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

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Title:
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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Rigor mortis   ( lcsh )
Muscles   ( lcsh )
Broilers (Poultry)   ( lcsh )
Food Science and Human Nutrition thesis Ph. D
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Thesis:
Thesis (Ph. D.)--University of Florida, 1987.
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Includes bibliographical references.
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by Alan Ray Sams.
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Typescript.
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Vita.

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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















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


I INTRODUCTION................................... 1

LI 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

II 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










V SUMMARY AND CONCLUSIONS... .....................78

APPENDIX REGRESSION EQUATIONS.......................... 81

REFERENCES................ .. ..............................82

BIOGRAPHICAL SKETCH. ..................................... 91















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 superficialis (PECT) held at
room temperature (240C) or chilled under
simulated commercial conditions (20 minutes,
240C; duration, 40C) ............................ 48

3-3 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 (240C) or chilled under
simulated commercial conditions (20 minutes,
240C; duration, 40C) .................... ......... 50















LIST OF FIGURES


Figure Page

3-1 Flow diagram of muscle sample collection
and analysis for the isotonic tension
development experiment........................... 42

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
pectoralis superficialis (PECT)..................67

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 superficialis (PECT).................. 72















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 (anterior

latissimus dorsi) was used as a model for the behavior of

red fibers while a homogeneous white muscle pectoraliss

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.


vii










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 fiDers than in white ones,


viii










suggesting that rigor mortis development in broiler muscles

could not 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 capital 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.





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










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









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 actually, 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










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

filamentouss actin) wrapped around each other forming a

helix. F-actin is actually a polymer of the more basic

action structure G-actin (globular actin). Attached to the

F-actin helix is the troponin-tropomyosin complex which

functions in the control mechanism of muscular









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+2) 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










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+2 ions. This serves to

inhibit the interaction of the thick and thin filaments.

As long as the Ca+2 concentration in the sarcoplasmic fluid

innervating the myofilaments is low (<10~6 M), interaction

of the filaments is prevented. Upon neurostimulation or

upon the inactivation of the SR pumping system, Ca+2 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 (Pi). 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










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










decrease to about 1 imole/g wet tissue at 20)C and is fully

developed at 0.1 pmole/g wet tissue (Honikel et al.,

1981a). The postmortem time when these conditions exist in

a fiber varies with antemortern 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+2 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.,

1969).



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, 1960; Stewart et al., 1984b). Locker (1960) and

Marsh (1975) both suggested that the extent of sarcomere

shortening (i.e., the degree of interdigitation 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 1000C (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









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










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,

1960; 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 (1960), 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.









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









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 230C.

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, 1960) or 30









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 actually 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









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 370C (at death) to about

100C, there is a continuous decrease in the rate of ATP

turnover through the normal influence of temperature on










biochemical reactions (Jolley et al., 1981). If the

temperature is further reduced to OOC, 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










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 (1960) 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









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, 1960). 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










than the rabbit, needing 2 to 5 hours (de Fremery and Pool,

1960; Whiting and Richards, 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










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










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










characters

muscle (CE

Sever

describe t

schemes he

various er

This has 7

in compare

histochemi

types knov.

and Ogata

was intern

capacity.

fiber type

dehydroger

classifica

dehydrogen

I while th

called typ

system als

intermedia

extremes.

a and ac)

underlying

account fc

difference


-ics of the individual fibers comprising that

sens and Cooper, 1971).

fiber classification schemes have been used to

various types observed in research. These

i been based on the presence or absence of

'mes or on the fiber's contractile properties.

1 to confusion in the literature and difficulty

; studies. Needham (1926) reported on the

il and contractile properties of the two fiber

at the time, as red and white. Ogata (1958)

id Mori (1964) observed a third fiber type that

late between red and white in oxidative

;tein and Padykula (1962) demonstrated three

(A, B, and C) based on the level of succinate

,e (SDH) in the fiber. Another histochemical

on was that fibers that were rich in

es and poor in phosphorylases were called type

e with the opposite enzyme compliment were

II (Dubowitz and Pearse, 1960a, b). This

accounted for a third fiber type that was

in its enzyme supply between these two

amaha et al. (1970) identified three types (a,

cording to their myosin-ATPase activity. The

law in these systems was that they failed to

both the biochemical and contractile

of fiber types in a single nomenclature system.









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 (8) based on contractile properties using the

myosin-ATPase activity. Thus, they proposed aW to be the

white, fast-twitch, anaerobic fibers and aR 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 acW, R, 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 (Cassens and Cooper,

1971; Ashmore and Addis, 1972). The BR fibers are just the

opposite while the aR fibers are intermediate in these









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 aR 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+2 ions

and a large supply of readily available energy, hence the

developed SR and the large supply of ATP, creatine










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 SR 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+2 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+2 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,










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










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









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










caused toughness. Also, the greater glycolytic capacity of

muscles of stress-susceptible pigs could cause toughening

because Essen-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 (1960) 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










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).










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, 1960) 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.












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 pectoraliss

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









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 00C and 277 minutes at 230C). Because

broiler carcasses are currently chilled to temperatures

below 4oC within 1 hour of death, low temperature has ample

time to exert its influence on rigor mortis development.










