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DEVELOPMENT AND TISSUE PLASTICITY
1Institut für Zoologie und Limnologie, Universität Innsbruck, A-6020 Innsbruck; and 2Institut für Zoologie, Universität Salzburg, A-5020 Salzburg, Austria
Submitted 4 March 2003 ; accepted in final form 30 April 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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interval training; endurance training; white muscle fiber; red muscle fiber; intermediate muscle fiber
Although many studies focus on the exercise physiology of adult animals, very little is known about the effect of swim training in larval fish. Is this plasticity already established in the earliest larval stages, so that swim training during early larval development results in the development of special "athletes," or is the initial part of the development mainly determined by genetic information, leaving little or no room for physiological adaptations? Environmental factors like oxygen availability and temperature have already been shown to have an impact on fiber size and fiber number (i.e., the cellularity of the muscle) in embryonic, larval, and adult fish, including for example salmonids, herring, and cyprinids (3, 18–21, 23, 24, 36, 39, 41).
In a recent study, zebrafish larvae were exposed to endurance training, and the results revealed that trained larvae consumed significantly less oxygen during swimming than untrained larvae, and trained larvae were much more resistant toward hypoxia (1). These observations suggest that in trained larvae the oxygen transport system and/or the efficiency of metabolic pathways were/was improved.
The oxygen transport system comprises oxygen uptake, convective oxygen transport in the blood, diffusive oxygen transport within the tissues, and oxidative metabolism, all of which might contribute to the observed effects. The present study therefore set out to identify the possible mechanisms that contribute to the improved performance of trained zebrafish. It was hypothesized that an improvement in the convective oxygen transport system, either due to an adaptation in cardiac performance or due to an increase in tissue capillarization, resulting in a reduction of the diffusion distance between blood and the muscle cells, may contribute to a better supply of oxygen to tissues and thus induce a higher resistance to oxygen deficiency. Alternatively, the improvement in the oxygen transport system might be achieved at the cellular level, for example by an increase in mitochondrial density and thus a reduction in the diffusion distance between mitochondria and capillaries within the muscle cells. To make behavioral adaptations to the water current more difficult and to test whether an altered training regime might have a differential effect on swimming muscle at the tissue and cellular level, an interval training was adopted in addition to the continuous training regime.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Experimental design. The training apparatus was a gravity-fed
system consisting of 12 separate treatment tubes, each allowing for fine
adjustment of water velocity. The treatment tubes, in which the larvae were
placed, had an inside diameter of 1.27 cm and a length of 23 cm, resulting in
a total volume of 30 ml of water. Fine nylon mesh on each end of the tubes
retained the larvae within the body of the tube. A filtering apparatus was
placed in the top water reservoir to ensure that large particulate matter did
not enter the gravity-fed water-flow system. A small bolus of dye injected
upstream from the first nylon mesh confirmed that water flow was laminar
through the tube. A desired water velocity was set for each tube by measuring
and adjusting the flow (ml/min) out of each tube and using the equation
Q/t = AV, where Q/t is
the flow (ml/min), A is the cross-sectional area of the tube
(cm2), and V is the velocity of the fluid (cm/min).
Velocities calculated in this manner were initially confirmed by timing the
movement of dye along a given distance in the tube.
To obtain a data set complementing the results of the previous study, the training regime followed the procedure described by Bagatto et al. (1).
Larvae were placed into the training tubes once they reached one of the two
defined developmental stages: "swim-up" larvae, 9 days
postfertilization (dpf) or "free-swimming" larvae, 21 dpf.
1) The swim-up larvae were trained until 15 dpf. At 9 dpf all larvae
had successfully made the transition from yolk-sac utilization to external
feeding. Because the training tubes were closed, the swim-up larvae could not
be fed during active training. Therefore, a regime of night training (18 h)
and daytime feeding was employed. The training regime for this age group was 6
days in length to encompass this nutritional state and avoid the significant
increase in mass that begins at 
17 dpf. 2) The experiments on
free-swimming larvae were terminated at 32 dpf. This age group was chosen to
encompass a period of significant growth. Due to the training system the same
regime of night training (18 h) and day feeding was used. Twenty to thirty
larvae of the same age were placed in each of the 12 tubes with either an
insignificant water velocity of <0.1 body lengths (BL)/s (control fish) or
5 BL/s (continuously trained fish). In addition to the continuous/endurance
training regime used by Bagatto et al.
