ELECTROPHORETIC COMPONENTS, pH,
AND CALCIUM, PHOSPHOROUS AND
MAGNESIUM CONTENT OF MUSCLE
AS RELATED TO TENDERNESS
DALE LINWOOD HUFFMAN
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE FrPQir r '.li TS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
The author sincerely appreciates the guidance and assistance
of Dr. A. Z. Palmer in the preparation of this dissertation and
throughout the course of his graduate study.
The author also expresses special thanks to members of his
advisory committee; Drs. J. W. Carpenter, R. L. Shirley, F. W. Knapp
and T. W. Stearns. The assistance of Dr. Marvin Koger in the sta-
tistical analysis of the experimental data is sincerely appreciated.
The author is deeply indebted to D. D. Hargrove, T. N. Meacham, R. E.
Deese, D. D. Bass, G. H. Taki, J. E. Martin, W. A. Tilton and other
graduate students who gave so generously of their time throughout
Gratitude is expressed to Mr. R. A. Newman and Mrs. Barbara
Sullivan for their dedicated assistance in the collection of experi-
mental data. The help and advice of personnel of the Meats Labora-
tory and Nutrition Laboratory is gratefully acknowledged.
Partial support furnished through a grant-in-aid from Armour
and Company, Chicago, Illinois is acknowledged.
Appreciation is extended to Mrs. Dorothy Hurd for the excellent
and accurate typing of this manuscript.
The author is deeply indebted to his wife Jo-Ann for her assist-
ance, encouragement and patience throughout the course of his graduate
TABLE OF CONTENTS
ACKNOWLEDGMENTS ................. ....... ii
INTRODUCTION . . 1
LITERATURE REVIEW . .. 5
The Relationship of Dietary Calcium and Phosphorus
and Tenderness . 5
The Relationship Between Tenderness and Calcium,
Phosphorus and Magnesium in Muscle .. 6
The Relationship Between Tenderness and Added
Phosphates .. . ... 8
The Relationship Between Tenderness and Muscle pH 8
The Relationship Between Electrophoretic Separa-
tion of Muscle Proteins and Tenderness 9
EXPERIMENTAL PROCEDURES . ... 13
Trial 1 . . 13
Trial 2 . . 15
Trial 3 . . .. 17
RESULTS AND DISCUSSION . . .. 29
Trial 1 . . 29
T iial 2 . . 31
Trial 3 . . 34
Effect of Dietary Calcium and Phosphorus on Weight
Gains and Feed Consumption of Mature Sheep 34
The Effect of Feeding a High Calcium Low Phos-
phorus Ration and a Low Calcium High Phos-
phorus Ration on Tenderness of Mutton Chops 34
The Effect of Ante-mortem Injection of Metaphos-
phate on the Tenderness of Mutton Chops 37
The Influence of Animal Age at Time of Slaughter
on the Tenderness of Mutton Chops 39
TABLE OF CONTENTS (Cont.)
The Relationship Between Muscle pH, Dietary Cal-
cium and Phosphorus, Ante-mortem Injection
of Phosphates and Tenderness ... 42
The Effect of Ante-mortem Injection of Metaphos-
phate and Pyrophosphate on Muscle pH 45
The Relationship Between Electrophoretic Muscle
Protein Components and Tenderness .... 47
The Effect of Dietary Calcium to Phosphorus Ratio
on Bone Breaking Strength ... 51
The Effect of Dietary Calcium to Phosphorus Ratio
on Muscle Calcium and the Relationship of
Muscle Calcium and Tenderness ... 54
The Effect of Dietary Calcium to Phosphorus Ratio
on Blood Serum Calcium and the Relationship
Between Blood Calcium and Tenderness 54
The Effect of Dietary Calcium to Phosphorus Ratio
on Muscle Phosphorus and the Relation of
Muscle Phosphorus to Tenderness .. 56
The Effect of Dietary Calcium to Phosphorus Ratio
on Bone Phosphorus and the Relationship of
Bone Phosphorus to Tenderness ... 59
The Effect of Dietary Calcium to Phosphorus Ratio
on Blood Serum Phosphorus and the Relation-
ship Between Blood Phosphorus and Tenderness 59
The Effect of Dietary Calcium to Phosphorus Ratio
on Muscle Magnesium and the Relationship of
Muscle Magnesium to Tenderness ...... 62
Regression Analysis of pH, Muscle Protein Frac-
tions and Mineral Constituents of Bone,
Muscle and Blood on Tenderness ...... 62
Proposed Mechanism of Phosphate Action .... 70
SUMMARY AND CONCLUSIONS . .... .72
Summary . . .... 72
Conclusions . . .. .. 78
APPENDIX . .. . 80
LITERATURE CITED . . 85
LIST OF TABLES
1. Percent Composition of Experimental Rations .
2. Calculated Analysis of Experimental Rations .
3. Calcium and Phosphorus Analysis of Experimental
Rations . . .
4. Calcium and Phosphorus Ratios of Experimental Rations
5. The Effect of Ante-mortem Injection of Sodium Meta-
phosphate on Mutton Tenderness -- Trial 1 .
6. The Effect of Ante-mortem Injection of Sodium Meta-
phosphate on the Tenderness of Mutton Chops Aged
48, 72, 96 and 120 Hours and Mutton Leg Steaks
Aged 48 Hours -- Trial 2 .. . .
7. The Effect of Dietary Calcium and Phosphorus Ratio
on Weight Gains and Feed Consumption -- Trial 3 35
8. The Effect of Dietary Calcium and Phosphorus and
Ante-mortem Injection of Metaphosphate and Pyro-
phosphate on the Tenderness of Mutton Chops --
Trial 3 . . 36
9. Simple Correlation Coefficients Between Tenderness
and Age, pH, Muscle Protein Fractions, Bone
Breaking Strength and Mineral Content in Muscle,
Bone and Blood (33 Animals) -- Trial 3 ..... .40
10. Simple Correlation Coefficients Between Tenderness
and Age, pH and Protein Fractions of Muscle (77
Animals) -- Trial 3 . .. .. 41
11. The Effect of Dietary Calcium and Phosphorus Ratios
and Pyrophosphate on Initial and Ultimate Muscle
pH -- Trial 3 . .... .43
12. The Effect of Feeding Varying Calcium to Phosphorus
Ratios and Ante-mortem Injection of Phosphates on
the Percent of Negative, Stationary and Positive
Protein Phases of Pre-rigor and Post-rigor Muscle
-- Trial 3 . .... .. 49
LIST OF TABLES (Cont.)
13. The Effect of Feeding Varying Calcium to Phosphorus
Ratios on Bone Breaking Strength -- Trial 3 .... 52
14. The Effect of Feeding Varying Calcium to Phosphorus
Ratios on the Calcium Content of Muscle -- Trial 3 55
15. The Effect of Feeding Varying Calcium to Phosphorus
Ratios on Blood Serum Calcium Levels -- Trial 3 57
16. The Effect of Feeding Varying Calcium to Phosphorus
Ratios on the Phosphorus Content of Muscle --
Trial 3 . ... .. 58
17. The Effect of Feeding Varying Calcium to Phosphorus
Ratios on the Phosphorus Content of Bone -- Trial 3 60
18. The Effect of Feeding Varying Calcium to Phosphorus
Ratios on Blood Serum Phosphorus Levels -- Trial 3 61
19. The Effect of Feeding Varying Calcium to Phosphorus
Ratios on the Magnesium Content of Muscle --
Trial 3 . ... .. 63
20. Coefficients For the Most Important Factors Influencing
and/or Associated with Taste Panel Tenderness
(33 Animals) -- Trial 3 . ... 64
21. Coefficients For the Most Important Factors Influencing
and/or Associated with Shear Tenderness (33 Animals)
Trial 3 . ... .. 65
22. Data Obtained from Individual Sheep on Trial 3 .. 81
Tenderness has been and is one of the more elusive palatability
characteristics to either subjectively or objectively identify. Even
in light of voluminous literature on the subject of tenderness, it is
still not possible to visibly distinguish a tender animal, a tender
carcass, or a tender cut on the basis of our present knowledge. Fed-
eral grade standards serve as a fairly reliable guide to tender beef,
but they are not infallible.
Genetic studies have shown that certain strains of cattle are
tender while others are tough. Tenderness has been found to be a
highly heritable trait. Research workers have found a means of tender-
izing beef through ante-mortem injection of enzymes. While these find-
ings are very important they fail to contribute directly to the basic
reason of why tenderness differences exist. Tenderization of each ani-
mal solves the problem for only that animal. Perhaps if the mode of
action of the enzymatic tenderization reaction was clearly understood,
more would be known about the problem of beef tenderness.
The need for research in the area of tenderness is self-evident;
the proper approach to the problem is not as clear. Early workers in
the field of tenderness thought that tenderness was a function of con-
nective tissue. More recently considerable evidence has been presented
to indicate that tenderness differences are less a function of connective
tissue than of the muscle plasma. Wierbicki (1954) defined muscle
plasma as that portion of muscle which consists of elongated cells,
containing jelly-like protoplasm.
Most authors accept that the character of the connective tissue
influences beef tenderness. Evidence indicating an association of
certain muscle plasma characteristics and tenderness was gained largely
through aging studies. It is easily demonstrated that connective tis-
sue changes very little, in most respects, during normal aging.
With this knowledge available then, it would seem that a logical
approach to the tenderness problem would center on the muscle plasma.
Three proteins: actin, myosin and actomyosin make up nearly 50 percent
of the muscle plasma. According to Szent-Gyorgi (1953), resting muscle
is extensible because actin and myosin are dissociated. When muscle
contracts, the actomyosin complex is formed. This is a reversible re-
action which requires enzymes, energy, oxygen and certain ions. Calcium
and magnesium are known to be essential.
When muscle dies, certain changes take place. Oxygen is no longer
available for glycolysis, hence, energy stores are exhausted. Glycogen
is converted to lactic acid which in turn lowers the pH below the opti-
mum level for enzyme activity. Certain changes take place in the ionic
balance which have not been elucidated as yet. The important change
from the standpoint of tenderness is the irreversible formation of the
actomyosin complex which confers upon muscle the characteristic rigid-
ity known as rigor mortis.
It has been shown that muscle is tender immediately after death,
then becomes tougher during rigor mortis. After rigor mortis, muscle
gradually returns to a tender state through the process of aging. The
mechanisms of many reactions that proceed during aging are only par-
tially understood. A challenging thought might be that if rigor mortis
did not occur the muscle would retain its initial degree of tenderness
and perhaps improve with aging.
One possible approach to the interruption of rigor mortis would
be to chelate the magnesium and calcium ions which are known to be
essential for rigor to occur. Such an approach was demonstrated to have
some merit by Kamstra and Saffle (1959) and Carpenter et al. (1961).
These workers made a pre-rigor infusion of metaphosphate and achieved
a marked degree of tenderness. It is possible that the chelation of
magnesium and calcium was not the reason for the tenderness they ob-
served. Perhaps the ionic environment was altered causing changes in
muscle proteins. The salt could have acted as a buffer or possibly the
phosphate was important and another polyphosphate would have had the
same effect. The important consideration is that this technique re-
sulted in an improvement in tenderness.
Since this approach appeared fruitful, it became of interest to
study this tenderizing technique in the live animal. Ante-mortem ad-
ministration of enzymes has been shown to be an excellent technique in
achieving uniform distribution of a tenderizing agent in tissue.
The primary objective of the study to be presented was to determine
the effect on tenderness of ante-mortem injection of sodium metaphosphate
(NaP03)6 (purified) and sodium pyrophosphate Na4P207.10 H20 (reagent).
Two different polyphosphates were used in order to compare their
tenderizing effects. It was considered of interest to find out if
the injection of phosphates brought about changes in the proteins of
the muscle plasma. An additional objective of this study was to frac-
tionate the muscle, immediately after slaughter and 48 hours after
slaughter. The fractionation, as accomplished with paper electrophoresis,
would allow a study of relationships among electrophoretic components,
initial and ultimate pH, and tenderness by taste panel and by Warner-
The second major objective of this study was to determine the
effect of ratios of dietary calcium to phosphorus on tenderness. This
area of study has received little attention judging from the literature.
