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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 gairdneri) 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 this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1965 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of • Br i t i sh Columbia., I agree that the Library shall make i t freely available for reference and study, I further agree that per-mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that:copying or publi-cation of this thesis for financial gain shall not be allowed without my written permission* The University of Brit ish Columbia, Vancouver 8, Canada Department of ABSTRACT The purpose of this study was to examine the effects of exercise of short duration, and the effects of re-exercise on carbohydrate metabolism. I f e e l that the levels of severe exercise studied approximate the levels of severe exercise which a rainbow trout probably experiences i n i t s natural environment. The l e v e l of blood la 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 unanesthetized, i n t a c t , one and one-half year old rainbow trout acclimated to 1 0 . j ? ° C . Samples were taken immediately afte r exercise of 3 seconds to 5 minutes, a f t e r recovery of 3 minutes to 6 0 minutes, and aft e r re-exercise of 3 seconds to 5 minutes. The results indicate that exercise of even the shortest duration studied causes an immediate increase i n the l e v e l of blood l a c t a t e , muscle lactate, and blood hemo-globin. Exercise also causes an immediate decrease i n muscle glycogen, but does not cause a change i n the l e v e l of l i v e r glycogen. Changes during the 60 minute recovery period are s l i g h t . In general, the effects of re-exercise a f t e r a 60 minute recovery period are additive. A c o r r e l a t i o n analysis between muscle glycogen and muscle lactate indicates that there i s a source of muscle lactate other than muscle glycogen at exercise levels of long duration. The source of this muscle lactate does not appear to come from l i v e r glycogen. The energy may be supplied by catabolism of protein or l i p i d , or by absorption of foodstuffs from the gut. This study provides evidence that rainbow trout are not well adapted for recovery from severe exercise of short duration. x i ACKNOWLEDGEMENTS Assistance for this project provided by the National Research Council of Canada through grant T-7 to Dr. E. C. Black i s g r a t e f u l l y acknowledged. The Fi s h and Game Branch of the B r i t i s h Columbia Department of Recreation and Conservation are thanked for provision of experimental animals, and the s t a f f at the Summerland Hatchery thanked for maintaining these animals. I express my sincere gratitude to Dr. E. C. Black for his encouragement, d i r e c t i o n , and assistance i n the present study. I am also indebted to Dr. Glen Manning for suggesting the research problem. Thanks are also due to the following for technical assistance: Mr. D. John Bosomworth, and Mr. S. J. Tredwell for assistance i n sampling and analysis of lactate; Miss G. Docherty for analysis of glycogen; Mr. K. Henze of the Department of Physiology, University of B r i t i s h Columbia and Miss Tomiko Hashimoto for assistance i n preparation of the manuscript. x i i I am also indebted to Dr. Dempster and the s t a f f of the Computing Center at U. B. C. for assistance 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 also thankful for encouragement and ideas from Professor D. H. Copp and from my fellow student, Dr. S. S. Shim. V TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 3 I . F I S H 3 I I . EXPERIMENTAL CONDITIONS 1|. A. U n e x e r c i s e d LL. B. E x e r c i s e d li. C. R e c o v e r y If. D. I n t e r m i t t e n t e x e r c i s e I4. I I I . SAMPLING- METHODS 5 A. B l o o d s a m p l e s 5 B. T i s s u e s a m p l e s o f m u s c l e and l i v e r 5 I V . • ANALYTICAL METHODS £ A. H e m o g l o b i n 6 B. L a c t a t e 7 C. Glycogen 7 V. HANDLING OF DATA 7 RESULTS AND DISCUSSION ' 11 I . HEMOGLOBIN 11 v i A. R e s u l t s 11 B. D i s c u s s i o n 17 I I . LACTATE 20 A. R e s u l t s - b l o o d l a c t a t e 20 B. R e s u l t s - m u s c l e l a c t a t e 2I4. C. D i s c u s s i o n 36 I I I . GLYCOGEN 55 A. R e s u l t s - m u s c l e g l y c o g e n 55 B. R e s u l t s - l i v e r g l y c o g e n . 61 C. D i s c u s s i o n 66 I V . RELATIONSHIP BETWEEN MUSCLE GLYCOGEN AND MUSCLE LACTATE . . 7 2 V. GENERAL DISCUSSION 78 CONCLUSIONS 82 SUMMARY 83 AREAS FOR FURTHER INVESTIGATION 86 BIBLIOGRAPHY 88 APPENDIX 98 v i i LIST OP TABLES TABLE Page 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 of times r e - e x e r c i s e d i n the present study. 9 I I . Time to o b t a i n and prepare blood and t i s s u e .. samples. 1 0 I I I . 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 continuous and d i s c o n t i n u o u s e x e r c i s e of the same t o t a l d u r a t i o n . 1 2 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 . 2 1 V. The l e v e l of blood 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 continuous and d i s c o n t i n u o u s 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 3 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 . 3 0 V I I . L e v e l s of blood l a c t a t e i n unexercised Salmo g a i r d n e r i . 3 7 V I I I . L e v e l s of muscle l a c t a t e i n unexercised f i s h . 38 IX. Maximal l e v e l s of blood and muscle l a c t a t e a f t e r e x e r c i s e i n f i s h , f r o g , and mammals. X. 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 from C h r i s t e n s e n ( 1 9 5 6 ) . 5 0 v i i i XI. A comparison of the r e s i s t a n c e to f a t i g u e between f i s h and man during i n t e r m i t t e n t strenous e x e r c i s e as i n d i c a t e d by the l e v e l of blood l a c t a t e . X I I . X I I I . XIV. XV. XVI. XVII. X V I I I . XIX. XX. XXI. 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 of muscle glycogen. 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 of 30 seconds on the l e v e l of l i v e r glycogen. 63 E f f e c t of 5 minutes severe e x e r i c s e , and 30 minutes recovery 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 glycogen. 61}. A n a l y s i s of variance of l i v e r glycogen data from Table XIV during and a f t e r 5 minutes severe e x e r c i s e . 65 Levels of muscle glycogen i n unexercised rainbow t r o u t . 67 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 muscle glycogen. 75 Sample s i z e , mean, and standard f o r each of 99 the v a r i a b l e s s t u d i e d . Time to ob t a i n and prepare blood t i s s u e samples. . 101 A c t u a l time taken to swim I4..3. meters (approximately 3 sec.) and a c t u a l time taken to swim 23 meters (approximately 15 sec.) 103 A n a l y s i s of variance and Duncan's new m u l t i p l e range t e s t on the data in.the l a t i n square design. 104 i x LIST OF FIGURES Each p o i n t i n every f i g u r e r e p r e s e n t s the mean of at l e a s t 6 samples from separate f i s h . FIGURE 1. Changes i n l e v e l s of blood hemoglobin c o n c e n t r a t i o n d u r i n g 5> minutes severe e x e r c i s e . 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. 3 . 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 c o n c e n t r a t i o n . 4 . 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 l e v e l s " of blood hemoglobin c o n c e n t r a t i o n . 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 . 6. Changes i n l e v e l s of blood l a c t a t e a f t e r continuous e x e r c i s e (continuous 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 of i n t e r m i t t e n t e x e r c i s e of V~> seconds on 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 e x e r c i s e of 30 seconds on the l e v e l s of blood l a c t a t e . 9. Changes i n the l e v e l 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 . 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. X 1 1 . 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 1 5 s e c o n d s o n t h e l e v e l s o f m u s c l e l a c t a t e . 1 2 . 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 3 0 s e c o n d s o n t h e s l e v e l s o f m u s c l e l a c t a t e . 1 3 . 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 5 m i n u t e s o n t h e l e v e l s o f m u s c l e l a c t a t e . Ii4.. C h a n g e s i n l e v e l s o f m u s c l e g l y c o g e 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 . 1 5 . C h a n g e s i n l e v e l s o f m u s c l e 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 , a n d 5 m i n u t e s . 1 6 . 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 1 5 s e c o n d s o n t h e l e v e l s o f m u s c l e g l y c o g e n . 1 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 3 0 s e c o n d s o n t h e l e v e l s o f m u s c l e g l y c o g e n . 1 8 . T h e r e l a t i o n s h i p b e t w e e n m u s c l e l a c t a t e a n d m u s c l e g l y c o g e n i n f i s h s a m p l e d i m m e d i a t e l y a f t e r e x e r c i s e . 1 9 . T h e e f f e c t o f o x y g e n s u p p l y o n c a r b o h y d r a t e m e t a b o l i s m . INTRODUCTION There have been many studies of the r e l a t i o n s h i p between severe exercise and concomitant changes i n carbohydrate metabolism i n f i s h (von Buddenbrock, 1938; Secondat and Diaz, 191+2; Black et a l , 1961; and C a l l l o u e t , 1961j.). In almost a l l of these studies, the f i s h were continuously exercised for 15 minutes. Exercise of this duration always elevated muscle lactate and blood l a c t a t e , depleted muscle glycogen, and i n general, produced a marked disturbance which i s referred to as muscular fatigue. There have been very few studies of the effects of discontinuous or intermittent severe exercise i n any intact animal, and none to my knowledge In f i s h . Christensen (1962) has demonstrated that man can do an optimum amount of exercise i n a given time by alternate exercise and rest periods of 30 seconds. His results showed that continuous exercise for 9 minutes elevated the l e v e l of blood lactate from 7 to 1$0 mg %, whereas discontinuous exercise f o r 30 minutes (alternate exercise and rest periods of 30 seconds) elevated the l e v e l of 2 blood lactate to only 20 mg %. Thus, the present study was an attempt to discover whether or not f i s h are adapted to tolerate discontinuous exercise as well as man i s with respect to accumulation of lact a t e . In other words, I presumed that energy for exercise i s supplied by degradation of muscle glycogen; and when demand for oxygen exceeded the supply, lactate accumulated. It was also the purpose of thi s study to examine the changes i n carbohydrate metabolism during severe exercise of extremely short duration. Moreover, I f e e l that discontinuous exercise and exercise of short duration probably simulate the exercise which a rainbow trout experiences i n i t s natural environment. 