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Increase in Diameter of Muscle Fibres in the Foetal Pig
Br. vet.
J.
(197 2 ),
128,
355
INCREASE IN DIAMETER OF MUSCLE FIBRES IN THE FOETAL PIG By D. C.
THURLEY
*
Central Veterinary Laboratory, Weybridge, Surrey
SUMMARY
Skeletal muscle fibres from foetal pigs between 45 days' gestation and 7 days after birth were examined and their growth in diameter measured by light microscopy of paraffin sections. Three developmental stages are described : A. Development oflarge myotubes. Up to day 55. B . Production of small, usually non-tubular, fibres between the myotubes. Days 55-100. C. Generalized growth in fibre diameter with disappearance of tubular forms. Mter day 100. INTRODUCTION
Though electronmicroscopy has probably contributed most to the recent advances in our understanding of the dynamics of foetal muscle formation (Boyd, 1960), light microscopy is still the principal tool used in studies on muscle morphology and in diagnostic pathology. Muscle histogenesis at the light microscope level is well documented in general terms (M aximow & Bloom, 1967; Le Gros Clark, 1965), but the time-scale of increasing mean fibre diameter (MFD ) in individual species has received rather less attention; in the case of the pig information is entirely lacking. As such information would be of interest and might be of help to the pathologist studying developmental myopathies of the pig, for example, congenital splayleg (Thurley, Gilbert, & Done, 1967), the opportunity to measure the fibre diameter in foetal pigs was taken when, for other purposes, a number of pregnant gilts and sows were slaughtered at known periods after conception. This paper describes some changes in morphology that occur from 45 d ays' gestation onwards. MATERIALS AND METHODS
Thirteen heterogeneous sows were served twice daily throughout oestrus by a single British Saddleback X Landrace boar. Two sows were allowed to farrow naturally and their litters killed at either 1 or 7 days of age. The remaining sows were killed and their litters harvested at the gestational ages shown in Fig. I. Conception was assumed to have occurred 2 days after the onset of oestrus. One piglet from each of the naturally-born litters, and two foetuses from each of the others, were available for this study. The piglets and foetuses were fixed by immersion in the Dubosq-Brazil • Present address: Wallaceville Animal Resear ch Centre, Private Bag, Wellington, New Zealand.
BRITISH VETERINARY JOURNAL, 128, 7
modification of Bouin's fluid (Lee, 1950) ; only as much superficial dissection of the larger animals was carried out as was necessary to ensure efficient penetration of the fixative. To obtain a good transverse section of the gracilis muscle, blocks were taken from the mid-femoral region, embedded in paraffin, cut at 5 flm and stained by haematoxylin and eosin. The MFD of each specimen was determined by measuring about 100 fibres traced out from a camera lucida projection. The space enclosed by the perimysium was not always filled by myofibrils. Measurements were made of the width of the area occupied in the section by myofibrils, but including the central cavity in myotubular forms. The gracilis muscle was the one measured in most cases, but occasionally another hind leg muscle had to be used. RESULTS
The development of muscle fibres in the foetal pig may be conveniently divided into three phases (Fig. I). Gestation al Age 45
Average fibre Diameter(tL)T 6·2
48
6·8
50
7·7
55
7·6
60
45
75
2·6
78 85
90
-'III
3·4
---
~
I
Phose B
3-4
3·4
97
4·5
104
5·9
IDoy 116 old 7 Doys 123
6·5
old
Phase A
t
Phase
---- 1 103
2-44 '77'094 11·714·116·5
Fibre diameter (fl.)
Fig.
I.
Changes in mean fibre diameter (MFD) of foetal muscle with age.
Phase A. Prevalence of large myotubes: up to day 55. During this phase the primitive elements differentiate to form large muscle fibres which grow from about 7 flm at 45 days' gestation to about I I flm at 55 days, a few reaching 14 flm or more. These are myotubes, and MFD measurements include the central cavity. Earlier in this gestation period, the "rind" of myofibrils is
PLATE
Fig .
