ABOMASAL INFUSION OF PROTEIN AND
GLUCOSE IN LACTATING COWS

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSlTY
LARS ViK- M0
197 3

 

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LIBRARY ""

Michigan State
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This is to certify that the

thesis entitled

Abomasal Infusion of Protein And
Glucose In Lactating Cows

presented by

Lars Vik—Mo

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Dairy Science

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17- 5/ [ZLZ/J

Major professor

Date 7/20/73

 

 

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This is to certify that the

thesis entitled

Abomasal Infusion of Protein And

Glucose In Lactating Cows

 

presented by

Lars Vik-Mo

has been accepted towards fulfillment
of the requirements for

Ph.D. Dairy Science

 

 

 

degree in
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Date 7/20/73 ‘

 

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ABSTRACT

ABOMASAL INFUSION OF PROTEIN AND
GLUCOSE IN LACTATING COWS

BV

Lars Vik—Mo

The influence of amino acid availability on milk
protein production in cows fed above NRC standards of
energy and protein was studied by infusions through
abomasal cannulas, using three and four Holsteins, re-
spectively, in two trial series (years). In every trial,
all of the cows received all treatments. Performance
during substrate infusion was compared to controls before
and after in four out of five trials.

Supplying 300g casein per day for six days increased
(P<.05, N=S cows) milk yield 1.1kg/day compared to con-
trols (15.3kg/day) and milk protein (NX6.38) production
increased (P<.01) 49g/day with slight change in milk
protein content (+0.07%, P>.05). During glucose infusion
(BOOg/day) milk yield increased slightly (0.9kg/day,
P<.05), and response in protein production was smaller

(P<.05) than with casein.

Lars Vik-Mo

Na-caseinate + 3% dl—methionine (K) at 75% of
milk protein output was compared to equicaloric amounts
of glucose (G) and a mixture (M), 1:1 of K+G, in a
3X3 Latin square design with saline infusions (0) before
and after. Periods were 7 days. K and M increased
(P<.05) daily milk yield 1.8kg over 0 (24.0:.7kg) and
1.9kg over G, while milk protein content was increased
(P<.05) 0.20% over 0 and 0.15% over G. Hence, daily
milk protein yield was increased more by K (101g, P/.01)
and M (799, P<.10) than by G (40g, NS) when compared to
adjacent controls (trial 2.1).

Caseinate + 3% dl-methionine infused at 50, 100,
and 200% of milk protein yield depressed (P<.Ol) feed
intake compared to saline controls in a 4X4 Latin square
design with 4-day periods and 2 days between periods
(trial 2.11). Milk yields (17.811.1kg/day) and protein
yields did not differ between treatments but estimated
true protein (ETP=(N-NPN)X6.38) content of milk increased
non—linearly (P<.05) with level of treatment.

Feed NPN at 38% and 14% of total N (120% of NRC
(1971) standards) in a cross over design (N=4 cows) did
not influence responses in milk protein to caseinate +
3% dl—methionine infusion which equalled 20% of the CP in
the feed (trial 2.111). This infusion increased milk

ETP content 0.25% (P<.Ol) and daily ETP yield 50g (P<.05)

Lars Vik-Mo

with a nonsignificant increase (0.4kg) in milk volume
over saline controls (12.9:1.3kg/day).

Multiple regression analyses showed responses in
milk protein yield to casein infusion (N=25) depended on
control yield (P=.002) and level of casein infused (P=.072)
(R=0.78, P<.01). The control protein yields eXplained
twice as much of the variance in responses in protein
yield as did level of infusion. The mean response of
64:69 milk protein per day was 11.6% above control yields
(556:25g/day) and accounted for 18% of the infused pro-
tein (360il7g/day).

While NPN content in milk generally increased
(P<.05) with protein infusion, this did not change the
ranking in traetment responses between milk CP and ETP.
Infusion treatments generally depressed milk fat content
0.2 to 0.3%. Plasma or blood urea N and plasma glucose
increased during protein infusion. Urea N was correlated
positively with NPN concentration in milk.

Plasma a amino N in trial series 1 and the molar
% ratio of essential to nonessential amino acids in trial
series 2 increased with protein infusion. In two dif—
ferent trials the molar % of threonine and phenylalanine
decreased (P<.05) by the protein infusion. Branched
chain amino acids were high and methionine increased (P<.Ol)
several fold from the lowest to the highest level of treat-

ment (trial 2.11). When relating the output of essential

Lars Vik—Mo

amino acids by milk protein to plasma concentration (trial
2.I11), phenylalanine appeared as the least abundant amino
acid.

The consistently higher responses in milk protein
production by abomasal protein than glucose infusion, and
a tendency towards more response with increasing levels of
infused protein, suggest that milk protein synthesis was
enhanced through improved amino acid supply. Blood

parameters support this interpretation.

ABOMASAL INFUSION OF PROTEIN AND

GLUCOSE IN LACTATING COWS

BY

Lars Vik-Mo

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Dairy Science

1973

:19 ACKNOWLEDGEMENTS

This thesis certainly could not have been com-
pleted without the generous help and support from numerous
peOple, whom I would like to acknowledge. Especially I
want to thank my major professor, Dr. J. T. Huber, for en-
couragement and help during the course of my graduate study,
and for assistance in correcting the thesis manuscript. 1
am also grateful to Drs. R. S. Emery, H. A. Tucker and
M. G. Yang for their willingness to serve on my guidance
committee. Among those in the Departments of Dairy Science
and Animal Husbandry who assisted me, I want to recognize
particularly Drs. R. Lichtenwalner, for help with parts of
the experiments, W. E. Bergen, for amino acid analysis, and
R. R. Neitzel, for aid with statistical calculations. Drs.
C. L. Miller, W. D. Oxdender and D. E. Ellis gave invaluable
assistance with the surgery of the cows, and the personnel
at the M.S.U. Dairy Barn are thanked for c00perative care
of the cows. Here I would also like to acknoledge my cousin,
Dr. H. A. Lillevik, for his aid in many different ways
during the years as a graduate student. Lastly, but not
least,1 want to express my appreciation for the financial
support from the W. K. Kellogg Foundation, which brought
me to Michigan State University and the continued support

from the Department of Dairy Science.

ii

INTRODUCTION

TABLE OF CONTENTS

0 Q 0 0 O o o o o o o o o 0 o 0

LITERATURE REVIEW . . . . . . . . . . . . .

1.

Assessment of protein demands in
lactating cows . . . . . . . . . . . . .

1.1

1.2

Protein quality: the concept of
biological value and essential
amino acids in lactating cows . . .
Factors influencing responses to
different protein supply for
lactating cows . . . . . . . . . .

The role of rumen metabolism in amino
supply to the host animal . . . . .

acid
2.1

2.2

2.3

The fate of protein and N in the
rumen . . . . . . . . . . . . . . .
Quantification of microbial
synthesis in the rumen . . . . . .
Quality of protein in the digesta .

Postrumen supply of proteins and amino
acids, and rumen bypass through
protective treatments . . . . . . . . .

3.1

Studies in sheep and growing cattle

a. Effects of chemically prepared
proteins . . . . . . . . . . .
b. Extraruminal protein or amino
acid supply and plasma amino
acid patterns . . . . . . . . .
Studies in lactating cows . . . . .
a. Methionine supplements bypassed
rumen . . . . . . . . . . . . .
b. Intravenous amino acid infusion
studies . . . . . . . . . . . .
c. Feed proteins introduced

postruminally . . . . . . . . .

iii

Page

10

l3

14

22
27

32
32

32

38
43
43
46

49

4. Milk protein content and dietary protein .

5. Summary of literature review with lead to
the experimental approach . . .

RESEARCH SECTION . . . . . . . . . . . . . .

I. First series of experiments (1970)

1. Methods and materials . . . . . . .

1.1

Hruhathka
O O O O

\immbww

1.8

Rationale for treatments and

design . . . . . . . . . . . . .
Animals and abomasal cannulation
Feeding and feed sampling . . . .
Infusion treatments . . . . . . .
Milking and milk sampling . . . .
Blood sampling . . . . . . . . .
Chemical analysis . . . . . . . .

a. Feed . . . . . . . . . .
b. Milk . . . . . . . . . . . .
c. Blood plasma (trial 11 only)

Calculations and statistical
analysis . . . . . . . . . . . .

2. Results . . . . . . . . . . . . . . .

2.1

Feed, energy and protein intake

a. Feed composition . . . .
b. Intakes . . . . . . . . . . .

Milk production and composition .

a. Trial I . . . . . . . . . . .
b. Trial II o o 0 Q o o g o 0 Q

Evaluation of the compounded
production data for trials

I and II . . . . . . . . . . . .
Blood parameters . . . . . . . .

a. Plasma urea N . . . . . . . .
b. Plasma a amino N . . . . . .

3. Discussion . . . . . . . . . . . . .

11. Second series of experiments (1971) . . .

i. Trial I 1971 o o o o o o o 0 Q o o 0 0

1.

Methods and materials . . . . .

iv

Page

54

55
56
56

56

56

59
63
65
67
68
68

68
69
70

7O

73
73

73
73

74

74
76

84
87

87
90

93
106
106
106

ii.

iii.

1.1 Rationale for treatment and
design . . . . . . . . . . .
1.2 Animals and abomasal
cannulation . . . . . . . . .
1.3 Feeding and feed sampling . .
1.4 Milking and milk sampling . .
1.5 Blood sampling . . . . . . .
1.6 Chemical assays . . . . . . .

a. Feed analyses . . . . . .
b. Milk analyses . . . . . .
c. Assays of blood constitu-

ents . . . . . . . . . .

1.7 Calculations . . . . . . . .
1.8 Statistical analyses . . . .

Results 0 O O O O O O O O O

2.1 Feed intakes . . .

2.2 Milk production and
composition . . . . . . . . .

2.3 Blood components . . . . . .

a. Blood urea N concentra-
tion (BUN) . . . . . . .

b. Plasma glucose concen-
tration (PG) . . . . .

Trial 11 1971 . . . . . . . . . . .

1.

Materialsand methods . . . . .

1.1 Rationale and planning . .
1.2 Arrangements and procedures .

Results . . . . . . . . . . . . .

2.1 Feed intakes . . . . . . . .
2.2 Milk production parameters
2.3 Blood parameters . . . . . .

Trial III 1971 . . . . . . . . . . .

1.

Methods and materials. . . . . . .

1.1 Rationale for the experiment
and design . . . . . . . . .
1.2 Arrangements and procedures .

Resu1ts . o 0 Q 0 o 0 0 o o O O O

2.1 Feed intakes . . . . . . . .
2.2 Milk production parameters .
2.3 Blood parameters . . . . . .

Page

106

110
112
113
113
113

113
114
115
116
117
119
119

121
127

127
130
131

131

131
134

136

136
138
141

143

143

143
144

148

148
150
153

iv. Discussion related to production

in 1971 trials . . . . . .

v. Protein production responses
described by regression analyses .

III. Plasma free amino acids (1971

1. Introductory remarks. . .

2. Methods and materials . .

3. Results and discussion. .
BIBLIOGRAPHY . . . . . . . . . . . . .

APPENDIX TELES O O O O O O O O O O 0

vi

trials)

Page

156

162

171

171

171

173

186

208

1.6

LIST OF TABLES

Trial I 1970. Treatment periods. . . . .
Trial 11 1970. Treatment periods . . . . .
Trials 1 and II 1970. Feed composition . .

Trials I and II 1970. Estimated net energy
and crude protein supply by the infusates
relative to total intakes (%) . . . . . . .

Trials 1 and II 1970. Production and
composition of milk and the main milk
constituents by periods . . . . . . . . . .

Trials 1 and II 1970. Combined data. Milk
production parameters during infusion (T)‘
versus control (0) periods. Means and

differences between means (d) . . . . . . .

Trials I and II 1970. Correlation
coefficients between milk, blood
and feed components . . . . . . . . . . . .

Trial 11 1970. Concentration of urea
N and a amino N in blood plasma (period
means and standard errors). . . . . . . . .

Trial 11 1970. Blood components: compari—
sons of mean values for different treatment
periods and bleeding times (mean and

difference between means) . . . . . . . . .

Trial 1 1971. Experimental design and
timing of periods . . . . . . . . . . . . .

vii

Page

58

60

64

66

75

80

83

88

91

108

Table

Page

Trial 1 1971. Substrate infusion rates
derived from pre—trial protein production . . . 110

Trial 1 1971. Consumption of dry matter,
crude protein and estimated net energy
(mean for treatment periods) . . . . . . . . . 120

Trial I 1971. Summary of feed intakes:
mean comparisons between treatment and
control periods . . . . . . . . . . . . . . . . 121

Trial I 1971. Milk production parameters;

treatment and control means, differences

between treatments and controls, and

results of statistiCal analyses . . . . . . . . 122

Trial I 1971. Comparison of production

results for the full 6 day periods and

adjacent controls to the last 3 days of

substrate infusion and closest 3 day

controls . . . . . . . . . . . . . . . . . . . 128

Trial 1 1971. Concentration of urea
nitrogen in blood (BUN) according to
sampling times and infusion treatments . . . . 129

Trial I 1971. Concentrations of glucose
in blood plasma . . . . . . . . . . . . . . . . 132

Trial 11 1971. Experimental design and
timing of periods . . . . . . . . . . . . . . . 133

Trial 11 1971. The levels of infused pro-
tein o o o o o o o 0 Q o o g o . . . . . . . g 135

Trial 11 1971. Summary of feed consumption:

intakes of dry material, crude protein, and
estimated net energy for different treatments

and periods . . . . . . . . . . . . . . . . . 137

Trial 11 1971. Milk production and composi—
tion by treatments (mean t SE) . . . . . . . . 140

Trial 11 1971. Concentrations of blood urea
nitrogen 0 0 0 0 0 I o o o o o o o o o o o o o 1'42

Trial 11 1971. Concentrations of glucose
in blood plasma . . . . . . . . . . . . . . . . 142

viii

Table Page

2.15 Trial III 1971. Experimental design and
timing of periods . . . . . . . . . . . . . . 145

2.16 Trial III 1971. Derivation of protein
qualtities infused, and comparisons to
crude protein (CP) consumed and put out
by the milk . . . . . . . . . . . . . . . . . . 145

2.17 Trial III 1971. Summary of feed intake:
daily consumption of dry matter, crude
protein, NPN and estimated net energy . . . . 149

2.18 Trial III 1971. Milk production para-
meters; summary of comparisons between
treatments 0 o o o o o o o o o o o o o o c Q o 151

2.19 Trial III 1971. Summary of blood urea
nitrogen and plasma glucose concentrations . . 154

2.20 Correlations (r x) between response to
abomasal infusion in milk protein (d=Y) and
protein production in control periods (O=X) . . 163

2.21 Excerpts from multiple regression analysis
of the relationship between protein produc—
tion responses, control production and
amount of casein infused . . . . . . . . . . 167

2.22 Regression equations for estimation of
response to abomasal casein infusion from
control production and amount infused . . . . . 168

3.1 Trial I 1971. Plasma free amino acids;
molar % distribution at different infusion
treatments 0 O O O I O O I I Q 0 O O O I Q 0 C 174

3.2 Trial 11 1971. Plasma free amino acids
concentrations at different treatments . . . . 175

3.3 Trial 11 1971. Plasma free amino acids,
molar % distribution at different
treatments . . . . . . . . . . . . . . . . . . 176

3.4 Trial III 1971. Plasma free amino acids;
concentrations and molar % distribution
during control and treatment. . . . . . . . . . 177

3.5 Trial III 1971. Relationship between the
output of amino acids with milk protein
(Maa) and the content of amino acids in
blood plasma (Paa) . . . . . . . . . . . . . . 183

ix

APPENDIX TABLES

TABLE Page

1.1 Trial I 1970. Average daily intake of feed
during each period . . . . . . . . . . .y. . . 208

1.2 Trial I 1970. Average daily consumption of
crude protein and estimated net energy during
each period; actual values and relative to
NRC standards . . . . . . . . . . . . . . . . . 209

1.3 Trial 11 1970. Average daily intake of feed
during each period . . . . . . . . . . . . . . 210

1.4 Trial 11 1970. Average daily intake of
crude protein and estimated net energy
during each period; actual values and
relative to NRC standards . . . . . . . . . . . 211

1.5 Trial I 1970. Observations in milk produc-
tion parameters . . . . . . . . . . . . . , . . 212

1.6 Trial I 1970. Differences in milk production
parameters between treatment (infusion) and
control periods . . . . . . . . . . . . . . . . 213

1.7 TrialIJ 1970. Milk production parameters;
basic observations . . . . . . . . . . . . . . 214

1.8 Trial 11 1970. Differences between treat—
ment (infusion) and control periods in milk
production parameters . . . . . . . . . . . . . 216

I.9 Trial 11 1970. Nonprotein nitrogen (NPN)
concentration in milk . . . . . . . . . . . . . 217

1.10 Trial 11 1970. Concentration of urea N
in blood plasma, mg/lOOml . . . . . . . . . . . 213

1.11 Trial 11 1970. a amino N in blood plasma,
uM/ml ' 0 O 0 O O O O O I o o o o o o o o o c Q 219

1.12 Trials I and II 1970. Analysis of variance
for milk production parameters . . . . . . . . 220

1.13 Trial 11 1970. Analysis of variance for
blood plasma parameters . . . . . . . . . . . . 221

 

 

 

Table

II.1

II.2

II.3

II.4

11.5

II.6

II.7

II.8

11.9

11.10

11.11

IIOlZ

II.13

II.14

II.15

Trial 1 1971. Feed composition and
estimated net energy values . . . . . . . . .

Trial I 1971. Feed offered and consumed
dry matter, estimated net energy for
lactation and crude protein for each cow

in each period . . . . . . . . . . . . . . .

Trial I 1971. Feed intake in treatment
and control periods averaged . . . . . . .

Trial I 1971. Intakes of estimated net
energy and crude protein relative to
NRC (1971) standards . . . . . . . . .

Trial 1 1971. Observations in milk
production parameters . . . . . . . . . . .

Trial I 1971. Concentrations of urea
nitrogen in blood . . . . . . . . . . . .

Trial 1 1971. Concentrations of glucose
in blood plasma . . . . . . . . . . . . . .

Trial I 1971. Layout of Anova I—l;
Latin square design applied to estimated
treatment responses . . . . . . . . . . .

Trial 1 1971. Layout for comparison of

each infusion treatment to adjacent

CODtI’OlS (Anova 1'2) 0 o o o o o o 0 o o o o 0
Trial 1 1971. Layout for Anova 1—3 . .‘.

Trial I 1971. Statistical analyses for
feed intake parameters . . . . . . . . . . . .

Trial I 1971. Statistical analyses for
milk production parameters; Anova I-l . . . .

Trial I 1971. Statistical analyses for
milk parameters; Anova 1—2 . . . . . . . . . .

Trial 1 1971. Statistical analyses for
milk parameters; Anova 1—3 . . . . . . . . . .

Trial I 1971. Statistical analyses for
blood parameters, Anova 1-3 . . . . . . . . .

xi

Page

222

223

224

225

226

228

229

230

231

232

233

234

235

236

237

Table

II.16

II.17

11.18

II.l9

II.20

II.21

II.22

11.23

II.24

II.25

II.26

II.27

II.28

II.29

II.30

II.31

Trial II 1971. Amounts of feed offered and
consumed, and total dry matter intake . . .

Trial 11 1971. Feed composition and
estimated net energy values . . . . . .

Trial 11 1971. Milk yield and concentra-
tion of milk constituents . . . . . . . .

Trial 11 1971. Milk production parameters:
period observations for each cow and
treatment and time—period means . . . . . .

Trial 11 1971. Concentration of blood
urea nitrogen, mg/lOOml . . . . . . . . . .

Trial 11 1971. Concentration of blood
plasma glucose, mg/lOOml . . . . . . . . . .

Trial 11 1971. Layout of Anova II . . . .

Trial 11 1971. Statistical analysis for
feed intake parameters . . . . . . . . . . .

Trial 11 1971. Statistical analysis for
milk production parameters . . . . . . . . .

Trial 11 1971.
blood parameters

Statistical analysis for

Trial III 1971. Ingredients in
concentrate mixtures . . . . . . . . . . . .

Trial III 1971. Feed composition and
estimated net energy values by periods . . .

Trial III 1971. Amounts of feed offered
and consumed as dry matter, and total
intakes of feed constituents per day . .

Trial III 1971. Intakes of crude protein
and estimated net energy for lactation
relative to NRC (1971) standards . . . . . .

Trial III 1971. Observations in milk
production parameters. . . . . . . . . . .

Trial III 1971. Blood urea N concentration
mg/lOOml . . . . . . . . . . . . . .

xii

Page

238

239

240

241

242

243

244

245

245

246

247

248

249

250

251

253

Table Page

11.32 Trial III 1971. Blood plasma glucose con—
centration, mg/lOOml . . . . . . . . . . . . . 254

11.33 Trial III 1971. Layout of Anova III . . . . . 255
11.34 Trial III 1971. AOV for feed intake . . . . . 256

11.35 Trial III 1971. Statistical analysis for
milk production parameters . . . . . . . . . . 257

11.36 Trial III 1971. Statistical analysis for
blood parameters . . . . . . . . . . . . . . . 258

xiii

LIST OF FIGURES

Figure Page

1. The response in milk protein yield to
abomasal protein infusion as related to
yield in control periods . . . . . . . . . . . . 165

xiv

 

01

of
th

an.

die

A. INTRODUCTION

In view of a rapidly increasing world population
it has been considered a matter of time whether production
of meat and milk based on cereals and high quality plant
proteins can continue as today. Already the proteins are
the most costly part of the feed for livestock.

These circumstances place ruminant nutrition in a
unique position due to the ruminant's ability to utilize
nonprotein nitrogen (NPN) to a far larger extent than
other farm animals. To what extent this relationship can
be drawn upon is a matter of economics for the educated
farmer. But it continues to be a great challenge for
animal scientists to eXpand the knowledge of ruminant
nutrition such that nonprotein nitrogen can be utilized
extensively with enhanced efficiency in overall protein
nutrition.

A most outstanding yet often forgotten feature
of protein nutrition in ruminants is its dualistic nature:
the microbial metabolism in the forestomachs on one hand,
and the metabolism in the remainder of the alimentary tract
and tissues of the animal on the other hand. Although

distinctive in place and character, the events of these

two phases will mutually influence each other. Thus the
fate of the crude protein fed will depend upon digestive
processes as well as the physiological status of the animal.

It is now well accepted that protein metabolism is
very dependent on energy metabolism. This also applies to
the rumen, as utilization of NPN is intimately linked to the
energy source. Thus, the other great asset of ruminant ani-
mals; the ability to thrive on substantial amounts of
cellulose for energy, is also influenced by the nitrogen
metabolism. Extensive utilization of NPN and cellulose,
however, are not totally compatible because highly digestible.
carbohydrates, preferably starch, are necessary for maximal
use of NPN; but bacterial growth on these readily avail-
able nutrients will depress digestion of cellulose. In
any event, there is a limit for how fast microbes in the
rumen can turn over; and this limits the amount for microbial
protein which can be supplied to the lower gut of the host
animal (Hungate 1966).

Some of the feed protein normally passes by the
rumen and supplements the microbial protein, but variable
amounts will be degraded in the rumen. The degradation
compounds can potentially be used for synthetic purposes in
the rumen but a rapid and extensive disintegration of feed
protein to ammonia will easily result in a loss of nitrogen

to the animal. All factors considered, it is hard to

assure that ruminants receive an amino acid mixture which
allows full expression of their capacity for protein syn-
thesis.

Since dairy cows produce large amounts of high
quality proteins, in the form of milk, it is questionable
whether the amino acid supply to the mammary gland is
sufficient for milk protein synthesis; even when cows
are fed a diet which, by conventional standards, is con-
sidered adequate. Apparently a more precise evaluation of
diets for dairy cows with better use of available resources
could be aided by greater knowledge of the cow's demands for
amino acids. Understanding the cow's potential for milk
protein synthesis is particularly important in view of the
high nutritive value of milk proteins.

It was the aim of this thesis work to study pro-
tein nutrition of lactating cows by delivering high
quality protein (casein) directly into the abomasum of
cows fed an apparently adequate diet and measuring

various response criteria.

B. LITERATURE REVIEW

1. Assessment of protein demands
in lactating cows

 

Earlier debates about appropriate terms for expressing
protein need and supply in ruminants reflect the qualitative
as well as quantitative aspects of protein nutrition of
dairy cows (Tyler, 1959). While crude protein (CP = N X
6.25) gradually has been accepted as a more appropriate term
than true proteins it still remains controversial how crude
protein should best be expressed; and statements about
level of protein supplement invariably raise the question
of quality. Variables in feed and physiological status of
the animal evidently should be considered in a discussion
of these relationships. This review, however, will only
briefly deal with the topic of protein quality in lactating
cows and mention the main factors critical in studies of
their protein supply. With reference to the tempering ef-
fect of rumen metabolism on absorbable amino acids, some
recent findings on rumen bypass of protein or amino acids
will be presented. Finally, there will be brief discussions
of nongenetic influence on milk protein content.

1.1 Protein quality: The concept of biological value and

 

essential amino acids in lactating cows.

 

The biological value (BV) of feed protein can, with

some qualifications, be obtained from an N balance

experiment. PrOperly, the term expresses the efficiency
of absorbed proteins in supplying amino acids needed for
the synthesis of body protein, thus taking account of
metabolic losses computed on the basis of truly digested
protein (Maynard & Loosli, 1962). It is now generally
accepted that BV of protein is determined primarily by
its content of essential amino acids (EAA) and, spe—
cifically, the content of that essential amino acid which
is in greatest deficit relative to the animal's require-
ment (Block & Mitchell, 1946). Due to digestive processes
in the rumen it is not valid to specify the BV of a given
feed protein for a ruminant. It is the EV of nitrogen
(N) in a given ration that is nutritionally significant
(McDonald, 1968). .

For a time following the work of Loosli, et_al.
(1949), showing that rumen microorganisms can synthesize
all the essential amino acids, it became a general feeling
that protein quality was relatively unimportant in ruminant

nutrition (Jacobson, et al., 1970; Purser, 1970). In

 

this period McNaught, et a1. (1954) found rumen microbial
protein to be of rather high nutritive value. Data for
amino acid composition of microorganisms presented by

Duncan, et a1. (1953) and Weller (1957) indicated small

 

variations even at different feeding regimens, an impres-

sion confirmed by later investigators (Purser, 1970).

Black, et_gl, (1955,1957) and Downes (1961), however, showed
that ruminant tissue is dependent upon an exogenous supply

of the same amino acids considered essential for other mammals.
According to the definition of Rose (1938), these are the
amino acids which must be supplied by the diet in order to
support the demands for protein anabolism.

Rose, gt_al. (Rose, 1938) singled out the essential
amino acids for the rat and dog by applying a "deletion tech-
nique" in which individual amino acids were successively re—
moved from the complete diet. Because of the comprehensive
microbial synthesis in the rumen, an indirect approach
making use of tracers was particularly valuable for identi-
fication of amino acids essential for ruminant tissues.
Furthermore, the rumen microbes make quantitative deter-
mination of EAA requirement by regular feeding trials a
difficult and hardly relevant task.

By intravenously injecting l4C-labelled glucose and
volatile fatty acids (VFA) to lactating cows Black, gt_al.
(1955, 1957) were able to largely circumvent the metabolism
in the rumen. The amount of 14C incorporated into various
amino acids ofmilk casein revealed two groups: those with
low levels of 14C, which corresponded well with the FAA, and
those with much higher levels which were generally the non-

essential amino acids.

Downes (1961) applied a similar method in a lactating
ewe and confirmed that tyrosine, phenylalanine, leucine,
isoleucine, valine, methionine, lysine, threonine and
histidine all had neglible radioactivity when isolated from
casein. The amino acids mentioned include 8 of 10 con—
sidered essential for the rat. Arginine is evidently not
essential for the sheep or the cow. Tyrosine is not classi—
fied as an essential amino acid from nutritional studies in
rat and dog. However, since it is synthesized in vivo by
the hydroxylation of phenylalanine, which is essential, no
l4C would be expected in tyrosine unless phenylalanine was
also radioactive (Downes, 1961).

This interpretation was confirmed by Black, et_§l,
(1972) who pulse-labelled jugular blood with U—C14-tagged
individual amino acids from casein and found tyrosine gained
10-12% of the original phenylalanine activity. Other BAA
did not label other amino acids, even NEAA remained largely
unlabelled. The NEAA, however, labelled each other in
characteristic pairs, but only for aspartate and glutamate
was interconversion extensive.

Trypt0phan and cystine Were not isolated in the first
experiments with Cl4 labelled precursors, but Marston
(1935) had already stimulated wool growth\by sub—

cutaneous injection of cystine which is present

in large amount in keratine. Other workers have

confirmed this exceptional role of cystine by postrumen
supply of protein and S-amino acids (Reis and Schinckel,
1964) and studies with BSS-cystine (Downes, 3331., 1970).
Land and Virtanen (1959) applied 15N to the feed of

two cows and observed less enrichment in BAA than other
amino acids in milk proteins. When the relative label of
glutamic acid was set to 100, valine was 59, lysine 54,
phenylalanine 50, arginine 41 and histidine 15. When 15N
was used in a cow adapted to purified diet with urea as

N source for 16 months there was more labelling of BAA
than observed earlier. Label introduced by (lsNH4)ZSO4

and 15

N-urea gave similar results. Virtanen (1966) sug-
gests the low label of histidine may indicate a united
supply of milk protein synthesis.

Because tryptophan is destroyed in commonly used
amino acid assays less data is available for this amino
acid, but according to Fenderson and Bergen (1972)
tryptophan is an essential amino acid in ruminants.
Studies by Piana and Piva (1969} according to Virtanen
1971) evidently showed a very low synthetic rate of

tryptOphan in the rumen of sheep when 15

NH4 was used as
a marker.
After years of shifting Opinions, the use of radio—

active tracers showed that milk proteins are largely (over

 

1A3 cited, the source not available.

90%) synthesized in the mammary gland from free amino acids
in the blood (Barry, 1964; Larson and Gillespie, 1957).
Arterio-venous (AV) differences of BAA over the mammary
gland have correlated more closely tx> output in milk
proteins than have the other amino acids (Mepham, 1971).

No single EAA, however, is present in milk proteins
in an extra high proportion. Thus, it is not simple to
identify any one as first limiting for maximum milk pro-
tein synthesis (Thomas and Clapperton, 1972). The most
abundant amino acid in milk proteins is glutamate (Porter,
gt al., 1968); and from experimental evidence, glutamate
has been suggested as a possible candidate for limiting
milk protein synthesis (Halfpenny, g£_al., 1969) although
it is not essential as defined above.

On the other hand, Verbeke and Peeters (1965) con-
cluded that a group of amino acids whose concentrations
in milk proteins showed a close linear relationship to
their AV differences should be considered essential for
the mammary gland. Included in this group was valine,
leucine, isoleucine, threonine, arginine and glutamate.
They did not succeed in determining a reliable AV dif-
ference for histidine. Tyrosine, phenylalanine, methionine
and alanine formed a group with some relation between milk

content and AV difference, but not as closely as the former

10

group. An eXplanation for these differences were not
suggested, but might be the consequence of various extent
of metabolism within the gland.

Using conventional figures for blood flow and milk
protein content Verbeke and Peeters (1965) calculated an
uptake of amino acids 15-25% short of the output by milk
protein. This discrepancy was probably due to a deficiency
in the blood sampling techinque (Mepham, 1971). Mepham
and Linzell (1966) measured simultaneously the AV difference
adn blood flow in goats with considerable attention to pos-
sible sampling errors. They found the uptake of EAA agreed |
closely with output of milk proteins. The uptake of total
amino N was apparently sufficient for the protein synthesis.

Discussing amino acid supply in cows (as well as
other animals), McCarthy, et al., (1970), suggested that
a distinction should be made between an amino acid de—
ficiency and what might be called an amino acid insuffi-
ciency. That is, a cow may be free of any deficiency
symptoms yet milk production may be limited by available
substrate rather than enzymatic reactions in the secretory
tissue (McCarthy, et al., 1970).

1.2 Factors influencing response to protein supply of

 

lactating cows.

 

Body protein reserves may buffer the effect of

various feed proteins in short term experiments (Reid,

ll

gt_§l., 1966, 1967) and prevent response to diet over a

wide range (lo-20%) of CP in the ration (Jacobson, e£_al.,
1970). Coppock, gt_al.,(l968) considered protein reserves quan-
titatively inferior to energy reserves, but Paquay, e£;al.,
(1972) found larger capacities for protein storing than

formerly thought possible; more than 15kg protein could

be lost and regained in cows around 600kg body weight.

Digestibility has been criticized as a misleading

 

description of protein availability in ruminants (Chalmers,
gt_al., 1954; Thomas, 1966) because extensive breakdown

to NH3 in the rumen may lead to large N losses in the
urine. Low N digestibility, however, as for heat damaged
forage (Thomas, gt_al., 1972) will be detrimental for
utilization of the whole ration. While the reliability of
N balances in ruminants have also been criticized (Agri-
cultural Research Council (ARC) 1965) these are neverthe—
less used as basis for estimation of the protein require-
ments expressed in feeding standards (ARC, 1965).

The level of energy intake, as well as its ratio

 

to protein, will generally influence protein requirements
(Reid, gt_al., 1966; Balch, 1967; Thomas,l966, 1971).
Protein needs can be adequately ascertained only after
energy requirements are met (Perkins, 1957), and several

experiments have shown that protein requirement

12

was minimal at a high energy allowance (Thomas, 1971).
Jacobson, et al. (1970) maintained that high levels of
protein encourage greater voluntary feed intake; and
while milk production was related more closely to net
energy than protein intake, the values for protein require-
ments (based on empirical results) have been confounded
with feed consumption. Balch (1967) advocated that dif-
ferent levels and qualities of protein should be tested
at different levels of energy. Gordon and Forbes (1970)
registered a greater response in milk yield to increased
dietary protein at an energy level 20% above standards
than at an energy level 20% below standards.

The_porportion of non protein nitrogen (NPN) in

 

the feed that will not depress milk yields evidently varies
with the protein need of the cow, which depends pri-

marily upon milk production (Huber, et al., 1967, 1972;
Conrad and Hibbs, 1968). Maximum levels of urea in lactating
cows therefore should be expressed on an absolute basis
rather than as a prOportion of the total dietary nitrogen
(Huber, et al., 1967; Conrad and Hibbs, 1968). Special
preparations can allow for higher than usual levels of

urea (Meyer, et al., 1967; Helmer, et al., 1970a,b; Con—

rad and Hibbs, 1968). These are based on a favourable

l3

timing of the release of NH3 and energy for protein

synthesis in the rumen (Chalupa, 1970), but the energy

source is still crucial for extensive NPN utilization
(Chalupa, 1970; Virtanen, 1966). A noticeable effect on
productivity and feed efficiency of source of NPN, apparently
through interaction with other feed constituents, has re—
sulted from NH3 additives to corn silage (Huber and Santana,

1972; Henderson, et al., 1972).

2. The role of rumen metabolism
in amino acid supply to
the host animal

 

 

 

In order to find the amount of amino acids avail-
able for absorption it is necessary to know: (1) the
flow rate of digesta from the rumen; and (2) the amino
acid content of the digesta. The latter would depend
upon the amount of microbial protein produced and its
quality as well as the amount of feed protein passing un—
altered by the rumen. Knowledge about the factors influ-
encing these entities apparently could be valuable for a
systematic manipulation of ruminant nutrition (Hutton and
Annison, 1972). Still, the importance of maintaining a
viable microbial population in order to obatin effective
digestion of forages should not be overlooked (Hutton

and Annison, 1972).

14

2.1 The fate of protein and N in the rumen.

 

The cycling of N between rumen and the tissues as
described by McDonald (1948) makes it difficult to assess
in quantitative terms the contribution made by dietary
protein versus microbial protein to the amino acid mixture
of the digesta in the small intestine (Ellinger and
Phillipson, 1964). When the dietary N (CP) level is low,
the recycling of N by urea to the rumen will lead to
conservation of N for the animal. However, when high N
levels are fed, the rumen NH3 and the blood urea concen—
trations are elevated, and increasing amounts of urea are
excreted by the urine. Ruminants thus appear to be less
efficient than mono-gastric animals in utilizing high
protein diets (McDonald, 1968). Weston and Hogan (1967)
found that the amount of microbial protein passing to the
lower gut in wethers was 8.8 and 8.1g per day when the
feed contained about 20 and 89 of CP. Clarke, et al.
(1966) reported similar results.

While it is well established that hydrolysis of
urea in the rumen usually proceeds at a faster rate than
NH3 assimilation into microbial protein, rumen bacterial

growth can also be limited by a low availability of NH -N;

3

thus, replacement of dietary protein with urea may in cer-

tain instances increase microbial growth (Allison, 1970).

15

The concentration of NH3 in the rumen critical for bacterial

growth has not been clearly defined, but optimal NH con-

3
centration will probably vary with shifts in microbial
pOpulations and growth rates (Allison (1970). Chalmers
(1971) implied that when grass or grass products are used
for feeding of dairy cows, there is hardly ever a limit

in bacterial growth due to low NH concentrations in the

3
rumen; thus, energy is the main limiting factor.

Waldo (1968) stated that the concentration of rumen
N fractions,size of the rumen pool, or N turnover rates,
have mathematical relationship to each other; and although
they are frequently measured and discussed as distinct
entities, it must not be ignored that a change in one of
these parameters is frequently a cause or a result of a
change in another. The turnover rates of N in the rumen,
however, are considerably below the growth potential of
most bacteria (Hungate, 1966). The rate of N removal in
the intestinal tract is influenced by the ratios of
particulate and soluble N, and the relative rates of
particle passage and water passage (Waldo, 1968).

Because bacteria and protozoa (Waldo, 1968) have different

retention times, it can be surmized that changes in the

16

relative biomass of the two types of microbes will have
impact on the amino acid supply to the animal beyond the
differences in protein quality for the two fractions.

Naturally, the pr0portion of feed protein reaching
the lower gut will be the inverse of the extent of NH3
production inthe rumen. Thus, McDonald (1952, 1954) fed
purified proteins to sheep and showed that a large part
(about 40%) of the highly insoluble protein zein passed
into the abomasum unchanged. Soluble casein, however, was
nearly all (about 96%) degraded and replaced by microbial
protein (McDonald and Hall, 1957). Feeding casein was
associated with far higher levels of NH3 in the rumen
than was observed for zein. Chalmers, gt_gl. (1954) con-
firmed these relationships by showing less NH3 formation
and less degradation of casein after making it less
soluble by treatment with sodium hydroxide and heating.
Herring meal also resulted in lower NH3 concentrations in
the rumen and higher N retention than did casein in grow-
ing lambs (Chalmers and Synge, 1954) and in lactating
goats and cows (Chalmers and Marshall, 1964).

Addition of starch or cereal meal reduced rumen
NH levels in animals fed highly-soluble groundnut meal

3
(McDonald, 1954; Annison, et al., 1954). Therefore,

 

differences in NH3 formation from different proteins need

not be of great practical importance when liberal

17

amounts of grain are fed, as with fattening of beef cattle
(Annison, et al., 1954).

Chalmers (1971) stated that no bench—test reflects
the rate of dissolution of a solid protein in the rumen.
Others reviewing work in this area, however, seem to agree
that solubility of feed protein is an important factor in
the rate of its breakdown and consequent NH3 accumulation
in the rumen (Waldo, 1968; Tillmand and Sidu, 1969; Smith,
1969). While Little, et al. (1963) found a poor associa-
tion in an artificial rumen system between microbial attack
on proteins and protein solubility in water or diluted
NaOH; a fairly close relationship prevailed between the
in vitro NH3 produciton and protein solubility in rumen
fluid at pH 7.

Proteins rapidly converted to NH3 also appeared
readily available as N sources for in vitro cellulose
digestion. Even though heated soybean meal was less
soluble than untreated meal, there was no difference in
growth rate of lambs on these protein supplements. On
the other hand, insufficient N-release for potential
protein synthesis in the rumen obviously hampered the
growth of lambs fed corn gluten meal. Adding urea to a
corn gluten diet markedly improved weight gains while
supplementing with lysine and methionine had little ef—
fect although corn gluten meal is low in lysine (Little,

et al., 1963).

