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Some aspects of exercise physiology in fish Kiceniuk, Joe Willie 1975

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SOME ASPECTS OF EXERCISE PHYSIOLOGY IN FISH by JOE WILLIE KICENIUK B.Sc, University of Alberta, 1969 M.Sc., University of B r i t i s h Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of ZOOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1975 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission 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 publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i ABSTRACT The l i m i t i n g factors of swimming performance were studied i n f i s h exer-c i s i n g i n a water tunnel. The relationship between (10 min) c r i t i c a l v e l o c i t y and body length determined i n 10 species of freshwater teleosts i s discussed with respect to the r a t i o of l a t e r a l red body musculature to t o t a l body weight. Electromyographic recording from red and white portions of the body musculature i n four species of f i s h showed that red muscle fibers alone are active during steady swimming at sustained speeds. White muscle fibers are active only during bursts of violent swimming, such as during rapid acceleration and for a b r i e f period preceding fatigue. Thus red muscle f i b e r s , generally accepted as having an aerobic metabolism, appear to be the major determinant of sustained swimming speed. To establish the time course of cardiovascular and respiratory changes during swimming; heart rate, v e n t i l a t i o n rate, dorsal a o r t i c , ventral a o r t i c , and right common cardinal blood pressures were monitored during steady swimming following abrupt changes i n water ve l o c i t y . Under these circumstances most of the heart rate increase occurred i n the f i r s t t h i r t y seconds and heart rate did not change further after 3-15 minutes at a given swimming speed. Ventila-tion rate tended to increase i n i t i a l l y and then decline, reaching a constant value after 15-30 minutes at a given swimming speed. Dorsal and ventral aortic blood pressure increased more slowly than heart rate, peaking after s i x minutes then declining to constant values after about 30 minutes. Blood pressure i n the common cardinal vein was constant during exercise. The animals were considered to be i n a steady state with regards to these circulatory and res-piratory variables after about 30 minutes. i i Oxygen consumption increased from a mean of 0.58 ml kg ^  min ^  at rest to a mean maximum of 4.34 ml kg 1 min \ Under the same circumstances cardiac output increased from a mean of 17.6 ml kg ^  min ^  at rest to a mean maximum of 52.6 ml kg ^  min \ The corresponding stroke volume was 0.46 ml kg ^  stroke at rest and 1.03 ml kg ^  stroke \ Arterio-venous oxygen difference at rest was 3.29 volumes % and increased to 8.3 volumes % as a result of a decrease i n venous saturation (to lower than 10% i n some cases) during exercise. Heart rate at rest was 31.75 min ^  and increased during exercise by a mean of 1.33 times. Ventral a o r t i c blood pressure rose from 38.8 Torr at rest to 61.7 Torr. The corresponding ventral a o r t i c pulse pressure rose from 11.6 Torr at rest to 26 Torr. Dorsal ao r t i c mean pressure at rest was 31 Torr and increased to 37 Torr with exercise, accompanied by an increase i n pulse pressure from 5.8 Torr at rest to 10 Torr. Ventilatory volume at rest was 211.4 ml kg * min ^ and increased to about 1700 ml kg" 1 min~l at maximal sustained swimming speed. The capacity rate r a t i o of oxygen exchange between water and blood increased from 0.6 at rest to 1.8 during exercise. A r t e r i a l blood of resting trout was 97% saturated with oxygen and % saturation did not change with exer-cise . Blood lactate at rest and at swimming speeds as high as 93% of c r i t i c a l v e l o c i t y was 0.5 ^ iM/ml. One minute after fatigue the blood lactate l e v e l had increased about f i v e f o l d and continued to increase, reaching a maximum value (6-10 uM/ml) 2 to 2.5 hours after fatigue. i i i TABLE OF CONTENTS Page Introduction 1 Section I. Recruitment of Red and White Myotomal Muscle During Swimming and i t s Relationship to C r i t i c a l V e l o c i t y Introduction 10 Materials and Methods 13 (1) The Water Tunnels 13 (a) The.laboratory tunnel 13 (b) The f i e l d tunnel 15 (2) The Fi s h 22 (3) Experimental Procedures 27 (a) Increasing v e l o c i t y tests 27 (b) Fixed v e l o c i t y tests 32 (c) Electromyography 32 (4) Analysis of Data 33 (a) Increasing v e l o c i t y tests 33 (b) Fixed v e l o c i t y tests 34 Results 36 Preliminary Experiments 36 (a) Increasing v e l o c i t y tests 36 (b) Fixed v e l o c i t y tests 36 (2) Increasing V e l o c i t y Tests 37 (3) Fixed V e l o c i t y Tests 37 (4) Ele c tromyo graphy 41 (a) Herring 41 i v (b) Carp (c) Pike (d) Trout Discussion Section I I . Changes i n Cardiovascular, Respiratory and Metabolic Variables Accompanying Prolonged Exercise i n Fish Introduction Materials and Methods II -(1) Animals (2) S u r g i c a l Procedures (3) Experimental Techniques and Equipment Used (a) Pressure measurement (b) Signal conditioning and recording (c) The water tunnel (d) Blood sampling procedures (e) Oxygen tension (f) Measurement of pH (g) Determination of oxygen consumption (h) Blood oxygen content ( i ) Hematocrit (j) Lactate determination (k) Blood i r o n concentration (1) V e n t i l a t i o n volume (m) Experimental procedure and data analysis Page Results 72 (1) Changes i n Cardiovascular and Respiratory Variables i n Response to Incremental Speed Increase , (Transient Responses) 72 (2) Cardiovascular, Respiratory and Metabolic Responses to Swimming at Steady State 75 (3) Recovery from Fatigue 90 Discussion 100 General Discussion 107 L i t e r a t u r e Cited Appendix 112 118 v i LIST OF TABLES Table Page Comparison between water v e l o c i t i e s i n the f i s h tunnel assumed from the Rev-counter or pump setting and actual measurements made with the Ott meter. Comparison between water v e l o c i t i e s i n the f i s h tunnel measured by the Aqua-log and the Ott meter. Cardiovascular and respiratory variables at rest and during exercise i n trout. 14 19 Tabulation of the K and e values for the equation V = KL e for 10 species of freshwater f i s h . 38 76 Cardiovascular and respiratory variables at rest and during exercise and after eighteen hours recovery i n i n d i v i d u a l trout. 119 v i i LIST OF FIGURES Figure Page 1 Photograph of the 25 cm diameter water tunnel used i n f i e l d experiments. 16 2 (a) Diagrammatic representation i n side view of the. flow p r o f i l e i n the f i e l d water tunnel (25 cm diameter) as determined from photographs of dye injections made over a range of water v e l o c i t i e s . 21 (b) Water v e l o c i t i e s at the periphery of the f i e l d tunnel expressed as a % of water v e l o c i t y i n the centre. 3 Photograph of the f i e l d apparatus moored i n position on the r i v e r . 24 Fatigue curves for 5 species of f i s h determined i n fixed v e l o c i t y tests. Electromyographs from carp myotomal musculature. M denotes record from mosaic muscle mass and R denotes that from red muscle mass. Electromyographs from pike red (R) and white (W) myotomal muscle masses. 40 Electromyographs recorded from herring with electrodes placed within the red myotomal muscle mass. 43 46 48 8 Electromyographs from trout red (R) and mosaic (M) myotomal musculature. 51 9 The relationship of length to c r i t i c a l v e l o c i t y (10 min). The points are for f i s h which were not represented by a s u f f i c i e n t l y large size class to derive a length v e l o c i t y regression equation. 54 10 Schematic diagram of the various instrumentation carried by the experimental f i s h . 69 11 Change i n dorsal a o r t i c and ventral a o r t i c blood pressures following an incremental increase i n swimming speed. 74 12 Cardiac output of trout during rest and exercise. 78 13 Arterio-venous oxygen differences i n trout at rest and during exercise plotted against V O 2 . 80 v i i i Figure Page 14 Stroke volume of trout heart at rest and during exercise normalized for weight of the f i s h and plotted against VC^. 15 Heart rate of trout at rest and during exercise plotted against water v e l o c i t y as a per cent of each individual's c r i t i c a l v e l o c i t y . 17. In vivo blood oxygen dissociation curve. Per cent saturation was derived from the measured ir o n concentration and measured blood oxygen content. 18 D i a s t o l i c and s y s t o l i c blood pressure i n both the dorsal (DA) and ventral (VA) aortae during exercise. 19 The relationship of v e n t i l a t i o n volume to oxygen consumption at rest and during exercise of incrementally increased intensity up to and including maximal 60 min sustained exercise. 82 84 16 Oxygen consumption of trout expressed per kilogram animal plotted against water v e l o c i t y as a per cent of each individual's c r i t i c a l v e l o c i t y . 87 89 92 94 20 Blood lactate levels of i n d i v i d u a l swimming trout at specified swimming speed and following fatigue. 96 21 Heart rate during recovery from fatigue. 98 22 E l e c t r i c a l model of f i s h circulatory system. 106 i x ACKNOWLEDGEMENT S I wish to thank my supervisor, Dr. Dave Jones, for h i s guidance through-out the course of my study. The f i s h swimming speed studies i n Section I were done j o i n t l y with Dr. Jones and Dr. 0. S. Bamford. Electromyography experi-ments were done with Dr. Q. Bone, and blood l a c t a t e analyses were done by Dr. W. R. Dri e d z i c . A l l conclusions drawn from t h i s work are my own and not nec e s s a r i l y those of my colleagues. I also wish to thank Dr. D. Randall f o r the generous loan of equipment to make t h i s study p o s s i b l e . I am g r a t e f u l to Dr. Bamford, Dr. J. Davis, Dr. J . P h i l l i p s , Dr. Randall, Dr. West, Lowell L a n g i l l e and B i l l Milsom for many i n s p i r i n g discussions and for reading the manuscript. The t e c h n i c a l assistance of Patty Crosson during the work i n Section II and help by Lorraine Smith with data processing i s also greatly appreciated. My sincere appreciation also goes to my wife, Carolyn, and my two daughters for t h e i r constant encouragement during t h i s study. Personal f i n a n c i a l support was provided by an F.R.B. student fellowship and teaching assistantships from U.B.C. The research was funded by Environment Canada Grants i n Aid of Research held by Dr. Jones. 1 INTRODUCTION The term exercise, i n the p h y s i o l o g i c a l sense, i s used to r e f e r to a c t i v i t y of s k e l e t a l muscle. Most vertebrates must use muscular a c t i v i t y i n t h e i r d a i l y l i v e s , to f i n d and consume food, escape predators, tend t h e i r o f f s p r i n g , and carry out other bodily functions. In addition muscles must be exercised p e r i o d i c a l l y or muscle f i b e r s w i l l degenerate and become nonfunction-a l ( F a l l s , 1968). Sk e l e t a l muscle i s composed of b a s i c a l l y two d i f f e r e n t types of muscle f i b e r s ; red type muscle f i b e r s which contract slowly and white type f i b e r s which have a fast or "twitch" contraction (Hess, 1970). The slow muscle f i b e r s derive t h e i r energy from aerobic metabolism of f a t and carbohydrate, whereas "twitch" f i b e r s metabolize glycogen anaerobically (George and Naik, 1958; Love, 1970). During exercise the contraction of muscle r e s u l t s i n an increase i n the animal's oxygen consumption. Oxygen to be consumed by an animal must be exchanged between the animal and i t s environment, transported to the tissues by the blood, and transferred from the blood i n t o the tissue where i t becomes the end acceptor of hydrogen i n the aerobic metabolic process. In such a sequential process any one of the steps could l i m i t the whole sequence. The possible l i m i t i n g factors of maximal oxygen consumption then are: (1) The rate of oxygen exchange between the animal and i t s environment (2) Rate of transport of oxygen by the blood (3) Rate of d i f f u s i o n of oxygen into the tissue (muscle) (4) A l i m i t a t i o n i n the a b i l i t y of muscle to further increase i t s metabolism (5) Other factors such as i n a b i l i t y to remove waste products (e.g. C*^, l a c t a t e , heat). 2 Increases i n the amount of oxygen exchanged at the gas exchange organ (lung or g i l l i n most vertebrates) can occur by increasing the amount of oxygen removed from each unit of medium, or by increasing the amount of medium breathed, or both. Transport of oxygen by the blood can be increased by increasing the amount of blood pumped (cardiac output), by increasing the amount of unloading of oxygen from the blood (increasing the arterio-venous oxygen difference), or both. Most vertebrate blood contains hemoglobin i n c e l l s or corpuscles. Hemoglobin not only increases the oxygen carrying capacity of blood but also because of i t s sigmoid dissociation curve and modu-l a t i o n by other molecules (CC^ and H+) allows the unloading of a large propor-tion of the oxygen from the blood without a large change i n oxygen tension. After unloading from the blood the d i f f u s i o n of oxygen into the tissue i s f a c i l i t a t e d by the presence, i n red muscle f i b e r s , of myoglobin, a compound sim i l a r to hemoglobin but having a higher a f f i n i t y for oxygen than hemoglobin (Wittenberg, 1970). A muscle contraction i s the result of the contraction of many in d i v i d u a l muscle f i b e r s . The int e n s i t y of contraction of a muscle (and therefore i t s metabolic rate) can be increased by recruitment of more muscle fibers to contract, or, since contraction of red fibers i s graded, by increasing the contraction of indi v i d u a l fibers (Hess, 1970). I t i s therefore evident that when both recruitment and contraction of a l l muscle fibers i s maximal the oxygen consumption of the muscle w i l l be maximal. In order to determine which of these factors may l i m i t an animal's maximal aerobic exercise capacity changes i n variables related to the possible l i m i t i n g factors must be studied i n r e l a t i o n to exercise. In mammals, pa r t i c u l a r l y man, many studies have been conducted on the effect of exercise on 3 o cardiovascular,, respiratory, and metabolic variables (Bevegardand Sheperd, 1967; Ekelund, 1967; Rowell, 1974). At the beginning of an exercise period oxygen consumption increases rapidly then plateaus after about 1 minute, at which time the animal i s said to be i n the "steady state" of exercise (Asmussen, 1965). In mammals a short exercise period (5 min) of increasing in t e n s i t y results i n increases i n oxygen consumption up'to a point beyond which oxygen consumption does not increase when int e n s i t y of exercise i s further increased. However, when long test periods are used Cl hour) no plateau i s observed i n oxygen consumption versus i n t e n s i t y of exercise. Under these circumstances maximal oxygen consumption corresponds to the highest exercise rate (Taylor, 1970, 1974). During exercise v e n t i l a t i o n volume increases i n proportion to oxygen con-sumption at low exercise levels but intense exercise causes v e n t i l a t i o n to increase more rapidly than oxygen consumption (Asmussen, 1967). Cardiac output rises with r i s i n g oxygen consumption, this i s accomplished by progres-sive increases i n stroke volume, heart rate, or both. In man stroke volume reaches the highest value at about 40 per cent of maximal oxygen consumption, the remaining increase i n cardiac output being the result of an increase i n heart rate. Arterio-venous oxygen difference increases with increasing oxygen consumption; at maximal oxygen consumption 80-85% of a l l available oxygen i n the blood i s extracted by the tissues. This high extraction i s the result of two adjustments; f i r s t l y , oxygen extraction by working muscles i s increased, secondly, blood flow to areas which normally have high blood flow rates but low oxygen extraction i s reduced. In man blood flow i s reduced to splanchnic tissue, kidney, and nonexercising muscle. Dogs, on the other hand, show only a s l i g h t change i n splanchnic blood flow even at maximal exercise levels 4 (Herrick et a l . , 1940; Rushmer et a l . , 1961; Lacroix and.Leusen, 1966; Van C i t t e r s and F r a n k l i n , 1969). However when cardiac output i s reduced i n dogs by heart block, blood r e d i s t r i b u t i o n s i m i l a r to that i n humans i s reported (Holtman, 1967; Vatner et a l . , 1971; M i l l a r d et a l . , 1972). The d i f f e r e n c e i n blood r e d i s t r i b u t i o n during exercise i n man and dogs may be the r e s u l t of the d i f f e r e n c e i n posture between the two animals but t h i s i s not c l e a r on the basis of e x i s t i n g evidence (Rowell, 1974). Oxygen removal from blood increases with exercise (one of the factors accounting for the increase i n a r t e r i o -venous oxygen difference) (Rowell, 1974). Since both oxygen consumption and oxygen extraction (A-VO2 d i f f ) are l a r g e r i n p h y s i c a l l y trained than i n un-trained i n d i v i d u a l s , oxygen ex t r a c t i o n from the blood (oxygen supply to tissue) i s not l i k e l y to be a l i m i t i n g f actor i n s e t t i n g maximal oxygen consumption i n mammals (Rowell, 1974). I f muscle i s not l i m i t e d i n i t s supply of oxygen the o v e r a l l oxygen con-sumption may s t i l l be l i m i t e d by the a b i l i t y of the muscle to increase i t s metabolism. One of the ways that the metabolic rate of muscle could be l i m i t e d i s by the amount of oxidative enzymes present i n the muscle f i b e r s . Holloszy (1967), on the basis of h i s findings that mitochondrial oxidative enzymes increase i n rats p h y s i c a l l y trained at near maximal exercise l e v e l s , considers t r a i n i n g to be a process of increasing muscle aerobic metabolic capacity and suggests that maximal oxygen consumption i s l i m i t e d by the t i s s u e . Another p o s s i b i l i t y (which i s not exclusive of the f i r s t ) i s that the maximal oxygen consumption i s determined by the amount of c o n t r a c t i l e protein i n red muscle. That i s to say that, at maximal oxygen consumption, a l l of the c o n t r a c t i l e proteins of red muscle are adequately supplied with ATP and are working to t h e i r maximal capacity. 