Skeletal muscle characteristics of reindeer (Rangifer tarandus L.)

CWI~.liin&m. Pfz~iof. Vol. 82A. No. 3. pp. 675.-679,1985

ll300-9629/W $3.00 + 0.00 (‘ 1985 Pergamon Press Ltd

Printed in Great Britain

SKELETAL MUSCLE CHARACTERISTICS REINDEER (RAlVG1lW? TARANDUS

OF L.)

B. E&N-GUSTAVSSON and C. REHBINDER Department of Medicine 1, Department of Pathology, Swedish University of Agricultural Sciences, S-750 07 Ilppsala. Sweden. Telephone: 018 171000

Abstract-l. Fibre type composition, fibre areas, capillaries, enzyme activities and intramuscular substrates were analysed on skeletal muscle samples from reindeer. 2. The muscles contained l&-20’% Type I fibres and a higher percentage of Type IIB (40-60x) than Type HA fibres (2&40x). All fibre types revealed medium or dark staining intensity for oxidative capacity. Glycolytic capacity was greatest in Type IIB fibres. All fibres stained for glycogen, while Type I and IIA fibres stained for lipids. 3. The mean number of capillaries in contact with fibres of each type, relative to fibre type area was high in all muscle types. 4. The metabolic profile of reindeer muscle indicates that energy, to a great extent. is produced through oxidative pathways.

JNTRODUCTJON Skeletal muscle consist of fibre types with different metabolic and contractile properties (Barnard et al., 1971; Close, 1972). The classification of muscle fibres into Types I and II is based on histochemical staining

for myofibrillar ATPase at pH 9.4 (Padykula and Herman, 1955; Engel, 1962). Physiologically it has been shown that Type I fibres have slow contraction times (slow twitch fibres, ST), while Type II fibres have fast contraction times (fast twitch fibres, FT) (Barany, 1967; Burke ef a/., 1973). Histochemically, Type II fibres can further be subgrouped into Types IIA and IIB fibres as they stain differently after acid preincubations (Brooke and Kaiser, 1969, 1970). Skeletal muscle is metabolically characterized by analysing enzyme activities representing different metabolic pathways and intramuscular substrates (Peter ef al., 1972; Es& et al., 1975). The metabolic characteristics of the different fibre types show that Type I fibres have a high oxidative, but a low glycolytic capacity, while Types IIA and IIB fibres both have a high glycolytic capacity. Large variations are seen in oxidative capacity among Type II fibres (Ashmore and Doerr, 1971; Reichmann and Pette, 1982). Most studies on skeletal muscle characteristics have been performed on laboratory or domestic animals such as rats, guinea pigs, rabbits, cats, dogs, pigs and horses. Furthermore, several studies have been performed on human skeletal muscle. The purpose of the present study was to investigate fibre types and enzyme activities in a semi-domestic animal like the reindeer.

MATERIALS AND

METHODS

Two eleven-month-old reindeer, one male and one female, were used in this study. The reindeer were kept in a corral together with 34 others at the reindeer station in Kuolpa. Nut~ents and water were supplied by means of commercial

reindeer fodder (Renfor) and snow. The reindeer were killed by means of a rifleshot in the head, when resting or ruminating and muscle samples were obtained within IOmin. Two small muscle pieces were cut out from each of four different muscle groups (triceps, gluteus, semitendinosus, longissimus). The samples for enzyme and substrate analyses were immediately frozen in liquid nitrogen, while the specimens for histochemical analyses were rolled in talcum powder before they were frozen. All muscle samples were transported to the laboratory in liquid nitrogen and then stored at -80-C until analysed.

The muscle tissue was freeze-dried during 24 hr and then put under a dissection microscope so that connective tissue. fat and blood could be removed. The muscle tissue was weighed and l-2 mg was homogenized in ice-cold, 0.1 M phosphate buffer (pH 7.3) with an ultrasound disintegrator and citrate synthase (CS). 3-OH-acylCoA dehydrogenase (HAD), trios; phosphate dehydrogenase (TPDH) aid lactate dehydrogenase (LDH) activities were analysed (Es& et al., 1980: Bass er al.. 1969). Glycogen was analysed in the form of glucose residues after l-2 mg of tissue had been boiled for 2 hr in I M HCI (Lowry anbpassaneau, 1970). For triglyceride analyses two or three muscle samples weighing 0.5 1.5 mg each were used and analysed according to Es&n cf al. (1978).

