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Pathogenesis of Ischaemic Pectoral Myopathy in the Domestic Turkey
/:J r. vel. } . ( 19 79 ), 135, 286
PATHOGENESIS OF ISCHAEMIC PECTORAL MYOPATHY IN THE DOMESTIC TURKEY J.
HENRICHS,
J.
C. L.
BERRY AND
M.
By K.
M. jONES, * SWASHt
The Institute if Pathology, The London Hospital Medical College, London, EIIBB and *The Food Research Institute, Norwich, NR4 7UA
SUMMARY
Micropaque-perfusion angiography of the pectoral muscles of domestic turkeys with green muscle disease has shown marked variability in the pattern of vascularization, with areas of ischaemia corresponding to the green infarcted lesions. These studies support the concept that pectoral myopathy in these birds is due to failure of perfusion of these muscles. Possible precipitating factors and ways of preventing the disease are discussed .
INTRODUCTION
Degeneration and green discolouration of part of the breast muscles (green muscle disease) of breeder turkeys was first recognized in the United States (Harper et aI., 1969) but it has now become a serious problem in the turkey industry in Britain (jones, 1974; Jones, King & Mulliner, 1974), and in other countries (Orr & Riddell, 1977 ). The abnormality usually develops in birds older than about 40 weeks. In some Rocks as many as a third of birds may be affected (Pettit & von Dreumel, 1973). The zones of necrosis found in the deep pectoral muscles (supracoracoideus muscles) are thought to be due to ischaemia (Harper et aI., 1975; Orr & Riddell, 1977) but its pathogenesis is unknown. In this paper we shall describe abnormalities found by micropaque perfusion radiography in the vasculature of the deep pectoral muscles of affected birds and shall correlate these with the distribution of the necrotic zones within these muscles. Comparison will be made with the blood supply of the pectoral muscles of unaffected turkeys from the same Rock. fRequests for reprints : Dr J. M . Jones, The Food Research Institute, Norwich, NR4 7UA.
ISCHAEMIC PECTORAL MYO P ATHY I N TURKEYS
287
MATERIALS AND METHODS
Eight female turkeys, aged 40 to 50 weeks were studied. Muscle samples were taken from the breast muscles of three birds for histological studies. Two of these were birds with obvious focal atrophy of the pectoral muscles and one was unaffected. Muscle samples, taken from the centre, from the edge of the atrophic zones and from homologous sites in the unaffected bird, were snap frozen in isopentane-nitrogen. Cryostat sections were prepared for histological examination by light microscopy using a standard series of stains and enzyme histochemical reactions including haematoxylin and eosin, modified Gomori trichrome, van Gieson, adenosine triphosphatase (preincubated at pH 4·3 and pH 9·5), oil red 0, nicotine adenine dinucleotide tetrazolium reductase (NADH tr) and periodic acid Schiff methods (Dubowitz&Brooke,1973).
Micropaque perfusion radiography oj the pectoral vasculature Five turkeys were investigated. Three had green muscle disease and two were unaffected. Laparotomy was performed under nembutal anaesthesia and each bird was exsanguinated via an aortic cannula. Thirty ml warm saline were injected into the vascular system through the cannula. The preparation was then maintained at 40°C by means of a water bath and perfusion was begun through the aortic cannula with a solution of barium sulphate (Micropaque, Nicholas laboratories) mixed with gelatine. A pulsed-flow pump was used. The mean perfusion pressure, recorded by a Devices pressure transducer, was 100 mmHg. After perfusion for I h each bird was placed in a refrigerator for 24 h to allow the intra-vascular Micropaque/gelatine mixture to set. The breast muscles of each animal were then excised and radiography performed. Subsequently the muscles were cut into thin slices and radiographs were made of each slice. Finally, in two birds, several of these slices of breast muscles of birds with zones of focal atrophy, were fixed in 10% neutral buffered formol-saline and paraffinembedded. Sections of these slices were stained with haematoxylin and eosin, van Gieson's and Mallory's phosphotungstic-acid haematoxylin methods and examined by light microscopy. RESULTS
The five birds with green muscle disease showed bilateral but asymmetrical depressions in their pectoral muscles. These depressions consisted of areas of green discolouration accompanied by hardness to palpation. The lesions were situated anteriorly in the thickest part of the pectoral musculature. They were usually bounded anteriorly and laterally by a fibrous interfascicular septum but caudally their margins were wider, and less clearly defined (Fig. 1).
