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0-7 639

IIIIIIIIIIIIIIIIII IIIIII IIIIIIIIIIII

3 1293 10752 20037 LIBRARY

Michigan State
University

 

 

This is to certify that the
thesis entitled
Protein Requirement and Non-Protein
Nitrogen for High Producing Cows in

Early Lactation

presented by

John Foldager

has been accepted towards fulfillment
of the requirements for

flv [/ degree in

 

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II: {II

PROTEIN REQUIREMENT AND NON-PROTEIN NITROGEN

FOR HIGH PRODUCING COWS IN EARLY LACTATION

BY

John Foldager

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Dairy Science

1977

ABSTRACT

PROTEIN REQUIREMENT AND NON-PROTEIN NITROGEN
FOR HIGH PRODUCING COWS IN EARLY LACTATION

BY
John Foldager

Recent reports have suggested that high producing cows should
be fed rations containing 15 to 17% crude protein (C?) in dry matter
(DM), and that only plant protein should supply the supplementary
nitrogen. Several studies have shown that milk yields in cows fed corn
silage treated with ammonia or urea at ensiling yielded as much or more
milk as those fed equal protein from soybean meal. However, none of
these studies were commenced at the beginning of lactation when milk
yields are highest and intakes, relative to needs, are lowest.

The requirement of protein and the feasibility of non—protein
nitrogen (NPN) as the supplementary nitrogen source were tested in
68 Holstein cows from weeks three through 20 post-partum. Identical
rations containing NPN were fed all cows from four weeks pre-partum
through the second week post-partum. Experimental rations were:
concentrate (.4 kg per kg milk, through week 10 post-partum and then
.33 kg from 11 to 20 weeks), corn silage gd_1ibitum, and 2.2 kg hay.

Seventeen cows were assigned to each of four treatment groups. The

 

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.- Irr

 

John Foldager

first two groups received rations of only plant protein; which con-
tained 12 to 13% CP in DM (group NC) and 15 to 16% (group PC),
respectively. The other two groups were also fed rations with 15 to
16% CP, but approximately 25% of total nitrogen came from NPN. Corn
silage treated with .65% urea (group U) or .40% ammonia (group AU) was
fed with concentrate containing 1.25% urea. Rumen and blood were
sampled two weeks pre-partum and at weeks 3, 10 and 18 post-partum.
Blood was also collected in week 6.

Treatment effects on intake of dry matter and nutrients were
not statistically significant, except for total CP, water soluble
nitrogen and CP content of DM. However, group NC tended to consume
more total DM and had highest intake of DM per 100 kg body weight.
Milk yields for all cows and adjusted yields for cows which produced
more than 25 kg per day during week two post-partum were 29.6, 32.6;
27.9, 34.8; 27.0, 32.1; and 27.7, 32.8 kg per day for the respective
groups. Neither milk yields, milk components, nor body weight gains
were significantly affected by treatments, except for slightly lower
adjusted yields for high producers in group U during weeks three
throughlsix.

Rumen volatile fatty acids, plasma glucose, plasma ammonia
and amino acids were not significantly affected by treatment. Rumen
ammonia and plasma urea nitrogen (PUN) were lowest for group NC,
highest for U, with PC and AU intermediate. Similar PUN in groups PC
and AU suggests equal losses of nitrogen from supplementing soybean

meal or corn silage treated with ammonia plus urea in the concentrate.

John Foldager

Lower PUN in group AU than U was attributed to prevention by ammonia
of plant protein proteolysis in corn silage.

Based on production results, body weight gains, supporting
evidence from rumen and blood metabolites, calculations for metaboli-
zable protein, and protein entering the abomasum, it was concluded that
high yielding cows fed rations of corn, corn silage, and limited hay
require no more than 13% CP in DM, which is equal to only 80 to 90%
of presently recommended standards (NRC 1971).

In the above and a previous experiment (also reported herein),
lowest plasma concentrations of essential amino acids coincided with
highest demands (high yielding, early lactation cows) and/or lowest
supply (low protein ration and zero and two hours after feeding).
Co-limiting amino acids identified by the two-phase method were
histidine, valine, and leucine. Milk yields at 42 days was also
positively correlated with these amino acids plus threonine and lysine.
Estimates of minimum blood supply to the mammary gland needed for
output of amino acids produced in milk identified methionine as the
first limiting amino acid in groups NC, PC, and U and phenylalanine
for group AU. However, the ranking of limiting amino acids by the
latter method could be easily changed by slight errors in arterio-
venous differences, since values were nearly equal for a number of

amino acids.

)- .I_-.

 

 

ACKNOWLEDGMENTS

This thesis could not have been completed without generous
help and support from numerous peeple, who I would like to acknowledge.
Especially, I want to thank my major professor, Dr. J. T. Huber, for
his support and encouragement throughout my graduate program. Thanks
are also given to Dr. W. G. Bergen for special interest in the project,
and for amino acid analysis. I also want to acknowledge the other
members of my advisory committee, Drs. J. L. Gill and R. M. Cook, for
their guidance and advise during the graduate study. Gratitude is also
extended to professor dr. med. vet. A. Neimann-Sorensen, Denmark, for
his encouragement and continued support to take upon the graduate
study. Thanks are also given to Dr. R. R. Neitzel, for aid with
statistical calculations. Here I would also like to express my appre-
ciation for the continued financial support from the Department of
Dairy Science. Last but not least I wish to extend my deepest and
most sincere gratitude to my wife, Karen, for her encouragement, love,
understanding and sacrifices during my course of study, research and

manuscript preparation.

ii

TABLE OF CONTENTS
Page
LIST OF TABLES . . . . . . . . . . . . . . . . . vi
LIST OF FIGURES. . . .. . . . . . . . . . . . . . xi

LIST OF ABBREVIATIONS. . . . . . . . . . . . . . . xiii

LITERATURE REVIEW . . . . . . . . . . . . . . . . l
1. Requirements and sources of protein in nations for dairy

cows. . . . . . . . . . . . . . . . . l
l. 1 Protein requirements. . . . . . . . . . . l
1.2 Nitrogen sources . . . . . . . . . . . . . . . 5
1.2.1 Catabolism of nitrogenous compounds in the rumen. . . . 7
1.2.2 Protein anabolism in the rumen. . . . . 9
1.3 Utilization of nonprotein nitrogen in dairy cattle rations . 12
1.4 Summary and conclusion of literature review on nitrOgen

utilization in dairy cows . . . . . . . . . . . 22

2. Physiological and biochemical factors in the synthesis of

milk lactose and protein and milk volume control . . . . 23
2.2 Lactose synthesis. . . . . . . . . . . . . . 24
2.2.1 Glucose metabolism by the mammary gland. . . . . . . 26
2.2.2 Lactose synthetase. . . . . . . . . . . . . . 30
L 3 Protein synthesis. . . . . . . . . . . . . . . 31
2.3.1 Protein fractions . . . . . . . . 31
2. 3.2 Amino acid metabolism by the mammary gland. . . . . . 33
2.3.3 Protein synthesis . . . . . . . . . . . . . 44
2. 4 Milk secretion. . . . . . . . . . . . . . . . 48
2.4.1 Protein and lactose secretion . . . . . . . . . . 48
2. 4.2 Milk volume . . . . . . . . . . . . . . . . 49
2.5 Mammary blood flow . . . . . . . . . . . . 50
2.5.1 Control of mammary blood flow . . . . . . . . . . 51

3. Effects of postruminal administration of energy (glucose)

and protein (casein and amino acids) on milk production. . 53
3.1 Effect on milk protein . . . . . . . . . . . . . 54
3. 2 Lactose and milk yield . . . . . . . . . . $9
3. 3 Intravenous infusion of graded levels of amino acids . . . 6O
3. 4 Effect of abomasal infusion on feed intake, dry matter

digestibility, and N-utilization . . . . . . . . . 61
3.5 Factors affecting blood metabolites in dairy cows . . . . 62

iii

Page
4. Limiting amino acids(s) for milk protein synthesis . . . . 64

5. Summary of interactive review on metabolic effects
on milk synthesis and production . . . . . . . . . 6S

6. Research proposal and objective . . . . . . . . . . 70
MATERIALS AND METHODS . . . . . . . . . . . . . . . 71
RESULTS. . . . . . . . . . . . . . . . . . . . 87
1. Nutrient content in feeds . . . . . . . . . . . . 87
2. Feed intake. . . . . . . . . . . . . . . . . 93
3. Nutrient intake. . . . . . . . . . . . . . . . 99
4. Milk yield and composition . . . . . . . . . . . . 108
5. Body weights and average daily gains. . . . . . . . . 119

6. Feed utilization and nutrient balance . . . . . . . . 119

7. Rumen pH, volatile fatty acids, and ammonia nitrogen . . . 127
8. Blood metabolites . . . . . . . . . . . . . . . 134
8.1 Plasma amino acids. . . . . . . . . . . 140
8.1.1 Initial experiment on factors affecting plasma amino

acid concentrations . . . . . . . . . . . . 140
8.1.2 Main study of factors affecting plasma amino acid

concentrations. . . . . . . . . . . . . 155
8.1.2.1 Effect of treatments for all cows 42 days post-partum. . 155
8.1.2.2 Effects of treatments and time from calving on plasma

amino acids in selected cows . . . . . . . . . 158
DISCUSSION. . . . . . . . . . . . . . . . . . . 162
l. Nutrients in feeds. . . . . . . . . . . . . . . 162
2. Feed and nutrient intakes . . . . . . . . . . . . 163
3. Production of milk and milk components . . . . . . . . 165
4. Rumen metabolities. . . . . . . . . . . . . . . 172

5. Blood metabolites . . . . . . . . . . . . . . . 17s

iv

Page
SUWARY AND CONCLUSIONS . . . . . . . . . . . . . . 188
BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . 193
APPENDIX A . . . . . . . . . . . . . . . . . . 209

APPENDIX B . . . . . . . . . . . . . . . . . . 225

Table

10.

11.

12.

13.

LIST OF TABLES

Required total protein in dry matter of dairy rations by
bodyweight, fat percent, dry matter intake, and milk
yield . . . . . . . . . . . . . . . .

Milk yield and content of fat, protein, lactose, ash and
solids non-fat for "normal" cows . . . . . . . .

The proteins of cow's milk and their percentage distri-
bution in raw skim milk protein . . . . . . . .

Amino acid composition of bovine milk protein (after
Whitney et al., 1976). . . . . . . . .

Gene frequency of milk protein variants ianolstein cows
(after Thompson & Farrell, 1974) . . . . . . . .

Amino acid changes due to genetic variants (after Whitney
at 81. , 1976) O O O O O O O O O O O O O 0

Limiting amino acids in milk protein production. . . .

Ration components and percent crude protein in the
different treatments . . . . . . . . . . . .

Ingredient composition of the concentrates used in this
stUdy , % O O O O O O O O O O O O O O 0

Analysis of variances used and degrees of freedom per
source of variation . . . . . . . . .

The dry matter content and the content of nutrients in
the dry matter of concentrate corn silage and hay, %

Changes in total nitrogen and nitrogen fractions between
harvest feeding in untreated and NPN treated corn
Silage. - O O O O O O O O O O O O O I O 0

Content of ethanol,-acetate, propionate, iso-butyrate,

butyrate, and lactate as well as pH in corn silage, %
in DM (89 observations per item) . . . . . . .

vi

Page

25

32

34

35

36

66

72

73

83

88

91

92

Table Page

14. IIntake of concentrate, corn silage, hay, total dry matter
and net energy for the different periods after
calving . . . . . . . . . . . . . . . . 97

15. Intake of crude protein, water soluble nitrogen, acid
detergent nitrogen (ADN), true protein, and percent
crude protein during treatment periods. . . . . . 103

16. Overall means and b-values for significant covariates . 108

17. Actual and adjusted milk, actual components yields, and
milk yield persistencies for the different periods
after calving . . . . . . . . . . . . . . 110

18. Adjusted yields of milk and milk components fer the
different periods in cows with more than 25 kg milk
per day in week two . . . . . . . . . . . . 113

19. The influence of protein source on milk composition in
periods from calving. . . . . . . . . . . . 115

20. Adjusted yields of fat corrected and solids corrected
milk in periods from calving for cows with more than
25 kg milk per day in week two . . . . . . . . 118

21. Average daily gains fer treatments befOre and after
minimum weight. . . . . . . . . . . . . . 121

22. Protein balance as percent of NRC (1971) requirement for
high yielding cows . . . . . . . . . . . . 126

23. Effects of time from calving on rumen pH and rumen concen-
trations of volatile fatty acids, m moles/liter. . . 129

24. Effect of protein sources on rumen pH and rumen concen-
trations of volatile fatty acids, m moles/liter; 68
observations per mean, . . . . . . . . . . . 130

25. Effect of treatment on molar distribution of rumen
volatile fatty acids, molar percent. . . . . . . 132

26. Effect of time on the molar distribution of rumen
volatile fatty acids, molar percent. . . . . . . 133

27. Effect of protein treatment and time from calving on
rumen concentrations of ammonia, mg/lOOml. . . . . 134

vii

 

Table Page

28. Effects of protein treatment and time from calving on
plasma concentrations of glucose, urea and ammonia
nitrogen, mg per 100 m1 . . . . . . . . . . . 136

29. Average content of urea and ammonia nitrogen in plasma
from selected cows for diurnal sampling, mg/100 ml . . 138

30. Protein in diets fed cows in initial experiment on
factors affecting plasma amino acids. . . . . . . 144

31. Experimental procedure for the initial study of factors
affecting plasma amino acids . . . . . . . . . 144

32. Initial experiment on effects of treatments, level of
milk production, week and hour of sampling on plasma
amino acid concentrations in dairy cows, uM/lOO m1 . . 145

33. Effects of treatments and week of sampling on plasma
concentrations of glycine, alanine, serine;
UM/IOO m1. 0 e e e e e I e e e e e e e e 152

34. Effects of treatments and hours of sampling on plasma
concentrations of valine and asparagine; uM/lOO m1 . . 152

35. Effects of treatments, weeks and hours of sampling on
plasma concentrations of arginine, histidine and
aspartic acid; uM/lOOml . . . . . . . . . . . 153

36. Distribution of samples assayed at days -14, 42, and 126
on breeding groups, blocks, and treatments. . . . . 156

37. Plasma concentrations of amino acids fer all cows at 42
days POSt-pal‘tum LIN/100 m1 0 e e e e e e e e e 156

38. Plasma amino acid concentrations (mM/liter) for treat-
ments and days from calving and/or by days within
treatment; uM/ml . . . . . . . . . . . . . 159

39. Intake of urea equivalents in the different periods from
calving by cows fed non-protein nitrogen supplemented
diets O O O O O O O O O O O O O O O O O 164

40. Urea fermentation potential (UFP; g urea/d) of high
concentrate, corn silage, limited hay rations used for
treatments in the different periods from calving. . . 168

41. Utilization of metabolizable protein fer milk protein

production'in dairy cows in the different_periods
from calving. . . . . . . . . . . . . . . 168

viii

Table

42.

43.

44.

45.

46.

A1.

A2.

A4.

A5.

A6.

A7.

A8.

Protein availability at the abomasal level, its relation
to present standards, and utilization for milk protein
production in dairy cows in the different periods from
calving . . . . . . . . . . .

Total protein and true protein available at the abomasal
level in dairy cows in weeks 7 through 10 post-partum.

Rumen ammonia nitrogen in dry cows at pasture and indoors
fed corn silage plus urea added at feeding (mg/100 ml
fluid) . . . . . . . . . . . . . . . ..

Amino acids potentially limiting milk protein production
of dairy cows in the initial and the main experiment,
as well as in abomasal protein . . . . . .

Estimated minimum flow of plasma to the mammary gland for
the output of amino acids in milk protein produced at
42 days post-partum in the main experiment, and the
ranking of amino acids . . . .

Amounts of corn silage harvested by year and silo and
its nutrient content. .

Date of birth, record intthe previous lactation, actual
date of calving, genetic group and assignment to
blocks within genetic group of cows used . .

Simple correlations among nutrient and quality measure-
ments on corn silage. . . . . . . . .

Partial correlations between the dependent variable and
independent variables selected by multiple regression
analysis. . . . . . . . . . . . .

Effects of breeding groups on feed and nutrient intake,
milk yield and composition and body weight

Interaction between breeding groups and treatments for
the solids non fat percentage in milk .

Interaction between breeding groups and treatments on
the average bodyweight . . . . . . . .

Simple correlation among milk parameters, body weight

and the balances for net energy and protein in high
yielding cows within periods .

ix

Page

169

172

174

184

186

209

210

214

215

216

217

217

218

 

Table

A9.

A10.

A11.

A12.

A13.

A14.

Page

Effects of breeding groups on rumen pH and rumen con-
centrations of volatile fatty acids and ammonia
nitrogen . . . . . . . . . . . . . . . . 219

Effect of breeding group on the rumen concentrations
of iso-butyrate and valerate, mMoles/liter. . . . . 220

Effect of breeding group on the molar distribution of
acetate, prepionate and valerate, molar percent . . . 221

Effects of breeding group on plasma concentrations of
glucose, urea and ammonia nitrogen; mg/lOOml . . . . 222

Plasma amino acid concentrations (uM/lOO ml) in breeding
groups and by treatment and/or days from calving
within breeding groups . . . . . . . . . . . 223

Minimum plasma flow at mammary gland for output of
individual amino acids in milk protein produced . . . 224

Figure

10.

11.

12.

LIST OF FIGURES

Milk response to added nitrogen from non-protein nitrogen
(NPN) or natural protein . . . . . . . . . . .

Arteriovenous differences fer amino acids as a percentage
of the arterial concentration . . . . . .

Daily intake of concentrate per treatment and week from
calving (kg/d; standard error 3,07)

Daily intake of corn silage per treatment and week from
calving (kg/d; standard error 31.02) . . . . . . .

Daily intake of total dry matter per treatment and week
from calving (kg/d; standard error 3,42). . . . . .

Daily intake of net energy for lactation per treatment
and week from calving (Mcal/d; standard error 3,812).

Daily intake of total crude protein per treatment and
week from calving (kg/d; standard error 3,06) . . .

Crude protein in dry matter per treatment and week from
calving (%, standard error 3,168). . . . . . .

Daily intake of water soluble nitrogen per treatment and
week from calving (kg/d; standard error 3,009) '

Daily actual milk yields per treatment and week from
calving (kg/d; standard error 39.16). .

Content of solids non fat in milk per week from calving
for treatments NC, PC, U and AU . . .

Average body weight for treatments per week from calving
(kg; standard error 33.6) . . . . . . . . .

xi

Page

19

38

94

95

96

100

102

105

107

109

116

120

Figure Page

13. Milk yield per kg dry matter for treatments by week from
calving (kg; standard error 3,040) . . . . . . . 122

14. Balance of energy intake relative to NRC (1971) standards
for treatments per week from calving (%; standard
error 3 1.7) . . . . . . . . . . . . . . 123

15. Balance of crude protein intake relative to NRC (1971)
standards for treatments per week from calving (%;
standard error 32.3). . . . . . . . . . . . 124

16. Effect of protein treatments on diurnal changes in plasma
urea nitrogen (average for 2 cows per treatment 59 and
112 days post-partum; mg/100 ml; + milking) . . . . 139

17. Effect of days from calving on diurnal plasma urea nitrogen
(8 cows/mean; mg/100ml; +milking) . . . . . . . 141

18. Effect of days from calving on diurnal plasma ammonia
nitrogen (8 cows/mean; mg/100 ml; + milking). . . . 142

19. Plasma valine and leucine responses to treatments in the
initial and the main experiment ._ . . . . . . . 180

20. Plasma threonine, lysine and isoleucine responses to
treatments in the initial and the main experiment . . 181

21. Plasma histidine and phenylalanine responses to treatments
in the initial and the main experiment. . . . . . 182

22. Plasma methionine and cysteine responses to treatments in
the initial and main experiment . . . . . . . . 183

Bl. Adjusted content of protein in.milk for breeding groups
per week from calving (standard error 3 .05). . . . 225

82. Concentrations of solids-non-fat in milk for breeding
groups per week from calving (standard error 3 .13) . 226

B3. Adjusted body weight for breeding groups per week from
calving (standard error 3 3.0) . . . . . . . 227

xii

ADH
ADN
ALA
ARG
ASN

ASP

A—V

BHBA

BW

16

CA

CP

CYS

DM

DNA

DP

GLN

LIST OF ABBREVIATIONS

acid detergent fiber
antidiarrhetic hormone

acid detergent nitrogen
alanine

arginine

asparagine

aspartate

group fed ration with ammonia
arterio-venous difference
B-hydroxy butyrate

body weight

fatty acid with 16 carbon units

casein

.crude protein

cysteine

dry matter
deoxyribonucleic acid
total dietary nitrogen
essential amino acid(s)

glutamine

xiii

cw
GLY
H18
18

115

IV

LG

LYS

MBF

MP

NEAA
NE

NPN

PAA
PAN
PC

pco2
PGF

PHE

glutamate
glycine
histidine
immunoglobulins
isoleucine
intravenous
lactalbumin
lactoglobulin
leucine

lysine
metabolizable amino acids

mammary blood flow

methionine

metabolizable protein

nitrogen

group fed the negative control ration
non-essential amino acid(s)

net energy for lactation

non-protein nitrogen

group fed ration containing ProSil (ammonia)
plasma amino acid(s)

plasma ammonia nitrogen

group fed the positive control ration
partial pressure of carbonioxide
prostaglandin F

20

phenylalanine

xiv

PP proteose-pentones

PRO = proline

PUN a plasma urea nitrogen

RAN = rumen ammonia nitrogen

RER = rough endoplasmic reticulum
RNA = ribonucleic acid

SA = serum albumin

SER = serine

SNF = solids-non-fat

SOLN = water soluble nitrogen

TDN = total digestible nutrients
THR = threonine

TN = total nitrogen

TP = true protein

TYR = tyrosine

U = group fed ration with urea
UFP = urea fermentation potential
VAL = valine

VFA = volatile fatty acid

YATP = yield of dry microbial cells per mole ATP

2? = group fed ration with twice the recommended level of ProSil
(ammonia) .

XV

LITERATURE REVIEW

1. Requirements and sources of protein in
rations for dairy cows

The synthesis of milk constituents and the milk volume in
dairy cows are under physiological and biochemical control, and the
controlling factors are to a large degree dependent upon the avail-
ability of absorbable nutrients. Studies utilizing abomasal and
intravenous (IV) infusion techniques have clearly shown positive
effects on the production of milk and milk constituents.

Protein is the most expensive major nutrient in dairy
cattle rations and to date has received less attention than energy.
In formulating the protein content of dairy rations the important
questions are: (1) How much protein does the cow require at various
production levels? (2) Which and in what combination can available

protein sources be used for the most lucrative production of milk?

1.1 Protein requirements

 

In Nutrient Requirements of Dairy Cows (1971) recommended
amounts of total protein (crude protein (CP) = N x 6.25) are 14, 15
and 16% of dry matter (DM) at milk yields less than 20, 20 to 30, and
more than 30 kg per day, respectively. The corresponding recommen-
dations for digestible crude protein are 10.5, 11.4, and 12.3% of

dry matter. Based on required daily amounts in relation to body

weight, milk yield, and milk composition, the recommended CP concen-
tration in dry matter (DM) will vary as shown in Table 1 depending

upon the daily DM intake per 100 kg body weight (BW). From the cal-
culations in Table l a high producing cow (50 kg/day) with a low DM
intake may require as much as 27.4 CP in the DM. The requirement
decreases with increased DM intake per unit BW, decreased fat percentage
and decreased milk yield.

The effect of protein content in DM on milk yield has recently
been studied by Schwab et al. (1971), Thomas (1971), Gardner and Parker
(1973), Sparrow et a1. (1973), Grieve et a1. (1974), Huber (1975),
Chandler et a1. (1976), Wallenius (1976), and Cressman et a1. (1977).
Cows producing about 30 kg milk per day had normal persistencies when
fed rations containing 12.5 to 13.6% CP in DM (Thomas, 1971). For
cows producing less than 20 kg milk per day, rations containing 10.5
to 11% CP appearred adequate (Thomas, 1971). Schwab et a1. (1971),
Huber (1975), Chandler et a1. (1976), and Wallenius (1976) obtained
equal milk productions in cows fed 12.8 to 14% CP rations versus 17
to 17.7% CP. Gardner and Parker (1973) showed that 305 day milk
yield increased when the ration CP was increased from 13.2 to 14.4
and 15.5%. In experiments conducted between parturition and 20 weeks
post-partum, milk yields were increased when the dietary protein was
increased from 13.5 to 18% of the DM (Sparrow et al., 1973; Grieve et
al., 1974; Cressman et al., 1977). Cressman et a1. (1977) obtained
the above response in multiparous cows. Heifers did not respond to
ration CP contents higher than 12.4%. In the trial by Grieve et a1.
(1974), the DM intake increased with the CP content. Cows fed rations

containing 15.1 or 16.1% CP showed no difference in milk yield (Lamb

by bodyweight, fat percent, dry

ions

1'.

1ry ra

a

Table l.-Required total protein in dry matter of da
matter intake, and milk yield.

 

 

 

c>
an
c>
<-
60
.x
c:
c> c:
\o to
1.:
.c
on
-H
m an
g‘ cm
a:
c>
or
In
H
O
<-
60 c:
.x to
C)
c:
in
a In
.= (V
on
-H
“i
'3 <3
6:
an
o
in
H
.n
2:
c:
p
a
0
u o
m H
IL 0
n.

 

14.2 16.4 18.6 23.0 27.4

12.0

2.80 13.5 16.1 18.9 21.4 26.7
3.00
3.20
3.40
3.60

3.80
4.00

3.5

13.3 15.3 17.4 21.5 25.6

11.2

15.1 17.5 20.0 24.9

12.6

12.4 14.4 16.3 20.2 24.0

10.5

14.1 16.4 18.8 23.4

11.8

11.7 13.5 15.3 19.0 22.6

9.9

13.3 15.5 17.6 22.0

11.1

11.1 12.8 14.5 17.9 21.3

9.4

12.6 14.6 16.7 20.8

10.5

12.1 13.7 17.0 20.3

10.5

8.9

11.9 13.8 15.8 19.7

9.9
9.5

10.0 11.5 13.0 16.1 19.2

8.4

13.2 15.0 18.7

11.3

13.8 15.8 17.9 22.1 26.3

11.7

2.80 13.1 15.6 18.1 20.6 25.6
3.00
3.20
3.40
3.60

3.80
4.00

3.0

12.8 14.8 16.7 20.6 24.5

10.9

14.5 16.9 19.2 23.9

12.2

12.0 13.9 15.7 19.3 23.0

10.2

13.6 15.8 18.0 22.4

11.4

11.3 13.0 14.8 18.2 21.6

9.6
9.1

12.8 14.9 16.9 21.1

10.8

12.3 13.9 17.2 20.4

10.7

12.1 14.1 16.0 19.9

10.2

10.1 11.7 13.2 16.3 19.3

8.6

11.5 13.3 15.2 18.8

9.6
9.2

9.6 11.1 12.5 15.5 18.4

8.2

12.7 14.4 17.9

10.9

 

aCalculated according to NRC (1971).

Dry matter intake as percent of bodyweight.

b

Milk yield, kg per day.

C

et al., 1974). Cows fed 16% CP rations and then dropped to 13% CP

at week 6 postpartum showed marked decreases in milk yield compared
to cows maintained at 13% CP throughout the 12 week trials (Huber,
1975). In a review of the protein supply in dairy cows, Satter and
Roffler (1975) made the suggestion to feed 17% CP rations in early
lactation, but Huber (1975) cautioned that it is unwise to routinely
recommend levels higher than 15% GP for dairy herd rations because of
expense and waste by low producers.

The described effects of CP content in dairy rations are
inconclusive. The variable results might be due to stage of lactation
when the study was initiated, dry matter intakes, and effects of
protein on energy utilization.

In the first 2 to 3 weeks postpartum the DM intake is lower
than during subsequent weeks (Huber, 1975). During this period of
reduced intake it has been theorized that the dietary protein content
should be increased (Table 1). However, research has not shown benefits
from this practice (Schwab et al., 1971; Huber, 1975) probably because
of the cows' abilities to use protein reserves to meet a small deficit
incurred in early lactation (Coppock, 1968; Paguay et al., 1972). In
cows fed .33 kg concentrate per kg milk and offered corn silage §g_
libitum the dry matter intake per 100 kg BW (range 2.46 to 3.96%) was
not related to days postpartum (which ranged 20 to 124 days) (Huber,
1975). However, milk yields were positively correlated with dry matter
intakes. Lamb et a1. (1974) found that increasing the rate of grain
feeding from .25 to .625 kg per kg milk led to decreased hay consumption
but increased total DM intake. Milk yields increased with the rate of

grain feeding, and could be attributed to the increased consumption

of net energy. In the trials reported by Sparrow et a1. (1973),
Grieve et a1. (1974), and Cressman et a1. (1977), dry matter intakes
increased with increased CP in the ration. Schwab et a1. (1971)
reported an increase in the apparent DM digestibility when the ration
CP content was increased from 12.8 to 17.7% by the addition of SBM. Moe
and Tyrrell (1977) fed cows rations containing 20, 17, and 14% CP
and showed that metabolizable energy was equal at 20 and 17% CP but
lower for the 14% CP diet.

Based on present knowledge, changes from NRC (1971) in the
protein content of rations are not warranted. However, further
research is needed where the protein is varied under conditions of

constant intake and net energy density of rations.

1.2 Nitrogen sources

 

To meet the recommended protein requirement in ruminants the
selection of natural proteins will rest mainly on the price and avail-
ability. There are very few data showing an advantage of one protein
source over another in practical dairy rations.

Numerous experiments have shown that nonprotein nitrogen (NPN)
can be utilized by rumen bacteria for microbial protein synthesis.

The microbial protein is then digested and utilized by the host animal.
The synthesis of microbial protein in the forestomachs of ruminants
places these species in a unique position in agricultural production,
that of being able to upgrade simple nitrogenous compounds to high
quality protein.

The concept that rumen bacteria can utilize NPN compounds

to form protein was presented as early as 1891 by Zuntz and Hagemann

(ref: Loosli et al., 1949; Chalmers, 1961). Loosli et a1. (1949) and
Duncan et a1. (1953) presented quantitative evidence that ovine and
bovine rumen microorganisms can utilize urea as their only source of
nitrogen for the synthesis of amino acids. Bryant and Robinson (1962,
1963) showed that 80 to 89% of freshly isolated strains of rumen
bacteria prefer to synthesize their cellular constituents from ammonia-N
linked with carbon sources other than amino acids. Feeding 15N-ammonium
sulfate or 15N-urea lead to the same degree of labeling of amino acids
by rumen microorganisms, and the incorporation of label increased

with the time of adaptation (Virtanen, 1966). Virtanen (1966) also
demonstrated that the amino acids synthesized in the rumen are used

for milk protein synthesis. Hungate (1965) hypothesized that the
limitation on microbial synthesis imposed by the anaerobic conditions

of the rumen limits the productivity of the host, and that increases

in the production by the host should be sought in increased synthesis

of microbial bodies in the rumen or by shunting of protein directly

to the abomasum.

Digestion and metabolism in ruminants has been reviewed
extensively (Lewis, 1961; Daugherty, 1965; Hungate, 1966; Phillipson,
1970; McDonald and Walner, 1975; Cole et al., 1976). Their findings
will not be recapitulated here. However, from all the studies it
becomes clear that the postruminal absorption in ruminants is as in
nonruminants except for a more continuous flow of digesta and a larger
degree of fermentation in the caecum and colon. The largest difference
between ruminants and nonruminants is the modifying effect by micro-
organisms inhabitating the rumen. The rumen microbes modify the feed

protein by catabolism and the production of microbial protein, which

has a fairly uniform amino acid composition, by anabolism. From a
cost-benefit point of view it is beneficial to replace natural protein
sources by a maximum amount of NPN. Therefore, this review will be
centered around the most efficient utilization of dietary nitrogen (N)
sources.
1.2.1 Catabolism of nitrogenous
compounds in the rumen

Degradation of dietary protein in the rumen depends largely
on its rate of passage and solubility (Phillipson, 1972; Miller, 1973;
Chalupa, 1975). It is often of no advantage to feed a highly soluble
protein, rich in essential amino acids (EAA), because of degradation
to ammonia and subsequent use of the nitrogen (N) for bacterial protein
and nucleic acid synthesis which is of lower digestibility and protein
quality than the original material (Smith, 1969). Rate of passage
is affected by feed intake, specific gravity of feed particles,
particle size of diet, concentrate to roughage ratio, rate of rumen
digestion, and water intake (Chalupa, 1975). Rumen bacterial proteases
(exo- and endo—peptidases) are bound at the cell surface, have free
access to substrate, and are not inhibited by addition of dietary
urea (Chalupa, 1975). Protozoa possess endogenous enzymes for protein
catabolism as seen in the digestion of engulfed bacteria (Allison,
1970; Stern et al., 1977a, b). In vitro solubility of proteins
increases with pH (Wohlt et al., 1973), and an average 60% of the
natural dietary protein is catabolized in the rumen (Smith, 1969; Smith
and McAllan, 1970; Al-Rabbat et al., 1971; Phillipson, 1972; Nolan
et al., 1973; Chalupa, 1975). The degree of protein degradation varies

from about 10% for proteins with low solubility (zein) to 90% for

proteins with a high solubility (casein). In the rumen of cattle the
half-life of casein was 5.6 to 21.5 minutes, but it was 175 minutes for
ovalbumin (Mangan, 1972). The solubility of albumins and globulins

is higher than for proteins composed primarily of prolamines and
glutelines (Wohlt et al., 1973).

The amount of dietary protein escaping rumen degradation (rumen
bypass) may be increased through a functional esophageal groove or by
processing. Rumen bypass of protein was reviewed by Allison (1970),
Phillipson (1972), and Chalupa (1975) and is summarized in the following
paragraph.

Protection of dietary protein by processing can be accomplished
by heat reaction between sugar aldehyde and free amino acid groups
which decreases protein solubility. It also destroys proteolytic
enzyme inhibitory factors, when present, facilitating digestion in
the intestine. Certain chemicals also decrease protein solubility
at the pH of the rumen by cross-linkages with amino acid amide groups.
These cross-linkages are destroyed by the acidity of the abomasum.

The optimum pH for rumen proteolysis is 6.5 and proteolytic
enzyme activity is not inducible (Blackburn, 1965). Amino acids and
nucleic acids released from natural protein are deaminated (Blackburn,
1965; Allison, 1970; Mangan, 1972; Chalupa, 1975). Half-lives for
free amino acids in the rumen are less than 2 hours (Chalupa, 1975).
Ammonia released by amino acid catabolism, feed NPN and salivary urea
enters a common rumen ammonia pool (Hutton, 1972; Nolan et al., 1973;

Satter and Roffler, 1975).

