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AN ANATOMICAL STUDY OF ADAPTIVE PROCESSES  
IN MUSCLE 
A thesis presented in partial fulfilment 
of the requirements for the Degree of 
Doctor of Philosophy at Massey University. 
NALLATHAMBY SIVACHELVAN 
1977 - 1980 
This work is dedicated to Dr W J Pryor , Assistant Director , 
Department of Primary Industry , Australia and former Dean 
of the Faculty of Veterinary Science , Massey University , 
who f irst  cleared a way for me in the direction of my 
aspirations . 
(ii ) 
Abstract of  a thesis  presented in partial fulfilment of the 
requirements of the Degree of Doctor of Philosophy . 
AN ANATOMICAL STUDY OF ADAPTIVE PROCESSES  IN MUSCLE 
b y  NALLATHAMBY S IVAC HELVAN 
The mechanisms involved in  the adaptability of muscle to 
functional changes have been tested using sheep as the 
experimental animal . 
( 1 )  A quantitative study of  the antenatal and postnatal 
development of  skeletal muscle was undertaken using the 
semitendinosus muscle of eighteen fetuses from 60 days 
gestation to birth and of  fif teen sheep from birth to 
adulthood . The muscle was also used as an experimental 
model to test antenatal anticipation of postnatal muscle 
func tions . The alkali-stabile myosin ATPase technique was 
used to ident ify and quantify the histochemical ATPase low 
(AL) and ATPase high (AH) fibres and to follow both fibre 
and fascicular growth within the muscle . Histochemical 
changes occurring within the developing fibres were also 
recognjzed . 
Presumptive AL f ibres had centrally occupying nuclei up to 
80 days . A smaller secondary fibre population formed the 
presumptive AH fibres during this period of development .  
From eighty days of gestation onwards , two distinct fibre 
types could be  observed histochemically in the muscle . The 
AL fibre stained pale while the AH f ibre stained dark . 
Areas of variable f ibre type population density were 
distinct within the muscle in all stages studied from 80 
(iii) 
days of  gestation. The highest population density of  
ATPase  low fibres was observed in the craniomedial aspect 
of the muscle (AL dense area ) . The lowest  AL f ibre 
population density was seen in the caudolateral aspec t of  the 
muscle  ( the AL sparse area ) .  The AL fibre percentage within 
the AL dense area of the muscle increased from about 10% to about 
30% from 80 days of  gestation to adulthood whereas in the AL 
sparse area the AL fibre type population density remained at  
about 4% throughout this period . 
Simultaneous electromyographic s tudies using the semitendinosus 
muscle  of three adult sheep suggested that the AL dense area 
is preferentially used for posture and during quiet  co-ordinated 
ac tivity ,  while the AL sparse  area is recrui ted only 
intermittently when the hip and stifle joints are less  
co-ordinated in movement .  By  using angiographic studies on 
the muscle , many large and c losely spaced blood vessels were 
seen to run in the AL dense area . Thus ,  a metabolic and 
nutritional prerequisite for the mechanically disadvantageous 
disposition of  the AL fibres in the deep part of  a muscle with 
a heterogeneous f ibre type d istribution has been suggested . 
A postural and propulsive involvement of  the muscle and an 
increase in percentage of AL fibres along with increasing 
body weight suggests a functional adaptatory change . 
The part of this change occurring antenatally is  expected 
to be the result of genetic  anticipation . 
A marked reduc tion in perimysial connective tissue occurred 
antenatally but no significant changes  occurred postnatally. 
The change involving endomysial connective tissue , on the 
(iv)  
other hand , was less  marked throughout the development . 
Also , the number of  f ibres per fascicle was constant both 
antenatally and postnatally . The perimysial enclosed fascicles 
increased in number antenatally but remained constant 
postnatally. This  suggests a role for the connective 
tissue framework of a muscle in constraining the growth 
in number of fascicles and f ibres . 
( 2 )  The function o f  one hind limb of  the sheep was modified 
to study the growth changes in the musculoskeletal sys tem . 
Seven lambs treated with one hind limb bound to the body 
with the hip fully flexed and the st ifle and hock fully 
extended were reared from the day af ter birth to about 
three months of  age , together with two untreated controls . 
A carcass dissection study was made of the treated and 
control lamb s .  Changes which occurred in the semitendinosus 
mu.scle were  studied using histochemistry and electron 
microscopy . 
All the supporting limbs of the treated lambs showed growth 
related changes induced by the treatment , suggesting a 
diagonal support of  body weight . The semitendinosus muscle 
of  the bound limb was heavier than that of  the controls and 
that of the supporting l imb , while the quadriceps muscle 
was heavier on the supporting side and lighter on the 
bound side . These resul ts from the semitendinosus 
and quadriceps muscles support the view that stretch is 
an important requirement of muscle hypertrophy . The 
semitendinosus muscle , stretched over two j oints ,  
hypertrophies to a greater extent than a muscle like the 
semimembranosus , stretched over one joint . The size 
increase of  other muscles  such as  the extensors of the 
stifle joint of the supporting hind limb may also have 
been due to  stretch . A trend was observed for the f emur 
(v) 
and tibia  to be more mineralised and thicker , but shorter , 
on the supporting than on the bound side of  the body . 
The histochemical study of the semitendinosus muscle 
indicated that both AL and AH f ibres are induced to grow 
by the stretch stimulus . An increase in the AL fibre type 
population as a percentage of  the regional f ibre population 
in the deep part of the semitendinosus muscle suggests that 
chronic abnormal usage of a muscle can produce an adaptive 
response . Thus ,  thi s  exaggerates a similar effect 
observable during normal growth of an animal because of 
its increase in body weight . During the growth of  a muscle 
under immobilized but s tretched conditions , the shape , the 
amount of  connective tissue and the size of fascicle and 
fibre components appear to maintain isometric proportions . 
Electron microscopically,  no abnormalities in the arrangement 
of the subcellular components  and in the proportions of  
myofibrillar , sarcoplasmic and mitochondrial e lements 
within the fibres of  the hypertrophied semitendinosus 
muscle were revealed . 
(vi ) 
TABLE OF CONTENTS 
ACKNOWLEDGEMENTS 
1 .  GENERAL INTRODUCTION 
2 . 0  PART 1 :  ANTENATAL AND POSTNATAL DEVELOPMENT 
OF THE SEMITENDINOSUS MUSCLE OF THE 
SHEEP 
2 . 1 INTRODUCTION 
2 . 2  MATERIALS AND METHODS 
2 . 2 . 1  SOURCE, NUMBER, SEX AND DEVELOPMENTAL STAGES 
OF ANIMALS USED 
2 . 2 . 2 PREPARATION OF HISTOLOGICAL MATERIAL 
2 . 2 . 2 . 1 Collection of samples 
2 . 2 . 2 . 2  Sectioning and staining procedure 
2 . 2 . 3 
2 . 2 . 4 
DETERMINATION OF THE PATTERN OF HISTOCHEMICAL 
FIBRE TYPE POPULATION DENSITY IN SECTIONS OF 
THE SEMITENDINOSUS MUSCLE 
DETERMINATION OF TOTAL FASCICULAR, TOTAL FIBRE, 
Page 
1 
4 
4 
1 2  
1 2  
1 3  
1 3  
1 3  
1 4  
FASCICULAR AND FIBRE TRANSVERSE SECTIONAL AREAS 1 5  
2 . 2 . 5  ELECTROMYOGRAPHIC STUDY 
2 . 2 . 5 . 1  Surgical procedure 
2 . 2 . 5 . 2  Recording procedure 
2 . 2 . 6  ANGIOGRAPHIC STUDIES 
2 . 2 . 6 . 1 Injection of resin 
2 . 2 . 6 . 2  Maceration 
2 . 2 . 6 . 3  Washing and trimming of the 
2 . 2 . 6 . 4 Pre para. tion of angiogram 
2 . 2 . 7 STATISTICAL METHODS 
RESULTS 
ca.st 
2 . 3  
2 . 3 . 1 ATTACHMENTS AND ORIENTATION OF THE 
SEMITENDINOSUS MUSCLE OF THE SHEEP 
2 . 3 . 2  MORPHOGENESIS AND DIFFERENTIATION OF MYOSIN 
ATPase FIBRE TYPES 
2 . 3 . 6 ATPase LOW FIBRE DISTRIBUTION PATTERN WITHIN 
THE SEMITENDINOSUS MUSCLE 
2 .  3 .  4 . QUANTITATIVE GROWTH ANALYSIS 
1 7  
1 7  
1 8  
19  
20 
20 
2 1  
2 1  
2 1  
22  
22  
23  
24 
25 
2 . 3 . 4 . 1  Fascicular growth 
2 . 3 . 4 . 2  Growth in fibre transverse sectional area 
2 . 3 . 4 . 3  Changes in connective tissue 
2 . 3 . 4 . 4  Changes in muscle shape and mechanics 
2 . 3 . 4 . 5  Estimation of total fibre number 
2 . 3 . 5  ELECTROMYOGRAPHIC STUDY 
2 . 3 . 6  ANGIOGRAPHIC STUDIES 
2 . 4 DISCUSSION 
2 • 4 .  1 ANALYTICAL METHODS 
2 . 4 . 2 GROWTH OF FIBRE AND FASCICULAR TRANSVERSE 
SECTIONAL AREAS 
2 . 4 . 3  
2 . 4 . 4  
2 . 4 . 5 
2 . 4 . 6  
2 . 4 . 7  
3 . 0  
3 . 1 
3 . 2  
3 .  2 . 1  
3 . 2 . 2  
3 . 2 . 3  
3 . 2 . 4  
3 . 2 . 5  
3 . 2 . 6  
3 . 2 . 7  
3 . 2 . 8  
3 . 2 . 9  
DIFFERENTIATION OF MUSCLE FIBRE TYPES 
MORPHOGENESIS OF ISOENZYMES OF MYOSIN 
THE SIGNIFICANCE OF FIBRE TYPE DISTRIBUTION 
WITHIN THE SEMITENDINOSUS MUSCLE 
CHANGES IN MYOSIN A TPase LOW FIBRE PROPORTION 
DEVELOPMENT OF FIBRE TYPE PATTERNS IN THE 
FETUS 
PART 2 :  EXPERIMENTAL INDUCTION OF RELATIVE 
GROWTH CHANGES IN THE MUSCULOSKELETAL 
SYSTEM OF THE SHEEP 
INTRODUCTION 
MATERIALS AND METHODS 
SOURCE OF MATERIAL 
BINDING OF LIMBS 
MANAGEMENT AND FEEDING 
SAMPLING PROCEDURE 
ELECTRON MICROSCOPIC STUDIES 
HISTOCHEMICAL AND QUANTITATIVE STUDIES 
CARCASS DISSECTION 'STUDIES 
BONE MEASUREMENTS 
STATISTICAL METHODS 
Page 
25 
26 
26 
2 7  
2 8  
2 8  
2 9  
2 9  
2 9  
3 1  
34 
35  
36  
4 1  
4 1  
44  
44  
49 
49  
49 
49 
50  
5 1  
5 3  
53  
53  
5 4  
3 . 3  
3 . 3 . 1  
3.3 . 2  
3 .  3 .  3 
3 .  3 . 4  
3 . 4 .  
3 . 4 . 1  
3 . 4 . 2  
3 . 4 . 3  
3 . 4 . 4 
3 . 4 . 5  
4 . 0 
RESULTS 
CARCASS DISSECTION STUDIES 
BONE MEASUREMENTS 
QUANTITATIVE ANALYSIS OF THE SEMITENDINOSUS 
MUSCLE 
ELECTRON MICROSCOPIC STUDIES 
DISCUSSION 
INCREASED GROWTH OF MUSCLES AND BONES DUE 
TO INCREASED WORK LOAD 
GROWTH OF MUSCLES DUE TO STRETCH AND 
RESTRICTED CONTRACTION 
GROWTH CHANGES IN BONES 
HISTOLOGICAL STUDIES 
ELECTRON MICROSCOPIC STUDIES 
GENERAL CONCLUSIONS 
TABLES 
FIGURES 
APPENDICES 
REFERENCES 
Page 
55 
55  
5 7  
5 8  
5 9  
5 9  
5 9  
6 1  
63 
64 
6 7  
69  
7 2 - 84 
85 - 120 
1 2 1  - 126  
1 2 7  - 141  
ACKNOWLEDGEMENTS 
I am deeply indebted to my chief supervisor Dr A S Davies for his 
guidance in research and for giving encouragement and everwilling 
co-operation throughout the research period , and to my eo-supervisor 
Professor R E Munford for his statistical advice as  well  as  providing 
facilities in the Department of Physiology and Anatomy , Massey 
University to carry out this research . The advice and help of Mr 
J C Newhook in the electromyographic studies is gratefully 
acknowledged . 
The sheep used for Part 1 were bred and supplied by Dr T E Broad 
of the Depar tment of Scientific and Industrial Research ,  
Palmerston North . 
The technical guidance offered by Mrs Pamela Slack of the Department 
of Pathology and Public Health , Massey University and Mr D Hopcroft  
o f  the D . S. I . R . , Palmerston North in acquainting myself with electron 
microscopic techniques is very much appreciated . Mrs Sharon Averill 
of the Meat Industry Research Institute , Hamilton , and Miss  Sandra 
Mallach of the Depar tment of Pathology and Public Health ,  Massey 
University , gave technical assistance in electron microscopy , and 
Messrs C G Fletcher and R P  Hansen in histochemistry . 
My sincere thanks are due to Messrs M Ratumaitavuki , N Ward and 
I Denby of  the Department of Physiology and Anatomy for their 
assistance in tending the lambs with altered limb function , and to 
Messrs M Bennett and I Denby for help with carcass  dissect ions . 
The help of  Dr M W  O ' Callaghan and Mr P Murphy in preparing the 
angiogram and corrosion cast ,  respectively, for Fig 21 of the 
thesis is appreciated . 
The assistance of Mr M J Birtles of  the Department of Physiology and 
Anatomy for taking photomicrographs ,  of Mr T G Law of the Faculty of 
Veterinary Science ,  Massey University and Mr G Bennett of D . S . I . R . , 
Palmerston North , for processing the photographs is kindly 
acknowledged . Mr T G Law taught me the techniques of  printing 
photographs . Mr G Trail,  a veterinary undergraduate , drew f igures 
21d and 23  used in the thesis . 
I wish to thank Dr E J Kirk , Mr M J Birtles , Dr A L Hart and 
Mrs Helen Pennington , the friendliness of  whomhas always been 
of inspiration to me . I also gratefully acknowledge the friendly 
co-operation extended by many people , academics, technicians , 
students  and others both within the faculty and outsid e ,  who have 
given my family and me many cherished memories of New Zealand . 
I owe a great deal of my progress to my wife  Malar and children 
Thayalan , Gunalan , Sudar and Bhama . My deep appreciation is 
due to them for extending their affection and moral support 
during this whole stimulating phase of my lif e .  
1 . 0 GENERAL INTRODUCTION 
A proportionate  and orderly distribution and growth of muscles 
throughout the body of an animal is maintained genetically 
under normal c ircumstances . Such a basic growth pattern may 
be modified applying an intensive selection programme , as used 
in improving meat production or athletic potential . Differences 
are seen in both muscle distribution and in growth pattern 
between species (Berg & Butterfield , 1 9 76) , breeds (Davies , 
1 9 74 ;  Gunn , 1 9 7 5 )  and sex (Lohse , 1 973 ;  Berg & Butterfield ,  
1 9 76 ) , and can be  attributed to differences in the func tional 
requirements of  genetically different types of  animals . 
Within a genetic framework , relative growth changes in muscles  
are  essential to  accommodate variation in  the functional needs 
activity patterns of an animal will make growth changes in 
muscles desirable . McMeekan ( 1 940)  stated that those parts of  
the body essential for life  processes and body function are 
relatively well  developed at birth and , as a cons�quence, they 
increase to a small extent postnatally while those organs 
connec ted with movement , storage or reserve are poorly developed 
at birth,  but grow more during postnatal life . Fowler ( 1 968 )  
proposed that an  animal tends to  adjust  to  environmental changes 
in such a way that the vital  functional relationships between 
essential body components are preserved ,  or modified to a form 
which gives the animal its  best  chance of survival and 
successful reproduction: Relative growth rates appear to differ 
between muscles depending on whether they are primarily postural 
in func tion (opposing the gravitational force on the body) , or 
propulsive in function (producing an accelerating force on 
1 
the body) (Davies, l 9 79 ) .  If a modification to the growth 
pattern of muscles is deemed essential , i t  can be anticipated 
to occur in such a way that a functional integrity of the 
whole body is preserved . 
Muscles are adaptable to conditions of altered structural 
integrity or to unexpec ted func tional changes . The postural 
muscles of  an amniote have to support the weight of the 
animal when it begins its  terrestrial mode of life . Various 
diseases affecting only a part of  an animal ' s  musculature can 
initiate adaptations in healthy muscles to restore function 
partially or fully . The se diseases may be intrinsic to the 
affected muscles , such as transport myopathy , muscular 
dystrophy , myositis ,  the Vitamin E - selenium deficiency 
diseases and disuse caused by immobilisation , or they may 
be extrinsic.  These lat ter include neurogenic diseases , for 
example polio myelitis and multiple sclerosis , or mechanical 
conditions resulting from arthritis , bone and j o int surgery 
or amputation , whereby disu·se of one part of the muscular 
system results in greater use elsewhere . The changes resulting 
from training programmes for physical fitness ,  athletics and 
body building can presumably also change the nature of muscles . 
The mechanisms and the types of  adaptation are ,  for most of  
these condi tions , still obscure . 
A muscle can be expected to  undergo both qualitative and 
quantitative changes as an adaptive response . The quantitative 
changes may involve the size of both the whole muscle or the 
f ibres within i t .  The qualitative changes may be  related to 
muscle metabolism, to the contractile properties of the muscle , 
or  to the myofibrillar and myofilamentous constituents in 
addition to the nature of  the connective tissue framework . 
In this thesis , two models  of  the adaptability of  muscle 
2 
have been used to identify the changes involved and to suggest 
the mechanism causing the changes in each case . In Part 1 ,  
qualitative changes in muscle occurring before , during and 
af ter the amniotic to terrestrial changes a t  b irth are 
described in one muscle of the sheep , namely the semitendinosus 
muscle . Contractile properties of the muscle are studied by 
using histochemistry and electromyography , and changes 
involved in the connective tissue components and the muscle 
fibre population are examined .  In Part 2,  quantitative 
and qualitative changes occurring as a result of  immobilization 
in an abnormal limb position and by chronic overload have 
been s tudied using sheep from b irth to 2-3 months of age . 
Quantitative changes are described for muscles and bones 
throughout the trunk and four limbs due to the al tered 
function of  one limb . Qualitative changes as  seen in a 
chronically stretched muscle have been studied using 
histochemistry and elec tron microscopy , again for the 
semitendinosus muscle . Changes in the fibre population 
and in connective tissue components of this muscle have also 
been analysed . 
The histochemical and electromyographic aspects of muscle 
from Part 1 above have been published as Sivachelvan ( 1 9 7 9 )  
and Sivachelvan & Davies ( 1980 , 198 1 ) . 
3 
2.0 PART 1. ANTENATAL AND POSTNATAL DEVELOPMENT OF THE 
SEMITENDINOSUS MUSCLE OF THE SHEEP 
2.1 INTRODUCTION 
postnatal periods . The present  study examines the antenatal 
adaptive changes occurring in a muscle performing a postural 
In early literature , there have been observations on the 
mechanisms which control the growth of  an organism . Aristotle 
made a general observation that growth can be explained in 
terms of completeness  of func tion (Nussbaum , 1 978 ) . Roux 
( 1 905 ) suggested that  the formation of structures in the 
"embryonic period" is predetermined , while the development 
of  structures formed during the "functional development 
period" is brought about by their "specific action" ( Singer , 
1959 ) . S imilar observations are also found in the literature 
relating to the growth of skeletal muscle . Hammond ( 1932 ) 
conducted growth s tudies in sheep and suggested that the 
changes in proportions of a muscle with age can be due to 
a)  function b )  heredity and c )  changes in the shape and 
relative proportions of the different bones . Bryden ( 1 969 , 
1 9 73 )  u sed the elephant seal to study changes in muscle 
distribution occurring in different postnatal growth phases 
and concluded that  postnatal growth changes in size occurring 
4 
in any muscle are governed by a functional adaptatory requirement 
Berg & Butterfield ( 1 9 7 6 )  suggested , from their observations 
based on relative muscle growth in cattle , that  !Jh.e antenatal 
phase of growth in a muscle is almost entirely under the 
influence of a genetic template ,  the immediate postnatal 
phase largely influenced by function , and the prepubertal 
and adolescent  phase a product of  genetic and func tional 
adjustment . Although genetic and environmental influences  
on  growth of skeletal muscle have thus been defined , f ew 
earlier s tudies have attempted to describe the phenotypic 
expression of  the genetic mechanism influencing antenatal 
muscle growth . Muscular adaptations in animal s  which walk 
5 
soon af ter birth , like the sheep (Hammond , 1932 ;  Palsson,  1955 ) ,  
must be greater at birth than at  any other s tage . Nevertheless , 
li ttle consideration has been given to the mechanism. of  this  
perinatal muscular adaptation . Because the f e tus  of  a 
mammalian quadruped grows as an amniotic aquanaut ,  its limbs 
are not used as postural struts . The gravitational force on 
the fetal body is opposed by a force of buoyancy .  The movements 
(Barcroft , Barron & Windle , 1936 ) and electrical ac tivity in 
muscles (AnggKrd & Ottoson , 1963 )  of the limbs of  sheep can 
be  detected during fetal stages ,  but such movements are not 
expected to be involved in pos ture . Af ter bir th ,  gravitational 
force is no longer opposed by an ef fective force of buoyancy, 
but by the animal's musculoskeletal system . The present study 
examines the extent of this postural awareness  antenatally 
in a muscle . 
Growth changes in f ibre size and number within skeletal 
muscle have been studied by several workers .  A constancy of  
f ibre number during postnatal growth has been suggested for 
the human (Maccallum , 1898 ) and for the pig (McMeekan , 
1940) . Joubert (1956a)  used the rectus f emoris ,  gastro-
cnemius and longissimus dorsi muscles of the sheep from 
birth to 290 days and observed that changes  in f ibre diameter 
correspond with changes  in muscle weight . He , therefore , 
suggested that muscular growth is  primarily a function of  
physiological age , and not strictly one of  chronological 
age . Enesco & Puddy ( 1964 ) found that,  in the biceps 
brachii , extensor carpi radiali s ,  gastrocnemius and cranial 
tibial muscles of the rat , fibre number is constant , while 
an increase in muscle weight during growth involves an 
increase in both muscle fibre size and connective tissue 
amount . They noticed no changes in the relative proportions 
of the perimysium, the endomysium and the muscle f ibres . 
Rowe & Goldspink ( 1 969 ) used the cranial tibial , biceps 
brachii , long digital extensor , soleus and s ternomastoideus 
muscles of the mouse , and noticed a constant f ibre number 
longissimus muscle of  the pig and suggested that in addition 
to a constancy in fibre number within postnatal muscles ,  
changes in histochemical fibre type proportions occur with 
growth to compensate for the strength insufficiency of  a 
muscle relat ive to the body mass . In contrast  to the above 
studies which indicated a constant number of f ibres 
postnatally,  Gunn ( 19 7 5 ,  1978 )  used the semitendinosus , 
diaphragm and transverse pectoral muscles of  the dog and 
horse and noticed a postnatal increase in total fibre number . 
His studies also showed that ,  in each muscle studied , 
differences in histochemical f ibre type proportions can be 
observed between non-athletic and a thletic breeds . There 
are also research reports to indicate a decrease in to tal 
f ibre number during postnatal growth . Layman , Hegarty 
& Swan ( 1980) , for example , used the soleus ,  plantaris , 
extensor digitorum longus and the biceps brachii muscles 
of  the rat to show that there is  a decrease in muscle fibre 
number during growth and suggested that fusion of muscle 
f ibres is  a possible explanation . 
Few workers}have studied changes involving the f ibre size 
of fetal muscles.  Joubert ( 1 955 ) used the longissimus , 
6 
rectus f emoris and gastrocnemius muscles of  the sheep , and 
observed changes in weight, dimensions and histology . 
His studies indicated that fetal fibres undergo relatively 
little change in diameter between approximately the 45th and 
103rd days of gestation , while during subsequent stages ,  
a considerable increase in f ibre diameter occurs . Ashmore , 
Robinson , Rattray & Doerr ( 1 9 7 2 )  used the semitendinosus 
muscle of the sheep between 50  days and 140 days of 
gestation , and observed changes in relative proportions 
of histochemical f ibre types  between 70 and 1 00 days 
gestation . Although they did not publish measurements , 
their observations indicated that, as growth progresses , 
there is an increase in the number of fibres per perimysial 
enclosed fascicle and in the size of fascicle s . Ashmore , 
Addis & Doerr ( 19 7 3 )  conducted a similar study to that of  
Ashmore et al ( 1 972 ) in the semitendinosus muscle of  the 
pig and noticed differences in growth rates between different 
histochemical fibre types . 
Thus ,  several studies  have concentrated on f ibre size changes 
during growth .  Nevertheless , no single study has examined 
the growth changes occurring in the fascicular , myofibre and 
the endomysial and perimysial connective tissue components 
of a muscle  covering the early fetal  period up to adulthood ,  
in any species. Such a study can determine the relationship 
between the growth in perimysial and endomysial connective 
tissue components and the total number of fascicles and 
fibres . The present study therefore endeavours to achieve 
this using whole muscle sections of the semitendinosus 
muscle of the sheep . It covers a period between 60 days 
gestation up to 5 years of  age . 
