DOCTOR OF PHILOSOPHY IN ANIMAL NUTRITION BY · His constant oasis of ideas and passions...
Transcript of DOCTOR OF PHILOSOPHY IN ANIMAL NUTRITION BY · His constant oasis of ideas and passions...
METABOLIZABLE PROTEIN AND ENERGY REQUIREMENTS
FOR LACTATING BUFFALOES FED ON
SILAGE BASED DIETS
THESIS SUBMITTED TO THE
ICAR-NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL
(DEEMED UNIVERSITY)
IN PARTIAL FULFILMENT OF THE REQUIREMENT
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
ANIMAL NUTRITION
BY
SONTAKKE UMESH BALAJI (M.V. Sc. Animal Nutrition)
DIVISION OF DAIRY CATTLE NUTRITION
ICAR- NATIONAL DAIRY RESEARCH INSTITUTE
(DEEMED UNIVERSITY)
KARNAL - 132001 (HARYANA), INDIA
2015
Regn. No. 1091109
Dedicated to
Respected Guide
Acknowledgement
In the first place I would like to record my gratitude to Dr. S. S.
Kundu, Principal Scientist, DCN Division for his supervision, advice and guidance
from the very early stage of my research as well as for giving me unflinching
encouragement and support in various ways. His constant oasis of ideas and
passions exceptionally inspired and enriched my growth as a student and
researcher. I feel lucky to get a mentor like him who helped me at every time
when I was in need of help and guidance of any and at any time. I am really
thankful to him for everything.
I gratefully acknowledge other Members of Advisory committee, Dr.
S. K. Tomar, Dr. Goutam Mondal, Dr. Nitin Tyagi, Dr. Manju Ashutosh and
Dr. R.K.Sharma for their timely help, valuable suggestions and constant
encouragement at various phases of my research work. I would like to express
my thanks to all the faculty members, Dairy Cattle Nutrition Division, for their
suggestion and timely help during my course of study.
I convey special acknowledgement to Dr. A. K. Srivastava, Director,
NDRI, Karnal for his indispensable help and providing necessary facilities for
carrying out this study and financial assistance in the form of NDRI
Institutional fellowship during my Ph.D programme.
I am grateful to Dr. J.P. Sehgal, Principal Scientist and Head, Dairy
Cattle Nutrition Division, for his critical suggestions, constant vigilance and
moral support to carry out this work successfully.
I owe deep sense of indebtedness and sincere gratitude to my seniors in
my lab namely Dr. Vijay Sharma, Dr. Munnendra Kumar, Dr. Asraf Hussain, Dr.
Sanjay Sawant, Dr. Avinash Ghule for their consistent encouragement and
sharing views.
I am thankful to Raj Bahadur ji, Sumit ji, Kalra ji, Vrinder ji, Rishipal ji,
Laxman ji, Amit ji, for all the help rendered during the experimental period. I
am also thankful to all the office staff members of DCN Division for their
timely help.
Thanks to my friends Dr. Sukhjinderjit Singh, Dr. Vishnu Kale, Dr. Anjila
Kujur and Dr. Sudheer Babu for their love and support. My beloved juniors
Bisitha, Tho, Papori, Hujaj, Amit Sharma, Suraj, Lalit Budhani deserve thanks,
whose company made me feel younger.
I would like to extend my special thanks to Mr. Bill Gates and Microsoft
Corp. for the true power of MS EXCEL, MS Word from grammar checks to
replace-all. Without this software, this thesis would not be written.
Indeed the words at my command are not adequate either in form of spirit
to convey the depth of my feelings and gratitude to my parents and brothers for
their blessings, affection, encouragement and unceasing moral support to
accomplish this study. I express my feeling for love, affections and
encouragement given to me by my mother Sou. Satyabhamabai Sontakke.
No appropriate words could be traced in the presently available lexicon to
express my indebtedness and gratitude to Dr.Sonali Prusty for whole hearted co-
operation during the process of research work.
Above all, I thank the almighty for giving me patience and strength to
overcome the difficulties, which crossed my way in accomplishment at this
endeavor.
Place: Karnal
Date: / / 2015 SONTAKKE UMESH BALAJI
CONTENTS
Chapter Title Page
1 Introduction 1-4
2 Review of Literature 5-41
2.1 Feeding value of silage in ruminants 7
2.1.1 Fermentation end products and their effect on silage
quality
7
2.1.2 Chemical composition of silage 9
2.1.3 In vitro study of silage 12
2.2 Enteric methane emissions from ruminants 13
2.2.1 Methane emissions from ruminants fed on silage or hay
based diets
14
2.2.2 Effect of feeding silage based diet on nutrient intake
and nutrient utilization
17
2.3 Metabolizable Energy Concept in ruminant 17
2.3.1 Energy requirements of buffaloes 18
2.3.2 Effect of different metabolizable energy on milk yield
and composition
21
2.3.3 Effect of different metabolizable energy on dry matter
intake of buffaloes
22
2.3.4 Effect of different metabolizable energy on body weight
change
23
2.4 Metabolizable protein concept 23
2.4.1 Metabolizable protein availability from feeds or feed
combinations
26
2.4.2 Protein requirements for maintenance 30
2.4.3 Metabolizable protein requirements for body weight
change in buffaloes
32
2.4.4 Protein requirements for lactation 33
2.4.3 Effect of metabolizable protein supply on milk yield 34
2.4.4 Urinary purine derivatives and creatinine excreation at
varying levels of protein and energy in diet
40
3 Materials and Methods 42-77
3.1 Preparation of silage in plastic jar and evaluation of
silage
42
3.1.1 Collection of fodders and silage preparation 42
3.1.2 Estimation of silage characteristics 42
3.1.3 Preparation of water extract of silage samples 42
3.1.4 Estimation of dry matter in fresh silage samples 43
3.1.5 Determination of lactic acid in silage samples 44
3.1.6 Determination of total-N and NH3-N in fresh silage
samples
46
3.1.7 Estimation of total volatile fatty acids and their
fractionation in fresh silage samples
47
3.2 Estimation of chemical composition and fibre fractions
of silage samples, experimental diets and dung
samples
48
3.2.1 Total ash (TA) 48
3.2.2 Organic matter (OM) 49
3.2.3 Crude protein (CP) 49
3.2.4 Ether extract (EE) 50
3.2.5 Estimation of cell wall constituents 50
3.2.5.1 Neutral detergent fibre (NDF) 50
3.2.5.2 Acid detergent fibre (ADF) 51
3.2.5.3 Cell contents and Hemicellulose 52
3.2.6 Acid detergent lignin (ADL) 53
3.2.7. Estimation of nitrogen 54
3.2.7.1 Determination of acid detergent insoluble nitrogen
(ADIN)
54
3.2.7.2 Determination of neutral detergent insoluble nitrogen
(NDIN)
54
3.2.7.3 Non protein nitrogen 54
3.3 Estimation of total digestible nutrient (% TDN) 55
3.4 In vitro gas production (IVGP) technique 56
3.4.1 Methane production 58
3.4.2 In vitro true dry matter and organic matter digestibility
(IVDMD and IVOMD)
59
3.5 Estimation of utilizable crude protein 60
3.5.1 Estimation of Intestinal digestibility of uCP 61
3.6 In vivo methane trial 62
3.6.1 Selection and grouping of animals 62
3.6.2 Feeding of animals 62
3.6.3 Digestibility trial 62
3.6.4 Collection of methane gas and estimation by SF6
tracer technique
63
3.7 Estimation of metabolizable energy (ME) requirements
for the lactating buffaloes fed on silage based diet
64
3.7.1 Selection, grouping and feeding of animals 64
3.7.2 Location of Experiment 66
3.7.3 Housing and Management of Animals 66
3.7.4 Body Weight and DM Intake 66
3.7.5. Daily Milk Yield 67
3.7.6 Milk Composition 67
3.7.7 Metabolism trial 67
3.7.8 Sampling, processing and storage 67
3.7.9 Analysis of feed, residue, faeces and urine 68
3.8 Estimation of metabolizable protein (MP) requirements
for the lactating buffaloes fed on silage based diet
68
3.8.1 Selection, grouping and feeding of animals 68
3.8.2 Housing and Management of Animals 70
3.8.3 Body Weight and DM Intake 70
3.8.4 Daily Milk Yield 70
3.8.5 Milk Composition 70
3.8.6 Metabolism trial 71
3.8.7 Sampling, processing and storage 71
3.8.8 Analysis of feed, residue, faeces and urine 71
3.8.9 Estimation of Urinary purine derivatives, cratinine and
microbial protein synthesis
72
3.8.9.1 Determination of allantoin 72
3.8.9.2 Determination of uric acid by uricase 74
3.8.9.3 Creatinine estimation 75
3.8.9.4 Calculation of absorbed of microbial purine
concentration
76
3.8.9.5 Calculation of intestinal flow of microbial N 76
3.9 Statistical Analysis 77
3.9.1 Statistical analysis to determine the energy and protein
requirements of Murrah buffaloes for maintenance and
6% FCM
77
4 RESULTS AND DISCUSSION 78-120
4.1 Evaluation of silage quality 78
4.1.1 Chemical composition and organoleptic characteristics
of maize, oat silage and fodders before ensiling
78
4.1.2 Fermentation characteristics of silages 802
4.1.3 In vitro total gas, methane production of maize and oat
silages and respective fodders
81
4.1.4 Estimation of utilizable crude protein (uCP), intestinal
digestibility of uCP and metabolizable protein
82
4.2. Phase II : Estimation of methane emissions from the
dry buffaloes fed on oat hay or silage
86
4.2.1 Chemical composition and nutritive value of
experimental oat hay and oat silage
86
4.2.2 Nutrient intake and digestibility of nutrients in buffaloes
fed on oat hay or silage
87
4.2.3 Energy loss through methane emissions in buffaloes
fed on oat hay or oat silage
89
4.2.4 ME intake at fortnight intervals and prediction of its
requirement for maintenance and body weight change
of non-lactating Murrah buffaloes fed on oat hay or
silage
93
4.2.5 TDN intake at fortnight intervals and prediction of its
requirement for maintenance and body weight change
94
of non-lactating Murrah buffaloes fed on oat hay or
silage
4.2.6 CP intake at fortnight intervals and prediction of its
requirement for maintenance and body weight change
of non-lactating Murrah buffaloes fed on oat hay or
silage
96
4.2.7 DCP intake at fortnight intervals and prediction of its
requirement for maintenance and body weight change
of non-lactating Murrah buffaloes fed on oat hay or
silage
97
4.2.8 MP intake at fortnight intervals and prediction of its
requirement for maintenance and body weight change
of non-lactating Murrah buffaloes fed on oat hay or
silage
99
4.3 Phase III: Estimation of metabolizable energy
requirements of Murrah buffaloes fed on silage
based diet
100
4.3.1 Chemical compositions of maize silage and varying
metabolizable energy level concentrates fed to
lactating buffaloes
101
4.3.2 Effect of varying metabolizable energy level in diet on
body weight lactating buffaloes
101
4.3.3 Fortnightly average dry matter intake (kg/d, kg/100Kg
BW and g/Kg W0.75) of lactating Murrah buffaloes
different levels of metabolizable energy (ME) in diet
102
4.3.4 Productive performance and % feed efficiency of milk
production in lactating Murrah buffaloes fed on varying
ME in the diet
103
4.3.5 Milk composition 103
4.3.5.1 Milk fat content 103
4.3.5.2 Milk protein content 104
4.3.5.3 Milk lactose content 104
4.3.5.4 Milk SNF content 104
4.3.5.5 Milk total solids content 104
4.3.6 Nitrogen Balance 105
4.3.7 Nutrient utilisation of lactating Murrah buffaloes fed
varying ME in diet
105
4.3.8 ME intake at fortnight intervals and prediction of its
requirement for maintenance and 6% FCM of Murrah
buffaloes
105
4.3.9 TDN intake at fortnight intervals and prediction of its
requirement for maintenance and 6% FCM of Murrah
buffaloes
107
4.4. Phase III: Estimation of metabolizable protein
requirements of Murrah buffaloes fed on silage
based diets
108
4.4.1 Chemical compositions of maize silage and varying
metabolizable protein level concentrates fed to
lactating buffaloes
108
4.4.2 Effect on body weight of lactating Murrah buffaloes fed
varying metabolizable protein in diet
109
4.4.3 Fortnightly average dry matter intake (kg/d, kg/100Kg
BW and g/Kg W0.75) of lactating Murrah buffaloes
different levels of metabolizable protein (MP) in diet
109
4.4.4 Productive performance and % feed efficiency of milk
production in lactating Murrah buffaloes fed on different
MP levels in diet
110
4.4.5 Milk composition 112
4.4.5.1 Milk fat content 112
4.4.5.2 Milk protein content 112
4.4.5.3 Milk lactose content 112
4.4.5.4 Milk SNF content 112
4.4.5.5 Milk total solids content 112
4.4.6 Effect of dietary protein levels on urinary purine
derivatives, creatinine and microbial N production in
lactating Murrah buffaloes
113
4.4.7 Nutrient digestibility coefficients in lactating Murrah
buffaloes fed on diets with varying levels of protein
114
4.4.8 Nitrogen dynamics in lactating Murrah buffaloes fed on
diets with varying levels of protein
114
4.4.9 MP intake at fortnight intervals and prediction of its
requirement for maintenance and 6% FCM of Murrah
buffaloes
115
4.4.10 DCP intake at fortnight intervals and prediction of its
requirement for maintenance and 6% FCM of Murrah
buffaloes
116
4.4.11 CP intake at fortnight intervals and prediction of its
requirement for maintenance and 6% FCM of Murrah
buffaloes
117
4.4.12 Comparison of predicted daily energy and protein
requirements of lactating buffaloes with ICAR, 2013
feeding standards
119
5 SUMMARY AND CONCLUSIONS 121-130
5.1 Chemical composition and organoleptic characteristics
of maize, oat silage and fodders before ensiling
122
5.1.2 Fermentation characteristics of silages 122
5.1.3 In vitro total gas, methane production of maize, oat
silages and respective fodders
122
5.1.4 Estimation of utilizable crude protein (uCP), intestinal
digestibility of uCP and metabolizable protein
123
5.2 Estimation of methane emissions from the dry
buffaloes fed on oat hay or silage
123
5.2.1 Energy loss from dry buffaloes through methane
emissions
123
5.2.2 Nutrient requirements of non-lactating Murrah buffaloes 124
5.2.3 Energy and protein requirements for maintenance in
non lactating Murrah buffaloes
124
5.2.4 Energy and protein requirements for body weight 125
change in Murrah buffaloes
5.3 Phase III: Estimation of metabolizable energy
requirements of Murrah buffaloes fed on silage based
diet
125
5.3.1 Effect of varying metabolizable energy level in diet on
body weight and nutrient intake of lactating buffaloes
125
5.3.2 Nutrient digestibility and nitrogen balance in lactating
buffaloes fed on varying ME in diets
125
5.3.3 Effect of varying ME in diets on milk production,
composition and % feed efficiency in lactating Murrah
buffaloes
126
5.3.4 Energy requirements of lactating Murrah buffaloes 126
5.4 Phase III: Estimation of metabolizable protein
requirements of Murrah buffaloes fed on silage based
diets
127
5.4.1 Effect of varying metabolizable Protein level in diet on
body weight and nutrient intake of lactating buffaloes
127
5.4.2 Nutrient digestibility and nitrogen balance in lactating
buffaloes fed on varying MP in diets
127
5.4.3 Effect of varying MP in diets on milk production,
composition and % feed efficiency in lactating Murrah
buffaloes
127
5.4.4 Effect of dietary protein levels on urinary purine
derivatives, creatinine and microbial N production in
lactating Murrah buffaloes
128
5.4.5 Protein requirements of lactating Murrah buffaloes 128
5.5 Conclusions 129
Bibliography i-xxix
LIST OF TABLES
Table No
Title Page No
3.1 Details of solutions for in vitro gas production technique 57
3.2 Detail of experimental non-lactating buffaloes fed on oat
hay or silage for methane emission study
63
3.3 Details of experimental lactating buffaloes fed on varying
metabolizable energy in the diets
63
3.4 Ingredients composition of concentrate varying
metabolizable energy level fed to lactating buffaloes
66
3.5 Ingredients composition of concentrate varying
metabolizable protein level fed to lactating buffaloes
68
3.6 Details of experimental lactating buffaloes fed on varying
metabolizable energy in the diets
69
4.1 Organoleptic characteristics of maize and oat silages
prepared in vitro
78
4.1.1 Chemical composition and energy value of maize, oat
and their silages (% DM)
79
4.1.2 Fermentation end products of maize and oat silages 80
4.1.3 In vitro dry matter digestibility (IVDMD) and methane
production (g/kg) in fodders and silages incubated for 48
hr.
83
4.1.4 Utilizable crude protein, intestinal digestibility (%) of uCP,
metabolizable protein of feeds and fodders
85
4.2.1 Chemical composition (%DM) of oat hay and oat silage
fed to buffaloes for estimation of methane emissions
86
4.2.2 Fortnightly body weight (kg) of dry Murrah buffaloes fed
on oat hay and oat silage
87
4.2.3 Effect of feeding oat hay or oat silage on fortnightly dry
matter intake in dry buffaloes
88
4.2.4 Effect of feeding oat hay or oat silage on intake and
digestibility of nutrients in dry buffaloes
88
4.2.5 Methane emissions and energy loss in buffaloes fed oat
hay or oat silage
89
4.2.6 Effect of feeding oat hay or oat silage on metabolizable
energy (MJ/d) in dry buffaloes
93
4.2.7 Effect of feeding oat hay or oat silage on total digestible
nutrient intake (kg/d) in dry buffaloes
95
4.2.8 Effect of feeding oat hay or oat silage on CP intake (kg/d)
in dry buffaloes
96
4.2.9 Effect of feeding oat hay or oat silage on DCP intake
(g/d) in dry buffaloes
97
4.2.10 Effect of feeding oat hay or oat silage on MP intake (g/d)
in dry buffaloes
99
4.3.1 Chemical compositions of maize silage and varying
metabolizable energy level concentrates fed to lactating
buffaloes
101*
4.3.2 Fortnightly body weight of lactating Murrah buffaloes fed
varying metabolizable energy in diet
101
4.3.3 Average fortnightly dry matter intakes (kg/d, kg/100Kg
BW and g/Kg W0.75) of lactating Murrah buffaloes fed
with varying metabolizable energy in diet
101*
4.3.4 Fortnightly average milk yield (kg/day) and 6% FCMY
(kg/day) in Murrah buffaloes fed with varying
metabolizable energy in diet
103*
4.3.5 Productive performances and feed efficiency for milk
production in Murrah buffaloes fed varying metabolizable
energy level
103*
4.3.6 Fortnightly milk composition in Murrah buffaloes fed with
varying metabolizable energy in diet
103*
4.3.7 Nitrogen balance (g/d) in lactating buffaloes fed on
varying ME in diet
105*
4.3.8 Nutrient digestibility (DM %) of Murrah buffaloes fed on
different ME level
105*
4.3.9 Metabolizable energy intake (MJ/d) in lactating Murrah 106
buffaloes fed on varying ME in the diets
4.3.10 Total digestible nutrient intake (kg/d) in lactating Murrah
buffaloes fed on varying ME in the diets
107
4.4.1 Chemical compositions of maize silage and varying
metabolizable protein level concentrates fed to lactating
buffaloes
109*
4.4.2 Fortnightly body weight of lactating Murrah buffaloes fed
varying metabolizable protein in diet
109
4.4.3 Average fortnightly dry matter intakes of lactating Murrah
buffaloes fed with varying metabolizable protein in diet
109*
4.4.4. Fortnightly average milk yield (kg/day) and 6% FCM yield
(kg/day) in Murrah buffaloes fed with varying
109*
4.4.5 Productive performances and feed efficiency for milk
production in Murrah buffaloes fed varying metabolizable
protein level
111*
4.4.6 Fortnightly milk composition in Murrah buffaloes fed with
varying metabolizable Protein in diet
113*
4.4.7 Effect of dietary metabolizable protein levels on urinary
purine derivatives and creatinine excretion and microbial
N production in lactating buffaloes
113*
4.4.8 Effect of dietary metabolizable protein levels on nutrient
digestibility coefficients (%) and nitrogen dynamics in
lactating buffaloes
115*
4.4.9 MP intake (g/d) in lactating Murrah buffaloes fed on
varying MP in the diets
115
4.4.10 Digestible crude protein intake (kg/d) in lactating Murrah
buffaloes fed on varying MP in the diets
117
4.4.11 Crude protein intake (kg/d) in lactating Murrah buffaloes
fed on varying MP in the diets
118
4.4.12 Comparison of predicted daily energy and protein
requirements of lactating buffaloes with ICAR, 2013
feeding standards
119
* indicates after page
LIST OF FIGURES
Figure No
Title Page No
2.1 Flow diagram of MP system (AFRC, 1992) 24
3.1 Standard curve for allantoin concentration 73
3.2 Standard curve for uric acid concentration 75
4.1 Relationship between neutral detergent fiber intakes
(NDFI), with methane emissions in buffaloes
92
4.2 Relationship between digestible neutral detergent fiber
(dNDFI with methane emissions in buffaloes
92
4.3 Relationship of ME intake (KJ/kg W0.75) with body weight
change (g/kg W0.75) of dry Murrah buffaloes
93*
4.4 Relationship of TDN intake (g/kg W0.75) with body weight
change (g/kg W0.75) of dry Murrah buffaloes
93*
4.5 Relationship of CP intake (g/kg W0.75) with body weight
change (g/kg W0.75) of dry Murrah buffaloes fed on oat
silage
97*
4.6 Relationship of DCP intake (g/kg W0.75) with body weight
change (g/kg W0.75) of dry Murrah buffaloes fed on oat
silage
97*
4.7 Relationship of MP intake (g/kg W0.75) with body weight
change (g/kg W0.75) of dry Murrah buffaloes
100
4.8 The relationship between ME intake (KJ/kg W0.75) and
6%FCM (kg/kg W0.75) in Murrah buffaloes fed on different
levels of ME in diet
107*
4.9 Relationship of TDN intake (g/kg W0.75) with 6%FCM
(kg/kg W0.75) of lactating Murrah buffaloes
107*
4.10 Relationship of MP intake (g/kg W0.75) with 6% FCM
(kg/kg W0.75) of Murrah buffaloes
115*
4.11 Relationship of DCP intake (g/kg W0.75) with 6% FCM
(g/kg W0.75) of Murrah buffaloes
115*
4.12 Relationship of CP intake (g/kg W0.75) with 6% FCM
(kg/kg W0.75) of Murrah buffaloes
118
* indicates after page
LIST OF PLATES
Plate No Title After Page No
3.1. Permeation tube and its parts 63
3.2. ECD detector for estimation of Sulphur hexafluoride
gas during methane estimation
63
3.3. Illustration of the SF6 tracer technique. 63
ABBREVIATIONS
AAT Amino acids absorbed in the small intestine
ADF Acid detergent fiber
ADFI Acid detergent fiber intake
ADG Average daily gain
ADICP Acid detergent insoluble crude protein
ADIN Acid detergent insoluble nitrogen
ADL Acid detergent lignin
ADS Acid detergent solution
AFRC Agriculture and Food Research Council
AH Alfalfa hay
AMP Adequate metabolizable protein
ARC Agricultural Research Council
BC Buffering capacity
BCP Bacterial crude protein
BF Berseem fodder
BG Barley grain
BMR hybrid Brown midrib hybrid corn
BP Beet pulp
bST Bovine somatotropin
BUN Blood urea nitrogen
BW Body weight
BW 0.75 Metabolic body weight
BWC Body weight change
CF Crude fiber
CG Corn grain
CNCPS Cornell net carbohydrate and protein system
CP Crude protein
CPI Crude protein intake
CS Corn silage
CSC Cotton seed cake
CSM Cotton seed meal
CTAB Cetyl trimethyl ammonium bromide
DCP Digestible crude protein
DDM Digestible dry matter
DE Digestible energy
DEI Digestible energy intake
DIP Degradable intake protein
DM Dry matter
DMI Dry matter intake
DMP Deficient in metabolizable protein
dNDF Digestible neutral detergent fiber
DORB Deoiled rice bran
DOMC Deoiled mustard seed cake
ECD Electron capture detector
EDTA Ethylene diamine tetracetic acid
EE Ether extract
ERDP Effective rumen degradable protein
EUN Endogenous urinary nitrogen
FA Fatty acid
FAO Food and Agricultural Council
FCM Fat corrected milk
FE Energy loss through faeces
FEG Fresh elephant grass
FID Flame ionization capture detectors
FM Fish meal
FME Fermentable metabolizable energy
GCM Gliricidia sepium leaf meal
GE Gross energy
GEI Gross energy intake
GLM General linear model
GNC Groundnut cake
GS Grass silage
HE/ HP High energy- high protein
HE/LP High energy-low protein
HED High energy density
HL High protein- low energy
HMPF High metabolizable protein feed
HPF High protein feed
IAEC Institutional Animal Ethics Committee
ICAR Indian council of Agricultural Research
IVDMD In vitro dry matter digestibility
IVGPT In vitro gas production technique
IVNDFD In vitro neutral detergent fiber digestibility
IVTD In vitro true digestibility
IVOMD In vitro dry matter digestibility
Kc Efficiency for growth of conceptus
Kgrowth Efficiency of ME utilization for growth
Klactation Efficiency of ME utilization for lactation
Kmaintenance Efficiency of ME utilization for maintenance
Kt Efficiency for utilization of mobilized body tissue for
lactation
LAB Lactic acid bacteria
LBP Lupin byproducts
LE/HP Low energy-high protein
LE/LP Low energy-low protein
LED Low energy density
LL Low protein-low energy
LMPF Low metabolizable protein feed
MBS Metabolic body size
MCP Microbial crude protein
ME Energy loss through methane
ME Metabolizable energy
MEI Metabolizable energy intake
MEm Metabolizable energy for maintenance
ME+10 Metabolizable energy 10% higher than ICAR (2013)
ME0 Metabolizable energy as per ICAR (2013)
ME-10 Metabolizable energy 10% less than ICAR (2013)
MED Medium energy density
MF Maize fodder
MFN Metabolic fecal nitrogen
MG Maize grain
MMPF Medium metabolic protein feed
MOC Mustard oil cake
MP Metabolizable protein
MP+10 Metabolizable protein 10% higher than ICAR (2013)
MP0 Metabolizable protein as per ICAR (2013)
MP-10 Metabolizable protein 10% less than ICAR (2013)
MPm Metabolizable protein for maintenance
MUN Milk urea nitrogen
NDF Neutral detergent fibre
NDICP Neutral detergent insoluble crude protein
NDIN Neutral detergent insoluble nitrogen
NDRI National Dairy Research Institute
NDS Neutral detergent solution
NEL Net energy lactation
NFC Non fibrous carbohydrate
NPN Non protein Nitrogen
NRC National research council
NSC Non structural carbohydrate
ODM Organic dry matter
OM Organic matter
PA Protein fraction A
PAF Processing adjustment factor
PB Purine bases
PD Purine derivative
PDIE Protein digested in small intestine, when ruminal
fermentable energy is limiting
PDIN Protein digested in small intestine, when ruminal
fermentable N is limiting
PF Partitioning factor
QDP Quickly degradable protein
RDP Rumen degradable protein
RL Rumen liquor
RUP Rumen undegradable protein
SBM Soybean meal
SDP Slowly degradable protein
SF Sorghum fodder
SF6 Sulfer hexafluoride
SNAN soluble non ammonia nitrogen
SNF Solid not fat
SOLP Soluble protein
SPV sweet potato vines
TA Total ash
TCA Trichloroacetic acid
TDN Total digestible nutrients
Tg Teragram
TMR Total mixed ration
TP Tomato pomace
uCP Utilizable crude protein
UDP Undegradable dietary protein
UE urinary energy loss
VFA Volatile fatty acids
WB wheat bran
WCR whole crop rice
WSC Water soluble carbohydrate
W0.75 Metabolic body weight
Abstract
The present study was conducted to determine the metabolizable energy (ME) and
protein (MP) requirements of lactating buffaloes fed on low methane emitting silage based diets.
The evaluation of silages prepared in jar (in vitro) showed non-significant differences in total and
individual volatile fatty acids (mM/100g DM) content of maize and oat silages. Total nitrogen
(%DM) was significantly (P<0.05) higher in oat silage (1.92) than maize silage (1.43) and In vitro
organic matter digestibility (IVOMD) was significantly (P<0.05) higher in oat fodder and silage
(80.53 and 88.07) than maize fodder and silage. The In vitro methane production (g/ kg IVDMD)
was highest (P<0.05) in oat fodder (39.23) and lowest in maize silage (35.24). The methane
production in silages were significantly lower (P<0.05) than their respective fodders. In phase II,
enteric methane emissions was estimated from dry buffaloes (n=8) fed oat hay (T1) or silage
(T2) solely using SF6 technique. No significant difference was observed in the DMI whereas
neutral detergent fiber (NDF) and acid detergent fiber (ADF) intake was significantly (P<0.05)
higher in T1 than T2. The digestibility coefficients of DM, OM, EE, NDF and ADF were
comparable between the groups, except CP digestibility which was higher in oat silage (60.59)
as compared to oat hay (58.28).Enteric CH4 emissions (L/d) was found significantly (P<0.05)
higher in T1 (341.35) than T2 (317.86) group. The ME and TDN requirements for maintenance
and BWC in dry buffaloes were 521.27 kJ, 34.455 g per kg W0.75; 32.651 kJ and 2.1581 g per kg
W0.75 respectively. CP, DCP and MP requirements for maintenance and BWC in dry buffaloes
were 5.26, 3.06 and 2.9866 g/kg W0.75; 0.3614, 0.2106 and 0.185 g for g BWC/ kg W0.75/day
respectively. In phase III, two separate experiments were conducted on lactating buffaloes, fed
on low methane emitting silage based diets to determine ME and MP requirements. In each trial
15 mid lactating Murrah buffaloes were divided into 3 groups based on milk yield (MY) and body
weight (BW), and fed on maize silage and concentrate in 60: 40 ratio. Both feeding trials were
conducted for 75 days. In first trial, buffaloes were fed on iso-nitrogenous diets varying in10% as
per ICAR (2013). Similarly, in second trial buffaloes were fed iso-caloric diets varying in 10%
MP levels. The results of 1st trial of varying metabolizable energy in diets did not show any
significant difference in milk yield and 6%FCM of ME0 and ME+10, (8.84,8.74 and 9.99, 9.88
kg/d) whereas it was significantly lower (P<0.05) in ME -10, (8.48; 9.64 kg/d) in comparison to
ME0 and ME+10. The DMI, BW, milk composition and nutrient digestibility did not differ among
the treatments (P>0.05). Based on regression equations, ME and TDN requirement for
maintenance and 6% FCM (per kg) during lactation were 533 KJ and 36.27g per kg BW0.75 and
6634 KJ and 438.51g, respectively. In 2nd feeding trial, DMI, BW, milk production (kg/d) and milk
composition did not differ (P>0.05) among the groups fed on different MP levels in the diets. CP
digestibility coefficient was found to be significantly higher in MP0 (66.23) and MP+10 (66.65) than
that of MP -10 (63.61). N excretion in faeces and N outgo in milk was not affected by the different
level of MP in the diet but the urinary excretion of nitrogen increased with the increase in N
intake in the diet. Based on data generated during 2nd trial and its regression analysis, the
maintenance requirements (g/kg W0.75) for MP, CP and DCP were 2.56, 5.02 and 3.19 g
respectively and the corresponding requirements for production of 1 kg of 6% FCM were 66.78,
116.05 and 71.77 g, respectively. Thus, it could be concluded that feeding of silage in ruminants
can reduce total enteric methane production by (6.86%) as compared to hay. The maintenance
and 6% FCM production requirement were 533 kJ/kgBW0.75 and 6634kJ/kg FCM for ME and
2.56g/kgBW0.75 and 66.78 g/kgFCM for MP, respectively.
ससलेज आधाररत आहार पर दधुारू भैंसों की उपापचयी ऊजाा व प्रोटीन की आवश्यकता
शोधार्थी मुख्य मार्ादशाक ववभार्
सोनटक्के यू. बी. डॉ. एस. एस. कुां डू डी. सी. एन.
