Sprint-training effects on trout (Oncorhynchus mykiss) - Canadian ...

Sprint-training effects on trout (Oncorhynchus mykiss) white muscle structure A. KURTGAMPERL'

AND

E. DONSTEVENS

Department of Zoology, University of Guelph, Guelph, Ont., Canada Nl G 2Wl Received February 8, 1991 GAMPERL, A. K., and STEVENS, E. D. 1991. Sprint-trainingeffects on trout (Oncorhynchusmykiss) white muscle structure. Can. J. Zool. 69: 2786-2790. In mammals, sprint-type exercise protocols induce muscular adaptation different from that caused by endurance training. Although there are many published studies on endurance training in fish, few have examined sprint (anaerobic) training. This study is an examination of whether sprint-training changes white muscle morphology in addition to its previously shown ability to improve trout fast-start accelerationperformance. Rainbow trout (Oncorhynchusmykiss) white muscle was sampled following 4,8, and 12 weeks of sprint training (30 s duration, every 2nd day). White muscle fiber cross-sectional area and perimeter were unchanged by the sprint-training regimen. The volume density of terminal cisternae, T-tubules, mitochondria, and lipid droplets were also not significantly different following training. A formula relating muscle fiber perimeter and area, derived from trout white muscle, appears to describe accurately the perimeter-area relationship for muscle fibers, regardless of species or fiber type. GAMPERL, A. K., et STEVENS, E. D. 1991. Sprint-training effects on trout (Oncorhynchus mykiss) white muscle structure. Can. J. Zool. 69 : 2786-2790. Chez les mammiferes, les protocoles d'exercices de type sprint entrainent des adaptations musculaires qui different de celles qui sont causCes par des exercices d'endurance. Alors que plusieurs publications traitent de l'endurance A l'exercice chez les poissons, il existe peu de travaux sur l'entrainement au sprint (anakrobique).Nous cherchons ici A savoir si, outre 1'amClioration de la performance d'accC1Cration au cours d'un dCpart rapide, effet dCjA connu, l'entrainement au sprint cause aussi des modificationsde la morphologie des muscles blancs. Du muscle blanc de Truites arc-en-ciel (Oncorhynchusmykiss) a CtC prClevC aprks 4, 8 et 12 semaines d'entrainement au sprint (durCe de 30 sec, tous les 2 jours). Le rCgime d'entrainement au sprint ne produit pas d'augmentations de la surface de la coupe transversale ou du pCrimktre du muscle blanc. La densit6 (en volume) des citernes terrninales, des tubules en T, des mitochondries et des gouttelettes de lipides se trouve Cgalement inchangCe aprks l'entrainement. Une formule mettant en relation le pCrimbre et la surface de coupe de la fibre musculaire dans le muscle blanc de la truite semble dCcrire assez justement la relation pCrimktre-surface des fibres musculaires des poissons en gCnCral, indipendamment de l'espkce ou du type de fibre. [Traduit par la rkdaction]

Introduction - In the past few decades extensive work has been carried out on the effect of sustained aerobic training on the physiology and biochemistry of salmonid fish muscle. Swim speeds of less than 3.5 body lengths per second (bus) cause changes in muscle fiber size (Greer Walker and Emerson 1978), capillary supply (Davie et al. 1986), metabolite levels (Davison and Goldspink 1977), and enzyme activity (Johnston and Moon 1980a). Recently, Gamperl et al. (1991) found that 9 weeks of sprint training increased trout acceleration during a shock-induced fast start. Similarly, Pearson et al. (1990) have shown that sprinttrained trout can sustain swimming speeds in excess of 7 bus for longer than untrained controls. However, the physiological adjustments associated with the improved performance are not known. In the present study we investigated whether the morphology of trout white muscle changes as a result of sprint training. Analysis focused on muscle fiber cross-sectional area, volume density of T-tubules and volume density of the terminal cistema because these parameters are good predictors of muscular performance. Volume density of the mitochondria and lipid droplets was also measured as a structural indicator of possible alterations in energy metabolism.

