The musculoskeletal system plays a major role in the athletic ability of horses. During show jumping, successful performance depends on an excellent combination of coordination, balance, and strength during the approach and takeoff. Most of the force for takeoff is provided by muscular power generated by the hind limbs.1,2
Muscular power is determined by fiber type composition and CSA of the muscle. Fiber type is usually defined by the MyHC isoforms present. The myosin isoform expressed is the major determinant of the maximum shortening velocity in muscle fibers.3 Generally, muscle fibers are categorized as type I (slow oxidative), IIa (fast oxidative-glycolytic), IId (or IIx; fast glycolytic), and IIad (a hybrid fiber).4-6 The CSA of each fiber type is a measure of power output of a muscle; muscle fibers with a large CSA generate more tension per CSA than muscle fibers with a small CSA.7 In horses, mean CSA of the fibers increases according to type: I < IIa < IId (including the hybrid fiber IIad).6
To maintain power output and prevent fatigue, a muscle depends on oxygen and various enzymes for oxidative capacity and excitability. Oxygen is delivered to muscle fibers via the capillary bed. The capillary supply varies with MyHC fiber type composition; the number of capillaries surrounding oxidative fibers is higher than that surrounding glycolytic fibers.6 A physiologically more relevant description for capillarity should include muscle fiber area because the area provides a more realistic indication of diffusion distances. The oxidative capacity of skeletal muscle can be assessed by measurement of citrate synthase activity. Citrate synthase catalyses the conversion of acetyl coenzyme-A and oxaloacetate into coenzyme-A and citrate during energy production. For maintenance of excitability and force of a muscle, Na+,K+-ATPase is present in the plasma membranes of muscle fibers.8 During excitation of the muscle fibers, action potentials are elicited by a rapid influx of Na+, followed by an efflux of K+. This leads to increases in intracellular Na+ and extracellular K+ concentrations that stimulate Na+,K+-ATPase activity. The pump restores the intra- and extracellular concentrations of the Na+ and K+ and protects the resting membrane's potential to maintain ongoing contractions.9
Skeletal muscle in horses develops quickly during the early postnatal period; the development is associated with changes in MyHC expression,5,10-12 fiber area,13,14 and oxidative capacity.14,15 To our knowledge, evaluations of Na+,K+-ATPase and capillary supply during the early postnatal period in horses have not been performed, but the activity of that enzyme and the distribution of the capillary supply are known to decrease in rat muscle during that period.16-18
In general, training enhances aerobic capacity, power output, and excitability of a muscle by increasing the proportion of oxidative fibers (ie, type I or IIa), CSA, capillarity, and oxidative enzyme activity19 with or without upregulation of Na+,K+-ATPase.9 Results of several studies5,11,12,20-22 have indicated that training influences muscle characteristics of foals and adolescent horses, but those studies focused on training for sport purposes other than show jumping. Training of Warm-bloods does not start before the horses are broken in at 3 years of age, and they are kept on pastures or in stables until that time. Previously, Santamaría et al23 reported results of the effect of early jumping training on jumping technique. That study revealed that compared with untrained horses, trained horses jumped more efficiently as indicated by lower acceleration during hind limb push and lower velocity at takeoff and by the fact that the trained horses positioned their center of gravity at the apex of the jump beyond the fence comparatively less.
The purpose of the study reported here was to investigate whether training for show jumping that is commenced early after birth affects the characteristics of equine locomotory muscle. The MyHC fiber type composition, fiber area, oxidative capacity, and Na+,K+-ATPase content of the gluteus medius muscle (which has a major role in propulsion as extensor of the hip joint) were evaluated in Dutch Warmblood horses that were raised conventionally or underwent jumping training from the age of 6 months.
Materials and Methods
Horses—The study group comprised 19 Dutch Warmblood horses that were expected to have reasonably good future jumping ability on the basis of the breeding values of their sires and dams. These study horses were part of a larger group that was used in a project to develop scientific criteria for selection and effective and injury-free training of show jumpers.24 The horses were kept at the Animal Sciences Group, Wageningen University and Research Centre, The Netherlands. After weaning, the horses were assigned to a control or a training group. Eight horses (mean ± SE body weight at 6 months, 317 ± 12 kg; at 1 year, 418 ± 14 kg; and at 3 years, 552 ± 17 kg) were included in the control group. Eleven horses (mean body weight at 6 months, 299 ± 10 kg; at 1 year, 400 ± 10 kg; and at 3 years, 531 ± 11 kg) were included in the training group.