Hall et al. (1944b) showed a slight improvement in tenderness of aged
roasts from cattle fed on a high phosphorus ration. A difference in
tenderness was not found when cattle were fed a high calcium ration.
It has been observed at the Florida Agricultural Experiment Sta-
tion that cattle from the branch stations seemed to vary in tenderness
depending on location. Some variation in tenderness was found even
when cattle of similar breeding and grade were compared. As previously
indicated, the purpose of this phase was to determine whether or not
dietary calcium or phosphorus affected tenderness. A further purpose
was to determine the effect of dietary calcium to phosphorus ratios
on muscle, bone and blood content of the minerals and to relate any
variations that might be found to tenderness. A study of the relation-
ships that might exist among electrophoretic components of the muscle,
muscle pH, mineral constituents and tenderness was a further purpose
of this study.
The Relationship of Dietary Calcium and Phosphorus and Tenderness.
A search of the literature pertaining to meat quality reveals
little information on the relationship of dietary calcium and phos-
phorus and tenderness. A rather intensive study was carried out at
the Kansas Station between 1929 and 1941 in this area. Hall et al.
(1944b) reported that feeding a high level of ground limestone to
cattle for 250 days resulted in no differences in tenderness. It was
noted in this study, however, that the high calcium lot had higher
blood serum phosphorus and a slightly lower blood serum calcium. Bone
mineral analysis showed that the high calcium cattle had slightly high-
er bone phosphorus and calcium. The breaking strength of cannon bones
from high calcium cattle was markedly higher than the controls.
In a similar study by Hall et al. (1944a) the effect of varying
levels of phosphorus on tenderness and minerals in bone and blood were
studied. Tenderness values for aged rib roasts by panel favored the
high phosphorus steers slightly but no differences were shown by Warner-
Bratzler shear. Blood serum phosphorus was considerably lower for low
phosphorus steers than controls. No difference was shown in blood serum
calcium. There was a marked difference in bone phosphorus and a slight
difference in bone calcium in favor of the high phosphorus steers. The
bone breaking strength was considerably greater for the high phosphorus
Investigations into the relationship between bone breaking strength
and tenderness have not been rewarding. Hall et al. (1944a) reported
both increased tenderness and increased bone breaking strength on one
ration, however, they did not indicate that a relationship existed.
Gilbreath (1959) found low, but significant, correlations between meta-
tarsal breaking strength and tenderness. He found little correlation
between metacarpal breaking strength and tenderness. Carpenter (1959)
reported that there were no significant differences in bone breaking
strength among Brahman, Shorthorn and Brahman Shorthorn crossbreds. He
did report a significant tenderness variation among these breed groups,
however. Alsmeyer (1960) found no significant relationship between
tenderness and breaking strength of metatarsal, however, he reported
a significant correlation between metacarpal breaking strength and ten-
The Relationship Between Tenderness and Calcium, Phosphorus and Magne-
sium in Muscle.
Meat scientists throughout the world have been interested in the
problem of water holding capacity for many years. It was recognized
early that the ionic atmosphere of meat was intimately associated with
protein hydration. More recently there has been speculation by Arnold
et al. (1956) and Locker (1962) that a close association between degree
of hydration and tenderness does exist. The literature does not con-
tain data to verify this speculation; however, Arnold et al. (1956)
states, "The ionic shifts may be related to the degree of hydration of
the muscle protein, and thereby related to tenderness since it is known
that the degree of hydration is related to tenderness."
Several workers have investigated the relationship between calcium,
phosphorus and magnesium and tenderness. Husaini et al. (1950a) indi-
cated that percent phosphorus in muscle ranged from 0.085 to 0.120 on
a wet basis. He reported that there was no correlation apparent be-
tween muscle phosphorus and tenderness. Arnold et al. (1956) used a
different approach to this problem. Using 32 cattle that differed
widely in grade they studied the absolute amounts of calcium and mag-
nesium in meat, water extracts and juice. These workers indicated that
there was a shift of these cations during post-mortem aging from the
protein to the juice. The correlation between the absolute amounts of
the two cations and tenderness at a given time was very low. At three
days post-mortem the correlation between calcium and tenderness was
-0.010 while at 13 days it was -0.287. While the correlations are
very low, it is worthy of note that the apparent shift of calcium from
protein to juice occurred simultaneously with an increase in tenderness.
The relationship did not appear to be as clear-cut with magnesium. Arnold
et al. (1956) concluded from this study that the addition of any electro-
lyte that will increase the overall charge of the meat protein or will
render insoluble the calcium ion, will increase the tenderness.
Wierbicki et al. (1954) reported that a strong relationship existed
between nitrogen content of muscle plasma extract and tenderness. He
was attempting to demonstrate that increased tenderness with post-mortem
age was a function of muscle plasma rather than connective tissue.
Swift and Berman (1959) reported the following significant correlations
between protein content of muscle and calcium 0.827, magnesium 0.806
and phosphorus 0.604.
The Relationship Between Tenderness and Added Phosphates.
Kamstra and Saffle (1959) have investigated the effect of pre-
rigor infusion of sodium hexametaphosphate on ham tenderness. They in-
fused one ham of 20 pairs with varying levels of the phosphate. A
significant increase in tenderness for the phosphate infused hams was
found. Carpenter et al. (1961) infused varying levels of metaphosphate
in pre-rigor cutter and canner cow rounds and found a significant in-
crease in tenderness.
The Relationship Between Tenderness and Muscle pH.
Numerous workers have linked pH with quality factors such as
color, tenderness and water holding capacity.
Bate-Smith (1948) has indicated that glycogen disappearance and
lactic acid increase continues after death until the pH falls too low
(approximately 5.4) for enzyme activity.
Briskey (1959) has suggested that pH may be coincidental with im-
provement in quality attributes rather than directly associated. Bris-
key (1959) further indicated that the initial pH was a result of the
death struggle while the ultimate pH was an indicator of the animals
state of fatigue and level of feeding. He also showed that the initial
pH was not correlated with the ultimate pH.
Husaini et al. (1950a and b) in two separate studies failed to
show a significant correlation between pH and tenderness at three and
13 days post-mortem.
Kamstra and Saffle (1959) and Carpenter et al. (1961) found phos-
phate infused meat to be more tender and also to have a higher pH than
control meat. In an effort to learn the relationship of pH and tender-
ness they combined lactic acid with the phosphate solution before in-
fusion. This technique lowered the ultimate pH but did not lower the
tenderness rating. This is in agreement with the findings of Briskey
(1959) who reported that a relationship between pH and tenderness might
The Relationship Between Electrophoretic Separation of Muscle Proteins
Early workers in the field of meat quality assumed the connective
tissue to be responsible for toughness in beef. In the middle forties
the evidence began to mount in favor of the proteins of the muscle
plasma (connective tissue-free muscle). Reports that tenderness was
not solely a function of connective tissue were published by Deatherage
and Reiman (1946), Deatherage and Harsham (1947), Ramsbottom and Stran-
dine (1949), Husaini et al. (1950a and b) and Winegarden et al. (1952).
Each group of workers approached the problem in a different manner but
with a similar purpose. Husaini et al. (1950b) reported there was
little relationship between the amino acid hydroxyproline, which is
high in connective tissue, and tenderness. It is noteworthy that in
an earlier study by Husaini et al. (1950a) no relationship was found
between tenderness and total nitrogen, non-protein nitrogen, trichloro-
acetic acid soluble nitrogen and heat coagulable water soluble nitrogen.
Husaini et al. (1950b) reported a significant correlation of 0.500
between muscle hemoglobin (presumably myoglobin) and tenderness. Ani-
mals used in this study varied widely in age, sex and grade which
would tend to make this relatively low correlation appear more reliable.
Wierbicki et al. (1954) prepared an actomyosin-free extract of
muscle plasma and determined nitrogen, refractive index and viscosity
and related these things to each other and to tenderness. The authors
point out that possibly more than actomyosin remained insoluble, how-
ever, their results suggest a relationship. The correlation between
percent nitrogen in the extract and tenderness was 0.507 which was high-
ly significant. Attempts to find a relationship between viscosity of
the extract and tenderness or percent nitrogen were not too fruitful.
These authors suggest that the extract might contain something other
than protein; they did not elaborate on this speculation, however. In-
terestingly there was a significant correlation of 0.565 between re-
fractive index and tenderness which would indicate that refractive
index was a function of nitrogen content and bore some relationship
The foregoing discussion suggests that protein fractionation may
provide valuable information about tenderness. New techniques are cur-
rently available for protein studies such as the ultracentrifuge, im-
proved microscopy, histochemical techniques, spectrophotometric devices
as well as numerous chromatographic and electrophoretic techniques.
Biochemists have long recognized electrophoretic mobility as a useful
tool in protein separations. Paper electrophoresis has found much
favor in studying blood proteins.
The scientific literature contains no references at the present
time on the use of paper electrophoresis to study meat tenderness.
Taki (1962) may be given credit as the first to attempt to approach
the problem of meat tenderness through the use of electrophoretic sepa-
rations. Work done by Taki (1962) has been an attempt to find a tech-
nique that can be applied to separation and characterization of muscle
proteins of the bovine.
The literature contains a wealth of information on the application
of paper electrophoresis to study various phenomenon in fish and mam-
mals. Reviews may be found in texts by Block, Durrum and Zweig (1958)
and Bier (1959).
Following an electrophoretic separation there remains the problem
of evaluation of the data obtained. The classical method is to cal-
culate the mobility of each phase. This may be accomplished by appli-
cation of an appropriate formula. A second method of evaluation lies
in the quantitative estimation of each component. This is accomplished
by use of the Analytrol and integrator mechanisms.
Jacob (1947) studying minced rabbit muscle at various buffer pH's
has shown that three general groups of constituents are apparent. Each
group of constituents is isoelectric at a different pH. As many as
eight components were found, although the peaks lacked the definition
necessary for accurate calculation of electrophoretic mobility. Jacob
(1948) later described a different buffer system and again grouped re-
sults on a quantitative rather than a mobility basis since it was not
possible to precisely identify each component and calculate mobility.
Nikkila and Linko (1955) studying species differences in fish
found a considerable difference between species as to muscle proteins.
These authors obtained highly reproducible results within species and
state that species of fish could be identified by a study of electro-
phoretic patterns. While the study provided little information about
bovine tenderness, it should be noted that it was possible to obtain
reproducible results and that species differences were reported. Since
these species differences do exist, care must be taken in comparing
one mammalian species to another without prior study.
Baechtel et al. (1957) found that paper electrophoresis was suf-
ficiently accurate to determine differences in water extractable
muscle proteins between pairs of rabbits differing in vitamin E in-
take. A quantitative determination was described by these authors
that involved cutting out each phase, eluting it, and reading the op-
Shields et al. (1960), studying muscle changes with cold acclima-
tion with the rat, found considerable variation in peaks between ani-
mals. To circumvent poor resolution of peaks the entire spread was
divided into divisions with the stationary component treated as a
single division. This procedure was similar to that of Baechtel et al.
(1957) and Jacob (1948) as previously discussed.
Twenty mature crossbred ewes used in this trial were fed a con-
centrate ration consisting of 75 percent ground snapped corn and 25
percent cottonseed meal and trace mineralized salt at the rate of one
half pound per head per day for 1 to 3 weeks before slaughter. Coastal
Bermuda grass hay was fed, free choice, and water was available at all
The purpose of this trial was to determine the effects of varying
levels of sodium metaphosphate, injected ante-mortem, on leg steak
and loin chop tenderness. An arterial occlusion technique suggested
by Beuk et al. (1959) was practiced on several animals until it could
be carried out with some degree of confidence. In using this technique,
one leg would have the circulation tied off and could not receive the
tenderizing agent injected in the jugular vein. In this manner, it
was possible to have a control leg and a treated leg from one animal
and thus avoid variations in inherent tenderness as well as a differ-
ential response to treatment by various animals. In the arterial tech-
nique that was used, the animal was immobilized by an injection of
nembutal in the jugular vein. An incision was made, parallel to the
L. dorsi, from the anterior edge of the pin bone to the 13th rib. The
aorta was located and a hemostat applied immediately posterior to the
junction of the common iliac and the aorta. The resulting blockage,
if properly applied, occluded most arterial circulation to the right
Preparation of Solution and Injection Technique
Sodium metaphosphate was combined with distilled water to make a
solution that contained 10 mg./ml. The pH of this solution was approx-
imately 7.4. No attempt was made to alter the pH of the solution. The
trial was divided into two phases. The first phase was designed to de-
termine the effects of differing amounts of injected metaphosphate upon
the tenderness of the injected leg as compared to the control leg. In
phase one 14 ewes were divided into seven lots with two animals per
lot. Two sheep were injected with 1.0 mg./lb., two with 1.5 mg./lb.,
two with 2.0 mg./lb., two with 2.5 mg./lb., two with 3.0 mg./lb., two
with 3.5 mg./lb. and two with 4.0 mg./lb. live weight of sodium meta-
phosphate. Taste panel tenderness scores of animals in phase one showed
that 3 mg./lb. live weight of sodium metaphosphate was the most desir-
able level to use. Phase two included six animals injected at the rate
of 3 mg./lb. live weight with sodium metaphosphate.