3 MATERIALS AND METHODS I. PISH One and one-half year old rainbow trout, Salmo  gairdneri, were reared from eggs obtained from a commercial trout hatchery In McLeary, Washington. They were raised at Summerland Trout Hatchery operated by B r i t i s h Columbia Depart-ment of Recreation and Conservation, Pish and Came Branch. Trout were fed twice d a i l y with pelleted f i s h food (J. R. Clarke Company, Salt Lake City, Utah). The water temperature remained constant throughout the summer, 10.5 to 11.5°C. At least two days p r i o r to experiment, f i s h were transferred from outside ponds to standard hatchery troughs (Lj-0 x 460 x 12 cm. deep). Fish were not starved before experiment and were kept covered to reduce external s t i m u l i . At the outset of the experiment furonculosis was prevalent. Diseased f i s h were not used i n the experiment and a l l f i s h were treated with sulfamerazine given with the food. Incidence of furonculosis was ne g l i g i b l e by July, and treatment was discontinued July 3« I I . EXPERIMENTAL CONDITIONS Pish were held i n covered troughs at least two days p r i o r to experiments, and feeding was continued as usual. Treatments were applied randomly and singly; and each f i s h used only once. A. Unexercised condition. The term unexercised rather than re s t i n g or basal l e v e l i s employed since the f i s h usually manifest some a c t i v i t y during sampling. Pish were captured with as l i t t l e disturbance as possible, air-dipped to the exercise trough, and sampled immediately. B. Exercised conditions. F i s h were captured with as l i t t l e disturbance as possible, air-dipped to the exercise trough, and exercised by splashing water i n the trough for a prescribed period of time (from 30 seconds to 5 minutes), or for a prescribed distance. F i s h were chased by two persons. C. Recovery condition. F i s h were exercised as above and then l e f t to rest a prescribed period of time, from 3 minutes to 60 minutes, i n the same trough. Movement was r e s t r i c t e d to one-half of the trough and the trough was covered during the recovery period. D. Intermittent exercised conditions. The f i s h was 5 exercised and l e f t to recover as above. After the r-ectfver-y period, the l i d was l i f t e d and the f i s h was re<^exercised. The sp e c i f i c levels of exercise and recovery times studied appear i n Table I. I I I . SAMPLING METHODS A. Blood samples. After treatment a f i s h was captured by hand as quickly and adeptly as possible, placed ventral side up i n a v-shaped trough which was kept submerged allowing the f i s h to respire i n water, and a blood sample was obtained by cardiac puncture. Blood was withdrawn into a 2 ml. syringe which had been previously water-proofed with parafin o i l , and rinsed with one drop of heparin solution. The blood sample was immediately ejected into a conical test tube; 0.02 ml. of the blood was transferred to 5.0 ml. of 0 .1 M hydrochloric acid for determination of hemoglobin concentration, and 0.5 ml. or 1.0 ml. was transferred to 9.0 ml. cold 10$ t r i c h l o r o -acetic acid for determination of lactate concentration. Hemo-globin concentration was determined within three hours of sampling. Lactate was determined on the deproteinized f i l t r a t e within three months. Time to capture the f i s h and time to withdraw the blood sample were recorded by stopwatch, and appear i n Table I I . B. Tissue samples of muscle and l i v e r . After treat-ment, the f i s h was captured as quickly as possible, k i l l e d by 6 breaking the neck, and placed on i t s side. The muscle sample was obtained by punching through the musculature immediately below the dorsal f i n with a # 9 cork borer. The l i v e r sample consisted of the whole l i v e r . Both muscle and l i v e r samples were frozen immediately i n dry ice and ethanol. Unwanted tissue (fat, skin, and bone) was removed, and the sample weighed on a t o r s i o n balance. The muscle sample was divided into two portions. One portion was homogenized with cold 10% t r i c h l o r o a c e t i c acid ( 6 . 0 ml. per gram) and the f i l t r a t e analyzed for lactate concentration within three months. The other portion of the muscle sample and the l i v e r sample were digested i n 60% potassium hydroxide ( 3 . 0 ml. per gram) for four hours at 100°C and the digests analyzed f o r glycogen concentration within 6 months. Time to capture, k i l l , obtain the muscle sample, and obtain the l i v e r sample were a l l recorded by stop-watch, and appear In Table I I . C. After sampling, the f i s h was weighed, and the sex determined by v i s u a l examination of the gonads. IV. ANALYTICAL METHODS A l l analyses were determined using a Klett-Summerson photoelectric colorimeter. A. Hemoglobin was analysed c o l o r i m e t r i c a l l y as acid hematin using a #5i|. f i l t e r and comparing the sample to a standard 7 supplied by Klett-Summerson. Hemoglobin i s reported i n g % (grams per 100 ml. blood). B. Lactate was determined c o l o r i m e t r i c a l l y on 1 ml. aliquots from deproteinized f i l t r a t e s of blood and muscle homogenates by the Barker-Summerson method (19J+1). I n t e r f e r i n g materials i n the f i l t r a t e were removed by the Van Slyke-Salkowski treatment of copper sulphate and calcium hydroxide (Hawk ejb a l , \95lx.). Lactic acid i s oxidized to acetaldehyde with concentrated sulphuric acid, the aldehyde i s coupled with a chromogenic agent, p-hydroxydiphenyl, and compared with a standard of li t h i u m l a c t a t e . Lactic acid i s reported i n mg % (mg per 100 ml. blood, or mg per 100 grams wet weight of muscle). C. Glycogen was determined c o l o r i m e t r i c a l l y by the anthrone method ( C a r r o l l et_ a l , 19^6) on potassium hydroxide digests of muscle and l i v e r which were diluted toa-known volume. Glycogen i s precipitated with ethanol, hydrolysed to glucose, and coupled with a chromogenic agent, anthrone. A standard curve i s determined for each analysis using three concentrations of glucose, and glycogen concentration deter-mined from the standard curve as glucose. 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 separately for each f i s h . Sample s i z e , mean, and standard deviation are given for each variable and each condition. Conditions were applied singly and randomly to each f i s h . Comparisons between conditions are made using the F-test, ANOV, and Duncan's new multiple range test (Steel and Torrie, I960). A l a t i n square design ( 9 x 9 x 9 ) was used i n one set of experiments (3 minutes, 8 minutes, and 30 minutes recovery from 1 minute, 2 minutes, and 5 minutes exercise) i n order to determine the effect of sampling sequence during the day, the effect of sampling sequence from day to day, and the effect of experimental conditions. A l l s t a t i s t i c s were calculated by the Computing Center, University of B r i t i s h Columbia. 9 Table I. Duration of exercise periods, duration of recovery periods, and number of times re-exercised i n the present study.. Duration of Exercise Duration of rest (minutes) Number of times re-exercised 0 (a) 3 0 , 60 0 3 seconds (b) 3 0 , 60 once afte r 60 min. rest 15 seconds (c) 3 0 , 60 twice a f t e r 60 min. rest 30 seconds 3 0 , 60 twice a f t e r 60 min. rest 1 minute 3 , 8, 30 once a f t e r each rest period 2 minutes 3 , 8, 30 once a f t e r each rest period 5 minutes 3 , 8 , 30 once a f t e r each rest period to exhaustion (d) 0 0 (a) unexercised condition (b) a c t u a l l y exercised 4 . 6 meters taking approximately 3 seconds (mean 3 « 4 seconds, range 2 .0 to 6 .0 seconds) (c) a c t u a l l y exercised 23 meters taking approximately 15 seconds (mean 15.0 seconds, range 10.0 to 3 6 . 0 seconds) (d) exercised to exhaustion. T y p i c a l l y the behavior of the f i s h changes following approximately 1 minute of exercise. At this time the escape behavior changes to a hiding behavior, (mean 67 .3 seconds, range 51 to 80 seconds) 10 Table I I . Time to obtain and prepare blood and tissue samples, Blood and tissue samples were obtained from separate f i s h . A l l times are given i n seconds. Sample Time n Mean + standard deviation blood to catch and p o s i t i o n f i s h 305 to withdraw blood sample 305 13.7 + I4-.I 31.4 + 23.9 tissue to catch f i s h to k i l l f i s h to remove muscle sample and place i n dry ice and ethanol to remove l i v e r and place i n dry i c e and ethanol 222 2.9 + 1.8 222 2.8 + 1.2 222 10.4 + 2.6 62 ll+.l + 6.k. 11 RESULTS AND DISCUSSION I. HEMOGLOBIN A. Results. The mean l e v e l of hemoglobin i n unexercised f i s h was 9 . 1 5 g %• The effects of exercise, recovery, and intermittent exercise are i l l u s t r a t e d i n P i g . 1 , 2 , 3» and 4. In general, exercise caused hemoconcentration. P ig. 1 i l l u s t r a t e s that the hemoconcentration response to exercise i s t r i p h a s i c . Exercise of short duration caused a marked increase i n hemoglobin which was followed by a marked decrease, and this i n turn was followed by a gradual increase. During recovery the hemoglobin continued to r i s e (Pig. 2 ) , The hemoglobin l e v e l decreased a f t e r a long recovery period. For example, the l e v e l had almost returned to pre-exercise l e v e l s a f t e r a 30 second exercise i n 60 minutes as indicated i n F i g . Ij.. Re-exercise caused further hemoconcentration as i l l u s t r a t e d i n Pig. 3 and ij.. The trip h a s i c curve persisted but to a lessor extent, when the f i s h were rechased a f t e r recovery periods of 30 minutes or 60 minutes. 12 Table I I I . Level of hemoglobin concentration during continuous and discontinuous exercise of the same t o t a l duration. T o t a l duration Condition Hemoglobin m (S %) of exercise E x e r c i s e R e c o v e r y Exercise , (seconds) (seconds) (minutes) (seconds)  30 30 0 0 9.70 15 60 15 11,00 60 60 0 0 9.75 30 60 30 10.53 120 120 0 0 10.05 60 3 60 11.82 60 8 60 12.96 60 30 60 12.00 1 3 F i g . 1 . Changes i n levels- of blood hemoglobin concentration during 5 minutes severe exercise. Ik F i g . 2. Eff e c t of rest a f t e r severe exercise of 1, 2, or 5 minutes on the l e v e l s of blood hemoglobin. 15 9^60-0 H — i i i I I 0 1 2 3 4 5 Duration of strenuous exercise in minutes P i g . 3 E f f e c t of exercise and re-exercise on the leve l s of blood hemoglobin concentration. 16 first exercise second exercise third exercise E O) o CT) o E a> X 11.60-1 11.00-10.40 9.80-9.20-o J 30 0 60 30 60 Duration of recovery time in minutes between intermittent exercise periods of 30 sec. F i g . k E f f e c t of intermittent exercise of 30 seconds on leve l s of blood hemoglobin concentration. 17 B. Discussion Korzhuev (I96I4.) found that centrifuging the acid hematin soluti o n reduced the hemoglobin values by 1 8 - 2 0 $ (the t u r b i d i t y of acid hematin solutions of f i s h blood i s due to the presence of n u c l e i i n the erythrocytes). Thus, the absolute values of hemoglobin concentration reported i n t h i s study are probably high, but comparisons between treatments are s t i l l v a l i d . The mean l e v e l of hemoglobin i n unexercised f i s h was 1.21+ g % higher than the value f o r 1 9 6 1 (Black, et_ al.) and 1 . 6 7 g % lower than the value f o r 1963 (Manning) f o r unexer-cised f i s h raised at the same hatchery, fed the same food, and kept at the same temperature. Schiffman and Promm ( 1 9 5 9 ) give the hemoglobin l e v e l of hatchery raised Salmo gairdneri as 6 . 3 1 g %• Some variable other than lo c a t i o n , type of feed, or temperature, must a f f e c t blood hemoglobin l e v e l s . Some p o s s i b i l i t i e s are stage of maturity, furonculosis, and sulfamethazine. Hemoconcentration associated with exercise of f i s h has been noted by others, but none has recorded a t r i p h a s i c response since, none to my knowledge has studied exercise of extremely short duration. Black ( 1 9 5 5 ) found a s i g n i f i c a n t hemoconcentration i n largemouth bass, but not i n kamloops trout, fine-scaled sucker, carp, squawfish, northern black 18 c a t f i s h , sockeye salmon (Black, 1957c) , or lake trout- (Black, 1957b) following 15 minutes severe exercise. There are three possible explanations for the observed hemoconcentration: 1. erythrocytes move into c i r c u l a t i n g blood, 2. water moves out of blood or, 3. both of the above. Black ejb a l . (1959) attempted to demonstrate the movement of water i n order to explain hemoconcentration of exercise, but observed no s i g n i f i c a n t difference between dry weight of blood before and afte r 15 minutes strenuous exercise. Hemocon-centration of exercise has been observed i n species other than f i s h : i n man (Bard 1956), and i n rats (Sieter 1958). Best and Taylor (1961), and Rakestraw (1921), indicate that exercise i n man results i n decreased plasma volume, increased plasma protein, increased red c e l l concentration, and increased hemoglobin concentration. G-regersen and Rawson (1959) observed a decrease i n t o t a l blood volume with exercise i n man. H a l l et a l . (1926) noted increased hemoglobin concentration with asphyxiation i n cod. Scholander et_ a l . (1962) noted c i r c u l a t o r y shut down with asphyxiation i n the grunion since there was an elevated muscle lactate but no elevated blood lactate. (Since lactate was not d i f f u s i n g from muscle to blood then blood flow to s k e l e t a l muscle must have been reduced.) Nakatani (1955) observed a s i g n i f i c a n t increase 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 shock i n rainbow 19 trout. Denyes and Joseph (1956) found an increased hemoglobin concentration i n g o l d f i s h and carp acclimated to higher temperatures. In species other than