Food Structure
Volume 7 | Number 2
Article 4
1988
Characterization of Hypercontracted Fibers in
Skeletal Muscle of Domestic Turkey (Meleagris
Gallopavo)
A. Sosnicki
R. G. Cassens
D. R. McIntyre
R. J. Vimini
M. L. Greaser
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Recommended Citation
Sosnicki, A.; Cassens, R. G.; McIntyre, D. R.; Vimini, R. J.; and Greaser, M. L. (1988) "Characterization of Hypercontracted Fibers in
Skeletal Muscle of Domestic Turkey (Meleagris Gallopavo)," Food Structure: Vol. 7: No. 2, Article 4.
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FOOD MICROSTRUCTURE, Vol. 7 (1988), pp. 147 - 152
Scanning Mi c roscopy Int ernat ional , Chicago (AMF O'Hare), IL 60666
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CHARACTERIZATION OF HYPERCONTRACTEO FIBERS IN SKELETAL MUSCLE OF
DO MESTIC TURKEY ( MELEAGR IS GALLOPAVO)
A . Sosnicki1,2, R.G. Cassens2,*, D.R. Mc lnt yre3, R . J. Vimini3 a nd M.L. Greaser2
LPresent address:
De p artment of Biology, Univ ers ity of Pennsylvania, Philadelphia, PA 19104
2Mu sc le Biology Laboratory , College of Agricultural a nd Life Sc iences, 1805 Linden Drive
Un iversity of Wisconsin, Madison, WI 53706, and
3oscar Mayer Foods Corporation, 910 Mayer Ave., Madison , WI 53707
Abstract
Int roduction
Several different muscles from a population of
thirt y Large Whit e ma le turk eys were s tudi ed by
hi s tological a nd hi s toc he mical me thod s, and a high
incidence of hypercontracted fibers was noted . The
fibers were charac t erized in cross-sec ti on by an
apparent swe lling, a rounded rather than polygonal
shape and a homogenous appearance.
They were
eosi nophilic, positive to Gomori-trichrome a nd
appea r ed to have an e leva t ed lipid co nt e nt.
An
unusual histochem ica l profile was not ed.
rt was
postulated tha t the hypercontracted fib ers were real ,
not ar tifnc tual, a nd resulted from some c hange
occuning in the muscle.
Hyperco ntrac tion is a.n excessive contraction of
myofibr il s a nd i s ren ec t ed by ab normally s hort
sarcomeres (Carpenter and Karpati, 1984). Hype r contrac ted fib e r s in cryos t at cross sec tion s appear
en larged and rounded ("giant" fibers). Because the
normal myofi brillar and intermyofibrillar sta ining
pattern is los t , the t erms "hyaline", "waxy" or
"opaque'' have also b een u sed to describe these fibers
(Wohlfart, 1937; Schm albru ch, 1973; 1975; Cull e n a nd
Fulthorpe, 1975; Carpenter and Karpati, 1984; Uchino
and Araki, 1986). Although the occurrence of hyper contrac t ed fib e rs in muscle ti ssu e in human and
domestic mammal s (pig, cattl e, sheep) has bee n note d
previously by many authors, the underl y in g causes of
the phe nomenon are controv e r sial.
Carpen ter and Karpati (1984) poin ted out that
hypercontraction of fibers seen in biopsies from
Duc henne muscu lar dystrophy pa tie nt s tak es pl ace
during b iopsy .
On t he other ha nd , Cu ll e n a n d
Fulthorpe (1975) a nd Schmalbruc h (1973, 1975) have
proposed that hy p e r contrac tion in human di seased
musc le is a patho log ical reaction of mu sc le ce ll s. In
animals, "g iant" fibers have been found in nor ma l
musc le o f wild and domesti c mammals i n both prerigor and pos t -r igor condi tions (Weatherspoon, 1969;
Linke, 1972; Dutson et al. , 1978; Schm idt and
Dumont, 1981; Handel and Stickland, 1986; Sink et
al.. 1986; Salomon and Eastridge, 1987). Scheper
(1979) and Schm idt and Dumont (1977) observed more
of this type of fib er in musc le of ca ttl e or sheep
which exhib it ed gross e nlargement or hypertrophy.