(1), an interval regime was
employed in which the animals were exposed to a water current of 5 BL/s for 15
min, followed by a 15-min period with stagnant water. This activity/rest cycle
was continued for a period of 18 h. During the remaining 6 h of the day, the
animals were fed ad libitum as described for the other two groups. Temperature
was maintained at 25°C.
PO2 was maintained at normoxic levels by vigorous aeration in the reservoir tanks. Even in free-swimming larvae, the ratio of total larval cross-sectional area to tube cross-sectional area did not exceed 1.0%. Thus solid blocking was not an issue in any age group, based on behavioral observations.
Imaging system. The imaging system consisted of an inverted microscope (Zeiss Axiovert 25) equipped with a 2/3-in. machine vision CCD camera (Hamamatsu C 2400 without infrared cut-off filter). The camera was connected to the luminance input of an SVHS video recorder (Sony S-9500). The VCR was remote controlled via the RS232 serial communication port. The settings of the video recorder as well as the recorded images were digitized by a monochrome frame grabber card (Imagination PX-610) with a personal computer (PIII 450 MHz). The illumination could be reduced to infrared light with a wavelength of 780 or 913 nm to prevent light-induced stress reactions of the animals. The microscope stage was temperature controlled (25°C).
Recording of cardiac activity. For measuring cardiac activity,
larvae were removed from the training tube, anesthetized with 0.08 mg/ml
tricaine (MS-222) neutralized with phosphate buffer, and immediately
transferred into the temperature-controlled incubation chamber of the
microscope stage. A comparison of anesthetized and free-swimming larvae
revealed that MS-222 at these early developmental stages has no influence on
cardiac activity. Heart rate was determined by measuring the time interval for
30 heart beats. Determination of stroke volume, using digital image analysis
(37), followed the method
described by Hou and Burggren
(16) and modified by Jacob et
al. (17). Video sequences of
the ventricle were saved in computer memory. The perimeter of the ventricle
image was outlined manually during end diastole and end systole using a mouse
or a graphic tablet. The perimeter was analyzed with a
"fit-to-ellipse" algorithm, which first calculated the center of
mass of the perimeter and subsequently the best fitting ellipse. The major and
minor axes of the ellipse were extracted and directly transferred into a
Microsoft Excel worksheet for calculation of stroke volume using the formula
for a prolate spheroid (4/3
ab2)
(16). For analysis five
diastoles and systoles were analyzed, and mean stroke volume was calculated as
the difference between diastolic and systolic ventricular volume.
Visualization of the vascular bed. A cast of the vascular bed was
obtained by accumulation of the shifting vectors of moving erythrocytes from a
number of subsequent difference pictures, as described in a previous study
(37). Briefly, by subtracting
the two fields of a video frame, any movement that occurred within the 20 ms
necessary for the acquisition of one field was visualized. The length of the
shifting vectors generated by this subtraction represented a direct
measurement for the velocity of a moving particle, i.e., an erythrocyte in the
vascular system. By accumulation of shifting vectors generated from several
subsequent video frames, a complete trace of the routes on which erythrocytes
moved was obtained. With this method vascular beds of the entire animal can be
visualized noninvasively. Typically, the difference pictures of 30
subsequent images were accumulated to obtain a complete cast of the vascular
bed.
In the tail fin, an essentially planar structure, vascular casts were digitally binarized and skeletonized. The length of the skeleton line provides the total vascular length in the tail fin. This method could not be used for the analysis of the vascularization in the muscle region, because the projection of vessels in the two-dimensional image produces artifacts. Therefore, vascularization of muscle tissues was assessed by determination of the vascularization index. In the axial tail muscle, vascular casts from 15- and 32-dpf zebrafish were patched from multiple images. In parallel to the spinal cord, three lines were placed equidistantly from each other between the spinal cord and the animal's back, beginning from the primary vascular loop in the tail and ending at the anus of the animal. Vessel intersections with the lines were counted by peak detection of the luminance spectrum along each line and summed from all lines to obtain a relative index of the vascularization.