During winter, both groups of horses were housed in an open stable with an adjacent paddock. During summer, all horses were kept on pasture. The horses were fed concentrates in the morning and afternoon and received additional straw and grass silage before noon. Water was available ad libitum. All horses were checked regularly by a veterinarian and received hoof care. All procedures were approved by the Institutional Care and Use Committee of Animal Sciences Group, Wageningen University and Research Centre, The Netherlands.
Training—Horses in the training group began training after weaning, and this continued until the horses were 3 years of age. Training included free jumping (2 d/wk) and a 20-minute period of exercise in a mechanical rotating (hot) walker (3 d/wk) performed on alternate days. The free-jumping course consisted of a 3-fence combination: a cross rail, vertical fence, and parallel oxer. At the start of training after weaning, the height of these fences was 40, 50, and 60 cm (width, 60 cm), respectively; when the horses were 3 years old, the fence heights were 80 to 90 cm. The distances between the fences were 6 to 7 m. During every session, the horses jumped the 3-fence combination 6 times; the mean speed per round of jumping was 5.6 m/s.
At the start of training after weaning, exercise in the mechanical walker (40 m in diameter) consisted of 6 sessions conducted within a 15-minute period. At 3 years of age, exercise in the mechanical walker consisted of approximately 18 sessions within a 20-minute period. In each session, horses walked (1.7 m/s) for 40 seconds, trotted (3.6 m/s) for 10 seconds, and cantered (5 to 5.6 m/s) for 10 seconds, followed by a period of walking when necessary.
Muscle biopsy procedures—At 0.5, 1, 2, and 3 years, muscle biopsy specimens were obtained percutaneously by use of a Bergström needlea (inner diameter, 4.00 mm) from each horse by 1 individual (EGD) according to the protocol5,25 of Lindholm and Piehl. Specimens were obtained from the deep part of the gluteus medius muscle after horses received local anesthesia via injection with lidocaine hydrochloride. To facilitate specimen collection, an imaginary line was drawn from the coxal tuber to the sacral tuber; at a third of the distance from the sacral tuber, the biopsy needle was inserted perpendicular to the skin as deep as possible (until resistance from the iliac wing was detected). All biopsy samples were stored at −80°C until analyzed.
Immunohistochemistry—Specific monoclonal antibodies were used to identify muscle fiber types according to their MyHC content.5 Monoclonal antibody 219-1D1 (1:25) reacts with type I fibers. Monoclonal antibody 332-3D4 (1:10) reacts with type IIa and IId fibers. Monoclonal antibody 333-7H1 (1:10) reacts with type IIa fibers. Monoclonal antibody 412-R1D5 (1:25) reacts with type I and IId fibers. Transverse serial sections (10 μm) of biopsy specimens were obtained and further processed as described previously.5,26
Assessments of MyHC fiber type composition, fiber area, and capillary supply—From each gluteus medius muscle specimen, a region of ≥ 200 contiguous fibers was collected for fiber typing, determination of fiber type proportions, assessment of fiber area, and counting of capillaries. Fiber perimeters and capillaries were identified by means of the A-amylase periodic acid–Schiff method.27 Images of the stained sections were digitized by use of a microscope and camera and analyzed by use of computer software.b,c
The muscle fibers of all 19 horses were classified as type I, IIa, IIad, or IId on the basis of monoclonal antibody reactions. In a subgroup of the 9 horses (ie, 4 control and 5 trained horses), fiber areas for each type of muscle fiber and the number of capillaries in the selected regions of muscle specimens were determined.