साराांश
इस वर्तमान अध्ययन का उद्देश्य दधुारू भैंसों की उपापचयी ऊर्ात व उपापचयी प्रोटीन की आवश्यकर्ा को कम ममथने उत्सर्तन वाले साइलेर् (silage) खिलाकर ननधातररर् करना था l र्ार में र्यैार मक्का व र्ई साइलेर् के मलू्याकन पर उनके कुल व वाष्पशील वसीय अम्ल (mM/ १०० ग्राम शषु्क पदाथत) में कोई महत्वपरू्त अरं्र नही पाया गया l हालााँकक कुल नाइट्रोर्न ( % शषु्क पदाथत) र्ई साइलेर् (१.९२) में मक्का साइलेर् (१.४३) की र्लुना में अधधक पाई गई (P<०.०५) और अरं्ःपात्र (in-vitro) कार्तननक पदाथत पचनीयर्ा (IVOMD) भी र्ई साइलेर् व चारे (८०.५३ व ८८.०७) में मक्का साइलेर् व चारे से अधधक पाई गई (P<०.०५) l अरं्ःपात्र (in-vitro) ममथेन उत्पादन (ग्राम / ककलो IVDMD) र्ई चारे (३९.२३ ) में सर्से अधधक (P<०.०५) और मक्का साइलेर् में सर्से कम पाई गई (३५.२४) l ममथेन उत्पादन साइलेर् में उनके सम्र्धंधर् चारे की र्लुना से (P<०.०५) काफी कम थी l कई र्रह के अनार्ों में uCP (% शषु्क पदाथत) र्ई में (९.९६) सर्से अधधक और र्ार्रा में (५.२१) सर्स े कम था l उपापचयी प्रोटीन ववमभन्न र्रह के चारे र्ैस े मक्के का चारा व इसकी साइलेर्, र्ई का चारा व इसकी साइलेर् और र्ई की सिूी घास में ६.३६ से लेकर ८.४६ % शषु्क पदाथत र्क पाया र्ार्ा है l दसुरे चरर् में, शषु्क (dry) भसैों को दो समहूों (प्रत्येक समहू में n = 8) में परूी र्रह र्ई की सिूी घास (T1) या र्ई का साइलेर् (T2) खिलाकर SF6 र्कनीकी द्वारा आन्र्ररक ममथेन उत्सर्तन का अनमुान लगाया गया l दोनों समहूों की भैंसों के प्रनर्ददन शषु्क पदाथत ग्रहर् (DMI) करने में कोई महत्वपरू्त अरं्र नही था , ककन्र् ुNDF और ADF T2 की र्लुना में T1
में र्हुर् अधधक था (P<०.०५)l शषु्क पदाथत (DM), कार्तननक पदाथत (OM), ईथरी सत्व (EE), NDF और ADF का पाच्य गरु्ांक दोनों समहूों में र्लुनात्मक था, लेककन CP का पाच्य गरु्ांक र्ई साइलेर् (६०.५९) में र्ई की सिूी घास (५८.२८) स ेअधधक था l समहू T1 (३४१.३५) में आन्र्ररक ममथेन उत्सर्तन (लीटर प्रनर्ददन) समहू T2 (३१७.८६) की र्लुना में र्हुर् अधधक (P<०.०५) पाया गया l शषु्क भैंसों में शारररक भार में र्दलाव (BWC) व रिरिाव के मलए उपापचयी ऊर्ात और कुल पाच्य पोषक र्त्व की अवश्यकर्ा क्रमशः ५२१.२७ kJ व ३४.४५५ ग्राम प्रनर् ककलो W0.75 और ३२.६५१ kJ व ०.२१५८१ ग्राम प्रनर् ककलो W0.75 पाई गई l शषु्क भैंसों में शारररक भार में र्दलाव ( BWC ) व रिरिाव के मलए CP, DCP व उपापचयी प्रोटीन (MP) क्रमशः ५.२६, ३.०६ व २.९८६६ ग्राम प्रनर् ककलो W0.75 और ०.३६१४, ०.२१०६ व ०.१८५ ग्राम प्रनर् ककलो BWC प्रनर् ककलो W0.75 प्रनर्ददन अननवायत पायी गई l र्ीसरे चरर् में, दधुारू भसैों में उपापचयी ऊर्ात व उपापचयी प्रोटीन की अवश्यकर्ा को ननधातररर् करने के मलए कम ममथेन उत्सर्तन वाले साइलेर् आधारीय आहार पर दो अलग – अलग प्रयोग ककय ेगये l प्रत्येक परीषण र् में दधु ध उत्पादन व शारररक भार के आधार पर १५ मध्य दधु ध स्त्त्रवर् वाली मरुातह भैंसों को ३ समहूों में ववभाजर्र् करके मक्का साइलेर् और दाना ६०:४० के अनपुार् में खिलाया गया l दोनों खिलाने वाले परीषण र्ों को ७५ ददनों के मलए आयोजर्र् ककया गया l पहले परीषण र् में भैंसों को सम- नाइट्रोर्न आहार खिला कर उपापचयी ऊर्ात स्त्र्र में १० % र्क र्दलाव ककया गया l ठीक उसी र्रह दसूरे परीषण र् में भैंसों को सम-उर्ातयकु्र् आहार खिलाकर
उपापचयी प्रोटीन में १० % र्क का र्दलाव ककया गया l पहले परीषण र् में र्र् की उपापचयी ऊर्ात (ME0 और ME+10) खिलाने के पररर्ामस्त्वरूप दधु ध उत्पादन और ६ % वसा पररवनर् तर् दधु ध (FCM) में (८.८४, ८.७४ व ् ९.९९,९.८८ ककलो/ ददन क्रमशः) कोई साथतक अरं्र नही पाया गया र्र्कक उपापचयी ऊर्ात (ME -10) खिलाने के कारर् दधु ध उत्पादन और ६ % वसा पररवनर् तर् दधु ध (FCM) में र्हुर् कमी पाई गई l इस प्रयोग के दौरान प्रनर्ददन शषु्क पदाथत ग्रहर्, शारीररक भार, दधु ध सघंटन और पोषक र्त्व की पचनीयर्ा में कोई अरं्र नही पाया गया (P<०.०५) l दधु ध स्त्त्रवर् के दौरान ६ % वसा पररवनर् तर् दधु ध (FCM) और रिरिाव के मलए समश्रायर् समीकरर् के आधार पर उपापचयी ऊर्ात और कुल पाच्य पोषक र्त्व क्रमशः ५३३ KJ व ३६.२७ ग्राम प्रनर् ककलो W0.75 और ६६३४ KJ व ४३८.५१ ग्राम की अवश्यकर्ा पायी गयी l दसुरे परीषण र् में, आहार में उपापचयी प्रोटीन स्त्र्र का समहूो में र्दलाव करने पर प्रनर्ददन शषु्क पदाथत ग्रहर्, शारीररक भार, दधु ध उत्पांदन (ककलो प्रनर्ददन) और दधु ध सघंटन में कोई अरं्र नही पाया गया l CP पाच्य गरु्ांक MP0 (६६.२३) और MP+10 (६६.६५) का MP-10 (६३.६१) की र्लुना में अधधक साथतक पाया गया l आहार में कई स्त्र्र के उपापचयी प्रोटीन देने से गोर्र में नाइट्रोर्न उत्सर्तन और दधू में नाइट्रोर्न व्यय पर कोई प्रभाव नही पाया गया, लेककन र्ैसे ही आहार में नाइट्रोर्न ग्रहर् अधधक हुआ वसेै ही मतू्र में नाइट्रोर्न उत्सर्तन भी अधधक पाया गया l दसुरे परीषण र् के दौरान प्राप्र् र्थ्यों और इसके समश्रायर् ववश्लेषर् (regression analysis) के आधार पर MP, CP और DCP की रिरिाव के मलए आवश्यकर्ा क्रमशः २.५६, ५.०४ और ३.१९ ग्राम प्रनर् ककलो W0.75 पाई गई र्था इसी के अनरुूप १ ककलो ६ % वसा पररवनर् तर् दधु ध उत्पांदन के मलए MP, CP और DCP की आवश्यकर्ा क्रमशः ६६.७८ , ११६.०५ और ७१.७७ ग्राम पाई गयी l इस प्रकार यह ननष्कषत ननकला र्ा सकर्ा है कक र्ुगाली करने वाले पशयुों के आहार में सिूे घास की र्लुना में साइलेर् खिलाने से कुल आन्र्ररक ममथेन उत्सर्तन को ६.८६ % र्क कम ककया र्ा सकर्ा है र्था रिरिाव व ६ % वसा पररवनर् तर् दधु ध (FCM) के मलए उपापचयी ऊर्ात व उपापचयी प्रोटीन की आवश्यकर्ा क्रमशः ५३३ kJ/kg BW0.75 व ६६३४ kJ/kg FCM और २.५६ g/ kg BW0.75 व ६६.७८ g/ kg FCM पायी गयी l
शोधार्थी मुख्य मार्ादशाक
CHAPTER – 1
Introduction
Page | 1
INTRODUCTION
Buffalo (Bubalus bubalis) is popular in several parts of the world because
of its superior quality of milk, better ability to adapt to hot and humid climate and
a greater capacity to use forages with high crude fiber (CF) content (ICAR,
2013). The buffalo is an important contributor to milk, meat, power, fuel and
leather production in India. The buffalo is being suggested as the future species
to meet continuously increasing demands for quality milk and meat. Global
buffalo population is estimated to be 185.29 million, out of which 97 % are in
Asia (FAO, 2008). India has 105.1 millions buffaloes; they comprise
approximately 56.7 percent of the total world buffalo population and 53.4
percentage of milk production in India (Planning Commission, 2012). Buffaloes
like other domesticated ruminants largely meet their protein and energy
requirements from rumen fermentation end products mainly, microbial protein
and volatile fatty acids. In recent past, comparative studies on the digestive
physiology and nutrient requirements of buffalo with other species such as cattle
and sheep have been reported (Puppo et al., 2002, Paul et al., 2003, Wanapat
and Rowlinson, 2007). A greater ruminal degradation of both fiber and protein
was noticed in buffaloes than in cattle and sheep. This unique ability to better
ferment fiber in buffaloes could be the result of adaptation because for years
they have been fed on low quality high fibrous feeds (Sarwar et al.,
2005).Particularly in India, energy and protein demands of buffaloes are being
mainly met by feeding them low-quality roughages, agricultural crop-residues
and industrial by-products which contain high levels of lingocellulosic materials,
low levels of fermentable carbohydrate and protein (Kundu et al., 2004)
The critical constraint in profitable buffalo production is the inadequacy of
quality forage (Touqir et al., 2007). In Asia, due to low per acre yield and lower
area under fodder production, the available fodder supply is much less than
actually needed (Sarwar et al., 2009). Low per acre fodder yield coupled with two
important fodder scarcity periods, one during summer and other during winter
months aggravates the fodder availability situations (Khan et al., 2006a). Regular
Introduction
Page | 2
supply of forage for buffaloes could be achieved by ensiling, when the fodders
are available in surplus.
During 1985-86 to 2005-2006, the country as a whole recorded 52.0,76.0
and 1.8% increase in crop residues (240.7 to 365.8 million tonnes (mt)),
concentrates (19.6 to 34.5 mt) and green forage (124.3 to 126.6 mt), respectively
(Planning Commission,2012). In spite of this, the country faces a net deficit of
62.8% green fodder, 23.5% dry crop residues and 65% compounded feeds
(ICAR, 2013). In India, bovines are mainly fed straw based diets supplemented
with the locally available concentrate feed ingredients. Such type of feeding
practices are likely to provide imbalanced rations in terms of protein and energy,
and however result into very inefficient nutrient utilization or over feeding and
imbalanced feeding may also cause drop in productivity and metabolic disorders
leading to significant economic losses. To harness optimum production or
reproduction performance in buffaloes, a balance nutrient supply and the
appropriate employing standards is of great importance.
Feed energy supply to ruminants often remains limiting under tropical
conditions. Total digestible nutrients (TDN), have been long in use to indicate
energy content of a feed as well as energy requirement of animals. The
estimations of crude fiber and nitrogen free extract in a feed are having inherent
analytical problems. A number of studies conducted in India and elsewhere
proved that the TDN determination by using cell wall fractions is more accurate
and it can also be used, for Indian feeds. The metabolizable energy (ME)
determination includes the corrections due to losses in urine as well as methane.
Thus ME adaptation may results to a more precise expression of energy
utilization efficiency. Thus in the present study ME has been chosen as the
criteria for determination of energy requirement.The existing feeding standard
(ICAR, 2013) have adopted the total digestible nutrient (TDN) and metabolizable
energy (ME) values for expressing nutrient requirement of cattle and buffaloes.
The concept of digestible crude protein (CP) for ruminants suffers due to
its limitations to define extensive degradation of dietary protein in rumen and
synthesis of substantial amount of microbial protein and availability of both
microbial protein and rumen undegradable protein (RUP) at intestinal level. NRC
(1996) defined metabolizable protein as the true protein which is absorbed by
Introduction
Page | 3
the intestine and supplied by both microbial protein and protein which escapes
degradation in the rumen; the protein which is available to the animal for
maintenance, growth, fetal growth during gestation and milk production.
Metabolizable protein systems (Burroughs et al., 1974; ARC, 1984) define the
animal’s requirement using estimates of available microbial and dietary escape
protein and thus are potentially more accurate than the digestible CP and CP
systems. In addition, MP system is also a better predictor of milk yield than CP
(Schwab and Ordway, 2004; Das et al., 2014). Replacing conventional CP
system with MP system provides better idea to define protein utilization and diet
formulation as this system fits with the biology of ruminants. Thus in the present
study MP has been chosen as the criteria for determination of protein
requirement in lactating buffaloes.
Ruminants produce methane during the fermentation of feed in the
rumen. Enteric methane emission from ruminants is a challenge currently being
faced by dairy industry as it causes not only dietary energy loss but also
contributes to green house gas levels. To mitigate the methane production from
ruminants and in particular from dairy animals, feeding strategies need to be
studied. Studies conducted on lactating buffaloes under different agro climatic
regions have revealed that ration balancing also helps in reducing the enteric
methane emission per kg of milk production (Kundu et al., 2015). Adequate
shortage of feeds and fodder during the extreme climate in India has resulted
increased demand of preserving feeds and fodder in the form of hay and silage
to maintain optimum production throughout the year. Most of the studies have
compared methane emissions on diets with different proportions of concentrates
and roughages (Pedreira et al., 2013). However, scanty literature is available on
effect of silage or hay as sole feed in ruminant. Therefore, the current study was
carried out to investigate the effect of feeding oat silage or oat hay on methane
emissions from dry buffaloes.
The scientific data to determine energy and protein requirements, their
utilization from different sources at various physiological stages in buffaloes is
scarce. In contrast to high producing western dairy cattle where much attention
has been paid to develop energy and protein standards and nutrient requirement
models, no such planned efforts have been made to establish protein or energy
Introduction
Page | 4
needs in buffaloes. Conflicting results have been reported by various workers on
the level of energy and protein required in buffalo diets during lactation and
growth (Puppo et al., 2002, Paul, 2011). ICAR, (2013) feeding standard gave the
similar nutrient requirements of cattle and buffaloes in terms of metabolizable
protein as still more validations through the different feeding trials are required to
establish separate standard for buffaloes. A precise knowledge of ME along with
MP requirements for maintenance and lactation are of prime importance for the
precise feeding of lactating buffaloes.
Taking into consideration of above facts, a research work was planned on
lactating buffaloes with following objectives
1. To evaluate different fodder silages by in-vitro techniques
2. To study the methane emission on feeding selected silage based diets
3. To determine metabolizable protein and energy requirements of lactating
buffaloes
CHAPTER – 2
Review of Literature
5 | P a g e
REVIEW OF LITERATURE
Buffalo (Bubalus bubalis), ruminant animal contributing to the integrated
farming systems, as a source of draft power, transportation, on-farm manure,
meat, milk and livelihood of the farmers. Buffaloes like other domesticated
ruminants meet their protein and energy requirements from fermentation end
products (microbial protein and volatile fatty acids). For many years, crude
protein (CP) content has been used in formulating diets for ruminants because
little was known regarding the response to dietary protein of varying quality. In
addition, many researchers postulated that the high quality microbial protein
(MCP) synthesized in the rumen would complement deficiencies in the quality of
dietary protein that escaped ruminal fermentation. However with advent of
sophisticated nutrition models like CNCPS, NRC, ARC, CPM-Dairy and Amino
Cow; ration formulation has moved from balancing diets from CP to MP, a
concept that describes the protein requirements of ruminants at intestinal level,
and which is available to animals for useful purposes. According to Van Soest
(1994); metabolizable protein is defined as the amount of true protein or amino
acids absorbed in the small intestine and specifically in ruminants, is represented
by the amount of amino acids or protein of microbial or dietary origin absorbed
from the intestine slender. The MP system represents the extent of protein
degradation in the rumen and the synthesis of microbial protein as variable
functions. The system also provides a more rational description of the energy
available for microbial growth (Beever and Cottrill, 1994). Another advantage
with MP system is that it provides a framework with which the net absorption of
amino acids from the small intestine can be computed in relation to the animal’s
requirement (Beever, 1996). So replacement of conventional CP system with MP
system seems to be a better idea to define and refine protein utilization and diet
formulation as this system fits with the biology of ruminants (NRC, 2001). Hence
formulating ruminant diets for MP, RDP and RUP instead of CP only emerges as
the most precise measure of protein nutrition (Varga, 2007).
Feeding standards for buffaloes are not clearly defined, there being wide
differences (as great as 40%) in nutrient requirements prescribed by various
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feeding standards. Most of the existing standards for buffaloes (Sen et al. 1978;
Kearl, 1982; Pathak and Verma, 1993) are based on one, or only a few feeding
trials. Because of their small, restricted database, these standards do not reflect
requirements for widely different planes of nutrition, quality of feed or individual
variation in animals requirements under tropical conditions. The scientific data to
explain energy and protein requirements, their utilization from different sources
at various physiological stages in buffaloes is scarce in comparison to high
producing western dairy cattle. Recent review highlights the topic shown below
2.1 Feeding value of silage in ruminants
2.1.1 Fermentation end products and their effect on silage quality
2.1.2 Chemical composition of silage
2.1.3 In vitro study of silage
2.2 Enteric methane emissions from ruminants
2.2.1 Methane emissions from ruminants fed on silage or hay based
diets
2.2.2 Effect of feeding silage based diet on nutrient intake and nutrient
utilization
2.3 Metabolizable Energy Concept in ruminant
2.3.1 Energy requirements of buffaloes
2.3.2 Effect of different metabolizable energy on milk yield and
composition
2.3.3 Effect of different metabolizable energy on dry matter intake of
buffaloes
2.3.4 Effect of different metabolizable energy on body weight change
2.4 Metabolizable protein concept
2.4.1 Metabolizable protein availability from feeds or feed combinations
2.4.2 Protein requirements for maintenance
2.4.3 Metabolizable protein requirements for body weight change in
buffaloes
2.4.4 Protein requirements for lactation
2.4.5 Effect of metabolizable protein supply on milk yield
2.4.6 Urinary purine derivatives and creatinine excretion at varying levels
of protein and energy in diet
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2.1 Feeding value of silage in ruminants
The critical constraint in profitable buffalo production is the inadequacy of
quality forage (Touqir et al., 2007). In Asia, due to low per acre yield and
minimum area under fodder production, the available fodder supply is much less
than actually needed. Low per acre fodder yield coupled with two important
fodder scarcity periods (one during summer and other during winter months)
further aggravates the fodder availability situations (Khan et al., 2006a,b,c).
Constant supply of forage for buffaloes could be achieved by ensiling when the
fodders are abundantly available (Sarwar et al., 2005). Silage making is a good
method of conserving green fodder. Silage is the material produced by controlled
fermentation of green forages with high moisture content. The purpose is to
preserve forages by natural fermentation by achieving anaerobic conditions and
discouraging clostridial growth. The ideal characteristics of material for silage
preservation are: an adequate level of fermentable substrate (8-10 per cent of
DM) in the form of water soluble carbohydrate (WSC); a relatively low buffering
capacity and DM content above 200 g/kg (Jianxin, 2003)
2.1.1 Fermentation end products and their effect on silage quality
One of the on-farm methods to estimate whether optimum fermentation
has taken place is to measure the pH of the silage. Normal pH values range
between 3.7 and 4.5, depending on certain crop characteristics. Maize silage
that is well preserved will have a pH range of 3.7-4.2 whereas Lucerne silage,
with a higher BC, will fall in the range of 4.2-5 (Seglar, 2003; Kung, 2001). A
low pH is an indication of good LAB fermentation that resulted in the optimum
production of lactic acid to inhibit the growth of unwanted microorganisms such
as clostridia and enterobacteria. Bad fermentation will therefore result in a high
end pH of up to 7.5. This can be due to whole array of factors such as lack of
WSC, growth of clostridia and enterobacteria or even cold conditions (Kung
and Stokes, 2001). A high pH due to clostridia will result in reduced intakes by
ruminants due to butyrate production. It was found that a low silage pH will
reduce intake. This is not due to the pH of the silage affecting the rumen pH
thereby reducing the cellulolytic activity, however, but rather to the organic
acids affecting the palatability of the silage. Silage pH however is affected by
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organic acids (Kung, 2001). Cherney et al. (2004) reported the pH of ensiled
samples was positively correlated with silage DM and increased 0.016 pH
units for each 1.0% increase in DM.
The organic acids, namely lactic, acetic, propionic and butyric acid also
have to be analysed to fully understand the type of fermentation that took
place. Lactic acid is the most important organic acid regarding silage quality
since it is the most abundant, contributing 65 to 70% to the total acids. It is
also stronger than the VFA’s and therefore has the greatest effect on the
silage pH (Kung, 2001; Seglar, 2003). It can range anywhere from 3-8% for
lucerne silage and 4-7% for maize silage on a DM basis. High moisture maize
on the other hand can have lactic acid concentrations ranging between 1-3%
(Kung and Stokes, 2001; Seglar, 2003). Acetic, propionic and butyric acids are
the VFAs contributing to the decline in pH during fermentation, giving silage its
characteristic smell and contributing to the aerobic stability of silage during the
feed-out phase. The VFA that has the biggest impact on aerobic stability is
acetic acid and is found at concentrations up to 3% (Kung, 2001; Danner et al.,
2003; Filya, 2003; Muck, 2010). A high level of acetic acid is not ideal since the
production thereof will result in a loss of carbon, thereby resulting in DM
losses. The ratio of lactic acid to acetic acid is an important factor to take into
consideration since lactic acid can be fermented to acetic acid (Filya, 2003). A
lactic acid to acetic acid ratio of 3:1 indicates that optimum fermentation took
place (Kung and Stokes, 2001). Propionic acid levels in well-fermented silage
will be less than 0.5%, with butyric acid being undetectable (< 0.1%). High
butyric acid levels, resulting in a rancid smell, are an indication of secondary
fermentations that will result in lower energy levels, reduced DM intakes and
also DM losses. It is also an indication that extensive protein degradation had
taken place, which would increase the fraction of soluble protein. There seems
to be controversy around the effects that these acids have on ruminant DMI,
with butyric acid being the first acid identified to reduce ruminant DMI. A lot of
studies were done to determine the effect of the other VFAs and lactic acid on
the DMI, but little correlation was found and results were inconsistent
(Charmley, 2001). Ethanol is another factor contributing to the acidity of silage
and is a result of yeast activity that can metabolise lactic acid, resulting in a
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high end pH. The amount of ethanol produced depends on the crop ensiled. In
maize, silage the optimum level is 1-3% with legume and grass silage being
<1.5% (Kung and Stokes, 2001; Seglar, 2003).
Ammonia concentration is also an important fermentation factor
contributing to silage quality; it is expressed as a percentage of the CP content
of the silage. Elevated levels of ammonia indicate that extensive protein
breakdown occurred, either due to a high end pH, which resulted in proteolytic
plant enzymes not being deactivated, or due to clostridial fermentation which,
again, will also result in butyric acid production. Lucerne and grass silage with
a higher CP level will have ammonia content of up to 15% of CP whereas
maize with lower CP will contain less than 10% of CP (Kung and Stokes 2001;
Seglar, 2003). Charmley (2001) found that an increase in ammonia will result in
reduced DM intakes in dairy cattle.
2.1.2 Chemical composition of silage
Moisture content is the first analysis done to characterise the
fermentation that has taken place and therefore the quality of the silage. The
optimum moisture content is critical for effective packing of the silo to exclude
air as fast as possible and for the effective growth of LAB (McDonald et al.,
2002; Knicky 2005). High moisture content (above 75%) can prolong
fermentation, which, in turn, will lower the energy content, increase the risk of
secondary fermentation and lead to excessive break down of plant proteins,
thereby increasing the non-protein-N (NPN) fraction. Excess air, on the other
hand, will be trapped in the silo if the moisture content is too low, which will
lead to secondary fermentation resulting in DM losses. The optimum DM for
maize at harvesting for silage can range from 30 to 45% with high-moisture
maize being as low as 25% (Beukes, 2013). Lehtomäki (2006) found that dry
matter (DM) and organic dry matter (ODM) concentrations of ensiled crops
were in general lower than those of fresh materials.
Oat forage intended for silage can be harvested at boot, milky dough or
soft dough stage. When cut at boot stage, oat silage has low DM, high
palatability, high energy and high protein content. Wilting is necessary to
reduce moisture and to prevent sewage during ensiling (Mickan, 2006). When
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cut at dough stage, oat silage has greater DM and higher energy value but
lower protein and palatability (Mickan, 2006). The choice of harvesting stage
should be guided by animal requirements: boot stage when high nutritive value
is required (such as for lactating dairy cows), and soft dough stage when
forage quantity is required (late pregnancy for instance) (Barnhart, 2011).
Wether to cut oat at milky dough stage or soft dough stage, is much debated
and seems to depend on local conditions and requirements.
When silage is done at dough stage, water soluble carbohydrates are
lower and fermentation does not start easily. Because the thickness and
hollowness of oat stems impedes the compressing necessary for anaerobic
conditions, it is recommended to chop oat forage to 10-20 mm length (Suttie et
al., 2004). Urea, enzymes and inoculants have been shown to improve aerobic
stability, pH drop, lactic acid production in oat silage and to improve feed
intake and milk production (Meeske et al., 2002).
Oat silage, like other whole-crop cereal silages, differs from grass silage
in that the NDF concentration does not increase after heading, but remains
constant or even decreases whereas starch content increases, resulting in OM
digestibility values that remain high after heading (Nadeau, 2007; Wallsten et
al., 2009 ). However, OM digestibility decreases with maturity (from 68% at
heading to 61-63% at early milk or early dough stage), which has been
explained by a decrease in NDF digestibility (from 70% at heading to 51% at
early dough stage). This is compensated by an increase in starch content from
7 to 14% DM (Wallsten et al., 2010). The DM intake in 350 kg dairy heifers fed
only oat silage increased with plant maturity (from 1.6 kg/100 kg LW at
heading or early milk stage to 2.0 kg/100 kg LW at early dough stage), due to
the low water content of the silage in the earlier stages (Wallsten et al., 2009).
The digestibility of oat silage is generally lower than that of barley silage,
as it was observed with sheep (McCartney et al., 1994), with dairy cows fed a
total mixed ration (50% concentrate, Khorasani et al., 1993), and with heifers
fed only silage ( Christensen et al., 1977b; Wallsten et al., 2010). The
differences in in vivo OM digestibility between oat and barley silages were
similar with sheep (61 vs 66%, McCartney et al., 1994) and heifers
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(63 vs 68%, Wallsten et al., 2010). This difference was attributed to a lower
digestibility of NDF, which could be due to a lower seed DM in oat compared to
barley (McCartney et al., 1994) and to a lower starch content (Nadeau, 2007).
Intakes of oat or barley silages were found to be similar, for instance in heifers
fed a limited amount of rolled barley (McCartney et al., 1994), and in mid-
lactation cows fed a total mixed ration (50% concentrate, Khorasani et al.,
1993). However, oat silage intake was lower than that of barley with sheep fed
only silage (McCartney et al., 1994) or with early lactation cows fed a total
mixed ration (Khorasani et al., 1993). In steers fed only silage, oat silage
intake could be higher than for barley silage (Christensen et al., 1977a). Milk
production of dairy cows was similar between oat or barley silage but milk
protein and lactose content were lower with oat silage (Khorasani et al., 1993).
Oat silage also gave lower average daily gains in growing steers (Oltjen et al.,
1980). It has been concluded that in contexts where oat outyields barley, oat
may be a more economical crop when supplemented with grain (McCartney et
al., 1994).
Compared with other whole crop silages, OM digestibility of oat silage
has been reported to be lower than that of triticale silage in sheep whereas oat
silage intake was generally higher in both heifers and sheep (McCartney et al.,
1994). However, in dairy cows fed a high amount of concentrate, no
differences in either OM digestibility or ingestion could be observed (Khorasani
et al., 1993). Oat silage had a lower in vitro OM digestibility compared to wheat
silage (Nadeau, 2007) but a higher ingestion in steers (Christensen et al.,
1977a). In dairy cows fed a high amount of concentrate, the digestibility and
intake of oat silage were lower than those of alfalfa silage (Khorasani et al.,
1993). In steers fed only silage, the digestibility of oat silage was lower than
that of maize silage (Christensen et al., 1977a) but higher than that of rye
silage (Christensen et al., 1977b).
Oat hay:
The digestibility of oat hay by sheep was comparable to that of triticale
hay, higher than that of rye hay and lower than that of barley hay harvested at
comparable stage. DM intake of oat and triticale hays was higher than that of
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barley and rye hays (Andueza et al., 2004). Oat hay fed alone can sustain
moderate weight gain in sheep (Umunna et al., 1995) and supplementation of
oat hay with moderate quantities of forage legume can help to increase intake,
body weight gain and N retention in sheep even though diet DM digestibility is
not affected.
2.1.3 In vitro study of silage
In vitro digestibility of corn silage ranges from 47-87% (Hutjens, 1998).
Beukes (2013) compared digestibility and rumen degradability of four diets
containing 0, 20, 50 and 70% maize silage using an in vitro study. The diet
containing no silage presented lower DM and NDF degradability than the
silage-based diets. In vitro true digestibility (IVTD) values did not differ
between the silage based diets, but was higher than the IVTD of diet
containing no silage. Silage-based diets had a higher potential degradable
fraction than diet containing no silage. As a result of the higher potential
degradable fraction of the silage-based diets, the effective degradability
was higher than the diet containing no silage. Differences in the potential
degradable fraction were also found for NDF degradability. The diet containing
no silage again had a lower potential degradable fraction than the silage-based
diets. The rate at which the potential degradable fraction disappeared was also
lower for the diet containing no silage. Effective degradability of neutral
detergent fibre was the highest for the 50% silage diet (62.59%) and the lowest
for the diet containing no silage (34.48%) and the 70% silage (35.48%) diet.
The In vitro dry matter disappearance was higher for the silage-based diets
than for the diet containing no silage. The rate at which the NDF in the silage-
based diets disappeared differed, however. Diets containing more than 20%
silage disappeared at a slower rate than the 20% silage diet. Ballard et al.
(2001) compared three corn hybrids Mycogen TMF94, Cargill F337 and
Pioneer 3861 in an in-vitro study. Cargill had the highest IVTDMD and
IVNDFD, which are attributed in part to the lower lignin content of this BMR
hybrid compared with Mycogen and Pioneer.
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2.2 Enteric methane emissions from ruminants
For most feeds consumed by ruminants, methanogenesis is the main
route of hydrogen disposal during anaerobic rumen fermentation (Beauchemin et
al., 2008). The methane resulting from methanogenesis represents a loss of
dietary energy to the animal (Johnson and Johnson, 1995) and it is contributing
significantly to greenhouse gas emissions (Steinfeld et al., 2006). It’s global
warming potential is reported 23 times as compared to carbon dioxide. Over the
last three centuries, the atmospheric methane (CH4) burden has grown 2.5-fold
and agricultural expansion contributed substantially to this burden (Lassey,
2007). Enteric fermentation from livestock was reported a major source of CH4
by Charmley et al. (2007). More than 90 per cent of livestock greenhouse gas
emissions arose from enteric rumen fermentation (Chhabra et al., 2009). As per
INCCA (2010) enteric fermentation emitted 10.09 million tones of methane and
was responsible for 73.3% of total methane emission from agriculture sector in
India. Livestock rearing is an integral part of the agriculture system in tropical
countries like India, Pakistan and Bangladesh which have the highest share in
largest livestock population in the world (FAO, 2013). Considering the large
livestock population, there is an apprehension that there is a serious
environmental hazard from the large amount of CH4 release from them. Large
ruminants, mainly cattle and buffalo contributed to more than 90% of the total
methane emission from livestock in these countries (Naqvi and Sejian 2011).
This methane production from buffalo and other large ruminants was not only a
problem to the environmental safety but also was related with the lower feed
utilization efficiency by them (Kennedy et al., 2012). Singhal et al. (2005)
estimated methane emission from enteric fermentation of Indian livestock using
dry matter intake approach. Indian livestock emitted about 10.08 Tg methane
due to enteric fermentation in the year 1994 in which crossbred cattle,
indigenous cattle and buffaloes emitted about 4.6, 48.5 and 39%, respectively.
Chhabra et al. (2009) reported 11.75 Tg of methane emission for the year 2003.
(Singhal et al., 2005) reported that the contribution of buffalo and cattle to total
livestock methane production was 39% and 53.1%, respectively. Further they
reported that among Indian livestock methane production from male buffalo
calves was quite higher. Buffaloes lost around 6% of their ingested energy as
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eructated methane (Johnson and Johnson, 1995). Holter and Young (1992)
reported that enteric methane emission factor was 39.35% in buffaloes, in
agreement with findings of Condor et al. (2008). Buffalo was the single largest
emitter of methane due to its higher methane emission coefficient i.e. 50
kg/animal/ year (NATCOM, 2004). Chhabra et al. (2009) reported that total
methane emission in India from buffalo constituted 42% of the emissions from
livestock sector for the year 2003 whereas 42.8% in the year 2010 has been
reported by Patra (2014).
The relative short life of CH4 in the atmosphere (10 -12 years) compared
to other green house gases (120 years for carbon dioxide) made the CH4
reduction strategy an effective mean of slowing global warming. Keeping the
above in mind lot of research has been done to combat the methane loss from
animal and its release into the environment. The strategies broadly classified into
nutritional and managemental. The nutritional strategies included altering the
proportion of structural and non structural carbohydrates in feed (Moss et al.,
2000), change in frequency of feeding, forage species and their stage of
maturity, processing of feeds and fodders, feeding of silages, complete feed
bock or total mixed ration (TMR) and manipulation of rumen fermentation by
supplementation of ionophores, addition of fats and oils, probiotics, propionate
enhancers (Mohammed et al., 2004), plant secondary metabolites like saponins,
tannins (Kamra et al., 2008; Patra et al., 2008; Patra and Saxena, 2010),
methane analogues, electron acceptors (Leng, 2008)., prebiotics and means of
defaunation, reductive acetogenesis (Atwood and McSweeney, 2008) and
methanotrophs introduction etc. The managemental strategy included selection
of high productive animals for rearing (Hegarty et. al., 2007; Zhou et. al., 2009).
2.2.1 Methane emissions from ruminants fed on silage or hay based
diets
There is limited information with regard to the effects of forage
preservation on CH4 production. Methane production (% of GEI) was shown to
be lower when forages were ensiled than when dried (Sundstol 1981). This is
because digestion is reduced in the rumen with ensiled forages due to the
extensive fermentation that occurs during silage making. Shingfield et al.
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(2002) observed that rumen fermentation of grass-silage-based diets was
characterized by a higher molar proportion of butyrate and a lower proportion
of acetate compared to hay-based diets. Total CH4 production (Mcal/d) was
depressed by 33% by the utilization of alfalfa silage instead of alfalfa hay,
using a mechanistic model approach to predict CH4 emissions from ruminants
(Benchaar et al. 2001). Kirkpatrick and Steen (1999) observed no differences
between forages conserved as silage vs. forages conserved by freezing
(directly after harvesting) on CH4 energy loss (% of GEI). Silage additives such
as bacterial inoculants and organic acids are used to enhance the quality and
palatability. Shingfield et al. (2002) also observed that the addition of
inoculants-enzyme preparation during ensiling lowered acetic acid and
increased propionate production as well as branched chained VFA in the
rumen when compared to formic acid. Thus, the addition of inoculant-enzyme
during silage making would seem to hold a greater potential for reducing
enteric CH4 emissions than the addition of formic acid.
It is known that methane production will decrease when silage inclusion
level increases. Silage based diets record less loss of energy via
methane, urine and faeces production (Beukes, 2013). A study conducted by
Moss et al., (1995) on sheep receiving a combination of concentrate and grass
silage found that methane production will decline if the silage fraction is
increased. Methanogenesis tends to be lower when forages are ensiled than
when they are dried (Martin et al., 2010). Some micronutrients necessary for
methanogens growth are often deficient in the silages. Maize silage has too
low nitrogen content for methanogens growth (Kalac, 2011). Some
bacteriocins are known to reduce CH4 production in vitro (Callaway et al.,
1997; Lee et al., 2002). Nisin is thought to act indirectly, affecting hydrogen-
producing microbes in a similar way to that of the ionophore antibiotic,
monensin (Callaway et al., 1997). There is a single in vivo result reporting a
significant 10% decrease of CH4 emissions in sheep with this bacteriocin
(Santoso et al., 2004). In contrast, the expected effect of nisin on the
improvement of nitrogen metabolism was not observed in other in vivo reports
(Russell and Mantovani, 2002; Santoso et al., 2006) implying that the same
may happen if CH4 was measured. These data indicate that more information
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is needed on the stability and effect of nisin in animals before considering its
application. In addition, nisin is widely used in the food industry as a
conservative and fears of microbial cross-adaptation might prevent its approval
as a feed additive. A bacteriocin obtained from a rumen bacterium, bovicin
HC5, decreased CH4 production in vitro up to 50% without inducing
methanogens’ adaptation (Lee et al., 2002). The reported inhibitory effect on
methanogenesis of spent culture from Lactobacillus plantarum 80 is also
probably induced by a bacteriocin or a similar compound (Nollet et al., 1998).
The compound(s) in question reduced numbers of methanogens, but, like
many other inhibitors that are efficient in vitro, the effect was lost in sheep after
continuous administration for a few days (Nollet et al., 1998). Klieve and
Hegarty (1999) also suggested the use of archaeal viruses to decrease the
population of methanogens. Methane decrease was more pronounced for a
hay diet than for a maize silage diet supplemented with linseeds in dairy cows
(Martin et al., 2008), and for a concentrate diet than for a forage diet
supplemented with coconut oil in beef heifers (Lovett et al., 2003).