Injections were made with an 18 gauge needle and a glass syringe.
The sheep were skinned, hanging from the rail to speed the slaughter
operation. Animals were slaughtered five minutes after the injection
was completed. All animals subjected to this technique were under vary-
ing degrees of stress prior to slaughter as judged by heart rate and
respiration rate. Carcasses were chilled at 340 to 360 for 48 hours.
Two one-inch thick steaks were cut from each leg by cutting per-
pendicular to the line of the shank and posterior to the aitch bone.
The steaks were broiled in electric ovens to a medium well-done degree.
Taste panel determinations were made by a two-member panel on the top
round muscle (Semimembranosus). The taste panel members rated each
sample on a one to nine scale with 1 designating too tough to be
edible; 2, extremely tough; 3, very tough; 4, below average in ten-
derness; 5, average tenderness; 6, above average -- tender; 7, very
tender -- chews easily; 8, extremely tender -- grainy; 9, mushy --
fibers not distinguishable. This tenderness rating scale was used
throughout the study.
Four one-inch thick butterfly chops were removed serially start-
ing at the anterior edge of the pin bone. The loin chops were broiled
in electric ovens to a medium well-done (approximately 1700 F.) degree
after aging for 48, 72, 96 and 120 hours. The chops were evaluated
by a two-member taste panel.
A statistical analysis of these data was not feasible since the
number of animals per treatment was not adequate.
Thirty-two mature crossbred ewes were used in this trial. The
ewes, obtained at the same time as those in trial 1, were fed and
cared for in the same manner as the animals used in trial 1.
This experiment was designed to provide data which would either
verify or reject differences noted in trial 1. Data obtained from the
eight animals receiving 3 mg./lb. sodium metaphosphate in trial 1 are
included in this trial also.
Four lots with eight animals per lot were included in this trial.
Animals were allotted to experimental lots by age and weight. The ani-
mals in lot 1 were not injected and served as a control. Lot 2 was
made up of eight animals from trial 1 that were injected with metaphos-
phate, 3 mg./lb. live weight, following nembutal injection and arterial
occlusion. The reason for including data from these eight animals was
to find out if the observed tenderness was due to metaphosphate or other
factors incidental to arterial occlusion. The third group of animals
received metaphosphate in the same total amount (3 mg./lb. live weight)
as other injected lots; however, the solution was divided into three
equal doses and administered 125, 65 and 5 minutes ante-mortem. The
animals in lot four received a single injection of metaphosphate, at
the rate of 3 mg./lb. live weight, administered 5 minutes ante-mortem.
It should be noted that the difference between lots 2 and 4 lies in
the fact that the sheep in lot 2 had undergone nembutal injection and
surgical blockage of the right leg while in lot 4 the animals had re-
ceived only the metaphosphate injection.
Preparation of Solution and Injection Technique
Sodium metaphosphate solution was prepared in exactly the same
manner as described for trial 1. It should be noted that solutions
were discarded after each use and new solutions prepared to avoid pos-
sible bacterial contamination and possible alteration of the phosphate
Injections were made as previously described. Sheep were slaugh-
tered and chilled at 340 to 36 F. for 48 hours.
Leg steaks and loin chops were removed in the same manner and at
the same time intervals after slaughter as indicated for trial 1. The
taste panel members were the same as those used in trial 1. Tenderness
rating was on the one to nine scale described for trial 1.
The tenderness data from the top round steaks were statistically
treated by analysis of variance as described by Snedecor (1956). Ten-
derness data from loin chops were statistically analyzed by analysis
of variance according to Henderson (1959).
One hundred crossbred sheep, ranging in age from one to five years
old were purchased for use in this trial. The sheep were predominately
wethers and rams, but a few barren ewes were included. Within a week
after the animals arrived at the University Meat Laboratory they were
shorn and given two injections of penicillin. Some death losses were
encountered due to the poor condition of the animals when purchased.
Prior to the feeding trial all the animals were given free access to
the same ration that was described for sheep in trials 1 and 2. The
only difference being that this group of sheep was moved to the Phys-
iology unit where pens in a pole barn were available.
Eleven sheep were allotted to each of seven experimental lots by
weight, sex and age. Animals included in lot 1 were designated as the
non-injected control lot and were fed the control ration. The lot 2
animals were fed a ration with a high calcium to phosphorus ratio.
The third group was fed a ration with a high phosphorus to calcium
ratio. Animals in lot 4 were injected with sodium metaphosphate at
the rate of 3 mg./lb. live weight, divided into three equal doses and
injected 3, 2 and 1 hours ante-mortem. The fifth lot received sodium
metaphosphate at the rate of 3 mg./lb. live weight, administered in
three equal doses 48, 24 and 3 hours ante-mortem. The sixth group
was injected with sodium pyrophosphate at the rate of 3 mg./lb. live
weight, divided into three equal doses and injected 3, 2 and 1 hours
ante-mortem. Group 7 animals were injected with sodium pyrophosphate
at the rate of 3 mg./lb. live weight, divided into three equal doses
and injected 48, 24 and 3 hours ante-mortem.
Feeding of Experimental Animals
The control lot as well as the injected lots 4, 5, 6 and 7 were
fed what will be designated as the control ration. This ration was
calculated by standards described by Morrison (1956) for mature sheep.
Animals in lot 2 were fed a ration which had a high calcium to phos-
phorus ratio. Lot 3 animals were fed a ration which had a high phos-
phorus to calcium ratio.
The rations were mixed by a portable mixer in order that cane
molasses could be added to provide palatability.
The composition of the three rations is shown in Table 1. Table
2 shows the calculated analysis as to protein, fat, crude fiber and
nitrogen-free extract for each experimental ration.
Calcium and phosphorus analyses were made on each experimental
ration by two laboratories. Results of calcium and phosphorus anal-
yses are shown in Table 3. Calcium to phosphorus ratios found at the
two laboratories are presented in Table 4.
Hay was not fed during the feeding trial since the concentrate
ration contained sufficient roughage. Fresh water was available at
all times. The animals were fed once daily, in the morning. Feed
was weighed carefully at each feeding. The amount of feed offered
was governed by the amount of weigh-back remaining among pens. The
average daily consumption per animal was four pounds. Feeding prob-
lems were not encountered other than that a slightly reduced intake
on extremely hot days was observed. Animals were weighed weekly.
Most animals gained steadily throughout the experiment and were in
very uniform condition at time of slaughter.
The experimental rations were fed for a nine day period prior
to the start of the feeding trial. The duration of the feeding trial
was 28 days.
PERCENT COMPOSITION OF EXPERIMENTAL RATIONS
Feed Control High High
Ration Calcium Phosphorus
Ground Snapped Corn 71.2 64.2 71.6
Cotton Seed Meal, 41% 23.8 21.3 23.9
Ground Limestone 0.5 10.0
Cane Molasses 4.0 4.0 4.0
Trace Mineralized Salt 1/ 0.5 0.5 0.5
SMinimum analysis: Mn 0.250%, Fe 0.270%, Cu 0.0337.,
Co 0.010%, I 0.007%, Zn 0.005%, NaCl 96.200%.
CALCULATED ANALYSIS OF EXPERIMENTAL RATIONS 1/
Item Control High High
% Protein 15.6 14.0 15.6
% Fat 3.5 3.2 3.5
% Crude Fiber 10.0 9.0 10.1
% Nitrogen-Free Extract 55.8 50.5 56.1
- Data taken from Morrison (1956).
CALCIUM AND PHOSPHORUS ANALYSIS OF EXPERIMENTAL RATIONS
Laboratory Control High Calcium High Phosphorus
Ration Ration Ration
% Ca % P % Ca % P % Ca % P
Nutrition Lab. 1 0.27 0.73 3.97 0.71 0.12 0.69
Fla. State Dept. of 0.40 0.60 4.20 0.50 0.03 0.50
Agriculture Lab. 1/
/ Calculated on an air dry basis.
CALCIUM AND PHOSPHORUS RATIOS
Nutrition Lab. 1/ 1:2.7
Fla. State Dept. of 1:1.5
Agriculture Lab. 1/
SCalculated on an air dry basis.
OF EXPERIMENTAL RATIONS
High Calcium High Phosphorus
Ca:P Ratio Ca:P Ratio
Preparation of Solutions
Sodium metaphosphate and sodium pyrophosphate solutions were pre-
pared in the same manner as described under trial 1. The pH of the
pyrophosphate solution and the metaphosphate solution was approximately
7.4. Solutions were prepared fresh daily for reasons previously indi-
Animals were injected in the jugular vein using a 19 gauge dis-
posable needle and a glass syringe. The disposable needles were found
to be superior to conventional stainless steel needles, being sharper
and therefore less difficult to insert when injecting.
Method of Blood Sampling
On the 28th day of the feeding trial, 15 ml. of blood was taken
from the jugular vein of all animals in lots 1, 2 and 3. This blood
was allowed to clot, centrifuged at 3000 r.p.m. for 30 minutes and the
serum decanted to a clean labeled test tube. The tubes were placed in
the freezer at 00 F. for calcium and phosphorus analysis.
The injected animals were slaughtered by the clock in order to
maintain control over the time interval between injection and slaugh-
ter. The sheep were skinned hanging on the rail to speed the slaugh-
ter operation. An exact interval of 30 minutes was maintained between
the time of slaughter and the time the carcass was rolled into the
chill cooler. The purpose of this was to minimize chilling rate dif-
ferences and thereby minimize the influence of temperature on rigor
development and pH. Carcasses were chilled for 48 hours at 340 to 360 F.
Initial pH readings were taken at exactly 15 minutes following
death. Additional readings were taken at 1, 2, 3, 4, 8, 12, 24 and
48 hours post-mortem. The reading taken at 48 hours will be referred
to as the initial pH. All readings were made with a Beckman portable
pH meter or a Beckman Zeromatic electric meter. The two instruments
were frequently checked against each other and against fresh buffer
solution. Beckman glass electrodes were used with both instruments.
Readings were taken on a cut surface of the top round (Semimem-
branosus) muscle. Care was taken to be certain a fresh surface was
cut immediately before each reading was taken.
Forty-eight hours following slaughter the entire loin was removed
from all carcasses, wrapped and frozen at 00 F. for later evaluation.
Twenty-four hours prior to the time the chops were to be cooked, the
loin was removed from the freezer and one-inch thick butterfly chops
were removed serially starting from the pin bone. Five chops were re-
moved and were designated A, B, C, D and E. Chops A, C and E were
broiled, on three different occasions, in electric ovens to a medium
well-done degree and evaluated for tenderness by a two member taste
panel on the one to nine scale described under trial 1. Chops B and
D were broiled to a medium well-done degree, cooled to room tempera-
ture and tested for tenderness with the Warner-Bratzler shear machine.
One one-half inch core was removed from each loin eye muscle or two
cores per butterfly chop. Each core was cut twice giving a total of
eight values per animal. These eight values were averaged to give the
tenderness score for the animal. The six taste panel scores were also
averaged to give one score for each animal.
Immediately after slaughter of the animal the skin was removed
while hanging from the rail. Before the carcass was eviscerated a
portion of the L. dorsi muscle was removed for electrophoresis. This
sample was taken from the left side between the tenth and sixth rib.
The sample was immediately trimmed free of all fat and connective
tissue, placed in a number 1 tall tin can, bathed in a stream of ni-
trogen and closed. The can was labeled and sharp frozen in acetone
and dry ice. The samples were then placed in the freezer at 0 F.
until determinations were made. The elapsed time between bleeding
and freezing the sample was approximately 15 minutes. This sample
will be designated as the fresh sample in future discussion.
