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‘ MGM-AN STATE UNWERSITY
KERN THUMAS JOKNS
1974 ‘

 

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This is to certify that the
thesis entitled

The Influence of Dietary and
Hormonal Factors on Growth

and Development in the Ruminant
presented by

John Thomas Johns

has been accepted towards fulfillment
of the requirements for

PI], . D . degree in Animal HQSb andry

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ABSTRACT

THE INFLUENCE OF DIETARY AND lDRMONAL FACTORS ON GROWTH
AND DEVELOPMENT IN THE RUMINANT

By
John Thomas Johns

Increasing the efficiency of production in ruminant animals is of
utmost importance. Hormonal regulation of growth would seem to be a
very promising method of accomplishing this but as yet no one is sure
of how hormones actually control growth. This study was designed to
determine normal relationships between hormones and growth and develop-
ment in the ruminant animal.

Forty-two lambs, half male and half female, at birth, 30, 60, 90
and 120 days of age were sacrificed for this experiment. .All lambs were
maintained on the ewe until slaughter or weaning. Six lambs were used
in each of the first three age groups. Remaining lambs were weaned
at 60 days and placed on either a 7 or 15 percent crude protein diet.
The dietary protein levels were used to establish two rates of growth.
Measurements were taken on tissue weights, plasma glucose and.urea
nitrogen, plasma and tissue free amino acids, tissue protein and nucleic
acids and serum and glandular hormone levels.

Liver, muscle and pituitary weights were increased on an absolute

basis by increasing dietary protein. Adrenal gland weights were not

John Thomas Johns

influenced on an absolute basis but as a percent of body weight were
greatly increased by feeding the low protein diet.

As expected, plasma glucose decreased and plasma urea nitrogen
increased with age. Increasing dietary protein had no influence on
plasma glucose but increased plasma urea nitrogen.

Plasma total free amino acids increased with age due to an
increase in the essential amino acids. Decreasing the dietary protein
decreased both essential and nonessential amino acids in plasma.

Neither age nor diet influenced essential or nonessential amino
acids in liver.

MDscle essential and nonessential amino acids increased with age
while decreasing the dietary protein decreased essential amino acids
only.

Liver and muscle RNA and DNA content increased until 120 days
of age when they decreased. Liver protein content followed the same
pattern while muscle protein continued to increase. Increasing the
dietary protein led to increases in each of the parameters.

Serum.growth hormone decreased with age while serum insulin
increased. Dietary protein level had no influence on growth hormone
but increasing protein led to increased serum insulin.

Neither diet nor age had any influence on serum or adrenal glu-
cocorticoid content or concentration.

Total pituitary growth hormone content increased with age but
was not influenced by diet. Neither diet nor age had any effect on

pituitary growth hormone concentration.

John Thomas Johns

Correlation coefficients indicate that growth is highly related
to muscle nucleic acid content and that hormones may exert their

influence on growth through nucleic acids.

THE INFLUENCE OF DIETARY AND HORMDNAL FACTORS ON GROWTH
AND DEVELOPMENT IN THE RUMINANT

By
John Thomas Johns

A D I SSERTAT ION

Submitted to
Michigan State University
in partial fulfillment of the requirenents
for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Hisbandry

1974

ACKNOWLEDGEMENTS

I would like to express my sincere thanks and appreciation to
the following people whose efforts have helped me in obtaining my
degree and the preparation of this manuscript.

Dr. werner G. Bergen for his invaluable help in the supervision
of my graduate program and the preparation of this manuscript.

Dr. Dorice Narins, Dr. Harold Henneman and Dr. J. T. HUber for
their corrections and suggestions which increased the clarity of this
manuscript and their participation in my graduate program.

Dr. R. H. Nelson for making the facilities at Michigan State
University available for this research.

Elizabeth Rimpau and Elaine Pink for their laboratory assistance
and Al Parr, Constantine Fenderson, John Shirley, Ken'McGuffey and

Steve Grigsby for their help in these experiments.

‘My wife Mary and my daughter Jennifer whose love and devotion

have meant so much.

ii

TABLE OF CONTENTS

Page

LIST OF TABLES .......................... iv
LIST OF APPENDIX TABLES ..................... vi
I. INTRODUCTION ....................... 1
II. LITERATURE REVIEW ..................... 2
Growth and Development in Animals ............. 2

The Influence of Age on Body Changes ........... S

The Influence of Diet on Body Changes ........... 9

The Mechanism of Protein Synthesis ............ 16
Hormonal Influences on Growth and Protein Synthesis. . . . 18

III. MATERIALS AND METHODS ................... 31
Design of Experiment ................... 31
Pre-Slaughter and Slaughter Procedures . ......... 31

Blood Sample Collection and Preparation .......... 33
Plasma Urea Nitrogen Determination . . . ......... 33
Plasma Glucose Determination ............... 35
Plasma Free Amino Acid Determination ........... 35

Serum Glucocorticoid Determination ............ 36
Insulin Determination. . . . ............... 37

Growth Hormone Determination ............... 39

Tissue Collection and Analysis Procedures ......... 40

IV. RESULTS .......................... 46
V. DISCUSSION ........................ 75
VI . GENERAL CONCLUSIONS ................ . . . . 86
VII. APPENDIX ......................... 88
VIII. BIBLIOGRAPHY ....................... 96

iii

Table

A

OmNO‘U‘I

10
11

12

13

14

15
16

LIST OF TABLES

Rations .........................
Average Daily Gain and Feed Intake ............

Effect of Age and Diet on Tissue weights .........

Effects of.Age, Diet and Slaughter Stress on Plasma

Glucose and Urea Nitrogen ...............
Effects of Age on Muscle Free Amino Acid Pools ......
Effects of Diet on Muscle Free Amino Acid Pools .....
Effects of Age on Liver Free Amino Acid Pools ......
Effects of Diet on Liver Free Amino Acid Pools ......
Effects of.Age on Plasma Free Amino Acid Pools ......

Effects of Diet on Plasma Free Amino.Acid Pools .....

Effects of Age and Diet on Liver and mscle Nucleic

Acid Concentration ..................

Effects of Age and Diet on Total Nucleic Content of

Liver and Miscle ......... . ..... . . . . .

Effect of Age and Diet on RNA/DNA Ratios of Liver and

Muscle ........................

Effects of Age and Diet on Serum Growth Hormone

and Insulin ........... . . . . . . .....

Effect of Age and Diet on Adrenal Glucocorticoids . . . .

Effect of Age, Diet and Slaughter Stress on Serum

Glucocorticoids ....................

iv

Page
32

47
48

SO
52
54
55
S7
S8
59

61

63

64

67

68

LIST OF TABLES (cont'd.)

T_ab_1_s_ Lass
17 Effect of Age and Diet on Pituitary Growth Hormone ..... 70
18 Effect of Age and Diet on Liver Protein .......... 71
19 Effect of Age and Diet on Miscle Protein .......... 72
20 Correlation Coefficients .................. 74

LIST OF APPENDIX TABLES

lab}: Page.

1 Glucose Oxidase Reagent .................. 88
2 Conposition of Reagents for Radioinummoassays ....... 89
3 Orcinol Reagent ...................... 91
4 Diphenylamine Reagent ................... 92

Acetaldehyde Solution ................... 93
6 BBU Vitamin and Mineral Mix Pl ............... 94
7 NSU Vitamin and Mineral Mix P2 ............... 95

vi

INTRODUCTION

As the world population becomes more affluent, the demand for
meat in the diet increases accordingly. The increased demand for beef
has meant a diversion of feedstuffs from the human population into
beef production. This has met with.much resistance from advocates
claiming that feedstuffs are utilized much.more efficiently by direct
human use. The human competition has greatly increased the cost of
feed to the beef producer. Still people want and demand red meat for
their diet. The solution to the problem would seem to be an increase
in the efficiency of beef production. To produce a beef animal accept-
able to the general population and to be able to decrease competition
for human foods is a goal that must be attained in animal agriculture.

Many experimental methods have been tried in an effort to reach
this goal. The most promising category would appear to be hormonal
regulation of growth. The use of synthetic hormones that act as
growth stimulants has greatly increased the efficiency of beef produc-
tion; however, this has met with resistance from human health advocates.
A.method to regulate hormonal control of growth without risking a
human health hazard is needed.

This study was designed to elucidate the relationships between
normal hormone patterns and growth rates so as to provide a basis for

further research into the area of hormonal regulation of growth.

1

LITERATURE REVIEW

Growth and Development in Animals

 

Growth and development are perhaps the most universal phenomena
that occur on earth. From the simplest microorganism to the most
complex individual, each has the ability to grow and develop. For
people in the livestock production, growth and development are the
most important aspects of their industry. The parameters of growth
and development are so complex that they cannot adequately be, and
perhaps should not be, distinguished from each other.

Many definitions and descriptions of growth and development
have been put forward, some more adequate than Others. Schloss
(1911) defined growth as a correlated increased in the mass of the
body in definite intervals of time in a way characteristic of the
species. This definition is good in that it implies a characteristic
rate of growth that is species dependent and includes development as
an integral part of growth. It does not, however, make any distinction
between.muscle and fat accretion. Brody (1945) has defined growth as
the production of new biochemical units brought about by cell division,
cell enlargement or incorporation of materials from the environment.
This definition does not necessarily imply an increase in physical

magnitude as it could be applied to only a maintenance situation.

Hammond (1952) and McMeekan (1959) have defined growth as merely an
increase in weight until a mature size is reached. .Again this makes
no distinction between.muscle and fat accretion and ignores development
as a part of growth. 'Maynard and Loosli (1969) have perhaps suggested
the most widely preferred description of growth. They indicate that
true growth involves an increase in the structural tissues such as
muscles and bone and also in the organs. They distinguished between
fat deposition in reserve tissues and characterized true growth pri-
marily as an increase in protein, mineral matter and water.

It does not appear important as to whether growth is defined
to include development or, as some have done, define develOpment to
include growth as long as the two are closely related. Lewis (1939)
has described development as a process involving growth, cellular
differentiation and/or development of form.

Palsson and Verges (1952) have reported that the order of tissue
growth and development follows an outward trend starting with the
central nervous system.and progressing through bone, tendon, muscle,
intermuscular fat and subutaneous fat. Growth and development of
the central nervous system is essentially complete at birth; there-
fore, postnatal growth is concerned primarily with increases in bone,
muscle and fat.

Berg and Butterfield (1968) have shown that bone, muscle and
fat growth occur in three overlapping phases. Bone growth is the
earliest developing while muscle tissue shows intermediate development

and fat deposition occurs later than both. These workers found that

factors such as level of nutrition, sex and breed differences could
affect the rate of growth of the tissue but not the order of develop-
ment.

Rate of growth of bone, muscle and fat is not the same as order
of development. Zinn (1967) has reported that over a 270 day feeding
period fat exhibited the greatest rate of growth, muscle an inter-
mediate rate and bone was the slowest growing tissue.

From a weight and economic viewpoint, muscle is the most
important body tissue. For this reason, some detail of muscle growth
and development will be presented. Stromer et_al, (1974) have
described the characteristics of mature skeletal muscle cells. These
cells are more elongated than normal, averaging 1 to 2 centimeters in
length. Each cell is multinucleated, containing from 100 to 200 nuclei.
These nuclei all lie immediately under the outer cell membrane. Each
of the muscle cells or fibers contain elongated protein threads with
their long axis oriented parallel with the long axis of the cell.

These threads serve as the contractile structure of the cell and are
called myofibrils. The myofibrils compose over 50 percent of the total
protein in.a mature skeletal muscle cell and are composed principally
of myosin or actin.

Ashmore et_al, (1972) and Ashmore (1974) use a slightly different
terminology. These workers describe the myofibrils as either a or B
fibers. They further classify the a or B fibers into red, white or
intermediate categories.

Regardless of terminology used, the sequence and thming of

events in skeletal muscle differentiation and developnent is known.

Mesoderm gives rise to a presumptive myoblast which yields a myoblast.
The myoblasts will fuse to form myotubes and through maturation and
differentiation myotubes will form mature myofibers (Straner et 11; ,
1974). Myoblast fusion ceases about the time of birth in domestic
animals; therefore, a newborn contains approximately all of the
skeletal muscle cells that it ever will. Postnatal muscle growth is
then acconplished by enlargement of existing cells. Ashmore gt _a_l_.
(1972) and Ashmore (1974) have described this enlargement beginning
in late fetal stages as a biphasic developnent. Beta fibers develop
first and a fibers later develop and expand around them. The number
of nuclei do increase during this enlargement but not because of
mitosis. They apparently result from the fusion of satellite cells
to the end of existing myofibrils (Stromer e; 31., 1974).

A differentiation in fiber type was observed (Ashmore _e_t_ a_l_. ,
1972) postnatally. Both a and B fibers are classed as red and are
adapted for aerobic metabolism in the newborn. Alpha fibers are
capable of changing phenotype to white and function to anerobic
metabolism depending upon nuscle activity. Red fibers predominate
in active nuscle while white fibers are generally in inactive muscles.
Fiber type is important in meat animal production as white fibers are
larger than red, thus influencing meat quantity but red fibers metabo-

lize and store more lipid than white, thus influencing meat quality.

The Inf luence of Age on Body Changes

 

Blood Metabolites

 

A decrease in blood glucose in the ruminant with increasing age

is well documented. Kennedy gt a1. (1939) reported a decrease in blood

glucose with age in.ca1ves from two days past partum to about one year.
The physiological factor triggering this decrease is still unknown.
'Murley et_al, (1952) measured a decrease in blood glucose values in
calves from birth to five weeks of age. Type of diet fed had no effect
on this response. Lambert et_a1, (1955) also found no relationship of
diet to a decrease in blood glucose measured from birth to eight weeks
of age in calves. Sterm et a1, (1971) also fOund a decrease in serum
glucose with age when comparing suckling, weanling and mature ruminants.

Lupien et a1. (1962) studied the effects of the digestive tract
in calves on an observed decrease in blood glucose from birth to eight
‘weeks of age. Removal of the rumen, reticulum, omasum and a portion of
the abomasum did not affect the Observed decrease. Nicolai and Stewart
(1965) Observed a decrease in blood glucose in calves from 92 mg% at one
week to 67 mg% at ninety days of age. These workers concluded that the
drop was not related to forestomach development or absorption of vola-
tile fatty acids. Young et_al, (1970), using milk fed calves, measured
an increase in blood glucose from birth to two days and then a decrease
to 100 days. Purser and Bergen (1969) and Ponto and Bergen (1974) have
concluded that the decrease in blood glucose may be a constitutive
change natural to the ruminant species as diet, rumen development and
volatile fatty acid absorption have no apparent relationship to the
decrease.