The capillary supply was expressed by the diffusion index (area supplied by 1 capillary [mm2 /capillary]) and calculated from the digital measurements. It was calculated as follows:
Assessment of citrate synthase activity in gluteus medius muscle—A portion of each muscle biopsy specimen was disintegrated ultrasonically (three 15-second pulses; amplitude, 22 Hz) in a buffered solution containing 8.6% sucrose, 2.3mM EDTA, 0.01M Tris, 2M HCl, and 25,000 units of heparin (pH, 7.4) and stored on ice. Citrate synthase activity was measured in duplicate by use of a spectrophotometerd (25°C; wavelength, 412 nm; measurement period of 9 minutes). A solution containing 1mM 5,5′-dithio-2-nitrobenzoate in 1M Tris (pH, 8.1) mixed with 7.5mM acetyl coenzyme A and 2% Triton X-100 was prepared; to establish background activity, this solution was equilibrated in the cuvette and homogenate (1%) was added and mixed. After 2 minutes, 5% oxaloacetate (10mM in 0.1M Tris [pH, 8.0]) was added and measurement continued. Citrated synthase activity was measured as the amount of enzyme in 1 mL of homogenate that converted 1 nmol of oxaloacetate/min. To standardize results, enzyme activity was expressed as milliunits per milligram of protein content of the homogenate.28
Quantification of Na+,K+-ATPase content—For 17 biopsy samples (obtained from horses in the control group [n = 8] and trained group [9]), Na+,K+-ATPase content was quantified by measuring the tritiated ouabain (3H-ouabain)-binding capacity of small muscle samples in the presence of vanadate.29 The values obtained corresponded to the total population of functional Na+,K+ -pumps.9,30 The method has also been validated for measurements in muscle biopsy specimens obtained from foals.31 Briefly, a ouabain concentration of 10−6M was used to allow saturation of most of the ouabain-binding sites.29 Biopsy specimens were incubated for 120 minutes at 37°C in buffer containing 3H-ouabain (0.6 MCi/mL) and unlabeled ouabain (final concentration, 10−6M). One set of biopsy samples was incubated with ouabain at a concentration of 10−3M to allow correction for the unspecific uptake of 3H-ouabain. On the basis of the specific activity of 3H-ouabain in the incubation medium, the amount of 3H-ouabain retained in the muscle samples (pmol/g of tissue wet wt) was calculated after correction for unspecific uptake and isotopic purity.
Statistical analysis—Statistical analyses were performed on general linear model repeated measurements by use of computer software,e with factors such as age (within) and group (control and training, between) An interaction between the age and group factors was considered indicative of an effect of early training. Data are expressed as mean ± SE. A value of P < 0.05 was considered significant.
Results
Development—From 0.5 to 3 years of age, there were significant changes in MyHC fiber type composition (specifically type IIa and IIad fibers) in horses of the control and training groups (Table 1). Also, in both groups, CSA of each fiber type increased (Table 2). The changes in MyHC fiber type composition of the gluteus medius muscle from 1 to 2 years were associated with an absolute increase (8%; P < 0.05) in type IIa fibers and a concomitant decrease (7%; P < 0.05) in type IIad fibers; from 2 to 3 years, changes in MyHC fiber type composition were associated with an increase (2%; P < 0.05) in type IIad fibers. These changes represented a relative change of 39%, 40%, and 16%, respectively. The relative proportions of fiber type I and IId did not change during postnatal development. Type I fibers accounted for approximately 35% and type IId fibers accounted for approximately 25% of the MyHC fiber type composition at all ages.
Myosin heavy chain composition of the gluteus medius muscle (expressed as mean ± SE frequencies [%] of type I, IIa, IIad, and IId fibers) in horses that did (training group; n = 11) or did not (control group; 8) undergo training from 0.5 to 3 years of age.
Mean ± SE CSAs (μm2) of MyHC fiber type I, IIa, IIad, and IId of the gluteus medius muscle in horses that did (training group; n = 5) or did not (control group; 4) undergo training from 0.5 to 3 years of age.
Diffusion index of the gluteus medius muscle was assessed in 5 trained horses and 4 control horses at 0.5, 1.0, and 3.0 years. At each time point, the diffusion index increased significantly (P < 0.05), compared with the previous value, in each group. At 0.5 years, the mean ± SE diffusion in the training and control groups was 0.88 ± 0.07 mm2/capillary and 0.77 ± 0.11 mm2/capillary (× 10−3), respectively; at 1 year, 1.01 ± 0.12 mm2/capillary and 1.16 ± 0.12 mm2/capillary (× 10−3), respectively; and at 3 years, 1.34 ± 0.12 mm2/capillary and 1.20 ± 0.12 mm2/capillary (× 10−3), respectively.
Citrate synthase activity of the gluteus medius muscle was assessed in 11 trained horses and 8 control horses at 0.5 and 3.0 years. At 3 years, citrate synthase activity was increased significantly (P < 0.05), compared with the value at 0.5 years, in each group. At 0.5 years, the mean ± SE activity in the training and control groups was 153 ± 12 mU/mg and 151 ± 13 mU/mg of protein, respectively; at 3 years, the values were 217 ± 14 mU/mg and 198 ± 14 mU/mg of protein, respectively.