18

A relatively low solubility of corn gluten meal
in rumen fluid was reported also by Chalupa, gt_al. (1963)
but they found isolated soy protein much less soluble than
did Little, et al.(l963); (7% vs. 63%). This discrepancy
might be related to processing methods, but Chalupa, e£_§l.
(1963) did not disclose the pH of the system they used
for testing.

Significant effects of rumen fluid acidity on
protein solubility was demonstrated in vitro by Isaacs and
Owen (1972), suggesting that ruminal pH influences the
bypass of proteins. They showed that within a pH range
of 5 to 7, the higher pH favoured solubility of casein
and soybean meal while corn protein appeared relatively
more soluble at the lower pH. The authors (Isaacs and
Owen, 1972) prOposed solubility curves for calculating
nitrogen availability to rumen microbes. Thus, for as-
sumed rates of rumen turnover it might be possible to
estimate protein degradation, and by difference, assess
the passage of intact protein through the rumen.

Protein degradation rates in vitro indicated to
Isaacs and Owen (1972) that rumen proteolytic enzymes are
saturated under common feeding conditions, although ruminal
proteolytic rate may decrease with time after feeding.

Since the concentration of free amino acids in

rumen fluid is quite low (McDonald, 1952; Blackburn, 1965),

 

the
dew
This
VarJ
in \
fast

of 6

capa
fluic
simii
0n th

three

(1955‘

19

the importance of free amino acids in ruminal microbial
metabolism is hard to assess. By concentrating rumen

fluid from sheep and removing its NH Lewis (1955) ob—

3,
served fifferent concentrations of individual amino acids.
These findings per se would not prove amino acids to be
intermediates in protein cataoolism; but the extent of
NH3 production following the introduction of amino acids
into rumen indicated hydrolysis is an intermediate step
that probably regulates the rate of protein breakdown
(Lewis, 1955).

Far lower rates of NH3 production occurred from
amino acids added to washed cell suspensions compared to
those in vivo (Lewis, 1955). Cells from casein-fed sheep
deaminated protein more vigorously than from hay-fed sheep.
This ranking among the diets was also shown in vivo.
Various rates for the different amino acids prevailed
in vitro as well as in vivo. Aspartate was attacked
fastest in both solutions with an Optimum in vitro pH
of 6.5 (Lewis, 1955).

Lewis and Emery (1962a) found the proteolytic
capacity of washed-cell suspensions infereior to rumen
fluid in vitro; but largely the same sequence of dis-
similation rates among individual amino acids persisted.
On the basis of resistance, the amino acids fell into

three groups; largely confirming the results of Lewis

(1955). Generally, d- and l-enantiomorphs differed in

20

catabolic resistance, but not so for serine and tryptOphan.
The production of NH3 resembled the rate of disappearance
of amino acids determined by chromatography (Lewis and
Emery, 1962a). The evolvement of NH3 from amino

acids in vitro fell with the pH of the medium from a
maximum at 6.5 to neglible release at 4.5 (Lewis and Emery,
1962b). Deamination rates in vivo (Lewis and Emery, 1962c)
paralleled the in vitro results. The highest NH3 level in
the rumen occurred 6 hours after arginine administration,
whereas tryptophan and lysine yielded smaller, yet de-
tectable, amounts of NH3. Lysine concentrations decreased
to 50% in 6 hours, but ruminal tryptOphan was not reduced
until 8 to 10 hours after it was supplied.

While arginine and lysine addition to the rumen
resulted in elevated plasma levels of severalamino acids
within one hour, plasma lysine itself did not increase
11nti1 four hours following the administration (Lewis
and Emery, 1962b). Conversion of arginine to ornithine
in the rumen yielded equal concentrations of these amino
acids 6 hours after introducing arginine (Lewis and Emery,
1962c).

Arginase activity in the rumen wall has been demon—
strated in vitro and in vivo (Harmeyer, gt_al., 1968).
Increased ornithine concentrations in ruminal venous

blood after arterial arginine injection was, however, not

21

detectable unless ample amount of starch was present in

the rumen (Harmeyer, gt_al., 1968). This observation is

in line with marked decrease of urea recycled to the rumen

of sheep deprived of easily fermentable carbohydrate (Houpt,
1959). Harmeyer, gt_al. (1968) suggested combined activities
of the arginase and urease associated with rumen mucosa par—
ticipate in regulating recycling of urea N to the rumen.

Actual transport of amino acids across rumen
epithelium has been demonstrated in steers and goats
(Cook, gt_gl., 1965), but Blackburn (1965) contends that
the low ruminal amino acid levels observed on continuous
feeding make it unlikely that direct absorption should
affect the nutrition of the animal. Even casein hydrolysate
placed into sheep rumen failed to increase‘ the a
amino N content in portal or arterial blood (Annison, 1956).
But Cook, gt_al.,(l965) state that amino N is not sufficiently
sensitive to reflect changes in ruminal absorption of amino
acids.

Cook, gt_al.,(l965) found added amino acids to be
quite stable in the rumen fluid for one to two hours. There-
after, concentrations fell which were accompanied by an in-
crease in the NH3 level. Emery (1971) found the mean half
life for 509 doses ofcnsmethionine to be 2.4 hours in the
rumen of mature cows, with no difference in persistency

between dl-methionine and other forms of the amino acid.

22

Thus, discrepancies in amino acid resistance to ruminal
destruction may largely beaaconsequence of differing con-
centrations. Large experimental dosages evidently
saturate catabolic pathways; for amino acids are rapdily
degraded at physiological concentrations (Emery, 1971).
Attempts to protect rumen amino acids with anti-
biotics carries potential hazards to vital fermentations;
but is, nevertheless, a challenging approach. Penicillin,
examined for this purpose in vitro, was not effective in
concentrations that prevented bloat, but levels ten-fold
higher markedly decreased amino acid disintegration (Lewis
and Emery, 1962a). Retarded ruminal deamination of methionine
and lysine has recently been reported after addition of
oxytetracycline hydrochloride (OTC), without noticeable
effects on the microbial activity (Schelling, gt_al., 1972).

2.2 Quantification of microbial synthesis in the rumen.

 

The extent to which food can be converted into
cell material is limited by anaerobiosis, and rarely ex—
ceeds 20% under such conditions (Hungate, 1966; Walker,
1965). Thus, as degradation rate of feed protein exceeds
the rate of re-synthesis in the rumen, its anaerobic
state imposes a thermodynamic limit on the extent of host
protein synthesis (Hungate, 1965, 1966).

In vitro cell yields have been related to ATP pro—

duced by pure cultures of bacteria (Bauchop and Elsden,

23

1960) and extended to in vivo conditions (Walker, 1965).
From these findings, Hutton and Annison (1972) concluded
that about 20g bacterial CP was synthesized per 1009 DM
digested.

Based on Hungate's (1966) anaerobic data, Purser
(1960) calculated that 18.3g digestible microbial protein
could be synthesized per Mcal feed digested. Chalupa
(1972) applied this value to commonly accepted figures for
feed intake and estimated that microbial protein can sup—
port maintenance and a daily production of 10kg milk.
However, research data indicate that the rate of microbial
synthesis might be higher than the theoretical one used
by Chalupa (Purser, 1970). This would allow support of
a larger productioncflfmilk on microbial protein alone.
The work of Virtanen (1966) also indicates a higher po-
tential for microbial protein synthesis than theoretical
calculations held possible.

Incubating rumen contents in vitro, Al-Rabbat,
gt_gl. (1971b) confirmed there is good agreement between
microbial growth and fermentation. However, microbial

protein syntehsis estimated by 15

N incorporation was
higher than calculated from VFA production, using 2 ATP/
mole VFA; but the rate of VFA production might have in-
fluenced the result. Tracer data showed 9.29 microbial

cells were synthesized from.®u1 per 100g digestible

3

C.

‘5

“9

ti!

Si:

ny]

Est

PEI

 

24

organic matter (DOM) fed (Al-Rabbat, et al., 1971a). This
was 61% of total cell production, the remaining N presumably
coming from amino acids and peptides. Similar figures have
been obtained by continuous ruminal infusion of 15NH4-salts
(Mathison and Milligan, 1971; Pilgrim et al., 1970) or
lsN-urea (Nolan and Leng, 1972); but recycling of NH3 may

obscure estimates by this approach (Mathison and Milligan,

1971).

Bucholtz (1972) measured cell synthesis from in-
corporation of 33P into phospholipids based on specified
properties of polar lipids in microbes. By frequently
sampling the rumen of sheep fed at 9 hour intervals com—
bined with in vitro incubations, microbial protein synthesis
was estimated at 269/1009 organic matter (OM) digested.

358 as a marker is based on its

Utilization of
incorporation in sulfur-containing amino acids (Walker and
Nader, 1968). In a study with lactating cows, Conrad,
et al. (1967a,b) combined 3SS-sulfide and fish mealsas
markers after finding 91% of the fish meal withstood
degradation in the rumen. Fish meal is rich in methionine,
and apparently should serve as an indicator of the frac-
tion of total rumen protein of feed origin. The high re-
sistance of fish meal to ruminal degradation, based on
nylon bag incubations, might have biased upwards the

estimates of total methionine synthesis; which were 1.59

per kg feed (Conrad, et al., 1967a,b). In the same

25

laboratory, Mugerwa (1969) found daily protein pynthesis
in rumen of a cow on a 70% NPN diet to be about 729 per
kg DM digested, regardless of whether cellulose or starch
was the main carbohydrate source.

Little NH3 is found in duodenal contents; and more
than 80% of the non—ammonia N has been accounted for as
amino acids (Clarke, gt_§l., 1966; Weston and Hogan, 1970).
The remaining is presumably found in the nucleic acids
(Ellis and Pfander, 1965; Weston and Hogan, 1970). Be-
cause nucleic acids entering rumen are not rapidly de—
graded, their concentration in digestsa can also estimate
the amount of microbial protein (Smith, 1969). Nucleic
acid N accounts for 14-19% of microbial N, mostly RNA
(Allison, 1970); and since RNA.associates directly with
protein sythesis and DNA levels vary considerably, it
appears that the RNA fraction is a better marker for micro—
bial cell growth (Smith, 1969).

Because a-e diamino pimelic acid (DAPA) is unique
to bacteria it also has been used to estimate bacterial
protein (Weller, gt_§l., 1958, 1962; Hogan and Weston,
1970) Hutton, gt_§l., 1971); although Synge (1953) showed
that DAPA/total N varied considerably between strains
of ruman bacteria. But Weller, gt_al. (1958) found the
ratio of DAPA/N on a fixed dietary regimen was quite con-

stant. The ratio of DAPA to non—ammonia N in digesta

26

leaving sheep's stomach, however, tended to decrease as the
crude protein in the diet increased (Hogan and Weston,
1970). This marker showed bacterial protein synthesized
corresponded to 3.79 N/lOOg OM apparently digested in

the stomach (Hogan and Weston, 1970) .

A decrease in the amount of DAPA was generally accom—
panied by decreased VFA production (Hogan and Weston, 1970);
another demonstration of the quantitative relation between
energy transactions and bacterial growth in the rumen.

The flow of total and bacterial protein, estimated by DAPA,
also has been found positively correlated with the molar %
of propionic acid in the rumen (Jackson, et al., 1971;
Ishague, et al., 1971) and circumstantial evidence sug-
gests this relationship may have more general applicability
(Thomas and Clapperton, 1972).

By summarizing a multitude of metabolic data in a
computer simulation system, Baldwin, et al. (1970) arrived
at 12 to 189 dry cells produced per 1009 DOM fed, con—
sistent with many direct experimental results.

Hutton, et al. (1971) stressed that an accurate
evaluation of the animals N economy requires information
on the magnitude of protozoal and endogenous N, as well
as bacterial N in the digesta. The ratio between protozoal
and bacterial N varies greatly with dietary conditions

from 1:10 on poor hay to 1:4 on lucerne (Weller, et al.,

27

1962). Values of 1:2.5 have been reported in grain-fed
animals (Hungate, 1966), and absence of protozoa was shown
on semipurified diets devoid of true proteins (Virtanen,
1966).

2.3 Quality of protein in the digesta.

 

Most researchers agree that 50 to 80% of dietary
N is converted to microbial N by the time the digesta
reaches the small intestine (Smith, 1969). Nevertheless,
quantity and quality of crude protein, as well as the over-
all ration markedly influence absorbable amino acid. This
point was confirmed in several studies with zein which largely
passes unaltered through rumen (McDonald, 1952, 1954; Ely,
et al., 1967; Amos, et al., 1971; Little, et al., 1968),
and is not well digested in the intestine either (Little
and Mitchell, 1967). Moreover, N-retention was lower
after zein administration per abomasum than per 03, while
the opposite was observed for casein, soybean and gelatin
(Little and Mitchell, 1967). Feeding of these proteins
yielded similar concentrations of amino acids in abomasal
hydrolysates bUt EAA in animals fed zein were quite
variable (Little, et al., 1968).

While the amino acid patterns for mixed abomasal
protein were similar on soybean meal and urea feeding
(Potter, et al., 1969), more of the total N in digesta was

present as protein when steers received soybean. Total N

28

was also higher on soybean than urea. This strengthened
the impression that the quantity of amino acids reaching
the lower gut, rather than the amino acid pattern, limits
performance on high NPN rations (Potter, et al., 1969).
However, adaptation to urea in lambs (Webb, et al., 1972)
increased total abomasal N from values lower than soy—
bean after 10 days feeding to higher levels after 20 days,
regardless of CP content (9 to 20%) inthe diet. Others
have shown a higher percent of the consumed N passing out
of rumen on low CP diets than high CP diets (Clarke, et al.,
1966; Hume, et al., 1970). Thus, the quantities of amino
acids passing out of the stomach may differ substantially
from those consumed, but differences between diets become
smaller, as one samples farther down the digestive tract
(Clarke, et al., 1966).

Confirming findings in sheep, Hale and Jacobson
(1972) reported the N flow through the abomasum of cows was
positively correlated with DM intakes, but recovery of
dietary protein in relation to level of feed consumed
was not reported. Because feed intakes in high yielding dairy
cows are higher than in other ruminants, digestive ob-
servations in other classes of livestock will not
describe ruminal protein bypass in high producing cows.

Amino acid balances in lactating cows combined with

observations of digesta in ewes on similar rations (Bigwood,

29

1964) revealed that ruminal synthesis was highest for
lysine, followed by leucine, which is particularly im—
portant when considering milk protein composition.
Methionine and phenylalanine were not increased in
digesta compared to the feed, but the adequacy of EAA in
digesta differed with the rations (Bigwood, 1964).

In recent studies with sheep the amount of individual
amino acids absorbed showed a high positive correlation with
the amounts entering the small intestine. Both parameters
were influenced by physical form and level of forage intake,
'(Coelbo-da Silva, et al., l972a,b) . Microbial N/total N in
digesta decreased with level of feeding.

Studies involving portal blood flow in wethers
indicated that with some exceptions amino acids were
absorbed in a ratio similar to their occurrence in rumen
bacterial protein (Hume, 1971b). These results apparently
contradict older ones (Clarke, et al., 1966) which led
Armstrong and Prescott (1971) to contend that the amino
acid content of duodenal digesta is not a satisfactory
indicator of absorbable amino acids.

Microbial protein quality cannot be assessed in
the ruminant animal under normal feeding conditions;
therefore, laboratory tests have been sought. Although
informative, such investigations can only approximate the

situation in well-fed ruminants (Smith, 1969).

30

McNaught, et a1. (1954) found the true digestibility in
rats was 75% for isolated bacteria and 90% for protozoa.
These coefficients have been confirmed by many other workers
(Purser, 1970), but values as low as 55% have been re-
ported for digestibility of bacteria (Hatfield, 1970).
Bergen, et al. (1967) found wide variations between
strains of bacteria grown in pure cultures. Changes in
microflora therefore may influence amino acids available
to the host. Likewise, low concentrations of protozoa
will adversely affect microbial protein quality (Klop-
fenstein, et al., 1965), but such impacts on amino acid
availability is not easily separated from digestion
in the rumen (Smith, 1969). Larger losses of fecal N in
ruminants than simple stomached animals, particularly at
low N intakes, have been related to the low digestibility
of bacteria (Smith, 1969). Their protein appears protected
by cell walls (Hoogenraad and Hird, 1970); which is also
responsible for the low digestibility of nucleic acids
(Smith, 1969).

Although microbial protein syntehsis leads to N
looses in the animal (Hatfield, 1970), this should not
be considered a serious problem in N economy if a cheap NPN
source supplies the NH3 for bacterial growth (Hungate,
1966; Purser, 1970). Likewise, the merits of conversion of
feed protein to microbial protein must be considered on
basis of feed protein quality in terms of absorbable amino

acids (Armstrong and Prescott, 1971).

31

Reviewing the topic of amino acids in protein of
ruminal microbiota, Purser (1970) concluded that the bulk
composition shows such uniformity that variations:h1animal
performance cannot be explained on this basis. Excellent
agreement was shown (Purser, 1970) between workers in
different countries, between strains of bacteria, and be-
tween microbial protein obtained under diverse dietary
conditons. Also bacterial and protozoal protein were
similar in this amino acid content, although protozoa
mere higher in lysine, leucine and phenylalanine (Purser,
1970).

The similarity in amino acid composition of bac-
terial and protozoa proteins suggests an equal BV. Reed,
et al. (1949) found BV for both types of microbes in
sheep to be slightly below 80 when fed to rats; and
McNaught, et al.,(l954) found BV 81 and 80 for bacterial
and protozoal proteins, respectively. These values
have been confirmed by others (Purser, 1970). Due to
higher digestibility, the protozoal protein will be of
higher NPU value (BVxN digestibility). Enzymatic digestion
in vitro (Bergen, et al., 1967) released from 2.5 to 52.6%
of EAA in protein of different strains of bacteria. Com—
pared to amino acids released from e99 protein the quality

of bacterial proteins ranged from 37 to 80% (NPUenz).

32

The data of Clarke, et a1. (1966) show higher
absorption of EAA than remaining amino acids in digesta.
These differences might reflect selective absorption
but could also be due to type of protein presented for
digestion (Abidi, et al., 1967; Purser, 1970).

3. Postrumen supply of
proteins and amino acids,

and rumen bypass through
protective treatments

 

 

 

 

3.1 Studies in sheep and growing cattle.

 

a. Effects of chemically prepared proteins.--Because

 

of differences in acidity between the rumen and abomasum,
Ferguson, et a1 . (1967) treated casein with formaldehyde
in order to channel it past the rumen for digestion in
the abomasum. A previous in vitro test showed treated
casein was virtually insoluble at pH 6 and highly pro-

tected against breakdown to NH during in vitro incubation

3
with rumen contents. Daily addition of 609 of formaldehyde
treated casein into a sheep rumen did not change ruminal
NH3 concentration, whereas similar amounts of untreated
casein resulted in a substantial increase. While formal-
dehyde thus had rendered casein resistent to ruminal
metabolism, the protein was still 80% digestible; and

good availability of amino acids from this supplement

was evident by more wool growth than controls fed un-

treated casein (Ferguson, et al., 1967).

33

Reis and Tunks (1969) confirmed the positive effects
of rumen bypass when sheep received threated or untreated
casein in the diet or casein infused into the abomasum
treated casein and casein per abomasum stimulated wool
growth equally well and far better than untreated casein in
the feed. Formaldehyde-treated casein in this trial was
90% digestible compared to 98 and 96% for untreated casein
per abomasum or per 03, respectively.

Sampling of the small intestine revealed that lambs
on a diet with 10% formalinized-casein digested 60% more
protein and 50% more starch in the lower gut than did lambs
on untreated casein (Faichney and Weston, 1971). Accordingly,
less organic material was digested in the rumen when casein
was treated with formaldehyde, and this diet provided 249
DCP to the intestine per 1009 DOM compared to 159 on the
casein diet. Similarly, a larger proportion of formaldehyde—
treated than untreated groundnut meal was digested in the
intestine (Miller, 1972). However, this difference was
observed for all components of the meal.

While 50% of soybean meal N could be accounted for
in the duodenum at a low level of feed intake, the recovery
was 80% after formaldehyde treatement (McLaughlin, et al.,
1972). Doubling the level of soybean meal increased re-
covery to 65% for untreated and 100% for formaldehyde pro-

tected protein.

34

An extensive digestion study involving formalinized
casein by Macrae, gt_al., (1972) provided quantitative data
on amino acid absorption from treated versus untreated casein
added to a basic grass diet. Twice as much of the amino acids
in treated casein passed into the small intestine and net
retention of supplementary N was increased by the formalde-
hyde treatment.

A corollary to these results are the findings of
Faichney and Weston (1971) that 9 amino N and insulin con-
centrations of blood plasma were increased and urea con-
centration was decreased in lambs on formaldehyde treated
casein compared to untreated controls. An enhanced flow of
protein into the intestine acted as a trigger for hormonal
mechanisms and might explain the depressed flow rate of
digesta in lambs on formalinized casein. Altered endocrine
balance might also partially explain differences in blood
levels of metabolites in lambs on treated versus nontreated
casein.

Downes, et_§l., (1970), however, demonstrated with
blood proteins labelled by 358 that the extent of formalin
treatment decisively influences the digestability of the
processed proteins. While wool growth involves only protein
with a very low demand for extra feed energy, body growth
cannot be stimulated by an elevated protein to energy ratio
beyond certain limits, which depends upon the protein quality

and physiological stage of the animal. Ruminants, after an

35

early age, will generally not respond to an excess of 189
absorbed amino acids per 1009 DOM. Any common ration, even
when low in N, will usually provide this amino acid to energy
ratio (CSIRO)2 (1971). Assuming a well-balanced amino acid
mixture, it is therefore not likely that addition of pro-
tected protein will produce the same relative increases in
weight gains as in wool growth.

Faichney (1971), however, found that lambs on a diet
with 10% formalinized casein gained significantly more than
lambs on similar diet with untreated casein (165.59 versus
154g/day); but growth of wool was not different in these
lambs. An experiment with calves (Faichney and Lloyd Davis,
1972) demonstrated that formaldehyde-treated peanut meal was
no better than untreated meal when fed at a dietary CP of
20%, while slightly higher growth rate and feed efficiencies
were observed in calves fed treated than untreated meal when
the diets contained 13% CP. The relatively small response
to protected peanut meal (Faichney and Lloyd Davis, 1972)
might be explained by the low BV for peanut meal and the
fact that it provided only 1/4 of the total protein in the
diet.

Nimrick, et_al., (1972) compared aldehyde-treated
fishmeal, rich in S-containing amino acids, and soybean

meal, for lambs. No metabolic difference was observed

 

2Commonwealth Scientific and Industrial Research
Organization, an Australian publication.

36

between the untreated proteins; but although treatment
lowered the digestibility, N retention was greater for the
treated fishmeal. A feedlot trial showed that treatment of
both protein sources improved growth rate and feed effi-
ciency in lambs fed ad libitum (Nimrick, et_al., 1972). The
authors point out that less response to treated protein
might be expected at ad libitum feeding than at restricted
feeding. Nevertheless, an improved pattern of absorbed
amino acids due to protected protein would result in higher
feed efficiencies.

Contrary to the preceding reports, Satter, g£_al.,
(1970) found formaldehyde-treated soybean meal inferior to
untreated meal or urea in promoting tissue and wool growth
in lambs. It may be speculated that too severe denatura-
tion by an overdose of formaldehyde caused the poor results.
The rate of treatment and the proportion of supplement in
the diet was not given in the abstract of this work.

Peter, et_al., (1971) found treatment with formalde-
hyde, glyoxal, and glutaraldehyde effectively protected
soybean meal as evidenced by increased N balances in lambs.
Nishimuta, gt_al., (1973) treated soybean meal with 19
formaldehyde per 1009 air-dry meal, and showed depressed
ruminal and postruminal digestion in lambs as indicated by

a lowered N retention. On the other hand, heat-treated

soybean meal increased N retention but depressed cellulose
digestibility. Tannic acid treatment (9% w/w) did not alter

digestibility or N retention compared to controls, but heat,

37

formaldehyde and tannic acid all increased the fraction of
digested N that was retained. Whereas total amino acids in
plasma did not differ compared to controls, all treatments
shifted the molar ratios to less glycine and alanine and
more of leucine, isoleucine, lysine and phenylalanine. Reis
and Tunks (1969) likewise observed lower glycine and higher
branched-chain amino acids levels in plasma of sheep fed
formalinized casein or infused abomasally with casein com-
pared to feeding the unaltered source.

Isaachs and Owens (1972) found casein treated with
1.2% (w/w) formaldehyde insoluble between pH 5 and 7 while
solubilities of unprotected casein and other proteins were
markedly influenced by pH. Insolubilization was suggested
as the way that formaldehyde prevents ruminal degradation.
Presumably, the effect of formaldehyde on digestion lies in
formation of methylene bridges or other cross-linkages
between chains of the proteins (Walker, 1964). For ruminant
nutrition the key feature of formalin treatment is the
stability of the formaldehyde-amino acid complex under close
to neutral conditions in the rumen; yet susceptibility to a
decreased pH and hydrolysing enzymes in the lower gut (Mills,
gt_al., 1972).

Prolonged time intervals between treatment of casein
with formaldehyde and feeding was found to increase the

14

Proportion 0f C of formaldehyde recovered in feces (Mills,

et al., 1972). Storing apparently rendered more 14C-

formaldehyde irreversibly linked to the protein such that

 

the COT

 

tract.
equal 1
to a f
Large 1
metabo
methan
degraé

1972).

goats
very

catin.
Aroum

passi:

313595
(1968M
diets
went L
infusi
feed j
the 15
of ur@
amino

LYSin‘]

 

 

38

the complex could not be degraded within the alimentary
tract. The actual preparation under study consisted of
equal parts of casein and safflower oil sprayed with formalin
to a final formaldehyde concentration of 1.5% by weight.
Large proportions (GO-80%) of ingested formaldehyde was
metabolized to CO2 and CH4. The appearance of 14C in
methane would imply that a portion of the formaldehyde was
degraded by methanogenic bacteria in the rumen (Mills, gt_al.,
1972).

Mills, gt_al., (1972) gave their feed to sheep and
goats for several weeks before the tracer studies, and a
very low content of 14C was found in tissue and milk; indi—
cating neglible break down of formaldehyde in the rumen.
Around 5% of 14C appeared in urine regardless of the amount
passing in the feces (11 to 27%).

b. Extraruminal protein or amino acid supply and

plasma amino acid patterns.--When Schelling and Hatfield

 

(1968) infused casein into abomasum of lambs on purified
diets containing urea as the sole N source, the feed intake
went up, and N retention was improved. Abomasal casein
infusion also resulted in higher N retention at controlled
feed intake; suggesting an inadequate amino acid supply in
the lambs on this purified diet. Isonitrogenous infusions
of urea, acid hydrolysed casein or a mixture of essential
amino acids gave lower N retention than casein infusion.

Lysine and glutamate infusion improved N retention to the

39

same extent as a mixture containing arginine, histidine,
lysine, phenylalanine and methionine. But neither phenyla-
lanine nor methionine increased the N retention; and the
absence of an effect from methionine contradicts other
studies involving this amino acid (Schelling and Hatfield,
1969).

Postruminal urea infusion in order to achieve isoni-
trogenous treatments in this type of experiments (Schelling
and Hatfield, 1969; Nimrick, et_al., l970a,b) can be criti-
cized on the basis of adversely affecting the motility of
the gut and the absorptive processes (Visek, 1966).

While Little and Mitchell (1968) found steers
retained more N of casein,soybean and gelatin infused into
abomasum than fed, digestibilities remained the same.
Fujihara and Tasaki (1973) confirmed in goats that casein
is equally well digested whether introduced into abomasum
or rumen. Addition of starch by either route of adiministra-
tion had no influence on the digestibility of casein.

Besides being isonitrogenous, test diets should also
be equal in energy. However, the utilization of energy can
also be influenced by route of administration; with the
metabolizable energy (MB) of postruminally administered
casein higher than orally fed casein (Blaxter and Martin,
1962). These points should receive close attention if

research is continued beyond the exploratory level.

40

By complete duodenal feeding of sheep and bottle-
feeding of goats, Potter, et_al., (1972) circumvented the
ruminal influence on protein quality and related this to
plasma levels of amino acids. The concentration of amino
acids known to be inadequate in test feed showed the largest
decrease relative to a reference pattern established with a
high quality protein (egg and casein). Application of this
approach to identify dietary amino acid imbalances would
require an optimal reference pattern established for each
species and physiological conditions (Potter, et_al., 1972).

Potter, gt_al., (1968) and Eskeland, gt_al., (1971)
showed i.v. glucose and VFA infusion depressed plasma amino
acids in characteristic patterns which apparently related
to the limiting amino acids for muscle protein synthesis.
But Potter, gt_al., (1972) could not confirm that this ap-
proach identified the amino acid most deficient in the diet.
Plasma amino acid data are generally difficult to interpret
(Purser, 1970; Jacobson, et_gl., 1970), but can aid in
explaining differences in animal performance in conditions
of amino acid stress (Young, et_al., 1973). Interactions in
ruminants between amino acids, energy yielding metabolites
(Potter, gt_al., 1968; Fenderson and Bergen, 1972) and
hormones (Hertelendy, et_al., 1969; McAtee and Trenkle, 1971;
Davis, 1972) appear to be similar to reactions in simple
stomached animals. Properties unique to ruminants may
become evident as amino acid and hormone relations are fur-

ther clarified.

41

Hatfield (1971) contended that a specific exogenous
amino acid supplied for maximum ruminant productivity
should fit the combined amino acid pattern of microbial and
residual dietary protein. Experiments at Illinois (Nimrick,
gt_al., 1970a,b, according to Hatfield, 1971) suggest the or-
der of limiting of amino acids in rumen microbial protein
produced on amino acid free diets is (l) methionine,

(2) lysine and (3) threonine. Infusions of these amino
acids increased N balance 60% over controls infused with
isonitrogenous levels of urea. Most balances went up sev-
eral times more than the amount of amino acid N infused,
indicating a stimulated protein synthesis rather than merely
storage of the infused amino nitrogen. Altered plasma amino
acid levels reflected the infusates, but threonine was
depressed by methionine infusion and still further by addi-
tional lysine (Nimrick, g£_al., 1970a). In another experi-
ment most plasma amino acids decreased linearly with
increasing methionine supplementation (Nimrick, gt_al.,
1970b). Plasma concentrations of methionine, however, rose
sharply with increasing methionine supplements beyond the
level which promoted maximum nitrogen retention (Nimrick,
gt_§l., 1970b).

Scott, e£_al., (1972) showed abomasal methionine
infusion in wethers elevated the plasma methionine concentra-
tions while depressing threonine, with no influence on other

plasma amino acid levels. These results paralleled those

42

of Wakeling, Annison and Lewis (1970)3 showing that threonine
followed methionine as the second limiting amino acid in
lambs fed a barley straw diet. The lack of effect of dietary
methionine on plasma amino acids, or on N balance is contrary
to abomasal supplementation and clearly demonstrates ruminal
degradation of orally supplied methionine (Scott, et;al,,
1972).

In steers, Steinacker, g£_al., (1970) found that
abomasally infused methionine increased N retention about
20% on a 12% CP diet with 40% of total N from urea when
compared to oral feeding of methionine or inorganic sulfur.
Chalupa, et_al., (1972), however, found inconsistent effects
of abomasally infused methionine on N retention in growing
steers. But retained N was doubled from 159/day by infusion
of casein + methionine, with no further increase when
tryptophan was added to the infusate. An BAA-mixture gave
results similar to casein + methionine.

In order to pass methionine through the rumen it has
been encapsulated with kaolin as a protecting substance.
Responses to coated methionine have also varied; and Mowat
and Deelstra (1972) found the effect dependent upon dietary
protein. Encapsulated methionine fed with soybean meal had
no influence on gains or feed efficiencies, but these para-

meters as well as carcass quality were improved by feeding

 

3A3 cited by Scott, et al., 1972, the source not
available.

43

encapsulated methionine with formalized soybean meal or
corn-urea. A growth trial revealed a cubic response in
weight gain to increasing levels of encapsulated methionine,
with indication of a toxic effect of the higher supplements

of methionine (Mowat and Deelstra, 1972).

3.2 Studies in lactating cows.

 

Regardless of the ideal energy to protein ratio for
milk production, high producing cows must absorb large
quantities of amino acids; and milking cows require a higher
ratio of protein to energy than finishing cattle. A suf-
ficiently high ratio may be hard to achieve even in cows on
high concentrate rations unless substantial amounts of feed
protein bypasses the rumen (Chalupa, 1971). Hence, the
favourable responses in wool and tissue growth to extra-
ruminal amino acids suggest good possibilities for beneficial
effects in lactating cows.

a. Methionine supplements in lactating cows.--
Although milk proteins are relatively low in methionine, the
exogenous supply of this amino acid has been studied more
extensively than any other in lactating cows as well as
other ruminants. The versatile involvement of methionine
in lipid and protein metabolism may render its availability
crucial in times of metabolic stress, as in early lactation

(McCarthy, et al., 1968).

44

As early as 1946 it was believed that methionine
might be nutritionally limiting in ruminants (Loosli and
Harris, 1946; Hungate, 1966); but Shaw (1946) did not show
methionine beneficial in treating bovine ketosis when admin-
istered orally or intravenously. McCarthy, et_al., (1968),
however, found that intravenous methionine followed by oral
doses of methionine hydroxy analog (MHA, 309/day for three
days) improved the health of ketotic cows. Further evidence
for a key role of methionine in lipid metabolism was indi-
cated by increased milk fat when methionine was supplied
orally as MHA.

Other researchers have in later years tested
methionine and its derivatives for ketosis with varying
results. Only slow changes toward normal levels of blood
metabolites and milk production were observed by Waterman
and Schulz (1972) who treated six cases of clinical ketosis
with 409 MHA per day. Pre-clinical conditions were not
reached over the three-week examination period. Fisher and
Erfle (1970) could not show that 409 methionine given intra-
venously over 24 hours alleviated symptoms of ketosis in
three ketotic cows.

Griel, gt_al., (1968) found 409 MHA supplement per
day from three weeks prepartum to eight weeks post-partum
increased FCM production compared to the controls. Eighty 9
MHA per day had different effects in cows of different
breeds but this was possibly due to an influence on feed

intake rather than specific metabolic reactions.

45

In a study conducted by Polan, e£_al., (1970) high
levels of MHA (909/day) frequently reduced the intake of
concentrate and corn silage, which offset any increase in
milk production. But the fall in milk volume was not con-
sidered to account for a linear increase in milk fat percent
with increasing levels of MHA. Altered composition of serum
lipid fractions during the MHA treatment further strengthened
the suggestion of an involvement of methionine in the cows'
lipid metabolism.

Bishop (1971) found MHA had no effect on the fat
test in heifers while there was a gradual increase with
advancing age in milk fat for MHA-treated cows versus con-
trols at a similar age. It appeared that MHA predominately
affected protein metabolism in the younger cows with a pro-
gressive involvement in lipid metabolism associated with
aging (Bishop, 1970). A trend towards a greater response
with the maturity of the cows was again reported by Bishop
and Murphy (1972) when the analog (2.29/kg concentrate) was
fed throughout a test year. The effect was evaluated by
comparing DHI production data with the preceding year's
records.

Kim, et_al., (1971) observed higher milk fat produc-
tion in cows while on MHA, but there was no effect on milk
volume or SNF. Neither did the treatment affect energy or
N digestibility, but cows on MHA lost more CH4 and urinary

N than did the controls. Neither Burgos and Olson (1970),

46

nor Whiting, gt_al., (1972) nor Begum and Jones (1972)
observed any increase in milk production by feeding differ-
ent levels of MHA. Digestibility of major feed constituents
and N retention was not influenced by MHA (Begum and Jones,
1970).

Broderick, et_al., (1970) found an increased
methionine to valine ratio as well as higher absolute
methionine levels in all of eight cows receiving 159
methionine per day in a kaolin-tristearate capsule. Milk
production and composition was not significantly influenced
by the supplement. An increase in the methionine/valine
ratio has been suggested to indicate an oversupply of
methionine, resulting in an imbalance to other EAA, parti-
cularly valine. Like other branch chained amino acids,
valine is slowly metabolized by the liver (Kaplan and Pitot,
1970). Neudoerffer, gt_gl., (1971) found about two-thirds
of methionine available for intestinal absorption in cattle
when the amino acid was encapsulated with kaolin and satu-
rated fat. About 30% of the substrate was broken down in
the rumen. The authors suggest that this method of nutrient
supply allows for close to full availability for intestinal
absorption.

b. Intravenous amino acid infusion studies in milking

 

cows.--Since amino acids interact in a competitive fashion
at absorption sites (Christensen, 1963) a gut overload of

one or several amino acids may result in a distorted plasma

47

pattern (Hume, 1972). Therefore, intravenous (i.v.) amino
acid administration may allow a more precise focusing on
metabolic reactions than is possible by supplying the gut.
Naturally though, the i.v. approach requires special
precautions and may still be hampered by complications.
Infusion of enzyme-hydrolyzed casein or fibrin by the jugu-
lar vein (Yousef, gt_§l., 1969) caused fever and depressed
milk production. Nevertheless, the arterio-venous (AV) con-
centration difference over the mammary gland increased during
infusion; 9% in three cows on a normal ration and 28% in
three cows on a high-grain ration demonstrated to stimulate'
milk protein synthesis. Fifty 9 acid-hydrolyzed casein per
day increased milk protein secretion 14%. Addition of glu-
cose to the protein hydrolysate helped control fever, and
combination of 509 glucose and 509 hydrolysate elevated milk
protein production 10-15% through increases in milk volume
and milk protein concentrations. When infusion of partially
hydrolysed fibrin + glucose (1:1)4 was compared to glucose,
no fever or discomfort in the cows (2 per treatment) was
seen during the first two days of the trial.5 As the treat-
ments were switched after two days break, however, severe

fever developed in the cows that received the protein

 

4AminosolR, Modified fibrin hydrolysate injection,

Low sodium U.S.P., from Abbot Laboratories, North Chicago,
Ill.

5Unpublished work for which the author held the main
responsibility.

48

hydrolysate, while only slight fever was detected in one of
the cows receiving glucose.

Since protein hydrolysates are used intravenously in
clinical nutrition they ought to be suitable for experimental
use in ruminants. However, the blood infusion technique
seems more suitable for studying single amino acids, and
several have conducted such experiments. Teichman, et_al.,
(1969) infused three levels of methionine and saline con—
tinuously for four days into eight cows, with the highest
rate of methionine equivalent to 20% of the expected output
in milk protein. No effect was observed on milk production.)
Fisher (1969) infused as much as 269 of di-methionine per
day, alone or together with 529 l-lysine hydrochloride, for
four days and did not observe any production response, even
though methionine and cystine in the plasma increased.

In later trials Fisher (1972) intravenously infused
methionine, histidine, and lysine at two levels in lactating
cows. Feed crude protein level was around 85% of theoreti-
cal requirement and urea supplied 85% of that N. Milk yield
was about 16kg per day and was not affected by any infusion
treatment. Milk protein concentrations tended to increase
as level of methionine infusion was increased. At the lower
rate of infusion, histidine increased feed consumption over
the saline controls, while feed consumption was depressed by
high histidine. Regardless of level of histidine infusion,

it lowered milk protein production (Fisher, 1972).

49

Fisher (1972) implies that stimulation of feed intake
by infusion of low levels of amino acids were due to sub-
optimal feed protein levels. The trend toward an adverse
effect at the higher levels of amino acid infusion might
suggest an amino acid imbalance, which was most obvious for
histidine. Valine, isoleucine and leucine in plasma fell as
methionine infusion increased, which would indicate critical
levels of these essential amino acids. Lysine did not alter
plasma concentrations of other essential amino acids, but
lysine itself was not determined in this study. From the
plasma levels, Fisher (1972) suggested methionine as the
most marginal amino acid in his experiment.

c. Feed proteins introduced postruminally.--Several

 

recent experiments where whole proteins have been supplied
postruminally have resulted in larger and more consistent
responses in lactating cows than single amino acid supplemen-
tation.