Only a few workers have studied the relationship of age with the
nitrogen components of blood. Leibholz (1965) reported a decrease in

total o-amino nitrogen concentration in calves from birth to 24 weeks

of age. She Observed significant decreases with age in the individual
amino acids serine, proline, glutamine, methionine, leucine, lysine and
histidine. Suprisingly, she also reported that plasma urea was signifi-
cantly higher at one week of age and decreased thereafter. Oltjen 2E.§l:
(1969) found no relationship of age with blood amino acids in bulls and
heifers except for an increase in asparagine, glutamine, glutamic acid
and citrulline. Bergen et_al, (1973) also reported no effect of age on
individual amino acids in the growing lamb from 60 to 120 days except
for lysine and leucine, and the response of the two to increasing age

‘was dietary dependent.

Tissue and Blood Hormone Levels

 

Curl et_al. (1968) reported that total content of growth hormone
in the pituitary of the bovine increased with age. Growth hormone
concentration, expressed either per unit of gland or per unit of body
weight, decreased with age. .A.positive correlation between.pituitary
growth hormone per unit of body weight and rate of gain was Observed.
Baker et 21, (1956) were not able to find any effect of age on pitu-
itary growth hormone content in holstein heifers. They did report a
decrease in growth hormone concentration with increasing age and a
positive correlation between growth rate and pituitary growth hormone
concentration. Purchas et_al, (1970) measured pituitary and plasma
growth hormone levels in bulls from birth to one year of age. Pitu-
itary growth hormone content and concentration, either as ug/mg of
gland or per unit of body weight, increased until fOur months of age

and then decreased to levels measured at birth and then remained

constant. Siers and Swiger (1971) reported a decrease in serum growth
hormone with age but concluded that the decrease was related to body
size instead of age. They found that pigs of similar size regardless
of age had similar serum growth hormone levels. .A negative correlation
between growth rate and serum growth hormone was also observed.

Regardless of the nature of the change in pituitary growth
hormone, it is apparent that pituitary growth hormone content and
concentration and plasma levels do change with age and are related in
some manner to growth rate.

Changes in levels of other hormones with age have been studied.
Macmillan and Hafs (1968) reported a decrease in pituitary luteinizing
hormone concentration from one month to a year. Pituitary total con-
tent as well as plasma concentration increased with age. Rawlings
et_§1, (1972) reported an increase in levels of blood and testicular
testosterone with age in holstein bulls. Dvorak (1972) reported a
decrease in plasma cortisol and corticosterone in.swine from one day

postnatally to maturity.

Tissue NUcleic Acid Content

 

Devi et_al, (1963) measured an increase in rat liver DNA and RNA
concentration immediately after birth but a decrease shortly thereafter.
Enesco and Puddy (1964) measured a 2-4 fold increase in muscle DNA
content in rats from suckling to young adults. Gordon.et_§l, (1966)
reported an increase in total DNA of rat quadriceps from birth to 90
days but no further increase after this point. Robinson and Lambourne

(1970) also reported a decrease in muscle DNA and RNA concentration in

the mouse. Buchanan and Pritchard (1970) found an increase in total

DNA content of tibialis anterior muscle of rats from birth to puberty.

 

Females increase were slightly less than those in.males. Howarth and
Baldwin (1971) have reported the same trends in the gastrocnemius of
rats. Gilbreath and Trout (1973) have also reported a decrease in

muscle DNA and RNA concentration with age in swine. Similar findings

have also been reported for cattle (LaFlamme et_al,, 1973).

The Influence of Diet on Body Changes

 

Blood Metabolites

 

Many contrasting results concerning the effects of dietary changes
on blood glucose levels in ruminants have been reported. In beef
heifers Stufflebeam et_al, (1969) could find no effect of level of
energy intake on blood glucose. Likewise Memon.et_al, (1969) reported
no change in plasma glucose of mature ewes with changes in dietary
protein or energy level. Howland et_al, (1966) found increased levels
of plasma glucose in ewes when dietary energy and protein were increased.
Jugular blood glucose in sheep increased significantly when the level
of corn in the ration fed was increased (Clary et_al,, 1967). Preston
and Burroughs (1958) found a decrease in serum glucose levels in lambs
with increasing levels of dietary protein even though feed consumption
was increased at higher protein levels. Although the differences may
be species related, TUmbleson et_al, (1969) using Hormel miniature swine
reported increased blood glucose when feeding a 16 percent versus 4
percent crude protein diet. .Although several contrasting reports have

been presented, it seems reasonable to believe that blood glucose levels

10

in ruminants nearing mature weight may be influenced by ration compo-
sition as well as total feed consumption.

Much work exists concerning the relationship of diet and the
nitrogen components of blood. Decreased protein intake, either by
starving (Leibholz and Cook, 1967) or decreasing the crude protein
content of the ration (Leibholz, 1969), lowered plasma urea nitrogen
in sheep.

Tagari et a1, (1964) suggested that plotting the change in blood
urea nitrogen with time after feeding may be a useful method for
assessing protein utilization in ruminants. They observed an increase
in blood urea nitrogen of sheep as ration protein was increased but
the time of maximum blood levels was delayed with increased protein.
Preston et a1. (1965) found that blood urea nitrogen could be quantified
‘with protein intake per unit of metabolic body weight (BW'75) and there-
fore proposed that the protein status of the animal could be partially
assessed by the blood urea nitrogen concentrations. Nimrick et a1.
(1971) also observed an increase in.plasma urea nitrogen of lambs as
the amount of soy in a corn-soy ration increased.

Abou Akkada and El Sayed Osman (1967) found that although blood
urea nitrogen increased as ration crude protein increased, it was more
closely related to changes in.rumen ammonia. These workers stated that
total nitrogen intake was probably not a major factor in controlling
blood urea nitrogen levels because of differing rumen solubilities of
the protein sources. Boling et_§l, (1972) observed an increase in

plasma urea nitrogen of cattle when increasing ration protein from 6

11

to 16 percent for either soy or urea. However, the magnitude of
increase was higher for urea. Little et_al, (1968) fed sheep the same
amount of nitrogen but varied the source. They reported lower plasma
urea nitrogen values when the protein source was soy or zein than
casein or gelatin.

Changes in the microflora and microfauna population of the rumen
can also affect blood urea nitrogen values. Defaunation will result
in a significantly increased rumen bacterial population (Klopfenstein
et al,, 1966). 'Males and Purser (1970) have suggested that the lower
blood urea values of defaunated sheep are a result of a greater rumen
ammonia utilization by the increased bacterial numbers.

It would appear that level of dietary crude protein does affect
blood urea nitrogen values. However, it seems likely that other
factors such as source of protein, ration energy density and rumen
microbial population.may be as important in determining blood urea
nitrogen levels as nitrogen intake alone.

Changes in ration protein may also affect blood amino acid
levels. Nimrick et_al. (1971) have reported significantly increased
levels of branched chain amino acids and a trend for all amino acids
to increase as dietary protein and feed consumption increased. Like-
wise, Weston (1971) and Hogan.et_al, (1968) have reported a decrease
in both plasma total essential and nonessential amino acids in sheep
fed purified diets ranging from 6 to 15 percent crude protein.

The source of dietary nitrogen has also been thought to influence
blood amino acid levels. Oltjen et_§l, (1969) reported a decrease in

blood essential amino acids of cattle fed a urea diet compared to

12

cattle fed an isolated soy diet. Boling et_§1, (1972) reported no
change in plasma amino acid concentration of cattle fed a corn silage
ration supplemented with either soy or urea. Bergen et_§1, (1973)
reported increased ration protein raised plasma total essential amino
acids with no change in total nonessential amino acids. Source of
nitrogen also influenced plasma amino acid patterns. Sheep fed fish
protein concentrate as the major protein had higher plasma amino acid
levels than sheep on other rations. Sheep fed a low protein basal
ration or an NPN containing ration tended to have lower branched

chain amino acids and phenylalanine than sheep on other rations. The
low lysine, high leucine content of zein was reflected in plasma levels
of sheep fed a corn protein ration. Schelling et_§l, (1967) and Nimrick
et_§1, (1970 A, B) have speculated that in ruminants total essential
amino acids may be limiting when urea or a highly soluble nitrogen
source is fed. These workers have reported changes in the blood amino
acid patterns by either supplementing the ration or abomasally infusing
certain essential amino acids. It seems likely that plasma amino acids
are controlled more by the amount of absorbable amino acid reaching

the lower gut than protein source per se. The amount of absorbable
amino acid reaching the lower gut will be influenced by ration energy

level and protein solubility in the rumen.

Tissue weights

 

Tumbleson 33 a1. (1969) and Elsley (1963) reported significantly

lighter adrenals, liver and gastrocnemius muscle in swine fed 4

13

versus 16 percent crude protein. Significantly heavier thyroid,
kidney, liver and pituitary weights were found in sheep when ration
protein was increased (Preston and Burroughs, 1958). Pituitary and
adrenal weights were increased in ewes receiving increased levels of
energy and protein (Howland et_al., 1966); however, Bellows et_al,
(1966) found no difference in pituitary weight of rats fed two levels
of dietary energy. Clarke (1969) reported an increase in rat adrenals
on either an absolute basis or as a percent of body weight when.dietary
crude protein was lowered to 4 percent. 'Memon.et_al, (1969) showed an
increase in pituitary weight of ewes when ration protein was increased.
The increased weight was apparently due to an increase in the size of
pituitary cells as measured by pituitary protein to DNA ratios.
Dietary protein intake apparently affects tissue weights more
than does dietary energy. The response in tissue weight to dietary

changes is variable and may be species dependent.

Tissue and Blood Hormone Levels

 

Armstrong and Hansel (1956) found no difference in pituitary
growth hormone content or concentration of holstein heifers grown on
a high and low plane of nutrition, although animals on the high plane
of nutrition grew much faster than on the low level. Stephan.et_al,
(1971) malnourished rat pups by underfeeding of dams during gestation
and lactation and found that growth hormone activity was greatly
reduced in the malnourished compared to the well-nourished pups.
Total growth hormone content of the gland in malnourished rats was

less than 25 percent of that in well-nourished rats. Sinha et_al, (1973)

14

also malnourished rats by placing 16 pups on a female compared to 4
pups for controls. Pituitary growth hormone concentration was signifi-
cantly decreased by malnourishment. Rate of growth hormone synthesis
was checked by label incorporation and found to be lower in the mal-
nourished animals. Plane of nutrition would appear to be an important
factor in controlling pituitary growth hormone levels in at least some
species. The true relationship of pituitary growth hormone content
and growth rate in maximally growing animals has yet to be elucidated.

Results of dietary alterations on blood growth hormone levels
have been variable. Stephan et_al, (1971) and Sinha et a1, (1973)
have reported decreased blood levels of growth hormone in malnourished
rat pups. Trenkle (1970) reported no effect of feeding high energy
rations to cattle on plasma growth hormone levels. 'McAtee and Trenkle
(1971), using cattle, and Trenkle (1971), using sheep, found no effect
of feeding, fasting or nutrient intake on plasma growth hormone levels.
Bassett et_al. (1971) reported that plasma growth hormone in sheep was
negatively related to digestable organic matter intake and the amount
of protein passing to the lower gut. The majority of the evidence
would indicate less than a direct influence of diet on plasma growth
hormone levels although there may be species differences.

Direct effects of dietary changes on blood insulin levels have
been reported. Trenkle (1966, 1970) found that plasma insulin
increased in cattle fed high energy finishing rations and appeared
to be related to consumption of the grain and supplement portion of
the ration. Trenkle (1966) also reported higher plasma insulin in

sheep fed rations in which the energy density was increased. In sheep

15

Bassett et a1, (1971) found high positive correlations between plasma
insulin and daily digestable organic matter intake as well as amount of
protein in the intestines. Borger Ethel, (1973 A) reported significantly
lower plasma insulin in steers fed a low protein ration than in steers
fed normally. It would appear that dietary changes in energy or protein
influence blood insulin levels. .An elevation of blood sugar in rumi-
nants increases insulin secretion (Manns and Boda, 1967) and increased
VFA from rumen fermentation is also thought to influence insulin levels
(Manns et_al., 1967). Amino acids also stimulate insulin secretion

(Frohman, 1969).

Tissue Nucleic.Acid Content

 

Much work on the relationships of dietary change and tissue
nucleic acid content has been reported. Borger et_al, (1973 B) found
no change in.muscle DNA or RNA of finishing cattle due to level of
protein fed. Umana (1965) reported an increase in DNA content and con-
centration in rat liver when either 5 percent protein or protein free
diets were compared to diets adequate in protein. Gilbreath and Trout
(1973) reported a significant decrease in muscle DNA and RNA content
of swine fed 5 percent protein. The low protein fed pigs had signifi-
cantly increased muscle DNA concentrations and decreased RNA concen-
trations. Apparently, cellular DNA content did not decrease as much
as other cellular constituents. Young and Alexis (1968) reported an
increase in skeletal muscle RNA content but a decrease in concentration
when rats were changed from a 3 to 18 percent protein diet. ‘Young et a1.

(1971) reported a decrease in skeletal muscle DNA and RNA content but

16

an increase in DNA concentration in rats fed a low protein diet. Ashley
and Fisher (1967) found similar results with protein depleted cocks.
Howarth and Bladwin (1971) reported a decrease in the rate of RNA and
DNA synthesis in rat muscle when food intake was decreased. Protein as
well as energy intake seems to affect both RNA and DNA content of

tissues, but RNA is more sensitive to nutrient changes.

The Mechanism of Protein Synthesis

 

Before one can discuss the influence of hormones on growth and
protein synthesis, an understanding of the mechanisms of protein
synthesis is essential. Therefore, a brief review will be presented.

The synthesis of all protein is related to and controlled by
the genetic information contained in cellular DNA. The information
may be thought of as flowing from DNA to RNA to proteins. Two major
processes are involved in the transmission of this genetic information
for protein synthesis (Lehninger, 1971) . The first process is tran-
scription, in which the genetic message contained in DNA is transcribed
into messenger RNA. The second is translation, the process in which
the genetic message is decoded and proteins are synthesized. Before any
protein can be synthesized these processes must occur.

Lehninger (1971) described the protein synthesis process in fig
as occurring in four major stages: (1) Amino acid activation,

(2) Initiation, (3) Elongation and (4) Termination.

Amino acid activation requires the preper amino acids, transfer
RNAs (tRNA) , aminoacyl-tRNA synthetases, ATP and Mg”. Amino acids are
enzymatically esterified to the respective tRNA utilizing energy from

ATP. The charged tRNA is now ready for later use in protein synthesis.

17

According to Lucas-Lenard and Lipmann (1971) initiation of the
protein Chain requires the initiating charged tRNA, messenger RNA.(mRNA),
GTP, Mg++, three initiation factors, 408 ribosomal subunit and a 608
ribosomal subunit. The initiating tRNA in bacteria is formylmethionyl-
tRNA and the initiating tRNA in.mammalian cells is thought to be
methionyl-tRNA also (Lucas-Lenard and Lipmann, 1971). Two forms of
methionyl-tRNA have been isolated from eukaryotic cells. One form.has
been found to supply only the N terminal methionine while the other
functions only internally in the growing peptide chain (Lucas-Lenard
and Lipmann, 1971). 'Messenger RNA is produced from DNA.by transcription
and acts as a template for protein synthesis. Energy is supplied from
GTP. An initiation complex is formed by the binding of mRNA, the 408
ribosomal subunit and the initiating charged tRNA. Initiation factors
and GTP are utilized. The 60S ribosomal subunit can now join the
complex to complete the ribosome formation.

