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UBC Theses and Dissertations

A study of the cardiovascular system of the rainbow trout (Salmo gairdneri) at rest and during swimming… Daxboeck, Charles 1981

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A STUDY OF THE CARDIOVASCULAR SYSTEM OF THE RAINBOW TROUT (Salmo gairdneri) AT REST AND DURING SWIMMING EXERCISE \^V*^y C h a r l e s Daxboeck B . S c , The U n i v e r s i t y o f T o r o n t o , 1975 M . S c , The U n i v e r s i t y o f T o r o n t o , 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (The Department o f Zool o g y ) We a c c e p t t h i s t h e s i s as con f o r m i n g t o t he r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA by June 1981 QCharles Daxboeck, 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 DE-6 (2/79) i i ABSTRACT The e f f e c t s o f s t e a d y - s t a t e , a e r o b i c swimming e x e r c i s e upon b l o o d volume and f l o w d i s t r i b u t i o n i n the rainbow t r o u t {Salmo gairdneri) were examined. I s o t o p i c Rubidium-86, and r a d i o l a b e l l e d m i c r o s p h e r e s were i n j e c t e d i n t o t r o u t f o r c e d to swim a g a i n s t a c u r r e n t a t 80% o f t h e i r c r i t i c a l v e l o c i t y ( U c r ^ t ) i n a B r e t t - t y p e water t u n n e l r e s p i r o m e t e r . The r e s u l t s g a t h e r e d from experiments u s i n g t h e s e r a d i o a c t i v e t r a c e r s w i t h i n the c i r c u l a t o r y system o f t he t r o u t i n d i c a t e d t h a t b l o o d f l o w d u r i n g e x e r c i s e was r e d i s t r i b u t e d t o f a v o u r working m u s c l e s , a t the expense o f d i m i n i s h e d b l o o d f l o w t o t h o s e organs and t i s s u e s i n the s y s t e m i c c i r c u l a t i o n which c o u l d t o l e r a t e p e r i o d s o f t r a n s i e n t h y p o x i a . A c t i v e hyperaemia i n the s k e l e t a l muscle and v a s o c o n s t r i c -t i o n o f the c o e l i a c o m e s e n t e r i c a r t e r y , v i a a d r e n e r g i c r e c e p t o r mechanisms a r e proposed as the main s i t e s o f the c o n t r o l f o r b l o o d volume and f l o w r e d i s t r i b u -t i o n i n the s y s t e m i c c i r c u l a t i o n i n t r o u t d u r i n g e x e r c i s e . The g i l l s o f t h e s e f i s h must be a b l e t o m a i n t a i n adequate gas t r a n s f e r i n o r d e r t o keep pace w i t h the i n c r e a s e d m e t a b o l i c demands o f the working muscles d u r i n g e x e r c i s e . An i s o l a t e d , s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n and a s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d whole t r o u t p r e p a r a t i o n were d e v e l o p e d i n o r d e r to s t u d y how i n c r e a s e s i n the p u l s a t i l i t y o f i n p u t and i n c r e a s e s i n the c a r d i a c o u t p u t through t h e s e g i l l s ; c a r d i o -v a s c u l a r a l t e r a t i o n s known t o o c c u r d u r i n g e x e r c i s e in vivo i n t h e s e f i s h , a f f e c t f l u i d f l o w d i s t r i b u t i o n t h r o u g h , and w i t h i n the b r a n c h i a l v a s c u l a t u r e , and gas exchange a c r o s s the g i l l s . Data from t h e s e p r e p a r a t i o n s i n d i c a t e d t h a t p u l s a t i l i t y o f f l o w i n c r e a s e d v e n o l y m p h a t i c f l u i d d r a i n a g e from w i t h i n g i l l t i s s u e s , as w e l l i n c r e a s i n g the f l u i d f l u x / r e f l u x a c r o s s the b r a n c h i a l m i c r o v a s c u l a t u r e . However, t h e s e changes i n f l u i d d i s t r i b u t i o n a s s o c i a t e d w i t h i n c r e a s e d p u l s e p r e s s u r e d i d n o t s i g n i f i c a n t l y change the r a t e o f gas t r a n s f e r a c r o s s the g i l l s . A l t h o u g h g i l l v a s c u l a r r e s i s t a n c e s to fl o w were 111 v e r y s e n s i t i v e t o a l t e r a t i o n s i n p u l s e p r e s s u r e and f l o w r a t e , o n l y p e r f u s i o n f l o w r a t e t h r o u g h the g i l l s c o u l d cause s i g n i f i c a n t changes i n the r a t e o f mass t r a n s f e r o f gases a c r o s s the g i l l s . The g i l l s o f t r o u t t h e r e f o r e were found t o be p e r f u s i o n and n o t d i f f u s i o n l i m i t e d f o r gas t r a n s f e r , under c o n d i t i o n s which s i m u l a t e d t h o s e found a t . r e s t and d u r i n g e x e r c i s e , in vivo. I t a l s o was shown t h a t , g i v e n oxygen uptake and c a r d i a c output:.data from the l i t e r a t u r e , combined w i t h t h o s e f o r b l o o d f l o w r e d i s t r i b u t i o n d u r i n g e x e r c i s e from the p r e s e n t s t u d y , the working m u s c l e s , which were o p e r a t i o n a l d u r i n g s t e a d y - s t a t e , a e r o b i c swimming e x e r c i s e i n rainbow t r o u t , c o u l d a c c o u n t f o r n e a r l y a l l the measured i n c r e a s e i n the t o t a l oxygen uptake a t t h i s l e v e l o f e x e r c i s e . The c i r c u l a t o r y system o f the rainbow t r o u t , both b r a n c h i a l and s y s t e m i c , was shown to.be q u i t e e f f i c i e n t i n i t s a b i l i t y t o take up and d i s t r i b u t e oxygen t o the t i s s u e s d u r i n g p r o l o n g e d , a e r o b i c swimming e x e r c i s e . The numerous c a r d i o r e s p i r a t o r y adjustments noted d u r i n g e x e r c i s e a c c o u n t f o r t h i s a n i m a l ' s a b i l i t y t o m a i n t a i n swimming a c t i v i t y i n the f a c e o f i n c r e a s e d oxygen demands put upon the c i r c u l a t i o n by e n f o r c e d a c t i v i t y . I V TABLE OF CONTENTS Page L i s t o f T a b l e s v i i i L i s t o f F i g u r e s x ACKNOWLEDGEMENTS — x i i GENERAL INTRODUCTION. 1 MATERIALS AND METHODS THE FISH 6 SWIMMING EXERCISE: The Apparatus and Method o f Performance Assessment .. 6 THE EXPERIMENTS. I. CARDIAC OUTPUT AND BLOOD VOLUME DISTRIBUTION P r e p a r a t i o n 9 Ex p e r i m e n t a l P r o t o c o l 10 L i q u i d S c i n t i l l a t i o n C o u n t i n g (LSC) T i s s u e S e l e c t i o n and P r e p a r a t i o n 11 P r e c a u t i o n s and C o r r e c t i o n s f o r L i q u i d S c i n t i l l a t i o n C o u n t i n g . . 13 Y - C o u n t i n g and R a d i o l a b e l l e d M i c r o s p h e r e s • • • 1 5 T i s s u e P r e p a r a t i o n and y - C o u n t i n g .18 V a l i d i t y o f T r a c e r Data 31 I I . ISOLATED, SALINE-PERFUSED TROUT HEADS 34 P e r f u s a t e and P e r f u s i o n 38 Ex p e r i m e n t a l P r o t o c o l 38 F l u i d Exchanges i n G i l l T i s s u e s 40 In vivo Marker T e s t s 40 Ethanol Washout Curves 41 I I I . SPONTANEOUSLY VENTILATING, BLOOD-PERFUSED TROUT PREPARATION A. CHARACTERIZATION OF THE PREPARATION Blood C o l l e c t i o n and P r e p a r a t i o n . . . . 44 S u r g i c a l P r o c e d u r e s . 45 Blood P e r f u s i o n 49 V Page B l o o d Sampling and A n a l y s i s 49 P r e s s u r e and Flow R e c o r d i n g . . 52 B. EFFECTS OF HAEMODYNAMIC CHANGES ON VASCULAR RESISTANCES TO FLOW, AND OXYGEN UPTAKE. 53 D i r e c t Measurement o f Oxygen Uptake ,... and V e n t i l a t o r y Flow A c r o s s t he G i l l s : A Comparison o f C a r d i a c Output by . D i r e c t and F i c k Methods... 54 SECTION I: BLOOD VOLUME AND FLOW DISTRIBUTION AT REST AND DURING EXERCISE IN THE RAINBOW TROUT INTRODUCTION I 58 RESULTS I Blo o d Volume and Flow D i s t r i b u t i o n . . . 59 E x h a u s t i o n 65 DISCUSSION I .. .. 69 SECTION I I : VASCULAR RESISTANCE TO FLOW IN SALINE- AND BLOOD-PERFUSED PREPARATIONS OF RAINBOW TROUT INTRODUCTION 80 RESULTS II E f f e c t s o f P u l s a t i l i t y o f Input on R e s i s t a n c e .to Flow, and F l u i d D i s t r i b u t i o n i n the G i l l s o f S a l i n e -P e r f u s e d T r o u t Heads... — 82 S t u d i e s o f G i l l B l o o d Flow i n a S p o n t a n e o u s l y V e n t i l a t i n g , B l o o d - P e r f u s e d T r o u t P r e p a r a t i o n : C h a r a c t e r i z a t i o n o f the P r e p a r a t i o n 83 C a r d i a c Output (Q) Changes 90 C a r d i a c Pump Frequency ( f ) and S t r o k e Volume (SV) M a n i p u l a t i o n s . 90 P u l s e P r e s s u r e (PP), Changes 92 Ha e m a t o c r i t (Hct) Changes.. 92 A d r e n a l i n e Exposure 95 • DISCUSSION I I : The E f f e c t s o f Haemodynamic A l t e r a t i o n s on Flow D i s t r i b u t i o n and B r a n c h i a l V a s c u l a r R e s i s t a n c e to Flow i n S a l i n e - P e r f u s e d T r o u t Head P r e p a r a t i o n s .....97 VI Page The S p o n t a n e o u s l y V e n t i l a t i n g , B l o o d -P e r f u s e d Whole T r o u t P r e p a r a t i o n a) How r e p r e s e n t a t i v e i s i t o f In. vivo C o n d i t i o n s ? ....99 Dorsal A o r t i c P r e s s u r e - DAP 102 Ve n t r a l A o r t i c ( i n p u t ) P r e s s u r e - VAP 101 B r a n c h i a l V a s c u l a r R e s i s t a n c e t o Flow - R 103 y S y s t e m i c V a s c u l a r . R e s i s t a n c e t o Flow - R " 104 b) Haemodynamic A l t e r a t i o n s i n Blood-, , Pe r f u s e d T r o u t . P r e p a r a t i o n s : E f f e c t s on V a s c u l a r R e s i s t a n c e t o Flow.106 SECTION I I I : GAS TRANSFER ACROSS THE GILLS OF SALINE- AND BLOOD-PERFUSED PREPARATIONS OF RAINBOW TROUT INTRODUCTION I I I 110 RESULTS I I I The E f f e c t s o f Haemodynamic A l t e r a t i o n s on Gas Exchange i n the B l o o d - P e r f u s e d T r o u t P r e p a r a t i o n 112 The F i c k E q u a t i o n , and the D i r e c t Measurement o f V e n t i l a t i o n Volume - V^ 116 The S a l i n e - P e r f u s e d T r o u t Head P r e p a r a t i o n : F a c t o r s a f f e c t i n g gas t r a n s f e r a c r o s s the g i l l s . 122 A n a l y s i s o f Water F l u x i n G i l l T i s s u e s : I n f l u x and E q u i l i b r a t i o n o f Ethanol (EtOH) 124 Compartmental A n a l y s i s o f EtOH Washout Curves.. 132 DISCUSSION I I I Gas Exchange i n the S p o n t a n e o u s l y V e n t i l a t i n g , B l o o d -P e r f u s e d T r o u t P r e p a r a t i o n : Comparisons w i t h in vivo d a t a . Blood Gas T e n s i o n s and C o n t e n t s : Input B l o o d 140 Dorsal A o r t i c ( a r t e r i a l ) B l o o d . 141 Oxygen Uptake A c r o s s the G i l l s - H O , 141 VI 1 Page Oxygen Uptake A c r o s s the Sys t e m i c C i r c u l a t i o n - M ^ . . . 142 Carbon D i o x i d e E x c r e t i o n A c r o s s t he G i l l s , and. P r o d u c t i o n by S y s t e m i c T i s s u e s . 142 Blood C h e m i s t r y . . 143 V e n t i l a t i o n Rate . , 143 Haemodynamic A l t e r a t i o n s and T h e i r E f f e c t s on Gas Exchange. .. 144 A p p l i c a b i l i t y o f the P i c k P r i n c i p l e . 148 Compartmental A n a l y s i s o f Washout Curves. 153 T o t a l Amount o f Ethanol Washed from G i l l T i s s u e s 154 SECTION IV: OXYGEN TRANSPORT DURING EXERCISE IN THE RAINBOW TROUT INTRODUCTION .IV 161 RESULTS AND DISCUSSION IV...; 167 LITERATURE CITED 174 APPENDIX A: EFFECTS OF CORONARY ARTERY ABLATION ON EXERCISE PERFORMANCE IN RAINBOW TROUT 188 APPENDIX B: A MODIFIED PUMP: M o d i f i c a t i o n s o f a P i s t o n - T y p e P e r f u s i o n Pump f o r D e l i v e r y o f Low Flow R a t e s . . . . . . 203 APPENDIX C: THE SPONTANEOUSLY VENTILATING, BLOOD-PERFUSED TROUT PREPARATION in situ 209 BIOGRAPHICAL INFORMATION. , 210 v i i i LIST OF TABLES Page TABLE 1. A n a l y s i s o f Vancouver tap water 7 TABLE 2. L i s t o f t i s s u e s sampled from rainbow t r o u t , t o determine c a r d i a c o u t p u t d i s t r i b t u i o n a t r e s t and d u r i n g e x e r c i s e , u s i n g Rb-86. .. 12 TABLE 3. Assessment o f c r o s s - i n t e r f e r e n c e i n energy s p e c t r a from the t h r e e i s o t o p e s counted s i m u l t a n e o u s l y i n t i s s u e samples from r e s t i n g and.swimming rainbow t r o u t 21 TABLE 4. B l o o d volume d i s t r i b u t i o n t o v a r i o u s t i s s u e s from n o r m a l , r e s t i n g rainbow t r o u t .61 TABLE 5. B l o o d volume d i s t r i b u t i o n t o v a r i o u s t i s s u e s from rainbow t r o u t swimming a t 80% U .. .62. . c n t TABLE 6. Pe r c e n t a g e o f c a r d i a c o u t p u t t o v a r i o u s t i s s u e s from rainbow t r o u t a t . r e s t , , e x p r e s s e d as p e r c e n t a g e o f t o t a l i n j e c t e d cpm 1 s. ; .. .63 TABLE 7. Percentage o f c a r d i a c o u t p u t t o v a r i o u s t i s s u e s from rainbow t r o u t swimming a t 80% IT .., e x p r e s s e d as p e r c e n t -age o f t o t a l . i n j e c t e d , cpm 1 s r. £ 6 TABLE 8. Summary o f d o r s a l a o r t i c p r e s s u r e (DAP) and h e a r t r a t e changes d u r i n g 80% U ... swimming e x e r c i s e i n rainbow t r o u t <:*]:..• £ 7 TABLE 9. Pe r c e n t a g e c a r d i a c o u t p u t t o v a r i o u s t i s s u e s from rainbow t r o u t swimming a t 80% U .. and a t p o i n t o f e x h a u s t i o n , e x p r e s s e d as percentage Co"f t o t a l i n j e c t e d cpm's £ 8 TABLE 10. Summary o f d o r s a l a o r t i c and a n t e r i o r venous o u t f l o w from the i s o l a t e d t r o u t head (Salmo gairdnevi) p r e p a r a t i o n d u r i n g p e r f u s i o n by t h r e e d i f f e r e n t i n p u t regimes £2a TABLE 11. Summary o f v a r i a b l e s f o r t h e n o r m a l , r e s t i n g s t a t e o f the s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d rainbow t r o u t a t 7 ° C £ 8 TABLE 12. Summary o f the d i f f e r e n c e s i n v a r i a b l e s a c r o s s t he g i l l ( A i n p u t - d o r s a l a o r t a ) and the s y s t e m i c ;(Adorsal a o r t i c -venous r e t u r n ) c i r c u l a t i o n s i n the r e s t i n g s t a t e o f s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d rainbow t r o u t , a t 7 ? C . . . .8.9 TABLE 13. Summary o f the e f f e c t s o f i n c r e a s e d and d e c r e a s e d c a r d i a c o u t p u t on c a r d i o v a s c u l a r v a r i a b l e s from normal, s p o n t a n e o u s l y v e n t i 1 a t i ng, b l o o d - p e r f u s e d t r o u t 51 TABLE 14. E f f e c t s o f c a r d i a c o u t p u t (Q), s t r o k e volume ( S V ) , p u l s e p r e s s u r e (PP) and c a r d i a c f r e q u e n c y ( f ) on g i l l r e s i s t a n c e change (AR % ) , i n r e s t i n g , b l o o d - p e r f u s e d t r o u t 93 i x LIST OF TABLES Page TABLE 1 5 . Summary o f the e f f e c t s o f v a r i a b l e i n p u t h a e m a t r o c r i t on c a r d i o v a s c u l a r parameters from normal s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t . 94 —6 TABLE 16. E f f e c t s o f 1 x 10" M a d r e n a l i n e on s e l e c t e d v a r i a b l e s i n b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n s 96 TABLE 17. Summary o f the e f f e c t s o f i n c r e a s e d and d e c r e a s e d c a r d i a c o u t p u t on b l o o d gases from normal., s p o n t a n e o u s l y v e n t i l a t i n g b l o o d - p e r f u s e d t r o u t . . . . . . . . . . 113 TABLE 18. Summary o f , t h e e f f e c t s o f v a r i a b l e i n p u t h a e m a t o c r i t on b l o o d gases f o r normal, s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d -p e r f u s e d t r o u t , 117 TABLE 19 . E f f e c t s o f c a r d i a c o u t p u t ( Q ) , s t r o k e volume ( S V ) , p u l s e p r e s s u r e (PP) and c a r d i a c f r e q u e n c y ( f ) on oxygen uptake (MgO£) i n r e s t i n g , b l o o d - p e r f u s e d t r o u t . . . 120 TABLE 2 0 . Summary o f the c a r d i o r e s p i r a t o r y v a r i a b l e s from normal s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t .in a m o d i f i e d van Dam a p p a r a t u s . 121 TABLE 2 1 . Summary o f gas exchange dat a i n the i s o l a t e d , s a l i n e -p e r f u s e d head o f S. gairdneri.. 123 TABLE 2 2 . E t h a n o l c o n c e n t r a t i o n s i n . w a t e r , d o r s a l a o r t i c p e r f u s a t e and a n t e r i o r venous p e r f u s a t e 129 TABLE 2 3 . Summary o f c a r d i o r e s p i r a t o r y changes a s s o c i a t e d w i t h submaxi mal, a e r o b i c , e x e r c i s e i n man and f i s h . . . 163 - 1 - 1 TABLE 2 4 . C a l c u l a t i o n o f muscle and s y s t e m i c b l o o d f l o w ('ml• min • kg ) d u r i n g r e s t and e x e r c i s e i n rainbow t r o u t 171 TABLE 2 5 . Oxygen uptake and d i s t r i b u t i o n to the s y s t e m i c c i r c u l a t i o n o f r e s t i n g and e x e r c i s i n g r a i n b o w . t r o u t 172 TABLE 2 6 . I n c r e a s e i n oxygen uptake by the whole f i s h , and by r e d muscle a t r e s t and d u r i n g e x e r c i s e a t 8 0 1 U £ ^ 173 TABLE 2 7 . E f f e c t s o f c o r o n a r y a r t e r y a b l a t i o n on swimming e x e r c i s e performance i n rainbow t r o u t 193 X LIST OF FIGURES Page FIGURE 1. Schematic r e p r e s e n t a t i o n o f t h e m i c r o s p h e r e i n j e c t i o n assembly 19 FIGURE 2. Energy spectrum d e r i v e d from a subsample o f Chromium-51 s t o c k s o l u t i o n used t o l a b e l r e d b l o o d c e l l s ...22 FIGURE 3. Energy spectrum d e r i v e d from a subsample o f Cerium-141-l a b e l l e d m i c r o s p h e r e s t o c k s u s p e n s i o n 24 FIGURE 4. Energy spectrum d e r i v e d from a subsample o f Niobium-SB-l a b e l l e d m i c r o s p h e r e s t o c k s u s p e n s i o n . . — ... 26 FIGURE 5. The d e c r e a s e i n known a c t i v i t y (100%) w i t h i n c r e a s i n g h e i g h t o f water added to s o u r c e , above bottom o f the gamma-counting tube. -28 FIGURE 6. Schematic r e p r e s e n t a t i o n o f the e x p e r i m e n t a l a p p a r a t u s used i n i s o l a t e d , s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n s . . . 36 FIGURE 7. Schematic c r o s s - s e c t i o n ^ t h r o u g h a secondary l a m e l l a and -f i l a m e n t o f - a t e l e o s t g i l l 42 FIGURE 8. Schematic s a g i t t a l s e c t i o n through t he h e a r t o f a t r o u t 47 FIGURE 9. Schematic r e p r e s e n t a t i o n o f the i n s t r u m e n t a t i o n used t o m o n i t o r v a r i a b l e s from b l o o d - p e r f u s e d t r o u t . ... .50 FIGURE 10. D i a g r a m a t i c r e p r e s e n t a t i o n of. t he m o d i f i e d van Dam a p p a r a t u s used i n the p r e s e n t e x p e r i m e n t s . 56 FIGURE 11. Summary o f the p o s s i b l e s i t e s o f d i r e c t n e u r a l , and c i r c u l a t i n g c a t e c h o l a m i n e a c t i o n i n the c i r c u l a t o r y system o f Salmo gairdneri....... ........'. 78 FIGURE 12. Records o f p r e s s u r e s and f l o w from s p o n t a n e o u s l y ven-t i l a t i n g , bloods-perfused t r o u t . . . . . . . 85 FIGURE 13. The e f f e c t s o f changes i n c a r d i a c o u t p u t on oxygen uptake and p^^2» a n c- carbon d i o x i d e e x c r e t i o n and P a ^ 2 a c r o s s the g i l l s o f s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t . 114 FIGURE 14. The e f f e c t o f v a r i a b l e i n p u t h a e m a t o c r i t on oxygen uptake a c r o s s the g i l l s o f s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d -p e r f u s e d t r o u t . . . . 118 FIGURE 15. T y p i c a l e t h a n o l c o n c e n t r a t i o n s i n the wat e r , d o r s a l a o r t i c p e r f u s a t e and a n t e r i o r venous p e r f u s a t e d u r i n g l o a d i n g o f g i l l t i s s u e s 125 FIGURE 16. R e p r e s e n t a t i o n o f the dat a p r e s e n t e d i n F i g . 15, . n o r m a l i z e d t o the f i n a l c o n c e n t r a t i o n s i n p e r f u s a t e a f t e r 15 min.. 127 XI LIST OF FIGURES Page FIGURE 17. Dorsal a o r t i c e t h a n o l (EtOH) c o n c e n t r a t i o n s d u r i n g washout from g i l l t i s s u e s 130 FIGURE 18. Record o f i n p u t p r e s s u r e d u r i n g p u l s a t i l e c o n s t a n t f l o w p e r f u s i o n . 133 FIGURE 19. A n t e r i o r venous e t h a n o l c o n c e n t r a t i o n s d u r i n g washout from g i l l t i s s u e s 135 FIGURE 20. Two-compartment models i l l u s t r a t i n g the r e s u l t s o f the a n a l y s i s o f the d o r s a l a o r t i c e t h a n o l washout c u r v e s d u r i n g p u l s a t i l e c o n s t a n t f l o w p e r f u s i o n and non-p u l s a t i l e c o n s t a n t p e r f u s i o n . . . . . 138 FIGURE 21. H y p o t h e t i c a l e t h a n o l c o n c e n t r a t i o n , p r o f i l e a c r o s s t he r e s p i r a t o r y b a r r i e r o f the g i l l s 155 FIGURE 22. Photographs o f sequence o f e v e n t s d u r i n g . t h e o p e r a t i o n where the c o r o n a r y a r t e r y was a b l a t e d . . 190 FIGURE 23. L o g a r i t h m i c - p r o b i t p l o t , o f c r i t i c a l swimming v e l o c i t y versus p e r c e n t f i s h f a t i g u e d a t t h a t v e l o c i t y . 194 FIGURE 24. L i n e a r r e p r e s e n t a t i o n o f , t h e r e l a t i o n s h i p between c r i t i c a l swimming v e l o c i t y (U .,) and f o r k l e n g t h , f o r i n t a c t and o p e r a t e d f i s h . . . rH? 196 FIGURE 25. C r o s s - s e c t i o n s through i n t a c t and o p e r a t e d bulbus ^ a r t e r i o s u s o f rainbow t r o u t . . .........199 FIGURE 26. M o d i f i e d clamping b l o c k and s y r i n g e b a r r e l assembly 205 FIGURE 27. Remote pumping head and c h e c k i n g mechanism f o r remote head..207 FIGURE 28. Photographs o f the a p p a r a t u s as used d u r i n g experiments c o n d u c t e d on s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t ...209 x i i ACKNOWLEDGEMENTS * D e d i c a t e d t o t h e memory o f Dr. George F r a n c i s H o l e t o n -a man who t a u g h t me t o "see t h i n g s which o t h e r s saw i n a way which o t h e r s d i d n ' t * I wish t o thank my r e s e a r c h s u p e r v i s o r , Dr. D.J. R a n d a l l , f o r h i s encouragement and a s s i s t a n c e t h r o u g h o u t my; work, and f o r s u f f e r i n g t h r o u g h the w r i t i n g o f the t h e s i s w i t h me. Dr. Steve F. P e r r y i s thanked f o r h i s c o n t i n u i n g f r i e n d s h i p . The b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n was d e v e l o p e d i n c o l l a b o r a t i o n w i t h Steve and Dr. P e t e r S. D a v i e . I t would have been i m p o s s i b l e even t o attempt t h i s work w i t h o u t t h e s e two i n d i v i d u a l s . As w e l l , P e t e r D a v i e was i n s t r u m e n t a l i n h e l p i n g d e v e l o p the s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n s and e x p e r i m e n t s . A g a i n , thank you Pete. K e l l y B a r n a r d , K r i s Wesbury and Tom Heming a r e thanked f o r t h e i r a s s i s t a n c e ' w i t h the c o r o n a r y a r t e r y a b l a t i o n / e x e r c i s e e x p e r i m e n t s . Drs. D.R. Jones and D.M. Hudson a l s o d e s e r v e a note o f thanks f o r t h e i r h e l p f u l a d v i s e w i t h the m i c r o s p h e r e s t u d i e s . I s i n c e r e l y a p p r e c i a t e the most u s e f u l comments and c r i t i c i s m s o f the t h e s i s from a l l members o f both my r e s e a r c h and examining committee. The f i n a l p r o d u c t w i t h o u t doubt b e n e f i t t e d from t h e s e c o n t r i b u t i o n s . H e a r t - f e l t tharks a l s o go t o a l l academic, non-academic and graduate s t u d e n t members, p a s t and p r e s e n t , who o v e r the y e a r s , have been a h e l p t o me i n numerous ways. These p e o p l e have made the Department o f Z o o l o g y a p l e a s u r e to be a s s o c i a t e d w i t h . Sandi i s thanked f o r her l o v e , u n d e r s t a n d i n g , t o l e r a n c e and encouragement t h r o u g h o u t the c o m p l e t i o n o f my r e s e a r c h and the w r i t i n g o f the t h e s i s . L a s t , but by no means l e a s t , I would l i k e t o thank by l o v i n g x i v p a r e n t s K a r l and T h e r e s i a , f o r the moral s u p p o r t and i n c r e d i b l e u n d e r s t a n d i n g t h e y have shown me ov e r a l l the y e a r s o f my " p r o f e s s i o n a l s t u d e n t s h i p " . I'm f i n a l l y f i n i s h e d ! W e l l , I guess I ' l l g e t a j o b now and s t a r t working f o r a 1 i v i n g . F i n a n c i a l s u p p o r t f o r my r e s e a r c h was p r o v i d e d through N.S.E.R.C. g r a n t s t o Dr. D.J. R a n d a l l . The Department o f Zool o g y p r o v i d e d t he f a c i l i t i e s t o conduct my e x p e r i m e n t s . I was f i n a n c i a l l y s u p p o r t e d through t e a c h i n g a s s i s t a n t s h i p s from the Department o f Z o o l o g y , U.B.C. Summer Graduate Research S c h o l a r s h i p s and N.S.E.R.C. Summer Research A s s i s t a n t s h i p s through D.J.R. GENERAL I N T R O D U C T I O N 1 GENERAL INTRODUCTION I n c r e a s e d s k e l e t a l muscle a c t i v i t y , o r e x e r c i s e , i s the most commonly e n c o u n t e r e d form o f s t r e s s i n most v e r t e b r a t e s . E x e r c i s e g e n e r a l l y i s a s s o c i a t e d w i t h i n c r e a s e s i n c a r d i a c o u t p u t , v e n t i l a t i o n volume and oxygen consumption. The c a r d i o r e s p i r a t o r y system o f mammals a d j u s t s t o mai n t a i n an adequate s u p p l y o f oxygen t o , and f o r the removal o f waste p r o d u c t s from the m e t a b o l i z i n g t i s s u e s d u r i n g s t e a d y - s t a t e e x e r c i s e c o n d i -t i o n s . B l o o d f l o w to these s k e l e t a l muscles.can i n c r e a s e 10 to 1 5 - f o l d d u r i n g maximal e x e r c i s e , w h i l e c a r d i a c o u t p u t i n c r e a s e s by o n l y f i v e - f o l d (Folkow and N e i l , 1971). There must be con c o m i t a n t b l o o d f l o w r e d i s t r i b u -t i o n elsewhere i n the body. The s p l a n c h n i c and r e n a l c i r c u l a t i o n i n man a t r e s t r e c e i v e s as much as 251 o f the t o t a l c a r d i a c o u t p u t (Rowell et a l . 3 1964; R o w e l l , 1974). During m i l d e x e r c i s e , the m e s e n t e r i c b l o o d f l o w d e c r e a s e s ( E k l u n d , 1967; Scher et a l . , 1972) and t h e r e f o r e , t h i s s p l a n c h n i c c i r c u l a t o r y b l o o d " p o o l " i s c o n s i d e r e d t o be the major s i t e o f c o n t r o l f o r b l o o d r e d i s t r i b u t i o n and a r t e r i a l b l o o d p r e s s u r e r e g u l a t i o n d u r i n g e x e r c i s e . Mean a r t e r i a l b l o o d p r e s s u r e i s i n c r e a s e d d u r i n g e x e r c i s e , even i n the f a c e o f an almost 50% d e c r e a s e i n the t o t a l p e r i p h e r a l r e s i s t a n c e to b l o o d f l o w once a s t e a d y - s t a t e c o n d i t i o n i s . a t t a i n e d . I n c r e a s e s i n the h e a r t r a t e d u r i n g t h i s e x e r c i s e s t a t e a r e p r o p o r t i o n a l t o the d e 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 ( B e v e r g a r d and Shephard, 1967). Numerous s t u d i e s have been conduct e d to e v a l u a t e . t h e e f f e c t s o f swimming e x e r c i s e on the c a r d i o r e s p i r a t o r y and m e t a b o l i c r e s p o n s e s o f f i s h e s t o t h i s n a t u r a l l y e n c o u n t e r e d form o f s t r e s s (see Fish Physiology, V o l . 7. W.S. Hoar and D.J. R a n d a l l , eds. Academic P r e s s , New York, f o r r e v i e w ) . In f i s h e s , some o f the p o s s i b l e l i m i t i n g f a c t o r s o f maximum oxygen consumption d u r i n g e x e r c i s e a r e : the r a t e and e f f i c i e n c y o f oxygen uptake a c r o s s the g i l l s , the r a t e o f oxygen d e l i v e r y t o the t i s s u e s by the c i r c u l a t i n g b l o o d , 2 or the r a t e and e f f i c i e n c y o f oxygen e x t r a c t i o n by the wor k i n g muscles. E x e r c i s e can be c a t e g o r i z e d i n t o b u r s t a c t i v i t y , and p r o l o n g e d e x e r c i s e a t maximal o r submaximal energy e x p e n d i t u r e . R e g a r d l e s s o f the type o f e x e r c i s e p e r f o r m e d , i t can be d i v i d e d f u r t h e r i n t o i n i t i a t i o n , s t e a d y - r s t a t e and r e c o v e r y phases. A l t h o u g h some o f the energy budget d u r i n g p r o l o n g e d , s t e a d y - s t a t e e x e r c i s e may be p r o v i d e d by a n a e r o b i o s i s , i t s c o n t r i b u t i o n has been" found t o be minimal i n salmonids- ( D r i e d z i c and K i c e n i u k j 19-76). There a l s o may be a l a r g e r r e l a t i v e a n a e r o b i c phase a t the o n s e t o f e x e r c i s e . , b ut once a s t e a d y - s t a e c o n d i t i o n i s e s t a b l i s h e d , n e a r l y , a l l m e t a b o l i s m i s a e r o b i c . The f i r s t s e c t i o n o f t h i s s t u d y examines the e f f e c t s o f p r o l o n g e d a e r o b i c e x e r c i s e on t h e c i r c u l a t o r y system.as a whole, i n v e s t i g a t i n g changes i n b l o o d volume and f l o w i n v a r i o u s t i s s u e s from r e s t , and t h e i r p o s s i b l e means o f r e g u l a t i o n . No s t u d i e s t o date however, have i n v e s t i g a t e d the e f f e c t s o f d i f f e r e n t p e r f u s i o n regimes on v a s c u l a r f l u i d d i s t r i b u t i o n i n the i n t a c t g i l l s and head o f t e l e o s t f i s h i n d e t a i l , a l t h o u g h the e f f e c t s o f p u l s e p r e s s u r e , mean p r e s s u r e and p e r f u s i o n f l o w on g i l l v a s c u l a r . r e s i s t a n c e i n i s o l a t e d h o l o b r a n c h s . have been s t u d i e d ( F a r r e l l et al:.,, 1979). S i m i l a r l y , the dynamics o f f l u i d f l o w through the r e c u r r e n t o r veno l y m p h a t i e c i r c u l a t i o n i n t e l e o s t g i l l s have n o t been examined to any e x t e n t . The anatomy o f the r e c u r r e n t b l o o d v e s s e l system however, has been d e s c r i b e d i n some d e t a i l . (Gannon e t a l . , 1973; L a u r e n t and Dunel, 1976; Vogel et al. , 1976; F a r r e l l , 1979). These d e s c r i p t i o n s i n d i c a t e t h a t the venolymphatie s i n u s compart-ment o f the g i l l s c o u l d c o n t a i n as much as 12% o f the t o t a l b l o o d volume o f the f i s h . Low h a e m a t o c r i t s have been r e p o r t e d f o r . t h i s r e c u r r e n t b l o o d space (Hughes and Wr i g h t , 1970; Booth, 1978). Plasma skimming a t the a n a s t o -moses between e f f e r e n t f i l a m e n t v e s s e l s and t h e r e c u r r e n t c i r c u l a t i o n c o u l d 3 account f o r the o b s e r v e d low h a e m a t o c r i t . How t h i s v e n o l y m p h a t i c / e x t r a -v a s c u l a r space behaves under d i f f e r e n t . p h y s i o l o g i c a l c o n d i t i o n s found in vivo a l s o has not been i n v e s t i g a t e d , a l t h o u g h p r e v i o u s s t u d i e s have i n d i c a t e d t h a t p u l s a t i l e p e r f u s i o n o f i s o l a t e d g i l l a r c h e s d e c r e a s e s v a s c u l a r r e s i s t a n c e t o f l o w . T h i s was thought t o be due, i n p a r t , t o i n -c r e a s e s i n ve n o l y m p h a t i c f l u i d c l e a r a n c e . ( F a r r e l T et al..., 1979).. In the second s e c t i o n , changes i n the g i l l c i r c u l a t i o n a r e d i s c u s s e d i n d e t a i l . In p a r t i c u l a r , I.examine the e f f e c t s o f changes i n p r e s s u r e and f l o w on g i l l v a s c u l a r r e s i s t a n c e and f l u i d d i s t r i b u t i o n d u r i n g e x e r c i s e . Gas exchange i n f i s h g i l l s has been examined i n some d e t a i l (see R a n d a l l , 1979). There a l s o have been e x t e n s i v e s t u d i e s , o f oxygen consumption o f i n t a c t f i s h (see. Brett:, 1972), and measurements o f oxygen uptake and gas t r a n s f e r a c r o s s t he g i l l s a r e r e l a t i v e l y common (see F i s h e r et'al..., 1969; Cameron and D a v i s , 1970; Davis and Cameron, 1971). From t h e s e s t u d i e s , f i s h g i l l s a r e assumed to be p r i m a r i l y d i f f u s i o n . l i m i t e d : f o r oxygen uptake. Because o f t h i s assumption, i t has been s u g g e s t e d t h a t c a r d i o r e s p i r a t o r y adjustments d u r i n g e x e r c i s e a re f o r the purpose o f d e c r e a s i n g the d i f f u s i o n b a r r i e r f o r ga s e s , such t h a t oxygen uptake, as w e l l as carbon d i o x i d e e x c r e -t i o n , are m a i n t a i n e d a t l e v e l s s u f f i c i e n t t o s u p p l y the a e r o b i c m e t a b o l i c r e q u i r e m e n t s . o f the f i s h d u r i n g swimming a c t i v i t y . C a r d i a c o u t p u t and v e n t i l a t i o n volume i n c r e a s e d u r i n g e x e r c i s e i n t r o u t . Oxygen : u p t a k e i s augmented by presumed d e c r e a s e s i n t h e d i f f u s i o n b a r r i e r and, t o a l e s s e r degree, g r e a t e r p e r f u s i o n o f a n . i n c r e a s e d r e s p i r a t o r y s u r f a c e a r e a , brought about by l a m e l l a r r e c r u i t m e n t (Jones and R a n d a l l , . 1978).. V e r t e b r a t e r e s p i r a t o r y v e s s e l s g e n e r a l l y a r e s u b j e c t e d t o b l o o d p r e s s u r e s which are much lower than i n any o t h e r p a r t o f the s y s t e m i c c i r c u l a t i o n . The c a p i l l a r y beds o f the r e s p i r a t o r y t i s s u e s o f f i s h g i l l s , the secondary l a m e l l a e , however, a r e e x c e p t i o n a l i n t h a t they have b l o o d 4 p r e s s u r e s which are a p p r e c i a b l y h i g h e r t h a n s y s t e m i c p r e s s u r e s (Johansen, 1972). These v e s s e l s a l s o have h i g h p u l s e p r e s s u r e s ( F a r r e l l , 1979); h i g h e r than i n any o t h e r b l o o d v e s s e l . o f s i m i l a r s i z e . . There a r e two d i s t i n c t b a r r i e r s t o f l u i d movement a c r o s s f i s h g i l l s ; the f i r s t o r b a s a l ( b l o o d - f a c i n g ) b a r r i e r , and the much more permeable w a t e r - f a c i n g b a r r i e r ( I s a i a et al.-., 1978). The s t r u c t u r e o f the blood/water, b a r r i e r o f f i s h g i l l s has been d e s c r i b e d i n d e t a i l by Hughes and Morgan (1973). B r i e f l y , an o u t e r e p i t h e l i a l c e l l l a y e r - s e p a r a t e s an i n t e r s t i t i a l f l u i d space from the e x t e r n a l environment. Beneath t h i s i n t e r s t i t i u m l i e s a basement membrane o v e r l y i n g the s u p p o r t i v e p i l l a r c e l l s o f the b l o o d s p a c e s . T h i s e p i t h e l i u m / basement m e m b r a n e / p i l l a r c e l l l a y e r complex i s r e f e r r e d t o as the f u n c t i o n a l r e s p i r a t o r y e p i t h e l i u m . Exchange o f f l u i d a c r o s s small v e s s e l s ( c a p i l l a r i e s and a l v e o l a r b l o o d v e s s e l s ) i s governed by a " S t a r l i n g e q u i l i b r i u m " (.1896), i n which f i l t r a t i o n r a t e i s p r o p o r t i o n a l t o the h y d r o s t a t i c p r e s s u r e o f the b l o o d and the p e r m e a b i l i t y o f the v e s s e l , w a l l ; ( L a n d i s and Pannenheimer, 1963). F i l t r a t i o n a c r o s s the w a l l a l s o has a p u l s a t i l e component, but i t s e f f e c t s a r e r e s t r i c t e d to a boundary l a y e r n e a r the b l o o d / v e s s e l w a l l i n t e r f a c e (Kenyon, 1979). Given t h a t f i s h l a m e l l a r b l o o d p r e s s u r e s a r e h i g h , compared to o t h e r v e r t e b r a t e r e s p i r a t o r y t i s s u e ( J o h a n s s e n , 1972), and the t h i c k n e s s o f the b l o o d / w a t e r b a r r i e r i s s m a l l , f l u i d movements a c r o s s the e n d o t h e l i u m o f f i s h g i l l s c o u l d be e x p e c t e d . t o be d i f f e r e n t from f l u i d movements a c r o s s o t h e r low p r e s s u r e c a p i l l a r y w a l l s . In S e c t i o n I I I , I,examine the e f f e c t s o f the changes i n p r e s s u r e and f l o w on gas t r a n s f e r a c r o s s . t h e g i l l s o f b l o o d - and s a l i n e - p e r f u s e d t r o u t . R e s u l t s from t h e s e s i m u l a t e d - e x e r c i s e , c a r d i o v a s c u l a r dynamics experiments l e a d . t o d i s c u s s i o n s o f t h e i r e f f e c t s on the d i f f u s i n g c a p a c i t y o f f i s h g i l l s d u r i n g e x e r c i s e , in vivo. 5 In the f i n a l s e c t i o n I have attempted t o b r i n g the r e s u l t s o f S e c t i o n I, II and I I I t o g e t h e r , to examine the r o l e o f the c i r c u l a t i o n i n gas t r a n s p o r t d u r i n g e x e r c i s e . The o b j e c t o f my s t u d y t h e r e f o r e i s t o examine v a r i o u s components o f the c a r d i o v a s c u l a r system o f the rainbow t r o u t (Salmo g a i r d n e r i ) , a t r e s t and d u r i n g s t e a d y - s t a t e , submaximal, a e r o b i c swimming e x e r c i s e . The adjustments and p o s s i b l e u n d e r l y i n g mechanisms r e s p o n s i b l e f o r the main-tenance o f adequate oxygen t r a n s f e r a c r o s s . t h e r e s p i r a t o r y o r g a n , the g i l l s , a r e i n v e s t i g a t e d . 5a M A T E R I A L S AND METHODS 6 MATERIALS AND METHODS THE FISH A l l e x p e r i m e n t s were c a r r i e d out on rainbow t r o u t {Salmo gairdnevi) o f both s e x e s , w e i g h i n g between 200 and 600 grams. F i s h were o b t a i n e d from the Sun V a l l e y T r o u t Farm, M i s s i o n , B.G. A f t e r t r a n s p o r t t o U.B.C, they were h e l d o u t d o o r s i n l a r g e f i b r e g l a s s tanks s u p p l i e d w i t h r u n n i n g , a e r a t e d and d e c h l o r i n a t e d Vancouver tap water (5 - 11°C, depending on s e a s o n ; see T a b l e 1 ) , and kept on ambient l i g h t c y c l e . F i s h were f e d a d a i l y d i e t o f d r i e d f i s h p e l l e t s ( M o o r e - C l a rke Co.) ad libitum, but were n o t f e d 24 hours b e f o r e o r d u r i n g e x p e r i m e n t a t i o n . A l l experiments were conducted a t the temperature t o which t he f i s h were a c c l i m a t e d . , SWIMMING EXERCISE: THE .APPARATUS AND METHOD OF PERFORMANCE ASSESSMENT . E x e r c i s e performance was a s s e s s e d f o r t r o u t d u r i n g s u s t a i n e d , a e r o b i c swimming a g a i n s t a water c u r r e n t i n a B r e t t (1964) r e s p i r o m e t e r / s w i m t u n n e l . The f i s h used f o r t h e s e e x p e r i m e n t s had been p r e - c o n d i t i o n e d t o swim by c o n t i n u o u s exposure t o a. mean water, v e l o c i t y o f 20 c m » s e c _ 1 ( z e r o c n r s e c - 1 i n c e n t r e ; 35-40 cm'sec" 1, a t edge) i n a l a r g e , c i r c u l a r h o l d i n g tank e q u i p -ped w i t h a pump. The time period.was never l e s s than 2 weeks. S e l e c t e d f i s h were i n t r o d u c e d i n t o the water t u n n e l through t he a c c e s s p o r t and a l l o w e d t o accommodate t o t h i s c o n f i n e m e n t f o r a t l e a s t 18 h a t a water v e l o c i t y o f a p p r o x i m a t e l y 8.7 cm*sec" 1, the lowes t speed on the pump, b e f o r e b e i n g t e s t e d . The r e s p i r o m e t e r / w a t e r t u n n e l i n c o r p o r a t e d a c y l i n d r i c a l P l e x i -g l a s s chamber (126.5 cm 2 c r o s s - s e c t i o n a l area) through which a v a r i a b l e -speed water pump c o u l d m a i n t a i n t he speed a t a d e s i r e d v a l u e (± 3 % ; " K i c e n i u k , 1975)... The water i n the p r e s e n t experiments was renewed a t a r a t e o f 1 L-min" 1. The temperature was c o n t r o l l e d (± 0.5°C) by a hea t exchanger system. The water t u n n e l used .in t he p r e s e n t experiments was i d e n t i c a l t o the one used by K i c e n i u k (1975), who g i v e s a d e t a i l e d d e s c r i p t i o n o f the 7 T a b l e 1. A n a l y s i s o f Vancouver tap water. S p r i n g 1971 A p r i l 1980 pH A l k a l i n i t y ( m g v l " 1 CaC0 3) (mg-L" 1) Mg 2 + (mg-L" 1) C o n d u c t i v i t y EDTA Hardness ( m g « L _ 1 CaC0 3) P 0 4 as P ( y g - L ^ 1 ) Fe ( y g . L - 1 ) N a + ( y g * m l _ 1 ) T o t a l O r g a n i c Carbon . (•yg.L- 1) 6.5 6.2 1 <1 23 3 0.1 c l <1 6.4 5.0 1 <1 17 5.4 3 0.1 <1 <1 C l ' ( v a r i a b l e between 0.015 - 0.03 y g . L " 1 ) 8 a p p a r a t u s . P a r t i a l p r e s s u r e o f oxygen o f the water f l o w i n g i n t o the r e s p i r o m e t e r was always g r e a t e r than o r equal t o 155 mm Hg. The c r i t i c a l v e l o c i t y swimming t e s t i s a measure o f e x e r c i s e performance. In the p r e s e n t e x p e r i m e n t s , where U ^ was d i r e c t l y d e t e r m i n e d , the f o r m u l a d e s c r i b e d by B r e t t (1964) was used as f o l l o w s : ^ c r i t = u l + ^ 1 ^ 2 x u 2 ^ ' w n e r e u i 1 S t n e h i g h e s t v e l o c i t y m a i n t a i n e d f o r the p r e s c r i b e d 30 min. p e r i o d (cm*sec - 1;).; i s the v e l o c i t y i n c r e m e n t (which was approx. 0.5*fork l e n g t h - s e c " 1 ( f l * s e c _ 1 ) ; t ^ i s the time (min) t h a t each f i s h swam a t the " f a t i g u e " v e l o c i t y ; and i s the p r e -s c r i b e d p e r i o d o f swimming (30 min).. In t h e s e e x p e r i m e n t s , f a t i g u e was d e f i n e d as t h e p o i n t a t which a f i s h was unable t o remove i t s e l f from the e l e c t r i f i e d downstream g r i d o f the water t u n n e l a f t e r r e p e a t e d e f f o r t s and e l e c t r i c a l s t i m u l a t i o n . I chose t o f o l l o w the p r o c e d u r e d e s c r i b e d by S m i t et a l . , (1971), whereby once a f i s h had f a l l e n a g a i n s t the g r i d , t he water v e l o c i t y . w a s reduced by 0.25 f l * s e c _ 1 . I f the, f i s h then c o n t i n u e d t o swim, even a f t e r the " f a t i g u e " v e l o c i t y was r e g a i n e d , the f i r s t f a i l u r e was i g n o r e d . However, the subsequent f a i l u r e t e r m i n a t e d t h a t e x p e riment and t h a t time t o " f a t i g u e " was r e c o r d e d . T h i s was the time used i n the c a l c u l a t i o n o f l l c r i t ' A l l swimming speed experiments were performed o v e r the same time p e r i o d o f the day, t o overcome p o s s i b l e d i e l d i f f e r e n c e s i n e x e r c i s e performance. S e v e r a l c o r r e c t i o n s t o the o b t a i n e d U v a l u e were made, o r a t c r i t l e a s t c o n s i d e r e d , b e f o r e U c r ^ t was r e p o r t e d . The s o l i d - b l o c k i n g e f f e c t (Pope and H a r p e r , 1966) r e s u l t i n g from t h e . d e c r e a s e i n t h e e f f e c t i v e c r o s s -s e c t i o n a l a r e a o f the t u n n e l due to the p r e s e n c e o f the f i s h , o b v i o u s l y v a r i e d w i t h the c r o s s - s e c t i o n a l a r e a o f each f i s h . I f the c r o s s - s e c t i o n o f the f i s h a t the l e v e l o f the p e c t o r a l f i n approximated an e l i p s e , a c o r r e c t i o n f a c t o r , k, was d e r i v e d from k Hr* , where r i s the i n s i d e n r z - nab 9 t u n n e l r a d i u s (6.35 cm) and a and b are the semi-axes o f the f i s h a t the w i d e s t p o i n t , f o r each f i s h . The c o r r e c t e d U .. v a l u e was e s t i m a t e d by c r i t k x U c H t . A " c o n d i t i o n f a c t o r " , . d e s c r i b e d by F u l t o n (1911, i n R i c k e r , 1975) was c o n s i d e r e d i n the p r e s e n t e x p e r i m e n t s , where C F . = Mass (grams) x f o r k l e n g t h - 3 (cm),. However, t h i s f a c t o r never a c c o u n t e d f o r more than a 1% i n c r e a s e i n t h e u n c o r r e c t e d v a l u e o b t a i n e d f o r U and t h e r e f o r e was c r i t c o n s i d e r e d r e l a t i v e l y unimportant t o the o v e r a l l e s t i m a t e o f e x e r c i s e performance. A l s o , d a t a p r e s e n t e d ; b y , B r e t t (1964, 1965a) i n d i c a t e t h a t t h e r e i s l i t t l e d i f f e r e n c e between t h e l e v e l s o f swimming a c t i v i t y a t t a i n e d by e i t h e r male o r female s a l m o n i d s , and,thus no se x u a l d i s t i n c t i o n was made f o r U ^ v a l u e s i n the p r e s e n t s t u d y . In the r a d i o l a b e l l e d e x p e r i m e n t s , U ..was not de t e r m i n e d c r i t d i r e c t l y . However, f i s h were e x e r c i s e d i n a manner s i m i l a r t o t h a t d e s c r i b e d above ( i . e . , 0.5»fl»sec" 1 v e l o c i t y i n c r e m e n t s a t 30 min time i n t e r v a l s ) , t o an e s t i m a t e d 80% U c r - j t - The U ^ . ^ v a l u e was determined from F i g u r e 9, \ K i c e n i u k (1975), but.was c o r r e c t e d i n o r d e r t o i n c l u d e t he added drag due to the d o r s a l a o r t i c cannula i n the water stream. T h e r e f o r e , the e s t i m a t e d U c r i t ' f r o m F i g ' ^' w a s r e d u c e d by 20% t o a c c o u n t f o r added r e s i s t a n c e (D.R: Jone s , p e r s o n a l communication), and then 80% o f t h a t v a l u e taken as the e s t i m a t e d 80% used f o r t h a t f i s h . THE EXPERIMENTS. I_. CARDIAC OUTPUT AND BLOOD VOLUME DISTRIBUTION  P r e p a r a t i o n P r e - t r a i n e d f i s h i n i t i a l l y were a n a e s t h e t i z e d i n a e r a t e d water c o n t a i n i n g 1:10,000 MS-222 (w/w methane t r i c a i n e s u l f o n a t e , a d j u s t e d t o pH 7.0 - 7.5 w i t h NaHCOg), and t r a n s f e r r e d t o an o p e r a t i n g t a b l e (Smith and B e l l . 1964, 1967). T h e r e , g i l l s were i r r i g a t e d c o n t i n u o u s l y w i t h a e r a t e d 1:15,000 MS-222 a t 4°C. F i s h were l a i d s u p i n e and a d o r s a l a o r t i c c a n n u l a (75 cm o f 10 PE 50 t u b i n g ( 0 . 5 8 mm I.D.,0.965 mm O.D.)) was i n s e r t e d through the r o o f o f the b u c c a l c a v i t y between g i l l a r c h e s 1 and 2. T h i s c a n n u l a was p r e -f i 1 l e d w i t h C o r t l a n d s a l i n e (Wolf, 1963) and plugged w i t h a p i n . The s u r g i c a l p r o c e d u r e f o l l o w e d has been d e s c r i b e d i n d e t a i l ' b y Smith (1978). T h i s c a n n u l a s e r v e d as the s i t e o f i n j e c t i o n f o r r a d i o - l a b e l l e d s u b s t a n c e s i n t o the c i r c u l a t i o n , as a b l o o d sampling p o r t , a p l a c e from which t o measure 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 (DAP) and a s i t e f o r t h e i n j e c t i o n o f s a t u r a t e d KC1 s o l u t i o n , used t o q u i c k l y a r r e s t c i r c u l a t i o n a t the end o f the e x p e r i m e n t s . , E x p e r i m e n t a l P r o t o c o l F o l l o w i n g surgery., a f i s h was p l a c e d i n s i d e the t u n n e l r e s p i r o m e t e r and a l l o w e d t o r e c o v e r f o r a t l e a s t 18 h (Houston and. Woods, 1972; Houston et a l . , 1971) a t 8.7 c m * s e c - 1 water f l o w : Water temperature d u r i n g t h e s e experiments was m a i n t a i n e d a t 4 - 5°C. Once r e c o v e r y was c o m p l e t e , DAP was r e c o r d e d f o r a t l e a s t one h o u r ' b e f o r e s t a r t i n g the t e s t i n g . DAP was r e c o r d e d from the c a n n u l a c o n n e c t e d t o a Statham P23 BB p r e s s u r e t r a n s d u c e r , which was m a n o m e t r i c a l l y c a l i b r a t e d a g a i n s t a s t a t i c column o f water. . The o u t p u t was d i s p l a y e d on a . G i l s o n MP5 pen r e c o r d e r . The p u l s a t i l e p r e s s u r e t r a c e s a l l o w e d h e a r t r a t e s t o be d e r i v e d . A p r e - e x e r c i s e b l o o d sample was w i t h -drawn, and h a e m a t o c r i t (Hct) d e t e r m i n e d . A t o t a l o f 6 f i s h (415.1 ± 4 0 . 3 g) were f o r c e d t o swim a g a i n s t a water c u r r e n t , i n 0 . 