5 The energy charge of blood perfused' maximally exercised^red muscle i s not known i n any animal. In mammals both the musculature and mode of locomotion are very complex; the proportion of red and white muscle f i b e r s i n d i f f e r e n t muscle masses i s not constant (some are pure red; others are mixed i n various proportions) and the number of muscles involved i n locomotion i s large. In fact i t i s not even c l e a r as to how many muscles are involved i n various kinds of t e r r e s t r i a l locomotion (Taylor, personal communication). Cardiovascular and re s p i r a t o r y phenomena have also been investigated i n bir d s during f l i g h t (Hart and Roy, 1967; Tucker, 1968; Berger_et a l . , 1970; Butler et a l . , 1975,). Birds are capable of somewhat higher maximal oxygen consumption rates than mammals (Lasiewski, 1960). Since the cost of f l y i n g (per unit distance) i s l e s s than the cost of running (Tucker, 1970) oxygen consumption during l e v e l f l i g h t i s probably comparable to that of running mammals. The cardiovascular and res p i r a t o r y responses to f l i g h t i n pigeons are s i m i l a r to those to exercise i n mammals (Butler et a l . , 1975). In birds the power for f l i g h t i s provided by b a s i c a l l y two muscle/,the p e c t o r a l i s and the supracoracoideus. The proportion of red type muscle f i b e r s i n the f l i g h t muscles of many species of birds has been investigated (George and Berger, 1966). The domestic chicken, which does not f l y , has a p e c t o r a l i s muscle composed predominantly of white type muscle f i b e r s , whereas very strong f l y e r s (house sparrow) and very a c t i v e birds (hummingbirds) have p e c t o r a l i s muscles composed predominantly (house sparrow) or ex c l u s i v e l y (hummingbird) of red f i b e r s . Those birds which soar have a predominance of intermediate type f i b e r s (George and Berger, 1966). The p h y s i o l o g i c a l s i g n i f i c a n c e of the pro-portions of d i f f e r e n t f i b e r types has not been investigated and i t can only be speculated that t h i s morphological d i s t r i b u t i o n i s rela t e d to aerobic metabolic 6 capacity. I t i s known that f l y i n g hummingbirds have a higher oxygen consump-tion per unit weight than has been recorded for any other vertebrate (Pearson, 1950; Lasiewski, 1960). No data available on the lactate accumulation during f l i g h t i n birds such as hummingbirds; an accumulation would s i g n i f y an insuf f i c i e n c y of oxygen supply at the tissue. The l i m i t a t i o n of maximal aerobic exercise i n mammals and birds i s not known; i t may be one of the factors discussed above or i t could be some other factor such as accumulation of heat. In some species of homeotherms body temperature, which increases as a result of exercise, has been shown to c o r r e l -ate favourably with the i n a b i l i t y of the animals to continue exercise. In the cheetah and rhea body temperature during exercise increases u n t i l the animal appears exhausted and refuses to run further. The body temperature of a cheetah during a run to exhaustion increases by 1.0 to 1.5°C (Taylor, 1974). Gazelles can run faster than the cheetah for a longer period of time. During exercise of this i n t e n s i t y a gazelle's body temperature increases by as much as 6°C. Under these conditions the brain i s maintained at a lower temperature than the rest of the body by a heat exchanger system located at the base of the s k u l l of the gazelle (Taylor, 1974). Thermoregulation during f l i g h t may be a l i m i t i n g factor i n birds f l y i n g at high ambient temperatures, under which c i r -cumstances the birds must re l y on evaporative cooling. Pigeons f l y i n g at an ambient temperature of 26°C increase the i r body temperature (at exhaustion) by about 2°C (Butler et a l . , 1975). At lower temperatures cooling by convection and radiation i s probably adequate and temperature regulation i s probably not a l i m i t i n g factor (Dawson and Hudson, 1970). Likewise heat accumulation i s not a problem i n small mammals (rats, mice) or i n long distance runners (African hunting dog, domestic dog) at submaximal exercise l e v e l s ; at maximal 7 exercise levels however this could be a l i m i t i n g factor but has hot been i n -vestigated (Taylor, 1974). The only other class of vertebrates i n which exercise studies have been conducted i s the teleost fishes. Fish as animals for the study of exercise have certain advantages. Because f i s h l i v e i n a medium with a large heat capacity and a low oxygen content, the g i l l s (during gas exchange) act as heat sinks, resulting i n blood being cooled to environmental temperatures. For this reason f i s h (except for a few which have heat exchangers to conserve heat i n parts of the body) have no problem with an/accumulation of heat i n their bodies during exercise. Another advantage i n studying f i s h i s that they have a single c i r c u i t c i r c u l a t o r y system i n which i t should be easier to determine the relationships of variables and thei r control than i t i s i n mammals and birds. Extensive studies have been carried out on the oxygen consumption of various species of f i s h i n r e l a t i o n to swimming speed (Brett, 1964; Brett and Sutherland, 1965; Rao, 1968; Kausch, 1968; Farmer and Beamish, 1969; Beamish, 1970). Oxygen consumption increases exponentially with increases i n swimming speed and a logarithmic transformation of oxygen consumption results i n a straight l i n e relationship of oxygen consumption to swimming speed (Brett, 1964) . Sockeye salmon show a decrement i n swimming speed and maximal oxygen consumption at temperatures i n excess of 15 C. { This i s attributed to a decrease j -< i n the oxygen content of water at higher temperatures (Brett, 1964). (^Jones (1971) hypothesized that the energy demand of the branchial pump w i l l l i m i t the amount of oxygen available to the tissue at high temperatures. The cardiovascular and respiratory responses of f i s h to exercise have been the subject of only a few studies. Smith, Brett and Davis (1967) described the 8 heart rate, v e n t i l a t o r y rate and dorsal a o r t i c blood pressure responses i n r e l a t i o n to swimming speed i n sockeye salmon. Stevens (1968) examined the responses of the cardiovascular and r e s p i r a t o r y systems of trout to exercise of short duration (5 min). S u t t e r l i n (1969) examined the e f f e c t of exercise on cardiac and v e n t i l a t o r y frequency i n pumpkinseed (Lepomis gibbosus), bullheads (Ictalurus nebulosus) and brown trout (Salmo t r u t t a ) . Priede (197^-) studied the e f f e c t s of exercise (up to maximal 30 min sustained) on heart rate and attempted to determine the r o l e of the vagus i n the c o n t r o l of heart rate i n rainbow trout. No studies have been conducted on the cardiovascular and r e s p i r a t o r y responses of f i s h to prolonged exercise. In those f i s h which swim with the t a i l (anguiliform mode) only the myoto-mal muscles are used for swimming. The myotomal musculature i n t e l e o s t s consists of a l a t e r a l muscle mass composed e n t i r e l y of red muscle f i b e r s and a much l a r g e r white or mosaic muscle mass composed of white f i b e r s (white mass) or of white and red or possibly_ intermediate type f i b e r s (mosaic mass) (Boddeke et a l . , 1958). I t i s known that the mosaic muscle mass of trout i s a c t i v e at low swimming speeds (Hudson, 1973) but the r e l a t i v e contributions of the two muscle masses at other submaximal and maximal sustained speeds i s not known. However t h i s morphological separation of red and white muscle f i b e r s (at l e a s t i n some f i s h ) provides a convenient means for a comparative physiolo-g i s t to study the recruitment of the d i f f e r e n t muscle f i b e r types during exercise i n r e l a t i o n to maximal sustained exercise. The f i r s t part of t h i s study was devoted to examining the i n t e r s p e c i f i c v a r i a b i l i t y i n swimming speeds of f i s h i n r e l a t i o n to the proportions of red and white muscle i n the myotome and the recruitment of the two muscle types during swimming. The second section deals with the cardiovascular, r e s p i r a t o r y 9 and metabolic changes which occur i n trout i n r e l a t i o n to thei r maximal exercise c a p a b i l i t y . 10 SECTION I Recruitment of Red and White Myotomal Muscle During Swimming and i t s Relationship to C r i t i c a l Velocity INTRODUCTION Since the attempt by Regnard (1893) to determine the maximum swimming speed of f i s h i n the laboratory, a large number of papers have presented data from both laboratory and f i e l d observations which allow assessment of t h i s variable. Brett (1964) established a fatigue curve for yearling sockeye salmon (Oncorhynchus nerka), recognising two d e f i n i t e t r a n s i t i o n points; one at 12—24 sec and another at 300 min. The t r a n s i t i o n point at 12-24 sec represents the termination of burst swimming and 300 min marks the end of steady performance. After t h i s l a t t e r t r a n s i t i o n the f i s h enters a period of sustained performance which can, t h e o r e t i c a l l y , be maintained i n d e f i n i t e l y . During burst swimming, speeds up to 3-4 times the sustained performance can be achieved. Data obtained by Blaxter and Dickson (1959) and Blaxter (personal communication i n Bain-bridge, 1960) and Bainbridge (1960, 1962) confirms the early t r a n s i t i o n from burst to steady performance for a variety of fishes. Gray (1953) reported that trout from a standing st a r t can accelerate to f u l l speed i n 50 msec. Consequent-l y there seems adequate experimental evidence that f i s h can reach extremely high forward v e l o c i t i e s very quickly for periods of several seconds and that some f i s h can swim for very long periods of time. The reasons for large int e r s p e c i f -i c variations i n sustained swimming speed have not be investigated. One of the possible explanations for these differences may be found i n the characteristics of the muscles generating the power for swimming. In most f i s h the myotomal musculature i s composed of two main types of muscle f i b e r s . The two f i b e r types are usually distinguished by the i r colour, 11 myoglobin and mitochondrial content, vascular supply and enzymatic properties, diameter, and, to some extent, innervation (Boddeke et al... 1959; Bone, 1966; Webb, 1969). White f i b e r s have been found to propagate muscle action poten-t i a l s , and are f a s t "twitch" .fibers, i n contrast to.the " t o n i c " red f i b e r s which do not propagate action potentials (Barets, 1961; Jansen et a l . , 1963; S t a n f i e l d , 1972). Red f i b e r s are w e l l supplied with blood c a p i l l a r i e s , have large numbers of mitochondria, usually contain f a t droplets, and are generally accepted as having an a e r o b i c a l l y based metabolism (Boddeke et a l . , 1959; Bone, 1966; Webb, 1969; Love, 1970). White f i b e r s are larger than the red ones, are more uniform i n s i z e , are poorly supplied with blood c a p i l l a r i e s , have few mitochondria, and no f a t reserves. On the basis of poor blood supply, glycogen depletion and l a c t a t e accumulation during t h e i r action, white f i b e r s are con-sidered to metabolize glycogen anaerobically (Boddeke et a l . , 1959; Bone, 1966; Webb, 1969; Hess, 1970; Love, 1970; P r i t c h a r d , 1971). Red muscle f i b e r s are always innervated by small motor nerves ending i n f i n e filaments with minute expansions on the ends (multiple innervation), whereas white type f i b e r s are innervated by larger nerve f i b e r s ending i n e i t h e r a s i n g l e end plate or multiple endings (Hess, 1970). In f i s h the f i b e r types are generally organized into a l a t e r a l s u p e r f i c i a l red muscle mass situated immediately below the skin at the l a t e r a l l i n e . This muscle mass i s composed e n t i r e l y of red type f i b e r s (Boddeke et a l . , 1959). The deeper myotomal musculature (often referred to as the white muscle mass) i s composed mostly or e n t i r e l y of white muscle f i b e r s . In many species (such as salmonids and carp) the "white muscle mass" also contains some intermediate type muscle f i b e r s . These are intermediate i n s i z e between red and white but contain f a t and are thought to be a slow f i b e r type (Boddeke et a l . , 1959). 12 The contra c t i l e and electrophysiological nature of these fibers has not been investigated. A muscle mass composed of red or intermediate type fibers and white fibers i s referred to as a mosaic muscle. Direct myographic recordings from electrodes inserted into red and white muscle masses i n sharks have shown that at low swimming speeds only the red fibers are active. E l e c t r i c a l a c t i v i t y from the white fibers i s observed only during bursts of swimming (Bone, 1966; Roymer and Kennan, 1967). Boddeke et a l . (1959) correlated the arrangement of the myotome i n a number of freshwater f i s h species to thei r feeding habits and related this to the type of swimming capability the various f i s h might be expected to have. The present experiments were designed to: (1) measure the maximum steady swimming performance which could be maintained for 10 minutes by a variety of teleost fishes; (2) examine the e l e c t r i c a l a c t i v i t y of the red and white portions of the myotome, and the proportion of red musculature i n some species, which differed widely i n swimming performance (in terms of maximum sustained v e l o c i t y ) , i n order to determine to what extent the action of the two muscle types can be related to the maximal sustained swimming speed. 