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The effect of intermittent exercise on carbohydrate metabolism in rainbow trout (Salmo gairdneri) Stevens, Ernest Donald 1965

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THE EFFECT OF INTERMITTENT EXERCISE ON  CARBOHYDRATE  METABOLISM IN RAINBOW TROUT (Salmo g a i r d n e r i ) by ERNEST DONALD STEVENS B. Sc., V i c t o r i a  College, 1963  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of PHYSIOLOGY  We accept t h i s t h e s i s as conforming t o the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1965  In presenting this thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of • B r i t i s h Columbia., I agree that the Library shall make i t available for reference and study,  freely  I further agree that per-  mission for extensive copying of this thesis f o r scholarly purposes may be granted by the Head of my Department or by his representatives.  It i s understood that:copying or publi-  cation of this thesis for financial gain shall not be allowed without my written permission*  Department of The University of B r i t i s h Columbia, Vancouver 8, Canada  ABSTRACT  The purpose of t h i s study was  to examine the e f f e c t s  of e x e r c i s e of s h o r t d u r a t i o n , and the e f f e c t s of r e - e x e r c i s e on carbohydrate metabolism.  I f e e l that the l e v e l s of severe  e x e r c i s e s t u d i e d approximate the l e v e l s o f severe e x e r c i s e which a rainbow t r o u t p r o b a b l y experiences i n i t s n a t u r a l environment.  The l e v e l of blood l a c t a t e , blood hemoglobin,  muscle  l a c t a t e , muscle glycogen, and l i v e r glycogen were determined i n u n a n e s t h e t i z e d , i n t a c t , one and o n e - h a l f year o l d rainbow t r o u t a c c l i m a t e d to 1 0 . j ? ° C .  Samples were taken immediately  a f t e r e x e r c i s e of 3 seconds t o 5 minutes, a f t e r r e c o v e r y o f 3 minutes to 6 0 minutes, and a f t e r r e - e x e r c i s e of 3 seconds to 5 minutes.  The r e s u l t s i n d i c a t e that e x e r c i s e of even the s h o r t e s t d u r a t i o n s t u d i e d causes an immediate  increase i n  the l e v e l of blood l a c t a t e , muscle l a c t a t e , and blood hemoglobin.  E x e r c i s e a l s o causes an immediate  glycogen, but does not cause a change  decrease i n muscle  i n the l e v e l of l i v e r  glycogen. slight.  Changes d u r i n g the 60 minute r e c o v e r y p e r i o d In g e n e r a l , the e f f e c t s of r e - e x e r c i s e a f t e r a  minute recovery  are 60  p e r i o d are a d d i t i v e .  A c o r r e l a t i o n a n a l y s i s between muscle glycogen  and  muscle l a c t a t e i n d i c a t e s that there i s a source of muscle l a c t a t e other than muscle glycogen at e x e r c i s e l e v e l s of l o n g The  duration.  source of t h i s muscle l a c t a t e does not appear to come from  l i v e r glycogen. p r o t e i n or l i p i d ,  The  energy may  or by a b s o r p t i o n of f o o d s t u f f s from the  T h i s study provides not w e l l adapted f o r recovery duration.  be s u p p l i e d by c a t a b o l i s m  of gut.  evidence that rainbow t r o u t are from severe e x e r c i s e of short  xi  ACKNOWLEDGEMENTS  A s s i s t a n c e f o r t h i s p r o j e c t provided by the N a t i o n a l Research C o u n c i l of Canada through grant T-7 t o Dr. E. C. B l a c k i s g r a t e f u l l y acknowledged. Game Branch and  of the B r i t i s h Columbia  C o n s e r v a t i o n are thanked  The F i s h and  Department of R e c r e a t i o n  f o r p r o v i s i o n of experimental  animals, and the s t a f f at the Summerland Hatchery  thanked  f o r m a i n t a i n i n g these animals.  I express my s i n c e r e g r a t i t u d e t o Dr. E. C. B l a c k f o r h i s encouragement, d i r e c t i o n , and a s s i s t a n c e i n the present study.  I am a l s o indebted t o Dr. Glen Manning f o r  suggesting the r e s e a r c h problem.  Thanks are a l s o due t o the f o l l o w i n g f o r t e c h n i c a l assistance:  Mr. D. John Bosomworth, and Mr. S. J . T r e d w e l l  f o r a s s i s t a n c e i n sampling and a n a l y s i s of l a c t a t e ; Miss G. Docherty  f o r a n a l y s i s of glycogen; Mr. K. Henze of the  Department of P h y s i o l o g y , U n i v e r s i t y of B r i t i s h Columbia and Miss Tomiko Hashimoto f o r a s s i s t a n c e i n p r e p a r a t i o n of the manuscript.  xii I am a l s o indebted  t o Dr. Dempster and the s t a f f  of the Computing Center at U. B. C. f o r a s s i s t a n c e i n the c a l c u l a t i o n of s t a t i s t i c s .  I am a l s o t h a n k f u l f o r encouragement and  ideas  from P r o f e s s o r D. H. Copp and from my f e l l o w student, S. S. Shim.  Dr.  V  T A B L E OF CONTENTS  Page INTRODUCTION  1  M A T E R I A L S AND METHODS  3  I. II.  III.  IV.  V.  FISH  3  EXPERIMENTAL CONDITIONS  1|.  Unexercised  B.  Exercised  li.  C.  Recovery  If.  D.  Intermittent exercise  I4.  SAMPLING- METHODS  5  A.  Blood  5  B.  Tissue  samples samples  • A N A L Y T I C A L METHODS  o f muscle  and l i v e r  5 £  A.  Hemoglobin  6  B.  Lactate  7  C.  Glycogen  7  HANDLING  OF DATA  R E S U L T S AND D I S C U S S I O N I.  LL.  A.  HEMOGLOBIN  '  7 11 11  vi  II.  III.  IV.  A.  Results  11  B.  Discussion  17 20  LACTATE A.  Results  - blood  B.  Results  - muscle  C.  Discussion  2I4.  lactate  36  GLYCOGEN  55  A.  Results  - muscle  B.  Results  - liver  C.  Discussion  glycogen  55 .  glycogen  R E L A T I O N S H I P BETWEEN M U S C L E G L Y C O G E N MUSCLE LACTATE .  V.  20  lactate  GENERAL D I S C U S S I O N  61 66  AND .  7  2 78  CONCLUSIONS  82  SUMMARY  83  AREAS FOR FURTHER I N V E S T I G A T I O N  86  BIBLIOGRAPHY  88  APPENDIX  98  vii  L I S T OP  TABLES  TABLE I.  II. .. III.  IV.  V.  VI.  VII.  VIII. IX.  X.  Page D u r a t i o n of e x e r c i s e p e r i o d s , d u r a t i o n r e c o v e r y p e r i o d s , and number o f t i m e s r e - e x e r c i s e d i n the present study. Time t o o b t a i n and samples.  prepare blood  and  of 9  tissue 10  L e v e l of hemoglobin c o n c e n t r a t i o n d u r i n g c o n t i n u o u s and d i s c o n t i n u o u s e x e r c i s e o f same t o t a l d u r a t i o n . E f f e c t o f e x e r c i s e and r e - e x e r c i s e on l e v e l of blood l a c t a t e .  the 12  the 21  The l e v e l o f b l o o d l a c t a t e c o n c e n t r a t i o n d u r i n g c o n t i n u o u s and d i s c o n t i n u o u s e x e r c i s e o f t h e same t o t a l d u r a t i o n .  23  E f f e c t o f e x e r c i s e and r e - e x e r c i s e on l e v e l of muscle l a c t a t e .  30  Levels of blood l a c t a t e i n Salmo g a i r d n e r i . Levels fish.  of muscle  lactate i n  Maximal l e v e l s of blood after exercise i n f i s h ,  the  unexercised 37 unexercised 38  and m u s c l e l a c t a t e f r o g , and mammals.  Blood l e v e l s of l a c t a t e a f t e r i n t e r m i t t e n t e x e r c i s e i n man f r o m C h r i s t e n s e n ( 1 9 5 6 ) .  50  viii  XI.  XII. XIII. XIV.  XV.  XVI. XVII.  XVIII. XIX. XX.  XXI.  A comparison of t h e r e s i s t a n c e t o f a t i g u e between f i s h and man d u r i n g i n t e r m i t t e n t s t r e n o u s e x e r c i s e as i n d i c a t e d by t h e l e v e l o f blood lactate. 52 E f f e c t of e x e r c i s e and r e - e x e r c i s e on the l e v e l o f muscle g l y c o g e n .  62  E f f e c t of e x e r c i s e and r e - e x e r c i s e o f 30 seconds on the l e v e l o f l i v e r g l y c o g e n .  63  E f f e c t o f 5 minutes severe e x e r i c s e , and 30 minutes r e c o v e r y a f t e r 5 minutes e x e r c i s e on the l e v e l s of l i v e r g l y c o g e n .  61}.  A n a l y s i s o f v a r i a n c e of l i v e r g l y c o g e n d a t a from T a b l e XIV d u r i n g and a f t e r 5 minutes severe e x e r c i s e .  65  L e v e l s of muscle g l y c o g e n i n u n e x e r c i s e d rainbow t r o u t .  67  The p e r c e n t of muscle l a c t a t e not accounted f o r by the decrease I n t h e l e v e l o f muscle glycogen.  75  Sample s i z e , mean, and standard the v a r i a b l e s s t u d i e d . Time t o o b t a i n and p r e p a r e b l o o d samples.  f o r each o f  99  tissue  A c t u a l time t a k e n t o swim I4..3. meters ( a p p r o x i m a t e l y 3 s e c . ) and a c t u a l time t a k e n t o swim 23 meters ( a p p r o x i m a t e l y 15 s e c . )  . 101  103  A n a l y s i s of v a r i a n c e and Duncan's new m u l t i p l e range t e s t on the d a t a i n . t h e l a t i n square design. 104  ix  L I S T OF FIGURES  Each p o i n t i n every f i g u r e represents 6 samples f r o m s e p a r a t e f i s h .  t h e mean o f a t  least  FIGURE 1.  Changes i n l e v e l s o f b l o o d h e m o g l o b i n d u r i n g 5> m i n u t e s s e v e r e e x e r c i s e .  2.  E f f e c t o f r e s t a f t e r s e v e r e e x e r c i s e o f 1, 2 , 5 m i n u t e s on t h e l e v e l s o f b l o o d h e m o g l o b i n .  3.  E f f e c t o f e x e r c i s e and r e - e x e r c i s e on t h e l e v e l s blood hemoglobin c o n c e n t r a t i o n .  4.  E f f e c t o f i n t e r m i t t e n t e x e r c i s e o f 30 s e c o n d s levels" of blood hemoglobin c o n c e n t r a t i o n .  5>.  E f f e c t of rest a f t e r of blood l a c t a t e .  6.  Changes i n l e v e l s o f b l o o d l a c t a t e a f t e r continuous e x e r c i s e ( c o n t i n u o u s c u r v e s ) , and a f t e r d i s c o n t i nuous e x e r c i s e ( d i s c o n t i n u o u s c u r v e s ) .  7.  E f f e c t o f i n t e r m i t t e n t e x e r c i s e o f V~> s e c o n d s the l e v e l s of blood l a c t a t e .  8.  E f f e c t of i n t e r m i t t e n t exercise the l e v e l s of blood l a c t a t e .  9.  Changes i n t h e l e v e l o f m u s c l e minutes severe e x e r c i s e .  10.  severe  concentration  e x e r c i s e on t h e  o f 30 lactate  Changes i n l e v e l s o f m u s c l e l a c t a t e r e c o v e r y f r o m s e v e r e e x e r c i s e o f 1, minutes.  or  on  levels  seconds during  during 2, and 5  of  on  on 5  X  1 1 . Effect levels 1  2  of intermittent exercise of muscle lactate.  of  1 5 seconds  . Effect of intermittent exercise the levels of muscle lactate.  of  3  0 seconds  on  the  on  s  13.  Effect of intermittent exercise the levels of muscle lactate.  Ii4..  Changes minutes  1 5 .  Changes i n l e v e l s o f muscle g l y c o g e n d u r i n g r e c o v e r y f r o m s e v e r e e x e r c i s e o f 1, 2 , and 5 m i n u t e s .  16.  Effect levels  of intermittent exercise of muscle glycogen.  of  1 5 seconds  17.  Effect of intermittent exercise the levels of muscle glycogen.  of  30  18.  The r e l a t i o n s h i p glycogen i n f i s h exercise.  19.  The e f f e c t o f metabolism.  i n levels of muscle severe exercise.  of 5  glycogen  minutes  during  supply  on  5  seconds  b e t w e e n m u s c l e l a c t a t e and sampled immediately after  oxygen  on  carbohydrate  on  the  on  muscle  INTRODUCTION  There have been many s t u d i e s o f the r e l a t i o n s h i p between severe e x e r c i s e and metabolism i n f i s h 191+2; B l a c k  concomitant changes i n carbohydrate  (von Buddenbrock, 1938; Secondat and  et a l , 1961; and  C a l l l o u e t , 1961j.).  of these s t u d i e s , the f i s h were c o n t i n u o u s l y minutes.  In almost a l l  e x e r c i s e d f o r 15  E x e r c i s e of t h i s d u r a t i o n always elevated  l a c t a t e and  blood  l a c t a t e , depleted  Diaz,  muscle  muscle glycogen, and  g e n e r a l , produced a marked d i s t u r b a n c e  in  which i s r e f e r r e d t o  as muscular f a t i g u e .  There have been very few discontinuous animal, and has  s t u d i e s o f the e f f e c t s o f  or i n t e r m i t t e n t severe e x e r c i s e i n any  none to my knowledge I n f i s h .  intact (1962)  Christensen  demonstrated that man can do an optimum amount of e x e r c i s e  i n a g i v e n time by a l t e r n a t e e x e r c i s e and seconds.  r e s t periods  of 30  His r e s u l t s showed that continuous e x e r c i s e f o r 9  minutes e l e v a t e d  the l e v e l of blood  whereas d i s c o n t i n u o u s e x e r c i s e and  l a c t a t e from 7 t o 1$0 mg %,  e x e r c i s e f o r 30 minutes ( a l t e r n a t e  rest periods  of 30 seconds) elevated  the l e v e l of  2 blood l a c t a t e t o only 20 mg %.  Thus, the present study was an attempt whether or not f i s h are adapted  to discover  to t o l e r a t e discontinuous  e x e r c i s e as w e l l as man i s w i t h r e s p e c t t o accumulation of lactate.  I n other words, I presumed t h a t energy  f o r exercise i s  s u p p l i e d by d e g r a d a t i o n of muscle glycogen; and when demand f o r oxygen exceeded the supply, l a c t a t e accumulated. the purpose metabolism  I t was a l s o  of t h i s study t o examine the changes i n carbohydrate d u r i n g severe e x e r c i s e of extremely s h o r t d u r a t i o n .  Moreover, I f e e l that d i s c o n t i n u o u s e x e r c i s e and e x e r c i s e of short d u r a t i o n probably s i m u l a t e the e x e r c i s e which a rainbow t r o u t experiences i n i t s n a t u r a l  environment.  3  MATERIALS AND METHODS  I. PISH One and one-half year o l d rainbow t r o u t , Salmo g a i r d n e r i , were reared from eggs obtained from a commercial t r o u t h a t c h e r y I n McLeary, Washington. Summerland Trout Hatchery  They were r a i s e d a t  operated by B r i t i s h Columbia  Depart-  ment of R e c r e a t i o n and Conservation, P i s h and Came Branch. Trout were f e d twice d a i l y w i t h p e l l e t e d C l a r k e Company, S a l t Lake C i t y , Utah). remained constant throughout  f i s h food The water  10.5  the summer,  to  At l e a s t two days p r i o r t o experiment,  temperature  11.5°C. f i s h were  t r a n s f e r r e d from o u t s i d e ponds t o standard hatchery (Lj-0 x 460 x 12 cm. deep). experiment  and were kept covered  Diseased  troughs  F i s h were not starved before t o reduce  At the outset of the experiment prevalent.  ( J . R.  f i s h were not used  external stimuli.  f u r o n c u l o s i s was  i n the experiment  and a l l f i s h were t r e a t e d w i t h sulfamerazine g i v e n with the food. Incidence o f f u r o n c u l o s i s was n e g l i g i b l e by J u l y , and treatment was d i s c o n t i n u e d J u l y 3«  II.  EXPERIMENTAL  CONDITIONS  P i s h were h e l d i n covered troughs at l e a s t two days p r i o r t o experiments, and f e e d i n g was continued  as u s u a l .  Treatments were a p p l i e d randomly and s i n g l y ; and each f i s h used only  once.  A.  Unexercised c o n d i t i o n .  