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Homogeneity of muscle fiber composition in the M gluteus medius of the horse
HOMOGENEITY OF MUSCLE FIBER COMPOSITION IN THE M GLUTEUS MEDIUS OF THE HORSE Craig H. Wood, PhD; 1 Tim T. Ross, PhD;2 Joe B. Armstrong, PhD; 2 David C. Hall, PhDa
SUMMARY
INTRODUCTION
Muscle biopsy samples (80 mg) were removed from 5 sites at the same depths (5 cm) within the gluteus medius of 5 Quarter Horse geldings. Muscle samples were rolled in glycerol, frozen in isopentane for 2 min, pre-cooled in liquid nitrogen (liq. N2) and sectioned to 10 Ix on a microtome cryostat. Sections were stained for myosin ATPase (pH 9A) and succinic dehydrogenase (SDH) activities and fiber types identified. Serial sections from each slide were counted 4 different times on different days to more accurately account for any operator differences. Coefficientof variation between counts on the same site was 3.52, 2.74 and 5.36 for fasttwitch high oxidative (FTH) fibers, fast twitch low oxidative (FT) fibers, and slow twitch high oxidative (ST) fibers, respectively. Muscle fiber composition was not different (P>.10) between sites within the area sampled on the same horse. However, there was a difference (P<.05) in muscle fiber composition between horses.
A number of studies have been conducted in the horse to determine a muscle's fiber type compositions,°.la,14.1sbut few studies have been designed to investigate the homogeneityof specific muscles. Researchers a have reported that muscle fiber composition was highly heritable for dizygous and monozygous human twins (estimated 95%) and that fiber composition did not vary significantly across or between superficial and deep areas of human leg muscle? Small coefficients of variations have been reported between duplicate samples taken from the same and contralateral muscles for myosin adenosine triphosphatase (pH 9.4) activity.14There was no significant variation between superficial and deep areas of a muscle in fiber composition. These researchers concluded that results obtained from small biopsy samples, counting as few as 200 fibers, could be areliable indication of fiber composition for that area of muscle. In contrast, it has been suggested that the proportion of fiber types can vary considerably between sites on the same muscle and between differentmuscles of the same animal. 14A recent studyla examined the variation in the distribution of muscle fiber types in the semitendinosus, soleus, longissimus dorsi and gluteus medius for a 4-year-old female Welsh pony.
Authors address: 1Department of Animal Sciences, College of Agriculture, University of Kentucky, Lexington, KY 40546-0215; ZDepartment of Animal and Range Sciences, New Mexico State University, Las Cruces, NM 88003 and aDepartment of Experimental Statistics, New Mexico State University, Las Cruces, NM 88003. Accepted for publication: March 25, 1988 294
EQUINE VETERINARY SCIENCE
TABLE 1 Coefficient of Variation (%) for Fiber T y p e s W i t h i n a Site a n d M u s c l e N
Coefficient of Variation (%)a
ST
5
FTH
5
5.36 2.74
FT
5
3.52
aCoefficientsof variationswere calculated using 4 differentcountsof the same slide within a site.
The longissimus dorsi and soleus exhibited no variation in muscle fiber type throughout supe~cial and middle areas and those areas closest to the bone. The semitendinosus and gluteus medius increased in slow twitch high oxidative (ST) fibers towards the deep areas of the muscle. However, in the gluteus medius, fast twitch low oxidative (FT) and fast twitch high oxidative (FTH) fibers showed the opposite trend, with the greatest percentage of these fibers found in superficial areas. Results of studies examining the distribution of muscle fiber types across equine skeletal muscle are conflicting. A lack of site difference between muscle fiber types would be advantageous because it would prevent the accumulation of scar tissue from repeated sampling on the same site. The objective of this study was to determine homogeneity of fiber type in the gluteus medius of the Quarter Horse over a specific area and depth. MATERIALS
AND METHODS
Muscle samples were removed from 5 sites within the gluteus medius of 5 Quarter Horse geldings. The initial site of removal was determined by measuring 8 cm posterior to the tuber coxae of the ilium and measuring up 40 °, where the middle gluteal muscle approaches the surface. The remaining 4 sites were determined by measuring 5 cm to the left and right of the initial site and up and down 3 cm (Figure 1). Xylocaine HCI (3 cc) was administered subcutaneously at each site as a local anesthetic. Stab incisions were made with a No. 10 surgicaI blade at each site. The biopsy needle 1°was inserted 5 cm into the belly of the muscle at each site and samples, approximately 80 rag, were removed. One suture was used to close the incision and a topical spray was applied to the surgical area. Samples were coated with glycerol and frozen for 2 minutes in isopentane (2-methylbutane) that was precooled in liq. N 2. Samples were stored in liq. N 2 for histochemical evaluation. Muscle samples were mountedon specimen holders with OCT embedding mediuma and sectioned to 10 ~t on a microtome cryosta¢. Serial sections were mounted on glass microscope slides and stained for myosin adenosine aTissueTek II, Miles Laboratory bTissueTek II, Lab Tek Products Volume 8, Number 4, 1988
Figure 1. Pattern and location where the 5 samples were removed from the m gluteus medius.