Elongation of the protein Chain requires specific charged tRNAs,
‘Mg++, GTP and two elongation factors (Haselkorn and RothmanrDenes,
1973). Elongation is carried out by the sequential addition of new
aminoacyl residues transferred from charged tRNAs specified by a code
located in the mRNA. After peptide bond formation, the mRNA and peptidyl-
tRNA chain are moved along the ribosome to bring the next mRNA code into
position. The processes require elongation factors and energy from GTP
(Lehninger, 1971).

Termination is accompanied by release of the protein chain from
the ribosome complex and requires the termination code in.mRNA, appar-

ently two releasing factors and GTP (Haselkorn and RothmanrDenes, 1973).

18

When the termination code is encountered and the releasing factors are
available, the protein chain is released from the complex. 'Messenger
RNA is now released, the intact ribosome dissociates into subunits and

associates randomly at initiation steps for continued protein synthesis.

Hormonal Influences on Growth and Protein Synthesis

Normal growth and development in domestic animals is dependent,
at least in part, on hormone action. .As a human food source, we are
primarily interested only in the muscle component of the animal carcass.
This portion of the review will then be concerned with the effects of
hormones on protein synthesis. Before actions of individual hormones
are considered, possible general modes of action for all hormones should
be briefly mentioned.

It is generally thought that growth and developmental hormones
exert their effects on protein synthesis via RNA.metabolism.
wannemacher and McCoy (1966) and Howarth (1972) have shown significant
and positive correlations between cellular RNA content and rates of
protein synthesis. ‘Manchester (1970) suggested three possible modes
of action of hormones in RNA.metabolism- (l) Hormones affect protein
synthesis by stimulating production of specific messenger RNA's.

(2) Hormones affect protein synthesis through a general increase in

all forms of cellular RNA due to a hormone induced increase in RNA
polymerase activity. (3) Hormones affect protein synthesis by
influencing the integrity and/or functional capacity of polysomes.
These are possibilities, individual hormones may function in any one or
a combination of the above methods. They may also function in yet a

different manner.

19

Growth Hormone

 

It is well established that hypophysectomy lessens and treatment
with growth hormone stimulates growth and protein synthesis in animals.
'Manchester (1970) indicated that protein and RNA content and rate of
synthesis decreases in a hypophsectomized rat. Treatment with growth
hormone reversed these findings. Tissue DNA content and rate of
synthesis is also decreased by pituitary gland removal, but returned
to normal by injections of growth hormone (Snipes, 1968; Cheek and
Hill, 1970; Trenkle, 1974).

.A lag period between tissue contact with growth hormone and the
observable increase in protein synthesis indicates that some metabolism
of the hormone is necessary (Rillema and Kostyo, 1971). Regardless of
the nature of the hormone metabolism, the increase in tissue protein
content is due to an increase in synthesis and not a decrease in
degradation rate (Goldberg, 1969).

Other factors affecting protein synthesis have also been studied
for their relationship with growth hormone. If amino acid levels
are limiting, protein synthesis rates will be decreased. Riggs and
walker (1960) observed that growth hormone treatment of hypophy-
sectomized rats increased the tissue uptake of a synthetic amino
acid almost immediately. Snipes (1967) reported a decrease in histi-
dine uptake by rat diaphragm from animals with the pituitary removed
due to lack of growth hormone. Snipes and Kostyo (1962) reported
similar results for alanine as well as histidine. When growth hormone

was administered, amino acid transport returned to normal.

20

Although it is apparent that growth hormone can stimulate amino
acid transport, this does not appear to be its main effect in the cell
(Kostyo, 1968) and appears unnecessary even for short term stimulation
of protein synthesis (Kostyo, 1964; Reeds et_§l,, 1971). However,
enhanced amino acid transport would seem necessary for a long term
general increase in protein synthesis.

.A hormone—induced increase in.mRNA or a general increase in all
species of RNA has been mentioned previously. Jefferson and KOrner
(1967) reported an increase in labeling of all nucleic acids from
[3H] orotic acid due to growth hormone stimulation. Sells and
Takahashi (1967), studying the labeling pattern of liver RNA in
hypophysectomized rats, reported the initial range of label incorpo-
ration to be 48 to 188. This range is characteristic of messenger
RNA. Additional time, however, revealed an increase label in ribosonal
RNA as well; so, no conclusion as to Wthh action was most important
for stimulating protein synthesis was reached. Salaman.et_§1, (1972)
reported the earliest observable effect of growth hormone at the cellu-
lar basis was an increase in 45-5 ribosomal precursor RNA in the nucle-
olus from rat liver. This appeared to be a secondary effect due to a
hormone induced increase in the activity of the enzyme RNA polymerase.
Widnell and Tata (1966) and Korner (1967) have also reported a growth
hormone induced increase in the activity of RNA polymerase from rat
liver nuclei. Growth hormone thus seems capable of stimulating
general RNA production and possibly that of specific messenger RNA.

Some actions of growth hormone occur so rapidly as to suggest a

mode of action other than increased RNA synthesis. 'Martin and

21

Young (1965), using diaphragm from hypophysectomized rats, and Korner
(1967) found that use of actinomycin D with growth hormone did not
block the hormone-stimulated increase in protein synthesis; therefore,
synthesis of new RNA is not necessary for initial growth hormone
action. It would seem reasonable, however, that for a long term
increase in protein synthesis, additional RNA would be necessary.

Considering that growth hormone may stimulate protein synthesis
without new synthesis of RNA, additional work has been done in an
attempt to elucidate the relationship of protein synthesis and growth
hormone. Korner (1967) found that the decrease in protein synthesis
in hypophysectomized rats was due to a decrease in the ability of the
liver microsome fraction to incorporate amino acids rather than a
decrease in the activation process itself or any defect in tRNA. The
change in the microsomal fraction was found in the ribosome itself.
Comparison of polysome profiles from hypophysectomized and normal rats
have not revealed any differences (Garren.et_al,, 1967; Kostyo and
Rillema, 1971). It would seem then that some hormone sensitive factor
controlling ribosome function or efficiency is the control point
(Garren.ethal., 1967). Kostyo and Rillema (1971) have suggested that
growth hormone stimulates the ability of the ribosome to promote
peptide bond synthesis (elongation) possibly due to an increased
activity of peptidyl transferase.

Other work has indicated that initiation rather than elongation
is the hormone control mechanism (Korner, 1968). This worker has con-

cluded that an attachment factor needed for combination of the ribosome

22

and messenger RNA either is not present or has impaired function.
Barden and Korner (1969), using hybridization studies have shown a
defect in the 408 ribosome from hypophysectomized rats. Tata (1968)
showed that growth hormone treatment increased the appearance of a 408
ribosomal precursor-messenger RNA particle just before the Observed
increase in protein synthesis. It would appear that growth hormone
can affect initiation via either initiation factor competency or by a
direct effect on the ribosome itself.

Growth hormone can cause rapid short term increases in protein
synthesis as well as more general long term effects. Several modes
of action for control have been discussed but it seems most reasonable
to believe that several factors serve as true controlling mechanisms

fer the hormone-induced increase in protein synthesis and body growth.

Insulin

Carbohydrate metabolism usually comes to mind when.metabolic
actions of insulin are discussed. However, insulin also can exert
profound influences on protein synthesis. Snipes (1968) reported that
the presence of insulin is necessary for the maximal response of hypo-
physectomized rats to treatment with growth hormone. 'Many different
modes of action for insulin stimulation of protein synthesis have been
suggested and many are similar to those discussed above for growth
hormone. The more important ones will be discussed briefly.

Without an adequate supply of amino acids for substrate, protein
synthesis rates would be greatly decreased. One action of insulin that

has been reported is the stimulation of tissue amino acid transport.

23

Guidotti et 31, (1968) reported that insulin administration stimulated
glycine and leucine transport in chick embryo heart. Manchester (1970)
reported an enhanced accumulation of alanine, histidine and.methionine
by rat diaphragm following insulin administration. Hider et_al, (1971)
reported an increase in glycine transport in rat skeletal muscle due
to insulin stimulation. Reeds et_al, (1971) reported that insulin
stimulated the transport of leucine, arginine, valine, lysine and
histidine into rabbit muscle. Growth hormone also enhanced the uptake
of these amino acids and the effects with insulin were more than
additive, suggesting that insulin may stimulate amino acid transport
in a different manner than growth hormone (Reeds et_§l,, 1971).

WOOl and Moyer (1964) reported a stimulation of amino acid
uptake by rat diaphragm due to insulin even in the presence of
actinomycin. Therefore, new RNA synthesis is not necessary for the
insulin enhancement of amino acid transport. Goldstein and Reddy
(1970) have suggested that the major mode of action for insulin
stimulation of protein synthesis is through an enhanced amino acid
transport. However, Manchester (1970) reported that puromycin, an
inhibitor of protein synthesis did not inhibit the insulin stimulation
of amino acid transport in diaphragm muscle. Thus, enhancement of
substrate supply would be important for long term increases in protein
synthesis but may not be a point of control for rapid adjustment of
protein synthesis rates.

Protein synthesis rates are decreased in diabetes mellitus and

Tragl and Reaven (1971) have proposed that it is due to a decreased

24

amount of messenger RNA resulting from the insulin deficiency.
‘Manchester (1970) and Pilkis and Salaman (1972) have reported increased
RNA.polymerase following insulin treatment. However, Eboue-Bonis et_al,
(1963) and WOOl and Cavicchi (1966) reported that RNA synthesis is not
necessary for insulin stimulation of protein synthesis. Protein syn-
thesis was necessary as both puromycin and cycloheximide prevented any
response to insulin addition (W601 and Cavicchi, 1966).

If synthesis of new RNA is not needed for an initial stimulus of
protein synthesis by insulin then the rapid control point must be some
factor in translation. WOol et_§l, (1966) and Leader et_§1. (1971)
have suggested that the difference in protein synthesis between diabetic
and control animals is due to a decreased capacity to initiate synthesis
via a factor in the cell sap. Tragl and Reaven (1972) reported a change
in the polysome profile from heavy polysomes to free ribosomes in an
insulin deficiency, indicating less binding of messenger RNA. This
may be due to a decreased amount or an impaired function of binding
factors in the cell. WOOl and Kurihara (1967) also Observed a change
in the polysome profile from heavy to light with insulin deficiency
and formulated a hypothesis of action for the hormones. They hypothe-
size that insulin first stimulates translation of an existing messenger
for a specific protein. This protein associates with the ribosome
and makes it more competent to bind messenger and form polysomes.

‘Martin and WOOl (1968) using hybridization studies and Castles et_§l,
(1971) have reported that the 605 ribosome of diabetic animals carries

a defect not allowing proper formation of polysanes.

25

Other factors may also be important in the insulin stimulation
of protein synthesis. WOOl et_§l, (1968) reported a decrease in the
activity of aminoacyl-tRNA synthetase in diabetic animals that could
be corrected with insulin additions. Davey and Manchester (1969)
reported that insulin increased labeling of leucyl and tyrosyl-tRNA
in 313:9, indicating that the hormone could increase charging. Other
work has indicated that uncharged tRNA may be able to actively inhibit
protein synthesis, thus the ratio of uncharged to charged tRNA.may be
a regulator of protein synthesis (Seeds and Conway, 1966; Levin and
Nirenberg, 1968).

Insulin can have many effects on protein synthesis, ranging
from charging to messenger binding to a general increase in RNA syn-
thesis to an increase in.amino acid transport. Perhaps all of these
factors in combination and others as yet unknown are needed for the

long term increase in protein synthesis due to insulin.

Glucocorticoids

 

The class of hormones synthesized by the adrenal gland known as
glucocorticoids can have profound and varied effects on tissue protein
synthesis. Palmer (1966) has indicated that glucocorticoids have
catabolic effects on skeletal muscle and anabolic effects on liver.
Bellamy (1964) reported a cessation of growth in rats given daily
injections of cortisol and Hafs et_al, (1971) reported that adrenal
and plasma levels of glucocorticoids were negatively related to rate
of growth in beef cattle. Adrenalectomy has been shown to increase

muscle amino acid uptake and rate of protein synthesis while treatment

26

with glucocorticoid has reversed this finding (Manchester, 1970).
‘Manchester (1970) also reported a decrease in thymus RNA polymerase
activity and ribosome function following glucocorticoid treatment.
Thus it would seem that glucocorticoids inhibit protein synthesis
more than stimulate it. However, these hormones are known to have
anabolic effects in liver at least. It is as yet unclear how gluco-
corticoids decrease muscle protein synthesis in some tissues, but
have the opposite effect on the liver. The evidence for mode of
action which does exist will be briefly reviewed.

Korner (1967) reported an increase in liver glutamic alanine
transaminase synthesis following corticosteroid treatment. This was
apparently due to new messenger synthesis as an increase in label
incorporation into liver RNA was observed before the increase in
enzyme synthesis began. Kenney (1970) has supported the above findings
by suggesting that glucocorticoids induce liver enzyme synthesis by
promoting specific transcriptions of DNA. Litwack and Singer (1972)
have found labeled cortisol complexed to rat liver nuclear histones
following injection of the hormone in_vivg. These workers suggested
that this interaction would allow more gene transcription. They also
reported an increase in the activity of DNA dependent RNA polymerase
activity which would also allow more messenger synthesis (Litwack
and Singer, 1972). In contrast to these findings, Tata (1968) reported
an increased appearance in liver of a 408 ribosomal precursor-
messenger RNA particle just before a hydrocortisone induced increase
in protein synthesis. Tata (1968) felt that the hormone was influencing

ribosome competency through an influence on.messenger binding.

27

Although all of the evidence does not agree, the predominant
portion suggests that glucocorticoids influence liver protein synthesis
by stimulation of specific messengers. The exact manner of increased
messenger synthesis and how one gene can be selected over another for

stimulation of transcription have yet to be elucidated.

Androgens

Androgens, as the male sex hormones, are usually thought of in
relation to sex organ development; however, they apparently play an
important role in general body development and protein synthesis.
Korner (1967) observed that castration decreased the protein synthetic
capacity of thigh muscle by decreasing the activity of ribosomes.
Treatment with testosterone restored the activity but administration
of actinomycin blocked the hormone response, indicating synthesis of
new RNA was necessary. Widne11 and Tata (1966) reported an increase
in RNA polymerase activity of nuclei isolated from castrated rat liver
following testosterone treatment. Autoradiographic studies have shown
administered testosterone to be located with the chromosomes (Manchester,
1970; Liao and Stumpf, 1968). The studies of Liao and Stumf (1968)
showed an enhanced nucleolar RNA synthesis that was inhibited by
actinomycin representing new RNA synthesis. Breuer and Florini (1966)
reported that treatment of castrate rats with testosterone propionate
increased RNA synthesis by increasing the priming efficiency of DNA,
leading to an increase synthesis of specific messenger RNA molecules.
Liao et a1. (1966) also suggested that testosterone acted through the

chromatin to increase RNA synthesis.