The Na+,K+-ATPase contents of the gluteus medius muscle samples collected from trained and control horses were assessed (Figure 1). At 2 years, the values in both groups were significantly (P < 0.05) different from the corresponding value at 0.5 years.
Mean ± SE Na+,K+-ATPase content (pmol/g of wet wt of muscle) of gluteus medius muscle samples (quantified as tritiated ouabain-binding capacity) obtained from horses that did (n = 9; white circles) or did not (8; black circles) undergo training from 0.5 to 3 years of age. *Value of the combined group is significantly (P < 0.05) different from the value at 0.5 years (ie, developmental effect). †Value is significantly (P < 0.05) different from the value at 1 year (ie, effect of early training as indicated by an interaction between the factors of age and group).
Citation: American Journal of Veterinary Research 68, 11; 10.2460/ajvr.68.11.1232
Mean ± SE Na+,K+-ATPase content (pmol/g of wet wt of muscle) of gluteus medius muscle samples (quantified as tritiated ouabain-binding capacity) obtained from horses that did (n = 9; white circles) or did not (8; black circles) undergo training from 0.5 to 3 years of age. *Value of the combined group is significantly (P < 0.05) different from the value at 0.5 years (ie, developmental effect). †Value is significantly (P < 0.05) different from the value at 1 year (ie, effect of early training as indicated by an interaction between the factors of age and group).
Citation: American Journal of Veterinary Research 68, 11; 10.2460/ajvr.68.11.1232
Mean ± SE Na+,K+-ATPase content (pmol/g of wet wt of muscle) of gluteus medius muscle samples (quantified as tritiated ouabain-binding capacity) obtained from horses that did (n = 9; white circles) or did not (8; black circles) undergo training from 0.5 to 3 years of age. *Value of the combined group is significantly (P < 0.05) different from the value at 0.5 years (ie, developmental effect). †Value is significantly (P < 0.05) different from the value at 1 year (ie, effect of early training as indicated by an interaction between the factors of age and group).
Citation: American Journal of Veterinary Research 68, 11; 10.2460/ajvr.68.11.1232
Exercise—From 1 to 2 years, there was a significant effect of early training on Na+,K+-ATPase content (Figure 1). The age-dependent decrease was partly prevented in the training group. At 2.0 years, the content of Na+,K+-ATPase in the trained horses (n = 9) was 132 ± 14 pmol/g of wet weight of muscle; the content in the control horses (8) was 105 ± 8 pmol/g of wet weight of muscle. This difference was significant (P < 0.05). A similar difference in Na+,K+-ATPase content between the 2 groups was also detected at 3 years. Early training had no effect on the MyHC fiber type composition, CSA, diffusion index, and citrate synthase activity of the gluteus muscle.
Discussion
In the present study, the effects of early training during postnatal growth on muscle characteristics in young Dutch Warmblood horses were investigated. We were interested in ascertaining whether previously identified effects of early training on jumping technique among young horses23 are also reflected in changes in muscular characteristics. With regard to developmental changes in the gluteus medius muscle, results of our study indicated that these included a change in MyHC fiber type composition (specifically, an increase in the proportion of type IIa fibers and a decrease in the proportion of type IIad fibers), an almost 2-fold increase in fiber area in all fiber types, increases in citrate synthase activity and diffusion index, and an age-dependent decrease in Na+,K+-ATPase content. In general, the MyHC fiber type composition included a high proportion of type I fibers and low proportion of type IId fibers. Surprisingly, there was no effect of early training on the different muscle variables, except for the Na+,K+-ATPase content.