The term  unexercised  r a t h e r than r e s t i n g or b a s a l l e v e l i s employed s i n c e the f i s h u s u a l l y manifest some a c t i v i t y during captured  w i t h as l i t t l e  sampling.  disturbance  as p o s s i b l e ,  to the e x e r c i s e trough, and sampled  immediately.  B. as l i t t l e  Exercised  disturbance  conditions.  P i s h were air-dipped  F i s h were captured  as p o s s i b l e , a i r - d i p p e d  with  t o the e x e r c i s e  trough, and e x e r c i s e d by s p l a s h i n g water i n the trough f o r a p r e s c r i b e d p e r i o d of time (from 30 seconds t o 5 minutes), or f o r a p r e s c r i b e d  distance.  F i s h were chased by two  persons.  C. and  then l e f t  Recovery c o n d i t i o n .  F i s h were e x e r c i s e d  to r e s t a p r e s c r i b e d p e r i o d  minutes to 60 minutes, i n the same trough. restricted  t o one-half  d u r i n g the recovery  D.  as above  of time, from 3 Movement  was  of the trough and the trough was covered  period.  Intermittent  exercised  conditions.  The f i s h was  5 e x e r c i s e d and l e f t t o r e c o v e r as above.  A f t e r the r-ectfver-y  p e r i o d , the l i d was l i f t e d and the f i s h was re<^exercised. specific  The  l e v e l s o f e x e r c i s e and r e c o v e r y times s t u d i e d appear  i n Table I .  III.  SAMPLING METHODS  A. Blood samples.  A f t e r treatment  a f i s h was captured  by hand as q u i c k l y and a d e p t l y as p o s s i b l e , p l a c e d v e n t r a l s i d e up i n a v-shaped trough which was kept submerged a l l o w i n g the f i s h t o r e s p i r e i n water, and a blood sample was obtained by c a r d i a c puncture.  Blood was withdrawn i n t o a 2 ml. s y r i n g e  which had been p r e v i o u s l y water-proofed  w i t h p a r a f i n o i l , and  r i n s e d w i t h one drop of h e p a r i n s o l u t i o n . was of  immediately  The blood  sample  e j e c t e d i n t o a c o n i c a l t e s t tube; 0 . 0 2 ml.  the blood was t r a n s f e r r e d t o 5.0  ml. of 0 . 1 M h y d r o c h l o r i c  a c i d f o r d e t e r m i n a t i o n of hemoglobin c o n c e n t r a t i o n , and 0.5 ml. or 1.0 ml. was t r a n s f e r r e d t o 9 . 0 ml. c o l d 10$ t r i c h l o r o a c e t i c a c i d f o r d e t e r m i n a t i o n of l a c t a t e c o n c e n t r a t i o n . g l o b i n c o n c e n t r a t i o n was determined sampling.  L a c t a t e was determined  w i t h i n three months.  Hemo-  w i t h i n three hours of  on the d e p r o t e i n i z e d f i l t r a t e  Time t o capture the f i s h and time t o  withdraw the blood sample were recorded by stopwatch, and appear i n Table I I .  B.  T i s s u e samples of muscle and l i v e r .  After  treat-  ment, the f i s h was captured as q u i c k l y as p o s s i b l e , k i l l e d by  6 b r e a k i n g the neck, and p l a c e d on i t s s i d e .  The muscle sample  was obtained by punching through the musculature immediately below the d o r s a l f i n w i t h a # 9 cork b o r e r . c o n s i s t e d of the whole l i v e r .  The l i v e r  Both muscle and l i v e r samples  were f r o z e n immediately i n dry i c e and e t h a n o l . (fat,  Unwanted t i s s u e  s k i n , and bone) was removed, and the sample weighed  on a t o r s i o n balance. two p o r t i o n s .  The muscle sample was d i v i d e d  One p o r t i o n was homogenized w i t h c o l d  t r i c h l o r o a c e t i c acid for  sample  into 10%  ( 6 . 0 ml. per gram) and the f i l t r a t e analyzed  l a c t a t e c o n c e n t r a t i o n w i t h i n three months.  The o t h e r  p o r t i o n of the muscle sample and the l i v e r sample were digested i n 60% potassium hydroxide at  100°C  ( 3 . 0 ml. per gram) f o r f o u r hours  and the d i g e s t s analyzed f o r glycogen c o n c e n t r a t i o n  w i t h i n 6 months.  Time to capture, k i l l ,  o b t a i n the muscle  sample, and o b t a i n the l i v e r sample were a l l recorded by stopwatch, and appear I n Table I I .  C. sex  A f t e r sampling, the f i s h was weighed, and the  determined by v i s u a l examination of the gonads.  IV.  ANALYTICAL METHODS  A l l analyses were determined u s i n g a Klett-Summerson photoelectric  A.  colorimeter.  Hemoglobin was analysed c o l o r i m e t r i c a l l y as a c i d  hematin u s i n g a #5i|.  f i l t e r and comparing the sample t o a standard  7  s u p p l i e d by Klett-Summerson.  Hemoglobin i s r e p o r t e d i n g %  (grams per 100 ml. b l o o d ) .  B. aliquots  L a c t a t e was determined  from d e p r o t e i n i z e d f i l t r a t e s  c o l o r i m e t r i c a l l y on 1 ml. of blood and muscle  homogenates by the Barker-Summerson method  (19J+1).  Interfering  m a t e r i a l s i n the f i l t r a t e were removed by the Van S l y k e Salkowski  treatment  o f copper  sulphate and c a l c i u m hydroxide  (Hawk ejb a l , \95lx.). L a c t i c a c i d i s o x i d i z e d t o acetaldehyde w i t h concentrated s u l p h u r i c a c i d , the aldehyde a chromogenic agent, p-hydroxydiphenyl, standard of l i t h i u m l a c t a t e .  i s coupled  with  and compared w i t h a  L a c t i c a c i d i s r e p o r t e d i n mg %  (mg per 100 ml. b l o o d , or mg per 100 grams wet weight o f muscle).  C. anthrone  Glycogen was determined  method  c o l o r i m e t r i c a l l y by the  ( C a r r o l l et_ a l , 19^6) on potassium  d i g e s t s o f muscle and l i v e r which were d i l u t e d volume.  Glycogen  i s precipitated  toa-known  with ethanol, hydrolysed  to glucose, and coupled w i t h a chromogenic agent, A standard curve i s determined  hydroxide  f o r each a n a l y s i s  anthrone. u s i n g three  c o n c e n t r a t i o n s o f glucose, and glycogen c o n c e n t r a t i o n d e t e r mined from the standard curve as g l u c o s e .  Glycogen  i s reported  i n mg % (mg of glycogen per 100 grams wet weight of t i s s u e ) .  V.  HANDLING OF DATA  No samples were pooled, a l l values were  determined  8 and recorded s e p a r a t e l y f o r each f i s h .  Sample s i z e , mean, and  standard d e v i a t i o n a r e g i v e n f o r each v a r i a b l e and each c o n d i t i o n . Conditions were a p p l i e d s i n g l y and randomly t o each f i s h . Comparisons  between c o n d i t i o n s are made u s i n g the F - t e s t , ANOV,  and Duncan's new m u l t i p l e range t e s t A l a t i n square d e s i g n ( 9 x 9 x 9 ) experiments  ( S t e e l and T o r r i e , I960).  was used i n one s e t of  (3 minutes, 8 minutes, and 30 minutes r e c o v e r y  from 1 minute, 2 minutes, and 5 minutes e x e r c i s e ) i n order t o determine the e f f e c t o f sampling sequence d u r i n g the day, the e f f e c t of sampling sequence from day to day, and the e f f e c t of experimental c o n d i t i o n s .  A l l s t a t i s t i c s were c a l c u l a t e d by  the Computing Center, U n i v e r s i t y of B r i t i s h  Columbia.  9  Table I .  D u r a t i o n of e x e r c i s e p e r i o d s , d u r a t i o n of r e c o v e r y  p e r i o d s , and number o f times r e - e x e r c i s e d i n the present study..  D u r a t i o n of  Duration of  Number o f times  Exercise  rest  re-exercised  (minutes)  0 (a)  3 0 , 60  0  3 seconds (b)  3 0 , 60  once a f t e r 60 min. r e s t  15 seconds (c)  3 0 , 60  twice a f t e r 60 min. r e s t  30 seconds  3 0 , 60  twice a f t e r 60 min. r e s t  1 minute  3,  8 , 30  once a f t e r each r e s t  2 minutes  3,  8 , 30  once a f t e r each r e s t p e r i o d  5 minutes  3,  8 , 30  once a f t e r each r e s t p e r i o d  to e x h a u s t i o n (d)  0  (a)  unexercised  (b)  actually  seconds (c)  (d)  0  condition  e x e r c i s e d 4 . 6 meters t a k i n g approximately 3  (mean 3 « 4 seconds, range 2 . 0 t o 6 . 0 seconds)  actually  seconds  period  e x e r c i s e d 23 meters t a k i n g approximately 15  (mean 1 5 . 0 seconds, range 1 0 . 0 t o 3 6 . 0 seconds)  exercised t o exhaustion.  T y p i c a l l y the b e h a v i o r of the  f i s h changes f o l l o w i n g approximately 1 minute o f e x e r c i s e . At  t h i s time the escape b e h a v i o r changes t o a h i d i n g b e h a v i o r ,  (mean 6 7 . 3 seconds, range 51 t o 80 seconds)  10  Table I I .  Time t o o b t a i n and prepare blood and  t i s s u e samples,  Blood and t i s s u e samples were obtained from separate  fish.  A l l times are g i v e n i n seconds.  Sample  Time  n  Mean + standard d e v i a t i o n  blood  to c a t c h and fish  position  t o withdraw blood sample  tissue  to c a t c h f i s h to k i l l  305  13.7  + I4-.I  305  31.4  +  23.9  222  2.9 +  1.8  222  2.8 +  1.2  fish  to remove muscle sample and p l a c e i n dry i c e and e t h a n o l to remove l i v e r and p l a c e i n dry i c e and e t h a n o l  222  10.4 + 2.6  62  ll+.l + 6.k.  11  RESULTS AND  I.  DISCUSSION  HEMOGLOBIN  A. R e s u l t s . The mean l e v e l o f hemoglobin 9.15  g %•  i n u n e x e r c i s e d f i s h was  The e f f e c t s of e x e r c i s e , r e c o v e r y , and i n t e r m i t t e n t  e x e r c i s e a r e i l l u s t r a t e d i n P i g . 1 , 2 , 3» and 4.  In g e n e r a l , e x e r c i s e caused hemoconcentration.  Pig. 1  i l l u s t r a t e s that the hemoconcentration response t o e x e r c i s e is triphasic.  E x e r c i s e of s h o r t d u r a t i o n caused a marked  i n c r e a s e i n hemoglobin which was f o l l o w e d by a marked decrease, and t h i s i n t u r n was f o l l o w e d by a g r a d u a l i n c r e a s e . r e c o v e r y the hemoglobin continued t o r i s e  (Pig. 2 ) ,  During The  hemoglobin l e v e l decreased a f t e r a l o n g r e c o v e r y p e r i o d . F o r example, the l e v e l had almost r e t u r n e d t o p r e - e x e r c i s e l e v e l s a f t e r a 3 0 second e x e r c i s e i n 60 minutes as i n d i c a t e d i n F i g . Ij..  R e - e x e r c i s e caused f u r t h e r hemoconcentration as  illustrated  i n P i g . 3 and ij..  The t r i p h a s i c curve p e r s i s t e d  but t o a l e s s o r e x t e n t , when the f i s h were rechased a f t e r r e c o v e r y p e r i o d s o f 3 0 minutes or 60 minutes.  12 Table I I I . and  L e v e l of hemoglobin c o n c e n t r a t i o n d u r i n g  discontinuous  e x e r c i s e of the same t o t a l d u r a t i o n .  Total duration of e x e r c i s e (seconds)  30  60  120  continuous  Condition  Hemoglobin  (S %)  m  E x e r c i s e R e c o v e r y Exercise , (seconds) (minutes) (seconds)  30  0  0  9.70  15  60  15  11,00  60  0  0  9.75  30  60  30  10.53  120  0  0  10.05  60  3  60  11.82  60  8  60  12.96  60  30  60  12.00  13  Fig. 1.  Changes i n l e v e l s - of blood hemoglobin d u r i n g 5 minutes severe e x e r c i s e .  concentration  Ik  F i g . 2.  E f f e c t of r e s t a f t e r severe e x e r c i s e of 1, 2, or 5 minutes on the l e v e l s of blood hemoglobin.  15  9^60-  0 H— 0  Pig. 3  i 1  i 2  i 3  I  4 Duration of strenuous exercise in minutes  I  5  E f f e c t of e x e r c i s e and r e - e x e r c i s e on the l e v e l s of blood  hemoglobin  concentration.  16  first exercise  second exercise  third exercise  11.60-1  11.00E  O)  10.40 o CT) o E a> X 9.80-  9.20oJ 30 0 60 30 60 Duration of recovery time in minutes between intermittent exercise periods of 3 0 sec.  Fig. k  E f f e c t o f i n t e r m i t t e n t e x e r c i s e o f 30 seconds on l e v e l s o f b l o o d hemoglobin  concentration.  17  B. D i s c u s s i o n Korzhuev (I96I4.) found t h a t c e n t r i f u g i n g the a c i d hematin s o l u t i o n reduced  the hemoglobin values by 1 8 - 2 0 $ (the t u r b i d i t y  of a c i d hematin s o l u t i o n s of f i s h blood i s due t o the  presence  of n u c l e i i n the e r y t h r o c y t e s ) . Thus, the a b s o l u t e v a l u e s of hemoglobin c o n c e n t r a t i o n r e p o r t e d i n t h i s study are p r o b a b l y h i g h , but comparisons between treatments  are  still  valid.  The mean l e v e l of hemoglobin i n u n e x e r c i s e d f i s h  was  1.21+ g % h i g h e r than the v a l u e f o r 1 9 6 1 (Black, et_ a l . ) and 1.67  g % lower than the v a l u e f o r 1 9 6 3 (Manning) f o r unexer-  c i s e d f i s h r a i s e d at the same h a t c h e r y , f e d the same f o o d , and kept a t the same temperature.  Schiffman and Promm ( 1 9 5 9 )  g i v e the hemoglobin l e v e l of hatchery r a i s e d Salmo g a i r d n e r i as 6 . 3 1 g %•  Some v a r i a b l e other than l o c a t i o n , type of f e e d ,  or temperature,  must a f f e c t blood hemoglobin l e v e l s .  p o s s i b i l i t i e s are stage of m a t u r i t y , f u r o n c u l o s i s ,  Some  and  sulfamethazine.  Hemoconcentration a s s o c i a t e d w i t h e x e r c i s e of f i s h  has  been noted by o t h e r s , but none has recorded a t r i p h a s i c response extremely  s i n c e , none t o my short d u r a t i o n .  hemoconcentration  knowledge has s t u d i e d e x e r c i s e of Black  i n largemouth  (1955)  found a s i g n i f i c a n t  bass, but not i n kamloops  t r o u t , f i n e - s c a l e d sucker, c a r p , squawfish, n o r t h e r n b l a c k  18 c a t f i s h , sockeye salmon (Black, 1 9 5 7 c ) , or l a k e trout- (Black, 1957b) f o l l o w i n g 15 minutes severe p o s s i b l e explanations  exercise.  There are three  f o r t h e observed hemoconcentration:  1.  e r y t h r o c y t e s move i n t o c i r c u l a t i n g  2.  water moves out of blood o r ,  3.  both of the above.  blood,  B l a c k ejb a l . (1959) attempted t o demonstrate the movement of water i n order t o e x p l a i n hemoconcentration of e x e r c i s e , but  observed no s i g n i f i c a n t d i f f e r e n c e between dry weight of  blood b e f o r e and a f t e r 15 minutes strenuous e x e r c i s e . c e n t r a t i o n of e x e r c i s e has been observed i n s p e c i e s than f i s h :  i n man (Bard  Best and T a y l o r  Hemocon-  other  1 9 5 6 ) , and i n r a t s ( S i e t e r 1 9 5 8 ) .  ( 1 9 6 1 ) , and Rakestraw (1921), i n d i c a t e t h a t  e x e r c i s e i n man r e s u l t s i n decreased  plasma volume, i n c r e a s e d  plasma p r o t e i n , i n c r e a s e d red c e l l c o n c e n t r a t i o n , and i n c r e a s e d hemoglobin c o n c e n t r a t i o n .  G-regersen and Rawson (1959) observed  a decrease i n t o t a l blood  volume with e x e r c i s e i n man.  H a l l et a l . (1926) noted i n c r e a s e d hemoglobin c o n c e n t r a t i o n w i t h a s p h y x i a t i o n i n cod. (1962) noted c i r c u l a t o r y shut grunion  Scholander et_ a l .  down with a s p h y x i a t i o n i n the  s i n c e there was an elevated muscle l a c t a t e but no  e l e v a t e d blood  lactate.  (Since l a c t a t e was not d i f f u s i n g from  muscle to blood  then blood  been reduced.)  