Red blood cell count. After accumulation of sufficient difference pictures to obtain a cast of the vascular bed, blood vessel diameter of defined sections of the vessels was measured, and the volume of a defined blood vessel section was calculated. Subsequently, erythrocytes within the defined section of the vessel were tracked on individual images, and a series of images showing individual blood cells was stored to the computer hard drive. Blood cells were automatically detected by their characteristic gray-scale value (color thresholding), marked with a red cross, and counted. The results were controlled by optical inspection of an image series, and misinterpretations were eliminated. This determination was repeated five times for each animal to take into account possible clustering of blood cells.
Electron microscopy and morphometry. For quantitative analysis, samples from the axial muscle of the anal region were dissected out and immersion-fixed in Karnovsky's paraformaldehyde-glutaraldehyde fixative (22) diluted to one-half concentration with cacodylate buffer, postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded into Epon 812 epoxy resin (for further details, see Ref. 40). For quantitative analysis, five individuals per experimental and age group were used. Ultrathin cross sections were cut on a Reichert Ultracut S ultramicrotome. All sections were taken at the same location and angle at the level of the anus. Sections were mounted on coated 200-mesh copper grids, contrasted with aqueous solutions of uranyl acetate and lead citrate, and viewed in a Zeiss EM 910. For stereological analysis of volume densities, 12 pictures per muscle fiber type and cross section with appropriate magnification were taken in consecutive frames of the copper grids, thus analyzing all red and intermediate fibers available on the individual cross section. The following cellular components were included: mitochondria, myofibrils, sarcotubular system, and sarcoplasm (for further details, see Ref. 32).
Data analysis. For comparison of two means, statistical significance was evaluated by unpaired Student's t-test. For multiple comparisons, one-way ANOVA followed by Student-Newman-Keuls multiple comparison test was used. Overall swimming activity effects on cellular components of the various muscle fiber types were tested for significance with one-way ANOVA followed by Student's t-test using the software package Minitab. Differences were considered significant at P < 0.05.
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RESULTS |
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Figure 2 summarizes the results obtained from the older larvae, trained between 21 and 32 dpf, and measured at 32 dpf. Again, no statistically significant differences in cardiac activity could be observed between trained and untrained animals.
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To test whether the swim activity induced an increase in the
oxygen-carrying capacity of the blood, the number of red blood cells per
nanoliter of blood was determined after the training period. As shown in
Fig. 3A, at 15 dpf the
concentration of red blood cells was 400 cells/nl of blood, and there was
no difference between the three experimental groups. In larvae of 32 dpf, the
concentration of red blood cells had increased to 600–700 cells/nl
blood, but again there was no significant difference between endurance-trained
or interval-trained animals and control animals raised in stagnant water
(Fig. 3B).
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Vascularization of tail fin and axial muscle tissue was analyzed from casts
of the vascular system. Compared with controls, the total length of blood
vessels in the tail fin was more than twice as high in continuously trained as
well as in interval-trained animals trained between 21 and 32 dpf
(Fig. 4A). In fish
trained at an earlier developmental stage, this difference was not apparent.
Similarly, the vascularization index determined in the muscle tissue caudal of
the anus in continuously trained as well as in interval-trained animals was
not different from control animals for the first age group trained between 9
and 15 dpf, while in animals trained between 21 and 32 dpf the vascularization
index increased by 30% (Fig.
4B).
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Regarding cellular components, stereological analysis of the muscle tissue revealed significant differences in red and intermediate muscle fibers. As shown in the schematic drawing of Fig. 5, the bulk of muscle is represented by white muscle fibers. Red muscle fibers are located superficially with a wedgelike insertion in the region of the horizontal septum. Intermediate fibers are located between red and white muscle fibers. The results for the younger age class trained between 9 and 15 dpf are summarized in Table 1. In red muscle fibers of both training groups, the volume density of mitochondria increased significantly (endurance training: P = 0.0076; interval training: P = 0.0008) during the training period. The volume density of myofibrils in turn was significantly reduced (endurance training: P = 0.048; interval training: P = 0.0074), so that fiber diameter remained relatively constant. No significant changes were observed in the volume densities of sarcotubular system and sarcoplasm.
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In intermediate muscle fibers, only an interval-training regime induced pronounced changes in the amount of all cell components analyzed. Compared with controls, mitochondrial content increased with a P = 0.014 and sarcoplasm with a P = 0.02. In contrast, the volume densities of sarcotubular system and myofibrils were significantly reduced (P = 0.0052 and P = 0.011, respectively). No significant changes were observed in white muscle fibers.