28

Fujii and Villee (1968) also reported increase in RNA synthesis
in young rats following testosterone treatment; however, they suggested
another action for the hormone. They propose that testosterone may
increase the transport of nuclear RNA to the cytOplasm, making more
available for protein synthesis. Recently, Palmiter and Haines (1973)
reported an increase in number of ribosomes and amount of messenger
RNA per cell in chick oviduct following dihydrotestosterone treatment.
They concluded that the increased messenger results from hormone stimu-
lation of RNA polymerase initiation on estrogen activated genes.

.Although clear evidence for the method of action of testosterone
is not known, it appears that the honmone stimulates new messenger RNA

synthesis by interaction with nuclear chromatin.

Estrogens

The estrogens, in accordance with their role in sexual differenti-
ation, influence protein synthesis in several tissues. Luck and Hamilton
(1972) overiectomized rats and reported a decrease in ribosomal RNA syn-
thesis. Treatment with estrogen returned the rate to normal and
increased the rate or efficiency of processing ribosome precursors.
Hamilton et_al, (1968) reported a decreased rate of synthesis of
nucleolar RNA in overiectomized rats. Treatment with estradiol 17 8
increased the rate of synthesis within 20 minutes. Use of actinomycin
D abolished this response, leading the workers to conclude that estrogen

acts by stimulating all forms of RNA synthesis.

29

Other work (Hamilton, 1968) has led to the conclusion that the
mode of action of estrogen on protein synthesis is more indirect,
reacting first with the nuclear chromatin to promote transcription.
Hamilton (1968) also suggested that estrogen accelerates the rate of
formation of ribosomal precursor particles and the transport of
particles with attached messenger to the cytoplasm. Pahmiter (1972)
administered estradiol to immature chicks and observed an increase in
oviduct protein synthesis. He concluded that the hormone mediated
protein synthesis primarily by influences on chain initiation and
increasing the amount of messenger RNA available.

'MOst workers have not found direct influences on the protein
synthetic process but have confined their conclusions to increased
RNA synthesis. Mbore and Hamilton (1964) and Teng and Hamilton
(1967) concluded the initial effect of estrogen on the overiectomized
rat uterus is gene activation allowing synthesis of new RNA leading
to increased polysome function and protein synthesis. Gorski and
Axman (1964), using cycloheximide, reported that protein synthesis
was necessary before the estrogen stimulation in RNA and protein
synthesis is seen. Gorski (1964) suggested that estrogen stimulates
synthesis of a specific protein that can stimulate RNA polymerase
activity thus explaining the observed increase in RNA synthesis.
Notides and Gorski (1966) added evidence to this when they showed
induction of a specific protein within 30 minutes after estrogen
treatment and prior to the increase in protein synthesis. They did
not, however, show a relation between the synthesized protein and RNA

polymerase. Knowler and Smellie (1971) concluded that estrogen first

30

stimulates production of a new messenger coding for a specific protein
that in turn leads to the stimulation of ribosomal RNA and protein
synthesis.

The protein synthesis stimulating effects of estrogen are
apparently indirect ones. It is suggested that the hormone acts at
the DNA level mediating synthesis of an intermediate compound which

then stimulates RNA and protein synthesis.

MATERIALS AND METHODS

Design of Experiment

 

A total of 42 lambs of the following ages: birth, 30, 60, 90
and 120 days were sacrificed in this experiment. Six lambs, half male
and half female, were taken from the ewe for slaughter in each of the
first three age groups. The 24 remaining lambs were weaned at 60 days
and placed on the experimental rations (Table l) . The rations were
designed to be isocaloric and 7 and 15 percent crude protein for the‘
low and high protein rations respectively. The 24 lambs were grouped
by weight and divided evenly between the two diets. Six high protein
and 6 low protein fed lambs were sacrified at both 90 and 120 days of

age.

Pre-Slaughter and Slaughter Procedures

 

New born lambs were removed from the ewe immediately after birth
and not allowed to suckle. All newborns were slaughtered within 12
hours of birth with a blood sample taken only at slaughter. Pre-
slaughter blood samples were not taken as it was assumed that the
stress of birth would mask any stress associated with slaughter
procedures. Pre-slaughter blood samples were taken from lambs of the

remaining age groups via jugular puncture. Heparin was used as an

31

32

 

 

 

TABLE 1
Rations
7% C.P. 15% C.P.
Ingredient % of Total % of Total
Corn, Dent, Yellow, grain, gr 2 US mm wt
54 (4) 4-02-931 40 40
Oats, grain (4) 4—03-309 10 ' ' 10-~.
Cerelose 10 6
Starch 17 10
Sugarcane molasses, (5) 5-04-604 10 10
Fish Protein Concentrate (80% Crude Protein) -- 8
Soybean, Seeds, Solv-extd, grnd, mx 7% fiber
(5) 5-04-604 -- 3
.Alfalfa, hay, S-C, mature 1-00-071 7 7
Mineral-Vitamin Mix 33 3b
Bed-O-Cobsc ___§ .3
100 100

 

a
BBU Vitamin Mineral Mix Pl, Composition in Appendix Table 6

b
NBU Vitamin Mineral Mix P2, Composition in Appendix Table 7

c
Andersons' No. 4 fines, The Andersons, Maumee, Ohio

33

anticoagulant and sodium fluoride at a concentration of 1 mg per ml of
blood was used to prevent glycolysis. .After bleeding, lambs were
trucked approximately 3 miles to the abbatoir and killed by exsanu-

iation without stunning.

Blood Sample Collection and Preparation

Plasma

 

Pre-slaughter blood samples were collected into heparinized tubes
via jugular puncture. Slaughter samples were taken by collecting
trunk blood into heparinized beakers. Both samples were centrifuged
at 4,080 x g for 10 minutes to separate plasma from red cells. The
plasma was transferred into small test tubes with disposable pasteur
pipettes and frozen (-70°) for later analysis of glucose and urea
nitrogen. .A protein free filtrate was prepared (as described later)

from a portion of the plasma and frozen fer amino acid analysis.

Serum

 

Pre-slaughter and slaughter blood samples were collected as
described above except that anticoagulant was not used. Blood.was
allowed to stand at room temperature for 1 hour and then overnight
in a coldroom at about 5°. The clot was rimmed and spun down at 2,200
x g for 30 minutes. The serum was transferred into small vials with

disposable pasteur pipettes and frozen (-70°) for hormone analysis.

Plasma Urea Nitrogen Determination
Plasma urea nitrogen was determined by the microdiffusion.method
of Conway (1960). .All plates were prepared by placing l'ml of glycerol

in the outer well, 1 m1 boric acid solution (.04N) in the inner

34

well, 0.5 ml of plasma and 0.5 ml of distilled water in one side of
the middle well and 0.5 ml urease solution (20 mg/ml) in the other side
of the middle well in order to hydrolyze the plasma urea to ammonia.
The lid was placed on the plate and rotated in the glycerol to provide
a seal and prevent ammonia escape. The plate was swirled gently to
mix the sample and enzyme and then placed on a rotator for one hour to
allow completion of the enzyme reaction. At completion of urea
hydrolysis 1 ml of potassium carbonate (K2003) solution (100% w/v) was
added to the middle well, the lid replaced and sealed and the plates
returned to the rotator for an additional hour. A water blank was
prepared in a similar manner for each group of samples. Each sample
was run in duplicate. At the end of the ammonia diffusion period the
content of the inner well of plates containing plasma appears, green
in color due to the trapped nitrogen. This content was titrated with
a standard solution of 0.04 N HCl until the color matched that of the
water blank (light pinkish red) and burette readings were recorded.
The grams of urea nitrogen per 100 ml of plasma were calculated by the
following equation: Grams of urea nitrogen per 100 ml plasma =

(A)(B)('0(1:4)(100) where A = m1 of acid used to titrate, B = normality

 

of the acid, C = ml of sample used.
All of the nitrogen detected was assumed to be in the form of
urea as previous experiments have shown the ammonia level in blood of

normal animals to be undetectable by this method.

35

Plasma Glucose Determination

 

Plasma glucose was determined by the glucose oxidase method of
Hugget and Nixon (1953) . Plasma was diluted with distilled water so
that 1 ml of diluted plasma contained 10-75 ugrams of glucose. One ml
of the diluted plasma was mixed with 2 ml of glucose oxidase reagent
(Appendix Table l) and incubated at 37° for 30 minutes. The incubation
was ended with the addition of 4 ml of 5 N HCl to each tube. After
mixing, the tubes were allowed to stand 20 minutes for maximum color
development. Optical density was read at a wavelength of 525 nm on
the Coleman Spectrophotometer model 620. A reagent blank containing
distilled water instead of diluted plasma was run in the same manner
as above. Aqueous glucose standard solutions containing glucose in
concentrations of 10, 20, 40, 60, 80 and 100 ugrams per ml were also
run as above for the construction of a standard curve. Glucose con-
centrations of the unknown samples were calculated from the standard

curve .

Plasma Free Amino Acid Determination

 

One mM norleucine was added to plasma used for free amino acid
analysis to act as an internal standard. Norleucine was added at the
rate of 0.1 ml per ml of plasma. This was followed by the addition
of 50% (w/v) sulfosalicylic acid (SSA) at the rate of 0.1 m1 SSA per
ml of plasma to precipitate plasma proteins. After placing in ice
for 30-60 minutes the mixture was centrifuged at 35,000 x g for 15
minutes. The supernatant (protein free filtrate) was removed with
a pasteur pipette and stored at -70° until a complete amino acid analy-

sis could be run (Bergen _e_t_ a_1_. , 1973; Bergen and Potter, 1971) .

36

Serum Glucocorticoid Determination

 

Extraction

 

Trimethylpentane (nanograde) was added to serum in a ratio of 1:5
and vortexed vigorously to wash out progestogens. The mixture is
frozen and stored at -20° for 1 hour and with caution to avoid thawing
the serum, the trimethylpentane layer containing progestogens is
decanted and discarded. For glucocorticoid extraction, the washed
serum was thawed, mixed with 2 ml of methylene chloride (reagent grade)
and vortexed vigorously for 1 minute. Two phases formed with the
methylene chloride glucocorticoid containing phase being the lower one.
This phase was transferred with a disposable pipette to a culture tube
and the methylene chloride was evaporated. The tube walls were rinsed 3
times drying between each rinse, with redistilled chloroform; methanol

(99:1) saturated with distilled water.

Competitive Protein Binding Assay

 

The isolated glucocorticoids were resuspended in redistilled
chloroformrmethanol (99:1) saturated with distilled water. .Aliquots
of 50 and 100 pl were transferred to disposable culture tubes and the
solvent evaporated. One ml of 1.25% dog plasma (Colorado Serum
Company) containing about 20,000 cpm/ml of 3H-cortisol was added to
each tube, vortexed and incubated for 12-16 hours at 5°. Bound and
free glucocorticoid were separated with the addition, while stirring,
of 0.5 ml of 0.05% dextran 150 (Pharmacia) and 0.5% carbon decolor-
izing neutral norit (Fisher Scientific Company) to each tube at a

temperature of 5°. Total time from addition of dextranrcoated charcoal

37

to the first tube until addition to the last tube should not exceed 10
minutes. The tubes were vortexed and centrifuged at 2,500 x g for 10
minutes. Radioactivity was determined by the addition of 0.5 ml of
supernatant to 10.0 ml of PCS scintillation fluid (Amersham Searle)

in a glass vial and counting in a Nuclear—Chicago liquid scintillation
counter model 6848. Glucocorticoid standards of concentrations 0.0,
0.1, 0.25, 0.5, 1.0, 1.5, 2.5, 5.0 and 10.0 ng/ml were treated as
described above for construction of a standard curve and calculation
of unknown glucocorticoid concentrations. Final concentrations were
corrected for procedural losses. Approximately 2,000 cpm of 3H-
glucocorticoid were placed in disposable culture tubes and unknown
serum (0.1 or 0.2 ml) added and allowed to equilibrate with the tracer
fOr 20 minutes. These samples were extracted and assayed as described

above and a recovery figure was calculated.

Insulin Determination

 

Serum insulin was determined by using the two antidoby radio-
immunoassay system of Grigsby (1973) modified from the prolactin assay
of Koprowski and Tucker (1971). The assay used guinea pig antibovine
insulin serum (GPABI) and sheep antiguinea pig gamma globulin (SAGPGG)
to form an isoluble complex with mass great enough to be precipitated
when centrifuged at 2,500 x g for 30 minutes. Compositions of all
reagents used are shown in.Appendix Table 2. The assay has been
validated by Grigsby (1973) and further validation was not considered
necessary. Standards were prepared from purified bovine insulin (Eli

Lilly and Company, Indianapolis, Indiana, lot 795372, 24.2 units per mg)

38

with 100 pl of each standard containing 0.04, 0.06, 0.08, 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 3.0, 4.0, or 5.0
ng of insulin.

In preparation for the insulin assay either 250 or 350 pl of 0.05
m phosphate buffered saline-1% bovine serum albumin, pH 7.4, were added
to all tubes prepared for serum samples. Four hundred ul of the above
buffer were added to the tubes for the bovine insulin standards. This
was done before volume was brought to 500 pl with serum samples (150
or 250 pl) or standards (100 pl) to prevent any binding of the hormone
to tube walls.

On day zero, 200 ul of GPABI diluted 1:105,000 in normal guinea
pig serum (NGPS) were added to each tube (except total counts), vor-
texed and incubated for 24 hours at 4°.

On day one, 100 pl of 125I-insulin containing about 15,000 cpm
were added to each tube, vortexed and incubated for 24 hours at 4°.

Two hundred ul of SAGPGG were added to each tube except total
counts on day two, vortexed and incubated for 96 hours at 4°.

At the completion of incubation on day six, 3 ml of 0.05 M
phosphate buffered saline were added to each tube except total counts
and all tubes were centrifuged at 2,500 x g for 30 minutes in a
refrigerated centrifuge with a swinging bucket rotor (Sorval MOdel
RC-3, Ivan Sorval, Inc., Norwalk, Connecticut). The supernatant was
decanted and the tubes inverted on absorbent paper for 30 minutes.

The tubes were then.wiped dry and counted for 10 minutes or 10,000

counts, whichever came first, in a Nuclear-Chicago'MOdel 4230

39

autogamma scintillation counter. The tube number and counting time
was simultaneously punched onto a paper tape (Teletype Corp., Skokie,
Illinois) which was later used in calculating unknown insulin con-
centrations. The insulin standards were used to construct a standard
curve based on the percent of labeled insulin bound. Regression
coefficients for the standard curve were calculated on the C.D.C. 3,600
and entered into an Olivetti calculator (Programma 101, Olivetti
Underwood, New York, New York) which corrected for dilution and auto-
matically calculated hormone concentrations of unknown sera as counting
time and tube number were entered via the punched tape editor (Beckman
'Model 6912 Tape Editor, Beckman Instruments, Inc., Fullerton,

California).