Serial sections (IOjtm) were cut in a cryostat and stained for myofibrillar ATPase after both acid (pH 4.3 for 3 min, and pH 4.6 for 7min at room temperature) and alkaline (pH 10.3 for 9 min at 37’C) pre-incubation (Brooke and Kaiser, 1970) a-glycerophosphate dehydrogenase (Wattenberg and Leong, 1960) and NADH dehydrogenase (Novikoff rf al., 1961). Thicker sections (20pm) were taken for the PAS (Glycogen) and OIL red (lipids) stains (Pearse, 1961). The stained ATPase sections were then photographed and fibre types were identified as Types I, IIA and IIB fibres according to Brooke and Kaiser (1970). Between 400-800 fibres were identified in each sample. These fibres were also subjectively classified as dark, medium or light according to the NADH dehydrogenase staining intensity.

B. ESS~N-~~sTAv.~~~

676

and C. REHBI~~E~

Table I. Fibre typecomposition, f&weareas. enzyme activities, giycogen and triglyceride levels in skeletal muscle of reindeer Gluteus male female

TriCeps male female

1 IIA IIB

22 25 53

22 20 58

30 26 44

44 21 35

cs HAD LDH TPDH

61

62

36 511 675

36 525 638

41 29 708 759

64 49 585

351

448

242

Triglyceride iP%‘g)

10.2

9.0

Capillaries were visualized by staining sections according to the amylase-PAS method (Andersen, 1975). The stained cross-sections were photographed and a large area containing a minimum of 225 fibres was framed. Within that area the capillary density (cap/mm2). the number of capillaries per fibre (capjfibre) and the mean number of capillaries in contact with iibres of each type (cc) were calculated as described by Andersen and Henriksson (1977). The total area of the framed amylase-PAS stained fibres and the individual fibre area was analysed with the MOP DIGIPLAN ANALYSER. RESULTS

Fihre type composition (Tub/e I) The percentage of T: #,e II fibres was high in all four muscles types studied (60-90”/0). The percentage of Type IIB fibres was almost double that of the Type IIA fibres in all muscles except for the longissimus where they were almost ::qual. Triceps had as much as 3(t45”, Type I fibres tvhile gluteus had about 2Oyi Type 1 fibres.

All the fibres showed a strong staining intensity for NADH dehydrogenase. No fibres stained lightly, while almost all of Types IIA and IIB fibres were medium and Type I fibres darkly stained. The greatest staining intensity for r-glycerophosphate dehydrogenase was found in Type IIB fibres and in some Type IIA fibres. The reverse pattern was seen for OIL red with the greatest staining intensity in Type I fibres and in some Type IIA fibres. All fibres stained

10.6

Longissnnus male female 14

Semitendinosus male female

40

16 33 51

17 32 51

IO lb 74

640

91 64 690 875

101 67 645 756

I03 82 818 996

63 36 488 572

331

3x5

517

523

464

46

7.2

13.8

9.3

l2.7

9.6

intensely for PAS. Some Type I fibres could be found which had a somewhat lesser staining intensity than the Type II fibres. Gl_vcogen and trigl_vceride (Tubk 1) Longissimus and semitendinosus both seemed to have a somewhat higher glycogen and triglyceride content compared with gluteus and triceps. Enzyme activities (Table 1) Citrate synthase (CS) was analysed as a marker for citric acid cycle activity 3-OH-acylCoA dehydrogenase (HAD) as a marker for lipid oxidation, triosephosphate dehydrogenase (TPDH) as a marker for glycolytic capacity and lactate dehydrogenase (LDH) as a marker for lactate production. CS and HAD activities were high in all muscle groups, but longissimus and semitendinosus had a somewhat higher (1.5 x ) activity than gluteus or triceps. TPDH and LDH activities also seemed to be highest in semitendinosus and longissimus. Cupilfuries (Tab 14* 2) In both reindeer, longissimus was the muscle that showed the most capillaries per fibre and per mm’, with 4-6 capillaries seen around each fibre type. In the other three muscle groups somewhat lower values were seen, with 3-4 capillaries found around each fibre type. The mean number of capillaries in contact with fibres of each type, relative to fibre type area, was very high in all muscle groups. The highest values (34) were seen for Type I fibres and the lowest (1.5-2) for Type IIB fibres. Type I fibres had the