13 MATERIALS AND METHODS (1) The Water Tunnels (a) The laboratory tunnel At U. B. C. f i s h were exercised i n a tunnel si m i l a r to that described by Brett (1964). B a s i c a l l y the respirometer was a r e c i r c u l a t i n g water tunnel 2 incorporating a c y l i n d r i c a l Plexiglass f i s h chamber (126.5 cm i n cross-sectioned area) connected to a pump through expansion and contraction cones. The maximum output of the pump was about 300 gal/min against a developed head of 40 f t , providing a maximum veloc i t y of 103 cm/sec,through the f i s h chamber. The t o t a l volume of the respirometer was 35 L. The expansion cone and three turbulence screens leading to the f i s h chamber were designed so that a r e l a t i v e -l y consistent, f l a t v e l o c i t y p r o f i l e occurred over the range of v e l o c i t i e s at which f i s h were forced to swim (10 cm/sec - 103 cm/sec). That the flow p r o f i l e was a streamlined flow of minutely turbulent water was checked by the use of dyes injected upstream of the pump but downstream of the f i s h tunnel. This procedure ensured adequate mixing of dye before i t reached the f i s h chamber. The water velo c i t y i n the tunnel was monitored continuously using a counter which monitored the revolutions of the pump shaft (Rev-counter) or by means of the setting on the pump's variable speed hydraulic gear (Setting). The Rev-counter and pump setting were calibrated i n terms of water v e l o c i t y using an Ott meter. At the end of this series of experiments the tunnel was recalibrated using the Ott meter and i t was found that,,except at the position where the Rev-counter was superseded by the pump sett i n g , values assessed from measure-ments made by these systems did not vary by more than 3% from those obtained using the Ott meter (Table 1). Replicate determinations made i n the same position of the tunnel over a series of several days agreed within ±2% of one 14 TABLE 1. Comparison between water v e l o c i t i e s i n the f i s h tunnel assumed from the Rev-counter or pump setting and actual measurements made with the Ott meter. A - Rev-counter/Setting (cm/sec) B - Measured ve l o c i t y (Ott meter) (cm/sec) % Error between A and B 24.37 24.73 -1% 51.77 50.45 +3% *77.7 73 +7% 102.8 100 +3% *Rev-counter superseded by pump setting at t h i s v e l o c i t y . another. Determinations of water ve l o c i t y at the centre, top, bottom and sides of the tunnel, with the Ott meter, gave values for water velo c i t y which were no more than 4% different between any two positions, thereby confirming the results of the dye injections i n that the vel o c i t y p r o f i l e was r e l a t i v e l y f l a t . The tunnel was dismantled and the turbulence screens cleaned p e r i o d i c a l l y to prevent scales and other detritus from blocking the grids and thereby reducing the maximum water ve l o c i t y i n the tunnel. As recommended by Brett (1964) a covered area at the front of the chamber was provided for the f i s h . Temperature was controlled within ±0.5°C by a flow of a refrigerant through a heat exchanger, counterbalanced by a 500-watt heater and relay. Fresh aerated water was fed 15 continuously into the system from a.reservoir located above the respirometer. The water was heated to a temperature s l i g h t l y above that i n the water tunnel and aerated to avoid problems of oxygen supersaturation of the introduced water. In the majority of experiments the water was renewed at a rate of 2 1/min. The oxygen tension of the water flushed out of the respirometer was monitored using a Radiometer or Beckman oxygen electrode and remained constant during any one experiment, (b) The f i e l d tunnel The exercise apparatus i n the f i e l d was considerably simpler i n design than that used i n the laboratory. As there was an abundant water supply a v a i l -able a one-pass flow system was designed. A gasoline powered trash pump was used to draw water from the r i v e r and t h i s was expelled through a 24 cm i n t e r n a l diameter P l e x i g l a s s f i s h tunnel (Fig. 3a). As i n the laboratory^the f i s h chamber had a forward covered area (Fig.::l). The pump, powered by a Ford engine, was rated at 40,000 gals/hr at zero head. In the 24 cm tube t h i s gave a maximum water v e l o c i t y of about 100 cm/sec. The suction hose and hoses connected to the f i s h tunnel were of 12.5 cm diameter, mating between them being achieved by use of an expansion cone. Three screens of 1 cm mesh were placed at the upstream end of the f i s h tunnel. An e l e c t r i f i e d g r i d was placed at the downstream end of the f i s h tunnel and a further contraction cone was used to mate the tunnel to the discharge hose (12.5 cm diameter). The g r i d was e l e c t r i f i e d at 5 v A.C. by using an i n v e r t e r which was powered from the battery on the pump engine. The turbulence screens were of considerably larger mesh s i z e than i s desirable i n th i s kind of tunnel; however, by s u i t a b l e o r i e n t a t i o n of the meshes i n each g r i d , r e l a t i v e to one another, i t was possible to achieve a f a i r l y f l a t , a l b e i t skewed, flow p r o f i l e across the tube over the range of 16 Figure 1 Photograph of the 25 cm diameter water tunnel used i n f i e l d experiments. 17 18 v e l o c i t i e s used (10 cm/sec to 100 cm/sec). Large mesh, sizes for the screens were chosen because i t was f e l t that t h i s might eliminate the need for continue a l dismantling of the tunnel to allow the turbulence screens to be cleaned. A T-piece and screw-valve was si t e d between the pump and the f i s h chamber. With the motor running at i d l i n g speed low flows i n the f i s h chamber could only be achieved by opening the valve and exhausting some of the pump's output to the r i v e r . Above water v e l o c i t i e s of 40 cm/sec the by-pass valve was shut and water ve l o c i t y i n the f i s h chamber controlled by the t h r o t t l e on the pump engine. In order to keep the f i s h chamber f i l l e d with water at low v e l o c i t i e s i t was necessary to raise the l e v e l of the discharge pipe above that of the ri v e r . As the water velo c i t y was increased the discharge pipe was lowered into the r i v e r . Water v e l o c i t i e s i n the tunnel were monitored using an Aqua-log boat speedometer, suitably modified to cover the range of water v e l o c i t i e s encount-ered during these experiments. Calibrations of the Aqua-log i n the f i e l d with the Ott meter (sited i n the mid-position of the tunnel) revealed considerable discrepancies at low speeds but above 40 cm/sec the difference between the two readings was s l i g h t (±3%) (Table 2). Measurements made with the Ott meter on two different days showed that by reading the Aqua-log alone the water velo c i t y i n the tunnel could be adjusted to ±1% of the required value. Since the water tunnel was f l o a t i n g on a dock surges of flow occurred when the l e v e l of the discharge hose changed; however, even violent fluctuations i n l e v e l (produced by jumping up and down on one end of the ra f t ) only caused fluctuations i n water ve l o c i t y of ±2% when v e l o c i t i e s were measured over a period of 100 sec with the Ott meter. In these tests no attempts were made to correct for these flow fluctuations by a l t e r i n g the t h r o t t l e control on the pump motor. 19 TABLE 2. Comparison between water v e l o c i t i e s i n the f i s h tunnel measured by the Aqua-log and the Ott meter. Aqua-log (cm/sec) B - Ott meter (cm/sec) % Error between A and B 10 11.46 -14% 20 26.5 -32% 30 33.4 -11% 40 41.27 -3% 50 50.26 0 60 58.9 +2%. 70 71.35 -2% 80 82.36 -3% 90 93 -3% 100 102.4 -2% Dye i n j e c t i o n s , made just downstream of the pump, revealed that the flow p r o f i l e was markedly different from the laboratory tunnel. The flow p r o f i l e was skewed i n such a way that the fastest flow v e l o c i t y occurred at the top and one side of the tunnel and the slowest v e l o c i t i e s at the bottom and opposite side ' (Fig.._2a) . I t was apparent that the sharp bend i n the tubing between the pump and the f i s h chamber was responsible for the shape of the flow p r o f i l e but the necessity for even weight d i s t r i b u t i o n on the f l o a t i n g dock prohibited any attempt at correcting this design f a u l t . The nature of the flow p r o f i l e was confirmed by measuring water v e l o c i t i e s at the sides, top and bottom of the tunnel with the Ott meter. Taking the value i n the centre of the tunnel as 20 Figure 2 Diagrammatic representation i n side view of the flow p r o f i l e i n the f i e l d water tunnel (25 cm diameter) as determined from photographs of dye injections made over a range of water v e l o c i t i e s . Water v e l o c i t i e s at the periphery of the f i e l d tunnel expressed as a % of water velo c i t y i n the centre. A l l determinations (average of 3 at each position) made with an Ott meter over 100 sec time periods. 21 s / \ \ \ \ \ \ \ \ \ \ \ 120% 118% 1 0 0 % 94% © 22 100% i t was found that v e l o c i t i e s at the top and l e f t side (looking into the flow) of the tunnel were some 118-120% of t h i s value whereas v e l o c i t i e s at the bottom and r i g h t hand side were some 91-93% of water v e l o c i t y i n the centre of the f i s h chamber (Fig...2b). C-In p r a c t i c e i t was noted that f i s h seldom swam at the top or l e f t hand side of the tunnel; the majority of animals, a f t e r the acclimation period, frequented the centre, bottom:.-or r i g h t hand side of the tube. Consequently values f o r c r i t i c a l v e l o c i t y determined i n the f i e l d may be overestimated by, at most, some 9%. Due to the f a c t that the spectacularly muddy L i a r d River enters the Mackenzie River j u s t upstream and on the same side as the i s l a n d on which Fort Simpson i s s i t e d , i t was impossible to locate the water tunnel i n the immediate v i c i n i t y of Fort Simpson. This seemed a wise choice i n that few f i s h are caught i n the L i a r d River water and i t therefore seemed unreasonable to expect f i s h to swim w e l l i n such a s i l t load. Furthermore since the Mackenzie River drops considerably i n l e v e l over the summer months i t was not f e a s i b l e to s i t e the apparatus on the bank opposite Fort Simpson. As a compromise the whole apparatus was assembled on a 12 f t by 10 f t r a f t and towed across the Mackenzie River and moored at the mouth of the Harris River (Fig..::3) . Due to t h i s procedure a r e l a t i v e l y c l e a r water supply was ensured throughout the period of the study. (2) The Fi s h Except for trout (Salmo g a i r d n e r i , Richardson), carp (Cyprinus carpio, L . ) , and her r i n g (Clupea harengus, L.) the f i s h species used i n t h i s study were supplied by the Fi s h e r i e s Service from Fort Simpson, Norman Wells, A r c t i c Red River and Aklavik. In the laboratory experiments i n d i v i d u a l s from a l l 4 f i e l d s t ations were used whereas the f i e l d tunnel only u t i l i s e d f i s h caught i n the 23 Figure 3 Photograph of the f i e l d apparatus moored i n position on the r i v e r . 24 25 v i c i n i t y of Fort Simpson. The f i s h were seined, g i l l - n e t t e d or caught on hook and l i n e . At the f i e l d operation the f i s h were placed i n holding pens s i t e d alongside the f l o a t i n g dock and were experimented upon 24 hours after capture. For the laboratory operation the f i s h were shipped to Vancouver by a i r freight after being held by the Fisheries Service i n pens for up to 3 days after capture. The f i s h were not fed before shipment. For shipping, the f i s h were placed i n clean double p l a s t i c bags, a layer of newspaper being placed between the bags i f the f i s h had spiny rays. This prevented puncture of the outside bag and subsequent loss of water. Water was placed i n the inner bag, s u f f i c i e n t just to cover the f i s h , and the a i r was squeezed out completely and a length of hose, connected to an oxygen cylinder, was used to i n f l a t e the inner bag to about 2-3 times the volume of the enclosed water. The bag was then twisted tight and taped. The outer bag was then t i e d i n the same manner. In v i r t u a l l y a l l shipments the f i s h were packed i n d i v i d u a l l y to avoid contamina-tion of specimens should one of a group die i n t r a n s i t . The bags were then placed i n styrofoam boxes which contained one or two freezer packs wrapped i n newspaper. The boxes were sealed with tape. As a general rule 1-2 large or 3-4 small f i s h could be adequately contained i n a single styrofoam box. Fish caught by stations at Aklavik and A r c t i c Red River were shipped v i a Northward Aviation to Inuvik, International Jet A i r to Whitehorse, and C. P. A i r to Vancouver. The t o t a l time spent i n t r a n s i t by t h i s routing was less than 12 hours. Unfortunately after only 3 shipments a labour dispute at C. P. A i r forced use of P. W. A. from Inuvik to Edmonton and thence to Vancouver. Under favourable circumstances (no delays) t r a n s i t time v i a this routing was 15 to 18 hours. In the l a t e r part of the study an attempt was made to further reduce the t r a n s i t time on this route by shipping the f i s h to Inuvik by a i r charter. 26 From Norman Wells and Fort Simpson f i s h were shipped by P. W. A. to Vancouver, v i a Edmonton, the t r a n s i t , time being of the order of 18 to 24 hours. Under favourable circumstances (no delays and care taken i n packing) mortality was low, being less than 10% of any shipment. I t was established that about 30% mortality occurred when tr a n s i t time exceeded 30 hours and 100% i f transit:, time exceeded 36-40 hours. On a r r i v a l at U. B. C. the f i s h were introduced to 2,000 1 c i r c u l a r tanks i n which water c i r c u l a t i o n was maintained by means of pumps. The water ve l o c i t y varied i n the tanks from zero at the centre to 35-40 cm/sec at the edge of the tank. Temperature was maintained at 12-13°C by means of thermo-s t a t i c a l l y controlled heaters working against the input water flow. The input water was at 9°C. After introduction to the tanks - this procedure taking about one hour to allow f i s h to adapt to the different water hardness between the Mackenzie River water and water at U. B. C. - pumps were placed i n the "mixing" mode and the f i s h treated with "nitrofurazone" (10 g/2,000 1). After 24 hours i n the tanks f i s h were offered trout p e l l e t s , artemia and minnows and the pumps were placed i n the c i r c u l a t i o n mode. Fish were fed every other day and after 3-5 days were used i n experiments, i f the water temperature i n the f i e l d was close to that i n the holding tanks. However, on some occasions, f i s h were l e f t i n the holding tanks for longer periods (5-10 days) to allow for thermal acclimation to the range of 12-13°C since they were taken i n the f i e l d at temperatures from 17-19°G. Fish were not fed for 24 hours before any series of experiments. Some f i s h were also acclimated, i n October, to a temperature of 7°C i n 1,000 1 refrigerated tanks. These f i s h were taken from the Mackenzie River at Fort Simpson, which was at a temperature of 8-9°C at that time. Water c i r c u l a -27 ti o n was maintained i n these tanks by means of submersible pumps. The f i s h were allowed, on average, about 15 days for the period of thermal acclimation before the star t of the experiments. These f i s h were offered trout p e l l e t s and minnows every other day before the star t of the experiments. Juvenile herring (Clupea harengus) were caught by seine netting i n the Georgia S t r a i t s and af t e r treatment with nitrofurazone (10 g/2,000 1) for 2 days they were held i n tanks through which aerated seawater'••(6-8°C) was circulated u n t i l they were used for experiments (one week). During the hold-ing period they were not fed. Female trout (Salmo gairdneri) were purchased from a commercial supplier (Trout Lodge, Ephrato, Washington, U. S. A.) and transported to U. B. C. by tank truck, where they were held i n large c y l i n d r i -c a l tanks (8000 1). Constant inflow of fresh dechlorinated water (9;-10°C) was maintained at a l l times. Carp (Cyprinus carpio) were caught by seine netting i n the Fraser Valley east of Vancouver, and held under the same conditions as the trout. Trout and carp used for experiments were physically trained for a minimum of two weeks. Training was accomplished by holding the f i s h i n 2000 1 c i r c u l a r tanks i n which water was kept i n motion by water j e t s driven by pumps. The water vel o c i t y varied from nearly 0 at the centre of the tank to 30-40 cm/sec at the circumference. The f i s h tended to swim constantly i n the high water vel o c i t y zone. Trout and carp were fed Clarke's Trout P e l l e t s s i x times weekly throughout the training period. (3) Experimental Procedures At the f i e l d station only increasing v e l o c i t y t e s t s , to allow determina-tion of c r i t i c a l v e l o c i t y , were carried out; whereas i n the laboratory fatigue t r i a l s were performed i n addition to the increasing ve l o c i t y tests. The experiments i n the f i e l d were performed from mid-July to the end of September 28 whereas the laboratory experiments ran from mid-May to mid-October, The effects of temperature on swimming performance were investigated both i n the laboratory and f i e l d . In the f i e l d a l l experiments were performed at ambient temperature (the tunnel of f e r i n g no opportunity for temperature regulation) and . .