Download response of rumen protected methionine and lysine

RESPONSE OF RUMEN PROTECTED
METHIONINE AND LYSINE SUPPLEMENTATION
ON LACTATION AND REPRODUCTIVE
PERFORMANCE IN PERIPARTURIENT DAIRY
COWS
THESIS SUBMITTED TO THE
NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
ANIMAL NUTRITION
BY
AMRUTKAR SUHAS ASHOKRAO
(M.V.Sc. Animal Nutrition)
DIVISION OF DAIRY CATTLE NUTRITION
NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY)
KARNAL-132 001 (HARYANA), INDIA
2012
Regn. No. 1080906
Division of Dairy Cattle Nutrition
National Dairy Research Institute
(Deemed University)
Karnal-132001 (Haryana), INDIA
_____________________________________________________________________
Dr. S. S. THAKUR
Principal Scientist
CERTIFICATE
This is to certify that the thesis entitled, “RESPONSE OF RUMEN
PROTECTED
METHIONINE
AND
LYSINE
SUPPLEMENTATION
ON
LACTATION AND REPRODUCTIVE PERFORMANCE IN PERIPARTURIENT
DAIRY COWS" submitted by Amrutkar Suhas Ashokrao towards the partial
fulfilment of the award of the degree of Doctor of Philosophy in Animal Nutrition of
the National Dairy Research Institute (Deemed University), Karnal (Haryana), India, is a
bonafide research work carried out by him under my supervision, and no part of the thesis
has been submitted for any other degree or diploma.
Dated:
23rd October, 2012
(S. S. Thakur)
Major Advisor & Chairman
DEDICATED TO
MY
BELOVED FRIEND
Late Dr. Nilesh Wankhede
ACKNOWLEDGEMENT
_______ ____________
__________________ _______________ _
First and foremost, I bow my head to thank the Almighty for having bestowed
me with all what I needed if not what I wanted and showing me the right path at
life’s cross roads.
I am extremely grateful and profoundly obliged to my major advisor, Dr. S. S.
Thakur ,Principal Scientist, Dairy Cattle Nutrition Division, National Dairy Research
Institute, Karnal, for his sagacious guidance, invaluable suggestions, constant
encouragement and constructive criticism during the entire course of this study, which
enabled me to bring the problem in hand to a successful end. My indebtedness is
reserved for his goodwill and patience during the entire period of my research work.
I would like to express my heartfelt thanks to Dr. (Mrs) Neelam Kewalramani,
Principal Scientist, DCN Division and Dr. A. K. Tyagi, Principal Scientist, DCN
Division, for their expertise, technical guidance, emphatic help, and pertinent
suggestions in planning and critical amendments made during the entire microbiological
study of this work.
My profound admiration and sincere gratefulness is extended to Dr. Mohinder
Singh, Principal Scientist, DCP Division, Dr. S. K. Sirohi, Principal Scientist, DCN
Division and Dr. Shivprasad, Principal Scientist, LPM Division for their
encouragement and precious, innovative and remedial suggestions during the course of
this study.
I sincerely pay my regards to Dr. S.S. Kundu, Head, DCN Division for his
valuable suggestions, support and providing mandatory facilities during the entire
study.
I am falling short of words to express my thanks to Dr. T. K. Walli and Dr S.
N. Rai, Retd. Head and Principal Scientist, DCN Division and Dr. Chander Datt,
Senior Scientist, DCN Division whose cooperation, suggestions, and moral support
enabled me in many ways to complete this research work.
I am deeply gratified to Dr. A. K. Srivastava, Director, NDRI Karnal for
providing requisite facilities and comfortable stay in the NDRI campus.
I wish to extend my sincere regards to Dr. G. R. Patil, Joint Director
(Academics) and Dr. S. L. Goswami, Joint Director (Research), NDRI Karnal for their
valuable inspirations and suggestions for this work.
Neither my language nor space is sufficient to express my heartfelt thanks to my
wife Dr. Manjushree Amrutkar for giving me moral support and encouragement during
the course of this study.
The homely atmosphere and cheerful company which I have enjoyed with my
friends namely Shivaji, Dr. Bhupendra, Dr. Deepak Sinha, Dr. Prokash Bala, Dr.
Sachin, Dr Satish and my seniors Dr. Prokash Bala and Dr Rijusmita Sarma and my
___ __________________
_
_ ____________________________
ABSTRACT
_______ ____
__________________________ _______________ ______
This study was conducted to investigate the effect of supplementing rumen protected
methionine (RPM) plus lysine (RPL) on milk production and its composition, nutrient utilization,
plasma metabolites and reproductive performance in peri parturient crossbred cows (Bos taurus
X Bos indicus). Eighteen crossbred cows were selected and divided into two groups (9 each) on
the basis of most probable production ability (MPPA) and lactation number. Animals in group 1
(MPPA - 4119 kg) were fed chopped wheat straw, chaffed green maize fodder and concentrate
mixture as per requirements (NRC, 2001) whereas, animals in supplemented group 2 (MPPA 4120 kg) were fed same ration as group 1 plus 5 gm RPM and 20 gm RPL, pre partum and 7 gm
RPM and 60 gm RPL, postpartum, respectively. The experimental period commenced 40 days
before expected date of parturition to 120 days post parturition.
During the pre-parturient period, rumen protected methionine and lysine supplemention
exhibited higher gross apparent changes in body condition score. Intakes of CP, RDP, RUP, MP,
TDN, ME and NE L were similar while duodenal supply of methionine and lysine was increased
on supplementation of rumen protected methionine and lysine in pre parturient cows . Plasma
triglycerides and VLDL concentration were increased (P<0.05) in prepartum cows on
supplementating rumen protected methionine plus lysine. Plasma glucose, phosphatidylcholine,
NEFA, vitamin E, cholesterol and BUN were not affected. Plasma amino acids profile of the pre
partum cows was not affected on supplementing rumen protected methionine and lysine while
plasma methionine, cysteine and lysine concentrations tended to increase on supplementation.
During post partum period, fortnightly changes in body weight revealed that there was
overall a net loss of 18.59 kg in group 1, whereas there was overall gain of 5.79 kg in group 2.
The DM, CP, MP, TDN, ME and NE L intake kg per day as well as per cent body weight was
higher in group 2 than that of group 1 lactating cows. Milk yield during supplementation period
in group 2 was 17.69 kg/d, which was 11.33 per cent higher (P<0.01) than that of group 1 (15.89
kg/d). There was no effect of supplementation on milk protein, solid not fat (SNF), milk choline
and MUN contents in both the groups, whereas milk fat content was higher (P<0.01) by 2.18 per
cent in group 2 (4.22%) than that of group 1 (4.13%). The efficiency of utilization of DM, CP,
MP, TDN, ME, NE L per kg milk or FCM yield was better in group 2 than that of group 1. The
saturated fatty acids, unsaturated fatty acids, mono and poly unsaturated fatty acids in milk fat
were similar in group 1 and group 2.
Plasma glucose (55.61 and 55.07 mg/dl), phosphatidylcholine (138.57 and 140.98 µg/ml)
NEFA (106.80 and 105.77 mg/L), vitamin E (1.01 and 0.90 µg/ ml), cholesterol (193.16 and
198.31 mg/dL) and BUN (18.13 and 18.01 mg/L) concentrations were similar in both the groups.
Plasma triglycerides and VLDL concentrations were 13.40 and 16.22 mg/dL and 2.68 and 3.24
mg/dL in group 1 and 2, respectively which were higher (P<0.01) in group 2 than in group 1.
Plasma aspartate, glycine, alanine, valine (P<0.05), methionine, cysteine, and lysine (P<0.01)
concentrations were increased in cows fed ration supplemented with RPM plus RPL. However,
RPM plus RPL supplementation lowered (P<0.05) plasma isoleucine concentration. The plasma
prolactin (79.68 and 80.39 ng/L) and growth hormone (6.63 and 6.63 ng/L) concentration were
similar in both the groups.
The body weight of calves at the time of birth was similar in both the groups. Two cases
of premature births were reported in group 1, however, one case was observed in group 2. Four
cases of retention of fetal membranes (RFM) were observed in group 1 while only one case was
observed in group 2. Higher incidence of metritis in group 1 (3 cases) were recorded whereas
only one case was observed in group 2. The time required for commencement of cyclicity was
similar (P>0.01) in both the groups. The service period was shorter (P<0.01) by 11.8 days in
group 2 than that of group 1, indicating that lesser time was required for the animals in group 2
for conception. AI required for conception was similar in both the groups. The conception rate
during the experimental period of 120 days was 55.55 % in group 1 and 66.66 % in group 2.
Supplementtion of rumen protected methionine and lysine to high yielding lactating
cows during periparturient period was found to be cost effective.
The results of the present study showed that the supplementation of rumen protected
methionine and lysine improved reproductive performance and lead to efficient use of nutrients
at early stage of lactation, which in turn manifested in higher milk yield. In addition, milk fat
content was increased, resulting in improvement in the commercial value of milk.
Keywords: Crossbred Cows, milk, protected methionine, protected lysine, milk fat
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Contents
Chapter
Title
Page No
1
INTRODUCTION
1
2
REVIEW OF LITERATURE
5
2.1
Sources contributing to metabolizable protein in ruminants
6
2.1.1
Endogenous crude protein
6
2.1.2
Microbial crude protein
7
2.1.3
Rumen-undegradable crude protein
9
2.2
Comparative Amino acid requirements in different species
11
2.3
Rumen Protected Proteins in Ruminant Nutrition
13
2.3.1
Degradation of Proteins in the Rumen
13
2.3.2
Protection of Protein from Ruminal Degradation
14
2.3.2.1
Heat Treatment
14
2.3.2.2
Formaldehyde Treatment
15
2.3.2.3
The Coating of Protein Source Particles with Insoluble
16
Substances
2.3.3
Advantages of Rumen Protection
17
2.4
Limiting amino acids
18
2.5
Amino acid supplementation
19
2.6
Rumen protected amino acids
20
2.6.1
Liquid sources of hydroxy analogs (chemically modified
21
molecules)
2.6.2
Surface coating or matrices of saturated fatty acids and
22
minerals
2.6.3
Surface coating with a fatty acid or pH-sensitive polymer
mixture
23
2.7
Responses to rumen protected methionine and Lysine dietary
23
supplementation
2.7.1
Effect on Milk Yield and Milk component yield
23
2.7.2
Effect on plasma amino acids
25
2.7.3
Effect on blood metabolites
26
2.8
Reproductive efficiency of animals fed on protected protein
28
and rumen protected amino acids
3
MATERIALS AND METHODS
33
3.1
Feed and fodder analysis
33
3.1.1
Proximate principles and cell wall constituents
33
3.1.2
Amino acids analysis of feed samples using HPLC
33
3.1.3
Estimation of tryptophan
36
3.2
Rumen protected methionine (RPM) and rumen protected
36
lysine (RPL) product evaluation
3.2.1
Methionine content in RPM
37
3.2.2
Lysine content in RPL
37
3.3
In vitro determination of microbial protein yield
38
3.3.1
Collection of rumen liquor
38
3.3.2
Preparation of substrates in the form of total mixed rations
38
(TMR’s)
3.3.3
In vitro determination of microbial N using nitrogen balance
39
technique
3.4
Separation of rumen bacteria from rumen liquor
42
3.5
Separation of the protozoa from rumen contents
42
3.6
In vitro estimation of RUP intestinal digestibility
42
3.7
Estimation of RDP and RUP of different feed stuffs
43
3.8
Estimation of rumen escape potential of commercial RPM and
44
RPL product
3.9
Location of experiment
45
3.10
Selection and distribution of animals
45
3.11
Estimation of most probable production ability (MPPA)
45
3.12
Feeding, housing and management of experimental animals
47
3.12.1
Green maize fodder
47
3.12.2
Concentrate mixture
47
3.12.3
Housing and management of animals
47
3.12.4
Watering of animals
48
3.13
Observations recorded during pre partum period
48
3.13.1
Body weight
48
3.13.2
Body Condition Score
48
3.13.3
Feed intake
49
3.14
Analysis of blood samples
49
3.14.1
Blood collection
49
3.14.2
Plasma amino acid analysis
49
3.14.2.1
Preparation of sample
49
3.14.3
Plasma choline estimation
50
3.14.4
Plasma glucose estimation
54
3.14.5
Non esterified fatty acids (NEFA) estimation
55
3.14.6
Triglycerides estimation
56
3.14.7
VLDL estimation
56
3.14.8
Estimation of plasma urea
56
3.14.9
Estimation of plasma cholesterol
58
3.14.10
Estimation of plasma vitamins E
58
3.14.11
Plasma growth hormone estimation
59
3.14.12
Plasma prolactin estimation
61
3.15
Reproduction study
64
3.15.1
Calf weight at the time of birth
64
3.15.2
Calf mortality within one month of parturition
64
3.15.3
Occurrence of reproductive disorders
64
3.16
Lactation Study
65
3.16.1
Housing and management
65
3.16.2
Milking of animals
65
3.16.3
Body weight and body condition score
65
3.16.4
Feed intake
65
3.16.5
Milk yield
66
3.16.6
Calculation of fat corrected milk (FCM)
66
3.16.7
Calculation of energy corrected milk (ECM)
66
3.17
Milk Composition
66
3.17.1
Fatty acid analysis of milk
66
3.17.2
Estimation of milk urea
69
3.18
Analysis of blood parameters
70
3.19
Statistical Analysis
70
4
RESULTS AND DISCUSSION
71
4.1
Effect of feeding rumen protected methionine and lysine
71
during pre partum period
4.1.1
Degree of Protection of Rumen Protected Methionine (RPM)
76
and Lysine (RPL)
4.1.2
RDP and RUP content of different feedstuffs
78
4.1.3
RUP Intestinal Digestibility
79
4.1.4
In vitro Microbial protein yield g per kg TDN fermented
80
4.1.5
Body weight changes
81
4.1.6
Body Condition Score
82
4.1.7
Nutrients Intake
83
4.1.8
Overall Plane of Nutrition in prepartum cows
87
4.1.9
Effect of RPM and RPL Supplementation on Certain Blood
87
Parameters
4.2
Effect of supplementing RPM and RPL during post parturient
95
period
4.2.1
Body weight change
95
4.2.2
Body Condition Score
98
4.2.3
Dry Matter Intake
99
4.2.4
Nutrients Intake
101
4.2.4.1
CP intake
101
4.2.4.2
RUP intake
103
4.2.4.3
RDP intake
104
4.2.4.4
MP intake
104
4.2.4.5
Duodenal Methionine supply (% of metabolizable protein)
105
4.2.4.6
Duodenal lysine supply (% of metabolizable protein)
106
4.2.4.7
TDN intake
107
4.2.4.8
ME intake
109
4.2.4.9
NE L intake
110
4.3
Effect
of
rumen
protected
methionine
and
lysine
112
supplementation on milk production and its composition
4.3.1
Milk Production
112
4.3.2
Milk Composition
116
a)
Milk fat
116
b)
Milk protein
118
c)
Milk lactose
121
d)
Milk solids not fat
123
e)
Milk total solid
123
f)
Milk Urea Nitrogen (MUN)
124
g)
Milk choline content
125
h)
Fatty acid profile of milk
126
4.3.3
Efficiency of nutrients for milk production
131
4.4
Overall Plane of Nutrition in lactating cows
133
4.5
Effect of Rumen Protected Fat and Protein Supplementation on
134
Some Lactation Related Blood Parameters
4.5.1
Blood Glucose Concentration
134
4.5.2
Plasma Phosphatidylcholine Concentration
135
4.5.3
Plasma NEFA Concentration
136
4.5.4
Plasma Triglycerides Concentration
138
4.5.5
Plasma VLDL Concentration
138
4.5.6
Plasma Vitamin Levels
140
4.5.7
Plasma cholesterol Levels
140
4.5.8
Blood Urea Nitrogen Levels
141
4.5.9
Plasma amino acid profile
142
4.5.10
Plasma Prolactin Concentration
150
4.5.11
Plasma Growth Hormone Concentration
151
4.6
Effect of RPM plus RPL supplementation on reproductive
154
performance in crossbred cows
4.6.1
Calving Performance
154
4.6.2
Reproductive abnormalities
154
4.6.3
Reproduction related parameters
157
4.7
Economics of feeding rumen protected methionine plus lysine
158
in crossbred cows
5
Summary and Conclusions
Bibliography
162
LIST OF TABLES
Table No.
Title
Page No.
3.1
Composition of TMRs
38
3.2
Details of experimental animals
46
3.3
Ingredients in concentrate mixture (parts)
48
4.1
Chemical composition of feed ingredients offered (% DM basis)
73
4.2
Amino Acid Profile (% of CP) of feed Ingredients
74
4.3
Characteristic of rumen protected methionine (RPM) and rumen
76
protected lysine (RPL) products
4.4
Different protein fractions, RUP (% of CP) of feed Ingredients
78
4.5
RUP Intestinal Digestibility (in vitro) (% DM basis) of feed
79
Ingredients
4.6
Microbial protein yield g per kg TDN fermented of different
80
TMRs
4.7
Fortnightly change in body weights (kg) of crossbred cows fed
81
ration with or without RPM plus RPL
4.8
Fortnightly body condition score of crossbred cows fed ration
82
with or without RPM plus RPL
4.9
Prepartum average nutrient intake of crossbred cows fed ration
84
with or without RPM plus RPL
4.10
Fortnightly average nutrient Intake (kg/100 kg body weight) in
86
crossbred cows fed ration with or without RPM plus RPL
4.11
Plane of nutrition of prepartum cows fed ration with or without
88
rumen protected methinone plus lysine
4.12
Average Plasma metabolites (Prepartum ) of crossbred cows fed
ration with or without RPM plus RPL
89
4.13
Average Plasma Amino Acid Profile (µmol/dl) of crossbred
93
cows (prepartum) fed ration with or without RPM plus RPL
4.14
Fortnightly average body weights (kg) of crossbred cows fed
96
ration with or without RPM plus RPL during postpartum period
4.15
Fortnightly change in Body Weight (kg) of crossbred cows fed
97
ration with or without RPM plus RPL
4.16
Percent change in body weights of crossbred cows fed ration
97
with or without RPM plus RPL
4.17
Fortnightly body condition score of crossbred cows fed ration
98
with or without RPM plus RPL
4.18
Fortnightly average dry matter intake (kg/d) in crossbred cows
100
fed ration with or without RPM plus RPL
4.19
Fortnightly average dry matter intake (kg/100 kg body weight)
100
in crossbred cows fed ration with or without RPM plus RPL
4.20
Fortnightly CP intake (kg/d) in crossbred cows fed ration with or
102
without RPM plus RPL
4.21
Fortnightly CP intake (kg/100 kg body weight) in crossbred
102
cows fed ration with or without RPM plus RPL
4.22
Fortnightly RUP intake (kg/d) in crossbred cows fed ration with
103
or without RPM plus RPL
4.23
Fortnightly RDP intake (kg/d) in crossbred cows fed ration with
104
or without RPM plus RPL
4.24
Fortnightly metabolizable protein intake (kg/d) in crossbred
105
cows fed ration with or without RPM plus RPL supplementation
4.25
Fortnightly duodenal methionine supply (% of metabolizable
106
protein) in crossbred cows fed ration with or without RPM plus
RPL
4.26
Fortnightly duodenal lysine supply (% of metabolizable protein)
107
in crossbred cows fed ration with or without RPM plus RPL
4.27
Fortnightly TDNI (kg/d) in crossbred cows fed ration with or
without RPM plus RPL
108
4.28
Fortnightly TDN intakes (kg/100 kg body weight) in crossbred
108
cows fed ration with or without RPM plus RPL
4.29
Fortnightly ME intake (Mcal/d) in crossbred cows fed ration
109
with or without RPM plus RPL
4.30
Fortnightly ME intake (Mcal/100 kg body weight) in crossbred
110
cows fed ration with or without RPM plus RPL
4.31
Fortnightly NE L intake (Mcal/d) in crossbred cows fed ration
111
with or without RPM plus RPL
4.32
Fortnightly NE L intake (Mcal/100 kg body weight) in crossbred
111
cows fed ration with or without RPM plus RPL
4.33
Fortnightly average milk yield (kg/d) of crossbred cows fed
113
ration with or without RPM plus RPL
4.34
Fortnightly average 4% fat corrected milk yield (kg/d) of
113
crossbred cows fed ration with or without RPM plus RPL
4.35
Fortnightly energy corrected milk yield (kg/d) in crossbred cows
114
supplemented with or without RPM plus RPL
4.36
Fortnightly milk fat content (%) in crossbred cows fed ration
116
with or without RPM plus RPL
4.37
Fortnightly milk fat yields (g/d) in crossbred cows fed ration
117
with or without RPM plus RPL
4.38
Fortnightly milk protein content (%) in crossbred cows fed
119
ration with or without RPM plus RPL
4.39
Fortnightly milk protein yield (g/d) in crossbred cows fed ration
120
with or without RPM plus RPL
4.40
Fortnightly milk lactose content (%) in crossbred cows fed ration
122
with or without RPM plus RPL
4.41
Fortnightly Milk Lactose yield (%) in crossbred cows fed ration
122
with or without RPM plus RPL
4.42
Fortnightly milk SNF content (%) in crossbred cows fed ration
with or without RPM plus RPL
123
4.43
Fortnightly TS content (%) in crossbred cows fed ration with or
124
without RPM plus RPL
4.44
Fortnightly MUN (mg/dl) in crossbred cows fed ration with or
125
without RPM plus RPL
4.45
Fortnightly milk choline (mg/dl) in crossbred cows fed ration
126
with or without RPM plus RPL
4.46
Fortnightly Fatty Acid Profile of Milk of crossbred cows fed
128
ration with or without RPM plus RPL
4.47
Overall fatty acid profile of milk of crossbred cows fed ration
130
with or without RPM plus RPL
4.48
Efficiency of utilization of nutrients of crossbred cows fed ration
131
with or without RPM plus RPL
4.49
Plane of nutrition of lactating cows fed with rumen protected
133
methinone plus lysine
4.50
Plasma glucose (mg/dl) concentration in crossbred cows fed
134
ration with or without RPM plus RPL
4.51
Plasma Phosphatidylcholine (µg/ml) concentration in crossbred
136
cows fed ration with or without RPM plus RPL
4.52
Plasma NEFA (mg/L) concentration in crossbred cows fed ration
137
with or without RPM plus RPL
4.53
Plasma triglycerides (mg/dl) concentration in crossbred cows fed
138
ration with or without RPM plus RPL
4.54
Fortnightly plasma VLDL (mg/dl) concentration in crossbred
139
cows fed ration with or without RPM plus RPL
4.55
Plasma vitamin E (µg/ml) concentration in crossbred cows fed
140
ration with or without RPM plus RPL
4.56
Fortnightly plasma Cholesterol (mg/dl) concentration in
141
crossbred cows fed ration with or without RPM plus RPL
4.57
Fortnightly plasma BUN (mg/L) concentration in crossbred
cows fed ration with or without RPM plus RPL
142
4.58
Postpartum plasma amino acid (µmol/dl) concentration in
142
crossbred cows fed ration with or without RPM plus RPL
4.59
Overall mean plasma amino acid (µmol/dl) concentration in
148
crossbred cows fed ration with or without RPM plus RPL
4.60
Weekly plasma prolactin (ng/ml) concentration in crossbred
150
cows fed ration with or without RPM plus RPL
4.61
Weekly plasma growth hormone (ng/ml) concentration in
151
crossbred cows fed ration with or without RPM plus RPL
4.62
Condensed information of lactation trial
152
4.63
Calving Performance of crossbred cows fed ration with or
155
without RPM and RPL
4.64
Reproductive abnormalities plus metabolic diseases observed in
156
crossbred cows fed RPM plus RPL
4.65
Reproductive parameters recorded in crossbred cows fed RPM
157
plus RPL
4.66
Economics of feeding rumen protected methionine plus lysine to
lactating crossbred cows
159
LIST OF FIGURES
Plate No.
Title
After Page
3.1
Chromatogram of standard amino acid profiile
34
3.2
Standard curve of choline
34
3.3
Standard curve of glucose
54
3.4
Standard curve of NEFA
54
3.5
Standard curve of BUN
56
3.6
Standard curve of cholesterol
56
3.7
Chromatogram of standard α-Tocopherol
58
3.8
Standard curve of milk urea nitrogen
58
3.9
Standard curve of growth hormone
60
3.10
Standard curve of prolactin
60
4.1
RUP Intestinal Digestibility (in vitro) (% DM basis)
80
4.2
Microbial protein yield g per kg TDN fermented of different
80
TMRs
4.3
Average fortnightly body weights (kg) of crossbred cows
96
4.4
Average fortnightly percent change in body weight (kg) of
96
crossbred cows
4.5
Fortnightly average body condition score of crossbred cows
98
4.6
Fortnightly average dry matter intake (kg/d) of crossbred
98
cows
4.7
Fortnightly average crude protein intake (kg/d) of crossbred
102
cows
4.8
Fortnightly average TDN intake (kg/d) of crossbred cows
102
4.9
Fortnightly average milk yield (kg/d) in crossbred cows
114
4.10
Fortnightly average 4% fat corrected milk yield (kg/d) in
114
crossbred cows
4.11
Fortnightly average milk fat (%) of crossbred cows
116
4.12
Average fortnightly milk composition
116
4.13
Average fortnightly fatty acid profile of milk of crossbred
130
cows
4.14
Average fortnightly plasma metabolites of crossbred cows
130
4.15
Average fortnightly plasma triglycerides and VLDL of
138
crossbred cows
4.16
Average fortnightly plasma amino acids of crossbred cows
138
4.17
Average weekly plasma prolactin of crossbred cows
150
4.18
Average weekly plasma growth hormone of crossbred cows
150
4.19
Reproductive abnormalities observed in crossbred cows
156
4.20
Reproductive parameters observed in crossbred cows
156
Abbreviations
__________________________________________
AA
Amino acid
EE
Ether extract
ACTH
Adrenocorticotropic hormone
ENS
ADF
Acid detergent fibre
Endogenous nitrogen
secretion
ADICP
Acid detergent insoluble
crude protein
EN
Endogenous nitrogen
FA
Fatty acids
AI
Artificial insemination
FCM
Fat corrected milk
Ala
Alanine
Glu
Glutamic acid
AP
Absorbed protein
Gly
Glycine
Arg
Arginine
GNC
Groundnut cake
Asp
Aspartic acid
His
Histidine
BCAA
Branched chain amino acid
HMB
BCS
Body condition score
2-hydroxy-4methylthiobutanoic acid
BHBA
ß- Hydroxyl butyric acid
HMBi
Isopropyl ester of HMB
BHT
Butylated hydroxytoluene
HPLC
High performance liquid
chromatography
BUN
Blood urea nitrogen
Ile
Isoleucine
BW
Body weight
LCFA
Long chain fatty acids
CNCPS
Cornell net carbohydrate and
protein system
Leu
Leucine
CP
Crude protein
LH
Luteinizing hormone
CPI
Crude protein Intake
Lys
Lysine
Cys
Cysteine
MCP
Microbial crude protein
DDG
Dried distillers grains
ME
Metabolizable energy
DIM
Days in milk
MEI
Metabolizable energy intake
DM
Dry matter
Met
Methionine
DMB
Dry matter basis
MP
Metabolizable protein
DMI
Dry matter intake
MPPA
Most probable production
ability
DOM
Digestible organic matter
MUFA
Mono unsatutated fatty acids
EAA
Essential amino acid
MUN
Milk urea nitrogen
ECM
Energy corrected milk
MY
Milk yield
ECP
Endogenous crude protein
NAN
Non ammonia nitrogen
Abbreviations
__________________________________________
NDF
Neutral detergent fibre
Thr
Threonine
NDICP
Neutral detergent insoluble
nitrogen
TMR
Total mixed ration
Trp
Tryptophan
NDRI
National Dairy Research
Institute
Tyr
Tyrosine
NEAA
Non essential amino acid
UFA
Unsaturated fatty acids
NEFA
Non esterified fatty acid
UDP
Undegradable protein
NE L
Net energy for lactation
Val
Valine
NE L I
NE L intake
VFA
Volatile fatty acids
NPN
Non protein nitrogen
VLDL
Very low density lipoprotein
NRC
National research council
OM
Organic matter
Phe
Phenylalanine
Pro
Proline
PUFA
Poly unsaturated fatty acids
RDN
Rumen degradable nitrogen
RDP
Rumen degradable protein
REP
Rumen escape potential
RPAA
Rumen protected amino acids
RPL
Rumen protected lysine
RPM
Rumen protected methionine
RPM+L
Rumen protected methionine
plus lysine
RUP
Rumen undegradable protein
RUP ID
RUP intestinal digestibility
Ser
Serine
SFA
Saturated fatty acids
SG
Specific gravity
SNF
Solid not fat
TDN
Total digestible nutrients
TDNI
Total digestible nutrients
intake
CHAPTER – 1
INTRODUCTION
1. Introduction
________________________________________________
The goal of ruminant protein nutrition is to achieve optimal rumen fermentation
efficiency and desired animal productivity (NRC, 2001). But there is still a large amount
of uncertainty, especially regarding protein and amino acid (AA) requirements for
ruminants. However, nutrient requirements are relatively well defined for most of the
domesticated monogastric species. Lysine (Lys) and Methionine (Met) have been
identified as first limiting essential amino acids in metabolizable protein (MP) supply to
dairy cattle (King et al., 1990; Schwab et al., 1992a and 1992b). Rumen undegradable
protein is the second important source of absorbable amino acid (AA) to animal after
microbial protein. But relative to concentrations in rumen bacteria, rumen undegradable
protein is low in Lys and/or Met. Most feedstuffs have lower amounts of Lys and Met,
particularly of Lys whereas contribution of Lys and Met to total essential amino acid in
body lean tissue and milk are similar. Production responses of lactating dairy cows to
increased supplies of Lys and Met in MP includes variable increase in content and yield
of protein in milk, milk yield and feed intake.
Feed CP can be divided into rumen-degradable protein (RDP), which is largely
incorporated into micro-organisms in the rumen when they synthesize microbial crude
protein (MCP) and rumen undegradable protein (RUP) which escapes rumen degradation
and passes from the rumen to abomasum and is digested in the small intestine. A lot of
the dietary CP that is ingested and absorbed is used for body protein synthesis which, in
mature animals, is mostly for replacing tissue (turnover). A part of absorbed CP gets
deaminated and is used for glucose synthesis. Therefore faecal and urinary N consists of
a mixture of undigested or unabsorbed dietary CP and detritus of metabolic processes.
Even though some of the protein is used by the cow for maintenance, growth,
health, milk production and reproductive processes, a large amount is excreted in urine
and faeces (Lapierre et al., 2002). Poor efficiency of CP use by ruminants may be due to
energy limitations, reduced growth of microorganisms in the rumen, catabolism and
partitioning of AA’s, imbalances in AA supply to the intestinal absorptive site and
genetic limitations (Bequette et al., 2002).
__
_________________ ___________ _
___________ 1
Introduction …
_______________________________________________________________________
Ruminants have the unique ability to transform low quality forage based diets,
partly indigestible by monogastric species, into high quality consumer products such as
milk, meat and fibre.
To improve the efficiency of CP use by ruminants, diets need to be balanced
according to the specific AA requirements of the animals. Balancing for post ruminal AA
delivery could allow use of lower CP rations because they would be balanced to supply
individual AA’s to the intestinal absorptive site. Metabolic costs of deamination of excess
AA’s and excretion of excess N would be lower, and removal of CP from the ration
leaves space to supply other nutrients, such as those that more efficiently supply energy
(Lapierre et al., 2002).
The nutritional value and chemical composition of feeds are greatly influenced by
the vegetative stage of the plant, weather during growth, time of day during harvest, soil
fertility, storage and even feed bunk management (Van Soest et al., 1994), and vary
widely in their proportions of protein and non-protein N (NPN), rate and extent of CP
degradation in the rumen, digestion in the intestine and the AA composition of
undegraded feed CP (NRC, 2001).
Since 1989, protein requirements have often been expressed in terms of absorbed
protein (AP) or total AA reaching the small intestine, which includes protein synthesized
by rumen micro-organisms and feed CP that escapes rumen degradation (Dugmore,
1995). Even though the NRC (1989) recognized that intestinal digestion of proteins
differed, they used a constant digestibility value of 800 g/kg for RUP in all feedstuffs due
to the lack of data to differentiate among feeds. Other shortcomings of this model, as
pointed out by several research groups (Satter 1986; Clark 1978), are the prediction of
increased milk yield when a protein source high in RDP is substituted by a source high in
RUP, when many research studies reported a lack of response. Possible reasons for this,
as reviewed by Santos et al (1998a), may be decreased microbial synthesis due to
removal of RDP from the diet, a poor AA profile, or low digestibility of the RUP source.
Some studies suggested that the source of RUP should have an AA profile to complement
that of MCP (Clark et al., 1992; Chen et al., 1993).
__
_________________ ___________ _
___________ 2
Introduction …
_______________________________________________________________________
Proteins digested to AA that are actually available for absorption in the small
intestine, are largely a combination of RUP, MCP and some endogenous secretions (ECP
- proteins secreted into the digestive tract) – collectively known as metabolizable protein
(MP) (CNCPS, 2000; NRC, 2001). As milk production increases, the proportion of the
total CP requirements met by MCP was predicted to decrease and substantial amounts of
dietary CP must escape rumen degradation to meet predicted protein needs (Santos et al.,
1998b). Higginbotham et al. (1989) and Taylor et al. (1991) showed that increasing the
amount of RUP in the ration could improve milk production, but only up to 30-40% of
total CP (Santos et al., 1998b), after which RDP becomes limiting, decreasing MCP
production and non ammonia N (NAN) supply to the intestine (Clark et al., 1992;
Ferguson et al., 2000). Further increases in RUP could also cause a reduction in diet
fermentability, dry matter intake (DMI) and milk production (Olmos Colmenero and
Broderick, 2006).
Research in poultry (NRC, 1994) and swine (NRC, 1998) revealed that each
physiological state in an animal requires a unique profile of absorbed AA. However,
these profiles still need to be established for ruminants.
Since Lys and Met are generally considered to be the most limiting AA for milk
production in ruminants, it is common to feed dairy cows by balancing diets to maximize
absorbable Lys and Met delivery. However, many RUP sources are low in Lys and/or
Met with AA profiles that are generally inferior to MCP, making it difficult to formulate
a ration to achieve the optimum concentration of both Lys and Met in MP, in order to
satisfy the animal’s requirement for limiting AA, without oversupplying N (Santos et al.,
1998a; Rode and Vazquez-Anon, 2006).
Further, Met is source of the methyl donor S- adenosyl Methionine, the metabolite
that provides methyl groups in variety of reactions including de novo synthesis of choline
from phosphatidylethanolamine, thus Met and choline metabolism are closely associated.
As much as 28% of the absorbed methionine is used for choline synthesis (Emmanuel
and Kennelly, 1984). Choline is involved in the transport of fat from liver and required
for the synthesis of phosphatidylcholine, a phosholipid found in the membranes of very
low density lipoprotein. Thus Met plays a role in VLDL synthesis and act to reduce
__
_________________ ___________ _
___________ 3
Introduction …
_______________________________________________________________________
plasma ketone bodies during early lactation. Increased percentage of fat in milk has been
reported with increased amount of rumen protected methionine and Met+Lys in diet.
This reality directed attention toward supplementing only the limiting AA in the
diet, leading to numerous studies to determine effects of adding ruminally protected, and
free AA to dairy rations, as well as infusing specific AA (or AA mixtures) to the
duodenum.
Genetic improvements in Indian dairy cows have lead to increased milk production,
which requires higher intake of dietary CP to meet the needs of milk protein synthesis.
Lys and Met have been suggested to be the most limiting AA for milk protein synthesis.
Balancing the concentration of Lys and/or Met in rations shows improvement in
efficiency of all the other amino acids. The efficient use of dietary AA in the dairy cow
can have immediate benefits such as improving milk production and yield of milk protein
and/or fat.
Although an increase in milk production, milk protein and/or milk fat in high
producing dairy cattle fed rumen protected Met and Lys has been reported, its effect on
hormonal responses, plasma metabolites and reproductive performance are not clearly
understood. Therefore, the present study has been planned to assess the effect of
supplemental rumen protected Met and Lys on feed utilization, milk production and its
composition, reproductive performance, plasma metabolites and hormonal responses in
periparturient dairy cows under Indian feeding regimen. The objectives of the present
study are
1.
To evaluate the effect of feeding rumen protected methionine (RPM) and rumen
protected lysine (RPL) on plasma metabolites and hormonal response of
periparturient dairy cows.
2.
To determine the effect of feeding RPM and RPL on feed intake, feed utilization,
milk production and its composition and on reproductive performance of dairy
cows.
3.
__
To calculate cost benefit ratio of RPM and RPL supplementation in dairy cows.
_________________ ___________ _
___________ 4
CHAPTER – 2
REVIEW OF LITERATURE
2. Review of Literature
__________________________________________________
Amino acids are the building blocks of all proteins. These are linked by dipeptide
bonds to form protein chains. Each protein chain has a specific sequence of AA that
determines its integrity and functionality such as production of enzymes, immunoglobins,
hormones and milk proteins, making them vital to the maintenance, growth, reproduction and
lactation of dairy cattle (Schwab, 1996; Rode and Vazquez-Anon, 2006).
The AA that are absorbed, but not used for protein synthesis, are catabolized and
serve as a source of metabolic energy when oxidized to CO 2 , while the amino groups are
used to synthesize other deficient AA. These can also be converted to fatty acids or serve as
precursors of other metabolites in pathways within the body, such as gluconeogenesis
(Vanhatalo et al., 2003; Rulquin et al., 2004). An estimated 110-180 g/kg of glucose flux was
synthesized from the glucogenic AA’s glutamic acid (Glu), aspartic acid (Asp), serine (Ser)
and glycine (Gly). Alanine (Ala) was quantitatively the most important AA (Wolff et al.,
1972), but it is much less efficiently used than propionate.
Methionine (Met) and lysine (Lys) are considered to be first limiting, but surprisingly
little is known of their metabolic fates. Methionine is a precursor for cysteine by donating a
sulfur group. As an intermediate in transmethylation reactions, it donates a methyl group to
synthesize choline, vitamin B 12 , phospholipids in cell membranes, creatine production for
energy storage and transfer, and the carnitine required in lipid metabolism and fatty acid
mobilization (Campbell and Farrell, 2003).
Lysine is an anomaly since it is almost always taken up by the udder in excess of
requirements. Excess Lys is oxidized to produce glutamate, an energy source for intestinal
mucosa cells (Windmueller and Spaeth, 1980) and a precursor for de novo Arg and Pro
synthesis (Bequette et al., 2002).
Amino acids synthesized by cells in the animal body, using metabolites from surplus
AA catabolism, are known as non-essential amino acids (NEAA) or dispensable AA and do
not necessarily have to be provided in the diet. Ten of the 20 primary AA in proteins are
classified as essential or indispensable amino acids (EAA) and need to be supplemented in
the diet (in the form of rumen escape protein) since they cannot be synthesized by animal
tissues in sufficient quantities to fulfil metabolic requirements for growth and high levels of
_______________________________________________________________________ 5
Review of Literature …
production. These include Lys, Met, arginine (Arg), histidine (His), isoleucine (Ile), leucine
(Leu), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) and valine (Val) (NRC, 2001).
Classification of AA as essential or non-essential was based on research conducted in
nonruminant animals, but was shown to be similar to that of ruminants (Black et al., 1957).
Essential AA are the focus of most nutritional studies, since there is little evidence that
NEAA profiles are important for efficiency, or that NEAA would ever become more limiting
than EAA (Schwab et al., 1976; NRC, 2001). A number of studies, where mixtures of AA
were administered post-ruminally, indicated that requirements for NEAA were met before
requirements for EAA, and that individual NEAA absorbed below requirements can be
synthesized from excess AA in adequate amounts to maintain animal performance (Oldham
et al., 1979; Fraser et al., 1991).
2.1
Sources contributing to metabolizable protein in ruminants
Amino acids utilized by the mammary gland are provided by MP, primarily
composed of:
•
Microbial CP containing an estimated 800 g/kg of true protein (the remainder
being nucleic acids), and with 800 g/kg digestibility about 640 g/kg of MCP is
therefore converted to MP (NRC, 1989; Verbic, 2002).
•
Rumen-undegradable protein, assumed to be 1000 g/kg true protein, but the
contribution to MP is variable depending on feed type since intestinal
digestibilities were assigned to each individual feedstuff range from 500 to
1000 g/kg (NRC, 1989).
•
Endogenous CP: Data on the proportion and digestibility of true protein in
ECP is extremely limited, but its true protein content is estimated to be 500
g/kg and digestibility is assumed to be 800 g/kg, resulting in a 400 g/kg
conversion to MP (NRC, 2001).
2.1.1
Endogenous crude protein
Endogenous CP originates from various sources (Tamminga et al., 1991):
•
Mucoproteins in saliva
___________________________________________________________________
6
Review of Literature …
•
Epithelial cells from the respiratory tract
•
Cellular debris abraded from the mouth
•
Cellular debris from the omasum and abomasum
•
Enzyme secretions into the abomasum
•
Enzyme secretions into the ileum
The first three don’t contribute to protein passage to the intestine since most is
probably degraded by rumen microorganisms (NRC, 2001). A number of studies to identify
the sources of endogenous N secretions (ENS) have been reported for sheep but, due to the
complexity of N exchanges, these studies are rare for dairy cows (Ouellet et al., 2007). It is
technically tedious to distinguish between endogenous, microbial and feed N in the duodenal
digesta, hampering attempts to determine passage of ECP to the small intestine. Most studies
ignore the contribution from these recycled materials, probably overestimating the ‘true’ AA
supply from the diet and MCP.
However, some approaches measured the flow of endogenous N (EN) through the
rumen and abomasum by using cows fed diets low in CP that were considered to be free of
RDP (Hannah et al., 1991; Lintzenich et al., 1995), or ruminants solely nourished by volatile
fatty acids (VFA) infused into the rumen (Ørskov et al., 1986). The NRC (2001) adopted an
average value of 1.9 g of N/kg of DMI based on data from these studies.
2.1.2
Microbial crude protein
Microbial CP synthesis involves degradation of RDP by proteases synthesized by
various strains and species of bacteria, protozoa and anaerobic fungi in the rumen, and
incorporation of the resulting peptides, AA and ammonia into microbial protein. It also
allows ruminants to convert external NPN sources, such as urea into ammonia and
subsequently into MCP. Bacteria are the most abundant micro-organism in the rumen and
protein degradation occurs through extra-cellular proteolysis, in which soluble or insoluble
proteins adsorb to bacteria (Nugent and Mangan, 1981; Wallace, 1985) which hydrolyzes it
to small peptides and free AA which are finally absorbed for further degradation and
utilization.
___________________________________________________________________
7
Review of Literature …
However, it was found that sequestration of protozoa in the rumen results in a lower
protozoa concentration in effluent than in corresponding rumen fluid, contributing too little
to outflow protein to significantly affect the composition of the total protein mixture. Unlike
bacteria, protozoa make use of intracellular hydrolysis of protein, obtained from ingesting
small feed particles, fungi or, primarily, bacteria. Amino acids are incorporated into
protozoal protein but they are not able to synthesize AA from ammonia as do bacteria. The
contribution of anaerobic fungi to protein degradation is considered negligible, due to
relatively low concentrations in rumen digesta (NRC, 2001).
Microbial CP is considered to be the most important and least expensive MP source
and it is the largest contributor of protein reaching the duodenum, providing about 100-150g
MCP/kg of DMI (Verbic, 2002). It has a high quality AA profile (Clark et al., 1992) and
apparent intestinal digestibility of about 800 g/kg (NRC, 1989). It has long been recognized
that the EAA profile of MCP is fairly constant, because EAA profiles between different
micro-organisms, and among predominant strains, vary little (Purser et al., 1966), and their
contribution to post-ruminal protein supply is not proportional to the respective rumen
biomass fractions (i.e., protozoa, bacteria and endogenous) (Harrison et al., 1979). The AA
composition of rumen bacteria was relatively constant regardless of sampling time post
feeding (Martin et al., 1996) and diet composition (Prestlokken and Harstad, 2001), but a few
studies reported large variations in AA composition of bacteria (Clark et al., 1992) at
different levels of DMI (Rodriquez et al., 2000). Regardless, MCP has a relatively high
proportion of NPN (200 g/kg nucleic acid-N) (NRC, 1989) and the AA composition of
microbial true protein is very similar to that of milk and lean body tissue, ensuring high
efficiency of AA utilization (Verbic, 2002). Microbial CP is mainly used for protein
synthesis in the mammary gland, but also acts as a precursor in gluconeogenesis for lactose
synthesis (Rode and Vazquez-Anon, 2006).
