c. Romm Presented a t the 25th Annual Reciprocal Meat Conference of the American Meat Science Association, 19p.

Transcription

1 211 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, ( 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 Presented a t the 25th Annual Reciprocal Meat Conference of the American Meat Science Association, 19p

2 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 -

3 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

4 214 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 400 ph 1040 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

5 215 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

6 216 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 loo), 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)

7 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 clearpermitting us t o describe 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

8 218 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

9 Figure 3 Section from semitendinosus of 100 day fetal lamb Myofibrillar ATPase Figure 4 Section from Semitendinosus of 125 day fetal lamb Myofibrillar ATPase

10 220 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 41) 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 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 41 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 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,

11 221 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

12 222 Figure 7 Section from leg muscle of 18 day chick embryo Myofibrillar ATPase

13 Figure 8 Section from leg muscle of 16 day chick embryo Myofibrillar ATPase after acid preincubation Staining pattern of cy and e fibers is reversed from that seen after alkaline preincubation (see Figure 6)

14 224 Figure 9 Section from leg muscle of 18 day chick embryo Myofibrillar ATPase after acid preincubation (see Figure 7)

15 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

16 226 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

17 Figure 10 Section from rrsmitendinorur of 4 month old normal calf SDH reaction Figure 11 Section from aemitendinosu8 of 4 month old "double mscled" calf SDH reaction

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