Histometric study on different fibre types in muscles of forelimb of the buffalo S C Dubal 1, K N Vyas 2 and Y L Vyas 3 Department of Anatomy,

College of Veterinary Science and Animal Husbandry, G.A.U., Campus Anand, Anand 388 001 (Gujrat ) India Key words: Fibre types, diameter, muscles, forelimb, buffalo

ABSTRACT The distribution of fibre types and their diameters, and fibre density were determined from succinic dehydrogenase activity in the muscles of forelimbs of apparently healthy six male Surti buffalo (4 6 month old with average 64.54 + 7.74 kg body weight and 5 10 years old adult with 518.4 + 10.93 kg body weight). The per cent population of red, intermediate and white fibres did not differ significantly (P> 0.05) between the age groups. The fibre diameter was significantly (P < 0.05) more in the adults than in the calves. Further, the fibre density was significantly (P < 0.01) lower in the adults than in the calves. Within the muscle the white fibres were significantly (P < 0.05) wider than the intermediate and red fibres. The muscles of proximal part of the limb had significantly more red fibres and fibre density than the distal one. It was concluded that the muscle profile of the forelimbs was more closely dependent upon the workload than the age of the animal and the buffalo was more suitable for heavy load bearing with slow speed. INTRODUCTION Muscles act as motor for power generation and are therefore composed of fibres with different contractile and metabolic properties depending upon the functional status in the animal body (Close, 1972). It is known that histochemically succnic dehydrogenase localization distinguishes three types of muscle fibres - red, intermediate and white. The red fibres are high-oxidative with slow-twitch and can sustain the activity for a long period, while the white fibres are low-oxidative with fast activity and can sustain the activity for a short period. Further the force generated ------------------------------------

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1

Assistant professor, 2 Retd. Principal and 3 Professor

is directly proportional to cross-sectional area of the fibre (Ikai and Fukunaga, 1968 ). Study on distribution of fibre typesand their diameters, therefore, will help in understanding the work ability of the animals Since the fore limbs support the body weight and initiate forward and upward movements (Rooney, 1969), it will be desirable to study the fibre type profile in the muscles of limbs of draught animals. There are few reports on the digital muscles of the buffalo and bullock ( Vyas and Vyas, 1986). The present work was therefore carried out in the muscles of forelimbs of the buffalo so that the study may help in constructing the structural bases of draught power. MATERIALS AND METHODS Muscle samples of forelimbs were obtained from apparently healthy six male Surti buffalo (4 6 months old calves with average 64.4 674 kg body weight and 5 10 years old adults with average 518.4 10.93 kg body weight ). The samples were immediately put in an ice - box. Within half an hour transverse sections of 10 m in thickness were obtained by cryostat microtome. Fibre types were distinguished after treating them for localization of succinic dehydrogenase (Nachlas et al., 1957). The red fibres stained intensively, intermediate fibres were moderately stained and the white fibres were relatively unstained. From the sections the percent population and the diameter of different fibre types, and fibre density were obtained. Data was analyzed statistically (Snedecor and Cochran, 1980 ). RESULTS

The findings of present work have been summarized in Tables 1 and 2. The mean values of per cent population of fibre types did not differ significantly (P> 0.05) between the age group. However, there was significant regional variation in the fibre

distribution. The muscles of proximal part of limbs had significantly (P < 0.05) higher percentage of red fibres than those of the distal one. Further, among the muscles of proximal parts, the muscles of pectoral girdle showed significantly (P < 0.05) high percentage of the red fibres. In general, the physiological extensor and antigravity (resisting the gravity in the quadruple standing position) muscles contained more red fibres than their antagonists. The fibre diameter was significantly (P < 0.05) more in the muscles of adult animals than the calves. The increment, irrespective of fibre types, was 24 3 % in the muscles of proximal part of the limbs which was significantly (P < 0.05) lower than that of the distal part (38 3 %). Further, the increase was more in the white fibres than others. In general, there was non significant variation in the diameters of fibre types of different muscle groups of the calves, however, the diameter of white fibres was significantly (P < 0.05) more in the muscles of distal parts of the limbs of adult buffalo. The fibre density was significantly (P < 0.01) lower in the adult animals than the calves. The fibre density of proximal part of the limb was also significantly (P < 0.05) higher than the distal part. DISCUSSION The main purpose of this study was to determine the distribution of fibre types and their diameters, and the fibre density to correlate the findings with workload. In human beings, the functional capacity of skeletal muscle (endurance, contraction

