211
PRENATAL DEVELOPMENT OF MUSCLE F I B E R TYPES
I N DOMESTIC ANIMALS*
c . Romm
ASHMORE
University of California, Davis
P. B. ADDIS
University of Minnesota, S t . Paul
INTRODUCTION
The area of study i n muscle biology concerned with l i g h t (anaerobic)
and dark (aerobic) muscle f i b e r s has expanded considerably in recent y e a r s .
For example, a t t h e f i r s t Wisconsin symposium, "Physiology and Biochemistry
of Muscle as a Food," held in 1965, no paper was presented which w a s
primarily concerned w i t h l i g h t and dark f i b e r s . A t the second symposium,
four years l a t e r , one e n t i r e session, including f i v e manuscripts, was
devoted completely t o t h e subject. Yet, i n s p i t e of t h i s increased
a t t e n t i o n t o , and increased information concerning muscle fiber types,
several questions of extreme importance t o animal s c i e n t i s t s remain
unanswered. They are: (1)Hat many d i f f e r e n t types of muscle fibers
e x i s t , and can a system of c l a s s i f i c a t i o n be applied which w i l l accurately
identify these ty-pes across species l i n e s as w e l l as under various
physiological and developmental s t a t e s ? ( 2 ) What i s t h e nature of t h e
embryonic development of fibers, and a t what time and place in myogenesis
a r e the a d u l t c h a r a c t e r i s t i c s of muscle f i b e r s determined? (3) What i s
the significance of t h i s information t o contemporary problems in meat
science?
This report w i l l (1)b r i e f l y review some of the e a r l y studies on light
and dark f i b e r s , (2) discuss the developnent of an improved system of
fiber-type c l a s s i f i c a t i o n , ( 3 ) review recent research on pre- and postnatal
development of muscle fibers, and ( 4 ) discuss and speculate upon the
implications of these findings t o m e a t science. W e w i l l report t h a t (1)
there a r e two fundamental fiber types in skeletal muscle, 01 and B, as
demonstrated histochemically by s t a i n i n g f o r myofibrillar adenosine
triphosphatase (ATPase) a c t i v i t y ; ( 2 ) l3 fibers are red f i b e r s , hence
@-red ( B R ) ; (3) CY fibers are red (aR) a t birth o r hatching but have the
capacity t o t r a n s f o m t o white ( W ) f i b e r s giving r i s e t o a t h i r d f i b e r
type; ( 4 ) during prenatal o r in ovo maturation, I3 fiber development
precedesthat of cr: fibers, the l a t t e r forming by p r o l i f e r a t i o n and f'usion
--
*
Supported in p a r t by grants from the Muscular Dystrophy Association of
America and by Public Health Service Grant ~~06138,
HSAA. S c i e n t i f i c
Journal Series Paper No. 8093, M i n n . A g r . Exp. Sta., S t . Paul, MLnn. 55101.
Presented a t the 25th Annual Reciprocal Meat Conference of the American
Meat Science Association, 19p.
of myoblasts along the surface of the primary myotube, t h e g f i b e r ; ( 5 )
it i s hypothesized that genetic selection f o r leanness and muscuhrity
i s accomplished by the deposition of additional a f i b e r s i n the bundle
and the transformation of greater numbers of 023 f i b e r s t o aW f i b e r s i n
affected muscles.
PROFERTIES OF FZD AND WHITE FIBERS
There i s l i t t l e doubt t h a t muscle color varies closely w i t h function.
Notions t o t h e contrary a r e dispelled by reading the account of the complex
architecture and fiber d i s t r i b u t i o n of pigeon pectoralis muscle summarized
by George and Berger (1966). Dark muscles are, most often, tonic, contract
slowly and usually perform postural functions. L i g h t muscles contract
quickly, w i t h more strength than dark muscles, but f a t i g u e more e a s i l y .
Metabolic c h a r a c t e r i s t i c s explain differences in r a t e s of f a t i g u e i n t h e
two f i b e r types. Red f i b e r s contain myoglobin, generate ATP by oxidation
of f a t and carbohydrates, and a r e generally smaller i n diameter w h i c h
f a c i l i t a t e s rapid exahange of substrates and t o x i c waste products. Due
t o such e f f i c i e n t metabolic mechanisms, they exhibit resistance t o f a t i g u e .
White f i b e r s depend p r i m w i l y upon anaerobic glycolysis t o produce ATP,
contain l i t t l e or no myoglobin and, therefore, t i r e quickly during strenuous
a c t i v i t y as endogenous glycogen s t o r e s a r e depleted (Close, 1972).
While t h e above patterns and differences a r e logical, some d i f f i c u l t y
a r i s e s when attempts are made t o correlate speed of contraction (twitch)
with color. The e a r l y work of Paukul (1904) demonstrated t h a t slow-twitch
muscles were invariably red; however, not a l l red muscles were slow-twitch.
