l a r g e i n c r e a s e i n b l o o d c a p a c i t y . S i n c e i t has been d e m o n s t r a t e d t h a t Mb does n o t c h a n g e a p p r e c i a b l y , t h e b l o o d c a p a c i t y i n c r e a s e s must be d e p e n d e n t upon t h e 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 o f h e m o g l o b i n . Thus t h e d e c r e a s e i n e f f e c t i v e n e s s i s r e l a t e d t o t h e n a t u r e o f t h e o x y g e n d i s s o c i a t i o n c u r v e o f t h e b l o o d . The p r e v i o u s d i s c u s s i o n c o n s i d e r e d e f f e c t i v e n e s s f r o m t h e a s p e c t o f b l o o d o x y g e n a t i o n . A n o t h e r c o n s i d e r a t i o n i s t h e e f f e c t i v e n e s s o f o x y g e n r e m o v a l f r o m t h e w a t e r . The e f f e c t i v e -n e s s o f o x y g e n r e m o v a l c a n be o b t a i n e d f r o m t h e f o l l o w i n g e x -p r e s s i o n : R 0 2 E f f e c t i v e n e s s o f r e m o v a l R 0 2 max p r e s 120 8 0 E F F E C T I V E N E S S 4 0 O O O X Y G E N LOADING BY B L O O D • O X Y G E N R E M O V A L F R O M RESPIRATORY O WATER O 4 0 8 0 I20 160 ENVIRONMENTAL P n M M HG u 2 - 59 -L egend f o r F i g u r e 24 Changes i n t h e e f f e c t i v e n e s s o f b l g g d o x y g e n a t i o n and t h e e f f e c t i v e n e s s o f o x y g e n r e m o v a l f r o m w a t e r o c c u r r i n g w i t h i n c r e a s i n g h y p o x i a . ! - 60 -R a t e o f 0- u p t a k e ml/min/Kg \7w SuToT, ( Two 2in - T b o 2 i n) Where RU^ max p r e s i s t h e maximum o r t o t a l u s e a b l e amount o f o x y g e n p r e s e n t e d t o t h e g i l l s u r f a c e . The e f f e c t s o f h y p o x i a upon e f f e c t i v e n e s s o f o x y g e n r e m o v a l f r o m i n s p i r e d w a t e r w i t h t h e r a i n b o w t r o u t have been c a l c u l a t e d u s i n g t h e i n f o r m a t i o n o b t a i n e d i n t h i s s t u d y and a r e i l l u s t r a t e d i n F i g u r e 24. W i t h t h e e x c e p t i o n o f v e r y h i g h (150 mm Hg PO^) and v e r y low (30 mm Hg PO2) e n v i r o n m e n t a l o x y g e n t e n s i o n s where a p a u c i t y o f d a t a r e n d e r s t h e v a l u e s l e s s r e l i a b l e , t h e e f f e c -t i v e n e s s o f o x y g e n r e m o v a l does n o t v a r y g r e a t l y f r o m 30%. The c o n s t a n c y o f t h e e f f e c t i v e n e s s o f t h e r e m o v a l o f o x y g e n f r o m i n s p i r e d w a t e r , c o u p l e d w i t h t h e k n o w l e d g e t h a t R0^ ( o x y -gen u p t a k e ) does n o t c h a n g e a p p r e c i a b l y , i n d i c a t e s t h a t a f a i r l y u n i f o r m q u a n t i t y o f u s e a b l e o x y g e n i s b e i n g p r e s e n t e d t o t h e g i l l s u r f a c e . T h i s i s a c c o m p l i s h e d by t h e i n c r e a s e i n v e n t i l a t i o n volume ( V w ) . The p e r c e n t a g e o f a v a i l a b l e o x y g e n i n i n s p i r e d w a t e r t h a t i s u t i l i z e d by t h e f i s h s e r v e s as an i n d e x o f t h e e f f i c i e n c y o f t h e g i l l s f o r o x y g e n u p t a k e (l/an Dam 1 9 3 8 ) . T h i s s t a t e m e n t i s i n c o m p l e t e w i t h o u t t h e a dded s t i p u l a t i o n t h a t t h e g i l l s must s t i l l be t a k i n g up s u f f i c i e n t o x y g e n t o keep t h e f i s h a l i v e . W i t h t h e r a i n b o w t r o u t i n t h i s s t u d y t h e a v e r a g e u t i l i -z a t i o n o f i n s p i r e d w a t e r d e c r e a s e d f r o m 5 5 % a t an e n v i r o n m e n t a l P 0 2 o f 160 mm Hg t o 2 0 % a t an e n v i r o n m e n t a l P 0 2 o f 100 mm Hg. B e l o w 100 mm Hg e n v i r o n m e n t a l P 0 2 t h e r e was no s i g n i f i c a n t d e p a r t u r e f r o m a u t i l i z a t i o n o f 20%. T h i s e f f e c t c a n be r e a d i l y s e e n by c o m p a r i n g o p e r c u l a r and b u c c a l w a t e r t e n s i o n s i l l u s t r a t e d i n F i g u r e 13. I t was o b s e r v e d t h a t t h e h i g h e s t - 61 -u t i l i z a t i o n s were o b t a i n e d by l a r g e f i s h w h i c h were e x c e p t i o n a l l y q u i e t . A f r i g h t o r b u r s t o f a c t i v i t y r e s u l t e d i n a l a r g e d r o p i n u t i l i z a t i o n and c o r r e s p o n d i n g l y a two t o f o u r f o l d i n c r e a s e i n v e n t i l a t i o n v o l u m e . Van Dam (1938 ) r e p o r t e d a u t i l i z a t i o n a s h i g h as 80/6 i n a r a i n b o w t r o u t r e s t i n g q u i e t l y i n w e l l o x y g e n a t e d w a t e r . A f o u r f o l d i n c r e a s e i n v e n t i l a t i o n volume r e s u l t e d i n a d r o p i n u t i l i z a t i o n f r o m 80/6 t o 60/6. These o b s e r v a t i o n s were b a s e d upon d e t e r m i n a t i o n s a t 10°C upon a s i n g l e 900 gram t r o u t w h i c h was k e p t i n a s m a l l (60 l i t e r s ) h o l d i n g t a n k p r i o r t o t e s t i n g . I n t h e p r e s e n t s t u d y a 945 gram f i s h t e s t e d A u g u s t 29, 1965 a t 16°C was o b s e r v e d t o have a r e s t i n g u t i l i z a t i o n o f 78%. The l o w e r i n g o f e n v i r o n m e n t a l o x y g e n t o 70 mm Hg P0^ r e s u l t e d i n an i n c r e a s e f r o m 7.75 t o 38.2 l i t e r s / h r / K g i n v e n t i l a t i o n v o lume and a d e c r e a s e ' i n u t i l i z a t i o n f r o m 78%> t o 34/6. Thus t h e b e h a v i o u r o f t h i s p a r -t i c u l a r f i s h was v e r y s i m i l a r t o t h e b e h a v i o u r o f t h e p a r t i -c u l a r f i s h upon w h i c h Van Dam ( 1 9 3 8 ) e x p e r i m e n t e d . Many o f t h e c a r d i o v a s c u l a r and r e s p i r a t o r y c h a r a c t e r i s -t i c s o f f i s h a p p e a r t o be h i g h l y v a r i a b l e . The t e c h n i q u e s u s e d t o measure t h e s e c h a r a c t e r i s t i c s a r e o f t e n p u s h e d t o t h e l i m i t s o f t h e i r a c c u r a c y . Under s u c h c i r c u m s t a n c e s t h e b e s t i n t e r e s t s o f a c c