The rate of rumen microbial growth and protein synthesis are affected by a number of
factors (Yang et al., 2001; Verbic, 2002) including:
•
Availability of rapidly fermentable carbohydrates: Energy supply in the
rumen is usually first limiting factor for microbial growth and the rate of
carbohydrate digestion in the rumen is the major factor controlling the amount
___________________________________________________________________
8
Review of Literature …
of energy available (Hoover and Stokes, 1991). At sub-optimal energy input
levels, microbial growth will increase with increased energy supply, but an
oversupply does not result in extra growth, only reduced efficiency (Dijkstra
et al., 1998) due to energy ‘spilling’ (Russell, 2007).
•
An adequate supply of N compounds: Peptides, AA and ammonia liberated
from RDP are incorporated into MCP by rumen bacteria. A deficiency of RDP
results in reduced MCP synthesis, fibre digestion, DMI and, ultimately,
reduced milk production. (Rode and Vazquez-Anon, 2006).
•
A suitable rumen environment: Low or high pH values can be deleterious to
microbial growth, reduce digestibility of fibre and divert energy in the rumen
towards non-growth functions to maintain or correct the pH.
•
Rumen outflow (turnover) rate: High DMI increases rumen outflow rate,
with microbes spending less time in the rumen. A faster turnover rate lowers
maintenance costs due to less N recycling. Higher DMI therefore increases
efficiency of MCP synthesis (Clark et al., 1992; Rodriquez et al., 2003) with
improved N digestion in the rumen (Rode et al., 1985).
Dry matter intake has the biggest influence of all dietary factors on passage of
microbial N to the small intestine, suggesting that CP in the diet should be determined
relative to DMI. The CP content could therefore be reduced in the diet of a cow eating large
amounts of DM without affecting microbial AA flow at the intestine or reducing milk yield
(Clark et al., 1992).
2.1.3
Rumen-undegradable crude protein
Microbial CP has a very high quality AA profile but, alone, it is insufficient to supply
adequate amounts of AA for optimum animal production (Rode and Vazquez-Anon, 2006).
Factors influencing the rate of passage of digesta include DMI, specific gravity (SG), feed
particle size and concentrate to forage ratio (Swanepoel, 2011). An alternative is the use of
feeds with naturally protected proteins that are relatively resistant to rumen degradation
(Clark et al., 1992) or feeds that have been chemically or physically treated to reduce protein
degradability and increase its RUP content.
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Review of Literature …
Heat processing causes the carbonyl groups of sugars to combine with the amino
groups of protein through the Maillard reaction, forming peptide links (i.e., proteincarbohydrate cross linkages) that are more resistant to enzymatic hydrolysis (Rode and
Vazquez-Anon, 2006). However, care should be taken during heat treatment since overheating reduces intestinal digestibility of RUP and leads to the destruction of AA such as
Cys, Arg and especially Lys.
Categories of chemical treatment include those that
•
Introduce cross-links by combining with proteins (e.g., aldehydes)
•
Alter protein structure through denaturation (e.g., acids, alkalis and ethanol)
•
Bind proteins without altering their structure (e.g., tannins)
However, the use of chemical treatments alone was accepted commercially, and leads
to combined chemical and heat treatments, which has been more effective in increasing the
amount of protein that escapes rumen degradation. One technique involves adding
lignosulfonate (i.e., a by-product of the wood products industry) to oilseed meals before heat
treatment (Borucki et al., 2007).
Most high quality grasses and legumes fed to lactating cows contain adequate
amounts of RDP, but are deficient in RUP, moving the focus of protein supplementation to
feedstuffs high in RUP (NRC, 2001). Common sources of RUP include animal and marine
by-products such as fishmeal and blood meal, dried distillers grains (DDG), brewers dried
grains and maize gluten meal (Rode and Vazquez-Anon, 2006). DDG are the solids that
remain after fermentation of grains such as maize during the ethanol production process.
Another challenge in diet formulation is to optimize the level of RUP reaching the
duodenum without reducing MCP synthesis, since low RDP levels have a potentially
negative effect on microbial growth due to inadequate available N supplies (Clark et al.,
1992; Ipharraguerre and Clark, 2005). No single source of RUP provides a balance of EAA
that matches the profile of milk, but proteins with the closest match are regarded as the
highest quality with the best nutritive value.
In high forage and soybean hull-based diets, where RUP intake is low, or where
animal-derived proteins make up most of the dietary RUP, Met is usually first limiting AA
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Review of Literature …
(Ahrar and Schingoethe, 1979; Schingoethe et al., 1988). In contrast, Lys has been identified
as first limiting when maize and maize by-products provide most of the RUP in the diet
(NRC, 2001). Microbial CP is low in Met, but relatively high in Lys, and the level of these
AA is lower in most feedstuffs. A deficiency in one of these AA can therefore be
exaggerated by feeding high levels of a single RUP source instead of combining several
sources with complementary AA profiles (Ferguson et al., 2000; NRC, 2001).
Optimum productivity can be achieved with the minimum amount of dietary CP when
rations are balanced to provide adequate amounts of RDP and RUP sources with desired,
complimentary, AA profiles (Clark et al., 1992; Ferguson et al., 2000; NRC, 2001). The
efficiency with which MP is used for protein synthesis depends on the amount of EAA in it
and how well the EAA profile in MP matches the AA profile required by animal metabolism
(NRC, 2001).
Increased milk production and protein yields require an increase in feed CP intake
and/or an improved postruminal supply of AA. Feedstuffs with low rumen degradability
and/or high quality protein with a well balanced AA profile (such as meat meal, bone meal,
fishmeal etc.) can be used to increase postruminal AA supply, but they are expensive and
legislatively banned in cattle feed in India. It is therefore becoming more difficult to
formulate rations that will provide the desired AA concentrations and ratios in MP.
2.2
Comparative Amino acid requirements in different species
There is remarkable consistency across species in amino acid composition of mixed
body proteins from fetal, growing and mature animals, suggesting that minimal qualitative
requirement for essential amino acids for growth will be similar. However there are few
exceptions. In fish, the composition of the tissue is also relatively constant across the fish
species, with the exception that lysine and arginine contents are higher in transgenic carp (Fu
et al. 2000), which may suggest that requirements for these are higher. In sheep, as expected,
wool is higher in the sulphur amino acids than average tissue proteins, but lower in lysine
and histidine, supporting recommendations that the sulphur amino acid requirement for wool
producing sheep is higher. Egg proteins appear to be similar in composition to body tissues
except for low level of lysine in eggs. These appears to be a consistent composition of milk
across several species ( human, sows, horses, cows, goat) despite the fact that the whey and
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Review of Literature …
casein contents vary across species (Davis et al. 1994). Compared to tissue, the composition
of cow and sow milk protein is adequate in most amino acids to support growth rate (Leibolz,
1982; Fligger et al., 1997). Reduced feed intake during the early weaning period may
exacerbate these deficiencies leading to the bacterial translocation, gut atrophy, mucosal
shedding and weight loss. Supplemental glutamate and glutamate plus arginine appear to
reverse these affects by enhancing total gut weight and preventing villus atrophy. Glutathione
plays an important role in maintaining the defence mechanisms of the gut mucosa against
peroxidative damage. Because glutamate, glycine and cysteine for glutathione synthesis are
derived mainly from the gut lumen (Reeds et al., 1997), glutathione synthesis may suffer at
the time of weaning when food intake is less than optimal.
There are metabolic differences between species that necessitates higher requirement
for certain amino acids to support maximum growth, thus these are designated as
conditionally essential. One species difference relates to expression of enzyme of the
ornithine - urea cycle. This cycle serves to dispose of excess ammonia, and it also plays a
critical role in the synthesis of the glutamate family of amino acids (proline, arginine,
citrulline, ornithine), in particular arginine, which can be degraded to form urea and
ornithine. The high concentration of the arginase, which catalyzes the hydrolysis of Larginine to produce L-ornithine and urea in the liver of rapidly growing animals, coupled
with the low rate of intestinal arginine synthesis, limits arginine for protein synthesis (Wu et
al., 1997). Arginine is required in cat’s diet because they lack the enzyme to synthesis
arginine and ornithine, plus they have the limited ability to convert glutamate into ornithine.
In the absence of these precursors, cats become comatose due to the buildup of toxic
ammonia. Symptoms can be reversed by supplemenating the diet with the arginine or its
precursor ornithione. Chickens also need dietary arginine because they do not have
functional urea cycle and the situation is amplified because of the high content of the
arginine in the feathers. Fish requires higher amount of arginine in the diet, not because they
do not have urea cycle, but because the main route of elimination of excess nitrogen is via
ammoniagenesis (transamination and deamination route). As a result, transfer of nitrogen
into the ornithine cycle is low with limited synthesis of arginine. In marine cartilagious fish,
the arginine requirent is higher and this probably reflects the need to synthesize urea to help
maintain buoyancy when salinity is low (Withers, 1998). Another example of species related
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Review of Literature …
requirements is taurine and phenylalanine/tyrosine in cats. Due to limited ability to convert
cysteine into taurine and the limited conversion of taurine via conjugation with cholic acid,
young cat require dietary taurine to prevent renal degeneration. Cat also requires higher
dietary levels of phenylalamine/tyrosine for melanine synthesis; otherwise, hair colour turns
from black to redish brown. Other species can convert phenylalamine into tyrosine via
phenylalanine hydroxylase, but this has not proven to be limitation. Wool growth is limited
by the supply of cysteine. Although there is synthesis of cysteine locally within the wool
follicle or skin via methionine transsulphuration with serine, the supply of methionine from
rumen microbial protein is generally limited. Moreover the transsuphuration pathway in the
skin may have to compete with polyamine synthesis and transmethylation reactions for
methionine.
2.3
Rumen Protected Proteins in Ruminant Nutrition
2.3.1
Degradation of Proteins in the Rumen
Feed proteins are hydrolyzed into peptides and amino acids by rumen micro-organisms.
However, most amino acids are rapidly degraded to organic acids, ammonia and carbon
dioxide. The ammonia produced is the primary nitrogenous nutrient for bacterial growth.
Some species of ruminal bacteria use peptides directly for synthesis of microbial protein.
Chalupa (1975) quoting several sources indicated that as little as 40 per cent or as high 80 per
cent of dietary protein normally might be degraded in the rumen and transferred into
microbial protein. Because rumen microbial protein production is an energy-dependent
mechanism, the amount of dietary protein transformed into microbial protein must be an
important aspect of nitrogen economy of the animal and should be an important factor in
whether or not to decrease ruminal degradation by artificial procedures. Although amino
acids are rapidly deaminated in the rumen under practical conditions, the rumen microbial
population derives 25-50 per cent of their nitrogen from sources other than ammonia. These
presumably intact amino acids or peptides which originate either directly from food protein,
from recycled nitrogen to rumen or from turnover of bacterial and protozoal protein within
rumen (Oldham, 1981). Ruminal degradation of proteins can be reduced by decreasing
retention time in the rumen. Factors known to influence this include level of food intake,
specific gravity, particle size of diet, concentrate to roughage ratio and rate of rumen
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Review of Literature …
digestion. Similar to other nutrients, the amount of protein that riches the small intestine
depends upon food intake. The breakdown of proteins by microorganisms as mentioned
earlier, gives rise also to intermediate products such as free amino acids in the rumen. The
low concentration of free amino acids in the rumen usually suggest rapid utilization, but
increased concentration after feeding imply that proteolysis occurs faster than subsequent
utilization of free amino acids (Chalupa, 1975). The free amino acids in the rumen can be
assimilated directly by microbes and be absorbed through the rumen but most are deaminated
to yield ammonia and other intermediate products (Hoover and Miller, 1991). The microbial
protein alone is likely sufficient to meet the needs of cattle at or near maintenance. Young
growing cattle and lactating cows need bypass protein in addition to microbial protein to
meet their metabolizable protein requirements. The international literature suggests that the
requirement for rumen degradable protein is 130 g kg-1 of digestible organic matter (DOM).
On the average, the amount of microbial protein synthesized is 130 g/kg of the DOM
(Klopfenstein, 1996).
2.3.2 Protection of Protein from Ruminal Degradation
It is possible to protect proteins using several procedures such as heat treatment,
chemical treatment/modification, and inhibition of proteolytic activity and identification of
naturally protected protein (Ferguson, 1975). The use of these techniques in comparison to
the usual sources of dietary proteins improves the supply of amino acids without an increase
in ammonia production, resulting in a better performance by the animal (Kaufman and
Lupping, 1982).
2.3.2.1 Heat Treatment
The effect of heat treatment during manufacturing or drying of forages in reducing
the rate of microbial fermentation is attributable to reduced solubility of the protein. In
feeding trials on heat treated protein, the rumen level of ammonia concentration is usually
very low and there is improvement in nitrogen balance or better growth particularly in sheep
and in some cases calves (Kaufman and Lupping, 1982). Increases in milk yield have also
been reported (Dijk et al.,1983). Heat treatment has been used to increase the undegradable
protein of common feedstuffs such as soybeans and grains (McNiven et al., 1994, Robinson
and McNiven, 1994, Prestlokken, 1999). However, high temperature and extended heating
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Review of Literature …
time have increased the acid detergent insoluble nitrogen content by the Maillard reaction
between sugars and amino acids (Satter, 1986, Broderic et al., 1991). Amino acids also form
peptide links with asparagine and glutamine (Belitz and Grosch, 1987). This resulting peptide
linkages from heating are more resistant to enzymatic hydrolysis. Excessive heating can
cause essential amino acids such as lysine, methionine, and cystine to be extensively
damaged (Kung, 1996). Although moderate heat may increase the protein flow to the small
intestine, excessive heat may decrease the quantity of some amino acids and lower the
digestibility of protein in the small intestine (McNiven et al., 2002).
The roasting of soybean meal increased the amount of soy protein escaping microbial
degradation in the rumen. Reduction of protein degradation occurs because of Maillard type
reaction between sugar aldehyde group and free amino groups (Dhiman et al., 1997).
Roasting of soybean and soybean meal resulted in the lowest in situ degradation rates of
protein 0.037 and 0.029 per hour, respectively (Faldet et al., 1991).
Walli, (2005) fine-tuned the heat treatment of GN-cake and soybean-cake and found
1500C for 2hr as the optimum temperature-time combination. Walli and Sirohi (2004) found
that the roasting of soybean at 1300C for 30 min protected its protein content from ruminal
degradation.
However, some disadvantages of dry processing of grains and feeds have also been
reported. Ayatse et al. (1983) reported that roasting of maize at 120°C to 130°C decreased
its nutritive value with loss of moisture, minerals and vitamins and reduced the essential
amino acid index. Roasting regular sunflower seeds tended to decrease the digestibility of fat
and energy (Adams and Jensen, 1985). The problem with ‘heat treatment’ is that it may not
be cost effective and moreover, it can also over-protect the protein. Formaldehyde treatment
has been used by several workers in India to reduce the protein degradability of high
degradable cakes and also to study the impact of its feeding on the productive performance of
dairy animals (Ramchandran and Sampath, 1995; Chatterjee and Walli, 2003).
2.3.2.2 Formaldehyde Treatment
Treatment of proteins with formaldehyde is the most widely used process at the
present time and it has been exploited commercially. Treatment of high quality proteins
result in the formation of cross-links with amino group and makes the protein less susceptible
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15
Review of Literature …
to microbial attack (Shelke et al., 2012). Such treatments of protein -rich feedstuffs has been
shown to increase the protein digested in the intestine and net nitrogen retention. The
concentration of amino acids in the plasma is generally increased depending on tissue
demands and the balance of amino acids supplied (Ferguson, 1975). The protection of rapidly
degradable proteins by either heat or formaldehyde might make more protein or amino acids
available for the host animal, but it might reduce the synthesis of microbial matter and
actually decrease productivity. Further to this, it has been observed that excessive heating or
over-treatment with formaldehyde of dietary protein can have a detrimental effect on the
nutritive value of the protein resulting in decreased digestibility (Vallejo, 1996) and poor
animal performance. A proportion of plant protein is already protected by its insolubility and
deliberate protection is pointless in this case. The excessive heating or overtreatment of
protein may also reduce the efficiency of energy utilization due to low ammonia N in the
rumen.
Antoniewicz et al. (1992) reported that the FT reactions with protein may be an
addition or condensation type in which non-ionic bands are formed between the active side
chain groups of amino acids like S–H, –OH, NH 3 etc. and the carbonyl (–C=O) group of FT.
Secondary cross-linking by methylene bridges may also be possible. Most of these reactions
are reversible under the action of diluted acid solution and do not affect amino acid
composition or post-abomasal protein digestibility.
Formaldehyde is available in several forms and concentrations. The form most often
used in feeds is formalin, a solution containing approximately 40 per cent formaldehyde. The
level of formaldehyde to be applied varies according to the type of feed and its protein
content. Treatment with higher dose of formaldehyde can have an adverse effect in causing
overprotection of the cake, which makes protein indigestible even in lower tract and can
hardly supply any RDN for rumen microbial growth. Thus, the level of HCHO is very
important and should be optimum due to its criticality.
2.3.2.3 The Coating of Protein Source Particles with Insoluble Substances
The coating of protein sources with insoluble substances like blood meal (Orskov,
1992) or fats (Sklan and Tinsky, 1993) have been used. The latter has drawn the attention of
researchers since fats, beside their effects on protein protection, are energy sources that could
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Review of Literature …
be important for high producing dairy cows during the first stage of lactation. The coating
soybean meal with lipid substances (Fish oil, beef tallow) is a protective method against
microbial degradation in the rumen. The coating of protein with lipid substances decreased
the ruminal degradability. The effect of protection increased with increasing amount of
coating agent (Manterola et al., 2001).
2.3.3 Advantages of Rumen Protection
In India, dairy animals by and large do not get their required energy content through
the normal feed which the animals are offered, as the feed is mostly devoid of any energy
rich grains. Success achieved in terms of increase in milk yield (volume) through the feeding
of bypass protein is essentially due to the supply of more energy to these energy deficient
animals, through the same feed, as the extra amino acids supplied through bypass protein
feeding are converted to glucose in liver. Thus, essentially the feeding of bypass protein
increases the efficiency of protein and energy utilization within the ruminant system.
Numbers of studies have been conducted on feeding of naturally occurring bypass protein
like cottonseed cake and maize gluten-meal to lactating ruminants, in India with most of
these experiments giving positive results (Ramchandra and Sampath, 1995; Chaturvedi and
Walli, 2000; Walli, 2005, Shelke et al., 2012).
Post ruminal utilization of nutrients eliminates energy losses associated with
fermentation and protein losses incurred in the transformation of dietary protein to microbial
protein. However, there are suggestions that protein digestion in the small intestine of the
ruminants may not be as complete as it is in monogastric, and that alteration of patterns of
absorbable nutrients produced by changing sites of digestion could also influence animal’s
endocrine status. Protection from ruminal degradation enables more amino acids to reach the
intestine and therefore provide more absorbable amino acids per unit of absorbable energy.
Heat treatment and the use of chemical treatment such as formaldehyde have been shown to
increase the percentage as well as the total of both dietary protein and amino acids escaping
degradation in the rumen. Provision of ruminally protected amino acids will depend very
much upon whether shortages of many or only a few amino acids are limiting production
(Chalupa, 1975). This is because the magnitude of ruminal destruction of dietary proteins
varies, and greatest responses can be expected when rumen soluble proteins are protected. To
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17
Review of Literature …
obtain responses with supplements of protected amino acids, one must know that amino acids
are in short supply for animals performing a particular physiological function. A lot of
studies showed that diet containing higher amounts of ruminally undegradable proteins or
ruminally protected amino acids resulted in increased milk production while other studies
show little or no response. The lack of response to ruminally undegradable protein is often
due to one of following reasons: (1) the rumen undegradable protein may have been bypassed
at the expense of ruminal microbial protein synthesis; (2) the ruminally undegradable protein
may have been poorly digested postruminally; and (3) the ruminally undegradable proteins
may have been deficient in the amino acids that limiting production (Schingoethe, 1996).
However, limited numbers of studies have been conducted on feeding of
formaldehyde treated cakes to lactating animals. Sampath et al., (1997) reported significantly
higher FCM yield in lactating crossbred cows fed formaldehyde treated GNC (7.8 vs. 9.4
kg/d). Walli and Sirohi (2004) also reported 15 % increase in milk yield on feeding of
formaldehyde protected mustard cake to crossbred cows. Sahoo and Walli, (2005) reported
that milk yield in mustard cake fed goats increased significantly from 1306 g/d in control
group to 1439 g/d in formaldehyde treated mustard cake fed group.
Positive response on milk production performance as a result of feeding
formaldehyde treated proteins has been observed by several workers (Madson, 1982;
Morgan, 1985). Garg et al., (2003) while comparing naturally protected protein (30 % UDP)
and processed (formaldehyde treated) sunflower seed meal supplement (optimal-bypass with
75 % UDP) in crossbred cows, found a significant increase in milk yield, milk fat and milk
protein percent. Yadav and Chaudhary, (2004) reported significantly increased milk yield
and FCM yield in medium producing cows on feeding formaldehyde treated GNC.
2.4 Limiting amino acids
Limiting AA refer to EAA that are in shortest supply relative to requirements. The
efficiency with which absorbed AA are utilized will likewise be determined by the supply of
the first limiting AA.
Methionine and Lys were identified, more than 30 years ago, as being the most
limiting AA for milk production in dairy cattle (Schwab et al., 1976), growth in steers (Burris
et al., 1976) and weaned dairy calves (Schwab et al., 1982), followed by Phe, Ile and Thr as
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Review of Literature …
most frequently limiting (Vik-Mo et al., 1974; Derrig et al., 1974; Nichols et al., 1998; Liu et
al., 2000). These findings have been confirmed by many other infusion studies. His and Arg
were recently identified as limiting when cows are fed grass-silage, barley and oat grain
based diets (Vanhatalo et al., 1999; Huhtanen et al., 2002) and that His may be the third
limiting AA in some maize-based rations.
The quantity and ratio of AA from MCP and dietary CP reaching the intestine may
determine whether animal production will respond to feeding rumen protected AA (RPAA).
Due to the low Lys content in maize products (16.5 g Lys/kg CP), the contribution of Lys to
AA passage to the intestine is reduced when higher levels of maize products are included in
the diet (Rogers et al., 1989). It has been suggested that large amounts of maize in rations
also reduces microbial growth, thereby reducing AA passage to the small intestine.
By supplementing, or infusing, various amounts of individual, or combinations, of
EAA into the abomasum and duodenum, researchers measured the effects of AA
supplementation on N retention to try and increase milk protein production (Pisulewski et al.,
1996; Huhtanen et al., 2002; Schei et al., 2007).
2.5 Amino acid supplementation
All AA exist as the isomers D and L which are chemically identical, the one being a
mirror image of the other. However AA in plant and animal proteins, as well as some
produced industrially, such as Lys, Thr and Trp, are in the L-form, while chemically
synthesized Met is a mixture of the two (i.e., DL-Met). Rumen protected methionine (RPM)
fed in the D-form are absorbed into the plasma but needs to be converted to the L-isomer
within tissues before it can be incorporated into animal proteins. Efficiency of conversion of
commercial Met products from the D to L form has been of some concern, but studies have
shown that the efficiency of use of D-Met, relative to L-Met, was 960 g/kg in growing steers
(Campbell et al., 1996).
Free AA are very sensitive to microbial degradation in the rumen and, due to
extensive deamination of hydrolyzed AA (Lewis and Emery, 1962), concentration of free AA
in rumen fluid is low (Velle et al., 1997; Volden et al., 2001). Supplementing free
(crystalline) AA to the diet has not been as effective in dairy cows as it has been in pigs and
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Review of Literature …
poultry, and studies with growing cattle have shown rapid degradation and minimal passage
of free AA to the duodenum when it is introduced into the rumen (Campbell et al., 1997).
Many studies have been conducted to increase the amount of AA that escape from the
rumen degradation by protecting EAA such as Lys and Met by chemical alteration or
physical protection.
2.6 Rumen protected amino acids
Use of protected amino acids, in contrast to protected proteins does not reduce the
excess of ammonia in the rumen and thus the load on the liver. Their effectiveness depends
upon the optimization of amino acids supply and pattern of amino acids reaching the intestine
(Kaufman and Lupping, 1982). When protected amino acids are used, response can mainly
be expected in the main production parameters such as milk yield and milk content, growth
rate etc. (Chalupa, 1975, Ferguson, 1975). For animals of low growth rate or milk
production, amino acid needs can be met by mainly microbial protein which has a good
amino acid balance. Amino acid deficiencies are only expected to occur in high performing
animals. As far as the question of which amino acid are limiting are concerned, this has been
a subject of several studies particularly with respect to lactation since amino acids output in
milk can be easily measured. Various techniques have been used in these studies, such as
comparison of the amino acid pattern of bacterial and milk protein, changes in the amino acid
content of blood after casein infusions and arteriovenous differences across the mammary
gland. The conclusion of these studies indicated that methionine is always included in the
group of most limiting amino acids, often closely followed by lysine, histidine, phenylalanine
and branched-chain amino acids (Kaufmann and Lupping, 1982). The primary methods
developed to prevent fermentative digestion of amino acids are structural manipulation to
produce amino acid analogs and coating with resistant materials (Chalupa et al., 1996).
Various analogs of amino acids have been tested for resistance to ruminal degradation
(Ayoade et al., 1982). One of the most tested amino acid is methionine hydroxy anolog. Test
results have been variable, with occasional improvements in milk production and milk fat
(Kung et al. 1996). Methionine and other amino acids can be protected from bacterial
degradation in the rumen by mechanical or chemical methods. The use of both processes has
led to commercially marketable products. Amino acids have been coated with polymeric
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20
Review of Literature …
compounds, formulized protein, fat, mixtures of fat and calcium, mixtures of fat and protein,
and with calcium salts of long chain fatty acids (Chalupa and Sniffen, 1991). Methionine and
lysine are limiting for milk yield and milk protein synthesis when cows are fed cornbased
diets. Post ruminal supply of specific amino acids can be increased by supplementing the diet
with polymerically encapsulated amino acids. In a number of experiments where protected
methionine and lysine have been used to increase protein content and milk yield, responses
have been small and variable in terms of milk yield. However, there was a small consistent
positive response in milk protein concentration (Donkin et al., 1989; Robinson et al., 1992;
Kincaid and Cronrath, 1993; Christensen et al., 1992; Martelli et al., 1993). In these
situations, the basal supply of amino acids could be different between experiments which
could account for the variable responses obtained. However, methionine supplementation has
failed on some occasions to produce the expected increase in the level of milk yield and
protein content or growth (Weber et al., 1992). Thus supplying protected amino acids to the
small intestine appears to offer some scope for increasing milk and protein concentration.
However, it may be necessary to supplement a number of the essential amino acids and not
just one or two, in order to get a significant response (Murphy and O’Mara, 1993).
Significant progress has been made in developing technologies to increase availability
and absorption of EAA by ruminants (NRC, 2001).
Protection methods currently used can be divided into three categories:
2.6.1 Liquid sources of hydroxy analogs (chemically modified molecules)
Analogs differ in chemical structure from their L-AA counterparts. They contain a
hydroxyl instead of an amino group and are therefore recognized by rumen microbes as an
organic acid (i.e., a fermentation end product) and not an AA. This aids in their rumen escape
potential since they are usually relatively reduced acids (compared to lactate which is more
oxidized) with only a selective group of microbes capable of extracting energy by fermenting
it. The Met analog may be a free acid in an aqueous solution or a Ca salt in a dry solid
(Koenig et al., 1999). As a liquid, it is easy to handle and can be incorporated into feed
pellets. The most studied Met hydroxy analog is DL-2-hydroxy-4-Methylthiobutanoic acid
(HMB) due to its successful use in monogastric animals. Rumen escape of HMB is a function
of the passage rate of the liquid phase from the rumen, the extent of degradation by
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Review of Literature …
microorganisms and, to some extent, absorption of HMB across the rumen wall (Koenig et
al., 1999). Absorption occurs by diffusion across the rumen (the portion degraded by
microbes), omasum and intestinal wall (the portion escaping rumen degradation) into the
blood stream. Productive tissues such as the mammary gland and muscle remove HMB from
circulation and convert it to L-Met (Rode and Vazquez-Anon, 2006). Esterification of HMB
to various alcohols, including the isopropyl ester of HMB (HMBi), decreases its rate and
extent of rumen degradation (St-Pierre and Sylvester, 2005).
Various in vitro (Vazquez-Anon et al., 2001) and in vivo (Koenig et al., 1999) studies
showed Met analog, HMB (DL-2-hydroxy-4-Methylthiobutanoic acid) was more resistant to
rumen degradation than Met, but controversy exists about its rumen escape rate. Values
range from as low as 10 g/kg of fed HMB recovered in the duodenum (Jones et al., 1988) to
as high as 500 g/kg, based on serum Met concentrations (Koenig et al., 1999). This variation
may be due to differences in dose levels, method of supplementation or physiological status
of the cows (Rulquin et al., 2006). Animal responses to HMB has mainly been an increase in
milk fat proportion, possibly due to its effect on rumen fermentation and VFA production,
increasing acetate production (Noftsger et al., 2003), with less consistent responses in milk
yield and none in milk protein (St-Pierre and Sylvester, 2005).
Approximately 500 g/kg of HMB escapes rumen degradation, determined by using
blood and milk true protein changes as bioavailability indicators (Noftsger et al., 2005),
regardless of whether it is supplemented in liquid or dry form (St-Pierre and Sylvester, 2005).
It is quickly absorbed in the small intestine, hydrolyzed into HMB and isopropyl and then
converted to Met and acetone, delivering about 480 g/kg Met to the cow (Graulet et al.,
2005).
2.6.2 Surface coating or matrices of saturated fatty acids and minerals
Development of a lipid-protected product requires identification of a process, and a
fat, to use for the matrix, or coating, that provides a reasonable degree of protection in the
rumen while allowing adequate intestinal release (NRC, 2001). Apparent bioavailability
depends on ruminal escape and intestinal release of AA. The products consist of DL-Met in a
matrix of Ca salts of long-chain fatty acids (LCFA), lauric acid, and a fatty acid (FA)
preservative; butylated hydroxytoluene (BHT).
___________________________________________________________________
22
Review of Literature …
Carbohydrate-protected products has a combination of coating materials applied to
ensure slow degradation in the rumen and slow release of Met in the intestine. It has a core,
consisting of DL-Met, coated with starch and several thin layers of stearic acid and
ethylcellulose. The latter minimizes enzymatic digestion and so the release of Met depends
on physical action and abrasion, wearing away the corners of the pellets (Lapierre et al.,
2002).
2.6.3
Surface coating with a fatty acid or pH-sensitive polymer mixture
This system involves protection of an AA core by coating it with a lipid/pH-sensitive
polymer. Release of AA do not depend on digestive enzymes, but rather a change in pH
between the rumen (i.e., pH 6.2 + 0.7), and the abomasum (i.e., pH 2.5 + 0.3). Here, DL-Met
is protected by a layer of ethylcellulose covered with a coating of stearic acid. It has
improved resistance to rumen degradation due to the presence of a copolymer, poly (2vinylpyridine-co-styrene), altering the steriochemistry of stearic acid. The copolymer
solubilizes at low pH, rapidly releasing Met in the abomasum.
2.7
Responses to rumen protected methionine and Lysine dietary supplementation
2.7.1
Effect on Milk Yield and Milk component yield
Low concentrations of Met in MP limited the responses to increasing concentrations
of Lys in MP and that low concentration of Lys in MP limited the responses to increasing
concentration of Met in MP (NRC, 2001). Amino acid concentrations in plasma, measured at
different time intervals after feeding the ruminally-protected product can be used to assess
and to compare AA availability among product. (Bach and Marshall, 2000).
Due to the high rumen protection and intestinal release coefficient of AA in the pH
sensitive products, it seems to be the most effective technology with the largest increases in
blood AA concentrations, but according to Watanabe et al., (2006), AA protected by a fat
coating are just as capable of improving production performances as the pH sensitive
polymer products.
Lara et al. (2006) reported no treatment effects of supplemented ruminally protected
methionine on DM intake (20.38 kg d-1), body weight (599.78 kg), body condition score
(2.51 units). However, milk production (35.8 kg d-1) and protein yield (3.16 kg d-1) were
___________________________________________________________________
23
Review of Literature …
increased with addition of RPM. Milk production responded quadratically to methionine
levels. The quadratic response in milk protein output was also reported by Guinard and
Rulquin (1995), effects which have been associated to a major synthesis of casein and a
reduction in urea nitrogen (Wu et al., 1997a,b; Dinn et al., 1998). Holstein cows with a mean
production of 35 kg d-1 milk require addition of ruminally protected methionine (16 g d-1) to
improve milk production (Lara et al., 2006).
Noftsger and St-Pierre, (2003) observed significant improvement in milk production
on dietary supplementation of rumen protected methionine (23.6 kg d-1) as compared to that
of control (21.7 kg d-1). Similarly, Bach and Marshall (2000) demonstrated an increase in
milk yield on supplementing rumen protected methionine to lactating Holsteins cows (45.9
vs 47.7 kg d-1). Similarly, Broderick et al. (2009) reported that supplementation of rumen
protected methionine and lysine enhanced the milk yield in crossbred cows (41.5 kg d-1) to
that of control (39.4 kg d-1) while Yang et al. (2010) reported increase (18.95 vs 21.55 kg d-1)
in milk yield on supplementation of rumen protected methionine and lysine. While Socha et
al. (2009) found that there was increase in milk production on abomasal infusion of
methionine and lysine during peak and early lactation while there was no difference in mid
lactation.
Multiparous cows showed increase in milk yield, milk protein and fat yield and
efficiency of dry matter utilization for milk production on rumen protected methionine
supplementation as compared to primiparous cows (Davidson et al. 2008). Broderick et al.
(2009) found that supplementation of rumen protected methionine in diet with reducing CP
level by replacing dietary soybean meal with high moisture corn, had no effect on DMI but
there was increase in milk yield, milk fat and protein yield.
However there are very few published articles involving supplementation of RPL
without concurrent supplementation of RPM. Rogers et al. (1989) fed three levels of RP Lys
(5.9, 13.5, and 21.1 g/d) to 3 groups of cows at different stages of lactation and reported
improved milk and milk protein production when cows were fed maize based diets, but not
when soybean meal was added to the ration. Plasma concentrations of Met and Lys were also
increased.
___________________________________________________________________
24
Review of Literature …
Xu et al. (1998) reported a positive response in milk yield and milk protein, that was
consistent through different stages of lactation, and an increase in milk fat content during
early lactation when RPL and RPM were supplemented to a ration limiting in metabolizable
Lys and Met.
Socha et al. (2005) studied the effect of RPM (10.2 g) and RPL (16.0 g)
supplementation at two level of dietary CP i.e. 18.5 and 16.0% and found that there was
increase in milk yield, milk fat, milk protein yield in both treatment groups while group
receiving diet with 16% CP and supplemented with RPM and RPL performed with same
efficiency compared to the group receiving 18.5% CP. Other studies also found that there
was increase in milk yield, milk fat yield and milk protein yield without affecting dry matter
intake on RPM and RPL supplementation. (Wu et al., 1997; Armentano et al., 1997;
Robinson et al., 1997).
In contrasts, many researchers Berthiaume et al. (2001), Misciattelli et al. (2003),
Girard et al. (2004), Noftsger et al., (2005), Broderick and Muck, (2008), Davidson et al.
(2008), Benefield et al. (2009) did not found any effect on milk production on supplementing
rumen protected methionine while Swanepoel et al. (2011) did not find any effect on milk
production on supplementing rumen protected lysine alone.
2.7.2
Effect on plasma amino acids
Increased supply to the small intestine of any AA is expected to change its
concentration in blood plasma and, possibly, improve availability of that AA for milk protein
synthesis. This was demonstrated by Blauwiekel et al. (1997) in an experiment in which
lysine supplementation increased duodenal lysine flow, as well as the concentration of lysine
in plasma, leading to an increased milk and milk protein yield.
Baumrucker (1985) explained that the transport systems for absorption of AA depend
on transport specificity and competition among AA. As feeding RPL provides more lysine to
cells with a Y+ (cationic) transport system, the uptake of AA sharing the same transport
system (i.e., arginine and isoleucine) may be reduced through competitive inhibition with
lysine.
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25
Review of Literature …
Supplementation with RPM caused an elevation in arterial concentration of Met
(Berthiaume et al., 2001, Overton et al., 1996, 1998; Blum et al., 1999) or the results were
same when Met infused postruminally (Guinard and Rulquin, 1995; Pisulewski et al., 1996;
Varvikko et al., 1999).
It is reported that there were decrease in plasma methionine concentrations (Rogers et
al., 1987) or decrease in most non-essential AA (NEAA) as well as threonine, leucine and
tryptophan (Swanepoel et al., 2011) when RPL was fed. Swanepoel et al. (2011) reported
that the plasma lysine concentration was only numerically increased with RPL feeding.
Results of Weekes et al. (2006) suggested that a decreased concentration of plasma AA
(especially histidine) without a positive milk protein response could be due to a more
efficient catabolism or deposition of AA into body protein, which might be supported by the
trend for lower BCS loss on RPL supplementation.
2.7.3
Effect on blood metabolites
Postruminal infusion of a basal amount of Lys and incremental amounts of Met
resulted in a linear increase in plasma BHBA (ß- Hydroxyl Butyric Acid) of cows entering
the second 100 d of lactation (Socha, 1994), but had no effect on plasma BHBA of cows
assigned to the treatments before 50 DIM (Pisulewski et al., 1996; Socha, 1994) or after 150
DIM (Socha, 1994). In these same infusion studies, increasing the intestinal supply of Met
linearly reduced plasma NEFA concentrations when the cows were assigned to the studies
before 50 DIM (Pisulewski et al., 1996; Socha, 1994) but not when they began receiving
treatments as they approached 100 DIM or after 150 DIM (Socha, 1994). One potential
reason why blood concentrations of energy metabolites such as NEFA, BHBA, and glucose
are not affected by improved Lys and Met nutrition in production studies (Chow et al., 1990;
Chapoutot et al., 1992; Xu et al., 1998) is that the effect may be transitory. In the production
studies, the experimental periods were 21 d or longer, with blood samples usually being
collected several weeks after initiation of treatments. This is in contrast to the infusion
experiments where the length of experimental periods was 10 to 14 d and all blood samples
were taken less than 2 wk after initiation of treatments (Socha, 1994; Pisulewski et al., 1996).
In contrast, Socha et al., (2005) found that there were no effects of RPM and RPM+L
supplementation on postpartum plasma NEFA and BHBA concentrations. While cows fed
___________________________________________________________________
26
Review of Literature …
RPM+L tended to have lower plasma glucose concentrations than cows fed only the basal
diet and RPM supplemented group. Feeding RPM reduced serum urea concentrations
compared with feeding on feeding RPM+L. They also reported that the effect of RPAA
supplementation on plasma glucose concentrations was dependent upon week postpartum.
Between wk 1 and 4 postpartum, plasma glucose concentrations of cows receiving RPM+L
declined at a more rapid rate than cows receiving either the basal diet or RPM. However,
between wk 4 and 7 postpartum, plasma glucose concentrations of RPM+L supplemented
cows increased, whereas plasma glucose concentrations of cows receiving no RPAA or RPM
remained essentially unchanged. Immediately following parturition, glucose concentrations
decreased in cows receiving 16 % CP in diet and 15 gm of RPM supplemented group, 16%
CP in diet and 6 gm of RPM plus 40 g/d of a RPM+L supplemented group, and 18.5% CP in
diet and 6 gm of RPM plus 40 g/d of a RPM+L supplemented group and increased in cows
receiving 18.5% CP and 15 gm of RPM supplemented group.
Similarly, in other reports, plasma NEFA, glucose and BHBA were not affected when
lactating dairy cows were fed RPM+L (Chow et al., 1990; Chapoutot et al., 1992; Xu et al.,
1998). Further, Misciattelli et al. (2003) did not found any effect on plasma NEFA
concentration on supplementation of rumen protected methionine and lysine, and Davidson et
al. (2008) on supplementation of rumen protected methionine. There was no significant
effect of abomasal infusion of methionine and lysine on plasma NEFA during mid lactation
(Socha et. al., 2008).
However, decrease in plasma nonesterified fatty acids concentrations in preruminant
calves (Auboiron et al., 1995; Chilliard et al., 1994) and lactating cows (Pisulewski et al.,
1996; Rulquin and Delaby, 1997) with increased amounts of Met in MP had been reported.
Similarly Socha et al. (2008) reported that there was decrease in plasma NEFA
concentration on abomasal infusion of rumen protected methionine and lysine during early
lactation and Bach et al. (2000) reported decreased plsma NEFA concentration on
supplementing rumen protected methionine and lysine.
Socha et al., (2005) found that there was no effect on BUN level on abomasal
infusion of methionine and lysine. While Berthiaume et al. (2001) and Bach et al. (2000)
___________________________________________________________________
27
Review of Literature …
found increased blood urea nitrogen concentration on supplementation of rumen protected
methionine.
Davidson et al., (2008) reported increased plasma triglyceride and VLDL level on
supplenentation of rumen protected methionine. The increase in milk fat percent may relate
to the role of AA in the intestinal and hepatic synthesis of chylomicrons and VLDL.
Required substrates for the synthesis of chylomicrons and VLDL, in addition to the presence
of the long-chain fatty acids that stimulate their formation, include apolipoproteins and
phospholipids (Bauchart et al., 1996). The synthesis of apolipoproteins requires AA. The
synthesis of phosphatidylcholine (lecithin), the most abundant phospholipid, requires choline.
It has been demonstrated that a portion of the dairy cows’ requirement for Met is as a methyl
donor for choline synthesis (Sharma and Erdman, 1989; Erdman, 1994), choline can be a
limiting nutrient for milk fat synthesis. That Met and Lys may sometimes be limiting for the
synthesis of chylomicrons or VLDL such that the availability of long chain fatty acids for
milk fat synthesis is reduced has not been demonstrated. However, there is limited evidence
that formation or secretion of these lipoproteins can be enhanced with improved Met and Lys
nutrition (Auboiron et al., 1995; Durand et al., 1992).
2.8 Reproductive efficiency of animals fed on protected protein and rumen protected
amino acids
Studies conducted at NDRI have shown that the feeding bypass protein to dairy
animals improves reproductive efficiency in both males and females. Feeding bypass protein
as such leads to higher growth rate in cattle/buffalo calves and goat kids. This improved
growth rate as such results in early maturity of both male and female animals, which has also
an over all impact on lifetime production of the animals. Apart from that, the feeding of
bypass protein improved the conception rate and reduced the number of days open in
crossbred cows (Kaur and Arora, 1995). Similarly, feeding bypass protein increased the
sexual behavior and seminal attributes in Mehsana buffalo bulls and also in crossbred bucks
(Walli, 2008). Thus, the improvement in reproductive efficiency of dairy animals is yet
another distinct advantage of feeding bypass protein to such animals.
Protein in excess of lactation requirements has been shown to have negative effects
on reproduction. Several workers have reported that feeding diets containing 19 percent or
___________________________________________________________________
28
Review of Literature …
more CP in diet DM lowered conception rates (Bruckental et al., 1989; Canfield et al., 1990;
Jordan and Swanson, 1979; McCormick et al., 1999). Others have observed that cows fed 20
– 23 percent CP diets (as compared to 12– 15 percent CP) had decreased uterine pH,
increased blood urea, and altered uterine fluid composition (Jordan et al., 1983; Elrod and
Butler, 1993). In a majority of the studies reviewed by Butler (1998), plasma progesterone
concentrations in early lactation cows were lower when diets contained 19– 20 percent CP
vs. lower concentrations of CP.