2.
r
PE 23 49. Transve rse sec tion of fo eta l pig muscl e a t 5 ' d ays' ges tation. H a ematox yli n and eosin . x 635.
F ig. 3. PE 245 I. T.S . fo e tal pig muscl e at 72 da ys' gesta tion. Haematoxy li n a nd eosin. x 635.
Thurley, B/,. vel .
J.
( 1972 ), 128, 7
PLATE II
Fig. 4. PE 2630. T.S . foeta l pig muscle at 93 days ' ges ta ti o n. H aema toxylin and eosin. x 635 .
Thu rley, Br. vet.
J. ( 1972), 128, 7
MUSCLE FIBRE DEVELOPMENT IN FOETAL PIGS
357
proportionately thinner than at the end of that period. Some "satellite" nuclei can be seen at the periphery of the myotubes, which are usually widely separated by very loose connective tissues (Fig. 2). Phase B. Increase in the number of small fibres: day 55-100. During the early part ofthis stage there is a dramatic rise in the number of small fibres (2-3 fLm ). Increasing numbers of small fibres which mayor m ay not be myotubes, and frequently have peripheral nuclei, can be found grouped around large myotubular forms. This proliferation causes a sharp fall in the mean diameter of all fibres, although the la rge myotubes are still as numerous as before (Fig. 3). During this phase the mean size of the large myotubes gradually decreases as the size of the smaller fibres increases. There is consequently rela tively little change in the overall MFD. The intercellular space within groups of muscle fibres is decreased, but that between the groups remains large. The connective tissue elements begin to organize into sheaths that enclose groups of fibres. Phase C. Growth of small fibres: from 100 days onward. In this phase there is probably little formation of new fibres, as judged by the proportion of fibres 3 fLm or less in diameter, but there is rapid growth of the existing forms. Recognizable myotubes become rare, and nearly all nuclei are peripheral. The distribution curve of fibre sizes becomes more flat topped. DIS CUSS ION
There are considerable difficulties in measuring muscle fibre diameters, and this is especially so about the time of birth when the perimysium does not always appear to be filled by myofibrils. The effect of different fixatives is also variable, and usually there is considerable shrinkage in the MFD (Goldspink, 1963). Different breeds of pigs vary considerably in mature body size, and the effect of this on the embryo, combined with the effects of maternal nutrition on the offspring, may all possibly lead to variation. These factors combine to allow little reliance to be put on the measurement of MFD in absolute terms, but by standardizing as many factors as possible, some accuracy may be claimed for the relative changes in fibre size, and for the morphological changes with age. ACKNOWLEDGEMENT
Part of this work was included in a thesis submitted for the degree of Ph.D. of London University. REFERENCES
BOYD,J. D. (1960). Structure and Function of Muscle, Vol. I, ed. Bourne, G. H. London: Academic Press. GOLDSPINK, G. (1963). Ph.D. Thesis, Trinity College, Dublin. LEE, B. (1950). The Microtomist's Vade-Mecum. 1 Ith edn., p. 325. London: Churchill. LE GROS CLARK, W . E. (1965). Tissues oj the Body, 5th edn. Oxford: Clarendon Press. MAXIM ow, A. G. & BLOOM, W. (1957). A Textbook ojHist%gy, 7th edn. Philadelphia : Saunders. THURLEY, D. C. GILBERT, F. R. & DONE, J. T. (1967). Vet. Rec. So, 302. (Acceptedjor publication 24 January 1972)
35 8
BRITISH VETERINARY JOURNAL,
12 9,
7