1.2.2 Protein anabolism in the rumen

Nitrogen Sources.--In reviews by Blackburn (1965), Allison

 

(1955, 1970), Smith (1969), and Chalupa (1975) it was concluded that
rumen ammonia probably is the main N source for bacterial growth,
whereas many protozoa require amino acids, pyrimidine and purine bases.
Oligopeptides have a stimulatory effect on growth of some bacterial
strains (Chalmers, 1961; Allison, 1970). Amino acids are synthesized
in the presence of exogenous amino acids which usually will not exert a
feedback inhibition (Allison, 1970). Supplementary amino acids had no
stimulatory effect on microbial growth when rations contained 65% of the
total N as NPN (Allison, 1970). However, Maeng et al. (1976a)

obtained maximum microbial growth on purified media when 75 and 25%

of the total N was supplemented as urea and amino acids, respectively.
A mixture of 18 amino acids supported greater growth than the 10 EAA or
8 nonessential amino acids (NEAA). Branched chain amino acids pg£_§g_
had no effect on growth. At the optimum mixture of urea and amino
acids 53% of the amino acids were incorporated into microbial cells,
14% into carbon dioxide and volatile fatty acids, and 33% remained

in the supernatant.

Activation of Ammonia.—-The ammonium ion (NHZ) is a substrate

 

for a major fixation enzyme (Glutamic dehydrogenase), and is the pre-

dominant form of nitrogen in near neutral solutions (Allison, 1970).

+

4
(Blackburn, 1965).

Moreover, NH is not absorbed as well across the rumen wall as is NH

3

The functional biochemical pathways in amino acid biosynthesis

by rumen bacteria are relatively unknown (Allison, 1970). Erfle et a1.

10

(1977) found that ammonia activation in mixed rumen bacteria is via

glutamine synthetase (Glu + NH. + ATP I GRn + ADP + Pi) and asparagine

3

synthetase (Asp + NH + ATP I Asn + AMP + PPi) when the ammonia concen-

3
tration is low (Km 1.8 and 4, respectively). At high ammonia concen-
trations, the activation is via glutamate dehydrogenase (a - KG + NH: +
NAD(P)H I Glu + NAD(P)+; Km 33). They concluded that rumen micro-
organisms have a glutamate synthesizing system which is more efficient

than in some nonrumen bacteria, and that the ammonium ion concentration

controls amino acid anabolism.

Nutritive Value of Microbial Protein.--Rumen bacteria contain

 

74 to 80% of their total N in the form of proteins, peptides or free
amino acids. Nucleic acids account for about 15% of the total N
(Smith, 1969; Allison, 1970; Smith and McAllan, 1970). The diversity
of the bacterial population makes it difficult to produce significant
changes in the quality of bacterial protein leaving the rumen, even
when there are rather large changes in the ration (Chalmers, 1961;
Bergen et al., 1968b; Allison, 1970; Fenderson and Bergen, 1972;
Chalupa, 1973; Burris et al., 1974). The biological value of the
mixture of rumen bacteria and protozoa is not affected by protein
source and is only slightly lower than for casein (80 to 85 versus 90)
(McNaught et al., 1954; Chalmers, 1961; Bergen et al., 1968a). Net
protein utilization by rats for bacteria, protozoa and casein were

75, 87, and 97% (Bergen et al., 1968a). The limiting amino acids in
bacterial protein compared with egg protein are methionine and iso-
leucine (Chalmers, 1961). The true digestibility of bacterial protein

was 76% (Hogan and Weston, 1970).

11

The Importance of Energy and Ammonia Nitrogen for Optional

Microbial Growth.--In continuous fermentation systems, the maximum

 

microbial cell yields depend on retention time, production of ATP
potentially formed during fermentation, and the ammonia nitrogen
available (Balch, 1961; Lewis, 1961; Hungate, 1965; Hogan and Weston,
1970; Al-Rabbat et al., 1971; Hogan, 1975; Satter and Roffler, 1975).
Microbial yield in ruminants is maximized when the cells
remain in the fermentation system 2 to 5 hours and is greatly depressed
for longer and shorter times (Hogan, 1975). This length of time is
sufficient for reproduction and development, and insures removal
before microbes enter the lag phase of the logistic pOpulation growth
curve and start to use substrate for maintenance and autolyses (Hogan,
1975). The microbial yield of dry cells per mole ATP (YATP) may change

when the dilution rate is increased from .1 h-1

to .3 to .5 h"1 (Hogan
and Weston, 1970). Determining factors in the movement of digesta
out of the omasum are feed intake, degree of fermentation, specific
gravity of particles in the rumen and rumen motility (Phillipson and
Ash, 1965; Balch and Campling, 1965). During an increased rate of
passage a decreased digestion of cellulose in the rumen may be com-
pensated for by an increased fermentation in the hind gut (Singleton,
1972).

During anaerobic fermentation energy is usually the primary
factor which determines microbial cell yields (Allison, 1965, 1970;

Al-Rabbat et al., 1971). Hungate (1965) found a Y yield of 10.5%

ATP
and suggested it was constant. Hogan and Weston (1970) found that

the YATP varies with bacterial species, substrate supply and dilution

rate. Recent in vivo estimates of microbial N flow to the abomasum

12

were summarized by Allen and Miller (1976) at approximately 32 g per
kg fermentable organic matter.

Rumen microorganisms have a limited capacity for ammonia
utilization, but a minimal rumen ammonia concentration is critical
for the microbial yield. When the rate of ammonia production exceeds
that of utilization by bacteria or when there is excessive NPN in the
diet, rumen ammonia accumulates without benefiting the host animal
(Smith, 1969; Allison, 1970; Conrad, 1972; Satter and Roffler, 1975).

In pure batch cultures the cell crop is proportional to the
ammonia concentration between 0.7 and 5.6 mg per 100 ml of fluid
(Allison, 1970). In continuous cultures ammonia is limiting below
5 to 6.4 mg per 100 ml (Allison, 1970; Satter and Slyter, 1972).

Bull et a1. (1975) obtained maximal protein synthesis at ammonia con-
centrations in excess of 12.6 mg per 100 m1 fluid. Above approxi-
mately 5 mg per 100 ml ammonia accumulates linearly with the supple-
mentary level of N, but without effect on the protein content of the
fermentor effluent. Rumen ammonia concentrations of 83 mg per 100 ml
had no adverse effect on cell yield (Chalupa, 1973; Satter and Slyter,
1972). In sheep, maximum rumen nonammonia nitrogen was obtained at
8.8 mg per 100 ml, but abomasal nonammonia nitrogen was not maximized
until rumen ammonia reached 13.3 to 16 mg per 100 ml (Hume et al.,

1970; Allen and Miller, 1976).

 

1.3 Utilization of nonprotein nitrogen
in dairy Cattle rations

 

Progress in NPN utilization by dairy cattle has recently been
reviewed by Burroughs et al. (1975a, b), Huber (1975), and Satter and

Roffler (1975). There is general agreement that NPN is well utilized

13

in dairy cows producing less than 20 kg milk per day. Cows producing
up to 20 kg milk on corn, corn silage, limited hay rations of 12 to
13% CP will maintain milk yields with all the supplementary N as NPN.
However, there are major disagreements concerning the use of NPN in
rations of cows producing more than 20 kg milk per day.

Burroughs et al. (1975a) and Satter and Roffler (1975) con-
cluded that NPN supplementation of dairy rations is without value for
cows producing more than 20 kg milk. Burroughs et al. (1975a, b)
calculated an imbalanced supply of absorbable amino acids from rations
supplemented with NPN. Satter and Roffler (1975) based their con-
clusion upon zero utilization of rumen ammonia when the ration CP
content exceeds 12 to 13%. Their conclusions were mainly based upon
theoretical considerations.

Burroughs et a1. (1973, 1974a, b, 1975a, b) termed a urea
fermentation potential (UFP) from estimates of metabolizable protein
(MP) and metabolizable amino acids (MAA). The above system for cal-
culating requirements was introduced as one which conforms more
closely with digestible protein in nonruminants than apparently
digestible protein and total protein (N x 6,25) for ruminants.
Metabolizable protein and MAA are defined as the quantity of protein
digested or amino acids absorbed in the postruminal portion of the
digestive tract of cattle and other ruminants. The UFP is defined as
the amount of urea that can be useful in any given cattle ration.

Calculation of MP, MAA, and UFP are by the equations in (l), (2), and

(3):

14

(1) MP = (91 x .90) + ([132 - 15.0] x .80)
(2) MAA = (.9 P1 x AA%P1)/100 + ([.8 92 - 12.0] x AA%P2)
(3) UFP = (1.04 TDN - P3)/2.8

where P1 is grams of undegraded a e amino protein entering the abomasum
from 1 kg feed DM; P2 is grams of abomasal microbial protein limited
to grams of rumen degraded protein and 1.04 times the grams of TDN in

1 kg feed DM; P is total grams of rumen degraded protein per kg feed

3
DM whose ammonia contributed to the total rumen pool; 15.0 is abomasal
protein required to satisfy the metabolic fecal protein of body origin;
.90 is the fraction of undegraded protein truly digested postruminally;
and .80 is the fraction of microbial protein truly digested postruminally.
The advantage of this system over those previously used is that it
recognizes that degradation of natural protein and synthesis of
microbial proteins in the rumen are variable. At the same time the
system avoids several potentially false assumptions. These are:

(a) Absorbable amino acids arise from that portion of the ration protein
undergoing ruminal destruction without resynthesis into microbial
protein. (b) That all the urea is utilized for protein synthesis in

the rumen when the ration has insufficient fermentable energy for
transforming the urea into microbial protein. Satter and Roffler (1975)
recognized the significant improvements in recommendations and feed
evaluations introduced by the MP-system compared to the unqualified

use of crude protein (N x 6.26). However, they found it had the dis-
advantage of being cumbersome and difficult to understand by livestock

producers. This led to an alternative method of calculating

15

metabolizable protein where more accurate information may be readily
accommodated as it becomes available. The assumptions of this system
are: (a) The amount of nitrogen recycled into the rumen-reticulum is
equal to 12% of the total dietary nitrogen intake (DP). (b) Eighty-
five percent of the dietary nitrogen intake in typical ruminant rations
supplemented with protein is true protein (TP) and the other 15% is

NPN from natural sources. (c) Forty percent of the dietary true protein
escapes degradation in the rumen and goes to the intestine while all

of the dietary NPN and recycled nitrogen passes through the ruminal
ammonia pool. (d) Ninety percent of all rumen ammonia N (RAN) pro-
duced is incorporated into microbial nitrogen when the ration fed

does not exceed the "upper limit" value for crude protein. (e) None
of the ruminal ammonia derived from dietary CP fed in excess of the
upper limit value for dietary crude protein is incorporated into
microbial nitrogen. (f) Eighty percent of microbial nitrogen is true
protein and 20% is nonutilizable NPN. (g) Eighty percent of the
microbial true protein will be absorbed. (h) Eighty-seven percent of
the dietary protein that escapes degradation in the rumen will be
absorbed. For rations where the rumen ammonia does not exceed the

critical value the calculation is as equation (4):

(4) MP = [DP x .85 x .40 x .87] + [(DP x .15) + (NPN) + (TP x .12) +

(DP x .85 x .60)] x .90 x .80 x .80

When the feed contains CP which will lead to rumen ammonia concen-
trations in excess of the critical value, these alterations are
involved. Metabolizable protein is calculated for CP less than the

critical value and the remaining N has to be supplemented as true

16

protein with a utilization of 30% (0.85 x 0.40 x 0.87). Satter and
Roffler (1975) found that expected RAN concentrations depend upon the

CP and TDN content in the total DM as in (5):

(5) RAN = 38.73 — 3.04 09 + .171 092 - .49 TDN + .0024 TDN2

By rearrangement of equation (5) the critical GP for zero utilization
of RAN may be calculated as in (6) and (7) when the critical RAN

value as well as TDN are given.

 

7 9
(6) cp = -(3.04) 1 7(3.04)“ - 4(-.171)(.49T0§_;.0024rnn‘ + RAN - 38.73)

 

 

 

2 x (-.171)
The formula in (6) may be reduced to (7):
(7) cp = 8.89 1 79.24 - (-.684)(.49T0N;.0024Tfifiz‘1'fixfi‘i“387737
(-.342)

The general concepts of the systems are improvements on the
system presently in use but some drawbacks for direct implementation
are evident. The major drawback to the system described and developed
by Burroughs et a1. (1973, 1974a, b, 1975a, b) is in establishing the
MP value of feeds (Bartley, 1976). Undegraded protein reaching the
abomasum is estimated for each feedstuff and is presently based on
limited information. Furthermore, the value will depend on the com-
position of the ration, its physical form, processing, treatment,
feeding rate, etc. (Bartley, 1976). Concerning the UFP, no con-
sideration is given to the rate of energy release and whether or not
this coincides with the release of ammonia (Bartley, 1976). The most
critical assumption in the method by Satter and Roffler (1975) is the

point of zero utilization of rumen ammonia. They use the value of 5 mg

17

per 100 ml established by in vitro methods (Satter and Slyter, 1972).
From (7) it may be calculated that a complete ration containing 75%
TDN has a critical value of 13.1%. If RAN can still be utilized for
microbial growth when the concentration is increased to 8.8 as sug-
gested by Allison (1970) then the critical CP value would be 15.2%.
Using the 15.2% value, a ration with 50% of the DM as corn silage and
50% as grain would have TDN (in DM) close to 80%, and could support
as high milk production when supplemented with NPN as by true protein.
This could be expected if the rate of ammonia release from the NPN
were decreased by its addition to silage at ensiling or by use of
improved forms of NPN in the grain portion of the feed.

The addition of NPN (urea and ammonia) to corn silage has been
investigated by workers at Michigan State University (Huber, 1975).
In five trials, where the initial milk yields were 23 to 30 kg per day
and one trial where initial milk was 34 kg per day, milk yield per-
sistencies were as high or higher for NPN rations as for rations sup-
plemented with natural protein. In these trials no negative control
group was included, but the unsupplemented ration DM would have con-
tained less than 12% CP. Approximately 60% of the supplemental N in
the treatment rations came from NPN and the remainder from soybean
meal. This indicates utilization of NPN in rations containing more
than 12% which was the limit suggested by Burroughs et al. (1975a, b)
and Satter and Roffler (1975); or that high yielding cows require less
total feed protein than recommended by the National Research Council
(1971).

Roffler and Satter (1975) utilized previously published

experiments to estimate zero response to added NPN. Most of their

18

calculations for cows producing over 20 kg milk per day were based
upon raw data rather than on milk yields adjusted for pretreatment
values. When data were recalculated on the basis of adjusted treat-
ment means, and after deletion of groups where factors other than NPN
had a major effect on the response (silage DM > 40%, or an extremely
high mineral level in silage which depressed intake) the point of zero
response was changed from 12 to l4-l4.5% CP (Huber, 1976). Further,
Roffler and Satter (1975) did not calculate the response to natural
protein supplementation from data collected in the trials which showed
a response curve identical to the one obtained for NPN supplemented
rations (Figure 1). Based on these results Huber (1975, 1976) con-
cluded that NPN can be used as a N supplement at higher crude protein
levels than estimated by Burroughs et al. (1975a, b) and Satter and
Roffler (1975). The disagreement may, to some degree, be due to a
slower and more synchronous release of ammonia from NPN-treated
silages than from urea added to unprocessed grains. The importance of
synchronous release of ammonia and energy has not been included in
any of the suggested MP systems.

Polan et a1. (1976) fed cows corn silage grain-based rations
for 140 days starting after a 4 week adaptation period initiated 30
days post-partum. The rations contained 9.4 to 16.2% CP with 0 to
40% of the total N as urea, which was added in the grain portion of
the feed. They concluded that urea resulted in less efficient nitrogen
utilization than would be expected from equal N supplementation from
natural protein. Urea was advantageous at low CP concentrations, but
had a negative effect as dietary CP increased. In this trial the

initial milk yield was 28.1 kg milk per day and treatment milk was

19

1

4O

NATURAL
’ PROTEIN

 

\\ e
\ °—""° NPN

h)
(D

65

E3

GD

J5

 

. . . . ‘8 .

0 “T e 9 IO M I2 )3 I4
CRUDE PROTEIN IN DRY o \
MATTER BEFORE N ADDITION,°/o \

IMPROVEMENT IN MILK YIELD DUE TO ADDED NITROGEN, °/o

I
£5

P

 

-8I.

Figure 1. Milk response to added nitrogen from non-protein nitrogen (NPN) or
natural protein.

Natural protein: Y - 203.7435 - 27.7573x + .9156xg; R . .91

20

23.8 kg. The milk yield increased 2.58 kg per unit increase in CP,
and decreased .22 kg per unit of urea substituted for CP. Feed intake
was highest for 12.8% CP and decreased at lower and higher levels, and
. by inclusion of urea. In this trial mean rumen ammonia was 27.5 mg
per 100 ml at 2 to 3 hours post feeding. This indicates a rapid
hydrolysis of the total daily urea intake and may be related to rapid
consumption. The negative effects encountered by Polan et al. (1976)
when urea was fed in the grain were not observed by Gardner et a1.
(1975) and Huber et a1. (1976) when methods for more synchronous
release of ammonia and energy or a decreased rate of ammonia release
were implemented. Gardner et a1. (1975) found equal production in the
first 126 days of lactation when cows, initially producing 30 kg milk,
were fed rations containing 15.5% CP from rations to which the follow-
ing were added to the grain: 1.5% urea + 2% soybean meal; 7.7% heat
processed corn urea; or 14.8% soybean meal. Huber et a1. (1976)
observed equal milk persistencies when cows 130 days post calving,
and initially producing 23 kg milk per milk per day, were given soybean
meal, urea or fermented ammoniated whey as the only supplemental
protein added to grain which was mixed with corn silage. In this
trial the highest producers (28.8 kg initial milk) responded as
favorably to NPN as to soybean meal supplementation and the persis-
tencies were significantly higher than for negative control cows.
Aitchison et a1. (1976) studied the effect of protein solu-
bility on utilization in dairy cows using three latin square experi-
ments. The solubility was varied by adding urea at 0, 8, l6 and 24%
of N in grain and the effects were analyzed according to Mertens

(1975). They calculated that 81% of insoluble N was utilized, while

21

utilization of soluble N was 29.3, 30.5, and 56.7% in trials 1, 2, and
3. Apparently soluble N was utilized more efficiently in trial 3 which
also had the highest producers. The utilization of N was relatively
constant across CP contents (12, 13, and 15%) and is in disagreement
with the system proposed by Satter and Roffler (1975). This led
Aitchison et a1. (1976) to conclude that the utilization of soluble N
may depend more upon the wash out effect associated with dry matter and
water intake than upon C? content of the ration or production of the
cow. Furthermore, they suggested that the solubility of N in the non-
urea portion of the ration may be decreased by urea addition.

These recent findings (Gardner et al., 1975, Huber et al.,
1976, and Aitchison et al., 1976) indicate NPN utilization at higher
CP content and milk yields than suggested by metabolizable protein
systems (Satter and Roffler, 1975; Burroughs et al., 1975) or the trial
of Polan et a1. (1976). The contrasting results may be due to rate of
feed consumption or the rate at which ammonia and energy are released.

Huber (1975) reviewed the utilization of improved forms of NPN
and the results may be summarized as follows. Silages treated with
ammonia and urea NPN have increased lactic acid and water insoluble N,
and are more stable when exposed to air. The increased stability is
due to an antifungal action of ammonia. The higher lactic acid content
is due to buffering by the ammonia. The increased content of water-
insoluble N appears related to decreased proteolysis of the original
plant protein and increased synthesis of microbial protein. These
effects are more pronounced for ammonia than urea treated silages.

An additional benefit of using ammonia instead of urea in the treatment

22

of corn silage is that cows fed ammoniated corn silage can tolerate more
NPN in the concentrate (1.4 to 1.5%) without depressing milk yields or
feed intake (Huber et al., 1975).

Other methods of increased NPN utilization have been sought by
heat expansion of grain in the presence of urea (Starea) and by
pelleting urea with alfalfa meal, dicalcium phosphate, sodium sulfate,
and sodium propionate (Dehy-lOO). Both methods have improved urea
utilization when compared with unprocessed urea plus grain.

1.4 Summary and conclusion of literature
review on nitrogen utilization in
dairy cows.

Recent studies of the protein requirement in high yielding
dairy cows have not shown conclusive evidence of a need for increases
in protein beyond the amounts recommended by the National Research
Council (1971). Further research in this area should be conducted
under conditions of constant DM intake and net energy density of the
feed.

The ability of rumen microbes to utilize ammonia for amino
acid and protein biosynthesis and the ruminants ability to utilize
microbial protein has been well documented. Much has been learned
concerning the metabolism of NPN by the rumen microbes and the host
animal. However, the specific pathways of ammonia activation for amino
acid biosynthesis and factors controlling rumen ammonia utilization
are still in their infancy.

In dairy cattle, calculation of requirements on the basis of
total crude protein (N x 6.25) has been used extensively, but neither

crude protein nor apparently digestible protein are totally'satisfactory,

23

nor are they as useful as digestible protein in nonruminants. Meta-
bolizable protein, metabolizable amino acids and urea fermentation
potential have been introduced as alternative methods for ruminants.
These methods (as presently interpreted) allow extensive use of NPN in
cows producing up to 20 kg milk per day. However, certain assumptions
render them useless in high producing dairy cows.

The data base for these assumptions is very weak. Furthermore,
recent studies and reevaluation of these critical assumptions suggest
an urgent need to ascertain the usefulness of NPN in high producing
dairy cows early in lactation. Limited data on NPN utilization in
high yielding dairy cows is due mainly to the initiation of production
trials in mid lactation. Only very limited information is available
for cows in beginning of lactation when milk yields and dietary needs
are highest and intakes are low. Moreover, most studies are of too
short duration and the number of cows is too few for objective evalu-
ation of treatment responses.

2. Physiological and biochemical factors in the
synthesis ofinilk lactose and protein
and milk volume control
2.1 Composition of normal milk

In dairy cows the milk yield per day (the lactation curve)
increases rapidly after calving, peaks 30 to 60 days postpartum, and
then decreases at a rather constant rate throughout the remainder of
the lactation. The relationship between the concentration (percentage)
of the solids and time is the inverse of the lactation curve. The

shape and the level of the curves depend on breed and genotype, the

24

assumed restrictions of age (lactation number), season of the year,
feeding, housing, etc. (Rook, 1961; Touchberry, 1974). The averages
and standard deviations in Table 2 may be used as a guideline for nor-
mal cows, but the possible deviation from the "normal" is large.
Several herds produce more than twice these amounts (Touchberry, 1974),
and the record for one Holstein cow (milked twice daily) is 25,247 kg
milk and 713 kg fat in 365 days (Meadows, 1976). At peak yield of
89 kg milk, the cow consumed 30 kg hay, 30 kg of a grain mixture with
15% crude protein (CP), and 190 liters of water.

Milk fat is synthesized from acetate, B-hydroxy butyrate
(BHBA), and plasma free fatty acids. Milk fatty acids shorter than 16

carbon units (C16) and one half of the C fatty acids are synthesized

16

from acetate and BHBA. The remaining C fatty acids and those longer

16
than C have their origin in plasma triglycerides. Further details on

16
the synthesis of milk fat may be obtained from Bickerstaffe et a1.
(1974), Bauman and Davis (1974), Davis and Bauman (1974), and Jeness
(1974).
The ash content of milk is relatively constant. Details on

its composition and the content of vitamins and other constituents

may be obtained from Jeness (1974).

2.2 Lactose synthesis

Milk lactose is synthesized from glucose absorbed from blood
by the mammary gland and is an important determinant of milk volume
(Bickerstaffe et al., 1974; Davis and Bauman, 1974; Hardwick et al.,
1961; Hartman and Kronfeld, 1973; Lindsay, 1971; Linzell, 1968, 1974;

Scott et al., 1976).

25

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.AamA .xooz AmL LAAmA .aeAaaom AAL xaAaA .Aaaanaaaoe AALa

Aaoemaflx eaueaao

“moueum coufica

“mane mom :« poer>A=co oneness

 

 

 

 

 

 

 

 

 

e e
AmL I m.» A. m.a I n.m I a.» - I g: eaoanaoam
ANL I m.m A. m.a I m.m I m.m I I g:
AAL om. A~.m I I am. ow.m ea. AA.m AmNA mmae m: Aaaaan
ANL I A.w A. m.a I A.» I m.» I I x:
ALL AN. ma.m I I om. AA.» mm. oA.m mmLA wmoA m: awaaaAoz
ANL - L; A. A: I a: I 9m I I LS
ALL «A. Ao.a I I «A. No.m ca. Am.a Nam mmAa m: Aaaaaaau
AAL I m.m A. o.m I a.m I o.a I I 3:
ALL om. mm.m I I mm. mm.m on. LA.a 5mm comm m: aawam :zoam
ANL I o.m A. A.v I t.m I A.v I I ax: :
AAL an. Am.m I I am. am.m Am. mm.n mam Aamm am: aawcaAA<
.u.m .>< a 3 am .o.m .>< .o.m .>< .o.m .><
00 o o o . om
a c a m m emzm Ea<IoaoaL * .efiauoam A .uaa ma .axAA: e um

 

.mzoo :Hmeuocz new ummIco: moonm can :me .omouomH .ewouonm .umm mo ucoucoo use voAx wazII.N oAneh

26

2.2.1 Glucose metabolism by the mammary_g1and.

Glucose uptake.--Simultaneous measurements of arterial and

 

venous glucose concentrations indicates that the glucose uptake re-
mains approximately constant as percent of the arterial concentration
(extraction coefficient) between 30 mg glucose per 100 ml blood in
fasting goats and 75 mg% in high yielding dairy cows (Annison et al.,
1974; Bickerstaffe et al., 1974; Derring et al., 1974; Hartman and
Kronfeld, 1973; Kronfeld et al., 1968; Linzell, 1967a). The mammary
blood flow (MBF) varied from 168 to 540 liters per hour. In fasting
animals and those treated with insulin the glucose uptake decreased
due to changes in the arterial concentration. However, the mammary
blood flow was also changed (Linzell, 1967a, 1974), and will be dis-
cussed later. Breed differences for glucose uptake have not proven
significant (Bickerstaffe et al., 1974), but glucose extraction by

the mammary gland is less efficient in late lactation (Linzell, 1974),
indicating that something other than substrate availability is
involved. The mechanism has not been clarified but progesterone and
estrogen have diabetogenic effects and lead to decreased glucose utili-
zation by maternal tissue (Lindsay, 1971). Glucocorticoids (dexame-
thasone) impair the mammary extraction of glucose even at hypergly-
cemic conditions and lead to redistribution of glucose to other tissues
and a decreased milk production (Hartman and Kronfeld, 1973). Hartman
and Kronfeld (1973) found that the decrease in milk yield was linearly
related to the pre-treated milk yield, and the cows returned to pre-
treatment production levels within five days of injection. Insulin

injection or infusion decreased the milk yield in cows and goats

27

(Linzell, 1967b; Kronfeld et al., 1963), but had no effect when the

animals received simultaneous intravenous infusions of glucose. These
results suggest that glucocorticoids, and maybe estrogen and progeste-
rone, have a direct effect on glucose extraction by the mammary gland

whereas insulin acts through arterial glucose concentrations.

Glucose entry rate into circulation and mammary gland upgake.--

 

The rate of glucose entry into the circulation in cows is of the order
of 3.3 to 4.0 kg per day and was highest in cows fed a high starch,
low roughage ration (Annison et al., 1974; Bickerstaffe et al., 1974).
In comparison, entry rates for acetate and plasma free fatty acids were
1.7 to 2.1 and 0.5 kg per day, respectively (Bickerstaffe et al., 1974).
The amount of glucose oxidized equals 4 to 11% of the total carbon
dioxide production and was again highest in cows fed a high starch,
low roughage ration (Bickerstaffe et al., 1974). Correspondingly,
acetate accounted for 32 to 50% of the carbon dioxide production
(Annison et al., 1974).

Of the total glucose entering the metabolic pool 60 to 90%
is metabolized by the mammary gland in goats and cows (Annison and
Linzell, 1964; Lindsay, 1971). The fraction of glucose taken up by
the mammary gland in goats and Jersey cows is lower than in Holstein
cows (Lindsay, 1971). These values are in good agreement with glucose
uptake in cows (Kronfeld et al., 1968; Annison and Linzell, 1964;

Hartman and Kronfeld, 1973).

Glucose metabolism within the mammary gland.--Within the

 

mammary gland, absorbed glucose is either phosphorylated to

28

g1ucose-6-phosphate or remains as free glucose (Davis and Bauman,
1974). Phosphorylation of glucose to g1ucose-6-phosphate is by the

low Km hexokinase (Davis and Bauman, 1974). The high Km glucokinase

is not fbund in mammary tissue suggesting that the mammary gland does
not experience high glucose concentrations. Hexose phosphates and free
glucose are not in equilibrium due to the low activity or absence of
g1ucose-6-phosphatase. Hence, the chance for glucose—é-phosphate being
changed back to glucose is eliminated (Davis and Bauman, 1974).

Glucose-6—phosphate can be metabolized via three routes: (1)
conversion to UDP-galactose via glucose-l-phosphate and then combine
with free glucose and form lactose, (2) generate triose phosphates
via fructose-o-phosphate and the glycolytic pathway, and (3) oxidation
via 6-phosphogluconate and the pentose—phosphate pathway with genera-
tion of NADPH for fatty acid synthesis (Bauman and Davis, 1974; Davis
and Bauman, 1974; Lindsay, 1971; Smith, 1971).

Smith (1971) hypothesized that glucose oxidation is limited to
NADPH generation via the pentose—phosphate pathway and that the only
other use of glucose, except for lactose synthesis, is to replace
intermediates removed from the tricarboxylic acid cycle by NEAA
synthesis.

Some of the galactose moity in lactose may be derived from
sources other than glucose (Lindsay, 1971). One is triose phosphates
which appear to recombine by the action of fructose-l, 6~diphosphatase
and pyruvate carboxykinase (Davis and Bauman, 1974). Also, some
glycerol was incorporated into lactose by in vitro incubation

of cow mammary gland; but other gluconeoganic substrates

29

(alanine, glutamate, lactate, pyruvate) were not used at either optimal
or suboptimal glucose levels (Scott et al., 1976). In goats, some
gluconeogenesis from glutamate carbon took place within the mammary
gland (Mepham and Linzell, 1974). Incorporation of gluconeogenic sub-
strates into glucose, regardless of the glucose concentration, is con-
sistent with the absence of glucose-6-phosphatase activity and the
lack of excess gluconeogenic substrate in the mammary gland (Scott et
al., 1976). The activities of pyruvate carboxylase, phosphoenol car-
boxykinase and fructose-l, 6-diphosphatese-are lower in the mammary
gland than in the liver (Scott et al., 1976).

Glucose does not contribute carbon units to fatty acid syn-
thesis in the bovine mammary gland due to low activities or the
absence of ATP, citrate, lyase and malate dehydrogenase (Smith, 1971).

Lactose biosynthesis accounted for 60 to 75%, oxidation for 20
to 30%, glycerol formation for 5%, and NEAA synthesis for 5% of the
absorbed glucose (Armstrong and Prescott, 1971; Annison and Linzell,
1964; Davis and Bauman, 1974; Lindsay, 1971; Linzell, 1974; Smith,
1971). Further, glucose accounts for 50% of the glycerol and citrate,
respectively (Linzell, 1974), and of the glucose entering the glucose-
6-phosphate pool 50 to 60% is used for lactose synthesis (Davis and
Bauman, 1974). In the goat the amount of glucose and acetate re-
maining after oxidation can account for all the lactose, all the gly-
cerol, and 17 to 45% of the fatty acids in milk fat (Annison and
Linzell, 1964). The NAD+:NADH ratio has been suggested as a major
factor regulating the metabolism of glucose via the glycolytic pathway

(Davis and Bauman, 1974). Mannose will not substitute for glucose in

30

lactose biosynthesis, but does stimulate oxygen consumption and
fatty acid synthesis from acetate to about the same extent as glucose

(Davis and Bauman, 1974).

 

2.2.2 Lactose synthetase

' The substrates for lactose biosynthesis are free glucose and
UDP-galactose, and the reaction is catalyzed by lactose synthetase.
The action of lactose synthetase has been reviewed by Ebner and
Schanbacker (1974), and Whitney et al. (1976), and is summarized below.

Lactose synthetase is a combination of the enzyme galactosyl-

transferase (A—protein) and the protein a — lactalbumin (a-LA;
B-protein). Galactosyltransferase is a nonspecific enzyme located in
the wall of the Golgi apparatus and is involved in sequential addition
of carbohydrates to an acceptor. It requires Mn2+, but is not affected
by Migz+ and Ca2+. The intracellular location of a-LA is less well
known, but it is probably synthesized on the ribosomes under strict
hormonal control. In the absence of a-LA, galactosyltransferase ads
galactose residues to the side chain of glycoproteins, and glucose is
not used as a galactosyl acceptor due to a high Km (1.4 M) for glucose.
During lactation a-LA is believed to pass into the tubules and vesicles
of the Golgi apparatus and form a short lived kinetic complex with
galactosyltransferase. The complex formation lowers the Km for glucose
(to 5 mM), and UDP-galactose is transferred to glucose instead of
N-acetylglucosamine. The dissociation of a-LA from galactosyltrans-
ferase occurs prior to product release. It is expected that a single
molecule of a-LA can interact with several molecules of galactosyl—

transferase as it passes through the Golgi apparatus and that control

31

of lactose biosynthesis is directly related to the concentration and
rate of flow of a-LA through the Golgi apparatus. The function of the
system assumes that the substrates, UDP—galactose and glucose, as well
as the UDP are permeable to the Golgi membrane whereas lactose is not.
Lactose synthetase activity in the mammary gland of dairy cows
is negligible 30 days pre-partum, increases to measurable levels by 7
days pre-partum and reaches an activity 2.5 to 4 times higher than at
7 days pre-partum between 7 days pre- and 40 days post-partum
(Mellenberger et al., 1973). The change in lactose synthetase activity
may now be related to a-LA synthesis. Even though this mechanism
appears to be wasteful, it is very effective and regulated via protein

synthesis suggested to be under normal hormonal control.