- the f i e l d experiments were carried out at temperatures between 12-13°C and 18-20°C. Since the f i s h were caught and held i n the same body of water as was passed through the tunnel i t was assumed that the f i s h were acclimated to the respective temperature regimes. The laboratory data gave c r i t i c a l v e l o c i t y determinations at 7-10°C and 12-13°C whereas the f i e l d data gave values for f i s h run at temperatures of 12-13°C and 18-20°C. Therefore comparison for any differences between laboratory and f i e l d data, for most species, could be made at 12-13°C whereas the effect on swimming performance of acclimation to higher and lower temperatures could be assessed by comparing the c r i t i c a l v e l o c i t i e s obtained with either the laboratory or f i e l d data at 12-13°C. In the f i e l d , following fatigue of the f i s h , i t was removed from the tunnel, weighed, measured (fork length and cross-sectional area) and tagged. The tagged individuals were returned to the tanks and after several days were used i n fatigue or temperature shock experiments. Following these experiments the f i s h were k i l l e d and opened i n the ventral midline to allow determination of the state of maturity or sex of the i n d i v i d u a l , (a) Increasing v e l o c i t y tests Fish were introduced into the respirometer by means of the access port and the downstream grid e l e c t r i f i e d with voltages varying from 3-10 v A.C. depending on the s e n s i t i v i t y of the f i s h . Great care was taken when introduc-ing f i s h to the tunnel to avoid undue excitement of the animal and to th i s end the transfer was usually achieved using a series of nets and water f i l l e d 29 buckets. The f i s h was allowed at l e a s t one hour's acclimation to the tunnel with the water v e l o c i t y at i t s lowest speed (9.5 cm/sec i n the laboratory tunnel and 11 cm/sec i n the f i e l d tunnel). I t was confirmed by v i s u a l observation that f i s h expended r e l a t i v e l y l i t t l e e f f o r t i n maintaining t h e i r p o s i t i o n i n the tunnel against t h i s water v e l o c i t y . Following t h i s introduc-tory period the f i s h were subjected to sequential water v e l o c i t y increments every 10 min. In the laboratory the water v e l o c i t y was increased i n steps of not more than 1/2 L/sec (L = fork length, cm) for small f i s h and 1/3 L/sec for large f i s h (>25 cm fork length). However, i n the f i e l d , the v e l o c i t y increments were of the order of 10 cm/sec regardless of the length of the f i s h . In order to introduce some standardization to the results^data from f i s h which f a i l e d to complete 3 v e l o c i t y increments i n the laboratory or 2 i n c r e -ments i n the f i e l d , was not used. As a r u l e only large f i s h were rejected on t h i s score and, i n many cases, t h e i r poor performance could often be r e l a t e d to prominent fungus growth or g i l l - n e t : marks. A f i s h was deemed to have become fatigued when i t could not remove i t s e l f from the e l e c t r i f i e d g r i d even when a voltage 3-4 times greater than that established i n the acclimation period was applied. Further confirmation of fatigue was obtained by t r y i n g to force the f i s h to remove i t s e l f from the g r i d i n response to strong mechanical s t i m u l i . The fatigue v e l o c i t y (Vf) and time taken f o r complete fatigue (t) at that v e l o c i t y was noted, along with the water v e l o c i t y applied before the fatigue v e l o c i t y was reached (Vp = penultimate v e l o c i t y ) . In the laboratory, i t proved possible to measure fork length and c r o s s - s e c t i o n a l area, weigh and tag the exhausted f i s h without the use of anaesthetics. Three hundred and s i x t y c r i t i c a l v e l o c i t y tests were made i n the f i e l d and three hundred and one i n the laboratory. 30 . Since f i s h almost always fatigued during the l a s t v e l o c i t y increment i n a time i n t e r v a l less than the desired time period (10 min) a measure of c r i t i c a l swimming speed was obtained empirically as has been described by Brett (1964), v i z : Cv = Vp + ([Vf - Vp] x t_) 10 where Cv = c r i t i c a l v e l o c i t y (cm/sec) Vp = penultimate vel o c i t y (cm/sec) Vf = f i n a l v e l o c i t y (cm/sec) t = time to fatigue (min) Because of the closed nature of the water tunnel, swimming f i s h experi-ence a drag higher than that expected at any given free-stream v e l o c i t y . For f i s h , corrections must be made for the extra drag a r i s i n g from horizontal buoyancy and solid-blocking effects (Pope and Harper, 1966) as wel l as a "propeller correction" (Webb, 1970). The horizontal buoyancy and propeller corrections are small and opposite i n effect to each other so they tend to cancel one another (Webb, 1971). However, the solid-blocking e f f e c t , which results from the decrease i n e f f e c t i v e cross-sectional area of the tunnel through which water can flow due to the presence of the f i s h , w i l l obviously vary depending upon the respective cross-sectional areas of the f i s h and tunnel. The increase i n water velo c i t y around the f i s h can be calculated from the following general formula - and must represent the actual v e l o c i t y at which the f i s h i s swimming i n the tube, v i z : C V 2 = [ 1 + f <AF^Af ) ] X C V 1 where Cv^ = uncorrected c r i t i c a l v e l o c i t y (cm/sec) 2 Af = maximum cross^sectional area of the f i s h (cm ) 31 2 At = cross-sectional area of the f i s h chamber (cm ) f = factor = corrected c r i t i c a l v e l o c i t y (cm/sec) Paulik and Delacy (1957) took f = 1 i n their calculations but Webb (1970) suggested that this gave erroneously high results unless the maximum cross-sectional area of the f i s h was a large proportion of the cross-sectional area of the tunnel. In the present experiments, to calculate the actual c r i t i c a l v e l o c i t y of the f i s h , f was taken as 0.8 and corrections were only mgcfe when the cross-sectional area of the f i s h was greater than 10% of the cross-section-a l area of the tunnel. Apart from 7 or 8 exceptions the maximum cross-sectional area of the f i s h was less than 30% of that of the laboratory tunnel meaning that even i f f = 1 the error between that value for c r i t i c a l v e l o c i t y and the one calculated, assuming f = 0.8, would be only 5%. For the f i e l d tunnel (24 cm in t e r n a l diameter) only 3 f i s h had maximum cross-sectional areas greater than 10% of the cross-sectional area of the tunnel and, for these animals, the error between assuming f = 0.8 and not 1.0 would be about 2.5%. Due to the large diameter of the f i e l d tunnel several f i s h were run i n each t r i a l but, i n the laboratory, only when very small animals were tested (10 cm fork length) was i t possible to run more than one f i s h at a time. As noted previously (Jones, 1971) interaction between individuals did not appear to affect swimming performance. Temperature was recorded throughout each experiment i n the f i e l d and laboratory and, i n addition, the oxygen tension of the water flowing from the laboratory respirometer was continuously monitored using a Radiometer or Beckman oxygen electrode. By appropriate adjustment of the water inflow i t was possible to keep the oxygen tension of the water constant during each test. 32 (b) Fixed vel o c i t y tests One hundred and thirty-two attempts were made i n the laboratory to establish fatigue curves for various species using a somewhat modified pro-cedure from that outlined by Brett (1967). A l l f i s h used i n these experiments had had their c r i t i c a l v e l o c i t i e s determined 5-10 days p r i o r to th i s test. After removal of the tag and introduction of the f i s h to the water tunnel, as outlined i n section (a), the speed was increased by small steps, following the one hour acclimation period, u n t i l a ve l o c i t y of 60-90% of the fish's previous-l y determined c r i t i c a l v e l o c i t y was obtained. Usually 5 steps were used to reach the test v e l o c i t y and a period of 3 min was allowed between each v e l o c i t y increment. When the desired test v e l o c i t y was reached the time to fatigue was measured using 100 min maximum. I f the f i s h did not fatigue i n 100 min the experiment was terminated. Fatigue, when i t occurred, was judged as outlined i n section (a). The temperature and oxygen tension of exhalent water from the respirometer was measured continuously. No marked changes i n either variable occurred during any one experiment. A l l experiments were conducted at the temperature to which the f i s h were acclimated (12-13°C). After fatigue, or at the termination of the experiment, the f i s h were removed from the respirometer, weighed, measured (fork length and maximum cross-sectional area) and re-tagged. (c) Electromyography To prepare f i s h for electromyography they were anaesthetized with MS222 (1:15000) and electrodes were inserted into the l a t e r a l red muscle, and the epaxial portion of the white muscle mass. Electrodes consisting of a pair of 40 swg insulated copper wires glued together with epoxy for a distance of 5 mm from the t i p , bent into a hook shape and bared at the end, were used i n the red 33 muscle of a l l species studied and for white muscle i n herring. In the other species the indiv i d u a l electrodes of each pair used for recording from white muscle were separated by a distance of 2-3 mm. After instrumentation the f i s h were allowed to recover from the anaes-t h e t i c . Herring were allowed to recover i n a bucket of seawater for 20-30 min; then i f the electrodes were functioning the f i s h were transferred to the water tunnel. Herring were found to be so delicate that i t proved impractical to allow a long recovery period after anaesthesia. The freshwater f i s h were allowed to recover for 18-20 hours i n the water tunnel before electromyographic recording was undertaken. Electromyograms were recorded from the f i s h at increasing swimming speeds u n t i l the maximum sustained speed of the ind i v i d u a l was reached. In recording from the freshwater f i s h (trout, carp, pike) signal to noise r a t i o was increas-ed by addition of small amounts of seawater to the water i n the water tunnel for the duration of the most c r i t i c a l recording periods. The water temperature i n the water tunnel was held at the same temperature as the water i n which the f i s h had been held, ±0.5°C. The potentials from the muscle were amplified by a Tektronix 122 preamplifier and recorded on a Brush 220 oscillographic pen recorder. After the experiments, some f i s h were k i l l e d , cooked i n b o i l i n g water, and the proportion of l a t e r a l red muscle to t o t a l body weight determined. This was done f i r s t weighing the cooked f i s h , then removing and weighing the lateral..red muscle. (4) Analysis of Data (a) Increasing v e l o c i t y tests The aim of the analysis was to provide information for the following formula for each species: 34 V = K L 6 (log V = log K + e log L) where V = c r i t i c a l v e l o c i t y (cm/sec) K = constant (y intercept) L = body length (cm) e = exponent (slope of the lin e ) Obviously not a l l species were examined i n s u f f i c i e n t numbers to give a re-gression equation i n which any confidence could be placed and, i n these cases, only mean values for various size classes i s presented. For reasons given previously i t was also necessary to define the effects of temperature of acclimation, and state or maturity, on swimming performance. Consequently a series of s t a t i s t i c a l tests was performed which ultimately led (or not, as the case may have been) to the f i n a l regression analysis. In a l l s t a t i s t i c a l procedures 5% was regarded as the f i d u c i a l l i m i t of significance. Analysis of co-variance was used to test for c r i t i c a l v e l o c i t y differences between immature, male and female individuals and individuals at different temperatures for each species with length as the co-variate. No s i g n i f i c a n t differences were found between the different groups i n any species. The data for each species were therefore pooled and a regression equation was calculated. Pearson product moment correlations were performed on log c r i t i c a l v e l o c i t y versus log length to assess the correlation of the two variables, (b) Fixed velooity tests The data are presented graphically as plots of time to fatigue (min) against swimming speed expressed as a per cent of the animal's previously determined c r i t i c a l v e l o c i t y . Since the l a t t e r represented the maximum speed that the animal could swim at for 10 min, a fixed point regression was perform-35 ed on the data with 10 min representing the fixed point of 100%. As the maximum time period of the experiment was 100 min, i f an unreasonably low speed was chosen for the f i s h to swim at, i t would be possible with t h i s type of analysis to obtain unreasonably low values, as a proportion of their previously determined c r i t i c a l v e l o c i t y , for sustained performance. Conse-quently, i t was a r b i t r a r i l y decided to eliminate a l l those 100 min points which were more than 10% below the mean of the two lowest percentages of c r i t i c a l v e l o c i t i e s at which any two f i s h had fatigued within the 100 min time period. 36 RESULTS (1) Preliminary Experiments (a) Increasing v e l o c i t y tests Increasing v e l o c i t y tests were structured to give the maximum steady performance for 10 min but i t has been claimed that longer periods between veloc i t y increments should be used i n t r i a l s of this type (e.g., 20-60 min) so, i n a preliminary series of experiments, 5 longnose suckers (Catostomus catostomus) and 3 a r c t i c grayling (Thymallus afcticus) were examined with both 10 and 20 min periods between velo c i t y increments. Although there was a s l i g h t reduction i n c r i t i c a l v e l o c i t i e s achieved, by the same f i s h , with the longer time period, the mean values for both species were not s i g n i f i c a n t l y d i f f e r e n t . Also, i t has been suggested that the period of acclimation to the respirometer before the s t a r t of the test may affect c r i t i c a l v e l o c i t y determined i n this manner (Brett, 1967), although i n a series of experiments with grayling and longnose suckers there was no s i g n i f i c a n t difference i n the c r i t i c a l v e l o c i t i e s achieved after 16, 12, 2 or 1 hour of acclimation to the water tunnel. (b) Fixed vel o c i t y tests Brett (1967) suggested that the minimum time period to termination of a fixed v e l o c i t y test should be 200 min, but i n the present series tests were terminated at 100 min as this represented 10 times the time period used to delimit the maximum steady performance. Consequently i n some fixed v e l o c i t y t r i a l s the ultimate time period was extended to 300 min, which represents the t r a n s i t i o n point to sustained performance i n sockeye (Oncorhynchus rierka) (Brett, 1964). The extended time period was imposed on 4 longnose suckers, 37 1 grayling and 1 burbot (Lota l o t a , L . ) . The suckers were swimming at 68,5 to 89% of t h e i r previously determined c r i t i c a l v e l o c i t i e s , the a r c t i c g rayling at 89%, and burbot at 96.6%. A l l animals except one longnose sucker, which was swimming at 83.5% of i t s previously determined c r i t i c a l v e l o c i t y , continued to swim throughout the extended time period, (2) Increasing V e l o c i t y Tests Increasing v e l o c i t y tests were performed on 179 longnose suckers (Catosto- mus catostomus, F o r s t e r ) , 20 white suckers (Catostomus commersoni, Lacepede), 169 humpback whi t e f i s h (Coregonus clupeafbrmis, M i t c h i l l ) , 24 broad whitefish (Coregonus nasus, P a l l a s ) , 105 a r c t i c g rayling (Thymallus a r c t i c u s , P a l l a s ) , 192 pike (Esox l u c i u s , L . ) , 54 yellow walleye (Stizostedion vitreum vitreum, M i t c h i l l ) , 53 burbot (L. l o t a ) , 34 carp (C. c a r p i o ) , 25 trout (S. g a i r d n e r i ) , and 11 a r c t i c char (Salvelinus alpinus, L . ) . The tests on burbot, broad wh i t e f i s h , trout, and carp were performed e x c l u s i v e l y i n the laboratory; a l l other species were tested both i n the laboratory and i n the f i e l d . Of the 11 species studied only 10 were represented by a s u f f i c i e n t l y wide s i z e c l a s s to obtain a regression equation (V = KL ). Table 3 shows the values f o r K and e of the 10 species along with .a p r o b a b i l i t y value for the c o r r e l a t i o n of c r i t i c a l v e l o c i t y on length for each of the species. (3) Fixed V e l o c i t y Tests Fixed v e l o c i t y tests were conducted on 5 species i n the laboratory at 12-13°C. A l l animals were acclimated to t h i s temperature. The data are i l l u s t r a t e d , for each species, i n Figure 4. I t i s of i n t e r e s t that the f i x e d point regression l i n e s are of the same slope for pike, longnose sucker and burbot, showing that, on the average, these f i s h can maintain 60% of t h e i r 10 min maximum performance for 100 min, (Fig...:4) . For both char and grayling 38 TABLE 3. Tabulation of the K and e values for the equation V = KL for 10 species of freshwater fish. Probability of V Species K e not being correlated to L Conditions pike 4.9 .55 .001 12-13°C yellow walleye 13.07 .51 .1 *F 18-20°C arctic grayling 36.2 .193 .02 F 12 1 12 F 20°C longnose sucker 11.03 .529 .0001 1 7 F 18-20°C white sucker 10.3 .552 .02 F 12, 19°C burbot 30.6 .07 .1 1 7, 12°C humpback whitefish 18.2 • 35 .0001 1 7 F 12, 19°C broad whitefish 9.7 .45 .003 1 12°C trout 12.4 .55 .0001 1 10°C carp 7.52 .65 .0001 1 10°C *I. -, F -Laboratory Field 39 Figure 4 Fatigue curves for 5 species of f i s h determined i n fixed v e l o c i t y tests. The tests were terminated at 100 min i f the f i s h f a i l e d to fatigue. PERCENT OF PREVIOSLY DETERMINED CRITICAL VELOCITY 41 the slopes of the regression l i n e s are twice as steep as those for the above 3 species. Char and grayling can therefore maintain about 80% of th e i r 10 min maximum performance for 100 min (Fig. 4). I t must also be pointed out that some of the individuals represented by these figures could maintain t h e i r 10 min c r i t i c a l v e l o c i t y for a period of 100 min or (as i s suggested by the one burbot swimming at 96.6% of c r i t i c a l v e l o c i t y for 300,min) much longer. In a l l of the species subjected to incremental increases i n water ve l o c i t y i t was observed (in about 80% of the cases) that f i s h tend to fatigue within the f i r s t 3 min of a new increment (regardless of the length of the test period). (4) Ele c trdmyo graphy (a) Herring In herring jswimming was of one of two d i s t i n c t patterns: either, the f i s h swam smoothly maintaining i t s position i n the water tunnel or, i t could not maintain i t s position while swimming smoothly but rather d r i f t e d backwards then accelerated up the tube with bursts of violent t a i l beats. During smooth swim-ming red muscle fibers alone were active (Fig. 5a, b & c). When the water veloc i t y was increased to the point at which the f i s h was unable to maintain position by swimming smoothly large potentials were recorded corresponding i n time to bursts of violent swimming (Fig. 5d). These large potentials are 4-5 times as large as the ones associated with smooth swimming and are thought to be white f i b e r action potentials. A period of such violent swimming always resulted i n fatigue i n 1-2 min. The red portion of the myotomal musculature was 5.6% of the body weight i n herring. (b) Carp Electromyographs were successfully recorded from the red and white portions of the myotome. E l e c t r i c a l a c t i v i t y was detected from the mosaic portion of 42 Figure 5 Electromyographs recorded from herring with electrodes placed within the red myotomal muscle mass. Lines a-c are records at increasing swimming speeds, d i s an electromyograph obtained from f i s h swimming with violent t a i l b e a t s . 