7 
in funct ion at b irth . The histochemical reaction for myosin 
adenosine triphosphatase (ATPase)  activity has been shown to 
indicate the intrinsic speed of contraction of individual 
f ibres . As developed by Padykula & Herman ( 1 955 ) , this method 
demonstrates a dark staining fibre high in myos in ATPase 
activity and a light s taining f ibre low in myos in ATPase 
activity (Guth & Samaha , 1969 ) . The studies of  Barany ( 1967 ) , 
Guth & Samaha ( 1 969 ) and Barany & Close ( 1 97 1 )  showed that 
the ATPase high fibres are fast  twitch while the ATPase low 
fibres are slow twitch in contrac tile properties . This idea 
was furthered by the histochemical and physiological studies 
of  Burke , Levine , Tsairis & Zajac  ( 1 973 )  in c lassifying motor 
units of the gastrocnemius muscle of the cat . Amongst  the 
8 
three types of motor units recognised by Burke et al, (loc . cit.), 
two groups of  fibres with short twitch contraction times are 
both ATPase high , while the third group (with f ibres showing 
long contrac tion time ) is ATPase low . Within each physiological 
type of f ibre,  namely aerobic , anaerobic or combined aerobic 
and anaerobic , all of the units  examined had the same 
histochemical profiles . In addition , Kugelberg ( 1 9 7 3 )  
identified the contractile proper ties of  the motor units o f  
ATPase high and ATPase low f ibre types . He demonstrated that 
the ATPase low 
·axidative activity show tolerance to 
intermittent bursts of  stimuli  for longer periods of  t ime 
without showing fatigue ,while the f ibres with low oxidat ive 
type of ac tivity are most fatiguable  and produce  brief but 
strong contractions . Thus , these  f indings support the 
concept that the dark ·staining alkali-stabile myosin ATPase 
high f ibre is  suited for high energy demanding , isotonic 
propulsive activity and that the light staining f ibre that 
is  low in myosin ATPase  activity is suited for low energy 
demanding , isometric postural ac tivity (Davies , 1 9 7 2 ;  Burke 
et al. 1973 ; Kugelberg ,  19 73 ) . 
The alkali-stabile myosin ATPase method has been used 
previously by Dubowitz  (1963 .; 1965 ) to follow the differentiation 
of  muscle f ibres in the trunk muscles of the guinea pig , hamster , 
rabbit , rat ,  mouse and human; by Fenichel (1966 )  in brachial and 
thigh muscles of the human;by Ashmore , Addis & Doerr (19 73 )  in ) 
the semitendinosus muscle of  the pig;  by Ashmore , Robinson , 
Rattray & Doerr (1972 )  in the semitendinosus muscle of the sheep; 
by Nystrom (1968 )  in the gastrocnemius and soleus muscles of  
the cat;  and Beermann , Cassens & Hausman (1978)  in  the semi­
tendinosus muscle of the pig . Of these studies , only that of  
Beermann et al. (1978)  compared the population densities of  f ibre 
types ante- and postnatally and then only for 2 1  days af ter 
birth .  Ashmore et al. (1972 , 19 73 )  suggested that the fibres 
which are to become ATPase low fibres are observed prior to the 
appearance of ATPase high f ibres and that these presumptive 
ATPase low fibres act as a structural framework upon which the 
ATPase high f ibre development subsequently occurs . Beermann 
et al. ( 19 78 )  noticed histochemically that the induction of  
acid-stabile myosin ATPase differentiation precedes that  of  
alkali-stabile ATPase and that the primary fibres (alkali­
stabile myosin ATPase low) exhibit  a positive reaction for 
acid-stabile myosin ATPase in the deep portion of  the semi­
tendinosus muscle but not in the superficial portion . Their 
studies also suggest  that f ibre type differentiation is  
neurally regulated. 
The present s tudy accepts  the concept that the alkali-stabile 
myosin ATPase fibres are predominantly postural in activity,  by 
opposing the gravitational force on the body , and the myos in 
ATPase high f ibres are predominantly propulsive in activity ,  by 
producing an accelerative force on the body . The semi tendinosus 
muscle of the sheep extends the hip joint against  the force of 
gravity,  and also retracts  the limb in propulsion . The 
development of a muscle with such a dual function might therefore 
incorporate characteristics relevant to both . Compared to 
the usual laboratory animals , the gestation period of  the 
sheep is long (about 1 50 days ) . Therefore , the successive 
stages of  growth are more easily distinguishable . In addition, 
the limb muscles of a sheep are functionally prepared to 
support  and propel the body from the day of  b irth . This model 
appears , therefore , to be suitable for an investigation of  the 
aspec ts of growth outlined . 
The existence of a heterogeneous distribution of  histochemical 
f ibre types within the semitendinosus muscles of  the dog and 
horse has been established by Gunn ( 1 975 ) . The deep parts  of  
these muscles have the highest percentage of alkali-stabile 
myosin ATPase  low f ibres . However , the functional significance 
for such a d istribution of histochemical fibre types has not 
been previously explored . The present study , therefore ,  
incorporates electromyographic and angiographic studies to  
determine any correlated structural or  functional regional 
differences . 
Electrical activity has been associated with muscle contraction 
ever since Galvani discovered 'Animal elec tricity ' in 1 7 7 1  
(Liddell , 1 9 60) . Vrbova ( 1 963 )  used the soleus , a slow muscle , 
and the tibialis cranialis ,  a fast  muscle of the rabbit  for 
electromyographic recordings and found that the slow twitch 
f ibres show a continuous type of  activity while the fast  twitch 
fibres show bursts of activity during spontaneous movement .  
In independent work published by Herring , Grimm & Grimm ( 1 9 79 ) , 
at  about the same time as  a preliminary communication by the 
present author (SivachelvanJ19 7 9 ) , both the alkali-stabile 
myosin ATPase technique  and electromyography were employed in 
an investigation of the masseter muscle of the miniature pig . 
They observed that the region of  the muscle having a higher 
percentage of ATPase low fibre shows activity throughout 
mastication, whereas the region with the lower percentage of 
1 0  
ATPase  low f ibres is involved only during a brief portion of the 
masticatory cycle . In the present study , a qualitative 
electromyographic investigation has been undertaken to test  for 
differences in activity between the areas of highest  and lowest 
ATPase low f ibre type population densi ties within the semi­
tendinosus muscle of the sheep . 
There is  a close relationship between the metabolic need of 
individual muscle f ibres and their vascularity (Romanul , 1965) . 
The f ibres with aerobic metabolic capacity have more blood 
capillaries than anaerobic f ibres . Furthermore , Myrhage & 
Eriksson ( 1 980) observed that  'slow ' muscles have a denser 
vascular network and a shorter average distance between the 
individual groups o f  secondary vessels than ' fast '  muscles . 
They concluded that  there is a direct correlation between the 
density of secondary vascular branches and the percentage of 
oxidative fibres in skeletal muscl e .  I f  this concept is  valid , 
studying the vascular patterns of  areas of  extreme histochemical 
f ibre types within a muscle could be expected to reveal 
regional differences between the areas . The possib ility o f  an 
accumulation of ATPase low fibres close to large and closely 
spaced blood vessels has been investigated in the present study . 
The growth of organs and tissues within an organism is 
multiplicative (Huxley , 1932 ) . The time independent allometric 
equation , y = a . xb (Huxley , 1932 ) , when transformed into a 
logarithmic form, log y = a + b . log x ,  produces a l inear graph 
( Seebeck , 1 968) , where b is the regression coefficient, x is 
the independent and y the dependent  variable , and a is  the 
intercept of  the regression line . In this  l inear form ,  it can 
be used to test a nul l  hypothesis , using a power function 
set for the b value relating x and y irrespective of their 
dimensions . The equation can also be used to calculate a 
.1..1. 
prediction of y at  any value of  x within the range studied, 
and the confidence limits of  this prediction ( Steel & Torrie , 
1 960 ) . Logarithmic regression has been previously used by 
many workers to study both carcass composition (Butterfield 
& Berg , 1966;  Seebeck , 1 967 ;  Davies ,  1 9 74 )  and changes 
occurring within individual muscles (Davies , 1 9 72;  Gunn , 1 9 75; 
Tan & Davies,  1 980 ) . This method has ,  therefore , been 
considered appropriate to study the relative growth of  various 
muscle component s .  
2 . 2  MATERIALS AND METHODS 
2 . 2 . 1 SOURCE, NUMBER�SEX AND DEVELOPMENTAL STAGES OF ANIMALS USED 
The semitendinosus muscle samples were collected between June 
1 9 7 7  and July 1978  from eighteen fetuses ( 1 0  females and 8 
males )  of ages at or near 60 , 70 , 80 , 100 , 120 and 140 days of 
gestation1and f if teen postnatal animals ( 1 2  females and 3 males 
aged 5 weeks , 5 months and 5 years old ( Table la ) . These ages 
were chosen to provide multiplicative growth increments in body 
weight appropriate  for logarithmic statistical analysis . The 
ewes and lambs used to provide the muscle samples were reared 
under controlled conditions at the Department of Scientific 
and Industrial Research ' s  property adjacent to the University 
campus . New Zealand Romney ewes grazed on pasture had been 
mated to Romney rams fitted with harnesses to detect mating . 
Gestation ages were calculated from the day of successful 
mating . The pregnant ewes were killed by exsanguination while 
under barbiturate anaesthesia . Wherever possible , samples of 
the left semitendinosus muscles (Figs. l  & 2 )  from both the dam 
and the fetus (es)  were collected for histological examination . 
The samples from the lambs were collected in the same manner 
as that for the ewes . 
1 2  
2 . 2 . 2  PREPARATION OF HISTOLOGICAL MATERIAL 
2 . 2 . 2 . 1  Collection of samples 
The semitendinosus muscle was obtained from each animal used 
in the study . As soon as a ewe was killed , the fetus was 
removed through an abdominal incision and weighed . Removal 
of the semitendinosus muscle was made via a skin incision at  
the caudolateral aspect of  the thigh . The muscle , which 
could be  identified easily because of its caudal position in 
the femoral region , was separated from its neighbours and i ts 
craniodorsal attachment to the gluteobiceps muscle severed . 
The muscle  was then removed in toto by detaching its  proximal 
and distal tendons . The distal stump of the nerve and blood 
vessels entering the hilus of  the muscle was left intact .  
The fully contracted muscle was cleaned of fat and weighed 
before being placed in a plastic bag prior to further 
processing . 
2 . 2 . 2 . 2  Sectioning and staining procedure 
Except for  samples from three fetal and two postnatal sheep 
listed below ,  sections were cut from slices of  the semitendinosus 
muscle on removal from the animal within 2 h of the animal ' s  
death . Problems relating to the availability of  a cryostat 
meant that the muscle samples belonging to animals number 7 ,  
1 6 , 1 7 , 1 9 , 2 9 ,  had to be sectioned af ter thawing for 1 h following 
storage at -50°C for up to one week . 
A complete transverse slice approximately 1 cm thick was 
obtained between the middle and distal thirds of the muscle 
immediately distal to the hilus where the branch of  the 
ischiatic nerve and its  accompanying blood vessels entered 
its  craniolateral aspect .  The sectioning a t  this  level made 
the orientation of  a slice  of muscle easier and the cryostat  
sectioning of  the fibres kept near the same transverse 
sectional level in all the muscles investigated . When the 
1 3  
slice was too large for complete cryostat sections to be  cut , 
it was d ivided into s ix smaller pieces , and Indian ink was 
injected into the craniomedial aspect of each piece to enable 
subsequent orientation . Immediately before sectioning , the 
slices were frozen as rapidly as possible in isopentane cooled 
to its freezing point (-1 600C)  with liquid nitrogen . Serial 
transverse , 10 �m thick , sections were cut f rom each of  them 
in a cryostat , mounted on cold slides , and then ei ther stained 
for alkal i-stabile myos in ATPase by the method of Padykula & 
Herman ( 1 9 5 5 )  as  modified by Davies & Gunn ( 1 9 7 2 )  (Appendix 1 )  or  
stained with PAS- hematoxylin . The unstained , mounted sections 
were left in a refrigerator for at least 30 minutes before 
staining for myosin ATPase to prevent the sections separating 
off the slides at later stages in the staining procedure . 
Preliminary tests showed that the concentration of calcium 
chloride is  an important criterion for the reaction . 
Whenever possible ,  sections of muscles from both a dam and its  
fetus were stained together for myosin ATPase to  facilitate 
comparison of antenatal and postnatal muscle . Five slides 
were stained from each sample and the best one was used for 
quantitative analysis . 
2 . 2 . 3  DETERMINATION OF THE PATTERN OF HISTOCHEMICAL FIBRE TYPE 
POPULATION DENSITY IN SECTIONS OF THE SEMITENDINOSUS MUSCLE 
By using the alkali-stabile myosin ATPase technique , a 
simple division into ATPase high (AH) or ATPase low (AL) was 
possible for any fibre relative to the overall activity of 
adjacent fibres in the semitendinosus muscle belonging to 
animals beginning at the 80  days fetal stage and extending 
I 
up to 5 years  postnatally .  The semitendinosus muscle had a 
heterogeneous distribution of  AL and AH f ibres within it . 
As the AL f ibres formed a smaller percentage population of  
the two f ibre types in any region of  the muscle ,  the AL f ibre 
14  
was preferable to the AH fibre for statistical analysis o f  
the relative . �!��s] of the two f ibre types . The 
populat ion density of AL f ibres.expressed as a percentage 
of the to tal number of fibres counted in each region1 
was est imated over the entire transverse sec t ional area o f  
the muscle using the method described below .  
A project ion microscope (Leit z ,  Wetzlar) was set up in a 
dark room to display the fibres within the stained sec tions 
on the opposite side of  a 
the case of antenatal muscles , fascicles from about 20  regions 
and in the postnatal ones up to 50 regions were examined . 
From each of  these regions 2 to 3 fascicles were selected 
for counting a total of  about 5000 to 7000 fibres . The regions 
were changed by shifting the slide in two directions using 
the mechanical stage control knobs to ensure that the same 
region was not viewed more than once . 
... - ·'· ·· _ .. . ... _ .. .... . <> . . . ..... ...... �·- • -· • '""'-- � .-.. .. . 
2 . 2 . 4  DETERMINATION OF TOTAL FASCICULAR, TOTAL FIBRE, FASCICULAR 
AND FIBRE TRANSVERSE SECTIONAL AREAS 
For the study of the to tal fascicular , to tal f ibre , fascicular 
and fibre transverse sec tional areas (TSA) within the 
semitendinosus muscle , outlines were traced on paper by means 
of a proj ection microscope , cut and weighed . This method is  
based on Delesse ' s  principle according to  which the area density 
of a profile on sect ion is , on the average , direc tly proportional 
to the frac tional volume of that component in the original 
solid body (Weibel , 1 9 7 9 ) . 
1 5  
A standard paper square of  known area and weight was 
compared with the weight of  an unknown area of an outline 
traced on the same grade of paper ( 1 10-1 1 5  g/m2, Carson , 
France ) . A slide obj ective micrometer (Olympus ,  Japan) of 
1 mm length and with 100 subdiv isions was used to draw the 
standard paper square at the desired magnification on the 
proj ec tion microscope . 
The outline of a whole stained transverse section of  the 
muscle  was traced directly from the slide on to paper and 
the area (referred to as  ac tual TSA) was obtained by cutting 
and weighing . In larger muscles ,  the tracings of  sections 
of pieces were oriented to form a mosaic of the whole muscle . 
To count and trace any trait  used in this histological 
analysis , a low magnification of the proj ection microscope 
was used for postnatal samples  and a high magnification for 
antenatal samples . For the purpose of  the present study , 
groups of f ibres with a mixed population of  presumptive or 
definite AL and AH f ibres and c ircumscribed by large spaces 
or by  definite  perimysial connective tissue boundaries , as 
seen from 70 days gestation onwards ,  were defined as  fascicles . 
For postnatal muscles , the sections were back projected at  a 
magnification of  250 t imes and the fascicles and the f ibres 
occupying the interior of  a square of 125  x 125 mm (effective 
area 0 . 5  x 0 . 5  mm) were traced and counted . Several squares 
were traced f rom different  regions using the same sampling 
method already described (vide supra) for establ ishing 
histochemical fibre type population density . The total area 
of the fascicles and f ibres occupied within the squares were 
estimated by cutting and weighing . As both the total number of 
fascicles and the fibres within these squares were known , an 
estimate for fascicular area and the f ibre area could be obtained.  
By using the difference between total area of  the squares 
16 
and the total area of  the fascicles , a percentage estimate 
for the perimysial connective t issue was obtained . Similarly , 
the difference between the to tal area of  fascicles and the 
total area of fibres was used to obtain a percentage estimate 
for the endomysial connective tissue . The percentage estimates 
of  perimysial and endomysial connective tissue components , so 
obtained,  were used to calculate the total fascicular TSA and 
total f ibre TSA respectively. 
For antenatal muscle s ,  the same procedure was followed but 
with following modifications : -
The fascicles and f ibres were traced within squares of  100  x 100  mm 
(effective area 0 .  25  x 0 .  25 mm) and at a magnification of  100 in 
the proj ection microscope . For 60  days gestation , however , no 
separate estimates  for endomysial and perimysial connective 
tissue components could be obtained . In muscle samples of  
the 60  and 70 days gestat ion periods , approximately 200 clearly 
observable presumptive AL f ibres were traced from each section 
to assess the s ize  changes involved during this growth period . 
In all ,  33 muscles were used in the study . About 100 sec tions 
were analysed , 400 , 000 f ibres counted , 4000 fascicles and 
60 , 000 fibres traced , cut and weighed in obtaining the data . 
2 . 2 . 5  ELECTROMYOGRAPHIC STUDY 
Two rams and one ewe were used in an electromyographic 
investigation of the possible functional significance of the 
observed variations in the proportions of  AH and AL fibres 
within the semitendinosus muscle . 
2 . 2 . 5.1 Surgical procedure 
Each animal under investigation was anaesthetised using 
thiopentone sodium solution (Pentothal , Abbot ) . Using aseptic 
precautions , a skin incision was made parallel to the long 
1 7  
axis of the femur . The deep part of the semitendinosus muscle 
was uncovered caudal to the gluteobiceps muscle , taking care 
to avoid damage to its main blood and nerve supply . Three 
insulated , mul tistrand, flexible grade , stainless  steel wires 
(0 . 08 mm diameter ; Cooner Wire Company , Chatsworth) , with 
their bared ends silver-soldered and bent into hooks to form 
recording electrodes , were implanted close to each other , distal 
to the hilus of  the muscle at  a depth of  approximately 10  mm . 
Three other wires were  similarly implanted caudolaterally 
at the same level in the superficial part  of the muscle . The 
two recording sites were thus in areas already established as  
having , respectively , dense or sparse d istributions of  AL fibres , 
and they were approximately 25-35 mm apart in the muscle . The 
two groups of wires were passed loosely through the subcutaneous 
fascia of the thigh region of the sheep and their  free ends were 
soldered on to a multi-j unction block taped to the gluteal region . 
In each instance the sheep was fed ad lib and looked bright 
and active from the following day . There was no evidence of  
distress resulting from the operation and no signs of infection 
were  observed . 
2 . 2 . 5 . 2  Recording procedure 
Each sheep was first  used for recording one week af ter the 
preparatory operation . The ac tion potentials from within the 
superficial and deep regions of the muscle were recorded 
simultaneously by using the two of  the three leads in each 
group which gave the c learest recording and the same two leads 
were used in recording muscle activities throughout a single 
session of the experiment . The third lead served as  the 
indifferent electrode . From the multi-junction block , each 
group of leads was connec ted via a screened multi-cable 
wire to a preamplifier ( Type 1 22 ,  Tektronix) and thence to 
one channel of  a cathode ray oscilloscope (56 1 B ,  Tektronix) 
with a four trace amplifier (3A74 ) .  Traces were recorded 
with a moving film camera ( Shackman AC2 /25 ) .  The two 
18  
differential preamplifier s  were set  to a frequency range 
(i . e .  band pass ) of 80 Hz to 10 KHz and an amplification 
of lOOOX. The amplitude setting on the oscilloscope was 200 
uV per division and the camera f ilm speed 5 am s-1• Events  
during the experiment were marked on the film by  switching 
a light emi tting diode in the camera viewing f ield . 
Recordings were taken with the animal standing , walking and 
bearing additional weight . The sheep was compelled to use 
the operated limb by having its contralateral hind limb held 
off the ground by an assis tant . I t  was induced to bear 
more weight on the operated limb by having an assistant press 
on the back of  the animal while raising the unoperated hind 
limb , to walk only on its  hind l imbs with an assis tant holding 
its two forelimbs in a raised position above the ground , and to 
kick backward with its operated limb in response to pinching 
the skin in the metatarsal region of the limb . 
The first recording session consisted of three series of  
trials in each of which each event was replicated five times . 
A further , similar set of  recordings was obtained from each 
animal one week later . 
At the end of  each experiment , the sheep was killed to check 
the actual placement of  the electrodes within the muscle , 
by  making a skin incision in the thigh region and following 
each electrode into the muscle to confirm its depth and the 
correctness of its position . 
2 , 2,6 ANGIOGRAPHIC STUDIES 
The distribution of the main vessels within the semitendinosus 
muscle of the adult sheep was studied by using corrosion 
casting and angiographic techniques .  
1 9  
The corrosion cast was prepared by following the general 
guidelines of Tompsett ( 1970) . The sheep was heparinized 
and then killed with a captive-bolt  gun . The hind limb was 
removed by sawing through the hip bone , and the semitendinosus 
muscle was exposed . The neighbouring muscles of the thigh 
region were removed ,  to leave the semitendinosus muscle and i ts 
femoral arterial branch entering its  distal part  intact . 
The part of  the limb distal to the hock j oint was removed 
and the semitendinosus , with its associated bones , was 
left  overnight.  A solution of 1 %  saline was inj ected into the 
artery supplying the muscle to ensure that the vascular 
passage way was clear . 
Batson's 1 7  anatomical resin compound (data sheet 1 05 , 
Polysciences , 1 9 78 )  was inj ected into the muscle on the 
following day. The f inal embedding mixture was composed 
of  10 ml of  monomer base solution A (methyl methacrylate) , 
1 . 2 to 2 ml of  catalyst B ,  1 drop of  Promoter C and� to 1 ml 
of red pigment .  
2 . 2 . 6 . 1  Injection of resin 
A polythene tube of  internal diameter 0 . 38 mm and external 
diameter 1 . 09 mm (Portex) was cut obl iquely,  was slid into the 
remaining portion of the femoral artery entering the muscle 
and was t ied in place . The resin was inj ected with moderate 
pressure using a 2 ml hypodermic syringe fitted via a 2 1 "  
gauge needle  t o  the polythene tubing . Leakages through other 
collateral branches of  the main vessels were promptly stopped 
by  using artery forceps . The resin was inj ected over a 90  to 
1 20 s period , after which the colour of the resin was apparent 
beneath the surface of  the muscle . 
2 . 2 . 6 . 2 Maceration 
The limb was left overnight for the resin to set  in the 
muscle . The muscle was then separated from the limb and 
20  
macerated by suspending it on a wire gauze in a solution 
of potassium hydroxide (340g t-1 of water) contained in a 
small stainless steel tank . The tank itself was left  for 
three days in a water bath maintained at a temperature of  
50°C by a tempette (Techne ) .  
2 . 2 . 6 , 3 Washing and trimming of the cast 
The corrosion cast , lying on the wire gauze ,  was lif ted out 
of the maceration tank and washed in water by means of  rubber 
tubing from a water tap connected via a plastic syringe 
f itted with a hypodermic needle , to remove any organic 
material remaining. By using f ine tweezers , the cast was 
trimmed off  to show the main vessel and its secondary and 
tertiary-- branches. 
2 . 2.6 . 4  Preparation of angiogram 
The venous drainage of  the semitendinosus muscle was 
demonstrated radiographically in a sheep , heparinized prior 
to it being killed . . The fresh muscle , with its  attachments 
to the hind limb intact , was exposed and the vein leaving 
the hilus cannulated and inj ected with meglumine lothalmate 
(Conroy 280) , a water-soluble radio-opaque fluid . Because 
of the resistance of  the venous valves ,  the inj ection and 
radiography proceeded in three stages using approximately 1 . 5 ml 
of the fluid initially and then an additional 1 ml each t ime 
to create a progressive back f low through the venous valves . 
2 .  2 .  7 STATISTICAL METHODS 
The allometric relationships estimated between variables were 
calculated for antenatal , postnatal and ante- plus postnatal 
periods using double logari thmic regressions (Table  2 ) . For 
any regression obtained,  x was the independent variable , y the 
dependent variable , sb the standard error and a the intercept . 
2 1  
The various relationships were used to suggest  changes in 
total f ibre number , in to tal fascicular number , in f ibre 
number within fascicles ,  in the growth of histochemical f ibre 
types and connective t issue components, and in the relative 
growth and shape of  the semitendinosus muscle of  the sheep , 
during the periods investigated . A b value of  1 was used to 
test a null  hypothesis within each pair of variables considered , 
except for isometric g rowth changes involving the shape of  the 
muscle where a b value of 0 . 66 7  was used to test  a 2 / 3 power 
relationship between area and weight of the muscle . 
Predictions for the total f ibre number at  60 days gestation ,  
birth and adulthood were made using each possible regression 
between mean fibre area (x) and total f ibre area (y) . A 
confidence limit at  a 95%  level was set in each case (Table 3 ) . 
When there was no overlap in these ranges between any two 
stage s ,  the effect was considered significant . 
Estimates of  the mean fascicular and mean fibre TSAs were 
obtained by weighing , together , the traced fascicles or f ibres 
sampled for each muscle  and then by dividing the calculated 
total area by the number of fascicles or f ibres included in 
the sample . Because the fascicles and f ibres were not , 
therefore , weighed individually , an estimate of  standard error 
for the mean could not be obtained . 
2 . 3  RESULTS 
2 . 3 . 1 ATTACHMENTS AND ORIENTATION OF THE SEMITENDINOSUS MUSCLE 
OF THE SHEEP 
Figure 1 shows the attachments of  the semitendinosus muscle 
of the sheep . The muscle originates from the lateral 
ischiatic tuber and runs caudal to the femur to insert on to 
the crest  of  the tibia . The attachment of  the muscle to the 
22 
gluteobiceps . near its  origin and its fascial connections to 
the gastrocnemius muscle are not shown . The semitendinosus 
muscle can ,  thus , extend the hip joint and flex the s tif le 
joint . Figure 2 shows a transverse section of  the thigh of 
the sheep and indicates the relative positions of  various  
muscles at  the level at  which the semitendinosus muscle was 
transected for histochemical s tudies . The blood vessels 
23 
and nerves entered the semitendinosus muscle in its  craniolateral 
aspect  (not shown ) . 