2.3 Protein synthesis

2.3.1 Protein fractions

 

Crude protein (Kjeldahl N x 6.38) in milk consists of appro-
ximately 95% true protein and 5% non-protein nitrogen (Larson and
Gillespie, 1957; Yousef et al., 1970). The major part of NPN in milk
is as free amino acids. True protein in milk consists of a-,K-,B-, and
6-casein (casein fraction) and the whey proteins; B—lactoglobulin
(B-LG), a-lactalbumin (a-LA), serum albumin (SA), immunoglobulins (1g)
and proteosepentones (PP) (Thompson and Farrell, 1974; Whitney et al.,
1976). The distribution of proteins in the various fractions is given

in Table 3.

32

 

nmmfi

 

 

 

 

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onmfi

..Hm um xocuwnz -- s: n. n.m m NH m.~ 5 mm ma mm aaawxmz
onmfi

..Hm go socumnz -- -- N. m.~ N A A. m mm m me asaaeaz

m
.aa um <a-a ustm <m <oso <u-m <u-y <0- 5
monogamom zmz :onOHm
Hocwz mcflouonm xoaz mcwomau

 

.:flopoum xflwa afixm 3mm :w :owuaafiaumwv mmmucoonom Hausa cam xafia m.3ou mo mcfiouonm onbnn.m oHpab

33

Caseins (CA) are phosphoproteins precipitated from raw skim
milk by acidification to pH 4.6 at 20 C. Whey proteins are those
remaining after CA precipitation (Whitney et al., 1976).

The caseins, B-LG and a-LA are homogeneous proteins synthesized
in the mammary gland from a pool of free amino acids in equilibrium
with the pool of free amino acids in the blood stream, and comprise
over 90% of the milk proteins (Larson and Gillespie, 1957; Linzell,
1968; Larson and Jorgensen, 1974; Davis and Bauman, 1974). Their
amino acid sequence has been determined and compositions are given in
Table 4. Genetic variants exist for all of these proteins. Their
frequency in Holstein cows, and related changes in the amino acid
sequences are given in Tables 5 and 6. Whether 6-CA is synthesized
in the mammary gland is still unknow, but the evidence suggests that
it is derived from B-CA by proteolysis (Jeness, 1974; Whitney et al.,
1976; Thompson and Farrell, 1974). Serum albumin, Ig and PP are
heterogeneous proteins and originate from the blood (Larson and
Jorgensen, 1974).

The evidence suggests that the amino acid composition of milk
protein remains constant, except for the presence of genetic variants
and variation in individual protein fractions. In cows fed rations
containing 9 to 18% protected protein the amino acid composition of

milk protein was unaltered (Broderick et al., 1974).

2.3.2 Amino acid metabolism by the mammary gland
The precursors of synthesized proteins are from a pool of free
amino acids in balance with the pool of free amino acids in blood, as

previously described. In dry cows the arterio-venous differences (A-V)

34

Table 4.--Amino acid Composition of bovine milk protein (after Whitney et al., 1976).

 

 

 

 

 

 

 

 

 

 

Casein (78-85‘) Whey Proteins‘l
6 (3.7%) 15-223
ms at B 6. 6, 6, SA B-LG 4-1.11 13 pp
4,9, 11,11 A; A} A; A} A} A: 4.3. A 3,c, 11,3.
Genetic s,c. A, a, A, n. a 0.11:1
variants. B 0,
nor E, C.
fractions as. 032 B-CA B-CA B—CA
6.. a... 29-209 106-209 108-209
ass
i of skim
.111: prot. 45.55 645 25-35 3-74 .7 7-12 2-5 1.9- .2-
Primary L 1.3 3.3 .3
structure- c
genetic 051-! I A A B
variance
Arg 6" 5 4 2 2 2 ... 3 1
111: s 3 s s 4 3 g 2 3
lie 11 13 1o 7 3 3 n 10 6
Lou 17 a 22 19 14 14 5 22 13
Lys 14 9 11 1 4 3 .. 1s 12
Met 5 2 6 6 4 4 n 4 1
an. s 4 9 9 s 5 ° 4 4
1'hr s 14 9 a 4 4 S 8 7
Trp 2 1 1 1 1 1 §_ 2 4
v.1 11 11 19 17 10 10 a 10 6
0
Ala 9 15 s 5 2 2 a 14 3
Asn a 7 s 3 1 1 s 12
Asp 7 4 4 4 2 2 L'. 11 9
Cys (1/2) -- 2 -- -- -- -- E s a
Gln 14 14b 22 22 11 11 a 9 s
Glu 25 13 17 1o 4 4 16 6
Gly 9 2 s 4 2 2 3 6
Pro 17 20 35 34 21 21 a 2
Set + SerP 8+8 12+l ll+5 l0+l 7+0 7+0 7+0 7+0
Tyr 10 9 4 4 3 3 4 4
Totll M-ml 199 169 209 131 104 102 162 123
1m:ca1cu- 23,613 19,005 23,930 18,362 14,174
' lated

 

‘Number of molecules per protein molecule.
bhereof l Pyroglu.
°Not point mutation

dSA is serum albumins. LG is lactoglobulin, LA is lactalbumin, lg is immunoglobulin.
and PP is proteones-pentones.

35

Table S.--Gene frequency of milk protein variants in Holstein cows8
(after Thompson 6 Farrell, 1974).

 

 

 

Casein
Variant

051 K 8 6 o-LA B-LG SA Ig
A 08 + .98 + + + - -
B .87 + .02 + + + - -
C .05 - 0 - - + - -
D + - 0 - - + .. -
Dr - - + - - + - -

 

aAbbreviations: a-LA a a-Lactalbumin, B-LG = B-Lactoglobulin,
SA = Bovine serum albumin, Ig = Immunoglobulin, + - found,
- = not found in dairy cattle.

36

Table 6.--Amino acid changes due to genetic variants (after Whitney
et al., 1976);

 

Residue Change

 

 

 

Protein Genetic Variant Change Amino Acid Number
as- CA B + A 13AA missing 14 to 26
C Glu + Gly 192
D Ala + Thr-P 53
K - CA B + A Lie + Thr 136
Ala + Asp 148
B - CA A; + A; Pro + His 67
A2 +* A His + Glu 106
A + B Pro + His 67
2 Ser + Arg 122
A + C Pro + His 67
Glu + Lys 37
2 Ser-P + Ser 35
A2 + E Glu + Lys 36
A 1+ 8 -Not available. Found in 305 Indicus only.
D -Not available. Found in 805 Indicus only.
8 - LG A + B Val +Ala 118
Asp_+ Gly 64
A D Glu + Gly 51
Asp + Gly 64
Val + Ala 118
A + Dr. AA composition as A
+ carbohydrate moieties
attached ratio
N-acetylneuraminie acid 1
glucosamine 3.4
galactosamine .9
mannose 1.9
galactose .8
a - LA 8 + A Arg‘+ Gln 10

 

37

for amino acids are negligible (Verbeke and Peeters, 1965), but in the
lactating cow, goat and sow the total uptake of N as amino acids is
sufficient to provide for all of the N of milk proteins synthesized by
the mammary gland (Mepham and Linzell, 1966; Linzell, 1968, 1974;
Bickerstaffe et al., 1974; Davis and Bauman, 1974; Yousef et al., 1970;

Verbeke and Peeters, 1965).

Uptake of amino acids.--In fasted goats the A-V for a-amino

 

acid N decreases as a percent of the arterial concentration, when
compared to fed goats (Linzell, 1967a). This led to a decrease in
actual uptake, which was further exaggerated by a decrease in MBF.

In goats given arterial infusions of amino acids the uptake was
increased as were milk and protein yields (Linzell and Mepham, 1974).
Milk protein yields were higher in cows fed a high concentrate, low
roughage ratio compared to a normal ration, but the A-V fbr a-amino
acid N were unchanged (Yousef et al., 1970).

Considerable differences exist for the uptake of individual
amino acids (Clark et al., 1974; Derring et al., 1974; Bickerstaffe
et al., 1974; Verbeke and Peeters, 1965; Mepham and Linzell, 1966;
Linzell and Mepham, 1974), and the uptake as a percentage of the
arterial concentration do not appear to be related (Figure 2). Some
of the reported differences may be related to the stage of lactation.
Chandler and Polan (1972) estimated that the extraction coefficients
decrease with decreasing milk yield when the MBF to milk ratio was
considered constant.

The uptake of BAA is in good agreement with the output of

individual BAA in milk and the extraction coefficients are high

 

38

 

 

 

 

 

 

 

 

 

100 ' Arginine 100 F Histidine
80 b 80 r-
60 r 0 60
:z 1 :' 0 F
9.40 r . 4o . .
[— ’ . e. .‘e
' e
520 - 20 - . ° "
8 e
5 1 1 J 1 J 1 L
U 1 2 3 4 5 1 2
1-3
5
a:
a:
an
310“ Isoleucine 100 F' Leucine
8
a, 8C3 80 p
m“
6C. 60 b .
§ 0 as e . g . .
E40 P e . . .. e 40 F p e ..e: e
u.
h! 8
1:
£20 1- 20 -
E 1 4 1 L a J J
>»
c') 1 2 1 3 4 5
H
E
E
10 Lysine 10 Methionine
8 8 .
O. . .
6 .3: 6 -, - :
4 . ° . 4
2 2
*1 l l l _] 1 l L L l
l 2 3 4 S l .2 .3 .4 .5
ARTERIAL CONCENTRATION, mg/lOOml
Figure 2. Arteriovenous differences for amino acids as a percentage

of the arterial concentration (adapted from Clark et al.
1972; Derring et a1. 1974; Bickerstaffe et a1. 1974;
Verbeke and Peeters 1965; Linzell and Mepham 1974a;
Mepham and Linzell 1966).

% OF ARTERIAL CONCENTRATION

ARTERIO—VENOUS DIFFERENCE,

10 Phenylalanine

 

 

 

 

8
6
e . .
4 e:e. 2..
2
l l
1.0 2.0
100 ' Valine
80 i-
60 R
40 _
.0 : .0 . ..
20 r- O O
1 1 1 l
l 2 3 4
10 Asparagine
8
6
4 e
2
L l
1.0 2.0
Figure 2. (cont.)

39

 

 

 

 

10%" Threonine
80.
6q. .
4..
d . . :-
20-
1 1
1:0 2(0'
10 Alanine
8
6
4
2 '.
e .e. .'
0 Cl 4
2.0 3.0
10 Aspartic acid
8
6 O
4 e
O ..
2 " '
1 1 1 EL .14
1 .2 3 .4 .4

100

80

60

40

20

100

80

6O

40

20

ARTERIO-VENOUS DIFFERENCE, % OF ARTERIAL CONCENTRATION

100 '

80

60

40

20

p

 

Cysteine

 

 

O‘P

Glutamine acid

 

 

1.0

Proline

1.0

Figure 2 (cont.).

2.0

1
2.0

AERTIAL CONCENTRATION, ng/lOO m1

.0

40

 

 

 

 

100 Glutamine
SG
60
4Q
20 . .
1 1 1, 1
l 2 3 4
10 Glycine
8
6
4
2
O
4"] I:l. l
2 4
10 Serine
8
6 O
4 . ’0
O
O O
2 e0 0
1 1 1 1
1.0 2.0

OF ARTERIAL CONCENTRATION

o
/o

ARTERIO-VENOUS DIFFERENCE,

100

80

6O

4O

20

100

80

60

4O

20

 

41

 

 

 

 

 

 

P Tyrosine 10oF Ornithine
L. sq.
. ' 60. -
. O ... O . .

I- . .. 40p .
L ° 20.

1 11 11 1

1.0 2.0 1.0 2.0
- Cittrulline
r
L.

.I. . J I

1 0 2.0

Figure

ARTERIAL CONCENTRATION, mg/100 ml

2. (cont.).

42

(Mepham and Linzell, 1966; Bickerstaffe et al., 1974; Derring et al.,
1974). Exceptions are arginine and valine whose uptake exceed the out-
put in milk. Ornithine and citrulline are extracted by the mammary
gland even though they are not found in milk protein (Mephan and
Linzell, 1966; Bickerstaffe et al., 1974; Verbeke and Peeters, 1965;
Derring et al., 1974; Clark et al., 1975).

The uptake of most NEAA cannot account for the respective con-
centrations in milk protein (Verbeke and Peeters, 1965; Mephan and
Linzell, 1966; Linzell, 1968; Bickerstaffe et al., 1974; Derring et
al., 1974; Clark et al., 1975). Data suggest that the mammary gland

has a capacity for NEAA anabolism (Verbeke and Peeters, 1965).

Amino Acid Catabolism and Anabolism by the Mammary Gland.--
Arginine extracted in excess of the output in milk protein is con-
verted to ornithine and urea, and the ornithine produced along with
that absorbed from the blood form a portion of the proline pool
(Mephan and Linzell, 1967; Linzell, 1968; Verbeke et al., 1968;
Derring et al., 1973; Clark et al., 1975; Davis and Bauman, 1974).
Arginine and ornithine also add to the pools of aspartate, glutamate,
urea and citrulline, but arginine is not formed from ornithine. It
was suggested that proline and glutamate are formed from ornithine via
an incomplete urea cycle (no argininosuccinate synthetase activity).
Verbeke et a1. (1968) found that 13 to 9% of the protein in casein
had its origin in plasma ornithine and arginine, respectively. Mepham
and Linzell (1967) suggested that 14% of the carbon in arginine is

transferred to urea.

43

Leucine was to a large extent incorporated in milk protein in
perfusion studies with DL[l-14C]-leucine. Catabolism of leucine was
equal to 1% of the recovered carbon dioxide (Verbeke et al., 1959).

Increased plasma levels of methionine could not overcome the
mammary glands requirement for cysteine (Davis and Bauman, 1974). The
low activity or absence of enzymes for the conversion of methionine to
cysteine is in agreement with the uptake: output ratios for cysteine
and methionine, which are high and low, respectively.

An active phenylalanine hydroxylase in the mammary gland con-
verts phenylalanine to tyrosine, and the rate of conversion was
increased 30 fold when the plasma phenylalanine concentration was
increased 4 times (Verbeke et al., 1972; Davis and Bauman, 1974).

Phynylalanine infusion did not lead to any production of C0 lactose

2.
and citric acid. This indicates that phenylalanine is not catabolized
by the mammary gland (Verbeke et al., 1972).

Threonine aldolase activity is found in the mammary gland and
threonine can add to the pools of glycine, glutamate and aspartate.

An amount equal to 0.4% of the expired CO was catabolized (Verbeke

2
et al., 1972).

Excess valine is converted to alanine, aspartate, glutamate
and glycine. This suggests it is metabolized via succinyl-coenzyme A
(Derring et al., 1973).

Alanine, asparagine, aspartate, glutamine, glutamate, glycine
and serine appear to be formed by transamination and amination from

glucose carbon with excesses of other amino acids as the nonspecific

nitrogen source (Linzell, 1968; Linzell and Mepham, 1968; Davis and

44

Bauman, 1974). In the goat mammary gland acetate was also a carbon
source, but its contribution to NEAA was insignificant (Linzell, 1968).
Isovalerate can contribute to the pools of glutamate and asparatate
(Verbeke et al., 1959). Smith (1971) concluded that milk protein
synthesis requires 4% of the absorbed glucose via the tricarboxylic
acid cycle, if allowance is made for NEAA synthesis from excess
arginine and ornithine.

Net synthesis of amino acids within the mammary gland has not
been established (Davis and Bauman, 1974). The rate of transmission
and amination of precursors appears to be affected by precursor-uptake
in relation to the output in milk; also by the degree to which the
precursor is used for systhesis of other amino acids (Davis and
Bauman, 1974).

This discussion of the amino acid metabolism suggests that the
catabolism of amino acids and the anabolism of NEAA follow known
pathways for amino acid metabolism in eucaryotic cells, except for the
incompleteness of the urea cycle and the apparent lack of a pathway to

convert methionine to cysteine.

2.3.3 Protein synthesis

 

The overall mechanism of protein synthesis in the mammary
gland is similar to the general pathways believed to exist in cells
in all organisms (Larson and Jorgensen, 1974; Keenan et al., 1974).
The site of polypeptide chain synthesis appears to be the ribosomes
of the rough endoplasmic reticulum (RER), with subsequent transport
through the passages of the RER to the Golgi region, and concentration

in the vacuoles (Larson and Jorgensen, 1974; Saache and Heald, 1974;

45

Topper and Oka, 1974). The addition of prostetic groups (phosphate
groups of CA and sugar moities of certain proteins) and the previously
described action of a-LA in lactose synthesis take place in the Golgi
region prior to their incorporation into vacuoles (Larson and
Jorgensen, 1974; Keenan et al., 1974).

Milk protein synthesis is under genetic, hormonal and specific
subcellular pathway control. Inherited genes control the sequence of
amino acid additions as seen in genetic variants in milk proteins (see
Tables 4 to 6) (Larson and Jorgensen, 1974; Thompson and Farrell, 1974;
Whitney et al., 1976).

Hormonal control of protein synthesis is seen in mammary cell
development, induction of lactation, and maintenance of lactation
(Larson and Jorgensen, 1974). Tucker (1974) hypothesized that lac-
tation is initiated when progesterone secretion is low, and the secre-
tion of prolactin and adrenal corticoids are elevated. In the bovine,
growth hormone and thyroxine appear to have a synergistic effect on the
latter hormones. Tucker (1974) also described the sequential events in
hormonal changes in cattle prior to or around parturition. Progeste-
rone levels are elevated during gestation and appear to block the
induction of a-LA and lactose synthesis. Estrogen stimulates pro-
lactin and ACTH secretion but its effects are inhibited by progesterone.
Shortly before parturition the circulating progesterone declines
markedly. Concurrently, estrogen levels increase 10 fold and achieve
a maximum two days pre-partum, then decline precipitously during the
last two days of pregnancy. Beginning about two days before calving

prolactin increases sharply, reaches a maximum one day before

46

parturition and then declines to basal levels by parturition. Adrenal
corticoids are released about 12 hours before parturition, peak at
parturition, and return to baseline levels 12 hours after calving.
Starting at calving growth hormone is released and remains elevated for
36 hours. Luteothropic hormone concentrations do not vary significantly
during this interval. The importance of these hormone spurts above
basal levels is unclear (Tucker, 1974) but the sequence of events is in
good agreement with those leading to lactogenesis in virgin mice mammary
tissue in vitro (Topper and Oka, 1974; Keenanet al., 1974). Prolactin
renders secretory cells sensitive to insulin. Upon insulin treatment
such cells undergo division with an accompanying increase in the syn-
thesis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Following insulin treatment glucocorticoids and prolactin increase RER
throughout the cytoplasm, translocate RER and the Golgi apparatus, and
secretory vesicles appear. These events lead to a marked increase in

CA synthesis and later a-LA emerges (Topper and Oka, 1974).

Maintenance of milk synthesis is closely associated with milk
removal, and requires prolactin, growth hormone, ACTH (or glucocorti-
coids), TSH (or thyroxine), and parathyroid hormone (Tucker, 1974).

In cows, prolactin appears to have little effect on milk yields, but
changes in its blood levels parallel changes in milk yield with
advancing lactation. Prolactin release can be elicited by washing the
udder without milk removal simulated venipuncture, washing the brisket,
feeding and TRH injection. Further, prolactin levels are higher at
night than during the day, and are higher during the summer than during

the winter (Tucker, 1974).

47

ACTH and gluco- and mineralocorticoids cause a temporary
decrease in milk production due to their effects on electrolyte, pro-
tein and carbohydrate metabolism (Tucker, 1974). ACTH is released by
milking and the ability to discharge it is not lost with progressing
lactation (Tucker, 1974). Adrenocorticosterone release is closely
related to the nucleic acid content of the mammary gland during
lactation (Tucker, 1974).

Thyroxine stimulates milk secretion if the feed intake is
increased concurrently, but the stimulatory effect is lost after two
to four months (Tucker, 1974). Thyroxine also stimulates the pro-
duction of corticosteroidbinding globulin and the release of pro-
lactin (Tucker, 1974).

In cows, insulin decreases milk yields when the blood glucose
concentration is not maintained (Kronfeld et al., 1963). Milking
causes insulin release (Tucker, 1974).

Estrogen inhibits lactation and its effects are enhanced by
progesterone (Tucker, 1974). Pregnancy causes reduction in milk
yield in late lactation but whether the cause is due to estrogen and
progesterone or an increased nutrient demand of the fetus is not
known (Tucker, 1974).

The described hormonal events during lactogenesis and main-
tenance of lactation suggests that prolactin, insulin and ACTH are
required for initiation of protein synthesis. These hormones are
also released at each milking and the release of prolactin parallels
changes in milk yield with advancing lactation. Therefore it may be
hypothesized that the hormones which initiate mammary synthesis and

secretion are also required for the maintenance of the synthesizing

48

and secretory activity concurrently with milk removal. A concurrent
pregnancy inhibits the effects of prolactin due to an increasing
estrogen and progesterone secretion.

At the cellular level, hormones, as well as cellular cycles,
product feedback inhibition, and variations in the intraalveolar
pressures must control protein synthesis (Larson and Jorgensen, 1974;
Saache and Health, 1974). Saache and Health (1974) suggested that
variations in the intraalveolar pressure, along with the residual milk
causing this pressure, are the most important factors governing
alveolar activity. This may be related to effects on the RNA content
and the number of secretory cells, since both of these factors are
reduced by omission of several milkings (Tucker, 1974). Subcellular
pathway control is seen before parturition where absence of protein
synthesis is due to a low concentration of tRNA with the proper tri—
nucleotide combination for amino-acyl-tRNA complex formation, or be-
cause the ribosomes are deficient in one or more of their components
and are unable to bind activated amino-acyl-tRNA complexes (Larson

and Jorgensen, 1974).

2.4 Milk secretion
The secretion of milk consists of the secretion of solids and

their dilution with water until it is isotonic with blood.

2.4.1 Protein and Lactose secretion

 

Protein synthesized in the ribosomes is transferred to the
Golgi region and concentrated in vacuoles as previously discussed.

During its passage a-LA leads to lactose synthesis. The rate of

49

lactose biosynthesis is suggested to be directly related to the con-
centration and rate of flow of orLA through the Golgi apparatus. This
is substantiated somewhat by the positive correlation between the con-
tent of lactose and a-LA in milk (Yousef et al., 1970; Brew, 1969).
Lactose and certain minerals are concentrated in the vacuoles along
with the proteins (Keenan et al., 1974). The vacuoles are pinched off,
migrate to the apical region of the cell where their membranes fuse with
the plasma membrane, and contents are discharged (Keenan et al., 1974).
This process supplies a continuous source of new plasma membrane to
restore that lost with the fat droplets, which are also released from
the cell; but are surrounded with plasma membrane and pieces of cell

contents (Larson and Jorgensen, 1974; Keenan et al., 1974).

2.4.2 Milk volume

 

Lactose is an osmoregulator and draws water from the cell
before or after its release into the alveolar lumen (Saache and
Heald, 1974; Linzell, 1974; Davis and Bauman, 1974).

In goats, milk secretion is not stimulated by acetate, small
amounts of glucose, fumarate or amino acids (Hardwick et al., 1961;
Linzell, 1968). The milk yield in fasting goats decreased to 90%
within 8 hours, decreased further to 56% after 24 hours of fasting,
and was then maintained at that level for 10 to 12 hours (Linzell,
1967a, a, 1971; Lindsay, 1971). Similar results were obtained within
two hours when goats and cows were treated with insulin (Linzell,
1967b; Kronfeld et al., 1963). Likewise, when glucose was omitted
from the perfusate in perfusion studies with mammary glands. the milk

volume was very small and very rich in fat and protein (Linzell, 1974;

50

Smith, 1971; Lindsay, 1971). In these studies lactose reappeared and
the milk volume was restored when fasted animals were refed or
received IV infusions of glucose; and when glucose was added to the
perfusate in perfusion studies.

The evidence presented suggests that secretion of protein,
lactose, fat and water are interrelated. Protein synthesis, under
hormonal control, is directly involved in the control of lactose bio-
synthesis and replenishment of the cell membrane after fat secretion.

Lactose, in turn, regulates the milk volume due to its osmotic effects.

2.5 Mammary blood flow.

In the mammary gland, secretory tissue is equal to 80 to 90%
of the total udder tissue (Linzell, 1974). In dry cowsthe blood flow
is similar in skin, teat capsule, fat and secretory tissue, but during
lactation the secretory tissue receives 2 to 3.5 times as much blood as
the other tissues (Linzell, 1974).

The mammary gland receives about 10% of the cardiac output,
which itself may increase 45% at parturition (Linzell, 1974). The
mammary blood flow (MBF) in goats and cows has been measured by the
thermodilution (Linzell, 1960b, 1971; Bickerstaffe et al., 1974;
Annison et al., 1974; Hartman and Kronfeld, 1973) and the antipyrine
methods (Kronfeld et al., 1968). By the antipyrine method the MBF to
milk ratio was 750:1 which is probably an overestimation (Linzell,
1974). By the themodilution method the ratio is approximately 500:1.
Under normal conditions in established lactations the MBF was not
affected by age, lactation number and stage of lactation when sub-

jected to analysis of variance (Linzell, 1974). The day to day and

51

hour to hour variation in MBF is of the order of 10% of the mean and
occasionally as high as 30%, but within the secretory tissue the blood
flow varies from 70 to 200% of the mean (Linzell, 1974). Even when
considering the variation in MBF within the secretory tissue a high
positive relationship exists between the overall MBF and the milk yield
in the above mentioned studies. The peak MBF coincides with peak milk
yield and declines steadily thereafter, but at a rate slower than the
rate of decline in milk yield (Linzell, 1974), and suggests that MBF is
a major determinant of quantitative differences in the mammary meta-
bolism. This is supported by the absence of a relationship between
A-V for glucose, acetate, BHBA and triglycerides and milk yield
(Bickerstaffe et al., 1974). The unsynchronized decrease in milk yield
and blood flow after peak yield leads to a highly inflated MBF to milk
ratio in late lactation (1000:l) (Linzell, 1974). The inflated MBF
to milk ratio and a decreased milk yield in late lactation may be.
related to the previously discussed hormonal effects.

The lymph flow from the mammary gland is linearly related to
the milk secretion but is less than 1/1000 of the MBF and can be

neglected in A-V studies (Linzell, 1974).

2.5.1 Control of mammary blood flow

The control of MBF can only be studied in conscious animals
(Linzell, 1974). The MBF is affected by feeding and tissue metabolite
concentrations and hormones, but the mechanisms involved (cause and

effect) are largely unknown (Linzell, 1974).

52

Effect of Feeding.--The MBF in goats was decreased to one-half

 

by fasting (Linzell, 1967a). In fasted cows MBF was also decreased, but
was similar in normal and spontaneous ketotic cows (Kronfeld et al.,
1968). The MBF was increased in cows fed a high starch, low roughage

diet (Annison et al., 1974).

Hormonal Effects on Mammary Blood Flow.--Two to three days
before parturition the MBF begins to increase, but the effects of
hormones are generally unknown. Potent vasoconstrictors and vasodi-
lators were discussed by Linzell (1974) and their effects on MBF are
summarized below.

Potential vasoconstrictors in MBF control are adrenalin,
noradrenalin, antidiarrhetic hormone (ADH), oxytocin, serotonin,
prostaglandins, partial C02 pressure (pCOz) and blood temperature.
Adrenalin and noradrenalin have no effect on the general blood
pressure in anesthesized dogs given one to 10 mg IV, but the MBF is
reduced, and leaves these hormones as serious contenders in MBF regu-
lation. The adrenalin released in insulin hypoglycemia in the goat
causes a serious reduction in MBF and could lower the milk yield
without altering the A-V for glucose. The quantities of ADH relase
for water balance regulation are below effective levels (10 mu/min)
for decreased MBF in cows and goats. Oxytocin in doses 10 times
larger than the quantities released in response to milking resulted in
a negligible reduction of MBF and small doses even caused an increase.
Serotonin is a mammary vasoconstrictor released from platelets as they
come in contact with foreign surfaces. It is active in vitro but the

in vivo effect is unknown. Prostaglandin (PGFZa) is also a potent

53

vasoconstrictor. In women the MBF is lowest during menstruation and in
goats the MBF was markedly reduced by infusion of 28 mg or more per
minute of PGF2 into the mammary artery.

The normal pCO2 in goats is approximately 50 mm Hg. In a hot
humid climate the pC02 may decrease, due to hyperventilation, and lead
to a decreased MBF. A high pC02 will also decrease the MBF, but is
unlikely under physiological conditions.

Mammary blood temperatures below 25 C lead to a marked but
reversible decrease in the MBF. However, milk yields are unaltered by
varying mammary blood temperatures between 34 and 39 c.

Potent vasodilators are acetylcholine, histamine, adenosine,
and bradykinin. Acetylcholine and histamine at 0.1 to 1.0 g levels
lead to vasodilation, but there is no evidence of both parasympathetic
and sypathetic innervation of the mammary gland. Adenosine has been
proposed to be involved in the control of microcirculation and to have

the same effects as adenosine phosphates and NaH P04. Bradykinin

2
causes vasodilation in vivo and is produced during zero flow. It also
caused vasoconstriction under in vitro conditions.
3. Effects of postruminal administration of energy
(glucose) and protein (casein and amino
acids) on milk production.

The discussion of milk synthesis and secretion suggests that
MBF and arterial metabolite concentrations are major determinants of
milk production. However, saturation experiments for maximum responses,
as used in non-ruminants, cannot be perfOrmed under normal conditions

due to the complicating factor of rumen fermentation and the lack of

adequate methods for rumen bypass. Hence, abomasal or intra arterial

54

and/or IV infusions must be used. Accumulated data obtained by these
methods will give the requirement at the abomasal level, or at the
level of the mammary gland. In combination with data on the maximal
abomasal flow of nutrients under various conditions of rumen fermen-
tation, the quantity of rumen bypass for maximum milk production may
be calculated. Only abomasal and IV infusion studies will be consid-
ered in this review.

The effects of abomal and IV infusions of glucose, casein, and
amino acids on the milk production in dairy cows have been studied by
Yousef et a1. (1969, 1970), Broderick et al. (1970), Hale and Jacobson
(1972), Hale et al. (1972), Derring et a1. (1972, 1974), Tyrrell et a1.
(1972), Vik-Mo et a1. (1972, 1974a, b), Spires et a1. (1973), Schwab
and Satter (1973, 1974), Schwab et a1. (1975, 1976), Fisher (1969,
1972), Fisher and Erfle (1974), Clark et a1. (1973), and Teichman et
a1. (1969), and have been reviewed by Chalupa and Chandler (1972) and

Clark (1975).

3.1 Effect on milk protein

When Yousef et a1. (1969) intravenously infused an enzymatic
hydrolysate of casein (50 g/day), the milk protein content increased
9 and 28% in cows fed normal and high grain rations, respectively.
However, the infusate led to high fever and a decreased milk yield,
and resulted in net decrease in milk protein synthesis. An acid
hydrolysate of casein (50 g/day) increased the protein content 14%.
Inclusion of glucose at a 1:1 ratio increased both milk yield and the
protein content, and fever was prevented. They concluded that both

amino acids and energy limit milk protein synthesis, and that extra

SS

grain in the diet of lactating cows should usually increase milk protein
synthesis.

Abomasal infusion of 200 to 1400 grams casein per day have
increased the milk protein yield when compared with saline controls
(Broderick et al., 1970; Tyrrell et al., 1972; Vik-Mo et al., 1972,
1974a; Schwab and Satter, 1974; Schwab et al., 1975, 1976), abomasal
infusion of isonitrogenous, isocaloric urea-glucose solutions (Spires
et al., 1973; Clark et al., 1973), urea treated corn silage pgr_§g_
(Hale et al., 1972; Vik-Mo et al., 1974a), or ruminal infusion of
casein (Derring et al., 1972, 1974). The daily protein yield
increased from -2.9 to + 18.4% and was due to an additive increase in
milk yield and the protein content. The increased protein synthesis
is usually seen immediately after infusion commences (Broderick et al.,
1970; Vik-Mo et al., 1974a). Such may be expected because of the high
amino acid turnover in lactating cows.

For cows fed above the NRC-standards (NRC 1971) for energy and
protein the response to abomasally infused casein was positively related
to both the protein yield in the control period and the amount infused
(Vik-Mo et al., 1974a). They concluded that the magnitude of the two
variables might depend on common causative factors such as the protein
synthesizing apparatus in the mammary gland, and the hormonal status
of the cows. Both IV and abomasal infusions of amino acids led to
elevated growth hormone and prolactin levels. Prolactin apparently has
an all or none effect in stimulating RNA and protein synthesis
(Vik—Mo et al., 1974a). Vik-Mo et al. (1974a) did not find any

interaction between feed NPN and infused casein in cows fed above the

S6

NRC-standard for energy and protein. Abomasally infused glucose and

a 1:1 mixture of glucose and casein also stimulate milk protein pro-
duction (from 1.7 to 17.1%). Responses to abomasal infusions of casein,
a casein—glucose mixture and glucose decreased in that order (Vik-Mo

et al., 1974a).

Productive N from abomasally infused casein was diverted with
84 and 16% towards milk protein and N-retention, respectively
(Derring et al., 1972). However, the increase in milk protein yield
amount to less than 20% of the infused casein (Broderick et al., 1970;
Vik—Mo et al., 1974a). After abomasal infusion of 433 to 860 grams of
casein per day, 12 to 24% of the infused casein was recovered as
increased milk protein (Tyrrell et al., 1972). The remainder of the
infused casein caused 20 to 30% increases in fecal, urinary, and
retained N. The amount of infused casein available for protein syn-
thesis is slightly higher than the observed increases and may be attri-
buted to differences among experiments and a "requirement of the
protein synthesizing apparatus." 0f the energy in abomasally infused
glucose (3.6 Mcal/day) 48% was excreted in the feces (Tyrell et al.,
1972).

Casein and glucose infusions increased true protein in milk
and to a lesser extent its NPN content (Broderick et al., 1970; Vik-
Mo et al., 1974a). Milk NPN was positively correlated with feed NPN
(Vik-Mo et al., 1974a), and may be due to an upper limit for the cows
ability of concentrate urea in urine (Vik-Mo et al. 1974a) with a

consequent Spillover in milk.

S7

Rook and Line (1961) and Yousef et a1. (1970) found that the
increase in protein by infusion led to an increased content of CA,
B-LG and a-LA. This suggests an increased protein synthesis within the
mammary gland and is in agreement with in vitro studies by Park et
a1. (1974) and Park and Chandler (1976). They found that the rate of
B-LG and CA synthesis increased linearly when the EAA complement was
increased five-fold. However, the increase in a-LA synthesis was
linear only from levels one to three. This may suggest uneven changes
in the synthesis of proteins form the various fractions, and is in
agreement with characteristic changes in the amino acid composition of
wool from sheep receiving abomasal infusions of sulfur amino acids
(Gillespie et al., 1969). Effects of non-synchronous changes in the
protein fractions in milk on the amino acid composition are doubtful
partly because a-LA is only a small fraction of the total protein, and
partly because the amino acid composition of milk protein was the same
in cows fed rations containing 9 and 18% protected protein (Broderick
et al., 1974). The previously discussed additive increase in milk
yield and protein content by abomasally infused nutrients may be due
to such a non synchronous change in the protein fractions. Yousef
et al. (1970) found a positive linear relationship between a—LA and
milk yield.