water at 2.5 Llmin. Prior to training, fish were fed to satiation twice daily with Martin's commercial trout pellets. Once training began, control and trained fish were pair-fed, trained fish to satiety, because sprint training depresses food consumption (Gamperl et al. 1988). Photoperiod was maintained at 12 h light : 12 h dark. Fish were trained every 2nd day for a period of 4, 8, or 12 weeks in an oval arena with a path length of 4.2 m and a width of 10 cm. Fish were individually transferred to the training arena and then vigorously chased for 30 s (Black 1957) with an electrified prod (5 V). Following training, fish were placed in an aerated recovery tank. Fish were not fed for 16 h before training. After 4, 8, or 12 weeks of training, eight trained and eight control fish were killed with a blow to the head, and a piece of white epaxial muscle was quickly removed from just posterior to the dorsal fin. After the skin and the most lateral muscle had been trimmed away, the remaining tissue was minced into 2 X 1 X 1 mm blocks. Muscle blocks were initially fixed for 2 h in several changes of 5% glutaraldehyde in Sorenson's buffer (0.015 M, pH 7.4). Samples were washed in several changes of phosphate buffer and postfixed for 2 h in 1% osmium tetroxide in phosphate buffer. Muscle blocks were dehydrated via an alcohol serial-dilution procedure (30% to absolute), cleared in propylene oxide, and embedded in Epon (Epoxy 8 12, Ernest F. Fullam Inc., Latham, N.Y.). During embedding, muscle samples were cut into 1-mmcubes and positioned in cross-sectionalor longitudinal orientation. Semithin (1 km thick) sections were cut approximately at right angles to the axis of muscle fibers, using glass knives. sections were Materials and methods stained with 1 % toluidine blue in 1% sodium borate (Mercer 1963). Rainbow trout (9.06 2.72 (SE) cm) were obtained from I-I~n~ber Photonegativeswere enlarged using a projector, and the fiber perimeter Springs Trout Farm, Orangeville, Ontario, and assigned to one of four was traced. A photonegative of the stage micrometer was projected treatments: control, 4 weeks' training, 8 weeks' training, or 12 weeks' between measurements to calculate magnification. Tracings were training. All tanks (70 L) were supplied with 11.6 + l.l°C aerated well subsequently digitized for cross-sectiona~area and perimeter of the fiber. Approximately 30 fibers from each fish were analyzed, for a total 'present address: Department of Biology, Dalhousie University, of 240 fibers (8 fish X 30 fibers) per treatment-time combination. Ultrathin sections of longitudinal muscle were cut with a diamond Halifax, N.S., Canada B3H 45 1.

*

2787

GAMPERL AND STEVENS

knife. Sections measuring approximately 60- 100 nm in thickness (silver-grey to gold interference colours) were collected on 200- and 300-mesh copper grids. The contrast of the sections was further enhanced by staining with 2% uranyl acetate in 50% ethanol (Watson 1958) and Reynolds' lead citrate (Reynolds 1963). From each block, enough ultrathin sections were obtained to allow electron micrography of 16 micrographs per fish. Therefore, 128 micrographs were analyzed to represent each treatment-time combination. Micrographs were obtained randomly by positioning the viewing screen in the comers of the supporting copper grid (Weibel et al. 1966). Final magnification for the measurement of volume density of the T-tubules and of the terminal cisternae was 36 500 X . The volume density of the intermyofibrillar mitochondria and lipid droplets was measured at 20 000 X . Subsarcolernmal (within 1 pm of the sarcolemma) mitochondria were not included in the analysis because intermyofibrillar mitochondria are considered the best indicators of fatigue resistance in glycolytic muscle fibers (Muller 1976). volume density, the volume of a parameter of interest relative to the total volume of the parameter and its surroundings, was calculated according to the method described by Weibel (1969): where Pi is the number of points falling on the desired parameter, i, and P, is the total number of test-grid points. The volume density of intermyofibrillar lipid droplets and mitochondria (PT = 120) was obtained using a square lattice system with lines every 0.75 pm. A square lattice test system with lines every 0.40 and 0.20 pm was used for measuring terminal cisternae (P - 100)and T-tubules (PT = 437), T respectively. Muscle parameters were compared among groups during each sampling period using a completely randomized design (CRD) with subsampling (Steel and Tonie 1980) This was necessary because multiple measurements were made on each fish. A CRD with subsampling was also performed to detect changes with successive weeks of training. To investigate the homogeneity of fiber area and perimeter distributions, medians (calculated for individual fish) were compared among groups at each time interval using t-tests.