AUgD1entation du dilunetre des fibres Dlusculaires chez Ie cochon foetal (Thurley) ResUDle. Entre les 45 jours de gestation et les 7 jours suivant la mise bas, les fibres musculaires striees du cochon foetal ont ete examinees et la croissance de leur diametre mesuree par sections histologiques paraffinees sous Ie microscope. Trois stages de developpement sont decrits: (a) Jusqu'au 55 ieme jour, developpement de larges tubes de tissu musculaire. (b) Du 55 ieme au 100 ieme jour, production de petites fibres, generalement non musculaires entre les tubes de tissu musculaire. (c) Apres Ie 100 ieme jour, croissance generalisee du diametre des fibres avec la disparition des formes tubulaires. Vergrosserung des DurchDlessers bei den Muskelfasern des Schweinefoetus (Thurley) Zusanunenfassung. Skelettmuskelfasern von Schweinen wurden vom 45. Tage der Trachtigkeit beim Foetus und beim Ferkel bis zum 7. Tage nach dem Wurfuntersucht und das Wachsen des Durchmessers histologisch in Paraffinschnitten rnikroskopisch gemessen. Drei Entwicklungsstadien werden beschrieben: (a) Entwicklung rohrenforrnigen muskularen Gewebes; bis zum 55. Tag. (b) Entstehung feiner, meistens nicht rohrenformiger Fasern zwischen dem rohrenformigen Muskelgewebe. 55.-100. Tag. (c) Allgemeines Wachstum des Durchmessers der Fasern und Ver~chwinden der rohrenfOrmigen Formen. Nach dem 100. Tag. AUDlento en diaDletro de las fibras Dlusculares del feto del cerdo (Thurley) ResUDlen. Se examinaron fibras musculares procedentes de fetos de cerdos entre el dia 45 de la gestaci6n y el dia 7 despues del nacimiento y se midi6 su crecimiento en diametro por microscopia simple de secciones de parafina. Se describen tres fases del desarrollo: (a) Desarrollo de grandes miotubulos. Hasta el dia 55. (b) Producci6n de fibras pequenas, generalmente no tubulares, entre los miotubulos, Dias 55100.
(c) Crecimiento generalizado en el diametro de las fibras con desaparici6n de las formas tubulares. Despues del dia 100.
J.
(197 2 ),
128,
355
INCREASE IN DIAMETER OF MUSCLE FIBRES IN THE FOETAL PIG By D. C.
THURLEY
*
Central Veterinary Laboratory, Weybridge, Surrey
SUMMARY
Skeletal muscle fibres from foetal pigs between 45 days' gestation and 7 days after birth were examined and their growth in diameter measured by light microscopy of paraffin sections. Three developmental stages are described : A. Development oflarge myotubes. Up to day 55. B . Production of small, usually non-tubular, fibres between the myotubes. Days 55-100. C. Generalized growth in fibre diameter with disappearance of tubular forms. Mter day 100. INTRODUCTION
Though electronmicroscopy has probably contributed most to the recent advances in our understanding of the dynamics of foetal muscle formation (Boyd, 1960), light microscopy is still the principal tool used in studies on muscle morphology and in diagnostic pathology. Muscle histogenesis at the light microscope level is well documented in general terms (M aximow & Bloom, 1967; Le Gros Clark, 1965), but the time-scale of increasing mean fibre diameter (MFD ) in individual species has received rather less attention; in the case of the pig information is entirely lacking. As such information would be of interest and might be of help to the pathologist studying developmental myopathies of the pig, for example, congenital splayleg (Thurley, Gilbert, & Done, 1967), the opportunity to measure the fibre diameter in foetal pigs was taken when, for other purposes, a number of pregnant gilts and sows were slaughtered at known periods after conception. This paper describes some changes in morphology that occur from 45 d ays' gestation onwards. MATERIALS AND METHODS
Thirteen heterogeneous sows were served twice daily throughout oestrus by a single British Saddleback X Landrace boar. Two sows were allowed to farrow naturally and their litters killed at either 1 or 7 days of age. The remaining sows were killed and their litters harvested at the gestational ages shown in Fig. I. Conception was assumed to have occurred 2 days after the onset of oestrus. One piglet from each of the naturally-born litters, and two foetuses from each of the others, were available for this study. The piglets and foetuses were fixed by immersion in the Dubosq-Brazil • Present address: Wallaceville Animal Resear ch Centre, Private Bag, Wellington, New Zealand.
