J. exp. Biol. (1977). 70. i - «Vith 2 figures

nted in Great Britain

THE EFFECT OF PROLONGED EXERCISE ON THELATERAL MUSCULATURE OF THE BROWN TROUT

(SALMO TRUTTA)

BY WILLIAM DAVISON* AND GEOFFREY GOLDSPINK

Muscle Research Laboratory, Department of Zoology,The University, Hull HU6 yRX, England

(Received 6 January 1977)

SUMMARY

Hatchery-reared brown trout (Salmo trutta L.) were exercised con-tinuously for periods of several weeks at swimming speeds of 1-5, 3-0 and4-5 body lengths/s and their rates of growth were determined. Changes inthe major muscle constituents were determined by biochemical analysis andchanges in muscle cells using histochemistry and electron microscopy.

At the lowest speed the fish grew much more rapidly, and converted foodinto fish flesh much more efficiently than their controls kept in still water.Large stores of glycogen and lipid were built up. Gross changes wereobserved mainly in red muscle cells, with enlargement of the mitochondriabeing very noticeable.

The fish swimming at intermediate speed showed greater growth than thecontrols, although the energy expended in swimming against the watercurrent led to an inefficient food conversion rate. Large stores of glycogenwere built up, but lipid levels fell, suggesting that this was the major fuelfor swimming at this speed. Changes in all the muscle fibre types wereobserved.

The energy required to maintain the fish in the water flow at the highestspeed was so great as to have serious detrimental effects on the fish, andmany did not survive. Those which did survive showed signs of grossdepletion.

INTRODUCTION

Exercise, and especially that regarded as athletic training, has received muchattention in recent years from mammalian physiologists. Many studies have shownthat prolonged exercise causes hypertrophy of muscle fibres (Goldspink, 1964;Walker, 1966; Kowalski et al. 1969; Goldspink & Howells, 1974), changes in fibretype composition (Barnard et al. 1971; Kiessling et al. 1975) and produces greaterresistance to fatigue (Holloszy et al. 1971; Baldwin et al. 1973). Supporting tissuessuch as the heart and blood system also change to be better able to supply the muscleswith nutrients and oxygen (Richter & Kellner, 1963; Rabinowitz & Zak, 1972).

• Present address: Department of Zoology and Comparative Physiology, University of BirminghamP.O. Box 363, Birmingham B15 aTT, England.

2 W. DAVISON AND G. G'OLDSPINK

The study of exercise in fish has received much less attention. Several studiesbeen carried out on sprint exercise (Bainbridge, 1958; Johnston & Goldspink, 1973 a;Wardle, 1975). These authors have shown that fish can rapidly accelerate up toextremely fast speeds, but that they can only maintain such velocities for a fewseconds. Other studies have looked at prolonged exercise where animals have beenforced to swim for several hours. Black et al. (1959, 1962) observed glycogen break-down with exercise as did several other workers (Beamish, 1968; Pritchard, Hunter &Lasker, 1971; Johnston & Goldspink, 19736). The other type of exercise, that oftraining animals for several weeks in order to observe adaptive changes caused bythe work load, has been much less studied. Several workers have observed the changesoccurring in salmonid fishes during the seasonal migration upstream to the spawninggrounds, although during this period the animals did not ingest any food, thus anyadaptive changes were masked by the effects of depletion (Brett, 1973). Training offish has mainly been carried out on members of the SaLmonidae. Hammond & Hickman(1966) showed that training trout resulted in an increase in the maximum swimmingspeed, this possibly being due to an increased tolerance to lactic acid build-up, asshown by Hochachka (1961). Trout have been shown to actively choose runningwater in preference to still water (Davidson, 1949).

The only work on the training of marine fish has been carried out on the coalfishby Greer Walker (1971) and Greer Walker & Pull (1973). These authors reportedthat exercise produced an increase in growth rate, provided that the water speed wasnot too great, and suggested roles for the different muscle types based on the degree ofhypertrophy of the fibres at different speeds.