After the carcass had aged for 48 hours a similar sample was re-
moved from the right L. dorsi muscle and frozen in the same manner as
the fresh sample. This sample will be designated as the aged sample.
At the time the electrophoretic determinations were to be made
the can was removed from the freezer, opened, the frozen sample minced
with a sharp knife and blended until homogenous in a Waring blender.
The samples did not thaw until they were blended. From this point the
method described by Taki (1962) was followed explicitly. Whatman 3 mm.
filter paper was the paper of choice. All samples were run at a con-
stant milliamperage of 10 for exactly 16 hours.
After paper strips were dried they were evaluated on the Spinco
Analytrol. By use of the integrator mechanism on the Analytrol the
total area under the curve was determined. Curves were then marked
by direct comparison with the dyed paper strips to determine the per-
cent of the total dyed protein area that did not migrate. This will
be called the stationary protein phase. When the total area and the
percent of the area in the stationary protein phase was known, it was
possible to calculate the area of the protein phase that migrated to
the left, or the negative protein phase, and area of the protein phase
that migrated to the right, or the positive protein phase. For the
sake of the regression analysis six protein phases were calculated:
negative, stationary and positive protein phases for the fresh samples
and negative, stationary and positive protein phases for the aged sam-
Bone Breaking Strength
The right metacarpus was removed from each carcass in lots 1, 2
and 3, 48 hours after slaughter, scraped clean of all flesh and con-
nective tissue and frozen. When determinations were to be made the
bones were thawed for 12 hours and spanned across two iron fulcrums,
placed one and three quarters inches apart. Bones were broken in the
center by applying a steady pressure supplied by a screw press with a
pound pressure recording dial. Breaking strength in pounds was re-
corded to the nearest pound. Bones were saved for subsequent
Phosphorus Analysis of Bone
The bones were placed in beakers in an air drying oven at 1100 C.
for 48 hours. The bones were then placed in extracting thimbles, and
the fat was ether extracted for 12 hours in a Goldfisch fat extrac-
tion apparatus. The bones were then weighed, placed in tared porce-
lain crucibles and ashed in the muffle furnace at 6000 C. for 48
hours. Crucibles were removed to a dessicator to cool; then the
ashed weight was taken.
Bone samples were placed under an exhaust hood and covered with
perchloric acid to digest. The samples were allowed to stand for
four days after which time triple distilled water was added, and the
water slowly evaporated on the hot plate until fuming started.
The samples were filtered through Whatman 41 filter paper and
brought to a volume of 250 ml. with triple distilled water.
Phosphorus determinations followed the photometric procedure
outlined by Fisk and Subbarow (1925). Calculations were expressed
as percent phosphorus on a dry, fat-free basis.
Calcium, Phosphorus and Magnesium Analysis of Muscle
Muscle tissue for mineral analysis was obtained from the L.
dorsi. The samples were blended in a Waring blender until homogenous.
Approximately 50 gm. samples were used for analysis.
Calcium determinations were carried out as described by Welcher
(1961). Percent calcium was calculated on a dry, fat-free basis and
on an ash basis.
The procedure used for phosphorus was the same as described
for bones, with the exception that the filtrate was made up
to 50 ml. instead of 250 ml.
Magnesium determinations were made following the procedure out-
lined by Welcher (1961). Calculations were expressed as percent mag-
nesium on an ash basis and on a dry, fat-free basis.
Calcium, Phosphorus Analysis of Blood
The method of obtaining blood serum has been outlined. The pro-
cedure outlined for blood serum by Welcher (1961) was followed for
calcium determinations. Determinations were expressed as mg. calcium/
100 ml. serum.
The colorimetric serum phosphorus method described by Fisk and
Subbarow (1925) was followed for serum phosphorus. Calculations were
expressed as mg. phosphorus/100 ml. serum.
Analysis of variance according to Snedecor (1956) was used to
determine if significant tenderness differences existed among treatment
groups. Duncan's Multiple Range Test according to Duncan (1955) was
used to compare treatment means with significant differences noted by
analysis of variance.
A stepwise regression analysis was also employed. The purpose of
this analysis was to determine the relationship, if any, of several
factors studied with tenderness. The data were analyzed in two pro-
grams. First, all 77 animals were included in the analysis. 'Age was
a continuous variable. The independent variables were initial pH, ulti-
mate pH and six electrophoretic protein phases. The dependent variable
was tenderness. Independent variables were correlated first with taste
panel tenderness, then with shear tenderness.
The second analysis included only the 33 animals from lots 1,
2 and 3. Age was a continuous variable. Independent variables were:
initial pH; ultimate pH; six electrophoretic protein phases; bone
breaking strength; percent calcium in muscle on a dry, fat-free basis;
percent calcium in muscle, ash basis; percent calcium in bone on a
dry, fat-free basis; percent calcium in bone on the ash basis; mg.
calcium/100 ml. serum; percent phosphorus in muscle on the dry fat-
free basis; percent phosphorus in bone on the dry, fat-free basis;
mg. phosphorus/100 ml. serum; percent magnesium in muscle on the dry,
fat-free basis; percent magnesium in muscle on the ash basis. The
dependent variable was tenderness. The program was repeated to obtain
correlations between tenderness by taste panel and tenderness by shear.
Data were analyzed by an IBM model 709 digital computer.
RESULTS AND DISCUSSION
Taste panel evaluations of top round steaks are presented in
Table 5. The first panel member preferred the tenderness of sodium
metaphosphate-injected legs from 12 of the 20 animals, the control
legs from 2 of 20 animals and had no preference on six animals. The
second panel member preferred sodium metaphosphate-injected legs
from 9 of 17 animals and had no preference on 2 of 17 animals. Av-
eraging the preferences, a 54 percent preference for sodium meta-
phosphate-injected legs was found, whereas there was found to be a
23 percent preference for control legs. No preference was indicated
for 23 percent of the legs.
These data tend to indicate that the sodium metaphosphate treated
legs were more desirable, from a tenderness standpoint, than the non-
treated legs. Tenderness differences noted between the two legs in-
dicated a need for further investigation of the suggested tenderizing
Panel tenderness evaluations on loin chops are shown in Table 5.
These chops were taken from an area of the carcass subject to effects
of the ante-mortem injection treatments. The purpose of testing the
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chops as to tenderness was to train the panelists in testing mutton
chops for tenderness and to determine the level of metaphosphate giving
the most promising tenderness response. On the basis of these data it
appeared that the 3 mg./lb. was the most desirable level to use in fu-
The results of the taste panel evaluation of top round steaks are
shown in Table 6. The treatment means appear to be sufficiently differ-
ent to be significant; however, such was not the case. Despite the lack
of statistical significance it should be noted that only two out of 24
animals in lots 2, 3 and 4 were below average in tenderness, while five
of the eight animals in the non-injected control lot were below average
in tenderness. A rather close agreement may be noted between the tender-
ness values for top round steak and loin chops.
The results of the taste panel evaluations of the loin chops are
shown in Table 6. Statistical analysis of these data showed a highly
significant difference in aging interval (PC.01). This analysis fur-
ther showed that animals, within treatment, were significantly different
(P ..01). The combination of these two effects precluded any significant
differences being found among treatments. The experimental design would
have been better if the chops had been frozen at 48 hours and panel
scores averaged to give a single estimate of tenderness for each animal.
The fact that aging of chops improved tenderness is of little value
since this is commonly accepted as a scientific fact. It would have
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been extremely interesting had there been a significant interaction
between injection treatment and aging. It was originally planned to
investigate this interaction since common practice in this type of
experiment is to evaluate for tenderness at only one degree of aging --
usually 48 hours.
The most marked tenderizing effect was in lot 2 where the animals
had received nembutal and were observed to have suffered severe shock
during the arterial occlusion operation. The tenderizing effect may
have been due, in part, to the nembutal or the physiological shock.
Such interpretation seems more significant in view of the fact that
this lot was identical with lot 4 in respect to ante-mortem injection
and lot 4 was not improved in tenderness over the control.
Lot 3 appeared to be greatly different in tenderness from the con-
trol. This was the only valuable finding from the standpoint of the
objectives of the experiment. It would appear that the metaphosphate
exerts an effect through a physiological change rather than strictly a
biochemical change. For this reason a longer interval between injection
and slaughter appeared to be a logical modification in method for future
While this trial failed to show conclusively that sodium metaphos-
phate improved tenderness when injected ante-mortem, the data give
some support to the hypothesis of a tenderizing effect. Additional
work should include a control group, a repeat of treatment 3 and a lot
of animals that would be injected for several days prior to slaughter.
Effect of Dietary Calcium and Phosphorus on Weight Gains and Feed
Consumption of Mature Sheep.
Average initial and final weights, average daily gain and feed
consumption data relative to the feeding of the experimental sheep
before slaughter are shown in Table 7. The initial weight data were
recorded after a nine day pre-trial feeding of experimental rations;
therefore, the weight gains shown should not reflect "fill".
Average daily gains were similar for the control and the calcium
lot. The fact that the high calcium ration contained 10 percent ground
limestone did not appear to affect feed palatability, weight gains or
feed consumption. Sheep in lot 6 gained faster than any uther lot,and
lot 2 which was fad the high phosphorus ration gained slowest, apparently
due to individual variations.
Efficiency of feed utilization was similar between groups.
The sheep had been on a maintenance ration prior to the pre-trial feed-
ing period. Further, the animals were rather mature and in very thin
flesh. The writer noted that the experimental rations appeared to be
very palatable judging from the apparent appetite of the animals; aver-
age daily consumption data confirm that observation.
The Effect of Feeding a High Calcium Low Phosphorus Ration and a Low
Calcium High Phosphorus Ration on Tenderness of Mutton Chops.
The average taste panel and shear scores for the feeding treatment
groups are shown in Table 8. The purpose of this portion of the study
was to study the effects of dietary calcium and phosphorus or the ratio
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between the two minerals on tenderness.
According to taste panel data, the feeding of the high calcium -
low phosphorus ration had neither a beneficial nor a harmful effect on
tenderness. By shear value data, the high calcium low phosphorus fed
group appeared slightly more tender than the control, although the dif-
ference was not significant statistically.
The high phosphorus to calcium ratio group provided chops that
were somewhat more tender than the control chops according to both the
panel and shear data. The difference in tenderness by taste panel was
not significant; the difference between the two groups in shear value
was significant at the .05 level.
These data appear to substantiate the observations of Hall et al.
(1944a) who first reported that high level dietary phosphorus slightly
improved the tenderness of beef.
It should be pointed out that the ration fed this lot was not ab-
normally high in phosphorus content, but rather it was low in calcium,
which developed the abnormal calcium to phosphorus ratio. The discussion
to follow will bring out some of the other relationships noted between
phosphorus levels in bone and muscle and tenderness.
The Effect of Ante-mortem Injection of Metaphosphate and Pyrophosphate
on the Tenderness of Mutton Chops.
Taste panel and shear averages for phosphate injected and control
sheep are shown in Table 8. Panel and shear evaluations of tenderness
are in rather close agreement although significant differences (P4.05)
were found only with the shear data. Group 7 chops were significantly
more tender (P<.05) by shear test than the control (lot 1) chops;
treatment effects were not significant according to taste panel data.
A most interesting observation is that the non-injected control
group was less tender than all injected lots. This trend has been noted
many times in this type of experiment, irrespective of the tenderizing
agent injected. This introduces the possibility that the stress in-
volved in injecting the solution may be a factor in tenderness.