2 . 3 . 2  MORPHOGENESIS AND DIFFERENTIATION OF MYOSIN ATPase FIBRE TYPES 
Figure 3 shows the morphological and histochemical changes 
occurring within the semitendinosus muscle between 60 and 70 
days gestation . Two types of  f ibres are observed within the 
muscle a t  60 days gestation . With PAS-hematoxylin s taining , 
many large fibres with central nuclei are seen to form c lusters 
(Fig, 3a) . With alkali-stabile ATPase staining , these large 
fibres have pale centres which are presumably uns tained ,  
nuclei o r  the spaces between nuclei , and have a darker periphery 
(Fig, 3b ) .  Some of these larger f ibres are closely associated 
with smaller sized f ibres (Figs . 3a , 3b) . By 70  days , the large 
fibres are more separated from each other and surrounded by more 
small sized f ibres than previously (Fig . 3c ) . Alkali-stabile 
myosin ATPase staining does not ,  however , clearly distinguish 
the two types of f ibres (Fig, 3 c ) . Each aggregated group of 
cells , with large sized f ibres located in the midst of  many 
small sized f ibres as seen in 70  days gestation , is defined 
as a fascicle . Fig. S shows sections of  the semitendinosus 
muscles , stained for alkali-stabile myosin ATPase , from 
80 days gestation (Sa , b) , 100  days gestation (Sc , d )  and adult 
(Se , f )  stages in the sheep . At 80 days gestation , the f ibres 
seen within individual fascicles have differentiated into 
distinct  pale and dark types with alkali-stab ile myosin ATPase 
staining . The pale  f ibres are identified as ATPase low (AL) 
and the fibres which appear homogeneously dark by this reaction 
are identified as  ATPase high (AH) fibres . These two distinct 
myosin ATPase f ibre types are observed within the muscle f rom the 
eightieth day of gestation onwards . 
During the period between 60 to 70  days gestation , the 
presumptive AL f ibres ac tually decrease  in size (Fig· 3b , c ) . 
Measurement of  a f ew selected presumptive AL fibres f rom 
myosin ATPase stained sections , indicated that the transverse 
area of  these cells  at 60 days is  approximately 325 �m2 
while at  70 days i t  is  approximately 150  �m2 
2 . 3. 3  ATPase LOW FIBRE DISTRIBUTION PATTERN WITHIN THE 
SEMITENDINOSUS MUSCLE 
The ATPase low (AL) fibre population density patterns of the 
whole muscle at fetal ages 80 ,  100 , 140 days and thos e of 
young and adult sheep are represented schematically in Figure 
6 .  Thi s three 
fibres is observed in the craniomedial a spect ( the AL dense 
area ) of the muscle and the lowe st density of AL fibres is  
seen in the caudolateral aspect ( the AL sparse area ) , 
Representative areas of AL sparse and AL dense regions have 
been shown at a low magnification in sections of the semi­
tendinosus muscle of  a sheep of  30 kg bodyweight (Fig. 4a , 4b ) and 
at a higher power magnification in those of 80 days gestation , 
lOO days gestation and adult (Figs . Sa , b , c , d , e , f) . Figure 7 
shows the relationship between log body weight and ATPase low 
fibre type density population , in AL dense and AL sparse 
area s within the semitendinosus muscle of the sheep from 80 
days gestation to adulthood . The percentage of AL fibres in 
24 
the AL sparse area i s  within the range o f  approximately  3%  
to 6%  throughout development ( Table lb , Figs , 6 & 7 ) . However , 
the AL fibre percentage in the AL dense area increa ses from 
approximately 10% in the fetal stages to about 20-30%  in the 
postnatal stages ( Table lB , Figs . 6-7 ) . The AL sparse areas 
are seen peripherally in both caudolateral and caudomedial 
regions of  the feta l  muscle but are restricted to the cauda­
lateral border in the postnatal muscles . There is  a shift 
in the AL dense area to a more central location during 
development . A study of the adult semitendinosus muscle 
proximal to the tendinous intersection in three muscles 
showed a similar pattern of  f ibre type di stribution to that 
found distall y .  
2 . 3 . 4  QUANTITATIVE GROWTH ANALYSIS 
Table la summarises the data obtained for the age , sex , and 
body weight of the animals and the weight of the semitendinosus 
muscle  used . It  also shows the quantitative measurements 
obtained for actual TSA , total fascicular TSA , total fibre TSA , 
mean fascicular TSA and mean fibre TSA of  each muscle analysed . 
Table lb shows the data related to the quantitative measurements 
obtained on fibres reacted hi stochemically to myosin ATPase 
classified as high (AH ) or low (AL)  reacting . The relevant 
data from these two tables ( l a ,  lb) have been analysed using 
logarithmic regressions to obtain information related to 
a )  growth in fascicles , fibres , perimysiurn and endomys ium 
b )  relative growth of the semitendinosus muscle and 
c )  dimensional changes including the shape and mechanics of 
the muscle ( Table 2 )  . The relationship between each pair 
of variables shown in Table 2 ,  have also been repre sented 
graphically (Figs . B-19) . 
2 . 3 . 4 . 1  Fascicular growth 
Fascicular growth was analysed using the regression o f  mean 
25  
fascicular TSA on total fascicular TSA (Table 2B ,  Fig. 8 ) . 
The growth in the mean fascicular TSA is significantly lower 
than that of the total fascicular TSA of the muscle 
antenatally (b= 1 . 25 ,  significantly greater than 1 ,  P < 0 . 05 ) . 
No s ignificant changes are shown postnatally (b= 0 . 74 ,  not 
significantly less  than 1 ,  P > 0 . 05 ) . This suggests  that 
there is an increase in to tal fascicular number antenatally 
while  it may even decrease postnatally .  
2 . 3 . 4 . 2  Growth in  fibre transverse sectional arsa 
) 
The growth in the fibre TSA within both the whole muscle and 
individual fascicles have been analysed (Table 2 , A , C ,  Figs 
9 , 10 ) . The regression of  mean fibre TSA on total f ibre TSA 
shows that the mean fibre TSA grows at a significantly lower 
rate than the growth of total f ibre TSA antenatally (b= 1 . 70 ,  
significantly greater than 1 ,  P <  0 . 0 1 ) . Postnatally,  no 
significant change is observed (b= 0 . 82, not significantly 
greater than 1 ,  P > 0 . 05 ) . Antenatally , therefore , there is  an 
increase in total fibre number1 while the number is possib ly 
constant postnatally . The regression of mean fib re TSA on 
mean fascicular TSA, on the other hand , shows a direc t 
proportionate relationship between these two variables 
(b= 1 . 20 ,  not significantly  different  from 1 ,  P > 0 . 05 ) . 
This suggests  that the f ibre number within individual 
fascicles does not change throughout the growth period studied . 
The growth in TSA of the AL f ibres is  not significantly 
different f rom that of the AH fibres in either AL sparse or 
AL dense areas (Table 2D , E 1 Figs . 1 1 & 1 2 ) . 
2 . 3 . 4 . 3  Changes in connective ti ssue 
Large areas of  perimysial connective t issue are present 
during early fetal  life . The regression of actual TSA on 
26 
total fascicular TSA (Table  2K,  Fig. 1 7 )  shows that there is  
a marked decrease in extrafascicular connective t issue 
antenatally (b= 1 . 28 , s ignificantly greater than 1 ,  P < 0 . 00 1 )  
but  no significant changes are observed postnatally (b= 1 . 00 , 
not significantly different from 1 ,  P > 0 . 05 ) . The reduction 
in endomysia! connec tive t issue (Table 2J ,  Fig 1 8 )  on the 
other hand is less marke� although this effect is  
s ignificant during the whole  period (b= l . 05, s ignificantly 
greater than 1 ,  P < 0 . 05 ) . The regression of  ac tual TSA on 
total f ibre TSA . also shows that the proportionate  changes in 
connective tissue within the whole muscle is  largely dependent 
on extra-fascicular connective tissue (b= 1 . 29 ,  significantly 
greater than 1 .  P < 0 . 00 1 )  than its endomysia! components 
(Table 2L ,  Fi� 1 9 ) . 
The changes in connective t issue proportions and in 
fascicular and f ibre number during antenatal growth suggests  
that the formation of new fascicles takes place . with a concurrent 
significant reduction in perimysial connective t issue . This is 
in preference to an addition of new f ibres within existing 
individual fascicles , for which no significant proportionate 
changes occur antenatally . 
2 . 3 . 4 . 4  Changes in muscle shape and mechanics 
The actual TSA, total fascicular TSA and total fibre TSA of  
the muscle and the weight of  the muscle show changes 
antenatally and postnatally which are more proportionate 
(b is  more closely equal to 0 . 66 7 )  when the connective tissue 
component of  the dependent variable is  reduced (Table  2G , H , I ,  
FigsJ 3 , 14 & 1 5 ) . Also the growth rate in the ac tual TSA i s  
significantly less (b= 0 . 5 6 ,  P < 0 . 00 1 ) ,  the total fascicular 
TSA is proportionate (b= 0 . 67 , P > 0 . 05 ) , and the total f ibre 
TSA is  significantly greater (b= 0 . 73 ,  P < 0 . 05 )  than 0 . 66 7 ,  
relative to the growth rate o f  muscle weight throughout 
2 7 
development .  The disproportionate  growth between the 
actual TSA and the weight of the muscle appears therefore 
to be due to a reduction in the connective tissue components , 
rather than due to changes in the component s  within the 
muscle f ibres . Rather than a decrease in the strength of the 
muscle , relative to its  weight , as might be inferred from its 
actual TSA (Table 2G) , the strength , as estimated by the to tal 
TSA of i ts  myofibres , appears to increase (Table 2I ) . 
The muscle grows faster during early antenatal stages (b� 1 . 1 1 ,  
significantly different from 1 ,  P < 0 . 05 )  relative to body 
weight (Table 2F, Fig . 1 6 )  than postnatally (b = 0 . 9 3 ,  not 
significantly less than 1 ,  P > 0 .  05 ) where growth is  
maintained at  a more proportionate rate . 
2 . 3 . 4 . 5  Estima tion of total fibre number 
Table 3 gives predictions of total f ibre number in the 
semitendinosus muscle , at the level of transection , at 60 
days of gestation , b irth and adulthood . Regressions of the 
total fibre TSA on the mean fibre TSA for the antenatal , ante­
and postnatal and postnatal periods (Table 2A) have been used 
in calculating the estimates . When the equation involving 
both antenatal and postnatal periods is used , there is a 
significant increase in fibre number for both growth stages . 
However ,  when the postnatal equation is  used , no significant 
change in f ibre number from birth to adulthood is shown . 
Postnatally , the variability between sheep is too high to 
e stablish a definite pattern of  changes in fibre number . 
Antenatally , however ,  whichever equation is used , there is  
approximately a three-fold increase in fibre number between 
60 days of gestation and birth . 
2 . 3 . 5  ELECTROMYOGRAPHIC STUDY 
Simultaneous electromyographic recordings from the superficial 
28 
(corresponding to AL sparse area) and the deep (AL dense 
area) parts of the semitendinosus muscle of a sheep obtained 
during various events in a single recording session are shown 
in Figure 20 . The upper tracing shows the activity recorded 
through the electrodes implanted in the superficial part of 
the muscle and the lower tracing is  from that of  the deep 
part . No muscle act ivity was apparent in the operated l imb 
when the animal was standing on its  own (a) , but when the 
operated l imb was forced to support  weight , there was activity 
in the deep part but not in the superficial part (b ) . When 
more powerful stimuli  to muscular effort were progressively 
applied , ac tivity was shown in both superficial and deep parts 
(c , d , e , ) .  The results  were consistent in all three animals 
used in the experiment . 
2 . 3 . 6 ANGIOGRAPHIC STUDIES 
The corr osion casts of  the part distal to the tendinous 
intersec tion of the semitendinosus muscle is shown in Figure 
21 b , c .  A d iagrammatic  representation of the branching pattern 
irr AL dense and AL sparse areas is shown in Fig . 2 1 d .  I t  is 
which are larger and closely placed . On the other hand , only a 
few widely placed large vessels enter the AL sparse area . The AL 
f ibres , which are aerobically active , are thus accumulated c lose 
to an abundant and immediate source of nutrients  and oxygen . The 
29  
angiogram showing the venous also indicates that 
2 . 4  DI SCUSSION 
2 . 4 . 1  ANALYTICAL METHODS 
The developmental stages of  both the f e tuses and postnatal 
sheep were chosen in such a way that the successive growth 
increases in body weight could be effectively represented 
in statistical logarithmic analysis . An additional s tage 
between 140 days gestation and the 5 weeks early postnatal 
period would be desirable . However , this  material was not 
available . All the postnatal animals , except for the three 
at 5 weeks of ageJ were females . The influence of sex on the 
measurements made is · expected to be small (Lohse , 1 9 73 ) . 
Five of the 33 semitendinosus muscles used ( 2 . 2 . 2 . 2 )  had 
to be f rozen and thawed before cryostat sectioning . These 
samples were not sufficient to analyse , statistically , 
whether there was a significant change observable in f ibre 
size of  these twice frozen samples , relative to the rest  of 
the samples which were frozen only once before cryostat 
sectioning . Errors such as  shrinkage , prerigor and thaw 
contracture , variation in response to treatment by different 
f ibre types ,  and the possible differences between fresh 
samples and those stored frozen before processing were 
inherent in the techniques used . These technical variables , 
together with the actual variation between individuals , 
contribute to the large overall variation observed (Table lA) . 
There i s ,  however , l ittle reason to suppose that this 
variation can bring about an incorrect conclusion from the 
statistical analysis . 
Problems o f  measuring f ibre size have been discussed by 
various workers (Hegarty & Naude , 1 9 70 ;  Goldspink , Gelder , 
Clapison & Overfield , 1 9 7 3 ;  Levine & Hegarty , 1 9 7 7 ; Clancy 
& Herlihy , 1978) . Hegarty & Naude (lac . cit . )  suggested that 
the peripheral fibres of  muscles f ixed postrigor are smaller 
than fixed prerigor . Goldspink et al. ( 19 73)  indicated , on 
the other hand , that the type of fixative used is more 
important in measuring fibre size than the effects of  rigor . 
Gunn ( 19 76 )  showed that storage of  muscles by freezing did 
30 
not alter the subsequent TSA of  his sections . I t  is  
evident that standardization of technique is necessary to 
reduce errors in measuring f ibre size . This was attempted , 
as  much as possible , in the present study . Errors in the 
measurement of individual f ibres were minimized by the estimation 
of the mean TSA of at least 2000 f ibres for each muscle , and 
the avoidance of  f ixatives before sectioning . 
In the present study, a paper cutting and weighing method 
was employed to measure total fascicular, total f ibre , mean 
fascicular and mean fibre TSAs of the muscle . Planimetry , line 
integration or point counting can be used to estimate structure 
parameters (Weibel , 1 9 79 ) . In planimetry , a direct estimation 
of the relative areas occupied by profiles is made ; in l ine 
integration , a test  line is applied to measure the intercept 
length ; and in point counting , a random poin t  grid or a 
systematic lattice is used . Planimetry can be employed by 
using a planimeter , by circle fitting , or by tracing the 
profile s ,  cutting them out and weighing them . Amongst these 
methods , the cut and weigh method has been shown to have the 
lowes t  error although it is the mos t  time consuming (Weibel ,  
1 9 7 9 ) . However , in the present study a projection microscope 
enabled direct tracings at various magnifications of the 
necessary profiles of  stained sections . The sampling of 
large numbers of areas was there fore easier . 
2 . 4 . 2  GROWTH OF FIBRE AND FASCICULAR TRANSVERSE SECTIONAL AREAS 
As discussed previously ( 2 . 1 . ) ,  various reports suggest that 
the postnatal growth of  muscle is  due to hypertrophy of  
f ibres present at  birth rather than an increase in number 
of fibres (t-iaccallum, 1 898 ;  Staun , 1 963 ; Enesco & Puddy , 1964 ; 
Rowe & Go ldspink , 1 969 ; Davies , 1 9 7 2 ;  Tan & Davies ,  1980) . 
However , there are also reports suggesting an increase (Rayne 
& Crawford , 1975 ; Gunn , 1 9 7 9 )  or a decrease (Layman , 
3 1  
Hegarty & Swan , 1 980)  in f ibre number postnatally . The 
present study suggests  that fibres increase in number during 
antenatal growth , but provides no evidence that the number 
of  fibres change postnatally . There is a wide variability 
in f ibre size amongst individuals at any particular stage of 
development (Table lA) and the estimates of  to tal f ibre 
number for a muscle at  any s tage depend on the developmental 
period used in calculating the estimate (Table 3 ) . A wide 
variability in f ibre size in the semitendinosus muscle during 
postnatal growth , between individuals ,  has also been 
observed by Suzuki ( 1 9 7 1 )  in the sheep and Gunn ( 1 9 7 9 )  in the 
dog and horse . Joubert ( 1 956b)  noticed a similarly wide range 
in f ibre size in three differ ent muscles in newborn lambs . 
The body weight of the fetal sheep increases approximately 
50  times and the mean f ibre TSA of the semitendinosus muscle 
six times , between 60 and 140 days of gestation (Table lA) . 
A re-analysis of  the data of  Joubert ( 1 955)  for 3 1  fetuses 
between 60 days and 129 days , using a double logarithmic 
regression between the mean f ibre TSA and the area of 
individual muscles ,  estimated by using a product of their 
widths and depths , suggested that the longissimus (b = 1 . 67 , 
sb = 0 . 3 1 )  has a smaller antenatal increase in f ibre number 
when compared with the gastrocnemius (b = 2 . 2 0 ,  Sb = 0 . 22 )  
and rectus femoris (b = 2 . 1 1 ,  sb = 0 . 35 ) . The mean f ibre 
area of these three muscles shows a three-fold increase 
during this period of growth ( Joubert loc. cit . ) . The 
fibre  area in the semitendinosus muscle in the sheep also 
shows a three-fold increase over an equivalent period in the 
present study . The two studies show a large difference in 
absolute fibre area ,  presumably due to the different tissue 
preparation techniques employed . 
As observed in the present study , al though the growth ranges 
in the antenatal sheep are appropriate for studies involving 
32 
changes in the fibre number , postnatally the fibre TSA 
increases only about three-fold and body weight only 
twenty-fold . A large sample size  would to some extent 
compensate for this small growth range . The pig , with a 
hundred-fold increase in body weight postnatally (Davies , 
1 974 )  is a better model to  test the constancy of postnatal 
muscle f ibre number . 
In a study involving growth related changes in connective 
tissue , the f ibrous architecture of muscle should be given 
due consideration . The semitendinosus muscle, being a strap 
muscle , should not show much variation in the distribut ion 
of connective tissue along its  length . Yet , it is possible 
that the most amount of connective t issue is found at the 
musculotendinous j unction with the least amount being 
deposited at the widest region of the belly . In the present 
study variation between samples was avoided by obtaining all 
samples from a f ixed region of the muscle in between these 
two extremes . 
! spaces in between myofibres and those outside the fascicles 
had been considered as connective t issue components ,  in the 
present study , to  estimate their proportions . But these 
spaces potent ially include , in addition to the normal 
connective t issue components ,  sectioning and staining 
artefacts and other extra-myofibre cells  like antenatal 
and postnatal satellite cel l s .  The changes in 
connective t issue components were studied using the same 
sections of muscles stained histochemically by the alkali­
tabile myosin ATPase technique to examine growth changes 
· n  the relevant histochemical fibre types . This technique , 
owever , does not clearly demonstrate the connective t issue 
omponents and their boundaries and also does not stain 
he proliferative myogenic cells . The artefact which was 
· nherent to the technique could have been greater in muscles  
rozen twice than those frozen once but the significance of  
, is  effect could not be ascertained for reasons mentioned � 2 . 4 . 1 .  However ,  visual examination did not reveal any 
33 
obvious d ifferences between sections frozen once and those frozen 
twice . The overall e ffect of  measuring potential spaces as 
connective tissue would be that the actual amount of connective 
tissue would have been overestimated and the total fascicular and 
fibre areas underestimated . The effect of  this overestimation 
of connective t issue which occurred in all the samples throughout 
the whole  period of investigation is thought to be negligible 
since this study is concerned with connective t issue proportions . 
The present study suggests that there is  a s ignificant 
proportionate reduction in perimysial connective tissue 
during antenatal growth , while postnatally the proportion 
appears to be constant . The endomysia! connective t issue , 
on the other hand , shows fewer changes than that of the 
perimys ial connective tissue . These results on postnatal 
growth of connective tissue elements agree with those of  
Enesco & Puddy ( 1 964 ) and Bridge & Allbrook (1 970) . The 
present study has extended this work by estimating the size and 
number of  fascicles during their entire development . The 
number of  fascicles increases antenatally while no significant 
changes occur postnatally . It  appears that there is no 
increase in f ibre number within a fascicle once it is  
enclosed by perimys ium . The fascicles , on the other hand , 
have the potential  to  grow in number within the muscle ante­
natally , with a corresponding proportionate decrease in 
perimysium . This ability is lost postnatally . Therefore , 
an antenatal increase in number of f ibres can be expected 
to be accompanied by newly forming fascicles rather than an 
increase in number within already existing fascicles . This 
observation is  the first to  suggest a pattern of f ibre and 
fascicular growth within a muscle . 
A collagenous matrix is shown to be  necessary for ' normal ' 
muscle differentiation in t issue culture (Hauschka & 
KBnigsberg , 1966 ) . The formation of  a basal lamina and 
33a 
associated collagenous connective t issue has been considered 
to serve as  a mechanism regulating the extent of fusion 
between myofibres and mononucleated myoblasts (Fischman, 1 9 70 ) . 
An excessive deposition of  collagen in the endomysium and peri­
mysium has been shown to be  associated with various forms of 
muscular diseases (Adams , Denny-Brown & Pearson , 1962 ; Pearson , 
1 963 ; Duance , Stephens , Dunn , Bailey & Dubowitz , 1980 ) . These 
studies and the present one indicate the importance of  the 
connective t issue framework in controlling normal muscle 
growth . 
2 . 4 . 3  DIFFERENTIATION OF MUSCLE FIBRE TYPES 
Wirsen & Larsson ( 1 964 ) considered that three generations of  
myotubes occur in the thoracic muscles of fetal mice  during 
prenatal growth between sixteen and nineteen days . A 
histochemical  technique using phosphorylase activity was used 
in their studies . This was criticised by Dubowitz ( 1 9 70 ) who 
considered that their f indings were due to variable staining 
intensities  of  the reaction depending on the number of 
polysaccharide units present in the synthetic glycogen formed . 
Dubowitz  ( 1 965 ) suggested that early fetal fibres originate 
as  a common pool , differentiating later into adult  muscle 
f ibre types .  His observations were based on studies using 
human limb and trunk muscles . However ,  Ashmore , Robinson , 
Rattray & Doerr ( 1972 ) , using the semitendinosus muscle of  
the sheep , demonstrated that the development of  muscle f ibres 
is  biphasic . A primary generation of  cells is  early to 
develop , and a secondary generation of cells originates later 
in the antenatal period . The postnatal fibre types evolve 
from these two basic types . The biphasic theory of  development 
of  skeletal muscles has been supported by Swatland & Cassens 
( 1 9 73 ) for fetal  porcine muscles . The myoblasts which are 
destined to form secondary muscle f ibres come to lie on the 
surface of the existing primary fetal myotubes . Fusion of  such 
34 
myoblasts results in the formation of secondary fe tal myofibres . 
Autoradiographic studies in chick muscles by Kikuchi ( 1 9 7 1 ) , 
and the present observations , support this theory . 
The f ibres of the primary generation are larger at  sixty 
days than at seventy days (Figs 3 ,  a , b , c ) . The photomicrographs 
of these f ibres in developing limb muscles published by 
Ashmore et al. ( 1 9 7 2 )  for the sheep and Beermann et al . 
( 1 978 )  for the pig show a similar size decrease , al though no 
reference was made to it . Bridge & Allbrook ( 1 970 )  suggest  
that this decrease in f ibre size  may be caused by f ixation 
artefact ,  shrinkage due to loss of f luid from f ibres as  they 
mature or smaller fibres splitting off the larger ones . 
This unusual observation of a decrease in size during normal 
growth is worthy of further investigation . 
Using the semitendinosus muscle of  the pig , Beermann et al. 
( 1 9 7 8 )  noticed that f ibres in the superficial and deep 
portions of the muscle show differences in histochemical 
differentiation .  When an acid-stabile myosin ATPase 
technique was used , only the primary fibres in the deep 
region , and not the superficial region, are positive at  60 
days . Again , only in the deep region and not the superficial 
region are some secondary f ibres later changed to a pos itive 
reaction . With alkali-stabile ATPase staining , all f ibres in 
the superficial region are positive at 90 days , whereas in 
the deep region primary fibres are negative . The present 
study did not use the acid-stabile myosin ATPase technique . 
However , both superficial and deep parts differentiate into 
alkali-stabile myosin ATPase f ibre types in a similar manner , 
beginning 80 days of  gestation in the sheep . 
2 . 4 . 4  MORPHOGENESIS OF ISOENZYMES OF MYOSIN 
Two suggestions have been made for the morphogenesis of  
35  
isoenzymes of  fast and slow myosin types . Masaki and 
Yoshizaki ( 19 74) , using a fluorescein-labelled antibody 
technique on the pectoralis muscle  of  the chicken , and 
Gauthier , Lowey & Hobbs ( 1978 ) , using an immunocytochemical 
technique on the diaphragm muscle of the rat ,  showed that 
fast and slow isoenzymes coexist in all early developing 
muscle fibre s .  · Rubinstein , Pepe & Holtzer ( 19 7 7 )  used 
immunodiffusion and electrophoretic techniques on the 
developing pectoralis and anterior latissimus dorsi muscles 
of the chicken and suggested a second scheme of  isoenzyme 
differentiation in which only fas t myosin is present during 
early development . They suggested that the synthesis of slow 
myosin is dependent upon exogenous factors such as innervation, 
while fast myosin formation is  endogenous . Either of  these 
suggestions could be appropriate to the present histochemical 
observations . Since the myosin ATPase reaction reveals a 
pattern of  f etal development consistent with adult differentiation , 
it  appears useful even though the nature of the enzymatic change 
in fetal AL fibres is in doub t .  
2 . 4 . 5 .  THE SIGNIFICANCE OF FIBRE TYPE DISTRIBUTION WITHIN 
THE SEMITENDINOSUS MUSCLE 
Previous electromyographic studies of  the semitendinosus muscle 
in the dog (Tokuriki , 19 73 ;  Wentink ,  1976)1  the horse U�entink , 19 78)  
and the cat (Wetzel ,  Atwater & S tuart ,  19 76) have shown activity 
to be confined almost entirely to the retraction phase during 
locomotion . These studies did not incorporate histochemical 
investigations . Conversely , Gunn ( 1978)  described a histochemical 
division within the semitendinosus muscle in both the horse and 
the dog , with the AL dense part of the muscle closest to the 
limb axis . He sugges ted a postural role for this region 
although no electromyographic  s tudies were undertaken . 