Van Gastelen et al (2015) studied the effects of replacing grass silage
(GS) with corn silage (CS) in dairy cow diets on enteric methane (CH4)
production, rumen volatile fatty acid concentrations, and milk fatty acid (FA)
composition. Multiparous lactating Holstein-Friesian cows (n=32) fed on four
dietary treatments, all having a roughage-to-concentrate ratio of 80:20 based
on dry matter (DM). The roughage consisted of either 100% GS, 67% GS and
33% CS, 33% GS and 67% CS, or 100% CS (all DM basis).They observed
that methane production (expressed as grams per day, grams per kilogram of
fat- and protein-corrected milk, and as a percent of gross energy intake)
decreased quadratically with increasing CS inclusion, and decreased linearly
when expressed as grams of CH4 per kilogram of DM intake. In comparison
with 100% GS, CH4 production was 11 and 8% reduced for the 100% CS diet
when expressed per unit of DM intake and per unit fat- and protein-corrected
milk, respectively. Replacing GS with CS in a common forage-based diet for
dairy cattle offers an effective strategy to decrease enteric CH4 production
without negatively affecting dairy cow performance, although a critical level of
starch in the diet seems to be needed.
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2.2.2 Effect of feeding silage based diet on nutrient intake and nutrient
utilization
Beukes (2013) compared digestibility of four diets containing 0, 20, 50
and 70% maize silage using an in vivo study in lambs. The 20% silage diet
presented the highest dry matter (DM) and organic matter (OM) digestibility.
No differences were found between NDF digestibility of the silage-based diets.
The diet containing no silage presented the lowest overall in vivo apparent
digestibility. Dry matter intake was the highest for the 20% silage diet and
therefore resulted in the highest energy intake and the best energy retention.
The 50 and 70% silage diet presented the lowest N retention while the 20%
silage diet had the highest N retention. Crude protein digestibility was higher
for the diet containing no silage (77.96%) and 20% silage (80.96%) diets and
differed significantly from the 50 (68.26%) and 70% (67.20%) diets. Acid
detergent and neutral detergent fiber digestion were the lowest for the diet
containing no silage. A study by Moran et al., (1988) on sheep receiving maize
silage-based diets had a DM digestibility of 72.2% for the 50 and 70% silage
diets. Beukes (2013) reported that maize silage can be included to up to 70%
in the finishing diets of Merino lambs. To improve the nitrogen retention, it is
important to supplement silage based diets with fermentable carbohydrate
sources such as cereal grains. This will provide the energy needed by the
rumen microorganisms to utilise the degradable nitrogen entering the rumen
(Stanley, 2003).
2.3 Metabolizable Energy Concept in ruminant
ME is the gross energy (GE) of the feed minus that of the faeces (FE),
urine (UE) and combustible gas (mostly methane, ME) and expressed as Mcal/kg
DM or Mcal/d. The ME value is derived using the following equation.
ME (Mcal/kg) = 1.01 x DE (Mcal/kg) -0.45
ME = GE – (FE + UE + ME)
ARC (1980) defined metabolizability of feed at maintenance (qm) as the
proportion of ME in the GE of that feed.
qm = ME/ GE
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The equations suggested to work out the efficiency of ME utilization for different
functions (ARC, 1980) were as follows;
Kmaintenance = 0.35qm+0.503
Kgrowth = 0.78qm+0.006
Klactation = 0.35qm+0.420
Lactating cows Kg = 0.95kl
Efficiency for growth of conceptus Kc = 0.133
Efficiency for utilization of mobilized body tissue for lactation Kt = 0.84
NRC (2001) used following equation to derive ME value
ME (Mcal/kg) = 1.01 x DE (Mcal/kg) -0.45 (NRC, 2001)
Indian feeding standards used TDN to express energy content and
requirements because of scantiness of information regarding ME values of
common Indian feedstuffs. Reports regarding utilization of energy using
calorimetric studies in buffaloes were limited (Tiwari et al., 2000). The French
feed unit system were based on ME content of feedstuffs and on the efficiencies
of ME utilization for maintenance, fattening and lactation. The ME content of
feedstuffs was calculated from their chemical composition, energy digestibility
and ME/DE ratio.
Metabolizable energy (ME) system of energy evaluation is more accurate
than total digestible nutrients (TDN) system because it takes into account the
losses through urine and combustible gases. Another advantage of ME system
is that efficiency of utilization of energy may be measured in this system. This
system can be used either to predict performance of animal from predetermined
ration or to formulate ration for specific performance.
2.3.1 Energy requirements of buffaloes
In buffaloes, the energy is required for the maintenance, growth,
development, reproduction and production performance. To determine energy
requirements, mainly values of metabolized energy (ME), total degradable
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nutrients (TDN), net energy lactation (NEL) have been widely used.The units
used in the feeding standards should ideally be in the same as those used in the
evaluation of feeds, hence, the existing feeding standards have adopted the total
digestible nutrient (TDN) and metabolizable energy (ME) values for expressing
nutrient requirement of buffaloes. TDN or ME system works well as is evident
from the fact that animal’s performance is closely related to TDN intake, when
the intakes of other nutrients are adequate. Sufficient data on NE content of feed
is not available and hence use of NE for feeding buffaloes cannot be adopted at
present.
Maintenance energy requirements for indigenous animals vary between
61 to 104 kcal/ kgW0.75 in dry cows and between113 to 160 kcal/ kgW0.75 during
lactation (Warth, 1926; Mullick and Kehar, 1952; Sen and Ray, 1964;
Shrivastava, 1970; Patle and Mudgal, 1975 and Ranjhan et al. 1975). ICAR
(1998) adopted the value of energy requirement for maintenance to be
equivalent to 122 kcal of ME/kgW0.75 both for cattle and buffaloes, based on
earlier reports. Katiyar (1972) and Patle and Mudgal (1975) estimated the ME
requirement for lactating Haryana cows and crossbred cattle (Brown Swiss X
Sahiwal) to be 127 and 131 kcal/ kgW0.75, respectively. Kearl (1982) adopted the
value of 118 kcal/ kgW0.75 for estimating the nutrient requirements of cattle
against 125 kcal/ kgW0.75 for buffaloes. Values for maintenance ME
requirements for buffaloes ranged between 121 to 178 kcal/ kgW0.75 by Siviaha
and Mudgal (1978) and Tiwari and Patle (1983), respectively. Paul et al. (2002)
reported a value of 128 kcal/ kgW0.75 and adopted similar ME requirements
values while recommending nutrient requirements of buffaloes (Paul and Lal,
2010).
Calorimetric studies have shown that fasting heat production is lower in
buffaloes than in cattle (284.5 kJ vs. 343, kJ per kg metabolic body size (MBS or
W); Maymone and Bergenzini, 1987). Khan et al. (1988) estimated fasting heat
production in adult non-pregnant buffaloes as 284.5 kJ /kg W 0.75. Estimates of
energy requirements for maintenance (g TDN/kg MBS) of different category of
buffaloes were recently reviewed by us (Paul and Lal, 2010) are as follows:
Adult, 27 to 29.78; growing, 27.5 to 52 g and lactating, 35.3-49.2. Huge variation
in these individual estimates is attributable mainly to difference in method of
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estimation. However, now estimates of maintenance requirements of energy by
meta-analysis of pooled data of long term feeding trials are available which are
as follows: Growing: 35-39.9 g TDN/kg MBS (Udeybir and Mandal., 2001);
Lactating: 35.3 g TDN/kg MBS (Paul et al., 2002). These values can be adopted
safely as guideline for feeding buffaloes.
The nutrient needs of lactating buffaloes depend upon the amount of milk
being produced and upon its composition. The milk yield depends primarily on
the type of breed. Buffalo milk contains more solids and fat than cow’s milk.
Generally the fat content ranges from 5.5-13.5%. Estimates of energy
requirement for milk production in buffaloes as reviewed by us recently (Paul and
Lal, 2010) ranges from 220 to 557 g TDN/kg 6% fat corrected milk (FCM). In an
earlier study, conducted at CIRB (Nabha), India, which was based on regression
analysis of the data of long term feeding trials conducted so far in India (35)
where energy was the sole limiting nutrient, energy requirement for milk
production was worked out 406.32 g TDN per kg 6% FCM (Paul et al., 2002).
This value can be adopted as safe guide for feeding buffaloes.
Patle and Mudgal (1976) found the value of 1183 Kcal/ kg FCM, whereas
Ranjhan et al. (1977) recorded 1039 Kcal/ kg FCM. Kearl, (1982) used 1144
Kcal ME/ kg FCM. The requirements proposed by Sen et al. (1978), and
Ranjhan (1991 and 1998) were based on 1188 Kcal ME/ kg FCM. In ICAR
(2013) values of cows and buffalo milk were computed as per fat and CP (3.5
and 4.5 % for cattle and buffaloes respectively) content in the milk which is
comparable to the earlier as well as new reports.
There are several studies focused on the possible effects of energy on
buffaloes. Paul et al., (2003) reported that the effect of energy utilization during
lactation was very high and for per kg corrected milk of 4% fat, 695.9 g TDN was
consumed, effectiveness of average gross energy was 30.53% and net energy
was 69.16%. Again, in buffaloes at this period while daily TDN requirement was
35.3 g/kg BW0.75 for maintenance, it was 406 g (1.47 Mcal/kg DM ME) for
producing per kg 6% fat-corrected milk and, for 1 kg body weight gain it was
1970 g (7.13 Mcal/kg DM ME) (Paul et al., 2002). Energy requirement for
pregnant buffaloes was 55.4 % TDN (2.00 Mcal/kg ME) in dry matter between
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240 and 270 days of gestation whereas it was 60.6 % TDN (2.19 Mcal/kg ME)
between 270 and 308 days (Paul et al., 2007).
With a high environmental temperature and humidity, mean TDN
requirement and utilization in buffaloes at lactation changed with respect to
months. The TDN requirement for buffaloe milk that contain 6 % fat was 6632 g
in August, 6179 g in November and 6030 g in March however the utilization of
TDN was 9848 g in August, 8154 g in November and 8610 g in March indication
that TDN utilization was higher than requirement (Hayashi et al., 2005).
2.3.2 Effect of different metabolizable energy on milk yield and
composition
Broderick (2003) demonstrated the importance of energy source in
determining N efficiency in lactating dairy cows. Increasing dietary energy, by
reducing proportion of forage (thereby NDF) and increasing the proportion of
shelled corn (mostlystarch),increased yields of total and true milk protein and
improved the efficiency of N use (defined earlier) from 0.25 to 0.30. In contrast,
reducing dietary CP concentrations from 184 to 151g/kg DM had little effect on
total milk protein yield and no effect on true milk protein yield, although efficiency
of N use was improved. The proportion of forage in the study was reduced from
0.75 to 0.50
Patton et al. (2006) who reported that increasing dietary energy up to
recommended level within 1st month of lactation enhanced milk yield.
Conversely, Grummer et al., (1995) found increase in milk yield by providing
extra dietary energy beyond NRC recommendation in lactating cows.
Jabbar et al., (2012) evaluated the effect of increasing energy density of
diets on production performance of bST administered lactating Nili-Ravi buffalo.
Animals were fed on low energy density (LED) 85 %; medium energy density
(MED), 100 %; and high energy density (HED) 115 % of the NRC, (1989)
standards. All the animals were injected with bST (every 14 days), and were fed
on a straw based TMR during a period of 90 days. Daily milk production was
highest 8.8 kg/day in animals fed high energy ration compared to 8.2 and 7.9 kg
of milk produced by MED and LED fed animals, respectively.
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Jabbar et al. (2013) confirmed that the NRC recommendations for large
dairy cows are suitable for Nili-Ravi buffaloes. Further, there was no advantage
to feed lactating buffaloes above NRC recommended level (E-120) for more milk
yield, while it is detrimental to feed below these levels (E-80) as milk production
decreased.
2.3.3 Effect of different metabolizable energy on dry matter intake of
buffaloes
Daily intake of dry matter (DM) in an animal defines its capability of feed
consumption. Feed consumption can be calculated from percentage of BW
and/or value derived from metabolic body weight (BW 0.75). Studies indicated
that daily DMI for the maintenance was 1.6-2.4% (Basra et al., 2003) and 1.2-
1.3% of BW and 59.9 g/kg BW 0.75 (Paul et al. 2002) and it was 2.2-2.6% of BW
in heifers (Terramoccia et al. 2005; Singhal et al. 2005). 2.5-3.0% of BW in
fattening buffaloes (Zicarelli, 2004; Ståhl-Högberg, 2003). In pregnant buffaloes
DMI starts to drop before parturition and levels up to 1.8-2.5% of BW (Bertoni et
al. 1994; Singhal et al. 2005) and and 68 g/kg BW 0.75 in dry period
(Mudgal,1988). Low level of DMI at the late stage of gestation does not show a
sharp increase when the milk production reaches the peak at the beginning of
lactation, in other words, despite the fast increase in milk yield following the
parturition upsurge in DMI is rather slow. Milk production of the animal usually
peaks 4 to 8 weeks postpartum. However feed consumption ability does not
immediately meet the demand for milk production. The highest feed consumption
capacity is reached at around 150 days after parturition (Terramoccia et al.,
2005). In accordance with this figure, daily DMI for buffaloes at lactation period is
2.0-2.2% of BW (Bartocci et al., 2002; Singhal et al., 2005) and 119.2-137 g/kg
BW 0.75 have been found (Mudgal, 1988; Paul et al., 2003). DM intake in
lactating Nili-Ravi buffaloes were unaffected by different levels of energy in the
diet (Jabbar et al., 2013). Aghaziarati et al. (2011) who reported that different
dietary energy density did not affect DM intake in Holstein cows. Similarly,
Broderick also reported that no effect of varying energy and protein levels on DM
intake. However, Vazquez Anon et al. (1997) observed that increasing dietary
energy density improved DM intake
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2.3.4 Effect of different metabolizable energy on body weight gain
Lalman et al. (2000) who found that weight change at early lactation stage
was not affected by increasing dietary energy density and Grummer et al. (1995)
who reported that body weight changes were not affected by dietary fat
supplementation postpartum. In contrast Brodericks (2003) reported that
increasing dietary energy density improved weight gain in lactating Holstein
cows. Jabbar et al. (2013) observed no significant change in body weight of
lactating Nili-Ravi buffaloes fed on different level of energy.
2.4 Metabolizable protein concept
The concept of metabolizable protein (MP) was first proposed by
Burroughs et al. (1974) in the USA. This concept was then developed into
systems to replace DCP, in UK by ARC (1984) and in France by INRA, later in
Scandinavian countries (Madsen, 1985) and USA. Several systems (Madsen,
1985; NRC, 2001; AFRC, 1993) used MP as a measure of protein quality. MP is
the true protein that is absorbed in the ruminant’s intestine that includes
estimates of available microbial and dietary escape protein and is potentially
more accurate than other protein systems. The goals of ruminant protein
nutrition is to provide adequate amounts of rumen degradable protein (RDP) for
optimal ruminal efficiency and to obtain the desired animal productivity with a
minimum amount of dietary CP. Selection of complementary feed protein and
non protein nitrogen (NPN) supplements provide the types and amounts of RDP
that would meet, but not exceed, the N needs of ruminal microorganisms for
maximal synthesis of MCP, and digestible RUP that would optimize the profile
and amounts of absorbed amino acid. Knowledge of the kinetics of ruminal
degradation of feed proteins was fundamental to formulate diets for adequate
amounts of RDP (Brito et al., 2006) for rumen microorganisms and adequate
amounts of RUP for the host animal. Microbial protein synthesis in the rumen
was often the main component of metabolizable protein supply in ruminants
(Moorby et al., 2006) and supplied 70 to 80% of the required amino acids to
ruminants (Chumpawadee et al., 2006). Bacterial crude protein (BCP) could
supply from 50% (NRC, 2000) to essentially all the MP required by beef cattle,
depending on the UIP (undegradable intake protein) content of the diet.
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Efficiency of synthesis of BCP is critical to meet the protein requirements of beef
cattle economically; therefore, prediction of BCP synthesis is an important
component of the MP system. In most cases, natural diets contained sufficient
DIP (degradable intake protein) to meet microbial needs for amino acids,
peptides, or branched chain amino acids. Deficiencies have not been reported in
practical feeding situations.
In CNCP system MP could be predicted if the CP and total carbohydrate
content, intake of CP and carbohydrate, fractional degradation rate and passage
rate of different carbohydrate and protein fractions of a feed or TMR are known.
NRC (2001) considered TDN (Total Digestible Nutrients) value of a feed in
estimating the MCP yield and further the MP availability. The MCP yield was
assumed 130g/ kg of TDN intake and the requirement for RDP was 1.18xMCP
yield. Therefore, yield of MCP was calculated as 0.130 x TDN when RDP intake
exceeded 1.18 x MCP yield. When RDP intake was less than 1.18 x TDN
predicted MCP, then MCP yield was calculated as 0.85xRDP intake.
Fig: 2.1 Metabolizable protein system (AFRC, 1993)
In AFRC (1992) key protein parameters i.e. quickly degradable protein
(QDP), slowly degradable protein (SDP) and rumen undegradable protein (UDP)
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were derived from measurements of the rates of protein degradation (dg) in the
rumen. The fractional rumen outflow rates per hour (r) varied from 0.02 to 0.08,
depending on the level of feeding.
Effective rumen degradable protein (ERDP) was a measure of the total N
supply that appeared to be actually captured and utilized by the rumen microbes
for their growth and synthesis purposes and digestible undegradable protein
(UDP) was that part of UDP of feed which was digested in lower tract/ intestine
of animal. The level of feeding (L) also influenced the ERDP and RUP values of
feed. Microbial protein synthesis was assumed to depend on several factors viz.
energy and nitrogen supply to the microbes, level of feeding to the animals
whereas energy supply was assumed the first limiting factor. For estimation of
rumen microbial crude protein (MCP) yields (y), fermentable metabolizable
energy (FME, Mcal/d or Mcal/kg DM) was used (AFRC, 1993). When nitrogen
supply to rumen microbes was limiting for microbial protein synthesis, MP
system increased the amounts of ERDP required to match the amount of FME
supplied by the diet.
The NRC (2001) proposed 80% digestibility of the microbial true protein
vs 85% proposed by AFRC (1993). Microbial true protein was set from MCP less
nucleic acid content which was considered to be 20% by NRC (2001) vs 25% by
AFRC (1993). Thus the percentage of MCP that truly contributed to MP was 64,
comparable to the value suggested by AFRC (63.75).
Zhao and Lebzien (2002) stated that the prediction of the total crude
protein supply at the duodenum was more accurate and simpler than the
separate prediction of the rumen microbial crude protein (MCP) and the rumen
undegraded dietary crude protein (UDP) and thus gave the concept of utilizable
crude protein (uCP). They found uCP determination more practical and accurate
than separate determination of UDP and MCP in evaluating dietary protein value
for ruminants. Zhao and Lebzien (2000) developed an in vitro incubation
technique for estimating uCP of feedstuffs for ruminants. We hypothesized in the
present study that when intestinal digestibility would be applied to uCP, in vitro
MP value of the feeds and fodders, would be obtained.
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2.4.1 Metabolizable protein availability from feeds or feed combinations
Knowledge of crude protein and total carbohydrate content of feed, crude
protein and carbohydrate intake, fractional degradation rate of different feed
fractions, passage rates of feed fractions, RDP and RUP values of feed,
microbial protein synthesis etc. are the major inputs in calculating the availability
of metabolizable protein from a feed or TMR (total mixed ration) in CNCP
system. NRC (2001) considered total digestible nutrients (TDN) value of a feed
in estimating the MP availability, while AFRC (1992) considered the fermentable
metabolizable energy (FME) content of feed in calculation of MP value of that
feed.
Tolkamp et al. (1998) experimented on dairy cows for their choice either
for metabolizable protein content of feed or RDP content of feed. In the process,
MP supply from various feeds was estimated as per AFRC, 1993. Silage used as
fodder source with 16.6% CP supplied 7.7% MP. A high protein feed (HPF) with
18.5% CP provided 11.4% CP, while MP supply from a low protein feed (LPF;
12.8% CP) was 7.4%. When HPF diet was supplemented with urea (22.5% CP),
it supplied 11.3% CP, while LPF diet along with urea (16.1% CP) supplied 8.9%
MP. Feeds prepared to supply high, low and medium MP i.e. HMPF, LMPF and
MMPF with 17.9, 16.7 and 17.3% CP provided 10.6, 10.8 and 10.7% of MP,
respectively. The study revealed that there was no specific selection for MP
content of feeds when feeds are adequate in their RDP content.
Vermeulen et al. (2001) calculated MP availability from range forage
winter having 4% CP and 49% TDN to be 81 g/d when intake was about 2.2% of
BW. MP supply from a high protein supplement (41% CP) when fed at the rate of
1.36 kg/cow/day was 158 g/d. Microbial MP availability was calculated to be 345
g/d. Thus the MP balance for the beef cows was predicted at -152 g/d. Four
experimental protein supplements were formulated to provide incremental level
of UIP (undegradable intake protein); a control supplements (C) and three other
supplements that provided additional UIP of 63 g/d (C+63), 126 g/d (C+126) and
189 g/d (C+129). All four supplements supplied similar DIP (degradable intake
protein, 396 g/d). The MP supplied from all four supplements was calculated to
be 190, 247, 303 and 360 g/d, respectively. When these supplements were fed
at the rate of 1.36 kg/d to four groups of early lactating cows grazing dormant
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native ranges, the MP supply from forages in four groups were 193 g/d (C), 191
g/d (C+63), 179 g/d (C+126) and 184 g/d (C+189). MP supply from protein
supplements were estimated as 111, 150, 180 and 217 g/d respectively in four
groups. Microbial MP supply was calculated as 373, 369, 346 and 355 g/d in four
groups, respectively.
Blouin et al. (2002) assesed the availability of metabolizable protein from
2 isoenergetic (1.62 Mcal/kg DM) and isonitrogenous diets (16.3% of DM).
These 2 diets supplied 1930 and 1654 g/d of MP due to variation in microbial
protein, RDP and RUP supply.
Raggio et al. (2004) estimated the metabolizable protein availability from
3 diets supplying similar energy, but having increased level of dietary CP content
ie. 12.7%, 14.7% and 16.6% of DM. The MP supply from these diets were 1992,
2264 and 2501 g/d respectively. The diets supplied similar energy i.e. 36.4
Mcal/d NEL.
Kabi et al. (2005) estimated MP availability from various feeds using
degradation characteristics of feed CP as per AFRC (1993) for early weaned,
growing and finishing beef bulls. In early weaned beef bulls, the MP availability
from fresh elephant grass (FEG; 10.8% CP) was 6.37%, when supplemented
with SPV (sweet potato vines). The contribution from SPV (19.7% CP) towards
MP availability was 10.74%. When FEG was fed along with GCM (Gliricidia
sepium leaf meal, CSC and wheat bran along with salt mixture; 20.1% CP), the
MP availability were 6.43 and 10.13% respectively. When FEG was
supplemented with commercial concentrate mixture (CCM; 20.4% CP), the
supply of MP were 6.27and 11.29% respectively. In growing beef bulls, MP
availability from FEG was 6.1%, when supplemented with SPV. The contribution
from SPV towards MP availability was 10.2%. When FEG was fed along with
GCM (19.9% CP), the MP availability were 6.8 and 10.7%, respectively. When
FEG (10.9% CP) was supplemented with CCM, the supplies of MP were 7.2 and
10.9%, respectively. In finishing beef bulls, MP availability from FEG (9.5% CP)
was 7.35%., when supplemented with GM (Gliricidia sepium leaf meal, maize
bran and salt; 19.5% CP). The contribution from GM towards MP availability was
10.32%. When FEG was fed along with GCM, the MP availability was 7.26 and
10.28% respectively. When FEG was supplemented with CM (CSC, maize bran
and salt), the supply of MP were 6.85 and 10.08%, respectively.
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Doepel and Lapierre (2006) evaluated two isonitrogenous and
isoenergetic diets in terms of MP availability. These two diets when fed at 26
kg/d (17.5% CP and 1.45 Mcal/kg NEL) supplied 2197 and 2674 g/d MP, which
suggested that there was no direct relationship between CP and MP. They also
evaluated two diets supplying similar MP (2676 and 2673 g/d) level, but found
that the diets supplied substantially different amounts of digestible histidine,
leucine and lysine; thus were different in terms of their protein quality.
Weiss and Wyatt (2006) estimated the metabolizable protein supply from
diets having 2 levels of CP content (Low CP and High CP) as per NRC (2001).
They used 2 types of diet, one with Brown midrib (BMR) hybrid corn silage and
the other with Dual purpose (DP) hybrid corn silage. The MP supply from low CP
(14.4%) and high CP (17.2%) diets having BMR silage were 2350 and 3000 g/d,
while the MP supply from low CP (14.2%) and high CP (17.1%) diets having DP
silage were 2370 and 2970 g/d.
Wang et al. (2007) fed 4 levels of dietary CP i.e. 11.9, 13, 14.2 and 15.4%
to Chinese Holstein cows and calculated the supply of metabolizable protein
from these diets. The MP availability was 1.75, 1.91, 2.09 and 2.16 kg/d with
corresponding DM intake to be 21.1, 21.4, 21.5 and 20.8 kg/d respectively. They
estimated that the above 4 diets were having MP: 8.3, 8.9, 9.7 and 10.4% of DM.
DiCostanzo (2007) compiled 128 treatment means containing results of
29 studies with 719 pens and 6362 heads of cattle. The cattle were fed for 141
days with average daily DMI of 20.98 lb/d. Average dietary CP was 14% and DIP
(degradable intake protein) averaged 53.1% of CP, while daily CP intake was 2.9
lb/d. The diet supplied 923 g/d of MP and 704 g/d of DIP, while the
corresponding requirements were 777 and 650 g/d.
Yu et al. (2008) compared availability of MP from three oat grain varieties
such as CDC Dancer, Derby and CDC SO-I (Super Oat) using NRC (2001)
model. The CP content of these varieties were 11.82, 11.10 and 12.81% of DM,
while the MP content of these varieties were estimated at 7.43, 7.16 and 8.13%
of DM, respectively.
Taghizadeh et al. (2008) used in situ method to determine metabolizable
protein of 10 test feeds such as corn grain (CG), cottonseed meal (CSM), barley
grain (BG), alfalfa hay- 3 cuts (AH), beet pulp (BP), tomato pomace (TP), lupin
byproducts (LBP) and fish meal (FM). Metabolizable protein of CG, CSM,BG,
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first cut AH, second cut AH, third cut AH, BP, TP, LBP and FM was 3.5098,
23.2197, 4.8509, 6.6067,6.3770,4.8044, 6.3005,16.3847 and 39.6774 g/kg DM.
They concluded that the degradability of CP of test feeds can be used in MP
determination and diet formulation.
Higgs (2009) evaluated MP availability from diet using two commercial
dairy herds in Western New York. When a herd (A) of 400 cows producing
average 35.8 kg/d milk was given a diet having 17.6% CP, the total MP supply
was 2950 g/d. When diet CP was reduced to 16.6%, the MP supply also reduced
to 2769 g/d, but milk yield increased to 36.3 kg/d. When another herd (B) of 600
cows yielding average 37.2 kg/d milk was offered a diet having 17.7% CP, the
total MP supply was 2646 g/d. On reducing the diet CP to 16.9%, the MP supply
slightly increased to 2690 g/d; but milk yield decreased to 36.3 kg/d. On both
herds, lowering ration CP resulted in improved N efficiency and decreased MUN
(milk urea nitrogen) level while maintaining herd milk production.
Islam et al. (2011) determined MP content of whole crop rice (WCR)
silage for dairy cows according to AFRC (1993). The estimated value of WCR
silage (CP 8.21%) was 4.29% of DM. They also found out that stage of maturity
of WCR was positively correlated with MP content and MP yield. It was also
established that out of the four botanical fractions of WCR (leaf blade, leaf
sheath, stem and head), portion of head was best related to MP content and MP
yield; thereby it could enhance the nutritive value of WCR silage.
Chase (2011) evaluated MP availability from six commercial dairy herd
rations. Ration A (14.3% CP) provided MP of 2600 g/d in cows yielding 41 kg/d
milk; the MP content of the diet being 10.5% of DM. Ration B (15.9% CP)
supplied MP of 3322 g/d to cows yielding 52.6 kg/d milk with diet MP
concentration being 12.2% of DM. MP availability from ration C (15.7% CP) was
2710 g/d in cows yielding 38.5 kg/d milk and the MP concentration was 11.1% of
DM. Diet D having 15.8% CP and MP of 11.2% of DM supplied MP of 2744 g/d
to cows yielding 43 kg/d milk. Similarly diet E having 15.5% CP and MP of 11.1%
of DM provided MP of 2720 g/d in cows yielding 40 kg/d milk. Diet F (16.2% CP
and MP of 12.1% of DM) given to cows producing 40.5 kg/d supplied MP of 2779
g/d.
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Lee et al. (2011) assigned randomly one of the following three diets to 36
cows; a diet with adequate MP balance (+44 g/d) and 16.7% CP concentration
(AMP); a diet deficient in MP (-156 g/d) and 14.8% CP concentration (DMP) or
DMP supplemented with approximately 500 g coconut oil/head/d (DMPCO;
14.7% CP concentration). The above three diets supplied 10.8, 9.7 and 9.5% of
MP (DMB). The AMP diet provided 10.6% RDP and 6.1and RUP, the DMP diet
provided 9.8% RDP and 4.9% RUP and the DMPCO diet provided 9.9% RDP
and 4.8% RUP. The MP balance of these three diets according to NRC (2001)
was 44, -156 and -288 g/d, respectively. However all diets provided similar
energy i.e. 1.64 Mcal/kg.
Das et al. (2014) determine the metabolizable protein (MP) content of
common indigenous feedstuffs used in ruminant nutrition using in situ method. It
was observed that the MP content of maize grain (MG), groundnut cake (GNC),
mustard oilcake (MOC), cottonseed cake (CSC), deoiled rice bran (DORB),
wheat bran (WB), berseem fodder (BF), maize fodder (MF) and sorghum fodder
(SF) was found to be 95.26, 156.41, 135.21, 125.06, 101.68, 107.11, 136.81,
72.01 and 76.65 g/kg DM, respectively. The corresponding ME (MJ/kg DM)
content of the test feeds was 13.66, 13.12, 13.65, 10.68, 9.08, 11.56, 9.64, 8.33
and 8.03, respectively. Among the test feeds, GNC contained the highest and
MF contained the lowest MP per kg DM.
2.4.2 Protein requirements for maintenance
The scientific data to explain protein requirements and their utilization
from different sources at various physiological stages in buffaloes is scarce. In
contrast to high producing western dairy cattle where much attention has been
paid to develop energy and protein standards and nutrient requirement models,
no such planned effort has been made to establish protein or energy needs in
buffaloes. Conflicting results have been reported by various workers on the level
of protein required in buffalo diets during lactation and growth (Verna et al.,
1994; Campanile, 1997; Terramoccia et al., 2000; Puppo et al., 2002). Protein
concentrations used in lactating buffalo diets can be equal to or below 12% on
DM basis, since these concentrations have little influence on the quantity and
quality of milk produced (Verna et al., 1992; Verna et al., 1994). Sivaiah and
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Mudgal (1978) suggested the administration of 166 to 126 g of digestible CP/100
g of milk protein produced, while according to Rai and Aggarwal (1991), the
concentration of CP on DM should be between 11 and 14%. A linear increase in
fiber digestibility, greater microbial counts and a linear reduction in N retention
was observed in buffalo bulls with increasing level of ruminally degradable
protein (RDP) (Javaid et al., 2008). In this study, ruminal ammonia, blood urea N
(BUN) and urinary N excretion were higher in bulls fed diets containing
increasing level of RDP. They concluded that no deleterious effects had been
noticed on ruminal parameters and digestibility of nutrients when 82% RDP (of
total 16% dietary CP) was fed to buffalo bulls. Nisa et al. (2008) reported that the
DM and NDF intakes were decreased while their total tract apparent digestibility
in lactating buffaloes was increased when RDP contents were increased from 50
to 82% in the dietary CP. Dietary RDP/RUP did not affect the CP digestibility in
lactating buffaloes. Milk yield and milk constituents (fat and protein) yields were
greater in buffaloes fed 50% RDP than those fed higher levels of RDP. Lower
conception rate and a linear decrease in N balance and milk yield of buffaloes
with increasing level of RDP:RUP was observed. From the published data it can
be concluded that supplementing buffalo diets with RUP can increase the
efficiency of N utilization by increasing the flow of N and amino acids to the small
intestine, supplying more amino acids for milk yield. Higher level of RDP in
buffalo ration causes excessive ruminal NH3 production in rumen which
ultimately results in an increase blood urea. Increase in ruminal NH3 and blood
urea decreases dry matter intake (DMI) and reduces conception rate in
buffaloes, similar to those observed in dairy cattle (Dhali et al., 2006).
Furthermore, excessive supply of protein and/or imbalance supply of RDP and
RUP in buffalo ration could cause negative energy balance associated with
metabolic disposal of excessive N escaping from the rumen
Kehar, (1944) determined the protein requirements for indigenous cattle
by factorial method. Endogenous urinary nitrogen (EUN) and metabolic fecal
nitrogen (MFN) can be determined using nitrogen free diets. The values can also
be derived using low nitrogen diets at certain levels and regression analysis of
the data at zero nitrogen intake(s). Endogenous urinary nitrogen excretion in
Indian cattle was 0.02 g/ kg B wt. (Mukherjee and Kehar, 1949). Sen et al.
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32 | P a g e
(1978) recommended 2.84g DCP/ kgW0.75 for Zebu and crossbred cattle and
buffaloes. Ranjhan (1980) reported DCP values ranging from 1.97 to 4.19 g/
kgW0.75. Kearl (1982) using Boran bullocks calculated EUN to be equal to 2.18 g
DP/ kgW0.75. Nitrogen losses thorough skin, hair and faeces would increase the
requirement substantially. Patle and Mudgal (1975) and Ranjhan et al. (1977)
found that crossbred cattle required 4.21 and 3.75 g DCP/ kgW0.75.
The data generated during last two decades reveals that DCP estimation
system itself is erroneous. It is well understood now that protein digestibility is
influenced significantly from the source of protein, energy and their dietary
levels. Thus, a single value of DCP requirement cannot be assigned with
reasonable accuracy (ARC, 1980). Therefore, the most scientific and accurate
metabolizable protein system, originally proposed by Burroughs et al. (1975) with
further refinements (ARC, 1980, NRC, 1989; SCA, 1990; NRC, 2001), has been
followed by ICAR, 2013. This system takes into account the rumen turnover rate
and level of feeding in predicting degradation of dietary protein and microbial
protein synthesis in the rumen. The efficiency of utilization of metabolizable
protein and hence estimates of requirements, can be early adjusted in
biologically logical fashion to take account of information relating to specific
feeding situation. Excretion of nitrogen through urine, feces and skin was
recommended to be 2.3g/ kgW0.75 (AFRC, 1993). It is also suggested that a
safety margin of 5% to be added at the time of diet formulation. The nitrogen
excretion through feces (MFN) depends on DM intake, and its fiber content.
Since tropical feeds have comparative more cell wall constituents, an additional
2% has been kept as safety margin in ICAR, (2013) document. Thus, the
equation has to be modified as MPm (g/d) = 2.46W0.75.