In the second age class, trained between 21 and 32 dpf, the training response was limited to mitochondria. Table 2 shows the summarized values for various components and muscle fiber types, respectively. Red and intermediate muscle fibers showed significant increases in mitochondrial volume density for both training protocols, endurance and interval training (red muscle fibers: P = 0.0045 and P = 0.01, respectively; intermediate muscle fibers: P = 0.0015 and P = 0.0004, respectively). In white muscle fibers, the volume density of mitochondria appeared to increase in endurance-trained and interval-trained animals, but these differences were not significant (endurance training: P = 0.062; interval training: P = 0.073).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Although Gallaugher et al. (8) conclude that in adult fish cardiovascular plasticity induced by endurance exercise is limited, several studies, including even this study, reported an increase in heart muscle mass and size as a consequence of continued swim training (6–8, 10, 11). In addition, an increase in axial muscle capillarity was found in some salmonids as well as cyprinids (4, 32, 35), in cyprinids not just in red and intermediate muscle fibers but also in white muscle fibers.
In larval zebrafish, endurance training was found to result in a significant reduction in energy expenditure during swimming (trained larvae consumed significantly less oxygen during swimming than untrained larvae) and also enhanced survival during exposure to extreme hypoxia in all age groups tested (1). As shown by Wieser (47), fish larvae generally swim more economically in hypoxic water (i.e., their cost of transport is lower) than in normoxic water. Thus the reduction in the critical PO2 could be due to an exercise-induced improvement in convective oxygen transport system, either due to an adaptation in cardiac performance, or due to an increase in tissue capillarization, resulting in a reduction of the diffusion distance between blood and the muscle cells.
The results of the present study, however, do not give any evidence for a modification in cardiac performance in either endurance-trained or interval-trained zebrafish larvae. Neither heart rate nor stroke volume was different in trained larvae compared with control animals. Similarly, in adult chinook salmon, although increasing relative ventricular mass, high-intensity exercise training did not affect cardiac output or stroke volume (8). This suggests that in fish, plasticity of cardiac muscle with respect to exercise training is quite limited. Despite an increase in cardiac muscle mass in trained animals, which has been reported for several fish species (see above), the physiological performance does not change.
Convective oxygen transport is also dependent on the oxygen-carrying capacity of the blood, but again, the concentration of red blood cells was not affected by either continuous or interval training in swim-up or free-swimming larvae. Similarly, no change in arterial oxygen content or in hematocrit was observed in adult trained chinook salmon (8), while in rainbow trout an increase in hematocrit was reported (11). It can be concluded that in zebrafish larvae, an exercise-induced increase in metabolic activity does not stimulate convective oxygen transport, either by stimulating cardiac activity or by increasing oxygen-carrying capacity of the blood.
However, hypoxic conditions do affect those parameters. In zebrafish larvae raised under chronic hypoxia (PO2 = 8.7 kPa) starting at 1 dpf until 15 dpf, cardiac output was increased and the concentration of red blood cells was significantly elevated compared with normoxic controls (17, 38). This clearly shows that the control mechanisms bringing about an elevated red blood cell concentration during hypoxic stimulation are established at this time of development in the zebrafish. Given that a lack of oxygen is the appropriate stimulus for the enhanced production of erythrocytes (30), it can be concluded that the increased metabolic activity during continuous or intermittent swimming at 5 BL/s did not reduce tissue PO2 to a level adequate for the stimulation of erythropoiesis. Accordingly, oxygen supply to the tissue, which is achieved by bulk diffusion at this stage of development in small larvae like zebrafish larvae or even Xenopus larvae (17, 31, 42), is sufficient not only for resting conditions but also for prolonged muscular activity.