36 
Other electromyographic studies on crural muscles of  the cat 
( Smith ,  Edgerton , Bet ts  & Colla tes , 1 9 7 7 ;  Walmsley , Hodgson 
& Burke , 1 978)  sugges t  that a division of labour exists  
between fast  and slow muscles , that they are recruited during 
different types and speeds of limb movement ,  and that the 
slow muscles are used more readily for postural funct ion and 
the fas t  muscles are active when more power is required . 
Gollnick et al . ( 1 9 74 ) , using the quadriceps f emoris muscle 
of the human and Sullivan & Armstrong ( 1 9 78) , the forelimb and 
hindlimb muscles of the rat ,  showed glycogen depletion 
during various activities . There was a selec tive depletion 
within muscles suggesting a differential recruitment of 
muscle fibres . According to Sullivan & Armstrong ( 1978 ) , 
as the ac tivity of  muscles becomes grea ter , there is a 
progressively greater reliance on fibres with lower 
oxidat ive capacities , situated in the more peripheral muscles 
or in the more peripheral areas of  a single muscle . A 
combined histochemical and electromyographic study made by  
Herring , Grimm & Grimm ( 1 9 7 9 )  showed that the electrical 
activity in different portions of  the masseter muscle of 
miniature pig varies systema tically during the various phases 
of mas t ication . The rostral portion ( which had 35% of AL 
fibres ) is  used throughout the masticatory contraction whereas 
the caudal portion (23%  AL fibres ) is used only during a 
brief portion of  the cycle . 
In the present s tudy ,  the superficial and deep parts  of  the 
semitendinosus muscle were  used to obtain simultaneous 
electromyographic recordings in order to tes t  for a functional 
difference between the areas confirmed as  having the lowest 
and the highest  AL f ibre type population densities  in this 
muscle . Although the fibres in the two regions run parallel 
to one another , no gross division occurs between the two 
region s .  Even though the two regions used for recordings 
37 
were about 25-35 mm apart from each other in the muscle , 
there is  no evidence that these two regions contained f ibres 
belonging to separate motor units . The results of  the present 
study are based on qualitative responses which need not be 
conclusive (Basmaj ian , 1 9 74 ) . Basmaj ian (loc . cit . )  states , 
however,  that two bellies or two heads in parallel within a 
muscle may show different electromyographic activities . 
Gans & Gorniak ( 1980)  discussed the repeatability and 
l imitations of electromyographic recordings and indicated 
that electromyographic recordings from physiologically and 
histochemically different fascicles of compound muscles can 
show differences in activity level . They also concluded that 
the e lectromyographic recordings obtained from reasonably 
s tandardized fine wire electrodes and standardized recording 
equipment and well-defined sites  in major subdivisions of  
muscles yield repeatable observations and permit predictions 
for equivalent events in the same muscle in other specimens . 
In the present study, motor units  of the slow ,  myosin ATPase 
low and the fast , myosin ATPase high f ibres , as  shown by 
Kugelberg ( 1 973 )  and Herring , Grimm & Grimm ( 1979 ) , appear 
to have been recruited in a selective manner during different 
activities . A functional significance for a regional difference 
in the myosin ATPase histochemical fibre type density within a 
muscle can therefore be postulated . The region with highest 
AL f ibre type population density becomes active readily in 
response to postural needs while the region with lowest AL 
fibre type population density i s  used only intermittently 
as  required during propulsive activity . 
Fibres  located closer to the j o ints over which a muscle acts 
have a lower torque about these pivots . Therefore , the AL 
f ibres lying close to the pivot suffer a mechanical 
disadvantage relative to the maj ority of  the AH f ibres . The 
explanation for the heterogeneous distribution of  AL f ibres 
38 
may invo'lve nutrition and energetics rather than mechanics . 
Firs t ,  AL fibres are oxidat ive and may be  expected to be 
most numerous in immediate proximity to the blood vessel 
bringing their nutrient and oxygen supply .  Electromyographic 
recordings in the present study and that of Herring et al. 
( 1 9 79 )  suggest  that the regions with high proportions of AL 
f ibres are ac tive in almost  every func tion of a muscle and this 
would require them to be  in need of an abundant and continuous 
supply of nutrients and oxygen . Secondly , during the 
production of a decelerating force , energy is transformed into 
heat (Feng , 1932 ; Carlson & Wilkie , 1974 ) . The production o f  
heat rather than mechanical work must  therefore be a feature 
of the posturally active AL f ibres . No t only metabolic 
waste , therefore , but also heat must  be  dissipated into 
adj oining blood  vessels . 
In earlier studies , Romanul ( 1 965 ) demonstrated the capillary 
supply of individual muscle f ibres within the gastrocnemius , 
plantaris and soleus muscles o f  the rabbit and showed that an 
aerobic fibre is  associated with a larger number of capillaries 
than an anaerobic f ibre . Myrhage & Eriksson ( 1 9 80) , showed 
that the ' slow ' soleus and medial heads of the gastrocnemius 
muscles of the cat have a denser vascular network with 
shorter average distance between the individual groups of 
secondary vessels compared to  the la teral head of  gastro­
cnemius , biceps femoris and tenuissimus muscles . The appearance 
of the corrosion cast  of the semitendinosus muscle of the 
sheep (Fig 2 1c )  suggests  that in the AL dense area , the 
vascular branches are thicker and more closely placed than 
in the AL sparse area , where the vessels  are narrower  and 
wider apart . It may be possible that AL f ibres occupy the 
part o f  the muscle where there is provision for ample blood 
supply to meet their nutritional and heat dissipation 
requirements . The semitendinosus muscle o f  the sheep was 
used in the present study for corrosion casting as this 
39  
muscle was already employed to study various other aspec ts . 
However , this muscle in the sheep is not suitable  to carry 
out an extensive study on vascular patterns and the dynamics 
of  blood flow between the AL dense and AL sparse areas within 
the muscle , because many AH fibres are also aerob ic ; they show 
a strong reaction for the aerobic metabolic enzyme succinate 
dehydrogenase (Fig. 22 ) . 
Previous studies indicate that blood flow pa tterns differ 
remarkably between slow and fast muscles . In the cat , the 
resting blood f low of the slow contracting soleus is 
approximately four times higher than that of the fast 
contracting medial head of the gastrocnemius ,  cranial tib ial 
and digital extensor muscles (Hilton , Jeffries & Vrbov� 1970 ) . 
During contraction , the blood flow through the fast muscles 
increases about seven times , while the change in the soleus 
muscle is much less . Hilton ( 1 972 )  deduced that inorganic 
phosphate , released by  fast muscles much more than the slow 
muscles during contrac tion , is responsible for the greater 
functional vasodilation in these muscles . It  is possible , 
therefore , that even within one muscle with distinct regions 
of ATPase low oxidative and ATP high glycolytic f ibre types , 
marked differences  in the vascular pattern and blood flow 
might be observed . The semitendinosus muscle of the pig 
should be suitable for such a study because it  has visually 
dis t inct ' red ' and 'white ' fibre areas which run parallel to 
each other for the length of the muscle and have the same 
origin and insertion (Tarrant , Hegarty & McLoughlin , 1 972 ) . 
Techniques involving radioactive microspheres (Ramsey & 
Donner , 1980) can be  used to s tudy the blood f low pattern 
between the red and white regions . In the same model , 
corrosion casting to show both arterial and capillary 
distribution in the two regions could be carried out . The 
validity of the hypothesis  advanced in the present study 
for the preferential accumulation of AL f ibres in a def inite 
40  
region of  a muscle could thus be  tested . Electromyography 
could also be employed . 
2 . 4 . 6 . CHANGES IN MYOSIN ATPase LOW FIBRE PROPORTION 
An increase in the percentage of  AL f ibres with the postnatal 
increase in body weight had been reported by Cardinet ,  Tunell 
& Fedde ( 1 9 7 1 )  in the pectineus muscle of  the dog , by 
Karpat i & Engel ( 19 6 7 )  in the soleus  muscle of  the rat and 
cat , by Davies ( 1 9 72 )  in the costal part of the diaphragm 
and in the thoracolumbar portion of  the longissimus muscle 
of the pig and by  Gunn ( 1 975)  in the semitendinosus and 
pectoralis  muscles of the dog . Kugelberg ( 1 9 76 ) , working 
on the soleus muscle of the rat and Swatland ( 1 9 7 7 ) , using 
the longissimus muscle of the pig , also reported a growth­
related shift  in ATPase activity . Holmes & Ashmore ( 19 7 2 )  
saw no s ignificant increase in the percentage o f  AL fibres 
in biopsies of  the semitendinosus muscle of cattle , but 
neither was such a change seen in the superficial part  o f  
this muscle in the present study . The observation of  
Swatland ( 1 9 78)  that the proportion of  AL f ibres decreases 
postnatally in the dark red area of the vastus medialis 
muscle of  the pig does not concur with the above studies . 
An increase . in the percentage of AL f ibres is  theoretically 
necessary postnatally if both isometric growth and postural 
ability are to be maintained (Davies , 1974 ) . Thus it is the 
deeper postural part of  the muscle that changes its AL 
f ibre proport ion during growth . Wherever possible ,  entire 
sections of muscle should be analysed if overall changes 
involving histochemical f ibre types are to be observed . 
Biopsies are l iable to give insufficient information . 
2 . 4 . 7 .  DEVELOPMENT OF FIBRE TYPE PATTERNS IN THE FETUS 
In the present study , regions of different proportions of  
ATPase low and ATPase high f ibre types ,  within the 
4 1  
semitendinosus muscle  of the sheep , are seen from the 8Q days 
gestation period onwards . There is an increase in the 
percentage of ATPase low fibres in the deep part of the 
muscle . Ashmore et al. ( 19 7 2 )  noticed an antenatal increase 
in the ratio of  AH f ibres relative to AL f ibres within the 
semitendinosus muscle of the sheep but they did not recognise 
dif ferences between superficial and deep parts of the muscle . 
Beermann et al. ( 1 9 78 ) , on the other hand , carried out a 
study on the semitendinosus muscle of the pig independent 
of that of  the present  study of  the sheep (Sivachelvan , 1 9 7 9 )  
and observed an antenatal increase in the AL f ibres in the 
deep but not in the superficial part of the muscle . The 
observations in the present study concur with those of 
Beermann et al . ( 1 9 7 8 ) . 
Activity of  fetal l imb muscles has been recorded in studies 
••  • 
such as those of Anggard & Ottoson ( 1 963 ) in the sheep and 
Bekoff , Stein & Hamburger ( 1975 ) in the chicken . 
. .  . 
Anggard & 
Ottoson ( 1 963)  noticed that electrical and mechanical 
responses in the gastrocnemius muscle increase with increasing 
fetal age and that these changes coincide with the 
myelination of nerve f ibres . Bekoff  et al. ( 1 9 7 5 )  recorded 
activities of  the gastrocnemius , peroneus and tib ialis 
muscles electromyographically and noticed that the flexor 
and extensor muscles are activated at different times while 
the activity of the synergists occur at  the same time . 
They suggested that the neural pattern generating circuits 
for selective activation of  muscles are established in the 
central nervous system without reliance on functional 
reflexes . The ac tivities  in the limb muscles of  an 
' amniotic aquanaut ' cannot be postural . The gravitational 
force acting on the fetal body is largely opposed by a force 
of  buoyancy . Thus , a precocious histochemical f ibre type 
differentiation and a regional distribution of the fibres within 
a fetal limb muscle are not due to a special mechanical 
42 
requirement . Both the f ibre type differentiation and the 
AL fibre type distribution pattern suggests an antenatal 
genetic influence on the semitendinosus muscle for its  
functional requirement at birth . 
43  
3 .0 PART 2 :  
3 .  1 ,  INTRODUCTION 
EXPERIMENTAL INDUCTION OF RELATIVE GROWTH 
CHANGES IN THE MUSCULOSKELETAL SYSTEM OF 
THE SHEEP . 
A muscle  may be altered in s ize  by experimental conditions .  
Increased work load and consequent increased size has been 
produced by surgical incapacitation of synergists by using 
denervation , tenotomy or removal methods by Crawford ( 19 6 1 )  in 
the cranial tibial muscle of the rabbit , by Denny-Brown ( 1 9 6 1 )  
in the soleus musc le  o f  the cat ,  by Reitsma ( 1969)  i n  the rectus 
femoris ,  plantaris and soleus muscles of the rat ,  by Binkhorst 
( 1 969 ) in the plantaris muscle of  the rat ,  by Jablecki & 
Kaufman ( 1 973)  in the soleus and plantaris muscles of  the rat and 
by Vaughan & Goldspink ( 19 7 9 )  and Hofmann ( 1 980)  in the soleus 
muscle of the mouse . Crawford (loc. cit . )  noticed that the 
increase in size of the cranial tibial muscle involves only the 
girth of the muscle  and not its  length . Denny-Brown (loc . cit. ) 
observed that the f ibre diameter of the hypertrophied soleus 
muscle  varies between 51 to 93 �m while that of the control soleus 
muscle  ranges between 38 to 87 �m . An increase in both the 
myofibrillar and sarcoplasmic components within hypertrophied 
f ibres has also been suggested in his studies . Reitsma (loc .  cit . )  
observed evidence for fibre splitting , an increase in the number 
of  nuclei ,  the formation of new capillaries and an increase in 
connec t ive tissue within muscles  hypertrophied by surgical 
removal of their synergists . Gutmann & Haj ek ( 19 7 1 )  induced 
hypertrophy in the extensor digitorum longus muscle of the rat 
by t enotomy of i ts  synergist , the cranial tibial muscle , and 
showed that the hypertrophied muscle has a prolongat ion of 
contraction time , a decrease in ATPase activity, and a relative 
increase of sarcoplasm but a decrease of  contractile proteins . 
Vaughan & Goldspink ( 1 9 79 )  noticed f ibre splitting within the 
soleus muscle hypertrophied by tenotomy of  its synergists . In 
44 
contrast to these experiments involving muscle hypertrophy, 
disu se of  a muscle following its  denervation , tenotomy or 
immobilization was used by Knowlton & Hines ( 1936)  to demonstrate 
size decrease in the gastrocnemius, soleus and plantaris muscles 
of  the rat , by Goldberg & Goodman ( 1969 ) and Rifenberick & Max 
( 1 9 7 4 )  in the soleus and plantaris muscles of  the rat and Gauthier 
& Dunn ( 1973)  in the semitendinosus muscle of the rat . 
Many denervation and immobilization studies , however , indicate 
that either an increase or a decrease in size can be produced 
in a part icular muscle depending on the state of stretch at which 
the muscle is kept during the period of  d isuse . This has , for 
example , been shown by Thomsen & Luco ( 1944 )  in the soleus and 
cranial tibial muscles of the cat , by Chang & Feng ( 1 962)  and 
Stewart ,  Sola & Martin ( 1 9 7 2 )  in the lat issimus dorsi  muscles of  
b ird s ,  by Stewart  ( 1968 )  in the gastrocnemius , plantaris and 
soleus muscles of the rat and by Williams & Goldspink ( 1 978)  in 
the soleus muscle of the mouse . Thomsen & Luco (loc . cit . )  
used a plaster cast to immobilize the tarsal joint o f  cats and 
noticed that when the j o int is in full extension there is an 
increase in the weight of  the cranial tibial  muscle while the 
soleus muscle decreases in weight . On the other hand , when the 
joint is fully flexed , an opposite effect with an increase in 
the weight of the soleus muscle and a decrease in that of the 
cranial tibial muscle is seen . Summers & Hines ( 1 95 1 )  
immobilized the hind limb of  the cat in neutral ,  shortened or 
stretched positions and noticed that the extent of  a trophy in 
the muscles immobilized in the shortened position is  greatest 
or least in the stretched or extended position respectively . 
Tabary , Tabary , Tardieu , Tardieu and Goldspink ( 19 7 2 )  
immobilized one hind limb of  the cat in lengthened , normal and 
shortened positions with a plaster cast , and measured muscle 
f ibre  length , sarcomere length and total number of sarcomeres 
within the soleus muscle in each instance , Contralateral muscles 
45  
were used as ' controls '  and no muscle weights were reported . 
Although f ibre length was similar between treated and supporting 
sides , there were more , shorter , sarcomeres in the soleus muscle 
of the plastered limb . Williams & Goldspink ( 1 978 )  noticed 
that in the soleus muscle of  the adult mouse , sarcomeres are lost 
in response to immobilization in the shortened position ,  whereas 
in the lengthened position , an addition of  sarcomeres occurs . 
Sola , Christensen & Martin ( 1 9 73)  showed that the cranial 
latissimus dorsi  muscle of  the chicken increases in size , whether 
the muscle is innervated or not ,  provided it is stretched . In an 
in vitro model using mechanical stretch on embryonic chicken 
muscle in culture , myotubes showed some of the biochemical changes  
seen in skeletal muscle hyper trophy (Vandenburgh & Kaufman , 1 9 79 ) . 
Thus the stimulus of stretch has a role in producing either an 
increase or a decrease in the size of a muscle (Thomsen & Luco , 1 944 ; 
Stewart ,  1972 ; Goss , 1 9 7 8 ;  Holly , Barnett , Ashmore , Taylor & Mole , 
1 980) . 
Of the experimental models used , only immobilization techniques 
maintain the anatomical integrity of  the animal . The results 
obtained by immobilization alone are therefore the easiest to  
interpret , provided the positions of j o ints over which affected 
muscles act are f ixed in a consistent manner .  Although the 
st imulus of stretch to growth will presumably be greatest for 
a muscle acting over two j o ints when both joints  are fixed in 
such a way as to stretch the muscle , no earlier study has compared 
a single�j oint muscle with a two-j oint muscle to show such an 
effect . Because immobilization of a l imb in a quadruped alters 
the stance of  the animal , there can be relative growth changes 
throughout the musculoskeletal system of  the trunk and limbs 
induced by alteration of  both muscle length and altered work 
load . Again , no earlier study on muscle hypertrophy using an 
immobilized l imb has attempted to examine the overall effects  on 
the components of the musculoskeletal system .  
46  
The present study was carried out on the sheep , a species of  
appropriate s ize for total and detailed musculoskeletal dissection 
and quantification . One hind limb was immobilized shortly af ter 
b irth to ensure stretch of  some muscles and contraction of others , 
for a period of  2-3 months . A poss ible differential response ,  
due to the influence of stretch , between single j o int and two 
j o int muscles was examined . An attempt was also made to analyse 
the relative growth changes throughout the musculoskeletal system 
of  the trunk and l imbs .  Alterations to growth patterns were 
studied by a comparison of treated and untreated sides of the 
animals , and by the study of untreated control animals . Changes 
in weight and ash content of  bones have been shown to be 
associated with changes in the usage of  muscles (Pottorf , 1 9 1 6 ;  
Gillespie , 1954 ; Kharrnosh & Saville , 1 965 ) . Thus , measurements of  
length, transverse sectional area and ash percentage of the 
femur and t ibia bones of the experimental lambs were undertaken 
to verify the repeatability of these observations . 
Changes in histochemical f ibre types during muscle hypertrophy 
have been examined by earlier workers .  Schiaffino & Bormiol i  
( 1 9 73 )  induced hypertrophy in the extensor digitorum longus 
and extensor hallucis longus muscles of  the rat by surgically 
removing the synergistic cranial tibial  muscle and noticed that 
the succinate dehydrogenase activity increases remarkably in 
the hypertrophied muscles , that the • white ' or mitochondria­
poor fibres are altogether absent compared to a proportion of 
about 40% in their control counterparts  and that the population 
of f ibres with low myosin ATPase activity increases in 
proportion . Yellin (1 9 7 4 )  used the hypertrophied , denervated 
hemidiaphragm of  the rat and observed that the ATPase low f ibres 
hypertrophy to the greatest extent , while wi thin the ATPase  
4 7  
h igh fibres ,  the ones which are purely anaerobic hypertrophy the least 
and for the briefest interval , and the ones which have combined 
aerobic and anaerobic capabilities respond in an intermediate  
fashion . Melichna & Gutmann ( 1974)  induced hypertrophy in  the 
extensor digitorum longus muscle of the rat using , in the same 
model , both immobilization of the limb in extension and denervation 
of the muscle . A decrease in myosin ATPase activity and an 
increase in succinate dehydrogenase ac tivity occurred . Holly 
et al . ( 1 980)  used a spring-loaded metallic tubular assembly to 
induce stretch hypertrophy in the wing muscles of  the chicken and 
observed that the oxidative enzyme activities increased 
substantially with stretch in the patagilis , a twitch muscle , 
but are relatively unchanged in the slow tonic cranial latissimus 
dorsi muscle . 
No previous histochemical study of f ibre types has used hypertrophy 
induced by stretching an immobilized muscle without other 
interference . The present s tudy , therefore , used the semitend­
inosus muscle , stretched in this way , to examine changes in the 
myosin ATPase low and high f ibre types . Changes in the shape 
of the muscle and in the number . of its  fascicles and muscle 
f ibre s ,  and the amount of connective tissue were s tudied in a 
manner similar to that employed in the study of this muscle 
during normal growth (Part 1 ) .  In this way , it was ascertained 
whether this type of hypertrophy occurs without al teration to 
the proportions of its  constituents . 
Electron microscopic studies by earlier workers have indicated 
that there can be disarranged sarcomeres and streaming of Z 
l ines (Tomanek , 1976 ; Vaughan & Goldspink, 1979 ) . The studies 
of  Vaughan & Goldspink ( 1 9 7 9 )  and Ho , Roy , Tweedle ,  Heusner , 
van Huss  & Carrow ( 1 980)  also indicated that splitting of f ibres 
occurs in muscle hypertrophy . However , their studies did not 
use immobilization . A limited electron microscopic investigation 
was included in the present study to observe the occurrence of  
these features in the semitendinosus muscle hypertrophied by  
immobilization technique . I t  is known that the tension 
developed by a muscle ul�imately depends on the transverse 
48 
49  
sectional area of  the myofibril content (Helander & Thulin ,  1962 ) . 
Therefore , an estimation of the proportions of  the mitochondrial , 
sarcoplasmic and myofibrillar components was undertaken to 
assess whether there were any changes at a subcellular level 
to indicate  that hypertrophy is associated with altered intrinsic 
propertie s . 
3 . 2  MATERIALS AND METHODS 
3 . 2 . 1 SOURCE OF MATERIAL 
Three male and six female , two-day old , Romney or Romney-Border 
Leicester cross lambs were used (Table 4 ) . The lambs were 
obtained during two lambing seasons , four in the first and f ive 
in the second season . Each lamb was separated from its dam two 
days af ter b irth and its body weight was recorded . Two lambs 
from the second lambing season were used as  untreated controls . 
The right hind limb of each of  the remainder was bound to the 
sternum for a period of two to three months. 
3 . 2 . 2  BINDING OF LIMBS 
Binding of the right hind limb of each lamb with adhesive tape 
was done immediately on its arrival in the experimental pen . 
One end of the tape was loosely wrapped around the metatarsal 
region of  the l imb , which was placed ventral to the sternum 
between the forelimbs , and secured by the tape around the 
thorax (Fig· 2 3 ) . Under these conditions , the lambs were able 
to support their body weights and walk on three l imbs . The 
b indings were changed every seven to ten days . 
3 . 2 . 3  MANAGEMENT AND FEEDING 
Foster milk (Glaxo) was introduced to each of  the treated and 
control lambs on the day of their arrival to the pen . The 
quantity of milk fed was gradually increased while reducing the 
frequency of  feeding f rom five times to twice daily . During 
the third week of the experiment ,  grass  and hay were introduced 
to supplement the milk diet . The animals were maintained in 
this manner throughout the rest of  the experimental period . 
All lambs were vacc inated against enterotoxaemia . The lambs 
5 0  
from each season were reared indoors , together in a pen 9 m2 in 
area . The pen was kept clean and warm . The wooden slatted f loors 
were not slippery . The animals were released from the pen at  
each feeding time and were made to  walk or run around a larger 
area for at least 30 minutes . The body weights of the lambs 
were recorded once a week. 
3 . 2 . 4 SAMPLING PROCEDURE 
lambs ,  the mean weight gain for the experimental lambs was 104±23 
g/day , less  but not significantly so (P > O . OS )  from that of  the 
controls , 1 4 1 ±39 g/day . The lambs were killed by stunning with 
a captive bolt and exsanguination . They were killed at f ive 
day intervals  to allow t ime for carcass dissection to be 
performed and for histochemical and , in the case of the animals  
belonging to the second lambing season (Nos . S  to  9 )1 for  electron 
microscopic specimens to be processed . 
Following stunning but before exsanguination , the semitendinosus 
muscle on each side was exposed and small strips from superf ic ial 
and deep parts  corresponding to AL dense and AL sparse areas of  
the muscle were obtained for electron microscopic studies . The 
s trips were immediately transferred into separate vials containing 
modified Karnovsky ' s  f luid (Appendix 3 ) . They were left in the 
refrigerator for at least one hour before further processing . 
Following slaughter , each lamb was skinned and eviscerated and 
its carcass weight (less head) was recorded . The semitendinosus 
muscle from each s ide was dissected out . A complete transverse 
slice was obtained and stored at -50°C for later histochemical 
studies . The remaining carcass was stored in a chiller for 
about three hours before muscles and bones from the limbs and 
trunk were dissected . 
3 .  2 .  5 ELECTRON MICROSCOPIC STUDIES 
The muscle strips , f ixed in modified Karnovsky ' s  f ixative 
(pH 7 , 2 ) ,  were transferred to petri dishes containing 
Karnovsky ' s  f ixative , With the aid of a dissection microscope , 
they were cut into 1 mm cubes using two halves of  a blade , 
making cutting strokes parallel to each other but in opposite  
directions . About ten selected cubes  were then left  overnight 
in a change of Karnovsky ' s  fixative . The cubes were then 
washed in 0 . 1  M phosphate buffered sucrose (pH 7 . 2 ,  Appendix 3 )  
and left in this solution at  4°C for  about four hours , changing 
the solution at least three times within this four hour p eriod . 
The cubes were post-f ixed in 1 %  Osmium tetroxide solution 
(Appendix 3 )  for one hour . Following post-fixation , they were 
washed again in phosphate-buffered sucrose solution , and left  
in a change of  that solution for about 30  minutes . An ascending 
series of alcohol of strengths 70% , 90% and 100% concentrations 
were used to dehydrate the specimens , which were agitated 
throughout the process .  The specimens were left for 10  minutes 
each in 70%  and 90% alcohol and were changed three t imes in 
absolute alcohol allowing 10 minutes for each change . The 
specimens were infiltrated first with two changes of 
propylene oxide , 1 0  minutes each , and then with 75% epoxy-resin 
(Appendix 3 )  in propylene oxide overnight . The caps of  the 
specimen vials were removed in order to a id evaporation of  the 
propylene oxide during infiltration by the resin .  The specimens 
were then transferred into fresh 1 00%  resin and left 6-8 hours . 