I t i s now believed t h a t t h e r a t e of hydrolysis of ATP limits the r a t e of
contraction. Thus, separate actomyosins might be expected t o e x i s t in fast
and slow f i b e r s . Guth and Samaha (1969) obtained cytochemical evidence f o r
the existence of d i f f e r e n t actomyosins between fast and slow f i b e r s . They
exposed t i s s u e sections t o acid and alkali p r i o r t o t h e ATPase reaction
medium, and were c l e a r l y able t o demonstrate differences between f i b e r s ;
slow fibers l o s t a c t i v i t y during alkali preincubation, but a c t i v i t y
persisted through acid treatment. The pattern was the reverse f o r f a s t
f i b e r s . They (Guth and Samaha, 1969) concluded that a t least two d i s t i n c t
actomyosins e x i s t , one f o r each type of fiber. Evidence obtained
biochemically on purified myosin has shown that myosins from slow and f a s t
muscle d i f f e r with respect t o s p e c i f i c ATPase a c t i v i t y , acid and alkali.
s t a b i l i t y , s u s c e p t i b i l i t y t o t r y p t i c digestion, and i n t h e number and
m l e c u l a r weights of l i g h t chain components (Saker e t al., 1971; S r e t e r
e t a l . , 1966; Barany e t al., 1965; Samaha e t a l . , 197Oa; Gergely e t a l . ,
196'5T. These findings a r e consistent with the hypothesis t h a t muscle
f i b e r s can d i f f e r w i t h regard t o speed of contraction as well as i n t h e i r
capacity t o resist fatigue, but importantly, these physiological characteri s t i c s a r e not necessarily c o r r e h t e d w i t h each other and are therefore
subject t o separate control mechanisms.
-
--
--
FTBEXi CLASSIFICATION
Several systems f o r t h e c l a s s i f i c a t i o n of muscle f i b e r s are currently
i n use. The finding of an "intermediate" f i b e r (Ogata, 1958), together
with the Friability of myologists t o segregate a l l f i b e r s i n t o a simple
d u a l system, fast-white and slow-red, has resulted in a p r o l i f e r a t i o n of
conventions ( t a b l e 1). S t e i n and Padykula (1962) i d e n t i f i e d t h r e e fiber
types "A", "B", and "C" based on the SDH stain. More recently, Samaha
e t a l . (1970) demonstrated three f i b e r types based on myosin ATPase h i s t o chemical assay. The three types were designated a, B, and (43. However,
t h e two systems were not compatible in a comparison of rat and c a t f i b e r
patterns (Yellin and Guth, 1970). The "B" fiber o r "intermediate" fiber
of both rat and cat contain allrali-labile ATPase (hence are B-fibers).
However, the "A" o r white f i b e r of the c a t contains acid-labile ATPase
(hence i s Cr) whereas in the "A" fiber of the ATPase i s intermediate i n pH
s t a b i l i t y (hence is 4 3 ) .
--
A study of postnatal development of f i b e r types in normal and dystrophic
chicks lead t o the d e v e l o p n t of a c l a s s i f i c a t i o n system which appeared
t o successfully combine t h e two systems (speed of contraction and metabolic
profi1e)into a single system f o r chick muscle. Ashmore and Doerr (1971)
studied the development of the pectoralis, s a r t o r i u s and adductor muscles
and found t h a t the two basic f i b e r types ( a and B), based on lqyosin ATPase
a c t i v i t y , axe i n i t i a l l y red (hence CYR and PR). "he CllR fiber has the
capacity, by modulating metabolic enzymes, t o transform t o a white (0%)
f i b e r . They f u r t h e r noted t h a t in the s a r t o r i u s , a muscle of mixed f i b e r
types, the small BR f i b e r s are located in t h e i n t e r i o r of the fiber bundles,
intermediate diameter CfR fibers a r e commonly adjacent t o the BR f i b e r s ,
and f i b e r s in t h e extreme periphery of bundles are nearly always c&J.
Table 2 summarizes the properties of PR, 03 and CN f i b e r s .
The p o t e n t i a l value of such a system of c l a s s i f i c a t i o n is greatly
enhanced i f it can be applied t o many species. Such a study w a s conducted
by Asbmore and Doerr (1971). Chick, muse, bovine and porcine red anh
white muscles were studied. I n muse muscle, SDR a c t i v i t y of BR f i b e r s
as assayed cytochemically, was l e s s than that of cX? f i b e r s , whereas in
the other species SDH a c t i v i t y tended t o be highest in BR fibers. However,
when the r a t i o SDH/phosphorylsse was calculated (a l o g i c a l index of aerobiosis)
BR f i b e r s of all species exhibited the highest r a t i o . It was concluded
that f o r the species studied, which include three species important t o meat
production, the BR, Qli, oiw system was compatible. It should be emphasized
that t h e Cy fiber population most often exhibits a continuum of oxidative
enzyme a c t i v i t y , r e f l e c t i n g the likelihood that a f i b e r s , w i t h respect t o
energy metabolism, are responsive t o physiological demand. The i d e n t i f i c a t i o n
of an CI f i b e r as R or W therefore may be arbitrary. This i s not generally
a major problem, however, since t h e u t i l i t y of muscle fiber c l a s s i f i c a t i o n
most o f t e n l i e s in r e l a t i v e canparisons rather than in absolute ones.
--
More recently, Burke e t a l . (1971)have designated f i b e r s as S (slowly
contracting), FR (fast contracting, and fatigue r e s i s t a n t ) , and FF (fast
contracting, fast fatigue). Their system is based on physiological features
214
TABU 1. COMPARISON OF PRevIOUS FIBER TYPE CIASSIFICATION SYSTEMS
TO THE ONE SUGGESTED BY ASHMORE AND DOERR, 1971
miteb
WhiteC
Whited
IIe
I1f
A@;
a
b
c
d
e
f
g
Red
Intermediate
Intermediate
I
Intermediate
Red
Red
I
I
B
I1
C
Ashmore and Doerr, 1971.