43 44 the myotome even at the lowest swimming speed (20 cm/sec) (Fig....6a) . At increased swimming speeds, the potentials from both portions of the myotome increased i n amplitude while decreasing i n duration. This increase i n ampli-tude suggests recruitment of more fibers at the higher swimming speed. Occasional rapid large potentials were observed (Fig. 6b) at intermediate swimming speeds. These were faster events than those which composed the remainder of the e l e c t r i c a l a c t i v i t y , and are presumably muscle action potentials of white muscle f i b e r s . At the maximum speed (10 min c r i t i c a l velocity) rapid potentials (Fig. 6c) formed the major part of the a c t i v i t y from the white muscle and were picked up by the electrodes located i n the red muscle mass. More information was obtained by alternately increasing and decreasing the speed s l i g h t l y while the f i s h was swimming at near to i t s c r i t i c a l v e l o c i t y . Under these conditions the f i s h could be induced to swim as did the herring, interspersing periods of steady swimming with a few violent t a i l beats while accelerating. During this type of swimming (Fig. 6d) two types of muscle discharges were recorded. Violent t a i l beats (indicated by asterisks i n Fig. 6d) resulted i n large potentials being recorded from the white muscle mass. These are presumably e x t r a c e l l u l a r l y recorded white f i b e r action potentials. In the period between violent t a i l beats e l e c t r i c a l a c t i v i t y from the white muscle mass consisted of small potentials resembling those recorded from the red f i b e r s . H i s t o l o g i c a l l y two types of muscle fibers were i d e n t i f i e d within the white muscle mass: a broad f i b e r and a narrow f i b e r . Lateral red muscu-lature i n carp i s 2.6% of the body weight, (c) Pike In pike there was no evidence of e l e c t r i c a l a c t i v i t y i n the white muscle mass at speeds of less than the individual's c r i t i c a l v e l o c i t y (Fig. 7a & b). 45 Figure 6 Electromyographs from carp myotomal musculature. M denotes record from mosaic muscle mass, and R denotes that from red muscle mass, a-c indicate records at increasing swimming speed from minimal to 10 min c r i t i c a l , d indicates record obtained when f i s h was swimming at near c r i t i c a l speed with interspersed violent tailbeats (denoted by * on record). 46 47 Figure 7 Electromyographs from pike red (R) and white (W) myotomal muscle masses, a-c indicate records at minimal to 10 min c r i t i c a l v e l o c ity. 48 49 At c r i t i c a l v e l o c i t y or i n excess of c r i t i c a l v e l o c i t y large fast potentials of the action potential type were recorded from the white muscle mass (Fig. 7c). The l a t e r a l red muscle i n pike was found to be only about 1% of the weight of the whole f i s h , (d) Trout The l a t e r a l red muscle mass i n trout forms 2,5% of the body weight. Red muscle was active at a l l swimming speeds but no e l e c t r i c a l a c t i v i t y was detectable i n the mosaic muscle at low speeds except for a few discharges at the beginning of some speed increments.when the f i s h was swimming unsteadily (Fig. 8a & b). During steady swimming at speeds up to 99% of the individual's c r i t i c a l v e l o c i t y no e l e c t r i c a l a c t i v i t y was observed i n the mosaic muscle. Water velo c i t y i n excess of c r i t i c a l v elocity resulted i n the f i s h using violent bursts of swimming during which large action potential type discharges were recorded from the mosaic musculature indicating that white muscle fibers were being used. This period of white muscle a c t i v i t y at high swimming speeds was observed i n a l l species studied and was followed by fatigue. 50 Figure 8 Electromyographs from trout red (R) and mosaic (M) myotomal muscu-lature, a-c are records at minimal swimming speed to 99% of 60 min c r i t i c a l v e l o c i t y , d i s a record at a velo c i t y i n excess of 60 min c r i t i c a l v e l o c i t y . 52 DISCUSSION There can be no doubt, for those species studied by both fixed and i n -creasing v e l o c i t y tests, that i n the 10 min increasing v e l o c i t y tests the animals were i n the zone of steady performance. I t would also appear reason-able to assume that this was true of a l l other species studied i n increasing v e l o c i t y tests alone. Bainbridge (1962) argued that there are major s p e c i f i c or f a m i l i a l differences i n the re l a t i o n of speed to si z e and Fry and Cox (1970) urged that this should be tested i n a thorough comparative study. For most salmonids the sustained speeds vary e s s e n t i a l l y as L^"^ (Bainbridge, 1960, 1962; Blaxter and Dickson, 1959; Brett, 1964, 1965; Fry and Cox, 1970), while for other species the exponents range from 1 for burst speeds of herring (Blaxter and Dickson, 1959) and dace (Bainbridge, 1960) to 0.31 for sustained speeds of pumpkinseed (Lepomis gibbosus) (Brett and Sutherland, 1965). In the present experiments for species showing a good length to swimming speed correla-t i o n , the range i n exponents was from 0.19 for grayling to 0.65 for carp. Figure 9 i s a graphical representation of the relationship of 10 min c r i t i c a l v e locity to length i n the different species studied, along with the proportion of l a t e r a l red muscle i n some of the species. Swimming speed i s more size dependent i n some species than others. This may be related to the feeding and migratory a c t i v i t y of the different species i n that small grayling (<20 cm), which feed i n the same r i f f l e areas as large grayling, have a higher c r i t i c a l v e l o c i t y than individuals of the same length -ftS- the other freshwater species studied. In general the larger proportion of red muscle i n the body muscula-ture (Fig. 9) the greater the c r i t i c a l v e l o c i t y . The present observations of electromyograms of red and white myotomal 5 3 Figure 9 The relationship of length to c r i t i c a l v elocity (10 min). The points are for f i s h which were not represented by a s u f f i c i e n t l y large size class to derive a length velo c i t y regression equation. Values i n parentheses represent the weight of l a t e r a l red myotomal musculature as a per cent of t o t a l body weight. 54 FISH LENGTH (CM) i T O U C a. 5) G r a y l i n g Loncnose Sucker White Sucker Carp (2.6) Tellow tSfalleye 105 \ 5 5 muscle masses of several f i s h species have shown that: 1. At low swimming v e l o c i t i e s red muscle fibers alone are active, not the white ones, i n a l l the species studied. 2. At speeds high enough to produce fatigue during the test period white muscle as well as the red i s active i n a l l of the species studied. In those f i s h which have only white fibers i n the white portion of the myotome (herring, pike) there i s no e l e c t r i c a l a c t i v i t y i n the white muscle i n the absence of violent t a i l beats. Mosaic muscle i n the physically trained trout used i n this study did not produce the small slow potentials t y p i c a l of red muscle, which were shown i n mosaic muscle of untrained trout by Hudson (1973). This difference between the two groups of trout (Hudson's and mine) may be the result of physical tr a i n i n g somehow changing the pattern of recruitment of the red type fib e r s i n the mosaic muscle or i t may be a difference between the two stocks of f i s h . The stock of f i s h used by Hudson (1973) have previously been shown to have a very low c r i t i c a l v e l o c i t y (about half of that of f i s h i n t h i s study) (Webb, 1971). Carp i n this study showed recruitment of the red fibers i n the white (mosaic) myotomal muscle mass at submaximal swimming speeds as was observed by Hudson (1973) i n trout. The narrow muscle fibers i n the mosaic muscle of trout contain myoglobin and fat and would therefore be expected to have an aerobically based metabolism. In examining the available evidence i t does not seem probable that white muscle operating anaerobically can or does contribute to the power output of a f i s h for periods longer than a few minutes without p r e c i p i t a t i n g fatigue. I t would also seem that the more red muscle a f i s h has i n the muscles which power swimming, the faster i t can swim on a sustained basis. 56 SECTION I I Changes i n Cardiovascular, Respiratory and Metabolic Variables Accompanying Prolonged Exercise i n Fish INTRODUCTION In Part I of t h i s study i t was demonstrated that during sustained swimming i n several teleosts only the red muscle fibers are involved and that the proportion of the l a t e r a l red muscle of the myotome i s related to the maximal sustained swimming speed. The oxygen consumption rate of f i s h during prolonged swimming periods has been extensively investigated and found to increase exponentially with swimming speed u n t i l the f i s h fatigues (Brett, 1964). Changes i n cardiovascular and respiratory variables during short duration (5 min) exercise i n f i s h have been studied (Stevens, 1968). In mammals the cardiovascular compensations to exercise change when exercise i s prolonged beyond 10-15 min. Under these conditions heart rate tends to r i s e and stroke volume decreases (both r e l a t i v e to the value at 10-15 min of exercise) (Cobb and Johnson, 1963; Ekelund and Holmgren, 1964; S a l t i n and Stenberg, 1964; Ekelund, 1967). This i s thought to be due to an increase i n blood flow to the skin for thermoregulatory purposes. I f this i s the case an animal which has no thermal load, such as f i s h , would not be expected to show such changes during prolonged exercise. The effect of prolonged exercise on cardiovascular and respiratory variables and th e i r relationship to metabolic variables has not been investigated i n f i s h . The experiments i n this section were designed to seek answers to the following questions: (a) What i s steady state with respect to cardiovascular and respiratory aspects of exercise i n fish? 57 (b) How do the cardiovascular and respiratory systems in. f i s h adjust to steady state exercise? (c) What determines, and causes termination of, maximal exercise ( i . e . , what i s the l i m i t i n g factor, and what i s fatigue)? 58 MATERIALS AND METHODS I I (1) Animals Trout (40-53 cm, 0.9-1.5 kg) were purchased from either the Trout Lodge, Ephrato, Washington, U. S. A. or Colebrook Trout Farm, Surrey, B, C. and transported to U. B. C. by tank truck where they were held as described i n Section I. A l l f i s h were barren females and came from the same stock ( i . e . , Colebrook Trout Farm bought f i s h from Trout Lodge). The experiments were carried out at water temperatures of 9-10.5°C on f i s h which had been trained for a minimum of two weeks (see Section I for t r a i n i n g procedure). (2) Surgical Procedures The f i s h was anaesthetized i n a bucket of MS222 Sandoz (1/15,000), weighed, measured, and placed on an operating table s i m i l a r to that of Smith the B e l l (1964). Flow of water or anaesthetic (MS222, 1/20,000) was maintained over the g i l l s during a l l s u r g ical procedures. Cannulae for blood sampling were placed i n the dorsal aorta, ventral aorta and common cardinal vein. The dorsal aorta was cannulated as described by Smith and B e l l (1964) using a 45 cm length of PE 60 tubing terminated with a 1 cm section of Huber point 21 G needle. Ventral a o r t i c cannulation was accomplished using a cannula s i m i l a r to the one for the dorsal aorta except that the needle end was 2 cm long and bent at a 60° angle 6 mm from the t i p . This cannula was inserted into the ventral aorta through the_ tongue at the l e v e l of the t h i r d g i l l arch. The ventral a o r t i c cannula was firmly sutured to the tongue and led straight out of the mouth. The right common cardinal vein cannula consisted of a 3 cm section of Huber pointed 18 G needle bent at 90° 18 mm from the t i p and attached to a 45 cm piece of PE 160 tubing (Clay Adams). This cannula was inserted perpen-59 dicular to the right side of the f i s h at a point about 3 mm posterior to the cliethrum and 3 mm ventral to the l a t e r a l l i n e . The cannula was then oriented so that the open end i n the vein was directed ventrallv before being sutured i n place. A l l blood vessel cannulae were f i l l e d with heparinized (10 u/ml) Courtland saline (Wolf, 1963) and plugged with pieces of wire of the appropri-ate diameter. Wires for ECG recording were inserted, one immediately, posterior to the middle-of the pectoral girdle and one medio-dorsallv posterior to the operculae. These wires were sutured to the skin at several locations along the right side of the f i s h up to the anterior edge of the dorsal f i n . At this point the wires together with a protective wire braid were firmly t i e d to the f i n . Any existing dorsal f i n damage was repaired by suturing to help prevent snagging of cannulae and wires. To measure v e n t i l a t i o n volume a s k i r t was made of the wrist portion of a disposable surgical glove and placed over the head of the f i s h to l i e i n contact with the body. After proper orientation the s k i r t was sutured i n place around the lower jaw and up over the operculae i n such a manner that the anterior of the s k i r t formed a close f i t to the head of the f i s h , whereas the posterior part was.loose to allow free movement of the operculae. The excess membrane was then trimmed o f f , leaving an area of membrane extending about 2 cm posterior to the operculum. A water sampling cannula. (PE 60) 60 cm long was placed with i t s open end ventro-medially anterior to the pectoral girdle (under the s k i r t ) , and sutured i n place, and then led up posteriorly to the l e f t pectoral f i n and sutured i n place dorsally. In order to measure respiration rate a buccal cannula (45 cm PE 60) was placed as described by Saunders (1961). After completion, of surgical procedures the f i s h were placed i n the water 60 tunnel and allowed 18-24 hours to recover from the anaesthetic and surgery. During t h i s time water v e l o c i t y i n the water tunnel was at the lowest s e t t i n g , about 10 cm/sec. (3) Experimental Techniques and Equipment Used (a) Pressure measurement Sanborn 267B tranducers were us'.ed for dorsal and v e n t r a l a o r t i c blood pressure measurements and a Statham P23v transducer was used to measure common card i n a l blood pressure. Buccal pressures were measured with a Sanborn 267B transducer. During the cardiovascular transients experiments i t was desirable to have an i n d i c a t o r of water v e l o c i t y ( i n the water tunnel) recorded along with the other v a r i a b l e s . One way that the v e l o c i t y of a f l u i d flowing i n a pipe may be measured i s by detecting the pressure change as the f l u i d passes around an elbow i n the pipe or through a c o n s t r i c t i o n . Since the water tunnel has, incorporated i n i t s design, two contraction cones ( e f f e c t i v e c o n s t r i c t i o n s ) the pressure change across one of these was measured using a Sanborn 268B d i f f e r e n t i a l pressure tranducer and the output recorded on the s t r i p chart along with the other v a r i a b l e s . To ensure that the amplitudes of the recorded blood pressure waves were accurately recorded the transducers and cannulae used f o r blood pressure recording were tested by the free v i b r a t i o n method described by Macdonald (1974). By t h i s method the n a t u r a l frequency of the Sanborn 267B transducers and cannula was found to be 15 Hz with a damping 35% of c r i t i c a l . The Statham transducer and cannula used for common ca r d i n a l pressure measurement had a natural frequency of 10 Hz and damping was 36% of c r i t i c a l . Pressure trans-ducers used for dorsal and v e n t r a l a o r t i c pressures were c a l i b r a t e d against a pressure head of 40 cm of s a l i n e (zero being the level., of the water tunnel). 