Normal and regenerating skeletal muscle fibres in Pietrain pigs

J. C:~~MP.

PATH.

1970. VOL. 80.

137

NORMAL SKELETAL

AND

MUSCLE

REGENERATING

FIBRES

IN

PIETRAIN

PIGS

B1’

A. R. Department

of .4natomy,

Royal (Dick)

h’IUIR

Sctmol qf I’eterina~s ,Studies, 1 hircrsity

of Edinburgh

INTRODCCTION

\ltllough Pietrain pigs contain a large proportion of muscle per unit of body wei-ght, the value of this breed is reduced by the poor quality of its meat, which is often pale, soft and exudative. Extensive reviews of studies on such watery pork Bendall and Lawrie, 1964; B&key, 1964 ; Briskey, Casscnsand Trautman, 1966) shotv that there are many etiological factors, including stress before slaughter, inethod of killing, speed and temperature of processing. A diagnostic feature is a rapid postmortem fall in muscle pH, due to accelerated rates of anaerobic glycolysis, which, because the temperature of the musculature is still high, leads to protein denaturation and so produces the other manifestations. The incidence in certain breeds, such as Landrace, Poland China and Pietrain, is higher even lvhen the other etiological factors are controlled, so there would appear to be a genetically determined muscular abnormality. The present study of the structure (of skeletal muscle from a susceptible breed, the Pietrain, was designed to determine whether any histological features, preceding the pale, soft and exudative condition, inigllt be related to such an abnormality. Earlier histological studies related to this l)robIcm have defined the nature of the postmortem changes in structure (Bendall ;lnd Wismer-Pedersen, 1962; Cassens,Briskey and Hoekstra, 1963a, 1~).

MATERIALS

AND

METHODS

l‘wenty two purebred Pietrain pigs were obtained from a closed herd maintained tjv the School of Agriculture, University of Newcastle-upon-Tyne. After weaning at six c\-eeksof age the pigs were penned in small groups within a controlled environment Ilouse and received a conventional meal diet (Table 1). This ration has been used for four years to supply the requirements of all pigs on performance test. The scale of rneal intake was either “to appetite” or “ad libitum”. Eighteen pigs, comprising nine gilts, seven boars and two castrated males, were slaughtered under commercial conditions. Material for examination was obtained within 15 minutes of electrical stunning followed by bleeding and scalding of the carcase. The growth rates of these pigs from 27 kg. bodyweight to slaughter at 90 kg. Ii\-eweight ranged from 0.56 to 0.72 kg./day. A further four pigs were killed in the laboratory by Etorphine (Reckitt and Sons, Hull) and chloroform anaesthesiaspecifically. to obtain antemortem samples; their serial numbers, sex, dates of slaughter, bodyweights and ageswere (63) boar, July, 58 kg., 162 days; (434) gilt, July, 56 kg., 167 days; (498) castrated male, October, 80 kg., 196 days; (497) castrated male, October, 77 kg., 196 days. At the abattoir, muscle sampleswere taken frorn longissimusdorsi, caudal to the last rib, while from the four pigs killed in the laboratory sampleswere taken from

138

PIETRAIN

PIG

SKELETAL

MUSCLE

longissimus dorsi, biceps femoris, gluteus medius, gracilis, triceps, latissimus dorsi, serratus ventralis thoracis, intercostales, flexor carpi ulnaris, sternocephalicus, cricothyroideus, and from the wall of the oesophagus. Thin strips of muscle (< 1 cm. thick) were taken, and when possible they were tied to splints before fixation and removal. For light microscopy, 4 per cent. formaldehyde buffered with phosphate to a pH of 7.2 was the fixative, and sections were stained with haematoxylin and eosin, Hollande’s chlorcarmine (Gatenby and Painter, 1937), Masson’s trichrome, methyl green-pyronin, cresyl fast violet and Wilder’s reticulin methods. For electron microscopy, strips (< 1 mm. thick) were teased from phosphate buffered 2 per cent. glutaraldehyde-fixed tissue, placed in 1 per cent. 0~0~ in phosphate buffer, and embedded in Araldite. Other thin strips were fixed in 1 per cent. 0~0, in cacodylate buffer. Sections, 1 pm. thick, were stained with toluidine blue and pyronin for light microscopy (Ito and Winchester, 1963) and thin sections were mounted on Athene 483 grids, without a supporting membrane, before staining with uranyl acetate-lead citrate (Reynolds, 1963) for examination in an AEI EMGB microscope. RESULTS