According to taste panel and shear data little difference was
found among the two metaphosphate lots and the 48, 24 and 3 hour pyro-
phosphate lot. This would indicate that the tenderizing effect noted
was a function of the phosphate rather than the chelating ability of
the metaphosphate as suggested by Kamstra and Saffle (1959) and Carpen-
ter et al. (1961). The purpose of using two polyphosphates in this
study was to determine if metaphosphate tenderized because of its che-
lating ability or because it was a polyphosphate.. Presumably pyrophos-
phate was less potent as a chelating agent than metaphosphate. It must
be assumed that the phosphates either interfered with the ionic balance
or that the excess phosphate per se was responsible for the observed
No difference was found between the metaphosphate treatments, thus
indicating that it made little difference if the metaphosphate was in-
jected ante-mortem in either 3 equal doses, 3, 2 and 1 hours before
slaughter or in 3 equal doses, 48, 24 and 3 hours before slaughter. The
difference between the pyrophosphate groups appeared to be of some mag-
nitude although the difference was not significant statistically. It
appeared that for the pyrophosphate to be effective it must be in-
jected over a longer period of time. The lot receiving daily injec-
tions of pyrophosphate for three days was significantly more tender
(P<.05) than the control. On the basis of these data it appears that
the tenderness of an animal may be altered by the proper level of a
phosphate solution injected at the proper time before slaughter. It
is evident that more work is indicated in this area before definite
conclusions may be drawn.
The Influence of Animal Age at Time of Slaughter on the Tenderness of
As previously indicated the animals were distributed arong the
seven treatment lots as uniformly as possible but with special con-
sideration of the variable age of the animals. When the data were com-
bined in the regression analysis, age was included as a continuous var-
iable. It was thereby possible to observe the influence of age on ten-
derness. Simple correlations of age with shear and taste panel tender-
ness shown in Tables 9 and 10 were very low on both programs. The
highest of these correlation coefficients was .24 between shear and
age of the 33 animals in groups 1, 2 and 3. This relationship accounted
for only 5.8 percent of the variability in the shear tenderness of loin
chops. The correlation coefficient between taste panel tenderness and
animal age was .07.
When considering the 77 animals from the seven treatment groups,
the correlation coefficient between age of animal and taste panel ten-
derness was -.02; the correlation coefficient between age of animal
and shear tenderness values was -.03. These low correlation coeffi-
cients indicate that age was not an important factor in the tenderness
SIMPLE CORRELATION COEFFICIENTS BETWEEN TENDERNESS AND AGE, pH, MUSCLE
PROTEIN FRACTIONS, BONE BREAKING STRENGTH AND MINERAL CONTENT IN
MUSCLE, BONE AND BLOOD (33 ANIMALS) -- TRIAL 3
Independent Variable Tenderness
Taste Panel Shear
1. Age .07 .24
2. Initial pH .40* .11
3. Ultimate pH -.38* -.13
4. Pre-rigor Muscle Negative Protein Phase .01 .08
5. Pre-rigor Muscle Stationary Protein Phase .37* -.12
6. Pre-rigor Muscle Positive Protein Phase .26 .01
7. Post-rigor Muscle Negative Protein Phase .26 -.27
8. Post-rigor Muscle Stationary Protein Phase .29 .04
9. Post-rigor Muscle Positive Protein Phase .06 .30
10. Bone Breaking Strength .20 .14
11. % Ca in Muscle; Dry, Fat-free Basis -.05 .01
12. % Ca in Muscle; Ash Basis .23 -.02
13. Mg. Ca/100 ml. Blood Serum .37* -.01
14. % P in Muscle; Dry, Fat-free Basis .39* .22
15. % P in Bone; Dry, Fat-free Basis .42* -.30
16. Mg. P/100 ml. Blood Serum .14 .01
17. % Mg in Muscle; Dry, Fat-free Basis .25 .15
18. % Mg in Muscle; Ash Basis .28 .12
* Significant at the .05 level of probability.
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of this group of animals.
The Relationship Between Muscle eH, Dietary Calcium and Phosphorus,
Ante-mortem Injection of Phosphates and Tenderness.
Average initial (pre-rigor) pH and ultimate (post-rigor) pH
values by treatment group are presented in Table 11. Among the three
groups fed varying calcium-phosphorus ratios, the control group car-
casses were significantly (P<.05) higher in initial muscle (Senimem-
branosus) pH than carcasses from animals fed the high phosphorus to
calcium ratio. A satisfactory explanation of this result is not forth-
coming since none of the animals in the first three lots were unduly
excited before slaughter. Differences among the post-rigor pH values
of the first three lots lacked significance.
The pre-rigor pH of lots 4 and 6 was significantly (P<.05) lower
than the control (lot 1) as shown in Table 11. Lots 4 and 6 were in-
jected ante-mortem with metaphosphate and pyrophosphate, respectively,
3, 2 and I hours before slaughter and were therefore excited at the
time of slaughter. According to Briskey (1959) the initial pH is a
function of the state of excitation of the animal at time of slaughter.
Lots 5 and 7 which had received the final injection six hours ante-
mortem, had a higher pre-rigor pH than lots 4 and 6 but a lower aver-
age pH than the control group which was rested at the time of slaughter.
The post-rigor pH of lots 6 and 7 which were injected ante-mortem
with pyrophosphate, was significantly (P<.05) higher than lot 4 and
slightly higher than lot 5. Neither the dietary treatments nor the
ante-mortem injection treatments influenced post-rigor pH when compared
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with the control group. The significant difference in pH, however,
between the pyrophosphate injected groups and group 3 (high phosphorus
to calcium ratio) and group 4 (metaphosphate injected) is noteworthy.
These data do not support the reports of Kamstra and Saffle (1959)
and Carpenter at al. (1961) who found that metaphosphate infusion
post-mortem, pre-rigor increased ultimate pH. In this present study
the phosphates were injected into the live animal which may account
for the difference between the findings of the South Dakota workers
and those presented here. Although lacking statistical significance
and verification, it appears that the pyrophosphates did influence
ultimate pH thereby corroborating to an extent the findings of Kamstra
and Saffle (1959) and Carpenter et al. (1961).
A highly significant (P<.01) negative correlation of -.92 was
found between initial pH and ultimate pH which does not agree with
the work reported by Briskey (1959) that indicated little relationship
between initial and ultimate pH.
The simple correlation coefficients between initial and ultimate
pH and panel tenderness shown in Table 9 were .40 and -.38 which were
significant (P<.05). Other correlation coefficients between pH and
tenderness shown in Tables 9 and 10 were low and non-significant. This
verifies reports by Husaini et al. (1950a and b), Kamstra and Saffle
(1959), Briskey (1959) and Carpenter et al. (1961) who have indicated
that the relationship between pH and quality attributes such as tender-
ness are largely coincidental.
Muscle phosphorus was found to be significantly (P<.01) correlated
with initial pH and ultimate pH with correlation coefficients of .91
and -.92 respectively. Correlation coefficients of this magnitude
suggest a very real relationship. It is doubtful if a direct relation-
ship exists between amount of phosphorus in muscle per se and pH,but
it is possible that phosphorus is a factor in altering a mechanism that
might contribute directly to pH change. Such a relationship has been
investigated by Lawrie (1953) who studied various phosphate esters as
they relate to pH and rigor mortis. Since phosphorus plays such a
vital role in intermediate metabolism,it is possible that absolute
amount of muscle phosphorus contribute directly to pH.
It is possible that the level of phosphorus in the tissue could
change the creatine phosphate stores. Lawrie (1953) has shown that pH
does not drop rapidly and rigor mortis does not enter the fast phase
until creatine phosphate stores are reduced to a given level. In this
manner Lawrie (1953) has shown a significant relationship among creatine
phosphate reserves, pH, rigor mortis and muscle function. It is possi-
ble that this was the mode of action of the injected phosphate in this
study. It is also possible that the advantage shown in tenderness for
pyrophosphate over metaphosphate injected animals reflects a differ-
ence in the availability of the phosphate radical for replenishing the
creatine phosphate stores.
The Effect of Ante-Mortem Injection of Metaphosphate and Pyrophosphate
on Muscle pH.
Figure 1 graphically represents the time and pH relationship. To
provide clarity the average pH of the two metaphosphate lots (lots 4
and 5) were averaged to provide a single curve. The pH values for the
two pyrophosphate lots (lots 6 and 7) were also averaged to give a
Lot 1. Control ---------
Lots 4 and 5. Metaphosphate injected ---------
Lots 6 and 7. Pyrophosphate injected
1 2 3 4 8 12 24
Hours after slaughter
Figure 1. --The effect of ante-mortem injection of meta-
phosphate and pyrophosphate on the pH of muscle.
single curve. The third curve is the average pH values for the con-
The objective of recording the muscle pH at these intervals was
to determine if the time course of rigor mortis had been influenced
by phosphate injection. It is generally accepted that the drop in pH
is a result of or associated with rigor. The foregoing discussion of
pH has dealt only with the initial and ultimate pH. Figure 1 shows
that pH differences among the three experimental groups are minimized
at the initial and ultimate points. Particularly striking is the
fact that the pH of the pyrophosphate lots remained considerably higher
than the metaphosphate lots and the control lot up to four hours post-
mortem. This wide difference may reflect a difference in the rate of
anaerobic glycolysis. The high pH of the pyrophosphate lots for the
first four hours cannot be explained by greater glycogen reserves. The
control group should have had the highest muscle glycogen reserve at
the time of slaughter due to less excitation prior to slaughter.
The Relationship Between Electrophoretic Muscle Protein Components and
Muscle plasma proteins were extracted from pre-rigor and post-
rigor muscle samples by the technique described by Taki (1962). The
plasma protein extract was then fractionated by paper electrophoresis.
Certain of the proteins (or protein complexes) were attracted to the
negative pole; other proteins were attracted to the positive pole; some
proteins migrated to neither the positive nor the negative pole. The
proteins migrating to negative were designated negative phase, the
proteins migrating to the positive were designated positive phase and
the proteins which did not migrate were designated the stationary
phase. The percentage of the muscle plasma protein that remained
stationary migrated to negative and migrated to positive were cal-
culated on samples taken pre-rigor and post-rigor.
Average negative, stationary and positive phase values on pre-
rigor and post-rigor samples are presented by treatment group in Table
Differences between lots in percent of either pre-rigor or post-
rigor negative, stationary and positive phases were not significant;
variations within lots were observed to be rather extreme.
Correlation coefficients between muscle protein phases and ten-
derness by panel and shear are shown in Tables 9 and 10, respectively.
Correlation coefficients shown in Table 9 based on only the 33 animals
from lots 1, 2 and 3 are small in general,although they are larger than
the coefficients in Table 10 which are based on all 77 animals. The
fact that the coefficients based on all animals were so much smaller
than the coefficients based on the 33 animals may be explained in part
by the fact that the phosphate injections may have had such a counter-
ing effect on the relationships of the various components with other
factors as to negate the relationships established on the smaller group
of animals. This confounding effect, if present, could have been re-
moved by calculating correlation coefficients on a within treatment
group basis. The higher correlations based on the smaller group could
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have been a result of chance.
The stationary protein phase of the pre-rigor muscle was signif-
icantly (P<.05) correlated with taste panel tenderness with a cor-
relation coefficient of .37 when considering only the 33 animals in
lots 1, 2 and 3. When the 77 animals were considered together the
same relationship was very low and non-significant with a coefficient
Positive protein phases from pre-rigor and the post-rigor muscle
samples were significantly (P<.01) correlated with a correlation
coefficient of .64 when considering the 33 animals in lots 1, 2 and 3.
A highly significant (P<.01) correlation coefficient of .58 was found
between the stationary phases of pre-rigor and post-rigor muscle sam-
ples; the correlation between the negative phases of the pre-rigor and
post-rigor samples was highly significant (P<.01). It would be noted
that a significant (PC.05) correlation coefficient of .39 was shown
between the pre-rigor stationary phase and the post-rigor positive
phase. Further, the relationship between the pre-rigor stationary
phase and the post-rigor negative phase was small and non-significant.
It is known that the post-rigor stationary phase is less than the pre-
rigor stationary phase. Considering all of these lines of reasoning,
it might seem logical to assume that the stationary protein phase mi-
grates, during rigor, to the positive phase.
Several valuable observations were made as a result of these
studies of muscle proteins. Considerable variation was found among
animals and such a finding should evoke further work to determine why
these differences exist. Further, caution should be exercised in
making generalizations concerning muscle proteins from studies based
on one or only a few animals.
A technique for holding muscle tissue samples in freezer storage
prior to electrophoretic analysis has been described. The technique
should facilitate future studies of this type since past studies have
been limited to a few animals due to the limitations of handling a
quantity of samples in such a way as to prevent protein denaturation.