As the restocking of ponds and rivers is being carried out on an increasinglylarge scale, it is important that information is obtained about the effect of transferringfish from one environment to another. The present work was carried out in orderto observe the effects of training trout at known water velocities, especially theeffects of training hatchery trout which had previously experienced only very slowlymoving water.

MATERIALS AND METHODS

Brown trout (Salmo trutta), 12-15 cm in length, were obtained from the YorkshireWater Authority Trout Hatchery at Pickering in North Yorkshire. They were allowedto acclimatize in an aquarium for 2 weeks before experimentation at 12 °C and a10/14 h light/dark photoperiod, with a daily diet of chopped liver.

EXERCISING METHODS

A flume of similar construction to those described by Greer Walker (1971) andJohnston & Goldspink (1973) was used in this study (Fig. 1). This consisted of atrough of dimensions 250 x 250 x 2500 mm through which a controlled flow ofwater could be moved. Water travelled through the trough from an upper reservoirto a lower one from which it was pumped back up to the upper one. Water speed wascontrolled by the rate of pumping, the gradient of the tunnel and the height of theweir. Flow rate was determined using a miniflow probe (George Kent (Stroud) Ltd.flTemperature was maintained at 12 °C by means of a refrigeration unit situated in

The effect of prolonged exercise

Upper grid

Shaded area

Lower grid(electrified)

Adjustable weir

Turbulence gridsWater pumps

Refrigeration unitLower reservoir

2h.pJ

Fig. i. The flume, the apparatus used to exercise the fish. The fish were kept in the troughbetween the upper and lower grids. Usually they remained in the shaded area.

the upper reservoir. Fish which fell back on to the lower restraining grid in theflume were induced to swim again by a mild electric shock (3-5 V a.c.) deliveredfrom the grid.

Three sets of experiments were carried out; 28 days at 1-5 body lengths per second(b.l./s), 28 days at 3-0 b.l./s, and 14 days at 4-5 b.l./s. These were started by placing thefish (in groups of ten) into the flume at a low water speed and allowing them tobecome acclimated to the new environment. This was taken as the time when theanimals began to accept food introduced into the flume and was usually 2 or 3 days.The water speed was then steadily increased until the desired speed was attained.At the lowest speed this was achieved after a few hours, at the intermediate speedafter 2 days, while at the highest speed, the final water flow was reached only after 7days. The trout were fed on chopped liver introduced into the top of the flume,and any excess liver was collected at the bottom with a hand net. All animals werefed once per day to satiation. The experiments were terminated by removing thefish from the water flow and killing them by a blow to the head. Blocks of muscletissue were then quickly removed from a point on the lateral line immediately belowthe dorsal fin for histochemical studies. The fish were then plunged into liquidnitrogen in order to prevent any post-mortem changes in the composition of themuscle.

4 W. DAVISON AND G. GOLDSPINK

HISTOCHEMISTRY

Muscle blocks were mounted on metal chucks and rapidly frozen in liquid freon(dichlorodifluoromethane I.C.I. Arcton 12) at —158 °C. Sections 7-12 /im thickwere then cut at — 20 °C in a cryostat (Bright Instruments Ltd.). These sections,mounted on coverslips, were stained for myofibrillar adenosine triphosphataseby the method of Padykula & Herman (1955) modified by Guth & Samaha (1970).The following modifications were made to the technique to make it suitable fortrout muscle. The sections were not prefixed as this was found to denature theenzyme rapidly. Preincubation was carried out at room temperature at a pH of 10-2for time periods ranging from 2 to 15 min (Johnston, Ward & Goldspink, 1975).Incubation was for 20 min at room temperature. Using this procedure the pinkmuscle could be differentiated from the white muscle tissue, although the red musclewas always unstained due to the instability of the red muscle enzyme at high pH.This tissue was stained by using techniques for lipid (Sudan Black B) and succinicdehydrogenase (Nachlas et al. 1957).

Using the slides thus produced, it was possible to count the total number of redfibres in the superficial muscle layer. This was achieved by using a projection micro-scope and a square grid system. The numbers of pink and of white fibres were notcounted. The diameters of all three types were measured using a graduated eyepiece.The diameter was found by taking the mean of two measurements on each fibre, thesecond being at right angles to the first. One hundred fibres were measured for eachfibre type for each animal.