By abomasal infusion of casein + methionine in
three cows Broderick, gt_al., (1970) found an improvement in
milk volume and production of all the main milk constituents.
The only significant effects, however, were in crude protein
(N%x6.38) concentration which increased about 6% (P < .01)
and protein production which increased almost 12% (P < .05).
The findings of altered EAA and NEAA levels agree with
changes associated with a general improvement in protein

status. A large increase in the plasma level of branched

50

chain amino acids during the abomasal infusion (Broderick,
gt_al,, 1970) paralleled observations in sheep which received
a similar treatment (Hogan, et_al., 1968).

In later studies Broderick, et_al., (1972) fed
formaldehyde treated casein to lactating cows on a corn-
based diet which contained 9% CP. The protein content was
raised by the additions of formalinized casein to give 12,
14, 16, and 18% CP. The casein supplements elevated milk
yield and protein concentration of the milk, with a maximum
protein production at 16% CP. Because increasing levels of
treated protein resulted in continuously higher plasma con-.
centrations of methionine, lysine, valine and isoleucine,
the authors considered these as amino acids limiting for
protein synthesis. Untreated protein fed to achieve a
higher level of CP% would have strengthened this study.

Spechter (1972) compared blood amino acid concentra-
tions in lactating cows during duodenal casein infusions
with those during saline infusion. He reasoned that
phenylalanine, histidine and methionine were most likely
to be marginal since these amino acids showed the greatest
concentration drop from control to casein treatment. He
assumed that a marked increase in milk protein output taxed
these amino acids harder than others. But Spechter (1972)
finally concluded that a general shortage of protein rather
than specific amino acids limited milk production on the

basal, unsupplemented diet, in which 40 to 45% of the N was

51

urea. The investigation (Spechter, 1972) was done with six
cows around the peak of lactation, producing between 20 and
35kg milk per day, and involved 2 week infusion periods.
Yields of both milk and protein rose substantially during
casein treatment compared to averaged pre- and post-casein
infusions.

During casein infusion, DM intakes increased almost
50% (4.2k9, P < .01) for two cows on the lowest casein sup-
plement while increases were less for the cows at the two
higher casein levels. Quite evidently, the increased feed
consumption might partially explain treatment responses.
Cows on low casein, which had the dramatic increase in DM
intake, showed the largest absolute and relative treatment
response in milk yield. Average protein production, however,
was increased most for the cows on medium level; and there
was a quadratic response in SCM yield (Spechter, 1972).

A nitrogen balance trial, with sample collections
in the latter week of each period, revealed a significant
linear effect of level of casein infusion on N utilization
(Spechter, 1972). There was marked difference in response
to treatment levels between the first and second week; pos-
sibly related to stress during the balance trial. Reasons
for the large responses (Spechter, 1972) observed in milk
protein yield might be the early stage of lactation of the

cows with a high milk yield potential; feeding of a ration

52

high in NPN, and the negative N balance in cows at the onset
of the experiment.

After the casein study, Spechter (1972) infused
glucose in a similar manner, but this had small and mostly
negative effects on milk production. At this time, however,
the cows were in positive N balance and feed intakes declined
with glucose infusion which commenced at about the thirteenth
week of lactation. The milk fat test dropped markedly
during the two higher casein infusions, continued to fall
thereafter, and fell still further with glucose infusion.
Despite the diverse effects of glucose' on feed intakes
they were positively correlated to milk production, but
could have been confounded with stage of lactation (Spechter,
1972).

Mugerwa (1969) found abomasal infusion of casein
enhanced the intake of a urea-cellulose ration by 27%, and
the intake of a urea-starch ration was increased 10%. On
the other hand, the consumption of urea treated corn silage
was not consistently affected by abomasal infusion of either
casein or gelatin.

Derring, gt_al., (1972), however, did not find that
abomasal or ruminal infusion of 4409 casein per day altered
the DM intake in milking cows; but the DM digestibility was
higher, the plasma urea was lower and milk N higher for
abomasal than ruminal infusions (Derring, et_al., 1972).

Milk yields did not differ significantly, but the fat

53

content of the milk was lower (P < .05) for the abomasal
route of infusion.

Hale and Jacobson (1972) fed or abomasally infused
casein, gelatin, partially delactosed whey (PDW) and zein
to cows without influence of source of protein on milk
production, but level of performance was quite low. Mugerwa
(1969) reported higher N utilization for casein than gelatin
when these proteins were infused into the abomasum; but milk
production data were not presented.

Extensive balance studies were carriedout by Tyrell,
et_al., (1972) during abomasal infusions of cows placed in
a respiration chamber. Generally, the infused casein was
utilized with low efficiency despite clear responses in milk
production. When 8609 casein was supplied to two cows
producing about 24kg per day, milk yields increased 3kg/day,
which was equivalent to 48% of the energy of the infusate.
However, less than 25% of the casein N was recovered in milk,
with 24% in feces, 21% in urine and 31% in positive tissue
balance. Glucose infused at a rate of 3.6Mcal per day
(a 9009) increased milk energy production equal to 16% of
that supplied while 48% was lost in feces (Tyrell, gt_al.,
1972). Spechter (1972) observed comparatively higher effi-
ciencies in his balance experiments where increased milk
protein could account for 75, 54, and 36% respectively of

27, 87, and 1459 of casein N infused daily; but N balances

were negative when the cows were on the basal diet.

54

4. Milk protein content
and dietary protein

 

 

Non-genetic factors influencing milk protein pro-
duction has been discussed in several reviews (Larson, 1958;
Huber and Boman, 1966; Kirchgessner, et_al., 1967). Total
protein yield tends to be more constant from day to day than
the yields of milk fat and lactose (Larson, 1958). While
the need for dietary protein varies directly with level of
milk production, it is also generally accepted that the con-
tent of protein in milk will not increase with excessive
protein allowance. The content of NPN, however, may in—
crease to some extent. High levels of energy and high
energy concentrations in feeds, on the other hand, usually
increase the concentration of protein in milk. This rela-
tionship is discussed further in section C.I.

Seasonal variations in milk protein concentrations
have been related to energy supply. German workers
(Kirchmeier, 1970) found that the amino acid composition of
casein varied more than could be explained by shift in the
ratio of the different caseins. The relative amound of non-
essential to essential amino acids of casein increased as
casein increased in seasons with ample nutritional supply.
These findings conflict with the concept that an invariable

mechanism is responsible for the biochemical replication of

this protein (Kirchmeier, 1970; Larson, 1958; Jenness, 1970).

55

 

 

5. Summary of literature review
which led to the experimental
approach

The optimal level and quality of feed protein for
milk production in general, and milk protein synthesis in
particular, is obscured by the metabolism in the rumen.
While ruminal synthesis of microbial protein is related to
energy transactions and can be estimated with fairly good
accuracy, the extent of rumen bypass of feed protein depends
upon quality and quantity of the total ration. A relation-
ship exists between absorbable amino acids and plasma free
amino acids, but more information is needed before plasma
concentrations can be used to identify an amino acid as
rate limiting for milk protein synthesis.

Postruminal introduction of proteins and amino acids
in non lactating animals have confirmed that ruminants
depend on feed protein for maximum production performance.
Responses to postrumen protein in lactating cows, however,
have mostly been obtained with rations deficient in protein;
and therefore the outcome has not been separated from the
general need for crude protein. Experimentally, direct
postrumen supply, as abomasal infusion, leaves out uncer-
tainties about the extent of rumen bypass. For study of a
general, nutritional effect, the intestinal route appears

more proper than intravenous infusion.

C. RESEARCH SECTION

I. FIRST SERIES OF EXPERIMENTS, 1970

1. Methods and Materials

 

1.1 Rationale for treatments
and design

 

 

The main objective of these experiments was to
study the influence of postrumen supply of amino acids on
production of milk and milk protein. No report on such
investigations had appeared in the literature before 1970.
Thus, the first trial was exploratory, although preceded
by a pilot study with one cow (No. 480).

After this cow had recovered from surgical install-
ment of an abomasal cannula 6209 of bovine albumen hydro-
lysate1 was infused over 50 hours. The cow was fed a
common ration considered to be adequate in protein and
energy according to NRC (1966) standards. Her daily milk
yield was around 14kg. During the infusion, the volume of
milk and its protein concentration were slightly increased
compared to the days before and after, and milk protein

production increased 11%.

 

lAminosolR 5%, Modified fibrin hydrolysate injec-
tion, Low sodium, U.S.P., from Abbott Laboratories, North
Chicago, Ill.

56

57

Since amino acids also have an energetic value,
and may serve as a substrate for gluconeogenesis, it was
decided to use an equicaloric infusion of glucose for
control to the protein in the experiments that followed.
Casein was chosen as the treatment protein as it is the
major milk protein and has a high biological value for
growth.

Still, it remained questionable how far the in-
fused protein might alter the pattern of amino acids
reaching the mammary gland, to improve conditions for
milk protein synthesis. Nevertheless, a desire to chal-
lenge the cows' ability to produce milk proteins sug-
gested a rather high treatment level. Partly based on
the pilot study it was decided to infuse casein at a rate
which would supply an equivalent to two-thirds of the daily
milk protein output. A cow producing 20-22 kg milk per
day should thus receive around 5009 casein. Because of
technical limitations it was possible to infuse only slightly
over 3009 casein per day in the first trial.

Only one infusion pump was available at the time
trial I commenced; but reasoning the study would be more
informative if the cows had fairly high production, the
experiment was started promptly. Two cows were used in
this trial; each was infused with casein before and after
a glucose infusion, with control periods interspacing

these treatments. During the first part of the trial

58

control observations were collected for one cow while the

other was infused, and vice-versa (Table 1.1).

Table l.1.--Trial I 1970. Treatment periods.

 

 

 

Period Treatment Cow No. Cow No.
No. 502 501
Days
1 Control 5/8-13 5/15-20
2 Casein infusion 5/14-19 5/21-26
3 Control 5/20-26a 5/27-6/1a
4 Glucose infusion 5/27-6a 6/2-7
5 Control 6/2-7 6/8-13b'C
6 Control 6/7-12b
7 Casein infusion 6/13-18 6/14—19d
8 Control 6/19-24 6/20-25

 

a . .
These periods served as post-casein as well as
pre-glucose control.

bFor cow No. 502, 6/2-7 was the post-glucose con-
trol, 6/7-12, was the pre-casein control. The delay was
partly because the abomasal cannula tended to slip out of
position.

CFor cow No. 501, 6/8-13 served as post-glucose as
well as pre-casein control.

dA new roller pump allowed simultaneous treatment
of the two cows in period 7.

The period length of six days was thought a minimum
for a production study although amino acids have a rapid

turnover rate (Black, et al., 1968; Munro, 1970), and

59

altered substrate availability may change the composition
of secreted milk within hours (Linzell, 1967). At any rate,
the total experimental timespan was intentionally kept as
short as possible since it was uncertain how durable the
stomach cannulation would be.

A switchback type of design was preferred because
it most efficiently removed the time trends which could
bias within-cow comparisons of milk production when only
few cows were receiving a sequence of treatments (Lucas,
1960). Since the same observations were post-treatment
controls for one infusion and pro-treatment controls for
a following infusion, treatment comparisons are not com-
pletely independent. This was not considred a major
problem, because it was assumed that treatments would not
have a carry-over effect; and two independent control
periods between infusions would have prolonged the trial.

Even with satisfactory controls, only two cows give
little power for statistical test. Therefore, with the
intention of possible pooling of data, a second trial
employing three cows was conducted similarly to trial I
despite apparent weaknesses in design (Table 1.2).

1.2 Animals and abomasal
cannulation
Three Holstein cows weighing 600-650k9 were fitted

with abomasal cannulas at the Michigan State University

6O

.mmmp
03¢ Hmnuosm now scamsmcw pmucm>mum ummu goddamn smmouufic < .h pOwHom so 300 was»
How mcoflum>ummno mo wasp Hmsuom m waso ohm muonu mane .popumomflp mmz m\m pom m\m

pom .mamuo>mm commons came» xHHE gnu .w\m so Hmumz usonufi3 was mom .oz 300m

.mmp use» once possessoo omv .02 so scamsmsfi may no pmoumomflp mmz ¢H\m hoop

.m\m Hens: pumum no: pasoo 300 men» CH sonSMcfl pom Umpumo
-meo on on one e\m oso m\m want on .m\o so ones oesunem use once owe .oz 3006
.mamseus mcexmma ou mop mumHmEoosH mmz coeuooaaoo moans .mom was How msoo
mom Honucoo ucmEummuuwum mm pm>uom mnemoumnu vlm\m cue? Hmnumgou AM was om\m mama
.xmp menu so m3oo may on pmsmummm mumz masses: mm popumomfip was H\m hoop

.sonSMsH mmoosam u 0

“GmesmsH sflmmmo u x undefium>uwmno Honusoo n o

"msowumw>onnm useEummHBm

 

 

ee-~e\o oeeum\m = s = = s = mom
oe-ee\o oeumxm e\ouom\m = = = = = Hem
oemnme\m omeuw\o o.ne\msom\m em-om\m mmuomxm menee\m mauo\m eum\o owe
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII mmmp IIIIIIIIIIIIIIIIIIIIIIIIIIIII: JNmIwa
o M o o 0 o M o "musoEummHB
m e o m e m N H uoz ooeuwe

 

 

.mooeume usosuoone .oeoe HH Hoeuenu.~.e ments

61

Veterinary Clinic2 early in their second or third lacta-
tions.

Feed was withdrawn for 24 hours prior to surgery,
which was performed with the cow laying on an elevated
surgery table. A vertical incision on the lower part of
abdomen was made. The abomasum was moved backwards and
upwards from its normal position, and sutured to the
abdominal muscle which surrounded the incision.

The first cow (No. 480) was given a tranquilizer
and operated upon with local anesthesia. A silastic
tubing3 was run about 15 cm into the abomasum and the
incision sutured close to the tubing. A 1mm silastic
sheet reinforced with dacron mesh4 about 5cm in diameter
was glued to the tubing to hold it in place. After closure
of the abdominal incision the tubing was run up on the side
of the cow and fastened with branding cement5 and surgical

tape.

 

2The surgical installation of abomasal cannula
was performed by Drs. W. D. Oxender and C. L. Miller,
aided by students in Large animal surgery and medicine at
the College of Vet. Med., Michigan State University.

3silasticR Medical-Grade Tubing, from Dow Corning
Corp., Midland, Mich.

4Dacron mesh from Dow Corning Corp., Midland,
Michigan.

5Branding cementR from Victor Business Forms Co.,
Lincoln, Neb.

62

Following surgery, the cow developed complete in-
appitance and dropped sharply in milk yield which never
completely recovered. About a week following surgery it
became apparent that the tubing would not stay in place
despite considerable effort to keep it in position. After
a few weeks the tubing came out from time to time and some
leakage from the abomasum occurred, and this cow (No. 480)
was not employed for the first trial.

In the other two cows (Nos. 501 and 502) a smaller

tubing (PE 260,6

2.5mm outside diameter) was fitted through
an abomasal stab wound. A purse string was fastened around
the tubing to keep it in position at the entrance of the l
stomach. It was then attached to the cow's side with brand—
ing cement and surgical tape. The tubing ran about 20-30cm
inside the stomach. A different anesthesia was used in
these cows, which went back on feed the day after surgery.
Likewise, milk production rapidly increased to presurgery
level.

A few weeks after surgery one of the latter tubings
broke at the entrance to the abdomen, and the other pulled
out. New tubings were established, but they tended to leak.
Before trial II, and about four months after the original

surgery, all three cows were fitted with fistula plugs made

by joining two Jarrett cannulas.7 By the end of trial II

 

6IntramedicalR Polyethylene Tubing, PE 260 ID .070"/
OD .110", From Clay Adams, Inc., New York.

7Jarrett cannula, from Australian Rubber Mill,
Aberham, South Australia (Jarrett, I.G., 1948, J. Council
Sci. Ind. Res. 21:311).

63

(periods 7 and 8) stomach contents did seep through the
fistula openings and the tissue became red and swollen.
This irritation was worst in cow No. 501, which was
slaughtered shortly after the termination of trial II.
Except for one occasion in trial II, however, when cow No.
480 lost her plug, leakage from the abomasum did not influ-

ence the feed intake or the health of the cows.

1.3 Feeding and feed sampling

 

Throughout both trials concentrate was fed in two
equal portions twice daily (~7:30 AM and 4 PM). At the
morning feeding the cows also received corn silage while
hay was fed in the afternoon. All feed was weighed out
and weighbacks were recorded daily. Feed refusals were
minimal in both trials and were not sampled for analysis.

Feed in trial I.--The ration consisted of common

 

feeds in their usual proportions (Appendix Table 1.1) but
was fairly high in urea (Table 1.3).

In addition to 4.5kg hay and 18kg corn silage,
concentrate (6.4 and 7.2kg for No. 501 and 502, respectively)
was fed to meet the cows' estimated requirement for energy
and protein (NRC 1966) at the beginning of the trial. The
level of feeding was not changed until the trial was termi-
nated, at which time the cows were being overfed (Appendix

Table I.5) .

64

Table l.3.--Trials I and II 1970. Feed composition.

 

 

 

 

 

 

 

Trial I Trial II

Dry Protein Esti- Dry Protein Esti-
Material in mated Material in mated

(DM) DM NE (DM) DM NE

Mcal Mcal

% % kg % % ‘k9_
Alfalfa hay ~90 ~l8 1.03 91.8 18.5 1.05
Corn silage 30.5 14.4a 0.50 33.1 10.2C 0.54

Concentrated 87.6 13.0b 1.78 89.0 18.5 1.82

 

aUrea added, 0.5%.

bUrea added, 1.0%.

CUrea added, ~0.3%.

dIngredients in the concentrates: Trial I: ground
shelled corn, 88.6%, molasses, 7.4%, urea, 1%, limestone,
1.6%, salt, 1%, gypsum, 0.4%; Trial II: ground shelled

corn, 68%, soybean meal (50% CP), 22.5%, molasses, 7.5%,
dicalcium phosphate, 1%, salt, 1%.

During trial I feed samples were obtained at
irregular intervals and since the hay quality changed, the
nutritive value assigned to the hay (Table 1.3) was not
always the same.

Feed in trial II.--In order to test the infusion

 

treatments under nutritive conditions which optimized milk
protein synthesis, the cows were changed to a high grain-
low roughage ration; reported to increase milk protein
content (Rook and Line, 1961; Huber and Boman, 1966;

Kirchgessner, et al., 1967; Yousef, et al., 1970). The

65

ration (Appendix Table I.3) was compsoed of 2.3kg hay and
4.5kg corn silage and respectively, 10.0 (No. 480), 11.4
(No. 501), and 12.7kg (No. 502) of a concentrate mixture
(Table 1.3). Crude protein greatly exceeded NRC (1966)
standards (Appendix Table 1.4) because the concentrate was
higher in protein than anticipated.

The feeds were sampled twice in each period and
samples were kept in sealed plastic bags until determina-

tion for DM and nitrogen.

1.4 Infusion treatments

 

A solution of casein was prepared by adding 2 to
2.59 NaOH to every 1009 casein:8 a 5% solution of NaOH was
mixed with casein in a mortar to form a dough which was
then placed in the appropriate amount of water in a bath
at 60°C. The casein (2.5% w/v) gradually dissolved over
several hours, hastened by occasional stirring. The glucose
solution was made from cerelose9 at the same strength as
casein, and an equal content of ME in the two substrates
was assumed. To be precise, however, the glucose solution
should have been stronger (Table 1.4, and comments), but the
correction required probably was smaller than errors due to

irregularities in the infusions. Relative to the total

 

8Casein from Nutritional Biochemicals, Cleveland,
Ohio.

9Cerelose (methyl dextrose), obtained from Corn
Industrial, CPC International, Englewood Cliffs, New
Jersey.

66

Table l.4.--Trials I and II 1970. Estimated net energy
and crude protein supply by the infusates
relative to total intakes (%).

 

 

 

 

 

 

 

 

 

Cow No.
501 502 480
CP ENE CP ENE CP ENE
%

Trial I. Infusion

First casein 11.5 4.7 11.5

Glucose -- 4.3 -- 4.3

Second casein 11.7 4.7 11.9 4.4
Trial II. Infusion

First casein 12.3 5.1 11.1 4.6 10.2 4.2

Glucose -- 4.8 -- 4.3 --

Second casein 11.6 5.1 10.6 4.3 10.0 .

 

a(Infusate value/feed value) x 100%.

Comments: Approximately 3009 (270-3309) casein
or glucose was infused per 24 hours. DM content of the
substrates was about 95%. Assumed energetic value of
casein, 4.5kcal/g; of glucose 3.8kcal/g (Maynard and
Loosli, 1962). Crude protein in casein was 88% (14.5% N
in DM x 6.38). Estimated supply: by casein, 2649 CP and
1.18Mcal, and by glucose, 1.08Mca1.

nutrients furnished, the infusates were quite minor, parti-
cularly for energy (Table 1.4).
In trial I, a double piston infusion withdrawal

lo 0 I I
pump was used for the continuous infu31on. Because a

 

10Harvard continuous automatic infusion-withdrawal
pump, series No. 950, from Harvard Apparatus Comp., Inc.,
150 Dover Road, Millis, Massachusetts.

67

casein solution stronger than 2.5% (w/v) would block the
pistons of the pump total infused casein was limited to
3009 per day (Table 1.4).

For trial II, two roller pumps11 were ready, but
for valid comparisons and combining of data for statisti-
cal analysis, the rate of infusion was kept the same as
in trial I (Table 1.4). The concentration of the sub-
strate solutions, however, were 4.6% (w/v).

As the level of feeding differed between and with-
in cows, the percent of total nutrients furnished by the
infusates also varied (Table 1.4); but the design of the
study allowed for each cow to serve as her own control
for all treatments imposed; thus, changes in the relative
level of infused nutrients were balanced among treatments.

The output by the pumps were recorded at frequent
intervals and if a tube was leaking, the loss was esti-
mated. These estimates admittedly were not exact. Thus,
a detailed presentation of infusion rates would have
little value. Small losses of infusate in the beginning
of the experiments occurred because the cows disrupted

the plastic tubings conveying the solutions.

1.5 Milking and milk sampling

 

In both trials the cows were milked shortly after

7:30 AM and again at 4 PM. The uneven milking intervals

 

llHolter multi-channel roller pump, model 911R,
from Extracorporeal Medical Specialties, Inc., Mt. Laurel
Township, New Jersey.

 

68
were necessary because of the working hours for the barn
personnel. Milk weights were recorded at every milking
and a milk sample (about 100ml) was collected into a bottle
with 0.3ml formalin for preservation (Association of Official
Agricultural Chemists, 1955). After warming these samples
to 38°C aliquots to the milk weights were taken to make
one composite sample representing two sequential days.
Thus, for every control and treatment period three milk

samples were analyzed for each cow.

1.6 Blood sampling

 

During both trials tail blood samples were obtained
every other day before the morning feeding and nine hours
later. Plasma was prepared by centrifuging at 2000xg for
20 min. During trial I several hours frequently elapsed
before processing of samples commenced, and the blood was
often clotted. Therefore, it was felt that data on blood
composition in trial I would have low validity, so samples

were not assayed.

1.7 Chemical analysis

 

a. Feed.--Dry matter was determined by drying at

 

100°C for at least 48 hours. Kjeldahl N was determined
on composite samples for each trial by the macro-Kjeldahl

procedure on wet silage and dried hay and grain. (AOAC,

1955). Net energy was estimated from NRC (1966) feed

 

69

tables by taking into account DM and N content and the
ingredients of the concentrates.
b. Milk.--Milk fat was determined according to

the Babcock method.12 Milk protein: During the first

 

part of trial I, a modification of the method of Lowry,

gt_al., (1951) was used for quantification of milk protein.

The milk was diluted 400 times with water and a casein

solution was used as a standard. Protein concentration

was calculated by a regression equation based on Kjeldahl N.
Apparently the Lowry method is not reliable if

the milk is not fresh, so it was decided to use Kjeldahl N

determinations (N x 6.38 = CP in milk) for trial II. This

was done by placing 3 ml of milk in a glass-stoppered

flask and quickly weighing on a balance. After pouring

the milk into a Kjeldahl flask, the weighing flask was

rinsed with distilled water and then with the 25ml sul-

furic acid to be added for digestion. A mixture of

59 K SO

and 19 CuSO was used as catalyst. If duplicates

2 4 4
deviated more than 0.05% crude protein (CP) another deter-
mination was performed.

Total solids in milk were determined in duplicates

 

by oven drying at 100°C for 4 hours. Two m1 milk was

pipetted for weighing in aluminum pans of 3cm diameter,

 

12The fat test was performed at the center for the
Dairy Herd Improvement Association, Forest Road, East
Lansing.

70

which were used for the drying. The content of solids
nonfat (SNF) was estimated as total solids % minus fat %.

Nonprotein N was assayed only in trial II, follow-

 

ing the procedure of Shahani and Sommer (1951). Initially
10ml of milk was weighed into a 100ml volumetric flask

and filled to the mark with 15% (w/v) trichloroacetic

acid (TCA), and shaken. After standing at room tempera-
ture for 2-3 hours, the supernatant was filtered and
stored frozen at -20°C until analyzed for N. The assay

13 with urea in

was done using a Technicon Auto Analyser
TCA solution as the standard. Due to low N concentrations
in the diluted samples, the sensitivity of the analysis
was less than desirable; but clear differences were dis-
cerned between samples, and duplicate samples agreed

quite well.

c. Blood p1asma.--a amino N was determined accord-

 

ing to Palmer and Peters (1969) on the freshly prepared
plasma.

Ammonia and urea were assayed in plasma stored

 

frozen for two to three months. The micro-diffusion
method of Conway (1960) was used, but there was not suf-
ficient NH3 present to be quantified.

1.8 Calculations and statis-
tical analysis

 

 

Responses to infusion treatments were, as pre-

viously indicated, estimated by comparing performance

 

13TechniconR Auto AnalyserR, Methodology N-36,
Kjeldahl Nitrogen, from Technicon Controls, Inc., Chauncey,
New York.

71

during the infusion period to that of averaged pre- and
post-infusion periods. For both trials the basic unit was
the mean daily production during a period although deter-
minations of milk constiuents were for two-day sub-periods.
But the repeated measurements can not be considered inde-
pendent observation and basis for estimated error mean square
in the analyses of variance.

The following scheme shows how the treatment versus
control differences (dp) were calculated, Yp indicating
performance in any parameter during a period (p):

first casein inqulon: d1 = Y2 - (Yl + Y3)/2

glucose lnquion: d2 Y4 - (Y3 + Y5)/2

second casein infusion: d3 Y7 - (Y6 + Y8)/2

<11 and d3 = dk' d2 = dG.

As mentioned, data for period 3 in trial I and II was
employed for two comparisons (d1 and d3). This was the
case also with period 5 for cow No. 501 in trial I (Table
1.1). Besides a possible carry-over effect of glucose in-
fusion on milk fat concentration (Appendix Tables 1.5 and
1.7) and plasma urea concentration (Appendix Table 1.11),
treatment effects were apparently not biased by the over-
lapping use of control periods.

The milk production results for these two trials
were similar and had homogenous variances; thus, one

statistical analysis of the data, involving all five cows,

72

was appropriate. Despite the time between the two trials
and differences in feeding, it can be objected that the two
cows re-used do not represent independent observations.
This point was not considered serious enough to abandon the
statistical evaluation.

Analysis of variance (AOV) (Appendix Table I.13)
was done by means of standard programs on an Olivettil4
desk computer, aided mainly by the textbooks of Sokal and
Rohlf (1969) and Cochran and Cox (1957).

Since the mean values for 9 amino N and urea in
blood plasma indicated distinct differences between AM and
PM samples the data for these parameters were analyzed
differently than those for milk production (Appendix Table
I.14). The unit for the analysis, however, was the mean
of two or three sampling days within a period, composited
for the same reasons that prompted neglect of the sub-
period observations in milk parameters. But the control
periods before and after an infusion were not averaged
for the blood parameters since it might be of interest
whether a difference between these periods was signifi-
cant or not; possibly influenced by the infusion treat-
ment. Presumably, there should not be a time trend
influencing the blood constituents in the way milk pro-

duction is affected. Because the AM and PM samples were

 

l4Olivetti, Underwood Programma 101, electronic
desk computer.

73

drawn repeatedly from any one cow on any one treatment
the AOV had to follow a split plot pattern (Gill and Hafs,

1971).

2. Results

2.1 Feed, energy and
protein intake

 

 

a. Feed composition.--Except for some variations

 

in the hay, the major feed traits were quite constant
based on random sampling (particularly in trial I). Only
average values for the main feed characteristics were ob-
served (Table l.3); but consumed dry matter, protein and
energy were calculated from the appropriate concentrations
for each period and average daily intake for each feed.

b. Intakes.--The feeds offered in both of these
trials were readily eaten, leading to equal feed consump-
tion for all periods (Appendix Table 1.1 and I.3). Since
the feed quality except for the hay, varied little, the
amounts of protein and energy consumed also remained quite
constant from period to period (Appendix Table 1.2 and 1.4)
and intakes of energy and protein stayed above standard
requirements.

The percent of total energy and protein supplied
by the infusates differed slightly between cows (Table
1.4). But the magnitude of these fractions were quite low
and responses to the infusions should be attributable to

the specific metabolites infused.

74

2.2 Milk production and
composition

 

 

a. Trial I.--Observations for the two cows used
in this trial are in Appendix Table 1.5. These figures
were pooled with the results for trial II (Table 1.5) for
statistical analysis.

Appendix Table 1.6 reveals that the milk produc-
tion was consistently higher during infusions than during
control periods, while composition was altered to a vari-
able extent. When casein was infused, the milk protein
yield was raised 5 to 11%; whereas the increase from glucose
was 8% over the mean of controls before and after. I

The response in protein production was greatest
for the casein treatment in the highest yielding cow (No.
502); who responded both by a higher milk protein level
and increased milk volume (Appendix Table 1.6). However,
an unusually steep fall after the last infusion period,
which had no specific explanation, exaggerated the dif-
ference between the treatment and the control periods.

The fat test tended to drop during infusions,
but the pattern was irregular. A decline in milk fat
content during the last casein treatment and associated
control periods might have been obscured by a rapid
decline in milk volume for both cows. Increased ambient
temperature (middle of June) might partially explain the

fall in milk production. Up to the last period the

75

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.o no HHmB mm m pofinom MOM poms duos mcoflum>ummoo dawn on» pom

pom omoosam swoSHon when m wage H Hmwuu ow pm: Hom .02 3000

 

 

 

 

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8 u m xH.z IuonB pofinmm
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HH Hoeue

 

 

sees some one one

xHHE mo coeuflmomfioo pom

.moofiuom mo mucmsufiumcoo
coHuUSUoum

.onaa HH pom H HMflHBII.m.H magma

76

decline in milk production was 1.5 to 1.7% per week which
is about normal (Hillman, gp_al., 1970).

Because fat content and milk yield changed in
opposite directions during infusions, FCM was not consis-
tently affected (Appendix Table 1.5 and 1.6).

While the estimated recovery of infused protein as
milk protein was between 10 and 15%, recovery of infused
energy in milk ranged from a negative value to a high of
75%.

The concentration of SNF did not show any consis-
tent treatment trend (Appendix Tables I.5 and 1.6), nor
was there a significant correlation between SNF and milk
protein concentrations (r = 0.11) (Table 1.7), although
change in SNF generally evolves in the proteins (Huber
and Boman, 1967). However, there was a significant nega-
tive correlation (r = 0.67, P < .01) between SNF and fat
percentage.

b. Trial II.--Comparing yield data for trial I
and II (Appendix Tables 1.5 and 1.7) readily shows that
the milk production had fallen considerably before trial
II commenced. Cow No. 501 reacted negatively to a change
from liberal to restricted roughage feeding which took
place over 10 to 12 days. Milk yield in this cow never

recovered after the changeover period even though she

consumed large amounts of feed.

77

The commonly observed depression in milk fat by
high concentrate and low roughage feeding did not develop
in these cows. Perhaps the late stage of lactation (6 to
8 months postpartum) and a declining milk production masked
the fat decline (Loosli, gp_§l., 1945).

The rapid consumption of allotted feed, the hungry
appearance of the cows, in addition to the decreases in
milk yield and high fat tests caused suspicion that mis-
takes were made in rationing of feed. Repeated checks of
intakes and of the man during feeding did not reveal this.
The high milk protein concentrations even from the begin-
ning of trial II agrees with previously demonstrated effects
of high energy rations (Rock and Line, 1961; Huber and
Boman, 1966).

Changes in protein percent with the infusions,
however, were small; but during casein infusion the trend
was towards an increase rather than decrease (Appendix
Tables 1.7 and 1.8). Thus, the production of milk protein
was increased over controls by casein infusion more than
by the glucose infusion (Appendix Table 1.8). The high
protein concentration of the milk prior to post-ruminal
infusion might have limited stimuation of milk protein
synthesis by the infusates. Again, the cow with the highest
production (No. 502) showed the greatest increase in milk
even though her response to the last casein infusion was

less than for the two earlier infusions.

78

Fat tests consistently decreased during the infusion
treatments without any obvious difference between the
effects of casein and glucose. The changes in SNF concen-
tration with the infusions were slight and less consistent
than protein concentration. However, there was a signifi-
cant correlation (r = 0.71, P < .01) between SNF and
protein level which was much higher than that (r = 0.11,
NS) in trial I (Table I.7). These correlation coefficients
were too far apart to allow a pooled correlation for the
two trials (Rohlf and Sokal, 1969).

Between SNF and fat content there was a low and
insignificant correlation (r = -O.29); much weaker than
between these parameters in trial I (r = -0.69, P < .01).
A composite correlation coefficient for SNF and fat levels
in the two trials, was low (r = -0.36) and not significant.
As in the first trial, there was no significant relation-
ship between the milk protein and fat (r = -0.05), and
the coefficient for the combined trials was also insigni-
ficant (r = -0.21).

Data for NPN in milk (Appendix Table I.9) showed
some variation with infusion treatments, but NPN concen-
trations during glucose infusion were significantly de-
pressed (P < .05, Table 1.6, row 9). Furthermore, the
response in NPN to glucose was significantly different
from the response to casein (P < .05). When NPN was

expressed as a fraction of total N in the milk there was

79

no influence of casein infusion. On the other hand, the
fractional expression confirmed that less of total N was
NPN during glucose infusion than during the other periods
of the trial (Appendix Table I.9).

Because of the different changes in NPN associated
with glucose and casein infusions the difference between
the two treatments in estimated true protein was less than
for milk crude protein (Table 1.6, row 3 and 10). However,
the influence of NPN on true protein production was minute
since NPN makes up such a small part of the total milk N.
Nevertheless, while the response in crude milk protein
production was significantly larger (P < .05) for casein
infusion than for glucose infusion (Table 1.6, row 6),
there was little difference in true protein production
response for the two treatments (Table 1.6, row 11).
Although the increase in milk protein (N x 6.38) production
with infusions were small, they were evidently not an
artifact due to increased NPN content. Thus, the NPN
data confirm greater milk protein synthesis during casein
and glucose infusions than during the appropriate control
periods.

The absolute differences in protein production
between treatment and control periods were smaller in
trial II than in trial I, probably because of the lower
milk production. However, treatment responses, as per-
centages of control production, did not differ much for

the two trials.

80

 

 

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81

 

 

 

 

 

 

 

 

 

 

 

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82

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83

Table l.7.--Trials I and II 1970. Correlation coefficients
between milk, blood and feed components.

 

 

 

 

 

 

 

Trial
Factors
I II I & II
milk fat (%) - milk SNF (%) -.67** —.29 —.36
milk cp (%) - milk SNF (%) .11 .71** a
milk CP (%) - milk fat (%) .12 .05 .21
milk cp (%) - milk NPN (mg%)b .25
Blood Sampling Time
AM PM Daily
Mean
plasma urea N (mg%) - __ __ 51**
milk NPN (mg%) °
plasma urea N (mg%) - .84** .59** .76**

pl. 0 am. N (um/ml)

plasma urea N (mg%) - 07 57**
feed cp (kg/day) ' '

plasma 0 am. N (pm/ml) ‘ 01 24
feed CP (kg/day) . .

 

aThe difference between trials significant (P < .05),
pooling of data not permissible.

bNPN (mg%) observed only in trial II.

84

2.3 Evaluation of the compounded

production data for trials
I and II

 

 

As mentioned, it was found allowable to combine
the production data in trials I and II. Results of the
pooled AOV are in Appendix Table 1.12. With the spread
and changes in yield and composition of milk through the
course of these trials, variances and standard errors
naturally were large (Table 1.5). By taking out varia-
tions due to cows and time trend, however, the experi-
mental errors were reduced so that some significant
differences between infusion and control periods were
detected (Table 1.6).

The statistical evaluation confirmed impressions
from each of the trials that increased milk yield rather
than altered protein concentration was the primary rea-
son for increased protein production during the infusion
treatments (Table 1.6). Although the increments in pro-
tein production due to casein infusion were not great,
they were consistent and significant (P < .05). For the
last casein infusion, protein content was also increased
significantly (P < .05) over the controls. However, two
of the five cows showed a marked fall in milk protein
after the last infusion.

While the effect of the casein treatment on milk
protein concentration was small, overall averages were

higher than during glucose infusion (Table 1.6, row 3).

85

Statistical tests of this and similar comparisons were
not carried out because of the unequal time span between
the glucose infusion and the first and second casein
infusions.

Differences in time after parturition might be the
reason for the slightly higher milk production averaged
for all glucose infusions compared to all casein infusions
(Table 1.6, row 1). Still, only the casein treatments
increased milk yields significantly (P < .05).

The infusion versus control differences in milk
yield for the casein and glucose treatments (dK vs. dG),
were almost identical (1.1 vs. 0.9kg, Table 1.6). Only
for protein production (N x 6.38) did the differences
between casein and glucose approach significance (.05 <
P < .10). This difference (dK - dG), of only 229, equalled
about 4% of the protein production during the control
periods, or about 7% of the infused casein, but its validity
is strengthened by the observation that response to glucose
infusion (dG) never was larger than that for the aver-
aged casein infusions (d1 +d2/2 = dK) in any of the five
cases studied.

Significant increments (P < .05) in true protein
production were also noticed for the casein infusions but
not for glucose; even though the casein increased and the
glucose decreased the NPN content of the milk. With the

few observations, however, the responses (dK vs. dG) were

86

not significantly different at the conventional level of
probability (P < .05).

The influence of treatment on milk yield is also
evident in SNF production. While differences in SNF con-
centrations between infusions and controls were nil, SNF
production was increased significantly (P < .05 by casein,
but not by glucose. The lack of a difference in SNF con-
centrations between infusion and control periods suggests
that lactose and protein concentrations varied in opposite
directions.

Although the fat depression during the glucose
infusion was larger than for either of the casein infusions,
it was not significant at the conventional level of prob-
ability (Table l.6, row 4). This was probably due to
large variations associated with the glucose treatment,
for a smaller fat decrease during the first casein infusion
was significant (P < .05). A small apparent fat depres-
sion during the second casein treatment, however, was
largely due to an increase after the infusion, when milk
volume fell off (Table 1.5).

During the glucose infusion (period 4) the fat per-
centage fell gradually for all five cows, and no other
parameter showed such a gradual response to treatment

(Appendix Tables 1.5 and I.7). The most marked time trends
were noticed for the highest yielding cows. In period 5

fat percentages for the two days immediately after glucose

infusion were on an average lower than for the remaining

87

4 days (Appendix Tables 1.5 and I.7). They continued to
rise during period 6 which suggests a carry-over effect
and the reason for the lack of nonsignificant treatment

effect to the glucose treatment.

2.4 Blood parameters

 

Changes in blood parameters generally reflect the
availability of metabolites for synthetic purposes. In
these experiments it was also surmized that the fate of
the infused substrates might be more completely explained
by observing crucial blood parameters. Many factors, how-.
ever, will influence the blood level of a metabolite; and
the impact of a particular treatment may thus be obscured.
For example, the diurnal variations evidently prevented
some changes in plasma urea and a amino N from reflecting
infusion treatments.

a. Plasma Urea N.--The levels of plasma urea N

 

in these cows (Table 1.8, Appendix Table 1.10) were in
the normal range (e.g., Barry, 1964). Between days, within
periods there was no particular concentration pattern.
In several periods one or more observations were missing
for a given day. Data from other cows were excluded for
that same observation in order to give equal numbers for
the period mean of all cows.