Histology The green lesions consisted of zones of pale fibres, which were not as rounded as in normal pectoral muscles. These lesions were difficult to section without causing fragmentation of fibres. With haematozylin and eosin stains, muscle fibres in these zones had a translucent, vitreous appearance (Fig. 2). Their nuclei were very small and
288
BRITISH VETERINARY JO URNAL , 135,3
only faintly basophilic. Intramuscular capillaries were absent, and interfascicular hbrous tissue was often fragmented (Fig. 2). These appearances are consistent with carnification following infarction. All the fibres in the lesions reacted for ATPase at pH 4·3 (Typ~ I fibres) but they reacted poorly and patchily for p.hosphorylase and NADH tr. . The lesions themselves were surrounded by a capsule of fibrous tissue, partly consisting of interfascicular septum and of small, compressed muscle fibres . This capsular region contained haemosiderin deposits. Blood vessels in the lesions were usually empty. Basophilic sarcoplasmic regeneration was not seen. Infiltration by macrophages was unusual and found only at the edges of the lesions. Two turkeys, thought to be unaffected, were found to have similar, but much smaller lesions in their pectoral muscles. Normal pectoral muscle in the turkey, studied in sections taken from unaffected parts 0 f these muscles, consists of rounded multinucleated fibres, strongly reactive for ATPase at pH 9·5 and 4·3 . These fib~es also showed moderate reactivity for phosphorylase. The NADH tr preparations showed fibres of either dark or light reactivity. Vasculature of the pectoral muscles The main arterial supply to the pectoral muscles consisting of the cranial and caudal pectoral arteries and the sternoclavicular artery, which supplies the deep surface of the supracoracoid muscle, are shown in Fig. 3. This diagram is based on the anatomical descriptions of Glenny (1951) and of Orr & Riddell (1977). Two of the five turkeys investigated by Micropaque-perfusion radiography showed asymmetrical pectoral lesions. These two birds and one other, without evident gross lesions in its pectoral muscles, showed the typical green lesions in their supracoracoid muscles. There were anomalies in the vascular supply of the pectoral muscles of all five birds studied and marked, differences were found in the pattern of the pectoral vasculature between the two sides in individual birds (Figs 4 and 5). The caudal pectoral artery varied in size and length in each muscle. In some birds this artery was derived from the subclavian artery, but in others it arose from the main trunk of the pectoral artery (Figs 3 and 4). In several pectoral muscles there was an extensive collateral circulation in the area supplied by the caudal pectoral artery (Fig. 6). In those pectoral muscles in which the caudal pectoral artery was particularly thin and short the muscular lesion was large, but in those in which it was a more prominent vessel the muscular lesion was small. There were also variations in the size and length of the cranial pectoral artery, and of the sternoclavicular artery. In all the breast muscles studied there was a striking lack of perfusion of the vasculature in the green muscular lesions themselves (Fig. 6). Variability in the vascular territory of the brachio-cephalic, carotid and axillary arteries, which were also perfused by the technique used, was not a feature. DISCUSSION
The histological features of naturally-occurring deep pectoral myopathy (green muscle disease) in the domestic turkey was consistent with infarction. Since perfusion
Fig. I . Gross appearance of the lesion of deep pectoral myopathy (asterix). Normal muscle to the right of the midline has been incised.