ANALYSES

Frozen fish were allowed to warm up to — 20 °C. Red and white muscle sampleswere then dissected out while still frozen, after which they were quickly cooledagain in liquid nitrogen. Several workers, e.g. Brandes & Dietrich (1953) and Blacket al. (1962) have shown that the chemical composition of teleosts alters with positionalong the length of the body, so all samples were taken from the same area, at theposition of the dorsal fin.

Water content of the tissue was found by drying to constant weight in a vacuumoven at 60 °C. Protein nitrogen levels were estimated by the Biuret method ofGornall, Bardawill & David (1949) after initial digestion in 30% KOH. Followingextraction by the method of Seiftere* al. (1949) the Carroll, Longley & Roe (1956)anthrone method was employed to determine the amount of glycogen stored in themuscle tissue. Lipid concentrations were determined by first extracting from themuscle tissue using the chloroform-methanol-water method of Bligh & Dyer (1959)modified by Hanson & Olley (1965), and then assaying with a Biochemica TestKit (Boehringer Mannheim) no. 15991.

ELECTRON MICROSCOPY

Small pieces of red and white muscle were fixed in glutaraldehyde and osmiumtetroxide and then embedded in Araldite. Ultrathin sections were then cut on a

The effect of prolonged exercise 5

Reichart ultramicrotome and mounted on collodion-coated grids. The grids werestained with uranyl acetate and lead citrate and then viewed using a JOEL JEM 7 Aelectron microscope.

RESULTS

Those fish swimming at 1*5 and 3-0 b.l./s quickly became adjusted to their newenvironment, with an average of 20 % of the animals becoming exhausted and fallingback on to the lower grid within the first 2 days of each experiment. This was notconsidered to be abnormal, as all experiments of this nature carried out on troutby the authors have yielded figures similar to this. Three experiments were carriedout at 4-5 b.l./s. In the first experiment all of the fish had become exhausted by theseventh day. By the seventh day in runs 2 and 3, the day on which the final speed wasreached, 50% had been removed. In the second experiment, at 14 days, this figurehad risen to 80 % with the remaining fish appearing very unhealthy and so the run wasterminated. At 14 days the third experiment had lost 70%.

Growth of the fish is shown in Table 1. Control fish, kept in still water, did notconsume much food and did not change much over the experimental period in eitherlength or weight. Fish swimming at the two lower speeds gave very different resultsfrom the controls. Those animals at 1-5 b.l./s appeared to be very healthy, whichwas shown at the end of the experiment by an 11 % increase in length and a 79 % gainin weight. At 3 b.l./s a much smaller length increase was observed (1-4%) and theweight had risen by only 7%. Animals surviving 14 days at the highest speed werevery unhealthy, and although they consumed large quantities of food there was aslight decrease in length and a 21 % loss in weight.

The efficiency of conversion of ingested food into fish flesh was recorded at thetwo lower speeds (Table 2). At the lowest speed, the conversion of food was muchmore efficient than for the controls, whereas at the intermediate speed the energyrequired for swimming greatly reduced the efficiency.

At all speeds involvement of the red muscle fibres was indicated by an increase inboth number and size (Table 3). A difference in fish from different years can immedi-ately be seen. The lowest-speed experiments were carried out in the spring of 1974with a single batch of animals. The red muscle was characterized by a relatively lownumber of fibres with a large mean diameter. Exercise produced hypertrophy inthis muscle by a small increase in fibre diameter (14%) and a large increase in thenumber of fibres (40%). In order to have the experiments at the same time of year,and with fish of equal age, the two higher-speed experiments were carried out in thespring of 1975, again using a single batch of animals. The red fibres in this groupwere characterized by a large number of small diameter fibres. Exercise produced amuch smaller increase in the number, 18% at the intermediate speed and 20% atthe highest. Increase in muscle mass at the intermediate speed was associated with a31% increase in fibre diameter, while at 4-5 b.l./s the result was complicated, sincehypertrophy of the fibres was apparently affected by the poor nutritional state of theanimals in the group, resulting in only a 13-5% increase.