velocity

and

strength),

have

been correlated with the distribution of fibre

types and fibre diameter (Tesch and Karlson, 1985). The endurance and contraction velocity are reflected by the red fibres and to some extent the intermediate fibres. The white fibres in general and the diameter in particular reflect the strength. The muscles of proximal

part of the forelimbs in general and the physiological extensors and antigravity muscles particularly, are constantly in isometric contraction (Rooney, 1969). These muscles have to sustain their function for a long duration. Hence theses muscles had significantly more red fibres than their opponents. This finding is in close agreement with that of Vyas and Vyas ( loc.cit. ). The present study showed that the distribution of fibre types was not affected by the age of the animals. However, training or exercise is known to influence the oxidative activity in the muscles of horses ( Roneus et al.,1992; Lopez-Rivero et al., 1992). The fibres that are recruited during exercise are influenced by the type, speed and duration of exercise and that the recruitment follows a specific order from Type I through Type IIA followed by Type IIB (Lindholm et al, 1974; Snow et al.,1982 ) The present findings showing no significant difference in muscle fibre types between the calves and the adults , could be attributed to inactivity of the adult buffaloes before slaughter. Muscles of buffaloes have to support weight and movements of the body, which require an increase in the strength of muscles with increased body weight and movements. Since the cross-sectional area of muscle fibre is directly proportional to the force exerted by the fibre, the fibre diameter of different fibres were therefore more in the adult buffaloes than the calves. Further, these activities were constantly in practice, the fibre diameter could remain more in the inactive adult buffalo than the calves. These observations are in close agreement with those of Vyas and Vyas(loc.cit).

Among the different fibre types, the white fibres had significantly large diameter followed by the intermediate and red fibres. According to Gottlieb et al. (1988) in horses, the oxidative capacity of the muscle is of decisive importance for fast trotting as well for draught load exercise. It is known that the red fibres are first fibres formed during myogenesis in the embryo, and they retain their slow-twitch aerobic capabilities throughout post-natal growth and development (Asmore et al.,

1972) Further, the white fibres are formed later during myogenesis, and although they have relatively slow contraction speed and are red or intermediate in neonate, their contraction speed increases shortly after birth from red to white (aerobic to anaerobic). In other words, the work induces metamorphosis of red fibres into white fibres. The increase in percent population and diameter of the white fibres provides the capability for muscle function (strength and resistance to fatigue) in the adult buffaloes. The workload (e.g. body weight and movements) seems to initiate the increase in the fibre diameter. This interpretation is in close agreement with the findings of Ramaswamy et al. 1992. They reported that there was a significant increase in the fibre diameter of logissimus dorsi muscle with an increase in body weight in barrows. The fibre density was significantly lower in the adult animals than in the calves. The over all decrease in the fibre was 3 % . Vyas and Vyas ( loc.cit.) also reported a 40 60 % decrease in the fibre density in the digital muscles of the adult animals from those of the calves. The decrease in the fibre density could be attributed to : (1) increase in the diameter of muscle fibres in the adult (Vyas and Vyas, loc. cit), (2) increase in connective tissues (Dubal et al., 1995) and (3) combination of these two factors. It is known that in horses the increase in diameter is workload related (Roneus et al., 1992). The muscles of fore limbs have to bear the body weight and sustain the movement. These two workloads

require an increase in diameter of muscle fibres and connective tissues; hence, the fibre density was reduced in the adult animals.