5 1  
They were f inally orientated at the bottom of a gelatine 
capsule , embedded in freshly prepared epoxy-res in and left  
in  an oven for about 48 hours for polymerization . 
The specimen blocks were trimmed in an ultratome (LKB) . 
Semithin sections o f  about 1 �m thickness were obtained and 
stained with 1 %  toluidine blue . The sections were examined under 
a light microscope and the best regions , with f ibres cut e ither 
longitudinally or transversely, were selected . The specimen 
block was f inally trimmed to use the best region for thin 
sectioning . Thin sections of about 70-90 nm thickness  were 
cut and picked up on both supported and unsupported grids . 
When the specimen grid was dry, it  was washed in d istilled 
52  
water by holding with curved forceps ,  face downwards .  It was 
blotted carefully with f ilter paper and immersed in a drop of  
uranyl acetate solution (Appendix 3 )  placed on paraf ilm , and 
stained for six minutes . The specimen was then washed in 50% 
alcohol and then in distilled water before counter-staining with 
a drop of lead citrate solution (Appendix 3) for s ix minutes . 
The specimen was washed and dried before it was viewed under an 
electron microscope . 
The thin sections were viewed under a Philips 200 electron 
microscope . Each section with about 3 to 5 f ibres was examined 
for possible  occurrence of a disorganisation of myofibrils , 
streaming of  Z lines and splitting of  f ibres . Electronmicrographs 
were taken at various  magnifications from appropriate regions . 
A point counting method was used to  analyse the electron­
micrographs . A square of 12 . 5  x 1 2 . 5  cm with 625 points  was 
used to calculate the proportions of the sarcoplasmic , 
myofibrillar and mitochondrial components within f ibres of  the 
semitendinosus muscle from both sides of the control lambs , 
and from the supporting and bound l imb sides of  the experimental 
lambs . A test point  distance of 5 mm was found to be best  to 
analyse the electronmicrographs of  1 2 . 5  x 12 . 5  cm area taken 
at a magnification of 1 7 , 500 . Electronmicrographs from 
transverse , longitudinal and oblique sections were used in 
determining the proportions of the subcellular components  
(Weibel , 1 9 79 ) . 
In total , four semitendinosus muscles from 2 control animals  
and 3 muscles each from supporting and bound limbs of  the 
experimental group were used for electron microscopy . 
were examined under the microscope to 
3 . 2 . 6  HISTOCHEMICAL AND QUANTITATIVE STUDIES 
53 
Stored , transverse slices of the semitendinosus muscle were thawed , 
sectioned and stained for alkali- stabile myosin ATPase following 
methods already described in Part 1 of this thesis . The transverse 
sectional area of the muscle , total fascicular area , total 
fibre area , mean fascicular area , mean f ibre area and the mean 
f ibre TSA of the AL and AH fibres in both AL dense and AL sparse 
areas were obtained ( Table 9 ) . 
3 , 2 , 7  CARCASS DISSECTION STUDIES 
The carcass dissect ion s tudy began about three hours af ter 
slaughter . The muscles were separated individually or in groups 
(Table 5 ) , cleaned of  extraneous fat and connective t is sue , and 
weighed , Bones were also cleaned and weighed . 
3 , 2 , 8 BONE MEASUREMENTS 
The length of  each femur , from intertrochanteric crest to 
intercondyloid fossa , and tibia , from its intercondyloid area 
to distal extremity, was measured . The individual bones 
were cut in the mid-shaft region using a band saw .  The cut 
surface of  the shaft was painted with ink and imprinted on 
paper . The outlines were traced and used to calculate the 
transverse sectional area (TSA) of both the shaft and cortex 
using a paper weighing method . The individual bones were 
then wrapped in aluminium foil and weighed . Contralateral 
bones f rom the same animal were placed together in a muffle 
furnace  and heated at  soo-ssooc for 10  to 15  hours until there 
was no significant further weight loss . Each aluminium foil , 
with and then without its contents ,  was weighed on removal 
from the furnace .  
3 . 2 . 9 STATISTICAL METHODS 
. 5 4  
8 ) . This comparison 
between supporting and bound sides v7ithin an individual excluded  
variations between animals . I t  was necessary to  estimate  the  ext:ent 
control lamb provided two sets of data (left and right ) . This 
enabled a mean to be compiled for control samples comparable in 
number with those of  the treated lambs (Table 7 ) . An analys is 
of the means , however ,  includes the variation between animals .  
Comparisons of  the weights  were expressed as a percentage of  
the carcass weight instead of the s laughter weight in order to 
eliminate the variable contributions of wool ,  skin and gut contents 
to slaughter weight (Table 7 ) . When a consistent trend could be 
observed between the muscles and bones of  the treated and untreated 
s ides of the experimental lambs1 this effect was considered to be 
due to the treatment regardless of the level of statistical 
s ignificance (Tables 6 , 7 ,  and 8 ) . 
The measurements obtained for the s emitendinosus muscle 
were subj ected to logarithmic regression analysis to  determine 
changes in the muscle of the bound side of the lambs relative to 
those in the muscle of the supporting side . The rationale for 
the use of  double logarithmic regression has already been 
outlined ( 2 . 1 . ,  2 . 2 . 7 ) . Both individual slopes for each animal 
and a common slope to include every experimental animal were 
incorporated in the analysis to test  the significance of the 
changes (Table 1 1 ) . This method of  analysis recognised 
separately f irst , the variation within individuals due to the 
treatment ,  and secondly the variat ion between individuals due 
to the possible effect of such factors as sex , b irth weight , 
slaughter weight and the length of  restraint . The difference 
between absolute area measurements of  each sampling pair of 
the semitendinosus muscles in the experimental lambs was 
analysed using the paired t-test ( Table  10) . The mean values 
of these area measurements for the treated and untreated sides 
of  the experimental lambs were compared with those for the 
controls to observe any significant decrease or increase in 
relative sizes of these  components . 
3 . 3  RESULTS 
3 , 3 . 1  CARCASS DISSECTION STUDIES 
Figure 23  is  a schematic representation of a lamb with 
altered hind limb function . Each of  the seven treated lambs 
had its right  hind limb bound beneath its sternum . The hip 
j oint was fully flexed and the s tif le and hock j o ints  were 
extended . In some respects , each treated lamb behaved 
differently from the controls . It  was less active , and when 
in sterna! recumbency ,  lay on the supporting hind limb side . 
When standing , its supporting hind l imb was kept  retracted 
and abducted . 
ss 
Table 4 gives data related to the breed , sex, age at  slaughter , 
l ive weight carcass weight , total carcass muscle  and total 
carcass bone for the seven experimental and two control lambs 
used in the experiment . Although many variables are observable  
between individuals ,  the mean growth rate of  the treated lambs , 
calculated a s  weight gain per day , was similar to those of  the 
controls . 
Table 5 includes  the measured values for weight of  muscles and 
bones of the experimental and control lambs used . Table  6 shows 
the muscles and bones of the experimental lambs ranked according 
to the mean weight differences  between the supporting and bound 
sides . The t value and its  level of significance ,  for each 
sampling pair,  obtained by using the paired ! test , is  also 
indicated . In the hind quarter , the quadriceps femoris , 
crural muscles , tensor fasciae latae , deep hip muscles and 
tibia are s ignificantly larger on the supporting side while 
the sartorius and gracilis , gluteus medius , semimembranosus 
and semitendinosus muscles and the hip bone are significantly 
larger on the bound side . In the forequarter , no muscles 
or bones are s ignificantly larger on the supporting side , 
while the scapula , humerus , and ribs and sternum are 
significantly larger on the bound side . In the forequarter , 
there is a trend for muscles and bones to increase in weight on 
the bound l imb side relative to the contralateral sid e .  The 
exceptions to this trend are the latissimus dorsi , neck 
and brachiocephalicus muscles which are expected to have 
minimal role  in supporting the body weight of the animal . 
In the hind quarter , on the other hand , there is  a trend 
for muscles and bones to increase in weight on the supporting 
limb side , relative to the contralateral side . This effect 
is  shown , in particular , in the extensors of  the stifle  
joint . The exceptions are the adductor femoris , gluteus medius , 
semimembranosus , semitendinosus (all of which are extensors of  
56  
the hip joint) , sartorius and gracilis (which adduct the hip 
and f lex the stifle)  and the hip bone . 
The mean percentage of  weights  of  pectoral , brachial ,  
antebrachial , femoral and crural muscles and bones  of  the treated 
lamb s ,  relative to the carcass weight ,  were tested for 
significance against those of  the control s  (Table  7 ) . In the 
treated lambs,  the scapula and the brachial muscles of both lef t 
and right forelimbs show a significant increase relative to 
5 7  
those  of  the controls . The bones and muscles of  the antebrachial 
region also show a significant increase on the bound side . 
The trend for all the muscles and bones of  both supporting and 
bound side forelimbs to increase in weight relative to the 
control is more marked on the bound limb side . In the hind limb , 
neither the femur nor the total side muscle on either side shows 
significant changes with respect to the controls .  In the crural 
region , however , the to tal muscles of the supporting side are 
heavier than the controls . The semitendinosus muscle is lighter ,  
al though not significantly,  than the control on  the supporting 
side , while it is significantly heavier on the bound side . This  
is in marked contrast  with the quadriceps group which is  
significantly heavier on  the supporting side and significantly 
l ighter on the bound side . Fig . 24 illustrates the semitendinosus 
mus cles  ob tained for the supporting and bound limb sides of an 
experimental lamb . 
3 . 3 . 2 BONE MEASUREMENTS 
Mean differences  calculated for length and transverse 
sectional area of  both the cortex alone and the whole mid shaf t ,  
together with the weight o f  ash and the percentage of ash, of  
the f emur and tib ia bones of the supporting and bound limbs of  
the experimental lambs are given in Table 8 .  The samples were 
tested for significance using the paired t test . The lash wei�ht of 
the tibia is  significantly greater in the supporting side . 
There is a general trend , for hind limb bones to be heavier , 
thicker and shorter on the supporting side than the 
corresponding bones of the bound l imb . 
3 . 3 . 3  QUANTITATIVE ANALYSIS OF THE SEMITENDINOSUS MUSCLE 
The semitendinosus muscle maintains the heterogeneous AL f ibre 
type population density pattern in both the supporting and 
bound limb s ,  as for normal muscles (2 . 3 . 3 ) . The AL f ibre type 
populatiog density , expressed as a percentage , in the AL dense 
area of the bound l imb muscles is  significantly greater 
58  
( t = 3 .  64 ,  P < 0 .  05 ) from that of  the supporting limb muscles as  
tested by the paired t test . There is  no significant difference 
in the AL sparse area ( t = 0 .  08 , P > 0 .  5 )  . The mean percentage 
of the AL f ibre type population density in both the AL dense 
area (control 23 . 7  ± 1 . 8 ,  supporting 20 . 9  ± 2 . 8 ,  bound 
28 . 4  ± 5 . 8  and the AL sparse area (control 3 . 80 ± 0 . 89 , supporting 
4 . 35 ± 1 . 8 1 ,  bound 4 . 1 7 ± 1 . 44 )  do not differ significantly 
between control , supporting and bound muscles . 
Table 9 shows the weight and area measurements of the 
semitendinosus muscle of the supporting and the bound sides of  
the treated lambs and the left  and right muscles of the controls . 
The paired t test was used to test  a null hypothesis , between 
the supporting and bound sides , for various components of  the 
muscle in the experimental lambs (Table  10) . The ac tual TSA , 
total fascicular TSA ,  total f ibre TSA , mean fascicular TSA , 
mean f ibre TSA , mean area of AL fibres in both AL dense and AL 
sparse areas and the mean area of  AH f ibres in the AL dense 
area show a significant increase on the bound side . The means 
of each of  these components for the supporting (n=7 )  sides were 
compared with the means for the controls (n=4 ) . No significant 
changes occur in any of these components in the experimental 
animals  relative to the controls , although there is  a general 
trend for the muscle components  on the bound side to be greater 
and those on the supporting side to be  smaller than those of  
the controls in all these cases . 
Double logarithmic analyses comparing transverse sectional 
areas and weights from the semitendinosus of the supporting 
and bound sides of  the experimental lambs were used to examine 
the relative changes in proportions due to the treatment 
(Table 1 1 ) .  These changes are graphically represented (Fig s .  
25-34 ) to  indicate variations both within individuals  due to 
treatment  (as shown by the slope of each line ) , and between 
individuals (as shown by the intercept of each line ) due to 
various factors previously mentioned (3 . 2 . 9 . ) . The control 
animals (Nos . 8 and 9 )  show, in each of these graphs 1 the nature 
of the variations found between the right and lef t muscles in 
normal animals .  No significant changes in connective t issue 
proportions , in fascicle number , in fibre number or in f ibre 
size dimensions occur within the muscle due to an increase in 
size (Table 1 1 ) . Thus , the almost  two-fold increase (Table 7 )  
in weight in the semitendinosus muscle i s  due to isometric 
growth of  the muscle ; the area of  fascicles and individual 
fibre s increases proportionately with those of the surrounding 
connective tissue components . Both AL and AH fibres are 
larger in the bound s ide when compared with the control or 
supporting side muscles (Fig. 34 , Table 10 ) . 
3 . 3 . 4  ELECTRON MICROSCOPIC STUDIES 
By an inspection of electronmicrographs , the sarcomere assembly,  
position of  triads and the Z lines were considered normal in 
their general arrangement (Figs . 35 and 36 ) . The proportions 
59  
of sarcoplasm, mitochondria and myofibrils were not signif icantly 
altered by the treatment (Table 1 2 ) . Thus , no ultrastructural 
component appears affected by the size increase due to the 
stretching of the muscle . 
3 . 4  DISCUSSION 
3 .  4 .  1 INCREASED GROWTH OF MUSCLES AND BONES DUE TO INCREASED WORK LOAD 
There have been several reports indicating that an increased 
work load on the hind limb muscles of  the rat produce s  an 
increase in muscle size . The work load was increased in muscles 
either by enforced exercise , or by the denervation , removal 
60 
or tenotomy of  their synergist s . Thus , Goldberg _ ( 1 96 7 )  used 
tenotomy of the gastrocnemius muscle and showed hypertrophy in 
the soleus and plantaris muscle s ;  Parizkova & Koutecky ( 1 968)  
used treadmill exercise and showed hypertrophy of the soleus 
muscle ;  Binkhorst ( 1 969)  used denervation of  the gastrocnemius 
and soleus muscles and treadmill  exercise , and showed hypertrophy 
of the plantaris muscle ; and Reitsma ( 1 969 )  used removal of 
synergist  muscles and showed hypertrophy in the rectus femoris 
and plantaris muscles .  
In the present study , the quadriceps femoris and tensor fasciae 
latae muscles increased in the supporting limb in relation to 
the muscles of  the bound limb . These muscles are extensors of  
the stifle join t .  They support the body weight by  preventing 
collapse of this joint . The extensors of  the shoulder , elbow 
and tarsus and flexors of the carpus could presumably also have 
been shown to enlarge ,  had the extensor and flexor muscles 
acting over these joints been separately analysed . The changes 
in the musculoskeletal components of  both the forelimbs and 
the supporting hind limbs suggest that the abnormal three-legged 
stance of  the animal stimulates growth of  muscles and bones of 
the supporting limbs . In particular , the pattern of  increase 
suggests  a diagonal support of  body weight using the contra­
lateral hind l imb and ipsilateral forelimb . There was a trend 
for the muscles of the crural l imb segment of the supporting 
hind l imb to show a greater increase than the thigh region . 
This may be due to the stance of  the treated lambs , with their 
supporting limb retracted and abducted , the contribution of 
thigh muscles and the femur being less than those of the crural 
components in supporting the body weight . 
The mech&nism of work load induced hypertrophy has been 
discussed by Stewart ( 1 9 72 ) , who suggested that the hypertrophy 
induced by enforced exercise , or by denervation , removal or  
tenotomy of  the synergists  of a muscle could be due to a 
' continuous active tension ' developing within the muscle . 
Nevertheless ,  in experiments involving teno tomy in the rat ,  
Mackova & Hnik ( 1 9 73 )  showed that the hypertrophy so produced 
in the muscles is due to a mechanical stretch of  these muscles 
rather than an excessive use of the muscles themselves . When 
the gastrocnemius muscle was tenotomised , the soleus muscle 
of the same side underwent hypertrophy . When the nerve supply 
of the antagonists of the soleus was cut along with teno tomy 
of the gastrocnemius ,  the soleus failed to hypertrophy . 
They obtained the same results when tenotomy of  the cranial 
tibial muscle was performed along with denervation of the 
antagonists of the long digital extensor muscles . Hofmann 
( 1 980) claimed that such a hypertrophy involved both a 
6 1  
stretching effect  due to antagonists and a neurogenic effec t .  
However , he used the values obtained for hyper trophy of soleus 
following unilateral tenotomy of synergists to evaluate the 
effects of b ilateral tenotomy of synergists but , since normal 
control animals were no t used , the results are difficult to 
interpret . The muscle hypertrophy produced in these experiments 
may be due to  the stretching rather than the chronic stimulation . 
3 . 4 . 2 .  GROWTH OF MUSCLES DUE TO STRETCH AND RESTRICTED CONTRACTION 
In various studies , it has been found that a muscle can undergo 
weight increase irrespective of intact  innervation , provided 
it is kept stretched by immobilization or an elast ic tensile 
apparatus (Thomsen & Luco , 1944 ; Chang & Feng , 1962 ; Sola , 
Christensen & Martin , 1 9 73 ; Williams & Goldspink, 1978 ; 
Holly et al . 1980 ) . 
In the present study , the hip extensors  of the bound hind 
limb were heavier than those of the supporting limb . All 
these muscles were kept stretched due to the f lexed posi tion 
of the hip during the experiment . Since the semitendinosus 
muscle is also a stifle  flexor ,  it  was maximally stretched 
and this could well explain why its hypertrophy was greater 
than that of  the other hip extensors . Thus , the present study 
shows that the stretch influence is greater for a muscle 
acting over two joints than for muscles acting over a single 
joint . All the extensors of the hip joint in the immobilized 
limb had an intact nerve supply.  The frequency of  stimulation 
was not measured , but it is expected that in such a bound 
position , an at tempt by the sheep would be frequently made to 
extend the hip in order to place the limb in a normal position . 
Neither stretch nor such a stimulation would be expected in 
the atrophied quadriceps muscl e .  This  muscle group was kept  
in a shortened position in the immobilized l imb . A significant  
reduction in its  growth rate may be due to  a lack of s tretch 
stimulus in it . By relating the treated lambs to the control 
lambs in the experiment , it  has been possible to demonstrate 
that the differences between the semitendinosus and the 
quadriceps muscles , due to the treatment ,  arise from a 
hypertrophy of  the semitendinosus in the bound side , and a 
combined atrophy on the bound side and hypertrophy on the 
supporting side for the quadriceps muscle . 
62  
Experiments involving chronic electrical stimulation (Pette , 
Smith,  Staudte & Vrbova, 1973 ; Pette , Ramirez , M�ller , Simon , 
Exner & Hildebrand , 19 75 ; Pette , M�ller , Leisner & Vrbov� , 1976 ; 
and Salmons & Sreter , 1 9 76 ) and cross  innervation (Buller , 
Eccles & Eccles , 1960 ;  Close , 1 969 ; Sreter , Gergely & Luff , 1 9 7 4 ;  
and Salmons & Sreter , 1 9 76 ) , show that the activity pattern of 
a muscle can change according to the type of  nervous stimulus 
it receives . As well as  producing changes in the contractile  
charac terist ics of  a muscle , a nerve can also modify its  
metabolic and histochemical properties , as demonstrated in 
cross  innervation experiments  (Dubowitz , 1 96 7 ;  B�r�ny & Close , 
1 9 7 1 ;  S almons & Sreter , 1 976 ) and by chronic  electrical 
stimulation (Pette  et al.  1975 ; Salmons & Sreter , 1 976 ) . 
Only a few of  the above mentioned reports demonstrate 
quanti tative changes in the muscles . Sreter et al. ( 1 9 74 )  
noticed loss of  weight in the cross innervated extensor 
digitorum longus muscle and in both the self innervated and 
cross innervated soleus muscle of the rat1 Pet te et al. 
( 1 9 75 ) , loss in mean fibre size in the intermittently 
stimulated cranial t ibial muscle of the rabbit , Pette  et al . 
( 1 9 76 ) , loss in weight in the chronically stimulated long 
digital extensor muscle of the rabbit , and Barany & Close 
( 1 9 7 1 ) ,  loss of  weight in the cross innervated soleus muscle 
of the rat . Thu s ,  there is no evidence to suggest a positive 
growth influence induced by a special pattern of  nerve activity . 
63  
Thus , stretch appears to be the mechanism producing size  changes 
in skeletal muscles kept in either a lengthened or a shortened 
position under immobilized conditions . The weight increase 
in muscles due to an excessive weight load may also be due to 
the same mechanism as observed in the extensors of the s tifle 
joint of  the supporting limb . The mus cles preventing the 
collapse of the j o int under gravity could have been hypertrophied 
due to stretch . It is  possible that the changes induced in the 
forelimbs of the sheep in the present experiment were also due 
to this mechanism because an excessive weight load or abnormal 
stance may have changed the position of various j o ints .  A 
detailed analysis of extensors and flexors of  the shoulder and 
elbow j oint would perhaps substantiate this . Stretch may also 
be an essential requirement for body building in man (Tanner , 
1952 ) , exercises for �.vhich are more successfully applied to the 
shoulder and arms than for the pelvic limb , in which j oint 
angles are more regulated by their weight supporting function . 
3 . 4 . 3 GROWTH CHANGES IN BONES 
Bones have been shown to lose weight and ash content with loss 
of  muscle activity (Pottorf , 1 9 16 ; Gillespie , 195 4 ;  Kharmosh 
bones tended to be l ighter ,  thinner and less mineralised than 
tho se of the supporting l imb . Both these bones , however ,  
showed a tendency t o  b e  longer than those of the supporting 
limb . Compression may , therefore ,  affect growth in bone length . 
3 . 4 . 4  HISTOLOGICAL STUDIES 
The increase in size of the semitendinosus muscle , as observed 
64 
in the model used , involved for many criteria only changes that 
would be expected during the normal growth o f  the muscle . First , 
the shape of the muscle is isometrically maintained ; the 
relation between weight and transverse sectional area is  
similar to that observed during normal postnatal growth (Table 
2 ) . Holly et al . ( 1 9 80 ) , using the patagilis ,  b iceps brachii ,  
long head of triceps brachii  and cranial latissimus dorsi mus cles 
showed that s tretch hypertrophy is  a result of both increased 
longitudinal and c ircumferential , growth . Longitudinal growth of  
the muscle is  maintained by an addition of  sarcomeres both in 
normal growth (Williams & Goldspink , 1 9 7 1 )  and muscles stretched 
in hypertrophy (Williams & Goldspink, 1 978) . 
Secondly, no significant change in fibre number occurs within 
the muscle under stretched conditions . Again , these effects  
are the same as  those observed in normal postnatal growth 
(Table 2 ) . Vaughan & Goldspink ( 1 9 7 9 )  also noticed that there 
were no significant changes in the fibre number of the soleus 
muscle following stretch . However , Reitsma ( 1 969 ) , using 
histological studies on the rectus femoris , plantaris and 
soleus muscles of the rat hypertrophied by surgical removal 
of synergists ,  and Sola , Christensen & Martin ( 19 73 )  using 
both denervation and stretching on the cranial latissimus 
dorsi muscle of  the chicken , c laimed that an increase in fibre 
number in addition to a f ibre s ize increase occurs under 
these conditions . They described an existence of newly 
forming f ibres with centrally positioned nuclei in these 
muscles . No such f ibres were observed in the present study 
(Fig . 34 ) . 
Thirdly , the connective tissue architecture shows no changes 
other than those commensurate with those seen during normal 
postnatal growth (Table  2 ) . Thus , the normal shape of  a 
stretched muscle appears to be  maintained by an increase in 
transverse sectional area due to a harmonious increase 
65 
between the fibre1 fascicular and connective tissue components and 
by an increase in length probably due to an addition of  
sarcomeres in series (Williams & Goldspink, 1 9 78 ) . 
In the present study , both AL and AH fibres of  the hypertrophied 
muscle show increase in size compared to those  of the semitend­
inosus muscle of the supporting l imb (Table 1 0 )  and the same 
trend when compared with those of  the control muscles . Yellin 
( 1 9 74 )  noticed tha t ,  in the stretched hemidiaphragm of the rat , 
AL f ibres increase to a greater extent than the AH fibres . 
Holly et al. ( 1 980)  also noticed that the growth response of 
the slow, cranial latissimus dorsi muscle is  more vigorous than 
that of the patagilis , a fast muscle . Although the present 
study does not provide conclusive evidence for such a preferential 
response of  the AL f ibres , this possibility is not excluded 
(Table 1 1 ) . Ho et al . ( 1 980)  used adult rats  in a weight 
lifting exercise programme and noticed hypertrophy in the long 
adductor muscle . Their studies suggested that the mean size 
of both AL and AH f ibres are s ignificantly smaller than those of  
the controls and that there is an increase in the number of f ibres 
per unit  of  transverse sectional area . These results  do not 
concur with those of the present study . The response to  the 
same s t imulus may differ between an actively growing muscle 
and a mature muscle . 
As ment ioned previously (3 . 4 . 2 ) ,  a change in contract ile 
propert ies  in muscle has been shown to occur as an adaptive 
response when there is a change in frequency of  s t imulation . 
This was demonstrated by cross innervation (Buller et al. 
66 
1960 � Dubowitz , 196 7 ;  Close , 1969 ; Sreter,  Gergely & Luf f ,  1 9 7 4 ;  
Salmons & Sreter , 1 9 7 6 )  and by chronic electrical s t imulation 
to produce a change in pattern of act ivity (Pette et al. 1 973 ; 
Pet te et al. 1 9 75 : Salmons & Sreter , 1 9 76) . Fast muscles 
subj ected to a continuous low frequency discharge acquire 
slow muscle properties , and slow muscles relieved of such a 
pattern of activation undergo reciprocal changes ( Salmons 
& Sr�ter , 1 9 7 6 ) . Accompanied by these changes in contractile 
properties , a change in myosin ATPase activity is  also 
observed . An increase in speed of  contraction parallels an 
increase in myosin ATPase  activity . When the muscle contract s  
more slowly the myosin ATPase activity is lower (Bar1ny , 1 9 6 7� 
r � , , Barany & Close··, 19 7 1 ;  Sreter , Gergely & Luff ,  1 9 74 : Vrbova , 
Gordon & Jones , 1 9 78 ) . These changes depend on the duration 
of  stimulation (Salmons & Sreter , 1 9 7 6 : Pette  et al . 1 9 7 6 : 
Vrbova, Gordon & Jones , 1 9 78) . Pette et al (loc. cit. ) used 
long intermittent (8h daily) or continuous (24h daily) 
st imulation with a frequency pattern resembling that of  a 
slow motoneurone to activate the fas t twitch cranial tibial 
and extensor digitorum longus muscles of  the rabbit . With 
intermittent stimulation , changes in the patterns of myosin 
light chain or alterations in the distribution of slow and 
fast fibres , as identif iable by histochemical myos in ATPase  
techniques , do  not occur within 40 days . However , these changes 
are observed af ter intermittent s t imulation period exceeding 
40 days or continuous s timulation periods longer than 20 days . 