f i s h , the following causes of hemoconcentration have been noted: e l e c t r i c shock or i n s u l i n shock i n man decreased plasma volume 10 to 20% (Bard 1956), and induced fever i n the dog increased the hemoglobin l e v e l (Kubicek 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 i n response to continuous infusion of adrenaline. Manning (unpublished) noted that severe exercise i n rainbow trout causes c i r c u l a t o r y shutdown, and thus plasma skimming occurs. A possible explanation for the observed results i s that the three phases of the hemoglobin response to exercise are associated with c i r c u l a t o r y changes. The i n i t i a l rapid increase and decrease are due to c i r c u l a t o r y changes i n response to an increase and decrease of adrenaline. The rapid increase i n hemoglobin i s due to plasma skimming, the rapid decrease i s due to erythrocyte sludging. The l a t e r slow increase i n hemoglobin i s due to c i r c u l a t o r y change i n response to the accumulation of metabolites causing the c h a r a c t e r i s t i c reactive hyperemic v a s o d i l i t a t i o n i n the muscle, and thus resolving the red c e l l sludging and restoring a condition of plasma skimming. A l t e r n a t i v e l y , the l a t e r slow increase i n hemoglobin could be due to a release of stored erythrocytes from storage 20 organs. Udvardy (unpublished) has demonstrated that erythrocyte storage organs i n salmon are i n the spleen and the anterior one-third of the kidney. Moreover, studies i n man have shown that release of erythrocytes i n exercise occurs slowly, and only i n severe exercise. The l a t e r slow increase i n hemoglobin concentration could also be due to water moving from blood to muscle tissue i n response to an osmotic gradient due to the increase i n the number of osmotically active p a r t i c l e s i n the muscle as a re s u l t of muscle contraction. I I . LACTATE A. Results - Blood Lactate. The mean l e v e l of blood lactate i n unexercised f i s h was 7.19 mg % (range 0.66 to 13*57 mg % ) . The effects of exercise, recovery, and intermittent exercise are i l l u s t r a t e d i n Pig. 5 , 6, 7, and 8. Exercise, even of the shortest duration, caused an elevated blood lactate. Allowing the f i s h to rest a f t e r the exercise always resulted i n a further increase i n blood lactate (Pig. 5). Fig . 8 i l l u s t r a t e s that a 60 minutes recovery period was i n s u f f i c i e n t for restoration of unexercised leve l s of blood lactate a f t e r exercise of only 15 seconds. However, the l e v e l of blood lactate reached a peak and started to decrease during the 60 minute recovery period. Table IV. E f f e c t of exercise and re-exercise 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 after exercise or re-exercise. Exercise time (seconds) 3 . 1 5 . 30 6P.. 60 60 120 . 120 120 3 ° 0 300 300 Recovery time (minutes) 60 .60 60 . 3 . 8 . 30 3 8 . 30 3 8 .30 Blood f i r s t chase .9 9. 111 .15 . 15 15. 21+ 24- 21+ 55 55 55 Lactate second chase lb W - 55 . 49. 61+ 86 79 115 « 6 98 120 mg % third chase 74 97 22 Re-exercise always caused a further increase i n blood lac t a t e , as indicated i n Table IV. A comparison of blood lactate levels of f i s h exer-cised for the same t o t a l length of time reveals that i n t e r m i t t -ent exercise caused greater increases than continuous exercise (Table V). However, from Table V i t can also be seen that the effect of continuous exercise i s to cause a greater increase i n the l e v e l of blood lactate than discontinuous exercise, i f the f i s h i s l e f t to rest a f t e r the i n i t i a l chase. The f i s h also appeared to respond better to chasing, and thus did more work when exercised intermittently than when exercised contin-uously. T y p i c a l l y the f i s h responds to chasing by evasive behavior for about 1 minute, and further chasing results i n the f i s h attempting to hide rather than to escape. The effect of thi s change i n behavior i s revealed by comparing values for exercise of 2 minutes, rest 3 minutes, (blood lactate of Sk- raS %) with 5 minutes of continuous exercise (blood lactate of 55 mg % ) . These values indicate that the f i s h ' s response during recovery from 2 minutes exercise was not as great as during the f i r s t 2 minutes of exercise. F i g . 6a compares the effect of continuous and discon-tinuous exercise of the same t o t a l duration (2 minutes). Examination of Fig . 6a reveals that exercise and re-exercise of 1 minute did not elevate the blood lactate as much as an 23 Table V. The l e v e l of blood lactate concentration during continuous and discontinuous exercise of the same t o t a l duration. Total duration Condition Blood of exercise exercise recovery re-exercise lactate mg "%" seconds s ec ond s minutes seconds 30 - 0 30 0 0 14 15 60 • 15 47 30 60 0 50 60 60 0 0 15 30 60 30 55 60 30 0 70 120 120 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 i n i t i a l continuous exercise of 2 minutes. But re-exercised f i s h were sampled immediately after the re-exercise, and had they been l e f t to recover, t h e i r blood lactate levels probably would have exceeded those of f i s h exercised continuously for 2 minutes. Pig. 6b compares the effect of s i m i l a r t o t a l dura-tions of exercise; 5 minutes continuous and 4 minutes discon-tinuous exercise. Examination of Pig. 6b reveals that exercise and re-exercise of 2 minutes elevated the blood lactate more than i n i t i a l continuous exercise of 5 minutes, even though re-exercised f i s h were sampled immediately a f t e r re-exercise. Fig. 7 and 8 i l l u s t r a t e the additive nature of i n t e r -mittent exercise on blood lactate and the lack of recovery i n rest periods of 60 minutes after exercise of only 15 or 30 seconds duration. B. Results - Muscle Lactate. The mean l e v e l of muscle lactate i n unexercised f i s h was 132.38 mg per 100 grams wet weight (range 57.19 to 199.05 mg % ) . The effects of exercise, recovery, and intermittent exercise are i l l u s t r a t e d i n Pig. 9 , 10, 11, 12, and 13. Exercise, even of the shortest duration, caused an immediate elevation i n muscle l a c t a t e . Restoration of unexer-cised levels of muscle lactate a f t e r a 30 minute rest period was s l i g h t (Pig. 9 ) . F i g . 10 i l l u s t r a t e s that muscle lactate i n 25 I Duration of strenuous exercise in minutes. Pig. 5 E f f e c t of rest after severe exercise on the levels of blood l a c t a t e . Pish were sampled immediately a f t e r exercise, 3 minutes a f t e r , 8 minutes a f t e r , and 30 minutes a f t e r exercise. 26 E o> E o o o O O m l O O - i 80-60 40-0 J 120 E O ) E 100 d> o O 80 T 3 O O CD 60 _ Exercised continuously X B 2 min. o A _ Exercised and re-exerc ised I min. Q _ E x e r c i s e d continuously I min. 1—1 I ! 0 3 8 30 Duration of recovery period after exercise ( c u r v e s B and C ) and between exercise p e r i o d s ( c u r v e A ) Exercised and re-exerc ised o A ' 2 min. Q _ Exerc ised continuously •x c N 5 m i n -E x e r c i s e d continuously 2 min. i—r-0 3 -r-8 — i 30 Duration of recovery period after e x e r c i s e ( c u r v e s B and C ) and between e x e r c i s e periods ( c u r v e A ) Pig. 6 . Changes i n levels of blood lactate a f t e r continuous exercise (continuous curves), and a f t e r discontinuous exercise (discontinuous curves). Note that f i s h exercised intermit-t e n t l y were sampled immediately aft e r the second exercise, whereas f i s h exercised continuously were sampled i n the recovery condition. 27 first second third ' exercise exercise exercise 80-1 0 30 60 30 60 Duration of recovery time in minutes between intermittent exercise periods of 15 sec. F i g . 7. E f f e c t of intermittent exercise of 15 seconds on the levels of blood lactate. 28 first second third exercise exercise exercise 0 30 60 30 60 Duration of recovery time in minutes between intermittent exercise periods of 30 sec. F i g . 8 E f f e c t of intermittent exercise of 30 seconds on the levels of blood l a c t a t e . 29 f i s h exercised only 1 minute continued to r i s e sharply for 3 minutes aft e r the exercise, whereas that of f i s h chased 2 minutes increased s l i g h t l y , and that of f i s h chased 5 minutes decreased rapidly. That muscle lactate f a i l e d to return to normal even after exercise of short duration i s i l l u s t r a t e d i n Pig. 9 , 11, and 12. This tendency for muscle lactate to remain elevated explains why blood lactate also f a i l e d to decrease to normal from short periods of exercise, and must be due to either 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 within muscle. The effects of re-exercise a f t e r 60 minutes rest appeared to be additive. However, there appeared to be an upper l i m i t (approximately 1170 mg %) which muscle lactate did not exceed (Pig. 13) . The effect of intermittent exercise of short duration, i l l u s t r a t e d i n Pig. 11 and 12, was to cause further increases i n muscle la c t a t e , again i n d i c a t i n g the f a i l u r e of muscle lactate to return to unexercised levels a f t e r exercise of short duration. The levels of muscle l a c t a t e , aft e r intermittent exercise appear i n Table VI. 30 Table VI. Eff e c t of exercise and re-exercise on the l e v e l of muscle la c t a t e . Pish rested 60 minutes between exercise periods, and were sampled immediately a f t e r exercise or re-exercise. Duration of exercise 15 seconds 30 seconds 5 minutes Muscle f i r s t chase 236 291 I4.68 lactate second chase 276 ' 3 6 6 I4.62 mg % t h i r d chase 3OI4. 358 31 Pig. 9. Changes i n the l e v e l of muscle lactate during 5 minutes severe exercise, and 30 minutes a f t e r the severe exercise. 32 Pig. 10. Changes i n levels of muscle lactate during recovery from severe exercise of 1, 2, and 5 minutes. 33 E CD E O o O _ l _CD O CO Z3 first exercise 320-, 200-120-o J s e c o n d exercise ~T~ 30 third exercise 0 30 60 60 Duration of recovery time in minutes between intermittent exercise periods of 15 s e c . Pig. 11. E f f e c t of intermittent exercise of 15 seconds on the level s of muscle la c t a t e . 3 4 f i r s t s e c o n d third e xercise exercise exercise Duration of r e c o v e r y time in minutes between intermittent exercise periods of 3 0 sec. F i g . 1 2 . E f f e c t of intermittent exercise of 3 0 seconds on the le v e l s of muscle lactate. 35 Duration of recovery time in minutes between intermittent exercise periods of 5 min. Pig. 13. E f f e c t of intermittent exercise of 5 minutes on the level s of muscle l a c t a t e . 36 C. Discussion of Blood and Muscle Lactate. Blood and muscle lactate are discussed together since muscle tissue i s the source of blood lactate, and thus changes i n levels of muscle lactate are r e f l e c t e d by changes i n levels of blood l a c t a t e . The r e s t i n g l e v e l of blood lactate varies from year to year i n the rainbow trout raised at Summerland, even though temperature and feeding conditions are s i m i l a r . Some probable reasons for this year to year v a r i a b i l i t y include the following: the h i s t o r y of the f i s h d i f f e r (for example the exact type of food), technique of catching the f i s h and withdrawing the sample vary from experimenter to experimenter; and most import-ant, there are d i s t i n c t races i n the species Salmo gairdneri. Levels of blood lactate i n unexercised Salmo gairdneri appear i n Table VII. These values are s i m i l a r to those reported for other species of f i s h (Secondat and Diaz, 194-2; Black, 1955* 1956, 1957a, 1957b; Parker et a l . , 1959; Gaillouet, 1964.; P h i l l i p s , 1958; Dean and Goodnight, 1964.; Lievestad, 1957; Heath and Pritchard, 1962), and s i m i l a r to those reported for man (Bock et a l . , 1932; Cook and Hurst, 1933; and Bang, 1936). The l e v e l of muscle lactate i n unexercised rainbow trout at Summerland i s much more variable than the l e v e l of blood l a c t a t e . Levels of muscle lactate i n unexercised f i s h appear i n Table VIII. 37 Table VII. Levels of blood lactate i n unexercised Salmo  gairdneri. Year Blood lactate mg% Age Reference years 1957 7-2 1 Black et a l . , 1959 1958 4 . 0 l i Black et a l . , 1962 1958 9.3 l-£ M i l l e r et a l . , 1959 1959 8.8 1-|- Black et a l . , 1962 1961 5 . 