2.4.3 Metabolizable protein requirements for body weight change in cows
The requirements of metabolizable protein is met from two sources i.e.
digestible microbial protein and undegraded dietary protein in ruminants. NRC
(2001) adopted factorial approach in estimating the metabolizable protein
requirements in dairy cattle. The net protein requirement includes that needed
for maintenance and production. The maintenance requirement consists of
urinary endogenous N, scurf N (skin, skin secretions and hair) and metabolic
Review of Literature
33 | P a g e
faecal N. The requirement for production includes the protein needed for
conceptus, growth and lactation etc. Average efficiency of converting MP to net
protein is around 67%, thus the protein requirements in MP units will be NP/0.67.
The MP requirement for maintenance of Nellore cattle was estimated as
4.0 g/kg BW0.75. NRC (1996) recommended MP requirement for maintenance as
3.8 g/kg BW0.75 in zebu cattle. Ezikiel (1987) obtained MP requirements for
maintenance of 1.72 and 4.28 g/kg BW0.75/d for Nellore and Holstein,
respectively. Valadares et al. (1997) calculated MP requirement for maintenance
of 4.13 g/kg BW0.75/d in zebu cattle. Hill (1998) estimated the value of 1.63 g/kg
BW0.75/d in Nellore. Vermeulen (2001) estimated MP requirements of beef cows
(avg. body wt. 499 kg) with a peak milk yield of 6.4 kg/d to be 734 g/d as per
NRC (1996).
2.4.4 Protein requirements for lactation
Protein concentrations used in lactating buffalo diets can be equal to or
below 12% on DM basis, since these concentrations have little influence on the
quantity and quality of milk produced (Verna et al., 1992; Verna et al., 1994).
Sivaiah and Mudgal (1978) suggested the administration of 166 to 126 g of
digestible CP/100 g of milk protein produced, while according to Rai and
Aggarwal (1991), the concentration of CP on DM should be between 11 and
14%. A linear increase in fiber digestibility, greater microbial counts and a linear
reduction in N retention was observed in buffalo bulls with increasing level of
ruminally degradable protein (RDP) (Javaid et al., 2008). In this study, ruminal
ammonia, blood urea N (BUN) and urinary N excretion were higher in bulls fed
diets containing increasing level of RDP. They concluded that no deleterious
effects had been noticed on ruminal parameters and digestibility of nutrients
when 82% RDP (of total 16% dietary CP) was fed to buffalo bulls. Nisa et al.
(2008) reported that the DM and NDF intakes were decreased while their total
tract apparent digestibility in lactating buffaloes was increased when RDP
contents were increased from 50 to 82% in the dietary CP. Dietary RDP/RUP did
not affect the CP digestibility in lactating buffaloes. Milk yield and milk
constituents (fat and protein) yields were greater in buffaloes fed 50% RDP than
those fed higher levels of RDP. Lower conception rate and a linear decrease in
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34 | P a g e
N balance and milk yield of buffaloes with increasing level of RDP: RUP was
observed. From the published data it can be concluded that supplementing
buffalo diets with RUP can increase the efficiency of N utilization by increasing
the flow of N and amino acids to the small intestine, supplying more amino acids
for milk yield. Higher level of RDP in buffalo ration causes excessive ruminal NH3
production in rumen which ultimately results in an increase blood urea. Increase
in ruminal NH3 and blood urea decreases dry matter intake (DMI) and reduces
conception rate in buffaloes, similar to those observed in dairy cattle (Dhali et al.,
2006). Furthermore, excessive supply of protein and/or imbalance supply of RDP
and RUP in buffalo ration could cause negative energy balance associated with
metabolic disposal of excessive N escaping from the rumen.
As per previous recommendations on requirements, a value (132g
digestible N/ 100g milk N) suggested by Sen et al. (1978) and Ranjhan (1980,
1992) is on lower side considering the data of nitrogen utilization efficiency in
cattle and buffaloes. MP utilization efficiency is 68 % therefore indicating that
DCP values in ICAR, (1998) were significantly lower and more studies on this
aspect are warranted. However, Kearl (1982) used a value of 55g DP/ kg 4%
FCM for cattle and Paul et al. (2002) found 55.2g DCP and 90.3g CP/ kg 6%
FCM for buffaloes. Considering the limitations of DCP, metabolizable protein
values were suggested by ICAR, 2013.
2.4.5 Effect of metabolizable protein supply on milk yield and milk
components
Smoler et al. (1998) examined the UK metabolizable protein system for its
suitability as potential predictor of milk protein concentration. The system was
evaluated based on the data from 163 cows offered five forage mixtures ad
libitum with concentrates at 3, 6 and 9 kg/d of DM. The system was again tested
on a separate data set of 100 cows offered seven forage mixtures ad libitum with
concentrates at 6 kg/d of DM. However this system was not found to be a
suitable prediction model of milk protein concentration in dairy cows.
Colin-Schoellen et al. (2000) evaluated the effects of three levels of MP
supply (PDIE: protein digested in small intestine, when ruminal fermentable
energy is limiting) in total mixed rations on milk production and composition in
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35 | P a g e
dairy cows in two trials with PDIN (protein digested in small intestine, when
ruminal fermentable N is limiting). In first trial, three diets were tested: diet having
13.8% CP supplying 10.8% PDIE with 1% PDIN deficit (high PDIE diet); diet
having 14.4% CP supplying 9.8% PDIE with no PDIN deficit (medium PDIE diet)
and diet having 12.5% CP supplying 9.8% PDIE with 1% PDIN deficit (medium
PDIE diet). In second trial four diets were tested; three medium PDIE diets and
one low PDIE diet. Medium PDIE diets were having 13.9, 12.6 and 11.1% CP
with 0, 1 and 2% PDIN deficits that supplied 9.6, 9.5 and 9.7% of PDIE on DMB.
The low PDIE diet having 10.9% CP and 1% PDIN deficit supplied 8.4% PDIE.
DMI, NE intake and milk yield were significantly higher with increasingly PDIE
level. However PDIE level of the diet did not affect milk true protein content. Fat
content decreased between low and medium PDIE levels and did not vary
between medium and high PDIE level. Milk NPN and MUN contents increased
with increased level of PDIE.
Raggio et al. (2004) studied the effect of three levels of metabolizable
protein supply on milk production and composition in six catheterized
multiparous lactating Holstein cows. Three diets, balanced to provide similar
energy intake (36.4 Mcal/d NEL) and increasing amount of MP (g/d) – low
(1992), medium (2264) and high (2501) were fed to those animals. Milk
production increased linearly with increasing supply of MP. Milk CP yield and
yield of each milk protein fraction increased linearly. Milk CP concentration
increased linearly, but the proportion of true protein decreased. Milk fat
concentration decreased linearly resulting in decrease in fat yield at the highest
MP level.
Schei et al. (2005) evaluated the effect of dietary energy and MP supply
on feed intake and milk production in Norwegian dairy cows. Three types of diets
were fed, (1) protein and energy supply according to Norwegian standard
recommendation (SS) for amino acids absorbed in the small intestine (AAT/MP)
and net energy lactation (NEL), (2) low protein-low energy (LL with 50% of the
concentrate energy of SS) and (3) high protein-low energy (HL with 135% of the
protein content of LL). Two types of concentrate mixtures (I and II) and grass
silage were offered, which supplied 19.4, 37.2 and 13.2% CP with corresponding
MP supply of 12.6, 22.5 and 7.5% AAT (MP) of DM. Concentrate mixtures I was
fed for cows that were fed the SS and LL diets, whereas concentrate mixtures II
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36 | P a g e
was used for cows fed the HL diet. Grass silage was provided to all groups ad
libitum. The total feed intake (kg/d) with SS, LL and HL diets were 18.7, 15.7 and
16.2%, respectively with three diets supplying AAT (MP) of 1910, 1417 and 1917
g/d and energy (NEL) of 129, 102 and 107 MJ/d, respectively. The milk yields
(kg/d) with above three diets were 26.1, 20.9 and 23.8, respectively.
Corresponding fat, protein and lactose yield (g/d) with these diets were 990, 829
and 1244, 852, 649 and 960 and 955, 753 and 1093 respectively.
Waterman et al. (2006) studied the effect of three supplements that
contained increasing amount of MP on post partum interval and nutrient
partitioning in two year old young beef cows. All three supplements provided
same dietary protein i.e. 327 g/d. However supplement 1 (RMP) supplied
required amount of MP according to NRC (2001); while supplement 2 (RMP+)
provided 31 g excess MP than the requirement. Supplement 3 (RMP+P)
contained 11% of calcium propionate and supplied 36 g excess MP than the
requirement. All supplements were fed at 908 g/d. As MP of diet with or without
propionate increased, a decrease was observed in post partum interval; but
there was no influence on pregnancy % by the treatments. BCS was slightly
improved on supplementation of 31 g of excess MP and there was increased
weaning wt. of calves.
Weiss and Wyatt (2006) evaluated the effect of supply of low or high MP
from both Brown midrib (BMR) hybrid corn silage and Dual purpose (DP) hybrid
corn silage based diets on milk production in Holstein cows. They observed that
milk yield (kg/d), energy corrected milk yield (kg/d), milk fat % and milk fat yield
(kg/d) were higher in high MP supply from both types of silage based diets. Milk
protein % and milk protein yield (kg/d) were similar in both low and high MP
supply from DP silage based diet, while these parameters increased in high MP
supply from BMR silage based diet.
Wang et al. (2007) studied the effect of 4 levels of metabolizable protein
(MP) on milk production and nitrogen utilization in 40 Chinese Holstein dairy
cows. The animals were offered with different levels of MP: 8.3% (Diet A), 8.9%
(Diet B), 9.7% (Diet C) and 10.4% (Diet D) of DM. The study revealed that milk
yield and milk protein % increased as the MP increased up to 9.7% of DM and
then leveled off. So it was concluded that the optimal dietary MP level was at
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37 | P a g e
9.7% of DM for Chinese Holstein dairy cows producing 30 kg of milk per day.
Daily milk yield per cow was 2.6 kg/d higher with the highest MP level compared
with the lowest MP level, equivalent to 1.2 kg/d for every 1%unit increase in the
MP content of the diet. But in contrast Milk yield did not increase when dietary
protein was increased from 17.2 to 19.0% (Sannes et al., 2002), from 16.8 to
19.4% (Davidson et al., 2003), from 16.7 to 18.4% (Broderick, 2003), and from
15 to 18.7% (Groff and Wu, 2005) in Holstein lactating cows.
Huhtanen et al. (2008) carried out meta analysis to evaluate the effect of
silage soluble N components on MP concentration from the study of 253
production data of cows with average 19.2 kg DMI/d (including average 11.5 Kg
DMI/d from silage). Average CP% of silage was 15.8%, while MP concentration
of that silage was 8.34%. The analysis showed that increased silage N solubility
was associated with reduced milk protein yield and efficiency of N utilization and
with increased MUN concentration. Proportion of SNAN (soluble non ammonia
nitrogen) in silage N had no effect on MP yield and consequently on the true
silage concentration.
Voltolini et al. (2008) evaluated the effects of increasing MP supply
beyond NRC (2001) recommendations for mid lactating dairy cows grazing
elephant grass pasture. Three concentrate diets were evaluated: control (17%
CP) was adjusted in relation to MP according to NRC (2001) and the other two
diets contained extra SBM to increase the CP content to 21.2% and 25%. The
diets supplied 3.06, 3.10 and 3.14 Mcal/kg of ME. Milk production, 3.5% FCM,
milk fat, protein, lactose and total solids contents were not affected by
treatments. Milk urea N and plasma urea N increased linearly as MP supply
increased. Treatments also did not affect BW gain and BCS of animals, which
indicated that CP content in the concentrate formulated according to NRC (2001)
was adequate for mid lactating cows grazing tropical pastures.
Weiss et al. (2009) designed fifteen types of diets having 50% forage on
DMB and 10.7% RDP on DMB; but diets differed in type of forage, digestible
RUP content, starch content and MP concentration. Diet having 25:75 alfalfa
silage and corn silage, 26% starch and 16.6% CP had a MP concentration of
10.4%. Similarly diet 32.3:67.7 alfalfa silage and corn silage, 23.2% starch and
15.4% CP had a MP concentration of 9.3%. The same diet with 17.4% CP
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38 | P a g e
contained 11.5% MP. When starch content of above diets changed to 28.8%, the
diets supplied same amount of CP and MP. At 50:50 alfalfa silage and corn
silage, the diet having 22% starch and 16.4% CP supplied 10.4% MP, while the
diet with 26% starch provided 8.8, 10.4 and 12% MP at 14.9, 16.2 and 17.7% of
CP content. At about 30% starch content and 15.9% CP content, the diet
provided 10.4% MP. At 67.8:23.2 alfalfa silage and corn silage; the diets with
23.2% starch supplied 9.3 and11.5% MP at CP contents of 15.3 and 17.4%
respectively; while the diets with 28.8% starch supplied same concentration of
MP at CP contents of 15.0 and 17.4% respectively. At 75:25 alfalfa silage and
corn silage, the diet supplied 10.4% MP having 16.6% CP and 26.0% starch.
Increasing the concentration of MP increased the digestibility of N. The diet DE
(digestible energy) content was also affected by forage type and MP
concentration. At low MP and high alfalfa silage, the diet DE concentration
reduced but at high MP, increasing amount of alfalfa increased diet DE
concentration. Increasing MP increased energy correlated milk yield and protein.
Nichols et al. (2010) evaluated the effect of feeding two levels of MP in
gestating two year old heifers. Animals were subjected to two types of dietary
treatments; one having 10.7% CP supplying 102% of MP requirements of NRC
(1996) and the other with 12.6% CP supplying 119% of MP requirements. Level
of MP had no effect on calf birth weight, ADG, age at weaning, cow BW at
calving and proportion of cows returning to conceive. So it was observed that
feeding MP in excess of NRC recommendations during mid to late gestation did
not enhance heifer productivity.
Rius et al. (2010) studied the interaction of energy and predicted MP in
determining N efficiency in lactating dairy cows. Forty mid lactation cows were
subjected to four types of treatments: high energy- high protein (HE/HP), high
energy-low protein (HE/LP), low energy- high protein (LE/HP), low energy- low
protein (LE/LP). Energy concentrations were 1.55 Mcal/kg NEL for high energy
diet and 1.44 Mcal/kg NEL for low energy diet. CP contents of HE/HP, HE/LP,
LE/HP and LE/LP were 18.7, 15.2, 19.1 and 15.5% and the MP supply from
these diets were 2986, 2356, 3074 and 2186 g/d, respectively.
Imaizumi et al. (2010) compared the efficiency of three types of diets on
performances of forty two lactating Holstein cows. The control diet was having
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39 | P a g e
16% CP, which supplied adequate RDP and MP according to NRC (2001). The
two test diets were having more protein (17.5% CP) by providing either extra
SBM and CSM (SBCS-17.5) or extra urea (U-17.5). The MP supplied from these
diets was 10.8% for control, 11.8% for SBCS-17.5 and 10.8% for U-17.5; though
the three diets supplied similar energy i.e. NEL of 1.54 to 1.57 Mcal/kg DM. DMI
was higher for U-17.5 diet than for the control diet. Milk and 3.5% FCM yields
were increased with SBCS-17.5 diet, but not by U-17.5 diet. Milk fat content and
yield were not affected by treatments. Higher Milk protein yields were observed
for SBCS-17.5 treatment, but it decreased when fed U-17.5 diet.
Long-term (up to 10 weeks), continuous design trials conducted with cows
producing 84 to 95 lbs milk/d at Penn State showed that decreasing NRC (2001)
estimated metabolizable protein supply to 8 to 13% below requirements may
depress DMI and/or decrease milk production. These diets were usually around
14% CP. In one study (Lee et al., 2012b), diets that were 12 to 13% deficient in
metabolizable protein (14% CP) but supplemented with rumen-protected amino
acids did not result in decreased production. In a recent trial with cows milking
around 95 to 99 lbs/d, 5 to 8% metabolizable protein deficient diets also did not
result in depressed DMI or milk production (Giallongo et al., 2014). It may be
important to point out that in these trials the calculated metabolizable protein
balance was based on the actual DMI and production of the cows. The correct
way of estimating metabolizable protein requirements – based on actual or
potential milk production and composition – may be debated. In some cases, this
could make a difference (for example, if estimated milk production is greater than
10 lbs more than actually produced), although in the Penn State trials, Giallongo
et al., (2014) found it of little relevance.
The NRC (2001) model over-predicts the effects of metabolizable protein
on milk production. In trials with metabolizable protein deficient diets, the NRC
(2001) protein model under-predicted milk response by about 5 lbs per 100 g of
metabolizable protein deficiency (Lee et al., 2012b). Similar trends were reported
by Lee et al. (2012a); on average, under-prediction of milk yield in the
metabolizable protein deficient groups of cows was 22.7 ± 1.7 lbs/d. In a more
recent trial with 60 cows in which DMI was not affected by the metabolizable
protein deficient diets, milk yield was under-predicted by NRC (2001) on average
by 7.7 ± 1.5 lbs/d (Giallongo et al., 2014). Possible reasons for these effects may
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40 | P a g e
include overestimation of RDP requirements, sufficient urea recycling, and
variable efficiency of conversion of metabolizable protein for metabolic functions.
2.4.6 Urinary purine derivatives and creatinine excreation at varying levels
of protein and energy in diet
Microbial enzymes in rumen rapidly degrade purines of dietary and
exogenous materials, so any purines present in the digesta in the small intestine
can be expected to be only of microbial origin and can be considered to be
specific markers for the microbial fraction (Nolan, 1999). Excretion rates of
purine derivatives (PD) in the urine reflect the duodenal absorption of purine
bases (PB) and thus predict the microbial N yield from the rumen (Topps and
Elliott, 1965). Since the method is simple and non-invasive it overcomes the
problems of earlier methods. The rate of allantoin and total PD excretion were
positively correlated with digestible organic matter intake in buffaloes (Pimpa,
2002; Dipu et al., 2006) and crossbred bulls (George et al., 2006). Buffaloes
have a lower plasma PD excretion rate via the renal route and a significant
proportion (22%) of the plasma PD loss is via the saliva (Pimpa et al., 2007).
Dipu et al., (2006) observed that the PD excretion (mmol/d or mmol/kg
W0.75/d) responded significantly to intake (kg/d or kg W0.75/d) of DM and DOM. A
significant (p<0.01) increase in allantoin and non-significant response in uric acid
excretion in urine was observed with respect to increase in feed intake. Similar
results observed by different earlier workers (Liang et al., 1994; Chen et al.,
1996; Nolan, 1999). The xanthine and hypoxanthine in buffalo urine not be
detected and their concentration might have been below the detectable levels
owing to higher activity of xanthine oxidase in the intestine and plasma (Chen et
al., 1996; Nolan, 1999; Pimpa et al., 2003).
Vercoe (1976) and Liang et al. (1993) who found lower PD excretion per
unit of feed intake in buffaloes in comparison to cattle. This might be due to a
higher non-renal route of PD disposal in buffaloes or due to a higher recycling of
plasma PD, but the mechanism of buffaloes excreting less PD in comparison to
cattle is yet to be understood (Chen and Orskov, 2003).
Daily allantoin execration observed by Liang et al. (1994) for Swamp
buffaloes (12.8 mmol) fed 1.5% DM of body weight, by Chen et al. (1996) for
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41 | P a g e
Murrah Swamp buffaloes (9.6 to 23.5 mmol) and by Pimpa et al. (2003) for
Malaysian Swamp buffaloes (10.9 to 20.6 mmol). The molar proportion of
allantoin: uric acid obtained in Murrah buffaloes by Dipu et al., (2006) was in
consistent with values of 0.90:0.10 (Chen et al., 1996) and 0.84:0.16 (Pimpa et
al., 2003) previously observed in buffaloes.
Urinary excretion of creatinine (mmol/kg W0.75/d) did not differ significantly
(p>0.05) among various treatments (Dipu et al., 2006). This is in agreement with
the findings in Malaysian Kedah Kelantan cattle (Pimpa et al., 2001) and
buffaloes (Chen et al., 1996). A similar study suggested that total daily creatinine
excretion in urine is breed/species specific and more closely correlated with
muscle mass than body weight (Narayanan and Appleton, 1980).
CHAPTER – 3
Materials and Methods
Page | 42
MATERIALS AND METHODS
The present study can be divided into three phases, in the first phase feed
ingredients, fodders and silages were evaluated for their chemical composition,
metabolizable protein and metabolizable energy and fermentation characteristics
which included their DM and OM digestibility and methane emission. In Phase II,
In vivo experiment was conducted to estimate methane production from
buffaloes fed on oat silage or oat hay. In phase III two feeding trials were
conducted to determine the requirements of metabolizable energy and protein of
lactating buffaloes fed on silage based diets. The techniques and methods
adopted during experimental period are described below.
3.1 Phase I: Preparation of silage in plastic jar and evaluation of silage
3.1.1 Collection of fodders and silage preparation
Maize and oat fodder was selected for preparation of silage in plastic jar.
Maize fodder was harvested at vegetative stage as it contains high protein and
low fiber. The material was tightly packed in the plastic jars and covered with
cap and sealed the cover with paraffin wax to maintain anaerobic condition.
Silages were open after sixty days of packing.
3.1.2 Estimation of silage characteristics
Silages were analysed for pH, colour, texture and dry matter. The pH was
measured immediately by taking a representative sample of silage in a glass
beaker along with little quantity of water using a digital pH
3.1.3 Preparation of water extract of silage samples
meter. Textures were
observed by pressing the silages between two fingers. Colours were observed
visually. Dry matter was estimated by toluene distillation method of Dewar and
McDonald (1961).
At the end of 60 days storage period, the caps of silage jars were opened.
Water extract of silage was prepared by adding 100 ml of distilled water to 10 g
of fresh samples in a beaker and homogenized by mechanical homogenizer. The
material was filtered through four layers of cheese cloth and stored in refrigerator
at 4oC. The pH of water extract was estimated by digital pH meter. A portion of
Materials & Methods
Page | 43
the sample was dried in a hot air oven at 70o
3.1.4 Estimation of dry matter in fresh silage samples
C for 48 hr and then ground in a
willey mill for determining cell wall components and dry matter degradability.
The fresh silage samples were analysed for dry matter by the method of
Dewar and McDonald (1961).
Chemicals required
1. Ethanol
2. Phenolphthalein indicator
3. NaOH 0.1 Molar solution
4. Toluene
Procedure
Weighed 70-80 g of fresh silage samples in the flask and added 400 ml
toluene to it. Heated the flask on a heating mantle and adjusted the heat
controller unit until toluene boils steadily. After 90 minutes and subsequently at
15 minutes intervals, noted the volume of aqueous phase in the receiver. When
two consecutive equal readings were obtained, heating was discontinued, the
receiver and its contents were allowed to be cooled to room temperature and the
volume of aqueous phase was recorded. Aqueous phase was collected in a
beaker and 100 ml of ethanol previously neutralized was added. This was
titrated with 0.1 M NaOH using phenolphthalein as indicator.
Calculations
The dry matter content in silage sample was obtained from:
DM = 100 – 99.8 (V– 0.00555 t)
W
Where
V = Total volume of aqueous phase (ml),
W = Weight of silage taken (g),
T = Titre (ml of 0.1 M NaOH used)
Materials & Methods
Page | 44
3.1.5 Determination of lactic acid in silage samples
The estimation of lactic acid in silage samples was done as per the
method of Barker and Summerson (1941) and modified by Barnett (1951).
Reagents
1. Standard lactic acid solution: Dissolved 0.213 g of dry lithium lactate
(A.R. grade) in about 100 ml of distilled water in a volumetric flask (1litre).
Added about 1 ml of concentrated H2SO4
2. Working standard solution: Diluted 5 ml of stock standard to 100 ml
water and mixed. This solution contains 0.01 mg of lactic acid per ml.
Prepared fresh standard solution at the time of analysis.
to it and diluted to the mark with
water. This 5 ml solution contains 1 mg of lactic acid and stable for an
indefinite period, if kept in refrigerator.
3. P-hydroxydiphenyl reagent: P-hydroxydiphenyl (1.5 g) was dissolved in
10 ml of 5 per cent (w/v) aqueous NaOH and made up to a litre with
distilled water and stored in amber coloured bottle.
4. Calcium hydroxide powder: A.R. grade.
5. Copper sulphate solutions: Two copper sulphate solutions were
required, one 0.8 M and another 0.16 M. These were prepared from
cupric sulphate A.R. grade.
6. Concentrated H2SO4: Extra pure H2SO4
Procedure
was used.
Of the representative fresh sample, two 5 g portions were placed in two
250 ml beakers and 100 ml of boiling distilled water was added and the whole
material allowed cooling for 5 minutes. Each preparation was treated in the
following way. The mixture was stirred mechanically very vigorously for 5
minutes during which 0.5 g of solid calcium hydroxide was added to assist in
subsequent filtration. The product was filtered through a hard filter paper and the
first 20 ml of filtrate was discarded. Two ml of this filtrate was taken and diluted
to 10 ml with distilled water. One ml out of this was placed in a small glass
stoppered bottle to which added in the following order; 8 ml of distilled water
from a burette, 1 ml of 0.8 M cupric sulphate solution and 1 g of calcium
hydroxide powder. A standard for comparison was prepared by using 5 ml of
Materials & Methods
Page | 45
standard lactic acid solution and made this up as described for test portion using
only 4 ml of distilled water. Similarly, a blank was prepared by using 1 ml of
distilled water instead of unknown. The four bottles were then stoppered and
shaken for 20 minutes in small mechanical shaker. The content was filtered
through small filter papers discarding the first 2 ml of the filtrate in each case.
The remainder of each filtrate was collected in small test tubes. Each filtrate was
treated in the following way: the filtrate (1 ml) was placed in a Pyrex tube and
mixed with 0.05 ml of 0.16 M CuSO4 and 6 ml of N2 free H2SO4 from a grease
free burette. The tubes were allowed to stand in boiling water for 5 minute and
then cooled in a stream of running water. To each tube was added 0.1 ml of P-
hydroxydiphenyl reagent and contents shaken laterally to ensure even
distribution of insoluble reagent. The tubes were then placed in water bath for 30
minutes at 30-330
Calculations
C being shaken at 10 minute intervals. Finally, the tubes were
heated for 2 minutes in a boiling water bath and cooled to room temperature.
Reading of colour development was taken on spectronic-20 at 560 nm.
As the 1 ml of copper containing portion of standard lactic acid contains
0.01 mg/ 1ml portion used, the concentration of lactic acid in the unknown is
given by the formula:
Per cent lactic acid in fresh silage
= Reading of unknown – blank X 1
Reading of standard – blank
Per cent lactic acid on DM basis
= Reading of unknown – blank X 100
Reading of standard – blank DM content (%)
Precautions
The reagents were very sensitive. Hence precautions were taken to
ensure absolute cleanliness of all glasswares. All the glasswares were washed
successively with dilute HCl, warm water, detergent, cold water and finally dried
in an oven.
Materials & Methods
Page | 46
3.1.6 Determination of total-N and NH3
Total nitrogen content was estimated in fresh silage samples by micro
Kjeldahl method. A weighed quantity of the sample (about 0.5-1 g) was digested
in digestion tubes with 20-30 ml concentrated H
-N in fresh silage samples
2SO4 in the presence of small
quantity (2-3 g) of digestion mixture (Sodium sulphate and copper sulphate in
ratio of 10:1) till the solution became colourless. After digestion, the contents
were cooled and volume was made to 100 ml. 10 ml of aliquot was distilled in
Kjeldahl distillation apparatus (KELPLUS Nitrogen Analyzer) after adding 10-15
ml of 40% NaOH solution to make the content alkaline. About 60-75 ml of
distillate (light green colour) was collected into an Erlenmeyer flask containing 10
ml of 2% boric acid solution with mixed Tashiro’s indicator (10 ml 0.2%
bromocresol green and 20 ml 0.1% methyl red indicator). The distillate was then
titrated against standard N/100 H2SO4 solution and the end point was recorded
when the colour changed to slight pinkish. Volume of N/100 H2SO4
Calculation
solution
used in titration was recorded. The crude protein content in the silage sample
was calculated by multiplying the nitrogen content with the factor 6.25.
Total N (%) =
0.00014 X Volume of N/100 H2 SO4
used X Total volume made (ml) X 100
Aliquot taken (ml) X Wt. of sample (g)
For estimating water soluble nitrogen, 2 ml of water extract was taken in a
100 ml digestion tube. To it was added 2 ml of concentrated H2SO4 and 0.5 g of
digestion mixture and mixture digested on a digestion bench. After digestion, the
contents were cooled and volume was made to 100 ml. 10 ml of aliquot was
distilled in Kjeldahl distillation apparatus (KELPLUS Nitrogen Analyzer) after
adding 10-15 ml of 40% NaOH solution to make the content alkaline. About 60-
75 ml of distillate (light green colour) was collected into an Erlenmeyer flask
containing 10 ml of 2% boric acid solution with mixed Tashiro’s indicator (10 ml
0.2% bromocresol green and 20 ml 0.1% methyl red indicator). The distillate was
then titrated against standard N/100 H2SO4 solution and the end point was
recorded when the colour changed to slight pinkish. Volume of N/100 H2SO4
Materials & Methods
Page | 47
solution used in titration was recorded. Water soluble nitrogen content calculated
as follows:
Calculation
Water soluble N (%) =
00014 X Volume of N/100 H2 SO4
2 X A
used X Total volume made (ml) X100
Where,
A = Weight of dried sample taken for water extract preparation.
Similarly, for NH3-N estimation, 2 ml of water extract was taken in micro-
kjeldahl apparatus and contents made alkaline with 40% NaOH solution. Steam
distillation was done using KEL PLUS - N analyzer (Pelican, India) and the NH3
evolved was collected in boric acid solution having mixed indicator and titrated
against N/100 H2SO4. NH3
NH
-N concentration was calculated as follows:
3
0.00014 X Volume of N/100 H
- N (%) =
2 SO4
2 X A
used X Total volume made (ml) X100
Where,
A = Weight of dried sample taken for water extract preparation.
3.1.7 Estimation of total volatile fatty acids and their fractionation in fresh
silage samples
One ml of water extract was mixed with one ml of oxalate buffer (10%
potassium oxalate solution and 5% oxalic acid solution mixed in the ration of 1:1)
and was taken in KEL PLUS - N analyzer (Pelican, India). 100 ml of the steam
distillate was collected in volumetric flask and titrated against standard 0.01 N
sodium hydroxide using phenolphthalein as indicator. The total volatile fatty acid
concentration was calculated by using the formula:
TVFA m Moles/100 g DM = Vol. Of 0.01 NaOH used x 100
A
Where,
A = Weight of dried sample taken for water extract preparation.
Materials & Methods
Page | 48
For determining individual VFA, 5 ml of water extract was taken in a
beaker and 1 ml of 25 % metaphosphoric acid (prepared in 5 N H2SO4
3.2 ESTIMATION OF CHEMICAL COMPOSITION AND FIBRE FRACTIONS
OF SILAGE SAMPLES, EXPERIMENTAL DIETS AND DUNG
SAMPLES
) was
added. The sample was kept overnight and centrifuged at 3500 - 4000 rpm for
15 - 20 minutes. The supernatant was injected in Gas chromatograph (Nucon
5700, Nucon Engineers, New Delhi) equipped with flame ionization detector and
stainless steel column packed with Chromosorb –101 (length 4’; o.d ¼”; i.d. 3
mm; mesh range 80-100) to serve as a stationary phase. Analytical conditions
for fractionation of VFA were as follows: Injection port temperature, 250ºC;
column temperature, 190ºC and detector temperature, 260ºC. The flow rate of
carrier gas (nitrogen) was 40 ml/min; hydrogen 30ml/min; air 300 ml/min.
Injection volume was 3 µl. The injection was performed by means of 10 µl
Hamilton syringe (Hamilton, Nevada, USA). Different VFA’s of the samples
were identified on the basis of their retention time and their concentration
(mM) was calculated by comparing the retention time as well as
the peak area of standards after deducting the corresponding blank values.
3.2.1 Total ash (TA)
Apparatus: Silica crucibles, hot plate, muffle furnace, electronic weighing
balance and tong.
Procedure
A known quantity of sample (about 2.5 - 5 g) was taken in pre-weighed
silica crucible. After charring the sample on heater (till the smoke disappeared),
the crucible was kept in muffle furnace for ignition at 550°C for 2-3 h. The
crucible was removed on cooling and kept in a desiccator and weighed again to
find out weight of ash. The ash content was calculated as given below:
(Wt. of crucible + ash after cooling - Wt. of crucible) X 100
Total ash (%) =
Wt. of sample (g)
Materials & Methods
Page | 49
3.2.2 Organic matter (OM)
Procedure:
OM was determined by subtracting the total ash content from 100.
OM (%) = 100 – total ash (%)
3.2.3 Crude protein (CP)
Apparatus: Digestion tubes, digestion unit, Kjeldahl distillation apparatus,
Erlenmeyer flasks, titration assembly, burette.
Reagents:
Digestion mixture (Na2SO4 and CuSO4 in the ratio of 10:1), 40% NaOH
solution (400 g NaOH pellets dissolved in distilled water and volume made to
1000 ml), concentrated H2SO4 (98% purity and specific gravity 1.84)), 2% boric
acid indicator solution (20 g boric acid dissolved to 1 L and added with 10 ml
0.2% bromocresol green and 20 ml 0.1% methyl red indicators), N/100 H2SO4
Procedure
solution.
Total nitrogen was measured by micro Kjeldahl method. A known
quantity of sample (about 0.5-1 g) was taken in Kjeldahl flask/digestion tubes
and digested with 20-30 ml concentrated H2SO4 and 2-3 g of digestion mixture
till the solution became colourless. After digestion, the contents were cooled and
volume was made to 100 ml. 10 ml of aliquot was distilled in Kjeldahl distillation
apparatus (KELPLUS Nitrogen Analyzer) after adding 10-15 ml of 40% NaOH
solution. About 60-75 ml of distillate (light green colour) was collected into an
Erlenmeyer flask containing 10 ml of 2% boric acid indicator solution. The
distillate was then titrated against standard N/100 H2SO4 solution and the end
point was recorded when the colour changed to slight pinkish. Volume of N/100
H2SO4
Calculation
solution used in titration was recorded.
N (%) = 0.00014 X Volume of N/100 H2SO4
Aliquot taken (ml) X wt. of sample (g)
used X Volume made (ml) X 100
Materials & Methods
Page | 50
The crude protein (%) of sample was calculated by multiplying the N
content with the factor 6.25. This was based on the principle that all the proteins
contain 16% nitrogen.
3.2.4 Ether extract (EE)
Apparatus: Soxhlet’s extraction apparatus, oil flask, thimble, hot air oven,
desiccator, weighing balance.
Reagent: Petroleum ether (boiling point = 40-60o
Procedure
C).
A known quantity of ground sample (about 3 g) was taken in a cellulose
thimble and extracted for 8 hours (condensing rate 5-6 drops per second) with
petroleum ether in Soxhlet’s extraction apparatus attached to a pre weighed oil
flask. The oil flask was removed and after evaporating the excess of ether, it was
dried overnight in a hot air oven (100±5°C). The flask was cooled in a desiccator
and weighed to a constant weight. The difference between two weights gave the
amount of ether extract in the sample.
Calculation
EE (%) = (Wt. of oil flask with ether extract – Wt. of empty oil flask) X 100
Wt. of sample
3.2.5 Estimation of cell wall constituents
The fraction of cell wall constituents such as NDF, ADF, hemicellulose,
cellulose and lignin were estimated (Van Soest et al., 1991).
3.2.5.1 Neutral detergent fibre (NDF)
Apparatus: Spoutless beakers, sintered glass crucible, vacuum pump, hot air
oven, muffle furnace, electronic balance, desiccator.