The increased synthesis of true protein by casein and glucose
infusion suggest an increased availability of amino acids. The plasma
concentrations of individual EAA was either unchanged or increased by
abomasally infused casein (Spires et al., 1973; Hale et al., 1972;

Broderick et al., 1970; Derring et al., 1974; Vik-Mo et al., 1974b).

58

The response in NEAA was more variable but usually the total essential
to total non-essential amino acid ratios were increased. Casein
infusion had no effect on plasma glucose and decreased the blood urea
nitrogen content in cows fed according to the NRC-standards (Derring

et al., 1972). In cows fed above these standards casein infusion
increased plasma glucose and blood urea nitrogen (Vik-Mo et al., 1974b).
In the latter cows, glucose infusion decreased the blood urea nitrogen.
Effects similar to those for glucose have been obtained in cows
receiving rumen infusions of proprionate, but not by acetate and buty-
rate (Rook and Balch, 1961; Rook et al., 1965; Armstrong, 1968). These
results suggest that extra protein (casein) increases availability of
plasma amino acids for milk synthesis and leads to increased gluconeo-
genesis from amino acids in the liver. The increased availability of
amino acids may lead to an increased protein synthesis and decrease the
demand for glucose for NEAA anabolism in the mammary gland. This,a1ong,
with an increased availability of glucose by gluconeogenesis from

amino acids in the liver may increase lactose synthesis. 0n the other
hand, extra glucose (or prOpionate) decreases the demand for gluco-
neogenesis from amino acids in the liver, which in turn may increase
the availability of amino acids for protein synthesis. The latter may
explain the protein-sparing effect of glucose and propionate as
discussed by Yousef et a1. (1970) and Huber and Boman (1966b). The
changes in blood (plasma) metabolite concentrations might not be
detectable by ordinary methods but even statistically non-significant
changes may be highly significant biologically, due to the large

multiplication factor from MBF. Whether a change in the plasma

59

glucose concentration affects the MBF per §g_in well fed and healthy
animals is unknown, but the MBF was decreased to one half in fasted
goats (Linzell, 1967a). Linzell (1974) discussed the possible impor—
tance of epinephrine as a vasoconstrictor in insulin hypoglycemia.
Whether or not an increased nutrient availability has a positive effect

on the integrity of the cells which synthesize milk is also unknown.

3.2 Lactose and milk yield

In the previously describe experiments, abomasal infusion of
casein and glucose in dairy cows increased milk yield an average of
4.4% (range -5.5 to +13.l%). The response to abomasal infusion of
casein tended to be larger in cows with high production fed below or
at the NRC-standards for energy and protein. In cows fed above these
standards the response is less and more variable (Vik-Mo et al.,
1974a). Low producing cows (5 to 6 kg milk per day) fed a urea con-
taining diet did not respond to abomasal infusions of casein, gelatin,
partially delactosed whey, zein or soybean meal. Milk yields were
increased in cows fed above the NRC-standards for energy and protein
and abomasally infused with 270 to 540 grams glucose per day or a
1:1 mixture of glucose and casein, and increased milk was positively
related to the amount of casein infused (Vik-Mo et al., 1974a). When
Derring et a1. (1974) infused casein into the abomasum, the milk
lactose content was unchanged but the lactose yield was increased
3.9% compared to controls receiving rumen infusions of casein. This
experiment is the only one where milk lactose was determined, but the

evidence suggests that increases in the solids-non-fat (SNF) content

60

may be attributed to an increased protein content, as suggested by
Rook and Line (1961) and Armstrong (1968).

The described effects of casein and glucose infusions on milk
lactose and milk volume are in agreement with the hypothesis that a
greater lactose biosynthesis increases milk secretion (Linzell, 1974).
An improved availability of amino acids increases a-LA (protein) syn-
thesis in the mammary gland which increases lactose biosynthesis from
the infused glucose or casein. The greater amount of lactose increases

milk volume, but lactose content remains relatively constant.

3.3 Intravenous infusion of graded levels of amino acids

Intravenous infusion of graded levels of amino acids have
been studied by Fisher (1969, 1972), Teichman et a1. (1969), and
Fisher and Erfle (1974). Fisher (1969) infused l3 and 26 g DL-
methionine or 26 g methionine plus 52 g L-lysine-HCL per day. The
treatments had no effect on milk yield, milk protein or feed intake.
Fisher (1972) also compared zero, low and high levels of methionine
(0, 11.2, 24.3 g/d), lysine (0, 21.2, 50.3 g/d) and histidine (o, 33.5,
and 65.9 g/d) in cows fed at 70 to 90% of the NRC-standard for CP.
Milk yields were not significantly affected by any of the treatments.
Cows receiving low methionine produced more protein than those
receiving none or the high level. At zero histidine, milk protein
content was higher than fer the low and high groups. Low level lysine
infusion tended to increase the milk protein yield, but no further
increase was obtained at the high level due to a decrease in milk
yield. For both methionine and lysine the protein content tended to

increase linearly with the level of infused amino acid. Treatment had

61

no effect on the milk lactose content. Fisher and Erfle (1974)
intravenously infused lysine (ng/d), lysine plus methionine (15 and
lOg/d, respectively), carnitine (30 g/d) or saline to cows fed 67% of
the NRC-standards for CP. The treatments had no significant effect on
milk yield and milk composition. Likewise IV infusion of methionine
at 5, 10, or 20% of its expected secretion rate did not affect milk
yields or composition (Teichman et al., 1969).
3.4 Effect of abomasal infusion on feed

intake, dry matter

digestibility, and
N-utilization

Feed Intake.--Mugerwa et al. (1969) hypothesized that an amino

 

acid imbalance might limit feed intake and N-utilization in cows. When
Broderick et al. (1970) infused 800 g casein plus 24 g methionine per
day, grain intake was decreased 10%. Vik-Mo et al. (1974a, trial 11
and III) found that the DM intakes decreased when more than 500 grams
casein per day were infused, but it was unaffected in experiments where
200 to 500 g casein were infused per day (Spires et al., 1973;

Mugerwa et al., 1969; Derring et al., 1972, 1974; Hale and Jacobson,
1972a; Vik-Mo et al., 1974a). In low producing cows, 200 g casein per
day tended to increase the DM intake (Hale et al., 1972; Hale and
Jacobson, 1972a). Vik-Mo et al. (1974a) suggested that a high postru-
minal protein load caused the decrease in feed intake at high infusion

rates .

Dry Matter Digestibility.--In cows fed concentrate and alfalfa

 

hay the DM digestibilities were increased significantly by abomasal

62

infusion of casein compared with ruminal infusion (Derring et al., 1972,
1974). However, Spires et a1. (1973) found no difference in the DM
digestibility when casein or a urea-glucose solution were infused into

the abomasum.

Nitrogen Utilization.-—Cuthbertson and Chalmers (1950) hypo-

 

thesized that entry of high quality protein into the rumen may not be
as beneficial to the host as entry into the abomasum. This has been
confirmed by Mugerwa et a1. (1969), Spires et al. (1973) and Derring
et a1. (1972, 1974). They found that the N-utilization in cows was
improved by casein infusion into the abomasum compared to abomasal
infusion of gelatin or urea, or ruminal infusion of casein, gelatin,
or urea. When casein was abomasally infused, fecal and urinary N was
decreased. The resulting increase in productive N led to an increase

in both milk N and N-retention (Derring et al., 1972, 1974).

3.5 Factors affecting blood metabolites in dairy cows

Diurnal Variation.--No consistent diurnal change in plasma

 

amino acid (PAA) concentrations was apparent in cows offered feed
twice daily at milking (Halfpenny et al., 1969; Vik-Mo et al., 1974b),
but blood urea nitrogen tended to increase and blood glucose decreased

with time after feeding (Vik-Mo et al., 1974b).

Stage of lactation.--A marked decrease in PAA concentrations was

 

noted between pregnancy and the sixth week of lactation (Halfpenny and
Rook, 1968; Halfpenny et al., 1969b). However, cows which were further

advanced in lactation increased in PAA concentrations between the

63

fourth and the ninth week of an experiment regardless of protein level
or source (natural protein negative and positive controls, urea and
ammonia corn silages). In this experiment total EAA concentrations

in plasma were consistently lower in high than low producing cows at
the fourth week of experimentation but differences had diminished by
the ninth week, as had also differences in milk yields. (Lichtenwalner

et al., 1971).

Energy.-—It was hypothesized that increased energy or ruminal
infusion of propionate lead to an increase in the plasma concentrations
of glucogenic NEAA, and a resultant decrease or no change in EAA (Half-
penny et al., 1969a, b; Huber and Boman, 1966a, b; Rook and Line, 1961).
Halfpenny et al. (1969a, b) theorized that an increased output of
amino acids in milk proteins results in a depression in the plasma
concentrations of the EAA and certain NEAA. Huber and Boman (1966b)
suggested that pr0pionate had a controlling effect on the amino acid

metabolism in the liver.

Protein Source and Level.--Intravenous infusion of various

 

amino acids (methionine, lysine, histidine) increased the plasma con-
centration of that particular amino acid (Fisher, 1969, 1972). Infu-
sion of 26 g DL-methionine per day increased plasma methionine and
cysteine, but inclusion of 52 g L-lysine-HCL per day had no effect on
plasma lysine. The infusions led to a decrease in blood ammonia but
had no significant effect on blood urea, citrulline and arginine.
When Fisher (1972) infused zero, low and high levels of methionine,

plasma methionine was higher for the low than for the zero and the

64

high levels of infusion. Histidine infusions resulted in a two-phase
response in plasma histidine. Plasma lysine was increased when only
lysine was infused; but the plasma concentrations were not given for
the zero group, so the response curve could not be established.

Broderick et a1. (1974) fed rations containing 11.2, 13.5,
15.7 and 18% CP in the DM to dairy cows where formaldehyde-treated
sodium caseinate, (0.8 w/w), was the sole prOtein supplement to a
basal ration of oat straw, concentrate and corn silage. The plasma
concentration of glycine decreased, total EAA increased slightly,
total NEAA was not affected and urea increased linearly with the
level of protein in the diet. Plasma concentrations of methionine,
lysine and valine also increased with the dietary protein level, but
no consistent changes were seen for phenylalanine, leucine, isoleu-
cine, tryptophan, threonine and histidine.

Up to 423 g urea per day had no effect on PAA profiles com-
pared to controls (81 g urea/day) (Van Horn et al., 1969), and NPN
did not affect total PAA and total NEAA in plasma compared to a posi-
tive control group. (Lichtenwalner et al., 1971). However, Polan
and Miller (1975) reported that CP (0 to 20% in DM) and urea (0 to
40% of CP) accounted for zero to 71% of the variation in PAA
concentrations.

4. Limiting amino acid(s) for milk
protein synthesis

Abomasal infusion of the 10 EAA in the same ratio as in casein
led to as high increases in milk and protein yields as casein infu-

sion (Schwab and Satter, 1973; Schwab et al., 1976). The limiting

65

amino acid for milk protein production in cows fed corn, corn silage and
alfalfa grass hay was then explored by successive delations form the
complete EAA mixture (Schwab and Satter, 1973, 1974; Schwab et al.,
1975, 1976). They concluded that lysine and methionine were the first
and second limiting or co-limiting amino acids for milk protein syn-
thesis. Under varying conditions and by various methods without
direct determination, the following amino acids have been termed the
first limiting: methionine (Chandler, 1970; Chandler and Polan, 1972;
Brown, 1969; Fisher, 1972; Broderick et al., 1974; Chalupa and
Chandler, 1972; Dingley et al., 1975; Mepham and Linzell, 1974),
phenylalanine (Derring et al., 1974; Vik-Mo et al., 1974b; Spires
et al., 1973; Clark et al., 1974), lysine (Jacobson et al., 1969;
Schwab et al., 1976), throenine (Derring et al., 1974; Broderick et
al., 1970), and histidine (Spires et al., 1973) (Table 7). Halfpenny
et al. (1969b) suggested that the supply of glutamate and proline may
limit the protein synthesis. However, this may be considered dis-
proved since IV infusion of NEAA had no effect on the milk protein
yield in goats (Mepham and Linzell, 1974). Schwab et a1. (1976)
obtained a response in protein synthesis only from abomasal infusion
of casein and a mixture of EAA with the same ratios as casein.

5. Summary of interactive review on metabolic

effects on milk synthesis
and production

The synthesis of milk protein and lactose and milk volume are
functions of the mammary blood flow, the uptake of glucose and amino
acids by the mammary gland, and the in situ metabolism of the absorbed

metabolites. The rate of synthesis appears to be under an overall

66

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67

hormonal control. The control of mammary blood flow is generally
unknown but the mammary blood flow to milk ratio remain constant at
(approximately 500:1 except for a relatively sharp increase in late
lactation because of a decrease in milk flow rate.

The uptake of glucose and amino acids appear to remain constant
as a percentage of arterial blood concentration; but the mechanisms of
extraction have not been established. The extraction coefficient for
glucose is impaired by glucocorticoids, whereas insulin appears to have
an indirect effect on blood glucose concentrations. It is speculated
that hormonal vasoconstrictors decrease blood glucose concentration by
reducing mammary blood flow. Overall the uptake of amino acids can
account for the synthesized protein, but large variations exist for
individual amino acids. Extraction coefficients for EAA are large and
the uptake equals the output in milk. The uptake of arginine and valine
is in escess of the output in milk, and ornithine and citrulline are
extracted even though they are not found in milk. The uptake of NEAA
is less than the output in milk which suggests NEAA anabolism in the
mammary gland.

Amino acid metabolism in the mammary gland conforms with
known pathways, except for an incomplete urea cycle (no arginino-
succinase activity), and an apparent lack of the enzymes for cysteine
generation from methionine. Some NEAA are generated from glucose with
excess amino acids as the non specific N source. The rates of amino
acid transamination and amination appear to depend on substrate con-
centrations and little is oxidized. Absorbed glucose is apparently
partitioned with relatively constant percentages towards lactose,

oxidation, glycerol and NEAA anabolism. It is unknown whether an

68

increased uptake of glycerol, NEAA, etc. affect the amount of glucose
utilized by the respective pathways. Little gluconeogenesis takes place
within the mammary gland, except for triose phosphate salvage.

Milk protein consists of caseins, the whey proteins (a-LA, B-LG,
SA, Ig, and PP). Serum albumin, Ig and PP are transferred from blood.
The caseins, a-LA and B-LG are synthesized from absorbed amino acids via
the general pathways believed to exist in all cells in all species.

This synthesis is under an overall hormonal control. The cause-effect
relationships and control mechanism governing milk protein synthesis
are generally unknown. It was hypothesized that the same hormones
(prolactin, insulin, and ACTH) are responsible for lactogenesis as are
involved in maintaining the integrity of the protein synthesizing appa-
ratus. The hormonal stimulation relating to the maintenance function is
dependent upon a regular removal of milk (milking). With progressing
lactation, the maintenance function is impaired by an increased release
of estrogen and progesterone, which decreases milk production. Lactose
is synthesized from UDP-galactose and a pool of free glucose. Its
biosynthesis is controlled by the concentration and rate of flow of
a-LA through the Golgi apparatus.

Milk protein, lactose and minerals are secreted via secretory
vesicles formed from the Golgi apparatus. The vesicle membrane re-
plenishes that lost with fat drOplets. Water is added to the solids
mainly by the osmotic effects of lactose. This suggests interrelated
processes in biosynthesis and secretion, but how strict the relation-

ships are, have not been determined.

69

The substrate availability in mammary blood affects the pro-
duction of protein and lactose and the milk volume. In dairy cows the
substrate concentrations of amino acids and glucose in mammary blood
have been increased by abomasal and intravenous infusions. This led to
an increased yield of protein, lactose, and milk and may be explained
by current knowledge of milk synthesis and secretion. An increased
protein intake (by abomasal infusion of casein or amino acids) in-
creases the availability of amino acids and increases the protein syn-
thesis. The increased protein intake also increases the availability of
glucose, by greater gluconeogenesis from amino acids in the liver. It
is unknown whether the synthesis of CA, a-LA and B-LG increase to the
same extent during increased protein synthesis, but an increased a—LA
production may increase the lactose synthesis when more glucose is
available. An increased energy intake (by infusion of glucose)
increases the availability of glucose for lactose biosynthesis and
leads to an increased milk yield. However, the protein yield is also
increased, and may be explained by an amino acid sparing effect of
glucose in the liver, and/or less NEAA anabolism from glucose in the
mammary gland.

Several amino acids have been suggested by various investigators
to limit milk protein synthesis, but there has not been a consensus on
the one which is most limiting. However, by different workers
methionine, lysine, phenylalanine and threonine have been mentioned as
those in shortest supply. This lack of consensus may be due to experi-

mental differences, but are also the result of the methods used for

7O

calculating the order of limitation of amino acids. Except for two
experiments, the limiting amino acid was determined by back calculations

instead of actual determination.

6. Research proposal and objective

The limited information on the feasibility of NPN substitution
for natural protein in feed rations for high yielding dairy cows
suggests the need for further research. Hence, this investigation will
study the utilization of NPN in rations of dairy cows in early lactation
when milk yields and nutrient demands are highest relative to nutrient
intake.

The objective of the proposed research will be: (1) Test
protein requirements and the feasibility of substituting two sources of
NPN for preformed protein in rations for high yielding dairy cows
early in lactation, and (2) Measure the effect of protein source on

critical rumen and blood metabolites.

MATERIALS AND METHODS

Sixty-eight lactating Holstein cows from the dairy herd at
Michigan State University were employed in an experiment to test pro-
tein requirements and the feasibility of nonprotein nitrogen (NPN)
substitution for proformed protein in dairy cattle rations in early

lactation.

Experimental.--The experimental variables and ration composi-

 

tions are given in Table 8. The only difference between the negative
control (NC) and the positive control (PC) treatments was the total
crude protein (CP) content in the dry matter (DM). Treatment NC
received 12 to 13% and treatment PC received 15 to 16% CP in the DM.
For both treatments the planned CP contents were obtained by varying
soybean meal and corn grain in the concentrate. In the two remaining
treatments the negative control ration was supplemented with either
urea (treatment U) or ammonia plus urea (treatment AU) to equal the
CP content of the positive control group. Treatment U cows received
corn silage treated with urea at ensiling and the NC concentrate
supplemented with 1.25% urea. Treatment AU cows were fed corn silage
treated with ammonia at ensiling plus the same grain used for treatment
U. The detailed composition of the concentrates used for treatment

NC, PC, U, and AU are given in Table 9.

71

72

Table 8.--Ration Components and percent crude protein in the different

 

 

 

 

treatments.
Corn silage
Concentrate CP (%) of
Treatment no.a No treatment Ureab Ammonia total DM

NC D-l99 + 12—13
PC D-200 + 15-16
U D-201 + 15-16
AU D-201 + 15-16

 

aConcentrate composition is given in Table 9.
b . .
NPN treatment at en5111ng.

CCP is crude protein (N * 6.25) and DM is dry matter.

Corn Silage.--The urea and ammonia treated corn silages were
stored in 3.1 x 12.2 m concrete stave silos. Untreated silage for the
NC and PC groups was stored in a similar silo in 1975 and a 4.3 x 12.2
m silo in 1976. Additional untreated corn silage was obtained from a
6.1 x 18.3 m concrete stave silo and a 11.0 x 24.1 x 4.0 m bunker silo.
The fresh corn plant was harvested with a 2-row conventional forage
harvester, cut to an average length of 1.0 to 1.5 cm, and delivered
into wagons with side unloaders. Each load was weighed and urea and
ammonia were added at 0.65% and .40% of the fresh weight, respectively.
The urea was applied in a uniform layer across the top of each load
before unloading. Ammonia was applied at the silo blower by the cold-
flow method, and amounts were controlled by flow meters. In 1975 the

quantity added was ascertained by weight differences of the supply

73

Table 9.—-Ingregient composition of the concentrates used in this
Study , %.

 

 

Treatment
Ingredient D-ISQa D-ggoa g-20Ig
Cornb 54.00 43.50 53.25
Oatsc 26.50 21.25 26.00
Soybean meal (50% CP) 11.50 27.25 11.50
Urea -- -— 1.25
Molasses 5.00 5.00 5.00
Dicalciumphosphate 1.50 1.50 1.50
~Limestone .50 .50 .50
TMSd 1.00 1.00 1.00

 

aHerd concentrate number.
bCoarsely ground shell corn.
cRolled.

dTrace mineralized salt contain a guaranteed minimum of:
.35% Zn, .12% Fe, .15% Mg, .03% Cu, .005% C0, .007% l,
and 96.00% NaCl.

8To all concentrates was added 4400 l.U. Vitamin A and
400 l.U. Vitamin D per kg.

74

tank, while in 1976 a flowmeter was connected to a small nursing tank
placed on a scale. The amount of corn ensiled, nutrient content at

ensiling, and level of added NPN are given in Table A1.

ng§,--The 68 lactating Holstein cows employed in this experi-
ment were from best, control and worst genetic groups (24, 16, and 28,
respectively). Within each genetic group cows were blocked in groups
of 4 based on age and expected date of calving. Cows within blocks
were randomly assigned to treatments. The milk production in the
previous lactation was not considered in the blocking of cows because
only a limited number of cows within each genetic group were available
at any given time. The cows' date of birth, record in the previous
lactation (age at calving, milk production and calving interval),
actual date of calving and assignment to blocks within genetic group
are given in Table A2.

Cows were housed in a stanchion barn and allowed to exercise
for about 5 hours each morning in a dry lot. The time spent exercising
was shortened to about 2 hours on cold and rainy days. The cows were
milked twice daily at 4 A.M. and 3 P.M. in a double eight herringbone
milking parlor.

From 28 days before calving and until 15 days after calving,
all cows were fed similarly. From April 15 to October 15, 1976 dry
cows grazed pasture and were also fed up to 23 kg/day of urea or
ammonia treated corn silage. During this period cows were moved to
indoor maternity pens about one week before expected calving. Dry
cows held indoors (for winter calvings) were fed up to 23 kg/day corn

silage to which 0.5% urea was added at feeding, 1.8 kg. concentrate

75

(D-201 in Table 9), and 2.3 kg alfalfa hay per day until 7 days before
the expected date of calving, at which time concentrate and the hay
were increased to 2.7 and 4.5 kg per day, respectively. After calving,
the amount of concentrate was gradually increased to 7.3 kg per day,
and then adjusted according to milk yield on a weekly basis. Concen-
trate was fed at 1 kg per 2.5 and 3 kg milk from day 8 to 70 and from
71 to 140, respectively. On day 15 post-partum cows were switched

to treatment rations as outlined in Table 8. Hay was fed at 4.5 kg per
day until day 15 and then reduced to 2.3 kg per day for the remainder
of the feeding trial. Corn silage was fed ad_libitum throughout
treatment at an amount frequently adjusted to insure 10% weigh back.
Corn silage and hay were fed once daily at approximately 0800 and

1300 hours, respectively. Concentrate was fed in two equal portions

by pouring on top of the silage at about 0900 and 1400 hours. Uncon-
sumed feed was weighed back the following morning between 0500 and 0700
hours. Refusals of concentrate, corn silage, and hay were separated as
well as possible and weighed separately. Daily weights were recorded

for components fed and weighed back and for milk.

Sampling.--Cows were weighed 14 and 28 days before the expected
calving date and biweekly, thereafter, starting the second week after
calving. Corn silage samples were taken Monday, Wednesday, and Friday
of each week and stored at 3C until processed. Concentrates and hay
were sampled biweekly. Beginning in the first week after calving,

composite milk samples of the afternoon and morning milking was taken

biweekly.

76

Rumen fluid and blood samples were collected 2 weeks before
calving, and 3, 10, and 18 weeks after calving. An additional blood
sample was taken 6 weeks after calving. Rumen and blood samples were
collected on Tuesdays of every week at 2 to 3 hours after the A.M.
concentrate feeding. Sampling of blood was attempted to approximate
days -l4, 21, 42, 70, and 126 after calving. Rumen contents were
sampled by stomach tube and strained immediately through 4 layers of
cheesecloth prior to placement in an icebath. At the time of sampling
ruman fluid was also prepared for ammonia determination according to
Kulasek (1976).

Blood samples were collected from the tail vein using single
draw needles1 with 15 ml vacutainer tubes.2 Each tube contained 30
mg potassium exalate and 37 mg sodium fluoride as anticoagulant and
glycolytic pathway inhibitor, respectively. After drawing blood, the
tube was inverted gently several times and placed in an ice bath until

processed.

Preparation of Samples and Analyses.--Corn silages, concen-

 

trates, and hay were composited on a biweekly, bimonthly and monthly
basis, respectively. To uniformly mix concentrates and hay were
ground through a 40 mesh screen and corn silages in a model 84142

Hobbart Silage Chopper.3 A portion of the corn silage composites

 

1Single Draw Vacutainer Needle ZOG. Becton-Dickinson,
Rutherford, New Jersey 07070.

2Vacutainer Evacuated Glass Tube. Becton-Dickinson,
Rutherford, New Jersey 07070.

3The Hobbart Manufacturing Company, Troy, Ohio.

77

was diluted with water (1:10) homogenized4 for 3 minutes in an icebath,
and filtered through four layers of cheesecloth. Readings for pH were
made from the filtered homogenate, which was then centrifuged at 27,000
x g for 10 minutes (at 0 to 5 C). The supernatent was frozen at -20 C ~
until analysis. All feed samples were analyzed for DM, total nitrogen,
acid detergent fiber (ADF), and acid detergent nitrogen (ADN). The
water extracts of silages were analyzed for soluble nitrogen (SOLN),
ammonia, urea, lactic acid, ethanol, and volatile fatty acids.

Dry matter was determined by drying in a forced air oven at
100 C for 48 hours. Total nitrogen and SOLN were determined by
Kjedahl (AOAC 1965) as modified by Wall and Gehrke (1975). Some of
the nitrogen readings were by ammonium electrode (Brenner and
Tabatabai, 1972). Acid detergent fiber and ADN were by the Van Soest
method (Goering and Van Soest, 1970). Samples for these analyses were
dried at 60°C for 48 hours in a forced air oven and then ground
thrOugh a 40-mesh screen. Urea and ammonia were analyzed by the method
of Okuda et a1. (1965), modified by Kulasek (1972, 1976) for rumen
ammonia and plasma urea. The method of Barker and Summerson (1941)
was used for lactic acid analysis.

Volatile fatty acids (acetate, propionate and butyrate) and
ethanol in the water extract of silages were measured using a Hewlett-
Packard gas liquid chromatograph model 5730A with flame ionization

detector.5 A glass colum (6 ft x 2 mm ID) was packed with 3% Carbowax

 

4Servall Omni mixer. Ivan Servall, Inc., Norwalk, Connecticut.

5Hewlett-Packard, Avondale, Pennsylvania 19311.

78

20 M, 0.5% HSPO on 60/80 Carbopack B.6 Nitrogen was the carrier gas

4
at a flow rate of 60 ml/min. The temperature program used was two
minutes beginning at 140 C with a temperature increase of 4°C/min until
180 C. The final temperature was maintained for 8 to 12 minutes.

Prior to injection, the samples (approximately 1.5 ml) were acidified
with one drop 9 N H2504.

fatty acids was calculated relating the areas under the peaks for the

Injection volume was 3 micro liters. Volatile

standards with the areas under the peaks of samples.

Fat, protein, and total solids in milk samples were determined
within 48 hours after sampling. Milk fat was determined by The
Michigan Dairy Herd Improvement Association Central Laboratory using
the Babcock method; milk protein by the Udy dye method (Udy, 1971); and
total solids by drying 3m1 at 90 C for 3 hours in a forced-air oven.

Upon arriving in the laboratory about one hour after sampling
rumen fluid samples were measured for pH; and then centrifuged at
27,000 x g (0-5 C) for 15 minutes. The supernatant was stored at -20 C
until analyses for content of volatile fatty acids and ammonia nitrogen.
Rumen volatile fatty acids (acetic, propionic, iso-bytyric, butyric,
2-methylbutyric, iso-valeric, and valeric) were determined by gas
liquid chromatography as described for water soluble extracts of
silages. Rumen ammonia nitrogen was analyzed by the method of Okuda
(1965) modified by Kulasek (1976). Rumen ammonia nitrogen was deter-
mined in all frozen samples and approximately 50% of the fresh samples.

Fresh samples were analyzed within 24 hours of sampling.

 

6Supleco Inc., Bellefonte, Pennsylvania 16823.

79

Two hours after sampling whole blood was centrifuged at
6800 x g (0-5 C) for 10 minutes. OneNhalf of the plasma was stored at
-20 C until analysis for urea ammonia and glucose. The remainder was
prepared for amino acid analysis by adding on the day of sampling .1 ml
(1 micromolar) nor—LeucineNPEC internal standard and .1 ml 50% sulfo-
salisylic acid per milliliter plasma. The percipitated protein was
removed by centrifugation at 34,800 x g (0-5 C) for 20 minutes and
supernatant was frozen at -20 C until analysis. Amino acids were deter-
mined on individual samples by ion exchange chromatography.7 Plasma
urea nitrogen and plasma ammonia nitrogen were determined as described
by Okuda (1965) and Kulasek (1972, 1976). Plasma glucose content was

determined using the coupled system of glucose oxidase and peroxidase.8

Statistics.--In each of the two years of harvest three types of

 

corn silage were placed in two or more silos. Nutrient contents were
determined on biweekly composites of samples taken at feeding. The
analysis of variance for each of the nutrients in corn silage was as a
2 x 3 factorial experiment with repeat measurements according to the

model in (8):
(8) Yijkl = u * A1 * Fj * (AF)ij * S(ij)k

is the observed value in the ith year, jth_feed, kth silo

where Yi' ___
within year and feed, and lth_measurement within silo;

jkl

u is the overall mean;

 

7Technicon TSM System, Technicon Instruments Corporation,
Tarrytown, New York.

8Worthington Biochemical Corporation, Freehold, New Jersey.

8O

Ai is the effect of the i£h_year; i

Fj is the effect of the j£h_feed; j

1.2;

1,2,3;

(AF) is the year by feed interaction;

13'
is the effect of the kth silo nested within year and
feed. 3 is error for A and F.

Sam
The four treatments were tested in 68 cows. Cows within three

breeding groups (best, control, worst) were blocked according to
expected date of calving and age. The number of blocks within best,
control and worst breeding groups were 6,.4 and 7, respectively. Feed
intake, milk yield and yield of milk components were calculated as
average per day and week from calving. Rumen and blood were sampled
and assayed for each cow at 14 days pre-partum, and 21, 70, and 126
days post-partum. Blood was also sampled at 42 days. The analysis of
variance for each dependent variable was a double-split plot design

with respect to space (blocks within breeding groups) and time from

calving, and was performed according to the model in (9):

‘93 Yijklm = u * Bi * D(i)j I Tk * BTik * DT(i)jk * C1 * BCi1 *
TCkl + E(ijkl)m
where Y..klm is the observed value of mgh cow at the lth_time on
1’ k£h_treatment, and jth_block within ith_breeding

group;
u is the overall mean;

B. is the effect of the ith_breeding group, i = 1,2,3;

D(.). is the effect of the j£h_block within the ith_breeding
1 3 group, j = 1,2. . .6 for i = l; j = 1,2. . .4 for
i = 2; j a 1,2. . .7 for i = 3. D is error for B:
T is the effect of the kth_treatment, k = 1,2. . .4;

81

BTik is the interaction between breeding group and treatment;

DT(i)'k is the interaction between block within breeding groups
3 and treatments. DT is error for T and BT;

C1 is the effect of the lth_time, l = 1,2. . .20;
BCi1 is the interaction between breeding group and time;
TCk1 is the interaction between treatment and time;
E(ijkl)m is the re51dual error.

Milk yields and yields of milk components were tested by
covariate analysis and the data were adjusted for significant covariates
before testing by the model in (9). The covariate analysis was accor-

ding to the model in (10):

(10) Yijk = u + Cov + 81 + D(i)j + Tk + BTik + DT(i)jk

where Yi’k is the average response fer weeks 3 through 20 to the
J kth_treatment, jth_block and ith_breeding group;

p is the overall mean;

Cov is the covariate and is equal to the daily response in
week two after calving; and

Bi’ D(i)j’ Tk’ BTik and DT(i)jk are as described in equation
(9).

Adjustment for significant covariates were according to (11):

(11) Y = Y - (Y - ij

ADJ Cov

where YADJ 15 the adjusted value.

Y is the observed value.

YCov is the average for the second week after calving.

”q

is the overall mean.

b is a constant.

82

The number of observations for time (C1) in (9) was 18 when

milk data were adjusted for covariates. For rumen and plasma para-

meters the number of observations with time were 4 and 5, respectively,

except for plasma amino acids 42 days post-partum, which were analyzed

statistically according to the model in (10) after removal of the

covariate. The sources of variation for the various models and the

degrees of freedom per source are summarized in Table 10.

An initial experiment on factors affecting plasma amino acid

concentrations was studied using 5 treatments in cows at two levels of

milk production. Cows were bled at three sampling hours at two stages

of the experiment. The analysis of variance for individual amino

acids was according to the model in (12) for a double split plot

factorial design with respect to time.

(12) Y

ijkl

= u + T1 + Lj + (TL)ij + Pk + (TP)ik + (LP)jk +

(TLP)ijk + H1 + (TH)i1 + (LH)jl + (TLP)ijl + (PH)k1 +

(TPH)ikl + (LPH)jkl + (TLPH)ijkl

where Yijkl

L5

(TL) 1 j

is the observed value at the lth_hour, kth period,
jth_level of milk production and ith_treatment;

is the overall mean
is the effect of the i£h_treatment, i = 1,2. . .5;

is the effect of the jth_level of milk production,
3 = 1,2;

is the interaction between treatment and level of milk
production. TL is error for T and L;

is the effect of the kth_period of sampling, k = 1,2;.

83

Table 10.--Analysis of variances used and degrees of freedom per
Source of Variation.

 

Degrees of Freedom

 

Sources of Model (9)a
variation vModelm Mode
Feedb Milkc Rumend Plasmae PAAf (10)g (10)fi

 

 

 

 

Breeding group (B) 2 2 2 2 2 2 2
Blocks within B (D) l4 l4 14 14 14 l4 14
Treatment (T) 3 3 3 3 3 3 3
B x T 6 6 6 6 6 6 6
D x T 42 42 42 42 12 41 42
Time (C) l9 l7 3 4 2 -- --
B x C 38 34 6 8 4 -- --
T x C 57 51 9 12 6 -- --
Residual error 1178 1054 184 298 39 -- ~-

 

8Model for analysis of variance.

bDependent variables not adjusted for significant covariate.
cDependent variables adjusted for significant covariate.
dRumen pH, volatile fatty acids and ammonia nitrogen.
ePlasma glucose, urea and ammonia nitrogen.

fPlasma amino acids.

gCovariate analysis for yield of milk components.

hPlasma amino acids at 42 days post partum.

