AMENO AGED REQUEREEEHTS AND -
EFFECT OF EXCESS BERRY QRUDE ~

' PROTEEN 0N VOLUNTARY FEED iNTAKE. '-
Aféi} NETROGEN MEMBQLESM

6F GRQ‘NWG STEERS ‘

Digsefiation for the Degree of Ph. 9‘1, . f ~

, MICHIGAN STATEUNI‘JERSETY .
CQNSTANTENE LLEWELLYN FENDERSON ‘
‘ ‘ - 1974‘ A ' -

This is to certify that the

thesis entitled

Amino Acid Requirements and Effect of Excess

Dietary Crude Protein on Voluntary Feed Intake

and Nitrogen Metabolism of Growing Steers
presented by

Constantine Llewellyn Fenderson

has been accepted towards fulfillment
of the requirements for

Ph. D. degreein Animal Husbandry and
Institute of Nutrition

 

 

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ABSTRACT

AMINO ACID REQUIREMENTS AND
EFFECT OF EXCESS DIETARY CRUDE PROTEIN ON VOLUNTARY FEED
INTAKE AND NITROGEN METABOLISM OF GROWING STEERS

By

Constantine Llewellyn Fenderson

This dissertation is concerned with two studies
which were conducted to determine the essential amino acid
requirements and the effect of excess dietary protein on
feed intake and nitrogen metabolism of growing steers.

In the first study, 7 growing Holstein steers (274
kg body weight) were fitted with soft plastic abomasal
cannulae and fed a 9.5% crude protein ration. Amino acids
were infused into the abomasum at incremental levels of 3.5,
7, 11 and 15 g per day for lysine (1ys), methionine (met)
and threonine (thr), and 1.7, 3.5, 7 and 11 g per day for
tryptophan (try). Changes in plasma amino acid concentra-
tions were used to evaluate amino acid requirements.
Nitrogen balances were conducted as a check of the plasma
amino acid method. Daily passage of nitrogen and amino acids
to the abomasum was: nitrogen 14.9, 1ys 4.5, cystine 1.5,
met 1.6, total sulfur amino acids (TSAA) 3.1, thr 3.7 and
try 0.6 g per kg of feed consumed or 105.3, 32.8, 10.7,

Constantine Llewellyn Fenderson
11.2, 21.9, 21.6 and 4.7 g per day, respectively. Plasma
‘met was constant until 7 g per day of met were infused and
thereafter increased with each higher infusion level.
Hence 7 g per day of infused met apparently met the require-
ments. Plasma 1ys, thr and try increased at all infusion
levels indicating that the requirements for these amino
acids were met by the digesta from.the rumen. Assuming a
digestibility of 70% for abomasal digesta, daily absorption
of met, cystine, TSAA, 1ys, thr and try reaching the small
intestine were calculated at 7.9, 7.4, 15.3, 22.5, 15.1 and
3.3g respectively. Daily amino acid requirements of the
slow growing 274 kg steers were calculated to be: met, 14.9;
TSAA, 22.3; 1ys, f 22.5; thr, f 15.1; try, 5 3.3g. The
above data were adjusted to fit the requirement pattern of
similar sized steers fed 9.5 or 12% crude protein ration .
By setting the determined TSAA requirements (22.3g per day)
at unity and assuming that cystine can fulfill 56% of the
need, and by comparing the 1973 NRC pattern of swine amino
acid requirements with the above data, the daily require-
ments of the growing 274 kg steer fed a 9.5% crude protein
ration were estimated to be TSAA 22.3, met 9.8, 1ys 31.2,
phenylalanine plus tyrosine 22.3, valine 22.3, isoleucine
-2.3, leucine 26.8, thr 20.1, try 5.8, histidine 8.0,
arginine 8.9 and total essential amino acids 177.5g per day.
The requirements for the respective amino acids of the

steer fed a 12% crude protein ration were estimated at 25.4,

Constantine Llewellyn Fenderson

11.2, 35.1, 25.4, 25.4, 25.4, 30.4, 22.8, 6.6, 9.8, 10.2
and 202.33 per day.

In the second study, Holstein steers (average body
‘weight, 300 kg) were allotted to a 4 x 4 Latin Square
design with 11 days for adjustment to a low protein ration
followed by a 14 day experimental period. Rations were
pelletted and composed of ground corn, oats, soybean meal,
isolated soy protein, minerals and vitamins. Crude protein
contents were 10.7, 20.2, 32.5 and 40.0% for rations 1, 2,
3 and 4 respectively. Steers were fed at 12 hour intervals
and total daily feed intakes were recorded. Blood samples
'were taken before feeding (To) and rumen samples taken at
T0 and 3 hours after feeding (T3) on days 1, 2, 3, 5, 7, 10
and 14 of the experimental period. Blood samples were
analysed for plasma urea nitrogen and free amino acid levels.
Rumen samples were fractionated and analysed for total
nitrogen, soluble nitrogen, insoluble nitrogen, ammonia
nitrogen, tungstic acid precipitable nitrogen, peptide and
amino acid nitrogen and non protein nitrogen concentrations.
Daily feed intake (kg) declined markedly as the dietary
crude protein increased (9.0, 8.4, 7.1 and 5.0, for rations
l, 2, 3 and 4 respectively) whereas daily protein intake
increased (1.0, 1.7, 2.3, 2.0 kg for the respective rations).
For rations 3 and 4 feed intake was high on the first day,
decreased drastically on the second and third day and then

increased over the remainder of the 14-day period.

Constantine Llewellyn Fenderson
The mechanism.of feed intake depression was not determined.
Steers fed rations 3 and 4 were nibblers while steers fed
rations 1 and 2 were meal eaters with no consistent pattern.
anen ammonia (mg/100 ml) increased with increased dietary
crude protein level (11.6, 39.8, 69.0, 81.9 for the res—
pective rations. Plasma urea nitrogen increased with
dietary protein but total plasma essential and nonessential

amino acids were not affected by treatment.

AMINO ACID REQUIREMENTS
AND
EFFECT OF EXCESS DIETARY CRUDE PROTEIN ON
VOLUNTARY FEED INTAKE AND NITROGEN METABOLISM
OF GROWING STEERS

By

Constantine Llewellyn Fenderson

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Animal Husbandry and Institute of Nutrition

1974

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude 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 and
his constant and vigilant supervision of my graduate program
and the preparation of this dissertation.

Dr. D.E. Ullrey, Dr. J.T. Huber and Dr. D. Narins
for their participation in my graduate program and their
corrections and suggestions which increased the clarity of
this manuscript.

Dr. W.T. Magee for his invaluable help in the
preparation and interpretation of the statistical analysis
of the data, Dr. R.H. Nelson for making funds available for
the project and my financial support throughout my 5 years
of graduate studies here at Michigan State University.
Elaine Fink, Phil Whether and Elizabeth Rimpau for laboratory
assistance, John Thomas Johns, Roger Crickenberger and Bill
Britt for their help in these experiments and Kim.Miller
for typing assistance.

My wife Una and son John whose constant love and

devotion I always relied on.

ii

TABLE OF CONTENTS

LIST OF TABLES .
LIST OF FIGURES . . . . . . . . .
I. INTRODUCTION . . . . . . . . . . . .
A. Protein Metabolism in the Ruminant .
Protein degradation and synthesis in the
rumen . . . . . . . . . . . . . . .
Protein and Amino Acid Reaching the Small

Intestine . . . . . . . . . . . . .

B. Plasma Amino Acids and What They Represent .
C. Amino Acid Requirements of Growing Annmals .

III. MATERIALS AND METHODS

A. Experiment One .
General design of experiment .
Cannula design .
Sample collection and preparation
Nitrogen determinations . .
Plasma amino acid determination
Chromium analysis .
Lignin determination . . . .

B. Experiment Two . . . . .

General design of experiment .

iii

Page

. vi
. viii

.13

19
26
26
26
30
30
32
32
34
34
35
35

TABLE OF CONTENTS (Continued . . . .)

Sample collection and preparation .
Nitrogen determination .
Rumen ammonia and blood urea nitrogen
determination . . . . . . . .
Volatile fatty acids determination
Statistical analysis
IV . RESULTS .
Experiment One
Plasma amino acid concentration changes
as an indicator of amino acid
requirement . . . . . . . . .
Nitrogen balance as an indicator of
amino acid requirement
Nitrogen and amino acid passage to the
abomasum.. . . . . . . . . . .
Quantitative aspects of amino acid
requirement .
V. DISCUSSION .
Amino acid requirements of growing steers

Experiment One

VI. RESULTS . . . . . . . . . . .
Experiment Two . . . . . . . . .
Intake parameters . . . . .

Ruminal nitrogenous fractions .

iv

Page
35
40

40
42
42
43
43

43

46

51

57
62

62
69
69
69
74

TABLE OF CONTENTS (Continued . . . .)

Page
Rumen volatile fatty acids . . . . . . . . . . 80
Plasma parameters . . . . . . . . . . . . . . 84
VII. DISCUSSION . . . . . . . . . . . . . . . . . . . 90
Effect of excess dietary crude protein on
voluntary feed intake and various ruminal
and plasma parameters, Experiment Two . . . . 90
VI. BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . 96

LIST OF TABLES

Table Page
1. Ration Used in Experiment One . . . . . . . . . 27
2. Experimental Design for Experiment Two . . . . 36
3. Rations Used in Experiment Two . . . . . . . . 37
4. Plasma Amino Acid Levels in Relation to the

Level of Amino Acid Infused, Experiment One . 44
5. Average Daily Feed Intake of Steer During

the Infusion Periods, Experiment One . . . . 45
6. Nitrogen Retention in Steers Abomasally

Infused With Different Levels of Amino

Acids . . . . . . . . . . . . . . . . . . . . 52
7. Ratios of Markers in Ration and Abomasal

Ingesta, Experiment One . . . . . . . . . . . 54
8. Nitrogen and Amino Acid Passage to the

Abomasum.in Steers Fed a 9.5% Crude Protein

Ration, Experiment One . . . . . . . . . . . 56
9. Quantitative Aspects of Methionine, Cystine

Total Sulfur Amino Acid, Lysine, Threonine

and Tryptophan Requirements, Experiment One . 58
10. Estimated Daily Essential Amino Acid

Requirement of Growing Steers,

Experiment One . . . . . . . . . . . . . . . . 60

vi

LIST OF TABLES (Continued . . . .)
Table Page
11. Effects of Various Levels of Dietary Crude
Protein on Feed Intake, Plasma Amino Acids
and Urea Levels, Ruminal VFAs and Ruminal
Nitrogenous Fractions, Experiment Two . . . . 70
12. Daily Feed and Protein Intake, Experiment
Two . . . . . . . . . . . . . . . . . . . . . 72
13. Average Daily Concentrations of Ruminal
Nitrogenous Fractions, Experiment Two . . . . 77
-14. Average Daily Rumen Volatile Fatty Acid
Concentrations, Experiment Two . . . . . . . 82
15. Plasma Urea Nitrogen and Amino Acid

Concentrations, Experiment Two . . . . . . . 85

vii

LIST OF FIGURES

Figure

1. Flow Chart of Experiment One .

2. Flow Chart of Fractionation of Roman
Contents . . . . . . . . . .

3. The Effect of Abomasal Methionine Infusions
on Plasma Methionine and Nitrogen Balance

4. The Effect of Abomasal Lysine Infusions on
Plasma Lysine and Nitrogen Balance .

5. The Effect of Abomasal Threonine Infusions
on Plasma Threonine and Nitrogen Balance .

6. The Effect of Abomasal Tryptophan Infusions
on Plasma Tryptophan and Nitrogen Balance

7. Fourteen Day Patterns of Daily Feed Intakes

8. Fourteen Day Patterns of Daily Protein Intake.

9. Fourteen Day Patterns of Plasma Urea Nitrogen
Concentrations .
10. Fourteen Day Patterns of Plasma TEAA

Concentrations .

viii

Page

. 29

. 41

. 47

. 48

. 49

. 50

. 73

. 75

. 86

. 88

INTRODUCTION

It is now well recognized that the rumen and its
symbiotic microbiota allow the ruminant animal to uniquely
utilize cellulose and non protein nitrogen. Adequate
'dietary nitrogen in one form or another is therefore
essential for the optimal growth. Many feeding systems
have been useful in increasing nitrogen utilization in the
ruminant. However, quantitative protein and amino acid
requirements of the growing ruminant have not been deter-
mined.

Ruminants, like all other classes of animals, have
a definite tissue requirement for essential amino acids.
Since extensive ruminal degradation and synthesis of pro-
tein dictate the quantity and pattern of amino acids that
reach the duodenum, it is impossible to determine these
requirements by merely manipulating dietary levels of indi-
vidual amino acids as in the case of non ruminants. It is
therefore necessary to develop a technique in which inter-
ferences by the rumen are completely eliminated. The first
experiment of the present study was designed to quantitate
the essential amino acid needs of growing steers. The
second experiment was to investigate the effects of very

high levels of dietary crude protein on feed intake, plasma

1

2
amino acid levels, plasma urea and the various ruminal
nitrogen fractions in growing steers. It is hoped that
the information obtained from these investigations will be

beneficial in the effort to further exploit the productive

capacities of ruminants.

LITERATURE REVIEW

A. Protein Metabolism.in the Ruminant
1. Protein Degradation and Synthesis in the Rumen

Protein metabolism in the ruminant has been a sub-
ject of considerable investigation over the years. The
use of non protein nitrogen for protein synthesis by rumen
microorganisms and the subsequent conversion of such pro-
tein to tissue protein by the host have made the ruminant
unique.