Lik ewise, "giant" fiber s were often found in pigs
having the c ondit ions of Por c ine Stress Syndrome
(PSS) , St ress-myopathy or Pal e, Soft and E xudativ e
(PSE) meat (Cassens et al., 1969; Dutson e t a l., 197 8;
Sosnicki a nd Domanski , 1983; Sosnicki, 1987).
Descr iption of the histochemical profil e of
hypercontracted fibers has also varied s ignifi can tl y.
They were observed as myosin ca•• - ATPase positive
(Cassens et a t. , 1969) or negative (Fenichel, 1963);
succinic de hydrogen ase (SOH) positive (De Bruin,
197 1; Handel a nd Stickland, 1986) or variab le (Dutson
e t al., 1978, Sink et al., 1986, Sosnick i, 1987); lactic
acid dehydrogenase (LOH) var iabl e (Sosnicki, 1987) or
posi t ive (Hraste et at., 1980).
lOosowska e t al. (1980) have noted the occurrence of "gia nt" fibers in muscle of chicke n. Grey
e t al., (1986) and Seemann e t a l., (1986) observed
what they t e r med " large rounded fib ers' 1 in a s tudy
of turkey breas t muscle and thought they we r e
Initi al paper received July 17, 1988
Manuscript received Sep t ember 08, 1988
Direct inquiries to ll.G. Cassens
Telephone number: 608 262 1792
Key words: Hypercontracted fibers, skele t al musc le,
htstopathology, domestic turkey
Person t o contac t :
*R .G. Cassens, Mu sc le Biology Labora tor y,
1805 Linden Driv e, University of Wisconsin,
Madison , lVI 53706;
Te lephon e No . (608) 262- 1792
147
A. Sosnicki, R.G. Cassens, D.R. Mcintyre, et al.
Figure 1 (a). Cross sec tion showing mu sc le fiber
undergoing necrosi s (large arrow) and several hyper contracted fibers (small arrows). (b). Cross section
show ing normal appeari ng fibers . M. biceps femoris
(Group A). H+E. Bar= 0.05 mm .
Results
associated with toughness of the mea t.
We have often obse rved hypercontracted fibers
during the course of our hi s toc hemi cal s tudies of
muscle of rapidl y grow ing turkey s . The purpose of
thi s work i s to present frequ e ncy data about the
hyperco nt racted fibers toge ther with some poss ibl e
ex pl a nation for the ir occurrence, and a description of
his tological and hi s toc hem ic al features is a ls o
provided.
Mater ial s and Methods
Histological and histochemical studies of "giant"
fibers were carried out on 30 Large White turkey
males that were 14 (group A), 16 (group B) and 18
(group C) week s of age (10 in eac h group). The
birds were selec t ed a t random from com me r c ial
flo c k s whi c h had been r a ised in light controll ed
hous es and fed a s t a nda r d cor n I soybean meal poultry
diet.
Samples for analysis we re collected immediately
post s laught er from 5 skeletal musc le s: m. pec torali s
thoracicus
(superficialis),
m.
supracoracoideus
(pectoralis deep) , m. biceps femoris , m. sem i tendinosus and m. femorotibialis medius. To prevent
trauma, muscle samp les were held at r es t length by
forceps and then rapidly frozen in isopent a ne cooled
with liquid nitrogen.
Serial cross and longitudinal sec tion s 8 mic ro meters thi ck were obtained with a cryostat. In the
analysis of histological t rait s , hematoxylin a nd eos in
(H&E), modifi ed Gomori-trichrome and Oil r e d 0
staining teclmiques were used.
Histochemical r e actions were conducted in order to show activ it y of
myosin ca+ + -ATPase (Guth and Samaha, 1970) ,
succinic dehydrogenase (SDH) (Bark a and Anderson,
1963), alkaline and acid phosphatases and
phosphorylases a and b (Chayen et al., 1973).
148
Whil e an exact quantification was not made, the
musc le in cidence was considered positive if hyper contracted fibers were present in a low power (25x)
of cross-sect ion ed muscle (1.68 mm2 area). A uniform pattern of incidence of the hype rcontracted
fibers in the particular birds was not seen nor were
cer tain muscles more prone to fiber hyp e r contrac ti on
than others. They a ppeare d to b e di s tribute d ran domly throughout any given musc le bundle and were
not located pre fere ntiall y in either peripheral or
central areas of the bundle. In som e ins t a nces, on l y
a few single, sca ttered hype rcontrac t ed fibers were
seen, but , in other cases, they appeared in c lose
proximity (being almo s t grouped) in number s of three
or more. In rare instances, up to twenty - five per ce nt of the fibers in a small area appeared affected.