triphosphatasel 2 (myosin-ATPase, pH 9.4) and succinic dehydrogenase (SDH) activities. 11Muscle fibers were identified using pfiotomicrographs and classified as ST, FT or FTH fibers. A total of 400 fibers were counted per sample to accurately estimate muscle fiber composition. Each sample per site was counted 4 times on different days to more accurately account for measurement error. Coefficients of variations were calculated based on 4 different counts using the formula CV = 100(s) Y where s is the standard deviation of the 4 different values and y is the mean. A completely randomized block design was used to evaluate the homogeneity of the muscle fiber composition of m gluteus medius with each horse being considered a replicate (block). RESULTS
AND DISCUSSION
The coefficients of variation for duplicate counts within the same site are shown in Table 1. Similar results have been reported by other researchers TMon ST, and FT fibers. However, in the present study the coefficient of variation for FT fibers was higher than those previously reported. The higher CV is probably due to a lack of differentiation by the staining technique. Table 2 shows the mean percentages for ST, FT and FTH fibers over the 5 sites. No difference (P>.10) was observed in muscle fiber composition between sites within the same muscle. The findings of no difference (P>. 10) in muscle fiber composition between sites within the same muscle agrees with other research reports.z,7,14However, there was a difference (P<.05) in the muscle fiber composition between horses. Other research has shown no difference in fiber composition between duplicate samples taken from the same and contralateral muscle, as well as from superficial and deep areas of the 295
chemical analysis not only identifies the 3 major fiber types
TABLE 2 Mean Percentages of Muscle Fiber T y p e s for Site Site 1 2 3 4 5 SE
Type Ia
Type IIAa
18.90 20.90 22.10 23,80 22.10 0.25
47.60 42.50 47.30 46.70 44.60 0.25
Type liBa 33.50 36.60 30.60 29,50 33.30 0.25
aMean percentages with no superscript did not differ(P>.05).
muscle. 14 Also, in man no differences have been reported between muscle fiber composition of superficial and deep areas of muscle/ A study involving the sampling o f a 4-year-old female W e l s h p o n y revealed that the gluteus medias varied in fiber distribution between superficial and deep areas as well as areas close to the bone. 13 However, there were no differences in the fast twitch low oxidative and fast twitch high oxidative fiber distribution between samples removed at the same depth, but there was a difference in slow twitch fibers. Otherresearch has shown that sampling o f the glutens medius from the tuber coxae to the head o f the femur at different depths resulted in variation o f muscle fiber composition between T y p e I and T y p e II fibers? Also, it has been hypothesized that the variation in muscle fiber distribution in a muscle is b y design and might allow different areas o f the muscle to function separately when called upon to perform certain tasks. 1.6 However, there are few physiological data available to support this hypothesis. The present study shows that within a defined area and depth the muscle fiber distribution is uniform. These findings would allow researchers the luxury of sampling a horse
repetitively over a small area of the muscle throughout a training or experimental period without the concern of being close to the previous site, provided sampling depth is the same. Horses would also benefit b y not having to continually be sampled at the same site, which would prevent accumulation o f scar tissue. The use o f muscle biopsies to aid in training and assessing a racehorse still holds some promise, because histo-
but also the amount o f glycogen stored and the aerobic capacity o f a fiber. The results o f this investigation show that no difference in muscle fiber composition existed within the area and depth sampled on the gluteus medius. Through further research, muscle biopsies might, more importantly, help to eliminate potentially poor performers before too much time and expense have been invested.