McDonald (1948) and Mangan (1972) have demonstrated
that proteins entering the rumen are first degraded by
rumen microorganisms to peptides, amino acids, and.ammonia.
Quantitatively, ammonia forms the largest fraction. The
extent of protein degradation depends on its solubility in
rumen fluid (el-Shazly, 1958; Blackburn and Hobson, 1960;
Hendrickaand Martin, 1963). Ingested non protein nitrogen
as well as urea entering the rumen from.saliva (McDonald,
1948; Somers, 1961) or diffusion across the rumen wall from
blood (Houpt, 1959; Juhasz, 1965; Decker £5 31. 1960) is
converted to ammonia by microbial urease. The ammonia is
then incorporated into microbial protein, but the ability
of the rumen microorganisms to utilize the ammonia is
dependent on the type and amount of carbohydrate present
(Lewis 1960). Thus, the nitrogenous nutrients absorbed by

3

4
the host are not merely those of the diet, as in the case
of the nonruminant, but a mixture of dietary constituents,
products of microbial metabolism in the rumen and amino
acids from the microorganisms themselves (McDonald, 1954).

Ammonia that is not reincorporated into microbial
protein will diffuse into the rumen vein, then to the
portal vein and finally to the liver which converts it to
urea (MCDonald, 1948; Cocimano and Leng, 1966). However,
very large excesses of ruminal ammonia will diffuse across
the rumen wall into the peripheral circulatory system
(Chalmers gt 31., 1972). Diffusion of ammonia across the
rumen wall into the circulatory system is recognized as
movement along a concentration gradient (Houpt, 1959).
Thus, ammonia toxicity is caused primarily by diffusion of
ammonia to the peripheral circulatory system.per se, so
that overloading of the liver is not a necessary condition
for toxicity (Chalmers 3; 31. 1972). Chalmers 35 31.
(1972) observed that there were no visible signs of ammonia
toxicity after administering 20 g of urea or 10 g of
ammonia to the abomasum.of sheep. However, when these
workers administered such treatments to the cecum, amonia
toxicity occurred within minutes. Payne and Morris (1969)
observed that there was no increase in plasma ammonia in
sheep which had been adapted to high dietary protein when
they Weredosed with 0.5 g of urea per kilogram of body

weight. However, when sheep maintained on low dietary crude

5
protein were subjected to the same treatment they exhibited
very high plasma ammonia and died. Since these workers
found increased levels of urea cycle enzymes without any
change in alkaline phosphatase activity in the liver of
the sheep maintained on the high dietary crude protein
they concluded that tolerance of ammonia toxicity in sheep
is at least partly a function of urea cycle enzymes in the
liver.

Significant positive correlations between dietary
nitrogen, rumen ammonia and circulating blood urea have been
reported (Lewis, 1957; Tagari g£‘§1., 1964; Preston 32 a1.,
1965; Cocimano and Leng, 1966; McIntyre, 1970). According
to Lewis (1957), the major factor controlling blood urea
level is the concentration of rumen ammonia, in that a
change in rumen ammonia concentration is usually accompanied
by a change in blood urea concentration. Cocimano and Leng
(1966) working with sheep, and Robins £5 31. (1974) working
with white tailed deer found that the plasma urea pool
size and urinary urea excretion rate increased as dietary
nitrogen intake increased.

Ruminal ammonia is the most important source of
nitrogen for rumen bacteria but is less important for
protozoa (Allison, 1969). Abe (1968) and Allison (1969)
observed that rumen ammonia is first incorporated into
bacterial cells and then into protozoal cells following the

ingestion of the bacteria by protozoa. Thus, the lower

6
concentration of rumen ammonia observed in defaunated lambs
than faunated lambs by Klopfenstein, Purser and Tyznik
(1966) and Males and Purser (1970) may be a direct result
of a greater concentration of bacteria in the rumen of
the defaunated animals.

The type of diet will quantitatively influence the
microbial protein reaching the small intestine (Bergen,
Purser and Cline, 1968). According to Bergen st 31. (1968),
microfauna represent a higher percentage of the total
microbial protein when the ruminant is fed a natural diet,
than on a semipurified diet where microflora make up most
of the microbial protein.

The extent to which rumen ammonia is incorporated
into microbial protein depends mainly on the available
energy, the caloric source and the level of sulfur present
in the rumen fluid (Lewis, 1960; Hungate, 1966; Hume, 1970).
Hume (1970a) reported that the addition of a mixture of
higher volatile fatty acids to a purified diet containing
non protein nitrogen as the sole source of nitrogen
significantly increased microbial protein production from
71 g per day to 81 g per day in sheep. Houpt (1959)
observed a significant increase in the utilization of rumen
ammonia in sheep fed increasing levels of a highly digest-
ible carbohydrate supplement.

The level of sulfur in rumen fluid will affect the

number of bacteria and protozoa in the rumen and not the

7

percentage of sulfur within these organisms. Thus, the
increase in rumen protein production from 82 to 94 g per
day, resulting from increasing the dietary sulfur from .61
to 1.95 g per day, was due to an increase in the rumen
microbial papulation (Hume and Bird, 1970). walker and
Nader (1968) determined the nitrogen to sulfur ratio in
bacteria to be 11:1. Bird (1973) found that the nitrogen
to sulfur ratio in bacteria was 20.2 to 21.8. He argued
that the high sulfur value found by Walker and Nader was
due to faulty technique in sulfur isolation from.bacteria.
Bird (1972) found that the ratio of the increment of
nitrogen:su1fur stored by sheep was 13.5:1 when sulfate
was added to sulfur deficient diet. He therefore concluded
that in order to maximize dietary nitrogen utilization in
sheep the nitrogen to sulfur ratio should not be greater
than 13.5:1. Mbir, Somers and Bray (1967) found maximum
nitrogen utilization in sheep when the nitrogen:su1fur
ratio was 10:1. Thus, according to Hume and Bird (1970)
there is a potential 69 g of microbial protein produced
from every gram of sulfur entering the rumen.

Several workers (Hungate, 1966; Hume, 1970b; Orskov,
Fraser and McDonald 1972; Walker and Nader, 1970; Bucholtz
and Bergen 1973) used a variety of techniques to quantitate
the amount of microbial protein that is synthesized in the
rumen. Hungate (1966) estimated that a maximum of 10 g

microbial protein can be synthesized from.100 g of digestible

8
organic matter (DOM). 0n the same basis, walker and Nader
(1970) calculated a yield of 14.4 g of microbial crude
protein while Hume (1970) with passage studies obtained a
mean yield of 13 g and suggested that maximum yield may
exceed 20 g. OrskOV'gE El. (1972) analyzed diaminopimelic
acid in abomasal digesta and estimated a rumen bacterial
protein synthesis of 15.6 g, and Bucholtz and Bergen (1973)
measured rumen microbial phospholipid synthesis and
calculated synthesis of true rumen microbial protein to be

16 g.

2. Protein and Amino Acid Reachinggthe Small Intestine

The capacity of the rumen and the metabolic activi-
ties of its microorganisms affect the flow and composition
of digesta passing to the small intestine of the ruminant
(Kay, 1969). If the bulk of the ingested nitrogen is from
a non protein or highly soluble protein source the protein
reaching the abomasum will be mainly of microbial origin
(Orskov, Fraser and McDonald, 1971). If, on the other hand,
the ingested protein is resistant to rumen degradation,
only a small percentage of the total protein reaching the
abomasum.will be of microbial origin (McDonald, 1954;
Orskov £5 21., 1971). Under normal dietary regimens, a high
proportion of the dietary nitrogen is converted to microbial
protein (McDonald, 1954). After feeding a diet of wheaten
hay to sheep, Pilgrim and Weller (1953) found that over a

9
16 hour post prandial period, 50% of the rumen nitrogen
was of microbial origin. Amos 2; 31. (1972) fed fish meal
and corn gluten meal to wethers and found that 55 to 60%
of the nitrogen in the rumen was associated with the
particulate portion and 40 to 55% in the liquid phase.
Since fish meal and corn gluten meal are fairly resistant
to rumen degradation, a large fraction of the particulate
portion was thought to be of dietary origin. Amos gt 5;.
(1971) found that the abomasal fluid of steers fed corn
gluten meal contained significantly higher levels of total
nitrogen, protein, nonessential amino acids and essential
amino acids than that of steers fed distillers dried solubles
or soybean meal. Soybean meal and distillers dried
solubles are more extensively degraded in the rumen,
resulting in a substantial loss of rumen ammonia; whereas,
most of the protein from the corn gluten meal reached the
abomasum.

The amino acid composition of rumen bacteria or
protozoa is not influenced by crude protein intake (Bergen
£5 31. 1968; Purser and Buechler, 1966; Fenderson and
Bergen, 1972; Burris, Bradley and Boling, 1974). ‘Bergen
£3 21. (1968) reported that there was no significant differ-
ence in the true digestibility, biological value or net
protein utilization of microbial proteins isolated from.the
rumens of sheep fed different rations.

Hogan and Weston (1967) investigated the passage of

nitrogen to the abomasum of sheep and found that the

10
quantity of non ammonia nitrogen passing to the abomasum
was similar whether the diet contained 7.8 or 19.8% crude
protein. This can be interpreted to mean that due to in-
sufficient energy a high percentage of the ammonia from
the high protein diet was not utilized by rumen micro-
organisms. This conclusion was substantiated by Orskov
gg‘gl. (1971) who observed that in the presence of adequate
dietary energy the amount of non.ammonia nitrogen reaching
the small intestine increased with increased dietary crude
protein intake up to 19.9%.

Burris gg‘al. (1974) used an 33,33552 pepsin-
pancreatin digestion procedure and found that the release
of threonine, valine, methionine, phenylalanine and lysine
from isolated microbial preparation varied significantly
with the source of ingested protein. These workers also
found that lysine had a greater rate of release from
bacteria obtained from the rumen of steers fed soybean meal
than steers fed fish meal. They concluded that the avail-
ability of certain amino acids to ruminants can vary with
the source of dietary crude protein. However, since there
are numerous interrelated factors affecting post ruminal
proteolysis such conclusions may not be valid under in 2323
conditions.

3 According to Bergen (1969) the release pattern of
amino acids may affect the digestibility of a particular

amino acid and influence absorption rates from the ruminant's

11
alimentary tract. In his review on amino acid nutrition
for the ruminant, Hatfield (1970) suggested that the pre-
ponderance of data in the literature indicates that the
qualitative pattern of essential amino acids at the
absorption sites is one of the rate limiting factors in
the growth of ruminants. Johns and Bergen (1973) and Hume,
Jacobson and Mitchell (1972) found significant competition
for transport binding sites among essential amino acids of
the same transport class. However, it is unlikely that
under normal in give conditions competitive inhibition
among amino acids would be a limiting factor in amino acid
absorption and utilization since Johns and Bergen (1973)
further suggested an unlimited absorptive capacity through-
out the length of the digestive tract.

According to Hume (1970) and Chalupa (1974) the
maintenance of optimum.rumen function along with the
maximization of rumen bypass of good quality dietary protein
or amino acids would be a feasible approach to meet the
amino acid requirements of ruminants. Thus, improved protein
utilization is possible by protecting dietary protein from
rumen microbial proteolysis. Feeding naturally resistant
protein or increasing the resistance of soluble protein and
amino acid by chemical treatments are suggested techniques.
Mowat and Deelstra (1972) found that addition of 0.45%
encapsulated methionine to the diet of lambs increased

weight gains by 11% and feed efficiency by 9% when the basal

12
diet was supplemented with corn-blood and feather meal.
Weight gains and feed efficiency were increased by 12%
and 10%, respectively, with urea in the basal diet. At
0.6%, encapsulated methionine the above diets severely
depressed gains and feed efficiency. Faichney (1971)
found that lambs fed the formaldehyde treated casein had a
faster growth rate and improved feed efficiency than those
fed the untreated casein. Macrae 35 31. (1972) observed
a significant increase in daily amounts of non ammonia
nitrogen and individual amino acids reaching the intestine
of sheep fed formaldehyde treated casein. Ferguson, Hemsley
and Reis (1967) fed formaldehyde treated casein as a
supplement to roughage-fed sheep and obtained substantial
increases in wool growth. These workers claimed that
formaldehyde forms acid reversible cross linkages with
amino and amide groups. Thus, the protein is insoluble at
rumen pH and soluble at abomasal pH. Faichney and weston
(1971) observed significant decreases in organic matter
digestion, VFA and ammonia production in the rumen and
significant increases in non ammonia nitrogen and starch
reaching the abomasum of sheep fed formaldehyde treated
casein.

Corn gluten meal and fish meal are protein sources
which are resistant to microbial degradation. Thus, when
these proteins bypass rumen degradation their amino acids
are reflected in plasma and tissue pools (Bergen, Henneman

and Magee, 1973). Amos st 31. (1971) observed a significantly

13
higher total nitrogen, isoleucine, leucine, methionine
and phenylalanine in abomasal fluid of steers fed corn

gluten meal than steers fed soybean meal or distillers

dried solubles.

B. Plasma Amino Acids and What They Represent

The investigation of plasma amino acids over the
years has yielded invaluable information on their role in
mammalian protein metabolism. According to Munro (1970)
the plasma free amino acid pool comprises a very small
proportion of the total free amino acid pools of the body.
Plasma free amino acids are rapidly renewed since the daily
amino acid intake is large in comparison to the plasma pool.
Changes in plasma free amino acids therefore may not reflect
changes in body free amino acids as a whole (Munro , 1970).
However, there is an interchange of free amino acids between
plasma and tissue pools. Munro (1970) suggested that the
body free amino acid pools have 3 metabolic outlets:
a) protein synthesis, b) synthesis of low molecular weight
compounds, and c) degradation through amino acid catabolism.