5 » f T ' s e c - 1 ' 3 0 m i n - 1 i n c r e m e n t s up t o an e s t i m a t e d 80% U c r i t " A f t e r 25 min swimming a t . t h e h i g h e s t v e l o c i t y , 0.05 mOi 8 6 R b C l (NEN) (0.30 ml C o r t l a n d s a l i n e v e h i c l e + 0.5 ml " c o l d " s a l i n e wash) were i n t r o -duced i n t o the c i r c u l a t i o n v i a the c a n n u l a . The small i n j e c t i o n volume d i d not appear t o a f f e c t the p r e s s u r e t r a c e i n any way. F i v e minutes were a l l o w -ed f o r t r a c e r e q u i l i b r a t i o n w i t h i n the body, and then the f i s h were s a c r i f i c e d by a 0.20 ml i n j e c t i o n o f KC1.'. 11 S i x o t h e r f i s h (384.4 ± 40.5 g) were p r e p a r e d as above, but were a l l o w e d t o r e c o v e r i n darkened Perspex h o l d i n g boxes, s u p p l i e d w i t h r u n n i n g a e r a t e d water a t the same temperature as swimming f i s h . A f t e r ' r e c o v e r y (18 h minimum), DAP was r e c o r d e d and h e a r t r a t e s determined f o r s e v e r a l h o u r s , and Hct taken as d e s c r i b e d b e f o r e . These " r e s t i n g " f i s h then were 86 i n j e c t e d w i t h Rb.and s a c r i f i c e d i n t h e same manner as d e s c r i b e d above. F i v e more f i s h (316.8 ± 36.6 g) were s a c r i f i e c e d by o v e r -a n a e s t h e s i a (1:10,000 MS-222, w/w) and f r e s h e x c i s i o n o f s e l e c t e d t i s s u e s was made, t o determine the r e l a t i v e p e r c e n t a g e o f the t o t a l b o d y w e i g h t accounted f o r by each sample. Care was taken t o b l o t e x c e s s b l o o d and o t h e r f l u i d s . f r o m a l l t i s s u e s . Gut c o n t e n t s were removed b e f o r e w e i g h i n g , as was the b l o o d c o n t a i n e d . w i t h i n the h e a r t . LIQUID SCINTILLATION COUNTING (LSC) T i s s u e s e l e c t i o n . a n d P r e p a r a t i o n Once each f i s h had been s a c r i f i c e d , 200 mg samples o f the t i s s u e s R d e s c r i b e d i n T a b l e 2 were c a r e f u l l y e x c i s e d and weighed i n t o NEN low potassium,, b o r o s i l i c a t e g l a s s s c i n t i l l a t i o n v i a l s (22 ml c a p a c i t y ) . Care was taken n o t t o cross-.contaminate any sample w i t h b l o o d or f l u i d from a n o t h e r . To each v i a l was added 1 . 5 - 2 . 0 ml o f P r o t o s o l t i s s u e s o l u b i l i z e r (0.5 M q u a t e r n a r y ammoniurn ;hydroxide s o l u t i o n ) . , and the capped c o n t e n t s were a l l o w e d t o d i g e s t a t 55°C f o r 18 - 24 h i n a t e m p e r a t u r e - c o n t r o l l e d s h a k i n g water bath. Once d i g e s t e d , c o l o u r e d s o l u t i o n s were p a r t i a l l y d e c o l o u r i z e d by the dropwise a d d i t i o n o f 0.1 - 0.5 ml 30% a t room t e m p e r a t u r e , then rewarmed and shaken f o r an a d d i t i o n a l 30 min a t 55°C. 5 0 y l g l a c i a l a c e t i c a c i d was added t o t h e s e v i a l s per 0.5 ml P r o t o s o l . Once c o o l e d a g a i n , 10 ml E c o n o f l u o r (NEN - a premixed s c i n t i l l a t i o n s o l u t i o n w i t h c h a r a c t e r i s t i c s s i m i l a r t o those o f t o l u e n e base c o c k t a i l s ) were added. Once mixed and 12 Table 2. L i s t of t issues sampled from rainbow t rout , to determine cardiac output d i s t r i bu t ion during rest and exerc ise , using Rb-86. Mosaic muscle - l e f t and r igh t side epaxial mass under dorsal f i n - l e f t and r ight side from the causal peduncle, with a l l red muscle ca re fu l l y removed, from under the region of the adipose f i n - l e f t and r ight cheek muscle (cephal ic port ion of adductor mandibularis) Red muscle - l e f t and r ight side la te ra l l i ne muscle, with la te ra l l i ne organ removed from; a) jus t caudad of the opercula b) the region under the dorsal f i n c) the caudal peduncle Organs: spleen, stomach (fundus and.pylor ic region sampled, with contents removed), in tes t ine (mid-region and highly vascular ized.absorpt ive reg ion, with contents removed), kidney (.cephalic region - "head kidney" - and from region under dorsal f i n ) , ven t r i c le (blood removed from lumen), gonad (whether mature or immature), b ra in , g i l l s ( l e f t and r ight.second arch). Duplicate 200 mg samples of each t issue in the above l i s t were taken and count rate determined. 13 shaken, v i a l s were kept i n dark b e f o r e c o u n t i n g , and were a l l o w e d t o remain i n the c o u n t e r f o r . a t l e a s t 60 min b e f o r e counts were begun. L i g h t e m i s s i o n s due t o 3 - i n t e r a t i o n s w i t h the f l u o r were m o n i t o r e d u s i n g a ISOCAP (Model 300, N u c l e a r Chicago) L i q u i d S c i n t i l l a t i o n c o u n t e r . Each sample was counted 32 f o r 10 min i n t h e P window s e t t i n g o f the c o u n t e r . Data were c o n v e r t e d t o counts per minute per gram t i s s u e (cpm.g" 1'). S i n c e the %age body w e i g h t o f each t i s s u e had been been d e t e r m i n e d p r e v i o u s l y , and t h e weight o f each t i s s u e sample was known ( u s u a l l y 200 mg), the t o t a l cpm's i n the f i s h c o u l d be e s t i m a t e d . T h i s v a l u e was compared t o the known i n j e c t i o n cpm's f o r each f i s h , s i n c e a s t a n d a r d f o r each i n j e c t i o n was counted w i t h each f i s h . A l l v a l u e s are e x p r e s s e d i n the t a b l e s as means ± S;E.M. w i t h the number o f o b s e r v a t i o n s (n) g i v e n . P r e c a u t i o n s and C o r r e c t i o n s f o r L i q u i d S c i n t i l l a t i o n C o u n t i n g (LSC) Rubidium-86. ( T ^ =• 18.7 days), i s a 3"-emitter (91% 1.77 MeV; 9% 0.69 meV), as w e l l as b e i n g a weak Y - e m i t t e r ( 9 % 1.08 MeV). Phosphorus-32 ^ 1 / 2 = c' a- v s) 1 S a p u r e ^ " - e m i t t e r (1.71 MeV). Rb e m i s s i o n counts 32 were m o n i t o r e d i n t h e . P window o f the ISOCAP s i n c e t h i s c o u n t e r had no d i r e c t p r o v i s i o n , f o r the measurement o f i s o t o p i c Rb a c t i v i t y . I n h e r e n t i n LSC a r e many i n t e r f e r i n g p r o c e s s e s . One o f t h e s e i s c h e m i l u m i f t e s e e n c e r e s u l t i n g from c h e m i c a l r e a c t i o n s between a d d i t i v e s or specimen and the components o f the f l u o r , . thus c a u s i n g more counts than would be due s o l e l y t o the e m i t t e r / f l u o r r e a c t i o n . The decay o f chemi- .. l u m i n e s c e n c e i s temperature dependent and proceeds f a s t e r a t h i g h e r tempera-t u r e s . T h e r e f o r e samples were kept a t l e a s t a t room temperature f o r a 3 hour p e r i o d b e f o r e c o u n t i n g , t o a l l o w t h i s i n t e r f e r e n c e t o decay. Add-i t i o n a l l y , c hemiluminescence can be d i m i n i s h e d o r c o m p l e t e l y a b o l i s h e d by a c i d i f i c a t i o n o f the P r o t o s o l w i t h g l a c i a l a c e t i c a c i d b e f o r e the a d d i t i o n o f f l u o r , as was done i n the p r e s e n t c a s e . 14 P h o s p h o r e s c e n c e i s a n o t h e r i n t e r f e r i n g p r o c e s s which c o u l d g i v e counts above those d i r e c t l y a t t r i b u t a b l e to the e m i t t e r i n t e r a c t i o n i t s e l f . Many p r o t e i n a c e o u s s u b s t a n c e s , e.g. l i v e r , when d i g e s t e d and d i l u t e d w i t h a t o l u e n e base s o l v e n t and exposed t o s u n l i g h t , or f l u o r e s c e n t T i g h t , emit l i g h t and thus g i v e h i g h count r a t e s , even i n the absence o f r a d i o a c t i v i t y ( L l o y d et a l . , 1962). T h i s problem was e l i m i n a t e d by k e e p i n g samples i n the dark and/or a c i d i f i c a t i o n o f the samples b e f o r e c o u n t i n g . Quenching i n t e r f e r e n c e s a r e those p r o c e s s e s i n which maximum photon y i e l d i s not a c h i e v e d f o r a g i v e n r a d i o a c t i v e s o u r c e . In t h e p r e s e n t s t u d y , c o l o u r quenching was the most p r e v a l e n t s o u r c e o f i n t e r f e r e n c e , e s p e c i a l l y from h i g h l y pigmented t i s s u e s , even a f t e r p a r t i a l d e c o l o u r i z a t i o n . T h i s was c o r r e c t e d by the c h a n n e l s - r a t i o method, s i n c e t h e r e was no i n t e r n a l s t a n d a r d i n the c o u n t e r . However, c o u n t i n g e f f e c i e n c y (e = cpm/dpm) was n e v e r d e t e r -mined, and t h e r e f o r e o n l y cpm's a r e r e p o r t e d i n t h i s s t u d y . S i n c e d i f f e r e n t t i s s u e s had d i f f e r e n t c o l o u r quenching c h a r a c t e r i s t i c s , i n d i v i d u a l c o l o u r quench c u r v e s were c o n s t r u c t e d f o r l i v e r , k i d n e y , s p l e e n , r e d m u scle, and w h i t e muscle and s i m i l a r . , l i g h t c o l o u r e d t i s s u e s . T h i s was a c c o m p l i s h e d by a d d i n g a constant'known cpm's s o u r c e t o u n c o l o u r e d , and v a r y i n g amounts o f t i s s u e d i g e s t s , and p l o t t i n g the c h a n n e l s - r a t i o s o b t a i n e d a g a i n s t the un-quenched counts ( 1 0 0 % ) . The ISOCAP p r i n t o u t p r e s e n t s s i m u l t a n e o u s d a t a f o r the count r a t e from the sample as w e l l as the c h a n n e l s - r a t i o a s s o c i a t e d w i t h t h a t sample, and t h e r e f o r e each sample c o u l d be c o r r e c t e d f o r quenching t o o b t a i n the, a c t u a l cpm's. S e v e r a l v i a l s c o n t a i n i n g o n l y the a p p r o p r i a t e volumes o f P r o t o s o l and E c o n o f l u o r , as used i n the e x p e r i m e n t s , were i n c l u d e d i n each s e r i e s t o determine the background count' r a t e i n h e r e n t i n t h e system from a v a r i e t y o f s o u r c e s . A l t h o u g h t h e s e s o u r c e s w i l l not be d i s c u s s e d , s u f f i c e t o say t h a t , a f t e r quench c o r r e c t i o n , background counts were s u b t r a c t e d from the cpm's o b t a i n e d . 15 A l t h o u g h c o u n t i n g " e f f i c i e n c y " was i n c r e a s e d , and c o r r e c t e d f o r by the methods o u t l i n e d above, no c o r r e c t i o n was made f o r any p o s s i b l e i n t e r f e r e n c e due t o Cerenkov r a d i a t i o n ( J e l l e y , 1956), from the h i g h l y 86 e n e r g e t i c 3 - e m i t t e r , Rb. y - C o u n t i n g and R a d i o l a b e l e d M i c r o s p h e r e s To determine t o t a l b l o o d volume and the b l o o d volumes o f v a r i o u s t i s s u e s i n r e s t i n g and e x e r c i s i n g rainbow t r o u t , 6 f i s h (371.0 ± 36.7 g) were m a i n t a i n e d a n d - c a n n u l a t e d as p r e v i o u s l y d e s c r i b e d . . Chromium-51 ( T ^ = 27.8 d a y s ) , s u p p l i e d as sodium chromate (NEN - 1 mCi'ml" 1 t o t a l a c t i v i t y ) was used t o l a b e l r e d b l o o d c e l l s by t h e method d e s c r i b e d by Conte et al., (1963). B r i e f l y , 5 - 6 ml b l o o d were withdrawn v i a v e n t r i c u l a r p u n c t u r e from donor f i s h o f the same s t o c k , and t r a n s f e r r e d from the s y r i n g e i n t o a 50 ml c o n i c a l tonometer f l a s k kept on i c e . 10 ml C o r t l a n d s a l i n e w i t h 1000 I»U sodium h e p a r i n was added, and the f l a s k s g e n t l y a g i t a t e d . 50 y C i t o t a l 51 Cr were added t o the b l o o d c e l l s u s p e n s i o n and i n c u b a t e d 1.5 - 2 h on i c e w i t h g e n j : l e a g i t a t i o n e v e r y 5 min. A f t e r i n c u b a t i o n , b l o o d was t r a n s f e r r e d t o round bottom t e s t tubes and c e n t r i f u g e d (.2,500 - 3,000 G's) f o r 2 min. The s u p e r n a t a n t was c a r e f u l l y a s p i r a t e d and d i s c a r d e d , e x c e p t f o r 3, 50 y l a l i q u o t s which were p l a c e d i n Y - c o u n t i n g tubes f o r subsequent a n a l y s i s . The low c e n t r i f u g a l f o r c e used d i d not pack the c e l l s t o o t i g h t l y , and they were r e a d i l y resuspended i n 10 - 15 ml c o l d s a l i n e , , f o r washing. A g a i n , the c e l l s u s p e n s i o n was c e n t r i f u g e d and t h e s u p e r n a t a n t t r e a t e d as b e f o r e . T h i s washing p r o c e d u r e was r e p e a t e d 2 - 3 t i m e s . A f t e r the f i n a l wash, the s u p e r n a t a n t and the top l a y e r o f c e l l s (white c e l l s and o t h e r d e b r i s ) was 51 d i s c a r d e d . The C r - l a b e l l e d r e d c e l l s were t r a n s f e r r e d t o an equal volume o f c o l d s a l i n e and resuspended. S e v e r a l 10 o r 25 y l a l i q u o t s o f t h i s f i n a l 51 b l o o d s u s p e n s i o n were removed, t o be a n a l y s e d f o r t o t a l Cr c o u n t r a t e , as w e l l as Hct. The Hct d e t e r m i n a t i o n a l s o a l l o w e d an assessment o f the 16 degree of b l o o d l y s i s due to the i n c u b a t i o n and mi x i n g p r o c e d u r e t o be made. L i t t l e damage was e v i d e n t p r i o r t o i n j e c t i o n o f t h i s b l o o d i n t o r e c i p i e n t f i s h . No r a d i o a c t i v i t y was found i n the s u p e r n a n t a n t f o l l o w i n g the second wash. One ml o f b l o o d was removed from t h e r e c i p i e n t f i s h and Hct 51 d e t e r m i n e d . T h i s volume was r e p l a c e d by 1 ml o f C r - l a b e l l e d b l o o d ( H c t = 52.3 + 10.1 %•.),• p r e p a r e d as d e s c r i b e d above, so as n o t t o change t o t a l b l o o d volume. From p r e l i m i n a r y e x p e r i m e n t s , i t , w a s found t h a t the a c t i v i t y i n the b l o o d , determined from s e q u e n t i a l , 50 yl d o r s a l a o r t i c samples, became c o n s t a n t 1 - 1.5 h.,after the i n j e c t i o n Of l a b e l l e d b l o o d . The l a b e l l e d b l o o d always was h i g h e r . i n Hct than normal. D o r s a l a o r t i c p r e s s u r e tended t o be e l e v a t e d above normal for. a p p r o x i m a t e l y 1 hour a f t e r i n j e c t i o n o f l a b e l l e d b l o o d , a f t e r which t h e p r e s s u r e d e c l i n e d t o normal l e v e l s . L a b e l l e d r e d c e l l s t h e r e f o r e were a l l o w e d t o c i r c u l a t e f o r a minimum o f 2 hours. A f t e r the e q u i l i b r a t i o n p e r i o d , a s m a l l sample o f d o r s a l a o r t i c b l o o d was taken and Hct d e t e r m i n e d , and compared w i t h the p r e - i n j e c t i o n v a l u e . As w e l l , 0.5 ml mixed r a d i o a c t i v e / c o l d DA b l o o d was taken and c o u n t e d , t o de t e r m i n e the b l o o d volume o f each f i s h . T h i s c o u l d be c a l c u l a t e d s i n c e the t o t a l i n j e c t e d count r a t e ( c o r r e c t e d f o r r e s i d u a l counts r e m a i n i n g i n the s y r i n g e and c a n n u l a ) was known, and the count r a t e i n 0.5 ml. mixed b l o o d a l s o was known. The d e c r e a s e i n the t o t a l count r a t e was due t o th e u n l a b e l l e d b l o o d volume d i l u t i n g and mi x i n g w i t h l a b e l l e d c e l l s . T h i s r a t i o (cpm*ml"Vtotal cpm i n j e c t e d ) gave t o t a l b l o o d volume ( m l ) , and s i n c e t o t a l body weight was known, the volume was e x p r e s s e d as p e r c e n t body w e i g h t , f o r comparison w i t h o t h e r p u b l i s h e d d a t a . R e g i o n a l d i s t r i b u t i o n o f c a r d i a c o u t p u t a t r e s t and d u r i n g swimming 141 95 e x e r c i s e was determined i n 7 f i s h , u s i n g Cerium- and N i o b i u m - l a b e l l e d m i c r o s p h e r e s , h a v i n g a d e n s i t y o f 1.3 g*cm"3 ( b l o o d = 1.05 g » c m ~ 3 ) . The 17 D supplier of these spheres (NENTRAC - New England Nuclear, Lachine, P.Q.) 141 provided size specifications with each batch ( Ce-spheres, 26.1 ± 1.1 ujn (±S..D.); ^Nb-spheres, 25.6 ± 1.3.nm (±S.D.) diam.). Hales et.al. (1979) and Jones et at. (1979) have examined s i m i l a r microspheres using haemocyto-metry, as well as S.E.M. to check manufacturer's stated sphere concentrations, size ranges etc., and have found no aggregation, i r r e g u l a r l y shaped spheres nor any other deviations from specifications. Each batch of spheres had 1 mCi total a c t i v i t y , supplied in 10 ml isotonic s a l i n e , to which 0.01% Tween 80 (a surfactant for bead dispersal) had been added. Supplier's data also indicated no appreciable leaching of a c t i v i t y from the spheres into saline. Subsamples were taken from the supplied stock v i a l s and diluted 5 4 with Cortland sali n e to give approximately 2,x 10 ± 5 x 10 spheres in 10 ml 141 95 total saline volume, f o r b o t h Ce- and Nb-labelled spheres. Before i n -j e c t i o n , the diluted sample v i a l s , containing a magnetic s t i r r i n g bar, were agitated on a vortex mixer.and then ul t r a s o n i f i e d for several minutes to evenly disperse the spheres. The vi a l s then were placed on a magnetic s t i r -rer and mixed continuously. An 0.25 ml sample was withdrawn into a 1 ml di s -posable syringe and attached to the sidearm of a p l a s t i c 3^way tap, fixed to a mechanical vibrator, to prevent spheres from s e t t l i n g out of suspension (see Fig. 1). Another syringe contained saline for rinsing residual spheres from cannulae (0.5 ml). 0.25 ml sphere suspension was injected prior to, and another 0.25 ml spheres were injected after 25 min.of swimming at the e s t i -51 mated 80% U for each f i s h . Since.1.0 ml Cr-labelled red c e l l s already c r i t J had been injected at rest, to determine blood volumes during exercise, 2.x 0.25 ml sphere volumes, followed, by 0.5 ml wash each, for a total 3.0 ml f l u i d were injected into each f i s h due to experimental protocol, an equivar lent 3.0 ml blood i n t i a l l y were removed.from each f i s h . Hct was determined and the rest of this blood was discarded, in order to keep.the tota l blood volume unchanged. Following 30 min exercise at 80% U . f , f i s h were 18 s a c r i f i c e d by i n j e c t i o n o f 0.2 ml s a t u r a t e d KC1 s o l u t i o n i n t o the c i r c u l a t i o n . U s u a l l y , 5, 10 o r 25 y l a l i q u o t s o f each s p h e r e s u s p e n s i o n were p r e p a r e d a t the time o f i n j e c t i o n , as a check f o r maximum p o s s i b l e i n j e c t e d a c t i v i t y ( a f t e r a p p r o p r i a t e m u l t i p l i c a t i o n t o c o r r e c t f o r volume d i f f e r e n c e s ) . In o r d e r t o det e r m i n e the t o t a l number o f i n j e c t e d c o u n t s , the e n t i r e assemble (see F i g . 1) was c u t up and p l a c e d i n t o c o u n t i n g t u b e s . R e s i d u a l counts i n the assembly as w e l l as t h o s e remaining i n the DA c a n n u l a e , were added up.and s u b t r a c t e d f r o m the measure o f the t o t a l p o s s i b l e i n j e c t e d a c t i v i t y , thus g i v i n g a v a l u e f o r the t o t a l counts i n j e c t e d . T i s s u e P r e p a r a t i o n and Gamma ; ( T ) - C o u n t i n g T i s s u e s sampled.were e s s e n t i a l l y t h e same as t h o s e l i s t e d i n T a b l e 2.. The e x c e p t i o n was t h a t , where p o s s i b l e , whole organ a c t i v i t y was co u n t e d , r a t h e r than o n l y 200 mg.samples, as i n the p r e v i o u s c a s e . As w e l l , s k i n samples taken from o v e r . t h e l a t e r a l l i n e were i n c l u d e d , t h e s e h a v i n g had a l l a d h e r i n g muscle c a r e f u l l y s c r a p e d away. T i s s u e s : w e r e e x c i s e d and weighed im m e d i a t e l y f o l l o w i n g s a c r i f i c i n g o f the f i s h . The wet weight s o f a l l t i s s u e s c o u l d be e x p r e s s e d a %age o f t o t a l body weight. ATI samples were d r i e d i h an oven' o v e r n i g h t .(,70 - 8 0 ° C ) , on. f o i l . Once d r i e d , a l l samples were p l a c e d i n c o u n t i n g tubes ( p o l y s t y r e n e ) and count r a t e s d e t e r m i n e d u s i n g 51 141 a N u c l e a r C h i c a g o m u l t i c h a n n e l p u l s e - h e i g h t Y - c o u n t e r . . C r - b l o o d , Ce-95 and Nb-microsphere r a d i a t i o n s were counted s i m u l t a n e o u s l y w i t h window s e t t i n g s s p a n n i n g a t l e a s t 85% o f the t o t a l photopeaks. These were de t e r m i n e d s e p a r a t e l y f o r each i s o t o p e (see F i g s . 2, 3 & 4 ) . The energy s p e c t r a were i d e n t i c a l t o th o s e found by Heymann et al. (1977) f o r the same i s o t o p e s . 95 There was c o n s i d e r a b l e s p i l l o v e r from the h i g h energy window (: Nb) i n t o 51 141 the o t h e r windows. As w e l l , Cr had a s p i l l o v e r component i n t o the Ce window. However, no i n t e r f e r i n g counts from the o t h e r two l a b e l s were 19 F i g u r e 1. Sc h e m a t i c r e p r e s e n t a t i o n o f the m i c r o s p h e r e i n j e c t i o n assembly. Numbers with a s s o c i a t e d arrows i n d i c a t e the sequence o f events i n the i n j e c t i o n o f any dose o f r a d i o -l a b e l l e d m i c r o s p h e r e s . 1, withdrawal o f 1 ml d o r s a l a o r t i c b l o o d and h o l d 2, slow and s t e a d y i n j e c t i o n o f 0.25 ml s p h e r e s i n t o DA 3, f l u s h o f c a n n u l a w i t h the p r e v i o u s 1 ml b l o o d 4, wash c a n n u l a o f b l o o d and any r e m a i n i n g s p h e r e s with 0.5 ml n o n - r a d i o a c t i v e C o r t l a n d s a l i n e . 20 0.25 ml spheres 0.5ml cold saline wash to fish in swim tube Mechanical vibrator 1 ml blood wash 21 T a b l e 3. Assessment o f c r o s s - i n t e r f e r e n c e i n energy s p e c t r a from the t h r e e i s o t o p e s counted s i m u l t a n e o u s l y i n t i s s u e samples from r e s t i n g and swimming rainbow t r o u t . Chromium-51 counts found i n Cerium-141 window Niobium-95 counts found i n Chromium-51 window Niobium-95 counts found i n Cerium-141 window = 20.34% 'Cr = 26.46% 9 5 N b = 22.39% 9 5 N b 22 F i g u r e 2. Energy spectrum d e r i v e d from a subsample o f Chromium-51 s t o c k s o l u t i o n used t o l a b e l r e d b l o o d c e l l s . The energy o f r a d i a t i o n was scanned from 0 through t o 1000 KeV f o r t h i s e m i t t i n g s o u r c e . 23 24 F i g u r e 3. Energy spectrum d e r i v e d from a subsample o f Cerium-141-l a b e l l e d m i c r o s p h e r e s s t o c k s u s p e n s i o n . The energy o f r a d i a t i o n was scanned from 0 through t o 1000 KeV f o r t h i s s o u r c e . 25 26 Figure 4 . Energy spectrum derived from a subsample of Niobium-95-labelled microspheres stock suspension. The energy of radiation was scanned from 0 through to 1000 KeV for this source. 27 28 F i g u r e 5. The d e c r e a s e i n known h e i g h t o f water added gamma-counting tube. a c t i v i t y (100%) w i t h i n c r e a s i n g t o s o u r c e , above bottom o f the 29 30 found i n the Nb window (see T a b l e 3 ) . 5 Samples were counted f o r 10 min o r u n t i l 1 x 10 counts had been accumulated, and then c o n v e r t e d t o cpm's. S e q u e n t i a l s p i l l o v e r c o r r e c t i o n s were made ( u s i n g T a b l e 3 ) , as w e l l as the c o r r e c t i o n f o r background count r a t e i n each window f o r each sample. C o u n t i n g geometry d i d n o t appear t o be a s i g n i f i c a n t d e t e r m i n i n g f a c t o r i n the o b t a i n e d count r a t e , s i n c e i n -dependent experiments i n d i c a t e d t h a t i d e n t i c a l counts were o b t a i n e d when a s t a n d a r d count r a t e was: a) a p p l i e d t o and wrapped i n f o i l and counted i n the bottom o f a dry c o u n t i n g t u b e , i b) wrapped i n f o i l and p l a c e d i n d i s t i l l e d water up t o a c r i t i c a l h e i g h t above the bottom o f t h e c o u n t i n g tube (see F i g . c) p l a c e d on wet t i s s u e w i t h or w i t h o u t f o i l w rapping, e i t h e r dry or i n water i n the t u b e , o r d) p l a c e d on d r i e d t i s s u e w i t h o r w i t h o u t f o i l w r a p p ing, e i t h e r dry o r i n water i n the tube. The % o f body weight r e p r e s e n t e d by each t i s s u e was c a l c u l a t e d , and the t o t a l counts i n each t i s s u e o r organ were c a l c u l a t e d from t h e t o t a l counts measured i n t h a t sample. These v a l u e s were added up and compared to the e s t i m a t e d t o t a l i n j e c t i o n c o u n t s , and then e x p r e s s e d as % r e c o v e r y . The % t o t a l counts found i n each t i s s u e was taken as the p e r c e n t a g e o f the t o t a l c a r d i a c o u t p u t ( a c t u a l v a l u e unknown) f l o w i n g t o t h a t t i s s u e d u r i n g 51 r e s t and e x e r c i s e . Where Cr measurements were s u f f i c i e n t i n any t i s s u e to be unobscured by the s p i l l o v e r from o t h e r windows, t i s s u e b l o o d volume d u r i n g e x e r c i s e a l s o c o u l d be d e t e r m i n e d . A d d i t i o n a l l y , a f t e r samples from 2 f i s h were removed and p r e p a r e d as d e s c r i b e d , t h e r e m a i n i n g t i s s u e was ashed c o m p l e t e l y i n a m u f f l e oven (1200 - 1 5 0 0 ° C ) , and the r e s i d u e counted. Thus, the t o t a l counts i n the whole body o b t a i n e d i n t h i s way were compared 31 w i t h t h a t v a l u e which was assumed t o have been i n j e c t e d i n i t i a l l y ( a f t e r c o r r e c t i o n s ) . D e s p i t e e x t e n s i v e smoking o f the a s h i n g t i s s u e , and the p r o b a b i l i t y t h a t t h i s smoke, as w e l l as heat c o n v e c t i o n may have c a r r i e d away some r a d i o ! a b e l l e d p a r t i c l e s , on average 78% o f the e s t i m a t e d t o t a l i n j e c t e d counts were r e c o v e r e d . S i n c e the number o f counts per sphere was d e t e r m i n e d , and the h a l f -l i f e ( T 1 / 2 ) °f a c t i v i t y was known, the number o f spheres a l s o was known f o r each t i s s u e sample. Only samples w i t h a t l e a s t 384 spheres a r e i n c l u d e d i n the r e s u l t s (Buckberg et a l . , 1971; Heymann et a l . , .1977).'• Count r a t e s from samples taken from l e f t and r i g h t s i d e s , o f f i s h were compared, t o t e s t f o r evenness o f m i c r o s p h e r e m i x i n g . No a p p a r e n t l e f t / r i g h t p r e f e r e n c e was noted f o r any t i s s u e so compared. G i l l t i s s u e and h e a r t samples were taken from each f i s h t o a s s e s s t h e degree o f sphere entrapment i n the p o s t -b r a n c h i a l a r t e r i a l system. Any counts i n the g i l l s o r h e a r t were an i n d i c a -t i o n o f c a p i l l a r y bypass. However, t h e s e counts never a c c o u n t e d f o r more than 0.5% o f the t o t a l a c t i v i t y i n the t i s s u e s and thus i t was c o n c l u d e d t h a t a r t e r i a l c a p i l l a r y entrapment o f the i n j e c t e d s p h e r e s was v e r y e f f e c t i v e . In a s u b s i d i a r y e x p e r i m e n t , 4 a d d i t i o n a l f i s h (534.5 ± 34.4 g) were e x e r c i s e d a t 80% U and i n j e c t e d . w i t h one dose o f l a b e l l e d s p h e r e s , as c r i t b e f o r e . These f i s h then were e x e r c i s e d f u r t h e r t o complete e x h a u s t i o n , and i n j e c t e d w i t h a second volume o f s p h e r e s . T i s s u e samples were t a k e n , p r e p a r e d and counted as d e s c r i b e d p r e v i o u s l y . V a l i d i t y o f T r a c e r Data 86 + E l e m e n t a l r u b i d i u m and i t s i s o t o p e Rb have b i o l o g i c a l p r o p e r t i e s which are v e r y s i m i l a r t o t h o s e o f p o t a s s i u m (Love et a l . , 1954). When QC RbCl o f known a c t i v i t y i s a d m i n i s t e r e d i n t r a v e n o u s l y as a s i n g l e i n j e c t i o n , the amount o f t h e ' i n d i c a t o r i n an organ remains e s s e n t i a l l y unchanged f o r an a p p r e c i a b l e time i n t e r v a l a f t e r the f i r s t c i r c u l a t i o n ( S a p i r s t e i n , 1956, 32 86 + 1958; Donato et a l . , 1964). S i n c e Rb e q u i l i b r a t e s and mixes w i t h the b l o o d , and i s d i s t r i b u t e d t h r o u g h o u t the body i n the same p r o p o r t i o n s as the b l o o d f l o w i t s e l f , the subsequent f r a c t i o n o f t h e t o t a l i n j e c t e d counts found i n any e x c i s e d t i s s u e sample w i l l be the same as t h a t f r a c t i o n o f the t o t a l b l o o d f l o w ( o r c a r d i a c , o u t p u t ) d i r e c t e d t o t h a t t i s s u e ( S a p i r s t e i n , 1967), under r e s t or e x e r c i s e c o n d i t i o n s . OC Data, o b t a i n e d from the Rb s t u d y , r e q u i r e c l o s e e x a m i n a t i o n as t o t h e i r v a l i d i t y , f o r the d e s c r i p t i o n o f c a r d i a c o u t p u t d i s t r i b u t i o n a t r e s t and d u r i n g e x e r c i s e i n the rainbow t r o u t . Even though a l l t i s s u e a c t i v i t i e s were c o r r e c t e d t o a c c o unt f o r most, s y s t e m a t i c e r r o r s , o t h e r e r r o r s cannot be a s s e s s e d . C o n s i d e r the f a c t t h a t a s m a l l volume o f s a t u r -a t e d KC1 s o l u t i o n was i n j e c t e d i n t o the d o r s a l a o r t i c c a n n u l a i n o r d e r t o q u i c k l y a r r e s t t h e h e a r t , and thus t h e c i r c u l a t i o n o f b l o o d , a t the t e r m i n a -t i o n o f an experiment. A f t e r t i s s u e uptake o f Rb , r e - e q u i l i b r a t i o n o f K and Rb p o o l s may occur,, l e a c h i n g Rb from some c e l l s and u l t i m a t e l y l e a d i n g t o an u n d e r e s t i m a t e o f the a c t u a l Rb r e t e n t i o n (Cameron, 1975). Furthermore, a l t h o u g h the c i r c u l a t i o n time o f rainbow t r o u t i s r e l a t i v e l y slow, compared t o t h a t o f mammals; i n the o r d e r o f one t o two minutes 86 + ( D a v i s , 1970; Itazawa, 1970)., a c o n s i d e r a b l e amount o f Rb r e d i s t r i b u t i o n may o c c u r f o l l o w i n g the i n i t i a l i n j e c t i o n and subsequent s a c r i f i c i n g o f the f i s h . A l s o , the amount o f counts c o n t a i n e d i n the b l o o d volume w i t h i n each t i s s u e sample s h o u l d have been taken i n t o a c c o u n t . D i f f e r e n c e s i n r e s i s t a n c e s o f v a r i o u s t i s s u e c e l l membranes to the t r a n s p o r t o f Rb , o r i n the e x t r a c t i o n r a t i o s f o r Rb /K c o u l d e x i s t i n f i s h , as have been found i n mammalian c e l l membranes (Sheehan and R e n k i n , 1972). Data f o r f i s h a r e n o t a v a i l a b l e , but i f such a s i t u a t i o n were the c a s e , i t c e r t a i n l y would l e a d t o e r r o r s i n the i n t e r p r e t a t i o n o f d i s t r i b u t i o n r e s u l t s , s i n c e a n a l y s e s r e l y on the assumption t h a t a l l t i s s u e s have s i m i l a r 33 membrane p r o p e r t i e s , w i t h r e s p e c t t o Rb uptake. The f a c t t h a t the e x t r a c -86 ~^ t i o n r a t i o f o r Rb o f i s o l a t e d , p e r f u s e d mammalian s k e l e t a l muscles appears t o be i n v e r s e l y r e l a t e d t o the r a t e o f b l o o d f l o w through t h a t t i s s u e (Friedman, 1968; Sheehan and R enkin, 1972) a l s o r a i s e s many q u e s t i o n s as t o the i n t e r p r e t a t i o n o f any Rb-based d a t a . I t a l s o i s a p p a r e n t t h a t the r e l a t i v e c o n t r i b u t i o n s o f . t h e s e p e r t u r b i n g f a c t o r s t o any d a t a a n a l y s e s a r e n o t r e a d i l y q u a n t i f i a b l e . The p o t e n t i a l problems e n c o u n t e r e d w i t h the use and e v a l u a t i o n 86 o f RbCl d a t a f o r . the s t u d y o f b l o o d f l o w p a t t e r n s i n the c i r c u l a t i o n were e l i m i n a t e d w i t h the use o f r a d i o - l a b e l l e d m i c r o s p h e r e s (see Rudolph and Heymann, 1967). The advantages o f t h i s method are obvious.. An a p p r o p r i a t e s i z e range o f s p heres was i n j e c t e d i n t o the c i r c u l a t o r y stream,, w i t h a known a c t i v i t y , and t h e s e s p h e r e s subsequently, became l o d g e d i n the c a p i l l a r y beds o f t i s s u e s . Because the s p heres d i d not exchange t h e i r r a d i o a c t i v i t y w i t h t h e t i s s u e s o r plasma, t h e r e was no problem o f r e d i s t r i b u -t i o n o f counts f o l l o w i n g t i s s u e , e n t r a p m e n t . The t o t a l • i n j e c t e d a c t i v i t y must remain d i s t r i b u t e d i n the f i s h , and g i v e n t h a t t h e s e p a r t i c l e s became u n i f o r m l y d i s t r i b u t e d . i n the c i r c u l a t i o n i f . i n j e c t e d a t a slow, c o n s t a n t . r a t e , the t i s s u e a c t i v i t i e s r e f l e c t the p r o p o r t i o n o f . t h e c a r d i a c o u t p u t d i r e c t e d t o any p a r t i c u l a r sample examined, a t r e s t : o r d u r i n g e x e r c i s e . Few s t u d i e s u s i n g m i c r o s p h e r e s f o r the e x a m i n a t i o n o f b l o o d f l o w p a t t e r n s i n . f i s h have been conducted (Cameron, 1974a,b, 1975), and a s t r i n g e n t e v a l u a t i o n o f haemodynamic consequences o f the i n j e c t i o n o f non-deformable p a r t i c l e s i n t o the c i r c u l a t o r y system o f f i s h i s n o t a v a i l a b l e . T h i s t e c h n i q u e , however, has been used e x t e n s i v e l y i n mammalian c i r c u l a t o r y i n v e s t i g a t i o n s (Rukasan and B l a h i t k a , 1974; Warren and Ledingham, .1974; M c D e v i t t and N i e s , 1976; F o s t e r and.Frydman, 1,978a,b). Data from numerous s t u d i e s c o n f i r m u n i f o r m i t y o f d i s t r i b u t i o n and n o n - d i s r u p t i v e haemodynamic e f f e c t s upon t h e c i r c u l a t i o n 34 d e s p i t e the i n j e c t i o n o f t h e s e p a r t i c l e s . Nor •. was t h e r e any i n d i c a t i o n o f s e r i o u s impairment o f t i s s u e f u n c t i o n from p a r t i a l c a p i l l a r y bed b l o c k -age by the s p h e r e s . Once e n t r a p p e d , t h e s e spheres d i d not change p o s i t i o n o v e r a l o n g p e r i o d o f time ( H a l e s a n d . C l i f f , 1977). Recent s t u d i e s by Mendall. and H o l l e n b e r g (1971) and F o s t e r and OC Frydman (1978a).have compared the Rb t r a c e r method w i t h the r a d i o l a b e l l e d m i c r o s p h e r e t e c h n i q u e f o r the e v a l u a t i o n o f b l o o d f l o w d i s t r i b u t i o n i n OC mammals. They have come t o t h e c o n c l u s i o n the Rb measurements have v e r y l i m i t e d q u a n t i t a t i v e v a l i d i t y , a t l e a s t i n . t h e i r e x p e r i m e n t s . I t t h e r e f o r e seems r a t h e r f o r t u i t o u s t h a t my Rb and m i c r o s p h e r e d a t a , d e s c r i b i n g a p p a r e n t c a r d i a c r e d i s t r i b u t i o n s dur-ing s t e a d y - s t a t e swimming e x e r c i s e i n the rainbow t r o u t are e q u a b l e . I t has been demonstrated t h a t the c h a r a c t e r i s t i c s o f b l o o d , e s p e c i a l l y h a e m a t o c r i t , change i n t h e i r passage through the g i l l v a s c u l a t u r e (see S e c t i o n , I I I ) . As w e l l , the r e d c e l l s d i f f e r f r o m d i f f e r e n t s e c t i o n s o f the mammalian s p l e e n (Groom et a l . , 1971).. The q u e s t i o n a r i s e s as t o whether the d o r s a l a o r t i c b l o o d samples were t r u l y r e p r e s e n t a t i v e or v a l i d f o r the r e f e r e n c e o f subsequent d e t e r m i n a t i o n s o f t i s s u e b l o o d volumes, 51 u s i n g C r - l a b e l l e d r e d b l o o d c e l l s . I f i n d e e d h a e m a t o e r i t s v a r y w i d e l y t h r o u g h o u t each i n d i v i d u a l c a p i l l a r y network o f each t i s s u e , how much c o n f i d e n c e can be p l a c e d on volume measurements from t h e s e d o r s a l a o r t i c b l o o d r e f e r e n c e samples? As I was unable t o a r r i v e a t any l o g i c a l a l t e r -n a t i v e r e f e r e n c e p o i n t , i f i n d e e d one e x i s t s f o r i n t a c t a n i m a l s , I have a c c e p t e d d a t a o b t a i n e d as b e i n g r e p r e s e n t a t i v e o f the in vivo c o n d i t i o n f o r e a c h . t i s s u e , w i t h the above r e s e r v a t i o n s noted. I I . ISOLATED SALINE-PERFUSED TROUT HEADS For s t u d i e s d e s i g n e d t o measure f l u i d d i s t r i b u t i o n i n g i l l s d u r i n g p e r f u s i o n by p u l s a t i l e and n o n - p u l s a t i l e f l o w , an i s o l a t e d s a l i n e - p e r f u s e d 35 t r o u t head p r e p a r a t i o n was used. T r o u t f o r t h i s s e r i e s o f experiments (271.1 ± 13.8 g) were o b t a i n e d and m a i n t a i n e d as d e s c r i b e d p r e v i o u s l y . Experiments were conducted a t h o l d i n g temperatures (7 - 9°C) and v a r i e d o n l y ± 0.5°G d u r i n g the c o u r s e o f any e x p e r i m e n t . F i s h were p r e p a r e d f o r and m a i n t a i n e d d u r i n g s u r g e r y as d e s c r i b e d p r e v i o u s l y (see P r e p a r a t i o n I ) . I s o l a t e d .heads were p r e p a r e d i n a manner s i m i l a r t o Wood (1974a), w i t h some m o d i f i c a t i o n s ( F i g . 6 ) . . B r i e f l y . , a c a t h e t e r (15 cm, PE 160 - 1 . 1 4 mm I.P. .x 1.57 mm O.D., Clay.Adams) was i n s e r t e d i n t o . t h e d o r s a l a o r t a , i n a. r e t r o g r a d e d i r e c t i o n , t o a p o i n t a p p r o x i m a t e l y 5 mm p o s t e r i o r o f the d o r s a l edge o f the o p e r c u l a r opening. T h i s c a t h e t e r was connected t o a wide bore tube (.6 .mm I.D.) and was m a i n t a i n e d a t the same l e v e l as the b a c k - p r e s s u r e r e s e r v o i r . (40 cm H^O). A s i d e -arm o f PE 50 (0.58 mm x 0.965 mm).tubing was a t t a c h e d t o t h i s c a t h e t e r near the p o i n t o f e n t r y i n t o the f i s h , t o f a c i l i t a t e s a m p l i n g o f d o r s a l a o r t i c p e r f u s a t e . o u t f l o w . In a d d i t i o n t o t h e v e n t r a l a o r t i c c a t h e t e r used f o r the i n p u t o f p e r f u s a t e to the g i l l s , a second c a n n u l a (PE 160) was t i e d t i g h t l y i n t o the v e n t r i c l e , and s e c u r e l y anchored by s u t u r e s t o the lower jaw. The m i d - v e n t r a l i n c i s i o n was c l o s e d c a r e f u l l y w i t h s u t u r e s . A s i d e - a r m , s i m i l a r t o t h a t i n the d o r s a l a o r t i c catheter, a l l o w e d s a m p l i n g o f s a l i n e from the a n t e r i o r venous f l o w which was g o i n g b a c k * i n t o the h e a r t . In a l l o t h e r r e s p e c t s , the p r e p a r a t i o n was as d e s c r i b e d by Wood (1974a).. A f l a r e d tube (9 mm I.D.), used t o d i r e c t water f l o w o v e r the g i l l s , was i n s e r t e d and s e a l e d i n t o the mouth w i t h p l a s t i c i n e . The jaws were s u t u r e d t i g h t l y c l o s e d around t h i s tube, u s i n g ,size-2 s i l k . V e n t i l a t o r y f l o w (.1 L^min" 1) thus was m a i n t a i n e d o v e r the g i l l s a t a l l t i m e s . . The L a t e x r u b b e r d e n t a l R dam s u r r o u n d i n g the body o f the f i s h was s e a l e d w i t h P o l y - G r i p ( B l o c k Drug Co., T o r o n t o ) . T h i s compound was v e r y e f f e c t i v e i n p r e v e n t i n g water l e a k a g e around the s u t u r e s used t o s e c u r e the dam t o the s k i n . 36 F i g u r e 6. S chematic r e p r e s e n t a t i o n o f the e x p e r i m e n t a l a p p a r a t u s used i n i s o l a t e d , s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n s . G i l l s were p e r f u s e d via the c a t h e t e r i n t h e v e n t r a l a o r t a . D o r s a l a o r t i c e f f l u e n t and a n t e r i o r venous e f f l u e n t were c o l l e c t e d from DA and AV c a t h e t e r s r e s p e c t i v e l y . Qp^, d o r s a l a o r t i c o u t f l o w Q^y, a n t e r i o r venous o u t f l o w P. , i n p u t p e r f u s i o n p r e s s u r e DAP, d o r s a l a o r t i c p r e s s u r e W , a d j u s t a b l e Windkessel gas m i x t u r e = 0.4% C 0 9 ; 4.1% 0 9 ; 95.5% N 9 37 38 P e r f u s a t e and P e r f u s i o n P e r f u s a t e used t h r o u g h o u t t h e s e e x p e r i m e n t s was d o u b l e - f i l t e r e d (0.45 urn) C o r t l a n d Salmonid s a l i n e (Wolf, 1963), t o which 40 g « L _ 1 PVP ( p o l y v i n y l p y r r o l i d i n e ; Matheson, Coleman and B e l l , Norwood, O h i o ; av. mol. wt. =» 40,000), a c o l l o i d o s m o t i c f i l l e r , had been added. T h i s c o n c e n t r a t i o n was chosen s i n c e i t approximated the plasma p r o t e i n c o n c e n t r a t i o n found i n f i s h (see Holmes and Donaldson, 1970). S a l i n e r e s e r v o i r s , k e pt a t the e x p e r i m e n t a l t e m p e r a t u r e , were v i g o r o u s l y bubbled w i t h gas m i x t u r e s s e t t o p h y s i o l o g i c a l venous gas t e n s i o n s , d e l i v e r e d v i a Wb'sthoff gas m i x i n g pumps. T h i s m i x t u r e (0.4% C 0 2 ; 4.1% 0 2 ; 95.5% Ng) gave P C Q and P Q v a l u e s s i m i l a r t o t h o s e r e p o r t e d by H o l e t o n and R a n d a l l (1967a) f o r v e n t r a l a o r t i c b l o o d o f S. gairdnevi. The t h r e e i n p u t regimes used t o p e r f u s e the i s o l a t e d t r o u t head p r e p a r a t i o n s were: 1) c o n s t a n t p u l s a t i l e f l o w d e l i v e r e d by a H a r v a r d B l o o d Pump Ha r v a r d Instrument Co., I n c . , Mass.), as m o d i f i e d by Davie and Daxboeck (1981) (see Appendix B). The p r e s s u r e and f l o w p r o f i l e s produced by t h i s pump were found t o be c o i n c i d e n t and s u p e r i m p o s a b l e (see F i g . 12), and s i m u l a t e d in vivo h e a r t a c t i o n . 2) c o n s t a n t p r e s s u r e p e r f u s i o n , from an a d j u s t a b l e p r e s s u r e head r e s e r v o i r , and 3) c o n s t a n t n o n - p u l s a t i l e f l o w , d e l i v e r e d v i a a H a r v a r d I n f u s i o n Pump (Model 976; H a r v a r d Apparatus Co., I n c . , Mass.). E x p e r i m e n t a l P r o t o c o l A f t e r the g i l l s had been c l e a r e d o f b l o o d (see F a r r e l l et a l . , 1979), the f i s h were p l a c e d i n the apparatus (see F i g . 6) and p e r f u s e d a t c o n s t a n t p u l s a t i l e f l o w (17.0 ± 1.0 m l « m i n " ^ k g " 1 ; K i c e n i u k and J o n e s , 1977), 39 a t a v e n t r a l a o r t i c p r e s s u r e o f 10 cm r^O ( s e t by v a r i a b l e W i n d k e s s e l ) , f o r 10 - 15 min. The p u l s e r a t e used was s e t a t 4 0 » m i n _ 1 . Input p r e s s u r e was m o n i t o r e d c o n t i n u o u s l y by a Statham P23Db p r e s s u r e t r a n s d u c e r ( H e w l e t t P a c k a r d ) . T h i s t r a n s d u c e r was c a l i b r a t e d a g a i n s t a s t a t i c column o f w a t e r . P r e s s u r e was r e c o r d e d on a G i l s o n c h a r t r e c o r d e r ( G i l s o n M e d i c a l E l e c t r o n i c s I n c . ) . A f t e r i n p u t p r e s s u r e s had s t a b i l i z e d , i n f l o w , d o r s a l a o r t i c o u t f l o w and a n t e r i o r venous o u t f l o w were c o l l e c t e d f o r f i v e min, and d e t e r m i n e d g r a v j m e t r i c a l l y on a M e t t l e r P1200 t o p - l o a d i n g b a l a n c e . P e r f u s i o n then was changed t o e i t h e r c o n s t a n t p r e s s u r e (regime 2 ) , o r c o n s t a n t non-p u l s a t i l e f l o w (regime 3 ) , and the same v a r i a b l e s measured. D u r i n g c o n s t a n t p r e s s u r e p e r f u s i o n , the i n p u t p r e s s u r e was a d j u s t e d t o match the mean i n p u t p r e s s u r e ( d i a s t o l i c + 1/3 p u l s e ; B u r t o n , 1972) produced by p u l s a t i l e p e r f u s i o n (regime 1). The f l o w r a t e s e l e c t e d f o r c o n s t a n t n o n - p u l s a t i l e f l o w was as near as p o s s i b l e t o 17.0 m l ' m i n - 1 • k g - 1 as c o u l d be o b t a i n e d from t h e range a v a i l a b l e on the H a r v a r d i n f u s i o n pump. F i s h were s e l e c t e d f o r s u i t a b l e w e i g h t s , t o be compatable w i t h f l o w s o b t a i n a b l e from the i n f u s i o n pump. S i n c e f l o w s and p r e s s u r e s were not s i g n i f i c a n t l y d i f f e r e n t f o r each o f the r e g i m e s , p u l s a t i l i t y o f f l o w and p r e s s u r e was the e x p e r i m e n t a l v a r i a b l e . The o r d e r o f p e r f u s i o n regimes was randomized d u r i n g d i f f e r e n t e x p e r i m e n t s . Changes from one p e r f u s i o n regime t o a n o t h e r were a c c o m p l i s h e d w i t h minimal d i s t u r b a n c e t o the p r e p a r a t i o n . In some e x p e r i m e n t s , samples drawn from the v e n t r a l and d o r s a l a o r t i c c a t h e t e r s and the a n t e r i o r venous c a t h e t e r were a n a l y s e d f o r pH, and P n , u s i n g an I.L. M i c r o 13 b l o o d gas a n a l y s e r ( I n s t r u m e n t a t i o n L a b o r a t o r i e s , u 2 Mass.). T o t a l carbon d i o x i d e (Crjc^) i n t h e s e samples as d e t e r m i n e d us the method o f Cameron (1971). P a r t i a l p r e s s u r e s o f CO2 (PQ Q ) were c a l c u l a t e d u s i n g a r e o r g a n i z a t i o n o f t h e H e n d e r s o n - H a s s e l b a c h e q u a t i o n as 40 f o l l o w s : C c o (mM) P l rco2  j ^ a n t i l o g (pH - pK 1) x a C 0 2 + otC0 2 The o p e r a t i o n a l pK 1 v a l u e s o f c a r b o n i c a c i d were o b t a i n e d from S e v e r i n g h a u s et a l . (1956) and the s o l u b i l i t y c o e f f i c i e n t o f C 0 2 ( o t C 0 2 ) was o b t a i n e d from A l b e r s (1970). Data are p r e s e n t e d as means, p l u s o r minus 1 s t a n d a r d e r r o r . R e s u l t s were a n a l y s e d by a one-way a n a l y s i s o f v a r i a n c e , combined w i t h Duncan's M u l t i p l e Range t e s t , and 5% c o n s i d e r e d t h e f i d u c i a l l i m i t o f s i g n i f i c a n c e . FLUID EXCHANGE IN GILL TISSUES For e x p e r i m e n t s d e s i g n e d t o examine f l u i d exchange between p e r -f u s a t e and r e s p i r a t o r y t i s s u e s d u r i n g f l o w and p r e s s u r e c o n d i t i o n s m i m i c k i n g t h o s e found in vivo, a s i m i l a r t r o u t head p r e p a r a t i o n as j u s t d e s c r i b e d above, was employed, w i t h the f o l l o w i n g m o d i f i c a t i o n . V e n t i l a t o r y water c o u l d be s w i t c h e d from the f l o w - t h r o u g h r e s e r v o i r s u p p l y (1 L » m i n _ 1 ) , t o t h e r e c i r c u l a t i o n o f 4 L o f water a t the same f l o w r a t e , w i t h no i n t e r r u p t i o n i n f l o w . T h i s f l o w was m a i n t a i n e d by an i n - l i n e i m p e l l e r - t y p e water pump (see F i g . 6 ) . P e r f u s a t e i n f l o w (Q\//\)> d o r s a l a o r t i c o u t f l o w (Qrj^K and a n t e r i o r venous o u t f l o w (Q^y) were determined as above, d u r i n g p e r f u s i o n w i t h c o n s t a n t p u l s a t i l e f l o w , and c o n s t a n t p r e s s u r e o n l y . In V i v o Marker T e s t s A number o f v i t a l dyes were used i n an a t t e m p t t o mark the e x t r a -v a s c u l a r f l u i d space o f t r o u t g i l l s (see F i g . 