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The effect of fiber type on the rate of rigor mortis development in broiler muscles

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The effect of fiber type on the rate of rigor mortis development in broiler muscles
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Sams, Alan Ray, 1960-
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ix, 91 leaves : ill. ; 28 cm.

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Birds ( jstor )
Cattle ( jstor )
Glycogen ( jstor )
Lipids ( jstor )
Meats ( jstor )
Metabolism ( jstor )
Muscles ( jstor )
Poultry ( jstor )
Rigor mortis ( jstor )
Toughness ( jstor )
Broilers (Poultry) ( lcsh )
Dissertations, Academic -- Food Science and Human Nutrition -- UF
Food Science and Human Nutrition thesis Ph. D
Muscles ( lcsh )
Rigor mortis ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1987.
Bibliography:
Includes bibliographical references.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Alan Ray Sams.

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THE EFFECT OF FIBER TYPE ON
THE RATE OF RIGOR MORTIS DEVELOPMENT
IN BROILER MUSCLES






BY






ALAN RAY SAMS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1 987




THE EFFECT OF FIBER TYPE ON
THE RATE OF RIGOR MORTIS DEVELOPMENT
IN BROILER MUSCLES
BY
ALAN RAY SAMS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA


ACKNOWLEDGMENTS
The author would like to express appreciation to his
committee chairman, Dr. D. M. Janky, for his assistance and
guidance throughout the author's graduate program and in
the preparation of this manuscript. Thanks are also
extended to Drs. D. D. Johnson, R. H. Schmidt, R. L. West
and S. A. Woodward for their time in serving on the
advisory committee and suggestions during this study.
A debt of gratitude is expressed to the Poultry
Science Department for funding the author's graduate
program and providing facilities and equipment for
conducting this study. The author thanks the Animal
Science and Dairy Science Departments and the J. H. Miller
Health Center Teaching Labs for the use of their facilities
and equipment. The author wishes to thank Gold Kist
Poultry, Inc. of Live Oak, Florida, for donating the
broilers used in the isotonic tension experiment.
Special thanks are extended to Drs. D. M. Janky, S. A.
Woodward, C. W. Comer and D. A. Comer, M. Dukes, J.
Eastridge and E. Hirchert for their technical assistance
and friendship throughout the completion of this study.
Deepest appreciation goes to the author's wife,
Kimberlee, for her love, friendship, and motivation
throughout his educational career.


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABSTRACT vii
CHAPTERS
I INTRODUCTION 1
II REVIEW OF THE LITERATURE 6
Rigor Mortis Development 6
The Importance of Rigor Mortis Development
to Meat Tenderness 11
The Relationship Between Postmortem
Metabolism, Rigor Mortis Development
and Meat Tenderness 16
Factors Affecting the Rate of Rigor Mortis
Development 19
Muscle Fiber Types 25
III RATE OF RIGOR MORTIS DEVELOPMENT AND
DISTRIBUTION OF FIBER TYPES IN FOUR
BROILER MUSCLES 36
Introduction 36
Materials and Methods 39
Results and Discussion 44
IV BIOCHEMICAL ASPECTS OF RIGOR MORTIS
DEVELOPMENT IN BROILER MUSCLES 52
Introduction 52
Materials and Methods 54
Results and Discussion 59
i i i


V SUMMARY AND CONCLUSIONS 78
APPENDIX REGRESSION EQUATIONS 8l
REFERENCES 82
BIOGRAPHICAL SKETCH 91
IV


LIST OF TABLES
Table Page
3-1 Fiber type distributions (in percent) of
broiler anterior latissimus dorsi (ALD),
sartorius (SART), posterior latissimus dorsi
(PLD) and pectoralis superficialis (PECT)... 45
3-2 Time (hours) required for development of
maximum tension by broiler anterior
latissimus dorsi (ALD), sartorius (SART),
posterior latissimus dorsi (PLD), and
pectoralis superficiales C~PECT) held at
room temperature (24C) or chilled under
simulated commercial conditions (20 minutes,
240C; duration, 4C) 48
3-3 Time (hours) required for development of
maximum tension by broiler anterior
latissimus dorsi (ALD), sartorius (SART),
posterior latissimus dorsi (PLD)~T~ and
pectoralis superficialis (PECT) held at
room temperature (24C) or chilled under
simulated commercial conditions (20 minutes,
24C; duration, 4C) 50
v


LIST OF FIGURES
Figure
Page
3-1
Flow diagram of muscle sample collection
and analysis for the isotonic tension
development experiment
4-1
Flow diagram of muscle sample collection
for the R value experiment
. . .56
4-2
Changes in R value over postmortem time in
the broiler anterior latissimus dorsi (ALD),
sartorius (SART), posterior latissimus dorsi
(PLD), and pectoralis superficialis (PECT)....
,60
4-3
Changes in glycogen concentration over
postmortem time in the broiler anterior
latissimus dorsi (ALD), sartorius (SART),
posterior latissimus dorsi (PLD), and
, ...67
pectoralis superficialis (PECT)
4-4
Changes in lactic acid concentration over
postmortem time in the broiler anterior
latissimus dorsi (ALD), sartorius (SART),
posterior latissimus dorsi (PLD), and
pectoralis superf icialis (PECT)
.... 72
vi


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE EFFECT OF FIBER TYPE ON
THE RATE OF RIGOR MORTIS DEVELOPMENT
IN BROILER MUSCLES
BY
ALAN RAY SAMS
December, 1987
Chairman: D. M. Janky
Major Department: Food Science and Human Nutrition
Two experiments were conducted to characterize the
development of rigor mortis by different fiber types in
broiler muscles. A homogeneous red muscle (anterlor
latissimus dorsi) was used as a model for the behavior of
red fibers while a homogeneous white muscle (pectoralis
superficial is) was used to characterize the behavior of
white fibers. The contribution of the different fiber
types in heterogeneous muscles was evaluated using a
predominantly red muscle (sartorius) and a predominantly
white muscle (posterior latissimus dorsi).
In the first experiment, isotonic tension development
was used to detect the development of rigor mortis while
histological examination indicated the fiber type
distribution in the four test muscles from male broilers.
vi 1


Rigor mortis development in the red, aerobic muscles was
significantly faster (P<0.05) than that in the white,
anaerobic muscles. Commercial-type chilling procedures
significantly (P<0.10) slowed rigor mortis development of
red muscles.
In the second experiment, the four test muscles were
removed from 128 male broilers at 0.33, 0.67, 1, 2, 3, 4, 6
or 8 hours postmortem and analyzed for R value. Glycogen
and lactic acid concentrations were each determined for one
of two additional groups of 16 male broilers that were
processed and sampled similarly to those in the R-value
analysis. R value was directly related to the aerobic
capacity while lactic acid and glycogen concentrations were
directly related to the anaerobic capacity of each of the
four test muscles. The aerobic muscles showed no change in
any of the biochemical parameters after 2 hours postmortem,
the anaerobic muscles showed continued metabolism beyond 2
hours and the pectoralis superficialis had substantial R
value and lactic acid increases up to 8 hours postmortem.
The rigor mortis development times from the tension
development experiment did not coincide with the
biochemical events thought to cause rigor mortis. Also,
continued energy metabolism in the pectoralis superficialis
was evident after maximum tension was achieved. These
results prompted the conclusions that rigor mortis
developed faster in red fibers than in white ones,
v i i i


suggesting
could not
that rigor mortis development in broiler muscles
be extrapolated from bovine or porcine muscles.


CHAPTER I
INTRODUCTION
In 1987, 77$ of the broilers produced in the United
States, almost 12 billion pounds, will be marketed as cut
up or in other further processed forms (Roenigk and
Pederson, 1987). This percentage is expected to continue
to increase to 87%, 13-5 billion pounds, by 1990. In 1990,
only one in every eight broilers will be marketed as a
whole carcass and further processed items will approach 30$
of all broiler sales. With these projections, United
States per capita broiler consumption will surpass that of
beef during 1989. Pork consumption was surpassed in 1985
by that of broilers.
In an effort to reduce the refrigeration space and
time needed for holding the carcasses for a traditional
aging period, many processors are boning or dismembering
carcasses immediately as they exit the chilling tanks, a
process known as chill boning. Consumer response and
published research, however, have indicated that much of
this meat is tough. In the last decade, with the shift in
the mix of poultry products from whole carcasses to cut-up
products, this toughness has become an increasing problem.
1


2
Current industry recommendations are that carcasses be
held for 4 hours between death and boning to maintain
tenderness. This time was determined experimentally as the
time needed for poultry muscle to decline to its ultimate
pH value, an event thought to signal the development of
rigor mortis (Stewart et al., 1984a, b). Four hours are
said to be required to reach the ultimate pH, despite the
fact that broilers, according to carcass conformation and
muscle turgidity, are in rigor mortis at 1 hour
postmortem. The fact that the pH continues to decline
after this 1-hour period indicates that there is some
factor of the relationship between postmortem muscle
metabolism and meat tenderness that current knowledge does
not explain.
The apparent difference in the usefulness of pH as an
indicator of rigor mortis between red meats and poultry
might be the result of the differing fiber type
compositions of the two muscle types. The use of pH has
been based on the theories that (1) the progression of
glycolysis is one of the major steps leading to rigor
mortis; and (2) that pH decline is the result of the
accumulation of lactic acid, a by-product of glycolysis.
These two relationships are probably valid but when one is
used as an indicator of the occurrence of the other,
inconsistencies result. Erroneous conclusions may be


3
reached when one uses the attainment of ultimate pH as an
indicator of the rigor mortis development time in poultry.
One idea that has yet to be investigated in poultry
meat is that the different muscle fiber types comprising a
muscle behave differently. The drastically different
metabolisms of the fiber types suggest that they have very
different capacities for survival in the anoxic conditions
of the postmortem environment. The "white" fibers, having
primarily an anaerobic metabolism, are well suited for the
postmortem anoxia. These fibers would therefore be
expected to proceed with their metabolism at a normal rate,
slowed only by decreasing temperature and substrate
concentration. The "red" fibers, having primarily an
aerobic metabolism, are not well adapted for the anoxic
conditions. Upon death it is expected that these fibers
continue to respire aerobically as long as possible. When
the oxygen is depleted, these fibers rapidly enter rigor
mortis. Thus, upon death, the red muscle fibers develop
rigor mortis first, followed by the white fibers. If a
muscle consisted of a mixture of fiber types, the rate of
rigor mortis development in that muscle would be
intermediate between that of red and white fibers. Each
fiber type present would have a significant effect on the
rate of rigor mortis development in a muscle.
If the two fiber types indeed had different rates of
rigor mortis development, it might explain the toughening


4
observed in chill boned meat. Rigor mortis development by
only one of the fiber types present in a muscle would make
that muscle appear, visually and tactually, in rigor
mortis. This would still leave one fiber type in the
prerigor state, susceptible to the toughening caused by
boning. Only after both fiber types develop rigor mortis
could boning be performed without toughening the meat. It
is conceivable, then, that chill boning meat one hour
postmortem toughens the meat by stimulating the fibers that
are not yet in rigor mortis. This toughness is seen at an
extreme in hot boned meat because none of the fiber types
are in rigor mortis at these early postmortem times.
A third muscle fiber type, called intermediate fibers,
is known to exist in broiler muscles (Ashmore and Doerr,
1971b). These fibers have high oxidative capacities like
the red fibers and also have glycolytic abilities not much
less than that of white fibers (Ashmore, 1974). Thus, the
presence of these fibers would help sustain the postmortem
reactivity of the muscle, whichever fiber type developed
rigor mortis faster.
Thus there exists the possibility that each of the
fiber types present in the broiler pectoralis superficialis
contribute to the rigor development rate observed for the
muscle as a whole. At the time of excision, one of the
fiber types could be in rigor, while the other fiber type
remains prerigor, provided the fiber types develop rigor


o
mortis at the same rate. This would explain why chill
boned broiler pectoralis superficialis is toughened, even
though it appears to be in rigor when deboned. Finally,
the observation of these fiber type effects in two
heterogeneous muscles of different fiber type
distributions, such as the pectoralis superficialis and the
sartorius, would suggest that these effects occur in all
skeletal muscles.
The objectives of this project were to determine
1 ) the relative rates of rigor mortis development by
red and white muscle fibers and their contributions
to the rate of rigor mortis development by the
heterogeneous muscles pectoralis superficialis and
sartorius;
2) the effect of a commercial chilling regimen on the
rate of rigor mortis development by red and white
muscle fibers;
3) the effect of fiber type on the pattern of muscle
ATP depletion, glycogen depletion and lactic acid
production during the development of rigor mortis.


CHAPTER II
REVIEW OF THE LITERATURE
Rigor Mortis Development
Rigor mortis, translated from Latin, is defined as
"the stiffness of death" (Stein, 1975) and has been
observed for centuries upon the death of an animal. Modern
meat scientists now know that this rigidity is caused by
the irreversible interaction of individual sarcomeres
within the body's muscles (Hamm, 1982).
Stryer (1981) reviewed two important changes in
postmortem muscle, contracture and rigor mortis that are
both currently thought to involve the interaction of the
thin and thick filaments of the sarcomere. Each sarcomere
is comprised of filaments oriented parallel to the fiber
and is bounded on both ends by a Z-line which is
perpendicular to the fiber direction. To each Z-line are
connected the thin actin filaments. The primary components
of these thin filaments are two strands of F-actin
(filamentous actin) wrapped around each other forming a
helix. F-actin is actually a polymer of the more basic
actin structure G-actin (globular actin). Attached to the
F-actin helix is the troponin-tropomyosin complex which
functions in the control mechanism of muscular
6


7
contraction. This complex covers the site on F-actin where
interaction with the thick filament occurs. The thick
filaments are composed of many myosin molecules. Each
myosin has a globular "head" and a filamentous "tail." The
myosins of an individual thick filament are connected to
each other by their tails. This arrangement places the
heads in close proximity to the thin filaments.
Innervating this system of filaments is the sarcoplasmic
reticulum (SR). The function of this network of membranous
vessicles is to sequester calcium ions (Ca+^) within its
vessicles. This is accomplished by an adenosine
triphosphate (ATP) driven pumping system located in the
membrane.
In a discussion of the role of energy metabolism in
rigor mortis development, Hamm (1982) reported that in
normal living muscle, ATP is the primary energy source and
is mainly produced by oxidative phosphorylation. When an
animal is killed, the lack of a fresh blood supply to the
muscle cells causes aerobic metabolism to decline. For a
short time, adenosine diphosphate (ADP) is converted to ATP
at the expense of creatine phosphate. The supply of
creatine phosphate is rapidly depleted and then the cell
becomes dependent on anaerobic glycolysis for ATP
production. One product of glycolysis is lactic acid which
accumulates and causes the pH of the muscle cells to
decrease from about 7.0 to about 5.5 several hours after


8
death. When the glycogen supply is depleted, ATP levels
begin to decline as the cell continues to use the energy of
ATP without further ATP production.
Hamm (1982) confirmed that in a living or dead
animal's muscles, as long as sufficient ATP is present, the
SR will continue to sequester Ca+^ ions. This serves to
inhibit the interaction of the thick and thin filaments.
As long as the Ca+^ concentration in the sarcoplasmic fluid
innervating the myofilaments is low (<10-^ M), interaction
of the filaments is prevented. Upon neurostimulation or
upon the inactivation of the SR pumping system, Ca+^ ions
are released into the sarcoplasm and bind to the troponin C
moiety of troponin. This oinding causes the tropomyosin to
move into a groove of the F-actin helix, exposing the site
of interaction of the actin molecule with the myosin
head. The myosin head then binds to the actin unit of the
thin filaments.
Reviewing the biochemistry of the contraction process
in skeletal muscle, Stryer (1981) reported that in the
resting state or early postmortem the thin and thick
filaments are not attached to each other and the myosin
head contains a tightly bound ADP and an inorganic
phosphate (Pj_)- Upon actin activation by the presence of
Ca+2, the myosin interacts with the actin. The release of
the ADP-Pi fcom the myosin causes the myosin head to tilt
toward the center of the sarcomere. This tilting pulls the


9
Z-lines closer together, thereby shortening the sarcomere
and causing contraction. If ATP is present, its binding to
the myosin causes the myosin to dissociate from the
actin. The hydrolysis of this bound ATP to ADP-P^ then
causes the myosin head to tilt back to its original
perpendicular orientation, completing the contraction
cycle. Therefore, ATP is required for the relaxation of a
contracted muscle (Stryer, 1981).
The ATP dependence of the relaxation process is
important to postmortem muscle (Hamm, 1982). Immediately
postmortem, sufficient ATP is present to support relaxation
and therefore the muscle is extensible (Harrington,
1979). As the cell continues to use ATP while ATP
production declines, less ATP is available for
relaxation. The decreasing relaxation is compounded by the
fact that the lower pH and lower ATP levels cause the
inactivation of the Ca+2 sequestering system of the SR.
Calcium ions are then released into the sarcoplasm,
activating more thin filaments (Cornforth et al., 1980;
Whiting, 1980). When insufficient ATP is present to
support relaxation, the overlapping thin and thick
filaments are irreversibly bound to each other. This
causes the muscle to lose its extensibility, thereby
developing rigor mortis. It is important to note that not
all fibers of a muscle will develop rigor at the same
time. Rigor begins to develop when the cellular ATP levels


1 o
decrease to about 1 pmole/g wet tissue at 20)C and is fully
developed at 0.1 ymole/g wet tissue (Honikel et al.,
1981a). The postmortem time when these conditions exist in
a fiber varies with antemortem conditions, fiber location
and postmortem processing practices.
Davies (1966) suggested that rigor mortis development
consisted of two closely related but quite different,
simultaneous processes. Postmortem muscle fibers shorten
or contract as they develop rigor mortis. This is a result
of insufficient ATP being present to prevent the
interactions between actin and myosin to form actomyosin.
The sarcomeres and muscle shorten with irreversible
"contraction" due to the presence of Ca+^ ions from a
leaking SR and/or mitochondria and the scarcity of the
myofibrillar plasticizer, ATP. This shortening, measurable
by the development of tension, causes the muscle to be more
rigid and therefore tougher. The other event proposed by
Davies was the loss of extensibility. Goll et al. (1969)
explained that the loss of extensibility could be equated
with the depletion of ATP. As the thin and thick filaments
lose the ability to freely slide over, each other, due to
increased interaction, muscle extensibility is lost. So
the course of ATP depletion induces first sarcomere
shortening at low ATP levels and then greatly reduced
extensibility in the absence of ATP.


Goll et al. (1969) suggested that some loss of
extensibility must accompany the shortening process because
the thin and thick filaments are becoming increasingly
attached to each other. The research of Huxley and Brown
(1967) and Huxley (1968) showed, however, that these two
processes are indeed distinct. These researchers reported
that in rigor-shortened muscle, only 20% of the myosin
heads of the thick filaments are attached to the thin
filaments. In contrast, nearly 100$ of the myosin heads
are attached to the thin filaments in muscle that has been
depleted of ATP and is, therefore, inextensible. Thus,
rigor-shortened muscle should be five times as extensible
as muscle that has completed its loss of ATP (Goll et al.,
1 969).
The Importance of Rigor Mortis Development
to Meat Tenderness
Meat tenderness is markedly influenced by the
development of rigor mortis. It has been well documented
that the onset of rigor mortis in a muscle drastically
toughens the resulting meat (Lowe, 1948; de Fremery and
Pool, I960; Stewart et al., 1984b). Locker (I960) and
Marsh (1975) both suggested that the extent of sarcomere
shortening (i.e., the degree of interdigitat ion of actin
and myosin filaments) in a muscle was the determining
factor in the extent of toughening resulting from rigor


mortis development. As the Z-lines are drawn closer
together by the interaction of the thick and thin
filaments, the sarcomere becomes more compact and dense
(Marsh et al., 1974). Hamm (1982) stated that shorter
sarcomeres resulting from the development of rigor mortis
could be interpreted as more cross-linkages between the
thin and thick filaments. More of these associations would
cause the muscle structure to be more rigid, increasing
cooked meat toughness. The shortened sarcomeres, being
more rigid, have more mass for teeth to shear than if the
sarcomeres were of normal length. Therefore the meat
produces the sensation of toughness when chewed. In fact,
Voyle (1969) suggested that, based on electron microscopy,
the myosin filaments buckle against (or pierce) the Z-line
upon shortening. It is then proposed that the myosin
filaments could conceivably link through the Z-lines,
forming a network in shortened muscle that would cause
toughening.
The connective tissues are becoming increasingly
recognized as the cause of toughness accompanying the onset
of rigor mortis, despite the widespread acceptance of
myofilament interdigitation. Voyle (1969) suggested the
importance of the muscle's connective and structural
components to meat tenderness. The gap filaments are
filamentous proteins which originate in one end of each
thick filament, extend out of the other end, through the


Z-line, and terminate in the distal end of the adjacent
sarcomere's thick filament (Locker and Leet, 1976). The
strength and elasticity of the gap filaments maintain their
integrity when tensile strain is applied to the muscle,
even to the point when the thin filaments tear out of the
Z-line (Locker, 1985). Because they are susceptible to
endogenous proteases such as calcium activated neutral
protease, the gap filaments or their component protein
connection (Locker, 1985) are degraded upon aging.
Therefore, the weakened gap filaments of postrigor muscle
are of less importance to meat toughness than the
interdigitation of actin and myosin.
Lawrie (1986) reported that when muscle is cooked for
4 hours at 100C (harsh enough treatment to degrade
collagen) while stretched enough to eliminate any overlap
of actin and myosin fibers, the resulting meat had 66$ of
the tensile strength of the unstretched control. In a
later publication reviewing the effects of heat on muscle
proteins, Locker (1985) stated that after an hour at 70C,
the gap filaments denatured, but remained strong and
elastic, capable of stretching when strained. Thus it is
conceivable that the gap filaments have an influence on
cooked meat toughness if the enzymatic degradation of the
muscle is sufficiently arrested.
Collagen has been suggested to be another contributor
to meat toughness (Bouton et al., 1973). As an animal


14
ages, the collagen fibers form crosslinkages with other
collagen molecules (Bailey, 1972). With time, these
collagen polymers become decreasingly heat and water
insoluble, thus forming a network of tough fibers within
the meat. However, since the poultry industry currently
markets broilers at the young age of 7 weeks, collagen is
not a major cause of toughness in broilers (Wangen and
Skala, 1968; Nakamura et al., 1975).
Interest in the rate of rigor mortis development
relates to the prerigor excision of the muscle, called hot
boning if done prior to chilling or chill boning if done
after chilling (Sams and Janky, 1986). Hot boning would
save the producer money in the areas of refrigeration,
space and labor. Seideman and Cross (1982) estimated that
50$ of the energy of beef processing plants could be saved
if hot-boning was implemented. Another advantage of hot-
boning is that prerigor meat has greater functional
properties, such as water holding capacity and emulsifying
capacity (Kramlich et al., 1973; West, 1983). Kramlich
et al. (1973) suggested that this was a result of the
primary emulsifying proteins, actin and myosin, being
easier to extract before rigor mortis. In the prerigor
state, actin and myosin have not substantially associated
into actomyosin, which would make their extraction
difficult. West (1983) stated that prerigor meat had a


1 5
higher pH which was further from the isoelectric point of
the myofibrillar proteins than in postrigor meat.
Consequently, this increases the proteins' charge,
increasing the protein/protein and protein/solvent
interactions responsible for functionality.
There is, however, one main disadvantage to hot-
boning. Several researchers have shown that excising
muscle prior to rigor mortis markedly decreases the
tenderness of the meat (Lowe, 1948; de Fremery and Pool,
I960; Stewart et al., 1984a; Sams and Janky, 1986). One
means by which hot boning increases toughness is by
increasing the muscle's susceptibility to cold shortening
(Locker and Hagyard, 1963). After excision, the muscle's
lack of surrounding tissue for insulation causes it to cool
more rapidly than if left on the carcass. This results in
more shortening and tougher meat. Another way prerigor
excision increases toughness is by removing the muscle from
its attachments. Locker (I960), Herring et al. (1967) and
Gillis and Henrickson (1969) all found that muscles excised
prerigor and permitted to contract freely were less tender
than those muscles restrained during rigor development.
The unattached muscle's unimpeded contraction allows more
shortening, both rigor shortening and cold shortening, and
therefore tougher meat.