REFERENCES 1. Botterman BR, Binder MD and Stuart DG: Functional anatomy of the association between motor units and muscle receptors. AmZoo/18:135, 1978. 2. Bruce V, and Turek RJ: Muscle fibre variation in the gluteus medius of the horse. Equine VetJ 17:317, 1985. 3. Edgerton VR, Smith JL and Simpson DR: Muscle fibre type populations of human leg muscles. Histochem J 7:259, 1975. 4. Essen B, Lindholm A, and Thornton J: Histochemical properties of muscle fiber types and enzyme activities in skeletal muscle of Standardbred trotters of different ages. Equine Vet J 12:259, 1980. 5. Guy PS and Snow DH: The effect of training and detraining on muscle composition in the horse. JPhysiol 269:33, 1977. 6. Henricksson-Larsen KB, Lexell J, and Sjostrom: Distribution of different fibre types in human skeletal muscles. I. Method for the preparation and analysis of cross-sections of whole tibialis anterior. Histochem J, 15:167, 1983. 7. Johnson MA, Polgan J, Weightman D, and Appleton D: Data on the distribution of fibre types in thirty-six human muscles. An autopsy study. J of Neurological Sci 18:111, 1973. 8. Komi PV, VitasoloJ, HasuH, Thortensson A, Sjodin B, and Karlson J: Skeletal muscle fibers and muscle enzyme activities in monozygous and dizygous twins of both sexes. Acta Physiol Scand 100:385, 1977. 9. Lindholm A, and Piehl K: Fiber compositions enzyme activity and concentration of metabolites and electrolytes in muscles of Standardbred horses. Acta Vet Scand 15:287, 1974. 10. May K: Percutaneous muscle biopsy needle for the horse---a new design and technique. SouthwesternVet 34(2):161, 1981. 11. Nachlas MM, Tsou K, Souza El), Chang C, and Suligman AM: Cytochemical demonstration of succinic dehydrogenase bythe use of a new p-mitrophenyl substituted ditetrazole. J Histochem Cytochem 5:420, 1975. 12. Padykula HA, and Herman E: Factors affecting the activity of adenosine triphosphatase as measured by histochemical technique. J Histochem Cytochem 3:161, 1955. 13. Raub RH, Bechtel PJ, and Lawrence LM: Variation in the distribution of muscle fiber types in equine skeletal muscles. JEquine Vet Sci 5:34, 1984. 14. Snow DH and Guy PS: Muscle fiber type compositionof a number of limb muscles in different types of horses. Res Vet Sci 28:137, 1980. 15. Snow DH and Guy PS.: Fiber type and enzyme activities of the gluteus medina in different breeds of horses. In: J. Poortmans and G. Niset (Ed.) Biochemistry of Exercise/V-B. P. 275. University Park Press, Baltimore, I98I.
CROSS PROTECTIVE AIIITISERUM Fights Gram negative endotoxemla in horses of all ages and corrects FPT (failure of passive transfer) in foals. Contains high levels of both IgG(t) and total IgG.
296
EQUINE VETERINARY SCIENCE
SUMMARY
INTRODUCTION
Muscle biopsy samples (80 mg) were removed from 5 sites at the same depths (5 cm) within the gluteus medius of 5 Quarter Horse geldings. Muscle samples were rolled in glycerol, frozen in isopentane for 2 min, pre-cooled in liquid nitrogen (liq. N2) and sectioned to 10 Ix on a microtome cryostat. Sections were stained for myosin ATPase (pH 9A) and succinic dehydrogenase (SDH) activities and fiber types identified. Serial sections from each slide were counted 4 different times on different days to more accurately account for any operator differences. Coefficientof variation between counts on the same site was 3.52, 2.74 and 5.36 for fasttwitch high oxidative (FTH) fibers, fast twitch low oxidative (FT) fibers, and slow twitch high oxidative (ST) fibers, respectively. Muscle fiber composition was not different (P>.10) between sites within the area sampled on the same horse. However, there was a difference (P<.05) in muscle fiber composition between horses.
A number of studies have been conducted in the horse to determine a muscle's fiber type compositions,°.la,14.1sbut few studies have been designed to investigate the homogeneityof specific muscles. Researchers a have reported that muscle fiber composition was highly heritable for dizygous and monozygous human twins (estimated 95%) and that fiber composition did not vary significantly across or between superficial and deep areas of human leg muscle? Small coefficients of variations have been reported between duplicate samples taken from the same and contralateral muscles for myosin adenosine triphosphatase (pH 9.4) activity.14There was no significant variation between superficial and deep areas of a muscle in fiber composition. These researchers concluded that results obtained from small biopsy samples, counting as few as 200 fibers, could be areliable indication of fiber composition for that area of muscle. In contrast, it has been suggested that the proportion of fiber types can vary considerably between sites on the same muscle and between differentmuscles of the same animal. 14A recent studyla examined the variation in the distribution of muscle fiber types in the semitendinosus, soleus, longissimus dorsi and gluteus medius for a 4-year-old female Welsh pony.