84

(TP)1k is the interaction between treatment and period;

(LP).k is the interaction between level of production and
J period;

(TLP)ijk is the interaction between treatment, level of pro-
duction and period. TLP is error for P, TP and LP;

H is the effect of the lth_hour of sampling, 1 = 1,2,3;

1
(TH)1 is the interaction between treatment and hour of
sampling;
(LH)jl is the interaction between level of production and

hour of sampling;

(TLP)i.1 is the interaction between treatment, level of produc-
3 tion and period. TLP is error for H, TH and LH;

(PH)k1 is the interaction between period and hour of sampling;

(TPH)ikl is the interaction between treatment, period and hour
of sampling;

(LPH).k1 is the interaction between level of production, period
3 and hour of sampling;
(TLPH) is the interaction between treatment, level of produc-
tion, period and hour of sampling. TLPH is error for
PH, TPH, and LPH.

ijkl

Diurnal changes in PUN and PAN were studied in two cows
selected from each treatment. Each cow was bled at 8 sampling hours on

two occasions and the analysis of variance was according to the model

in (l3):
(l3) Yijkl = u + T1 + C(i)j + PR + (TP)ik + (Tcp)(i)jk + H1 +
(TH)il * (TCH)(i)jl * (PH)k1 I (TP“)ikl E(ijk1)
where Yijkl is the observed value for the jth cow within the ith

treatment, at the kth time from— calving, and the 1th
hour of sampling;

u is the overall mean;

85

T. is the effect of the i£h_treatment. i = 1,2,3,4;

C(i)’ is the effect of the jth_cow within the ith_treatment,
J j = 1,2. C is error for T;

P is the effect of the kth_time from calving, k = 1,2;
(TP)i is the interaction between treatment and period;

(TCP)(i)'k is the interaction between treatment, cow and time.
J TCP is error for P and TP;

H1 is the effect of the lth_hour of sampling, 1 = 1,2. . .8;
(TH)i1 is the interaction between treatment and hour of .
sampling;

(TCH)(i).1 is the interaction between treatment, cow and hour of
J sampling. TCH is error for H and TH;

(PH)k1 is the interaction between days from calving and hour
of sampling;

(TPH)ikl is the interaction between treatment, days from calving
and hour of sampling;

B(ijkl) is the residual error.
Differences between treatment means were only tested for signi-

ficant sources of variation. Main effects with an equal number of

observations per group were tested by orthogonal contrasts (group NC

13, PC, U, AU; group PC 35, U, AU; group U vs: AU). Bonferroni's

t-test was used when additional contrasts were tested.

Ca1Culations.--Nutrient intakes, milk yields, yields of milk

 

components, and estimated nutrient balances (percent of NRC (1971)
requirement consumed) per day and week from calving for individual
cows were estimated utilizing a computer program which matched

feed analysis arranged by week of the year with intakes and yields
arranged by days from calving. Intakes were calculated on a daily

basis and then averaged per 7 days from calving. Analysis of

86

variances, other statistical analysis and plots of responses

utilized standard programs (Nie et al., 1975; Cohen and Burns, 1976;
Tuccy, 1976; STAT Systems Group, 1974) and were performed on the
Control Data Corporation 6500 Computer, Computer Laboratory, Michigan

State University.

RESULTS

Main emphasis has been placed on the effects of treatments and
time. However, variables affected by breeding groups and its inter-
actions with treatments and time are reported at the end of each

section.
1. Nutrient content in feeds

Concentrates.--The concentrates were prepared as described in
Table 9 and analysis are given in Table 11. Dry matter (DM), acid
detergent nitrogen (ADN), calculated net energy for lactation (NE1)’
and acid detergent fiber (ADF) in DM were essentially equal in the
three concentrates. Total nitrogen (TN) and crude protein (N * 6.25;
CP) in DM was increased from 2.52 and 15.73% in mixture D-199 to 3.66
and 22.89% in D-200 by replacement of corn and oats (2:1) for soybean
oil meal. The nitrogen content of D-201 was elevated to 3.11% by
replacement of corn and oats fer urea. In D-201 urea nitrogen amounted

to 19.0% of the total nitrogen.

Corn silage.--The nutrient content in corn silage is also
summarized in Table 11. Differences between feeds and years was

analyzed statistically according to the model in (8).

87

88

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Nmz ao< oesou m om: NW N N no
HH oH H H a 3 oanHom Roam: Hench xwmmz .oz

 

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89

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NH
HH

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NN oN NNN .62

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m:0Huoenm cowouqu

 

.NosuNoeou--.NN oNNNN

90

The overall DM content in the corn silage was 34.15% and tended
to be lower (P<.10) in silage harvested in 1975 than in 1976 (30.83 vs.
36.75%).

The TN content of silages was increased by NPN treatments
(P<.001), and was higher for the urea than the ammonia treated silage
(P<.001). The lower content of TN in the ammonia treated silage was
in part due to a lower content in the fresh untreated material
(1.31 vs. 1.42 in DM), but mainly to a higher loss as vapors during
ensiling and great difficulties in adjusting the rate of ammonia
application which was by the cold flow method (Kjelgard et al., 1973).

The amounts of total NPN and ammonia nitrogen in water soluble
fractions (SOLN) were determined and the amount of urea nitrogen was
calculated by difference between the two fractions (Table 11). Urea
nitrogen was set at zero when total NPN was equal to or less than
ammonia nitrogen. Urea nitrogen in dry matter1averaged .33% for the
urea treated silage and was equal to 48.3 of the added which is similar
to previous reports (e.g., Huber et al., 1973). Urea was not detected
in the control nor the ammonia treated silages.

The content of ADN in silages did not differ among years but
was higher in NPN treated than untreated silages (P<.05). Also, the
ADN content was higher in the ammonia than the urea treated corn
silage (P<.05).

The ADF content did not differ among years nor treatments at
ensiling.

The change in TN and the nitrogen fractions between harvest
and feeding of silages is given in Table 12. In control silage the

total nitrogen content decreased 7.5% with a redistribution from the

91

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.332 .. 2 - NU a CNN

 

 

 

 

 

 

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N8 HNH N5 N8 5 H8 Q a: 3
N weHueom um0>uam * mcHuoom umo>um= N mcHuoom ume>ua=
owcanu emcenu omcmgu
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2c :H N 2: :H a Zn aH N comouuaz
«Hcoea< «on: oooz .

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92

water insoluble to the water soluble fractions. Insoluble proteins are
hydrolyzed and to a large degree deaminated during silage fermentation.
The decrease in protein nitrogen during fermentation was inhibited some
by urea treatment but more by ammonia. Ammonia treatment also increased
the ADN content during fermentation.

Quality measurements on the corn silages included ethanol,
acetate, propionate, iso-butyrate, lactate, and pH (Table 13). None
of the measurements were significantly affected by treatment at
ensiling, and only the ethanol (P<.01) and pr0pionate (P<.05) content
differed among years. The obtained values are all characteristic 0f
high quality silage.
Table l3.--Content of ethanol, acetate, propionate, iso-butyrate,

butyrate, and lactate as well as pH in corn silage, % in
DM (89 observations per item).

 

 

Standard
Item Average Error
Ethanol .260 .037
Acetate 1.734 .118
Prepionate .050 .005
Isa-butyrate .007 .001
Butyrate .071 .024
Lactate 4.862 .230

pH 4.510 .090

 

93

The simple and significant correlations among nutrient and
quality measurements are given in Table A3. All the correlations,
except between TN and SOLN were low and explained less than 20% of the
variation of each other. A stepwise multiple regression was used to
estimate the dependence of a quality measurement on the nutrient con-
tent. The results are given by partial and multiple correlations in
Table A4. Thirty-five percent of the variation in the ethanol content
was explained by the DM content and the concentrations of SOLN and ADF
in DM. The content of acetate in DM was only related to the ADF
content which in turn only explained 5% of the variation. Propionate,
iso-butyrate, and lactate were not related to any of the nutrient
content measurements. The content of butyrate was negatively related
to DM and ADF in DM and positively related to ADN in DM. The three
variables explained 33% of the variation in the butyrate content in

silage DM.

2 . Feed intake

Concentrate, corn silage, hay, and total dry matter.--The‘
average intake per day by week from calving of concentrates, corn
silage and total dry matter is given in Figures 3 to 5 and for periods
(weeks 3 through 6, 7 through 10, 11 through 15, and 16 through 20)
in Table 14. Intake of the various feed components did not differ
between treatments.

The intake of concentrate increased throughout the first six
weeks post-partum and then remained constant through week 10 (Figure 3
and Table 14). The sharp decrease in concentrate intake 10 weeks after

calving is due to a change in the rate of concentrate feeding from

94

15.00

 

 

 

 

 

 

)—
0:
C3
0:
1.1.1
0.
(.5
I
11.1
1..
I
a:
1..
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LIJ
2
o SOZOOV
U
4.
”EH”
+ Trt. NC
2.60!»
-.- Trt. PC
J. -.- Trt. U
--I- Trt. AU
0 4 8 12 18

HEEKS FROM CHLVING

Figure 3. Daily intake of concentrate per treatment and week from

calving (kg/d; standard error :.07).

20

95

 

 

 

 

 

‘QOT
).
I
o 41-
M
11.1
0.
(D
.3: ‘20“
11.1
(.5
(I
_J
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2
M
8
104
110nm
+ Trt. NC
3* * Trt. PC
+ Trt. U
-IF- Trt. AU
0* 4 0 12 18

HEEKS FROM CHLVING

Figure 4. Daily intake of corn silage per treatment and week from

calving (kg/d; standard error 11.02).

 

96

 

 

 

 

 

 

30.00
1r
25.000”
>- ‘P
(I
D
a: 20.00017
U
(L
0 4P
1:
I 150000“
11.1
p.
y.
c: 4F
2:
)-
0: 10.0000-
D
4»
LEKMP
-dr-T&T. NC
8.00 -l- Trt. PC
dfih Trt.lJ
-I- Trt. AU
0 4 0 I! 10 20

WEEKS FROH CHLV 1N0

Figure 5. Daily intake of total dry matter per treatment and week

from calving (kg/d; standard error 21.42).

97

Table 14.--Intake of concentrate, corn silage, hay, total dry matter
and net energy for the different periods after calving..

 

 

 

Treatment
Period s.e.a
NC PC U AU

Concentratec;kg/d:

Weeks 3 through 6 12.49 11.00 10.32 11.42 .62
" 7 " 10 12.88 11.74 11.09 ,11.83 .70
" ll " 15 9.81 9.35 9.10 9.26 .56
" 16 " 20 8.83 8.52 8.22 8.30 .54

Corn silagec; kg/d:

Weeks 3 through 6 21.62 19.62 20.61 20.46 1.48
" 7 " 10 23.26 23.03 23.13 21.29 1.57
" ll " 15 25.28 25.99 25.14 22.94 1.62
" l6 " 20 27.61 25.34 24.32 24.57 1.36

Hayes kg/d:

Weeks 3 through 6 2.21 2.18 2.19 2.29 .14
" 7 " 10 2.18 2.12 2.18 2.19 .09
" ll " 15 2.20 2.12 2.18 2.17 .05
" l6 " 20 2.17 2.09 2.15 2.10 .06

Dry Matterc;kg/d;

Weeks 3 through 6 19.75 17.78 17.52 18.55 .79
" 7 " 10 20.86 19.72 19.31 19.28 .83
" ll " 15 18.93 18.68 18.56 17.93 .77
" l6 " 20 18.93 17.74 18.00 17.70 .75

Net energy; Mcal/d:

Weeks 3 through 6d 37.35 33.18 31.74 34.57 1.54
" 7 " 10 39.40 36.75 35.13 36.01 1.65
" 11 " 15 34.96 34.15 33.67 32.80 1.48
" l6 " 20 34.61 32.29 32.58 32.12 1.42

 

3Standard error.

b

17 cows per mean.

cNone of the differences between means within a row are

significant.

dBy orthogonal contrast: Treatment NC different than treatments

PC, U, and AU. No other difference within a row was significant

(p<.05).

98

1 kg per 2.5 kg milk to 1 kg per 3 kg milk. The corn silage intake
increased throughout the experiment (P<.001) and the rate of increase
was approximately constant (Figure 4 and Table 14). Hay was fed at a
constant rate of 2.3 kg per day after the second week post-partum.

The dry matter intake increased from calving to six weeks post-partum,
and remained constant until weeks 10 to 11. After the change in the
rate of concentrate feeding, the intake of DM decreased slightly, but
remained approximately constant throughout the last 10 weeks of the
experiment (Figure 5 and Table 14).

For concentrate intake there was a significant interaction
between treatment and time (P<.05). However, this interaction was not
significant for corn silage and it only approached significance for
total dry matter (P<.25). The significant interaction between treat-
ment and time for concentrate but not for total DM may be due to a
difference in change of milk yields between treatments. Hence, the
fixed ratio between milk and concentrate would lead to the significant
interaction observed. Also cows on reduced amounts of concentrate
compensate by increased intake of corn silage. The total dry matter
is the sum of concentrate, corn silage and hay eaten and any tendency
towards compensatory intake of feed components fed §d_libitum, when
restricted ingredients are reduced, would decrease differences due to
time.

Dry matter intake averaged 3.05% of body weight during the
third week after calving, increased to a peak of 3.5% during the
seventh week, and remained at that level until week 11. When con-
centrate was reduced at the end of the tenth week, intakes dropped

to 3.3% and then declined at approximately .03% per week to 3.0% at

99

20 weeks post-partum. Only the changes with time were significant
(P<.001), but cows on treatment NC tended to eat more dry matter per
unit body weight than those on the other three treatments, particularly

from weeks 2 to 10 and 16 to 20.

Breeding g10ups.--The average intake of concentrate, corn
silage, hay and total DM in breeding groups is given in Table A5, but
none of the differences were significant. However, the interaction
between breeding group and time was significant (P<.001) for concentrate,
corn silage and hay, and approached significance for total DM (P<.10).
The significant interaction for individual feed components but not for
total DM may be due to compensatory intake of corn silage when the

concentrate was decreased.

3. Nutrient intake

Energy.--The intake of NE was estimated utilizing standard

1
values from NRC (1971). The averages per day by week and periods from

calving are shown in Figure 6 and 14, respectively. The NE intake

1
was not significantly affected by treatments but did change with time
(P<.001), and the interaction between treatment and time was signifi-
cant (P<.025). The time trend and the interactions with time may be
explained by the previously described changes in the intake of indi-
vidual feed components and a compensatory increase in corn silage when
the amount of concentrate was reduced. Furthermore, the time by
treatment effect is enhanced by crossover for treatment PC and AU six

weeks after calving, and the decrease in intake fer treatment U during

weeks 6 and 7.

100

 

 

 

 

 

 

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)-

a: 4»

D

M

m 45.000“

0..

_J

c: 1r

0

I:

T 40000 P

z 01

O

1- v

a:

1..

U

a: 35.0001?

.1

M

o .. N In

L1. 9 ‘.

>—

0 30.00%

K

MJ

2

11.1 3' I I

1...

U 1 + Trt. NC

2 25.0000
* Trt. PC

0 -¢— Trt. U
*Trt. AU
20.000? 4 a 5 4 5 TL 4 a if
0 4 0 12 10 20
WEEKS FROM CHLVING
Figure 6. Daily intake of net energy for lactation per treatment and

week from calving (Meal/d; standard error 1.812).

101

Crude protein and nitrogen fractions.--The daily intake of

 

total CP by week from calving and the averages for periods are given
in Figure 7 and Table 15, respectively. The only difference between
treatments was the lower (P<.001) intake for group NC than for other
groups. The trend for the CP intake with time was significant (P<.001),
and may be attributed to the trends in total DM intake and changes in
intake of the different feed components. The interaction between
treatment and time was also significant (P<.001). This is probably in
part due to the initial decrease for treatment NC when the cows were
changed to treatment diets. Furthermore, the crossover between
treatment PC and U at 11 and 13 weeks as well as the uneven increases
for treatment AU during weeks 5 through 10 attributed to the inter-
actions.

The CP content in DM remained approximately constant at 12.5%
for treatment NC, and at 15 to 17% for PC, U and AU (Figure 8 and
Table 15); and was affected by treatment (P<.001), time (P<.001) and
the interaction between treatment and time (P<.001). The CP content
was higher for U than AU. The significance of the interactions
between treatments and time may be attributed to none parallel changes
and crossovers. For group NC the CP content in DM remained relatively
constant at 12.5%. For PC the initial content was approximately 17.5%
and decreased with time to 15%. The CP content remained constant
between 15.5 and 16% for U, whereas it decreased from 16 to 15% for AU.
The larger changes with time fer treatments PC and AU than for U may
be attributed to a larger difference in GP between the concentrate and

corn silage for these two treatments.

102

 

KG PER DRY

3-56
umnu
7’ + Trt. NC
-.- Trt. PC
30300"
+ Trt. u
.4;. TrL.AU
.L
3.040» N 4
a
._ 1 v‘ ..
20780" ‘

 

CRUDE PROTEIN.
A
I‘

 

 

 

ZOEZOT’
4 /
2.260-
2400 4 i J: i 4 Jr # i i
0 4 8 12 16 20

WEEKS FROH CHLVING

Figure 7. Daily intake of total crude protein per treatment and week
from calving (kg/d; standard error I306):

103

 

 

 

 

 

 

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104

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PERCENT

105

 

20.00
Lfilfll
J} .5. Trt. NC
-I- Trt. PC
18.000" "" Trt- U
-I- Trt. AU
{h
/
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20

a:
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.—
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10.00 £ 1* 4 4 4 4 + 4 4T
0 4 8 12 16
NEEKS FRO" CRLVING

Figure 8. Crude protein in dry matter per treatment and week from

calving (96, standard error 1.168).

106

The intake of SOLN was calculated as the sum of urea in con-
centrate and SOLN in the corn silage. The results are shown in
Figure 9 and Table 15, and the effects of treatments and time were
significant (P<.001). The intake of SOLN was the same for NC and PC,
but less than for U and AU. Also, cows on AU consumed less than on U.

The intake of ADN was 35 to 45 grams per day for all four
treatments (Table 15) and was only affected by time from calving
(P<.001).

The intake of "true protein" was calculated as the difference
between C? and SOLN plus ADN and is shown in Table 15. "True protein"
in periods was equal for NC and U, and slightly less than for AU. Cows
on PC consumed more "true protein" than the other groups.

The intake of ADP increased with time (P<.001), from 2.5 to
3.8 kg per day. The treatment by time interaction approached signifi-
cance (P<.10), and may be explained by changes in the intake of feed
components as described previously.

The intake of acetate and lactate increased (P<.001) as the
intake of corn silage increased. Acetate intake ranged from 95 to
168 g per day, and lactate from 249 to 515 g. Treatments approached
significance for acetate (P<.10), with NC and PC consuming less than U

and AD. Treatment effects for lactic acid were not significant.

Breeding_groups.--The averages fer daily intake of nutrients
and CP in DM are given in Table A5. Differences between breeding
groups only approached significance for CP in DM. However, all
variables reported showed significant interactions between breeding
group and time, and may be attributed to significant interactions for

individual components.

.2000

J.

E
I.

D .16000 ‘

m I

u /

0... 4r

(.5 .

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DJ

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+

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3 +Trt. PCv
” +Trt. u

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0 T 1‘ I is j 112 I ‘16 T
HEEKS FRDH CRLVING
Figure 9. Daily intake of water soluble nitrogen per treatment and

107

 

 

 

week from calving (kg/d; standard error _+_ . 009).

 

20

108

Table l6.--Overall means and b-values for significant covariates.

 

 

Overall Significance
Item Mean b-value of ba

Milk. kg/d. 27.837 .6851 ***
Protein, % 3.260 .2424 **
Fat. kg/day .975 .1572 *

Protein, kg/day .393 .4556 4**
Total solids, kg/day 3,397 .5593 44*
Solids non fat. kg/day 2.426 .4849 ***
Fat corrected milk, kg/day 25.766 .3607 ***
Solids corrected milk, kg/day 25.819 .4788 ***
Body weight. kg. 574.598 .8477 ***

 

aSignificance levels: *, P<.05; **, P<.Ol; ***, P<.001.

4. Milk yield and composition

For the yields of milk and milk components reported in this
section the analysis of variance was carried out as follows. An
average was calculated for weeks 3 through 20. The averages were
subjected to covariate analysis according to the model in (10),
utilizing week two as the covariate. The data was adjusted for signi-
ficant covariates according to (11), and analyzed statistically
according to the full model in (9). The overall mean and the values

for significant covariates are given in Table 16.

Milk yield.--The average unadjusted milk yield per week and
per period are shown in Figure 10 and Table 17. The analysis of

variance for adjusted data showed that only changes with time were

109

 

 

 

 

 

4
4
90+ 4". \
I” I “i.
\
>- “.
CE in
o T ‘5 g”...
0.
CD .
x 20
l:
.J
H D
I: 1
. 101+ .
+ Trt. NC
4}. * Trt PC
+ Trt U
-iI-‘Trt AU

HEEKS FROM CHLV ING

Figure 10. Daily actual milk yields per treatment and week from
calving (kg/d; standard error 11.16).

110

Table l7.--Actual and adjusted milk, actual components yields, and milk
yield persistencies for the different periods after calving.f

 

 

 

 

 

 

 

 

Treatment
Period
NC PC U AU s.e.

Actual milk, kg/d.

Week 2 29.80 25.92 26.35 27.66 1.59

Weeks 3 through 6 32.39 29.53 28.79 30.28 1.84
" 7 " 10 31.68 29.70 28.77 29.64 1.81
" ll " 15 28.55 27.42 26.72 26.49 1.67
" 16 " 20 28.87 25.00 23.90 24.29 1.54

Adjusted milk, kgld,

Weeks 3 through 6 31.05 30.85 29.81 30.40 1.84
" 7 " 10 30.33 31.02 29.80 29.76 1.81
" 11 " 15 27.21 28.76 27.94 26.60 1.67
" 16 " 20 24.53 26.31 24.92 24.41 1.54

Fatz kg/d.b

Week 2 1.47 1.30 1.40 1.36 .12

Weeks 3 through 6 1.20 1.11 1.06 1.08 .09
" 7 " 10 1.08 1.06 1.00 .96 .08
" 11 " 15 .96 .95 .97 .88 .06
" 16 " 20 .90 .87 .86 .83 .05

Protein, kg/d.b

Week 2 1.06 .98 .96 .98 .06

Weeks 3 through 6 .99 .92 .88 .96 .06
" 7 " 10 .99 .93 .90 .95 .05
" ll " 15 .91 .91 .87 .87 .05
" l6 " 20 .86 .85 .82 .82 .05

Total solids,»kg/d.b

Week 2 4.11 3.58 3.72 3.73 .24

Weeks 3 through 6 3.97 3.60 3.56 3.65 .23
" 7 " 10 3.80 3.57 3.49 3.54 .22
" 11 " 15 3.44 3.35 3.35 3.20 .19
" 16 " 20 3.15 3.10 3.03 2.96 .18

Solids non fat, kgéd,b

Week 2 2.64 2.28 2.33 2.37 .16

Weeks 3 through 6 2.78 2.50 2.50 2.58 .16
" .7 " 10 2.72 2.52 2.50 2.59 .16
" ll " 15 2.49 2.40 2.38 2.32 .14
" 16 " 20 2.25 2.24 2.17 2.14 .14

111

Table l7.--Continued.

 

 

 

 

Treatment
Period

NC PC U AU s.e.

Persistency of milk yieldsc
Weeks 3 through 6 1.08 1.13 1.11 1.10 .03
" 7 " 10 1.06 1.14 1.12 1.09 .04
" 11 " 15 .96 1.06 1.04 .97 .04
" 16 " 20 .88 .97 .94 .89 .04

 

aStandard error.

bNone of the differences within a row are significant.

cPersistency = Milk per day in treatment period/Milk in week
two 0

dWithin rows P<.25 for the difference between treatments PC,
U and AU (added nitrogen). Treatment PC vs. U, Au, and U vs. Au were
not significant.

aTreatment differences within periods were not significant.

fl7 cows per mean.

112

significant (P<.001). Persistencies for milk production, also given
in Table 17, tended to be lower for treatment NC than for PC, U and AU,
but did not differ between treatment PC and the NPN groups nor between
the NPN groups. The apparent lower persistencies for NC and AU are
largely due to higher milk yields during the second week after calving
when cows were fed similar rations. The validity of using only week
two of lactation as a standardization period is questionable and will
be discussed later. Treatment differences for milk yields in week two
approached significance (P<.10). The average age at calving for the
four treatments was 38.516, 44.6:l.2, 39.1337, and 38.5:37 months.

The literature review indicated that protein levels and protein
sources are most critical in cows producing more than 25 kg milk per
day. Therefore, cows producing more than 25 kg milk per day during the
second week after calving were compared after covariate analysis. The
crude protein content in dry matter for these cows was slightly higher
than average values. For treatments NC, U and AU the difference was
less than .3% and it decreased from period one to feur. For treatment
PC the higher producers received .66, .78, .52, and .32% more CP than
the treatment average. The increased crude protein content may be
attributed to more grain fed high yielding cows. The covariate was
significant for all 4 periods (P<.05), but only in period one did
differences between treatments approach significant (P<.10) (Table 18).
The low protein group (NC) did not differ significantly from those of
high protein (treatment PC, U, AU), but PC cows produced more than
U, (P<.05) and the difference between the NPN groups was not significant.
The overall adjusted milk yields fer periods 2 through 4 were

37.23:,68, 30.59:,75, and 25.461363, respectively.

113

Table l8.--Adjusted yields of milk and milk components for the differ-
ent periods in cows with more than 25 kg milk per day in

 

 

 

 

 

 

week two.
Treatment
Period 5 a SigébOf
NC PC u AU °°'
'No. of Cows 13 10 9 9
Milk, kg/d: cd c d cd
Week 3 through 6 38.88 40.44 37.78 39.08 .70 P<.10
" 7 " 10 36.92 39.25 35.56 37.09 1.38 NS
" 11 " 15 29.88 32.69 30.11 29.75 1.53 NS
" l6 " 20 24.65 26.94 25.00 25.44 1.27 NS
Fat, kg/d: cd c d d
Week 3 through 6 1.25 1.31 1.15 1.19 .06 P<.10
" 7 " 10 .98 1.13 .96 .96 .08 NS
" ll " 15 1.02 1.10 1.11 .98 .06 NS
" l6 " 20 .96 .96 .99 .90 .04 NS
Protein,_kg/d:
Week 3 through 6 1.06°d 1.06Cd .99c 1.09d .04 p<.1o
" 7 " 10 1.06 1.06 .98 1.09 .04 P<.25
" 11 " 15 .93 1.00 .92 .94 .04 NS
" l6 " 20 .82 .86 .82 .84 .04 NS
Total solids, kg/d: c c d c
Week 3 through 6 4.29 4.38 4.02 4.31 .12 P<.25
" 7 " 10 3.84 4.06 3.63 3.96 .17 NS
" ll " 15 3.30 3.59 3.40 3.36 .17 NS
" 16 " 20 2.85 3.14 2.99 2.99 .15 NS
Solidgnon fat, kgld;
Week 3 through 6 3.26 3.07 2.87 3.12 -- --
" 7 " 10 2.86 2.93 2.67 3.00 -- ~-
" ll " 15 2.28 2.49 2.29 2.38 -- --
" l6 " 20 1.89 2.18 2.00 2.15 -- --

 

8Standard error for 10 cows per group.
bSignificance of treatments.

CdMeans within a row without a common superscript are different
(P<.05).

114

Fat, protein, total solids and solids non fat production.--The
covariates for the production of milk fat, protein, total solids and
solids non fat were significant (P<.001). Only the differences in time
were significant (P<.001) when the adjusted data were subjected to
analysis of variance. The average production of the four milk com-
ponents is shown in Table 17. The production of the four components
was highest in period one and decreased with time.

The production of milk fat, protein and total solids in periods
in high yielding cows was analyzed by covariate analysis. The covariate
was significant fer all variables and periods, except for milk fat in
periods 3 and 4. Table 18 gives component yields according to treat-
ment and period, but only significant differences will be discussed.
Production of milk fat was greater (P<.05) for treatment PC than U and
AU in period one. In periods one and two cows on AU produced more milk
protein (P<.05) than those on U with NC and PC intermediate. In period
one total solids production was less (P<.05) for cows on U than other
groups.

The contents of fat, protein, total solids and solids non fat
in periods are given in Table 19. Time significantly affected con-
centrations of all milk components (P<.01), but treatment differences
were not significant. Interactions between treatments and time for
the SNF percentage approached significance (P<.25) and was due to

frequent crossovers among treatments (Figure 11).

Fat corrected and solids corrected milk yields.--The production
of fat corrected (FCM) and solids corrected (SCM) milk was signifi-
cantly correlated with the initial production (Table 16). For both

estimates adjusted yields were significantly affected by time (P<.001).

115

Table 19.--The influence of protein source on milk composition in
periods from calving.bc

 

 

 

 

 

 

 

Treatment
Period a Overall
NC PC U AU s.e. Mean
fat, %
Weeks 3 through 6 3.62 3.66 3.67 3.66 .17 3.65
" 7 " 10 3.46 3.56 3.41 3.29 .17 3.43
" 11 " 15 3.39 3.54 3.63 3.37 .13 3.48
" l6 " 20 3.51 3.56 3.60 3.50 .13 3.54
Protein, %:
Weeks 3 through 6 3.04 3.14 3.05 3.21 .08 3.11
" 7 " 10 3.14 3.17 3.16 3.25 .07 3.18
" ll " 15 3.21 3.36 3.28 3.32 .08 3.29
" l6 " 20 3.33 3.42 3.45 3.44 .08 3.41
Total Solids, 96:
Weeks 3 through 6 12.20 12.12 12.40 12.19 .18 12.23
" 7 " 10 12.00 12.10 12.10 12.05 .18 12.06
" ll " 15 12.11 12.31 12.55 12.20 .18 12.29
" 16 " 20 12.23 12.50 12.71 12.32 .18 12.44
Solidsnon fat, 8:
Weeks 3 through 6 8.58 8.46 8.73 8.53 .14 8.58
" 7 " 10 8.53 8.54 8.69 8.76 .13 8.63
" ll " 15 8.71 8.77 8.92 8.83 .09 8.81
" l6 " 20 8.72 8.94 9.11 8.82 .12 8.90
aStandard error.
bTreatment differences within periods were not significant.

c17 cows per mean.

PERCENT

HILK SOLIDS NON FRT.

12.00

116

 

11.000

10.000

9.000

0.000

V

r

V

 

7.00

 

i

«U-

 

 

__L

 

Figure 11.

0 P4 8

1 J
I 1 I

Jr

'12 18 20

WEEKS FRO" CRLVING

Content of solids non fat in milk per week from calving

for treatments NC, PC, U and AU.
3316.)

(96; standard error

117

The overall production of FCM and SCM was 28.78 and 28.39 in period 1,
27.33 and 27.27 in period 2, 24.87 and 24.24 in period 3, and 22.86
and 23.20 kg per day in period 4.

In periods one and two the production of FCM and SCM by high
yielding cows was correlated with the initial yields (P<.01), but for
periods three and four the correlation only approached significance
(P<.10). Adjusted treatment means in periods are given in Table 20.
Treatment differences in FCM in period one were significant (P<.05),
and approached significance (P<.25) in period two. Differences in SCM

approached significance in period one (P<.10).

Breeding group.--Milk yields and milk composition for breeding
groups are given in Table A5, but only the protein percent was signifi-
cantly affected (P<.01). The interaction between breeding groups and
time was significant for the SNF percentage and is shown in Table A6.
The interaction between breeding group and time was significant for
the persistency of milk yield (P<.001), protein percent (P<.05;

Figure 81), and the SNF percent (P<.05; Figure 82); and approached
significance for the yields of total solids (P<.25) and FCM (P<.25).

The effect of breeding groups on yields of milk and milk com-
ponents by high yielding cows in periods are given in Table A5. Milk
yields were not different, but differences in the yields of fat, total
solids and FCM approached significance in periods one (P<.25), two
(P<.25) and three (P<.10), and were significant in period four (P<.05).
Differences in protein yields approached significance in period two
(P<.25). The differences in SCM yields approached significance in

periods one and two (P<.25) and were significant in periods three

118

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.mueeaueonu we ooeeufiMMeuflmn

.ueoapeonu non mzou ca mom muouuo oneneeume

 

 

 

 

 

oz o~.~ ~o.m~ om.o~ ou.o~ oo.m~ ou : on :
mz o~.H om.a~ oo.o~ ao.o~ ao.a~ on = on :
oz on." oH.o~ oo.m~ vm.o~ Ho.au oz : a :
oz.vo oo.o ono.~n ooo.o~ omo.mm om~.~n o snooze» m, memo:
uo\uz .xzoa oouuonuoo moozom
oz No.” o~.m~ oo.m~ oo.m~ om.m~ o~ : on =
oz om.~ Nn.A~ me.o~ v~.o~ oo.- on = on :
m~.vo we.o ou.a~ Hv.o~ vo.on om.a~ on = a :
mo.vo oo.H oom.~m o~o.om uoo.nn oao.~n o zuooueom memo:
uo\wx 4x~oa oouoouuoo pea
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pom mcw>amu Scum

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muowuom an xafie wouoounoo menace one wouoonhoo pew mo mvaewx woumonu<uu.o~ ozone

119

(P<.Ol) and four (P<.05). The interaction between breeding groups and
treatments approached significance (P<.10) for the yield of milk fat

in period one.

5. Body weights and average daily gains

The average body weight fer all cows during the experiment was
576 kg and treatment means are shown in Figure 12. Changes with time
from calving were significant (P<.001) but neither the differences
between treatments nor the treatment by time interaction was significant.

Average daily gains were also subjected to analysis of variance
and only the time effect was significant (P<.001). Table 21 shows the
number of weeks from calving to minimum BW and daily gains before and
after minimum was attained. Group U attained minimum BW in week eight
whereas this occurred in week 4 for the other groups. Weight losses
were least rapid for group U and most for group AU, with NC and PC
intermediate. After minimum weight daily gains were highest for group U

and followed in a descending order for PC, AU and NC.