Pink muscle fibres did not show any hypertrophy at the lowest speed, but at thetoitermediate speed there was an 11 % increase in fibre diameter. At the highest speed,this result was again apparently a consequence of the poor state of the fish, and a

6 W. DAVISON AND G. GOLDSPINK

Table i. Changes in length and weight of the fish over the l-month experimental period

Control (30)

Experimental (25)

Control (30)

Experimental (23)

Control (30)

Experimental (5)

Length (cm)Weight (g)

Length (cm)Weight (g)

Length (cm)Weight (g)

Length (cm)Weight (g)

Length (cm)Weight (g)

Length (cm)Weight (g)

Initial

12-2824-23

12-942725

12-2824-26

13-15314812282426

I3-I52500

Final

12-6325-O5I4-3848-94

12-2523-84

1333338012-252384

13-03I9-85

Change(%)

280331

11137960

- 0 2 4-I-70

i-37737

— 0-24- 1 70

- o - 8 o— 20-76

Overall change,exptl.-control (%)

8-337629

—

161907

—

- 0 5 6—1906

12% decrease in pink fibre diameter was observed. White muscle fibres increased insize at all swimming speeds; by 7% at the lowest, by 16-5% at the intermediate, andby 6*5 % at the highest. The low value at the highest speed was believed to be due tothe lack of available energy matter rather than a direct effect of swimming at thisspeed.

At the lowest speed the fish almost doubled their body weight. This was reflectedin the lateral muscles by large increases in glycogen and lipid, and by a rise in theprotein concentration (Table 4). Glycogen stores were very much increased, risingby 419% m t n e r ed muscle and by 395% in the white. Lipid changes were smaller at57% and 33% respectively. Protein concentrations rose by 12% and 40% in redand white muscle. The large increase in lipid stored in the red muscle was reflected bya drop in the water content of this tissue. There was no change in the water contentof white muscle.

At the intermediate swimming speed, the glycogen stores were 251 % and 290 %greater than the controls for red and white muscle respectively. Although a lessmarked effect than at the lowest speed, these still represented large increases. Themajor source of energy at this speed appeared to be lipid; although glycogen wasbeing accumulated, the lipid was being utilized, both that ingested and that whichwas already present stored in the muscle. The amount of lipid stored in the musclefell by 14% and 27% in red and white tissue respectively. The water content ofboth muscles did not change and neither did the protein content.

The poor condition of the animals at the highest speed was indicated by the verylow concentrations of stored material in the muscles. Glycogen had dropped by 77 %and 84% respectively in the red and white muscles, the levels being hardly detect-able. Much of the stored muscle lipid had been mobilized, as shown by an 85%drop in red muscle and a 60 % drop in the white. The water content of both muscleshad increased, while protein levels in the red muscle had dropped only slightlycompared with the white tissue which had lost 30% of its initial value.

Journal of Experimental Biology, Vol. 70 Fig. 2

Fig. 2(a). Longitudinal section of part of a red muscle fibre from a control trout. Severalmyofibrils can be seen. Note the size of the mitochondria and associated lipid droplet. X 25 000.(6) Longitudinal section of part of a muscle fibre from red muscle of an exercised fish (lowestswimming speed). Note the enlargement of the mitochondria and lipid droplets. The darkgranules are stores of glycogen. X 17500.

W. DAVISON AND G. GOLDSPINK (Facing p. 7)

The effect of prolonged exercise 7

Table 2. Food conversion rates of trout

Food conversion rate is expressed here as a ratio of wet weight of food ingested over the weight increasein wet weight of the fish. The smaller the figure, the more efficient the conversion of available food intofish flesh (Ricker 1968).