Reagents: Neutral detergent solution (NDS), amylase solution, acetone
Neutral detergent solution (NDS)
Sodium lauryl sulphate - 30.00 g
Disodium ethylene diamino tetra acetate (EDTA) - 18.61 g
Sodium borate decahydrate - 6.81 g
Materials & Methods
Page | 51
Disodium hydrogen phosphate (anhydrous) - 4.56 g
Triethylene glycol - 10 ml
Distilled water - 990 ml
EDTA and sodium borate decahydrate were put together in a large beaker
with some distilled water and heated on hot plate until dissolved. Similarly,
sodium lauryl sulphate was dissolved in distilled water and triethylene glycol was
added to it. The solution of sodium lauryl sulphate and triethylene glycol was
added to the previous solution. Disodium hydrogen phosphate was taken in
another beaker and some amount of distilled water was added and the contents
were heated until dissolved. Then, it was added to solution containing other
ingredients and volume was made up to one litre with distilled water.
Amylase solution: Dis s olve 2 gm α–amylase enzyme in 90 ml water, filter
through Whatman 54 paper and stored at 5°C.
Procedure
Approximately 1 g sample was taken in spoutless beaker of 1 L capacity.
To this, 100 ml NDS and 0.5g of sodium sulphite were added. The contents of
spoutless beaker were refluxed for half an hour. Thirty minutes after onset of
boiling, beaker was removed and 2 ml of enzyme solution was added. One hour
after initial boiling, the contents of beaker were filtered through pre-weighed 50
ml sintered glass crucible using oil-free vacuum pump. The contents were
washed repeatedly with hot boiling water and then acetone to remove all salts.
The sintered crucible containing residue was dried in hot air oven (100±5°C)
overnight, cooled and weighed to a constant value. Then ashing was done and
crucible along with ash was weighed again.
The NDF (ash free) was calculated as follows:
(Wt. of crucible with residue – wt. of crucible with residual ash) ×100
NDF (%) =
Wt. of sample taken
3.2.5.2 Acid detergent fibre (ADF)
Apparatus: Spoutless beaker, sintered glass crucible, vacuum pump, hot air
oven, electronic balance, desiccator.
Materials & Methods
Page | 52
Reagents: Acid detergent solution (ADS), acetone, hot boiling water.
Acid detergent solution (ADS): 20 g cetyl trimethyl ammonium bromide
(CTAB) was dissolved in one litre of 1 N H2SO4
Procedure
.
Approximately 1 g of sample was taken in a spoutless beaker of 1 L
capacity. To this, 100 ml ADS was added and the contents were refluxed for
exactly 1 hour. After refluxing, the residue was filtered through pre-weighed
sintered glass crucible using vacuum pump and washed with hot water 2-3 times
followed by acetone to remove all salts. The sintered crucible containing residue
was dried in hot air oven (100 ± 5°C) and weighed again. The ADF was
calculated as follows:
(Wt. of crucible with residue – Wt. of empty crucible)
ADF (%) = x 100
Wt. of sample taken
3.2.5.3 Cell contents and Hemicellulose
Cell contents (%) = 100- NDF content (%)
Hemicellulose was soluble in ADS and thereby calculated by subtraction
of ADF from NDF as: Hemicellulose (%) = NDF (%) – ADF (%)
Cellulose
Principle
For estimation of cellulose, ADF procedure was used as a preparatory
step. The ADF residue consisted of cellulose, lignin, cutin and acid insoluble ash
(mainly silica). Cellulose was dissolved by 72% H2SO4
Apparatus: Same as that of ADF estimation, enamel tray.
(w/w) treatment of ADF.
Reagents: ADS, 72% H2SO4
Preparation of 72% H
(w/w), hot boiling water.
2SO4 (w/w basis): Reagent grade H2SO4 (specific
gravity 1.84 and 98% purity) was standardized to specific gravity 1.634 at 200C
or 12 M. For this, 1200 g H2SO4 (about 654 ml reagent grade H2SO4) was
added to 440 ml distilled water in 1L capacity volumetric flask while cooling
Materials & Methods
Page | 53
under running tap water. The weight was standardized to 1634 g/L at 200C by
removing solution and adding distilled water or H2SO4
Procedure
as required.
Sintered glass crucible (G-I) containing ADF contents was weighed and
then placed in enamel tray in such a manner that one end of the enamel tray
was at about 2 cm height than the other end, so that acid could drain away from
the crucible. The crucible was then filled with 72% H2SO4 (w/w basis) and the
contents were stirred with glass rod to break all the lumps. The crucible was
refilled with 72% H2SO4
W
after 1 hour interval. After 3 hours, the crucible was
removed from the tray and filtration of acid was done by using vacuum pump.
The material was washed with hot water until it became free from acid and kept
in the oven (100 ± 5°C) overnight and weighed.
1 - W Cellulose (%) = x 100
2
Y
Where,
W1
W
= wt. of crucible + residue (before acid extraction)
2
Y = wt. of initial sample (g)
= wt. of crucible + residue (after acid extraction)
3.2.6 Acid detergent lignin (ADL)
Apparatus and reagents: Same as that of ADF estimation.
Procedure
The procedure for estimation of ADL content was exactly same up to the
filtering and drying of ADF contents of sintered crucible after treating with 72%
H2SO4 (w/w) in the cellulose estimation procedure. Then, the crucible with dry
residue was kept in muffle furnace for ignition at 550 to 6000
ADL (%) = (Wt. of crucible with dry residue – Wt. of crucible with ash)
C for 2-3 hrs, cooled
and weighed again. The acid detergent lignin was calculated as follows:
X 100
Wt. of sample (g)
Materials & Methods
Page | 54
3.2.7. Estimation of nitrogen
Acid detergent insoluble nitrogen (ADIN), neutral detergent insoluble
nitrogen (NDIN), non protein nitrogen and soluble protein were estimated as per
Licitra et al. (1996)
3.2.7.1. Determination of acid detergent insoluble nitrogen (ADIN)
Nitrogen in ADF residue was estimated following the standard kjeldahl
procedure. ADIN of sample was expressed as percent of total nitrogen or as N ×
6.25.
3.2.7.2. Determination of neutral detergent insoluble nitrogen (NDIN)
Nitrogen in NDF residue was estimated following the standard kjeldahl
procedure. NDIN of sample was expressed as percent of total nitrogen or as N ×
6.25.
3.2.7.3. Non protein nitrogen
Apparatus:
Erlenmeyer flask (125ml), whatman filter paper 54 or 541, analytical
balance, water bath, vacuum source, filter manifold fitted with conical funnel (50
ml), kjeldahl apparatus
Reagents: Trichloroacetic acid solution in water 10% (W/V) was kept
refrigerated.
Procedure:
0.5 gm ground dry sample was weighed into a 125 Erlenmeyer flask. To
it 50 ml of distilled water was added and allowed to stand for 30 min. To it 10 ml
of 10 % trichloroacetic acid was added and let stand for 20 -30 min. Then filtered
through whatman paper 54 or 541 by gravity and washed twice with
trichloroacetic acid solution. Paper was transferred to kjeldahl flask and residual
nitrogen was determined. NPN was calculated by subtracting residual nitrogen
from total nitrogen. Value may be expressed as crude protein (N x 6.25) or as
percent of total feed nitrogen.
Materials & Methods
Page | 55
3.3. Estimation of total digestible nutrient (% TDN)
(1) Truly digestible NFC (tdNFC)= 0.98 (100 - [(NDF - NDICP) + CP + EE +
Ash]) X PAF
(2) Truly digestible CP for forages (tdCPf) = CP X exp [-1.2 X (ADICP/CP)]
(3) Truly digestible CP for concentrates (tdCPc) = [1 - (0.4 X (ADICP/CP))] X
CP
(4) Truly digestible FA (tdFA) = FA Note: If EE < 1, then FA = 0
(5) Truly digestible NDF (tdNDF) = 0.75 X (NDFn - L) X [1 - (L/NDFn) 0.667
Where, NDICP = neutral detergent insoluble N X 6.25, PAF = 1 for
fodders, ADICP = acid detergent insoluble N X 6.25, FA = fatty acids (i.e., EE -
1), L = lignin, and NDFn = NDF - NDICP. All values are expressed as a per cent
of dry matter (DM).
]
Equations 1, 2, 3, 4 and 5 are based on true digestibility, but TDN is
based on apparent digestibility; therefore, metabolic fecal TDN was subtracted
from the sum of the digestible fractions. Weiss et al. (1992) determined that, on
average, metabolic fecal TDN is equal to 7. The TDN was calculated using
equation 6.
(6) TDN (%) = tdNFC + tdCP + (tdFA X 2.25) + tdNDF – 7
The TDN was also calculated from digestibility data of metabolism trial
using the equation 7.
(7) TDN (%) = (% dig NFC) + (% dig CP) + (% dig. FA) X 2.25 + (% dig NDF)
Where % dig nutrients (NFC, CP, FA and NDF) were calculated as below;
% nutrient in fodder X digestibility (%)
% dig nutrient =
100
Total digestible nutrients (TDN) and ME of maize silage were estimated
using the following equations given by Clemson University (1996) and Penn
State University respectively.
TDN% in silage = 85.65 + (CP x 0.362) - (ADF x 0.825)
ME (MJ/kg DM) =.1642 X TDN%
Materials & Methods
Page | 56
3.4 In vitro gas production (IVGP) technique
In vitro gas production was measured as discussed by Menke and
Steingass (1988).
Preparation of solutions:
Micro mineral solution
CaCl
a
2.2H2
MnCl
O 13.2 g
2.4H2
CoCl
O 10.0 g
2.6H2
FeCl
O 1.0 g
3.6H2
Dissolved in 100 ml of water
O 8.0 g
Rumen buffer solution
NH
b
4HCO3
NaHCO
4.0 g
3
Dissolved in 1000 ml of water
35.0 g
Macro mineral solution
Na
c
2HPO4
KH
anhydrous 5.70 g
2PO4
MgSO
anhydrous 6.20 g
4.7H2
Dissolved in 1000 ml of water
O 0.60 g
Resazurine solutiond
Reducing solution
0.10 % w/v
1N NaOH 4.0 ml
e
Na2S.9H2
Water 95 ml
O 625 mg
The details of solutions and the order in which they were added prior to
the filling in syringes are as follows:
Materials & Methods
Page | 57
Table 3.1: Details of solutions for in vitro gas production technique
Items 30 syringes 45 syringes 60 syringes
Solution:I
Distilled water 365 ml 550 ml 730 ml
Micro mineral solutiona
Rumen buffer solution
0.1 ml 0.15 ml 0.185 ml
b
Macro mineral solution
183 ml 275 ml 365 ml
c
Resazurine solution
183 ml 275 ml 365 ml
d
Solution:II
0.95 ml 1.45 ml 1.90 ml
1N NaOH 1.6 ml 2.4 ml 3.1 ml
e
Na2S.7H2
Distilled water 37 ml 55 ml 73 ml
O 220 mg 330 mg 440 mg
Solution:III
Rumen liquor 330 ml 500 ml 660 ml
The incubation of feed samples were carried out in 100 ml calibrated
glass syringes (Haberle, Germany) as described by Menke and Steingass
(1988). The sample was weighed on a plastic boat with removable stem and
placed into the bottom of the glass syringe without sticking it to the sides of
syringe. The piston was lubricated with petroleum jelly and pushed inside the
glass syringe. Each substrate was taken in triplicate. The syringes were kept in
an incubator at 39±0.5oC overnight. To incubate 200 mg sample, the
composition of solutions were as; 10 ml strained rumen liquor (RL) and buffer
mixture solutions like, 5 ml bicarbonate buffer, 5 ml macro minerals, 0.002 ml
micro minerals, 0.02 ml resazurine and 10 ml distilled water (total 30 ml). The
buffer solution was prepared and kept overnight at 39±0.5oC and then bubbled
with CO2.
Rumen liquor was collected from donor buffaloes, fitted with permanent
rumen fistula. Donor animals were fed on roughage and concentrate based diet
(2.5 kg concentrate mixture, 30 kg maize fodder and 3 kg wheat straw). Rumen
The blue color of solution first changed to pink and finally became
colorless.
Materials & Methods
Page | 58
liquor was collected before feeding and watering of the animals and filtered
through 4 layers of muslin cloth and kept into the pre-warmed thermo-flask and
brought to the laboratory. The rumen liquor was bubbled with CO2
After medium became colorless, the required amount of SRL was added.
The ratio of medium to rumen liquor was 2:1. Then, 30 ml of incubation medium
was injected into the each syringe using auto pipette. The syringes were shaken
gently and 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±0.5
for about 1
minute.
oC). The syringes were shaken 4 to 5 times during incubation.
Gas produced (ml) during fermentation was measured after 24 h in all feeds
except green and dry roughages where it was measured after 48 h. Net gas
produced (mM/200 mg substrate) were calculated after deducting the blank and
considering the volume of 1 mM of gas at 39o
3.4.1 Methane production
C in Karnal is equal to 24.97 ml.
After the incubation of feeds, suitable aliquot of gas was withdrawn from
the tip of the syringe using Hamilton gas tight syringe and was analyzed for its
CH4 using Gas chromatograph (Nucon 5700, India) fitted with stainless steel
column packed with Porapak-q (length 6’; o.d. 1/8”; i.d. 2 mm; mesh range 80-
100) and thermal conductivity detector. The temperature of injection port, column
and detector was 150, 60 and 130oC, respectively. The flow rate of carrier gas
(N2) through the column was 40 ml/ min. The standard gas used for CH4
estimation (Spantech Calibration gas, Surrey, England) composed of 50% CH4
and 50% CO2. The peak of CH4 gas was identified based on of retention time of
standard CH4 gas and the area obtained was used to calculate CH4
Area of sample
percentage
in the gas sample.
CH4
Area of Standard
(%) = x 50
CH4 produced from the substrate during incubation was corrected for
the blank values. The net volume of CH4 (ml) produced was calculated and then
the amount of CH4 (g/kg DM incubated, g/kg IVDMD and g/kg IVOMD.
Materials & Methods
Page | 59
Total gas produced (ml) X % CH4
CH
in the sample
4
100
(ml) =
Net CH4 (ml) = CH4 in sample - CH4
in blank
Net CH4
CH
(ml) X 1000 X16
4
DM incubated (g) X 24.97 X 1000
(g/Kg DM incubated) =
Net CH4
n CH
(ml) X 1000 X16
4
TDMD (g) X 24.97 X 1000
(g/Kg IVDMD) =
Net CH4
CH
(ml) X 1000 X16
4
TOMD (g) X 24.97 X 1000
(g/Kg IVOMD) =
3.4.2 In vitro true dry matter and organic matter digestibility (IVDMD and
IVOMD)
The method of Goering and Van Soest (1970) for determination of truly
degradable feed was used. Substrate (500 mg) was taken in triplicates in bottles
(100 ml). The rumen fluid (660 ml) was added to warm (about 390C) and
reduced medium consisting of 1095 ml distilled water, 730 ml rumen buffer
solution (35.0 g NaHCO3 and 4 g NH4HCO3 made up to 1 litre with distilled
water), 365 ml macro mineral solution (6.2 g KH2PO4, 5.7 g Na2HPO4, 2.22 g
NaCl and 0.6 g MgSO4. 7H2O made up to 1 litre with distilled water), 0.23 ml
micro mineral solution (10.0 g MnCl2.4H2O, 13.2 g CaCl2. 2H2O, 1 g CoCl2
.6H2O, 8.0 g FeCl2. 6H2O and made up to 100 ml with distilled water), 1 ml
resazurine (0.1 g made up to 100 ml with distilled water) and 60 ml freshly
prepared reduction solution containing 580 mg Na2S. 9H2O and 3.7 ml 1 M-
NaOH (Makkar et al. 1995).The mixture was kept stirred under CO2, at 390C
using a magnetic stirrer fitted with a hot plate. A portion (40 ml) of the rumen-
fluid medium was transferred into each bottle and incubated in a water bath at
390C for 24 h (all feeds except dry roughages, which were incubated for 48 h)
duration. After incubation entire bottle content was taken into 1000 ml spoutless
Materials & Methods
Page | 60
beaker. The bottles were washed with 40 ml of double strength NDS and
refluxed at 100oC for 1 h. Contents were filtered through the sintered glass
crucible (G-1) and kept in hot air oven at 90oC for drying. The residue in each
crucible was ashed in muffle furnace at 550o
IVDMD (%) = Wt. of DM incubated – Wt. of NDF residue x 100
C for 2 h to determine the OM
content. The following equations were used to determine in vitro DMD and OMD.
Wt. of DM incubated
IVOMD (%) = Wt. of OM incubated – Wt. of OM residue x 100
Wt. of OM incubated
3.5 Estimation of utilizable crude protein
The incubation procedure was based on the in vitro incubation technique
of Zhao and Lebzien (2000). Each sample was incubated in six replicates. Three
samples were processed for uCP estimation whereas remaining three were
freeze dried after incubation. From those three freeze dried samples were
digested further by pepsin and pancreatin (Calsamiglia and Stern (1995) to
determine the intestinal digestibility of uCP.
Collection of rumen liquor: The rumen liquor from the rumen of fistulated
buffalo was collected into thermos pre-wormed to 380
Preparation of buffer
C, before its morning
feeding and was filtered through 4 layers of surgical gauze. The fistulated animal
was fed on NDRI concentrate mixture along with maize green ad lib and had free
access to water.
Buffer I: Na2HPO4. 12H2O (23.5 g), NaHCO3 (15 g) and NH4HCO3 (9.5 g) were
dissolved in 400 ml distilled water. NH4HCO3
Buffer II: NaCl (23.5 g), KCl (28.5 g), MgCl
was used as nitrogen source for
the microbes.
2. 6H2O (6 g) and CaCl2. 2H2
Fifty ml buffer II was mixed with 400 ml buffer I. An adequate amount of
distilled water was added to the buffer mixture to yield a final volume of 500 ml.
Then 250 ml of the mixed buffer was diluted with 1000 ml distilled water and
O (2.63
g) were dissolved in 1000 ml distilled water.
Materials & Methods
Page | 61
prewormed to 380C. Then 312.5 ml rumen fluid was added and continuously
gased with CO2. Sample weighing 0.5 gm was taken in 100 ml glass bottle. For
each sample 6 replicates were taken. To it 50 ml of buffer rumen fluid mixture
was transferred. Each bottle was sealed with rubber stopper and then kept at
380
Ammonia estimation by distillation: The contents of the syringes were transferred
into Kjeldahl flasks, in which 15 ml of 0.25 M phosphate buffer (90 g of
Na
C in a water bath. Additionally 6 blanks without any feedstuff were incubated.
After 24 h of incubation, all the bottles were taken out of the water bath and the
pH was measured immediately. Three replicates from each sample and blank
were freeze dried for further amino acid analysis. The samples in the remaining
replicates were filtered through ashless filter paper (no. 42) and washed with
distilled water. The volume of the liquid was recorded and 25 ml liquid was
sampled for N determination. The liquid sample was distilled to release excess
ammonia. The solid material and the filter paper were collected into kjeldahl flask
for N determination.
2HPO4. 12H2O/l of distilled water; pH = 11.0 adjusted with sodium hydroxide)
was added to 15 ml of each sample to achieve a pH between 10.0 and 10.5.
This pH level was chosen to cast out all NH4+ as NH3 of basic environment and
simultaneously minimise alkaline-caused release of NH3 out of non-ammonia
nitrogen substances during degradation. Distilled NH3
[NH
was then collected in 3%
(w/v) boric acid and titrated with 0.05 M hydrochloric acid solution.
3-Nblank (mg) + Nsample(mg) - NH3-Nsample
uCP (g/kg DM)=
(mg)]x 6:25 x100 000
Sample weight (mg) - Sample DM (%)
3.5.1. Estimation of Intestinal digestibility of uCP
A three step in vitro procedure developed by Calsamiglia and Stern
(1995) was adopted for estimating intestinal digestibility of the RUP (rumen
undegradable protein) fraction of feed proteins. In the present study instead of
RUP uCP residue was considered for analysis. The uCP 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
Materials & Methods
Page | 62
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
uCP Intestinal Digestibility = x 100
Undegraded N
Phase II
3.6. In vivo methane trial: Estimation of methane production from buffaloes
fed on oat silage or oat hay
3.6.1. Selection and grouping of animals
Sixteen dry Murrah buffaloes (566.5 ± 12.5 kg body weight) were selected
from cattle yard herd of National Dairy Research Institute, Karnal and distributed
randomly into two equal groups based on body weight (Table 3.2) All the
experimental procedures including animals were approved by Institutional
Animal Ethics Committee of NDRI (IAEC/21/14 dated 04.01.2014).
3.6.2. Feeding of animals
The animals of group I (T1) and group II (T2
3.6.3 Digestibility trial:
) were fed on oat hay and oat
silage solely as a nutrient supplement. The silage was prepared in tower silos
from oats (Avena sativa) harvested at milk stage. The ensiling material contained
29%DM. Oat silage samples were collected daily during the experimental
feeding and the DM content was determined by toluene distillation method
(Dewar and McDonald,1961). Oat fodder was harvested for hay making at
kernels in the soft dough stage of maturity. Animals were offered ad-libitum fresh
drinking water twice daily in morning at 10:00 h and evening at 17:30 h.
A digestibility trial of 7 days duration was conducted after 60 days of
preliminary feeding period. Animals were weighed before and after the trial
consecutively for 2 days. Individual record of dry matter intake (DMI) was
maintained for each animal. Representative samples of fodders, residue, and
faeces were collected daily from each animal and processed for chemical
analysis. The samples were analyzed for organic matter (OM), crude protein
Materials & Methods
Page | 63
(CP) and ether extract (EE) (AOAC 2005). Total digestible nutrients (TDN), gross
energy (GE) and metabolizable energy (ME) values were calculated using
equations of National Research Council (2001).
Table 3.2: Detail of experimental non-lactating buffaloes fed on oat hay
or silage for methane emission study
Treatment Animal No. Body wt. (Kg)
Oat hay feeding
( T1 )
5766 693
5895 601
5973 564
6122 557
5754 564
6011 520
6008 495
296 538
Average 566±21.29
Oat silage Feeding
(T2)
5701 669
5784 598
5946 572
5743 653
5841 605
5848 522
495 418
6040 498
Average 567±29.65
3.6.4 Collection of methane gas and estimation by SF6
Methane production by the animals was measured by SF
tracer technique
6 tracer
technique (Johnson et al. 1994). For individual animal measurement, a
permeation tube containing SF6 of known release rate is placed in the rumen
prior to an experiment. Releasing rate of SF6 from permeation tube was
standardized by weighing at regular intervals for 6 weeks. Each animal was fitted
with a halter, which supports a capillary tubing (SS Capillary tubing 0.0635” X
Plate 3.1. Permeation tube and its parts
Plate 3.2. ECD detector for estimation of Sulphur hexafluoride gas during
methane estimation
Plate 3.3 Illustration of the SF6 tracer technique.
Materials & Methods
Page | 64
0.004”) in such a way that it’s open close to the nose. The eructed gas from nose
and mouth was collected evacuated canister. Five successful collections of 24-h
durations were done for each animal. Methane and SF6
Methane emission rate was calculated as follows;
concentrations were
estimated in the collected sample with the help of gas chromatograph (Nucon-
5700) fitted with flame ionization and electron capture detectors (FID and ECD)
respectively.
QSF6 - CH4
QCH
conc
4
SF
=
6
Where, Q CH
conc
4
Q SF
= methane production rate (g=min),
6 = SF6
CH
release rate (μg=min),
4 = methane concentration in collected sample (μg=m3
SF
) and
6 = SF6 concentration in collected sample (μg=m3
)
3.7. Estimation of metabolizable energy (ME) requirements for the
lactating buffaloes fed on silage based diet
3.7.1 Selection, grouping and feeding of animals
Fifteen Murrah buffaloes in mid lactation were selected from cattle yard
National Dairy Research Institute, Karnal and divided into three groups based on
their body weight, milk production and lactation number. A seventy five days
feeding trial was conducted. These animals were randomly divided into three
groups i.e. ME-10, ME0 and ME+10 with five animals in each group. Three
concentrate mixture were formulated being iso -nitrogenous but having varying
energy levels, viz; ME-10, ME0 and ME+10; ME reflecting metabolizable energy
followed by percentage energy i.e. 10% variation in relation to ICAR, (2013)
recommended levels for lactating buffaloes. ME0 group was fed on diet that
meet ME as per ICAR (2013) recommendations while ME-10 and ME+10 groups
were fed diet containing 10% less and 10% more ME as compared to ME0. The
ration consisted of concentrate mixture (40%) and maize silage (60%). All the
experimental procedures including animals were approved by Institutional
Animal Ethics Committee of NDRI (IAEC/21/14 dated 04.01.2014).
Materials & Methods
Page | 65
Table 3.3: Details of experimental lactating buffaloes fed on varying
metabolizable energy in the diets
Sr. No. Animal No Parity Body Wt (kg) Milk Yield (kg)
ME90 (10% less than ICAR, 2013)
1 6154 1 534 11.50
2 6108 1 512 9.50
3 6010 1 649 8.50
4 5642 4 564 8.00
5 5992 1 568 6.00
Mean ±SE 1.60±0.67 565±23.28 8.70±0.90
ME100 ( As per ICAR, 2013)
1 6205 1 607 11.50
2 5959 1 557 8.50
3 5835 2 516 8.50
4 6138 1 581 8.00
5 5801 3 576 8.00
Mean ±SE 1.60±0.43 567±15.13 8.90±0.66
ME110 (10% more than ICAR, 2013)
1 5855 3 629 13.00
2 6085 1 538 8.50
3 5914 2 556 7.50
4 5940 2 583 8.50
5 6160 1 547 6.50
Mean ±SE 1.80±0.23 570±16.43 8.80±0.43
Materials & Methods
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Table 3.4: Ingredients composition of concentrate varying metabolizable
energy level fed to lactating buffaloes
Ingredient ME ME90
ME100
110
Maize -- -- 15.62
Pearl millet 40.62 17.62 --
Mustard oil cake -- 12.70 15.70
Cotton seed cake 16.70 -- --
Soybean meal 18.12 18.12 18.12
Wheat bran 11.46 23.46 23.46
Deoiled rice bran 10.10 25.10 24.10
Mineral mix. 2.00 2.00 2.00
Salt 1.00 1.00 1.00
Total 100 100 100
3.7.2 Location of Experiment
The study was conducted in an experimental animal shed of National
Dairy Research Institute, Karnal, (India), located at 29°41′N 76°59′E / 29.68°N
76.98°E. It has an elevation 250 meters (748 feet) from sea level.
3.7.3 Housing and Management of Animals
All the experimental buffaloes were housed in a well-ventilated animal
shed having the arrangement for individual animal feeding without having access
to the other animal’s diet. The animal shed was 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 to keep the animals away from infection.
3.7.4 Body Weight and DM Intake
The animals were weighed before feeding and watering in the morning on
two consecutive days at the start of experimental feeding and thereafter at
fortnightly intervals during the experimental period of 75 days. DM intake was
recorded daily by subtracting the residual DM from the quantity of DM offered.
Materials & Methods
Page | 67
3.7.5 Daily Milk Yield
Milking was done twice daily i.e. morning at 5:30 a.m.and evening at 6:00
p.m. The milk was collected in milking vessels after screening through muslin
cloth.
3.7.6 Milk Composition
Milk samples were collected for the estimation of milk parameters every
fortnightly. Milk fat, protein, lactose and SNF were determined using automatic
milk analyzer (Lactostar).
3.7.7. Metabolism trial
A metabolism trial for a period of 7 days was conducted to determine the
nutrient digestibility and intake and N balance. Animals were shifted 3 days prior
to the metabolism trial to adjust with the environment of the metabolism shed.
Animals were weighed consecutively for 2 days before and after the trial. Feed
was offered daily at 9.00 h in the morning. Fresh drinking water was provided
twice a day and the quantity was measured each time to calculate the total water
intake.
3.7.8. Sampling, processing and storage
The feeds and fodders were weighed before offering. DM content of
rations offered was measured daily. The residues of previous day were collected
daily at 8.00 h before offering feed and weighed. The feed, fodder samples
before offering and residue samples after compiling were collected daily and
dried to find out their DM content. At the end of 7th
Faeces voided during 24 h were collected daily for 7 days and weighed at
8:30 h daily and a small sample (2% of total sample on fresh weight basis) was
pooled and processed in a similar manner as that for feeds and residue.
Simultaneously, total urine voided was also measured daily and an aliquot (1%
of total volume) was collected and stored in plastic containers containing 2 ml of
25% H
day collection the dried
samples were mixed ground to pass through 1 mm sieve and stored in air tight
containers.
2SO4.
Materials & Methods
Page | 68
3.7.9. Analysis of feed, residue, faeces and urine
The samples were analyzed for proximate composition (AOAC, 2005), cell
wall frcations (Van Soest et al., 1991) and fiber bound protein fractions such as
NDF and ADF bound CP (NDICP and ADICP) (Licitra et al., 1996). Total-N
content of urine samples were estimated (AOAC, 2005). The TDN, DE and ME
value of the fodders was calculated using chemical composition based formulae
suggested by NRC (2001).
3.8 Estimation of metabolizable protein (MP) requirements for the
lactating buffaloes fed on silage based diet
3.8.1 Selection, grouping and feeding of animals
Fifteen Murrah buffaloes in mid lactation were selected from cattle yard
National Dairy Research Institute, Karnal and divided into three groups based on
their body weight, milk production, lactation number. All the experimental
procedures including animals were approved by Institutional Animal Ethics
Committee of NDRI (IAEC/21/14 dated 04.01.2014).
Table 3.5: Ingredients composition of concentrate varying metabolizable
protein level fed to lactating buffaloes
Ingredient MP MP90
MP100
110
Maize 30 25 22
Bajra 10 10 10
MC-deoiled 10 15 18
CSC 17 20 25
SBM -- 3 6
DORB 30 24 16
Mineral Mixture 2 2 2
Salt 1 1 1
Total 100 100 100
Materials & Methods
Page | 69
Table 3.6: Details of experimental lactating buffaloes fed on varying
metabolizable energy in the diets
Sr. No. Animal No Parity Body Wt (kg) Milk Yield (kg)
MP90 (10% less than ICAR, 2013)
1 5743 1 558 8.00
2 6617 2 488 9.50
3 5476 1 569 7.00
4 6093 1 528 7.00
5 5950 1 543 6.00
Mean ±SE 1.2±0.20 537.20±14.11 7.50±0.59
MP100 ( As per ICAR, 2013)
1 6010 3 615 7.50
2 449 1 552 8.00
3 6006 1 486 5.00
4 5590 2 531 7.00
5 5614 2 526 9.00
Mean ±SE 1.8±0.43 542±21.14 7.30±0.66
MP110 (10% more than ICAR, 2013)
1 5826 2 589 7.50
2 5801 2 570 8.00
3 6093 1 498 5.00
4 5590 1 529 7.00
5 5992 1 515 9.00
Mean ±SE 1.40±0.24 540±17.05 7.30±0.66
Materials & Methods
Page | 70
A seventy five days feeding trial was conducted. These animals were
randomly divided into three groups i.e. MP-10, MP0 and MP+10 with five animals in
each group. Three diets were formulated being iso-caloric but having varying
metabolizable protein levels, viz; MP-10, MP0 and MP+10 ; MP reflecting
metabolizable protein followed by percentage protein i.e. 10% variation in
relation to ICAR, (2013) recommended levels for lactating buffaloes. MP0 group
was fed on diet that meet MP as per ICAR (2013) recommendations while MP-10
and MP+10 groups were fed diet containing 10% less and 10% more MP as
compared to MP0.
3.8.2 Housing and Management of Animals
in relation to ICAR (2013) recommended levels for lactating
buffaloes. The ration consisted of concentrate mixture (40%) and maize silage
(60%).
All the experimental buffaloes were housed in a well-ventilated animal
shed having the arrangement for individual animal feeding without having access
to the other animal’s feed. The animals shed was 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 to keep the animals away from infection.
3.8.3 Body Weight and DM Intake
The animals were weighed before feeding and watering in the morning for
two consecutive days at the start of experimental feeding and thereafter at
fortnightly intervals during the experimental period of 75 days. DM intake was
recorded daily by subtracting the residual DM from the quantity of DM offered.
3.8.4 Daily Milk Yield
Milking was done twice daily i.e. morning at 5:30 a.m.and evening at 6:00
p.m. The milk was collected in milking vessels after screening through muslin
cloth.
3.8.5 Milk Composition
Milk samples were collected for the estimation of milk parameters every
fortnightly. Milk fat, protein, lactose and SNF were determined using automatic
milk analyzer (Lactostar).
Materials & Methods
Page | 71
3.8.6. Metabolism trial
A metabolism trial for a period of 7 days was conducted to determine the
nutrient digestibility, intake and N balance. Animals were shifted 3 days prior to
the metabolism trial to adjust with the environment of the metabolism shed.
Animals were weighed consecutively for 2 days before and after the trial. Feed
was offered daily at 9.00 h in the morning. Fresh drinking water was provided
twice a day and the quantity was measured each time to calculate the total water
intake.
3.8.7. Sampling, processing and storage
The feeds and fodders were weighed before offering. DM content of
rations offered was measured daily. The residues of previous day were collected
daily at 8.00 h before offering feed and weighed. The feed, fodder samples
before offering and residue samples after compiling were collected daily and
dried to find out their DM content. At the end of 7th
Faeces voided during 24 h were collected daily for 7 days and weighed at
8:30 h daily and a small sample (2% of total sample on fresh weight basis) was
pooled and processed in a similar manner as that for feeds and residue.
Simultaneously, total urine voided was also measured daily and an aliquot (1%
of total volume) was collected and stored in plastic containers containing 2 ml of
25% H
day collection the dried
samples were mixed, ground to pass through 1 mm sieve and stored in air tight
containers.
2SO4.
3.8.8. Analysis of feed, residue, faeces and urine
The samples were analyzed for proximate composition (AOAC, 2005), cell
wall frcations (Van Soest et al., 1991) and fiber bound protein fractions such as
NDF and ADF bound CP (NDICP and ADICP) (Licitra et al., 1996). Total-N
content of urine samples were estimated (AOAC, 2005). The TDN, DE and ME
value of the fodders was estimated using chemical composition based formulae
as suggested by NRC (2001).
Materials & Methods
Page | 72
3.8.9 Estimation of Urinary purine derivatives, cratinine and microbial
protein synthesis
The procedures for collection, preservation, analysis and calculation of
urinary purine derivatives, described by IAEA (1997), were followed during this
study.
3.8.9.1. Determination of allantoin
The urine samples which had been previously diluted before storage
needed further dilution. The dilution was made using distilled water in such a
fashion that allantoin concentration remain within the range of standard’s
concentration, which were as follows:
a) Standard solution of allantoin
Allantoin (HiMedia, India) stock solution (100 mg/ L) was prepared. It was
diluted to give working concentration of 10, 20, 30, 40, 50 and 60 mg/ L and
stored at -200
b) Procedure
C.
1. One ml each of diluted urine sample, standard and distilled water (blank)
was taken in duplicate in different tubes (15 ml).
2. Five ml distilled water and 1 ml 0.5 M NaOH were added and mixed well
using vortex mixer.
3. All tubes were placed in boiling water bath for 7 min, thereafter cooled in
cold water bath.
4. One ml HCl (0.5 M) added to each tube and pH was checked (pH 2-3).
5. One ml of phenyl hydrazine solution was added, mixed well and tubes
were transferred to boiling water for exactly 7 min.
6. Tubes were removed from the boiling water and placed immediately in the
icy alcohol bath for several minutes.
7. Three ml concentrate HCl and 1 ml potassium ferricyanide was added to
each tube within the shortest possible time span.