16
The Relationship Between Postmortem Metabolism,
Rigor Mortis Development and Meat Tenderness
Red meat scientists have long reported a very good
correlation between muscle pH and the shear force of the
resulting cooked meat (Lundberg et al., 1987). Several
attempts have been made to apply this relationship to
poultry processing, but with only limited success. Stewart
et al. (1984b) and Sams and Janky (1986) both reported
lower, nonsignificant correlations between pH at time of
boning and shear force. Sams and Janky (1986) observed
nonsignificant correlation coefficients, ranging from -0.24
to 0.20 for three postmortem boning times studied. Even
when these three groupings were subdivided into nine
groupings of very narrow pH ranges, the correlations were
still nonsignificant. These authors did, however, report a
significant correlation after pooling all the boning
times. This significance was expected because pooling the
three sets of pH values produced by boning meat at three
different times creates an inherent correlation. Stewart
et al. (1984b) reported a nonsignificant correlation
coefficient of 0.19 after pooling the data from the six
boning times used. This lack of correlation was probably
because, by pooling six boning times instead of three (as
in the previous study), less correlation was caused by the
pooling process. The common factor in these studies was


1 7
the lack of correlation between pH at muscle excision and
cooked meat shear force.
Much literature has been published reporting the
increase in meat toughness and changes (acceleration or
deceleration) in glycolysis resulting from prerigor
excision of muscle (Tarrant, 1977; Stewart et al., 1984a,
b). It therefore seems indisputable that some relationship
exists between pH and cooked meat shear force. Stewart
et al. (1984b) suggested that these two parameters in
themselves were not directly, through cause-and-effect,
related. They were, though, probably indirectly related
through their common association with postmortem
metabolism.
By definition, the development of rigor mortis is
characterized by muscle shortening, loss of extensibility
and development of tension. Using isometric tension
development, Wood and Richards (1974a, b) reported rigor
mortis development times of 3 to 5 hours postmortem for
broiler pectoralis superficialis maintained at 23C.
However, one can observe broilers on the processing line
whose muscles have developed tension as early as 20 to 30
minutes postmortem. Legs and wings have drawn close to the
body and the breast muscles have become turgid. Such
observations prompted researchers to report that broilers
are in rigor mortis by 10 minutes (Shrimpton, I960) or 30


18
minutes (D.L. Fletcher, 1985, personal communication) after
death.
This is contrasted by reports stating that the
ultimate pH in chilled broiler breast meat is not attained
until more than 4 hours postmortem (Stewart et al.,
1984a). This obvious inconsistency is grounds to doubt the
accuracy of ultimate pH as an indicator of rigor mortis and
therefore as a guideline for the time to bone poultry meat
without toughening it. Stewart et al. (1984b) boned breast
meat at 6 postmortem times and observed that as long as the
pH had not reached its ultimate level prior to boning,
objectionable toughening resulted. Research has shown that
breast meat boned 1 hour postmortem, after the carcasses
have been chilled, is tougher than meat boned after 24
hours of aging (Sams and Janky, 1986; Thompson and Janky,
1986). This was despite the fact that the muscles were
visually and tactually in rigor mortis although the
ultimate pH had not been achieved. This is not the case in
the red meat field in which a pH of 5.8 is considered a
level at or below which muscle excision will not result in
objectionable toughness (Bendall, 1975). In this respect,
poultry does not conform to the theories of red meat.
It seems obvious from this evidence and from the
preceding discussions that the traditional view of poultry
meat tenderness as a function of rigor development rate,
indicated by pH decline, is insufficient to explain the


1 9
experimental results being observed with chill boned
poultry meat. Therefore, there must be other factors
influencing the relationship between rigor development rate
and subsequent meat tenderness.
Factors Affecting the Rate of Rigor Mortis Development
The rate of rigor mortis development in a muscle has a
profound influence on the tenderness of the resulting
muscle. Tarrant (1977) suggested that exceptionally fast
or slow rates of rigor mortis development could toughen
beef. De Fremery (1966) and Ma and Addis (1973) have also
reported that increasing the rate of postmortem metabolism
and rigor onset toughens poultry meat. De Fremery and Pool
(1960) showed that treatments producing accelerated ATP
loss and pH decline, indicating faster rigor mortis
development, resulted in increased toughness in broiler
breast meat. Treatments included prarigor muscle excision,
high postmortem environmental temperature, freezing and
thawing of prerigor muscle, exhaustive electrical
stimulation, mechanical beating and electron irradiation.
One of the most important factors in the rate of rigor
mortis development is the postmortem environmental
temperature of the muscle. As the ambient temperature of
postmortem muscle decreases from 37C (at death) to about
10C, there is a continuous decrease in the rate of ATP
turnover through the normal influence of temperature on


20
biochemical reactions (Jolley et al., 1981). If the
temperature is further reduced to 0C, energy metabolism is
accelerated through a process called cold shortening of
sarcomeres. First observed in red meats (Locker and
Hagyard, 1963; Marsh and Leet, 1966), this phenomenon has
also been reported in avian muscle (Smith et al., 1969;
Whiting and Richards, 1975; Wood and Richards, 1974a). It
would be expected that since cold shortening accelerates
postmortem metabolism and the postmortem metabolic rate
determines the rate of rigor onset, that cold shortening
would accelerate the rate of rigor mortis development.
Because in commercial poultry processing plants, prerigor
poultry is rapidly chilled to temperatures near freezing,
cold shortening is a possibility.
The mechanism of cold shortening is fairly well
understood although there still are some obscure points.
Cold shortening can be explained by changes at early-
postmortem low temperatures in the lipoprotein systems of
membranes. Hamm (1982) postulated that low temperatures
inactivate the Ca+2 sequestering ability of the SR and/or
increase the permeability of the mitochondria to Ca+2.
This causes Ca+2 ions to "leak" into the myofibrillar
spaces. The increased concentration of Ca+2 in the
sarcoplasm coupled with the considerable amount of ATP
still present at this early postmortem time results in


21
muscular contracture prior to rigor mortis onset (Davey and
Gilbert, 1974; Cornforth et al., 1980; Locker, 1985).
Marsh and Leet (1966) reported that the shortening/
toughening relationship was not linear. The increases in
toughness resulting from decreasing sarcomere length were
small at low degrees of shortening but increased rapidly
thereafter. At 40$ shortening the meat was 4 times the
toughness of the unshortened control. Above 40$
shortening, sarcomere length decreases actually decreased
toughness by disrupting cellular structure.
Despite the fact that prerigor excision toughens meat,
there is some debate on whether it does so by actually
accelerating the onset of rigor mortis. De Fremery and
Pool (I960) and Peterson and Lilyblade (1979) showed that
prerigor muscle excision was accompanied by an accelerated
rate of ATP depletion and pH decline, leading to a faster
onset of rigor mortis. These authors thought that the
toughness of the resulting meat was caused by the
accelerated postmortem metabolism. Their ideas were
supported by Tarrant (1977) who, working with beef,
reported that hot-boning increased the rate of glycolysis
and toughened the meat.
Conversely, Stewart et al. (1984a) reported that
prerigor excision actually slowed the rate of pH decline
which indicated a slower rigor development rate. These
authors still reported a toughening effect with prerigor


22
excision, probably as a result of increased sarcomere
shortening. They proposed that the removal of the muscle's
attachments allows the muscle to contract unimpeded,
temporarily reducing the need for ATP and thus reducing the
glycolysis needed to produce it.
The rate of rigor mortis development is also
influenced by antemortem electrical stunning. This
widespread processing technique is used to reduce carcass
blood retention and as a means of immobilization to improve
the cutting efficiency of the killing machine (Kuenzel and
Walther, 1978; Kuenzel et al., 1978). Ma and Addis (1973)
reported that pectoralis superficialis from stunned turkeys
required more than twice as long to develop rigor mortis as
that from freely struggling controls. Because stunning
eliminates much of the death struggle, it slows postmortem
ATP depletion and therefore slows rigor development
(de Fremery, 1966).
Another consideration in rigor mortis development rate
is the specie involved. In general, the larger the
species, the slower will be its rigor mortis development
rate (de Fremery and Pool, I960). Considering mammals,
rabbits develop rigor mortis at 1.5 to 4 hours postmortem
(Bendall, 1951) while the whale requires 14 to 50 hours
(Marsh, 1952), beef 10 hours (Marsh, 1954) and pork 6 to 9
hours (Busch et al.,
1972). The broiler is slightly slower


23
than the rabbit, needing 2 to 5 hours (de Fremery and Pool,
I960; Whiting and Ricnards, 1975).
It is interesting to note that, as these species get
larger and their corresponding rigor development rates get
slower, there is a general trend for their muscles to have
increasingly red fiber type distributions (George and
Berger, 1966). This comparison across species depends, of
course, on the muscles being compared as the fiber type
composition of an animal varies between its muscles (George
and Berger, 1966; Cassens and Cooper, 1971).
Rigor mortis development has been measured in many
ways, yielding varying results. Busch et al. (1972)
reported that isotonic and isometric tension development by
prerigor muscles commenced and maximized simultaneously.
Direct measurements of loss of extensibility or increased
shortening, by isometric tension, isotonic tension or
tactile evaluation, are complex and time consuming.
Therefore, easier, indirect methods such as pH decline or
ATP depletion have been used as estimates. Very little
research has been done using either direct or indirect
methods, on the development of rigor mortis in broilers
processed in a commercial-type manner (i.e., electrical
stunning and exsanguination). Due to the considerable
effect of antemortem stunning on the rigor mortis
development rate, it would not be prudent to use data from


24
studies on unstunned birds to explain the behavior of
muscles from today's stunned broilers.
Immediately postmortem, there is a period in which no
shortening or extensibility loss occurs called the delay
phase (Schmidt et al., 1968). After this comes the onset
phase in which the muscle shortens and loses
extensibility. After the maximum isometric tension is
reached, a gradual decline occurs, signifying the
resolution phase of rigor mortis.
The upward sloping of the onset phase of a plot of
isometric tension-vs-postmortem time illustrates the
statement by Hamm (1982) that all fibers in a muscle do not
develop rigor mortis at the same time. The ATP depletion
rate, and therefore the rate of rigor development, for an
individual fiber depends mostly on that fiber's location
and type. The superficial fibers would be expected to cool
faster and therefore have slower rates of metabolism and
rigor development simply due to the effect of cooling on
biochemical reactions. Also, because the different fiber
types have drastically different capacities for anaerobic
metabolism (George and Berger, 1966; Cassens and Cooper,
1971), their potential for survival in the postmortem
period would also be expected to be different. In the plot
of isometric tension-vs-postmortera time, the differences in
maximum tension developed between muscles are mainly due to
the fiber type compositions of the muscles with


25
predominantly red muscles developing more tension than
predominantly white ones (Busch et al., 1972; Whiting and
Richards, 1975). With rigor development so dependent on
the fiber types of the muscle involved, it seems necessary
to study rigor development in the individual fiber types
instead of muscles having simply a majority of one fiber
type or the other.
Muscle Fiber Types
The existence of different kinds of muscles, based on
their color, is commonly accepted. An example would be the
common reference to light and dark poultry meat.
Lorenzini, in 1678, is credited with being the first to
describe the difference in muscles due to their color
(Cassens and Cooper, 1971). By the time of Ranvier in
1874, these muscles were also found to have different
contractile properties (Cassens and Cooper, 1971). He
associated redness with quickness of contraction and
whiteness with slowness. Classifying muscles as red or
white based on gross examination leads to erroneous
conclusions because this classification neglects the more
important structural and biochemical differences between
the muscle fibers (George and Berger, 1966). Therefore it
should be considered that the color of a muscle is due to
its degree of heterogeneity and is a reflection of the


26
characteri
;ics of the individual fibers comprising that
muscle (Ce
>ens and Cooper, 1971).
Sever
. fiber classification schemes have been used to
describe t
; various types observed in research. These
schemes he
j been based on the presence or absence of
various er
'tnes or on the fiber's contractile properties.
This has ]
1 to confusion in the literature and difficulty
in compari
l studies. Needham (1926) reported on the
histochemi
il and contractile properties of the two fiber
types knov.
at the time, as red and white. Ogata (1958)
and Ogata
id Mori (1964) observed a third fiber type that
was intern
iiate between red and white in oxidative
capacity.
Itein and Padykula (1962) demonstrated three
fiber type
(A, B, and C) based on the level of succinate
dehydroger
e (SDH) in the fiber. Another histochemical
classifica
on was that fibers that were rich in
dehydrogen
es and poor in phosphorylases were called type
I while th
e with the opposite enzyme compliment were
called typ
II (Dubowitz and Pearse, 1960a, b). This
system als
accounted for a third fiber type that was
intermedia
in its enzyme supply between these two
extremes.
amaha et al. (1970) identified three types (a,
6 and afJ)
cording to their myosin-ATPase activity. The
underlying
law in these systems was that they failed to
account fc
both the biochemical and contractile
difference
of fiber types in a single nomenclature system.


27
Ashmore and Doerr (1971a) proposed a classification
system in which a fiber's contractile and histochemical
characteristics were both represented. This system
combined the red (R) and white (W) typing based on
metabolism using the SDH activity, with fast-twitch (a) and
slow-tonic (0) based on contractile properties using the
myosin-ATPase activity. Thus, they proposed aW to be the
white, fast-twitch, anaerobic fibers and 0R to be the red,
slow-tonic, aerobic fibers. The aR designation was given
to the "intermediate" fibers denoting their intermediate
properties. Because this system was designed for studying
chickens and because previous systems were found to be
limited in their applicability across specie lines, Ashmore
and Doerr (1971b) studied the fibers of chicken, mouse,
bovine and porcine muscle and found the aW, 3R, aR system
to be valid. Since these studies, the Ashmore and Doerr
(1971a) nomenclature system has become the system of choice
for most muscle fiber type researchers.
Regardless of the nomenclature system used, the
differences between the three main fiber types can be
segregated into three areas: structural, biochemical, and
physiological. Structurally, the aW fibers are large in
diameter, have a well developed sarcoplasmic reticulum and
are poorly supplied with capillaries (Gassens and Cooper,
1971; Ashmore and Addis, 1972). The 0R fibers are just the
opposite while the aR fibers are intermediate in these


28
characteristics. These differences are logical since the
aW fibers do not depend on oxygen for their metabolism.
Thus, blood supply and oxygen diffusion are minimized in
the aW fibers by the lack of capillaries and large
diameter, respectively. The opposite is the case for the
BR fibers. Also, mitochondria, the primary site of
oxidative metabolism, are scarce in the aW fibers but are
abundant in the BR fibers. Because the aW fibers are fast
contracting, they would be expected to, and indeed do, have
a well developed system for rapidly dispersing large
quantities of the agent initiating contraction, Ca+2
ions. This is why the aW fibers have an extensive SR while
the BR do not.
Biochemically, the aW fibers are rich in glycolytic
enzymes, glycogen, ATP and creatine phosphate while being
poor in myoglobin, oxidative enzymes and lipids with BR
fibers being the opposite (George and Berger, 1966; Cassens
and Cooper, 1971; Ashmore and Addis, 1972). The
physiological characteristics of the fiber types are a
result of the structural and biochemical features. Muscles
requiring rapid but short-lived activity have a high
proportion of aW fibers. These fibers are suited for this
kind of activity. For a fiber to contract rapidly, it
would need to have a means of quickly dispersing Ca+^ ions
and a large supply of readily available energy, hence the
developed SR and the large supply of ATP, creatine


29
phosphate and glycogen. As would be expected, however, in
a fiber that depends on glycolysis for rapid contraction,
the lactic acid resulting from extensive glycolysis
severely limits the length of the sustainable
contraction. Likewise, the muscles requiring sustained but
not necessarily fast contraction would have a predominance
of BR fibers. These fibers are more suited for a gradual
tension development that can be maintained. With little
lactic acid production and plentiful lipid, the rich source
of energy for ATP production, the aerobic metabolism can be
continued at length. The slow release of Ca ions from
the less extensive (than the aW) SR in conjunction with the
aerobic metabolism impart the BR fibers with their slow-
tonic contractile properties.
In view of the vast metabolic and functional
differences between fiber types, it would be expected that
a muscle's fiber types would also have dissimilar
postmortem behavior. An example of one such distinction is
the difference in cold shortening between red and white
muscles. Bendall (1975) reported that red muscles are more
susceptible to cold shortening. The red muscles have a
predominance of red fibers which are richer in mitochondria
than white fibers. Because at least some of the Ca+^ ion
release causing cold shortening is from the mitochondria
(Hamm, 1982), the red fibers, having more mitochondria,
would be more prone to cold-shortening. Furthermore,


30
Lawrie (1986) postulated that whatever Ca + 2 ions were
released would be less effectively removed from the
sarcoplasm in a fiber whose SR was less extensively
developed. Fawcett and Revel (1961) reported that the red
fiber SR was less developed than the white fiber SR. Also,
Newbold (1980) showed that the SR of the red fiber
accumulated Ca + 2 ions less efficiently than that of the
white fibers.
An alternate theory to explain the greater cold
shortening in red fibers results from the work of Graeser
(1977) and Newbold (1979). They reported that the
involvement of the Ca+2 release from the mitochondria was
less significant to cold shortening than the exchanges of
Ca+2 between the sarcoplasm and the SR. Newbold and Tume
(1977), however, reported that this cold induced release of
Ca+2 from the SR was suppressed by inorganic phosphate.
Newbold (1979) later observed that white fibers had more
inorganic phosphate than red fibers and therefore were less
susceptible to cold shortening.
Although there is clear evidence for the involvement
of both theories, more research in this area is needed to
determine the exact mechanism of cold shortening and which
fibers (or muscles) it affects. This need becomes apparent
when one considers that Bendall (1975) has observed severe
cold shortening in porcine longissimus dorsi and Wood and


31
Richards (1974b) reported cold shortening in broiler
pectoralis superficialis, both predominantly white muscles.
Various facets of rigor mortis' development are
expected to differ between red and white fibers. Working
with whole or partial muscles, several researchers have
noted such differences. Several studies have investigated
these differences in relation to stress susceptibility in
pigs, a condition causing pale, soft exudative (PSE)
pork. Cooper et al. (1969) and Sair et al. (1972) found
stress susceptibility to be closely related to an increased
proportion of intermediate fibers. Using porcine vastus
lateralis as a red muscle and longissimus dorsi as a white
muscle, Schmidt et al. (1970) found that the white muscle
had a longer delay phase and a slower rate of tension
development in the onset phase of rigor mortis than the red
fibers. This would seem logical because the white fibers
would not be as adversely affected by the postmortem
anaerobic conditions as the red fibers.
Conversely, Briskey et al. (1952) and Beecher et al.
(1965) reported that porcine red fibers had a longer delay
phase of rigor mortis than white ones. The red fibers
could metabolize for a short time after death, depleting
the oxygen and creatine phosphate, prior to converting to
glycolysis. Furthermore, these red fibers would be less
efficient at glycolysis and therefore would not deplete


32
their glycogen as quickly as the white fibers. Beecher
et al. ( 1 965) also observed lower initial glycogen levels
and faster postmortem glycolysis in white fibers than in
red fibers. These findings were incompatible with the
rigor development results of the same study.
Research on the relationship between fiber type and
rigor mortis development in avian muscle is very limited.
Whiting and Richards (1975) reported faster rigor mortis
development in the broiler pectoralis superficialis (a
predominantly white muscle) than in the biceps femoris (a
predominantly red muscle).
Little research has been done relating a muscle's
fiber type distribution to the subsequent meat quality. In
the research that has been reported, the primary area of
concern is the role of fiber types in the development of
PSE pork. Hegarty et al. (1963) and Didley et al. (1970)
both reported increased toughness in PSE pork. The
influence of fiber type in this relationship may be two
fold. First, because PSE pork has more intermediate fibers
than normal pork (Cooper et al., 1969; Sair et al., 1972)
it is logical that PSE pork was found to have a larger
average fiber diameter and a greater glycolytic capacity
(Didley et al., 1970; Sair et al., 1972). The larger fiber
diameter would be expected to contribute to the toughness
of PSE pork as Herring et al. (1965) and Seideman and
Crouse (1986) both reported that increased fiber diameter


33
caused toughness. Also, the greater glycolytic capacity of
muscles of stress-susceptible pigs could cause toughening
because Essn-Gustavsson and Fjelkner-Modig (1985) reported
that an elevation in this capacity resulted in tough pork.
In order to study the effects of the individual fiber
types on the overall properties of heterogeneous muscles
such as the broiler pectoralis superficialis and sartorius,
it is necessary to study the isolated typed fibers of those
muscles. This must be done using a model system because
determining a fiber's type is a process that destroys the
fiber's functional ability. Fedde (1972) reported a model
system in which the contractile properties of two
homogeneous muscles were used as indicators of the behavior
of their respective fiber types in heterogeneous muscles.
The anterior latissimus dorsi was used as the homogeneous
red muscle and the posterior latissimus dorsi was used as
the homogeneous white muscle. The homogeneity of these
muscles was previously confirmed by Ginsborg (I960) and
Hess (1961). However, since Fedde's work nearly fifteen
years ago, many changes have occurred in the commercial
broiler. Genetic selection practices may have
inadvertently altered the fiber type composition of these
model muscles. This would invalidate the use of these
muscles as models. Ashmore et al. (1972) and Ashmore
(1974) reported that the common genetic selection practices
used in livestock production to maximize growth rate and


34
meat yield achieved these goals by increasing the
proportion of intermediate and white fibers.
The system of Fedde (1972) was designed to study
living muscles by disconnecting the latissimus dorsi
muscles from their origins while not severing the nerve or
blood supplies. This same system could be used to study
rigor mortis development in postmortem muscle by
disconnecting the blood flow to the muscle completely.
Since rigor mortis is characterized by the development of
isometric tension, directly measuring this development
would give a more accurate indication of rigor than making
indirect measurements such as pH.
In order to relate the rigor development data from the
homogeneous muscles to the tenderness of meat harvested
early postmortem, it is necessary to know the fiber type
distribution and tension development rates for the muscles
used as meat. In the broiler, the principle muscle of meat
interest is the pectoralis superficialis. Furthermore,
this was the muscle used in the chill boning experiments
which resulted in the previously discussed inconsistencies
in the relationship between muscle pH, rigor mortis
development and meat tenderness (Sams and Janky, 1986;
Thompson and Janky, 1987). This muscle, although primarily
comprised of white fibers, has been found to be
heterogeneous (Chandra-Bose et al., 1964; Ashmore and
Doerr, 1971b).


35
Past and current research has not considered the role
of fiber types in poultry meat quality. As with PSE pork,
fiber types could conceivably have an important effect.
The more rapid rate of rigor mortis development in poultry
than in beef or pork (de Fremery and Pool, I960) suggests
that avian muscle behavior should not be considered as
simply an extrapolation of that of bovine and porcine
muscles. The commercial implementation of such toughening
procedures as hot and chill boning of poultry reinforces
the need for a complete understanding of all factors
affecting meat tenderness.