Nakatani  flow t o s k e l e t a l muscle must have  (1955) observed a s i g n i f i c a n t  i n hemoglobin l e v e l as a r e s u l t of e l e c t r i c  increase  shock i n rainbow  19 trout.  Denyes and  Joseph (1956) found an i n c r e a s e d hemoglobin  concentration i n g o l d f i s h temperatures.  and  carp acclimated  In s p e c i e s other than f i s h , the f o l l o w i n g causes  of hemoconcentration have been noted: shock i n man and  induced  (Kubicek  to h i g h e r  electric  plasma volume 10 to 20%  decreased  f e v e r i n the dog  shock or  insulin  (Bard 1956),  i n c r e a s e d the hemoglobin l e v e l  et a l . 1959).  Barcroft  (1956) observed a t r i p h a s i c response i n blood  flow to s k e l e t a l muscle of man of a d r e n a l i n e .  i n response to continuous  Manning (unpublished)  noted t h a t  infusion  severe  e x e r c i s e i n rainbow t r o u t causes c i r c u l a t o r y shutdown, and thus plasma skimming  occurs.  A p o s s i b l e e x p l a n a t i o n f o r the observed r e s u l t s i s that the three phases of the hemoglobin response to e x e r c i s e are a s s o c i a t e d with c i r c u l a t o r y changes. i n c r e a s e and  decrease are due  response to an i n c r e a s e and  accumulation  to c i r c u l a t o r y changes i n  The  rapid  l a t e r slow i n c r e a s e  to c i r c u l a t o r y change i n response to the  of m e t a b o l i t e s  c a u s i n g the c h a r a c t e r i s t i c r e a c t i v e  hyperemic v a s o d i l i t a t i o n i n the muscle, and the red c e l l s l u d g i n g and skimming.  The  to plasma skimming, the r a p i d  to e r y t h r o c y t e s l u d g i n g .  i n hemoglobin i s due  i n i t i a l rapid  decrease of a d r e n a l i n e .  i n c r e a s e i n hemoglobin i s due decrease i s due  The  thus r e s o l v i n g  r e s t o r i n g a c o n d i t i o n of plasma  A l t e r n a t i v e l y , the l a t e r slow i n c r e a s e i n hemoglobin  could be due  to a r e l e a s e of stored e r y t h r o c y t e s from  storage  20 organs. storage  Udvardy  (unpublished)  has demonstrated t h a t  organs i n salmon a r e i n the s p l e e n and the a n t e r i o r  o n e - t h i r d o f the kidney.  Moreover, s t u d i e s i n man have  shown that r e l e a s e of e r y t h r o c y t e s i n e x e r c i s e occurs and  erythrocyte  only i n severe  exercise.  slowly,  The l a t e r slow i n c r e a s e i n  hemoglobin c o n c e n t r a t i o n could a l s o be due t o water moving from blood due  t o muscle t i s s u e i n response t o an osmotic  gradient  t o the i n c r e a s e i n the number of o s m o t i c a l l y a c t i v e  p a r t i c l e s i n the muscle as a r e s u l t of muscle c o n t r a c t i o n .  I I . LACTATE A.  Results - Blood  Lactate.  The mean l e v e l of blood  l a c t a t e i n unexercised  f i s h was 7.19 mg % (range 0.66 t o 13*57 mg % ) . e x e r c i s e , recovery,  The e f f e c t s of  and i n t e r m i t t e n t e x e r c i s e a r e i l l u s t r a t e d  i n P i g . 5 , 6, 7 , and 8.  E x e r c i s e , even o f the s h o r t e s t d u r a t i o n , caused an elevated blood  lactate.  A l l o w i n g the f i s h t o r e s t a f t e r the  e x e r c i s e always r e s u l t e d i n a f u r t h e r i n c r e a s e i n blood lactate  ( P i g . 5 ) . F i g . 8 i l l u s t r a t e s t h a t a 60 minutes  r e c o v e r y p e r i o d was i n s u f f i c i e n t f o r r e s t o r a t i o n of unexercised l e v e l s o f blood  l a c t a t e a f t e r e x e r c i s e of only 15 seconds.  However, the l e v e l of blood  l a c t a t e reached a peak and s t a r t e d  to decrease d u r i n g the 60 minute r e c o v e r y  period.  T a b l e IV.  E f f e c t of e x e r c i s e and r e - e x e r c i s e on the l e v e l of blood l a c t a t e .  In  each case f i s h were sampled immediately a f t e r e x e r c i s e or r e - e x e r c i s e .  E x e r c i s e time  (seconds)  3  .15.  30  Recovery time  (minutes)  60  .60  60  Blood  first  chase  Lactate  second  mg %  t h i r d chase  chase  .9 lb  74  .  111  9. W  6P..  -  55 97  .  120  . 120  120  300  300  60  60  . 8 .  30  3  8  . 30  3  8  .30  .15 .  15  15.  21+  24-  21+  55  55  55  49.  61+  86  79  «6  98  120  3  115  3°0  22 Re-exercise  always caused a f u r t h e r i n c r e a s e i n blood  l a c t a t e , as i n d i c a t e d i n Table IV.  A comparison of blood  l a c t a t e l e v e l s of f i s h  exer-  c i s e d f o r the same t o t a l l e n g t h of time r e v e a l s that i n t e r m i t t ent e x e r c i s e caused g r e a t e r i n c r e a s e s than continuous (Table V ) . the  exercise  However, from Table V i t can a l s o be seen that  e f f e c t of continuous e x e r c i s e i s t o cause a g r e a t e r  in  the l e v e l of blood  l a c t a t e than d i s c o n t i n u o u s  if  the f i s h i s l e f t to r e s t a f t e r the i n i t i a l  a l s o appeared t o respond b e t t e r t o chasing, work when e x e r c i s e d  increase  exercise,  chase.  The f i s h  and thus d i d more  i n t e r m i t t e n t l y than when e x e r c i s e d  contin-  uously.  T y p i c a l l y the f i s h responds t o chasing by evasive  behavior  f o r about 1 minute, and f u r t h e r c h a s i n g r e s u l t s  i n the f i s h attempting  to h i d e r a t h e r than t o escape.  e f f e c t of t h i s change i n behavior values  i s revealed  by comparing  f o r e x e r c i s e of 2 minutes, r e s t 3 minutes,  of Sk- S %) w i t h 5 minutes o f continuous e x e r c i s e ra  of 55 mg % ) .  These values  during recovery  tinuous  (blood l a c t a t e (blood l a c t a t e  i n d i c a t e t h a t the f i s h ' s response  from 2 minutes e x e r c i s e was not as great  d u r i n g the f i r s t  Fig.  The  as  2 minutes of e x e r c i s e .  6a compares the e f f e c t of continuous and d i s c o n -  e x e r c i s e of the same t o t a l d u r a t i o n  (2 minutes).  Examination of F i g . 6a r e v e a l s t h a t e x e r c i s e and r e - e x e r c i s e of 1 minute d i d not e l e v a t e the blood  l a c t a t e as much as an  23  Table V.  The l e v e l o f blood  lactate concentration  continuous and discontinuous total  seconds  30  60  120  -0  e x e r c i s e of the same  duration.  Blood  Condition  Total duration of e x e r c i s e  during  exercise  recovery  re-exercise  s ec ond s  minutes  seconds  30  0  15  60  30  lactate mg "%"  0  14  15  47  60  0  50  60  0  0  15  30  60  30  55  60  30  0  70  120  0  0  24  60  3  60  49  120  3  0  54  60  8  60  64  120  8  0  74  60  30  60  86  120  30  0  100  •  24 initial  continuous e x e r c i s e of 2 minutes.  But  re-exercised  f i s h were sampled immediately a f t e r the r e - e x e r c i s e , and they been l e f t to r e c o v e r ,  t h e i r blood  l a c t a t e l e v e l s probably  would have exceeded those of f i s h e x e r c i s e d 2 minutes.  P i g . 6b compares the  and  exercise.  continuously  for  e f f e c t of s i m i l a r t o t a l dura-  t i o n s of e x e r c i s e ; 5 minutes continuous and tinuous  had  4 minutes  discon-  Examination of P i g . 6b r e v e a l s that  r e - e x e r c i s e of 2 minutes elevated  the blood  exercise  l a c t a t e more  than i n i t i a l continuous e x e r c i s e of 5 minutes, even though re-exercised  f i s h were sampled immediately a f t e r r e - e x e r c i s e .  Fig. mittent  was mg  l a c t a t e and  B.  Results  The  mean l e v e l of muscle l a c t a t e i n unexercised  The  per  100  - Muscle  30  grams wet  Lactate.  weight  (range 57.19  e f f e c t s of e x e r c i s e , recovery, i n P i g . 9,  10,  11,  and 12,  immediate e l e v a t i o n i n muscle l a c t a t e .  fish  to 199.05  intermittent and  E x e r c i s e , even of the s h o r t e s t d u r a t i o n ,  slight  or  in  duration.  e x e r c i s e are i l l u s t r a t e d  cised  inter-  the l a c k of recovery  of 60 minutes a f t e r e x e r c i s e of only 15  132.38 mg %).  8 i l l u s t r a t e the a d d i t i v e nature of  e x e r c i s e on blood  r e s t periods seconds  7 and  Restoration  13.  caused  an  of unexer-  l e v e l s of muscle l a c t a t e a f t e r a 30 minute r e s t p e r i o d (Pig. 9).  F i g . 10  i l l u s t r a t e s that muscle l a c t a t e i n  was  25  I  Duration of strenuous exercise in minutes.  Pig. 5  E f f e c t of r e s t a f t e r severe e x e r c i s e on the l e v e l s of  blood l a c t a t e .  P i s h were sampled immediately a f t e r  3 minutes a f t e r , 8 minutes a f t e r , and 30 minutes exercise.  exercise,  after  26  lOO-i  E o> 80E o o o  _ E x e r c i s e d continuously 2 min.  X B  o A _ E x e r c i s e d and r e - e x e r c i s e d I min. Q _ E x e r c i s e d continuously I min.  60  O 40O  m  0  J  1—1  I  !  0 3  8  30  Duration of r e c o v e r y period after  exercise  (curves  exercise  B and C )  periods ( c u r v e  and between  A )  120  o  E O)  E  d> o  O  •x  100  E x e r c i s e d and r e - e x e r c i s e d 2 min. Q _ E x e r c i s e d continuously  A  '  c  5 N  min  -  Exercised continuously 2 min.  80  T3  O O  60  CD  i—r- -r—i 30 0 3 8 Duration of r e c o v e r y period after e x e r c i s e  ( curves periods  Pig.  6.  exercise  B and C ) and between  exercise  (curve A )  Changes i n l e v e l s of blood l a c t a t e a f t e r continuous (continuous c u r v e s ) , and a f t e r d i s c o n t i n u o u s  (discontinuous c u r v e s ) .  Note that f i s h e x e r c i s e d  exercise  intermit-  t e n t l y were sampled immediately a f t e r the second e x e r c i s e , whereas f i s h e x e r c i s e d c o n t i n u o u s l y were sampled i n the recovery condition.  27  first exercise  second exercise  third ' exercise  80-1  0  30  60  30  60  Duration of recovery time in minutes between intermittent  F i g . 7.  exercise periods of  E f f e c t of i n t e r m i t t e n t  l e v e l s of blood  lactate.  15 sec.  e x e r c i s e of 15  seconds on the  28  first exercise  0  second exercise  30  60  third exercise  30  60  Duration of recovery time in minutes between intermittent exercise periods of 30 sec.  Fig. 8  E f f e c t of i n t e r m i t t e n t e x e r c i s e of 30 seconds on the  l e v e l s of blood  lactate.  29 f i s h e x e r c i s e d only 1 minute continued  to r i s e s h a r p l y f o r  3 minutes a f t e r the e x e r c i s e , whereas t h a t of f i s h chased 2 minutes i n c r e a s e d  slightly,  and t h a t of f i s h chased 5  minutes decreased r a p i d l y .  That muscle l a c t a t e f a i l e d  t o r e t u r n t o normal even i n P i g . 9,  a f t e r e x e r c i s e of short d u r a t i o n i s i l l u s t r a t e d 11,  and 12.  T h i s tendency f o r muscle l a c t a t e t o remain elevated  e x p l a i n s why blood short p e r i o d s  lactate also failed  t o decrease to normal from  of e x e r c i s e , and must be due to e i t h e r a d i f f u s i o n  b a r r i e r between muscle and blood,  or due to impaired  blood  c i r c u l a t i o n w i t h i n muscle.  The e f f e c t s of r e - e x e r c i s e a f t e r 60 minutes r e s t appeared to be a d d i t i v e . upper l i m i t not  exceed  However, there appeared t o be an  (approximately (Pig. 13).  1170 mg %) which muscle l a c t a t e d i d  The e f f e c t of i n t e r m i t t e n t e x e r c i s e of  short d u r a t i o n , i l l u s t r a t e d  i n P i g . 11  and 12,  was  to cause  f u r t h e r i n c r e a s e s i n muscle l a c t a t e , again i n d i c a t i n g the failure  of muscle l a c t a t e to r e t u r n to unexercised  a f t e r e x e r c i s e of s h o r t d u r a t i o n .  levels  The l e v e l s of muscle l a c t a t e ,  a f t e r i n t e r m i t t e n t e x e r c i s e appear i n Table VI.  30  Table VI.  E f f e c t of e x e r c i s e and r e - e x e r c i s e on the l e v e l  of muscle l a c t a t e . periods,  P i s h r e s t e d 60 minutes between e x e r c i s e  and were sampled  immediately a f t e r e x e r c i s e or  re-exercise.  D u r a t i o n of e x e r c i s e chase  15 seconds  30 seconds  236  Muscle  first  lactate  second chase  276  mg %  t h i r d chase  3OI4.  '  5 minutes  291  I4.68  366  I4.62  358  31  P i g . 9.  Changes i n the l e v e l of muscle l a c t a t e during 5 minutes  severe e x e r c i s e , and 3 0 minutes a f t e r the severe  exercise.  32  Pig.  10.  Changes i n l e v e l s of muscle l a c t a t e d u r i n g r e c o v e r y  from severe e x e r c i s e of 1, 2,  and 5 minutes.  33  E  320-,  first exercise  second exercise  third exercise  CD  E O  o  200-  O _l  _CD  O CO Z3  120-  o  J  ~T~  0  30  60  30  60  Duration of recovery time in minutes between intermittent exercise periods of 15 sec.  P i g . 11.  E f f e c t of i n t e r m i t t e n t  l e v e l s of muscle  lactate.  e x e r c i s e of 15  seconds on the  3 4  first exercise  second exercise  third exercise  Duration of recovery time in minutes between intermittent exercise periods of 3 0 sec.  Fig.  1 2 .  E f f e c t of i n t e r m i t t e n t  l e v e l s of muscle  lactate.  e x e r c i s e of 3 0 seconds on the  35  Duration of recovery time in minutes between intermittent exercise periods of 5 min.  Pig.  13.  E f f e c t of i n t e r m i t t e n t  l e v e l s of muscle  lactate.  e x e r c i s e of 5 minutes on the  36 C.  D i s c u s s i o n of Blood and Muscle L a c t a t e .  Blood and muscle l a c t a t e are d i s c u s s e d  together  s i n c e muscle t i s s u e i s the source of blood l a c t a t e , and  thus  changes i n l e v e l s of muscle l a c t a t e are r e f l e c t e d by changes i n l e v e l s of blood  lactate.  The r e s t i n g l e v e l of blood l a c t a t e v a r i e s from year to year i n the rainbow t r o u t r a i s e d at Summerland, even though temperature and reasons  f e e d i n g c o n d i t i o n s are s i m i l a r .  f o r t h i s year to year v a r i a b i l i t y i n c l u d e the f o l l o w i n g :  the h i s t o r y of the f i s h d i f f e r f o o d ) , technique  ( f o r example the exact type of  of c a t c h i n g the f i s h and withdrawing the  sample vary from experimenter ant, there are d i s t i n c t  to experimenter;  i n Table V I I .  (Secondat  Salmo g a i r d n e r i appear  and D i a z , 194-2; Black, 1955*  1957a, 1957b; Parker et a l . , 1959;  Phillips,  1958;  Heath and  P r i t c h a r d , 1 9 6 2 ) , and  Dean and  (Bock et a l . , 1932;  The  import-  These values are s i m i l a r to those r e p o r t e d f o r  other s p e c i e s of f i s h  man  and most  races i n the s p e c i e s Salmo g a i r d n e r i .  L e v e l s of blood l a c t a t e i n unexercised  1956,  Some probable  G a i l l o u e t , 1964.;  Goodnight, 1964.; L i e v e s t a d ,  1957;  s i m i l a r to those r e p o r t e d f o r  Cook and Hurst, 1933;  and Bang, 1 9 3 6 ) .  