Growth Hormone Determination

 

The assay used for growth hormone (GH) was the double antibody
radioimmunoassay of Purchas (1969). The assay used guinea pig anti—
bovine growth hormone serum (GPABGH) and sheep antiguinea pig gamma
globulin (SAGPGG) to form an insoluble complex that would precipitate
when centrifuged at 2,500 x g for 30 minutes. Standards were prepared
from NIH-GH-B with 100 pl of each standard containing 0.1, 0.3, 0.5,
0.8, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0 ng of GH.

In preparation for the GH assay either 250 or 350 pl of 0.05 M
phosphate buffered saline-1% bovine serum albumin, pH 7.4, were added
to all tubes prepared for serum. Both standards and serum samples were
handled in the manner described previously for the insulin assay. This

'was considered as day zero.

40

On day one, 200 pl of GPABGH diluted 1:3200 were added to all
tubes except total counts, vortexed and incubated for 24 hours at 4°.

On day two, 100 pl of 125I-GH containing about 30,000 cpm were
added to each tube, vortexed and incubated for 24 hours at 4°.

Two hundred ul of SAGPGG were added to each tube except total
counts on day three, vortexed and incubated for 72 hours at 4°.

.At the completion of the incubation on day six, additions and
handling procedures were identical with those described earlier for

the insulin assay. The calculation of results were also identical

to the method described for the insulin assay.

Tissue Collection and Analysis Procedures

 

Following slaughter the liver was immediately removed from the
animal and weighed. Subsamples were taken from each lobe and frozen
in liquid nitrogen and stored at -70° for later analysis.

Samples of liver were taken from the freezer and allowed to
thaw. Approximately 2 grams of liver were weighed and homogenized in
ice cold distilled water. The homogenate was made up to a volume of
40 ml with cold distilled water. Two ml of the homogenate were analyzed
for total nitrogen by the Kjeldahl method using copper as a catalyst.
Eighteen m1 of the homogenate were mixed with 2 ml of 50% (w/v) SSA,
vortexed, placed in ice for 30-60 minutes and centrifuged at 35,000 x
g for 15 minutes. The supernatant was made up to a volume of 20 m1 and

a 5 ml sample taken for Kjeldahl nitrogen analysis. This fraction

41

represents the soluble or non protein nitrogen fraction. The remainder
of the homogenate was frozen for DNA-RNA analysis.

Liver was also analyzed for free amino acids. Approximately 0.5
grams of liver was homogenized in 5 ml of 5% (w/v) SSA with 0.7 ml of
norleucine (1 mM) added to act as an internal standard. The homogenate
‘was placed in ice for 30-60 minutes and centrifuged at 35,000 x g for
15 minutes. The supernatant was evaporated to near dryness and then
resuspended in 2 ml of a pH 2.0 buffer. The samples were frozen and
stored at -70° until a complete amino acid analysis could be run.

A.modification of the method of Munro and Fleck (1969) was used
to determine RNA and DNA. Two ml of the original liver homogenate were
pipetted into glass centrifuge tubes and 10 ml of 2:1 methanol-
chloroform (v/v) were added. The tubes were vortexed, stoppered and
agitated on a rotator for 18 hours. .At the end of agitation, the
tubes were vortexed and centrifuged at 39,000 x g for 15 minutes. The
supernatant was decanted and discarded and the tubes drained upside
down on absorbent paper under the hood to allow evaporation of solvent
fumes. The pellet was broken up with a small wooden applicator stick
and 5 ml of cold 2.5% (w/v) perchloric acid (PCA) were added. Tubes
were vortexed, placed in ice for 10 minutes, vortexed and centrifuged
at 39,000 x g for 15 minutes. The supernatant was decanted and dis-
carded. The pellet was broken up as before and 5 m1 of cold 1.0%

(w/v) PCA were added. Tubes were vortexed and centrifuged at 39,000
x g for 15 minutes and the supernatants discarded. The pellet was

broken up and 4 ml of 0.3 N potassium hydroxide were added and the

42

tubes vortexed. Tubes were incubated at 37° in a water bath for 3
hours, being agitated several times during the incubation. .After
incubation, tubes were vortexed and placed in ice until cold. Five
ml of cold 5.0% (w/v) PCA were added and the tubes vortexed and
placed in ice for 15 minutes after which the tubes were vortexed and
centrifuged at 39,000 x g for 10 minutes. The supernatant was
decanted into 25 m1 graduated tubes and saved. The pellet was washed
twice with 5 m1 of cold 5.0% (w/v) PCA, vortexing and centrifuging
at 39,000 x g for 10 minutes each time. The washings were added to
the 25 m1 graduated tubes and total volume was brought to 20 ml with
5.0% (w/v) PCA. This fraction represented RNA and was saved. The
pellets were saved for DNA extraction and analysis.

DNA extraction was begun by breaking up the pellets as described
above and adding 5.0 ml of 10% (w/v) PCA to each tube. The samples
were vortexed and marbles placed on top to act as condensers. The
samples were then digested in a 70° water bath for 25 minutes. The
samples were agitated frequently during the digestion. .After digestion,
the samples were vortexed, placed in ice until cold and centrifuged at
39,000 x g for 10 minutes. The supernatant was decanted into tubes
calibrated at 10 ml. The pellet was washed with 4.85 ml of cold 10.0%
(w/v) PCA, vortexed and centrifuged as above with the supernatant added
to the 10 m1 calibrated tubes. The total volume was made up to 10 ml
with 10.0% (w/v) PCA and saved. This fraction represented total DNA.

RNA concentration was determined by a colorimetric procedure

utilizing orcinol. Two ml of the RNA fraction were pipetted into a

43

test tube and 2 m1 of a 1.0% (w/v) orcinol reagent (Appendix Table 3)
were added and mixed. Marbles were placed on top to act as a condenser
and the tubes were placed in a boiling water bath for 30 minutes. A
reagent blank of 2 ml of 5.0% (w/v) PCA instead of sample and RNA
standards of 12.5, 25.0, 37.5 and 50.0 mg per ml were treated in the
same manner. After boiling, the tubes were cooled in running cold
water, allowed to reach room temperature and read immediately in the
Coleman SpectrOphotometer model 620 at a wavelength of 680 nm.

DNA concentration was determined by a colorimetric procedure
utilizing diphenylamine and acetaldehyde. Two ml of the DNA fraction
'were pipetted into a test tube and 2 ml of 4.0% (w/v) diphenylamine
in glacial acetic acid (Appendix Table 4) and 0.1 ml of acetaldehyde
solution (Appendix Table 5) were added and mixed. 'Marbles were
added to the top to act as condensers and the samples incubated over-
night at 30° in a water bath. .A reagent blank containing 2 m1 of
10.0% (w/v) PCA instead of sample and DNA standards of 12.5, 25.0, 37.5,
and 50.0 mg per ml were treated in the same manner. The samples were
removed from the water bath and cooled to room temperature and read in

the Coleman Spectrophotometer model 620 at a wavelength of 595 nm.

M2

The gastrocnemius muscle was removed immediately after slaughter,
weighed, frozen whole in liquid nitrogen and stored at -70° for later
analysis.

Muscle samples weighing 2 grams were homogenized and analyzed

for nitrogen as described previously for liver. ‘Muscle was also

44

analyzed for free amino acids. Approximately 0.5 grams of muscle were
homogenized in 20.0 ml of 5.0% (w/v) SSA. The homogenate was placed
in ice for 30-60 minutes and centrifuged at 35,000 x g for 15 minutes.
Five m1 of the supernatant were mixed with 0.7 m1 of 1 mM norleucine
as an internal standard and evaporated to near dryness. The sample
was resuspended in 1.0 m1 of a pH 2.0 buffer and frozen until a complete
amino acid analysis could be run.

Muscle DNA and RNA were analyzed as described for liver with one
exception. The length of the potassium hydroxide digestion was decreased
from 3 to 2 hours duration. The remainder of the analysis was as previ-

ously described.

Adrenal Glands

 

Both adrenal glands were removed immediately after slaughter,
weighed, frozen whole in liquid nitrogen and stored at -70° for later
hormone analysis.

The glands were homogenized for approximately 15 seconds with a
Polytron (Brinkmann Instruments, Inc., westbury, New York). Total
volume of the homogenate was made up to 10.0 ml with distilled water.
The extraction and analysis of glucocorticoids was as described previ-

ously for serum.

Anterior Pituitary

 

The head was severed from the animal and the anterior pituitary
removed, weighed, frozen whole in liquid nitrogen and stored at -70°

for later analysis of growth hormone content.

45

The pituitary was homogenized for approximately 15 seconds in
sufficient volume of 0.05 M phosphate buffered saline, pH 7.4, to
give a concentration of 10.0 mg pituitary per m1 of buffer. Growth

hormone was determined as previously described for serum.

RESULTS

Average daily gain, daily feed intake and feed to gain ratios for
rams and ewes are presented in Table 2. Increasing the percent crude
protein in the diet significantly (P<.05) increased average daily gain
and daily feed intake for rams and ewes at both 90 and 120 days of age.
Feed to gain ratios were significantly (P<.05) decreased in all cases
by the increase in dietary protein except for rams at 120 days of age.
Feed conversion was excellent in all cases for the 15% crude protein
diet, always being less than 3.0.

The effects of diet on adrenal, pituitary, liver and gastrocnemius
muscle are presented in Table 3. Organ weights would normally increase
with age; therefore, the statistical analysis for effects of age was
not performed. Increasing the dietary protein had no significant
(P>.05) effect on adrenal weight, all glands averaging between 0.8 and
0.9 grams.

Pituitary weights were significantly (P<.05) increased by the
increase in dietary protein. Glands from lambs fed the high protein
diet averaged 0.49 grams vs 0.21 grams for glands from lambs on the
low protein diet.

Increasing the dietary crude protein led to a large and highly

significant (P<.01) increase in liver weights. Lambs fed the high

46

47

TABLE 2

Average Daily Gain and Feed Intake

 

 

 

 

Rams Ewes
1 2 3 1 2 3
ADC Intake F/G ADG Intake F/G
90 da 7% 41" 513A 12.5A 36A 409A 11.2A
90 da 15% 309B 781B 2.5B 318B 649B 2.0B
120 da 7% 95A 463A 4.9A 27A 373A 13.7A
120 da 15% 340B 1003B 2.9A 254B 722B 2.8B

 

1

Grams per sheep, mean of 6 animals per group.

2

Grams per day per sheep, mean of 6 animals per group.

3

Grams of feed/gram of gain.

A,B

Nbans differing in superscripts differ significantly between diets

P<.05.

48

TABLE 3

Effect of Age and Diet on Tissue Weights1

 

 

 

 

 

Age Left Adrenal Right Adrenal Pituitary Liver ‘Muscle
Birth .46 .41 .ll 96 20
30 .66 .66 .17 280 56
60 .58 .50 .39 455 102
90 .84 .82 .27 924 156
120 .96 .92 .71 941 172
SEM .02 .03 .03 16 2
Diet Left Adrenal Right Adrenal Pituitary Liver Miscle
Low Protein .88A .80A .er 532a 122a
High Protein .90A .87A .49B 933b 164b
SEM .03 .03 .04 19 3

 

1
Least Square Means in grams, 12 animals/mean for diet.

a b
,'Means in columns differing in superscripts differ significantly,
P<.01; A:BP<.05.

49

protein diet yielded livers averaging 933 grams while livers of low
protein fed lambs averaged only 532 grams.

Gastrocnemius muscle weights also increased significantly (P<.01)
with the additional dietary crude protein. ‘Muscles from lambs fed the
high protein diet averaged 164 grams while lambs on the low protein
diet yielded muscles averaging only 122 grams.

Age, diet and slaughter stress effects on plasma glucose and urea
nitrogen are presented in Table 4. The stress associated with handling
procedures prior to slaughter led to a significant (P<.01) increase in
plasma glucose levels in 90 and 120 day old lambs. Plasma glucose
levels increased from 65 to 75 mg/100 ml with slaughter in 90 day old
lambs and from 58 to 90 mg/100 ml in 120 day old lambs. No effect of
stress on glucose values was observed in either 30 or 60 day old lambs.
As stated previously, pre-slaughter blood samples were not taken from
lambs at birth; therefore, no assessment of stress for this age group
could be made.

Pre-slaughter plasma glucose values decreased significantly (P<.01)
with age at 30, 60, and 90 days, averaging 100 to 85 to 67 mg/100 ml
respectively. The pre-slaughter value of 58 mg/100 ml taken at 120
days was numerically but not significantly smaller than the value at
90 days. Slaughter glucose values exhibited the same general trends
in decreasing with age as the pre-slaughter samples except from birth
to 30 days of age. .A significant (P<.01) increase from 73 to 108 mg/100
ml was observed from birth to 30 days.

The increase in dietary protein exerted no effect on pre-slaughter

plasma glucose levels, being 60 and 62 mg/100 ml for low and high protein

50

TABLE 4

Effects of Age, Diet and Slaughter Stress on
Plasma Glucose and Urea Nitrogen

 

 

 

 

 

 

 

Glucose Urea Nitrogen

Age Preslaughter Slaughter Preslaughter Slaughter
Birth -------- 73:10B ------ lsirA

30 100:2A2 108:4A2 15:1A2 17iTA2

6O 85:4B2 80:4B2 ZZiZBZ 22:1B2

90 67:2C2 75:4B3 21:2B2 24:2B2
120 58:2C2 90:833 20:1B2 24:1B2
Diet
Low Protein 60:3a 70:33 4:.73 5:.7a
High Protein 62:2a 82i5b 20i1b 24:1b

Means : Standard Error, mg/100 ml, 6 animals/mean for age and 12/mean
for diet.

Glucose
Means differing in supersc ipts differ significantly,
2:3slaughter stress P<.Ol; a, diet P<.05.

A’Bage P<.01;

Urea Nitrogen A B
'Means differing in supergcripts differ significantly, ’ age P<.01;
slaughter stress P>.OS; a, diet P<.01.

51

fed lambs respectively. .A significant (P<.05) increase from 70 mg/100
ml for low protein fed lambs to 82 mg/100 ml for high protein fed lambs
was observed in samples taken at slaughter.

The stress of pro—slaughter handling procedures had no effect
on levels of plasma urea nitrogen of any age group.

Pre-slaughter urea nitrogen levels increased significantly
(P<.01) from 15 mg/100 ml at 30 days to 22 mg/100 ml at 60 days. No
further increases were observed as levels were 21 and 20 mg/100 ml
for 90 and 120 days respectively.

Plasma urea nitrogen from samples taken at slaughter exhibited
the same trends as pre-slaughter samples, being significantly (P<.01)
lower at birth and 30 days than any other age group.