Feeding more than the recommended amounts of protein or inclusion of excess
degradable protein in the diet adversely affects animal reproduction. On such diets, it is likely
that relatively more ammonia is released in the rumen through the process of deamination by
bacterial enzymes. The excess ammonia or urea produced in the rumen can diffuse from the
alimentary tract to the peripheral circulation and appear in the uterine secretions (Tamminga,
2006), thereby disturbing endocrine functions (Ferguson et al., 1986) and the corpus luteum
(Garwacki et al., 1979), and may also reduce motility and survival of sperms as a result of a
changed uterine environment.
In a review on effects of protein on reproduction, Butler (1998) concluded that
excessive amounts of either RDP or RUP could be responsible for lowered reproductive
performance. However, intakes of ‘‘digestible’’ RUP in amounts required to adversely affect
reproduction without a coinciding surplus of RDP would be uncommon. In most of the
studies, reviewed by Butler (1998), excessive RDP rather than excessive RUP was associated
with decreased conception rates. Canfield et al. (1990) showed that feeding diets containing
RUP to meet requirements while feeding RDP in excess of requirements resulted in
decreased conception rates. Garcia-Bojalil et al. (1998) reported that RDP fed in excess (15.7
percent of DM) of recommendations decreased the amount of luteal tissue in ovaries of early
lactation cows.
Although most studies have indicated an adverse effect on reproductive performance
of feeding high CP diets, others indicate no effect of diet CP on reproduction. Carroll et al.
(1988) observed no differences in pregnancy rate or first service conception rates of dairy
cows fed 20 percent CP and 13 percent CP diets. Howard et al. (1987) reported no difference
in fertility between cows in second and greater lactation fed 15 percent CP or 20 percent CP
___________________________________________________________________
29
Review of Literature …
diets. There are many theories as to why excess dietary CP decreases reproductive
performance (Barton, 1996a, 1996b; Butler, 1998; Ferguson and Chalupa, 1989). The first
theory relates to the energy costs associated with metabolic disposal of excess N. To the
extent that additional energy may be required for this purpose, this energy may be taken from
body reserves in early lactation to support milk production. Delayed ovulation (Beam and
Butler, 1997; Staples et al., 1990) and reduced fertility (Butler, 1998) have been associated
with negative energy status. Another effect of negative energy status is decreased plasma
progesterone concentrations (Butler, 1998).
Another theory is that excessive blood urea N (BUN) concentrations could have a
toxic effect on sperm, ova, or embryos, resulting in a decrease in fertility (Canfield et al.,
1990). High BUN concentrations have also been shown to decrease uterine pH and
prostaglandin production (Butler, 1998). High BUN may also reduce the binding of
leutinizing hormone to ovarian receptors, leading to decreases in serum progesterone
concentration and fertility (Barton, 1996a). Ferguson and Chalupa (1989) reported that byproducts of N metabolism may alter the function of the hypophysealpituitary-ovarian axis,
therefore decreasing reproductive performance. And last, high levels of circulating ammonia
may depress the immune system and, therefore, may result in a decline in reproductive
performance (Anderson and Barton, 1988).
Milk urea nitrogen (MUN) and blood urea nitrogen (BUN) are both indicators of urea
production by the liver. Milk urea N concentrations greater than 19 mg/dl have been
associated with decreased fertility (Butler et al., 1995). Likewise, BUN concentrations
greater than 20 mg/ dl have been linked with reduced conception rates in lactating cows
(Ferguson et al., 1988). Bruckental et al. (1989) found that BUN levels increased when diet
CP was increased from 17 to 21.6 percent and pregnancy rate decreased by 13 percentage
units. In a case study, Ferguson et al. (1988) observed that cows with BUN levels higher than
20 mg/dl were three times less likely to conceive than cows with lower BUN concentrations.
Although high BUN concentrations have been associated with decreased reproductive
performance, others have reported no adverse effects on pregnancy rate, services per
conception, or days open with BUN levels above 20 mg/dl (Oldick and Firkins, 1996).
___________________________________________________________________
30
Review of Literature …
Studies by Carroll et al. (1988) and Howard et al. (1987) indicated that maintaining a
strict reproductive management protocol can reduce the negative effects of excess protein
intake on reproduction. Barton (1996a) demonstrated that an intense reproductive program
could be used to reach reproductive success regardless of diet CP level or plasma urea N
concentrations. These studies highlight the idea that dietary protein is just one of many things
that have an effect on reproductive performance. Protein intake, along with other factors such
as reproductive management, energy status, milk yield, and health status all have an effect on
reproductive performance in dairy cattle.
The toxic effects of ammonia, urea or other unidentified nitrogenous compounds
influence the survival of ova, sperms and early developing embryos (Jordan et al., 1983;
Sahlu et al., 1992). Excess protein, irrespective of protein source or degradability, decreases
uterine pH on day 7 of the estrous cycle (Elrod and Butler, 1993) by decreasing the uterine
Mg, K and PO 4 concentrations during the luteal phase (Jordan et al., 1983) which can in turn
inhibit the endometrial carbonic anhydrase activity (Rowlett et al., 1991). The decrease in pH
in the uterus adversely affects the activity and survivability of sperms in the reproductive
tract (White, 1974). Ferguson et al., (1988) suggested that dietary protein intake producing
serum urea-N greater than 200 mg/l were indicative of excess protein intake, particularly
RDP.
High protein intake may also influence the reproductive system as a result of
activation of different mechanisms in the liver for hepatic detoxification and the increase of
energy demand for deamination of excess amino acids (Visek, 1984). Excess ammonia may
also disturb intermediary metabolism and thus increase blood concentrations of urea,
glucose, non-esterified fatty acids and insulin. Smith (1986) reported that some nitrogenous
end products might alter the functioning of the hypophyseal-pituitary-ovarian axis by
decreasing LH pulse frequency and amplitude.
Scanty literature is available on the effect of supplemental rumen protected Met and
Lys on reproductive performance of cows. Berthiaume et al. (2001) and Bach et al. (2000)
reported decreased blood urea nitrogen on supplementation of rumen protected methionine,
but they did not study its effect on reproduction.
___________________________________________________________________
31
Review of Literature …
Ardalan et al. (2009) reported that there was significant decrease in days to first
oestruos, open days, services per conception and increase in pregnancy rate in Holstein dairy
cows fed 18 gm RPM from 4 week prepartum to 20 weeks postpartum.
Xu et al. (1998) reported the overall incidence of health related disorders were
numerically lowest for the dairy cows fed high amount of rumen protected methionine and
lysine.
In contrast, Polan et al. (1991) reported that supplementation of rumen protected form
of Methionine and Lysine had no significant effect on days to first service, services per
conception and calving interval in dairy cows.
Ardalan et al. (2010) reported that there was increase in milk yield while incidences
of postpartum reproductive and metabolic diseases viz. retained placenta, mastitis, uterine
problem, dystokia, were significantly reduced in Holstein dairy cow fed RPM 18 g/d from 4
week prepartum to 14 weeks postpartum.
Reviewing the recent information on the protein nutrition of transition cows suggest
that high producing dairy animals, especially during early lactation, DMI is decreased which
adversely affects the productivity. Where as high milk yield during early lactation retards
development of ovarian follicles, prolonging the postpartum interval to first ovulation. High
yield is antagonistic to the expression of estrus and is associated with reduced conception
rate. Reproductive disorders are one of the major factors reducing the milk yield and
affecting the production potential of cows. Therefore, this research project was planned to
incorporate the rumen protected methionine and lysine in the ration of transition cows to
avoid the negative N balance and to enhance the milk productivity with desirable
composition, which will have far reaching benefits on their reproductive performance.
___________________________________________________________________
32
CHAPTER – 3
MATERIALS AND METHODS
3. Materials and Methods
_________________________________________________
PHASE – I – Feed, fodder and product evaluation
3.1
Feed and fodder analysis
3.1.1
Proximate principles and cell wall constituents
Feeds, fodders and total mixed rations were dried to constant weight at 80oC and
ground (1 mm) using a Willey mill. The ground samples were stored in air tight
polyethylene bags for further analysis. Dry matter, proximate analysis of feed i.e. crude
protein (CP), ether extract (EE), crude fibre (CF) and ash was carried out as per AOAC
(2005) and cell wall fractions i.e. neutral detergent fiber (NDF), acid detergent fiber (ADF)
and acid detergent lignin were done as per modified method of Van Soest et al. (1991)
using amylase.
TDN of feeds and fodders was calculated as per formulae given in NRC (2001). For
feeds that are not animal proteins or fats and that contain some NDF (forages, many byproducts, concentrates), the following equations are used:
TDN = tdNFC + tdCP + (tdFA X 2.25) + tdNDF - 7
Where,
tdNFC (Truly digestible NFC) = 0.98 (100 - [(NDF-NDICP) + CP + EE + Ash])
X PAF
tdCPf (Truly digestible CP for forages) = CP X exp[ -1.2 X (ADICP/CP)]
tdCPc (Truly digestible CP for concentrates) = 1- (0.4 X (ADICP/CP))] X CP
tdFA (Truly digestible FA) = FA Note: If EE < 1, then FA = 0
tdNDF (Truly digestible NDF) = 0.75 X (NDFn – L) X [1 – (L/NDFn) 0.667]
3.1.2
Amino acids analysis of feed samples using HPLC (AOAC, 2005)
Amino acids profile of common feedstuffs were analysed using High Performance
Liquid Chromatography (HPLC) except tryptophan.
_______________________________________________________________________ 33
Materials and methods …
A.
Reagents
a) Redrying solution: In a ratio of 2:2:1 (v/v) methanol, water and triethylamine was mixed
to formulate the redrying solution.
b) Derivatization reagent: This was prepared by mixing methanol: triethylamine: water:
phenyl isothiocyanate in a 7: 1: 1:1 ratio (v/v).
c) Sample diluents: Disodium hydrogen phosphate 710 mg was dissolved in 1 litre of
HPLC grade water. pH of the solution was adjusted to 7.4 by adding 10% aqueous
phosphoric acid. Then acetonitrile was added @ 5% by volume (v/v) to the resultant
solution.
d) Eluent A: Sodium acetate trihydrate 19.0 g was dissolved in 1 litre of HPLC grade water,
to this 0.5 ml triethylamine was added and mixed properly. The pH of the solution was
adjusted to 6.4 by adding glacial acetic acid and was filtered through 0.45 µ Millipore filter
paper. From this solution, 940 ml was taken in a 1 litre volumetric flask and volume was
made to 1 litre by adding HPLC grade acetonitrile. It was degassed by sonicating few
second under vacuum.
e) Eluent B: Acetonitrile 600 ml and HPLC grade water 400 ml were mixed and filtered
through 0.45 µ Millipore filter paper and degassed by sonicating under vacuum pressure.
B.
Preparation of protein hydrolysate
Feed sample (75 mg) was taken in duplicate in 30 ml capacity vials and 10.0 ml 6N
HCl was added. The tube was sealed under constant supply of nitrogen gas. The sealed vial
was kept at 1100C for 24 h for complete hydrolysis of protein. At the time of analysis, the
seal was broken and filtered through whatman no. 1 filter paper and the filtrate was filtered
through 0.45 µ Millipore filter paper.
C.
Derivatization
An aliquot of 5 µl of protein hydrolysate was taken in a reaction vial and mixed with
20 µl redrying solution. It was dried under vacuum and several additional drying were
carried out. After drying, 20 µl derivatization solution was added and mixed gently. The
tube was kept at room temperature for 20 minutes and then again dried under vacuum using
______________________________________________________________________ 34
Fig. 3.1 Chromatogram of standard amino acids profile
Fig 3.2 Standard curve of choline
Materials and methods …
PICO tag assembly. Then the sample was diluted to 100 µl using sample diluents and was
used for amino acids analysis.
D.
Standard preparation
In a sample vial, 5 µl of amino acid standard was taken and dried, redried and
derivatized as described above for sample.
E.
Analytical procedure
An aliquot of 10 µl of derivatized sample was injected in to the HPLC system fitted
with absorbance detector adjustable at 254 nm and PICO tag column (15.0 cm × 3.9 mm).
The gradient controller programming for eluents with respect to time is in table given under.
Gradient controller programming for eluents used for amino acids analysis
Time (min)
Flow
Eluent A
Eluent B
Initial
1.0
100 %
0%
10.0
1.0
54 %
46 %
10.5
1.0
0%
100 %
11.5
1.5
100 %
0%
12.0
1.5
0%
100 %
12.5
1.5
100 %
0%
20.0
1.5
100 %
0%
20.5
1.0
100 %
0%
The derivatized standard mixture of amino acids and unknown sample was injected
under identical condition and their areas were compared with the standard (Fig 3.1).
______________________________________________________________________ 35
Materials and methods …
3.1.3
Estimation of tryptophan
Tryptophan was analysed by colorimetric method given by Mertz et al. (1975).
Composition of reaction mixtures
A. Papain solution
Dissolve 400 mg technical grade papain in 100 ml 0.1 N sodium acetate buffer pH
7.0. Fresh solution was prepared every day.
B. Reagent A
Dissolved 135mg FeCl 3 .6H 2 O in 0.25 ml water and diluted to 500 ml with glacial
acetic acid containing 2% acetic anhydride.
C. Reagent B
30 N H 2 SO 4
D. Reagent C
Mixed equal volumes of reagent A and B about one hour before use.
E. Standard Tryptophan
Dissolved 5 mg tryptophan in 100 ml water (50 ug/ml).
Air-dried, powdered and defatted feed samples (100 mg) were weighed into a small
test tubes. 5 ml papain solution was added, shaked well. The tube were closed and incubated
at 65°C overnight. The digested sample was cooled to room temperature, centrifuged and
collected the clear supernatant. 1ml supernatant was taken in test tube and 4ml reagent C
was added. Then it was mixed in a vortex mixer and incubated at 65°C for 15 min. the
digest was cool to room temperature and read the orange-red colour at 545 nm. A blank was
set with 5ml papain alone. 0, 0.2, 0.4, 0.6, 0.8 and 1ml standard tryptophan were pipetted
out and made up to 1 ml with water.
3.2
Rumen protected methionine (RPM) and rumen protected lysine (RPL)
product evaluation
The RPM and RPL were purchased from market. Both products were in the form of
______________________________________________________________________ 36
Materials and methods …
encapsulation with fatty acids and prepared by spray freeze drying technology.
3.2.1
Methionine content in RPM
Methionine in RPM product was estimated by Kjeldahl method. 0.5 gm RPM sample
in triplicate was digested in the digestion tube with conc. H 2 SO 4 in presence of digestion
mixture (Na 2 SO 4 and CuSO 4 in the ratio of 9:1) over a hot plate. After completion of
digestion, the contents were cooled and transferred to 100 ml volumetric flask and total
volume was made up to the mark with distilled water. An aliquot (10 ml) was transferred to
Micro kjeldahl apparatus and sufficient amount of 40 % of NaOH solution was added to
make the contents alkaline. The distillate was collected in a conical flask containing 20 ml
of 2 % boric acid with mixed indicator (20 g boric acid powder, 0.1 percent solutions of
each methyl red and bromocresol green in 2:1 proportion was added and the volume was
made to 1000 ml in distilled water). The distillate was titrated against standard 0.01 N
H 2 SO 4 . This was the total nitrogen content. The Methionine content in the sample was
calculated by multiplying nitrogen content with 10.65.
Volume of H2SO4 used x Normality x 0.0014 x Volume made
Methionine % =
x 10.65 x 100
Weight of sample x Volume taken
3.2.2
Lysine content in RPL
The total nitrogen was estimated in 3.2.1. The Lysine content in the sample was
calculated by multiplying nitrogen content with 6.52.
Volume of H2SO4 used x Normality x 0.0014 x Volume made
Lysine %
=
x 6.52 x 100
Weight of sample x Volume taken
______________________________________________________________________ 37
Materials and methods …
PHASE – II – In vitro study
3.3
In vitro determination of microbial protein yield
3.3.1
Collection of rumen liquor
The rumen liquor was collected from three rumen fistulated cattle before feeding and
watering in the morning after removing the ice cap. Rumen liquor was collected from
different sides of rumen and at different depths by to and fro movement of the plastic tube in
order to get a representative and homogenous sample. The samples were mixed and strained
through four layered muslin cloth into a thermos flask which was previously flushed with
CO 2 to maintain anaerobic conditions and brought to laboratory immediately for in vitro
studies.
3.3.2
Preparation of substrates in the form of total mixed rations (TMR’s)
Concentrate mixture, green maize and wheat straw were dried in hot air oven at 70oC
for overnight. Samples were ground separately to reduce particle size (1mm) and mixed
weighed quantities of concentrate and roughage in the different proportions. The sample
was again mixed by a mixer to avoid sample variation. Three TMRs were prepared by
taking concentrate to roughages ratio as 60:40, 50:50 and 60:40 respectively for three
groups as shown in table 3.1. In roughage, wheat straw and green maize were taken in the
ratio of 40:60.
Table 3.1 Composition of TMRs
Ingredients
TMR-1
TMR-2
TMR-3
Wheat Straw
16 parts
20 parts
24 parts
Maize fodder
24 parts
30 parts
36 parts
Concentrate Mixture
60 parts
50 parts
40 parts
19.8 parts
16.5 parts
13.2 parts
Maize
______________________________________________________________________ 38
Materials and methods …
GNC
3.3.3
12.6 parts
10.5 parts
8.4 parts
Mustard cake
7.2 parts
6.0 parts
4.8 parts
Deoiled rice bran
6.6 parts
5.5 parts
4.4 parts
Wheat bran
12.0 parts
10.0 parts
8.0 parts
Mineral Mixture
1.2 parts
1.0 parts
0.8 parts
Salt
0.6 parts
0.5 parts
0.4 parts
In vitro determination of microbial N using nitrogen balance technique
The incubations were carried out in 100 ml calibrated Glass syringes (Leur make) as
described by Menke and Steingass (1988). The syringes were incubated in incubator at 39 ±
0.5oC. The substrate (500 mg) was weighed on a plastic boat with removable stem and
placed into the bottom of the glass syringe without sticking to the sides of the syringes. The
piston was lubricated with petroleum jelly and pushed inside the syringe. The syringes were
incubated at 39 ± 0.5oC up to 48hr.
The medium was prepared by mixing 400 ml distilled water, 200 ml rumen buffer
solution and 200 ml macro minerals solution, 1 ml Resazurine solution, 0.1 ml micro
minerals solution and 40 ml reducing solution (prepared fresh and added just prior to
incubation). The medium was pre-warmed to 39oC and bubbled with CO 2 till the blue color
of the medium vanished.
The details of solutions and the order in which these were added prior to the filling in
syringes.
Items
30 syringes
45 syringes
60 syringes
Distilled water
365 ml
550 ml
730 ml
Micromineral solutiona
0.1 ml
0.15 ml
0.185 ml
Solution-I
______________________________________________________________________ 39
Materials and methods …
Rumen buffer solutionb
183 ml
275 ml
365 ml
Macromineral solutionc
183 ml
275 ml
365 ml
Resazurine solutiond
0.95 ml
1.45 ml
1.90 ml
1N NaOH
1.6 ml
2.4 ml
3.1 ml
Na 2 S.7H 2 O
220 mg
330 mg
440 mg
Distilled water
37 ml
55 ml
73 ml
330 ml
500 ml
660 ml
Solution-IIe
Solution-III
Rumen liquor
a
Micromineral solution
CaCl 2 .2H 2 O
13.2 g
MnCl 2 .4H 2 O
10.0 g
CoCl 2 .6H 2 O
1.0 g
FeCl 3 .6H 2 O
8.0 g
Dissolved in 100 ml of water.
b
Rumen buffer solution
NH 4 HCO 3
NaHCO 3
4.0 g
35.0 g
Dissolved in 1000 ml of water.
c
Macromineral solution
Na 2 HPO 4 anhydrous
5.70 g
KH 2 PO 4 anhydrous
6.20 g
MgSO 4 .7H 2 O
0.60 g
Dissolved in 1000 ml of water.
______________________________________________________________________ 40
Materials and methods …
d
Resazurine solution
e
0.10 % w/v
Reducing solution
1N NaOH
Na 2 S, 9H 2 O
Water
4.0 ml
625 mg
95 ml
Rumen liquor was collected from three donor bulls, fitted with permanent rumen
fistula. Donor animals were fed on roughage and concentrate based diet (2.0 kg concentrate
mixture and wheat straw ad libitum). Rumen liquor was collected before feeding the donor
animal into a pre-warmed thermo-flask and brought to the laboratory. The rumen liquor was
bubbled with CO 2 for about 2 minutes and filtered through 4 layers of muslin cloth.
After medium became colourless, the required amount of strained rumen liquor
(SRL) was added. The ratio of medium to rumen liquor was 2:1. Then, 30 ml of incubation
medium was injected to each syringe using auto pipette. The syringes were shaken gently,
residual air or air bubble, if any, was removed and the outlet was closed. The level of piston
was recorded and the syringes were placed in an incubator (39 ± 0.5oC). The syringes were
shaken every 30 minutes for first 2 h from the start of the incubation and thereafter every 2 h
up to 24 h of incubation. After 24 hr incubation, the content of the syringe was centrifuged
at 8000 rpm for 15 min at 4oC. N content of the pellet were estimated and corrected for N in
blank. The pellet obtained from second tube was treated with NDS for 24 hr. Neutral
Detergent insoluble nitrogen were estimated by Kjeldahl method.
MN = Pellet N (24 hr) – Pellet N in blank (0 hr) – NDIN
Where
MN – Microbial Nitrogen
NDIN – Neutral Detergent Insoluble Nitrogen
______________________________________________________________________ 41
Materials and methods …
3.4
Separation of rumen bacteria from rumen liquor
The strained rumen liquor was centrifuged in duplicate at 1000 rpm for 2 min. to
remove feed particles. The supernatant were centrifuged at 8000 rpm for 15 min at 4oC.
Supernatant was discarded to separate bacterial pellet. Bacterial pellets were washed with
Bridge detergent solution and centrifuged. And then pellet was treated with 10% TCA
solution and centrifuged. N and amino acids content of the bacteria were estimated as
discussed above in 3.1.
3.5
Separation of the protozoa from rumen contents
The strained rumen liquor was strained through six layers of surgical gauze without
squeezing, then scraped the deposit from the gauze, resuspended it in distilled water and restrained through clean gauze. The combined filtrates were then allowed to stand for atleast 1
hr. in boiling-tubes filled to the top, when the larger, whiter, and heavier protozoa gradually
sank to the bottom. The supernatant was carefully decanted, the deposit washed several
times by decantation with 0.003 M phosphate buffer (pH 6.8) and finally with distilled
water. The final deposits consisted almost entirely of protozoa. Again supernatant was
carefully decanted and protozoa were dried at 550C in hot air oven. N and amino acids
content of the bacteria were estimated as discussed above in 3.1.
3.6
In vitro estimation of RUP intestinal digestibility
A three step in vitro procedure developed by Calsamiglia and Stern (1995) were
adopted for estimating intestinal digestibility of the RUP fraction of feed proteins.
Ruminally undegraded feed residue containing about 15 mg residual N were incubated for 1
hr in 10 ml 0.1 N HCl solution containing 1 g/L of pepsin. The mixture were neutralized
with 0.5 ml 1N NaOH and 13.5 ml of pancreatin solution followed by 24 h incubation. The
undigested protein as precipitated with TCA solution and RUP intestinal digestibility was
calculated as follows,
TCA Soluble N
x 100
RUP Intestinal Digestibility =
Undegraded N
______________________________________________________________________ 42
Materials and methods …
PHASE –III - in sacco Study
3.7
Estimation of RDP and RUP of different feed stuffs
RDP and RUP of different feed stuffs were estimated by in sacco nylon bag
technique as described by Orskov and McDonald (1979).
Three rumen cannulated bulls (18 to 24 months of age) were stalled in the well
ventilated animal shed of Cattle Yard, NDRI, Karnal. The animals were kept on a constant
diet of wheat straw and concentrate mixture (65:35) for one month of preliminary feeding
period, before commencing the experiment. The concentrate mixture consisted of maize,
barley, GNC, mustard cake, DORB, wheat bran, mineral mixture and salt in proportion
same as listed in Table 3.2. The animals were also offered clean drinking water ad libitum
twice a day.
The method of Mehrez and Orskov (1977) was used for suspension and removal of
nylon bags in the rumen.
Pre-weighed nylon bags (9 x 15 cm with 40 µ pore size) containing 3 g of different
ground feedstuffs were tied with nylon threads and fastened tightly to three iron chains. The
chains containing the bags were then placed in the rumen of three fistulated animals so that
the observation for each ingredient could be recorded, in triplicate, in three animals.
The bags were taken out at regular intervals of time viz., 0, 2, 4, 6, 8, 12, and 24h.
After removal from the rumen, the bags were washed thoroughly by swirling vigorously in
plastic trough filled with a continuous supply of fresh water, till the water in the trough was
clear. The bags were drained off to get rid of excess water and then dried in oven at 550C for
48 h. The lumps formed inside the bags were broken manually and the material was
thoroughly mixed, followed by drying at 550C till constant weight. The dried bags after
desiccation were weighed again in order to determine the dry matter loss at different hours
for each of the samples. The residue left in each bag was subjected to nitrogen estimation.
From the degradability data obtained at various hours, the time versus degradation
curves were drawn and the constants a, b and c was measured by computer analysis using
______________________________________________________________________ 43
Materials and methods …
the following expression given by Mehrez and Orskov (1977):
P = a + b (1- e –ct)
Where as, P = Degradability
t = Time
a = Intercept on Y-axis
b = Potentially degradable fraction and
c = Degradation rate or rate constant
RDP and RUP were calculated as follows.
RDP = A + B [kd / (kd + kp)]
RUP = B [kp / (kd + kp)] + C
Here,
Fraction A is the percentage of total CP that is NPN (i.e., assumed to be instantly
degraded) and a small amount of true protein that rapidly escapes from the in
situ bag because of high solubility or very small particle size.
Fraction B is the rest of the CP and includes the proteins that are potentially degradable.
Fraction C is the percentage of CP that is completely undegradable; this fraction generally
is determined as the feed CP remaining in the bag at a defined end-point of
degradation.
3.8
Estimation of rumen escape potential of commercial RPM and RPL product
Rumen escape potential of the RPM and RPL were determined using an in situ
nylon-bag technique (Mehrez and Orskov, 1977; Osuji et al, 1993; Nozière and MichaletDoreau, 2000).
Pre-weighed nylon bags (9 x 15 cm with 40 µ pore size) containing 3 g of RPM or
RPL were tied with nylon threads and fastened tightly to three iron chains. The chains
containing the bags were then placed in the rumen of three fistulated animals so that the
observation for each ingredient could be recorded, in triplicate, in three animals. After
______________________________________________________________________ 44
Materials and methods …
removal of bags, these were rinsed with cold water, dried at 550C for 48 hours and weighed.
Contents of the bags were subjected to N analysis (AOAC, 2005) by Kjeldahl Method.
Data obtained at various hours from the degradability, the time versus degradation
curves were drawn and the constants a, b and c was measured by computer analysis using
the following expression given by Mehrez and Orskov (1977). Effective degradability was
calculated as per equations in 3.7.
PHASE – IV - In vivo Study
Eighteen crossbred cows (Karan Fries) in second to fourth lactation with most
probable production ability (MPPA) of around 4500 lit milk production were selected from
the cattle herd maintained at NDRI, Karnal. The experimental animals were randomly
divided into two dietary groups of 9 each in control and experimental group on the basis of
MPPA and lactation number.
3.9
Location of experiment
The study was conducted in the experimental cattle shed of National Dairy Research
Institute, Karnal, India located at 29° 42″ 20 sec N and 76° 58″ 52.5 sec E at an altitude of 834
feet amsl. Minimum and maximum ambient temperature ranged from near freezing point in
winter to 45° C in summer with annual rainfall of 700 mm. The experiment was conducted in
May, 2011 to April, 2012 with daily minimum and maximum temperature averaging 5.6° C
and 40° C.
3.10
Selection and distribution of animals
The animals were put on experiment around 40 days before parturition and given
adaptation period of ten days. The duration of experiment was thirty days before calving to
120 days after parturition. The details of the experimental animals are given in table 3.2.
3.11
Estimation of most probable production ability (MPPA)
The Most Probable Production Ability (MPPA) or Expected Producing Ability
(EPA) was computed on the basis of formula given by Lush (1945) as follows:
nr
MPPA = A +
x (P-A)
1 + (n-1)
______________________________________________________________________ 45
Materials and methods …
Where, A – Population mean
n – Total number of animals
r – Repeatability of lactation milk record
P – Milk yield in previous lactation
Table 3.2 Details of experimental animals
Group 1 (Control)
Group
2
(RPM
plus
RPL
supplementation)
Sr.
Animal
No.
No.
1
KF-6918
2
Lactation
Sr
Animal
No.
No.
No.
No.
5034
3
1
KF-6774 4757
4
KF-7068
4234
2
2
KF-6969 3952
1
3
KF-6997
3949
1
3
KF-7083 3975
1
4
KF-7034
4770
1
4
KF-7045 4010
1
5
KF-6960
4037
1
5
KF-7088 4643
1
6
KF-7013
4160
1
6
KF-6944 3985
2
7
KF-7085
3394
1
7
KF-7000 3654
1
8
KF-7106
4028
1
8
KF-7052 4111
1
9
KF-6998
3569
1
9
KF-7108 3992
1
Avg.
MPPA
4119 ± 173
Avg.
MPPA
Lactation
4120 ± 117
______________________________________________________________________ 46
Materials and methods …
3.12
Feeding, housing and management of experimental animals
All the eighteen cross bred cows were fed as follows:
A. Control group (T1)
Animals in control group fed chopped wheat straw (particle size- 1.5 to 2.0 cm),
chaffed green maize fodder (particle size- 2.0 to 2.5 cm) and compounded concentrate
mixture as per NRC (2001) requirements. Before diet formulation; the concentrate mixture,
wheat straw and maize fodder were analyzed for proximate composition, ADF and NDF.
The daily ration was offered to individual cows three times a day before each milking.
A. Treatment group (T2)
Animals in group 2 (treatment group) were fed same ration as control group plus 5
gm RPM and 20 gm RPL prepartum and 7 gm RPM and 60 gm RPL postpartum,
respectively. RPM and RPL is mixed in concentrate feed at the time of feeding of individual
animal.
3.12.1 Green maize fodder
Green maize was supplied daily by Farm Maintenance Section, National Dairy
Research Institute, Karnal. After chopping on the chaff cutter, it was fed to experimental
animals. The dry matter contents of green maize were estimated at weekly intervals through
out the experimental period.
3.12.2 Concentrate mixture
The various ingredients of compounded concentrate mixture fed to cows of both
groups were shown in Table 3.3.
3.12.3 Housing and management of animals
All the crossbred cows were housed in well-ventilated shed in Cattle Yard of NDRI,
Karnal, having the arrangement for individual animal feeding. The animals shed were
washed twice daily and thoroughly cleaned to remove faeces and dirt. All the animals were
maintained under clean and hygienic conditions. Antiseptic solution containing phenyl was
applied at regular intervals on the floor of the shed to keep the animals away from infection.
______________________________________________________________________ 47
Materials and methods …
Table 3.3 Ingredients in concentrate mixture (parts)
Feed ingredients
Parts
Maize grain
33
GNC (Exp)
21
Mustard cake
12
DORB
11
Wheat Bran
20
Mineral mixture
02
Salt
01
DORB= Deoiled rice bran; Exp= Expeller; GNC= Ground nut cake
3.12.4 Watering of animals
The animals were provided with fresh and clean tap water free of choice three times
daily at 8.00 hrs, 12.00 hrs and 18.30 hrs.
PRE PARTUM STUDY
3.13
Observations recorded during pre partum period
3.13.1 Body weight
Body Weight of the animals was recorded at fortnightly intervals using
computerized weight management system from Leotronic Scales Pvt. Ltd. The maximum
capacity of the system was 1500 kg and minimum capacity of 4 kg, the error was ± 200 g.
The animals were weighed for two consecutive days in the morning before offering feed and
water. The average of two days was considered as body weight for that fortnight.
3.13.2 Body Condition Score
For recording the body condition of the animals, following points were taken into
______________________________________________________________________ 48
Materials and methods …
account:
i.
Vertebral column (chine, lion and rump) flesh covering at the spinous processes of
these regions.
ii.
Spinous processes: their prominence and sharpness.
iii.
Tail head region: prominence of depression between backbone and pins and between
pin and hook bones.
iv.
Ribs: their flesh covering.
Considering the above points, NRC (2001) presented a score chart, which was
adopted in the present study. Body condition score of experimental cows were scored on
every fortnight up to parturition. The condition-scoring chart has been presented in
Annexure I.
3.13.3 Feed intake
Daily DM intake was calculated by recording the daily feed offered and residue from
40 days before parturition to calving day. The DM of different feed ingredients was
recorded once every week. Fortnightly intake of nutrients viz CP, RDP, RUP, MP, TDN,
ME, NE L of each cow was calculated by using formulae of NRC (2001) (Annexure II).
3.14
Analysis of blood samples
3.14.1 Blood collection
Blood Samples were collected at fortnightly intervals (-30 and -15th day) for
estimation of phosphatidylcholine, amino acid, VLDL, NEFA, glucose and vitamin E and at
weekly intervals (-15 and -7th day) before calving for estimation of prolactin and growth
hormone, respectively. The blood samples from individual animals were collected by
jugular vein puncture into heparinized vaccutainer tubes, 16 X 100 mm (Becton Dickinson,
Rutherford, NJ). The plasma was separated by centrifugation of the blood samples at 2400
rpm for 15 min and it was stored in plasma vials at -200 C.
3.14.2 Plasma amino acid
3.14.2.1 Preparation of sample
______________________________________________________________________ 49
Materials and methods …
Plasma samples (0.1 ml) were mixed with ice-cold methanol (400 μl) while
vortexing and allowed to stand for 15 minutes in an ice bucket before centrifuging (5000
rpm / 15 minutes) and the supernatant was collected. The protein free supernatants were
processed immediately for assaying total amino acids by HPLC analysis as mentioned above
in 3.1.7.
3.14.3 Plasma choline estimation
A. Principle
The test portion was acid digested at 70°C, thereby releasing the majority of the
predominantly bound choline. Following pH adjustment, residual choline phospholipids
were cleaved with phospholipase D and free choline moiety subjected to choline oxidase
with consequent liberation of hydrogen peroxide. In the presence of peroxidase, phenol is
oxidized and a quinoneimine chromophore formed with 4-aminoantipyrine. Both enzymatic
and color-forming reactions are formatted to occur concurrently. Absorbance was measured
at 505 nm and choline content calculated by interpolation from a multilevel calibration. For
this procedure, choline is defined and reported as the hydroxide.
B. Apparatus
(a) Spectrophotometer.—Digital readout to 0.001 absorbance at 505 nm. Glass cuvettes (10
mm) or flow-through cell.
(b) Covered water baths.—37° ± 2°C and 70° ± 4°C.
(c) pH meter.—2 dp, with calibration buffers, 4.0 and 7.0.
(d) Pipettes.—Calibrated glass, or preferably auto-pipettes for 0.100 and 3.00 ml.
(e) Volumetric flasks.—10, 50, and 100 ml and 1 L.
(f) Digestion vessels.—Conical flasks, 100 or 150 ml, with stoppers.
(g) Test tubes.—10 ml with glass stoppers, plus rack. Disposable polypropylene or
polystyrene plastic tubes fitted with screw-capped lids may also be used for convenience.
(h) Filter paper.—Medium speed, porosity 1.4–2.9 µm, Whatman No. 2 or equivalent.
______________________________________________________________________ 50
Materials and methods …
C. Reagents
All the reagents used were of AR grade. All enzymes and 4-aminoantipyrine were stored at
4°C or as instructed by the supplier.
(a) Water.—Purified to >18 MS resistivity.
(b) Hydrochloric acid (HCl)—Concentrated (37% w/v).
(c) Hydrochloric acid (1.0 M).—85 ml of concentrated HCl was taken into 1 L volumetric
flask and filled to mark with water. Standardization was not required.
(d) Sodium hydroxide (NaOH, 50% w/v).— 50 g of NaOH was introduced into 100 ml
graduated cylinder. It was carefully dissolved in 80 ml water. After cooling, it was filled to
mark with water.
(e) Choline bitartrate.—Sigma C-2654 or equivalent. About 0.7–1.0 g of choline bitartrate
was dried at 102°C, to constant weight and stored in desiccator at ambient laboratory
temperature.
(1) Stock choline standard (2500 µg/ml as choline hydroxide).
Into l00 ml volumetric flask, 523 mg dry choline bitartrate was introduced. It was
dissolved in water and filled to mark. Stock solution was refrigerated at 4° ± 2°C.
(2) Working choline standard (250 µg/ml as choline hydroxide).
10 ml of stock solution was pipetted in 100 ml volumetric flask and filled up to l00
ml with distilled water. Working solution was prepared fresh daily.
(f) Tris (hydroxymethyl) aminomethane (Trizma) buffer (0.05M, pH 8.0).
Into 1 L volumetric flask containing 500 ml water, 6.057 g tris (hydroxymethyl)
aminomethane (Sigma T-1503 or equivalent) was introduced. The pH was brought to 8.0
with 1M HCl, and filled to mark with water. It was stored at 4 0 C. This solution was stable
for 1 month.
(g) Phospholipase D, Sigma Type VI, P-8023, from Streptomyces chromofuscus, 150
units/mg. Unit definition: one unit will liberate 1.0 µmol choline from L-"-phosphatidyl
______________________________________________________________________ 51
Materials and methods …
choline (egg yolk)/h at pH 5.0 at 30°C. This form of phospholipase D was preferred to
others due to its activity for sphingomyelins and lysophospholipids.
(h) Choline oxidase.—Sigma, C-5896, from Alcaligenes species, 10 units/mg. Unit
definition: one unit will form 1.0 µmol H2O2 with oxidation of 1.0 µmol choline to betaine
aldehyde per min at pH 8.0 at 37°C.
(i) Peroxidase.—Sigma Type I, P-8125, from horseradish, 80 units/mg. Unit definition: one
unit will form 1.0 mg purpurogallin from pyrogallol in 20 s at pH 6.0 at 20°C.
(j) 4-Aminoantipyrine.—Sigma A4382 or equivalent.
(k) Phenol.
(l) Chromogenic reagent
Into l00 mL volumetric flask, sequentially weighed 75–100 units phospholipase D,
100–120 units choline oxidase, 250–280 units peroxidase, 15 mg 4-aminoantipyrine, and 50
mg phenol. It was dissolve and filled to volume with 0.05M Trizma buffer.
D. Procedure
All steps of the assay were performed under incandescent lighting in the absence of
fluorescent lighting or direct sunlight.
(a) Preparation of test sample
5.00 g sample was weighted into 100 ml conical flask (or boiling tube). 30 ml of 1.0
M HCl was added, immediately stoppered, and mixed by shaking until well dispersed. The
flasks were placed into water bath at 70°C for 3 h, shaked occasionally. The stoppers were
loosened or removed during early heating stage to avoid excessive pressure build-up.
Further periodic pressure release was necessary. After 3 h, it was cooled to ambient
temperature. The pH was adjusted to 3.5 – 4.0 with 50% NaOH, and quantitatively
transferred discolored solution to 50 ml volumetric flask, filled to mark with water. The
hydrolysate was filtered through Whatman filter paper No 2, and filtrate was collected after
discarding the first 5 – 10 ml. It was ensured that filtrate was free of particulates, otherwise
re-filtered. This filtrate at this stage was stable and could be stored in the dark at 4°C for up
______________________________________________________________________ 52
Materials and methods …
to 3 days prior to analysis. The filtrate was allowed to return to ambient temperature before
proceeding with the assay. Two 10 ml test tubes were separately labeled for each sample as
Sample Test Solution (tube 1) and Sample Blank (tube 2). Into each tube, 0.100 ml aliquot
of sample filtrate was dispensed.
(b) Preparation of standard curve
2, 4, 6, and 8 ml aliquots of working standard (250 µg/mL) were pipetted into four
10 ml volumetric flasks and filled to mark with water. The fifth level calibrant was the
undiluted working standard. The 5-level standard curve (50, 100, 150, 200, and 250 µg/ml)
was prepared by dispensing 0.100 ml of each standard into separately labeled 10 ml test
tubes. A Reagent Blank tube (0 µg/mL) was also included by substituting 0.100 ml water in
place of choline. (Table 3.2)
(c) Enzymatic determination
To each Sample Blank (tube 2), 3.00 ml water was added. 3.00 mL chromogenic
reagent was added to each Sample Test Solution (tube 1), the 5 standards, and the Reagent
Blank. The test tubes were placed in covered water bath at 37° ± 2°C for 15 min during
which time a pink/red color was developed. After 15 min, tubes were removed from water
bath and cooled to ambient temperature for 15 min. Without delay, contents of each tube
were transferred into 10 mm optical cells, or a flow-through cuvette. The spectrophotometer
was set to zero at 505 nm against water and measured the absorbance of Standards (Astd),
Sample Test Solutions (Asmp), Sample Blanks (Abl), and Reagent Blank (Areag).
E. Calculation
Standard calibration of net absorbance (Astd –Areag) vs choline concentration
(µg/ml) was created using linear least mean squares regression analysis, with forced origin.
Absorbance of Sample Blanks and Reagent Blank was subtracted from each Sample
Test Solution to eliminate nonspecific spectral background:
Net absorbance of sample (A) = Asmp – Abl – Areag
The choline concentration in test sample was determined and expressed as choline
hydroxide:
______________________________________________________________________ 53
Materials and methods …
Choline hydroxide, mg/dL =
where ,
A
x
S
V
x
100
W
1000
A = net absorbance of sample;
S = slope of standard graph;
V = volume in ml of hydrolysates (50 ml);
W = weight in grams of sample (5.00 g);
100 = conversion to l00 g basis;
1000 = conversion µg to mg. The data was reported to one decimal place.
3.14.4 Plasma glucose
Principle:
Glucose is reduced in the presence of O-toluidine solution to emit a bluish-green
color which can be measured at 625 nm.
Reagent:
1. Trichloroacetic acid solution (3% w/v) with distilled water.
2. O-toluidine solution (6% v/v) with glacial acid.
3. Distilled water (DW)
3. Glucose standard solution.
4. Working standard = 1.2 mg/ml
Procedure:
The plasma samples, standard and blank were run according to the protocol given below.
Reagents
Blank
Sample
(ml)
Plasma
-
0.20
Standard
1
2
3
4
5
-
-
-
-
-
______________________________________________________________________ 54
Fig 3.3 Standard curve of glucose
Fig 3. 4 Standard curve of NEFA
Materials and methods …
Standard
-
-
0.04
0.08
0.12
0.16
0.20
DW
0.20
-
0.16
0.12
0.08
0.04
0.00
O-toluidine
3.00
3.00
3.00
3.00
3.00
3.00
3.00
reagent
Tubes were boiled in water bath for 8 minutes and then cooled under tap water.
Absorbance was read at 625 nm against reagent blank.
Standard curve (Fig 3.3) was plotted and read the concentration of unknown sample.
Calculation:
Glucose (mg/100ml) =
OD unknown
OD standard
x concentration of standard x dilution fact.x 100
3.14.5 Non esterified fatty acids (NEFA)
The non-esterified fatty acids (NEFA) concentration in the plasma samples was
standardized using the extraction method of Itaya and Ui (1965) and the colour development
procedure of Duncombe (1963) as follows:
Reagents used
i) Redistilled chloroform of Analar grade.
ii) Phosphate buffer, pH 6.4, 33 mmol/ 1 was prepared by mixing 2 volumes of
potassium dihydrogen phosphate (4.53 g/1) and 1 volume of disodium hydrogen
phosphate dehydrate (5.938 g/l) solutions.
iii) Copper-ethanolamine solution: 1 M triethanolamine; 1 N acetic acid and 6.45 per
cent of Cu (NO 3 ) 2 .3H 2 O in 9:1:10 (v/v/v).
iv) 0.5 per cent Na-diethyl dithiocarbamate solution in n-butanol.
v) Standard palmitic acid (MW 256.43) was dissolved in chloroform to prepare
______________________________________________________________________ 55
Materials and methods …
standard solution in the range of 20 to 200 mg/1.