The muscles of proximal parts of the limbs have to perform their functions in a single contraction, which bulky muscles can do (Alexander and Goldspink, 1977.). Hence, these muscles are bulky in comparison to the distal ones. These muscles had higher fibre density. Further, Dubal et al.(1995) reported that the non-muscular tissues in the muscles of the proximal part of the fore limbs of the buffalo, was significantly more than the distal part. Further more, it is known that the non-muscular tissues can store about 50 % of elastic strain energy, which is of particularly importance in locomotion (Alexander, 1984). Thus, from the present study, it can be concluded that the distribution of different fibre types and their diameters and the fibre density were more closely related with workload than the age of the animals. The over all view of muscle fibre profile of the muscles of forelimbs of the buffalo was more suitable for heavy loading with slow speed.

REFERENCES

Alexander, R McN. (1984). Elastic energy store in running vertebrates. American Journal of Zoology. 24: 85 94Alexander, R. McN. and Goldspinks, (1977). Asmore, C.R., Robinson, D.W., Rattray, P. and Doerr, L. (1972). Biphasic

development of muscle fibres in fetal lamb. Experimental Neurology. 37: 241-55. Close, R. I . (1972). Dynamic properties of mammalian skeletal muscles. Physiology Review. 52 : 129 97. Dubal, S.C., Vyas, K.N. and Vyas, Y.L (1995). Age related increase in the nonmuscular tissues in muscles of forelimbs of the buffalo. Orissa Veterinary Journal. 20: 38-43

Gottlieb, M., Essen-Gustavason, B., Lindholm, A. and Pearson, S.G.B. (1988).Circulatory and muscle metabolic responces to draught work compared to increasing trotting velocities. Equine Veterinary Journal. 20: 430 - 484 Ikai M and Fukunaga T.(1968). Calculation of muscle strength per unit cross sectional area of human muscles by means of ultrasonic measurements Arbeistsphysiol.26:26. Lindholm A, Bjemeld A B and Stalin B. 1974.Acta Physiologia Scandavania .90 : 475 84. Lopez-Rivero, J. L., Aguera, E., Monlerde, J. G., Vivo, J. and Rodriguez-Barbudo, M.V.(1992). Skeletal muscle fibre size in untrained and endurance trained horses.American Journal Veterinary Research .53 : 847 50 Nachlas, M. M., Tson, K. C., Sonza, F., Chang, C. S. and Seligman, A. M. (1957). Enzymelocalization. Journal Histochemistry and Cytochemistry. 5: 565 Roneus, M. essen-Gustavason, B., Lindholm, A. and Pearson, S.G.B. (1992). Skeletal muscle characteristics in young trained and untrained Standardbred. Equine Veterinary Journal. 25 : 143 46. Ramaswami, A.M., Jayaprasad, A., Radhakrishnan, K.J. and Kanan, G. (1992).

Influence of slaughter weight on fibre diameter, sarcomere length, shear force value and tenderness score in large white Yorkshire barrows. Indian Journal of Animal Research. 26: 67 - 74 Rooney, J. M. (1969). Biomechanics of Lameness in Horses. The Williams and Wikins Co., Baltimore Snedecor, G. W. and Cochran, W. G. (1980). Statistical Methods. 7 th edn. Oxford and I.B.H. co., New Delhi Snow D H, Kerr M G, Nimemo M A and Abbott E A. 1982. Vet Rec. 110 : 377 84.

Tesch, P. A. and Karlson, J. (1985). Muscle fibre types and size in trained and untrained muscles of elite athletics. Journal of Applied Physiology. 59 : 1716 20. Vyas, Y. L. and Vyas, K. N. (1986). Histochemical and histometric observations on different fibre types in the digital extensor and flexor muscles of Surti buffalo and Kankrej bullocks. Indian Journal Animal Sciences .56 : 190 93.