CHAPTER III
RATE OF RIGOR MORTIS DEVELOPMENT AND DISTRIBUTION
OF FIBER TYPES IN FOUR BROILER MUSCLES
Introduction
There is conflicting evidence regarding the rates of
rigor mortis development in red and white muscle fibers.
Working with porcine muscles, Briskey et al. (1962) and
Beecher et al. (1965) reported that red fibers developed
rigor mortis faster than white fibers. Conversely, Schmidt
et al. (1970) observed that white porcine fibers developed
rigor mortis sooner than red fibers. The conflict might
have resulted in that these researchers selected strips
from muscles of "predominantly" one fiber type on the basis
of muscle color alone. Therefore, the actual fiber type
distribution of the test muscles was not known.
Furthermore, the test muscles used in the conflicting
studies were not the same. Briskey et al. (1962) and
Beecher et al. (1965) both used samples from the light and
dark portions of the semitendinosus while Schmidt et al.
(1970) used the vastus lateralis and the longissimus.
Variations in the proportions of the fibers present should
alter the rate of rigor mortis development in the muscle.
36


37
Literature on the rate of rigor mortis development in
the individual fiber types of avian muscle is very
limited. Using muscle strips, Whiting and Richards (1975)
reported that both the "white" muscle (pectoralis
superficialis) and the "red" muscle (biceps femoris) began
developed rigor mortis shortly after death. The white
muscle required 3 hours while the red muscle required 5
hours to develop maximum tension.
The use of strips from heterogeneous muscles for
determining rigor mortis development does not yield results
that are representative of the true rates of rigor mortis
development of individual fiber types. Each test muscle is
a mixture of fiber types in varying proportions and has a
rigor mortis development rate that is a composite of all
the fiber types present. A more accurate estimation of
fiber type rigor mortis development rates would involve
comparison of two homogeneous muscles, one entirely
composed of red fibers and one entirely composed of white
fibers. Such a comparison would determine the true fiber
type rigor development rates and the difference between
them would most probably be larger than when comparing
fiber rigor mortis development rates in heterogeneous
muscles. It would still be necessary to know the rigor
development rate of the heterogeneous muscles used for


38
meat. Thus fiber type homogeneous muscles would be used
simply as indicators of the behavior of respective fiber
types in heterogeneous muscles.
Fedde (1972) reported the use of a model system in
which the contractile properties of the white fiber
homogeneous muscle, posterior latissimus dorsi, and the red
fiber homogeneous muscle, anterior latissimus dorsi, were
used as fiber type indicators. This system was designed to
study living muscles by disconnecting the muscles from
their origins while not severing their nerve or blood
supply. This same model system could be used to study
rigor development in fiber types of postrigor muscles by
discontinuing the blood flow to the muscle or by excising
the muscle completely. Because rigor mortis is, by
definition, the development of tension, directly measuring
this development would provide an accurate indication of
rigor mortis.
Postmortem temperature is also important to a muscle's
and, presumably, an individual fiber's rigor mortis
development rate. Wood and Richards (1974b) observed
considerable differences in time to maximum tension due to
muscle temperature with broiler pectoralis superficialis
(3 minutes at 0C and 277 minutes at 23C). Because
broiler carcasses are currently chilled to temperatures
below 4C within 1 hour of death, low temperature has ample
time to exert its influence on rigor mortis development.


39
It would appear that research on rigor mortis development
rates should consider the effect of temperature; however,
published studies on fiber or muscle type rigor mortis
development rates were all performed at temperatures above
20C. No literature is available regarding the nature of
rigor mortis development in the fiber types of the
commercially processed broiler tissues. This information
is essential to the interpretation of muscle behavior in
terms of the rigor mortis development of individual fiber
types and muscles.
The objectives of this experiment were to determine
the rate of isotonic tension development and the rate of
rigor mortis development in the broiler anterior and
posterior latissimus dorsi, pectoralis superficialis and
sartorius; the effect of cold temperatures similar to those
encountered with commercial-type chilling on the rates of
rigor mortis development in these same four muscles; and
the actual fiber type distribution of these same four
muscles.
Materials and Methods
Broiler Selection and Processing
On 4 separate occasions (4 trials), a uniform
population of 10, seven-week-old male broilers (variation
in body weight: < 250 g or 10%) were obtained from a


40
commercial broiler processing plant and transported to the
University of Florida Poultry Science Department where they
were housed for up to 7 days in litter-covered floor pens
and fed a commercial-type, corn-soy finishing diet.
Following a 12-hour feed-withdrawal periods, birds were
individually processed by electrical stunning and
exsanguination.
The anterior and posterior latissimus dorsi, the
sartorius and a pectoralis superficialis strip (10 x 50 x
10 mm), located 2.54 cm from the anterior end of the
muscle, were removed from the right side of each bird
immediately after the completion of a 90-second bleeding
period. The pectoralis strip was cut parallel to the
direction of the muscle fibers. The location of pectoralis
sampling was chosen to approximate the site of normal shear
force sampling. Care was taken to prevent stretching of
the muscles and strip during excision and preparation. All
muscle samples were dissected and attached to the
contraction apparatus within 20 minutes postmortem.
Isotonic tension development patterns of all four
samples were obtained concurrently, immediately after
excision using four channels and transducers on an E&M 6-
channel Physiograph (Narco Bio-Systems, Inc., Houston,
Texas), using a combined method from Busch et al. (1972)
and Wood and Richards (1974b). One end of each muscle
sample was placed in a clamp attached to a glass rod which


41
was fixed approximately 5 cm off the bottom of a
polycarbonate cylinder filled with a phosphate buffer (pH
7.0, 0.15 M). This buffer was recommended over a Tris-
acetate buffer and distilled water by Wood and Richards
(1974b). The other end of each muscle sample was placed in
a clamp which was attached to an isotonic transducer by
means of surgical cotton thread. Tension was maintained on
the muscle sample throughout the tension development using
a 20 g counterweight. Time to maximum shortening was
determined from graphs obtained for each muscle sample.
For each group of 10 birds, two birds were
individually killed and analyzed on each of five
consecutive days. On each day, one bird's samples were
analyzed with the buffer maintained at 24C. The phosphate
buffer for the samples dissected from the second bird
analyzed each day was maintained at 24C for 20 minutes,
cooled, and maintained at 4C for the duration of tension
development to simulate commercial broiler chilling
procedures (Figure 3-1). Buffer temperature changes and
maintenance were achieved by using a closed-system heat
exchange unit immersed in a constant temperature water bath
adjusted to the appropriate temperature. Buffer cooling
rate was approximately 1C per minute.
This design resulted in four trials of 10 birds each
in two simultaneous and overlapping completely randomized
designs. Two chilling treatments were compared in one


42
10 broilers (in each of 4
replications)
feed withdrawn (12 hours)
stun(Cervin model FS, Minneapolis,
(90 seconds) bleed Minnesota, setting 4)
dissection
attachment
^ (15 min.)
! o
room temperature (24 C)
(5 min)
/ \
5 broilers
J
rigor mortis development
at 24C
5 broilers
chilled to 4 C within
one hour postmortem
rigor mortis development
at <4C
Figure 3-1 Flow diagram of muscle sample collection and
analysis for the isotonic tension development
experiment.


43
design while four muscles were compared in the other
design. Data were subjected to analysis of variance and
differences between means within temperature treatment were
tested for significance with Duncans Multiple Range Test,
while differences between means within muscle were tested
for significance with the independent t-test. All
statistical analyses were performed using the computer
programs in the Statistical Analysis System (SAS Institute,
Inc., 1985).
Fiber Typing
From the first trial of 10 birds, the corresponding
muscles or strips from the left side of each bird were tied
to wooden splints at their original lengths, powdered with
talcum, excised, wrapped in aluminum foil and frozen in
liquid nitrogen. Blocks of muscle (1 cm^) were taken from
each muscle sample and mounted for sectioning on a
Damon/IEC Cryostat (International Equipment Company,
Needham, Massachusetts) at -20C. Sections (12 pm) from
each block were cut, mounted on glass slides, and stained
for acid-stable myosin adenosine triphosphatase (ATPase)
and reduced nicotinamide adenine dinucleotide (NADH)
tetrazolium reductase enzymes with the combination method
of Solomon and Dunn (1986) using the following
modifications for avian muscle: sections were acid-
preincubated at pH 4.15, stained for the reductase enzyme
and then stained for the ATPase enzyme.


44
Red, intermediate, and white fibers were identified by
observing stained sections under an American Optical
microscope (model 1086; Buffalo, New York). Fiber type
composition of each muscle was determined by counting the
number of fibers of each type in ten fiber bundles from
sections of each muscle sample. Fiber type percentage
distributions were calculated and averaged for each muscle
from all carcasses.
Results and Discussion
Fiber Type Distribution
Fedde (1972) reported that the anterior latissimus
dorsi was homogeneously red and the posterior latissimus
dorsi was homogeneously white in their muscle fiber type
distributions. The results of the present experiment
indicated that although the anterior latissimus dorsi was
essentially comprised of only red fibers, the posterior
latissimus dorsi contained a substantial amount of
intermediate fibers (Table 3~1) This deviation from the
results of Fedde (1972) may have been caused by 15 years of
genetic selection or by the use of different strains of
bird. Fowler et al. (1980) reported that genetic selection
for rapid growth in Japanese quail caused a change in the
distribution of fiber types in the muscles of the birds.
Also, Aberle et al. (1983) observed that the distribution
of fiber types in the muscles of broiler-type birds was


45
Table 3-1. Fiber type distributions (in percent) of
broiler anterior latissimus dorsi (ALD),
sartorius (SART), posterior latissimus dorsi
(PLD) and pectoralis superficialis (PECT).
Muscle
Fiber types
Red
Intermediate
White
ALD
>99
<1
0
SART
32
53
1 5
PLD
2
1 7
81
PECT
0
<1
>99


46
different from that in layer-type birds. Fedde (1972) did
not specify the strain of bird used, so he could have used
layer-type birds whereas broiler-type birds were used in
the present experiment.
For maintenance of the model system for studying rigor
mortis development, the pectoralis superficialis proved to
be essentially comprised of only white fibers (Table
3-1). The differences between these results and earlier
reports of pectoralis superficialis muscle fiber type
heterogeneity (Chandra-Bose et al., 1964; Ashmore and
Doerr, 1971b) might have been caused by the same genetic
selection and strain effects previously discussed for the
posterior latissimus dorsi. The homogeneity of the
pectoralis superficialis allowed it to model the behavior
of a white fiber while the anterior latissimus dorsi
modeled the behavior of a red fiber.
The broad distribution of fiber types observed in the
sartorius (Table 3~1 ) allowed this muscle to indicate the
behavior of a heterogeneous muscle that was predominantly
comprised of red fibers. Conversely, the posterior
latissimus dorsi would have exemplified the behavior of a
heterogeneous muscle that was predominantly comprised of
white fibers.
The four muscles tested represented a range of
possible fiber type distributions, from the entirely red
anterior latissimus dorsi, through the two heterogeneous


47
muscles of opposite predominances, to the entirely white
pectoralis superficialis (Table 3~1) Because different
fiber types have different metabolisms (George and Berger,
1966; Ashmore and Doerr, 1971b; Cassens and Cooper, 1971)
muscles that had different compliments of these fiber types
were also expected to have different metabolisms.
Isotonic Tension Development
Within each temperature level, the time required to
develop maximum shortening (i.e., to develop rigor mortis)
was significantly shorter for the predominantly red,
aerobic muscles than for the predominantly white, anaerobic
muscles (Table 3_2). These results agreed with those of
Schmidt et al. (1970) who studied porcine vastus lateralus
as a predominantly red muscle and porcine longissimus dorsi
as a predominantly white muscle. White fibers and muscles
predominating in white fibers have been reported to have
greater glycogen supplies and glycolytic capacities than
red fibers and muscles predominating in red fibers (George
and Berger, 1966; Cassens and Cooper, 1971). The more
anaerobic white muscles would, therefore, be able to
continue producing sufficient adenosine triphosphate (ATP)
to sustain themselves in the absence of bloodflow and
oxygen. Conversely, once the oxygen supply was depleted,
the lack of adequate glycolytic potential in the red fibers
would leave them with decreased means of ATP production,
causing them to enter rigor mortis.


48
Table 3~2. Time (hours) required for development of
maximum tension by broiler anterior latissimus
dorsi (ALD), sartorius (SART), posterior
latissimus dorsi (PLD), and pectoralis
superficialis (PECT) held at room temperature
(24C) or chilled under simulated commercial
conditions (20 minutes, 24C; duration, 4C).
Muscle
Temperature
treatment
ALD
SART
PLD
PECT
Room temperature
3.52b
3 55b
5.31a
5.43a
Chilled
4.1 8b
4.65ab
5.67ab
6.08a
a,b Means within a temperature treatment with different
superscripts are significantly different (p<0.05).


49
Simulated commercial-type chilling significantly
slowed rigor mortis development in red muscles but not in
white muscles (Table 3~3) This slowed rate of rigor
mortis development was expected due to the normal slowing
effect that temperature reduction has on biochemical
reactions (Hamm, 1982). These results were consistent with
the findings of Jolley et al. (1981), who observed that
bovine muscle had slower postmortem metabolism, as
indicated by a slower pH decline, at 5C than at 30C.
Hamm (1982) interpreted this slower metabolism as a slower
rate of ATP depletion and therefore a slowed rate of rigor
mortis development.
Cold shortening and its accompanying acceleration of
postmortem metabolism was probably not an important factor
because the muscle temperature was maintained at 4C.
Honikel et al. (1981b) reported that no cold shortening
occurred in bovine muscles held at or above 4C.
The fact that rigor mortis development was slowed more
in red muscles than in white muscles indicated that aerobic
metabolism was affected more than anaerobic metabolism by
the temperature reduction. An explanation for this
observation might involve differences in cellular
organization between red and white muscle cells. Aerobic
metabolism occurs partially in the cytoplasm and partially
in the mitochondrial matrix (Stryer, 1981). However,


50
Table 3_3. Time (hours) required for development of
maximum tension by broiler anterior latissimus
dorsi (ALD), sartorius (SART), posterior
latissimus dorsi (PLOT, and pectoralis
superficialis (PECT) held at room temperature
(24C) or chilled under simulated commercial
conditions (20 minutes, 24C; duration, 4C).
Muscle
Temperature
treatment
ALD
SART
PLD
PECT
Room temperature
3- 52b
3.55b
5.31a
5.43a
Chilled
4.1 8a
4.65a
5.67a
6.08a
a,b Means within a muscle with different superscripts are
significantly different (p<0.10).


51
anaerobic metabolism occurs only in the cytoplasm (Stryer,
1981). Thus, aerobic metabolites must cross the
mitochondrial membranes as well as overcome diffusion
limitations in order to react. This is contrasted by the
anaerobic metabolites which are only diffusion limited
within the cytoplasm. Stryer (1981) furthermore reported
that low temperatures decreased the fluidity of biological
membranes by partially solidifying their lipids. This
decreased fluidity could decrease permeability, thereby
slowing aerobic metabolism without affecting the rate of
anaerobic metabolism. Further evidence for low temperature
membrane changes was provided by Hamm (1982) who reported
that disrupting the lipoprotein systems of membranes at
temperatures near 0C increased the permeability of
membranes. If the temperature was low enough to allow some
membrane lipid solidification but not so low as to disrupt
the actual membrane structure, the permeability of a
membrane would decrease. Honikel et al. (1981b) and Jolley
et al. (1981) reported that 4C was the temperature below
which postmortem metabolism was drastically accelerated and
cold shortening of sarcomeres was evident. It was possible
that while 4C was not low enough to increase membrane
permeability by membrane disruption, it was low enough to
decrease membrane permeability by lipid solidification.


CHAPTER IV
BIOCHEMICAL ASPECTS OF RIGOR MORTIS DEVELOPMENT
IN BROILER MUSCLES
Introduction
Lowe (1948), de Fremery and Pool (I960), and many
others reported that prerigor muscle excision caused meat
toughness, as measured by increased shear force. Red meat
scientists have reported very good correlations between
muscle pH at time of boning and shear force, and
successfully used pH as an indicator of the development of
rigor mortis and subsequent meat tenderness (Hamm, 1982;
Lundberg et al., 1987). Stewart et al. (1984b), using the
spear probe method, and Sams and Janky (1986), using the
iodoacetate method of pH determination, both reported
nonsignfiicant correlations between pH and shear force in
broiler pectoralis superficialis. These findings suggest
that the postmortem metabolism determining rigor mortis
development rate is different for red meat than for poultry
breast meat.
Postmortem metabolism differences between red and
poultry meats would not be surprising considering that they
are drastically different in fiber type distribution
(Ashmore and Doerr, 1971a, b). Red meat, possessing a
52


53
large percentage of aerobic fibers, would operate much
differently in the anaerobic environment of postmortem
muscle than poultry muscle, observed in Chapter III (Table
3-1) to be mainly comprised of anaerobic fibers.
In postmortem muscle, ATP is continually depleted as
the muscle cells strive to survive. Some researchers have
defined a specific muscle adenosine triphosphate (ATP)
concentration as the point at which the cell develops rigor
mortis (Honikel et al., 1931 a). The anaerobic conditions
associated with postmortem muscles force cells to depend on
glycolysis for replenishing ATP. Because glycogen is
metabolized to lactic acid during glycolysis, lactic acid
production, usually measured as pH, has frequently been
used as a convenient indicator of the endpoint of the
entire spectrum of postmortem biochemical metabolism. Such
"blanket" indicators are frequently not as accurate or
applicable as their users imply. The application of a
"blanket" indicator across fiber types is not very prudent
as these fiber types have very different metabolisms.
Measuring the levels of the principle metabolites in
postmortem muscle (ATP, glycogen and lactic acid) would be
more accurate than measurement of the "blanket" indicator,
pH. A difference between red and white fibers in their
rate of metabolite change over the time course of rigor
mortis development would be expected and might, at least
partially, explain the differences in correlations in pH,


54
shear force, and rigor mortis development between red and
poultry meats.
The objective of this experiment was to determine the
postmortem pattern of ATP depletion, glycogen depletion and
lactic acid production, within white and red fiber types
and in heterogeneous broiler muscles of different fiber
type composition.
Materials and Methods
Broiler Rearing, Selection, and Processing
Cobb, feather-sexed, male broilers were reared in
litter-covered floor pens and fed a commercial-type corn-
soy diet. At 55 days of age, broilers weighing between
1800 and 2300 g were cooped (8 broilers/coop) and held for
12 hours prior to processing, to simulate commercial feed
withdrawal procedures. Sixty-four birds (in 4 replicates
of 16 birds each) were electrically stunned, exsanguinated,
scalded, picked and eviscerated.
R Value
Immediately following evisceration (20 minutes
postmortem), the anterior and posterior latissimus dorsi
muscles, the sartorius, and a pectoralis superficialis
strip (10x50x10 mm), located 2.54 cm from the anterior end
of the muscle, were excised from both sides of two
carcasses per replication. These samples were immediately
minced, identified, placed in plastic bags, frozen in


55
liquid nitrogen and held at -40C for later analysis. The
remaining 14 carcasses per replication were chilled in
agitated tap water at 21 C for 20 minutes and 1 C for 20
minutes. Chilling water agitation was achieved by pumping
the water from the bottom of the chill tanks directly to
the top of the chill tanks. Samples from the same four
muscles as previously described were removed from two
chilled carcasses (40 minutes postmortem) and treated as
described above. The remaining chilled carcasses were
packed in crushed ice and held at 4C. Sampling was
repeated on two additional carcasses per replication at 1,
2, 3, 4, 6 and 8 hours postmortem (Figure 4-1).
Adenosine triphosphate depletion was monitored by
analyzing each liquid nitrogen-frozen sample for ATP and
inosine monophosphate (IMP) using methods of Khan and Frey
(1971) and Honikel and Fischer (1977) as modified for
poultry by Thompson and Janky (1987). Approximately 3
grams of frozen, powdered meat were homogenized in 20 ml of
1 M perchloric acid for 1 minute using a Virtis 23
(Gardiner, New York) homogenizer at 13,800 rpm. Following
gravity filtration (6 pm paper), 0.1 ml of the acid
filtrate was added to 4 ml of 0.1 M phosphate buffer
(pH 7.0). Absorbances at 250 and 260 nm were measured on a
Hitachi Perkin-Elmer spectrophotometer (Maywood, Illinois)
with a slit width of 0.5 mm. R values were calculated as
absorbance at 250 nm divided by the absorbance at 260 nm.


16 broilers (in each of 8
replications)
(90 seconds) bleed
_ pi ck
2 ^carcasses r
sampled ^
(20 mi n)
feed withdrawn (12 hours)
stun (Cervin model FS, Minneapolis,
2 carcasses
sampl ed^l
(40 min)
2 carcasses-^
sampled
(1 hour)
2 carcasses'
sampled
(2 hours)
2 carcasses
sampled
(3 hours)
pre-chi 11
(tap HO)
1
chill (1C)
packed in ice

! carcasses
sampled
(4 hours)
Minnesota, setting 4)
2 carcasses
sampled
(8 hours)
2 carcasses
sampled
(6 hours)
Figure 4-1. Flow diagram of muscle sample collection for
the R value experiment.


57
The experiment was repeated, resulting in two trials
of 64 birds each. Four muscle types were sampled at each
of 8 different postmortem times. Changes in R value over
postmortem time were subjected to stepwise linear
regression to characterize the pattern of change using
programs available in the Statistical Analysis System (SAS
Institute, Inc., 1985). Because there was no significant
sampling time x trial interaction, data from the two trials
were pooled.
Glycogen and Lactic Acid Concentrations
Broiler processing and muscle sampling procedures
described above were repeated on two additional groups,
each of 16 birds, similar to the birds used above. Samples
from one group of birds were analyzed for lactic acid
concentration at the previously described postmortem
sampling times while those from the other group of birds
were analyzed for glycogen at these same sampling times.
For each metabolite, two birds were sampled at each of the
eight sampling times.
Lactic acid levels were measured with a lactic acid
kit and procedure available from Boehringer Mannheim
Biochemicals (Indianapolis, Indiana). Lactic acid (L-
lactate) was oxidized by nicotinamide adenine dinucleotide
(NAD) to pyruvate by L-lactate dehydrogenase. The
equilibrium of the reaction, however, was almost completely
toward the lactate. By removing the pyruvate from the


58
reaction, the equilibrium was shifted to the right.
Therefore, pyruvate was subsequently involved in the
transamination of L-glutamate to form L-alanine and a-
oxogluterate, which was catalyzed by glutamate-pyruvate
transaminase (GPT). The amount of reduced nicotinamide
adenine dinucleotide (NADH) formed was stoichiometric with
the amount of L-lactic acid in the sample. The increase in
NADH was indicated by the absorbance change at 3^0 nm.
Glycogen concentrations were measured with a starch
analysis kit and procedure available from Boehringer
Mannheim Biochemicals (Indianapolis, Indiana). This kit
utilized amyloglucosi dase to hydrolyze the glycogen to
glucose which was changed to glucose-6-phosphate (G-6-P) by
hexokinase. In the presence of G-6-P dehydrogenase, G-6-P
was oxidized by nicotinamide adenine dinucleotide phosphate
(NADP) to gluconate-6-phosphate while NADP was reduced to
reduced nicotinamide adenine dinucleotide phosphate
(NADPH). The amount of NADPH formed in this third reaction
was stoichiometric with the amount of glucose in the
sample. The amount of NADPH formed was determined by
measuring the absorbance change at 3^0 nm.
Changes in glycogen and lactic acid over postmortem
time were subjected to stepwise linear regression to
characterize the pattern of change using programs available
in the Statistical Analysis System (SAS Institute, Inc.,
1 985) .