It has been shown by several workers (Munro .SE.El-:
1962; Crofford, Felts and Lacy, 1964; Potter, Purser and
Cline, 1968; Bergen and Purser, 1968; Halfpenny, Rook and
Smith, 1969; Knipfel 33 31., 1969) that the administration
of energy to a fasted animal causes a significant depression
of plasma free essential amino acids. Such depressions in

plasma free essential amino acids have been interpreted to

14
be a direct result of tissue uptake of these amino acids.
Such interpretation is substantiated by the data of
Halfpenny £2 51. (1969) which showed that an increase in
energy to lactating cows caused a significant decrease in
plasma essential amino acids and a substantial increase in
milk protein. Call £5 a1. (1972) showed that the injection
of insulin into sheep caused a severe depression in plasma
levels of glucose, acetate and both essential and non-
essential amino acids. Hence, it appears that infusion of
energy does not directly depress plasma free amino acids,
but induces the release of insulin which directly stimulates
the uptake of amino acids (Munro, 1964; Potter st 21., 1968;
Preston gt 31., 1973). Such a conclusion can be sub-
stantiated by the fact that fat as an energy source does not
stimulate amino acid uptake, mainly because it does not in-
fluence insulin secretion (Munro, 1964).

Plasma amino acids will reflect protein intake only
in the sense that an increase in dietary protein will be
accompanied by an increase in plasma amino acid level during
the absorptive phase. An exception is when one amino acid
is severely limiting the utilization of the other amino
acids present in large quantities (Zimmerman and Scott,
1965; Dean and Scott, 1965). Purser (1970) in his review
on amino acid requirements of ruminants, pointed out that
plasma concentrations of a specific amino acid do not always

reflect the nutritional status of an animal unless that

15

amino acid is substantially limiting. McLaughlan (1963)
reported that the plasma amino acid concentrations of rats
increased after a meal of good quality protein, but the
duration of the increase was dependent on the amount and
composition of the fed protein. According to Cecyre,
Jones and Gandreau (1973) plasma amino acid level decrease
gradually with time after feeding. Hogan, Weston and
Lindsay (1968) observed that the total plasma essential
amino acids of sheep increased with each successive level
of casein infused into the abomasum, but as a proportion
of the total essential amino acids only valine, leucine
and phenylalanine increased. The sulfur amino acids
remained unchanged and histidine and arginine decreased.
In an effort to explain plasma amino acid response to
dietary protein intake in ruminants, Bergen gt'al. (1973)
pointed out that the changes observed in plasma amino acid
parameters in response to various rations in ruminants are
related primarily to the quantity of protein reaching the
small intestine and not to dietary protein per se. With
the exception of methionine and histidine, plasma amino
acids, for the first 4 days of infusion, did not reflect
the amino acid pattern of protein sources infused into the
duodenum of sheep (Potter, Purser and Bergen, 1972).

The metabolic state of the animal will influence
the plasma amino acid level independently of dietary protein

(Munro, 1964). During starvation or fasting, plasma level

16
of the essential amino acids increased significantly
(Leibholz and Cook, 1967; Zimmerman and Scott, 1967; Hogan
‘ggigl., 1968; Leibholz, 1970; Bloxam, 1971). Leibholz and
Cook (1967) attributed this large increase in plasma free
amino acids to tissue breakdown and amino acid release
during starvation. According to Bloxam (1971) there is a
general fIOW’Of most amino acids from extra hepatic tissues
to the liver, whereas lysine and the branched chain amino
acids flow from the liver to extra hepatic tissues during
starvation. This out flow of branched chain amino acids
from the liver to extra hepatic tissue may be due in part
to the inability of the liver to deaminate branched chain
amino acids (Leibholz, 1970; Neale, 1972). During
starvation Leibholz (1970) observed an increase in the
essential to nonessential amino acid ratio from 0.35 to
0.56. This may be explained by the degradation of tissue
protein during starvation and the ultimate deamination of
the circulating amino acids, other than the branched chain
amino acids, by the liver. Thus, the increased level of
circulating branched chain amino acids were the major con-
tributor to the increased plasma amino acid ratio. However,
this may only be partially true during extended fast since
Brown 25 31. (1961) measured plasma amino acid levels of
cattle during an 88 hour fast and found approximately a
two-fold increase in glycine, a significant increase in the

aromatic and branched chain amino acids, lysine and

17

threonine. There was a decrease in serine and alanine and
no significant changes in glutamic acid, cystine, histidine
and arginine. Zimmerman and Scott (1967) observed that
plasma lysine, methionine, leucine, isoleucine, tyrosine,
phenylalanine and histidine increased with each extension
of fasting in chickens, whereas plasma cystine was decreased
and proline, glutamic acid and arginine were unaffected.

Interrelationships between the different plasma
amino acids in several species have been well documented.
Several workers (Snyderman and Holt, 1970; Clark, Umezawa
and Swendseid, 1973; Keiichiro, Takeuchi and Sakurae, 1973;
McLaughlan, Karsrud and Knipfel, 1973) observed a reciprocal
relationship between the different branched chain amino
acids in plasma. Snyderman and Holt (1970) reported a
significant decrease in plasma valine, isoleucine tyrosine
and threonine after loading the animal with leucine. Clark
25 31; (1973) observed very high plasma levels of isoleucine
and valine after feeding a diet devoid of leucine to rats.
Keiichiro £3 51. (1973) and McLaughlan.g£Igl. (1973)
observed that an increase in plasma lysine above requirement
caused a significant decrease in plasma threonine.
Zimmerman and Scott (1965) reported that severe deficiencies
of either lysine or arginine and large excesses of lysine
or valine caused a substantial increase in plasma threonine;
also, plasma lysine accumulated when dietary arginine was

deficient. Zimmerman and Scott (1965) concluded that a

18

decrease in plasma threonine should not be interpreted that
threonine is limiting.

Reis, Tunks and Sharry (1973) found that infusion
of 4.9 to 10 g of methionine per day into the abomasum.of
sheep significantly increased plasma methionine, cystine,
taurine and cystathionine and significantly decreased plasma
levels of the branched chain amino acids. However, the
infusion of equimolar cystine significantly increased
plasma cystine, taurine and cystathionine, significantly
decreased plasma branched chain amino acid levels but had no
effect on plasma methionine level. ‘The results of these
experiments agree with the 1973 NRC pig requirement report
that methionine can fulfill the total sulfur amino acid
need whereas cystine can only fulfill a part of such need.
Byington and Howe (1972) fed a 70:30 ratio of methionine to
cystine to rats and found high levels of plasma methionine,
alanine, isoleucine, valine and a-aminobutyric acid and low
levels of threonine and taurine. However, reversing the
ratio resulted in high plasma levels of threonine and
taurine. Shannon, Howe and Clark (1972) observed that rats
fed 4.8 or 3.6 millimoles of sulfur amino acids consumed
comparable amounts of food and had similar weight gains
regardless of whether 100% methionine was fed or 25 or 50%
of the methionine was replaced by cystine. However,
replacement of 75% of the methionine by cystine significantly
depressed feed intake and decreased weight gains to 25% of

normal.

19
The effects of insulin, protein intake and
metabolic state of the animal on plasma amino acids along
with the constant turning over and interrelationships of
plasma amino acids, imply that an authentic overall inter-
pretation of plasma amino acid is difficult unless these

interrelated factors are given due consideration.

C. The Amino Acid Requirement of Growing Animals

The amino acid requirements of man, pigs, chickens,
sheep and rats have been directly determined with a variety
of techniques. Such techniques include plasma amino acid
response curves (Zimmerman and Scott, 1965; Young 35 31.,
1971; Tontisirin £3 31., 1972; Young 25 21., 1972;
McLaughlan 95 al., 1973), nitrogen retention (Nimrick gtflgl.,
1970a; Nimrick gt al., 1970b; Boila and Devlin, 1972),
weight gain (Stockland £3 31., 1970; Boomgaardt and Baker,
1973a; Boomgaardt and Baker, 1973b), oxidation rates of
amino acids (Brooks, Owens and Garrigus, 1972) and plasma
urea concentration (Puchall gt 31., 1962; Christensen £5 31.,
1972; Mercer and Miller, 1973; Brown and Cline, 1974).
Regardless of the method used the fundamental criterion
for any amino acid requirement determination is that the
amino acid under investigation be limiting. The limiting
amino acid may be defined as that amino acid which, by
quantity, is least able to satisfy the animal's requirement.

The concept of a limiting amino acid implies that the

20
other amino acids (which are in excess relative to that
which is limiting) will be utilized in quantity as dictated
by the most limiting amino acid (Velu, Baker and Scott,
1971).

The approach of feeding incremental levels of an
essential amino acid and quantitatively measuring its
appearance in plasma has been proven to be quite satisfactory
in non ruminants (Zimmerman and Scott, 1965; Velu 35.31.,
1971; Young 25 31., 1971; Young g£_gl., 1972). ‘Working
with chickens, Zimmerman and Scott (1965) found that the
first limiting amino acid in the diet remained at a low and
constant level in plasma irrespective of the severity of
the deficiency. The break point at which the amino acid
began to accumulate was regarded as the requirement level.
These workers concluded that the plasma technique can be
used to determine the amino acid requirement. However,
such technique cannot be utilized in the ruminants because
of amino acid degradation by ruminal microorganisms.

Thus, the determination of the essential amino acid
requirement of the ruminant animal can be achieved only if
the amino acid under investigation is infused directly into
the abomasum of animals where the quantity of that amino
acid passing from the rumen is limiting (Wakeling, Annison
and Lewis, 1970), The response curve of plasma amino acids
versus the quantity of amino acid passing the duodenum

should show a rapid increase in plasma level beyond the

21
requirement level. Thus, the point of inflection on the
plasma amino acid response curve denotes the requirement
level (Wakeling 25 $1., 1970). McLaughlan and Illman
(1967) defined the amino acid requirement as that dietary
level at which the corresponding plasma amino acid is equal
to normal fasting level. Tontisirin gt 31. (1974)
explained that the amino acid requirement should be con-
sidered as the intake of that amino acid which is capable
of just maintaining higher blood plasma amino acid plateau
level. Working with young men, Young 35 31. (1971) regarded
the level of dietary tryptophan above which a linear
increase in plasma tryptophan occurred as the tryptophan
requirement. Tontisirin ggwgl. (1972) found that both pre
and post prandial plasma free tryptOphan levels in children
became constant at a tryptophan intake of 4 mg per kg body
weight and below, but they increased linearly between 4 and
7 mg per kg body weight. These workers concluded that the
tryptophan requirement of children 7 to 13 years old is
4 mg per kg body weight per day. Young g£_gl. (1972)
observed that free valine in plasma of young men remained
constant below valine intakes of 14-16 mg per kg body weight
per day and increased with intakes above this level. The
lysine requirement of rats was determined by MbLaughlan st 31.
(1973) who found that plasma lysine remained low until
supplemental lysine exceeded .12% of the diet then increased

rapidly beyond this point of intake. These workers regarded

22

the point of lysine intake at which the plasma lysine curve
deflected upward as the lysine requirement. Tontisirin
st 21. (1973) observed that after fasting or 3 1/2 hour post
prandial, plasma tryptophan levels of elderly people
decreased as daily tryptOphan intake was lowered to 2 mg
per kg body weight, below that level plasma tryptophan
remained constant. Thus they concluded that the tryptophan
requirement of elderly people (65 years and over) was 2 mg
per kg body weight per day. After compiling their data on
the tryptophan requirement of children, young men and
elderly people (4, 3, 2 mg/kg body weight/day) these workers
concluded that the minimum.tryptophan requirement per unit
of body weight in humans decreases with increasing age.

Several workers (Nimrick gt $1., 1970a; Nimrick
35 31., 1970b; Nimrick and Kaminiski, 1970; Boila and Devlin,
1972; Schelling, Chandler and Scott, 1973) have used
nitrogen retention as an indicator of amino acid require-
ments. The level of amino acid intake or intake plus
infusion which coincided with the point of inflection on
the nitrogen retention curve was regarded as the requirement
of that amino acid. Nitrogen retention was maximized in
lambs at 0.40, 0.10, 0.16 and 0.10% of the diet for glutamic
acid, methionine, lysine-HCl and threonine, respectively
(Nimrick 22.2l-: 1970a). These workers further found a
relationship between nitrogen retention, plasma free

methionine and methionine supplementation. Plasma methionine

23
remained low and constant as nitrogen retention increased with
increased methionine supplementation and then increased rapid-
ly after nitrogen retention was maximized. Schelling gt al.,
(1973) carried out a series of abomasal methionine infusions
in sheep and found that 2 to 3 g of methionine per sheep per
day were adequate for optimal nitrogen retention. Nimrick and
Kaminiski (1970) infused combinations of lysine-HC1, methionine,
threonine and urea into the abomasum.of sheep at 0.25, 0.18,
0.11 and 0.12% of feed intake and found the mean daily nitrogen
retentions to be 2.76, 0.52, 0.89, 0.94 and 1.76 g for lysine-
HCl plus methionine, lysine-HCl plus threonine, methionine plus
threonine, lysine-HC1 and methionine respectively. Scrimshaw
£2 21. (1973) reported that lysine supplementation of 2.25% of
the total protein intake significantly improved nitrogen balance
in young men.

Maximum weight gain has been used as an indicator in amino
acid requirement in growing animals (Stockland £5 31., 1970;
Boomgaardt and Baker, 1973a; Boomgaardt and Baker, 1973b).

The level of amino acid intake or infusion which corresponds
to the point of inflection on the weight gain curve is the
requirement of that amino acid. Thus Stockland st 31. (1970)
found the lysine requirement for maximum weight gain in

rats to be 0.6% of a diet containing 10% dietary crude
protein. Boomgaardt and Baker (1973a) reported that the
lysine requirement for maximum weight gain in chickens

fed 14, 18.5 and 23% dietary protein was

24

4.73, 4.72 and 4.62% of the protein or 0.66, 0.88 and
1.05% of the diet, respectively. Boomgaardt and Baker
(1973b) found the lysine requirement for maximum growth
rate in chickens to be a constant 4.62% of the dietary
protein with increasing age.