Results The fiber cross-sectional area of trained fish was 2.3, 8, and 7.3% at 4, and l 2 weeks, but these differences were not significant (Table 1). Over the course of the experiment, mean fiber cross-sectional area increased in control and trained groups by 36 and 30%, respectively; ahnost all the increase occurred between 4 and 8 weeks. Changes in muscle fiber perimeter reflected changes in fiber cross-sectional area. Fiber perimeter increased by 10% in control fish and 9% in trained fish between 4 and 12 weeks. Although the muscle fiber perimeter of control fish was slightly greater than that of trained fish at all sampling periods, the difference was not significant (P > 0.13). The equation 7'

[l]

perimeter = 2

where X is the ratio of the major axis to the minor axis of an ellipse, was found to accurately describe the perimeter-area relationship for trout white muscle fibers. A best fit was given by X = 2.103 (Fig. 1). There was no difference in the frequency distribution of myofiber cross-sectional areas between control and sprint-trained trout after 4,8, or 12 weeks of training (e.g., Fig. 2). At all three sampling times, more than 50% of the fiber areas were smaller than 1500 p,m2. At 4 weeks, the only size classes that composed greater than 10% of the total fibers were those below 1500 p,m2 in cross-sectional area. In contrast, at 8 and 12 weeks, fibers larger than 5000 p,m2 constituted between 10 and 15% of the

TABLE1. Median and mean cross-sectional area and perimeter of white muscle fibers from control and trained fish at 4, 8, and 12 weeks Area (pm2) Mean

Median

Perimeter (pm) Mean

-

Median -

-

4 weeks Control Trained

16932130 1655k 126

12442197 14922212

15927.4 15125.5

144212 158210


2274+ 156 21052 148

16692 142 15132 181

17426.2 16225.4

16428 15627


23072153 2151 + 138

13492124 1483295

17225.8 16425.4

14827 15826

NOTE:Values are given as the mean 2 SE. 550

Fiber Area x 1000

(yrn ')

FIG. 1. Relationship between fiber cross-sectional area and fiber perimeter for rainbow trout white muscle fibers. The broken line represents the perimeter-area relationship for a circle. The solid line with open triangles indicates the actual perimeter-area relationship for muscle fibers ranging from 0 to 8000 pm2in cross-sectional area. (n 2 6), the squares indicate this relationship for individual muscle fibers ranging from 10 000 to 16 000 pm2 in cross-sectiona~area, and the dotted line represents this relationship as determined by our fitted equation for an ellipse (see text).

total fibers. At all sampling periods the control group had a slightly greater percentage of fibers in excess of 5000 p,m2 than the sprint-trained group. Median values for fiber perimeter and area were not significantly different between groups after 4, 8, or 12 weeks of training (Table 1). This provides further evidence that sprint training had no effect on the frequency distribution of fiber cross-sectional area or perimeter. As the experiment progressed, the control fish became increasingly heavier and longer than the trained fish (Table 2), but there was no difference in condition factor between the two groups (P = 0.87). No relation existed between fiber crosssectional area and size of the fish at the time of sampling. The volume density of terminal cisternae, T-tubules, lipid droplets, and mitochondria was not significantly different among groups after 8 or 12 weeks of training (Table 3).