61 The Statham P23v transducer used for common cardinal blood pressures was calibrated against a head of 15 cm of saline, as was the transducer used for buccal pressure recording. A l l calibrations were checked frequently. (b) Signal conditioning and recording An EKG analog ratemeter was used (triggered by the QRS wave of the ECG) to obtain beat to beat heart rate i n the study of heart rate transients, Recording of a l l electronic signals was done on either a Brush model 220 two channel pen recorder or a Techni-rite 8 channel recorder, model number TR8-88; both recorders w r i t i n g on r e c t i l i n e a r co-ordinates. (c) The water tunnel The water tunnel used was b a s i c a l l y the same design described by Brett (1964). For a complete description of t h i s equipment refer to Section I. (d) Blood sampling procedures Blood samples were taken from the cannulae after f i r s t bleeding the saline out of the cannula. A sample of blood for oxygen content determination was then taken from the cannula by inserting the needle of a needle-tipped micro buret (previously heparinized and dried) into the end of the cannula and allowing the buret to f i l l the required amount by blood pressure (dorsal and ventral aortic samples) or by siphoning (common cardinal vein). A blood sample for pH and PO2 was taken from the cannula with a 1 ml heparinized syringe and a three-way valve arrangement. The valve and syringe were rinsed with a small amount of blood (rinse blood saved), the sample (0.5 ml) taken, and immediately injected into the previously calibrated pH and PO2 equipment. A sample (0.3-1 ml) of blood was taken i n a s i m i l a r manner on occasions when lactate was to be determined and immediately diluted 1.0:3.5 v/v i n cold 8% HC10,., 62 (e) Oxygen tension Oxygen tensions were measured with a Radiometer type E5046 electrode i n a type D616 thermostated c e l l . The zero s e t t i n g was established, using a 0.01 M Na2B0^ s o l u t i o n with 5 mg/20 ml Na2S0^ added, twice d a i l y . The span was set before each sample or group of samples i n the case of duplicates using a i r e q u i l i b r a t e d water at the ambient water temperature. Both c a l i b r a -tions were reproducible to 0.5 Torr over the period of the measurements. (f) Measurement of pH Measurement of pH was done with a Radiometer type G297/G2 blood pH e l e c -trode c a l i b r a t e d using p r e c i s i o n buffers (type S1500 and S1510). One or both c a l i b r a t i o n s were done before each blood sample depending on the s t a b i l i t y of the electrode. Readout from the oxygen and pH electrodes was on a Radiometer Acid-Base Analyzer. (g) Determination of oxygen consumption Before each oxygen consumption determination the water tunnel was checked fo r trapped a i r bubbles. Any a i r bubbles that were found were removed. Oxygen consumption was determined by c l o s i n g the system (shutting o f f the input of fresh water) and measuring the rate of change of the oxygen tension of the water c i r c u l a t i n g i n the water tunnel. This was done by taking a water sample with a 5 ml syringe from the water tunnel at the same time that the inflowing water was shut o f f and another sample at the end of a su i t a b l e time period. The length of time (5-45 min) between the samples was determined by the s i z e and i n t e n s i t y of swimming of the f i s h . During t h i s time the oxygen tension of the water dropped about 10-15 Torr. The oxygen tension of the water samples was measured i n duplicate immediately a f t e r each was taken. Oxygen consumption was then calculated using the formula: 63 V0 2 = APCyV-a t VC>2 - oxygen consumption (ml ATPS/min) A P O 2 - change i n oxygen tension (Torr) V - volume (34.51) a - s o l u b i l i t y c o e f f i c i e n t of 0 2 i n H^ O (ml C^/l ^O-Torr) t - time (min) (h) Blood oxygen content Oxygen content of a r t e r i a l and venous blood was determined by the method of Tucker (1967). The chamber used was larger (3 ml) than that described by Tucker and was used i n conjunction with a Radiometer type E5046 oxygen elec-trode. The volume of the chamber varied depending on how deep the electrode was inserted, and was measured da i l y . A l l oxygen content determinations were done at 32°C as described by Tucker except that the blood volumes were larger (50 y l for venous and 25 y l for a r t e r i a l blood) because the chamber was larger. The blood samples were pipetted into the chamber within 30 sec of sampling. A r t e r i a l and venous samples were done s e r i a l l y 5-10 min apart. (i) Hematocrit The blood remaining i n the micro buret after the oxygen content determina-tion was transferred into three 20 y l micro pipettes sealed with Seal-ease (Clay Adams). The excess length of pipette was then cut o f f , the samples were spun i n a commercial micro hematocrit centrifuge and measured. After hemato-c r i t determination the samples were labelled and frozen for blood iron deter-mination. 64 (j) Lactate determination Lactate determinations were done by a co-worker (W, Driedzic) i n the following manner: the sample (see under blood sampling) was centrifuged to remove protein and the supernatant was neutralized with 3 M K^CO^ containing 0.5 M triethanolamine. KCIO^ was removed by centrifugation and an aliquot of the supernatant was analyzed for lactate enzymatically (Sigma b u l l e t i n #826) . Assays were carried out on a Unicam SP 1800 dual beam spectrophotometer connect-ed to a s t r i p chart recorder, (k) Blood i r o n concentration Micropipettes containing the 20 y l blood samples saved (frozen) from hematocrit determinations were rinsed on the outside and the pipettes broken up inside clean b o r o s i l i c a t e glass s c i n t i l l a t i o n v i a l s . After an overnight drying period at 80°C the v i a l s and contents were placed i n a muffle furnace and ashed at 680°C for a minimum of 8 hours or u n t i l only a white powder remained i n the pipette sections i n the v i a l s . Upon cooling to room temperature the v i a l s were re-labelled and the contents dissolved i n 0.2 N HC1. Recovery of sample was checked by an i d e n t i c a l treatment of standard solutions i n pipettes, and found to be 100%. The samples were analyzed on a Tectron model AA120 atomic absorp-tion spectrophotometer using a wavelength of 248.3 mm from a Varian FeCo hollow cathode lamp against standards ranging from 0 to 10 mg Fe/1. The standard was made by dissolving 0.1 g of Iron Powder (99+ Fisher chemicals) i n a minimum quantity of Analar HC1 and d i l u t i n g i n 0.2 N HC1 to the appropriate concentra-tions . (1) Ventilation volume Ventilation volume can be determined by measuring the oxygen tension of the inhalent and exhalent water and the oxygen consumption of a f i s h . From the 65 above information and the s o l u b i l i t y c o e f f i c i e n t of oxygen i n water the v e n t i l a -t i o n volume can be calculated by the Fick equation: Vg = V0 2/(P I0 2 - P E0 2)a where V0 2 = oxygen consumption (ml 02/min) Vg = v e n t i l a t i o n volume (ml H20/min) P-j-02 = oxygen tension of inhalent water (Torr) Pg0 2 = oxygen tension of exhalent water (Torr) a = s o l u b i l i t y c o e f f i c i e n t of C»2 i n water (ml 0 2/ml H20 Torr) One way to measure the oxygen tension of exhaled water i s by sampling water from within the opercular cavity by means of opercular or c l i e t h r a l cannulae. Both of these methods have been found to have low r e l i a b i l i t y (Davis and Watters, 1970). The observation of B a l l i n t i j n and Hughes (1965) that the pressure i n the opercular cavity i s negative u n t i l the end of the opercular stroke, together with v i s u a l observations of the movement of the opercular valve, indicated that there i s a backflow of water from outside into the opercular cavity when the operculum opens f u l l y . Water sampled by an opercular or c l i e t h r a l cannula would therefore be of higher oxygen tension than the water which has just come across the g i l l s . A method was devised of attaching a rubber s k i r t to loosely cover the area at the opening of the operculae (described under surgical procedures), thus providing a buffer zone of exhaled water between the opercu-l a r cavity and the outside water. This i s e s s e n t i a l l y a modification of the method of Davis and Cameron (1970) for use on free-swimming f i s h . Davis and Watters theorized that the reason for the large v a r i a b i l i t y i n the oxygen tensions of water samples taken from cannulae i n different locations i n the opercular cavity was variations i n oxygen extraction i n d i f f e r -66 ent regions of the g i l l s . I f such i s the case the large volume ( r e l a t i v e to opercular volume) under the s k i r t should act as a mixing chamber as wel l as a buffer for any backflow which occurs during the respiratory cycle. To deter-mine whether water samples taken from different locations under the s k i r t and the same PO^ ,, and whether the; s k i r t imposed a load on the opercular muscula-ture, the following procedure was used: The resting and VC^ max (VC^ at c r i t i c a l velocity) of a trout were measured before any sur g i c a l procedures were carried out (a normal trout). A s k i r t was then attached to the trout (as described under su r g i c a l procedures) with cannulae attached at the l a t e r a l and latero-ventral positions as well as i n the ventral position. After 18-24 hours recovery the VC^ and oxygen tensions of exhaled water collected from the three sampling cannulae were measured at rest and at various swimming speeds up to c r i t i c a l v e l o c i t y . The measurement of water samples taken from the cannula i n different positions i n two f i s h at rest and during swimming at various speeds showed no detectable difference (42 samples). Since there was no difference i n the oxygen tension of the water samples from different posi-tions on the f i r s t two f i s h a ventral cannula alone was used on the two remain-ing f i s h . Resting VC^ of four f i s h before and after attaching the s k i r t s were not s i g n i f i c a n t l y d i f f e r e n t . The VC^ max of each of these individuals was the same before and after attaching the s k i r t . C r i t i c a l v e l o c i t y (60 min) was decreased by an average of 15 cm/sec over the values obtained for the same individuals previous to i n s t a l l a t i o n or af t e r removal of the s k i r t . This effect on c r i t i c a l v e l o c i t y was also obtained by attaching dummy wires and cannulae (of the type used for blood sampling and ECG recording) and i s presumably the effect of increased drag on the f i s h . 67 (m) Experimental procedure and data analysis Fish were instrumented as described i n Surgical Procedures and allowed to recover i n the water tunnel for 18-24 hours. Figure 10 i s an i l l u s t r a t i o n of a f u l l y instrumented trout. Such a f i s h did not e x i s t ; for instance, determina-tions of v e n t i l a t i o n volume were not done on the same individuals as measure-ment of cardiovascular variables (except heart rate i n two cases), Venous cannulation was either of the ventral aorta or the common cardinal vein, not both. Ba s i c a l l y three types of experiments were performed: (1) Transients experiments (2) Blood and cardiovascular experiments (3) Ven t i l a t i o n experiments. In the transients experiments only heart rate, v e n t i l a t i o n rate and blood pressure i n the dorsal aorta, ventral aorta, and common cardinal vein were monitored, along with the ve l o c i t y of the water i n the water tunnel. After recording the different variables at rest the water vel o c i t y was increased abruptly by about 1/6 of c r i t i c a l v e l o c i t y and maintained at that v e l o c i t y for 60 min. The variables were recorded after 0.5, 1, 3, 6, 10, 15, 30, 45, and 60 min, at which time the ve l o c i t y was increased again and the measurements repeated. The variables were measured at each test time i n each t r i a l . Results were analyzed i n the following way: Heart rate for each time i n each t r i a l was expressed as the heart rate at that time divided by the heart rate immediately before the beginning of the ve l o c i t y increment. In the case of blood pressure the differences between the pressure at each time and the pressure before the start of the increment were computed. Respiratory rate was treated as a rate per min for each time. Means and standard errors were then calculated for the normalized variables. 68 Figure 10 Schematic diagram of the various instrumentation carried by the experimental f i s h . 70 In the experiments investigating the blood and cardiovascular variables measurements of heart rate, arterial^oxygen content,^venous oxygen tension, venous and a r t e r i a l hematocrit, venous and a r t e r i a l C0 2 contents, blood pressure, a r t e r i a l and venous pH, lactate, oxygen tension of inhaled water and oxygen consumption, were attempted on each i n d i v i d u a l . In th i s set of experi-ments 89 trout were cannulated. During recovery several f i s h died due to in t e r n a l bleeding as a result of cannulation; i n some one or both cannulae became non-functional, and i n others the hematocrit was too low as a result of inter n a l bleeding. On the remaining f i s h as much information as possible was obtained. Oxygen consumption was determined on 25 ind i v i d u a l s , blood pressure i n 10, lactate i n 7, v e n t i l a t i o n volume i n 5 and heart rate i n 30. The samples of blood were taken from the resting f i s h ( s i t t i n g q uietly on the bottom of the water tunnel at a water ve l o c i t y of about 10 cm/sec) and analyzed as previously described. When a l l other variables had been measured the V0 2 of the f i s h was deter-mined. After V0 2 determination at rest the water ve l o c i t y i n the water tunnel was increased by about 1/4 to 1/3 of the estimated c r i t i c a l v e l o c i t y of the part i c u l a r i n d i v i d u a l . The f i s h was checked p e r i o d i c a l l y or continuously to make certain that i t was swimming constantly and not tangling on the cannulae and wires throughout the test period. After swimming for 50 min of the test increment blood sampling commenced and a l l the variables were measured before the oxygen consumption was determined and swimming speed again increased. At maximal swimming speed when i t was suspected that the f i s h was about to fatigue the sampling procedure was sometimes carried out before the 50 min time. The data from these experiments were analyzed . r e l a t i v e to c r i t i c a l v e l o c i -ty or to V0 2 max of each i n d i v i d u a l . This treatment was necessary since 71 swimming speed i s affected by drag, which i s determined by the amount and type of instrumentation attached to the f i s h , whereas the c r i t i c a l v e l o c i t y and VC>2 max of an i n d i v i d u a l represent that individual's maximal aerobic capacity. Ventilation experiments were carried out using the same test period (£0 min) as the blood and cardiovascular system experiments. Exhaled water samples were taken throughout the exercise periods as were samples of inhaled water. Towards the end of each exercise period oxygen consumption was deter-mined. Per cent u t i l i z a t i o n (%U = P-r°2 ~ PE°2y''E>I02^ a n c* v e n til a t i o n volume (Vg = V02/(PJ02 - P E0 2)a) were calculated. 72 RESULTS (1) Changes In Cardiovascular and Respiratory Variables i n Response  to Incremental Speed Increase (Transient Responses) In order to determine the length of time to reach steady state during pro-longed exercise i n trout f i v e variables (heart rate, v e n t i l a t i o n rate, and blood pressure i n the dorsal aorta, ventral aorta, and common cardinal vein) were monitored i n 5 f i s h exposed to increases i n swimming speed. Heart rate was found to be steady after about 15 minutes. Ventilation rate was extremely variable even at rest and the v a r i a b i l i t y increased during exercise. The ve n t i l a t i o n rate tended to increase at the beginning of an exercise period and to decline by about 15-30 minutes. Dorsal aortic and ventral a o r t i c blood pressures were steady after about 30 min.