59
Results and Discussion
R Value
The anterior latissimus dorsi, composed of only red,
aerobic fibers (Chapter III), had the highest initial R
value (Figure 4-2). The two muscles with intermediate
fiber type distributions had intermediate initial R values
with the more red, aerobic sartorius having a higher R
value than the more white, anaerobic posterior latissimus
dorsi. The pectoralis superficialis, entirely comprised of
white fibers, had the lowest initial R value.
After the death of an animal its muscle cells continue
to hydrolyze adenosine triphosphate (ATP) in an effort to
sustain themselves (Hamm, 1982). This hydrolysis, through
various intermediate steps, eventually results in the
production of inosine, inosine monophosphate (IMP) and
hypoxanthine (Khan and Frey, 1971; Calkins et al., 1983).
The postmortem conversion of the adenosine compounds to
inosine compounds continues until insufficient ATP is
present to prevent actomyosin formation (Hamm, 1982). This
formation causes sarcomere shortening, tension development
in intact muscles and is thought to indicate rigor mortis
development (Busch et al., 1972; Hamm, 1982).
R value is a ratio of two absorbances and is used as
an indicator of the general energy status of a muscle cell
(Calkins et al., 1983). The absorbance maxima of adenosine


R VALUE (A250/A260)
Figure 4-2. Changes in R value over postmortem time in the broiler anterior latissimus
dorsi (ALD), sartorius (SART), posterior latissimus dorsi (PLDT, and
pectoralis superficialis (PECT). (Arrows indicate the time required for
maximum isotonic tension development by the respective, chilled muscles (from
Table 3-2).)
CTv
O


61
nucleotides are at 260 nm and the absorbance maxima of
inosine nucleotides are at 250 nm (Calkins et al., 1983).
Because adenosine is converted to inosine over postmortem
time, R value, the ratio of the absorbance at 250 nm to the
absorbance at 260 nm increases with postmortem time (Khan
and Frey, 1971; Calkins et al., 1983).
The initial R value levels for the various muscles
tested (Figure 4-2) reflected the predominance of aerobic
or anaerobic metabolism in the muscles. Aerobic metabolism
yields substantially more ATP molecules from a single
glucose molecule than does anaerobic metabolism (Stryer,
1981). Therefore, aerobic fibers require less ATP,
relative to the amount of inosine nucleotides, because they
are capable of rapidly producing ATP. This is contrasted
by higher levels of ATP, relative to the level of inosine
nucleotides, in white fibers because their anaerobic
metabolism is less efficient in ATP production. This
contrast would have resulted in aerobic fibers having
greater initial R values than anaerobic fibers. The
relationship between predominant metabolism and R value
could be extended to the level of the whole muscle by
considering the fiber type distributions of the muscles
(Figure 4-2). The muscle with the greatest aerobic
capacity, the anterior latissimus dorsi, had the highest R
value while the most anaerobic muscle, the pectoralis
superficialis had the lowest R value. The two muscles that


62
were intermediate in their metabolisms were also
intermediate in their R value, with the predominantly
aerobic sartorius being greater than the more anaerobic
posterior latissimus dorsi.
Early postmortem, the R values for all muscles
increased (Figure 4-2). In the anterior latissimus dorsi,
sartorius, and posterior latissimus dorsi, the R values
plateaued at approximately 2 hours postmortem and remained
constant for the duration of the test period. However, the
R value for the pectoralis superficialis continued to
increase throughout the test period. The R value changes
over postmortem time (Figure 4-2) indicated that the
depletion of oxygen and cessation of aerobic metabolism
occurred between 1 and 2 hours postmortem. This could be
observed by the location of inflection points at this time
in the R-value lines for the anterior latissimus dorsi and
sartorius. The R values for these muscles showed no
further change after this time due to their dependence on
aerobic metabolism. The pectoralis superficialis and
posterior latissimus dorsi continued to be metabolically
active in the absence of oxygen because of their anaerobic
capacities. Eventually, the R value of the posterior
latissimus dorsi stopped increasing when its anaerobic
capacity had been exceeded. The R value of the pectoralis
superficialis continued to increase throughout the 8-hour
test period. The positive slope of the pectoralis


63
superficialis R value line at 8 hours suggested that it
would have continued to increase well after the test period
ended.
When the respective rigor mortis completion times from
Chapter III (Table 3~1) for chilled muscles (indicated by
the arrows in Figure 4-2) were plotted in combination with
the R value changes in each muscle, a discrepancy between R
values and rigor mortis development times was apparent.
The anterior latissimus dorsi and sartorius, because of
their aerobic natures, would have been the avian muscles
most like bovine and porcine muscles. The R-value lines of
the anterior latissimus dorsi and sartorius showed no
change in R value after 2 hours postmortem. However, rigor
mortis development was not complete until more than 2 hours
later. Discussing bovine muscle metabolism, Hamm (1982)
reported that when the cellular ATP level fell to 1 umole/g
wet tissue, rigor mortis development began. He continued
that this development was complete when the ATP level
reached 0.1 umole/g wet tissue. Thus, rigor mortis
development and completion were associated with decreasing
levels of ATP. Briskey et al. (1962) reported that the
red, aerobic portion of the porcine semitendinosus
completed rigor mortis development, as measured by
extensibility, at approximately 2 hours postmortem. This
corresponds to the time in the present experiment when
oxygen depletion and cessation of aerobic metabolism were


64
evident by inflections in the anterior latissimus dorsi and
sartorius R-value lines. Red porcine muscles therefore
develop rigor mortis when aerobic metabolism stops and ATP
levels fall, all according to the widely accepted red meat
theories of Hamm (1982). The fact that rigor mortis
development in the avian anterior latissimus dorsi did not
occur until over 2 hours after aerobic metabolism stopped
indicated that poultry red muscles did not develop rigor
mortis according to the same metabolic principles as bovine
and porcine muscles. Some undetermined element of
metabolism was apparently able to sustain the red muscles
of the broiler over 2 hours after the stabilization of ATP
concentration. This delay in rigor mortis development was
also observed in the sartorius, another predominantly
aerobic muscle. Even the mainly anaerobic posterior
latissimus dorsi showed evidence of a similar delay with 2
to 3 hours passing between the last substantial R-value
change and the development of rigor mortis.
The pectoralis superficialis continued to exhibit
substantial R-value changes well after the completion of
rigor mortis development. This was inconsistent with the
accepted theory of rigor mortis development in bovine and
porcine muscles (Hamm, 1982). This theory is based on the
contention that the depletion of ATP causes rigor mortis
development. The results of the present experiment
contradict this theory by clearly showing that substantial


65
ATP degradation occurs after rigor mortis development.
Rigor mortis development in avian muscles, then, was not
characterized by the same biochemical events and principles
as noted for bovine and porcine muscles. This suggested
the existence of unknown enzyme or metabolite systems or
new relationships among those already known. Different
metabolisms for avian and mammalian muscles would not be
surprising, considering their evolutionary, physiological,
and biochemical differences. Such a simple difference as
body temperature suggests differences in enzyme activities
and tolerances. The normal broiler body temperature (42C)
is higher than that of such mammals as cows (38C) or pigs
(39C) (Hafez, 1968). Therefore, either avian metabolic
systems are adapted for optimum functioning at elevated
temperatures, or distinct metabolite differences exist
between mammals and birds. An example of the latter is the
differing ways mammals and birds excrete amino nitrogenous
wastes. Mammals excrete these wastes as the water soluble
compound, urea, while birds synthesize purines from their
excess amino nitrogen and then degrade them to form urate
which is excreted as the water insoluble compound, uric
acid (Stryer, 1981).
Differences in the mechanism of rigor mortis
development might well explain the discrepancy between
birds and mammals with regard to the relationship between


66
postmortem metabolism and meat tenderness. Lundberg et al.
(1987) reported that correlations between pH and rigor
mortis development in porcine, ovine and bovine muscles
were so high that only pH needed to be monitored to
indicate rigor mortis development. These authors reported
that, by indicating rigor mortis development, pH alone
could indicate when a muscle could be boned without
increasing toughness. In contrast, Stewart et al. (1984b)
and Sams and Janky (1986) reported nonsignificant
correlations between broiler muscle pH at boning and
subsequent meat tenderness. Stewart et al. (1984b)
suggested that broiler muscle pH and meat tenderness were
not directly related, except through their common
association with postmortem metabolism.
Glycogen Depletion
Initial glycogen concentration was higher in the
anaerobic muscles, pectoralis superficialis and posterior
latissimus dorsi, than in the aerobic muscles, sartorius
and anterior latissimus dorsi (Figure 4-3). The pectoralis
superficialis with its greater predominance of white fibers
had a higher glycogen content than the posterior latissimus
dorsi. The glycogen levels of the two aerobic muscles were
quite similar, despite the differing fiber type
distributions of these muscles.


GLYCOGEN (mg/g MEAT)
POSTMORTEM TIME (HR)
Figure 4-3. Changes in glycogen concentration over postmortem time in the broiler
anterior latissimus dorsi (ALD), sartorius (SART), posterior latissimus dorsi
(PLD)", and "pectoralis super ficialis (PECTT. (Arrows indicate the time
required for maximum isotonic tension development by the respective, chilled
muscles (from Table 3~2).)


68
Muscle cells maintain stored supplies of energy for
sustenance during times of fasting. When muscular activity
is required by the animal, this energy store is mobilized
and used as fuel for the contraction process. Depending on
the principal mode of cellular metabolism, the energy may
be stored in different forms to compliment the metabolism
and contractile properties of the cell (Hamm, 1982).
Aerobic fibers not only contract slowly with much
force but also are capable of sustaining a contraction for
a long period of time, such as in the maintenance of
posture (Ashmore, 1974). Aerobic fibers use lipid as their
primary energy store (George and Berger, 1966; Cassens and
Cooper, 1971). The energy yield from lipid is much higher
than from carbohydrates so that a large amount of energy
could be derived from a relatively small amount of lipid
(Stryer, 1981). Another aspect of the oxidative use of
lipid as an energy store is more complex and requires more
time than the use of carbohydrates (Stryer, 1981). Thus,
aerobic fibers use lipid as a rich source of energy that is
gradually consumed during chronic activity.
Anaerobic fibers contract very rapidly but with less
force than aerobic fibers (George and Berger, 1966)
maintaining their tension for short times. Because of this
they have been termed "twitch" fibers (George and Berger,
1966). Anaerobic fibers use glycogen as their primary
energy store (George and Berger, 1956; Cassens and Cooper,


69
1971). Glycogen is a carbohydrate and therefore has a much
lower energy yield than lipid (Stryer, 1981). Glycogen
therefore cannot sustain chronic tension but is adequate
for supporting brief contractions. To compensate for its
low energy yield, glycogen, as a carbohydrate, can be
rapidly broken down to its component glucose molecules, an
immediate source of energy (Stryer, 1981). Thus, glycogen
provides the necessary volume of readily available energy
to support a brief contraction.
The initial glycogen levels of the various muscles
tested (Figure 4-3) reflected the predominance of aerobic
or anaerobic metabolism in the muscles. Because glycogen
is the primary energy store of anaerobic fibers, muscles
with a predominance of these fibers had high glycogen
levels. In contrast, predominantly aerobic muscles had low
glycogen levels as aerobic fibers use lipid as their main
energy store.
With the progression of postmortem time, glycogen
stores in the test muscles decreased, with the exception of
the anterior latissimus dorsi which remained unchanged
(Figure 4-3). The glycogen level in the pectoralis
superficialis rapidly decreased, reaching its minimum level
in only 3 hours. Because of its lower initial
concentration, the posterior latissimus dorsi glycogen
supply required only 2 hours to be depleted. The small
amount of glycogen present in the sartorius was depleted in


70
2 hours whereas the anterior latissimus dorsi showed no
change in glycogen concentration throughout the test
period.
The changes that occurred in glycogen levels (Figure
4-3) reflected the predominant postmortem metabolisms of
the muscles. Rapid depletion of the glycogen stores were
observed in the pectoralis superficialis and posterior
latissimus dorsi. This indicated ongoing metabolic
activity by the cell to sustain itself in the anoxic,
postmortem condition. Only slight glycogen level changes
were observed in the partially anaerobic sartorius while no
glycogen metabolism was evident in the anterior latissimus
dorsi.
Plotting the rigor mortis development times from
Chapter III (Table 3-2) in combination with the postmortem
changes in glycogen levels revealed that the time of rigor
mortis development did not coincide with glycogen
depletion. The current theory of muscle biology holds that
once aerobic metabolism stops, glycolysis is the only means
of support for a cell (Hamm, 1982). When glycolysis
ceases, the cell cannot adequately supply itself with ATP
and therefore develops rigor mortis. In the present
experiment the muscles with little glycolytic capacity,
anterior latissimus dorsi and sartorius, were able to
survive for over 4 hours postmortem. This delay in rigor
mortis development occurred despite minimal glycogen use.


71
According to the R value results, no ATP degradation
occurred after 2 hours. Similar results were seen for the
pectoralis superficialis and posterior latissimus dorsi;
however, their high glycogen levels and anaerobic
capacities allowed increased glycolytic activity. Even
with the glycolysis in these anaerobic fibers, about 2.5
hours elasped between the last substantial decrease in
glycogen and the development of rigor mortis.
These findings reinforced the suggestion presented in
the R value discussion that rigor mortis development in
avian muscles fibers was different than that in bovine or
porcine fibers. The possibility for the existence of
unknown metabolites (e.g., energy supplies) or of unknown
relationships between known metabolites was clearly
indicated.
Lactic Acid Production
Initial lactic acid concentration was highest in the
pectoralis superficialis and slightly lower in the less
predominantly anaerobic posterior latissimus dorsi (Figure
4-4). Still lower in initial lactic acid content was the
sartorius. The least anaerobic muscle, anterior latissimus
dorsi, had the lowest initial lactic acid level.
Stryer (1981) reviewed the metabolic role of pyruvate
and lactate in skeletal muscles. The main product of
glycolysis is pyruvate which, under aerobic conditions, is
used by mitochondria in the tricarboxylic acid (TCA) cycle


140
0$ , ,
012345678
POSTMORTEM TIME (HR)
Figure Changes in lactic acid concentration over postmortem time in the broiler
anterior latissimus dorsi (ALD), sartori us (SART), posterior latissimus dorsi
(PLD), and pectoralis superficialis (PECfT. (Arrows indicate the time
required for maximum isotonic tension development by the respective, chilled
muscles (from Table 3-2).)


73
to produce ATP. However, under anaerobic conditions, such
as occur with prolonged intense muscular activity or after
the death of an animal, pyruvate is reduced to lactate that
accumulates in the cell. In the living animal, this
lactate can be removed by the circulating blood and
converted back to pyruvate in the liver.
The accumulation of lactate, as lactic acid, decreases
cellular pH and has resulted in the use of pH as an
indicator of postmortem metabolism. The use of pH as such
an indicator in poultry muscle has caused some confusion as
was discussed in Chapter II. Because of this confusion,
the actual metabolite thought to be responsible for the pH
change, lactic acid/lactate, was measured in the present
experiment instead of the more general measurement of pH.
Throughout the test period, the lactic acid
concentration of the pectoralis superficialis steadily
increased (Figure 4-4). The lactic acid levels in the
posterior latissimus dorsi and sartorius increased rapidly
early postmortem but plateaued after 2 hours and remained
constant for the rest of the test period. As with the
glycogen results (Figure 4-3) the anterior latissimus dorsi
had no change in lactic acid concentration for the duration
of the experiment.
The changes that occurred in lactic acid levels
(Figure 4-4) also reflected the predominant metabolisms of
the muscles. No significant increase in lactic acid was


74
observed in the anterior latissimus dorsi over the 8-hour
test period. This was expected because of this muscle's
low glycolytic capacity. The sartorius and posterior
latissimus dorsi had moderate increases in their lactic
acid levels with the rapid, early increase lasting longer
for the more anaerobic posterior latissimus dorsi than for
the more aerobic sartorius. The entirely anaerobic
pectoralis superficial is had the largest increase of all
muscles tested, as was expected due to its large glycolytic
capacity. Unlike the other muscles, the lactic acid level
in the pectoralis superficialis continued to increase
throughout the 8-hour test period. The positive slope of
the pectoralis superficialis line at the end of the test
period suggested that it would have continued to increase
well after 8 hours postmortem. This extended lactic acid
production is similar to the extended ATP metabolism by
this same muscle as indicated by the R value results
(Figure 4-2).
The continued increase in lactic acid levels is in
conflict with the previously discussed glycogen results.
Glycolysis is commonly thought to be the major source of
lactic acid in postmortem muscle (Hamm, 1982). The
glycogen analysis showed no significant change in
pectoralis superficialis glycogen after 3 hours postmortem
while the lactic acid levels continued to increase up to


75
8 hours. Therefore, some other metabolite besides glycogen
was serving as a source of lactic acid.
The similarities between the lactic acid and R value
curves of the pectoralis superficialis suggested a link
between these two parameters. The continued production of
lactic acid could somehow have been used to produce a
continued supply of ATP, the hydrolysis of which was
reflected in the extended increase in R value. Such a
metabolic pathway has not yet been elucidated in anaerobic
systems, but the current knowledge has been primarily based
on mammalian muscles. The possibility of different enzymes
and pathways in avian and mammalian muscles has already
been discussed.
In addition to different enzymes and pathways, the
relationships between known metabolic systems may also be
different for avian and mammalian muscles. The discussions
of Stryer (1981) contain currently accepted biochemical
events that can plausibly be reorganized to explain the
suspected link between the lactic acid and R value
increases observed in the present experiment. In living
animals, proteins are degraded to amino acids of which the
3-carbon species undergo transamination and/or deamination
to form pyruvate. A similar proteolysis is thought to
occur after the death of an animal (Davey, 1983; Dayton
et al., 1 983; Goll et al., 1983). Stryer (1981) continued
that in the absence of aerobic metabolism, as occurs in the


76
postmortem animal, pyruvate would be reduced to lactate
with the simultaneous oxidation of reduced nicotinamide
adenine dinucleotide (NADH) to nicotinamide adenine
dinucleotide (NAD+). The formation of NAD+ is important
because it creates oxidative potential which drives
anaerobic, ATP-yielding pathways such as glycolysis.
Although the supply of the usual substrate for glycolysis
would be expectedly low postmortem, the possibility still
exists for the utilization of the oxidative potential of
NAD+ through some shortened or modified form of glycolysis.
In a discussion of lipid degradation, Stryer (1981)
reported that glycerol and glycolytic intermediates such as
glyceraldehyde 3-phosphate were readily interconvertable.
Thus, lipolysis could serve as a source of glyceraldehyde
3-phosphate, a potential substrate for a shortened form of
glycolysis. Such a system would utilize the oxidative
potential of the NAD+ to produce two ATP molecules. The
NAD+ could then be regenerated by reducing pyruvate to
lactate.
The limitations of this lipid-fueled glycolysis in the
avian pectoralis superficialis are the low level of
intracellular lipid in this muscle and the locations of any
extracellular lipid deposits. The pectoralis superficialis
is an anaerobic muscle and therefore its cells store only a
small amount of their energy supply as lipid (George and
Berger, 1966; Cassens and Cooper, 1971). The intracellular


77
lipid content of this muscle would therefore be expected to
be low, with some lipids existing in the form of
membranes. Furthermore, because avian muscles possess
little intercellular lipid, most of the lipid deposits are
intermuscular. In the postmortem animal without blood
flow, the location of most of the lipid deposits would
require the lipolysis products to diffuse across one or
more membranes, to reach the cytoplasm of the muscle cell
and the site of metabolism. These limitations pose
questions to be answered but do not eliminate the
possibility that yet undetermined entities or relationships
exist which might influence postmortem muscle behavior.
An example of a new relationship might be the enhanced
activity of a compound like pyruvate. Pyruvate is thought
to participate in many metabolic pathways such as the
anabolism and catabolism of carbohydrates, lipids, and
proteins (Stryer, 1981). This compound is frequently the
mode of entry into, exit from, and exchange between these
systems, the possibility of additional metabolic roles for
pyruvate or related compounds does not seem so remote.


CHAPTER V
SUMMARY AND CONCLUSIONS
Red muscle fibers developed rigor mortis faster than
white fibers, probably due to the latter's greater
anaerobic capacity. This trend was also evident in
heterogeneous muscles with predominantly red muscles
developing rigor mortis faster than predominantly white
muscles. These findings may be useful to poultry
processors who must wait until broiler muscles are in rigor
mortis to prevent toughening of the meat during boning. To
increase production efficiency, dark portions of broiler
carcasses could be aged for a different length of time
before boning than the white portions. Broiler breast meat
that is toughened by boning immediately after chiLling (1
hour postmortem) is probably toughened because all of the
muscle's fiber types are still prerigor at 1 hour
postmortem.
Commercial-type chilling significantly slowed the
development of rigor mortis in predominantly aerobic
muscles but only slightly slowed it in predominantly
anaerobic muscles. Differences between the fiber types in
cellular organization may have accounted for the differing
effects of chilling on the fiber types. The slowing
78


79
effects of chilling on rigor mortis development indicated
that results of previous research obtained without
commercial-type chilling should not be used to describe
rigor mortis development in commercially processed
broilers.
R value, glycogen, and lactic acid changes in the four
test muscles were consistent with the postmortem metabolism
of each muscle. These metabolic parameters rapidly
stabilized in the aerobic muscles while they showed
evidence of continued metabolism in the anaerobic
muscles. This indicated that aerobic metabolism stopped
soon after death but anaerobic metabolism continued long
after oxygen depletion.
Adenosine triphosphate depletion, glycogen utilization
and lactic acid accumulation, three events thought to be
directly related to rigor mortis development in bovine and
porcine muscles, were not well related to rigor mortis
development in broiler muscles as measured by isotonic
tension development. In aerobic muscles, rigor mortis was
delayed several hours after the apparent cessation of
metabolism. Conversely, the pectoralis superficialis
provided clear evidence for continued metabolism well after
the completion of rigor mortis development, an event
thought to signify the conclusion of all substantial energy
metabolism.


80
These discrepancies suggested that rigor mortis
development in broiler muscles was not characterized by the
same biochemical events or processes as in bovine or
porcine muscles. This could have accounted for pH/shear
force correlation differences that have been observed
between avian and mammalian muscles. It was possible that
yet undetermined enzymes or metabolic pathways were active
in avian muscle rigor mortis development. Also possible
were the alteration or reorganization of existing pathways,
such as glycolysis, and the enhanced activities of such
important metabolites as pyruvate. Whatever the
explanation, it seems clear from these results that rigor
mortis development in broiler muscles should not be
considered merely an extrapolation of this development in
bovine or porcine muscles.


APPENDIX
REGRESSION EQUATIONS
Regression equations for the curves in Figures 4-2, 4-3 and
4-4.
Figure
Muscle
Regression Equation
C\J
1
ALD
InR
value =
-0.051 85-0.01 1 99*1 /time
SART
InR
value =
-0.0781 5-0.01 1 30*1 /time
PLD
InR
value =
-0.08032-0.02 3475*1 / time
PECT
InR
value =
-0.1737+0.02934*lntime
4-3
ALD
glycogen = 97.6041-5.6053*time
SART
glycogen = 9
. 4951 +30.0296*1 /time
PLD
glycogen = 1
0. 9765 + 71 51 34*1 /time
PECT
glycogen = 1
71 31 36+239.81 29*1 /time
4-4
ALD
lactic acid
= 23.441 3+0.8054*time
SART
lactic acid
= 40.022+5.721*lntime
PLD
lactic acid
= 55.659+5.287*lntime
PECT
lnlactic aci
d = 3.9746+0.1178*time
81


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shortening" effect in avian muscle. J. Food Sci.
34:42.
Solomon, M.B., and M.C. Dunn. 1986. A technique for the
simultaneous determination of three fiber types in a
single section of ovine skeletal muscle. Stain
Technol. (In Press).
Stein, J. 1975. The Random House College Dictionary.
Random House, New York, NY.
Stein, J.M., and H.A. Padykula. 1962. Histochemical
classification of individual skeletal muscle fibers of
the rat. Amer. J. Anat. 110:103.
Stewart, M.K., D.L. Fletcher, D. Hamm and J.E. Thomson.
1984a. The effect of hot boning broiler breast meat
muscle on postmortem pH decline. Poultry Sci.
63:2181.
Stewart, M.K., D.L. Fletcher, D. Hamm and J.E. Thomson.
1984b. The influence of hot boning broiler breast
muscle on pH decline and toughening. Poultry Sci.
63:1935.


Stryer, L. 1981. Biochemistry (2nd ed.). W.H. Freeman
and Co., San Francisco, CA.
Tarrant, P.V. 1977. The effect of hot-boning on
glycolysis in beef muscle. J. Sci. Fd. Agrie. 28:927
Thompson, L.D., and D.M. Janky. 1987. The effect of
postmortem electrical stimulation on the texture of
hot-boned, chill-boned, and age-boned broiler breast
fillets. Poultry Sci. (in press).
Voyle, C.A. 1969. Some observations on the histology of
cold shortened muscle. J. Food Technol. 4:275.
Wangen, R.M., and J.H. Skala. 1968. Tenderness and
maturity in relation to certain muscle components of
White Leghorn fowl. J. Food Sci. 33:613.
West, R.L. 1983. Functional characteristics of hot-boned
meat. Food Technol. 37,5:57.
Whiting, R.C. 1980. Calcium uptake of bovine muscle
mitochondria and sarcoplasmic reticulum. J. Food Sci
45:288.
Whiting, R.C., and J.F. Richards. 1975. Thaw rigor
induced isometric tension and shortening in broiler-
type chicken muscles. J. Food Sci. 40:960.
Wood, D.F., and J.F. Richards. 1974a. Cold shortening in
chicken broiler pectoralis major. J. Food Sci.
39:530.
Wood, D.F., and J.F. Richards. 1974b. Isometric tension
studies on chicken pectoralis major muscle. J. Food
Sci. 39:525.