As already mentioned, tissue capillarization is another variable contributing to oxygen supply of working muscle. An increased vascularization is often observed in trained muscles, and hypoxia appears to be the stimulus eliciting this response (25). In the older larvae, total length of the tail fin blood vessels as well as the vascularization index of the axial tail muscle increased significantly after 11 days of continuous or interval swim training, suggesting an improvement in the blood supply to the muscle cells. Although this response was highly significant in the older larvae, in larvae trained between 9 and 15 dpf no differences could be detected. As in the older larvae, also in adult fish like rainbow trout (4) and some cyprinid species like Chalcalburnus chalcoides mento, Chondrostoma nasus, and Leuciscus cephalus (32, 35), endurance exercise training induced a significant increase in muscle capillarity. These results indicate that angiogenesis is stimulated in fish during exercise training, but this effect is not observed in very early developmental stages, i.e., within the first 2 wk of development in the zebrafish. It is suggested that, at this earlier stage of development, a modification of the vascular bed is not yet possible or not necessary. This would be in line with the observation that during the first 2 wk of development, the circulatory system is not necessary to supply oxygen to the tissues under resting conditions (17, 31). Another possible explanation would be that the stimulus was not adequate to elicit a response at this early stage of development.
An increase in capillary density in muscle tissue increases diffusional surface area and thus reduces the mean distance between capillaries and mitochondria (43, 45). Morphometric analysis revealed that in muscle tissue, capillaries are designed to match maximal oxygen demand (43, 46) and that the mitochondrial content can be used as an index for the oxygen demand of the tissue. The observed increase in capillarization therefore suggests that the aerobic capacity of the muscle tissue is enhanced in trained animals and will meet the demand for oxygen indicated by the observed significantly higher mitochondrial volume density.
According to the continuous-training protocol, the swimming activity will
only be fueled by aerobic metabolism, even for the interval-trained fish. If
lactate had accumulated during the 15-min period of swimming activity, another
15 min would have been available for recovery. Due to the extended time course
of recovery from lactacidosis, this appears not to be sufficient to eliminate
accumulated lactate (26,
27,
44). Thus the intermittent
training protocol would have resulted in a more or less continuous
accumulation of lactate over a period of 18 h, which does not appear
possible. In addition, significant glycogen stores, which would be necessary
to fuel anaerobic metabolism, could not be detected in larval zebrafish (M.
Drexel, G. Krumschnabl, and B. Pelster, unpublished results).
Finally, an improvement in the oxygen transport system and aerobic potential can be achieved at the cellular level by an increase in mitochondrial content, resulting in a reduction of diffusion distances. In the present study, both training regimes induced an increase in mitochondrial volume densities of red and intermediate muscle fibers in the second age class. This feature is well known from adult fish, although species specific (5, 32). In adult fish like rainbow trout and Danube bleak, sustained swim training can induce an increase in aerobic capacity even in white muscle fibers (4, 35), but in our study no significant changes were observed, although mitochondrial density of white muscle fibers of endurance-trained and interval-trained animals appeared to be higher than in untrained animals. In mammals, endurance exercise may even induce a fiber-type transition in white muscle cells (29). Such an adaptational change in white muscle fibers would enable the fish to use the more profitable aerobic pathways for energy production, prevent local fatigue, and enhance the removal of metabolic products like lactate.
White muscle of swim-up larvae were unaffected by the training regimes, as were the intermediate muscle fibers in endurance-trained fish. It was just the red muscle that was affected by both training regimes. That white muscle did not show that trend of increased mitochondrial volume densities may be due to a rather high content of these cell components per se (8% compared with 3% in the older age class). This does not hold for the intermediate muscle population with a similar amount of mitochondria in controls (11% in swim-up larvae compared with 10% in the second age class). It may be concluded that, during this early stage of development, it is mainly the red and intermediate muscle portion recruited at the endurance swimming activity employed in the present study.
The results presented here demonstrate that muscle cell plasticity occurs even in very early developmental stages, i.e., within the first 2 wk of development, and that interval training provoked similar effects to a continuous training regime. Larval zebrafish forced into either continuous or interval swimming activity show no adaptation with respect to cardiac activity and oxygen-carrying capacity in the blood. Thus, at the level of total oxygen transport in the blood, neither endurance nor interval training provokes any improvement during the first 32 days of development. In later developmental stages (i.e., between 21 and 32 dpf), however, continuous and interval training enhances both the axial muscle tissue and tail fin capillary supply. In addition, mitochondrial density is increased significantly in red and intermediate muscle fibers in both age classes. Even white muscle is affected by both training regimes in the older age class, although the changes were not significant. Such increases in capillarization and mitochondrial density enhance the aerobic capacity of the muscle. These adaptations at tissue and cellular levels, therefore, may well contribute to the increase in swimming efficiency and the increased resistance to hypoxic conditions observed in trained zebrafish larvae (1).
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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