Antemortem

Material

Most of the hbres show the normal features of mammalian skeletal muscle. Cross striations are evident in stained preparations, in Polarized light (Fig. l), and in teased fresh fibres. The diameter of the fibres, as measured on fixed embedded material, varies in different muscles, the cricothymid (range 24 to 40 pm.) being the narrowest and the biceps femoris (range 36 to 120 pm.) the thickest of the muscles examined. TABLE MEAL

Wheatfeed Barley meal White fish meal Soya bean meal Molasses Added per ton Di-calcium phosphate Zinc carbonate Salt (NaCl) Vitamin premix Mineral premix

1 DIET

% by weight 25 50 I’: 5

30 4 + 5 5

lb. lb. lb. lb. lb.

The large muscles of the limbs and trunk contain fibres up to 120 pm. in diameter, and these have 1 to 6 nuclei in a single transverse section. These muscles contain two types of fibre which can be distinguished in the osmium plastic embedded specimens (Figs. 2, 3 and 4). One type contains few mitochondria and is equivalent to the white fibres of Ranvier (1873); the other red type of fibre, which accounts for a small proportion, is narrower and has columns of mitochondria between the myofibrils (Figs. 2 and 4). In the Chester White breed, Moody and Cassens (1968) showed that the red fibre content of the longissimus dorsi was

A.

R.

MUIR

139

about 30 per cent. while 60 per cent. of the fibres in the trapezius were of this type. The sarcomere structure of both fibre types is entirely typical having A-bands 1.5 pm. long which are bisected by M-bands (Fig. 3). No unusual features are noted in the mitochondria when they had been satisfactorily preserved, but in many preparations they are distended and indistinct. The mitochondrial content of the white fibres is very low, but their random location in groups under the sarcolemma, often at the poles of the nuclei, would make quantitation by electron microscopic methods very tedious. The sarcoplasmic reticulum is profusely distributed around the myofibrils of the white fibres, and a noticeable feature, which has not been reported for other mammals but which is seen in other breeds of pig, is the presence of electron dense granules in the terminal cisternae (Fig. 5). A small proportion of the fibres in many muscles from all four animals show the histological features of muscle regeneration. Necrotic fibres contain eosinophilic masses of myofibrillar material and varying numbers of macrophages with large pale vesicular nuclei (Fig. 6). Other endomysial tubes are filled with small round, intensely basophilic cells with dark nuclei containing prominent nucleoli (Fig. 7) ; these cells are identified as myoblasts. Myotubes are present consisting of columns of closely packed nuclei surrounded by a few myofibrils whose striations may only be detected in polarized light (Fig. 10). Slender cross striated fibres with many nuclei, most of which are subsarcolemmal, are also seen (Fig. 11). In transverse section there are a few faintly basophilic fibres which are slightly thinner than those of the rest of the muscle. Some are heavily nucleated (Fig. 9), and others contain single clear cells (Fig. 8)+ Although a regenerating fibre has not been included in the samples examined by electron microscopy, one such sample contained a binucleate satellite cell (Fig. 12). The nuclear material is clumped and, midway between the two nuclei, a zone of cytoplasm clear of organelles contains signs of bisecting membranes. This is interpreted as a telophase of a satellite cell division. The various stages of regeneration can be seen in the above sequence along the length of a single fibre. A basophilic stain for cytoplasmic RNA such as cresyl fast violet, used on transverse sections, is the most effective method fol identifying regeneration. The regenerating fibres are scattered randomly throughout the muscle; usually a single fibre is affected, but pairs of adjacent fibres are more common than the concentration would predict. No cellular or other reaction is evident in the extracellular space around the affected fibres. Of the muscles examined, the thoracic ventral serrate contains the highest proportion of regenerating fibres and, including all the stages listed above, about 1 per cent. of the fibres are affected. Definite evidence of regeneration is also detected in the biceps femoris, longissimus dorsi, gracilis, gluteus medius, intercostal muscles, but it is not seen in the cricothyroideus or the wall of the oesophagus. Postmortem