Full Text
THE EFFECT OF FIBER TYPE ON
THE RATE OF RIGOR MORTIS DEVELOPMENT
IN BROILER MUSCLES
BY
ALAN RAY SAMS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA

ACKNOWLEDGMENTS
The author would like to express appreciation to his
committee chairman, Dr. D. M. Janky, for his assistance and
guidance throughout the author's graduate program and in
the preparation of this manuscript. Thanks are also
extended to Drs. D. D. Johnson, R. H. Schmidt, R. L. West
and S. A. Woodward for their time in serving on the
advisory committee and suggestions during this study.
A debt of gratitude is expressed to the Poultry
Science Department for funding the author's graduate
program and providing facilities and equipment for
conducting this study. The author thanks the Animal
Science and Dairy Science Departments and the J. H. Miller
Health Center Teaching Labs for the use of their facilities
and equipment. The author wishes to thank Gold Kist
Poultry, Inc. of Live Oak, Florida, for donating the
broilers used in the isotonic tension experiment.
Special thanks are extended to Drs. D. M. Janky, S. A.
Woodward, C. W. Comer and D. A. Comer, M. Dukes, J.
Eastridge and E. Hirchert for their technical assistance
and friendship throughout the completion of this study.
Deepest appreciation goes to the author's wife,
Kimberlee, for her love, friendship, and motivation
throughout his educational career.

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF FIGURES vi
ABSTRACT vii
CHAPTERS
I INTRODUCTION 1
II REVIEW OF THE LITERATURE 6
Rigor Mortis Development 6
The Importance of Rigor Mortis Development
to Meat Tenderness 11
The Relationship Between Postmortem
Metabolism, Rigor Mortis Development
and Meat Tenderness 16
Factors Affecting the Rate of Rigor Mortis
Development 19
Muscle Fiber Types 25
III RATE OF RIGOR MORTIS DEVELOPMENT AND
DISTRIBUTION OF FIBER TYPES IN FOUR
BROILER MUSCLES 36
Introduction 36
Materials and Methods 39
Results and Discussion 44
IV BIOCHEMICAL ASPECTS OF RIGOR MORTIS
DEVELOPMENT IN BROILER MUSCLES 52
Introduction 52
Materials and Methods 54
Results and Discussion 59
i i i

V SUMMARY AND CONCLUSIONS 78
APPENDIX REGRESSION EQUATIONS 8l
REFERENCES 82
BIOGRAPHICAL SKETCH 91
IV

LIST OF TABLES
Table Page
3-1 Fiber type distributions (in percent) of
broiler anterior latissimus dorsi (ALD),
sartorius (SART), posterior latissimus dorsi
(PLD) and pectoralis superficialis (PECT)... 45
3-2 Time (hours) required for development of
maximum tension by broiler anterior
latissimus dorsi (ALD), sartorius (SART),
posterior latissimus dorsi (PLD), and
pectoralis superf icialis C~PECT) held at
room temperature (24°C) or chilled under
simulated commercial conditions (20 minutes,
240C; duration, 4°C) 48
3-3 Time (hours) required for development of
maximum tension by broiler anterior
latissimus dorsi (ALD), sartorius (SART),
posterior latissimus dorsi (PLD)~T~ and
pectoralis superficialis (PECT) held at
room temperature (24°C) or chilled under
simulated commercial conditions (20 minutes,
24°C; duration, 4°C) 50
v

LIST OF FIGURES
Figure
Page
3-1
Flow diagram of muscle sample collection
and analysis for the isotonic tension
development experiment
4-1
Flow diagram of muscle sample collection
for the R value experiment
. . . .56
4-2
Changes in R value over postmortem time in
the broiler anterior latissimus dorsi (ALD),
sartorius (SART), posterior latissimus dorsi
(PLD), and pectoralis superficialis (PECT)....
,60
4-3
Changes in glycogen concentration over
postmortem time in the broiler anterior
latissimus dorsi (ALD), sartorius (SART),
posterior latissimus dorsi (PLD), and
, ...67
pectoralis superficialis (PECT)
4-4
Changes in lactic acid concentration over
postmortem time in the broiler anterior
latissimus dorsi (ALD), sartorius (SART),
posterior latissimus dorsi (PLD), and
pectoralis superf icialis (PECT)
.... 72
vi

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
THE EFFECT OF FIBER TYPE ON
THE RATE OF RIGOR MORTIS DEVELOPMENT
IN BROILER MUSCLES
BY
ALAN RAY SAMS
December, 1987
Chairman: D. M. Janky
Major Department: Food Science and Human Nutrition
Two experiments were conducted to characterize the
development of rigor mortis by different fiber types in
broiler muscles. A homogeneous red muscle (anterlor
latissimus dorsi) was used as a model for the behavior of
red fibers while a homogeneous white muscle (pectoralis
superficialis) was used to characterize the behavior of
white fibers. The contribution of the different fiber
types in heterogeneous muscles was evaluated using a
predominantly red muscle (sartorius) and a predominantly
white muscle (posterior latissimus dorsi).
In the first experiment, isotonic tension development
was used to detect the development of rigor mortis while
histological examination indicated the fiber type
distribution in the four test muscles from male broilers.
vi 1

Rigor mortis development in the red, aerobic muscles was
significantly faster (P<0.05) than that in the white,
anaerobic muscles. Commercial-type chilling procedures
significantly (PC0.10) slowed rigor mortis development of
red muscles.
In the second experiment, the four test muscles were
removed from 128 male broilers at 0.33, 0.67, 1, 2, 3, 4, 6
or 8 hours postmortem and analyzed for R value. Glycogen
and lactic acid concentrations were each determined for one
of two additional groups of 16 male broilers that were
processed and sampled similarly to those in the R-value
analysis. R value was directly related to the aerobic
capacity while lactic acid and glycogen concentrations were
directly related to the anaerobic capacity of each of the
four test muscles. The aerobic muscles showed no change in
any of the biochemical parameters after 2 hours postmortem,
the anaerobic muscles showed continued metabolism beyond 2
hours and the pectoralis superficialis had substantial R
value and lactic acid increases up to 8 hours postmortem.
The rigor mortis development times from the tension
development experiment did not coincide with the
biochemical events thought to cause rigor mortis. Also,
continued energy metabolism in the pectoralis superficialis
was evident after maximum tension was achieved. These
results prompted the conclusions that rigor mortis
developed faster in red fibers than in white ones,
v i i i

suggesting
could not
that rigor mortis development in broiler muscles
be extrapolated from bovine or porcine muscles.

CHAPTER I
INTRODUCTION
In 1987, 77$ of the broilers produced in the United
States, almost 12 billion pounds, will be marketed as cut¬
up or in other further processed forms (Roenigk and
Pederson, 1987). This percentage is expected to continue
to increase to 87%, 13-5 billion pounds, by 1990. In 1990,
only one in every eight broilers will be marketed as a
whole carcass and further processed items will approach 30$
of all broiler sales. With these projections, United
States per capita broiler consumption will surpass that of
beef during 1989. Pork consumption was surpassed in 1985
by that of broilers.
In an effort to reduce the refrigeration space and
time needed for holding the carcasses for a traditional
aging period, many processors are boning or dismembering
carcasses immediately as they exit the chilling tanks, a
process known as chill boning. Consumer response and
published research, however, have indicated that much of
this meat is tough. In the last decade, with the shift in
the mix of poultry products from whole carcasses to cut-up
products, this toughness has become an increasing problem.
1

2
Current industry recommendations are that carcasses be
held for 4 hours between death and boning to maintain
tenderness. This time was determined experimentally as the
time needed for poultry muscle to decline to its ultimate
pH value, an event thought to signal the development of
rigor mortis (Stewart et al., 1984a, b). Four hours are
said to be required to reach the ultimate pH, despite the
fact that broilers, according to carcass conformation and
muscle turgidity, are in rigor mortis at 1 hour
postmortem. The fact that the pH continues to decline
after this 1-hour period indicates that there is some
factor of the relationship between postmortem muscle
metabolism and meat tenderness that current knowledge does
not explain.
The apparent difference in the usefulness of pH as an
indicator of rigor mortis between red meats and poultry
might be the result of the differing fiber type
compositions of the two muscle types. The use of pH has
been based on the theories that (1) the progression of
glycolysis is one of the major steps leading to rigor
mortis; and (2) that pH decline is the result of the
accumulation of lactic acid, a by-product of glycolysis.
These two relationships are probably valid but when one is
used as an indicator of the occurrence of the other,
inconsistencies result. Erroneous conclusions may be

3
reached when one uses the attainment of ultimate pH as an
indicator of the rigor mortis development time in poultry.
One idea that has yet to be investigated in poultry
meat is that the different muscle fiber types comprising a
muscle behave differently. The drastically different
metabolisms of the fiber types suggest that they have very
different capacities for survival in the anoxic conditions
of the postmortem environment. The "white" fibers, having
primarily an anaerobic metabolism, are well suited for the
postmortem anoxia. These fibers would therefore be
expected to proceed with their metabolism at a normal rate,
slowed only by decreasing temperature and substrate
concentration. The "red" fibers, having primarily an
aerobic metabolism, are not well adapted for the anoxic
conditions. Upon death it is expected that these fibers
continue to respire aerobically as long as possible. When
the oxygen is depleted, these fibers rapidly enter rigor
mortis. Thus, upon death, the red muscle fibers develop
rigor mortis first, followed by the white fibers. If a
muscle consisted of a mixture of fiber types, the rate of
rigor mortis development in that muscle would be
intermediate between that of red and white fibers. Each
fiber type present would have a significant effect on the
rate of rigor mortis development in a muscle.
If the two fiber types indeed had different rates of
rigor mortis development, it might explain the toughening

4
observed in chill boned meat. Rigor mortis development by
only one of the fiber types present in a muscle would make
that muscle appear, visually and tactually, in rigor
mortis. This would still leave one fiber type in the
prerigor state, susceptible to the toughening caused by
boning. Only after both fiber types develop rigor mortis
could boning be performed without toughening the meat. It
is conceivable, then, that chill boning meat one hour
postmortem toughens the meat by stimulating the fibers that
are not yet in rigor mortis. This toughness is seen at an
extreme in hot boned meat because none of the fiber types
are in rigor mortis at these early postmortem times.
A third muscle fiber type, called intermediate fibers,
is known to exist in broiler muscles (Ashmore and Doerr,
1971b). These fibers have high oxidative capacities like
the red fibers and also have glycolytic abilities not much
less than that of white fibers (Ashmore, 1974). Thus, the
presence of these fibers would help sustain the postmortem
reactivity of the muscle, whichever fiber type developed
rigor mortis faster.
Thus there exists the possibility that each of the
fiber types present in the broiler pectoralis superficialis
contribute to the rigor development rate observed for the
muscle as a whole. At the time of excision, one of the
fiber types could be in rigor, while the other fiber type
remains prerigor, provided the fiber types develop rigor

o
mortis at the same rate. This would explain why chill¬
boned broiler pectoralis superficialis is toughened, even
though it appears to be in rigor when deboned. Finally,
the observation of these fiber type effects in two
heterogeneous muscles of different fiber type
distributions, such as the pectoralis superficialis and the
sartorius, would suggest that these effects occur in all
skeletal muscles.
The objectives of this project were to determine
1 ) the relative rates of rigor mortis development by
red and white muscle fibers and their contributions
to the rate of rigor mortis development by the
heterogeneous muscles pectoralis superficialis and
sartorius;
2) the effect of a commercial chilling regimen on the
rate of rigor mortis development by red and white
muscle fibers;
3) the effect of fiber type on the pattern of muscle
ATP depletion, glycogen depletion and lactic acid
production during the development of rigor mortis.

CHAPTER II
REVIEW OF THE LITERATURE
Rigor Mortis Development
Rigor mortis, translated from Latin, is defined as
"the stiffness of death" (Stein, 1975) and has been
observed for centuries upon the death of an animal. Modern
meat scientists now know that this rigidity is caused by
the irreversible interaction of individual sarcomeres
within the body's muscles (Hamm, 1982).
Stryer (1981) reviewed two important changes in
postmortem muscle, contracture and rigor mortis that are
both currently thought to involve the interaction of the
thin and thick filaments of the sarcomere. Each sarcomere
is comprised of filaments oriented parallel to the fiber
and is bounded on both ends by a Z-line which is
perpendicular to the fiber direction. To each Z-line are
connected the thin actin filaments. The primary components
of these thin filaments are two strands of F-actin
(filamentous actin) wrapped around each other forming a
helix. F-actin is actually a polymer of the more basic
actin structure G-actin (globular actin). Attached to the
F-actin helix is the troponin-tropomyosin complex which
functions in the control mechanism of muscular
6

7
contraction. This complex covers the site on F-actin where
interaction with the thick filament occurs. The thick
filaments are composed of many myosin molecules. Each
myosin has a globular "head" and a filamentous "tail." The
myosins of an individual thick filament are connected to
each other by their tails. This arrangement places the
heads in close proximity to the thin filaments.
Innervating this system of filaments is the sarcoplasmic
reticulum (SR). The function of this network of membranous
vessicles is to sequester calcium ions (Ca+^) within its
vessicles. This is accomplished by an adenosine
triphosphate (ATP) driven pumping system located in the
membrane.
In a discussion of the role of energy metabolism in
rigor mortis development, Hamm (1982) reported that in
normal living muscle, ATP is the primary energy source and
is mainly produced by oxidative phosphorylation. When an
animal is killed, the lack of a fresh blood supply to the
muscle cells causes aerobic metabolism to decline. For a
short time, adenosine diphosphate (ADP) is converted to ATP
at the expense of creatine phosphate. The supply of
creatine phosphate is rapidly depleted and then the cell
becomes dependent on anaerobic glycolysis for ATP
production. One product of glycolysis is lactic acid which
accumulates and causes the pH of the muscle cells to
decrease from about 7.0 to about 5.5 several hours after

8
death. When the glycogen supply is depleted, ATP levels
begin to decline as the cell continues to use the energy of
ATP without further ATP production.
Hamm (1982) confirmed that in a living or dead
animal's muscles, as long as sufficient ATP is present, the
SR will continue to sequester Ca+^ ions. This serves to
inhibit the interaction of the thick and thin filaments.
As long as the Ca+^ concentration in the sarcoplasmic fluid
innervating the myofilaments is low (<10-^ M), interaction
of the filaments is prevented. Upon neurostimulation or
upon the inactivation of the SR pumping system, Ca+^ ions
are released into the sarcoplasm and bind to the troponin C
moiety of troponin. This oinding causes the tropomyosin to
move into a groove of the F-actin helix, exposing the site
of interaction of the actin molecule with the myosin
head. The myosin head then binds to the actin unit of the
thin filaments.
Reviewing the biochemistry of the contraction process
in skeletal muscle, Stryer (1981) reported that in the
resting state or early postmortem the thin and thick
filaments are not attached to each other and the myosin
head contains a tightly bound ADP and an inorganic
phosphate (Pj_)- Upon actin activation by the presence of
Ca+2, the myosin interacts with the actin. The release of
the ADP-Pi from the myosin causes the myosin head to tilt
toward the center of the sarcomere. This tilting pulls the

9
Z-lines closer together, thereby shortening the sarcomere
and causing contraction. If ATP is present, its binding to
the myosin causes the myosin to dissociate from the
actin. The hydrolysis of this bound ATP to ADP-P^ then
causes the myosin head to tilt back to its original
perpendicular orientation, completing the contraction
cycle. Therefore, ATP is required for the relaxation of a
contracted muscle (Stryer, 1981).
The ATP dependence of the relaxation process is
important to postmortem muscle (Hamm, 1982). Immediately
postmortem, sufficient ATP is present to support relaxation
and therefore the muscle is extensible (Harrington,
1979). As the cell continues to use ATP while ATP
production declines, less ATP is available for
relaxation. The decreasing relaxation is compounded by the
fact that the lower pH and lower ATP levels cause the
inactivation of the Ca+2 sequestering system of the SR.
Calcium ions are then released into the sarcoplasm,
activating more thin filaments (Cornforth et al., 1980;
Whiting, 1980). When insufficient ATP is present to
support relaxation, the overlapping thin and thick
filaments are irreversibly bound to each other. This
causes the muscle to lose its extensibility, thereby
developing rigor mortis. It is important to note that not
all fibers of a muscle will develop rigor at the same
time. Rigor begins to develop when the cellular ATP levels

1 o
decrease to about 1 pmole/g wet tissue at 20)C and is fully
developed at 0.1 ymole/g wet tissue (Honikel et al.,
1981 a). The postmortem time when these conditions exist in
a fiber varies with antemortem conditions, fiber location
and postmortem processing practices.
Davies (1966) suggested that rigor mortis development
consisted of two closely related but quite different,
simultaneous processes. Postmortem muscle fibers shorten
or contract as they develop rigor mortis. This is a result
of insufficient ATP being present to prevent the
interactions between actin and myosin to form actomyosin.
The sarcomeres and muscle shorten with irreversible
"contraction" due to the presence of Ca+^ ions from a
leaking SR and/or mitochondria and the scarcity of the
myofibrillar plasticizer, ATP. This shortening, measurable
by the development of tension, causes the muscle to be more
rigid and therefore tougher. The other event proposed by
Davies was the loss of extensibility. Goll et al. (1969)
explained that the loss of extensibility could be equated
with the depletion of ATP. As the thin and thick filaments
lose the ability to freely slide over, each other, due to
increased interaction, muscle extensibility is lost. So
the course of ATP depletion induces first sarcomere
shortening at low ATP levels and then greatly reduced
extensibility in the absence of ATP.

Goll et al. (1969) suggested that some loss of
extensibility must accompany the shortening process because
the thin and thick filaments are becoming increasingly
attached to each other. The research of Huxley and Brown
(1967) and Huxley (1968) showed, however, that these two
processes are indeed distinct. These researchers reported
that in rigor-shortened muscle, only 20$ of the myosin
heads of the thick filaments are attached to the thin
filaments. In contrast, nearly 100$ of the myosin heads
are attached to the thin filaments in muscle that has been
depleted of ATP and is, therefore, inextensible. Thus,
rigor-shortened muscle should be five times as extensible
as muscle that has completed its loss of ATP (Goll et al.,
1 969).
The Importance of Rigor Mortis Development
to Meat Tenderness
Meat tenderness is markedly influenced by the
development of rigor mortis. It has been well documented
that the onset of rigor mortis in a muscle drastically
toughens the resulting meat (Lowe, 1948; de Fremery and
Pool, I960; Stewart et al., 1984b). Locker (I960) and
Marsh (1975) both suggested that the extent of sarcomere
shortening (i.e., the degree of interdigitat ion of actin
and myosin filaments) in a muscle was the determining
factor in the extent of toughening resulting from rigor

mortis development. As the Z-lines are drawn closer
together by the interaction of the thick and thin
filaments, the sarcomere becomes more compact and dense
(Marsh et al., 1974). Hamm (1982) stated that shorter
sarcomeres resulting from the development of rigor mortis
could be interpreted as more cross-linkages between the
thin and thick filaments. More of these associations would
cause the muscle structure to be more rigid, increasing
cooked meat toughness. The shortened sarcomeres, being
more rigid, have more mass for teeth to shear than if the
sarcomeres were of normal length. Therefore the meat
produces the sensation of toughness when chewed. In fact,
Voyle (1969) suggested that, based on electron microscopy,
the myosin filaments buckle against (or pierce) the Z-line
upon shortening. It is then proposed that the myosin
filaments could conceivably link through the Z-lines,
forming a network in shortened muscle that would cause
toughening.
The connective tissues are becoming increasingly
recognized as the cause of toughness accompanying the onset
of rigor mortis, despite the widespread acceptance of
myofilament interdigitation. Voyle (1969) suggested the
importance of the muscle's connective and structural
components to meat tenderness. The gap filaments are
filamentous proteins which originate in one end of each
thick filament, extend out of the other end, through the

Z-line, and terminate in the distal end of the adjacent
sarcomere's thick filament (Locker and Leet, 1976). The
strength and elasticity of the gap filaments maintain their
integrity when tensile strain is applied to the muscle,
even to the point when the thin filaments tear out of the
Z-line (Locker, 1985). Because they are susceptible to
endogenous proteases such as calcium activated neutral
protease, the gap filaments or their component protein
connection (Locker, 1985) are degraded upon aging.
Therefore, the weakened gap filaments of postrigor muscle
are of less importance to meat toughness than the
interdigitation of actin and myosin.
Lawrie (1986) reported that when muscle is cooked for
4 hours at 100°C (harsh enough treatment to degrade
collagen) while stretched enough to eliminate any overlap
of actin and myosin fibers, the resulting meat had 66$ of
the tensile strength of the unstretched control. In a
later publication reviewing the effects of heat on muscle
proteins, Locker (1985) stated that after an hour at 70°C,
the gap filaments denatured, but remained strong and
elastic, capable of stretching when strained. Thus it is
conceivable that the gap filaments have an influence on
cooked meat toughness if the enzymatic degradation of the
muscle is sufficiently arrested.
Collagen has been suggested to be another contributor
to meat toughness (Bouton et al., 1973). As an animal

14
ages, the collagen fibers form crosslinkages with other
collagen molecules (Bailey, 1972). With time, these
collagen polymers become decreasingly heat and water
insoluble, thus forming a network of tough fibers within
the meat. However, since the poultry industry currently
markets broilers at the young age of 7 weeks, collagen is
not a major cause of toughness in broilers (Wangen and
Skala, 1968; Nakamura et al., 1975).
Interest in the rate of rigor mortis development
relates to the prerigor excision of the muscle, called hot
boning if done prior to chilling or chill boning if done
after chilling (Sams and Janky, 1986). Hot boning would
save the producer money in the areas of refrigeration,
space and labor. Seideman and Cross (1982) estimated that
50$ of the energy of beef processing plants could be saved
if hot-boning was implemented. Another advantage of hot-
boning is that prerigor meat has greater functional
properties, such as water holding capacity and emulsifying
capacity (Kramlich et al., 1973; West, 1983). Kramlich
et al. (1973) suggested that this was a result of the
primary emulsifying proteins, actin and myosin, being
easier to extract before rigor mortis. In the prerigor
state, actin and myosin have not substantially associated
into actomyosin, which would make their extraction
difficult. West (1983) stated that prerigor meat had a

1 5
higher pH which was further from the isoelectric point of
the myofibrillar proteins than in postrigor meat.
Consequently, this increases the proteins' charge,
increasing the protein/protein and protein/solvent
interactions responsible for functionality.
There is, however, one main disadvantage to hot-
boning. Several researchers have shown that excising
muscle prior to rigor mortis markedly decreases the
tenderness of the meat (Lowe, 1948; de Fremery and Pool,
I960; Stewart et al., 1984a; Sams and Janky, 1986). One
means by which hot boning increases toughness is by
increasing the muscle's susceptibility to cold shortening
(Locker and Hagyard, 1963). After excision, the muscle's
lack of surrounding tissue for insulation causes it to cool
more rapidly than if left on the carcass. This results in
more shortening and tougher meat. Another way prerigor
excision increases toughness is by removing the muscle from
its attachments. Locker (I960), Herring et al. (1967) and
Gillis and Henrickson (1969) all found that muscles excised
prerigor and permitted to contract freely were less tender
than those muscles restrained during rigor development.
The unattached muscle's unimpeded contraction allows more
shortening, both rigor shortening and cold shortening, and
therefore tougher meat.