Padykula and Gauthier, 1967, a s applied t o mice, rats, and guinea p i g s .
B i d , as applied t o domestic animals and man.
George and Berger, 1966, as applied t o avian muscle.
Engle, 1962, as determined by the SDH histochemical assay.
Ibid, a s determined by t h e ATPase histochemical assay.
S t e i n and padykula, 1962.
COMPARISON OF CHARACTERISTICS AMONG BR, 03 and olw FIBERS
TABLE 2.
~
~~
Character i st i c
Diameter
Color
Blood supply
Mpglob Fn
Glycogen
Phosphorylase
Succinate dehydro enase (SDH)b
SDH / phos phory las e
Myofibrillar ATPase
pH 4.00
pH 10.40
Contraction speed
8!
narrow
red
high
hi@
law
lUW
hi@
narrow-intermediat e
red
intermediate
high
high
intermediate
intermediate
intemdiate
+
-
slaw
fast
-
+
broad
white
low
low
hi*
hi€$
low
low
-
+
fast
a C l a s s i f i c a t i o n system suggested by A s h o r e and Doerr, 1971.
b In t h e mouse and c e r t a i n other species of laboratory animals, SDH
a c t i v i t y as demonstrated cytochemically, of c91 exceeds that of 8R
f i b e r . However, i f the r a t i o SDH/Phosphorylase (an index of a e r o b i o s i s )
is calculated and the same comparisons m d e , PR i s c l a s s i f i e d as more
aerobic than t h e 023.
215
of muscle f i b e r s , but the cytochemical features, described by them, a r e
the sa= as those described by us f o r BR, CS, and aiW fibers, respectively.
--
Barnard e t a l . (1971)have used the terms slow-twitch intermediate,
fast-twitch red, and fast-twitch white t o i d e n t i f y f i b e r s on the basis o f
a c o n t r a c t i l e c h a r a c t e r i s t i c and on a metabolic c h a r a c t e r i s t i c . However,
their system of c l a s s i f i c a t i o n , developed from studies on mice and guinea
pigs, cannot be applied t o most other species, since i n t h e l a t t e r ,
"intermediate" f i b e r s (based on SDH a c t i v i t y ) are fast fibers, not slow
fibers (Ashmore and Doerr, 1971a, b )
E'urther, i d e n t i f i c a t i o n of a
c o n t r a c t i l e c h a r a c t e r i s t i c on the b a s i s of a histochemical assay
(myofibrillar ATPase a c t i v i t y ) i s hazardous since it has been shown that
'tslowltfibers i n an e a r l y developmental s t a t e demonstrate an intense
histochemical reaction f o r myofibrillar ATPase a c t i v i t y (Ashmore e t a l . ,
1972) and could therefore be erroneously c l a s s i f i e d as "fast" f i b e r s .
Guth and Samba (1972)have demnstrated a lack of correlation between
histochemical and biochemical assays for myofibrillar ATPase a c t i v i t y in
neonatal rat muscle.
.
DFVELOFMEZlT OF MUSCW FIBER TYPES
Postnatal maturation of muscle f i b e r s is concerned w i t h the development
of preprogrammed p o t e n t i a l s f o r growth, contraction, and energy generation.
Each of these i s a multifaceted t r a i t and opt-1
expression depends upon
correct envlronmntal interactions with genetically determined p o t e n t i a l .
Muscle contraction consumes considerable quantities of ATP and muscle
fibers, therefore, require r a t h e r e f f i c i e n t mechanisms f o r generation of
high energy intermediates. In terms of metabolic pathways, some f i b e r s
demnstrate a d i s t i n c t dependence upon e i t h e r the aerobic pathways (Kreb's
cycle enzymes and electron transport enzymes of mitochondria) or the
anaerobic pathway (enzymes associated with t h e breakdown of glycogen t o
glucose-6-phosphate and then t o l a c t i c a c i d ) . Other fibers exhibit a
s u b s t a n t i a l capacity t o generate ATP by both pathways, using the aerobic
pathway when s u f f i c i e n t oxygen i s available and the anaerobic pathway when
the requirement f o r ATP exceeds t h e capacity of oxygen and aerobic metabolism
t o meet t h e demand.
The development of aerobic metabolic capacity precedes the development
of anaerobic capacity s o that i n i t i a l l y a l l fibers could be described as
being "red". The f3 fiber population never develops any s u b s t a n t i a l capacity
f o r anaerobic metabolism and s o remains red throughout i t s lifespan. I n
support of t h i s type of metabolism, f3 fibers remain r e l a t i v e l y small in
diameter and a r e supplied w i t h a l i b e r a l number of c a p i l l a r i e s . The a
f i b e r population begins t o exhibit an increasing capacity f o r anaerobic
metabolism approximately near b i r t h . In a proportion of the (31 f i b e r
population, t h i s increase is decidedly more rapid and i s acco-nied
by a
rapid increase tn fiber growth and a d i l u t i o n of mitochondrial density.
Indeed, not only the s p e c i f i c a c t i v i t y of mitochondrial enzymes changes
but the pattern of mitochondrial enzymes as well as the mrphology of the
216
organelles a r e a l t e r e d during the transformation of c91 t o aW f i b e r s .
"he regulatory signals and mechanisms which induce t h i s transformation
a r e not known, but it seems possible that it occurs in c1 f i b e r s in which
f'unctional a c t i v i t y occurs a t a l e v e l below that which i s required t o
maintain a high aerobic capacity.