Breeding groups.--Average daily gains according to breeding
groups are given in Table A5, but none of the differences are signifi-
cant. However, the interaction between breeding groups and treatments
approached significance (P<.10; Table A7), and the breeding group by
time interaction was significant (P<.001). Adjusted weekly body

weights are shown in Figure 83.

6. Feed utilization and nutrient balance

Feed utilization.--Mild yields per kg of DM intake of the

 

different treatments are shown in Figure 13. The overall feed

120

 

 

 

 

 

(b
3:
J:
p.
(D
H
DJ
E!
>.
CD
CD
e:
um
1’ 'ni-Trt. NC
8201’ *Irt. PC
+Trt. U
9 *Trt. AU
30' :4 If* €> 4? i? #77 4: <4 4%
0‘ 4 8 12 18
WEEKS FROM CRLVING
Figure 12. Average body weight for treatments per week from calving

(kg; standard error 33.6).

 

121

Table 21.-—Average daily gains for treatments before and after minimum

 

 

 

 

weight.
Treatment

NC PC U AU
Minimum weight,
weeks from calving: 4 4 8 4
Dailygain, kg/d:
Before minimum 8W3 -.954 -.843 -.692 -1.200
After minimum 13wa +.l89 +.274 +.359 +.215

 

aBody weight.

bl7 cows per mean.

utilization was 1.74 kg milk per kg dry matter during the second week
after calving and decreased linearly at a rate of .02 per week through-
out the remainder of the experiment (P<.001). Treatment differences
and the interactions between treatments and time were not significant.
The yield of FCM per kg of DM intake was also analyzed and gave results

similar to those observed for milk yields.

Nutrient balance.--Intakes for NE1 and crude protein were
calculated as a percent of published requirements (NRC, 1971), and the
results are shown in Figures 14 and 15. Figure 14 shows that all
treatments reached 100% of NRC requirements fer energy at approximately
three weeks after calving. Intakes increased to about 20% in excess
of requirements between weeks 3 and 7, and remained constant there-
after. Only the changes with time were significant (P<.001). Treat-

ment differences in high yielding cows also were not significant.

122

 

 

 

 

 

 

2:00
i
(b 1 .BOOT
a:
M
u.I "
...
....
a:
t l 080%?
)-
M
C)
qp
(D
K
a:
DJ 1 ~4001P
o.
a:
..l
H 1’
Z
1.200iP ... Trt. NC
* Trt. PC
a} + T1": U
* Trt. AU
1.00 ‘r 4 + i 4 + 4 4r 4-
0 4 0 12 18
WEEKS FROH CRLVING
Figure 13. Milk yield per kg dry matter for treatments by week from

calving (kg; standard error 1.040).

123

 

 

 

 

S

x 130"

c:

...

....

2: q,

H

...

2:

u:

2

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0.

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L)

2: 1,

CE

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C:

an

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nu
+Trt. NC

1’. *Trt. PC
+1.“. U
*Trt. AU
7 5: .4, —# 1 4? i 4: 1
0 4 O 12 18
WEEKS FRO" CRLVING
Figure 14. Balance of energy intake relative to NRC (1971) standards

for treatments per week from calving (%; standard error

: 1.7)0'

 

 

124

 

 

 

12

ca "

Lu 1'

a: .

O 110- A

.2 1P “ \ ~9 ‘

F‘ .

.— , /

z 1004

DJ

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a_ 'H‘P Trt NC
7°“ + Trt. PC

... Trt U
1’ + Trt. AU
6 4 44 *4r 4* 4 4 4 4 944
0 4 8 12 16
WEEKS FROM CRLVING
Figure 15. Balance of crude protein intake relative to NRC (1971)

standards for treatments per week from calving (%;
standard error :2.3).

 

 

125

Data in Figure 15 shows that for treatments PC, U and AD the
cows were consuming 100% of the NRC protein standard 3 to 4 weeks after
calving. From that time and throughout the remainder of the trial
these cows received crude protein from zero to 15% in excess of NRC
requirements. For treatment NC the crude protein intake was 10 to 20%
below the recommended requirements and was lower than for the other
treatments (P<.001). The described changes with time, the differences
between treatments, as well as the time by treatment interaction were
significant (P<.001; P<.001 and P<.05, respectively). .Average percents
' of requirements fer treatments NC, PC, U and AU in weeks 3 through 10
and 11 through 20 were 83.9, 85.2; 108.9, 104.8; 106.3, 110.6; and!
103.6, 105.0, respectively, with a standard error for treatments of
:7.9%. Except for treatments U and AU in period one, protein intakes
of high yielding cows exceeded estimated requirements for all supple-
mented treatments (Table 22). None of the differences between treat-
ments PC, U and AU were significant. Treatment NC was significantly
lower (P<.001) than supplemented treatments.

Simple correlations between production responses, body weights,
and the balances for net energy and protein for high yielding cows are
shown in Appendix Table A8. The correlation between milk yield and
yield of milk constituents was high, but will not be further discussed.
Milk yields and the yield of milk constituents were positively related
to BW in period one. In period two, only the yield of milk constituents
was related to the BW, and all correlations with BW were not signifi-
cant after week 10. Generally milk yields and yield and content of
milk constitutents were negatively correlated with the energy balance

in periods one and two. In period three milk yield, and the yield of

126

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.euuuo>
(e

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=< oo oz

 

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nzaozo ooz we eeeeeeo mo coroner eooeoeo--.- eoooe

127

fat, total solids, SNF, FCM, and SCM were negatively related to

estimated energy balances but none of these correlations were signifi-
cant in period fbur. The yields of fat, SNF, FCM and SCM were negatively
correlated with the protein balance in periods one and two, but these
relationships were not significant in periods 3 and 4. The correlation
between milk yield and the protein balance was negative in period 3,

but the protein percentage in period 3 and the SNF percentage in

period 4 were positively related to the protein balance.

Breeding ggoups.--Averages for milk per kg of DM, energy and CP
balances are given in Table A5, but only the interaction between
breeding groups and time for milk per kg of DM was significant (P<.01).
7. Rumen pH, volatile fatty acids, and
ammonia nitrogen

Rumen pH, volatile fatty acids (VFA), and ammonia nitrogen
(RAN) were measured in samples taken by stomach tube two weeks before
calving and 21, 70, and 126 days after calving. The results were

analyzed statistically according to the model in (9).

Rumen pH.--Pre-partum pH was higher (P<.001) than the post-
partum values (Table 23). After calving the pH tended to decrease
with time probably because of increased concentrate intake but the
differences are not significant. Treatments did not significantly

affect rumen pH.

Volatile fatty_acid concentration (millimoles/liter).--Rumen

 

VFA's measured included acetate, propionate, iso-butyrate, butyrate,

2-methy1-butrate, iso-valerate, valerate, and total acids. The effect

128

of time was significant or approached significance for all acids
(Table 23). Rumen acetate was lower (P<.005) two weeks before and
three weeks after calving than at 10 and 18 weeks post-partum. This
change may be related to an increasing amount of corn silage in the
diet. Pro-partum prOpionate concentrations were lower than the post-
partum values (P<.05), and there was a trend toward highest levels at
70 days after calving. Propionate was lower (P<.05) for treatments NC
and U than for PC and AU (Table 24).

Iso-butyrate was lowest 21 days post-partum (P<.05), and tended
to be highest at 70 days and then returned to the pre-partum level at
126 days. Concentrations were higher for treatment PC than fer others
(P<.05). For butyrate, only the time was significant with lowest con-
centrations pre-partum. There was only a slight trend towards in-
creasing values with time from calving. Concentration of 2-methyl-
butyrate was lower prior to than after calving (P<.05). Isa-valerate
levels were highest 70 days after calving (P<.05); and greater for PC
than for NC, U or AU (P<.05). Valerate concentrations increases form
pre-partum to 70 days post-partum and then decreased at 126 to a level
comparable to that shown at 21 days. Group U was lower in valerate
than others (P<.05). Rumen total acids total increased (P<.05) until

70 days post-partum and then remained-constant.

Molar distribution of rumen volatile fatty acids.--The molar
distributions of VFA were also calculated because salivary secretions
may dilute the rumen sample when collected by stomach tube, thereby
increasing the variation and making detection of differences more

difficult.

129

Table 23.--Effects of time from calving on rumen pH and rumen concen-
trations of volatile fatty acids, m moles/liter.a

 

 

 

Days from calving Signifi-

-14 21 70 126 s.e.b of‘iiiie

pH 7.388 7.01f 6.98f 6.96f .04 P<.001
Acetate 39.4o° 40.286 45.91f 45.15f 1.90 P<.10
Propionate 10.54e 15.59f 17.86f 15.30f .85 p<.001
ISO-butyrate .49e .45f .54° .50° .02 p<.1o
Butyrate 6.73° 8.09f 8.54e 8.s9° .42 p<.01
2-methyl-butyrate .45e .56f .60f .55f .03 P<.025
Iso—valerate .37° .35° .45f .40° .02 p<.os
Valerate .67° .93f 1.09d .eof .06 P<.001
Total acids 58.65e 66.04d° 75.00f 71.39df 3.05 p<.oos

 

368 cows per time mean.

bStandard error.
cDetermined by analysis of variance.

d’°'fMeans within a row without a common superscript are
different (P<.05).

130

Table 24.-—Effect of protein sources on rumen pH and rumen concentra-

tions of volatile
tions per mean.

fatty acids, m moles/liter; 68 observa-

 

Treatment

 

Sig of
NC PC U AU s.e. trt.a

 

pH

Acetate
Propionate
Isa-butyrate
Butyrate
2-methyl-butyrate
Isa-valerate
Valerate

Total volatile fatty acids

7.05 7.03 7.15 7.09 .06 NS

40.32 43.71 42.84 43.87 2.32 NS

d d

13.77c 15.77 12.91c 16.64 .99 p<.01

.46° .sod .51d .45c .03 P<.10
7.39 8.33 8.01 8.22 .48 NS
.52 .59 .50 .54 .04 NS
.38° .48d .38° .55° .03 P<.Ol
.87c .99 .07 P<.25

63.70 70.42 65.95 71.00 3.97 NS

 

aSignificance of treatment determined by analysis of variance.

c’dMeans within a row without a common superscript are

different (P<.05).

131

Acetate was a bigger fraction of the total acids in group U cows
than in cows for the other three groups (P<.05), and the reverse was
observed for propionate and valerate (P<.05; Table 25). For proprionate
it was found that the molar fraction was larger for AU than for NC
(P<.05). Iso-butyrate values were higher for treatments NC, PC and U
than for AU (P<.05). The level of 2-methy1-butyrate was higher
(P<.05) in cows on treatments NC and PC than on treatments U and AU.
Iso-valerate was higher (P<.05) for NC, PC and U than for AU; and
higher for PC than NC and U (P<.05).

The effect of time on the molar fractions of individual acids
was significant for all acids except 2-methyl-butyrate and iso—valerate
(Table 26). Pro-partum values were highest for acetate, iso-butyrate,
and iso-valerate; whereas, the reverse was observed for the other

acids.

Rumen ammonia nitroggg,--Rumen ammonia nitrogen (RAN) was lower
(P<.05) for group NC than for diets higher in CF (PC, U, AU), and was
lower (P<.05) for PC than for the NPN treatments with little difference
between U and AU (Table 27). The pro-partum value was lower for PC
than the other three treatments, whereas the post-partum responses
followed the overall averages. Changes with time were significant only
for NC which showed a marked decrease (P<.05) between pre- and post-
partum samplings; however, there was a trend towards increased responses

for treatment AU.

Breeding groups.--Rumen pH and concentrations of VFA and RAN,
as well as the molar distribution of VFA for breeding groups are given

in Table A9. Differences in molar concentrations of iso-butyrate and

132

Table 25.--Effect of treatment on molar distribution of rumen volatile
fatty acids, molar percent.a

 

 

 

Treatment - Significance

ACid NC PC U AU s.e.b trermentc
Acetate 62.87e 62.43e 65.17f 62.499 .50 P<.001
Propionate 20.99° 21.85eg 19.55f 22.868 .48 P<.001
ISO—butyrate .75e .81e .79° .oof .02 p<.01
Butyrate 11.60° 11.90e 11.97° 11.39° .23 NS
2-methy1-butyrate .81° .84e .76f .77f .03 P<.25
Iso-valerate .62°g .71f .58g .47d .03 p<.001
Valerate 1.37° 1.45e 1.19f l.36° .07 p<.1o

 

a68 observations per treatment mean.

bStandard error.

cDetermined by analysis of variance.

d’e'f’gMeans within a row without a common superscript are
different (P<.05).

133

Table 26.--Effect of time on the molar distribution of rumen volatile
fatty acids, molar percent.

 

 

 

Days from calving Significance
Acid b of
-14 21 70 126 s.e. trmec
Acetate 67.39d 61.596 61.49° 65.49f .51 p<.001
Propionate 17.94d 22.79° 23.44a 21.08f .54 P<.OOl
Isa-butyrate .84d .72° .73° .73° .02 p<.001
Butyrate 11.27d 12.11° 11.43d 12.05° .22 P<.025
2-methyl-butyrate .77d .85° .80d .77d .05 p<.2s
Iso-valerate .64d .55° .62d .578 .05 P<.10
Valerate 1.17d 1.4o° 1.so° 1.31° .08 p<.025

 

a68 observations per mean.

bStandard error.

cDetermined by analysis of variance.

d’e’fMeans within a row without a common superscript are

different (P<.05).

134

Table 27.—-Effect of protein treatment and time from calving on rumen
concentrations of ammonia, mg/lOOml.a

 

 

 

Days from Treatment
°31Ving NC PC u AU
-14 13.43c 10.85d 13.97c 16.36c
21 4.12c 7.14d 16.50° 13.256
70 5.31c 12.98d 16.81d 14.69d
126 6.38c 9.35d 17.056 13.976

 

817 cows per mean.
bStandard error.

c’d’eMeans within a row without a common superscript are
different (P<.05).
the interaction between breeding group and time for valerate approached
significance (P<.10) (Table A10). Effects of breeding group, the
breeding group by treatment interaction and the interaction between
breeding group and time approached significance for molar percentages

of acetate, propionate and valerate (Table All).

8. Blood metabolites

Blood metabolite analysis reported here include glucose, urea
and ammonia nitrogen, and amino acid concentrations in plasma. Plasma
glucose, urea-nitrogen (PUN), and ammonia nitrogen (PAN) were deter-
mined for all cows two weeks before expected calving, and again 21,
42, 70 and 126 days after calving. In 8 cows selected for high milk
yield blood was collected at 1430, 1530, 1900, 2300, 0330, 0430, 0800

and 1200 hours at approximately 59 and 112 days post-partum and samples

135

were assayed for PUN and PAN. Plasma amino acids are reported for a
preliminary study conducted in 1971 as well as for all cows in this
study at 42 days post-partum. For days 14 pre-partum and 126 post-
partum plasma amino acids were assayed in samples from cows in the

first seven blocks on experiment.

Plasma glucose.--Plasma glucose was highest before calving

 

(P<.05), lowest 21 days after calving (P<.05), and remained constant
for the last three samplings (Table 28). Treatment differences were.
not significant. The treatment by time interaction approached signifi-

cance (P<.25) and may be attributed to crossovers between treatments.

Plasma urea- and ammonia nitrogen.--Values for PUN were lower

 

(P<.05) for NC than all other treatments and PC was lower (P<.05) than
U and NU (Table 28). Concentrations were lowest 21 days after calving
(P<.05). The treatment by time interaction (P<.001) may in part be
attributed to the decrease in treatment NC and slight increases of
supplemented diets between pre- and post-partum samplings.

Plasma ammonia nitrogen was significantly affected by treatments
and by time. The PAN concentrations were lower for groups NC, PC, and
AU than for group U (P<.05). The PAN concentration was highest at the
pro-partum sampling and decreased gradually as the cows progressed into
lactation.

The hours of sampling for diurnal variation in PUN and PAN were
one-half hour before and after milking at 1500 and 0400 hours and
every 4 hours thereafter. On the two days of sampling the averages
of milk, fat, protein, total solids and solids-not-fat were 33.6:l.4,

29.211, kg; 3.62:.16, 3.551.294; 3.321.08, 3.031.088, 12.45:.14,

136

Table 28.-~Bffects of protein treatment and time from calving on
plasma concentrations of glucose, urea and ammonia
nitrogen, mg per 100 ml.3.

 

 

 

 

 

Treatment
Days from
calving NC PC U AU
Glucose
-14 57.45 58.30 61.69 63.47
21 45.71 49.54 45.14 50.83
42 53.42 56.68 50.00 54.68
70 53.44 56.72 57.11 50.21
126 52.93 51.64 53.61 49.86
Urea nitrogen:
-14 13.58c 13.51c 14.05c 14.48c
21 6.06c 11.42d 14.73e 13.61de
42 5.67c 14.98d 17.106 13.54d
70 8.049 14.27d 16.16d 16.09d
126 7.17c 13.426 17.546 14.59(1
Ammonia nitrogen:
-14 .37 .40 .43 .34
21 .27 .35 .38 .33
42 .33 .30 .41 .33
7o .27 .30 .35 .29
126 .27 .27 .31 .26

 

aStandard errors: glucose 8 :3.18, urea-N = :398, ammonia - N

bl7 cows per mean.

c’d’eMeans within a row without a common superscript are
different (P<.05).

137

12.431.278 and 8.83, 8.88%, respectively. The analysis of variance

was according to the model in (13). Plasma urea nitrogen was affected
by all factors included in the model, except the interaction between
treatment and days from calving. The three way interaction for
treatments, days from calving and hour of sampling only approached
significance and will not be discussed. Plasma ammonia nitrogen was
significantly affected by days from calving and the interaction between
days from calving and hours of sampling. The effect of hours of
sampling and the three-way interaction for treatment, days from calving
and hours of sampling approached significance.

The overall means for treatments and days from calving are
given in Table 29. The PUN was lower (P<.05) fer NC than for the other
three treatments, and was lower (P<.05) at 112 than 59 days after
calving. The PAN showed no particular trend for treatments but the
averages were lower (P<.05) at 59 than 112 days after calving.

The overall change in PUN with hours of sampling was non-
significant except for higher concentrations at 1200 than 0800 hours
with a slight increase until 1900 hours and relatively constant con-
centrations, thereafter. The only significant differences were between
the samplings at 0800 hours and those at 1900 and 0330 hours. The
changes in PUN concentrations with time within treatments are given in
Figure 16. ‘Figure 16 shows that the PUN concentrations remained
approximately constant and parallel at levels characteristic for each
treatment except fer a temporary increase for U at 1900 and 2300 hours.
At those hours PUN was higher for U than fer PC and AU. PUN concentra-

tions tended to increase after milking.

138

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139

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140

The interaction between days from calving and hours of
sampling for PUN and PAN are illustrated in Figures 17 and 18,
respectively. None of the differences between PUN concentration Within
a given day of sampling were significant. However, the PUN concentra-
tions were lower (P<.05) at 112 than at 59 days after calving for
samples taken between 1200 and 0330 hours. The PAN concentrations
remained relatively constant throughout the 24-hour sampling period at
59 days after calving, and none of the differences were significant.
By 112 days after calving PAN concentrations at 0330, 0430, and 1200
hours were higher than at 59 days (P<.05) and the concentrations at
consecutive hours of sampling within 112 days after calving were
different between 1900 and 1200 hours (P<.05). The described changes
in PAN concentrations indicate a diurnal rhythm at 112 days after
calving, but it is doubtful whether any rhythm was present for PUN

concentrations.

Breeding ggoups.-—Average concentrations of glucose, urea and
ammonia nitrogen in plasma for breeding groups are given in Table All,

but none of the differences were significant.

8.1 Plasma amino acids
Plasma amino acid analysis to be reported include an initial

experiment and the main experiment.

8.1.1 Initial experiment on factors affectingfiplasma amino acid
concentrations

 

Plasma amino acid profiles were determined in 30 cows divided
into two equal groups according to milk yield. The average milk

yield in the two groups was 31.2 (high) and 21.0 (low) kg per day.

141

vs

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143

Within these two groups an equal number of cows were assigned to each
of five treatments (Table 30). Two rations with all natural protein
containing untreated corn silage were fed. One used a concentrate with
8% CP (group NC) and the other with 18% CP (group PC). Three diets
contained NPN and consisted of corn silage treated with .5% urea

(group U), 2% (group P), and 4% (group 2P) Prosil9 at ensiling. The
concentrate fed groups U and P contained 13% CP and that fed group 2P
had 8% CP. All cows were bled at zero, 2 and 5 hours post-feeding
after 4 and 9 weeks on trial. For the respective bleeding times plasma
was pooled for the three cows within each production group within each
treatment and then prepared as previously described. The experimental
procedure is-sunmarized in Table 31. Production data were reported by
Huber et a1. (1973). Responses for individual amino acids presented

in Table 32 were statistically analyzed according to the model in (12).

Proline was not affected by any of the factors studied.

Effects of treatments.--Plasma concentrations of VAL, LEU, GLU
(P<.05), LYS, ILE, HIS, CYS, SER, and ASP (P<.25) were affected by
treatments. However, interactions between treatments and other vari-
ables were observed for several amino acids. The interaction between
treatment and week of sampling affected ARG (P<.01), GLY, ALA (P<.05)
and SER (P<.25). The treatment by hour of sampling interaction
approached significance for VAL, ARG, ASN and ASP (P<.25). Finally
the three-way interaction between treatments, week of sampling and

and hour of sampling approached significance for ARG, HIS (P<.25) and

 

9A mixture containing 13.6% N (as NH3), 55.1% molasses, 5.68%
NaCl, plus Ca, P, 5, Mg, Zn, Cu, Co, 1. Trade name, Prosil.

144

Table 30.--Protein in diets fed cows in initial experiment_on factors
affecting plasma amino acids.

 

 

 

 

 

Crude protein, % in DM Treatment
NC 2P U P PC
Concentrate 8.4 8.6 13.8 14.0 19.1
Corn silage 9.1 16.5 13.6 12.9 9.1
Total ration 9.5 13.2 13.9 13.7 14.1
. b a b

NPN added to Silage % - 4.00 .50 2.00 -

aUrea.

b

A mixture containing 13.6% N (as NH3) 55.1% molasses, 5.68%
NaCl, plus Ca, P, S, Mg, Zn, Cu, Co, 1. Trade name, Prosil.

Table 31.--Experimental procedure for the initial study of factors
affecting plasma amdno acids.

 

Cows used: 30 Lactating dairy cows

Outcome groups: High producers averaged 31.2 kg/day
Low producers averaged 21.0 kg/day

Silage treatments: 1) Negative Control (8% C.P. Conc)
2) Positive Control (18% C.P. Conc)
3) Urea (13% C.P. Conc)
4) Pro-Sil (13% C.P. Conc)
5) Pro-sil 9 2X (13% C.P. Conc)

Bleeding sChedule: 0, 2, and 5 hrs post-feeding after 4 and 9
weeks on trial

 

145

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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148

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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151

ASP (P<910). The changes in amino acids will be described in the
order of increasing complexity with respect to interactions.

Treatment was the only factor affecting LEU, LYS, CYS and GLU.
Leucine was lowest (P<.OS) for treatment NC and increased approximately
linearly for treatments 2P, U, PC and 2P. Lysine remained constant for
treatments NC, U and P and then increased (P<.OS) for PC and 2P.
Isoleucine increased (P<.05) from group NC to 2P to U and then
remained constant for groups P and PC. Cystine was higher (P<.05)
for group PC than others, whereas GLU was highest (P<.05) for Group P.

The effects of treatments and week of sampling on GLY, ALA and
SER are shown in Table 33. In week four none of the treatment differ-
ences were significant for SER, but GLY was higher (P<.05) for group
2P than others, and ALA was higher (P<.05) for groups 2? and PC than
others. In week 9 treatment differences for GLY and ALA were non-
significant, but SER was higher (P<.05) in groups NC and 2P than in
others.

The interaction between treatments and hour of sampling
affected VAL and ASN (P<.25) and the results are given in Table 34.
Plasma VAL was lower (P<.05) in group NC than others, but at 2 hours
none of the treatment differences were significant. At 5 hours VAL
was lower (P<.05) for groups NC, 2P and U than P and PC. The ASP
response at zero hours was higher (P<.05) for group 2P than others,
but none of the differences at 2 hours were significant, and at 5
hours it was lower (P<.05) in groups NC, 2P and U than P and PC.

The three-way interaction between treatments, week and hours
of sampling for ARG, HIS and ASP are shown in Table 35. None of the

treatment differences within hours of sampling for these three amino

152

Table 33.-~Effects of treatments and week of sampling on plasma con-
centrations of glycine, alanine, serine; uM/lOO ml.

 

 

 

Week of Treatment
Amino experi-
acid ment NC 2P U P PC
Glycine: Week 4 32.19: 44.90: 27.84: 31.37 29.58:
Week 9 33.60 33.98 32.35 27.85 27.92
Alanine: Week 4 19.76: 25.62: 20.95: 25.93a 21.52a
Week 9 24.64 24.23 24.81 24.62 24.908
Serine: Week 4 5.47a 6.30a 5.98: 5.42 5.35
Week 9 7.02a 8.09a 5.71 5.87 6.08

 

a,b
ent (P<.05).

Means within a row without a common superscript are differ-

Table 34.--Effects of treatments and hours of sampling on plasma con-

centrations of valine and asparagine; uM/lOO ml.

 

 

 

Hour Treatment
Amino of
acid sampling NC 2? U P PC
Valine: 0 hours 12.95: 17.04: 19.86: 17.09: 20.54:
2 hours 14.75a 15.743 16.368 17.30b 17.51b
5 hours 16.29 15.99 16.59 19.95 22.03
Asparagine: 0 hours 3.75 5.31: 4.33: 3.64: 3.96:
2 hours 3.22a 3.73a 3.75a 3.75b 3.70b
5 hours 3.93 3.59 3.34 4.00 4.32

 

a,b
ent (P<.05).

Means within a row without a common superscript are differ—

153

Table 35.—-Effects of treatments, weeks and hours of sampling on plasma
concentrations of arginine, histidine and aspartic acid;

 

 

 

 

 

 

 

101/1001111.c
Week of Hours Treatment

Amino from .

acid feeding NC 2P U P PC

Arginine; 0 3.64: 5.24: 4.17: 4.63: 4.91:
2 4.17 5.38 4.48 4.76 4.07
5 3.73a 3.36a 7.06b 3.96a 3.88a
0 4.688 7.60b 5.608 5.008 7.865
2 7.498 7.938 4.96b 7.338 7.118
s 6.828 6.678 6.798 8.618 4.38b

Histidine: 0 4.92: 5.34: 4.71: 4.83: 5.53:
2 4.31 5.44 4.99 4.16 3.78
5 5.02a 4.58a 5.06a 4.24a 5.06a
0 6.968 8.188 5.49D 5.555 6.718
2 7.578 6.988 4.82 7.068 6.188
5 6.678 7.108 5.788 6.708 5.668

Aspartic acid: 0 .49: .95: .76: 1.04: .87:
2 .46a .43a .48a .58a .55a
5 .71 .87 .65 .76 .69
0 .83: 1.168 .848‘ .888* .85a
2 1.26 1.28: 1.10; .94:b 1.23:
5 .98 1.04 1.87 1.37 2.13

 

 

a’bMeans within a row without a common superscript are differ-

ent (P<.05).

cStandard error: Arginine I.°733 Histidine :_.49; Aspartic

acid :_.17.

154

acids was significant in week 4. In week 9 ARG was higher (P<.OS) at
zero hours for groups 2P and PC than others, lower (P<L05) at 2 hours
for group U than others, and lower at 5 hours for group PC than others.
Histidine was lower (P<.05) at zero hours for groups U and P than
others, and group U remained lower (P<.OS) than others at 2 hours, but
none of the differences at 5 hours were significant. For ASP the only
significant differences were at 5 hours where groups U and PC were

higher (P<.05) than others.

Effects of level of milk production, week and hour of samplin .--

 

The level of milk production affected GLY (P<.01); LEU, LYS (P<.05);
VAL, ILE, HIS, SER (P<.10); and GLY (P<.25). week of sampling affected
all amino acids studied except THR, CYS and PRO, and the interaction
between level of production and week of sampling affected LEU, ARG
(P<.05), VAL, PHE, GLY (P<.10) and ILE (P<.25). Results are given in
Table 32. Generally, EAA concentrations were lower in high than low
yielding cows, and lower in week 4 than week 9 when the milk yield had
decreased. For amino acids with interactions between level of pro-
duction and week of sampling, the general trend was similar con-
centrations in weeks 4 and 9 in low yielding cows. High yielding cows
had lower concentrations in week 4 than low yielders, but by 9 weeks
the concentrations had increased to or above the measurement for low
producers in week 4. For NEAA the trends tend to be the reverse of
those observed for BAA, but the responses are more variable.

The responses with hours from feeding affected all amino acids
except LYS, ARG, HIS, GLY, ALA, PRO, and SER, and the results are

shown in Table 32. Generally, all amino acids remained constant or

155

decreased between zero and two hours and then increased at 5 hours.
However, MET was lower (P<.05) at S than at zero and two hours in high
producers and remained low (P<.OS) in low producers. The interaction
between week and hour of sampling was significant for GLU, SER and ASP
(P<.OS) and approached significance for ILE, CYS, GLY, and TYR (P<.25).
The results are not reported but the trends are in agreement with those

reported for high and low producers.

8.1.2 Main study of factors affecting_p1asam amino acid concentrations
All cows in the project were bled at 14 days before expected
calving (day -l4) and at days 21, 42, 70 and 126 post-partum, and
individual samples were prepared for amino acid analysis. However,
the data is incomplete due to limitations on the capacity for amino
acid analysis, and only selected samples were assayed. Day 42 after
calving was selected to coincide with peak milk yield. All samples for
that period were assayed, and the results were analyzed according to
the model in (10) after removal of the corvariate. Additional samples
at days -14 and 126 were assayed for cows in the first 7 blocks on
experiment, and their distribution within breeding groups is given in
Table 36. Results for these cows were analyzed statistically according

to the model in (9).

8.1.2.1 Effect of treatments for all cows 42 days post—partum

Plasma concentrations for the complete data at 42 days post-
partum are given in Table 37. The data includes 17 cows per group for
treatments NC and PC and 14 and 15 cows in groups U and AD, respectively.
One cow in group U was deleted because of very high concentrations

coinciding with diagnosed ketosis. None of the observed treatment

156

Table 36.——Distribution of samples assayed at days -14, 42, and 126 on
breeding groups, blocks, and treatments.

 

Days from calving

 

 

 

 

 

Block -14 42 126
Treatment NC PC U AU NC PC U AU NC PC U AU
Best 1 + + + + + + + + + + +
n 2 + + + + + + + + + + + +
H 3 + + + + + + + + + + + +
H 4 + + + + + + + + +
Control I’ + + + + + + + + + + +
Worst 1 + + + + + + + + + + + +
'v 2 + ~+ + + + + + + + +
Number of cows ?77 5 6 6 7 7 6* 7 7 7'17 6

 

 

Table 37.--Plasma concentrations of amino acids for all cows at 42 days
post-partum uM/100 ml.

 

 

 

Amino acid Treatment

NC 90 0 AU s.e.a
Number of cows 17 17 14 15
Valine 20.23 23.58 18.93 19.78 1.76
Leucine 13.56 16.59 13.39 14.05 1.33
Threonine 8.82 8.60 7.81 9.23 .75
Lysine 7.67 7.54 6.97 6.68 .47
Isoleucine 11.78 11.54 10.03 11.18 1.03
Arginine 9.94 7.81 8.08 7.10 1.29
Histidine 8.52 7.21 7.79 7.23 .46
Phenylalanine 6.08 5.86 4.66 5.11 .74
Methionine 2.35 2.91 2.22 2.73 .27
Cysteine 1.90 1.81 1.84 1.67 .17
Glycine 38.22 34.01 32.30 35.53 4.48
Alanine 23.94 22.37 19.92 23.04 2.42
Praline 8.91b 8.78b 8.73 9.96 1.17
Glutamate 17.28 19.49 21.96c 22.65c 1.75
Serine 9.50 9.27 9.91 10.46 .83
Tyrosine 4.89 5.10 5.05 5.47 .44
Aspartate 2.56 3.26 2.70 3.19 .39

 

aStandard error for 17 cows per treatment mean.

b’cTreatment means within a row without a common superscript are
different (P<.05).

157

means were significantly different at (P<.25), except for glutamate and
tyrosine. Glutamate concentrations were lower for groups NC and PC
than for groups U and AU (P<.05).

The relationship between milk production and plasma amino acid
concentrations was studied by the use of stepwise multiple regression
analysis. Milk yields and milk composition values used were the
average for weeks 6 and 7 post-partum. The list of independent vari-
ables to be selected included all amino acids assayed, plasma urea and
ammonia nitrogen and plasma glucose. The determined model for milk
yield is in (14) and explained 61% of the variation in milk yield.

When all essential amino acids were forced into the prediction equation
the amount of explainable variation was only increased to 65% and

details of the results are not reported.

(14) Milk = 47.27 + .6119 LEU + .8435 THR - 1.2290 LYS + 1.0770

HIS - 1.9439 MET - 2.7331 CYS - .5584 GLU - .2049 GLC; R = .78 (P<.005).

Partial Min. Max.

Where Milk is 1111111 yield, kg/d __— 1_3.49 4871
LEU " leucine, uM/lOO ml .39 6.90 43.22
THR " threonine, uM/lOO ml .30 3.42 21.56
LYS " lysine, uM/100 ml -.35 4.06 78.39
HIS " histidine, uM/lOO ml .31 1.93 79.37
MET " methionine, uM/IOO ml -.33 1.14 5.57
CYA " cysteine, uM/100 ml -.27 .62 5.83
GLU " glutamate, uM/lOO ml -.66 8.22 44.97
GLC " glucose, mg/100 ml -.44 ? 70.38

47.27 " a constant .86

The milk fat percentage was not related to any of the variables studied.
Of the variation in the milk protein percentage 18% was explained by
the constant (r=.91; P<.001) plus PHE (r-.28; P<.05) and glucose

(r=.32; P<.01). 0f the variation of milk protein yield 43% was

explained by a constant (r=.84; P<.001) plus GLY (r=.31; P<.05),

158

GLU (r=.62; P<.001) and GLC (r=-.25). The prediction equation for SNF
explained 59% of the variation and included the same independent vari-
ables listed fbr milk yield. A prediction equation for the yield of
calculated milk lactose plus ash was similar to the ones determined for
the yields of milk and SNF.

8.1.2.2 Effects of treatments and time from calving_ongplasma amino
acids in selected cows

 

 

Treatment means, averages for days -l4, 42, and 126, and their
interactions are given in Table 38. Significant effects of breeding
groups and its interaction with treatment and time are shown in
Table A13.