u r a c y a r e s e r v e d by p o o l i n g t h e i n f o r m a t i o n f r o m a l a r g e number o f e x p e r i m e n t s . I n f o r m a t i o n o b t a i n e d f r o m a s i n g l e o r few d e t e r m i n a t i o n s o f a h i g h l y v a r i a b l e p a r a m e t e r c a n o f t e n be m i s l e a d i n g . To i l l u s t r a t e t h i s p o i n t , S a u n d e r s ( 1 9 6 2 ) r e p o r t e d an 82 f o l d i n c r e a s e i n v e n t i l a t i o n v o lume f o r t h e s u c k e r i n r e s p o n s e t o h y p o x i a . T h i s i s b a s e d on a c a l c u l a t e d i n c r e a s e i n v e n t i l a t i o n volume f r o m 1.8 m l / s e c o n d t o 143.0 m l / s e c o n d . The s u c k e r w e i g h e d l e s s t h a n 250 grams and y e t - 62 -was r e p o r t e d l y a b l e t o move 143 m l / s e c w i t h i t s r e s p i r a t o r y pumps. T h i s w o r k s o u t t o a p p r o x i m a t e l y 70 t o 100 ml e a c h b r e a t h i n g c y c l e l T h i s h i g h v e n t i l a t i o n volume i s b a s e d i n t u r n upon a d e t e r m i n e d u t i l i z a t i o n o f 3.3/6. I f t h e a c c u r a c y o f t h e o x y g e n d e t e r m i n a t i o n t e c h n i q u e was o n l y p l u s o r minus 1.5% i t w o u l d be e a s y t o i n c u r an e r r o r o v e r t e n f o l d i n t h e v e n t i l a t i o n v o l u m e . S u m m a r i z i n g , i t i s s u g g e s t e d t h a t t h e b r a n c h i a l pump o f t h e r a i n b o w t r o u t manages, t h o u g h a t p r o g r e s s i v e l y h i g h e r e n e r g y c o s t s , t o m a i n t a i n a u n i f o r m s u p p l y o f o x y g e n t o t h e g i l l s u r f a c e i n t h e f a c e o f i n c r e a s i n g h y p o x i a . The c i r c u l a -t o r y s y s t e m r e s p o n d s t o t h e h y p o x i a by i n c r e a s i n g i t s a b i l i t y t o c a r r y t h e o x y g e n away f r o m t h e g i l l s . A c h a n g e i n t h e c a p a c i t y r a t e r a t i o o f b l o o d (\/b x Sbo2) and w a t e r (l/w x Swo^) w o u l d a l t e r t h e e f f e c t i v e n e s s o f gas e x c h a n g e (Hughes & S h e l t o n 1962, Hughes 1 9 6 4 ) . The e f f e c t o f h y p o x i a on t h e c a p a c i t y r a t e r a t i o o f b l o o d and w a t e r was c a l -c u l a t e d and i s s u m m a r i z e d i n F i g u r e 25. I t was o b s e r v e d t h a t t h e c a p a c i t y r a t e r a t i o i n c r e a s e d f r o m a b o u t 0.30 a t e n v i r o n -m e n t a l o x y g e n t e n s i o n s f r o m 140 t o 100 mm Hg t o more t h a n 0.60 a t e n v i r o n m e n t a l l e v e l s l e s s t h a n 60 mm Hg PO^. As Hughes and S h e l t o n ( 1 9 6 2 ) have p o i n t e d o u t , t h e c a p a c i t y r a t e r a t i o c a n have a s t r o n g i n f l u e n c e upon e f f e c t i v e n e s s . As t h e c a p a c i t y r a t e r a t i o i n c r e a s e s t h e e f f e c t i v e n e s s o f o x y g e n l o a d i n g o f t h e b l o o d w i l l d e c r e a s e (Hughes and S h e l t o n 1962, Hughes 1 9 6 4 ) . D u r i n g h y p o x i a t h e c a p a c i t y r a t e r a t i o o f t h e t r o u t i n c r e a s e s ( 0 . 3 t o 0.6) and as m i g h t be e x p e c t e d t h e e f -f e c t i v e n e s s o f t r a n s f e r d e c r e a s e s . - 63 -L e g e n d f o r F i g u r e 25 C hanges i n c a p a c i t y r a t e r a t i o o f b l o o d and w a t e r o c c u r r i n g w i t h i n c r e a s i n g h y p o x i a . _ 64 -The d i f f u s i o n c a p a c i t y o r t r a n s f e r f a c t o r (Ruch and P a t t o n 1965) w i l l a l s o a f f e c t t h e r a t e o f gas e x c h a n g e a c r o s s t h e g i l l s . I f t h e s u r f a c e a r e a o f t h e g i l l s a v a i l a b l e f o r gas e x c h a n g e i n c r e a s e s , o r i f t h e d i f f u s i o n d i s t a n c e b e t ween b l o o d and w a t e r d e c r e a s e s , t h e e f f e c t i v e n e s s o f o x y g e n l o a d i n g o f t h e b l o o d w i l l be i n c r e a s e d . The t e r m t r a n s f e r f a c t o r ( R u c h & P a t t o n 1965) i s d e f i n e d a s : R a t e o f 0 u p t a k e ml/min/Kg °2 ~ Mean p r e s s u r e g r a d i e n t a c r o s s g i l l e p i t h e l i u m RO °2 ~ ( T w o 2 i n + T__^ou\) {Tbo^~~ ThiT^Q~') 2 ~ 2 The e f f e c t o f h y p o x i a i n t h e medium on t h e t r a n s f e r f a c t o r was c a l c u l a t e d and i s s u m m a r i z e d i n F i g u r e 26. T h e r e i s a marked i n c r e a s e i n t h e t r a n s f e r f a c t o r o f t h e g i l l s o f t r o u t i n r e s p o n s e t o h y p o x i c c o n d i t i o n s " i n t h e e n v i r o n m e n t . S t e e n and K r u y s s e ( 1 9 6 4 ) d e m o n s t r a t e d t h e p r e s e n c e o f a l t e r n a t e c i r c u l a t o r y p a t h w a y s i n t h e g i l l s o f t h e e e l and t e n o t h e r t e l e o s t s , i n c l u d i n g t h e r a i n b o w t r o u t . They d e m o n s t r a t e d ^ t h a t s h u n t i n g c h a n g e s c o u l d a f f e c t t h e d e g r e e o f o x y g e n a t i o n o f t h e b l o o d o f t h e e e l . They i n t i m a t e t h a t most i f n o t a l l t e l e o s t f i s h have t h e c a p a c i t y t o c h a n g e t h e f u n c t i o n a l c a p a -c i t y o f t h e i r g i l l s f o r gas e x c h a n g e by means o f v a s c u l a r c h a n g e s w i t h i n t h e g i l l s . The i n c r e a s e d t r a n s f e r f a c t o r i s p r o b a b l y r e l a t e d t o a d e c r e a s e i n t h e d i f f u s i o n d i s t a n c e b e t w e e n b l o o d and w a t e r r a t h e r t h a n an i n c r e a s e i n t h e g i l l a r e a a v a i l a b l e f o r gas e x c h a n g e . A c h a n g e i n t h e d i f f u s i o n d i s t a n c e b e t w e e n b l o o d and w a t e r w o u l d be i n d i c a t e d by a - 65 -L e g e n d f o r F i g u r e 26 Changes i n t r a n s f e r f a c t o r (To^) o f t h e g i l l s d u r i n g i n c r e a s i n g h y p o x i a . - 66 -chan g e i n t h e p a t t e r n o f b l o o d f l o w t h r o u g h t h e g i l l s ( F i g . 