The developmental acquisition of specific c h a r a c t e r i s t i c s by d i f f e r e n t
muscle f i b e r s is a matter of some importance t o meat s c i e n t i s t s since it i s
c l e a r that t h e c h a r a c t e r i s t i c s of meat a r e simply r e f l e c t i o n s of the sum of
the characteristics of each of the muscle f i b e r s of which meat i s composed.
We have recently discussed some of the possible e f f e c t s on meat quantity
and quality that might be expected t o r e s u l t from v a r i a t i o n of the proport i o n a l i t y of muscle f i b e r types (Ashmore e t al., 1972). L e t us turn t o
some of our recent observations obtained in studies of f e t a l muscle.
Some e a r l i e r studies (see Cassens e t al., 1969, f o r review) had shown
f e t a l muscle f i b e r s t o be i n i t i a l l y indistinguishable from each other, and
t h a t the development of specific f i b e r types occurred a t d i f f e r e n t ages in
d i f f e r e n t species. This i s especially apparent w i t h regard t o energy
metabolism since, as discussed above, a l l f i b e r s a r e i n i t i a l l y red, with
subsequent development of CIW f i b e r s from 023 f i b e r s occurring in most species
just before o r j u s t after b i r t h . However, d i s t i n c t c1 and B f i b e r populations
can be observed during t h e e a r l y stages of myogenesis w e l l before any
s u b s t a n t i a l amount of muscular a c t i v i t y .
--
I n t h e experiment t o be reviewed (Ashmore e t al., l972), muscle o f
f e t a l Lambs was examined a t various times during gestation, which, f o r
the sheep is 147 days, Muscle biopsies were placed in ice-cold s a l i n e
f o r 1 min, then quick-frozen i n dry-ice/acetone and sections (10 u ) were
prepared and reacted f o r lqyosin ATPase (pH lO.O), succinic dehydrogenase
(SDH), phosphorylase, and glycogen. The r e s u l t s obtained on the semitendinosus muscle w i l l be presented.
A t 50 days gestation the muscles a r e characterized by widely scattered
f i b e r s approximately uniform in cross-sectional a r e a . A l l f i b e r s react f o r
myosin ATPase but a r e negative f o r both phosphorylase and SDH. Morphologically, a l l f i b e r s exhibit one or more large vacuoles in the center of
the f i b e r . Most often t h e e n t i r e c e n t r a l area of the f i b e r i s devoid of
myofibrillar staining material. These f i b e r s are c a l l e d presumptive f3
fibers
.
At
55 days, more presumptive B f i b e r s e x i s t than a t 5 0 .
A t 60 days of development, the presumptive B f i b e r s a r e s l i g h t l y
increased in averege cross-sectional area. I n addition, a second population
of fiSers has begun t o appear. They a r e smaller than the presumptive l3
f i b e r s were a t 50 days. I n many cases d i r e c t contact occurs between the
two types. The smaller f i b e r s a l s o exhibit myosin ATPase a c t i v i t y but a t
Lhis age cannot be c l e a r l y differazttated cytochemically from the l a r g e r
f'ibers by t h i s reaction alone (figure 1).
A t 70 days, t h e number of small f i b e r s around the presumptive B
fibers has roughly doubled. The difference i n staining of myosin ATPase
a c t i v i t y now is quite clear--permitting us t o des.cribe the large f i b e r s
as P, the small f i b e r s a r e CY (figure 2 ) .
A t 100 days, nearly a l l of t h e B f i b e r s a r e completely f i l l e d w i t h
myofibrils. The number of a fibers has again doubled. Large, e a r l y
formed CY fibers have been displaced outwsrd toward t h e periphery of the
bundle by the small n e w l y formed a fibers ( f i g u r e 3 )
A t 125 days, bundles and f i b e r s a r e nore t i g h t l y packed. Growth in
s i z e of both types of f i b e r s has occurred t o a s u b s t a n t i a l l y greater
degree than has growth in numbers of f i b e r s . It i s c l e a r l y seen that the
l a r g e s t and smallest f i b e r s a r e the a type. B fibers a r e of uniform s i z e
(figure 4 ) . Phosphorylase a c t i v i t y is uniformly high and low i n a and B
f i b e r s , respectively, a t 125 days.
Some small a f i b e r s a r e s t i l l being formed a t 130 days. A t 140 days,
however, t h i s process has stopped and f u r t h e r growth occurs only by
enlargement of existing f i b e r s .
A t 140 days, the SDH s t a i n i n g pattern i s most intense i n the B
f i b e r s and those a f i b e r s i n the center of the bundle.
Therefore, these findings suggest that t h e d i f f e r e n t i a t i o n of
IC
and B
f i b e r s (those destined t o be f a s t and slow i n mature muscles) would not
seem t o be related t o t h e q m n t i t y of muscle contraction. The f3 f i b e r s ,
referred t o as primary myotubes i n a recent electron microscope study o f
embryonic chick muscle (Ukuchi, 1971) serve as a s t r u c t u r a l framework
upon w h i c h myoblast p r o l i f e r a t i o n and fusion continue w i t h t h e subsequent
generation of secondary mlyotubes. These secondary generations of' myotubes
a r e those destined t o be the a f i b e r population. The a f i b e r s a r e
progressively formed, released, and displaced outward. Thus, t h e first c*:
f i b e r s formed i n f e t a l lamb muscle are those w h i c h commonly reside on t h e
periphery of f i b e r bundles i n mature muscle. The f3 f i b e r population i s
completed i n a r e l a t i v e l y short period of t i m e , precedes t h e fcmnation of
the c1 f i b e r population, and serves some kind of r o l e i n t h e organization
of' muscle f i b e r bundles snd therefore t h e muscle as a whole. The observation
t h a t f3 f i b e r s serve as "nuclei" f o r t h e development of cx fibers explains
the i n t e r n a l location of red f i b e r s (BR) in a d u l t f i b e r bundles.