Material

Less than 15 minutes after stunning, the pH of Pietrain muscle as measured in the iongissimus dorsi has dropped to 5.85 to 6.3. More than 70 per cent. of the fibres in most samples fixed at this time, with this acidity, show gross disorganization of muscle structure (Fig. 13). In these fibres a few myofibrils, with normal banding pattern, remain, but examination with polarized light shows that most of

140

PIETRAIN

PIG

SKELETAL

MUSCLE

the cross-striated material has become disorganized. Irregular coarse transverse banding resembling retraction clots, has appeared; these dark bands may I-UJ~ partially or completely across the fibre and they vary in thickness from ipm. to 15pm. A portion of a single fibre may be affected (Fig. 14). As the bands she\\ faint birefringence which is oriented differently from that of the surviving myofibrils, polarized light provides a convenient method of identification. Electron microscopic examination shows that in the region of the transverse dark bands, myofibrillar organization is disrupted with a loss of Z-disc material. Even a mildly affected fibre (Fig. 15) has lost the normal parallel alignment of myofibrils, in register with each other, and the transverse bands contain fibrillar material from coagulated myofibrils and probably include adsorbed and trapped denaturtscl sarcoplasmic proteins (Bendall and Wismer-Pedersen, 1962). Glycogen granules can be identified in the sarcoplasm of the affected fibres, indicating that glycolysis is not complete at the time of muscle disorganization. In this disorganized postmortem tissue, with the distorted non-coagulated fibres between the coagulated ones, muscle regeneration can be identified with difficulty. Clumps of myoblasts and areas containing macrophages and eosinophilic debris can be seen in 15 of the 18 longissimus dorsi samples taken. DISCUSSION

Histological changes of the type found in Pietrain muscle after slaughter can be induced in the living muscle and after death by a variety of chemical, thermal and physical agents (Fishback and Fishback, 1932). Such coagulation of myofibrillar and sarcoplasmic proteins is a non-specific reaction to injury, and so the notable feature in this breed is merely the extent and speed of these postmortem changes. This is caused by an unusually rapid fall in muscle pH after slaughter. Examination of watery pork from other breeds shows a similar pattern of disorganization and transverse banding (Bendall and Wismer-Pedersen, 1962 ; Cassens et al., 1963b) and so these histological features can be added to the pallor, poor water-binding properties and changes in texture which are already associated with the condition. Using such histological criteria in association with direct pH measurements, it is evident that the irreversible changes of wateriness are established in the Pietrain within a few minutes of slaughter. This conclusion has commercial implications, but it also means that studies with these animals which attempt to define the cytological cause of the degeneration cannot be performed on slaughterhouse material. The present morphological studies on antemortem material have not revealed any unusual features to account for the rapid postmortem changes. In relation to the number observed in smaller mammals, the mitochondria in the predominant white type of Pietrain muscle fibre account for a very small proportion of the sarcoplasmic volume, but Gauthier and Padykula (1966) have shown that the mitochondrial content of diaphragm muscle fibres declines as the size of the animal increases. In the absence of histochemical and biochemical information it is unlikely that fine structural studies will define the cause of this condition. However, the more rapid development of wateriness in white fibres (Briskey, 1964) implies that a low mitochondrial, aerobic oxidative enzyme content may be a

A.

R.