l e v e l of muscle l a c t a t e i n unexercised  rainbow  t r o u t at Summerland i s much more v a r i a b l e than the l e v e l of blood l a c t a t e .  L e v e l s of muscle l a c t a t e i n unexercised  appear i n Table V I I I .  fish  37  Table V I I .  L e v e l s of blood l a c t a t e i n unexercised  Salmo  gairdneri.  Year  Blood  l a c t a t e mg%  Age  Reference  years 1957  7-2  1  B l a c k et a l . , 1959  1958  4.0  li  B l a c k e t a l . , 1962  1958  9.3  l-£  M i l l e r et a l . , 1959  1959  8.8  1-|-  B l a c k e t a l . , 1962  1961  5.1  l i  Manning,  1964  7.2  1§-  present  1963 study  38  Table VIII.  Year  L e v e l s of muscle l a c t a t e  Muscle  Species  i n unexercised  fish.  Reference  lactate  mg^  150  1927  haddock  1927  cod  80  1927  hake  50  1957  cod  5  1957  steelhead  1958  rainbow  trout  66  Black  et a l . ,  1962  1959  rainbow  trout  187  Black  et a l . ,  1962  1962  grunion  196J4.  rainbow  trout  174  50 trout  132  Ritchie  to  50  Leivestad  e_t a l .  Nakatani,  1957  Scholander present  study  39 The values of muscle l a c t a t e i n r e s t i n g f i s h are much h i g h e r than those r e p o r t e d f o r the f r o g  (8 mg %, Sacks  et a l . , 1954) and o r the c a t (15 mg %, Sacks and Morton, f  1 9 5 ° ) > but the f r o g was p i t h e d and the c a t was a n e s t h e t i z e d , thus e l i m i n a t i n g a c t i v i t y d u r i n g the sampling  The  procedure.  source o f r e s t i n g blood l a c t a t e i s probably  muscle, but Evans  (1934)  n  a  s  s t a t e d that i n the h e a r t - l u n g  p r e p a r a t i o n of a dog, the lungs produce enough l a c t a t e t o account  f o r r e s t i n g blood l a c t a t e p r o d u c t i o n .  (1933) s t a t e d that the source i s g l y c o l y s i s . Bernard  (1931) observed  Cook e_t a l . , Leopold and  t h a t r e s t i n g blood l a c t a t e was not  c o r r e l a t e d w i t h r e s t i n g blood glucose i n c h i l d r e n .  A c c o r d i n g t o Huckabee ( 1 9 5 6 ) , the d i s t r i b u t i o n of l a c t a t e between the e r y t h r o c y t e s and the plasma i s equal. Decker (1942) observed  t h a t the d i s t r i b u t i o n i s equal only  i n the r e s t i n g c o n d i t i o n , and that at h i g h e r c o n c e n t r a t i o n s of l a c t a t e , the c o n c e n t r a t i o n of l a c t a t e i s g r e a t e r i n the plasma than i n the red c e l l s .  Many v a r i a b l e s other than e x e r c i s e a l t e r blood and muscle l e v e l s o f l a c t a t e .  Scholander  (1962) u s i n g the grunion,  L:eivestad at a l . (1957) u s i n g the cod, and C a i l l o u e t  (1964)  u s i n g the channel c a t f i s h , showed that asphyxia, induced by removing the f i s h from the water, e l e v a t e d muscle l a c t a t e . They found t h a t the blood l a c t a t e d i d not r i s e u n t i l the f i s h  was returned  t o the water and concluded that blood was  being  d i v e r t e d away from muscle t i s s u e when the f i s h was out of water.  I n mammals, Himwich (1932) elevated blood with the f o l l o w i n g :  adrenaline  injection,alkali  e v i s c e r a t i o n , anaemia, and hemorrhage.  blood  (1959)  l a c t a t e i n dogs.  and Evans (1930) showed that h y p o v e n t i l a t i o n decreased  lactate.  bicarbonate  Huckabee (1958) showed that h y p e r v e n t i l a t i o n ,  i n f u s i o n , pyruvate i n f u s i o n , and glucose  a l l e l e v a t e blood elevated blood The m a j o r i t y  lactate.  infusion  Huckabee (1961) a l s o found an  l a c t a t e i n many r e s t i n g h o s p i t a l p a t i e n t s .  of these elevated blood  buted to e i t h e r elevated blood  l a c t a t e s could be  the elevated blood  attri-  pyruvates or t o hypoxemia o f  r e s p i r a t o r y or c a r d i o v a s c u l a r d i s e a s e . for  injection,  Kubicek ejt a l .  showed that induced f e v e r i n c r e a s e d blood Eggleton  lactate  No e x p l a n a t i o n was given  l a c t a t e i n the remaining cases,  and each  of these p a t i e n t s died o f a c i d o s i s .  Secondat and Diaz e x e r c i s e e l e v a t e s blood first The  (191+2) were the f i r s t  t o show that  l a c t a t e i n f i s h ; and Nakatahi  (1957)  t o show that e x e r c i s e e l e v a t e s muscle l a c t a t e i n f i s h .  l a c t a t e response to e x e r c i s e was f i r s t  F l e t c h e r and Hopkins ( 1 9 0 7 ) .  demonstrated by  They a l s o proposed that the  f a i l u r e of muscle to c o n t r a c t i n s p i t e o f adequate s t i m u l a t i o n was due t o muscle l a c t a t e r e a c h i n g  a c e r t a i n maximal v a l u e .  Meyerhof (1920) demonstrated that the source of l a c t a t e was muscle glycogen.  The has  response of blood  l a c t a t e d u r i n g severe e x e r c i s e  been studied i n many species of f i s h  1942;  Black  I960;  M i l l e r et a l . , 1959;  a l . , 1959;  1957  a, b, c; Nakatani, 1957; Parker and  Jonas e_fc a l . , 1962;  C a i l l o u e t , 1964;  Dean,  C o l l i n s and  Black and  Elling  (I960)  and  1959;  1957;  Barrett, Connor,  only two  fold.  e l e v a t e s blood  1962).  C o l l i n s et a l . , ( 1 9 6 4 )  fishway increased  blood l a c t a t e  glycogen reserves  the e f f e c t of t r a i n i n g on  i n rainbow t r o u t .  He  t h a t the t r a i n e d group could accumulate an oxygen debt times that of the untrained  f a s t e r during recovery,  the t r a i n e d f i s h . et. a l . ,  (I960)  o c c u r i n g In man  Lactate  found three  group, that the t r a i n e d group  used more of t h e i r muscle glycogen reserves it  exercise  man.  Hochachka (1961) analyzed oxygen debt and  trout  Huckabee (1956) r e p o r t s that even mild lactate in  -  Parker et  found that prolonged moderate e x e r c i s e of steelhead (Salmo g a i r d n e r i ) i n an endless  Diaz,  Black et a l . 1958  Black,  B a r r e t t and  1962;  (Secondat and  and  and  resynthesized  that i t took longer to exhaust  l e v e l s were not measure.  Anderson  found that the maximum l e v e l s of blood l a c t a t e a f t e r e x e r c i s e were the same f o r a t h l e t e s  and  non-athletes.  Denyes and  Joseph (1956) observed t h a t l a r g e r bass  tended to produce more l a c t a t e than s m a l l e r bass, but no f i c a n t c o r r e l a t i o n between s i z e and  l a c t a t e l e v e l s was  signi-  found  h-2 i n the p r e s e n t study.  B l a c k et al.(1962) observed a d i f f e r e n c e i n the l a c t a t e response to e x e r c i s e between sexes i n rainbow The  l e v e l of muscle  l a c t a t e was  g r e a t e r i n the female than  the male d u r i n g e x e r c i s e and r e c o v e r y . a maximum l e v e l a f t e r 2 minutes  trout.  Muscle l a c t a t e reached  of severe e x e r c i s e i n the  female, whereas i t took 9 minutes  i n the male.  Anderson  (I960)  observed t h a t the l a c t a t e response was  g r e a t e r i n women than  men  Caillouet  f o r the same amount of work done.  (196i+)  observed no s i g n i f i c a n t d i f f e r e n c e s between sexes i n the l a c t a t e response d u r i n g e x e r c i s e and r e c o v e r y i n the channel c a t f i s h . No c o n s i s t e n t s i g n i f i c a n t d i f f e r e n c e s were observed between sexes i n the p r e s e n t experiment.  The e f f e c t of age or stage of s e x u a l m a t u r i t y on the l a c t a t e response to e x e r c i s e has not been e x p e r i m e n t a l l y determined age was  i n rainbow t r o u t .  Anderson  (I960) observed  that  a s i g n i f i c a n t f a c t o r i n determining the l a c t a t e  response to e x e r c i s e i n man.  In the present study, the gonads  of every f i s h were examined.  We  i n gonad development  observed an obvious i n c r e a s e  d u r i n g the summer, but the s e x u a l matur-  a t i o n d i d not appear to a f f e c t the l a c t a t e response to e x e r c i s e i n any c o n s i s t e n t manner.  Maximal l e v e l s of blood and muscle  lactate  after  1+3 e x e r c i s e vary from s p e c i e s to s p e c i e s throughout  the  kingdom.  Most workers studying the l a c t a t e response  have used  15 minutes continuous  observed  severe e x e r c i s e .  t h a t the maximal blood l a c t a t e was  e x e r c i s e f o r 1 minute i n man.  (approximately 1 minutes), and  I4.OO meters f a i l e d lactate.  produced  by  to r i s e a f t e r e x e r c i s e i n f i s h 1957 > Second.at and D i a z , 191+2).  sprinting further  I960).  Black  that blood l a c t a t e  than  after  continues  (Black e_t a l . , 1962; T h i s has a l s o been (1957a) observed  l a c t a t e r i s e s f o r 3 i hours a f t e r 15 minutes a f t e r  Nakatani, observed  t h a t blood severe  e x e r c i s e , whereas the delayed i n c r e a s e only l a s t s 2-5 (Anderson,  that  IX.  Others have observed  i n man  severe  by s p r i n t i n g lj.00  Maximal l e v e l s of blood and muscle l a c t a t e  (Anderson,  (1936)  Bang  to cause a g r e a t e r i n c r e a s e i n blood  e x e r c i s e appear i n Table  i n man  in fish  Anderson (I960) observed  maximal blood l a c t a t e l e v e l s were produced meters  animal  I960).  minutes  There are at l e a s t three p o s s i b l e  e x p l a n a t i o n s f o r the continued r i s e i n blood  lactate:  1.  that the muscle continues to produce l a c t a t e ,  2.  that there i s an e f f i c i e n t d i f f u s i o n b a r r i e r  between muscle and b l o o d , or 3.  that c i r c u l a t i o n to the muscle i s impaired.  Anderson (I960) has a t t r i b u t e d l a c t a t e i n man  the continued r i s e of blood  to p o s t - e x e r c i s e i n c r e a s e d tonus a s s o c i a t e d  Table IX. exercise  Maximal l e v e l s of blood and muscle l a c t a t e a f t e r  i n fish,  Species  f r o g , and mammals.  Duration of exercise minutes  Maximum blood lactate  S %  Maximum muscle lactate mg %  m  Referenc e  rainbow  trout  15  77.9  415  B l a c k et a l . , 1962  rainbow  trout  15  54.0  295  Black et a l . , 1962  rainbow  trout  15  32.6  rainbow  trout  15  69.7  rainbow  trout  steelhead  trout  M i l l e r et a l . ,  1959  54-6 8.5  Manning, 1963  468  present  389  Nakatani,  study 1957  tench  15  53  Secondat and Diaz,  channel c a t f i s h  15  59.1  C a i l l o u e t , 1964  black c a t f i s h  15  34-9  Black,  1955  sucker  15  55.3  Black,  1955  carp  15  54.3  B l a c k , 1955  squawfish  15  94-2  Black, 1955  bass  15  76.2  Black, 1955  77  Heath and P r i t c h a r d  sunfish  6 - 45  1962  87  frog  322  cat man  19-42  l  126  Sacks e t a l . , 1954 Sacks and Sacks,  1956  Bang, 1936.  45 with hyperexcitability muscle l a c t a t e but  decreased  that  immediately  blood.  been s t u d i e d  to a c i d o s i s .  continues to r i s e  i s , 5 minutes.  m u s c l e and  due  after  after  In the present  of 1 or 2  exercise  exercise  rate  of d i f f u s i o n  b e t w e e n e r y t h r o c y t e s and  o f l a c t a t e has  plasma.  Johnson  (1945) showed t h a t l a c t a t e d i f f u s i o n i n mammalian b l o o d passive  and  unrestricted diffusion  extremely  temperature  i n c r e a s e d f r o m 2 t o 30  ( t h e Qio  fo  decrease  o f 25-36 t o 0-14  observed  that  r  but  degrees).  Margaria  the d e l a y of d i f f u s i o n  blood  i s greatest during severe exercise.  noted  that  shutdown. blood  I t i s most p r o b a b l e  lactate  i s due  Another increase  of blood  the e f f e c t showed no two  Joseph  lactate  and  was  temperature  to  (unpublished) circulatory  increase i n  factors.  c o n t r i b u t e s t o delayed--  to the slowness  on t h e l a c t a t e  differences  et a l , ,  from muscle  causes  the delayed  f a c t o r which p o s s i b l y  significant  of recovery i s  response.  of l a c t a t e  Black  (1957a)  production at  o f a c c l i m a t i o n i n Kamloops t r o u t .  Denyes  and  (1956) f o u n d t h e h i g h e s t i n d i v i d u a l v a l u e s and g r e a t e s t  variability lower  to a l l three  of temperature  temperatures  that  only  e_t a l . (1933)  Manning  trout  between  dependant  for a  of l a c t a t e  severe e x e r c i s e i n rainbow  minutes,  of longer d u r a t i o n ,  Some d i f f u s i o n b a r r i e r must e x i s t The  study  among f i s h was  temperature  associated  with f i s h  i n l a r g e mouth b a s s .  Dean and  acclimated to Goodnight  (1964) f o u n d a h i g h e r l a c t a t e p r o d u c t i o n i n w h i t e c r a p p i e , and b l a c k b u l l h e a d when a c c l i m a t e d t o l o w e r  temperatures.  In  l a r g e mouth bass, blood l a c t a t e was temperatures  lower at lower a c c l i m a t i o n  f o r u n e x e r c i s e d f i s h , but h i g h e r at lower  mation temperatures f o r e x e r c i s e d f i s h . blood l a c t a t e was for  In the  h i g h e r at lower a c c l i m a t i o n  accli-  bluegill,  temperatures  u n e x e r c i s e d f i s h , but lower at lower a c c l i m a t i o n temper-  atures f o r e x e r c i s e d f i s h . lower temperatures  They a l s o concluded that at  of a c c l i m a t i o n , f i s h r e l y on the pentose  shunt f o r energy p r o d u c t i o n . that glucose-6-phosphate  Ekberg  (1962)  demonstrated  dehydrogenase i s not a l t e r e d  changing e x t e r n a l temperature  by  of the c a r p , whereas 6-phospho-  g l u c o n i c dehydrogenase and a l d o l a s e are more a c t i v e i n c o l d adapted  fish.  He a l s o found that r e s i s t a n c e to i o d o a c e t a t e  poisoning i s greater i n g i l l s  from c o l d adapted g o l d f i s h , but  r e s i s t a n c e to cyanide p o i s o n i n g i s g r e a t e r i n warm adapted fish. to  Hart and Heroux  e x e r c i s e was  as i t was the  ( 1 9 5 4 ) showed that the l a c t a t e  the same i n deer mice a c c l i m a t e d to 0 degrees  i n those a c c l i m a t e d to 22 degrees.  l a c t a t e response was  They found  that  o n l y a l t e r e d by temperature at  extremely low temperatures, - 1 0 (1964)  response  to -i|.0 degrees.  Caillouet  found that l e v e l s of blood l a c t a t e of u n e x e r c i s e d  channel c a t f i s h were h i g h e r i n f i s h a c c l i m a t e d to h i g h e r temperatures, and t h a t s i m i l a r trends were noted i n e x e r c i s e d channel c a t f i s h .  Caillouet  values and great v a r i a b i l i t y to h i g h e r temperatures. Pry  and H a r t , 194$)  a  n  d  e r y t h r o c y t e s to plasma  (1964) a l s o noted h i g h e r i n d i v i d u a l among i n d i v i d u a l carp a c c l i m a t e d  A c t i v i t y i n fishes  (Pry, 1 9 4 7 J  the r a t e of d i f f u s i o n " o f l a c t a t e (Johnson e_t a l . , 1945)  from  have been shown  '  to  47  i n c r e a s e with i n c r e a s e i n temperatures.  