BRITISH VETERINARY JOURNAL, 128, 7
modification of Bouin's fluid (Lee, 1950) ; only as much superficial dissection of the larger animals was carried out as was necessary to ensure efficient penetration of the fixative. To obtain a good transverse section of the gracilis muscle, blocks were taken from the mid-femoral region, embedded in paraffin, cut at 5 flm and stained by haematoxylin and eosin. The MFD of each specimen was determined by measuring about 100 fibres traced out from a camera lucida projection. The space enclosed by the perimysium was not always filled by myofibrils. Measurements were made of the width of the area occupied in the section by myofibrils, but including the central cavity in myotubular forms. The gracilis muscle was the one measured in most cases, but occasionally another hind leg muscle had to be used. RESULTS
The development of muscle fibres in the foetal pig may be conveniently divided into three phases (Fig. I). Gestation al Age 45
Average fibre Diameter(tL)T 6·2
48
6·8
50
7·7
55
7·6
60
45
75
2·6
78 85
90
-'III
3·4
---
~
I
Phose B
3-4
3·4
97
4·5
104
5·9
IDoy 116 old 7 Doys 123
6·5
old
Phase A
t
Phase
---- 1 103
2-44 '77'094 11·714·116·5
Fibre diameter (fl.)
Fig.
I.
Changes in mean fibre diameter (MFD) of foetal muscle with age.
Phase A. Prevalence of large myotubes: up to day 55. During this phase the primitive elements differentiate to form large muscle fibres which grow from about 7 flm at 45 days' gestation to about I I flm at 55 days, a few reaching 14 flm or more. These are myotubes, and MFD measurements include the central cavity. Earlier in this gestation period, the "rind" of myofibrils is
PLATE
Fig .
2.
r
PE 23 49. Transve rse sec tion of fo eta l pig muscl e a t 5 ' d ays' ges tation. H a ematox yli n and eosin . x 635.
F ig. 3. PE 245 I. T.S . fo e tal pig muscl e at 72 da ys' gesta tion. Haematoxy li n a nd eosin. x 635.
Thurley, B/,. vel .
J.
( 1972 ), 128, 7
PLATE II
Fig. 4. PE 2630. T.S . foeta l pig muscle at 93 days ' ges ta ti o n. H aema toxylin and eosin. x 635 .
Thu rley, Br. vet.
J. ( 1972), 128, 7
MUSCLE FIBRE DEVELOPMENT IN FOETAL PIGS
357
proportionately thinner than at the end of that period. Some "satellite" nuclei can be seen at the periphery of the myotubes, which are usually widely separated by very loose connective tissues (Fig. 2). Phase B. Increase in the number of small fibres: day 55-100. During the early part ofthis stage there is a dramatic rise in the number of small fibres (2-3 fLm ). Increasing numbers of small fibres which mayor m ay not be myotubes, and frequently have peripheral nuclei, can be found grouped around large myotubular forms. This proliferation causes a sharp fall in the mean diameter of all fibres, although the la rge myotubes are still as numerous as before (Fig. 3). During this phase the mean size of the large myotubes gradually decreases as the size of the smaller fibres increases. There is consequently rela tively little change in the overall MFD. The intercellular space within groups of muscle fibres is decreased, but that between the groups remains large. The connective tissue elements begin to organize into sheaths that enclose groups of fibres. Phase C. Growth of small fibres: from 100 days onward. In this phase there is probably little formation of new fibres, as judged by the proportion of fibres 3 fLm or less in diameter, but there is rapid growth of the existing forms. Recognizable myotubes become rare, and nearly all nuclei are peripheral. The distribution curve of fibre sizes becomes more flat topped. DIS CUSS ION