7 f o r a d e s c r i p t i o n o f the anato-my). These dyes were i n j e c t e d v i a a d o r s a l a o r t i c c a n n u l a , t o a p p r o x i m a t e a plasma c o n c e n t r a t i o n o f 0.1%. A l t e r n a t e l y , dyes were added t o the e x t e r n a l medium a t a c o n c e n t r a t i o n o f 0.1% and a l l o w e d t o be i r r i g a t e d by the f i s h 41 f o r 1 hour. H i s t o l o g i c a l e x a m i n a t i o n u n d e r " l i g h t m i c r o s c o p y o f f i x e d g i l l t i s s u e s ( S e r r a ' s f i x a t i v e ) , which had been c l e a r e d and embedded i n p a r a f i n ( s e c t i o n a t 10 u m ) , . r e v e a l e d t h a t none of. the dyes t e s t e d was an adequate e x t r a v a s c u l a r marker, r e g a r d l e s s o f a d m i n i s t r a t i o n method. E t h a n o l (EtOH) then was c o n s i d e r e d as a marker because i t i s r a p i d l y taken up by t i s s u e s and e a s i l y m o n i t o r e d . However, e q u i l i b r a t i o n o f e t h a n o l t h r o u g h o u t a l l body t i s s u e s i n g o l d f i s h can t a k e between 3 and 60 hours (E. S h o u b r i d g e , p e r s o n a l communication). The in vivo uptake o f e t h a n o l a c r o s s t he g i l l s o f f i v e rainbow t r o u t was measured from d o r s a l a o r t i c b l o o d samples, removed via c h r o n i c c a n n u l a e , a t 45s, 60s, 90s, 120s, 5 min, and 15 min i n t e r v a l s f o l l o w i n g t h e a d d i t i o n o f e t h a n o l t o the water to make a c o n c e n t r a t i o n o f 3 mM (95% EtOH u s e d ) . A f t e r 60s, plasma e t h a n o l c o n c e n t r a t i o n was a p p r o x i m a t e l y 45% o f water c o n c e n t r a t i o n . A f t e r 15 min, bl o o d e t h a n o l c o n c e n t r a t i o n was 58.4 ± 5.2% o f water c o n c e n t r a t i o n . These d a t a show t h a t measurable amounts o f e t h a n o l were taken up a c r o s s t he g i l l s o f i n t a c t f i s h i n l e s s time than one c i r c u l a t i o n (1 - 2 min) o f b l o o d . through the body ( D a v i s , 1970; Itazawa, 1970). E t h a n o l Washout Curves G i l l t i s s u e s were l o a d e d w i t h e t h a n o l by i n t r o d u c i n g 10 ml 95% EtOH i n t o the r e c i r c u l a t i n g v e n t i l a t o r y water o f . t h e s a l i n e - p e r f u s e d head p r e p a r a t i o n . A f t e r 15 min, v e n t i l a t o r y water was changed from r e c i r c u l a t i n g e t h a n o l s o l u t i o n , t o water from t h e f r e s h water r e s e r v o i r . Samples (.0.3 ml each) o f " i n s p i r a t o r y " water, d o r s a l a o r t i c o u t f l o w , and a n t e r i o r venous o u t f l o w were taken a t time z e r o (.15 min a f t e r EtOH i n t r o d u c t i o n ) , 30s, 60s, 90s, 120s, 5 min, 10 min, and 15 min a f t e r the r e t u r n o f f l o w - t h r o u g h water. These samples were f r o z e n and s t o r e d f o r subsequent d e t e r m i n a t i o n o f e t h a n o l c o n c e n t r a t i o n s . Input p e r f u s a t e ( v e n t r a l a o r t i c ) samples a l s o were t a k e n , and s e r v e d as b l a n k s . 42 F i g u r e 7. Schematic c r o s s - s e c t i o n through t h e s e c o n d a r y l a m e l l a e and f i l a m e n t o f a t e l e o s t g i l l , t o show v a s c u l a r and e x t r a -v a s c u l a r spaces which c o u l d have c o n t a i n e d t h e v i t a l dyes t e s t e d (redrawn from R a n d a l l et a l . , 1981). 43 S E C T I O N T H R O U G H A S E C O N D A R Y L A M E L L A epithel'a blood channel with R B C flanged pillar cell extracellular or lymph space chloride and mucous cells body of filament pocket valve epithelium around central veno-lymphatie sinus 44 Et h a n o l was assayed a c c o r d i n g t o the method o f B e r n t and Gutmann (1974), on d u p l i c a t e 50 y l samples. The f o r m a t i o n o f a c e t a l d e h y d e from e t h a n o l was f o l l o w e d by measuring the i n c r e a s e i n a b s o r p t i o n a t 340 nm on a s p e c t r o p h o t o m e t e r (PYE Unicam SP3-200 U.V.), due t o the f o r m a t i o n o f 3NADH (see 1). (1) E t h a n o l + 3NAD ^==================^ A c e t a l d e h y d e + BNADH + H + (see f o o t n o t e 1) Chemicals used i n the above assay were o b t a i n e d from Sigma Chemical Company. Washout c u r v e s were p l o t t e d on s e m i l o g graph paper and s t a n d a r d c u r v e - p e e l i n g t e c h n i q u e s were a p p l i e d ( S h i p p l e y and C l a r k , 1972). A l l EtOH c o n c e n t r a t i o n s were n o r m a l i z e d , the i n i t i a l c o n c e n t r a t i o n ( a f t e r 15 min EtOH i n t r o d u c t i o n ) , b e i n g s e t t o equal 1.0, t o f a c i l i t a t e compartmental a n a l y s i s . Data are p r e s e n t e d as mean ± s t a n d a r d e r r o r o f the mean. The f i d u c i a l l i m i t o f s i g n i f i c a n c e was s e t a t 5% f o r a l l s t a t i s t i c a l t e s t s . I I I . SPONTANEOUSLY VENTILATING, BLOOD-PERFUSED TROUT PREPARATION A. CHARACTERIZATION OF THE PREPARATION A l l f i s h used were the same as those d e s c r i b e d p r e v i o u s l y . Experiments were c a r r i e d out a t 7°C. Blood C o l l e c t i o n and P r e p a r a t i o n Donor f i s h were a n a e s t h e t i z e d w i t h 1:15,000 w/w a e r a t e d MS 222 s o l u t i o n (pH a d j u s t e d t o 7.0 - 7.5 w i t h Sodium b i c a r b o n a t e ) and then t r a n s f e r r e d t o an o p e r a t i n g t a b l e (Smith and B e l l , 1964), as i n p r e v i o u s c a s e s . To f a c i l i t a t e b l o o d w i t h d r a w a l , f i s h were i m p l a n t e d w i t h c h r o n i c i n d w e l l i n g d o r s a l a o r t i c cannulae ( S m i t h , 1978), and a l l o w e d t o r e c o v e r f o r at l e a s t 24 hr i n darkened Perspex boxes, s u p p l i e d w i t h r u n n i n g a e r a t e d F o o t n o t e 1: ADH = NAD = NADH = a l c o h o l dehydrogenase n i c o t i n e a m i d e adenine d i n u c l e o t i d e r e d u c e d NAD 45 water (7°C). G e n e r a l l y , 12 f i s h were c a n n u l a t e d and would s u p p l y enough b l o o d f o r two p e r f u s i o n e x p e r i m e n t s . B l o o d was c o l l e c t e d from donor f i s h i m m e d i a t e l y p r i o r to each p e r f u s i o n i n t h e f o l l o w i n g manner. A p p r o x i m a t e l y 3 ml C o r t l a n d s a l i n e (as d e s c r i b e d p r e v i o u s l y ) c o n t a i n i n g 2000 U.S.P. u n i t s o f sodium h e p a r i n were i n j e c t e d i n t o t h e d o r s a l a o r t i c c a n n u l a , and f o l l o w i n g a 5 min m i x i n g p e r i o d , b l o o d was withdrawn a n a e r o b i c a l l y . T y p i c a l l y , 10 ml o f b l o o d c o u l d be o b t a i n e d from each f i s h . u s i n g t h i s t e c h n i q u e . A f i n a l b l o o d volume o f a p p r o x i m a t e l y 100 ml u s u a l l y was r e -q u i r e d f o r a s i n g l e p e r f u s i o n p r e p a r a t i o n ' and was p r e p a r e d by d i l u t i n g donor b l o o d w i t h s a l i n e t o a d e s i r e d h a e m a t o c r i t o f 10 - 12%. B l o o d then was d i v i d e d i n t o t h r e e o r f o u r tonometer f l a s k s . These were shaken . c o n t i n u o u s l y ( B u r r e l l w r i s t - a c t i o n s h a k e r ) w i t h g a s m i x t u r e s (o.4% CO2 i n a i r ; see Haswell et a l . , 1979) c l o s e l y r e s e m b l i n g t r o u t venous b l o o d gas t e n s i o n s . These m i x t u r e s were s u p p l i e d by W o s t h o f f gas m i x i n g pumps. S u r g i c a l P r o c e d u r e s A f i s h c a n n u l a t e d the previous'day and which had a p a t e n t d o r s a l a o r t i c c a n n u l a was a n a e s t h e t i z e d i n a 1:15,000 MS 222 s o l u t i o n . A second c a n n u l a (PE 50) was i m p l a n t e d i n t h e b u c c a l c a v i t y as d e s c r i b e d by Saunders (1961) t o m o n i t o r v e n t i l a t o r y movements. The f i s h was t r a n s f e r r e d t o the o p e r a t i n g t a b l e where the g i l l s were, i r r i g a t e d t h r o u g h o u t the o p e r a t i o n w i t h a e r a t e d 1:20,000 MS 222 e i t h e r o r t h o g r a d e from a tube i n the mouth, o r r e t r o g r a d e , from tubes i n the o p e r c u l a r o p e n i n g s . . The f i s h was l a i d s u p i n e and the p e r i c a r d i u m exposed b y . c u t t i n g the s k i n above the h e a r t and c a r e f u l l y p a r t i n g the h y p a x i a l m u s c u l a t u r e down the m i d l i n e . Any s m a l l v e s s e l s which b t e d i n t o the opening were c a u t e r i z e d c l o s e d w i t h a B r i t c h e r e l e c t r o s e c t i 1 i s u n i t ( B i r t c h e r Corp., Los A n g e l e s ) . The p e r i c a r d i u m was opened and two 3-0 s i l k t h r e a d s were p l a c e d around the bulbous a r t e r i o s u s . H e r p a r i n (2000 U.S.P. u n i t s i n 2 ml 46 s a l i n e ) then was i n j e c t e d i n t o the b l o o d v i a the d o r s a l a o r t i c c a n n u l a and a l l o w e d to c i r c u l a t e f o r 5 min. As much b l o o d as p o s s i b l e then was w i t h -drawn from the d o r s a l a o r t a and d i s c a r d e d . The v e n t r a l a o r t i c i n p u t c a t h e t e r c o n s i s t e d o f 2.5.cm o f s i l a s t i c r u b b e r t u b i n g (1.45 mm x 2.30 mm; S a r g e n t Welch S c i e n t i f i c ) a t t a c h e d t o a cu r v e d 13 gauge hypodermic .needle s h a f t . The c a t h e t e r was co n n e c t e d t o a r e s e r v o i r o f C o r t l a n d s a l i n e (on i c e ) t o which 40 g * L _ 1 P.V;P. and 1 x 10"^ M n o r a d r e n a l i n e ( f r e e base, Sigma Chem. Co.) had been added. As b e f o r e , s a l i n e was f i l t e r e d through Whatman No. 5 paper and M i l l i p o r e d i s c s (0.45 ym). The bulbus was c u t j u s t caudad o f the midpoint: and the v e n t r a l a o r t i c c a t h e t e r i n s e r t e d ( F i g . 8) and t i e d i n p l a c e w i t h one o f the t h r e a d s , u s i n g a 2+2+1 knot ( T e r a and AuberJ>, 1976). The p e r f u s i o n f l o w was s t a r t e d and the f i s h was p e r f u s e d a t a p r e s s u r e o f 50 cm H^O, t o c l e a r a l l v e s s e l s o f b l o o d . A f t e r e x s a n g u i n a t i o n , the p e r f u s i o n f l o w was redu c e d and t h e r e t r o -grade (venous r e t u r n ) c a t h e t e r i n s e r t e d . The venous r e t u r n c a t h e t e r was a h e a t - f l a r e d p i e c e o f PE 200 t u b i n g (1.4 mm x 1.9 mm) which.was i n s e r t e d i n t o the bulbus through the same c u t as the i n p u t c a t h e t e r and t i e d i n t o p l a c e w i t h a s i m i l a r knot as b e f o r e ( F i g . 8 ) . T h i s c a t h e t e r was found t o o f f e r v e r y l i t t l e r e s i s t a n c e t o "venous" b l o o d r e t u r n and "normal" h e a r t f u n c t i o n , under the p r e s e n t c i r c u m s t a n c e s . . P e r f u s i o n was resumed w h i l e c a t h e t e r s were anchored t o the body w a l l a n d t h e i n c i s i o n c l o s e d w i t h s u t u r e s . The f i s h : was t r a n s f e r r e d t o a van Dam-type box (see Davis and Cameron, 1971), and b l o o d p e r f u s i o n was s t a r t e d . Water f l o w o v e r the g i l l s was m a i n t a i n e d d u r i n g r e c o v e r y . f r o m t h e o p e r a t i o n by.a tube i n the mouth. The o p e r a t i o n took, on average, 45 min. I n t e r r u p t i o n s t o p e r f u s i o n w i t h e i t h e r s a l i n e o r b l o o d were l e s s than one minute. D u r i n g s a l i n e p e r f u s i o n on the o p e r a t i n g t a b l e , f i s h o f t e n e x h i b i t v e n t i l a t o r y movements, a 47 F i g u r e 8. Schematic s a g i t t a l s e c t i o n through the h e a r t o f the t r o u t , t o i l l u s t r a t e c a t h e t e r p o s i t i o n s used f o r b l o o d - p e r f u s i o n e x p e r i m e n t s . Top o f t h e page i s d o r s a l , r i g h t i s c e p h a l i d . A , a t r i u m B , bulbus a r t e r i o s u s IC , i n p u t c a t h e t e r i n t o v e n t r a l a o r t a , l e a d i n g t o g i l l s VRC, venous r e t u r n c a t h e t e r , f l o w a i d e d by v e n t r i c u l a r c o n t r a c t i o n s Arrows w i t h i n lumena o f tubes and h e a r t chambers i n d i c a t e d i r e c t i o n o f b l o o d f l o w s . 48 49 p a r t i c u l a r l y c o u g h i n g . Once p l a c e d i n t o the box and p e r f u s e d w i t h b l o o d , e q u i l i b r i u m was r e g a i n e d and r e g u l a r v e n t i l a t i o n resumed w i t h i n 30 min. F i s h were l e f t 2 - 3 h t o r e c o v e r from t h e acu t e e f f e c t s o f a n a e s t h e s i a b e f o r e any experiments commenced. Blo o d P e r f u s i o n B l o o d was h e l d i n t h r e e or f o u r tonometer f l a s k s . Each t o n o -meter, c o n t a i n i n g a p p r o x i m a t e l y 30 ml o f b l o o d y had a p o l y e t h y l e n e tube (PE 160) l e a d i n g t o a s e t o f 3-way taps t o e n a b l e b l o o d t o be drawn i n t o a c a r d i a c pump. These taps a l l o w e d s w i t c h i n g from a n y . f l a s k t o any o t h e r w i t h o u t i n t e r r u p t i o n o f p e r f u s i o n . The c a r d i a c pump used, as d e s c r i b e d i n the p r e v i o u s methods (II),, w a s . a c c u r a t e t o w i t h i n 1% o f g r a v i m e t r i c e s t i m a t e s o f the f l o w r a t e (Q)., and was n o t p r e s s u r e s e n s i t i v e . T h i s pump a l l o w e d independent adjustments o f f r e q u e n c y and s t r o k e volume (SV) t o be made, w i t h o u t i n t e r r u p t i o n s t o the f l o w . P u l s e p r e s s u r e was a d j u s t e d by c h a n g i n g t he s i z e o f the gas space a t the top o f a wide-bore s i d e - a r m ( W i n d k e s s e l ) i n the p e r f u s i o n l i n e ( F i g . 9 ) . Bl o o d was pumped i n t o t h e v e n t r a l a o r t a through the i n p u t c a t h e t e r and c i r c u l a t e d through the e n t i r e body. D e s p i t e two c a t h e t e r s i n the b u l b u s , v e n t r i c u l a r c o n t r a c t i o n s were m a i n t a i n e d and pumped venous r e t u r n b l o o d back t o the tonometers via the wide-bore s i l a s t i c r u b b e r tube (1.97 mm x 3.05 mm). P e r f u s i o n f l o w was a d j u s t e d by a l t e r i n g s t r o k e volume a t a c a r d i a c pump f r e q u e n c y o f 40 s t r o k e s * m i n " 1 , t o t h a t n e c e s s a r y t o m a i n t a i n d o r s a l a o r t i c p r e s s u r e a t 40 cm h^O. T h i s f l o w r a t e was always about 1 6 - 1 7 m l * m i n " 1 , k g - 1 . Blood Sampling and A n a l y s i s A p p r o x i m a t e l y 0.7 ml o f b l o o d were withdrawn s i m u l t a n e o u s l y from each o f the t h r e e sampling s i t e s ( i n p u t , DA and VR). Samples were s e a l e d 50 F i g u r e 9. Schematic r e p r e s e n t a t i o n o f t h e i n s t r u m e n t a t i o n used to m o n i t o r v a r i a b l e s from b l o o d - p e r f u s e d t r o u t . Q, f l o w r e c o r d from c a r d i a c pump d i s p l a y e d on c h a r t r e c o r d e r P, p r e s s u r e r e c o r d s , from top t o bottom, r e c o r d e d from: i n p u t c a t h e t e r ( v e n t r a l a o r t a , VAP.) d o r s a l a o r t a (DAP) b u c c a l p r e s s u r e ( f o r b r e a t h i n g r a t e ) venous r e t u r n c a t h e t e r ( f o r i n t r i n s i c h e a r t r a t e ) d i s p l a y e d on c h a r t r e c o r d e r S,- b l o o d s a m p l i n g s i t e s VR, venous r e t u r n t o tonometer f l a s k s c o n t a i n i n g b l o o d W,- Windkessel t o a d j u s t p u l s e p r e s s u r e o f i n p u t Arrows i n d i c a t e d i r e c t i o n o f b l o o d f l o w . Note: See Appendix C f o r photographs o f s e t u p in situ. 51 52 and s t o r e d on i c e d u r i n g t h e a n a l y s i s p e r i o d . G e n e r a l l y , i n p u t b l o o d was a n a l y s e d f i r s t , f o l l o w e d by d o r s a l a o r t a and venous r e t u r n . A n a l y s i s was completed w i t h i n 15 min o f samp l i n g and no measured b l o o d v a r i a b l e was found t o change d u r i n g t h i s p e r i o d . pH and P n measurements were made u 2 u t i l i z i n g an I n s t r u m e n t a t i o n L a b o r a t o r i e s M i c r o 13 pH/blood gas a n a l y s e r . T o t a l carbon d i o x i d e (C^Q ) and t o t a l oxygen (Cg ) c o n t e n t s were d e t e r -mined u s i n g the methods o f Cameron (1971) and T u c k e r (1967) r e s p e c t i v e l y , w i t h a Radiometer Copenhagen PHM 71 d i g i t a l a c i d / b a s e a n a l y s e r and : a s s o c i a t e d C O 2 and p o l a r o g r a p h i c C^ e l e c t r o d e s . A l l pH and PQ measurements were performed a t ambient water t e m p e r a t u r e , w h i l e c o r r e s p o n d i n g C^Q and C n d e t e r m i n a t i o n s were made a t 45°G, t o speed up the re s p o n s e time o f the u 2 e l e c t r o d e s used. P a r t i a l p r e s s u r e o f carbon d i o x i d e (P^Q ) and b i c a r b o n a t e c o n c e n t r a t i o n s were c a l c u l a t e d u s i n g the measured pH and C^Q v a l u e s , and the r e o r g a n i z a t i o n o f the Henderson-Hasselbach e q u a t i o n as d e s c r i b e d p r e v i o u s l y on page 4 0 . For s i m p l i c i t y , o n l y C^Q v a l u e s a r e r e p o r t e d i n the r e s u l t s , w h i l e HCO^" v a l u e s have been o m i t t e d . A t p h y s i o l o g i c a l pH, HCOg" c o n c e n t r a t i o n (where [[HCO^-!! = C^Q - ((oi-C^) x (P'^Q )) ) comprises a p p r o x i m a t e l y 95% o f b l o o d C^Q , and as such C^Q i s a r e a s o n a b l e a p p r o x i -mation o f b i c a r b o n a t e c o n c e n t r a t i o n . F o l l o w i n g a n a l y s i s , a sm a l l p o r t i o n o f b l o o d was used t o dete r m i n e h a e m a t r o c r i t (Hct) and any r e m a i n i n g b l o o d was r e t u r n e d t o the tonometer, or c e n t r i f u g e d and f r o z e n , so t h a t plasma samples c o u l d be a n a l y s e d a t a l a t e r d a t e . Plasma c h l o r i d e was determined w i t h a B u c h l e r - C o t l o v e amperometric t i t r a t o r and o s m o l a r i t y was measured u s i n g an Osmette f r e e z i n g p o i n t d e p r e s s i o n osmometer. P r e s s u r e and Flow R e c o r d i n g P r e s s u r e s ( i n p u t , d o r s a l a o r t i c , v e n t r i c u l a r and b u c c a l ) a l l were measured u s i n g Statham P23Db p r e s s u r e t r a n s d u c e r s , c a l i b r a t e d a g a i n s t a s t a t i c c o l u m r o f water. The water l e v e l above the f i s h was taken as z e r o . 53 Mean p r e s s u r e s were c a l c u l a t e d as i n t h e p r e v i o u s s e c t i o n . The p r e s s u r e drop a c r o s s the i n p u t c a t h e t e r was measured, w i t h l i g a t u r e s s t i l l i n p l a c e , a f t e r each experiment. C a t h e t e r " r e s i s t a n c e " was used to c o r r e c t measured i n p u t p r e s s u r e s f o r each p r e p a r a t i o n . I n f l o w was measured w i t h an IVM b l o o d f l o w t r a n s d u c e r and B i o -t r o n i x 610, e l e c t r o m a g n e t i c p u l s e d - l o g i c a m p l i f i e r s (In V i v o M e t r i c Systems, Los Angeles B i o t r o n i x Lab., M a r y l a n d , U:S;A.). Each o f the p r e s s u r e and f l o w s i g n a l s was a m p l i f i e d and d i s p l a y e d on. a Brush s i x - c h a n n e l r e c o r d e r (Gould I n c . , C l e v e l a n d , Ohio) (See F i g . 9 ) . A l l o f the p r e s e n t experiments were conducted a t 7. ± 1°C and temperature d i d not change d u r i n g any one e x p e r i m e n t . A l l d a t a are p r e s e n t e d as means ± s t a n d a r d e r r o r , from the mean. Where a p p r o p r i a t e , r e s u l t s were s t a t i s t i c a l l y a n a l y s e d u s i n g S t u d e n t ' s t - t e s t , and 10% o r 5% was t a k e n as the f i d u c i a l : l i m i t o f s i g n i f i c a n c e . B.„ EFFECTS OF HAEMODYNAMIC CHANGES ON RESISTANCES AND OXYGEN UPTAKE F i s h used f o r t h i s s e r i e s o f e x p e r i m e n t s were m a i n t a i n e d and s u r g i c a l l y p r e p a r e d as. d e s c r i b e d above. The c a r d i a c pump a l l o w e d the way i n which b l o o d was d e l i v e r e d t o the g i l l s t o be a l t e r e d . A s e t o f normal b l o o d samples was taken and a n a l y s e d b e f o r e any haemodynamic m a n i p u l a t i o n s . Then, changes were made w i t h no i n t e r r u p t i o n i n p e r f u s i o n f l o w , and a 5 min e q u i l i b r a t i o n . and s t a b i l i z a t i o n p e r i o d was a l l o w e d b e f o r e a n o t h e r s e t o f samples was taken f o r a n a l y s i s . The s a m p l i n g method and a n a l y s e s were the same as d e s c r i b e d i n 'the above s e c t i o n . The a l t e r a t i o n s performed were as f o l l o w s : 1) i n c r e a s i n g c a r d i a c o u t p u t (Q) by 1.5 times normal, by d e c r e a s i n g s t r o k e volume ( S V ) , w h i l e the d e l i v e r y f r e q u e n c y was kept normal ( f = 40 bpm)*; 2) d e c r e a s i n g c a r d i a c o u t p u t by 0.5 times normal, by d e c r e a s i n g s t r o k e volume, w h i l e the pump f r e q u e n c y was kept n o r m a l * ; 54 3) i n c r e a s i n g s t r o k e volume and d e c r e a s i n g t he pump f r e q u e n c y , so t h a t c a r d i a c o u t p u t remained c l o s e t o normal ( f = 20 bpm; SV = 2 x S V n ) , thus s i m u l a t i n g h y p o x i c b r a d y c a r d i a i n normoxic water*; 4) d e c r e a s i n g s t r o k e volume and i n c r e a s i n g pump f r e q u e n c y so t h a t c a r d i a c o u t p u t remained c l o s e t o normal ( f = 60 bpm; SV = 0.5 -x SV . ) * ; 5) i n c r e a s i n g i n p u t p u l s e p r e s s u r e to a p p r o x i m a t e l y 2 times normal p u l s e p r e s s u r e r e c o r d e d from t h e v e n t r a l a o r t a , independent o f any changes i n c a r d i a c o u t p u t o r pump f r e q u e n c y (40 bpm); 6) i n c r e a s i n g i n p u t h a e m a t o c r i t a p p r o x i m a t e l y 2 times t h a t o f "normal" b l o o d (2 x-Hct )•., w h i l e k e e p i n g a l l o t h e r c o n d i t i o n s normal, by adding r e d b l o o d c e l l s ( r b c ' s ) t o a tonometer; 7) d e c r e a s i n g i n p u t h a e m o t o c r i t t o a p p r o x i m a t e l y 4%, w h i l e keeping a l l o t h e r c o n d i t i o n s normal, by removing r b c ' s and ad d i n g plasma t o a tonometer; 8) a d d i n g 1 x 10" M f i n a l cone, o f a d r e n a l i n e (Sigma; f r e e base) t o a tonometer, w h i l e c a r d i a c o u t p u t and method o f d e l i v e r y were kept normal. The above sequence o f haemodynamic a l t e r a t i o n was a l t e r n a t e d among the p r e p a r a t i o n s , and no d i f f e r e n c e s due t o s e q u e n c i n g was no t e d i n any measured b l o o d v a r i a b l e . A e r a t e d water f l o w t o t h e . p r e p a r a t i o n , was m a i n t a i n e d t h r o u g h -out t h e s e m a n i p u l a t i o n s , s u p p l i e d from.a f l o w - t h r o u g h r e s e r v o i r ( 7 ° C ) . D i r e c t Measurement o f Oxygen Uptake and V e n t i l a t o r y Flow A c r o s s the G i l l s : A Comparison o f C a r d i a c Output by D i r e c t and F i c k Methods F i s h used f o r t h e s e experiments (N = 4; 340.1 ± 13.2 g) were m a i n t a i n e d and s u r g i c a l l y p r e p a r e d as above, w i t h the f o l l o w i n g e x c e p t i o n . In o r d e r t o measure v e n t i l a t o r y water f l o w (V ) d i r e c t l y , a t h i n L a t e x surgeon's g l o v e 08 1/2) was s u t u r e d (3-0 s i l k ) around the mouth o f a f i s h i n o r d e r t o a v o i d i n t e r f e r i n g w i t h movements o f the b r a n c h i a l a p p a r a t u s . * P u l s e p r e s s u r e s were not a d j u s t e d t o a c o n s t a n t normal l e v e l . 55 When a f i s h was c o n f i n e d w i t h i n the h o l d i n g a p p a r a t u s , t h i s dam e f f e c t i v e l y s e p a r a t e d i n s p i r e d and e x p i r e d water, and thus v e n t i l a t o r y f l o w c o u l d be measured v o l u m e t r i c a l l y from t h e o v e r f l o w i n the caudal compartment ( F i g . 1 0 ) . During r e c o v e r y from a n a e s t h e s i a , the water l e v e l i n the c e p h a l i c compartment was r a i s e d a p p r o x i m a t e l y 2 cm above t h a t i n the caudal compartment, t h e r e b y m a i n t a i n i n g a p o s i t i v e water f l o w (approx... 750 ml •min"1:) o v e r t he g i l l s . When dye was added to the water i n the c e p h a l i c compartment t h e r e was no i n d i c a t i o n ^ ' o f leakage around t he s u t u r e s between the dam and s k i n . Once r e g u l a r spontaneous v e n t i l a t o r y movements were e s t a b l i s h e d , water l e v e l s i n the two compartments were kept equal f o r t he d u r a t i o n o f e x p e r i m e n t a t i o n . In a l l other, r e s p e c t s , the a p p r a r a t u s was i d e n t i c a l t o t h a t a l r e a d y d e s c r i b e d by Davis and Cameron . ( 1 9 7 1 ) . I n s p i r e d water ( f o r P T measurements) was t a k e n one cm i n f r o n t X 0 2 o f the mouth o f the f i s h . E x p i r e d water samples ( f o r P F measurements) were drawn from one cm i n s i d e t h e gap a t the back o f the'box c o n f i n i n g the f i s h . Other e x p i r e d water s a m p l i n g s i t e s have been found t o be u n s a t i s -f a c t o r y (see Davis and M a t t e r s , 1970; Davis and Cameron, 1971) . Once out o f the operculum, e x p i r e d water c o u l d emerge on:ly from t h i s gap, as the t i g h t l y f i t t i n g c o v e r o v e r the f i s h allowed,no exchange w i t h water from the r e s t o f the a p p a r a t u s . A t t h e same tim e as water samples were t a k e n , b l o o d samples a l s o were withdrawn and a n a l y s e d as b e f o r e . Vg and the r e s p i r a t o r y f r e q u e n c y ( f ) were determined a t t h i s time as w e l l . 9 I n f l o w i n g water passed through a gas exhange column, and a i r was bubbled through the water (.P[Q" * = 151.4 ± 0 . 8 mm Hg).. A l l d a t a a r e a n a l y s e d and p r e s e n t e d as d e s c r i b e d i n the p r e v i o u s s e c t i o n . 56 F i g u r e 10. Diagrammatic r e p r e s e n t a t i o n o f the m o d i f i e d van Dam a p p a r a t u s used i n the p r e s e n t e x p e r i m e n t s . The L a t e x dam s e p a r a t e d i n s p i r a t o r y water from e x p i r e d w a t e r , such t h a t the o v e r f l o w i n the caudal compartment o f the h o l d i n g chamber was t h a t volume o f water which the f i s h v e n t i l a t e d o v e r i t s g i l l s (Vg). A c o n s t a n t s u p p l y o f a e r a t e d water a t a r a t e f a r i n e x c e s s o f the v e n t i l a t i o n volume was m a i n t a i n e d . 57 overflow = Vg 5 cm 57a S E C T I O N I BLOOD VOLUME AND FLOW D I S T R I B U T I O N DURING E X E R C I S E 58 INTRODUCTION: SECTION I In the g e n e r a l i n t r o d u c t i o n I p o i n t e d out t h a t i n mammals d u r i n g e x e r c i s e , b l o o d f l o w t o the working s k e l e t a l muscles can i n c r e a s e two t o t h r e e times above the i n c r e a s e measured i n the t o t a l c a r d i a c o u t p u t . That t h e r e must be r e d i s t r i b u t i o n o f b l o o d f l o w e l s e w h e r e i n the body i s o b v i o u s , and the s p l a n c h n i c c i r c u l a t i o n was noted as th e most p r o b a b l e s i t e o f blo o d f l o w and p r e s s u r e c o n t r o l d u r i n g e x e r c i s e . P r e v i o u s l y , Stevens and R a n d a l l (1967a) had r e c o r d e d b l o o d p r e s s u r e and f l o w changes i n the sub-i n t e s t i n a l v e i n o f rainbow t r o u t d u r i n g e x e r c i s e , and a t t h a t time s u g g e s t e d t h a t t h e s e changes a l s o were a s s o c i a t e d w i t h the s h u n t i n g o f b l o o d away from one t i s s u e towards a n o t h e r . Stevens (1968a) n o n e t h e l e s s found no s i g n i f i c a n t changes i n the b l o o d volume d i s t r i b u t i o n t o d i f f e r e n t organs between r e s t i n g and e x e r c i s i n g t r o u t , e x c e p t f o r a volume r e d u c t i o n i n the s p l e e n . In c o n t r a s t , no s t u d i e s have been c o n d u c t e d , t o d a t e , which i n v e s t i g a t e the p o s s i b i l i t y o f b l o o d flow r e d i s t r i b u t i o n d u r i n g e x e r c i s e i n f i s h , as i s known to o c c u r i n mammals. The e x p e r i m e n t s i n t h i s s e c t i o n t h e r e f o r e a r e d e s i g n e d to examine not o n l y the d i s t r i b u t i o n o f t i s s u e b l o o d volume, b u t a l s o t h e d i s t r i b u t i o n o f c a r d i a c o u t p u t ( b l o o d . f l o w ) t o v a r i o u s organs i n the s y s t e m i c c i r c u l a t i o n o f rainbow t r o u t a t r e s t , and d u r i n g a e r o b i c , s t e a d y - s t a t e e x e r c i s e a t 80% o f t h e i r e s t i m a t e d c r i t i c a l swimming speed. These experiments were c a r r i e d out u s i n g . v a r i o u s r a d i o t r a c e r t e c h n i q u e s , t o a s s e s s the c i r c u l a t o r y p a t t e r n o f t r o u t f o r c e d t o swim a g a i n s t a water c u r r e n t i n a B r e t t - t y p e t u n n e l r e s p i r o m e t e r . From t h e d a t a o b t a i n e d , p o s s i b l e u n d e r l y i n g mechanisms f o r the c o n t r o l o f s y s t e m i c c i r c u l a t i o n a t r e s t and d u r i n g swimming e x e r c i s e i n rainbow t r o u t , Salmo gaivdnevi, a r e d i s c u s s e d . 59 RESULTS I B l o o d Volumes and Flow D i s t r i b u t i o n T a b l e 4 l i s t s r e p r e s e n t a t i v e p e r c e n t a g e s o f t o t a l body w e i g h t OC f o r v a r i o u s t i s s u e s sampled^ from f i s h used i n the R b - i n j e c t i o n , c r i t i c a l swimming v e l o c i t y e x p e r i m e n t s . Whether v a l u e s were determined d i r e c t l y ( g i v e n as means ± S.E.M.), o r taken from the l i t e r a t u r e , those l i s t e d i n t h i s T a b l e were used i n the p r e s e n t s t u d y f o r t h e c a l c u l a t i o n s o f t o t a l counts per minute (cpm). i n each whole t i s s u e and w i t h i n the whole body, a t r e s t and 51 d u r i n g e x e r c i s e . Normal r e s t i n g t r o u t were i n j e c t e d w i t h 1.0 ml C r - l a b e l l e d b l o o d c e l l s u s p e n s i o n which had a n . h a e m a t o c r i t o f 52.3 ± 10.1%, and a t o t a l a c t i v i t y o f 70,094 ± 8,723 cpm: The i n j e c t i o n o f ^ G r - l a b e l l e d r e d b l o o d .. c e l l s a l l o w e d the blood.volume d i s t r i b u t i o n i n the v a r i o u s t i s s u e s t o be a s s e s s e d , a t r e s t . These d a t a i n d i c a t e d a r e s t i n g b l o o d volume o f 6.08 ± . 1.31% o f t o t a l body we i g h t , as d e t e r m i n e d by the degree o f d i l u t i o n o f r a d i o a c t i v i t y i n the d o r s a l a o r t i c b l o o d samples a f t e r 2 hours o f e q u i l i b r a -t i o n . A l s o , the i n j e c t i o n o f 1 ml o f h i g h h a e m a t o c r i t , l a b e l l e d b l o o d had minimal e f f e c t s on the measured p o s t - e q u i l i b r a t i o n d o r s a l a o r t i c Hct ( T a b l e 4 ) . A t o t a l o f 87.3% o f the body weight o f the f i s h was a c c o u n t e d f o r from measurements made and p r e s e n t e d i n T a b l e 4. From t h i s , t he p e r c e n t a g e o f t o t a l b l o o d volume acc o u n t e d f o r by the t i s s u e s l i s t e d was 2.43% o f body weight. T h i s volume however, d i d n o t take i n t o account the b l o o d volume c o n t a i n e d w i t h i n the h e a r t c a v i t i e s , v e n t r a l a o r t a , a r t e r i e s , the C u v e r i a n s i n u s and the v e i n s . A n o t h e r 12.7% o f the body weight a l s o was s t i l l un-a c c o u n t e d f o r . I f t h i s 12.7% o f the r e m a i n i n g t i s s u e s were as p o o r l y v a s c u l a r i z e d as the mosaic muscle, then o n l y 2.54% body wei g h t v a l u e f o r the t o t a l b l o o d volume i s i n d i c a t e d . However, t h i s v a l u e o n l y r e p r e s e n t s a p p r o x i m a t e l y 40% o f the t o t a l b l o o d volume, c o n t a i n e d i n t h e c a p i l l a r y n e t -work, a r t e r i o l e s and s m a l l v e i n s o f the sampled t i s s u e s . When t h i s was 60 taken i n t o a c c o u n t , then the c o r r e c t e d t o t a l b l o o d volume from the f i s h used h e r e , u s i n g i n f o r m a t i o n from T a b l e 4, was 6.35% body w e i g h t , o r 6.35 ml-lOOg" 1. T h i s v a l u e compared w e l l w i t h independent v a l u e s o b t a i n e d from 51 d o r s a l a o r t i c b l o o d samples and Cr a c t i v i t y i n the b l o o d (6.08 m l » 1 0 0 g _ 1 ) . The d a t a i n T a b l e 4 a l s o i n d i c a t e d t h a t r e d muscle a t r e s t c o n t a i n e d an. average 2.6 times the b l o o d volume p e r gram t i s s u e o f mosaic muscle. A l s o i n d i c a t e d was t h e . f a c t t h a t the s p l e e n c o n t a i n e d the most b l o o d p e r gram t i s s u e o f any o t h e r organ, and approximated 0.7% o f the c a l c u l a t e d t o t a l b l o o d volume. T h i s organ o n l y a c c o u n t e d f o r 0.23% o f the body weight. There was a measurable d e c r e a s e i n the a p p a r e n t t o t a l b l o o d volume d u r i n g e x e r c i s e , which was accompanied by. a c o n c o m i t a n t h a e m o c o n c e n t r a t i o n , as i n d i c a t e d by an i n c r e a s e i n the. d o r s a l a o r t i c h a e m a t o c r i t o f 32.9 ± 9.0% above t h a t found a t r e s t . F i s h which were f o r c e d t o swim a t 80% o f t h e i r 51 c r i t i c a l v e l o c i t y were, i n j e c t e d . n o t o n l y w i t h C r - l a b e l l e d r e d c e l l s so t h a t t i s s u e b l o o d volumes c o u l d . b e d e t e r m i n e d a t 80% U ^ ^ , but a l s o were i n j e c t e d w i t h m i c r o s p h e r e s , f o r the d e t e r m i n a t i o n o f b l o o d f l o w d i s t r i b u t i o n s . However, because o f c r o s s - c h a n n e l counts i n t e r f e r e n c e (see T a b l e 3 ) , o n l y t h o s e counts above t h i s i n t e r f e r e n c e c o u l d be i n c l u d e d i n T a b l e 5. These d a t a i n d i c a t e d t h a t b l o o d volumes i n the l i v e r , . s p l e e n , k i d n e y and stomach were lower d u r i n g e x e r c i s e than they were a t r e s t . N o n e t h e l e s s , o n l y s p l e n i c and r e d muscle b l o o d volumes appeared t o change s i g n i f i c a n t l y d u r i n g e x e r c i s e a t 80% U c r i - t , w h i l e o t h e r t i s s u e b l o o d volumes remained unchanged from r e s t i n g v a l u e s . The p e r c e n t o f c a r d i a c o u t p u t d i s t r i b u t e d ' t o v a r i o u s t i s s u e s i n the r e s t i n g rainbow t r o u t , as d e t e r m i n e d by the p e r c e n t a g e of. the t o t a l i n j e c t e d counts found i n each t i s s u e sampled, i s summarized f o r both t r a c e r methods i n T a b l e 6. These values, were d e r i v e d u s i n g d a t a i n T a b l e 4. T h e r e f o r e , d a t a i n T a b l e 6 r e p r e s e n t the a b s o l u t e p e r c e n t a g e o f c a r d i a c o u t p u t o r f l o w 61 T a b l e 4. B l o o d volume a d i s t r i b t u i o n to v a r i o u s t i s s u e s from normal, r e s t i n g rainbow t r o u t (N = 6; 371.0 ± 36.7 g ) . T i s s u e B l o o d Volume raTg"1 P e r c e n t Body Weight P e r c e n t T o t a l Volume :mT100 g" B l o o d •1 Mosaic muscle 0.0084 ±0.002 6 6 b 0.512 L a t e r a l r e d muscle 0.0211 ±0.040 2 . 5 C 0.053 G i l l s (2nd a r c h ) 0.1594 ±0.040 3.9 b 0.622 L i v e r 0.2255 ±0.060 1.156 ±0.245 0.261 Spleen 3.0883 ±0.820 0.227 ±0.09 0.700 V e n t r i c l e (emptied) 0.0647 ±0.014 .0.136 0.014 0.009 Pseudobranch 0.256.6 ±0.072 0.12 ±0.06 0.031 Stomach and esophagus 0.0204 ±0.006 1.275 ±0.055 0.026 I n t e s t i n e and c a e c a 0.0329 ±0.008 1.645 ±0.282 0.054 Gonads 0.0238 ±0.007 4.218 : ±1.571 0.100 S k i n 0.0164 ±0.007 4.0 b 0.066 Eyes 0.0296 ±0.006 1.12 ±0.17 0.034 P r e - i n j e c t i o n Hct - 16.25 ± 1.92% ( a t time = 0) Sample H c t - 17.72 ± 2.62% ( a t time = +2 hours) B l o o d volume - 6.08 ± 1.31% body weight A l l volumes d e t e r m i n e d from d o r s a l a o r t i c ( a r t e r i a l ) b l o o d sample r e f e r e n c e cpm's. b S t e v e n s (1968a). K i c e n i u k and Jones (1977). 62 T a b l e 5.. B l o o d volume a d i s t r i b u t i o n t o v a r i o u s t i s s u e s from rainbow t r o u t swimming a t .80% U ._t (N =• 7; 445.0 ± 27.7 g ) . T i s s u e R e s t i n g b l o o d volume E x e r c i s e b l o o d volume (n f o r e x e r c i s e ) m l » g _ 1 m l » g _ 1 White muscle 0.0084 ±0.002 0.0085 ±0.001 L a t e r a l r e d muscle (n = 1) 0.0211 , _ ^ ±0.003 ( n " 3 6 ) 0.1250 L i v e r 0.2255 ±0.820 0.0654 ±0.022 S p l e e n 3.0883 ±0.820 1.546 ±0.253 Kidney 0.3265 ±0.101 0.1174 ±0.026 Stomach and esophagus 0.0204 ±0.006 0.0122 ±0.008 I n s t e s t i n e and cae c a 0.0329 ±0.008 0.0340 ±0.004 S k i n 0.0164 ±0.007 0.0183 ±0.007 H a e m a t o c r i t 20.5 ± 1.66% 26.4 ± ( a t t= a t 80% T o t a l b l o o d volume 6.08 ± 1.31% 3.72 ± c r i V body w e i g h t ( a t t= 25 min a t 80% U ..) c r i t1 a A l l volumes d e t e r m i n e d from d o r s a l a o r t i c ( a r t e r i a l ) b l o o d sample r e f e r e n c e cpm's. 63. T a b l e 6. P e r c e n t a g e o f c a r d i a c o u t p u t t o v a r i o u s t i s s u e s from rainbow t r o u t a t r e s t , e x p r e s s e d as p e r c e n t a g e o f t o t a l i n j e c t e d cpm's. -o cu cu s~ cu 13 > CO cu X I o : 3 cu cu o s : SC c •r— +-> cu O S -o 3 o 4-> >> cu - C •r— s : cu ta CO -a cu c: •r-3 CO C D n3 cu cu B CU ca c cu •i— c -•r— CO -o i— > o + J £ " O cu ra •<- B CU o cu C L • r— + J • c O i . CL 2 : cc co _ l oo 1—1 CJ3 o C Q (g) 6 24 36 12 12 12 12 12 12 12 12 USING ^ R u b i d i u m X 384.4 36.4 6.51 0.74 8.73 1.86 5.82 1.99 9.26 0.16 0.02 82.6 ±S.E.M. 40.5 8.0 0.53 0.32 .2.88 0.47 1.34 0.67 3.46 0.05 0.01 5.4 7.68 ( m o s t l y ±1.75 immature) ±S.E. USING RADIOLABELLED MICROSPHERES . s k i n n J 28 14 7 7 7 14 12 14 X 429.4 49.0 11.2. 1.06 4.5 7.40 10.0 4.5 8.3 89.3 M. 26.4 7.6 2.6 0.40 1.9 3.10 4.9 ( m o s t l y mature) 2.0 2.1 7.9 64 d i r e c t e d t o the whole t i s s u e l i s t e d a t rest.- Had the a c t u a l c a r d i a c o u t p u t been known, then the a b s o l u t e b l o o d f l o w t o each t i s s u e or organ ( i n m l - m i n - 1 ) c o u l d have been c a l c u l a t e d s i m p l y by m u l t i p l i c a t i o n o f the c a r d i a c o u t p u t by the p r o p o r t i o n o f a b s o l u t e c a r d i a c o u t p u t t o t h a t t i s s u e . N o n e t h e l e s s , the two d a t a s e t s are comparable, and i n d i c a t e t h a t r e d and mosaic muscle masses, which c o n s t i t u t e a p p r o x i m a t e l y 70% o f the t o t a l body weight o f the f i s h a l s o r e c e i v e d the l a r g e s t p r o p o r t i o n o f the b l o o d f l o w (.43 t o 60% o f the c a r d i a c o u t p u t ) a t r e s t . A t r e s t , mosaic muscle r e c e i v e d o n l y 4.4 t o 5.6 times more o f the t o t a l b l o o d f l o w t h a n . d i d the r e d muscle, y e t mosaic muscle a c c o u n t e d f o r 26.4 times more o f t h e t o t a l body mass. These d a t a i n d i c a t e d t h a t , a t r e s t , r e d muscle r e c e i v e d almost 5 times the amount o f b l o o d f l o w on a p e r gram b a s i s than does mosaic muscle. The r e m a i n i n g b l o o d f l o w was more o r l e s s e v e n l y d i v i d e d between f l o w through t h e c o e l i a -c o m e s e n t e r i c a r t e r y , to s u p p l y t h e v i s c e r a , and f l o w v i a the d o r s a l a o r t a t o s u p p l y the k i d n e y , s k i n and gonads. D u r i n g swimming e x e r c i s e a t 80% I T , ^ , t h e r e was an a p p a r e n t r e d i s t r i b u t i o n , o f b l o o d f l o w , as i n d i c a t e d by d a t a p r e s e n t e d i n T a b l e 7. A t t h i s l e v e l o f e x e r c i s e , r e d and mosaic muscle a c c o u n t e d f o r 56 t o 82% o f the t o t a l c a r d i a c o u t p u t . P r o p o r t i o n a l b l o o d f l o w t o r e d muscle now was i n c r e a s e d o v e r t h a t d i r e c t e d t o mosaic muscle, which a c t u a l l y showed a d e c r e a s e i n the p r o p o r t i o n o f t o t a l c a r d i a c o u t p u t f l o w i n g t o t h i s t i s s u e mass, compared t o the r e s t i n g c o n d i t i o n . However, i t must be remembered t h a t the a b s o l u t e c a r d i a c o u t p u t a t t h i s l e v e l o f swimming c o u l d have been i n c r e a s e d by 3 - f o l d o v e r that, found a t r e s t i n t r o u t ( K i c e n i u k and J o n e s , 1977), and thus the a b s o l u t e b l o o d f l o w t o r e d as w e l l as mosaic muscle was i n c r e a s e d above t h a t at. r e s t . N o n e t h e l e s s , the d a t a i n d i c a t e d t h a t the red muscle, under t h e s e c i r c u m s t a n c e s , r e c e i v e d a p p r o x i m a t e l y 43 times the amount o f b l o o d f l o w per gram t i s s u e o v e r mosaic muscle f l o w . P r o p o r t i o n a t e 65 b l o o d f l o w t o the s p l e e n , l i v e r and gut was c o n s i s t e n t l y lower d u r i n g e x e r c i s e than f l o w measured a t r e s t . . I t i s i n t e r e s t i n g t o note t h a t , 86 whether u s i n g Rb or m i c r o s p h e r e t e c h n i q u e s to a s s e s s b l o o d f l o w d i s t r i b u -t i o n d u r i n g e x e r c i s e , the p e r c e n t a g e change from r e s t i n t h o s e organs s u p p l i e d b y t h e ' c o d i a c o m e s e n t e r i c a r t e r y was the same f o r each organ ( T a b l e 7 ) . The d a t a f o r b l o o d f l o w changes i n . k i d n e y c i r c u l a t i o n i n d i c a t e an i n c r e a s e d p e r f u s i o n o f t h i s organ d u r i n g e x e r c i s e , w h i l e the s k i n c i r c u l a t i o n a p p a r e n t l y was d e c r e a s e d . Other t i s s u e s showed some degree o f v a r i a b i l i t y i n f l o w ( T a b l e 7),. w i t h no c o n s i s t e n t t r e n d s b e i n g o f n o t e . I n i t i a l l y , d o r s a l a o r t i c p r e s s u r e (DAP) was r e c o r d e d from r e s t i n g and e x e r c i s i n g f i s h , and t h e s e d a t a a r e p r e s e n t e d i n T a b l e 8. However, 8 6 t h e s e d a t a were c o l l e c t e d o n l y d u r i n g the Rb s t u d i e s , but not f o r m i c r o -s p h e r e e x p e r i m e n t s . Data from T a b l e 8 i n d i c a t e d t h a t d u r i n g swimming e x e r c i s e a t 80% U ^ . ^ , h e a r t r a t e was not changed from r e s t i n g l e v e l s . However., d o r s a l a o r t i c p r e s s u r e was e l e v a t e d .some 41% above the r e s t i n g l e v e l . I f c a r d i a c o u t p u t was i n c r e a s e d d u r i n g e x e r c i s e , then i n the p r e s e n t c a s e , t h e s e f i s h o b v i o u s l y a c c o m p l i s h e d . t h i s by i n c r e a s i n g t h e i r s t r o k e volume r a t h e r than the h e a r t r a t e . As a consequence, t h e d o r s a l a o r t i c p r e s s u r e was e l e v a t e d d u r i n g e x e r c i s e . E x h a u s t i o n Complete e x h a u s t i o n a f t e r s t r e n u o u s swimming e x e r c i s e r e s u l t e d i n f u r t h e r changes i n the c a r d i a c o u t p u t d i s t r i b u t i o n t o v a r i o u s t i s s u e s from the p a t t e r n e s t a b l i s h e d a t 80% U -t. The d a t a i n T a b l e 9 i n d i c a t e a d e c r e a s e i n the p r o p o r t i o n a l c a r d i a c o u t p u t d i s t r i b u t i o n t o most t i s s u e s sampled, compared t o t h a t found d u r i n g s t e a d y - s t a t e swimming a t 80% U^.^. The o n l y e x c e p t i o n was the a p p a r e n t p r o p o r t i o n a l i n c r e a s e i n mosaic muscle b l o o d f l o w , a t the expense o f l e s s f l o w t o r e d muscle. N o n e t h e l e s s , r e d and mosaic muscle v a s c u l a t u r e s s t i l l c o n s t i t u t e d the major p r o p o r t i o n o f the T a b l e 7. P e r c e n t a g e c a r d i a c o u t p u t t o v a r i o u s t i s s u e s from Rainbow T r o u t swimming a t 80% U ^ . ^ , e x p r e s s e d as %age o f t o t a l i n j e c t e d cpm's. USING 8 6 R b - BLOOD wt wh i t e r e d % cpm g(6) muscle muscle s p l e e n l i v e r stomach i n t e s t i n e gonad ki d n e y cheek b r a i n i n j e c t e d (24) (36) (12). (12) • (12) (12) (12) (12) (24) (12) r e c o v e r y 415 .1 21.0 35.4 0.37 5.49 1.32 4.55 1.13 11.93 0.21 0.017 87.4 40.3 2.7 2.5 0.15 1.30 0.37 i 1.15 1 0.28 1.77 0.04 0.007 4.6 1 5 - i - '.. .68 (mostly 1 . 4 3 immature) A % Of rest % iS.E.M. - 3 0 . 0 1 5 . 4 +.465.2 7 0 . 0 - 5 0 . 0 9 . 8 - 3 7 . 1 1 9 . 7 - 3 7 . 7 1 6 . 0 - 2 8 . 0 1 7 . 2 + 2 8 . 8 1 5 . 2 + 3 6 . 4 - 2 2 . 7 1 5 . 2 7 . 2 USING RADIO-LABELLED SPHERES skin ( n i 7 14 14 7 7 7 14 12 14 X 4 2 9 . . 4 3 2 . 2 4 9 . 5 0 . 0 9 0 . 7 7 3 . 4 0 8 . 3 2 2 . 6 2 2 . 7 0 8 6 . 9 iS.E.M. 2 6 . . 4 8 . 5 8 . 2 0 . 0 2 0 . 6 6 2 . 6 0 5 . 6 0 (mostly mature) 0 . 8 0 0 . 5 0 9 . 4 A % of - 4 3 . 8 + 4 1 2 . 8 - r 8 3 . 5 - 7 8 . 4 - 8 3 . 0 - 6 . 4 8 - 1 2 . 6 - 5 6 . 3 rest % iS.E.M. 9 . 5 5 5 . 7 7 . 6 1 2 . 5 1 4 . 4 3 7 . 6 2 3 . 8 9 . 9 67 T a b l e 8. Summary, o f d o r s a l a o r t i c p r e s s u r e (DAP) and h e a r t r a t e changes d u r i n g 80% U ^ s w 1' m i T 1'' r i9 e x e r c i s e i n rainbow t r o u t RESTING DAP (cm H 20) 29.8 ± 0.63 H e a r t r a t e (bpm) 37.4 ± 0 . 