1 1 ) , and i n d e e d t h i s i s i n d i c a t e d by t h e c h a n g e s i n v a s c u l a r r e s i s t a n c e a s s o c i a t e d w i t h h y p o x i a , c o n c l u d e d f r o m an e x a m i n a -t i o n o f t h e c h a n g e s i n b l o o d p r e s s u r e r e c o r d e d e f f e r e n t and a f f e r e n t t o t h e g i l l s o f t h e t r o u t . An i n c r e a s e i n t h e a r e a f o r d i f f u s i o n r a t h e r t h a n a d e c r e a s e i n t h e d i s t a n c e o f d i f -f u s i o n i s u n l i k e l y b e c a u s e t h e i n c r e a s e i n w a t e r v e l o c i t y a s s o -c i a t e d w i t h h y p o x i a w o u l d most l i k e l y d i s t u r b t h e a r r a n g e m e n t o f t h e g i l l l a m e l l a e and so r e d u c e t h e a r e a o f t h e g i l l s p r e -s e n t e d t o t h e w a t e r f o r gas e x c h a n g e . The i n c r e a s e i n t r a n s f e r f a c t o r d u r i n g h y p o x i a i s p r o b a b l y a l o w e s t i m a t e . B i j t e l ( 1 9 4 7 , 1949) s t a t e d t h a t d u r i n g q u i e t r e s p i r a t i o n t h e t i p s o f t h e g i l l f i l a m e n t s were i n c o n t a c t and t h a t a l l r e s p i r a t o r y w a t e r c i r c u l a t e d o v e r l a m e l l a r r e g i o n s . A t h i g h v e n t i l a t i o n , S a u n d e r s ( 1 9 6 1 ) showed t h a t t h e t i p s o f t h e f i l a m e n t s o f s e v e r a l f r e s h w a t e r t e l e o s t s s e p a r a t e d some-w h a t . T h i s w o u l d mean t h a t some p o r t i o n o f t h e r e s p i r a t o r y w a t e r was p a s s i n g by t h e g i l l s w i t h o u t c o m i n g i n c l o s e c o n t a c t w i t h t h e r e s p i r a t o r y s u r f a c e . I f t h i s i s t h e c a s e , t h e n w a t e r e f f e r e n t t o t h e l a m e l l a r s u r f a c e i s p r o b a b l y o f l o w e r t e n s i o n ( O j ) t h a n i s t h e e f f e r e n t w a t e r s a m p l e d i n t h e o p e r -c u l a r chamber. T h i s i n t u r n w o u l d mean t h a t a t h i g h e r v e n t i -l a t i o n l e v e l s t h e o x y g e n t e n s i o n g r a d i e n t o v e r t h e g i l l s u r -f a c e i s l i k e l y s m a l l e r t h a n e s t i m a t e d a n d t h e t r a n s f e r f a c t o r i s c o r r e s p o n d i n g l y l a r g e r t h a n e s t i m a t e d . T h e r e was a l o o s e i n v e r s e c o r r e l a t i o n b e t w e e n t h e hema-t o c r i t o f t h e f i s h and t h e o x y g e n t e n s i o n a t w h i c h t h e f i s h - 67 -began t o show s i g n s o f r e s p i r a t o r y o r c a r d i a c f a i l u r e . F i g u r e 18 shows t h i s r e l a t i o n s h i p b etween t h e i n i t i a l h e m a t o c r i t o f t h e f i s h and t h e o x y g e n t e n s i o n a t w h i c h t h e v a r i o u s e x p e r i -ments were t e r m i n a t e d . I t w o u l d a p p e a r t h a t t h e h e m a t o c r i t a nd c o r r e s p o n d i n g l y t h e u n i t c a p a c i t y o f t h e b l o o d o f t h e f i s h i s o f c o n s i d e r a b l e i m p o r t a n c e t o t h e f i s h i n s u r v i v a l i n h y p o x i c c o n d i t i o n s . SUMMARY OF PART THREE 1. T h e r e were no s i g n i f i c a n t c h a n g e s i n o x y g e n u p t a k e d u r i n g h y p o x i a i n t h e medium. V a l u e s o f b e t w e e n 100 - 125 mg/hr/Kg were o b t a i n e d f o r t h e r a i n b o w t r o u t d u r i n g t h e s e e x p e r i m e n t s ' ; 2. The b l o o d l a c t a t e o f f o u r f i s h e x a m i n e d i n c r e a s e d 2.7 f o l d d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t s . 3. V e n t i l a t i o n volume i n c r e a s e d 13 f o l d i n r e s p o n s e t o h y p o x i a . The a v e r a g e v e n t i l a t i o n volume i n c r e a s e d f r o m a r e s t i n g l e v e l o f 274 m l / m i n / K g t o a l e v e l o f 3560 m l / m i n / K g i n r e s p o n s e t o h y p o x i a . 4. C a r d i a c o u t p u t e s t i m a t e d by t h e F i c k p r i n c i p l e d i d n o t c h a n g e s i g n i f i c a n t l y . The a v e r a g e v a l u e f o r c a r d i a c o u t p u t v a r i e d f r o m 65 m l / m i n / K g t o 100 m l / m i n / K g . 5. S t r o k e v o l u m e o f t h e h e a r t i n c r e a s e d f r o m a p p r o x i -m a t e l y 1 m l / s t r o k e t o 2 - 5 m l / s t r o k e i n r e s p o n s e t o h y p o x i a . 6. E f f e c t i v e n e s s o f b l o o d o x y g e n a t i o n d e c r e a s e d f r o m a p p r o x i m a t e l y 1 0 0 % t o a p p r o x i m a t e l y 3 0 - 4 0 % a s a - 68 r e s u l t o f t h e b l o o d c a p a c i t y i n c r e a s e w h i c h o c c u r r e d a t l o w e r o x y g e n t e n s i o n s . 7. E f f e c t i v e n e s s o f o x y g e n r e m o v a l f r o m i n s p i r e d w a t e r t e n d e d t o r e m a i n f a i r l y u n i f o r m a t a b o u t 3 0 % i n d i -c a t i n g t h a t a u n i f o r m q u a n t i t y o f u s e a b l e o x y g e n i s b e i n g p r e s e n t e d t o t h e g i l l s . 8. P e r c e n t a g e u t i l i z a t i o n o f o x y g e n f r o m i n s p i r e d w a t e r d e c r e a s e d , b e t w e e n o x y g e n l e v e l s i n t h e e n v i r o n m e n t ( f r o m 160 mm Hg P 0 2 t o 100 mm Hg P 0 2 , t o 2 0 % t o 5 5 % ) . Below e n v i r o n m e n t a l P 0 2 l e v e l s o f 100 mm Hg t h e p e r -c e n t a g e u t i l i z a t i o n d i d n o t v a r y s i g n i f i c a n t l y from' 2 0%. 9. C a p a c i t y r a t e r a t i o ( C a p a c i t y o f b l o o d / C a p a c i t y o f w a t e r ) i n c r e a s e d f r o m a p p r o x i m a t e l y 0.30 t o more t h a n 0.60 i n r e s p o n s e t o h y p o x i a . 