Authors address: 1Department of Animal Sciences, College of Agriculture, University of Kentucky, Lexington, KY 40546-0215; ZDepartment of Animal and Range Sciences, New Mexico State University, Las Cruces, NM 88003 and aDepartment of Experimental Statistics, New Mexico State University, Las Cruces, NM 88003. Accepted for publication: March 25, 1988 294
EQUINE VETERINARY SCIENCE
TABLE 1 Coefficient of Variation (%) for Fiber T y p e s W i t h i n a Site a n d M u s c l e N
Coefficient of Variation (%)a
ST
5
FTH
5
5.36 2.74
FT
5
3.52
aCoefficientsof variationswere calculated using 4 differentcountsof the same slide within a site.
The longissimus dorsi and soleus exhibited no variation in muscle fiber type throughout supe~cial and middle areas and those areas closest to the bone. The semitendinosus and gluteus medius increased in slow twitch high oxidative (ST) fibers towards the deep areas of the muscle. However, in the gluteus medius, fast twitch low oxidative (FT) and fast twitch high oxidative (FTH) fibers showed the opposite trend, with the greatest percentage of these fibers found in superficial areas. Results of studies examining the distribution of muscle fiber types across equine skeletal muscle are conflicting. A lack of site difference between muscle fiber types would be advantageous because it would prevent the accumulation of scar tissue from repeated sampling on the same site. The objective of this study was to determine homogeneity of fiber type in the gluteus medius of the Quarter Horse over a specific area and depth. MATERIALS
AND METHODS
Muscle samples were removed from 5 sites within the gluteus medius of 5 Quarter Horse geldings. The initial site of removal was determined by measuring 8 cm posterior to the tuber coxae of the ilium and measuring up 40 °, where the middle gluteal muscle approaches the surface. The remaining 4 sites were determined by measuring 5 cm to the left and right of the initial site and up and down 3 cm (Figure 1). Xylocaine HCI (3 cc) was administered subcutaneously at each site as a local anesthetic. Stab incisions were made with a No. 10 surgicaI blade at each site. The biopsy needle 1°was inserted 5 cm into the belly of the muscle at each site and samples, approximately 80 rag, were removed. One suture was used to close the incision and a topical spray was applied to the surgical area. Samples were coated with glycerol and frozen for 2 minutes in isopentane (2-methylbutane) that was precooled in liq. N 2. Samples were stored in liq. N 2 for histochemical evaluation. Muscle samples were mountedon specimen holders with OCT embedding mediuma and sectioned to 10 ~t on a microtome cryosta¢. Serial sections were mounted on glass microscope slides and stained for myosin adenosine aTissueTek II, Miles Laboratory bTissueTek II, Lab Tek Products Volume 8, Number 4, 1988
Figure 1. Pattern and location where the 5 samples were removed from the m gluteus medius.
triphosphatasel 2 (myosin-ATPase, pH 9.4) and succinic dehydrogenase (SDH) activities. 11Muscle fibers were identified using pfiotomicrographs and classified as ST, FT or FTH fibers. A total of 400 fibers were counted per sample to accurately estimate muscle fiber composition. Each sample per site was counted 4 times on different days to more accurately account for measurement error. Coefficients of variations were calculated based on 4 different counts using the formula CV = 100(s) Y where s is the standard deviation of the 4 different values and y is the mean. A completely randomized block design was used to evaluate the homogeneity of the muscle fiber composition of m gluteus medius with each horse being considered a replicate (block). RESULTS
AND DISCUSSION
The coefficients of variation for duplicate counts within the same site are shown in Table 1. Similar results have been reported by other researchers TMon ST, and FT fibers. However, in the present study the coefficient of variation for FT fibers was higher than those previously reported. The higher CV is probably due to a lack of differentiation by the staining technique. Table 2 shows the mean percentages for ST, FT and FTH fibers over the 5 sites. No difference (P>.10) was observed in muscle fiber composition between sites within the same muscle. The findings of no difference (P>. 10) in muscle fiber composition between sites within the same muscle agrees with other research reports.z,7,14However, there was a difference (P<.05) in the muscle fiber composition between horses. Other research has shown no difference in fiber composition between duplicate samples taken from the same and contralateral muscle, as well as from superficial and deep areas of the 295
chemical analysis not only identifies the 3 major fiber types
TABLE 2 Mean Percentages of Muscle Fiber T y p e s for Site Site 1 2 3 4 5 SE
Type Ia
Type IIAa
18.90 20.90 22.10 23,80 22.10 0.25
47.60 42.50 47.30 46.70 44.60 0.25
Type liBa 33.50 36.60 30.60 29,50 33.30 0.25
aMean percentages with no superscript did not differ(P>.05).