Facing page 288
Fig. 2. Haematoxylin and eosin TS x 160 .The muscle fibres are pal e and an ucl eate. They are separated from each o lh er by clear spaces devoid of cap illaries. The interfascicula r fibrous tissue is disrupted.
Fig. 3. The blood supply of the pectoral muscles of the turkey: modified from Orr & Riddell (19 77). A pectoral artery, B cranial branch of pectoral artery, C caudal branch of pectoral artery, D sternoclavicular artery.
Fig. 4. Micropaque- perfusion angiography. The vascular pattern in the pectoral muscles of a normal lurkey. There are differences in the distribution of the long caudal branches on the two sides. but neither pectoral muscle was infarcted.
Fig. 5. Micropaque- perfusion angiography. Variability in vascularization of the pectoral muscles. Note differences between the two sides and the long co urse of the caudal pectoral vessels. There was an infarcted. green zone in the avascular area (asterix).
Fig. 6. (a ) Radiograph of sagittal slice after Micropaque perfusion: collateral circulation in distribution of cauda l pectoral artery. Note ischaemic area (asterix). (b ) PTAH preparation. The pale area represents infarction in the distribution of the avascular area shown in (a ).
289
ISCHAEMIC PECTORAL MYOPATHY IN TURKEYS
of the infarcted zones is not restored the dead tissue becomes carnified, assuming a characteristic hard texture. The green colour, and the presence of haemosiderin in the margins of the lesion suggests that haemorrhage is a feature of an early stage. Indeed, an area of reddish discolouration and oedema has been observed in an early lesion (Harper et at., 1975). The lesions are defined by their vascular territory : for examp le, they always abut interfascicular fibrous septa. Orr & Riddell ( 1977 ) tried to reproduce the green lesion by ligating the cranial and caudal pectoral arteries , but they could not produce consistent results and were thus unable to suggest a mechanism for the appearance of the naturally-occurring myopathy. However, Siller et at. (1978 ) have recently produced similar lesions in White Leghorn fowls by ligation of the subclavian artery on one side. The simil arity of the gross and microscopical appearances of the lesions of green muscle disease to those found in infarction of muscle in the anterior tibial compartment syndrome in man have been noted by others {Orr & Riddell , 197 In the latter syndrome infarction of the muscles of the anterior tibial compartment occurs in untrained subjects during prolonged strenuous exercise (Hughes , 1948). It has been suggested that unaccustomed exertion leads to trauma to muscle fibres . The resulting intramuscular oedema causes a rise in pressure in the anterior tibial compartment, with impairment of its venous drainage and arterial perfusion, and so leads to haemorrhagic infarction (Hughes, 1948 ; Carter, Richards & Zachary, 1949). I n man there is a marked predominance of Type I fast-contracting, oxidative, ATPasedependent fibres in these muscles {Johnson et at., 1973). These fibres may be likely to undergo swelling and infarction if subjected to ischaemia. The white pectoral muscles of the turkey consist of fibres of a simi lar histochemical type. The blood supply of the pectoral muscles of the turkey is dependent on long vessels which enter the anterior parts of these muscles (Fig. 3) and traverse it in a caudal direction. These vessels are vulnerable to compression as they pass through the dense fibrous septa which divide the muscle into large fascic uli (Fig. 1). The fascicular pattern of the muscle is well developed and corresponds to the zones of infarction found in birds with pectoral myopathy (Figs 5 and 6). Variability in the size and length of the caudal and cranial pectoral vessels, and of the sternoclavicular artery may lead to local vulnerability of parts of these muscles situated at the furthest extent of the arterial tree of these vessels. We suggest that in these inbred birds, which are not normally allowed to range freely or to attempt flight, these muscles, as in untrained military recruits, may be vulnerable to infarction during sudden muscular effort. The vascular anomalies and the thick fibrous interfascicular septa provide anatomical predisposing factors. The vulnerability of the modern domestic turkey to ischaemic pectoral myopathy is thus probably the result of genetic factors, which have led to the development of very large pectoral muscles with a large and tenuous blood supply, comb ined with environmental factors . It is not known whether it might be possible to prevent the development of pectoral muscle infarction by allowing these birds free range, and thus more exercise, during development. However, since we have observed marked variability in the pattern of the vasculature of the pectoral muscles it might also be possible to prevent the disease by selective breeding from birds known not to be susceptible.