Hayes  Hochachka and  (1962) have shown that t r o u t a c c l i m a t e d to low  temperatur  r e l y more on the pentose shunt f o r energy p r o d u c t i o n than the Embden-Meyerhoff pathway.  I t i s probable that a c t i v i t y ,  l i s m , and d i f f u s i o n are temperature trout. at  A l l of the experiments  the same temperature,  10.5  dependant i n the  metabo  rainbow  i n the p r e s e n t study were done degrees  C.  The major d i f f e r e n c e between f i s h and mammals with r e s p e c t to the l a c t a t e response i s the r a t e of r e c o v e r y of blood l a c t a t e .  A l p e r t and Root  (1954) showed that hfi% of  l a c t a t e produced by severe e x e r c i s e i n man  was  utilized  within  10 minutes; whereas B l a c k e_t a l . , (1962) showed that \xQ% of the l a c t a t e produced utilized  by severe e x e r c i s e i n rainbow  In approximately 8 hours.  trout  The slow r e c o v e r y of  blood l a c t a t e i n f i s h has been noted by others (Secondat D i a z , 191+2; B l a c k et a l . , 1 9 5 9 ) . has shown t h a t blood l a c t a t e was resting levels after exercise.  was  I n man, the l a s t  Anderson  and  (I960)  value to r e t u r n to  Heart r a t e , oxygen i n t a k e ,  carbon d i o x i d e output, and pulmonary v e n t i l a t i o n each returned to  unexercised l e v e l s b e f o r e blood  The  lactate.  f a t e of l a c t a t e produced  has been the s u b j e c t of much study.  by strenuous  Cori  exercise  (1929) observed  l a c t a t e g i v e n e i t h e r by mouth or subcutaneously enhanced glycogenosis.  that liver  That the l i v e r removes l a c t a t e was c o r r o b o r a t e d  48 ' by  other workers  ( E g g l e t o n and E v a n s ,  1930;  E v a n s e t a l . , 1930;  I v y a n d C r a n d a l l 1941:  Himwich e t a l . , 1930;  Vennesland  eta l . ,  1942.)  E g g l e t o n and E v a n s  lactate  and observed  Himwich  (1932) and M c G i n t y (1931) showed t h a t t h e h e a r t o x i d i z e s  lactate.  Evans  (1930) p e r f u s e d f r o g m u s c l e w i t h  no i n c r e a s e i n m u s c l e g l y c o g e n e s i s .  (1933), Himwich (1932), a n d A s h f o r d  showed t h a t b r a i n m e t a b o l i z e s the recovery o f l a c t a t e rapid  lactate.  Hill  (1931)  (1924) d e s c r i b e d  as a b i p h a s i c p r o c e s s ;  the i n i t i a l  p h a s e was t h e o x i d a t i o n o f m u s c l e l a c t a t e ,  and t h e l a t e r  s l o w p h a s e was t h e o x i d a t i o n o f l a c t a t e w h i c h e s c a p e d muscle t o t h e b l o o d . lactate that  Wells  (1957) r e l a t e d t h e r e c o v e r y c u r v e o f  t o t h a t o f o x y g e n c o n s u m p t i o n a n d on t h i s  r e c o v e r y was a t r i p h a s i c  basis  was r e s y n t h e s i z e d i n t o g l y c o g e n .  t i o n and g l y c o g e n e s i s a r e p r o c e s s e s which o c c u r both r e q u i r e o x y g e n . of  E g g l e t o n and E v a n s  removal o f l a c t a t e  mg f p e r m i n u t e a f t e r 0  by any  Drury  and W i c k  lactate  oxida-  i n t h e l i v e r and  found  that the rate  2 mg % p e r m i n u t e t o  removing the l i v e r .  i s resynthesized into glycogen appears  that lactate  hypothesized  decreasedfrom  (1930  Both  Recent  0.6  observations  (1956, a n d 1953} i n d i c a t e t h a t v e r y l i t t l e i f  Most o f t h e l a c t a t e observed  strenuous  (1920) t h e o r i z e d t h a t £ o f t h e l a c t a t e was  exercise, Meyerhoff and 3/4  concluded  process.  On t h e b a s i s o f o x y g e n c o n s u m p t i o n a f t e r  oxidized,  from t h e  i n the i n t a c t  as e x p i r e d carbon  dioxide.  i s o x i d i z e d f a s t e r than glucose  that lactate  a c t s as a quick f u e l  in  rabbit. They  also  and t h e y  emergencies.  4-9  There are very few e x e r c i s e i n man,  and  s t u d i e s of i n t e r m i t t e n t strenuous  a review of the l i t e r a t u r e r e v e a l e d  on i n t e r m i t t e n t strenuous e x e r c i s e i n f i s h . found t h a t a l t e r n a t i n g p e r i o d s  Christensen  of e x e r c i s e and  recovery  seconds f o r one hour d i d not cause a s i g n i f i c a n t i n blood  l a c t a t e to l£0 mg %. d u r a t i o n of work and  of 3 0  increase  i n 9 minutes and  elevated  blood  C h r i s t e n s e n a l s o showed that i f the r e s t p e r i o d s was  lactate increased.  i n Table  i n c r e a s e d , then the  Some of Christensen's  data appear  X.  Astrand  e_t a l . (I960) u s i n g experimental  s i m i l a r to C h r i s t e n s e n blood  (1956)  l a c t a t e , whereas continuous e x e r c i s e at the same  i n t e n s i t y caused exhaustion  blood  none  showed that the major f a c t o r  l a c t a t e d u r i n g i n t e r m i t t e n t e x e r c i s e was  of the e x e r c i s e p e r i o d , and p e r i o d was  conditions  the  determining duration  t h a t the d u r a t i o n of the  recovery  of secondary importance.  Man  can t o l e r a t e strenuous i n t e r m i t t e n t e x e r c i s e at  l e a s t 2 0 times longer than f i s h ,  s i n c e he recovers  from  e x e r c i s e much f a s t e r .  F l o c k e_t a l . (1939) i n t e r m i t t e n t l y e l e c t r i c a l l y stimulated  the muscle of an a n e s t h e t i z e d  that a recovery  mouse.  They found  time of g r e a t e r than 2 8 minutes was  required  between e l e c t r i c a l s t i m u l i l a s t i n g 1 minute to prevent i n c r e a s e i n muscle l a c t a t e .  an  50 Table X. i n man  Blood  l e v e l s of l a c t a t e a f t e r i n t e r m i t t e n t e x e r c i s e  from C h r i s t e n s e n  D u r a t i o n of exercise 30 seconds  (1956)•  D u r a t i o n of  D u r a t i o n of t o t a l  r e s t p e r i o d e x e r c i s e time  Blood mg  lactate %  30 seconds  30 minutes  20  1 minute  1 minute  30 minutes  J4.5  2 minutes  2 minutes  30 minutes  95  3 minutes  3 minutes  30 minutes  120  9 minutes  150  9 minutes  51 Astrand  and C h r i s t e n s e n  the b a s i s of myoglobin.  E x e r c i s e i s done a e r o b i c a l l y u s i n g  oxygen bound t o myoglobin. myoglobin i s depleted, and  e x p l a i n t h e i r r e s u l t s on  When the oxygen s t o r e bound t o  then the source of energy i s anaerobic  the r e s u l t i s l a c t a t e formation.  On t h i s b a s i s ,  r e s u l t s show that the myoglobin w i l l provide for  sufficient  oxygen  30 seconds strenuous e x e r c i s e , and that only 30 seconds  recovery The  their  time i s r e q u i r e d  t o r e l o a d the myoglobin w i t h oxygen.  r e s u l t s of C h r i s t e n s e n  and those of the present  study  demonstrate the marked d i f f e r e n c e i n the response between f i s h and man to i n t e r m i t t e n t e x e r c i s e , and are compared i n Table X I .  An  explanation  of the obvious d i s c r e p a n c y  between  the response of f i s h and man to i n t e r m i t t e n t e x e r c i s e i s difficult.  Yamaguchi and Matsuura (1961) have c h a r a c t e r i z e d  myoglobin o f tuna, and concluded that the p r o s t h e t i c group of hemoglobin and myoglobin of f i s h i n g e n e r a l the same as that of mammals.  Matsuura and Hashimoto  have c h a r a c t e r i z e d myoglobin i n s e v e r a l species and  i s probably (1956)  of f i s h  (tuna  swordfish) and found that myoglobin o f f i s h i s s i m i l a r  to that of horse h e a r t .  Matsuura and Hashimoto  (1955)  demonstrated that the i r o n content i n myoglobin of the red muscle o f f i s h greater  (Paranthunnus s i b i and Thunnus o r i e n t a l i s ) i s  than that found i n horse h e a r t .  myoglobin i s present  Assuming that  i n t r o u t muscle, then the l o n g time  52  Table XI. f i s h and  A comparison of the r e s i s t a n c e to f a t i g u e between man  d u r i n g i n t e r m i t t e n t strenuous e x e r c i s e  as  i  i n d i c a t e d by the  Animal  l e v e l of blood  Duration  Duration  of  of  exercise  rest period  .  lactate  Number of  Total  Blood  of times  time  lactate  exercised  exercised  mg  _  Reference  % .  man  30  sec.  30  sec.  60  30  min.  20  fish  30 sec.  60  min.  3  1.5  min.  97  Christensen present study  53 r e q u i r e d f o r recovery can only be explained by a slower c i r c u l a t i o n time, or a slower d i f f u s i o n time of l a c t a t e  from  muscle t o b l o o d .  The c i r c u l a t i o n time i s probably much l e s s i n f i s h than i n man.  Mott  (1957) r e p o r t s t h a t the c i r c u l a t i o n time  i n the t o a d f i s h i s about 2 minutes, whereas that of man i s only 15 t o 18 seconds  (Fishman,  1963).  Assuming that  myoglobin  i s a f u n c t i o n a l component of f i s h muscle and assuming that the c i r c u l a t i o n time i n f i s h i s 10 times that of man, then recovery from strenuous e x e r c i s e of 30 seconds' should take 10 times as long i n f i s h as i t takes i n man.  But, the r e s u l t s of the  present study show that f i s h can not r e s i s t  intermittent  strenuous e x e r c i s e when the r e c o v e r y p e r i o d Is 120 times as long.  As p r e s e n t , there appears  t o be no e x p l a n a t i o n f o r the  d i s c r e p a n c y between the response to strenuous  intermittent  e x e r c i s e between f i s h and man.  The s i g n i f i c a n t r e l a t i o n s h i p between e x e r c i s e and m o r t a l i t y i n f i s h has been reviewed by B l a c k ( 1 9 5 8 ) . as a r e s u l t of h y p e r a c t i v i t y has been observed  Mortality  i n cod and dab  (von Buddenbrock, 1 9 3 8 ) , tench (Secondat and D i a z , 191+2), s t r i p e d bass  ( L i t t , 1954-) » chinook salmon f i n g e r l i n g s  (Annonymous, 1 9 5 7 ) ,  sockeye  salmon (Black, 1957c) coho and  chinook salmon (Parker and B l a c k , 1 9 5 9 ) , ( C a i l l o u e t , 1964).  and channel c a t f i s h  I n most of these s t u d i e s m o r t a l i t y was  54 a s s o c i a t e d w i t h h i g h l e v e l s of blood  lactate.  I n the present  study there were no m o r t a l i t i e s produced by e x e r c i s e . a search  of the l i t e r a t u r e has f a i l e d  t o produce any evidence  f o r m o r t a l i t y i n rainbow t r o u t as a r e s u l t of severe Huckabee (1961) has d e s c r i b e d  The elevated blood  exercise.  a f a t a l pathological condition  i n humans i n which m o r t a l i t y i s a s s o c i a t e d acidosis.  Moreover,  with l a c t a t e  l a c t a t e i n t h i s case i s not  a s s o c i a t e d with h y p e r a c t i v i t y , b u t r a t h e r w i t h impaired pheral  peri-  circulation.  Other parameters have been a s s o c i a t e d elevated  blood  l a c t a t e of h y p e r a c t i v i t y .  observed a decreased blood w i t h an elevated  blood  w i t h the  Secondat  (1950)  oxygen c a p a c i t y which was c o r r e l a t e d  l a c t a t e i n the carp.  Jonas e_t a l .  (1962) showed that m o r t a l i t y i n rainbow t r o u t could be produced by i n j e c t i o n s of l a c t i c a c i d but not w i t h sodium l a c t a t e .  (1942) observed that  Auvergnat and Secondat  15  minutes e x e r c i s e caused a decreased pH i n carp b l o o d .  The pH  d i d not r e t u r n to normal a f t e r a 6 hours r e s t p e r i o d .  They  a t t r i b u t e d t h i s a c i d i f i c a t i o n of blood lactic  a c i d from muscle t o blood.  to the r e l e a s e of  A decrease i n blood  carbon  d i o x i d e accompanied the decrease i n pH, and the carbon d i o x i d e was s t i l l  decreasing  6 hours a f t e r the e x e r c i s e .  noted that the osmotic pressure  of plasma was  They a l s o  elevated  immediately a f t e r the e x e r c i s e , and remained elevated  f o r 4 hours.  55 The i n c r e a s e d osmotic p r e s s u r e of plasma was a t t r i b u t e d presence of by-products  of muscle metabolism.  (1959) made s i m i l a r s t u d i e s i n rainbow that both blood pH and blood first  3 minutes  trout.  t o the  B l a c k e_fc a l . They observed  carbonate i n c r e a s e d d u r i n g the  of strenuous e x e r c i s e , and then both decreased  d u r i n g the remaining 12 minutes  of e x e r c i s e .  Blood pH c o n t i -  the l e v e l b e i n g s i g n i f i c a n t l y depressed 2 hours  nued t o f a l l ,  a f t e r the e x e r c i s e .  The h i g h e s t l e v e l of carbonate occurred  8 hours a f t e r the e x e r c i s e .  B l a c k noted that the c h a r a c t e r i s t i c  change i n b e h a v i o r of e x e r c i s e d t r o u t occurred at the same time as the maximum l e v e l s of hydrogen blood carbonate.  Thus, l a c t i c  i o n and maximum l e v e l s of  a c i d i s n e u t r a l i z e d a t the  expense of plasma b i c a r b o n a t e , but a s i g n i f i c a n t decrease i n blood pH s t i l l  occurs a f t e r 15 minutes  (Auvergnat and Secondat,  In  of severe e x e r c i s e  19^2).  summary; the l e v e l of blood l a c t a t e , and thus of  muscle l a c t a t e , i s the r e s u l t of a great number of d y n a m i c a l l y operating factors.  Those f a c t o r s which probably c o n t r i b u t e  most t o the v a r i a b i l i t y  i n the observed data are sex and age.  III. A.  R e s u l t s - Muscle  GLYCOGEN Glycogen  The mean l e v e l of muscle glycogen i n unexercised f i s h was 251 mg % (mg per 100 grams wet weight  of t i s s u e ) .  56 The  e f f e c t s of e x e r c i s e , recovery, i n P i g . IIL., 15,  are i l l u s t r a t e d  The  16,  and and  intermittent 17.  l e v e l of muscle glycogen i n unexercised  decreased d u r i n g the course of the summer from 2 5 l mg  exercise  to  fish 213  %.  E x e r c i s e , even of the s h o r t e s t d u r a t i o n , immediate decrease i n muscle glycogen. that one-half  caused  P i g . llj. i l l u s t r a t e s  of the muscle glycogen present  in  unexercised  as a r e s u l t of only. 30 seconds e x e r c i s e .  f i s h i s depleted  an  The  r a t e of u t i l i z a t i o n of muscle glycogen decreased with f u r t h e r exercise.  15  Pig. exercised  illustrates  1 minute and  that muscle glycogen i n f i s h  2 minutes continued  to decrease f o r 3  minutes a f t e r the e x e r c i s e , whereas that of f i s h e x e r c i s e d minutes decreased only s l i g h t l y . exercised  2 and  5 minutes f a i l e d  5  Muscle glycogen of f i s h to recover  i n 30 minutes  a f t e r the e x e r c i s e , whereas that of f i s h e x e r c i s e d  1 minute  did  to r e t u r n  recover  slightly.  to unexercised illustrated  l e v e l s even a f t e r extremely short d u r a t i o n i s  i n P i g . 16  The  That muscle glycogen f a i l e d  and  17.  