Period means (Table 1.8) reveal a distinct effect
of treatment on plasma urea levels. There was a moderate

increase during the first casein infusion, then a dramatic

88

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89

decrease during the glucose infusion followed by a slow
recovery. After the last casein infusion, when the level
barely rose above the preceding control, there was again
a drastic fall which has no explanation. The elevated
level during casein infusion, however, can be regarded as
an expected consequence of deamination of infused protein.
The depressed urea level (P < .01), during glucose infu-
sion suggests less amino acid deamination because the
readily available glucose promoted protein synthesis (e.g.,
Potter, gp_al., 1968) or retarded protein mobilization.
Alternatively, the augmented glucose available to the
liver may have stimulated synthesis Of nonessential amino
acids. However, this probably would not alone have caused
the large Observed decrease in the plasma urea levels. In
any event, it is puzzling that such a marked and lasting
depression in plasma urea could result from the modest
glucose infusion in cows fed an abundance Of protein.

The higher plasma urea in the afternoon (PM
bleeding) than before the morning feeding (AM bleeding)
is probably a reflection of diurnal eating patterns be-
cause most of the feed was consumed between 8 AM and 5 PM.
Thus, absorption Of nutrients would be much lower in the
early morning hours than in the afternoon. Changes in

plasma urea are known to follow the deamination of feed
amino acids, with peak levels occurring a few hours after

a protein load (Knott, et al., 1972).

90

The interaction of casein infusion and bleeding
time on plasma urea was most Obvious for the first infu-
sion study and probably resulted from a load Of absorbed
amino acids during the post-fed state. Before feeding,
the infused amino acids were apparently handled without
increasing the plasma urea levels, but the addition of
infused protein in the postprandial state augmented the
urea synthesis and increased plasma concentrations. With
only the AM samples it would not have been possible to
detect changes in plasma urea due to casein infusions;
neither would the Observations have inferred the fate Of
the infused amino acids. Regardless the presence of a
treatment and bleeding time interaction, the PM samples
clearly suggest deamination Of infused amino acids.

b. Plasma 9 amino N.--Whi1e no quantitative

 

estimate Of infused amino acids was made, the concentra—
tion of 9 amino N in plasma (Appendix Table 1.11) did
increase during the casein infusion (Tables 1.8 and 1.9).
This would mean that the availability of at least some
amino acids for milk protein synthesis was increased.
Generally, the levels Obtained agree with those reported
for sheep (Fenderson and Bergen, 1972) and lactating cows
(Rook and Line, 1961).

For different reasons one or more Observations were
lacking for a sampling day in five of the eight periods;
and similar tO urea N data, these days were omitted to

permit an equal sample size for all cows in each period.

921

Table l.9.--Trial II 1970. Blood components: comparisons Of mean values for different
treatment periods and bleeding times (mean and difference between means

 

 

 

 

 

 

 

 

 

a
(diSEd).
T Col. NO. 1 Col. NO. 2 Col. NO. 3 Col. NO. 4 Col. No. 5
reat-
Parameter ment ControlsATO)
lst Casein Glucose 2nd Casein Cas. vs. Glc. only
mg% mg% mg% mg%
Urea N Tb 21.6 9.7 17.2 KC 19.4
g 18.4 15.8 15.1 G 9.7 h
d 3 2:1 49 -6.lil.1** 2.1:2 5 9.713.l
AM 16.9 13.4 13.0 14.0 14.3
PM 22.1 i 14.1 18.7 18.1 i 17.6 .
“a =§Tizo.8 ’UTSil.3 ‘37721.1** ‘ITIio.6 — . 2.67J
um/ml um/ml um/ml um/ml
0 amino N Tb 2.60 2.18 2.48 Re 2.47
O 2.49 2.24 2.13 G 2.18
.1lt.10 -.06t.06 .35t.19 .29t.02*
AM 2.85 2.35 2.31 2.46 2.43
PM 2.38 i 2.07 2.11 2.22 2.16 .
“a ‘TI7:.08 '7282.o4* ‘720:.11* ‘TII:.13 ‘TIV:.063

 

aStandard error of a difference between two means (see Table 1.6).

bThe average of control before (01) and after (02) infusion.
CThe average of first casein (K1) and second casein (K2) infusion.
dOnly control after infusion (02) was considered because change in the assay.

eOnly the second casein infusion (K2) used for comparison because change in
the assay.

fAsterisk indicates level of significance (see Table 1.6 gnuion, Appendix

Table I.14).
gSignificant, P < .05, but also significant treatment x bleeding interaction.
hSignificant, P < .01, but also significant treatment x bleeding interaction.
iSignificant, P < .001, but also significant treatment x bleeding interaction.

JSignificant, P < .01, but also significant bleeding x period interaction.

92

The 9 amino N concentration for all cows varied
considerably from day to day within a period (Appendix
Table 1.11), and period means for cows also differed in
levels as well as diurnal trends. These diversities are
expressed in an interaction between periods and bleeding
times for the first casein infusion (P < .01) and for
control periods (P < .001). For undiscovered reasons,
the 9 amino N levels in period 1 increased from the morn-
ing to the afternoon, but in no other period was this
trend noted.

Omitting the first sampling day for periods 4, 5
and 7, gave largely the same results for the treatment
versus control comparisons as obtained with the complete
set of Observations; indicating no systematic carry-over
effects.

For the second casein and the glucose infusions,
the AM values were significantly (P < .05) higher than
the PM values, and no interaction between sampling time
and treatments was shown. The high concentrations in a
amino N before feeding (Table 1.9) may have been due to
a low availability of energy yielding metabolites, mainly
volatile fatty acids (VFA). Diurnal patterns in plasma
9 amino N levels in sheep reflected the energy supply
(Fenderson and Bergen, 1972). Still it seems contradictory

that the 9 amino N level was high in the morning when

plasma urea N was low, and vice versa. It might be that

93

mobilization of protein for gluconeOgenesis in the pre-fed
state resulted in an elevated amino acid level, but this
possibility seems remote. Rook and Line (1961) generally
found higher a amino N levels in cows 5 to 8 than 2 hours
after feeding, with a peak at 5 hours. The difference
between 2 and 8 hour samples were more marked in well-fed
than under—fed cows (Rook and Line, 1961).

Although two samples a day is hardly indicative
of an average daily value, the infusion Of casein at
least tended to raise 9 amino N concentration above con-
trol values, while glucose infusion failed to show this

tendency.

3. Discussion

 

Although abomasal casein infusions in these ex-
periments did consistently raise milk and protein production
(P < .05 or lower, Table 1.6), the increments were not
dramatic relative to control values. More data are needed
to verify that improved amino acid supply to the mammary
gland was the reason for observed responses. The validity
Of this hypothesis has been supported by more recent
experiments in our own laboratory and elsewhere. Shortly
after these experiments were completed, Broderick, e£_al.,
(1970) reported positive response to abomasal infusion of
casein. They used three cows in a similar change over

design. The cows averaged 31kg milk per day, and the

94

infusates furnished about 8009 casein + 249 methionine daily
for one week. Treatments increased milk protein produc-
tion (N x 6.38) llOg/day, or 11.6% (P < .05); compared to
an average of 509, or a 9% increase (P < .05 or lower) for
casein infusions of 3009/day in our experiments with cows
averaging 16kg per day. They reported a nonsignificant
increase in milk yield 1.2kg/day) compared to 1.2 (P < .05)
and 0.9kg (P < .05) increments for casein infusions in our
study. Milk protein in our experiments increased only 0.07
percent compared to 0.20 percent (P < .10) Observed by
Broderick, ep_al., (1970).

Although the Wisconsin workers noticed a 10% (P <
.5) decrease in grain intake during casein infusion, con-
sumption during control periods was not sufficient to meet
the cows' energy requirements according to common standards.

On the other hand, Broderick, gp_al., (1970) noted
no response in protein production or percent or in milk
yield to infusion of glucose and urea designed to be
isocaloric and isonitrogenous to the casein and methionine
infusion. In the present experiments, however, the milk
yield was increased about as much by glucose as by casein
infusion while the influence Of glucose treatment on pro-
tein content was nil (-0.3% change). Negative responses

in milk protein content, milk yields, and protein produc-

tion resulted from duodenal infusion of glucose in the

studies of Spechter (1972). Increases of about 30% in

95

milk protein production were observed by Spechter (1972)
when casein was infused into early lactation cows on a
ration with 40-45% of total N as NPN. A negative N
balance and deficit of natural protein in the ration may
explain the greater responses Obtained in that study.

As might be expected, the efficiency of conversion
of postruminally infused protein to milk proteins have
usually been inversely related to the level of treatment.
Thus, for the lowest rate of casein infusion in Spechter's
(1972) experiment, 74% was recovered as milk protein while
the fractions for the medium and high treatments over 54
and 36%, respectively. Even the latter value is higher
than apparent recovery Of 17% in our experiment (supplying
3009/casein per day), and 13% in that of Broderick, g£_gl.,
(1970); where 8009 protein was infused.

Tyrell, gp_al., (1972) recovered 24% of 8609
abomasally infused casein in two cows producing approxi-
mately 24kg milk per day. The treatment increased milk
yield by about 3kg/day. Infusing 433g casein in one cow
(Tyrell, gp_al., 1972) increased milk yield 2kg/day, but
only 12% of the protein was recovered in the milk. Judging
from these reports, the extent of recovery of the infused
supplement depends on nutrient adequacy of the ration and
physiological status of the animal, as well as level Of

protein infusion.

96

It should be noticed that the milk protein con-
tent in the studies discussed here refer to N x 6.38 with
the exception of Spechter (1972), who applied an infrared
spectrum analyser to determine true protein. While it is
well documented that feed protein above accepted standards
will not change SNF or protein content of milk (Huber and
Boman, 1966), the NPN fraction may increase significantly
by high levels of digestible protein (Storry and Rook, 1962;
Senft and Klobasa, 1969).

Casein infusions slightly raised (P < .05) milk NPN;
whereas, glucose infusion depressed this entity (P < .05).'
Others have not reported fractionation of N components in
studies where postruminal infusions have enhanced milk
protein secretion.1

The differential changes in milk yield and protein
concentrations for the various studies suggest that the
consistent increments in milk protein production originated
by different routes. Thus, the infusates apparently
elicited different metabolic or secretory mechanisms in the
cows in different experiments. For example, cows in both
Wisconsin (Broderick, gp_al., (1970) and Canadian (Spechter,
1972) studies responded to casein infusion with larger in-
creases in milk protein concentration; perhaps because of

a lower intake Of dietary protein relative to needs, than

in the current experiments.

 

1After completing this manuscript it was learned that
Broderick (1972) measured milk NPN when feeding formalinized
casein.

97

Milk protein concentration per se apparently does
not exert a strong feedback influence on its synthesis as
Rook and Line (1965) found substantial increases in protein
concentrations and unchanged protein yield when milk volume
was lowered in insulin-treated cows with depressed plasma
glucose levels. In vitro studies, on the other hand, sug-
gested an end product inhibition of a lactalbumin synthesis
in bovine mammary cells (Larson, 1969).

The depression in milk fat in our study was of a
magnitude similar to that reported by Derring, gp_al.,
(1972) and Spechter (1972), but there was no fat decrease
in the experiment Of Broderick, gp_al., (1970). Apparently
a lower threshold for dietary influences on milk fat con-
tent exists in cows producing large amounts Of milk on a
gluconeogenic metabolism (Orskov, gE_al., 1969).

The increased plasma urea level during casein in—
fusion in our second trial suggests increased gluconeo-
genesis resulted from a greater absorption of amino acids.
This seems very likely according to the scheme Of Krebs
(1963). The study Of Derring, gp_al., (1972) also indicates
that infused amino acids were deaminated for further cata-
bolism. Plasma glucose, however, was not higher during
abomasal than ruminal casein infusion in the experiment Of

Derring, et al., (1972), but this agrees with Wright, et al.,
(1966) who showed that 35kg sheep may handle 3509 exogenous
glucose per day without a raise in blood glucose concentra-

tions.

98

In lowering milk fat content the abomasally infused
casein resembled a high starch-low fiber diet. Such a
ration characteristically yields a depressed acetate to
propionate ratio in the rumen, enhances rumen bypass of
starch, and probably increases glucose absorption (Van
Soest, 1963; Wright, ep_al., 1966; Orskov, gE_al., 1969).
Moreover, such a change in metabolism usually depresses
milk fat and increases milk protein (Rook and Line, 1961;
Huber and Boman, 1966). Ruminal additions of propionate
have increased milk protein and depressed milk fat (Rook
and Balch, 1961; Storry and Rook, 1962; Halfpenny, gE_al.,
1969); but when ruminally infused propionate replaced 15%
Of ME for 6 weeks in a fat-depressing ration, milk protein
content as well as milk yield were lowered compared to the
basal ration (Orskov, gp_al., 1969).

An effect Of energy form, generally starch versus
cellulose, is often hard to separate from that of energy
level since a high rate of energy supply usually is achieved
by higher grain feeding (Huber and Boman, 1966). Rock and
Balch (1961) imply that high energy levels will increase
milk SNF and protein when "additional" energy from grain
amounts UO4000kcal or more per cow. However, Yousef,
gp_gl., (1970), found increased milk protein concentration,

particularly the a casein and B lactoglobulin fractions,

resulted from increased energy concentration in the ration.

They also demonstrated that ruminal VFA changes do not

99

always accompany the milk protein increments. Addition of
sodium bicarbonate and magnesium oxide in the concentrate
for cows on high-grain, low-roughage ration changed the
ruminal acetate/propionate ratio toward that of normal
feeding, and partly corrected a milk fat depression, but
the milk protein content still remained as high as on the
ration not supplemented with the salts.

These studies (Yousef, ep_al., 1970) suggested a
greater capacity for protein synthesis by the mammary gland
of cows on a high grain ration; apparently independent Of
a high rumen propionate. The swiftness of this reaction
is not known; but Rock (1971), on the other hand, contended
that increased protein secretion by propionate infusion has
a lag phase of 2-3 weeks. Such a long term induction would
indicate another mechanism than that seen in the experi-
ments where postruminal protein infusion spontaneously
increased the milk protein production. Still, the different
experiments do not exclude an impact Of a glucogenic type
Of metabolism as defined by Orskov, ep_al., (1969). Even
though Yousef, gp_al., (1970) imply that propionic acid
was not critical for raising milk protein, the relatively
lower propionate to acetate ratio in the rumen after feeding
NaHCO and MgO may have been accompanied by a greater

3
glucose absorption from the postruminal digestive tract

(Wright, et al., 1966).

100

Armstrong and Prescott (1971) concluded in a review
article that the stimulating effect of propionate on milk
protein secretion probably is mediated through the sparing
of amino acids for gluconeogenesis by the liver. The same
authors (Armstrong and Prescott, 1971) also pointed out that
glucose and propionate initiate different endocrine actions
(Lindsay, 1970): whereas glucose stimulates insulin secre-
tion; propionate stimulates both insulin and glucagon. The
impact of route of introduction of these metabolites can
also be extended from the work of Fisher and Elliot (1966)
where intravenous infusion of propionate and glucose failed
to increase milk protein; but both treatments lowered the
milk fat content. On an equicaloric basis, glucose caused
a more severe fat depression than did propionate. Since
these infusions lasted four days, an effect on protein
secretion might not be expected (Rook, 1971).

Although abomasal glucose infusion in our experi-
ments increased the milk yield short of significance
(P > .05), its effect resembles that Of the intravenous in-
fusions of glucose and propionate (Fisher and Elliot, 1966)
which significantly (P < .05) increased milk yield and
lactose concentrations (P < .10). Ruminal propionate, how-
ever, failed to raise the yield of milk while milk protein

secretion was increased (Rook and Balch, 1961; Rook, et al.,

1965).

101

Volatile fatty acids added to concentrate tend to
depress intake, but Jones (1971) demonstrated that ME and
feed protein were most efficiently used for milk production
at maximal acetate levels. Although 25-30% of the glucose
taken up by the lactating cows' mammary gland may be
oxidized (Annison and Linzell, 1964; W-od, e£_al., 1965)
acetate is apparently a more critical energy substrate
(Rook and Hopwood, 1970). Propionate, on the other hand,
inhibits acetate utilization by the sheep liver (Pennington,
1957); and may thus influence the mammary metabolism
although hardly any propionate reaches the udder.

The specific effect of ruminal propionate in in-
creasing milk protein secretion was independent Of ration
composition in one experiment by Rook, §£_al,, (1965). In
the two trials reported herein, response in protein yield
to casein infusion was similar on a normal or a high-grain,
low-forage ration. However, our increases in milk protein
were due largely to higher milk yields and not to increased
protein concentration as reported (Rook, gp_al., 1965) after
infusion Of propionate into the rumen.

In discussing changes in concentrations of the
major milk components; Wiegner's law, according to Kirch-
gessner, gp_al., (1967), implies that the concentration

stability of a milk component is inversely proportional to
its degree Of dispersion. Thus, the content Of fat, the

least dispersible component, is most easily altered,

102

followed by casein, the other milk proteins, lactose, and
salts. Consequently, a metabolic change affecting syn-
thesis Of all milk components appears most easily in the
fat secretion, and the lactose will be more stable than
protein.

A more modern View on milk secretion, stated in
biochemical terms, contends that the rate of milk-fat
secretion may vary independently from that of the other
constituents (Silcock and Patton, 1972). However, Silcock
and Patton (1972) found closely related secretion rates for
milk protein, lactose, and ionic potassium. Supported by.
related observations these authors (Silcock and Patton,
1972) suggest that lactose, protein, and K+ are secreted
together from the Golgi apparatus of the alveolar cell.
This, however, seemingly would not need to exclude a dif-
ferent rate of synthesis of protein and lactose if the
substrate, energy or hormonal exposure of the mammary cell
were varied.

Rook (1971) has pointed out that the rate of uptake
Of nutrients by the gland may modify milk secretion not
only through a specific precursor-product relationships,
but also by altered supply of substrate for ATP production.
German workers have found that pyruvate concentration of

the mammary gland changes with the season in positive cor-

relation with the content Of casein in the milk (Wald-

schmidt, 1973) as influenced by nutrition (Kirchmeier, 1970).

103

Moreover, the content of nonessential amino acids increased
with the casein content of the milk (Kirchmeier, 1970).

Regardless of metabolic mechanisms involved, an
increased protein output means more amino acids lost from
the animal, and, consequently, more amino acids were re-
moved from the blood by the mammary gland. Yousef, g£_al.,
(1970), however, did not find increased AV differences Of
9 amino N when milk protein production went up on a high—
grain, low-roughage ration. But such differences are of
limited value if not accompanied with blood flow data
(Linzell, 1971). Since milk yield largely determines the_
mammary blood flow (Linzell, 1971), a substrate's AV con-
centration difference may stay fairly constant even though
the gland's actual uptake differs due to changes in pro-
duction.

In simple terms, an increase in plasma amino acid
concentration should indicate improved conditions for
protein synthesis (Munro, 1970). In the second trial here,
when 0 amino N was measured, its concentration did tend to
increase in tail blood plasma during casein infusion com-
pared to the control; and it was significantly higher
(P < .05) during casein than during glucose infusion. Like-
wise, Rook and Line (1961) found elevated levels of 9 amino

N in jugular venous plasma when feeding a high energy
ration that promoted increased milk protein production. It

can be calculated from the data of Broderick, et al.,

104

(1970) and Spechter (1972) that abomasal infusion in both
studies increased the level Of total amino acids in plasma
and whole blood. More noteworthy, though, was a higher
ratio of essential to non-essential amino acids, a trend
known to indicate an improved amino acid status in ruminants
(Oltjen and Putnam, 1966) as well as single-stomached
animals (Munro, 1970).

Dietary supply influences plasma free amino acid
concentrations despite a high buffering capacity through
continuous protein catabolism (Wannemacher and Allison,
1968) and hormonal regulations (Munro, 1970). Because of-
selective membrane transport mechanisms, absolute and rela-
tive concentrations of amino acids may vary widely between
tissues and plasma (Wannemacher and Allison, 1968; Munro,
1970). As an augmented milk protein output causes a
stronger drain on the amino acid pools, the plasma level of
particular amino acids may be lowered, at least on a molar
basis because those presented to the gland will not fit the
pattern demanded for milk protein synthesis. Furthermore,
diurnal variations in cows' plasma amino acid levels may
be substantial (Halfpenny, gE_al,, 1969) although less than
in simple stomached animals (Champredon, ep_al., 1969).

Because milk secretion rates are quite constant
over normal intervals and production levels (Linzell, 1960;
Tucker, gp_al., 1961), and in view of the numerous factors

affecting supply and demand of amino acids, there probably

105

is a regulated uptake by the mammary cell; as suggested by
Rook (1971). As yet, amino acid transport into mammary
cells has received little attention.

Regardless of complicated systems regulating amino
acid availability, enhanced efficiency of protein nutrition
by amino acid supplement requires that the limiting amino
acids be introduced in a quantitatively tailored manner
(Allison, 1963). Identification of critical amino acids

therefore becomes a central part of this topic.

 

 

 

II. SECOND SERIES OF EXPERIMENTS (1971)

i. Trial I 1971

1. Methods and material

 

1.1 Rationale for treatments and design.

 

While the first series of experiments indicated the
availability of amino acids was more critical for milk pro-
duction than was glucose, a stimulating effect of improved|
glucose supply could not be excluded, and the estimated
responses were possibly due to a combined effect Of glucose
and amino acids. Thus, the likelihood for an interaction
effect of the two substrates was tested in an experiment
aimed at verifying the results Of the first series Of
experiments.

Intending to relate to our earlier findings, the
mode of treatments were kept as previously, but the rate
Of supplementation was according to milk production.

Thus, abomasal protein infusion equalled 75% Of the milk
protein output in one treatment, with equicaloric glucose
infusion in another; and the third was a mixture Of
equicaloric amounts of protein and glucose. The mixture
was infused at the same rate as the individual substrates,

although for testing an interaction effect it might have

106

107

been more apprOpriate to keep the infusion rate Of each
substrate unchanged. The reciprocal influence on the
availability Of the two substrates, however, suggested

the total supplement ought to be equal for all treatments.
Originally six cows were available for placement of
abomasal cannula. But because two cows were lost shortly
after surgery and there was doubt as to the availability

Of a third cow, only three out of six cows were used
initially. In order to include a control treatment and the
three supplements described, the regular Latin square design
was modified to that presented in Table 2.1.

Experimental periods lasted seven days, with the
first day for transition and then two three-day sub-
periods. When the fourth cow (NO. 603) was recovered
from surgery, she received the casein infusion because
it was of primary interest. Although not included in
means and statistical analyses, observations from this
cow have been recorded in order to strengthen the overall
conclusions from the data.

Casein is relatively poor in methionine and other
S-containing amino acids; and methionine supplementation
greatly improves the EV of casein for growth (Allison, pp
31;, 1959). Moreover some experiments have shown that
feeding methionine was beneficial to lactating cows

(McCarthy, et al., 1968; Polan, et al., 1970; BishOp and

108

Table 2.1-—Trial I 1971. Experimental design and timing
of periods.

 

 

Sub Obsv. Cow No.
period per. beg. end 604 606 607 603a
NO. No. days Infusion Treatment
1 1 6/24-6/26
2 6/26-6/29 O O O
2 1 7/2-7/4
2 7/5-7/7 G K K
3 1 7/9-7/11
2 7/12-7/14 O O O
4 1 7/16-7/18
2 7/19-7/20b M G K
5 1 7/24-7/26 o 0 0 o
2 7/27-7/29
6 1 7/31-8/2
2 8/2-8/5C K M G K
7 1 8/10-8/12
2 8/13-8/15 O 0 O O

 

Symbols (infusion treatment):

0 = saline control (volume as for the other
infusions)

K = caseinate + 3% dl—methioni equicaloric,

G = glucose fl} together sym-

M = mixture (50/50) of K and G bolized T

8Results for cow No. 603 are not included in any
mean or statistical analysis.

bDay 7/21 was discarded due tO improper milking.

CA third 3—day infusion followed, but only 2 sub-
periods were used: No. 604 was Obviously not well 8/1—2,
infusion was faulty in 603 and 606 8/2-5, 607 was in heat
8/7-8; the affected subperiods thus discarded.

109

Murphy, 1970), but contrasting results were shown
(Broderick, et al., 1970; Burgos and Olson, 1970;
Williams, et al., 1970; Begum and Jones, 1972). There-
fore the caseinate was enriched with 3% dl-methionine2
which was similar to the supplement used for abomasal
infusion by Broderick, et al.,(l970).

For all the following experiments sodium case-
inatel was dissolved in tap water by heating to
55-60°C and stirring occasionally. The glucose solution
was made from cerelose.3

With the ME Of casein protein being 4.6kca1/g and '
that of glucose 3.8kcal/g (Maynard and Loosli, 1962, p.
322), and sodium caseinate 85% protein, equal weights case-
inate and cerelose yield that same ME.

In control periods saline (0.9% W/V NaCl) was in-
fused at a volume similar to the substrates. The infusion
rates were derived from milk protein production for three
days preceding the trial, and are shown in Table 2.2.

The substrate concentration for each cow was
regulated to fit a volume Of 10-121 for 24 hours. In-
fusion was achieved with multichannel pumps,3 each serving

two cows. Ruptured tubes and other malfunctions of the

 

lSodium caseinate, from Nutritional Biochemicals,

Cleveland, Ohio. Typical analysis 5% moisture and 92.5%
protein (NXG.38) in dry material.

2Obtained from Nutritional Biochemicals, Cleve-
land, Oh§o.

The same as in 1970, see section I, 1.1.

110

Table 2.2-—Trial I 1971. Substrate infusion rates derived
from pre-trial protein production.

 

Amount infused

 

Protein
Cow Milk Protein yield Total Energy
yield content (NXG.38) Proteina solutionb (est.ME)
kg % g g g Mcal
604 25.9 2.7 700 525 650 2.37
606 22.2 2.7 600 450 550 2.09
607 23.6 3.0 700 525 650 2.37
603 18.1 3.1 580 435 550 2.09

 

a75% of daily protein production.

bThe same for Na-casinate and cerelose, Na-
caseinate being 85% crude protein.

infusion system occurred at times, particularly in the
beginning,but the total infusion over 24 hours was usually
very close to that intended.

1.2 Animals and abomasal cannulation.

 

Six cows were bought for these eXperiments, four
within a month after calving. One was a first-calf heifer,
the others were starting their fourth to eight lactations.

Because it was desirable to use the same cows for
experiments requiring abomasal sampling, a cannula with
31 mm outer and 22 mm inner diameter was used.

The cannula was made from liquid plastic4 which

was heated to ~80°C before being poured into a form.

 

4Plastisol, liquid plastic material from U.S. Stone-
ware, Inc., P.O. Box 350, Akron, Ohio.

111

Air bubbles in the liquid material were removed by vacuum
and gradual cooling was necessary for satisfactory quality.
Consisting of one piece of pliable plastic, the cannula
had an 8cm flange that prevented it from being forced out
of the fistula Opening.

The cows did not receive any feed for 24 hours pre—
ceding surgical insertion of the cannulas at the MSU
Veterinary Clinc.5 One attempt to Operate on a cow (No.
603) while standing was abandoned because the abomasum
could not be moved to a desirable position. Later this
cow was cannulated as the other cows, but an infection
in the abdominal wall after the first Operation delayed
the second incision.

Surgery was similar to the previous year, but the
cows were laid down on a foam-covered floor using nitrous
oxide as anesthesia. The first cow Operated upon in this
manner (No. 602) did not recover from the anesthesia
until three hours after the Operation; and she had a poor
appetite for several days thereafter. A few weeks later
this cow ruptured and lost her cannula to the interior
Of the abomasum; so she was slaughtered.

In Operating on Cow NO. 605 a second incision was
required to locate the abomasum. Apparently this cow had

not been deprived of food for the prOper period before

 

5Dr. D. J. Ellis supervised the surgery aided by
staff and students in Large Animal Surgery and Medicine.

112

surgery. She was very weak and showed inappetance after
the Operation; still milk production stayed up quite well.
However, she contracted severe mastitis and never became
fit for eXperimental use.

The surgery in cows NO. 604, 606 and 607 was with—
out complication, lasting around two hours.

l.3 Feeding and feed sampling.

 

Both net energy and total protein intake were in-
tended to be higher than NRC (1971) standards. Since the
smallest cow (No. 604) produced more milk than the other
two, the same amount of feed was offered to all three cows
(Appendix Table 11.1). The daily concentrate ration was
devided in two feedings and fed at milking (at 8AM and
4 PM). Corn silage was fed around 10 AM and hay around
4 PM.

Samples of feeds were Obtained on days 2, 4, and
6 Of each period, and DM was determined immediately by
oven drying. Period composites Of corn silage were frozen
at -20C; and hay and concentrate composites were dried,
ground and stored for chemical analysis. Feed refusals
were weighed and sampled as the feeds. However, the same
DM values were used for the refused as for the fed corn
silage, because this was always mixed with concentrate.

A few DM values were Obtained for concentrate refusals

which were averaged for calculating intakes.

113

1.4 Milking and milk sampling.

 

Milk sampling and handling of the samples was
similar to earlier experiments (section I, 1.5). Two
composite milk samples were prepared, one for the first
three days and the other for the last three days Of each
period.

1.5 Blood sampling.

 

On the last day of each period tail blood was
sampled at three times; before the AM feeding (BO z 7:30 AM),
3 to 3.5 hours after feeding (B3 a 10:30 - 11:00 AM), and
8.5 to 9 hours after the morning feeding (89 z 4:30 - 5 PM).
The blood was drawn into vacuum tubes without anticoagulants;
but was immediately poured into 50ml centrifuge tubes con-
taining 40mg potassium oxalate and 50mg sodium fluoride.
The centrifuge tube lwas stoppered and placed on ice until
further processing in the laboratory, which usually com-
menced within 30 minutes following the sampling. After
whole blood was sampled for the urea determination, plasma
was Obtained by centrifuging at 5000Xg for 10 minutes.

1.6 Chemical assays.

 

Chemical assays were generally as for the 1970
trials, and only additions or differences from previous
practice will be mentioned.

(a) Feed analyses.—-Crude fiber (CF) was determined

 

on hay and corn silage only. This assay and N in hay and

114

corn silage were carried out by the Forage Analysis
Laboratory in the MSU Department of Biochemistry.

(b) Milk analyses.-—Total solids were determined

 

gravimetrically by drying according to AOAC (1955). After
weighing about 2ml milk in drying pans as previously (sec—
tion I, 1.8b), the pans were placed on a steaming water
bath for 30 minutes before drying in oven at 100°C for four
hours.

Total nitrogen was determined by the macro Kjeldahl

 

procedure (AOAC, 1955) using 3m1 milk in triplicates for
each sample.

Non protein nitrogen (NPN) was separated from the

 

protein N by trichloro acetic acid (TCA) precipitation as
described by Mahan, et al. (1971). Eight m1 of milk was
weighed in a 50ml centrifuge tube. After adding 24ml 15%
(W/V) TCA, it stood at room temperature for 1 hour and was
then centrifuged at BOOOXg for 10 minutes. The super-
natant was filtered over aWhatman No. 42 filter into a
50ml volumetric flask. Ten m1 more TCA was added to the
precipitate which was stirred, recentrifuged, and the
supernatant filtered into the same flask. The volume was
finally brought to 50ml with additional washings of 15% TCA.
The protein free filtrate was frozen until assayed

on an auto analyser,6 but the filtrate had to be concentrated

 

6TechniconR Auto Analyser,R see section I, 1.8.

115

4 times by evaporation on a steam water bath in order to
reach a detectable N concentration. This inconvenience
could have been avoided by using a more concentrated TCA
:Mflution to a larger amount of milk to achieve ~ 10% TCA
in the final volume.

Lactose was determined by a method according to
Hinton and Macara (1928), modified after Ling (1956). A
lactose carrying filtrate was obtained by tungstic acid
precipitation and stored frozen until the lactose content
of the filtrate was quantified by iodometric titration.
The lactose determination in this trial was not as precise
as desired. Despite repeated determinations on the
triplicates obtained from each milk sample the variations
were large within sample (day) and between days in period
for any cow, and results for period 1 was discarded.

(c) Assays of blood constituents.-~Urea N was
determined according to Coulombe and Favreau (1963). One
ml oxalated blood was pipetted into 25ml centrifuge tubes
with addition of 9ml of tungstic acid reagent, made from
8 volumes N/12 sulfuric acid + 1 volume 10% (W/V) sodium
tungstate just before use. After standing 10 minutes
upon shaking the tubes were centrifuged for 15 minutes at
8000Xg, the supernatant decanted off and frozen until

final assay.

116

Then, 0.4m1 of the blood filtrate was taken out and
mixed with 10ml of "reagent A" prepared just before use
from 10 volume 60% (W/W) ortho phosphoric acid (H3PO4) and
2 volume DAM-TSC solution (0.6g diacetyl monoxide + 0.03g
thiamine carbazide in 100ml water). Urea water standard
solutions of appropriate concentration were prepared simi-
larly. Sample, standard and reagent blank preparations were
boiled together for 20 minutes as the tubes were sealed with
glass beads. After a quick cooling in cold water the Optical
density was determined by spectrOphotometry read at 540nm.7
Urea N concentrations were calculated by regression formulas
derived from the standards. Plasma glucose was deter-
mined enzymatically7’8 in protein free filtrate of plasma

prepared at time of sampling, frozen until the final assay

2—3 months later. Plasma free amino acid determination will

 

be described in section III.
1.7 Calculations.

Estimated net energy (ENEL) for corn silage and
hay in each period was calculated from CP and CF in DM
according to values in NRC (1971) feed tables. For the
concentrate mixture, each ingredient was assigned a NE

L
value according to NRC (1971) feed tables.

 

7Using a Gilford SpectrOphotometer, model 2000, Serial
650; Gilford Instrument Lab., Inc., Oberlln, Ohlo.

8GlucostatR, enzymatic glucose determination, from
Wortington Biochm. Corp., Freehold,New Jersey.

117

Milkyproduction parameters.——From the production of
milk constituents for every sub—period a weighted mean was
derived for the concentration in the milk for the whole
period. For the concentrations of milk constituents as
well as production, the averages for saline periods before
and after casein, glucose and mixture were used as con—
trols. Since this leads to repeated use of periods 3 and
5, the control periods as presented in Table 2.1 are not
completely independent.

Because the fraction of NPN/total N in milk were
all around 5%, these values were transformed by the arcsin
function (Rohlf and Sokal, 1969, Table K) for statistical
analyses.

1.8 Statistical analysis.

 

Since the order of the three infusion treatments
were randomly allotted to three cows (Table 2.1), these
treatements as well as the differences (dT) between the
treatments and averaged pre- and post-treatment controls
form a 3X3 Latin square (e.g., Cochran and Cox, 1957).

Thus, the estimated responses (dT) were used for an analysis
of variance (AOV) according to the Latin square design
(Anova I-l, Appendix Table II.8) although this renders

only two degrees of freedom (df) for the

118

error mean square (EMS) and a very low power of the F-test.
Still the magnitudes of the F's for different parameters
and the relative size of mean squares should indicate the
impact of the different treatments as sources of variation
(Appendix Tables II.11a and 11.12).

The absolute production levels were used for two
additional AOV, attempting to draw benefit from the several
measurements in each animal to enhance the df for the EMS,
thus strenghtening the power of the test. Furthermore, it
was desirable to single out an effect of bleeding times.
Hence, each of the three substrate treatments were com—
pared to adjacent controls as outlined in Appendix Table
II.9 (Anova I-2).

Although Anova I-2 yields the variance among con-
trols before and after treatment as well as the variance
among treatments and controls (orthogonal contrasts), the
repeated use of control periods implies that the difference
between controls has no relevance. Furthermore, because
the cows received the specific infusions in different
periods, this AOV assumes no effect of time and treat—
ment sequence.

Direct comparison of the actual performance at
each treatment and averaged adjacent controls was carried
out according to the arrangement in Appendix Table II.10.

Again it had to be assumed that the overlapping of control

119

periods had a neglible effect on the estimated variances.
In addition it was assumed that there is no interaction
between cows and sequence of treatment, neither that
treatments and associated controls interacted with sequence
or cows. The controls for different treatments naturally
are not randomly distributed, but this objection was not
considered serious since the difference between controls
was of no interest in itself. Considering three separate
controls, however, gave a balanced design.

Since all three bleedings occurred in each treat-
ment and control period, a split plot pattern was used

(Anova I-3, Appendix Table II.10b).

2. Results

2.1 Feed intakes.

 

Since the same amount of feeds were offered through-
out the trial (Appendix Tables II.2 and II.3) variations
in consumption of dry matter (DM) and nutrients reflect
feed acceptability and composition (Appendix Table II.1).
The hay quality (Appendix Table II.1) varied from
extremely poor in period 4 to very good in period 7. For
the latter period the corn silage was also more acceptable
than earlier as indicated by higher consumption. Thus,
the intakes of CP and ENE were higher during period 7 than

other periods (Table 2.3).

120

Table 2.3-—Tria1 I 1971. Consumption of dry matter, crude
protein and estimated net energy (mean for
treatment periods).

 

 

Period 1 2 3 4 5 6 7

 

Control/Treatment Oa Tb O T O T 0

Dry matter, kg/day 16.2 15.5 16.3 16.2 15.8 16.1 17.0
Crude protein, kg/day 2.38 2.46 2.44 2.11 2.35 2.47 2.67
Est. NEL, Mcal/day 28.8 26.8 28.8 28.0 27.6 28.8 30.5

 

aSaline. bSubstrate infusion (see Table 2.1).

Generally, the cows refused much of their corn
silage, but the intakes of CP and ENE (Table 2.4) still
surpassed the NRC (1971) standards (Appendix Table 11.4).
Except for period 7 there was no real tendency towards
greater overfeeding at the end of the trial and milk
production remained stable.

Despite time trends and feed quality changes there
was a small but consistent tendency for lower intakes during
treatment than control periods (Table 2.4), and the
F value for all treatments versus all controls approached
significance (P ~.10).

Analyzing estimated treatment responses (dT) by
Latin square (Anova I-l, Appendix Table II.11a) showed a
significant period effect (P<.05) on CP intake, evidently
dueto low intakes in period 4 when the hay quality was
low. ENE was affected similarly but to a smaller degree.

Adding the infused protein to CP eaten for cows No. 604

121

Table 2.4--Trial I 1971. Summary of feed intakes: mean
comparisons between treatment and control

 

 

periods.

Infusion Dry matter Crude protein Est. NEL
Treatment kg/daya kg/daya Mcal/daya
(N=3)

K 15.9 2.55 28.0

OK 16.4 2.47 29.2

d -.5:.57 -.08:.12 -l.2:l.22

G 15.3 2.29 27.1

()G 15.8 2.41 28.2

d -.5i.46 -.12i.12 -.§i.98

M 15.8 2.40 28.4

OM 16.0 2.44 28.6 _

d -.2:.78 .04:.13 -.2i1.03
(N=9)
All T 15.7i.23 2.35:.06 27.8:.50
A11 0 16.1:.21 2.44:.03 28.7:.41

-.4 -.09 -.9

 

aMean values, and SEd = standard error for the
difference between two means for each infusion study, and
SE = standard error of the composited means. See also
Appendix Table 11.11.

and 607 in period 4 elevated total protein supplied to
above the standard requirements.

2.2 Milk production and composition.

 

Milk yield.--Milk yields were consistently in-

 

creased by treatments, with the casein effect greater than

in previous experiments. Because of the few replicates, however,
responses (dT) were not significant for any particular

treatment (Table 2.5; Appendix Table II.12), but dK and dM

exceeded dG (P<.05, Table 2.5, Appendix Table II.12).