Materials & Methods
Page | 73
c) Each tube was mixed thoroughly and absorbance was recorded exactly
after 20 min at 522 nm using spectrophotometer (SPECORD 200,
Germany made)
d) Standard curve
A linear regression between the known allantoin concentrations
(Standard= X) and the corresponding absorbance (Y) was fitted (figure 3.1) as Y
= a + b X
Fig. 3.1. Standard curve for allantoin concentration
e) Calculation
Concentration of allantoin in the samples was measured from the
absorbance Y
C = (Y – a) ÷b x F.
Where, C was the concentration of unknown,
Y was the absorbance of the unknown,
‘a’ and ‘b’ were the intercept and slope of the standard curve, respectively
and
F was the dilution factor
y = 0.125x - 1.736 R² = 0.949
-1
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60 70
OD
at
522 n
m
Allantoin concentration (mg/l)
Materials & Methods
Page | 74
3.8.9.2. Determination of uric acid by uricase
Urine samples were further diluted accordingly so that uric acid
concentrations in the samples were within the standard uric acid concentration.
a) Standard
Uric acid (HiMedia, India) stock solution (100 mg/ L) was prepared. It was
diluted to give working concentration of 5, 10, 20, 30 and 40 mg/ L.
b) Procedure
1. One ml each of diluted urine, standard and blank (distilled water) was
taken in duplicate in different tubes (10 ml). Phosphate buffer (2.5 ml) was
added to each tube and mixed well. Two sets of tubes were prepared.
2. To one set, 150 µl buffer and in other 150 µl uricase (Sigma USA) was
added and mixed well.
3. Tubes were incubated at 370
4. Tubes were removed from water bath and contents were mixed and
transferred to cuvette. Absorbance was recorded at 293 nm using
spectrophotometer.
C for 120 min in a water bath.
Standard curve was drawn using absorbance of uric acid without addition
of uricase. Concentrations of standard (X) and corresponding absorbance (Y)
were transformed to natural log (Ln) functions. Ln(Y) was linearly correlated to
Ln(X) as shown in figure 3.3. The relationship was used for the calculation of the
concentration of uric acid in urine samples from their absorbance.
Ln (Y) = a + b Ln (X)
Where, ‘a’ was the intercept and ‘b’ was the slope of regression
c) Calculation
The net reduction in absorbance (∆OD) was calculated for the samples
due to uricase treatment.
∆OD = OD without uricase – OD with uricase.
Uric acid concentration was calculated from ∆OD based on the following
established standard equation.
Materials & Methods
Page | 75
C = Exp [(Ln (∆OD) – a) ÷b] x F
Where, C was the concentration of unknown,
∆OD was the net reduction in optical density after uricase treatment of the
unknown and F was the dilution factor
Fig. 3.2 Standard curve for uric acid concentration
3.8.9.3. Creatinine estimation
Creatinine was estimated by creatinine test kit with modified Jaffe’s reaction
Reagents: Reagent 1- Picrate reagent
Reagent 2- Sodium hydroxide
Reagent 3- Creatinine standard
Reagents 1 and 2 are stable at room temp (15-30°C) and reagent 3 is stable at
2-8°C.
Working reagent preparation: Working reagent was prepared by mixing equal
volume of reagent 1 with reagent 2. It is stable for 7 days at 2-8°C.
y = 1.2539x - 3.556 R² = 0.962
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
LN
(O
D a
t 293n
m)
LN Uric acid concentration (mg/l)
Materials & Methods
Page | 76
Procedure
Pipetted into tubes marked Standard Test
urine - 100μ
Reagent 3 100μ -
Working creatinine reagent 100μ 100μ
The above reagents were mixed well. Then the creatinine was analysed in
autoanalyzer at an optical wavelength of 505 nm. The readings were recorded
then.
3.8.8.4. Calculation of absorbed of microbial purine concentration
Following equation, suggested for buffaloes by Dipu et al. (2006) was
used to describe the quantitative relationship between absorbed microbial purine
absorption (X, mmol/ d), and the excreted PD in urine (Y, mmol/ d):
Y = 0.74X+0.117 kg W
Purine derivative concentration (mmol/lit) = Allantoin (mmol/lit) + Uric acid
(mmol/lit)
0.75
Creatinine excretion (mmol/ kg W0.75
PDC index = [PD] / [Creatinine] ×W
/day) = 0.98
Estimated purine derivative excretion (mmol/day) = PDC Index x Creatinine
excretion (mmol/W
0.75
0.75
3.8.8.5. Calculation of intestinal flow of microbial N
/day)
The following equation was used to calculate intestinal flow of microbial N
(g N/d) from the microbial purine absorbed.
X (mM/d) x 70
Microbial N (g N/d) = = 0.727 X
0.116 x 0.83 x 1000
Materials & Methods
Page | 77
The factors used in the equation were:
1. Digestibility of microbial purine was assumed to be 0.83, which was taken
as the mean digestibility value for microbial nucleic acids.
2. The N content of purine was 70 mg N/ mM.
3. The ratio of purine N and Total N in mixed rumen microbes was measured
as 11.6: 100.
The above method for the calculation of protein supply from purine
absorption presumes that the purine: protein ratio in mixed rumen microbes
remains unchanged by dietary treatments (IAEA, 1999).
3.9. Statistical Analysis
All the data generated during study were subjected to the statistical
analysis as per Snedecor and Cochran 1994. Analysis of variance (ANOVA) was
done to find out the significant difference between groups using SAS System
('Local', W32_7PRO), version 9.3. Data was presented as mean ±
3.9.1. Statistical analysis to determine the energy and protein requirements
of Murrah buffaloes for maintenance and 6% FCM
SE.
To analyze the maintenance and 6%FCM requirements of buffaloes
general linear model (GLM) procedure of SAS, version 9.3 was used. The model
was as follows
Y = b0 + b1
Where, constant b
X + ε
0
Coefficient b
was the intercept,
1
ε was the error term/residual that was not explained by the variables in
the model
was the parameter estimate for the variable X and
CHAPTER – 4
Results and Discussion
Results & Discussion
Page | 78
RESULTS AND DISCUSSION
The present study has been carried out in three phases; Phase-I,
preparation of silages in lab and its evaluation in terms of silage quality, chemical
composition, in vitro rumen fermentation and estimation of utilizable crude
protein (uCP), metabolizable protein in feeds. Phase-II includes estimation of
methane emissions from dry buffaloes fed on oat silage or oat hay by SF6
technique. In phase III, two separate experiments were conducted in lactating
buffaloes to estimate ME and MP requirements, respectively. The results
obtained during the course of this study have been presented and discussed in
respective sections.
Phase – I
4.1 EVALUATION OF SILAGE QUALITY
4.1.1 Chemical composition and organoleptic characteristics of maize, oat
silage and fodders before ensiling
Data pertaining to chemical composition (% DM basis) of maize and oat
silages and their respective fodders have been presented in Table 4.1.1 The CP,
TDN and ME (MJ/kg DM) contents of maize fodder, maize silage, oat fodder,
oat silage and oat hay were 8.59, 8.86, 11.85, 11.97 and 9.20; 57.80, 56.78,
63.15,62.68 and 53.10; 8.89,8.70,9.88,9.80 and 8.01 respectively. Maize silage
was greenish yellow in color while oat silage was golden yellow in color. Both
silages were soft, non viscous in texture and had slightly acidic, vinegar smell
(Table 4.1).
Table 4.1 Organoleptic characteristics of maize and oat silages prepared
in vitro
Parameter Maize Silage Oat Silage
Colour Greenish yellow Golden yellow
Smell Slightly acidic and vinegar , Pleasant
Slightly acidic and vinegar
Texture Loose , soft and non viscous Loose, soft, non viscous
Results & Discussion
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Table 4.1.1 Chemical composition and energy value of maize, oat and their silages (% DM)
Forage CP EE NDF ADF NDICP ADICP Ash ADL TDN DE, MJ/kg ME, MJ/kg
Maize
Fodder 8.59±0.02 2.11±0.01 59.48±0.84 33.95±0.28 6.02±0.21 2.00±0.05 6.05±0.17 5.78±0.51 57.80±0.67 10.66±0.04 8.89±0.01
Maize
silage 8.86±0.05 1.96±0.09 54.59±0.42 34.45±0.19 1.60±0.19 1.90±0.01 7.90±0.34 4.42±0.48 56.78±0.51 10.47±0.76 8.70±0.16
Oat fodder 11.85±0.06 2.15±0.02 44.93±0.48 32.28±0.57 3.97±0.05 1.33±0.67 9.76±0.12 5.64±0.17 63.15±0.11 11.65±0.54 9.88±0.02
Oat silage 11.97±0.09 1.97±0.01 47.77±0.52 32.61±0.31 2.10±0.15 1.00±0.63 9.21±0.02 5.10±0.39 62.68±0.72 11.56±0.12 9.80±0.01
Oat hay 9.20±0.22 1.82±0.02 63.41±0.41 38.00±0.27 1.30±0.34 0.6±0.24 8.90±0.31 8.29±0.65 53.10±0.61 9.80±0.03 8.01±0.05
Results & Discussion
Page | 80
4.1.2 Fermentation characteristics of silages
The data on fermentation characteristics of maize and oat silages
prepared in lab have been presented in the Table 4.1.2. Silages of maize and
oat had pH of 3.80 and 3.71, respectively. The lactic acid contents of maize
silage and oat silage were 6.80 and 5.19 per cent, respectively. The comparable
TVFA content of maize and oat silages were 37.20 and 35.11 mM/100g DM,
respectively was recorded. The propionate (mM/100g DM) was 4.48 and 3.78 in
maize silage and oat silage, respectively. The acetate content was 32.01, 30.77
and butyrate content 0.71, and 0.49 mM/100g DM, respectively. There was
variation in nitrogen fractions (%DM) of maize and oat silages. The average
value of total nitrogen (%DM) content of maize and oat silage was 1.43 and 1.92,
respectively, which was significantly (P<0.05) higher in oat silage.
Table 4.1.2 : Fermentation end products of maize and oat silages
Parameter Maize silage Oat Silage
DM 31.13±0.30 34.26±0.89
pH 3.80±0.01 3.71±0.03
Lactic acid(g/100g DM) 6.80 ± 0.74 5.19 ± 0.69
Volatile fatty acid fractions
TVFA (mM /100g DM) 37.20±1.91 35.11±1.54
Acetate (mM/100 g DM) 32.01±1.50 30.77±1.48
Acetate (% DM) 1.92±0.09 1.85±0.09
Propionate (mM/100 g DM) 4.48±0.35 3.78±0.14
Propionate (% DM) 0.33±0.02 0.28 ± 0.11
Butyrate (mM/100 g DM) 0.71±0.05 0.49±0.05
Butyrate (%DM) 0.06±0.005 0.04±0.004
Nitrogen fractions
Total-N (g/100g DM) 1.43b± 0.02 1.92a± 0.01
NH3-N ( g/100 g DM) 0.10±0.01 0.11± 0.01
Means bearing different superscripts in a row differ significantly ( P < 0.05)
Results & Discussion
Page | 81
Kung and Stoke (2001) reported pH values in range from 3.7- 4.2 for
maize silage. Gupta et al. (1981) concluded that silage with pH value 4.2 or less
could be considered as good silage. Similar results were obtained by Church
(1991), Etman et al. (1994), McDonald et al. (1995), Sheperd and Kung (1996),
and they reported pH values for maize silage ranging from 3.42 to 4.20. The pH
values of silage prepared from maize and oat fodder in present study were within
the range reported by earlier workers for good silage. Lactic acid is the most
abundant acid (75% of the total acids contained in silage) and also stronger than
the volatile fatty acids (VFA) and thus has the greatest effect on silage pH (Kung,
2001; Seglar, 2003). It ranged between 4-7% for maize silage (Seglar, 2003).
The lactic acid content of both silages was within the range and resulted in an
optimum pH. Duo et al. (2002) tested the content of lactobacillus in oat wrapped
silage and oats+vetch mixture wrapped silage. The results showed that the
content of lactobacillus in the two wrapped silages was similar in winter, but
increased at different rates in spring. The content of lactobacillus increased more
in the oats wrapped silage than in oats+vetch mixture wrapped silage mostly
because of much higher water content in vetch. The VFA that has the biggest
impact on aerobic stability is acetic acid, which is found in concentrations of up
to 3% (Danner et al., 2003; Filya, 2003; Kung, 2001; Muck, 2010). The acetic
acid content of all the silages fell within the normal range. Seglar (2003)
observed that propionic acid in well fermented maize silage was less than 0.5%,
with butyric acid being undetectable (< 0.1%) and similar pattern was observed
in present study. Langston et al. (1958) stated that high quality silage is
characterized by low NH3-N concentration. Sheperd and Kung (1996) postulated
that NH3-N concentration of corn silage ranged between 0.04 and 0.15% of DM.
The ammonia-N content of the maize silage and oat silage in the present study
fell within the range. High pH silage favors secondary fermentation, leading to an
increase in ammonia-N (McDonald et al., 1991).
4.1.3 In vitro total gas, methane production of maize and oat silages and
respective fodders
The In vitro gas and methane production study was carried out and the
average values are presented in Table 4.1.3. The mean values of in vitro dry
matter digestibility (IVDMD) (%) of maize fodder and silage; oat fodder and oat
silage were 65.50 and 67.00; 75.05 and 76.31 respectively (Table 4.1.3). The in
Results & Discussion
Page | 82
vitro organic matter digestibility (IVOMD) was significantly higher in oat fodder
and silage (80.53 and 88.07) than maize fodder and silage. The mean values of
in vitro methane production (g/ kg IVDMD) of maize fodder and its silage; oat
fodder and its silage were 38.47 and 35.24; 39.23 and 36.52, respectively. It was
highest (P<0.05) in oat fodder and lowest in maize silage. The methane
production in silages were significantly lower (P<0.05) than respective fodders.
Methanogenesis tends to be lower when forages were ensiled than when they
were dried (Martin et al., 2010). Maize silage has too low nitrogen content for
methanogens growth (Kalac, 2011). Some bacteriocins are known to reduce
CH4 production in vitro (Callaway et al., 1997; Lee et al., 2002). Nisin is
thought to act indirectly, affecting hydrogen-producing microbes in a similar
way to that of the ionophore antibiotic, monensin (Callaway et al., 1997)
The results of the in vitro gas production (IVGP) in present study were in
line with those of Calsamigila et al. (2007) and Calabro et al. (2007) who
reported IVGP (ml/g DM) on corn silage to be 122.85 in 24 hr and 389-402 at 48
hr incubation period respectively. Similar results were also reported by Rapetti et
al. (2005). The results of in vitro methane production (IVMP) in present study
were in line with that of Varadyova et al. (2010). Kamble et al. (2011) reported
higher CH4 production (ml/g DDM) from maize fodder (66.61) compared to
barley (52.2), oats (53.9) and wheat fodder (50.57). They reported that methane
production was unique to different forages, which may probably be due to
presence of specific secondary metabolites.
4.1.4 Estimation of utilizable crude protein (uCP), intestinal digestibility of
uCP and metabolizable protein
The utilizable crude protein (uCP), intestinal digestibility and metabolizable
protein in feeds were presented in the Table 4.1.4. Among the grains the uCP
(%DM) content was highest in oat (9.96) and lowest in pearl millet (5.21). The
intestinal digestibility of uCP (%) among grains ranged from 73.89-87.61. The
MP content was 8.24, 8.11, 7.36 and 4.26 % of DM in maize, barley, oat and
pearl millet. Among the grains MP content was lowest in pearl millet. The uCP
content was 26.17, 18.25 and 32.56 %DM in DOMC, CSC and SBM,
respectively which differ among each other significantly (P<0.05). Among the
cakes, intestinal digestibility (%) was towards lower side in DOMC (73.19) than
the CSC and SBM.
Results & Discussion
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Table: 4.1.3 In vitro dry matter digestibility (IVDMD) and methane production (g/kg) in fodders and silages incubated
for 48 hr.
Feed IVDMD% IVOMD%
Gas production
(ml/200mg)
Gas production
(mM/200mgDM)
Methane
(g/kg DM incubated)
Methane (g/kg IVDMD)
Maize Fodder 65.50b±0.08 67.67b±0.43 31.20b±0.77 1.11b±0.01 25.19c±0.45 38.47a±1.12
Maize Silage 67.00b±0.16 70.06b±0.18 23.29b±0.20 1.05c±0.04 23.6d±0.18 35.24c±0.08
Oat Fodder 75.05a±0.38 80.53a±0.23 38.38a±0.59 1.40a±0.18 29.42a±0.98 39.23a±0.12
Oat Silage 76.31a±0.13 88.07a±0.48 37.41a±0.87 1.36a±0.02 28.29b±1.08 36.52b±0.18
Means bearing different superscripts in a row differ significantly (P < 0.05)
Results & Discussion
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The MP contents were 27.88, 14.82 and 19.15%DM in DOMC, CSC and
SBM respectively and differed among each other significantly (P<0.05). The uCP
and MP (%DM) in the wheat bran and rice bran was similar. The uCP (%DM)
content among the fodders was highest in oat fodder (11.24) compare to oat
silage (10.32), oat hay (10.01), maize fodder (9.11) and maize silage (8.99). The
intestinal digestibility of oat hay (63.56) was significantly lower (P<0.05) than
maize fodder (76.34), maize silage (72.59), oat fodder (75.29) and oat silage
(73.81) respectively. The MP content among the fodders ranged between the
6.36-8.46 % DM. Among fodders, MP (%DM) content in was highest (8.46) in
oats and lowest in oat hay (6.36).
The uCP of feeds analyzed by Zhao and Lebzien (2002) were in
corroboration with present findings. The uCP (%DM) of maize, barley, CSC and
SBM estimated from in vitro incubation with rumen liquor of cattle was 15.4, 16.4
and 28.9 whereas that estimated with rumen liquor of sheep was 14.4, 14.2 and
24.2, respectively (Zhao and Lebzien, 2002). The small intestinal digestibility of
feed ingredients viz. cotton seed meal and SBM, feed analyzed by in vivo
methods ranged from 73.7- 90.4% and 96.6-98.4%respectively depending on the
sources of ingredients (Moloney et al., 2001). Above feeds when analyzed in
vitro showed small intestinal digestibility in the range of 55.9-71.5% and 81.7-
88% respectively (Moloney et al., 2001). Das et al. (2014) reported similar MP of
maize grain, CSC and wheat bran to present study, using nylon bag technique
(AFRC, 1992). Taghizadeh et al. (2008) used in situ degradability method to
analyze MP, reported lower MP of maize (3.51%), and barley (4.85%) and higher
MP of cottonseed meal (23.22%) compared to present findings. Calsamiglia and
Stern (1995) reported similar (85-90%) post ruminal digestion of SBM. Promkot
and Wanapat (2003) found higher intestinal digestibility of CP (% of rumen
residual CP) of SBM compared to CSC but in present finding the intestinal
digestibility of these feeds was similar. The intestinal digestibility of undegraded
CP resulting from this simulation for corn silage was 70% indicated by Ve´ rite´ et
al. (1987) and same assumed by the National Research Council (NRC 2001)
which was almost comparable with that of present findings.
Results & Discussion
Page | 85
Table 4.1.4 Utilizable crude protein, intestinal digestibility (%) of uCP, metabolizable protein of feeds and fodders
Feed CP (%DM) uCP (% DM) Intestinal digestibility (%) MP (%DM)
Grains
Maize 9.89b±0.37 9.41b±1.14 87.61a±0.32 8.24a±0.72
Barley 9.10b±0.13 9.72b±0.11 83.45a±1.14 8.11a±0.03
Oat 11.68a±0.19 9.96a±1.09 73.89c±0.96 7.36a±0.31
Pearl millet 10.63a±0.16 5.21c±0.31 81.73a±0.56 4.26b±0.34
Agro-industrial byproduct
Wheat bran 14.75±0.49 12.45a±0.39 76.31a±0.47 9.50a±0.36
Rice bran 14.72±1.11 11.96a±1.31 79.87b±0.61 9.55a±1.21
Cake
DOMC 39.24b±0.53 26.17b±0.76 73.19b±1.12 19.15b±1.14
CSC 24.15c±0.62 18.25c±0.21 81.23a±1.35 14.82c±0.79
SBM 44.38a±0.60 32.56a±1.18 85.62a±0.73 27.88a±0.36
Fodder
Maize fodder 8.59c±0.02 9.11c±1.03 76.34a±0.31 6.95c±0.14
Maize silage 8.86c±0.05 8.99c±0.54 72.59a±1.01 6.53c±1.09
Oat fodder 11.85a±0.06 11.24a±1.41 75.29a±0.78 8.46a±0.91
Oat silage 11.97a±0.09 10.32b±0.97 73.81a±0.91 7.62b±0.67
Oat hay 9.20b±0.22 10.01b±0.49 63.56b±0.74 6.36d±0.25
Means bearing different superscripts in a row differ significantly (P < 0.05)
Results & Discussion
Page | 86
Phase II
Estimation of methane emissions from the dry buffaloes fed on oat hay or
silage
4.2.1 Chemical composition and nutritive value of experimental oat hay
and oat silage
Detailed chemical composition (% DM) of oat silage and oat hay is given
in table 4.2.1. A relatively higher content of CP was observed in oat silage
compare to oat hay, 11.97 and 9.20, respectively. The NDF and ADF content in
oat hay (56.17 and 43.16) were higher than the oat silage (47.40 and 32.35).
Table: 4.2.1 Chemical composition (%DM) of oat hay and oat silage fed to
buffaloes for estimation of methane emissions
Parameter Oat hay Oat silage
DM 87.00±0.72 26.72±0.80
OM 91.10±0.15 90.79±0.12
CP 9.21± 0.22 11.97±0.09
EE 1.82±0.02 1.97±0.01
NDF 56.17±0.41 47.40±0.52
ADF 43.16±0.27 32.35±0.31
Ash 8.90±0.19 9.21±0.29
NDICP 1.3±0.07 2.1±0.09
ADICP 0.6±0.03 1.0±0.06
Hemicellulose 13.02±0.69 15.05±0.43
Cellulose 40.53±0.23 41.11±0.31
ADL 8.29±0.65 6.01±0.39
TDN % 54.61±0.61 56.83±0.72
DE (MJ/kg) 10.07±0.03 10.48±0.12
ME (MJ/kg) 8.29±0.05 8.71±0.01
Results & Discussion
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4.2.2 Nutrient intake and digestibility of nutrients in buffaloes fed on oat
hay or silage
The average fortnightly dry matter intake in T1 and T2 groups were
presented in the Table no. 4.2.3. The mean (DMI kg/day) was 8.30 and 8.34
respectively in T1 and T2 groups which was non significant (P>0.05).Neutral
detergent fiber intake (NDFI) and acid detergent fiber intake (ADFI) was
significantly (P<0.05) higher in T1 than T2 while the CP intake was significantly
(P<0.05) higher in T2 than T1group. Wallsten et al. (2009) reported that the DM
intake increased in heifers fed oat silage with plant maturity. The increase was
from 1.6 kg/100 kg LW at early milk stage to 2.0 kg/100 kg LW at dough stage,
possibly due to the low water content of the silage in the earlier stages. Data
pertaining to the nutrient digestibility of buffaloes fed on oat hay or silage were
given in Table 4.2.4. The digestibility coefficients of all nutrients were similar
between the groups except that of CP. The CP digestibility (%) was significantly
(P<0.05) higher in T2 than T1. The DM and CP digestibility coefficients were
68.87 and 69.09; 58.28 and 60.59 in T1 and T2 respectively. Silage protein
usually serve as rumen degradable protein (RDP) since a lot of the plant
proteins are broken down during fermentation to non-protein nitrogen, like
ammonia nitrogen, and is therefore readily available to rumen micro-
organisms (Papadopoulos and Mckersie, 1983). This resulted in plant protein
reaching the abomasums by 10% for digestion and thus silage mainly serves
as a source of RDP in ruminant diets (Wilkinson, 2005). In steers fed only
silage, the digestibility of oat silage was lower than that of maize silage
(Christensen et al., 1977a) but higher than that of rye silage (Christensen et al.,
1977b).
Table: 4.2.2 Fortnightly body weight (kg) of dry Murrah buffaloes fed on
oat hay and oat silage
Fortnight Oat hay (T1) Oat silage (T2)
1 569.88±21.15 570.50±29.70
2 571.88±21.38 573.13±29.07
3 573.50±21.41 575.13±29.01
Mean 571.75±1.05 572.92±1.34
Results & Discussion
Page | 88
Table: 4.2.3 Effect of feeding oat hay or oat silage on fortnightly dry matter
intake in dry buffaloes
Fortnight
DMI (kg/day) DMI (kg/100Kg BW) DMI (g/Kg W0.75)
Oat hay
(T1)
Oat silage (T2)
Oat hay (T1)
Oat silage (T2)
Oat hay (T1)
Oat silage (T2)
1 8.51±0.18 8.56±0.31 1.50±0.03 1.51±0.03 73.13±1.25 73.55±0.70
2 8. 26±0.43 8.28 ±0.27 1.45±0.06 1.46±0.07 70.59±2.92 71.29±2.62
3 8.13±0.44 8.17±0.33 1.42±0.07 1.43±0.05 69.47±3.25 69.80±1.78
Mean 8.30±0.19 8.34±0.20 1.46±0.01 1.47±0.01 71.07±0.66 71.55±0.67
Table:4.2.4 Effect of feeding oat hay or oat silage on intake and
digestibility of nutrients in dry buffaloes
Parameter Oat hay group (T1) Oat silage group (T2)
BW, kg 566±21.29 567±29.65
Final BW, kg 571.75±1.64 572.92±1.82
DM intake, kg 8.30±0.19 8.34±0.20
DMI intake, (kg/100kg BW)
1.46±0.01 1.47±0.01
DMI intake, W0.75 71.07±0.66 71.55±0.67
CP intake, kg 0.82b±0.04 1.06a±0.07
CP intake, g/kgW0.75 6.54b±0.06 8.73a±0.08
EE intake, kg 0.15±0.03 0.16±0.01
NDF intake, kg 4.66a±0.04 3.95b±0.21
ADF intake, kg 3.58a±0.03 2.70b±0.02
Digestibility coefficient (%)
DM 68.87±2.19 69.09±1.76
OM 70.44±3.33 69.51±1.88
CP 58.28b±1.78 60.59a±2.72
EE 80.18±2.92 82.99±3.11
NDF 64.50±2.22 67.99±1.89
ADF 61.31±1.41 61.46±1.12
Means bearing different superscripts in a row differ significantly (P < 0.05)
Results & Discussion
Page | 89
4.2.3. Energy loss through methane emissions in buffaloes fed on oat hay
or oat silage
The energy intake and loss of energy in the form of methane from
buffaloes fed on oat hay or silage is presented in table 4.2.5. The energy intake
in terms of gross energy (GE), digestible energy (DE) was similar between the
groups. The methane (g/day) was 219.27 and 204.18 in T1 and T2 respectively.
The CH4 (L/day) and CH4 (g/kg DMI) were 341.35, 317.86 and 24.36, 21.79 in
T1, T2 respectively.
Table 4.2.5: Methane emissions and energy loss in buffaloes fed oat hay
or oat silage
Parameter Oat hay group (T1) Oat silage group (T2)
DMI, kg/d 9.00±0.26 9.37±0.19
GEI, MJ/d 162.00±1.68 168.66±2.79
DEI, MJ/d 90.63±1.57 98.20±4.85
MEI, MJ/d 74.61b±1.31 81.61a±1.57
CH4, L/d 341.35a±3.44 317.86b±6.28
CH4, g/d 219.27a±2.21 204.18b±4.03
CH4, g/kg DMI 24.36a±0.88 21.79b±0.82
CH4, g/kg NDFI 35.68a±0.22 34.52b±0.37
CH4, MJ/d 12.24a±0.12 11.40b±0.23
CH4, MJ/kg DM 1.36a±0.05 1.21b±0.02
CH4, MJ/kg DDM 1.77a±0.13 1.65b±0.03
CH4 loss as %GE 7.56a±0.05 6.76b±0.08
CH4 loss as % DE 13.51a±0.07 11.61b±0.14
CH4 loss as %ME 16.41a±0.10 13.97b±0.19
Means bearing different superscripts in a row differ significantly (P < 0.05)
Results & Discussion
Page | 90
The overall methane production was significantly lower (P<0.05) in oat
silage group than oat hay fed group. The highest methane emission (g/kg DM,
MJ/kg DM and g/kg NDF intake) was in T1 (24.36, 1.36 and 35.68) compared to
T2. Methane loss as percentage of DE and ME energy intake was 13.51,
16.41and11.61, 13.97 in T1 and T2 respectively, which is significantly (P<0.05)
differ between the group. Total CH4 production (MJ/d) was depressed by 6.86%
in dry buffaloes fed on oat silage instead of oat hay. Methane release in buffalo
in relation to NDF and dNDF intake is depicted in figure 4.1 and 4.2 respectively.
Significantly high correlation coefficients were observed between methane
emissions and NDF intake (R2 = 0.61, p<0.05).
Methane production (% of GEI) was shown to be lower when forages
were ensiled than when dried (Sundstol, 1981). This could be because of
reduced digestion in the rumen with ensiled forages due to the extensive
fermentation that had occurred during silage making. Total CH4 production
(Mcal/d) was depressed by 33% by the utilization of alfalfa silage instead of
alfalfa hay, using a mechanistic model approach to predict CH4 emissions
from ruminants (Benchaar et al. 2001). Kirkpatrick and Steen (1999) observed
no differences between forages conserved as silage vs forages conserved by
freezing (directly after harvesting) on CH4 energy loss (% of GEI). Silage
based diets record less loss of energy via methane, urine and faeces
production (Beukes, 2013). Methanogenesis tends to be lower when forages
were ensiled than when they were dried (Martin et al., 2010). In dairy cows,
less CH4 production per kilogram of DMI with a corn silage diet was reported
than with a hay diet in two successive experiments conducted by Martin et al.
(2008). Van Gastelen et al (2015) observed that the methane production was
11% reduced in 100% corn silage diet than 100% grass silage diet in lactating
Holstein-Friesian cows, when expressed per unit of DM intake. McCourt et al.
(2007) observed less CH4 production in dairy cows when fed a grass silage as
compared with a corn silage diet, whereas Van Vugt et al. (2005) did not observe
any change in CH4 production between cows fed a corn silage vs a white clover
silage diet. In addition, Kirchgessner et al. (1994) showed that diets based on
corn silage and fed to dairy cows produced more CH4 per day than diets based
on grass hay or silage, but no information was available on the percentage of
Results & Discussion
Page | 91
concentrates in these diets. Waugh et al. (2005) found that methane yield (g/kg
DM intake) increased when pasture forage was substituted with up to 35% maize
silage in dairy cows fed at similar intakes. Other literature suggests that methane
yield in cattle respond in a quadratic manner when pasture forage is substituted
with maize silage or grain (Blaxter and Wainman 1964; Arndt et al. 2010;
Hassanat et al. 2012). Doreau et al (2011) reported that daily enteric CH4
production (g/d) was similar for the natural grassland hay (49% in diet) and corn
silage (63% in diet) but was less for the corn grain (70% diet) diet fed to beef
cattle. He observed that gross energy intake loss as CH4 averaged 6.9% for the
hay and corn silage diets and 3.2% for the corn grain diet (P < 0.001).
Blaxter and Clapperton (1965) found that both feed intake and digestibility
were important factors affecting CH4 yield (% of GEI) and that at the
maintenance level of feeding CH4 yield increased with higher digestibility of the
diet. In the present study, where buffaloes were fed close to maintenance,
neither feed intake nor feed digestibility was related to CH4 yield (% of GEI),
whereas CH4 production (g/d) was related to NDF intake, but not to diet
digestibility. This is supported by McCaughey et al. (1997, 1999) who reported
that feed intake rather than digestibility is the major determinant of CH4
production. In present study, the intake of the digestible fraction of NDF (dNDFI)
had the positive correlation with CH4 production. A similar relationship was also
found in small ruminants (sheep and alpaca) fed forages ad libitum indoors or
grazed (Pinares-Patiño et al., 2003), confirming the concept that CH4 production
was mainly a function of the amount of cell walls digested in the rumen (Moe and
Tyrrell 1980). Singh et al. (2011) suggested the variation in CH4 production from
dry roughages might be attributed to significant difference in the NDF, ADF,
carbohydrate and protein fractions. Woodward et al. (2001) found CH4
production of 26.9 and 35.1 g/kg DMI in lactating dairy cows fed on silages made
from Lotus corniculatus (CT- 0.026 %DM) and pasture, respectively. Moss and
Givens (2002) divided sheep into four groups and gradually replaced grass
silage (25%, 50% and 75%) with SBM in three groups apart control. They
observed increased volume of methane production per day with decreased
proportion of grass silage and on subsequent replacement with SBM.
Results & Discussion
Page | 92
Fig 4.1: Relationship between neutral detergent fiber intakes (NDFI), with
methane emissions in buffaloes
Fig 4.2: Relationship between digestible neutral detergent fiber (dNDFI
with methane emissions in buffaloes
265.00
285.00
305.00
325.00
345.00
365.00
385.00
405.00
425.00
445.00
5.00 5.50 6.00 6.50 7.00
CH
4 (L
/d)
NDF intake (kg/d)
250.00
270.00
290.00
310.00
330.00
350.00
370.00
390.00
410.00
430.00
3.50 3.70 3.90 4.10 4.30 4.50 4.70
CH
4 (
L/d
)
dNDF intake (kg/d)
Results & Discussion
Page | 93
4.2.4 ME intake at fortnight intervals and prediction of its requirement for
maintenance and body weight change of non-lactating Murrah
buffaloes fed on oat hay or silage
The ME intake (MJ/d) of the non lactating Murrah buffaloes at fortnight
intervals is presented in table 4.2.6. The overall mean fortnightly metabolizable
energy intake in oat hay group and oat silage group were 68.79±0.56 and
72.61±0.62 respectively, which differ significantly (P<0.05) between the groups.
Table: 4.2.6 Effect of feeding oat hay or oat silage on metabolizable
energy (MJ/d) in dry buffaloes
Fortnight Oat hay (T1) Oat silage (T2)
1 70.52±1.53 74.56±2.72
2 68.44±3.53 72.12±2.39
3 67.42±3.66 71.15±2.89
Mean 68.79b±0.56 72.61a±0.62
Means bearing different superscripts in a row differ significantly (* P < 0.05)
The ME intake (kJ/kg W0.75) was regressed linearly upon body weight
change (BWC, g/kg W0.75) to determine the energy requirements for
maintenance and body weight change. The relationship between ME intake
(kJ/kg W0.75) and body weight change (BWC, g/kg W0.75 is shown in figure 4.3.
and the regression equation developed was as follows; y = 32.651x + 521.27 (R2
= 0.867, P<0.01, n = 48)
Where, y= ME intake (kJ/kg W0.75) of buffaloes and x= BWC (g/kg W0.75)
In absence of any body weight change i.e. no loss or gain in body weight,
ME intake was 521.27 kJ/kg W0.75 or 124.58 kcal/kg W0.75 which was the ME
requirements for maintenance (MEm) of non-lactating buffaloes per day and the
ME requirement for body weight change in buffaloes was 32.651 kJ or 7.80 kcal
per g BWC/ kg W0.75/day.