Arginine, MET, PRO and TYR were not affected by any of the
factors studied. Treatments affected CYS, ASP (P<.001) and LYS
(P<.25). Lysine was higher (P<.05) for groups NC and PC than the NPN
groups, and CYS was higher (P<505) for group NC than others. Aspartate
was higher (P<.05) for group AU than others.

Changes with time from calving were significant for PHE, GLY
(P<.001); ILE (P<.01); LEU, THR, SER, ASP (P<.05); CYS (P<.10); LYS,
ALA and GLU (P<.25). Leucine, GLY, ALA, SER, and ASP were highest
(P<.05) at 42 days post-partum. Threonine was lowest (P<.05) pre-
partum. Lysine, ILE, PHE and GLU were highest (P<.05) pre-partum, and
then remained low or increased slightly between days 42 and 126.
Cysteine was highest (P<.05) at 126 days.

The treatment by time interaction affected PHE (P<.001), THR
and ILE (P<.25) and the responses are given in Table 38. Phenylalanine
was higher (P<.05) for group NC than AU at the pre-partum sampling.

However, at 42 days PHE was higher (P<.05) for group PC than others

159

 

 

 

 

 

 

 

 

 

 

 

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160

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161

and differences at 126 were non-significant. Pro-partum differences
in THR were not significant, but at 42 days it was higher (P<.05) for
group AU than others and at 126 days groups U and AU tend to have
higher concentrations than groups NC and PC. Isoleucine was lower
(P<.05) at day -14 for groups U and AU than for NC and PC, but at 42

and 126 days groups PC and AU tend to have higher concentrations than

groups NC and U.

 

DISCUSSION

1. Nutrients in feeds

The assayed nutrient content in concentrate was in good
agreement with that planned by the use of NRC (1971) feed tables.

Hay used was mainly of low quality. These feeds will not be discussed
any further.

Urea and ammonia treatment of corn at ensiling increased total
nitrogen 48 and 38%, respectively (Table 11). The application of crude
protein equivalents was planned to be equal for the two NPN sources.
Lower values for ammonia than urea treated silage may be due to loss
as vapor and method of application. A flow meter for field application
of liquid ammonia would be improved if the dial had finer graduations
at the low end of the scale. An increase in the rate of unloading
after initial matching with ammonia flow could also lower the amount
applied.

The quality of nitrogen in corn silage resulting from NPN
treatment at ensiling was estimated by changes in the water insoluble
and water soluble fractions (Table 11). water insoluble nitrogen
decreased 32 to 33% in untreated and urea treated silages versus a
11% drop in that treated with ammonia. Bergen et al. (1974) suggested
that the higher water-insoluble N in ammonia treated silage was due

to decreased proteolysis of the original plant protein. This

162

163

conclusion has recently been confirmed in studies with 15N-ammonia
treatment of corn silage (Huber, 1977). In urea treated silage 48%
of the added urea was present in that form at feeding; the remainder
of the urea nitrogen was apparently changed to ammonia and other
nitrogenous compounds. These data agree with previous reports (Huber
et al., 1968).

Silages at feeding were all of high quality and were not
adversely affected by NPN treatment. Huber et a1. (1973) found
increased lactic acid in NPN treated silages due to a buffering by
ammonia. Lactic acid in silage BM in the present study average 3.94,
4.70 and 4.35% for untreated, urea and ammonia treated silages, but
the differences were not significant. The low values and a greater
than usual variation may have contributed to the lack of significant

differences between treatment in lactate content.

2. Feed and nutrient intakes

The changes in intake of individual feeds with time after
calving were different between treatments, but for total dry matter
this interaction only approached significance. As suggested, the
change from a significant interaction for individual feeds to a less
pronounced effect for total dry matter was due to fixed feeding of
concentrate and a compensatory intake of corn silage. However, it
should be noted in Figures 6 to 8 that group NC, which received most
concentrate (because of highest milk yields) also ate slightly more
corn silage than other groups; whereas AU, which received the second
largest quantity of concentrate, ate the least corn silage. Intake

of total ON in group NC (12.5% CP) was slightly higher than in other

164

groups (15 to 16% CP) throughout the entire 20 weeks. These results
are in agreement with those of Polan et al. (1976) who found highest
intakes at 12.8% CP, but in contrast with others (Sparrow et al., 1973;
Grieve et al., 1974; Cressman et al., 1977) who reported increased
intake up to 17% CP. In the present study, cows fed the urea supple-
mented ration (U) received equivalent to 247 g urea per day (Table 39).
The amount of urea consumed by the U group, intake of NPN, and the
fraction of total N fed as urea or ammonia all approached the upper
limits recommended by NRC (1971).

Table 39.--Intake of urea equivalents in the different periods from
calving by cows fed non-protein nitrogen supplemented

 

 

 

 

diets.

Urea equivalents, Urea equivalents
g/d from silage, %

Period

Group U Group AU Group U Group AU

Weeks 3 through 6 225 208 47.4 37.0
" 7 " 10 247 216 48.5 37.1
" ll " 15 234 192 55.5 44.8
" l6 " 20 220 188 57.2 49.3

 

None of the sources of supplementary N in groups PC, U and AU
significantly affected DM intakes (Table 14) even though urea fed cows
consumed less dry matter than other groups during weeks two to seven.

Dry matter intake per unit body weight also were not signifi-
cantly affected by treatments and peaked at 3.5% during weeks 7

through 11. At no time form weeks 3 to 20 did average intakes fall

165

below 3% of BW. By this method of calculation group NC also tended
to have highest intakes.

The intake of crude protein was approximately 500 g per day
lower for group NC than others, but "true protein" (TN-SOLN-ADN) intake
was equal for NC and U, with AU 100 g higher due to the suggested
reduction by ammonia of plant protein proteolysis. Group PC received

400 to 500 g more true protein than other groups.

3. Production of milk and milk components

Treatments did not significantly affect the production of milk
or milk components, except for a depressed production in the urea group
for weeks 3 through 6 in the high producing cows. The decrease may
have been due to slightly lower intake of cows fed urea. The longer
period of body weight losses of the urea-fed cows also suggested more
energy stress.

The similar production of high yielding cows fed approximately
12.5 and 16% CP (NC vs. PC and AU) is in sharp contrast to NRC
recommendations (NRC, 1971) and with results or suggestions of Gardner
and Parker (1973), Sparrow et a1. (1973), Grieve et a1. (1974),
Cressman et al. (1977) and Satter and Roffler (1975). However, these
data agree with Thomas (1971) who reported normal persistencies in cows
producing about 30 kg milk per day fed rations containing 12.5 to 13.6%
CP. Also, Chandler et a1. (1976) obtained similar milk yields in cows
fed 12.5 and 15.5% CP in ration DM. In the present experiment, dry
matter intakes and energy densities were not significantly affected by
treatments. In trials by Sparrow et al. (1973), Grieve et al. (1974),

and Cressman et al. (1977) the energy intakes increased with increasing

166

CP in the ration DM. Lamb et al. (1974) showed increased milk yields
when the rate of grain feeding was increased from .25 to .63 kg per kg
milk, thus raising the intake of total dry matter. Schwab et al.
(1971) and Moe and Tyrrell (1977) reported increased digestibilities
with increased protein in the diet.

The urea fermentation potential (UFP), as defined by Burroughs
et al. (1973, 1974a,b, 197Sa,b), are estimated for rations used in the
present experiment in Table 40. These calculations suggest that 244
to 267 g urea could be utilized if bypass of true protein is 40%, but
UFP is decreased to 237 to 259 g urea if rumen bypass of true protein
is decreased to 10%. Tables 39 to 40 shows that only group U in weeks
7 through 10 received urea equal to the UFP if 10% bypass is considered.

The system of metabolizable protein suggested by Satter and
Roffler (1975) was applied to results of the present study. Crude
protein in DM for group NC was less than 13% and RAN values were in
agreement with the suggested critical value for rumen ammonia utiliza-
tion. Thus metabolizable protein for group NC may be calculated
according to (4). Satter and Roffler (1975) suggested zero utiliza-
tion of supplementary NPN if RAN concentrations exceeded 5 mg per 100
ml fluid, whereas 30% of true protein may be utilized. Therefore,

metabolizable protein for groups PC, U, and AU was calculated as in

(15):
= _. iv
(15) MP MPNC + (TP TPNC) .30.
where MP is metabolizable protein for groups PC, U or AU;

MPNC is metabolizable protein for group NC;

TP is "true protein" intake for groups PC, U or AU (Table 15);

167

TP is "true protein" intake for group NC; and .30 is a

NC
constant

(see Satter and Roffler, 1975).

Metabolizable protein used for milk protein synthesis is calculated
in Table 41. By this method, coefficients may be expected equal for
all diets. However, Table 41 shows that the nutritive value of the
plant protein (groups PC, U and AU) was depressed when protein in DM
exceeded 13%. Moreover, protein was less efficiently utilized in cows
receiving the most natural protein (PC) compared to those on NPN (U
and AU).

Production results of this experiment suggest that the CP
requirement of high yielding dairy cows fed corn, corn silage, limited
hay rations is not more than 13% CP in DM. Protein reserves, released
early in lactation (COppock et al., 1968; and Paquay et al., 1972), may
have compensated for a dietary deficit, but weight changes before and
after minimum BW and the number of weeks until minimum weight was
attained were not different for groups NC, PC and AU. Had the NC cows
incurred a protein deficit of 3% of the dry matter intake, compared
to others, then body weight changes should have been sensitive enough
to detect the differences. For example, to increase dietary CP from
12.5 to 15.5% for group NC would require approximately 600 g CP per day
innweeks 3 through 20. If the efficiency of utilization of dietary
protein for milk protein synthesis is 33%, as in this experiment
(Table 42), then protein already assimilated should be approximately
three times more efficient for milk protein production. Thus, the net
tissue protein loss that would equal a 600 g/d dietary deficiency

might be estimated at 200 g per day. If body weight were 20% protein

Table 40.--Urea fermentation potential (UFP;a g urea/d) of high

168

concentrate, corn silage, limited hay rations used for

treatments in the different periods from calving.

 

 

 

 

 

Treatment
Period

NC PC U AU

40% bypass of true protein:
Weeks 3 through 6 264 262 263 261
" 7 " 10 267 263 259 258
" ll " 15 263 258 252 251
" 16 " 20 259 256 244 248

10% bypass of true protein:
Weeks 3 through 6 259 247 252 248
" 7 " 10 256 245 248 246
" 11 " 15 253 245 242 239
" l6 " 20 253 244 234 237

 

aBurroughs et al. (197Sa,b).

Table 41.--Utilizationa of metabolizable protein? for milk protein
production in dairy cows in the different periods from

calving; %.

 

 

 

 

Treatment
NC PC U AU
All cows:
Weeks 3 through 6 S4 46 49 51
" 7 " 10 51 42 47 48
" 11 " 15 54 49 51 50
" l6 " 20 51 47 49 48
High producers:
Weeks 3 through 6 53 48 49 53
" 7 " 10 52 46 47 51
" 11 " 15 51 48 50 50
" 16 " 20 55 53 54 54

 

a(Milk protein*100)/(metabolizable protein).
bSatter and Roffler (1975).

169

Table 42.--Protein availability at the abomasal level, its relation to
present standards, and utilization for milk protein pro-
duction in dairy cows in the different periods from calving.

 

 

 

 

 

 

 

Treatment
Period
NC PC U AU

Protein at abomasum,a kgld}
Weeks 3 through 6 3.03 2.99 2.87 2.96

" 7 " 10 3.17 3.24 2.81 2.83

" 11 " 15 2.78 2.95 2.70 2.63

" 16 " 20 2.75 2.77 2.50 2.50
Protein balance,b %:
WeEks 3 through 6 105 101 93 103

" 7 " 10 105 111 97 105

" ll " 15 100 108 100 100

" l6 " 20 105 108 110 102
Utilization of crudeproteinj,c %:
Weeks 3 through 6 39 30 31 33

" 7 " 10 38 28 29 31

" ll " 15 39 32 30 32

" 16 " 20 37 32 30 31
Utilization of protein at abomasum,E_%:
Weeks 3 through 6 33 31 31 32

" 7 " 10 31 29 32 34

" ll " 15 33 31 32 33

" 16 " 20 31 31 33 33

 

a16.5 g microbial protein per 100 g digestible organic matter
(Bucholtz and Bergen, 1973).

bBalance = (protein per abomasum)/(NRC(l9/l) requirement).

cUtilization = (milk protein)/(avai1able protein).

170

(Huber, 1975) then a weight loss of 1000 g might be incurred for every
200 g of protein loss. The weight loss in the first four weeks
approached the above value for group NC but similar losses were also
observed for the other groups. However, weight gains were encountered
for groups NC, PC and AU after the second week on trial. Therefore

one might conclude that the protein deficits for group NC was less

than estimated, or that changes in tissue composition actually occurred
which were not detected. The important question of how dependent are
early lactation cows on body reserves to sustain the milk protein
yields still remains unanswered.

The in vivg_production of microbial protein have been estimated
at 32 g per kg fermentable organic matter (e.g., Allen and Miller,
1976). Bucholtz and Bergen (1973) suggested 16.5 g microbial protein
per 100 g organic matter digested when the rumen turnover of protein
was at 25%. Protein at abomasum in the present study were calculated
as in (16) and (17):

3
(16) DOM = 1:1 (IT* ON * OM * TDN)

(17) ABPR = [DOM - (TP * BP)] * .165 + (TP * BP)

where DOM is intake of digestible organic matter, kg;
i is 1 for concentrate; 2 for corn silage; and 3 for hay;
IT is intake of feed on as is basis, kg;
DM is dry matter in feed, %;
OM is dry matter minus ash %;
TDN is total digestible nutrients, %;

ABPR is total protein at abomasal level, kg;

171

TP is true protein, kg;

BP is plant protein bypassing rumen fermentation, %; and

.165 is kg microbial protein per kg DOM.
Estimates of total protein available at the abomasal level as well as
the balance of abomasal protein in relation to recommended standards
(NRC 1971), and the utilization of CP and abomasal protein for milk
protein synthesis are given in Table 42. By this method of calculation
available protein and its utilization for milk protein synthesis were
equal for all groups, and were equal to or slightly higher than
present standards. However, for group NC available protein exceeds the
CP intake and cannot occur if the pool of body protein is constant.
Utilization of CP and protein at the abomasal level was the same in
groups PC, AU and U; but in group NC the CP was utilized more effi-
ciently than protein at abomasum. The discrepancy may be explained by
the amount of plant protein bypassing rumen fermentation (Table 43).
If only 10% true protein bypass the rumen, then protein at the abomasum
would be equal to the CP intake for group NC. In groups PC, U and AU
protein at the abomasum is equal to NC when 10 to 20% of true protein
bypass the rumen, and the utilization for milk production is 40% for
all groups.

The production results obtained and metabolic factors con-
sidered all support the previous statement that high yielding cows in
early lactation fed corn, corn silage, limited hay rations require no
more than 13% CP in the diet. This conclusion is supported by Thomas
(1971), Chandler et al. (1976), and Huber (1976). The value of NPN
in early lactation rations still remains unsolved; but Roffler and

Satter (1975b), Burroughs (l975a,b) and Huber (1975, 1976) all

172

Table 43.--Total protein and true protein available at the abomasal
level in dairy cows in weeks 7 through 10 post-partum.

 

 

 

 

 

True protein bypassing Treatment
the rumen, % NC PC U AU
Totalpprotein at abomasum, kg/d:
0 2.48 2.35 2.23 2.23
10 2.65 2.57 2.40 2.42
20 2.82 2.79 2.56 2.60
30 3.00 3.02 2.73 2.78
40 3.17 3.24 2.90 2.96
True plant protein at abomasum, kELQE
0 0 0 0
10 .21 .27 .20 .22
20 .41 .54 .40 .43
30 .62 .80 .60 .65
40 .82 1.07 .80 .87

 

reported that the utilization of some NPN in rations containing 12 to

13% CP was as high as for plant protein, particularly when high energy

rations are fed. Huber (1975) provided evidence that high yielding

cows fed less than 12% CP rations had lesser persistencies than those

fed 13 to 14% CP. These trials also showed that milk production in

cows fed rations with ammonia treated corn silage was as persistent

as in cows given plant protein as the supplementary N source.

Polan

et al. (1976) reported that urea supplementation was advantageous at

low levels of CP (9.4%) in the basal ration, but had a negative effect

at increased protein.

4. Rumen metabolities

The molar concentration of total rumen VPA's increased from

the pro-partum sampling until 70 days post-partum and then decreased

slightly. The increase was due to increased concentrations of all

173

acids. The molar percentage of acetate decreased between pre— and
post-partum sampling, and other acids increased. The observed changes
are characteristic for changes from all forage to high grain rations.
Generally, molar concentrations of rumen VFA's and total acids were
highest for group PC and AU. However, iso-butyrate and iso-valerate
for group AU were equal to those in groups NC and U. The increased
iso-acid concentrations for group PC may be due to greater deamination
of amino acids from soybean meal.

Rumen ammonia nitrogen (RAN) in pro-partum samples was similar
for groups NC, U and AU and higher than in group PC. This may be
explained by the distribution of calvings. Table 44 shows that RAN
was lower in dry cows at pasture than in those fed indoors, and that
the lower pre-calving value for group PC can be attributed to unequal
numbers in the groups. The values for dry cows indoors fed corn silage
with added urea are in agreement with treatment values reported by
Polan et al. (1976), and higher than all post-partum values found in
the present trial (Table 27).

Post-partum concentrations of RAN for group NC was in close
agreement with the critical value of 5 mg per 100 ml suggested by
Roffler and Satter (1975). For supplemented groups, RAN was lowest
fer group PC and highest for group U with AU intermediate. The
slightly higher RAN concentrations for group PC than NC at days 21
and 126 suggests that considerable amounts of the supplementary soy-
bean meal given group PC escaped rumen degradation. A greater rumen
bypass of plant protein on PC than NC is in contrast to the calcula—

tions for protein available at the abomasal level (Table 42). This

174

Table 44.--Rumen ammonia nitrogen in dry cows at pasture and indoors
fed corn silage plus urea added at feeding (mg/100 ml fluid).

 

 

 

Treatment All
NC PC U AU °°"5
Pasture:a
Number of cows 9 12 10 10 41
Average 5.59 4.87 7.66 7.96 6.46
Standard error 1.76 1.34 3.12 2.51 1.10
Indoors:b
Number of cows 8 5 6 7 26
Average 19.64 24.71 27.57 27.65 24.60
Standard error 2.50 3.30 4.92 2.32 1.68

 

aSamples taken before October 23, 1976.

bSamples taken after October 23, 1976.

discrepancy may be related to time of sampling in relation to time of
feeding.

Despite the fact that total NPN intake was higher for group U
and AU after than before calving, RAN concentrations were higher pre-
partum. This was probably because more grain was fed post-partum which
increased efficiency of microbial utilization of ammonia. The
insoluble nitrogen levels in silage treated with NPN at ensiling as
opposed to those treated at feeding might also have contributed to the
lower RAN in post-partum cows.

The RAN values for NPN diets in the present study did not
exceed those reported by Roffler and Satter (1975b) for all plant
protein diets with comparable CP contents. The relatively low RAN

concentrations should not be attributed to a decreased rate of feed

175

consumption, but to a synchronous release of ammonia and energy for

microbial utilization.

5. Blood metabolites

Plasma glucose was lowest at 21 days post-partum but was not
affected by treatments nor the interaction between treatment and time
after calving. Plasma glucose concentrations in the present experiment
are in agreement with arterial concentrations reported by Bickerstaffe
et al. (1974) and Annison et a1. (1974), but lower than those reported
by Derring et a1. (1974) and Vik-Mo et al. (1974a) who infused glucose
and/or casein per abomasum. The concentrations at 21 days post-partum,
when the energy intake was still slightly less than the NRC recommenda-
tions, approached values for fasting cows (Kronfeld et al., 1968).

Post-partum PUN values were lowest fer group NC, highest for
group U with groups PC and AU intermediate. The low PUN concentrations
for group NC are in agreement with lowest RAN values and highest
coefficients for utilization of metabolizable protein. Furthermore,
PUN values for group NC are in agreement with those reported by Huber
and Thomas (1971) when cows were fed corn silage treated with urea at
ensiling. Comparable PUN concentrations for groups PC and AU, despite
differences in RAN concentrations indicate similar losses in urine
whether the supplementary protein source is soybean meal or NPN.
Whether the discrepancy in RAN and PUN concentrations for group PC is
due to deamination of amino acids from soybean meal bypassing rumen
degradation, or is an effect of sampling hour, is unknown. Highest
PUN concentrations in group U indicate less efficient utilization of

urea than ammonia when added at ensiling, and may be related to the

176

presence of non-hydrolyzed urea in the silage and less protection of
plant protein. Plasma urea nitrogen (PUN) concentrations in the
selected cows were in general agreement with overall treatment values
and showed little diurnal variation. The PUN concentrations were
less sensitive than RAN to time of sampling or to differences in
feeding.

Plasma ammonia nitrogen (PAN) in selected cows tended to show
diurnal rhythm at 112 days post-partum with lowest concentrations at
0800 hours (Figure 21). Group U maintained slightly higher PAN con-
centrations than the other groups for the samplings at 1530, 1900 and
2300 hours. Pro—partum PAN were higher than post-partum concentrations
and decreased throughout lactation. Differences between treatments
were small but group U tended to have the highest concentrations.

This is in agreement with treatment effects on PUN. Similar PAN con-
centrations for groups PC and AU suggests similar utilizations of N in
soybean meal and corn silage treated with ammonia at ensiling. Highest
PAN values for group U support the suggested decreased utilization of
urea in corn silage when compared to ammonia treated silage.

The higher PUN concentrations for groups PC and AU than NC are
in agreement with more efficient use of dietary protein for production
of milk protein. Elevated PUN levels were also reported for cows
receiving high levels of abomasally infused casein (e.g., Vik-Mo et al.,
1974b). The high PUN and PAN levels in plasma and the lower coeffi-
cients of utilization of metabolizable protein for the groups fed
supplementary protein reemphasize our suggestion that about 13% CP is
sufficient for high yielding cows in early lactation consuming the

type of diet fed in these studies. The observed discrepancies between

177

RAN and PUN concentrations for cows fed supplemental N suggest that
PUN might be a better measurement of nitrogen utilization in dairy
cows than RAN as suggested by Roffler and Satter (197Sa,b). Derring
et al. (1972) observed decreased PUN when cows fed according to NRC
feed standards received abomasally infused casein. However, Vik-Mo
et al. (1974b) fed cows above NRC feed standards and observed in-
creased PUN when casein was infused into the abomasum.

Factors affecting plasma amino acid concentrations were
studied in an initial experiment and the main experiment. Factors
studied included treatments, level of milk production, stage of
lactation and sampling hours in relation to feeding. In the initial
experiment VAL, LEU, THR, LYS, ILE, ARG, HIS, PHE, ALA, GLU, ASN and
ASP were lowest for high yielding cows or for the samples taken during
week 4 ver§g§_week 9 of treatment. Methionine, GLY, SER and TYR
decreased, whereas, CYS and PRO remained constant. In the main
experiment amino acids were generally highest at the pro-partum
sampling, decreased at 42 days and then increased at 126 days. How-
ever, LEU, GLY, SER and ASP were highest at 42 days, and PHE continued
to decrease throughout lactation.

Plasma amino acid concentrations arise from the balance
between rates of digestion, absorption, and synthesis, as well as
tissue demand and breakdown (Albanese, 1959; Gitler, 1964; Harper,
1968; Munro, 1970; McLaughlan, 1974). Lowest PAA concentrations in
high yielding cows and at 42 days post-partum are in agreement with
highest demands for milk synthesis. However, dramatic increases with
decreasing milk yield are prevented by normal decreases in feed intake

with advancing lactation. Decreasing plasma concentrations of some

178

amino acids with decreasing milk yield may be related to an increased
availability of the limiting amino acid relative to milk yield; hence,
an increased demand for these amino acids would arise. The constant
CYS concentrations cannot be explained by either a decreased demand or
an increased availability.

Neither Halfpenny et al. (1969a,b) nor Vik-Mo et a1. (1974)
observed diurnal rhythms for PAA in dairy cows milked twice daily.

In the initial experiment PAA concentrations generally remained con-
stant or decreased slightly at two hours when compared with samples
taken before feeding, and then increased at 5 hours. The described
trends with time may be attributed to changes in demand for milk
synthesis or in the availability of amino acids.

In the initial experiment treatments significantly affected
plasma concentrations of VAL, LEU, LYS, ILE, HIS and CYS and non-
significant trends were observed for other EAA. None Of the treatment
differences in the main experiment at 42 days post-partum were sta-
tistically significant. Increased PAA concentrations have been related
to increased availability of amino acids in cows given abomasal
infusions of casein (Spires et al., 1973; Hale et al., 1972; Broderick
et al., 1970; Derring et al., 1974; Vik-Mo et al., 1974b) and IV
infusion of individual amino acids (Fisher, 1969, 1972; Teichman
et al., 1969; Fisher and Erfle, 1974).

Interpretation of amino acid profiles without quantitative
estimates of the availability of amino acids may be improved by
adaptation of the methods used in amino acid requirement studies in
nonruminants. In such experiments the amino acid under investigation

remains constant in plasma until the requirement is met and then

179

increases linearly (two-phase response; e.g., Zimmerman and Scott,
1965). The feasibility of this method in ruminants was demonstrated
by Fenderson and Bergen (1975) in steers given graded levels of an
amino acid per abomasum. However, it should be emphasized that the
method may give erroneous results if the availability and the demand
are changed simultaneously.

The plasma concentrations of EAA in high and low yielding cows
in the initial experiment and at 42 days post-partum in the main study
are plotted in Figures 19 to 22. In these figures arrangement of
treatments on the abcissa was in order of ascending plasma concentra-
tions, and an assumed increased availability with increasing plasma
concentrations. Figure 19 to 22 shows that plasma concentrations of
these amino acids in the main experiment were higher than in both high
and low yielding cows in the initial experiment, except for THR, MET
and CYS. In the initial experiment THR, MET and HIS were lower in low
than high yielding cows, and CYS tended to remain constant. Tentative
two-phase responses are seen fer VAL, LEU and LYS in high yielding
cows in the initial experiment; and in the main study VAL, LEU and HIS
may be implemented. None of the amino acids showed two-phase responses
in low yielding cows.

Those three or four amino acids which might be limiting in the
various groups are in Table 45. In the initial study VAL or LEU
appeared co-limiting in the negative control and NPN groups, except
for group P. Lysine was limiting or co-limiting for groups NC, U and
P in the initial experiment. Histidine was identified as limiting in
group PC in both experiments and co-limiting in group AU. The

limiting amino acids identified by the above method are also those

180

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

24 F
_
Valine
22 -
b
p
Eézo -' "
g; '7
fl - -
~181-
5' 7 '1 F'
I:
< P
>
H
§§I6 -
15
O.
p
'4 hil—ihfithJL—H—L
2p NC 0 P PC NC 2P P 0 PC 0 AU NC PC

TREATMENT

'6 Leucine
E-
E
\14.
m
5' 1. r57
5:12 - F' F' F'
5
15
CL

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NC 0 2P PG P 0 NC AU PC
TREATMENT

Figure i9. Plasma valine and leucine responses to treatments in the initial and the
main experiment.

181

3

PLASMA THREONINE, ,uM/IOO 1111
\J to
I I r U

E l

Threonine

\O

 

 

 

 

 

 

 

 

 

 

| .11...

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2P NC U PC P NC PC 2P P U U PC NC AU
TREATMENT
8
[ Lysine
'E - r. F1 '1
'§ F'
\ 7 '- I
E "l
as n 1 I
U: n
2
5;
:1 6 F I
g «undul-
5 NC P u 2P PC P u NC Pc 2P AU 0 PC NC
0- TREATMENT
__l2
5 F isoleucine H H
O
O 1.
z _ "l
3
l0 P
u: H 1
SE
8 r- '1
LLI
..J
£3 8
5 MUM—d
o- .
2P NC PC U . P NC 2P U P PC U AU PC NC
TREATMENT

Figure 20. Plasma threonine, lysine and isoleucine responses to treatments in the
initial and the main experiment.

182

 

 

 

 

 

 

 

 

 

 

 

' l"
Histidine
a l- "‘—
1 W

_7 - l T
E
O
O
{I
z
16
111‘
E
E3
p.
(0
5:5
§ 1
U)
5 U PC P NC 2P P U PC 2P NC PC AU U NC
“- TREATMENT

6 -

Phenylalanine F
5 r I.

A

PLASMA PHENYLALANINE,)uM/|00 ml
Ld

 

 

 

 

 

 

 

 

 

 

 

U 2P NC P PC 2P NC PC P U U AU PC NC
TREATMENT

Figure 2|. Plasma histidine and phenylalanine responses to treatments in the
initial and the main experiment.

PLASMA METHIONINE

PLASMA CVSTEINE

183

 

 

 

 

 

 

 

 

S P Methignine
E4. F
83' i“ "
g2. i' F’FF r'
l P
Lila-1.10

 

 

 

 

 

 

 

 

 

 

 

 

 

U PC P NC 2P NC P PC 2P U U NC AU PC
TREATMENT

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cysteine
rrrr F”
'E
§§ i.fl..LHi.lLL..LJs.JaJ
\
33 P 2P 0 NC PC AU PC u AU
TREATMENT

Figure 22. Plasma methionine and cysteine responses to treatments in the

initial and main experiment.

184

Table 45.-~Amino acids potentially limiting milk protein production of
dairy cows in the initial and the main experiment, as well
as in abomasal protein.

 

Amino acid
Treatment

 

VAL LEU THR LYS ILE ARG HIS PHE MET CYS?

 

Initial experimenta:

 

 

 

 

m * * * * *

2P * * 'k *

U * * * *

P *

PC * *

Main experimenta:

m 'k *

PC *

U 'k *

AU * * *

Main experimentb:

NC (213(3) (1) (1?)
PC (2) (3) (1) (1?)
U (3) (2) (1) (1?)
All (2) (1) (3) (1?)
Calculatedcz

Bacteria (3) (2) (1)

Corn (3) (1) (2)

SW 6 (3) (2) (1)
75%+25%f (2) (3) (l)

75%+25% (2) (l) (3)

 

8Determined by two-phase response in plasma amino acid con-
centrations.

bDetermined by minimum.plasma flow at the mammary gland for
output in milk.

cDetermined by the ratio between amino acid in "feed protein"
and milk protein.

dSoybean meal.
eAbomasal protein is 75% microbial protein and 25% corn protein.

£Abomasal protein is 75% microbial protein and 25% soybean meal
protein.

gRanking of limiting amino acids (one first limiting).

185

which are generally in least supply when bacterial protein constitute
the major fraction of protein available at the abomasal level, or when
abomasal protein consists of 75% bacterial protein plus 25% corn or

soy protein (Table 45). However, the amino acids identified by this
method are in contrast to previous investigations (Table 7); except for
LYS and H18 reported by Spires et al. (1973).

These discrepancies may be related to arterio-venous differ—
ences. Verbeke and Peters (1965), Clark et al. (1974), Derring et a1.
(1974), and Bickerstaffe et al. (1974) reported similar ANV for indi-
vidual amino acids, but their research also showed highly variable A-V
among amino acids. Chandler and Polan (1972) suggested minimum
transfer coefficients as a method of comparison for the availability
of amino acids for milk protein synthesis. In the present main study
the drain on the amino acid pool was considered largest at 42 days
post-partum. The minimum plasma supply for amino acid output in milk
was calculated form average A-V for individual amino acids (Verbeke and
Peters, 1965; Clark et al., 1974; Derring et al., 1974; and Bicker-
staffe et al., 1974), amino acid composition of milk (NRC, 1969; NDC,
1965; Smith, 1971), and determined plasma amino acid concentrations
and milk protein yields. The calculations are given in Table A14 and
Table 46. Table 46 shows that MET was the most limiting in groups NC,
U and PC, whereas PHE was indicated for group AU. These amino acids
are also those most often indicated by other researches (see Table 7).
However, it should be noted from Table 46 that the required plasma
flow per day for several amino acids are very close to those for the
amino acid identified as first limiting. Thus only slight changes in

A-V may rearrange the three or four most limiting amino acids. Davis

186

Table 46.--Estimated minimum flow of plasma to the mammary gland fer

the output of amino acids in milk protein produced at 42
days post-partum in the main experiment, and the ranking

 

 

 

 

 

 

 

 

 

of amino acids.
Treatment
Amino acid NC PC

MPFa Rankb MPF Rank MPF Rank MPF Rank
Essential amino acids:
Valine 91 6 75 7 88 6 90 6
Leucine 111 2 70 8 108 3 104 4
Threonine 98 3 97 2 100 4 91 5
Lysine 96 4 93 4 96 5 107 2
Isoleucine 83 7 81 6 88 7 84 7
Arginine? 36 9 44 9 40 9 49 9
Histidine 74 8 83 5 73 8 81 8
Pheynlalanine 95 5 94 3 111 2 110 l
Methionine 122 1 99 l 121 1 105 3
Non-essential amino acids:
Cysteine I40 140 132 156
Glycine 102 109 109 106
Alanine 123 126 134 124
Proline 346 338 319 300
Glutamic acid 136 115 97 101
Serine 150 148 130 132
Tyrosine 126 117 110 110
Aspartic acid 624 472 532 490

aMiammary plasma flow; hl/d.
bRanking of amino acids according to required MPF. .One for

highest flow.

187

and Bauman (1974) failed to identify a pathway for the generation of
CYS from MET. The required plasma flow for the output of CYS in milk
was larger than for any of the EAA, but the flow also exceeded that
expected for the output of milk if one were to assume a constant of
500 liters of blood per liter milk and a packed cell volume of 30%.
The limiting amino acids identified by plasma amino acid
concentrations were also those included in a stepwise multiple

regression (l4). Amino acids with positive partial correlations

 

(HIS, LEU, THR) indicate they are limiting, whereas those with negative
partial correlations are those removed from plasma when the avail-
ability of the limiting nutrient is increased. Negative effects of
increased methionine have previously been reported by Fisher (1972),

Vik-Mo et a1. (1974), and Schwab et al. (1976).

SUMMARY AND CONCLUSIONS

The review of literature showed that mammary blood flow,
arterial metabolite concentrations, and the in_§itg metabolism are
major determinants of milk yield. The mammary blood flow to milk ratio
remain constant at approximately 500:1. The uptake of glucose and
amino acids remain constant as a percentage of arterial blood con-
centrations. The biosynthesis of milk constituents appear to be inter-
related, and the yield was increased during increased substrate
availability (abomasal and/or intravenous infusion); but there has not
been a concensus on the nutrient which is most limiting. Recent
studies of the protein requirement in high yielding cows have not
shown conclusive evidence of increased yields by protein supplementa-
tion beyond the amounts recommended by the National Research Council
(1971). Crude protein has been used extensively in dairy cattle ration
formulation but metabolizable protein and urea fermentation potential
have been introduced as alternatives which are more in agreement with
digestible protein in nonruminants. These methods allow extensive use
of NPN in cows producing less than 20 kg milk per day, but certain
assumptions and a weak data base render them useless in high producing
dairy cows.