The use of blood urea concentration as an indicator
of amino acid requirement in growing animals is promising
although not extensively used. The rationale underlying
this technique is that plasma urea level will decrease to a
minimum when maximum utilization of the amino acid in
question is achieved. Working with pigs, Christensen st 31.,
(1972) observed that serum.urea concentration decreased to
a.minimum as dietary methionine supplementation increased
from 0.25 to 0.45% of the diet. These workers used plasma
amino acid and weight gain techniques to test their results
and obtained identical values for the methionine require-
ment of .46 and .45% of the diet respectively. Puchall
st 31. (1962) found that plasma urea concentration was
proportional to feed per gain ratio and inversely propor-
tional to gain when pigs were fed proteins of different
quality. Mercer and Miller (1973) investigated the valid-
ity of using plasma urea concentration as an indicator of
amino acid requirement and found that the methionine require-
ment of lambs growing at a mean rate of 154 g per day was

35

2.63g per day. Using urinary S as an indicator to con-

firm their results these workers obtained a value of 2.35g

25

of methionine per day.

The oxidation rate of an amino acid as an indicator
of its requirement was investigated in rats by Brooks,
Owens and Garrigus (1972). These workers used radioactive
CO2 as an index for the amount of lysine oxidized and
found that the oxidation rate of lysine did not increase
markedly until the dietary lysine intake was increased above
that level at which the average daily gain and gain per feed
ratio were maximal. They concluded that the oxidation of
amino acid technique is a very good method for determining

the dietary amino acid requirement of a growing rat.

MATERIALS AND METHODS

A. Experiment One

1. General Design of Experiment

 

Seven Holstein steers with an average body weight of
274 Kg were fitted with soft plastic abomasal cannulae.
These steers were fed a 9.5% crude protein ration (Table 1)
at 12 hour intervals at 3% of their body weight. Steers
were housed individually in 91 cm x 244 cm metal metabolism
collection stalls and given free access to water.

Amino acids (dissolved in 500 ml of water and pH
adjusted to 2.5) were infused into the abomasum with a
Harvard peristaltic pump at the rate of 0.42 ml per minute.
Methionine, lysine and threonine were infused at incremental
levels of 3.5, 7, 11 or 15 g per day and tryptophan at 1.7,
3.5, 7.0 or 11 g per day. Each level of amino acid was
infused for seven days during which time a nitrogen balance
study was conducted (Figure 1). Nitrogen balance was
expressed as total nitrogen intake (dietary and infused)
minus the total nitrogen excreted (urine and feces).

Abomasal samples were collected prior to each amino
acid infusion period to determine the quantity of each amino
acid (per Kg of feed consumed per day) reaching the abomasum.

Chromic oxide and lignin were used as insoluble markers to

26

27

TABLE 1

Ration Used in Experiment One

 

 

Ingredients %
Oats, grain (4) 4-03-309 10.00
Wheat, bran, (4) 4-05-191 5.00
Corn, dent yellow grain, gr 2 US

mm wt 54, (4) 4-02-931 51.55
Soybean, seeds, sov-ext. grnd, mx 7%

fiber, (5) 5-04-604 3.75
Corn, cobs, grnd, (1) 1-02-782 20.00

Sugarcane, molasses, mm 48% invert
sugar mn 79.5 degrees br/x, (4)
4-04-696 5-00

Urea (45% N) 0.25
Limestone, grnd, mn 33% calcium, (6)

6-02-632a 1.45
Trace mineral salt be 2.00
Chromic oxide-flour 1.00
Crude protein 9.50
Vitamin Ade 2,000,000
Vitamin Def 250,000
Vitamin E98 55,000

 

aCalciumCarbonate Co., Quincy Illinois.

bContained in %: Zn, mm 0.35; Mn, mm 0.2; Fe, mn 0.2; Mg,

3% 0.15; Cu, mn 0.03; Co, mm 0.05; 12, mm 0.007; NaCl, mx
‘ .5.

28

Table 1 (continued . . .)

cInternational Mineral Co.

dVitamin A Palmitate (Pfizer Co., Terre Haute, Indiana).
eInternational Units

f

Ergocaldiferol (Fleichman Irradiated Dried Yeast).

gAlpha tocopherol acetate (Eastman Kodak, Rochester, N.Y.).

29

FIGURE 1

Flow Chart of Experiment One

Steers with abomasal fistulae

14 day feed adaptation period

Experimental periods

Diys 1 to 3 - Abomasal sample collection

D s 4 to 6 - Rest days

Da 3 7 to 14 - Amino acid infusion - Level 1 and Nitrogen
alance

Day 15 - Rest day

Days 16 to 23 - Amino acid infusion - Level 2 and Nitrogen

Jbalance
D y 24 - Rest day

D s 25 to 32 - Amino acid infusion - Level 3 and Nitrogen
alance

Da 33 - Rest day

Days 34 to 41 - Amino acid infusion - Level 4 and Nitrogen
alance

:Jdays - Rest period

omasal sample collection and infusion of next amino acid

30

quantitate the total nitrogen passage to the abomasum” To
this end, chromic oxide was mixed with wheat flour (ratio
of 1:4) into a paste with water and baked in an oven at
100°C. The baked mixture was ground in a Wiley mill and
added to the ration (1%) Orskov £3 a1. (1971).

Amino acids were obtained from General Biochemicals
(Laboratory Park, Chagrin Falls, Ohio). They were of the
L-isomer and NRC grade containing less than 0.3% moisture.

Lysine was lysine hydrochloride.

2. Cannula Design

Cannulae were made from clear plastisol (obtained
from.Norton's Plastic and Synthetics Division, Akron, Ohio)
in a brass mould obtained from Dr. L.D. Satter of the
university of Wisconsin. The plastisol was placed under
strong vacuum for one hour to remove air bubbles, after
which it was poured into the preheated mould and allowed to
be baked in an oven at 190°C for approximately 20 minutes.
After cooking the mold was cooled by immersing it in cold
‘water for a few minutes. Properly prepared cannulae were

pliable,transparent, amber in color and free of air bubbles.

3. Sample Collection and Preparation
a. Abomasal samples
Abomasal samples were collected twice daily at 6 hour
intervals over a 3 day period. The abomasal contents were

agitated with air from.a small air pump and approximately

31
200 ml of the digesta were collected in a beaker. A pre—
liminary study revealed that a representative sample could
be obtained by agitating the abomasal contents during
sampling. Samples from each steer were pooled and freeze-
dried. Dry matter, nitrogen, acid lignin, chromium and
amino acids were then determined.
b. Blood samples

Blood samples were collected immediately before the
morning feeding on days 4 and 8 of each infusion period.
A preliminary study of sampling on days 1, 2, 3, 4, 5, 6, 7
and 8 revealed that days 4 and 8 were the best. At each
sampling time 10 ml of blood was drawn from the right
jugular vein of each steer into a heparinized syringe and
placed into a bucket of ice until all steers were sampled.
Plasma was prepared for amino acid analysis according to
Bergen g£_§l. (1973) and stored at -70°C.

c. Urine samples

Total urine from each steer was collected in a plastic
carboy containing 200 ml of 18 N sulfuric acid. The sulfuric
acid was added to prevent liberation of ammonia or other
nitrogen compounds from.the urine. The carboy was emptied
once per day and the urine volume measured, diluted to 10
liters with water and a 1 liter aliquot stored in the cooler.
The remaining dilute urine was discarded. At the end of
each 7 day collection period samples from each steer were

pooled into a composite sample and properly mixed. A 10%

32
subsample was then secured for nitrogen determination.
d. Fecal samples

Total feces were allowed to pass through a wide
spaced steel grid in the floor immediately behind each
steer and were collected in large plastic containers in the
pit below the collection stalls. Feces were removed once
daily and the total output for each steer weighed, properly
‘mixed and a representative sample (10% of the total weight)
secured by taking several small subsamples from different
points in the mixture. At the end of each 7 day collection
period, samples from each steer were properly mixed and '
into a composite sample and a 10% subsample retained for

nitrogen analysis.

4. Nitrogen Determinations

Feed, abomasal contents, fecal and urinary nitrogens
were determined with a standard semi-micro Kjeldahl method
(copper catalyst) using Aminco system and Sargent Spectro-

Electro Titrator.

5. Plasma Amino Acid Determinations
a. Plasma lysine, methionine and threonine
Plasma lysine, methionine and threonine were deter-
mined from.the protein free filtrate byion exchange
chromatography (Technicon Amino Acid Analyser) as detailed
by Bergen and Potter (1971) and Bergen 23 El- (1973).
b. Plasma tryptophan
Plasma tryptophan was assayed using the simplified

33
spectrofluorometric micromethod of wapnir and Stevenson
(1969) as modified by Fenderson and Bergen (1972). The
detailed procedure was outlined by Fenderson (1972).
c. Abomasal lysine, methionine and threonine

Abomasal lysine, methionine and threonine were
determined from acid hydrolysates of the freeze-dried
abomasal samples on the amino acid autoanalyzer. The crude
protein content of the sample was first determdned by the
micro Kjeldahl procedure. A sample containing approximately
10 mg of protein was weighed into a 35 m1 screw capped test
tube and 1 ml of 1 mM norleucine, l‘ml 12 N HCl and 8 ml of
6N HCl were added. The tube was flushed with nitrogen (to
exclude air) capped and autoclaved for 16 hours at 121°C.
The contents of the tube were filtered through Whatman No.2
filter into an evaporating flask. The filtrate was
evaporated to dryness under vacuum (at 60°C) washed twice
with approximately 10 ml of deionized distilled water,
evaporated to dryness and finally resuspended in 4'ml of
pH 2 (.3 lithium..05 citrate) buffer. The buffered filtrate
was then analyzed on the amino acid analyzer as previously
outlined in section 5a.

d. Abomasal tryptophan

Tryptophan determination of the freeze-dried abomasal

samples was done by a modified Ba(OH)2 protein hydrolysis

procedure as outlined by Fenderson (1972).

34
6. Chromium Analysis

 

Chromium content of feed and abomasal samples was
determined by nitric-perchloric acid digestion followed by
atomic emission spectrophotometry using the I.L. 453 Atomic
Absorption/Emission Spectrophotometer.

Between 200 and 500 mg of the finely ground sample
were digested in 60 ml of concentrated nitric acid in a
250 ml Phillips beaker on a hot plate until the volume was
l-2 ml. The flask was then cooled to room temperature and
the digest was oxidized by adding 7‘ml of 72% perchloric
acid and heating it on the hot plate until 1-2 ml of the
solution was left in the flask. The flask was again cooled
and the content diluted to 100 ml with deionized distilled
water. The oxidized diluted samples were then read on the
atomic emission spectrophotometer at a wave length of
425.4 nm, scale of 2.5, slit width of 80, photomultiplier
voltage at 700 volts, acetylene flame, hollow cathode
chromium lamp and nitricoxide solid burner. A chromium
standard curve was obtained by subjecting ammonium.chromate
solutions of 1, 2, 5, 10 and 15 ppm.chromium to the same

treatments as the samples.

7. Lignin Determination
Acid lignin content of feed and abomasal freeze-
dried samples was determined by the standard Van Soest

procedure (Van Soest, 1963; Van Soest and Wine, 1965).

35

B. Experiment Two
1. General Design of Experiment

Four growing, rumen fistulated, Holstein steers
with an average body weight of 328 Kg were randomly
allotted to a 4 x 4 Latin Square experiment (Table 2). In
this experiment four rations containing different levels
of crude protein (Table 3) were fed to the steers in four
14 day periods. Steers were housed indoors (temperature
between 55 and 70°F) in 91 cm x 244 cm metal stalls, given
free access to water and fed twice daily at ad libitumm
Uhconsumed feed was weighed back to quantitate daily feed
intake.

In each experimental period, blood samples were
collected immediately before the morning feeding (To) on
days 1, 2, 3, 5, 7, 10 and 14 and rumen samples were taken
immediately before the morning feeding and 3 hours after
feeding (T3) on days 1, 2, 3, 5, 7, 10 and 14. Each treat-
ment period was preceded by an 11 day adjustment period
during which the steers were fed ration 1 (low protein

control diet).

2. Sample Collection and Preparation
a. Blood samples
Blood samples were collected from.the right jugular
vein of each steer and prepared for amino acid analysis as

previously outlined in section 3b of experiment 1. Three m1

Experimental Design for Experiment Two

36

TABLE 2

 

Steer NUmber

 

 

 

Period 1 2 3 4
l A B C D
2 B C D A
3 D A B C
4 C D A B
Treatments: A = RATION l
B = RATION 2
C = RATION 3
D

RATION 4

 

Mn MN VH m <rHs

37

 

oom.- oom.N~ oom.- oom.- sum caawua>
ooo.n~H ooo.m~a ooo.n~a ooo.n~H mun :aauua>
ooo.ooo.H ooo.ooo.a ooo.ooo.H ooo.ooo.a mo<.cflawua>
o.o¢ m.~m ~.o~ N.oa cwououm ousuu
o.~ o.~ o.~ o.~ co uaom Houoafia.oooua
o.~ o.~ o.~ o.~ Nm~-~o-H Adv .ucum .mnoo .auou
o; o4 oA o4 Go .3333 Nam as .ufimpwmmmmmmmfi
o.oa u u u oANomv Gaououm somehow woumHOmH
o.om o.o~ - - «Asoav compose smashes woumaoaH
m.- m.~m 0.0m o.m nonwm Nu x8 .ocum .vuxou>aom .wwmeOHmemwom
o.o~ o.om o.om o.mn cm u3_aa m: N um .aawuw .3OHHWMmummmw MNWOU
o.n o.m o.n o.n soo-so-n Anv .mmmmmaoa acmoummsm
m.N m.N 0.0H o.oH monumqu adv cwmuw .mumo

N N N N

d m N H mucowvouwcH

 

039 uaoaauomxm cw womb mcowumm

m mdm<H

38

.Axuo» 3oz .Houmonoom .xmvoM amaummmv ououooo Hononmooou «sedan

.Aummow vowun woumfivoHHH cuanuumamv Hoummwoaooowumw

muHcD Hmcoaumahouch

.AmcwauaH .oudmm ouuma ..ou Hmuwmmv mumuwaamm < cflamuw>m

.mwonHHHH .oHHH>%uHonHA ..ou Hmuocfiz HmcoaumcuouaHv

. . . . . . . .m.mm as .Homz “Noo.o as .NH
Mno.0 as oo .mo.o as so .mH.o as m2 .~.o as we mNa as .ez “mm.o as .:N "N as umgamuaooo

.mHoGHHHH .hocaao ..ou ouoconumo_aawoamon

.wcmHvaH .Hauwomn .mhom kuuauom

 

A. . . omnaauaoov m «Home

39
of plasma from each sample were frozen (-70°C) for plasma
urea nitrogen determinations.
b. Rumen samples

Rumen samples were collected at specified times by
inserting the full length of the arm through the cannula
into the rumen and mixing the contents thoroughly. A 200 cc
sample of rumen contents was then removed. The rumen
samples were then immediately frozen and stored at -70°C
for further processing at a later date. At the time of
processing, 25 g of thawed, well mixed sample (Figure 2)
were strained through 2 layers of cheese cloth to obtain
rumen liquor. Four ml of the rumen liquor were used for
the ammonia determination and another 5 ml portion was
treated with 1 ml of 257. metaphosphoric acid to precipitate
the soluble proteins. The metaphosphate-treated rumen
liquor was then centrifuged at 12,100 x g for 15 minutes
and the supernatant was saved for volatile fatty acid (VFA)
determination .