1 l i Manning, 1963 1964 7.2 1§- present study 38 Table V I I I . L e v e l s of muscle l a c t a t e i n unexercised f i s h . Year Species Muscle l a c t a t e mg^ Reference 1927 haddock 150 R i t c h i e 1927 cod 80 1927 hake 50 1957 cod 5 to 50 L e i v e s t a d e_t a l . 1957 s t e e l h e a d t r o u t 174 Nakatani, 1957 1958 rainbow t r o u t 66 B l a c k et a l . , 1962 1959 rainbow t r o u t 187 Black et a l . , 1962 1962 grunion 50 Scholander 196J4. rainbow t r o u t 132 present study 39 The values of muscle lactate i n r e s t i n g f i s h are much higher than those reported f o r the frog (8 mg %, Sacks et a l . , 1954) and f o r the cat (15 mg %, Sacks and Morton, 195°)> but the frog was pithed and the cat was anesthetized, thus eliminating a c t i v i t y during the sampling procedure. The source of res t i n g blood lactate i s probably muscle, but Evans (1934) n a s stated that i n the heart-lung preparation of a dog, the lungs produce enough lactate to account for r e s t i n g blood lactate production. Cook e_t a l . , (1933) stated that the source i s g l y c o l y s i s . Leopold and Bernard (1931) observed that r e s t i n g blood lactate was not correlated with re s t i n g blood glucose i n children. According to Huckabee (1956), the d i s t r i b u t i o n of lactate between the erythrocytes and the plasma i s equal. Decker (1942) observed that the d i s t r i b u t i o n i s equal only i n the re s t i n g condition, and that at higher concentrations of lactate, the concentration of lactate i s greater i n the plasma than i n the red c e l l s . Many variables other than exercise a l t e r blood and muscle levels of lac t a t e . Scholander (1962) using the grunion, L:eivestad at a l . (1957) using the cod, and Caillouet (1964) using the channel c a t f i s h , showed that asphyxia, induced by removing the f i s h from the water, elevated muscle l a c t a t e . They found that the blood lactate did not r i s e u n t i l the f i s h was returned to the water and concluded that blood was being diverted away from muscle tissue when the f i s h was out of water. In mammals, Himwich (1932) elevated blood lactate with the following: adrenaline i n j e c t i o n , a l k a l i i n j e c t i o n , evisceration, anaemia, and hemorrhage. Kubicek ejt a l . (1959) showed that induced fever increased blood lactate i n dogs. Eggleton and Evans (1930) showed that hypoventilation decreased blood l a c t a t e . Huckabee (1958) showed that hyperventilation, bicarbonate infusion, pyruvate infusion, and glucose infusion a l l elevate blood l a c t a t e . Huckabee (1961) also found an elevated blood lactate i n many r e s t i n g h o s p i t a l patients. The majority of these elevated blood lactates could be a t t r i -buted to either elevated blood pyruvates or to hypoxemia of respiratory or cardiovascular disease. No explanation was given for the elevated blood lactate i n the remaining cases, and each of these patients died of acidosis. Secondat and Diaz (191+2) were the f i r s t to show that exercise elevates blood lactate i n f i s h ; and Nakatahi (1957) f i r s t to show that exercise elevates muscle lactate i n f i s h . The lactate response to exercise was f i r s t demonstrated by Fletcher and Hopkins (1907). They also proposed that the f a i l u r e of muscle to contract i n spite of adequate stimulation was due to muscle lactate reaching a ce r t a i n maximal value. Meyerhof (1920) demonstrated that the source of lactate was muscle glycogen. The response of blood lactate during severe exercise has been studied i n many species of f i s h (Secondat and Diaz, 1942; Black 1957 a, b, c; Nakatani, 1957; Black et a l . 1958 -I 9 6 0 ; M i l l e r et a l . , 1959; Parker and Black, 1959; Parker et a l . , 1959; Jonas e_fc a l . , 1962; Black and Barrett, 1957; C a i l l o u e t , 1964; Dean, 1 9 6 2 ; Barrett and Connor, 1 9 6 2 ) . Collins and E l l i n g ( I 9 6 0 ) and C o l l i n s et al. , ( 1 9 6 4 ) found that prolonged moderate exercise of steelhead trout (Salmo gairdneri ) i n an endless fishway increased blood lactate only two f o l d . Huckabee (1956) reports that even mild exercise elevates blood l a c t a t e i n man. Hochachka (1961) analyzed the effect of t r a i n i n g on oxygen debt and glycogen reserves i n rainbow trout. He found that the trained group could accumulate an oxygen debt three times that of the untrained group, that the trained group used more of t h e i r muscle glycogen reserves and resynthesized i t f a ster during recovery, and that i t took longer to exhaust the trained f i s h . Lactate levels were not measure. Anderson et. a l . , ( I 9 6 0 ) found that the maximum levels of blood lactate occuring In man a f t e r exercise were the same for athletes and non-athletes. Denyes and Joseph (1956) observed that larger bass tended to produce more lactate than smaller bass, but no s i g n i -f i c a n t c o r r e l a t i o n between size and lactate levels was found h-2 i n the present study. Black et al.(1962) observed a difference i n the lactate response to exercise between sexes i n rainbow trout. The l e v e l of muscle lactate was greater i n the female than the male during exercise and recovery. Muscle lactate reached a maximum l e v e l a f t e r 2 minutes of severe exercise i n the female, whereas i t took 9 minutes i n the male. Anderson (I960) observed that the lactate response was greater i n women than men for the same amount of work done. Caillouet (196i+) observed no s i g n i f i c a n t differences between sexes i n the lactate response during exercise and recovery i n the channel c a t f i s h . No consistent s i g n i f i c a n t differences were observed between sexes i n the present experiment. The effect of age or stage of sexual maturity on the lactate response to exercise has not been experimentally determined i n rainbow trout. Anderson (I960) observed that age was a s i g n i f i c a n t factor i n determining the lactate response to exercise i n man. In the present study, the gonads of every f i s h were examined. We observed an obvious increase i n gonad development during the summer, but the sexual matur-ation did not appear to a f f e c t the lactate response to exercise i n any consistent manner. Maximal levels of blood and muscle lactate after 1+3 exercise vary from species to species throughout the animal kingdom. Most workers studying the lactate response i n f i s h have used 15 minutes continuous severe exercise. Bang (1936) observed that the maximal blood lactate was produced by severe exercise for 1 minute i n man. Anderson (I960) observed that maximal blood lactate levels were produced by spr i n t i n g lj.00 meters (approximately 1 minutes), and s p r i n t i n g further than I4.OO meters f a i l e d to cause a greater increase i n blood l a c t a t e . Maximal leve l s of blood and muscle lactate a f t e r exercise appear i n Table IX. Others have observed that blood lactate continues to r i s e a f t e r exercise i n f i s h (Black e_t a l . , 1962; Nakatani, 1957 > Second.at and Diaz, 191+2). This has also been observed i n man (Anderson, I960). Black (1957a) observed that blood lactate r i s e s for 3 i hours aft e r 15 minutes a f t e r severe exercise, whereas the delayed increase only l a s t s 2-5 minutes i n man (Anderson, I960). There are at least three possible explanations for the continued r i s e i n blood l a c t a t e : 1. that the muscle continues to produce lactate, 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 blood, or 3 . that c i r c u l a t i o n to the muscle i s impaired. Anderson (I960) has attributed the continued r i s e of blood lactate i n man to post-exercise increased tonus associated Table IX. Maximal levels of blood and muscle lactate a f t e r exercise i n f i s h , frog, and mammals. Species Duration of exercise minutes Maximum blood lactate mS % Maximum muscle lactate mg % Referenc e rainbow trout 15 77.9 415 Black et a l . , 1962 rainbow trout 15 5 4 . 0 295 Black et a l . , 1962 rainbow trout 15 32.6 M i l l e r et a l . , 1959 rainbow trout 15 69.7 Manning, 1963 rainbow trout 54-6 468 present study steelhead trout 8.5 389 Nakatani, 1957 tench 15 53 Secondat and Diaz, 19-42 channel c a t f i s h 15 5 9 . 1 Caillouet, 1964 black c a t f i s h 15 34-9 Black, 1955 sucker 15 55.3 Black, 1955 carp 15 54.3 Black, 1955 squawfish 15 94-2 Black, 1955 bass 15 76.2 Black, 1955 sunfish 6 - 45 77 Heath and Pritchard 1962 frog 87 Sacks et a l . , 1954 cat 322 Sacks and Sacks, 1956 man l 126 Bang, 1936. 45 w i t h h y p e r e x c i t a b i l i t y due to a c i d o s i s . In the present study muscle l a c t a t e continues to r i s e a f t e r e x e r c i s e of 1 or 2 minutes, but decreased immediately a f t e r e x e r c i s e of longer d u r a t i o n , t h a t i s , 5 minutes. Some d i f f u s i o n b a r r i e r must e x i s t between muscle and blood. The r a t e of d i f f u s i o n of l a c t a t e has only been s t u d i e d between e r y t h r o c y t e s and plasma. Johnson et a l , , (1945) showed that l a c t a t e d i f f u s i o n i n mammalian blood was p a s s i v e and u n r e s t r i c t e d but extremely temperature dependant (the Qio f o r d i f f u s i o n i n c r e a s e d from 2 to 30 f o r a temperature decrease of 25-36 to 0-14 degrees). Margaria e_t a l . (1933) observed t h a t the d e l a y of d i f f u s i o n of l a c t a t e from muscle to blood i s g r e a t e s t d u r i n g severe e x e r c i s e . Manning (unpublished) noted that 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. I t i s most probable t h a t the delayed i n c r e a s e i n blood l a c t a t e i s due to a l l three f a c t o r s . Another f a c t o r which p o s s i b l y c o n t r i b u t e s to delayed--i n c r e a s e of blood l a c t a t e and to the slowness of r e c o v e r y i s the e f f e c t of temperature on the l a c t a t e response. Black (1957a) showed no s i g n i f i c a n t d i f f e r e n c e s of l a c t a t e p r o d u c t i o n at two temperatures of a c c l i m a t i o n i n Kamloops t r o u t . Denyes and Joseph (1956) found the h i g h e s t i n d i v i d u a l values and g r e a t e s t v a r i a b i l i t y among f i s h was a s s o c i a t e d w i t h f i s h a c c l i m a t e d to lower temperature i n l a r g e mouth bass. Dean and Goodnight (1964) found 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 white c r a p p i e , and b l a c k b u l l h e a d when ac c l i m a t e d to lower temperatures. I n large mouth bass, blood lactate was lower at lower acclimation temperatures for unexercised f i s h , but higher at lower a c c l i -mation temperatures for exercised f i s h . In the b l u e g i l l , blood lactate was higher at lower acclimation temperatures for unexercised f i s h , but lower at lower acclimation temper-atures for exercised f i s h . They also concluded that at lower temperatures of acclimation, f i s h r e l y on the pentose shunt for energy production. Ekberg (1962) demonstrated that glucose-6-phosphate dehydrogenase i s not altered by changing external temperature of the carp, whereas 6-phospho-gluconic dehydrogenase and aldolase are more active i n cold adapted f i s h . He also found that resistance to iodoacetate poisoning i s greater i n g i l l s from cold adapted goldfish, but resistance to cyanide poisoning i s greater i n warm adapted f i s h . Hart and Heroux (1954) showed that the lactate response to exercise was the same i n deer mice acclimated to 0 degrees as i t was i n those acclimated to 22 degrees. They found that the lactate response was only altered by temperature at extremely low temperatures, -10 to -i|.0 degrees. Caillouet (1964) found that levels of blood lactate of unexercised channel c a t f i s h were higher i n f i s h acclimated to higher temperatures, and that s i m i l a r trends were noted i n exercised channel c a t f i s h . Caillouet (1964) also noted higher i n d i v i d u a l values and great v a r i a b i l i t y among i n d i v i d u a l carp acclimated to higher temperatures. A c t i v i t y i n fishes (Pry, 1947J Pry and Hart, 194$) a n d the rate of di f f u s i o n " o f lactate from erythrocytes to plasma (Johnson e_t a l . , 1945) have been shown ' 4 7 to increase with increase i n temperatures. Hochachka and Hayes (1962) have shown that trout acclimated to low temperatur r e l y more on the pentose