e f f e c t s of r e - e x e r c i s e a f t e r 60 minutes  recovery  are to cause f u r t h e r decrease i n l e v e l s of muscle glycogen,  57  Pig. severe  Changes exercise.  i n levels  of muscle  glycogen during  5  minutes  58  P i g . 15.  Changes i n l e v e l s of muscle glycogen d u r i n g r e c o v e r y  from severe e x e r c i s e of 1,  2,  and 5  minutes.  59  third exercise  second exercise  first exercise 250-1 *  E o CD  o o >> O  200-  150-  0) 100-  o  CO  50/  o-J r  r~ r~ 60 30 o 60 Duration of recovery in minutes between —  —  30  intermittent exercise periods of. 15 sec.  F i g . 16 levels  Effect  of i n t e r m i t t e n t  of muscle glycogen.  exercise  of 15  seconds on the  60  first exercise  second exercise  third exercise  250-1  200-  E CD E c 150CD O) O O  o  100-  o CO 3  0 30 60 30 60 Duration of recovery between exercise and re - exercise.  Pig. the  17.  E f f e c t of i n t e r m i t t e n t e x e r c i s e of 30  l e v e l s of muscle glycogen.  seconds  on  61  but there appeared  t o be a lower l i m i t  which muscle glycogen d i d not exceed  (approximately 20 mg fo)  ( F i g . 16 and 1 7 ) .  The  l e v e l s of muscle glycogen a f t e r i n t e r m i t t e n t e x e r c i s e appear i n the Table X I I .  B. R e s u l t s - L i v e r  Glycogen  The mean l e v e l of l i v e r glycogen i n unexercised f i s h was 4 . 9 0 g % (g per 100 g wet weight).  The  l e v e l o f l i v e r glycogen decreased  d u r i n g the  summer as d i d muscle glycogen.  The changes i n the l e v e l s of l i v e r glycogen d u r i n g e x e r c i s e , d u r i n g r e c o v e r y , and d u r i n g i n t e r m i t t e n t e x e r c i s e were not s t a t i s t i c a l l y s i g n i f i c a n t i n the present study. C h a r a c t e r i s t i c a l l y , the l i v e r glycogen samples have a very l a r g e Inherent v a r i a b i l i t y .  L e v e l s of glycogen a f t e r e x e r c i s e and  r e - e x e r c i s e of 30 seconds  appear i n Table X I I I .  The changes  i n l e v e l s i n l i v e r glycogen d u r i n g 5 minutes severe e x e r c i s e appear i n Table XIV.  The r e s u l t s o f an A n a l y s i s of Variance  on the data i n Table XIV appear i n Table XV.  Changes i n  l e v e l s of l i v e r glycogen d u r i n g 5 minutes severe e x e r c i s e were not s i g n i f i c a n t .  62  Table X I I .  Effect  of e x e r c i s e and r e - e x e r c i s e  of muscle glycogen. periods,  on the l e v e l  P i s h r e s t e d 60 minutes between e x e r c i s e  and were sampled  immediately a f t e r exercise, or  re-exercise.  D u r a t i o n of. .exercise Muscle  first  chase  glycogen  second chase  mg %  t h i r d chase-  15 s e c .  3.0. sec.  5 - min.  113  i+9  Til  85  2J4.  83  59  161  63 Table X I I I .  E f f e c t of e x e r c i s e and r e - e x e r c i s e of  on the l e v e l of l i v e r glycogen. between e x e r c i s e p e r i o d s ,  seconds  3 0  P i s h r e s t e d 60 minutes  and were sampled  immediately a f t e r  e x e r c i s e or r e - e x e r c i s e .  L i v e r glycogen  (9$%  mean first  chase  2  .  1  second chase  2  .  third  3  .  chase  +  .  9 0  (grams  0  7  8 2  4 7  %)  confidence i n t e r v a l )  (1.592  to  3 . 2 2 2 )  (2.225 to  3 . 7 4 3 )  (3.027  to  4  .  O  7  4  )  64  Table XIV.  E f f e c t of £ minutes severe e x e r c i s e , and 30 minutes  r e c o v e r y a f t e r 5 minutes severe e x e r c i s e on the l e v e l s of liver  glycogen.  l i v e r glycogen  Condition  (grams %)  mean ± standard d e v i a t i o n unexercised  4.90  +  1.40  chase 1  min.  4.54  +  2.11  chase 2  min.  4.83  +  1.30  chase 5  min.  5.21  +  1.46  3.36  +  0.88  chase 5 min.,  r e s t 30  rain.  65  Table XV.  A n a l y s i s of v a r i a n c e of l i v e r glycogen data from  Table XIV d u r i n g and a f t e r 5 minutes severe e x e r c i s e .  Source o f  degrees o f  Sum of  Mean  variation  , freedom  squares  square  total treatment error  38 LL 3k  P  F  . 0 5  95-7162 15.81J.75  3.96  79.8687  2.35  1.69.  2.65  66  C.  D i s c u s s i o n - Muscle and L i v e r  Glycogen.  The l e v e l s of muscle glycogen r e p o r t e d i n the present study are s i m i l a r to v a l u e s . i n the l i t e r a t u r e f o r Salmo g a i r d n e r i i . c i s e d rainbow  The l e v e l s o f muscle glycogen i n unexer-  t r o u t appear i n Table XVI.  Claude Bernard  (1876) was the f i r s t  presence of glycogen i n f i s h . glycogen content i n f i s h  t o r e p o r t the  Other e a r l y workers a l s o noted  (Schondorff and Wachholder, 191i|.;  K i l b o r n and MacLeod, 1919; and D i l l ,  1921).  I n g e n e r a l , the  l e v e l of muscle glycogen i s lower i n f i s h than i n mammals (West and Todd r e p o r t the l e v e l of muscle glycogen i n man to be O.ij. to 0.6 g % ) .  The l e v e l of l i v e r glycogen i s  s i m i l a r I n man and rainbow  trout.  Prom year t o year there i s a'marked v a r i a t i o n i n the l e v e l s of muscle glycogen among f i s h t r e a t e d at  Summer l a n d .  similarly  M i l l e r §t_ a l . (195-9) have shown that changes i n  d i e t can cause changes i n l e v e l of muscle glycogen.  In  Kamloops t r o u t , B l a c k et_ al_. (1962) found that the l e v e l of muscle glycogen was h i g h e r i n males than i n females d u r i n g r e s t , e x e r c i s e , and d u r i n g r e c o v e r y from e x e r c i s e . s i g n i f i c a n c e that B l a c k and h i s co-workers  only  I t i s of  demonstrated  t h i s sex differenceorjP-ej i n l a t e r s t u d i e s the sex d i f f e r e n c e was  not s i g n i f i c a n t .  I t i s probable that the e f f e c t of  67  Table XVI.  L e v e l s of muscle glycogen I n unexercised  rainbow  trout.  Muscle glycogen S.fo  .  .  Diet . .  .  Reference ......  0.085  ground mammalian l i v e r and f i s h v i s c e r a  B l a c k e_t a l . , I960  0.118  beef l i v e r  Miller  0.178  C l a r k ' s t r o u t feed  M i l l e r et a l . , 1959  0.251  C l a r k ' s t r o u t feed  present  et a l . , 1959  study  68  severe e x e r c i s e or carbohydrate metabolism  w i l l produce  r e s u l t s between the sexes only i n s e x u a l l y mature Hochachka (1961) has  different  fish.  shown that age, stage of maturation,  and  t r a i n i n g each a f f e c t the l e v e l of muscle glycogen i n t r o u t . Moreover, g l y c o l y s i s d u r i n g sampling w i l l d e p l e t e the muscle glycogen by 3 0 $ i n 5 minutes at room temperature  (Black et a l . ,  1960).  Recently, the a n a l y t i c a l method f o r glycogen has been criticized  ( O r r e l l and Bueding,  1964).  These workers i n d i c a t e  t h a t values of muscle glycogen obtained w i t h potassium  hydroxide  d i g e s t s are r...similars rt 6,1. c.n the values obtained by other procedures.  The  first  experimental study of the d e p l e t i o n of  muscle glycogen i n f i s h d u r i n g e x e r c i s e was et  a l . (I960).  M i l l e r et a l .  (1959)  done by B l a c k  have a l s o s t u d i e d the  e f f e c t of e x e r c i s e on muscle on muscle glycogen i n f i s h . B l a c k and M i l l e r observed  that one-half of the muscle  i s depleted by 2 minutes severe e x e r c i s e . to  Both  glycogen  This i s i n contrast  the f i n d i n g s of the present study i n which one-half of the  muscle glycogen was  utilized  that over two-thirds was  w i t h 30 seconds  utilized  i n 2 minutes severe  B l a c k e_fc a l . (I960) showed that about glycogen was  utilized  exercise,  80$  and exercise.  of the muscle  i n 1 5 minutes severe e x e r c i s e , whereas  i n the present study 80$ was  utilized  i n 5 minutes severe  69 exercise.  There are two p o s s i b l e e x p l a n a t i o n s f o r the  r e p o r t e d above.  First,  results  that the previous workers did not  determine the l e v e l of muscle glycogen a f t e r 30 seconds exercise.  And  the unexercised  severe  second, that the l e v e l of muscle glycogen i n f i s h was  h i g h e r i n the f i s h t h a t I s t u d i e d than  i n the f i s h s t u d i e d by Black or M i l l e r .  B l a c k e_t a l . , (1961) have r e l a t e d i n muscle glycogen and  the r a p i d  decrease  to the r a p i d i n c r e a s e i n muscle l a c t a t e  to the change i n b e h a v i o r .  They noted  that f i s h  swim r a p i d l y a f t e r 2 minutes severe e x e r c i s e .  rarely  S i m i l a r changes  i n behavior occurred i n the present study, but the changes i n swimming v e l o c i t y were not q u a n t i t a t i v e l y  determined.  Hochachka (1961) pointed out t h a t muscle i s r a r e l y depleted to zero, no matter how the e x e r c i s e .  Muscle glycogen was  i n the present experiment.  lactic  severe or how  never completely  presence  a c i d i n the t i s s u e s w i l l i n c r e a s e the hydrogen i o n  enzyme i n the g l y c o g e n o l y t i c or g l y c o l y t i c Cori  depleted  For example, the  content which could cause i n a c t i v a t i o n of some a c i d  and  long  A possible explanation f o r this i s  that a p r o t e c t i v e mechanism e x i s t s . of  glycogen  chain.  sensitive Keller  (1955) have shown that the enzyme phosphorylase  i n a c t i v a t e d i n the presence  is  of h i g h hydrogen i o n c o n c e n t r a -  t i o n , and Hers e_t a l . (1961+) have shown that a m y l o - 1 , 6 g l u c o s i d a s e i s i n a c t i v a t e d at about pH  1+.5.  70 B l a c k et_ a l . (1962) has observed that the l e v e l of f o r 1$  muscle glycogen i n rainbow t r o u t s e v e r e l y e x e r c i s e d minutes d i d not r e t u r n to normal d u r i n g a Zlx. hour period.  I n the present  study the l e v e l of muscle glycogen  did not r e t u r n t o the unexercised recovery  recovery  l e v e l d u r i n g a 60 minute  p e r i o d from only 15 seconds severe e x e r c i s e .  Black  et a l . (I960) have a l s o shown t h a t the muscle glycogen recovers  f a s t e r I f the f i s h are f e d d u r i n g r e c o v e r y .  glycogen r e t u r n s The  Muscle  t o normal f a s t e r i n mammals than i n f i s h .  slow r e s y n t h e s i s  of muscle glycogen i n f i s h could be due  to any o f the f o l l o w i n g : 1.  Enzyme i n h i b i t i o n due to the elevated  hydrogen c o n c e n t r a t i o n elevation of l a c t i c 2.  associated  w i t h the prolonged  a c i d i n the muscle.  The metabolism of the f i s h i s lower s i n c e  the temperature i s lower than that of mammals. 3.  The l a c k of n u t r i e n t s r e q u i r e d  s i z e t h e muscle glycogen due t o impaired would slow glycogen  t o resynthecirculation  synthesis.  To my knowledge, there have been no previous s t u d i e s on the e f f e c t of i n t e r m i t t e n t e x e r c i s e on f i s h , and, no  s t u d i e s on the e f f e c t of ^'intermittent e x e r c i s e on muscle  glycogen l e v e l s i n any i n t a c t animal.  F l o c k e_t a l . ( 1 9 3 9 )  i n t e r m i t t e n t l y e l e c t r i c a l l y stimulated  the l e g muscle of mice  and  measured l a c t a t e and glycogen l e v e l s .  They found that the  71 muscle glycogen was almost completely depleted a f t e r  stimu-  l a t i o n f o r 1 minute, and t h a t the l e v e l of muscle glycogen changed l i t t l e t h e r e a f t e r even though the muscle continued to  contract.  These workers t h e r e f o r e p o s t u l a t e d that  glycogen serves as the i n i t i a l source of energy, but that there must a l s o be some other source of energy. is  the major source of energy  I f muscle glycogen  f o r muscle c o n t r a c t i o n d u r i n g  severe e x e r c i s e i n t r o u t , then the present study  indicates  that muscle glycogen r e s e r v e s a r e p r o b a b l y the l i m i t i n g in  exercise.  factor  Moreover, the frequency o f severe e x e r c i s e of  short d u r a t i o n must be v e r y low--that i s , only a few times per day.  Heath and P r i t c h a r d consumption remains  (1962) demonstrated  e l e v a t e d f o r about  2l\. hours  that oxygen  after  e x e r c i s e of 6 t o l\S minutes i n the b l u e g i l l s u n f i s h . a l s o measured blood l a c t a t e l e v e l s and observed  severe They  that the  l a t t e r r e t u r n e d t o p r e - e x e r c i s e l e v e l s w i t h i n 10 hours.  It  i s probable that the e l e v a t e d oxygen consumption d u r i n g the first  10 hours  produced  i s a s s o c i a t e d w i t h o x i d a t i o n o f the l a c t a t e  by e x e r c i s e , and that the e l e v a t e d oxygen consumption  from 10 t o 2i|, hours glycogen.  i s a s s o c i a t e d w i t h r e - s y n t h e s i s of muscle  72  IV.  The R e l a t i o n s h i p  between Glycogen and L a c t a t e i n Muscle*.  Meyerhoff (1920) was the f i r s t  t o demonstrate  that muscle glycogen was the source o f l a c t a t e d u r i n g Black  (1957c) presumed that the source of blood l a c t a t e i n  exercised  t r o u t was muscle glycogen.  demonstrated that and  exercise.  Black e_t a_l. (I960)  the r e l a t i o n s h i p between muscle glycogen  blood l a c t a t e i s c u r v i l i n e a r .  My data  substantiates  B l a c k ' s data s i n c e i n the present study the r e l a t i o n s h i p between blood and muscle l a c t a t e i s c u r v i l i n e a r . extrapolation  Thus, an  from Black's graphs would y i e l d a l i n e a r r e l a -  t i o n s h i p between muscle glycogen and muscle l a c t a t e .  To demonstrate the r e l a t i o n s h i p between l a c t a t e and  glycogen i n muscle a c o r r e l a t i o n a n a l y s i s  the means o f 11 samples. 0.934) i n d i c a t e d variables  that  i s highly  (the c a l c u l a t e d  The c o r r e l a t i o n c o e f f i c i e n t (r = -  the r e l a t i o n s h i p between these two  significant.  regression  The observed  glycogen appear as 180 mg. o f l a c t a t e .  the p o i n t s  162 mg. of  At lower l e v e l s of  (that i s , low l a c t a t e and h i g h glycogen f i t the t h e o r e t i c a l l i n e very w e l l .  h i g h e r l e v e l s of e x e r c i s e  levels) But, at  (that i s , low glycogen and h i g h  l a c t a t e l e v e l s ) a l l of the p o i n t s line.  relationship  l i n e ) i s compared w i t h the theore-  t i c a l r e l a t i o n s h i p i n P i g . 18 assuming that  exercise  was done on  are  above the t h e o r e t i c a l  These l a t t e r r e s u l t s i n d i c a t e that  at the h i g h e r  73  i  1  0  50  1  100  1 150  1 200  1 250  Muscle Glycogen mgm.96  F i g . 18.  The r e l a t i o n s h i p between muscle l a c t a t e and muscle  glycogen i n f i s h sampled immediately  after  exercise.  Ik l e v e l s of e x e r c i s e s t u d i e d i n the present experiment i s a source of muscle l a c t a t e other than muscle The  there  glycogen.  