Decreasing the dietary protein led to a significant (P<.01)
decrease in plasma urea nitrogen levels for both pre-slaughter and
slaughter samples. Lambs fed the low protein diet averaged 4 to 5
mg/100 ml while those fed the high protein diet averaged 20 to 24
mg/100 ml plasma urea nitrogen.

The changes in muscle free amino acid pools with increases in
age are presented in Table 5. Total essential amino acid (TEAA)
levels decreased numerically but not significantly from birth to 60
days. A significant (P<.01) increase in TEAA levels was observed at
90 days and again (P<.01) at 120 days of age. The same pattern of
change was observed with total nonessential amino acids (TNEAA).
Ratios of nonessential to essential (N/E) amino acids were signifi-

cantly (P<.01) greater at 60 days than at any other age. The N/E

52

TNEES

Effects of Age on Muscle Free Amino Acid Pools1

 

 

 

 

Amino Acids Birth 30 60 90 120 SEM
Lysine .04A. .06A .03A .05A .04A .01
Histidine .10A .09A .09A .09A .07A .03
Arginine .15a .08b .08b .05C .oobC .02
Threonine .48a .oob .07b .04b .04b .02
Valine .04a .06b .08C .07bC .07bC .02
Methionine .03A .03A .03-A .03A .03A .008
Isoleucine .04a .05de .oobd .04Cd .05d .01
Leucine .05a .07b .07b .07b .08e .02
Phenylalanine .osab .03a .03b .03C .02d .006
TEAA .96a .58a .53a 1.68b 4.36C .15
TNEAA 3.033b 1.77a 2.243 4.84b 10.81C .47
N/E 3.30a 3.09ac 4.18b 2.65Cd 2.48d .13
1

umoles of amino acid per gram of wet tissue, Least Square Means.

a,b,c,d,e
Means in a row differingAin superscripts differ significantly
between age groups P<.01; P<.OS.

53

ratio decreased at both 90 and 120 days, reaching a value of 2.48 at
120 days which was significantly (P<.01) less than any other ratio
obtained.

Dietary effects on muscle free amino acid pools are presented
in Table 6. TEAA levels of muscle from lambs fed the high protein diet
were significantly (P<.05) greater than levels measured in muscle of
lambs consuming the low protein diet. No significant (P>.05) dietary
effect on TNEAA was observed. N/E ratios were significantly (P<.01)
increased in muscle from lambs on the low protein diet, reaching a
value of 4.35.

Dietary effects on individual essential amino acids were not
constant. No change was observed in the level of lysine, phenyl—
alanine and histidine. ‘Methionine significantly (P<.01) increased
'with the increase in dietary crude protein. The branched chain amino
acids (valine, leucine and isoleucine) also exhibited a significant
(P<.01) increase when dietary crude protein was increased.

Changes in liver free amino acid pools with changes in age are
presented in Table 7. Liver TEAA levels were significantly (P<.01)
greater at 60 days than at any other age. Levels at all other ages
were not statistically different. Liver TNEAA significantly (P<.01)
increased at 30 and again (P<.01) at 60 days. A significant (P<.01)
decrease to levels measured at birth was observed at 90 and 120 days.
No significant (P>.05) change in liver N/E ratios was found with

changes in age.

Effects of Diet on MUscle Free Amino Acid Pools1

54

TABLE 6

 

 

 

 

Amino Acids High Protein Low Protein SEM
Lysine .04A .04A .04
Histidine .08A .11A .05
Arginine .06A .05A .05
Threonine .04a .03b .01
Valine .07a .04b .02
Methionine .03a .02b .01
Isoleucine .05a .04b .01
Leucine .07a .05b .02
Phenylalanine .02A .02A .008
TEAA 3.02A 2.0613 .19
TNEAA 7 . 82A 8 .45A .71
N/E 2.56a 4.35b .15

umoles of amino acid per gram of wet tissue, Least Square Means.

a,b

‘Means in a row di
between diets P<.01;

ffiefiing in superscripts differ significantly
: P<.05.

55

TABLE 7

Effects of Age on Liver Free Amino Acid Pools1

 

 

 

 

Amino Acids Birth 30 6O 90 120 SEM
Lysine .28a .33a .51b .30a .31a .01
Histidine .37a .57b .65C .68C .58b .01
Arginine .003 .06C .14b .08C .06C .005
Threonine 1.97A 1.63A 2. 52A 1.00A .90A .19
Valine .323 .44C1 .84b .52e .64C .02
Methionine .23A .15C .23A .20B .21B .008
Isoleucine .193 .24C1 .43b .zod .32C .008
Leucine .50a .64b 1.05d .ogb .84C .02
Phenylalanine .15a .16a .25b .15a .22b .008
TEAA 4.02a 4.233 6.63b 3.87a 4.10a .19
TNEAA 15.95a 20.44b 23.63C 15.073 16.843 .58
%/_B 3.99A 4.90A 3.76A 25.93A 4.11A .17

umoles of amino acid per gram of wet tissue, Least Square Means.

a,b,c,d,e
Means in a row differing in superscripts differ significantly
between age groups P<.01; :B»CP<.05.

56

The effects of changing dietary crude protein level on liver
free amino acid pools are presented in Table 8. Increasing the dietary
crude protein had no effect on liver TEAA. Liver TNEAA increased
numerically but not significantly (P>.OS) in lambs consuming the low
protein diet. A nonsignificant (P>.05) increase in the liver N/E
ratio of lambs eating the low protein diet was also found. As was the
case in muscle, lysine and phenylalamine levels did not change and
histidine significantly (P<.01) decreased with the increase in
dietary crude protein. 'Methionine levels also remained constant with
the increasing level of crude protein in the diet. unlike muscle,
only one of the branched chain amino acids, valine, significantly
(P<.05) increased with increasing levels of dietary crude protein.
Isoleucine and leucine levels remained constant.

Plasma free amino acid pool changes with increases in age are
shown in Table 9. Plasma TEAA levels measured at birth were signifi-
cantly (P<.01) lower than levels measured at any other age. Levels
measured at all other ages were relatively constant. Plasma levels
of TNEAA decreased numerically but nonsignificantly (P>.05) with each
increase in age. The plasma N/E ratio was significantly (P<.01) higher
at birth than any other time, with all other ages being statistically
identical.

Changes in plasma free amino acid pools with increased dietary
crude protein are presented in Table 10. Lambs consuming the high
protein diet exhibited significantly (P<.01) higher TEAA than lambs

consuming low protein. Plasma TNEAA levels were the reverse, being

57

TABLE 8

Effects of Diet on Liver Free Amino Acid Pools1

 

 

 

 

Amino Acids High Protein Low Protein SEM
Lysine .30A .30A .02
Histidine .63a .81b .02
Arginine .07a .12b .006
Threonine .95A .80A .04
Val ine .58A .49B .02
Methionine . 20A . 19A .01
Isoleucine .29A .27A .01
Leucine .77A .78A .03
Phenylalanine .18A .20A .01
TEAA 3.98A 3.96A .11
TNEAA 15.96A 21.20A .98
ill/B 4.02A 5.45A .28

umoles of amino acid per gram of wet tissue, Least Square Means.

a,b
Nbans in a row differing in superscripts differ significantly
between diets P<.01;‘A2BP<.OS.

58

TABLE 9

Effects of Age on Plasma Free Amino Acid Pools1

 

 

 

Amino Acids Birth 30 60 90 120 SEM
Lysine 4.83a 16.00b 13.17b 15.50b 15.67b .006
Histidine 8.50A 7.00B 5.67C 4.83C 7.83AB .004
Arginine 6.83a 15.50b 21.33C 9.67a 15.33b .009
Threonine 30.33a 26.33b 21.83C 16.33d 14.33d .009
Valine 13.17a 34.83bC 35.17bC 33.17b 40.178 .020
Methionine 1.003 4.17b 4.33b 6.00C ' 7.67d .002
Isoleucine 4.83a 17.83b 12.67C 18.33b 12.33C .008
Leucine 8.00a 17.67b 23.17C 20.83bC 28.50d .009

Phenylalanine 5.67A 8.67A 6.83A 8.00A 9.17A .004

 

TEAA 83.17a 148.00bC 144.17bC 132.67b 151.00C .040
TNEAA 178.33A 167.33A 164.67A 151.33A 151.00A .050
N/E 2.20a 1.15b 1.14b 1.15b 1.00b .040
1

umoles of amino acid per 100 m1 of plasma, Least Square Means.

a,b,c,d,e
Means in a row differing iB guperscripts differ significantly
between age groups P<.Ol; A, 1 P<.05.

Effects of Diet on Plasma Free Amino Acid Pools

59

TABLE 10

l

 

 

 

 

Amino Acids High Protein Low Protein SEM
Lysine 15.58a 8.42b .005
Histidine 6.333 8.58b .004
Arginine 12.50a 6.50b .008
Threonine 15.33a 8.17b .006
Valine 36.67a 19.25b .010
‘Methionine 6.833 3.00b .002
Isoleucine 15.33a 10.25b .004
Leucine 24.67a 16.33b .009
Phenylalanine 8.58a 5.33b .003
TEAA 141.83a 85.83b .020
TNEAA 151.58A 167.50B .030
N/E 1.08a 1.97b .050

umoles of amino acid per 100 ml of plasma, Least Square Means.

a,b

Means in a row differing in superscripts differ significantly

between diets P<.01;‘A,BP<.05.

60

significantly (P<.05) higher in lambs consuming the low protein diet.
Hence, the plasma N/E ratio was significantly (P<.01) increased in
lambs consuming the low protein diet. Individually, all essential
amino acids measured significantly (P<.01) increased except histidine
which significantly (P<.01) decreased when lambs were fed the high
protein diet.

The effects of age and diet on liver and muscle nucleic acid
concentrations are presented in Table 11. Liver RNA concentrations
decreased significantly (P<.01) from birth and 30 days to 60 and 90
days. A further significant (P<.01) decrease to the lowest value
measured of 6.3 mg/gram was found at 120 days of age. Increasing the
dietary crude protein led to a significant (P<.01) decrease in liver
RNA concentration.

Liver DNA concentrations decreased significantly (P<.01) with
each increase in age. Values dropped from 6.88 mg/gram at birth to
2.7 mg/gram at 120 days. Liver DNA concentrations also decreased
significantly (P<.01) with the increase in dietary crude protein.

Miscle RNA concentrations also decreased significantly (P<.01)
'with age, dropping from 8.98 mg/gram at birth to 3.47 mg/gram.at 120
days. The increased dietary crude protein exerted no significant
(P>.05) effect on.muscle RNA concentrations.

Muscle DNA concentrations followed the same trends as RNA,
decreasing significantly (P<.01) from 3.64 mg/gram at birth to 1.16
mg/gram at 120 days. No significant (P>.05) effect on.muscle DNA
concentration was observed when increasing dietary crude protein

content .

61

TABLE 11

Effects of Age and Diet on Liver and
Muscle Nucleic Acid Concentrationl

 

 

 

 

 

 

Age Liver RNA Liver DNA Muscle RNA Muscle DNA
Birth 24.56a 6.88a 8.98a 3.648

30 24.218 5.64b 5.49b 2.03b

6O 17.53b 4.26C 4.19C 1.54C

90 18.54b 3.62d 4.52C 1.53C
120 6.30C 2.70e 3.47d 1.16d
SEM .68 .16 .17 .07
Diet Liver RNA Liver DNA Muscle RNA Muscle DNA
Low Protein 17.083 4.123 3.66A 1.49A
High Protein 12.42b 3.16b 3.99A 1.34A
gem .36 .10 .24 .06

Least Square Means, 6 animals/mean for age, lZ/mean for diet;
mg/ gram fresh weight.

a,b

Means differing in superscripts differ significantly P<.01;

A»BP<.05.

62

Age and dietary effects on total nucleic acid content of liver
and muscle are presented in Table 12. Liver RNA content was signifi—
cantly (P<.01) increased over levels measured at birth for both 30
and 60 day old lambs. .A further significant (P<.01) increase was also
measured at 90 days. Liver RNA content at 120 days was significantly
(P<.01) decreased compared to levels Obtained at 30 and 60 days.

Liver RNA content was numerically but not statistically decreased by
the decrease in dietary protein.

Liver DNA content tended to follow the same pattern as liver
RNA. Levels at 30 and 60 days were significantly (P<.01) higher than
at birth and a further increase (P<.01) was shown at 90 days, followed
by a drop at 120 days (P<.01). Unlike RNA, liver DNA content was
significantly (P<.01) raised by the increase in dietary crude protein.

Muscle RNA content followed the trend observed for liver.

Total content significantly (P<.01) increased with age up to 90 days,
with a decrease at 120 days. Total content of muscle RNA was increased
significantly (P<.01) by the increase in dietary crude protein.

'Muscle DNA content followed the same pattern as RNA with signifi-
cant (P<.01) increases up to 90 days and then a decrease at 120 days.
No significant (P>.05) effect of diet on muscle DNA content was found.

The effects of age and diet on liver and muscle RNA/DNA ratios
are presented in Table 13. Liver ratios increased from birth to 90
days (P<.01) but decreased at 120 days (P<.01). The increased dietary
crude protein had no significant (P>.05) effect on liver RNA/DNA

ratios.

63

TABLE 12

Effects of Age and Diet on Total Nucleic
Content of Liver and Muscle

 

 

 

 

 

Age Liver RNA Liver DNA 'Muscle RNA Muscle DNA
Birth 2.34s.26a .65i.08a .18s.02a .07s.006a
30 6.71s.62b 1.571.09b .30i.03ab .11i.OlOab
60 8.10:.97b 1,971.25bC .42i.04bC .16i.008bC
90 17.17s1.27C 3.30r.16d .70s.05d .24s.009d
120 5.93s.51b 2.52s.1sC .61i.1OCd .20:.030Cd
Diet Liver RNA Liver DNA Miscle RNA Muscle DNA
Low Protein 9.32:1.56A 2.19i.19a .44i.05a .18i.01‘A
High Protein 11.55:l.8lA 2.91:.16b .65s.06b .zzi.02A

 

1

Means 1 Standard Error, 6 animals/mean for age, 12/mean for diet,

Grams / organ.

a,b

Means differing

A»BP<.05.

in superscripts differ significantly, P<.Ol;

64

TABLE 13

Effect of Age and Diet on RNA/DNA Ratios of Liver and Miscle1

 

 

 

 

 

 

Age Liver Muscle
Birth 3.69i.24aA 2.52:.12A
30 4.33s.3oabAh 2.82:.21A
60 4.22i.30aA 2.74s.14A
90 5.22:.23bB 2.98:.17A
120 2.34i.06C 3.08s.21A
Diet Liver Muscle
Low Protein 4.10s.34A 2.51i.17A
High Protein 3.78:.32A 3.04:.13B

'Mean 1 Standard Error, 6 animals/mean for age and 12 animals/mean for
diet.

a’b’CMeans in ech column differing in superscripts differ signifi-
cantly, P<.01; ’ P<.05.