shunt for energy production than the Embden-Meyerhoff pathway. It i s probable that a c t i v i t y , metabo lism, and d i f f u s i o n are temperature dependant i n the rainbow trout. A l l of the experiments i n the present study were done at the same temperature, 10.5 degrees C. The major difference between f i s h and mammals with respect to the lactate response i s the rate of recovery of blood lactate. Alpert and Root (1954) showed that hfi% of lactate produced by severe exercise i n man was u t i l i z e d within 10 minutes; whereas Black e_t a l . , (1962) showed that \xQ% of the lactate produced by severe exercise i n rainbow trout was u t i l i z e d In approximately 8 hours. The slow recovery of blood lactate i n f i s h has been noted by others (Secondat and Diaz, 191+2; Black et a l . , 1959). In man, Anderson (I960) has shown that blood lactate was the l a s t value to return to r e s t i n g levels a f t e r exercise. Heart rate, oxygen intake, carbon dioxide output, and pulmonary v e n t i l a t i o n each returned to unexercised levels before blood lactate. The fate of lactate produced by strenuous exercise has been the subject of much study. Cori (1929) observed that lactate given either by mouth or subcutaneously enhanced l i v e r glycogenosis. That the l i v e r removes lactate was corroborated 48 ' by other workers (Eggleton and Evans, 1930; Evans et a l . , 1930; Himwich et a l . , 1930; I v y and C r a n d a l l 1941: Vennesland et a l . , 1942.) E g g l e t o n and Evans (1930) perfused f r o g muscle w i t h l a c t a t e and observed no i n c r e a s e i n muscle g l y c o g e n e s i s . Himwich (1932) and McGinty (1931) showed t h a t the heart o x i d i z e s l a c t a t e . Evans (1933), Himwich (1932), and Ashford (1931) showed t h a t b r a i n metabolizes l a c t a t e . H i l l (1924) d e s c r i b e d the r e c o v e r y o f l a c t a t e as a b i p h a s i c process; the i n i t i a l r a p i d phase was the o x i d a t i o n of muscle l a c t a t e , and the l a t e r slow phase was the o x i d a t i o n of l a c t a t e which escaped from the muscle t o the b l o o d . W e l l s (1957) r e l a t e d the r e c o v e r y curve of l a c t a t e t o t h a t o f oxygen consumption and on t h i s b a s i s concluded t h a t r e c o v e r y was a t r i p h a s i c p r o c e s s . On the b a s i s of oxygen consumption a f t e r strenuous e x e r c i s e , Meyerhoff (1920) t h e o r i z e d t h a t £ of the l a c t a t e was o x i d i z e d , and 3/4 was r e s y n t h e s i z e d i n t o glycogen. Both oxida-t i o n and g l y c o g e n e s i s are processes which occur i n the l i v e r and both r e q u i r e oxygen. E g g l e t o n and Evans (1930 found t h a t the r a t e of removal of l a c t a t e decreasedfrom 2 mg % per minute t o 0 .6 mg f0 per minute a f t e r removing the l i v e r . Recent o b s e r v a t i o n s by Drury and Wick (1956, and 1953} i n d i c a t e t h a t very l i t t l e i f any l a c t a t e i s 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 i n the i n t a c t r a b b i t . Most of the l a c t a t e appears as e x p i r e d carbon d i o x i d e . They a l s o observed t h a t l a c t a t e i s o x i d i z e d f a s t e r t han glucose and they h y p o t h e s i z e d t h a t l a c t a t e a c t s as a quick f u e l i n emergencies. 4-9 There are very few studies of intermittent strenuous exercise i n man, and a review of the l i t e r a t u r e revealed none on intermittent strenuous exercise i n f i s h . Christensen (1956) found that alternating periods of exercise and recovery of 3 0 seconds for one hour did not cause a s i g n i f i c a n t increase i n blood l a c t a t e , whereas continuous exercise at the same in t e n s i t y caused exhaustion i n 9 minutes and elevated blood lactate to l£0 mg %. Christensen also showed that i f the duration of work and rest periods was increased, then the blood lactate increased. Some of Christensen's data appear i n Table X. Astrand e_t a l . (I960) using experimental conditions si m i l a r to Christensen showed that the major factor determining blood lactate during intermittent exercise was the duration of the exercise period, and that the duration of the recovery period was of secondary importance. Man can tolerate strenuous intermittent exercise at least 2 0 times longer than f i s h , since he recovers from exercise much faster. Flock e_t a l . (1939) intermittently e l e c t r i c a l l y stimulated the muscle of an anesthetized mouse. They found that a recovery time of greater 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 an increase i n muscle l a c t a t e . 50 Table X. Blood levels of lactate after intermittent exercise i n man from Christensen (1956)• Duration of Duration of Duration of t o t a l Blood lactate exercise rest p e r i o d e x e r c i s e time mg % 30 seconds 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 9 minutes 150 51 Astrand and Christensen explain t h e i r results on the basis of myoglobin. Exercise i s done aerobically using oxygen bound to myoglobin. When the oxygen store bound to myoglobin i s depleted, then the source of energy i s anaerobic and the re s u l t i s lactate formation. On this basis, t h e i r r e s u l t s show that the myoglobin w i l l provide s u f f i c i e n t oxygen for 30 seconds strenuous exercise, and that only 30 seconds recovery time i s required to reload the myoglobin with oxygen. The r e s u l t s of Christensen and those of the present study demonstrate the marked difference i n the response between f i s h and man to intermittent exercise, and are compared i n Table XI. An explanation of the obvious discrepancy between the response of f i s h and man to intermittent exercise i s d i f f i c u l t . Yamaguchi and Matsuura (1961) have characterized myoglobin of tuna, and concluded that the prosthetic group of hemoglobin and myoglobin of f i s h i n general i s probably the same as that of mammals. Matsuura and Hashimoto (1956) have characterized myoglobin i n several species of f i s h (tuna and swordfish) and found that myoglobin of f i s h i s sim i l a r to that of horse heart. Matsuura and Hashimoto (1955) demonstrated that the i r o n content i n myoglobin of the red muscle of f i s h (Paranthunnus s i b i and Thunnus o r i e n t a l i s ) i s greater than that found i n horse heart. Assuming that myoglobin i s present i n trout muscle, then the long time 52 Table XI. A comparison of the resistance to fatigue between f i s h and man during intermittent strenuous exercise as i indicated by the l e v e l of blood lactate Animal Duration Duration Number of Total Blood Reference of of of times time lactate exercise rest exercised exercised mg % period . _ . man 30 sec. 30 sec. f i s h 30 sec. 60 min. 60 30 min. 3 1.5 min. 20 Christensen 9 7 present study 53 required for 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 lactate from muscle to blood. The c i r c u l a t i o n time i s probably much less i n f i s h than i n man. Mott (1957) reports that the c i r c u l a t i o n time i n the toadfish i s about 2 minutes, whereas that of man i s only 15 to 18 seconds (Fishman, 1963). Assuming that myoglobin i s a functional 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 exercise of 30 seconds' should take 10 times as long i n f i s h as i t takes i n man. But, the results of the present study show that f i s h can not r e s i s t intermittent strenuous exercise when the recovery period Is 120 times as long. As present, there appears to be no explanation for the discrepancy between the response to strenuous intermittent exercise 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 exercise and mortality i n f i s h has been reviewed by Black (1958). Mortality as a r e s u l t of hyperactivity has been observed i n cod and dab (von Buddenbrock, 1938), tench (Secondat and Diaz, 191+2), striped bass ( L i t t , 1954-) » chinook salmon f i n g e r l i n g s (Annonymous, 1957), sockeye salmon (Black, 1957c) coho and chinook salmon (Parker and Black, 1959), and channel c a t f i s h (Caillouet, 1964). In most of these studies mortality was 54 associated with high levels of blood l a c t a t e . In the present study there were no mo r t a l i t i e s produced by exercise. Moreover, a search of the l i t e r a t u r e has failed to produce any evidence for mortality i n rainbow trout as a r e s u l t of severe exercise. Huckabee (1961) has described a f a t a l pathological condition i n humans i n which mortality i s associated with lactate acidosis. The elevated blood lactate i n this case i s not associated with hyperactivity,but rather with impaired p e r i -pheral c i r c u l a t i o n . Other parameters have been associated with the elevated blood lactate of hyperactivity. Secondat (1950) observed a decreased blood oxygen capacity which was correlated with an elevated blood lactate i n the carp. Jonas e_t a l . (1962) showed that mortality i n rainbow trout could be produced by in j e c t i o n s of l a c t i c acid but not with sodium la c t a t e . Auvergnat and Secondat (1942) observed that 15 minutes exercise caused a decreased pH i n carp blood. The pH did not return to normal aft e r a 6 hours rest period. They attributed t h i s a c i d i f i c a t i o n of blood to the release of l a c t i c acid from muscle to blood. A decrease i n blood carbon dioxide accompanied the decrease i n pH, and the carbon dioxide was s t i l l decreasing 6 hours a f t e r the exercise. They also noted that the osmotic pressure of plasma was elevated immediately af t e r the exercise, and remained elevated for 4 hours. 55 The increased osmotic pressure of plasma was attributed to the presence of by-products of muscle metabolism. Black e_fc a l . (1959) made si m i l a r studies i n rainbow trout. They observed that both blood pH and blood carbonate increased during the f i r s t 3 minutes of strenuous exercise, and then both decreased during the remaining 12 minutes of exercise. Blood pH c o n t i -nued to f a l l , the l e v e l being s i g n i f i c a n t l y depressed 2 hours a f t e r the exercise. The highest l e v e l of carbonate occurred 8 hours a f t e r the exercise. Black noted that the c h a r a c t e r i s t i c change i n behavior of exercised trout occurred at the same time as the maximum levels of hydrogen ion and maximum level s of blood carbonate. Thus, l a c t i c acid i s neutralized at the expense of plasma bicarbonate, 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 of severe exercise (Auvergnat and Secondat, 19^2). In summary; the l e v e l of blood l a c t a t e , and thus of muscle lactate, i s the r e s u l t of a great number of dynamically operating factors. Those factors which probably contribute most to the v a r i a b i l i t y i n the observed data are sex and age. I I I . GLYCOGEN A. Results - Muscle 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 effects of exercise, recovery, and intermittent exercise are i l l u s t r a t e d i n Pig. IIL., 15, 16, and 17. The l e v e l of muscle glycogen i n unexercised f i s h decreased during the course of the summer from 2 5 l to 213 mg %. Exercise, even of the shortest duration, caused an immediate decrease i n muscle glycogen. Pig. llj. i l l u s t r a t e s that one-half of the muscle glycogen present i n unexercised f i s h i s depleted as a r e s u l t of only. 30 seconds exercise. The rate of u t i l i z a t i o n of muscle glycogen decreased with further exercise. Pig. 15 i l l u s t r a t e s that muscle glycogen i n f i s h exercised 1 minute and 2 minutes continued to decrease for 3 minutes a f t e r the exercise, whereas that of f i s h exercised 5 minutes decreased only s l i g h t l y . Muscle glycogen of f i s h exercised 2 and 5 minutes f a i l e d to recover i n 30 minutes af t e r the exercise, whereas that of f i s h exercised 1 minute did recover s l i g h t l y . That muscle glycogen f a i l e d to return to unexercised levels even a f t e r extremely short duration i s i l l u s t r a t e d i n Pig. 16 and 17. The effects of re-exercise a f t e r 60 minutes recovery are to cause further decrease i n levels of muscle glycogen, 57 P i g . C h a n g e s i n l e v e l s o f m u s c l e g l y c o g e 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 . 58 Pig. 15. Changes i n levels of muscle glycogen during recovery from severe exercise of 1, 2, and 5 minutes. 