Conversion rate

Controli-Sb.l.s"1

30 b.l. 8"1

11

318

•6544•48

ELECTRON MICROSCOPE STUDIES

The ultrastructure of the red and white myotomal muscles of teleost fish has beenwell documented (Nag, 1972; Patterson & Goldspink, 1972). The brown troutexamined in the present study had a similar structure. At all speeds, exercise did notappear to affect the structure of the white muscle; that is, any changes that did occur,such as hypertrophy of the fibres, were not noticeable under the electron microscope.In contrast to this, very noticeable changes occurred in the red muscle fibres at allspeeds. At the lower speeds, large changes in the amounts of stored metabolitescould often be seen. Fig. z(a, b), from a control fish (a) and a fish exercised at thelowest speed (b), shows these changes. In addition to this, exercise affected the oxida-tive capacity of this muscle which was seen by marked enlargement of the mito-chondria (Fig. za, b). This happened at all speeds, even the highest.

DISCUSSION

During the course of this study, in almost every experiment, approximately 20 %of the trout died. This always occurred during the early stages when the animalswere at a low swimming speed. It appeared that these animals were simply unable toadjust to their new environment of continuously flowing water and fell back ex-hausted on to the lower grid. It is well known that hatchery-reared salmonids do nothave a high survival rate when transferred into running water. Needham & Slater(1945) found that 66% and 44% of two populations of rainbow trout were lost aftertransplantation, and Miller (1951) noted that 33% of a 3-year-old, and nearly allof a 2-year-old cutthroat trout population died within the first 2 weeks. Many factorsare involved in the survival of hatchery trout after relocation, such as availability offood, acceptance of the new food, predation, etc. This study indicates that one factor,the change in environment from the relatively still water of the hatchery pond to theflowing water of the stream, probably accounts for about 20% of the population.It would be interesting to study the effects of exercise on very young trout fry todetermine the mortality occurring in these fish and to see whether this inability toadjust to a flowing water environment was a feature possessed by specific members ofa population.

The control fish kept in still water did not grow well and some of the fish actuallylost weight. This was in marked contrast to the animals kept in running water at

k '5 b.l./s, which ingested much food, grew very rapidly and made a much moreEfficient use of the food than the controls (Table 2). After the 28-day period they had

0 m i -vl 2 :;?=;

e: + 1 + * + 1 r\ nfl b * Q l E

The effect of prolonged exercise 9

almost doubled their weight. This appeared to be achieved by much protein depo-sition in white muscle and by a build-up of large stores of both lipid and glycogen.In addition, the mitochondria increased in size quite significantly. This is because thered muscle is the active tissue at this speed, although the hypertrophy was alsoobserved at the other two speeds indicating that it was also active there.

At the intermediate speed, 3 b.l./s, much food was ingested but very little growthresulted. At this speed, which was sufficient to involve the white muscle, most of theingested material was being utilized to provide energy for swimming. The majority ofenergy was apparently being provided by the oxidation of lipid, as indicated by thehypertrophy of the mitochondria. In addition, whereas glycogen was being built up,the amount of lipid was decreasing despite the amounts taken in each day. Thus itwould appear that lipid is the major fuel at this speed in both red and white muscle.The large drop in the lipid content of the white muscle is interesting as this muscletype is very anaerobic. However, it may be that at this speed, which is much lowerthan the critical speed of 4-5 b.l./s for these fish (Davison, 1976), the white muscleis not being used to any great degree and thus if the very small fibres of the mosaicare the ones in use (Davison & Goldspink, 1977), enough oxygen could be reachingthe white tissue to allow oxidation of lipid to occur. In a recent report, Cooper &Hudlidca (1976) showed that long-term stimulation of a mammalian muscle in-creased the capillary density allowing much more blood to reach the muscle fibres.This may well have occurred in trout white muscle at this speed and is at presentbeing investigated.

At the highest speed many of the animals were unable to survive. Depletion of thetissues was very obvious, and at this speed glycogen appeared to be very important,as indicated by the almost non-existent levels. This is presumably because at such ahigh speed more energy would be required than could be obtained using the amount ofoxygen available in the blood, thus anaerobic respiration would be required. Muchprotein had been mobilized, but only in the white muscle, leaving the red tissuerelatively unaffected. This may have been connected with the production of lactatein the white muscle, as protein breakdown is known to have a role in buffering theacidic effects of lactic acid (Kutty, 1972). This, however, is a wasteful process asvaluable energy in the form of amino acids is lost by excretion. This process is alsovery unhealthy for the fish as at this speed the white muscle would be doing most ofthe work, so if the muscle-protein, which is mainly contractile, was being mobilized,then the fish would become progressively less able to work against the water current.Thus the large drop in protein content is no doubt a final resort as far as energyproduction is concerned.