Further studies on embryonic chick muscle have canfirmed the f i n d i n g s
obtained on lamb muscle (Addis and Ashmore, 1972). Embryos were examined
d a i l y from 7 days t o hatching (21 days). A t least 10 embryos were studied
each day. Several embryo muscles w e r e studied, including those o f the
pectoral, cervical, and femural regions. The methods used f o r chick
biopsy preparation were e s s e n t i a l l y t h e sane as those used f o r lamb t i s s u e
except that t h e samples were not c h i l l e d in saline solution p r i o r t o
f r e e z l n g . DFle t o the small s i z e and f r a g i l i t y of chick embryo muscle
t i s s u e , the muscles were not removed from t h e embryo, but instead, the
e n t i r e femural region was mounted. This permitted sectioning thrcxlgh the
218
Figure 1.
Section from 8&teUd%nOSU8
of 60 day fetal lamb.
Myofibrillar ATPase.
Figure 2.
Section from semitendinosus of 70 day fetal lamb.
Myofibrillar ATPase.
Figure 3.
Section from semitendinosus of 100 day fetal lamb.
Myofibrillar ATPase.
Figure 4.
Section from Semitendinosus of 125 day fetal lamb.
Myofibrillar ATPase.
220
e n t i r e femur and, consequently, the histochemically staining of several
muscles simultaneously. Sections (10 and 1 6 ~ )
were prepared and stained
w i t h hematoxylin and eosin and f o r myofibrillar ATPase (pH 10 and pH 4.1).
A l t h o u g h some myofibrillar ATPase a c t i v i t y i s present as e a r l y a s 7
days, organizational p a t t e r n s and myotubes are discernable only a f t e r about
12-14 days in ovo incubation.
--
The development of embryonic chick muscle is, i n general, similar t o
t h a t noted in lamb muscle. The p a t t e r n i s c l e a r l y biphasic i n nature w i t h
I3 f i b e r s forming during the i n i t i a l stages of myoblast fusion followed by
p r o l i f e r a t i o n of c1 f i b e r s from the surface o f t h e primary myotube during
t h e l a t t e r stages of in ovo developnent (figures 5-7).
--
Figures 8 and 9 are comparable t o figures 6 and 7, respectively, but
the myofibrillar ATPase reaction was performed a f t e r preincubating t h e
sections a t pH 4.1 f o r 10 minutes. In t h i s case the j3 fibers stain
intensely whereas the a c t i v i t y in the a f i b e r s i s inhibited. This i s the
preferred technique f o r i d e n t i f i c a t i o n of f3 fibers i n f e t a l and embryonic
muscle since imnature f3 f i b e r s a l s o stain intensely a f t e r alkaline
preincubation (figure 2 and 5 ) . Indeed, in M. complexus of t h e chick, B
fibers cannot be i d e n t i f i e d adequately by the alkaline preincubation
technique u n t i l 1 week after hatching (Ashmore e t al., 1972).
SIGNIFICANCE TO MUSCLE A S A FOOD
The reader is referred t o several reviews concerning t h e r e l a t i o n of
f i b e r tw, morphology alld postnatal development t o ultimate properties
of muscle as a food (Hegarty, 1971; Kauffman, 1971; S w a t l s s d , 1971; Topel,
1971; V a n Sickle, 1971; Cassens and Cooper, 1970; Cassens e t al., 1969)
The biphasic development of a and @ fibers observed in the f e t a l Lamb
and embryonic chick raises several questions of significance t o meat animal
development. For example, can an animal physiologically support only a
limited number of red f i b e r s (j3R and a)?If so, then any f u r t h e r increase
i n muscularity of meat animals which i s achieved by increasing t h e t u t a l
number of muscle f i b e r s would r e s u l t s p e c i f i c a l l y i n an increase i n the
number of aW fibers.
Just as the subject of light and dark muscle f i b e r s has received much
a t t e n t i o n from researchers, the relationship of red: white fiber r a t i o t o
muscle q u a l i t y has been thoroughly investigated. Nevertheless, considerable
disagreement e x i s t s in t h e literature. We believe that cansideration of the
organization, development and biochemical properties of BR, 03 and
fibers
as presented in t h e foregoing discussion, may provide an explanation f o r
these discordant viewpoints.
Numerous s t u d i e s have been published on the histology, histochemistry
and biochemistry of light and dark muscles, areas of muscles, o r fibers i
n
r e l a t i o n t o postmortem properties. It is l o g i c a l t o expect white muscles,
221
Figure 5 .
Section from leg muscle of 14 day chick embryo.
Myofibrillar ATPase.
Figure 6.
Section from leg muscle of 16 day chick embryo.
Myofibrillar ATPase.
222
Figure 7.
Section from leg muscle of 18 day chick embryo.
Myofibrillar ATPase.
Figure 8.
Section from l e g muscle of 16 day chick embryo.