Method
To 0.2 ml plasma samples in glass stoppered test tubes (15 x 150 mm), 6.0 ml of
chloroform and 1 ml of phosphate buffer (pH 6.4), were added and the mixture was shaken
for 2 min. After a settling period of 20 min, the upper layer along with the protein
precipitate was aspirated with a fine tipped glass syringe and was discarded. Thereafter 3 ml
of Cu-Triethanolamine solution was added in the tube and the mixture was hand shaken
again for 2 min. The tubes were thereafter centrifuged for 5-10 min at 1,000 g to separate
the phases clearly. The Cu-triethanolamine solution was aspirated with a fine tipped syringe
taking care that all the Cu-reagent was removed from the solution. Three ml of chloroform
solution was pipetted into clean dry tube; care was taken not to touch the inner wall of the
tube as traces of Cu-containing aqueous phase might be transferred. Then 0.5 ml of diethyl
dithiocarbamate reagent was added to chloroform solution and the solutions after mixing
were read immediately at 440 nm in 1 cm light path cell against the blank (Fig. 3.4).
3.14.6 Estimation of plasma Triglycerides
Estimation of triglycerides was done using GPO-PAP liquid Gold Kit purchased
from Cogent Span Diagnostic Ltd., Surat, India.
3.14.7 Estimation of plasma VLDL
Estimation of VLDL was calculated from the value of triglycerides as per the
formula given by Friedewald et al. (1972).
VLDL (mg/dL) = triglycerides (mg/dL) ÷ 5
3.14.8 Estimation of plasma urea
The plasma urea was estimated according to the procedure described by
Rahmatullah and Boyde (1980).
Principle
Urea forms a complex with the Diacetyl monoxime reagent under hot (90oC, acidic
conditions). The chemical reaction is stimulated by ferric ions and is stabilized by
______________________________________________________________________ 56
Fig 3.5 Standard curve of BUN
Fig 3.6 Standard curve of cholesterol
1.4
Absorbance
1.2
1
0.8
0.6
0.4
0.2
0
0
0.5
1
1.5
Concentration (ug/ml)
2
2.5
3
Materials and methods …
thiosemicarbazide.
Urea + DMAB
H3PO4 (90oC)
Thiosemicarbazide + Fe+
Red colour complex
Colour was determined by spectrophotometer at 525 nm.
Reagents
a)
Acid ferric solution
Added 100 ml concentrated phosphoric acid to 300 ml of concentrated Sulphuric
acid and 600 ml distilled water. Dissolved 100 mg ferric chloride in this solution.
b)
Diacetyl monoxime (DAMO) thiosemicarbazide (TSC) solution
500 mg DAMO and 10 mg TSC were dissolved in distilled water and diluted to 100
ml volume.
c)
Chromogenic reagent
Mixed two parts of reagent 1 with one part of reagent 2 immediately before use.
Procedure
a)
Deproteinisation of samples
To 0.2 ml of serum sample, added 2 ml of TCA solution of 5 per cent concentration
in a centrifuge tube. Mixed well and centrifuged the samples for 5 to 10 minutes at 1,500 to
2,000 rpm. After centrifugation, removed the precipitate and collected the deproteinised
samples in test tube.
b)
Plasma urea concentration
Took 0.2 ml of serum sample and added 3 ml of chromogenic reagent. Mixed well
and boiled in water bath for 5 minutes. Cooled it room temperature and read absorbance at
525 nm against a blank comprised of distilled water (0.2 ml) and chromogenic reagent (3
ml).
______________________________________________________________________ 57
Materials and methods …
c)
Preparation of standard curve
Stock standard urea solution (500 µg/ ml) was prepared by dissolving 50 mg urea in
100 ml distilled water. Working standard (50 µg) was prepared by diluting 1 ml of stock
with 9 ml of distilled water. Different volumes of working standard 0.02 ml, 0.04 ml, 0.06
ml, 0.08 and 0.10 ml were taken separately in test tube and distilled water 0.18 ml, 0.16 ml,
0.14 ml, 0.12 ml and 0.10 ml, respectively was added to make the volume 0.2 ml. Then 3 ml
of chromogenic reagent was added into each test tube and mix well. The blank contained 0.2
ml distilled water and 3 ml chromogenic reagent. Tubes are incubated in boiling water bath
for 5 minutes exactly and then cooled to room temperature and absorbance was read against
the blank at 525 nm. Absorbance was plotted against concentration to prepare the standard
curve (Fig 3.5).
3.14.9 Estimation of plasma cholesterol
Plasma cholesterol concentration of blood samples was done using CHOD-PAP,
Liquid Gold Kit purchased from Cogent, Span Diagnostic Ltd., Surat, India. (Fig 3.6)
3.14.10 Estimation of plasma vitamins E
A. Extraction of plasma samples:
0.5 ml of plasma was deproteinized with an equal volume of 95% ethanol containing
5% ascorbic acid. Three extractions were carried out using 2 ml petroleum ether (40-600C)
each time by keeping the test tube in the ice salt mixture. The ether layer was decanted to
amber coloured test tubes.
B. Preparation of Standards
Stock solutions of α-tocopherol (Sigma) 2 mg/ml were prepared in 100 % ethanol.
Requisite aliquots of individual stock solutions were taken in amber coloured tubes and
were dried under nitrogen at room temperature. The dried standards were reconstituted in
mobile phase. A working standard solution containing 20 µg/ml α-tocopherol was prepared
at the time of use.
C. HPLC system and procedure:
The mobile phase consisted of acetonitrile and HPLC water in the ratio of 95:5. The
HPLC system (Waters) consisted of a model 510 pump, UV visible absorbance detector
______________________________________________________________________ 58
Fig. 3.7 Chromatogram of standard α-Tocopherol
Fig 3.8 Standard curve of milk urea nitrogen
1.4
1.2
Absorbance
1
0.8
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
Concentration (ug/ml)
1.2
1.4
1.6
Materials and methods …
486, rheodyne injector with 20 µl loop, using multiwavelengh detector. A reverse phase
Discovery C-18 (15 cm x 4.6 mm) column was used. The flow rate was 1.5 ml/minute. 20 µl
of standard/ sample was injected in HPLC column for chromatographic separation and the
run time was 10 minutes per sample (chromatograph for vitamin E given in Fig 3.7). A
specific programme was adopted for the separation of α-tocopherol using Millennium
software. Program for HPLC analysis of α-tocopherol on was as follows:
Wavelength
Change time
Retention time
(nm)
(min)
(min)
290
4.00
6.70
Vitamin
α-tocopherol
3.14.11 Plasma growth hormone estimation
Estimation of plasma growth hormone was done using Bovine Growth Hormone
Elisa Test Kit manufactured by Endocrine Technologies Inc., USA.
Principle:
The rGH quantitative test kit is based on the principle of a solid phase enzyme linked
immunosorbent assay (ELISA). The assay system utilizes a mouse anti- rGH specific
antibody for solid phase (microtiter wells) immobilization and high affinity anti-mousebGH antibody in the antibody-enzyme (horseradish peroxidase) conjugate solution. The test
sample was allowed to react simultaneously with the antibodies, resulting in bGH molecules
being sandwich between the solid phase and enzyme linked antibodies. After 3 hours of
incubation at 37oC, the wells were washed with water to remove unbound-labelled
antibodies. A solution of TMB was added and incubated for 20 minutes, resulting in the
development of a blue colour. The colour development was stopped with the addition of
stop solution, and the colour was changed to yellow and measured spectrophotometrically at
450 nm. The concentration of bGH is directly proportional to the colour intensity of the test
samples.
Materials provided:
1. Antibody coated micro-titer wells, 96 well plate.
______________________________________________________________________ 59
Materials and methods …
2. Enzyme conjugate reagent (12 ml).
3. Reference standards (0, 1.0, 2.5, 5.0, 10, 25 ng/ml).
4. TMB colour reagent (12 ml).
5. Stop solution (2N HCl) 6 ml.
6. 20x washing buffer (20 ml).
Material required:
1. Precision pipettes: 50 µl, 100 µl, 200 µl and 1 ml.
2. Disposable pipette tips
3. Distilled water
4. Glass tubes or flasks to prepare TMB solution
5. Vortex mixture or equivalent
6. Absorbent paper
7. Graph paper
8. Microtiter plate reader
Reagent preparation:
All reagents in kit were brought to room temperature (18-250 C) before use.
Ready to use reference standards were provided with the kit.
Assay procedure:
1. Secure desire no of coated wells in the holder
2. Dispensed 100 µl of standards, specimens and controls into appropriate wells
3. Dispensed 100 µl of enzyme conjugate reagent into each well. Shook the plate for 30
seconds. It was very important to shake the plate very well at this step.
4. Incubated at room temperature (18-250 C) for 3 hours.
5. Removed the incubation mixture by dumping plate contents into a waste container.
______________________________________________________________________ 60
Fig 3.9 Standard curve of growth hormone
4.5
4
Absorbance
3.5
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
Concentration (ng/ml)
Fig 3.10 Standard curve of prolactin
Concentration (ng/ml))
C
2500
2000
1500
1000
500
0
0
0.5
1
1.5
Abosorbance
2
2.5
3
Materials and methods …
6. Rinsed and dumped the microtititer wells five (5) times (200-300 µl) with dilute
wash buffer.
7. Dispensed 100 µl of TMB solution into each well. Gently mixed for 10 seconds.
8. Incubated at room temperature for 20 minutes in the dark.
9. Stopped reaction by adding 50 µl of stop solution (2N HCl) to each well.
10. Gently mixed for 30 seconds. It was important to observe a colour change from blue
to yellow.
11. Read optical density at 450 nm with a microtiter well reader.
Calculations of the results
Calculated the mean absorbance value (A450) for each set of reference standards,
specimens, controls and patient samples. Construct the standard curve by plotting the mean
absorbancy value on the vertical or Y axis and the concentration on the horizontal or X axis
(Fig 3.9). Use the mean absorbency values for each specimen to determine the
corresponding concentration of bGH (ng/ml) from the standard curve.
3.15.12 Plasma prolactin estimation
Estimation of blood prolactin was done by using enzyme-linked immunosorbent
assay kit for bovine prolactin (PRL) purchased from the Uscn Life Science Inc.
Principle
The microtiter plate provided in this kit has been pre-coated with a monoclonal
antibody specific to prolactin. Standards or samples were then added to the appropriate
microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for
PRL. Next, Avidin conjugated to horseradish peroxidase was added to each microplate well
and incubated. After TMB substrate solution was added, only those wells that contains PRL,
biotin conjugated antibody and enzyme conjugated Avidin will exhibit a change in colour.
The enzyme substrate reaction was terminated by the addition of sulfuric acid solution and
the colour change was measured spectrophotometrically at a wavelength 450 ± 10 nm. The
______________________________________________________________________ 61
Materials and methods …
concentration of PRL in the sample is then determined by comparing the optical density
(OD) of the samples to the standard curve.
Material required
Microplate reader 450 ± 10 nm filter
Precision single and multichannel pipettes and disposable tips
Eppendorf tubes for diluting samples
Deionized or distilled water
Absorbent paper for blotting the microtiter plate
Container for wash solution
PBS solution (pH = 7.0 to 7.2)
Reagent preparation
All kit components and samples were brought to room temperature (18 to 290 C)
before use.
1. Standard preparation
The standard was reconstituted with 1 ml of Standard Diluent (kept for 10 min. at
room temperature) and shaken gently (not to foam). The concentration of the standard in the
stock solution was 2000 pg/ml. Seven tubes were prepared containing 0.5 ml Standard
Diluent and produced a double dilution series. Each tube was thoroughly mixed before the
next transfer. 7 points of the diluent standards such as 2000, 1000, 500, 250, 125, 62.5, 31.2
pg/ml were set and at the last EP tube with the standard diluent was kept the blank as 0
pg/ml.
2. Assay Diluent A and Assay Diluent B:
Assay Diluent A or B concentrate (2X) were diluted with 6 ml of deioniozed water
to prepare 12 ml of Assay Diluent A or B. The prepared working dilution was used
immediately.
3. Detection Reagent A and Detection Reagent B
______________________________________________________________________ 62
Materials and methods …
The stock Detection A and Detection B were briefly vortexed before use. It was
diluted to the working concentration with working Assay Diluent A or B, respectively
(1:100)
4. Wash solution
20 ml of wash solution concentration (30X) was diluted with 580 ml of deionized
water to prepare 600 ml of wash solution (1X).
5. TMB substrate
The needed dosage of the solution was aspirated with sterilized tips.
Sample preparation
Plasma samples were diluted by 100 fold. 10 µl of sample was mixed with 990 µl
PBS (0.02 mol/L PBS).
Assay procedure
1. 7 wells for standard and 1 well for blank were prepared. 100 µl each of dilutions of
standard, blank and samples were added into the appropriate wells. Plate was
covered with the plate sealer and incubated for the 2 hours at 37 0 C.
2. The liquid of each well was removed.
3. 100 µl of Detection Reagent A working solution was added to each well and
incubated for 1 hour at 37 0 C after covering it with the plate sealer.
4. The solution was aspirated and washed with 350 µl of 1X wash solution to each well
using multichannel pipette and allowed to stand for 1-2 minutes. The remaining
liquid from all wells was removed completely by snapping the plate onto absorbent
paper. Totally there were 3 washings. After the last wash, remaining any wash buffer
was removed by aspirating or decanting. The plate was inverted and blotted it
against absorbent paper.
5. 100 µl of Detection Reagent B working solution was added to each well and
incubated for 30 min at 37 0 C after covering it with plate sealer.
6. The aspiration/wash process was repeated for total 5 times as conducted in step 4.
______________________________________________________________________ 63
Materials and methods …
7. 90 µl of substrate solution was added to each well and covered with new plate sealer.
The microplate was incubated for 15-25 minutes at 370C (did not exceed 30
minutes). The liquid turned blue by the addition of substrate solution in each well.
8. 50 µl of stop solution was added to each well. The liquid turned yellow by the
addition of the stop solution. The liquid was mixed by tapping the side of the plate
for proper mixing.
9. Any drop of water and fingerprint on the bottom of the plate were removed and
conformed that there was no bubble on the surface of the liquid. Then, the
microplate was measurement at 450 nm immediately.
Calculation
The readings for each standards, control and samples subtracted from the average
zero standard OD. Create the standard curve on log-log graph paper, with prolactin
concentration on the Y axis and absorbance on the X- axis (Fig 3.10). Draw a best fit strait
line through the standard points and it can be determined by regression analysis.
3.15
Reproduction study
All animals was transferred to calving pens 10 days before the expected calving date
in calving shed of Cattle Yard, NDRI, Karnal. Separate calving pen were provided to each
animal and feeding and management of all animals was same as described earlier during
prepartum study.
Following parturition related parameters were noted during the study:
3.15.1 Calf weight at the time of birth
The Body weight of the calves was measured at the time of birth using Avery
Platform balance with round display panel. The maximum capacity of the balance was 300
kg and error was ± 500 g.
3. 15.2 Calf mortality within one month of parturition
3.15.3 Occurrence of reproductive disorders
The occurrences of reproductive disorders like dystokia, retained placenta, metritis,
______________________________________________________________________ 64
Materials and methods …
mastitis were recorded. For this the help of experts in the field of veterinary medicine and
veterinary surgery in the Cattle Yard, NDRI was obtained for disease diagnosis and
treatment.
POSTPARTUM STUDY
The postpartum study was divided into four parts:
A. Lactation study
B. Reproduction study
3.16
Lactation Study
3.16.1 Housing and management
Housing, management and watering of animals was same as described in pre partum
studies.
3.16.2 Milking of animals
Animals were hand milked trice a day i.e. early in morning (5.00 am), noon (11.30
am) and in the evening (6.00 pm).
3.16.3 Body weight and body condition score
Body Weight of the animals was recorded at fortnightly intervals by using
computerized weight management system from Leotronic Scales Pvt. Ltd as described in the
pre partum study. The animals were weighed for two consecutive days in the morning
before offering feed and water and after milking. The average of two days was considered as
body weight for that fortnight. Body condition score was recorded as per described in
annexure I.
3.16.4 Feed intake
Daily DM intake was observed by recording the daily feed offered and residue
throughout the experiment period of 60 days pre-partum to 210 days post-partum. The DM
of different feed ingredients was recorded once every week. Fortnightly intake of nutrients
viz CP, RDP, RUP, MP, TDN, ME, NE L of each cow was calculated by using formulae of
NRC (2001) (Annexure II).
______________________________________________________________________ 65
Materials and methods …
3.16.5 Milk yield
Daily milk yield was recorded for individual animal by using circular dial type
spring balance, with the capacity of 20 kg and an accuracy of ± 0.05 kg, at each milking
time.
3.16.6 Calculation of fat corrected milk (FCM)
For the conversion of whole milk into 4 per cent FCM, the following equation was
adopted.
4% FCM = 0.4M + 15F
Whereas, M = Milk yield (kg)
F = Milk fat (Kg)
3.16.7 Calculation of energy corrected milk (ECM)
For the conversion of whole milk into ECM, the following equation given by Sjaunja
et al. (1990) was adopted.
ECM (kg/d) = Milk Yield (kg/d) x ((38.3 x Fat (g/kg) + 24.2 x Protein (g/kg) + 16.54 x
Lactose (g/kg) + 20.7))/3140
3.17
Milk Composition
Milk samples from individual animals were collected and analyzed for milk
composition at fortnightly intervals throughout the experimental period. The samples
collected twice from milking of the day for each animal were proportionately pooled to
represent milk sample of that animal. Representative samples were analyzed for its
composition (fat, Protein, lactose and SNF) using precalibrated Milk Analyzer (LactoStar,
FUNKE GERBER, Article No 3510, Berlin). The total solids in milk were calculated by
adding milk fat % and SNF %.
3.17.1 Fatty acid analysis of milk
From the pooled milk samples of individual animal, during early, mid and late
lactation the fatty acid analysis of milk was done using saponification method (Gulati and
Ashes, 2000) as follows:
______________________________________________________________________ 66
Materials and methods …
1.
Preparation of methyl esters
a) Saponification
1. Pipetted 2 ml of milk sample into a 25 ml stoppered test tube
2. Added 1 ml of Ethanol
3. Added 1 ml of 5 N sodium hydroxide (NaOH)
4. Shook well
5. Covered with foil
6. Placed into oven at 80oC for 1.5 – 2 h
Removed from oven and allowed to cool a little
b) Acidification
1. Added 5 N Hydrochloric Acid (HCL) approx. 2 ml.
2. Inverted test tube with care
3. pH was checked for each sample using pH paper (must be pink)
4. Cooled the samples sufficiently
5. Extract Fatty Acids – added 4 ml of Petroleum Ether shaked well, pipetted the
supernatant into a labeled 15 ml test tube
6. Repeated step-4 – pooling the extracts
7. Evaporated PE extracts to dryness in a warm water bath under a stream of Nitrogen
c) Methylation
1. To the dried sample, added 3 ml of 1% Sulphuric acid in methanol
2. Refluxed on a heating block at 50-60oC for 1.5 h
3. Added 3 ml of 5% NaCl (salt solution)
4. Added 3 ml of PE
5. Cooled, shook well
______________________________________________________________________ 67
Materials and methods …
6. Centrifuged at 1500 rpm
7. Decanted supernatant into GLC vial and capped the vial
2.
Reagents needed
1. Ethanol (absolute)
2. 5 N Sodium Hydroxide (NaOH) (200 g of Na OH pellets/ 1 litre of distilled water)
3. 5 N Hydrochloric Acid (HCL) (500 ml of concentrated acid added to 500 ml of
distilled water)
4. Petroleum Ether (PE) (Boiling range 40-60) Hexane can be used as an alternative
5. 1% sulphuric acid in Methanol:
6. 5% salt solution (Na Cl) (50 g to 1 litre of distilled water)
3.
Fatty acid analysis by gas liquid chromatography
Analysis was carried out on GLC fitted with Flame ionization detector and 50 m
length of capillary column. Initial temperature of the column was 140ºC. The RAMP rate
was 2 ºC / min. The other conditions were as follows:
Stationary Phase
-
Fused silica column
Injector temperature
-
210 ºC
Delay Time
-
0.00
Sampling Rate
-
12.50pts/sec
Carrier Gas
-
Helium
Flow Rate
-
11.2 psig
Ignition Gas
-
Air plus H 2
Identification of peaks was made through retention time of the reference standards
purchased from Supelco, Bellefonte PA, USA.
______________________________________________________________________ 68
Materials and methods …
3.18.2 Estimation of milk urea
Milk urea content was determined by a modified colorimetric DMAB (pDimethylaminobenzaldehyde) assay (Bector et al., 1998).
Principle
Urea forms a yellow complex with the p-Dimethylaminobenzaldehyde reagent in an
alcohol low acidic solution at room temperature. The intensity of colour was measured by
spectrophotometer at 425 nm. The standard curve is presented in Fig. 3.8.
Alcohol
Urea + DMAB
Yellow color complex
HCl
Reagents
a) Trichloroacetic acid (TCA) 12% (s/v) solution
120 gm TCA was dissolved in the distilled water and the volume was made to 1000
ml.
b) Phosphate buffer, pH 7.0
Anhydrous potassium dihydrogen orthophosphate (3.403 gm) and anhydrous
dipotassium monohydrogen orthophosphate (4.355 gm) were dissolved in distilled water
and volume was made to 1000 ml.
c) Diluting reagent
Equal volumes of 12% (w/v) TCA and phosphate buffer (pH 7.0) were mixed to
make the diluting reagent.
d) Coloring reagent (1.6% DMAB solution)
1.6 gm DMAB dissolved in ethyl alcohol, 10 ml concentrated HCL was added into it
and the volume was made to 100 ml.
Procedure
a) Preparation of milk samples
______________________________________________________________________ 69
Materials and methods …
Milk samples were warmed at room temperature (27oC to 30oC) and mixed well. 10
ml of milk was mixed with 10 ml of 12% TCA solution and filtered through Whatman filter
paper no. 1. Filtrates were then centrifuged at 3,000 rpm for 15 to 20 min. and supernatants
were collected.
b) Estimation of urea
2 ml of coloring reagent (1.6% DMAB solution) was added to 2 ml of supernatant to
develop the yellow color complex. The absorbance was read at 425 nm against a blank
containing 2 ml of diluting reagent and 2 ml of coloring reagent (1.6% DMAB solution).
c) Preparation of standard curve
Standard urea solution (1 mg/ml) was prepared in phosphate buffer (pH 7.0).
Different concentrations of urea solution (0.25 mg, 0.50 mg, 0.75 mg, 1.00 mg, 1.25 mg and
1.50 mg) were separately taken into different test tubes and the total volume was made to 2
ml of each case with diluting reagent solution. Two ml of 1.6% DMAB solution was added
to each test tube to develop the yellow colour complex. The absorbance was read at 425 nm
against a blank containing 2 ml of diluting reagent and 2 ml of 1.6% DMAB solution. The
values were plotted against concentration (Fig. 3.3).
3.18
Analysis of blood parameters
Blood Samples were collected at fortnightly intervals up to 45 days of parturition
and thereafter at monthly intervals up to end of experiment. The blood samples from
individual animals were collected by jugular vein puncture into heparinized vaccutainer
tubes, 16 X 100 mm (Becton Dickinson, Rutherford, NJ). The plasma was separated by
centrifugation of the blood samples at 2400 rpm for 15 min and it is stored in plasma vials at
-20
0
C for subsequent analysis of amino acids, choline, glucose, non esterified fatty acid
(NEFA), triglycerides, blood urea nitrogen (BUN) and VLDL as described in pre partum
study section.
3.19
Statistical Analysis
Statistical analysis was carried out by Three Way ANOVA using SigmaStat for
Windows version 3.10.
______________________________________________________________________ 70
CHAPTER – 4
RESULTs AND DISCUSSION
4. Results and Discussion
_______________________________________________
To investigate the effect of supplementing rumen protected methionine (RPL)
and lysine (RPL) on pre and post parturient performance such as calving, lactation,
nutrient utilization and reproduction, eighteen crossbred cows were selected and divided
into two groups (9 each) on the basis of Most Probable Production Ability (MPPA) and
lactation number (Table 3.2). Animals in group 1 (control group) were fed chopped
wheat straw, chaffed green maize fodder and compounded concentrate mixture as per
requirements (NRC, 2001). However, animals in group 2 (treatment group) were fed
same ration as control group plus 5 gm RPM and 20 gm RPL prepartum and 7 gm RPM
and 60 gm RPL postpartum, respectively. The experimental period was from 40 days
before expected date of parturition to 120 days post parturition. The results obtained
during the course of this study have been presented and discussed in respective sections.
4.1
EFFECT OF FEEDING RUMEN PROTECTED METHIONINE AND
LYSINE DURING PRE PARTUM PERIOD
The physical composition of concentrate mixture used in the experiment is
presented in Table 3.3. Percent composition of the concentrate mixture was: maize 33,
groundnut cake 21, mustard cake 12, wheat bran 20, deoiled rice bran 11, mineral
mixture 2 and common salt 1.
Chemical composition of feeds and fodders used in the experiment is presented
in Table 4.1. Concentrate mixture contained 21.00, 3.75, 35.96, 11.23, 24.73, 3.97, 0.94,
4.1, 8.74 and 69.24 per cent CP, EE, NDF, ADF, hemicellulose, NDICP, ADICP, lignin,
Ash and TDN, respectively. The corresponding values for green maize fodder were
10.52, 1.67, 48.64, 25.26, 23.38, 1.38, 1.08, 2.89, 4.36 and 65.26; for wheat straw these
values were 3.50, 1.32, 73.45, 50.65, 22.80, 2.18, 1.42, 9.30, 7.66 and 46.57,
respectively. The chemical composition of green maize forage and wheat straw was
within the normal range (Ranjhan, 1998).
Maize contained 9.63, 5.50, 10.21, 3.24, 6.97, 0.83, 0.40, 1.11, 2.54 and 85.22
per cent CP, EE, NDF, ADF, hemicellulose, NDICP, ADICP, lignin, Ash and TDN,
respectively. The corresponding values for ground nut cake were 45.06, 7.03, 22.42,
12.54, 9.88, 5.82, 1.11, 4.71, 5.76 and 83.79. The corresponding values for mustard cake
__________________________________________________________________
71
Results and Discussion …
_______________________________________________
were 35.44, 9.50, 29.54, 21.60, 7.94, 6.23, 2.51, 7.86, 7.29 and 74.94. The
corresponding values for deoiled rice bran were 14.38, 1.25, 26.86 , 4.70, 12.16, 3.6,
0.45, 4.71, 10.53 and 66.53. The corresponding values for wheat bran were 14.94, 4.30,
43.05, 15.75, 27.30, 2.91, 1.47, 4.42, 6.36 and 69.05.
Amino acid profile of feed and fodder used in the experiment is presented in
Table 4.2. Essential amino acid conetnts of concentrate mixture viz. arginine, cysteine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan
and valine were 5.82, 0.89, 1.64, 2.92, 5.71, 3.43, 1.50, 3.68, 2.74, 1.07 and 4.23 per
cent of CP, respectively.
The corresponding values for green maize fodder were 4.43, 0.71, 1.24, 3.83,
6.62, 3.60, 1.45, 4.26, 3.29, 0.52 and 5.04; for wheat straw these values were 5.53, 1.18,
1.66, 3.11, 5.10, 3.11, 1.23, 4.03, 2.64, 1.57 and 4.48, respectively. Non - essential
amino acid profile of concentrate mixture viz. alanine, aspartic acid, glutamic acid,
glycine, proline, serine and tyrosine was 3.68, 7.36, 11.50, 3.78, 6.10, 3.65 and 3.56 per
cent of CP, respectively. The corresponding values for maize green fodder were 5.15,
7.94, 8.79, 4.09, 1.24, 5.19, 3.46 and 3.72; for wheat straw these values were 3.42, 5.37,
14.97, 3.80, 1.66, 6.76, 3.27 and 3.35, respectively. The amino acid profile of other
common feed stuffs which are commonly used in feed formulation is presented in table
4.2. Essential amino acid contents of bacteria viz. arginine, cysteine, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine
were 4.48, 0.91, 0.63, 3.39, 3.76, 7.23, 2.65, 3.18, 4.30, 2.58 and 4.72 per cent of CP,
respectively. The corresponding values for protozoa were 3.43, 2.01, 1.57, 2.27, 4.40,
5.16, 2.62, 3.06, 3.15, 1.87 and 3.22. Both bacteria and protozoa are the good source of
methionine and lysine. But all the feed stuffs are either difficient in methionine and/or
lysine.
__________________________________________________________________
72
Table 4.1 Chemical composition of feed ingredients offered (% DM basis)
Ingredient
CP
EE
NDF
ADF
Maize fodder
10.52
1.67
48.64
25.26
Wheat straw
3.50
1.32
73.45
Concentrate
21.00
3.75
Maize grain
9.63
GNC
Hemicellul-
NDICP
ADICP
Lignin
Ash
TDN%*
23.38
1.38
1.08
2.89
4.36
65.26
50.65
22.8
2.18
1.42
9.3
7.66
46.57
35.96
11.23
24.73
3.97
0.94
4.1
8.74
69.24
5.50
10.21
3.24
6.97
0.83
0.40
1.11
2.54
85.22
45.06
7.03
22.42
12.54
9.88
5.82
1.11
4.71
5.76
83.79
Mustard Cake
35.44
9.50
29.54
21.6
7.94
6.23
2.51
7.86
7.29
74.94
Deoiled Rice Bran
14.38
1.25
26.86
14.70
12.16
3.6
0.45
4.71
10.53
66.53
Wheat Bran
14.94
4.30
43.05
15.75
27.3
2.91
1.47
4.42
6.36
69.05
ose
* Calculated as per formula given in NRC (2001)
73
Table 4.2 Amino Acid Profile (% of CP) of feed Ingredients
Feed
Essential Amino Acids (% of CP)
ARG
Non Essential Amino Acids (% of CP)
CYS
HIS
ILE
LEU
LYS
MET
PHE
THR
TRP
VAL
ALA
ASP
GLU
GLY
PRO
SER
TYR
Experimental Diet
Wheat Straw
5.33
1.18
1.66
3.11
5.10
3.11
1.23
4.03
2.64
1.57
4.48
3.42
5.37
14.97
3.80
6.76
3.27
3.35
Maize Fodder
4.43
0.71
1.24
3.83
6.62
2.60
1.45
4.26
3.29
0.52
5.04
5.15
7.94
8.79
4.09
5.19
3.46
3.72
Concentrate
5.82
0.89
1.64
2.92
5.71
3.43
1.50
3.68
2.74
1.07
4.23
3.68
7.36
11.50
3.78
6.10
3.65
3.56
Common Feed Ingredients
Oat fodder
2.13
0.78
1.97
5.53
6.61
3.51
1.87
4.74
4.10
1.58
4.10
8.90
7.01
1.60
9.32
12.29
4.02
10.10
fodder
4.13
1.07
2.14
3.93
7.05
4.17
1.43
4.86
4.06
1.51
4.96
7.28
8.63
5.74
7.43
13.35
5.77
6.99
Maize grain
4.40
0.94
1.46
2.48
6.27
1.35
1.63
3.43
2.35
0.81
4.14
3.75
5.48
9.29
2.73
6.79
3.03
3.89
Barley grain
5.40
2.19
2.31
3.44
6.85
3.59
1.66
5.00
3.35
1.37
4.84
6.40
6.00
7.36
6.37
9.14
9.44
6.25
Pearl Millet
4.56
1.85
4.00
4.31
4.18
5.44
2.04
5.31
3.54
1.39
5.92
6.55
5.39
8.39
8.13
7.63
10.2
5.25
Oat grain
6.79
2.84
2.43
3.73
7.27
4.16
1.70
5.14
3.45
1.21
5.08
10.38
5.53
6.96
6.02
8.00
8.18
4.58
GNC
8.54
0.49
1.70
2.75
4.91
3.94
1.55
4.09
2.14
1.07
3.36
2.79
8.83
13.78
4.30
4.10
3.76
3.46
Berseem
Continued….
Feed
Essential Amino Acids (% CP)
Non Essential Amino Acids (% CP)
ARG
CYS
HIS
ILE
LEU
LYS
MET
PHE
THR
TRP
VAL
ALA
ASP
GLU
GLY
PRO
SER
TYR
Mustard Cake
7.12
1.15
2.28
4.05
7.16
5.13
1.48
4.48
4.14
1.57
5.29
3.72
6.44
16.02
4.91
7.38
4.06
4.06
Raw Soybean
7.05
1.45
2.77
4.38
7.32
5.92
1.46
4.92
3.91
1.32
4.35
5.02
9.12
6.08
4.24
2.71
7.76
10.35
Extr. Soybean
6.62
1.42
2.63
4.15
6.95
5.87
1.39
4.23
3.65
1.24
4.52
5.02
8.90
5.96
4.71
2.54
7.65
10.49
Soybean meal
7.11
1.59
2.61
4.27
7.93
6.46
1.38
4.85
4.69
1.29
6.42
3.82
6.89
5.73
3.51
1.64
7.39
10.99
Whole Cott. S.
6.88
2.79
3.62
3.51
5.57
4.88
1.63
5.07
3.69
1.29
4.45
4.96
9.52
3.77
10.92
5.40
7.02
4.83
Cotton S. C.
7.92
1.75
2.88
3.07
6.29
4.08
2.74
5.44
3.07
1.28
4.28
9.59
4.59
10.00
5.22
7.94
2.49
5.79
Clusterbean M
5.86
1.24
2.63
3.94
5.29
4.30
2.67
4.36
3.97
1.11
3.40
2.77
4.55
3.86
12.90
4.38
3.20
2.19
Maize gluten
3.21
1.80
2.14
4.12
16.83
1.69
2.38
6.37
3.39
0.50
4.65
3.03
4.63
2.13
4.22
5.73
3.17
14.69
Rice Bran
7.95
1.08
2.11
3.58
6.79
3.75
1.11
4.70
3.75
1.33
5.92
5.28
8.62
12.53
4.88
6.88
4.42
4.22
Wheat Bran
4.21
1.11
1.46
3.24
5.02
3.97
1.67
3.19
3.02
1.52
4.39
4.15
9.89
11.18
4.27
6.79
4.43
2.98
Rumen microbes
Protozoa
3.43
2.01
1.57
2.27
4.40
5.16
2.62
3.06
3.15
1.87
3.22
4.05
11.25
3.96
15.68
4.37
4.39
0.97
Bacteria
4.48
0.91
0.63
3.39
3.76
7.23
2.65
3.18
4.30
2.58
4.72
9.45
7.29
5.01
4.46
2.92
3.76
1.26
Results and Discussion …
4.1.1
Degree of Protection of Rumen Protected Methionine (RPM) and Lysine
(RPL)
The total methionine content in the supplemental RPM was 52.68 per cent and
the ether extract content was 44.90 percent. The rumen escape potential of RPM from
rumen hydrolysis was 75.20 per cent. The total lysine content in the supplemental
RPL was 40.20 per cent and the ether extract content was 49.00 percent. The rumen
escape potential of RPL from rumen hydrolysis was 54.97 per cent. The effective
degradability of RPM and RPL was 24.80 and 45.03 per cent , respectively. (Table
4.3).
Table 4.3 Characteristic of rumen protected methionine (RPM) and rumen
protected lysine (RPL) products
Particular
RPM
RPL
52.68±0.04
0
0
40.20±0.46
44.90±0.32
49.00±0.50
0 hr
0.77 ± 0.28
15.34 ± 0.88
3 hr
20.25 ± 0.91
44.64 ± 0.52
6 hr
22.01 ± 0.60
46.41 ± 0.16
12 hr
28.37 ± 0.68
48.04 ± 0.25
24 hr
44.17 ± 2.28
48.99 ± 0.16
ED
24.80 ± 0.57
45.03 ± 0.22
REP
75.20 ± 1.04
54.97 ± 0.79
Methionine (% of DM)
Lysine (% of DM)
Fat (% of DM)
Rumen Escape Potential (REP)
For Met in the RPM and Lys in the RPL to have an impact on milk protein
synthesis, it needs to be delivered to the small intestine without being degraded in the
rumen. To determine how much of the Met and Lys would escape from the rumen
_________________________________________________________________ 76
Results and Discussion …
hydrolysis, it is necessary to consider the rate at which the particles are degraded and
the amount of time that they spend in the rumen. Rumen degradability can then be
calculated as Kp/(Kd + Kp) (McDonald et al, 2002) where Kp = rate of passage and Kd
= rate of digestion. Using the results obtained from the degradation study on the RPM
and RPL particles, the kd was 0.045/h. If a rate of passage of 0.05/h is assumed, which
is the passage rate used for animals fed a diet between maintenance (0.02/h) and high
production (0.08/h), 75.20 of the RPM and 54.97% of the RPL particles would have
been delivered to the duodenum.
4.1.2
RDP and RUP content of different feedstuffs
This section deals with the estimation of RDP and RUP content (% of CP)
from nylon bag over a period of time and degradation constants (Protein – A, Protein
– B and Protein -C values).
Protein Fraction A is the percentage of total CP that is NPN (i.e. assumed to
be instantly degraded) and a small amount of true protein that rapidly escapes from
the in situ bag because of high solubility or very small particle size. As presented in
table 4.4, protein A was found highest in GNC (62.11) followed by mustard cake
(39.74) and found lowest in wheat straw (9.39). Fraction B is that percent of the
proteins which are potentially degradable. Protein B fraction was found highest in
maize (69.26) and lowest in GNC (35.60). Only the B fraction is considered to be
affected by relative rates of passage; all of fraction A is considered to be degraded and
all of fraction C is considered to pass to the small intestine. The amount of fraction B
that is degraded in the rumen was determined by the fractional rate of degradation that
was determined in the study for fraction B and an estimate of fractional rates of
passage. Both A and B were calculated by computer analysis using the expression
given by Mehrez and Orskov (1977) i.e. P = a + b (1- e –ct). While Protein Fraction C
is the percentage of CP that is completely undegradable; this fraction generally is
determined as the feed CP remaining in the bag at a defined end-point of degradation
was calculated by difference. Fraction C was found highest in wheat straw (36.00)
and lowest in GNC (2.29) and mustard cake (5.46).
The RDP and RUP values for a feedstuff (percent of CP) using this model
were computed using the equations RDP= A + B [kd / (kd+ kp)] and RUP = B [kp /
_________________________________________________________________ 77
Results and Discussion …
(kd + kp)] + C. GNC had the highest RDP (89.27% of CP), followed by mustard cake
(83.58) and lowest for wheat straw (31.88). While highest RUP per cent observed in
wheat straw (68.12) followed by maize (44.82) and deoiled rice bran (40.01).
Numerous factors affect the amount of CP in feeds that will be degraded in the
rumen. The chemistry of feed CP is the single most important factor. The two most
important considerations of feed CP chemistry are: (1) the proportional concentrations
of NPN and true protein, and (2) the physical and chemical characteristics of the
proteins that comprise the true protein fraction of the feedstuff. Non protein N
compounds are degraded so quickly in the rumen (>300%/h) that degradation is
assumed to be 100 percent (Sniffen et al., 1992).
Table 4.4 Different protein fractions, RUP (% of CP) of feed Ingredients
Feed
Protein – A
Protein – B
Protein – C
RDP
RUP
Wheat straw
9.39 ± 0.42
54.61 ± 0.65
36.00 ± 0.73 31.88 ± 0.47 68.12 ± 0.47
Maize
fodder
34.39 ± 1.16 52.43 ± 0.47
13.18 ± 0.75 60.61 ± 0.95 39.39 ± 0.95
Concentrate
33.00 ± 1.02 51.64 ± 0.82
15.36 ± 0.67 69.41 ± 0.69 30.59 ± 0.69
Maize
20.90 ± 0.35 69.26 ± 0.50
9.84 ± 0.44
55.18 ± 0.31 44.82 ± 0.31
GNC
62.11 ± 0.55 35.60 ± 0.83
2.29 ± 0.64
89.27 ± 0.51 10.73 ± 0.51
Mustard
Cake
39.74 ± 0.33 54.80 ± 0.76
5.46 ± 0.79
83.58 ± 0.66 16.42 ± 0.66
Deoiled
Rice Bran
34.11 ± 0.66 51.75 ± 0.83
14.13 ± 0.80 59.99 ± 0.60 40.01 ± 0.60
Wheat bran
32.82 ± 0.76 56.08 ± 1.09
11.10 ± 0.88 77.68 ± 0.74 22.32 ± 0.74
Differences in 3-dimensional structure and chemical bonding (i.e., cross-links)
that occur both within and between protein molecules and between proteins and
carbohydrates are functions of source as well as processing. These aspects of structure
affect microbial access to the proteins, which apparently is the most important factor
affecting the rate and extent of degradation of proteins in the rumen. Proteins that
_________________________________________________________________ 78
Results and Discussion …
possess extensive cross-linking, such as the disulfide bonding in albumins and
immunoglobulins or cross-links caused by chemical or heat treatment, are less
accessible to proteolytic enzymes and are degraded more slowly (Ferguson, 1975;
Hurrell and Finot, 1985; Mahadevan et al., 1980; Mangan, 1972; Nugent and
Mangan, 1978; Nugent et al., 1983; Wallace, 1983).
4.1.3 RUP Intestinal Digestibility
RUP intestinal digestibility values of wheat straw, maize fodder and
concentrate were 64.40, 70.47 and 79.84 percent, respectively. While maize,
groundnut cake, mustard cake, rice bran and wheat bran had 89.60, 73.73, 91.47,
75.13 and 65.33 percent RUP intestinal digestibility, respectively. (Table 4.5; Fig 4.1)
Table 4.5 RUP Intestinal Digestibility (in vitro) (% DM basis) of feed Ingredients
Ingredients
RUP ID %
Wheat straw
64.40 ± 0.67
Maize fodder
70.47 ± 0.29
Concentrate
79.84 ± 1.34
Maize
89.60 ± 1.83
GNC
73.73 ± 2.38
Mustard Cake
91.47 ± 0.89
Rice Bran
75.13 ± 0.57
Wheat bran
65.33 ± 1.57
Ruminally undegraded feed CP is assumed to be 100 percent true protein
(National Research Council, 1989). Estimates of intestinal digestibility have been
assigned to the RUP fraction of each feedstuff; assigned values vary from 50 to 100
percent (NRC, 2001). Therefore, the contribution of RUP to MP is variable and
dependent on feed type.
_________________________________________________________________ 79
Results and Discussion …
4.1.4 In vitro Microbial protein yield per kg TDN fermented
The microbial protein yield per kg TDN fermented was 127.84, 119.62 and
110.04 g in different TMRs containing concentrate to roughage ratio of 60:40, 50:50
and 40:60, respectively (Table 4.6; Fig 4.2). The microbial protein yield (g) per kg
TDN fermented was higher (P<0.01) in T1 than T2 and T3 and in T2 than T3
(P<0.01). The result of the present in vitro study revealed that the relative availability
of nitrogen for fermentation is an important factor affecting efficiency of microbial
protein synthesis.
Table 4.6 Microbial protein yield g per kg TDN fermented of different TMRs
Trials
C:R 60:40
C:R 50:50
C:R 40:60
(TDN – 64.66%)
(TDN – 63.51%)
(TDN – 62.37%)
1
125.46
117.79
106.29
2
124.91
124.38
109.98
3
128.32
114.43
110.29
4
128.93
120.65
114.35
5
130.46
119.56
105.42
6
128.95
120.90
113.93
Average
127.84 ± 0.89
119.62 ± 1.36
110.04 ± 1.52
a, b,c
Means having different superscripts in a row differ significantly (P<0.01)
In roughage portion, wheat straw and maize fodder was in the ratio 40:60 on DMB.
Microbial protein is the protein of the ruminal bacteria, protozoa, and fungi
that pass to the small intestine. Bacteria provide most of the microbial protein leaving
the rumen. Protozoa contribute significantly to the microbial biomass of ruminal
contents. However, because they are more extensively recycled in the rumen than
bacteria (Ffoulkes and Leng, 1988; Leng et al., 1986; Punia et al., 1992,), protozoa do
not contribute to postruminal protein supply in proportion to their contributions to the
total microbial biomass in the rumen (NRC, 2001). Ruminally synthesized microbial
_________________________________________________________________ 80
Fig 4.1 RUP Intestinal Digestibility (in vitro) (% DM basis)
Fig 4.2 Microbial protein yield g per kg TDN fermented of different
TMRs
Results and Discussion …
protein typically supplies a majority of the AA flowing to the small intestine of
growing cattle (Titgemeyer and Merchen, 1990) and dairy cows (Clark et al., 1992).