The development-associated changes in MyHC fiber type composition were primarily detected from 1 until 3 years of age in IIa and IIad fibers; the proportion of type IIa fibers increased and the proportion of type IIad fibers decreased, whereas the proportions of type I and IId fibers remained constant. This suggests a transition of type IIad fibers into type IIa fibers.5,14 Evidence for this concept of fiber transition was provided by results of a study by Eizema et al,32 which indicated that after birth, equine muscle develops towards a slower type of MyHC (according to the so-called nearest-neighbor rule for MyHC fiber type transitions). Additionally, it has been determined that in adult horses, fibers co-expressing IIa and IId MyHC protein predominantly express mRNA for the IIa isoform; these hybrid fibers are probably converting to the type corresponding to the expressed mRNA.33 The prevailing view is that hybrid fibers enable a muscle to fine-tune the wide range of forces and velocities it is required to generate.34
The proportions of type I (35%) and type IId (25%) fibers in the horses of the present study are in contrast with results of other studies12,14,35 involving horses of approximately the same age. In those studies, the proportion of type I fibers ranged from 15% to 20% and the proportion of type IId fibers ranged from 35% to 60%. An explanation for this discrepancy in type I fiber proportions could be a result of differences in tissue sample depth,36,37 staining method (ATPase vs immunohistochemistry),20 or breed. However, in a study20 involving horses of the same breed from which tissue samples were collected at the depth and stained by use of the same method as that used for the present study, proportions of type I and IId fibers were 20% and 38%, respectively, at 22 weeks. The differences between the findings of these similar studies could partly be ascribed to differences in genetic background between both study groups38 because the horses of the present study were especially selected for show jumping according to studbook criteria.
Other studies23,39 have revealed that training results in a jumping technique that is more balanced and requires less force impulses generated by the hind limbs, compared with the jumping technique of inexperienced horses. By relating these results to the MyHC fiber type composition and knowing that adjustments to exercise always occur in the fast to slow direction,40 we expected that early training would result in a more aerobic fiber type composition in the training group, compared with findings in the control group, in the present study. Also, previously reported results of the effects of training of horses at young ages have indicated that the proportion of MyHC fiber type I or IIa increases and that of fiber type IId decreases.12,21,22 The training protocols used in those studies were designed to improve the physiologic adaptation to performance at high speed and were more intense (maximal speed, 16 m/s) than the protocol used in the present study (maximal speed, 5.6 m/s). However, the increase in Na+,K+-ATPase content of gluteus medius muscle in the horses of the present study indicated that the jumping training was intense enough to initiate a reaction in that muscle. Also, in other studies of foals that were trained before weaning, the muscle content of Na+,K+-ATPase was higher in the trained group,31 compared with the untrained group, whereas there were no changes in the MyHC fiber type composition.5 The lack of training effects on MyHC fiber type composition can probably be explained, at least in part, by the adaptive range of each fiber type, which depends on the basal protein isoform profile and hence the position of that fiber type within the fast-slow spectrum.41 At the start of the present study, the horses in both the training and control groups already had an aerobic fiber type composition; therefore, training could not induce an aerobic transition in the fiber types.
Although training did not lead to adjustments in CSA of fibers in the horses of the present study, development clearly resulted in an almost 2-fold increase in CSA of all fiber types. The increase in fiber CSA associated with development is similar to findings of previous investigations in horses.12,14,35 In comparison to findings of those studies, CSA of the individual fiber types in the present study, especially the type IId fibers, were relatively small (3,000 to 7,000 μm2 vs 2,149 to 4,796 μm2, respectively). This is probably a breed-related characteristic associated with the athletic ability of the breed used in the present study because the large fiber size coincided with a high proportion of MyHC IId fibers and the small fiber size coincided with a low proportion of the IId fibers. Typically, training results in increases in fiber CSAs19 and enhances power output. However, the results of early jumping training on the jumping technique23 indicated that trained horses generated less power at takeoff than did the untrained horses. This could suggest that fiber CSA would decrease, but this was not evident in the present study. Probably, the enlargement of fibers during natural maturation of muscle outweighed the effects of training.
In the horses of the present study, the diffusion index also significantly increased (ie, the area that 1 capillary has to supply was increased) with age, but there was no effect of early training. The increase during development was likely caused by an increase in muscle size as capillary proliferation lags behind. This has been identified in rats, in which capillary density decreases during normal muscle growth.17,18 Generally, in adult horses, capillaries proliferate in muscle to improve oxygen supply and removal of waste products in response to training.19,42 In the present study, training did not induce proliferation of capillaries. Again, we speculate that the maturation process outweighed the demand of exercise.
Similarly, in the horses of this report, citrate synthase activity in gluteus medius muscle increased with age but was not affected by training. The increase in citrate synthase activity reflects developmental changes towards a more pronounced aerobic fiber type composition. In a few studies,43,44 a decrease in oxidative capacity of skeletal muscle of horses was detected, but this change occurred before the horses were 1 year old. Between 0.5 and 3 years of age, the oxidative capacity of gluteus medius muscle in the horses of the present study increased by 37%. This increase in activity cannot be explained by an increase in the proportion of oxidative fibers (type I, IIa, and IIad). Possibly, changes in oxidative capacity of the different fiber types would have been revealed by succinate dehydrogenase activity measurements, as in other studies.14,21 Results of previous studies15,19,21,22 on the effect of training in horses have indicated an enhancement of oxidative capacity. Therefore, a greater increase in citrate synthase activity was expected in the training group of the present study, but the increase in citrate synthase activity during development was probably sufficiently large to supply energy for the increased demand associated with training.