Table 1: Mean S.E. of percentage of different fibre types in muscles of forelimbs of Surti buffalo. Percentage of fibre types Name of muscle Calves Adult Red Intermediate White* Red Intermediate White* A.Pectoral Girdle 1.Trapezius 37 2 36 1 27 1 35 2 37 3 29 2 2.Rhomboideus 38 3 34 1 28 1 35 1 35 3 30 3 3.Superficial pectoral 36 2 37 1 27 1 33 2 39 3 28 3 4.Deep pectoral 34 2 36 1 30 2 32 1 38 3 30 2 5.Serratus ventralis 43 3 31 1 27 1 44 1 33 2 23 2 6.brachio-cephalicus 27 2 41 2 33 2 23 1 41 3 36 2 7.Omotrans-versarius 29 2 40 2 31 1 29 1 45 3 37 2 8.Latissimus dorsi 29 2 40 1 31 1 29 3 41 1 30 3 B.Shoulder 1.Supraspinatus 35 2 35 1 30 2 34 2 36 2 30 2 2.Infraspinatus 34 2 37 1 30 2 32 1 38 3 29 3 3.Terse minor 26 1 41 1 33 3 27 1 40 2 33 2 4.Deltoides 23 1 43 1 34 4 22 1 43 3 35 3 5.Subscapularis 31 2 37 1 31 4 29 1 39 3 33 3 6.Teres major 30 1 40 1 30 4 26 1 43 3 31 3 7.Coracobrachialis 29 1 39 1 33 3 28 2 40 2 32 2 C.Arm 1.Long head of triceps 39 2 33 1 28 4 36 1 34 3 28 2 brachii 2.Medial head of triceps 19 1 44 2 37 3 20 1 45 1 36 1 brachii 3.Lateral head of triceps 29. 2 41 1 31 4 27 2 41 3 32 3 brachii 4.Biceps brachii 28 2 41 1 32 3 26 1 43 3 31 3 5.Brachialis 27 2 41 1 32 4 25 1 43 2 33 2

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6.Anconeus 21 1 43 2 36 2 20 D.Forearm and manus 1.Exrensor carpii radialis 23 1 41 1 36 1 22 2.Lateral digital extensor 12 1 47 1 41 5 15 3.Commondigitalextensor 13 1 47 2 40 5 15 4.Medial digital extensor 16 1 44 2 40 5 18 5.Flexor carpii radialis 15 1 46 2 38 4 12 6.Flexor carpii ulnaris 21 1 44 2 35 3 18 7.Ulnaris lateralis 15 1 44 2 41 3 14 8.Superficial digital flexor 12 1 49 2 39 4 13 9.Deep digital flexor 15 1 45 1 41 4 16 * The per cent population of the white fibres differed significantly ( that of the red fibres.

41 2

39 2 34 38 40 39 40 38 39 38 40 2 2 2 3 2 2 3 3 2

1 44 3 1 47 2 1 45 2 1 43 3 1 48 2 1 44 2 1 47 3 1 50 3 1 44 3 P < 0.05 ) from

Table 2: Mean S.E. of diameters ( m ) of different fibre types in muscles of forelimbs of Surti buffalo. Name of muscle Red A.Pectoral Girdle 1.Trapezius 2.Rhom boideus 3.Superficial pectoral 4.Deep pectoral 5.Serratus ventralis 6.brachio-cephalicus 7.Omotrans-versarius 8.Latissimus dorsii B. Shoulder 1.Supraspinatus 2.Infraspinatus
3.Terse minor 4.Deltoides 5.Subscapularis 6.Teres major 7.Coracobrachialis C.Arm 1.Long head of triceps brachii 2 .Medial head of triceps brachii 3.Lateral head of triceps brachii