4.1.5
Body weight changes
Fortnightly changes in body weights of pre parturient cows are presented in
Table 4.7; Fig 4.3 and Fig 4.4. In the first fortnight (from day -30th to -15th days pre
parturient), the experimental animals gained 7.6 and 6.7 kg body weight in groups 1
and 2, respectively, while in the 2nd fortnight (from day -15th to the partuirition), the
loss in weight was 58.2 and 56.1 kg, respectively due to parturition and release of the
foetal membranes. The changes in body weight were not different between the groups.
Table 4.7 Fortnightly change in body weights (kg) of crossbred cows fed ration
with or without RPM plus RPL
Day
Group 1
Group 2
-30
509.8 ± 22.9
498.6 ± 10.1
-15
517.4 ± 23.0
505.3 ± 9.6
1*
459.2 ± 20.3
442.7 ± 11.5
-15
+ 7.6 ± 0.6
+ 6.7 ± 0.9
1*
-58.2 ± 4.3
-56.1 ± 3.1
-15
+ 1.5 ± 0.1
+ 1.4 ± 0.2
1*
-11.2 ± 0.7
-11.2 ± 0.7
Change in Body weight
% Change in body weight
Group 1: Cows fed control diet
Group 2: Cows fed control diet + RPM 5 gm (Effective Methionine was 1.98 g ) & RPL 20
gm (Effective lysine was 4.42 g)
* : Day of parturition
During the transition period, feed intake decreases at a time when the protein
and energy requirements increase due to rapid growth of the conceptus, leading to the
_________________________________________________________________ 81
Results and Discussion …
increased demand of methionine and lysine. Consequently, in order to prevent a
change in N balance, the deodenal flow of methionine and lysine should be optimum.
Thus, due to supplementation of rumen protected methionine and lysine to the
treatment group, the duodenal supply of methionine and lysine from the consumed
ration were increased. But in the present study, there were no significant effect on
body weight change while decreasing trend in body weight loss was observed in
treatment group. Similar findings were also observed by Socha et al. (2005) on
supplementation of rumen protected methionine.
4.1.6
Body Condition Score
Body condition score (BCS) is a logistic tool for assessment of nutritional
status of dairy cows and their management for optimal performance. The maintenance
of an optimal body condition score relative to lactation stage, milk yield, nutrition and
health status is perhaps the most important aspect of dairy cow management that
facilitates a healthy transition from pregnancy to lactation.
Table 4.8 Fortnightly body condition score of crossbred cows fed ration with or
without RPM plus RPL
Day
Group 1
Group 2
-30
3.8 ± 0.05
3.8 ± 0.06
-15
3.9 ± 0.05
4.0 ± 0.04
1
3.5a ± 0.11
3.7b ± 0.06
Body condition score reflected changes in the body weight in both
supplemented and control groups. The body condition score during pre parturient
period ranged from 3.84 to 3.88 in group 1, and from 3.81 to 4.00 in group 2 (Table
4.8; Fig 4.5). The initial BCS of group 1 and 2 was 3.84 and 3.81, respectively. The
pre partum changes in body weights were not conspicuously reflected in BCS changes
which were higher in supplemented group than control group before parturition.
However, supplemented group exhibited higher gross apparent difference of changes
in body condition score than control group due to higher energy intake. Body
condition score at calving is a reliable indicator of reproductive performance
_________________________________________________________________ 82
Results and Discussion …
(Baruselli et al., 2001). Cows that are having high body condition score at calving or
those losing excess body weight are more likely to have a prolonged interval to first
oestrus; thereby prolonging days open (Roche, 2006). Major increases in or loss of
BCS has been found to be undesirable and the optimum reproductive efficiency
observed when BCS loss was at or below 0.5 units during the transition period
(Roche, 2006). The findings of the present study are in agreement with those of Socha
et al. (2005) who studied the effect of rumen protected methionine and lysine in
transition cows.
4.1.7
Nutrients Intake
The average DMI (Table 4.9, Fig, 4.6) in the first fortnight (from day -30th to
-15th days pre parturient) was 9.33 and 8.87 (kg/d) and in the second fortnight (day 15th to the day of parturition) it was 8.21 and 7.75 kg/d in group 1 and 2, respectively.
However, DMI/100 kg body weight (Table 4.10, Fig. 4.6) in first and second fortnight
was 1.83 and 1.59 kg/d in group 1 and 1.78 and 1.53 kg/d in group 2, respectively.
There was no difference (P>0.05) among two groups in average DMI/ 100 kg body
weight before parturition. The results of present study indicated that there was no
effect of rumen protected methionine and lysine supplementation on DMI of
prepartum cows.
The average CPI (Table 4.9, Fig, 4.7) in the first fortnight (from day -30th to 15th days pre parturient)) was 1.17 and 1.14 (kg/d) and in the second fortnight (day 15th to the day of parturition) it was 1.09 and 1.04 (kg/d) in group 1 and 2,
respectively. However, CP intake/ 100 kg body weight (Table 4.11, Fig. 4.6) in first
and second fortnight was 0.23 and 0.21 kg/d in group 1 and 0.23 a 0.23 kg/d in group
2, respectively. There was no difference (P>0.05) between two groups in average
prepartum CP intake/ 100 kg body weight.
The average RUP and RDP intakes (Table 4.9) in the first fortnight (from day
-30th to -15th days pre parturient) were 0.40 and 0.39; 077 and 0.75 (kg/d) and in the
second fortnight (day -15th to the day of parturition) they were 0.36 and 0.34 and 0.72
and 0.70 (kg/d) in group 1 and 2, respectively.
_________________________________________________________________ 83
Results and Discussion …
Table 4.9 Prepartum average nutrient intake of crossbred cows fed ration with
or without RPM plus RPL
Parameter
Fortnight
Group 1
Group 2
DM (kg/d)
-2
9.33 ± 0.50
8.87 ± 0.28
-1
8.21 ± 0.54
7.75 ± 0.24
-2
1.17 ± 0.04
1.14 ±0.04
-1
1.09 ± 0.05
1.04 ±0.03
-2
0.40 ± 0.02
0.39 ± 0.01
-1
0.36 ± 0.02
0.34 ± 0.02
-2
0.77 ± 0.03
0.75 ± 0.02
-1
0.72 ± 0.03
0.70 ± 0.03
-2
0.76 ± 0.03
0.74 ± 0.02
-1
0.70 ± 0.03
0.67 ± 0.03
Methionine Duodenal
-2
1.90 a ± 0.03
2.22 b ± 0.02
Flow (% of MP)
-1
1.97 a ± 0.02
2.30 b ± 0.02
Lysine Duodenal
-2
6.44a ± 0.03
7.09b ± 0.04
Flow (% of MP)
-1
6.57 a ± 0.06
7.35b ± 0.07
TDN (kg/d)
-2
5.90 ± 0.20
5.67 ± 0.18
-1
5.14 ± 0.31
4.66 ± 0.18
-2
21.78 ± 0.90
21.28 ± 0.63
-1
19.78 ± 1.00
18.50 ± 0.80
-2
13.59 ± 0.55
13.33 ± 0.39
-1
12.36 ± 0.61
11.60 ± 0.50
CP (kg/d)
RUP (kg/d)
RDP (kg/d)
MP (kg/d)
ME (Mcal/d)
NE L (Mcal/d)
a, b
Means having different superscripts in a row differ significantly (P<0.01)
_________________________________________________________________ 84
Results and Discussion …
The average MP Intake (Table 4.9) in the first fortnight (from day -30th to 15th days pre parturient) was 0.76 and 0.74 (kg/d) and in the second fortnight (-15th to
the day of parturition) it was 0.70 and 0.67 (kg/d) in group 1 and 2, respectively.
The average TDNI (Table 4.9; Fig 4.8) in the first fortnight (from day -30th to
-15th days pre parturient) was 5.90 and 5.67 (kg/d) and in the second fortnight (-15th to
the day of parturition) it was 5.14 and 4.66 (kg/d) in group 1 and 2, respectively. TDN
intake/100 kg body weight (Table 4.10) in first and second fortnight was 1.14 and
1.01 kg/d in group 1 and 1.14 and 0.97 kg/d in group 2, respectively. There was no
difference (P>0.05) among two groups in average TDN/ 100 kg body weight before
parturition.
The average ME Intake (Table 4.9) in the first fortnight (from day -30th to 15th days pre parturient) was 21.78 and 21.28 (Mcal/d) and in the second fortnight (15th to the day of parturition), it was 19.78 and 18.50 (Mcal/d) in group 1 and 2,
respectively. ME intake/ 100 kg body weight (Table 4.10) in first and second fortnight
was 4.28 and 3.83 Mcal/d in group 1 and 4.28 and 3.67 Mcal/d in group 2
respectively. There was no difference (P>0.05) among two groups in average
prepartum ME intake/ 100 kg body weight.
The average NE L Intake (Table 4.9) in the first fortnight (from day -30th to 15th days pre parturient) was 13.59 and 13.33 (Mcal/d) and in the second fortnight (15th to the day of parturition) it was 12.36 and 11.60 (Mcal/d) in group 1 and 2,
respectively. NE L intake/ 100 kg body weight (Table 4.10) in first and second
fortnight was 2.67 and 2.40 Mcal/d in group 1 and 2.68 and 2.30 Mcal/d in group 2,
respectively. There was no difference (P>0.05) among two groups in average
prepartum NE L intake/ 100 kg body weight.
The nutrient intake was within the normal range as per NRC (2001) and no
difference was observed between the two groups.
The average duodenal methionine flow (Table 4.9) in the first fortnight (from
day -30th to -15th days pre parturient) was 1.90 and 2.22 (% of MP/d) and in the
second fortnight (-15th to the day of parturition) was 1.97 and 2.30 (kg/d) in group 1
and 2, respectively. The average duodenal lysine flow (Table 4.9) in the first fortnight
(from day -30th to -15th days pre parturient) was 6.44 and 7.09 (kg/d) and in the
_________________________________________________________________ 85
Results and Discussion …
second fortnight (-15th to the day of parturition) was 6.57 and 7.35(kg/d) in group 1
and 2, respectively. Duodenal methionine and lysine flow in group 2 was higher
(P<0.01) than group 1 due to supplementation of RPM and RPL.
Table 4.10 Fortnightly average nutrient Intake (kg/100 kg body weight) in
crossbred cows fed ration with or without RPM plus RPL
Parameter
DM (kg/100 kg body weight)
TDN (kg/100 kg body weight)
CP (kg/100 kg body weight)
ME (Mcal/100 kg body weight)
NE L (Mcal/100 kg body
weight)
Fortnight
Group 1
Group 2
-2
1.83 ± 0.05
1.78 ± 0.06
-1
1.59 ± 0.08
1.53 ± 0.03
-2
1.14 ± 0.03
1.14 ± 0.04
-1
1.01 ± 0.04
0.97 ± 0.05
-2
0.23 ± 0.01
0.23 ± 0.01
-1
0.21 ± 0.01
0.21 ± 0.01
-2
4.28 ± 0.09
4.28 ± 0.15
-1
3.83 ± 0.14
3.67 ± 0.19
-2
2.67 ± 0.06
2.68 ± 0.09
-1
2.40 ± 0.08
2.30 ± 0.12
The diets formulated in this experiment was designed to cover energy and
protein requirements of dairy cows as per NRC (2001) except for Met and Lys as a
percentage of Metabolizable protein. Based on calculations made as suggested by
NRC (2001), it was estimated that Met represented 1.90 % of MP in the small
intestine in control group, which represented 79.2 % of the recommended level
(2.30% of MP) and Lysine supply was 6.44 % of MP which was lower than the
recommended level (7.2 % of MP) by 10.55 %. The addition of RPM (5 g/d) and Lys
(20 gm) fulfilled the requirement of methionine and lysine as per NRC (2001).
The results of the present study indicated that there was no effect on DMI in
RPM plus RPL supplemented group in the period before calving in cows. These
_________________________________________________________________ 86
Results and Discussion …
results are in agreement with those of Socha et al. (2005) who reported there was no
effect of pre partum rumen protected methionine supplementation on DMI of cows.
In most of the cases, e.g. Christensen et al., 1994; Piepenbrink et al., 1998;
Polan et al., 1991, DMI was depressed by feeding RPM, but this depression was
reversed when RPL was also supplemented. In contrast, RPL supplementation
depressed DMI by 0.68 kg/d on being fed @ 16 g of lysine/day (Watanabe et al.,
2006). Reductions in DMI might also be attributed to lower microbial activity, and
therefore digestion in the rumen. However, in the present study analysis of the RPM
and RPL products didn’t reveal any contaminants that could affect microbial growth.
4.1.8
Overall Plane of Nutrition in prepartum cows
The overall plane of nutrition of prepartum cows is presented in Table 4.11.
The actual nutrient intake was compared with nutrient requirement as per NRC
(2001). DM intake was undersupplied by 10.51 percent and 13.44 percent than
requirements in group 1 and 2, respectively. The CPI was 7.62 percent higher than
requirement in group 1 and 3.81 percent higher in case of group 2. TDN intake was
12.65 percent and 5.51 percent higher than requirements in group 1 and 2,
respectively whereas NE L intake was 13.77 percent and 11.25 percent higher in group
1 and group 2, respectively. The plane of nutrition of cows of both the groups
indicated that both the groups were adequately fed, thereby fulfilling the requirements
as per NRC (2001).
4.1.9
Effect of RPM and RPL Supplementation on Certain Blood Parameters
a) Blood glucose concentration
The blood glucose concentration (Table 4.12) on -30th day of parturition was
57.19 and 56.06 (mg/dL) and on -15th day of parturition, it was 56.55 and 54.14
(mg/dL) in group 1 and 2, respectively while it was decreased (P<0.05) at the day of
parturition. The blood glucose concentration remained within the normal range and no
difference was observed at any stage of experiment. The reason may be a high
metabolic rate of utilization of glucose and homeostatic mechanism of animal body
that does not allow appreciable change in glucose level.
_________________________________________________________________ 87
Results and Discussion …
In contrast, Socha et al. (2005) reported decrease in blood glucose level on
supplementing rumen protected methionine and lysine to prepartum cows.
Table 4.11 Plane of nutrition of prepartum cows fed ration with or without
rumen protected methinone plus lysine
Parameter
DM
CP
TDN
NE L
Group 1
Group 2
Intake (kg/d)
8.77
8.31
Requirement * (kg/d)
9.80
9.60
Deficit in DMI %
10.51
13.44
Intake (kg/d)
1.13
1.09
Requirement * (kg/d)
1.05
1.05
Extra CP intake (%)
7.62
3.81
Intake (kg/d)
5.52
5.17
Requirement * (kg/d)
4.90
4.90
Extra TDN intake (%)
12.65
5.51
Intake (Mcal/d)
12.97
12.46
Requirement * (Mcal/d)
11.4
11.2
Extra NE L intake (%)
13.77
11.25
* Requirements as per NRC (2001)
b) Plasma phoshatidylcholine levels
The blood phosphatidylcholine concentration (Table 4.12) on -30th day of
parturition was 152.10 and 149.39 (µg/ml) and on -15th day of parturition, it was
122.35 and 133.26 (µg/ml) in group 1 and 2, respectively. There was increase
(P<0.05) in plasma phosphatidylcholine levels on the day of parturition on
supplemention
of
rumen
protected
methionine
and
lysine.
The
plasma
phosphatidylcholine concentration remained within the normal range and no
difference was observed on -30th and -15th of parturition.
_________________________________________________________________ 88
Results and Discussion …
The increase in plasma phosphatidylcholine on the day of parturition may be
due to utilization of the absorbed methionine for de nova synthesis of choline
(Emmanuel and Kennely, 1984).
C) Plasma NEFA level
The plasma NEFA concentration (Table 4.12) on -30th day of parturition was
69.53 and 67.63 (mg/L) and on -15th day of parturition, it was 91.89 and 77.58
(mg/L) in group 1 and 2, respectively. While the plasma NEFA concentration on the
day of parturition was 115.94 and 112.14 in group1 and group 2, respectively. The
plasma NEFA concentration remained within the normal range and no difference was
observed in both the groups at any stage of experiment. These results are in agreement
with that of Socha et al. (2005). NEFA concentration increased in both the groups as
the pregnancy advanced.
Higher plasma NEFA concentration observed in multifarious cows towards
parturition suggested that they were mobilizing more stored energy to support
increasing demand of growing conceptus.
Table 4.12 Average Plasma metabolites (Prepartum ) of crossbred cows fed
ration with or without RPM plus RPL
Parameter
Day
Group 1
Group 2
Glucose, mg/dL
-30
57.19 ± 1.20
56.06 ± 0.86
-15
56.55 ± 0.85
54.14 ± 0.82
1
46.02 ± 1.36
45.86 ± 0.53
Phosphatidylcholine
-30
152.10 ± 5.49
149.39 ± 5.80
, µg/ml
-15
122.35 ± 2.54
133.26 ± 7.26
1
95.58 a ± 2.31
109.00 b ± 5.57
-30
69.53 ± 1.14
67.63 ± 1.05
-15
91.89 ± 1.32
77.58 ± 2.89
1
115.94 ± 1.74
112.14 ± 2.55
NEFA, mg/L
_________________________________________________________________ 89
Results and Discussion …
Triglycerides, mg/dL
VLDL, mg/dL
Vitamin E, µg/ml
Cholesterol, mg/dL
BUN, mg/L
a, b
-30
17.30 ± 0.84
18.66 ± 0.31
-15
16.27a ± 0.58
18.24b ± 0.23
1
16.12a ± 0.67
17.89b ± 0.43
-30
3.46 ± 0.17
3.73 ± 0.05
-15
3.25a ± 0.12
3.65b ± 0.05
1
3.22a ± 0.13
3.58b ± 0.09
-30
1.07 ± 0.11
1.03 ± 0.03
-15
0.81 ± 0.07
0.79 ± 0.04
1
0.68 ± 0.03
0.66 ± 0.03
-30
166.80 ± 6.13
167.37 ± 5.66
-15
167.25 ± 5.90
172.74 ± 4.57
1
171.33 ± 5.53
173.88 ± 5.97
-30
14.08 ± 0.20
13.38 ± 0.42
-15
19.39 ± 0.87
19.03 ± 0.55
1
20.34 ± 0.90
20.75 ± 0.34
Means having different superscripts in a row differ significantly (P<0.05)
D) Plasma Triglycerides levels
The plasma triglycerides concentration (Table 4.12) on -30th day of parturition
was 17.30 and 18.66 (mg/dL) and on -15th day of parturition, it was 16.27 and 18.24
(mg/dL) in group 1 and 2, respectively. There was increase (P<0.05) in plasma
triglycerides on the -15th day of parturition and on the day of parturition in RPM plus
RPL supplemented group.
In contrast, Fahey et al. (2002) and Delbecchi et al. (2001) reported no
difference in plasma triglyceride concentration in Holstein cows fed TMR
supplemented with 4.8% canola meal or 4.8% formaldehyde protected canola seeds.
_________________________________________________________________ 90
Results and Discussion …
E) Plasma VLDL levels
The plasma VLDL concentration (Table 4.12) on -30th day of parturition was
3.46 and 3.73 (mg/dL) and on -15th day of parturition, it was 3.25 and 3.65 (mg/dL) in
group 1 and 2, respectively. There was increase (P<0.05) in plasma VLDL on the 15th day of parturition and on the day of parturition in RPM plus RPL supplemented
group.
F) Plasma vitamin E levels
The plasma vitamin E concentration (Table 4.12) on -30th day of parturition
was 1.07 and 1.03 (µg/ml) and on -15th day of parturition, it was 0.81 and 0.79
(µg/ml) in group 1 and 2, respectively. The plasma vitamin E concentration remained
within the normal range and no difference was observed at any stage of experiment.
G) Plasma cholesterol levels
The plasma cholesterol concentration (Table 4.12) on -30th day of parturition
was 166.80 and 167. 37 (µg/ml) and on -15th day of parturition, it was 167.25 and
172.74 (µg/ml) in group 1 and 2, respectively. The plasma cholesterol concentration
remained within the normal range and no difference was observed at any stage of
experiment.
Higher cholesterol concentration is associated with better reproductive
performance in high yielding dairy cows, as it acts as a precursor of steroid hormones
(Son et al. 1996). However in the present experiment, higher cholesterol
concentrations were not reported between two groups.
H) Blood urea nitrogen levels
The blood urea nitrogen concentration (Table 4.12) on -30th day of parturition
was 14.08 and 13.38 (mg/dL) and on -15th day of parturition, it was 19.39 and 19.03
(mg/dL) in group 1 and 2, respectively. The concentrations of plasma urea N did not
differ in response to dietary treatment, suggesting that overall N utilization was
similar in both the groups. But there was increasing trend in plasma BUN
concentration as the cows progressed towards parturition. It may be due to the
decreased dry matter intake.
_________________________________________________________________ 91
Results and Discussion …
Feeding of rumen protected methionine and lysine not only results in more
supply of methionine and lysine, but also saves energy wasted in urea synthesis due to
optimum use of amino acids. Blood urea concentration is an indicator of efficient
protein balance (Campanile et al., 1998; Dhali et al., 2006) and is typically increased
in cows deficient in energy.
Similar results were reported by Tiwari and Yadava, (1994) on feeding
formaldehyde treated mustard cake to buffalo calves and Sahoo and Walli, (2005) to
lactating goats. Similarly, non significant difference in BUN on supplementing rumen
protected methionine and lysine was reported by Socha et al. (2005) in prepartum
cows.
I)
Plasma amino acid profile
Prepartum fortnightly plasma amino acids viz. aspartate, glutamate, serine,
glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine,
cysteine, leucine, isoleucine, Phenylalanine and lysine concentrations are presented
in Table 4.13. The plasma concentration of different amino acids on -15 day of
parturition and on the day of parturition were 20.28 and 21.97 µmol/dl in group 1 and
21.99 and 22.91 µmol/dl in group 2 (aspartate), 24.12 and 23.58 µmol/dl in group 1
and 23.85 and 24.48 µmol/dl in group 2 (glutamate), 13.37 and 12.59 µmol/dl in
group 1 and 13.38 and 13,72 µmol/dl in group 2 (serine), 19.46 and 18.68 µmol/dl in
group 1 and 20.13 and 20.72 in group 2 (glycine), 4.95 and 4.66 µmol/dl in group 1
and 4.41 and 4.31 µmol/dl in group 2 (histidine), 7.84 and 7.63 µmol/dl in group 1
and 8.01 and 8.29 µmol/dl in group 2 (arginine), 12.26 and 11.84 µmol/dl in group 1
and 12.45 and 12.50 µmol/dl in group 2 (threonine), 23.14 and 22.82 µmol/dl in
group 1 and 22.83 and 24.03 µmol/dl in group 2 (alanine), 8.85 and 9.14 µmol/dl in
group 1 and 8.81 and 9.04 µmol/dl in group 2 (proline), 7.01 and 7.49 µmol/dl in
group 1 and 7.17 and 7.58 µmol/dl in group 2 (tyrosine), 15.66 and 15.21 µmol/dl in
group 1 and 16.32 and 16.20 µmol/dl in group 2 (valine), 5.54 and 5.39 µmol/dl in
group 1 and 5.95 and 5.93 µmol/dl in group 2 (methionine), 1.68 and 161 µmol/dl in
group 1 and 2.43 and 2.11 µmol/dl in group 2 (cysteine), 11.24 and 10.79 µmol/dl in
group 1 and 9.44 and 9.54 µmol/dl in group 2 (isoleucine), 15.19 and 15.11 µmol/dl
in group 1 and 15.36 and 16.18 in group 2 (leucine), 7.74 and 8.26 µmol/dl in group
_________________________________________________________________ 92
Results and Discussion …
1 and 7.92 and 9.40 µmol/dl in group 2 (phenylalanine) and 13.26 and 13.28 µmol/dl
in group 1 and 14.49 and 14.25 µmol/dl in group 2 (lysine), respectively.
Table 4.13 Average Plasma Amino Acid Profile (µmol/dl) of crossbred cows
(prepartum) fed ration with or without RPM plus RPL
Amino Acid
Day
Group 1
Group 2
Aspartate
-30
21.24 ± 1.56
21.22 ± 2.71
-15
20.28 ± 1.17
21.99 ± 1.33
1
21.97 ± 1.11
22.91 ± 0.76
-30
25.81 ± 1.94
21.52 ± 2.78
-15
24.12 ± 1.50
23.85 ± 0.87
1
23.58 ± 1.18
24.48 ± 1.00
-30
13.10 ± 0.93
13.05 ± 0.42
-15
13.37 ± 0.88
13.38 ± 0.32
1
12.59 ± 0.69
13.72 ± 0.35
-30
19.31 ± 1.05
19.66 ± 0.56
-15
19.46 ± 1.16
20.13 ± 0.51
1
18.68 ± 0.98
20.72 ± 0.56
-30
4.91 ± 0.56
4.59 ± 0.30
-15
4.95 ± 0.55
4.41 ± 0.38
1
4.66 ± 0.49
4.31 ± 0.43
-30
7.69 ± 0.68
8.02 ± 0.45
-15
7.84 ± 0.52
8.01 ± 0.24
1
7.63 ± 0.56
8.29 ± 0.21
-30
11.84 ± 0.91
11.14 ± 0.92
Glutamate
Serine
Glycine
Histidine
Arginine
Threonine
_________________________________________________________________ 93
Results and Discussion …
Alanine
Proline
Tyrosine
Valine
Methionine
Cysteine
Isoleucine
-15
12.26 ± 0.71
12.45 ± 0.70
1
11.84 ± 0.61
12.50 ± 0.85
-30
22.85 ± 1.77
23.03 ± 1.18
-15
23.14 ± 1.88
22.83 ± 1.00
1
22.82 ± 1.79
24.03 ± 0.62
-30
9.15 ± 0.73
8.28 ± 0.38
-15
8.85 ± 0.45
8.81 ± 0.37
1
9.14 ± 0.59
9.04 ± 0.38
-30
6.90 ± 0.61
6.86 ± 0.19
-15
7.01 ± 0.51
7.17 ± 0.36
1
7.49 ± 0.66
7.58 ± 0.41
-30
14.93 ± 1.15
14.46 ± 0.91
-15
15.66 ± 1.11
16.32 ± 0.41
1
15.21 ± 0.91
16.20 ± 0.53
-30
5.34 ± 0.54
5.63 ± 0.11
-15
5.55 ± 0.50
5.95 ± 0.20
1
5.39 ± 0.32
5.93 ± 0.27
-30
1.57 ± 0.43
1.66 ± 2.32
-15
1.68 ± 0.42
2.43 ± 0.40
1
1.61 ± 0.45
2.11 ± 0.29
-30
11.15 ± 0.94
8.24 ± 1.17
-15
11.24 ± 1.08
9.44 ± 0.69
1
10.79 ± 0.83
9.54 ± 0.72
_________________________________________________________________ 94
Results and Discussion …
Leucine
Phenylalanine
Lysine
-30
14.67 ± 1.13
13.43 ± 1.95
-15
15.19 ± 1.25
15.36 ± 0.84
1
15.11 ± 1.08
16.18 ± 0.61
-30
8.04 ± 0.78
7.11 ± 0.94
-15
7.74 ± 0.63
7.92 ± 0.42
1
8.26 ± 0.62
9.40 ± 0.87
-30
12.97 ± 1.06
13.09 ± 1.94
-15
13.36 ± 1.18
14.49 ± 0.73
1
13.28 ± 0.86
14.25 ± 0.84
There were no effect of supplementation of rumen protected methionine and
lysine on prepartum plasma amino acid profile of cows while increasing trends had
been observed in plasma methionine, cysteine and lysine concentration in cows
supplemented with RPM plus RPL.
4.2
EFFECT OF SUPPLEMENTING RPM PLUS RPL DURING POST
PARTURIENT PERIOD
4.2.1
Body weight changes
The initial body weights of animals at day one after parturition were 459.2 and
442.7 kg in group 1 and 2, respectively and after 120 days of feeding, final body
weight were 440.6 and 448.4 kg (Table 4.14, Fig 4.3). There was a declining trend in
body weights of cows of both the groups due to mobilization of body reserves to
support lactation. This trend was evident up to fourth fortnight in both the groups
(Table 4.15). Perusal of the fortnightly changes in body weight revealed that there
was overall a net loss of 18.59 kg in group 1, whereas there was overall gain of 5.79
kg in group 2. There was a net loss of 15.5 kg in group 1 at first fortnight, whereas it
was 10.6 kg in group 2 owing to decrease in DMI during first fortnight and
mobilization of body reserves to support lactation. In first fortnight, there was
decrease in DMI owing to calving stress.
_________________________________________________________________ 95
Results and Discussion …
Table 4.14 Fortnightly average body weights (kg) of lactating crossbred cows fed
ration with or without RPM plus RPL during postpartum period
Fortnight
Group 1
Group 2
1 day*
459.2 ± 20.3
442.7 ± 11.5
1
443.7 ± 20.8
432.0 ± 12.2
2
431.5 ± 20.8
423.8 ± 12.6
3
420.4 ± 20.2
416.8 ± 12.7
4
418.6 ± 21.3
415.1 ± 12.7
5
422.7 ± 21.7
423.7 ± 13.2
6
427.9 ± 21.2
432.8 ± 13.1
7
434.2 ± 20.9
441.0 ± 12.7
8
440.6 ± 20.6
448.4 ± 12.4
Group 1: Cows fed control diet
Group 2: Cows fed control diet + RPM 7 gm (Effective Methionine was 2.77 g ) & RPL 60
gm (Effective lysine was 13.26 g)
* : Day of parturition
Perusal of body weight changes as per cent change in body weight (Table
4.16, Fig. 4.5) revealed that there was overall a net loss of 4.07 percent in group 1,
whereas there was overall net gain of 1.32 per cent in group 2. The impact of negative
energy balance appeared to be less severe in group 2 as the loss in weight was 2.4 kg
in group 2 as compared to 3.5 kg loss in group 1 in the first fortnight. Cows in group 1
and group 2 attained positive energy balance from fourth fortnight onwards as was
evident from increasing trend in body weights.
Supplementation of RPM and RPL to group 2 cows was effective in reducing
the extent and duration of body weight loss as compared to group 1 cows. Socha et al.
(2005) reported higher post partum body weight and body condition scores of cows
supplemented with rumen protected methionine and lysine.
_________________________________________________________________ 96
Fig 4.3 Average fortnightly body weights (kg) of
crossbred cows
Fig 4.4 Average fortnightly percent change in body weight (kg)
of crossbred cows
Results and Discussion …
Table 4.15 Fortnightly change in body weight (kg) of lactating crossbred cows
fed ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
-15.5a ± 1.3
-10.6b ± 1.4
2
-12.1a ± 1.8
-8.2b ± 1.0
3
-11.1a ± 2.6
-7.0b ± 1.6
4
-1.9 ± 3.0
-1.8 ± 2.2
5
4.1a ± 1.4
8.7b ± 1.7
6
5.2a ± 1.2
9.0b ± 1.1
7
6.4a ± 1.8
8.3b ± 1.6
8
6.4a ± 1.5
7.4b ± 1.4
Overall change
-18.59a ± 6.32
+ 5.79b ± 5.11
Means having different superscripts in a row differ significantly (P<0.01)
Table 4.16 Percent change in body weights of lactating crossbred cows fed ration
with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
-3.5a ± 0.4
-2.4b ± 0.4
2
-2.8a ± 0.5
-1.9b ± 0.3
3
-2.5a ± 0.6
-1.7b ± 0.4
4
-0.5 ± 0.7
-0.4 ± 0.6
5
1.0a ± 0.3
2.1b ± 0.4
6
1.3a ± 0.4
2.2b ± 0.3
7
1.6a ± 0.5
2.0b ± 0.4
8
1.5 ± 0.4
1.7 ± 0.3
Overall change
-4.07a ± 1.30
+ 1.32b ± 1.16
Means having different superscripts in a row differ significantly (P<0.01)
_________________________________________________________________ 97
Results and Discussion …
However, many researchers found that on supplementation of rumen protected
methionine alone, there was no effect on postpartum body weight change. (Noftsger
and St-Pierre, 2003, Berthiaume et al., 2001, Girard et al., 2005, Noftsger et al., 2005,
Benefield et al., 2009 and Davidson et al., 2008).
4.2.2
Body Condition Score
Body condition score (BCS) is a tool to adjust feeding and management
practices in order to maximize the potential for milk production and minimize
reproductive disorders. In early lactation, dairy cows frequently produce far more
milk than can be supported by feed intake alone. They do this by drawing on body
reserves that were built up before calving. BCS influences productivity, reproduction,
health, and longevity of the dairy cattle and buffaloes.
Table 4.17 Fortnightly body condition score of lactating crossbred cows fed
ration with or without RPM plus RPL
a, b
Day
Group 1
Group 2
1
3.5a ± 0.11
3.7b ± 0.06
15
3.2a ± 0.12
3.3b ± 0.08
30
3.0 ± 0.09
3.1 ± 0.04
45
2.9 ± 0.07
3.0 ± 0.07
60
2.8 ± 0.08
2.9 ± 0.07
75
2.7a ± 0.07
2.9a ± 0.06
90
2.7a ± 0.07
2.9a ± 0.06
105
2.8 ± 0.06
2.9 ± 0.04
120
3.0 ± 0.03
3.1 ± 0.04
Overall Mean
3.0a ± 0.08
3.1b ± 0.06
Means having different superscripts in a row differ significantly (P<0.01)
Thinness or fatness can be a clue to underlying nutritional deficiencies, health
problems, or improper herd management. Thus, BCS of the lactating cows on a
_________________________________________________________________ 98
Fig 4.5 Fortnightly average body condition score of
crossbred cows
Fig 4.6 Fortnightly average dry matter intake (kg/d) of
crossbred cows
Results and Discussion …
routine basis is an excellent tool to help manage the herd more effectively and reduce
the incidence of metabolic disorders during early lactation.
Body condition score reflected changes in the body weight in both
supplemented and control groups (Table 4.17; Fig 4.5). The BCS after parturition was
3.47 and 3.69 in group 1 and 2, respectively. The body condition score during post
parturient period ranged from 2.72 to 3.16 in group 1, and from 2.92 to 3.33 in group
2 during different fortnights. In first fortnight, BCS of group 1 and 2 was 3.16 and
3.33, respectively. There was higher body weight change (Table 4.16, Fig 4.4) during
first fortnight which reflected in BCS of this fortnight. Overall average BCS of
different fortnights was 2.95 and 3.09 in group 1 and 2, respectively, which was
slightly higher in group 2 due to higher energy intake by this group that led to less
negative energy balance. The results of present study indicated that supplementation
of rumen protected methionine and lysine during early lactation lead to less
mobilization of body fat to support milk production.
4.2.3
Dry Matter Intake
The DMI (Table 4.18, Fig 4.6) ranged from 11.24 to 13.59 kg/d in group 1 and
11.26 to 14.39 kg/d in group 2 during different fortnights. In 2nd, 7th and 8th fortnight,
the DMI per day was higher in group 2 than that of group 1. The overall mean DMI
was 13.01 kg and 13.36 kg/d in group 1 and 2, respectively, and higher (P<0.05) DMI
was recorded in group 2. The highest DMI was observed in group 2 during eighth
fortnight (14.39 kg/d) but in case of control group, it was 13.59 kg/d. It appears that
animals in group 2 were able to withstand the effect of post parturient stress in a better
way due to supply of rumen protected methionine and lysine. DMI/100 kg body
weight (Table 4.19) ranged from 2.48 to 3.23 kg/d in group 1 and 2.55 to 3.30 kg/d in
group 2. In all fortnights except 6th forthnight, the DMI per 100 kg body weight was
higher in group 2 than that of group 1. The overall average DMI was 3.03 and 3.13
kg/100 kg body weight in group 1 and 2, respectively. The overall mean DMI/100 kg
body weight was higher (P< 0.05) in group 2 than group 1, due to higher body weight
recorded in group 2 (Table 4.19).
_________________________________________________________________ 99
Results and Discussion …
Table 4.18 Fortnightly average dry matter intake (kg/d) in lactating crossbred
cows fed ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
11.24 ± 0.50
11.26 ± 0.43
2
12.67a ± 0.51
12.85b ± 0.44
3
12.88 ± 0.53
13.38 ± 0.50
4
13.16 ± 0.59
13.64 ± 0.48
5
13.43 ± 0.60
13.69 ± 0.44
6
13.58 ± 0.63
13.60 ± 0.56
7
13.49a ± 0.64
14.08b ± 0.56
8
13.59a ± 0.64
14.39a ± 0.51
Overall Mean
13.01a ± 0.28
13.36b ± 0.34
Means having different superscripts in a row differ significantly (P<0.01)
Table 4.19 Fortnightly average dry matter intake (kg/100 kg body weight) in
lactating crossbred cows fed ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
2.48a ± 0.14
2.55b ± 0.08
2
2.86 a ± 0.11
2.98b ± 0.10
3
3.01a ± 0.12
3.16b ± 0.08
4
3.15a ± 0.12
3.27b ± 0.06
5
3.22a ± 0.07
3.30b ± 0.05
6
3.23 ± 0.09
3.24 ± 0.07
7
3.17a ± 0.09
3.28b ± 0.07
8
3.14a ± 0.08
3.30b ± 0.04
Overall Mean
3.03a ± 0.09
3.13b ± 0.09
Means having different superscripts in a row differ significantly (P<0.01)
_________________________________________________________________ 100
Results and Discussion …
The results of the present study are in agreement with Benefield et al. (2009)
and Broderick et al. (2009) who found that there was increase in DMI on
supplementation of rumen protected methionine.
In contrast, Girard et al. (2005) supplemented rumen protected methionine (18
g/head/day) to lactating dairy cows and observed no effect on DMI. Similarly,
Berthiaume et al. (2001) observed non-significant effect on DMI on supplementing
rumen protected methionine to dairy cows. Similarly, Bach et al. (2000), Noftsger and
St-Pierre (2003), Davidson et al. (2008) and Swanepoel et al. (2011) reported that
there was no effect on DMI on supplementing rumen protected methionine and lysine
to lactating cows. Socha et al. (2008) observed no effect of abomasal infusion of
methionine and lysine to lactating cows on DMI in peak and mid lactation while in
there was increase in DMI in early lactation. Lara et al. (2006) reported no treatment
effects of supplemented ruminally protected methionine on DM intake (20.38 kg d-1),
body weight (599.78 kg), and body condition score (2.51 units).
The results of the present study indicated that there was significant increase in
DMI in lactating cows on rumen protected methionine and lysine supplementation.
4.2.4
Nutrients Intake
4.2.4.1 CP intake
The CP intake ranged from 1.86 to 2.20 kg/d in group 1 and 1.96 to 2.26 kg/d
in group 2 (Table 4.20, Fig 4.7). In 1st, 3rd and 8th fortnight, CP intake was higher
(P<0.05) in group 2 than that of group 1. The overall average mean CPI was 2.11 and
2.17 kg/d in the group 1 and group 2, respectively, which was higher (P< 0.01) in
group 2 than that of group 1, due to higher intake of DMI. The lowest CPI within the
group was observed in first fortnight in both the groups which may be due to reduced
DMI in the first fortnight. Average CP intake on percent body weight basis (Table
4.21) ranged from 0.41 kg to 0.52 kg/d in group 1 and 0.44 to 0.53 kg/d in group 2.
Fortnightly average CP intake per 100 kg body weight was higher in group 2 than that
of group 1 except 6th and 7th fortnight.
_________________________________________________________________ 101
Results and Discussion …
Table 4.20 Fortnightly CP intake (kg/d) in lactating crossbred cows fed ration
with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
1.86a ± 0.08
1.96b ± 0.06
2
2.06 ± 0.11
2.12 ± 0.07
3
2.10a ± 0.08
2.24a ± 0.07
4
2.16 ± 0.10
2.19 ± 0.07
5
2.18 ± 0.10
2.20 ± 0.08
6
2.20 ± 0.11
2.17 ± 0.09
7
2.16 ± 0.11
2.23 ± 0.08
8
2.16a ± 0.11
2.26a ± 0.07
Overall Mean
2.11a ± 0.10
2.17b ± 0.07
Means having different superscripts in a row differ significantly (P<0.01)
Table 4.21 Fortnightly CP intake (kg/100 kg body weight) in lactating crossbred
cows fed ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
0.41a ± 0.02
0.44b ± 0.01
2
0.47a ± 0.02
0.49b ± 0.01
3
0.49a ± 0.02
0.53b ± 0.02
4
0.51a ± 0.01
0.53b ± 0.01
5
0.52a ± 0.01
0.53b ± 0.01
6
0.52 ± 0.01
0.52 ± 0.01
7
0.51 ± 0.01
0.52 ± 0.01
8
0.50a ± 0.01
0.52b ± 0.01
Overall Mean
0.49a ± 0.01
0.51b ± 0.01
Means having different superscripts in a row differ significantly (P<0.01)
_________________________________________________________________ 102
Fig 4.7 Fortnightly average crude protein intake (kg/d) of
crossbred cows
Fig 4.8 Fortnightly average TDN intake (kg/d) of crossbred
cows
Results and Discussion …
The overall average CP intake/ 100kg BW during the experimental period was 0.49
and 0.51 kg/d in group 1 and group 2, respectively, which was higher (P<0.05) in
group 2 than that of group 1. The faster regain in body weight of the cows in group 2
and higher DMI might be responsible for the higher CP intake observed in group 2.
4.2.4.2 RUP intake
The RUP intake (Table 4.22) ranged from 0.61 to 0.76 kg/d in group 1 and
0.64 to 0.80 kg/d in group 2 during different fortnights. In 1st, 3rd, 7th and 8th fortnight,
RUP intake was higher (P<0.05) in group 2 than that of group 1. The overall average
mean RUP intake was 0.72 and 0.74 kg/d in the group 1 and group 2, respectively,
which was higher (P< 0.05) in group 2 than that of group 1, due to higher intake of
DMI and CPI. The lowest RUP intake within the group was observed in first fortnight
in both the groups which may be due to reduced DMI in the first fortnight.
Table 4.22 Fortnightly RUP intake (kg/d) in lactating crossbred cows fed ration
with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
0.61a ± 0.03
0.64b ±0.02
2
0.68 ± 0.04
0.68 ± 0.03
3
0.71a ± 0.03
0.77b ± 0.03
4
0.73 ± 0.03
0.75 ± 0.03
5
0.75 ± 0.03
0.76 ± 0.03
6
0.76 ± 0.04
0.75 ± 0.03
7
0.74a ± 0.04
0.78b ± 0.03
8
0.74a ± 0.04
0.80b ± 0.03
Overall Mean
0.72a ± 0.04
0.74b ± 0.03
Means having different superscripts in a row differ significantly (P<0.05)
_________________________________________________________________ 103
Results and Discussion …
4.2.4.3 RDP intake
The RDP intake (Table 4.23) ranged from 1.25 to 1.44 kg/d in group 1 and
1.32 to 1.47 kg/d in group 2 during different fortnights. In 1st, 3rd and 8th fortnight,
RDP intake was higher (P<0.05) in group 2 than that of group 1. The overall average
mean RDP intake was 1.40 and 1.43 kg/d in the group 1 and group 2, respectively,
which was higher (P< 0.05) in group 2 than that of group 1, due to higher intake of
DMI and CPI. The lowest RDP intake within the group was observed in first fortnight
in both the groups which may be due to reduced DMI in the first fortnight.
Table 4.23 Fortnightly RDP intake (kg/d) in lactating crossbred cows fed ration
with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
1.25a ± 0.06
1.32b ± 0.04
2
1.37 ± 0.07
1.41 ± 0.04
3
1.41a ± 0.07
1.47b ± 0.04
4
1.43 ± 0.07
1.44 ± 0.05
5
1.44 ± 0.07
1.44 ± 0.05
6
1.44 ± 0.07
1.42 ± 0.05
7
1.42 ± 0.07
1.45 ± 0.05
8
1.42a ± 0.07
1.47b ± 0.05
Overall Mean
1.40a ± 0.07
1.43b ± 0.05
Means having different superscripts in a row differ significantly (P<0.05)
4.2.4.4 MP intake
The MP intake (Table 4.24) ranged from 1.15 to 1.37 kg/d in group 1 and 1.19
to 1.44 kg/d in group 2 during different fortnights. The MP intake was higher in group
2 than that of group 1 in 3rd, 7th and 8th fortnights. The overall average mean MP
intake was 1.31 and 1.35 kg/d in the group 1 and group 2, respectively, which was
higher (P< 0.01) in group 2 than that of group 1, due to higher intake of DMI and CPI.