Myofibrillar ATPase a f t e r acid preincubation.
pattern of cy and
e
Staining
f i b e r s is reversed from that seen
a f t e r a l k a l i n e preincubation (see Figure 6 ) .
224
Figure 9.
Section from leg muscle of 18 day chick embryo.
Myofibrillar ATPase after acid preincubation
(see Figure 7).
areas o r f i b e r s t o exhibit rapid pH decline compared t o t h e i r red counterparts since upon exsanguination anoxic conditions a r e established m r e
quickly in white muscle tissue. A t the cessation of oxidative phosphoryl a t i o n , as dictated by t h e "Pasteur Effect" (Van Eys, 1961), anaerobic
glycolysis would commence. Beecher e t a l . (1965a) determined t h a t the
rate of pH decline in light area of t h e semitendinosus exceeded t h a t of
the dark area of the same muscle. Beecher e t al. (1965b) determined light
and dark f i b e r r a t i o in seven porcine muscles. Sudan Black B ( a l i p i d
s t a i n ) was u t i l i z e d t o d i f f e r e n t i a t e f i b e r s . Red muscles contained more
red f i b e r s , greater myoglobin concentrations and had longer sarcomeres
than white muscles. Further studies (Beecher e t a l . , 1969) noted t h a t
gluteus medius and longissimus d o r s i muscles exhibited more rapid l o s s of
ATP and more rapid pH decline than i n the red rectus femoris muscle.
I a c t a t e dehydrogenase (LDH) a c t i v i t y of white muscle exceeded (P < .01)
t h a t of red muscle. LDH isoenzyme patterns corresponded t o these data
w i t h more of an anaerobic d i s t r i b u t i o n among white muscles. The r e s u l t s
of t h e above studies were canfinned by Addis and AUen (1970). They noted
t h a t l i g h t muscles (1. dorsi, l i g h t b. femris, light semitendinosus and
g . medius) exhibited greater t o t a l LDH a c t i v i t y (per mg soluble protein)
than dark muscles (dark b. femoris, semitendinosus and trapezius )
IDH
isoenzyme V, the muscle type, also s e g r e s t e d light muscles from d a r k
muscles.
--
--
.
The question of greater importance than comparisons among muscles
within the same breed are comparisons between breeds and s t r a i n s known
t o d i f f e r in degree of s t r e s s - s u s c e p t i b i l i t y . The f i r s t study t o demons t r a t e an interaction among environmental treatment, l i g h t : dark f i b e r
r a t i o and postmortem glycolytic rate w a s Howe e t a l . (1968). They u t i l i z e d
psychrometric chambers t o r e a r animals under four d i f f e r e n t environmental
Forty " s t r e s s -susceptible" Poland China barruws were used.
treatments
Sudan black B was used t o stain muscles. Emrironment during growth
s i g n i f i c a n t l y affected the 1ight:dark f i b e r r a t i o ; a high r a t i o was found
i n the treatment w h i c h e l i c i t e d the most rapid pH decline of the four
treatments studied.
c-
.
The f F r s t d i r e c t comparison of fiber d i s t r i b u t i o n between stressr e s i s t a n t and stress-susceptible breeds was conducted by Sair e t a l .
(1970). Unexpectedly, their findings suggested that the s t r e s s - r e s i s t a n t
breed displayed a higher r a t i o of 1ight:dark f i b e r s than the susceptible
breed.
--
--
Cooper e t a l . (1969), using histochemical assays f o r ATPase, amylophosphorylase, and an oxidative enzyme system stain, further characterized
differences in muscle from s t r e s s - r e s i s t a n t and stress-susceptible p i g s .
Their findings indicated that muscle from stress-susceptible animals
contained more intermediate f i b e r s . Under the s y s t e m of c l a s s i f i c a t i o n
used by us, these fibers w o u l d l i k e l y correspcmd t o 03 fibers. Muscles
from t h e two breeds d i d not d i f f e r Fn t h e capillary/fiber r a t i o .
Dildey e t a l . (1970)grouped f o r t y barrows (Hampshire-Yorkshire )
according to-(irpostmortem muscle color-structure, and ( 2 ) muscuhrity
Histological sections were taken from t h e 1. d o r s i and stained f o r Sudan
.
226
black B . S t a t i s t i c a l analysis of histochemical data revealed that PSE
pigs exhibited a higher (P < .01) l i g h t : dark fiber r a t i o than animals
exhibiting normal muscle color-morphology
.
From the foregoing discussion it i s apparent t h a t no clear consensus
e x i s t s amng researchers concerning the histochemical c h a r a c t e r i s t i c s of
muscle in r e l a t i o n t o the degree of s t r e s s - s u s c e p t i b i l i t y in the animal.
These questions a r e of extreme importance in r e l a t i o n t o the larger question
of genetic aspects of hypenrmscularity, s t r e s s - s u s c e p t i b i l i t y and postmortem
muscle quality. In light of the new laboratory procedures, f i b e r c l a s s i f i cation and nomenclature information, and the improvement in our understanding
of f i b e r interrelationships reported e a r l i e r i n t h i s paper, it would be
extremely i n t e r e s t i n g t o reexamine some of the above mentioned research
projects. For example, the work of Howe e t a l . (1968)was completed p r i o r
t o research demonstrating the dynamic i n t e r r e l a t i o n s h i p between C
B and CW
fibers. Thus, it i s possible that t h e i r r e s u l t s reflected the conversion
of some 023 f i b e r s i n t o aW f i b e r s as e l i c i t e d by the same growing environment
which w a s detrimental t o muscle properties. The importance of using
myofibrillar ATPase and SDH histochemical assays on s e r i a l sections t o
determine the d i s t r i b u t i o n s of BR, a(R and aW fibers, now t h a t such
techniques a r e available, cannot be over-emphasized.