(see Fig. 11) while common cardinal blood pressure showed no change at any time. On the basis of the transient response of these variables the animals are i n steady state after about 30 minutes. Apart from defining the length of time needed to reach steady state the heart rate and blood pressure transients are worthy of detailed description. During the time that swimming speed was being increased (10 sec) bradycardia was observed, followed by tachycardia. Often several cycles of bradycardia and tachycardia were observed i n the f i r s t 30 seconds of a new ve l o c i t y increment. This apparent hunting disappeared a f t e r about 30 sec while the heart rate continued to increase at a somewhat slower rate than at the begin-ning of the increment. Heart rate reached a maximum value for that p a r t i c u l a r swimming speed at 3-15 minutes. The heart rate response described above was ty p i c a l of a l l incremental v e l o c i t y increases up to about 90% of c r i t i c a l 73 Figure 11 Change i n dorsal a o r t i c and ventral a o r t i c blood pressures following an incremental increase i n swimming speed. The pressure at any given time minus the pressure before the increase i n swimming speed was calculated for each time; the means (points) and standard errors (horizontal bars) were then calculated for each pressure at each time. Numbers above the records represent the number of determinations. 75 v e l o c i t y , above which heart rate was maximal and did not change with further increases i n swimming speed. The cardiac i n t e r v a l at steady state was constant. Dorsal a o r t i c blood pressure (both s y s t o l i c and d i a s t o l i c ) showed a transient increase (Fig. 11) following an incremental increase i n swimming speed. This increase was present immediately (5 sec) a f t e r the water v e l o c i t y was increased and peaked at about 6 min, then diminished. Pulse pressure i n the dorsal aorta did not change s i g n i f i c a n t l y . Ventral a o r t i c s y s t o l i c and d i a s t o l i c pressure showed the same transient response as did the dorsal pressures (Fig. 11). Common c a r d i n a l blood pressure did not change during or following increases i n swimming speed. (2) Cardiovascular, Respiratory and Metabolic Responses to Swimming  at Steady State Table 4 and Figures 12-14 contain the r e s u l t s of d e t a i l e d measurements (on 6 i n d i v i d u a l trout) of cardiovascular parameters at steady state. Resting heart rate i n cannulated animals tended to be higher (37.8 ±1.505 min n=9) than the mean f o r a l l trout studied (31.75 ±0.98 min" 1, n=32) (Fig. 15). Heart rate i n a l l cases increased with swimming u n t i l a maximum heart rate was reached. Mean maximum heart rate for cannulated animals was 51.4 ±2.478 (n=4) with a mean maximum increase of 1.33X r e s t i n g . Ca02 at rest was 10.4 ±0.544 v% (n=6) and changed l i t t l e during increased swimming. Mean a r t e r i a l oxygen saturation was 97.0 ±1.29% (n=8) at rest and did not change with i n t e n s i t y of exercise (Table 4). Venous oxygen content decreased with increasing swimming a c t i v i t y , r e s u l t i n g i n increased A-V0 2 differences with exercise. A-V0 2 difference at rest was 3.29 ±0.266 v% (n=8) and increased to a mean maximum of 8.30 ±0.507 v% (n=4) (an increase of 2.84X) (Fig. 13). TABLE 4. Cardiovascular and respiratory variables at rest and during exercise i n trout. Speed H.R. Ca0 2 Cv0 2 Pa0 2 Pv0 2 Hcta Hctv pHa pHv A-V0 2 vo 2 Q S.V. PIO2 Sa0 2 %Cv min v% v% Torr Torr % % v% ml kg ml kg ^ ml kg 1 1 Torr % -1 , -1 ST. min min rest 37.8± 10.4+ 7.1± 137± .33.2± 22.6+ 24.2± 7.932 7.959 3.29± 0.56± 17.6± 0.46+ 152.9± 97.0 1.505 0.544 0.715 4.23 3.056 1.02 1.809 +7.991 +8.025 0.266 0.025 1.095 0.021 1.959 ±1.291 n=9 n=9 n=9 n=8 n=8 n=9 n=8 -7.879 -7.902 n=9 n=9 n=9 n=9 n=8 n=9 41- 42.7+ 9.8± 4.4± 123.5+ 63% 3.18 0.737 0.833 7.5 Cv n=3 n=3 ' n=3 n=2 22.7± 24.451 1.386 1.05 n=3 n=2 5.4± 1.52± 28.4± 0.62+ 0.1 0.245 4.996 0.079 n=3 n=3 n=3 n=3 152.0± 96.0 2.309 +5.00 n=3 n=3 70-78% Cv 49.0± 1.00 n=5 9.02+ 0.497 n=5 3.4± 0.391 n=5 123.0± 4.203 n=4 23.5+ 2.062 n=4 20.34± 1.391 n=5 21.85± 7.924 2.447 +8.046 n=4 -7.829 n=3 7.988 +8.081 -7.911 n=3 5.6± 1.9+ 34.8± 0.7± 155.75± 98.75 0.585 0.276 4.809 0.091 0.947 ±1.03 n=5 n=5 n=5 n=5 n=4 n=5 81- 51.3± 10.2+ 2.9± 128.0± 29.3± 22.5± 25.8± 7.859 7.883 7.3± 3.12± 42.9± 0.86± 146.7± 99.7 91% 4.667 1.31 1.477 5.033 6.173 1.348 0.939 +7.970 +7.950 0.492 0.379 5.446 0.157 0.666 ±0.667 Cv n=3 n=3 n=3 n=3 n=3 n=3 n=3 -7.770 -7.825 n=3 n=3 n=3 n=3 n=3 n=3 n=2 n=2 max. 51.4± 9.7± 1.35+ 126.0± 16.0+ 25.7± 27.4+ 7.610 7,548 8,3± 4.34± 52,6+ l.Q3± 151.8± 98.5 (92%- 2.478 0.732 0.413 5.431 2.121 0.882 1.181+7.620 +7,630 0,5068 0,1687 2.16Q 0.Q74 2.658 ±0.866 Cv) n=4 n=4 n=4 n=4 n=4 n=4 n=4 -7.600 -7.480 n=4 n=4 n=4 n=4 n=4 n=4 n=2 n=2 77 Figure 12 Cardiac output of trout during rest and exercise. The values are single determinations normalized for weight of the f i s h and plotted against V0 9. Thirty-one determinations were done on 6 trout. 79 Figure 13 Arterio-venous oxygen differences i n trout at rest and during exercise plotted against VO,,. Thirty-one determinations were done on 6 f i s h . 80 A-VOS (ML/10CMJ 81 Figure 14 Stroke volume of trout heart at rest and during exercise normalized for weight of the f i s h and plotted against VC^. Thirty-one determinations were done on 6 f i s h . 82 83 Figure 15 Heart rate of trout at rest and during exercise plotted against water v e l o c i t y as a per cent of each individual's c r i t i c a l v e l o c i t y . Point at less than 25% CV i s for animals which were resting. The points are means of in d i v i d u a l determinations, the horizontal bars denote one standard error, and the numbers above the points indicate the numbers of determinations on 29 animals. 84 . HEART R A T E (/MIN) 8 8 Y J . k 85 -1 -1 Q (cardiac output) at rest was 17 .6 ±1.095 ml kg min (n=9) and increased with swimming by 2.86X to a mean maximum of 52 .6 +2.160 ml kg 1 min 1 (n=4) (Fig. 12).. Concomitant with the r i s e i n Q, stroke volume (SV) increased from a r e s t i n g value of 0.46 ±0.021 ml k g - 1 s t r o k e - 1 (n=9) by 2.12X to 1.03 ±0.074 ml k g " 1 s t r o k e " 1 (n=4) (Fig. 14). Oxygen consumption (V0 2) increased exponentially with swimming speed -1 (Fig. 16). Mean r e s t i n g V0 2 f o r the cannulated animals was 0.56 ±0.025 ml kg -1 -1 -1 min (n=7) as compared to 0.580 ±0.010 ml kg min (n=31) for a l l r e s t i n g V0 2 determinations (Fig. 16). V0 2 max for the cannulated trout was 4.34 ±0.168 (n=4) ml kg 1 min 1 as compared to 4.344 ±0.070 ml kg 1 min 1 (n=13) for a l l trout. The mean maximum V0 2 increase f o r the cannulated trout (n=4) was 7.71X. V0 2 max whenever determined was remarkably constant regardless of how the i n d i v i d u a l was instrumented ( i . e . , no instrumentation, ECG electrodes only, cannulae, opercular s k i r t ) as can be seen from the small amount of sca t t e r of the points i n Figure 16 as 100% CV i s approached. However when swimming speed rather than %CV i s used as the x axis the scatte r of points i s very large. A r t e r i a l oxygen tension i n the s i x trout examined i n d e t a i l was 137 ±4.23 (n=6) Torr at r e s t , whereas the mean f o r a l l Pa0 2 determinations done on re s t i n g f i s h was 134 .4 ±2.14 Torr (n=21). The a r t e r i a l oxygen tensions did not change s i g n i f i c a n t l y with exercise. Venous and a r t e r i a l oxygen tensions p l o t t e d against per cent saturation (as determined from oxygen content and blood i r o n concentrations) showed a sigmoidal i n vivo oxygen d i s s o c i a t i o n curve ( F i g . 17). Neither a r t e r i a l nor venous hematocrit showed a s t a t i s t i c a l l y s i g n i f i c a n t change during swimming but showed a trend towards an increase i n i n d i v i d u a l s 86 Figure 16 Oxygen consumption of trout expressed per kilogram animal plotted against water v e l o c i t y as a per cent of each individual's c r i t i c a l v elocity. Points at v e l o c i t i e s of less than 25% CV are for animals at rest. One hundred and one determinations are presented on 25 in d i v i d u a l trout. 87 V05 (ML/ftG*MIN) 88 Figure 17 In vivo blood oxygen dissociation curve. Per cent saturation was derived from the measured iron concentration and measured blood oxygen content. The 61 determinations were performed on 20 f i s h and a curve f i t t e d by eye. 89 90 at maximal exercise l e v e l s (see Table 5 i n Appendix ). A r t e r i a l and venous pH tended to decrease with exercise. Blood pressure i n the v e n t r a l aorta increased with increasing i n t e n s i t y of exercise (Fig. 18). Mean pressure i n the v e n t r a l aorta at re s t was 38.8 Torr and rose to a mean of 61.7 Torr at 80-100% of c r i t i c a l v e l o c i t y . The corresponding pulse pressure was 11.5 Torr and 26 Torr. Blood pressure i n the dorsal aorta (Fig. 18) showed a smaller increase over the same speed range. Pulse pressure i n the dorsal aorta at rest was 5.8 Torr and increased to 10 Torr during exercise, while mean pressure increased from 31 Torr to 37 Torr. Blood pressure i n the r i g h t common ca r d i n a l vein at rest was 1.4 ±0.281 Torr (n=4), and increased by an i n s i g n i f i c a n t amount to 1.9 ±0.375 Torr (n=4) at c r i t i c a l v e l o c i t y . No increases i n venous pressure were observed at i n t e r -mediate speeds. G i l l v e n t i l a t i o n volume (Vg) increased with increased oxygen consumption -1 -1 during swimming (Fig. 19).- The mean r e s t i n g Vg was 211.4 ±5.81 ml kg min - 1 - 1 • (n=5) and increased to about 1700 ml kg min at maximal VO^. Per cent u t i l i z a t i o n remained constant at a mean value of 33.0 ±0.43% (n=74) during r e s t and swimming at speeds up to 99% of c r i t i c a l . Blood l a c t a t e i n trout remained about 0.5 umoles/ml during r e s t and swim-ming up to 93% of c r i t i c a l v e l o c i t y . One minute a f t e r fatigue blood l a c t a t e had increased to 5X the r e s t i n g l e v e l (Fig. 20). (3) Recovery from Fatigue Following fatigue blood pressure remained constant f o r about 2 min, then decreased gradually, reaching a r e s t i n g value a f t e r about 120 min. Heart rate (Fig. 21) remained at the maximal value for 10-30 min then declined slowly to 91 Figure 18 D i a s t o l i c and s y s t o l i c blood pressure i n both the dorsal (DA) and ventral (VA) aortae during exercise. Horizontal bars indicate one standard error of the mean and the number above each point indicates the sample s i z e . Horizontal axis i s the swimming speed expressed as a per cent of the individual's c r i t i c a l v e l o c i t y . 92 BLOOD PRESSURE (TORR) 8 * 8 - 8 3 8 & g & L r* r" .• .• 5 93 Figure 19 The relationship of v e n t i l a t i o n volume to oxygen consumption at rest and during exercise of incrementally increased i n t e n s i t y up to and including maximal 60 min sustained exercise. Twenty-one determinations were done on 5 trout. 94 V T J 2 ( M L / K G - M I N ) 95 Figure 20 Blood lactate levels of indi v i d u a l swimming trout at specified swimming speed and following fatigue. Multiple points at a given percentage c r i t i c a l v e l o c i t y are representative of repeat runs on different days. A l l blood samples taken from dorsal aorta except one represented by 0 which i s from ventral aorta. The curve i s a regression l i n e drawn through a l l points prior to fatigue Thirty determina-tions were done on four f i s h . _0> o £ © • • «Q 0.2 1 Q n2 u 3 a 4°- b'u 60. 70. 80. ~ 9 6 ~ l c o . i 2 P e r c e n t a g e C r i t i c a l V e l o c i t y T i m e Post Fat igue (min.) 97 Figure 21 Heart rate during recovery from fatigue. Curve f i t t e d by eye. 99 resting l e v e l over a period of 12-18 hours. Blood lactate levels increased after fatigue, reached a maximum of 6-10 umoles/ml at 2—2-1/2 hours and declined thereafter to resting levels after 11-17 hours. Seventeen to 18 hours after fatigue heart rate, a r t e r i a l and venous pH, A-VO2 difference, cardiac output, and stroke volume had returned to pre-exercise l e v e l s . 100 DISCUSSION The response of heart rate to changes i n swimming speed i n trout was rapid, as reported by Priede (1973) and S u t t e r l i n (1969), There was con-siderable o s c i l l a t i o n i n heart rate before a new plateau was reached after an increment increase i n swimming speed. This o s c i l l a t i o n may be due to hunting i n the control system or i t may be due to variations i n the int e n s i t y of swim-ming of the individuals at the beginning of an incremental increase i n water velocity. Unlike the case i n mammals, where heart rate rises continuously (Cobb and Johnson, 1963; Eklund, 1967; Eklund and Holmgren, 1964; Smith_et a l . , 1952) during periods of prolonged exercise (more than 10-15 min), trout reach a steady state with regards to heart rate after some 15--30 minutes. Blood pressure did not show any evidence of hunting. Both ventral and dorsal a o r t i c blood pressures increased smoothly, peaked, and then decreased gradually to reach a steady state after about 45 min. In mammals prolonged exercise causes a reduction i n systemic blood pressure (Eklund, 1967; Erikson _ e t _ a l . , 1971; Smith _et. a l . , 1952). Eklund_e_f_al. (1964) suggest that t h i s change i s due to increased peripheral vasodilation which results i n a reduced cardiac f i l l i n g pressure and resultant decrease i n stroke volume. The p e r i -pheral vasodilation may be partly that of the cutaneous c i r c u l a t i o n for purposes of thermoregulation (Christensen et a l . , 1942; Z i t i n k and Lorenz, 1969). In f i s h there i s no requirement for cutaneous thermoregulation by vasodilation, therefore the observed blood pressure transient must be due to other factors. The i n i t i a l increase i n cardiac output at the onset of an increase i n exercise in t e n s i t y may be i n excess of the required cardiac output and a slow compensatory decrease may follow to optimize the system, or an 101 increase i n cardiac output which i s faster than decreases i n peripheral r e s i s -tance (in muscle) may be the cause of the pressure transient. The relationship of the variables which govern oxygen transport by the blood to oxygen consump-tion (VX^) i s stated by the Fick equation: V0 2 = Q(A-V02) and Q = HR x s.v. As oxygen consumption increases during increased a c t i v i t y cardiac output can be varied by increasing either heart rate or stroke volume or both. In the present experiments heart rate reaches a maximum at about 90% of c r i t i c a l v e l o c i t y but oxygen consumption continues to r i s e with increasing swimming speed u n t i l c r i t i c a l v e l o c i t y i s reached. At speeds i n excess of 90% of c r i t i c a l v e l o c i t y the increases i n oxygen transport must be by increases i n stroke volume and A-V02 difference. Mean stroke volume at V0 2 max was 1.03 ml kg 1 stroke 1 and mean venous saturation was 16.5%. I t i s unlikely however that either of these variables i s l i m i t i n g V0 2 max since stroke volumes as high as 1.23 ml kg 1 stroke 1 and venous saturations lower than 10% were observed (not i n the same animal). Individuals which were not cannulated had the same V0 2 max as the cannulated individuals i n spite of the fact that the former individuals had hematocrit values of 30% or greater as compared to 25% or less for cannulated individuals. This together with the high a r t e r i a l oxygen saturation values observed i s further evidence that V0 2 max i s not set by the exchange rate or transport of oxygen to the tissue. Studies of blood lactate levels during swimming at increasing speeds showed that blood lactate levels do not increase appreciably at swimming speeds up to 93% of c r i t i c a l . This i s not surprising since electromyography (see Section I) shows that white muscle i s not involved to any extent i n swimming at speeds of less than c r i t i c a l v e l o c i t y i n this group of animals. The large 102 increase i n blood lactate following fatigue suggests either a large washout of accumulated lactate from the white muscle following fatigue or a very rapid production of lactate immediately preceding fatigue. Since white muscle a c t i v i t y was always observed immediately before fatigue (Section I) and blood pressure was maintained following fatigue, i t i s unlikely that the sudden appearance of large quantities of lactate i n the blood immediately a f t e r fatigue i s the res u l t of a large washout of lactate accumulated over a long period of time but therefore due to a large production p r e c i p i t a t i n g fatigue. During swimming at speeds less than 91% of c r i t i c a l v e l o c i t y a r t e r i a l and venous pH dropped s l i g h t l y . Swimming at speeds i n excess of 91% of c r i t i c a l velocity caused a large decrease i n both a r t e r i a l and venous pH. This suggests either an accumulation of C O 2 or some other acid i n the blood. Measurement of whole blood and plasma C O 2 content was attempted by Cameron's method (1971) but i n our hands t h i s method did not work very w e l l on blood sampled from f i s h . From those results which were obtained i t does not seem l i k e l y that a net accumula-t i o n of C O 2 i s the cause of the change i n blood pH. Whatever the cause of the low pH i t did not affect the loading of hemoglobin with oxygen since a r t e r i a l blood remained 98.5% saturated at the maximal exercise l e v e l . Since percent u t i l i z a t i o n remained constant during exercise, v e n t i l a t i o n volume increased i n proportion to oxygen consumption. Ven t i l a t i o n perfusion r a t i o (Vg/Q) was 12:1 at rest and increased to 32:1 at V O 2 max. The capacity rate r a t i o given by the formula VgeCW^/^BO^(Hughes and Shelton, 1962), where < oc±s the s o l u b i l i t y coeff:'cient for 