There is scanty literature available on the study on basal metabolism and
maintenance requirements of adult non-pregnant buffaloes. Khan et al. (1988)
concluded that the MEm requirement of buffaloes was 451 kJ/ kg W 0.75 which
Fig 4.3: Relationship of ME intake (KJ/kg W0.75) with body weight change (g/kg
W0.75) of dry Murrah buffaloes
Fig 4.4: Relationship of TDN intake (g/kg W0.75) with body weight change (g/kg
W0.75) of dry Murrah buffaloes
y = 32.651x + 521.27 R² = 0.867
350
400
450
500
550
600
650
700
-4 -2 0 2 4
ME
I (k
J/ k
g W
0.7
5)
BWC (g/ kg W0.75)
y = 2.1581x + 34.455 R² = 0.867
15
20
25
30
35
40
45
-4 -3 -2 -1 0 1 2 3 4
TD
N g
/W0.7
5
BWC (g/ kg W0.75)
Results & Discussion
Page | 94
was lower than the present study. The current MEm requirement of Murrah
buffaloes is almost similar to Kearl (1982) recommendation (523 kJ/ kg W 0.75)
and Ranjhan (1992) recommendations (510 kJ/ kg W 0.75). MEm of lactating
buffaloes was reported to be 540.3 kJ/ kg W 0.75(Paul et al 2003), 508 kJ/kg W
0.75 (Siviah and Mudgal, 1978), 534 kJ/ kg W 0.75 (ICAR, 1998) and 521 kJ/kg
W0.75 calculated using the equations of AFRC (1990).
The present requirements of MEm of Murrah buffaloes was comparable
with Paul et al. (2002) reported value 535.55 kJ/ kg W0.75 but marginally lower
than ICAR, 2013 (550 kJ/ kg W 0.75). Variation in these individual estimates is
attributable mainly to difference in method of estimation. Khan et al. (1988)
reported fasting heat production in adult non-pregnant Murrah buffaloes as 284.5
kJ /kg W 0.75 and in a swamp buffaloes as 287 kJ/ kg W 0.75 (Kawashima et al.,
2006). Calorimetric studies have shown that fasting heat production was lower in
buffaloes than in cattle (284.5 vs. 343, kJ/kg W0.75); Maymone and Bergenzini,
(1960). Generally, estimates of nutrient requirements reported from feeding trial
data using regression method were likely to be marginally higher and close to
practical requirements than the values reported from calorimetric studies in any
species.
While MEm(kJ/kg W0.75) value of Indian lactating cattle was 572.2, 575.7
and 546.9 reported by Patel and Mudgal (1977), Paul et al. (2003) and Patel and
Mudgal (1976), respectively. Yan et al. (1997) concluded that MEm requirements
in lactating cattle ranged from 490-640 kJ/kg W0.75 based on regression analysis
of calorimetric data reported by different authors.
The ME requirements for body weight change (MEBWC; kJ/g BWC/ kg
W0.75) were estimated to be 32.651 in present study was lower than 34.26 in
buffaloes (Paul et al., 2003) but with range 30.85-35.52 in cattle (Siviah and
Mudgal, 1978).
4.2.5. TDN intake at fortnight intervals and prediction of its requirement for
maintenance and body weight change of non-lactating Murrah
buffaloes fed on oat hay or silage
The TDN intake (kg/d) at fortnight intervals of the Murrah buffaloes fed on
oat hay or oat silage is presented in table 4.2.7. The overall mean fortnightly
Results & Discussion
Page | 95
TDN intake in oat hay group and oat silage group were 4.53±0.04 and 4.75±0.05
respectively, which differed significantly (P<0.05) between the groups. The TDN
intake (g/kg W0.75) was regressed linearly upon body weight change (BWC, g/kg
W0.75) to determine the TDN requirements for maintenance and body weight
change. The relationship between TDN intake (g/kg W0.75) and body weight
change (BWC, g/kg W0.75 is shown in figure 4.4. and the regression equation
developed was as follows;
y = 2.1581x + 34.455 (R² = 0.867, P<0.01, n = 48)
Where,
y = TDN intake (g/kg W0.75) of buffaloes
x = BWC (g/kg W0.75)
Table 4.2.7 Effect of feeding oat hay or oat silage on total digestible
nutrient intake (kg/d) in dry buffaloes
Fortnight Oat hay (T1) Oat silage (T2)
1 4.65±0.10 4.86±0.18
2 4.51±0.23 4.71±0.16
3 4.44±0.24 4.64±0.19
Mean 4.53b±0.04 4.74a±0.05
Means bearing different superscripts in a row differ significantly (P < 0.05)
The TDN requirement of non-lactating buffaloes for maintenance was
34.455 g/ kg W0.75 per day when the body weight change was zero and TDN
requirement for body weight change in buffaloes was 2.1581 g per g BWC/ kg
W0.75/day.
The present energy requirements for maintenance (g TDN/kg W0.75) of
adult buffaloes was comparable with ICAR, 2013 and as reviewed by Paul and
Lal, (2010). It ranged from 27 to 29.78 while lower than lactating buffaloes 35.3 g
TDN/kg W0.75 (Paul et al., 2002). The current TDN requirement (g per g BWC/ kg
W0.75) was within the range of gain reported in literature 0.78 to 2.23 g TDN/g
gain by Paul and Lal, (2010).
Results & Discussion
Page | 96
4.2.6 CP intake at fortnight intervals and prediction of its requirement for
maintenance and body weight change of non-lactating Murrah
buffaloes fed on oat hay or silage
The CP intake (kg/ d) at fortnight intervals of the Murrah buffaloes fed on
oat hay or oat silage is presented in table 4.2.8. The overall mean fortnightly
crude protein intake in oat hay group and oat silage group were 0.76 ±0.01 and
1.00±0.02 respectively, which differ significantly (P<0.05) between the groups
Table: 4.2.8 Effect of feeding oat hay or oat silage on CP intake (kg/d) in
dry buffaloes
Fortnight Oat hay (T1) Oat silage (T2)
1 0.78b±0.02 1.02a±0.04
2 0.76b±0.04 0.99a±0.03
3 0.75b±0.04 0.98a±0.04
Mean 0.76b±0.01 1.00a±0.02
Means bearing different superscripts in a row differ significantly (P < 0.05)
The CP intake (g/kg W0.75) was regressed linearly upon body weight
change (BWC, g/kg W0.75) to determine the CP requirements for maintenance
and body weight change. The regression equation developed was as below
equation (1) but the R2 value of this equation was very less so CP intake (g/kg
W0.75) of oat hay and silage group was regressed linearly upon respective body
weight change (BWC, g/kg W0.75) separately and regression equation developed
was as below equation (2), (3) respectively.
Equation 1(T1+T2): y = 0.4248x + 6.0846 (R² = 0.271, P<0.01, n = 48)
Equation 2 (T1): y = 0.3836x + 7.0564 (R² = 0.3126, P<0.01, n = 24)
Equation 3 (T2): y =0.3614x + 5.2642(R² = 0.8721, P<0.01, n = 24)
Where,
y = CP intake (g/kg W0.75) of buffaloes
x = BWC (g/kg W0.75)
Results & Discussion
Page | 97
The equation 3 was having highest R2 value (0.8721) as compared to
equation 1 and 2. So equation 3 predicts the CP requirements of buffaloes more
accurately as compare to other equation. The relationship between CP intake
(g/kg W0.75) and body weight change (BWC, g/kg W0.75) in oat silage fed
buffaloes was shown in figure 4.5.
Based on the prediction equation the CP intake at zero BWC was 5.2642
g/ kg W0.75 which was the CP requirements for maintenance of non-lactating
Murrah buffaloes per day and the CP requirement for BWC of buffaloes was
0.3614 g per g BWC/ kg W0.75/day. Daily CP requirements for maintenance (g/kg
W0.75) were recorded to be higher in present study as compared to the ICAR,
(2013) but lower than Paul et al., (2002),Tiwari and Patle (1983) which were
4.87, 5.43 and 5.84 respectively.
4.2.7 DCP intake at fortnight intervals and prediction of its requirement for
maintenance and body weight change of non-lactating Murrah
buffaloes fed on oat hay or silage
The DCP intake (g/d) of Murrah buffaloes at fortnight intervals is
presented in table 4.2.9. The overall mean fortnightly crude protein intake in oat
hay group and oat silage group were 444.93±3.61 and 604.60±5.17 respectively,
which differ significantly (P<0.05) between the groups. The DCP intake (g/kg
W0.75) was regressed linearly upon body weight change (BWC, g/kg W0.75) to
determine the DCP requirements for maintenance and body weight change.
Table: 4.2.9 Effect of feeding oat hay or oat silage on DCP intake (g/d) in
dry buffaloes
Fortnight Oat hay (T1) Oat silage (T2)
1 456.10b±9.88 620.84a±22.61
2 442.63b±22.85 600.51a±19.90
3 436.07b±23.65 592.46a±24.11
Mean 444.93b±3.61 604.60a±5.17
Means bearing different superscripts in a row differ significantly (P < 0.05)
Fig 4.5: Relationship of CP intake (g/kg W0.75) with body weight change (g/kg
W0.75) of dry Murrah buffaloes fed on oat silage
Fig 4.6: Relationship of DCP intake (g/kg W0.75) with body weight change (g/kg
W0.75) of dry Murrah buffaloes fed on oat silage
y = 0.3614x + 5.2642 R² = 0.8721
0
1
2
3
4
5
6
7
8
-4 -3 -2 -1 0 1 2 3 4
CP
I (g
/ k
g W
0.7
5)
BWC (g/ kg W0.75)
y = 0.2106x + 3.068 R² = 0.8571
1
1.5
2
2.5
3
3.5
4
4.5
-4 -3 -2 -1 0 1 2 3 4
BW
C (
g/
kg
W0.7
5)
BWC (g/ kg W0.75)
Results & Discussion
Page | 98
The DCP intake (g/kg W0.75) was regressed linearly upon body weight
change (BWC, g/kg W0.75) to determine the DCP requirements for maintenance
and body weight change. The regression equation developed was as below for
Equation (1) based on T1+T2 the R2 value of this equation was low so DCP
intake (g/kg W0.75) of oat hay and oat silage group was regressed linearly upon
respective body weight change (BWC, g/kg W0.75) separately in and regression
equation developed was as below equation (2), (3) respectively.
Equation 1(T1+T2): y = 0.26x + 3.5711 (R² = 0.3217, P<0.01, n = 48)
Equation 2 (T1): y = 0.2293x + 4.1896 (R² = 0.5014, P<0.01, n = 24)
Equation 3 (T2): y = 0.2106x + 3.068 (R² = 0.8571, P<0.01, n = 24)
Where,
y = DCP intake (g/kg W0.75) of buffaloes and
x = BWC (g/kg W0.75)
The relationship between DCP intake (g/kg W0.75) and body weight
change (BWC, g/kg W0.75) in oat silage group was shown in figure 4.6. Equation
3 was having highest R² and thus more accurately predicted the DCP
requirements of buffaloes.
DCP intake at zero BWC was 3.068 g/kgW0.75 which were the DCP
requirement for maintenance of non-lactating Murrah buffaloes per day and the
DCP requirement for BWC of buffaloes were 0.2106 g per g BWC/ kgW0.75/day.
The DCP requirements of dry buffaloes in present study was comparatively
higher than earlier reports by Gupta et al (1966), Kurar and Mudgal (1981) and
Singh (1965), was 2.84, 2.48 and 2.09, respectively. While DCP requirements of
lactating buffaloes were 3.20, 3.47, 3.00 and 3.14 reported by Mudgal and
Kumar (1978), Siviah and Mudgal (1978), Tiwari and Patle (1983) and Paul et al
(2002), respectively.
The present DCP requirement for BWC of buffaloes were comparable to
values 0.20, 0.19 and 0.20 reported by Tiwari and Patle (1983), Paul et al.(2003)
and Kurar and Mudgal (1981), respectively.
Results & Discussion
Page | 99
4.2.8 MP intake at fortnight intervals and prediction of its requirement for
maintenance and body weight change of non-lactating Murrah
buffaloes fed on oat hay or silage
The MP intake (g/d) of Murrah buffaloes at fortnight intervals is presented
in table 4.2.10. The overall mean fortnightly MP intake in oat hay group and oat
silage group were 527.77 ±4.28 and 635.23±5.43 respectively, which differ
significantly (P<0.05) between the groups
Table 4.2.10 Effect of feeding oat hay or oat silage on MP intake (g/d) in dry
buffaloes
Fortnight Oat hay (T1) Oat silage (T2)
1 541.01b±11.72 652.29a±23.76
2 525.04b±27.10 630.93a±20.90
3 517.26b±28.05 622.47a±25.33
Mean 527.77b±4.28 635.23a±5.43
Means bearing different superscripts in a row differ significantly (P < 0.05)
The MP intake (g/kg W0.75) was regressed linearly upon body weight
change (BWC, g/kg W0.75) to determine the MP requirements for maintenance
and body weight change. The regression equation (1) developed using all data
together (T1+T2) but the R2 value of this equation was lower so MP intake (g/kg
W0.75) in oat hay and silage group was regressed linearly upon respective body
weight change (BWC, g/kg W0.75) separately in and regression equation
developed was as below equation (2), (3) respectively.
Equation 1(T1+T2): y = 0.2286x + 3.3449 (R² = 0.4104, P<0.01, n = 48)
Equation 2 (T1): y = 0.201x + 3.6798 (R² = 0.3104, P<0.01, n = 24)
Equation 3 (T2): y = 0.185x + 2.9866 (R² = 0.7782, P<0.01, n = 24)
Where,
Y = MP intake (g/kg W0.75) of buffaloes
x = BWC (g/kg W0.75)
Results & Discussion
Page | 100
The relationship between MP intake (g/kg W0.75) and body weight change
(BWC, g/kg W0.75) in oat silage group was shown in figure 4.7. Equation 3 was
having highest R² and more accurately predicts the MP requirements of
buffaloes.
Fig 4.7 : Relationship of MP intake (g/kg W0.75) with body weight change
(g/kg W0.75) of dry Murrah buffaloes
MP intake at zero BWC was 2.98 g/kgW0.75 which was the MP
requirement for maintenance of non-lactating Murrah buffaloes per day and the
MP requirement for BWC of buffaloes was 0.185 g per g BWC/ kgW0.75/day. The
present requirement of MP for maintenance was comparable with that of ICAR,
(2013) feeding standard.
Phase III
Estimation of metabolizable energy requirements of Murrah buffaloes fed
on silage based diet
The experiment was conducted on Murrah buffaloes to determine their
metabolizable energy requirement. The results obtained during the course of this
study have been presented and discussed in following sections.
y = 0.185x + 2.9866 R² = 0.7782
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
-4 -3 -2 -1 0 1 2 3 4
MP
I (g
/kg
W0.7
5)
BWC (g/kg W0.75)
Results & Discussion
Page | 101
4.3.1 Chemical compositions of maize silage and varying metabolizable
energy level concentrates fed to lactating buffaloes
The ration consisted of concentrate mixture (40%) and maize silage (60%).
Detailed chemical composition of feeds and fodder is given in Table 4.3.1. ME
(MJ/kg DM) content of concentrate mixture was 10.00±0.12, 11.13±0.03 and
11.84±0.11 in ME -10, ME0 and ME +10 groups, respectively. The concentrate
mixtures were iso nitrogenous in nature.
4.3.2 Effect of varying metabolizable energy level in diet on body weight
lactating buffaloes
The average body weights of buffaloes at day one of trial period were 565,
567 and 570 kg in ME -10, ME0 and ME +10 groups, respectively. Fortnightly
record of buffaloes body weight is presented in Table 4.3.2. After 75 days of
feeding the final body wt. were 569.39±25.31, 571.24±13.35 and 570.32±15.04
in ME -10, ME0 and ME +10 groups. Mean body weights of lactating Murrah
buffaloes did not show any significant difference among different treatments.
Table 4.3.2: Fortnightly body weight of lactating Murrah buffaloes fed
varying metabolizable energy in diet
Fortnight ME -10
ME0
ME+10
1 565.20±23.14 568.40±11.60 567.60±18.08
2 567.40±24.77 570.80±11.60 571.40±15.73
3 568.20±26.37 570.60±16.22 569.20±13.15
4 571.20±27.65 572.60±12.52 571.80±13.40
5 574.80±26.89 573.80±12.35 574.60±13.55
Mean 569.39±25.31 571.24±13.35 570.32±15.04
Our results are in agreement with Jabbar et al. (2013) who observed a
non significant difference in body weight change of lactating Nili-Ravi buffaloes
fed on different level of energy. Lalman et al. (2000) reported a non significant
effect in body weight change of animals which were fed on increasing dietary
Table 4.3.1: Chemical compositions of maize silage and varying metabolizable
energy level concentrates fed to lactating buffaloes
Parameter Maize silage
Concentrate mixture
ME -10
ME0
ME+10
Dry matter 29.01±0.02 87.93±0.63 90.43±0.16 89.32±0.78
OM 86.13±1.08 90.81±0.69 91.75±0.12 90.13±0.57
CP 9.57±0.05 20.14±0.03 20.21±0.08 20.40±0.09
EE 2.18±0.23 3.09±0.87 3.26±0.04 3.85±0.43
Ash 13.87±1.08 9.19±0.69 8.25±0.12 9.87±0.57
NDF 54.19±0.59 29.39±0.76 25.07±0.09 22.86±0.72
ADF 34.45±0.13 12.29±0.01 11.87±0.08 12.78±0.10
NDICP 1.60±0.11 2.19±0.14 3.40±0.18 3.39±0.13
ADICP 0.9±0.03 1.12±0.01 1.17±0.06 1.11±0.04
Hemicellulose 19.74±0.55 17.10±0.58 13.20±0.69 10.08±0.62
Cellulose 22.04 ± 0.98 6.84±0.57 7.82±0.12 6.98±0.27
ADL 3.35 ± 0.01 5.26±0.21 6.06±0.01 5.30±0.04
TDN (%) 60.69±0.02 64.83±0.25 70.44±0.01 76.21±0.14
ME(MJ/kg) 9.97±0.03 10.16±0.12 11.24±0.03 12.32±0.11
Table 4.3.3 : Average fortnightly dry matter intakes (kg/d, kg/100Kg BW and g/Kg W0.75) of lactating Murrah buffaloes
fed with varying metabolizable energy in diet
Fortnight
DMI (kg/day) DMI (kg/100Kg BW) DMI (g/Kg W0.75)
ME -10
ME0
ME+10
ME -10
ME0
ME+10
ME -10
ME0
ME+10
1 14.61±0.53 14.44±0.36 14.59±0.44 2.58±0.18 2.54±0.13 2.57±0.33 126.02±7.87 124.02±5.31 125.44±5.45
2 14.66±0.60 14.73±0.40 14.74±0.43 2.58±0.17 2.58±0.17 2.58±0.16 126.08±7.07 126.11±7.01 126.14±6.00
3 15.04±0.46 14.95±0.40 14.69±0.43 2.65±0.15 2.62±0.16 2.58±0.14 129.28±6.47 128.05±6.57 126.02±5.44
4 14.68±0.43 15.04±0.37 14.60±0.47 2.57±0.15 2.63±0.12 2.55±0.12 125.64±6.52 128.51±5.15 124.89±4.93
5 14.60±0.49 14.88±0.37 15.10±0.44 2.54±0.15 2.59±0.12 2.63±0.13 124.33±6.34 126.91±4.97 128.64±5.09
Overall Mean
14.72±0.02 14.81±0.03 14.74±0.02 2.58±0.02 2.59±0.02 2.59±0.01 126.26±0.76 126.72±0.74 126.33±0.53
Results & Discussion
Page | 102
energy density during early lactation stage. Similarly, Grummer et al. (1995) also
reported that body weight changes were not affected by dietary fat
supplementation postpartum. However, in contrast to present study Brodericks
(2003) reported that increasing dietary energy density improved weight gain in
lactating Holstein cows.
4.3.3 Fortnightly average dry matter intake of lactating Murrah buffaloes
different levels of metabolizable energy (ME) in diet
Periodic observations of total DMI (kg/d, kg/100 kg BW and g/kg W0.75)
during trial period are presented in Table 4.3.3. During the 1st fortnight of trial
period the average DMI was 14.61±0.53, 14.44±0.36 and 14.59±0.44 kg/day
which was calculated to be 2.58, 2.54 and 2.57 percent of the body weight ME -
10, ME0 and ME +10 groups respectively.
The overall mean DMI was 14.72±0.02 kg/day (2.58% BW), 14.81±0.03
kg/day (2.59% BW) and 14.74± 0.02 (2.59% BW) in ME -10, ME0 and ME +10
groups respectively. The DMI (g/kg W0.75) during the 1st fortnight were
126.02±7.87, 124.02±5.31 and 125.44±5.45 for ME-10, ME0 and ME+10 groups
respectively. At the end of trail i.e. during 5th fortnight DMI (g/kg W0.75) were
124.33±6.34, 126.91±4.97 and 128.64±5.09 in ME -10, ME0 and ME +10 groups
respectively. DMI (g/kg W0.75) among different treatments were not significantly
affected during different fortnights in lactating buffaloes.
The results of present study are in agreement with those reported by
Aghaziarati et al. (2011), who reported that different dietary energy density did
not affect DM intake in Holstein cows. Similarly, Broderick (2003) also reported
no effect of varying energy and protein levels on DM intake. Different dietary
energy levels did not influence DM intake in lactating Nili-Ravi buffaloes (Jabbar
et al. 2013). However, contradictory to our findings, Vazquez Anon et al. (1997)
observed that increasing dietary energy density improved DM intake. The range
of values for DMI (%BW) in lactating buffaloes reported by different researchers
is 2.5-3.25% of BW (Paul and Lal, 2010) which was corroborated with results of
present study.
Results & Discussion
Page | 103
4.3.4 Productive performance and % feed efficiency of milk production in
lactating Murrah buffaloes fed on varying ME in the diet
During the 1st fortnight of trial period the average milk production was
8.78±0.77, 8.88±0.52 and 8.88±1.11 kg/day which was ME -10, ME0 and ME +10
groups respectively. The overall mean milk yield (Table 4.3.4) up to 75 day
period was 8.48±0.09, 8.84±0.02, 8.74±0.04 kg/d in respective groups. With
advancing of lactation, decreasing trends in milk production was observed
among all the groups but significantly low milk production observed in the ME -10
as comparison to ME0 and ME +10. The milk production decreased in the groups
might be due to stage of lactation, but less effect was observed on buffaloes fed
on ME as per ICAR, (2013) and 10% higher ME.
Data pertaining to the 6% FCM (kg/day) was presented in the table
no.4.3.4 The overall mean 6% FCM (kg/day) were 9.64, 9.99 and 9.88 in ME -10,
ME0 and ME +10 respectively which was significantly (P<0.05) lower in ME -10 as
compare to the ME0 and ME+10. These results agreed with those obtained by
Broderick (2003) who found that increasing dietary protein and energy gave
linear increases in milk yield and FCM. Feeding greater amounts of more
fermentable NFC would be expected to improve milk yield (Ekinci and
Broderick, 1997; Wilkerson et al. 1997; Kebreab et al. 2000 and Valadares et al.
2000). El-Ashry et al. (2003) found that buffaloes fed the high energy level
showed higher milk yield and 7% FCM.
Percent feed conversation efficiency for milk production was 57.98, 60.27
and 59.61 in ME-10, ME0 and ME+10 respectively which was significantly higher
(P<0.05) in ME0 than ME+10 than ME-10 (Table 4.3.5). These findings are partly
in accordance with the earlier studies (Jabbar et al. 2013; Broderick, 2003) who
reported that increasing dietary energy in diet up to recommended level
increased feed efficiency (milk yield/DMI) in lactating cows. El-Ashry et al.
(2003) found that buffaloes fed the high energy level showed the best feed
efficiency.
4.3.5. Milk composition
4.3.5.1. Milk fat content
The fortnightly milk fat percent and fat yield of three experimental groups
Table 4.3.4 : Fortnightly average milk yield (kg/day) and 6% FCMY (kg/day) in Murrah buffaloes fed with varying
metabolizable energy in diet
Fortnight
Milk yield (kg/day) 6% FCM yield (kg/day)
ME -10
ME0
ME+10
ME -10
ME0
ME+10
1 8.78±0.70 8.88±0.52 8.88±1.11 9.90±0.74 9.88±0.56 9.85±1.17
2 8.54±0.78 8.75±0.55 8.65±1.06 9.70±0.84 9.90±0.55 9.80±1.09
3 8.44±0.66 8.82±0.79 8.69±1.03 9.63±0.73 10.01±0.82 9.87±1.07
4 8.31±0.74 8.89±0.74 8.74±1.01 9.52±0.85 10.11±0.78 9.96±1.03
5 8.30±0.54 8.84±0.80 8.72±1.04 9.47±0.61 10.04±0.80 9.93±1.13
Overall
mean 8.48b±0.09 8.84a±0.02 8.74a±0.04 9.64 b±0.08 9.99a ±0.04 9.88 a ±0.03
Means bearing different superscripts in a row differ significantly (P < 0.05)
Table 4.3.5 : Productive performances and feed efficiency for milk
production in Murrah buffaloes fed varying metabolizable
energy level
Means bearing different superscripts in a row differ significantly (P < 0.05)
Particular ME -10
ME0
ME+10
Number of Animal 5 5 5
Avg. Initial body wt.(kg) 565±23.28 567±15.13 570±16.43
Avg. Final body wt.(kg) 569.39±25.31 571.24±13.55 570.32±15.04
DMI (kg/day) 14.72±0.02 14.81±0.03 14.74±0.02
DMI (kg/100Kg BW) 2.58±0.02 2.59±0.02 2.59±0.01
MEI (MJ/day) 129.22c±0.73 135.87 b±0.97 143.13a±0.90
CPI (kg/ day) 1.84±0.01 1.83±0.03 1.85±0.01
TDNI (kg/day) 8.89c ±0.05 9.28b ±0.07 9.58a ±0.06
Avg. milk yield (kg/animal/day) 8.48b±0.09 8.84a±0.02 8.74a±0.04
6 % FCM yield (kg/day/ animal) 9.64b±0.08 9.99a ±0.04 9.88 a ±0.03
% Feed efficiency (kg milk production × 100 /kg DM intake)
57.98b±0.66 60.27 a±0.14 59.61a± 0.33
Table 4.3.6: Fortnightly milk composition in Murrah buffaloes fed with varying metabolizable energy in diet
Variable (%) Group 1 2 3 4 5 Mean
Fat ME -10
7.12±0.09 7.19±0.05 7.23±0.04 7.25±0.02 7.23±0.03 7.20±0.02
ME0 6.99±0.12 7.16±0.11 7.20±0.12 7.22±0.12 7.21±0.14 7.16±0.04
ME+10
6.97±0.11 7.20±0.14 7.22±0.13 7.24±0.16 7.23±0.12 7.17±0.05
Protein ME -10
4.24±0.02 4.40±0.04 4.27±0.07 3.99±0.04 4.16±0.02 4.21±0.02
ME0 4.17±0.07 4.32±0.05 4.18±0.06 4.23±0.06 4.39±0.06 4.25±0.01
ME+10
4.19±0.03 4.23±0.09 4.15±0.06 4.31±0.06 4.16±0.08 4.21±0.02
Lactose ME -10
5.09±0.03 5.10±0.04 5.04±0.06 5.16±0.04 5.14±0.05 5.11±0.02
ME0 5.10±0.04 5.09±0.04 5.07±0.06 5.18±0.05 5.16±0.03 5.12±0.03
ME+10
5.09±0.04 5.15±0.05 5.11±0.07 5.17±0.05 5.17±0.05 5.14±0.01
SNF ME -10
11.23±0.04 11.34±0.09 11.51±0.04 11.14±0.07 11.25±0.06 11.29±0.01
ME0 11.25±0.07 11.33±0.05 11.31±0.03 11.27±0.07 11.30±0.04 11.29 ±0.03
ME+10
11.03±0.07 11.39±0.06 11.39±0.07 11.00±0.08 10.88±0.08 11.14 ±0.04
Total Solid ME -10
18.35±0.09 18.53±0.11 18.74±0.04 18.39±0.07 18.48±0.04 18.50 ±0.01
ME0 18.24±0.14 18.48±0.12 18.50±0.10 18.49±0.13 18.51±0.16 18.45±0.06
ME+10
18.00±0.12 18.59±0.18 18.61±0.16 18.24±0.17 18.11±0.15 18.31 ±0.05
Results & Discussion
Page | 104
along with their average is depicted in Table 4.3.6. Milk fat ranged from 7.12 to
7.25 in T1, 6.99 to 7.22 and 6.97 to 7.24 in T2 and T3 group, respectively. On an
average, the milk fat was 7.20, 7.16 and 7.17 percent in ME -10, ME0 and ME +10
groups, respectively. The fat contents among all the groups were similar.
4.3.5.2 Milk protein content
Milk protein ranged from 4.16 to 4.40, 4.17 to 4.39 and 4.15 to 4.31
percent in ME -10, ME0 and ME +10 groups, respectively (Table 4.3.6). Overall
mean for protein value was 4.21, 4.25 and 4.21% in respective group. The milk
protein values varied non significantly among the groups.
4.3.5.3. Milk lactose content
The overall mean lactose values were 5.11, 5.12 and 5.14 % in three
respective groups (Table 4.3.6). Milk lactose content did not differ among the
groups
4.3.5.4. Milk SNF content
The average fortnightly SNF content of milk (%) is depicted in Table 4.3.6.
The SNF content ranged from 11.14 to 11.51, 11.25 to 11.33 and 11.03 to 11.39
percent in T1, T2 and T3 group, respectively. On an average, the milk SNF
content (%) was 11.29, 11.29 and 11.14 in three respective groups. No
significant (P<0.001) difference was observed between ME -10, ME0 and ME +10.
4.3.5.5. Milk total solids content
The total solids content of milk (%) is depicted in Table 4.3.6. The total
solids ranged from 18.35 to 18.74, 18.24 to 18.51 and 18.00 to 18.69 % in ME -
10, ME0 and ME +10 group, respectively in different fortnights of experiment. The
overall mean total solids content was 18.50, 18.45 and 18.31 % in the three
respective groups.
The contents of milk fat, protein, lactose, solids not fat and total solids
were not influenced (P>0.05) by varying dietary energy levels in lactating Murrah
buffaloes. The results are in agreement with the findings of Aghaziarati et al.
(2011) who concluded that enriched dietary energy and protein with varying
milking frequency did not affect milk fat and protein percent. Likewise,
Komaragiri et al. (1998) also found that milk production and composition were
Results & Discussion
Page | 105
not affected by feeding an energy-rich diet (added dietary fat) to Holstein cows.
However, Vazquez-Anon et al. (1997) studied the effect of high energy diet
during mid to late lactation and concluded that increasing dietary energy density
enhanced milk protein yield. Broderick (2003) found that increasing dietary
protein and energy increased all milk components except fat which decreased
with increasing dietary energy. El-Ashry et al. (2003) found that buffaloes fed the
high energy level showed higher fat, protein, lactose, SNF, TS and ash
percentages.
4.3.6 Nitrogen Balance
The mean values of N intake, N outgo in faeces, N outgo in Urine, N outgo
in milk, Total out go and nitrogen balance is illustrated in Table 4.3.7. N excretion
in faeces nitrogen excretion via urine and N outgo in milk was not affected by the
different level of ME in diet. Nitrogen balance (g/d) was 22.82, 22.60 and 24.01
in ME -10, ME0 and ME +10 group, respectively which similar among the groups.
4.3.7 Nutrient utilisation in lactating Murrah buffaloes fed varying ME in
diet
The digestibility coefficients of DM, OM, CP, EE, NDF and ADF are
presented in Table 4.3.8. The digestibility of DM was 65.79, 66.46 and 66.83 in
ME -10, ME0 and ME +10 respectively which did not differ significantly among the
groups. Similar trend was observed in the digestibility of other nutrients. The
digestibility coefficients of OM, CP, EE, NDF and ADF were 68.93,60.83,
70.50,57.51 and 44.14 in ME -10; 69.57,61.13,72.64,58.01 and 44.76 in ME0 ;
70.11,60.76,73.29,58.73 and 45.10 in ME+10 respectively. But El-Ashry et al.
(2003) showed that buffaloes fed the highest energy level recorded the highest
digestibility of DM, OM, CP, CF and EE. In present study energy variation was
not very large, thus the digestibility might not differ significantly.
4.3.8 ME intake at fortnight intervals and prediction of its requirement for
maintenance and 6% FCM of Murrah buffaloes
The ME intake (MJ/d) of the lactating Murrah buffaloes at fortnight
intervals fed on different ME levels is presented in table 4.3.9. The overall mean
fortnightly metabolizable energy intake (MJ/d) was 129.22±0.73, 135.87±0.97
and 143.13±0.90 in ME -10, ME0 and ME +10 group respectively, which differed
Table 4.3.7 Nitrogen balance (g/d) in lactating buffaloes fed on varying ME in
diet
Particular ME -10
ME0 ME
+10
N intake 302.04±4.06 294.16±6.28 298.51±2.31
N Outgo in faeces 120.43±5.12 118.56±2.46 117.21±4.19
N Outgo in Urine 101.33±3.53 94.12±7.31 99.62±5.82
N Outgo in Milk 57.46±2.19 58.88±2.86 57.67±3.05
Total Outgo 279.22±8.41 271.56±8.31 274.50±7.51
Nitrogen balance 22.82±2.46 22.60±4.96 24.01±3.11
Table 4.3.8 Nutrient digestibility (DM %) of Murrah buffaloes fed on different
ME level
Parameter ME -10
ME0
ME+10
DM 65.79±1.23 66.46±1.01 66.83±1.14
OM 68.93±1.05 69.57±1.03 70.11±0.93
CP 60.83±0.26 61.13±0.32 60.76±0.54
EE 70.50±2.06 72.64± 1.86 73.29±1.62
NDF 57.51± 1.60 58.01± 2.00 58.73± 1.85
ADF 44.14±1.99 44.76±1.05 45.10±2.49
Results & Discussion
Page | 106
significantly (P<0.05) among the groups. The relationship between ME intake
(KJ/kg W0.75) and 6%FCM (kg/kg W0.75) is shown in figure 4.8. The ME intake
(KJ/kg W0.75) was regressed linearly upon 6%FCM (kg/kg W0.75) to determine the
energy requirements for 6%FCM and maintenance.
Table 4.3.9: Metabolizable energy intake (MJ/d) in lactating Murrah
buffaloes fed on varying ME in the diets
Fortnight ME -10
ME0 ME
+10
1 128.25±4.83 132.48±4.65 141.61±10.55
2 128.70±5.49 135.13±4.78 143.12±9.72
3 132.09±4.37 137.18±6.06 142.57±9.33
4 128.89±4.98 138.03±5.64 141.77±9.38
5 128.14±3.97 136.53±6.24 146.57±10.03
Mean± SE 129.22c±0.73 135.87
b±0.97 143.13
a±0.90
Means bearing different superscripts in a row differ significantly (P < 0.05)
The relation is depicted in form of graph in figure 4.8 and the regression
equation developed was as follows;
y = 6634.2x + 533.65 (R2 = 0.4898, P<0.01, n = 50)
Where,
Y = ME intake (kJ/kg W0.75) of buffaloes
X = 6% FCM (kg/kg W0.75)
The ME intake 533.65 kJ/kg W0.75 was the ME requirements for
maintenance of lactating buffaloes per day and the ME requirement for per kg
6% FCM in was 6634.2 kJ. The current maintenance requirement of Murrah
buffaloes is marginally higher than Ranjhan, (1992) recommendations (510 kJ/
kg W 0.75) and comparable to Kearl, (1982) recommendation (523 kJ/ kg W 0.75),
respectively. MEm of lactating buffaloes was reported to be 540.3 kJ/ kg W
0.75(Paul et al 2003), 508 kJ/kg W 0.75 (Siviah and Mudgal, 1978), 534 kJ/ kg W
0.75 (ICAR, 1998) and 521 kJ/kg W0.75 calculated using the equations of AFRC
(1990). The variation in these individual estimates is attributable mainly to
Results & Discussion
Page | 107
difference in method of estimation. Generally, estimates of nutrient requirements
reported from feeding trial data using regression method are likely to be higher
than the values reported from calorimetric studies in any species. While MEm
(kJ/kg W0.75) value of Indian lactating cattle was 572.2, 575.7 and 546.9 reported
by Patel and Mudgal (1977), Paul et al. (2003) and Patel and Mudgal (1976),
respectively. Yan et al. (1997) concluded that MEm requirements in lactating
cattle ranged from 490-640 kJ/kg W0.75 based on regression analysis of
calorimetric data reported by different authors.