Chronic abnormal usage of a muscle , as  utilized in the present  
s tudy , may also  produce changes in contractile properties . 
Thete is a s ignificant increase in AL fibre type proportions 
seen in the AL dense area . of the semitendinosus muscle in 
the bound l imb side . Changes in the activity pat tern of  this 
muscle could have been ei ther due to a change in frequency 
of  the stimulus applied or due to long term activity of  
the muscle . Since the nerve supply was not interfered 
with in this experiment ,  it is unlikely that the frequency 
pattern of the muscle s t imulation was altered by the 
treatment . However , the muscle might well have , over a large 
part of  the treatment interval , been stimulated to generate 
a force always opposed by the bindings , in an at tempt by the 
sheep to restore it s bound l imb to its normal position . The 
present experiment has shown that an increase in AL f ibre 
percentage within a muscle occurs even without the muscle 
being involved in the support of  body weight . This postural 
function was invoked to explain the effect of  an increase in 
AL f ibre percentage during postnatal growth ( 2 . 4 . 6 ) . The 
method of  strapping up the limb appears to have exaggerated 
the stimulation and the response that occurs during normal 
growth of  an animal . 
3 . 4 . 5 .  ELECTRON MICROSCOPIC STUDIES 
Earlier workers have shown that in muscle hypertrophied by 
tenotomy or by weight lifting exercises , ' disarrangement ' of 
sarcomeres , ' streaming ' of Z lines and ' split ting ' of f ibres 
are poss ible abnormalities expected in the muscles (Tomanek , 
1 9 76 , Vaughan & Goldspink , 1 9 79 , Ho et al. 1980 ) . The present 
study provided no evidence for these changes .  
Morphometric analysis of  skeletal muscle f ibres imposes many 
limitations because of  an ordered organisation of the fibres  
and a periodicity shown by  the sarcomeres (Williams , 1 9 7 9 ) . 
In using the point counting method for obtaining absolute 
measurements , the sampling method and the number of samples 
used , in addition to the magnification of the electron· 
micrographs , the number of test  points , the distance of  the 
test point in the square lattice employed and the 
orientation of the lattice have to be given careful 
consideration (Weibel  & Bolender ,  197 3 ,  Williams , 1 9 7 7 , 
Weib�l ,  19 79) . 
6 7  
In the presen t  s tudy , the proportionate changes involving the 
myofibrillar , sarcoplasmic and mitochondrial subcellular 
components  were considered quantitatively . In spi te of the 
material was 
are essent ially normal in this type of hypertrophy . Stretch 
hypertrophy need not be produced by a process o ther than 
normal myofibrillar growth . 
68 
4 . 0  GENERAL CONCLUSIONS 
The changes involved in skeletal muscle during both normal 
and altered growth have been studied . The results  suggest 
that the qualitative changes seen antenatally in a muscle 
are governed genetically . The growth pattern of  fascicles 
and f ibres within the semitendinosus mus cle of  the sheep 
suggests that endomysial and perimysial connective tissue 
control the number of fascicles and f ibres during normal 
growth of the muscle . The study also supports the idea that , 
within a muscle with a heterogeneous f ibre type distribution , 
the region which shows a more frequent postural type of 
activity has a h igh proportion of alkali-stabile myosin ATPase 
low (AL) fibres while the region which is involved in a more 
intermittent propulsive type of ac tivity has a low proportion 
o f  this f ibre type . A hypothesis is advanced that an 
accumulation of  AL fibres in the deeper part of a muscle with 
a heterogeneous fibre type population is  due to the special 
metabolic and heat dissipation requirements of  AL f ibres . 
By studying the regions of  extreme f ibre type distribution , 
the histochemical properties of a particular region of a 
69  
muscle has been shown to be altered during normal growth and by  
abnormal use . The transformation appears to  be due to  chronic 
stimulation rather than to the frequency of motoneurone disharge , 
since the latter should not have been af fected by  the altered 
conditions . 
Stretch appears to be a stimulus for muscle growth . The 
present study provides evidence that a muscle acting over two 
j o ints shows greater growth response to a stretch stimulus 
than a single j o int muscle . The model used has provided further 
evidence that a muscle can change its normal growth pattern , 
when the necessary stimulus is applied to it , irrespective of  
whether the muscle is  immobilized or not .  The increased 
growth of the muscle under immobilized but stretched 
conditions appears to occur isometrically , and without a 
change in intracellular composition . An immobilized muscle 
without stre tching becomes slower growing despite its  intact 
innervat ion . 
The semitendinosus muscle of  the sheep has therefore been a 
useful model in demonstrating new aspects of muscle growth . 
By the use of  certain extensions and modificat ions , the model 
can be used to explore muscle growth further . Chemical 
analysis involving dry mat ter , hydroxyproline and intra­
muscular fat  of the semitendinosus muscle will  extend the 
results  related to both normal growth changes and in hypertrophy . 
Electromyographic studies can be used in various muscles and in 
different species to establish a general concept distinguishing 
the postural and propulsive involvement of myos in ATPase low and 
high fibre types . Electrical activity of muscles kept 
immobilised can also be recorded to ascertain the behaviour of  
muscles during abnormal usage . Other extensions to the present 
model such as long term but intermittent immobilization , 
restoration of  limb act ivity after long term immobilization , 
or using a series of lambs at different time intervals ,  will 
establish the time sequence of stretch hypertrophy . Adult 
sheep can be used for immobilization to ascertain whether there 
are differences in stretch response between an actively growing 
muscle and one in which normal growth has been completed . As 
an alternative procedure , the hind limb may be immobilised with 
both the hip and stifle joints f lexed . In this instance , the 
semitendinosus muscle remains at about normal length while the 
semimembranosus muscle , ac ting over the hip alon� ,  lengthens 
as in the present model . Thus , the relative hypertrophy between 
the two muscles should be different from that already shown . 
It  was found in the present s tudy that the experimental lamb 
transmitted more weight on to the forelimbs than on to 
the supporting hind limb . An immobilised forel imb may , 
therefore , demonstrate bet ter than an immobilised hind l imb 
70 
the effects  on muscle weight distribution and histochemical 
f ibre type composition in the supporting contralateral l imb . 
One forelimb can be immobilised with both shoulder and elbow 
j o ints flexed . Changes in muscles such as biceps brachii and 
triceps brachii can be expected . These experiments  can be 
extended further to create immobilization of  limbs in utero , 
in order to demonstrate whether s tretch also acts  as a s t imulus 
to the antenatal growth of muscles . 
7 1  
Tab-le l a :  Data  related t o  the semitendinosus muscle o f  3 3  sheep . 1 8  from 60 days gestat ion to birth , · and 1 5  from birth to adulthood . 
TSA = Transverse sectional area . 
Sheep No . Age Sex Body weight Muscle weight Actual TSA Total Fasc icular TSA Total Fibre TSA Mean Fascicular TSA Mean Fibre TSA 
(kg) (g)  ( cm2 ) ( cm2 ) (cm2 ) (xl0311m2 ) ( \1m2 ) 
Antenatal 2eriod 
1 60 days F 0 . 08 0 . 1 8 0 . 1 5 - 0 . 04 - 1 10 
2 60 days F 0 . 05 0 . 06 0 . 1 2  - 0 . 02 - 1 1 3  
3 6 1  days F 0 . 04 0 . 05 0 . 1 9  - 0 . 05 - 98 
4 70 days M 0 . 12 0 . 18 0 . 24 0 . 1 0  0 . 08 2 . 6 1  1 00 
5 70 days F 0 . 18 0 . 30 0 . 2 1  0 . 06 0 . 05 2 . 35 66 
6 70 days M 0 . 20 0 . 29 0 . 2 5  0 .  1 2  0 . 10 4 . 45 70 
7 80 days M 0 . 34 0 .  72  0 . 9 2  0 . 55 0 . 33  6 . 35 1 86 
8 80 days F 0 . 35 0 . 78 0 . 44 0 . 25 0 . 15 4 . 73 1 1 5  
9 80 days F 0 . 33 0 . 69 0 . 63 0 . 35 0 . 20 5 . 14 138  
10  102 days F 1 .  04 2 . 75 1 . 48 0 . 8 1  0 . 52 10 . 30 202 
1 1  102 days M 1 .  06 2 . 9 7  1 . 02 0 . 80 0 . 53 1 1 . 38 2 5 7  
1 2  1 04  days M 1 . 24 2 . 40 0 . 96 0 . 5 1  0 . 3 1  10 . 7 1 1 7 1  
1 3  1 19 days F 3 . 04 5 . 50 2 . 9 1  2 . 1 2 1 . 42 26 . 1 9 309 
14  1 22  days M 2 . 98 5 . 8 1  3 . 02 2 . 16 1 . 05 26 . 60 263 
15  122  days M 3 . 42 5 . 14 2 . 37  1 . 96 1 .  2 1  42 . 74 735 
16  140  days F 1 . 69 5 . 90 1 . 66 1 . 30 1 . 18 20 . 65 464 
1 7  140  days F 1 .  73 7 . 30 1 . 44 1 .  22  1 . 02 1 8 . 3 1  468 
18 1 3 7  days M 5 . 0 1  1 0 . 50 1 .  73  1 . 39 1 . 08 20 . 59 5 1 5  
Postnatal Eeriod 
1 9  5 weeks M 1 0 . 90 24 . 00 2 . 85 2 . 68 2 . 32 55 . 99 932 
20 5 weeks M 1 1 . 8 1  34 . 00 4 . 22  3 . 78 3 . 35 55 .40 1053 
2 1  5 weeks M 1 3 . 1 8  33 . 70 4 . 73 4 . 14 3 . 45 52 . 80 8 1 5  
22 5 months F 2 6 . 80 58 . 00 7 . 06 6 . 5 7 5 . 79 1 3 7 . 02 2426 
23  5 months F 29 . 96 60 . 50 9 . 75 8 . 58  7 . 9 1  149 . 80 2830 
24 5 months F 30 . 00 85 . 9 1  8 . 56 7 . 94 6 . 45 1 72 . 89 1 5 73  
25  5 months F 35 . 30 1 56 . 30 9 . 1 9 7 . 80 7 . 10 1 82 . 84 2 700 
26 5 months F 3 7 . 50 86 . 04 5 . 40 4 . 86 4 . 46 1 34 . 43 2085 
2 7  5 months F 39 . 30 68 . 70 8 . 36 7 . 95 6 . 85 1 8 1 . 76 2293  
28  5 months F 39 . 50 1 00 .  10  7 . 12 6 . 20 6 . 03 1 60 . 47 1 8 47 
29  5 years F 45 . 40 93 . 50 8 . 93  8 . 25 7 . 38 208 . 0 1  3042 
30 5 years F 49 . 00 1 1 9 . 50 8 . 9 1  8 . 4 1  6 . 56 1 75 . 67 1 630 
3 1  5 years F 52 . 27 1 37 . 00 1 1 . 69 10 . 52 9 . 62 202 . 35 2 705 
32 5 years F 59 . 00 1 24 . 00 12 . 06 1 0 . 98 9 . 22 1 74 . 58 3066 
33 5 years F 6 3 . 00 1 22 . 00 9 . 45 9 . 07 8 . 28 139 . 40 2525  
-...J 1\) 
Table 1b : Data related to the semitendinosus muscle of  2 7  sheep . 12  from 80 days gestat ion to  b irth , and 1 5  from 
birth to adulthood . Measurements are made on f ibres reacted histochemically to  myosin ATPase , 
classified as  high (AH) or low (AL) reacting . 
Sheep No . FIBRE TYPE MEAN TRANSVERSE SECTIONAL AREA AL FIBRE NUMBER AS PERCENTAGE OF TOTAL FIBRES 
(�m2 ) 
AL dense area AL sparse area AL sparse area AL dense area 
AL AH AL AH 
7 1 68  1 37  288 1 8 7  4 . 4  12 . 8  
8 1 5 6  1 3 1  1 78 140  4 . 1  10 . 8  
9 1 5 2 148  1 68  1 25 5 . 8  10 . 8  
1 0  2 2 4  280 1 78 268  6 . 2  9 . 6  
1 1  22/+ 2 56  1 64  229  4 . 4 1 1 . 0  
12  286  2 6 6  185 253 3 . 8  1 2 . 2  
1 3  240 390 220  370  4 . 2 1 3 . 5  
14 350 5 53  2 5 7  329  2 . 8  1 5 . 5  
15  600  960  520  780  2 . 5  1 9 . 1  
1 6  482  423  454 4 1 5  5 . 5  14 . 1  
1 7  454 4 15 482 423  ·s . 8 13 . 6  
18  404 6 6 7  653  882  6 . 4  1 6 . 2  
1 9  9 9 6  1334  804  984  5 . 5 2 1 . 5  
20 831  1 1 75 7 6 1  1395  4 . 8  19 . 3  
2 1  695  1 0 1 5  657  820 3 . 7  1 7 . 5  
2 2  2358  2687  2696  2 7 76 5 . 5  23 . 5  
23  2488 2 788 2234  2990 6 . 3  22 . 8  
24 1 380 1840 1590  1960  3 . 6  26 . 0  
25  2730  2 740  2 690  2930 3 . 9  24 . 7  
26  1660  2 240 1 580 1940 4 . 1 25 . 0  
2 7  2023 ' 2673  1878  2948  6 . 9  26 . 6  
28  2223  2 1 54  2353  1 9 1 4  2 . 8 21+ . 2 
29  2 1 53  2666  1 8 7 1  3095 4 . 3 2 6 . 8  
30 2590  2 750  2320  2280 4 . 1 25 . 3  
3 1  2569  3706  2 1 60 3040 3 . 6  30 , 0  
32 2400 2 732 2546  305 7 3 . 5  29 . 0  
33 1 865 J R49 2275  2 600 4 . 4 2 7 . 8  """ \Jol 
Table 2 :  Regressions o f  the form log y ; a + b log x ,  comparing transverse sect ional areas (TSA) and weights from M .  Semitendinosus o f  the 
sheep , with ages ranging from 60 days antenatally to five years postnatally . n ; number of animals included in calculating each 
regression : sb ; Standard error of the regression coefficient b .  Values of  b significantly different ( P  < 0 . 05 )  from 1 or 0 . 67 ( G,H,I )1 
as appropriate,  are marked with an asterisk. 
VARIABLES ANTENATAL PERIOD ANTE AND POSTNATAL PERIOD POSTNATAL PERIOD 
b sb log a n b Sb log a n b Sb log a n 
A Total f ibre number 
x ; mean f ibre TSA (�m2 ) 1 . 70** 0 . 23 +3 . 55 18  1 . 3 7*** 0 . 08 +4 . 28 33  0 . 82 0 . 12  +6 . 08 15 
y - total fibre TSA (um2 ) 
B Total fascicular number 
x ; mean fascicular TSA (um2 ) 1 . 25* 0 . 10 +2 . 7 7  1 5  1 . 16 0 . 10 +3 . 0 1 30 0 . 74 0 . 1 1  +5 . 02 15 y ; total fascicular TSA (um2 ) 
c Fibre number within fascicle 
x ; mean f ibre TSA (um2 ) 1 . 12 0 . 1 3  +1 . 40 1 5  1 . 20 0 . 06 +1 . 18 30 0 . 95 0 . 15 +1 . 99 15  
y ; mean fasicular TSA (um2 ) 
D Fibre types in AL sparse area 
x ; mean TSA of AL fibres (um2 ) 1 . 0 1  0 . 20 +0 . 03 12  1 . 02 0 . 05 +o .0 1  2 7  0 . 8 1  0 . 1 1 +0 . 7 1  15  y ; mean TSA of  AH fibres (�m2 ) 
E Fibre types in AL dense area 
x ; mean TSA of AL f ibres (um2 ) 1 . 25  0 . 1 7 -0 . 55 1 2  1 .02 0 . 04 +0 .0 1  2 7  0 . 84 0 . 13 +0 . 58 1 5  
y ; mean TSA of  AH f ibres 
F Relative growth of M. Semitendinosus 
x ; body weight (g)  1 . 1 1* 0 . 05 +0 . 01 1 8  1 . 05"'  0 . 02 +0 . 1 4. 33  0 . 93 0 . 1 1  +0.  7 1  1 5  y ; muscle weight (mg) 
G Muscle shape and mechanics 
x ; muscle  weight (mg) o . 5s-:.- 0 . 05 +6 . 10 18  0 . 56*** 0 . 02 +6 . 14 33  0 . 6 1  0 . 10 +5 . 18 1 5  
y ; Actual TSA (um2 ) 
H Muscle shape and mechanics 
x ; muscle weight (mg) 0. 76 0 . 08 +5 .29  1 5  0 . 6 7  0 . 03 +5 . 55 30 0 . 6 1  0 . 10 +5 . 87 15  
y ; total fascicular (TSA (um2 ) 
I Muscle shape and mechanics 
x = muscle weight (mg) 0. 75 0 . 05 +5 . 14 1 8  0 . 73* 0 . 02 +5 . 20 33  0 .63  0 . 10 +5. 65 1 5  y = total f ibre TSA (um2) 
J Endomysia! connective t issue 
x = total fascicular TSA (um2) 0 . 95 0 . 04 +0 . 23 1 5  1 . 05* 0 . 02 -0 . 56 30 1 . 04 0 . 0 7  -0 . 42 15  y = total fibre TSA (um2 ) 
K Perimysial connective tissue 
x = Actual TSA (um2 ) 1 . 28*** 0 . 05 -2 .46  1 5  1 . 2 1*** 0 . 02 - 1 . 88 30 1 . 00 0 . 02 -0 . 05 15  
y = total fasc icular TSA (um2 ) 
L Perimysial and endomysia! tissue --...) 
x = Actual TSA (um2 ) ... 
y = total f ibre TSA (um2 ) 1 . 26*** 0 . 06 -2 . 46 18  1 . 29*** 0 . 03 -2 . 7 1 33  0 . 98 0 . 2 7  +0 . 10 15  
75  
Table 3 :  Predictions of  total fibre number in the semitendinosus 
muscle at the level o f  transection , from the equations 
in Table 2A . 
Age Equation for period Prediction 95% confidence l imits 
60 days Antenatal 89 . 1  X 103 56 . 1  X 103 - 141 X 103 
Antenatal & Postnatal 105  X . 103 75 . 8  X 103 - 146 X 103 
Antenatal 348 X 103 169 X 103 - 7 16 X 10 3 
birth Antenatal & Postnatal 2 1 7  X 103 177  X 10 3 - 265  X 103 
Postnatal 368 X 10 3 2 7 5  X 103 - 498 X 10 3 
Adul t  
Antenatal & Postnatal 3 7 2  X 103 268 X 10 3 - 516  X 103 
Postnatal 283  X 103 243  X 103 - 3 3 3  X 10 3 
Table 4 :  
Lamb No . 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Data related to the lambs used . Lambs 1 to  7 had their right hind limbs bound beneath their sternum . 
Lambs 8 and 9 were controls .  
Breed Sex Age at . slaughter T ,ive weight Carcass weight Total carca!:':s  Total  carcass 
(Days ) (kg) (kg) muscle bone 
(kg) (kg ) 
Romney Male 95 1 7 . 4  8 . 7 3 6 . 5 2 1 . 64 
Romney x Male 86 1 6 . 5  -7 . 30 5 . 5 1  1 . 4 1  
Border-Leicester 
Romney Male 63  10 . 0  4 . 08 2 . 74 . 1 . 04 
Romney Female 68 1 0 . 1  4 . 2 7 3 . 18 1 . 03 
Romney x Female 86  14 . 3  5 . 96 4 . 24 1 . 29  
Border-Leicester 
Romney Female 99 1 3 . 8  5 . 76 3 . 94 1 . 2 7  
Romney Female 79  1 3 . 3  5 . 1 8 3 . 87 1 . 1 4 
Romncy Female 79  1 6 . 3  6 . 80 4 . 64 1 . 40 
Romney Female 92  1 5 . 5  6 . 38 4 . 59 1 . 28 
'-1 0'\ 
Table 5 : Measured values for weights ,in gramrnes 1of muscles and bones of the lambs used . Lambs 1 to 7 had their right limbs bound beneath their sternum . Lambs 8 and 9 were controls.  L = left ; R = right . 
Neck muscles 
Brachiocephalicus 
Trapezius 
Latissimus dorsi 
Serratus ventralis 
Rhombideus 
Vertebral & rib muscles 
Pectoral muscles 
Brachial muscles 
Antebrachial muscles 
Diaphragm 
Longissimus system 
Sublurnbar muscles 
Abdominal muscles 
L 
129 
46 ,4  
27 .4  
63 , 4  
144 
201 
144 
392 
95 . 5  
45 . 1  
260 
87 . 8  
206 
Gluteus medius 120 
Deep hip muscles * 47 . 2 
Tensor fasciae latae 63 . 6  
Gluteobiceps 162 
Quadriceps femoris 191 
Semitendinosus 48. 2 
Semimembranosus 138 
Adductor femoris 68 . 4  
Sartorius & Gracilis 41 . 2  
Crural muscles 178 
Cutaneous & scrap muscles 333 
TOtal side muscle 3232 
Scapula 
Humerus 
Radius & ulna 
Rib & sternum 
Cervical vertebrae 
Thoracic vertebrae 
Lumbar vertebrae 
Sac rum 
Hip bone 
Femur 
Patella 
Tibia 
Total side bone 
• 
5 3 . 6  
60 . 5  
5 0 . 1  
153  
85 . 0  
79 . 0  
84 . 0  
31 . 0  
6 3 . 6  
79 . 2  
5 . 5  
63 . 9  
808 
1 
R 
121  
50 .4  
3 1 . 1· 
64 . 2  
164 
232 
174 
410 
101 
36 . 4  
284 
90 . 2  
208 
126 
41 . 3  
25 . 8  
144 
139 
83 . 1 
162 
64 . 6  
39. 8  
148 
346 
3285 
54 . 1  
6 3 . 7  
52 . 4  
173  
85 . 0  
7 9 . 0  
84 . 0  
3 1 . 0  
68 . 5  
7 7 . 1  
5 . 2  
5 7 . 3  
830 
L 
97 . 5  
47 . 5  
22 , 7  
46 . 8  
105 
222 
101 
339  
95 . 7  
30 . 5  
253 
72 . 5  
166 
93 . 8  
27 . 4  
3 0 . 4  
125 
177 
41 . 9  
106 
52 . 2  
26 . 6  
165 
307 
2751 
39. 3 
51 . 3  
43 . 1  
129 
76. 0 
81 . 0  
80 . 0  
19 . 5  
50. 7 
66 . 1  
4 . 7  
56. 3 
697 
2 
R 
81 . 7  
43 . 2  
24 ,4  
45 . 1  
1 18 
273 
109 
365 
97 . 2  
3 1 . 7  
256 
71 . 3  
174 
110 
25 . 9  
23 . 0  
135  
126 
59 . 8  
111 
5 0 . 1  
33 . 1  
129 
266 
2758 
41 . 8  
5 2 . 8  
44 . 9  
144 
76. 0 
54 . 5  
80 . 0  
19 . 5  
57 . 0  
66 . 3  
4 . 0  
47 . 7  
715 
L 
88 . 9  
29 . 3  
18 . 0  
24 . 3  
57 . 9  
50 . 8  
49 . 0  
156 
48 . 2  
17 . 0  
87 . 4  
33 . 2  
93 . 5  
3 
R 
87 . 0  
29 . 1  
18 . 4  
23 . 2  
68 . 3  
61 . 8  
50 . 1  
160 
5 1 . 2  
1 5 . 7  
92 . 4  
24 . 3  
81 . 6  
43 . 0  56 . 5  
19. 2 1 5 . 4  
15 . 9  5 . 80 
57 . 2  62 . 4  
97 . 1  64 . 0  
23 . 0  3 9 . 0  
43 . 9  68 . 7  
20 , 3  2 1 . 6  
1 2 . 1  21 . 9  
74 . 6  60. 7  
140 262 
1300 1441 
2 9 . 7  
41 . 1  
40 . 2 
97 . 7  
60 . 0  
54 . 5  
47 . 0  
1 3 . 6  
35 . 7  
50. 4  
3 . 7  
43 . 0  
517 
3 1 . 1  
43 . 2  
40. 6 
100 
60 . 0  
54 . 5  
47 . 0  
13 . 6  
38 . 5  
50 . 2 
3 . 4  
38 . 8  
521 
L 
106 
2 0 . 6  
2 1 , 4  
34 . 0  
71 . 9  
56 . 0  
73 . 6  
180 
58 . 8  
28 . 4  
111  
48 . 1  
119  
51 . 5  
23 . 6  
17 . 8  
69 .7  
97 , 6  
25 . 7  
64 . 7  
30 . 3  
20 . 1  
99. 4 
144 
1573 
27 . 5  
39 . 3  
35 . 1  
92 . 8  
56 . 1  
5 2 . 0  
5 2 . 0  
1 5 . 0  
37 . 4  
52 . 5  
4 . 4  
44 . 8  
509 
4 
R 
123  
2 3 . 4  
18 . 5  
28. 4  
69 . 9  
4 7 . 0  
69. 0 
170 
52 . 2  
30. 2  
126 
3 7 . 0  
108 
58 . 5  
21 . 0  
8 . 80 
7 3 . 3  
62 . 9  
45 . 5  
81 . 9  
29 . 0  
24 . 3  
82 . 0  
216 
1605 
26 .8  
39 . 3  
3 5 . 6  
92 . 8  
56. 1 
52 . 0  
52 . 0  
15 . 0  
40 . 8  
55 . 2  
3 . 9  
4 1 . 3  
517 
L 
143 
2 5 . 5  
16 . 5  
39 . 0  
88 . 0  
43 . 5  
58 . 5  
238 
67 . 0  
26 . 5  
161 
45 . 0  
135  
73 . 5  
49 . 0  
26. 0 
101 
168 
36 . 5  
86. 0  
3 5 . 0  
23 . 5  
114 
291 
2190 
3 5 . 0  
5 2 . 0  
43 . 5  
99 . 0  
67 . 5  
62 . 0  
56 . 5  
19 . 5  
50 . 0  
67 . 0  
3 . 5  
59 . 0  
632 
5 
R 
118 
26 . 0  
15 . 5 
34. 5 
92 . 0  
28 . 5  
lOO 
248 
69 . 0  
30 . 0  
149 
49 . 0  
1 18 
81 . 0  
4 0 . 0  
10 . 0  
77 . 0  
77 . 0  
71 . 0  
112 
44 . 0  
31 . 5  
92 . 0  
341 
2053 
40 . 0  
56. 0 
46. 0 
116 
67 . 5  
62 . 0  
56 . 5  
19 . 5  
59 . 0  
67 . 0  
5 . 5  
52 . 0  
662 
L 
132  
27 . 0  
17 . 5  
37 . 5  
87 . 0  
108 
80 . 0  
235 
64 . 0  
29 . 0 
140 
39 . 0  
119  
57 . 0  
40 . 0  
1 9 . 0  
82 . 0  
137 
35 . 0  
75 . 0  
3 5 . 0  
17 . 0  
104 
234 
1950 
35 . 5  
4 9 . 0  
41 . 5  
131 
67 . 0  
70 . 5 
6 1 . 5  
19 . 0  
41 . 0  
6 3 . 0  
3 . 0  
54 . 0  
633 
Deep hip muscles include Pectineus , Deep gluteal , Obturator and Gemelli  muscles . 