Marked differences in the extractability of pre-rigor and post-
rigor muscle were noted. The protein extract obtained from pre-rigor
muscle was extremely viscous and difficult to separate with centrifuga-
tion and filtration. The post-rigor muscle separated very easily with
centrifugation and yielded a large volume of clear fluid. The writer
observed that if a pre-rigor muscle sample was allowed to remain at
room temperature for several hours or if a pre-rigor sample was cen-
trifuged at high speed for 30 minutes or more, the resultant extract
would be clear and as easily separated as post-rigor muscle. It was
apparent that in either case a degree of denaturation or dissociation
of proteins had taken place. Since the pre-rigor muscle was difficult
to separate it would be interesting to know the comparative tenderness
values of pre-rigor and post-rigor muscle.
The Effect of Dietary Calcium to Phosphorus Ratio on Bone Breaking
Average bone breaking strength by treatment groups is presented
in Table 13. Analysis of variance showed that differences between treat-
THE EFFECT OF FEEDING VARYING CALCIUM TO PHOSPHORUS RATIOS ON BONE
BREAKING STRENGTH -- TRIAL 3
Treatment Number Average Bone Breaking
of Strength in Pounds Li
High Dietary Ca:P Ratio
High Dietary P:Ca Ratio
No significant differences were
found by analysis of variance.
ments were not significant.
Simple correlations between bone breaking strength and panel and
shear tenderness, shown in Table 9, were .20 and .14, respectively.
The lack of a significant relationship between bone breaking strength
and tenderness is in agreement with reports by Alsmeyer (1960) and
The relationship between age and breaking strength was not as
great as expected although a significant (P<.05) coefficient of .41
Correlations between muscle phosphorus and magnesium and bone
breaking strength were highly significant (P<.01); the correlations
were .76 and .54, respectively.
The correlation coefficients between bone breaking strength and
blood calcium and phosphorus were .37 and .63, respectively, which
were significant at the .05 and .01 levels of probability. The cor-
relation coefficient between bone breaking strength and bone phos-
phorus was .37 which was significant (P<.05). A relationship between
bone calcium and bone breaking strength was not established. This
means that blood calcium and phosphorus, which reportedly remain at
fairly constant levels under normal conditions, are better indicators
of bone breaking strength than bone calcium and phophorus levels.
Such a finding might have some interesting clinical implications if
The Effect of Dietary Calcium to Phosphorus Ratio on Muscle Calcium
and The Relationship of Muscle Calcium and Tenderness.
The correlation coefficients between muscle calcium and tender-
ness are shown in Table 9. These correlations are too low to be of
Table 14 shows the treatment means for muscle calcium in the
three treatment groups. The fact that the high calcium lot was sig-
nificantly higher in muscle calcium was not expected on the basis of
the literature. It is an accepted textbook principle that mineral
constituents of muscle and blood will be affected only after severe
depletion or by dietary imbalances. Further, it was not expected
that muscle calcium would be significantly altered in only a 28 day
The correlation coefficient between dry, fat-free calcium and
ash calcium was .83 and highly significant (P<.01). This correla-
tion was not as high as the similar correlation found between bone
calcium by two methods of calculating. This might be partially ex-
plained by the fact that such a small amount of calcium is present in
The Effect of Dietary Calcium to Phosphorus Ratio on Blood Serum Cal-
cium and the Relationship Between Blood Calcium and Tenderness.
The correlation coefficients between blood calcium and tenderness
are shown in Table 9. The correlation coefficient with taste panel
tenderness was significant (P<.05) but low (r .37). The correlation
coefficient between serum calcium and shear tenderness was -.01.
THE EFFECT OF FEEDING VARYING CALCIUM TO PHOSPHORUS RATIOS ON THE
CALCIUM CONTENT OF MUSCLE -- TRIAL 3
Treatment Number Average Percent Muscle
of Calcium- Dry Fat-free
Sheep Basis 1!
1. Control 11 .041
2. High Dietary Ca:P Ratio 11 .069
3. High Dietary P:Ca Ratio 11 .041
11 2 was significantly higher than 1 and 3 (P<.01).
The lot means for serum calciunrare presented in Table 15. Anal-
ysis of variance revealed that the high dietary phosphorus to calcium
ratio was significantly (P<.05) lower in serum calcium than either the
high calcium to phosphorus ratio lot or the control group. The high
calcium lot was higher in serum calcium than the control, but this dif-
ference was not significant.
The Effect of Dietary Calcium to Phosphorus Ratio on Muscle Phosphorus
and the Relation of Muscle Phosphorus to Tenderness.
The simple correlation coefficient of .39 between taste panel ten-
derness and muscle phosphorus was significant (P<.05). The relation-
ship between muscle phosphorus and shear tenderness was too low to be
Highly significant (P<.01) correlation coefficients of .92 and
-.93 were found between muscle phosphorus and initial and ultimate pH,
respectively. Since inorganic phosphorus and phosphate esters are in-
volved in glycolysis, it is conceivable that a direct relationship
does exist between muscle phosphorus and pH as these data reflect.
Since injected phosphates tend to increase tenderness, it is possible
that this inter-relationship might partially explain the so-called
"coincidental" relationship between pH and tenderness. Averages for
muscle phosphorus by treatment groups are shown in Table 16. Appar-
ently there was a very small amount of variation in muscle phosphorus
of any of the animals irrespective of treatment. Analysis of the
muscle phosphorus data by treatment group showed that the differences
shown were not significant.
THE EFFECT OF FEEDING VARYING CALCIUM TO PHOSPHORUS RATIOS ON BLOOD
SERUM CALCIUM LEVELS -- TRIAL 3
Treatment Number Average mg. Calcium/100
of ml. Blood Serum ./
1. Control 11 10.2
2. High Dietary Ca:P Ratio 11 11.1
3. High Dietary P:Ca Ratio 11 8.6
1/ 3 was significantly lower than 1 and 2 (P<.05).
THE EFFECT OF FEEDING VARYING CALCIUM TO PHOSPHORUS RATIOS ON THE
PHOSPHORUS CONTENT OF MUSCLE -- TRIAL 3
Treatment Number Average Percent Muscle
of Phosphorus; Dry
Sheep Fat-free Basis-
1. Control 11 3.5
2. High Dietary Ca:P Ratio 11 3.4
3. High Dietary P:Ca Ratio 11 3.4
1/ No significant differences were found by analysis of variance.
The Effect of Dietary Calcium to Phosphorus Ratio on Bone Phosphorus
and the Relationship of Bone Phosphorus to Tenderness.
The correlation coefficient of .42 (Table 9) between bone phos-
phorus and taste panel tenderness was significant (P<.05), however,
the correlation coefficient between bone phosphorus and shear tender-
ness of -.30 was low and non-significant. It should be noted that
although these correlation coefficients are not large that they are
larger than any of the other determinations made on these animals.
Average phosphorus content of bones by treatment group is pre-
sented in Table 17. Lots 2 (high calcium to phosphorus ratio) and
3 (high phosphorus to calcium ratio) were significantly (P<.05)
higher in bone phosphorus than the control lot. It has been suggested
that the feeding of a high calcium to phosphorus feed might interfere
with the absorption and normal deposition of bone phosphorus. If
this is true, then it does not seem logical ctht ch. high aalciumr lot
had the highest level of phosphorus in the bone
The Effect of Dietary Calcium to Phosphorus Ratio on Blood Serum Phos-
phorus and the Relationship Between Blood Phosphorus and Tenderness.
While the high phosphorus to calcium ratio lot had the highest
average blood serum phosphorus according to Table 18 the difference
was not significant. The relationship between serum phosphorus and
tenderness values was low and lacked significance.
THE EFFECT OF FEEDING VARYING CALCIUM TO PHOSPHORUS RATIOS ON THE
PHOSPHORUS CONTENT OF BONE -- TRIAL 3
Treatment Number Average Percent
of Phosphorus; Dry Fat-
Sheep free Basis 1/
1. Control 11 5.70
2. High Dietary Ca:P Ratio 11 7.44
3. High Dietary P:Ca Ratio 11 7.24
11 2 and 3 were significantly higher than
THE EFFECT OF FEEDING VARYING CALCIUM TO PHOSPHORUS RATIOS ON BLOOD
SERUM PHOSPHORUS LEVELS -- TRIAL 3
Treatment Number Average mg. Phosphorus/
of -100 ml. of Blood
Sheep Serum 1/
1. Control 11 16.9
2. High Dietary Ca:P Ratio 11 17.2
3. High Dietary P:Ca Ratio 11 19.7
No significant differences were found by analysis of variance.
The Effect of Dietary Calcium to Phosphorus Ratio on Muscle Magnesium
and the Relationship of Muscle Magnesium to Tenderness.
The treatment means for muscle magnesium are presented in Table
19. Significant differences were not found among the means by anal-
ysis of variance. The high calcium to phosphorus ratio lot was the
lowest in muscle magnesium content, however, considerable individual
variation was noted. Muscle magnesium had little relationship to taste
panel or shear tenderness. Correlation coefficients are presented in
Regression Analysis of pH, Muscle Protein Fractions and Mineral Con-
stituents of Bone, Muscle and Blood on Tenderness.
Tables 20 and 21 present the simple, partial and multiple correla-
tion coefficients for the most important factors studied on the 33 ani-
mals in lots 1, 2 and 3, influencing or associated with taste panel and
shear tenderness. The stepwise regression program which was used picks
out the independent variable having the highest simple correlation with
the dependent variable, then proceeds in a stepwise manner to add one
independent variable at a time which, when combined with previous in-
dependent variables give the best estimate of the variation in the
dependent variable. The order in which independent variables enter
the program depends on which variables are in the program; therefore,
if the most highly correlated independent variable were to be deleted
from the program, an entirely different sequence of independent vari-
able influence on the dependent variable would likely evolve.
The highest of the simple correlation coefficients listed in
THE EFFECT OF VARYING CALCIUM TO PHOSPHORUS RATIOS ON THE MAGNESIUM
CONTENT OF MUSCLE -- TRIAL 3
Treatment Number Average Percent
of Magnesium; Dry
Sheep Fat-free Basis 1/
1. Control 11 .13
2. High Dietary Ca:P Ratio 11 .11
3. High P:Ca Ratio 11 .14
SNo significant differences were found by analysis of variance.
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Table 9 was the coefficient between taste panel tenderness and bone
phosphorus which was the first variable to enter the stepwise re-
gression. Bone phosphorus accounted for 17.6 percent of the variabil-
ity in taste panel tenderness according to this study. If the pre-
dicted value of taste panel tenderness was Y then the predictive equa-
tion for Y from only the knowledge of bone phosphorus (X17) becomes:
= 3.62 + .24 X17. This means that as bone phosphorus changes one
percent, taste panel tenderness increases .24 units on the basis of
The second step in the regression analysis included pre-rigor
muscle positive protein phase (X6), which had a simple correlation
coefficient with taste panel of .26 according to Table 20 and gave
a multiple correlation coefficient of .54 when combined with bone
phosphorus. Knowledge of bone phosphorus levels and percent protein
in the positive phase of pre-rigor muscle would account for 29.2 per-
cent of the variability in taste panel tenderness. The predictive
equation would become: f 2.66 + .27 X17 + .04 X6. If percent pro-
tein in the pre-rigor positive phase (X6) were held constant one per-
cent change in bone phosphorus (X17) would change the predicted value
of taste panel tenderness by .27 units, and conversely if bone phos-
phorus were held constant a one percent change in pre-rigor positive
protein phase would give a change in predicted taste panel tenderness
of only .04 units.
In step 3 the independent variable added according to Table 20
was percent positive phase in post-rigor muscle (Xg) which increased
the multiple correlation coefficient from .54 to .62 but failed to
increase the estimate of variability in tenderness to any great extent.
Adding percent positive phase in post-rigormuscle (Xg) to X17 and X6
gives the predictive equation: Y = 2.90 + .32 X17 + .06 X6- .04 Xg.
If X6 and X9 are held constant one percent change in bone phosphorus
will give a change of .32 in predicted taste panel tenderness. Hold-
ing bone phosphorus (X17) and either X6 or X constant results in only
a .06 and .04 unit change in predicted taste panel tenderness for one
percent change in either X6 (pre-rigor positive protein phase) and Xg
(post-rigor positive protein phase), respectively.