5 2 Weight (g) 384.4 ± 40.5 n = 59 o b s e r v a t i o n s o f 6 f i s h EXERCISE DAP (cm H 20) 42.1 ± 1.46 H e a r t r a t e (bpm) 37.9 ± 1.46 Weight (g) 415.1 ± 40.3 n = 18 o b s e r v a t i o n s o f 6 f i s h Table.9. Percentage cardiac output to various tissues from Rainbow Trout swimming at 80% U .. and at point of exhaustion, expressed as %age of total injected cpm's. 80% U - Using Radiolabeled Microspheres wt(g) whi te muscle red muscle spleen l i v e r guts gonad kidney skin % cpm injected recovery (4) (24) (16) (4) (4) (4) (4) (4) (21) 534.5 20.8 60.7 0.97 2.35 4.12 3.37 2.82 3 .18 95.5 34.4 7.2 10.1 0.95 2.00 3 .14 2.82 0.88 1.02 12.3 EXHAUSTION 534.5 31.8 44.6 0.08 0.28 0 .13 2.39 1.90 90.5 34.4 15.7 13.2 0.05 0 .10 0.07 1.00 0.66 5.1 69 t o t a l b l o o d f l o w . I n t e r e s t i n g l y , a t e x h a u s t i o n , o n l y 81.2% o f the c a r d i a c o u t p u t d i s t r i b u t i o n c o u l d be a c c o u n t e d f o r i n the c a p i l l a r y networks o f the t i s s u e s sampled. T h i s d e c r e a s e d from a v a l u e o f 98.3% found when t i s s u e s were sampled from f i s h swimming a t 80% o f t h e i r c r i t i c a l v e l o c i t y . DISCUSSION I_ I t i s known t h a t e x e r c i s e causes a d i u r e s i s i n t r o u t (Wood and R a n d a l l , 1973) and the measured i n c r e a s e i n d o r s a l a o r t i c p r e s s u r e d u r i n g e x e r c i s e i n my e x periments c o u l d e x p l a i n the o b s e r v e d i n c r e a s e s i n the k i d n e y u l t r a f i l t r a t i o n r a t e . A l s o * d a t a from t h e Rb i n v e s t i g a t i o n i n d i c a t e a 29% i n c r e a s e , i n the b l o o d f l o w t o the kidney d u r i n g e x e r c i s e , o v e r t h a t a t r e s t . I n c r e a s e d k i d n e y f u n c t i o n t h e r e f o r e can account, f o r the measured d e c r e a s e i n the t o t a l b l o o d volume as w e l l as the h a e m o c o n c e n t r a t i o n d u r i n g e x e r c i s e i n the p r e s e n t study.. During e x e r c i s e , an i n c r e a s e i n the water f l u x a c r o s s the g i l l t i s s u e s i n t o the b l o o d (Wood and R a n d a l l , 1973) imposes an i n c r e a s e d water l o a d on the f i s h , but t h i s excess water i s e l i m i n a t e d by i n c r e a s e d u r i n e p r o d u c t i o n by. the k i d n e y s , as w e l l as p o s s i b l e water l o s s t o the t i s s u e s (oedema) from the b l o o d . M i c r o s p h e r e d a t a i n d i c a t e a 13% d e c r e a s e i n the p r o p o r t i o n : o f , c a r d i a c o u t p u t d i s t r i b u t e d t o the k i d n e y s under the same e x e r c i s e c o n d i t i o n s . I f t h e r e i s an e x e r c i s e d i u r e s i s , then k i d n e y v a s c u l a r d i l a t i o n c o u l d l e a d t o . t h e noted d e c r e a s e i n the e n t r a p -ment o f the i n j e c t e d m i c r o s p h e r e s , ; compared t o r e s t i n g v a l u e s . My d a t a a l s o i n d i c a t e t h a t the l a t e r a l r e d muscle has a v e r y s u b s t a n t i a l i n c r e a s e i n the p e r c e n t c a r d i a c o u t p u t d u r i n g s t e a d y - s t a t e e x e r c i s e . T h e , p r o p o r t i o n o f c a r d i a c o u t p u t t o mosaic muscle i s d e c r e a s e d below t h a t found a t r e s t , but as p o i n t e d out p r e v i o u s l y , the a b s o l u t e f l o w t o t h i s t i s s u e mass i s i n f a c t i n c r e a s e d as w e l l , s i n c e c a r d i a c o u t p u t a t t h i s l e v e l o f e x e r c i s e i n rainbow t r o u t can i n c r e a s e by a t l e a s t 3 - f o l d above t h a t found a t r e s t ( K i c e n i u k and J o n e s , 1977). Working muscles t h e r e f o r e 70 are p r e f e r e n t i a l l y p e r f u s e d over o t h e r t i s s u e s , as i s found t o o c c u r i n . mammals d u r i n g e x e r c i s e . Most t e l e o s t f i s h e s have two types o f s k e l e t a l muscle f i b r e s . I t i s g e n e r a l l y a c c e p t e d t h a t the h i g h l y v a s c u l a r i z e d r e d muscle o f f i s h e s , e s p e c i a l l y s a l m o n i d s , i s used p r i m a r i l y f o r s u s t a i n e d swimming, as d u r i n g m i g r a t o r y p e r i o d s ( B i l i n s k i , 1974). Mosaic muscle c o n t a i n s a s m a l l p e r c e n t -age o f red f i b r e s i n rainbow t r o u t (Webb, 1975) and i s p o o r l y v a s c u l a r i z e d , but comprises the l a r g e s t b u lk o f the body mass. I t i s thought t o a c t as a powerful but temporary p r o p u l s i v e f o r c e f o r b u r s t swimming a c t i v i t y . The c o n t r a c t i l i t y o f r e d m u s c l e s , used f o r p r o l o n g e d swimming e x e r c i s e , i s main-t a i n e d by energy s u p p l i e d by a e r o b i c metabolism. White muscle on the o t h e r hand, i s geared t o have i t s m e t a b o l i c r e q u i r e m e n t s met p r i m a r i l y from a n a e r o b i c p r o c e s s e s ( D r i e d z i c and Hochachka, 1978). C e r t a i n l y the d i f f e r e n c e i n muscle c a p i l l a r i e s ' between r e d and white f i b r e s i s i n d i c a t i v e o f t h e i r modes o f metabolism. The c a p i l l a r y d e n s i t i e s o f f i s h r e d muscle a r e some 2 t o 10 times t h o s e o f white muscle (Cameron and Cech, 1970; Mosse, 1978), depend-i n g upon s p e c i e s . As such, b l o o d volume per u n i t weight i n r e s t i n g r e d muscle o f rainbow t r o u t i s 2 t o 3 times t h a t o f mosaic muscle. A l s o i t has been shown t h a t anywhere from 5% (Krogh, 1919) t o 50% (Wright and S o n n e n s c h e i n , 1965) o f the muscle c a p i l l a r i e s o f f i s h a re p a t e n t a t r e s t . D u r i n g e x e r c i s e , a range o f from 2 t o 20 times i n c r e a s e i n t h e p o t e n t i a l b l o o d volume t h e r e f o r e c o u l d be e x p e c t e d , g i v e n t h a t a l l c a p i l l a r i e s a r e p e r f u s e d . In the p r e s e n t s t u d y , a 6 - f o l d i n c r e a s e i n r e d muscle b l o o d volume over t h a t measured a t r e s t , i s i n d i c a t e d d u r i n g e x e r c i s e i n the rainbow t r o u t . Mosaic muscle b l o o d volume however d i d not show- any change a t the same l e v e l o f e x e r c i s e . White muscle, however, has been shown t o be a c t i v e d u r i n g p r o l o n g -ed swimming. In.brook t r o u t {Salvelinus • f'ontinalis) f o r example the t h r e s -71 h o l d f o r the r e c r u i t m e n t o f w h i t e muscle f i b r e s , based on E.M.G. r e c o r d i n g s , was a p p r o x i m a t e l y 1.8 f o r k l e n g t l v s e c ! 1 ( J o h n s t o n and Moon, 1980a). T h i s swimming speed, i n the p r e s e n t e x p e r i m e n t s , i s a l m o s t e q u i v a l e n t t o the 80% U .. e x e r c i s e l e v e l f o r rainbow t r o u t . T h e r e f o r e , both muscle f i b r e t ypes c r i t J V c o u l d have been f u n c t i o n a l d u r i n g e x e r c i s e i n the rainbow t r o u t d u r i n g my e x p e r i m e n t s . Davison and G o l d s p i n k (1977) and Walker and Emerson (1978) found t h a t w i t h e x e r c i s e f o r 3 t o 4 weeks, rainbow t r o u t n o t o n l y showed h y p e r t r o p h y o f r e d muscle f i b r e s , both i n terms o f f i b r e d i a m e t e r s and l e n g t h s , but w h i t e f i b r e s a l s o hypertrophied., t o an even g r e a t e r e x t e n t than r e d f i b r e s . Such muscle f i b r e h y p e r t r o p h y i n d i c a t e s t h a t w h i t e muscle may be u t i l i z e d d u r i n g e x e r c i s e t r a i n i n g . J o h n s t o n and Moon (1980b) c o n s i d e r e d t h a t the a e r o b i c c a p a c i t y o f w h i t e muscle was s u f f i c i e n t t o s u p p l y the energy r e q u i r e m e n t s o f t h i s t i s s u e d u r i n g s u s t a i n e d swimming-, s i n c e t h e y were unable t o d e t e c t a n y i n c r e a s e i n b l o o d l a c t a t e (an i n d i c a t i o n o f a n a e r o b i c metabolism) d u r i n g e x e r c i s e . D r i e d z i c and K i c e n i u k (1976) a l s o f a i l e d t o note any i n c r e a s e i n b l o o d l a c t a t e i n rainbow t r o u t swimming a t v e l o c i t i e s as h i g h as 93% U c r - j t - They c o n c l u d e d , however, t h a t the g i l l s were a b l e t o o x i d i z e any l a c t a t e p r e s e n t i n the b l o o d , due to w h i t e muscle a n a e r o b i o s i s , so t h a t i t would not be e x p r e s s e d i n the d o r s a l a o r t i c b l o o d . . A d d i t i o n a l l y , H u l b e r t and Moon (1978) have shown t h a t e e l (Anguilla anguilla) r e d muscle can perform a m e t a b o l i c r e c y c l i n g f u n c t i o n , i n a d d i t i o n t o c o n t r a c t i o n (in vitro), so t h a t a g l u c o n e o g e n i c p r o c e s s , which c o n v e r t s w h i t e muscle l a c t a t e t o g l u c o s e i s i n e x i s t e n c e i n t h i s t i s s u e . There i s no r e a s o n however, t o assume t h a t white muscle c a p i l l a r i e s s u p p l y r e d muscle f i b r e s in vivo i n the rainbow t r o u t . The l a c k o f any measured i n c r e a s e i n b l o o d l a c t a t e d u r i n g e x e r c i s e i s more l i k e l y t o i n d i c a t e p r i m a r i l y an a e r o b i c m e t a b o l i c r o u t e f o r the energy o f t h e muscles. The e r r a t i c b u r s t -g l i d e swimming b e h a v i o u r e x h i b i t e d by t r o u t as they near 100% U .. i s 72 i n d i c a t i v e o f i n c r e a s e d w h i t e muscle a c t i v i t y . The p o s s i b i l i t i e s o f m e t a b o l i c a c i d o s i s b e i n g e x p r e s s e d i n the b l o o d as the a e r o b i c c a p a c i t y o f w h i t e muscle i s exceeded a r e t h e r e f o r e h i g h a t t h i s l e v e l o f e x e r c i s e . Muscle c e l l s o f some f i s h ( i . e . Pleuronectes plates so), however appear t o po s s e s s the a b i l i t y t o r e g u l a t e t he r e l e a s e o r r e t e n t i o n o f t h e i r l a c t a t e l o a d t o b l o o d d u r i n g e x e r c i s e , and i n c r e a s e d b l o o d f l o w t o t h e s e muscles does n o t a f f e c t l a c t a t e r e l e a s e (Wardle, 1978). The r e l e a s e appears t o be a c t i v a t e d by a c a t e c h o l a -mine ( a s . y e t u n i d e n t i f i e d ) c i r c u l a t e d i n t h e b l o o d stream o n l y f o l l o w i n g the t e r m i n a t i o n o f s t r e s s . Whether such a mechanism i s o p e r a t i o n a l i n t r o u t i n o r d e r t o m i n i m i z e the e f f e c t s o f m e t a b o l i c a c i d o s i s i n the g e n e r a l c i r c u l a -t i o n d u r i n g p r o l o n g e d e x e r c i s e , i s n o t known. I f an organ can be e f f e c t i v e l y i s o l a t e d from t h e g e n e r a l c i r c u l a t i o n by c o n s t r i c t i o n o f i t s a f f e r e n t and/or e f f e r e n t b l o o d pathway d u r i n g e x e r c i s e , then even though t h e r e may have been no measurable volume change, t h e r e w i l l be d r a m a t i c changes i n b l o o d f l o w through t h a t organ. Data p r e s e n t e d i n T a b l e s 6 and 8 o f t h i s t h e s i s i n d i c a t e t h a t such i s in d e e d the case f o r e x e r c i s i n g rainbow t r o u t . S p l e n i c b l o o d volume and f l o w , as w e l l as the f l o w through the o t h e r organs s u p p l i e d by the c o e l i a c o m e s e n t e r i c a r t e r y a r e reduced d u r i n g e x e r c i s e . T h a t the c o e l i a c o m e s e n t e r i c a r t e r y i s the main s i t e o f c o n t r o l f o r f l o w i n the s p l e e n , l i v e r , stomach and i n t e s t i n e s i s based on o b s e r v a t i o n s from both Rubidium and m i c r o s p h e r e s t u d i e s , t h a t the pe r c e n t a g e d e c r e a s e i n c a r d i a c o u t p u t d i s t r i b u t i o n t o thes e organs d u r i n g e x e r c i s e i s o f . t h e same magnitude. The s i t u a t i o n w h e r e b y . t h i s c i r c u l a t i o n i s c u r t a i l e d d u r i n g e x e r c i s e i n the rainbow t r o u t , i n f a v o u r o f i n c r e a s e d b l o o d f l o w t o the working muscles i s v e r y s i m i l a r t o the s p l a n c h n i c b l o o d " p o o l " i n man (Rowell et al., 1964).. T h i s c i r c u l a t i o n i n f i s h p r o b a b l y f u n c t i o n s i n a s i m i l a r c a p a c i t y d u r i n g e x e r c i s e . The u l t i m a t e r e s u l t o f the o v e r a l l b l o o d s h u n t i n g i s t o i n c r e a s e the e f f e c t i v e c i r c u l a t i n g b l o o d volume, 73 t o d e c r e a s e the oxygen consumption o f . t i s s u e s not d i r e c t l y r e l a t e d t o the maintenance o f swimming, and t o augment oxygen d e l i v e r y t o the working muscles, through i n c r e a s e s i n ; haematocr.it and haemoglobin c o n c e n t r a t i o n s d u r i n g e x e r c i s e (see S t e v e n s , 1968b). There s t i l l appears t o be c o n s i d e r a b l e c o n t r a v e r s y over the mode o f s y s t e m i c vasomotor tone maintenance i n r e s t i n g f i s h e s . I t has, however, been r e p e a t e d l y demonstrated t h a t at l e a s t . i t i s under g e n e r a l , a - a d r e n e r g i c c o n t r o l , a l t h o u g h i t i s not known whether i t i s n e u r o n a l i y o r h o r m o n a l l y mediated. W a l h q v i s t and N i l s s o n (1977) s t a t e t h a t a d r e n e r g i c tonus i n Gadus i s m a i n t a i n e d by c i r c u l a t i n g c a t e c h o l a m i n e s r a t h e r than a d r e n e r g i c n e r v e s . S a n t e r (1977).has shown t h a t t h e c h r o m a f f i n system i n f i s h , which i n f u n c t i o n i s s i m i l a r t o the a d r e n a l g l a n d s o f , h i g h e r v e r t e b r a t e s , i s much b e t t e r d e v e l o p e d than i s the s y m p a t h e t i c nervous system. T h i s l e d him t o c o n c l u d e t h a t c i r c u l a t i n g (hormonal) a d r e n e r g i c c o n t r o l i s o f g r e a t e r importance f o r the maintenance o f s y s t e m i c v a s c u l a r . t o n e . To t h i s end, Vanhoutte (1978) has noted t h a t most, b l o o d v e s s e l s c o n t a i n v e s i c l e s which s t o r e or s e q u e s t e r dopamine and c o n v e r t t h i s t o n o r a d r e n a l i n e , or. a c t i v e l y take up n o r a d r e n a l i n e d i r e c t l y from the blood.. A l t h o u g h ,the l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s i n s a l m o n i d s a r e r e p o r t e d t o i n c r e a s e d u r i n g s t r e s s (Nakano and T o m l i n s o n , 1967), t h e s e c a t e c h o l a m i n e s have a l o n g h a l f - l i f e i n the b l o o d , and so i t seemed u n l i k e l y t h a t t h e s e c o u l d m a i n t a i n f i n e enough c o n t r o l o f v a s c u l a r tonus i n t h e s e f i s h . Thus Wood and S h e l t o n (.1975) c o n c l u d e t h a t vasomotor tone i s m a i n t a i n e d by autonomic i n n e r v a t i o n , i n S. gaivdneri. The p e r s i s t -ence o f Mayer waves (measured as c y c l i c a l v a r i a t i o n s i n the mean 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 ) i n rainbow t r o u t s u g g e s t s t h a t tone i s r e f l e x l y r e g u l a t e d through a n e g a t i v e feedback c o n t r o l . f o r b l o o d p r e s s u r e maintenance, via the s y m p a t h e t i c nervous system (Wood, 1974b). T h e r e f o r e , i t seems l i k e l y t h a t s y s t e m i c v a s c u l a r r e s i s t a n c e (tone) i s m a i n t a i n e d i n r e s t i n g f i s h t hrough a - a d r e n e r g i c c o n s t r i c t o r r e s p o n s e s ( R a n d a l l and S t e v e n s , 1969; Helgason and 74 N i l s s o n , 1973; Wood, 1976; W a h l q v i s t , 1980; Wood and She!ton, 1980a). As w e l l , t h e r e may be ^ d i l a t o r y a d r e n o c e p t o r s i n f i s h e s , as i n the s y s t e m i c v a s c u l a t u r e o f mammals. However, t h e s e d i l a t o r y r e s p o n s e s a r e thought t o be seen o n l y a g a i n s t a background of. p r i m a r i l y a-adrenergic c o n s t r i c t o r responses (Wood, 1976,, 1977). Whether 3 - a d r e n e r g i c v a s o d i l a t i o n i s o p e r a -t i v e i n t r o u t d u r i n g e x e r c i s e s t i l l i s not r e s o l v e d . In elasmobranch f i s h e s , i n the absence o f any s i g n i f i c a n t sympath-e t i c nervous system c o n t r o l , the c h r o m a f f i n t i s s u e s r e l e a s e s u b s t a n t i a l amounts o f a d r e n a l i n e , and more i m p o r t a n t l y n o r a d r e n a l i n e i n t o the c i r c u l a t o r y system. These c a t e c h o l a m i n e r g i c s p e c i e s a r e a s p i r a t e d d i r e c t l y i n t o the. g e n e r a l c i r c u l a t i o n w i t h each beat o f - t h e h e a r t . V a s c u l a r tone i n t h e s e f i s h o b v i o u s l y i s c o n t r o l l e d by c i r c u l a t i n g c a t e c h o l a m i n e s a t a l l times ( S a t c h e l ! , 1971; Capra and S a t c h e l ! , 1977). The c i r c u l a t o r y system o f elasmobranchs a l s o i s thought t o be d e v o i d o f any d i r e c t n e u r a l means by which they can c o n t r o l . b l o o d f l o w d i s t r i b t u i o n t o the m u s c u l a t u r e , . e v e n d u r i n g e x e r c i s e , e x c e p t p o s s i b l y by a u t o r e g u l a t i o n o f the m i e r o v a s c u l a t u r e (Opdyke et al., 1979). The e f f e c t s o f e x e r c i s e on b l o o d p r e s s u r e and h e a r t r a t e i n f i s h e s have been w e l l documented (see Jones and R a n d a l l , 1978 f o r r e v i e w ) . G e n e r a l -l y , mean d o r s a l a o r t i c p r e s s u r e s are found t o i n c r e a s e w i t h a c t i v i t y . , as do v e n t r a l a o r t i c mean and p u l s e p r e s s u r e , venous ( s u b i n t e s t i n a l ) , p r e s s u r e s , and c a r d i a c output.. S i n c e the" d o r s a l : a o r t a and:.vessels s u p p l y i n g - blood' to .the :body m u s c u l a t u r e i n t r o u t appear t o be a l s o d e v o i d o f any a d r e n e r g i c i n n e r v a t i o n (Gannon, 1972), d i r e c t nervous v a s o d i l a t o r y a c t i o n was m i n i m a l . The measured i n c r e a s e i n r e g i o n a l b l o o d f l o w t o t h e s e t i s s u e s must r e f l e c t , to some degree, the p a s s i v e d i l a t i o n o f c a p a c i t a n c e , v e s s e l s , brought about by the i n c r e a s e d p r e s s u r e head r e a c h i n g t h e s e v e s s e l s d u r i n g e x e r c i s e . I t has been s u g g e s t e d t h a t i n t e r c a p i l l a r y anastomoses i n mammalian s k e l e t a l 75 muscles p r o v i d e a l t e r n a t i v e pathways around t r a n s i e n t o b s t r u c t i o n s i n the c a p i l l a r y network, due to c o n t r a c t e d m u s c l e s , (Honig et al., 1977) i n a d d i t i o n t o i n c r e a s i n g oxygen s u p p l y t o t h e s e muscles through i n c r e a s e s i n b l o o d volume and f l o w . A u t o r e g u l a t i o n o f m i c r o c i r c u l a t i o n i s thought t o i n v o l v e such t h i n g s as m e t a b o l i c v a s o d i l a t o r s u b s t a n c e s which l e a d t o a c t i v e hyperaemia, and myogenic tonus a c t i v i t y w i t h i n t h e s e v e s s e l s (Jones and Berne, 1963; Green and Rapela,-1964; Johnson, 1964; S t a i n s b y , 1964; S t r a n d e l l and Shephard, 1967; Vanhoutte and J a n s s e n s , 1978). A t lower venous P n , the s e n s i t i v i t y o f u 2 mammalian h i n d limb r e s i s t a n c e v e s s e l s ( t h e c a p i l l a r i e s ) t o m e t a b o l i c and haemodynamic d i s t u r b a n c e s i n c r e a s e s (Granger et al. , 1976), and t h e s e v e s s e l s are more s e n s i t i v e than any o t h e r v e s s e l w i t h i n the c i r c u l a t o r y system ( H e ! l a n d e r , 1978). T h i s can be o f o b v i o u s importance t o the o b s e r v e d f l o w changes i n t r o u t muscle d u r i n g e x e r c i s e , s i n c e i t s v a s c u l a t u r e appears t o l a c k any n e u r o n a l pathway, f o r v a s o d i l a t i o n . R e s i s t a n c e v e s s e l s a l s o s u g g e s t -ed t o have the a b i l i t y t o escape from t h e c o n s t r i c t o r a c t i o n o f c a t e -c holamines when t i s s u e o x y g e n a t i o n i s lower than normal ( M e l l a n d e r , 1978). T h e r e f o r e , the v a s o c o n s t r i c t o r e f f e c t s o f any i n c r e a s e i n t h e l e v e l o f c i r c u l a t i n g c a t e c h o l a m i n e s , s h o u l d t h i s i n f a c t o c c u r d u r i n g e x e r c i s e i n t r o u t , c o u l d be o v e r r i d d e n i n the muscle v a s c u l a t u r e by such a mechanism. T h i s would i n d i c a t e t h a t s u b s t r a t e changes i n the m e t a b o l i c a l l y a c t i v e muscle t i s s u e s are r e s p o n s i b l e f o r v a s o d i l a t i o n i n t h e s e t i s s u e s d u r i n g e x e r c i s e . In t h i s manner, red and mosaic muscle b l o o d f l o w c o u l d be i n c r e a s e d , as was measured i n my e x p e r i m e n t s , w i t h i n a background o f g e n e r a l c a t e c h o l a m i n e r g i c v a s o c o n s t r i c t i o n e l s e w h e r e i n the body d u r i n g e x e r c i s e . Smith (1978) has shown t h a t a - a d r e n e r g i c c o n s t r ' c t i o n o f s y s t e m i c v e s s e l s a t t h e o n s e t o f swimming e x e r c i s e i n rainbow t r o u t (.5 min a t 2 f l ' s e c " 1 , from a s t a n d i n g s t a r t ) c o n t r o l s the measured changes i n b l o o d p r e s s u r e . Smith however, may 76 have been measuring t h e " s t a r t - u p " r e s p o n s e s , i n i t i a t e d by nervous s t i m u l a t i o n , w h i l e i n my e x p e r i m e n t s , the o b s e r v e d f l o w and volume changes, u s i n g the g r a d u a l i n c r e a s e i n e x e r c i s e l e v e l . , may have been m a i n t a i n e d through c i r c u l a t i n g c a t e c h o l a m i n e s i n t h i s s t e a d y - s t a t e c o n d i t i o n . A l t h o u g h the d e t a i l s o f the mechanisms r e s p o n s i b l e f o r the mainten-ance o f b l o o d volume and f l o w r e d i s t r i b u t i o n d u r i n g e x e r c i s e i n f i s h a r e n o t f u l l y u n d e r s t o o d , i t appears t h a t a - a d r e n o c e p t o r s t i m u l a t i o n o f the c o e l i a -c o m e s e n t e r i c a r t e r y , and l o c a l hyperaemia i n muscle v a s c u l a t u r e a r e the main s i t e s f o r t h i s c o n t r o l . Gonadal b l o o d f l o w i n .immature t r o u t i s d e c r e a s e d t o a g r e a t e r and l e s s v a r i a b l e degree than i s t h e case f o r mature t r o u t under the same swim-ming e x e r c i s e c o n d i t i o n s (see T a b l e 8 ) . The high v a r i a b i l i t y i n d a t a from mature f i s h may r e f l e c t a m i g r a t o r y s t r a t e g y o f m a i n t a i n i n g adequate b l o o d s u p p l y t o the gonads f o r t h e u l t i m a t e purpose o f spawning a t the end o f a long p e r i o d o f m i g r a t i o n . A l t h o u g h the d a t a are t e n t a t i v e f o r the experiments where t r o u t were e x e r c i s e d t o complete e x h a u s t i o n , they do i n d i c a t e t h a t e x h a u s t i o n may be a s s o c i a t e d w i t h p e r i o d i c c e s s a t i o n s o f the c i r c u l a t i o n ( i . e . h e a r t s t o p p a g e ) . S i n c e m i c r o s p h e r e s are s l i g h t l y more dense than r e d b l o o d c e l l s , t hey have a s t r o n g e r tendency t o s e t t l e out o f s u s p e n s i o n i f n o t kept i n c o n t i n u o u s motion. I f t h e c i r c u l a t i o n were t o be t e m p o r a r i l y s t o p p e d d u r i n g the i n f u s i o n p e r i o d a t e x h a u s t i o n , as i n the p r e s e n t s t u d y , then an uneven d i s t r i b u t i o n o f spheres i n the c i r c u l a t i o n would be e x p e c t e d . I f some spheres s e t t l e d out i n t o l a r g e a r t e r i e s and/or v e i n s , and were n o t e n t r a p p e d i n the m i c r o c i r c u l a t i o n o f the t i s s u e s a t the time when the animaT was s a c r i f i c e d , then a decreased, p e r c e n t o f the t o t a l i n j e c t e d counts which a r e r e c o v e r e d i n the sample t i s s u e s can be e x p e c t e d . Only 8"f;% o f t h e c a r d i a c o u t p u t i s a c c o u n t e d f o r i n the t i s s u e s samples from f i s h which were e x h a u s t e d , as 77 opposed t o 98% i n the t i s s u e s a t 80% U c n - t - Because s h o r t b u r s t a c t i v i t y i s q u i t e prominant b e f o r e e x h a u s t i o n , i t i s not s u r p r i s i n g t h a t w h i t e (mosaic) muscle b l o o d f l o w i s i n c r e a s e d o v e r t h a t found a t 80% U ... The c n t f a i l u r e o f the oxygen t r a n s p o r t system t o m a i n t a i n adequate s u p p l y f o r a e r o b i metabolism,may be i n d i c a t e d by the d e c r e a s e d r e l a t i v e f l o w t o the r e d muscle, compared t o t h a t found a t 80% I t ^ . - I f i n d e e d massive c i r c u l a t o r y c a t e -cholamine r e l e a s e i s s t r e s s r e l a t e d (Mazeaud et al., 1977), and the s y s t e m i c v a s c u l a t u r e i s under a - c o n s t r i c t o r vasomotor c o n t r o l , then t i s s u e f l o w t o a l l o t h e r c a p i l l a r y networks, as measured by m i c r o s p h e r e entrapment, w i l l be d r a s t i c a l l y reduced a t e x h a u s t i o n , o v e r t he flows found a t 80%'U However, t h i s a r e a o f r e s e a r c h needs f u r t h e r i n v e s t i g a t i o n b e f o r e a p r e c i s e d e s c r i p t i o n o f the c a r d i o v a s c u l a r e v e n t s w h i c h . p a r t i c i p a t e - i n -the' i n a b i l i t y o f t he t r o u t t o m a i n t a i n any f u r t h e r swimming a c t i v i t y can be g i v e n . The p r e v i o u s d i s c u s s i o n s u g g e s t s t h a t s y s t e m i c v a s c u l a r motor c o n t r o l o f Salmo gaivdnevi, a l t h o u g h . g e n e r a l l y u n d e r s t o o d , i s r a t h e r d i f f i c u l t o d e s c r i b e p r e c i s e l y . F i g u r e 11 i s p r e s e n t e d as a g e n e r a l summary o f some o f the c o n t r o l mechanisms which, have been found t o e x i s t i n t h e rainbow t r o u t The p l a s t i c i t y and v a r i a b i l i t y o f the r e l a t i v e e x t e n t o f n e u r o n a l versus humoral ( h o r m o n a l ) r e s p o n s e s . i n the v a s c u l a t u r e a re n o t r e a d i l y q u a n t i f i a b l e f o r the r e s t i n g c o n d i t i o n , and appear t o be even l e s s so d u r i n g s t e a d y - s t a t e , a e r o b i c e x e r c i s e i n t h e s e f i s h . 78 F i g u r e 11: Summary o f the p o s s i b l e s i t e s o f d i r e c t n e u r a l , and c i r c u l a t i n g c a t e c h o l a m i n e a c t i o n i n the c i r c u l a t o r y system o f Salmo gaivdneri. The v a s c u l a t u r e can be d i v i d e d i n t o b r a n c h i a l and c a r d i a c , and. g e n e r a l s y s t e m i c r e g i o n s . D i r e c t i n n e r v a t i o n i s from c r a n i a l n e r v e s IX and X t o the b r a n c h i a l v a s c u l a t u r e , stomach, s p l e e n , h e a r t , h e p a t i c and c o e l i a c o m e s e n t e r i c a r t e r i e s . C h + = c h o l i n e r g i c c o n s t r i c t i o n Ch~ = c h o l i n e r g i c d i l a t i o n / i n h i b i t o r y c a r d i a c ( c h r o n o t r o p i c ) a + = a d r e n e r g i c c o n s t r u c t i o n 3 + = 3 - a d r e n e r g i c s t i m u l a t i o n ( i n o t r o p i c c a r d i a c ) 3" = 3-j o r 3 2 - a d r e n o c e p t o r d i l a t i o n 3-j =• n e u r o n a l 3 - a d r e n o c e p t o r s Rn = humoral 3 - a d r e n o c e p t o r s ( A d r e n o c e p t o r t e r m i n o l o g y a f t e r A l q u i s t , 1948) 7 9 Autonomic nerves- Chromaffin tissue - kidney, terminal release of cardiac, venous-c i rcu la t ing catecholamines to catecholamines to stimulate muscle /B 79a S E C T I O N I I VASCULAR R E S I S T A N C E TO FLOW IN S A L I N E -AND B L O O D - P E R F U S E D P R E P A R A T I O N S OF RAINBOW TROUT 80 INTRODUCTION: SECTION II In the p r e v i o u s s e c t i o n , I have shown t h a t i n i n t a c t t r o u t , system-i c b l o o d f l o w d u r i n g s t e a d y - s t a t e swimming e x e r c i s e i s r e d i s t r i b u t e d t o f a v o u r working muscles. B l o o d p r e s s u r e and c a r d i a c o u t p u t i n c r e a s e d u r i n g e x e r c i s e (see Jones and R a n d a l l , 1978) a s s o c i a t e d w i t h s y s t e m i c b l o o d f l o w r e d i s t r i b u -t i o n . The g i l l s m a i n t a i n the n e c e s s a r y gas t r a n s f e r t o meet the i n c r e a s e d a e r o b i c m e t a b o l i c r e q u i r e m e n t s o f the muscles. The i n c r e a s e d b l o o d p r e s s u r e and f l o w a s s o c i a t e d w i t h swimming e x e r c i s e c o u l d have p r o f o u n d e f f e c t s upon the r e s i s t a n c e t o f l o w through the g i l l s , and upon the p a t t e r n o f f l u i d d i s t r i b u t i o n w i t h i n the g i l l s , g i v e n t h a t t h e s e t i s s u e s , a t r e s t , a l r e a d y f u n c t i o n under e l e v a t e d h y d r o s t a t i c ( b l o o d ) p r e s s u r e . One o b j e c t o f t h i s s t u d y t h e r e f o r e , i s t o examine how changes i n i n p u t p r e s s u r e and f l o w t o the g i l l s c o u l d a l t e r t h e i r f u n c t i o n d u r i n g e x e r c i s e i n the rainbow t r o u t . In o r d e r t o examine g i l l f u n c t i o n i n f i s h e s , a v a r i e t y o f in vitro p e r f u s i o n t e c h n i q u e s have been employed. Most animals seek t o m a i n t a i n c o n s t a n t a r t e r i a l p r e s s u r e by changing b l o o d f l o w o r v a s c u l a r r e s i s t a n c e ( M e l l a n d e r and Johansson, 1968). Most g i l l p r e p a r a t i o n s t h e r e f o r e have been p e r f u s e d u s i n g e i t h e r c o n s t a n t i n p u t p r e s s u r e (Keys, 1931; Rankin and Maetz, 1971; S t r a y - P e d e r s o n and S t e e n , 1975; Payan and M atty, 1975) o r c o n s t a n t i n p u t f l o w (Keys and Bateman, 1932; K i r s c h n e r , 1969; S h u t t l e w o r t h , 1972; Bergman et.al., 1974; Wood, 1974a; Smith, 1977; C l a i r b o r n e and Evans, 1980). However, Bergman et al. (1974) have s t a t e d t h a t p e r f u s i o n o f rainbow t r o u t g i l l s w i t h c o n s t a n t p u l s a t i l e flow.was q u a l i t a t i v e l y s u p e r i o r t o p e r f u s i o n a t c o n s t a n t p r e s s u r e . F u r t h e r m o r e , few p r e p a r a t i o n s have used p h y s i o l o g i c a l d o r s a l a o r t i c p r e s s u r e , even.though the e f f e c t s o f a l t e r a t i o n s i n d o r s a l a o r t i c p r e s s u r e on g i l l v a s c u l a r r e s i s t a n c e t o f l o w can be p r o f o u n d (Wood, 1974a). In the f o l l o w i n g s e c t i o n , an i s o l a t e d , s a l i n e - p e r f u s e d rainbow t r o u t head p r e p a r a t i o n i s d e s c r i b e d . T h i s p r e p a r a t i o n i s used s i n c e i t 81 allows the measurement of f l u i d d i s t r i bu t ion in the g i l l s to be made during perfusion with three d i f fe rent input regimes. These data allow the ef fects of absolute and pu l sa t i l e pressure on recurrent flows and g i l l resistances to be assessed. Haemodynamic a l te ra t ions mimicking those known to occur during exercise in vivo3 and. their e f fects on i n vitro f l u i d d i s t r i bu t ion and g i l l vascular resistance to flow are discussed with respect to the i r possible roles in g i l l function at r es t , and during exercise in the in tac t t rout . A spontaneously v e n t i l a t i n g , blood-perfused whole trout preparation also i s described in d e t a i l . - This preparation overcomes many of the problems inherent in the i n vitro, sal ine-perfused studies. This blood-perfused preparation i s characterized and, in th is sec t i on , i t s s u i t a b i l i t y for the study of the ef fects of• haemodynamic changes on.branchial and systemic vascular resistances to blood flow in f i s h , i s assessed. 82 RESULTS II E f f e c t o f P u l s a t i l i t y o f I n p u t on R e s i s t a n c e t o f l o w and F I u i d Flow D i s t r i b u - t i o n In the G i l l s o f S a l i n e - P e r f u s e d T r o u t Heads A d e s c r i p t i o n o f the s u r g i c a l p r o c e d u r e s used f o r the p r e p a r a t i o n o f the s a l i n e - p e r f u s e d t r o u t heads, has been,{given i n the M a t e r i a l s and Methods s e c t i o n on pages 34 to.35. A t o t a l o f 24 f i s h were used, but o n l y r e s u l t s from p r e p a r a t i o n s which-gave c o n t i n u o u s and s t a b l e f l o w from both d o r s a l and a n t e r i o r venous c a t h e t e r s t h r o u g h o u t a l l e x p e r i m e n t s a r e p r e s e n t e d . T w o - t h i r d s o f . t h e s e p r e p a r a t i o n s f a i l e d t o s a t i s f y t h e s e c r i t e r i a , l e a v i n g e i g h t complete s e t s o f d a t a . Where time p e r m i t t e d , r e p e a t measurements were made'\w'i-t'ft"* p u l s a t i l e and/or c o n s t a n t p r e s s u r e p e r f u s i o n . I n i t i a l measurements were no t s i g n i f i c a n t l y , d i f f e r e n t from t h o s e taken l a t e r * f o r any o f the p e r f u s i o n regimes t e s t e d , d e s p i t e the n o t i c e a b l e s w e l l i n g , ( t i s s u e oedema) i n the p r e p a r a t i o n s a t the end o f the 1.5 t o 2. hours o f e x p e r i m e n t a t i o n . S a l i n e was bubbled w i t h a gas m i x t u r e which s i m u l a t e d gas t e n s i o n s i n venous b l o o d o f i n t a c t and r e s t i n g rainbow t r o u t . Haswell et al. (1978) have r e p o r t e d t h a t low oxygen t e n s i o n , i n the s a l i n e p r o l o n g s p r e p a r a t i o n v i a b i l i t y , and i s q u a l i t a t i v e l y b e t t e r f o r the p e r f u s i o n o f i s o l a t e d f i s h head p r e p a r a t i o n s . T a b l e 10 summarizes the v a l u e s f o r f l o w s and the e s t i m a t e s o f o t h e r parameters o b t a i n e d from the s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n s . These d a t a i n d i c a t e t h a t o n l y a n t e r i o r venous o u t f l o w (Q^y) was i n c r e a s e d s i g n i f i -c a n t l y d u r i n g p u l s a t i l e p e r f u s i o n , over v a l u e s found w i t h e i t h e r c o n s t a n t p r e s s u r e o r c o n s t a n t f l o w p e r f u s i o n . Other v a r i a b l e s and parameters showed no s i g n i f i c a n t d i f f e r e n c e s among d i f f e r e n t regimes. G i l l v a s c u l a r r e s i s t a n c e t o f l o w (R .) was c a l c u l a t e d from the p r e s s u r e d i f f e r e n c e between the d o r s a l and v e n t r a l a o r t a e , d i v i d e d by the r a t e o f i n p u t p e r f u s i o n t Q ^ ) - V e n t r a l a o r t i c p r e s s u r e was t a k e n a s the measured i n p u t p r e s s u r e minus the p r e s s u r e drop a c r o s s the c a t h e t e r a l o n e , a t the same f l o w r a t e (see Wood and S h e l t o n , 82a T a b l e 10. Summary, o f d o r s a l a o r t i c and a n t e r i o r venous o u t f l o w from the i s o l a t e d t r o u t head .[Salmo-gaivdneri) p r e p a r a t i o n d u r i n g p e r f u s i o n by t h r e e d i f f e r e n t i n p u t regimes. N r e p r e s e n t s number o f o b s e r v a t i o n s . P e r f u s i o n Regime C o n s t a n t p u l s a t i l e flOW (1) N. = 16 C o n s t a n t Flow (2) N = 12 Cons t a n t n o n - p u l s a t i l e Flow (3) N = 8 V a r i a b l e % A (ml"min ) 1.54 ± 0 . 1 3 '1 .43 ± 0 .22 1.34 ± 0 . 1 8 QAV , (ml'min ) 1.39 ± 0 . 0 9 T .09 ± 0.11 1.01 ± 0.11 R g , (Cm H^O-rnl i" mi n ' l O O g - 1 ) 14.41 ± 1 .36 18 .11 ± 2 . 8 5 * 2 3 . 5 3 ± 5 . 2 2 R A V . (Cm H 2 0*ml" * m i n ' l O O g - 1 ) 11 . 67 ± 0 . 9 8 14 . 97 ± 1 .83 1 7 . 8 3 ± 2 . 6 0 Q V A (ml"min" 1) 4 . 7 9 ± 0 . 1 9 4 .41 ± 0 . 29 4 .62 ± 0 . 1 3 % S a l i n e l o s s 38.71 ± 3 .08. 42 .26 ± 3 .75 4 8 . 8 6 ± 4 . 3 3 U n d e r l i n e d v a l u e i s the o n l y one which was s i g n i f i c a n t l y d i f f e r e n t due t o changes i n the p e r f u s i o n regime used. * n o t e : Rg (Wood, 1974a) a t same d o r s a l a o r t i c p r e s s u r e , and f l o w r a t e was 18.8 Cm H ? 0 " m l _ 1 * m i n " l O O g " 1 . 83 1975). R e s i s t a n c e o f the a n t e r i o r venous c i r c u l a t i o n was c a l c u l a t e d from the p r e s s u r e d i f f e r e n c e between the d o r s a l a o r t a and:the a n t e r i o r venous c a t h e t e r ( s e t t o z e r o p r e s s u r e ) , d i v i d e d by Q^.. R e s i s t a n c e v a l u e s were e x p r e s s e d i n u n i t s o f cm HgO'mT.^mtn'lOOg" 1". These v a l u e s were not d i f f e r e n t among the p e r f u s i o n regimes. P e r c e n t o f p e r f u s a t e l o s t from each p r e p a r a t i o n was d e t e r m i n e d and was found t o remain c o n s t a n t a t a p p r o x i m a t e l y 43% o f the i n f l o w . P e r c e n t l o s s was n o t s i g n i f i c a n t l y d i f f e r e n t among the p e r f u s i o n regimes t e s t e d . A dye ( p a t e n t B l u e ; a - Z u r i n e , Sigma) was i n t r o -duced i n t o the s a l i n e t o check how e v e n l y the g i l l s were p e r f u s e d , and t o a s s e s s the degree o f v a s c u l a r l e a k a g e . V i s u a l i n s p e c t i o n o f the g i l l s d u r i n g dye i n f u s i o n showed even c o l o u r a t i o n d u r i n g a l l p e r f u s i o n regimes. The t i p s o f g i l l f i l a m e n t s were the l a s t a r e a s t o c o l o u r w i t h dye. O c c a s i o n a l l y , s m a l l l e a k s were o b s e r v e d from the g i l I s , o f t e n where mec h a n i c a l damage was e v i d e n t . However, most o f the s a l i n e l o s s seen d u r i n g dye i n f u s i o n was from the h y p o b r a n c h i a l a r e a , which was c u t d u r i n g c a t h e t e r i z a t i o n o f the h e a r t . S t u d i e s o f G i l l . B l o o d Flow i n a^  S p o n t a n e o u s l y V e n t i l a t i n g , B l o o d - P e r f u s e d  T r o u t P r e p a r a t i o n : C h a r a c t e r i z a t i o n o f the p r e p a r a t i o n , D e s c r i p t i o n o f the s u r g i c a l p r o c e d u r e s used d u r i n g the p r e p a r a t i o n o f b l o o d - p e r f u s e d t r o u t have been g i v e n i n the M a t e r i a l s and Methods s e c t i o n , pages 45 t o 49. B l o o d used f o r p e r f u s i o n was taken from donor f i s h which had r e c o v e r e d from the e f f e c t s o f c a n n u l a t i o n o f t h e i r d o r s a l a o r t a e f o r a t l e a s t 14 hours. T h i s was done i n o r d e r t h a t the b l o o d which was used t o p e r f u s e the r e c i p i e n t ^ f i s h was f r e e o f a n a e s t h e t i c . Donor f i s h a l s o d i d n o t appear t o be a g i t a t e d d u r i n g the withdrawal o f t h e i r b l o o d . T h i s a v o i d e d p o s s i b l e i n c r e a s e s i n the l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s , known t o be r e l e a s e d i n t o the b l o o d d u r i n g s t r e s s i n f i s h (see Nakano and T o m l i n s o n , 1967). S u r g i c a l l y p r e p a r e d f i s h s t a r t e d to v e n t i l a t e s p o n s t a n e o u s l y w i t h i n 20 - 30 minutes o f the commencement o f b l o o d p e r f u s i o n . Once v e n t i l a t i o n 84 became r e g u l a r , the mouth tube which a s s i s t e d , water f l o w o v e r the g i l l s was removed and the f i s h then i r r i g a t e d t h e i r own g i l l s a t 69 ± 1 v e n t i l a -t i o n s - m i n " 1 . By t h i s t i m e , f i s h had r e g a i n e d r i g h t i n g and v i s u a l t r a c k i n g r e f l e x e s , l o s t d u r i n g a n a e s t h e s i a . Some f i s h became a g i t a t e d and attempted t o swim d u r i n g t he i n i t i a l r e c o v e r y p e r i o d . V i s u a l d i s t u r b a n c e s from t he s u r r o u n d i n g s were e l i m i n a t e d , i n p a r t , by masking the h o l d i n g box w i t h b l a c k p l a s t i c s h e e t s . T y p i c a l l y , e x periments l a s t e d 4 - 6 h o u r s , which, when combined w i t h 2 - 3 hours f o r r e c o v e r y from o p e r a t i v e p r o c e d u r e s , meant-that measure-ments were taken from f i s h which were p e r f u s e d f o r no more than 9 hours. Over t h i s time p e r i o d , t h e r e appeared to. be no a p p r e c i a b l e d e t e r i o r a t i o n o f b l o o d , as i n d i c a t e d by the l a c k o f c e l l l y s i s , o r o f the f i s h , as i n d i c a t e d by c o n t i n u o u s and s t a b l e oxygen uptake r a t e s , as w e l l . a s carbon d i o x i d e e x c r e t i o n r a t e s a c r o s s t he g i l l s . . Some p r e p a r a t i o n s were m a i n t a i n e d f o r up t o 18 hours under r e s t i n g , normal c o n d i t i o n s , without, any e x p e r i m e n t a l m a n i p u l a t i o n s . G e n e r a l l y , f a i l u r e o f t he p r e p a r a t i o n was due t o , m e c h a n i c a l problems a s s o c i a t e d . w i t h the c a r d i a c pump s y r i n g e p l u n g e r , r a t h e r than d e t e r i o r a t i o n o f the f i s h . T h r e e , 0.7 ml. b l o o d samples were withdrawn s i l u l t a n e o u s l y from the tonometer, d o r s a l a o r t a and venous r e t u r n l i n e . No d i f f e r e n c e s were o b s e r v e d i n pH o r gas t e n s i o n s o f b l o o d from t h e tonometers or the i n p u t c a t h e t e r . T h e r e f o r e , b l o o d samples from the tonometer were taken as r e p r e s e n t a t i v e o f v e n t r a l a o r t i c i n p u t v a l u e s . An advantage o f e x t r a c o r p o r e a l r e s e r v o i r s i s t h a t r e p e a t e d s a m p l i n g does not d e p l e t e the b l o o d volume i n the a n i m a l . C o n s e q u e n t l y , more experiments c o u l d be performed on each b l o o d - p e r f u s e d p r e p a r a t i o n than on i n t a c t f i s h . T y p i c a l s i m u l t a n e o u s r e c o r d i n g s o f c a r d i o r e s p i r a t o r y v a r i a b l e s o b t a i n e d from a p r e p a r a t i o n are shown i n F i g . 12. V e n t i l a t o r y i n t e r a c t i o n 85 F i g u r e 12. Records o f p r e s s u r e s and f l o w from s p o n t a n e o u s l y v e n t i l a t i n g b l o o d - p e r f u s e d t r o u t . A. Simultaneous r e c o r d s o f f l o w and p r e s s u r e from a p r e p a r a t i o n which d i s p l a y e d c a r d i o r e s p i r a t o r y i n t e r a c t i o n s . Arrow heads show i n t e r a c t i o n s o f r e s p i r a t o r y movements on the p r e s s u r e and f l o w t r a c e s . Q, p e r f u s i o n f l o w r a t e ; VAP, v e n t r a l a o r t i c ( i n p u t ) p r e s s u r e ; DAP, d o r s a l a o r t i c p r e s s u r e ; b u c c a l p r e s s u r e r e c o r d o f v e n t i l a t o r y movements; v e n t r i c u l a r p r e s s u r e r e c o r d o f i n t r i n s i c h e a r t a c t i v i t y . Arrow i n d i c a t e s c o n t r a c t i o n . B. Record o f VAP d u r i n g i n c r e a s e d p u l s e p r e s s u r e . Note t h a t the h e a r t - l i k e p r e s s u r e s i g n a l i s m a i n t a i n e d . C. Record o f v e n t r i c u l a r c o n t r a c t i o n d u r i n g exposure o f f i s h t o h y p o x i c w a t e r , showing the 'on' re s p o n s e ( b r a d y c a r d i a ) and a f t e r 180 seconds e x p o s u r e , and the ' o f f res p o n s e ( p o s t - h y p o x i c t a c h y c a r d i a ) a f t e r resumption o f normoxic water f l o w . Note t h a t t he p r e s s u r e d e v e l o p e d by the v e n t r i c l e i n c r e a s e d d u r i n g h y p o x i c exposure. T h i s p r e s s u r e was not c a l i b r a t e d . 86 ml-min A 10 | -,1 5 l O O r VAP c m H ^ 1 0 « -ioor DAP c m H 2 0 SOI 101-+5| BUCCAL P. c m H 2 0 C - 5 B A A A / V W -TIME,SEC N 2 O N • 180 S E C . T CQ CC < UJ X CO z E lUUUUUUt 10SEC 10SEC JlLk. J i INTRINSIC^ HEART BEAT I \ If I u J I VU 10 S E C . • AIR ON + 280 S E C . TIME, S E C . 87 on the i n p u t p r e s s u r e t r a c e were o b s e r v e d f r e q u e n t l y , e s p e c i a l l y d u r i n g a r e s p i r a t o r y "cough" (Hughes and Ardeny, 1977) (see F i g . 12A). T h i s p u l s e o f i n c r e a s e d p r e s s u r e , whether i n phase w i t h the c a r d i a c pump c y c l e o r n o t , u s u a l l y was t r a n s m i t t e d through the g i l l v a s c u l a t u r e t o some e x t e n t (see a l s o Wood and S h e l t o n , 1980a), and was e v i d e n t i n the d o r s a l a o r t i c p r e s s u r e t r a c e o b t a i n e d i n the p r e s e n t s t u d y . F i g u r e 12B shows a p o r t i o n o f i n p u t t r a c e from a f i s h i n which the p u l s e p r e s s u r e has been r a i s e d by a d j u s t i n g the s i z e o f the a i r space i n the Windkessel (see F i g . 9 ) . B r a d y c a r d i a i s a s s o c i a t e d w i t h exposure o f t r o u t t o h y p o x i c w a t e r , and i s one o f t h e b e t t e r d e s c r i b e d c a r d i o v a s c u l a r r e f l e x e s i n f i s h ( s e e Dax'boeck and H o l e t o n , 1978; Smith and J o n e s , 1978). B l o o d - p e r f u s e d t r o u t p r e p a r a t i o n s a l s o showed t y p i c a l h y p o x i c b r a d y c a r d i a r e s p o n s e s when n i t r o g e n gas was bubbled through the i n s p i r e d water t o . l o w e r the oxygen t e n s i o n ( F i g . 12C). Both the "on" r e s p o n s e ( b r a d y c a r d i a ) and the " o f f " response ( p o s t - h y p o x i c t a c h y c a r d i a ) were o b s e r v e d . These r e s u l t s i n d i c a t e d t h a t b l o o d -p e r f u s e d t r o u t had o p e r a t i o n a l a f f e r e n t and e f f e r e n t r e f l e x pathways a v a i l a b l e , by which c a r d i o v a s c u l a r adjustments may be made. V a r i a b l e s which were measured r o u t i n e l y from u n d i s t u r b e d and r e s t i n g , b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n s a r e summarized i n T a b l e s 11 and 12. These d a t a were c o n s i d e r e d t o be "normal" f o r t h i s p r e p a r a t i o n , as d e f i n e d by the c r i t e r i a s e t out e a r l i e r i n the M a t e r i a l s and Methods S e c t i o n o f t h i s t h e s i s . Under t h e s e c o n d i t i o n s , a l l f i s h m a i n t a i n e d c o n s i s t e n t oxygen uptake (Mg0 2) and carbon d i o x i d e e x c r e t i o n r a t e s (MgCOg) a c r o s s the g i l l s . : A c r o s s the s y s t e m i c c i r c u l a t i o n , oxygen was e x t r a c t e d ( M ^ ) , and carbon d i o x i d e produced by the m e t a b o l i z i n g t i s s u e s ( M C ^ ) . F u r t h e r t r e a t m e n t o f the d a t a c o n c e r n i n g gas t r a n s f e r i n t h e s e p r e p a r a t i o n s i s p r e s e n t e d i n S e c t i o n I I I i n d e t a i l , and w i l l n o t be d i s c u s s e d i n t h i s s e c t i o n . T a b l e 11. Summary o f v a r i a b l e s f o r the normal, r e s t i n g s t a t e o f the s p o n t a n e o u s l y v e n t i l i n g , b l o o d - p e r f u s e d rainbow t r o u t a t 7 C (N = 15 f i s h ; 306.2 ± 9.3 g ) . P r e s s u r e ( c m H^O) mean* p u l s e C a r d i a c pump f(bpm) SV(ml) ml Q . • -1. mm Hct Acid/Base P 0 2 C0 o ' l O O g - 1 % H + nM (pH) mm Hg nM p r c o 2 mm Hg c c o 2 mM Plasma O s m o l a r i t y mOsm C I " mM INPUT (VENOUS) BLOOD X 58.8 10.7 40 0.125 1.619 10.3 17.66 (7.76) 24.9 0.90 3.36 10.28 275.4 111.9 S.E.M. 2.0 0.2 0 0.003 0.025 0.2 . 0.40 1.1 0.05 0.15 0.23 0.62 0.70 n 45 45 45 45 45 45 44 44 44 44 45 5 ( p o o l e d ) 5 DORSAL AORTIC BLOOD X 34.8 2.05 8.80.19.72 (7.72)103.4 1.58 3.66 9.02 265.7 115.8 S.E.M. 0.95 0.13 n o t a p p l i c a b l e 0.2 0.71 2.4 0.08 0.16 0.20 0.30 0.60 n 45 45 42 44 44 44 44 45 5 ( p o o l e d ) 5 f J ' m i n _, I n t r i n s i c ) h e a r t r a t e (bpm) VENOUS RETURN BLOOD X 9 69:4 48.5 9.4 17.55 (7.76) 13.0 0.42 3.65 10.01 273.8 108.2 S.E.M. 0.96 0.99 n o t measured 0.6 0.83 0.5 0.04 0.18 0.20 0.69 1.60 n 42 37 39 42 41 39 41 42 5 ( p o o l e d ) 5 Mean p r e s s u r e s have been c a l c u l a t e d as (1 s y s t o l i c + 2 d i a s t o l i c ) / 3 ( B u r t o n , 1972), T a b l e 12. Summary o f the d i f f e r e n c e s i n v a r i a b l e s a c r o s s the g i l l ( A i n p u t - d o r s a l a o r t a ) and the s y s t e m i c ( A d o r s a l a o r t a - venous r e t u r n ) c i r c u l a t i o n s i n the r e s t i n g s t a t e o f s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d rainbow t r o u t . a t 7°C (N = 15 f i s h ; 306.2 ± 9.3 g ) . AINPUT - DORSAL AORTA OO H + H + P0 o P0 o C 0 o CO 2 r"2 ^2 ^2 PC0„ PC0 9 C G 0 C. n AP Rg M 0 o M CO RO nM % mmHg % mM % mm rig % Z Sfc 9 _ u n. ,-1. . . l n A -1 M. - I 9 2 -1 9-5 _ i • cm H 20 cm H 20-ml -min'lOOg"- 1 yM'min" ' 100g _ J- y M ' m i n " 1 , 1 0 0 g _ i M g r n /Mg n g 2 u 2 X +2.06 +13.6 +78.7 +347 +0.69 +79.6 +0.05 +2.32 -1.25 -11.93 23.1 14.23 1.17 2.05 1.85 ±S.E.M. 0.61 3.4 2.4 20.8 0.05 5.8 0.12 3.36 0.09 0.77 1.75 1.14 0.08 0.15 0.12 n 44 44 44 44 44 43 43 44 45 45 45 44 44 44 43 ADORSAL AORTA - VENOUS RETURN flPs Rs "s°2 id). RQ s 2 ^s X - 1 . 3 6 - 6 . 3 6 - 8 9 . 3 - 8 7 . 4 - 1 . 2 0 - 7 4 . 6 + 0 . 0 7 + 4 . 2 7 + 1 . 0 1 + 1 1 . 