There are considerable difficulties in measuring muscle fibre diameters, and this is especially so about the time of birth when the perimysium does not always appear to be filled by myofibrils. The effect of different fixatives is also variable, and usually there is considerable shrinkage in the MFD (Goldspink, 1963). Different breeds of pigs vary considerably in mature body size, and the effect of this on the embryo, combined with the effects of maternal nutrition on the offspring, may all possibly lead to variation. These factors combine to allow little reliance to be put on the measurement of MFD in absolute terms, but by standardizing as many factors as possible, some accuracy may be claimed for the relative changes in fibre size, and for the morphological changes with age. ACKNOWLEDGEMENT
Part of this work was included in a thesis submitted for the degree of Ph.D. of London University. REFERENCES
BOYD,J. D. (1960). Structure and Function of Muscle, Vol. I, ed. Bourne, G. H. London: Academic Press. GOLDSPINK, G. (1963). Ph.D. Thesis, Trinity College, Dublin. LEE, B. (1950). The Microtomist's Vade-Mecum. 1 Ith edn., p. 325. London: Churchill. LE GROS CLARK, W . E. (1965). Tissues oj the Body, 5th edn. Oxford: Clarendon Press. MAXIM ow, A. G. & BLOOM, W. (1957). A Textbook ojHist%gy, 7th edn. Philadelphia : Saunders. THURLEY, D. C. GILBERT, F. R. & DONE, J. T. (1967). Vet. Rec. So, 302. (Acceptedjor publication 24 January 1972)
35 8
BRITISH VETERINARY JOURNAL,
12 9,
7
AUgD1entation du dilunetre des fibres Dlusculaires chez Ie cochon foetal (Thurley) ResUDle. Entre les 45 jours de gestation et les 7 jours suivant la mise bas, les fibres musculaires striees du cochon foetal ont ete examinees et la croissance de leur diametre mesuree par sections histologiques paraffinees sous Ie microscope. Trois stages de developpement sont decrits: (a) Jusqu'au 55 ieme jour, developpement de larges tubes de tissu musculaire. (b) Du 55 ieme au 100 ieme jour, production de petites fibres, generalement non musculaires entre les tubes de tissu musculaire. (c) Apres Ie 100 ieme jour, croissance generalisee du diametre des fibres avec la disparition des formes tubulaires. Vergrosserung des DurchDlessers bei den Muskelfasern des Schweinefoetus (Thurley) Zusanunenfassung. Skelettmuskelfasern von Schweinen wurden vom 45. Tage der Trachtigkeit beim Foetus und beim Ferkel bis zum 7. Tage nach dem Wurfuntersucht und das Wachsen des Durchmessers histologisch in Paraffinschnitten rnikroskopisch gemessen. Drei Entwicklungsstadien werden beschrieben: (a) Entwicklung rohrenforrnigen muskularen Gewebes; bis zum 55. Tag. (b) Entstehung feiner, meistens nicht rohrenformiger Fasern zwischen dem rohrenformigen Muskelgewebe. 55.-100. Tag. (c) Allgemeines Wachstum des Durchmessers der Fasern und Ver~chwinden der rohrenfOrmigen Formen. Nach dem 100. Tag. AUDlento en diaDletro de las fibras Dlusculares del feto del cerdo (Thurley) ResUDlen. Se examinaron fibras musculares procedentes de fetos de cerdos entre el dia 45 de la gestaci6n y el dia 7 despues del nacimiento y se midi6 su crecimiento en diametro por microscopia simple de secciones de parafina. Se describen tres fases del desarrollo: (a) Desarrollo de grandes miotubulos. Hasta el dia 55. (b) Producci6n de fibras pequenas, generalmente no tubulares, entre los miotubulos, Dias 55100.
(c) Crecimiento generalizado en el diametro de las fibras con desaparici6n de las formas tubulares. Despues del dia 100.