n
290
BRITISH VETERINARY JOURNAL, 135,3 REFERENCES
CARTER, A. B., RICHARDS, R. L. & ZACHARY, R. B. (J949). Lancet ii, 928. D UBOWITZ, V. & BROOKE, M. H. (J973). Muscle Biopsy: A Modem Approach. London : W . B. Saunders. GLENNY, F. H. (195 I). Ohiojoumal of Science 51,47 . HARPER,]. A., BERNIER, P. E., HELFER, D. H . & SCHMITZ,]' A . (1975).joumal ofHeredity 66,362. HARPER,]. A ., BERNIER, P. E., STEVENS,]. 0. & DICKINSON, E. M . (J969). Poultry Science 48, 1816. H UGHES,]. R. (J948).joumal of Bone andjoint Surgery 30B, 58!. J OHNSON, M . A ., POLGAR, ]., WEIGHTMAN, D. & ApPLETON, D . (1973). joumal of Neurological Science 18, iii. J ONES,]. M. (1974). Turkeys 22,20. JONES,]. M ., KING, N . R. & MULLINER, M. ( 1974). Poultry Science 15, 19!. ORR,]. P. & RIDDELL, C . ( 1977). Americanjoumal of Veterinary Research 38, 1237 . PETTIT,]. R. & VAN DREUMEL, A. A. (1973 ). Canadian Veterinaryjoumal14, 198 . SILLER, W . G ., WIGHT, P . A ., MARTINDALE, L. & BANNISTER, D . W . (J978 ). Research in Veterinary Science 24, 267.
(Acceptedforpublication 5 September 1978)
PATHOGENESIS OF ISCHAEMIC PECTORAL MYOPATHY IN THE DOMESTIC TURKEY J.
HENRICHS,
J.
C. L.
BERRY AND
M.
By K.
M. jONES, * SWASHt
The Institute if Pathology, The London Hospital Medical College, London, EIIBB and *The Food Research Institute, Norwich, NR4 7UA
SUMMARY
Micropaque-perfusion angiography of the pectoral muscles of domestic turkeys with green muscle disease has shown marked variability in the pattern of vascularization, with areas of ischaemia corresponding to the green infarcted lesions. These studies support the concept that pectoral myopathy in these birds is due to failure of perfusion of these muscles. Possible precipitating factors and ways of preventing the disease are discussed .
INTRODUCTION
Degeneration and green discolouration of part of the breast muscles (green muscle disease) of breeder turkeys was first recognized in the United States (Harper et aI., 1969) but it has now become a serious problem in the turkey industry in Britain (jones, 1974; Jones, King & Mulliner, 1974), and in other countries (Orr & Riddell, 1977 ). The abnormality usually develops in birds older than about 40 weeks. In some Rocks as many as a third of birds may be affected (Pettit & von Dreumel, 1973). The zones of necrosis found in the deep pectoral muscles (supracoracoideus muscles) are thought to be due to ischaemia (Harper et aI., 1975; Orr & Riddell, 1977) but its pathogenesis is unknown. In this paper we shall describe abnormalities found by micropaque perfusion radiography in the vasculature of the deep pectoral muscles of affected birds and shall correlate these with the distribution of the necrotic zones within these muscles. Comparison will be made with the blood supply of the pectoral muscles of unaffected turkeys from the same Rock. fRequests for reprints : Dr J. M . Jones, The Food Research Institute, Norwich, NR4 7UA.