6 6 31.1 19.21 1.97 1.63 0.83 ±S.E.M. 0.64 3.1 2.2 0.55 0.06 1.67 0.14 4.06 0.06 0.83 0.95 0.88 0.11 0.09 0.09 n 41 41 41 41 39 39 41 41 42 42 45 44 39 44 39 90 The d a t a i n T a b l e 11 a l s o i n d i c a t e t h a t b l o o d was d i l u t e d i n i t s passage through the g i l l s o f b l o o d - p e r f u s e d t r o u t , as h a e m a t o c r i t f e l l by 24%, and plasma o s m o l a r i t y d e c r e a s e d by 3.5%. These changes c o u l d be caused by a number o f f a c t o r s , i n c l u d i n g c e l l volume changes a s s o c i a t e d w i t h COg e x c r e t i o n , water uptake a c r o s s the g i l l e p i t h e l i u m , o r s i m p l y as an a r t i f a c t o f the s a m p l i n g p r o c e d u r e . B r a n c h i a l v a s c u l a r r e s i s t a n c e (Rg) comprised 43% o f the t o t a l v a s c u l a r r e s i s t a n c e t o f l o w i n t h e s e p r e p a r a t i o n s ( T a b l e 12). Data p r e s e n t e d i n T a b l e s 11 and 12, taken from s p o n t a n e o u s l y v e n t i l a t i n g , , b l o o d - p e r f u s e d whole t r o u t p r e p a r a t i o n s , - a r e comparable t o t h o s e a v a i l a b l e from i n t a c t and r e s t i n g rainbow t r o u t (see Jones and R a n d a l l , 1978). The p r e v i o u s l y d e s c r i b e d p r e p a r a t i o n t h e r e f o r e was used t o a s s e s s the e f f e c t s o f s p e c i f i c changes i n c a r d i o v a s c u l a r parameters which a r e known t o o c c u r d u r i n g e x e r c i s e i n vivo, on g i l l and s y s t e m i c v a s c u l a r r e s i s t a n c e s , but under normoxic, r e s t i n g c o n d i t i o n s . C a r d i a c Output (Q) Changes C a r d i a c o u t p u t i n c r e a s e s i n i n t a c t t r o u t d u r i n g swimming. T a b l e 13 summarizes the e f f e c t s o f changes i n the p e r f u s i o n f l o w r a t e (Q) on the measured v a s c u l a r v a r i a b l e s i n s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t a t r e s t . Input p r e s s u r e (VAP), and d o r s a l a o r t i c p r e s s u r e (DAP) i n c r e a s e d above normal v a l u e s by s m a l l , , but n o n - s i g n i f i c a n t amounts, w i t h i n c r e a s i n g Q. As a r e s u l t , w i t h i n c r e a s e d f l o w , both the c a l c u l a t e d g i l l r e s i s t a n c e (Rg)» a n d the s y s t e m i c r e s i s t a n c e ( R g ) t o f l o w were d e c r e a s e d from v a l u e s c a l c u l a t e d under normal c o n d i t i o n s . C o n v e r s e l y , t h e s e r e s i s t a n c e s i n c r e a s e d as the r a t e o f p e r f u s i o n was d e c r e a s e d below normal, and both VAP and DAP were d e c r e a s e d from normal v a l u e s and d e c r e a s i n g Q. C a r d i a c Pump Frequency (f_) and S t r o k e Volume (SV) M a n i p u l a t i o n s S i m u l a t e d e x e r c i s e t a c h y c a r d i a , but w i t h no i n c r e a s e i n Q, had no T a b l e 13. Summary o f the e f f e c t s o f i n c r e a s e d and d e c r e a s e d c a r d i a c o u t p u t on c a r d i o v a s c u l a r v a r i a b l e s from normal, s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t (N = 7; 292.7 ± 14.3 g ) . VAP (cm H 20) DAP (cm H 20) Q ml ' m i n - 1 Hct ( i n p u t ) Hct (DA) R9 R s cor mean p u l s e mean p u l s e % % cm H 20 ' m r 1'min'100g" 1 cm HgC ' m r^min'lOOg" 1 :MAL X 54.1 11.1 34.4 2.0 4.80 9.6 8.5 12.1 18.5 O ± S. E, .M. 5.1 0.3 1.9 0.2 0.20 0.4 0.5 2.8 2.9 cr re X 56.5 15.8 36.3 2.5 7.20 9.6 8.5 8.20 13.2 i—i rc ± s. E. .M.- 4.2 0.7 2.4 0.5 0.40 0.5 0.6 1.40 0.7 cor X 42.9 7.8 29.4 1.4 2.76 9.34 7.8 14.3 26.9 O 1 ± s. E, ,M. 3.5 0.5 2.0 0.2 0.24 0.5 0.5 2.9 2.0 92 e f f e c t upon any o f the measured v a r i a b l e s , e x c e p t t h a t the v e n t r a l and 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 s were d e c r e a s e d . However, u n l i k e most i n t a c t t r o u t which have been e x e r c i s e d (see Jones and R a n d a l l , 1978), my f i s h d i d n o t show on i n c r e a s e i n :heart r a t e ( T a b l e 8 ) . S i m u l a t e d b r a d y c a r d i a i n normoxic water however, s i g n i f i c a n t l y d e c r e a s e d g i l l r e s i s t a n c e ( T a b l e 14), d e s p i t e a s l i g h t d e c r e a s e i n Q. T h i s p e r f u s i o n c o n d i t i o n a l s o s i g n i f i c a n t l y i n c r e a s e d VAP and DAP. .The a m p l i t u d e o f the p u l s e p r e s s u r e was reduced by the same amount i n i t s passage through the g i l l s d u r i n g s i m u l a t e d b r a d y c a r d i a and t a c h y c a r d i a , a t c o n s t a n t p e r f u s i o n f l o w r a t e s . A l l o t h e r v a r i a b l e s measured changed v e r y T i t t l e from normal v a l u e s . P u l s e P r e s s u r e (PP) Changes I n c r e a s e d i n p u t p u l s e p r e s s u r e a s o c c u r s in vivo d u r i n g e x e r c i s e , but w i t h no accompanying change i n pump f r e q u e n c y o r s t r o k e volume, caused no s i g n i f i c a n t changes i n any o f the v a r i a b l e s measured, compared t o v a l u e s found i n T a b l e 12. A l t h o u g h 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 was i n c r e a s e d above normal, the a m p l i t u d e o f t h e 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 d i d n o t i n c r e a s e by the same amount, i n d i c a t i v e o f p r e s s u r e damping w i t h i n the g i l l v a s c u l a t u r e . D e c r e a s e s . i n 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 (3 f i s h o n l y ) a l s o produced no s i g n i f i c a n t changes i n any o f the v a r i a b l e s measured, a l t h o u g h an i n c r e a s e i n the g i l l r e s i s t a n c e was o b s e r v e d ( T a b l e 14). H a e m a t o c r i t (Hct) Changes R e g a r d l e s s o f whether h i g h o r low h a e m a t o c r i t b l o o d was p e r f u s e d through the g i l l s o f b l o o d - p e r f u s e d t r o u t , VAP was i n c r e a s e d t o the same l e v e l above the normal v a l u e . C o n v e r s e l y , DAP i n both i n s t a n c e s f e l l t o s i m i l a r l e v e l s below t h a t found i n the normal s i t u a t i o n ( T a b l e 15). S i n c e the p r e s s u r e d i f f e r e n t i a l a c r o s s the g i l l s . w a s i n c r e a s e d i n both c a s e s , w i t h no accompanying changes i n Q, the c a l c u l a t e d g i l l r e s i s t a n c e was i n c r e a s e d . 93 T a b l e 14. E f f e c t s o f c a r d i a c o u t p u t (Q), s t r o k e volume ( S V ) , p u l s e p r e s s u r e (PP) and c a r d i a c f r e q u e n c y ( f ) on g i l l r e s i s t a n c e change (AR % ) , y i n r e s t i n g , b l o o d - p e r f u s e d t r o u t (0 = no change from normaa1; + = i n c r e a s e f r o m n o r m a l ; - = d e c r e a s e from n o r m a l ) . Means ±S.E.M. TREATMENT N (from n o r m a l ) . (1) SV +; PP +; f 0; Q + 7 -22.91 ± 9.09 (2) SV +; PP +: f -; Q 0 8 -25.22 ( b r a d y c a r d i a ) ± 8 . 9 8 (3) SV -; PP -; f 0; Q - 7 -35.38 ±17.96 (4) SV - ; PP.-J f +; Q 0 8 + 4.48 ( t a c h y c a r d i a ) ±18.63 (5) SV 0; PP + ; f 0; Q 0 8 +15.37 ±11.16 (6) SV 0; PP -; f 0; Q 0 3 +14.99 ±15.89 T a b l e 15. Summary o f the e f f e c t s o f v a r i a b l e i n p u t h a e m o t o c r i t on c a r d i o v a s c u l a r parameters from normal s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t (N = 6 f i s h ; 320.2 ± 17.2 g and c o n s t a n t Q = 5.20 ± 0.04 m l ' m i n - 1 ) . VAP (cm H 20) mean p u l s e DAP mean (cm H 20) p u l s e Hct ( i n p u t ) % Hct (DA) % cm H 20"ml ^'min'lOOg R s cm H 20 - m r 1 ,min -100g" 1 X 60.6 10.8 40.2 2.2 11.3 9.3 12.6 22.3 + s. .E.M. ; l . 7 0.5 2.6 0.4 0.5 0.5 1.5 1.8 X 76.2 10.7 28.3 1.4 4.3 3.9 29.6 15.1 + s. ,E.M. 7.7 0.7 1.9 0.3 0.4 0.5 4.9 1.4 X 76 . 1 11.8 33.8 1.7 20.2 16.5 26 . 1 18.4 ± s, .E.M. 6.9 0.9 3 . 1 0.2 1.6 1.2 4.3 1.7 95 As a consequence o f the d e c r e a s e d DAP, s y s t e m i c r e s i s t a n c e was a f f e c t e d , b e i n g lower than normal l e v e l s . No o t h e r v a s c u l a r v a r i a b l e s were a f f e c t e d by changes i n Hct. A l l d a t a p r e s e n t e d i n T a b l e 15 a r e comparable t o normal v a l u e s o b t a i n e d f r o m s i m i l a r p r e p a r a t i o n s (see T a b l e 11). A d r e n a l i n e Exposure The l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s a r e thought t o i n c r e a s e d u r i n g e x e r c i s e i n i n t a c t t r o u t . The e f f e c t s o f the a d d i t i o n o f a d r e n a l i n e to a f i n a l b l o o d c o n c e n t r a t i o n o f 1 x.1.0" M on v a s c u l a r v a r i a b l e s i n b l o o d -p e r f u s e d p r e p a r a t i o n s a r e summarized i n T a b l e 16. T h i s c o n c e n t r a t i o n may have been s l i g h t l y h i g h e r than in vivo. P r e p a r a t i o n s showed s i g n i f i c a n t (P<:0.10) d e c r e a s e s i n g i l l v a s c u l a r r e s i s t a n c e t o f l o w o f a p p r o x i m a t e l y 38%, a s s o c i a t e d w i t h a l a r g e r r i s e i n DAP than i n VAP. S y s t e m i c v a s c u l a r r e s i s t -ance was i n c r e a s e d s i g n i f i c a n t l y (P<0.10) by 56% above normal i n t h i s s i t u a t i o n . The f a l l i n plasma o s m o l a r i t y a c r o s s t h e g i l l s was t w i c e as l a r g e as o b s e r v e d . i n normal b l o o d - p e r f u s e d p r e p a r a t i o n s . This, was e q u i v a l e n t t o and a p p r o x i m a t e . 2 - f o l d i n c r e a s e i n . t h e c a l c u l a t e d n e t water i n f l u x a c r o s s the g i l l s d u r i n g a d r e n a l i n e e x p o s u r e . T h i s r e s u l t was c o n s i s t e n t w i t h changes ob s e r v e d i n i s o l a t e d , s a l i n e - p e r f u s e d t r o u t heads exposed t o an e q u i v a l e n t l e v e l o f a d r e n a l i n e ( I s a i a , Maetz and Haywood, 1978). 96 T a b l e 16. E f f e c t s o f 1 x 10 M a d r e n a l i n e on s e l e c t e d v a r i a b l e s i n b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n s (N = 4 f i s h ) . VAP DAP R D 9 R, s cm H o0 ,mr i'min"100g"- L cm ti^O'ml_1"min' 100g" (cm H ?0) (cm hLO) , , i _1 NORMAL X, 40.4 30.5 6.48 16.38 ± S.E.M. 5.4 5.3 0.82 3.35 ADRENALINE X 51.3 44.6 4.01 25.06 + S.E.M. 4.7 3.1 1.02 1.97 97 DISCUSSION IT: The E f f e c t s o f Haemodynamic A l t e r a t i o n s , on Flow D i s t r i b u t i o n  and B r a n c h i a l V a s c u l a r R e s i s t a n c e t o Flow i n S a l i n e - P e r f u s e d  T r o u t Head P r e p a r a t i o n s -S a l i n e - p e r f u s e d head p r e p a r a t i o n s were p e r f u s e d a t f l o w r a t e s s i m i l a r t o t h o s e used f o r b l o o d - p e r f u s e d t r o u t , w i t h a d o r s a l a o r t i c p r e s s u r e m a i n t a i n e d a t 40 cnrH^O. These v a l u e s were chosen t o mimic those found i n -i n t a c t , u n r e s t r a i n e d rainbow t r o u t a t r e s t (Stevens and R a n d a l l , 1967a; K i c e n i u k and J o n e s , 1977). A n t e r i o r venous f l o w (Q^y) from s a l i n e - p e r f u s e d t r o u t heads was g r e a t e r d u r i n g p u l s a t i l e p e r f u s i o n than d u r i n g e i t h e r o f the n o n - p u l s a t i l e p e r f u s i o n regimes. None o f t h e o t h e r v a r i a b l e s were a l t e r e d s i g n i f i c a n t l y by p e r f u s i o n w i t h d i f f e r e n t regimes. The a n t e r i o r venous c i r c u l a t i o n i s comprised o f head v e s s e l s , as w e l l as g i l l v enolymphatie v e s s e l s . P u l s a t i l i t y i n h i g h p r e s s u r e g i l l a r t e r i e s may have been t r a n s m i t t e d t o the s u r r o u n d i n g low p r e s s u r e v e n o l y m p h a t i e system. T h i s mechanical i n t e r a c t i o n t h e r e f o r e may be i m p o r t a n t i n g e n e r a t i n g the o b s e r v e d i n c r e a s e i n d u r i n g p u l s a t i l e p e r f u s i o n o f the g i l l s . P u l s a t i l e p r e s s u r e s i n v e n o l y m p h a t i e v e s s e l s found in vivo i n the l i n g c o d , Ophiodon elongatus ( F a r r e l l , 1979) s u p p o r t t o n o t i o n o f t r a n s m i s s i o n o f p u l s e p r e s s u r e from a r t e r i e s t o v e n o l y m p h a t i e v e s s e l s . T h i s i s a c l o s e p r o x i m i t y o f v e s s e l s t o f a c i l i t a t e m e c h a n i c a l i n t e r a c t i o n s , s i n c e the v enolymphatie p a r a l l e l the fi.lamenta 1 a r t e r i e s , and o f t e n s u r r o u n d the l a m e l l a r a r t e r i e s i n d o u g h n u t - l i k e s t r u c t u r e s ( p e r s o n a l o b s e r v a t i o n s from polymer c a s t s o f g i l l s o f Amia oalva; see a l s o Daxboeck et al.., 1981). These r e g u l a r p r e s s u r e p u l s a t i o n s i n the e x t e n s i v e l y v a l v e d v e n o l y m p h a t i e v e s s e l s (Gannon, u n p u b l i s h e d o b s e r v a t i o n s ) w i l l augment l y m p h a t i c space d r a i n a g e . Thus, the p u l s a t i l i t y , r a t h e r than the a b s o l u t e p r e s s u r e , causes the i n c r e a s e s i n measured i n the s a l i n e - p e r f u s e d t r o u t head, by i n c r e a s i n g v e n o l y m p h a t i e f l o w , p r i m a r i l y from the g i l l s . 98 E x e r c i s e i n f i s h i s a n a t u r a l c o n d i t i o n w i t h which d r a m a t i c i n c r e a s e s i n b l o o d p u l s e p r e s s u r e a r e a s s o c i a t e d ( K i c e n i u k and J o n e s , 1977). D u r i n g e x e r c i s e , rainbow t r o u t show i n c r e a s e s i n v e n t r a l , and t o a l e s s e r e x t e n t , d o r s a l a o r t i c p r e s s u r e s (Stevens and R a n d a l l , 1967a; K i c e n i u k and J o n e s , 1977) as w e l l . The q u e s t i o n i s : : What r o l e c o u l d i n c r e a s e d v e n o l y m p h a t i c f l o w p l a y i n g i l l f u n c t i o n , when the r e s p i r a t o r y gas exchange demands p l a c e d on the g i l l s a r e h i g h e r than normal? The n u t r i t i v e f u n c t i o n o f t h e s e v e s s e l s be-coming more i m p o r t a n t as more o f t h e g i l l , t i s s u e becomes i n v o l v e d i n gas t r a n s f e r , because o f l a m e l l a r r e c r u i t m e n t ( P a r r e l 1 et al., 1979), i s one p o s s i b l e e x p l a n a t i o n . The o t h e r c o n t r i b u t i o n t o v e n o l y m p h a t i c f l o w i s lymph i t s e l f . I t i s p o s s i b l e .that p u l s a t i l e f l o w and/or p r e s s u r e i n c r e a s e s lymph f o r m a t i o n a n d . i t s c l e a r a n c e f r o m ; i n t e r s t i t i a l s p a c e s , i n t o the l y m p h a t i c v e s s e l s , t h e r e b y r e d u c i n g g i l l t i s s u e oedema d u r i n g e x e r c i s e in vivo. Other consequences o f t h e s e f l o w changes brought about by i n c r e a s e d p u l s a t i l i t y on gas t r a n s f e r a c r o s s t he g i l l s o f rainbow t r o u t a t r e s t , and d u r i n g swimming e x e r c i s e w i l l be e x p l o r e d i n S e c t i o n I I I o f t h i s t h e s i s . The v a s c u l a r r e s i s t a n c e t o f l o w through t r o u t g i l l s , in vivo, i s lower than i s found from in vitro measurements i n the p r e s e n t p r e p a r a t i o n s , r e g a r d l e s s o f whether the g i l l s a r e p e r f u s e d w i t h p u l s a t i l e o r n o n - p u l s a t i l e f l o w . Rg v a l u e s from i s o l a t e d , s a l i n e - p e r f u s e d p r e p a r a t i o n s a re h i g h e r than. t h o s e measured in vivo p r i n c i p a l l y as a r e s u l t o f e l e v a t e d t r a n s - g i l l p r e s s u r e d i f f e r e n t i a l s , r a t h e r than low,flow r a t e s . Wood (1974a) has shown t h a t e l e v a t i o n o f d o r s a l a o r t i c p r e s s u r e from 0 t o 40 cm H 20 i n i s o l a t e d t r o u t g i l l p r e p a r a t i o n s can reduce R by as much as 50%, a t f l o w r a t e s o f y a p p r o x i m a t e l y 17 m l • m i n " 1 * k g " 1 . S a l i n e p e r f u s i o n "haemodynamics" t h e r e f o r e , do not mimic e x a c t l y the in vivo s i t u a t i o n . However, d a t a o b t a i n e d from t h e s e i s o l a t e d p r e p a r a t i o n s s t i l l are u s e f u l s i n c e they d e s c r i b e q u a l i t a t i v e l y , p o s s i b l e b l o o d and l y m p h a t i c f l o w d i s t r i b u t i o n p a t t e r n s , and some u n d e r l y i n g 99 mechanisms r e s p o n s i b l e f o r t h e s e , d u r i n g p e r i o d s o f i n c r e a s e d i n p u t p r e s s u r e p u l s a t i l i t y t o the g i l l s , as d u r i n g e x e r c i s e , in vivo. In a d d i t i o n , i f the purpose o f p e r f u s e d g i l l p r e p a r a t i o n s i s t o mimic the in vivo c o n d i t i o n , then from the p r e s e n t r e s u l t s , p u l s a t i l e c o n s t a n t f l o w i s c o n s i d e r e d the most a p p r o p r i a t e method, and a p h y s i o l o g i c a l d o r s a l a o r t i c p r e s s u r e must be main-t a i n e d i n o r d e r t o o b t a i n r e p r e s e n t a t i v e d a t a . The S p o n t a n e o u s l y V e n t i l a t i n g , B l o o d - P e r f u s e d Whole T r o u t P r e p a r a t i o n : a) How r e p r e s e n t a t i v e i s i t o f In Vivo c o n d i t i o n s ? S p o n s t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t show c a r d i o -v a s c u l a r dynamics which a r e e s s e n t i a l l y , i d e n t i c a l . t o t h o s e o f i n t a c t f i s h . S i m i l a r l y gas exchange a t the g i l l s , and t i s s u e s i s v e r y s i m i l a r to d a t a a v a i l a b l e from i n vivo measurements, d e s p i t e the f a c t t h a t t h e h a e m a t o c r i t o f b l o o d used f o r p e r f u s i o n i n t h e s e p r e p a r a t i o n s i s lower than n o r m a l l y found i n i n t a c t f i s h . B lood was d i l u t e d w i t h s a l i n e so t h a t fewer f i s h need t o be s a c r i f i c e d f o r each p r e p a r a t i o n . T r o u t have been shown to be q u i t e c a p a b l e o f s u r v i v i n g w i t h h a e m a t o c r i t s as low as 3%, and v a l u e s o f 15% a r e not uncommon f o r normal t r o u t in vivo{See Wood and S h e l t o n , 1980a). An Hct o f 10 - 12%, as i s used i n my p r e p a r a t i o n s , can be c o n s i d e r e d as a r e a s o n a b l e compromise between economy and s i m u l a t i o n o f in vivo c o n d i t i o n s . iy B l o o d - p e r f u s e d f i s h behave i n a manner s i m i l a r t o i n t a c t a n i m a l s . They are c a p a b l e o f m a i n t a i n i n g e q u i l i b r i u m i n the water, o f v i s u a l t r a c k i n g , o f swimming motions, and show a t y p i c a l b r a d y c a r d i a r e s p o n s e when exposed t o a q u a t i c h y p o x i a . O c c a s i o n a l l y , when b l e e d i n g i s o b s e r v e d from s u t u r e s which c l o s e the i n c i s i o n and anchor c a t h e t e r s t o the body w a l l , t h e s e s m a l l l e a k s tend t o c l o t w i t h t i m e , w h i l e l a r g e r l e a k s u s u a l l y are i n d i c a t i v e o f a dislodge'd' c a t h e t e r . C a n n u l a t i o n o f the d o r s a l a o r t a 24 hours b e f o r e e x p e r i -m e n t a t i o n e l i m i n a t e s leakage from around t h i s p o i n t o f i n s e r t i o n . In no s u c c e s s f u l p r e p a r a t i o n i s l e a k a g e so l a r g e as t o r e q u i r e a d d i t i o n o f more 100 b l o o d to the tonometers. The l e v e l s o f th e a n a e s t h e t i c , MS-222, r e m a i n i n g i n the body o f ' f i s h p e r f u s e d w i t h b l o o d c l e a r l y were too low t o m a i n t a i n a n a e s t h e s i a , a l t h o u g h some unmeasured e f f e c t s o f a n a e s t h e s i a on the f i s h , s t i l l may have p e r s i s t e d a f t e r o n l y 2 - 3 hours o f r e c o v e r y from s u r g e r y i n my experiments (Houston et al., 1971). Had any a n a e s t h e t i c been p r e s e n t i n t h e s e t i s s u e s , and s u b s e q u e n t l y r e a p p e a r e d i n the b l o o d , t h e n . i t c o u l d s t i l l , be e l i m i n a t e d r a p i d y from the b l o o d , a c r o s s the g i l l s i n t o the water (see Daxboeck and H o i e t o n , - 1 9 8 0 ) , a n d . i t s e f f e c t s on the p r e p a r a t i o n would have been i n s i g n i -f i c a n t . There i s a l a r g e number o f p u b l i c a t i o n s which r e p o r t some o f the v a r i a b l e s i n T a b l e s 12 and 13, w h i l e few.workers have measured a s i g n i f i c a n t number o f th e s e v a r i a b l e s s i m u l t a n e o u s l y . For c o m p a r a t i v e p u r p o s e s , o n l y t h o s e s e t s o f d a t a i n , w h i c h as many o f the above mentioned v a r i a b l e s have been measured s i m u l t a n e o u s l y , were chosen f o r the f o l l o w i n g d i s c u s s i o n o f i n d i v i d u a l . v a r i a b l e s from b l o o d - p e r f u s e d t r o u t . Flow Rate - Q Mean p e r f u s i o n , r a t e was 16.2 m l ' m i n - ' 1 " k g - 1 i n the b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n s . C a r d i a c o u t p u t (Q), measured d i r e c t l y i n o t h e r f i s h s p e c i e s , range between 6 and 21'ml."min" 1'kg - 1 (Ophiodon elongatus 6.0, Stevens et al., 1972; Gadus morhua 20.8 m l ' m i n - 1 , Jones et al., 1974).. From t h e s e l i m i t e d d a t a for, d i r e c t measurements o f c a r d i a c o u t p u t s from o t h e r r e s t i n g , i n t a c t f i s h , i t i s a p p a r e n t t h a t Q f o r the b l o o d - p e r f u s e d f i s h approximateV-m vivo v a l u e s . C a r d i a c o u t p u t a l s o has been measured d i r e c t l y i n r e s t i n g : r a i n b o w t r o u t by Wood and S h e l t o n (1980a). These a u t h o r s r e p o r t a v a l u e o f 36.7 ml' m i n " 1 ' k g - 1 , whereas c a r d i a c o u t p u t o f r e s t i n g rainbow t r o u t , e s t i m a t e d by the -1 -1 F i c k p r i n c i p l e are 21.6 ml'min 'kg (Stevens and R a n d a l l , 1967b) and ":. , -1 -1 17.6 ml'min 'kg ( K i c e n i u k and J o n e s , 1977). Thus, mean r e s t i n g c a r d i a c 101 o u t p u t r e p o r t e d by Wood and S h e l t o n (1980a) i s a t l e a s t t w i c e t h a t o f e s t i m a t e s u s i n g . t h e F i c k p r i n c i p l e , d e s p i t e the f a c t t h a t t h i s method f o r the c a l c u l a t i o n o f c a r d i a c o u t p u t i s known t o g i v e o v e r e s t i m a t e s o f the a c t u a l f l o w (Wood et.al., 1978; see a l s o S e c t i o n I I I ) . S t r o k e volumes c a l c u l a t e d from a l l a v a i l a b l e in..vivo d a t a f o r t r o u t a re s i m i l a r ( a p p r o x i m a t e l y 0.46 m l " k g " 1 ) . C o n s e q u e n t l y , the above d i f f e r e n c e s i n Q from rainbow t r o u t appear t o a r i s e from h e a r t r a t e d i f f e r e n c e s . The 5°C temperature d i f f e r e n c e between the s t u d i e s o f Wood and S h e l t o n (198oa), and those o f K i c e n i u k and Jones (1977) and Stevens and R a n d a l l . (1976b) i s i n s u f f i c i e n t t o . a c c o u n t f o r the hi g h h e a r t r a t e s r e c o r d e d by; Wood and S h e l t o n . V e n t r a l A o r t i c ( i n p u t ) P r e s s u r e - VAP Mean VAP i n normal b l o o d - p e r f u s e d f i s h was 58.8 cm HgO. Other s p e c i e s i n which r e s t i n g v e n t r a l a o r t i c p r e s s u r e s have been measured [Gadus movhua 58 cm.H^O, Helgason and N i l s s o n , 1973; Ophiddon^elbngatus, 59.2 cm HgO, Stevens et al., 1972) g i v e v a l u e s comparable t o those measured i n b l o o d - p e r f u s e d t r o u t . However, t h i s v a l u e appears t o be s l i g h t l y , h i g h f o r measurements from i n t a c t rainbow t r o u t (47.1 cm H^O, Stevens and R a n d a l l , 1967b; 52.7 cm H 20, K i c e n i u k and Jo n e s , 1977; 42.5 cm H'20, Wood and S h e l t o n , 1980a). T h i s p o s s i b l y i s i n d i c a t i v e o f i n p u t f l o w , o r g i l l v a s c u l a r r e s i s t a n c e t o f l o w b e i n g s l i g h t l y h i g h e r i n b l o o d - p e r f u s e d t r o u t , ' t h a n found i n i n t a c t ones, However, v e n t r a l a o r t i c p r e s s u r e s i n b l o o d - p e r f u s e d f i s h s t i l l f a l l , w i t h i n a range o f v a l u e s o b t a i n e d from in vivo s t u d i e s . Venous p r e s s u r e was no t measured i n b l o o d - p e r f u s e d f i s h , but was assumed to be 4 cm H^O (Wood and S h e l t o n , 1980a) f o r the purpose o f s y s t e m i c v a s c u l a r r e s i s t a n c e c a l c u l a t i o n s . 102 D o r s a l A o r t i c P r e s s u r e - DAP Q i n i t i a l l y was a d j u s t e d t o a c h i e v e a DAP o f 40 cm H^O, because f i s h , l i k e most o t h e r v e r t e b r a t e s , p r o b a b l y r e g u l a t e p r e s s u r e by a l t e r -i n g Q and v a s c u l a r r e s i s t a n c e ( W a l q v i s t and N i l s s o n , 1977; 1980; Smith, 1979; Wood and S h e l t o n , 1980b). . As DAP r e c o r d s from t r o u t a r e r e l a t i v e l y common, and the d o r s a l a o r t a l i e s between the two major v a s c u l a r r e s i s t -ance s i t e s , I chose to s e t o t h e r c a r d i o v a s c u l a r parameters around t h i s p o i n t . Once Q was s e t , DAP f e l V d u r i n g the l i f e o f a. p r e p a r a t i o n , to g i v e a mean DAP o f 34.8 ± 1.0 em H^O. However, DAP's measured i n r e s t -i n g i n t a c t t r o u t range from 34 t o 42 em H^O (Stevens and R a n d a l l , 1967a,b; K i c e n i u k and J o n e s , 1977; Wood and S h e l t o n , 1980a; T a b l e 9, S e c t i o n I) and o t h e r s p e c i e s i n which DAP.'s have been measured in vivo have g i v e n s i m i l a r v a l u e s (see Stevens et.al-., 1972 ; Helgason and N i l s s o n , 1973; Davie and F o r s t e r , 1979). Mean DAP i n b l o o d - p e r f u s e d f i s h compares w e l l w i t h p u b l i s h e d in vivo d a t a . 103 B r a n c h i a l V a s c u l a r R e s i s t a n c e t o Flow - Rg The p r e s s u r e drop a c r o s s the g i l l s d i v i d e d by the f l o w r a t e g i v e s a n u m e r i c a l v a l u e f o r the v a s c u l a r r e s i s t a n c e o f the g i l l v e s s e l s t o f l u i d f l o w through them. Mean Rg o f b l o o d - p e r f u s e d f i s h g i l l s i s 14.2 cm H^O'ml" 1* m i n « 1 0 0 g _ 1 . In vivo Rg v a l u e s however, are t y p i c a l l y around 6 cm H^O ' m l ' 1 * min«100 g " 1 ( S t e v e n s and R a n d a l l , 1967b, 3.4; K i c e n i u k and J o n e s , 1977,6.02; and Wood and S h e l t o n , 1980a, 3.4 'cm H^O'inl"^min* 1 0 0 g _ 1 ) . 'The d i f f e r e n c e s . •; • a r i s e from h i g h e r g i l l r e s i s t a n c e v a l u e s found i n b l o o d p e r f u s e d f i s h , o r o v e r -e s t i m a t e s : o f ; Q i n . the o t h e r -in vivo, studie&. Whfin.,.bTood-perfus.i!Oh p r e p a r a -t i o n s were exposed t o 1 x 10" M a d r e n a l i n e , t h e r e was a s i g n i f i c a n t d e c r e a s e i n b r a n c h i a l v a s c u l a r r e s i s t a n c e , and a s i g n i f i c a n t r i s e i n s y s t e m i c v a s u l a r r e s i s t a n c e t o f l o w (see T a b l e 18);- in vivo measurements may have been taken from f i s h i n which t h e r e was l i t t l e v a s c u l a r tone. P h a r m a c o l o g i c a l experiments d e s i g n e d t o e l i c i t . f a l l s i n R g i n i n t a c t f i s h (eg. i n j e c t i o n o f i s o p r e n a l i n e , a s p e c i f i c 3 - a d r e n e r g i c a g o n i s t , Wood and S h e l t o n , 1980a) showed o n l y s m a l l e f f e c t s , i n d i c a t i n g low v a s c u l a r tone i n the g i l l s o f t h e s e a n i m a l s . U n f o r t u n a t e l y t h e s e a u t h o r s d i d not measure both Q and VAP i n the same a n i m a l s , which make t h e i r v a l u e s more d i f f i c u l t t o compare to the ones from the p r e s e n t . s t u d y . fn vivo measurements o f Rg g e n e r a l l y have been take n from f i s h o f g r e a t e r w e i g h t than t h o s e used f o r b l o o d p e r f u s i o n i n . t h e p r e s e n t e x p e r i m e n t s . Wood and S h e l t o n (1975) and Payan and Matty (1975) have shown i n v e r s e r e l a t i o n s h i p s between weight and v a s c u l a r r e s i s t a n c e i n i s o l a t e d s a l i n e -p e r f u s e d p r e p a r a t i o n s . The l a r g e r s i z e o f f i s h used f o r in vivo measurements may p a r t l y e x p l a i n , w h y t h e s e c a l c u l a t e d R g v a l u e s a r e lower than t h o s e from b l o o d - p e r f u s e d f i s h . Over the range o f Q, VAP and DAP o f normal b l o o d -p e r f u s e d p r e p a r a t i o n s , Rg was s i g n i f i c a n t l y c o r r e l a t e d o n l y w i t h VAP. R e g r e s s i o n o f R g a g a i n s t VAP showed a h i g h l y s i g n i f i c a n t l i n e a r r e l a t i o n s h i p (p<> 0.001) as f o l l o w s : 104 •R (em H g O -raT^min-lOOg" 1) = 0.649 x VAP (cm H 20) - 24.07 Sy s t e m i c V a s c u l a r R e s i s t a n c e t o FTow - R g Mean s y s t e m i c v a s c u l a r r e s i s t a n c e t o f l o w i n b l o o d - p e r f u s e d f i s h i s 19.2 cm H 2 0 * m l - 1 » m i n v l 0 0 g " 1 . T h i s v a l u e i s s i m i l a r t o t h o s e measured by S t e v e n s . a n d R a n d a l l (1967b) o f 16.6, and K i c e n i u k and Jones (1977) o f 22.8 cm H 2 0 « m r 1 r m i n ' 1 0 0 g " 1 . The v a l u e o f R g from Wood and S h e l t o n (1980a) o f 7.1 cm H 2 0 * m l " 1 * m i n ' 1 0 0 g - 1 however, i s lower than i s found i n the b l o o d - p e r f u s e d p r e p a r a t i o n s . The l a t t e r v a l u e i s low p r i n c i p a l l y as a r e s u l t o f s m a l l e r p r e s s u r e d i f f e r e n t i a l s a c r o s s t h e body, r a t h e r than t h e i r h i g h e r measured c a r d i a c o u t p u t . S u f f i c e t o say the R e c a l c u l a t e d f o r b l o o d - p e r f u s e d p r e p a r a t i o n s i s s i m i l a r t o t h o s e measured in vivo. In g e n e r a l s y s t e m i c v a s c u l a r r e s i s t a n c e s measured in vivo, and in vitro from e i t h e r b l o o d -p e r f u s e d or s a l i n e - p e r f u s e d p r e p a r a t i o n s a r e i n good agreement:, b e t t e r than s e e n i h . t h e comparisons o f the g i l l r e s i s t a n c e v a l u e s from t h e s e types o f p r e p a r a t i o n s . S t r e s s B l o o d - p e r f u s e d f i s h u ndoubtedly a r e s u b j e c t t o a g r e a t d e a l o f s t r e s s . Acute s t r e s s evokes a number o f r e s p o n s e s (see Mazeaud et al. , 1977), many o f which are mediated through the r e l e a s e o f c a t e c h o l a m i n e s i n t o the b l o o d , However the l a r g e t o t a l b l o o d volume,taken from r e s t i n g f i s h which had r e c o v e r e d from the s t r e s s o f the c a n n u l a t i o n p r o c e d u r e f o r a t l e a s t 14 hours e n s u r e d t h a t low l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s would be p r e s e n t i n t h i s b l o o d . The f r e q u e n t s w i t c h i n g from one tonometer t o a n o t h e r meant t h a t , t o e l e v a t e b l o o d c a t e c h o l a m i n e l e v e l s by 15 times o v e r r e s t i n g l e v e l s , as seen d u r i n g s t r e s s i n some f i s h (Nakano and T o m l i n s o n , 1967; Mazeaud et al. , 1977), a l a r g e amount o f c a t e c h o l a m i n e s would have had to be r e l e a s e d i n t o the b l o o d by the e x p e r i m e n t a l animal i n the p r e s e n t s t u d y . D i s c a r d i n g the b l o o d o f o p e r a t e d a n i m a l s , and s a l i n e - p e r f u s i o n p r i o r t o the commencement o f b l o o d -105 p e r f u s i o n meant t h a t i n i t i a l c o n c e n t r a t i o n s o f endogenous c i r c u l a t i n g c a t e c h o l a m i n e s were s m a l l i n e x p e r i m e n t a l a n i m a l s . I f the l e v e l s o f c a t e c h o l a m i n e s i n the b l o o d however, were e l e v a t e d above a "normal" c o n c e n t r a t i o n then one c o u l d e x p e c t a number o f r e s p o n s e s i n the c a r d i o v a s c u l a r system o f t h e . b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n . C a t e c h o l a m i n e s a r e known t o cause s y s t e m i c v a s o c o n s t r i c t i o n i n t r o u t . System-i c r e s i s t a n c e t o f l o w however, d e c r e a s e d r a t h e r than i n c r e a s e d d u r i n g the l i f e o f any i n d i v i d u a l p r e p a r a t i o n . H e a r t . r a t e and v e n t i l a t o r y r e s p o n s e s t o e l e v a t e d l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s a l s o . w e r e not a p p a r e n t . P r e p a r a t i o n s were a b l e t o respond t o p h y s i o l o g i c a l doses o f a d m i n i s t e r e d a d r e n a l i n e i n a normal m a n n e r , ( i . e . b r a n c h i a l v a s c u l a r r e s i s t a n c e d e c r e a s e ; s y s t e m i c v a s c u l a r r e s i s t a n c e to f l o w i n c r e a s e s ) , i n d i c a t i n g t h a t b l o o d l e v e l s o f c a t e c h o l a m i n e s were not e l e v a t e d unduly. I t was c o n c l u d e d t h a t s t r e s s r e s p o n s e s i n b l o o d - p e r f u s e d p r e p a r a t i o n s were d i m i n i s h e d because o f the e x p e r i m e n t a l p r o t o c o l . e m p l o y e d , and p r o b a b l y d i d n o t a f f e c t . t h e r e s u l t s s i g n i f i c a n t l y , 1 The c a r d i o v a s c u l a r system o f b l o o d - p e r f u r s e d t r o u t t h e r e f o r e e x h i b i t p r e s s u r e s , f l o w s a n d . v a s c u l a r r e s i s t a n c e s i n t e r m e d i a t e between those in vivo and i s o l a t e d , s a l i n e - p e r f u s e d s t u d i e s . L a r g e d i f f e r e n c e s between d a t a s e t s u s u a l l y a r e e x p l i c a b l e i n . t e r m s o f the p a r t i c u l a r e x p e r i m e n t a l t e c h n i q u e s . Where t e c h n i q u e s were the same, comparisons r e v e a l good a g r e e -ment between d a t a which. I have p r e s e n t e d , and t h o s e o f o t h e r s t u d i e s . The sp o n t a n e o u s l y v e n t i l a t i n g , bloodTperfused t r o u t p r e p a r a t i o n i s deemed -s u i t a b l e f o r , the e x a m i n a t i o n o f the- e f f e c t s o f c a r d i o v a s c u l a r haemodynamics on a v a r i e t y o f c a r d i o r e s p i r a t o r y problems, as w e l l as f o r the s t u d y o f gas t r a n s f e r under a number o f d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s . These i n v e s t i g a t i o n s a r e made l e s s c o m p l i c a t e d than i n i n t a c t f i s h , s i n c e c a r d i a c o u t p u t (Q), and venous b l o o d gas t e n s i o n s , and i t s chemical makeup 106 are e x p e r i m e n t a l parameters which can be c o n t r o l l e d and m a n i p u l a t e d , r a t h e r than b e i n g measured or e s t i m a t e d v a r i a b l e s . b) Haemodynamic A l t e r a t i o n s i n B l o o d - P e r f u s e d T r o u t P r e p a r a t i o n s : E f f e c t s on v a s c u l a r r e s i s t a n c e to f l o w The s i x t r e a t m e n t s i n v o l v i n g m a n i p u l a t i o n s o f p e r f u s i o n f r e q u e n c y / s t r o k e v olume/pulse p r e s s u r e , and f l o w r a t e ( Q), and t h e i r e f f e c t s on g i l l r e s i s t a n c e (-R .) to f l o w i n b l o o d - p e r f u s e d p r e p a r a t i o n s have been p r e s e n t e d i n T a b l e 16. As had been n o t e d d u r i n g e x e r c i s e i n i n t a c t t r o u t from my e x p e r i m e n t s , i n c r e a s e d b l o o d f l o w ( i . e . c a r d i a c o u t p u t ) , s h o u l d i t i n f a c t be i n c r e a s e d above r e s t i n g v a l u e s , can be a c h i e v e d by i n c r e a s i n g o n l y the s t r o k e volume while, the h e a r t r a t e remains a t a normal l e v e l (see T a b l e 8). T h i s was accompanied by an i n c r e a s e i n p u l s e p r e s s u r e . S i m u l a t i o n o f t h i s in vivo c o n d i t i o n i n the r e s t i n g , b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n r e s u l t e d i n a s i g n i f i c a n t d e c r e a s e i n R . E i t h e r t he s t r o k e volume i n c r e a s e , the p u l s e p r e s s u r e i n c r e a s e , the i n c r e a s e i n Q, o r any c o m b i n a t i o n o f t h e s e f a c t o r s c o u l d have caused the o b s e r v e d f a l l i n Rg. When p u l s e p r e s s u r e a l o n e was i n c r e a s e d however, w h i l e o t h e r parameters were kept c o n s t a n t , Rg i n c r e a s e d o n l y s l i g h t l y . Thus, i n c r e a s e d p u l s e p r e s s u r e a l o n e cannot e x p l a i n the d e c r e a s e i n Rg. However, p u l s e p r e s s u r e changes a s s o c i a t e d w i t h s t r o k e volume i n c r e a s e s and c a r d i a c pump f r e q u e n c y d e c r e a s e s , w h i l e f l o w r a t e was kept c o n s t a n t , were c a p a b l e o f c a u s i n g much l a r g e r d e c r e a s e d i n R . Thus, y the d e c r e a s e i n g i l l v a s c u l a r r e s i s t a n c e t o f l o w can be ac c o u n t e d f o r by l a r g e i n c r e a s e s i n s t o k e volume, o r d e c r e a s e s i n h e a r t r a t e , b u t i t appears more l i k e l y t h a t t h i s d e c r e a s e i n Rg was a r e s u l t o f i n c r e a s e d s t r o k e volume, and the a t t e n d a n t i n c r e a s e i n p u l s e p r e s s u r e . Data f r o m . b l o o d - p e r f u s i o n t r o u t p r e p a r a t i o n s t h e r e f o r e i n d i c a t e t h a t the p r i n c i p l e p h y s i o l o g i c a l e f f e c t o f i n c r e a s e d p u l s e p r e s s u r e i s t o lower v e n t r a l a o r t i c mean p r e s s u r e below t h a t found under normal p e r f u s i o n c o n d i t i o n s 107 As l o n g as ..-a:< c o n s t a n t - p e r f us i o n "flow rate' i's m a i n t a i n e d at.normal . l e v e l s , the g i l l v a s c u l a r r e s i s t a n c e t o f l o w i s d e c r e a s e d . And, a l t h o u g h i n c r e a s e d 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 i n i t s e l f has no e f f e c t on b l o o d -p e r f u s e d g i l l s r e s i s t a n c e t o f l o w , . d e c r e a s e d v e n t r a l a o r t i c p u l s e r e s u l t s i n l a r g e i n c r e a s e s i n Rg. The most p r o b a b l e cause o f t h i s r e s i s t a n c e change when i n p u t p u l s e t o the g i l l s i s d e c r e a s e d below normal, i s the c o l l a p s e o f some v e s s e l s whose c r i t i c a l c l o s i n g p r e s s u r e s (.15 - 25 cm H 20 H 20; Wood, 1974a: F a r r e l l et al., .1979) a r e n o t exceeded by peak s y s t o l i c p r e s s u r e . Decreases i n s t r o k e volume and/or p u l s e p r e s s u r e , o r a d e c r e a s e i n the p e r f u s i o n f l o w . r a t e a l s o r e s u l t i n an i n c r e a s e i n , Rg. These c o n d i t i o n s tend a l s o t o d e c r e a s e v e n t r a l a o r t i c peak p r e s s u r e , and i n the absence o f any c o n c o m i t a n t c h a n g e s . i n the s t r o k e volume or f l o w r a t e from n o rmal, a d e c r e a s e i n p u l s e p r e s s u r e a l o n e can r e s u l t i n an i n c r e a s e i n R g. P u l s e p r e s s u r e t h e r e f o r e may be i m p o r t a n t i n m a i n t a i n i n g normal g i l l v a s c u l a r r e s i s t a n c e t o f l o w , by a s s i s t i n g e x t r a v a s c u l a r f l u i d c l e a r a n c e from g i l l t i s s u e s , as p o i n t e d out i n the s a l i n e - p e r f u s e d t r o u t heads, but appears t o be o f l i t t l e i mportance by i t s e l f , i n a c t u a l l y r e d u c i n g Rg. C o n c l u s i o n s which can be.drawn from t h e s e experiments are t e n t a t i v e but do i n d i c a t e t h a t i n c r e a s e s i n s t r o k e volume, which a r e known t o accompany i n c r e a s e s i n c a r d i a c o u t p u t d u r i n g e x e r c i s e in vivo, can d e c r e a s e g i l l v a s c u l a r r e s i s t a n c e t o f l o w , . w h i l e d e c r e a s e s i n p u l s e p r e s s u r e i n c r e a s e R , y a t l e a s t i n b l o o d - p e r f u s e d p r e p a r a t i o n s a t r e s t . Decreased c a r d i a c f r e q u e n c y ( b r a d y c a r d i a ) , and i n c r e a s e d f l o w r a t e a l s o p l a y a r o l e i n l o w e r i n g g i l l r e s i s t a n c e , a l t h o u g h f r e q u e n c y changes i n themselves are o f l i t t l e consequence. In s i t u a t i o n s where c a r d i a c o u t p u t i n c r e a s e s above r e s t i n g l e v e l s , as d u r i n g e x e r c i s e i n i n t a c t t r o u t , i t may a c t u a l l y be advantageous t o a c h i e v e t h i s by s t r o k e volume r a t h e r than j u s t by f r e q u e n c y i n c r e a s e s . I f f r e q u e n c y a l o n e were t o be used t o i n c r e a s e Q d u r i n g e x e r c i s e , then g i l l r e s i s t a n c e c o u l d be e x p e c t e d t o r i s e , and the competence o f the r e s p i r a t o r y t i s s u e s c o u l d be 1 0 8 t h r e a t e n e d by e x c e s s i v e l y h i g h v e n t r a l a o r t i c p r e s s u r e s , l e a d i n g t o g i l l t i s s u e oedema. As I have p o i n t e d o u t i n S e c t i o n I, s y s t e m i c vasomotor tone i n t r o u t i s u n d e r , g e n e r a l a - a d r e n e r g i e v a s o c o n s t r i c t o r c o n t r o l . S t r e s s i s known to i n c r e a s e the l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s i n f i s h (Mazeaud et al., 1977), and perhaps t h i s a l s o o c c u r s d u r i n g e x e r c i s e i n vivo. The p r i n c i p l e e f f e c t s o f i n c r e a s e d l e v e l s o f a d r e n a l i n e i n the b l o o d o f b l o o d - , p e r f u s e d t r o u t were upon v a s c u l a r r e s i s t a n c e and a r i s e i n s y s t e m i c v a s c u l a r r e s i s t a n c e to f l o w . The c o n c e n t r a t i o n o f a d r e n a l i n e used i n t h e s e e x p e r i -ments was chosen to i l l u s t r a t e the. d i f f e r e n c e s i n s e n s i t i v i t y o f b r a n c h i a l and s y s t e m i c v a s c u l a r beds t o t h i s c a t e c h o l a m i n e , which i s found in vivo in t r o u t (Wood and S h e l t o n , 1975; Wood, 1976). B l o o d - p e r f u s e d f i s h showed a p e r c e n t a g e f a l l i n which was i d e n t i c a l t o t h a t o b s e r v e d i n p e r f u s e d t r o u t heads exposed to the same l e v e l o f a d r e n a l i n e (Wood, 1974a). The r i s e i n s y s t e m i c v a s c u l a r r e s i s t a n c e i n the p r e s e n t p r e p a r a t i o n was o n l y o n e - h a l f t h a t seen i n i s o l a t e d s a l i n e - p e r f u s e d t r o u t t r u n k s (Wood and S h e l t o n , 1975a). B l o o d - p e r f u s e d f i s h p r o b a b l y had s i g n i f i c a n t s y s t e m i c v a s c u l a r t o n e , and c o n s e q u e n t l y some a - c o n s t r i c t i o n may have been masked by B g - a d renoceptor v a s o d i l a t i o n o f the s y s t e m i c c i r c u l a t i o n . The e f f e c t s o f a d r e n a l i n e on v a s c u l a r r e s i s t a n c e i n b l o o d - p e r f u s e d t r o u t t h e r e f o r e , are p r e c i s e l y t h o s e which would have been p r e d i c t e d from p r e v i o u s s t u d i e s . I t appears t h a t the g i l l s o f i s o l a t e d , . s a l i n e - p e r f u s e d t r o u t are more s e n s i t i v e t o a l t e r a t i o n s i n s t r o k e volume and p u l s e p r e s s u r e than t o any o t h e r haemodynamic.variable. The e f f e c t s o f t h e s e changes on f l u i d f l o w d i s t r i b t u i o n w i t h i n the b r a n c h i a l v a s c u l a t u r e , as brought about by c o n d i t i o n s which mimic thos e found i n the c a r d i o v a s c u l a r system i n vivo, and t h e i r . e f f e c t s on v a s c u l a r r e s i s t a n c e s have been d i s c u s s e d . The r e l e v a n c e 109 o f t h e s e f i n d i n g s t o the f u n c t i o n o f the g i l l s f o r gas exchange w i l l be examined i n t h e f o l l o w i n g s e c t i o n . 109a S E C T I O N I I I GAS T R A N S F E R ACROSS THE G I L L S OF S A L I N E - AND B L O O D - P E R F U S E D P R E P A R A T I O N S OF RAINBOW TROUT 110 INTRODUCTION: SECTION I I I In the p r e v i o u s s e c t i o n , I.have p r o v i d e d d a t a which show t h a t haemodynamic a l t e r a t i o n s which mimic those found t o o c c u r d u r i n g e x e r c i s e in vivo, can have p r o f o u n d e f f e c t s upon v a s c u l a r r e s i s t a n c e s and f l u i d f l o w d i s t r i b u t i o n s w i t h i n the b r a n c h i a l c i r c u l a t i o n o f t r o u t . T h a t t h e s e changes a l s o may have e f f e c t s upon gas. t r a n s f e r a c r o s s t he g i l l s i s i n v e s t i g a t e d i n t h e f o l l o w i n g s e c t i o n . S t u d i e s o f the amount o f oxygen taken up by the bl o o d i t s e l f i n f i s h a t r e s t , o r d u r i n g e x e r c i s e however, a r e r e l a t i v e l y few i n number (S t e v e n s and R a n d a l l , 1967b; Eddy et al., .1977; K i c e n i u k and Jone s , 1977). S t u d i e s i n v o l v i n g i n t a c t , f i s h a r e l i m i t e d by the d i f f i c u l t y i n o b t a i n i n g a p p r o p r i a t e s i m u l t a n e o u s b l o o d samples, and the low b l o o d volumes o f f i s h (Holmes and Donaldson, 1970). Low b l o o d volumes n e c e s s a r i l y l i m i t t h e number o f b l o o d samples which can.be withdrawn from a s i n g l e a n i m a l . Moreover, c a r d i a c o u t p u t s u s u a l l y have been c a l c u l a t e d , from oxygen consump-t i o n by t h e whole f i s h , by t h e 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 . Where c a r d i a c o u t p u t has been measured d i r e c t l y . , b l o o d gas d a t a n e c e s s a r y f o r c a l c u l a t i o n s o f oxygen uptake and.carbon d i o x i d e e x c r e t i o n r a t e s by t h e b l o o d are l a c k i n g . These t e c h n i c a l d i f f i c u l t i e s have l e d i n v e s t i g a t o r s t o use i s o l a t e d , s a l i n e - p e r f u s e d p r e p a r a t i o n s i n o r d e r t o study g i l l f u n c t i o n i n f i s h (Wood, 1974a; Wood and S h e l t o n , 1975; Payan and Matty, 1975; Wood et al., 1978). However, i t has been shown t h a t t h e s e s a l i n e - p e r f u s e d p r e p a r a t i o n s can be l i m i t e d i n t h e i r u s e f u l n e s s f o r s t u d i e s o f gas t r a n s f e r i n f i s h g i l l s ( s e e Wood et al., 1978). The s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d whole t r o u t p r e p a r a t i o n (see S e c t i o n I I ) overcomes the problem o f in a d e q u a t e r a t e s o f gas t r a n s f e r a c r o s s the g i l l s , a p e r t u r b i n g consequence o f s a l i n e - p e r f u s e d p r e p a r a t i o n s , and e n a b l e s the s i m u l t a n e o u s measurements o f all the v a r i a b l e s n e c e s s a r y f o r the d e t e r m i n a t i o n o f b l o o d , as w e l l as v e n t i l a t o r y oxygen up-take to be made. The e f f e c t s o f haemodynamic a l t e r a t i o n s , which s i m u l a t e I l l t h o s e found t o o c c u r d u r i n g e x e r c i s e i n t r o u t , in vivo, upon oxygen uptake a c r o s s the g i l l s o f r e s t i n g , b l o o d - p e r f u s i n g rainbow t r o u t a l s o are a s s e s s e d . Because c a r d i a c o u t p u t i s s e t p r e c i s e l y by an e x t e r n a l p e r f u s i o n pump, the b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n a l s o p r e s e n t s a unique o p p o r t u n i t y t o t e s t the a p p l i c a b i l i t y o f . t h e F i c k p r i n c i p l e i n p r e d i c t i n g the c a r d i a c o u t p u t , g i v e n the measured oxygen uptake r a t e a c r o s s the g i l l s , and by the b l o o d i t s e l f , as i t passes t h r o u g h . t h e g i l l s . The consequences o f the f i n d -i n g s from t h e s e experiments a r e p r e s e n t e d i n some d e t a i l , as the F i c k p r i n c i p l e i s used w i d e l y . i n c a r d i o r e s p i r a t o r y p h y s i o l o g y f o r e s t i m a t i n g c a r d i a c o u t p u t and f l o w s i n v a r i o u s o r g a n s , w i t h . a minimum o f s u r g i c a l i n t e r v e n t i o n . P r e c a u t i o n s f o r i t s use, and recommendations f o r the i n t e r -p r e t a t i o n o f n u m e r i c a l v a l u e s o b t a i n e d w i t h the use o f the F i c k p r i n c i p l e are d i s c u s s e d . From the d a t a c o l l e c t e d from the above e x p e r i m e n t s , the c o n t e n t i o n . t h a t f i s h g i l l s a r e d i f f u s i o n l i m i t e d f o r gas t r a n s f e r i s examined i n d e t a i l . D u r i n g e x e r c i s e in, vivo, mean and p u l s e p r e s s u r e s .in the g i l l s o f f i s h i n c r e a s e . In t h i s s e c t i o n , the i s o l a t e d , s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n i s used i n o r d e r a l s o t o i n v e s t i g a t e the e f f e c t s o f the p u l s a t i -l i t y o f c o n s t a n t p e r f u s i o n f l o w r a t e on t h e n a t u r e o f f l u i d exchange a c r o s s the b r a n c h i a l v a s c u l a t u r e o f f i s h . , u s i n g an e t h a n o l l o a d i n g / t i s s u e washout t e c h n i q u e . In a d d i t i o n , t h i s s a l i n e - p e r f u s e d p r e p a r a t i o n i s used to examine the e f f e c t s o f p e r f u s i o n regime a l t e r a t i o n ( i . e . p u l s a t i l i t y o f f l o w ) upon gas t r a n s f e r a c r o s s the g i l l s o f t h e s e t r o u t heads i n normoxia. The d a t a from t h e s e s t u d i e s l e a d t o d i s c u s s i o n s o f the p o s s i b l e e f f e c t s o f f l u i d exchange and f l o w d i s t r i b u t i o n p a t t e r n s w i t h i n g i l l t i s s u e s upon gas t r a n s f e r e f f i c i e n c y i n t r o u t , in vivo, a t r e s t and d u r i n g swimming e x e r c i s e . V 112 RESULTS I I I : The E f f e c t s o f Haemodynamic A l t e r a t i o n s on Gas Exchange i n the  Bloods-Perfused T r o u t P r e p a r a t i o n The e f f e c t s o f changes i n c a r d i a c o u t p u t on the r e s t i n g , b l o o d -p e r f u s e d t r o u t p r e p a r a t i o n a re summarized i n T a b l e 17. D e s c r i p t i o n o f the s u r g i c a l p r o c e d u r e s used f o r the. p r e p a r a t i o n o f t h e s e f i s h have been g i v e n i n the M a t e r i a l s and Methods.pages 45 to.49. The d a t a . i n d i c a t e d a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between the. r a t e o f p e r f u s i o n and both oxygen uptake (MgOg), and carbon d i o x i d e excretion"(MgCO^) a c r o s s the g i l l s ( F i g . 