Discussion Although trout that are sprint trained using our protocol attain swimming speeds in excess of 9 bus (Pearson et al. 1990; Gamperl et al. 1988) and are clearly exhausted at the end of each

CAN. J. ZOOL. VOL. 69, 1991

2788

fiber diameter (hypertrophy) in contributing to the growth of epaxial muscle. Eisenberg et al. (1974) assumed that myofibers are irregular polyhedrons approximately circular in cross section. Although this description is qualitatively descriptive, is is not accurate in terms of fiber morphometrics (Fig. 1). The present study clearly shows that the relationship between fiber cross-sectional area and fiber perimeter is accurately described by an elliptical function. Although the elliptical function describing the myofiber area-perimeter relationship was derived using the white muscle of trout, it is applicable to other species and fiber types. Using data for the hagfish (Myxine glutinosa L.) (Korneliussen and Nicolaysen 1975) and the air-breathing catfish (Clarias mosambicus) (Johnston et al. 1983), the perimeter values of red, white, and intermediate fibers, calculated using the mean fiber crosssectional area, deviated from the measured perimeter values by an average of 3.2% (range 0.4-10.0%). The volume density of T-tubules (0.25%) and terminal cisternae (1.9%) is in agreement with the values obtained with perch by Akster (1981) and Akster et al. (1985) (2.1% for terminal cisternae, 0.26% for T-tubules), but are decidedly lower than those obtained with rainbow trout by Nag (1972) (4.0% for terminal cisternae, 0.4% for T-tubules). In the present study and those of Akster (1981) and Akster et al. (1985) the volume density of T-tubules and terminal cisternae was determined using point-counting methods. Nag (1972) used the formulas [2] p, = w,x L, x LJS and [3] PC= 2wt x LC x LaIS

-84

, 0 to 500

,

, 1000 to 1500

,

, 2000 to 2500

,

,

, 3000 to 3500

Flbre Area

(

, 4000 to 4500

,

, 5000

,

I

+

2)

'l m

FIG. 2. Fiber cross-sectional area classes (percent frequency) for rainbow trout white muscle following 8 weeks of sprint training. Control fish were not trained. Values for trained fish minus those for control fish are shown in c.

30-s training session, no change in muscle structure was seen over time. This is in contrast to endurance training of fish, which causes hypertrophy of both red and white muscle fibers (Greer Walker and Emerson 1978; Johnston and Moon 1980b), but is in agreement with sprint-training studies in man, where no change in the cross-sectional area of either slow-twitch or fast-twitch fibers is observed (Jacobs et al. 1987; Costill et al. 1979; Thorstensson 1975). At all sampling periods, muscle fibers smaller than 500 pm2 composed between 20 and 25% of the myofibers, whereas fibers greater than 5000 pm2constituted approximately 10%. The continued presence of a large number of myofibers smaller than 500 pm2 implies that the recruitment of fibers was a major contributor to muscle growth at all sampling periods. Luquet and Durand (1970), using the weight-specific DNA content of trout muscle, postulated that in young fish, about 70% of the increase in muscle cross-sectional area was due to hyperplasia. Similarly, Weatherly et al. (1980) reported that below 18-20 cm, the input of new fibers is of much greater importance than increases in