59 first exercise second exercise third exercise 250-1 * E o CD o o >> O 0) o CO 200-150-100-50-/ o-J r o 30 — r ~ 60 — r ~ 30 60 Duration of recovery in minutes between intermittent exercise periods of. 15 sec. F i g . 16 Eff e c t of intermittent exercise of 15 seconds on the level s of muscle glycogen. 60 first exercise second exercise third exercise 250-1 E CD E c CD O ) O O o o CO 3 200-150-100-0 30 60 30 60 Duration of recovery between exercise and re - exercise. Pig. 17. E f f e c t of intermittent exercise of 30 seconds on the levels of muscle glycogen. 61 but there appeared to be a lower l i m i t (approximately 20 mg fo) which muscle glycogen did not exceed (Fig. 16 and 1 7 ) . The levels of muscle glycogen aft e r intermittent exercise appear i n the Table XII. B. Results - Liver 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 of l i v e r glycogen decreased during the summer as did muscle glycogen. The changes i n the levels of l i v e r glycogen during exercise, during recovery, and during intermittent exercise 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 large Inherent v a r i a b i l i t y . Levels of glycogen aft e r exercise and re-exercise of 30 seconds appear i n Table XIII. The changes i n levels i n l i v e r glycogen during 5 minutes severe exercise appear i n Table XIV. The results of an Analysis of Variance on the data i n Table XIV appear i n Table XV. Changes i n levels of l i v e r glycogen during 5 minutes severe exercise were not s i g n i f i c a n t . 62 Table XII. E f f e c t of exercise and re-exercise on the l e v e l of muscle glycogen. Pish rested 60 minutes between exercise periods, and were sampled immediately afte r exercise, or re-exercise. Duration of. .exercise 15 sec. 3.0. sec. 5 - min. Muscle f i r s t chase 161 113 i+9 glycogen second chase T i l 85 2J4. mg % t h i r d chase- 83 59 63 Table X I I I . E f f e c t of exercise and re-exercise of 3 0 seconds on the l e v e l of l i v e r glycogen. Pish rested 60 minutes between exercise periods, and were sampled immediately aft e r exercise or re-exercise. Liver glycogen (grams %) mean (9$% confidence in t e r v a l ) f i r s t chase 2 . 1 + . 0 7 second chase 2 . 9 8 4 t h i r d chase 3 . 0 2 7 (1.592 to 3 . 2 2 2 ) (2.225 to 3 . 7 4 3 ) ( 3 . 0 2 7 to 4 . O 7 4 ) 64 Table XIV. E f f e c t of £ minutes severe exercise, and 30 minutes recovery a f t e r 5 minutes severe exercise on the lev e l s of l i v e r glycogen. Condition l i v e r glycogen (grams %) mean ± standard deviation unexercised chase 1 min. chase 2 min. chase 5 min. chase 5 min., rest 30 rain. 4 .90 + 1.40 4 .54 + 2 .11 4 .83 + 1.30 5 .21 + 1.46 3.36 + 0 .88 65 Table XV. Analysis of variance of l i v e r glycogen data from Table XIV during and af t e r 5 minutes severe exercise. Source of degrees of Sum of Mean P F . 0 5 v a r i a t i o n , freedom squares square t o t a l 38 95-7162 treatment LL 15.81J.75 3.96 1 . 6 9 . 2.65 error 3k 79.8687 2.35 66 C. Discussion - Muscle and Liver Glycogen. The levels of muscle glycogen reported i n the present study are si m i l a r to values.in the l i t e r a t u r e for Salmo g a i r d n e r i i . The levels of muscle glycogen i n unexer-cised rainbow trout appear i n Table XVI. Claude Bernard (1876) was the f i r s t to report the presence of glycogen i n f i s h . Other early workers also noted glycogen content i n f i s h (Schondorff and Wachholder, 191i|.; Kilborn and MacLeod, 1919; and D i l l , 1921). In general, 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 report 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 simi l a r In man and rainbow trout. Prom year to year there i s a'marked v a r i a t i o n i n the levels of muscle glycogen among f i s h treated s i m i l a r l y at Summer land. M i l l e r §t_ a l . (195-9) have shown that changes i n diet can cause changes i n l e v e l of muscle glycogen. In Kamloops trout, Black et_ al_. (1962) found that the l e v e l of muscle glycogen was higher i n males than i n females during res t , exercise, and during recovery from exercise. It i s of signi f i c a n c e that Black and his co-workers only demonstrated th i s sex differenceorjP-ej i n l a t e r studies the sex difference was not s i g n i f i c a n t . It i s probable that the effect of 6 7 Table XVI. Levels of muscle glycogen In unexercised rainbow trout. Muscle glycogen Diet Reference S.fo . . . . . ...... 0.085 ground mammalian l i v e r Black e_t a l . , I960 and f i s h viscera 0.118 beef l i v e r M i l l e r et a l . , 1959 0.178 Clark's trout feed M i l l e r et a l . , 1959 0.251 Clark's trout feed present study 6 8 severe exercise or carbohydrate metabolism w i l l produce different results between the sexes only i n sexually mature f i s h . Hochachka (1961) has shown that age, stage of maturation, and t r a i n i n g each af f e c t the l e v e l of muscle glycogen i n trout. Moreover, gl y c o l y s i s during sampling w i l l deplete 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 for glycogen has been c r i t i c i z e d ( O r r e l l and Bueding, 1 9 6 4 ) . These workers indicate that values of muscle glycogen obtained with potassium hydroxide digests are r...similars rt 6,1. c.n the values obtained by other procedures. The f i r s t experimental study of the depletion of muscle glycogen i n f i s h during exercise was done by Black et a l . (I960). M i l l e r et a l . ( 1 9 5 9 ) have also studied the effect of exercise on muscle on muscle glycogen i n f i s h . Both Black and M i l l e r observed that one-half of the muscle glycogen i s depleted by 2 minutes severe exercise. This i s i n contrast to the findings of the present study i n which one-half of the muscle glycogen was u t i l i z e d with 30 seconds exercise, and that over two-thirds was u t i l i z e d i n 2 minutes severe exercise. Black e_fc a l . (I960) showed that about 80$ of the muscle glycogen was u t i l i z e d i n 1 5 minutes severe exercise, whereas i n the present study 80$ was u t i l i z e d i n 5 minutes severe 69 exercise. There are two possible explanations for the results reported above. F i r s t , that the previous workers did not determine the l e v e l of muscle glycogen aft e r 30 seconds severe exercise. And second, that the l e v e l of muscle glycogen i n the unexercised f i s h was higher i n the f i s h that I studied than i n the f i s h studied by Black or M i l l e r . Black e_t a l . , (1961) have related the rapid decrease i n muscle glycogen to the rapid increase i n muscle lactate and to the change i n behavior. They noted that f i s h r a r e l y swim rapidl y a f t e r 2 minutes severe exercise. Similar 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 quantitatively determined. Hochachka (1961) pointed out that muscle glycogen i s r a r e l y depleted to zero, no matter how severe or how long the exercise. Muscle glycogen was never completely depleted i n the present experiment. A possible explanation for this i s that a protective mechanism exi s t s . For example, the presence of l a c t i c acid i n the tissues w i l l increase the hydrogen ion content which could cause i n a c t i v a t i o n of some acid sensitive enzyme i n the glycogenolytic or g l y c o l y t i c chain. K e l l e r and Cori (1955) have shown that the enzyme phosphorylase i s inactivated i n the presence of high hydrogen ion concentra-t i o n , and Hers e_t a l . (1961+) have shown that amylo - 1 , 6 -glucosidase i s inactivated at about pH 1+.5. 70 Black et_ a l . (1962) has observed that the l e v e l of muscle glycogen i n rainbow trout severely exercised for 1$ minutes did not return to normal during a Zlx. hour recovery period. In the present study the l e v e l of muscle glycogen did not return to the unexercised l e v e l during a 60 minute recovery period from only 15 seconds severe exercise. Black et a l . (I960) have also shown that the muscle glycogen recovers faster I f the f i s h are fed during recovery. Muscle glycogen returns to normal faster i n mammals than i n f i s h . The slow resynthesis of muscle glycogen i n f i s h could be due to any of the following: 1. Enzyme i n h i b i t i o n due to the elevated hydrogen concentration associated with the prolonged elevation of l a c t i c acid i n the muscle. 2. The metabolism of the f i s h i s lower since the temperature i s lower than that of mammals. 3 . The lack of nutrients required to resynthe-size the muscle glycogen due to impaired c i r c u l a t i o n would slow glycogen synthesis. To my knowledge, there have been no previous studies on the effect of intermittent exercise on f i s h , and, no studies on the effect of ^ 'intermittent exercise on muscle glycogen levels i n any inta c t animal. Flock 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 leg muscle of mice and measured lactate and glycogen l e v e l s . They found that the 71 muscle glycogen was almost completely depleted aft e r stimu-l a t i o n for 1 minute, and that the l e v e l of muscle glycogen changed l i t t l e thereafter even though the muscle continued to contract. These workers therefore postulated that glycogen serves as the i n i t i a l source of energy, but that there must also be some other source of energy. I f muscle glycogen i s the major source of energy for muscle contraction during severe exercise i n trout, then the present study indicates that muscle glycogen reserves are probably the l i m i t i n g factor i n exercise. Moreover, the frequency of severe exercise of short duration must be very low--that i s , only a few times per day. Heath and Pritchard (1962) demonstrated that oxygen consumption remains elevated for about 2l\. hours aft e r severe exercise of 6 to l\S minutes i n the b l u e g i l l sunfish. They also measured blood lactate levels and observed that the l a t t e r returned to pre-exercise levels within 10 hours. It i s probable that the elevated oxygen consumption during the f i r s t 10 hours i s associated with oxidation of the lactate produced by exercise, and that the elevated oxygen consumption from 10 to 2i|, hours i s associated with re-synthesis of muscle glycogen. 72 IV. The Relationship between Glycogen and Lactate i n Muscle*. Meyerhoff (1920) was the f i r s t to demonstrate that muscle glycogen was the source of lactate during exercise. Black (1957c) presumed that the source of blood lactate i n exercised trout was muscle glycogen. Black e_t a_l. (I960) demonstrated that the relationship between muscle glycogen and blood lactate i s c u r v i l i n e a r . My data substantiates Black's data since i n the present study the rel a t i o n s h i p between blood and muscle lactate i s c u r v i l i n e a r . Thus, an extrapolation from Black's graphs would y i e l d a l i n e a r r e l a -tionship between muscle glycogen and muscle lactate. To demonstrate the rel a t i o n s h i p between lactate and glycogen i n muscle a c o r r e l a t i o n analysis was done on the means of 11 samples. The c o r r e l a t i o n c o e f f i c i e n t (r = -0 .934) indicated that the relat i o n s h i p between these two variables i s highly s i g n i f i c a n t . The observed rela t i o n s h i p (the calculated regression l i n e ) i s compared with the theore-t i c a l r e l a t i o n s h i p i n Pig. 18 assuming that 162 mg. of glycogen appear as 180 mg. of lactate. At lower levels of exercise (that i s , low lactate and high glycogen levels) the points f i t the t h e o r e t i c a l l i n e very well. But, at higher levels of exercise (that i s , low glycogen and high lactate levels) a l l of the points are above the t h e o r e t i c a l l i n e . These l a t t e r results indicate that at the higher 73 i 1 1 1 1 1 0 50 100 150 2 0 0 2 5 0 Muscle Glycogen mgm.96 F i g . 18. The relationship between muscle lactate and muscle glycogen i n f i s h sampled immediately aft e r exercise. Ik levels of exercise studied i n the present experiment there i s a source of muscle lactate other than muscle glycogen. The slope of the l i n e of the t h e o r e t i c a l relationship l i e s within the 95$ confidence l i m i t s of the slope of the calculated regression l i n e , but not within the 99$ confidence l i m i t s of the slope of the calculated regression l i n e . The slope of the l i n e of the t h e o r e t i c a l relationship i s -1.11. The 95$ confidence 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 .82. In Table XVII, the percent of lactate not accounted for by the decrease i n glycogen i s high only for severe exercise l a s t i n g for more than one minute. There are four' possible explanations for the results recorded i n F i g . 