s l o p e of the l i n e of the t h e o r e t i c a l r e l a t i o n s h i p  lies  w i t h i n the 95$ confidence l i m i t s of the slope o f the c a l c u l a t e d r e g r e s s i o n l i n e , but not w i t h i n the 99$ confidence l i m i t s of the slope of the c a l c u l a t e d r e g r e s s i o n l i n e .  The slope of  the l i n e o f the t h e o r e t i c a l r e l a t i o n s h i p i s -1.11.  The 95$  c o n f i d e n c e l i m i t s are -1.05 to -1.91, and the 99$ confidence l i m i t s are - l . l l f . to - 1 . 8 2 .  In Table XVII, the percent of l a c t a t e not accounted f o r by the decrease i n glycogen i s h i g h only f o r severe e x e r c i s e l a s t i n g f o r more than one minute.  There are f o u r '  p o s s i b l e e x p l a n a t i o n s f o r the r e s u l t s recorded i n F i g . 18 and Table  XVII.  1.  A c t u a l glycogen present i s g r e a t e r than  that measured. 2.  A c t u a l l a c t a t e present i s l e s s than that  measured. 3.  Glycogen  i s being r a p i d l y synthesized  during exercise. ij..  There i s a source of l a c t a t e other than  glycogen d u r i n g the l a t e r stages of prolonged severe muscular e x e r c i s e .  Table XVII.  The percent of muscle l a c t a t e not accounted f o r by the decrease i n the  l e v e l of muse l e glycogen. ...... Exercise condition  L e v e l of muscle glycogen mg %  ....  Decrease i n muscle glycogen levels mg %  L e v e l of muscle lactate mg %  L Increase i n muscle lactate. levels mg %  T Theoretical increase i n l e v e l of muscle l a c t a t e mg fo  (T-L) : . L. .  unexercised  251  0  132  0  0  0  15 s e c .  161  90  236  104.  100  4  30 s e c .  113  138  291  159  153  k  15 sec. (twice)  111  14.0  276  144  156  0  15 s e c . (3 times)  83  168  304  172  187  0  1 min.  9?,  159  306  174  177  0  30 s e c . (twice)  85  166  366  234  184  21  30 s e c . (3 times)  59  192  358  226  213  6  2 min.  74  177  387  255  197  23  5 min.  49  202  468  336 .  224  33  5 min. (twice)  24  227  462  330'  252  24  (T-L) ~~L~  x 100$  • •  •  Percent of muscle l a c t a t e not accounted f o r by the decrease In the l e v e l of muscle glycogen assuming that 162 mg of glycogen appear as 180 mg of l a c t a t e .  76  The a n a l y t i c a l methods f o r d e t e r m i n a t i o n of glycogen have been r e c e n t l y c r i t i c i z e d  by O r r e l and Bueding  (19614.).  These workers r e p o r t e d t h a t e x t r a c t i o n w i t h hot a l k a l i  yields  a b s o l u t e values s i m i l a r t o other methods of e x t r a c t i o n ; but t h a t the glycogen i s o l a t e d w i t h a l k a l i has d i f f e r e n t c h a r a c t e r istics  than t h a t obtained by l e s s severe e x t r a c t i o n techniques.  The method used to determine was  lactate concentration  c r i t i c a l l y examined by Barker and Summerson (19I4.I) i n the  o r i g i n a l paper substances  on t h i s method.  I t i s u n l i k e l y that  any  i n t e r f e r e with lactate determination i n s u f f i c i e n t  q u a n t i t i e s to i n v a l i d a t e the evidence f o r a source of l a c t a t e other than muscle glycogen.  There i s no  evidence, to my  knowledge, f o r or a g a i n s t  s y n t h e s i s of muscle glycogen d u r i n g e x e r c i s e i n f i s h . measurements of the r a t e of r e s t o r a t i o n of muscle  But,  glycogen  a f t e r 15 minutes severe e x e r c i s e by B l a c k et a l . (I960) i n d i c a t e that muscle glycogen s y n t h e s i s i s a slow process i n fish.  T h e r e f o r e i t i s probable that there i s a source of  l a c t a t e other than muscle glycogen d u r i n g prolonged  muscular  exercise.  If  liver  I would expect two 1.  glycogen i s a source of muscle l a c t a t e , results: a d e p l e t i o n of l i v e r  glycogen d u r i n g  then  77  e x e r c i s e , and 2.  an elevated blood  M i l l e r e_fc a_l. ( 1 9 5 9 ) i s i n f a c t depleted  glucose  during exercise.  demonstrated that l i v e r  d u r i n g 15 minutes e x e r c i s e i n t r o u t i f  the i n i t i a l l e v e l of muscle and l i v e r glycogen and  Goodnight  i s low.  decreased  l i v e r glycogen  a f t e r d e p l e t i o n of muscle glycogen  stores.  significantly  The l e v e l s of l i v e r  d i d not 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 or  d u r i n g i n t e r m i t t e n t e x e r c i s e i n the present  E l e v a t i o n o f blood  glucose  study.  d u r i n g e x e r c i s e has not  been demonstrated i n rainbow t r o u t .  Secondat  t h a t e x e r c i s e u s u a l l y elevated blood  glucose  the i n c r e a s e was not c o n s i s t e n t . found t h a t blood  glucose  (1950) found i n carp, but that (1957b)  I n lake t r o u t B l a c k  i n c r e a s e d a f t e r 15 minutes  severe  e x e r c i s e and remained elevated throughout the 24 hour period  studied.  Goodnight  (1964) found that 15 minutes e x e r c i s e always  I f glucose  involves adrenaline.  a d r e n a l i n e i n c r e a s e s blood glucose  excitement  glucose  does i n c r e a s e i n response to e x e r c i s e ,  the mechanism probably  blood  recovery  I n bass, c r a p p i e , and b u l l h e a d , Dean and  produced a s t a t i s t i c a l s i g n i f i c a n t i n c r e a s e i n blood level.  Dean  (1964) found that 15 minutes e x e r c i s e i n bass,  c r a p p i e , and b u l l h e a d  glycogen  glycogen  glucose  In mammals,  l e v e l s , and the e l e v a t e d  d u r i n g muscular a c t i v i t y appears t o be r e l a t e d t o  (Peters and Van S l y k e , 1 9 4 6 ) .  78 Other p o s s i b l e sources of l a c t a t e i n c l u d e c a t a b o l i s m lipid  and p r o t e i n from blood or from the muscle i t s e l f ,  a b s o r p t i o n of f o o d s t u f f s from the gut.. examined i n the present study.  None of these were  Fontaine and Hatey (1953) have  shown t h a t both f a t and p r o t e i n are metabolized upstream m i g r a t i o n of A t l a n t i c  and by-  salmon.  Blazka  d u r i n g the (1958) has  r e p o r t e d that e x e r c i s e e l e v a t e s ammonia and n o n - p r o t e i n n i t r o g e n i n carp b l o o d . Hypoxia e l e v a t e s non e s t e r i f i e d (but  does not e l e v a t e l a c t a t e ) i n t r o u t blood  In  f a t t y acids (Blazka, 1958.)  summary, the present study p r o v i d e s  evidence  that there i s a source of muscle l a c t a t e other than muscle glycogen, and t h a t t h i s source of energy of  lipid  i s probably c a t a b o l i s m  or p r o t e i n .  V. General D i s c u s s i o n I t i s evident t h a t i n f i s h the changes of carbohydrate metabolism d u r i n g severe e x e r c i s e a r e s t i l l not completely understood.  In g e n e r a l , metabolism appears  t o be s i m i l a r i n  f i s h and mammals i n t h a t glycogen i s broken down d u r i n g severe e x e r c i s e .  When the supply of oxygen does not meet the demand, then the reduced  forms of  the Kreb's c y c l e f a i l s  DPNH2  A  N  D  FADH2  accumulate.  Thus,  t o operate due to l a c k of supply of the  79  o x i d i z e d forms of DPN and PAD.  In t u r n , pyruvate backs up i n  the chemical c h a i n to form l a c t a t e . of and  As a r e s u l t ,  accumulation  l a c t a t e i n d i c a t e s the i n s u f f i c i e n c y of the oxygen supply a l s o muscular e x e r t i o n .  hydrogen acceptor f o r  L a c t a t e a l s o serves as an  DPWH2'  During recovery from severe e x e r c i s e , l a c t a t e i s e i t h e r excreted or converted back t o pyruvate.  The pyruvate  so formed i s then o x i d i z e d to carbon d i o x i d e and water, or r e s y n t h e s i z e d t o glycogen.  Resynthesis  of glycogen i s  p r o b a b l y from blood glucose r a t h e r than from muscle and  i s a slow process i n f i s h .  lactate,  The above scheme i s i l l u s t -  rated i n F i g . 19.  I t i s p o s s i b l e t o e x p l a i n the slowness of recovery from severe e x e r c i s e on t h e b a s i s of the c i r c u l a t o r y system u s i n g 30 seconds severe e x e r c i s e as an example.  1.  Severe  e x e r c i s e f o r 30 seconds e l e v a t e s muscle  l a c t a t e 160 mg per 100 g of muscle above the unexercised level. of  Since 1 ml of oxygen i s r e q u i r e d to convert 8 . 0 3 mg  l a c t a t e t o pyruvate, then 160 mg of l a c t a t e r e q u i r e s 20  ml o f oxygen.  2.  I n the present study the hemoglobin c o n c e n t r a t i o n  was s u f f i c i e n t t o t r a n s f e r a maxium o f 11 ml o f oxygen  80 per  100 ml b l o o d .  Thus t o t r a n s f e r 20 ml of oxygen,  182  ml of blood i s r e q u i r e d .  3.  The c a r d i a c output i n f i s h i s s m a l l , and i s  probably about 1 ml/100 g body weight/minute f o r rainbow trout  (Hart, 191+3).  In order to pump 182 ml of blood/100 g  body weight, the f i s h would r e q u i r e 182 minutes, or approxi m a t e l y 3 hours to r e c o v e r from 30 seconds severe e x e r c i s e .  81 IN THE PRESENCE OF ADEQUATE OXYGEN - UNEXERCISED CONDITION muscle  I  glycogen  ^  _  muscle pyruvate ^ muscle l a c t a t e ^ ^-oxygen muscle carbon d i o x i d e  ~blood  lactate  • b l o o d carbon d i o x i d e  IN THE PRESENCE OF AN INSUFFICIENT OXYGEN SUPPLY - SEVERE EXERCISE muscle  I  glycogen  _  muscle pyruvate"" - y muscle l a c t a t e ^ ^-oxygen muscle carbon d i o x i d e  , blood  lactate  RECOVERY FROM AN INSUFFICIENT OXYGEN SUPPLY - RECOVERY FROM SEVERE EXERCISE excreted"^  blood^lactate  r  tissue  •liver  glycogen  lactate  t i s s u e pyruvate ^-oxygen t i s s u e carbon d i o x i d e — • b l o o d carbon d i o x i d e  Fig. 19.  The e f f e c t of oxygen supply on carbohydrate  metabolism.  82  CONCLUSIONS  Some g e n e r a l c o n c l u s i o n s present  study.  can be made from the  I n rainbow t r o u t , severe e x e r c i s e  muscle glycogen extremely r a p i d l y .  Moreover, i n f i s h , the  r e s t o r a t i o n of muscle glycogen t o p r e - e x e r c i s e slow p r o c e s s .  depletes  l e v e l s i s a.  I n c o n t r a s t t o t h i s , the r a t e of d e p l e t i o n  of muscle glycogen d u r i n g moderate e x e r c i s e i s s m a l l et a l . I960). elevated  Blood  l a c t a t e continues  (Black  t o r i s e and remains  f o r a t l e a s t 30 minutes f o l l o w i n g e x e r c i s e of  extremely short d u r a t i o n , t h a t i s , 3 seconds. severe e x e r c i s e a f t e r recovery f u r t h e r i n c r e a s e s i n blood  periods  Intermittent  of 60 minutes causes  and muscle l a c t a t e , and f u r t h e r  decreases i n muscle glycogen.  I n c o n c l u s i o n , i t appears  that rainbow t r o u t are not w e l l adapted t o t o l e r a t e severe exercise.  83  SUMMARY  1.  The e f f e c t s of i n t e r m i t t e n t e x e r c i s e and  e x e r c i s e of short d u r a t i o n  ( 3 seconds to 1 minute) on  carbohydrate metabolism have been s t u d i e d i n rainbow t r o u t . 2.  The e f f e c t of severe e x e r c i s e o f short  duration  was t o cause the f o l l o w i n g : a.  an immediate decrease i n muscle glycogen  levels.  One-half of the muscle glycogen was  utilized  d u r i n g 3 0 seconds severe e x e r c i s e , and  Q0% u t i l i z e d b. levels.  d u r i n g 5 minutes e x e r c i s e .  an immediate i n c r e a s e i n muscle l a c t a t e Muscle l a c t a t e i n c r e a s e d more than two  f o l d d u r i n g 3 0 seconds severe e x e r c i s e , and i n c r e a s e d more than three and one-half  fold  during  5 minutes severe e x e r c i s e . c. levels.  an immediate i n c r e a s e i n blood l a c t a t e Blood  l a c t a t e l e v e l s increased  two f o l d  d u r i n g 3 0 seconds severe e x e r c i s e , and i n c r e a s e d more than t e n f o l d d u r i n g 5 minutes severe e x e r c i s e . d.  an i n i t i a l r a p i d i n c r e a s e i n hemoglobin  c o n c e n t r a t i o n , followed by a r a p i d decrease i n hemoglobin c o n c e n t r a t i o n , and  t h i s i n t u r n followed  by a slow i n c r e a s e i n hemoglobin c o n c e n t r a t i o n . 3. severe  During  recovery periods  of 3 to 60  minutes  after  e x e r c i s e the f o l l o w i n g o c c u r r e d . a.  Muscle glycogen  l e v e l s changed very  b.  Muscle l a c t a t e l e v e l s changed very  c.  Blood  l a c t a t e l e v e l s continued  the recovery periods  little. little.  to r i s e  during  even i n f i s h chased 3 seconds.  T h i s i s i n complete c o n t r a d i s t i n c t i o n to f i n d i n g s i n mammals. d.  Hemoglobin l e v e l s continued  to r i s e  during  the r e c o v e r y p e r i o d s even i n f i s h chased 3 seconds. The  e f f e c t of r e - e x e r c i s e was  to cause the  a.  an immediate f u r t h e r decrease i n muscle  following:  glycogen  l e v e l s u n t i l the muscle glycogen  depleted  to approximately  b.  20 mg  was  %.  an immediate f u r t h e r i n c r e a s e i n muscle  l a c t a t e l e v e l s u n t i l muscle l a c t a t e l e v e l reached approximately c. of blood 5.  20 mg  %.  an immediate f u r t h e r i n c r e a s e i n l e v e l s l a c t a t e and hemoglobin c o n c e n t r a t i o n .  L e v e l s of l i v e r glycogen  d u r i n g e x e r c i s e , recovery,  did not appear to change  or r e - e x e r c i s e .  L i v e r glycogen  are c h a r a c t e r i z e d by t h e i r l a r g e v a r i a b i l i t y .  values  85 6. exercise. to  sex,  No  No  consistent  weight, 7.  mortalities  probably a source  of muscle  a p p e a r t o be  were n o t e d  o r sample  i s presented  glycogen d u r i n g 5 minutes does n o t  differences  c a t c h time,  Evidence  o c c u r r e d as a r e s u l t  lactate  severe  the source  of  severe  with respect  time.  which i n d i c a t e s other than  exercise. of t h i s  muscle  Liver  muscle  there i s  glycogen  lactate.  86  AREAS FOR  I t would be using  steelhead  species  but  FURTHER INVESTIGATION  i n t e r e s t i n g to repeat the  t r o u t , which i s a d i f f e r e n t race of the same  generally  thought to be a more a c t i v e  Much of the v a r i a b i l i t y i s probably due controlled. of age,  s i z e , sex,  and  studied.  r e v e a l the  findings  i n man  i n the  by  Christensen  the c i r c u l a t o r y system  c i r c u l a t i o n time are r e q u i r e d .  i t i s e s s e n t i a l that the  of the venous r e s e r v o i r s and c e l l r e s e r v o i r s be  fish  In p a r t i c u l a r an accurate estimate of  output and  exercise,  of  are  c i r c u l a t o r y systems of  e x p l a i n the observed changes i n blood hemoglobin during  effects  d i f f e r e n c e s between f i n d i n g s  i t i s of importance that  the c a r d i a c  study  maturity.  