MUIR

141

predisposing structural characteristic. On the other hand, it is possible that an increase in postmortem utilization of high energy phosphates is due to the calciumpumping activity of the profuse sarcoplasmic reticulum which surrounds the large mass of myofibrils in the white fibres. The demonstration of regenerating muscle in apparently healthy animals of this species is a new observation of significance. During a histochemical study of postmortem changes, Bodwelf, Pearson and Fennel1 (1965) noted foci of acid phosphatase activity in the longissimus dorsi muscle of pigs and concluded that these represent sites of active degeneration (Beckett and Bourne, 1958; Fennel1 and West, 1963). The features of regeneration, originally described by Waldeyer ( 1865), are effectively summarized in the reviews of Godman (1958), Field (1960) and Adams, Denny-Brown and Pearson (1962), and the possible role of satellite cells has recently been discussed (Muir, Kanji and Allbrook, 1965; Church, 1968). All these features are clearly observable in the present material and they establish that a dynamic process of regeneration has been continuing in these muscles for march than three weeks before biopsy. Urine assays of creatine/creatinine ratios (Blaxter and McGill, 1955) or plasma glutamic-oxaloacetic transaminase levels (Briskey, 1964) should establish the e.utent and rate of muscle damage. Preliminary studies of glutamic-oxaloacetic transaminase levels in breeds susceptible to rapid postmortem glycolysis did not reveal any significant changes from non-susceptible breeds (Hopkins, Sayre and Briskey, unpublished; quoted Briskey, 1964). Negative results from such tests do not however dismiss the clear histological evidence, and they can be explained bv the relatively small amount of damage and regeneration. The cause of the damage which initiates the regenerative process cannot be defined from the present study, but its presence in all the antemortem material and most of the postmortem samples justifies a consideration of possible factors. Local trauma and infection are unlikely causes because of the distribution and sequence of regenerating fibres and the absence of an inflammatory reaction. Hence an inherent muscular disease of dietary, toxic or genetic origin seems more likely Muscular myopathies in pigs have been reviewed by Blaxter and McGill (1955), and by Briskey (1964), and they refer to dystrophies due to vitamin E and selenium deficiencies. From the limited evidence available, a supply of 1.5 mg. total tocopherols/kg. dry matter in the diet is estimated to be sufficient to prevent signs of vitamin E deficiency (A.R.C. 1967). Since the present diet supplied 2.3 mg. total tocopherols/kg. dry matter and did not contain added fat, this level of vitamin E should have been sufficient to meet requirements. Certain cytotoxic substanes such as plasmocid, 8 - (3 diethylamino-propylamino) .- 6 - methoxyquinoline dihydrochloride, and a food-colouring dye, Brown FK, selectively damage striated muscle (Grasso, Muir, Golberg and Batstone, 1968). The regenerative process following plasmocid administration in rats and mice is histologically almost identical to the present material (Adams et al., 1962). No potentially cytotoxic agents could be identified in the diet of the Pietrain herd and no viral infection of porcine muscle has yet been identified. Certain idiopathic or genetic dystrophies cause similar regenerative patterns. K

142

PIETRAIN

PIG

SKELETAL

MUSCLE

Gilbert and Hazard (1965), studying human dermatomyositis, illustrate regenerating fibres similar to the those in the present study. The postmortem degeneration producing wateriness has been described under various names, many of which such as “muscle degeneration disease” (Ludvigsen, 1954), “la myopathie exudative depigmentaire du port” (Henry, Romani and Joubert, 1958), and “so-called white muscle disease” (Lawrie, 1960; Briskey, Bray, Hoekstra, Grummer and Phillips, 1959) have pathological connotations. Bendall and Lawrie (1964), critic&se the use of such titles and the generic term “dystrophy” for a condition which manifests itself only after death. The present demonstration of in vivo muscle degeneration and subsequent regeneration noes localizes a true myopathy in a breed susceptible to postmortem degeneration, but no correlation between these events has been established. Further studies should show whether this antemortem myopathy and the postmortem degeneration share genetical and biochemical etiological factors, or whether the association is fortuitous. As there are many causes for the damage which initiates regeneration, an extensive and carefully controlled survey of its occurrence in healthy animals of different breeds and species is necessary before its role in the normal economy of muscle can be established. YUMhfARY