1122

 

 

 

 

 

(Betw.dT)

Table 2.5--Tria1 I 1971. Milk production parameters; treatment and control means (m:SE),
differences between treatments and controls (dtSEd), and results of statistical
analyses.‘3

Column: 1 2 3 4 5 6
Est. True Protein
ANOVA Milk yield FCM rod. Prot conc. Prot prod. conc. prod.
for F test Trtm. m t SE m SE m t SE m t SE m 1 SE m SE
(N=3) kg kg 8 g 3 9
K 26.03 11.00 21.3 i .83 3.19 t 12 833::60 3 02.t.12 788 t 56
0x 24.37 21.39 39.8 21.19 3.00 2.11 731 :51 2.86 1.10 697 t 47
1‘2 d 1.66 t .80 .5 i .98 .19 1.051* 101 122** .16 1.054. 91 i 22“
(d%) 7.0 2.5 6 14 6 13
G 23.85 31.29 18.8 11.27 3.03 2.13 722 137 2.89 1.12 686 t 31
06 23.28 1 .79 19.3 11.61 2.95 2.13 682 :22 2.81 t. 12 651 t 21
d .57 t .64 -.§ 11.03 .08 1.052*) 40 :26 .08 i .043‘) 35 t 24
I—2 (d%) 2.5 -2.5 3 6 3 5
M 25.66 $1.50 19.9 t .99 3.17 814 :59 3.01 :.11 773 z 59
0M 24.52 11.41 20.4! 1.87 3.00 735 :47 2.85 .06 669 t 47
I-2 d 1.14 2 .65 -.53 .97 .17 t.069* 79 137*) .16 1.044* 74 z 38*)
(d%) 4.5 -2.5 6 11 6 11
(N=9)
T 25.18 1 .72 20.0 t .69 3.13 2.065 790 131 2.97 1.061 750 1 30
O 24.04 i .71 20.2 t .82 2.98 3.050 716 :23 2.84 $.04? 682 t 12
I-3 d 1.14 t .69* .2 t .83 .13 t.041** 74 :29** .13 t.041** 68 t 28"
(d%) 4.5 -1 5 9 5 10
1-3 (Betw. 'r)KM>G* KM>GH KM>G** KM>G** KM>G**
I - 1 (Betw.dT)KM>G* KM>G' KM>G'
aSymbols for probable significance: :)= p<. 10
= P<. 05
8* = P<. 01
"* = P<.001
Table 2.5--Continued.
ANOVA Column: 7 8 9 10 11 12
for F test NPN conc. NPN/Total N Fat conc.Lactose conc. SNF conc. SNF prod
m t SE m 1 SE m 1 SE m 1 SE m SE
(N=3) mg/100m1 8 8 g 8 g
n 27.4 5.5 2.80 4,37 8.47 2207
OK 21.6 4.6 3.03 5.07 8.42 2054
I-2 8.5 t.29*‘* .9! 19‘** - .23! .16 L_26 1_11 . $.11 153 164')
(d8) 27 7. 5 —4 <1 7.5
G 22.9 5.1 2.58 4.85 8. 43 2008
CG . 21.1 4.7 2.85 5.01 8. 34 1935
I—2 1. 2.96 .4t.46 -.27 1.17 3716 ‘.14155 1.06 73 143
(as) 8.5 8.5 -9 2.5 4
M 25.5 5.2 2.50 4.94 8.59 2208
06 21.5 4.6 2.85 5.02 8.35 2050
I—2 d 4.0 11.27* .62.29*) -.35 2.25 ZZU§'¢,04 . 3.16 158 i80')
(d%) 18.5 13 -12 3 .
(N=9)
T 25.3 $1.16 5. 3t. 22 2.63 1.07 4,33 1,04 8.50 1.10 2164 171
O 21. 4 -0. 62 4.61.10 2.91 1.09 5,03 3,04 8.37 3.10 2013 166
I-3 d 3.311.16*** .7 8* —.28 i.15*-.]Z 1,11 .13 t.09*) _1513 72*
(d8) 18. 5 15 -9.5 -3 1.5 7.5
I-3 (Betw.T) KM>G*' K>M KM>G"
I-l

 

bThe response in lactose production (dT ) was larger (P<. 05) for K (33g/day) and M
(39g/day) than G (-1Zg/day)

123

However, statistical analysis according to Anova I-3
(Appendix Table II.14) clearly shows differences between
the substrate infusions and the controls. The overall
difference of 1.1kg also was significant (P<.05), and
protein infusions promoted higher (P<.05) milk output
than glucose. Milk yields during full protein infusion
were slightly higher than when mixture was infused. More—
over, responses for the three treatments above control
tended to increase linearly' as the level of protein in-
fused increased. The largest increase in milk yield from
casein infusion, 2.7kg for cow No. 607, however, was
partially due to a steep decline in yield after infusion.

The extra cow (No. 603) that received casein in-
fusion also produced more milk (+2kg) than during control
periods.

Protein concentration (NXG.38).--The two infusions

 

with protein (K and M) gave almost identical responses in
crude protein concentration of milk compared to controls
(P<.05). Individually, however, the cows responded some-
what differently; No. 604 showed a higher response to
glucose than the mixture but greatest to the full protein
infusion; No. 607 reaponded most to the mixture; and 606
responded equally to the mixture and full protein, but not
to glucose. In no case was protein concentration lower

during substrate infusion than during the appropriate

124

control periods, but the mean for control period 5 was
equal to the preceding treatment period (Appendix Table
11.5).

While the milk protein levels during infusion with
protein were higher than during glucose infusion, re-
sponses (dT) were not significantly different.

Protein (Nx6.38) production.--The concentration of

 

any milk constituent will be influenced by changes in other
constituents as well as milk volume, and only the actual
yield reflects the true rate of secretion. Since the in—
fusions stimulated milk yield as well as protein concen-
tration, crude protein production was higher during treat-
ment than control periods. Greatest responses were shown
for the casein infusion (P<.01) which also caused the
largest increase in milk yields. A relatively large error
attached to the estimated response to the mixture render
this difference barely significant (P<.10). Responses (dT)
to protein were also higher (P<.10) than for the glucose
treatment.

Estimated true protein (ETP).--The difference in

 

true protein production between casein and the mixture
was less than for crude protein and reflects the larger
increase in NPN during casein infusion. Being such a
small fraction of total N, however, changes in NPN must

be large to have an impact on total protein.

125

For the glucose infusion no differences between
treatment and controls were noted for CP and ETP produc-
tion, suggesting no influence of glucose infusion on milk
NPN even though total N went up. The reason for the lower
milk NPNfor cow No. 606 on glucose (period 4) may have been
due to the low protein intake compared to her needs (Appen-
dix Table II.4). With the addition of infused protein (K
or M) the other cows received an adequate N intake also in
period 4.

Statistical analyses of production of estimated
true protein revealed essentially a pattern similar to crude
protein; despite the significant changes in NPN concentra—
tion of milk.

Milk 1actose.--All lactose values for period 1 were

 

discarded as too high due to improper assay. Moreover,
parallel determinations for lactose were often in poor
agreement, so mean lactose levels had large experimental
errors. Nevertheless, there was a trend towards slightly
lower milk lactose during the treatment than control

periods. Despite apparently depressed lactose concentra—
tions during protein infusions the lactose output was in-
creased due to increased milk volume. Increases in

lactose yield due to protein infusions were different (P<.05)
from the slightly negative responses to glucose (Table 2.5,

col. l4).

126

Solid non fat (SNF).--Milk SNF concentration increased

 

for the separate substrates compared to controls, but differ-
ences were small with rather large errors (Table 2.5, col.
ll). Variance among treatments were larger than among con-
trols (Appendix Table II.14). Similar to data for protein
production, the multiple effect of milk volume and concen—
tration made changes in SNF production more dramatic than
those in concentration; but still the increments to pro—
tein infusion barely approached significance (P<.10).
The overall treatment response was less significant than
for protein production,but SNF yields during protein in-
fusions were definitely higher than during glucose infusions.

Milk fat.--Although changes in milk fat content
from control to substrate infusions were consistently
negative (Appendix Table 11.5, item (6)), the large errors
attached to differences between treatment means eXplain
why none were significant. The largest depression in fat
content was noted with the mixture, but no physiological
reason for this appears evident. While variances among
treatments were larger than among controls (Anova I-3)
Appendix Table II.14), the fat content for all treatments
were significantly lower than for the controls (P<.05).

As fat percent drOpped and milk yield increased
due to treatments, responses in fat production and FCM

were small and standard errors for the responses large.

127

Hence, noneof the differences among infusion treatments ap-
proached significanceand milk energy output was not sys-
tematically changed due to the substrate infusions. The
energy supplied with these infusions should theoretically
suffice for 1.5 - 1.8kg FCM (NRC 1971).

Supporting the findings in previous trials our
data (Table 2.6) reveal that means and responses were
similar for the full period and the last three-day sub~
period indicating that treatment effects were instantaneous.
2.3 Blood components.

a. Blood urea nitrogen concentration (BUN).-—
Blood urea nitrogen (BUN) was not determined the first
control period (period 1). Thus comparison during sub-
strate infusions to pre- and post-treatment controls is
not possible. Using the control following any infusion
as its particular control, statistical analyses were
determined according to Anova I-3 (Appendix Table II.15).

Concentration at different bleedings within a
day for individual cows varied considerably, as did
the levels between days (Appendix Table 11.6); thus,
standard errors were relatively large (Table 2.7).

Still, the BUN concentration during protein infusions
were higher (P<.05) than during glucose infusion, which

was slightly lower than for control periods.

128

Table 2.6—-Tria1 I 1971. Comparison of production results
for the full 6 day periods and adjacent con-
trols to last 3 day of substrate infusion and
closest 3 day controls.a

 

_.. .—-.___ _-_'_. _——.... —
_. _._ ._——.———‘_._ ~..— __—_. . --

Means for cows No. 604, 606 & 607

‘3".

 

_.- ..-.___—._

 

Treat- Milk Yield Prot. Conc. Prot. Prod. Fat Conc.
ment 6d 3d 6d 3d 6d 3d 6d 3d
kg %
K 26.03 26.28 3.19 3.17 833 834 2.80 2.87
0K 24.37 24.50 3.00 3.00 732 732 3.03 3.10
d 1.66 1.78 .19 .17 101 102 -.23 -.23
G 23.85 23.20 3.03 3.00 722 694 2.58 2.57
0G 23.28 22.85 2.95 2.93 682 666 2.85 2.88
d .57 .35 .08 .07 40 28 -.27 -.31
M 25.66 25.69 3.17 3.18 814 818 2.50 2.37.
0M 24.52 24.76 3.00 2.99 735 742 2.85 2.83
1.14 .93 .17 .19 79 76 -.35 -.46

 

 

 

 

aControl period before and after
as usual.

infusion averaged

Including the control periods in the analysis
(Anova I-3) showed significant (P<.05) decline in BUN
throughtout the day (Table 2.7). This diurnal trend is
opposite the usual (Knott, et al., 1972) but may reflect
the cows' eating pattern. In no case was BUN influenced
significantly by a treatment X bleeding hours interaction.
But when the control periods were taken into account (Anova
I-3) there was a highly significant (P<.Ol) interaction be-
tween treatment sequence (periods) and bleeding hour. No
physiological explanation for this appears evident; but
during sequence 2 (periods 4 and 5, Appendix Table II.6),

the BUN was higher than before and after.

129

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130
Opposite to expected, BUN levels were highest

before AM feeding and fell linearly throughout the day
(Table 2.7). Only during full protein treatment (K) did
the BUN concentrations increase after the AM feeding as
often observed in this parameter. However, the prefeed—
ing and late PM levels during the mixture treatment were
as high as peak levels during casein infusion (Table 2.7).
Influence of different eating patterns may be the reason
for the different diurnal trends in BUN between treat-
ments, as well as the deviation from the common pattern
in control periods.

The overall increase in the BUN level during pro-
tein infusion confirms earlier results suggesting deamina-
tion of infused amino acids, A slight tendency to lower
BUN level during glucose infusion than during controls
also is in accordance with earlier findings; indicating
improved N (amino acid) utilization or an amino acid
sparing effect on the glucose supplement.

The BUN levels at 3 hr postfeeding were signifi-
cantly correlated (r = 0.57, P < .05) with the NPN level
in the milk. Although BUN was as high before feeding as
3 hrs later, prefeeding BUN and milk NPN were not signifi-
cantly correlated (r = 0.30, NS) and BUN at 9 hrs post-
feeding showed no relationship to milk NPN (r = 0.06).

b. Plasma glucose concentration (PG).—- Con-

 

centrations of PG for substrate treatments were sig-

nificantly (P < .05) higher than for the controls

131

(Table 2.8). Variations among treatments also were larger
than among controls; but the substrates were not signifi—
cantly different in PG, although higher levels were noted
during infusions with protein (K & M) than with glucose
(G). Observations in PG are in Appendix Table

II.7. Generally, the PG concentrations tended to be

higher before than after feeding (Table 2.8).

ii. Trial II 1971

1. Material and Methods

 

1.1 Rationale and planning.——One of the main re—
sults of trial I 1971 was a higher response in milk
protein production on casein + methionine (K) than on
the mixture with glucose (M) which provided half as much
protein as K. This difference suggested that graded
treatments of protein infusion ought to be tested fur—
ther. Although the recovery of infused protein as
increased milk protein was lower for K than M (20 vs.
30%), still higher infusion levels of protein were
desirable to fully challenge the milk protein synthesiz—
ing capacity. Thus, in a 4x4 Latin square experiment
casein was infused at 50—, 100—, and 200% of daily milk
protein production with saline infusion as the control
treatment (Table 2.9).

Because responses in trial I 1971 were equal
during both sub—periods (Table 2.6), infusions were for

only 4 days with the first day as a change-over.

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132

 

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133

Table 2.9.--Trial II 1971. Experimental design and timing
of periods.

 

 

 

 

 

Bleeding hour No.a
Per. 1 2 3 1 2 3 l 2 3 l 2 3
No.b Days
Cow 607 Cow 604 Cow 603 Cow 606
1 9/9-12 O L M H
2 9/15-18 L O H M
3 9/21-25C M H o L
4 9/29-10/2d H M ' L 0
Symbols:
C = cows
T = Infusion treatments Number of factors
0 = saline control C: c=4
K = caseinate + 3%dl-methionine P: p=4 r=4
Level of K-relative to T: t=4
milk production B: b=3
L = low = 1/2 X
M = medium = l X
H = high = 2 X

aBleeding hours (B) relative to morning feeding:
l = before feeding; 2 = 3 hours post-feeding; 3 = 9 hours
post-feeding.

bPeriods (P) lasted 4 days with day 1 used for
transition. There were also 2 days of saline infusion
between actual periods.

CInfusion lasted through 9/25 because cows 604 and
607 were in heat 9/24 and 9/22 respectively, which were
omitted; and 9/25 was not included for cows 603 and 606.

dThe start of the last period was delayed because
No. 606 for unknown reasons apparently was not well
(temperature 101°F) on 9/27 and 9/28.

134

Additionally, two days of saline infusion interspaced
each period.

Levels of casein for infusion were derived from
milk protein production just prior to the trial (Table
2.10), and the caseinate was fortified with 3% methionine
as in trial I 1971.

1.2 Arrangements and procedures.-—Infusion
arrangements, sampling techniques and chemical assays

were similar to trial I 1971. The cows were also the

 

same as used in trial I 1971. They were in their 5th to
7th month of lactation so milk yields were lower than

for the previous trial. Some difficulties were encoun-
tered in stabilizing the cows on their rations, so treat—
ments had to be delayed 2-3 weeks. During treatments
each cow received 6.8 kg hay per day and concentrate to
furnish about 110% of NRC (1966) standards for energy

and protein.

Feed samples were obtained twice during each

 

period, on day 1 and 3, and they were handled as pre-
viously. Feed analyses are presented in Appendix Table
II.17. Milking and milk sampling were also done as
previously described but only one composite sample was
taken for determination of milk constituents for each
cow and period.

.2199d was sampled three times on the last day of
each period and handled as in trial I 1971. Chemical

analyses were performed as in the previous trial with the

135

Table 2.10--Trial II 1971. The levels of infused protein.

 

 

Cow Milkd Prot.d Prot. prod. Level of infusiona
No Yield Cons. i 5% range L M b c H
' protein '

k9 % 9 ------- 9/day -------
603 16 3.2 480-540 255 510 1020
604 21 3.3 660-730 350 700 1400
606 18 3.1 530-590 280 560 1120
607 19 3.3 590-660 315 630 1260

 

low level = 1/2 X milk protein
medium level==l x milk protein K (casein)
high level == 2 x milk protein

EL1.
M
H

’bThe actual amount of sodium caseinate infused
derived as protein (K) X 100/85; as the caseinate was 85%
protein.

cDL—methionine added; 3% of the protein infused.

dMeasured for a couple of days just prior to the
trial.

following exceptions: the NPN content of the milk protein-
free filtrate was assayed by semi—micro Kjeldahl rather
than the autoanalyzer because of more precise duplication
of results. Also, better agreement between duplicates in
the lactose determination than for trial I 1971 apparently
resulted from greater care with the assay.

The statistical analysis followed the outline for

 

a Latin square design (e.g., Cochran and Cox, 1956), with

additional splitting of the main plots for the bleeding

136

times (Appendix Table II.22). No interaction between
factors related to the main plots was assumed, but the
two—way interactions between bleeding times and the three
factors of the main plots were estimated (Appendix Table

II.22).

2. Results

2.1 Feed intakes.--Amounts for feed offered and
consumed for each cow are in Appendix Table II.16. Cow
No. 603 received 9.1kg concentrate per day, while 11.3kg
was allotted to the other three cows. These amounts pro-
vided at least 15% more energy and protein than required
according to NRC (1971) standards at the onset of the
trial.

The intake of energy apparently was influenced by
changing feed quality. Thus, the hay quality in period 2
was lower than for other periods (Appendix Table II.17).
The CP content of concentrate also varied more than

expected.

Increasing protein infusions in this trial de-

pressed feed intakes almost linearly (Table 2.11), although

differences between levels of treatments were not signifi—
cant (P > .05, Appendix Table II.23). Similar trends
appeared for CP and ENE intakes (Table 2.11), despite
variations in feed quality. The similarity in DM intakes

between control periods and days between treatments (Table

137

Table 2.ll—-Tria1 II 1971. Summary of feed consumption:
intakes of dry material, crude protein, and
estimated net energy for different treatments
and periods, means per day.

 

 

Dry material Crude Esti.

Hay Cons. Total protein NEL

Treatment kg kg kg kg Mcal
o 5.1 8.4 13.5a 2.19a 23.2a

L 5.0 7.7 12.7 2.04 21.6

K M 5.5 6.9 12.4 1.99 20.5

H 5.3 6.5 11.8 1.91 19.6

Period

1 5.1 7.4 12.5b 2.16b 21.4b‘

2 5.1 7.4 12.5 1.82 20.8

3 . . 13.6 2.28 23.1

4 4.9 6.8 11.7 1.89 19.7

ssdc 0.37 0.60 0.77

Days of change of trtm.

(saline inf. between periods) 13.5 i 0.4

 

ao > K, P < .01

bPeriods differed significantly, P < .05 or lower.

SEd = standard error of a difference between two
means.

138

2.11) suggests no serious overlapping of infusion effects
on feed intake.

2.2 Milk parameters.--Milk production data for
individual cows are in Appendix Table 11.18 and 11.19,
and results of AOV are in Table II.24.

Milk yields within cows varied much more than in
earlier trials, probably because of fluctuating feed in—
takes. While milk yields did not increase as protein in-
fusion increased, concentration of protein in milk was
related to level of infusion; although there was no
difference between the L— and M treatments (Table 2.12).
Only cow No. 607 showed a linear trend of increased milk
protein with level of infusion (Appendix Table II.19).
Cow No. 606 had as low milk protein concentrations during
the M and H treatments as during 0, with the L causing a
slight increase. Perhaps this was due to low DM intakes
for No. 606 during infusion of the higher protein levels
(Appendix Table II.16).

Because milk yields varied so much between treat-
ments, protein production did not show any marked trend
with level of infusion (Table 2.12).

Similar to earlier trials, concentrations of NPN
in milk increased significantly (P < .01) with each level
of protein infusion (Table 2.12). But these increments
were not large enough to render the ranking between treat-
ments in estimated true protein (ETP) different from that

for crude protein.

139

The concentration of NPN in milk was again cor-
related with blood urea nitrogen with r = 0.77 (P < .01)
at 3 hours postfeeding, and 0.48 (P a .05) at prefeeding.
This relationship agrees with the concept that urea, the
major constituent of milk NPN, diffuses readily into milk;
and that high blood urea levels, as attained in this trial,
substantially increases total milk NPN. Lactose concen-
trations were not affected by treatments and variations
were low as demonstrated by small standard errors (Table
2.12).

Treatment means for SNF showed the same pattern
as for milk protein. Statistical analysis of SNF data
were not performed because loss of samples during period
1. Milk fat concentration did not show any trend with
level of protein infusion, nor was there a significant
difference between control and overall treatments; but
the mean fat percent was slightly higher during the
saline than other treatments.

Fat production as well as fat corrected milk were
also highest during saline infusion. Thus, the energetic
efficiency<mfmilk production was decreased by the infused
protein for total energy from feed consumed and infusate was

not lower than the energy intake in control periods.

The adverse effect of high levels of protein in-
fusion on feed intakes and the fluctuating milk yields in
this trial greatly obscured the intended test of cows'

ability to utilize high levels of infused amino acids for

140

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141

milk protein synthesis. Just the effect on feed intake,
however, may indicate that the amino acid load surpassed
the cows' capacities for amino acid metabolism, possibly
resulting in an amino acid toxicity (Harper, 1959).

2.3 Blood parameters.——Blood urea nitrogen (BUN)

 

increased with level of protein infusion, but only between
control and overall protein treatments were differences
significant (P < .01). Observations are in Appendix Table
11,20. Concentrations of BUN before feeding and 3 hrs,
thereafter were essentially identical, but the diurnal
trend differed for the different treatments (Table 2.13).
Neither bleeding times and treatment, nor any other fac-
tors tended to have an interaction effect on the BUN
levels (AOV in Appendix Table II.25).

Plasma glucose concentrations in this trial were

 

slightly but significantly (P < .05) higher during the
control than protein infusions (Table 2.14). This is
contradictory to trial I 1971 (Table 2.8); and the dis-
crepancy may reflect the more severe depression of feed
intake by substrate infusions in trial II.

The medium level of infusion resulted in lowest
blood glucose (Table 2.14), and this corresponded with
minimum milk yields. However, the plasma glucose levels
were all (Appendix Table II.21) above those (~40mg%) con-
sidered critical for milk secretion (Linzell, 1967).
Diurnal trends in plasma glucose varied from period to

period (Appendix Table II.21); but concentrations were

142

Table 2.13.--Trial II 1971. Concentrations of blood urea
nitrogen.

 

 

Bleeding times

 

 

Treat- b
MeantSE(N)
ment 1 2 3a
(0 hr) (3.5 hr) (9 hr)
mg/loomI’
o 28.0 27.0 31.5 28.3:2.7 (10)C
L 30.8 39.3 39.0 35.8i2.l (10)
M 38.0 37.3 47.6 37.6i3.4 (10)
H 47.5 43.0 42.7 44.6i3.8 (11)
m SE 36.1i3.l 36.612.3 41.2i4.0 36.6
(N) (16) (16) (10) (42)

 

aNot included in AOV because of missing samples.
bSEd for treatments = 2.6.
CK > o, P < .01.

Table 2.14.--Trial II 1971. Concentrations of glucose in
blood plasma.

 

 

Bleeding times

 

 

 

Trzifi- MeaniSE(N=12)a
m 1 2 3
(0 hr) (3.5 hr) (9 hr)
mg/lOOml
o 70.2 68.5 68.9 69.2:o.8C
L 70.3 67.4 68.3 68.6i1.1
M 68.7 64.4 67.0 66.7iO.9
H 70.0 68.3 67.5 68.6il.2
miSE
(N=16)b 69.8:1.l 67.1iO.8 67.910.8 68.3
aSEd for treatments = 1.1.
bSEd for bleedings = 1.3. B1 > B2 + B3, P < .05.

Co > K, P < .05; H > M, P < .05; B > B P < .01.

1 2+3'

143

higher before the AM feeding than later in the day (P < .01,
Table 2.14) although differences were only 2—3mg%. The
interaction between bleeding times and periods (Appendix
Table II.25) also was significant (P < .05), thus complicat-
ing the interpretation of diurnal changes.

iii. Trial III 1971

1. Methods and materials

 

1.1 Rationale for the experiment and design.--
Milk production may not reach its full potential when high-
yielding cows receive rations with a large fraction of CP
in the form of NPN. Crucial limits have been set to 1/3'
of total N as NPN; but apparently a more appropriate limit
is “0.459 urea (= 0.2g NPN) per kg of body weight (Huber,
et al., 1967; Conrad and Hibbs, 1968). In any event, high
proportions of NPN demand an adequate supply of carbohy—
drates (starch). Improved performance observed when sub-
stituting NPN with plant portein may result from rumen by—
pass of feed amino acids (Oltjen, 1967; Chalupa, 1972).
Accordingly, a postrumen supply of amino acids, as with
abomasal protein infusion, might increase milk protein
synthesis in cows on high NPN rations.

By the time this experiment commenced, the cows
were seven to nine months in lactation and produced less
than lSkg/day. Thus, they were not as metabolically
sensitive to high NPN and postrumen protein as higher
producers. Nevertheless, it was decided to test the NPN

affect and to accumulate more data on the infusion of

144

casein + methionine. An experiment was planned with two
diets, supplying a low (~15%) and a high (~40%) fraction of
the CP as NPN (Table 2.15). For a comparison of NPN levels
in cows at similar production level and body weight it seems
appropriate to express NPN as % of total N although the limit
for NPN utilization is better defined in relation to body
weight (Huber, et al., 1967). A crossover of the diets
tested abomasal infusion of casein at both NPN levels in

four cows. Infusing protein before and after the saline con—
trol in half of the cases would have been more correct
statistically, but for practical reasons all infusions

were conducted in parallel. More serious, it seems, for

the statistical analysis, was the low power of the test
resulting from fractionation of degrees of freedom.
Nevertheless, the interaction between infusion treatments

and level of NPN was of particular interest, and the de-

sign estimated this effect.

1.2 Arrangements and Operations of the trial.--
Animals. The four cows used in this trial were the same
as employed in trials I 1971 and II 1971. No particular
problem with their abomasal cannula were encountered,
although scar tissue at the fistula was predominant in
No. 606 toward termination of the experiment.

Infusion periods lasted 5 days with day l of each
period omitted from the results for transition. Infused
casein was intended to equal 20% of the feed CP; which again

exceeded NRC standards. Dl-methionine was anew included at
3% of the caseinate, and the infusate was 76 to 109% of milk

protein output (Table 2.16).

Table 2.

145

15.--Trial III 1971. Experimental design and
timing of periods.

 

 

Bleeding Time No.
l 2 3 l 2 3 l 2 3 l 2 3

 

 

Seqs. Per. Da 5 Inf.
No.a No.b y Tretm.
Cow No.
603 607 604 606
Feeding
l l ll/28-12/2 01 H L L
12/3-7 K H H L L
12/8-12 02 H H L L
2 1/10-14 01 L L H H
1/15-20 K L L H H
6 1/21-25 02 L L H H

 

aAbout 4 weeks elapsed between the two sequences

with change over of feed rations.

tion.

Symbols:

bPeriods lasted 5 days, but day l was for transi-

Infusion treatments (T, t=3):

01 = saline before casein + methionine.
02 = saline after casein + methionine.
K = caseinate + 3%dl-methionine, intentionally 20%

of feed CP.
Feeding; i.e., NPN level in the feed (F, f=2):
H = high level.
L = low level.
Bleeding times relative to the AM feeding (B, b=2):
l = just before feeding.
2
3

3-3.5 hours after feeding.

~9 hours after feeding.

146

Table 2.16.--Trial III 1971. Derivation of protein
quantities infused and comparison to crude
protein (CP) consumed and put out by the

 

 

 

 

 

milk.
Columna Column Column Column Column Column
No. 1 No. 2 No. 3 No. 4 No. 5 No. 6
Cow ———————— ______. ______.
Est. CP 20% of Na-cas. CP inf. CP inf. E-ME of
fed CP fed inf. CP int. Milk CP inf. CP
9 g g % % Mcal
603 1970 350 400 22 109 1.62
604 2260 450 520 20 76 2.09
606 1860 370 425 23 81 1.71
607 1970 400 460 19 106 1.85

 

aComment for each column follows as assigned:

(1) Estimated crude protein consumption at the
onset of the trial.

(2) Infused protein (casein) was intended to be
20% of consumed protein, and protein fed (1) = potentially
consumed. Additional dl-methionine was 3% of infused
casein.

(3) Sodium caseinate was 85% casein.

(4) Crude protein infused in % of crude protein
intake in control periods.

(5) Crude protein infused in % of milk crude
protein production in control periods.

(6) Estimated metabolizable energy of the infused
protein (4.6 kcal ME/g casein).

147

Feed! feed sampling and analysis. About three

 

weeks adaptation to the high NPN ration was allowed before
the trial started; but urea—treated corn silage was fed
for several weeks before introduction of urea containing
concentrate. Feed composition data are in Appendix Table
II.27. Corn silage treated with urea ran out before the
last sequence of infusions and the cows then received
ProSil-treated1 silage. The mean N content of the two
silages, determined simultaneously for another experiment,
was identical. The mean values for silage N and NPN were
employed here since samplings did not follow experimental
periods. The NPN content of an aqueous silage extract
was determined after water soluble protein was precipiv
tated with sulfosalicylic acid (SSA). Dry matter and
crude fiber in corn silage were determined as previously
described on samples obtained twice in each period. Hay
and concentrate mixtures also were sampled and handled as
described previously.

The formulas for the concentrate mixtures, with
and without urea, are in Appendix Table II.26.

Milking, milk sampling, handling of the samples,
and analyses were all done as previously mentioned. Milk
constituents were determined on samples composited for two

consecutive days, but a period mean weighted for the

 

lProSil, ammonia, minerals and molasses additive,
manufactured by Ruminant Nitrogen Products Co., Adrian,
Michigan.

148

amount of milk in the sub—periods was used for further

calculations.

Since the preceding two trials showed that lactose
concentrations were not particularly informative, lactose
was not determined.

Blood samples were obtained and handled as earlier,

 

and assays of blood components were analyzed by the same
methods. However, plasma-free amino acids were not
determined for the samples obtained 3 to 3.5 hrs post—
feeding (BZ) (section B.III).

Statistical analysis followed the outline in

 

Appendix Table II.33 (Anova III).

2. Results

2.1 Feed intakes.--Generally the urea-containing
concentrate was readily accepted, but cow No. 603 had a
low intake during the first control period (Appendix Table
II.22). Concentrate consumption for this cow also dropped
during protein infusion while on hte low NPN ration in
sequence 2. But there was no overall trend towards lower
feed intakes during protein infusion, nor any effect on
intakes due to feed NPN levels (Table 2.17, Appendix
Table II.28 and 11.34).

Variable intakes of different feeds resulted in
estimated NPN fractions below 40% for the high NPN ration
(Table 2.17). The absolute amount of NPN (g/day, Table
2.17), however, was equivalent to a urea level considered
maximum in lactating cows (Huber, et al., 1967; Conrad and

Hibbs, 1968).

Table 2.17.--Tria1 III 1971.

149

Summary of feed intake:

daily consumption of dry matter, crude
protein, NPN and estimated net energy.

 

 

NPN level fed

 

 

 

 

 

 

Parameter
Inf. Trtm. . Both feeds
High Low (N=8)
k9
Dry matter
01 10.9 11.6 11.3 r .69
K 11.4 11.1 11.3 + .71
02 11.6 11.8 11.7 i .51
A11 inf. (N=12) 11.3 i .51 11.5 i .52
Crude protein kg
01 1.77 1.89 1.83 t .112
K 1.88 1.84 1.86 i .107
02 1.91 1.85 1.93 i .077
All Inf. (N=12) 1.85 i .082 1.89 i .079
NPNa (L/H)%
O1 109 44 40
K 116 41 35
O2 118 44 37
A11 Inf. (N=12) 114.2 42.9 37.6
NPN/Total N (L/H)%
01 38.3 14.3 37.3
K 38.0 13.8 36.3
02 38.5 14.0 36.4
All Inf. (N=12) 38.3 14.0 36.6
Mcal
Estimated
Net energy
01 19.5 21.0 20.25: 1.32
K 20.6 20.1 20.351:1.26
02 21.0 21.5 21.231: .93
All Inf. (N=12) 20.37 .96 20.85 .93

 

a . . . . .
Minimum values; NPN 1n Silage + urea N in con-

centrate.

150

Intakes of crude protein (CP) and NE exceeded

L
NRC (1971) standards, particularly for period 6 (Appendix
Table II.29). For a test of nutritional stress imposed by
high NPN fractions the total CP intakes probably were too
high.

2.2 Milk production parameters.—-Much as expected
for reasons related to the CP intakes, feed NPN levels had
no effect on any milk production parameter (Table 2.18,
Appendix Table II.30). Neither was there a tendency for
interaction between infusion treatments and feeds (T x F),
(Appendix Table II.35).

Despite the low production levels and high level
of feeding, the infusion of protein increased milk protein
production significantly (P < .05, Table 2.18) as in
earlier trials. Milk yield was only slightly higher dur-
ing casein + methionine infusion than during the controls
(+0.5kg), but the concentration of protein (N x 6.38), in-
creased significantly (P < .005). Nominally, the esti-
mated response of 0.3 per cent was even higher than in
trial I 1971. The mean increases in crude protein con-
centration were identical for the two infusion sequences.

Estimated true protein increased slightly less on
both feeding regimens than did crude protein, but the
casein infusion response of 0.25% ETP was clearly signifi-
cant (P < .01). NPN as a fraction of total N was slightly
but not significantly higher during protein than saline

infusion (5.68 vs. 5.28%), like noted earlier.

151

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152

For both sequences there was a tendency for the
CP as well as ETP content to be higher before than after
protein infusion, but the difference was not great and
is not readily explained.

Mean ETP yield increased significantly (P < .025),
as shown in earlier trials, but recovery of infused pro-
tein was lower than in trial I 1971. This might be
expected with a higher rate of infusion relative to milk
production in this trial.

The difference in SNF% between protein and saline
infusions in sequence 1 approached the difference in pro—
tein content, but with far larger standard errors, More-
over, in sequence 2 the apparent response (dT) in SNF
percent was nil (Appendix Table II.30, item 4). Varia-
tions within cow and periods also were larger in SNF than
in protein. Although not estimated, it seems that
analytical errors were larger for milk SNF than for
nitrogen.

Milk fat also varied substantially within cows
and periods, possibly due to different butterfat testers.
The tendency for lower fat percent during protein infu—
sion (Table 2.18) agrees with the 1970 experiments (Table
1.6) and trial I 1971 (Table 2.5).

Although milk NPN concentrations as well as BUN
were increased significantly by protein infusions, these

entities were not correlated for any of the three bleeding

153

times (r = 0.35 or lower), Contradictory to findings in
our earlier experiments, this lack of correlation may
merely reflect peculiar variations in these parameters.

2.3 Blood parameters.——Blood urea nitrogen con-

 

centrations observed in trial III are in Table II.31.
Contrary to milk production, the BUN level was signifi—
cantly (P < .05, Table 2.19) influenced by the NPN level
of the feed. The higher BUN at high (H) than at low (L)
NPN ration (36.1 vs. 32.5mg%) might be expected; but this
relationship was not observed by Knott, g£_31, (1972).

BUN increased significantly (P < .05) during pro-
tein infusions compared to saline controls (37.3 vs.
32.8mg%), a trend similar to earlier experiments. The
influence of infusions seemed more obvious in sequence 1
than sequence 2, although overall means for the two
sequences were practically identical (Appendix Table
II.31). The BUN concentrations before and after protein
infusion also were similar, and there was no real inter-
action between infusion and factors of higher order
(Appendix Table II.36). Infusion treatments and bleed-
ing times, however, tended to interact (P < .10) on BUN
and resulted in a higher BUN level 9 hrs post—feeding
for protein than control infusions.

With the restrictions due to the interactions,
the bleeding times had a significant (P < .001) influence

on the BUN level. A lower concentration before feeding

154

Table 2.19.--Trial III 1971. Summary of blood urga N
and plasma glucose concentrations.a'

 

 

NPN level in the feed (F)

 

 

 

Bleeding
times (B)
. Both feeds _
High Low (N = 24) (N — 24)
mg/lOOml
Blood urea N
Infusion (T)
01 33.9 33.8 33.8a B1 30.3a
K 40.2 34.3 37.3b B2 35.9b
02 33.8 29.7 31.8a B3 36.6b
SEd-T=4.l SEd-B=4.2
All inf. _
(N=36) 36.1a 32.5b SEd F—2.3
mg/lOOml
Plasma glucose
Infusion (T)
01 62.4 60.2 61.3a B1 66.4a
K 65.7 64.7 65.2b B2 60.4b
02 63.8 60.7 62.3a B3 61.9b,c
SEd-T=2.03 SEd-B=2.04
All inf. _
(N=36) 64.0 61.8 SEd F—3.8

 

aMeans for the main factors and standard errors
for a difference between two means (SE ), as assigned:
T = infusion treatments; F = level of PN in feed; B =
bleeding times relative to AM feeding.

bFigures with different superscript differ signi-
ficantly, P < .05 or lower.

155

(30.3mg%) than later in the day (average 36.5mg%) was
also observed in trial II 1971, while BUN in trial I was
higher before feeding.

The high BUN levels generally fit the high N
intakes and indicates that the capacity to clear urea
from blood by excretion or recycling approached its upper
limit (Mugerwa nad Conrad, 1971).

Plasma glucose (PG) observations for this trial

 

are in Appendix Table II.32. The concentrations were
somewhat higher during high than low NPN feeding (Table
2.19), but the difference wasnnot significant and has no
obvious explanation. Markedly higher PG levels before
than after the AM feeding (P < .001, Table 2.19) simulate
the pattern of the previous trials. This diurnal trend
occurred free of interaction between infusion treatment
and level of NPN fed.

Protein infusions clearly elevated PG, but infu-
sion treatments interacted (P < .05) with treatment
sequences. During the second protein infusion the PG
rose to a higher level than in the first and remained
high during the control period that followed. While
markedly influenced by the interaction (T x S), the mean
PG level for sequence 2 tended to be higher than for
sequence 1 (P < .10). At the time of the second protein
infusion (period 5, January, 1972) ambient temperatures were
lower, milk yield had fallen, and the cows were eating dif—

ferent silages. These changes might have influenced the

156

responses in PG to protein infusion, but the change of
silage occurred before sequence 2 started. Elicited by
the cold weather, endocrine mechanisms might have prompted
the high PG concentrations during the last two periods of
sequence 2. Hormonal involvements might also explain the
decrease in PG levels in the post—fed state when an abun-

dance of substrate is available for glucogeogenesis.

iv. Discussion of 1971 trials

Consistently positive responses, yet of variable
magnitude, in milk protein production by abomasal protein
infusion in trials I and III emerge as the main feature of
this series of experiments. Despite different production
levels in the two trials, the increments in protein produc-
tion were identical; 12.3 i 1.8% and 12.3 i 1.1% for all
protein infusions in trials I and III, respectively. In
trial I, reponse to full protein treatment (K, 13.8 i 2.1%)
slightly exceeded that for the mixture (M, 10.7 i 3.0%),
which supplied half as much protein, while increments to
glucose infusion were small and variable (5.8 i 2.3%).

These results support our earlier studies and those of
Broderick, et a1. (1970), Spechter (1972), Derring, et a1.
(1970), and Tyrrell, et al. (1972).

Because the response to full protein treatment
(K) in trial I tended to be greater than to the mixture
(M) (1019, P < .01 vs. 799, P < .10), with an insignifi-

cant response to glucose alone (409, NS), an improved

157

supply of amino acids again seems the reason for the
increased milk protein yields.

While the cows in trial II failed to respond with
an increased protein production, the content of protein
in the milk increased with level of infused protein,
although not linearly (Table 2.12). Fluctuating milk
yields evidently prevented an augmented protein output in
trial II as the protein infusion depressed feed intakes
(P < .01).