The fourth step in the stepwise regression analysis included
muscle magnesium (X20). The multiple correlation coefficient was not
increased appreciably by adding muscle magnesium. The predictive equa-
tion became Y = 2.99 + .30 X17 + .06X6 .05 X + .38 X20 which meant
that holding X17 (bone phosphorus), X6 (positive protein phase, pre-
rigor muscle) and X9 (positive protein phase, post-rigor muscle) con-
stant a one percent change in muscle magnesium (X20) resulted in a .38
change in predicted taste panel tenderness. Holding X6, X9, and X20
constant a one percent change in bone phosphorus caused a change of
.30 in taste panel tenderness.
If X17' X6, X9, and X20 were known it would be possible to account
for only 43.6 percent of the variability in taste panel tenderness. The
addition of more independent variables did not result in a significant
increase in the estimate of taste panel tenderness. Further, the pre-
dictive equation would not be markedly improved because the error for
the next added independent variable was as great as the added predic-
The highest of the simple correlations listed in Table 10 was the
coefficient between shear tenderness and bone phosphorus (X17) which
was the first variable to enter the stepwise regression. Bone phos-
phorus accounted for nine percent of the variability in shear tender-
ness according to this study. If the predicted value of shear tender-
ness was Y then the predictive equation for Y from the knowledge of
bone phosphorus (X17) becomes: Y = 16.41 .46 X17. This means that
as bone phosphorus changes one percent, shear tenderness changes .46
units according to these data.
The second step in the regression analysis included X9 (post-
rigor muscle, positive protein phase), which had a simple correlation
coefficient with taste panel of .30, according to Table 21, and gave
a multiple correlation of .41 when combined with bone phosphorus (X17).
Knowledge of bone phosphorus and percent protein in the positive phase
of post-rigor muscle would account for 16.8 percent of the variability
in shear tenderness. The predictive equation would become: Y 14.12 -
.42 X17 + .07 X9. If X9 were held constant a one percent change in
X17 (bone phosphorus) would change the predicted value of shear tender-
ness by .42 units and conversely, if X17 were held constant a one per-
cent change in X9 (percent of protein in post-rigor muscle positive
phase) would change the predicted value of shear tenderness by .07
The third variable added to the analysis was X6 (percent protein
in positive phase pre-rigor muscle) which increased the multiple corre-
lation coefficient from .41 to .55 but failed to increase the estimate
of variability in tenderness to any great extent. The predictive equa-
tion for shear tenderness now becomes: Y = 16.30 .66 X17 + .15 X9 -
.12 X6. Holding X9 and X6 constant, a percent change in X17 (bone
phosphorus) will result in a change of .66 in predicted value of shear
tenderness. Holding X17 and X6 constant, a percent change in Xg will
result in changing the predicted value of tenderness by only .15 units.
Holding X17 and X6 constant, a percent change in X9 (post-rigor positive
phase) will cause a change of .12 in the predictive value of shear ten-
The fourth step included initial pH (X2) which changed the multiple
correlation coefficient from .55 to .59. The predictive equation then
became: Y = 5.05 .69 X17 + .17 X9 .16 X6 + 1.66 X2. Holding X17
(bone phosphorus), Xg (positive protein phase of post-rigor muscle) and
X6 (positive protein phase of pre-rigor muscle) constant a change in X2
of one pH unit would change the predictive value of shear tenderness
by 1.66 units.
If X17, X9, X6 and X2 were known it would be possible to account
for only 34.8 percent of the variability in shear tenderness. The addi-
tion of more independent variables did not result in a significant in-
crease in the estimate of shear tenderness.
Proposed Mechanism of Phosphate Action.
Sodium metaphosphate and sodium pyrophosphate were injected
ante-mortem in an effort to improve tenderness. In trials 2 and 3
phosphate-injected lots were consistently more tender than the non-
injected control. In trial 3 one pyrophosphate-injected lot was sig-
nificantly more tender than the control. The conclusion drawn from
this study was that phosphate injection tended to improve tenderness,
but not to the extent that would permit a definite conclusion. Since
the sodium metaphosphate and sodium pyrophosphate injections did not
provide the tenderizing effect anticipated, it became of interest to
speculate what the fate of the phosphates was in vivo. If the injected
phosphate was completely combined with magnesium and calcium in the
blood stream, it would not be available to the tissue as sodium meta-
phosphate or sodium pyrophosphate.
It was assumed that a 100 pound animal had fiver percent of its
body weight as blood, of which 50 percent was serum. It may also be
assumed the calcium content of blood serum was 10 mg./100 ml. or 113.5
mg. of calcium, but probably only 60 percent (68.1 mg.) of the calcium
was in the diffusable form. If the magnesium content of blood serum
was 2 mg./100 ml. there was 13.6 mg. of magnesium in the blood serum
of the 100 pound animal.
The following reactions were proposed for sodium metaphosphate
and sodium pyrophosphate with calcium and magnesium ions:
(Na P03)6 + 3 Ca++ -- 3 Ca (PO3)2 + 6 Na+
(Na P03)6 + 3 Mg+ -- 3 Mg (P03)2 + 6 Na+
Na4P207 10 H20 + 2 Ca++ -- Ca2P207 5 H20 + 4 Na+ + 5 H20
Na4P207 10 H20 + 2 Mg++ -3 Mg2P207 3 H20 + 4 Na + 7 H20
The formula weight for sodium metaphosphate was 611.80 and that
for calcium 120, therefore 5.1 mg. metaphosphate would be required for
each mg. calcium chelated. If 300 mg. metaphosphate were available from
the injection this would chelate 58.9 mg. of calcium leaving 9.2 mg. of
calcium and 13.6 mg. of magnesium, assuming that calcium was preferen-
The formula weight for sodium pyrophosphate was 446.11 and that
for calcium 120; therefore 5.6 mg. pyrophosphate would be required for
each mg. calcium chelated. If 300 mg. of pyrophosphate were injected,
this would chelate 53.7 mg. of calcium, leaving 14.4 mg. of calcium and
13.6 mg. of magnesium, again assuming that calcium was preferentially
From these theoretical calculations it would seem possible that
the metaphosphate or pyrophosphate could have combined in the blood to
form the calcium or magnesium salt. If this was true, then the ob-
served tenderizing effect probably was not due to the chelating ability
of the phosphate at the tissue level, but rather to the effect of cal-
cium or magnesium phosphate salt or to some as yet unidentified sec-
SUMMARY AND CONCLUSIONS
Two preliminary trials were conducted to develop experimental
techniques and gain knowledge concerning the usefulness of sodium
metaphosphate as a tenderizing agent when injected ante-mortem.
The first preliminary trial with mature sheep provided an oppor-
tunity to study an arterial occlusion technique which made it possible
to have an internal control, thereby holding constant inherent tender-
ness differences. The arterial occlusion technique made it possible
to gain more information from a small number of animals than would
otherwise be possible.
In trial I it was learned that loin chops from sheep were more
uniform in tenderness and thereby permitted a more valid estimate of
the tenderness of an animal than leg steaks.
Data obtained from the first trial suggested that when sodium
metaphosphate was injected ante-mortem at the rate of 3 mg./lb.live
weight tenderness of mutton was improved. Further, it was observed
that the animals used in this trial were under severe shock that
could be confounding the effect of injected metaphosphate.
The second preliminary trial also utilized sheep as experimental
animals. The arterial occlusion technique was not used, however, data
obtained from eight animals in the first trial were included as one
experimental treatment group to compare the suspected effect of phys-
iological shock on tenderness. The other two treated lots both re-
ceived the same level of sodium metaphosphate, but at different times
before slaughter. A fourth lot was included to serve as non-injected
control. Each of the four lots in trial 2 contained eight animals.
Data from broiled leg steaks indicated a marked though non-significant,
tenderizing effect in favor of the three sodium metaphosphate-injected
groups. Similar effects were noted from broiled loin chops. The most
pronounced tenderizing effect was noted in the lot that had received
the arterial occlusion and resultant physiological shock. Lack of
statistical significance in this preliminary trial may have been due,
in part, to improper design. Data obtained in trial 2 provided addi-
tional support for the theory that ante-mortem injection of sodium meta-
phosphate at the proper level and time before slaughter will improve
The third trial involved 77 sheep divided into seven lots with
11 animals per lot. This trial had several objectives. One objective
was to attempt to establish whether or not sodium metaphosphate or
sodium pyrophosphate would improve tenderness when injected ante-mortem.
A second major objective of this trial was to study the effect of feed-
ing abnormal calcium to phosphorus ratios on tenderness. Incidental
to these primary objectives another purpose of this study was to in-
vestigate the relationship between tenderness and muscle pH, and
mineral constituents of bone, muscle and blood and several electro-
phoretic protein fractions of muscle.
Animals included in lot I were designated as the non-injected
control and were fed the control ration. Lot 2 animals were fed a
ration with a high calcium to phosphorus ratio. The third group of
animals received a ration with a high phosphorus to calcium ratio.
Animals in lot 4 were injected with metaphosphate at the rate of
3 mg./lb. live weight,divided into three equal doses and injected
3, 2 and 1 hours ante-mortem. The fifth lot received metaphosphate
at the rate of 3 mg./lb. live weight,administered in three equal doses
48, 24 and 3 hours ante-mortem. The sixth group was injected with
pyrophosphate at the rate of 3 mg./lb. live weight,divided into three
equal doses and administered 3, 2 and 1 hours ante-mortem. The ani-
mals in lot 7 received 3 mg. of pyrophosphate per pound live weight,
divided into three equal doses and injected 48, 24 and 3 hours ante-
All animals were fed for a 28 day feeding period prior to slaugh-
ter. Rations fed lots 1, 2 and 3 have been listed. Lots 4, 5, 6 and
7 received the control ration during the feeding period. Following
the feeding period lots 4, 5, 6 and 7 were injected in the prescribed
manner, animals were slaughtered and carcasses chilled for 48 hours.
Muscle pH was recorded 15 minutes post-mortem,then at intervals 1, 2,
3, 4, 8, 12, 24 and 48 hours post-mortem. A fresh pre-rigor sample
was removed from the L. dorsi immediately after slaughter and an aged
post-rigor sample was removed 48 hours after slaughter for electro-
phoretic protein fractionation. These samples were sharp frozen for
later analysis. At 48 hours post-mortem, bones were removed for
breaking strength determinations and mineral analysis, muscle tissue
was removed from the L. dorsi for mineral analysis and a section of
the loin was removed for tenderness determinations.
Analysis of taste panel data on loin chops failed to show that
a significant difference existed, however, it was apparent that the
injected lots were more tender than the control; further, the high
dietary phosphorus ratio lot was also more tender than the control.
The high dietary calcium ratio lot was only slightly more tender than
the control. According to the panel determinations the greatest im-
provements in tenderness were in the high dietary phosphorus to cal-
cium ratio lot, the lot that received mataphosphate 48, 24 and 3 hours
ante-mortem and the lot that received pyrophosphate 48, 24 and 3 hours
Analysis of variance on the Warner-Bratzler shear determinations
revealed a highly significant (Pc.01) tenderness difference. There
was very close agreement between the panel and shear determinations
among lots. As a result of these two methods of tenderness determina-
tion it appears that injected phosphates will play a significant role
in meat tenderness if properly administered. It may also be said
that on the basis of these data there appears to be a relationship
between dietary phosphorus and tenderness. Further, a high dietary
calcium does not appear to have an adverse effect on meat tenderness;
nor does dietary calcium improve tenderness on the basis of these data.
Analysis of pH data revealed that the control lot had the highest
initial pH level, followed by the high calcium lot and the injected
lots that were injected 48, 24 and 3 hours ante-mortem. Lower pH
values were recorded initially for the high dietary phosphorus to
calcium ratio lot and the two injected lots that were injected 3, 2
and 1 hours ante-mortem. Analysis of variance indicated significance
(PC.05) in the initial pH values. It is likely that excitation was
a factor in the two injected lots having a low initial pH, however,
it is difficult to explain the reason why the high phosphorus lot had
a lower pH.
When ultimate post-rigor pH values were analyzed a significant
(PC.05) difference was found to exist. Pyrophosphate-injected ani-
mals had carcasses with the highest ultimate pH. Significant (P< .05)
but low correlation coefficients were found between initial and ultimate
pH and tenderness.