In view of this distribution, we me rel y counted a
bird positive if one or more hypercontracted fibers
were prese nt (see above for area viewed).
From
Tab le 1, it a ppea r s that the olde r birds had a higher
inc ide n ce .
Typi ca l hyp e r contrac t ed and norm al appea ring
muscle fibers a r e illus trat ed in Figures Ia and 1b
resp ec tively. In comparis on to normal mu sc le cells ,
an apparent swelli ng of hypercontracte d fibers was
observed, and in cross-sec tion they had a rounded
rather than polygonal shape. They had a homogene ous appearance (described typically as "waxy" or
" hyaline"), were eos inophilic and stained a uniform
dark-red with the Gomori-trichrome procedure . The
content of intracellular fat was also highe r in comparison to that present in normal muscle cell s (Fig ure 2). However , int erpretation of this may b e confuse d by a c hange of the histologica l staining appearance du e to the contrac tion (an apparent s trong e r staining intensity may re sult from the den seness
due to contrac tion) . The hypercontrac ted fibers als o
had a characteristic a ppearance in longitudinal section (Figure 3). In some areas a strong contr action
was obvious with associated tearing and separation of
the contents of the fibers.
Open areas were ob served in many cases surrounding the hypercontracted fiber s both in longitudinal (Figure 3) and cross
Hypercontraction of musc le fibers
~·
A higher con t ent of intracellular fat in
hypercontracted fib er s (arrows) as shown with oil
Red 0 s t aining. M. pectoralis thoracicus (Group A).
Figure 3. Longitudinal section showi ng a 11 Compresse 11
appearance of hyp ercontrac ted fibers (arrow). M.
pectoralis thoraci cus (Group C). H+E.
~ · Multifocal hypercontrac tion (arrows) withundergoing necrosis. M. supracoracoideus
(Group C). Gomori - trichrome.
~r
~·
Hypercontrac ted fibers showing high SDH
reaction (arrows); ot he r muscle fibers show a weak
activity. M. femorotibial is medius (Group B).
Figure 6. ca++ _ATPase reaction (pH 4.60) showing a
high activity in hypercontracted fibers (arrows). M.
semitendinosus (Group B).
Bar (for all Figures) = 0.05 mm .
149
A . Sosnicki , R . G. Cassens, D.R . Mc int yre, et a l.
Table L. Number of birds in whic h t he hypercontraction of musc le fibers was r ecorde d
Group s 1/Age in wee k s
Musc le
A I 14
B I 16
c
I 18
Pec torali s thoracicus
Supraco rncoid eus
10
Bi ce ps femoris
Semitendinosus
Femorotibialis medi us
1 All groups contained 10 birds
sec tio ns (see al so Figure 6). In other in s tances an
appar ent d egeneration was observed in association
with the hypercontracted fibe r s (Figure 4).
Because the reac tion pattern of the hy p er co ntract ed fibers was not typical , the hi s tochemical
profile was somew ha t c onfusi ng (Table 2). A s trong
positive SOH react ion was exhibited (Figure 5) an d a
positive r eaction was fo un d for myosi n ca++ _ATPase
after either alkalin e (pH 10 . 4) or acid pre incub ation
(pH 4.6) ( see Figure 6) . For phos phorylase a and b,
some cases of weak reac tion wer e found b ut the
ma jority of hype r con trac t ed fibers possessed hig h
ac tivity. The r efor e, a s impl e c lass ifi ca tion of the
hypercontrac t ed fibers as s low -ox idative (Type l) ,
fast - g lyco lyt ic (Type I fb) or fa s t oxida t ive- glyco lyt ic
(Type I Ia) was n ot possible. Pos itive, b ut diffuse,
reacti on s for acid and alk aline phosphatase were
observed.
mononu c lear cell invasion were often see n . In addition , we noted a positive acid phos phatase ac tivit y in
hype r contracted fiber s which can b e t ake n as evi dence of necrosis. In the final s t ep of breakdown of
hyper contrac ted fibers fatty ti ssu e replacemen t u sual ly occ urred (Cullen and Fulthorpe, 1975) . Similar
symptoms we r e observed in our s tudy.