By infusing casein + methionine at a rate similar
to the higher levels in trial II, Broderick, g£_§l. (1970)
also observed lowered grain consumption than in control
periods. Considering potential gluconeogenesis from amino
acids, it is noticeable that intravenous infusion of
glucose or propionate in lactating cows, at rates approach-
ing the higher treatment levels in trial II, did not sup-
press appetite (Fisher and Elliott, 1966). And several
different experiments in sheep and goats dismissed glucose
as a mediator of intake regulation in ruminants in general
(Baile and Mayer, 1970).

Apparently, the high loads of protein post—
ruminally stressed the cows' capacity for amino acid
metabolism. Treatment for only four days might have been
too short for an adaptation, but intakes returned to
normal during the two days between protein infusions,

High protein diets lead to inappetance in rats, but this

158

was overcome as amino acid catabolic enzyme activities
increased (Anderson, e£_al., 1968). Induction of these
enzymes developed in two days after high protein consump—
tion commenced.

The elevated levels of blood urea during protein
infusions, particularly in trial II, suggest extensive
catabolism of infused amino acids. High blood urea
generally means increased N losses in the urine (Ford and
Milligan, 1969), but there evidently prevails an upper
limit for the cow's capacity to concentrate urea into
the urine (Mugerwa and Conrad, 1971). This limitation
was also linked to depressed appetite (Mugerwa and Conrad,
1971) as observed at high NPN feeding. Toxicity at
extremely high NPN intakes relates to high levels of
ammonia in body fluids (Visek, 1972), but blood ammonia
was not determined in these experiments.

High BUN levels even in control periods and an
excessive CP intake relative to NRC (1971) standards sug-
gest excessive nitrogen intakes by these cows. A lowered
efficiency of supplementary protein utilization through
urinary N losses (Knott, gt_al., 1972), as indicated by
high blood urea concentrations (Ford and Milligan, 1969),
has been discussed earlier. However, blood urea in trial
I tended to be higher on the mixture than on full casein
(K) (29 vs. 26 mg%), although the latter provided twice

as much infused protein. Nevertheless, the recovery of

159

infused protein in the form of increased milk protein
production tended to be higher for the mixture (M) than
the casein (K) treatment (22—46 vs. l6—24%).

Both protein infusions (K and M) in trial I as
well as trial III (51 and 82) raised the blood glucose
level which indicated augmented gluconeogenesis from
amino acids; but this was not observed in trial II. The
crucial role of glucose as a precursor of lactose in milk
secretion (Linzell, 1967) has been mentioned earlier.
However, contradictory to earlier studies (Linzell, 1960),
Linzell (1967) found a marked arteriovenous difference
over the udder even at very low blood glucose concentra-
tions (< 30mg%). From this it was concluded that the
glucose uptake at low arterial levels is governed by the
mammary blood flow.

Apparently plasma glucose in these experiments
stayed above concentrations critical for milk secretion.
Experimenting with insulin and other treatments, Kronfeld,
gt_al. (1963) arrived at 60mg% and Rook and Hopwood (1970)
at 40mg% as plasma glucose levels under which milk secre-
tion rates decreased. Bartley and Black (1966) found as
much as 1.5 kg glucose per day infused duodenally in lac-
tating cows did not increase the rate of glucose oxidation.
Following a peak a few hours after initiation of infusion,
plasma glucose plateaued at 80mg%. The two cows in their

experiment produced less than 10kg milk per day, and the

160

influence of the glucose load on volume and composition
of milk was not reported. But Bartley and Black (1966)
contended from their isotOpe dilution data that lactating
cows encounter a relative glucose deficiency.

Fisher and Elliott (1966) found 1.8 to 3.3 Mcal
glucose and prOpionate infused intravenously increased
milk yields nad lactose concentrations of the milk, al-
though only glucose increased blood glucose concentrations
(from 40 to about 50mg%). Milk protein concentration was
not affected by these treatments, while glucose was more
effective than prOpionate in depressing milk fat percent,
as discussed earlier. An association between increased
plasma glucose and enhanced milk protein production in
these trials seems plausible, and our data and those of
others does not exclude the possibility that the protein
infusion directly stimulates milk protein synthesis by
improved glucose availability. Our consistently higher
responses to protein than glucose, however, imply that
amino acids were more crucial metabolites in this connec-
tion than was glucose.

The effect of the substrate infusions might in
any event have involved altered hormonal status of the
cows. Carstairs (1972) found growth hormone was increased
in two cows abomasally infused with casein. Both cows
had higher milk production during casein infusion than

during the control or glucose infusions. The infusion

161

treatments did not consistently change milk composition
but one cow also suffered large body weight losses.

A stimulatory effect of growth hormone on milk
secretion has been documented (Meites, 1961). Hutton
(1957) reported a highly significant linear relationship
between the log weight of injected growth hormone and
increases in milk yield. While the fat percent remained
unchanged, there was a negative relationship between log
level of growth hormone and solids nonfat of the milk.
Radloff and Miyake (1969) reported STH significantly
increased milk yields, while ACTH lowered milk yields
and increased concentrations of fat and SNF.

Prolactin secretion in ruminants has also been
stimulated by infusion of amino acids (Davis, 1972;
McAtee and Trenkle, 1971), and one of two cows in the
study of Carstairs (1972) showed elevated plasma prolac-
tin from abomasal casein infusion. A galactOpoietic
action of prolactin in cows has not been fully established
(Schmidt, 1971); but Keenan, et al. (1970) concluded that
the present knowledge on the influence of prolactin on
secretory events in the mammary cell indicate an all or
none effect of this hormone in stimulating RNA and pro—

tein synthesis.

162

Due to lack of hormone data in these experiments,
it can only be theorized that increased milk production by
post—ruminal infusion of amino acids was mediated by an
endocrine influence on metabolite interaction. An improved
supply of essential amino acids to the mammary cell might,
nevertheless, have been crucial for the observed increase

in milk protein synthesis.

v. Protein production responses described by regression
analysis -

 

In trial I 1970 it was noticed that the cow with
highest protein production in control periods showed the
greatest response to abomasal casein infusion. With pos—
sible influence of repeated measurements on the same
animals, the 1970 data combined showed responses in pro—
tein production significantly correlated (r = 0.82, P<:.01,
Table 2.20) to production in control periods.

This relationship was not seen in trial I 1970
where the amount of protein infused tended to show a
graded response, even though the level of treatment was
related to the level of production. However, combining
all casein infusions in trials I and III 1971 (n = 14)
revealed a significant correlation (r = 0.70, P < .01)
between response and baseline production. The relation-
ship (r = 0.72) was also significant (P < .05) for trial
III alone. With the small number of observations, the

correlation coefficients for the two trials in 1971 were

163

Table 2.20.--Correlations (r ) between response to

. . X . . .
abomasal inquign in milk protein (d)a
and protein production in control periods

 

 

 

 

 

 

(o)b
d & O as d & O as
Year Trial Treat- (N) CP(Nx6.38) ETPC
ment
9/day g/day
ryx P yx P

1970 I 8 II K1 5 0.673

I 8 II K2 5 0.863 <.05

I 8 II K1 10 0.824 <.01

II K1 6 0.795

II G 5 0.259
1971 I K 8 M 6 0.323 0.336

III K 8 0.718 <.05 0.710 <.05

I 8 III K6 14 0.703 <.01 0.685 <.01

I 8 III Ke 15f 0.658 <.01
1970
8 '71 All above Ke 24f 0.711 <.01

All above K8 25 0.643 <.01

All above G 8 0.396
(1972) Spechterg K 6 0.547

All aboveh K9 31 0.616

a _

dT — Y
bo = x

trial.

cEstimated true protein in milk; (N-NPN)x6.38.

dInfusion treatments K
methionine, G =
to K and G.

casein or casein plus

eAlso including M in trial I 1971.

glucose and M = mixture K + G equicaloric

fIncluding Cow No. 603 (1971) receiving K in an
extra infusion (Appendix Table 11.5).

9The cows were in negative N balance before the

With X =

h

g casein infused r =

0.909,

With X = g casein infused r = 0.808, P <

P <

.05.
.01.

164

not significantly different, neither did the coefficients
for the two years differ significantly.

A plot of responses to casein infusion (Y) versus
control production (X) including both years' data (Figure
1) showed great spread. However, when control production
(X1) and level of casein infused (X2) were included as
linear and quadratic factors in a multiple regression
analysis1 (Table 2.21) a regression coefficient (R) of
0.79 (P < .01), k = 4, n = 24) was obtained, The cor—
responding R2 (goodness of fit = 0.62) accordingly
revealed that the factors included accounted for 62% of‘
the variation around the mean (Table 2.21). Including
cow No. 603, which showed great response to casein infu-
sion at the end of trial I 1971 (Table II), resulted in
an R2 of 0.61 (n = 25).

Successive deletion of the least significant
(P < .10) factor from the model (n = 25, Table 2.21)
left only the linear components, and the fit remained
essentially unchanged (R2 = 0.60). With n = 24, the
deletion of casein infused (X2) as well as the quadratic
effects showed P = .169 (Table 2.21). The B-weights for
the two factors left in the regression (Table 2.22) con-
firm that the level of control production was relatively

more important than the amount of casein infused for

explanation of the responses shown here, but level of

 

lLS routine program on CDC 3600 computer.

165

observations

 

 

 

A I970 trials
0 trial I I97I
response (d ) in milk protein ' "'a'm'97'
yield above control
g/day Y - response (d)
'40 .. X. 8 control yield (0)
X28 casein inf. (q/day)
. a
Y =-63.6+0.I38 x,+o.l44 x2 a
lace 57-21.: mas
—o— 9.x,at x2 -356
D
IOO -
80 -
.,§
60 r
40 -
2 O " A
3?
(file I I l i 1 L 1
300 400 500 600 700 800

milk protein yield in control periods (0). q/d

Figure l. 0)!

The response in milk protein yield to abomasal protein infusion
as related to yield in control periods

166

infusion was constant for the 1970 data (10 of 25 obser-
vations). This relationship as well as the regression
coefficients changed slightly by excluding the extra
observation in cow No. 603 (n = 24, Table 2.22).

Employing only the 1971 data (n = 15) when graded
levels of casein were infused, the quadratic effect of
infused casein was the second most important factor (Tables
2.21 and 2.22). However, its contribution to the fitting
of the regression was far from significant in the small
material.

An analysis of data from Spechter (1972) did not
yield a regression equation with significant coefficients.
Moreover, infusion level (X2) alone showed almost as good
fit as inclusion of yield level (X1), the second most
important factor (Table 2.21). The cows in the Canadian
study were in early lactation and negative N balances,
which might eXplain the difference in response functions
compared to cows in our trials which were fed above standards.

Spechter (1972) contended that the large responses to
abomasal casein in his study indicated an inadequate amino
acid supply. Although the regression of responses to in-
fused casein on protein yield failed to be significant in
his study with only six observations, the higher R2 than for
our data suggests that in our experiments factors other

than levels of production and treatment limited responses

167

Table 2.21.--Excerpts from multiple regression analysis
of the relationship between protein produc-
tion responses, control production and amount
of casein infused.a

 

 

Factors deletedb Regr. about mean

 

 

Trials
Year (N)

Analyzed Xi P R2 F P

I & II 1970 24 none .620 7.8 .001

and X3 .871 .620 10.9 .0005

I & III 1971 X3X4 .955 .619 17.1 .0005
X3X4X2 .160 .509 22.5 .0005
X3X4X1 .001 .400 4.1 .055

I & II 1970 25c none .610 7.8 .001

and X .857 .609 10.9 .0005

I & III 1971 X4X3 .812 .601 16.6 .0005
X4X3X2 .072 .451 18.9 .0005
X4X3X1 .002 .191 5.4 .029

I 8 III 1971 15C none .572 3.3 .056
X2 .812 .569 4.8 .022
XZX3 .789 .552 7.3 .008
X2X3X4 .402 .443 9.9 .008
X2X3Xl .091 .205 3.4 .090

Spechter (1972) 6 none .984 14.9 .101
X4 .958 .984 39.7 .025
X4X3 .316 .835 7.6 .067
X4X3Xl .386 .827 19.1 .012
X4X3X2 .194 .300 1.7 .261

 

aAll data in g crude protein per day. Y = re-
sponse (d) to abomasal infusion above control; X1 =
control production (01 + 02/2); X2 = amount of casein
infused; x = x ; x = x2.
3 l 4 2
bSuccessive deletions of the factor or factor
combination least significant at each stage of factor

numbers (k).

cCow No. 603 (1971) included.

168

Table 2.22.--Regression equations for estimation of re-
sponse to abomasal casein infusion from
control production and amount infused.a

 

 

Trials Factor Sign. RegreSSion fit

 

Year (N) Regression
Analyzed P 8 wt. R2 R p

 

I & II 1970 24 y=bo+b X +b X .619 .787 <.01

and 1 1 2 2
I 8 III 1971 b -59.3040
of 0.13992 .001 .682
b2 0.12350 .160 .338
s- 20.27
y
I 8 II 1970 25 y=b +b x +b x .610 .781 <.01
and o l l 2 2 p
I 8 III 1971 b -63.6377
b? 0.13718 .002 .643
b2 0.14376 .072 .389
- 21.07
5y
_ 2
I 8 III 1971 15 y-bo+blxl+b4X2 .620 .788 <.01
b0 —18.8274
bl 0.11458 .070 .589
b4 0.00016155 .400 .350
s- 24.19
y
Spechter (l972)6 y=bo+b2X2 .827 .909 <.05
b0 35.3024
62 0.32956 .194 .909
s- 56.33

 

aEquations derived at by multiple regression as
outlined in Table 2.21.

169

to postruminal amino acids. Including additional factors
in the model probably could help explain more of the
variability in our studies. Since the cows were fed above
standards, metabolic data might be more informative than
nutrient intake, although interactions between metabolites
(e.g., amino acids) might tend to mask their importance.

Physiologically, the correlation between yield
response as the dependent (Y) and control production as
the independent (X) variables probably is an oversimpli-
fication. Rather, the magnitude of the response and the
control production might depend on common causative face
tors and therefore they are correlated.

The difference in response functions in ours and
the Canadian study did not warrant an analysis of combined
data. Even a well—fitting mathematical expression may not
be physiologically feasible (Kleiber, 1950). However, the
importance of control production on responses in well-fed
cows is supported by in yitrg studies with mammary tissue.
Emery, gt_al. (1970) reported the capacity for protein
synthesis by mammary slices ranked according to milk
production for the donor cows. Larson (1972) found mam-
mary cell cultures from individual cows differed in total
protein synthesis as well as in response to methionine.
Schingoethe, g£_31. (1967) showed that the rate of
synthesis of B-casein by a mammary cell culture could be

augmented by amino acid concentrations above the normal

170

physiological level, abomasal protein infusion may have
created such a situation in_yiyg.

In any event, the best fitting regression equa-
tions in Table 2.22 are presented to show the relative
importance of factors in describing response variations
in this material rather than for the purpose of predic-
tion. If the latter was intended, pre—infusion produc—

tion would be a more logical base line.

III. PLASMA FREE AMINO ACIDS (1971 TRIALS)

1. Introductory Remarks

 

Plasma free amino acids (PAA) are generally con-
sidered the currency for protein metabolism (Munro, 1970;
Allison, 1964), but erythrocytes and proteins have been sug—
gested (Elwyn, 1970) as additional means of amino acid
transport. While the PAA pool is a small part (0.5%) of
the body's total free amino acid, the PAA reflect the body's
supply and demand for amino acid, being influenced by
dynamic metabolic regulations (Munro, 1970). An essential
amino acid (EAA) will not accumulate in plasma unless it is
supplied in excess relative to other EAA or requirement
(Almquist, 1954). In this study PAA were measured in an
attempt to further explain the effect of postruminal protein

on milk protein synthesis.

2. Materials and Methods

 

Plasma free amino acids (PAA) were determined on a
protein free filtrate obtained by mixing Sml plasma, 0.5ml
50% (w/w) sulfo salicylic acid (SSA), and 0.5ml of a nor-
1eucine (nle) solution containing lpM nle/ml as an internal
standard. The plasma reagent mixture was shaken and kept

on ice for about 2hr before centrifugation at 15,000xg for

171

172

30 min. Supernatant thus obtained was decanted into glass
tubes, sealed with a cork, and kept frozen until final
assay.l For periods 1 through 5 of trial I, no nle was
detected at assay, and nle had to be added.

Concentrations of individual amino acids were cal-
culated by relating areas under traced curves to those for
a standard, and the area of the nle curves related the
sample areas to known concentrations for the standard. A
standard curve was obtained for every 30-35 samples.

Generally, concentrations of total PAA were in the
range found previously in this laboratory and by others
(Jacobson, et_§l., 1970; Fisher, 1972) in lactating cows.
influence of the period of assay was evident; for a part of

the samples in trial I which showed very low concentrations

was assayed at a time when the traced areas were very small.

This probably increased the error on the measurements. The
reason for the low peaks is not known. Since all amino
acids were low it was assumed that the molar ratios were
unaffected; hence a molar % distribution was calculated for
each sample (Table 3.1).

Since amino acids are incorporated into proteins in

given molar ratios, and the metabolism of amino acids are

interrelated, the molar % expression has merits as an expres—

sion for relative amino acid availability (Scott, et al.,

 

lAssay was done on a Technicon TSM Amino Acid
Analyzer in the Department of Animal Husbandry.

173

1972; Boling g£_31., 1972). Molar % ratios were also cal-
culated for trials II and III, though concentrations for
these trials did not fluctuate as in trial I.

Further calculations and statistical analysis fol-
lowed the outline for blood urea and plasma glucose for each
trial. However, because the molar percentages all were
below 30, they were transformed by the arcsin function
(Rohlf and Sokal, 1969) to achieve a normal distribution for
the AOV.2 For trial III, the samples obtained 3hr post-
feeding (B2) were not analyzed for amino acids. Diurnal
variations and bleeding x infusion interactions generally

were small and are not considered in the summarizing pre-

sentation of the PAA results.
3. Results and Discussion

Concentrations of amino acids (pM/l) in trials II
and III (Tables 3.2 and 3.4) tended to increase with the
protein infusion as observed by others in similar studies
with lactating cows (Broderick, gt_gl., 1970; Spechter,
1972) and sheep (Hogan, §E_al., 1968). Generally, this
trend indicates an improved amino acid status, but can also
suggest an imbalance (Young, gt_§l., 1973). In trial II the

levels of branched chain amino acids (BrAA = valine,

 

2 . .

Calculations to molar %, transformations and
statistical analysis were done on a CDC 3600 computer,
programmed by Dr. R. R. Neitzel.

174

Table 3.1. Trial I 1971. Plasma free amino acids; molar %
distribution at different infusion treatments.

 

 

 

Amino a b a b a b

Acid 0G a P oM M P oK K P
----% -------- % -------- %--—-

LysC 3.9 4.1 3.7 4.1 3 6 4.0

His 2.5 3.2 2.5 2.8 <.05 2.3 2.8

Thr 6.0 5.4 6.2 5.4 <.05 6.1 4.7 <.05

Val 13.1 11.6 13.3 13.4 13.0 15.1 <.10

Gle 5.9 7.0 6.1 6.9 5.8 6.9

Leu 8.9 7.8 8.5 8.5 8.5 9.4

Met 2.1 3.0 2.0 3.5 <.10 2.0 3.9 <.10

Cys 1.5 1.3 1.6 1.1 1.5 1.1

Phe 2.5 3.1 2.6 3.1 <.05 2.6 2.9

Tyr 2.6 3.9 <.10 2.8 3.6 2.8 3.7

Arg 3.7 3.5 ‘ 3.6 4.1 3.5 3.5

Asp 0.8 0.2 0.7 0.3 <.10 0.7 0.2 <.05

Glu 4.2 4.2 4.2 3.9 3.8 4.3

Pro 6.7 6.0 7.1 5.8 7.2 6.2

Ser 4.9 5.0 5.1 5.4 5.2 4.4

Gly 18.2 17.6 17.5 15.2 18.6 13.3 <.10

Ala 12.4 13.1 12.2 13.0 12.9 13.4

E/N ratio 0.82 0.83 0.82 0.91 0.78 0.99 <.10

 

aAverage of pre- and post-treatment.

bProbability level in test of significance by Anova
I-2 (Appendix Table II. 9d), OT vs. T by ortogonal contrast.
9 observations behind T, 18 behind OT'

CThe names of the amino acids are given fU11Y in
Table 3.4, which also indicates essential (E) and non-

essential amino acids (N) as conventional for rat growth.
Tryptophan was not determined for these trials.

175

Table 3.2.--Trial II 1971. Plasma free amino acid
concentrations.

 

 

 

 

 

Treatment

Amino acid 0 L M H Pe
Lysf 89a 116b 97° 85d <.01
His 64 70 60 60
Thr 114 116 100 114
Val 310a 332b 410° 486° <.01
Ile 132a 139b 1730 174C
Leu 191a 205b 252C 293d <.01
Met 31 51 66 496 <.01g
Cys 31 39 37 29
Phe 56 57 58 54
Tyr 58a 68b 63a 70a
Arg 77 84 65a 51b <.01
Asp 16 16 13 19 <.05
Glu 106a 117b 98b 88c
Pro 116 159 173 251 <.05
Ser 95 96 79 97
Gly 266a 239b 156° 160° <.01
Ala 243 283 275 269
EAA 987 1086 1216 1762
NEAA 1008 a 1101 b 959 C 1034 d
E/N 0.98 0.99 1.27 1.70 <.01

a,b,c,d

The figures with different superscript are
significantly different.

eProbability level in test of significance by Anova II
(Appendix Table II.22) when comparing all treatments. The
different superscripts indicate significant difference by
ortogonal contrasts; 0 vs. LMH, L vs. MH and M vs. H. 12
observations per plot.

fThe name of the amino acids are given fully in
Table 3.4 which also indicates essential (E) and nonessential
(N) amino acids.

9P < .001 for M vs. H.

176

Table 3.3.--Trial II 1971. Plasma free amino acids, molar %
distribution.a

 

 

Treatment
Amino acid

 

O L M H

 

0‘

O
l
l
I
I

00

l
l
l
l

Lys
His
Thr
Val
Ile
Leu
Met
Cys
Phe
Tyr
Arg
Asp
Glu
Pro
Ser
Gly
Ala
E/N ratio

g.»

ONw-bU'IU'IOwNNl-‘l—‘WO‘U'IU'Iw-k
l-‘

owoeqmowwNI-atoxommmwm
H

l—‘NflwmbOWNNl-‘WI—‘mmbwb
|-‘

H

:ooxmcucumcncnosaqcoovoxooxmcn
FJH

l—‘KOWWKOUJOHNl-‘l-‘Qomxlbtvw

\OkaO-bwubflmHmmw-bwaNw

\owwoooowooxoxooommmmmqwm

ZZZZZZZZWZL‘UMHWME’JM

HFH

Hra

H
~qovqunclaslmtnunoslwhohuakao

 

aTable 3.2 presents significance for difference
between treatments in actual concentrations (uM/l).

bThe name of the amino acids are spelled fully in
Table 3.4.

C O U 0 O
E = essential, N = nonessential amino aCid.

 

 

 

 

Table 3.4.--Trial III 1971.

177

control and treatment infusions.

Plasma free amino acids at

 

 

 

Amino acid oa K 01 K 02 Pb
----uM/1---- --%----
Lysine EC 86 99 5.3 5.3 5.1
Histidine E 65 71 3.8 3.8 4.1
Threonine E 75 85 4.4 4.6 4.6
Valine E 193 271 11.6 14.5 12.0 <.05
Isoleucine E 103 121 6.2 6.5 6.1
Leucine E 140 180 8.5 9.6 8.6
Methionine Ed 30 49 1.7 2.6 1.9 <.01
Cystine N 35 38 2.1 2.0 2.2
Phenylalanine E 42 41 2.6 2.2 2.5 <.05
Tyrosine N 48 52 3.0 2.8 2.9
Arginine N 76 82 4.7 4.4 4.6
Aspartic acid N 13 12 0.7 0.6 0.7
Glutaric acid N 129 110 8.9 5.9 6.8 <.05
Proline N 101 152 6.0 8.1 6.2
Serine N 72 72 4.2 3.9 4.5
Glycine N 224 193 12.8 10.3 14.3 <.01
Alanine N 214 241 13.4 12.9 13.1
EAA° 734 917 44.1 49.1 44.9
NEAAd 912 952 55.8 50.9 55.3
E/N ratio 0.80 0.96 0.80 0.97 0.81 <.05

 

a
The
methionine in

bProbability level in test of significance by

control before (0
fusion (K) averagéd.

) and after (02) casein +

Anova III (Appendix Table 11.33) for 0 vs. K by ortogonal

contrast. 16

CE

dN

observations per plot.
essential amino acid.

nonessential amino acid.

 

178

isoleucine and leucine) were high, even at the control, com-
pared to trial III and other studies in lactating cows
(Jacobson, et_al., 1970; Fisher, 1972). Apparently the

level did not decline to the normal between protein infu-
sions which increased (P < .05)valine and isoleucine in
particular. In rats on a high protein diet, the plasma

BrAA remained high while there was an adaptation in cata-
bolic capacity for other amino acids (Anderson, et_al., 1968).

In all trials the casein and methionine infusions
tended to increase the molar % of EAA relative to NEAA
(Tables 3.1, 3.2 and 3.4), but the E/N ratio for the lowest
level of infusion (L) in trial II was identicalwith the
control. The glucose infusion in trial I did not change
the E/N ratio. An increase in this ratio results from
lower rate of catabolism of EAA relative to NEAA (Kaplan and
Pitot, 1970), and generally it indicates an improved amino
acid status (Munro, 1970).

In trial II the E/N ratio increased from treatment
level L to M mainly because the BrAA increased, while the
increase from M to H was due to a dramatic increase in
methionine. The actual level of BrAA also increased from
M to H, but in molar proportion this increase was offset by
the large increase in methionine. Thus, the molar % com-

parison fails to show some changes which may have had

metabolic significance.

 

179

Methionine and BrAA are metabolized largely by the
muscles and at a slower rate than other EAA (Kaplan and
Pitot, 1970). Inappetance has been imposed on rats by im-
balances between leucine and isoleucine and by toxicity from
high levels of methionine (Harper, 1958); and Broderick,
gt_al., (1970) observed decreased grain intake during
abomasal infusion of casein + methionine which increased
BrAA and methionine. Because feed intakes were depressed by
all protein treatments and did not differ significantly
between levels of infusion, a direct relationship between
the inappetance observed in trial II and a methionine toxi-
city was not apparent, deSpite the dramatic increase in
methionine from M to H.

In trial II the molar % of certain EAA were depressed
at the higher infusion levels compared to the control, due
to increased BrAA and methionine. In trials I and III, how-
ever, the molar % increased or remained unchanged for most
EAA, except threonine which was lowered (P < .05) by M and K
in trial I, and phenylalanine which was decreased (P < .05)
by the casein + methionine infusion in trial III. Moreover,
phenylalanine was the only EAA.which concentration (uM/l)
did not tend to be higher during treatment than control.

As plasma concentration of the first limiting amino
acid may decline when abundant supply of other EAA stimulate

protein synthesis (Munro, 1970), threonine in trial I and

phenylalanine in trial III might be considered the first

180

limiting when protein was infused in these two trials,
respectively. In trial I the molar % of threonine, together
with valine and leucine, tended to decline also by the

glucose infusion; thus strengthening the impression of a
relative threonine deficiency during the control as well as
treatment period. Abomasal infusion of methionine, fre-
quently considered the first limiting amino acid in ruminants,
depressed plasma concentration of threonine in growing sheep
(Scott, g£_gl., 1972; Nimrick, et_§l., 1970a) while the level
of methionine itself increased.

Different catabolic rates among amino acids, however,
may obscure the significance of plasma concentration changes.
Threonine dehydratase increased in rat liver with increased
protein intakes (Anderson, et_§l., 1968). Thus, threonine
may be lowered for reasons other than stressing demand by
increased protein synthesis. But Harper (1968) implied that
enzymes involved in metabolism of NEAA adapt according to
intake while enzymes that catabolize EAA adapt to their
intake as it relates to amino acid requirements.

Falling levels of threonine and phenylalanine and
other amino acids by post-ruminal supply of protein in
lactating cows were also observed by Spechter (1972) and
Broderick (1972; Broderick, gt_al,, 1972). However, their
media, blood and plasma reSpectively, as well as their in—
terpretations differed. Spechter (1972) concluded histidine,

phenylalanine and methionine were limiting at the highest

181

level of infusion since the blood concentration of these
amino acids then were lower than at the control. Broderick
(1972) implicated methionine, valine and lysine as limiting
amino acids because the plasma level of these were increased
by formalinized casein supplements. Potter, et_al., (1972)
found that indices, as applied by Spechter (1972), did not
identify the limiting amino acids in sheep as was earlier
suggested (Potter, et_al., 1968).

Spechter (1972) as well as Broderick (1972) supplied
insufficient levels of crude protein during control periods
which make search for a first limiting amino acid irrelevant
in that dietary situation. By observing amino acid con-
centrations at graded levels of supplements, however, the
limiting amino acids may be appropriately indicated at a
total protein level which meets or exceeds suggested
standards. In any event, blood concentrations no more than
implicate the critical availability of specific amino acids.
Separate experiments with appropriate additions of these
amino acids are required in order to verify a limiting supply.

Young, gt_al., (1973) concluded that excessive
plasma levels of amino acids may mask specific amino acid
deficiencies. At low dietary protein levels, however, they
found highly significant correlations between the plasma

levels of most EAA and daily gains in steers. Thus, plasma
amino acid levels during postruminal protein supply above

standard feed requirements may be unsuitable to identify the

 

182

rate limiting amino acid. On the other hand, this treatment
will express the potential for amino acid utilization more
explicitly than supplementation of substandard diets.

Taking synthetic demands as well as availability of
amino acids into account, Chandler and Polan (1972) related
the concentration of EAA in blood serum to milk protein out-
put by calculating a "minimum transfer efficiency" at an
assumed blood flow rate (4501 per 1 of milk produced). With
a higher transfer efficiency than any other EAA at yields
between 16 and 37kg milk per day, methionine was the most
critical amino acid while the ranking among four other amino
acids (lysine, phenylalanine, tyrosine and threonine) shifted
with level of production. Transfer efficiency decreased with
level of production (Chandler and Polan, 1972), as might be
expected.

The data of trial III was evaluated similarly to the
approach of Chandler and Polan (1972). The output of each
amino acid by milk protein (Maa) was related to the content
of this amino acid in plasma (Paa) (Table 3.5). The ratio,
[Maa (g/day): Paa (g/1)] expresses how many 1 plasma must
be "cleared" [C, (l/daY)] to furnish the amino acid in milk
protein but neglects differences in uptake efficiency by the
mammary cells. Our data and that of Chandler and Polan

(1972) ranked essentially the same amino acids as critical,
although their order differs somewhat. Here phenylalanine

was the EAA of lowest availability in plasma relative to

 

183

Table 3.5.--Trial III 1971. The relationship between output
of amino acids in milk protein (Maa)a and plasma
free amino acids (Paa).

 

 

"Clearance" (C)c (C/AVdd)x100

Amino

aCld Oe K Oe K

 

 

l/day (rank)f l/day (rank) l/day (rank) l/day (rank)

Lys 2792 (2) 2580 (2) 5369 4962
His 1125 (8) 1130 (7) 4892 4913
Thr 2226 (5) 2099 (3) 6953 (2) 6559 (2)
Val 1276 (7) 965 (8) 6079 (3) 4595
Ile 1860 (6) 1742 (5) 5635 5279 (3)
Leu 2292 (4) 1922 (4) 4982 4178
Met 2448 (3) 1653 (6) 4707 3179
Phe 3102 (l) 3463 (1) 7754 (1) 8860 (1)
Tyr 2500 2596 5209 5408

 

aMaa = the amount of any amino acid (aa) put out by
estimated true protein (ETP, g/day) in milk (Appendix Table
II.30). Maa = ETP x F, where F = the fraction (weight %) of
each amino acid in total milk proteins derived from Porter,
et al., (1968); i.e., Lys 7.60, his 2.53, thr 4.32, val 6.19,
ile 5.54, leu 9.10, met 2.44, phe 4.60, tyr 4.78.

bPaa = the content of any amino acid in plasma (g/l);
Paa = uM/l x MW x 10‘5, where MW is the molecular weight
(Damm, et al., 1966).

CClearance (C) = Maa/Paa (l/day) for any amino acid;
i.e., the plasma volume that must be cleared to furnish the
output of an amino acid by milk protein.

dAVd = the arterio-venous concentration difference
over cows' udder as a fraction of arterial concentration,
derived from Verbeke and Peeters (1964); i.e., in %, lys 52,
his 23, arg 42, thr 32, val 21, ile 33, leu 46, met 52, phe
40, tyr 48.

eAverage control before and after casein + methionine
(K) infusion.

fAmong EAA.

184

demand, followed by lysine, in the control (0) as well as
the treatment (K) periods. While methionine was ranked the
third least available in the control situation, casein +
methionine infusion increased methionine concentrations and
rendered it among the more abundant EAA. Threonine was the
third most critical during protein infusion. Tyrosine, which
was among the critical amino acids in the Chandler and Polan
(1972) study, had a high clearance value, close to lysine.
This high value for tyrosine emphasizes the low availability
of phenylalanine. Moreover, phenylalanine was the only EAA
for which molar % decreased (P < .05) from control to treat-
ment periods. This amino acid stimulated milk protein
synthesis more than other amino acids in an in vitro system
(Emery, g£_§1., 1970).

When dividing the clearance values by arteriovenous
differences (AVd) observed in cows (Verbeke and Peeters,
1964) (Table 3.5), neglecting that a small part (”10%) of
milk proteins are not synthesized by the udder, phenylalanine
still appears as the least available amino acid, followed by
threonine. Applying the AVd values obtained in goats (Mepham
and Linzell, 1966) showed lysine as the least abundant EAA,
followed by phenylalanine. Lysine was the only EAA that had
lower AVd (%) in the goat study (Mepham and Linzell, 1966)
than in the cow study (Verbeke and Peeters, 1964). However,
it is a crucial question how far the efficiency of mammary

amino acid uptake changes with the amino acid availability

(Rock, 1971).

 

185

The high AVd observed in goats (Mepham and Linzell,
1966) for methionine, phenylalanine, threonine and leucine
was suggested (Mepham, 1971) to indicate critical supply of
these EAA. Experimental evidence shows transport into and
out of cells generally provides one potent means for regula-
tion of protein metabolism (Munro, 1970). And an extracel-
1ular excess of one amino acid may affect the entry of that
or other amino acids into certain tissues (Munro, 1970).
Possible amino acid transport to the udder by proteins and
enythrocytes (Elwyn, 1970) have been neglected in this dis-
cussion, but variation in these sources might also influence
the availability of amino acids for milk protein synthesis.

The irregularity in actual PAA levels in trial I and
the fluctuating production in trial II discourage evalua-
tion by clearance values for these experiments.

While the PAA data point to phenylalanine as the
most critical amino acid in trial III and threonine in
trial I, the dependency of response on treatment level
(Table 2.22) may suggest that more than one EAA was re-
sponsible for the increase in milk protein production. The
statement of Munro (1970) that "we know very little about
the effect of lack of specific amino acids in the diet on
synthesis of protein in tissues other than the liver" appar-

ently is valid also for the cow's mammary gland.

 

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203

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204

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Yousef,

Yousef,

207

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APPENDIX TABLES

208

 

 

m.vH v.w N.mH o.o N.va v.m «.mH m.m O m
m.va m.m N.ma m.v m.mH o.o m.ma m.v x b
m.mH m.m N.mH m.v II II II II 0 m
v.mH m.m o.mH m.v o.mH ¢.m N.mH m.v O m
m.mH m.m «.ma m.v o.mH v.0 N.mH m.v o v
m.ma m.m ~.ma m.v H.mH m.m m.ma o.o o m
m.mH o.h N.mH m.¢ H.mH v.m N.mH m.v M N
m.ma H.h m.ma m.v m.va o.o «.ma oa.v o a
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209

 

 

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mm m.mm mHH mm.m baa o.mm nma mm.m w v
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210

 

 

 

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2112

 

 

 

Appendix Table I. 5--Trial I 1970. Observations in milk production parameters.
Sub- Milk yield Prat. conc.a Fat conc.a SNF conc.a
Per Trtm per 501 502 m 501 502 m 501 502 m 501 502 m
kg/day 8 8 0
1 0 l 19.10 22.60 3.22 3.29 4.1 --b 9.40 -_
2 19.25 22.45 3.37 3.32 --b 4.8 -- 9.65
_§ 18.95 21.90 3.19 3.26 4.0 4. 2.05 19.99
m 19.10 22.32 20.71 3.262 3.291 3.277 .07 4.44 4.26 9.22 9.81 9.54
2 K 1 19.00 22.60 3.32 3.41 4.2 3.8 8.80 9.60
2 20.25 22.95 3.22 3.58 --b 3.9b 9.20 9.40
_9 19.60 22.95 3.34 . 3.8 —- —-
m 19.62 22.83 21.25 3.293 3.518 3.406 3.96 3.83 3.89 9.19 9.50 9.35
3 0 l 17.65 21.47 3.26 3.35 4.0 3.9 9.45 9.10
2 16.80 21.60 3.46 3.37 3.6 4.2 9.55 8.70
_g 17.35 22.25 3.82 3.22 3.2 4.0 9.65 9.30
m 17.27 21.77 19.50 3.512 3.311 3.412 3.84 4.03 3.95 9.55 9.04 9.27
4 G l 17.25 21.35 3.75 3.07 4.2 3.9 9.20 9.10
2 16.55 21.65 3.81 3.43 2.9 3.4 10.30 9.10
_9 17.25 22.85 78 3 50 jLJ__j4A_
m 17.02 21.95 19.49 3.781 3.338 3.560 3.27 3.56 3.44 9.90 9.13 9.47
5 0 l 16.00 21.45 3.82 3.52 3.2 3.5 10.00 9.55
2 16.00 20.35 3.77 3.54 4.3 2.4 9.30 10.70
_; 14.80 20.50 ;,§6 3.34 4.4 --b 8.§0 --
m 15.60 20.77 18.19 3.752 3.467 3.610 3.96 2.98 3.40 9.32 10.14 9.79
6 0 l 16.00 20.75 3.82 3.43 3.2 3.2 10.00 10.10
2 16.00 20.30 3.77 3.75 4.3 2.5 9.30 10.40
_2 14.80 17.80 3.66 3.70 4.4 3.8 8.60 9.85
m 15.60 19.62 ‘17.61 3.752 3.623 3.688 3.96 3.14 3.50 9.32 10.12 9.77
7 K 1 17.70 19.75 3.68 3.90 3.8 4.0 9.25 9.30
2 17.20 19.95 3.84 3.70 3.8 4.0 9.70 8.50
_; 15.55 20.45 3.75 3.67 4.4 3.2 2.30 9.70
m 16.82 20.05 18.44 3.756 3.756 3 756 3.99 3.73 3.86 9.42 9.17
8 0 l 15.30 16.75 3.73 3.68 4.5 4.8 9.40 8.65
2 14.95 18.60 3.62 3.60 4.8 4.5 9.10 8.80
_§ 14.95 17.70 3.50 3.69 4.4 3.6 8.80 9.25
m 15.07 17.68 16.38 3.617 3.655 3.636 4.57 4.29 4.42 9.10 8.90
aMeans (m) for each cow period weighted by the yield of milk.

b

Fat test observation missing.

Appendix Table I.6--Tria1 I 1970.

213

Differences in milk pro-
duction parameters between treatment
(infusion) and control periods.