10. The t r a n s f e r f a c t o r o f t h e g i l l s i n c r e a s e d by a f a c -t o r o f a t l e a s t t h r e e , and i t i s l i k e l y t h a t t h i s e s t i m a t e d i n c r e a s e i s l o w . T h e r e i s a s t r o n g s u g -g e s t i o n t h a t t h e r e i s a s h o r t e n i n g i n t h e d i s t a n c e i n t h e d i f f u s i o n p a thway f o r o x y g e n a c r o s s t h e g i l l membrane a s a r e s u l t o f v a s c u l a r c h a n g e s w i t h i n t h e g i l l s . T h i s s u g g e s t i o n i s s u p p o r t e d by i n d i c a t i o n s o f r e s i s t a n c e c h a n g e s t o b l o o d f l o w t h r o u g h t h e g i l l s . - 69 -GENERAL DISCUSSION In t h e p r e s e n t s t u d y t h e f i s h were u n d e r g o i n g o n l y t r a n -s i e n t t e s t s o f t h e i r r e s p o n s e t o h y p o x i a . I t i s l i k e l y t h a t most o f t h e f i s h t e s t e d c o u l d n o t have s u r v i v e d a t t h e l o w e r o x y g e n l e v e l s , i f s u b j e c t e d t o t h e s e f o r a l o n g p e r i o d o f t i m e . The main p u r p o s e o f t h e e x p e r i m e n t s was t o e x a m i n e t h e s h o r t t e r m r e s p o n s e s o f t h e c a r d i o v a s c u l a r and b r e a t h i n g s y s t e m s t o h y p o x i a . The b u i l d u p o f c a r b o n d i o x i d e i n t h e r e s -p i r o m e t e r f r o m t h e f i s h ' s r e s p i r a t i o n was a c o m p l i c a t i n g f a c -t o r , b u t t h e l e v e l s were lo w ( o f t h e o r d e r o f a few mm Hg). I t i s p o s s i b l e t h a t t h e c a r b o n d i o x i d e may have a f f e c t e d t h e t e n s i o n s a t w h i c h t h e v a r i o u s r e s p o n s e s o c c u r r e d , b u t i t i s h i g h l y u n l i k e l y t h a t t h e n a t u r e and g e n e r a l o r d e r o f o c c u r r -e n c e o f t h e r e s p o n s e s w o u l d be g r e a t l y a f f e c t e d . T e l e o s t s a r e a v e r y d i v e r s e g r o u p and t h e r e s p o n s e s o f t h e r a i n b o w t r o u t t o h y p o x i a a r e n o t n e c e s s a r i l y r e p r e s e n t a -t i v e o f t h e w h o l e g r o u p . The t r o u t i s a r a t h e r u n s p e c i a l i z e d f i s h w h i c h l i v e s , g e n e r a l l y , i n e n v i r o n m e n t s o f h i g h o x y g e n c o n t e n t . S i n c e i t a p p e a r s t h a t t r o u t a r e a c t i v e , and t h e i r c l o s e r e l a t i v e s , t h e s a l m o n , d e m o n s t r a t e t r e m e n d o u s a b i l i t y f o r a c t i v i t y , i t i s r e a s o n a b l e t o assume t h a t t h e t r o u t p r o -b a b l y h ave a w i d e s c o p e f o r i n c r e a s i n g t h e i r o x y g e n u p t a k e . The p r e s e n t e x a m i n a t i o n has d e m o n s t r a t e d t h a t t h e u p t a k e o f o x y g e n c a n , a t l e a s t i n p a r t , be m a i n t a i n e d d e s p i t e l o w e r -ed e n v i r o n m e n t a l o x y g e n l e v e l s . The f i s h were t o some e x t e n t r e s o r t i n g t o a n a e r o b i c means f o r t h e i r m e t a b o l i c e n e r g y s u p p l y as i s e v i d e n c e d by t h e r i s e i n l a c t a t e l e v e l s i n t h e b l o o d , b e t w e e n t h e b e g i n n i n g and t h e end o f t h e e x p e r i m e n t s . - 70 -I t was assumed t h a t u n d e r h y p o x i c c o n d i t i o n s t h e f i s h w o u l d need some o x y g e n and w o u l d t a k e s t e p s t o o b t a i n i t . U n l e s s t h e minimum amount o f o x y g e n r e q u i r e d t o s u s t a i n t h e f i s h i s v e r y s m a l l , t h e f i s h w o u l d have t o i n c r e a s e t h e i r e f f o r t t o e x t r a c t o x y g e n f r o m t h e e n v i r o n m e n t . I f t h i s i s t h e c a s e t h e e x t r a c t i n g m a c h i n e r y w i l l r e q u i r e i n c r e a s e d o x y g e n t o r u n i t . T h i s w o u l d c r e a t e a p r o b l e m o f d i m i n i s h i n g r e t u r n s p e r u n i t e f f o r t f o r e x t r a c t i o n as t h e e n v i r o n m e n t a l o x y g e n d e c r e a s e s . F u n d a m e n t a l l y t h e f i s h c a n t a k e c o m p e n s a t o r y m e a s u r e s i n two m a n n e r s ; p a s s i v e and a c t i v e . F i r s t l y t h e f i s h c a n p a s s i v e l y d e c r e a s e t h e amount o f o x y g e n u p t a k e needed f o r s u s t e n a n c e . S e c o n d l y m e a s u r e s c a n be t a k e n t o m a i n t a i n t h e amount o f o x y g e n t a k e n i n by t h e f i s h . To c o u n t e r a c t t h e e f f e c t s o f h y p o x i a by p a s s i v e means, t h e f i s h c o u l d do t h e f o l l o w i n g : a. Reduce t h e i r m e t a b o l i s m g e n e r a l l y , r e s o r t i n g t o a n a e r o b i c m e t a b o l i s m where n e c e s s a r y . b. S h u t down o x i d a t i v e m e t a b o l i s m i n n o n - v i t a l t i s s u e s . c. D e c r e a s e m u s c u l a r a c t i v i t y , e s p e c i a l l y a c t i v i t y n o t a s s o c i a t e d w i t h o x y g e n e x t r a c t i o n . The r i s e i n l a c t a t e i n t h e b l o o d o f t h e e x p e r i m e n t a l f i s h w o u l d s u g g e s t t h a t t h e f i s h were r e s o r t i n g t o a n a e r o b i c r e s p i r a t i o n t o some e x t e n t . T h e r e was no r e l i a b l e way o f m e a s u r i n g w h e t h e r o r n o t t h e f i s h were t a k i n g a d v a n t a g e o f t h e l a s t two m e a s u r e s . A c t i v e m e a s u r e s t a k e n by t h e f i s h w h i c h w i l l i n c r e a s e t h e r a t e o f d i f f u s i o n o f o x y g e n a c r o s s t h e r e s p i r a t o r y mem-b r a n e s c a n be l i s t e d u n d e r t h e f o l l o w i n g c a t e g o r i e s : - 71 -a. The t e n s i o n g r a d i e n t s c o u l d be 1 i n c r e a s e d . b. The a r e a o f t h e e x c h a n g e s u r f a c e c o u l d be i n -c r e a s e d . c. The d i s t a n c e f o r d i f f u s i o n c o u l d be d e c r e a s e d . d. The t i m i n g and a r r a n g e m e n t o f f l o w s o f l i q u i d s p a s t t h e r e s p i r a t o r y e p i t h e l i u m c o u l d be r e l a t e d so as t o o b t a i n an o p t i m a l f l o w a r r a n g e m e n t f o r t h e e x c h a n g e o f g a s e s . V a r i a b l e s w h i c h w i l l a f f e c t t h e t e n s i o n g r a d i e n t f o r o x y g e n a c r o s s t h e g i l l membrane a r e ; t h e t e n s i o n o f t h e en-v i r o n m e n t a l o x y g e n , t h e o x y g e