Black et al. (1962) have shown that at high speeds the glycogen content of whitemuscle in salmonids rapidly goes down. As the animals used for analysis were stillalive after 14 days of high-speed swimming, then all stored glycogen in the whitetissue would have been used up long before the end of the experiment. So to keepfunctioning, the white muscle must have been obtaining energy from other sources.It is possible that the protein depletion was due to the animal obtaining energy thisway, though as breakdown of protein involves the consumption of much oxygenthis seems unlikely. It is probable that fuel was received as glucose via the bloodsystem. If this was so, then the proposal of Wittenberger (1972, 1973) that red

io W. DAVISON AND G. GOLDSPINK

muscle (in addition to other stores such as the liver) supplies the white with carbo-hydrate might in this case be valid, as the red muscle would have little locomotoryfunction at this speed.

A weight drop of 21% was observed in trout at the highest speed. In starving fishthis is by no means severe, as several authors have noted much greater depletion;e.g. Creach & Serfaty (1965) noted that starved Cyprinus carpio lost 47% of theirinitial weight, and Boethius (cited by Love, 1970) reported that an eel had lastedmore than 4 years without feeding, losing no less than 76% of its body weight.However, the major difference between these studies and the present one is thatthey were carried out on quiescent animals. Starved fish become very inactive andthus save energy. The fish in the flume were forced to be active, and it is believed thatthe observed weight loss was critical, and it is unlikely that they would have survivedmuch longer, as indicated by the protein measurements. Certainly it would seemthat an active animal cannot afford to lose as much weight as a quiescent one.

The number of fibres in most mammalian muscles is fixed at birth so that anygrowth of a muscle is achieved by enlargements of the existing fibres (Goldspink,1972). An increase in fibre number can occur, but only if the muscle is subjectedto an extreme load (Rowe & Goldspink, 1968; Hall-Craggs, 1970). This situationdoes not occur in fish muscle tissue. Greer Walker (1970) studied changes in the redand white myotomal muscles of the cod Gadus morhua with growth and found thatthe total number of fibres increased with the size of the fish. He also noted that thefibres tended to reach a maximum size after which increase in muscle mass wasachieved by an increase in fibre number. In the present study similar observationswere also made in the red muscle of the trout. The two populations of fish had verydifferent growth patterns when kept in the still water of the hatchery ponds, onehaving fewer larger diameter fibres and the other having many smaller diameterfibres. Exercise produced hypertrophy of the red muscle in both groups, but indifferent ways. The size of the cells appeared to have almost reached maximum sizein the first group; although they were smaller than neighbouring white fibres, thiswas necessarily so, to allow efficient aerobic metabolism. Hypertrophy was thusattained by a large increase in the number of fibres. In the second group the fibreswere much below the maximum size and so in this case increase in red muscle masswas achieved mainly by simple increase in the myofibril content of the existing cells.Exactly how the increase in cell number is achieved is not exactly clear. In grosslyoverloaded mammalian tissue, increase in cell number is thought to be achieved bysplitting of existing ones, producing two smaller daughter cells, although this is anabnormal increase. At the light microscope level in trout, it appeared as though some ofthe cells were in the process of splitting. Under the electron microscope very smallfibres could occasionally be found in both red and white muscles. This tends toindicate that normal fibre number increase may be brought about by the productionof new fibres and not by the splitting of existing ones.

It is apparent from the results presented here that exercise does have profoundeffects on the growth of muscle tissue of fish and the quantification of these changesat the cellular level is obviously important. The interplay between diet and the levelof exercise is complex and further work is needed to establish the optimum levelsfor other species of fish, particularly those which are farmed commercially.

The effect of prolonged exercise 11

This work was carried out while one of the authors (W. D.) was in receipt of anNERC studentship.

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12 W. DAVISON AND G. GOLDSPINK

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