ISCHAEMIC PECTORAL MYO P ATHY I N TURKEYS
287
MATERIALS AND METHODS
Eight female turkeys, aged 40 to 50 weeks were studied. Muscle samples were taken from the breast muscles of three birds for histological studies. Two of these were birds with obvious focal atrophy of the pectoral muscles and one was unaffected. Muscle samples, taken from the centre, from the edge of the atrophic zones and from homologous sites in the unaffected bird, were snap frozen in isopentane-nitrogen. Cryostat sections were prepared for histological examination by light microscopy using a standard series of stains and enzyme histochemical reactions including haematoxylin and eosin, modified Gomori trichrome, van Gieson, adenosine triphosphatase (preincubated at pH 4·3 and pH 9·5), oil red 0, nicotine adenine dinucleotide tetrazolium reductase (NADH tr) and periodic acid Schiff methods (Dubowitz&Brooke,1973).
Micropaque perfusion radiography oj the pectoral vasculature Five turkeys were investigated. Three had green muscle disease and two were unaffected. Laparotomy was performed under nembutal anaesthesia and each bird was exsanguinated via an aortic cannula. Thirty ml warm saline were injected into the vascular system through the cannula. The preparation was then maintained at 40°C by means of a water bath and perfusion was begun through the aortic cannula with a solution of barium sulphate (Micropaque, Nicholas laboratories) mixed with gelatine. A pulsed-flow pump was used. The mean perfusion pressure, recorded by a Devices pressure transducer, was 100 mmHg. After perfusion for I h each bird was placed in a refrigerator for 24 h to allow the intra-vascular Micropaque/gelatine mixture to set. The breast muscles of each animal were then excised and radiography performed. Subsequently the muscles were cut into thin slices and radiographs were made of each slice. Finally, in two birds, several of these slices of breast muscles of birds with zones of focal atrophy, were fixed in 10% neutral buffered formol-saline and paraffinembedded. Sections of these slices were stained with haematoxylin and eosin, van Gieson's and Mallory's phosphotungstic-acid haematoxylin methods and examined by light microscopy. RESULTS
The five birds with green muscle disease showed bilateral but asymmetrical depressions in their pectoral muscles. These depressions consisted of areas of green discolouration accompanied by hardness to palpation. The lesions were situated anteriorly in the thickest part of the pectoral musculature. They were usually bounded anteriorly and laterally by a fibrous interfascicular septum but caudally their margins were wider, and less clearly defined (Fig. 1).
Histology The green lesions consisted of zones of pale fibres, which were not as rounded as in normal pectoral muscles. These lesions were difficult to section without causing fragmentation of fibres. With haematozylin and eosin stains, muscle fibres in these zones had a translucent, vitreous appearance (Fig. 2). Their nuclei were very small and
288
BRITISH VETERINARY JO URNAL , 135,3
only faintly basophilic. Intramuscular capillaries were absent, and interfascicular hbrous tissue was often fragmented (Fig. 2). These appearances are consistent with carnification following infarction. All the fibres in the lesions reacted for ATPase at pH 4·3 (Typ~ I fibres) but they reacted poorly and patchily for p.hosphorylase and NADH tr. . The lesions themselves were surrounded by a capsule of fibrous tissue, partly consisting of interfascicular septum and of small, compressed muscle fibres . This capsular region contained haemosiderin deposits. Blood vessels in the lesions were usually empty. Basophilic sarcoplasmic regeneration was not seen. Infiltration by macrophages was unusual and found only at the edges of the lesions. Two