to calculate the volume density of T-tubules and terminal cisternae, where W, is triad width, L, is triad dimension parallel to the fiber axis, LC is the mean longitudinal dimension of the terminal cisternae, La is the length of interfibrilla space per fiber cross-sectional area, and S is the mean sarcomere length. Because Hilliard and Cahn (1961) and Weibel (1979) indicate that point-counting methods afford the greatest accuracy in estimating structural parameters, 0.26 and 2.0% for the volume density of T-tubules and terminal cisternae, respectively, should be used as the standard for fish white muscle. The estimates of Nag (1972) are less accurate because (i) the membrane system components were measured on a limited number of micrographs; (ii) the size of individual stereological parameters varied; and (iii) four stereological measurements, each with individual measurement errors, were used to calculate the volume density of a membrane component. The absence of alterations in the volume density of intermyofibrillar lipid droplets or mitochondria suggests that no changes in lipid or oxidative metabolism accompanied sprint training. This is in agreement with Roberts et al. (1982), who reported increases in glycolytic enzyme activities but not in oxidative enzyme activities following 5 weeks of sprint training in men. Pearson et al. (1990) attributed the large postexercise drop in white muscle lactate content of trained fish to increases in efflux, oxidation, and (or) glyconeogenesis. The absence of increases in intermyofibrillar mitochondria1 volume density following 9 weeks of sprint training suggests that increased lactate oxidation within white muscle could only have occurred through an increase in the efficiency of individual mitochondria. For fish of similar size, maximum swimming speed depends on muscle contraction time and stride length (Wardle 1975), stride length being dependent on the force produced by the axial

GAMPERL AND STEVENS

TABLE2. Length, weight, and condition factor for sprint-trained and control rainbow trout sampled for muscle morphometrical analysis after 4, 8, and 12 weeks of training Control

Trained

4 weeks Length Weight Condition factor 8 weeks Length Weight Condition factor 12 weeks Length Weight Condition factor NOTE: Control fish were not trained. Significant differences betweengroupsareindicatedas follows: *, P < 0.1; **, P < 0.05.

TABLE3. Volume density (%) of white muscle terminal cisternae, T-tubules, mitochondria, and lipid droplets in control (nontrained) and sprint-trained rainbow trout Control

Trained

8 weeks Mitochondria Lipid droplets Terminal cisternae T-tubules 12 weeks Mitochondria Lipid droplets Terminal cisternae T-tubules NOTE: Values are given as the mean 2 SE. The volume density of mitochondria and lipid droplets was measured in the intermyofibrillar area only. Control fish were not significantly different from trained fish for any parameter.

musculature. According to Josephson (1975) and Webb and Johnsrude (1988), the maximum force exerted by a muscle, other things being equal, is determined by the muscle's total crosssectional area, the proportion of muscle occupied by myofibrils, and the amount of overlap between the actin and myosin filaments. In the present study there was no change in new fiber recruitment, no increase in fiber cross-sectional area, and no observable alterations in myofibrillar packing. These observations suggest that alterations in maximum force production were not responsible for the improved acceleration performance of sprint-trained trout reported by Gamperl et al. (1991). Calcium released from the sarcoplasmic reticulum (SR) terminal cisternae initiates contraction, and thus there is a strong positive correlation between the rate of force development and the amount of terminal cisternae; fast muscles have a greater volume of terminal cisternae (Akster et al. 1985). Although sprint training increased acceleration and velocity during stage 1 of a fast start (Gamperl et al. 1991), this increase in performance was not correlated with any detectable increase in the volume of SR terminal cisternae. Increases in the rate of cross-bridge cycling, the remaining

2789

parameter determining contraction speed, possibly caused the observed improvement in fast-start acceleration performance reported by Gamperl et al. (1991). Although trout white muscle M g 2 + - ~ ~ p aactivity se is unaffected by endurance training (Johnston and Moon 1980b), the lack of change in myofibrillar ATPase activity probably relates to the low level of training intensity (3 blls). Belcastro et al. (1984) found alterations in rat skeletal and cardiac muscle actomyosin ATPase following a single bout of exercise to exhaustion, and Thorstensson et al. (1975) reported a 30% increase in M g 2 + - ~ ~ p aactivity se following 8 weeks of sprint training in men. In the study of Gamperl et al. (199 I), because fish were exhausted during each high-intensity training bout, it is not unrealistic to expect alterations in white muscle actomyosin ATPase activity.