The re may be a local in ab ility of the sa r comeres to r elax, which imp lies a defec t of mitochondri a or sar coplasmic r eticulum (Cull e n and Fult horpe,
1975).
In other word s, hypercontrac tion may be
cause d by an influx of ex tracellular calc ium into the
musc le fiber s (Carpe nt er and Karpati, 1984). Indeed,
the high enzy me reac tion of hypercont r ac t ed fiber s
see n in the prev ious, a nd present s tudy (see Table
2), may r esult from fragmen t break s in t he surface
membrane in the vici nity of the observed hypercontrac tion (Carpen t er and Karpati, 1984; Handel and
Stickland, 1986, Levin et al. , 1981, Sink et al., 1986).
Furthermore, functional s t ressing of mu sc le fibers
(i.e. a s trong exerc ise) has been shown to affect calc ium ion uptake and muscle rel axa tion, and conse que ntly the fibe r s ar e t hought to be more prone to
post-mor tem lysis (Sa lomons and He nrik sson, 198 1).
On the other hand, ev ide n ce is also ava il able to sup por t the concep t tha t the subcellular component s of
ab normal fibers (porcine PS E musc le) are more s u scep tibl e to the effec t s of fr eeze - thaw con trac tion
(Cloke et al., 1981).
Finally , regarding an exp lana ti on for the basis
of occ urren ce of hy pe r contrac t ed fi bers, we cannot
make a definit e conc lu s ion. We favor the ex p la nation that they are r ea l and may be associated with
ac tual c hanges occurring in the mu sc le . We con clude
thi s becau se we d id observe so me areas o f apparen t
degener ation and fatty ti ssue r eplacement in longi tudinal sec tion s ofhypercontrac t ed fibers. Moreover,
the positive react ion for ac id phosphatase in the
hypercontracted fibers and an unusual histochemical
profil e support the idea that they are rea l and a r e
assoc iated with a deve loping or present musc le
pa tho logy.
Discussion
The ques tion of artifac t alw ays deserves con s id e ration , a nd hypercontracted fibers are a case in
point - are they artifactual or r e fl ec t iv e of an in
vivo patho logical reaction?
Carpenter and Karpati (1984) r e po rted t hat
hy pe r contrac t ed fibers, see n in a lmost all biopsies
from Duche nne dystrophic patients, did not show
features of necros i s, rarel y occurred in groups, a nd
t ransitional s t ages between hyp ercontra c ti on and tru e
n ec ros is were not observed. Conversely , Cull en and
Fulthorpe (197 5) d esc r ibed the process of hy per c on traction as resulting in the contrac til e filam e nt s
forming a homogenous mass (hyaline degeneration) .
The next s t ep of degeneration was described as tru e
necros is of hype r contrac ted fibers (Cull e n and
Fu lthorpe, 1975). Lik ewise, Schmalbruc h (197 5) repor ted some of the hyp er contrac t ed fibers con ta in ed
phagocytes a nd macrophages, and they s howed c lose ly
attached regener ating fiber s with norma l sarcomere
spacing.
The r e is t he ques tion if the hypercont racted
fibe r s we observed are an intermedia te s t age between
normal fibers and those undergoing a n ec rotic
change.
We did not observe obv iou s regenerative
changes. However. parts of a hyper co nt rac t ed fi ber
in c lose proximity to a nec roti c fiber and an area of
Acknow ledgeme nt s
This work was supported by the College of Agricult ura l and Life Scien ces, Unive r s ity of Wisco n sin ,
Madison and by Oscar Maye r Foods Corpora tion.
Musc le Biology Laboratory Manu scri pt number 236.
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Hypercon traction of mu scle fibers
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T he charac te ristic staining and reaction pattern of normal and hypercontracted musc le fiber s
Type of staining
Type of musc le fiber s
or reaction
I
H
& E
Gomori - trichrome
slow-oxidative
II
fast-glycolyt ic
hypercontracted
positive (pink)
positive (pink)
eos inophili c (red)
positive (green)
po s itiv e (green)
positive (dark r ed )
high
Oil Red 0
high
low
ATPase, p H 9 .4
low
high
high
ATPase, p H 4.6
high
low
high
diffuse or h igh
SDH
high
low
AI kaline phosp ha ta se
no ac tivity
no ac tiv it y
d iffuse
Ac i d pOOsphatase
no activ it y
no act ivit y
diffuse
high
high
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low
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Reviewer IV : How man y hypercont rac t ed fibers a r e
found per bird and does the fr equ en cy in crease with
time?