Twenty five g of the original rumen sample (Figure
2) were fractionated into total ruminal nitrogen, soluble
nitrogen, insoluble nitrogen, nonprotein nitrogen, soluble
tungstic acid precipitable nitrogen and peptide nitrogen
as follows. One hundred ml of distilled water were added
to the 25 g of original rumen contents and the mixture was
homogenized for 2 minutes in an Omnimixer placed in an ice

bucket. Two 2 ml aliquots of the homogenate were used for

40

the total rumen content nitrogen determination. Forty five
ml of the remaining homogenate were centrifuged at
18,000 x g for 15 minutes and two 1 ml portions of the
supernatant were kept for soluble rumen nitrogen determina-
tion. Twenty ml of the supernatant were mixed with 5'ml of
1.07 N sulfuric acid and 5 ml of 10% sodium.tungstate (w/r)
(Winter gg,al., 1964) and left to precipitate the protein
over night in the cold room. The mixture was then centri-
fuged at 13,000 x g for 10 minutes and two 2 ml portions of
the supernatant were used for non-protein nitrogen analysis.

Insoluble nitrogen, soluble precipitable nitrogen
and peptide nitrogen were calculated as the difference be-
tween total and soluble nitrogens, soluble and non-protein
nitrogens and non-protein nitrogen and ammonia nitrogen

respectively.

3. Nitrogen Determination

 

Total, soluble and non-protein nitrogens in rumen
samples were determined as outlined in section 4 of experi-

ment 1.

4. Rumen Ammonia and Blood Urea Nitrogen Determination

Rumen ammonia and blood urea nitrogens were deter-
Inined by the microdiffusion method of Conway (1960) as out-
lined by Fenderson (1972).

41

FIGURE 2
Flow Chart of Fractionation of Rumen Contents,

Experiment Two

 

 

ElmiCmtmts\
25ng 25g+100m1water
(21ayers) cheesecloth \
l W
RimenLiquor foerimtes
I 4mltotalRumn/
AmnxniaDeternfinatim NDeterminatim
(L w
5m1+lm1257. 45mloe1trifugedat
me phoric acid 18,000xg for 1.5
I: \
Centrafuged at 12 . 000 Superhuman Discarded

ngorljmirmtes

/ \ fimmm

VFA DeterufiImfim Residue discarded soluble N determinatim

 

W
20ml+5mll.07NH2304
+5m1107.Sodiun‘mngstate.

precipitate in cold overnight

Centrifugedat 13,000ngor15mirutes

Non protein Nitrogen detern'fination Residue discarded

42

5. Volatile Fatty Acids Determination

Rumen VFA concentrations were determined by gas
liquid chromatography using a Packard G.L.C. (Model 840).
The column (183 cm long and 2 mm internal diameter, teflon
tubing) was packed with Chromosorb 101 (Anspec Co., Inc.,
Ann Arbor, Michigan), the flow rate of the carrier gas
(Nitrogen) was 40 ml/minute and the column temperature was
maintained at 185°C (Ponto and Bergen, 1974). Volatile
fatty acids in rumen fluid samples were identified quanti-
tatively by comparison with a standard solution of volatile

fatty acids.

6. Statistical Analysis

Data were statistically analyzed by the usual
analysis of variance technique on a CDC 6500 computer at
Michigan State University Computer Laboratory. Difference
between means were determined by the "Duncan's New Multiple
Range Test" on the CDC 6500 computer.

RESULTS

Experiment One
1. Plasma Amino Acid Concentration Changes As An Indicator
of Amino Acid Requirements.

In the present study, plasma amino acid changes in
response to incremental abomasally infused amino acid
levels were used as a response criterion to determine the
essential amino acid requirements of growing steers. Plasma
level of the infused amino acid was expected to remain
constant until the animal's requirement for that amino acid
was satisfied and then increase thereafter with higher
infusion levels. The infused level, at which the plasma
concentration began to deflect upward, along with the
quantity entering the small intestine was regarded as the
requirement.

The effects of incremental abomasal methionine,
lysine, threonine and tryptophan infusions on their respec-
tive plasma concentrations are presented in Table 4 and
average daily feed intake in Table 5.

Plasma methionine concentration, on both day 4 and
day 8 did not change until 7 g of methionine were infused.
Thereafter a large linear increase was observed with each

successive increment of infused methionine. A graphical

43

44

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m

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45
TABLE 5
Average Daily Feed Intake of Steers During

Infusion Periods, Experiment One

 

 

Infusion Period No. of Av . Daily Feed Intake
Steers Kg§100 Kg B.W.

Methionine 7 2.6

Lysine 7 2.6

Lys + met 7 2.6

Threonine 2 2.3

Tryp 2 2.7

 

representation of plasma methionine levels in relation to
~the level of infused methionine are presented in Figure 3.
The point of inflection on days 4 and 8 of plasma
methionine occurred when 7 g of methionine were infused.
Plasma lysine increased linearly with each succes-
sive infusion increment of lysine indicating that the
animal's requirement for lysine was satisfied by the quantity
leaving the rumen. A graphical representation of these
results is presented in Figure 4. Incremental levels of
lysine along with 11 grams of methionine were infused into
the abomasum.of 3 larger steers (311 kg) to determine if
lysine was the second limiting amino acid. Under these
conditions, plasma lysine increased immediately with the

first infusion level (3.5g of lysine) and then continued to

in

in

_ 46
increase rapidly with each successive increment. This
indicated that lysine was not limiting in the presence of
adequate methionine and that lysine passing to the small
intestine from the rumen-reticulum.met the requirement of
this amino acid.

Plasma threonine level at both sampling times
increased with the first infusion level of 3.5g of
threonine and with each successive increase in infused
threonine. These results indicated that the animal's
threonine requirement was met by the level of threonine in
the ingesta passing to the small intestine. A graphical
representation of these results is presented in Figure 5.

Plasma tryptophan at both sampling times increased
immediately upon tryptophan infusion and at each successive
increment of infused tryptophan into the abomasuml This
also indicated that the animal's tryptOphan requirement was
satisfied by the tryptOphan leaving the rumen. A graphical

representation of these results is presented in Figure 6.

2. Nitrogen Balance As An Indicator of Animp Acid Requirement
Results of nitrogen balance studies carried on two
steers during the infusion of each amino acid are presented
in Table 6 with graphical representations accompanying the
specific amino acid in Figures 3, 4, 5 and 6.
The quantity of nitrogen retained in one steer

infused with methionine increased linearly until after 7 g

47

I Methionine D4. Y1 = .03x + 2.78. (r = .55)
Y2 = .47x + (-.46). (r = -.99)
O Methionine D8' Y3 = .12x + 3.13. (r = .96)
Y4 = .36x + (-1.6l). (r 8v.99)

‘ Nitrogen balance

 

 

 

8 V 300

3 7 .4...

E

O

S 6 4- .. 250 1.3

\ V

m

w 3

'3 5‘“ Y .5

5 as
3

2 4 .4, . 200 u

«4

S 3 1 Y 80

‘5 1 u

0) «U

E 0H

m 2 ~r .. 150 ‘3

a H

m m

‘“ ‘8

3: la. E4

i i i if
3.5 7 11 15

Methionine infused (g)

FIGURE 3. The effect of abomasal methionine infusions on

plasma methionine and nitrogen balance.

48
. lysine D4. Y1 = .44x + 6.2. (r = .99)
O lysine D8.Y2 = .30x + 4.91. (r - .85)
X lysine + methionine. Y3 = .42x + 6.2. (r = .99)

‘ Nitrogen balance. Y4 *3 -3.l4x + 285. (r = -.75)

 

 

 

 

13 'T
12 -. 300
. t Y

11... 4 7.
Q A
E! 10 .L. ‘ - 250 3.9
O
O "U
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Cd
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3 s 4- «r- 200 “
2 Y i
'5} 7 1* f;
5" s
3 6 Y2 0 ~- 150 .4
a) d
{U u
3: Sub I» IS
4 J 100
: : : i
3.5 7 11 15

Lysine infused (g)

FIGURE 4. The effect of abomasal lysine infusions on plasma

lysine and nitrogen balance.

49

I Threonine D4. Y1 .80x + 5.1. (r = .9)

O Threonine D8' Y2 = .80x + 3.96. (r = .96)

‘ Nitrogen balance. Y3 = 6.18x + (-212). (r= -.99)

 

 

 

 

 

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16 -1. 250
14 ..

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1 4. 1 1
3.5 7 11 15

Threonine infused (g)

FIGURE 5. The effect of abomasal threonine infusions on

plasma threonine and nitrogen balance.

50

I Tryptophan D4. Y1 = .09x + .78. (r .98)

.97)

. Tryptophan D8' Y2 = .08x + .77. (r

‘ Nitrogen balance. Y = -6.05x + 222. (r= '-97)

4 at “F250

 

 

Plasma tryptOphan (mg/100 ml)
Total nitrogen retained (g)

 

 

 

i 4 : ‘r —
1.7 3.5 7 11

Tryptophan infused (g)

'FIGURE 6. The effect of abomasal tryptophan infusions<xl

plasma tryptophan and nitrogen balance.

51
of methionine were infused and then plateaued. However,
that of the second steer infused with methionine decreased
with each successive level of infusion. This is not
surprising, since this steer had diarrhea throughout the
infusion period. However, plasma responses to the
methionine infusion were apparently not affected.

The quantity of nitrogen retained during the lysine,
threonine and tryptophan infusion periods decreased linearly
as their respective plasma concentrations increased with
each increment of infusion. These results support those of
the plasma studies and show that the animal's requirements
for lysine, threonine and tryptophan were met by the
quantity of each amino acid reaching the small intestine

from.the rumen.

3. Nitrogen and Amino Acid Passage to the Abomasum

Acid lignin/nitrogen and chromium/nitrogen ratios
were used to quantitate the nitrogen passage to the abomasum
(Table 7). The percentages of lignin, chromium.and nitrogen
in the experimental ration were 2.61, 0.13 and 1.49,
respectively. The lignin,chromium.and nitrogen contents in
the abomasal ingesta of steers fed the experimental ration
was 3.79, 0.16 and 2.12 percent respectively. The lignin
to nitrOgen ratios in the ration and abomasal ingesta
(1.75 and 1.79 respectively) indicated that there was no

significant net ruminal nitrogen loss (2%) under these

w MNwHMHAVQ'H.

52

 

 

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nonficoonnh

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HmN OOH NHN ¢NN u mNmO N\w cowouuwc Nassau:

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omcanownuoz

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m “NON mHHNON OHHOON N soeN u mva N\w cowouuwo Hmoom

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55
particular circumstances. The chromium/nitrogen ratio in
the ration, 0.09 and in the abomasal ingesta 0.08, and
lignin/chromium.ratio in the ration 20.08 and in the
abomasal ingesta 23.69 tend to indicate that chromiumuwas
reaching the abomasum at a slightly faster rate than lignin
and nitrogen.

The quantities of nitrogen and each amino acid
reaching the abomasum.are presented in Table 8. The average
quantities of nitrogen, lysine, cystine, methionine, total
sulfur amino acids, threonine and tryptophan reaching the
abomasum.per kg of feed consumed were determined by the use
of nitrogen to marker ratios. The quantity (g) of each
amino acid per kg of feed consumed (lysine 4.5, cystine 1.5,
methionine 1.6, total sulfur amino acid 3.1, threonine 3.7
and tryptophan 0.6) was obtained by multiplying the quantity
of each amino acid per g of abomasal nitrogen by g of nitro-
gen passing to the abomasum.for each Kg of feed. The
average daily quantity of.each amino acid reaching the
abomasum was determined by multiplying the average quantity
of each.amino acid reaching the abomasum.per Kg of feed
consumed by the average daily feed intake. These values
are 103.3, 32.3, 10.7, 11.2, 21.9, 21.6 and 4.7 g per day
for nitrogen, lysine, cystine, methionine, total sulfur

amino acids, threonine and tryptophan, respectively.