65

Muscle RNA/DNA ratios tended to increase with age but no
significant (P>.05) effect was found. Increasing the level of dietary
crude protein significantly (P<.05) increased the muscle RNA/DNA
ratio.

Age and dietary effects on levels of serum growth hormone and
insulin are presented in Table 14. Levels of growth hormone were not
significantly (P>.05) different from birth to 90 days, although the
trend was to increase from birth to 60 days and decrease thereafter.
.A significant (P<.05) decrease, resulting in the lowest value measured,
occurred at 120 days. Changing the level of dietary protein had no
significant (P>.05) effect on serum growth hormone levels.

Serum insulin was somewhat erratic but tended to increase
with age to 90 days and then decrease. The level of 86 uunits/ml
measured at 90 days was significantly (P<.05) larger than values
obtained at any other time. The increase in dietary protein levels
led to a significant (P<.01) increase in insulin from 34 uunits to 69
uunits/ml.

The effects of changes in age and diet on total content and
concentration of adrenal glucocorticoids are presented in Table 15.
Although both total content and concentration tended to decrease with
age and increase with increasing levels of dietary crude protein, no
significant (P>.05) difference was found.

Changes in serum glucocorticoids as related to age, diet and
slaughter stress are presented in Table 16. No significant (P>.05)
effect was recorded in either pre-slaughter or slaughter samples for

any change in age or dietary crude protein level. The stress of

66

TABLE 14

Effects of Age and Diet on Serum Growth Hormone and Insulin

 

 

 

 

 

 

Age Growth Hormone2 Insulin3
Birth 5.4:1.IA 27:3A
30 7.5:1.4A 41:5B
60 8.8:z.0A 28:4A
90 4.8: .6A 86:18C
120 2.4: .2B 52:6B
Diet
Low Protein 4.3: .73 34:3a
High Protein 3.8: .43 69:10b
1Means 1 Standard Error, 6 animals/mean for age and 12 animals/mean
for diet.
2Growth Hormone, Ng/ml
Means differing in superscripts differ significantly, A’Bage P<.05;

adiet P> 05.

3Insulin, uunits/ml. A B C
bMeans differing in superscripts differ significantly, ’ 9 age P<.05;
3» diet P<.01.

67

TABLE 15

Effect of Age and Diet on Adrenal Glucocorticoidsl

 

 

 

 

 

 

Age Ng/totalgland Ng/lOO mg wet weight

Birth 217sA 549A

30 2068A 329A

60 1004A 238A

90 1525A 201A
120 1029A 111A
SEM 260 50
Diet
Low protein 881A 112A
High Protein 1277A 156A
saw 202 27

Least Square Means, 6 animals/mean for age, lZ/mean for diet.

.A

'Means in each column differing in superscripts differ significantly,

P<.05.

68

TABLE 16

Effect of Age, Diet and Slaughter Stress on Serum.Glucocorticoids1

 

 

 

 

 

Age Preslaughter Slaughter
Birth ----- 64:21
30 15:3a 2015a
60 12:23 11:2a
90 6:23 19:5b
120 13:2a 14:3a
Diet
Low Protein 7:1 1211
High Protein 9:2 16:3

 

I_.

'Means t Standard Error, Ng/ml, 6 animals/mean for age and stress,
12/mean for diet.

a,b
Means differing in superscripts differ significantly due to
slaughter stress P<.Ol; no significant effect of age and diet P>.05.

69

pre-slaughter handling procedures had no effect except at 90 days
when a significant (P<.01) increase in hormone level was found in
blood samples taken at slaughter.

Table 17 shows the relationship of age and diet to pituitary
gland growth hormone content and concentration. Growth hormone
concentration expressed per mg of pituitary or on a body weight basis
exhibited no significant Change (P>.OS) with age or level of dietary
protein. However, growth hormone concentration per mg of pituitary
was lowest for new born lambs. Total growth hormone content per gland
increased significantly (P<.01) with age after birth with maximum
values at 90 and 120 days of age. No significant (P>.OS) effect of
dietary protein level on total growth hormone content was found
although a large numerical increase was observed at the higher protein
level.

The effects of age and diet on liver protein content and con-
centration expressed as grams per gram of liver are presented in
Table 18. Total liver protein increased significantly (P<.01) with
each increase in age and with the increase in dietary protein level.
The concentration of liver protein increased significantly (P<.01)
from birth to 60 days and then decreased significantly (P<.01) to 120
days, reaching values obtained in the neWborn. Level of dietary
protein had no influence on liver protein concentration.

Table 19 presents the effects of age and diet on muscle protein
content and concentration. Changes in.muscle total protein were

identical to changes observed with liver, a significant increase with

70

TABLE 17

Effect of Age and Diet on Pituitary Growth Hormone1

 

 

 

 

 

 

ug/mg ug/total ug/lOO lbs
Age wet weight gland body weight
Birth 1. 32A 1803‘
30 5. 22A 899b 3849A
60 3.74A 1293b 4013A
90 6.34A 3200C 2306A
120 5.04A 3682C 3484A
Diet .56 SEM 206 SEM 490
Low Protein 6. 26A 2025A 2814A
High Protein 5.08A 3596A 3298A
1 . 71 SEM 410 SEM 532

Least Square Means, 6 animals/mean for age, lZ/mean for diet.

AMeans inbeach column differing in superscripts differ significantly,
P<.05; a, P<.01.

71

TABLE 18

Effect of Age and Diet on Liver Protein1

 

 

 

 

 

 

.Age Total Content2 Concentration3
Birth 12.8a .13a
30 52.5b .19b
60 97.2C .22C
90 184.8e .20b
120 133.0d .14a
Diet 2.95 SEM .004
Low Protein 92.3a .17A
High Protein 158.9b .17A
4.5 SEM .005
1Least Square Means, 6 animals/mean for age, lZ/mean for diet.
2Grams.
3Grams/Grams.

AMeans inba column differing in superscripts differ significantly,
P<.05; ’ P<.01.

72

TABLE 19

Effect of Age and Diet on Miscle Protein1

 

 

 

 

 

Age Total ContentZ Concentration
Birth 1.8a .09a
30 9.8b .17b
60 19.1C .18b
90 24.7d .16b
120 27.58 .l6b
Diet .58 SEM .005
Low Protein 18.6a .16A
High Protein 26 . lb . 16A
.68 SEM .003

 

1

Least Square Means, 6 animals/mean for age, 12/mean for diet.

2Grams .

3 Grams/ Grams .

AMeans i a column differing in superscripts differ significantly,

P<.05; 3’ P<.01.

73

age and level of dietary protein. Miscle protein concentrations
increased significantly (P<.01) from birth to 30 days and remained
constant thereafter. Level of dietary protein had no effect on
muscle protein concentrations.

The correlation coefficients of some selected parameters are
presented in Table 20. Growth, measured in this study as average
daily gain, was positively correlated (P<.01) with muscle DNA and
RNA content. Pituitary growth hormone content showed only a small
positive correlation (P>.05) with average daily gain. However,
pituitary growth hormone content was positively correlated (P<.01)
with muscle DNA and RNA content. Serum insulin was positively
correlated (P<.01) with average daily gain as well as muscle DNA
(P<.05) and RNA (P<.01) contents. Pituitary growth hormone concen-
tration expressed per mg of gland was negatively correlated (P>.OS)
with average daily gain. .A.small positive correlation (P>.OS) with
muscle DNA content was observed. A.positive correlation (P<.05) was
established between pituitary growth hormone concentration and.muscle
RNA content. .A small negative correlation (P>.05) between serum growth

hormone and muscle DNA and RNA was noted.

74

TABLE 20

Correlation Coefficients

 

 

 

 

2 PGH Serum
1 PGH Serum. Concen- Growth
Parameter ADG Content Insulin tration Hormone
ADG .13 .643 -.19 .09
MDNA4 Content .553 .53a .37A .24 -.26
MRNAS Content .543 .623 .523 .33A -.19
1
Average Daily Gain
2
Pituitary Growth Hormone
3
ug/mg of Gland
4
Muscle DNA
5
Muscle RNA
A
P<.05
a

P<.01

DISCUSSION

An objective of this study was to elucidate hormonal relation-
ships with observed growth rates in order to learn.more of hormonal
influences that control growth. Two growth rates, normal and reduced,
were needed in order to determine which hormone-growth rate relation-
ships were important in normal growth. For this reason sheep were
maintained on a ration permitting normal growth (high protein) and
on a ration only sufficient in protein for maintenance of body weight.

Voluntary food intake in cattle (Elliott, 1967 A) and sheep
(Elliott, 1967 B) has been shown to be influenced by level of dietary
protein. Therefore, decreasing the level of dietary protein from 15
percent to 7 percent was chosen as the method to control growth in
this study. The method was effective (Table 2) as both rams and
ewes at 90 and 120 days of age fed the low protein diet gained and
ate significantly less than those on high protein. Feed efficiency
was excellent on the 15 percent ration with a feed ratio conversion
of less than 3 to 1. It must be emphasized that in the remaining
discussion, results reflecting dietary changes are a function not
only of decreased protein intake but also of decreased energy intake

as well.

75

76

Blood glucose (Table 4) values increased from birth to 30 days
and then decreased to 120 days. The peak increase occurs before 30
days in calves (Mirley et 31:, 1952; Kennedy 93.3.1.” 1939; Young _e_1_;_ 31.,
1970) and was probably missed in this experiment. Factors causing
this change are as yet still unclear. The beginning of the decrease
tends to coincide with the time when the ruminant is changing
metabolism from carbohydrate-lipid (milk) to volatile fatty acid
dependent. Jarrett 23,213,1964 and webb §t_ai,,1969 reported a
decrease in the glucose utilization rate as ruminants increased in
age, thus it would appear that the change in energy dependence from
glucose to fatty acid would precipitate the decrease in blood sugar.
However, other workers have shown the decrease in blood glucose to
occur regardless of diet or rumen function (Lupien gt_al,, 1962;
Nicolai and Stewart, 1965; Lambert et al,, 1955). Ponto and Bergen
(1974) studied changes in blood glucose in both germfree and con-
ventional ruminants and found a decrease in blood glucose regardless
of diet or germfree status. They concluded the decrease was constitu-
tive to the ruminant animal and apparently unrelated to rumen function
or VOIatile fatty acid production. It can be concluded that the
change in blood glucose reported in the current study is a normal
occurrence in ruminants but the cause is as yet unknown.

In an effort to measure the effects of pre-slaughter handling
procedures on levels of blood metabolites, blood samples were taken
via jugular puncture with the animals in a resting state and trunk

blood was collected at the slaughter house. At 30 and 60 days there

77

was no effect of stress on blood glucose (Table 4); however, at 90

and 120 days glucose was significantly increased by stress. The 90

and 120 day old lambs were much larger and more difficult to load and
handle than younger groups which prObably accounts for the additional
stress. The increase may have been mediated through increased gluco-
corticoids. Edwards (1969) has indicated that increased glucocorticoids
will lead to increased blood glucose in lambs. Bassett (1963) Observed
an increase in blood glucose as plasma cortisol increased in sheep.
Serum glucocorticoids (Table 16) were significantly (P<.01) elevated

at 90 days and although not significantly different were still high

at 120 days in this study. It is possible that the increased dif—
ficulty in handling was manifested by an increased secretion of
glucocorticoid which in turn mediated the rise in blood glucose.

Increasing the level of dietary protein in this study did not
influence plasma glucose values in pro-slaughter blood samples but
significantly increased glucose values in samples Obtained at slaughter.
The reason for the difference in glucose levels due to time of sampling
is not readily apparent although it could represent an interaction of
stress and diet.

Alterations in diet have been shown to influence glucose values
in the ruminant. Clary gt_al, (1967) reported increased blood glucose
in sheep when corn in the ration was increased. Likewise, Howland
gt_§l, (1966) reported increased blood glucose in ewes when dietary
protein and energy were increased. Bassett gt a}, (1971) reported a
positive and significant correlation between blood glucose and

digestable organic matter intake in sheep. Other reports have

78

indicated either no effect (Stufflebeam et_ai,, 1969; Memon et al,,
1969) or a decrease (Preston and Burroughs, 1958) in plasma glucose of
ruminants following an increase in dietary energy or protein. As
pointed out previously (Clary gt_§i,, 1967), increased carbohydrate

in the diet can lead to increased blood glucose in ruminants probably
through rumen bypass; therefore, it might be expected tht if the
increased feed intake was sufficient to provide rumen bypass of carbo-
hydrate, an increased plasma glucose should have been detected. The
results unfortunately are not sufficient to allow a definitive con-
clusion concerning plasma glucose and dietary relationships in the
ruminant.

Blood urea nitrogen (Table 4) exhibited significant increases
from birth and 30 days to older ages. As the animals increased in
age and size, feed intake (nitrogen intake) increased. Preston
e£_ai, (1965) and Nimrick et_al, (1971) have reported an increase in
plasma urea nitrogen as amount of protein being consumed increased.
This is a reasonable explanation for the increase in urea nitrogen
with increasing age reported in this study.

Differences in urea nitrogen values in the low and high protein
fed animals reflect differences in ration protein content and animal
protein intake. Others have shown that blood urea nitrogen increases
in the ruminant as protein intake increases (Preston gt_al,, 1965;
Nimrick et ai., 1971; Tagari §t_al,, 1964). However, factors other
than ration protein content could have influenced blood urea values
in this experiment and must be considered. Dietary nitrogen, due

to differences in rumen solubility, will influence the amount of

 

 

79

ammonia escaping the rumen and consequently the blood urea levels
(Little gt_al,, 1968; Ely e£_§l,, 1969; Abou Akkada and Osman, 1967).
Ration energy availability through the effects on rumen.micrdbial
protein synthesis will also influence blood urea values; consequently,
low TDN containing rations can lead to increases in blood urea
(Leibholz, 1969; Dror et_ai,, 1969). Although ration protein sources
varied, the rations were isocaloric and the low protein sheep apparently
used the small amount of nitrogen received very efficiently. Thus, it
seems reasonable to assume that differences in plasma urea nitrogen
values were a direct result of the differences in nitrogen intake.

Tables 5, 7 and 9 show individual and total amino acid responses
in muscle, liver and plasma to increases in age. With the exception
of threonine, individual plasma amino acids increased with age.
Bergen et_§i, (1973), with a ration corresponding closely to the one
used in this study, reported no time dependent changes in plasma amino
acids other than an increase in leucine and lysine. Examination of
Table 9 will show significant differences as early as 30 days in the
present study. The sheep used by Bergen §t_§i, (1973) were not
sampled before weaning which may account for the contrasting results.
Leibholz (1965) reported that in calves from birth to 24 weeks of age
plasma methionine, leucine, lysine and histidine decrease with age
while other amino acids did not change.

Similar to this study Bergen §t_§l, (1973) also reported no
change in liver free amino acids.