a t t r i b u t e d to d i f f e r e n c e s  i n f i s h be  i n data of the present  A more complex d e s i g n may  the present study and  man,  fish.  to many of the v a r i a b l e s which were not  Because many of the  and  experiment  the  ascertained.  concentration  l o c a t i o n and  l o c a t i o n and  To  control  c o n t r o l of  red  87 I t would a l s o be p r o f i t a b l e to study the r o l e of myoglobin  i n rainbow t r o u t .  That i s , to determine  t r a t i o n and i t s d i s s o c i a t i o n curve i n order to whether or not i t i s f u n c t i o n a l i n t h i s  i t s concen-  demonstrate  animal.  To e x p l a i n the unknown source of muscle l a c t a t e d u r i n g 5 minutes severe e x e r c i s e , i t i s necessary to study changes i n l e v e l s of p r o t e i n s and the blood.  lipids  i n the muscle and  I t would a l s o be of i n t e r e s t to determine  the  r a t e of turnover o f g l u c o s e d u r i n g e x e r c i s e and r e c o v e r y .  88 BIBLIOGRAPHY  A l p e r t , N. A. and W. S. R o o t . 1954. R e l a t i o n s h i p between e x c e s s r e s p i r a t o r y m e t a b o l i s m and u t i l i z a t i o n o f i n t r a v e n o u s l y i n f u s e d s o d i u m r a c e m i c l a c t a t e and s o d i u m L ( - ) lactate. A m e r i c a n J o u r n a l o f P h y s i o l o g y 177: 4 5 5 - 4 6 2 . , A n d e r s o n , K. L . I960. 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B r a d s t r e e t and W. F. Garey. 1962. L a c t i c a c i d response In the grunion. Comparative B i o c h e m i s t r y and P h y s i o l o g y 6: 2 0 1 - 2 0 3 . Schondorff, B., and K. Wachholder. 1914* Glykogenstoffwechsel der F i s h e . I. Die Glykogengehalt von Susswasserfischen. P f l u g e r s A r c h i v f u r d i e Gasamte P h y s i o l o g i e 157: 147-64. d  e  n  Secondat, M. 1950a. I n f l u e n c e de l ' e x e r c i c e musculaire sur l a v a l e u r de l a glycBme de l a carpe (Cyprinus c a r p i o L . ) . Comptes Rendus Academie des Sciences 233: 796-797• Secondat, M. 1950b. I n f l u e n c e de l ' e x e r c i c e musculaire sur l a c a p a c i t e pour 1'oxygene du sang de l a carpe (Cyprinus c a r p i o L . ) . Compte Rendus Academie des Sciences 230: 1787-1788. Secondat, M., and D. D i a z . 1942. Recherches sur l a l a c t a c i d emie chez l e p o i s s o n d'eau douce. Comptes Rendus Academie des Sciences 215: 71-73» S r e t e r , F. A. and S. M. Friedman. 1958. The e f f e c t of muscular e x e r c i s e on plasma sodium and potassium i n the rat. Canadian J o u r n a l of B i o c h e m i s t r y and P h y s i o l o g y  36:  333-338.  97 S t e e l , R. G. D. and J . H. T o r r i e . I960. P r i n c i p l e s and procedures of s t a t i s t i c s . McGraw-Hill Co., New York. Udvardy, M. D. P. Unpublished  data.  Vennesland, B., A. K. Solomon, J . M. Buchanan, R. 0. Cramer and R. B. H a s t i n g s . 191*2. . Metabolism of l a c t i c a c i d containing, r a d i o a c t i v e carbon i n the «* and B positions. J o u r n a l of B i o l o g i c a l Chemistry 11*2: 371-377. Wells, J . G., B. Balke and D. D. Van Possan. 1957* Lactic a c i d accumulation d u r i n g work. A suggested standardi z a t i o n of work c l a s s i f i c a t i o n . J o u r n a l of A p p l i e d P h y s i o l o g y 10: 51-55• West, E. S. and W. R. Todd. 1961. Textbook of b i o c h e m i s t r y . 3rd ed. MacMillan Co., New York. Yamaguchi, Y. and P. Matsuura. 1961. On the p r o s t h e t i c group of hemoglobin and myoglobin of tuna. B u l l e t i n of the Japanese S o c i e t y of S c i e n t i f i c F i s h e r i e s - 27 ( 1 ) : 38-1*1.  98  99 Table XVIII. Sample S i z e , mean, and s t a n d a r d d e v i a t i o n f o r each of the v a r i a b l e s studied. Blood and t i s s u e samples were o b t a i n e d from tseparate f i s h . Exercise chase rain.  condition  rest min.  chase min.  Blood n  weight grams mean + Sd  1  3  275 +  2  9  252 + 56.9  5  9  1  3  <  samples  lactate mg % mean + Sd  Muscle hemoglobin g % mean + Sd  n  weight grams mean + Sd  samples  lactate mg % mean + Sd  glycogen mg Jo. ' mean ± Sd  15 +  3.6  9 . 7 5 + 1 .11  9  223 + 2 7 . 0  306 ±  69-4  92 ± 4 1 . 3  6.2  10.05 + 1 .09  337 ±  33.1  74 ± 7 5 - 4  9.5  1 0 . 7 4 + 1 .09  9 8  255 ± 2 9 - 4  219 + 2 7 . 3  + 55 +  260 + 64.3  463±  50.7  49 ± 1 5 . 7  9  252 +  55.0  43 +  7.7  1 0 . 9 6 + 1 .01  9  245 ± 4 7 . 5  362 + 1 0 7 . 4  46 ± 2 9 . 7  33.0  1 0 . 4 2 + 1 .05 11.00 + 1 .04  9  220 + 4 0 . 6  400 ±  90.0  49 ± 4 3 . 3  9  213 ± 4 3 . 4  394 ±  35.6  33 ± 4 3 . 7  69.5  2  ^  2  •3  9  266 +  5  3  9  256 +  57.3  54 + 3 . 6 74 + 1 4 . 9  1  8  9  243 +  54.6  49 + 1 1 . 5  1 1 . 0 5 + 1. . 2 2  9 9  272 +  75-9  73 ± 5 6 . 3  9  1 1 . 2 0 + 0 ..99  242 + 4 9 . 5  74 + 5 . 3 73 + 1 6 . 7  270 ± 54.4  413+  71.0  43 ± 4 0 . 0  1 .75  9  223 ± 3 4 . 3  424 ±  62.5  33 ± 4 5 . 6  70 + 1 2 . 9  1 1 . 1 9 + 1 .35  9  207 ± 4 6 . 3  256 +  55.2  9  244± 59.9  33© + 1 3 2 . 7  34 ± 2 2 . 3  9  230 ± 7 6 . 5 303 + 6 2 . 2  402 + 403 ±  91.1  35 ± 2 4 . 5  41.0  19 ± 1 3 . 6  265 + 4 3 . 6  462 +  42.4  24 ± 1 1 . 0  5  *  9  267 + 4 4 . 4 274 + 39.3  1  30  9  237 +  9  5  30 30  5  60  6  233 + 260 + 301 +  1  1  6  359 +  40.2  49 +  5.4  1 1 . 3 2 + 1 .49  2  6  272 +  29.2  79 + 10.3  1 2 . 1 4 + 0 ..73  5  3 3 3  5  6  336 + 1 0 0 . 5  36 +  6.7  1 2 . 5 9 + 0 ,.59  1  8  1  6  313 +  55.0  64 +  5.1  1 2 . 9 6 + 1 .26  2  a  2  6  293 +  49.7  36 +  7.6  1 1 . 3 2 + 1 ., 2 2  5  6  356 +  59.5  93 + 1 0 . 4  1 2 . 3 4 + 1 .23 1 2 . 0 0 + 1 .25  2  2  2  5  9  93.6 53.5  100 +  47.0  107 + 25.3  61.1  144 + 27.6  7.1  1  30  1  6  293 +  34.9  36 + 1 4 . 1  2  30  2  6  349 +  71.3  115 + 1 4 . 7  5  30  5  290 +  77.3  120 + 1 9 . 0  5  60  •5  6 6  294 +  41.1  143 + 16.5  11.56  1 1 . 4 7 + 1. . 7 0 1 2 . 1 7 + 1 .1© 1 0 . 3 1 + 0 ..35  + 0 .93 12.32 + 1 .71 1 1 . 4 9 + 0 .94  6  103 ± 7 6 . 1  11.37  Table X V I I I . ( c o n t i n u e d ) .  Exercise condition  Blood  chase r e s t chase r e s t chase s e c . min. s e c . min. sec.  weight grams mean + Sd  7 247 + 33.6  3 3 3  60  3  60  3  8 286 + 55.7 6 265 38.4 6 262 + 53.3 6 261 + 3 3 . 8 6 306 + 56.9 6 295 40.9 6 227 + 4 6 . 2  30  15 15  30  15  60  15  60  15  15  60  15  30  15  60 60  15  60  15  60  15  n  15  30 30  30  30 30  60 60  30  30  60  30  30  30 30  60  30  60  60  30  60  0  30  0  60  to e x h a u s t i o n unexercised  lactate mg fo mean + Sd  9 +  hemo g l o b i n g 5'o mean  n  weight grams mean + Sd  lactate mg % mean + Sd  1.22  6  259 + 4 2 . 8 306 + 53.3 304 + 51.0  236 +  0.76  6 6  1.9  10.74  + 0.46  4.5  10.83  0.50  19 + 17.5 16 + 6 . 9  10.78  24  samples g l yc og en mg % mean + Sd  + 0.78 10.32 + 0.71 51.0  161 + 3 1 . 2 148 + 6 4 . 0  47 + 20.5  + 10.65 + 10.13 + 11.00 +  1.23  6  243 + 58.0  193 + 2 0 . 7 240 + 114.5 276 + 56.O  6 6 6  251 + 50.4 302 + 44.0  177 196 +  40.8  134 + 4 2 . 2  95.9  289 + 4 2 . 9  304 +  83.7  132 + 30 .5 83 + 4 4 . 0  6  291 +  61.3  271 + 179 +  77.3 32.0  9 db 2 . 8 34 + 8.0  25 +  9.2  10.15  0.75  162 + 3 0 . 3 111 + 62.9  5 289 + 61.3 7 301 + 2 5 . 1 6 298 + 40.2  76 +  5.8  11.42 + 0.61  63 +  20 J.  11.53 + 1.20 11.52 + 1.07  7 308 + 36.3  14 +  3.0  9.70 + 1.13  6 270 + 34.9 6 258 + 58.8  49 + 11.1 50 + 15.6 55 + 12.1  + 0.98 10.04 + 0 . 7 4 10.53 + 1.04  6 6  287 + 6 5 . 5 263 + 4 5 . 7 256 + 6 2 . 6  6  253 + 39.6  366 +  70.7  6 308 + 58.3 102 + 15.5 6 274 + 4 2 . 2 88 + 13.1  11.34 + 0.97  6  294 + 53.5 304 + 1 9 . 8  309 + 320 +  36.6 68.9  81 + 2 5 . 7 81 + 2 3 . 6  268  68.3  358 +  36.I  59 + 11.6  270 + 68.9 221 + 4 9 . 2  388 + 132 +  75.4 49.7  78 + 21.2  6 300 + 2 7 . 1  30  Muscle  samples  74 + 23.0  6 292 + 40.3  97 + 19.1  6 323 + 2 9 . 7 6 259 + 50.5  16 + 11.7  -  -  8 310 + 53.0  14 +  7.6  -  7 +  10.87  + 0.94 11.50 + 0.54 10.82  + 0.55 10.60 + 1.07  157 + 38.5 85 + 2 1 . 1  10.70  -  4.1  6 6  113 + 1 2 . 5 127 + 5 6 . 2  9.15 + 1.40  .0  9  251 + 48.8  101 Table XIX. Time to obtain and prepare blood and t i s s u e  Exercise condition chase .n.  rest min.  chase min.  samples  Blood samples (time i n seconds) catch time mean + Sd  time to obtain blood mean + Sd  14.4 + 5.18 11.7 + 3.67 1 1 . 8 + 4.35  74.3 + 44.46 51.2 + 31.12 113.2 + 67.31  3  1 6 . 1 + 3.26  5  3 3  19.1 + 4.37 17.4 + 5.98  28.0 + 14.88 50.2 + 74.66 2 0 . 2 + 13.17  1  8  2 4 . 0 + 8.9©  2  8 8  17.3 ± 2.45 1 8 . 1 + 4.65  30 30  1 2 5 1 2  5  5 5  30  20.0 + 4.44 1 9 . 1 + 5.37 20.1 + 4 . 3 1  60  • 9 . 3 ± 2.33  1 2  3 3  1 2  5  3  5  1 2  8  1 2  1 2  5  5 1 2  3© 30  1 2  5  3© 60  5  5  5  22.5 + 1 0 . 5 5 19.2 13.76 3 7 . 0 + 26.62 4 4 . 1 + 38.92 2 8 . 1 + 8.10 23.6 + 11.17 16.08 16.5  6 . 2 ± 0.75 4 . 7 ± 1.75 4 . 8 + 1.17  1 4 . 7 + 15.38 1 1 . 8 + 2.86 10.7 +  3.14  5.2 + 1.33  17.8 +  8.28  4 . 8 + 0.75 6 . 2 + 1.17  12.5 +  5.5 + 1.22 4.7 + 0.81 5 . 8 + 2.31 6.5 + 2.07  24.3 + 30.94 1 3 . 0 + 5.93 13.0 + 12.79 38.0 + 33.64  Muscle samples (time i n seconds) catch time mean + Sd  k i l l time mean + Sd  muscle time mean + Sd  l i v e r time mean + Sd  2 . 9 + 1.05 2.1 + 0-33 2.0 + 0.93  2 . 8 + 1.09  1 4 . 8 + 2.44 1 5 . 9 + 5.33 1 6 . 1 + 5.19  1 6 . 8 + 11.29 2 0 . 8 + 12.69 16.5 + 5.78  3.3 + 2.7 + 3.8 ± 1 '4.1 +  2.82  3.2 + 0.67  1.00  2 . 6 + 1.24  10.7 + 2.35 1 0 . 6 + 2.65  3.77  2 . 9 + 1.05  1 1 . 4 + 2.50  3.02  2.4 + 0.88 3 . 0 + 1.58  1 0 . 2 + 1.20  2 . 7 +.2.33 2.3 + 1.00 3-1 ± ! « 3 3 . 0 + 1.32 d  3 . 1 + 2.15 1.7 + 0 . 8 2  3 . 8 + 2.53 3 . 0 + 1.07  2 . 6 + 1.13 3 . 3 + 1.12 3 . 6 + 1.33 3 . 0 + 1.00 2 . 7 + 1.21  1  1 0 . 1 + 3.1© 1 0 . 1 + 2.80 10.2 + 1.30 11.0 + 2.06 9 . 6 + 2.83 3.3 + 4.26  -  11.4 j t  -  6.59 16.5 + 16.56  -  \ I 1.2 + 0 . 4 1  1.5 + 0 . 3 4  8.3 + 0 . 5 2  3.16  102  Table X I X .  (continued)  Exercise condition ^^^^^^^^  Blood samples  Muscle samples (time i n seconds)  (seconds)  i  -  k i l l time mean + Sd  muscle time mean + Sd  c a t c h time mean + Sd  3  16.7  + 6.37  35.6 + 20.03  ±  2.62  24.6 + 25.63  + 7.99 21.3 + 13.92  33-0 + 26.25  10.3 +  5.20  16.3  + 11.32  3 . 2 + 1.47  2 . 2 + 1.17  9 . 5 + 1.87  17.5 + 3.56 2 8 . 0 + 15.62  21.0  ±  16.05  3 . 5 + 2.0.7  3.0 + 0.89  8 . 8 + 1.33  61.5  + 37.57  4.7 + 4.76  3.5 + I . 6 4  13.8 +  2.23  26.7  22.61  2.8 + 0.75  3 . 2 + 1.33  8.2 + 1.47  3.51  24.8 + 3 3 . 6 9  1.8 + 0.75  3.0 + 1.67  8 . 8 + 1.47  24.4  22.30  + 12.73  3 . 0 + O.63 3 . 3 + 1.89  9.5 + 2.43  17.8  6 . 2 + 8.33 3 . 4 + 1.62  0.52 2.3 2 . 8 + 1.72  10.77  + I.63 3.0 + 2.28 3.0 + 2.28  19.32  2.3  1.03  3.0  8.38  5 . 3 + 2.42  2.2  64.52  1.7 + 1.21  2.7  22.29  1.8 + 0 . 7 5  1.7  + 0.83  3.4  3  30  13.4  3  60  21.7  3  60  3  15 15  30  15  60  15  60  15  15  60  15  30  8.6  15  60  15  60  10.7  15  60  15  60  15  + 4.61 12.5 + 5.21 9.3 +  30  2.81  time to o b t a i n b l o o d mean + Sd  c a t c h time mean + Sd  chase r e s t chase r e s t chase sec. min. sec. min. sec.  25.5 + 25.52  30  1 4 . 8 + 5 . 00  31.2  30  60  14.5 +  2.17  20.5  30  60  30  11.8 +  3-43  17.8  30  60  30  30  20.2  30  60  30  60  30  60  30  60  + 0 . 82 8 . 8 + 1.72 9.2 + 2.93  unexercised 0 0  30 60  to e x h a u s t i o n  30  19.5 18.2 + 14.8 +  28.25  25.7  30  7.7  59.2 29.3  + + + + + +  23.05  7.19  4Y7.9 + 3 9 . 6 8  3.65  2 3 . 8 + 17.72 3 0 . 5 + 19.89  4.17  l i v e r time mean + Sd  1  2.7  2.2  2.0 + 0.67  2.7  + + + + +  0.82 1.41 0.75 1.21 0.52 2.00  2.0 + 0.67  10.2  2.48  7.9'+ 0 . 9 0 9.2  2.40  + 9.0 + 7.8 + 8.2 +  4.49  0.75  8.5  2.26  10.1  + 3.38  2.97 1.33  8.5 + 1.64 13.8  12.3  6.98  8.6 + 2 . 2 7  1 0 . 3 + 1.34  11.1  + I.84  11.0 ± 6.50  103  Table XX.  A c t u a l time taken t o swim 4.3 meters (approximately  3 sec.) and a c t u a l time taken t o swim 23 meters (approximately 15 sec.)  Exercise  ACTUAL TIME TAKEN (seconds) Blood samples  condition  chase rest. chase r e s t chase sec. min. sec. min. sec.  f i r s t chase mean + Sd 2.86  3 3  30  2.75  3  60  3-33  3  60  3  +  second chase t h i r d chase mean + Sd mean + Sd :  0.38 0.71  + +  1.37 1.97  4.00 + 1.27  + 1.17 13.00 + 2.10 14.33 + 1.21 15.33 + 4* 84 15.60 + 5.46 16.43 + 2.70 14.83 + 3.97  17.83 + 2.43  3.33 13.83  15 15  30  15  60  15  60  15  15  60  15  30  15  60  15  60  15  60  15  60  15  12.60  +2.50  15.29+  2.14  14.83+ 1.17  16.67  + 3.20  Mnsrl R samnl es f i r s t chase second chase t h i r d chase mean + Sd mean + Sd mean + Sd 15.85  15 15  30  13.35  15  60  17.50  15  60  15  15  60  15  15  60  15  60  + + +  2.48 0.52 2.43  17.50  +1.48  15.00  + 1.09  30  16.65  1.03  14.15  ± 0.41  15  60  14.50  +  1.22  15.85  + 1.17  15  60  15.00  + 0.82  15.70  + 0.69  15  15.00  + 0.82  104 Table XXI.  A n a l y s i s o f v a r i a n c e and Duncan's new  multiple  range t e s t on the data i n the l a t i n square d e s i g n .  The  purpose  o f t h i s d e s i g n was t o demonstrate the e f f e c t o f v a r i a b i l i t y d u r i n g any one day, from day t o day, and t h e treatment e f f e c t .  A.  The experimental c o n d i t i o n s  Treatment number  B.  Condition  Day numb er  Date June  Time  exercise minutes  recovery minutes  1  1  3  1  3  AM  2  1  8  2  4  AM  3  1  30  3  4  PM  4  2  3  4  5  AM  5  2  8  5  5  PM  6  2  30  6  24  AM  7  5  3  7  24  PM  8  5  8  8  25  AM  9  5  30  9  25  PM  Analysis of Variance.  source  C a l c u l a t e d F v a l u e s f o r each v a r i a b l e weight catch sample hemoglobin time time  F ^ Q ^ ? Q± t  lactate  d u r i n g the day  2.31  1.09  1.17  1.43  2.30  between days  1.56  2.83  1.28  4.65  1.02  treatment  0.84  2.13  1.19  1.36  26.65  2.12  2.87  105 B.  Duncan's New M u l t i p l e Range T e s t on means w i t h  F values.  significant  Any two means not underscored by t h e same l i n e a r e  significantly different  from each o t h e r a t t h e 5$ l e v e l . Any  two means which a r e underscored by the same l i n e a r e not significantly different  a t the 5% l e v e l .  The t e s t s a r e a p p l i e d  i n t h e o r d e r from t h e most s i g n i f i c a n t to the l e a s t  1. Treatment - l a c t a t e - h i g h l y s i g n i f i c a n t . number 1 2 4 3 5 7 mean 43 49 54 70 74 74 2. Between days - hemoglobin - h i g h l y number 3 9 2 1 5 mean 9 . 5 4 1 0 . 4 6 11.14 11.39 11.54  78  significant.  8  significant. 8 7 11.62 11.64  3. Between days - c a t c h time - s i g n i f i c a n t . number 5 3 2 4 1 9 mean 1 4 . 1 17.0 17.3 18.4 19.3 19.8  6 21.5  4. During t h e day - weight - s i g n i f i c a n t . number 5 2 4 8 7 mean 224 232 235 260 262 266  9  5. During t h e day - l a c t a t e - s i g n i f i c a n t . number 5 8 9 3 4 mean 65 66 67 69 72 73  2  6. Treatment - c a t c h time - s i g n i f i c a n t . number 1 5 7 8 6 4 mean 16.1 17.3 17.5 18.1 19.1 19.1  3 278  78  6  3 20.0  6 100  11.78  9 107  4  6 11.90  8 21.7  7 22.2  1 288  6 299  1  7 84  78  9 20.1  2 24.0  

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