16
The Relationship Between Postmortem Metabolism,
Rigor Mortis Development and Meat Tenderness
Red meat scientists have long reported a very good
correlation between muscle pH and the shear force of the
resulting cooked meat (Lundberg et al., 1987). Several
attempts have been made to apply this relationship to
poultry processing, but with only limited success. Stewart
et al. (1984b) and Sams and Janky (1986) both reported
lower, nonsignificant correlations between pH at time of
boning and shear force. Sams and Janky (1986) observed
nonsignificant correlation coefficients, ranging from -0.24
to 0.20 for three postmortem boning times studied. Even
when these three groupings were subdivided into nine
groupings of very narrow pH ranges, the correlations were
still nonsignificant. These authors did, however, report a
significant correlation after pooling all the boning
times. This significance was expected because pooling the
three sets of pH values produced by boning meat at three
different times creates an inherent correlation. Stewart
et al. (1984b) reported a nonsignificant correlation
coefficient of 0.19 after pooling the data from the six
boning times used. This lack of correlation was probably
because, by pooling six boning times instead of three (as
in the previous study), less correlation was caused by the
pooling process. The common factor in these studies was

1 7
the lack of correlation between pH at muscle excision and
cooked meat shear force.
Much literature has been published reporting the
increase in meat toughness and changes (acceleration or
deceleration) in glycolysis resulting from prerigor
excision of muscle (Tarrant, 1977; Stewart et al., 1984a,
b). It therefore seems indisputable that some relationship
exists between pH and cooked meat shear force. Stewart
et al. (1984b) suggested that these two parameters in
themselves were not directly, through cause-and-effect,
related. They were, though, probably indirectly related
through their common association with postmortem
metabolism.
By definition, the development of rigor mortis is
characterized by muscle shortening, loss of extensibility
and development of tension. Using isometric tension
development, Wood and Richards (1974a, b) reported rigor
mortis development times of 3 to 5 hours postmortem for
broiler pectoralis superficialis maintained at 23°C.
However, one can observe broilers on the processing line
whose muscles have developed tension as early as 20 to 30
minutes postmortem. Legs and wings have drawn close to the
body and the breast muscles have become turgid. Such
observations prompted researchers to report that broilers
are in rigor mortis by 10 minutes (Shrimpton, I960) or 30

18
minutes (D.L. Fletcher, 1985, personal communication) after
death.
This is contrasted by reports stating that the
ultimate pH in chilled broiler breast meat is not attained
until more than 4 hours postmortem (Stewart et al.,
1984a). This obvious inconsistency is grounds to doubt the
accuracy of ultimate pH as an indicator of rigor mortis and
therefore as a guideline for the time to bone poultry meat
without toughening it. Stewart et al. (1984b) boned breast
meat at 6 postmortem times and observed that as long as the
pH had not reached its ultimate level prior to boning,
objectionable toughening resulted. Research has shown that
breast meat boned 1 hour postmortem, after the carcasses
have been chilled, is tougher than meat boned after 24
hours of aging (Sams and Janky, 1986; Thompson and Janky,
1986). This was despite the fact that the muscles were
visually and tactually in rigor mortis although the
ultimate pH had not been achieved. This is not the case in
the red meat field in which a pH of 5.8 is considered a
level at or below which muscle excision will not result in
objectionable toughness (Bendall, 1975). In this respect,
poultry does not conform to the theories of red meat.
It seems obvious from this evidence and from the
preceding discussions that the traditional view of poultry
meat tenderness as a function of rigor development rate,
indicated by pH decline, is insufficient to explain the

1 9
experimental results being observed with chill boned
poultry meat. Therefore, there must be other factors
influencing the relationship between rigor development rate
and subsequent meat tenderness.
Factors Affecting the Rate of Rigor Mortis Development
The rate of rigor mortis development in a muscle has a
profound influence on the tenderness of the resulting
muscle. Tarrant (1977) suggested that exceptionally fast
or slow rates of rigor mortis development could toughen
beef. De Fremery (1966) and Ma and Addis (1973) have also
reported that increasing the rate of postmortem metabolism
and rigor onset toughens poultry meat. De Fremery and Pool
(1960) showed that treatments producing accelerated ATP
loss and pH decline, indicating faster rigor mortis
development, resulted in increased toughness in broiler
breast meat. Treatments included prarigor muscle excision,
high postmortem environmental temperature, freezing and
thawing of prerigor muscle, exhaustive electrical
stimulation, mechanical beating and electron irradiation.
One of the most important factors in the rate of rigor
mortis development is the postmortem environmental
temperature of the muscle. As the ambient temperature of
postmortem muscle decreases from 37°C (at death) to about
10°C, there is a continuous decrease in the rate of ATP
turnover through the normal influence of temperature on

20
biochemical reactions (Jolley et al., 1981). If the
temperature is further reduced to 0°C, energy metabolism is
accelerated through a process called cold shortening of
sarcomeres. First observed in red meats (Locker and
Hagyard, 1963; Marsh and Leet, 1966), this phenomenon has
also been reported in avian muscle (Smith et al., 1969;
Whiting and Richards, 1975; Wood and Richards, 1974a). It
would be expected that since cold shortening accelerates
postmortem metabolism and the postmortem metabolic rate
determines the rate of rigor onset, that cold shortening
would accelerate the rate of rigor mortis development.
Because in commercial poultry processing plants, prerigor
poultry is rapidly chilled to temperatures near freezing,
cold shortening is a possibility.
The mechanism of cold shortening is fairly well
understood although there still are some obscure points.
Cold shortening can be explained by changes at early-
postmortem low temperatures in the lipoprotein systems of
membranes. Hamm (1982) postulated that low temperatures
inactivate the Ca+2 sequestering ability of the SR and/or
increase the permeability of the mitochondria to Ca+2.
This causes Ca+2 ions to "leak" into the myofibrillar
spaces. The increased concentration of Ca+2 in the
sarcoplasm coupled with the considerable amount of ATP
still present at this early postmortem time results in

21
muscular contracture prior to rigor mortis onset (Davey and
Gilbert, 1974; Cornforth et al., 1980; Locker, 1985).
Marsh and Leet (1966) reported that the shortening/
toughening relationship was not linear. The increases in
toughness resulting from decreasing sarcomere length were
small at low degrees of shortening but increased rapidly
thereafter. At 40$ shortening the meat was 4 times the
toughness of the unshortened control. Above 40$
shortening, sarcomere length decreases actually decreased
toughness by disrupting cellular structure.
Despite the fact that prerigor excision toughens meat,
there is some debate on whether it does so by actually
accelerating the onset of rigor mortis. De Fremery and
Pool (I960) and Peterson and Lilyblade (1979) showed that
prerigor muscle excision was accompanied by an accelerated
rate of ATP depletion and pH decline, leading to a faster
onset of rigor mortis. These authors thought that the
toughness of the resulting meat was caused by the
accelerated postmortem metabolism. Their ideas were
supported by Tarrant (1977) who, working with beef,
reported that hot-boning increased the rate of glycolysis
and toughened the meat.
Conversely, Stewart et al. (1984a) reported that
prerigor excision actually slowed the rate of pH decline
which indicated a slower rigor development rate. These
authors still reported a toughening effect with prerigor

22
excision, probably as a result of increased sarcomere
shortening. They proposed that the removal of the muscle's
attachments allows the muscle to contract unimpeded,
temporarily reducing the need for ATP and thus reducing the
glycolysis needed to produce it.
The rate of rigor mortis development is also
influenced by antemortem electrical stunning. This
widespread processing technique is used to reduce carcass
blood retention and as a means of immobilization to improve
the cutting efficiency of the killing machine (Kuenzel and
Walther, 1978; Kuenzel et al., 1978). Ma and Addis (1973)
reported that pectoralis superficialis from stunned turkeys
required more than twice as long to develop rigor mortis as
that from freely struggling controls. Because stunning
eliminates much of the death struggle, it slows postmortem
ATP depletion and therefore slows rigor development
(de Fremery, 1966).
Another consideration in rigor mortis development rate
is the specie involved. In general, the larger the
species, the slower will be its rigor mortis development
rate (de Fremery and Pool, I960). Considering mammals,
rabbits develop rigor mortis at 1.5 to 4 hours postmortem
(Bendall, 1951) while the whale requires 14 to 50 hours
(Marsh, 1952), beef 10 hours (Marsh, 1954) and pork 6 to 9
hours (Busch et al.,
1972). The broiler is slightly slower

23
than the rabbit, needing 2 to 5 hours (de Fremery and Pool,
I960; Whiting and Ricnards, 1975).
It is interesting to note that, as these species get
larger and their corresponding rigor development rates get
slower, there is a general trend for their muscles to have
increasingly red fiber type distributions (George and
Berger, 1966). This comparison across species depends, of
course, on the muscles being compared as the fiber type
composition of an animal varies between its muscles (George
and Berger, 1966; Cassens and Cooper, 1971).
Rigor mortis development has been measured in many
ways, yielding varying results. Busch et al. (1972)
reported that isotonic and isometric tension development by
prerigor muscles commenced and maximized simultaneously.
Direct measurements of loss of extensibility or increased
shortening, by isometric tension, isotonic tension or
tactile evaluation, are complex and time consuming.
Therefore, easier, indirect methods such as pH decline or
ATP depletion have been used as estimates. Very little
research has been done using either direct or indirect
methods, on the development of rigor mortis in broilers
processed in a commercial-type manner (i.e., electrical
stunning and exsanguination). Due to the considerable
effect of antemortem stunning on the rigor mortis
development rate, it would not be prudent to use data from

24
studies on unstunned birds to explain the behavior of
muscles from today's stunned broilers.
Immediately postmortem, there is a period in which no
shortening or extensibility loss occurs called the delay
phase (Schmidt et al., 1968). After this comes the onset
phase in which the muscle shortens and loses
extensibility. After the maximum isometric tension is
reached, a gradual decline occurs, signifying the
resolution phase of rigor mortis.
The upward sloping of the onset phase of a plot of
isometric tension-vs-postmortem time illustrates the
statement by Hamm (1982) that all fibers in a muscle do not
develop rigor mortis at the same time. The ATP depletion
rate, and therefore the rate of rigor development, for an
individual fiber depends mostly on that fiber's location
and type. The superficial fibers would be expected to cool
faster and therefore have slower rates of metabolism and
rigor development simply due to the effect of cooling on
biochemical reactions. Also, because the different fiber
types have drastically different capacities for anaerobic
metabolism (George and Berger, 1966; Cassens and Cooper,
1971), their potential for survival in the postmortem
period would also be expected to be different. In the plot
of isometric tension-vs-postmortem time, the differences in
maximum tension developed between muscles are mainly due to
the fiber type compositions of the muscles with

25
predominantly red muscles developing more tension than
predominantly white ones (Busch et al., 1972; Whiting and
Richards, 1975). With rigor development so dependent on
the fiber types of the muscle involved, it seems necessary
to study rigor development in the individual fiber types
instead of muscles having simply a majority of one fiber
type or the other.
Muscle Fiber Types
The existence of different kinds of muscles, based on
their color, is commonly accepted. An example would be the
common reference to light and dark poultry meat.
Lorenzini, in 1678, is credited with being the first to
describe the difference in muscles due to their color
(Cassens and Cooper, 1971). By the time of Ranvier in
187-4, these muscles were also found to have different
contractile properties (Cassens and Cooper, 1971). He
associated redness with quickness of contraction and
whiteness with slowness. Classifying muscles as red or
white based on gross examination leads to erroneous
conclusions because this classification neglects the more
important structural and biochemical differences between
the muscle fibers (George and Berger, 1966). Therefore it
should be considered that the color of a muscle is due to
its degree of heterogeneity and is a reflection of the

26
characteri
;ics of the individual fibers comprising that
muscle (Ce
sens and Cooper, 1971).
Sever
. fiber classification schemes have been used to
describe t
: various types observed in research. These
schemes he
j been based on the presence or absence of
various er
â– 'tnes or on the fiber's contractile properties.
This has ]
i to confusion in the literature and difficulty
in compari
l studies. Needham (1926) reported on the
histochemi
il and contractile properties of the two fiber
types knov.
at the time, as red and white. Ogata (1958)
and Ogata
id Mori (1964) observed a third fiber type that
was intern
iiate between red and white in oxidative
capacity.
Itein and Padykula (1962) demonstrated three
fiber type
(A, B, and C) based on the level of succinate
dehydroger
•e (SDH) in the fiber. Another histochemical
classifica
on was that fibers that were rich in
dehydrogen
es and poor in phosphorylases were called type
I while th
e with the opposite enzyme compliment were
called typ
II (Dubowitz and Pearse, 1960a, b). This
system als
accounted for a third fiber type that was
intermedia
in its enzyme supply between these two
extremes.
amaha et al. (1970) identified three types (a,
6 and afJ)
cording to their myosin-ATPase activity. The
underlying
law in these systems was that they failed to
account fc
both the biochemical and contractile
difference
of fiber types in a single nomenclature system.

27
Ashmore and Doerr (1971a) proposed a classification
system in which a fiber's contractile and histochemical
characteristics were both represented. This system
combined the red (R) and white (W) typing based on
metabolism using the SDH activity, with fast-twitch (a) and
slow-tonic (0) based on contractile properties using the
myosin-ATPase activity. Thus, they proposed aW to be the
white, fast-twitch, anaerobic fibers and 0R to be the red,
slow-tonic, aerobic fibers. The aR designation was given
to the "intermediate" fibers denoting their intermediate
properties. Because this system was designed for studying
chickens and because previous systems were found to be
limited in their applicability across specie lines, Ashmore
and Doerr (1971b) studied the fibers of chicken, mouse,
bovine and porcine muscle and found the aW, 3R, aR system
to be valid. Since these studies, the Ashmore and Doerr
(1971a) nomenclature system has become the system of choice
for most muscle fiber type researchers.
Regardless of the nomenclature system used, the
differences between the three main fiber types can be
segregated into three areas: structural, biochemical, and
physiological. Structurally, the aW fibers are large in
diameter, have a well developed sarcoplasmic reticulum and
are poorly supplied with capillaries (Gassens and Cooper,
1971; Ashmore and Addis, 1972). The 0R fibers are just the
opposite while the aR fibers are intermediate in these

28
characteristics. These differences are logical since the
aW fibers do not depend on oxygen for their metabolism.
Thus, blood supply and oxygen diffusion are minimized in
the aW fibers by the lack of capillaries and large
diameter, respectively. The opposite is the case for the
BR fibers. Also, mitochondria, the primary site of
oxidative metabolism, are scarce in the aW fibers but are
abundant in the BR fibers. Because the aW fibers are fast
contracting, they would be expected to, and indeed do, have
a well developed system for rapidly dispersing large
quantities of the agent initiating contraction, Ca+2
ions. This is why the aW fibers have an extensive SR while
the BR do not.
Biochemically, the aW fibers are rich in glycolytic
enzymes, glycogen, ATP and creatine phosphate while being
poor in myoglobin, oxidative enzymes and lipids with BR
fibers being the opposite (George and Berger, 1966; Cassens
and Cooper, 1971; Ashmore and Addis, 1972). The
physiological characteristics of the fiber types are a
result of the structural and biochemical features. Muscles
requiring rapid but short-lived activity have a high
proportion of aW fibers. These fibers are suited for this
kind of activity. For a fiber to contract rapidly, it
would need to have a means of quickly dispersing Ca+^ ions
and a large supply of readily available energy, hence the
developed SR and the large supply of ATP, creatine

29
phosphate and glycogen. As would be expected, however, in
a fiber that depends on glycolysis for rapid contraction,
the lactic acid resulting from extensive glycolysis
severely limits the length of the sustainable
contraction. Likewise, the muscles requiring sustained but
not necessarily fast contraction would have a predominance
of BR fibers. These fibers are more suited for a gradual
tension development that can be maintained. With little
lactic acid production and plentiful lipid, the rich source
of energy for ATP production, the aerobic metabolism can be
continued at length. The slow release of Ca ions from
the less extensive (than the aW) SR in conjunction with the
aerobic metabolism impart the BR fibers with their slow-
tonic contractile properties.
In view of the vast metabolic and functional
differences between fiber types, it would be expected that
a muscle's fiber types would also have dissimilar
postmortem behavior. An example of one such distinction is
the difference in cold shortening between red and white
muscles. Bendall (1975) reported that red muscles are more
susceptible to cold shortening. The red muscles have a
predominance of red fibers which are richer in mitochondria
than white fibers. Because at least some of the Ca+^ ion
release causing cold shortening is from the mitochondria
(Hamm, 1982), the red fibers, having more mitochondria,
would be more prone to cold-shortening. Furthermore,

30
Lawrie (1986) postulated that whatever Ca + 2 ions were
released would be less effectively removed from the
sarcoplasm in a fiber whose SR was less extensively
developed. Fawcett and Revel (1961) reported that the red
fiber SR was less developed than the white fiber SR. Also,
Newbold (1980) showed that the SR of the red fiber
accumulated Ca + 2 ions less efficiently than that of the
white fibers.
An alternate theory to explain the greater cold
shortening in red fibers results from the work of Graeser
(1977) and Newbold (1979). They reported that the
involvement of the Ca+2 release from the mitochondria was
less significant to cold shortening than the exchanges of
Ca+2 between the sarcoplasm and the SR. Newbold and Tume
(1977), however, reported that this cold induced release of
Ca+2 from the SR was suppressed by inorganic phosphate.
Newbold (1979) later observed that white fibers had more
inorganic phosphate than red fibers and therefore were less
susceptible to cold shortening.
Although there is clear evidence for the involvement
of both theories, more research in this area is needed to
determine the exact mechanism of cold shortening and which
fibers (or muscles) it affects. This need becomes apparent
when one considers that Bendall (1975) has observed severe
cold shortening in porcine longissimus dorsi and Wood and

31
Richards (1974b) reported cold shortening in broiler
pectoralis superficialis, both predominantly white muscles.
Various facets of rigor mortis' development are
expected to differ between red and white fibers. Working
with whole or partial muscles, several researchers have
noted such differences. Several studies have investigated
these differences in relation to stress susceptibility in
pigs, a condition causing pale, soft exudative (PSE)
pork. Cooper et al. (1969) and Sair et al. (1972) found
stress susceptibility to be closely related to an increased
proportion of intermediate fibers. Using porcine vastus
lateralis as a red muscle and longissimus dorsi as a white
muscle, Schmidt et al. (1970) found that the white muscle
had a longer delay phase and a slower rate of tension
development in the onset phase of rigor mortis than the red
fibers. This would seem logical because the white fibers
would not be as adversely affected by the postmortem
anaerobic conditions as the red fibers.
Conversely, Briskey et al. (1962) and Beecher et al.
(1965) reported that porcine red fibers had a longer delay
phase of rigor mortis than white ones. The red fibers
could metabolize for a short time after death, depleting
the oxygen and creatine phosphate, prior to converting to
glycolysis. Furthermore, these red fibers would be less
efficient at glycolysis and therefore would not deplete

32
their glycogen as quickly as the white fibers. Beecher
et al. (1965) also observed lower initial glycogen levels
and faster postmortem glycolysis in white fibers than in
red fibers. These findings were incompatible with the
rigor development results of the same study.
Research on the relationship between fiber type and
rigor mortis development in avian muscle is very limited.
Whiting and Richards (1975) reported faster rigor mortis
development in the broiler pectoralis superficialis (a
predominantly white muscle) than in the biceps femoris (a
predominantly red muscle).
Little research has been done relating a muscle's
fiber type distribution to the subsequent meat quality. In
the research that has been reported, the primary area of
concern is the role of fiber types in the development of
PSE pork. Hegarty et al. (1963) and Didley et al. (1970)
both reported increased toughness in PSE pork. The
influence of fiber type in this relationship may be two¬
fold. First, because PSE pork has more intermediate fibers
than normal pork (Cooper et al., 1969; Sair et al., 1972)
it is logical that PSE pork was found to have a larger
average fiber diameter and a greater glycolytic capacity
(Didley et al., 1970; Sair et al., 1972). The larger fiber
diameter would be expected to contribute to the toughness
of PSE pork as Herring et al. (1965) and Seideman and
Crouse (1986) both reported that increased fiber diameter

33
caused toughness. Also, the greater glycolytic capacity of
muscles of stress-susceptible pigs could cause toughening
because Essén-Gustavsson and Fjelkner-Modig (1985) reported
that an elevation in this capacity resulted in tough pork.
In order to study the effects of the individual fiber
types on the overall properties of heterogeneous muscles
such as the broiler pectoralis superficialis and sartorius,
it is necessary to study the isolated typed fibers of those
muscles. This must be done using a model system because
determining a fiber's type is a process that destroys the
fiber's functional ability. Fedde (1972) reported a model
system in which the contractile properties of two
homogeneous muscles were used as indicators of the behavior
of their respective fiber types in heterogeneous muscles.
The anterior latissimus dorsi was used as the homogeneous
red muscle and the posterior latissimus dorsi was used as
the homogeneous white muscle. The homogeneity of these
muscles was previously confirmed by Ginsborg (I960) and
Hess (1961). However, since Fedde's work nearly fifteen
years ago, many changes have occurred in the commercial
broiler. Genetic selection practices may have
inadvertently altered the fiber type composition of these
model muscles. This would invalidate the use of these
muscles as models. Ashmore et al. (1972) and Ashmore
(1974) reported that the common genetic selection practices
used in livestock production to maximize growth rate and

34
meat yield achieved these goals by increasing the
proportion of intermediate and white fibers.
The system of Fedde (1972) was designed to study
living muscles by disconnecting the latissimus dorsi
muscles from their origins while not severing the nerve or
blood supplies. This same system could be used to study
rigor mortis development in postmortem muscle by
disconnecting the blood flow to the muscle completely.
Since rigor mortis is characterized by the development of
isometric tension, directly measuring this development
would give a more accurate indication of rigor than making
indirect measurements such as pH.
In order to relate the rigor development data from the
homogeneous muscles to the tenderness of meat harvested
early postmortem, it is necessary to know the fiber type
distribution and tension development rates for the muscles
used as meat. In the broiler, the principle muscle of meat
interest is the pectoralis superficialis. Furthermore,
this was the muscle used in the chill boning experiments
which resulted in the previously discussed inconsistencies
in the relationship between muscle pH, rigor mortis
development and meat tenderness (Sams and Janky, 1986;
Thompson and Janky, 1987). This muscle, although primarily
comprised of white fibers, has been found to be
heterogeneous (Chandra-Bose et al., 1964; Ashmore and
Doerr, 1971b).

35
Past and current research has not considered the role
of fiber types in poultry meat quality. As with PSE pork,
fiber types could conceivably have an important effect.
The more rapid rate of rigor mortis development in poultry
than in beef or pork (de Fremery and Pool, I960) suggests
that avian muscle behavior should not be considered as
simply an extrapolation of that of bovine and porcine
muscles. The commercial implementation of such toughening
procedures as hot and chill boning of poultry reinforces
the need for a complete understanding of all factors
affecting meat tenderness.