n t e n s i o n o f ve n o u s b l o o d , t h e f l o w r a t e s o f b l o o d and w a t e r p a s t t h e e x c h a n g e s u r f a c e , and t h e o x y g e n c a p a c i t y o f t h e b l o o d and t h e w a t e r . The maximum g r a d i e n t w h i c h c a n be a c h i e v e d , i s t h e d i f -f e r e n c e i n o x y g e n t e n s i o n b e t w e e n t h e e n v i r o n m e n t and v e n o u s b l o o d . W i t h p r o g r e s s i v e l y d e e p e n i n g h y p o x i a t h e maximum p o s -s i b l e g r a d i e n t i s d i m i n i s h i n g . I n c r e a s i n g t h e f l o w s o f b l o o d and w a t e r p a s t t h e g i l l s u r f a c e w i l l s e r v e t o m a i n t a i n t h e g r a d i e n t a t a l e v e l a s n e a r t h e t h e o r e t i c a l maximum as p o s s i b l e . H o wever, i n c r e a s i n g t h e f l o w s i n d e f i n i t e l y w i l l b r i n g s t e a d i l y d i m i n i s h i n g r e t u r n s a s t h e g r a d i e n t n e a r s i t s l i m i t i n g m a x i -mum. An i n c r e a s e i n t h e c a p a c i t y o f t h e l i q u i d s f o r o x y g e n w i l l a l l o w t h e o x y g e n t e n s i o n g r a d i e n t t o be m a i n t a i n e d w i t h l o w e r f l o w r a t e s . W i t h t h e p r e s e n t s t u d y , i t i s o b v i o u s t h a t t h e o x y g e n t e n s i o n o f t h e e n v i r o n m e n t a l w a t e r i s d r o p p i n g t h r o u g h o u t t h e c o u r s e o f t h e e x p e r i m e n t . I n o t h e r w o r d s , t h e f i s h i s p r e s e n t e d w i t h t h e p r o b l e m o f c o n t i n u a l l y d e c r e a s i n g maximum - 72 -p o s s i b l e d i f f u s i o n g r a d i e n t s f o r o x y g e n o v e r i t s r e s p i r a t o r y s u r f a c e . To c o u n t e r a c t t h i s t h e f i s h have no means o f a l t e r -i n g t h e c a p a c i t y o f t h e w a t e r , but t h e y c a n , and do, i n c r e a s e t h e f l o w o f w a t e r o v e r t h e g i l l s some t h i r t e e n f o l d . The a r t e r i a l and v e n o u s o x y g e n t e n s i o n s and t h e 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 o f t h e b l o o d a r e s u c h t h a t t h e f u n c t i o n a l c a p a c i t y o f t h e b l o o d i s i n c r e a s e d w i t h t h e h y p o x i a even t h o u g h t h e t o t a l c a p a c i t y i s d i m i n i s h e d . As p o i n t e d o u t p r e v i o u s l y i n t h i s d i s c u s s i o n , i t i s e n e r g e t i c a l l y more e c o n o m i c a l t o i n -c r e a s e t h e c a p a c i t y o f t h e f l u i d s t h a n t o i n c r e a s e t h e f l o w s . The t r o u t s t u d i e d i n t h i s work were o b s e r v e d t o i n c r e a s e t h e f u n c t i o n a l c a p a c i t y o f t h e i r b l o o d ; a t t h e same t i m e t h e r e was no s i g n i f i c a n t c h a n g e i n t h e e s t i m a t e s o f c a r d i a c o u t p u t . Thus i t a p p e a r s t h a t a t l e a s t w i t h a s p e c i a l i z e d f l u i d , t h e b l o o d , t h e f i s h a r e a b l e t o o b s e r v e t h e economy r e s u l t i n g f r o m c a p a c i t y i n c r e a s e s i n m a i n t a i n i n g a s h i g h a g r a d i e n t a s p o s s i b l e f o r o x y g e n t e n s i o n s o v e r t h e r e s p i r a t o r y membrane. I t i s i n t e r e s t i n g t o n o t e t h a t w i t h t h e t r o u t s t u d i e d , a l t h o u g h t h e r e i s a l a r g e i n c r e a s e i n t h e amount o f w a t e r p r e s e n t e d t o t h e g i l l s i n r e s p o n s e t o h y p o x i a , t h e amount o f u s e a b l e o x y g e n i n t h e w a t e r had i n t h e same i n t e r v a l become p r o p o r t i o n a t e l y l e s s . W i t h t h e b l o o d , t h e s i t u a t i o n was t h e r e v e r s e . The f l o w d i d n o t c h a n g e s i g n i f i c a n t l y b u t t h e u n i t c a p a c i t y i n c r e a s e d a s a r e s u l t o f t h e 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 o f t h e b l o o d . T h u s , as Hughes and S h e l t o n , 1962, and H u g h e s , 1964, s u g g e s t e d , t h e r e i s a f a i r l y c l o s e c o u p l i n g o f t h e c a p a c i t y x r a t e r a t i o b e t w e e n t h e b l o o d and t h e w a t e r p a s s i n g o v e r t h e g i l l s , i n r e s p o n s e t o h y p o x i a . - 73 -The p r i n c i p l e o f f u n c t i o n a l l y s h o r t e n i n g t h e d i f f u s i o n p a t h t h r o u g h t h e g i l l membrane r e p r e s e n t s a v e r y e f f e c t i v e may o f a u g m e n t i n g t h e r a t e o f gas movement t h r o u g h t h e g i l l membrane. The s u g g e s t i o n o f S t e e n and K r u y s s e ( 1 9 6 4 ) t h a t b l o o d c a n be c h a n n e l l e d c l o s e r t o t h e s u r f a c e o f t h e g i l l s i s v e r y i n t e r e s t i n g . S t e e n and K r u y s s e ( 1 9 6 4 ) have demon-s t r a t e d t h a t t h e s t r u c t u r a l r e q u i r e m e n t s f o r s u c h a s h u n t i n g s y s t e m a r e p r e s e n t i n a v a r i e t y o f f r e s h w a t e r t e l e o s t s . They a l s o d e m o n s t r a t e d t h a t a d r e n a l i n c o u l d i n c r e a s e t h e d e g r e e o f o x y g e n a t i o n o f e f f e r e n t b l o o d . E v i d e n c e f r o m t h e p r e -s e n t s t u d y s u g g e s t s t h a t t h e r e a r e v a s c u l a r c h a n g e s i n t h e g i l l s a s s o c i a t e d w i t h h y p o x i a w h i c h a r e c a u s i n g an i n c r e a s e i n r e s i s t a n c e o v e r t h e g i l l s t o t h e f l o w o f b l o o d . T h i s o c c u r r e n c e , c o u p l e d w i t h t h e i n c r e a s e i n t r a n s f e r f a c t o r o f t h e g i l l s w i t h i n c r e a s i n g h y p o x i a , p r o v i d e s good e v i d e n c e f o r t h e f u n c t i o n i n g o f a s y s t e m whereby t h e d i f f u s i o n c h a r -a c t e r i s t i c s o f t h e g i l l s a r e c h a n g e d . T h e r e i s a s t r o n g p o s s i b i l i t y t h a t b l o o d i s b e i n g p r e s e n t e d c l o s e r t o t h e w a t