18 and Table XVII. 1. Actual glycogen present i s greater than that measured. 2. Actual lactate present i s less than that measured. 3. Glycogen i s being rapi d l y synthesized during exercise. ij.. There i s a source of lactate other than glycogen during the l a t e r stages of prolonged severe muscular exercise. Table XVII. The percent of muscle lactate not accounted for by the decrease i n the l e v e l of muse le glycogen. ...... .... L T Exercise Level of Decrease Level of Increase Theoretical (T-L) : condition muscle i n muscle muscle i n muscle increase i n . L. . glycogen glycogen lactate lactate. l e v e l of mg % levels mg % levels muscle lactate mg % mg % mg fo unexercised 251 0 132 0 0 0 15 sec. 161 90 236 104. 100 4 30 sec. 113 138 291 159 153 k 15 sec. (twice) 111 14.0 276 144 156 0 15 sec. (3 times) 83 168 304 172 187 0 1 min. 9?, 159 306 174 177 0 30 sec. (twice) 85 166 366 234 184 21 30 sec. (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 3 3 0 ' • • • 252 24 (T-L) x 100$ ~ ~ L ~ Percent of muscle lactate not accounted for by the decrease In the l e v e l of muscle glycogen assuming that 162 mg of glycogen appear as 180 mg of lactate. 7 6 The a n a l y t i c a l methods for determination of glycogen have been recently c r i t i c i z e d by Orrel and Bueding (19614.). These workers reported that extraction with hot a l k a l i yields absolute values si m i l a r to other methods of extraction; but that the glycogen iso l a t e d with a l k a l i has d i f f e r e n t character-i s t i c s than that obtained by less severe extraction techniques. The method used to determine lactate concentration was 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 on this method. It i s u n l i k e l y that any substances i n t e r f e r e with lactate determination i n s u f f i c i e n t quantities to inval i d a t e the evidence for a source of lactate other than muscle glycogen. There i s no evidence, to my knowledge, for or against synthesis of muscle glycogen during exercise i n f i s h . But, measurements of the rate of restoration of muscle glycogen a f t e r 15 minutes severe exercise by Black et a l . (I960) indicate that muscle glycogen synthesis i s a slow process i n f i s h . Therefore i t i s probable that there i s a source of lactate other than muscle glycogen during prolonged muscular exercise. If l i v e r glycogen i s a source of muscle lac t a t e , then I would expect two r e s u l t s : 1. a depletion of l i v e r glycogen during 77 exercise, and 2. an elevated blood glucose during exercise. M i l l e r e_fc a_l. ( 1 9 5 9 ) demonstrated that l i v e r glycogen i s i n fact depleted during 15 minutes exercise i n trout i f the i n i t i a l l e v e l of muscle and l i v e r glycogen i s low. Dean and Goodnight (1964) found that 15 minutes exercise i n bass, crappie, and bullhead decreased l i v e r glycogen s i g n i f i c a n t l y a f t e r depletion of muscle glycogen stores. The levels of l i v e r glycogen did not change s i g n i f i c a n t l y during exercise or during intermittent exercise i n the present study. Elevation of blood glucose during exercise has not been demonstrated i n rainbow trout. Secondat (1950) found that exercise usually elevated blood glucose i n carp, but that the increase was not consistent. In lake trout Black ( 1 9 5 7 b ) found that blood glucose increased af t e r 15 minutes severe exercise and remained elevated throughout the 24 hour recovery period studied. In bass, crappie, and bullhead, Dean and Goodnight (1964) found that 15 minutes exercise always produced a s t a t i s t i c a l s i g n i f i c a n t increase i n blood glucose l e v e l . I f glucose does increase i n response to exercise, the mechanism probably involves adrenaline. In mammals, adrenaline increases blood glucose l e v e l s , and the elevated blood glucose during muscular a c t i v i t y appears to be related to excitement (Peters and Van Slyke, 1946). 78 Other possible sources of lactate include catabolism l i p i d and protein from blood or from the muscle i t s e l f , and by-absorption of foodstuffs from the gut.. None of these were examined i n the present study. Fontaine and Hatey (1953) have shown that both fat and protein are metabolized during the upstream migration of A t l a n t i c salmon. Blazka (1958) has reported that exercise elevates ammonia and non-protein nitrogen i n carp blood. Hypoxia elevates non e s t e r i f i e d f a t t y acids (but does not elevate lactate) i n trout blood (Blazka, 1958.) In summary, the present study provides evidence that there i s a source of muscle lactate other than muscle glycogen, and that this source of energy i s probably catabolism of l i p i d or protein. V. General Discussion It i s evident that i n f i s h the changes of carbohydrate metabolism during severe exercise are s t i l l not completely understood. In general, metabolism appears to be si m i l a r i n f i s h and mammals i n that glycogen i s broken down during severe exercise. When the supply of oxygen does not meet the demand, then the reduced forms of D P N H 2 A N D F A D H 2 accumulate. Thus, the Kreb's cycle f a i l s to operate due to lack of supply of the 7 9 oxidized forms of DPN and PAD. In turn, pyruvate backs up i n the chemical chain to form lactate. As a r e s u l t , accumulation of lactate indicates the i n s u f f i c i e n c y of the oxygen supply and also muscular exertion. Lactate also serves as an hydrogen acceptor for D P W H 2 ' During recovery from severe exercise, lactate i s either excreted or converted back to pyruvate. The pyruvate so formed i s then oxidized to carbon dioxide and water, or resynthesized to glycogen. Resynthesis of glycogen i s probably from blood glucose rather than from muscle lac t a t e , and i s a slow process i n f i s h . The above scheme i s i l l u s t -rated i n F i g . 19. It i s possible to explain the slowness of recovery from severe exercise on thebasis of the c i r c u l a t o r y system using 30 seconds severe exercise as an example. 1. Severe exercise for 30 seconds elevates muscle lactate 160 mg per 100 g of muscle above the unexercised l e v e l . Since 1 ml of oxygen i s required to convert 8.03 mg of lactate to pyruvate, then 160 mg of lactate requires 20 ml of oxygen. 2. In the present study the hemoglobin concentration was s u f f i c i e n t to transfer a maxium of 11 ml of oxygen 80 per 100 ml blood. Thus to transfer 20 ml of oxygen, 182 ml of blood i s required. 3 . The cardiac output i n f i s h i s small, and i s probably about 1 ml/100 g body weight/minute for rainbow trout (Hart, 191+3). In order to pump 182 ml of blood/100 g body weight, the f i s h would require 182 minutes, or approx-imately 3 hours to recover from 30 seconds severe exercise. 81 IN THE PRESENCE OF ADEQUATE OXYGEN - UNEXERCISED CONDITION muscle glycogen I ^ _ muscle pyruvate ^  muscle l a c t a t e ^ ~blood lactate ^-oxygen muscle carbon dioxide • b l o o d carbon dioxide IN THE PRESENCE OF AN INSUFFICIENT OXYGEN SUPPLY - SEVERE EXERCISE muscle glycogen I _ muscle pyruvate"" - y muscle lactate ^ , blood lactat e ^-oxygen muscle carbon dioxide RECOVERY FROM AN INSUFFICIENT OXYGEN SUPPLY - RECOVERY FROM SEVERE EXERCISE excreted"^ blood^lactate • l i v e r glycogen tissue lactate r tissue pyruvate ^-oxygen tissue carbon d i o x i d e — • b l o o d carbon dioxide F i g . 1 9 . The effect of oxygen supply on carbohydrate metabolism. 82 CONCLUSIONS Some general conclusions can be made from the present study. In rainbow trout, severe exercise depletes muscle glycogen extremely r a p i d l y . Moreover, i n f i s h , the resto r a t i o n of muscle glycogen to pre-exercise levels i s a. slow process. In contrast to t h i s , the rate of depletion of muscle glycogen during moderate exercise i s small (Black et a l . I960). Blood lactate continues to r i s e and remains elevated for at least 30 minutes following exercise of extremely short duration, that i s , 3 seconds. Intermittent severe exercise a f t e r recovery periods of 60 minutes causes further increases i n blood and muscle l a c t a t e , and further decreases i n muscle glycogen. In conclusion, i t appears that rainbow trout are not well adapted to tolerate severe exercise. 8 3 SUMMARY 1 . The effects of intermittent exercise and exercise of short duration ( 3 seconds to 1 minute) on carbohydrate metabolism have been studied i n rainbow trout. 2. The effect of severe exercise of short duration was to cause the following: a. an immediate decrease i n muscle glycogen l e v e l s . One-half of the muscle glycogen was u t i l i z e d during 3 0 seconds severe exercise, and Q0% u t i l i z e d during 5 minutes exercise. b. an immediate increase i n muscle lactate l e v e l s . Muscle lactate increased more than two fold during 3 0 seconds severe exercise, and increased more than three and one-half fold during 5 minutes severe exercise. c. an immediate increase i n blood lactate l e v e l s . Blood lactate levels increased two fo l d during 3 0 seconds severe exercise, and increased more than ten fold during 5 minutes severe exercise. d. an i n i t i a l rapid increase i n hemoglobin concentration, followed by a rapid decrease i n hemoglobin concentration, and this i n turn followed by a slow increase i n hemoglobin concentration. 3. During recovery periods of 3 to 60 minutes after severe exercise the following occurred. a. Muscle glycogen levels changed very l i t t l e . b. Muscle lactate levels changed very l i t t l e . c. Blood lactate levels continued to r i s e during the recovery periods even i n f i s h chased 3 seconds. This i s i n complete c o n t r a d i s t i n c t i o n to findings i n mammals. d. Hemoglobin levels continued to r i s e during the recovery periods even i n f i s h chased 3 seconds. The effect of re-exercise was to cause the following: a. an immediate further decrease i n muscle glycogen levels u n t i l the muscle glycogen was depleted to approximately 20 mg %. b. an immediate further increase i n muscle lactate levels u n t i l muscle lactate l e v e l reached approximately 20 mg %. c. an immediate further increase i n levels of blood lactate and hemoglobin concentration. 5. Levels of l i v e r glycogen did not appear to change during exercise, recovery, or re-exercise. Liver glycogen values are characterized by t h e i r large v a r i a b i l i t y . 85 6 . No m o r t a l i t i e s occurred as a r e s u l t of severe e x e r c i s e . No c o n s i s t e n t d i f f e r e n c e s were noted with r e s p e c t to sex, weight, c a t c h time, or sample time. 7. Evidence i s presented which i n d i c a t e s there i s probably a source of muscle l a c t a t e other than muscle glycogen d u r i n g 5 minutes severe e x e r c i s e . L i v e r glycogen does not appear to be the source of t h i s muscle l a c t a t e . 86 AREAS FOR FURTHER INVESTIGATION It would be i n t e r e s t i n g to repeat the experiment using steelhead trout, which i s a d i f f e r e n t race of the same species but generally thought to be a more active f i s h . Much of the v a r i a b i l i t y i n data of the present study i s probably due to many of the variables which were not controlled. A more complex design may reveal the effects of age, s i z e , sex, and maturity. Because many of the differences between findings of the present study and findings i n man by Christensen are attributed to differences i n the c i r c u l a t o r y systems of f i s h and man, i t i s of importance that the c i r c u l a t o r y system i n f i s h be studied. In p a r t i c u l a r an accurate estimate of the cardiac output and c i r c u l a t i o n time are required. To explain the observed changes i n blood hemoglobin concentration during exercise, i t i s e s s e n t i a l that the l o c a t i o n and control of the venous reservoirs and the location and control of red c e l l reservoirs be ascertained. 87 It would also be p r o f i t a b l e to study the role of myoglobin i n rainbow trout. That i s , to determine i t s concen-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 demonstrate whether or not i t i s functional i n this animal. 