CHAPTER III
RATE OF RIGOR MORTIS DEVELOPMENT AND DISTRIBUTION
OF FIBER TYPES IN FOUR BROILER MUSCLES
Introduction
There is conflicting evidence regarding the rates of
rigor mortis development in red and white muscle fibers.
Working with porcine muscles, Briskey et al. (1962) and
Beecher et al. (1965) reported that red fibers developed
rigor mortis faster than white fibers. Conversely, Schmidt
et al. (1970) observed that white porcine fibers developed
rigor mortis sooner than red fibers. The conflict might
have resulted in that these researchers selected strips
from muscles of "predominantly" one fiber type on the basis
of muscle color alone. Therefore, the actual fiber type
distribution of the test muscles was not known.
Furthermore, the test muscles used in the conflicting
studies were not the same. Briskey et al. (1962) and
Beecher et al. (1965) both used samples from the light and
dark portions of the semitendinosus while Schmidt et al.
(1970) used the vastus lateralis and the longissimus.
Variations in the proportions of the fibers present should
alter the rate of rigor mortis development in the muscle.
36

37
Literature on the rate of rigor mortis development in
the individual fiber types of avian muscle is very
limited. Using muscle strips, Whiting and Richards (1975)
reported that both the "white" muscle (pectoralis
superficialis) and the "red" muscle (biceps femoris) began
developed rigor mortis shortly after death. The white
muscle required 3 hours while the red muscle required 5
hours to develop maximum tension.
The use of strips from heterogeneous muscles for
determining rigor mortis development does not yield results
that are representative of the true rates of rigor mortis
development of individual fiber types. Each test muscle is
a mixture of fiber types in varying proportions and has a
rigor mortis development rate that is a composite of all
the fiber types present. A more accurate estimation of
fiber type rigor mortis development rates would involve
comparison of two homogeneous muscles, one entirely
composed of red fibers and one entirely composed of white
fibers. Such a comparison would determine the true fiber
type rigor development rates and the difference between
them would most probably be larger than when comparing
fiber rigor mortis development rates in heterogeneous
muscles. It would still be necessary to know the rigor
development rate of the heterogeneous muscles used for

38
meat. Thus fiber type homogeneous muscles would be used
simply as indicators of the behavior of respective fiber
types in heterogeneous muscles.
Fedde (1972) reported the use of a model system in
which the contractile properties of the white fiber
homogeneous muscle, posterior latissimus dorsi, and the red
fiber homogeneous muscle, anterior latissimus dorsi, were
used as fiber type indicators. This system was designed to
study living muscles by disconnecting the muscles from
their origins while not severing their nerve or blood
supply. This same model system could be used to study
rigor development in fiber types of postrigor muscles by
discontinuing the blood flow to the muscle or by excising
the muscle completely. Because rigor mortis is, by
definition, the development of tension, directly measuring
this development would provide an accurate indication of
rigor mortis.
Postmortem temperature is also important to a muscle's
and, presumably, an individual fiber's rigor mortis
development rate. Wood and Richards (1974b) observed
considerable differences in time to maximum tension due to
muscle temperature with broiler pectoralis superficialis
(3 minutes at 0°C and 277 minutes at 23°C). Because
broiler carcasses are currently chilled to temperatures
below 4°C within 1 hour of death, low temperature has ample
time to exert its influence on rigor mortis development.

39
It would appear that research on rigor mortis development
rates should consider the effect of temperature; however,
published studies on fiber or muscle type rigor mortis
development rates were all performed at temperatures above
20°C. No literature is available regarding the nature of
rigor mortis development in the fiber types of the
commercially processed broiler tissues. This information
is essential to the interpretation of muscle behavior in
terms of the rigor mortis development of individual fiber
types and muscles.
The objectives of this experiment were to determine
the rate of isotonic tension development and the rate of
rigor mortis development in the broiler anterior and
posterior latissimus dorsi, pectoralis superficialis and
sartorius; the effect of cold temperatures similar to those
encountered with commercial-type chilling on the rates of
rigor mortis development in these same four muscles; and
the actual fiber type distribution of these same four
muscles.
Materials and Methods
Broiler Selection and Processing
On 4 separate occasions (4 trials), a uniform
population of 10, seven-week-old male broilers (variation
in body weight: < 250 g or 10%) were obtained from a

40
commercial broiler processing plant and transported to the
University of Florida Poultry Science Department where they
were housed for up to 7 days in litter-covered floor pens
and fed a commercial-type, corn-soy finishing diet.
Following a 12-hour feed-withdrawal periods, birds were
individually processed by electrical stunning and
exsanguination.
The anterior and posterior latissimus dorsi, the
sartorius and a pectoralis superficialis strip (10 x 50 x
10 mm), located 2.54 cm from the anterior end of the
muscle, were removed from the right side of each bird
immediately after the completion of a 90-second bleeding
period. The pectoralis strip was cut parallel to the
direction of the muscle fibers. The location of pectoralis
sampling was chosen to approximate the site of normal shear
force sampling. Care was taken to prevent stretching of
the muscles and strip during excision and preparation. All
muscle samples were dissected and attached to the
contraction apparatus within 20 minutes postmortem.
Isotonic tension development patterns of all four
samples were obtained concurrently, immediately after
excision using four channels and transducers on an E&M 6-
channel Physiograph (Narco Bio-Systems, Inc., Houston,
Texas), using a combined method from Busch et al. (1972)
and Wood and Richards (1974b). One end of each muscle
sample was placed in a clamp attached to a glass rod which

41
was fixed approximately 5 cm off the bottom of a
polycarbonate cylinder filled with a phosphate buffer (pH
7.0, 0.15 M). This buffer was recommended over a Tris-
acetate buffer and distilled water by Wood and Richards
(1974b). The other end of each muscle sample was placed in
a clamp which was attached to an isotonic transducer by
means of surgical cotton thread. Tension was maintained on
the muscle sample throughout the tension development using
a 20 g counterweight. Time to maximum shortening was
determined from graphs obtained for each muscle sample.
For each group of 10 birds, two birds were
individually killed and analyzed on each of five
consecutive days. On each day, one bird's samples were
analyzed with the buffer maintained at 24°C. The phosphate
buffer for the samples dissected from the second bird
analyzed each day was maintained at 24°C for 20 minutes,
cooled, and maintained at 4°C for the duration of tension
development to simulate commercial broiler chilling
procedures (Figure 3-1). Buffer temperature changes and
maintenance were achieved by using a closed-system heat
exchange unit immersed in a constant temperature water bath
adjusted to the appropriate temperature. Buffer cooling
rate was approximately 1°C per minute.
This design resulted in four trials of 10 birds each
in two simultaneous and overlapping completely randomized
designs. Two chilling treatments were compared in one

42
10 broilers (in each of 4
replications)
feed withdrawn (12 hours)
stun(Cervin model FS, Minneapolis,
(90 seconds) bleed Minnesota, setting 4)
dissection
attachment
^ (15 min.)
1 o
room temperature (24 C)
(5 min)
/ \
5 broilers
J
rigor mortis development
at 24°C
5 broilers
chilled to 4 C within
one hour postmortem
rigor mortis development
at <4°C
Figure 3-1• Flow diagram of muscle sample collection and
analysis for the isotonic tension development
experiment.

43
design while four muscles were compared in the other
design. Data were subjected to analysis of variance and
differences between means within temperature treatment were
tested for significance with Duncan’s Multiple Range Test,
while differences between means within muscle were tested
for significance with the independent t-test. All
statistical analyses were performed using the computer
programs in the Statistical Analysis System (SAS Institute,
Inc., 1985).
Fiber Typing
From the first trial of 10 birds, the corresponding
muscles or strips from the left side of each bird were tied
to wooden splints at their original lengths, powdered with
talcum, excised, wrapped in aluminum foil and frozen in
liquid nitrogen. Blocks of muscle (1 cm^) were taken from
each muscle sample and mounted for sectioning on a
Damon/IEC Cryostat (International Equipment Company,
Needham, Massachusetts) at -20°C. Sections (12 pm) from
each block were cut, mounted on glass slides, and stained
for acid-stable myosin adenosine triphosphatase (ATPase)
and reduced nicotinamide adenine dinucleotide (NADH)
tetrazolium reductase enzymes with the combination method
of Solomon and Dunn (1986) using the following
modifications for avian muscle: sections were acid-
preincubated at pH 4.15, stained for the reductase enzyme
and then stained for the ATPase enzyme.

44
Red, intermediate, and white fibers were identified by
observing stained sections under an American Optical
microscope (model 1086; Buffalo, New York). Fiber type
composition of each muscle was determined by counting the
number of fibers of each type in ten fiber bundles from
sections of each muscle sample. Fiber type percentage
distributions were calculated and averaged for each muscle
from all carcasses.
Results and Discussion
Fiber Type Distribution
Fedde (1972) reported that the anterior latissimus
dorsi was homogeneously red and the posterior latissimus
dorsi was homogeneously white in their muscle fiber type
distributions. The results of the present experiment
indicated that although the anterior latissimus dorsi was
essentially comprised of only red fibers, the posterior
latissimus dorsi contained a substantial amount of
intermediate fibers (Table 3~1)• This deviation from the
results of Fedde (1972) may have been caused by 15 years of
genetic selection or by the use of different strains of
bird. Fowler et al. (1980) reported that genetic selection
for rapid growth in Japanese quail caused a change in the
distribution of fiber types in the muscles of the birds.
Also, Aberle et al. (1983) observed that the distribution
of fiber types in the muscles of broiler-type birds was

45
Table 3-1. Fiber type distributions (in percent) of
broiler anterior latissimus dorsi (ALD),
sartorius (SART), posterior latissimus dorsi
(PLD) and pectoralis superficialis (PECT).
Muscle
Fiber types
Red
Intermediate
White
ALD
>99
<1
0
SART
32
53
1 5
PLD
2
1 7
81
PECT
0
<1
>99

46
different from that in layer-type birds. Fedde (1972) did
not specify the strain of bird used, so he could have used
layer-type birds whereas broiler-type birds were used in
the present experiment.
For maintenance of the model system for studying rigor
mortis development, the pectoralis superficialis proved to
be essentially comprised of only white fibers (Table
3-1). The differences between these results and earlier
reports of pectoralis superficialis muscle fiber type
heterogeneity (Chandra-Bose et al., 1964; Ashmore and
Doerr, 1971b) might have been caused by the same genetic
selection and strain effects previously discussed for the
posterior latissimus dorsi. The homogeneity of the
pectoralis superficialis allowed it to model the behavior
of a white fiber while the anterior latissimus dorsi
modeled the behavior of a red fiber.
The broad distribution of fiber types observed in the
sartorius (Table 3~1 ) allowed this muscle to indicate the
behavior of a heterogeneous muscle that was predominantly
comprised of red fibers. Conversely, the posterior
latissimus dorsi would have exemplified the behavior of a
heterogeneous muscle that was predominantly comprised of
white fibers.
The four muscles tested represented a range of
possible fiber type distributions, from the entirely red
anterior latissimus dorsi, through the two heterogeneous

47
muscles of opposite predominances, to the entirely white
pectoralis superficialis (Table 3~1)• Because different
fiber types have different metabolisms (George and Berger,
1966; Ashmore and Doerr, 1971b; Cassens and Cooper, 1971)
muscles that had different compliments of these fiber types
were also expected to have different metabolisms.
Isotonic Tension Development
Within each temperature level, the time required to
develop maximum shortening (i.e., to develop rigor mortis)
was significantly shorter for the predominantly red,
aerobic muscles than for the predominantly white, anaerobic
muscles (Table 3_2). These results agreed with those of
Schmidt et al. (1970) who studied porcine vastus lateralus
as a predominantly red muscle and porcine longissimus dorsi
as a predominantly white muscle. White fibers and muscles
predominating in white fibers have been reported to have
greater glycogen supplies and glycolytic capacities than
red fibers and muscles predominating in red fibers (George
and Berger, 1966; Cassens and Cooper, 1971). The more
anaerobic white muscles would, therefore, be able to
continue producing sufficient adenosine triphosphate (ATP)
to sustain themselves in the absence of bloodflow and
oxygen. Conversely, once the oxygen supply was depleted,
the lack of adequate glycolytic potential in the red fibers
would leave them with decreased means of ATP production,
causing them to enter rigor mortis.

48
Table 3~2. Time (hours) required for development of
maximum tension by broiler anterior latissimus
dorsi (ALD), sartorius (SART), posterior
latissimus dorsi (PLD), and pectoralis
superficialis (PECT) held at room temperature
(24°C) or chilled under simulated commercial
conditions (20 minutes, 24°C; duration, 4°C).
Muscle
Temperature
treatment
ALD
SART
PLD
PECT
Room temperature
3.52b
3 • 55b
5.31a
5.43a
Chilled
4.1 8b
4.65ab
5.67ab
6.08a
a,b Means within a temperature treatment with different
superscripts are significantly different (p<0.05).

49
Simulated commercial-type chilling significantly
slowed rigor mortis development in red muscles but not in
white muscles (Table 3~3)« This slowed rate of rigor
mortis development was expected due to the normal slowing
effect that temperature reduction has on biochemical
reactions (Hamm, 1982). These results were consistent with
the findings of Jolley et al. (1981), who observed that
bovine muscle had slower postmortem metabolism, as
indicated by a slower pH decline, at 5°C than at 30°C.
Hamm (1982) interpreted this slower metabolism as a slower
rate of ATP depletion and therefore a slowed rate of rigor
mortis development.
Cold shortening and its accompanying acceleration of
postmortem metabolism was probably not an important factor
because the muscle temperature was maintained at 4°C.
Honikel et al. (1981b) reported that no cold shortening
occurred in bovine muscles held at or above 4°C.
The fact that rigor mortis development was slowed more
in red muscles than in white muscles indicated that aerobic
metabolism was affected more than anaerobic metabolism by
the temperature reduction. An explanation for this
observation might involve differences in cellular
organization between red and white muscle cells. Aerobic
metabolism occurs partially in the cytoplasm and partially
in the mitochondrial matrix (Stryer, 1981). However,

50
Table 3_3. Time (hours) required for development of
maximum tension by broiler anterior latissimus
dorsi (ALD), sartorius (SART), posterior
latissimus dorsi (PLOT, and pectoralis
superficialis (PECT) held at room temperature
(24°C) or chilled under simulated commercial
conditions (20 minutes, 24°C; duration, 4°C).
Muscle
Temperature
treatment
ALD
SART
PLD
PECT
Room temperature
3.52b
3.55b
5.31a
5.43a
Chilled
4.1 8a
4.65a
5.67a
6.08a
a,b Means within a muscle with different superscripts are
significantly different (p<0.10).

51
anaerobic metabolism occurs only in the cytoplasm (Stryer,
1981). Thus, aerobic metabolites must cross the
mitochondrial membranes as well as overcome diffusion
limitations in order to react. This is contrasted by the
anaerobic metabolites which are only diffusion limited
within the cytoplasm. Stryer (1981) furthermore reported
that low temperatures decreased the fluidity of biological
membranes by partially solidifying their lipids. This
decreased fluidity could decrease permeability, thereby
slowing aerobic metabolism without affecting the rate of
anaerobic metabolism. Further evidence for low temperature
membrane changes was provided by Hamm (1982) who reported
that disrupting the lipoprotein systems of membranes at
temperatures near 0°C increased the permeability of
membranes. If the temperature was low enough to allow some
membrane lipid solidification but not so low as to disrupt
the actual membrane structure, the permeability of a
membrane would decrease. Honikel et al. (1981b) and Jolley
et al. (1981) reported that 4°C was the temperature below
which postmortem metabolism was drastically accelerated and
cold shortening of sarcomeres was evident. It was possible
that while 4°C was not low enough to increase membrane
permeability by membrane disruption, it was low enough to
decrease membrane permeability by lipid solidification.

CHAPTER IV
BIOCHEMICAL ASPECTS OF RIGOR MORTIS DEVELOPMENT
IN BROILER MUSCLES
Introduction
Lowe (1948), de Fremery and Pool (I960), and many
others reported that prerigor muscle excision caused meat
toughness, as measured by increased shear force. Red meat
scientists have reported very good correlations between
muscle pH at time of boning and shear force, and
successfully used pH as an indicator of the development of
rigor mortis and subsequent meat tenderness (Hamm, 1982;
Lundberg et al., 1987). Stewart et al. (1984b), using the
spear probe method, and Sams and Janky (1986), using the
iodoacetate method of pH determination, both reported
nonsignfiicant correlations between pH and shear force in
broiler pectoralis superficialis. These findings suggest
that the postmortem metabolism determining rigor mortis
development rate is different for red meat than for poultry
breast meat.
Postmortem metabolism differences between red and
poultry meats would not be surprising considering that they
are drastically different in fiber type distribution
(Ashmore and Doerr, 1971a, b). Red meat, possessing a
52

53
large percentage of aerobic fibers, would operate much
differently in the anaerobic environment of postmortem
muscle than poultry muscle, observed in Chapter III (Table
3-1) to be mainly comprised of anaerobic fibers.
In postmortem muscle, ATP is continually depleted as
the muscle cells strive to survive. Some researchers have
defined a specific muscle adenosine triphosphate (ATP)
concentration as the point at which the cell develops rigor
mortis (Honikel et al., 1931 a). The anaerobic conditions
associated with postmortem muscles force cells to depend on
glycolysis for replenishing ATP. Because glycogen is
metabolized to lactic acid during glycolysis, lactic acid
production, usually measured as pH, has frequently been
used as a convenient indicator of the endpoint of the
entire spectrum of postmortem biochemical metabolism. Such
"blanket" indicators are frequently not as accurate or
applicable as their users imply. The application of a
"blanket" indicator across fiber types is not very prudent
as these fiber types have very different metabolisms.
Measuring the levels of the principle metabolites in
postmortem muscle (ATP, glycogen and lactic acid) would be
more accurate than measurement of the "blanket" indicator,
pH. A difference between red and white fibers in their
rate of metabolite change over the time course of rigor
mortis development would be expected and might, at least
partially, explain the differences in correlations in pH,

54
shear force, and rigor mortis development between red and
poultry meats.
The objective of this experiment was to determine the
postmortem pattern of ATP depletion, glycogen depletion and
lactic acid production, within white and red fiber types
and in heterogeneous broiler muscles of different fiber
type composition.
Materials and Methods
Broiler Rearing, Selection, and Processing
Cobb, feather-sexed, male broilers were reared in
litter-covered floor pens and fed a commercial-type corn-
soy diet. At 55 days of age, broilers weighing between
1800 and 2300 g were cooped (8 broilers/coop) and held for
12 hours prior to processing, to simulate commercial feed
withdrawal procedures. Sixty-four birds (in 4 replicates
of 16 birds each) were electrically stunned, exsanguinated,
scalded, picked and eviscerated.
R Value
Immediately following evisceration (20 minutes
postmortem), the anterior and posterior latissimus dorsi
muscles, the sartorius, and a pectoralis superficialis
strip (10x50x10 mm), located 2.54 cm from the anterior end
of the muscle, were excised from both sides of two
carcasses per replication. These samples were immediately
minced, identified, placed in plastic bags, frozen in

55
liquid nitrogen and held at -40°C for later analysis. The
remaining 14 carcasses per replication were chilled in
agitated tap water at 21 °C for 20 minutes and 1 °C for 20
minutes. Chilling water agitation was achieved by pumping
the water from the bottom of the chill tanks directly to
the top of the chill tanks. Samples from the same four
muscles as previously described were removed from two
chilled carcasses (40 minutes postmortem) and treated as
described above. The remaining chilled carcasses were
packed in crushed ice and held at 4°C. Sampling was
repeated on two additional carcasses per replication at 1,
2, 3, 4, 6 and 8 hours postmortem (Figure 4-1).
Adenosine triphosphate depletion was monitored by
analyzing each liquid nitrogen-frozen sample for ATP and
inosine monophosphate (IMP) using methods of Khan and Frey
(1971) and Honikel and Fischer (1977) as modified for
poultry by Thompson and Janky (1987). Approximately 3
grams of frozen, powdered meat were homogenized in 20 ml of
1 M perchloric acid for 1 minute using a Virtis 23
(Gardiner, New York) homogenizer at 13,800 rpm. Following
gravity filtration (6 pm paper), 0.1 ml of the acid
filtrate was added to 4 ml of 0.1 M phosphate buffer
(pH 7.0). Absorbances at 250 and 260 nm were measured on a
Hitachi Perkin-Elmer spectrophotometer (Maywood, Illinois)
with a slit width of 0.5 mm. R values were calculated as
absorbance at 250 nm divided by the absorbance at 260 nm.

16 broilers (in each of 8
replications)
(90 seconds) bleed
_ pi ck
2 ^carcasses r â– 
sampled ^
(20 mi n)
feed withdrawn (12 hours)
stun (Cervin model FS, Minneapolis,
2 carcasses
sampl ed^l
(40 min)
2 carcasses-^
sampled
(1 hour)
2 carcasses'
sampled
(2 hours)
2 carcasses
sampled
(3 hours)
pre-chi 11
(tap HO)
1
chill (1°C)
packed in ice
â–¼
! carcasses
sampled
(4 hours)
Minnesota, setting 4)
2 carcasses
sampled
(8 hours)
2 carcasses
sampled
(6 hours)
Figure 4-1. Flow diagram of muscle sample collection for
the R value experiment.

57
The experiment was repeated, resulting in two trials
of 64 birds each. Four muscle types were sampled at each
of 8 different postmortem times. Changes in R value over
postmortem time were subjected to stepwise linear
regression to characterize the pattern of change using
programs available in the Statistical Analysis System (SAS
Institute, Inc., 1985). Because there was no significant
sampling time x trial interaction, data from the two trials
were pooled.
Glycogen and Lactic Acid Concentrations
Broiler processing and muscle sampling procedures
described above were repeated on two additional groups,
each of 16 birds, similar to the birds used above. Samples
from one group of birds were analyzed for lactic acid
concentration at the previously described postmortem
sampling times while those from the other group of birds
were analyzed for glycogen at these same sampling times.
For each metabolite, two birds were sampled at each of the
eight sampling times.
Lactic acid levels were measured with a lactic acid
kit and procedure available from Boehringer Mannheim
Biochemicals (Indianapolis, Indiana). Lactic acid (L-
lactate) was oxidized by nicotinamide adenine dinucleotide
(NAD) to pyruvate by L-lactate dehydrogenase. The
equilibrium of the reaction, however, was almost completely
toward the lactate. By removing the pyruvate from the

58
reaction, the equilibrium was shifted to the right.
Therefore, pyruvate was subsequently involved in the
transamination of L-glutamate to form L-alanine and a-
oxogluterate, which was catalyzed by glutamate-pyruvate
transaminase (GPT). The amount of reduced nicotinamide
adenine dinucleotide (NADH) formed was stoichiometric with
the amount of L-lactic acid in the sample. The increase in
NADH was indicated by the absorbance change at 3^0 nm.
Glycogen concentrations were measured with a starch
analysis kit and procedure available from Boehringer
Mannheim Biochemicals (Indianapolis, Indiana). This kit
utilized amyloglucosi dase to hydrolyze the glycogen to
glucose which was changed to glucose-6-phosphate (G-6-P) by
hexokinase. In the presence of G-6-P dehydrogenase, G-6-P
was oxidized by nicotinamide adenine dinucleotide phosphate
(NADP) to gluconate-6-phosphate while NADP was reduced to
reduced nicotinamide adenine dinucleotide phosphate
(NADPH). The amount of NADPH formed in this third reaction
was stoichiometric with the amount of glucose in the
sample. The amount of NADPH formed was determined by
measuring the absorbance change at 3^0 nm.
Changes in glycogen and lactic acid over postmortem
time were subjected to stepwise linear regression to
characterize the pattern of change using programs available
in the Statistical Analysis System (SAS Institute, Inc.,
1 985) .