turkeys, thought to be unaffected, were found to have similar, but much smaller lesions in their pectoral muscles. Normal pectoral muscle in the turkey, studied in sections taken from unaffected parts 0 f these muscles, consists of rounded multinucleated fibres, strongly reactive for ATPase at pH 9·5 and 4·3 . These fib~es also showed moderate reactivity for phosphorylase. The NADH tr preparations showed fibres of either dark or light reactivity. Vasculature of the pectoral muscles The main arterial supply to the pectoral muscles consisting of the cranial and caudal pectoral arteries and the sternoclavicular artery, which supplies the deep surface of the supracoracoid muscle, are shown in Fig. 3. This diagram is based on the anatomical descriptions of Glenny (1951) and of Orr & Riddell (1977). Two of the five turkeys investigated by Micropaque-perfusion radiography showed asymmetrical pectoral lesions. These two birds and one other, without evident gross lesions in its pectoral muscles, showed the typical green lesions in their supracoracoid muscles. There were anomalies in the vascular supply of the pectoral muscles of all five birds studied and marked, differences were found in the pattern of the pectoral vasculature between the two sides in individual birds (Figs 4 and 5). The caudal pectoral artery varied in size and length in each muscle. In some birds this artery was derived from the subclavian artery, but in others it arose from the main trunk of the pectoral artery (Figs 3 and 4). In several pectoral muscles there was an extensive collateral circulation in the area supplied by the caudal pectoral artery (Fig. 6). In those pectoral muscles in which the caudal pectoral artery was particularly thin and short the muscular lesion was large, but in those in which it was a more prominent vessel the muscular lesion was small. There were also variations in the size and length of the cranial pectoral artery, and of the sternoclavicular artery. In all the breast muscles studied there was a striking lack of perfusion of the vasculature in the green muscular lesions themselves (Fig. 6). Variability in the vascular territory of the brachio-cephalic, carotid and axillary arteries, which were also perfused by the technique used, was not a feature. DISCUSSION
The histological features of naturally-occurring deep pectoral myopathy (green muscle disease) in the domestic turkey was consistent with infarction. Since perfusion
Fig. I . Gross appearance of the lesion of deep pectoral myopathy (asterix). Normal muscle to the right of the midline has been incised.
Facing page 288
Fig. 2. Haematoxylin and eosin TS x 160 .The muscle fibres are pal e and an ucl eate. They are separated from each o lh er by clear spaces devoid of cap illaries. The interfascicula r fibrous tissue is disrupted.
Fig. 3. The blood supply of the pectoral muscles of the turkey: modified from Orr & Riddell (19 77). A pectoral artery, B cranial branch of pectoral artery, C caudal branch of pectoral artery, D sternoclavicular artery.
Fig. 4. Micropaque- perfusion angiography. The vascular pattern in the pectoral muscles of a normal lurkey. There are differences in the distribution of the long caudal branches on the two sides. but neither pectoral muscle was infarcted.
Fig. 5. Micropaque- perfusion angiography. Variability in vascularization of the pectoral muscles. Note differences between the two sides and the long co urse of the caudal pectoral vessels. There was an infarcted. green zone in the avascular area (asterix).
Fig. 6. (a ) Radiograph of sagittal slice after Micropaque perfusion: collateral circulation in distribution of cauda l pectoral artery. Note ischaemic area (asterix). (b ) PTAH preparation. The pale area represents infarction in the distribution of the avascular area shown in (a ).