Acknowledgements We thank Russ Hopcroft, Sandra Frombach, and Cameron Ackerley for their technical assistance. We are grateful to Dr. R. G. Boutilier and Dr. J. A. Nelson for criticisms of earlier drafts of the manuscript. This project was supported by a Natural Sciences and Engineering Research Council of Canada operating grant to Dr. E. D. Stevens. AKSTER,H. A. 1981. Ultrastructure of muscle fibers in head and axial muscles of the perch (Perca fluviatilis L.). Cell Tissue Res. 219: 111-131. AKSTER,H. A., GRANZIER, L. M., OSSE,J. W. M., and TERLOW,A. 1985. Muscle fiber types and muscle function in the fish. Fortschr. Zool. 30: 27-30. M., SECORD,D., and BELCASTRO, A. N., TURCO'ITE,R., ROSSITER, MAYBANK, P. E. 1984. Myofibril ATPase activity of cardiac and skeletal muscle of exhaustively exercised rats. Int. J. Biochem. 16: 297-303. BLACK,E. C. 1957. Alterations in blood level of lactic acid in certain salmonid fishes following muscular activity. I. Kamloops trout, Salmo gairdneri. J. Fish. Res. Board Can. 14: 117- 134. COSTILL,D. L., COYLE,E. F., FINK, W. F., LESMES,G. R., and WITZMANN, F. A. 1979. Adaptations in skeletal muscle following strength training. J. Appl. Physiol. 46: 96-99. DAVIE,P. S., WELLS,R. M. G., and TETENS,V. M. 1986. Effects of sustained swimming on rainbow trout muscle structure, blood oxygen transport, and lactate dehydrogenase isoenzymes: evidence for increased aerobic capacity of white muscle. J. Exp. Zool. 237: 159-171. DAVIDSON, W., and GOLDSPINK, G. 1977. The effect of prolonged exercise on the lateral muscle structure of the brown trout (Salmo trutta). J. Exp. Biol. 70: 1-12. EISENBERG, B. R., KUDA,A. M., and PETER,J. B. 1974. Stereological analysis of mammalian skeletal muscle. I. Soleus of the adult guinea pig. J. Cell Biol. 60: 732-754. GAMPERL, A. K., BRYANT, J., and STEVENS, E. D. 1988. Effect of a sprint training protocol on growth rate, conversion efficiency, food consumption and body composition in the rainbow trout Salmo gairdneri Richardson. J. Fish Biol. 33: 86 1-870. GAMPERL, A. K., SCHNURR, D. L., and STEVENS, E. D. 1991. Effect of a sprint-training protocol on acceleration performance in rainbow trout (Salmo gairdneri). Can. J. Zool. 69: 578-582. GOLDSPINK, G. 1971. Ultrastructural changes in striated muscle fibers during contraction and growth with particular reference to the mechanism of myofibril splitting. J. Cell Sci. 9: 123- 138. 1983.Alterations in myofibril size and structure during growth, exercise, and changes in environmental temperature. In Handbook of physiology. Sect. 10. Skeletal muscle. Edited by L. D. Peachey. American Physiologial Society, Bethesda, MD. pp. 539-554. GREERWALKER,M., and EMERSON, L. 1978. Sustained swimming speeds and myotomal function in the trout, Salmo gairdneri. J. Fish Biol. 13: 475-481.