--
Studies on b o v b e hypermuscularity, although limited i n number,
suggest t h a t an a l t e r a t i o n has occurred in f i b e r d i s t r i b u t i o n . Recent
studies in our laboratory confirm t h e increase in the r e l a t i v e proportion
of clw fibers (figures 10 and 11) reported e a r l i e r f o r muscles of the
"double muscled" bovine mutant (Ashmore and Robinson, 1969), but i n
addition provide evidence f o r an increase in t h e t o t a l number of f i b e r s
as w e l l as a reduction i n the percentage of the red f i b e r popuhtion
( H o b s and Aehmore, 1972).
SUMMARY
The studies reported herein provide good evidence f o r the following:
(1) the embryonic d e v e l o p n t of a and f3 f i b e r s occurs sequentially, and
( 2 ) a major determiaant of muscle quantity and quality i s the r a t e and
time of CI f i b e r formation in the second phase of myogenesis. Finally,
and perhaps most importantly, t h e observation of phasic development of
f3 f i b e r populations makes possible the design of new types of
experiments not heretofore encouraged by t h e thought that a l l f e t a l muscle
f i b e r s were i n i t i a l l y equivalent.
cx and
Figure 10.
Figure 11.
Section from rrsmitendinorur of 4 month old normal calf.
SDH reaction.
Section from aemitendinosu8 of 4 month o l d "double mscled" calf.
SDH reaction.
228
REFERENCES
Addis, P . B. and C. R. Ashmore.
fibers.
J. h i m . S c i . 35:lB.
1972. Rnbryonic development of a and
(Abstr.)
f3
1970. Oxidation of p y r u v a t e - 2 d 4 , and Lactate
dehydrogenase a c t i v i t y and isoenzyme d i s t r i b u t i o n of f i v e porcine muscles.
J. Food S C ~ 35:207.
.
Addis, P. B. and E. Allen.
Ashmore, C . R., P. B o Addis, L. Doerr and H. Stokes. 1972. Development
of muscle f i b e r s i n t h e comrplexus muscle of normal and dystrophic
chicks. J Histochem Cytochem. (Submitted )
.
.
Ashmore, C . R. and L. Doerr. 197la. P o s t n a t a l d e v e l o p n t of f i b e r types
i n normal and dystrophic skeletal muscle of t h e chick. Exp Neurol.
.
30:431.
Ashmore, C . R. and L. Doerr. 1971b. Comparative aspects of muscle f i b e r
types in d i f f e r e n t species. Exp. Neurol. 31:405.
A s b r e , C . R . and D . W . Robinson. 1969. Hereditary muscular hypertrophy
i n t h e bovine. I H i s t o l o g i c a l and biochemical characterization.
Proc. SOC. Exp. Biol. Med. 132:548.
.
.
Barany, M., K. Barany, T. Reckard and A . Volpe. 1965. Myosin of fast
and slow muscles of the r a b b i t . Arch. Biochem. Biophys 109:185.
Bernard, R . J., V . R. Edgerton, T. F w u k a w i a and J. B. P e t e r . 1971.
Histochemical, biochemical and c o n t r a c t i l e properties of red, white,
and ictermediate f i b e r s . h e r . J. Physiol. 220:410.
Beecher, G. R., E. J. Briskey, W . G. Hoekstra. 1965a. A comparison of
glycolysis and associated changes In l i g h t and dark portions of the
porcine semitendinosis. J. Food S c i . 30:477.
Beecher, G . R., R. G. Cassens, W. G. Hoekstra, E. J . Briskey. 1965b.
Red and white f i b e r ccmtent and associated post-mortem properties of
seven porcine muscles. J. Food S c i . 30:969.
Beecher, G . R., L. L. Kastenschmidt, W. G. Hoekstra, R. G . Cassens and
E . J . Briskey. 1969. Energy metaboldtes in red and white striated
muscles of t h e p i g . A g r . Food Chem. l7:29.
Burke, R . E., D . N . Levine, F. E. Zajoe, 111, P. Tsairis, and W . K. Engel.
1971. Mammalian and motor units : physiological-histochemical c o r r e k t i o n
Fn t h r e e types in c a t gastrocnemius. Science 174:7@.
Cassens, R . G. and C
Res. 1g:l.
..
C
Cooper.
1969. Red and white muscle
Adv. Food
., . .
Cassens, R . G
C C Cooper and S Morita. 1969. D i f f e r e n t i a t i o n of
muscle f i b e r s during growth and development. In: Proc 22nd Ann. Rec
Meat Conf. (Amer. Meat Sci. Assoc). National Livestock and Meat Board,
Chicago, Ill. p. 101.
.
.
Close, R. I. 1972. Dynamic properties of mmmalian s k e l e t a l muscles.
Physiological Rev. 52:Ug.
Cooper, C . C., R. G. Cassena. and E. J. Briskey. 1969. Capillary
d i s t r i b u t i o n and f i b e r c h a r a c t e r i s t i c s in s k e l e t a l muscle of s t r e s s susceptible animals. J. Food S c i . 34:299.