The present experiment was conducted: (1) to test protein

requirements and the feasibility of substituting two sources of

188

189

nonprotein nitrogen (NPN) for natural protein in rations for high
yielding cows early in lactation, and (2) to measure the effect of
protein source on critical rumen and blood metabolites. This was
tested in 68 lactating Holstein cows in weeks 3 through 20 post-partum.
All cows were fed the same ration, containing NPN, from four weeks
pre-partum through the second post-partum. An equal number of cows
were assigned to four treatments. The first two groups received plant
protein as the only nitrogen source, one 12 to 13% CP (group NC) and
the other 15 to 16% (group PC). Two groups were also fed rations with
15 to 16% CP with a approximately 25% of total nitrogen as NPN. Corn
silage treated with .65% urea (group,U) or .40% ammonia (group AU)
at ensiling was fed with concentrate containing 1.25% urea. Corn
silage was fed gd_libitum, hay was restricted to 2.2 kg per day, and
grain was fed at one kg per 2.5 kg milk in the first 10 weeks post—
partum and then reduced to one per 3 kg milk during weeks 11 to 20.
Dry matter and nutrient intakes were not statistically differ-
ent between treatments except for crude protein, water soluble nitrogen
and crude protein content in dry matter. However, group NC tended to
have highest intakes of total dry matter and of dry matter per unit
body weight. Actual milk yields for all cows average 29.9, 27.9, 27.0
and 27.7 kg per day for groups NC, PC, U and AU, respectively.
Adjusted average milk for cows producing more than 25 kg milk per day
in the second week post-partum.were 32.6, 34.8, 32.1 and 32.8 kg per
day in groups NC, PC, U and AU. Neither milk yield nor milk components
were affected by treatments except for slightly lower yields for high

producers in group U during weeks 3 through 6, post-partum.

190

Because average production for groups did not differ it was
concluded that the crude protein requirement of high yielding cows fed
corn, corn silage, and limited hay does not exceed 13% CP in DM. This
conclusion was supported by rumen and blood metabolite concentrations.
This study did not solve the question of the feasibility of using NPN
as a substitute for plant protein in rations for high yielding cows.
However, numerous experiments and theoretical considerations have
demonstrated that NPN and plant protein are equally beneficial in
high energy rations containing less than 13% crude protein in dry
matter.

Rumen volatile fatty acids, plasma glucose, plasma ammonia
nitrogen and amino acids were not significantly affected by protein
treatments. Rumen ammonia and plasma urea nitrogen were lowest in
group NC highest in U with PC and AU intermediate. The higher values
for group U than AU were attributed to higher losses of ammonia during
ensiling and to the presence of more plant protein in ammonia than urea
treated silages. Plasma urea nitrogen in groups PC and AU were equal,
despite lower rumen ammonia nitrogen in PC than AU. The calculated
supply of protein at the abomasal level was equal in all groups when
10 to 20% of true protein consumed bypasses rumen fermentation. The
equal plasma urea nitrogen concentrations in groups PC and AD,
irrespective of the site of’deamination, suggest similar losses of
urea, whether supplementary nitrogen was from soybean oil meal or
ammonia added to corn at ensiling plus urea in the grain. Based upon
these observations and the effects of site and hours of sampling upon

rumen ammonia nitrogen, it is concluded that plasma urea nitrogen is

191

superior to rumen ammonia nitrogen as a measurement of protein avail-
ability.

Factors affecting plasma amino acid concentrations were studied
in two experiments. Essential amino acid concentrations were generally
lowest when the demand was highest (high vg£§n§_low producers; and
early verggg late lactation) and/or the supply was lowest (one vs,
five hours after feeding). The method used in amino acid requirement
studies with non-ruminants was adapted to these experiments. Based
upon this method histidine and threonine were limiting or co-limiting
on all treatments during the initial experiment.and for PC and AU in the
main experiment. Valine and leucine were co-limiting in all groups fed
negative control or NPN rations except one in the initial experiment.
In the initial experiment, lysine was also co-limiting for cows fed
the negative control diet and those supplemented with urea- or ammonia-
treated silages.

From the ratio of an amino acid in abomasal and milk protein,
histidine is first limiting when rumen bypass of plant protein is less
than 25%. Milk production was regressed on plasma amino acid con-
centrations and positive partial correlations were found only for
histidine, valine and threonine. Lysine, methionine, cysteine and
glutamic acid concentrations were negatively correlated with milk
yield. The minimum plasma supply at the mammary gland for the output
of amino acids in milk protein produced was calculated for results
at 42 days post-partum in the main experiment. By this method the first
limiting amino acid was methionine in groups NC, PC and U, but
phenylalaline in group AU. The failure to clearly identify the

limiting amino acid for a given treatment may be related to a nearly

192

identical minimum blood flow to the mammary gland fer several essential
amino acids and slight changes in the arterlovenous differences would

change the rankings and suggested degree of limitation.

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Tyrrell, H. F., D. J. Bolt, P. W. Mae and H. Swan. 1972. Abomasal
infusion of water, casein, or glucose in Holstein cows. J.
Anim. Sci. 35:277 (Abstr.).

Udy, D. C. 1971. Improved dye method for estimating protein. J. Am.
Oil Chemists Sac. 48:29A.

Van Horn, H. H., D. R. Jacobson and A. P. Graden. 1969. Influence
of level and source of nitrogen on milk production and blood
components. J. Dairy Sci. 52:1395.

Verbeke, R., M. Lauryssene1 G. Peeters and A. T. James. 1959. Incor-
poration of DL-[l- 4C] leucine and [1-14C] isavaleric acid into
milk constituents by the perfused cows udder. Biochem J.
73:24.

Verbeke, R. and G. Peeters. 1965. Uptake of free plasma amino acids
by the lactating cows udder and amino acid composition of
udder lumph. Biochem. J. 94:183.

Verbeke, R., G. Peeters, A. M. Massart-leen and G. Cacquyt. 1968.
Incorporation of DL-[2-14C] ornithine and DL-[5-14C] arginine
in milk constituents by the isolated lactating sheep udder.
Biochem. J. 106:719.

Verbeke, R., E. Raets A. M. Massart-leen and G. Peeters. 1972. Meta-
bolism of [U-i4C]-L-threanine and [U-14C]-L-phenylalanine by
the isolated perfused udder. J. Dairy Res. 39:239.

Vik-Ma, L., J. T. Huber and R. E. Lichtenwalner. 1972. Abomasal
infusion of casein, glucose, and casein + glucose, and
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55:7ll (Abstr.).

Vik-Ma, L., R. S. Emery and J. T. Huber. 1974a. Milk protein pra-
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Vik-Mo, L., J. T. Huber, W. G. Bergen, R. E. Lichtenwalner and R. S.
Emery. 1974b. Blood metabolites in cows abomasally infused
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208

Whitney, R. McL., J. R. Brunner, K. E. Ebner, H. M. Farrell, Jr.,
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Yousef, I. M., J. T. Huber and R. S. Emery. 1969. Action of high

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52:943 (Abstr.). .

Yousef, I. M., J. T. Huber and R. S. Emery. 1970. Milk protein
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Dairy Sci. 53:734. '

Zimmerman, R. A. and H. M. Scott. 1965. Interrelationship of plasma
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by suboptimal and superaptimal dietary concentrations of
single amino acids. J. Nutr. 87:13.

APPENDIX A

 

 

.uoowm unannouoo uao<

.voemehoaeo uoz

 

 

 

 

 

 

 

 

 

 

 

.eNououm ocean”
”MUM H ewmnv ewcoaae no «on: me eaves peoHa>Nseo :«ouoan among:
.2 eHosHoN 1 2 Hence "ooeooommHe Nae
v¢.mN mm.on ow.nN mm.oN NH.nN oN.mN om.mN ~m.v~ o~.mN on: vumn<
om.- Hm.eH nn.o~ oo.NH NN.NN wo1v~ cc.o~ vo.m~ HN.@ an.» ammo vouuomxm
Nv.v NN.o vm.o oo.m wN.v Nm.m ow.o VN.o 1- 11 ammmu
we.N mm.» av.m oc1N mv.m om1w v~.m om.» HN.m mm.w : unwououm ovshu
mam. mNm. 0N~.~ wa. Nno.~ ooc.~ cam. moc.~ Avo.~ cNa. : m.o~oaflancu
mmN. ave. mom. NmN. HNn. new. ovm. mmn. mnv. .emv. : annouam
eoH.H eNN.H NHN.H oNH.H NNN.H NHe.H Noe.H eNe.H eNe.H eNN.H an :H N .Heaoe "euNoHqu
~.ov H.~n v.0N m.oN n.mn .N.Hn 0.0N ~.NN o.cn H.0N * .uouuufl Nun
N.m~ m.n~ N.m~ o.v~ N.o~ m.nu n.m~ Q.N~ m.cn a.N~ .u .Houuml Aha
m.Nn v.me v.mm N.vm n.Nv N.Nv m.Nm N.vo m.NN~ o.mo .u .u£u«03 :moum
N n N m m o a v N o .o: auwm
nH\m N\m mH\m m~\m nn\m N\a wH\m Nu\m N\m1~\m mH\m uno>ua£ me open
onn mNmH onH mNmn ona mNmH uno>Hun ma Hoe»
«Neo3a< «on: oeoz ueoloeoub

 

.ocoueoa aeowuune new one ouwa use nae» Na uoumo>Hac oueHNm once we muesoe<11.u< oHoeN

209

210

 

 

 

 

 

 

 

N 2 o.wH moN mmHN man one eeHH NN1m1m mN1mN-HH eN1eH-H NmeH

o 3 m.NH NON «Ham cmm mmm eweH NN1oN-N 0N1N-m mN1wN1H ommH

m 2 e.oN mNN mmmm NmN omm moHH oN1NH-HH mN1m-NH mN1e-HH NHeH

e 3 w.NN mmN mNHN mHm Hon mHoH 0N1Hm1oH mN-m1HH NN1oN1m NomH

m z m.mH HmN QNHN Nwm owe NNNH oN1N1HH mN-mN1w NN1m1N mmmH

N z 1 1 1 - 1 oHN 0N1wN-m 1 vN1NH1o vaH

H 3 - 1 1 1 1 com oN1m1N 1 VN1NN-m oeeH

e u m.0N mMN NeHN men Hoe omHH NN1om1H 0N10N-NH mN-wN1oH mHeH

m u o.HN NwN oveo Now How mmNH oN1m1NH mN1m1NH mN1NH-N mmmH

N u 1 1 1 1 - mmm oN1mH-oH - eN1m1N ooeH

H o H.eH mmH one mom Nmm momH 0N1NN1o mm-oN1mi-NN-pmeH oemH

o m o.HN. mNN memo mHm on momH NN1nH1H oN1m1H NN1om1HH emmH

m m N.NN oeN omoo oom mmm HmHH oN-m1NH mNeoH1NH mN1HH1oH woeH

e m 1 1 1 1 1 meN oN1N1N 1 vN-mN1o NmeH

m m N.HN NON wNmm NNN ONN ova oN-N1o mN1mH1N HN1NH1m eNNH

N m o.mH cmN eHHo mmm omm Nem oN1N-o mN1mH1m mN1m1HH ommH

H m m.wH mHN mmeo New New mom 0N1N1m mN1oN1m mN-m1HH HmmH
oz econ—pea;

macaw .Ho omooao mx mx mx xHHz oHe> o.>Heu Heaoo< .>oum :oHHm .oz sou

:chHz oHuaeoo oNHHHz Hem xHHz eH «Nan -HooeH Hanuo< we open
xaon .>Heu on ow< .>Heu we open,

HHoHueuoeH meager.

 

.eom: maoo mo mooaw oHuoeom cHnqu mxaoHo op peoacuHmme use
mocha aHoocom .mcH>Heo me open Heaven .eoHoeuaeH mooH>aHm any cH oaoaon .gunHo mo open-1.N< oHoeN

211

 

 

 

 

 

 

 

 

 

 

 

N 2 H.wN oem Nva Non mNN oNMN NN1m1m 0N1eN1N ON1oN-oH mmHH

o 2 w.oH NmH wNHm can con wNmH NN-N-H 0N1N1H NN1NN-cH NNmH

m 2 - 1 - 1 1 mom oN1oH1oH 1 «N1eN1N NovH

e 2 m.mH wwH oeNm va Nam wme oN-NH-HH mN1NH1oH HN1NH1oH moNH

m 2 e.NH HmN ammo mom oNv «ONH 0N1NN1oH mN1mN1w mN1HH1N cmmH

N 2 1 - 1 1 1 Hmo oN1H1N 1 eN1oH1w oovH

H 2 - - 1 - 1 HMN. chum-o - erN1o omeH

e no m.NN HoN vaN mHn «mm vaH NN-HH1H mN1eN1NH mN-eN-HH HNeH

m u o.NH NaH chm NNN wnn ONHN oN1H1oH mN1mN-oH oN1NH1NH NONH

N u 1 1 1 1 1 HoN oN1oH1oH 1 «N1oH1m moeH

H u N.eH ooH NNHv HmN -wmm, mHmH.MNwN1o mN1NH1o HN-wJMi. oNNH

o m m.HN HmN ONoo mom mmn NemN NN1NN1N oN-V1N ON1m-m vaH

m m e.mH NHN vam omN mmm oHNH 0N1oN1oH mN1w-HH mN-oN1m monH

e m 1 1 - 1 1 eNN oN1o1N 1 VN1N1N mmvH

m m - 1 - 1 1 mmN oN1mN1m - eN1cm1e eva

N m 1 1 1 - - mom oN1cN1m 1, eN1a1n wmvH

H m H.mH owH cove va com NcwH ON-oN1m mN1N1oH HN1HN1cH voNH
um anonymous

Adana .Ho macaw ax ax NH xHHz oHe> .>Heu Heaua< .>ono spawn .oz sou

:chH2 wHuoeou o\xHHz new xHHz :H mNea 1HoucH Heaua< me open
xaon .>Heu on ow< .>Heu mo aueo

COM HGHUNA mDOM>01Hm

 

.eoueHueoo11.N< oHoea

212

 

 

 

 

 

 

N e2 1 1 - 1 1 NmN NN-H-H 1 «N1NN1cH vaH

o 2 m.mH NmH mmoe HoN mmm mNeH oN-NN1NH oN-eN1H mN1mN1H mmmH

m 2 1 - 1 1 1 com oN1wH-m 1 vN1oN1H emeH

v 2 m.eH oNH vae Hon mmn NHmH oN-HH1HH mN-mH1HH NN1oH1m HNmH

N 2 m.HN an mme HNm eHv mmNH oN-NH1oH mN1mN1w MN1o1m onH

N 2 1 - - 1 1 one oN1mH1m 1 NN1wN1oH oHeH

H 2 1 1 1 1 1 Now-.mmeHuNi 1 eroH1o HmeH

e u o.oH oNH ova NNN New omoH oN-eH-HH mN1N1NH MN1vH-HH mHvH

n u o.HN mmN mHeN mmn new aHmH NN1HH1H mN1eN1oH NN1mH1H NwNH

N u N.NN NmN mmNo new con mNHH oN1mH1m mN1HN1m NN1mN1o wmvH

H u - 1 - - - HNa oNumH1o 1 -iMN-m1NH eNeH

o m N.wH oON comm «Nn man onH NN1N1m 0N1N1N NN1H1H Nva

m m N.aH NNN Nch men NHv MHNH 0N1wN1oH mN1N-m NN1m1N Nme

e m - 1 1 1 1 mMN oN1N1N 1 «N1mN-o omvH

m m 1 1 - 1 own omHH oN-mH1o mN1mH1N NN1NN1v eNmH

N m o.HN NQN mncN mnm ch omHH oN1NH-m mN1n-e NN1o1N mmmH

H m H.NN NwN ammo on cHe mvNH oN1H-m mN1NN1N HN-H1NH wHNH

a peanueoah

moon» .ao macho ax an ax HHH: aHe> o.>Heo Heauo< .>oam :ohHm .oz sou

eHAoH2 meoeou o\xHHz you xHN: :H «Non 1aoueN Heaoa< we open
Hoon .>Heu on ou< .>Heu me even

 

HHoHue poo..— 335.5

 

6355:8122 oHooN

 

#1»de i-ii .ilhluiJ-Ihululixp—GK 1
COMUNHONA WSOM>OHQ .

 

N< aunts

 

 

213

 

.mNeOa

 

 

 

 

 

 

 

 

 

.3oa umuo2o
zoo enema

N 2 - 1 1 - 1 NwN ON1NH1NH 1 «N1NN1OH «NaH

O 2 O.mN NNN HOON NON own HNNH NN1m1H oN1mN1H NN1NN1O OOvH

m 2 o.HN OON Hmwm HNN own OOOH oN-N1HH mN1N1NH NN1m1HH OHeH

e 2 O.NH oNH meNv NmN Own Nme oN1eN1HH mN1N-NH HN1ON1O OONH

N 2 1 1 1 1 1 NNOH ON1m-N 1 NN1vH1O eOeH

N 2 N.OH ewH Once oeN mNm NOHH ON1OH-N mN1ON1O NN1N1N oONH

H 2 N.eH NNH mmom mmN mam -MNHH ON1mH1m MNwOH1O MN1mwmi, comm

emu N.OH HNN omHo HNN HHv mmOH NN1NH1N ON1HN1H «N1OH1N mmeH

N ou m.NH NON ONem won NNm ONaH ON-m-NH mN-ON1HH HN-mN-w emNH

N u 1 1 1 1 1 HHN ON-NH1OH 1 «N1VN1N moeH

H on O.oH OmH NNme .ime ONN NOOH 0N1n-n mN-N1o .mmhwmwm- NonH

o m w.mN NON NHNm mom Nmn ONmH NN1ON-N 0N1mN1N NN-mN-OH HmmH

m m «.mH mVN Nva OO¢ Oee HNNH oN1mN1OH mN1NH-w NN-N1m NNNH

v m 1 1 1 - 1 com ON1¢H1NH - «N-H1m movH

m m 1 1 1 1 1 HNN. oN1o1N 1 «N1NN-m OHeH

N m 1 1 1 1 1 HNm ON1m1o 1 NN-NH1OH HHvH

H O e.NN mmN NONN HHN wen onNH oN-mH1m mN-N1o NN1oN1NH menH

:2 unusueone

mocha .uo caecum m2 ax mx waz aHe>_ o.>Heu Heoua< .>oum :uuHm .oz 300
sHeon uHuoeeo eNNHHz sen HHH: oH mNeo 1HoueH Heouo< me open

Hoon .>Heu we ou< .>Heu mo oven

:oHoeuaeH mooH>oad

 

.eesaHosou11.N< oHoee

.oueuaeH .uaeH HoHNNNunn .em HoueuNunn1omH .52-H
Hooeeonoam .HN Houeuoae .o< HHocecuo .um “Hoon peomuouoo oHoe .mO< .eouoHuH: unannouoo OHoe

.2O< HcowouoHc oHoaHom Hope: .zqom Heououuwe Hepoo .ZN Humouea New .za "meowuew>oaoo<o
.OoooHoO one: mcoHoeHoaaoo

Hmzv pceoHMNcmHm :oz .mmO.voV peeaHchuHm one NON.+ meHooooxo mpeoHaHmmooo coHuaHoHHoue

 

214

 

 

-- Nz NN.- Nz NN. N2 N2 N2 N2 N2 N2 HoeH
- NN. N2 N2 HN. He.- oN. NN. N2 N2 5N
- N2 N2 N2 N2 N2 N2 N2 N2 so-H
-- NN. oN. N2 N2 N2 Nz Nz Ha
- Nz NN. N2 N2 N2 N2 o<
- N2 N2 N8. NN. NN.- um
- N2 N2 HN. NN. un<
- N2 N2 N2 zo<
- eN. NN.- ZHON
- Ne.- 2N
- "so eH N
sees an so-H so o< um Hn< za< zHom 2e N
2o
2o eH

 

o.e

.oueHHm :Noa co mucoEonameoa NuHHese one oeoHHuac moose meoHueHouaoa onaNm11.n< oHoeN

215

oaoz Nose one

.OmO.vm we oneoHMHeuHm uoz

a

.Hmc.vm ma vopoHoo

omO.vo NH :oHoeauo coHuowooun on» cH OoosHoaH ouoz moHoeHae> oeoocomoOeHo

 

 

N2 N2 N2 N2 N2 N2 N2 22 N2 : .eaeuueH

Noo.ve NN. NN. Ne. HN.- NN. N2 N2 NN.- = .ouaHNasm

22 N2 N2 N2 N2 22 N2 N2 mz = .ouuHNusa1onH

N2 N2 N2 N2 N2 N2 N2 N2 N2 = .ooaeoHeona

HNo.12 No. NN. No.1 NN. 22 N2 22 mz 22 2H N .ouuuou<

Noco.va NN. NN. oe. oN.- Nz oN. oN2 NN.- :2 2H N .Hoeueum
mo owcea N2 2 mMMWm mn< za< 240m 2N 2w ANV oHooHue>
1HmHeme aeoueoooO

 

aHxV moHoeHne> peooeomoocH

 

VOHOOHOW mOHDdMHdN'

e.mHmNHeea eonmoRuou onHana No

ueooeoaoucH use oHpeNHe> unaccomoo on» coozooo meoHoeHouuoo HeHuHed-1.e< oHneN

216

.m:w>2mu scam Nxmv :H 0529 was “unusuaouu NM a museum wcwuoonn N“ m

n

.9309» Hon N300 vN you 20990 vamucaumm

 

 

 

 

 

 

a

No ounuu2N2meN

 

macaw unaccoum

mz m2 mz NH.m .o02 .mm .mm 022 mo a oucaaan :aouonm
N2 N2 N2 oo.o .222 .N22 .222 umz No 2 oona2mn wuuoam
2oo.va N2.v2 N2 .NN .2NN .NNN .VNN .Nw .NANMoz 2uoN
Nc.vm No.vm N2 2N. o2.N NN.N NN.N N .uum no: Nu22oN
m~.vm m2 m2 we. uv.~2 mn.N2 Hv.~2 * . NvMHON 2NHOH
No.vm N2 2o.v2 N2. NN.N NN.N NN.N N . :2ouo22
N2 N2 N2 N2. 22.N NN.N «N.N N . 2am
wz Nz wz om.n n~.oN NN.NN No.mN v\mx .2225 vouoouaou NvMHON
mN.vm wz mz ov.e nv.o~ m2.n~ mm.¢~ v\mx .xufia vouoonuoo awn
m2 mz mz NN. NN.N NN.N NN.N v\mx .uam no: NuaHON
mm.vm mz mz m2. N¢.n mm.n m~.n2 v\mx .vauom munch
N2 N2 N2 22. NN. NN. NN. vax .c2opoum
N2 N2 N2 N2. Nc.2 No.2 NN. u\Nx .uam
N2 N2 N2 o«.e ~¢.h~ NN.NN 2N.o~ m\N2 .2222
:o2pusvoum,x222
No.vm N~.v2 N~.vm NN. No.22 NN.N2 so.e2 «.22 :2 =2ouo22 «Nana
c2.vm N2 N2 22. NN.N NN.N NN.N o\N2 .Na<
N2 N2 N2 N2o. NNN. Nae. «No. vxux .cuuoua2: o2n=2oN
N2 2o.vm N2 22. NN.N N5.~ NN.N u\N2 .a2ouo22 ousuu
N2 N2.v2 N2 N2.N ~2.vn NN.NN NN.NN .u\2auz .2Nuogo uoz
N2 N2.v2 N2 N2.e NN.N2 NN.N2 NN.N2 N\N2 .uouuaa 222
N2 2oo.v2 N2 NN. NN.N 22.~ NN.N uxux .2“:
N2 2oo.vm N2 NN.N NN.NN 2N.2~ NN.NN uxux .oNu22N :uou
N2 2oo.vm N2 22.N NN.N N¢.o2 NN.N uxux .ouunucoucou
"caduca uaowhuan nqu noon
N2 N2 «2 Nzou mo nonasz

oawexm has a N.o.n away: Houucou amen

flown

 

.2222, 28.2

can :ofiufimomaoo van @2022 x22a .oxuucw ucufiuuac van noon co Nmsouu uamuoonn mo muuommm-n.m< oupwh

217

Table A6.--Interaction between breeding groups and treauments for the
solids non fat percentage in milk.3

 

 

 

 

 

 

Treatment Overall
Breeding b c d
group No. NC PC U AU Av. s.e.
Best 24 8.64 8.85 8.70 8.92 8.78 .21
Control 16 8.82 8.53 8.79 8.95 8.77 .26
werst 28 8.60 8.67 9.07 8.47 8.70 .20
Overall Av. 68 8.67 8.70 8.87 8.74 8.75 --
s.e. -.32 .32 .32 .32 -- .20
al7 Cows per treatment mean.
bNumber of cows per breeding group.
cAverage.
d

Standard error.

Table A7.-~Interaction between breeding groups and treatments on the
average bodyweight}.c

 

 

 

 

 

Treatment Overall
Breeding b
group No. NC PC U AU Av. s.e.
Best 24 591 561 564 542 564 33
Control 16 587 574 575 593 582 40
werst 28 568 601 560 596 581 30
Overall Av. 68 581 S81 S65 S76 --

s.e. ' 23 23 23 23 -- 23

 

317 cows per treatment group.
bNumber of cows per breeding group.

cThe number of cows per breeding group within treatment are 6
best, 4 control, and 7 worst and the standard errors are 39, 47 and
36 kg, respectively.

 

218

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you 2252 €308.30 new .uem 2822 N323 £323 neueu 222020.222 Jam one 3n .9 .622 .25 .m... ... .2. 3622.23.32?»

.2o.va Non.uw .No.va NNNNMN co2ua>uoano New

$222.53 noun. goo: 222303 22.2 2262qu300
.oa-o2 a: new N2 x2uunu ee2ue2onueu mo o2ueu2uu 293626
.22-22 22: new N2 .222qu 282320.223 mo 02923.5 2222220
.22 .2: .2622 N2 222.52. 2822.22.23 No 222252.22 .2223

 

 

 

 

....
.o-n 22.. you N2 222.2»:- 2822222823 mo 02982.3 22225.-
~2.- no... - en. - .. - - - a - mn- - - - u N 2223922.
NN.- NN. NN. - - - - - - - - - - - - - 2 22 cue-2.2
NN. NN. - - - - - - - - - - - NN. - - N2 22
oo. NN. - NN.- - - NN. 2N. NN. 22. ON. NN. - - - NN. e222 :ua
NN. 2N. - NN.- - NN. - N2. 2N. NN. om. - - - - NN. e222 22N
NN. N2. - 2N.- - NN. NN. - 2N. 2N. 24. NN. - - NN.- NN. e222 N2
NN. NN. - NN.- - NN. 2N. 2N. - NN. 20. NN. - - NN.- NN. e222 N2
22. No. - - - NN. NN. NN. om. - NN. - - - NN.- NN. e222 2
N2. NN. - 22.- - NN. NN. 22. 22. N2. - - NN. - 2v. 2N. e222 2
N2. N2. - - - - - - - - - - 22. - - - Nazm
NN. O2. - - - - - - - - 2.. ca. - NN. No. NN.- 2N2
2.. 22. NN. cc. - - - - - NN. - o2. No. - 2.. 22.- 22
22.- NN.- - - - on. 22. - - - co. NN. NN. 2c. - oN.- 22
NN. oo. 02.- 22.- - 22. mm. .2». NN. NN. NN. NN.- mm.- .- - - .N2N2-222u
a2-N2 N2-22 eo-o2 2:
a: a: Human: ‘
No.- N2. - Ne. - NN.- - 22.- - NN.- - - - - - 2 =2ououa
N2. NN. N2. - - 24.- NN.- NN.- 2N.- - NN.- - NN.- - NN.- NN.- 2 22 Nun-2.2
22. 22. - 22.- - NN. 2N. - N2. 22. NN. - - - - - N2 .22
no. 22. N2.- 2..- N2. - NN. NN. NN. NN. 2N. - NN. - NN. NN. e222 xuN
3. 3. 22.. NN.- 3. 8. - N2. NN. 22. NN. - NN. - NN. «N. 322 .92
NN. c2. - - NN. NN. v2. - NN. NN. NN. - - - - NN. 2222 22N
No. 22. NN.- NN.- 2v. NN. NN. NN. - NN. NN. - NN. - 22. «N. 2222 N2
oo. 2o. - - 22. N2. NN. NN. NN. - NN. - NN. - - oN. e222 2
22. NN. NN.- 2a.- 22. NN. 22. NN. NN. NN. - - 2N. 4N. N2. 2N. e222 2
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Table A9.--Effects of breeding groups on rumen pH and rumen concentra-
tions of volatile fatty acids and ammonia nitrogen.

 

Breeding group

 

Significance of

 

 

 

 

Fatty acid

Best Control werst s.e. B B*T B*Time
Number of cows 24 16 28 P<.10 NS NS
Rumen pH. 7.03 7.08 7.13 .11 NS NS NS
Millimoles per liter
Acetate 44.50 40.14 42.59 3.28 NS NS NS
Propionate 15.44 15.04 14.06 1.91 NS NS NS
Isa-butyrate .53 .45 .50 .04 P<.10
Butyrate 8.26 7.57 8.00 .69 NS NS NS
2-methyl-butyrate .55 .52 ..54 .06 NS NS NS
Isa-valerate .40 .36 .41 .05 NS NS NS
Valerate .88 .94 .89 .10 NS NS P<.10
Total acids 70.45 65.14 67.06 5.73 NS NS NS
Molar_present:
Acetate 63.63 61.98 64.25 1.17 P<.10 P<.O5 P<.25
Propionate 21.42 22.83 20.38 1.30 P<.10 P<.05 P<.25
Isa-butyrate .76 .72 .77 .04 NS NS NS
Butyrate 11.66 11.60 11.82 .44 NS NS NS
Z-methyl-butyrate .77 .81 .81 .07 NS NS NS
Isa-valerate .57 .57 .63 .06 NS NS NS
Valerate 1.25 1.49 1.34 .02 P<.25 P<.25 P<.25
Rumen ammonia-N
mg/lOOml. 10.67 13.31 12.37 2.50 NS NS NS

 

 

220

Table A10.--Effect of breeding group on the rumen concentrations of
iso-butyrate and valerate, mMoles/liter.

 

 

 

 

f
Iso-buty- Valerate
Breeding rate° Days from calving
group
No8 Av.c s.e.d Nob -14 21 70 126 s.e.
Best 96 .538 .oz 24 .6381 .83313 1.1681‘ .90gj .10

Control 64 .45 .02 16 .64gi 1.26hj .96hk .87gik .12

Werst 112 .50g .02 28 .71g1 .8181 1.1133 .92gi .09

 

 

3Number of observations per mean for iso-butyrate.
bNumber of observations per mean within a row for valerate.

cAverage.

dStandard error.

ep<.lO by analysis of variance.

fp<.10 by analysis of variance.'

g’hMeans within the column without a common superscript are
different (P<.05).

1’J’kMeans within a row for valerate within a common superscript
are different (P<.05).

221

Table A11.--Effect of breeding group on the molar distribution of
acetate, propionate and valerate, molar percent.

 

Breeding group

 

 

Acid Significance of
Best Control Worst breeding groupd
Number of obs.8 96 64 112
Acetate Av.b 63.63° 61.98f 64.2s° p<.1o
s.e.c .59 .72 .54
Prepionate Av. 21.42°f 22.as° 20.38f p<.1o
s.e. .65 .80 .60
Valerate Av. 1.2s° 1.49f 1.34Bf p<.25
s.e. .07 .09 .07

 

8Number of observations per breeding group.
bAverage.

cStandard error.

dDetermined by analysis of variance.

e’f‘Means within a row without a common superscript are different
(P<.05).

222

Table A12.--Effects of breeding group on plasma concentrations of
glucose, urea and ammonia nitrogen; mg/lOOml.

 

Breeding group Significance ofb

 

 

Best Control worst s.e. B B*T B*Time

 

NUmber of cows

per mean 24 16 28

Glucose 53.51 52.59 54.26 2.37 NS NS NS
Urea nitrogen 12.51 13.72 12.94 1.39 NS NS NS
Ammonia-N .33 .32 .33 .06 NS NS NS

 

 

aStandard error fer 24 cows per mean.

b

calving.

B is breeding group; T is treatment and Time is days from

223

Table A13.--P1asma amino acid concentrations (uM/lOO ml) in breeding
groups and by treatment and/or days from calving within
breeding groups.

 

 

 

 

Amino DFC/ Breeding group Amino Breeding group

acid TRT' B C W ac1d B C W

VAL 18.71 18.05 20.65 GLY -14 31.66 30.46 34.45
-14 20.43 18.42 18.61 42 30.54 46.83 32.93
42 17.24 18.85 22.18 126 22.00 23.23 23.98

 

126 18.86 17.17 21.12

 

 

SER -14 8.88 6.87 9.80

 

 

LEU 12.44 12.50 14.05 42 8.60 11.85 9.26
NC 13.28 14.58 11.70 126 7.43 7.59 8.28
PC 12.92 10.27 17.05
U 11.61 12.06 13.62
AU 11.70 13.09 13.78 ASP NC 3.03 2.51 2.41

 

PC 2.40 2.47 3.00

 

 

 

 

 

 

ILE 11.06 10.23 12.37 AU 2:65 4.85 2.87
-14 2.43 2.08 2.68
His NC 7.80 7.84 6.56 42 2.98 3.26 3.19
PC 6.31 7.45 6.80 126 2.47 3.81 2.56
U 6.88 7.81 6.60
AU 6.75 6.88 7.39
PHE 5.63 4.76 5.13
CYS NC 1.79 2.80 1.76
PC 1.63 1.31 1.76
U 1.73 2.08 1.70
AB 1.55 1.46 1.47

 

224

 

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APPEND IX 8

 

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0 4 8 12 16
WEEKS FROM CFILVING

Figure Bl. Adjusted content of protein in milk for breeding groups

per week from calving (standard error _+_ .05).

225

20

 

226

 

 

 

 

 

 

 

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WEEKS FROM CHLVING

Figure 82. Concentrations of solids-non-fat in milk for breeding
groups per week from calving (standard error ~_I~_ .13).

 

227

 

 

 

 

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WEEKS FROM CRLV ING

Figure 83. Adjusted body weight for breeding groups per week from
calving (standard error i 3.0).

 

20

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