 

 

 

Cow No. 501 Cow No. 502
Infusion Infusion

Parameter lst CAS GLC 2nd CAS lst CAS GLC 2nd CAS
(1) Milk yield Trt 19.62 17.02 16.82 22.83 21.95 20.05
kg/day Ctr 18.19 16.44 15.34 22.05 21.27 18.65

d 1.43 .58 1.48 .79 .68 1.40

(2) Prot cons Trt 3.29 3.78 3.76 3.52 3.34 3.76
% Ctr 3.39 3.63 3.68 3.30 3.39 3.64

d -.10 .15 .08 .22 -.05 .12

(3) Fat cons Trt 3.96 3.27 3.99 3.83 3.56 3.73
% Ctr 3.95 3.90 4.26 4.23 3.50 3.72

d .01 -.63 -.27 -.40 .06 .01

(4) SNF cons Trt 9.19 9.90 9.42 9.50 9.13 9.17
% Ctr 9.37 9.43 9.21 9.43 9.57 9.55
d -.18 .37 .21 .07 .07 -.38

(5) Prot prod Trt 646 643 632 803 733 753
g/day Ctr 614 596 565 727 721 679

d 32 47 67 76 12 74

(6) Fat prod Trt 778 557 670 875 782 747
g/day Ctr 720 640 653 934 748 688

d 58 -83 17 -59 34 59

(7) SNF prod Trt 1802 1684 1584 2169 2005 1839
g/day Ctr 1705 1552 1413 2079 2037 1780

d 97 132 171 90 -32 59
(8) FCM prod Trt 19.52 15.16 16.78 22.26 20.51 19.20
kg/day Ctr 18.08 16.18 15.93 22.83 19.73 17.80
d 1.44 -1.02 .85 -.57 .78 1.40

 

Appendix Table I.7--Tria1 II 1970.

2114

Milk production parameters;

basic observations.

 

 

 

 

 

 

 

 

 

 

 

 

Sub- Milk yield Protein conc.a Fat 6000-3
Per.Trtm.peL480 501 502 m 480 501 502 m 480 501 502 m
------ kg/day------- —-------8------- -------8--—----
1 0 1 12.03 12.00 17.13 3.64 4.02 3.76 5.1 4.9 4.0
2 12.65 11.38 15.60 3.50 3.91 3.82 4.6 4.6 4.0
_3 12.10 11.47 15.60 4.5 14.7, 414
. m 12,25 11.62 16.11 13.33 3.526 3.887 3.780 3.731 4.73 4.74 4.13 4.49
2 K 1 12.77 13.16 19.03 3.58 3.77 3.77 4.1 4.3 4.0
2 12.66 13.29 18.30 3.59 3.89 3.79 4.4 3.8 4.0
3 12.82 11.84 17.76 3.49 . 4.2 417 319 .
"m 12775——TIT76__18736 14.62 3.553 3.826 3.790 3.723 4.23 4.25 3.97 4.13
3 0 1 12.53 11.65 15.99 3.45 3.82 3.73 4.4 4.9 4.1
2 12.97 12.40 17.12 3.46 3.79 3.65 4.5 4.6 4.1
_2 12.77 11.90 14.76 3.38 4.6 4.1 4.2
m Ij;75 11.98’ 15.96 13.57 3.431 3.844 3.691 3.655 4.50 4.53 4.13 4.36
4 G 1 12.86 12.73 17.92 3.41 3.83 3.46 4.4 4.2 3.8
2 13,07 12.50 17.97 3.41 3.79 3.52 4.1 3.8 3,4
3 11.95 12.02 17.5 3.68 3.97 3.85 4.3 4.2 3.0
'fi , . 17.81 14.28 3.498 3.860 3.608 3.655 4.27 4.07 3.40 3.85
S 0 1 10.77 11.84 16.12 3.65 3.80 3.95 4.4 4.2 3.6
2 12.40 12.25 14.33 3.60 3.92 3.95 4.4 3.6 4.6
_3 11.98 11.64 14.77 3.60 3.95 4.00 4.7 4.6 0
m . 1 15.10 12.91 .6 3 3.890 3.967 3.823 4T50 4.12 4.05 4.21
6 0 1 11.75 11.64 14.77 3.68 3.95 4.00 4.2 4.6 4.0
2 10,35 11.43 14.20 3.47 3.95 4.02 --a 4.0 3.0
_3 10.89 11.93 16.33 3.75 3.90 5.93 4.5 4.8 4-0
m _17 . 15.10 12.64 .6 3 3.934 4.018 3.862 4.33 4.73 3.94 4.30
7 K 1 10.61 12.17 16.02 3.75 4.03 3.95 4.5 4.5a 3.88
2 11,75 11.72 15.06 3.81 3.93 3.97 4.5 -- --
3 11.76 10.84 15.24 3.86 4.05 3,81
‘fi , . 15.44 12.80 .9 4.005 3.910 3.909 4.57 4.53 ,4.13 4.38
8 0 1 11.20 11.12 14.56 3.65 3.91 3.87 5.0 4.9 4.3
2 11.88 10.12 12.77 3.58 3.85 3.80 4.9 4.6 4.7
_§ 11.25 10.25 14.62 3.60 3.81 3.70 4.3 4.9 4.2
m . . . 8 11.97 3.615 3.862 3.79 3.756 4.74 4.81 4.39 4.62

 

aMeans (m) for each cow period weighted by yield of milk.

b

Fat test observation missing.

2315

Appendix Table I.7--Continued.

 

 

 

 

 

 

 

 

 

 

SNF conc.a Production of milk constituents
Per. Trtm.Subp. 480 501 502 m Per, Trtm , 480 501 502 m
.......... §—-——----- -—-protein, g/day--
1 0 1 9.04 9.20 9 40 1 0 432 450 609 497
2 9_24 9 50 9 62 2 K 453 488 696 546
3 9.37 9‘44 9‘55 3 _ o 433 461 590 496
'5 9.22 9.38 9.52 9 39 4 G ‘41 ‘79 643 521
' 5 0 423 463 599 495
2 K 1 9.66 9.34 9 48 6 0 406 459 606 490
2 9.38 9.95 9.40 7 K 434 464 604 501
_§ 9.46 9 50 9-76 8 0 413 405 530 449
m 9.50 9 60 9.54 9.55 ----fat. g/day _____ .
3 O 1 9.26 9 40 9.70
2 9.21 9.58 9 59 1 0 580 550 665 598
3 9.18 10.22 9.66 2 K 539 542 729 603
‘E 9.22 9.72 9.65 9 54 3 0 574 543 559 592
4 G 539 505 606 550
4 G 1 9.25 9 70 9.55 5 0 528 491 611 543
l 9.37 9 75 9.40 6 o 484 552 595 544
_; 9.37 9 60 9.50, 7 x 520 525 637 561
m 9.33 9 69 9.48 9 50 8 0 542 504 613 553
S 0 1 9.15 9 40 .26
2 9.37 --a 3.15 "”SNF' 9/d‘Y """
3 9.48 9.87 9-77 1 O 1130 1089 1534 1251
”a 9.34 9.62 9.55 9.51 2 K 1212 1226 1752 1397
b 3 O 1176 1166 1540 1294
6 0 1 9.37 -- 10.10 4 G 1178 1203 1689 1357
3 9.48 9.03 9.70 s o 1094 1146 1441 1227
__ 9.17 9.61 9.50 6 o 1043 1109 1474 1209
m 9-34 9-51 9-75 9-56 7 x 1091 1105 1476 1224
7 K 1 9.50 9. 50b 9. 70b 8 0 Egzgpcn'l 196/day-£§.?.§ 1171
2 9.55 -- " 1 0 13.6 12.9 16.4 14.3
.3 9-72 9-60 9-37 2 x 13.2 13.2 19.3 14.9
m 9.59 9.55 9.56 9.56 3 0 13,7 12,9 16,3 14.3
8 0 1 9.42 9.68 9.60 4 G 13.1 12.5 16.2 14.0
2 9.40 9.97 9"3 5 O 12.6 12.1 15.2 13.3
3 9.46 9.39 9.45 6 O 11.7 13.0 15.0 13.2
1 9.43 9.68 9.50 9.53 7 X 12.4 12.5 15.7 13.5
8 0 12.7 11.8 14.8 13.1

 

aMeans (m) for each cow period weighted by the yield of milk.

bFat test observation missing.

2115

Appendix Table I.8--Trial II 1970. Differences between treatment (infusion) and control
periods in milk production parameters.

 

Cow No. 480 Cow No. 501 Cow No. 502
Infusion Study Infusion Study Infusion Study
First Second First Second First Second
(row) Parameter Casein Glucose Casein Casein Glucose Casein Casein Glucose Casein

 

(1) Milk yield Trt 12.75 12.63 11.37 12.76 12.42 11.58 18.36 17.81 15.44
kg/day Ctr 12.51 12.24 11.31 11.80 11.95 11.09 16.04 15.53 14.54
d .24 .39 .06 .96 .47 .49 2.32 2.29 .80

(2) Prot. conc. ‘Trt 3.55 3.50 3.81 3.83 3.86 4.01 3.79 3.61 3.91
% Ctr 3.48 3.52 3.62 3.86 3.87 3.90 3.74 3.83 3.90
d .07 -.02 .19 -.03 -.01 .11 .05 -.22 . .01
(3) Est.True Trt 3.33 3.35 3.66 3.61 3.69 3.78 3.56 3.43 3.71
prot.a conc,Ctr 3.27 3.33 3.41 3.63 3.65 3.68 3.53 3.62 3.70
g d .06 .02 .25 -.02 .04 .10 .03 -.19 .01
(4) Fat conc. Trt 4.23 4.27 4.57 4.25 4.07 4.53 3.97 3.40 4.13
8 Ctr 4.62 4.50 4.53 4.63 4.33 4.77 4.13 4.09 4.16
d -.39 -.23 .04 -.38 -.26 -.24 -.16 -.69 -.03
(5) SNF Conc. Trt 9.51 9.33 9.60 9.61 9.69 9.55 9.54 9.48 9.56
% Ctr 9.22 9.27 9.38 9.56 9.68 9.58 9.58 9.60 9.63
d .29 .06 .22 .05 .01 -.03 -.04 -.12 .07
(6) Prot. prod. Trt 453 441 434 488 479 464 696 643 604
g/day Ctr 435 431 410 455 462 432 599 595 568
d 18 10 24 33 17 32 97 48 36
(7) Est. True Trt 424 422 416 461 458 437 654 611 574
prot, prod. Ctr 408 407 386 428 436 409 567 633 539
g/day d 16 15 30 33 22 28 84 -22 35
(8) Fat prod. Trt 539 539 520 542 505 525 729 606 637
g/day Ctr 577 551 513 547 517 528 662 635 604
d -38 -12 7 -5 -12 —3 67 -29 33'
(9) SNF prod. Trt 1212 1178 1091 1226 1203 1105 1752 1689 1476
g/day Ctr 1153 1135 1061 1128 1156 1063 1537 1491 1401
d 59 43 31 98 47 42 115 198 75
(10) FCM prod. Trt 13.19 13.14 12.35 13.23 12.54 12.51 18.28 16.21 15.73
kg/day Ctr 13.66 13.16 12.22 12.92 12.54 12.36 16.35 15.74 14.88
d -.47 -.04 .13 .31 .00 .15 1.93 .47 .85

 

a(Tota1 Kjeldahl N-NPN) x 6.38.

217

Appendix Table I.9--Trial II 1970. Non Protein Nitrogen
(NPN) concentration in milk.

 

 

 

Cow No. Total
Parameter Per Trtm 480 501 502 meaniSE
----------- mg/100m1----------—-
Non Protein 1 0 34.1 37.7 30.5 34.1 i 2.08
Nitrogen (NPN) 2 K 35.0 34.1 35.9 35.0 t 0.52
3 0 33.3 35.0 32.4 33.6 t 0.76
4 G 23.4 26.9 28.7 26.3 t 1.56
S 0 28.7 32.3 31.4 30.8 t 1.08
6 0 31.4 35.0 36.7 34.1 t 1.56
7 K 23.4 35.9 31.5 30.3 t 3.66
8 0 35.0 32.2 26.1 31.1 i 2.63
C.V. 12.5
............... g--_--_--__-----
NPN as a frac- l 0 6.2 6.2 5.1 5.83 i .36
tion of total N 2 K 6.3 5.7 6.0 6.00 i .17
3 0 6.2 5.8 5.6 5.87 i .17
4 G 4.3 4.4 5.0 4.57 t .22
5 0 5.1 5.3 5.0 5.13 t .08
6 0 5.5 5.7 5.8 5.67 i .08
7 K 3.9 5.7 5.1 4.90 i .53
8 0 6.2 5.3 4.4 5.30 i .52

 

 

218

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Appendix Table I.12--Trial I 6 II 1970.

222C)

Analysis of variance for milk production para-

 

 

 

 

 

 

 

meters.

Infusion Study: lst Casein Glucose 2nd Casein dCAS.vs.dGLC.

Source Test Test Test Test
of of of of of

Parameter Variance 55 DP sign. SS DF sign. SS DF sign. SS DP sign.

Milk yield Cows 149.5337 4 119.4019 4 92.4331 4 3.17814 4

(kg) Trtm 3.4164 1 P<.05 1.9492 1 P<.10 1.8879 1 P<0-5 .03969 1 us
Error 1.1866 4 1.2526 4 0.7243 4 .71441 4
Total 154.1367 9 122.6037 9 95.0453 9 3.93224 9

Protein Cows 0.38665 4 .30719 4 .11579 4 .037267 4

conc (%) Trtm .00473 1 NS .0023? 1 .02406 1 P<.05 .025553 1 NS
Error .02806 4 .03475 4 .00892 4 .055022 4
Total .41944 9 .34431 9 .14877 9 .117822 9

Fat Cows .43032 4 1.15841 4 1.11277 4 .143243 4

cone (5) Trtm .17450 1 P<.05 .30520 1 P<.10 .02520 1 (P<.25) .069806 1 NS
Error .06728 4 .18817 4 .04232 4 .259507 4
Total .67210 9 1.65178 9 1.18029 9 .472556 9

Protein Cows 14l,901.l 4 114,415.6 4 115,786.8 4 2,369.00 4

produc- Trtm 6,451.6 1 P<.05 1,849.6 1 P<.05 5,475.6 1 P<.01 1,166.40 1 P~10

tion (9) Error 2,240.9 4 732.4 4 1,004.2 4 1,119.85 4
Total 150,593.6 9 116,997.6 9 122,266.6 9 4,665.25 9

Fat pro- Cows 176,376.5 4 76,547.0 4 59,882.2 4 8,104.91 4

duction Trtm 55.2 1 NS 1,040.4 1 NS 1,292.6 1 P=.10 2,222.41 1 NS

(9) Error 6,343.9 4 3,546.6 4 1,197.2 4 2,915.56 4
Total 182,775.6 9 81,134.0 9 62,379.0 9 13,242.88 9

FCM (kg) Cows 125.606 4 71.633 4 55.345 4 1.3862 4
Trtm 0.702 1 NS 0.002 1 NS 1.056 1 P<.05 0.7840 1 NS
Error 2.476 4 1.839 4 0.495 4 4.1768 4
Total 128.784 9 73.474 9 56.896 9 4.1768 9

Appendix Table I.12--Continued.

Infusion Study: lst Casein Glucose 2nd Casein dCAS.vs.dGLC.
Source Test Test Test Test

of of of of of

Parameter Variance 55 DF sign. SS DE sign. SS DE sign. 55 or sign.

SNF cons. Cows .133693 4 .245644 4 .124039 4 4

(8) Trtm .003055 1 NS .000118 1 NS .000513 1 NS 1 NS
Error .059439 4 .210075 4 .115959 4 4
Total .196187 9 .455837 9 .240511 9 9

SNF pro— Cows l,301,503.4 4 1,025,523.6 4 764,333.4 4b 4

duction Trtm 31,379.1 l P<.025 15,173.4 1 (P<.25)14,276.3 1 1 P<.05

(9) Error 7,233.6 4 15,793.9 4 6,284.2 4 4
Total 1,340,116.1 9 l,056,490.9 9 784,893.9 9 9

NPN cons Cows 2.416 2 12.711 2 41.610 2 44.530 2

(mg%)(Tr. Trtm 2.042 1 NS 51.333 1 P<.05 9.127 1 NS 47.602 1 P<.05

11 only) Error 11.235 2 5.448 2 41.543 2 4.923 2
Total 15.693 5 69.492 5 92.280 5 97.055 5

NPN/T.N Cows .5200 2 0.0850 2 1.006 2 0.5842 2

Tr.II only Trtm .0520 1 NS 2.2571 1 P<.10 0.928 1 NS 2.0184 1 P<.10

on trans- Error .3500 2 0.5433 2 2.509 2 0.2548 2

formedidataTotal .9220 5 2.8854 4.444 5 2.8574 5

True pro- Cows 32,679.25 2 44,104.08 2 28,511.33 2 40,585.58 2

tein pro- Trtm 1,683.38 1 p<.10 3,151.04 1 us 1,536.00 1a 77.04 1 us

duction (9) Error 273.25 2 1,383.08 2 25.00 2 215.58 2

Trial II Total 34,635.88 5 48,638.20 5 30,511.33 5 40,878.20 5

only and values
Cows .11421 2 .11185 2 .04377 2 .035,536 2
Trtm .00091 1 NS .00322 1 NS .02042 1 NS .020,651 1 NS
Error .00189 2 .01772 2 .01556 2 .012,007 2
Total .11701 5 .13279 5 .07975 5 .068,194 5

aP<.05.

bp<.01.

221

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223

Appendix Table II.2--Tria1 I 1971. Feed offered, and consumed
dry matter (DM), estimated net energy for
lactation (E-NEL) and crude protein (CP)

for each cow in each period.

 

 

Period
Item 1 2 3 4 5 6 7

Feed Offered, kg/daya

 

 

 

 

Hay 5.4 5.4 5.4 5.4 5.4 5.4 5.4
Corn Silage 9.0 8.5 9.0 9.0 9.0 9.0 9.0
Concentrate 11.3 11.3 11.3 11.3 11.3 11.3 11.3
Intakes, DM and CPkg/day, ENELMcal/day
No. 604 Trtm. G M K
DM Hay 4.0 4.7 4.8 4.7 4.5 4.5 4.7
C.S. 1.5 1.4 1.6 2.0 1.8 2.0 3.3
C.tr. 10.9 _§.7 10.1 10.1 10.1 10.2 10.1
DM Total 15.5 15.8 16.4 16.8 16.4 16.7 18.1
CP Total 2.31 2.40 2.41 2.15 2.39 2.46 2.80
E-NEL Total 27.8 26.0 28.5 28.6 28.3 29.2 32.4
No. 606 Trtm. K G M
DM Hay 4.4 4.6 4.6 4.6 4.5 4.4 4.6
C.S. 1.8 1.7 1.6 1.4 0.8 1.0 2.1
C.tr. 9.6 8.5 9.7 9.1 9.4 9.9 9.3
DM Total 15.8 1418 15.9 15.1 14.77515.3 16f0
CP Total 2.32 2.41 2.42 1.99 2.24 2.46 2.55
E-NEL Total 27.8 25.7 28.2 26.0 25.8 28.1 28.7
No. 607 Trtm. M K G
DM Hay 4.4 3.3 4.0 4.1 3.9 4.3 4.4
C.S. 2.9 2.5 2.4 2.3 2.4 2.1 2.6
C.tr. 12.0 10.2 10.2 10.2 9.9 9.8 9.9
DM Total 7.3 16.0 16.6 16.6 16.2 16.2 16.9
CP Total 2.51 2.58 2.48 2.19 2.41 2.49 2.66
E-NEL Total 30.7 28.6 29.7 29.3 28.7 29.2 30.5
No. 603 Trtm.b K
DM Hay 4.5 4.5 4.7
C.S. 3.2 3.0 3.4
C.tr. 8.1 8.2 8.1
DM Total 15.8 15.7 16.2
CP Total 2.34 2.38 2.53
E-NEL Total 27.2 27.7 28.7

 

aFor cows No. 604, 606 and 607.

bCow No. 603 received only the K treatment. Her
daily feed was 5.4kg hay, 9.0kg corn silage and 9.1kg
concentrate.

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.Ummwum>m mpoflumm

Houucoo was mUOHHmm pcmEsmoHu CH mMMuCH pooh

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225

Appendix Table II.4--Trial I 1971. Intakes of estimated net
energy and crude protein relative (%)
to NRC (1971) standards.

 

 

Cow No.

 

 

 

 

Trtm. Meana
604 606 607 603
Crude Protein intake (%)
K 101d 107 86 126 106
OK 106 108 103 135 103
G 103 95 112 109
0G 100 108 119 104
M 90d 117 105 98
0M 98 119 101 106
Estimated NE intake (%)
K 117 108 110 137 117
OK 120 121 116 142 119
G 108 118 124 119
0G 114 118 129 120
M 119 126 111 112
OM 112 127 115 118
Paramtr. Period No.a
1 2 3 4 5 6 7 A11
Ob T O T O T O O T
CP (%) 103 105 102 90 103 110 125 107 119
E-NEL (%) 118 109 115 116 116 122 135 102 116
aCow No. 603 (received only the K trtm.) not included.
b = control; saline infusion.
C = treatment; substrat infusion.

dIn period 5 (after M, before K) the CP intake was

96 (%).

2226

Appendix Table II.5-—Trial I 1971. Observations in milk production parameters.

 

 

 

 

 

 

 

 

 

 

 

 

Item: (1) (2)
Cow: 604 606 607 603 Mean8 604 606 607 603 Meana'b
Per. Subper. Milk Yield Protein (Nx6.38) concentration
1 ------------ kg/day ------------------------ 3‘ ------
1 26.04 22.14 23.95 2.85 2.74 3.15
2 24.48 22.65 24.60 2.72 3.04 2.88
m 25.26 22.40 24.28 23.98 2.787 2.891 3.0137 2.90
2 G K M G K M
1 26.72 23.69 25.96 3.04 3.00 3.35
2 25.80 25.07 26.14 2.92 2.89 3.39
m 26.26 24.38 26.05 25.56 3.000 2.943 3.370 3.10
3
1 25.13 23.81 24.49 2.80 2.69 3.20
2 27.15 23.31 .24.53 2.93 2.74 3.10
m 26:14 23.56 24.51 24.74 2. . . 5 2.91
4 M G K M G K
1 27.84 23.54 26.11 3.09 2.82 3.32
2 28.35 23.27 25.56 3.10 2.83 3.37
m 28.04 23.43 25.89 25.79 3.094 2.824 3.340 3.09
5 1 27.88 22.89 21.89 17.28 3.11 2.89 3.14 3.07
2 27.78 22.85 21.88 17.96 3.09 2.96 3.20 2.92
m 27.83 22.87 21.88 17.62 24.19 3.100 2.925 3.170 2.992 3.07
6 K M G K K M G K
1 27.46 23.20 23.20 19.82 3.34 3.04 3.29 3.32
2 28.20 22.59 20.52 19.02 3.25 3.05 3.27 3.21
7 m 0 l. . 24.20 3.294 3.045 3.28023.266 3.20
1 25.90 21.61 19.41 17.69 2.92 2.88 3.23 3.07
2 26.28 21.39 20.14 16.75 3.02 2.93 3.23 3.06
m 26.09 21.50 19.78 17.22 22.46 2.970 2.906 3.230 3.065 3.04

 

aCow No. 603 not included in the means.

bPeriod mean for each cow weighted by the amount of milk in subperiods.

Appendix Table II.5—-Continued.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Item (3) b (4) a b
Cow: 604 606 607 603 Meana' 604 606 607 603 Mean '
Per. Subper. NPN concentration3 Fat concentration
1 ----------- mg/lOOml ----------------------------- 8 ----------------
1 3.0 3.0 2.9
2 20.05 18.35 19.20 3.1 2.9 3.1
m 20T05“I§T3§"I9720 19.20 3.05 72.95 3.00 3.00
2 c x M G x M
1 -- 24.60 24.50 2.8 2.9 2.7
2 23.05 24.00 23.65 2.7 2.6 2.5
m 23.05 24.29 24.07’ 23.80 2175"'IT75“"2700 2.70
3 1 19.35 18.35 21.65 3.5 3.2 3.0
2 19.70 19.35 21.85 2.9 2.7 3.5
m 19.53 18.84 21.75 20.04 3.19 2.95 3.25 3.13
4 M c x M c x
1 23.60 17.45 27.60 2.4 2.3 2.7
2 23.95 19.55 28.60 2.3 2.3 3.1
m 23.82 18.47 28.04 23.44 2.35 2130 2.89' 2.51
5 1 22.05 18.20 23.90 31.35 3.2 2.5 2.8 2.2
2 23.65 22.40 22.70 31.70 2.6 2.5 2.9 2.0
m 22.85 20.30 23:30 22.15 2.90 2:50 2.85 2.10 2.75
6 x M G x x M G x
1 30.08 30.60 28.46 39.80 2.6 2.8 2.7 2.5
2 29.45 26.80 25.70 -- 2.9 2.3 2.7 2.3
m 29.76 28.72 27.16 39280' 28.55 2.75 2.55 2.70 2.40 2.67
7 1 24.00 24.95 24.40 -- 3.3 2.3 2.6 2.6
2 24.70 26.00 24.80 31.20 3.2 2.29» 2.5 2.5
m 24.35 25.47 24.60 31.20 24.81 3 25 2.25 2.55 2.55 2.68

 

222'7

Appendix II.S--Continued.

 

 

 

 

 

 

 

 

 

 

 

 

 

Item (5) a b (5) b
Cow: 604 606 607 603 Mean ' 604 606 607 603 Meana'
Per. Subper. -------- Lactose conc., 8- ------------------- sup conc., g .............
1 8.32 8.30 8.65
2C 8.40 7.93 8.20
1 m 8.36 81117'78.42 8.30
c G x M
1 4.80 -- 5.01 8.50 8.20 9.15
2 4.56 4.82 5.03 8.38 8.24 8.89
2 m 4.68 4.82 5.02 4.84 8.44 8.22 9.02 8.56
1 5.16 5.35 4.57 8.25 7.90 8.73
2 5.04 5.08 5.62 8.25 7.88 8.70 8.29
3 m 5.10 5.22 5.10 5.14 8.25 777289 8172
M G K
1 5.14 4.62 5.11 8.45 8.10 8.74
2 4.99 5.24 4.80 8.56 8.04 8.35
4 m 5.07 4.92 4.95 4.98 8.52 8.06 8.53 8.37
1 5.12 4.86 4.57 5.24 8.42 8.02 8.62 8.37
2 5.00 4.80 5.03 5.06 8.64 8.18 8.63 8.56
5 m 5.06 4.83 4.80 5.15 4.90 8.53 8.10 '78.63 8.47 8.42
K M G K K M G K
1 4.79 4.76 4.86 4.88 8.61 8.21 8.93 8.60
2 4.87 4.67 5.03 4.86 8.70 8.26 8.96 8.83
6 m 4.832 4.72 4.94 4.87 4.83 8.66 8.23 8.78 8.78 8.56
1 5.08 4.93 4.95 5.03 8.78 8.14 8.94 8.72
2 4.89 4.93 5.02 5.13 8.50 8.02 8.70 8.70
7 m 4.98 4.93 4.98 5.08 4.96 8.64 8.08 8.82 8f71 8.51

 

CAssayed values discarded, disparting unacceptably from the common values and
wide variation between parallels.

Appendix Table II.5—-Continued.

 

 

 

 

 

 

Item: (7) (8) a
Cow: 604 606 607 603 Mean“ 604 606 607 603 Mean
Per. Subper. Protein Production Est. True Prot. Prod.
1 ------------ s/day ----------------------------- q/day -------------
1 742 607 754
2 666 689 709
m WIT—“732’ 695 m 665
2 c x M c x M
1 812 711 870
2 753 725 886
m m8 795 711—W 754
3 1 704 641 784
2 796 639 760
m 750 640 1 772 721 7T8"“609“"73T 687
4 M c x M a x
1 860 664 867
2 878 659 861
m 839 332 365 799 325 537 322 762
5 1 867 662 687 531
2 858 676 700 524
m 863““669"“691“"528 742 823"”339""5€I"“197 708
6 x M c x x M c x
1 917 705 763 658
2 917 689 671 611
m 917““697““7I7“"630 777 864 655 680 586 733
7 1 756 622 627 543
2 794 627 654 513
m 775 625 639 528 680 734 583 608 494 642

 

aCow No. 603 not included in the means.

228

Appendix Table II.6——Tria1 I 1971. Concentrations of urea nitrogen in blood (BUN).

 

Total period, Total seq.

 

 

 

 

 

 

 

Bleeding C0“ (the three first cows)
Sequence Perioda (B) 604 606 607 603 mp ms
------------------------ mg/100m1—-------—------------—---------
Trtm G K M
1 2T 1 25 19 27 23.7
2 13 22 32 22.3
3 20 39 28 29.0
all 19.3 26.7 29.0 25.0
30 l 15 16 17 16.0
2 15 21 19 18.3
3 l3 14 15 14.0
all 14.3 17.0 17.0 16.1 20.6
M G K
2 4T 1 30 21 29 26.7
2 21 16 34 23.7
3 37 14 18 23.0
all 29.3 17.0 27.0 24.4
0 136 OK 0
SO 1 27 33 34 30 31.3
2 24 25 32 b 32 27.0
3 14 27 [21] 31 (20.5)
all 21.7 28.3 (33.0) 31 26.3 25.4
K M G K
3 er 1 21 34 20 [29]b 25.0
2 37 23 b 27 25 29,0
3 11 [23] 17 14 (14.0)
all 23.0 (28.5) 21.3 (19.5) 22.7
70 OK Oh CG 0’
1 30 18 28 35 25.3
2 22 20 12 31 18.0
3 12 19 14 16 15.0
all 21.3 19.0 18.0 27.3 19.4 21.6
Total for cow mc 21.1 22.4 23.7 26.7

 

aSamples for period 1 lost in storage.

bl ]: stipulated according to Cochran and Cox (1957, p. 125), used for
for statistical analyses, not included in mean (m).

2229

Appendix Table II.7--Trial I 1971. Concentrations of glucose in blood plasma.

 

Total period Total seq.

 

 

 

 

 

 

 

Trtm. a Bleeding Cow (the three first cows)
Sequence Period (B) 604 606 607 603 mp ms
----------------------- mg/100m1--------------------------------
1 Trtm. G K ’4
2T 1 68.7 66.0 55,3 55,7
2 69.4 66.8 65.3 67.2
3 74.0 72.7 73.2 73.3
all 70.7 68.5 67.9 69.0
3o 05 OK 0M
1 63.8 63.8 64.4 64.0
2 67.6 63.8 66.0 65.8
3 67.1 64.0 62.5 64.5
all 66.2 63.9 64.3 64.8 66.9
M G 82‘
2 4T 1 72.2 74.8 65.5 70.0
2 74.5 67.2 61.7 67.8
3 66.5 58.5 73.7 66.2
all 69.3 68.6 58.4 68.3
0M 06 0x o
50 1 67.0 71.0 64.2 65.8 67.4
2 71.0 65.7 53.0 60.5 63.2
3 70.0 69.2 58.0 60.8 65.7
all 69.3 68.6 58.4 62.4 65.5 66.9
K M G K
3 6T 1 71.5 71.1 72.3 68.4 71.6
2 66.2 67.2 58.5 55.0 64.0
3 71.5 69.0 58.8 62.6 66.4
all 69.7 69.1 63.2 62.0 67.3
70 0x 0M 06 0
1 70.5 65.5 69.0 65.2 68.3
2 69.8 67.2 64.0 61.0 67.0
3 67.5 70.4 57.2 67.5 65.0
all 69.3 67.7 64.2 64.6 66.8 67.1
Total for cow mC 69.4 67.4 64.0

 

8Samples for period 1 lost in storage.

230

.Appendix Table II.8a--Trial I 1971. Layout of ANOVA I—l; Latin

Square design applied to estimated treat—
ment responses (dT).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cow: 604 606 607
Treatments and estimated

Period treatment responses (dT)a
2 G K M
1+3/2 oG 0K oM
(dT) dG dK dM
4 M G K
2+5/2 oM oG 0K
(dT> dM do dx
6 K M G
5+7/2 OK OM OK
Table II.8b--Anova I-l.

Source of Degrees of freedom
Variance (symbol) No.
Cows (c-l) 2
Periods (p-l) 2
Treatments (t-l) 2
Orthogonal contrasts

dG vs.deK

dK vs. d 1
Error (rest (r-1)(r-2) 2

Total (r2-1) 8

aSymbols

c=3, for cows (c).

p=3, for period (P) sequences.

t=3, for treatment (T) vs. control (0) differences.
r=3, for the square side units.

Appendix Table II.9a--Trial I 1971.

121311

controls (ANOVA 1-2).

Layout for comparison
of each infusion treatment to adjacent

 

 

 

 

Cow: 604 606 607
Bleeding hours

Period 0 3 0 3 9 0 3
1 061 0K1 °M1
2 c x M
3 062 0x2 0M2
3 °M1 061 0x1
4 M c x
5 0M2 OG2 0K2
5 0K1 0M1 001
6 x M G
7 0x2 0M2 002

 

Appendix Table

II.9b--Periods for each cow employed in the
AOV of the respective infusion treat-

 

 

ment studies (ANOVA I-2).
Study: K G M
Cow
604 5 6 l 2 3 3 4 5
606 l 2 3 4 5 5 6 7
607 3 4 5 6 7 1 2 3

 

Appendix Table II.9c--Example of a complete set of plots
utilized in ANOVA I-2;

infusion study.

the G (glucose)

 

 

Cow 604 606 607
Bleeding hours
0 3 0 9 0 3 9
Infusion period of infusion treatment
061 3 S
G 2 4 6
062 5 7

 

Appendix Table II.9d--Trial I 1971.
variance and degree of freedom-

Anova 5'2:

sources of

 

Source of

Degrees of freedom

 

Variation symbols No.
a)Whole plot
(between cows (ct-1) 8
and infusions)

Cows (c—l) 2
Infusion trtm. (t-l) 2
Orthogonal contrasts

T vs. 0 1
Error a (rest) (c-1)(t—l) 4
b)Sp1it plot

(bleeding times

within cows and

infusions) (ctb-1)-(ct-1) 18
Bleeding times
orthogonal contrasts (b-l) 2

31 vs. 82 6 83 1

82 vs. 83 1
interactions

8 x C (b-1)(c-1) 4

a x T (b-1)(t-1) 4
Error b (BXCXT, rest) (b-1(c-l)(t—1) 8

Total 26

 

232

Appendix Table II.10a--Tria1 I 1971. Layout for ANOVA I—3.

 

 

 

Cow: 604 606 507
Bleeding hour No.
l 2 3 1 2 3 l 2 3 p
Trtm. . f
Sequence Pers. Infu31on Treatment
1+3/2 0G OK OM .‘
3 6 K M G
5+7/2 OK OM 0G

 

Appendix Table II.10b--ANOVA I-3.

 

 

Source of Degrees of freedom F-test
Variance symbols No. MS
a)Whole plots (cst—l) l7

Cows (c-l 2
Sequences (s—1) 2
Treatments (t-l 5
orth contrasts
T vs. 0 1
Betw. O-s 2
Betw. T-s 2
KM vs. G 1
K vs. M 1
Error a (residual) 8
b)Sp1it plot (bcst-l)-(cst-l)36
Bleedings (b-l) 2
orth contrasts
B vs. 8 B 1
8% vs. 8% 3 1
BXC 4
BXS 4
BXT 10

Error b (residual) 16

 

2233

Appendix Table II.11a—-Trial I 1971. Statistical analyses for feed intake
parameters, Anova I—l.

 

 

Var. Msa r F sign. Ms F F sign. MS F P sign.
Source DF DM Intake (Kg/day) CP Intake (kg/day) E—NEL Intake (Meal/day)
Par Mt.

Cows 2 .02781 .00166 .09023
Para. 2 1.03448 7.3 .08929 20.1 P<.05 2.99320 5.3
Trtm. 2 .11443 <1 .00745 .96053 1.7
KM v3.6 1 .09386 .00445 .49999
K vs.M 1 .13500 .01049 1.42107
Error 2 .14117 .00445 .56250
Total (SS)

8(2.63576) (.20573) (9.21000)

 

aTotal SS in brackets to be discerned from MS for sources of variance.

Appendix Table II.11b--Tria1 I 1971. Statistical analysis for feed intake
parameters, Anova I—2.a

 

 

Eiiiflitii gasein Inf. (K) Glucose inf. (G) Mixture inf. (M)
var. DP MS F F sign- MS P P sign. MS P P sign.
DM Intake (kg/day)
Cows 1.24000 1.44333 1.00811
Trtm. .58333 1.2 .30333 <1 .05445 <1
T vs.0 .49999 .60499 .06722
betw 0's .66664 .00016 .04167
Error (rest) .49334 .31667 .91552
Total (SS) (6.0200) (4.76000) (4.10223)
CP Intake (kg/day)
Cows .03221 .06903 .03204
Trtm. .02974 1.4 .01563 <1 .00674 <1
T vs.O .02722 .02644 .00222
betw O's .03226 .00482 .01126
Error (rest) .02215 .02517 .02113
Total (SS) (.21249) (.27000) (.16209)
ENE Intake (Meal/day)
Cows 6.004 6.281 3.431
Trtm. 2.268 ~1 1.401 ~1 .275 <1
T v3.0 2.494 2.801 .067
betw 0's 2.042 .001 .482
Error (rest) 2.216 1.450 1.598
Total $8) (25.409) (21.163) (13.803)

 

aOnly F for treatments presented for sake of simplification.

bTotal SS in brackets to discern from MS for sources of variance.

234

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235

Appendix Table II.13--Tria1 I 1971. Statistical analysis for milk parameters, Anova I-2.a

 

 

 

 

 

Parameter Casein infusion (K) Glucose infusion (G) Mixture infusion (M)
Source of Var. DP MS F F sign- MS F F sign. MS F F sign.
Milk Yield (kg)
Cows 2 12.424 16.725 18.110
Trtms. 2 3.598 2.8 .666 ~1 1.329 2.2
T vs. 0 1 5.478 .724 2.607
Betw. Ch 1 1.717 .551 .050
Error (rest)b 4 1.276 .611 .611
Total ($5) 8 (37.146) (37.214) (41.522)
29M Prod. (kg)
Cows 2 9.7655 19.5886 22.2642
Trtms. 2 .8790 <1 1.2505 <1 .2544 <1
T vs 0 1 .5067 .5033 .4672
Betw.0's 1 1.2512 2.0068 .0416
Error (rest) 4 1.8848 1.5897 1.4181
Total ($8) 8 (28.8278) (48.0461) (50.7095)
Prot. Cons. (8)
Cows 2 .11034 .14262 .03826
Trtms. 2 .04406 11.4 <.025 .01755 4.3 <.10 .04130 11.4 <.025
T vs. 0 1 .07450 16.9 <.025 .01458 3.6 .06207 16.9 <.025
Betw. 0's 1 .01363 3.1 .02042 5.1 <.10 .02053 3.1
Error (rest) 4 .00385 .00404 .00722
Total (SS) 8 (.32419) (.33652) (.18800)
Prot. Prod. (g)
Cows 2 25941.2 6481.3 22295.2
Trtms. 2 12786.6 17.0 <.025 1620.1 1.6 7296.9 3.5
T vs. 0 1 20550.4 27.3 <.01 3173.4 3.2 12624.6 6.0 <.10
Betw. 0's 1 5022.8 6.7 66.7 <1 1969.3 <1
Error (rest) 4 752.6 1000.9 2103.8
Total ($5) 8 (79466.2) (20206.3) (67599.3)

 

80n1y P for treatments presented for sake of simplicity.
bSS for total in brackets to discern from NS for variance sources.

Appendix Table II.13--Continued.