289
ISCHAEMIC PECTORAL MYOPATHY IN TURKEYS
of the infarcted zones is not restored the dead tissue becomes carnified, assuming a characteristic hard texture. The green colour, and the presence of haemosiderin in the margins of the lesion suggests that haemorrhage is a feature of an early stage. Indeed, an area of reddish discolouration and oedema has been observed in an early lesion (Harper et at., 1975). The lesions are defined by their vascular territory : for examp le, they always abut interfascicular fibrous septa. Orr & Riddell ( 1977 ) tried to reproduce the green lesion by ligating the cranial and caudal pectoral arteries , but they could not produce consistent results and were thus unable to suggest a mechanism for the appearance of the naturally-occurring myopathy. However, Siller et at. (1978 ) have recently produced similar lesions in White Leghorn fowls by ligation of the subclavian artery on one side. The simil arity of the gross and microscopical appearances of the lesions of green muscle disease to those found in infarction of muscle in the anterior tibial compartment syndrome in man have been noted by others {Orr & Riddell , 197 In the latter syndrome infarction of the muscles of the anterior tibial compartment occurs in untrained subjects during prolonged strenuous exercise (Hughes , 1948). It has been suggested that unaccustomed exertion leads to trauma to muscle fibres . The resulting intramuscular oedema causes a rise in pressure in the anterior tibial compartment, with impairment of its venous drainage and arterial perfusion, and so leads to haemorrhagic infarction (Hughes, 1948 ; Carter, Richards & Zachary, 1949). I n man there is a marked predominance of Type I fast-contracting, oxidative, ATPasedependent fibres in these muscles {Johnson et at., 1973). These fibres may be likely to undergo swelling and infarction if subjected to ischaemia. The white pectoral muscles of the turkey consist of fibres of a simi lar histochemical type. The blood supply of the pectoral muscles of the turkey is dependent on long vessels which enter the anterior parts of these muscles (Fig. 3) and traverse it in a caudal direction. These vessels are vulnerable to compression as they pass through the dense fibrous septa which divide the muscle into large fascic uli (Fig. 1). The fascicular pattern of the muscle is well developed and corresponds to the zones of infarction found in birds with pectoral myopathy (Figs 5 and 6). Variability in the size and length of the caudal and cranial pectoral vessels, and of the sternoclavicular artery may lead to local vulnerability of parts of these muscles situated at the furthest extent of the arterial tree of these vessels. We suggest that in these inbred birds, which are not normally allowed to range freely or to attempt flight, these muscles, as in untrained military recruits, may be vulnerable to infarction during sudden muscular effort. The vascular anomalies and the thick fibrous interfascicular septa provide anatomical predisposing factors. The vulnerability of the modern domestic turkey to ischaemic pectoral myopathy is thus probably the result of genetic factors, which have led to the development of very large pectoral muscles with a large and tenuous blood supply, comb ined with environmental factors . It is not known whether it might be possible to prevent the development of pectoral muscle infarction by allowing these birds free range, and thus more exercise, during development. However, since we have observed marked variability in the pattern of the vasculature of the pectoral muscles it might also be possible to prevent the disease by selective breeding from birds known not to be susceptible.
n
290
BRITISH VETERINARY JOURNAL, 135,3 REFERENCES
CARTER, A. B., RICHARDS, R. L. & ZACHARY, R. B. (J949). Lancet ii, 928. D UBOWITZ, V. & BROOKE, M. H. (J973). Muscle Biopsy: A Modem Approach. London : W . B. Saunders. GLENNY, F. H. (195 I). Ohiojoumal of Science 51,47 . HARPER,]. A., BERNIER, P. E., HELFER, D. H . & SCHMITZ,]' A . (1975).joumal ofHeredity 66,362. HARPER,]. A ., BERNIER, P. E., STEVENS,]. 0. & DICKINSON, E. M . (J969). Poultry Science 48, 1816. H UGHES,]. R. (J948).joumal of Bone andjoint Surgery 30B, 58!. J OHNSON, M . A ., POLGAR, ]., WEIGHTMAN, D. & ApPLETON, D . (1973). joumal of Neurological Science 18, iii. J ONES,]. M. (1974). Turkeys 22,20. JONES,]. M ., KING, N . R. & MULLINER, M. ( 1974). Poultry Science 15, 19!. ORR,]. P. & RIDDELL, C . ( 1977). Americanjoumal of Veterinary Research 38, 1237 . PETTIT,]. R. & VAN DREUMEL, A. A. (1973 ). Canadian Veterinaryjoumal14, 198 . SILLER, W . G ., WIGHT, P . A ., MARTINDALE, L. & BANNISTER, D . W . (J978 ). Research in Veterinary Science 24, 267.
(Acceptedforpublication 5 September 1978)