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

200C. 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










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










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 240C. The phosphate

buffer for the samples dissected from the second bird

analyzed each day was maintained at 240C for 20 minutes,

cooled, and maintained at 40C 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 10C 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



















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.)


room tempi



5 broilers


rigor mortis development
at 24 C


Figure 3-1.


erature (24"C)
(5 min)


5 broilers


chilled to 4 C within
one hour postmortem

rigor mortis development
at <4 C


Flow diagram of muscle sample collection and
analysis for the isotonic tension development
experiment.









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 cm3) were taken from

each muscle sample and mounted for sectioning on a

Damon/IEC Cryostat (International Equipment Company,

Needham, Massachusetts) at -200C. 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.










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 fioer 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













Table 3-1. Fiber type distributions (in percent) of
broiler anterior latissimus dorsi (ALD),
sartorius (SART), posterior latissimus dorsi
(PLD) and pectoralis superficialis (PECT).


Fiber types

Muscle Red Intermediate White


ALD >99 <1 0

SART 32 53 15

PLD 2 17 81

PECT 0 <1 >99










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










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.












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
(240C) or chilled under simulated commercial
conditions (20 minutes, 240C; duration, 4OC).


Muscle
Temperature
treatment ALD SART PLD PECT


Room temperature 3.52b 3.55b 5.31a 5.43a

Chilled 4.18b 4.65ab 5.67ab 6.08a

a,b Means within a temperature treatment with different
superscripts are significantly different (p<0.05).










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 50C than at 300C.

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 40C.

Honikel et al. (1981b) reported that no cold shortening

occurred in bovine muscles held at or above 40C.

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,













Table 3-3.


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
(240C) or chilled under simulated commercial
conditions (20 minutes, 240C; duration, 40C).


Muscle
Temperature
treatment ALD SART PLD PECT


Room temperature 3.52b 3.55b 5.31a 5.43a

Chilled 4.18a 4.65a 5.67a 6.08a

a,b Means within a muscle with different superscripts are
significantly different (p<0.10).










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 00C 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 40C was the temperature below

which postmortem metabolism was drastically accelerated and

cold shortening of sarcomeres was evident. It was possible

that while 4oC 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 (1960), 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










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., 1981a). 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,










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










liquid nitrogen and held at -400C for later analysis. The

remaining 14 carcasses per replication were chilled in

agitated tap water at 210C for 20 minutes and 10C 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 40C. 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)


feed withdrawn (12 hours)
stun (Cervin model FS, Minneapolis,
(90 seconds) bleed Minnesota, setting 4)
2 carcasses pick
sampled 4--
(20 min) pre-chill
2 carcasses (tap H20)
sampled-----
(40 min) chill (10C)


2 carcassess-- ----
sampled packed in ici
(1 hour)
2 carcasses
sampled
(2 hours)
2 carcasses
sampled
(3 hours)
2 carcasses
sampled
(4 hours)


Figure 4-1.


2 carcasses
sampled
(8 hours)


carcasses
sampled
(6 hours)


Flow diagram of muscle sample collection for
the R value experiment.










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










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 340 nm.

Glycogen concentrations were measured with a starch

analysis kit and procedure available from Boehringer

Mannheim Biochemicals (Indianapolis, Indiana). This kit

utilized amyloglucosidase 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 340 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.,

1985).










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






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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










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










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 pmole/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









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










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 (420C)

is higher than that of such mammals as cows (380C) or pigs

(390C) (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











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.
















































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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, 1966; Cassens and Cooper,









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










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.










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







































- (0







-r

-

2


I-


0

*" H
0
a-


l-o


0 0
00 0

(1V3W B/WAn) a13V 3113V1


*r-4

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j a)
00
0 -
- *) -I
0 0 aC



ar-' 3 4-'




a)) :
L" i s co

0 0 rO
.-Q 0 c )



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* .-I 0 ( Q


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0


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0 0
S*.O




O- C


L E >

> L. z1M O



- 4 I- C
** -4 C*



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0 --* oC




0 1 0 *~J
0 6



X3 ( I

0 O 0 *=






c 0 S
so eo
CO OV j Q c
a) -- [ ) o


Xr:C 3
U~~ tf^l, S


'I----- ---------- ---- ---Y~,,,~,.;,,,,


o o


1. 1










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










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 superficialis 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










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., 1983; Goll et al., 1983). Stryer (1981) continued

that in the absence of aerobic metabolism, as occurs in the










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










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










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.










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



Figure Muscle Regression Equation


4-2 ALD InR value = -0.05185-0.01199*1/time
SART InR value = -0.07815-0.01130*1/time
PLD InR value = -0.08032-0.023475*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 = 10.9765+71.5134*1/time
PECT glycogen = 171 .3136+239.8129*1 /time


4-4 ALD lactic acid = 23.4413+0.8054*time
SART lactic acid = 40.022+5.721*lntime
PLD lactic acid = 55.659+5.287*lntime
PECT Inlactic acid = 3.9746+0.1178*time















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BIOGRAPHICAL SKETCH


Alan Ray Sams was born on November 1, 1960, 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.