13). That i s , Mg^ as w e l l as M ^ i n c r e a s e d as Q i s ra i s e d . . A l i n e a r r e l a t i o n s h i p between c a r d i a c o u t p u t . a n d oxygen uptake i s r e p r e s e n t e d by e q u a t i o n 3, (3) M g 0 2 .(uM'-mirf M O O g ' 1 ) =' 0.63. x Q ( m l - m i r T M O O g - 1 ) - 0.16 (p^O.Ol; n = 21). A comparable r e l a t i o n s h i p a l s o can be d e r i v e d f o r carbon d i o x i d e e x c r e t i o n , and i s r e p r e s e n t e d by e q u a t i o n 4, (4) M gC0 2 (yM-min'^lOOg- 1) = 1.04.3'x Q ( m l - m i n _ l « 1 0 0 g _ l ) - 0.13 (p<0. 01.; n = 21). P 0 o rema.ined c o n s t a n t (106.0 ± 2.0 mm Hg; n = 21) o v e r a c the range o f Q used i n thes e e x p e r i m e n t s , i n d i c a t i n g t h a t b l o o d oxygen s a t u r a -t i o n was m a i n t a i n e d ( F i g . 13). P C0 o a l s o remained unchanged (3.43 ± 0.12 mm a c Hg; n = 21) d u r i n g changes i n the p e r f u s i o n f l o w r a t e through the g i l l s o f t h i s p r e p a r a t i o n . The r a t e o f b l o o d d e l i v e r y t o the s y s t e m i c c i r c u l a t i o n a l s o appeared to l i m i t t h e oxygen consumption o f the s y s t e m i c t i s s u e s , s i n c e a c o n s t a n t 87% d e c r e a s e i n P ^ * and a c o n s t a n t 76% o f the oxygen c o n t e n t (CgOg) were e x t r a c t e d by. t h e s e t i s s u e s , r e g a r d l e s s o f the r a t e o f b l o o d f l o w through them. Of c o u r s e i n c r e a s e d b l o o d f l o w d u r i n g e x e r c i s e , in vivo, i n v o l v e s g r e a t e r 0^ u t i l i z a t i o n by t i s s u e s , and 0^ e x t r a c t i o n i n c r e a s e s . As the r a t e o f p e r f u s i o n was d e c r e a s e d , a l o n g e r e q u i l i b r a t i o n time was a f f o r d e d the b l o o d i n the tonometer, and t h i s was r e f l e c t e d by i n c r e a s e s 113 T a b l e 17. Summary o f the e f f e c t s o f i n c r e a s e d and d e c r e a s e d c a r d i a c o u t p u t on b l o o d gases from normal s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d -p e r f u s e d t r o u t (N = 7 f i s h ; 292.7 ± 14.3 g ) . Q Hct P. C. Hct P a C D n D n i n 0 2 1 n 0 2 (DA) a 0 2 a 0 2 R Q g R Q s ml'min" 1 . % mm Hg fnM % mm Hg JnM X 4.80 9.6 28.7 0.761 8.5 103.6 1.34 1.65 1.06 ±S.E.M. 0.20 0.4 1.9 0.052 0.5 4.7 0.09 0.34 0.17 o-o ^ X 7.20 9.6 21.6 0.740 8.5 109.5 1.42 1.34 0.82 CD = ±S.E.M. 0.04 0.5 1.6 0.60 0.6 3.1 0.13 0.33 0.08 o- X 2.76 9.34 41.3 1.06 7.8 104.9 1.46 2.82 1.23 zs. 3 ±S.E.M. 0.24 0.5 3.0 0.14 0.5 2.4 0.15 0.87 0.19 114 FIGURE 13. The e f f e c t s o f changes i n c a r d i a c o u t p u t on oxygen uptake and P 0 o ( A ) , and carbon d i o x i d e e x c r e t i o n and P,C0 o (B) a d. a c a c r o s s the g i l l s o f s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d -p e r f u s e d t r o u t . 115 116 i n both i n p u t PQ and CQ ( T a b l e 17), a l t h o u g h a l l o t h e r i n p u t b l o o d v a r i a b l e s remained unchanged. The e f f e c t s o f c h a n g i n g h a e m a t o c r i t on the gas t r a n s f e r a c r o s s the g i l l s o f the b l o o d - p e r f u s e d , p r e p a r a t i o n s a r e shown i n T a b l e 18. Oxygen uptake i n c r e a s e d as Hct was r a i s e d ( F i g . 14), w h i l e RQg was d e c r e a s e d . Input b l o o d oxygen c o n t e n t ( C . ^ ) was n e g a t i v e l y c o r r e l a t e d w i t h H c t , w h i l e i n p u t P n i n c r e a s e d w i t h d e c r e a s i n g Hct. N o n e t h e l e s s , P 0 o and AP0 o a c r o s s the g i l l 0 2 a 2 2 3 c i r c u l a t i o n (venous - a r t e r i a l ) were not a f f e c t e d s i g n i f i c a n t l y by changes i n Hct. The e f f e c t s o f changes i n the p e r f u s i o n r a t e , s t r o k e volume and p u l s e p r e s s u r e on oxygen uptake a r e summarized i n T a b l e 19. These da t a i n d i c a t e t h a t , as l o n g as Q was m a i n t a i n e d a t a c o n s t a n t normal r a t e , a l l o t h e r changes had n o n - s i g n i f i c a n t e f f e c t s upon the measured v a l u e o f Mg0 2-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 changes a s s o c i a t e d w i t h t h e s e m a n i p u l a t i o n s have been p r e s e n t e d e l s e w h e r e (see T a b l e 14). Oxygen uptake a c r o s s the g i l l s o f b l o o d - p e r f u s e d t r o u t (n = 4) was 1.10 ± 0.23 y M « m i n " 1 * 1 0 0 g " 1 , and was n o t changed by the a d d i t i o n o f 1 x 10" 6 M a d r e n a l i n e t o the b l o o d (1.30 ± 0.25 y M ' m i n ' S l O O g " 1 ) . The e f f e c t s o f a d r e n a l i n e exposure a t t h i s c o n c e n t r a t i o n on the p r e s s u r e and r e s i s t a n c e t o f l o w i n the g i l l s o f t h e s e b l o o d - p e r f u s e d p r e p a r a t i o n s a l s o have been p r e s e n t -ed e l s e w h e r e (see T a b l e 16). The F i c k E q u a t i o n , and the D i r e c t Measurement o f V e n t i l a t i o n Volume - V^ T a b l e 20 summarizes t h e d a t a o b t a i n e d from b l o o d - p e r f u s e d t r o u t i n normoxia, u s i n g the m o d i f i e d van Dam apparatus (see M a t e r i a l s and Methods, pages 54 t o 5 7 ) . These d a t a , when compared w i t h d a t a o b t a i n e d from normal p r e p a r a t i o n s w i t h o u t the van Dam m o d i f i c a t i o n (see T a b l e s 11 and 12), r e v e a l t h a t a l l v a r i a b l e s are e s s e n t i a l l y i d e n t i c a l . However, a most i n t e r e s t i n g f i n d i n g from the p r e s e n t experiments was t h a t the amount o f oxygen removed T a b l e 18. Summary o f the e f f e c t s o f v a r i a b l e i n p u t h a e m a t o c r i t on b l o o d gases f o r normal, s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t (N = 6 f i s h ; 320.2 ± 17.2 g and c o n s t a n t Q = 5.20 ± 0.04 m l " m i n " 1 ) . Hct K i n 0 2 mm Hg G. m 0 2 mM mm Hg C a Q -2 mM Mg 0 u2 y M ' m i n " L 1 0 0 g " 1 R Q g Hct ( i n p u t ) % Hct (DA) % NORMAL X 20.3 0.95 95.3 1.77 1.34 2.0 0.67 11.3 9.3 NORMAL + S. E.M. 2.5 0.20 6.6 0.20 0.19 0.1 0.11 0.5 0.5 4 Hct X 39.4 0.59 108.7 0.87 0.46 2.9 0.85 4.3 3.9 o _i j_> + S. E.M. 4.2 0.03 5.8 0.12 0.18 0.4 0.44 0.4 0.5 HIGH He" X 16.3 1.37 91.6 2.88 2.45 1.7 0.87 20.2 16.5 HIGH He" + s . E.M. 2.6 0.26 13.2 0.40 0.40 0.3 0.16 1.6 1.2 118 F i g u r e 14. The e f f e c t o f v a r i a b l e i n p u t h a e m a t o c r i t on oxygen uptake a c r o s s the g i l l s o f s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d -p e r f u s e d t r o u t . 119 120 T a b l e 19. E f f e c t s o f c a r d i a c o u t p u t (Q), s t r o k e volume ( S V ) , p u l s e p r e s s u r e (PP) and c a r d i a c f r e q u e n c y ( f ) on oxygen uptake (MgO?) i n r e s t i n g , b l o o d - p e r f u s e d t r o u t (0 = no change from normal; + = i n c r e a s e from normal; - = d e c r e a s e from n o r m a l ) . Means ± S.E.M. TREATMENT n % 2 uM-min'^lOOg" 1 (1) SV +; PP +; f 0; Q + 7 * 1.41 .±0.28 (2) SV +; PP +; f -; Q 0 8 1.07 ( b r a d y c a r d i a ) ±0.31 (3) SV -; PP -; f 0; Q - 7 0.38* ±0.06 (4) SV -; PP -; f +; Q 0 8 0.99 ( t a c h y c a r d i a ) ±0.26 (5) SV 0; PP +; f 0; Q 0 8 0.73 ±0.18 (6) SV 0; PP -; f 0; Q 0 3 0.84 ±0.15 Normal 44 .1.17 ±0.08 * S i g n i f i c a n t l y d i f f e r e n t from normal ( 5 % ) . T a b l e 20. Summary o f the c a r d i o r e s p i r a t o r y v a r i a b l e s from normal s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t i n a m o d i f i e d van Dam appa r a t u s (n = 10 o b s e r v a t i o n s on 4 f i s h ; 340.1 ± 13.2 g. P C Q = 151.4 ± 0.8 mm Hg). X ± S.E.M. VAP DAP cm H£0 cm H 2 O Q ( a c t u a l ) ml'min~*'k g - * ml Q ( F i c k ) -1 'min "kg V„ V n : Q •1 9. -1 9 ml ,min M 9 0 2 yM'min'^lOOg" 1 • V . 9 0 2 yM'min ' H o O g " " 1 C a o u 2 mM C V R 0 2 mM 52.6 35.4 1.1 2.4 16.81 0.48 25.12 2.18 57.4 9.47 6.42 0.82 1.083 0.103 1.653 0.229 1.26 0.07 0.315 0.04 ACo2 (V-M) yM'min _ 1'100g" v., 1 cm H 20-ml min' 1 0 0 g _ 1 cm R S - l H 20'ml min'lOOg •1 AC C 0 2 (v-a) RQ„ AC r n , g c o 2 (a - VR) RQ S 0.726 0.184 8.80 0.99 17.58 1.33 -14.4% 1.33 2.55 +12.7% 1.3 0.30 1.08 0.07 122 from the water which was v e n t i l a t e d a c r o s s the g i l l s ( V g g ) was c o n s i s t e n t l y i n e x c e s s (by 0.726 ± 0.184 y M « m i n ~ 1 - 1 0 0 g " 1 ) o f t h a t amount o f 0 2 uptake which was measured i n the b l o o d a c r o s s the g i l l s (M ). A v e r y i m p o r t a n t consequence o f the " e x t r a " oxygen uptake, which was not e x p r e s s e d i n the b l o o d , was found when the c a l c u l a t e d c a r d i a c o u t p u t , u s i n g the F i c k e q u a t i o n ( 5 ) . V n ( y M , m i n " 1 ' k g " 1 ) (5) Q ( m l - m i n ' ^ k g " 1 ) = -g 0 u 2 C a - C (yM) a 0 2 v 0 2 was compared w i t h the a c t u a l c a r d i a c o u t p u t ( c o n t r o l l e d by the c a r d i a c pump o f the a p p a r a t u s used i n t h e e x p e r i m e n t s ) . A c o n s i s t e n t o v e r e s t i m a t e o f 49.2 ± 11.4% o f the a c t u a l Q w a s i n d i c a t e d , when u s i n g the F i c k e q u a t i o n . The S a l i n e - P e r f u s e d T r o u t Head P r e p a r a t i o n : F a c t o r s a f f e c t i n g gas t r a n s f e r a c r o s s the g i l l s The e f f e c t s o f v a r i a b l e i n p u t f l o w c h a r a c t e r i s t i c s on the gas t r a n s f e r a c r o s s the g i l l s o f i s o l a t e d , s a l i n e - p e r f u s e d t r o u t heads (see M a t e r i a l s and Methods, pp.34-5; are p r e s e n t e d i n T a b l e 21. In g e n e r a l , changes a c r o s s the g i l l s (VA - DA d i f f e r e n c e s ) were s m a l l e r than changes a c r o s s the head c i r c u l a t i o n (DA - AV d i f f e r e n c e s ) . None o f the changes o f any o f the v a r i a b l e s was s i g n i f i c a n t f o r e i t h e r c i r c u l a t i o n . Changes i n P n a c r o s s t h e g i l l s were comparable t o t h o s e changes u 2 o b s e r v e d by Wood et al. (1978) under s i m i l a r e x p e r i m e n t a l c o n d i t i o n s . C o n s i s t e n t C 0 2 e x c r e t i o n , as measured by changes i n t o t a l C 0 2 , a c r o s s the l a m e l l a r c i r c u i t was o b s e r v e d d u r i n g a l l p e r f u s i o n regimes i n the p r e s e n t e x p e r i m e n t s . The s m a l l i n c r e a s e in.pH, e x c e p t d u r i n g p e r f u s i o n w i t h c o n s t a n t p r e s s u r e , i s c o m p a t i b l e w i t h C 0 2 l o s s a c r o s s the g i l l s . D u r i n g p e r f u s i o n a t c o n s t a n t p r e s s u r e , a s m a l l change i n C^g a c r o s s the g i l l c i r c u i t was accompanied by a d e c r e a s e i n pH. P e r f u s a t e pH and P n d e c r e a s e d , w h i l e u 2 P C Q ( c a l c u l a t e d ) i n c r e a s e d as i t p e r f u s e d the head v a s c u l a r bed. T h i s was T a b l e 21. Summary o f gas exchange d a t a i n the i s o l a t e d , s a l i n e - p e r f u s e d head o f s.;gdirdneri (pH d e t e r -m i n a t i o n s i n c l u d e d f o r r e f e r e n c e } P e r f u s i o n Regime V a r i a b l e C o n s t a n t Q ( p u l s a t i l e ) O ) MEAN ± SE Constant P r e s s u r e (mean o f p u l s e ) ( 2 ) MEAN ± SE Constant Q ( n o n - p u l s a t i l e ) ( 3 ) MEAN ± SE VA DA . VA DA VA DA RCULATION \ (mm Hg) 45.1 ±2.6 (13) 42.5 ±2.5 48.7 ±2.6 (8) 51.4 ±2.9 50.2 ±3.5 (7) 51.1 ±4.7 GILL CI PH 7.73 ±0.27 . (13) 7.82 ±0.02 7.85 ±0.04 (8) 7.82 ±0.02 7.74 ±0.02 (7) 7.82 ±0.04 DA AV DA AV DA AV CULATION \ (mm Hg) 42.5 ±2.5 (13) 29.9 ±2.5 51.4 ±2.9 (7) 30.0 ±3.7 51.1 ±4.7 (7) 28.6 ±4.3 HEAD CIR pH 7.82 ±0.02 (13) 7.61 ±0.03 7.82 ±0.02 (8) 7.77 ±0.04 7.82 ±0.04 (7) 7.68 ±0.04 numbers a s s o c i a t e d w i t h d a t a a r e o b s e r v a t i o n s 124 an e x p e c t e d s i t u a t i o n , g i v e n an a c t i v e l y . m e t a b l o l i z i n g p r e p a r a t i o n . However, o n l y the e f f e c t s o f haemodynamic a l t e r a t i o n s upon oxygen uptake w i l l be d i s c u s s e d s u b s e q u e n t l y . A n a l y s i s o f Water Flux, i n G i l l T i s s u e s  I n f l u x and e q u i l i b r a t i o n o f e t h a n o l (EtOH) F i g u r e s 15 and 16 d e p i c t t y p i c a l EtOH c o n c e n t r a t i o n s i n the r e -c i r c u l a t e d v e n t i l a t o r y w a t e r , d o r s a l a o r t i c , and a n t e r i o r venous o u t f l o w s d u r i n g l o a d i n g o f the g i l l s o f an i s o l a t e d , s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n (see M a t e r i a l s and Methods, pp.34-44).During t h i s p e r i o d o f EtOH l o a d i n g , the p r e p a r a t i o n was p e r f u s e d w i t h c o n s t a n t p u l s a t i l e f l o w . The h i g h e r EtOH c o n c e n t r a t i o n s i n the water f o r the f i r s t 4 - 5 minutes p r o b a b l y r e f l e c t s m i x i n g o f the added EtOH w i t h the 4 L r e c i r c u l a t i n g water volume. D o r s a l a o r t i c EtOH c o n c e n t r a t i o n s f o l l o w c l o s e l y t h e water EtOH c o n c e n t r a t i o n . The r a t e o f f a l l o f EtOH i n the water was the same as t h a t found i n the d o r s a l a o r t i c o u t f l o w . A f t e r 15 min, d o r s a l a o r t i c and a n t e r i o r venous EtOH c o n c e n t r a t i o n s both were a p p r o x i m a t e l y 1/20 o f t h a t f o u n d : i n the water (see T a b l e 2 2 ) . T h i s T a b l e , a l s o i n d i c a t e s t h a t the c o n c e n t r a t i o n o f EtOH i n the d o r s a l a o r t i c o u t f l o w was n o t s t a t i s t i c a l l y d i f f e r e n t ( S t u d e n t ' s t - t e s t ) f o r the two p e r f u s i o n regimes used i n t h e s e e x p e r i m e n t s . F i g u r e 17 i l l u s t r a t e s EtOH c o n c e n t r a t i o n i n the d o r s a l a o r t i c e f f l u e n t d u r i n g washout, w h i l e the head was p e r f u s e d a t c o n s t a n t p u l s a t i l e f l o w (17.0 m l ' m i n " 1 « k g " x ) , and c o n s t a n t p r e s s u r e ( f l o w - 16.2 m l * m i n " 1 , k g " x ) . Flows and p r e s s u r e s from p r e p a r a t i o n s used i n t h e s e e x p e r i m e n t s were no t s i g n i f i c a n t l y d i f f e r e n t from those r e p o r t e d i n T a b l e 10 ( S t u d e n t ' s t - t e s t ) . T h i s f i g u r e i l l u s t r a t e s t h a t the EtOH was washed from the g i l l s more complete-l y and more r a p i d l y d u r i n g p u l s a t i l e p e r f u s i o n than d u r i n g n o n - p u l s a t i l e p e r f u s i o n . A d d i t i o n o f EtOH t o the water, and u l t i m a t e l y t o the p e r f u s a t e by t r a n s e p i t h e l i a l d i f f u s i o n , d i d not i n any way a l t e r the v a s c u l a r r e s i s t -125 F i g u r e 15. T y p i c a l e t h a n o l c o n c e n t r a t i o n s i n the w a t e r , d o r s a l a o r t i c p e r f u s a t e and a n t e r i o r venous p e r f u s a t e d u r i n g l o a d i n g o f g i l l t i s s u e s . The d e c l i n e i n d o r s a l a o r t i c EtOH c o n c e n t r a t i o n a f t e r 5 min f o l l o w s the f a l l i n water EtOH c o n c e n t r a t i o n . A f t e r 15 min o f l o a d i n g , d o r s a l a o r t i c and a n t e r i o r venous EtOH c o n c e n t r a t i o n s a r e e q u a l . I n f l u x r e f e r s t o n e t move-ment o f EtOH a c r o s s the t i l l s , from water t o p e r f u s a t e i n the v a s c u l a t u r e . Water EtOH c o n c e n t r a t i o n D o r s a l a o r t i c EtOH c o n c e n t r a t i o n A n t e r i o r venous EtOH c o n c e n t r a t i o n 126 127 F i g u r e 16. R e p r e s e n t a t i o n o f the d a t a p r e s e n t e d i n F i g . 15, n o r m a l i z e d to the f i n a l c o n c e n t r a t i o n s i n p e r f u s a t e a f t e r 15 min. T h i s f i g u r e i n d i c a t e s t h a t d o r s a l a o r t i c l o a d i n g proceeds a t a f a s t e r r a t e than does the a n t e r i o r venous o u t f l o w , but they r e a c h e q u i l i b r a t i o n a t the same time span chosen f o r l o a d i n g . Rest o f l e g e n d as i n F i g . 15. 1 Oo ico 129 T a b l e 22. Ethano l c o n c e n t r a t i o n s i n w a t e r , d o r s a l a o r t i c p e r f u s a t e and a n t e r i o r venous p e r f u s a t e . P r e p a r a t i o n s were exposed t o e t h a n o l f o r 15 minutes to l o a d g i l l t i s s u e s d u r i n g p e r f u s i o n by e i t h e r c o n s t a n t p u l s a t i l e f l o w o r c o n s t a n t n o n - p u l s a t i l e p r e s s u r e p e r f u s i o n regimes, (n = 5 ) . Water D o r s a l a o r t i c A n t e r i o r venous p e r f u s a t e p e r f u s a t e mM mM mM P u l s a t i l e P e r f u s i o n 3.57 ± 0.74 0.15 ± 0.05 0.13 ± 0.03 N o n - p u l s a t i l e p e r f u s i o n 3.33 ± K 0 4 0.19 ± 0.08 0.15 ± 0.08 130 F i g u r e 17. Dorsal a o r t i c e t h a n o l (EtOH) c o n c e n t r a t i o n s d u r i n g washout from g i l l t i s s u e s . EtOH i s washed from g i l l s more r a p i d l y and more c o m p l e t e l y d u r i n g p u l s a t i l e p e r f u s i o n , compared t o n o n - p u l s a t i l e p e r f u s i o n , o f the same mean p r e s s u r e . • P u l s a t i l e c o n s t a n t f l o w N o n - p u l s a t i l e c o n s t a n t p r e s s u r e Normalized [EtOH] P cn b cn 132 ance o f the g i l l s , o r the p r e s s u r e p r o f i l e o f the p e r f u s i o n p u l s e ( s e e F i g . 1 Ethanol c o n c e n t r a t i o n s i n the a n t e r i o r venous p e r f u s a t e d u r i n g washout are shown i n F i g . 19. The p o i n t s on these c u r v e s f o r washout d u r i n g the two regimes a r e not s i g n i f i c a n t l y d i f f e r e n t , a l t h o u g h the c o n c e n t r a t i o n d u r i n g p u l s a t i l e p e r f u s i o n a p p e a r s t o be f a l l i n g more r a p i d l y toward the end o f the 15 min p e r i o d . Compartmental A n a l y s i s o f EtOH Washout Curves S e m i l o g p l o t s o f EtOH washout c u r v e s from d o r s a l a o r t i c samples r e v e a l e d two d i s t i n c t compartments. By curv e p e e l i n g , s t r a i g h t l i n e s were f i t t e d t o p l o t s o f the n a t u r a l l o g s ( In) o f the e t h a n o l c o n c e n t r a t i o n s , versus time. The n o r m a l i z e d e q u a t i o n f o r the EtOH washout c u r v e d u r i n g p u l s a t i l e p e r f u s i o n i s ( 1 ) . (1) Amount o f EtOH a t time = t = Q 8 7 2 E - ' L - 2 8 T + Q 1 2 7 e ~ ° ' 2 8 t Amount o f EtOH a t time = 0 The r 2 v a l u e s ( c o e f f i c i e n t s o f the d e t e r m i n a t i o n ) f o r both l i n e s g e n e r a t e d from the d a t a were 0.98. The e q u a t i o n f o r the c u r v e o f EtOH washout d u r i n g n o n - p u l s a t i l e f l o w i s ( 2 ) . (2) 0 . 6 0 6 e " ° - 2 0 3 t + 0 . 3 9 4 e - 0 - 0 3 5 2 t , h a v i n g r 2 v a l u e s o f 0.96 and 0.99, f o r the f a s t and slow components r e s p e c t i v e l y . S i n c e EtOH c o n c e n t r a t i o n s were measured o n l y i n the second, o r downstream ( f l u i d v a s c u l a r space) compartment, a complete a n a l y s i s would not n o r m a l l y have been p o s s i b l e . However, t h e s e arguments a p p l y o n l y t o the l o a d i n g o f t i s s u e s . As g i l l t i s s u e s f i r s t were e q u i l i b r a t e d w i t h EtOH and then i t s subsequent washout measured, i t was p o s s i b l e to c o m p l e t e l y a n a l y s e the system f o r a l l r a t e c o n s t a n t s and volumes o f d i s t r i b u t i o n . The model used f o r t h e . a n a l y s i s i s p r e s e n t e d i n F i g . 20. I m p l i c i t i n t h i s model a r e the assumptions t h a t ; d u r i n g washout, p a r t o f the EtOH i n the t i s s u e s i s washed out i n t o the e x t e r n a l medium, and, t h e r e i s 133 F i g u r e 18. Record o f i n p u t p r e s s u r e d u r i n g p u l s a t i l e c o n s t a n t f l o w p e r f u s i o n . Note t h a t the s y s t o l e phase l a s t s f o r one t h i r d and d i a s t o l e phase two t h i r d s o f each pump c y c l e . I n t r o -d u c t i o n o f e t h a n o l (EtOH, a t arrow) t o the water has no e f f e c t on i n p u t p r e s s u r e . 134 135 F i g u r e 19. A n t e r i o r venous e t h a n o l c o n c e n t r a t i o n s d u r i n g washout from g i l l t i s s u e s . These were n o r m a l i z e d t o the f i n a l p e r f u s a t e c o n c e n t r a t i o n a f t e r 15 min l o a d i n g w i t h EtOH. P u l s a t i l e p e r f u s i o n had no s i g n i f i c a n t e f f e c t on the washout o f EtOH from the a n t e r i o r venous c i r c u l a t i o n . • • P u l s a t i l e c o n s t a n t f l o w — A — N o n - p u l s a t i l e c o n s t a n t p r e s s u r e Normalized [EtOH] O _ i _ L <* b 01 ' I I 137 exchange o f EtOH between g i l l t i s s u e s and the p e r f u s a t e w i t h i n the g i l l v e s s e l s . A l t h o u g h a n a t o m i c a l i d e n t i f i c a t i o n o f the two compartments i s not p o s s i b l e , i t i s p o s t u l a t e d t h a t t he f a s t compartment i s the v a s c u l a r s pace, and t h a t t he slow compartment i s comprised o f c e l l s , and perhaps the e x t r a c e l l u l a r / e x t r a v a s c u l a r . v o l u m e . F i g u r e 20 i l l u s t r a t e s a number, o f d i f f e r e n c e s i n the c u r v e s p r e s e n t e d i n F i g . 17. F i r s t l y , the o v e r a l l washout r a t e ( r a t e c o n s t a n t s ) oa d u r i n g p u l s a t i l e p e r f u s i o n i s some 12 times g r e a t e r than t h a t f o r non-p u l s a t i l e p e r f u s i o n . The r e l a t i v e volumes o f the f a s t and slow compartments d u r i n g p u l s a t i l e p e r f u s i o n show t h a t 7/8 o f the EtOH i n the g i l l s i s i n the f a s t o r v a s c u l a r compartment. D u r i n g n o n - p u l s a t i l e p e r f u s i o n however, o n l y 2/3 o f the EtOH i s i n t h i s f a s t compartment. P r o b a b l y the most i m p o r t a n t f e a t u r e o f t h i s a n a l y s i s i s t h a t exchange between the two compartments ( r a t e c o n s t a n t s and k ^) i s four, times g r e a t e r d u r i n g p u l s a t i l e p e r f u s i o n as compared t o n o n - p u l s a t i l e p e r f u s i o n . Ethanol c o n c e n t r a t i o n s i n v e n t i l a t o r y water d u i r n g washout o f EtOH from the g i l l s show t h a t t h e r e was l e s s than 50% o f the i n i t i a l e t h a n o l i n the water a f t e r 30 seconds, and none was d e t e c t a b l e a f t e r 90 s e c . 138 F i g u r e 20. Two-compartment models i l l u s t r a t i n g the r e s u l t s o f the a n a l y s i s o f the d o r s a l a o r t i c e t h a n o l washout c u r v e s d u r i n g : (A) P u l s a t i l e c o n s t a n t f l o w p e r f u s i o n , and (B) n o n - p u l s a t i l e c o n s t a n t p r e s s u r e p e r f u s i o n . D u r i n g p u l s a t i l e p e r f u s i o n EtOH i s l o s t more r a p i d l y from the g i l l s ( k o a ) , and exchanges between t h e f a s t and slow compartments ( k ^ ; k ^ ) are g r e a t e r . A l s o d u r i n g p u l s a t i l e p e r f u s i o n , the f a s t ( v a s c u l a r ) compartment i s r e l a t i v e l y l a r g e r than the slow compartment, when compared t o the compartment s i z e s d u r i n g n o n - p u l s a t i l e p e r f u s i o n . 139 To water m k b a 0.27 k a b 0.41 3.12 ml k o a 0.88 Slow compartments B Fast compartments To water 0.89 ml k b a 0.06 1.37 ml k o a 0.07 140 DISCUSSION I I I : Gas Exchange i n , the S p o n t a n e o u s l y V e n t i l a t i n g , . B l o o d - P e r f u s e d T r o u t P r e p a r a t i o n : Comparisons w i t h in vivo d a t a . B l o o d Gas T e n s i o n s and Contents  Input Blood B l o o d i n the tonometers was exposed to gas c o n t a i n i n g 0.4% C O 2 i n 40% a i r (PQQ = 3.0 mm Hg; P Q = 60 mm Hg, P^ = 697 mm Hg). Measured mean P h o f the i n p u t b l o o d was 24.9 mm Hg, p r i n c i p a l l y because venous r e t u r n •2 b l o o d had a low PQ . V e n t r a l a o r t i c PQ i n r e s t i n g rainbow t r o u t , , i n .vivo. i s . a r o u n d 30 mm Hg (Cameron and D a v i s , 1970; Eddy et al., 1977; K i c e n i u k and J o n e s , 1977), a l t h o u g h l o w e r v a l u e s have been measured (19.0 mm Hg, Stevens and R a n d a l l , 1967a). T h e r e f o r e , i n p u t P n v a l u e s from the b l o o d -p e r f u s e d p r e p a r a t i o n s were i n . t h e p h y s i o l o g i c a l range measured in vivo. Mean i n p u t oxygen c o n t e n t was 0.9 mMX -* f o r the b l o o d - p e r f u s e d f i s h . K i c e n i u k and Jones (1977) measure oxygen c o n t e n t s o f v e n t r a l a o r t i c b l o o d in vivo, from r e s t i n g rainbow t r o u t o f "about 3.2 mM'L Given t h a t the b l o o d - p e r f u s e d p r e p a r a t i o n s a r e p e r f u s e d w i t h b l o o d o f lower Hct (10.3% compared t o 24.2% f o r the f i s h used by K i c e n i u k and J o n e s ) , and t h a t my P n i s lower by 25%, C n o f the i n p u t b l o o d . i n my e x p e r i m e n t s : i s x l o s e to u2 u2 t h a t p r e d i c t e d from a v a i l a b l e d a t a (see H o l e t o n and R a n d a l l , 1967a). In vivo venous PQQ v a l u e s range between 1 and 5 mm Hg i n r e s t i n g rainbow t r o u t ( H o l e t o n and R a n d a l l , 1967a; Stevens and R a n d a l l , 1967b; Eddy et al., 1977; Wood et al. , i n p r e s s ) . The c a l c u l a t e d v a l u e f o r i n p u t P c 0 (3.36 ± 0.15 mm Hg) i n b l o o d - p e r f u s e d f i s h more c l o s e l y resembled t h a t o f the gas m i x t u r e . CO^ c o n t e n t g e n e r a l l y was h i g h e r i n the i n p u t b l o o d than has been r e p o r t e d , i n the l i t e r a t u r e (Eddy et al., 1977). However, r e c e n t v a l u e s (Wood et al., i n p r e s s ; P e r r y and Heming, i n p r e s s ) are v e r y c l o s e to those r e p o r t e d here. 141 D o r s a l A o r t i c ( a r t e r i a l ) B l o o d Mean d o r s a l a o r t i c b l o o d P n i n b l o o d - p e r f u s e d f i s h was 103 mm Hg u 2 and C 0 o was 1.58 mM.L"1.. For a mean Hct o f 8.8%, t h i s i n d i c a t e d o v e r 98% a c oxygen s a t u r a t i o n (from C 0 o = 0.311 x Hct + 0 . 7 ( V o l . % ) , H o l e t o n and a c R a n d a l l , 1967b). was lower than some i n y i y o v a l u e s (133.2 mm Hg, Cameron and D a v i s , 1970; 117 mm Hg, Eddy et al., 1977; 137 mm Hg f o r t r o u t i n a swim tube, K i c e n i u k and J o n e s , 1977), y e t h i g h e r than t h o s e o f Stevens and R a n d a l l (1967b) o f 85..mm Hg. . However, each o f . t h e above mentioned P 0, v a l u e s a L. i n d i c a t e s b e t t e r than 95% s a t u r d a t i o n o f the b l o o d i n t h e i r p a r t i c u l a r cases as wel1. C C 0 o measurements and c a l c u l a t e d P C 0 o v a l u e s from b l o o d - p e r f u s e d a 2 a 2 v f i s h were s i m i l a r t o i n vivo v a l u e s , a l t h o u g h v a r i a b i l i t y o f t h e i n vivo d a t a p r e c l u d e s any d e t a i l e d comparison. Oxygen Uptake A c r o s s the G i l l s - M^Og There are no d i r e c t in vivo measurements o f M 0 o a v a i l a b l e i n the l i t e r a t u r e . However, Stevens and R a n d a l l (1967b) and K i c e n i u k and Jones (1977) measured N n , the oxygen consumption by the whole f i s h i n a r e s p i r o -u 2 meter, f o r rainbow t r o u t , and found v a l u e s o f 2.68 and 2.5uM O g T u i n ' ^ l O O g - 1 , r e s p e c t i v e l y . Mean oxygen uptake by the b l o o d during, i t s passage through the g i l l s i n b l o o d - p e r f u s e d f i s h was 1.17|iM*min" 1»100g" 1. A f t e r c o r r e c t i o n f o r temperature d i f f e r e n c e s beween in vivo experiments and mine, the i n vivo v a l u e s f o r M 0 o s t i l l were some 60% h i g h e r . g 2 a B l o o d - p e r f u s e d p r e p a r a t i o n s may have shown d i f f e r e n t Mg0 o v a l u e s than t h o s e : f r o m i n vivo experiments f o r the f o l l o w i n g r e a s o n s . Tonometering b l o o d w i t h the p a r t i c u l a r m i x t u r e o f gases used tended t o i n c r e a s e i n p u t P n from 13 mm Hg (measured mean v a l u e f o r venous r e t u r n blood) t o 25 mm Hg, u 2 which i n c r e a s e d i n p u t C n t w o - f o l d . Had no attempt been made t o r a i s e i n p u t u 2 P n t o what were c o n s i d e r e d p h y s i o l o g i c a l l e v e l s (in vivo v a l u e s from the u 2 142 l i t e r a t u r e ) , then blood entering the g i l l s would have had a greater capacity for oxygen uptake, by virtue of an increased diffusion gradient for oxygen across the g i l l s . As dorsal aortic blood always was greater than 98% oxygen saturated in these preparations., M CL may have been limited by high .input y £ C n • Oxygen consumption by the skin (Kirsch and.Nonnotte, 1977) and the 2 . g i l l tissues (see Section. I l l ) in N n (or V 0 9) determinations also could ^2 g c-have contributed to the noted differences between M 0 o values obtained from blood-perfused and in '•vivo experiments. Oxygen Uptake across the Systemic Circulation - M 0 2 Oxygen extraction by the systemic tissues was some 1.75 times the measured value for the oxygen uptake rate across the g i l l s . This finding was a. consequence of raising input P n by tonometry, rather than allowing u2 input blood to reach i t s own P n level from venous return blood alone. Had 2 this approach been used, then one.could presume that M 0 ? would increase by y an appropriate factor to accommodate the measured Ms02> since these values were much closer to the estimates from in- vivo experiments. Systemic tissues nonetheless extracted a very large proportion of the delivered blood oxygen content. Carbon Dioxide Excreti on Across the G i l l s , and Production by Systemic Tissues Mean carbon dioxide excretion across the g i l l s of blood-perfused trout was 2.05|iM'min~1*100g~1. As venous C^Q and a r t e r i a l C^g have yet to be measured simultaneously with cardiac output (Q) in.vivo, no values for comparison of M 0 ? are available from the l i t e r a t u r e . Eddy et al. (1977) y £ have described a 27% f a l l in C^Q across the g i l l s of two f i s h , . where both venous and a r t e r i a l C^Q were measured. Although mean C^Q change across the g i l l s of blood-perfused.fish was 12%, some individual f i s h did show upwards of a 28%.decrease in C^Q under normal conditions. Blood-perfused 2 f i s h were able to excrete s u f f i c i e n t net CO2 to keep pace with i t s producti on 143 by the s y s t e m i c t i s s u e s . The mean r a t i o o f COg e x c r e t i o n o v e r Og uptake a c r o s s the g i l l s (RQg) o f b l o o d - p e r f u s e d f i s h was 1.85. T h i s h i g h v a l u e r e s u l t e d from low M 0 ? measurements, which i n t u r n were the r e s u l t o f the e l e v a t i o n o f i n p u t y £-PQ and CQ .. Mean r a t i o o f COg p r o d u c t i o n by the s y s t e m i c t i s s u e s , over the Og u t i l i z a t i o n by t h e s e t i s s u e s (RQ $) was 0.83. T y p i c a l l y , t h i s r e p r e s e n t s f a t c a t a b o l i s m ( R o b i n s o n , 1974). However, the m e t a b o l i c sub-s t r a t e s used by the p r e s e n t p r e p a r a t i o n a r e unknown. Blo o d C h e m i s t r y Plasma o s m o l a r i t y and CI" c o n c e n t r a t i o n s were s i m i l a r t o , t h o s e r e p o r t e d by Holmes and Donaldson.(1970), and to.measurements made by P e r r y and Heming ( i n p r e s s ) . T a b l e 16 i n d i c a t e s some d i l u t i o n o f the b l o o d as i t passes through the g i l l s o f b l o o d ^ p e r f u s e d f i s h . Hct f e l l by 14%. There are a number o f p o s s i b l e e x p l a n a t i o n s , i n c l u d i n g d i l u t i o n d u r i n g s a m p l i n g . Water uptake r a t e s , c a l c u l a t e d from the plasma o s m o l a r i t y d i f f e r e n c e between i n p u t and d o r s a l a o r t i c b l o o d o f my p r e p a r a t i o n s , were i n the o r d e r o f 0.06 ml*min" 1•1OOg" 1. Net water i n f l u x a c r o s s t r o u t g i l l s , measured i n o t h e r p r e p a r a t i o n s , i s a p p r o x i m a t e l y 0.17 m l • m i n " 1 , 1 0 0 g " 1 ( I s a i a et al., 1978). G i l l s o f b l o o d - p e r f u s e d t r o u t behave as they do in vivo, w i t h r e s p e c t t o t r a n s e p i t h e l i a l water f l u x . V e n t i l a t i o n Rate The mean v e n t i l a t i o n r a t e o f b l o o d - p e r f u s e d t r o u t was 69.4 b r e a t h s * m i n - 1 . The w e a l t h o f d a t a a v a i l a b l e from i n t a c t f i s h i n d i c a t e s t h a t a t the temperature o f the p r e s e n t p r e p a r a t i o n s ( 7 ° C ) , t h i s r a t e i s normal ( H o l e t o n and R a n d a l l , 1967a; Davis and Cameron, 1971; Daxboeck and H o l e t o n , 1978, 1980). I n t e r a c t i o n s between r e s p i r a t o r y : movements and b l o o d p r e s s u r e were e v i d e n t i n t he t r a c e s from b l o o d - p e r f u s e d p r e p a r a t i o n s ( F i g . 1 2 ) , and were s i m i l a r t o those d e s c r i b e d by Wood and S h e l t o n (1980a) from t r o u t in vivo.' These-144 i n t e r a c t i o n s can cause moderate i n c r e a s e s ,in b l o o d f l o w through the g i l l s , as proposed by Johansen et al. (1966), and have been d i s c u s s e d i n d e t a i l by Hughes (1973). Haemodynamic A l t e r a t i o n s and T h e i r E f f e c t s on Gas Exchange The g i l l s o f f i s h , in vivo, are presumed t o be d i f f u s i o n l i m i t e d f o r gas t r a n s f e r . As s u c h , haemodynamic changes which c o u l d p o s s i b l y d e c r e a s e the g i l l d i f f u s i o n b a r r i e r t o gases s h o u l d . t h e r e b y i n c r e a s e oxygen uptake measured a c r o s s t h e s e g i l l s . Data c o l l e c t e d from s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t i n d i c a t e t h a t g i l l , r e s i s t a n c e to f l o w , and not the mass t r a n s f e r o f oxygen (MgC^) i s the v a r i a b l e most s e n s i t i v e t o haemodynamic a l t e r a t i o n s , o t h e r than c a r d i a c o u t p u t . The d a t a from the b l o o d - p e r f u s e d s t u d y show t h a t the g i l l s o f rainbow t r o u t , a t r e s t a r e PERFUSION LIMITED f o r gas t r a n s f e r . u n d e r t h e s e c o n d i t i o n s . For g i l l s t o be termed p e r f u s i o n l i m i t e d , oxygen uptake by the b l o o d (MgO^) must i n c r e a s e as the r a t e o f p e r f u s i o n through the r e s p i r a t o r y c a p i l l a r y network i n c r e a s e s . Such i s the case i n t h e s e p r e p a r a t i o n s (see F i g . 13), and carbon d i o x i d e e x c r e t i o n '(MgOg) shows a s i m i l a r r e l a t i o n s h i p (see F i g . 13). The r e s i d e n c e time o f b l o o d . i n the g i l l s o v e r the range o f f l o w r a t e s t e s t e d a l s o i s s u f f i c i e n t f o r b l o o d oxygen s a t u r a t i o n , s i n c e P 0 o remains c o n s t a n t a t a a c. l e v e l e q u i v a l e n t t o 98% s a t u r a t i o n . A s . w e l l , P g C ^ i s u n a f f e c t e d by changes i n p e r f u s i o n flow.; and a c o n s t a n t g r a d i e n t between b l o o d and water i s main-t a i n e d o v e r a wide range o f Q. . I f the c a r r y i n g c a p a c i t y o f the b l o o d i s d e c r e a s e d , by d e c r e a s i n g H c t . t h e n a c o r r e s p o n d i n g d e c r e a s e i n the oxygen uptake r a t e a c r o s s the g i l l s i s n o ted i n . t h e b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n , g i v e n the p e r f u s i o n r a t e remains c o n s t a n t . D o r s a l a o r t i c PQ , as w e l l as the change i n PQ between i n p u t and a r t e r i a l b l o o d a c r o s s the g i l l s however a r e not s i g n i f i c a n t l y d i f f e r e n t from o t h e r Hct c o n d i t i o n s , even w i t h p e r f u s i o n w i t h an Hct as low 145 as 4%. The m a i n t a i n e d l e v e l o f P 0, i s due, i n p a r t , t o an i n c r e a s e d a <L c o n t r i b u t i o n by d i s s o l v e d Og i n a l a r g e r volume o f plasma,, augmented by the presence o f the r e d b l o o d c e l l s . U n l i k e s e v e r e l y anaemic t r o u t , which i n c r e a s e c a r d i a c o u t p u t i n response t o t h i s s t r e s s in. vivo (Wood et al. 1979; Wood and S h e l t o n , 1980a), i n o r d e r t o m a i n t a i n r e l a t i v e l y c o n s t a n t oxygen uptake r a t e s , a c o n s t a n t c a r d i a c o u t p u t i n the p r e p a r a t i o n s which I used e l i m i n a t e s any p o s s i b l e masking e f f e c t s due t o changes i n Q. A l t h o u g h t h e r e are g i l l v a s c u l a r r e s i s t a n c e changes t o f l o w w i t h e i t h e r h i g h e r than o r lower than "normal" Hct b l o o d p e r f u s i o n o f whole t r o u t p r e p a r a t i o n s , t h e s e p r e s s u r e changes by themselves have no e f f e c t on gas t r a n s f e r , as p r e v i o u s l y d i s c u s s e d . T h e r e f o r e , the r e s u l t s w i t h v a r i a b l e h a e m a t o c r i t p e r f u s i o n r e f l e c t only, the c o n t r i b u t i o n o f the number o f e r y t h r o c y t e s upon Og t r a n s f e r a c r o s s the g i l l s . The gas exchange e f f i c i e n c y o f t r o u t g i l l s i s s a i d t o be i n c r e a s e d d u r i n g e x e r c i s e , in vivo. ( K i c e n i u k and J o n e s , 1977), due t o a t t e n d a n t c a r d i o r e s p i r a t o r y a d j u s t m e n t s . From the d a t a c o l l e c t e d d u r i n g m a n i p u l a t i o n s o f c a r d i a c f r e q u e n c y / s t r o k e volume/pulse p r e s s u r e i n the b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n s a t , r e s t , i n normoxia, w h i l e Q i s kept c o n s t a n t , n e g l i g a b l e changes i n oxygen uptake, and carbon d i o x i d e e x c r e t i o n r a t e s a c r o s s the g i l l s are n o t e d . N e i t h e r was oxygen uptake a c r o s s the g i l l s from the water (VgOg), o r by the b l o o d (MgOg) changed from normal when b r a d y c a r d i a was s i m u l a t e d . . As d o r s a l a o r t i c b l o o d always i s g r e a t e r than 98% Og s a t u r a t e d , i n c r e a s e s i n MgOg o n l y c o u l d have a r i s e n from i n c r e a s e s i n the amount o f oxygen d i s s o l v e d i n the plasma, whether the g i l l s a r e d i f f u s i o n o r p e r f u s i o n l i m i t e d . As plasma, o r s a l i n e oxygen c a r r y i n g c a p a c i t y i s s m a l l , compared to t h a t o f the r e d c e l l s , no s i g n i f i c a n t changes i n MgOg can be e x p e c t e d under t h e s e p e r f u s i o n c o n d i t i o n s : a s i t u a t i o n analogous t o the s a l i n e -p e r f u s e d p r e p a r a t i o n s . What t h e s e d a t a do i n d i c a t e however, i s t h a t the 146 proposed changes i n g i l l d i f f u s i o n c a p a c i t y f o r g a s e s , a s s o c i a t e d w i t h changes i n g i l l b l o o d f l o w and p r e s s u r e ( F a r r e l l et al..3 1979, 1980; d a t a p r e s e n t e d f o r i s o l a t e d head p r e p a r a t i o n s i n t h i s t h e s i s ) . a r e r e l a t i v e l y un-i m p o r t a n t t o gas t r a n s f e r . These d a t a a g a i n i n d i c a t e t h a t t he g i l l s o f r a i n -bow t r o u t a t r e s t , under the e x p e r i m e n t a l c o n d i t i o n s , a r e p e r f u s i o n l i m i t e d f o r oxygen uptake, as w e l l as carbon d i o x i d e e x c r e t i o n , independent o f mean and p u l s e p r e s s u r e . One may s t i l l argue t h a t gas t r a n s f e r a t the g i l l s i s d i f f u s i o n r a t h e r than p e r f u s i o n l i m i t e d . Because o f t h e r a t h e r low "normal" haemato-c r i t o f the b l o o d used i n the b l o o d - p e r f u s i o n e x p e r i m e n t s , and the a t t e n d a n t i n c r e a s e s i n i n p u t PQ and P C Q ( r e a s o n s f o r t h i s have been d i s c u s s e d p r e -v i o u s l y ) , a h i g h e r p e r c e n t 0 2 s a t u r a t i o n f o r the i n p u t i s measured, h i g h e r than may may have been measured had the b l o o d not been tonometered w i t h the the chosen gas m i x t u r e u s e d . As a consequence, a lower change i n the P n between i n p u t and d o r s a l a o r t i c b l o o d i s measured, than p o t e n t i a l l y c o u l d have been a c h i e v e d d u r i n g haemodynamic a l t e r a t i o n s , had Hct been "more r e p r e -s e n t a t i v e " o f t h a t found in vivo, and PQ and PQQ .been a l l o w e d t o f a l l t o whatever l e v e l they c o u l d have a t t a i n e d . A l s o , one c o u l d s u g g e s t t h a t , s i n c e a d i f f u s i o n l i m i t e d exchange system i s dependent upon the a b i l i t y o f t h a t system t o somehow i n c r e a s e the d i f f u s i o n a l a r e a as w e l l , a l l t he se c o n d a r y l a m e l l a e i n the b l o o d - p e r f u s e d p r e p a r a t i o n s a r e b e i n g p e r f u s e d a l r e a d y , and thus any p o s s i b i l i t y o f demon-s t r a t i n g changes i n d i f f u s i o n c a p a c i t y by t h i s avenue a l r e a d y i s s a t u r a t e d . Steen and Kruysse (1964) and Payan, G i r a r d , Peyraud and Waitzenegger (un-p u b l i s h e d o b s e r v a t i o n s r e p o r t e d by G i r a r d and Payan, 1980) note an i n c r e a s e i n b r a n c h i a l Or, p e r m e a b i l i t y i n t r o u t exposed t o a d r e n a l i n e . These d a t a i n d i c a t e p o s s i b l e d e c r e a s e s i n d i f f u s i o n b a r r i e r s as a r e s u l t o f the adren-a l i n e e x p osure. A l t h o u g h v e n t i l a t i o n r a t e s (Peyraud-Waitzenegger, 1979), and i n t r i n s i c h e a r t r a t e s (Gannon, 1971) might be e x p e c t e d t o r i s e d u r i n g 147 a d r e n a l i n e e x p o s u r e , n e i t h e r o f t h e s e e v e n t s o c c u r i n the b l o o d - p e r f u s e d p r e p a r a t i o n s . S i n c e tha major e f f e c t o f a d r e n a l i n e on. t r o u t h e a r t s i s i n o -t r o p i c , i n c r e a s e s i n the magnitude o f v e n t r i c u l a r p r e s s u r e s are o b s e r v e d d u r i n g the a d m i n i s t r a t i o n o f a d r e n a l i n e i n my p r e p a r a t i o n s , but no changes • • • i n P a n o r Can „are measured. N e i t h e r a r e M 0 o nor M C 0 o s i g n i f i c a n t l y 0 2 0 o g 2 g 2 3 J a l t e r e d d u r i n g a d r e n a l i n e exposure i n t h e s e p r e p a r a t i o n s . An i n c r e a s e i n • Mg0 2 however, may n o t have been e x p r e s s e d i n the p r e s e n t a d r e n a l i n e expo-s u r e e x p e r i m e n t s because, u n l i k e in vivo, the c a r d i a c pump which r e p l a c e s the a c t u a l h e a r t can not make the a p p r o p r i a t e r e s p o n s e s t o t h i s c a t e c h o l -amine. I n c r e a s e s i n oxygen uptake in vivo d u r i n g p e r i o d s o f i n c r e a s e d l e v e l s o f c i r c u l a t i n g c a t e c h o l a m i n e s , as presumed d u r i n g e x e r c i s e i n t r o u t , t h e r e -f o r e may not be due t o the d i r e c t e f f e c t o f changes i n the d i f f u s i o n b a r r i e r a t the g i l l s , b ut as a r e s u l t o f the changes which a d r e n a l i n e promote i n c a u s i n g an i n c r e a s e i n the r a t e o f b l o o d f l o w through the g i l l s . The r e s u l t s w i t h t h e a d d i t i o n o f a d r e n a l i n e t o t h e b l o o d i n my experiments n e v e r g i v e o c c a s s i o n t o presume t h a t the b l o o d - p e r f u s e d p r e p a r a t i o n demonstrates any s i g n i f i c a n t d i f f u s i o n l i m i t a t i o n s . I f s t r e s s had i n d u c e d an above normal amount o f mucous t o be produced to c o v e r the g i l l s o f b l o o d - p e r f u s e d f i s h , a s i t u a t i o n o f t e n noted w i t h t h e s a l i n e - p e r f u s e d p r e p a r a t i o n s , i t has been demonstrated t h a t , w i t h i n l i m i t s , t h i s mucous l a y e r i s p r i m a r i l y a r e s i s t a n c e b a r r i e r f o r i o n i c exchange r a t h e r than f o r gas d i f f u s i o n ( U l t s c h and Gros, 1979). I f t he h a e m a t o c r i t were t o i n c r e a s e t o such an e x t e n t t h a t t h e b l o o d was so v i s c o u s t h a t the r e d b l o o d c e l l s would a c t u a l l y i n t e r f e r e w i t h each o t h e r , c a u s i n g d i f f u s i v e gas exchange problems, n o t w i t h s t a n d i n g t h e p r e s s u r e and f l o w e f f e c t s b r o u g h t about by the i n c r e a s e d v i s c o s i t y , then 0 2 uptake measurements under t h e s e c o n d i t i o n s c o u l d l e a d t o the c o n c l u s i o n t h a t the g i l l s a r e s t i l l d i f f u s i o n l i m i t e d . D u r i n g e x e r c i s e , Hct does r i s e , but o b v i o u s l y not t o a l e v e l which i s d e t r i m e n t a l t o the e f f e c t i v e n e s s - ••-, 148 o f gas t r a n s f e r under t he p h y s i o l o g i c a l c o n d i t i o n s which promote t h i s s i t u -a t i o n . A l s o , i f c a r d i a c o u t p u t were t o i n c r e a s e t o the p o i n t where b l o o d r e s i d e n c e time i n the g i l l s was so a b b r e v i a t e d t h a t the d i f f u s i v e p r o c e s s o f haemoglobin o x y g e n a t i o n were the l i m i t i n g s t e p , the g i l l s a r e then d i f f u s i o n l i m i t e d . However, the v a s c u l a r e f f e c t s o f such a s i t u a t i o n would s u r e l y be c o m p l e t e l y d i s r u p t i v e , and so o u t s i d e t h e scope o f r e a s o n a b l e p h y s i o l o g i c a l f u n c t i o n , t h a t such a c o n d i t i o n i s u n t e n a b l e . A p p l i c a b i l i t y o f the F i c k P r i n c i p l e The b l o o d - p e r f u s e d p r e p a r a t i o n a f f o r d s t h e o p p o r t u n i t y to t e s t t h e a p p l i c a b i l i t y o f t h e