6 
R 
131  
21 . 5  
21 . 0  
36 . 0  
93 . 5  
120 
92 . 0  
248 
69 . 0  
26 . 0  
136 
37 . 5  
129 
66 . 0  
25 . 5 
11 . 0  
71 . 5  
77 . 5  
66 . 0  
95 . 0  
39 . 0  
23 . 5  
84 . 5  
269 
1989 
36 . 5  
52 . 0  
44 . 0  
130  
67 . 0  
70. 5 
61 . 5  
19 . 0  
44 . 5  
59 . 5  
2 . 9  
47 . 5  
634 
L 
126 
24 . 5  
12 . 0  
28 . 0  
81 . 0  
111 
59 . 0  
222 
54 . 0  
28 . 0  
145 
43 . 0  
133  
58 . 0  
37 . 5  
2 2 . 0  
82 . 5  
138 
26 . 0  
70 . 5  
35 . 0  
20 . 0  
112  
240 
1908 
28 . 0  
42 . 0  
35 . 5  
105 
61 . 0  
57 . 0  
4 9 . 0  
16 . 0  
3 3 . 0  
5 5 . 5  
2 . 0  
47 . 5  
532 
7 
R 
134 
27 . 0  
14 . 0  
28 . 0  
7 9 . 0  
123  
7 2 . 0  
234 
59 . 0  
29 . 0  
151  
38 . 0  
134 
58 . 5  
28 . 0  
29 . 0  
64 . 5  
6 1 . 0  
47 . 5  
7 7 . 0  
29 . 0  
24 . 0  
76 . 0  
343 
1959 
30 . 5  
43 . 5  
35 . 5  
111 
61. 0 
57 . 0  
49 . 0  
16 . 0  
34 . 0  
50 . 0  
2 . 0  
4 0 . 0  
530 
L 
158 
35 . 0  
15 . 0  
37 . 0  
105 
153 
88 . 0  
245 
70 . 0  
31 . 5  
202 
50 . 5  
156 
81 . 5  
4 1 . 0  
16 . 0  
108 
136 
46 . 0  
128 
52 . 5  
24 . 0  
124 
259 
2362 
36. 5 
54 . 5  
44 . 5  
145 
70 . 5  
78 . 5  
67 . 5  
20 . 5  
45 . 0  
74 . 0  
4 . 0  
56 . 5  
696 
8 
R 
156 
30 . 0  
13 . 0  
32 . 0  
102 
147 
89 . 0  
243 
72 . 5  
30 . 0  
191 
52 . 5  
159 
68 . 0  
46 . 0  
15 . 0 
108 
136 
44 . 5  
122 
52 . 5 
24 . 5  
120 
228 
2281 
38. 0 
55 . 5  
44 . 0  
155 
7 0 . 5  
78 . 5  
67 . 5  
20 . 5  
4 5 . 0  
71 . 0  
4 . 0  
52 . 5  
701 
L 
161 
28 . 5  
16. 5 
32 . 0  
96 . 5  
116 
75 . 5  
238 
65 . 0  
26 . 5  
186 
48 . 0  
180 
77 . 5  
45 . 0  
16 . 0  
104 
151  
4 2 . 0  
1 14 
5 3 . 0  
23 . 0 
110  
272 
2277 
29 . 0  
51 . 0  
3 9 . 0  
137 
75 . 5  
7 5 . 0  
53 . 5  
19 . 0  
42 . 5  
68. 5 
1 . 0  
5 2 . 5  
643 
9 
R 
143 
30 . 5  
15 . 5  
3 1 . 5  
9 5 . 0  
123 
81 . 0 
238 
66 . 0  
30 . 0  
177 
47 . 0  
164 
77 . 0  
43 . 0  
14 . 0  
199 
150 
42 . 5  
112 
5 2 . 0  
24 . 5  
110 
247 
2312 
30 . 0  
5 0 . 0  
39 . 0  
132  
75 . 5  
7 5 . 0  
53 . 5  
19 . 0  
41 . 0  
67 . 5  
1 . 5  
52 . 5  
636 
"'l 
"'l 
Table �: Muscles and bones ranked according to the mean weight difference (supporting side - bound side) , 
for seven lambs with bound right hind limbs . 
MUSCLES Mean difference 
I 
Larger 
on left 
Quadriceps femoris 
Crural muscles 
Tensor fasciae latae 
Gluteobiceps 
Deep hip muscles 
Sublumbar muscles 
Abdominal muscles 
(supporting) Latissimus dorsi Diaphragm 
side 
Larger 
on right 
(bound) 
side . 
l 
Neck muscles 
Brachiocephalicus 
Trapezius 
Adductor femoris 
Antebrachial muscles 
Brachial muscles 
Longissimus system 
Sartorius & Gracilis 
Serratus ventralis & 
Rhomboideus 
Gluteus medius 
Vertebral & rib muscles 
Pectoral muscles 
Semimembranosus 
Semitendinosus 
(g) 
+ 56 . 90 
+ 25 . 00 
+ 14 . 20 
+ 7 . 3 1  
+ 6 . 86 
+ 3 . 04 
+ 2 .  70 
+ 1 . 94 
+ 1 . 30 
+ 0 . 9 1 
+ 0 . 1 7  
1 . 06 
1 . 87 
2 . 20 
3 . 00 
5 . 29 
5 . 37 
7 . 1 3 
8 . 53 
- 1 3 . 50 
- 14 . 40 
- 1 7 . 64 
- 25 . 82 
t+ 
+ 7 . 1 1*** 
+ 7 . 35*** 
+ 3 . 48* 
+ 1 . 44 
+ 4 . 09** 
+ 1 . 44 
+ 0 . 68 
+ 2 . 22 
+ 0 . 88 
+ 0 . 15 
+ c;l . 12 
- 1 . 1 7 
.,. 1 . 06 
- 1 . 39 
- 0 . 5 1  
- 1 . 19 
- 3 . 93** 
- 2 . 34 
- 4 . 40** 
- 1 . 57  
- 2 . 36 
- 5 . 38** 
- 9 . 52*** 
BONES Mean difference 
(g)  
Tibia + 5 . 91  
Femur + 1 . 20 
Patella + 0 . 56 
Radius & Ulna - 1 . 43 
Scapula - 1 . 74 
Humerus - 2 . 19 
Hip bone - 4 .4 1  
Ribs & Sternum - 9 . 29 
+ Values of Student ' s  t for null hypothesis that supporting side weight - bound side weight 
Significant values are marked with an asterisk (* = P<O . OS; ** = P < O . O l ;  "!<** = P < O . OO l )  
t+ 
+ 8 . 30*** 
+ 1 . 1 7 
+ 2 . 18 
- 1 . 4 1  
- 2 . 52*  
- 4 . 34** 
- 4 . 46** 
- 3 . 41*  
0 --.;) 00 
PECTORAL 
BRACHIAL 
79 
Table 7 :  Comparison of  the weights  of  var ious l imb regions . The 
supporting  side  and bound s i de of  seven t reated lambs are  
expressen as  a percentage of  the carcass we igh t ,  and compared 
with that of t he two controls . Each control lamb provided two 
se ts  of da t a  ( r ight and lef t ) .  Va lues  showing sign i f icant 
d i f ferences  f rom the con trols  (P < 0 . 05 )  are shown wi th asterisks . 
Scapula 
Huscles  
Humerus 
Muscles  
Control  lambs 
(n=4 ) 
Mean s . d .  
0 . 2 1 0 0 . 0 1 9  
0 . 524  0 . 023  
0 . 332  0 . 006 
1 . 5 1 6  0 . 0 1 5  
Treat ed lambs 
(n=7 )  
support ing s ide  
Mean s d .  
0 . 2 6 1 *  0 . 032  
0 . 585 0 . 142  
0 . 356 0 . 034 
1 . 8 1 2 *  0 . 2 30  
bound s ide  
Mean s . n .  
0 . 2 7 3*  0 . 028  
0 . 6 7 9  0 . 1 49 
0 . 363  0 . 037  
1 . 8 7 7 *  0 . 2 66 
Radius & Ulna 
ANTEBRACHIAL 
0 . 2 62 0 . 0 1 0  0 . 3 1 0  0 . 046  0 . 320* 0 . 045 
Muscles 0 . 430 0 . 0 1 0  0 . 505 0 . 062  0 . 5 1 8* 0 . 047  
Femur 0 . 442  0 . 008 0 . 460 0 . 040  0 . 453  0 . 054  
Total muscle 3 . 324  0 . 226  3 . 264 0 . 383  3 . 09 1  0 . 4 1 9  
FEMORAL 
Semitendinosus 0 . 2 74  0 . 004 0 . 245 0 . 0 24 0 . 430f<  0 . 055  
Quadriceps 0 . 903  0 . 069 1 . 045*  0 . 0 7 1  0 . 626*  0 . 1 1 3 
Tibia 0 . 3 3 7  0 . 009 0 . 384 0 . 049  0 . 342  0 . 04 7  
CRURAL 
Muscles 0 .  7 29  0 . 0 1 6  0 . 879*  0 . 1 1 3  0 . 697  0 . 1 06 
rable 8 :  Values for mean differences and Student ' s  t obtained 
by using the paired t test  for various measurements  
o f  the femur and tibia bones for seven lambs  with 
bound right hind limb . 
Femur Tibia 
Mean d ifference t+ Mean difference + t 
Length of  bone -0 . 30 -1 . 22 -0 . 16 -0 . 33 
( cm) 
TSA of cortex +0 . 09 +0 . 4 1  +0 . 0 1  +0 . 45 
(cm2 ) 
TSA of shaft +0 . 16 +0 . 7 1 +0 . 003 +0 . 06 
( cm2 ) 
80  
Weight of  ash +0 . 36 +0 . 32 +2 . 20 +3 . 16* 
(g)  
Percentage of  ash +0 . 34 +0 . 22 + 1 . 11� +0 . 77  
+ Values of  Student ' s  t for the null hypothesis , support ing 
side - bound side = 0 .  Significant value is  marked with an 
asterisk ( *= P < 0 .  05 )  • 
TSA = Transverse sect ional area . 
Table 9 :  
Sheep 
number 
L 
R 
2 L R 
3 L R 
4 L R 
5 L R 
6 L R 
L 
7 R 
8 
L 
R 
9 L R 
Weight of the semitendinosus muscle and area measurements  of its  struc tural components ,  of supporting (left)  and bound (right ) sides,  of 
lambs used in experimental induction of al tered hind limb function . 
TSA = Transverse sectional area . 
Lambs 1 to 7 are treated animals and 8 and 9 are controls .  
AL dense area AL sparse area 
Muscle Actual Total Total Mean Mean total AL fibre AH f ibre AL f ibre AH f ibre 
weight TSA fascicular f ibre fascicular fibre TSA TSA TSA TSA 
TSA TSA TSA TSA 
(g) (cm2 ) (cm2 ) (cm2 ) (\1m2 ) (\1m2 ) (\1m2 ) (1Jm2) (\1m2 ) (1Jm2 ) 
48 . 2  5 . 60 5 . 2 1  4 . 37 70700 1 100 1690 1 500 1060 1 1 70 
83 . 1  8 . 52 7 . 84 6 . 90 73 100 1820 2 1 10 2000 2950 2070 
4 1 . 9  5 . 08 4 . 37 3 . 45 6 1400 1 190 1680 1 380 10 10  1 240 
59 . 8  6 . 07  5 . 33  4 . 78 68300 1260 2400 1300 2890 1 380 
2 3 . 0  4 . 43  3 . 63 2 . 97  44800 494 462 579 480 435 
39 . 0  5 . 07  4 . 45 3 . 63 50300 583 889 698 782 580 
25 . 7  3 . 35 2 . 87 2 . 2 7  39000 764 826 . 1 0 10  63 1  682 
45 . 5  4 . 20 . 3 .  78 3 . 02 50500 1 18 1  1 220 1 300 989 1 000 
36 . 5  3 . 7 1 3 . 0 1 2 . 38 76800 1420 1 340 1 5 10 1 390 1 780 
7 1 . 0  7 . 46 6 . 88 5 . 18 96 100 1 980 3650 1 9 10 2 360 1540 
35 . 0  5 . 12 4 . 00 3 . 37 37300 750 608 695 61 1 885 
66 . 0  7 . 05 6 . 46 5 . 52 68500 1 1 1 0  1 630 1 390 1 550 1 330 
26 . 0  3 . 5 7  3 . 07 2 . 33 43 100 7 78 733 830 694 785 
4 7 . 5  5 . 8 1  5 .  1 3  3 . 96 55 100 1 1 00 1680 1 020 1 100 1050 
46 . 0  4 . 62 l. . 04 3 . 3 1 36400 773  9 13  857  869 928 
44 . 5  4 . 5 1  3 . 74 3 . 1 8 38300 843 938 1020 926 1 060 
42 . 0  4 . 89 4 . 29 3 . 54  54900 1 1 50 1 1 90 999 1430 1030 
42 . 5  4 . 74 4 . 3 1 3 . 70 52700 1040 1 1 10 1070 1040 920 
(X) 
� 
Table 10 : Values for mean differences and Student ' s  t ,  
obtained by using the paired t test for structural 
components  of  the semit endinosus muscle , for seven 
lambs with bound right hind l imbs . 
Mean difference t value+ 
Actual TSA . -1 . 90 -4 . 33** 
(cm2 ) 
Total fascicular TSA - 1 . 96 -4 . 56** 
(cm2 ) 
Total fibre TSA -1 . 69 -5 . 33** 
(cm2 ) 
Mean fascicular TSA -1 2685 -3 . 4 2* 
(]..lm2 ) 
Mean f ibre TSA -363 -4 . 08** 
(J..lm2 ) 
AL den·se area 
AL fibre TSA -89 1 -3 . 49* 
(]..lm2 ) 
AH fibre TSA -302 -3 . 1 1* 
(J..lm2 ) 
AL S:earse area 
AL fibre TSA -964 -3 . 7 2** 
(J..lm2 ) 
AH fibre TSA -282 -2 . 1 5 
(J..lm2 ) 
+ Values of Student ' s  t for the null hypothesis that 
supporting side area - bound side area = 0 .  Significant 
values are marked with an asterisk (* = P < 0 . 05 ,  
** = p < 0 .  0 1 )  . 
TSA = Transverse sectional area . 
82 
Table 1 1 : Double logarithmic regre ssion analysis+ comparing 
M .  semitendinosus of  the supporting and bound sides 
of seven lambs with bound right hind limbs . 
a )  Changes in shape 
x muscle weight (g )  
y = actual TSA ( cm2 ) 
b) Changes in connective tissue 
i )  perimysium 
x actual TSA ( cm2 ) 
y = total fascicular TSA ( cm 2) 
ii )  perimysium and endomysium 
x = actual TSA ( cm2 ) 
y = total fibre TSA (cm2 ) 
iii )  endomysium 
x = total fascicular TSA (�m2)  
y = total fibre TSA (�m2 ) 
c )  Changes in fasc icle number 
x = mean fascicular TSA (�m2 ) 
y = total fascicular TSA (�m2 ) 
d) Changes in fibre number 
i )  within muscle 
x mean fibre TSA (�m2 ) 
y = total fibre TSA (�m2 ) 
ii )  within fascicle 
x = mean fibre TSA (�m2 ) 
y = mean fascicular TSA (�m2 ) 
e )  Changes in fibre size dimensions 
i) AL sparse area 
x AL fibre TSA (�m2 ) 
y = AH fibre TSA (�m2 ) 
ii)  AL dense area 
x AL fibre TSA (�m2 ) 
y = AH fibre TSA (�m2 ) 
b t 
0 . 63  0 . 2 1  0 . 16 . ( 0 .  6 7 )  
1 . 18 0 . 40  0 . 4 5  ( 1 . 00 )  
1 . 2 1 0 . 39 0 . 54 ( 1 . 00 )  
1 . 06 0 . 34 0 . 17  ( 1 . 00 )  
1 . 82 0 . 56 1 . 46 ( 1 . 00 )  
1 . 35 0 . 4 2  0 . 8 3  ( 1 . 00 )  
0 .  7 2  0 . 45  0 .  64 ( 1 .  00 )  
0 . 39 0 . 33 1 . 8 3  ( 1 . 00 )  
0 . 40  0 . 32 1 . 88 ( 1 .  00 )  
+ The rate of increase of y with respect to x for each sheep was 
calculated and the value of  the common slope b was tested for a 
significant difference from 1 . 00  or 0 . 67 , as indicated , using 
Student ' s  t test . sb standard error o f  b .  None o f  the values 
is significant . TSA = Transverse sectional area . 
83  
Tabl e 12 : Measurements + re lated to ul trastructural components 
of the semi tendino sus mus c l e s  of the control l ambs 
and of the supporti ng and bound s ides o f  experimental 
lambs with a ltered l imb function . 
Control Supporting side Bound s ide 
Mean s . e n Mean s . e .  n Mean s . e .  n 
Sarcoplasm 19 . 1  4 . 2  8 - 19 . 4  4 . 2  7 17 . 3  2 . 1  7 
Mi tochondria 2 . 1  1 . 6 8 3 . 9 2 . 4 7 3 . 8  2 . 1  7 
Myo f ibril lar 78 . 7  5 . 0  8 7 6 . 7  5 . 2  7 80 . 1  3 . 5  7 
componen t  
+ Proportionate a r e a  of the sarcoplasm ,  mi tochondria and 
the myo f ibri l l ar component expres sed as a percentage of the 
area examined . n = number o f  f ibre s analysed . 
s . e .  = standard e rror of the mean . None of the value s 
i s  s ignificant . 
84 
F i gure 1 :  Attachments o f  the semi te ndinosus mu scle o f  the sheep . 
Sartorius 
M ED IAL  
CRAN IAL 
Tensor fasc iae latae 
�---Vastus latera l is  
++---+-- Vastus intermedius  
�-1--.,._-----FEMUR  
LATERAL 
4-----1------t'<>Pi itea I fat 
�-----ll----- B iceps femoris 
em ite n d i n o s u s 
CAU DAL 
F i gure 2 :  Schematic repre sentation o f  the transverse section o f  
the th i gh o f  the adult sheep . The propor t ionate 
d i s tribution o f  myo s i n  ATPase low f ibres as a 
percentage of to tal f ibre popu lat ion , in d i f ferent 
regions o f  the s emi tend inosus mu scle , is i nd icated 
by d i f ferent shade s . Wh i te repre sents a AL f ibre 
population den s i ty of 3-6% o f  all f ibre s ; sma l l  dot s  
a den s i ty o f  7 -9% , large dot s  a den s i ty o f  10-19% ; 
and black , an AL f ibre dens i ty of 2 0- 30% o f  a l l  f ibre s . 
a 
b 
c 
Figure 3 .  Frozen transverse sections of the semitendinosus 
muscle of  the sheep , showing early differentiation of  fibre 
types . x 650 
87  
a .  PAS-hematoxylin �taining o f  6 0  days fetal muscle . Clusters 
of , large , primary generation cells with central nuclei are 
seen . Arrows indicate small sized secondary generation cells . 
b .  Alkali-stabile myosin ATPase staining of 60  days fetal muscle . 
The large cells show a pale central area . Arrows indicate 
the secondary generation cells . 
c .  Alkali-stabile myosin ATPase staining of 70 days fetal muscle . 
The large primary generation cells are widely separated and 
have a paler centre and a darker periphery (arrows ) .  Many 
smaller cells are seen to surround individual primary 
generation cells . 
a 
b 
Figure 4 .  Frozen transverse sec t ions of the semitendinosus 
muscle of  a 30 kg live weight sheep , s tained for myosin 
ATPase in regions of  extreme difference in ATPase low 
(AL) fibre population density within the muscle . x 1 60 .  
a .  AL sparse area , including the superficial lateral border of  
the muscle . 
b .  AL dense area from the deep medial part of the muscle . 
88 
a 
c 
b 
F igure 5 :  Frozen transverse sec tions of the semitendinos u s  mus c le o f  
the sheep , s tained f o r  myo s in ATPa se i n  regio n s  o f  extreme 
d i f ference in myos in A TPa s e  low ( AL) fibre popul ation dens i ty 
wi th in the mus cle a t  three deve lopmental s tage s .  Top row , 
80  days ge s tat ion ; midd l e  row , l O O  days ge s t a t ion ; 
bot tom row , adul t . Le ft s ide : AL sparse are a ;  r ight s ide : 
AL dense area . x 350 . 
I 
' 90  
CRAN IAL  
80  DAYS \ Body wt . 0 .4 kg 0 
1 cm I 
100 DAYS 
Body wt. 0 .8 kg 
140 DAYS 
Body wt . 1 . 8 kg 
LAMB  
Body wt . 25  kg 
ADULT 
Rody wt . 50 kg 
0 
1 cm 
0 
1 cm 
1 cm 
1 cm 
M E D IAL  LATERAL  
CAU DAL  0 0.::>:: []··=·= 11 · . · . . . . . · . . . . . 
. . : ;. 
. . . 
3 - 6  7- 9 10 -19  20 -30  
Figure 6 :  Schematic representation of  regional changes involving 
ATPase low fibre density within the semitendinosus muscle of the sheep at 80 , 1 00 ,  140  days gestation , and postnatally at 5 months and adult stages based on data from at least three sheep at each stage . Left  column represents the transverse sectional size of 
the muscle between the middle and dis tal thirds . Right column 
represents the patterns of ATPase low fibre density,  shaded as  
in Figure 2 .  
Percentage of )) x- ATPase Low SJnrse Area •-ATPase Low Dense Area 
ATPase low 
Fi bres 
20 
• 
10 , 
t "" 
,. "' . 
. . 
1 
• t 
" 
r 1( 
. . . 
·.  
""( 
.. . 
J( 
• t • 
I 
• K " 
• 
'• 
��. ,-----��----- ---�1�.--�--����, -----������A 
Body Wei ght ( Kg ) bi r tn 
F i gure 7 :  Rela tionship between log body we ight and ATPase low 
f ibre type de n s i ty population , expre s sed as a pe rcentage , 
in AL dense and AL sparse areas w i thin the semi tendinosus 
muscle of the sheep from 80  days ge station to adulthood . 
9·0 • • 
•• • • • 
• 
Loa 8 .5  
'l'otal 
faaclc'll u 
'I' SA 
( ,_2 ) 
8 .  
llloh period : 
los 7 • 1 . 16 los x • 3 .01 
Aatenatal period 1 
?.0  loa 7 • 1 .25 loa  x • 2 .17 
• 
6 .5�-
-----�------�------��------�------� 
3.0 3.5  .. . o 4 .5 5 . 0  5 .5  
Los •••• faac lcvlar TSA ( pa2 ) � 
F igure 8 :  Change s in tota l fa scicular number wi thin the 
semi tendinosus mu s c l e  of the sheep . 
Lo! total 
fi bre TSA 
( ,..2 ) 
9 .0  
8 .5 
• 
B . o  • 
7 . 5 
• • 
Whole period : 
• 
... . """' • 
• 
l og y z 1 . 37 log x + lt .28 
Awtenatal period 
?.0  log y • 1 .?0 log  x + 3 . 55 
• 
6 .0+---------�--------.--------,,,-------� 
1 .5 2 .0 2 .5 3 .0 
2 birth Log •eaa fibre TSA ( p• ) 
Figure 9 :  Changes in total f ibre number within the 
semi tendinosus mu scle o f  the sheep . 
4 . 5j Los , 
•eaa faecicular : 
'!'SA 
( p.rl ) 
• • 
Whole per iod : 
los y : 1 . 20 lo! z + 1 . 18 
Aatenatal period 
log y % 1 . 12 lo! z + 1 . 40 
3 . 0�------�----------------�------� 
1 . 5 2 .0 
Log •eaa fibre '!'SA ( �2 ) bi rth 
Figure 10 : Change s in fibre number wi thin i ndividual fascicles 
o f  the semitend inosus mu scle o f  the sheep . 
Los TSA or  
AH f ibres 
( ,,..2 ) 
� .(' 
2 . 5 
2 . 0  
? .0 
Loo: TSA 
• 
• 
• 
• 
or AL ri breo 
• 
? . 5  
( I'• 2 
• 
• 
llbole period ' 
• 
• 
log 1 • 1 . 02 log z + 0.01 
Antenatal period : 
log '! = 1 .01  log x + 0.03 
' . 0  
'b'l"rtii 
• 
F i gure 11 : Re l ative change s in transve r se sectional area o f  f ibre 
type s : AL spa rse area . 
-, 
, 
F i gure 12 . 
IAI TIA of 
AI ttllno 
( ,.z ) 
• 
- -------------· 
• 
• 
• 
• • 
ftole ,.rl .. 1 
lot 1 • , .oz 101 a - o.o, 
AatoMtal ,.rl.. 1 
101 , • , .25 101 • - 0.55 
z.o +--------.--------�-------� 
2 .0 '·5 
Re lat ive change s i n  transverse sectional area o f  f ibre 
type s : AL dense are a . 
1 ••
• 
.. : _.  