Authors: We hav e not don e a s t ati s ti cal quantific a tion but merely r eported that the hy pe r c ontracted
fib er s appeared quit e fr equ entl y in the birds examin e d. Our work was direc t ed more at an exa min ation
of the prope rties of t he fiber s in an a tt empt to determin e if the y wer e r eal or a rtifac tu al . One field
und er low powe r mag nification (25x ) is e qu al to
about 1.65 mm2 , a nd the approximate ar ea of our
c ryos tat sec tion s was 1 c m2. The refore, we u su all y
evaluat ed about 60 fields for a giv en mu sc le and
bec ause the s amples wer e coll ec t ed fro m 5 different
muscles, about 300 fi elds wer e evalu a te d pe r bird.
Discuss ion with Reviewer s
Reviewe r I: Wa s all the biops ied mat erial froze n in
tsopentane cool ed with liquid nitrogen? Althoug h
froze n tissue is necessa r y for hi s toc hem ic al s tudies
a nd an H&E s tain don e on thi s mat eri al prov ides a
"control 11 for hi s toche mi cal s tudi es, the bas ic
morphology of the mu sc le see n by H & E s tain might
bett e r be done on formalin fixe d ti s sue.
Author s:
All the muscle samples s tudi ed in thi s
experiment wer e frozen in isopentane cooled with
liquid nitrogen. We agree that a be tter morpOOlogy
would undoubt edly have been seen if H& E s t aining
ha d b ee n don e on formalin fixe d ti ssue r athe r tha n
on unfixed froze n ti ssue . However , fo r malin fi xed
mu sc l e fibe r s usu ally have a more roun de d sha pe in
con t r ast to the more angul ar or pol ygon al s ha pe of
musc l e fibe r s in froze n sec tion. The r oun ded sha pe
might hav e cause d some confus ion in int erp ret a tion
o f the hype r contract ed fiber s .
Rev iewer III: If nec ros is has occ urred , would one
expec t to fmd regenerativ e c hange s?
Authors:
Yes , the d ege ne r ation an d regen eration
processes u su all y occ ur toge ther and we woul d expec t
to obse rve evide nce o f r ege ne r ation --but suc h was
not the case . Necros is i s not alw ays accompanied b y
r egeneration , and , fo r exam pl e, is unlik e ly u nde r
i sc hemi c condition s .
Reviewe r Ill: Wha t about the poss ibility of lysosomal
protea ses deg r ading the sa r c ol emma?
Authors: This is not an eas y que s tion because the re
ts no direct evidence on the matter. One ex plana tion for hypercontrac tion i s a n in c r e ase of available
c alcium in the fibe r whi c h could r esult from damage
to the fiber during the preslaught er time or during
co ll ection and fr eez ing of the s ampl e . Our observa tion s on ac id phos pha tase s how ed a pos itive but dif fu se ac tivit y . We be liev e lysosomal prot eases do not
direc tl y cau se hyper contrac tion of fib e r s but may ac t
in a sec onda r y way .
Some experime nt s to be cons ider e d
Reviewer 11:
would be the e ffect of trauma at the time of
s laught er whe r e one might compare a le tha l dose of
Ne mbutal to exsanguination.
Also, to an swe r the
question of grow th hypertrophy to the appear ance of
hy percontracted fibers, on e might suppl ement the
turk e y' s die t with s t eroid s .
Author s : We did not s tudy sac rifi ce by Nembutol in JeC tion or the e ffec t of s t e roid s in thi s e xperim ent.
Tr au ma during tran s port and s laug ht e r is alw ays a
c once rn. The r eason s for beli eving tha t the hy per contract ed fib e r s ere not a rtifac tual ar e ex pl aine d in
the t ex t.
Reviewe r IV:
Were the mu sc les r es t ra ined be for e
r emoval from the ca r cass?
Authors : No . They were r emove d a n d the n an at temp t was made to ad ju s t the sam ple to approximate
r est length and r es tra in it with for cep s during
freez ing.
Rev iewer Ill : In regard to quantitation of h yp er contract e d fiber s , how many fie ld s per musc le pe r
bird we r e counted and what was meant b y "a s lig htl y
hi ghe r in cide nc e of these fibers"?
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