F i c k e q u a t i o n f o r c a l c u l a t i n g c a r d i a c o u t p u t , as the a c t u a l Q a l r e a d y i s known. When such a comparison i s made, any measurement o f oxygen uptake from t h e water a c r o s s the g i l l s (VgC^) c o n s i s t e n t l y i s i n excess o f the amount o f oxygen which i s a c t u a l l y p i c k e d up by the bl o o d (H O J . T h i s d i s c r e p a n c y i s due t o the f a c t t h a t g i l l t i s s u e s a r e meta-y £ b o l i c a l l y a c t i v e and t h e r e f o r e consume a s i g n i f i c a n t amount o f oxygen from the water and b l o o d . Data from experiments conducted by Johansen and P e t t e r s -son (1981), u s i n g i s o l a t e d p e r f u s e d g i l l a r c h e s , have i n d i c a t e d t h a t a p p r o x i -m ately 40% o f the m e t a b o l i c oxygen demand o f g i l l t i s s u e s i s met by d i r e c t uptake and u t i l i z a t i o n from t he water. The d i f f e r e n c e between V 0 o and M 0 o g 2 g 2 i n the b l o o d - p e r f u s e d p r e p a r a t i o n s (0.726 ± 0.184 yM 0 2'min ' 100 g body w e i g h t 1 ) r e p r e s e n t s t h e measured 0 2 consumption o f the g i l l s . S i n c e g i l l t i s s u e s c o n s t i t u t e 3.9% o f the t o t a l body w e i g h t i n t r o u t ( S t e v e n s , 1968a), a v a l u e o f 18.6 yM 0 2* min 1 - 100 grams o f g i l l t i s s u e - 1 i s d e r i v e d f o r norm-o x i c rainbow t r o u t g i l l s . T h i s v a l u e compares w e l l w i t h the a v a i l a b l e d a t a f o r o t h e r f i s h g i l l s (Amazonian f i s h - measurements c o r r e c t e d t o 7°C: Hoplias mdlabavious 3 10.0; Osteoglosswn bieirrhosum, 12.8; Hoplerythr-inus unitaenia-tus3 13.3; and Avapaima gigas3 21.3 yM 0 2 ' min 1 - lOOg t i s s u e *; P.W. Hochach-ka, u n p u b l i s h e d d a t a ) . One can e n v i s a g e t h a t under normal c o n d i t i o n s , g i l l 149 t i s s u e s w i l l a c c o u n t f o r a c e r t a i n p r o p o r t i o n o f VgOg. Then and o n l y then can the remainder be ex p r e s s e d i n the b l o o d as MgOg. D u r i n g e x e r c i s e , Og consump-t i o n by g i l l t i s s u e s becomes p r o p o r t i o n a t e l y l e s s because consumption by the whole f i s h may i n c r e a s e by an o r d e r o f magnitude o v e r t h a t a t r e s t . Thus, g i l l oxygen consumption becomes almos t i n s i g n i f i c a n t compared w i t h the measured oxygen uptake d u r i n g e x e r c i s e in vivo. I t i s VgOg v a l u e s which are used i n the F i c k e q u a t i o n t o c a l c u l a t e the c a r d i a c o u t p u t , d e s p i t e t h e s e v a l u e s b e i n g d i f f e r e n t from the amount o f oxygen a c t u a l l y e n t e r i n g the b l o o d . T h i s d i f f e r e n c e can be accou n t e d f o r by g i l l m e t a b o l i s m , as w e l l as ha v i n g some o f the d i f f e r e n c e a c c o u n t e d f o r by oxygen consumption by the s k i n , i n some methods o f measurement ( K i r s c h and Nonnotte, 1977). Because V^Og i s found t o be an o v e r e s t i m a t e o f the a c t u a l oxygen uptake by the b l o o d , the mean c a r d i a c o u t p u t ( Q ) v a l u e d e r i v e d from F i c k i s a 49.2 ± 11.4% (n = 9) o v e r e s t i m a t e o f the a c t u a l Q s e t by the e x t e r -• • nal c a r d i a c pump. The median MQ f o r g i l l t i s s u e s i s 27% o f the r e s t i n g V Og. 2 9 T h i s o b s e r v a t i o n i s i n c o n t r a s t w i t h the f i n d i n g s made by Hughes et al. (1980), who have made s i m i l a r measurements from e e l s . These a u t h o r s found t h a t the a c t u a l Q exceeded tho s e o b t a i n e d from F i c k measurements by 39%, and a t t r i b u t e d the d i f f e r e n c e t o t h e f a c t t h a t n e a r l y 30% o f the c a r d i a c o u t p u t i n e e l s i s n o t i n v o l v e d i n gas exchange a t the g i l l s . Such a s i t u a t i o n i s u n l i k e t h a t found i n t r o u t , where n e a r l y a l l b l o o d must pass through the s e c o n d a r y l a m e l -l a e t o be oxygenated. Johansen and P e t t e r s s o n (1981) recommended t h a t e s t i -mates o f c a r d i a c o u t p u t from F i c k a l s o be c o r r e c t e d , t o a c c o u n t f o r the amount o f b l o o d Og c o n t e n t removed by the g i l l t i s s u e s between venous and a r t e r i a l b l o o d . Such a c o r r e c t i o n o b v i o u s l y i s n o t n e c e s s a r y , s i n c e i n h e r e n t i n the use o f the F i c k e q u a t i o n i s the use o f the a c t u a l a r t e r i a l / v e n o u s oxygen c o n t e n t d i f f e r e n c e . T h i s measured v a l u e a l r e a d y has had whatever Og c o n t e n t needed by m e t a b o l i z i n g g i l l t i s s u e s removed. Needless to s a y , from the e v i -dence p r e s e n t e d , i t i s q u i t e a p p a r e n t t h a t c o n s i d e r a b l e c a r e must by e x e r c i s e d 150 when a p p l y i n g the F i c k e q u a t i o n t o a q u a t i c a n i m a l s . The arguments which I have p r e s e n t e d c l e a r l y demonstrate t h a t the g i l l s o f b l o o d - p e r f u s e d t r o u t , under the c o n d i t i o n s o f my e x p e r i m e n t s , and p r o b a b l y a l s o in vivo, a r e p e r f u s i o n l i m i t e d f o r gas t r a n s f e r . I m p l i c i t i n t h i s c o n t e n t i o n i s the f a c t t h a t haemoglobin oxygen b i n d i n g i s n o t the r a t e l i m i t i n g s t e p i n oxygen uptake, but t h a t mass t r a n s f e r o f oxygen, as w e l l as carbon d i o x i d e e x c r e t i o n a r e p r i m a r i l y dependent upon the r a t e o f p e r f u s i o n o f the g i l l s , i n f i s h . The d a t a from s a l i n e - p e r f u s e d p r e p a r a t i o n s , w h i l e showing t h a t p u l s a t i l i t y o f i n p u t f l o w i n c r e a s e s v e n o l y m p h a t i c c l e a r a n c e o f g i l l and head spaces i n the t r o u t , however, do not p r o v i d e any c l e a r e v i d e n c e f o r o r a g a i n s t t h e s e g i l l s b e i n g d i f f u s i o n l i m i t e d f o r gas t r a n s f e r . The low oxygen uptake r a t e by s a l i n e - p e r f u s e d g i l l s , r e g a r d l e s s o f the t y p e o f p e r f u s i o n regime used, i s l a r g e l y the r e s u l t o f low oxygen c a r r y i n g c a p a c i t y o f the s a l i n e , and the h i g h m e t a b o l i c r a t e o f the g i l l t i s s u e s . The gas exchange d a t a o b t a i n e d from s a l i n e - p e r f u s e d g i l l s i n my e x p e r i m e n t s i n d i c a t e t h a t , under a l l p e r f u s i o n r e g i m e s , n e t COg e x c r e t i o n i s m a i n t a i n e d t o a c e r t a i n d e g r e e , w h i l e n e t oxygen uptake i s n o t always accomplished.. PQQ and C^Q d e c r e a s e a c r o s s the g i l l s as p e r f u s a t e pH r i s e s . S i m i l a r l y , i n the g e n e r a l head c i r c u l a t i o n , what oxygen i s p r e s e n t e d t o the t i s s u e s via the d o r s a l a o r t i c s a l i n e f l o w i s used by the m e t a b o l i z i n g t i s s u e s . T h i s i s i n d i c a t e d by c o n s i s t e n t d e c r e a s e s i n PQ and pH, w i t h a t t e n d a n t i n c r e a s e s i n the PQQ o f the venous r e t u r n p e r f u s a t e i n a l l regimes used. The v e n o l y m p h a t i c v e s s e l s i n the g i l l s o f e i t h e r s a l i n e - p e r f u s e d o r b l o o d - p e r f u s e d p r e p a r a t i o n s c o u l d a c t as f l u i d " s h u n t s " , a l l o w i n g some blo o d to pass through the g i l l s t w i c e , w i t h o u t g o i n g through a s y s t e m i c v a s c u l a r bed. The purpose o f t h i s c o u l d be t o i n c r e a s e the a r t e r i a l oxygen t e n s i o n , o r to lower the a r t e r i a l carbon d i o x i d e t e n s i o n i n the p e r f u s i n g f l u i d , so t h a t the net r e s u l t would be more t r u l y " a r t e r i a l " b l o o d b e i n g 151 d e l i v e r e d t o the s y s t e m i c c i r c u l a t i o n . N e i t h e r o f t h e s e e x p l a n a t i o n s seem p l a u s i b l e , as d o r s a l a o r t i c b l o o d i s c l o s e t o f u l l y s a t u r a t e d w i t h oxygen a f t e r a s i n g l e pass through the g i l l s , as i l l u s t r a t e d i n the b l o o d - p e r f u s e d p r e p a r a t i o n . A d d i t i o n a l l y , the CO^ produced as s a l i n e passes through the a n t e r i o r venous c i r c u i t i n the i s o l a t e d head i s g r e a t e r than t h a t l o s t a c r o s s the g i l l s . T h e r e f o r e , no b e n e f i t to enhanced gas t r a n s f e r i s a p p a r e n t , i f such s h u n t i n g o f b l o o d were t o o c c u r in .vivo. I n c r e a s e d v e n o l y m p h a t i e c l e a r a n c e from g i l l t i s s u e s c o u l d however, d e c r e a s e the d i f f u s i o n d i s t a n c e through which gases must pass t o be p i c k e d up by the b l o o d . T h i s would be o f some s i g n i f i c a n c e d u r i n g e x e r c i s e , when i n p u t p u l s e i s i n c r e a s e d . In t h i s s e c t i o n , I.have shown t h a t haemodynamic a l t e r a t i o n s which mimic tho s e r e c o r d e d d u r i n g e x e r c i s e i n t r o u t , in vivo, can change the p a t t e r n o f f l o w through the b r a n c h i a l c i r c u l a t i o n , and.can a f f e c t v a s c u l a r r e s i s t a n c e t o f l o w . I n c r e a s e s i n c a r d i a c o u t p u t and 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 f f e c t n o t o n l y b r a n c h i a l v a s c u l a r r e s i s t a n c e s , but a l s o r e s u l t i n measureable i n c r e a s e s i n oxygen uptake r a t e s i n b l o o d - p e r f u s e d p r e p a r a t i o n s . And, a l t h o u g h I have shown g i l l s t o be p r i m a r i l y p e r f u s i o n l i m i t e d f o r gas t r a n s -f e r , a s m a l l d i f f u s i o n l i m i t a t i o n s t i l l c o u l d e x i s t i n t h e s e t i s s u e s , g i v e n the complex n a t u r e o f the i n t r a - and e x t r a v a s c u l a r f l u i d spaces w i t h i n t h e s e r e s p i r a t o r y t i s s u e s . Water f l u x a c r o s s the g i l l s i s known t o i n c r e a s e d u r i n g swimming e x e r c i s e i n t r o u t (Wood and R a n d a l l , 1973), and an i n c r e a s e i n t h i s f l u x may i n d i c a t e t h a t the c o n c o m i t a n t i n c r e a s e i n f l u x can i n some way be a f f e c t e d by the n a t u r e o f the f l u i d exchange a c r o s s t h e s e t i s s u e s d u r i n g e x e r c i s e . The n a t u r e o f t h i s f l u i d exchange w i t h i n g i l l t i s s u e s d u r i n g simu-l a t e d e x e r c i s e c o n d i t i o n s was i n v e s t i g a t e d w i t h t h e s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n , and an e t h a n o l l o a d i n g / w a s h o u t t e c h n i q u e . The c h o i c e o f e t h a n o l as a marker o f f l u i d spaces i n g i l l t i s s u e s was made f o r the f o l l o w ^ i n g r e a s o n s . Data from i n d e p e n d e n t in vivo s t u d i e s have shown t h a t s u f f i c i e n t 152 eth a n o l c r o s s e s i n t o the v a s c u l a r space through t h e e p i t h e l i a from the water w i t h i n one pass o f the p e r f u s a t e through t he g i l l s , t o y i e l d measureable EtOH c o n c e n t r a t i o n s . As the s a l i n e p e r f u s a t e i s presumed t o pass through the g i l l s o n l y once, i t i s e s s e n t i a l t h a t s u f f i c i e n t e t h a n o l be taken i n t o t h e v a s c u l a r space d u r i n g t h i s s i n g l e p a s s , and e q u i l i b r a t e t h r o u g h o u t t h e g i l l and head t i s s u e s o v e r the time p e r i o d chosen f o r l o a d i n g i n t h e s e e x p e r i m e n t s . Ethanol c o n c e n t r a t i o n s a re measured e a s i l y , t o a h i g h degree o f a c c u r a c y , even a t v e r y low c o n c e n t r a t i o n s . The e n z y m a t i c a s s a y used t o deter m i n e t h e s e c o n c e n t r a t i o n s o b v i a t e s the use o f r a d i o a c t i v e t r a c e r s , w i t h t h e i r a t t e n d a n t problems. S t r a y - P e d e r s e n and Steen (1975) have d e r i v e d v a l u e s f o r the p e r m e a b i l i t y c o n s t a n t o f e e l g i l l s to w a t e r , e t h a n o l and a v a r i e t y o f o t h e r s u b s t a n c e s . Steen and S t r a y - P e d e r s e n ( 1 9 7 5 ) . a l s o have shown t h a t t h e s e g i l l s have p e r m e a b i l i t i e s to water and e t h a n o l which are al m o s t i d e n t i c a l , and t h e s e p e r m e a b i l i t i e s change i n the same manner i n response t o p e r t u r b a t i o n s . F o r t h e s e r e a s o n s , e t h a n o l was a marker o f e x t r a -and i n t r a v a s c u l a r space i n the g i l l t i s s u e o f the s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n , and was judged s u p e r i o r t o e i t h e r d y e s , o r r a d i o t r a c e r methods. The p e r m e a b i l i t y c o n s t a n t o f e t h a n o l a c r o s s t r o u t g i l l s from the p r e s e n t s t u d y i s c a l c u l a t e d i n a manner s i m i l a r t o t h a t d e s c r i b e d by S t r a y -Pedersen and S t e e n (1975). V a l u e s f o r the p e r m e a b i l i t y c o n s t a n t , k, d u r i n g -8 -1 p u l s a t i l e p e r f u s i o n and n o n - p u l s a t i l e p e r f u s i o n a re 4.12 x 10 cm«sec~ and 5.30 x 10" cm*sec" r e s p e c t i v e l y . The o i l / w a t e r p a r t i t i o n c o e f f i c i e n t o f e t h a n o l i s 0.022. The t h i c k n e s s o f the r e s p i r a t o r y b a r r i e r i s taken t o equal T h e r e f o r e t he d i f f u s i o n c o e f f i c i e n t f o r e t h a n o l ( D ^ tQ H) can be -9 2 -1 c a l c u l a t e d . D u r i n g p u l s a t i l e p e r f u s i o n , D^Q^ = 1.12 x 10 cm -sec , and -9 2 -1 f o r n o n - p u l s a t i l e p e r f u s i o n , D^^Q^ = 1.44 x 10 cm «sec . These v a l u e s a re -9 2 i n c l o s e agreement w i t h t h o s e c a l c u l a t e d f o r t h e e e l g i l l (7.3 x 10 cm • s e c " 1 ) by S t r a y - P e d e r s e n and Steen (1975). I t must be p o i n t e d o u t t h a t t h e s e 153 a u t h o r s used v e r y low p e r f u s i o n r a t e s and p r e s s u r e s , which may a c c o u n t f o r the d i f f e r e n c e " b e t w e e n t h e i r v a l u e f o r D ^ Q ^ and the ones p r e s e n t e d f o r t h e s a l i n e - p e r f u s e d head, n o t w i t h s t a n d i n g any s p e c i e s d i f f e r e n c e s . As e t h a n o l p e r m e a b i l i t y a c r o s s t r o u t g i l l s i n my experiments i s s i m i l a r t o t h a t found f o r e e l g i l l s , e t h a n o l movements p r o b a b l y mimic water movements i n t h e s e t i s s u e s as wel1. A f t e r l o a d i n g the p r e s e n t p r e p a r a t i o n w i t h e t h a n o l f o r 15 min, d o r s a l a o r t i c and a n t e r i o r venous e f f l u e n t e t h a n o l c o n c e n t r a t i o n s a r e n o t d i f f e r e n t . T h i s time p e r i o d t h e r e f o r e i s s u f f i c i e n t t o a l l o w f o r complete e q u i l i b r a t i o n o f g i l l and head t i s s u e s w i t h e t h a n o l . As e t h a n o l e n t e r s c e l l s and a l l o t h e r f l u i d spaces (Goodman and Gilman, 1975), subsequent washout o f t h i s marker i s from a l l the p o s s i b l e compartments i n the p e r f u s e d head p r e p a r a t i o n . Compartmental a n a l y s i s o f Washout Curves G i l l v a s c u l a r ( b l o o d ) volume i n S. gaivdneri has been found t o be a p p r o x i m a t e l y 0.16 m l ^ g " 1 o f g i l l t i s s u e (see T a b l e 4.); G i l l t i s s u e s con-s t i t u t e 3.9% o f the t o t a l body w e i g h t o f t r o u t ( S t e v e n s , 1968a). S i n c e the f i s h used i n t h i s s t u d y had an average w e i g h t o f 289 g, the c a l c u l a t e d g i l l v a s c u l a r volume i s 1.8 ml. D u r i n g n o n - p u l s a t i l e p e r f u s i o n , when i t i s p r o -posed t h a t l i t t l e exchange ( r e f l u x ) o f f l u i d between the f a s t and slow compartments o c c u r s (see l a t e r d i s c u s s i o n ) , the f a s t compartment s h o u l d approximate the v a s c u l a r volume. The volume, d u r i n g n o n - p u l s a t i l e p e r f u s i o n was c a l c u l a t e d t o be 1.37 m l , which i s a r e a s o n a b l e e s t i m a t e , c o n s i d e r i n g . t h a t i n the absence o f p u l s a t i l e p r e s s u r e , fewer than 100% o f the g i l l l a m e l l a e would be p e r f u s e d (see F a r r e l l et al., 1979). The volume o f the f a s t compartment, as measured by EtOH washout a n a l y s i s d u r i n g p u l s a t i l e p e r f u s i o n c o u l d be e x p e c t e d t o be l a r g e r than i n d e p e n d e n t e s t i m a t e s o f v a s c u -l a r volume because o f the e x t e n s i v e r e f l u x o f f l u i d between v a s c u l a r and 154 e x t r a v a s c u l a r f l u i d spaces as p r e s s u r e r i s e s and f a l l s w i t h each p a s s i n g pump c y c l e . The f a s t compartment d u r i n g p u l s a t i l e p e r f u s i o n was c a l c u l a t e d to be 3.12 ml, and t h i s . v o l u m e i s a l m o s t t w i c e t h a t o f i ndependent e s t i m a t e s . T o t a l Amount o f Ethan o l Washed from the G i l l T i s s u e s U s i n g the measured d o r s a l a o r t i c o u t f l o w r a t e o f 1.54 ml'min" 1, and the e q u a t i o n s f o r the e t h a n o l c o n c e n t r a t i o n i n the d o r s a l a o r t i c e f f l u e n t (1 and 2 ) , the t o t a l amount o f e t h a n o l washed from the g i l l s via t h i s a r t e r i a l r o u t e c o u l d be c a l c u l a t e d . D u r i n g p u l s a t i l e p e r f u s i o n t h e r e a r e 5.54 x 10"^ moles o f EtOH, and f o r n o n - p u l s a t i l e t h e r e a r e 7.52 x 10" moles o f EtOH washed from the t i s s u e f l u i d spaces i n t o the v a s c u l a r space. Given t h a t the same g i l l s were used from t h e t i s s u e f l u i d s paces i n t o t h e v a s c u l a r s pace. Given t h a t the same g i l l s were used f o r both p e r f u s i o n r e g i m e s , and t h a t the i n p u t f l o w r a t e s were not d i f f e r e n t , then the 1 4 - f o l d d i f f e r e n c e i n the above e t h a n o l v a l u e s must have r e s u l t e d e i t h e r from d i f f e r e n c e s i n the c o n c e n t r a t i o n o f e t h a n o l i n the g i l l v e s s e l s a t time = 0, o r i n d i f f e r e n t volumes o f d i s t r i b t u i o n and/or c o n c e n t r a t i o n w i t h i n the g i l l t i s s u e s . S i n c e c o n c e n t r a t i o n i n the d o r s a l a o r t a ( f a s t compartment) was n o t d i f f e r e n t w i t h p e r f u s i o n by e i t h e r p u l s a t i l e o r n o n p u l s a t i l e f l o w , i t i s d i f f i c u l t to imagine how v a s c u l a r o r e x t r a v a s c u l a r volumes c o u l d have changed by more than a 2 - f o l d i n c r e a s e , as s u g g e s t e d from the p r e v i o u s c a l c u l a t i o n o f v a s c u l a r , volumes. I t t h e r e f o r e appears t h a t the e t h a n o l c o n c e n t r a t i o n i n the t i s s u e s (slow compartment), i s d i f f e r e n t f o r the two p e r f u s i o n regimes. The proposed model o f the e t h a n o l c o n c e n t r a t i o n g r a d i e n t s a c r o s s the r e s p i r a t o r y b a r r i e r i n the g i l l s i s i l l u s t r a t e d i n F i g . 21. T h i s model i n c l u d e s the two b a r r i e r s to water movement, i n which the b a s a l , o r b l o o d -f a c i n g b a r r i e r i s e i g h t times l e s s permeable than the a p i c a l o r water-f a c i n g b a r r i e r ( T s a i a et al., 1978). In the model, the amount o f e t h a n o l i n the r e s p i r a t o r y b a r r i e r , washed i n t o the p e r f u s a t e , t h e r e f o r e i s one-155 F i g u r e 21. H y p o t i i e t i c a l e t h a n o l c o n c e n t r a t i o n p r o f i l e a c r o s s the r e s p i r a t o r y b a r r i e r o f the g i l l s . As the b a s a l ( b l o o d -f a c i n g ) b a r r i e r i s e i g h t times l e s s permeable than the a p i c a l ( w a t e r - f a c i n g ) b a r r i e r , e t h a n o l i n the t i s s u e s t o the l e f t o f the heavy v e r t i c a l dashed l i n e i s assumed to be l o s t t o the water, w h i l e t h a t on the r i g h t i s assumed t o be removed by the p e r f u s a t e i n the b l o o d v e s s e l s . I f exchange between f a s t ( v a s c u l a r ) and slow ( t i s s u e ) compartments i s r a p i d , and extends a c r o s s the b a s a l b a r r i e r , then the s o l i d l i n e b e t t e r r e p r e s e n t s the e t h a n o l c o n c e n t r a t i o n . When exchange i s slow and extends o n l y t o the b a s a l b a r r i e r , then the d o t t e d l i n e b e t t e r r e p r e s e n t s the e t h a n o l c o n c e n t r a t i o n p r o f i l e . From the f i g u r e , the amount o f e t h a n o l i n the t i s s u e s d u r i n g n o n - p u l s a t i l e p e r f u s i o n i s some 11 times g r e a t e r than d u r i n g p u l s a t i l e p e r f u s i o n . See the t e x t f o r f u r t h e r e x p l a n a t i o n . 156 157 e i g h t h o f the t o t a l e t h a n o l i n the b a r r i e r . W i t h i n the space c l e a r e d to the b l o o d ( p e r f u s a t e ) s i d e , the amount o f e t h a n o l i s some e l e v e n times g r e a t e r d u r i n g n o n - p u l s a t i l e p e r f u s i o n , when t h e r e i s a p p a r e n t l y l e s s exchange between b l o o d and t i s s u e s , as can be e s t i m a t e d by the summation o f the a r e a s under the c u r v e s i n F i g . 21. T h i s h i g h e t h a n o l c o n c e n t r a t i o n i n the slow compartment, combined w i t h the r e l a t i v e i n c r e a s e i n volume d u r i n g n o n p u l s a t i l e p e r f u s i o n (see F i g . 1 8 ) , can be shown t o a c c o u n t f o r the d i f f e r e n c e i n the c a l c u l a t e d , amounts o f e t h a n o l washed from the p r e p a r a t i o n when p e r f u s e d by d i f f e r e n t regimes. I have shown t h a t p u l s a t i l e p e r f u s i o n i n c r e a s e s c l e a r a n c e o f e t h a n o l from s a l i n e - p e r f u s e d t r o u t g i l l s , p r o b a b l y because o f i n c r e a s e d exchange between t i s s u e and p e r f u s a t e . What i s not c l e a r i s the mechanism by which p u l s a t i l e p e r f u s i o n c o u l d i n c r e a s e e t h a n o l , and most p r o b a b l y body f l u i d ( i . e . lymph and water) c l e a r a n c e . I n c r e a s e d c l e a r a n c e o f t h e s e f l u i d s from g i l l t i s s u e i n t e r s t i t i a , e s p e c i a l l y d u r i n g e x e r c i s e , when mean and p u l s e p r e s s u r e s w i t h i n g i l l s a r e i n c r e a s e d , would reduce oedema, and p o s s i b l y the d i f f u s i o n b a r r i e r t o gas t r a n s f e r . In a d e s c r i p t i o n o f water f l u x e s through the w a l l s o f b l o o d v e s s e l s which are s u b j e c t e d t o h i g h mean and p u l s e p r e s s u r e s ( a r t e r i e s , i t appears t h a t the s t r e s s o f the a p p l i e d p r e s s u r e i s s h a r e d by the t i s s u e water and the t i s s u e m a t r i x o r s k e l e t o n ( H a r r i s o n and.Massaro, 1976; Kenyon, 1979). Kenyon (1979) a n a l y s e d the water movements i n a r t e r y w a l l s d u r i n g a p p l i c a t i o n o f p r e s s u r e p u l s e s i n the lumen. His a n a l y s i s a l l o w s p r e d i c t i o n s t o be made about the p r e s s u r e g r a d i e n t s a c r o s s t h e a r t e r y . From t h i s , and w i t h the a p p l i c a t i o n s o f d i f f u s i o n e q u a t i o n s , f l u x e s o f water through a r t e r i a l t i s s u e s can be e s t i m a t e d . Kenyon a l s o c a l c u l a t e s the boundary l a y e r i n the v e s s e l w a l l , 6, through which water must move, and a c r o s s which p r e s s u r e g r a d i e n t s can be e s t a b l i s h e d d u r i n g a p p l i c a t i o n o f p u l s e d p r e s s u r e . The boundary l a y e r t h i c k n e s s i s c a l c u l a t e d by u s i n g D/f , where D = the 158 2 -1 d i f f u s i o n c o e f f i c i e n t (cm 'sec ), and f = the f r e q u e n c y o f p u l s e s ( H z ) . From my p r e v i o u s c a l c u l a t i o n s , the d i f f u s i o n c o e f f i c i e n t f o r e t h a n o l ( o r -9 2 - 1 water) a c r o s s t he whole t r o u t g i l l i s a p p r o x i m a t e l y 1 x 10 cm 'sec 'D^ g -8 2 -1 2 i n t h e s e r e s p i r a t o r y v e s s e l s w i l l be a p p r o x i m a t e l y 8 x 10~ cm " s e c " , i f one assumes t h a t 7^8 o f the b a r r i e r t o water movement i s l o c a t e d . n e a r the b l o o d , and f = 30 bpm (.0.5 Hz) a t r e s t , : in vivo ( K i c e n i u k and J o n e s , 1977). The boundary l a y e r t h i c k n e s s i n the l a m e l l a r v e s s e l s o f the f i s h g i l l then i s equal to a p p r o x i m a t e l y 4,ym a t r e s t . D u r i n g e x e r c i s e , when f = 0.9 Hz ( K i c e n i u k . a n d J o n e s , 1977), the t h i c k n e s s o f the boundary l a y e r i s a p p r o x i -mately 3 urn. These e s t i m a t e s i n d i c a t e t h a t p r e s s u r e g r a d i e n t s and t r a n s -e p i t h e l i a l f l u i d f l o w s , caused b y . p u l s e p r e s s u r e s , c o u l d a c t o v e r a s i g n i -f i c a n t p r o p o r t i o n o f the t o t a l b l o o d / w a t e r r e s p i r a t o r y b a r r i e r in vivo. In vivo b l o o d p r e s s u r e , a p p l i e d t o an a r t e r y w a l l , i s composed o f a p u l s a t i l e P^, and a n o n - p u l s a t i l e o r mean component, P^, such t h a t t he t o t a l p r e s s u r e a p p l i e d , P = P^ + P ' ^ s i n t t t . From Darcy's LaW o f f i l t r a t i o n t hrough porous membranes, the f i l t r a t i o n r a t e caused by.the mean p r e s s u r e a l o n g , W i s equal to.(k/u.) x ( P ^ / h ) , where F^/h = the p r e s s u r e g r a d i e n t a c r o s s the a r t e r y wall., and k/u = the h y d r a u l i c r e s i s t a n c e o f the v e s s e l w a l l (see Vargas et al., 1979). For the p u l s e d p o r t i o n o f the p r e s s u r e , the f i l t r a t i o n r a t e , |W | = (k/u) x P A''R 2'h'S, where P^'R i s the p u l s a t i l e p r e s s u r e g r a d i e n t a c r o s s the boundary l a y e r . By rearrangement o f t h e s e two components o f the f i l t r a t i o n r a t e a c r o s s the i w a l l , i t can be seen t h a t | W | =. W'R'PA'h o r 159 W | = W x R"P A 2'S:.PA In r e s t i n g t r o u t g i l l s , t h e r e a r e e s t i m a t e s o f each o f t h e s e p a r a m e t e r s : -4 R = the r a d i u s o f the l a m e l l a r v a s c u l a r " v e s s e l s " o f 8.36 x 10 cm ( F a r r e l l , 1979) f = 30 beats p er minute o r 0.5 Hz ( K i c e n i u k and J o n e s , 1977) P A = mean p u l s e p r e s s u r e i n the l a m e l l a e = 1/6 p' = 5/6 P' AVA DA = 10.8 cm HgO ( c a l c u l a t e d from K i c e n i u k and J o n e s , 1977) 6 = boundary l a y e r through which f l u i d moves w i t h each h e a r t beat = v T O T f , as d e f i n e d above -4 = 4 x 10 cm, and P A = 43.8 cm H o0 ( K i c e n i u k and J o n e s , 1977). T h e r e f o r e , |. W j' = W x 0.25. That i s , t h e r e i s 1/4 o f the n e t water f l u x a c r o s s t he g i l l e n d o t h e l i u m moving a c r o s s t h i s l a y e r and back d u r i n g each b e a t o f the h e a r t . C o n s i d e r now the e f f e c t s o f chang i n g h e a r t r a t e and mean and p u l s e p r e s s u r e s as d u r i n g e x e r c i s e . A f t e r 50 minutes o f swimming e x e r c i s e a t 80% U .T,- P A = 55.8 cm HgO f = 52 bpm o r 0.86 Hz P A = 16.97 cm H 20 R = 9.197 x 1 0 " 4 cm, and 6 =• 3 x 10"^ cm (from K i c e n i u k and J o n e s , 1977). In t h i s i n s t a n c e , |W j'= W x 0.46. Now, al m o s t h a l f o f the n e t f l u i d f l u x a c r o s s t he g i l l e n d o t h e l i u m i s moving a c r o s s t h i s l a y e r and back w i t h each h e a r t beat. These d a t a p r o v i d e a mechanism f o r the o b s e r v e d i n c r e a s e i n the c l e a r a n c e o f e t h a n o l from g i l l t i s s u e s d u r i n g p u l s a t i l e p e r f u s i o n . 160 The " s l o s h i n g " o f p e r f u s a t e back and f o r t h a c r o s s the l a m e l l a r e n d o t h e l i u m may p l a y an i m p o r t a n t r o l e i n the exchange o f s o l u t e s w i t h i n g i l l t i s s u e s . D i f f u s i o n , i s the p r i n c i p a l . p r o c e s s whereby t r a n s c a p i l l a r y exchange o f s o l u t e s t a k e s . p l a c e . However, Lundgren and M e l l a n d e r (1967) have shown t h a t i n c r e a s e d n n e t c a p i l l a r y f i l t r a t i o n r a t e a l s o i n c r e a s e d the r a t e o f t r a c e r (Iodine,, Rubidium, Potassium) t r a n s f e r a c r o s s c a p i l l a r i e s . A l l . measurements o f p r e s s u r e s i n c a p i l l a r i e s have been shown t o be p u l s a t i l e ( I n t a g l i e t t a et al. , 1970; C o k e l e t * 1380), and a r e n o t s t a t i c . As most measurements o f c a p i l l a r y p e r m e a b i l i t y and f i l t r a t i o n r a t e s have been made d u r i n g n o n - p u l s a t i l e p e r f u s i o n o r p r e s s u r e a p p l i c a t i o n , perhaps an i m p o r t a n t p a r a - c e l l u l a r pathway f o r s o l u t e exchange has been i g n o r e d . I t i s w e l l known.that r e s t i n g t r o u t do n o t p e r f u s e a l l l a m e l l a e a t a l l times ( B o o t h , 1978)y in vivo. Non-perfused l a m e l l a e would, have r e d u c e d , i f not z e r o , p u l s e p r e s s u r e , and the exchange o f s o l u t e s w i t h i n the g i l l t i s s u e s o f n o n - p e r f u s e d l a m e l l a e may be much reduced. T h i s would u l t i m a t e l y r e s u l t i n the c o n s e r v a t i o n o f water and i o n s , depending on the medium, by d e c r e a s i n g exchange e f f i c i e n c y , and s t i l l may p l a y an as y e t u n r e s o l v e d r o l e i n d e t e r m i n i n g the d i f f u s i o n b a r r i e r t o gas t r a n s f e r i n g i l l s , even though g i l l s a r e p r i m a r i l y p e r f u s i o n l i m i t e d . 160a SECTION IV OXYGEN TRANSPORT DURING EXERCISE IN THE RAINBOW TROUT 161 INTRODUCTION: SECTION IV E x e r c i s e i n f i s h can be c a t e g o r i z e d as s u s t a i n e d , p r o l o n g e d o r b u r s t swimming (Hoar and Randall., 1978). F u r t h e r d i s c u s s i o n . however, w i l l c o n c e n t r a t e on those . c a r d i o v a s c u l a r changes i n the rainbow t r o u t , Salmo gaivdneri, a s s o c i a t e d w i t h p r o l o n g e d swimming e x e r c i s e a t 80% o f t h e i r c r i t i c a l v e l o c i t y (80%-U n - t ) . T h i s l e v e l o f s t e a d y - s t a t e e x e r c i s e i s c h a r a c t e r i z e d by a e r o b i c metabolism. When rainbow t r o u t a r e f o r c e d t o swim a t l e v e l s which approach t h e i r c r i t i c a l v e l o c i t y , oxygen uptake i n c r e a s e s by a f a c t o r o f 6 to 12 times the v a l u e found a t . r e s t . The a r t e r i a l - v e n o u s oxygen c o n t e n t d i f f e r e n c e i n c r e a s e s by.--2 to 3 t i m e s , and the c a r d i a c o u t p u t i n c r e a s e s by 3 times the r e s t i n g v a l u e ("Kiceniuk-.-and;Jones,': 1977)..'. V e n t i l a t i o n .volume:and .rate.'also i n c r e a s e , w h i l e oxygen u t i l i z a t i o n from the water f l o w a c r o s s the g i l l s drops from an average 80% a t r e s t , t o as low as 10 - 30% d u r i n g e x e r c i s e ( S h e l t o n , 1970). The oxygen t r a n s p o r t system o f the e x e r c i s i n g t r o u t however, s t i l l has the c a p a b i l i t y o f i n c r e a s i n g i t s d e l i v e r y up t o 12 times t h a t o f the s t a n d a r d m e t a b o l i c r a t e ( B r e t t , 1964, 1965, 1972), through c a r d i o r e s p i r a t o r y a l t e r a t i o n s . At the o n s e t o f e x e r c i s e , t h e r e i s a l a r g e i n c r e a s e i n the mean and.; p u l s e i n p u t b l o o d p r e s s u r e t o the g i l l s . These p r e s s u r e s peak between 10 t o 15 minutes a f t e r the o n s e t o f e x e r c i s e and then d e c l i n e to l e v e l s which s t i l l a r e s e v e r a l mm Hg above the r e s t i n g v a l u e (Stevens and R a n d a l l , 1967a). S i m i l a r but s m a l l e r changes a r e o b s e r v e d i n the d o r s a l a o r t a . A l t h o u g h v e n t r a l a o r t i c p r e s s u r e i s e l e v a t e d d u r i n g e x e r c i s e , and the g i l l v a s c u l a r bed i s e l a s t i c and d i s t e n s i b l e ( F a r r e l l et aZ.,,1979), t h e r e i s l i t t l e change i n g i l l r e s i s t a n c e t o f l o w . There i s , however, a l a r g e r e d u c t i o n i n s y s t e m i c v a s c u l a r r e s i s t a n c e t o f l o w , a s s o c i a t e d w i t h a r i s e i n b l o o d f l o w to the w o r k i n g muscles d u r i n g e x e r c i s e . Haemoglobin c o n t e n t , plasma p r o t e i n 162 l e v e l s ( S t e v e n s , 1968a) and h a e m a t r o c r i t a l l i n c r e a s e d u r i n g e x e r c i s e , p o s s i b l y a s s o c i a t e d w i t h a r e l e a s e o f e r y t h r o c y t e s from the s p l e e n , and a r e d u c t i o n i n plasma volume due t o an i n c r e a s e i n u r i n e f o r m a t i o n (Wood and R a n d a l l , 1 9 7 3 ) . An i n c r e a s e i n haemoglobin l e v e l would augment the oxygen c a r r y i n g c a p a c i t y o f the b l o o d d u r i n g e x e r c i s e . These and o t h e r changes i n the c a r d i o r e s p i r a t o r y systems o f f i s h d u r i n g e x e r c i s e have been p r e s e n t e d p r e v i o u s l y (see T a b l e 2 3 ) , and are reviewed by Jones and R a n d a l l (1978),, There a r e s u r p r i s i n g l y few q u a l i t a t i v e d i f f e r e n c e s between the adjustments made by man and f i s h i n o r d e r t o cope with, t h i s s t r e s s . The i n c r e a s e i n c a r d i a c output, i s a c h i e v e d by an i n c r e a s e i n both h e a r t r a t e and s t r o k e volume, mediated- i n p a r t by a r e l e a s e o f v a g a l .. c h o l i n e r g i c t o n e , and perhaps an i n c r e a s e . i n c i r c u l a t i n g c a t e c h o l a m i n e s and s y m p a t h e t i c t o n e , a s s o c i a t e d w i t h a r i s e i n venous p r e s s u r e and f l o w r e t u r n i n g to the h e a r t . Because o f the s i n g l e - c i r c u i t d e s i g n o f the f i s h c a r d i o v a s c u l a r system, a l l t h i s c a r d i a c o u t p u t must p r o b a b l y pass from the h e a r t d i r e c t l y through the g i l l s ; the s i t e o f gas exchange. The g i l l s t h e r e -f o r e must be a b l e to a d j u s t i n such a manner so as t o e n s u r e adequate b l o o d o x y g e n a t i o n , i n o r d e r t o meet the i n c r e a s e d m e t a b o l i c demands o f the f i s h d u r i n g e x e r c i s e . At r e s t , o n l y about 60% o f the g i l l s e c o ndary l a m e l l a e a r e not p e r f u s e d i n a rainbow t r o u t (Booth, 1978). I t . i s ^ k n o w n i f l a m e l l a r r e c r u i t -ment o c c u r s d u r i n g e x e r c i s e . However, i t seems p r o b a b l e t h a t a l l l a m e l l a e w i l l be p e r f u s e d c o n t i n u o u s l y d u r i n g e x e r c i s e , as a r i s e i n v e n t r a l a o r t i c p r e s s u r e i s known t o cause l a m e l l a r r e c r u i t m e n t in vitro ( F a r r e l l et al., 1979), and i n c r e a s e s . i n b l o o d c a t e c h o l a m i n e s cause l a m e l l a r r e c r u i t m e n t in vivo (Booth, 1979). I t i s not,known i f c i r c u l a t i n g c a t e c h o l a m i n e l e v e l s i n c r e a s e and remain e l e v a t e d above normal d u r i n g p r o l o n g e d e x e r c i s e . However, i t i s known t h a t they do so i f f i s h a r e s t r e s s e d (Nakano and T o m l i n s o n , 1967; Mazeaud et al., 1977; W a h l q v i s t and N i l s s o n , 1977). I n c r e a s e s i n v e n t r a l a o r t i c p r e s s u r e , changes i n b l o o d c a t e c h o l a m i n e s , o r a c t i v i t y i n a d r e n e r g i c 163 T a b l e 23. Summary o f the c a r d i o r e s p i r a t o r y changes a s s o c i a t e d w i t h submaximal, a e r o b i c e x e r c i s e i n man and f i s h . IN MAN IN FISH (from numerous s o u r c e s ) (from numerous s o u r c e s ) H e a r t Rate . : 1 C a r d i a c Output A ^ S t r o k e Volume 1 A r t e r i a l P r e s s u r e S y s t o l i c 1 D i a s t o l i c V e n t r a l A o r t i c V S y s t o l i c • • 1 1 Blood P r e s s u r e D i a s t o l i c 11 D o r s a l A o r t i c S y s t o l i c 1 Blo o d P r e s s u r e D i a s t o l i c 1 A r t e r i a l pH _ i _ A H a e m a t o c r i t 1 1 Haemoglobin . ^ C o n c e n t r a t i o n ^ Oxygen ^ /\ Consumption V e n t i l a t i o n Rate 1 ^ V e n t i l a t i o n Volume A ^ A-V 0 2 D i f f e r e n c e s 1 1 Blood Gas T e n s i o n s ( P a 0 2 ) _A _^ ( P V O 2 ) >| i C i r c u l a t i n g C a t h e c h o l amines 1 1 T o t a l P e r i p h e r i a l R e s i s t a n c e t o Blood vj \J Flow Blood Flow D i s t r i b u t i o n t o Working Muscles ^ 1 To S p l a n c h n i c Region j .To Gas Exchange Organ /\ /\ To H e a r t and B r a i n _A ? 1 6 4 f i b r e s i n n e r v a t i n g the g i l l v a s c u l a t u r e (Gannon, 1972) t h e r e f o r e may cause l a m e l l a r r e c r u i t m e n t i n e x e r c i s i n g t r o u t . The r i s e i n v e n t r a l a o r t i c p r e s s u r e p r o b a b l y r e s u l t s i n a r i s e i n l a m e l l a r b l o o d p r e s s u r e as w e l l , and t h i s may be enhanced.by d i l a t i o n .-of the a f f e r e n t l a m e l l a r a r t e r i o l e s due to p o s s i b l e e f f e c t s o f i n c r e a s e d a d r e n e r g i c a c t i v i t y o r c i r c u l a t i n g c a t e - . cholamines i n d i l a t i n g the smooth muscle s p h i n c t e r around the a f f e r e n t l a m e l l a r a r t e r i o l e s ( F a r r e l l : , T98T). An i n c r e a s e i n l a m e l l a r p r e s s u r e a l s o causes a more even, d i s t r i b u t i o n o f b l o o d . i n t h e s e condary l a m e l l a e , and an i n c r e a s e d p e r f u s i o n o f t h e exposed a r e a o f the l a m e l l a e ( F a r r e l l et al.t 1980). In a d d i t i o n , t h e r e may be some t h i n n i n g o f the b l o o d / w a t e r b a r r i e r as a r e s u l t o f e x p a n s i o n o f the l a m e l l a r b l o o d s h e e t , a s s o c i a t e d w i t h a r i s e i n l a m e l l a r pressure.. There i s no d i r e c t e v i d e n c e f o r t h e s e changes i n e x e r c i s i n g f i s h , but i f they do o c c u r , then the d i f f u s i o n c a p a c i t y o f the g i l l s f o r oxygen s h o u l d i n c r e a s e d u r i n g e x e r c i s e . The c a r b o n monoxide d i f f u s i n g c a p a c i t y o f the c a t f i s h g i l l (ictalurus nebulosus) ©n the o t h e r hand, has been measured, and has been shown t o i n c r e a s e i n h y p o x i a , compared w i t h t h a t measured i n normoxia ( F i s h e r et al., 1969). The mass t r a n s f e r o f carbon monoxide (M^Q) .across the g i l l s o f I. nebulosus a l s o i s found to be p r o p o r t i o n a l t o t h e p a r t i a l , p r e s s u r e o f carbon monoxide i n the water. Based on t h e s e , and o t h e r o b s e r v a t i o n s , i t was c o n c l u d e d t h a t oxygen uptake, l i k e M C Q , i n f i s h g i l l s i s d i f f u s i o n l i m i t e d (Hughes, 1972, 1980). The t h i c k e r e p i t h e l i u m o f the g i l l , compared t o t h a t o f l u n g , and the boundary l a y e r o f u n s t i r r e d water next t o the r e s p i r a t o r y s u r f a c e o f f i s h impose a d d i t i o n a l , d i f f u s i o n l i m i t a t i o n s ( H i l l s and Hughes, 1970). Thus the d i f f u s i o n d i s t a n c e between the medium and b l o o d i s even l a r g e r than s i m p l y the t h i c k n e s s o f the e p i t h e l i u m i n f i s h g i l l s . Indeed i t i s o b v i o u s from the l i t e r a t u r e t h a t b l o o d i n f i s h e s n e ver a c h i e v e s e q u i l i b r a t i o n w i t h the p a r t i a l p r e s s u r e o f oxygen i n the water ( R a n d a l l , 1970). There a l s o i s a l a r g e oxygen c o n t e n t d i f f e r e n c e between a i r (209.5 m l ' L - 1 " S T P ) and water (6.34 ml'L A f r e s h water 165 a t 20 C). T h e r e f o r e , f i s h must v e n t i l a t e a t l e a s t 30 times more water minute volume t o o b t a i n an e q u i v a l e n t amount o f oxygen as a i r b r e a t h i n g a n i m a l s . But, i t has been shown t h a t d u r i n g a q u a t i c h y p o x i a , when the h e a r t r a t e d e c l i n e s , but the c a r d i a c o u t p u t remains c o n s t a n t and v e n t i l a t i o n ; i n c r e a s e s , gas t r a n s f e r a c r o s s the g i l l s o f t r o u t i s " f a c i l i t a t e d " ( R a n d a l l . , et al., 1967,; H o l e t o n and Randall., 1967a,b). T h e r e f o r e , a l t h o u g h t h e r e are d i f f e r e n c e s i n h e a r t r a t e and c a r d i a c o u t p u t between e x e r c i s e and h y p o x i c exposure i n t r o u t , both s t r o k e volume and v e n t r a l a r o t i c p r e s s u r e s i n c r e a s e , and this,may l e a d t o a r i s e i n t h e g i l l oxygen d i f f u s i n g c a p a c i t y , shown i n the h y p o x i c c a t f i s h , and supposed i n e x e r c i s i n g rainbow t r o u t . . D o r s a l a o r t i c b l o o d i n normoxic f i s h however, i s a l w a y s - g r e a t e r than 95% oxygen s a t u r a t e d , d e s p i t e the f a c t t h a t a r t e r i a l oxygen p a r t i a l p r e s s u r e i s never equal to t h a t o f t h e s u r r o u n d i n g water. A d d i t i o n a l l y , d u r i n g swimming e x e r c i s e , when the c a r d i a c o u t p u t and v e n t i l a t i o n a r e i n c r e a s e d , the measured oxygen uptake i s augmented, not o n l y by presumed d e c r e a s e s i n the d i f f u s i o n b a r r i e r t o gases a c r o s s the g i l l s , but a l s o , t o a l e s s e r e x t e n t , an i n c r e a s e d p e r f u s i o n o f a g r e a t e r r e s p i r a t o r y s u r f a c e a r e a , brought about by p o s s i b l e l a m e l l a r r e c r u i t m e n t . I t i s s u g g e s t e d t h a t c a r d i o v a s c u l a r adjustments during, t h i s imposed s t r e s s a r e f o r the purpose o f d e c r e a s i n g the d i f f u s i o n b a r r i e r , such t h a t oxygen uptake can be m a i n t a i n e d a t s u f f i c i e n t l e v e l s t o meet the m e t a b o l i c r e q u i r e m e n t s o f the f i s h . B l o o d t r a n s i t times through the s e c o n d a r y l a m e l l a e a r e between 1 and 3 seconds i n f i s h (Hughes et al. , 1981; R a n d a l l , 1981). From l a m e l l a r volumes, 51 d e r i v e d from p l a s t i c c a s t s , o f the g i l l s , and my G r - l a b e l l e d r e d c e l l b l o o d volume,measurements, and u s i n g the c a r d i a c o u t p u t v a l u e s from K i c e n i u k and Jones (1977), c o r r e c t e d f o r a median 27% o f r e s t i n g oxygen uptake b e i n g u t i l i z e d by the g i l l t i s s u e s and n o t e n t e r i n g the b l o o d (see D i s c u s s i o n , S e c t i o n I I I ) , then the c a l c u l a t e d b l o o d t r a n s i t time i n r e s t i n g t r o u t i s 3 seconds, i f a l l l a m e l l a e a r e p e r f u s e d . I f however, o n l y 2/3 o f t h e s e 1 6 6 l a m e l l a e are p e r f u s e d ; a more p r o b a b l e s i t u a t i o n ( f o r in vivo d a t a , see Booth, 1978; in vitro d a t a , see F a r r e l l et dl.-, 1979), then t h i s time i s reduced t o 2 seconds. I t w i l l be reduced even f u r t h e r , to one second d u r i n g e x e r c i s e , when i t i s assumed t h a t a l l the secondary l a m e l l a e a r e b e i n g p e r f u s e d . . S i n c e i t appears t h a t . t h e s t r o k e volume o f the h e a r t p r o b a b l y n e v e r exceeds g i l l l a m e l l a r , b l o o d volume, even d u r i n g maximal e x e r c i s e , a complete c a r d i a c c y c l e w i l l o c c u r w i t h i n the time f o r b l o o d to f l o w through the l a m e l l a e , even though b l o o d v e l o c i t y changes d u r i n g each h e a r t beat. As. l o n g as a l l o f the c a r d i a c o u t p u t p a s s e s through t h e s e secondary l a m e l l a e , and the c a r d i a c o u t p u t and volume o f p e r f u s e d l a m e l l a e a r e known, t r a n s i t t i m e s can be a c c u r a t e l y d e t e r m i n e d . However, the f a t e o f t h i s c a r d i a c o u t p u t d i s t r i b t u i o n once out o f the g i l l s s t i l l i s a matter, f o r s p e c u l a t i o n , s i n c e n o t al1 c a r d i a c o u t p u t goes d i r e c t l y to the d o r s a l , a o r t a . For /example, the g e n e r a l head c i r c u l a t i o n , the h y p o b r a n c h i a l m u s c l e s , and "the h e a r t must a l s o be s u p p l i e d from e f f e r e n t b r a n c h i a l , a r t e r i e s . A r t e r i a l oxygen t e n s i o n s n o n e t h e l e s s remain w i t h i n t h e same range o v e r a wide range o f p r o l o n g e d swimmings and t h e b l o o d remains more than 95% oxygen s a t u r a t e d . Even a t t h e most r a p i d g i l l t r a n s i t t i m e s , the d i f f u s i o n c a p a c i t y o f t h e g i l l s . t h e r e f o r e must be adequate t o a c h i e v e haemoglobin oxygen s a t u r a t i o n . That a r t e r i a l b l o o d n e v e r e q u i l i b r a t e s w i t h i n f l o w i n g water P n has b e e n . p o i n t e d out p r e v i o u s l y . The reasons f o r t h i s s t i l l a r e u 2 not c l e a r , but must i n p a r t be due t o a l a r g e d i f f u s i o n b a r r i e r , c o n s i s t i n g o f .the u n s t i r r e d water l a y e r , the g i l l e p i t h e l i u m , the p i l l a r c e l l and the b l o o d space. T h i s e p i t h e l i u m i s much t h i c k e r than t h a t i n the l u n g presumably because the c e l l l a y e r i s much more a c t i v e ; b e i n g i n v o l v e d i n i o n and water movements as w e l l as gas t r a n s f e r . . 