_________________________________________________________________ 104
Results and Discussion …
The lowest MP intake within the group was observed in first fortnight in both the
groups which may be due to reduced DMI in the first fortnight.
Table 4.24 Fortnightly metabolizable protein intake (kg/d) in lactating crossbred
cows fed ration with or without RPM plus RPL supplementation
a, b
Fortnight
Group 1
Group 2
1
1.15 ± 0.05
1.19 ± 0.04
2
1.26 ± 0.06
1.29 ± 0.05
3
1.30a ± 0.05
1.40b ± 0.05
4
1.33 ± 0.06
1.36 ± 0.05
5
1.35 ± 0.06
1.38 ± 0.05
6
1.37 ± 0.07
1.36 ± 0.09
7
1.36a ± 0.07
1.41b ± 0.05
8
1.36a ± 0.07
1.44b ± 0.05
Overall Mean
1.31a ± 0.06
1.35b ± 0.05
Means having different superscripts in a row differ significantly (P<0.01)
4.2.4.5 Duodenal Methionine supply (% of metabolizable protein)
The fortnightly duodenal methionine supply (Table 4.25) ranged from 1.78 to
1.90 % of metabolizable protein, in group 1 and 2.26 to 2.33 % of metabolizable
protein, in group 2 during different fortnights. The overall mean duodenal methionine
supply was 1.86 % and 2.27 % of metabolizable protein per day in the group 1 and
group 2, respectively, which was higher (P< 0.01) in group 2 than that of group 1, due
to supplementation of rumen protected methionine in group 2. (Table 4.25)
Estimation of duodenal amino acid flow suggests that lysine was the first
limiting amino acid and methionine the second, however after addition of RPL,
methionine may be the first one. In the experiment conducted by Wu et al. (1997),
increased lysine supplied to 15.2% of essential amino acids improved performance in
cows during the first stage of lactation. Schwab (1996) and Schwab et al. (1992a,b)
_________________________________________________________________ 105
Results and Discussion …
estimated the requirements as a percentage of essential amino acids, at 5.3%
methionine and 15.2% lysine during the first third of lactation.
Table 4.25 Fortnightly duodenal methionine supply (% of metabolizable protein)
in lactating crossbred cows fed ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
1.90a ± 0.01
2.33b ± 0.01
2
1.88 a ± 0.01
2.29 b ± 0.01
3
1.88 a ± 0.00
2.27 b ± 0.01
4
1.87 a ± 0.01
2.27 b ± 0.01
5
1.87 a ± 0.00
2.27 b ± 0.01
6
1.87 a ± 0.00
2.28 b ± 0.01
7
1.87 a ± 0.09
2.26 b ± 0.01
8
1.87 a ± 0.01
2.26 b ± 0.01
Overall Mean
1.86 a ± 0.02
2.27 b ± 0.01
Means having different superscripts in a row differ significantly (P<0.01)
4.2.4.6 Duodenal lysine supply (% of metabolizable protein)
The fortnightly duodenal lysine supply (Table 4.26) ranged from 6.11 to 6.27
% of metabolizable protein (MP), in group 1 and 7.04 to 7.33 % of MP in group 2
during different fortnights. The overall mean duodenal lysine supply was 6.15 and
7.12 % of metabolizable protein per day in the group 1 and group 2, respectively,
which was higher (P< 0.01) in group 2 than that of group 1, due to supplementation of
rumen protected lysine in group 2. (Table 4.26)
_________________________________________________________________ 106
Results and Discussion …
Table 4.26 Fortnightly duodenal lysine supply (% of metabolizable protein) in
lactating crossbred cows fed ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
6.27a ± 0.03
7.33b ± 0.05
2
6.19 a ± 0.03
7.18 b ± 0.05
3
6.14 a ± 0.02
7.06 b ± 0.06
4
6.12 a ± 0.02
7.10 b ±0.05
5
6.11 a ± 0.01
7.08 b ± 0.05
6
6.12 a ± 0.02
7.11 b ± 0.05
7
6.14 a ± 0.02
7.06 b ± 0.05
8
6.14 a ± 0.02
7.04 b ± 0.04
Overall Mean
6.15 a ± 0.02
7.12 b ± 0.05
Means having different superscripts in a row differ significantly (P<0.01)
4.2.4.7 TDN intake
TDN intake (Table 4.27, Fig. 4.8) ranged from 7.75 to 9.18 kg/d in group 1
and 7.97 to 9.70 kg/d in group 2. In 3rd, 7th and 8th fortnight, TDN intake was higher
(P<0.05) in group 2 than that of group 1. Overall average TDNI was 8.76 kg and 9.05
kg/d in group 1 and 2, respectively. The average TDNI was higher (P<0.05) by 3.31
per cent in group 2 over that of group 1. The TDN intake on percent body weight
basis (Table 4.29) ranged from 1.71 to 2.18 kg/d in group 1 and 1.80 to 2.22 kg/d in
group 2. The TDN intake per 100 kg body weight was higher (P<0.01) in group 2 than
that of group 1 in all the fortnights except in 6th fortnight. The overall average TDN
intake/ 100 kg BW (Table 4.28) was 2.04 kg/d in group 1 and 2.12 kg/d in group 2.
TDN intake/100 kg BW was higher (P<0.05) by 3.92 % in group 2 than that of group
1.
_________________________________________________________________ 107
Results and Discussion …
Table 4.27 Fortnightly TDNI (kg/d) in lactating crossbred cows fed ration with
or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
7.75 ± 0.29
7.97 ± 0.24
2
8.47 ± 0.41
8.61 ± 0.31
3
8.65a ± 0.36
9.30b ± 0.33
4
8.84 ± 0.40
9.08 ± 0.32
5
9.00 ± 0.42
9.21 ± 0.31
6
9.18 ± 0.45
9.09 ± 0.41
7
9.09a ± 0.45
9.48b ± 0.36
8
9.10a ± 0.46
9.70b ± 0.31
Overall Mean
8.76a ± 0.40
9.05b ± 0.32
Means having different superscripts in a row differ significantly (P<0.05)
Table 4.28 Fortnightly TDN intakes (kg/100 kg body weight) in lactating
crossbred cows fed ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
1.71a ± 0.08
1.80b ± 0.04
2
1.92a ± 0.08
2.00b ± 0.06
3
2.02a ± 0.08
2.20b ± 0.08
4
2.11a ± 0.06
2.18b ± 0.04
5
2.16a ± 0.05
2.22b ± 0.04
6
2.18 ± 0.06
2.16 ± 0.06
7
2.13a ± 0.07
2.21b ± 0.05
8
2.10a ± 0.06
2.22b ± =0.04
Overall Mean
2.04a ± 0.07
2.12b ± 0.05
Means having different superscripts in a row differ significantly (P<0.05)
_________________________________________________________________ 108
Results and Discussion …
4.2.4.8 ME intake
ME intake (Table 4.29) ranged from 28.98 to 33.71 in group 1 and 29.76 to
35.41 Mcal/d in group 2. In 3rd, 7th and 8th fortnight, ME intake was higher (P<0.05)
in group 2 than that of group 1.
Table 4.29 Fortnightly ME intake (Mcal/d) in lactating crossbred cows fed
ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
28.98 ± 1.06
29.76 ± 0.86
2
31.44 ± 1.47
31.88 ± 1.07
3
32.01a ± 1.27
34.21a ± 1.11
4
32.55 ± 1.39
33.40 ± 1.11
5
33.11 ± 1.50
33.76 ± 1.08
6
33.71 ± 1.58
33.38 ± 1.39
7
33.39a ± 1.58
34.66b ± 1.24
8
33.49a ± 1.65
35.41b ± 1.07
Overall Mean
32.33a ± 1.44
33.31b ± 1.12
Means having different superscripts in a row differ significantly (P<0.05)
Overall average ME intake was 32.33 and 33.31 Mcal/d in group 1 and 2,
respectively. The average ME intake was higher (P<0.05) by 3.03 per cent in group 2
over that of group 1. The ME intake on percent body weight basis (Table 4.30) ranged
from 6.37 to 8.01 Mcal in group 1 and 6.73 to 8.14 Mcal/d in group 2. The ME intake
per 100 kg body weight was higher (P<0.01) in group 2 than that of group 1 in all the
fortnights except in 6th fortnight. The overall average ME intake/ 100 kg BW was 7.53
Mcal/d in group 1 and 7.82 Mcal in group 2. ME intake/100 kg BW was higher
(P<0.01) in group 2 than that of group 1.
_________________________________________________________________ 109
Results and Discussion …
Table 4.30
Fortnightly ME intake (Mcal/100 kg body weight) in lactating
crossbred cows fed ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
6.37a ± 0.28
6.73b ± 0.13
2
7.13a ± 0.27
7.40b ± 0.22
3
7.48a ± 0.28
8.11b ± 0.28
4
7.78a ± 0.22
8.01b ± 0.12
5
7.94 ± 0.16
8.14 ± 0.13
6
8.01 ± 0.20
7.94 ± 0.20
7
7.83a ± 0.22
8.08b ± 0.16
8
7.73a ± 0.19
8.12b ± 0.13
Overall Mean
7.53a ± 0.19
7.82b ± 0.18
Means having different superscripts in a row differ significantly (P<0.01)
4.2.4.9 NE L intake
NE L intake (Table 4.31) ranged from 18.19 to 22.21 in group 1 and 18.69 to 22.51
Mcal/d in group 2. In 3rd, 7th and 8th fortnight, NE L intake was higher (P<0.05) in
group 2 than that of group 1. Overall average NE L intake was 20.58 and 21.00 Mcal/d
in group 1 and 2, respectively. The average NE L intake was higher (P<0.01) by 2.04
per cent in group 2 over that of group 1.
The NE L intake on percent body weight basis (Table 4.32) ranged from 4.00
to 5.01 Mcal in group 1 and 4.23 to 5.08 Mcal in group 2. The NE L intake per 100 kg
body weight was higher (P<0.01) in group 2 than that of group 1 in all the fortnights
except in 6th fortnight. The overall average NE L intake/ 100 kg BW was 4.72 Mcal in
group 1 and 4.89 Mcal in group 2. NE L intake/100 kg BW was higher (P<0.01) in
group 2 than that of group 1.
_________________________________________________________________ 110
Results and Discussion …
Table 4.31 Fortnightly NE L intake (Mcal/d) in lactating crossbred cows fed
ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
18.19 ± 0.67
18.69 ± 0.53
2
19.71 ± 3.20
19.99 ± 0.67
3
20.09a ± 0.81
22.51b ± 1.34
4
20.44 ± 0.89
20.92 ± 0.69
5
20.74 ± 0.94
21.13 ± 0.67
6
21.10 ± 0.99
20.89 ± 0.87
7
20.93 ± 0.99
21.70 ± 0.78
8
20.95a ± 1.03
22.16b ± 0.68
Overall Mean
20.58a ± 1.19
21.00b ± 0.78
Means having different superscripts in a row differ significantly (P<0.05)
Table 4.32 Fortnightly NE L intake (Mcal/100 kg body weight) in lactating
crossbred cows fed ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
4.00a ± 0.18
4.23b ± 0.08
2
4.47a ± 0.17
4.64b ± 0.13
3
4.69a ± 0.17
5.07b ± 0.17
4
4.88a ± 0.13
5.02b ± 0.08
5
4.97 ± 0.10
5.10 ± 0.08
6
5.01 ± 0.12
4.97 ± 0.13
7
4.91a ± 0.14
5.06b ± 0.10
8
4.83a ± 0.12
5.08b ± 0.08
Overall Mean
4.72a ± 0.12
4.89b ± 0.11
Means having different superscripts in a row differ significantly (P<0.01)
_________________________________________________________________ 111
Results and Discussion …
The results of the present study indicated that there was significant increase in
DMI on rumen protected methionine and lysine supplementation in lactating cows.
The result of the present study are in agreement those of with Benefield et al. (2009)
and Broderick et al. (2009) who also found that there was increase in DMI on
supplementation of rumen protected methionine. This lead to an increased TDN, ME
or NE L intake in RPM plus RPL supplemented group with that of control group
4.3
EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE
SUPPLEMENTATION
ON
MILK
PRODUCTION
AND
ITS
COMPOSITION
4.3.1
Milk Production
Average daily milk production (Table 4.33, Fig 4.9) ranged from 14.23 to
16.48 kg/d in group 1 and 16.70 to 18.12 kg/d in group 2 in different fortnights on
supplementation of rumen protected methionine and lysine. Milk production was
higher in group 2 than that of group 1 in all the fortnights. Average milk production
during supplementation period was 15.89 kg/d in group 1 and 17.69 kg/d in group 2,
which was 11.33 per cent higher (P<0.01) in group 2 over that of group 1.
Fortnightly average 4 % FCM yield (Table 4.34; Fig 4.10) ranged from 14.52
to 16.91 in group 1 and 17.40 to 18.67 in group 2 in different fortnights on
supplementation of rumen protected methionine and lysine. 4 % FCM yield was
higher in group 2 than that of group 1 in all the fortnights. Average daily 4 % FCM
yield was 16.21 in group 1 and 18.24 kg in group 2. Group 2 had 12.52 per cent
higher (P<0.01) FCM yield over that of group 1.
Fortnightly average energy corrected milk (ECM) yield (Table 4.35) ranged
from 14.55 to 16.88 in group 1 and 17.40 to 18.63 in group 2 in different fortnights on
supplementation of rumen protected methionine and lysine. ECM yield was higher in
group 2 than that of group 1 in all the fortnights. Average daily ECM yield was 16.20
in group 1 and 18.11 kg in group 2. Group 2 had 11.79 per cent higher (P<0.01) ECM
yield over that of group 1.
_________________________________________________________________ 112
Results and Discussion …
Table 4.33 Fortnightly average milk yield (kg/d) of lactating crossbred cows fed
ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
14.23a ± 1.81
16.70b ± 1.11
2
16.17a ± 1.87
18.01b ± 1.18
3
16.46a ± 1.60
18.12b ± 1.15
4
15.99a ± 1.37
17.99b ± 1.07
5
16.15a ± 1.64
18.09b ± 1.16
6
15.47a ± 1.99
17.93b ± 1.37
7
16.48a ± 1.48
17.62b ± 1.18
8
16.19a ± 1.27
17.02b ± 1.30
Overall Mean
15.89a ± 0.26
17.69b ± 0.19
Means having different superscripts in a row differ significantly (P<0.01)
Table 4.34 Fortnightly average 4% fat corrected milk yield (kg/d) of lactating
crossbred cows fed ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
14.52a ± 1.91
17.40b ± 1.12
2
16.54a ± 2.00
18.67b ± 1.21
3
16.73a ± 1.66
18.60b ± 1.18
4
16.17a ± 1.40
18.46b ± 1.14
5
16.44a ± 1.68
18.60b ± 1.21
6
15.76a ± 2.03
18.44b ± 1.40
7
16.91a ± 1.52
18.19b ± 1.20
8
16.65a ± 1.30
17.65b ± 1.36
Overall Mean
16.21a ± 0.27
18.24b± 0.17
Means having different superscripts in a row differ significantly (P<0.01)
_________________________________________________________________ 113
Results and Discussion …
Lara et al. (2006) observed increased milk production (31.4 vs 35.8 kg d-1) and
protein yield (3.35 vs 3.58 kg d-1) on addition of RPM in diet. Milk production
responded quadratically to added methionine levels. The quadratic response in milk
protein output was also reported by Guinard and Rulquin (1995), effects which have
been associated to a major synthesis of casein and a reduction in urea nitrogen (Wu et
al., 1997a,b; Dinn et al., 1998). Holstein cows with a mean production of 35 kg d-1
milk require addition of ruminally protected methionine (16 g d-1) to improve milk
production (Lara et al., 2006).
Dairy cows fed with alfalfa hay and heat treated soybean meal may meet
lysine requirement but not of methionine (Armentano et al., 1997); diets with soybean
meal or cotton seed are deficient in methionine and lysine, therefore, a positive
response in milk production and composition can be observed on ruminally protected
amino acids supplementation (Rulquin and Delaby, 1997). The same response can be
expected in rations with moderate amounts of blood meal, fishmeal, or meat meal,
which presumably are low in bypass methionine (Xu et al., 1998).
Table 4.35 Fortnightly energy corrected milk yield (kg/d) in lactating crossbred
cows supplemented with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
14.55a ± 1.89
17.40b ± 1.03
2
16.54a ± 1.98
18.63b ± 1.12
3
16.69a ± 1.63
18.57b ± 1.10
4
16.18a ± 1.40
18.45b ± 1.05
5
16.39a ± 1.67
18.55b ± 1.14
6
15.73a ± 2.05
18.14b ± 1.29
7
16.88a ± 1.53
17.99b ± 1.10
8
16.62a ± 1.30
17.11b ± 1.27
Overall Mean
16.20a ± 0.27
18.11b ± 0.20
Means having different superscripts in a row differ significantly (P<0.01)
_________________________________________________________________ 114
Fig 4.9 Fortnightly average milk yield (kg/d) in crossbred cows
Fig 4.10 Fortnightly average 4% fat corrected milk yield (kg/d) in
crossbred cows
Results and Discussion …
Maximum milk yield was recorded in seventh fortnight in group 1 and in third
fortnight of group 2 (16.48 kg in group 1 and 18.12 kg/d in group 2). The two major
reasons for higher milk production in group 2 may be, higher fortnightly TDN intake
observed in this group due to fortification of the diet with rumen protected methionine
and lysine and occurrence of less number of cases of RFM and metritis in this group
(Table 4. 61). The lesser post-parturient stress was also manifested by trend observed
in fortnightly body weight changes (Table 4.21) as animals in group 2 had lower body
weight change.
The significant improvement in milk production on supplementation of rumen
protected methionine and lysine is in line with the findings of many researchers.
Noftsger and St-Pierre, (2003) observed significant improvement in milk production
on dietary supplementation of rumen protected methionine (23.6 kg d-1) as compared
to that of control (21.7 kg d-1). Similarly, Bach et al. (2000) demonstrated an increase
in milk yield on supplementing rumen protected methionine to lactating Holsteins
cows (45.9 vs 47.7 kg d-1). Similarly, Broderick et al. (2009) reported that
supplementation of rumen protected methionine and lysine enhanced the milk yield in
crossbred cows (41.5 kg d-1) to that of control (39.4 kg d-1) while Yang et al. (2010)
reported increase (18.95 vs 21.55 kg d-1) in milk yield on supplementation of rumen
protected methionine and lysine. While Socha et al. (2008) found that there was
increase in milk production on abomasal infusion of methionine and lysine during
peak and early lactation while there was no difference in mid lactation.
In contrast, many researchers (Berthiaume et al., 2001; Misciattelli et al.,
2003; Girard et al, 2005; Noftsger et al., 2005; Broderick and Muck, 2001; Davidson
et al., 2008 and Benefield et al., 2009) did not found any effect on milk production on
supplementing rumen protected methionine while Swanepoel et al. (2011) did not find
any effect on milk production on supplementing rumen protected lysine alone.
Christensen et al. (1994) concluded that addition of fat along with ruminally
protected amino acids to the diet of lactating cows, increased the production of milk
and 4% FCM yield.
_________________________________________________________________ 115
Results and Discussion …
In the present study, the higher incidences of calving related abnormalities
delayed the cows in group 1 to reach the peak yield and also lowered milk production
at peak, which in turn reduced the milk production during experimental period.
Thus, the present study establishes that supplementation of rumen protected
methionine and lysine to high yielding crossbred cows was beneficial as it increased
milk and FCM production.
4.3.2
Milk Composition
a)
Milk Fat
The milk fat content (Table 4.36, Fig 4.11) ranged from 4.07 to 4.19 per cent
in group 1 and 4.17 to 4.29 per cent in group 2 in different fortnights. Milk fat per
cent was higher (P<0.01) in group 2 than that of group 1 in all the fortnights. The
overall average milk fat per cent was higher (P<0.01) by 2.18 per cent in group 2
(4.22%) than that of group 1 (4.13%).
Table 4.36 Fortnightly milk fat content (%) in lactating crossbred cows fed
ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
4.11 a ± 0.07
4.29 b ± 0.05
2
4.12a ± 0.07
4.25b ± 0.06
3
4.10a ± 0.05
4.18b ± 0.05
4
4.07a ± 0.05
4.17b ± 0.05
5
4.12a ± 0.05
4.18b ± 0.05
6
4.13a ± 0.04
4.22b ± 0.05
7
4.17a ± 0.02
4.24b ± 0.03
8
4.19a ± 0.03
4.27b ± 0.03
Overall Mean
4.13 a ± 0.05
4.22 b ± 0.05
Means having different superscripts in a row differ significantly (P<0.01)
_________________________________________________________________ 116
Fig 4.11 Fortnightly average milk fat (%) of crossbred cows
Fig 4.12 Average fortnightly milk composition
*
Results and Discussion …
The milk fat yield (Table 4.37) ranged from 588.50 to 687.59 g/d in group 1
and 714.43 to 764.51 g/d in group 2 in different fortnights. Milk fat yield was higher
(P<0.05) in group 2 than that of group 1. The overall average milk fat yield was
657.18 g/d in group 1 and 745.32 g/d in group 2 which was higher (P<0.01) by 13.41
per cent in group 2 than that of group 1.
In this experiment, feeding RPM plus RPL increased milk fat percentage over
the entire treatment period; however, differences in milk fat yield were not significant
when compared for each fortnight. Reported effects of Met and Lys supplementation
on milk fat percentage and yield have been inconsistent. Socha et al. (2008) reported
that duodenal infusion of Met increased milk fat percentage and yield in cows during
early lactation (~15 wk of lactation), but not during peak (~5 wk of lactation) or mid
lactation (~21 wk of lactation). Misciatteilli et al. (2003) also concluded that early
lactation cows fed RP-Met had increased milk fat percentage compared with control
cows; however, supplementation did not affect milk fat yield. Overton et al. (1996)
reported that RP-Met supplementation during the transition period and early lactation
increased yield of 3.5% FCM during the first 105 d of lactation.
Table 4.37 Fortnightly Milk Fat yields (g/d) in lactating crossbred cows fed
ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
588.50a ± 78.92
714.43b ± 45.20
2
671.29a ± 83.63
764.51b ± 49.47
3
676.52a ± 67.99
757.11b ± 48.31
4
651.57a ± 57.32
750.58b ± 47.56
5
665.14a ± 68.64
757.25b ± 49.96
6
638.36a ± 82.32
745.00b ± 56.96
7
687.59a ± 62.13
740.06b ± 48.54
8
678.43a ± 53.11
705.99b ± 56.03
Overall Mean
657.18a ± 65.38
745.32b ± 46.29
Means having different superscripts in a row differ significantly (P<0.01)
_________________________________________________________________ 117
Results and Discussion …
Moreover, other research showed that supplementing diets with rumenprotected methionine (RPM) improved yields of milk, fat, and protein (Bach et al.,
2000; Noftsger and St-Pierre, 2003; Misciattelli et al., 2003; Broderick et al., 2008,
2009; Benefield et al., 2009; Yang et al., 2010; Swanepoel et al., 2011).
In contrast, in several other studies (Berthiaume et al., 2001; Leonardi et al.,
2003; Noftsger and St-Pierre, 2003; Noftsger et al., 2005; Davidson et al., 2008;
Broderick et al., 2008; Preynat et al., 2009) RP-Met supplementation did not affect
milk fat percentage or yield.
It is not clear why increased amounts of Met and Lys in MP may sometimes
increase fat content of milk. One reason may relate to the role of AA in the intestinal
and hepatic synthesis of chylomicrons and very low density lipoproteins (VLDL).
Required substrates for the synthesis of chylomicrons and VLDL, in addition to the
presence of the long-chain fatty acids that stimulate their formation, include
apolipoproteins and phospholipids (Bauchart et al., 1996). The synthesis of
apolipoproteins requires AA. The synthesis of phosphatidylcholine (lecithin), the
most abundant phospholipid, requires choline. It has been demonstrated that a portion
of the dairy cows’ requirement for Met is as a methyl donor for choline synthesis
(Sharma and Erdman, 1989; Erdman, 1994), choline can be a limiting nutrient for
milk fat synthesis. That Met and Lys may sometimes be limiting for the synthesis of
chylomicrons or VLDL such that the availability of long chain fatty acids for milk fat
synthesis is reduced has not been demonstrated. However, there is limited evidence
that formation or secretion of these lipoproteins can be enhanced with improved Met
and Lys nutrition (Auboiron et al., 1995; Durand et al., 1992).
In the present experiment, plasma VLDL concentration had been increased
and that may be the reason for the higher time-related average milk fat content of milk
exhibited by the experimental group 2 than in the group 1 (P<0.01).
b)
Milk Protein
Fortnightly milk protein content is presented in Table 4.38 and the same is
graphically presented in Fig 4.12. The milk protein content ranged from 3.21 to 3.32
per cent in group 1 and 3.22 to 3.35 per cent in group 2 in different fortnights. The
_________________________________________________________________ 118
Results and Discussion …
overall average milk protein was 3.27 and 3.28 per cent in group 1 and group 2
respectively which was similar.
The milk protein yield (Table 4.39) ranged from 470.12 to 538.73 g per day in
group 1 and 539.57 to 594.77 g/d in group 2 in different fortnights. Milk protein yield
in all the fortnights was significantly higher (P<0.01) than that of group 1.The overall
average milk protein yield was 518.68 g/d in group 1 and 578.63 g/d in group 2
exhibiting higher (P<0.01) milk protein by 11.56 per cent in group 2 than that of
group 1.
Table 4.38 Fortnightly milk protein content (%) in lactating crossbred cows fed
ration with or without RPM plus RPL
Fortnight
Group 1
Group 2
1
3.32 ± 0.03
3.35 ± 0.04
2
3.29 ± 0.03
3.30 ± 0.02
3
3.25 ± 0.03
3.28 ± 0.02
4
3.27± 0.03
3.31 ± 0.02
5
3.24 ± 0.02
3.26 ± 0.02
6
3.21 ± 0.03
3.22 ± 0.01
7
3.26 ± 0.02
3.24 ± 0.02
8
3.28 ± 0.02
3.26 ± 0.03
Overall Mean
3.27 ± 0.03
3.28 ± 0.02
The results of the present experiment are is in agreement with those of
Berthiaume et al. (2001); Girard et al. (2005); Noftsger et al. (2005); Davidson et al.
(2008) and Yang et al. (2010) who reported no effect of rumen protected methionine
fortification on milk protein content while Swanepoel et al. (2011) found that there
was no effect of rumen protected lysine on milk protein percent.
In contrast, several other workers (Noftsger and St-Pierre, 2003; Bach et al.,
2000; Misciattelli et al., 2003; Brydl et al., 2005; Broderick and Muck, 2008;
_________________________________________________________________ 119
Results and Discussion …
Benefield et al., 2009; Broderick et al., 2009) reported that RP-Met supplementation
increased milk protein percentage or its yield.
Studies have suggested that Lys and Met are the most limiting AA for milk
protein synthesis in dairy cows fed corn silage and alfalfa-based commercial diets
typically used in the United States and that optimal concentrations of these limiting
AA are required in MP to increase milk protein synthesis (Schwab et al., 1976). Thus,
balancing the ration for these AA is of practical significance as a means to enhance
milk protein production and to improve efficiency of milk protein synthesis (Cho et
al., 2007).
Table 4.39 Fortnightly milk protein yield (g/d) in lactating crossbred cows fed
ration with or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
470.12a ± 58.38
557.47b ± 33.13
2
532.12a ± 61.64
593.70b ± 37.26
3
533.87a ± 50.42
593.61b ± 36.73
4
522.02a ± 43.87
594.77b ± 34.65
5
521.64a ± 52.57
590.79b ± 38.47
6
500.02a ± 66.96
569.92b ± 43.18
7
538.73a ± 50.18
565.90b ± 36.03
8
530.90a ± 42.23
539.57b ± 44.13
Overall Mean
518.68a ± 50.30
578.63b ± 35.43
Means having different superscripts in a row differ significantly (P<0.01)
In contrast, the infusion of graded amounts of Met (0, 3.5, 7.0, 10.5, and 16.0
g/d) into the duodenum of post peak lactation cows fed a corn-based diet
supplemented with soybean products and blood meal increased percentages in milk of
both fat (3.73, 3.86, 3.78, 3.91, and 4.15) and true protein (3.00, 3.07, 3.09, 3.13, and
3.15) (Socha et al., 1994b). However, when the same cows fed the same feedstuffs
_________________________________________________________________ 120
Results and Discussion …
were infused with similar amounts of Met during peak lactation (Socha et al., 1994c)
or mid lactation (Socha et al., 1994a), percentages of fat in milk did not change but
protein in milk increased.
As reviewed by the NRC (2001) and generally accepted, Met is the most
limiting AA or is co-limiting with Lys for milk protein production when cows are fed
green maize fodder and alfalfa diets. Furthermore, the NRC (2001) reported that
relationships of both with content and yield of milk protein with digestible Met
supply, expressed as a percentage of MP, were best described by rectilinear models
(straight line at lower digestible Met supplies leading to a breakpoint as digestible
Met supply increases). Leonardi et al. (2003) determined that equal amounts of RPMet increased milk protein percentage for cows fed both high- and low-CP diets,
although milk production and milk protein yield were not affected by treatments.
In the present study, supplementation with RPM plus RPM did not increase
milk protein but milk protein yield was significantly increased due to increased milk
production.
c)
Milk Lactose
Fortnightly milk lactose content is presented in Table 4.40 and the same is
graphically depicted in Fig 4.12. The milk lactose content ranged from 4.86 to 4.89
per cent in group 1 and 4.85 to 4.87 per cent in group 2 in different fortnights. The
overall average milk lactose was 4.88 and 4.86 per cent in group 1 and group 2,
respectively which was similar in both the groups.
The milk lactose yield (Table 4.41) ranged from 694.38 to 803.13 g per day in
group 1 and 802.34 to 881.69 g/d in group 2 in different fortnights. Milk lactose yield
in all the fortnights was significantly higher (P<0.01) than that of group 1.The overall
average milk lactose yield was 774.60 g/d in group 1 and 858.08 g/d in group 2 which
was significantly higher (P<0.01) by 10.77 per cent in group 2 than that of group 1.
This was due to increased milk yield.
_________________________________________________________________ 121
Results and Discussion …
Table 4.40 Fortnightly milk lactose content (%) in lactating crossbred cows fed
ration with or without RPM plus RPL
Fortnight
Group 1
Group 2
1
4.88 ± 0.01
4.86 ± 0.01
2
4.87 ± 0.01
4.86 ± 0.01
3
4.87 ± 0.01
4.87 ± 0.01
4
4.87 ± 0.01
4.85 ± 0.02
5
4.88 ± 0.01
4.87 ± 0.01
6
4.89 ± 0.01
4.87 ± 0.01
7
4.88 ± 0.01
4.87 ± 0.02
8
4.86 ± 0.01
4.86 ± 0.02
Overall Mean
4.88 ± 0.01
4.86 ± 0.02
Table 4.41 Fortnightly Milk Lactose yield (%) in crossbred cows fed ration with
or without RPM plus RPL
a, b
Fortnight
Group 1
Group 2
1
694.38a ± 88.44
812.15b ± 54.01
2
787.41a ± 90.98
875.78b ± 57.26
3
801.05a ± 77.09
881.58b ± 56.42
4
779.51a ± 67.48
872.21b ± 51.91
5
787.57a ± 79.70
881.69b ± 57.08
6
756.95a ± 97.86
862.86b ± 66.57
7
803.13a ± 70.78
851.69b ± 57.94
8
786.80a ± 61.13
802.34b ± 61.99
Overall Mean
774.60a ± 74.83
858.08b ± 53.95
Means having different superscripts in a row differ significantly (P<0.01)
_________________________________________________________________ 122
Results and Discussion …
d)
Milk Solids-not-fat (SNF) content
The SNF content (Table 4.42; Fig 4.12) ranged from 8.83 to 9.00 per cent in
group 1 and from 8.86 to 8.98 per cent in group 2. Mean SNF content was the same
(8.91) in groups 1 and 2. There was no difference in average SNF contents between
the groups.
Table 4.42 Fortnightly milk SNF content (%) in lactating crossbred cows fed
ration with or without RPM plus RPL
e)
Fortnight
Group 1
Group 2
1
9.00 ± 0.03
8.98 ± 0.04
2
8.97 ± 0.03
8.94 ± 0.03
3
8.87 ± 0.02
8.91 ± 0.02
4
8.93 ± 0.03
8.93 ± 0.03
5
8.88 ± 0.02
8.91 ± 0.03
6
8.83 ± 0.05
8.86 ± 0.02
7
8.90 ± 0.02
8.88 ± 0.03
8
8.90 ± 0.02
8.88 ± 0.03
Overall Mean
8.91 ± 0.03
8.91 ± 0.03
Milk Total Solids contents
Total solids content of milk is given in Table 4.43 and graphically represented
in Fig 4.12. The total solids ranged from 12.91 to 13.10 in group 1 and from 13.09 to
13.27 per cent in group 2 in different fortnights. The milk TS content was higher
(P<0.05) in group 2 than that of group 1 in all the fortnights. The mean total solids
content in group 1 and 2 were 13.03 and 13.14 per cent respectively. The total solid
content of group 2 was higher (P<0.05) than that of group 1. The higher milk total
solid was mainly due the increased milk fat content in dairy cows fed ration
supplemented with RPM plus RPL.
_________________________________________________________________ 123
Results and Discussion …
Table 4.43 Fortnightly TS content (%) in lactating crossbred cows fed ration
with or without RPM plus RPL
a, b
f)
Fortnight
Group 1
Group 2
1
13.10a ± 0.07
13.27b ± 0.07
2
13.09a ± 0.09
13.19b ± 0.08
3
12.99a ± 0.06
13.09b ± 0.05
4
13.00a ± 0.07
13.10b ± 0.04
5
12.91a ± 0.11
13.10b ± 0.06
6
12.96a ± 0.08
13.09b ± 0.05
7
13.07a ± 0.03
13.13b ± 0.06
8
13.10a ± 0.05
13.17b ± 0.07
Overall Mean
13.03 a ± 0.07
13.14 b ± 0.06
Means having different superscripts in a row differ significantly (P<0.01)
Milk Urea Nitrogen (MUN)
Milk urea nitrogen ranged from 17.76 to 18.95 in group 1 and 17.06 to 19.79
mg/dL in group 2 (Table 4.44) in different fortnights. The overall average of MUN
levels was 18.57 and 18.44 mg/dL in group 1 and 2, respectively. The average
fortnightly MUN was similar in group 1 cows to that of group 2.
Milk urea nitrogen (MUN) measurement can be a useful diagnostic tool in
evaluating the N efficiency of diets. Milk urea nitrogen is an indicator of ammonia
levels in the rumen. High MUN levels would suggest excess ruminal ammonia,
perhaps due to overfeeding of degradable protein and/or not enough fermentable
carbohydrate, while low MUN levels would indicate low ruminal ammonia levels.
Also, there is an additional energy cost for excreting excess unusable ammonia, which
can reduce milk production. It has been suggested that milk urea nitrogen levels might
provide guidance about proper ration formulation. Thus BUN is the major end
product of N metabolism in ruminants and high concentrations of it are indicative of
_________________________________________________________________ 124
Results and Discussion …
an inefficient utilization of dietary nitrogen. It is well established that urea
equilibrates rapidly with body fluids including milk (Brodrick and Clayton, 1997; Hof
et al., 1997). Variance in MUN has been shown to be related to extent of CP
degradation in rumen and the amount of ammonia in excess of microbial nitrogen
requirements (Ropstad et al., 1989; Hof et al., 1997). However, Bach et al. 2000,
reported greater MUN concentration on the 18% CP rations than the 15% CP rations,
which paralleled arterial urea concentrations. In the present study, there were no
differences in CP percent of the ration of both control as well as treatment group.
Similar results had been reported by Noftsger et al. (2005); Benefield et al. (2009)
and Swanepoel et al. (2011).
Table 4.44 Fortnightly MUN (mg/dl) in lactating crossbred cows fed ration with
or without RPM plus RPL
g)
Fortnight
Group 1
Group 2
1
18.59 ± 0.55
18.68 ± 0.71
2
18.39 ± 0.68
19.61 ± 0.55
3
18.68 ± 0.61
17.63 ± 0.29
4
18.94 ± 0.81
18.84 ± 0.62
5
18.82 ± 0.72
17.06 ± 0.53
6
18.42 ± 0.67
18.22 ± 0.62
7
17.76 ± 0.42
17.72 ± 0.57
8
18.95 ± 0.76
19.79 ± 0.51
Overall Mean
18.57 ± 0.65
18.44 ± 0.55
Milk choline content
Milk choline levels ranged from 83.25 to 91.75 in group 1 and 83.25 to 92.75
mg/dL in group 2 (Table 4.45) in different fortnights. The overall average of milk
choline was 87.69 and 88.63 mg/dL in group 1 and 2, respectively. The average
fortnightly choline was similar in group 1 cows to that of group 2.
_________________________________________________________________ 125
Results and Discussion …
Table 4.45 Fortnightly milk choline (mg/dl) in lactating crossbred cows fed
ration with or without RPM plus RPL
h)
Fortnight
Group 1
Group 2
1
84.25 ± 3.62
87.25 ± 3.66
2
90.25 ± 2.81
90.75 ± 1.70
3
91.75 ± 2.50
88.25 ± 3.59
4
86.75 ± 1.77
88.75 ± 2.42
5
83.25 ± 3.02
87.75 ± 2.69
6
85.75 ± 1.70
90.25 ± 3.02
7
91.25 ± 2.15
83.25 ± 1.27
8
88.25 ± 3.76
92.75 ± 1.70
Overall Mean
87.69 ± 2.67
88.63 ± 2.51
Fatty Acid Profile of Milk
The fortnightly fatty acid profile of milk of group 1 and group 2 cows are
presented in Table 4.46, table 4.47 and Fig 4.13.
Milk saturated fatty acids ranged from 66.89 to 70.11 % of fatty acids in
group 1 and 66.63 to 70.68 % of fatty acids in group 2 (Table 4.47) in different
fortnights. The overall average of milk saturated fatty acid was 68.83 and 68.33 % of
fatty acids in group 1 and 2, respectively. The average fortnightly saturated fatty acid
contents was similar in group 1 cows to that of group 2.
Milk unsaturated fatty acid (as per cent of total FA) ranged from 29.91 to
33.11 % in group 1 and 29.33 to 33.39 % in group 2 (Table 4.47) in different
fortnights. The overall average of milk unsaturated fatty acid contents were 31.30 and
31.67 % of fatty acid in group 1 and 2, respectively. The average fortnightly saturated
fatty acids was similar in group 1 cows to that of group 2. Mono unsaturated fatty acid
(MUFA) (as per cent of total FA) ranged from 27.12 to 30.12 % in group 1 and 26.26
to 30.39 % in group 2 (Table 4.47) in different fortnights. The overall average of milk
_________________________________________________________________ 126
Results and Discussion …
mono unsaturated fatty acid contents were 28.21 and 28.70 % of fatty acid in group 1
and 2, respectively. The average fortnightly mono saturated fatty acid contents were
similar in group 1 cows to that of group 2. Poly unsaturated fatty acid (PUFA) (as per
cent of total FA) ranged from 2.44 to 4.48 % in group 1 and 2.42 to 4.47 % in group 2
(Table 4.47) in different fortnights. The overall average of milk poly unsaturated fatty
acid was 3.09 and 2.97 % of fatty acid in group 1 and 2, respectively. The average
fortnightly poly un saturated fatty acid was similar in group 1 cows to that of group 2.
The findings of the present study are in agreement with those of Casper et al.,
1987; Chow et al., 1990; Karunanandaa et al., 1994; Kowalski et al., 1999; Rulquin
and Delaby, 1997; Varvikko et al., 1999 who also did not observe any effect of
increased postruminal supplies of Met on fatty acid composition of milk fat.
In contrast, Pisulewski et al. (1996) demonstrated that the infusion of Met into
the duodenum of early lactation cows increased the proportions of short- and mediumchain fatty acids and decreased proportions of long-chain fatty acids in milk fat.
Christensen et al. (1994) reported a similar trend in the fatty acid composition of milk
when lactating cows were fed ruminally protected Met and Lys and suggested that
increased in milk fat percent may involve a possible effect of Met on de novo
synthesis of short- and medium-chain fatty acids in the mammary gland.