2790

CAN. J. ZOOL. VOL. 69, 1991

HILLIARD, J. E., and CAHN,R. 1961. Counting and sizing of particles in transmission microscopy. Trans. Metall. Soc. AIME, 224: 906. JACOBS,I., ESBJORNSSON, M., SYLVEN, C., HOLM,I., and JANSSON, E. 1987. Sprint training effects on muscle myoglobin, enzymes, fiber types, and blood lactate. Med. Sci. Sports Exercise, 19: 368-374. JOHNSTON, I. A., and MOON,T. W. 1980a. Exercise training in skeletal muscle of brook trout (Salvelinus fontinalis). J. Exp. Biol. 87: 177-194. 1980b. Endurance exercise training in the fast and slow muscles of a teleost fish (Pollachius virens). J. Comp. Physiol. 135: 147- 156. L. M., and MALOIY,G. M. 1983. Aquatic JOHNSTON, I. A., BERNARD, and aerial respiration rates, muscle capillary supply and mitochondrial volume density in the air-breathing catfish (Clarias mossambicus) acclimated to either aerated or hypoxic water. J. Exp. Biol. 105: 3 17-338. JOSEPHSON, R. K. 1975. Extrinsic and intrinsic factors determining the performance of striated muscle. J. Exp. Zool. 194: 135- 153. KORNELIUSSEN, H., and NICOLAYSEN, K. 1975. Distribution and dimension of the T-system in different muscle fiber types in the atlantic hagfish (Myxine glutinosa L.). Cell Tissue Res. 157: 1-16. LUQUET, P., and DURAND, C. T. 1970. Evolution de la teneur en acides nuclkiques de la musculature epaxiale au cours de la croissance chez la truite arc-en-ciel (Salmo gairdneri): r8les respectifs de la multiplication et du grandissement cellulaires. Ann. Biol. Anim. Biochim. Biophys. 10: 48 1-492. MERCER,E. H. 1963. A scheme for section staining in electron microscopy. J. R. Microsc. Soc. 81: 179- 186. MULLER,W. 1976. Subsarcolemmal mitochondria and capillarization in soleus muscle fibers m young rats subjected to an endurance training: a morphometric study of semithin sections. Cell Tissue Res. 174: 367-389. NAG,A. C. 1972. Ultrastructure and adenosine triposphatase activity of red and white muscle fibers of the caudal region of a fish, Salmo gairdneri. J. Cell Biol. 55: 42-57. PEARSON, M. P., SPRIET,L. L., and STEVENS,E. D. 1990. Effect of a sprint training protocol on swim performance and white muscle - metabolism during exercise and recovery in rainbow trout (Salmo gairdneri Richardson). J. Exp. Biol. 149: 45-60.

REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17: 208-212. ROBERTS,A. D., BILLETER,R., and HOWALD,H. 1982. Anaerobic muscle enzyme changes after interval training. Int. J. Sports Med. 3: 18-21. STEEL,R. G. D., and TORRIE,J. H. 1980. Principles and procedures of statistics: a biometrical approach. 2nd ed. McGraw-Hill Inc., New York. THORSTENSSON, A., SJODIN,B., and KARLSSON,J. 1975. Enzyme activities and muscle strength after "sprint training" in man. Acta. Physiol. Scand. 94: 3 13-3 18. WARDLE,C. S. 1975. Limit of fish swimming speed. Nature (London), 255: 725-726. WATSON,T. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 5: 475-478. WEATHERLEY, A. H., and GILL,H. S. 1984. Growth dynamics of white myotomal muscle fibres in the bluntnose minnow, Pimephales notatus Rafinesque, and comparison with rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 25: 13-24. WEATHERLEY, A. H., GILL,H. S., and ROGERS,S. C. 1980. Growth dynamics of mosaic muscle fibres in fingerling rainbow trout (Salmo gairdneri) in relation to somatic growth rate. Can. J. Zool. 58: 1535-1541. WEBB,P. W., and JOHNSRUDE, C. L. 1988. The effect of size on the mechanical properties of the myotomal-skeletal system of the rainbow trout (Salmogairdneri). Fish Physiol. Biochem. 5: 163- 171. WEIBEL,E. R. 1969. Stereological principles for morphometry in electron microscopic cytology. Int. Rev. Cytol. 26: 235. 1979. Stereological methods 1. Practical methods for biological morphometry. Academic Press, Toronto. WEIBEL,E. R., KISTLER,G. S., and SCHERLE,W. F. 1966. Practical stereological methods for morphometric cytology. J. Cell Biol. 30: 23. WINEGRAD,S. 1970. The intracellular site of calcium activation of contraction in frog skeletal muscle. J. Gen. Physiol. 55: 77-88.