Table 2. In each fibre type (I, IIA, IIB). the staining intensity f$x NADH dehydro~~nase was rated

as dark, medium and light. The values are given as per cent of dark. medium and light stained tibres within Type I. IIA and IIB fibre type populatmn Gluteus male female dark medium light

100 -

Type IIA

dark medium light

I 99

Type IIB

dark medium Liaht

100

Type

1

Triceps male female

90 IO

84 I6

91 9

I

45 55

23 77 ._

99 _ I 99

100

I 99

Longissimus female male

33 67 4 96

Semitendinosus male female

57 43

89 II

39 61

3 97 _..

2 98

17 83

1

I

-

IO

Y9 _

99 -

100 -

90

Reindeer skeletal muscle

smallest fibre area. Type HA fibrcs were 1.4x and Type IIB fibres I .6 x larger than the Type I fibres. DISCUSSION

The muscle of reindeer show a high percentage of Type II fibres which can be subgrouped into types IIA and IIB fibres. Longissimus and semitendinosus have a higher percentage of Type II fibres than gluteus and triceps and similar results have recently been obtained in pigs (Essen et al., 1980), horses (Esstn et al., 1980) and deer (unpublished observation). While in most humans and animals studied Types I and IIA fibres are more oxidative than Type IIB fibres, the reindeer muscles show a remarkably high oxidative capacity in all fibre types. There were only minor differences between tibres rated dark or medium in the NADH stain, and it was evident that all fibres revealed a high staining intensity (Fig. 1, Table 2). In some other non-domestic animals like mice (Sher and Cardasis, 1976, Reichmann and Pette, 1982), roedeer, fahow deer, red deer and moose (unpublished observation), a high oxidative capacity has also been found in Type IIB fibres. The enzyme activities furthermore show that reindeer muscles have a large oxidative potential, the activities of citrate synthase and 3-OH-acytCoA dehydrogenase being similar to those seen in racehorses, greyhounds (unpublished observation), and elite human athletes (E&en-Gustavsson and Henriksson, 1984). Muscles which show a high oxidative capacity often contain a very low percentage of Type IIB fibres or none at all. Racehorses have a higher Type IIAjIIB ratio compared with inactive horses, and greyhound muscle only contains Type IIA fibres (unpublished observation). In addition human subjects performing endurance types of activity have a much lower percentage of Type IIB fibres compared with untrained subjects (Saltin et a/., 1977; Jansson and Kaiser, 1977; Es&n-Gustavsson and Henriksson, 1984). It has been shown in both humans and thoroughbred horses that the Type IIA/IIB ratio will increase with training (Andersson and Henriksson, 1977; Lindholm et al., 1983). All these data indicate that physical activity seems to be an important factor influencing not only the muscle fibres oxidative capacity but also the proportion of Type IIAjIIB fibres. The reindeer is a physically active animal in the free state and is exposed to a considerable amount of endurance activity. This includes selective grazing activity when reindeer move frequently (Skjenneberg and Slagsvold, 1968) especially long-distance migratory movements. An interesting finding of the reindeer muscle, then, is that in spite of its very high oxidative capacity it still contains a high percentage of Type IIB fibres. In addition to being active a common characteristic for reindeer, as well as other wild prey animals, seems to be a strong flight and avoidance preparedness against most kinds of disturbances, particularly against predators. It is generally believed that the oxidative Types I and IIA fibres are fatigue-resistant and recruited during steady and continous types of activity, whereas the glycolytic Type IIB fibres are fast-fatiguable and largely involved in maximal efforts (Burke et al., 1973). In flight situations, all