_________________________________________________________________ 127
Table 4.46 Fortnightly Fatty Acid Profile of Milk of lactating crossbred cows fed ration with or without RPM plus RPL
1
Fatty Acid (% of fatty acid)
2
3
4
Group 1
Group 2
Group 1
Group 2
Group 1
Group 2
Group 1
Group 2
Caproic Acid (C6:0)
0.95 ± 0.07
1.23 ± 0.23
1.01 ± 0.10
0.91 ± 0.06
1.11 ± 0.07
1.27 ± 0.13
1.10 ± 0.11
1.02 ± 0.18
Caprylic Acid (C8:0)
2.96 ±0.37
2.75 ± 0.61
3.09 ± 0.16
3.19 ± 0.25
2.57 ± 0.44
2.88 ± 0.91
2.87± 0.08
3.18 ± 0.16
Capric Acid (C10:0)
3.75 ± 0.12
3.56 ± 0.14
3.32 ± 0.35
3.31 ± 0.36
3.39 ± 0.35
3.88 ± 0.94
3.53 ± 0.08
3.92 ± 0.28
Lauric Acid (C12:0)
3.65 ± 0.08
3.53 ± 0.08
3.59 ± 0.10
3.58 ± 0.09
3.74 ± 0.19
3.59 ± 0.11
3.42 ± 0.13
3.18± 0.15
Myristic Acid (C14:0)
9.86 ± 0.32
10.56 ± 0.30
9.88 ± 0.19
10.10 ± 0.28
9.75 ± 0.18
9.60 ± 0.43
10.40± 0.08
9.34± 0.17
Myristoleic Acid (C14:1)
1.64 ± 1.03
0.73 ± 0.24
2.82 ± 1.34
2.81 ± 1.34
1.61 ± 0.46
1.53 ± 0.39
1.44 ± 0.47
2.47 ± 0.00
Palmitic Acid (C16:0)
28.04 ± 0.74
27.64 ± 0.46
25.66 ± 1.05
26.17 ± 1.41
27.79 ± 0.49
29.91± 0.68
28.45 ± 0.56
26.47 ± 0.62
Palmitoleic Acid (C16:1)
1.50 ± 0.11
1.36 ± 0.19
1.44 ± 0.17
1.43 ± 0.16
1.54 ± 0.06
1.48 ± 0.04
1.47 ± 0.13
1.21± 0.04
Hepiadecanoic Acid (C17:0)
0.56 ± 0.06
0.50 ± 0.09
0.62 ± 0.03
0.62 ± 0.03
0.52 ± 0.01
0.50 ± 0.01
0.68 ± 0.11
0.54± 0.09
Stearic Acid (C18:0)
14.16 ± 0.18
14.51 ± 0.30
13.52 ± 0.23
13.25 ± 0.26
14.14 ± 0.64
12.11 ± 0.93
14.11 ± 0.50
13.73± 0.17
Oleic Acid (C18:1)
23.04± 0.81
22.19 ± 0.87
21.19 ± 0.20
21.12 ± 0.26
20.43 ± 0.63
19.77 ± 1.09
20.94 ± 0.52
23.49 ± 0.21
Elaidic Acid (C18:1)
1.87 ± 0.27
2.14 ± 0.22
1.97 ± 0.18
1.82 ± 0.17
2.29 ± 0.04
2.20 ± 0.04
1.84 ± 0.12
2.09 ± 0.14
Linoleic Acid (C18:2)
1.77 ± 0.12
1.81 ± 0.09
1.87 ± 0.18
1.86 ± 0.18
1.93 ± 0.07
1.84 ± 0.05
1.76 ± 0.40
1.87 ± 0.00
α-linolenic Acid (C18:3)
0.72 ± 0.12
0.63 ± 0.07
0.62 ± 0.33
2.61 ± 2.03
0.90 ± 0.21
0.87 ± 0.22
0.62 ± 0.08
0.87 ± 0.08
Arachidic Acid (C20:0)
0.45 ± 0.03
0.46 ± 0.03
0.46 ± 0.07
0.45 ± 0.07
0.46 ± 0.11
0.44 ± 0.11
0.48 ± 0.02
0.40± 0.04
∑SFA
68.92 ± 1.66
70.16 ± 0.68
66.89 ± 0.88
67.15 ± 0.99
69.61 ± 0.40
70.68 ± 1.08
70.11 ± 0.85
66.63 ± 0.53
∑MUFA
29.04 ± 0.30
27.39 ± 0.72
28.63 ± 1.32
28.39 ± 1.23
27.30 ± 0.13
26.36 ± 0.75
27.12 ± 0.90
30.39 ± 0.32
∑PUFA
3.12 ± 0.65
2.44 ± 0.05
4.48 ± 2.20
4.47 ± 2.21
3.09 ± 0.28
2.97 ± 0.34
2.79 ± 0.51
3.00 ± 0.31
∑UFA
32.17 ± 0.57
29.83 ± 0.67
33.11 ± 0.89
32.86 ± 1.00
30.39 ± 0.40
29.33 ± 1.09
29.91 ± 0.84
33.39 ± 0.54
∑UFA/∑SFA
0.47 ± 0.02
0.43 ± 0.01
0.50 ± 0.02
0.49 ± 0.02
0.44 ± 0.01
0.42 ± 0.02
0.43 ± 0.02
0.50 ± 0.01
(Table continued……)
5
Fatty Acid (% of fatty acid)
6
7
8
Group 1
Group 2
Group 1
Group 2
Group 1
Group 2
Group 1
Group 2
Caproic Acid (C6:0)
1.09 ± 0.05
1.11 ± 0.08
0.95 ± 0.02
1.04 ± 0.08
0.98 ± 0.10
1.06 ± 0.07
1.18 ± 0.13
1.36 ± 0.49
Caprylic Acid (C8:0)
2.47 ± 0.57
3.15 ± 0.21
3.02 ± 0.13
3.24 ± 0.10
3.22 ± 0.07
2.97 ± 0.21
2.58 ± 0.55
3.01 ± 0.26
Capric Acid (C10:0)
2.81 ± 0.58
3.64 ± 0.10
3.08 ± 0.26
3.42 ± 0.13
4.34 ± 0.21
3.58 ± 0.13
3.48 ± 0.09
3.62 ± 0.10
Lauric Acid (C12:0)
3.60 ± 0.22
3.30 ± 0.14
3.62 ± 0.07
3.42 ± 0.20
3.60 ± 0.04
3.21 ± 0.16
3.47 ± 0.03
3.57 ± 0.06
Myristic Acid (C14:0)
10.46 ± 0.55
9.67 ± 0.05
9.84 ± 0.27
9.73 ± 0.36
10.05 ± 0.75
10.04 ± 0.67
10.87 ± 0.25
10.39 ± 0.45
Myristoleic Acid (C14:1)
0.97 ± 0.02
1.98 ± 0.49
0.93 ± 0.04
1.47 ± 0.50
0.99 ± 0.01
1.99 ± 0.49
0.98 ± 0.01
0.75 ± 0.25
Palmitic Acid (C16:0)
26.24 ± 0.72
27.70 ± 0.49
28.36 ± 1.08
27.99 ± 1.43
27.54 ± 0.20
27.02 ± 0.12
27.08 ± 0.18
27.38 ± 0.62
Palmitoleic Acid (C16:1)
1.50 ± 0.27
1.48 ± 0.19
1.11 ± 0.26
1.12 ± 0.26
0.88 ± 0.25
1.28 ± 0.10
1.30 ± 0.15
1.53 ± 0.06
Hepiadecanoic Acid (C17:0)
0.57 ± 0.04
0.55 ± 0.06
0.88 ± 0.40
0.47 ± 0.05
0.53 ± 0.09
0.56 ± 0.06
0.48 ± 0.08
0.59 ± 0.02
Stearic Acid (C18:0)
14.20 ± 0.28
13.65 ± 0.15
13.38 ± 0.38
13.86 ± 0.11
13.65 ± 0.28
13.61 ± 0.23
14.26 ± 0.55
13.57 ± 0.27
Oleic Acid (C18:1)
23.25 ± 0.35
22.63 ± 0.74
22.28 ± 0.71
22.77 ± 0.83
22.95 ± 0.79
23.65 ± 0.25
22.46 ± 1.01
23.48 ± 0.21
Elaidic Acid (C18:1)
2.06 ± 0.06
2.13 ± 0.17
2.12 ± 0.26
2.03 ± 0.17
2.01 ± 0.13
1.92 ± 0.08
2.13 ± 0.22
2.00 ± 0.14
Linoleic Acid (C18:2)
1.90 ± 0.15
1.66 ± 0.13
2.08 ± 0.25
1.86 ± 0.10
2.00 ± 0.03
1.83 ± 0.07
1.73 ± 0.06
1.78 ± 0.09
α-linolenic Acid (C18:3)
1.07 ± 0.36
0.87 ± 0.08
0.82 ± 0.15
0.93 ± 0.12
0.71 ± 0.13
0.87 ± 0.08
0.71 ± 0.01
0.64 ± 0.08
Arachidic Acid (C20:0)
0.53 ± 0.04
0.41 ± 0.04
0.35 ± 0.06
0.30 ± 0.02
0.46 ± 0.04
0.52 ± 0.03
0.49 ± 0.04
0.46 ± 0.04
∑SFA
66.90 ± 0.25
67.56 ± 0.44
69.17 ± 0.69
68.20 ± 1.43
69.34 ± 0.52
67.37 ± 0.55
69.74 ± 0.88
68.86 ± 0.45
∑MUFA
30.12 ± 0.55
29.89 ± 0.40
27.74 ± 1.01
28.57 ± 1.48
27.92 ± 0.40
29.90 ± 0.42
27.83 ± 0.92
28.72 ± 0.47
∑PUFA
2.97 ± 0.44
2.53 ± 0.14
3.09 ± 0.38
3.23 ± 0.42
2.73 ± 0.18
2.71 ± 0.15
2.44 ± 0.06
2.42 ± 0.01
∑UFA
33.09 ± 0.25
32.42 ± 0.45
30.83 ± 0.67
31.81 ± 1.43
30.66 ± 0.54
32.61 ± 0.56
30.27 ± 0.87
31.13 ± 0.46
∑UFA/∑SFA
0.49 ± 0.01
0.48 ± 0.01
0.45 ± 0.01
0.47 ± 0.03
0.44 ± 0.01
0.48 ± 0.01
0.43 ± 0.02
0.45 ±0.01
Table 4.47 Overall fatty acid profile of milk of lactating crossbred cows fed
ration with or without RPM plus RPL
Fatty Acid
Group 1
Group 2
Caproic Acid (C6:0)
1.04 ± 0.03
1.13 ± 0.05
Caprylic Acid (C8:0)
2.85 ± 0.10
3.05 ± 0.06
Capric Acid (C10:0)
3.46 ± 0.16
3.62 ± 0.07
Lauric Acid (C12:0)
3.59b ± 0.04
3.42a ± 0.06
Myristic Acid (C14:0)
10.14 ± 0.14
9.93 ± 0.15
Myristoleic Acid (C14:1)
1.42 ± 0.23
1.72 ± 0.26
Palmitic Acid (C16:0)
27.40 ± 0.36
27.54 ± 0.40
Palmitoleic Acid (C16:1)
1.34 ± 0.08
1.36 ± 0.05
Hepiadecanoic Acid (C17:0)
0.60 ± 0.05
0.54 ± 0.02
Stearic Acid (C18:0)
13.93 ± 0.12
13.54 ± 0.24
Oleic Acid (C18:1)
22.07 ± 0.38
22.39 ± 0.48
Elaidic Acid (C18:1)
2.04 ± 0.05
2.04 ± 0.04
Linoleic Acid (C18:2)
1.88 ± 0.04
1.81 ± 0.03
α-linolenic Acid (C18:3)
0.77 ± 0.23
0.80 ± 0.23
Arachidic Acid (C20:0)
0.46 ± 0.02
0.43 ± 0.02
∑SFA
68.83 ± 0.44
68.33 ± 0.52
∑MUFA
28.21 ± 0.35
28.70 ± 0.48
∑PUFA
3.09 ± 0.22
2.97 ± 0.24
∑UFA
31.30 ± 0.46
31.67 ± 0.52
∑UFA/∑SFA
0.46 ± 0.01
0.46 ± 0.01
(% of fatty acids)
a, b
: Means having different superscripts in a row differ significantly (P<0.05)
__________________________________________________________________130
Fig 4.11 Fortnightly average milk fat (%) of crossbred cows
Fig 4.12 Average fortnightly milk composition
*
Results and Discussion …
4.3.3
Efficiency of nutrients for milk production
Efficiency of utilization of DM, CP and TDN for milk production is presented
in Table 4.48. The efficiency of utilizing DMI, kg per kg milk production, was 0.82
and 0.76 and for 4 % FCM, it was 0.80 and 0.73 for group 1 and 2, respectively. The
DM was utilized better (P<0.01) for milk production on supplementation of rumen
protected methionine and lysine in group 2 than that of group 1. The rumen protected
methionine and lysine supplementation increased the methionine and lysine supply in
metabolizable protein and hence resulted in more milk production which may be the
reason for increased efficiency for DM utilization for milk production.
Table 4.48 Efficiency of utilization of nutrients of lactating crossbred cows fed
ration with or without RPM plus RPL
Parameter
Group 1
Group 2
DMI, kg/kg Milk Yield
0.82a
±
0.01
0.76b
±
0.02
DMI, kg/kg FCM
0.80 a
±
0.02
0.73 b
±
0.02
CPI, g/kg Milk Yield
132.81 a
±
1.72
122.18 b
±
1.79
CPI, g/kg FCM
130.18 a
±
1.75
128.99 b
±
1.76
MPI, g/kg Milk Yield
82.37 a
±
1.19
76.62 b
±
1.56
MPI, g/kg FCM
80.73 a
±
1.18
74.73 b
±
1.53
TDN, kg/kg Milk Yield
0.55 a
±
0.01
0.51 b
±
0.01
TDN kg/kg FCM
0.54 a
±
0.01
0.50 b
±
0.01
MEI, Mcal/kg Milk Yield
2.04 a
±
0.03
1.88 b
±
0.04
MEI, Mcal/kg FCM
2.00 a
±
0.03
1.83b
±
0.03
NE L I, Mcal/kg Milk Yield
1.28 c
±
0.02
1.19d
±
0.02
NE L I, Mcal/kg FCM
1.25 a
±
0.02
1.15b
±
0.02
a, b
Means having different superscripts in a row differ significantly (P<0.01)
c, d
Means having different superscripts in a row differ significantly (P<0.05)
__________________________________________________________________131
Results and Discussion …
The CP intake, kg per g milk production, was also better (P<0.05) in group 2
than that of group 1. These values for milk production were 132.81 and 122.18 g and
for 4 % FCM production, these were 130.19 and 128.99 g in group 1 and 2,
respectively. The CP intake (kg) per kg 4 % FCM production was also better (P<0.01)
in group 2 than that of group 1. The better CP utilization for milk production in group
2 may be due to higher availability of methionine and lysine from rumen protected
methionine and lysine.
The rumen protected methionine and lysine supplementation in group 2
increased supply of these amino acids in the intestine for absorption and subsequently
their uptake by mammary gland with a greater efficiency, which resulted in an
increased efficiency of protein utilization in the group 2.
The metabolizable protein intake, g per kg milk yield, was 82.37 g in group 1
and 76.62 g in group 2. MPI, g per kg FCM yield, was 80.73 and 74.73 in group 1 and
2, respectively. Group 2 exhibited better utilization (P<0.01) of metabolizable protein
for milk and FCM production than that of group 1.
The TDN intake, kg per kg milk yield, was 0.55 in group 1 and 0.51 in group
2 and TDNI, kg per kg FCM yield, was 0.54 and 0.50 in group 1 and 2, respectively.
Group 2 exhibited better utilization (P<0.01) of TDN for milk and FCM production
than that of group 1.
The metabolizable energy intake, Mcal per kg milk yield, was 2.04 in group 1
and 1.88 in group 2 and MEI, Mcal per kg FCM yield, was 2.00 and 1.83 in group 1
and 2, respectively. Group 2 exhibited better utilization (P<0.01) of metabolizable
energy for milk and FCM production than that of group 1.
The NE L intake, Mcal per kg milk yield, was 1.28 in group 1 and 1.19 in
group 2 and MEI, Mcal per kg FCM yield, was 1.25 and 1.15 in group 1 and 2,
respectively. Group 2 exhibited better utilization (P<0.01) of metabolizable energy for
FCM production than that of group 1.
Thus, the results of the present study on efficiency of utilization of nutrients
exhibited that rumen protected methionine and lysine supplemented group could
utilize the available nutrients towards milk production more efficiently.
__________________________________________________________________132
Results and Discussion …
4.4
Overall Plane of Nutrition in lactating cows
The overall plane of nutrition of cows is presented in Table 4.49. The actual
nutrient intake was compared with nutrient requirement as per NRC (2001).
Table 4.49
Plane of nutrition of lactating cows fed with rumen protected
methinone plus lysine
Parameter
DM
CP
TDN
NE L
Group 1
Group 2
Intake (kg/d)
13.01
13.36
Predicted * (kg/d)
13.60
14.40
Deficit in DMI
4.34
7.22
Intake (kg/d)
2.10
2.17
Requirement * (kg/d)
1.77
1.92
Extra CP intake (%)
18.64
13.02
Intake (kg/d)
8.76
9.05
Requirement * (kg/d)
8.21
8.75
Extra TDN intake (%)
6.70
3.43
Intake (Mcal/d)
20.58
21.00
Requirement * (Mcal/d)
19.70
21.30
Extra NE L intake (%)
4.47
- 1.41
* Requirements as per NRC (2001)
The DMI was undersupplied by 4.34 percent than requirement in group 1 and
7.22 percent in group 2. The CPI was 18.64 percent higher than requirement in group
1 and 13.02 percent higher in case of group 2. TDN intake was 6.70 percent and 3.43
__________________________________________________________________133
Results and Discussion …
percent higher than requirements in group 1 and 2, respectively whereas NE L intake
was 4.47 percent higher in group 1 while 1.41 percent less in group 2. The plane of
nutrition of cows of both the groups indicated that both the groups were adequately
fed, thereby fulfilling the requirements as per NRC (2001).
4.5
Effect of Rumen Protected Methionine and Lysine Supplementation on
Some Lactation Related Plasma Parameters
4.5.1
Blood Glucose Concentration
The blood glucose concentration (Table 4.50, Fig 4.14) in different fortnights
varied from 46.02 to 58.71 and 47.15 to 57.91 in group 1 and 2, respectively. The
overall average was 55.61 and 55.07 mg/dl in group 1 and 2, respectively. The lowest
level of blood glucose was found at the day of parturition in both the groups. It may
be due to parturition related stress and decreased dry matter intake, but it did not fall
below the lower limit of the physiological range (40-60 mg/dL) in either group.
Table 4.50 Plasma glucose (mg/dl) concentration in lactating crossbred cows fed
ration with or without RPM plus RPL
Day
Group 1
Group 2
1
46.02 ± 1.36
47.15 ± 0.66
15
56.80 ± 1.07
56.55 ± 1.50
30
57.20 ± 1.04
54.70 ± 1.00
45
57.91 ± 0.62
57.27 ± 1.33
75
57.03 ± 1.23
56.87 ± 0.77
105
58.71 ± 1.26
57.91 ± 0.35
Overall Mean
55.61 ± 1.10
55.07 ± 0.92
The blood glucose concentration remained within the normal range and no
difference was observed at any stage of experiment. The reason may be a high
metabolic rate of utilization of glucose and homeostatic mechanism of animal body
__________________________________________________________________134
Results and Discussion …
that does not allow appreciable change in glucose level. The results of the present
study are similar to that of Bach et al. (2000) on supplementing rumen protected
methionine and Socha et al. (2008) on supplementing rumen protected methionine
and lysine in peak and mid lactation but increase was observed in early lactation.
However, Berthiaume et al. (2001) found increased blood glucose level on
supplementing rumen protected methionine.
Socha et al., (2005) also reported that the effect of RPAA supplementation on
plasma glucose concentrations was dependent upon period after parturition. Between
wk 1 and 4 postpartum, plasma glucose concentrations of cows receiving RPM+L
declined at a more rapid rate than cows receiving either the basal diet or RPM.
However, between wk 4 and 7 postpartum, plasma glucose concentrations of RPM+L
supplemented cows increased, whereas plasma glucose concentrations of cows
receiving no RPAA or RPM remained essentially unchanged. Immediately following
parturition, glucose concentrations decreased in cows receiving 16 % CP in diet and
15 gm of RPM supplemented group, 16% CP in diet and 6 gm of RPM plus 40 g/d of
a RPM+L supplemented group, and 18.5% CP in diet and 6 gm of RPM plus 40 g/d of
a RPM+L supplemented group and increased in cows receiving 18.5% CP and15 gm
of RPM supplemented group.
4.5.2
Plasma Phosphatidylcholine Concentration
The plasma phosphatidylcholine concentration (Table 4.51, Fig 4.14) in
different fortnights varied from 95.58 to 164.16 and 109.00 to 163.90 µg/ml in group
1 and 2, respectively. The overall average was 138.57 and 140.98 µg/ml in group 1
and 2, respectively.
Met is source of the methyl donor S- adenosyl Methionine, the metabolite that
provides methyl groups in variety of reactions including de novo synthesis of choline
from phosphatidylethanolamine, thus Met and choline metabolism are closely
associated. Choline is a nutrient involved with the transport of fat from liver. Choline
is synthesised in part from methionine and required for the synthesis of
phosphatidylcholine, a phospholipid found in the membrane of VLDL. As much as
28% of the absorbed methionine is used for choline synthesis (Emmanuel and
Kennely, 1984). Thus, Methionine plays a direct role in VLDL synthesis in bovine
__________________________________________________________________135
Results and Discussion …
and acts to reduce plasma ketone bodies during early lactation. (Durrand et al., 1992).
Methionine is also involved in many pathways including the synthesis of
carnitine, creatinine, polyamines and energy metabolism.
Table
4.51
Plasma Phosphatidylcholine (µg/ml) concentration in lactating
crossbred cows fed ration with or without RPM plus RPL
a, b
Day
Group 1
Group 2
1
95.58 a ± 2.31
109.00 b ± 5.57
15
121.19 a ± 3.81
129.52 b ± 2.79
30
133.32 ± 2.99
135.71 ± 1.82
45
155.19 ± 5.07
147.19 ± 3.37
75
161.97 ± 3.46
160.55 ± 1.25
105
164.16 ± 4.09
163.90 ± 2.73
Overall Mean
138.57 ± 3.72
140.98 ± 3.83
Means having different superscripts in a row differ significantly (P<0.05)
In the present study, there was increase in plasma phoshatidylcholine during
first fortnight on supplementation of rumen protected methionine and lysine which
might be due to contribution of methionine in de novo synthesis of choline.
4.5.3
Plasma NEFA Concentration
The plasma NEFA concentration (Table 4.52, Fig 4.14) in different fortnights
varied from 96.14 to 118.17 and 100.28 to 112.14 mg/L in group 1 and 2,
respectively. The overall average was 106.80 and 105.77 mg/L in group 1 and 2,
respectively.
The NEFA concentration was higher in both the groups at the day of
parturition (P=0.3626). The NEFA concentration decreased (P<0.05) during first
__________________________________________________________________136
Results and Discussion …
fortnight on supplementation of rumen protected methionine and lysine. But overall
plasma NEFA average was statistically similar in both the groups.
Table 4.52 Plasma NEFA (mg/L) concentration in lactating crossbred cows fed
ration with or without RPM plus RPL
Day
Group 1
Group 2
1
115.94 ± 1.74
112.14 ± 2.55
15
118.17 a ± 2.03
109.64 b ± 3.81
30
103.61 ± 1.45
102.67 ± 1.18
45
103.78 ± 1.13
106.39 ± 0.80
75
103.17 ± 0.95
103.50 ± 0.66
105
96.14 ± 1.78
100.28 ± 1.14
Overall Mean
106.80 ± 3.46
105.77 ± 1.83
Comparative decreases in plasma nonesterified fatty acids concentrations in
first fortnight in RPM plus RPL supplemented group reflected reduced mobilization
of fatty acids from body reserves. Similarly, Socha et al. (2008) reported that there
was decrease in plasma NEFA concentration on abomasal infusion of rumen protected
methionine and lysine during early lactation. Bach et al. (2000) reported decreased
plsma NEFA concentration on supplementing rumen protected methionine and lysine.
Whereas, Misciattelli et al. (2003) did not found any effect on plasma NEFA
concentration on supplementation of rumen protected methionine and lysine, and
Davidson et al. (2008) on supplementation of rumen protected methionine. There was
no effect of abomasal infusion of methionine and lysine on plasma NEFA level during
mid lactation (Socha et. al., 2008).
__________________________________________________________________137
Results and Discussion …
4.5.4
Plasma Triglycerides Concentration
The plasma triglycerides concentration (Table 4.53, Fig 4.15) in different
fortnights varied from 11.87 to 16.12 and 13.77 to 17.89 mg/dL in group 1 and 2,
respectively. It was higher (P<0.01) in group 2 in all the forthnights. The overall
average was 13.40 and 16.22 mg/dL in group 1 and 2, respectively, which was higher
(P<0.01) in RPM plus RPL supplemented group.
Table 4.53 Plasma triglycerides (mg/dl) concentration in lactating crossbred
cows fed ration with or without RPM plus RPL
Day
Group 1
Group 2
1
16.12c ± 0.67
17.89d ± 0.43
15
14.62 a ± 0.26
17.89 b ± 0.43
30
12.81 a ± 0.33
16.84 b ± 0.72
45
12.58 a ± 0.24
15.79 b ± 0.66
75
12.42 a ± 0.23
15.16 b ± 0.62
105
11.87 a ± 0.14
13.77 b ± 0.53
Overall Mean
13.40 a ± 0.34
16.22 b ± 0.54
a, b
Means having different superscripts in a row differ significantly (P<0.01)
c, d
Means having different superscripts in a row differ significantly (P<0.05)
4.5.5
Plasma VLDL Concentration
The plasma VLDL concentration (Table 4.54, Fig 4.15) in different fortnights
varied from 2.37 to 3.22 and 2.75 to 3.58 mg/dL in group 1 and 2, respectively. The
overall average was 2.68 and 3.24 mg/dL in group 1 and 2, respectively, which was
higher (P<0.01) in RPM plus RPL supplemented group.
The increase in milk fat percent may relate to the role of AA in the intestinal
and hepatic synthesis of chylomicrons and VLDL. Apolipoproteins and phospholipids
__________________________________________________________________138
Fig 4.13 Average fortnightly fatty acid profile of milk of
crossbred cows
Fig 4.14 Average fortnightly plasma metabolites of crossbred
cows
Results and Discussion …
are the required substrates for the synthesis of chylomicrons and VLDL, in addition to
the presence of the long-chain fatty acids that stimulate their formation (Bauchart et
al., 1996). The synthesis of apolipoproteins requires AA. The synthesis of
phosphatidylcholine (lecithin), the most abundant phospholipid, requires choline. It
has been demonstrated that a portion of the dairy cows’ requirement for choline was
accomplished by Met as Met is as a methyl donor for choline synthesis (Sharma and
Erdman, 1989; Erdman, 1994), choline can be a limiting nutrient for milk fat
synthesis.
Table
4.54
Fortnightly plasma VLDL (mg/dl) concentration in lactating
crossbred cows fed ration with or without RPM plus RPL
Day
Group 1
Group 2
1
3.22c ± 0.13
3.58d ± 0.09
15
2.92 a ± 0.09
3.58b ± 0.06
30
2.56 a ± 0.07
3.37 b ± 0.14
45
2.52 a ± 0.05
3.16 b ± 0.13
75
2.48 a ± 0.05
3.03 b ± 0.12
105
2.37 a ± 0.03
2.75 b ± 0.11
Overall Mean
2.68 a ± 0.11
3.24 b ± 0.06
a, b
Means having different superscripts in a row differ significantly (P<0.01)
c, d
Means having different superscripts in a row differ significantly (P<0.05)
That Met and Lys may sometimes be limiting for the synthesis of
chylomicrons or VLDL such that the availability of long chain fatty acids for milk fat
synthesis is reduced has not been demonstrated. However, there is limited evidence
that formation or secretion of these lipoproteins can be enhanced with improved Met
and Lys nutrition (Auboiron et al., 1995; Durand et al., 1992).
__________________________________________________________________139
Results and Discussion …
4.5.6
Plasma Vitamin E levels
Plasma vitamin E concentration (Table 4.55, Fig 4.14) in different fortnights
ranged between 0.68 to 1.33 µg/ml in group 1 and 0.66 to 1.17 µg/ml in group 2. The
average values were 1.01 and 0.90 µg/ml in group 1 and 2, respectively, which were
similar in both the groups.
Table 4.55 Plasma vitamin E (µg/ml) concentration in lactating crossbred cows
fed ration with or without RPM plus RPL
a, b
Day
Group 1
Group 2
1
0.68 ± 0.03
0.66 ± 0.03
15
0.82 ± 0.06
0.75 ± 0.03
30
0.94 ± 0.10
0.82 ± 0.05
45
1.05 ± 0.08
0.96 ± 0.02
75
1.25a ± 0.06
1.06 b ± 0.05
105
1.33 a ± 0.04
1.17 b ± 0.03
Overall Mean
1.01 ± 0.07
0.90 ± 0.04
Means having different superscripts in a row differ significantly (P<0.05)
4.5.7
Plasma cholesterol levels
Plasma cholesterol concentration (Table 4.56, Fig 4.14) in different fortnights
ranged between 171.33 to 237.07 mg/dL in group 1 and 173.29 to 229.27 mg/dL in
group 2. The average values were 193.16 and 198.31 mg/dL in group 1 and 2,
respectively, were similar in both the groups.
__________________________________________________________________140
Results and Discussion …
Table 4.56 Fortnightly plasma Cholesterol (mg/dl) concentration in lactating
crossbred cows fed ration with or without RPM plus RPL
Day
Group 1
Group 2
1
171.33 ± 5.53
173.88 ± 5.97
15
171.86 ± 5.31
173.29 ± 5.24
30
183.92 ± 6.66
191.42 ± 5.90
45
184.45 ± 7.34
203.80 ± 8.41
75
210.32 ± 7.57
218.19 ± 4.78
105
237.07 ± 9.17
229.27 ± 5.32
Overall Mean
193.16 ± 6.93
198.31 ± 5.94
4.5.8
Blood urea nitrogen levels
Blood urea nitrogen (BUN) concentration (Table 4.57 Fig 4.14) in different
fortnights ranged between 15.28 to 20.34 mg/L in group 1 and 14.33 to 21.51 mg/L in
group 2. The average values were 18.13 and 18.01 mg/L in group 1 and 2,
respectively.
The plasma urea concentration observed in the present study was within the
normal range in cows (Borghese, 2005). Norton et al. (1979) reported lower blood
urea concentrations in cattle than buffaloes. The BUN level remained similar in both
the groups. Similar results were reported by Socha et al., (2005) by abomasal infusion
of methionine and lysine. While Berthiaume et al. (2001) and Bach et al. (2000)
found decreased blood urea nitrogen on supplementation of rumen protected
methionine. But no significant difference was observed on BUN level in present study
on supplementation of rumen protected methionine plus lysine.
__________________________________________________________________141
Results and Discussion …
Table 4.57 Fortnightly plasma BUN (mg/L) concentration in lactating crossbred
cows fed ration with or without RPM plus RPL
Day
Group 1
Group 2
1
20.34 ± 0.90
20.75 ± 0.34
15
19.58 ± 0.80
21.51 ± 1.29
30
18.11 ± 0.31
17.19 ± 0.39
45
17.77 ± 0.27
17.47 ± 0.30
75
17.67 ± 0.38
16.80 ± 0.58
105
15.28 ± 0.47
14.33 ± 0.27
Overall Mean
18.13 ± 0.52
18.01 ± 0.53
4.5.9 Plasma amino acid profile
Fortnightly plasma amino acids viz. aspartate, glutamate, serine, glycine,
histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cysteine,
leucine, isoleucine, phenylalanine and lysine concentration are presented in Table
4.58, 4.59 and Fig 4.16.
Table 4.58 Postpartum plasma amino acid (µmol/dl) lactating concentration in
crossbred cows fed ration with or without RPM plus RPL
Amino Acid
Aspartate
Day
Group 1
Group 2
1
21.97 ± 1.11
22.91 ± 0.76
15
21.53 ± 0.97
23.24 ± 1.48
30
24.77 ± 1.08
24.00 ± 0.85
__________________________________________________________________142
Results and Discussion …
Glutamate
Serine
Glycine
45
21.34 ± 1.89
23.07 ± 1.18
75
20.56 ± 1.48
22.02 ± 1.64
105
20.72 ± 1.20
22.20 ± 1.05
1
23.58 ± 1.18
24.48 ± 1.00
15
22.58 ± 1.04
24.60 ± 1.00
30
24.83 ± 0.49
24.12 ± 1.10
45
22.99 ± 2.31
24.29 ± 0.83
75
22.83 ± 1.89
22.14 ± 0.94
105
24.00 ± 1.63
23.20 ±1.17
1
12.59 ± 0.69
13.72 ± 0.35
15
12.99 ± 0.56
13.45 ± 0.42
30
13.69 ± 0.82
13.06 ± 0.47
45
12.43 ± 1.01
13.32 ± 0.39
75
12.26 ± 0.61
12.94 ± 0.65
105
13.92 ± 1.14
12.59 ± 0.52
1
18.68 ± 0.98
20.72 ± 0.56
15
19.39 ± 0.53
20.35 ± 0.68
30
20.11 ± 0.71
19.93 ± 0.71
45
18.13 ± 1.55
20.26 ± 0.60
75
16.71 ± 2.09
19.76 ± 1.04
__________________________________________________________________143
Results and Discussion …
Histidine
Arginine
Threonine
Alanine
105
19.28 ± 0.96
19.15 ± 0.81
1
4.66 ± 0.49
4.31 ± 0.43
15
4.69 ± 0.52
4.44 ± 0.39
30
4.98 ± 0.61
4.79 ± 0.29
45
4.16 ± 0.34
4.55 ± 0.39
75
4.12 ± 0.32
4.24 ± 0.46
105
4.62 ± 0.48
4.60 ± 0.57
1
7.63 ± 0.56
8.29 ± 0.21
15
7.66 ± 0.51
8.02 ± 0.22
30
8.28 ± 0.63
7.93 ± 0.30
45
7.72 ± 0.33
8.02 ± 0.22
75
7.34 ± 0.30
7.54 ± 0.25
105
7.50 ± 0.54
7.51 ± 0.33
1
11.84 ± 0.61
12.50 ± 0.85
15
11.28 ± 0.59
11.46 ± 0.71
30
12.16 ± 0.86
11.69 ± 0.66
45
11.15 ± 0.97
11.82 ± 0.94
75
11.53 ± 0.61
11.90 ± 0.76
105
11.88 ± 0.83
11.34 ± 0.68
1
22.82 ± 1.79
24.03 ± 0.62
__________________________________________________________________144
Results and Discussion …
Proline
Tyrosine
Valine
15
23.81 ± 3.33
23.68 ± 0.85
30
23.27 ± 1.56
24.04 ± 1.16
45
22.64 ± 3.28
23.63 ± 0.98
75
22.41 ± 2.16
22.79 ± 1.47
105
21.13 ± 1.15
21.79 ± 0.97
1
9.14 ± 0.59
9.04 ± 0.38
15
9.03 ± 0.48
9.02 ± 0.41
30
8.87 ± 0.56
8.83 ± 0.39
45
9.14 ± 0.47
8.99 ± 0.36
75
8.41 ± 0.40
8.76 ± 0.49
105
8.65 ± 0.56
8.76 ± 0.43
1
7.49 ± 0.66
7.58 ± 0.41
15
7.36 ± 0.50
7.02 ± 0.32
30
6.80 ± 0.45
6.87 ± 0.31
45
7.03 ± 0.46
7.06 ± 0.29
75
7.09 ± 0.35
7.13 ± 0.46
105
6.82 ± 0.54
6.82 ± 0.28
1
15.21 ± 0.91
16.20 ± 0.53
15
15.55 ± 0.79
16.53 ± 0.60
30
16.42 ± 0.98
16.33 ± 0.56
__________________________________________________________________145
Results and Discussion …
Methionine
Cysteine
Isoleucine
45
14.29 ± 1.09
15.52 ± 0.63
75
15.04 ± 0.85
16.00 ± 0.85
105
15.15 ± 0.71
15.63 ± 0.72
1
5.39a ± 0.32
5.93b ± 0.27
15
6.11 ± 0.53
6.40 ± 0.18
30
5.92 ± 0.24
6.10 ± 0.22
45
5.71 ± 0.33
6.04 ± 0.21
75
5.57 ± 0.18
5.90 ± 0.13
105
5.60 ± 0.36
5.94 ± 0.19
1
1.61a ± 0.45
2.11b ± 0.29
15
1.83 ± 0.32
2.21 ± 0.21
30
1.84 ± 0.48
2.10 ± 0.31
45
2.01 ± 0.43
2.24 ± 0.36
75
2.09 ± 0.46
2.17 ± 0.30
105
1.85 ± 0.43
1.92 ± 0.22
1
10.79 ± 0.83
9.54 ± 0.72
15
10.63 ± 0.76
10.23 ± 0.74
30
11.34 ± 1.00
10.18 ± 0.75
45
10.65 ± 0.75
10.23 ± 0.80
75
10.50 ± 0.88
10.30 ± 0.87
__________________________________________________________________146
Results and Discussion …
Leucine
Phenylalanine
Lysine
a, b
105
11.66 ± 0.90
11.20 ± 0.96
1
15.11 ± 1.08
16.18 ± 0.61
15
14.22 ± 1.77
15.52 ± 1.00
30
14.40 ± 2.05
15.11 ± 1.03
45
12.68 ± 1.92
13.14 ± 0.93
75
13.20 ± 1.65
14.64 ± 0.80
105
14.74 ± 1.06
14.91 v 0.68
1
8.26 ± 0.62
9.40 ± 0.87
15
8.66 ± 0.78
9.12 ± 1.01
30
8.93 ± 0.82
9.14 ± 1.02
45
7.51 ± 0.47
9.14 ± 0.99
75
7.84 ± 0.37
7.95 ± 0.37
105
8.60 ± 0.75
8.81 ± 1.01
1
13.28 ± 0.86
14.25 ± 0.84
15
13.50 ± 0.93
14.00 ± 0.68
30
15.47a ± 0.92
16.63b ± 0.88
45
14.39a ± 0.68
16.68b ± 1.01
75
13.93 ± 1.83
14.93 ± 0.51
105
12.89 ± 0.78
13.97 ± 0.86
Means having different superscripts in a row differ significantly (P<0.01)
__________________________________________________________________147
Results and Discussion …
Table 4.59 Overall mean plasma amino acid (µmol/dl) concentration in
lactating crossbred cows fed ration with or without RPM plus RPL
Amino acid
Group 1
Group 2
Asparate
21.81a ± 1.31
22.91b ± 1.37
Glutamate
23.47 ± 1.50
23.80 ± 1.21
Serine
12.98 ± 0.83
13.18 ± 0.44
Glycine
18.72 c ± 1.13
20.03 d ± 0.68
Histidine
4.54 ± 0.48
4.41 ± 0.40
Arginine
7.69 ± 0.51
7.88 ± 0.28
Threonine
11.64 ± 0.76
11.79 ± 0.78
Alanine
22.68 c ± 2.12
23.33 d ± 1.03
Proline
8.88 ± 0.53
8.90 ± 0.40
Tyrosine
7.10 ± 0.51
7.08 ± 0.33
Valine
15.28 c ± 0.95
16.04 d ± 0.65
Methionine
5.71 a ± 0.38
6.05 b ± 0.19
Cysteine
1.87 c ± 0.43
2.13 d ± 0.55
Isoleucine
10.93d ± 0.89
10.28 c ± 0.84
Leucine
14.06 ± 1.49
14.92 ± 0.98
Phenylalanine
8.30 ± 0.65
8.93 ± 0.83
13.91 a ± 1.03
15.08 b ± 0.93
Lysine
a, b
c, d
Means having different superscripts in a row differ significantly (P<0.01)
Means having different superscripts in a row differ significantly (P<0.05)
__________________________________________________________________148
Results and Discussion …
Overall average of free plasma amino acid concentrations are presented in
Table 4.59; Fig 4.16. Aspartate, glycine, alanine, valine, methionine, cysteine, and
lysine concentrations were increased (P<0.01 or P<0.05) in cows fed ration
supplemented with RPM plus RPL. However, RPM plus RPL supplementation
lowered (P<0.05) plasma isoleucine concentration. All other free plasma amino acids
i.e. glutamate, serine, histidine, arginine, threonine, proline, tyrosine, leucine and
phenylalanine were unaffected by the treatment.
Increased supply to the small intestine of any AA is expected to change its
concentration in blood plasma and, possibly, improve availability of that AA for milk
protein synthesis. This was demonstrated by Blauwiekel et al. (1997) in an
experiment in which lysine supplementation increased duodenal lysine flow, as well
as the concentration of lysine in plasma, leading to an increase in milk and milk
protein yield.
Baumrucker (1985) explained that the transport systems for absorption of AA
depend on transport specificity and competition among AA. As feeding RPL provides
more lysine to cells with a Y+ (cationic) transport system, the uptake of AA sharing
the same transport system (i.e., arginine and isoleucine) may be reduced through
competitive inhibition with lysine.
Supplementation with RPM caused an elevation in arterial concentration of
Met (Berthiaume et al., 2001, Overton et al., 1996, 1998; Blum et al., 1999) or the
results were same when Met infused postruminally (Guinard and Rulquin, 1995;
Pisulewski et al., 1996; Varvikko et al., 1999)
It is reported that there were decrease in plasma methionine concentrations
(Rogers et al., 1987) or decrease in most non-essential AA (NEAA) as well as
threonine, leucine and tryptophan (Swanepoel et al., 2011) when RPL was fed.
Swanepoel et al. (2011) reported that the plasma lysine concentration was only
numerically increased with RPL feeding. Results of Weekes et al. (2006) suggest that
a decreased concentration of plasma AA (especially histidine) without a positive milk
protein response could be due to a more efficient catabolism or deposition of AA into
body protein, which might be supported by the trend to lower BCS loss on RPL
supplementation.
__________________________________________________________________149
Results and Discussion …
Abomasal Met infusions of 56.5 g/d (Seymour et al., 1990) and duodenal Met
infusions of 0, 6, 12, 18, and 24 g/d (Pisulewski et al., 1996) elevated blood and
plasma Met concentrations linearly. Similarly, dietary supplementation with rumenprotected Met generally is associated with higher blood or plasma Met concentrations
(Nichols et al., 1998; Blum et al., 1999) but not in all instances (Colin-Schoellen et
al., 1995; Xu et al., 1998). Sylvester et al. (2003) reported increased plasma Met
concentration on diets supplemented with HMBi. Bequette et al. (1997) compared
unidirectional fluxes of AA across the mammary gland to their secretion in milk
protein. Based on their results, milk protein synthesis must compete with other
metabolic processes within the mammary gland for the use of some AA.
4.5.10 Plasma prolactin levels
Plasma Prolactin concentrations (Table 4.60 Fig 4.17) in different weeks
ranged between 13.50 to 215.41 ng/ml in group 1 and 13.29 to 216.26 ng/ml in group
2. The average values were 18.13 and 18.01 ng/L in group 1 and 2, respectively.
There was no effect of rumen protected methionine and lysine supplementation on
plasma prolactin concentration.
Table
4.60
Weekly plasma prolactin (ng/ml) concentration in lactating
crossbred cows fed ration with or without RPM plus RPL
Day
Group 1
Group 2
-15
25.58 ± 4.60
26.08 ± 4.63
-7
118.31 ± 14.00
120.10 ± 14.32
1
215.41 ± 23.85
216.26 ± 23.99
7
25.59 ± 2.27
26.22 ± 2.22
15
13.50 ± 0.65
13.29 ± 0.63
Overall Mean
79.68 ± 9.07
80.39 ± 9.16
The hormones involved in lactogenesis include increased secretion of
prolactin, adrenocorticotropic hormone (ACTH) and estrogen, and a decrease or
virtual absence of progesterone. Prolactin concentration in cattle does not change
__________________________________________________________________150
Fig. 4.17 Average weekly plasma prolactin of crossbred cows
Fig. 4.18 Average weekly plasma growth hormone of
crossbred cows
Results and Discussion …
appreciably during gestation, but a major increase occurs just before parturition.
Prolactin induces gene expression in mammary tissue for casein synthesis. Once the
lactation has been established in cows, the concentration of basal circulating prolactin
and the release of prolactin at milking can be reduced to low level without affecting
milk yield. (Reece, 2004)
The results of the present study indicated that there was increase in level of
prolactin on day of parturition and thereafter sudden decrease in prolactin
concentration with the progression of lactation.
4.5.11 Plasma growth hormone levels
Plasma growth hormone concentration (Table 4.61; Fig 4.18) of different
weeks ranged between 6.25 to 7.01 ng/ml in group 1 and 6.22 to 6.97 ng/ml in group
2. The average values were similar (6.63 ng/L) in group 1 and 2, respectively. There
was no effect of rumen protected methionine and lysine on plasma growth hormone
concentration. Similarly Misciattelli et al. (2003) also did not observe any effect of
rumen protected methionine on plasma growth hormone level.
Table 4.61 Weekly plasma growth hormone (ng/ml) concentration in lactating
crossbred cows fed ration with or without RPM plus RPL
Day
Group 1
Group 2
-15
6.36 ± 0.07
6.33 ± 0.08
-7
6.25 ± 0.08
6.22 ± 0.09
1
6.84 ± 0.10
6.95 ± 0.06
7
6.67 ± 0.08
6.67 ± 0.07
15
7.01 ± 0.06
6.97 ± 0.07
Overall Mean
6.63 ± 0.08
6.63 ± 0.07
Growth hormone is more important in the maintenance of cow’s lactation as it
is galactopoeitic in cattle. A surge in growth hormone from the anterior pituitary
occurs just before parturition, perhaps assuming its role of directing nutrients to the
__________________________________________________________________151
Results and Discussion …
mammary gland for milk synthesis. It has been shown that plasma growth hormone
concentration is significantly higher in high yielding cows than in low yielding cows,
and that a significant reduction occurs when high yielding cows cease lactating.
(Reece, 2004).
The results of the present study showed that there was increase in
concentration on plasma growth hormone after parturition in both the groups.
Table 4.62 Condensed information of lactation trial
Sr. No.