Percentage of fibre types Calves Intermediate White Red 56 2 57 1 59 54 52 54 55 52 58 54 40 55 45 60 62 1 1 2 1 2 3 2 2 2 1 1 2 1 58 2 60 3 61 62 55 62 62 57 58 59 42 64 53 60 64 2 3 3 2 2 2 3 4 3 2 3 4 5 56 3 57 2 60 51 66 48 42 56 60 53 42 50 62 60 60 3 3 4 2 3 3 2 2 1 3 4 1 1

Adult* Intermediate 66 3 67 2 71 66 66 63 59 64 4 3 4 3 1 2

White 70 3 70 3 74 74 71 70 71 66 70 70 52 84 75 80 86 4 2 4 1 3 3 3 3 2 4 3 5 3

48 2 48 1 50 41 49 40 38 41 2 1 1 1 2 2

51 1 43 1 36 1 40 1 41 2 53 1 52 1 45 2 38 1 34 1 37 1 39 2 40 2 36 1 35 2 36 2 40 40 39 40 40 2 3 3 2 2

69 3 66 3 50 2 66 2 57 3 68 2 67 1 72 3 66 3 55 2 57 3 59 2 66 2 63 4 74 3 75 4 70 71 71 65 69 5 4 5 4 4

56 2 56 2 46 3 44 3 47 2 52 3 43 3 53 5 40 5 53 53 53 51 52 4 4 4 3 3

60 3 53 3 45 2 50 3 55 4 50 4 45 2 60 4 55 4 60 62 62 57 59 5 5 5 5 4

56 2 49 2 41 1 48 2 48 2 48 1 54 5 53 4 53 4 52 52 52 50 51 3 3 2 2 2

70 3 70 5 55 2 65 4 72 4 62 2 70 5 89 5 78 4 82 84 83 74 81 5 5 4 4 4

4.Biceps brachii 5.Brachialis 6.Anconeus D.Forearm and manus 1.Exrensor carpi radialis 2.Lateral digital extensor 3.Common digital extensor 4.Medial digital extensor 5.Flexor carpii radialis 6.Flexor carpii ulnaris 7.Ulnaris lateralis 8.Superficial digital flexor 9.Deep digital flexor

39 2

52 3

55 3

49 2

65 3

75 5

The diameter of fibre types was than the buffaloes the calves

significantly ( P < 0.05 ) more in the adult

Table 3: Mean S.E. of fibre density in muscles of forelimbs of buffalo. Mane of muscle Calves A.Pectoral Girdle 1.Trapezius 2.Rhomboideus 3.Superficial pectoral 4.Deep pectoral 5.Serratus ventralis 6.brachio-cephalicus 7.Omotrans-versarius 8.Latissimus dorsi B.Shoulder 1.Supraspinatus 2.Infraspinatus 3.Terse minor 4.Deltoides 5.Subscapularis 6.Teres major 7.Coracobrachialis C.Arm 1.Long head of triceps brachii 2.Medial head of triceps brachii 3.Lateral head of triceps brachii 4.Biceps brachii 5.Brachialis 6.Anconeus D.Forearm and manus 1.Exrensor carpii radialis 2.Lateral digital extensor 3.Common digital extensor 4.Medial digital extensor 5.Flexor carpii radialis 6.Flexor carpii ulnaris 7.Ulnaris lateralis 8.Superficial digital flexor 9.Deep digital flexor 327 319 315 313 370 310 312 332 327 325 310 315 365 322 300 359 350 317 340 317 315 346 308 341 321 318 313 335 323 315 18 15 12 15 20 14 14 17 15 16 14 10 20 17 14 16 21 15 18 19 20 22 19 16 22 17 16 18 23 20 Buffalo Adults* 156 155 148 146 165 152 154 163 153 152 137 142 144 137 126 133 135 140 135 155 158 154 136 147 131 130 134 121 122 154 11 12 11 8 7 10 12 13 10 10 7 9 8 9 6 9 8 10 9 12 11 10 9 10 6 7 9 7 6 10

The adult aminals had significantly (P < 0.01) lower values of fibre density than the calves.