 

 

 

 

 

Parameter Casein infusion (K) Glucose infusion (G) Mixture infusion (H)
Source of Var. DP MS P F sign. MS P P sign. HS P 7 sign.
Est. True Prot. Cons. (9)
Cows .03493 .12162 .04657
Trtms. 2 .09626 8.1 <.05 .01547 5.7 <.10 .02979 3.4
T vs. 0 1 .05413 12.6 <.025 .01091 4.0 .05078 5.7 <.10
Betw. 0's 1 .01571 <1 .0200? 7.4 <.10 .00882 ~l
Error (rest) 4 .00430 .00273 .00890
Total ($5) 8 (.27955) (.28510) (.18831)
Est. True Prot. Prod. (g)
Cows 2 22026.7 5558.5 22502.3
Trtms. 2 10888.4 15.3 <.025 1324.2 1.6 5931.6 2.7
T vs. 0 1 16701.8 23.5 <.01 2563.3 3.0 10775.1 4.9 <.10
Betw 0's 1 5075.0 7.1 <.10 85.1 <1 1008.1 <1
Error (rest) 4 711.4 851.7 2191.9
Total (SS) 8 (68675.8) (17172.3) (65635.5)
NPN Cons. (%)
Cows 2 21.642 25.989 51.710
Trtms. 2 34.555 272.1 <.001 3.343 1.3 16.174 5.9 <.10
T vs. 0 1 67.009 527.0 <.001 6.396 2.6 .195 <1
Betw. 0's 1 2.100 16.5 <.025 .789 <1 15.980 11.6 <.05
Error (rest) 4 .127 2.419 1.375
Total ($8) 8 (112.901) (68.338) (73.384)
NPN/Tot.N(8)c
Cows 2 .41053 .89105 .70935
Trtms. 2 1.03903 35.7 <.01 .25943 <1 .52528 4.1 :.10
T vs. 0 1 1.77347 61.0 -.001 .41404 1.3 .48166 3.8
Betw. 0's 1 .30459 10.5 ~.025 .10482 <1 .56890 4.4 <.10
Error (rest) 4 .02907 .31091 .12840
Total (SS) 8 (3.01560) (3.54460) (2.98283)

 

CAOV on values obtained by aresin

transformation (Rohlf a Sohal,

1969, p.

129).

236

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cHuoue >n vocHsuno sesHe> so ¢>oz<o

.socsHHs> no moonsOM new m2 sown chooewo on as on susxoeun cH mm Hsuoan

.coHusOHuHHdEHe mo exec new concussed acoEusouu now u uHcov

 

 

Hm.HNNNHH. HmmHNN.OHV Hv.mmm4mHv HH.HNHNmHV Hon.va NHHmmV Hsuoa
4.NoNH vemvN. .oH.v N.uomN «.mmNH oNo.H NHueeuvuouum
Hv m.m4m N.ovaH Hv m.va H z.e> u
moo.v N.4H m.mmmNH m.mNoo moo.v v.wH m.voooN H o.e> xx
mNo.v m.N o.moom Hv mmmNo. oH.v m.mmNoH mNo.v v.o o.m>moH N e.a 390m
m.H N.NHNN Hv mHmmH. N.momm H.N N.¢NmN N e.o sued
moo.v «.mH N.NoNoN Ho.v o.mH ovaN.m o.oomN moo.v 4.NH a.wvmvN H o .e> e
Ho.v H.N o.ommm o.m mvmmN. «.mmvv Ho.v H.N m.mvHoH m.N aoN.N m .eEuua
H.Noo NNNo4.N H.4mmo H.oNo moH.4 N .noom
4.4o4Hm mmoNo. m.NmH4m N.mNomm NHH.vN N usou
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Anamom.v .mmm.mmHv HHNHNH.HV Hmnamm.v HHmN.mNV NHHmmo Heuos
HmNoo. mNo.N Novmo. NmNoo. NHN. «Humouvuouum
Hv mNooo. m.N moo.m oH.v oHNmH. Hv ooooo. Hv mON. H z.e> x
Ho.v H.NH 4mNmo. Ho.v m.NH oom.mN memoo. moo.v N.oH oomvo. Ho.: H.HH Nmm.N H o.e>zx
mNo.v o.o oooHo. mNo.v m.n mmH.mH mNoNo. Ho.v m.o omHNo. N.N mmo.N N e.9 390m
Hv mmHoo. Hv NmH. vNHmo. H- omNoo. o.N vov.H N «.0 390m
Hoo.v 4.Hm 4NmNo. Hoo.v m.mm omm.Nm mNo.v mvmmm. Hoo.v N.NN mmmmo. mmo.v H.o «Hm.m H o .e> a
moo.v N.N oomNo. moo.v N.N mmm.aH OH.v NNNHH. moo.v v.HH 44NNo. mo.v N.v Nmm.m m .seuua
NomHo. NN4.NN HNNoo. oH4No. NNN.N N .ooom
HmHNH. NmN.N vaHH. vmomH. mNN.mN N e300
Hay .ecou.uoum one .uem ANmEV .mcoo zmz How .wcoo uem Nov .ncoo cHeuoum mev oHoH» xHHZ .Houoesusm
ooHo N N m: ooHn N N m: ooHn N N m: ooHn N N m: 86H4 N N on: No wwwawm

 

m.m|H s>oc< .musuosdusm

coHuoooonN xHHe now nHmNHooo HooHHNHuoum .HNNH .H Hona-4H.HH «Home xHoooaoo

237

Appendix Table II.15--Tria1 I 1971. Statistical analysis
for blood parameters, Anova I-3.

 

 

Parameter: Blood urea N(mg/100m1)P1asmaglucose(mg/100ml)
Source of F F
Variance DF MS F sign.DF MS F sign;_
a)Whole plots 1? 17
Cows 2 17.055 2 131.760
Seqs. 2 117.166 2 .019
Infs. 5 126.077 2.7 “.01 5 33.111 2.5
Contrasts
T vs. 0 1 188.907 4.1 <.10 1 87.911 6.7 <.05
amg.O 2 24.481 <1 2 13.775 ~1
amg.T 2 196.259 4.3 <.10 2 25.055 *2
KM vs.G 1 357.796 7.8 <.05 1
K vs.M 1 34.722 <1 1
Error a(rest) 8 46.084 8 13.111
b)Split plots 34 36 .
Bleedgs. 2 111.722 6.0 <.025 2 24.221 2.1
Contrasts
B1 vs.BzaB3 1 126.750 6.6 <.025 1 38.521 3.4
B2 vs. 33 1 96.696 5.3 <.05 1 9.923 <1
B C 4 34.111 4 14.310 1.3
B S 4 170.830 9.4 <.001 4 40.821 3.6 ”.05
B I 10 36.655 10 21.406 1.9
Error b(rest)a 14 18.160 16 11.442
Total (SS) 51 (2935.500) 53 1200.435

 

aError b for blood urea N has only 14 DF because
2 plots were missing.

238

Appendix Table II.16--Tria1 II 1971. Amounts of feed
offered and consumed, and total dry
matter intake.

 

 

 

 

 

Cow/Period if? 1 2Consumed3 4
kg kg kg kg kg

607 Trtm. 0 L M H
Hay 6.8 5.7 6.1 6.8 6.2
Cons. 11.3 10.2 10.5 10.2 7.5

DM Total 14.9 14.6 15.0 12.3
604 Trtm. L O H M
Hay 6.8 5.1 5.5 6.3 5.1
Cons. 11.3 9.3 10.5 8.4 8.5

DM Total 12.8 14.1 13.2 12.4
603 Trtm. M H O L
Hay . 6.5 5.3 6.3 4.2
Cons. . 6.2 6.4 8.1 6.1

DM Total 11.3 10.5 12.8 10.0
606 Trtm. H M L O
Hay 6.8 6.6 6.8 6.6 5.6
Cons. 11.3 5.8 5.2 8.2 7.7

DM Total 11.1 10.8 13.3 12.2

 

239

Appendix Table II.17--Tria1 II 1971. Feed composition and
estimated net energy values.

 

 

Average

Period 1 2 3 4 feed/refusal
Hay

Dry matter, % 84.7 85.7 86.9 87.6 86.2 / 80
crude prot.

in DM,% 18.0 13.2 17.7 17.3 16.6
Crude fiber

in DM, % 28.4 35.5 28.8 30.9 30.9

Est. Net

Energy, Mcal/kga 1.25 1.15 1.25 1.20 1.21
Concentrateb

Dry matter, % 88.8 88.3 89.0 88.5 88.7 /'82
crude prot.

in DM, % 16.7 15.5 16.1 15.1

Est.Net

Energy, Mcal/kg DMC the same value used 2.02

throughout

 

aEstimated out from CP and CF content using
NRC (1971) feed tables.

bFor ingredients of the concentrate mixture see
Table II.1,footnote b.

CCalculated from values assigned to the ingre-
dients (NRC 1971) as for trial I 1971.

240

Appendix Table II.18--Tria1 II 1971. Milk yield and con-
centration of milk constituents.

 

 

Period 607 604 603 606

l Trtm. O L M H m
Milk yield, kg. 17.99 20.61 15.27 16.83 17.68
N cons., % .50893 .52163 .52465 .47210

NPN cons., % .03005 .03279 .04024 .03420

ETP cons., % 3.055 3.119 3.091 2.794

Fat Cons., % 2.97 3.13 2.73 2.90

Lactose cons., % 4.92 4.75 4.52 4.61

SNF cons. 88 --- -—- --- ---

2 Trtm. L O H M

Milk yield, kg. 17.27 21.15 16.60 14.45 17.37
N cons., .55125 .53966 .56082 .47571

NPN cons. .03196 .02674 .03948 .02859

ETP cons., % 3.313 3.272 3.326 2.849

Fat Cons., % 3.00 3.00 2.70 3.30

Lactose cons. 4.77 4.87 4.65 4.79

SNF conc. 8.83 8.68 8.75 7.97

3 Trtm. M H O L

Milk yield, kg 16.93 21.94 16.76 17.22 18.21
N cons., % .57476 .59357 .51411 .48871

NPN cons., % .03478 .03993 .03030 .02867

ETP cons., % 3.445 3.523 3.087 2.935

Fat cons., % 3.00 2.94 2.57 3.15

Lactose cons., % 4.95 4.79 4.55 4.83

SNF cons., % 9.36 8.83 8.80 8.59

4 Trtm. H M L 0

Milk yield, kg 16.57 21.62 16.33 16.33 17.68
N cons., % .58244 .54216 .53135 .47226

NPN cons., % .03952 .03515 .02954 .02019

ETP cons., % 3.464 3.235 3.202 2.884

Fat cons., % 3.03 3.13 2.10 3.20

Lactose cons., % 4.96 4.79 4.66 4.77

SNF cons., % 9.40 9.09 9.30 8.53

 

aObservation missing period 1.

241

Appendix Table II.19--Trial II 1971. Milk production
parameters: period observations
for each cow and treatment, and
time period means.

 

 

 

 

 

 

 

 

 

 

Treatment Period
Trtm./Cow 603 604 606 607 means (NoJmeans
Milk yield kg/day
O 16.8 21.2 16.3 18.0 18.1 (1) 17.7
L 16.3 20.6 17.2 17.3 17.9 (2) 17.4
M 15.3 21.6 14.5 16.9 17.1 (3) 18.2
H 16.6 21.9 16.8 16.5 18.0 (4) 17.7
Protein cone,
0 3.28 3.44 3.01 3.25 3.25 (1) 3.23
L 3.39 3.33 3.12 3.52 3.34 (2) 3.39
M 3.34 3.46 3.03 3.67 3.38 (3) 3.47
H 3.58 3.79 3.01 3.72 3.53 (4) 3.40
Protein prod. g/day
O 549 728 492 584 588 (l) 572
L 553 685 529 608 594 (2) 592
M 510 748 438 621 579 (3) 632
H 594 830 507 616 637 (4) 602
NPN conc. mg%
0 30.3 26.7 20.2 30.1 26.8 (1) 34.3
L 29.5 32.8 28.7 32.0 30.7 (2) 31.7
M 40.2 35.2 28.6 34.8 34.7 (3) 33.4
H 39.5 39.9 34.2 39.5 38.3 (4) 31.1
ETP conc. %
O 3.09 3.27 2.88 3.06 3.07 (l) 3.01
L 3.20 3.12 2.94 3.31 3.14 (2) 3.19
M 3.09 3.24 2.85 3.45 3.16 (3) 3.25
H 3.33 3.53 2.79 3.46 3.28 (4) 3.20
ETngrod. g/day
O 517 692 471 550 558 (1) 534
L 523 643 505 572 561 (2) 557
M 472 699 412 583 541 (3) 595
H 552 775 470 574 592 (4) 567
Fat Conc. %
O 2.57 3.00 3.20 2.97 2.94 (1) 2.93
L 2.10 3.13 3.15 3.00 2.85 (2) 3.00
M 2.73 3.13 3.30 3.00 3.04 (3) 2.92
H 2.70 2.94 2.90 3.03 2.89 (4) 2.87
Lactose conc. %
O 4.95 4.87 4.66 4.62 4.78 (1) 4.70
L 4.96 4.75 4.55 4.79 4.76 (2) 4.77
M 4.92 4.79 4.65 4.83 4.80 (3) 4.78
H 4.77 4.79 4.52 4.77 4.71 (4) 4.80

 

242

Appendix Table II.20--Tria1 II 1971. Concentrations of
blood urea nitrogen, mg/100m1.

 

_.-——_~_.. ,...-...._-_. 1-... .-——-..—. H. - —..—- ————._..__ .H—‘H .-
-. ”—N---_.._.——=Z.. --"~-_.._....- ..~—..._ ..___.__ ”*2 -

period Bleeding 607 604 603 606 mb m b

 

P
1 Trtm. O L M H
1 24 34 26 32 29.0
2 30C 39 41 33 35.8 32.4
3 -- 35 59 20 38.0
2 Trtm. L O H M
1 21 44 55 36 39.0
2 43 18 44 25 32.5 35.7
3 __ -- -_ __
3 Trtm. M H O L
1 53 44 19 31 36.8
2 38 40 38 34 37.5 37.2
3 46 49 36 43 43.5
4 Trtm. H M L O
1 59 37 37 25 39.5
2 55 45 41 22 40.8 40.2
3 59 38 -- 27 41.3
Cow m 40.4 37.6 37.6 29.8

 

aBecause so many plots are missing of bleeding
3 the statistical analyses employed bleeding 1 and 2 only.

bB1 & B2 only behind the period and cow means.

CPlot observation missing.

Appendix Table II.21--Tria1 II 1971.

243

Concentrations of

blood plasma glucose, mg/lOOml.

 

 

 

 

 

 

Cow
Period Bleeding 607 604 603 606 m
1 Trtm. O L M H
1 69.6 72.5 68.0 68.8 69.7
2 67.1 70.4 63.5 65.3 66.6
3 68.4a 69.3 69.3 68.8 69.0
m 68.3 70.7 66.9 67.3 68.4
2 Trtm. L O H M
1 66.5 72.2 70.0 65.6 68.6
2 65.1 71.7 66.7 63.5 66.8
3 67.5 70.3 70.5 67.0 68.8
m 66.4 71.4 69.1 65.4 68.1
3 Trtm. M H O L -
1 75.2 75.2 74.0 78.0 75.6
2 63.5 73.0 70.0 69.0 68.9
3 66.5 71.5 71.0 70.0 69.8
m 68.4’ 73.2 271.7' 72.3 71.4
4 Trtm. H M L O
1 66.0 66.0 64.0 65.0 65.3
2 68.2 67.2 65.0 65.2 66.4
3 59.0 65.0 66.3 65.8 64.3
m 64.4 6671 65.1 65.3 65.2
Cow total m 66.7 70.4 68.2 67.7

 

aMissing observation; the value estimated by
formulas of Cochran and Cox (1956), p. 125, employing

data for bleeding 3 only.

The value not included in AOV.

Appendix Table II.22--Tria1

Source of

244

II 1971.

Degrees of freedom

Layout of ANOVA II.

 

 

Variance symbol No.
a)The Latin Square rz-l 15
Cows c-l 3
Periods p—l 3
Treatments t-l 3
orth.contrasts
K vs. 0 1
L vs. MH 1
M vs. H 1
Error a (rest) (r-1)(r-2) 6,
b)Within LSQ plots (bEZ-l)
-(r -1) _3__2_
Bleedings b-l 2
orth. contrasts
Blvs.B2B3 1
B2 VS. B3 1
BxT (b-1)(t-l) 6
EXP (b-l)(p-1) 6
BxC (b—l)(c-l) 6
Error b (rest) 12
Total (brz-l) 47

 

245

Appendix Table II.23-—Tria1 II 1971. Statistical analysis for feed intake

 

 

 

parameters.
Source of
Variance DF MS F F sign. MS F F sign. MS F F sign.
A. Trial II DM Intake (kg/day) CP Intake (kg/day) ENE Intake (Meal/day)
Cows 3 7.4270 27.4 .18686 26.0 30.1233 25.4
Pers. 3 2.3558 8.7 .19310 26.8 8.1303 6.9
Inf.Trtm. 3 2.0839 7.7 <.025 .05430 7.6 <.025 9.6224 8.1 <.025
0 vs. K 1 4.6128 17.0 <.025 .12710 17.7 <.01 20.5408 17.3 <.01
L vs. MH 1 .9720 3.6 .02220 3.1 6.3860 5.4 <.10
M vs. H 1 .6730 2.5 .01361 1.9 1.9405 1.6
Error 6 .2707 .00719 1.1859
Total (SS) 15 (37.2246) (1.34595) (150.7432)

 

Appendix Table II.24——Tria1 II 1971. Statistical analyses for milk production

 

 

parameters.
Source of
Variance DF MS F F sign. MS F 8 sign. MS F P sign.
Parameter: Milk Yield (kg/day) Fat Cons (%) Prot. Cons. (%)
Cows 3 23.7716 .30149 .206842
Pers. 3 .4933 .01246 .038742
Inf.Trtm. 3 .8148 1.2 .00277 <1 .054092 10.2
0 vs. K 1 .085008 15.8
L vs. MH 1 .032267 6.0
M vs. H 1 .045000 8.3
Error(rest) 6 .7061 .04184 .005358
Total (88) 15 (79.4740) 1.27684 (.931175)
Parameter: Prot. Prod. (g/day) NPN Cons. (mg/100ml) NPN/Tot. N(%)
Cows 3 47940.7 40.671 .4863
Pers. 3 2561.1 8.933 .7576
Inf. Trtm. 3 2632.1 3.6 <.10 98.030 29.9 <.001 2.2281 15.6 <.005
0 vs. K 1 678.0 <1 180.226 54.9 <.001 4.3440 30.5 <.005
L vs. MH 1 427.7 <1 113.864 34.7 <.005 2.0242 14.2 <.01
M vs. H 1 6675.9 9.0 <.025 88.052 26.8 <.005 .3160 2.2
Error(rest) 6 739.6 3.282 .1426
Total (88) 15 (163839.4) (462.591) (10.7852)
Parameter: Est. True Prot. (8) Est. True Prot. (g/day) Lactose Cons. (%)
Cows 3 .172139 4173.61 .064691
Pers. 3 .041783 2600.51 .007088
Inf.Trtm. 3 .029043 5.1 <.05 1842.57 2.7 .005088 <1
0 vs. K 1 .041478 7.3 <.05
L vs. MH 1 .014900 2.6
M vs. H 1 .030752 5.4 <.10

Error(rest) 6 .005697 671.90 .006225
Total 15 (.763.082) (142680.83) (.267975)

 

246

.ooueEHumo uon oco monsoon mco an House mo mo COHuospmmn

.msmmm cH ooHHOQm
mmHmEMM Hmuo>mm monsoon oommouo Ammo mEHu mCHHmEMm pooHn puHsu scam

 

 

LONN.oHoo o4 HNHN.NNNNV HN
HNN.N nHH N4N.4HH 4 Has wonwm
Hv NN4.N o H4 moN.Ho N are
mo.v 0.4 NNH.NH o Hv NHH.NN N Nxm
o.H N4N.o o Hv NHH.N4 N oxm
H.H omN.4 H N Na .65 mm
Ho.v 4.4H 4oo.4m H non.n> N
NNo.v N.N NNo.NN N-I H. HNN.N II o.noooon
HN oH uoHN uHHNmHn
N4H.N o N44.Nm o Hoe nouns
NNo.v o.OH 0N4.HN H 4.4 Nom.NNN H N .n> z
N.N NNN.N H N.4 HNo.HNN H m: .n> H
mo.v N.e 444.NH H Ho.v N.NH HoN.4NN H N .n> o
mNo.v N.4 NNN.4H N mNo.v N.N HNN.NN4 N .nsuns .NoH
NoN.oN N NNo.NN N .6006
mHo.oN mn- HNo.NoH N-I nzoo
NH NH uoHN oHorz Ho
.omHn N N w: No .omHn N N m2 No

AHE00H\mEV .ocoo omoosHm mEMMHmHHEo0H\mEV .ocoo .2 mos: GOOHm

 

.mHoooeoHom oooHo Now mHmmHooo HooHumHuoum .HNNH HH HoHss-NN.HH 0Hnos MHoomana

247

Appendix Table II.26—-Tria1 III 1971. Ingredients in
concentrate mixtures.

 

 

 

Mixture A Mixture B
(D-122) (D-123)
_________ %______--__a
Ground shelled corn 50.2 63.5
Oats 24.0 24.0
Urea 0 2.4
Soybean meal (50%) 16.9 1.2
Molasses (cane) 7.2 7.2
Trace min. salt 0.5 0.5
Dicalcium phosphate 1.0 1.0
Sodium sulfate 0.2 0.2
Added per kg
Vitamin A 4400 10 4400 IU
Vitamin D 2200 I0 2200 IU

 

aOn wet weight basis as mixed. Urea was 2.74%
of dry matter (87.5%) in mixture B.

248

Appendix Table II.27--Tria1 III 1971. Feed composition
and estimated net energy values by

 

 

periods.
DM CP CF ENELa
Feed Seqs. Per. in dry matter
% % % Mcal/kg
Hay 1 1 85.0 16.7 30.8 1.30
2 85.6 17.7 29.5 1.30
3 83.8 15.6 34.0 1.25
2 4 84.7 15.8 33.1 1.25
5 84.0 18.0 34.4 1.25
6 84.7 18.9 28.3 1.35
Corn Silage lb 1 34.2 14.2 22.6 1.7
2 33.1 14.2 20.4 1.7
3 34.9 14.2 21.7 1.7
2C 4 34.6 14.2 18.6 1.7
5 33.2 14.2 19.8 1.7
6 32.7 14.2 20.9 1.7
Concentrate 1 1 88.0 17.6 2.05
mixture A; 2 87.9 17.6 2.05
without 3 88.3 17.6 2.05
urea (”’122) 2 4 87.7 17.7 2.05
5 88.1 17.7 2.05
6 87.9 17.7 2.05
Concentrate 1 1 87.4 17.5 2.04
mixture B; 2 87.7 17.5 2.04
with 2.5% 3 87.6 17.5 2.04
urea (0‘123) 2 4 87.1 17.6 2.04
5 87.6 17.6 2.04
6 87.3 17.6 2.04

 

aEstimated net energy for lactation (ENE
derived from NRC (1971) feed tables based on crud

)

2 pro—

tein (CP) and crude fiber (CF) in dry matter (DM) of
hay and corn silage, and on assigned values for the
ingredients of concentrates (formulations in Appendix

Table II.26).

bUrea added at ensiling.

cProSil added at ensiling.

245)

Appendix Table II.28.--Tria1 III 1971. Amounts of feed offered and consumed
as dry matter, and total intakes of feed constituents

 

per day.
Feeds Sum intake
Corn Cons. Cons. Urea in NPN in
Cow S F P T Hay sil. mix.A mix.B DM CP ENE Cons.B Tot.N

 

603 1 H ana 2.3 9.1 6.3 / 6.3
1 o 1.9 3.1 3.2 8.2 1.32 14.3 88 80 38
2 x 1.9 3 o 5.4 10.3 1.71 18.6 148 106 39
3 o 1.9 3 2 5.6 10.6 1.73 19.1 153 111 40
2 L 4 o 1.8 3.1 4.7 9.6 1.56 17.2 32 13
5 x 1.6 2.4 3.9 7.6 1.32 14.1 25 . 12
6 o 1.9 2.9 5.4 10.3 1.74 18.6 30 11
607 a11a 2.3 13.6 7.3 / 7.3
1 H 1 o 1.9 4.7 5.6 12.1 1.96 21.8 153 130 41
2 x 1.9 4.5 5.6 12.0 1.96 21.6 153 127 40
3 o 1.9 4.8 5.6 12.0 1.95 21.8 153 131 42
2 L 4 o 1.9 4.7 5.6 12.2 1.95 21.8 49 16
5 x 1.8 4.5 5.6 11.9 1.96 21.4 47 15
6 o 1.9 4.5 5.6 11.9 1.98 21.4 47 15
604 a11a 2.3 13.6 8.2 / 8 2
1 L 1 o 1.9 4.4 7.2 13.5 2.21 24.6 57 16
2 x 1.9 4.4 7.2 13.6 2.23 24.8 57 16
3 o 1.9 4.8 7.2 13.9 2.25 25.2 62 17
2 H 4 o 1.9 4.7 7.1 13.6 2.21 24.7 194 136 38
5 K 1.8 4.5 7.2 13.5 2.22 24.5 197 136 38
6 0 1.9 4.4 7.1 13.5 2.24 24.7 194 133 37
606 311a 2.3 9.1 7.2 / 7.2
1 L 1 o 1.9 2.8 6.4 11.1 1.85 20.3 36 12
2 x 1.9 2.7 6.4 11.1 1.85 20.2 35 12
3 o 1.9 2.9 6.4 11.2 1.84 20.4 38 13
2 H 4 o 1.9 2.7 5.1 9.7 1.58 17.3 140 91 36
5 x 1.9 2.5 5.4 9.8 1.64 17.6 148 93 35
6 o 1.9 2.6 5.6 10.1 1.71 18.4 153 96 35

 

aAmou t of feed offered, wet basis. Figures for feeds in each period
are DM consumed.

250

Appendix Table II.29-~Tria1 III 1971.
protein (CP) and estimated net
energy for lactation (ENEL) rela-
tive to NRC (1971) standards (%).

Intakes of crude

 

 

 

CP intake ENEL intakes
Cow: 603 607 604 606 603 607 604 606
......... %_-_.....___ -_-_.._....%-..........- ..
Feed: H H L L H H L L
Per. Inf. Trtm.
1 O1 100 121 110 115 92 115 113 111
2 K 121 121 115 120 113 117 117 112
3 02 123 120 114 113 126 115 118 111
Feed: L L H H L L H H
4 O1 128 131 130 100 119 124 131 97
5 K 109 130 125 113 98 121 125 105
6 O2 146 136 133 119 131 125 134 111
Means CP ENEL CP ENEL
Inf. Trtm. Feeding
O1 117 113 H 119 115
K 119 114 L 122 117
O ' 126 121

2

 

Appendix Table II.30--Trial III 1971.

2551.

Observations

in milk production parameters.

 

 

 

 

 

 

 

 

(Item)= (1) M118 yield. kqlday (2) Nitrogen Conc., 4“
Sub- Cow No. d Cow No. d
Seqs. Per. per. Trtm. 603 607 604 606 m (d8) 603 607 604 606 m (68)
Ha H L L H H L L
1 1 01 9 53 11,53 20_12 14,02 .53900 .55125 .49525 .53200
2 3,30 12,02 20,45 14.11 .56000 .55475 .52063 .53288
1 m 9770 II709 20779 I0707 13.84 (3.504) (3.544) (3.261)(3.394) 3.42
1 x 10.50 12.07 20.57 14.56 .57750 .58625 .56175 .57575
2 10.95 11.05 19.80 14.31 :57925 .58800 .56000 1.56525 0.28
2 m 10773 II757 907I9 10700 14. 23(3. 3)(3. 000) (3 742T 197075)(3.039Y 3.66(8.3)
1 02 10.39 10.98 18.57 13.88 .54355 .53760 .50400 .52255
2 10.12 11.34 20.21 14.22 .51870 .52920 .50750 .52745
3 m I0790 II7I0 I9709 IT705 13.71 (370907 (37905Y (5793IY T37950)3.35
Ld L H H L L H H
2 1 01 9.16 10.59 17.51 13.13 .57750 .54600 .52325 .53550
g 8.55 10.57 17.12 13.27 .58800 .53550 .52325 .53900
4 m 8.85 10750 17707 I0790 12.49 (3.716) 13.450) (5:340)(5.400T 3.48
1 x 8.39 10.50 17.96 13.02 .60550 .57730 .57050 .56700
2 8.39 10.86 18.35 12.88 0.50 .63700 .57750 .54950 .58450 0.28
S m 0799 I0700 r0710 I0795 12.50(4.2) (57900) (52000) ($7573)(§70707 3.73 (8.1)
1 02 8 35 10.41 16.78 12.36 .55125 .53783 .48650 .53463
2 7 77 8.75 15.60 11.95 .55125 .54425 .51538 .55563
6 m 8706 9.58 I67I6 I27I5 11.50 (3.520) (3.137) ( . . 3.41
Appendix Table II.30--Continued.c
NPN Cons., mg/100m1a (3) (4) SN? Concentration, 45 (5) Fat concentration, ‘a,b
Cow No. d Cow No. d Cow No. d
603 607 604 606 m (d4)603 607 604 606 m (as) 603 607 604 606 m (69)
H H L L H H L L H H L L
25.43 29.74 27.07 29.33 9.00 9.04 8.66 8.49 3.7 3. 6 3.3 3.1
26.96 28.18 28.03 30.51 8.98 8.88 8.71 8.45 3.7 3. 7 3.4 3.4
26718 28.93 27.55 29.91 28.2 9700 0790 0709 0707 8.78 . 5. 05 9105 9795 3.49
34.40 31.01 35.50 32.94 9.06 9.07 8.92 8.60 3.6 3 4 3 1 3. 0
29.94 30.64 33.80 32.59 4.9 2129 2199 8152 0.31 3.5 3. 6 3 0 2. 9 -0.
09711 3070I 00700 99770 32.6(17.7)9.12 9.03 8.99 8.59 8.93(3.6)3.54 5.49 9700 9795 3. 26 (- -9. 30)
26.65 25.90 27.58 27.42 8.80 8.71 8.52 8.37 3.8 4.1 L 5 3. 6
30.36 26.17 26.59 27.02 8.80 8.70 8.62 9.27 3.6 4. 0 3.4
20707 20700 97700’77777 27.2 8.80 8.71 8.57 8.32 8.60 - 3 15 3; 16 3 66
L L H n L L H H L L M
29.93 26.65 28.08 25.97 9.12 9.39 9.40 7.86 2.8 3.2 2.7 3. 5
28.96 28.35 29.15 29.24 8.84 2,93 2119 £12! 3.0 3. 3 3.0
29700 97750 . 2770I 28.3 8.99 9.21 9.27 8.07 8.89 . . 2705 5 15 3.10
29.77 35.03 34.80 31.13 (8.79)9.18 9.25 8.93 2.7 3.0 2. 8 2. 6
32.40 34.66 35.98 31.31 4 0 45 8,91 2191 8.84 0.16 3.6 3 7 3 1 . -0. 06
01709 00700 35709 0I799 33.1(13. 7)9. 45 9.04 9.13 8L89 9.13(1.8)§7I5 9770 9795‘9775 3. 07 (- -1. 9)
29.92 30.19 30.82 29.50 9.17 8. 68 9. 57 8.98 3.2 3.5 3 0 2 9
29. 81 29.70 28.70 30.19 mg ‘ngg 277% 9.24 . 3.1 3.6 2. 9
99707 99797 29705 2 .85 29.9 9. 07 8. 8.87 9.04 3715 3.55 I. 93 2. 95 3.16

 

a
Means for each cow and period are weighted by the milk volume for each subperiod.

bPat test values for subperiod 1 in the last period (6) are lacking although
samples were submitted for testing; the assigned values are average of the first day of

infusion and the second subperiod.

cThe arrangement is identical with that for the first part of table (Seqs., Per.,

Subper., Trtm. ).
d

The feed level of NPN; H = high, L a low.

252

Appendix Table II.30-~Continued.e

 

 

 

 

Cow No. d
Seq. Per. Trtm. m (d%)
603 607 604 606
Crude Prot. prod., g/day
H H L L
1 O1 321 419 662 478 470
1 2 K 396 433 722 526 519 56
3 O2 348 380 626 471 456 (12.1)
L L H H
4 O1 329 365 578 453 431
2 5 K 332 393 649 476 460 51
6 O2 284 329 517 420 387 (12.5)
Est. True Prot. conc., %
H H L L
1 01 3.34 3.34 3.07 3.21 3.238
1 2 K 3.48 3.55 3.35 3.43 3.454 ‘0.25
3 02 3.21 3.24 3.05 3.18 3.169 (7.8)
L L H H
4 01 3.53 3.27 3.16 3.25 3.303
2 5 K 3.77 3.46 3.35 3.47 3.512 0.25
6 02 3.33 3.26 3.01 3.29 3.221 (7.7)
Est. True Prot. prod., g/day
H H L L
1 O1 306 395 623 452 444
1 2 K 373 411 676 495 489 51
3 O2 329 362 591 447 432 (11.6)
L L H H
4 O1 312 346 547 429 -409
2 5 K 316 370 608 449 436 48
6 O2 269 312 486 400 367 (12.4)
FCM prod., kg/day
H H L L
1 01 8.75 11.21 18.31 12.49 12.69 -0.26
l 2 K 10.00 10.69 17.31 12.16 12.54 (-2.1)
3 02 9.77 11.24 17.79 12.78 12.90
L L H H
4 01 7.39 9.39 14.32 12.11 10.80 0.29
2 5 K 7.32 9.65 15.30 10.52 10.70 (2.7)
6 02 7.03 8.39 13.63 10.24 9.82

 

eDerived values; period means (m) only.

253

Appendix Table II.31--Trial III 1971. Blood urea N concen-
tration, mg/lOOml.

 

 

 

Cow No.
Seq. Per. Inf. Bldg. 603 607 604 606 mB :mP mS
H H L L
1 1 0l 1 30 25 35 18 27.0
2 35 36 35 39 36.3
3 42 34 34 30 35.0 32.8
2 K 1 25 43 36 36 35.0
2 25 46 35 32 34.5
3 48 43 40 46 44.3 37.9
3 O2 1 29 24 26 27 26.5
2 38 42 . 32 37 37.3
3 37 44 29 30 35.0 32.9 34.5
L L H H
2 4 0l 1 30 34 19 28 27.8
2 38 41 43 39 40.3
3 34 37 42 34 36.8 34.9
5 K 1 26 26 40 37 32.3
2 36 33 38 41 37.0
3 30 34 44 55 40.8 36.7
6 02 l 38 34 25 37 33.5
2 30 29 28 34 30.3
3 13 31 37 31 28.0 30.6 34.1
Means
Feeds: H 36.1 Infusions: 01 33.8 Bleedings: Bl 30.3
L 32.5 K 37.3 B2 35.9
02 31.8 B3 36.6
01+2 32.8 B2+3 36.3

 

254

Appendix Table II.32--Trial III 1971.
concentration, mg/lOOml.

 

 

—— .—,.——_ ..‘_.,.,__.__-___

Cow No.

Blood plasma glucose

 

Seq. Per. Inf. Bldg. 603 607 604 606 m3 mP mS
I H H L L
1 0l 1 66 65 55 66 63.0
2 67 56 61 57 60.3
3 61 59 60 61 60.3 61.2
K 1 67 67 70 64 67.0
2 63 60 60 59 60.5
3 59 55 65 56 58.8 62.1
02 1 61 67 60 65 63.3
2 59 56 56 51 55.5
3 59 64 61 55 59.8 59.5 60.9
L L H H
2 0l 1 66 66 65 63 65.0
2 63 48 60 61 58.0
3 63 56 64 62 61.3 61.4
K 1 69 72 72 69 70.5
2 71 55 74 66 66.5
3 72 63 70 66 67.8 68.3
02 1 68 69 69 73 69.8
2 68 55 64 60 61.8
3 61 59 68 66 63.5 65.0 64.9
Means
Feeds: 64.0 Infusions: 01 61.3 Bleedings: B1 66.4
61.8 K 65.2 B2 60.4
02 62.3 B3 61.9
O1+2 61.8 B2+3 61.1

 

255

Appendix Table II.33--Trial III 1971. Layout of ANOVA III.

 

Source of

Degrees of freedom

 

Variance symbol No.
a) Between main plots;
comprizing C,S,&F (cs-l) _Z
Cows (c-l) 3
Sequences (s-1) 1
Feedings (f-l) 1
Error a (rest) 2
b) Infusion treatments Df for milk paramt.
w/in CSF (split plot) (tcs-l)-(cs—l) 01 & 02 averaged
16 8
Treatments (t—l) ——2 - 1
orth.contrasts
K vs.G's 1
01 vs. 02 1
TXF (t-l)(t-l) 2 l
TxS (t—l)(s-1) 2 1
TxC (t—l)(c-1) 6 3
Error b (rest) 4 2
c)B1eedings w/in T (btcs-l)
(split-split plot) -(tcs-l) 24
Bleedings (b-l) 2
orth contrasts
B vs. B B l
B1 vs. B3 3 1
841 4
BxF 2
8x8 2
BxC 6
Error 0 (rest) 16
Total (btcs-l) 71

 

Appendix Table

II.34.--Trial III 1971.

256

AOV for feed intake.

 

B. Trial III.

'DM Intake (kg/day)

 

var. source df MS F
a)Whole plot 1
Cows 3 59.641 36.0
Seqs. 1 1.550 2.8
Feeds 1 .220 <1
Error a 2 1.104
b) Split plot lg
Inf. Trtm. 2 1.206 1.2
K vs. 0
01 vs. 02
T x C 6 2.038 <1
T x S 2 1.211 <1
T x F 2 1.106 <1
Error b 4 2.054
Total (SS) 23 (70.130)

 

Appendix Table II.35--Tria1 III 1971.

2557

Statistical analysis for milk production

 

 

 

 

 

parameters.
DF MS F F sign. MS F F sign. MS F F sign.
a)Whole plot 1 Milk Yield (kg/day) Prot. Conc. (%) NPN Conc. (mg/100ml)
Cows 3 67.6862 .04782 1.684
Segs. 1 12.0930 .01488 3.151
Feeds 1 .2782 <1 .00319 <1 2.176 1.3
Error a 2 .3144 .01874 1.666
b)Split plot g
Inf.Trtm. 1 1.0215 2.9 .30636 301.8 <.005 82.356 34.4 <.05
IxC 3 .1016 <1 .00108 1.1 3.268 1.2
1x5 1 .0109 <1 -- .600 <l
IxF 1 .1464 <1 .00099 <1 .176 <1
Error b 2 .3547 .00102 2.395
Total (55) 175’(218.1536) (.51156) (111.425)
FCM Yield (kg/day) Prot. Prod. (g/day) NPN/Tot. N (8)
a)Whole plot 1
Cows 3 43.655 68433.3 .4936
Seqs. 1 18.104 12210.2 .0203
Feeds 1 .112 <1 324.0 <1 .0637
Error a 2 1.440 410.4 .2547
b)Split plot g
Inf.Trtm. 1 .003 l 11990.2 52.0 .025 .9653 2.6
IxC 3 .195 <1 544.3 2.4 .0088 ’1
1x8 1 .326 <1 9.0 <1 .0203 1
IxF 1 .136 <1 72.3 <1 --
Error b _2 .426 226.4 .3669
Total (SS) 15(152.525) (232812.0) (3.820)
Appendix Table II.3S—-Continued.
DF MS F F sign. MS F F sign. MS F F sign-
Est. True Prot. (%) SNF Cone. (%) Fat Conc. (%)
a)Whole plot 1 .05335 .67572 .20212
Cows -3 .01363 .22801 .41281
Seqs. 1 .00243 2.0 .02176 1.1 .03516 1.7
Feeds 1 .00121 .01948 .02088
Error a 2
b) Split Plot 2
Inf. Trtm. 1 .25075 113.2 <.01 .16201 3.3 .14631 5.4 <.25
IxC 3 .00094 <1 .01777 <1 .08462 1.0
1x8 1 -— .00764 <1 .13637 5.0
IxF 1 .00098 <1 .00949 <1 .00530 <1
Error b 2 .00222 .00959 .02723
Total (85) I5 (.47355) (1.29650) (1.41425)
a)Whole plot 7 Est. True Prot. (g/day) SNF Prod. (g/day) Fat Prod.(g/day)
Cows 3 59106.3 S37055.2 49452.3
Seqs. 1 10816.0 59902.6 36576.6
Feeds 1 256.0 <1 105.1 <1 67.2 <l
Error a 2 401.0 1447.1 3088.4
b)Split plot g
Inf.Trtm. 1 10000.0 84.7 .025 22788.9 14.7 519.8 <1
IxC 3 454.8 3.9 583.2 <1 1977.6 <1
1x5 1 6.3 <1 2141.8 1.4 1242.6 <1
IxF 1 72.3 <1 1897.1 1.2 204.5 <1
Error b 2 118.1 1553.8 1149.8
Total (53) I5(200871.8) (l698370.9) (194333.0)

 

 

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