A.differential effect with age in levels of essential amino

acids was noted in muscle, liver and plasma. Total essential amino

80

acids decreased from birth to 60 days and increased from 60 to 120
days in muscle; increased from birth to 120 days in plasma; increased
to 60 days in liver and then decreased. This pattern of Change in
the three tissues might indicate a less than Optimal supply of energy
and/or amino acids for protein synthesis. The muscle N/E ratios
(Table 5) which increased until 60 days and decreased thereafter also
suggest that nutrient intake was not sufficient at 60 days of age
(Bergen gt_§i,, 1973).

The low protein ration significantly decreased muscle (Table 6)
and plasma (Table 10) total essential amino acids but caused no Change
in liver (Table 8) essential amino acids. In contrast with our data
Boling gt_ai, (1972) reported no change in plasma.amino acids of
steers when dietary crude protein Changed from 6 to 16 percent. How-
ever, most workers have reported an increase in plasma essential amino
acids when protein intake increased (weston, 1971; Hogan.§t_§i,, 1968;
Schelling et_§i,, 1967; Cecyre et_al,, 1973). Feed intake also affects
plasma amino acids (weston, 1971; Nimrick et al,, 1971). Hence, the
decreased plasma pools observed on low dietary protein could be parti-
ally attributed to feed intake.

Mbscle total essential amino acids decreased and the N/E ratio
increased when dietary protein was decreased (Table 6). No Change in
liver free pools with diet changes were measured (Table 8). In a
protein-energy deficiency, muscle will be degraded to supply amino
acids. The bulk of the resulting amino acids would be taken.up by the
liver for catabolimm and could account for the lack of change in liver

free amino acid pools Observed in our study.

81

When protein sources having low rumen solubility are used in
feeding studies or before the rumen becomes totally functional, the
amino acid balance of the protein source may be reflected in plasma
(Leibholz, 1965; Bergen _e_t_ EL, 1973). Fish protein is limiting in
methionine and histidine (Makdani §t_§i,, 1971) but this balance was
not reflected in free amino acid pools in our study possibly because
protein sources in addition to fish protein concentrate were also
used in the ration (Table 1).

Organ and gland and body weights increased with age (Table 3).
The rate of increase was highest at younger ages, decreasing as the
animals neared a market age of 120 days. Purchas gt a1, (1970)
reported a similar pattern for body weights in bulls.

Liver and muscle were smaller on the low protein diet as was
previously reported in studies with swine (Elsley, 1963; TUmbleson gt-
‘ai., 1969). Pituitary glands were smaller on the low protein diet,
but adrenal weights were not altered. Bellows et_§l, (1966) reported
no change in pituitary weight of rats fed an energy restricted diet.
Energy intake was also decreased in the present study because of
decreased feed intake (Table 2). As a percent of body weight, there
was no difference in pituitary weight, whiCh agrees with the work of
Bellows gt El: (1966).

On an absolute basis, no difference in adrenal weight due to
diet changes were measured; however, when.measured as a percent of
body weight adrenals from low protein lambs were much larger than
those from high protein fed lambs, which supports the work of Clarke

(1969) who reported adrenal hypertrophy in rats fed a 4 percent crude

82

protein diet. The adrenal hypertrophy is most readily explained as
the animal response to a stress situation even though there was no
increase in adrenal (Table 15) or serum (Table 16) glucocorticoid
content due to diet. It is possible for adrenal hypertrophy to occur
without an increase in glucocorticoid synthesis (Clarke, 1969).

An interest in domestic meat animal production forces one to
ask why a lower protein diet resulted in decreased liver and muscle
weights. Robinson (1971) proposed that cell size may be determined
by either the protein or RNA to DNA ratio. An increase in either
ratio would indicate an increase in cell size. The muscle RNA/DNA
ratio (Table 13) was significantly lower in low protein lambs sug-
gesting a smaller cell size in these muscles. Liver RNA/DNA ratios
did not change with diet; however, the liver protein to DNA ratio
of 42 for low protein and 55 for high protein groups indicate a
smaller liver cell in lambs fed the low protein ration.

The decreased tissue weight is due to a decrease in cell size
(RObinson, 1971) and cell number. Tissue DNA content is an indicator
of cell number in mononucleate cells. 'Muscles apparently do not
increase in cell number after birth (Hedrick, 1968; Stromer et_al,,
1974; Enesco and Puddy, 1964; Rowe and Goldspink, 1969); therefore,
any increase in.muscle size is probably due to a larger cell size
because of increased cellular constituents. Protein is the major
cellular dry matter constituent thus any factor influencing protein

synthesis will influence mature muscle size.

83

Nucleic acids are essential for protein synthesis to occur.
Protein synthesis is known to be highly correlated with cellular
RNA content (Howarth, 1972; wannemacher and MCCoy, 1966). Feeding a
low protein diet will decrease protein synthesis (waterlow and
Stephen, 1966; Young and Alexis, 1968; Young gt_§l,, 1971) possibly by
decreasing the RNA content (Young §t_al,, 1971; Gilbreath and Trout,
1973; Trenkle, 1974). Total tissue protein was decreased in this
study and the decreased cell size discussed earlier indicates less
protein per cell. Total RNA content, as well as DNA, was also
decreased in the low protein animals (Table 12), thus it would appear
that a decrease in protein synthesis occurred in the low protein
lambs and resulted in decreased tissue size.

As discussed in the literature review, hormones influencing
growth and deveIOpment can have a significant effect on protein
synthesis. It now becomes important in domestic meat animal pro-
duction to determine which hormone-protein synthesis relationships
are most important and how these relationships are mediated.

The hormones Chosen for study in this experiment were growth
hormone, insulin and glucocorticoids. Growth hormone and insulin
are known to have positive influences on muscle protein synthesis
(Manchester, 1970; Goldberg, 1969; W601 et_al,, 1968) possibly at
the ribosome level (Manchester, 1970; Wool and CavicChi, 1966).
Although glucocorticoids have a catabolic effect on skeletal muscle,
they stimulate liver protein synthesis (Palmer, 1966) prObably through
some influence on RNA (Tata, 1968). Positive correlations between

pituitary growth hormone concentration and growth rate have been

84

observed in the bovine (Curl gt_§1,, 1968; Armstrong and Hansel, 1956)
consequently'measurement of glandular levels of hormones were also
made in this study.

Serum growth hormone concentration decreased with age (Table 14)
while pituitary content and concentration (Table 17) increased. If
plasma clearance rate of growth hormone does not change with age
(Trenkle, 1971) secretion rate must have decreased. At 120 days of
age serum growth hormone concentration, liver protein, RNA and DNA
and muscle RNA and DNA content were all decreasing, even though liver
and muscle weights were still increasing. Ribosome activity decreases
with age (Breuer and Florini, 1965), and so does protein synthesis.

If degradation rates remained constant and synthesis decreased, then
a decrease in cellular constituents would occur. However, tissue
weight could have been maintained or increased by an increase in fat
content. As discussed above, growth hormone positively influences
protein synthesis; therefore, the decrease in growth hormone may have
caused the decrease in cellular constituents. It has been suggested
that the cessation of rapid growth in animals is due to a dilution

of growth hormone per unit of body weight (Curl et_ai,, 1968; Baird
et_ai,, 1952; Baker gt_ai,, 1956).

The association of growth and growth hormone becomes less clear
when the effects of diet are examined. Feeding the low protein diet
did not significantly alter serum or pituitary growth hormone levels
although liver and muscle RNA, DNA and protein levels were decreased.
.Although other work has established a positive relationship between

serum and pituitary growth hormone and induced growth rates

8S

(Stephan gt_ai,, 1971; Sinha §t_al,, 1973), no relationship could
be established in the present study.

A relationship between plasma insulin and induced growth rate
can be established. Feeding the high protein diet increased liver
and muscle protein, RNA and DNA content and also increased serum
insulin (Table 14). Insulin would seem to more directly influence
growth rate in this study than growth hormone.

No clear relationship of serum or adrenal glucocorticoids and
growth rate could be established as there was no significant effect
of diet on adrenal (Table 15) or serum (Table 16) glucocorticoids.

The correlation coefficients presented in Table 20 emphasize
previous points. No clear relationship of pituitary or serum growth
hormone and growth measured as average daily gain were established.
Average daily gain and serum insulin were significantly correlated
with muscle nucleic acid content. Serum insulin also was directly
correlated with average daily gain. Thus, it can be postulated that
growth measured as average daily gain is influenced by muscle RNA
and DNA content and that hormonal influences on growth may be medi-

ated through a relationship with nucleic acids.

GENERAL CONCLUSIONS

1. Feeding a low protein diet to growing lambs will create a
stress situation resulting in decreased liver and muscle weights but
in adrenal hypertrophy.

2. A decrease in plasma glucose and increase in plasma urea
nitrogen with increasing age are normal responses in ruminants. A
clear relationship of dietary protein level and plasma glucose is
not known but plasma urea nitrogen increases as dietary protein intake
increases.

3. Nhscle total free amino acids, both essential and non-
essential, increase with age. Decreasing dietary protein does not
influence total amino acids but essential amino acids are decreased.

4. Liver total free amino acids, both essential and nonessential,
are not influenced by age or diet.

5. Plasma total free amino acids increased with age due to an
increase in the essential amino acids. Feeding the low protein diet
decreased total amino acids, both essential and nonessential.

6. Low protein intake will lead to decreased tissue protein and
nucleic acid content. No clear relationship of diet and growth hormone
can be established but insulin varies directly with dietary protein

intake.

86

87

7. Growth measured as average daily gain is directly influenced
by muscle nucleic acid content and this may be the route of hormonal
influence on growth. Insulin apparently also has another more direct

relationship with growth rate and needs further investigation.

APPENDIX

88

APPENDIX
TABLE 1

Glucose Oxidase Reagent

 

 

Tris-phosphate-glycerol buffer ---------------------------------- 100 ml
Glucose oxidase (Boehringer, New York, N.Y.) -------------------- 30 mg
Horseradish perosidase (Boehringer, New York, N.Y.) ------------- 3 mg
O-dianisidine dihydrochloride (Sigma, St. Louis, Mo.) ----------- 10 mg

Dissolve and store at 4°C

Tris-phosphate-glycerol buffer

Tris ------------------------------------------------------------ 36.3g
NaHzm4.H20 """""""""""""""""""""""""""" 50 .0g
Glycerol -------------------------------------------------------- 400 ml

Add water to 1 liter and adjust pH to 7.0 by addition of solid
NEIHZPO4 'HzO

 

89

APPENDIX
TABLE 2

Composition of Reagents for Radioimmunoassays

 

 

0.05 M PBS-1% BSA pH 7.4

NaCl ------------------------------------------------------- 9.0 g
Dissolve with 1 liter of Buffer A1
Buffer A1
NaH PO4'2HZO ----------------------------------------------- 6.2 g
'Mer hiolate ------------------------------------------------ 0.25g
BSA (Fraction V, Sterils, 35% solution serological, NBC,

Cleveland, Ohio) ---------------------------------------- 14.6 ml

Add 950 ml distilled water
pH to 7.5 with S N NaOH
Dilute to 1 liter

Guinea Pig Anti-bovine Insulin and Guinea Pig.Anti-bovine Growth
Hormone

Antisera diluted 1:400 with 0.05 m
PBS-EDTA, pH 7.0. On day of use, dilute 1:400 antisera to
required concentration using 1:400 NGPS as diluent

0.05 M PBS-EDTA pH 7.0

Disodium EDTA ---------------------------------------------- 18.612 g
Add about 950 m1 PBS

Adjust pH to 7.0 with 5 N NaOH

Dilute to 1 liter

0.01 M phosphate buffered saline, pH 7.0 (PBS)

NaCl ------------------------------------------------------- 143 g
‘Monobasic phosphate ---------------------------------------- 120 m1
Dibasic phosphate ------------------------------------------ 240 m1
Merthiolate ------------------------------------------------ 1.75 g

Dissolve in distilled water and transfer to large container
Dilute to 17.5 liters with distilled water
Adjust pH to 7.0 with NaOH if necessary

H.

90

APPENDIX TABLE 2 (cont'd.)
Monobasic phosphate (0.5m)

NaH PO 'HZO ---------------------------------------------- 69.05 g
Disgol3e in distilled water and dilute to 1 liter

Dibasic phosphate (0.5m)
NazHPO4 -------------------------------------------------- 70.98 g
Dissolve in distilled water, heat to dissolve and dilute

to 1 liter

1:400 Normal Guinea Pig Serum (NGPS)

Obtain blood from guinea pigs not used for antibody production
Clot the blood, recover serum and store
Add 2.5 ml of serum to 1 liter volumetric flask and dilute

to 1 liter with 0.05 m PBS-EDTA and store

Sheep Anti-Guinea Pig Gamma Globulin Antibody (SAGPGG)

Dissolve 50 mg guinea pig gamma globulin (Pentex, Kankakee,
Illinois, Fraction II) in 5 ml of .85% sterile saline

Emulsify in 5 m1 Freund's complete adjuvant by continous flux
through an 18 guage needle. (Emulsified when a droplet
retains a bead form when dropped on a water surface)

Antigen is injected subcutaneously in 6-8 sites on side of
animal

Injections repeated every two weeks with Freund's incomplete
adjuvant substituted for the complete

Antisera is collected about six weeks after first injection
(collect about 600 ml blood from a 70 Kg sheep)

Guinea Pig Anti-Bovine Growth Hormone Antibody (GPABGH)

Two mg of bovine GH is dissolved in 0.5 ml saline and emulsified
with Freund's complete adjuvant as described above

Injections were started as above with subsequent injections
of 0.5 mg emulsified in Freund's incomplete adjuvant made
every two weeks for up to seven injections

Blood is collected by heart puncture and serum recovered

 

91

APPENDIX
Table 3

Orcinol Reagent

 

 

Make a stock solution of 0.1% FeC12'6HZO in concentrated HCl.
Before each use, prepare a 1.0% orcinol solution using the
stock solution.

92

APPENDIX
TABLE 4

Diphenylamine Reagent

 

 

Prepare a 4.0% solution of diphenylamine in glacial acetic
acid. Store at 4°C.

 

93

APPENDIX
TABLE 5

Acetaldehyde Solution

 

 

Add 0.4 ml of acetaldehyde to a 250 ml volumetric flask.
Finish filling with distilled water and store at 4°C.

 

94

APPENDIX
TABLE 6

MSU Vitamin and Mineral Mix Pl

 

 

 

Ingredient % of Total
Dicalcium Phosphate 47.38 (26.5% Ca and 20.5% P)
Trace Mineral Salt (High Zn) 47.42
NaZSO4 4.78 (22.5% S)

Vitamin A (10,000 Iu/g) .32

Vitamin D (9,000 IU/g) .10

 

95

APPENDIX
TABLE 7

MSU Vitamin and Mineral Mix P2

 

 

 

Ingredient % of Total
Dicalcium Phosphate 42.29 (26.5% Ca and 20.5% P)
Trace Mineral Salt (High Zn) 42.29
NaZSO4 15.00 (22.5% S)

Vitamin A (10,000 IU/g) .32

Vitamin D (9,000 Iu/g) .10

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