56

TABLE 8
Nitrogen and Amino Acid Passage to the Abomasum in Steers

Fed a 9.5% Crude Protein Rationa, Experiment One

 

 

Parameter g/Kg Feed g/day

Nitrogenbc 14.9 105.3:6.2
Lysine b° 4.5 32.1:o.9
Cystinebc 1.5 10.7:1.1
Methioninebc 1.6 11.210.8
Total sulfur amino acidbc 3.1 21.9

Threoninede 3.7 21.610.5
Tryptophandf .6 4.7tO.9

 

aFed at 37. of body weight

bMean and standard error of 7 steers

cSteers consumed 7.07t.4 Kg/feed/day
dMean and standard error of 2 steers
eSteers consumed 5.88 Kg feed/day

fSteers consumed 7.55 Kg feed/day

57
4. The Quantitative Aspects of Amino Acid Requirement

The quantitative aspects of lysine, cystine,
methionine, total sulfur amino acids, threonine and
tryptophan are presented in Table 9. According to Hogan
(1973) postruminal protein has an average digestibility
coefficient of approximately 0.70. Thus, the quantity of
each absorbable amino acid reaching the small intestine
was obtained by multiplying the quantity of each amino acid
that was reaching the abomasum.by the coefficient of digest-
ibility. These values were for methionine, 7.9; cystine,
7.4; total sulfur amino acid, 15.3; lysine, 22.5; threonine,
15.1; and tryptophan, 3.3.

The daily requirement for each amino acid was
obtained as the sum of the absorbable amino acid and the
determined infusion requirement level. These values were
for methionine, 14.9 and the total sulfur amino acids,
22.3g per day. Since the animal's lysine, threonine and
tryptOphan requirements were satisfied by the quantity of
these amino acids in the digesta reaching the small
intestine, their daily requirements were regarded as equal to
or less than the absorbable amount.

During the infusion periods the steers were growing
very slowly or in spurts and were fed a dietary protein
level that would not support maximal growth. These data
were used to derive an essential amino acid requirement

pattern for the same size steers growing continuously and

 

58

 

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mhmmum N. MO .HOHHO UHGUCNUm USN fimmzm

 

 

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was ocwcooush

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.ocamma .wwo< ocwa< unmadm kuoH .ocwumhu .mcwcownuoz mo muoomm<Vo>Humuwucmso

m mgm<fi

59
fed a 9.5 or 12% crude protein ration at approximately 2.5%
of body weight. Results of these tabulations are given in
Table 10.

In order to calculate these amino acid requirement
patterns, the experimentally determined total sulfur amino
acid requirement (22.3 g per day) was taken as unity and
was then multiplied by the amino acid ratios of the 1973
NRC pig requirement pattern (using the pig's total sulfur
amino acid requirement as unity). On this basis cystine
can fulfill 56% of the total sulfur amino acid need (NRC
pig requirement, 1973) and the methionine requirement in
the presence of adequate cystine for the 274 kg growing
steer fed 9.5% crude protein was calculated to be 9.8 g per
day. The other essential amino acid requirements were
calculated for lysine 31.2, phenylalanine plus tyrosine
22.3, valine 22.3, isoleucine 22.3, leucine 26.8,
threonine 20.1, tryptophan 5.8, histidine 8.0, arginine 8.9
g per day.

The total sulfur amino acid requirement for the
274 Kg steer fed the 12% ration was calculated in relation
to the determined total sulfur amino acid requirement of
the 9.5% ration, with the assumption that only 90% of the
nitrogen from the 12% ration is reaching the abomasum.(see
discussion). The individual essential amino acids were
then calculated in a manner similar to that of the 9.5%

ration. These values for the respective amino acids were

 

60

TABLE 10
Estimated Daily Essential Amino Acid Requirement of Growing

Steers, Experiment One

 

Amino Acid Present study 274 Kg steer Chalupa (1973)

 

 

9.5% C.P. 12% C.P. (300 Kg steer)
a

TSAAa 22.3 25.4" -

Met. 9.8b 11.2b 12.0
Lysine 31.2b 35.1b 36.5
Phe & Tyr 22.3b 25.4b 21.0
Valine 22.3b 25.4b 25.0
Isoleucine 22.3b 25.4b 25.0
Leucine 26.8b 30.4b 39.0
Thr. 20.1b 22.8b 20.5
Try. 5.8b 6.6b 3.0
His. 3.0b 9.8b 13.5
Arginine 8.9b 10.2b 30.0
TEAAC 177.5b 202.3b 225.5

 

aTotal sulfur amino acids

bCalculations based on pig requirement (NRC 1973) pattern

cTotal essential amino acids

 

61
25.4, 11.2, 35.1, 25.4, 25.4, 25.4, 30.4, 22.8, 6.6, 9.8,
10.2 and 202.3g per dayg'With the exception of arginine
and leucine these values compare favorably with those of
Chalupa (1974) who based his calculations on estimation of
the total absorbable protein that was bypassing ruminal
degradation, total ruminal protein synthesis and post

ruminal infusion data.

 

DISCUSSION

Amino Acid Requirements of Growing Steers,

Experiment One

Numerous authors (Zimmerman and Scott, 1965;
McLaughlan and Illman, 1967; Mitchell et al., 1968;
Stockland gt 21., 1970; Brave £2 31., 1970; Wakeling 3; 31.,
1970; Young st 31., 1971; Keith gg‘gl., 1971; Young_gt_a1.,
1972; Tontisirin gt 31., 1972) have reported that changes
in plasma amino acid levels in response to amino acid intake
or infusion can be used as an indicator of essential amino
acid requirements. However, such a technique will give
valid results only if the amino acid under investigation is
the first limiting amino acid in the ingesta reaching the
small intestine (Zimmerman and Scott, 1965; wakeling £5 21,,
1970). Results of the present study shows that methionine
was the first limiting amino acid in the ingesta reaching
the small intestine of these steers. Similar results have
been obtained in sheep by Wakeling 25 31. (1970). However,
because of wool growth, the sulfur amino acid requirements
of sheep are generally higher than those of steers. The
observed changes in plasma methionine levels in response to
methionine infusion substantiates the observations of

Almquist (1954), Morrison EEHEl- (1961), Zimmerman and

62

63

Scott (1965), Mitchell £5 31. (1968), Wakeling st 31. (1970)
and Keith g£_al. (1971) that plasma level of the limiting
amino acid remains constant until the animal's requirement
is met and increases rapidly as intake or infusion levels
are increased. The immediate and linear increase in plasma
lysine during the lysine infusion indicates that lysine was
not limiting in the ingesta reaching the small intestine
and neither was it made limiting when infused in the pre-
sence of adequate methionine. A similar result has been

obtained in sheep by Wakeling st 31. (1970). Also, threonine

 

and tryptophan were not found limiting in the ingesta reach-
ing the small intestine. This can be attributed to the slow
growth rate of the steers due to the low protein diet and
stresses arising from the confinement in the metabolism
stalls. No attempt was made to determine if these amino
acids would be limiting in the presence of adequate methio-
nine.

Nitrogen balance has been successfully used as an
indicator of amino acid requirements (Nimrick gt 31., 1970a;
Nimrick gt $1., 1970b; Nimrick and Kaminiski, 1970;
Schelling 35.31., 1973). However, little can be concluded
from.the nitrogen balance during the methionine infusion
period since there was only one animal on this treatment.
The observed decrease in nitrogen retention during the
lysine, threonine, and tryptophan infusions was contrary to

the expected plateau (Nimrick §£_gl., 1970a).

64

Fecal nitrogen excretion remained constant through-
out each nitrogen balance trial indicating that all the
infused nitrogen was absorbed. However, the increase in
urinary nitrogen was greater than the increase of nitrogen
from infused amino acids. This suggests that an increased
nitrogen load was placed on the animals either by excessive
amino acids or other stresses such as extended confinement
in the metabolism stalls or both. Similar results have
been reported by Boila and Devlin (1972) who observed an
11% decrease in nitrogen retention after infusing 93 per day
of lysine into the abomasum of steers. These workers claimed
that the decrease in nitrogen retention was due to degradation
of the very high level of plasma free lysine resulting from
infusion. However, this is not an adequate explanation,
because if the high level of plasma lysine balanced the
amount of lysine infused, nitrogen retained should not have
changed.

Since ruminal activities dictate the quantity and
pattern of amino acids reaching the small intestine, it was
imperative that the quantity of each amino acid in the abo-
masal ingesta be determined. The lignin to nitrogen ratios
in feed and abomasal ingesta showed that there was no net
ruminal nitrogen loss° This was expected, since the ration
contained only 9.5% crude protein. Using lignin and
chromic oxide as markers, similar results have been obtained

by Weller, Pilgrim and Gray (1971) in sheep fed 8-10% crude

 

65

protein ration. Usually, when large amounts of soluble
protein are fed, the nitrogen reaching the small intestine
is less than dietary intake. Conversely, when a high
energy, low protein ration is fed to the ruminant the
nitrogen reaching the small intestine tends to be equal or
higher than the dietary intake (Clarke 25 21., 1966). F
There is considerable controversy concerning the use of
chromic oxide as an insoluble marker for the determination

of nutrient digestibility. Drennan, Holmes and Garrett
(1969) and Faichney (1972) after finding that chromic oxide

 

behaved independently of the particulate matter in the
digesta, concluded that chromic oxide is not a satisfactory
indicator of flow of digesta through the gastrointestinal
tract. However, Orskov, Frazer and MCDonald (1970), Offer,
Axford and Evans (1971), Offer, Evans and Axford (1971a),
Offer, Evans and Axford (1971b) and weller 25 51. (1971)
obtained satisfactory results with chromic oxide. Although
fairly good results were obtained with chromic oxide in the
present study, the slight differences between the lignin]
chromium ratio of the ration and the lignin/chromium ratio
of the abomasal ingesta indicates that chromic oxide was
moving through the alimentary tract independent of lignin
and nitrogen. However, regardless of the marker used the
key to good passage studies is sampling technique (Weller
g5 _a_1_., 1971; Axford 95 31., 1971). I

66

In order to relate the determined sulfur amino acid
requirement of steers in the present study to growing
steers of the same body weight and consuming 12% protein
at 2.5% of their body weight daily, it was necessary to
make 3 basic assumptions: (1) That the tissue requirements
for essential amino acids for pigs and cattle are similar
(Black 25 al., 1957; Downes, 1961; Hutton and.Annison, 1972).
This assumption is based on a similar gain in carcass
nitrogen expressed as a percentage of live weight gain
(2.64) for pigs (Woodman and Evans, 1951) and steers (2.40)
(Agricultural Research Council, 1965). Although these find-

 

ings are frmm early data, no new data are available to
challenge their authenticity. Another basis for the above
assumption is that the amino acid composition of pig and
beef muscles are similar (Black g£_gl., 1957; Downes, 1961).
After observing that isoleucine, leucine, lysine, methionine,
phenylalanine, threonine, valine and histidine were
‘metabolically essential to growing and mature sheep, Downes
(1961) concluded that tissue amino acid metabolism of sheep
is similar to that of non ruminants. (2) That the require-
ment for each amino acid per 16g of nitrogen remains un-
changed regardless of protein intake. This assumption is
based on the findings of Boomgaardt and Baker (1973a) who
observed that the lysine requirement for maximum weight
gain in chickens remained a constant percentage of dietary

protein whether the diet contained 14, 18.5 or 23% crude

67
protein. (3) That at least 90% of the nitrogen from a 12%
protein ration would reach the abomasum. This assumption
is well within reasonable limits. Weller £5 31. (1971)
reported that when sheep were fed 8-10% crude protein, the
amount of nitrogen reaching the abomasum.was approximately
100% of the dietary intake. In the present study it was
found that 98% of the dietary nitrogen reached the abomasum.
Clarke 35 31. (1966) found that at low protein intakes the
amount of nitrogen reaching the duodenum was equal to or

greater than the dietary intake but as the nitrogen intake

 

increased the amount reaching the duodenum decreased
pr0portionally.

According to the 1973 NRC pig requirement report,
methionine can fulfill the total sulfur amino need whereas
cystine can meet only 56% of the total sulfur amino acid
requirement. The results of the present study indicated that
methionine fulfilled part of the cystine need. Hutton and
Annison (1972) used the pig amino acid requirement pattern
to calculate the essential amino acid requirement for a
growing 200 Kg steer. However, these workers did not con-
sider that cystine can supply at least 56% of the sulfur
amino acid requirement, consequently most of their calculated
requirement values were low when compared to those of the
present study.

It can be concluded from this study that the
method used to determine the essential amino acid require-

ments for growing steers was satisfactory. However, it

68
would be advisable to use a lower crude protein ration so
that the requirement for several amino acids would not be
satisfied by the quantity leaving the rumen. Also, since
age and body weight influence protein and essential amino
acid requirements (Young EE.E$-’ 1971; Tontisirin gt 31.,
1972; Tontisirin st 31., 1973), future experiments should
include different ages and body weights under more favor-
able growth conditions. Steers of different fatness should

also be studied.

 

RESULTS

Experiment Two

This experiment was designed to study the effects
of dietary crude protein content (10.7, 20.2, 32.5 and
40.0% for rations l, 2, 3 and 4 respectively) on voluntary
feed intake, protein intake, plasma amino acids, plasma

urea nitrogen, rumen volatile fatty acids (VFA) and the

 

various nitrogenous fractions in the rumen. ’

Intake Parameters

 

The results from.the feed intake study are pre-
sented in Table 11. Feed intake decreased non significantly
(P>.05) with each increment of dietary crude protein. The
fourteen day patterns of feed intake of steers fed the four
rations are presented in Table 12 and graphically in
Figure 7. There were no significant changes in daily feed
intake of steers fed ration 1 throughout the 14 day
experimental period. Daily feed intake of steers fed
ration 2 decreased on day 2 and then increased significantly
(P<.05) for the remainder of the period. Daily feed intake
of steers fed rations 3 and 4 started to decrease on day 2
and was significantly depressed (P<.01) by day 3. There-
after, feed intake of steers fed these two rations increased
throughout the rest of the period.