The Na+,K+-ATPase content of equine gluteus medius muscle (measured as [3H]ouabain capacity and expressed in pmol/g of wet wt of muscle) in the present study was similar to findings of other studies31,45 in foals (100 to 200 pmol/g of wet wt of muscle). During postnatal development, an age-dependent decrease in Na+,K+-ATPase content was detected in the foals of this report, which is in agreement with results of an earlier study16 in rats and mice. This decrease can be explained by the enlargement of fiber CSAs, which resulted in larger fiber dimensions and a subsequent reduction in surface-to-volume ratio.9 Training partly prevented the age-dependent decrease in the Na+,K+-ATPase content. Training is considered a long-term regulatory factor for upregulation of Na+,K+-pumps and is, in general, associated with a concomitant upregulation of the oxidative potential.9 In the present study, upregulation of the Na+,K+-pump content in the trained group was not associated with an increase in citrate synthase activity. However, one has to take into account that the horses were in a rapid growth phase and that, probably, the increase in citrate synthase activity during development was sufficiently large to supply energy for the increased training-associated demand. However, Na+,K+-pumps account for only 4% to 10% of the total energy turnover of muscle both at rest and during contraction46; thus, it is not surprising that we did not detect an increase in citrate synthase activity attributable to an increased activity of Na+,K+-ATPase in muscle samples from the horses of the present study.
Although early training of young horses induced an effect on the Na+,K+-pump content of locomotory muscle in our study, the question remains whether this effect is permanent and will be beneficial when conventional training starts at 3 years of age. It is known that the initial advantage in jumping technique derived from early training disappears when inexperienced horses also start training.23 The Na+,K+-pump content in equine muscle increases by 20% to 50% with training at different ages,31,45,47 and it would be likely that the Na+,K+-pump content would also become upregulated in inexperienced horses by training that starts at 3 years of age. The question that remains is whether the horses that underwent early jumping training will have an additional increase in Na+,K+-ATPase content when training is continued at 3 years of age and will maintain a higher content of Na+,K+-pumps, compared with horses that have had no previous training prior to 3 years of age. If so, these early-trained horses would have an advantage in muscle excitability, compared with formerly untrained horses.
In addition to the effect on skeletal muscle, it is also relevant to know whether early training has a beneficial or detrimental effect on other components of the musculoskeletal system (eg, articular cartilage, bone, and tendons). In previous studies48-50 of the effects of training at a young age in horses, it was concluded that a certain amount of exercise (when it is well balanced) is essential for development. However, exercise should not be excessive because there are strong indications that too much exercise or wrongly balanced combinations of types of exercise may be deleterious and may even have negative long-term effects.
The results of our study have indicated that training of show jumpers at a young age, which can result in a positive effect on jumping technique,23 induces an increase in Na+,K+-ATPase content of the gluteus medius muscle. It is known that those Na+,K+-ATPase pumps are important for the maintenance of excitability and force of the muscle. The lack of training effects in the other muscular characteristics that were evaluated can partly be explained by the presence of an appropriate (aerobic) fiber type composition of the muscle at the start of training. In addition, it also suggests that the developmental changes in muscle represent sufficient adaptation to meet the demands of the training. Finally, the importance of training for show jumping in horses seems to be more on the level of coordination and balance than on the level of muscle characteristics.
ABBREVIATIONS
| CSA | Cross-sectional area |
| MyHC | Myosin heavy chain |
Custom-made Bergström needle, Faculty of Veterinary Medicine, University Utrecht, Utrecht, The Netherlands.
Database IM500, version 1.2, release 19, Leica Microsystems AG, Heerbrugg, Switzerland.
Qwin standard, version 2.7, Leica microsystems imaging solutions LTD, Cambridge, UK.
Ultrospec 200, GE Healthcare Bio-sciences, Piscataway, NJ.
SPSS, version 12.0.1 for Windows, SPSS Inc, Chicago, Ill.
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