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Lactic and pyruvic acid relations .In. contracting mammalian muscle. American Journal of physiology I06: 221-223. Sacks, J. and W. C. Sacks. 1937* Blood and muscle l a c t i c acid i n the steady state. American. Journal of Physiology 118: 697-702. Schiffman, R. H. and P. 0. Fromm. 1959. Measurement of some parameters i n rainbow trout (Salmo gai r d n e r i ; ) . Canadian Journal of Zoology 37": 2"5"-32. Scholander, P. F., E. Bradstreet and W. F. Garey. 1962. Lactic acid response In the grunion. Comparative Biochemistry and Physiology 6: 201-203. Schondorff, B., and K. Wachholder. 1914* d e n Glykogenstoffwechsel der Fishe. I. Die Glykogengehalt von Susswasserfischen. Pflugers Archiv fur die Gasamte Physiologie 157: 147-64. Secondat, M. 1950a. Influence de l'exercice musculaire sur l a valeur de l a glycBme de l a carpe (Cyprinus carpio L.). Comptes Rendus Academie des Sciences 233 : 796-797• Secondat, M. 1950b. Influence de l'exercice musculaire sur l a capacite pour 1'oxygene du sang de l a carpe (Cyprinus carpio L.). Compte Rendus Academie des Sciences 230: 1787-1788. Secondat, M., and D. Diaz. 1942. Recherches sur l a l a c t a c i d -emie chez le poisson d'eau douce. Comptes Rendus Academie des Sciences 215: 71-73» Sreter, F. A. and S. M. Friedman. 1958. The e f f e c t of muscular exercise on plasma sodium and potassium i n the rat . Canadian Journal of Biochemistry and Physiology 36: 333-338. 97 Steel, R. G. D. and J. H. Torrie. 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. Hastings. 191*2. . Metabolism of l a c t i c acid containing, radioactive carbon i n the «* and B positions. Journal 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 acid accumulation during work. A suggested standard-i z a t i o n of work c l a s s i f i c a t i o n . Journal of Applied Physiology 10: 51-55• West, E. S. and W. R. Todd. 1961. Textbook of biochemistry. 3rd ed. MacMillan Co., New York. Yamaguchi, Y. and P. Matsuura. 1961. On the prosthetic group of hemoglobin and myoglobin of tuna. B u l l e t i n of the Japanese Society of S c i e n t i f i c Fisheries- 27 (1 ) : 38-1*1. 98 9 9 Table XVIII. Sample Size, mean, and standard deviation f o r each of the variables studied. Blood and ti s s u e samples were obtained from tseparate f i s h . Exercise condition Blood samples chase rest chase rain. min. min. n weight grams mean + Sd la c t a t e mg % mean + Sd hemoglobin g % mean + Sd 1 3 2 7 5 + 6 9 . 5 1 5 + 3 . 6 9 . 7 5 + 1 . 1 1 2 9 2 5 2 + 5 6 . 9 2 ^ + 6 . 2 1 0 . 0 5 + 1 . 0 9 5 9 219 + 2 7 . 3 55 + 9 . 5 1 0 . 7 4 + 1 . 0 9 1 3 < 9 2 5 2 + 5 5 . 0 4 3 + 7 . 7 1 0 . 9 6 + 1 . 0 1 2 •3 9 2 6 6 + 3 3 . 0 5 4 + 3 . 6 1 0 . 4 2 + 1 .05 5 3 9 2 5 6 + 5 7 . 3 7 4 + 1 4 . 9 1 1 . 0 0 + 1 . 0 4 1 8 9 2 4 3 + 5 4 . 6 4 9 + 1 1 . 5 1 1 . 0 5 + 1. . 2 2 2 9 2 6 7 + 4 4 . 4 74 + 5 . 3 1 1 . 2 0 + 0 . . 9 9 5 * 9 2 7 4 + 3 9 . 3 7 3 + 1 6 . 7 1 1 . 5 6 1 .75 1 3 0 9 2 3 7 + 9 3 . 6 7 0 + 1 2 . 9 1 1 . 1 9 + 1 .35 2 30 9 233 + 5 3 . 5 1 0 0 + 7 . 1 1 1 . 4 7 + 1. . 7 0 5 30 9 2 6 0 + 4 7 . 0 1 0 7 + 2 5 . 3 1 2 . 1 7 + 1 . 1 © 5 6 0 6 301 + 6 1 . 1 1 4 4 + 2 7 . 6 1 0 . 3 1 + 0 . . 3 5 1 3 1 6 3 5 9 + 4 0 . 2 4 9 + 5 . 4 1 1 . 3 2 + 1 . 4 9 2 3 2 6 2 7 2 + 2 9 . 2 7 9 + 1 0 . 3 1 2 . 1 4 + 0 . . 7 3 5 3 5 6 3 3 6 + 1 0 0 . 5 3 6 + 6 . 7 1 2 . 5 9 + 0 , . 5 9 1 8 1 6 3 1 3 + 5 5 . 0 6 4 + 5 . 1 1 2 . 9 6 + 1 . 2 6 2 a 2 6 2 9 3 + 4 9 . 7 3 6 + 7 . 6 1 1 . 3 2 + 1. , 2 2 5 5 6 3 5 6 + 5 9 . 5 9 3 + 1 0 . 4 1 2 . 3 4 + 1 . 2 3 1 30 1 6 2 9 3 + 3 4 . 9 3 6 + 1 4 . 1 1 2 . 0 0 + 1 .25 2 30 2 6 3 4 9 + 7 1 . 3 115 + 1 4 . 7 1 1 . 3 7 + 0 . 9 3 5 30 5 6 2 9 0 + 7 7 . 3 1 2 0 + 1 9 . 0 1 2 . 3 2 + 1 . 7 1 5 6 0 •5 6 2 9 4 + 4 1 . 1 1 4 3 + 1 6 . 5 1 1 . 4 9 + 0 . 9 4 Muscle samples n weight lactate glycogen grams mg % mg Jo. ' mean + Sd mean + Sd mean ± Sd 9 223 + 2 7 . 0 306 ± 6 9 - 4 92 ± 41.3 9 255 ± 2 9 - 4 337 ± 3 3 . 1 74 ± 7 5 -4 8 2 6 0 + 6 4 . 3 4 6 3 ± 50.7 49 ± 15.7 9 245 ± 4 7 . 5 362 + 1 0 7 . 4 46 ± 29.7 9 220 + 40.6 400 ± 9 0 . 0 49 ± 43 .3 9 213 ± 4 3 . 4 394 ± 3 5 . 6 33 ± 43 .7 9 242 + 49.5 272 + 75-9 73 ± 5 6 . 3 9 2 7 0 ± 5 4 . 4 4 1 3 + 7 1 . 0 43 ± 4 0 . 0 9 223 ± 34.3 424 ± 6 2 . 5 33 ± 4 5 . 6 9 207 ± 4 6 . 3 256 + 5 5 . 2 103 ± 7 6 . 1 9 2 4 4 ± 5 9 . 9 33© + 132.7 34 ± 2 2 . 3 9 230 ± 7 6 .5 402 + 9 1 . 1 35 ± 24 .5 6 303 + 6 2 . 2 403 ± 4 1 . 0 19 ± 1 3 . 6 265 + 43.6 462 + 42 .4 24 ± 11.0 Table XVIII. (continued). Exercise condition Blood samples chase rest chase rest sec. min. sec. min. chase sec. n weight lactate hemo grams mg fo g 5 mean + Sd mean + Sd mean globin 'o 3 7 247 + 33.6 9 + 1.9 10.74 + 0.46 3 30 8 286 + 55.7 24 4.5 10.83 0.50 3 60 6 265 38.4 19 + 17.5 10.78 + 0.78 3 60 3 6 262 + 53.3 16 + 6 .9 10.32 + 0.71 15 6 261 + 33.8 9 db 2.8 10.15 + 0.75 15 30 6 306 + 56.9 34 + 8.0 10.65 + 0.76 15 60 6 295 40.9 25 + 9 .2 10.13 + 1.22 15 60 15 6 227 + 46.2 47 + 20 .5 11.00 + 1.23 15 60 15 30 5 289 + 61 .3 76 + 5.8 11.42 + 0 .61 15 60 15 60 7 301 + 25.1 63 + 20 J. 11.53 + 1.20 15 60 15 60 15 6 298 + 40.2 74 + 23 .0 11.52 + 1.07 30 7 308 + 36.3 14 + 3 .0 9.70 + 1.13 30 30 6 270 + 34.9 49 + 11.1 10.87 + 0 .98 30 60 6 258 + 58 .8 50 + 15.6 10.04 + 0 .74 30 60 30 6 300 + 27 .1 55 + 12.1 10.53 + 1.04 30 60 30 30 6 308 + 58.3 102 + 15.5 11.34 + 0 .97 30 60 30 60 6 274 + 42 .2 88 + 13 .1 10.82 + 0 .94 30 60 30 60 30 6 292 + 40.3 97 + 19.1 11.50 + 0 .54 0 30 6 323 + 29 .7 16 + 11.7 10.70 + 0.55 0 60 6 259 + 50 .5 14 + 7.6 10.60 + 1.07 to exhaustion - - - -unexercised 8 310 + 53.0 7 + 4.1 9.15 + 1.40 Muscle samples n weight lactate gl yc og en grams mg % mg % mean + Sd mean + Sd mean + Sd 6 259 + 4 2 . 8 236 + 51.0 161 + 31.2 6 306 + 53.3 193 + 2 0 . 7 148 + 64 .0 6 304 + 51.0 240 + 114.5 162 + 30.3 6 243 + 58.0 276 + 56.O 111 + 62.9 6 251 + 50.4 177 40 .8 134 + 42.2 6 302 + 44.0 196 + 95.9 132 + 30 .5 6 289 + 42.9 304 + 83.7 83 + 44.0 6 287 + 65.5 291 + 61.3 113 + 12.5 6 263 + 45.7 271 + 77.3 127 + 56.2 6 256 + 62.6 179 + 32.0 157 + 38.5 6 253 + 39.6 366 + 70.7 85 + 21.1 6 294 + 53.5 309 + 36.6 81 + 25 . 7 6 304 + 19.8 320 + 6 8 . 9 81 + 23.6 6 268 68.3 358 + 3 6 . I 59 + 11.6 .0 270 + 68 .9 388 + 75.4 78 + 21.2 9 221 + 49.2 132 + 49.7 251 + 48 .8 101 Table XIX. Time to obtain and prepare blood and tissue samples Exercise condition Blood samples (time i n seconds) chase rest chase catch time time to obtain blood .n. min. min. mean + Sd mean + Sd 1 14.4 + 5.18 74.3 + 44.46 2 11.7 + 3.67 51.2 + 31.12 5 11.8 + 4.35 113.2 + 67.31 1 3 16.1 + 3.26 28 .0 + 14 .88 2 3 19.1 + 4.37 5 0 . 2 + 74.66 5 3 17.4 + 5 .98 2 0 . 2 + 13.17 1 8 24 .0 + 8.9© 22.5 + 10.55 2 8 17.3 ± 2.45 19.2 13.76 5 8 18.1 + 4.65 37 .0 + 26.62 1 30 20.0 + 4 . 4 4 4 4 . 1 + 38.92 2 30 19.1 + 5.37 28.1 + 8.10 5 30 20 .1 + 4.31 23.6 + 11.17 5 60 • 9 . 3 ± 2.33 16.5 16 .08 1 3 1 6 .2 ± 0.75 14.7 + 15 .38 2 3 2 4 . 7 ± 1.75 11.8 + 2 .86 5 3 5 4 . 8 + 1.17 10.7 + 3.14 1 8 1 5.2 + 1.33 17 .8 + 8 . 2 8 2 2 4 . 8 + 0.75 12.5 + 6 . 5 9 5 5 6.2 + 1.17 16.5 + 16.56 1 3© 1 5.5 + 1.22 24.3 + 30.94 2 30 2 4 .7 + 0 . 8 1 13.0 + 5.93 5 3© 5 5 .8 + 2.31 13.0 + 12.79 5 60 5 6.5 + 2.07 38.0 + 33.64 Muscle samples (time in seconds) catch time k i l l time muscle time l i v e r time mean + Sd mean + Sd mean + Sd mean + Sd 2.9 + 1.05 2 .8 + 1.09 14.8 + 2.44 1 6 . 8 + 11.29 2.1 + 0-33 3 . 8 + 2.53 15.9 + 5.33 2 0 . 8 + 12.69 2.0 + 0 .93 3 . 0 + 1.07 1 16.1 + 5.19 16.5 + 5.78 3 . 3 + 2.82 3.2 + 0.67 10.7 + 2.35 -2.7 + 1.00 2.6 + 1.24 10.6 + 2.65 -3 . 8 1 ± 3.77 2.9 + 1.05 11.4 + 2.50 -'4 .1 + 3 .02 2 .4 + 0 . 8 8 10.2 + 1.20 -2.7 +.2.33 3 . 0 + 1.58 10.1 + 3.1© -2.3 + 1.00 2.6 + 1.13 10.1 + 2.80 -3-1 ± ! « d 3 3.3 + 1.12 10.2 + 1.30 -3.0 + 1.32 3.6 + 1.33 11.0 + 2.06 -3.1 + 2.15 3 .0 + 1.00 9.6 + 2.83 11.4 j t 3.16 1.7 + 0 . 8 2 2.7 + 1.21 3 . 3 + 4.26 -\ I 1.2 + 0.41 1.5 + 0 .34 8.3 + 0 .52 -102 Table XIX. (continued) Exerc i se condi t ion Blood samples (seconds) ^^^^^^^^ i -chase rest chase r e s t chase catch time time to obtain blood sec. min. sec. min. sec . mean + Sd mean + Sd 3 16.7 + 6 .37 35.6 + 20.03 3 30 13 .4 ± 2.62 24.6 + 25.63 3 60 21.7 + 7.99 33-0 + 26.25 3 60 3 21.3 + 13.92 25.5 + 25.52 15 10.3 + 5 .20 16.3 + 11.32 15 30 17.5 + 3.56 21.0 ± 16.05 15 60 28.0 + 15.62 61.5 + 37.57 15 60 15 1 3 . 8 + 2.23 26.7 22.61 15 60 15 30 8.6 3 .51 24.8 + 33.69 15 60 15 60 10.7 + 4.61 2 4 . 4 22.30 15 60 15 60 15 12.5 + 5.21 17 .8 + 12.73 30 9 .3 + 2.81 25.7 28.25 30 30 14 .8 + 5. 00 31.2 + 23.05 30 60 14.5 + 2.17 20.5 + 10.77 30 60 30 11.8 + 3-43 17 .8 + 19.32 30 60 30 30 7.7 + 0 . 82 20.2 + 8.38 30 60 30 60 8 . 8 + 1.72 59.2 + 64.52 30 60 30 60 30 9.2 + 2.93 29.3 + 22.29 unexercised 19.5 7.19 4Y7.9 + 39.68 0 30 18.2 + 3.65 2 3 . 8 + 17.72 0 6 0 14 .8 + 4 .17 30.5 + 19.89 to exhaustion -1 Muscle samples (time i n seconds) catch time k i l l time muscle time l i v e r time mean + Sd mean + Sd mean + Sd mean + Sd 3 . 2 + 1.47 3.5 + 2.0.7 4 . 7 + 4.76 2 . 8 + 0.75 1.8 + 0.75 6 . 2 + 8.33 3 . 4 + 1.62 2.7 + I . 6 3 3 . 0 + 2.28 3 . 0 + 2.28 2.3 1.03 5.3 + 2.42 1.7 + 1.21 1.8 + 0.75 2.2 + 0.83 2.0 + 0.67 2.2 + 1.17 3 . 0 + 0.89 3.5 + I . 6 4 3 . 2 + 1.33 3 .0 + 1.67 3 . 0 + O .63 3 .3 + 1.89 2.3 0 .52 2 .8 + 1.72 2.7 + 0.82 3.0 + 1.41 2.2 + 0.75 2.7 + 1.21 1.7 + 0 . 5 2 3 .4 2.00 2.0 + 0.67 9.5 + 1.87 8 .8 + 1.33 10.2 2.48 8.2 + 1.47 8.8 + 1.47 9.5 + 2.43 7.9' + 0.90 9 .2 2.40 10.1 + 4.49 9 .0 + 2.97 7 .8 + 1.33 8.2 + 0.75 8.5 2.26 8.5 + 1.64 13.8 6 . 9 8 8.6 + 2.27 12.3 + 3 . 3 8 10.3 + 1.34 11.1 + I . 8 4 11.0 ± 6.50 103 Table XX. Actual time taken to swim 4.3 meters (approximately 3 sec.) and actual time taken to swim 23 meters (approximately 15 sec.) Exercise condition ACTUAL TIME TAKEN (seconds) Blood samples chase rest. chase rest chase f i r s t chase second chase t h i r d chase sec. min. sec. min. sec. mean + Sd mean:+ Sd mean + Sd 3 2.86 + 0.38 3 30 2.75 0.71 3 60 3-33 + 1.37 3 60 3 3.33 + 1.97 4.00 + 1.27 15 13.83 + 1.17 15 30 13.00 + 2.10 15 60 14.33 + 1.21 15 60 15 15.33 + 4* 84 17.83 + 2.43 15 60 15 30 15.60 + 5.46 12.60 +2.50 15 60 15 60 16.43 + 2.70 15.29+ 2.14 15 60 15 60 15 14.83 + 3.97 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 15.85 + 2.48 15 30 13.35 + 0.52 15 60 17.50 + 2.43 15 60 15 17.50 +1.48 15.00 + 1.09 15 60 15 30 16.65 1.03 14.15 ± 0.41 15 60 15 60 14.50 + 1.22 15.85 + 1.17 15 60 15 60 15 15.00 + 0.82 15.70 + 0.69 15.00 + 0.82 104 Table XXI. Analysis of variance and Duncan's new multiple range test on the data i n the l a t i n square design. The purpose of t h i s design was to demonstrate the effect of v a r i a b i l i t y during any one day, from day to day, and the treatment e f f e c t . A. The experimental conditions Treatment Condition Day Date Time number numb er June exercise recovery minutes 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 B. Analysis of Variance. source Calculated F values f o r each variable F ^ Q ^ ?tQ± weight catch sample hemoglobin l a c t a t e time time  during the day 2.31 1.09 1.17 1.43 2.30 between days 1.56 2.83 1.28 4.65 1.02 2.12 2.87 treatment 0 .84 2.13 1.19 1.36 26.65 105 B. Duncan's New Multiple Range Test on means with s i g n i f i c a n t F values. Any two means not underscored by the same l i n e are s i g n i f i c a n t l y d i f f e r e n t from each other at the 5$ l e v e l . Any two means which are underscored by the same l i n e are not s i g n i f i c a n t l y d i f f e r e n t at the 5% l e v e l . The te s t s are applied i n the order from the most s i g n i f i c a n t to the le a s t s i g n i f i c a n t . 1. Treatment - l a c t a t e - highly s i g n i f i c a n t . number 1 2 4 3 5 7 8 6 9 mean 43 49 54 70 74 74 78 100 107 2. Between days - hemoglobin - highly s i g n i f i c a n t . number 3 9 2 1 5 8 7 4 6 mean 9 .54 10.46 11.14 11.39 11.54 11.62 11.64 11.78 11.90 3 . Between days - catch time - s i g n i f i c a n t . number 5 3 2 4 1 9 6 8 7 mean 14.1 17.0 17 .3 18 .4 19 .3 19 .8 21.5 21.7 22.2 4 . During the day - weight - s i g n i f i c a n t . number 5 2 4 8 7 9 3 1 6 mean 224 232 235 260 262 266 278 288 299 5. During the 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 2 6 1 7 mean 65 66 67 69 72 73 78 78 84 6. Treatment - catch time - s i g n i f i c a n t . number 1 5 7 8 6 4 3 9 2 mean 16.1 17.3 17.5 18.1 19.1 19.1 20.0 20.1 24.0 

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