69

 

70

 

 

 

 

m.o sma.m an. s Hm.q N. m muuuoHu>
H.o us.s one. n smo.e MN. m mumuuHu>omH
¢.H m0.2 an. mH s.o~ mm. mH «sausage
o.~ <mH.~N H<MH. Hm mum.~e m<unw. mm mumaOHaoum
m.H mm.sm «a.mo ma.ns N. as assuuoa
nm¢m> aoanm

m.s ps.em nm.om o.Hm we. so «HnmuHaHumum
N.¢ aam.aH <smHHN «a H. on mos. es eHu< oaaa< a osHuaum
m.m oum.Hm umoo.ao mama. mm «as. HH «Heoaa<
H.m moiHOH mono.Hm m<no.oe <uo.on eHuuoum aoz
s.oe mm.Hos mm. mmm ma.omn an. ass uHssHomcH
m.m moo.moH Howe. HmH m<n¢.omH awn. oNH OHHaHom
¢.Hs mm.sos so. Hen o. HsH so. awn Hayes
Naowouuaz cuaum

e.w mm.w mw.~ mm.“ mm.m 53.m we ooHHHsmomw we
N . . . N . . Gamuou Han
H.H m<Mo.n mmH.H m<pwe.w amo.m H Human aHHun
QKNUGH
.m.m s m N H

nouuaouwm

maowuwm

 

039 ucmBHHomxm .maowuomum mnoaowouuaz Hoadanm was m<m> Hmnfiaum .mHo>oA so»:
was mowed oaHa< seesaw .oxouaH pooh do aflououm dunno humuoaa mo mao>oa mdowum> mo muoowmm

HH mqde

71

wllrill.in

.Ho.vm om< “ mo.vm o.n.m

.ucouowmav kaucmowmwcme one son mama onu cw umaHomHoQSm udouommav £uw3_monao>

Hoaowa awash as you <h> o>wuoommou 0:» mo moaoaouoaz

ucouaoo smash HE ooa Hon cowouuaa wz

«Human as you moaoaouoHZn

mamwam vooan Ha.OOH Hon ammouuwc w2¢

m

N

mop Hon woman you wMH

 

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m<<mz Houoa
<¢m HouOH
«comequ won:

seesaw

 

.m.m

 

maowumm

Houoawumm

 

H. . . . suseHueoov HH «Heme

72

Hoovm 90$ .mo.vm U.U.D.N

ucoHoMMHv hHuconMHame one sou same can cH mumHuomuoaam uaonHMHv nuH3 mosHm>

mom you Hoops Hon cHououm mo .mx omauo><

map you humus Hon voom mo .wM owuuo><

N

H

 

 

 

 

 

 

H.o o.m m.N H.N O.N m.o m.H m.N q nOHumm
H.w amm.m noomw.N UMM.m omMm.m <Mm.# m<wm.H noun.N m coHumm
H. . . . . . .H m.H N aoHumM
H.o «Mo.H <MO.H «nMo.H <nwm.o <nwm.o <Mm.o «an.o H coHumm
oxmucH aHouon_
mno ass a osmHHo muonmnm om<onmne «an N m<nmmnm omsonanm e coHuma
m o mnm w mnN w new 5 m<nm m <00 m <oH m m<nm n m coHumm
m.o nmm.m nun.m pom.¢ nN.w nwm.n m¢.o nwm.n N aoHumm
m.o w¢.m mN.m mm.m m~.w mm.» mo.w mm.m H GOHumm
HoxmucH comm
.m.m wH .DH «h m .m. N H
mesa Houoawumm
039 unmaHuomxm .oxwuca aHououm was pooh hHHon

NH mHm<H

73

 

oxmuaH poem zHHmv mo mcuouuma New cmouusom .N MMDme

 

 

       
 

mw<o
«H OH N m m N H o
P h b n P b b
q q u q u q 1
L. H
4. N
aoHumm C
nOHumM 4V m
coHumm o .u q
coHumm 3 8. m
//+; m
II N

 

 

 

 

 

 

7?

0H

(Kev/9x) axenUI peas

74

Daily protein consumptions of steers fed the four
rations are presented in Table 11. Protein intake was
highest among the steers fed ration 3 (P<.01). Steers fed
rations 3 and 4 had a significantly higher daily protein
intake than steers fed ration l (P<.05) but protein intakes
of steers fed rations l and 2 were not significantly
different at P<.05. Differences in protein intakes of
steers fed rations 2, 3 and 4 were not significant (P>.05).
The fourteen-day patterns of protein intake for steers fed
the four rations (Table 12 and Figure 8) are similar to
those of total feed intake.
Ruminal Nitrogenous Fractions

Total ruminal nitrogen concentrations of steers fed
the four rations are presented in Table 11. Total ruminal
nitrogen concentration was highest in steers fed ration 3
and lowest on ration 1. However, differences between treat-
ments were not significant. The fourteen day patterns of
total ruminal nitrogen for steers fed the four rations are
presented in Table 13. Rumen nitrogen of steers fed ration
1 did not differ during the 14 day period (P>.05). Rumen
nitrogen of steers fed rations 2 and 3 increased linearly
(P<.05) throughout the period. Rumen nitrogen of steers fed
ration 4 decreased significantly on day 3 (P<.Ol) and
increased (P<.01) thereafter throughout the remainder of the

period.

 

75

«pi

.mxmuaH cHouoHa NHva mo announce hop noouuaom

«no
H

mw<a

.DIn

um

1H“

.w MMDUHm

de

 

s aOHumm

m aOHumm
N cOHumm

H coaumm

 

 

O ‘Q 1.

X

\\N
\\
\\.

 

(Kep/By) axenuI uranoxd

76

Soluble rumen nitrogen of steers fed the four
rations are presented in Table 11. Concentrations were
highest in steers fed ration 4 and lowest for ration l
(P<.01). The fourteen day patterns of soluble rumen
nitrogen for steers fed the four rations are presented in
Table 13. There were no significant changes in the patterns
of steers fed ration 1. Although soluble rumen nitrogen
of steers fed rations 2 and 3 increased significantly
(P<.05) after day l and changed markedly during the period,

there was no obvious pattern of change. Steers fed ration 4

 

had a decrease in rumen soluble nitrogen on day 3. There-
after, concentrations increased linearly (P<.Ol) for the
rest of the period.

Insoluble rumen nitrogen (Table 11) was highest in
steers fed ration 2 and lowest in steers fed ration 4. How-
ever, differences were not significant at P<.05. The four-
teen day patterns of insoluble rumen nitrogen of steers fed
the four rations are presented in Table 13. Insoluble
ruminal nitrogen of steers fed rations 3 and 4 decreased on
days 2 and 3, respectively, and then increased linearly
throughout the rest of the period, while that of those fed
rations l and 2 increased over the entire 14 day period,
but daily differences were not significant.

Ruminal non protein nitrogen (rumen NPN) of steers
fed the four rations are presented in Table 11. Rumen NPN

was highest in steers fed ration 4 (P<.01) and lowest in

77

 

 

 

 

 

 

 

onm moqu maommH QUHUNHH unvomOH m<nm¢N omonom <dNn s GOHumm
o m oHH NDH qHH Hm «w oN No m aoHumm
. um mono . oopo om<pop um<onm m<nw <m
o m nmmw nmNN nmw anw pmmm nuON own N GOHuom
O.N mNm mHm mHm mHm an «on «an H aoHuum
aumoHqu :Hououm doz
. momNm Hangman maunmen Haunsmme «uon flnmNNN Hausumne s =0Huum
H NN nomvnmnm auvmmo apoHN m<nmmm¢ m¢w¢Nq §<wNH¢ U.HsHNs m sOHHmm
H.NN some mNmo anon mHam mmnn mHNn anon N :oHumm
H.NN ”mos MNNm Mon MNNm ”mos MNHe ”mam H coHuam
nowouqu cuanm oHnaHomaH
mud avaN nvmmN QUHVoNNN nomoOHN m<annH om<onowH <womH s cOHumm
w s mON OHN «NN me «NH ooH mmH m aoHuum
. omoo once on om<on undue m<nm <m
m e NHONH m esH m< anH NHNQH mnoNH m<m mnH aaOHH N aoHumN
N.s mHH msNH meNH mNHH NHH soNH H =0Humm
cowouqu Guaam oHnsHom
N. NN oonm omonq¢N umonmoN o<onomo muqu m<dNNm o<nmomn q aoHumm
N. NN omMmom omLOHm onoem m<smNo <swoo «mem <ono m nOHumm
N.NN pmNm nHNw mNmN mmnN memN msNN mmHo N noHuom
N.NN sown nose Mmmo Home MNmm ”Hmn MnHm H GOHumm
cuMoHqu aoanm HmuoH
.m.m HH 0H N m m N .LH
when Houoaauom

 

039 uuoaHuomxm .

mGoHuomHm mnocomouqu Hmcfiaum mo meHumHunooaoo NHHmn omeo><

m

H MHm<H

78

 

HO.Vm W.Q.U.m.< .mo.vm O.U.O.Q.w

ucmumMMHv NHuCMOHMHcmHm mum 3o» 03¢: 0:» GH mumHuomumanm unouuMMHv nuHs mmsHm>

.uamuaoo swan» Ha ooH Ham cowouuHc wzH

 

 

 

 

 

 

H.m m¢0H wqu moHH «mm wmw mam «mm ¢ aOHuwm
H.m swam HNOH OOH HmHN puma puma «NN m aoHuum
H.m mnNm HHHm m<qu anew mpmm m<nmm <uHm N cOHumm
H.m mmo umN «me mom «mm «mm wNN H :OHuwm
cowouqu mHnuuHmwumum

m.N wN oH ¢H HH oH NH «m w GOHumm
m.~ «HMNN EmHH «Mom «MHN <HMHH «HMHH «MNH m coHuum
m.N «Hm mmN moq mNm mnm mNm mN¢ N GOHumm
m.N mmq qu wwq mom mow m¢¢ moq H GOHumm
auwouqu vHo< oafia<_vam mvHumwm

mHN avONH QHUmHH novowm QUONm mnNm umnmo <mmH q GOHumm
m N «a Hm ¢m ON ON m¢ nH m :OHumm

. 0v ova ow omun¢ omen mp 4w co
m N am HwHH mmH mHH mNH mNH umH H GOHumm
«Haoaa< amaum
.m.m qH 0H N m m, N H

mhwn Hmugawumm

 

Ac

. . umsaHucouV nH «Hana

79

steers fed ration l (P<.01). Fourteen day patterns are
presented in Table 13. There were no significant daily
changes in rumen NPN of steers fed ration 1 throughout the
14 day period while that of steers fed rations 2 and 3
increased over the entire 14 day period but decreased
slightly on day 10. Rumen NPN of steers fed ration 4
decreased on day 3, then increased linearly throughout the
rest of the period and was closely related to the pattern of
protein intake.

As presented in Table 11, rumen ammonia nitrogen
was highest for steers fed ration 4 (P<.Ol) and lowest for
steers fed ration 1 (P<.01). Steers fed ration 4 had
significantly higher concentrations (P<.OS) than those fed
1 and 2, and steers fed ration 3 higher (P<.05) than those
fed rations l and 2, but differences between rations 3 and 4
were not significant. Fourteen day patterns are presented
in Table 13. Differences in daily rumen ammonia nitrogen
were not significant for steers fed rations 1 and 2. Rumen
ammonia of steers fed rations 3 and 4 increased linearly
(P<.05) throughout the 14 day period with a slight depression
on day 3 for steers fed ration 4.

Rumen peptide and amino acid nitrogen (RPAAN) of
steers fed the four rations are presented in Table 11.
Steers fed ration 1 had higher RPAAN (P<.05) than those fed
rations 3 and 4 but not significantly higher than those fed
ration 2. Also, steers fed ration 2 had higher RPAAN

 

80

(P<.05) than those fed ration 4 but not significantly
higher than steers fed ration 3. Fourteen day RPAAN
patterns are presented in Table 13. Differences in daily
RPAAN of steers fed rations 1 and 2 were not significant.
Steers fed rations 3 and 4 had their highest RPAAN on day l
(P<.Ol) with a significant depression (P<.05) on day 4 for
steers fed ration 4.

Rumen tungstic acid precipitable nitrogen (RPN) of
steers fed the four rations are presented in Table 11.
RPN nitrogen was highest in steers fed ration 4 and lowest
in steers fed ration 1 (P<.05) but differences for steers
fed rations 2, 3 and 4 were not significant. Fourteen day
patterns are presented in Table 13. Differences in daily
RPN of steers fed rations l and 4 were not significant.
However, the random significant differences noted in steers
fed rations 2 and 3 were not part of any consistent pattern.
Rumen Volatile Fatty Acids

Rumen acetate concentrations of steers fed the four
rations are presented in Table 11. Although rumen acetate
was higher in steers fed rations 2 and 3, differences among
treatments were not significant. Fourteen day patterns are
presented in Table 14. There were no significant differ-
ences in daily rumen acetate of steers fed ration l but
that of steers fed rations 2, 3 and 4 increased daily
throughout the entire 14 day period with the exception of a

significant depression on day 3 for steers fed ration 4.

 

81

Rumen propionate of steers fed the four rations are
presented in Table ll. Steers fed ration 2 had higher
(P<.05) rummn.propionate than steers fed rations 3 or 4 but
the differences between that of steers fed rations 2 and 1
were not significant. Fourteen day patterns are presented
in Table 14. Differences in the daily ruminal propionate
of steers fed ration 2 were not significant but steers fed

ration 1 had their highest rumen propionate level on day 14

 

(P<.05). Rumen propionate of steers fed rations 3 and 4

decreased significantly on day 3 (P<.OS) and then increased

1 .

daily during the remainder of the period.

Rumen butyrate of steers fed the four rations are
presented in Table 11. Steers fed ration 2 had the highest
rumen butyrate (P<.05) but differences in concentrations for
steers fed the other 3 rations were not significant. The
fourteen day patterns are presented in Table 14. Differences
in daily rumen butyrate of steers fed rations 1 and 2 were
not significant. Rumen butyrate in steers fed rations 3 and
4 decreased slightly on days 2 and 3, but increased linearly
(P<.05) throughout the remainder of the period.