Antemortem material was obtained from Pietrain pigs during killing by anaesthetics and postmortem samples were obtained from the slaughterhouse. The light and electron microscopic antemortem structure of red and white skeletal muscle fibres is normal. Evidence of in vivo degeneration and subsequent regeneration is presented. After fixation, within 15 minutes of stunning, the myofibrillar and sarcoplasmic proteins in most of the fibres have coagulated. At present no connection between these phenomena has been established. It is clear that with this breed of pigs information about degenerative and regenerative processes in life cannot be obtained from a study of samples taken some time after death. ACRNOWLEDGbfENTS

Part of the cost of this study was borne by the Pig Industry Development Authority. Dr. W. C. Smith made the pH measurements and provided the material and much helpful advice. Skilled technical help was given by Mr. N. Smith and Mr. J. Keillor. REFERENCES

Adams, R. D., Denny-Brown, D., and Pearson, C. M. (1962). Diseases CJ~Muscle, 2nd Edit., Kimpton; London. (1967). The Nutrient Requirements of Farm Livestock, Part 3, Pigs, Agric. Res. Count.; London. Beckett, E. B., and Bourne, G. H. (1958). Acta Amt., 35, 326. Bendall, J. R., and Wismer-Pedersen,J. (1962). J. Food Sci., 27, 144. Bendall, J. R., and Lawrie, R. A. (1964). Animal Breeding Abstracts, 32, 1. Blaxter, K. L., and McGill, R. F. (1955). Vet. Rev. Ann., 1, 91. Bodwell, C. E., Pearson, A. M., and FenneIl, R. A. (1965). J. Food Sci., 30, 944. Briskey, E. J. (1964). Adv. Food Res., 13, 89.

A.

R.

143

MUIR

Briskey, E. J., Bray, R. W., Hoekstra, W. G., Grummer, R. H., and Phillips, P. II. (1959). J. animal Sci., 18, 153. Briskey, E. J., Cassens,R. G., and Trautman, J. C. (1966). The Physiology and Biochemistry of Muscle as a Food, University of Wisconsin Press; Madison, Wisconsin. Cassens,R. G., Briskey, E. J., and Hoekstra, W. G. (1963a). J. Food Sci., 28, 680; (1963b). Biodynamics, 9, 165. Church, J. C. T. (1968). M.D. Thesis, Cambridge. Fennell, R. A., and West, W. T. (1963). J. Histochem. Cytochem., 11, 374. Field, E. J. (1960). in The Structure and Function of Muscle, 3, Ed. G. H. Bourne, Academic Press;New York, London. Fishback, D. K., and Fishback, H. R. (1932). Amer. J. Path., 8, 193. Gatenby, J. B., and Painter, T. S. (1937). The Microtomists Vade Mccum, 10th Ed., p. 144. Churchill; London. (Gauthier, G. F., and Padykula, H. A. (1966). J. cell Biol., 28, 333. Gilbert, R. K., and Hazard, J. B. (1965). J. Path. Bact., 89, 503. Godman, G. C. (1958). in Frontiers in Cytology, p. 381. Ed. S. L. Palay, Yale University Press; New Haven. (Grasso,P., Muir, A. Golberg, L., and Batstone, E. (1968). Fd. Cosmct. Toxicol., 6, 13. Henry, M., Romani, J. D., and Joubert, L. (1958). Rev. Path. gen. Physiol. clin., 58. 355. Ito, S., and Winchester, R. J. (1963). J. cell Biol., 16, 541. Lawrie, R. A. (1960). J. camp. Path., 70, 273. Ludvigsen, J. (1954). 272. Bretn. Forsogslab. (Kbh.), 112 pp. Moody, W. G., and Cassens,R. G. (1968). J. animal Sci., 27, 961. Muir, A. R., Kanji, A. H. M., and Allbrook, D. (1965). J. Anat., 99, 435. Ranvier, L. (1873). Compt. rend. Sot. biol., Paris, 77, 1030. Reynolds, E. S. (1963). J. cell Biol., 17, 208. Waldeyer, W. (1865). Arch. Path. Anat. Physiol. klin. Med., 34, 473. [Received

for publication,

May

27th, 19691