Electrophoretic protein fractionation failed to provide informa-
tion that would indicate a relationship existed between any of the
fractions separated and tenderness. This study did, however, elucidate
a technique for the preservation of muscle samples in a relatively
static state by sharp freezing. This technique will enable future in-
vestigators to use larger numbers of animals in this type of protein
studies with some confidence that the frozen samples will not be de-
natured if properly handled. A distinct difference was noted in the
extractability of the fresh and aged meat samples when the same buffer
was used. This phenomenon, that had previously been suspected in
mammalian muscle, has been confirmed by this study.
Age at time of slaughter was found to be totally unrelated to
tenderness in this study. Similarly, bone breaking strength was not
found to be related to tenderness. Both of these findings are in
agreement with recent reports in the literature.
Muscle calcium was found to be significantly (P<.01) higher in
the high dietary calcium to phosphorus ratio lot than in the control
or the high dietary phosphorus to calcium ratio lot. A relation be-
tween muscle calcium and tenderness was not found.
Serum calcium was significantly (P <.05) higher in the high
dietary calcium lot than in either the control or the high phosphorus
ratio lot. A consistent relation was not established between blood
serum calcium and tenderness, however, a significant (P< .05) corre-
lation did exist between these two variables.
Bone phosphorus was found to be significantly (P<.05) lower in
the control than in the high dietary phosphorus ratio or the high
dietary calcium ratio lots, a fact that cannot be explained on the
basis of the feed fed these animals. Bone phosphorus was found to be
more highly correlated with tenderness than any of the other variables
studied. This fact substantiates the general findings throughout this
study, namely that phosphorus and tenderness were intimately related.
One cannot say whether this was a direct or indirect relationship.
Muscle phosphorus did not vary significantly among the three
feeding lots. Muscle phosphorus was significantly (P< .05) related
to tenderness but the correlation coefficient was small. It is of in-
terest that a rather high correlation was found between muscle phosphorus
and initial and ultimate pH. It has been suggested that this might
possibly account partially for the relationship that is frequently evi-
dent between pH and tenderness.
Analysis of blood serum phosphorus by analysis of variance did
not reveal a significant difference. The high dietary phosphorus to
calcium ratio lots had a higher average than either of the other lots
but the variation within lots, was high. The correlation coefficient
between blood phosphorus and tenderness was low and non-significant.
Muscle magnesium varied slightly among the three feeding lots.
The relationship found between muscle magnesium and tenderness was
The hypothesis that ante-mortem injected phosphates will tenderize
meat animals has been supported by this study. Definite proof of the
hypothesis will require further experimentation. While the present
study did not prove that ante-mortem injection of phosphates will ten-
derize meat animals, it did provide evidence that sodium pyrophosphate
injected in the manner indicated, tenderized mature sheep.
This study also provided evidence in favor of the hypothesis that
a high dietary calcium to phosphorus ratio appears to favor increased
tenderness in mature sheep. Further, the theory that a high dietary
calcium to phosphorus ratio results in less tender carcasses is not
supported by this study since no tenderness differences were noted be-
tween the control and the group fed a high dietary calcium to phosphorus
The theory that the relationship between tenderness and pH of
muscle is coincidental was supported in this study. Lot 7 (pyrophos-
phate-injected 48, 24 and 3 hours ante-mortem) was the most tender lot
and had the highest 48 hour pH of all lots; however, the correlation
coefficients between tenderness and pre-rigor and post-rigor pH were
Attempts to isolate a single electrophoretic protein phase that
was highly correlated with tenderness met with little success. It
was observed that wide individual variation exists in electrophoretic
mobility. Muscle extracts from pre-rigor and post-rigor samples dif-
fer in volume, ease of separation and viscosity. Denatured, pre-rigor
muscle extract is very similar to post-rigor muscle extract, which
suggests denaturation and dissociation of muscle proteins during the
first 48 hours post-mortem.
Phosphorus levels in body tissue appear to be intimately related
to tenderness. This observation is not confirmed in the present study,
however, more work in this area is indicated.
Calcium and magnesium levels in body tissues do not appear to be
factors in tenderness.
Further work should include a more detailed study of the inter-
relationships of dietary and injected phosphorus on muscle tenderness.
Such an investigation should include determinations of organic phos-
phate esters in muscle as well as inorganic phosphorus.
DATA OBTAINED FROM INDIVIDUAL SHEEP ON TRIAL 3
.4' a) (I) i)a
i~4- 4-3 -H 3 i
4-) 4- 0 P4 4->
5 l-( Pi
::D an V a to Ego f
c o l o r o 8* a oa -
< ri CQ < M li M CQ D3C Kili M Mt
C O4 r4 .rI
Cl a W tt SC I-i U O
Q 1^ sC
9 to to Enu ~ (
I2 ta ED i to n ( co 0Q In to to
Ci (jD 1c U)i -,-I CD H^ 0 ag o, s pql
> ~- ?-l *p *^ i *^ l ~- O i-l P- ii t- t
> En ,I to F-4 En E-4
< ati Fc Zf< ti
Ell a4 PQ U) Q tp- g
1 .. 1
TABLE 22 (Cont.)
c *i a H
4 6a 5 4- 5.70 CO 4 46 a 34 4S 1
i o -H^ 4- 1 +o a
4 9 B 3 o a .) C8 m 1
16 4 M 4 126 140 6.2 5.8 20.7 42.9 36.4 48.0 33.1 18.9 5.8 9.25
31 4 M 3 88 936.0 5.7 20.0 46.3 33.7 55.8 28.7 15.4 6.3 9-94
34 4 M 2 91 101 6.4 5.7 17.3 41.8 4o.8 58.7 26.8 14.5 5.5 12.09
36 4 F 1 54 63 6.4 5.8 14.4 40.7 44.9 57.4 29.3 13.3 5.5 14.41
49 4 M 1 75 84 6.4 5.5 26.3 43.1 30.6 53.1 25.8 21.1 5.7 14.00
66 4 M 2 65 75 6.2 5.7 36.9 o4.6 22.5 33.4 23.9 42.6 4.7 14.31
77 4 M 4 75 85 6.0 5.1 17.0 41.5 41.5 42.9 21.8 35.4 5.0 13.16
79 4 M 1 64 71 6.7 5.8 42.3 39.3 18.4 50.7 32.5 16.7 6.5 10.66
81 4 F 3 73 79 6.8 5.9 35.9 49.2 14.9 46.0 32.8 21.3 6.2 11.19
87 4 M 4 96 100 6.4 5.8 22.6 42.9 34.5 49.5 29.4 21.1 4.7 14.53
88 4 M 2 73 73 6.6 5.9 52.8 36.4 10.8 45.0 32.3 22.7 4.2 15.28
23 5 M 1 70 76 7.3 5.7 23.6 43.0 33.3 22.6 48.8 28.6 7.0 8.19
39 5 M 4 106 113 7.1 5.7 23.2 51.2 25.6 45.1 31.5 23.4 7.0 7.41
47 5 M 3 72 78 6.9 5.1 21.6 40.2 38.2 16.4 32.9 50.6 5.7 12.94
59 5 M 1 90 101 6.7 5.8 21.4 57.1 21.4 17.4 38.4 44.2 5.2 14.47
60 5 M 4 100 108 6.3 5.8 15.6 52.4 32.0 48.4 41.2 10.3 3.8 16.31
53 5 F 5 69 80 6.3 5.9 31.6 50.9 17.5 20.1 31.6 48.3 6.7 11.50
76 5 M 2 68 76 6.5 5.8 37.9 40.7 21.4 40.5 35.4 24.1 4.8 16.88
82 5 M 1 58 59 6.5 5.8 22.2 60.6 17.2 24.1 35.3 40.5 4.8 19.59
89 5 M 2 74 80 6.2 5.8 17.5 52.6 29.8 20.5 33.7 45.8 6.0 11.59
93 5 M 5 82 93 6.1 5.8 25.4 59.3 15.3 24.5 33.0 42.5 5.2 12.19
95 5 M 2 72 79 6.3 6.2 12.3 58.5 29.2 20.2 28.6 51.2 6.7 10.59
TABLE 22 (Cont.)
8 6 8 9 6.3 59 13.8 51 35. 5.6 35.1 1.3 .8 3.
43 6 M 3 81 107 6.6 5.8 22.6 64.5 12.9 17.C O 1.4 41.4 3.8 16.25
63 6 M 4 88 03 6. .0 138. 51.0 30.6 13.8 37.1 49.1 5-5 1 .81
32 6 F 5 114 134 6.6 5. 213.3 58.4 20.2 48.8 32.6 18.6 5.53 7.94
43 6 M 3 81 107 67 6.1 22.6 64.95 125.79 17.2 4.4 4.4 3.8 16.25
57 6 M 2 75 1098 6.4 5.9 23.5 57.8 18.6 22.0 3 41.8 5.5 12.97
92 6 M 3 78 103 6.4 6.0 51.0 30 17.5 13.3 37.1 49.1 5.7 115.981
96 6 M 1 67 90 6.2 5.6 23.3 50.0 2.7 213.< 48.3 28.7 5.5 16.41
75 6 M 1 63 876.6 6.1 21.3 62.9 15.7 1 38.8 45.3 4o.4 5.2 14.41
80 6 M 2 77 97 6.6 5.9 33.1 521.65 14.4 41.4 35.5 17.1 4.2 17.22
92 6 M 3 78 104 6.8 6.0 22.2 60.3 17.5 21.6 48.1 31.9 4.7 15.91
96 6 M 1 68 90 6.5 6.1 42.2 49.5 4.3 13.2 38.3 48.o 4.0 16.41
13 7 M 2 81 105 6.6 6.0 32.2 46.7 21.1 38.3 45.2 15.9 5.3 13.84
15 7 M 5 71 98 6.5 5.9 34.1 51.6 14.3 1?.1 32.5 48.4 5.5 10.22
19 7 M 4 94 6.6 6.0 48.7 46.2 5.1 32.3 4.2 27.0 5.5 13.78
45 7 M 3 79 93 6.9 6.0 27.2 51.1 21.7 43.5 35-7 20.8 4.3 13.69
54 7 M 1 65 101 6.8 5.6 15.2 43.9 40.9 45.4 35.5 19.1 4.2 16.59
58 7 M 4 79 106 6.7 6.1 46.2 37.5 14.3 34.2 47.9 17.8 5.7 9.44
64 7 M 3 98 12 6.2 6.1 25.4 50.0 24.6 49.2 38.3 12.5 5.3 11.56
68 7 M 3 73 88 6.6 5.8 27.4 51.9 20.8 19.3 44.3 36.4 6.s 8.91
72 7 M 1 111 127 6.7 5.8 14.8 54.7 30.5 16.6 37.7 45.7 6.3 9.13
83 7 F 1 69 81 6.7 6.0 21.3 53.3 25.3 20.0 43.8 36.2 7.2 6.47
97 7 M 2 86 105 6.1 5.9 41.5 52.1 6.4 42.8 32.1 25.1 6.3 8.78
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The author, Dale Linwood Huffman, was born July 23, 1931, in
Churchville, Virginia. He graduated from Chautauqua Central School,
Chautauqua, New York in June, 1949.
In September, 1950, he enrolled as a student at Bridgewater College,
Bridgewater, Virginia, and attended that institution until June, 1952.
In August, 1952, he entered the United States Air Force and was
released from active duty in August, 1956, with the rank of Staff
In September, 1956, he enrolled as a student at Cornell University
and received the Bachelor of Science degree from that institution in
February, 1959. As an undergraduate at Cornell University, he was a
member of the University Meats Judging Team..
He entered the University of Florida as a graduate assistant in
the Animal Science Department in February, 1959. He received the
degree Master of Science in Agriculture in June, 1960.
The author is a member of Alpha Zeta, Gamma Sigma Delta, American
Society of Animal Science, Institute of Food Technologists, and the
Reciprocal Meat Conference.
He is now a candidate for the degree of Doctor of Philosophy.
This dissertation was prepared under the direction of the chairman
of the candidate's supervisory committee and has been approved by all
members of that committee. It was submitted to the Dean of the College
of Agriculture and to the Graduate Council, and was approved as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
August 11, 1962
Dean, College of Agriculture
Dean, Graduate School
UNIVERSITY OF FLORIDA
II Il1262 553III 90IIII79
3 1262 08553 9079