Rumen isovalerate (Table 11) was higher (P<.05) in
steers fed ration 4 than steers fed rations 1 and 2, but was
not significantly different from steers fed ration 3. Rumen
isovalerate of steers fed ration 3 was higher than that of
steers fed ration l (P<.OS) but not significantly higher than

that of steers fed ration 2. Differences in concentrations

82

 

 

 

 

 

 

m.o sm.o «H.N «O.N mm.N s¢.m «0.0 sm.m a coHumm
m.o mo.o mo.o an.o aN5. an.n mo.m an.¢ m aoHumm
m.o mo.¢ «0.4 sm.¢ «N.m mo.m no.4 sN...” N noHumm
«.0 ma.H «O.N m¢.m sm.N an.m mm.¢ sH.s H aoHumm
OumeHNNrOQH cam
mno ouHN omwoNH omaunmmH m<naoH awn m<nma omaunuNH s aoHuam
N o o NN omnON DHNN masmH «uoH asoH HH4.....NH m aoHuum
m.o “4N sHN «ON sHN sHN m0N uNH N aoHusm
m.o moH mNH mNH «NH unH moH mnH H coHumm
oumuhuam nuanm
m.H ans qu was «ms was «ms was N aoHumm
m.H pas swam puma nst swam mmN mwN H coHusm
oumconoum coanm
O.N we so me an Na Na mm a coHuum
O.N mme mmNN muma m<MMwo <ann “was «New m aoHuum
O.N nNN AON ammo ammo nmNo swam «an N aoHumm
O.N was mNs was was «an mNm «on H aOHumm
oumuoo< coaum
.m.m 4H oH x» m m N H

 

m%wn

Houuawumm

 

039 unmawuogxm .

maowumuuooucoo owo< huuwm

«H mumdfi

oHHuwHo> Goanm magma owwuo>¢

83

 

Hoovm UNQN¢ .nO.VnH 0.9.“
uaouommwo maunmowmacmam mum 30H mean on”. a.“ unfiuomummam ucouowmao £33 $45.3,

mono: c955 no He won $9 mo moaoaouodf

 

 

 

«H m ms.q m¢.m mm.m mm.N «N.m sn.s a aoHumm
as m an.m mH.m mN.N mm.m sm.m mm.s N aOHumm
mo.m «mus scum moms «Nuq «mum «Hus N aoHuum
mumuoam> swam

«H OH N m m N H

 

whoa nouoamumm

 

A. . . uoscHucoov «H «Hana

84
between steers fed ration l and ration 2 were not
significant. Fourteen day patterns for steers fed the 4
rations are presented in Table 14. Daily differences in
concentrations for steers fed each ration were not
significant.

Rumen valerate of steers fed the four rations are
presented in Table 11. Steers fed ration 3 had higher
rumen valerate (P<.05) than steers fed ration l but differ-
ences between steers fed the other rations were not
significant. The fourteen day patterns for steers fed the
four rations are presented in Table 14. Daily differences
in concentrations of steers fed each ration were not
significant.

Plasma Parameters

Plasma urea nitrogen (PUN) of steers fed the four
rations are presented in Table 11. Steers fed ration 4 had
higher PUN (P<.05) than those fed rations l and 2 but not
significantly higher (P<.05) than steers fed ration 3.
Steers fed ration 3 had higher PUN (P<.05) than steers fed
rations l and 2 but differences between steers fed rations l
and 2 were not significant. The fourteen day patterns of
PUN are presented in Table 15 and Figure 9. There were no
significant differences in the daily PUN of steers fed
ration 1. FUN of steers fed rations 2 and 3 increased
significantly (P<.01)_on day 3 and then remained constant

during the remainder of the 14 day period; whereas that of

 

”7. “_§' 0

I
a

85

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U

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2593 HE Mom moaoaouoazd Samoan H0093 H8 2: Hon dowouuaa ms.”

00.0 00.H 00.H NH.H 00.0 00.0 00.0 HN.0 0 soHumm
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0. . . . . H0. 0N.0 N aOHsmm
00.0 0MH0.0 0ENS. H 0as”: 0 0«”090 0«”30 0<M0N.0 0M00.0 H :oHuum,
0<<mzN<<m

00.0 N0.0 0N.0 00.0 00.0 00.H 00.0 NH.H 0 coHuam
00.0 0wmw00.0 «MoN.0 mmme.0 0““30 m«MM00.0 0«mu00.0 MpNH.H 0 aoHuum
00.0 m09H 000. 0 0H0. 0 m09H HH. H mN90 u 0.H N aoHumm
00.0 mNN.H M00. H M00. H 0N0.H M0H. H mH9H m09H H aoHuum
N<<mz H0000
00.0 0N.H 00.0 00.H H0.0 H0.0 NN. 0 NN.0 0 aoHuum
00.0 M0M.0 “NH.H 0M00.0 0MWN.0 0wmw. w “MM. m “00.0 0 onuqm
00.0 0 .H 0.H 00.H H.H . H0.0 N oHumm
00.0 0. 0.H 0...M09H MH.0M0N0 0<M00.0 0<M0N. 0 0”050 <M00.0 H.coHumm[
N040 Hsuoa
0.0 NN 00NN 00N 0N 000H 0N N 0 coaumm
0.0 0W0N fieN 000 00N 0meN fl0N 0MMMH 0M0 0 000000
0.0 0H %NH 00H 0H 0NH H 0 N :oHumm
0.0 0WN fiN momN 00M0 0MN 0MN 0M0 H coHumm+
Hnowouuwz «on: 3595
.m.0 \NH, 0H N 0» 0 N H

whoa Houoawumm

 

03m. udofiwHonm .wcowuouudoocoo 38¢ 055 on.» cowouuwz no.5 mammam

Wag

86

mcowumuuaoocoo Gmwouuwc won: mammam mo mauouuma how coouudom

 

 

 

 

 

 

 

 

 

 

.m MMDUHm
mafia
dH 0 n m m N H
PT, m u u a. u? I.
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87

steers fed ration 4 increased significantly (P<.Ol)
throughout the period.

Total plasma essential amino acid concentrations
(TEAA) of steers fed the four rations are presented in
Table 11. Differences in TEAA of steers fed the four rations
were not significant. The fourteen day patterns are pre-
sented in Table 15 and Figure 10. There were no significant
differences in daily TEAA of steers fed ration l or ration

3, but concentrations in steers fed ration 2 increased

 

throughout the 14 day period with a significant difference
(P<.05) only between days 1 and 14. There was no consist-
ent pattern for steers fed ration 4.

Total plasma nonessential amino acid concentrations
(TNEAA) of steers fed the four rations are presented in
Table 11. Differences in TNEAA of steers fed the four
rations were not significant whereas TNEAA of steers fed
rations 3 and 4 were highest on day l (P<.05) but decreased
gradually during the remainder of the period.

The plasma essential amino acid nonessential amino
acid (E/N) ratios are presented in Table 11. Steers fed
ration 2 had a significantly higher (P<.05) plasma E/N
ratio than steers fed ration 1, but differences in the
ratios of steers fed ration 2 and rations 3 and 4 were not
significant. The fourteen day patterns are presented in
Table 15. Daily differences in.the plasma E/N ratios of

steers fed ration l were not significant. Daily E/N ratios

88

3

.mcoHumuusooaoo <¢m9 mammaa mo mcuouuwm 5mo GoouHSOm

 

0H

 

1'

H N Md“

‘I

coaumm I
coaumm 4

cowumm I

Gowumm “

j.

. OH mmDUHm

 

(emsstd '[m/ satomoxorw) W31

89
in steers fed rations 2, 3 and 4 increased throughout the
period with their highest values (with the exception of

steers fed ration 3) appearing on day 14.

 

DISCUSSION

Effect of Excess Dietary C rude Protein on Voluntary Feed In-
take and Various Ruminal and Plasma Parameters,

Experiment Two

The present study was conducted with the basic
premise that the large quantity of ammonia produced in the
rumen by microbial degradation of excess protein would exert
a feedback saturation effect on the proteolytic and de-
aminating enzymes, so that a greater amount of undergraded
protein and amino acids would be reaching the lower gut.
Chalmers and Hughes (1969) and Emery (1971) showed that when
a large quantity of a single amino acid was administered to
the rumen, the rate of deamination of that amino acid was
substantially reduced. Thus, a greater quantity of the
amino acid was absorbed from the small intestine as
evidenced by the increased plasma level (Chalmers and Hughes,
1969). The results of this study (Table 11) demonstrated
that there was no apparent upper limit to ruminal proteolysis
and deamination.

It was observed that steers fed the high protein
rations (3 and 4) were nibblers (eating a very small quan-
tity of feed at any one time throughout the day) while
steers fed the low protein rations (l and 2) were meal

90

 

91

eaters. Such an irregular pattern of feed consumption of
steers fed rations 3 and 4 completely nullified the ex-
pected effects. This problem along with an apparent
dilution of rumen volume by an observed increased water
intake in steers fed high protein rations might have caused
the failure to demonstrate an upper limit in ruminal
proteolysis and deamination in steers fed the high protein. an
That is, ruminal amino acid and ammonia concentrations
might not have been high enough to impose a feedback

saturation effect on the proteolytic and deaminating enzymes.

 

The overall average daily voluntary feed intake ;~*
(although not statistically significant) decreased as the
protein content of the ration was increased (Table 11).
Protein intake, on the other hand, was highest for the high
protein rations. An explanation of this is that the high
protein content of rations 3 and 4 more than compensated for
the decrease in total feed intake. The non significant
decrease in feed and protein intakes in steers fed ration 2
on day 2 and the significant decrease in these two para-
meters in steers fed rations 3 and 4 on day 3 require care-
ful consideration. Palatability is generally one of the
contributing factors in intake phenomenon. However, since
intake of the high crude protein rations was high on day 1
it is not likely that palatability exerted any significant
influence on feed intake in this study. Therefore, some

feedback mechanisms might have triggered the depression in

92
feed intakes on day 3. It is possible that high protein,
high ruminal ammonia and high blood urea concentrations
inhibited enzyme systems of the rumen or the liver; or they
might have stimulated the satiety center of the brain.
Russek (1971) suggested that the presence of hepatic
receptors which monitor glucose and ammonia levels might be
major contributory factors in the reduced voluntary feed
intake of high protein rations. Peng st 31. (1974), working
with rats, explained control of voluntary food intake by

protein level as shifts in tissue free amino acid equilibrium

 

towards anabolism.when the rats ate low protein diet and
towards catabolism when the high protein diet was consumed.
Almquist (1954) working with chickens, Mellenkoff (1957)
working with humans, Harper st 31. (1964) and Harper (1965)
working with rats all suggested that voluntary feed intake
is monitored by plasma.amino acid concentrations. This is
not a completely valid explanation for the results of the
present study since differences in plasma essential or non-
essential amino acid concentrations of steers fed the four
rations were not significant. However, differences in amino
acid passage to the small intestine were not assessed. It
is therefore difficult to pinpoint an aminostatic control
that was monitoring the intake phenomenon in this study.
Although total protein intake was significantly
higher for the high protein rations, differences in total

rumen nitrogen of steers fed the four rations were not

93
significant. The most logical explanation of this leveling
off effect of total ruminal nitrogen is that there was an
extensive loss of rumen nitrogen in steers fed rations 3
and 4 due to rapid proteolysis and deamination. This is
evidenced by the significantly higher rumen ammonia and
plasma urea nitrogen levels in steers fed rations 3 and 4.
The fourteen day patterns of daily total rumen nitrogen
were similar to those of the daily protein intake. The
decrease in total rumen nitrogen of steers fed rations 3 and
4 on day 3 was in response to the decrease in feed and pro-
tein intake.

The increase in soluble rumen nitrogen as the level
of dietary protein increased in rations 3 and 4 is an indi-
cation of the high solubility of the isolated soy protein
of these two rations as compared with the high concentration
of corn protein in rations 1 and 2. Since rumen ammonia
indicate the extent of ruminal protein degradation, and the
extent of ruminal protein degradation depends on its
solubility in rumen fluid (el-Shazly, 1958; Blackburn and
Hobson, 1960; Hendrickx and Martin, 1963), the higher rumen
ammonia of steers fed rations 3 and 4 was a function of both
the high level of protein and the high solubility of the
isolated soy protein. The higher rumen ammonia in the
steers fed the high protein rations confirmed the findings
of Lewis (1957), Tagari 35 El- (1964), Preston 22.2;-

(1965), Cocimano and Leng (1966) and McIntyre (1970) who

 

”flu-c9 n - J-
.
l

94
reported a high positive correlation between protein intake
and rumen ammonia nitrogen. RPAAN decreased as the crude
protein content of the ration increased. Since rations l
and 2 contained a large proportion of corn and since zein
is not extensively degraded by ruminal proteolysis
(McDonald 1954), it is possible that a fair amount of the .
zein might have undergone partial proteolysis resulting in a [“1
greater quantity of peptides and small molecular weight
compounds which increased the quantity of RPAAN fraction of
steers fed rations 1 and 2. The higher RPN of steers fed

 

the high protein rations reflects the high quantity of
undegraded soluble soy protein of these rations. According
to Hume (1974) only 60% of soy protein is degraded in the
rumen.

The significant increase in ruminal isovaleric con-
centration with increased dietary protein can be explained
as the result of the larger proportion of branched chain
carbon skeletons resulting from.the deamination of the
higher level of branched chain amino acids found in the
isolated soy protein of rations 3 and 4 (Drskov and Oltjen,
1967).

The significant increase in PUN with increased
dietary protein is in agreement with the findings of Lewis
(1957); Tagari gt El- (1964); Preston gg‘gl. (1965) and
Fenderson (1972) who observed a significant positive
correlation between dietary protein, rumen ammonia and PUN

concentrations.

95

It can be concluded from this study that a control
mechanism was monitoring the depression of feed intake in
steers fed rations 3 and 4 on days 2 and 3. However, since
daily feed consumption in steers fed these two rations was
most irregular, no obvious mechanism was evident. The
results also imply that it is not economical to feed high
protein rations to growing steers unless ruminal proteolysis *

and deamination can be controlled.

 

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