e r . One p o s s i b l e r e a s o n t h a t t h e f i s h does n o t l e a v e t h e t r a n s f e r f a c t o r o f t h e g i l l s a t t h e h i g h e r l e v e l s a s s o -c i a t e d w i t h h y p o x i a i s t h a t t h e s u p p o s e d l y s h o r t e r d i f f u s i o n p a t h p r o b a b l y i n c r e a s e s t h e i o n i c o r o s m o t i c p r o b l e m s t h a t t h e f i s h has t o c o n t e n d w i t h , a p r o b l e m w h i c h w o u l d t h r o w a h e a v y work l o a d upon t h e k i d n e y s and t h e g i l l s . The i n c r e a s e i n t r a n s f e r f a c t o r i s l i k e l y t o be an e m e r g e n c y r e s p o n s e t o augment t h e u p t a k e o f o x y g e n u n d e r t h e p e n a l t y o f i n c r e a s e d i o n i c o r o s m o t i c p r o b l e m s . - 74 -T h e r e i s a c o u n t e r c u r r e n t f l o w a r r a n g e m e n t o f b l o o d and w a t e r p a s s i n g t h e e x c h a n g e s u r f a c e w h i c h a i d s i n m a i n t a i n i n g t h e h i g h e s t gas t e n s i o n g r a d i e n t s u n d e r most o f t h e r e a s o n a b l e f l o w r a t e s . E v i d e n c e o f s u c h a s y s t e m has been p r e v i o u s l y d i s c u s s e d i n t h e s e c o n d p a r t o f t h e p r e s e n t s t u d y . Rainbow t r o u t a p p a r e n t l y do have a c o u n t e r c u r r e n t f l o w a r r a n g e m e n t o f b l o o d and w a t e r p a s t t h e r e s p i r a t o r y s u r f a c e . A n o t h e r means by w h i c h f i s h augment t h e e x c h a n g e p r o c e s s i s by a marked s y n c h r o n y b e t ween t h e b r e a t h i n g movements and t h e h e a r t b e a t s . T h i s has been o b s e r v e d i n a w i d e v a r i e t y o f f i s h d u r i n g h y p o x i a ( H u g h e s , 1961; S h e l t o n and R a n d a l l , 1962; S a t c h e l l , 1 9 6 0 ) . Hughes 1964, has a m p l y p o i n t e d o u t t h e v a l u e o f c o u p l i n g t h e b l o o d and w a t e r movements. W i t h t h e r a i n b o w t r o u t t h e p o i n t a t w h i c h t h e homeo-s t a t i c mechanisms c a n no l o n g e r c o m p e n s a t e f o r t h e r e d u c e d o x y g e n s u p p l y i s t h e b e g i n n i n g o f a c a t a c l y s m i c c o l l a p s e . The r e s p i r a t o r y and h e a r t m u s c l e w i l l b e g i n t o f a i l i f o x y g e n s u p p l i e s f a l l t o o l o w , and t o do so w i l l f u r t h e r r e d u c e t h e u p t a k e o f o x y g e n w h i c h i n t u r n w i l l f u r t h e r r e d u c e t h e e f f e c t -i v e n e s s o f t h e e x t r a c t i n g m a c h i n e r y e v e n more s o . An i n t e r e s t i n g f u n c t i o n a l a s p e c t o f t h e b l o o d p i g m e n t o f t h e r a i n b o w t r o u t i s t h a t i t s e r v e s a s a r e s e r v o i r f o r f u n c t i o n a l b l o o d o x y g e n c a p a c i t y i n c r e a s e s . The v e n o u s b l o o d p i g m e n t o f t h e r a i n b o w t r o u t i s p r a c t i c a l l y a l l o x y h e m o g l o b i n when t h e e n v i r o n m e n t a l o x y g e n t e n s i o n s a r e h i g h . T h i s i s i n s h a r p c o n t r a s t w i t h t h e a s s u m p t i o n s o f some ( M o t t , 1957; S a u n d e r s , 1962) t h a t t h e v e n o u s b l o o d i s , i n most c a s e s , c o m p l e t e l y d e o x y g e n a t e d . I n g e n e r a l t h e h e m o g l o b i n i s n o t - 75 -u s e d t o much e x t e n t when t h e f i s h i s r e s t i n g i n a w e l l o x y g e n a t e d e n v i r o n m e n t . W i t h some s p e c i e s o f C h a e n o c e -p h a l u s f r o m a n t a r c t i c r e g i o n s t h e r e a r e no m a n i f e s t a t i o n s o f b l o o d p i g m e n t s a t a l l (Ruud, 1 9 5 4 ) . A n t h o n y ( 1 9 6 1 ) r e p o r t e d t h a t g o l d f i s h c o u l d s u r v i v e i n a medium w i t h h i g h t e n s i o n s o f c a r b o n m o n o x i d e , and c o n c l u d e d t h a t h e m o g l o b i n was n o t a b s o l u t e l y n e c e s s a r y t o t h e r e s t i n g g o l d f i s h . I t w o u l d a p p e a r f r o m t h e p r e s e n t s t u d y and e a r l i e r work t h a t t h e b l o o d p i g m e n t o f r a i n b o w t r o u t does n o t s e r v e as a m a j o r c a r r i e r o f o x y g e n e x c e p t u n d e r c o n d i t i o n s o f s t r e s s o r h i g h o x y g e n demand. I n s t e a d i t a p p e a r s t h a t t h e p i g m e n t s e r v e s t o c a r r y some o f t h e i n c r e a s e d l o a d s upon t h e gas u p t a k e m echanism r a t h e r t h a n t h e h e a r t . I t w o u l d be o f a d v a n t a g e t o a v o i d i n c r e a s i n g c a r d i a c o u t p u t . I t w o u l d seem r e a s o n a b l e t o assume t h a t t h e h e a r t does n o t have a v e r y l a r g e s c o p e f o r i n c r e a s i n g i t s work l o a d , s i n c e i t must work c o n t i n u o u s -l y . T h e r e a l r e a d y i s an i n c r e a s e d work l o a d on t h e h e a r t d u r i n g h y p o x i a , s i n c e t h e c a r d i a c o u t p u t does n o t c h a n g e and t h e b l o o d p r e s s u r e s n e a r l y d o u b l e . The amount o f h e m o g l o b i n i n t h e b l o o d w o u l d r e f l e c t t h e c a p a b i l i t y f o r i n c r e a s i n g t h e c a p a c i t y o f t h e b l o o d , and t h e a b i l i t y t o e a s e t h e work done by t h e h e a r t . T h i s s u g g e s t i o n i s s u p p o r t e d by t h e l o o s e c o r r e l a t i o n o b s e r v e d b e t w e e n t h e h e m a t o c r i t o f t h e b l o o d and t h e t e n s i o n o f t h e w a t e r a t w h i c h t h e v a r i o u s e x p e r i m e n t s were t e r m i n a t e d . P i s h w i t h h i g h e r h e m a t o c r i t s were g e n e r a l l y a b l e t o s u s t a i n l o w e r e n v i r o n m e n t a l t e n s i o n s t h a n f i s h w i t h l o w h e m a t o c r i t s . The b r a d y c a r d i a a s s o c i a t e d w i t h t h e h y p o x i a r e m a i n s an i n t e r e s t i n g p r o b l e m . The b r a d y c a r d i a i s a