muscle. 14 Also, in man no differences have been reported between muscle fiber composition of superficial and deep areas of muscle/ A study involving the sampling o f a 4-year-old female W e l s h p o n y revealed that the gluteus medias varied in fiber distribution between superficial and deep areas as well as areas close to the bone. 13 However, there were no differences in the fast twitch low oxidative and fast twitch high oxidative fiber distribution between samples removed at the same depth, but there was a difference in slow twitch fibers. Otherresearch has shown that sampling o f the glutens medius from the tuber coxae to the head o f the femur at different depths resulted in variation o f muscle fiber composition between T y p e I and T y p e II fibers? Also, it has been hypothesized that the variation in muscle fiber distribution in a muscle is b y design and might allow different areas o f the muscle to function separately when called upon to perform certain tasks. 1.6 However, there are few physiological data available to support this hypothesis. The present study shows that within a defined area and depth the muscle fiber distribution is uniform. These findings would allow researchers the luxury of sampling a horse
repetitively over a small area of the muscle throughout a training or experimental period without the concern of being close to the previous site, provided sampling depth is the same. Horses would also benefit b y not having to continually be sampled at the same site, which would prevent accumulation o f scar tissue. The use o f muscle biopsies to aid in training and assessing a racehorse still holds some promise, because histo-
but also the amount o f glycogen stored and the aerobic capacity o f a fiber. The results o f this investigation show that no difference in muscle fiber composition existed within the area and depth sampled on the gluteus medius. Through further research, muscle biopsies might, more importantly, help to eliminate potentially poor performers before too much time and expense have been invested.
REFERENCES 1. Botterman BR, Binder MD and Stuart DG: Functional anatomy of the association between motor units and muscle receptors. AmZoo/18:135, 1978. 2. Bruce V, and Turek RJ: Muscle fibre variation in the gluteus medius of the horse. Equine VetJ 17:317, 1985. 3. Edgerton VR, Smith JL and Simpson DR: Muscle fibre type populations of human leg muscles. Histochem J 7:259, 1975. 4. Essen B, Lindholm A, and Thornton J: Histochemical properties of muscle fiber types and enzyme activities in skeletal muscle of Standardbred trotters of different ages. Equine Vet J 12:259, 1980. 5. Guy PS and Snow DH: The effect of training and detraining on muscle composition in the horse. JPhysiol 269:33, 1977. 6. Henricksson-Larsen KB, Lexell J, and Sjostrom: Distribution of different fibre types in human skeletal muscles. I. Method for the preparation and analysis of cross-sections of whole tibialis anterior. Histochem J, 15:167, 1983. 7. Johnson MA, Polgan J, Weightman D, and Appleton D: Data on the distribution of fibre types in thirty-six human muscles. An autopsy study. J of Neurological Sci 18:111, 1973. 8. Komi PV, VitasoloJ, HasuH, Thortensson A, Sjodin B, and Karlson J: Skeletal muscle fibers and muscle enzyme activities in monozygous and dizygous twins of both sexes. Acta Physiol Scand 100:385, 1977. 9. Lindholm A, and Piehl K: Fiber compositions enzyme activity and concentration of metabolites and electrolytes in muscles of Standardbred horses. Acta Vet Scand 15:287, 1974. 10. May K: Percutaneous muscle biopsy needle for the horse---a new design and technique. SouthwesternVet 34(2):161, 1981. 11. Nachlas MM, Tsou K, Souza El), Chang C, and Suligman AM: Cytochemical demonstration of succinic dehydrogenase bythe use of a new p-mitrophenyl substituted ditetrazole. J Histochem Cytochem 5:420, 1975. 12. Padykula HA, and Herman E: Factors affecting the activity of adenosine triphosphatase as measured by histochemical technique. J Histochem Cytochem 3:161, 1955. 13. Raub RH, Bechtel PJ, and Lawrence LM: Variation in the distribution of muscle fiber types in equine skeletal muscles. JEquine Vet Sci 5:34, 1984. 14. Snow DH and Guy PS: Muscle fiber type compositionof a number of limb muscles in different types of horses. Res Vet Sci 28:137, 1980. 15. Snow DH and Guy PS.: Fiber type and enzyme activities of the gluteus medina in different breeds of horses. In: J. Poortmans and G. Niset (Ed.) Biochemistry of Exercise/V-B. P. 275. University Park Press, Baltimore, I98I.
CROSS PROTECTIVE AIIITISERUM Fights Gram negative endotoxemla in horses of all ages and corrects FPT (failure of passive transfer) in foals. Contains high levels of both IgG(t) and total IgG.
296
EQUINE VETERINARY SCIENCE