Dildey, D . D., E . D. Aberle, J. C . Forrest and M. D. Judge. 1970. Porcine
muscularity and properties associated w i t h pale, s o f t , exudative muscle.
J . Anim. Sci. 3 l : a l .
Engle, W. K. 1962. The e s s e n t i a l i t y of h i s t o - and cytochemical s t u d i e s
of s k e l e t a l muscle i n the investigation of neuromuscular disease.
Neurol 12 :778
.
George, J. C . and A. J. Berger.
New York.
1966. Avian Myology-.
Academic Press.
Gergely, J., D . Pragay, A . F. Scholz, J. C. Seidel, F. A. S r e t e r and M. M.
Thompson. 1965. In: Molecular Biology of Muscular Contraction.
S. Ebashi, F. Oosawa, T. Sekine and Y. Tonomvra (ed.) Elsevier, New
York. p . 145.
Guth, L. and F. J. Samaha. 1969. Qualitative differences between actomyosin
ATPase of slow and fast mammlian muscle. Exp. Neurol. 25:138.
Guth, L. and F. J. Samaha. 1972. Erroneous i n t e r p r e t a t i o n s which m y
r e s u l t from a p p l i c a t i o n of the "Myofibrillar ATPase" histochemical
procedure t o developing muscle. Exp. Neurol. 34:465.
.
Hegarty, P .V.J.
1971. Muscle f i b e r g r o w t h and development. In: Proc
24th Ann. Rec. Meat Conf. (Amer. Meat S c i . Assoc.). National Livestock
and Meat Board, Chicago, Ill. p . 319.
.
.
.
Holmes, J. H G . and C R. Ashmore. 19'72. A histochemical study of
development of muscle f i b e r s i n normal and "double muscled" c a t t l e .
Growth. (In press )
Kauffman, R. G. 1971. Variation in g r o s s composition of meat animals.
In: Proc. 24th Ann. Rec. Meat Conf. (Amer. Meat S c i . Assoc.).
National Livestock and Meat Board, Chicago, Ill. p . 297.
Kikuchi, T. 1971. Studies on development and d i f f e r e n t i a t i o n of muscle.
111. Especially on t h e mode of increase i n t h e number of muscle c e l l s .
Tohoku J. Agric. Res. 22:l.
Ogata, T.
19%.
Three types of muscle f i b e r s .
Acta Med. O k a y a m a ~ 2 1 6 .
Padykula, H . A . and G. F. Gauthier. 1967. Morphological and cytochemical
c h a r a c t e r i s t i c s of f i b e r types in normal mammalian s k e l e t a l muscle. In:
Exploration Concepts in Muscular Dystrophy and Related Disorders.
A T Milhorst (ea. ), Ekcerpta Medico, New York.
..
Paukul, E. 1904. Die zucktmgformen von kaninchenmuskeln verschiedener
farbe and s t r u c t u r . Arch. Physiol. S. 100.
S a i r , R . A . , D. L i s t e r , W. G. Moody, R. G. Cassens, W. G. Hoekstra and
E. J. Briskey. 1970. Action of curare and magnesium on s t r i a t e d
muscle of s t r e s s - s u s c e p t i b l e pigs. h e r . J. Physiol. 2 l 8 : l m .
.
.
Albers
1970~. Differences between
slow and f a s t muscle myosin: AWase a c t i v i t y and r e l e a s e of associated
proteins by p-chloramercuri-phenylsulfonate J. Biol. Chem. 245:219.
Sarnsha, F. J., L. Guth and R. W
Samaha, F. J., L. Guth and R. W. Albers.
1970b. Phenotypic differences
between t h e actomyosin ATPase of the t h r e e f i b e r types of m R n r m t L l i a n
s k e l e t a l muscle. Exy. Neurol. 26:120
S r e t e r , F. A., J. C. Seidel and J. Gergely. 1966. Studies on myosin
from red and white s k e l e t a l muscles of the r a b b i t . I. Adenosine
J Biol. Chem. 241: 5772
triphosphatase a c t i v i t y
.
Stein, J. M. and H. A . Padykula. 1962. Histochemical c l a s s i f i c a t i o n of
individual skeletal muscle f i b e r s of t h e rat. Amer. J. Anat. 110:103.
.
.
Swatland, H. J . 1971. Morphology of the motor innervation of muscle.
In: Proc. 24th Ann. Rec. Meat Conf. ( h e r Meat S c i . Assoc ). National
Livestock and Meat Board, Chicago, Ill. p . 400.
. .
.
Topel, D G
1971. Physical and chemical changes during growth and
development of f a t and muscular pigs. In: Proc 24th Ann. Rec Meat
Conf. (Amer Meat S c i . Assoc. ) Natianal Livestock and Meat Board,
Chicago, Ill. p. 304.
.
V a n Sickle, D. C. 1971. Neurotrophic influences on p o s t n a t a l s k e l e t a l
(American Meat S e i . Assoc )
muscle. In: Proc 24th Ann. Rec Meat Conf
National Livestock and Meat Board, Chicago, Ill. p . 390.
.
.
.
.
.
..
1961. Regulatory mechanisms in energy metabolism. In: Control
Mechanism in C e l l u l a r Processes. D . M Bonner (ed. )
The Ronald
Press Company, N e w York.
Van Eys
.
Yellin, H. and L. Guth. 1970. The histochemical c l a s s i f i c a t i o n of muscle
f i b e r s . Exp. Neurol. 26:424.