,,l 
9.0 
• 
• 8, •• 
• 
8,0 • 
'· 
• -· .-. .. I 
1 .. , . ..  " 1 .. • •  '·" • 
• .. ... tal ..... .  1• r • o,,. 1• • • '· • 
• 
' ;:�,------,,To------,�
.,
--�--,�.or-----,-.r
,
------.T.o----� • . �,----�,�.:.-----,:� 
Figure 13 : Changes in shape o f  the semitendino sus mus c l e  o f  ' the sheep . 
Q ,C.  
Lo� 
totsl  
fsec i cular 
TSA 
( pr/ 8 .5 
s .o 
7 .0  
Whole period 1 
log J c 0 .67 log X + 5 .55 
Aatenatal peri od 
log  1 ; 0.76 log x + 5 .29 
• 
6 •
5�------
2
�
5
-------
,
�
o
-------
,
�
5
-------
4�o
r------4�
.
�
5
----
--�5�.o�----�5�.5 ? ,0 • • • • 
Log muecle weight mg  ) biTtii 
Figure 14 : Change s in shape o f  the semitend i nosus musc l e  of the sheep . 
9 .0 
8 .5  
Log total 
fibre TSA 
2 ( ]»" ) 
8 . 0  
7.5  
7 .0  
• 
• 
• 
• 
• 
Whole period 1 
log 1 • 0.7} log x + 5 .20 
Antenetel period : 
log 1 • 0.75 log x + 5.1'  
6 .�----- �----��--��----��----.. 4�----.  4�-----5�0-----5� 1 .5 2 .0 2 . 5  3 .0  3 .5  .o .5 • • 
F i gure 15 : Changes in shape o f  the semitend inosus musc le o f  
the sheep . 
100 
---���---------------------------�
4 . 0  
Log muacle 
weight  ( 11g ) 
' · 5  
2 . 5  
2 .0 
Log body wetght  ( g 
• 
• 
•• 
Whole period : 
log  y = 1 .05 log x + 0 . 1 4  
Antenatal period 
log y = 1 . 1 1  log x + 0.01 
birth 
Figure 16 : Weight changes with growth of  the semitendinosus 
muscle of the sheep . 
Los 
9 .0 -
I 
8 . 5  
8.o-
total 
faacicular 
TSA ? •5  
( p•2 ) 
Whole period : 
l os y • 1 .21  los z - 1 . 88 
Anteaatal period 
los y • 1 .28 log x - 2 .46 ' ·] 6.�--------�------�--- -�--------�----� 
7 .0  7.5  s . o  8 .5 9 .0  9 . 5  
2 
Los ac tual TSA ( p• ) 
F i gure 17 : Change s  in connect ive t i s sue skeleton Perimy s ium . 
9.0 
Los total 
fibre '!'SA 
s .o 
( ,..2 ) 
?.5 
?.0 
• 
• 
• 
Dole perlo4 1 
101 1 • 1 .05 101 K • 0.56 
AateMtal .. l'lM I 
101 1 • 0o95 l01 K + 002) 
6 .�--��----�----�----�----��--� 
6.5 ?.0 ?.5 8.0 8.5 
Figure 18 : Change s i n  connective tis sue ske l e ton Endomys i um .  
Loc total 
U'bro �SA 
( ,_
2 
) 
''l 
I 
e.
� 
e .� 
7.� 
• 
7 .o. 
Wllole period 1 
loc 1 • 1 .29 loc x - 2.71 
, . Aatoutal period 1 
loa 1 • 1 .26 loc x - 2.'6 
6 .�--------r-----��--------.--------r-------� 1 .0 1.5  e .o P .5 9 .0  
tti=Til 
Figure 19 : Change s  in connec tive tis sue skeleton 
and Endomysium .  
Per imy s ium 
( a )  operated l imb 
not in use 
(b) operated l imb 
supporting 
we ight 
( c )  sheep walking 
quietly 
(d) sheep wa lking 
hind legs 
( e )  sheep k icking 
200  )lVL 
0 . 2  s 
AL sparse area 
AL dense area 
AL sparse area 
AL dense area 
AL sparse area 
AL dense area 
AL sparse area 
AL dense area 
AL sparse area 
AL dense area 
F igure 20 : S imul taneous e l ec tromyographic recordings in extreme reg ions 
of f ibre type population density i n  the semi tendinosus 
mus c l e  o f  adul t sheep dur ing varied l imb activi t ie s . 
a .  
b .  
c .  
d .  
e .  
Sheep 
Sheep 
Sheep 
Sheep 
Sheep 
standi ng quietl y .  Operated l imb . not favoured . 
made to use op:era ted l imb . Only the AL derise area i s · act ive . .  
walking quietly . Ac tivity now extends to the AL sparse area . 
made to walk on hind legs . Both areas show enhanced activity . 
k icking . Both areas are active . 
a 
b 
c 
d 
c:mdl-lateral 
crano-medial 
A - A L SJX1rse k'efJ. 
..6. - A L Dense Area 
F igure 21 : Blood suppl y  to the portion di s tal to the tendinous 
septum of the semitendinosus m� scle of the sheep . 
a .  Angiogram showing the venous drainage . 
b .  Cor ro s ion cast showing the arte r ia l  supply . 
c .  Corro s ion cast sculptured to show the ma in ve ssel s .  
d .  Diagramatic repre sentation o f  AL sparse and AL dense areas of the 
mus c l e  in rela tion to the arterial suppl y .  The portion o f  the 
mus c l e  proximal to the tendinou s septum i s  i nd i cated by shading . 
a 
b 
Figure 2 2 : ¥rozen serial  sec tions o f  the semi tendinosus muscle  o f  
the adult  sheep  s ta ined f or  alkali-s tabile  myosin ATPase 
and succ ina te  dehydrogenase according to the me thod of 
Nachlas M . M .  et  al . 1 9 5 7  (Appendix 2 ) .  x 1 60 .  
About  30% of  the f ibres  wi thin the fascicle  have a low 
myosin ATPase  react ion (a ) whi le about 60% o f  them show 
high SDHase  ac t ivity  (b ) . 
106 
Figure 23 : A schematic repre sentation o f  a lamb with al tered hind 
l imb func tion . Each o f  the seven treated l ambs had 
i ts r ight hind l imb bound beneath i t s  s ternum with ful l y  
fl exed h i p  and ex tended stifle and hock j o i n ts , from 
the seco nd day a fter birth to about 3 months of age . 
F i gure 24 : The semi tendinosus musc l e s  from the suppor ting ( le f t )  
and bound ( righ t )  l imb sides o f  a l amb with a l tered 
hind l imb func tion . The muscle o f  the bound hind l imb 
i s  larger than the one o f  the suppor ting l imb . 
1 . 0 
0 .9 
o . 8  
Log TSA o f  
IIIUI!!C l e  
( u2)0 . ?  
o . 6  
o •
• 3 
3 
Left Number of 
supporting ) Sheep 
side  
Log  •uscle wei gh t  ( g ) 
R ight  
( bouad 
side 
2 . 0  
F i gure 25 : Shape change s  in the s emi tendi no su s  mus c l e  o f  the sheep 
due to chronic s tre tching . 
9.0  
8 . 9  
Log total 
faseieular 
TSA 
( )1112 ) 8 .  
8 . ?  
8 ,6 
8 .5 
Left 
supporting 
side 
' 
Right 
bound ) 
side 
8 , 4L-------,-------�-------r------�----�-r------� 
8. 4  8 .5 8 .6 8 . ?  8 .8 8 .9 9.0  
Log  ac tual TSA  �m2  l 
F i gure 26 : Changes in connective t i s sue content o f  the 
semi tendinosus musc l e  o f  the sheep due to chronic 
stretching : Per imysium . 
l 
· ··r , 
I 
B .&  
8 . '1 
I 
Left 
eupportias 
side 
N1111ber ot 
Sheep 
Log total 1 fi bre TSA 
( p•2 i 
8 .4 
8 .J 
B .  
B ,,_ __ _ _L_ 
B .4 B . 5  8 . 6  B . ?  
Log ac tual TSA ( p•2 ) 
Risht 
( bouad 
side 
B . B  B . 9  9.0 
F igure 2 7 : Changes in connec tive tissue content o f  the 
semi tendino sus mus cle o f  the sheep due to chroni c  
stretching : Per imysium a n d  Endomysium .  
Log total 
fibre TSA 
2 ( ,... ) 
8 , 8 .  
8 . 7 ... 
8 .  
Left 
supporting 
side 
Number of 
Sheep 
8 ,'lf-----.---
8 . � 8 , r;  8 . 6  
Lo� totsl  fsecicular TSA ( 
Right  
{ bound 
dde 
8 , 8  8 . 9  9 . 0  
F igure 28 :  Changes i n  connective tis sue content o f  the 
semitendino sus muscle o f  the sheep due to chronic 
s tretching : Endomysium . 
Los total 
fa acicular 
TSA ( ,..2 ) 
8 .9  
I s .sr 
8 .J 
8 .  
Left 
aupporUos 
aide 
' 
Nu•'bar of 
Sheep 
Risht 
( bouod 
eide 
8 .�----�------�------�----�------� 
� . 5  � . 6  � .? 4 .8 
Los •••o foocicular TSA ( pm? ) 
Figure 2 9 :  Changes in fascicle number of the semi tendinosus 
mu scle o f  the sheep due to chronic stre tching . 
8 .9 
8 . 8  
8 . 7  
Left Nu•her ot 
supporting Sheep 
aide 
Log total 
f'i bre TSA 
( ,,l ) 
8 , 6  
8 ,4 
8 . :51------- --4- __ , 
2 . 7  2 ,8  2 . 9  
Log mean fibre TSA 
Right 
( bound 
side 
� . 1  J , 2  
F igure 30 : Changes in f ibre number of the semitendinosus muscle 
o f  the sheep du� to chronic s tretching . 
0 � • 
5 .0 Left Number of Right 
supporting Sheep ( bound 
side side 
4 . 9  
.... 
1 
4 . 8  
Mean faeeieular 
2 TSA ( p111 ) 
4 .? 
3 
4 .  
4 .5•r-------�------��------�------�--------�-------r 
2 . 7  2 . 8  2 . 9  3 .0 3 . 1  3 . 2  3 . 3  
Mean fi bre TSA ( pm2 
Figure 3 1 : Changes in fibre number within individual fascicles 
of the semitendinosus muscle of the sheep due to 
chronic stretching . 
l l5 
'·'  
,.2  
Lo1 AI TSA 
1Aft W.Mr of 
( -pportiBC ) 9 .. p 
• u. 
a1su 
( ... .. ) 
.u • 
( ,.• ) ' · '  
F i gure 3 2 : 
,.o 
2.9 
2.8 
/ / 
� ::+-----2�.�?-----.�.�8�---.�.70-----,�.70-----,�.7,----�,� . •.----,�.�,-----.,�.,----.� 
Lo� AL TSA ( JA•2 ) 
Change s in f ibre s i ze proportions in the AL sparse area 
o f  the semitendinosus mu scle of the sheep due to chroni c  
s tre tch ing . 
' ·' 
,.o 
Loa .u 
, 
Loft -loo• of 
( ... , ...  u., ) .... , 
•1•• 
TSA ( ,_2 ) 
2 .e 
er
_______... 
� 
2.7 
2.6�------------�------�----�------�----��------------�----�------� 2.6 2.7 2 .8 2.9 ,.o ,. ,  '·' '·'  '·' '·' '·' 
Figure 3 3 :  
Loa .u. !SA ( ,_2 l 
Changes in fibre s i z e  proportions in the AL dense area 
of the semi tendino sus mu scle o f  the sheep due to chronic 
s tretching . 
Figure 34 : Frozen transverse sec tions of the semitendinosus muscle 
stained for alkali-stabile myosin ATPase . x 1 60 . 
Top row, control ; middle row,  supporting limb ; and 
bottom row, bound limb . Lef t  side ,  AL sparse area ; 
right side , AL dense area . 
l l� 
Figure 35 : Low power electron micrographs o f  fibres from the 
semi tendinosus muscle of the sheep . x 6400 . 
Top row , control ; mi ddle row , supporting l imb ; and 
bottom row , bound l imb . Le ft s ide , longitud inal sec tion ; 
r ight s ide , transve r se section . 
F igure 36 : High power e l ec tron mic rographs o f  f ibre s  from the 
semitend ino su s  muscl e  of the sheep . x 17500 
Top row , supporting side ; bottom row , bound s ide . 
Left s ide , longitud inal section ; r ight s ide , 
transverse sec tion . 
Appendix 1 .  
Alkali-stabile Myosin ATPase Technique 
Substrate 
1 . 0 M Tris- (hydroxymethyl ) - aminomethane 
(M . W .  1 2 1 . 1 4 )  
0 . 1 8 M Calcium Chloride CaCl2 . 6H20 
Adenosine triphosphate (disodium dihydrogen salt )  
Distilled water up to  
8 ml 
4 ml 
60 mg 
30 ml 
1 2 1  
Adj ust pH t o  9 . 5 with 0 . 1 M HCl . Add dist illed water t o  make u p  a f inal 
volume of 40 ml . Incubate for 20 minutes . 
Method 
( 1 )  Cut fresh frozen sections at 10-15  )Jm thickness  
( 2 )  Mount on  slides (and leave in refrigerator for  at least 30  min . 
This prevents the sect ion peeling off  from the slide later ) . 
( 3 )  Fix in 4%  Formaldehyde buffered with cacodylate (see below) 
pH 6 . 8  for 2 minutes (exactly) . 
(4 ) Wash in two changes of  dist illed water . 
( S )  Incubate for 20  minutes in the above substrate 
( 6 )  Wash in dist illed water 
( 7 )  Treat with 2 %  Cobalt Chloride solution for 2-3 minutes 
(8 )  Develop in dilute ( 1 %)  yellow Ammonium Sulphide solut ion for 
30  seconds . 
(9 )  Wash , mount in aqueous mountant . 
(Note :  Concentrations of calcium chloride seems critical in getting 
good react ion) . 
Cacodylate buffered Formaldehyde 
2 . 14 g Sodium cacodylate . 3H20 in 50 ml distilled water 
50  ml of above solut ion + 6 . 3  ml 0 . 2M HCl 
+ 20 ml 40% W/V Formaldehyde 
Make up to 200 ml with distilled water.  Final pH 6 . 8 .  
Appendix 2 
Succinate Dehydro�enase  Histochemical Technique 
Special reagents required 
( 1 )  Substrate 
0 . 1  M Phosphate buffer pH 7 . 6  (see below) 
0 . 2  M Sodium succinate (M .W .  2 70 . 1 5 )  
Nitro blue tetrazolium ( 1mg/ml ) 
(needs to be made fresh each t ime ) 
( 2 )  Phosphate buffe.r (pH 7 , 6 )  
7 . 5  m1 
7 . 5  ml 
15 ml 
Potassium dihydrogen orthophosphate KH2P04 ( 1 . 36g / 1 00 ml ) 
( 1 .  78g/ 100 ml) 
122 . 
1 0  ml 
90  ml Disodium hydrogen orthophosphate Na2HP04 
Distilled water to 500 ml 
( 3 )  1 0%  Formal ( Stock solution) 
Method 
1 .  Fresh frozen sect ions cut at 10-15  �m are mounted on coverslips . 
2 .  Incubate at 3 7°C in the substrate ( 1 )  for 20 minutes . 
3 .  Wash in two changes of  distilled water . 
4 .  Dry the sections to eliminate gas bubbles .  
5 .  Fix in 10%  formalin for 10 minutes . 
6 .  Wash and mount in glycerine j elly . 
Appendix 3 
Electron microscopy technique 
a) Fixat ives and embedding media 
1 )  Phosphate buffer (0 . 1  M, pH 7 . 2 ) 
36 ml of  0 . 2  M Na2HP04 1 2H20 ( 7 1 . 64 g dissolved in distilled water 
to make 1 £ ) . 
1 4  ml of  0 . 2  M NaH2Po4 . 2H20 (3 1 . 2 1  g dissolved in distilled water 
to make 1 £ ) . 
Made up to 100 ml with distilled water . 
2 )  Phosphate buffered sucrose (pH 7 . 2- 7 . 4 ) 
Dissolve 35 . 0  g of sucrose in 300 ml of  0 . 1 M phosphate buffer . 
3 )  Modified Karnovsky ' s fixative 
2% Formaldehyde 
3% Glutaraldehyde 
in 0 . 1 M phosphate buffer pH 7 . 2  
To make 100 ml : 
i )  Heat 2 g of  paraformaldehyde in 80 ml  water to  60-70°C 
ii)  Slowly add 1 N NaOH dropwise until milky solution clears . 
Leave for 5 minutes at room temperature . 
iii)  Add buffer salts 2 . 5 1 g Na2HP04 . 12H20 
0 . 4 1  g KH2Po4 
iv) Add 6 ml of  50%  Glutaraldehyde solut ion 
v)  Make up  to  100  ml . Store in  refrigerator . 
4 )  Osmium tetroxide f ixat ive 
1 %  Osmium tetroxide ( 1  g of Oso4 in 0 . 1 M phosphate buffer to make 100 ml) . 
(Work in a fume cupboard ; score the Os04 ampoule about its circumference 
with a file ; place the vial in a strong brown glass-stoppered bottle 
and shake it to break the vial ; introduce correct volume of 0 . 1 M 
phosphate buffer ) . 
5 )  Durcupan ACM epoxy-resin 
To make 20  ml : 
1 0  ml Component A ( 1 0 . 94 g )  - Epoxy resin 
1 0  ml Component B ( 9 . 58 g) - Hardener (Anhydride of a dicarboxyl ic acid 
aliphatic , side chain) . 
124  
0 . 4  ml Component C - Accelerator (Phenol derivative with amino group) 
0 . 2  ml Component D - Plasticizer (Di-n-butyl phthalate)  
(Use 30 ml  disposable glass vial ; measure components A & B by  weight 
using a top-loading balance ; mix components  by invers ion of the vial ; 
warm with hot air blower or at 60°C in an over and mix again ; add 
components C & D using disposable plastic 1 ml syringes ) . 
b)  Staining solut ions 
1 )  Uranyl acetate stain 
Saturated Uranyl acetate in 50% Ethanol 
(Add uranyl acetate to 50% ethanol until it will no longer dissolve ; 
centrifuge and store supernatant in a brown glass bottle ) . 
2 )  Lead c itrate stain 
0 . 025 g lead citrate 
10  ml dist illed water 
0 . 1  ml 10 N NaOH 
Shake vigorously until dissolved . 
JvfYOS IN  
M .N .  S iv�! chelvan 
D�Dartment of Physiology m;..-l A natom 1-·. }.1 assey Uni,·ersiTy, 
Palmas!un lhr£h, New Zrala!td 
The myosir.. aden o�; ine t:-ir.hospho!8�c (myo<;i n ATPasc) h ist ocherr.:ca! 
tcch i t ; •_J :le \vas c::np ) c.yed in a qu.:n : i t �: t 1vc study on a n tcnat & l  and  
pos t ; �;; � a !  d':'!vclopmcnt of  t he �.tn , i t c n d i ; !oS t<s mvsclc 0f  the  she•:p .  Ti 1 i >  
enabkd the  pal ttrn of developmc: 1:t  of t !� c  fibres ?. nd ! he ir  sequenc;: of  
diffc�ent iat ion into funct i •m<: : Jy d i , t i nc t  f1brr. �yp:s to  be determined ,  
i �1 ferring the  morphot,Ci1C5 is of  �rcrific j�,,en 7ymc:s of myos in .  
T w o  d is t inct gencratio:1s of fibr�s cl e'.•e !Oil early in foe tal development .  
The pri t &} a ry genera t i o n  of cel l s  is rec0gn isabie al fo�ty d ays gestat ion  
while the second�!)' grnei';. t ion  o[ rc lh bcg i ;1 t o  upp·:ar a t  s ix ty  d ays.  U p  
to seventy days of gestat ion,  both  gen,:r; , t ions  o f  cc �is $�;: ! n  Jark h' i t h  
myos in  /\TPasc sta in .  A t  sc.vcnty days  f2Wi t ion ,  t h e  pr i r�l<� r:v· ;enera t i n 1 1  
of  cc:lls <!re larger i n  s i ze (mc[!n t r <H;$vcr sc s-::ct io:1:'. l ;u..;a (TSA) o f  I :;o 
1.un2)with pale CC!1t r;; l cores conta in ing nuclei and o(·t:J pyi ng  a rr.ore 
ccntn:l po:; i i ion  i n  th-:  r,rwly forming  p:: rimysial cpc: Jo:.cd fa$ciclrs The 
seconda ry generation of c · l ls shows a ho:�10gencous d :� rk  appearance 
with the sta i n ,  and h"ve a mean TSA of 1 (>0 ; .. on2 • The fasc:iclcs at th i s  
stage have a mea n TS/\ of 3 ,500 prn2• 
From eighty days of  r,cs� a t ion  omv;s rds, two d istinct  myos i n  isoc nzym-:: 
fi brc types are observcd hi�l ocl tr  mically i n  the musck. The prim a ry 
generat ion  of cells presumably  has lo�l  it� dark sta in ing  c h a rac:te r i� : ic 
to bccotnc pale stain ing ATI-�ase low  (A L) fibre.:; "· !-:et  ea� t h:; seconda ry 
generat ion  of cel ls  c o n t i n u e s  to retai n its d;nk s ta ir. inc, ab i l i ty  and form 
the  ATPase h igh (A H )  fibre popuUion .  , Jhe d is t ribu t ion  0f  the  ftb�e 
types is heterogeneous from this pct io�i oi �1du l thood .  Regions of hig i 1cq 
and l owest AL fibre de nsity arc rccogni � ;� blc i n  ail t h_-:sc s tages a nd a : e  
referred t o  it s  the AL  dense and the  A L  � parse areas o f  the muscle. The  
percent age of AL fibres in  t h e  AL  d�nsc r; re<·s increases from 1 0  pcrcsnt  :lt 
eighty days to 30 percent in thc  ad u l t ,  wbik: no appreciable change occt:rs 
in  _the p-:rcentage of AL fi bres (4 percent )  in  t he AL spar�c a rea .  The 
mean fihrc TSAs Clf the AL and A H fi o�es in both AL dense ;:;nd A L  
spa rse <t t eas d o  not  di ffer �: ignifican t !y  i n  t i le adult ,  with a value o f  3 ,500 
pm2• The mean fa�cicubi· TSA ult imotr:!y a tta ins a value  of 1 75 ,000 pm�.  
These f: nd ings ind icate that  the spccific i ty  of  the myosin isoenzymt.:s is  
not r c:\·ca led Juring early foet a l deve lopm.:.' n t .  Uy e ighty days foe t al sta£e 
the f ibres beg in  to  show spccific i ty. shown by others to be due  to loss of an  
i n it ia l  d imorphic i socnzyme activity.  The  presence of an  ATPase dense 
and an  ATPa�e sparse area i n  the muscle  i s  re':.1ted to  the p0-; tura l and the 
p ropuhive funct ional  rcqu i re !l l en ts  of the postnatal  semi t end inosus 
muscle,  as shO\vn in  clectromyor.raphic s tud ies. Th� a n tcil a t a l  a nd 
post natal i ncrease in AL.  fibre pe rcc n t :1 gc i n  part of the  musc le with  a 
predomi nant ly post ur:1l  funct ion rctlects both antenati\ I an t icipation 
.
of, 
and postnatal  adaptat ion fo r, 1 1ostural  dcm:wd on t he muscle dunng 
�rowth.  
125  
International Conference on Fibrous Proteins , Palmerston North , 
1 979 • Book of  abstrac ts  , 47-48 • 
At-ii [W;TAL ANT I C I P,\T ! ON Or �lUSCLE !"'OSTI\ATAL FUNCT I ON 
t.: . N .  S I V!ICHF.LVAN 11nd A . S . DtW I ES 
M-:lssey Un i vers i ty 
The sem i tend i nc:stJs  of the sheep was used ns an exper i menta l mode l to 
test 1 h'c; antenzto l genet i c  un 1" i c i p0t i on of musc l n.  to i t s pos1·nata l 
funct ionil l  dern;;;nds . The a l k;t l i ne rr.yos i n  AT?ase h i s�·ochcr.. : Ca l  
techn i que l·,a s used to i dent i f y and q•Jon t i � y  p-:Jstura l ,  AT!\'Jse l ow (ALl· , 
and proru l s i "c ,  _ATPase h i nh ( r.H l , f i bres . Areas of  h i ghest and 
l owest  AL f i bre dens i ty were recogn i sab l e  w i th i n  the musc i o  i n  a l l 
stnges o f  deve l opment  f rom 80 days gestat i on .  The num�er of 1\L f i bres,  
as  a per·ccntage of  to1 a l  f i bres i n  t he AL dense a ;-ea , i ncrea sed f rom 
1 0% at  80 days gcs1 at i on up to 30% i n  the adu l t , •,;hcre;as i r , the t,L 
spa rse area the AL dens i ty rema i ned a t  aoout 4% throughou1 1 h i s  
g r·owth per· i od .  
E l ect romyag:aph i c  reC'.)rci i r.�s m<�_de s i rw l tanl·c�.;s l y  i n  the A L  dense area 
and  the Al S;'a rs'.l a r·e'l of th<;) sem i tend i nosus musc l e  i n  adu l t  s�1eep 
proved that 1 he Al dense area i s  ac t i ve postura l ! y  and AL sparse 
area i s  ect i va i n  p ropu l s i on .  
fhe p:-esence o f  postura l and propu l s i 't0 pads w i th i n  the musc l e  ond 
the i nc re?.se i n  percen1 age of AL f i bres a l ong w i i h i ncrea s i ng body 
we i ght  du r i ng post na 1 a l  growth suqgcsts  a f unct i ona l a<iap1·atory 
ch3ng•3 .  I n  1 hP. ant ena �·a ! per i od , however ,  t he fetus i s  a n  amn i ot i c  
aqu�naut su�ported b y  forces o f  buoyancy , and  does not exper i ence 
adaptatory i nf I uc nce . Thus 1 he prus�..r1cc of postu ra I and  pr·opu I s  i ve 
reg ions cven du r i ng a .,te;:atz l g row t h  anc' an accompany i n!J i ncrea se; I n  
AL f i brc dens i ty w i th i n  the po�tu ra I reg ion i nd i cates a n  ar 1 tenaTa I 
gene t i c  a nt i c i pat ion of musc l e  to i ts postnata l f unct i ona l demands.  
Department of Phy s i o l ogy and  A.na1or.-,y 
M . N .  S l vacho l van 
Proceedings of  the Anatomical Society of Australia and New 
Zeala•d , 1 9?9 • Journal of Anatomy , 1ZQ , 203-204 • 
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