27% o f the oxygen l e a v i n g the water a t r e s t i n t r o u t i s u t i l i z e d b y t h e g i l l e p i t h e l i u m and o n l y 73% e n t e r s the b l o o d to be d i s t r i b u t e d t o the s y s t e m i c c i r c u l a t i o n . The u t i l i z a t i o n o f 167 oxygen by the g i l l t i s s u e s a l s o w i l l i n c r e a s e the a p p a r e n t d i f f u s i o n b a r r i e r between water and b l o o d . Why some f i s h have a P 0, o f 120 mm Hg, a c. w h i l e o t h e r s have 95 t o 100 i s p r o b a b l y a r e f l e c t i o n o f the s i z e o f the d i f f u s i o n b a r r i e r and the r a t e o f oxygen consumption by the g i l l t i s s u e s . S i n c e h y p e r c a p n i a r e s u l t s i n a marked i n c r e a s e i n P ^ i n the d o g f i s h ( R a n d a l l al., 1976), t h i s c o u l d i n d i c a t e a r e d u c t i o n i n g i l l m e t abolism due t o the p r e v a i l i n g a c i d i c c o n d i t i o n s a s s o c i a t e d w i t h h y p e r c a p n i a . RESULTS AND DISCUSSION IV In the s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n s , an i n c r e a s e . i n b l o o d f l o w through the g i l l s was 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 oxygen uptake r a t e , but not w i t h any change i n a r t e r i a l oxygen t e n s i o n . These g i l l s t h e r e f o r e appear t o be p e r f u s i o n l i m i t e d f o r oxygen uptake. The d i f f u s i o n c a p a c i t y o f the g i l l s f o r oxygen i s p r o b a b l y reduced i h i . r e s t -i n g f i s h t o a m e l i o r a t e water and i o n t r a n s f e r , as d i s c u s s e d i n S e c t i o n I I I . D u r i n g e x e r c i s e , any p o s s i b l e i n c r e a s e i n t h e d i f f u s i n g c a p a c i t y a s s o c i a t e d w i t h a r i s e i n c a r d i a c o u t p u t , and a r e d u c t i o n i n venous oxygen c o n t e n t p e r m i t s a marked r i s e i n oxygen uptake a c r o s s the g i l l s . An i n c r e a s e i n d i f f u s i n g c a p a c i t y t h e r e f o r e may be o n l y o f s i g n i f i c a n c e a t h i g h l e v e l s o f e x e r c i s e , because m a n i p u l a t i o n s o f i n p u t p r e s s u r e i n r e s t i n g , b l o o d - p e r f u s e d t r o u t n e i t h e r i n c r e a s e s i n mean nor p u l s e p r e s s u r e s had any e f f e c t on oxygen uptake r a t e o r P 0 o . a L The i n p u t b l o o d to the s y s t e m i c c i r c u l a t i o n t h e r e f o r e remains n e a r l y oxygen s a t u r a t e d o v e r a wide range o f p r o l o n g e d swimming speeds, and g e n e r a l l y shows s m a l l i n c r e a s e s i n mean and p u l s e p r e s s u r e d u r i n g e x e r c i s e . T h e r e i s however, a presumed marked i n c r e a s e i n b l o o d f l o w t o r e d muscle d u r i n g e x e r c i s e , a s m a l l e r i n c r e a s e i n k i d n e y b l o o d f l o w , and f l o w t o the "white" o r mosaic muscle a c t u a l l y i n c r e a s e s d u r i n g swimming i n the rainbow t r o u t . T h i s w h i t e muscle mass c o n s t i t u t e s 66% o f the t o t a l body we i g h t (B.W.) 168 ( S t e v e n s , 1968a), c o n s i s t i n g o f 64.5% B.W. w h i t e f i b r e s and 1.5% B.W. r e d f i b r e s (Webb, 1975). T h e r e f o r e , the t r o u t . h a s 2.5% B.W. red f i b r e s i n the l a t e r a l r e d muscle and 1.5% B.W. embedded i n the w h i t e muscle. I f one assumes the i n c r e a s e i n b l o o d f l o w t o be the same t o a l l r e d muscle f i b r e s d u r i n g e x e r c i s e , then more than a l l o f the f l o w i n c r e a s e measured w i t h i n the mosaic muscle i s d i r e c t e d towards the r e d f i b r e s . , and f l o w t o t h e w h i t e f i b r e s may be s e v e r e l y c u r t a i l e d a t . t h i s l e v e l o f a c t i v i t y (80% U c r i t ) . That the f a c t o r s l e a d i n g t o b l o o d r e d i s t r i b u t i o n d u r i n g s t e a d y ^ s t a t e e x e r c i s e are q u i t e complex and as y e t , u n r e s o l v e d , has been p o i n t e d out e a r l i e r ( S e c t i o n I ) . In most o t h e r s t u d i e s , the r i s e i n d o r s a l a o r t i c p r e s s u r e i s s m a l l (Jones and R a n d a l l , 1978). The e l a s t i c i t y o f the s y s t e m i c c i r c u i t would.have t o be v e r y high t o respond t o such s m a l l changes i n i n p u t p r e s s u r e i n o r d e r t o a l l o w s y s t e m i c r e s i s t a n c e t o f a l l i n p r o p o r t i o n to the i n c r e a s e i n f l o w . However, I-o b s e r v e d l a r g e r changes i n d o r s a l a o r t i c p r e s s u r e than e i t h e r K i c e n i u k and Jones (1977) o r Stevens and R a n d a l l ( 1 9 6 7 a ) , on the same s p e c i e s . There were d i f f e r e n c e s i n t h e e x e r c i s e regime and the e x t e n t o f c a n n u l . a t i o n , and t h e s e may e x p l a i n some o f the o b s e r v e d d i f f e r e n c e s . A l t h o u g h the l a r g e r i n c r e a s e i n s y s t e m i c i n p u t p r e s s u r e o b s e r v e d i n my e x p e r i m e n t s c e r t a i n l y w i l l p l a y some r o l e i n i n c r e a s i n g s y s t e m i c b l o o d flow., i t p r o b a b l y does n o t account f o r . a l l t h e i n c r e a s e , n o r can i t a c c o u n t f o r the o b s e r v e d , r e d i s t r i b t u i o n s among o t h e r t i s s u e s . The d e c r e a s e i n l i v e r , stomach and s p l e e n b l o o d f l o w can be a c c o u n t -ed f o r by c o n s t r i c t i o n o f the c o e l i a c o m e s e n t e r i c a r t e r y (Hoimgen a n d , N i l s s o n , 1974; Johansson, 1979). An a d r e n e r g i c a l . l y i n n e r v a t e d muscle s p h i n c t e r a t the base o f . t h i s a r t e r y has been shown t o e x i s t (Gannon, 1972). C o n s t r i c t i o n o f the s p h i n c t e r t h e r e f o r e would a c c o u n t f o r the f l o w changes o b s e r v e d d u r i n g e x e r c i s e . C o n t r a c t i o n o f the m u s c u l a r s p l e e n i t s e l f , which a l s o i s under a d r e n e r g i c c o n t r o l ( N i l s s o n and Grove, 1974; Holmgren and N i l s s o n , 1975) 169 would account f o r the n o t e d r e d u c t i o n i n s p l e n i c volume. T h i s s p l e n i c c o n t r a c t i o n t h e r e f o r e i s a s m a l l c o n t r i b u t i n g cause t o the o b s e r v e d h a e m o c o n t r a e t i o n d u r i n g e x e r c i s e , by a d d i n g r e d b l o o d c e l l s t o the g e n e r a l c i r c u l a t i o n . I t has been d i s c u s s e d p r e v i o u s l y t h a t i t i s d i f f i c u l t t o a s c r i b e causes t o the i n c r e a s e i n muscle b l o o d f l o w d u r i n g e x e r c i s e . The t r u n k v a s c u l a t u r e o f t h e . t r o u t shows powerful, a-adrenergic c o n s t r i c t o r responses to i n f u s e d c a t e c h o l a m i n e s (Wood et al., 1978). The muscle v a s c u l a t u r e l a c k s any d i r e c t , a d r e n e r g i c nerve i n n e r v a t i o n . T h e r e f o r e , i f t h e s e r e c e p t o r s are l o c a t e d w i t h i n muscle b l o o d v e s s e l s , they must respond to c i r c u l a t i n g c a t e c h o l a m i n e s . . These a r e known t o . i n c r e a s e i n s t r e s s e d f i s h , , and may i n c r e a s e i n . e x e r c i s i n g f i s h , t h e r e b y c a u s i n g s y s t e m i c v a s o c o n s t r i c -t i o n . What i s o b s e r v e d i n i n t a c t f i s h , however, i s a v e r y s i g n i f c a n t vaso-d i l a t i o n a s s o c i a t e d w i t h ^ i n c r e a s e d b l o o d f l o w to r e d muscle f i b r e s . Davie (1981) h a s . i n v e s t i g a t e d the r e s p o n s e s o f a p e r f u s e d , i s o l a t e d e e l t a i l p r e p a r a t i o n to c a t e c h o l a m i n e s and r e p o r t e d the e x i s t e n c e o f both a- and a d r e n o c e p t o r s . Davie s u g g e s t e d t h a t the B g - r e c e p t o r s , which caused vaso-d i l a t i o n , might be l o c a t e d i n r e d muscle and the o t h e r s c o u l d be a s s o c i a t e d w i t h w h i t e f i b r e b l o o d v e s s e l s , A d r e n e r g i c n e r v e s and/or c i r c u l a t i n g c a t e c h o l a m i n e s t h e r e f o r e may be i n v o l v e d i n c o e l i a c o m e s e n t e r i c and s p l e n i c c o n s t r i c t i o n , p o s s i b l e v a s o c o n s t r i c t i o n i n white muscle v a s c u l a t u r e , and v a s o d i l a t i o n i n . r e d muscle. However, i t i s u n l i k e l y t h a t t h i s i s a complete d e s c r i p t i o n o f the c o n t r o l mechanisms i n v o l v e d , s i n c e i n f u s i o n o f c a t e -cholamines do n o t mimic the s t e a d y - s t a t e s y s t e m i c response seen i n i n t a c t t r o u t d u r i n g e x e r c i s e . A c t i v e hyperaemia p r o b a b l y i s the major cause o f the marked r e d u c t i o n i n v a s c u l a r r e s i s t a n c e t o f l o w i n r e d muscle. Both muscle c o n t r a c t i o n i t s e l f , and movements o f the t a i l a l s o may augment muscle b l o o d f l o w (see R a n d a l l 1 9 6 8 , f o r d e t a i l s o f mechanisms) d u r i n g swimming e x e r c i s e i n t r o u t . 170 E x e r c i s e a t 8 0 1 U, i n t r o u t i s l a r g e l y a e r o b i c and i s a s s o c i a t e d w i t h a l a r g e i n c r e a s e i n oxygen uptake, most o f i t presumably b e i n g u t i l i z e d by the working muscles. The oxygen i s d e l i v e r e d t o t h e s e working muscles ( p r i m a r i l y the r e d muscle f i b r e s ) by the i n c r e a s e d b l o o d f l o w . The c a l c u -l a t i o n s p r e s e n t e d i n T a b l e s 24, 25 ;and 26, based on the f l o w d i s t r i b u t i o n s r e p o r t e d p r e v i o u s l y i n t h i s t h e s i s , i n d i c a t e t h a t the i n c r e a s e i n f l o w i s adequate.to s u p p l y the i n c r e a s e d oxygen r e q u i r e m e n t s o f the muscles. 93% o f the i n c r e a s e i n oxygen taken up by t h e f i s h a t 80% U c r i - t i s d e l i v e r e d to to the muscle, and r e d muscle oxygen u t i l i z a t i o n i n c r e a s e s by a f a c t o r o f 12 a t t h i s l e v e l of'swimming a c t i v i t y . The c a l c u l a t i o n s , assuming r e s t i n g r a t e s o f oxygen e x t r a c t i o n from non-muscle t i s s u e s d u r i n g e x e r c i s e , i n d i c a t e 96% oxygen u t i l i z a t i o n from b l o o d f l o w i n g through r e d muscle.„ T h i s would mean t h a t r e d muscle P n must be reduced t o o n l y 1 o r 2 mm Hg d u r i n g u 2 e x e r c i s e . The r e s u l t i n g c a l c u l a t e d mixed venous oxygen c o n t e n t d u r i n g e x e r c i s e shows v e r y c l o s e a g r e e m e n t . w i t h . r e c o r d e d v a l u e s a t the same swimming speed, on the same s p e c i e s ( T a b l e 25). I have demonstrated that: the c i r c u l a t o r y system o f the rainbow t r o u t , Salmo gairdnevi, i s e f f i c i e n t i n i t s a b i l i t y t o d i s t r i b u t e the n e c e s s a r y oxygen t o the t i s s u e s d u r i n g p r o l o n g e d , a e r o b i c swimming e x e r c i s e . The numerous c a r d i o r e s p i r a t o r y a d j u s t m e n t s , i n v o l v i n g both s y s t e m i c b l o o d f l o w r e d i s t r i b t u i o n . , and changes w i t h i n the b r a n c h i a l v a s c u l a t u r e d u r i n g e x e r c i s e a c c o u n t f o r t h i s a n i m a l ' s a b i l i t y t o m a i n t a i n swimming a c t i v i t y i n the f a c e o f i n c r e a s e d oxygen demands by t h e working muscle. 171 T a b l e 24. C a l c u l a t i o n o f muscle and s y s t e m i c b l o o d f l o w ( m l ' m i n ' . ^ k g - 1 ) d u r i n g r e s t and e x e r c i s e i n rainbow t r o u t . REST EXERCISE (80% U ..) x c r i t ' Red + White muscle 5.49 10.15 White m u s c l e 9 4.81 0.42 Red m u s c l e 3 ( i n w h i t e muscle) 0.68 9.73 Red Muscle ( l a t e r a l ) 1.14 16.21 Rest o f Body 6.22 11.79 T o t a l b l o o d f l o w b ( c a r d i a c o u t p u t ) 12.85 38.15 Assuming t h a t w h i t e p l u s r e d muscle (66% o f body weight - Stevens (1968a) c o n s i s t s o f 64.5% B.W. white muscle and 1.5% B.W: r e d muscle (Webb, 1975), and t h a t r e d muscle f l o w i n t h i s t i s s u e i s the same as c a l c u l a t e d f o r l a t e r a l r e d muscle. ^Data from K i c e n i u k and Jones (1977), c o r r e c t e d f o r 27% o f r e s t i n g oxygen uptake by g i l l s when c a l c u l a t i n g c a r d i a c o u t p u t u s i n g t he F i c k p r i n c i p l e . 172 T a b l e 25. Oxygen uptake and d i s t r i b u t i o n t o the s y s t e m i c c i r c u l a t i o n o f r e s t i n g and e x e r c i s i n g rainbow t r o u t . REST EXERCISE (.80% U c r i t ) T o t a l 0 o Uptake' (ml«nrin - 1*kg"*) 0.409 2.969 A r t e r i a l 09 c o n t e n t ( v o l %) c Venous 09 content* 3 ( v o l %•) c T i s s u e 0 o e x t r a c t i o n (%) 2 Op uptake by r e s t o f body d u r i n g e x e r c i s e , assuming 31.7% 0 2 e x t r a c t i o n from b l o o d Og uptake by muscles o n l y T o t a l Og d e l i v e r e d by b l o o d t o muscies d u r i n g e x e r c i s e Oxygen e x t r a c t i o n from b l o o d (%) Mean venous oxygen c o n t e n t d u r i n g e x e r c i s e ( c a l c u l a t e d . ) 10.4 7.1 31.7 10.2 2.9 71.6 = 0.381 m l • m i n " 1 . k g - 1 = 2.588 m l » m i n ' 1 - k g " 1 = 2.689 ml-min" 1'-kg' 1 = 96.2 = 2.42 Mean v a l u e ( v o l . %) 2.9 ± 1.47 a d a t a from K i c e n i u k and Jones (1977) c o r r e c t e d f o r 27% 0g uptake by g i l l s i n c a l c u l a t i n g 0g uptake by b l o o d , ^Data from K i c e n i u k and Jones (1977). 173 T a b l e 26. I n c r e a s e i n oxygen uptake by the whole f i s h and by r e d muscle a t r e s t and d u r i n g e x e r c i s e a t 80% U c r i t ' REST EXERCISE Og uptake ( m l ' m i n " 1 » k g " 1 ) Og uptake by muscle ( m l « m i n " 1 * k g " 1 ) I n c r e a s e i n 0 ? uptake by muscle ( f a c t o r ) I n c r e a s e i n Og uptake by muscle ( m l - m i n " x , k g " 1 ) I n c r e a s e i n 0 ? uptake by whole f i s h a t 80% U ( m l * m i n " 1 , k g " 1 ) c r i t %0p i n c r e a s e g o i n g t o working muscle a t 80% U -t 0.409 0.219 c = x 12 = 2.369 = 2.56 = 93% 2.969 2.588 aAssumes an e x t r a c t i o n r a t e o f 31.7% i . e . the same as t h a t f o r whole animal a t r e s t . b D a t a d e r i v e d from K i c e n i u k and Jones (1977). 173a LITERATURE CITED 174 LITERATURE CITED A h l q u i s t , R.P. 1948. A stu d y o f the a d r e n o t r o p i c r e c e p t o r s . Am. J . P h y s i o l . 153: 586 - 600. A l b e r s , C. 1970. A c i d - b a s e b a l a n c e . 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P h a r m a c o l o g i c a l p r o p e r t i e s o f the a d r e n e r g i c r e c e p t o r s r e g u l a t i n g s y s t e m i c v a s c u l a r r e s i s t a n c e i n the rainbow t r o u t . J . Comp. P h y s i o l . 107: 211 - 228. Wood, C M . 1977. C h o l i n e r g i c mechanisms and the response t o ATP i n the s y s t e m i c v a s c u l a t u r e o f the rainbow t r o u t . J . Comp. P h y s i o l . 122: 325 - 347. Wood, C M . and D.J. R a n d a l l . 1973. The i n f l u e n c e o f swimming a c t i v i t y on water b a l a n c e i n the rainbow t r o u t {Salmo gairdneri). J . Comp. P h y s i o l . 82: 257 - 276. Wood, C M . and G. S h e l t o n . 1975. P h y s i c a l and a d r e n e r g i c f a c t o r s a f f e c t i n g s y s t e m i c v a s c u l a r r e s i s t a n c e i n the rainbow t r o u t : A comparison w i t h b r a n c h i a l v a s c u l a r r e s i s t a n c e . J . Exp. B i o l . 63: 505 - 523. Wood, C M . and G. S h e l t o n . 1980a. The r e f l e x c o n t r o l o f h e a r t r a t e and c a r d i a c o u t p u t i n the rainbow t r o u t : I n t e r a c t i v e i n f l u e n c e s o f hy p o x i a , haemorrhage, and s y s t e m i c vasomotor tone. J . Exp. B i o l . 87: 271 - 284. Wood, C M . and G. S h e l t o n . 1980b. C a r d i o v a s c u l a r dynamics and a d r e n e r g i c r e s p o n s e s o f rainbow t r o u t in vivo. J . Exp. B i o l . 87: 247 - 270. Wood, CM., B.R. McMahon and D.G. McDonald. 1978. Oxygen exchange and v a s c u l a r r e s i s t a n c e i n the t o t a l l y p e r f u s e d rainbow t r o u t . Am. J . P h y s i o l . 234: 201 - 208. Wood, CM., B.R. McMahon and D.G. McDonald. 1979. R e s p i r a t o r y , v e n t i l a t o r y and c a r d i o v a s c u l a r r e s p o n s e s t o e x p e r i m e n t a l anaemia i n the s t a r r y f l o u n d e r , Platiehthys stellatus. J . Exp. B i o l . 82: 139 - 162. Wood, CM., D.G. McDonald and B.R. McMahon. 1981. The i n f l u e n c e o f e x p e r i -mental anaemia on b l o o d a c i d - b a s e r e g u l a t i o n in vivo and in vitro i n t h e s t a r r y f l o u n d e r {Platiehthys stellatus) and t h e rainbow t r o u t , ( i n p r e s s ) . W r i g h t , D.L. and R.R. Sonn e n s c h e i n . 1965. R e l a t i o n s among a c t i v i t y , b l o o d f l o w , and v a s c u l a r s t a t e i n s k e l e t a l muscle. Am. J . P h y s i o l . 208: 782 - 789. 187a APPENDIX A EFFECTS OF CORONARY ARTERY ABLATION ON EXERCISE PERFORMANCE IN RAINBOW TROUT 188 INTRODUCTION: APPENDIX A F i s h h e a r t s commonly a r e r e f e r r e d t o as bei n g venous h e a r t s , s i n c e they o n l y pump venous b l o o d around a " s i m p l e " , s i n g l e c i r c u l a t i o n ; the v a s c u l a r beds b e i n g a r r a n g e d i n s e r i e s w i t h each o t h e r . The spongy, t r a -b e c u l a t e d i n n e r myocardium i s s u p p l i e d by l a c u n a r venous b l o o d , w h i l e the compact c o r t i c a l myocardium i s r i c h l y s u p p l i e d w i t h c o r o n a r y v e s s e l s ( O s t a d a l et a l . 3 1970). An e s t i m a t e d 39% o f the v e n t r i c u l a r mass i s comprised o f t h i s compact c o r t i c a l l a y e r i n Salmo gairdneri ( S a n t e r and Walker, 1980). The co r o n a r y a r t e r i a l b l o o d s u p p l y t o the c o r t i c a l l a y e r i s d e r i v e d from the second e f f e r e n t h y p o b r a n c h i a l a r t e r i e s and the d o r s a l a o r t a , and i s comprised o f two branches (Watson and Cobb, 1979). One branch s u p p l i e s the p e r i c a r d i u m and the whole a d v e n t i t i a o f the b u l b u s , w h i l e the o t h e r runs a l o n g the v e n t r a l s u r f a c e o f the v e n t r a l a o r t a ( t h e prominant c o r o n a r y a r t e r y seen i n t r o u t ) . Both run t o g e t h e r t o s u p p l y a r t e r i a l b l o o d t o the v e n t r i c l e . The h e a r t i s an a e r o b i c t i s s u e i n most v e r t e b r a t e s , a l t h o u g h t h o s e s p e c i e s which appear l e s s a c t i v e than t r o u t , and have h i g h e r t o l e r a n c e s t o hy p o x i a , can m a i n t a i n m y o c a r d i a l c o n t r a c t i l i t y l o n g e r in vitro, a t low l e v e l s o f oxygen ( G e s s e r , 1977). On the b a s i s o f th e weight o f th e v e n t r i c l e , and assuming t h i s t i s s u e t o have an 0^ demand s i m i l a r t o t h a t o f s t i m u l a t e d r e d muscle in vitro, o r t h a t o f the human h e a r t , Cameron (1975) e s t i m a t e d t h a t the h e a r t ' s oxygen consumption has an upper l i m i t o f 4% o f th e t o t a l oxygen uptake o f th e f i s h a t r e s t . The q u e s t i o n however s t i l l remains as to the f u n c t i o n a l advantage o f havi n g the b u l k o f the c o n t r a c t i l e t i s s u e o f t r o u t d e v o i d o f an a r t e r i a l c o r o n a r y b l o o d s u p p l y , w h i l e the c o r t i c a l l a y e r i s so r i c h l y v a s c u l a r i z e d . I f the patency o f an a r t e r i a l b l o o d s u p p l y i s n e c e s s a r y f o r the maintenance o f m y o c a r d i a l c o n t r a c t i l i t y i n an a c t i v e f i s h l i k e t he rainbow t r o u t , then g i v e n the h e a r t must work h a r d e r d u r i n g e x e r c i s e , a b l a t i o n o f t h i s c o r o n a r y a r t e r y may a f f e c t the l e v e l o f e x e r c i s e performed by the f i s h . The h e a r t may be unable t o i n c r e a s e i n work c a p a c i t y d u r i n g 189 p e r i o d s o f i n c r e a s e d demand, as d u r i n g e x e r c i s e , because o f a d e c r e a s e i n the s u p p l y o f a r t e r i a l oxygen t o the myocardium, The f o l l o w i n g experiments were performed i n an attempt t o t e s t the p r e v i o u s h y p o t h e s i s on e x e r c i s i n g rainbow t r o u t . MATERIALS AND METHODS A t o t a l o f 26 rainbow t r o u t (316 + 15.6 g) were used f o r t h e s e e x p e r i m e n t s . The c r i t i c a l swimming v e l o c i t y o f f i s h i n a t u n n e l r e s p i r o m e t e r was d e t e r m i n e d as d e s c r i b e d p r e v i o u s l y (see page 6 ). Once U had been det e r m i n e d f o r each f i s h , 10 o f t h e s e f i s h (292.0 + 9.0 g) then were s e l e c t e d a t random and s u r g i c a l l y p r e p a r e d as f o l l o w s . Each f i s h was a n a e s t h e t i z e d and p l a c e d s u p i n e on an o p e r a t i n g t a b l e (as d e s c r i b e d i n M a t e r i a l s and Methods pp. 9 t o i o ) . A small m i d - v e n t r a l i n c i s i o n was made through t he s k i n and u n d e r l y i n g p e c t o r a l m u s c u l a t u r e ( F i g . 22A) t o expose t he p e r i c a r d i u m c o n t a i n -i n g the h e a r t ( F i g . 22B). Care was taken not t o d i s r u p t t he p e c t o r a l g i r d l e symphasis. The p e r i c a r d i u m was opened c a r e f u l l y t o expose t he c o r o n a r y a r t e r y ( F i g . 22C) seen on the v e n t r a l s u r f a c e o f the v e n t r a l a o r t a ( a t a r r o w ) . T h i s v e s s e l was a b l a t e d by e l e c t r o c a u t e r y ( B i r t c h e r E l e c t r o s t a s i s U n i t ) . Thus the main a r t e r i a l s u p p l y t o the v e n t r i c l e was d i s r u p t e d . The muscula-t u r e was s u t u r e d c l o s e d , and the s k i n then was c l o s e d s e p a r a t e l y ( F i g . 22D and 22E). The o p e r a t e d f i s h were a l l o w e d to r e c o v e r and heal f o r 5 - 7 days b e f o r e the U -t p r o c e d u r e was r e p e a t e d . Immediately a f t e r f i s h had r e g a i n e d e q u i l i b r i u m f o l l o w i n g a n a e s t h e s i a , t h e y s t a r t e d t o use t h e i r p e c t o r a l f i n s i n a normal manner, i n d i c a t i v e o f l i t t l e impairment o f muscle f u n c t i o n due t o s u r g i c a l p r o c e d u r e s . A l s o , s i x o f the p r e v i o u s 26 f i s h (342.0 + 21.0 g) were sham-operated i n a manner s i m i l a r t o t h a t hust d e s c r i b e d , e x c e p t t h a t the c o r o n a r y c i r c u l a t i o n was l e f t i n t a c t . Recovery and subsequent U r e - e v a l -u a t i o n were the same as thos e o f o p e r a t e d f i s h . I n t a c t , o p e r a t e d and sham-operated h e a r t s were examined v i s u a l l y • 190 F i g u r e 22. Photographs o f the sequence o f e v e n t s d u r i n g t he o p e r a t i o n where the c o r o n a r y a r t e r y was a b l a t e d . A. Small i n c i s i o n made i n s k i n and e p a x i a l p e c t o r a l m u s c u l a t u r e , r e t r a c t e d t o expose p e r i c a r d i u m B. P e r i c a r d i u m c a r e f u l l y opened t o expose h e a r t w i t h c o r o n a r y a r t e r y c l e a r l y v i s a b l e on v e n t r a l s u r f a c e o f v e n t r i c l e a n d b u l b u s , r u n n i n g t o v e n t r i c u l a r mass C. Coronary a r t e r i a l s u p p l y t o v e n f r i c l e i s c a u t e r i z e d and no e v i d e n c e o f any l a r g e p a t e n t v e s s e l i s v i s a b l e D. P e c t o r a l m u s c u l a t u r e s u t u r e d c l o s e d o v e r the h e a r t E. S k i n o v e r h e a r t i s c l o s e d s e p a r a t e l y from m u s c u l a t u r e A f t e r 5 - 7 days r e c o v e r y , f i s h show l i t t l e s i g n o f , t i s s u e n e c r o s i s due t o t h e o p e r a t i o n a l p r o c e d u r e and h e a l i n g i s complete. 191 192 i m m e d i a t e l y a f t e r e x e r c i s e . F i s h were s a c r i f i c e d f o l l o w i n g f a t i g u e , a f t e r b e i n g removed from the downstream e l e c t r i f i e d g r i d o f the r e s p i r o m e t e r , by a sharp blow t o the head. In post-mortem a u t o p s y , h e a r t s were e x c i s e d and p l a c e d i n 20 ml o f 2% g l u t a r a l d e h y d e i n 0.14 M 5 o r e n s e n ' s phosphate b u f f e r f o r a t l e a s t 24 h, so as t o f i x the t i s s u e . Whole h e a r t s were s u b j e c t e d t o p r o g r e s s i v e a l c o h o l d e h y d r a t i o n , through x y l e n e , and embedded i n p a r a f f i n . These t i s s u e s then were s e c t i o n e d ( a t 8/xm) on a s t e e l b l a d e microtome and s e c t i o n s were mounted on warmed g l a s s s l i d e s . These were s t a i n e d w i t h h a e m o t o x y l i n and e o s i n , and a l l s e c t i o n s were viewed and s e l e c t e d s e c t i o n s photographed u s i n g a Z e i s s P h o tomicroscope. RESULTS F o l l o w i n g 5 - 7 days r e c o v e r y from s u r g i c a l p r o c e d u r e s f o r c o r o n -a r y a b l a t i o n , U . j t v a l u e s were not d i f f e r e n t ( p<0.05) from v a l u e s o b t a i n e d from f i s h which were sham-operated, o r c o m p l e t e l y i n t a c t ( T a b l e 27). B i g g e r f i s h a t t a i n e d g r e a t e r a b s o l u t e speeds ( c m - s e c - 1 ) , but a l l f i s h t e s t e d a t t a i n e d the same r e l a t i v e U c r i t ( f l - s e c - 1 ) . The d a t a comparing i n t a c t and o p e r a t e d f i s h ' s swimming performance are g r a p h i c a l l y p r e s e n t e d i n F i g s . 23 and 24. The l o g U ( c m - s e c - 1 ) versus cummulative p e r c e n t a g e o f f i s h " f a t i g u e d " (on p r o b i t s c a l e ) , both from o r i g i n a l d a t a ( F i g . 23A), o r from data t r a n s f r o m a t i o n where c r i t i c a l v e l o c i t i e s were grouped i n t o 10 c m - s e c - 1 i n t e r v a l s , i n d i c a t e no d i f f e r e n c e s ( p<0.01) between the two l i n e s g e n e r a t e d . In a d d i t i o n , a s i g n i f i c a n t l i n e a r r e l a t i o n s h i p between f o r k l e n g t h and ^ c r i t ( ^ ' s e c w a s found ( p < 0.05), even o v e r the v e r y l i m i t e d range o f f o r k l e n g t h s f o r the f i s h used i n t h i s s t u d y . T h i s r e l a t i o n s h i p ( F i g . 24) i n d i c a t e d t h a t the s m a l l e r f i s h had h i g h e r U .. v a l u e s than d i d l a r g e r f i s h , 3 c r i t 3 when e x p r e s s e d i n f o r k l e n g t h s - s e c - 1 (see a l s o B r e t t and G l a s s , 1973). Post-mortem e x a m i n a t i o n o f the p e r i c a r d i a l c a v i t y o f the rainbow t r o u t i n d i c a t e d t h a t the c a u t e r i z e d c o r o n a r y a r t e r y d i d remain c l o s e d d u r i n g 193 E f f e c t s o f c o r o n a r y a r t e r y a b l a t i o n on swimming e x e r c i s e performance i n rainbow t r o u t . Fork l e n g t h Weight U c r U U c r i t ( f l - cm) g cm'sec" 1 f l ' s e c - 1 _ NORMAL (N = 26) x_ 29.2 316.4 73.8 2.53 l : 0-44 15.6 1.6 0.06 SHAM OPERATED (N = 6) x 31.2 342.0 82.4 2.62 ± S.E.M. 0.15 21.0 2.8 0.05 X 27.8 ± S.E.M. 0.33 CORONARY ABLATION (N = 292.0 69.8 9.04 2.5 10) 2.50 0.08 194 F i g u r e 23. L o g a r i t h m i c - p r o b i t p l o t o f c r i t i c a l swimming v e l o c i t y v e r s u s p r e c e n t f i s h f a t i g u e d a t t h a t v e l o c i t y . Upper frame r e p r e s e n t s the o r i g i n a l d a t a , and i n d i c a t e s t h a t the o p e r a t e d f i s h B and the i n t a c t f i s h 0 l i e a l o n g the same s t r a i g h t l i n e . In t h e lower frame, the o r i g i n a l d a t a were grouped i n t o 10 cm'sec 1 i n t e r v a l s , w i t h the a t t e n d a n t n numbers i n c l u d e d . The two l i n e s a r e not d i f f e r e n t . Rest o f . f r a m e as i n upper one. 195 196 F i g u r e 24. L i n e a r r e p r e s e n t a t i o n o f the r e l a t i o n s h i p between c r i t i c a l swimming v e l o c i t y ( U c r i t ) and f o r k l e n g t h f o r i n t a c t • , and o p e r a t e d f i s h ( f i ) . The e q u a t i o n o f the l i n e i s o n l y f o r d a t a from i n t a c t , f i s h (:N = 26) w h i l e the o p e r a t e d f i s h data (N = 10) i s p r e s e n t e d t o show t h a t they t o o l i e w i t h i n t he range d e s c r i b e d f o r i n t a c t f i s h . 197 Fork length, cm 198 e x e r c i s e , whereas a f t e r e x e r c i s e i n i n t a c t f i s h , t h i s v e s s e l was v i s i b l y d i -l a t e d , and p a t e n t . H i s t o l o g i c a l e x a m i n a t i o n o f c r o s s - s e c t i o n s o f the v e n t r a l a o r t a / b u l b u s i n d i c a t e d t h a t the c o r o n a r y v e s s e l was the dominant and appeared t o be th e o n l y v e s s e l v i s i b l e i n a l l s e c t i o n s i n " i n t a c t " f i s h ( F i g . 25-1 and - 2 ) . Once the main a r t e r i a l s u p p l y was c a u t e r i z e d however, numerous s m a l l e r v e s s e l s around t h e o u t e r l a y e r o f t h e v e n t r a l a o r t a appeared t o ope up, wi t h r e d b l o o d c e l l s i n s i d e t h e i r lumena ( F i g . 25-3, -4, and - 5 ) . These c o u l d be t r a c e d a l o n g t h e i r c o u r s e t o the myocardium. No attempt was made t o q u a n t i f y the t o t a l c r o s s - s e c t i o n a l a r e a c o n t r i b u t e d by t h e s e "new" v e s s e l s , compared t o t h a t o f the p a t e n t c o r o n a r y a r t e r y . Nor was any attempt made t o a s c e r t a i n the time c o u r s e o f "new" v e s s e l p a t e n c y , f o l l o w i n g c o r o n a r y a r t e r y a b l a t i o n . DISCUSSION Swimming e x e r c i s e i n t r o u t i s a s s o c i a t e d w i t h a 3 to 6 - f o l d i n c r e a s e i n the c a r d i a c o u t p u t , o v e r t h a t found a t r e s t ( K i c e n i u k and J o n e s , 1977). A l t h o u g h the h e a r t must work h a r d e r d u r i n g e x e r c i s e , the a b l a t i o n o f the c o r o n a r y a r t e r y has no e f f e c t on e x e r c i s e performance i n t h e s e f i s h , compared t o i n t a c t t r o u t . The i n n e r m y o c a r d i a l mass a c q u i r e s t h e n e c e s s a r y m e t a b o l i c oxygen f o r c o n t r a c t i l i t y from the venous b l o o d pool w i t h i n the v e n t r i c u l a r lumen. I t a l s o has been shown t h a t t h i s i n n e r myocardium o f the t r o u t {Sal-velinus fontinalis) has the a b i l i t y t o a e r o b i c a l l y m e t a b o l i z e l a c t a t e , p r o -duced as a by p r o d u c t o f a n a e r o b i c m etabolism ( L a n c t i n et a l . s 1980) i n the myotomal mass o f t h e s e f i s h . The venous PQ i s much lower d u r i n g e x e r c i s e than a t r e s t i n t r o u t ( K i c e n i u k and J o n e s , 1977) and t h e r e f o r e , t h e v e n t r i c l e must i n c r e a s e oxygen e x t r a c t i o n e f f i c i e n c y d u r i n g e x e r c i s e ( R a n d a l l , 1970). Oxygen t o mammalian h e a r t s i s s u p p l i e d p r i m a r i l y v i a an e x t e n s i v e c o r o n a r y c i r c u l a t i o n . I f man has a sm a l l c o r o n a r y a r t e r i o l a r b l o c k a g e , he runs the r i s k o f m y o c a r d i a l i n f a r c t i o n , due t o l o c a l i s c h a e m i a . E x e r c i s e w i t h •199 F i g u r e 25. A ( l ) . C r o s s - s e c t i o n through the bulbus a r t e r i o s u s o f the rainbow t r o u t , t o show the prominant c o r o n a r y a r t e r y (CA) on the v e n t r a l s u r f a c e . In t h e normal t r o u t , no p a t e n t v e s s e l s a r e v i s i b l e i n the a d v e n t i t i a o f the b u lbus ( 2 ) , o t h e r than the c o r o n a r y a r t e r y . B ( l , 2 , 3 ) . The b u l b u s , 5 - 7 days r e c o v e r y from h a v i n g the main, c o r o n a r y a r t e r y s u p p l y c o m p l e t e l y a b l a t e d u s i n g e l e c t r o c a u t e r y . In t h i s c a s e , the main c o r o n a r y s u p p l y i s no l o n g e r p a t e n t , but s m a l l v e s s e l s i n . t h e a d v e n t i t i a ( a t a rrows) a r e open, and t h e s e can.be t r a c e d to the v e n t r i c u l a r myocardium. S e c t i o n s c u t a t 8 ym, s t a i n e d w i t h H and E. 200 201 an a r t e r i a l b l o o d s u p p l y t o the h e a r t which i s i n t e r r u p t e d , t h e r e f o r e c o u l d l e a d t o some r a t h e r s e r i o u s consequences. Given t he d i f f e r e n c e s between myo-c a r d i a l oxygen d e l i v e r y i n f i s h and man, but t h a t i n a l l o t h e r r e s p e c t s , c a r d i o r e s p i r a t o r y adjustments t o e x e r c i s e a r e q u a l i t a t i v e l y s i m i l a r , one a l s o might have e x p e c t e d some change i n the l e v e l o f e x e r c i s e performance i n t r o u t w i t h an a b l a t e d c o r o n a r y a r t e r y . S i n c e t h i s i s not the case when the main a r t e r i a l oxygen s u p p l y t o the t r o u t h e a r t i s i n t e r r u p t e d , t he h e a r t may have adopted an a l t e r n a t e m e t a b o l i c - p a t h w a y , a n a e r o b i o s i s , t o s u p p l y t he en-ergy r e q u i r e m e n t s f o r m a i n t a i n e d c o n t r a c t i l i t y . T h i s seems u n l i k e l y s i n c e G e s s e r (1977) has shown t h a t t r o u t h e a r t s , in v i t r o , a r e e s p e c i a l l y i n t o l e r -a n t o f low oxygen l e v e l s , and r a p i d l y d i m i n i s h i n t h e i r s t r e n g t h o f c o n t r a c -t i o n when exposed t o such c o n d i t i o n s . I t i s noted however, t h a t when the c o r o n a r y a r t e r y i s a b l a t e d i n t r o u t , s m a l l a r t e r i o l e s i n the a d v e n t i t i a o f the v e n t r a l a o r t a / b u l b u s appear t o become p a t e n t , f o l l o w i n g 5 t o 7 days o f r e c o v e r y from s u r g e r y . These t h e r e f o r e m a i n t a i n a r t e r i a l b l o o d s u p p l y t o the v e n t r i c u l a r c o r t e x , i n p l a c e o f the main b l o o d pathway. While t he p r e c i s e r e a s o n f o r the a r t e r i a l v a s c u l a r arrangement o f the t r o u t h e a r t i s not c l e a r , i t does seem t h a t t he maintenance o f c o r o n a r y a r t e r i a l b l o o d f l o w i s impor-t a n t t o t h e s e f i s h a t a l l t i m e s . What i s not known from t h i s study i s whether t h e s e t r o u t would have been a b l e to a t t a i n t he same l e v e l o f e x e r c i s e performance d u r i n g t he i n t e r m e d i a t e d p e r i o d , j u s t f o l l o w i n g c o r o n a r y a r t e r y a b l a t i o n , assuming the s u r g e r y i t s e l f would not have been a p e r t u r b i n g f a c t o r i n t h e s e t e s t s . The f i s h i n the p r e s e n t s t u d y were a l l o w e d t o r e c o v e r f o r s e v e r a l days b e f o r e b e i n g t e s t e d . The sm a l l p a t e n t v e s s e l s noted a f t e r t h i s time i n my e x p e r i -ments may have taken some time t o open up f o l l o w i n g a b l a t i o n o f the main c o r o n a r y a r t e r i a l b l o o d s u p p l y . In t h i s c a s e , t he h e a r t may have used the a l t e r n a t e m e t a b o l i c pathway, a l l u d e d t o e a r l i e r , t o s u s t a i n c o n t r a c t i l i t y . However, a n a e r o b i c c a p a c i t y o f the myocardium o f t r o u t i s v e r y l i m i t e d , 202 and f i s h may not have been a b l e t o e x e r c i s e a t a l l i n t h e s e e a r l y s t a g e s o f r e c o v e r y . A l t e r n a t i v e l y , t h e s e small v e s s e l s i m m e d i a t e l y became p a t e n t , so t h a t a r t e r i a l b l o o d f l o w t o the h e a r t was c o n t i n u o u s , even when the c o r o n a r y a r t e r y i s a b l a t e d . However, t h e end r e s u l t i s t h a t t h e t r o u t h e a r t does have the c a p a c i t y t o have i t s v a s c u l a r b l o o d s u p p l y a d j u s t o v e r a p e r i o d o f s e v e r a l days i n o r d e r t h a t a r t e r i a l b l o o d oxygen d e l i v e r y i s m a i n t a i n e d , and e x e r c i s e performance i s not a f f e c t e d by c o r o n a r y a r t e r y a b l a t i o n . 202a APPENDIX B A MODIFIED PERFUSION PUMP 203 MODIFICATION OF A PISTON-TYPE PERFUSION PUMP FOR DELIVERY OF LOW FLOW RATES Har v a r d B l o o d Pumps (model 1405; H a r v a r d Apparatus Co., I n c . , Mass. U.S.A.) a r e i n s e n s i t i v e t o back p r e s s u r e , and produce f l o w i n a f i x e d phase o f s y s t o l e : d i a s t o l e o f 1:2*. However, t h e s e pumps and t h e i r a s s o c i a t e d v a l v e s were found t o not o p e r a t e w e l l a t low f l o w r a t e s , when s t r o k e volumes a l s o were s m a l l . T h e r e f o r e , a r e p l a c e m e n t p i s t o n system, w i t h newly d e s i g n e d v a l v e s was c o n s t r u c t e d , t o a l l o w the d e l i v e r y o f low minute f l o w , w i t h s t r o k e volumes i n the range o f 0.05 - 0.3 ml per s t r o k e . The e x i s t i n g c o n n e c t i n g s h a f t was a t t a c h e d t o the p l u n g e r o f a d i s p o s a b l e 1 cc s y r i n g e ( T u b e r c u l i n ; B & D Co., M i s s i s s a u g a , Canada) by a U-shaped s t e e l c o n n e c t o r ( F i g . 26). The b a r r e l o f the s y r i n g e was c u t o f f a t the 1.0 c c mark and clamped i n t o a machined Perspex b l o c k ( F i g . 2 6 ) . T h i s b l o c k , w i t h the s y r i n g e b a r r e l , then was a t t a c h e d t o the e x i s t i n g v a l v e mounting b r a c k e t on the pump c h a s s i s by the machine b o l t s p r o v i d e d . The s y r i n g e b a r r e l was c o n n e c t e d t o a r e d e s i g n e d remote pumping head by a l e n g t h PE 240 t u b i n g (1.67 x 2.42 mm) ( C l a y Adams, I n t r a m e d i c ) . F i g u r e 27A i s a diagram o f t h i s pumping head, which i s s i m i l a r i n c o n c e p t t o t h a t a v a i l a b l e from H a r v a r d Apparatus Co. Newly d e s i g n e d v a l v e s , which a t t a c h t o i n p u t and o u t p u t Luer-Lok n e e d l e s on the r e d e s i g n e d remote pumping head, a r e shown i n F i g . 27B. The c h e c k i n g mechanism o f t h e s e u n i d i r e c t i o n a l v a l v e s was p r o v i d e d by a p i e c e o f L a t e x r u b b e r d e n t a l dam s t r e t c h e d l i g h t l y o v e r a c o l l a r c u t from a 1.5 ml Eppendorf c e n t r i f u g e tube (Brinkmann Co.). The c o l l a r and dam ( v a l v e assem-b l y ) was p r e s s - f i t t e d o v e r the t i p o f a 5 cc d i s p o s a b l e Luer-Lok s y r i n g e (B & D C o . ) , and then t h i s assembly was mounted i n s i d e a h o u s i n g c o n s t r u c t e d from the t i p o f two 10 c c d i s p o s a b l e Luer-Lok s y r i n g e s (B & D C o . ) . When assembled and a t t a c h e d t o a Harvard Blood Pump, t h e new remote pumping head and v a l v e u n i t a l l o w e d o n l y u n i d i r e c t i o n a l f l o w , r e g a r d -l e s s o f i t s o r i e n t a t i o n , and f l o w r a t e s were independent o f o u t f l o w p r e s s u r e 204 a t s t r o k e volumes as small as 0.05 ml. A l s o , t h e r e was no d i s c e r n a b l e f l u i d b a c k f l o w d u r i n g the s u c t i o n ( d i a s t o l i c ) phase o f the pumping c y c l e . A l l o t h e r f e a t u r e s o f the Harvard Blood Pump ( i . e . s y s t o l e : d i a s t o l e r a t i o , p r e s -s u r e p r o f i l e , f r e q u e n c y and s t r o k e volume adj u s t m e n t s ) were u n a f f e c t e d by t h e s e m o d i f i c a t i o n s . V a l v e s were t h e r e f o r e i n e x p e n s i v e and e a s i l y c o n s t r u c -t e d . '''"Operating and Maintenance I n s t r u c t i o n s f o r S e r i e s 1400 P u l s a t i l e Blood Pumps." H a r v a r d Apparatus Co., Inc. 150 Dover Road, M i l l i s , Mass., U.S.A. 02054. 1980. 205 F i g u r e 26. M o d i f i e d c l a m ping b l o c k and s y r i n g e b a r r e l assembly. A. S i d e view B. Top view 206 clamping block for 10-32 bolts 1 cc syringe HARVARD pump shaft 2 cm 1— ^-d 1 1 1 < / — I I ! 1 I I 1 cm for chassis guide-pin 207 F i g u r e 27. A. S i d e (a) and f r o n t a l (b) views o f the r e d e s i g n e d remote pumping head. T h i s was manufactured from machined Perspex. B. The c h e c k i n g mechanism (a) and the u n i d i r e c t i o n a l v a l v e s (b) i n e x p l o d e d view, which were a t t a c h e d to i n f l o w and o u t f l o w o f the remote pumping head. A l l j o i n t s and i n s e r t s were s e a l e d w i t h A r a T d i t e epoxy cement. 208 -2- DAXBOECK RESEARCH INTERESTS: Comparative p h y s i o l o g y o f lower v e r t e b r a t e s : c o n t r o l s arid r e g u l a t i o n s o f c a r d i o r e s p i r a t o r y a d j u s t m e n t s a f f e c t i n g gas t r a n s f e r d u r i n g e x e r c i s e i n f i s h . A s p e c t s o f gas exchange i n b i m o d a l l y b r e a t h i n g f i s h e s . PUBLICATIONS: Daxboeck, C. 1977. A Study o f P e r i p h e r a l / S u p e r f i c i a l Oxygen-S e n s i t i v e Chemoreceptors i n the Rainbow T r o u t (Salmo G a i r d n e r i ) . M.Sc. T h e s i s , U n i v e r s i t y o f T o r o n t o , 120 pp. T e l f o r d , M . and C. Daxboeck. 1978. P o r c e l l a n a sayana Leach (Crustacea:Anomura) s y m b i o t i c w i t h Stfombus g i g a s ( L i n n a e u s ) (Gastropoda'-.Strombidae) and w i t h t h r e e s p e c i e s o f h e r m i t c r a b s (Anomura:Diogenidae) i n Barbados. B u l l . M a r i n e . S c i . 28: 202-205. Daxboeck, C. and G.F. H o l e t o n . 1978. Oxygen r e c e p t o r s i n the rainbow t r o u t , Salmo g a i r d n e r i . Can. J . Z o o l . 56: 1254-1259. F a r r e l l , A.P., C. Daxboeck and D.J. R a n d a l l . 1979. The e f f e c t o f i n p u t p r e s s u r e and f l o w on t h e p a t t e r n and r e s i s t a n c e t o f l o w i n t h e i s o l a t e d p e r f u s e d q i l l o f a t e l e o s t f i s h . J . Comp. P h y s i o l . 133: 233-240. Daxboeck, C. 1979. A s p e c t s o f bimodal b r e a t h i n g i n t h e b o w f i n , Ami a c a l v a L. Am. Z o o l . 1 9 ( 4 ) : A b s t r a c t No. 48, p. 863. Daxboeck, C. and G.F. H o l e t o n . 1980. The e f f e c t o f MS-222 on the hy p o x i c r e s p o n s e o f rainbow t r o u t (Salmo g a i r d n e r i ) . Comp. Biochem. P h y s i o l . 56C: 117-121. Daxboeck, C. and T.A. Heming. 1980. E f f e c t o f c o r o n a r y a r t e r y a b l a t i o n on e x e r c i s e performance i n Salmo g a i r d n e r i . Am. Z o o l . 2 0 ( 4 ) : A b s t r a c t No. 392, p. 800. Daxboeck, C , D.K. B a r n a r d and D.J. R a n d a l l . 1981. F u n c t i o n a l morphology o f t h e g i l l s o f t h e b o w f i n , Ami a c a l v a L., w i t h s p e c i a l r e f e r e n c e t o t h e i r s i g n i f i c a n c e d u r i n g a i r e x p o s u r e . R e s p i r . P h y s i o l . 43: 349-364. R a n d a l l , D.J., J.N. Cameron, C. Daxboeck and N. Smatresk. 1981. A s p e c t s o f bimodal gas exchange i n t h e b o w f i n , Amia c a l v a L. ( A c t i n o p t e r y g i i - . A m i i f o r m e s ) . R e s p i r . P h y s i o l . 43: 339-348. D a v i e , P.S. and C. Daxboeck. 1981. M o d i f i c a t i o n o f a p i s t o n - t y p e p e r f u s i o n pump f o r d e l i v e r y o f low f l o w r a t e s . E x p e r i m e n t i a ( i n p r e s s ) . F a r r e l l , A.P. and C. Daxboeck. 1981. Oxygen uptake o f the l i n g c o d , Ophiodon e l o n g a t u s d u r i n g p r o g r e s s i v e h y p o x i a . Can. J . Z o o l . ( i n p r e s s ) Daxboeck, C. 1981. A Study o f the C a r d i o v a s c u l a r System o f the Rainbow T r o u t (Salmo g a i r d n e r i ) a t Re s t and D u r i n g Swimming E x e r c i s e . Ph.D. T h e s i s . U n i v e r s i t y o f B r i t i s h Columbia, 210 pp. -3- DAXBOECK (Con t i n u e d ) Daxboeck, C. and P.S. D a v i e . The e f f e c t s o f p u l s e p r e s s u r e on f l o w d i s t r i b u t i o n w i t h i n an i s o l a t e d , s a l i n e - p e r f u s e d t r o u t head p r e p a r a t i o n . (MS s u b m i t t e d t o Can. J . Z o o l . ) D a v i e , P.S. and C. Daxboeck. E f f e c t s o f p u l s e p r e s s u r e on f l u i d exchange between b l o o d and t i s s u e i n t r o u t g i l l s . (MS s u b m i t t e d t o Can. J . Z o o l . ) Daxboeck, C. The e f f e c t o f c o r o n a r y a r t e r y o c c l u s i o n on e x e r c i s e . performance i n the rainbow t r o u t . (MS s u b m i t t e d t o Can. J . Zool.)-. R a n d a l l , D.J. and C. Daxboeck. C i r c u l a t o r y changes d u r i n g e x e r c i s e i n f i s h . George H o l e t o n Memorial Symposium: R e s p i r a t o r y P h y s i o l o g y o f F i s h and Amphibia. To be p u b l i s h e d i n s p e c i a l i s s u e o f the Can. J . Z o o l . ( s u b m i t t e d ) . D a v i e , P.S., C. Daxboeck, S.F. P e r r y and D.J. R a n d a l l . A s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n : A new approach t o the s t u d y o f gas t r a n s f e r i n f i s h e s . (MS s u b m i t t e d t o J . exp. B i o l . ) Daxboeck, C , P.S. D a v i e , S.F. P e r r y and D.J. R a n d a l l . Oxygen uptake i n a s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n . (MS s u b m i t t e d to J . exp. B i o l . ) . P e r r y , S.F., P.S. D a v i e , C. Daxboeck and D.J. R a n d a l l . A comparison o f C02 e x c r e t i o n i n the s p o n t a n e o u s l y v e n t i l a t i n g , b l o o d - p e r f u s e d t r o u t p r e p a r a t i o n and s a l i n e - p e r f u s e d g i l l p r e p a r a t i o n s : C o n t r i b u t i o n s o f the b r a n c h i a l e p i t h e l i u m and r e d b l o o d c e l l . (MS s u b m i t t e d t o J . exp. B i o l . ) . Daxboeck, C , D.J. R a n d a l l and D.R. J o n e s . C a r d i a c o u t p u t d i s t r i b u t i o n d u r i n g r e s t and s t e a d y - s t a t e swimming e x e r c i s e i n the rainbow t r o u t : A comparison o f Rubidium-86 and r a d i o l a b e l l e d m i c r o s p h e r e t r a c e r t e c h n i q u e s . (MS s u b m i t t e d t o Am. J . P h y s i o l . ) . R a n d a l l , D.J., C. Daxboeck, A.P. F a r r e l l and P. D a v i e . The e f f e c t of.changes i n p r e s s u r e and f l o w on b l o o d d i s t r i b u t i o n w i t h i n t h e g i l l s . P r o c . F i s h G i l l Symposium, G i a r r e , S i c i l y (1980). i n m a n u s c r i p t . R a n d a l l , D.J. and C. Daxboeck. B l o o d f l o w t h r o u g h g i l l s . In: F i s h P h y s i o l o g y , V o l . X. W.S. Hoar and D.J. R a n d a l l , eds. Academic p r e s s , New York. In p r e p a r a t i o n . Approved f o r p r e s s i n 1982. P e r r y , S.F., P.S. Davie and C. Daxboeck. 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A b s t r a c t e d . ... . Annual M e e t i n g o f Can. Soc. Z o o l . , V i c t o r i a , B.C. O x y g e n - s e n s i t i v e r e c e p t o r s i n rainbow t r o u t . 1977. A b s t r a c t e d . Annual M e e t i n g o f Can. Soc. Z o o l . , London, Ont. B l o o d f l o w d i s t r i b u t i o n d u r i n g r e s t and e x e r c i s e i n rainbow t r o u t . 1978. A b s t r a c t e d . Vancouver Aquarium Soc. Guest L e c t u r e . " B i t e s and S t i n g s and Poisonous T h i n g s i n F i g i i a n Waters" - Marine B i o t o x i n s . March, 1979. Annual M e e t i n g o f Can. Soc. Z o o l . , Quebec C i t y , P.Q. A s p e c t s o f a q u a t i c and a e r i a l r e s p i r a t i o n i n the b o w f i n , Ami a c a l v a L. 1979. A b s t r a c t e d . ' Annual M e e t i n g o f Am. Soc. Z o o l . , Tampa, F l a . A s p e c t s o f bimodal b r e a t h i n g i n t h e . b o w f i n , Ami a c a l v a L. 1979. A b s t r a c t e d . Annual Meeting o f Can. Soc. Z o o l . , B a n f f , A l t a . The e f f e c t o f changes i n p r e s s u r e and f l o w on b l o o d d i s t r i b u t i o n w i t h i n the g i l l s o f f i s h . 1980. A b s t r a c t e d . Annual meeting o f Am. Soc. Z o o l . , S e a t t l e , Wash. The e f f e c t o f c o r o n a r y a r t e r y a b l a t i o n on e x e r c i s e performance i n Salmo  g a i r d n e r i . 1980. A b s t r a c t e d . Annual M e e t i n g o f Soc. Exp. B i o l . Med. (N.W. S e c t i o n ) , Vancouver, B.C. 1) P u l s a t i l e p e r f u s i o n i n c r e a s e s c l e a r a n c e o f e t h a n o l from g i l l t i s s u e s i n i s o l a t e d rainbow t r o u t heads. A b s t r a c t e d . 2) A e s t i v a t i o n i n the t o a d , Bufo marinus. A b s t r a c t e d : w i t h a s h o r t 16mm c i n e f i l m p r e s e n t a t i o n . 1980. Annual M e e t i n g o f Can. Soc. Z o o l . , W a t e r l o o , Ont. I n v i t e d s p e a k e r a t G.F. H o l e t o n Memorial Symposium. 1) C i r c u l a t o r y changes d u r i n g e x e r c i s e i n f i s h . A b s t r a c t e d . 2) Gas exchange i n s p o n t a n e o u s l y - v e n t i l a t i n g , b l o o d p e r f u s e d t r o u t . A b s t r a c t e d . 3) A e r i a l r e s p i r a t i o n i n the s t i c h a e i d f i s h , X i p h i s t e r  a t r o p u r p u r e u s . A b s t r a c t e d . 

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