1
2
Parameter
Average Initial Body Weight (After
Calving)
Average Final Body Weight (at 120
Days)
Group 1
Group 2
459.2 ± 20.3
442.7 ± 11.5
440.6 ± 20.6
448.4 ± 12.4
3
BCS (After Calving)
3.47 ± 0.11
3.69 ± 0.06
4
BCS (at 120 days)
2.97 ± 0.03
3.06 ± 0.04
5
Average Nutrients Intake (kg/d)
a
DMI
13.01a ± 0.28
13.36b ± 0.34
b
CPI
1.91a ± 0.09
1.95b ± 0.07
c
TDNI
8.76a ± 0.40
9.05b ± 0.32
6
Milk Yield (120 Days)
a
Average MY (kg/d)
15.89a ± 0.26
17.69b ± 0.19
b
Average FCM Yield (kg/d)
16.21a ± 1.59
18.24b± 1.13
c
Average ECM yield (kg/d)
16.20a ± 0.27
18.11b ± 0.20
__________________________________________________________________152
Results and Discussion …
7
Average Milk Composition
a
Total Solids (%)
13.03 a ± 0.07
13.14 b ± 0.06
b
Milk Fat (%)
4.13 a ± 0.05
4.22 b ± 0.05
c
Milk Protein (%)
3.27 ± 0.03
3.28 ± 0.02
d
Milk Lactose (%)
4.88 a ± 0.01
4.86 b ± 0.02
e
Solid-Not-Fat (%)
8.91 ± 0.03
8.91 ± 0.03
f
Milk Urea Nitrogen (mg/dl)
18.57 ± 0.65
18.44 ± 0.55
g
Milk Choline
87.69 ± 2.67
88.63 ± 2.51
8
Efficiency of Nutrient Utilization per kg milk yield
a
DM (kg/kg MY)
b
CP (g/kg MY)
c
TDN (kg/kg MY)
9
Efficiency of Nutrient Utilization per kg FCM
a
DM (kg/kg FCM)
b
CP (g/kg FCM)
c
TDN (kg/kg FCM)
10
Blood Parameters
a
Blood Glucose (mg/dl)
b
Vitamin E (µg/ml)
0.82a ± 0.01
0.76b ± 0.02
132.81 a ± 1.72 122.18 b ± 1.79
0.86 ± 0.01
0.80 a ± 0.02
0.85 ± 0.01
0.73 b ± 0.02
130.18 a ± 1.75 128.99 b ± 1.76
0.54 a ± 0.01
0.50 b ± 0.01
55.61 ± 1.10
55.07 ± 0.92
1.01 ± 0.07
0.90 ± 0.004
__________________________________________________________________153
Results and Discussion …
c
NEFA (mg/l)
106.80 ± 3.46
105.77 ± 1.83
d
Phosphatidylcholine (µg/ml)
138.57 ± 3.72
140.98 ± 3.83
e
Triglycerides (mg/dl)
13.40 a ± 0.34
16.22 b ± 0.54
f
VLDL (mg/dl)
2.68 a ± 0.11
3.24 b ± 0.06
g
Cholesterol (mg/dl)
193.16 ± 6.93
198.31 ± 5.94
h
BUN (mg/L)
18.13 ± 0.52
18.01 ± 0.53
a, b
Means having different superscripts in the same row differ significantly (P<0.01)
4.6
EFFECT
OF
RPM
PLUS
RPL
SUPPLEMENTATION
ON
REPRODUCTIVE PERFORMANCE IN CROSSBRED COWS
4.6.1
Calving Performance
The effect of feeding RPM and RPL on the calving performance of animals is
presented in Table 4.63. The average body weight of calves at the time of birth was
25.72 kg in group 1 and 26.00 kg in group 2 which was not different. Incidence of
premature births was higher in group 1 than that in group 2. As maximum growth of
the foetus takes place in the last trimester of the pregnancy, adequately supply of
energy and protein are crucial for the normal development of foetus.
4.6.2
Reproductive abnormalities
The reproductive abnormalities observed during the course of experiment are
presented in Table 4.64; Fig 4.19. There were two cases of premature births in group
1, However, one case was observed in group 2. No case of still birth was reported in
both the groups. Four cases of retention of fetal membranes (RFM) were observed in
group 1 while one case was observed in group 2. Generally, the RFM leads to
metritis, endometritis and pyometra in animals. It may be reason behind higher
incidence of metritis in group 1 in the post parturient period than that of group 2
cows. Dystokia and milk fever cases did not occur in both the groups. Two case of
mastitis was observed in group 1, whereas, one such case was observed in group 2
cows during post partum experimental period of 120 days. The major problem
__________________________________________________________________154
Results and Discussion …
associated with such calving related abnormalities is delay in reaching the peak yield
and also lower milk production at peak, which in turn reduced the milk production
during experimental period as observed in present study.
Table 4.63
Calving Performance of crossbred cows fed ration with or without
RPM and RPL
Dam No.
Date of
Calf No.
Calf sex
Parturition
Calf Birth weight
(kg)
Group 1
6918
11.06.2011
7500
Female
26
7068
16.06.2011
7597
Male
22.5
6997
20.06.2011
7598
Male
38
7034
27.06.2011
7504
Female
32
6960
02.08.2011
7606
Male
28
7013
10.08.2011
Died
Male
8
7085
18.08.2011
7510
Female
29
7106
08.11.2011
Died
Female
18
6998
03.01.2012
7632
Male
30
Average
25.72 ± 2.91
Group 2
6774
22.06.2011
7502
Female
27
6969
24.06..2011
7600
Male
25
7083
24.07..2011
Died
Female
14
__________________________________________________________________155
Results and Discussion …
7045
09.08.2011
7608
Female
28
7088
18.08..2011
7511
Female
25
6944
25.08..2011
7612
Male
28
7000
02.09.2011
7513
Female
26
7052
22.09.2011
7617
Male
31
7108
02.01.2012
7631
Male
30
Average
26.00 ± 1.65
Table 4.64 Reproductive abnormalities plus metabolic diseases observed in
crossbred cows fed RPM plus RPL
S. No.
Parameter
Group 1
Group 2
1
Still Birth
0
0
2.
Premature Birth
2
1
3.
RFM
4
1
4.
Metritis
3
1
5.
Dystokia
0
0
6.
Milk fever
0
0
7
Mastitis
2
1
RFM= Retention of fetal membranes
Four cases of retention of fetal membranes (RFM) were observed in group 1
while one case was observed in group 2. Generally, the RFM leads to metritis,
endometritis and pyometra in animals. It may be reason behind higher incidence of
__________________________________________________________________156
Fig 4.19 Reproductive abnormalities observed in crossbred cows
Fig 4.20 Reproductive parameters observed in crossbred
cows
Results and Discussion …
metritis in group 1 in the post parturient period than that of group 2 cows. Dystokia
and milk fever cases did not occur in both the groups. Two case of mastitis was
observed in group 1, whereas, one such case was observed in group 2 cows during
post partum experimental period of 120 days. The major problem associated with
such calving related abnormalities is delay in reaching the peak yield and also lower
milk production at peak, which in turn reduced the milk production during
experimental period as observed in present study.
The results of the present study are similar to that of Ardalan et al. (2010) who
reported that incidences of postpartum reproductive and metabolic diseases viz.
retained placenta, mastitis, metritis, dystokia, were significantly reduced in Holstein
dairy cows fed RPM 18 g/d from 4 week prepartum to 14 weeks postpartum.
4.6.3
Reproduction related parameters
The reproduction related parameters are presented in Table 4.65. The average
duration for commencement of cyclicity was 65.13 days in group 1 and 64.11 days in
group 2. The time required for commencement of cyclicity was similar (P>0.01) in
both the groups.
Table 4.65 Reproductive parameters recorded in crossbred cows fed RPM plus
RPL
Parameter
Group 1
Group 2
Commencement of cyclicity (Days )
65.13
±
2.26
64.11
±
1.02
Days Open (Days )
164.2
±
9.53
152.4
±
6.64
AI/Conception
2.60
±
0.81
2.67
±
0.51
Conception rate
55.55
66.66
The days open or service period was calculated on the basis of five animals
that conceived in group 1 and six animals in group 2 during the experimental duration
of 120 days. It was 164.2 and 152.4 days in group 1 and 2, respectively. The service
period was shorter by 11.8 days in group 2 than that of group 1, indicating that lesser
__________________________________________________________________157
Results and Discussion …
time was required for the animals in group 2 for conception. Similarly, Ardalan et al.
(2009) reported that there was significant decrease in days to first estrous and open
days in Holstein dairy cows fed 18 gm RPM from 4 week pre partum to 20 weeks
postpartum.
Cows of Group 1 required 2.60 AIs per conception while group 2 required
2.67 AIs per conception. There were no effect of supplementation of RPM plus RPL
on no of AI required for conception.
The conception rate during the experimental period of 120 days was 55.55 %
in group 1 and 66.66 % in group 2. The results of the present study indicate that RPM
plus PRL supplementation increased the conception rate in cows. Similar results were
reported Ardalan et al. (2009) on supplementing rumen protected methionine and
lysine in the diet of high yielding lactating dairy cows.
Xu et al. (1998) reported the overall incidence of health related disorders were
numerically lowest for the dairy cows fed high amount of rumen protected methionine
and lysine.
In contrast, Polan et al. (1991) reported that supplementation of rumen
protected form of methionine and lysine had no significant effect on days to first
service, services per conception and calving interval in dairy cows.
4.7
ECONOMICS OF FEEDING RUMEN PROTECTED METHIONINE PLUS
LYSINE IN CROSSBRED COWS
The economics of feeding rumen protected methionine and lysine to lactating
cows throughout the trial period (120 days) has been shown in Table 4.66.
The cost of rumen protected methionine and lysine was Rs 450 and Rs 250 per
kg, respectively. RPM and RPL used as a supplement in the concentrate at the rate of
7 and 60 gm, respectively, the additional cost of supplement was Rs 18.15 per day
postpartum. Based on the prevalent price of different ingredients, the cost of
concentrate mixtures worked out to be Rs. 1640.
Net return over feed cost of milk yield per animal per day in control and
treatment group was Rs. 192.1 and 215.8, respectively, indicating that it was higher
by Rs. 23.7 in treatment group over that of control group. The feed cost (Rs.) per kg
__________________________________________________________________158
Results and Discussion …
FCM produced was Rs. 10.94 and Rs. 10.92 respectively in group 1 and group 2.
While cost benefit ratio was similar in both the groups.
Table 4.66 Economics of feeding rumen protected methionine plus lysine to
lactating crossbred cows throughout the whole experimental period (120days)
Attributes
Group 1
Group 2
13.01 ± 0.28
13.36 ± 0.34
33
34
35640
36720
1
1.2
1080
1296
Concentrate intake (kg/d/animal)
8.62
8.75
Total concentrate intake in 120 days trial by 9
9310
9450
Total cost for green maize (@ Rs. 100.00/qtl.)
35640
36720
Total cost for wheat straw (@ Rs. 300.00/qtl.)
3240
3888
Total cost for concentrate (@ Rs. 1640.00/qtl.)
152677.4
154980.00
Total feed cost (Rs.)
191557.4
195588.00
-
3.15
DMI (kg/day)
Feed intake (fresh basis)
Green maize intake (kg/d/animal)
Total green maize intake in 120 days trial by 9
animals (kg)
Wheat straw intake (kg/d/animal)
Total wheat straw intake in 120 days trial by 9
animals (kg)
animals (kg)
Feed cost (Rs.)
Cost of postpartum supplementation of rumen
__________________________________________________________________159
Results and Discussion …
protected Methionine (7 g/d/animal for 120 days
@ Rs. 450/kg)
Cost of postpartum supplementation of rumen
-
15
-
3402.0
-
16200.0
191557.4
215190.0
Feed cost (Rs/animal/day)
177.37
199.25
Average milk yield (kg/d/animal)
15.89
17.69
Average FCM milk yield ((kg/d/animal)
16.21
18.24
Average milk fat %
4.13
4.22
Average Milk SNF %
8.91
8.91
Total milk yield in 120 days by 9 animals (kg)
17161.2
19105.2
Total FCM yield in 120 days by 9 animals (kg)
17506.8
19699.2
10.94
10.92
23.25
23.46
399072.6
448249.9
protected Lysine (60 g/d/animal for 120 days @
Rs. 250/kg)
Additional cost of Rumen protected Methionine
supplementation
Additional Cost of Rumen protected Lysine
supplementation
Total feed cost with RPM plus RPL (Rs.)
Feed cost (Rs.) per kg FCM produced
Market price per kg of milk (@ Rs. 230.93/ kg
milk fat and Rs 153.95/ kg of milk SNF)
Gross income (Rs.) from selling of milk (@ Rs.
230.93/ kg milk fat and Rs 153.95/ kg of milk
SNF)
__________________________________________________________________160
Results and Discussion …
Net return in 120 days from 9 animals (Rs.)
Net return (Rs./animal/day)
Additional profit in 120 days from 9 animals on
207515.2
233059.9
192.1
215.8
-
supplementation of RPM plus RPL (Rs)
Additional
profit
per
day
per
cow
on
25544.6
-
supplementation of RPM plus RPL (Rs)
Benefit cost ratio
1.08
23.7
1.08
The net return from experimental ration was markedly higher by 12.30% than
that of the control ration. As a result, additional profit in 120 days from 9 cows on
supplementation of rumen protected methionine and lysine was Rs. 25544.6.
Thus, based on the results of the present study, it could be concluded that
feeding rumen protected methionine and lysine to high yielding lactating cows during
early lactation was cost effective.
__________________________________________________________________161
CHAPTER – 5
SUMMARY AND CONCLUSIONs
5. SUMMARY AND CONCLUSIONS
_________
_______
To investigate the effect of supplementing rumen protected methionine (RPL)
and lysine (RPL) on pre and post parturient performance such as calving, lactation,
nutrient utilization and reproduction, eighteen crossbred cows were selected and
divided into two groups (9 each) on the basis of Most Probable Production Ability
(MPPA) and lactation number. Animals in group 1 (control group) were fed chopped
wheat straw, chaffed green maize fodder and compounded concentrate mixture as per
requirements (NRC, 2001), whereas, animals in group 2 (treatment group) were fed
the same ration as to control group plus 5 gm RPM and 20 gm RPL during prepartum
period and 7 gm RPM and 60 gm RPL during postpartum period, respectively. The
experimental period started from 40 days before expected date of parturition to 120
days post parturition. The results obtained during the course of this study have been
summarized.
5.1
In vitro and in sacco study
5.1.1
Product evaluation
Rumen escape potential of RPM and RPL from rumen hydrolysis was 75.20
and 54.97 per cent, respectively.
5.1.2
RDP and RUP contents of feed ingredients
RDP content of wheat straw, maize fodder and concentrate were 31.88, 60.61
and 69.41 per cent of CP, respectively. Whereas RUP content of wheat straw, maize
fodder and concentrate were 68.12, 39.39 and 30.59 per cent of CP, respectively.
Groundnut cake had the highest RDP (89.27% of CP), followed by mustard
cake (83.58) and lowest for wheat straw (31.88). While highest RUP was observed in
wheat straw (68.12) followed by maize (44.82) and deoiled rice bran (40.01%).
5.1.3
Microbial protein yield
The microbial protein yield g per kg TDN fermented was 127.84, 119.62 and
110.04 g in different TMRs containing concentrate to roughage ratio 60:40, 50:50 and
40:60, respectively.
5.1.4
RUP digestibility
RUP intestinal digestibility values of wheat straw, maize fodder and
162
Summary and Conclusions…
___________________ ______________________________________
concentrate were 64.40, 70.47 and 79.84 percent, respectively.
5.2
EFFECT OF FEEDING RUMEN PROTECTED METHIONINE AND
LYSINE DURING PRE PARTURIENT PERIOD
5.2.1
Body weight
In the first fortnight (from day -30th to -15th day pre parturient), the
experimental animals gained 7.6 and 6.7 kg body weight in groups 1 and 2,
respectively, while in the 2nd fortnight (from day -15th to the day of parturition), the
loss in weight was 58.2 and 56.1 kg, respectively due to parturition and release of the
foetal membranes. The changes in body weight were not statistically different
between the groups.
5.2.2 Body condition score
The body condition score during pre parturient period ranged from 3.84 to
3.88 in group 1, and from 3.81 to 4.00 in group 2. The initial BCS of group 1 and 2
was 3.84 and 3.81, respectively. The pre partum changes in body weights were not
conspicuously reflected in BCS changes which were higher in supplemented group
than control group before parturition.
5.2.3
Nutrients intake
The average DMI in the first fortnight (from day -30th to -15th day pre
parturient) was 9.33 and 8.87 (kg/d) and in the second fortnight (day -15th to the day
of parturition), it was 8.21 and 7.75 kg/d in group 1 and 2, respectively. DMI/100 kg
body weight in first and second fortnight was 1.83 and 1.59 kg/d in group 1 and 1.78
to 1.53 kg/d in group 2 respectively. There was no difference among two groups in
average DMI/d as well as DMI/100 kg body weight before parturition.
The average CPI and TDNI in the first fortnight (from day -30th to -15th day
pre parturient) was 1.17 and 1.14 (kg/d) and 5.90 and 5.67 (kg/d) and in the second
fortnight (day -15th to the day of parturition) was 1.09 and 1.04 (kg/d) and 5.14 and
4.66 (kg/d) in group 1 and 2, respectively, which were similar. CP intake/100 kg body
weight and TDN intake/100 kg body weight in first and second fortnight was 0.23 and
0.21 kg/d and 1.14 and 1.01 kg/d in group 1 and 0.23 and 0.23 kg/d and 1.14 and 0.97
kg/d in group 2 respectively, which were also similar in both the groups before
parurition.
__
_____________________________
_ _ __
163
Summary and Conclusions…
___________________ ______________________________________
The average RUP and RDP intakes in the first fortnight (from day -30th to 15th day pre parturient) were 0.40 and 0.39 (kg/d) and 077 and 0.75 (kg/d) and in the
second fortnight (day -15th to the day of parturition) they were 0.36 and 0.34 and 0.72
and 0.70 (kg/d) in group 1 and 2, respectively.
The average MP Intake in the first fortnight (from day -30th to -15th day pre
parturient) was 0.76 and 0.74 (kg/d) and in the second fortnight (-15th to the day of
parturition), it was 0.70 and 0.67 (kg/d) in group 1 and 2, respectively. It was
statistically similar.
The average ME and NE L Intake in the first fortnight (from day -30th to -15th
day pre parturient) was 21.78 and 21.28 (Mcal/d) and 13.59 and 13.33 (Mcal/d) and in
the second fortnight (-15th to the day of parturition), these were 19.78 and 18.50
(Mcal/d) and 12.36 and 11.60 (Mcal/d) in group 1 and 2, respectively. ME and NE L
intake/ 100 kg body weight in first and second fortnight was 4.28 and 3.83 Mcal/d
and 2.67 and 2.40 Mcal/d in group 1 and 4.28 and 3.67 Mcal/d and 2.68 and 2.30
Mcal/d in group 2, in group 2 respectively. There was no difference among two
groups in average prepartum ME and NE L intake per day aswelll as per100 kg body
weight.
The average duodenal methionine flow in the first fortnight (from day -30th to
-15th day pre parturient) was 1.90 and 2.22 (% of MP/d) and in the second fortnight (15th to the day of parturition) was 1.97 and 2.30 (kg/d) in group 1 and 2, respectively.
The average duodenal lysine flow in the first fortnight (from day -30th to -15th day pre
parturient) was 6.44 and 7.09 (kg/d) and in the second fortnight (-15th to the day of
parturition) was 6.57 and 7.35(kg/d) in group 1 and 2, respectively. Duodenal
methionine and lysine flow in group 2 was higher (p<0.01) than group 1 due to
supplementation of RPM and RPL.
5.2.4
Overall plane of nutrition in prepartum cows
On comparison of the actual nutrient intake with that of nutrient requirement
as per NRC (2001), DM intake was deficient by 10.51 percent and 13.44 percent than
requirements in group 1 and 2, respectively. The CPI was 7.62 percent higher than
requirement in group 1 and 3.81 percent higher in case of group 2. TDN intake was
12.65 percent and 5.51 percent higher than requirements in group 1 and 2,
respectively and NE L intake was 13.77 percent and 11.25 percent higher in group 1
__
_____________________________
_ _ __
164
Summary and Conclusions…
___________________ ______________________________________
and group 2, respectively. The plane of nutrition of cows of both the groups indicated
that both the groups were adequately fed, thereby fulfilling the requirements as per
NRC (2001).
5.2.5
Effect of rumen protected methionine and lysine supplementation on
certain blood parameters of prepartum cows
The blood glucose concentration on -30th day of parturition was 57.19 and
56.06 (mg/dL) and on -15th day of parturition, it was 56.55 and 54.14 (mg/dL) in
groups 1 and 2, respectively while it was decreased (p<0.01) at the day of parturition
in both the groups.
The blood phosphatidylcholine concentration on -30th day of parturition was
152.10 and 149.39 (µg/ml) and on -15th day of parturition, it was 122.35 and 133.26
(µg/ml) in groups 1 and 2, respectively. There was increase in plasma
phosphatidylcholine level on the day of parturition on supplemention of rumen
protected methionine and lysine.
The plasma NEFA concentration on -30th day of parturition was 69.53 and
67.63 (mg/L) and on -15th day of parturition, it was 91.89 and 77.58 (mg/L) in group 1
and 2, respectively. Plasma NEFA concentration on the day of parturition was 115.94
and 112.14 in group1 and group 2, respectively. No difference was observed in both
the groups at any stage of experiment.
The plasma triglycerides and VLDL concentration on -30th day of parturition
was 17.30 and 18.66 (mg/dL) and 3.46 and 3.73 (mg/dL) and on -15th day of
parturition was 16.27 and 18.24 (mg/dL) and 3.25 and 3.65 (mg/dL) in group 1 and 2,
respectively. There was increase (P<0.05) in plasma triglycerides and VLDL on the 15th day of parturition and on the day of parturition in RPM plus RPL supplemented
group.
The plasma vitamin E and cholesterol concentration on -30th day of parturition
was 1.07 and 1.03 (µg/ml) and 166.80 and 167. 37 (µg/ml) and on -15th day of
parturition, it was 0.81 and 0.79 (µg/ml) and 167.25 and 172.74 (µg/ml) in group 1
and 2, respectively. The plasma vitamin E and cholesterol concentration remained
within the normal range and no difference was observed at any stage of experiment.
__
_____________________________
_ _ __
165
Summary and Conclusions…
___________________ ______________________________________
The blood urea nitrogen concentration on -30th day of parturition was 14.08
and 13.38 (mg/dL) and on -15th day of parturition, it was 19.39 and 19.03 (mg/dL) in
group 1 and 2, respectively. The concentrations of plasma urea N did not differ in
response to dietary treatment, suggesting that overall N utilization was similar in both
the groups.
5.2.5
Plasma amino acid profile
Prepartum fortnightly plasma amino acids viz. aspartate, glutamate, serine,
glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine,
cysteine, leucine, isoleucine, phenylamine and lysine concentration were not affected
by supplementation of rumen protected methionine and lysine while increasing trends
were observed in plasma methionine and lysine concentration in cows supplemnted
with RPM plus RPL.
5.3
EFFECT OF FEEDING RUMEN PROTECTED METHIONINE AND
LYSINE DURING POST PARTURIENT PERIOD
5.3.1
Body weight changes
The initial body weights of animals at day one after parturition were 459.2 and
442.7 kg in group 1 and 2, respectively and after 120 days of feeding, final body
weight were 440.7 and 448.4 kg. Perusal of the fortnightly changes in body weight
revealed that there was overall a net loss of 18.59 kg in group 1, whereas there was
overall gain of 5.79 kg in group 2.
5.3.2
Body condition score
BCS after parturition was 3.47 and 3.69 in group 1 and 2, respectively. The
body condition score during post parturient period ranged from 2.72 to 3.16 in group
1, and from 2.92 to 3.33 in group 2 during different fortnights. Overall average BCS
of different fortnights was 2.95 and 3.09 in group 1 and 2, respectively, which was
higher (P<0.01) in group 2 due to higher energy intake by this group that lead to less
negative energy balance.
5.3.3
Nutrient Intake
The overall mean DMI was 13.01 kg and 13.36 kg/d in group 1 and 2,
respectively, and higher (P<0.05) DMI was recorded in group 2. The average
DMI/100 kg body weight was 3.03 and 3.13 kg/d in group 1 and 2, respectively. The
__
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166
Summary and Conclusions…
___________________ ______________________________________
average CPI was 2.11 and 2.17 kg/d in the group 1 and group 2, respectively, which
was higher (P< 0.01) in group 2 than that of group 1. The overall average CP intake/
100 kg BW during the experimental period was 0.49 and 0.51 kg/d in group 1 and
group 2, respectively, which was higher in group 2 than that of group 1. Average
TDNI was 8.76 kg and 9.05 kg/d in group 1 and 2, respectively. The average TDNI
was higher (P<0.01) by 3.31 per cent in group 2 over that of group 1. The overall
average TDN intake/ 100 kg BW was 2.04 kg/d in group 1 and 2.12 kg/d in group 2.
The average RDP and RUP intake was 1.40 and 1.43 kg/d and 0.72 and 0.74
kg/d in the group 1 and group 2, respectively, which were higher (P<0.05) in group 2
than that of group 1. The average MP intake was 1.31 and 1.35 kg/d in the group 1
and group 2, respectively, which was higher (P< 0.01) in group 2 than that of group 1.
The overall mean duodenal methionine and lysine supply was 1.86 and 6.15%
and 2.27 and 7.12 % of metabolizable protein per day in the group 1 and group 2,
respectively, which was higher (P<0.01) in group 2 than that of group 1, due to
supplementation of rumen protected methionine and lysine in group 2.
Average ME and NE L intake was 32.33 and 20.58 Mcal/d in group 1 and
33.31 and 21.00 Mcal/d in group 2, respectively. The average ME and NE L intake
was higher (P<0.05) by 3.03 and 2.04 per cent in group 2 over that of group 1,
respectively. Average ME intake/ 100 kg BW was 7.53 and 4.72 Mcal/d in group 1
and 7.82 Mcal and 4.89 in group 2. ME and NE L intake/100 kg BW was higher
(P<0.05) in group 2 than that of group 1.
5.4
EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE
SUPPLEMENTATION
ON
MILK
PRODUCTION
AND
COMPOSITION
5.4.1
Milk production
Average milk production during supplementation period was 15.89 kg/d in
group 1 and 17.69 kg/d in group 2, which was 11.33 per cent higher (P<0.01) in group
2 over that of group 1. Average daily 4 % FCM yield was 16.21 in group 1 and 18.24
kg in group 2. Group 2 had 12.52 per cent higher (P<0.01) FCM yield over that of
group 1. Average daily ECM yield was 16.20 in group 1 and 18.11 kg in group 2.
__
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_ _ __
167
Summary and Conclusions…
___________________ ______________________________________
Rumen protected methionine and lysine supplemented group exhibited 11.79 per cent
higher (P<0.01) ECM yield over that of control group.
5.4.2
Milk composition
The overall average milk fat per cent was higher (P<0.01) by 2.18 per cent in
group 2 (4.22%) over that of group 1 (4.13%). Average milk fat yield was 657.18 g/d
in group 1 and 745.32 g/d in group 2 which was higher (P<0.01) by 13.41 per cent in
group 2 than that of group 1.
The average milk protein and lactose content was 3.27 and 3.28 per cent and
4.88 and 4.86 per cent in group 1 and group 2, respectively. Average milk protein and
lactose yield was 518.68 and 578.63 and 774.60 and 858.08 g/d in groups 1 and 2,
respectively showing higher (P<0.01) milk protein and lactose yield by 11.56 and
10.77 per cent in group 2 than that of group 1, respectively.
Mean SNF content was similar (8.91 vs 8.91 %) in both the groups. Mean total
solids content in group 1 and 2 were 13.03 and 13.14 per cent, respectively which
were higher (P<0.01) in group 2 than that of group 1.
The overall average of MUN and milk choline contents were 18.57 and 18.44
mg/dL and 87.69 and 88.63 mg/dL in group 1 and 2, respectively, which were similar
in groups 1 and 2.
Average milk saturated fatty acids contents were 68.83 and 68.33 % of fatty
acids in group 1 and 2, respectively. Average milk unsaturated fatty acids contents
were 31.30 and 31.67 % of fatty acids in group 1 and 2, respectively. The average
milk mono unsaturated fatty acids content were 28.21 and 28.70 % of fatty acids in
group 1 and 2, respectively. Average milk poly unsaturated fatty acids content was
3.09 and 2.97 % of fatty acids in group 1 and 2, respectively. The saturated acids,
unsaturated acids, MUFA and PUFA content of the milk were similar in group 1 and
group 2.
5.4.3
Efficiency of milk production
The efficiency of utilizing DMI (kg) per kg milk production was 0.82 and 0.76
and for 4 % FCM, it was 0.80 and 0.73 for groups 1 and 2, respectively. The CP
intake (kg) per kg milk production was also better (P<0.01) in group 2 than that of
group 1. The TDN intake kg per kg milk yield was 0.55 in group 1 and 0.51 in group
__
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_ _ __
168
Summary and Conclusions…
___________________ ______________________________________
2 and TDNI kg per kg FCM yield was 0.54 and 0.50 in group 1 and 2, respectively.
Group 2 exhibited better utilization (P<0.01) of TDN for milk production as well as
FCM production than that of group 1.
The ME and NE L intake, Mcal per kg milk yield, was 2.04 and 1.28 in group 1
and 1.88 and 1.19 in group 2, respectively. ME and NE L Intake, Mcal per kg FCM
yield, was 2.00 and 1.83 Mcal and 1.25 and 1.15 Mcal in group 1 and 2, respectively.
Group 2 exhibited better utilization (P<0.01) of ME and NE L for milk and FCM
production than that of group 1.
5.5
EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE
SUPPLEMENTATION ON SOME BLOOD PARAMETERS
Average blood glucose levels were similar in both the groups (55.61 and 55.07
mg/dl). Average phosphatidylcholine levels were 138.57 and 140.98 µg/ml in group 1
and 2, respectively, which were also similar in the two groups. Average plasma NEFA
and vitamin E concentrations were 106.80 and 105.77 mg/L and 1.01 and 0.90 µg/ ml
in group 1 and 2, respectively. The average values of plasma cholesterol and BUN
were 193.16 and 198.31 mg/dL and 18.13 and 18.01 mg/L in group 1 and 2,
respectively. Plasma NEFA, vitamin E, cholesterol and BUN concentrations in both
the groups were similar.
Average plasma triglycerides and VLDL concentrations were 13.40 and 16.22
mg/dL and 2.68 and 3.24 mg/dL in group 1 and 2, respectively which were higher
(P<0.01) in group 2 than in group 1.
5.6
EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE
SUPPLEMENTATION ON PLASMA AMINO ACIDS
Aspartate, glycine, alanine, valine, methionine, cysteine, and lysine
concentrations in plasma were increased (P<0.05) in cows fed ration supplemented
with RPM plus RPL. However, RPM plus RPL supplementation lowered (P<0.05)
plasma isoleucine concentration. All other free plasma amino acids i.e. glutamate,
serine, histidine, arginine, threonine, proline, tyrosine, leucine and phenylamine were
unaffected by RPM plus RPL supplementation.
5.7
EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE
SUPPLEMENTATION ON PLASMA HORMONAL PROFILE
__
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_ _ __
169
Summary and Conclusions…
___________________ ______________________________________
The average weekly plasma prolactin and growth hormone concentration were
79.68 and 80.39 ng/L and 6.63 and 6.63 ng/L in group 1 and 2, respectively. There
was no effect of rumen protected methionine and lysine supplementation on plasma
prolactin and growth hormone concentration.
5.8
EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE
SUPPLEMENTATION ON REPRODUCTIVE PERFORMANCE IN
COWS
5.8.1
Calving performance
The average body weight of calves at the time of birth was 25.72 kg in group 1
and 26.00 kg in group 2 which was not affected on supplementing rumen protected
methionine and lysine.
5.8.2
Reproductive abnormalities
There were two cases of premature births in group 1, however, one case was
observed in group 2. Four cases of retention of fetal membranes (RFM) were
observed in group 1 while one case was observed in group 2. Higher incidence of
metritis in group 1 (3 cases) were recorded whereas only one case was observed in
group 2. Two cases of mastitis were observed in group 1, whereas, one such case was
observed in crossbred cows of group 2 during experimental period of 120 days post
partum.
5.8.3
Reproduction related parameters
The average duration for commencement of cyclicity was 65.13 days in group
1 and 64.11 days in group 2, which was similar in both the groups. The service period
was 164.2 and 152.4 days in group 1 and 2, respectively. The service period was
shorter (P<0.01) by 11.8 days in group 2 than that of group 1, indicating that lesser
time was required for the animals in group 2 for conception. Cows of Group 1
required 2.60 AIs per conception while group 2 required 2.67 AIs per conception.
There were no effect of supplementation of RPM plus RPL on no of AI required for
conception. The conception rate during the experimental period of 120 days was
55.55 % in group 1 and 66.66 % in group 2.
5.9
ECONOMICS OF FEEDING RUMEN PROECTED METHIONINE AND
LYSINE IN COWS
__
_____________________________
_ _ __
170
Summary and Conclusions…
___________________ ______________________________________
Net return over feed cost of milk yield per animal per day in control and
treatment group was Rs. 192.1 and 215.8, respectively, indicating that it was higher
by Rs. 23.7 in treatment group over that of control group. The feed cost (Rs.) per kg
FCM produced was Rs. 10.94 and Rs. 10.92, respectively in group 1 and group 2.
While cost benefit ratio was similar (1.08) in both the groups. The net return on
experimental ration was markedly higher by 12.30% than that of the control ration. As
a result, net profit in 120 days from 9 cows on supplementation of rumen protected
methionine and lysine was Rs. 25544.6 over that observed in group1.
CONCLUSIONS
The following conclusions were drawn on the basis of results obtained in the
present study on supplementation of rumen protected methionine and lysine to
crossbred cows during pre and post parturient period.
Prepartum
•
Rumen protected methionine (5g/d) and lysine (20g/d) supplementation exhibited
higher gross apparent difference of changes in body condition score.
•
Intakes of CP, RDP, RUP, MP, TDN, ME and NE L in prepartum cows were
similar while duodenal supply of methionine and lysine was increased on
supplementation of rumen protected methionine and lysine.
•
Plasma triglycerides and VLDL concentration were increased in prepartum cows
on supplementating rumen protected methionine and lysine. Plasma glucose,
phosphatidylcholine, NEFA, vitamin E, cholesterol and BUN were not affected.
•
Plasma amino acids profile of the prepartum cows was not affected on
supplementing rumen protected methionine and lysine while plasma methionine,
cysteine and lysine concentrations tended to increase on supplementation.
Postpartum
•
Body weight loss during early lactation was reduced on supplementation of rumen
protected Methionine (@ 7 gm/d/animal) and rumen protected lysine (@ 60 gm
/d/animal).
•
Improvement in CP, RDP, RUP, MP, TDN, ME and NE L intakes were observed
on supplementation of rumen protected methionine and lysine.
__
_____________________________
_ _ __
171
Summary and Conclusions…
___________________ ______________________________________
•
Average daily milk, FCM and ECM yields were increased on supplementation of
rumen protected methionine and lysine.
•
The efficiency of utilization of DM, CP and TDN for milk and FCM production
was improved in rumen protected methionine and lysine fortified cows.
•
Milk fat and total solid content was increased, whereas milk protein, SNF, lactose,
BUN and milk choline content was similar in both groups on supplementation of
rumen protected methionine and lysine.
•
The proportions of saturated fatty acids, unsaturated fatty acids, MUFA, PUFA in
milk fat were not affected in cows fed ration fortified with rumen protected
methionine and lysine.
•
Plasma triglycerides and VLDL concentration were increased in cows on
supplementating rumen protected methionine and lysine.
•
Plasma aspartate, glycine, alanine, valine, methionine, cysteine, and lysine
concentrations were increased on supplementation of rumen protected methionine
and lysine whereas plasma isoleucine concentration was lowered.
•
Periparturient plasma prolactin and growth hormone concentration were not
affected on supplementing rumen protected methionine and lysine.
•
The birth weight of calves was not affected while cases of premature births were
lowered on supplementation of rumen protected methionine and lysine.
•
Occurrence of parturition related abnormalities like RFM and metritis were
reduced on supplementing rumen protected methionine and lysine.
•
The initiation of cyclicity was not affected but service period was reduced on
supplementing rumen protected methionine and lysine.
•
Feeding rumen protected methionine and lysine to high yielding lactating cows
during early lactation was cost effective.
__
_____________________________
_ _ __
172
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ANNEXURE
Annexure I
Annexure II
NRC 2001 Model
The NRC 2001 model was used for the calculation of NEL, ME, TDN, CP, RDP, UDP,
MP intake. The model is divided into two major components: prediction of requirements and
supply of nutrients. Within this structure, there are submodels for young calves, maintenance,
pregnancy, growth, lactation, dry matter intake, minerals, reserves, energy and protein supply,
amino acids, and diet evaluation. A glossary of the terms used in the equations is given in NRC
(2001).
TDN Calculation for feed and forages
For feeds that are not animal proteins or fats and that do contain some NDF (forages, many byproducts, concentrates), the following equations are used:
TDN = tdNFC + tdCP + (tdFA X 2.25) + tdNDF - 7
Where,
Truly digestible NFC (tdNFC) = 0.98 (100 - [(NDF-NDICP) + CP + EE + Ash]) X PAF
Truly digestible CP for forages (tdCPf) = CP X exp[ -1.2 X (ADICP/CP)]
Truly digestible CP for concentrates (tdCPc) = 1- (0.4 X (ADICP/CP))] X CP
Truly digestible FA (tdFA) = FA Note: If EE < 1, then FA = 0
Truly digestible NDF (tdNDF) = 0.75 X (NDFn – L) X [1 – (L/NDFn) 0.667]
ME and NE L for feed and fodders
If Fat ≥ 3 and if the animal is a dry cow or a lactating cow, then
ME = (1.01 X DiscDE) - 0.45 + (0.0046 X (Fat - 3))
Net energy for lactation for feeds having more than 3% fat is computed.
NE L = (0.703 X ME) - 0.19 + ((((0.097 X ME) + 0.19) /97) X (Fat - 3))
If the feeds have < 3% fat, the equation to compute ME for lactating and dry cows is
ME = (1.01 X DiscDE) - 0.45
The equation to compute the NE L of low fat feeds is:
Annexure II
NE L = (0.703 X ME) - 0.19
Calculations for Protein Supply
Microbial yield (MCP Total) is calculated as a percentage of discounted TDN (TDN_Act_Total):
MCP_Total = 0.13 X TDN_Act_Total
The following equation is used to calculate the amount of crude protein from each feed.
CP X = (Feed X CP /100) X (DMFed X 1000)
To calculate the site of digestion of protein, both passage (kp) and digestion (kd) rates are
needed. Separate passage equations are used for concentrates, dry forages, and wet forages.
Concentrate
Kp = 2.904 + (1.375 X BW_DMI) - (0.02 X PercentConc)
Dry Forage
Kp = 3.362 + (0.479 X BW_DMI) -(0.017 X Feed X NDF) - (0.007 X PercentConc)
Wet Forage
Kp = 3.054 + (0.614 X BW_DMI)
The amount of RDP in a specific feed is calculated using the following equation. It is assumed
that all of Protein A is ruminally available and that none of Protein C is degraded in the
rumen.Thus, only Protein B is affected by digestion and passage rates.
If (Feed X .Kd + Kp) > 0 Then
RDP X = ((Feed X .Kd /(Feed X .Kd + Kp)) X ((((Feed X .PrtB /100) X (Feed X CP /100)) X
Feed X DMFed))) +(((Feed X PrtA /100) X (Feed X CP /100)) X Feed X DMFed)
Otherwise, RDP X _ 0
The amount of ruminally-undegraded proteinis obtained by subtraction:
RUP X = (CP X - (RDP X X 1000)) /1000
If RUP_Total > 0, then DietRUPDigest= Total Digested RUP /RUP_Total
Otherwise, Diet RUPDigest = 0.
Annexure II
The supply for RDP and RUP is calculated in the following equation.
RDPSup =TotalDMFed X 1000 X DietCP X CP_RDP
RUPSup = CP Total - RDPSup
The efficiency of microbial crude protein synthesis can not exceed 0.85.
If MCP_Total > (0.85 X (RDP_Total X 1000)), then
MCP_Total = (0.85 X (RDP_Total X 1000))
MPBalance = (((MPFeed X 1000) + MPBact + MPEndo) - (MPMaint + MPPreg + MPLact +
MPGrowth))
Amino Acids supply
The amino acid supply is calculated using the following equation with Methionine (Met) as an
example. The structure of this equation is similar for all of the amino acids that are considered in
the model.
TMet = TMet + (((DMFed /TotalDMFed) X (CP /100) X ((RUP X X 1000) /CP X )
x (Met /100) X TotalDMFed) X 1000)
Where TMet = Total Metionine, DMFed = quantity of feed X fed,
TotalDMFed =Total dry matter fed,
CP = % Crude Protein, RUP X = RUP in feed X ,
CP X = Crude protein in feed X.
The next step is to calculate the total digestible supply of each amino acid. Below is the equation
for Dig TMet. The equations for the other amino acids have the same format.
Dig TMet = Dig_TMet + (((DMFed /TotalDMFed) X (CP /100) X ((RUP X X 1000) /
CP X ) X (Feed X RUPDigest /100) X (Met /100) X TotalDMFed) X 1000)
Where Dig_TMet = Total digestible Methionine,
RUPDigest = RUP digestibility of feed X
Annexure II
The total essential amino acid supply before the contribution of the microbial protein has been
added (EAATotal Before MP) is calculated.
EAA Total Before MP = (TArg+This+Tile+Tleu + TLys + TMet + TPhe + TThr + TTrp + TVal)
The variables x1and x2 areused in the following sets of calculation softhe total amount of each
amino acid supplied. The equations tocal culate the total amounts of each amino acid follow. In
all equations, it is assumed that:
If EAATotal BeforeMP > 0 then
x1= ((TMet (or other amino acid)/EAATotal Before MP X 100)
Otherwise x1 = 0
If ((RUP_Total X 1000) + EndCP + MCP_Total) > 0 then
x2 = ((RUP_Total X 1000) /((RUP_Total X 1000) + EndCP + MCP_Total)) x 100
Otherwise, x2 = 0
TotalArg = 7.31 + (0.251 X x1)
TotalHis = 2.07 + (0.393 X x1) + (0.0122 X x2)
TotalIle = 7.59 + (0.391 X x1) - (0.0123 X x2)
TotalLeu = 8.53 + (0.41 X x1) + (0.0746 X x2)
TotalLys = 13.66 + (0.3276 X x1) - (0.07497 X x2)
TotalMet = 2.9 + (0.391 X x1) - (0.00742 X x2)
TotalPhe = 7.32 + (0.244 X x1) + (0.029 X x2)
TotalThr = 7.55 + (0.45 X x1) - (0.0212 X x2)
TotalVal = 8.68 + (0.314 X x1)
The total essential amino acid supply is calculated below:
Total EAA = 30.9 + (0.863 X EAATotal Before MP) + (0.433 X MCP_Total)
Total flows of RUP of specific amino acids are calculated below:
TotalRUPArgFlow = 0.863 X TArg
Annexure II
TotalRUPHisFlow = 0.863 X THis
TotalRUPIleFlow = 0.863 X TIle
TotalRUPLeuFlow=0.863 X TLeu
TotalRUPLysFlow = 0.863 X TLys
TotalRUPMetFlow = 0.863 X TMet
TotalRUPPheFlow = 0.863 X TPhe
TotalRUPThrFlow = 0.863 X TThr
TotalRUPTrpFlow = 0.863 X TTrp
TotalRUPValFlow = 0.863 X TVal
Duodenal flow(g/day) is calculated using an equation of the form below for each amino acid.
Methionine is given as an example.
Met Flow = (TotalMet /100) X TotalEAA
The contribution of microbial crude protein and endogenous protein to the amino acid supply is
calculated as follows. The form of this equation is similar for all amino acids.
TotalMCPEndMetFlow = Met_Flow - TotalRUPMetFlow
The next step is to calculate the supply of each amino acid in RUP that is digestible. The form of
the equation for each amino acid is similar to that given for Metinine below:
If TMet > 0, then dTotalRUPMet=TotalRUPMetFlow X (Dig_TMet /TMet)
Otherwise, dTotalRUPMet = 0
The amount of a specific amino acid that is digestible and is of microbial or endogenous origin
then is calculated. Metinine is used as the example but similar calculations are made for all
amino acids.
dTotalMCPEndMet = 0.8 X TotalMCPEndMetFlow
The flow of digestible Metinine, or other amino acids, then is calculated.
Dig_Met _Flow = dTotalRUPMet + dTotalMCPEndMet
Annexure II
The protein in the duodenum must be converted from crude protein to a metabolizable protein
basis. Microbial crude proteinis converted to metabolizable protein with an efficiency of 0.64:
MPBact = 0.64 X MCP_Total
MPFeed = TotalDigestedRUP
MPEndo = 0.4 X EndCP
The next computation is to determine the percent of a specific amino acid of metabolizable
protein.The Methionine equation is similar to those of the other amino acids.
If (MPBact+(MPFeedX1000)+MPEndo) > 0, then
MetPctMP = 100 X (Dig_Met_Flow /(MPBact+ (MPFeed X 1000) + MPEndo))
Otherwise, MetPctMP = 0