0 2 i n water (W) and blood (B), was 0.6 at rest and increased to 1.8 at maximal exercise. The mean gradient ( 4 P G ) for d i f f u s i o n from one side to the other i n a counter-103 current exchanger such as the g i l l s i s given by the formula APG = l/2(Pj.O + P_0-)-l/2(PQ0„ + P 0 o) (Jones et al., 1970). During exercise the mean gradient I i Z a / v L __ -— for d i f f u s i o n of oxygen increased from 120 Torr at rest to 136 Torr at V0 2 max. This increase i n mean gradient i s reflected i n the change i n transfer factor for oxygen: T0 2 = VO^/APG. The transfer factor for oxygen increased only 5.92X, from 0.013 at rest to 0.077 at V0 2 max, while V0 2 increased by 7.75X. Per cent u t i l i z a t i o n (%U = P j 0 ^ - Pg0 2 x 100) remained constant (33%) of a l l Pl°2 values of V0 2 during exercise, whereas effectiveness for oxygen removal from the water, E = P-j-02 - P E 0 2 x 100 PI°2 " Pv°2 decreased from 42.2% at rest to 36.9% at V0 2 max as a result of a decrease i n P v0 2 > Effectiveness values of 11-30% have been obtained i n other studies for oxygen removal from water by Randall et a l . (1967), who thought that t h i s low effectiveness was the result of the f i s h facing into a water stream. In view of the c r i t i c i s m by Davis and Watters (1970) of opercular cannulation as a method of sampling expired water and observations of backflow of water into the opercular cavity by myself, i t seems more l i k e l y to attribute these low values to technical d i f f i c u l t i e s associated with water sampling. Per cent u t i l i z a t i o n as determined i n my study (33%) i s higher than the values obtained by opercular cannulation (10%) (Stevens, 1968), but lower than those reported for restrained f i s h (oral membrane method, 46%) (Cameron and Davis, 1970). The f i s h i n the Cameron and Davis study had a low p a 0 2 (105 Torr) suggesting that they were not v e n t i l a t i n g adequately, thus increasing the per cent u t i l i z a t i o n . Using the data on cardiac output and mean blood pressures i n the ventral 104 aorta, dorsal aorta, and common c a r d i n a l vein, the resistances to blood flow were calculated. The resistance to blood flow i n the g i l l s was 0.5 Torr min/ ml at rest and decreased to 0.46 Torr min/ml (0.92X) at maximal exercise. Resistance to blood flow i n the systemic c i r c u l a t i o n decreased from a r e s t i n g value of 1.68 Torr min/ml to 0.68 Torr min/ml (0.41X) when maximal exercise was imposed on the f i s h . P u l s a t i l i t y , defined as pulse pressure/mean pressure, increases i n both the v e n t r a l and dorsal aortae despite the increase i n heart rate (1.33X) which, i n the absence of other f a c t o r s , should reduce d i a s t o l i c run-off and hence reduce p u l s a t i l i t y . These anomalous increases i n p u l s a t i l i t y cannot be explained on the basis of the observed changes i n g i l l and systemic resistances and are most e a s i l y described by appealing to an e l e c t r i c a l model (Fig..::22). In t h i s model the heart i s represented by an e l e c t r i c a l generator, vascular compliances by t h e i r e l e c t r i c a l analogue, capacitors, and the g i l l and systemic resistances by r e s i s t o r s . As frequency (heart rate) increases the impedance of the dorsal a o r t i c capacitance (Cda) drops (since impedance varies as the inverse of frequency f o r a c a p a c i t o r ) . At the same time p e r i p h e r a l re s i s t a n c e (Rs) decreases r e s u l t i n g i n a further decrease i n the impedance across which ventral a o r t i c capacitance (Cva) discharges ( p a r a l l e l combination of Cda and Rs) allowing the v e n t r a l a o r t i c capacitance to discharge more each d i a s t o l e . Thus the e f f e c t of increases i n heart rate on capa c i t i v e impedance overcompensates f o r the reduced duration of the cardiac cycle and v e n t r a l a o r t i c pressure and flow p u l s a t i l i t y increase. In the dorsal aorta there i s an increased inflow p u l s a t i l i t y r e s u l t i n g from the increased p u l s a t i l i t y i n the v e n t r a l aorta. Consequently the increased p u l s a t i l i t y i n the c i r c u l a t o r y system during exercise i s rel a t e d to the e f f e c t s of elevated heart rate on the f l u i d impedance of the v e n t r a l and dorsal a o r t i c compliances. 105 Figure 22 E l e c t r i c a l model of f i s h circulatory system (Satchell, 1971). 1 0 6 g i l l r e s i s t a n c e s y s t e m i c r e s i s t a n c e g e n e r a t o r w v v • W W — i v e n t r a l a o r t i c c a p a c i t a n c e d o r s a l a o r t i c c a p a c i t a n c e 107 GENERAL DISCUSSION Steady state aerobic exercise i s a state of dynamic equilibrium. Oxygen i s being consumed by red muscle at a rate dependent on the amount of red muscle which i s being used, which i n turn i s dependent on the amount of power required for that p a r t i c u l a r intensity of exercise. During exercise the respiratory and cardiovascular systems increase the uptake and transport of oxygen to the tissue to meet the increased demand. The amount of oxygen removed from the water per unit volume remains the same as v e n t i l a t i o n volume i s increased. Cardiac output increases as a result of increases i n both heart rate and stroke volume and i n addition, venous saturation i s decreased. Aerobic exercise capacity (VC^ max) does not appear to be l i m i t e d by the a b i l i t y of the r e s p i r a -tory and cardiovascular systems to supply oxygen at 9°C. Different species of f i s h d i f f e r greatly i n th e i r sustained swimming a b i l i t y . The proportion of l a t e r a l red musculature to t o t a l body weight i s related to swimming a b i l i t y i n the species studied. Electromyographic studies on several species of teleosts showed that white muscle i s not continuously active during sustained swimming, but i s active during violent swimming such as that observed before the f i s h fatigues. After fatigue f i s h can s t i l l swim at a reduced speed but under no circumstances were fatigued f i s h observed to struggle, I t therefore seems that fatigue i s a r e s u l t of a loss of function of the white muscle system rather than the red. Since blood supply i s sparse and the white muscle fibers are large (Boddeke et a l . , 1959) i t i s unlikely that white muscle can depend on the circulatory system for supply of nutrients during periods of contraction. This i s supported by the finding of Stevens and.'iBlack (1966) ( i n trout) that mosaic muscle glycogen i s depleted during intense swimming, that 108 lactate accumulates at the same rate as glycogen i s depleted, and that replen-ishing of glycogen a f t e r severe exercise i s very slow. In mackerel which has a white rather than mosaic muscle mass Pritchard (1971) found a near t o t a l depletion of white muscle glycogen at fatigue. I t therefore seems that fatigue of the white muscle i s related to a depletion of i t s glycogen reserve. The rate of appearance of lactate i n the blood following fatigue may be . explained on the basis of a poor blood supply to the mosaic muscle mass. A rapid i n i t i a l washout of lactate as observed may be from the white muscle fibers closest to the blood c a p i l l a r i e s followed by a slower d i f f u s i o n of lactate from deeper areas. A number of problems have come to mind during this study, one of which has been the subject of much work i n mammalian exercise physiology, namely, what i s the nature of the stimulus which i n i t i a t e s the cardiovascular and respiratory responses char a c t e r i s t i c of muscular exercise. Asmussen and Nielsen (1964) found that bicycle ergometer exercise i n humans during occlusion of muscle blood flow produced respiratory and cardiac responses equal to or greater than observed without occlusion even though there was no change i n oxygen consump-tion . They attributed th e i r findings to a chemoreceptor located i n the muscle. Asmussen (1967) proposed that the i n i t i a t i n g "work factor" i n regulation of respiration was of peripheral nervous o r i g i n closely correlated with aerobic metabolism and somehow dependent on the mechanical condition-;of the muscle. Coote _et a l . (1971), working on anaesthetized and decerebrate cats i n which the hindlimbs were induced to exercise (by e l e c t r i c a l stimulation of the ventral roots L6-S1), obtained increases i n systemic blood pressure and heart rate. The responses increased with increasing tension i n the muscle. A b o l i t i o n of muscle contraction by gallamine or section of dorsal roots abolished the 109 response, confirming the r e f l e x nature of the i n i t i a t o r stimulus. Occlusion of the le g c i r c u l a t i o n potentiated the responses. The authors concluded that the stimulus was chemical rather than mechanical and that the receptors were the free endings of group I I I and IV sensory nerve fibers around the blood vessels i n the muscle. McCloskey and Mi t c h e l l (1972) found that cessation of isometric exercise during blood occlusion resulted i n a small decrease i n the cardiovascular and respiratory responses followed by a much larger decrease at the release of occlusion. They concluded that mechano- as well as chemoreceptors were involved. Comroe and Schmidt (1943) found that passive movement of the knee j o i n t i n anaesthetized dogs even when a l l the muscles were cut produces hyperpnoea. The response was t o t a l l y abolished when procaine was injected into the j o i n t . Similar observations were made by Barron and Coote (1973) but the contribution of the a r t i c u l a r receptors to the r e f l e x i s small (1/4-1/2) compared to the response produced by e l e c t r i c a l stimulation of the motor nerves to the muscles (as i n Coote e t a l . , 1971). Kao et a l . (1967) using anaesthetized cross perfused dogs demonstrated both a neural stimulus for i n i t i a t i o n of increases i n cardiac output and v e n t i l a t i o n volume, and a humoral one. The authors suggest the humoral factor to be CO2, since the effect of hypercapnia on an anaesthetized dog i s si m i l a r to that of exercise of the companion dog. Neural and humoral factors were additive. Freyschuss (1970), by paralysing arm muscles of human subjects and record-ing cardiovascular response during attempted handgrip exercise, found that paralysis reduced the response s l i g h t l y but did not abolish i t . Mediation of the cardiovascular response by the central nervous system with possible feed-back from the periphery (muscles) i s suggested. 110 Since f i s h have a simpler circulatory system the controls of cardiovascu-l a r responses to exercise may be more e a s i l y understood. S u t t e r l i n (1969) observed that the i n i t i a t i o n of tachycardia at the onset of exercise was not due to the stimulus of water flow past the f i s h since water flow past non-swimming f i s h produced an i n i t i a l bradycardia. Increases i n heart rate and stroke volume due to increased venous return (by muscle pumping) are ruled out on the basis of the response being too slow, the pressures required are too high (13, Torr ( S u t t e r l i n , 1969) as compared with 2 Torr observed i n t h i s study), and there i s no increase i n venous pressure during exercise i n trout. The p o s s i b i l i t y of receptors i n the muscle i n i t i a t i n g the cardiac and ventilatory responses to exercise has been investigated i n trout. Passive movement of the body i n trout, under MS222 or urethane anaesthesia produced acceleration of ventilatory rate but no change i n heart rate (West, 1975, personal communication). The resting heart rates tended to be high (40-60 min) and may have masked any possible cardiac response. The ventilatory response was eventually found to be a generalized one to any disturbance, such as turn-ing on the apparatus without any contact with the f i s h and s t i l l occurred after transection of the spinal cord and l a t e r a l l i n e nerves.(West, 1975, personal communication). I t i s therefore doubtful whether the i n i t i a t i o n of this response has anything to do with exercise. Another approach might be to examine changes i n heart rate and blood pressure and flow during spontaneous swimming i n a f i s h which tends to swim for only a b r i e f period of time (such as sole and rockfish). The analysis of the time courses of the cardiovascular responses to short periods of a c t i v i t y (5-10 sec) may provide some information on the nature of the i n i t i a t i o n of the exercise response. I l l Likewise the cause of the blood pressure transient observed at the beginning of exercise increments could be investigated by measuring blood flow i n the ventral aorta as well as heart rate and blood pressures during long swimming periods. Blood pressures (dorsal ao r t i c and venous) and blood flow could then be used to calculate peripheral resistance at different times to determine whether the observed blood pressure transient i s the result of an overshoot i n the increase of cardiac output followed by a s l i g h t decrease i n cardiac output, or to an increase i n cardiac output which leads the decrease i n peripheral resistance In siimmafyg ^ cardiovascular and respiratory variables i n trout reach a constant l e v e l (steady state) about 30 min after an incremental increase i n swimming speed. Heart rate, cardiac output, stroke volume, and A-VO^ difference a l l increase with increased exercise in t e n s i t y . Respiratory volume increases i n proportion to oxygen consumption. The cardiovascular and respiratory systems do not appear toe l i m i t maximal oxygen consumption, but rather the maximal oxygen consumption and maximal sustained swimming speed appear to be deterimined by the amount of red muscle i n the body musculature of f i s h . Thus when a l l available red muscle i s recruited during swimming no further increase i n power output can be accomplished without recruitment of white muscle f i b e r s . The use of white muscle fibers results i n a depletion of their glycogen reserves and an accomulation of lactate, one or both of which result i n fatigue of the white muscle. 112 LITERATURE CITED Asmussen, E. 1965. Muscular exercise. In: Handbook of Physiology. Respira-t i o n . Washington, D. C. Am. Physiol. S o c , sect. 3, v o l . I I , chapt. 36, 939-978. Asmussen, E. 1967. Exercise and the regulation of v e n t i l a t i o n . Suppl. I. C i r c . Res. 20, 21: 1-132 - 1-145. 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S. & Lorenz, R. R. 1969. S e n s i t i v i t y of methods for detection of active changes i n venous w a l l tension. Am. J. Cardiol. 24: 220-223. 118 APPENDIX TABLE 5. Cardiovascular and respiratory variables at rest and during exercise and after eighteen hours recovery i n in d i v i d u a l trout. Fish T r i a l Speed %Cv H.R. . -1 min Ca0 2 v% Cv0 2 v% A~V02 v% vo2 ml kg 1 . -1 min Q ml kg . -1 min S'.V. ml kg 1 ST _ 1 Hcta % Sv0 2 % Pv0 2 Torr Pa0 2 Torr Sal % T32 1 15 37 8.4 4.2* 4.2 .68 16.2 .44 18 5 0 a ) 23 138 41 39 8.4 2.8* 5.6 1.10 19.6 .50 20 3 3 a ) 22 137 70 48 8.35 2.55* 5.8 2.05 35.3 .80 19 3 1 a ) 17 136 T70 1 18 44 9.95 6.76 3.19 .57 17.9 .41 22.5 68 35 144 99 78 52 7.96 2.89 5.08 2.26 44.5 .86 18 36 24 114 99 98 53 9.15 .20 8.95 4.30 48 .91 23.5 2 11 133 97 2 * venous blood from ventral aorta (1) per cent saturation calculated assuming that Ca0 2 i s 100% (no iron content done on blood of t h i s i n d i v i d u a l ) . Fish T r i a l Speed H.R. CaC>2 CvC>2 %Cv min - 1 v% v% T65 1 16 38 12.1 9.2 16 38 12.3 10.2 16 38 12.3 9.8 61 40 10.9 5.6 § 81 50 12.1 5.6 T65 2 16 38 8 5.8 81 44 10.9 2.7 100 44 10.7 1.9 16 44 3.4 1.6 T71 1 17 35 9.9 6.8 74 49 10.1 4.2 98 54 10.0 1.3 17 36 8.8 6.0 Table 5 (cont'd) A-V02 V0 2 Q v% ml kg 1 ml kg . -1 . -: min min 2.9 .51 17.6 2.1 .48 22.9 2.5 .50 20 5.3 1.95 36.9 6.5 3.35 51.5 2.2 .56 25.5 8.2 3.63 44.3 8.8 4.74 53.9 1.8 .60 33.3 3.1 .51 16.5 5.9 2.38 40.3 8.7 4.40 50,6 2.8 .49 17.5 S.V. Hcta SvO ml k g - 1 % % ST _ 1 .46 25 81 .60 23.7 81 .51 24 81 .77 23.7 47 1.03 25 46 .67 18.5 64 1.01 20,4 25 1.23 26,5 18 .76 7.3 53 .47 25 68 .82 24,3 42 .93 25,2 13 .49 20.1 68 Pv0 2 Pa0 2 SaO, Torr Torr % 39 140 99 43 138 99 40 142 101 37 131 91 35 124 101 37 120 89 35 122 99 20 123 97 19 134 103 20 139 99 18 134 101 14 136 101 21 138 100 Table 5 (cont'd) Fish T r i a l Speed H.R. CaC>2 Cv0 2 A-V02 VC>2 Q S.V. Hcta Sv0 2 Pv0 2 Pa0 2 Sa0 2 %Cv min" 1 v% v% v% ml kg" 1 ml kg" 1 ml kg" 1 % % Torr Torr % • "! • -1 0 -1 min mm ST T69 1 17 37 9.9 6 3.9 .63 16.2 .43 25.2 60 21 150 99 63 49 10.1 4.8 5,3 1.52 28.7 .59 24.5 48 116 101 101 55 8.8 2.0 6.8 3.92 58 1.05 27.6 23 19 112 100 T69 2 17 55 8.75 3.58 5.17 .59 11.4 .21 17,1 41 21 140 99 91 60 7.76 .50 7,26 2,38 32.8 ,55 22 6 17 138 99 17 55 5.57 1.99 3,58 .58 16.2 .29 10,6 36 131 100 T72 1 19 29 11.14 6.57 4.57 .51 11.2 ,39 23 59 32.5 110 93 70 46 10.35 2.88 7.47 1.24 16.6 .36 23 43 28 124 96 T72 2 19 44 7.96 4.77 3.19 .64 20,1 ,46 17 57 36 133 95 73 50 8.36 4.48 3.88 1.45 37.4 .75 17.4 54 24 120 99 108 55 6.76 — — • 16,5 -- — -19 34 7.40 1.89 5,51 .43 7.8 .23 15,0 26 22 107 100 

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