v e r y w i d e -- 76 -s p r e a d r e s p o n s e o b s e r v e d i n d i v i n g b i r d s , mammals, and many p o i k i l o t h e r m s . I t has more o r l e s s been assumed t h a t t h e b r a d y c a r d i a r e s u l t s i n a d e c r e a s e i n t h e h e a r t o u t p u t . Hughes ( 1 9 6 4 ) s u g g e s t s t h a t t h e b r a d y c a r d i a s e r v e s t o l e n g t h e n t h e r e s i d e n c e t i m e f o r b l o o d i n t h e g i l l s , by m e r i t o f de-c r e a s e d o u t p u t . T h i s w o u l d p r o v i d e f o r more c o m p l e t e l o a d i n g o f t h e e f f e r e n t b l o o d and m a i n t a i n i n g t h e t i s s u e o x y g e n l e v e l s What r e n d e r s t h e p r e s e n t s t u d y i n t e r e s t i n g i s t h a t t h e r e does n o t a p p e a r t o be any d e c r e a s e i n c a r d i a c o u t p u t c o n c o m i t a n t w i t h t h e h y p o x i a - i n d u c e d b r a d y c a r d i a . T h e r e i s i n s t e a d , a marked i n c r e a s e i n s t r o k e volume w h i c h i s o f a b o u t t h e same m a g n i t u d e a s t h e s e v e r i t y o f t h e b r a d y c a r d i a . Why t h e n i s t h e r e a b r a d y c a r d i a a t a l l ? Some known f a c t o r s a r e , t h a t t h e work l o a d on t h e h e a r t i s i n c r e a s i n g and t h e o x y g e n un-l o a d i n g t e n s i o n s a t t h e t i s s u e l e v e l a r e l o w e r . In o t h e r w o rds t h e h e a r t w i l l need more o x y g e n and a t t h e same t i m e t h e d i f f u s i o n g r a d i e n t s d r i v i n g t h e o x y g e n a r e l o w e r . P o s s i b l y t h e b r a d y c a r d i a s e r v e s t o p r o v i d e a l o n g e r p e r i o d f o r o x y g e n and m e t a b o l i t e s t o be r e s t o r e d i n c a r d i a c m u s c l e b e t w e e n e a c h w o r k i n g s t r o k e . The s u g g e s t i o n i s t h a t c a r d i a c m u s c l e o f f i s h , and p o s s i b l y o t h e r v e r t e b r a t e s , may be a b l e t o o p e r a t e more e f f i c i e n t l y t h r o u g h l o n g s p a c e d s t r o k e s t h a n t h r o u g h s h o r t s p a c e d o n e s . At any r a t e i t i s h a r d t o v i s u a l i z e any b e n e f i t f r o m t h e i n c r e a s e d p u l s a t i l i t y i n p r e s -s u r e s and p r e s u m a b l y g i l l b l o o d f l o w s , w h i c h r e s u l t f r o m t h e b r a d y c a r d i a . - 77 -REFERENCES A n t h o n y , E.H. ( 1 9 6 1 ) . S u r v i v a l o f g o l d f i s h i n p r e s e n c e o f c a r b o n m o n o x i d e . J o u r n a l o f E x p e r i m e n t a l B i o l o g y , 3_8: 109-126. B e a m i s h , F.lAl. ( 1 9 6 3 ) - I I I . R e s p i r a t i o n o f f i s h e s w i t h s p e c i a l e m p h a s i s on s t a n d a r d o x y g e n c o n s u m p t i o n , I I I - I n f l u e n c e o f o x y g e n . C a n a d i a n J o u r n a l o f Z o o l o g y , 4 2 : ( 1 9 6 4 ) . B e r g , T., and J.B. S t e e n , ( 1 9 6 5 ) . P h y s i o l o g i c a l mechanisms f o r a e r i a l r e s p i r a t i o n i n t h e e e l . Comp. B i o c h e m . P h y s i o l . 1 5 : 469-484. B i j t e l , H. ( 1 9 4 7 ) . The mechanism o f movement o f t h e g i l l f i l a m e n t s i n t e l e o s t e i . E x p e r i e n t i a . V o l . H I / 4 : 158-166. B l a c k . E.C. ( 1 9 6 5 ) . B l o o d l e v e l s o f h e m o g l o b i n and l a c t i c a c i d i n some f r e s h w a t e r f i s h e s f o l l o w i n g e x e r c i s e . J . F i s h . R e s . Bd. C a n a d a . 1 2 ( 6 ) : 9 1 7-929. B l a c k , E.C. ( 1 9 5 7 ) . A l t e r a t i o n s i n t h e b l o o d l e v e l s o f l a c t i c a c i d i n c e r t a i n s a l m o n i d f i s h e s f o l l o w i n g m u s c u l a r a c t i v i t y . I , K a m l o o p s t r o u t , Salmo p a i r d n e r i . J . F i s h . R es. Bd. C a n a d a , 1 4 ( 2 ) : 117-134. B l a c k , E.C., Anne R o b e r t s o n C o n n o r , Kwok-Cheung Lam, and Wing-Gay C h i u . ( 1 9 6 2 ) . Cha nges i n g l y c o g e n , p y r u v a t e , and l a c t a t e i n r a i n b o w t r o u t ( Salmo q a i r d n e r i ) d u r i n g and f o l l o w i n g m u s c u l a r a c t i v i t y . J . F i s h . Res. Bd. Canada. 1 9 ( 3 ) : 4 0 9 - 4 3 6 . B l a c k , E.C., D o n a l d K i r k p a t r i c k , and H a r o l d H. T u c k e r . ( 1 9 6 6 ) . Oxygen d i s s o c i a t i o n c u r v e s o f t h e b l o o d o f b r o o k 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 ) a c c l i m a t e d t o summer and w i n t e r t e m p e r a t u r e s . J . F i s h . R es. Bd. C a n a d a . 23(1 ) : 1966. B r e t t , J.R. ( 1 9 6 4 ) . The r e s p i r a t o r y m e t a b o l i s m and swimming p e r f o r m a n c e o f young s o c k e y e s a l m o n . J . F i s h . Res. Bd. C a n a d a . 21 ( 5 ) : 1 183-1226. B r e t t , J.R. and D.B. S u t h e r l a n d . ( 1 9 6 5 ) . R e s p i r a t o r y meta-b o l i s m o f p u m p k i n s e e d ( L e p o m i s q i b b o s u s ) i n r e l a t i o n t o swimming s p e e d . J . F i s h . R es. Bd. C a n a d a . 2 2 ( 2 ) : 4 0 5 -409. B u r g e r , J.W., and S.E. B r a d l e y . ( 1 9 5 1 ) . The g e n e r a l f o r m o f t h e c i r c u l a t i o n i n t h e d o g f i s h S g u a l u s a c a n t h i a s . J . C e l l u l a r Comp. P h y s i o l . 3_7 : 3 8 9 - 4 0 2 . F e r g u s o n , J.K.W., and E.C. B l a c k . (1941 ) . The t r a n s p o r t o f c a r b o n d i o x i d e i n t h e b l o o d o f c e r t a i n f r e s h w a t e r f i s h e s . B i o l o g i c a l B u l l e t i n . 8_0: 139-1 52. - 78 -F r y , F . E . J . ( 1 9 5 7 ) . The a q u a t i c r e s p i r a t i o n o f f i s h ( p a g e s 1 55 i n t h e P h y s i o l o g y o f F i s h e s , e d. by IY|. E. B r o w n ) . G o l d s t e i n , L., Roy P. F o r s t e r , and A.M. F a n e l l i J r . ( 1 9 6 4 ) . G i l l b l o o d f l o w and ammonia e x c r e t i o n i n t h e m a r i n e t e 1 e o s t , M y o x o c e p h a l u s s c o r p i u s . Comp. B i o c h e m . P h y s i o l 12: 489-499. G r e e n e , C . w1. ( 1 9 0 4 ) . P h y s i o l o g i c a l s t u d i e s o f t h e c h i n o o k s a l m o n . B u l l . U.S. B u r . F i s h e r i e s . 