677

fibre types are likely to be recruited and it would than

be favourable for an animal to have oxidative Type IIB fibres to provide a high speed with great endurance. In trained muscles of both humans and horses Type IIB fibres are also shown to be oxidative (Sjoogard Edal.. 1980; Es&n et al., 1980). In inactive animals, such as pigs, muscles contain a high proportion of low-oxidative Type IIB fibres, but some Type IIB fibres are usually also highly oxidative (EssenGustavsson and Lindholm, 1984). Thus, oxidativc capacity can vary markedly in Type IIB fibres both between different species and within muscles of the same species. The metabolic profile of a fibre is therefore not necessarily correlated to its contractile properties. When fibres are classified as Types I, 11.4 and IIB in various muscles it is essential to note that the individual fiber types may differ greatly in both contractile and metabolic characteristics between animals. Furthermore, it is important to realize that histochemical methods only are semi-quantitative. The metabolic profile of reindeer muscle shows that all fibre types have a high oxidative capacity and the glycogen and triglyceride stores indicate that both carbohydrates and lipids are important for energy production. However, the results from the staining of intramuscular substrates indicate that the metabolism may vary somewhat between the fibres. From Fig. I it can be seen that Type I fibres, and to some cxtcnt Type IIA fibres, have large amounts of intraceilularly stored lipid, whereas Type JIB fibres seem to contain much less lipid. On the other hand, all fibres contain glycogen, with Types IIA and IIB fibres possessing stores of a somewhat higher magnitude. This data suggests that oxidation of lipids might be more important in Types I and IIA fibres, whereas glycogen probably can be utilized in all fibre types. However, the a-glycerophosphate dehydrogenase stain shows the greatest intensity in Type IIB fibres indicating that these fibres have a higher glycolytic capacity. Since Type IIB fibres show only a minor lipid storage but a high oxidative capacity, this probably suggests that the oxidation of pyruvate plays an extremely important role in these fibres. The activities of triosephosphate dehydrogenase and lactate dehydrogenase in reindeer muscle are both about 3--4 times lower than those observed in pigs (Es& et al., 1980), horses (Lindholm ;‘t tr!., 1983) and bulls (unpublished observation). The low lactate dehydrogenase activity further indicates that energy is generated, to the greatest extent, through oxidative pathways in reindeer muscles. The high oxidative capacity of all fibres in reindeer may in part be related to the small areas of the fibrcs, since this may favour a smaller diffusion distance for oxygen. Furthermore, the reindeer muscle is highly capillarized (Table 3). It is interesting that the number of capillaries per fibre (l&2.5), and the number of capillaries around each fibre type (4-6) do not differ greatly from that seen in both trained and untrained human muscle (Andersson and Henriksson, 1977; Schantz, 1983). However, due to the very small fibre areas in reindeer the number of capillaries per mm2 are as much as 34 times greater than in human muscle. Thus, the small fibre areas and the many capillaries might not only facilitate the uptake of oxygen and blood-born substrates like

B. ESS~N-CIJSTAVSSON and C. REHBIWEK

678

Fig. 1. Serial sections of longissimus muscle of reindeer stained for myofibrillar ATPase after alkaline (pH 10.3) = a and acid (pH 4.6) = b. (pH 4.3) = c pre-incubation OIL RED = d, NADH dehydrogenase = e and a-glycerophosphate dehydrogenase = f. The section stained for PAS = g is not serial.

Table 3. Mean fibre area, number of capillaries in contact with each fibre (CC), CC relatwe to fibre area, capillaries per mm2 in four different muscle WOUDS of reindeer

auteus Fibre type Fibre area Number of capillaries in contact with each fibre (CC) CC relative to libre area gm-’ x IO .j Capjfibre

Cap;mm”

1 IIA IIB

male

I263 1485 1774

female 935 1407 I460

Triceps male female --_.

1477 1827 I892

Longissimus inale female

1280 1403 1630

231; 1513 I148

per libre and capillaries ~mitendinosus female male 1319 1445 2055

I245 1372 2072

1 HA IIB

4.1 45 4.2

2.8 3.6 3.4

3.3 3.5 2.9

SO 5.4 6.0

4.2 4.8 4.9

4.1 4.1 4.2

3.5 3.7 4.4

I

3.3 3.0 2.4

3.0 2.6 2.3

2.2 .-.

1.5

3.9 3.9 3.1

3.2 3.2 2.8

3.1 2.9 2.0

2.8 2.7 2.1

1.5

1.1

_.

1.1

IIA IIB

990

820

1.9

__

594

2.1 1105

1.8 1071

1.4 810

1.5 RSJ

Reindeer

skeletal

glucose and fatty acids, but may also facilitate the release of waste products like lactate. Thus, muscles of reindeer are composed of Types I, IIA and IIB fibres which all have a high oxidative capacity. The metabolic profile indicates that energy, to a great extent, is produced through oxidative pathways. A~knowle(lXement-This study was supported from the Swedish Council for Forestry and Research.

by a grant Agricultural

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