59
Results and Discussion
R Value
The anterior latissimus dorsi, composed of only red,
aerobic fibers (Chapter III), had the highest initial R
value (Figure 4-2). The two muscles with intermediate
fiber type distributions had intermediate initial R values
with the more red, aerobic sartorius having a higher R
value than the more white, anaerobic posterior latissimus
dorsi. The pectoralis superficialis, entirely comprised of
white fibers, had the lowest initial R value.
After the death of an animal its muscle cells continue
to hydrolyze adenosine triphosphate (ATP) in an effort to
sustain themselves (Hamm, 1982). This hydrolysis, through
various intermediate steps, eventually results in the
production of inosine, inosine monophosphate (IMP) and
hypoxanthine (Khan and Frey, 1971; Calkins et al., 1983).
The postmortem conversion of the adenosine compounds to
inosine compounds continues until insufficient ATP is
present to prevent actomyosin formation (Hamm, 1982). This
formation causes sarcomere shortening, tension development
in intact muscles and is thought to indicate rigor mortis
development (Busch et al., 1972; Hamm, 1982).
R value is a ratio of two absorbances and is used as
an indicator of the general energy status of a muscle cell
(Calkins et al., 1983). The absorbance maxima of adenosine

R VALUE (A250/A260)
Figure 4-2. Changes in R value over postmortem time in the broiler anterior latissimus
dorsi (ALD), sartorius (SART), posterior latissimus dorsi (PLDT, and
pectoral is superficial is (PECT). (Arrows indicate the time required for
maximum isotonic tension development by the respective, chilled muscles (from
Table 3-2).)
CTv
O

61
nucleotides are at 260 nm and the absorbance maxima of
inosine nucleotides are at 250 nm (Calkins et al., 1983).
Because adenosine is converted to inosine over postmortem
time, R value, the ratio of the absorbance at 250 nm to the
absorbance at 260 nm increases with postmortem time (Khan
and Frey, 1971; Calkins et al., 1983).
The initial R value levels for the various muscles
tested (Figure 4-2) reflected the predominance of aerobic
or anaerobic metabolism in the muscles. Aerobic metabolism
yields substantially more ATP molecules from a single
glucose molecule than does anaerobic metabolism (Stryer,
1981). Therefore, aerobic fibers require less ATP,
relative to the amount of inosine nucleotides, because they
are capable of rapidly producing ATP. This is contrasted
by higher levels of ATP, relative to the level of inosine
nucleotides, in white fibers because their anaerobic
metabolism is less efficient in ATP production. This
contrast would have resulted in aerobic fibers having
greater initial R values than anaerobic fibers. The
relationship between predominant metabolism and R value
could be extended to the level of the whole muscle by
considering the fiber type distributions of the muscles
(Figure 4-2). The muscle with the greatest aerobic
capacity, the anterior latissimus dorsi, had the highest R
value while the most anaerobic muscle, the pectoralis
superficialis had the lowest R value. The two muscles that

62
were intermediate in their metabolisms were also
intermediate in their R value, with the predominantly
aerobic sartorius being greater than the more anaerobic
posterior latissimus dorsi.
Early postmortem, the R values for all muscles
increased (Figure 4-2). In the anterior latissimus dorsi,
sartorius, and posterior latissimus dorsi, the R values
plateaued at approximately 2 hours postmortem and remained
constant for the duration of the test period. However, the
R value for the pectoralis superficialis continued to
increase throughout the test period. The R value changes
over postmortem time (Figure 4-2) indicated that the
depletion of oxygen and cessation of aerobic metabolism
occurred between 1 and 2 hours postmortem. This could be
observed by the location of inflection points at this time
in the R-value lines for the anterior latissimus dorsi and
sartorius. The R values for these muscles showed no
further change after this time due to their dependence on
aerobic metabolism. The pectoralis superficialis and
posterior latissimus dorsi continued to be metabolically
active in the absence of oxygen because of their anaerobic
capacities. Eventually, the R value of the posterior
latissimus dorsi stopped increasing when its anaerobic
capacity had been exceeded. The R value of the pectoralis
superficialis continued to increase throughout the 8-hour
test period. The positive slope of the pectoralis

63
superficialis R value line at 8 hours suggested that it
would have continued to increase well after the test period
ended.
When the respective rigor mortis completion times from
Chapter III (Table 3~1) for chilled muscles (indicated by
the arrows in Figure 4-2) were plotted in combination with
the R value changes in each muscle, a discrepancy between R
values and rigor mortis development times was apparent.
The anterior latissimus dorsi and sartorius, because of
their aerobic natures, would have been the avian muscles
most like bovine and porcine muscles. The R-value lines of
the anterior latissimus dorsi and sartorius showed no
change in R value after 2 hours postmortem. However, rigor
mortis development was not complete until more than 2 hours
later. Discussing bovine muscle metabolism, Hamm (1982)
reported that when the cellular ATP level fell to 1 umole/g
wet tissue, rigor mortis development began. He continued
that this development was complete when the ATP level
reached 0.1 umole/g wet tissue. Thus, rigor mortis
development and completion were associated with decreasing
levels of ATP. Briskey et al. (1962) reported that the
red, aerobic portion of the porcine semitendinosus
completed rigor mortis development, as measured by
extensibility, at approximately 2 hours postmortem. This
corresponds to the time in the present experiment when
oxygen depletion and cessation of aerobic metabolism were

64
evident by inflections in the anterior latissimus dorsi and
sartorius R-value lines. Red porcine muscles therefore
develop rigor mortis when aerobic metabolism stops and ATP
levels fall, all according to the widely accepted red meat
theories of Hamm (1982). The fact that rigor mortis
development in the avian anterior latissimus dorsi did not
occur until over 2 hours after aerobic metabolism stopped
indicated that poultry red muscles did not develop rigor
mortis according to the same metabolic principles as bovine
and porcine muscles. Some undetermined element of
metabolism was apparently able to sustain the red muscles
of the broiler over 2 hours after the stabilization of ATP
concentration. This delay in rigor mortis development was
also observed in the sartorius, another predominantly
aerobic muscle. Even the mainly anaerobic posterior
latissimus dorsi showed evidence of a similar delay with 2
to 3 hours passing between the last substantial R-value
change and the development of rigor mortis.
The pectoralis superficialis continued to exhibit
substantial R-value changes well after the completion of
rigor mortis development. This was inconsistent with the
accepted theory of rigor mortis development in bovine and
porcine muscles (Hamm, 1982). This theory is based on the
contention that the depletion of ATP causes rigor mortis
development. The results of the present experiment
contradict this theory by clearly showing that substantial

65
ATP degradation occurs after rigor mortis development.
Rigor mortis development in avian muscles, then, was not
characterized by the same biochemical events and principles
as noted for bovine and porcine muscles. This suggested
the existence of unknown enzyme or metabolite systems or
new relationships among those already known. Different
metabolisms for avian and mammalian muscles would not be
surprising, considering their evolutionary, physiological,
and biochemical differences. Such a simple difference as
body temperature suggests differences in enzyme activities
and tolerances. The normal broiler body temperature (42°C)
is higher than that of such mammals as cows (38°C) or pigs
(39°C) (Hafez, 1968). Therefore, either avian metabolic
systems are adapted for optimum functioning at elevated
temperatures, or distinct metabolite differences exist
between mammals and birds. An example of the latter is the
differing ways mammals and birds excrete amino nitrogenous
wastes. Mammals excrete these wastes as the water soluble
compound, urea, while birds synthesize purines from their
excess amino nitrogen and then degrade them to form urate
which is excreted as the water insoluble compound, uric
acid (Stryer, 1981).
Differences in the mechanism of rigor mortis
development might well explain the discrepancy between
birds and mammals with regard to the relationship between

66
postmortem metabolism and meat tenderness. Lundberg et al.
(1987) reported that correlations between pH and rigor
mortis development in porcine, ovine and bovine muscles
were so high that only pH needed to be monitored to
indicate rigor mortis development. These authors reported
that, by indicating rigor mortis development, pH alone
could indicate when a muscle could be boned without
increasing toughness. In contrast, Stewart et al. (1984b)
and Sams and Janky (1986) reported nonsignificant
correlations between broiler muscle pH at boning and
subsequent meat tenderness. Stewart et al. (1984b)
suggested that broiler muscle pH and meat tenderness were
not directly related, except through their common
association with postmortem metabolism.
Glycogen Depletion
Initial glycogen concentration was higher in the
anaerobic muscles, pectoralis superficialis and posterior
latissimus dorsi, than in the aerobic muscles, sartorius
and anterior latissimus dorsi (Figure 4-3). The pectoralis
superficialis with its greater predominance of white fibers
had a higher glycogen content than the posterior latissimus
dorsi. The glycogen levels of the two aerobic muscles were
quite similar, despite the differing fiber type
distributions of these muscles.

GLYCOGEN (mg/g MEAT)
POSTMORTEM TIME (HR)
Figure 4-3. Changes in glycogen concentration over postmortem time in the broiler
anterior latissimus dorsi (ALD), sartorius (SART), posterior latissimus dorsi
(PLD)", and ~pectoralis super ficialis (PECTT. (Arrows indicate the time
required for maximum isotonic tension development by the respective, chilled
muscles (from Table 3-2).)

68
Muscle cells maintain stored supplies of energy for
sustenance during times of fasting. When muscular activity
is required by the animal, this energy store is mobilized
and used as fuel for the contraction process. Depending on
the principal mode of cellular metabolism, the energy may
be stored in different forms to compliment the metabolism
and contractile properties of the cell (Hamm, 1982).
Aerobic fibers not only contract slowly with much
force but also are capable of sustaining a contraction for
a long period of time, such as in the maintenance of
posture (Ashmore, 1974). Aerobic fibers use lipid as their
primary energy store (George and Berger, 1966; Cassens and
Cooper, 1971). The energy yield from lipid is much higher
than from carbohydrates so that a large amount of energy
could be derived from a relatively small amount of lipid
(Stryer, 1981). Another aspect of the oxidative use of
lipid as an energy store is more complex and requires more
time than the use of carbohydrates (Stryer, 1981). Thus,
aerobic fibers use lipid as a rich source of energy that is
gradually consumed during chronic activity.
Anaerobic fibers contract very rapidly but with less
force than aerobic fibers (George and Berger, 1966)
maintaining their tension for short times. Because of this
they have been termed "twitch" fibers (George and Berger,
1966). Anaerobic fibers use glycogen as their primary
energy store (George and Berger, 1956; Cassens and Cooper,

69
1971). Glycogen is a carbohydrate and therefore has a much
lower energy yield than lipid (Stryer, 1981). Glycogen
therefore cannot sustain chronic tension but is adequate
for supporting brief contractions. To compensate for its
low energy yield, glycogen, as a carbohydrate, can be
rapidly broken down to its component glucose molecules, an
immediate source of energy (Stryer, 1981). Thus, glycogen
provides the necessary volume of readily available energy
to support a brief contraction.
The initial glycogen levels of the various muscles
tested (Figure 4-3) reflected the predominance of aerobic
or anaerobic metabolism in the muscles. Because glycogen
is the primary energy store of anaerobic fibers, muscles
with a predominance of these fibers had high glycogen
levels. In contrast, predominantly aerobic muscles had low
glycogen levels as aerobic fibers use lipid as their main
energy store.
With the progression of postmortem time, glycogen
stores in the test muscles decreased, with the exception of
the anterior latissimus dorsi which remained unchanged
(Figure 4-3). The glycogen level in the pectoralis
superficialis rapidly decreased, reaching its minimum level
in only 3 hours. Because of its lower initial
concentration, the posterior latissimus dorsi glycogen
supply required only 2 hours to be depleted. The small
amount of glycogen present in the sartorius was depleted in

70
2 hours whereas the anterior latissimus dorsi showed no
change in glycogen concentration throughout the test
period.
The changes that occurred in glycogen levels (Figure
4-3) reflected the predominant postmortem metabolisms of
the muscles. Rapid depletion of the glycogen stores were
observed in the pectoralis superficialis and posterior
latissimus dorsi. This indicated ongoing metabolic
activity by the cell to sustain itself in the anoxic,
postmortem condition. Only slight glycogen level changes
were observed in the partially anaerobic sartorius while no
glycogen metabolism was evident in the anterior latissimus
dorsi.
Plotting the rigor mortis development times from
Chapter III (Table 3-2) in combination with the postmortem
changes in glycogen levels revealed that the time of rigor
mortis development did not coincide with glycogen
depletion. The current theory of muscle biology holds that
once aerobic metabolism stops, glycolysis is the only means
of support for a cell (Hamm, 1982). When glycolysis
ceases, the cell cannot adequately supply itself with ATP
and therefore develops rigor mortis. In the present
experiment the muscles with little glycolytic capacity,
anterior latissimus dorsi and sartorius, were able to
survive for over 4 hours postmortem. This delay in rigor
mortis development occurred despite minimal glycogen use.

71
According to the R value results, no ATP degradation
occurred after 2 hours. Similar results were seen for the
pectoralis superficialis and posterior latissimus dorsi;
however, their high glycogen levels and anaerobic
capacities allowed increased glycolytic activity. Even
with the glycolysis in these anaerobic fibers, about 2.5
hours elasped between the last substantial decrease in
glycogen and the development of rigor mortis.
These findings reinforced the suggestion presented in
the R value discussion that rigor mortis development in
avian muscles fibers was different than that in bovine or
porcine fibers. The possibility for the existence of
unknown metabolites (e.g., energy supplies) or of unknown
relationships between known metabolites was clearly
indicated.
Lactic Acid Production
Initial lactic acid concentration was highest in the
pectoralis superficialis and slightly lower in the less
predominantly anaerobic posterior latissimus dorsi (Figure
4-4). Still lower in initial lactic acid content was the
sartorius. The least anaerobic muscle, anterior latissimus
dorsi, had the lowest initial lactic acid level.
Stryer (1981) reviewed the metabolic role of pyruvate
and lactate in skeletal muscles. The main product of
glycolysis is pyruvate which, under aerobic conditions, is
used by mitochondria in the tricarboxylic acid (TCA) cycle

140
0$ , , , ,
012345678
POSTMORTEM TIME (HR)
Figure Changes in lactic acid concentration over postmortem time in the broiler
anterior latissimus dorsi (ALD), sartori us (SART), posterior latissimus dorsi
(PLD), and pectoralis superficiales (PECfT. (Arrows indicate the time
required for maximum isotonic tension development by the respective, chilled
muscles (from Table 3-2).)

73
to produce ATP. However, under anaerobic conditions, such
as occur with prolonged intense muscular activity or after
the death of an animal, pyruvate is reduced to lactate that
accumulates in the cell. In the living animal, this
lactate can be removed by the circulating blood and
converted back to pyruvate in the liver.
The accumulation of lactate, as lactic acid, decreases
cellular pH and has resulted in the use of pH as an
indicator of postmortem metabolism. The use of pH as such
an indicator in poultry muscle has caused some confusion as
was discussed in Chapter II. Because of this confusion,
the actual metabolite thought to be responsible for the pH
change, lactic acid/lactate, was measured in the present
experiment instead of the more general measurement of pH.
Throughout the test period, the lactic acid
concentration of the pectoralis superficialis steadily
increased (Figure 4-4). The lactic acid levels in the
posterior latissimus dorsi and sartorius increased rapidly
early postmortem but plateaued after 2 hours and remained
constant for the rest of the test period. As with the
glycogen results (Figure 4-3) the anterior latissimus dorsi
had no change in lactic acid concentration for the duration
of the experiment.
The changes that occurred in lactic acid levels
(Figure 4-4) also reflected the predominant metabolisms of
the muscles. No significant increase in lactic acid was

74
observed in the anterior latissimus dorsi over the 8-hour
test period. This was expected because of this muscle's
low glycolytic capacity. The sartorius and posterior
latissimus dorsi had moderate increases in their lactic
acid levels with the rapid, early increase lasting longer
for the more anaerobic posterior latissimus dorsi than for
the more aerobic sartorius. The entirely anaerobic
pectoralis superficial is had the largest increase of all
muscles tested, as was expected due to its large glycolytic
capacity. Unlike the other muscles, the lactic acid level
in the pectoralis superficialis continued to increase
throughout the 8-hour test period. The positive slope of
the pectoralis superficialis line at the end of the test
period suggested that it would have continued to increase
well after 8 hours postmortem. This extended lactic acid
production is similar to the extended ATP metabolism by
this same muscle as indicated by the R value results
(Figure 4-2).
The continued increase in lactic acid levels is in
conflict with the previously discussed glycogen results.
Glycolysis is commonly thought to be the major source of
lactic acid in postmortem muscle (Hamm, 1982). The
glycogen analysis showed no significant change in
pectoralis superficialis glycogen after 3 hours postmortem
while the lactic acid levels continued to increase up to

75
8 hours. Therefore, some other metabolite besides glycogen
was serving as a source of lactic acid.
The similarities between the lactic acid and R value
curves of the pectoralis superficialis suggested a link
between these two parameters. The continued production of
lactic acid could somehow have been used to produce a
continued supply of ATP, the hydrolysis of which was
reflected in the extended increase in R value. Such a
metabolic pathway has not yet been elucidated in anaerobic
systems, but the current knowledge has been primarily based
on mammalian muscles. The possibility of different enzymes
and pathways in avian and mammalian muscles has already
been discussed.
In addition to different enzymes and pathways, the
relationships between known metabolic systems may also be
different for avian and mammalian muscles. The discussions
of Stryer (1981) contain currently accepted biochemical
events that can plausibly be reorganized to explain the
suspected link between the lactic acid and R value
increases observed in the present experiment. In living
animals, proteins are degraded to amino acids of which the
3-carbon species undergo transamination and/or deamination
to form pyruvate. A similar proteolysis is thought to
occur after the death of an animal (Davey, 1983; Dayton
et al., 1 983; Goll et al., 1983). Stryer (1981) continued
that in the absence of aerobic metabolism, as occurs in the

76
postmortem animal, pyruvate would be reduced to lactate
with the simultaneous oxidation of reduced nicotinamide
adenine dinucleotide (NADH) to nicotinamide adenine
dinucleotide (NAD+). The formation of NAD+ is important
because it creates oxidative potential which drives
anaerobic, ATP-yielding pathways such as glycolysis.
Although the supply of the usual substrate for glycolysis
would be expectedly low postmortem, the possibility still
exists for the utilization of the oxidative potential of
NAD+ through some shortened or modified form of glycolysis.
In a discussion of lipid degradation, Stryer (1981)
reported that glycerol and glycolytic intermediates such as
glyceraldehyde 3-phosphate were readily interconvertable.
Thus, lipolysis could serve as a source of glyceraldehyde
3-phosphate, a potential substrate for a shortened form of
glycolysis. Such a system would utilize the oxidative
potential of the NAD+ to produce two ATP molecules. The
NAD+ could then be regenerated by reducing pyruvate to
lactate.
The limitations of this lipid-fueled glycolysis in the
avian pectoralis superficialis are the low level of
intracellular lipid in this muscle and the locations of any
extracellular lipid deposits. The pectoralis superficialis
is an anaerobic muscle and therefore its cells store only a
small amount of their energy supply as lipid (George and
Berger, 1966; Cassens and Cooper, 1971). The intracellular

77
lipid content of this muscle would therefore be expected to
be low, with some lipids existing in the form of
membranes. Furthermore, because avian muscles possess
little intercellular lipid, most of the lipid deposits are
intermuscular. In the postmortem animal without blood
flow, the location of most of the lipid deposits would
require the lipolysis products to diffuse across one or
more membranes, to reach the cytoplasm of the muscle cell
and the site of metabolism. These limitations pose
questions to be answered but do not eliminate the
possibility that yet undetermined entities or relationships
exist which might influence postmortem muscle behavior.
An example of a new relationship might be the enhanced
activity of a compound like pyruvate. Pyruvate is thought
to participate in many metabolic pathways such as the
anabolism and catabolism of carbohydrates, lipids, and
proteins (Stryer, 1981). This compound is frequently the
mode of entry into, exit from, and exchange between these
systems, the possibility of additional metabolic roles for
pyruvate or related compounds does not seem so remote.

CHAPTER V
SUMMARY AND CONCLUSIONS
Red muscle fibers developed rigor mortis faster than
white fibers, probably due to the latter's greater
anaerobic capacity. This trend was also evident in
heterogeneous muscles with predominantly red muscles
developing rigor mortis faster than predominantly white
muscles. These findings may be useful to poultry
processors who must wait until broiler muscles are in rigor
mortis to prevent toughening of the meat during boning. To
increase production efficiency, dark portions of broiler
carcasses could be aged for a different length of time
before boning than the white portions. Broiler breast meat
that is toughened by boning immediately after chiLling (1
hour postmortem) is probably toughened because all of the
muscle's fiber types are still prerigor at 1 hour
postmortem.
Commercial-type chilling significantly slowed the
development of rigor mortis in predominantly aerobic
muscles but only slightly slowed it in predominantly
anaerobic muscles. Differences between the fiber types in
cellular organization may have accounted for the differing
effects of chilling on the fiber types. The slowing
78

79
effects of chilling on rigor mortis development indicated
that results of previous research obtained without
commercial-type chilling should not be used to describe
rigor mortis development in commercially processed
broilers.
R value, glycogen, and lactic acid changes in the four
test muscles were consistent with the postmortem metabolism
of each muscle. These metabolic parameters rapidly
stabilized in the aerobic muscles while they showed
evidence of continued metabolism in the anaerobic
muscles. This indicated that aerobic metabolism stopped
soon after death but anaerobic metabolism continued long
after oxygen depletion.
Adenosine triphosphate depletion, glycogen utilization
and lactic acid accumulation, three events thought to be
directly related to rigor mortis development in bovine and
porcine muscles, were not well related to rigor mortis
development in broiler muscles as measured by isotonic
tension development. In aerobic muscles, rigor mortis was
delayed several hours after the apparent cessation of
metabolism. Conversely, the pectoralis superficialis
provided clear evidence for continued metabolism well after
the completion of rigor mortis development, an event
thought to signify the conclusion of all substantial energy
metabolism.

80
These discrepancies suggested that rigor mortis
development in broiler muscles was not characterized by the
same biochemical events or processes as in bovine or
porcine muscles. This could have accounted for pH/shear
force correlation differences that have been observed
between avian and mammalian muscles. It was possible that
yet undetermined enzymes or metabolic pathways were active
in avian muscle rigor mortis development. Also possible
were the alteration or reorganization of existing pathways,
such as glycolysis, and the enhanced activities of such
important metabolites as pyruvate. Whatever the
explanation, it seems clear from these results that rigor
mortis development in broiler muscles should not be
considered merely an extrapolation of this development in
bovine or porcine muscles.

APPENDIX
REGRESSION EQUATIONS
Regression equations for the curves in Figures 4-2, 4-3 and
4-4.
Figure
Muscle
Regression Equation
4-2
ALD
InR
value =
-0.051 85-0.01 1 99*1 /time
SART
InR
value =
-0.0781 5-0.01 1 30*1 /time
PLD
InR
value =
-0.08032-0.02 3475*1 / time
PECT
InR
value =
-0.1737+0.02934*lntime
4-3
ALD
glycogen = 97.6041-5.6053*time
SART
glycogen = 9
. 4951 +30.0296*1 /time
PLD
glycogen = 1
0. 9765 + 71 . 51 34*1 /time
PECT
glycogen = 1
71 . 31 36+239.81 29*1 /time
4-4
ALD
lactic acid
= 23.441 3+0.8054*time
SART
lactic acid
= 40.022+5.721*lntime
PLD
lactic acid
= 55.659+5.287*lntime
PECT
lnlactic aci
d = 3.9746+0.1178*time
81

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BIOGRAPHICAL SKETCH
Alan Ray Sams was born on November 1, I960, in Grand
Rapids, Michigan, to Chester Lee and Shirley Joan Sams.
After moving to Florida in 1967, the author graduated from
Fort Pierce Central High School in 1978.
Four years later, with a poultry science major and a
food and resource economics co-major, the author received
his Bachelor of Science degree from the University of
Florida. Following his graduation, the author remained at
the University of Florida for one year as a post¬
baccalaureate student in the Poultry Science Department.
The author then began work on a Master of Science degree
which he received one year later with a major in poultry
science and a minor in food science and human nutrition.
After working as a quality control operations analyst
for Gold Kist Poultry, Inc. for one year, the author
returned to the University of Florida to pursue a Ph.D.
degree in food science and human nutrition. The author is
now a candidate for the degree of Doctor of Philosophy.
The author is married to the former Kimberlee Kay
Koppenhoefer of Fort Pierce, Florida. He is also a member
of Poultry Science Association, Institute of Food
Technologists, Sigma Xi, Alpha Zeta, Gamma Sigma Delta, and
Phi Kappa Phi.
91

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
r
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d
D. Janky/, Chairman
Professor of Food S'cience and
Human Nutrition
I certify that
opinion it conforms
presentation and is
I have read this study and that in my
to acceptable standards of scholarly
fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
9Í-
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D. D. Johnson
Assistant /Professor of Animal
Science
I ce
opinion i
presentat
as a diss
rtify that
t conforms
ion and is
I have read this
to acceptable st
fully adequate,
ertation for the degree of
study and that in my
andards of scholarly
in scope and quality,
Doctor of Philosophy.
R. H. Schmidt
Professor of Food Science and
Human Nutrition
I certify
opinion it conf
presentation an
as a dissertati
that I have read thi
orms to acceptable s
d is fully adequate,
on for the degree of
s study and that in my
tandards of scholarly
in scope and quality,
Doctor of Philosophy.
R. L. West
Professor of Food Science and
Human Nutrition

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
yL.j, ’ 7 v ■' V C C cX-i-t iX
SV A. Woodward
Assistant Professor of Poultry
Science
This dissertation was submitted to the Graduate Faculty of
the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
December, 1987
Dean, Graduate School

UNIVERSITY OF FLORIDA



UNIVERSITY OF FLORIDA



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