24: 431-447. H a n i n g , Q.C., and A l a n M. Thompson. ( 1 9 6 5 ) . A c o m p a r a t i v e s t u d y o f t i s s u e c a r b o n d i o x i d e i n v e r t e b r a t e s . Comp. B i o c h e m . P h y s i o l . 1_5: 17-26. H a r t , J . S . ( 1 9 4 3 ) . The c a r d i a c o u t p u t o f f o u r f r e s h w a t e r f i s h C a n a d i a n J o u r n a l o f R e s e a r c h . V o l . 21 (D) #4: 77-84. Hughes, G.M., and S h e l t o n , G. ( 1 9 6 2 ) . R e s p i r a t o r y mechanisms and t h e i r n e r v o u s c o n t r o l i n f i s h . A d v a n c e s i n Compara-t i v e P h y s i o l o g y and B i o c h e m i s t r y . V o l I : 275-364. Hughes, G.M. ( 1 9 6 4 ) . F i s h r e s p i r a t o r y h o m e o s t a s i s . X V I I I TW S p o s i u m o f t h e S o c i e t y f o r E x p e r i m e n t a l B i o l o g y , C a m b r i d g I r v i n g , L. , E.C. B l a c k , and V i r g i n i a S a f f o r d . (1941 ) . The i n f l u e n c e o f t e m p e r a t u r e upon t h e c o m b i n a t i o n o f o x y g e n w i t h t h e b l o o d o f t r o u t . B i o l o g i c a l B u l l e t i n , V o l . LXXX, No. 1: 1-17. Feb. 1941. I t a z a w a , Y. ( 1 9 5 7 ) . Gas c o n t e n t o f t h e b l o o d i n r e s p o n s e t o t h a t o f Medium w a t e r i n f i s h . B u l l , o f t h e J a p a n e s e So c . o f S c i . F i s h e r i e s , V o l . 23, No. 2: 71-80. J o h a n s e n , K. ( 1 9 6 2 ) . C a r d i a c o u t p u t and p u l s a t i l e a o r t i c f l o w i n t h e t e l e o s t , Gadus morhua. Comp. B i o c h e m . P h y s i o l . V o l . 7: 169-174. J o h a n s e n , K, (19 6 3) . C a r d i o v a s c u l a r d y n a m i c s i n t h e a m p h i -b i a n , Amphiuma t r i d a c t y l u m . N o r w e g i a n Monographs on M e d i c a l S c i e n c e , S c a n d . U n i v . B o o k s . U n i v e r s i t e t s f o r l a -g e t . J o h a n s e n , K. ( 1 9 6 5 ) . C a r d i a c o u t p u t i n t h e l a r g e c e p h a l o p o d O c t o p u s d o f l e i n i . J . Exp. B i o l . 42: 47 5 - 4 8 0 . K a y s , lAJ. M. , and L o n d o n , A.L. ( 1 9 5 8 ) . Compact h e a t e x c h a n g e r s . New Y g r k , McGraw H i l l Pub. K r o g h , A. ( 1 9 4 1 ) . C o m p a r a t i v e p h y s i o l o g y o f r e s p i r a t o r y mech-a n i s m s . U n i v e r s i t y o f P e n n s y l v a n i a P r e s s , 1941. (172 p a g e s ) . - 79 -M o t t , J.C. ( 1 9 5 7 ) . The c a r d i o v a s c u l a r s y s t e m ( i n P h y s i o l o g y o f F i s h e s , M.E. Brown, Ed., A c a d e m i c P r e s s I n c . " IM. Y . 1957. ) Pages 81-108. M o t t , J.C. ( 1 9 5 0 ) . R a d i o l o g i c a l o b s e r v a t i o n s on t h e c a r d i o v a s -c u l a r s y s t e m i n A n g u i l l a a n o u i l l a . J . E x p t l . B i o l . 2 7 ; 324 - 3 3 3 . Ogden, E. ( 1 9 4 5 ) . R e s p i r a t o r y f l o w i n M u s t e l u s . Am. J . P h y s i o l . 1 4 5 ( 2 ) : 134-139. P r o s s e r , C L . and Brown, F.A. (1961 s e c o n d E d . ) . C o m p a r a t i v e A n i m a l P h y s i o l o g y . W.B. S a u n d e r s Co., P h i l a . - L o n d . R a n d a l l , D.J. and S h e l t o n , G. (19 63) . The e f f e c t s o f c h a n g e s i n e n v i r o n m e n t a l gas c o n c e n t r a t i o n s on t h e b r e a t h i n g and h e a r t r a t e o f a t e l e o s t f i s h . Comp. B i o c h e m . and P h y s i o l . 9: 229-239. R a n d a l l , D.J., L.S. S m i t h and J.R. B r e t t . ( 1 9 6 5 ) . D o r s a l a o r t i c b l o o d p r e s s u r e s r e c o r d e r f r o m t h e r a i n b o w t r o u t , ( S a l mo g a i r d n e r i ) . C a n a d i a n J g u r n a l o f Z o o l o g y , V o l . 43 : 8 6 3 - 8 7 2 . R u c h , T.C. and P a t t o n ( E d s . ) ( 1 9 6 6 ) . P h y s i o l o g y and B i o p h y s i c s . 1 9 t h Ed. S a u n d e r s & Co. Rushmer, R.F. and S m i t h , O.A. J r . ( 1 9 5 9 ) . C a r d i a c C o n t r o l . P h y s i o l . Rev. 3 9 : 41-68. Ruud, J.T. ( 1 9 5 4 ) . V e r t e b r a t e s w i t h o u t e r y t h r o c y t e s and b l o o d p i g m e n t . N a t u r e , May 8, 1954: 8 4 8 - 8 5 0 . S a t c h e l l , G.H. ( i 9 6 0 ) . The r e f l e x c o o r d i n a t i o n o f t h e h e a r t b e a t w i t h r e s p i r a t i o n i n t h e d o g f i s h . J . Exp. B i o l . 37: 7 1 9 - 7 3 1 . S a t c h e l l , G.H. (1961 ) . The r e s p o n s e o f t h e d o g f i s h t o a n o x i a . J . Exp. B i o l . 3 8 : 531-543. S a u n d e r s . R.L. ( 1 9 6 1 ) - I . The i r r i g a t i o n o f t h e g i l l s i n f i s h e s ( I ) . Can. J . Z o o l . 3 9 : 637-653. S a u n d e r s , R.L. ( 1 9 6 2 ) - I I . The i r r i g a t i o n o f t h e g i l l s i n f i s h e s . Can. J . Z o o l . 4 0 : 8 1 7 - 8 6 2 . S h e l t o n , G. and R a n d a l l , D.J. ( 1 9 6 2 ) . The R e l a t i o n s h i p b e t w e e n h e a r t b e a t and r e s p i r a t i o n i n t e l e o s t f i s h . Comp. B i o c h e m . P h y s i o l . 7: 2 3 7 - 2 5 0 . S h e l t o n , G. and D.R. J o n e s . ( 1 9 6 5 ) . C e n t r a l b l o o d p r e s s u r e and h e a r t o u t p u t i n s u r f a c e d and s u b m e r g e d f r o g s . J . Exp. B i o l . 4 2 : 33 9 - 3 5 7 . - 80 -S m i t h , L.S. and G.R. B e l l . ( 1 9 6 3 ) . A t e c h n i q u e f o r p r o l o n g e d b l o o d s a m p l i n g i n f r e e - s w i m m i n g s a l m o n . J . F i s h . R es. Bd. Canada. , 2 1 ( 4 ) : 7 11-717. S t e e n , J.B. and A. K r u y s s e . ( 1 9 6 4 ) . The r e s p i r a t o r y f u n c t i o n o f t e l e o s t e a n g i l l s . Comp. B i o c h e m . P h y s i o l . 1_2_: 127-142. W o l f , K. ( 1 9 6 3 ) . P h y s i o l o g i c a l s a l i n e s f o r f r e s h w a t e r t e l e o s t s . P r o g r e s s i v e F i s h C u l t u r i s t , 2 5 ( 3 ) : 135-140.
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Factors influencing gas exhange in the rainbow trout (Salmo gairdneri)
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