4.3.9 TDN intake at fortnight intervals and prediction of its requirement for
maintenance and 6% FCM of Murrah buffaloes
The TDN intake (kg/d) of the lactating Murrah buffaloes at fortnight
intervals fed on different ME levels is presented in table 4.3.10. The overall
mean fortnightly TDN intake (kg/d) were 8.89±0.05, 9.28±0.07 and 9.58±0.06 in
ME -10, ME0 and ME +10 group respectively, which differ significantly (P<0.05)
among the groups. The relationship between TDN intake (g/kg W0.75) and
6%FCM (kg/kg W0.75) is shown in figure 4.9. TDN intake (g/kg W0.75) was
regressed linearly upon 6%FCM (kg/kg W0.75) to determine the energy
requirements for 6%FCM and maintenance.
Table 4.3.10: Total digestible nutrient intake (kg/d) in lactating Murrah
buffaloes fed on varying ME in the diets
Fortnight ME -10
ME0
ME+10
1 8.82±0.32 9.04±0.30 9.48±0.69
2 8.85±0.36 9.23±0.31 9.58±0.64
3 9.09±0.29 9.37±0.40 9.54±0.61
4 8.87±0.33 9.42±0.37 9.49±0.61
5 8.82±0.26 9.32±0.41 9.81±0.66
Mean± SE 8.89c±0.05 9.28b±0.07 9.58a±0.06
Means bearing different superscripts in a row differ significantly (P < 0.05)
Fig 4.8: The relationship between ME intake (KJ/kg W0.75) and 6%FCM (kg/kg
W0.75) in Murrah buffaloes fed on different levels of ME in diet
Fig 4.9: Relationship of TDN intake (g/kg W0.75) with 6%FCM (kg/kg W0.75) of
lactating Murrah buffaloes
y = 6634.2x + 533.65 R² = 0.4898
200
400
600
800
1000
1200
1400
1600
0.04 0.06 0.08 0.10 0.12
kJ
/ W
0.7
5
6%FCM (kg/ kg W0.75)
y = 438.51x + 35.27 R² = 0.4916
20
30
40
50
60
70
80
90
100
0.05 0.07 0.09 0.11 0.13
TD
NI (g
/ W
0.7
5)
6%FCM (kg/ kg W0.75)
Results & Discussion
Page | 108
The relation is depicted in form of graph in figure 4.9 and the regression
equation developed was as follows;
y = 438.51x + 35.27 (R2 = 0.4916, P<0.01, n = 50)
Where,
Y = TDN intake (kJ/kg W0.75) of buffaloes
X = 6% FCM (kg/kg W0.75)
The TDN requirement of lactating buffaloes for maintenance was 35.27 g/
kg W0.75 per day when the body weight change was zero and TDN requirement
for 6%FCM in buffaloes was 438.51 g per kg 6%FCM. The present energy
requirements for maintenance (g TDN/kg W0.75) of lactating buffaloes was
comparable with ICAR, 2013 and Paul et al., (2002).
Phase III
Estimation of metabolizable protein requirements of Murrah buffaloes fed
on silage based diets
The experiment was conducted on Murrah buffaloes to investigate the effect
of different metabolizable protein levels in diet on milk production, milk
composition and MP requirements. The results obtained during the course of
this study have been presented and discussed in following respective sections.
4.4.1 Chemical compositions of maize silage and varying metabolizable
protein level concentrates fed to lactating buffaloes
The ration consisted of concentrate mixture (40%) and maize silage
(60%). Detailed chemical composition of feeds and fodder is given in Table
4.4.1. MP (%DM) content of concentrate mixture was 9.98±0.03, 11.09±0.05 and
12.21±0.15 in MP-10, MP0 and MP+10 groups, respectively. The concentrate
mixtures were varying in crude protein content 18.50,19.33 and 20.84 in MP-10,
MP0 and MP+10 groups, respectively while almost similar in energy (MJ/kg)
content.
Results & Discussion
Page | 109
4.4.2 Effect on body weight of lactating Murrah buffaloes fed varying
metabolizable protein in diet
The average body weights of buffaloes at day one of trial period were
537±14.11, 542±21.14 and 540±17.05 kg in MP-10, MP0 and MP+10 groups.
Fortnightly record of buffalo’s body weight is presented in Table 4.4.2. Overall
final body wt. was 545.92±2.04, 548.64±2.34 and 547.92±1.85 in MP-10, MP0 and
MP+10 groups respectively. A non significant difference was observed between
the mean body weights of MP -10, MP0 and MP +10, respectively.
Table 4.4.2: Fortnightly body weight of lactating Murrah buffaloes fed
varying metabolizable protein in diet
Fortnight MP-10
MP0
MP+10
1 539.80±15.10 541.60±25.59 543.80±18.24
2 543.40±15.10 545.80±22.96 544.20±19.67
3 546.00±15.22 548.60±22.60 548.20±19.93
4 549.00±15.62 552.40±21.84 549.60±18.32
5 551.40±15.80 554.80±21.39 553.80±17.48
Mean 545.92±2.04 548.64 ±2.34 547.92±1.85
4.4.3 Fortnightly average dry matter intake of lactating Murrah buffaloes
different levels of metabolizable protein (MP) in diet
Periodic observations of total DMI (kg/d, kg/100 kg BW and g/kg W0.75)
during trial period are presented in Table 4.4.3. During the 1st fortnight of trial
period the average DMI was 13.06±0.34, 13.05±0.30 and 13.31±0.23 kg/day
which was calculated to be 2.42, 2.41 and 2.43 percent of the body weight in
MP -10, MP0 and MP+10 groups respectively.
The overall mean DMI was 13.24±0.01 kg/day (2.43% BW), 13.29±0.01
kg/day (2.42% BW) and 13.26± 0.04 (2.42% BW) in MP-10, MP0 and MP+10
groups respectively. The DMI (g/kg W0.75) during the 1st fortnight was
116.87±2.79, 116.34±2.85 and 117.43±2.83 for MP -10, MP0 and MP +10 groups
Table 4.4.1: Chemical compositions of maize silage and varying metabolizable protein
level concentrates fed to lactating buffaloes
Parameter Maize silage Concentrate mixture
MP-10
MP0 MP
+10
DM 32.41±0.05 89.30±0.53 91.14±0.23 91.63±0.67
OM 83.72±0.15 92.69±0.05 92.87±0.01 92.53±0.33
CP 10.65±0.03 18.50±0.31 19.33±0.02 20.84±0.22
EE 2.41±0.01 4.12±0.02 4.30±0.01 4.57±0.05
Ash 16.84±0.05 7.31±0.05 7.13±0.07 7.47±0.23
NDF 60.37±0.05 33.25±0.15 29.64±0.84 32.60±0.43
ADF 36.42±0.01 11.26±0.12 11.46±0.28 12.07±0.09
NDICP 1.90±0.21 2.19±0.15 2.40±0.11 2.39±0.14
ADICP 1.60±0.09 1.32±0.03 1.17±0.06 1.11±0.07
Hemicellulose 23.95±0.01 21.99±0.10 18.17±0.55 20.53±0.35
Cellulose 22.78±0.25 6.98±0.45 6.84±0.50 7.82±1.03
ADL 3.68±0.05 3.30±0.39 4.26±0.65 4.06±0.14
TDN 59.46±0.15 72.89±1.03 72.92±0.69 72.50±0.05
ME(MJ/kg) 9.76±0.01 11.67±0.13 11.69±0.32 11.61±0.43
MP (%DM) 6.53±1.09 9.98±0.03 11.09±0.05 12.21±0.15
Table 4.4.3 : Average fortnightly dry matter intakes of lactating Murrah buffaloes fed with varying metabolizable protein in
diet
Fortnight
DMI (kg/day) DMI (kg/100Kg BW) DMI (g/Kg W0.75)
MP-10
MP0
MP+10
MP-10
MP0
MP+10
MP-10
MP0
MP+10
1 13.06±0.34 13.05±0.30 13.31±0.23 2.42±0.14 2.41±0.05 2.43±0.07 116.87±2.79 116.34±2.85 117.43±2.83
2 13.26±0.32 13.26±0.28 13.12±0.28 2.44±0.14 2.43±0.06 2.41±0.08 117.90±2.84 116.94±3.06 115.27±2.82
3 13.27±0.33 13.39±0.28 13.38±0.15 2.43±0.14 2.44±0.05 2.44±0.08 117.78±2.77 117.13±2.84 116.68±2.77
4 13.29±0.33 13.31±0.26 13.25±0.22 2.42±0.14 2.41±0.06 2.41±0.06 118.33±2.72 115.87±2.90 114.95±2.74
5 13.34±0.35 13.43±0.24 13.35±0.18 2.42±0.14 2.42±0.06 2.41±0.07 118.72±2.85 116.46±3.14 115.41±2.69
Overall Mean
13.24±0.01 13.29±0.01 13.26±0.04 2.43±0.01 2.42±0.01 2.42±0.02 117.93±0.18 116.55±0.09 115.95±0.36
Table4.4. 4: Fortnightly average milk yield (kg/day) and 6% FCM yield (kg/day) in Murrah buffaloes fed with varying
metabolizable protein in diet
Fortnight
Milk yield (kg/day) 6% FCM yield (kg/day)
MP-10
MP0
MP+10
MP-10
MP0
MP+10
1 7.34±0.44 7.23±0.64 7.25±0.27 9.10±0.53 8.92±0.78 9.05±0.29
2 7.27±0.35 7.34±0.65 7.29±0.44 8.84±0.37 9.04±0.78 8.96±0.53
3 7.31±0.33 7.31±0.53 7.34±0.42 8.88±0.42 8.94±0.69 9.02±0.47
4 7.27±0.35 7.26±0.58 7.37±0.37 9.07±0.39 9.01±0.71 9.15±0.45
5 7.32±0.37 7.28±0.51 7.31±0.32 9.03±0.44 8.97±0.63 9.05±0.40
Overall
mean 7.30±0.01 7.28±0.02 7.31±0.02 8.98±0.05 8.98±0.02 9.05±0.03
Results & Discussion
Page | 110
respectively. At the end of trail period that is during 5th fortnight DMI (g/kg
W0.75) was 118.72±2.85, 116.46±3.14 and 115.41±2.69 for MP-10, MP0 and
MP+10 group which were non significant (P>0.05) among the treatments.
The average DMI (kg/day) in present study was comparable in all the
three groups. Similar results were observed by Bovera et al. (2002) in buffaloes
fed on surplus of energy and protein in their diet. In another study Wang et al.
(2007) in Chinese Holstein dairy cows fed four different levels of metabolizable
protein in the diet. But significantly higher DMI (kg/d) was found with increasing
PDIE (protein digested in the small intestine when rumen-fermentable energy is
limiting) in cows when fed three levels of metabolizable protein supply (108, 98–
95 and 85 g PDIE kg DM) in diet (Colin-Schoellen et al. 2000). Trials with high
producing dairy cows at Penn State have shown a variable effect of decreasing
dietary CP or metabolizable protein on DMI. DMI decreased when animals were
fed the metabolizable protein deficient diets and milk production also decreased
(Lee et al., 2011; Lee et al., 2012a). On the contrary, when DMI did not
decrease, milk production was also not different from diets with adequate
metabolizable protein (Lee et al., 2012b; Giallongo et al., 2014).
In agreements with the our findings, Cunningham et al. (1996); Leonardi
et al. (2003); Colmenero and Broderick (2006) observed no effect of dietary CP
content on DMI when dietary CP was increased from 16.5 to 18.5% and from
16.1 to 18.9%, respectively. However Broderick (2003) reported a linear
increase in DMI when dietary CP was increased from 15.1 to 16.7 and 18.3%.
4.4.4 Productive performance and feed utilization efficiency for milk
production in lactating Murrah buffaloes fed on different MP levels in
diet
Average daily milk production (Table 4.4.4) ranged from 7.27 to 7.34, 7.23
to 7.34 and 7.25 to 7.37 kg/d in MP-10, MP0 and MP+10 groups, respectively in
different fortnights. Overall average milk production was 7.30, 7.28 and 7.31 kg/d
in MP-10, MP0 and MP+10 groups respectively which was non-significant (P>0.05)
among the groups. Average 6%FCM was 8.98, 8.98 and 9.05 kg/d in MP-10, MP0
and MP+10 groups respectively. Percent feed utilization efficiency for milk
Results & Discussion
Page | 111
production was 55.13, 54.79 and 55.13 in MP-10, MP0 and MP+10 respectively
which was non-significant (P>0.05) among the groups (Table 4.4.5).
The effect of dietary MP level on milk production and 6% FCM of
buffaloes was non significant indicating that no effect of 10% increase or
decrease in MP than ICAR, (2013) recommendation in the diet of lactating
buffaloes. Bovera et al. (2002) observed non significant changes in milk
production (kg/d) when buffaloes were fed higher amount of energy and protein
as calculated by CPM –dairy software. Similar results were reported when
buffaloes fed on differing in energy and protein concentrations (diet A: 6.1 MJ/kg
DM of NEl, 112.5 g/kg DM MP, vs. diet B: 6.4 MJ, 95.1 g of MP, 78.9 g of PDI)
(Bovera et al. 2007). As MP level increase, the milk yield and protein yield
increased in cattle producing milk more than 25-30 kg/d (Aboozar, 2012). Wang
et al. (2007) reported increase milk yield, 4% FCM and milk protein % but
decreasing milk fat % with increasing dietary MP in Chinese Holstein cows
producing average 30.2 kg/d milk. Similarly increased milk yield from 33.90 to
36.20 kg/d with increasing MP from 8.1 to 10.2% was reported (Raggio et al.
2004). Voltolini et al. (2008) evaluated the effects of increasing MP supply
beyond NRC (2001) recommendations for mid lactating dairy cows grazing
elephant grass pasture. They reported that milk production, 3.5% FCM were not
affected by treatment.
Cows milking around 95 to 99 lbs/d, when fed metabolizable protein
deficient by 5to 10%, diets did not result in depressed DMI or milk production
(Giallongo et al., 2014). Olmos Colmenero and Broderick (2006) observed non
significant effect on milk yield and FCM in lactating Holstein cows fed on
varying CP in diet. They also concluded that feeding diets with about 16.0% or
less CP (DM basis) provides insufficient MP for maximal milk synthesis, but
feeding diets with more than 17.0% CP does not improve milk yield. Milk yield
did not increase when dietary protein was increased from 17.2 to 19.0%
(Sannes et al., 2002), from 16.8 to 19.4% (Davidson et al., 2003), from 16.7 to
18.4% (Broderick, 2003), and from 15 to 18.7% (Groff and Wu, 2005) in
Holstein lactating cows.
Table 4.4.5: Productive performances and feed efficiency for milk production
in Murrah buffaloes fed varying metabolizable protein level
Means bearing different superscripts in a row differ significantly (* P < 0.05)
Particular MP-10
MP0
MP+10
Number of Animal 5 5 5
Avg. Initial body wt.(kg) 537.20±14.11 542±21.14 540±17.05
Avg. final body wt.(kg) 545.92±2.04 548.64±2.34 547.92±1.85
DMI (kg/day/ animal) 13.24±0.01 13.29±0.01 13.26±0.04
DMI (kg/100Kg BW) 2.43±0.01 2.42±0.01 2.42±0.02
MP intake (g/day/animal) 887.98c±6.58 940.56b±6.52 996.80a±6.08
CP intake (kg/day/animal) 1.76c±0.03 1.86b±0.01 1.97a±0.02
DCP intake (kg/day/animal) 1.12c±0.02 1.23b±0.01 1.32a±0.02
ME intake (MJ/day/animal) 134.70 ±2.18 135.74 ±1.22 135.92 ±1.13
Avg. milk yield (kg//animal/day) 7.30±0.01 7.28±0.02 7.31±0.02
6 % FCM Milk yield (kg/day/ animal) 8.98±0.05 8.98±0.02 9.05±0.03
%Feed efficiency (6% FCM ×100/kg DM intake)
55.13±2.10 54.79±1.85 55.13±2.05
Results & Discussion
Page | 112
4.4.5. Milk composition
4.4.5.1. Milk fat content
The fortnightly milk fat percent of three experimental groups along with
their average is depicted in Table 4.4.6. Milk fat ranged from 7.06 to 7.36 in MP
-10, 7.11 to 7.29 and 7.19 to 7.36 in MP0 and MP +10 groups, respectively. On an
average, the milk fat was 7.20, 7.21 and 7.26 percent in MP -10, MP0 and MP +10
groups, respectively. The fat content among all the groups was similar.
4.4.5.2 Milk protein content
Milk protein ranged from 4.03 to 4.24, 4.13 to 4.30 and 4.14 to 4.37
percent in MP -10, MP0 and MP+10 groups, respectively (Table 4.4.6). Overall
mean for protein value was 4.15, 4.22 and 4.20% in respective group. The milk
protein values varied non significant among groups.
4.4.5.3. Milk lactose content
The overall mean lactose values were 5.13, 5.14 and 5.13 % in three respective
groups (Table 4.4.6). Milk lactose content did not differ among the groups
4.4.5.4. Milk SNF content
The average fortnightly SNF content of milk (%) is depicted in Table 4.4.6.
The SNF content ranged from 11.15 to 11.25, 11.21 to 11.30 and 11.16 to 11.23
percent in MP -10, MP0 and MP+10 groups, respectively. On an average, the milk
SNF content (%) was 11.20, 11.25 and 11.21 in three respective groups. No
significant (P<0.001) difference was observed among MP -10, MP0 and MP+10
groups.
4.4.5.5. Milk total solids content
The total solids content of milk (%) is depicted in Table 4.4.6. The total
solids ranged from 18.21 to 18.55, 18.32 to 18.53 and 18.35 to 18.53 % in MP -
10, MP0 and MP+10 groups, respectively in different fortnights of experiment. The
overall mean total solids content was 18.40, 18.47 and 18.48% in the MP -10,
MP0 and MP+10 respective groups
No effect of protein supply on milk or milk component yield was observed
in cows fed at 85% and 115% of predicted MP supply (Weiss and Wyatt, 2006).
Imaizumi et al. (2010) observed that milk fat % was not affected but increased
Results & Discussion
Page | 113
milk protein in (extra protein by soyabean meal and cotton seed) SBCS-17.5
(MP, 11.8% DM) treatment, but it decreased when fed (extra protein through
urea) U-17.5 diet than control (MP, 10.8% DM). Voltolini et al. (2008) evaluated
the effects of increasing MP supply beyond NRC (2001) recommendations for
mid lactating dairy cows grazing elephant grass pasture. They reported that milk
production, 3.5% FCM, milk fat, protein, lactose and total solids contents were
not affected by treatments.
Leonardi et al. (2003) found that milk protein content decreased (3.25 and
3.18%) when CP was increased from 16.1 to 18.9%; however, fat content
increased significantly in response to dietary CP in cows. Several studies have
reported no improvement in milk and protein production when dietary CP was
increased from 16.1–16.7% to 18.4–18.9% in cows (Cunningham et al., 1996;
Broderick, 2003; Leonardi et al., 2003)
4.4.6 Effect of dietary protein levels on urinary purine derivatives,
creatinine and microbial N production in lactating Murrah buffaloes
The data on urinary purine derivatives (PD) and creatinine excretion and
microbial N production in lactating Murrah buffaloes is provided in table 4.4.7.
There was no significant effect of varying protein levels on allantoin, uric acid,
creatinine, total purine derivatives and microbial N production. Allantoin
constituted the principal PD in the urine. Allantoin and uric acid ranged from 4.51
to 4.62 mmol/l and 2.32 to 3.49 mmol/l, respectively. Total PD varied from
203.38 to 214.06 mmol/day. The microbial N productions (g/d) were 135.47,
141.83 and 142.94 in MP-10, MP0 and MP+10 respectively.
Urinary excretion of purine derivatives, of which allantoin is the major
component, reflects microbial nucleic acid absorption from the small intestine
and is related to microbial protein formation in the rumen (Stangassinger et al.,
1995). Urinary excretion of creatinine did not differ (p>0.05) between animals fed
at different levels of dietary protein as observed by earlier workers (Dipu et al.,
2006 and George et al., 2006). Excretion rate of creatinine was relatively
constant in healthy animals and remained independent of level of feed intake
(Dipu et al., 2006). Jetana et al. (2009) reported significantly lower (p<0.01)
urinary purine derivatives (PD) and the creatinine (Cr) excretion by swamp
buffaloes than Brahman cattle. The in vitro ruminal microbial N synthesis per kg
Table 4.4.6: Fortnightly milk composition in Murrah buffaloes fed with varying metabolizable Protein in diet
Variable (%) Group I II III IV V Mean
Fat MP-10
7.28±0.05 7.08±0.07 7.06±0.07 7.36±0.07 7.23±0.08 7.20±0.06
MP0 7.24±0.09 7.21±0.08 7.11±0.07 7.29±0.05 7.22±0.08 7.21±0.03
MP+10
7.36±0.07 7.19±0.08 7.19±0.07 7.29±0.04 7.27±0.03 7.26±0.03
Protein MP-10
4.03±0.05 4.18±0.03 4.18±0.06 4.12±0.02 4.24±0.02 4.15±0.04
MP0 4.30±0.06 4.28±0.02 4.20±0.06 4.13±0.05 4.23±0.06 4.22±0.03
MP+10
4.37±0.04 4.16±0.08 4.17±0.05 4.14±0.05 4.19±0.03 4.20±0.04
Lactose MP-10
5.16±0.05 5.14±0.05 5.11±0.03 5.13±0.04 5.09±0.03 5.13±0.01
MP0 5.20±0.05 5.16±0.03 5.14±0.02 5.12±0.06 5.10±0.04 5.14±0.02
MP+10
5.15±0.03 5.17±0.05 5.13±0.02 5.13±0.05 5.09±0.04 5.13±0.01
SNF MP-10
11.15±0.07 11.25±0.06 11.15±0.04 11.19±0.04 11.23±0.04 11.20±0.02
MP0 11.24±0.07 11.30±0.04 11.21±0.06 11.24±0.04 11.25±0.07 11.25±0.02
MP+10
11.17±0.05 11.27±0.20 11.16±0.06 11.22±0.09 11.23±0.03 11.21±0.02
Total Solid MP-10
18.43±0.10 18.34±0.05 18.21±0.10 18.55±0.06 18.46±0.09 18.40±0.06
MP0 18.49±0.13 18.52±0.06 18.32±0.02 18.53±0.5 18.48±0.14 18.47±0.04
MP+10
18.53±0.10 18.47±0.25 18.35±0.07 18.52±0.12 18.51±0.04 18.48±0.03
Table4.4.7: Effect of dietary metabolizable protein levels on urinary purine
derivatives and creatinine excretion and microbial N production in lactating
buffaloes
Parameter MP-10
MP0
MP+10
Average metabolic body wt. 110.57±2.85 114.11±3.10 113.25±2.69
Avg. DMI (kg/d) 13.24±0.01 13.29±0.01 13.26±0.04
Allantoin (mmol/l) 4.51±0.14 4.59±0.14 4.62±0.08
Uric acid (mmol/l) 0.49±0.02 0.53±0.05 0.53±0.05
Purine derivative concentration (mmol/l)
5.01±0.14 5.12±0.12 5.15±0.11
Creatinine concentration (mmol/l)
2.67±0.08 2.69±0.07 2.67±0.09
PDC index 207.53±5.83 217.01±5.73 218.43±4.72
Total PD excreted (mmol/d) 203.38±5.71 212.67±5.62 214.06±4.62
Absorbed purine (mmol/d) 185.89±5.39 194.63±5.38 196.16±4.63
Microbial N, g 135.47±3.93 141.83±3.91 142.94±3.36
g MN/ kg DOMI 23.18±0.74 24.31±0.98 24.48±0.76
Results & Discussion
Page | 114
OMTD (truly digestible OM) was reported to be 26.3 - 30.5g (Blummel and
Lebzien, 2001).
Purine derivative (allantoin plus uric acid) excretion showed a linear trend
in response to increasing CP content of the diets of Holstein cows (Olmos
Colmenero and Broderick, 2006)
4.4.7 Nutrient digestibility coefficients in lactating Murrah buffaloes fed on
diets with varying levels of protein
The digestibility coefficient (Table 4.4.8) of DM was 62.36, 62.62 and 63.
57 per cent in MP -10, MP0 and MP+10 groups respectively, and did not differ
significantly among the groups. Similar trend was observed in the digestibility of
other nutrients except CP in which was significantly higher in MP0 and MP+10
than that of MP -10. The digestibility coefficients of OM, CP, EE, NDF and ADF
were 64.82, 63.61, 69.79, 56.38 and 43.53 per cent in MP -10;
65.60,66.23,70.48,55.78 and 44.53 per cent in MP0 and 65.71, 66.65, 71.65,
56.71and 44.76 per cent in MP+10, respectively.
Trials with high producing dairy cows at Penn State have shown a
variable effect of decreasing dietary CP or metabolizable protein on nutrient
digestibility. Total tract apparent neutral detergent fiber (NDF) digestibility was
decreased (6 to 20%) by the low CP, metabolizable protein deficient diets by
Lee et al., (2011); Lee et al., (2012b); Giallongo et al., (2014).Christensen et al.
(1993) did not detect improvement in intake or apparent ruminal digestibility of
OM, NDF and ADF by increasing the CP content of the diet from 16.4 to 19.6%
of DM. Total tract digestibility of CP showed a linear and quadratic response to
dietary CP and its maximum was observed on the 19.4% CP diet.
4.4.8 Nitrogen dynamics in lactating Murrah buffaloes fed on diets with
varying levels of protein
The mean values of N intake, N outgo in faeces, N outgo in Urine, N
outgo in Milk, Total out go and nitrogen balance is presented in Table 4.4.8. N
intake (g/d) was 284.43, 298.11 and 309.42 in MP -10, MP0 and MP+10,
respectively, and differed significantly (P<0.05) among the groups. N excretion in
faeces and N outgo in milk was not affected by the different level of MP in the
diet but the urinary excretion of nitrogen increased with the increase in N intake
Results & Discussion
Page | 115
in the diet. Urinary nitrogen excretion (g/d) was 94.29, 106.27 and 114.70 in MP -
10, MP0 and MP+10, respectively which differ significantly (P<0.05) among the
groups. Overall N balance (g/d) was 23.18, 24.31 and 24.48 in MP -10, MP0 and
MP+10, respectively which was non-significant among groups.
Similar to present study, urinary nitrogen excretion decreased by the diet
deficient in metabolizable protein than high MP diets (+200 to -250 g/d; i.e. 12
to 13% variation in MP) reported by Lee et al., (2011); Lee et al., (2012b);
Giallongo et al., (2014). Colmenero and Broderick (2006) observed that
concentrations of MUN, urine volume, and urinary excretion of total N and urea
N all increased significantly in response to dietary CP content in Holstein cows
fed on five varying CP in diets. Castillo et al.(2001),from an extensive review of
published studies, reported that on average, 72% of the N consumed by dairy
cows was excreted in faeces and urine and that there was a linear relationship
between N intake and N excreted in faeces and urine.
4.4.9 MP intake at fortnight intervals and prediction of its requirement for
maintenance and 6% FCM of Murrah buffaloes
The MP intake (g/d) of the lactating Murrah buffaloes at fortnight intervals fed on
different MP levels is presented in table 4.4.9. The relationship between MP
intake (g/kg W0.75) and 6%FCM (kg/kg W0.75) is shown in figure 4.10. The MP
intake (g/kg W0.75) was regressed linearly upon 6%FCM (kg/kg W0.75) to
determine the MP requirements for 6%FCM and maintenance.
Table 4.4.9: MP intake (g/d) in lactating Murrah buffaloes fed on varying
MP in the diets
Fortnight MP -10
MP0 MP
+10
1 865.14±26.42 916.73±54.63 983.58±23.20
2 883.03±18.06 937.83±54.73 981.84±37.31
3 903.09±21.57 947.40±49.77 1008.30±34.09
4 891.86±17.23 946.28±49.91 1011.03±32.71
5 896.78±20.77 954.58±44.30 999.25±32.43
Mean± SE 887.98c
±6.58 940.56b
±6.52 996.80a
±6.08
Means bearing different superscripts in a row differ significantly (P < 0.05)
Fig 4.10: Relationship of MP intake (g/kg W0.75) with 6% FCM (kg/kg W0.75)
of Murrah buffaloes
Fig 4.11: Relationship of DCP intake (g/kg W0.75) with 6% FCM (g/kg W0.75)
of Murrah buffaloes
y = 66.781x + 2.5691 R² = 0.4967
2
3
4
5
6
7
8
9
10
0.05 0.06 0.07 0.08 0.09 0.1
MP
(g
/ k
g W
0.7
5)
6% FCM (kg/kg W0.75)
y = 71.774x + 3.191 R² = 0.478
2
3
4
5
6
7
8
9
10
11
12
0.04 0.05 0.06 0.07 0.08 0.09 0.1
DC
P (
g/k
g B
W 0
.75)
6% FCM (kg/kg BW 0.75)
Table4.4.8: Effect of dietary metabolizable protein levels on nutrient
digestibility coefficients (%) and nitrogen dynamics in lactating buffaloes
Particular MP-10
MP0 MP
+10
Nutrient digestibility coefficient (%)
DM 62.36±0.60 62.62±0.52 63.57±0.49
OM 64.82±0.65 65.60±0.56 65.71±0.58
CP 63.61
b
±0.34 66.23a
±1.50 66.65a
±0.77
EE 69.79±0.95 70.48±1.03 71.65±0.44
NDF 56.38±0.53 55.78±0.57 56.71±0.66
ADF 43.53±0.91 44.72±0.17 44.76±0.56
Nitrogen Balance (g/d)
N intake 284.43c ±4.57 298.11b ±5.08 309.42a ±6.03
N Outgo in faeces 120.58±2.63 121.30±6.01 124.67±9.02
N Outgo in Urine 94.29c ±3.17 106.27b ±1.36 114.70a±1.27
N Outgo in Milk 48.06±6.19 48.27±6.18 48.23±2.20
Total Outgo 262.93±5.16 275.84±8.06 287.6±7.12
Nitrogen balance 21.50±6.32 22.27±6.98 21.82±3.44
Results & Discussion
Page | 116
The relation is depicted in form of graph in figure 4.10 and the regression
equation developed was as follows;
y = 66.781x + 2.5691 (R2 = 0.4967, P<0.01, n = 75)
Where,
Y = MP intake (g/kg W0.75) of buffaloes
X = 6% FCM (kg/kg W0.75)
The MP requirement of lactating buffaloes for maintenance was 2.5691 g/
kg W0.75 per day and MP requirement for 6%FCM in buffaloes was 66.781 g per
kg 6%FCM. The present MP requirement for maintenance (g/kg W0.75) of
lactating buffaloes was comparable with ICAR, (2013). NRC (1996)
recommended MP requirement for maintenance as 3.8 g/kg BW0.75 in zebu
cattle. Ezikiel (1987) obtained MP requirements for maintenance of 1.72 and
4.28 g/kg BW0.75/d for Nellore and Holstein, respectively. Valadares et al. (1997)
calculated MP requirement for maintenance as 4.13 g/kg BW0.75/d in zebu cattle.
Hill (1998) estimated the value of 1.63 g/kg BW0.75/d in Nellore. Vermeulen
(2001) estimated MP requirements of beef cows (avg. body wt. 499 kg) with a
peak milk yield of 6.4 kg/d to be 734 g/d as per NRC (1996).
4.4.10 DCP intake at fortnight intervals and prediction of its requirement for
maintenance and 6% FCM of Murrah buffaloes
The DCP intake (kg/d) of the lactating Murrah buffaloes at fortnight
intervals fed on different MP levels is presented in table 4.4.10. The relationship
between DCP intake (g/kg W0.75) and 6%FCM (kg/kg W0.75) is shown in figure
4.11. The DCP intake (g/kg W0.75) was regressed linearly upon 6%FCM (kg/kg
W0.75) to determine the DCP requirements for 6%FCM and maintenance.
The relation is depicted in form of graph in figure 4.11 and the regression
equation developed was as follows;
y = 71.774x + 3.191 (R2 = 0.478, P<0.01, n = 75)
Where,
Y = DCP intake (g/kg W0.75) of buffaloes
X = 6% FCM (kg/kg W0.75)
Results & Discussion
Page | 117
Table 4.4.10: Digestible crude protein intake (kg/d) in lactating Murrah
buffaloes fed on varying MP in the diets
Fortnight MP -10
MP0 MP
+10
1 1.09±0.03 1.20±0.07 1.30±0.03
2 1.11±0.02 1.23±0.07 1.30±0.04
3 1.14±0.02 1.24±0.06 1.33±0.04
4 1.12±0.02 1.24±0.06 1.33±0.04
5 1.13±0.02 1.25±0.06 1.32±0.04
Mean± SE 1.12c
±0.02 1.23b
±0.01 1.32a
±0.02
Means bearing different superscripts in a row differ significantly (* P < 0.05)
DCP intake 3.191 g/kgW0.75 was the DCP requirement for maintenance of
lactating Murrah buffaloes and the DCP requirement for 6% FCM was 71.774 g
per kg. Whereas DCP requirement for maintenance reported 3.20, 3.47, 3.00
and 3.14 by Mudgal and Kumar (1978), Siviah and Mudgal (1978), Tiwari and
Patle (1983) and Paul et al (2002), respectively and these were comparable with
present findings.
4.4.11 CP intake at fortnight intervals and prediction of its requirement
for maintenance and 6% FCM of Murrah buffaloes
The CP intake (kg/d) of the lactating Murrah buffaloes at fortnight intervals
fed on different MP levels is presented in table 4.4.11. The relationship between
CP intake (g/kg W0.75) and 6%FCM (kg/kg W0.75) is shown in figure 4.12. The CP
intake (g/kg W0.75) was regressed linearly upon 6%FCM (kg/kg W0.75) to
determine the CP requirements for 6%FCM and maintenance.
The relation is depicted in form of graph in figure 4.12 and the regression
equation developed was as follows;
y = 116.05x + 5.0204 (R2 = 0.4789, P<0.01, n = 75)
Where,
Y = CP intake (g/kg W0.75) of buffaloes
x = 6% FCM (kg/kg W0.75)
Results & Discussion
Page | 118
Based on the prediction equation the CP intake 5.0204 g/ kg W0.75 which
was the CP requirements for maintenance of lactating Murrah buffaloes and the
CP requirement for 6% FCM of buffaloes was 116.05 g per kg 6% FCM.
Table 4.4.11: Crude protein intake (kg/d) in lactating Murrah buffaloes fed
on varying MP in the diets
Fortnight MP -10
MP0 MP
+10
1 1.71±0.05 1.81±0.10 1.95±0.04
2 1.75±0.03 1.85±0.11 1.94±0.07
3 1.79±0.04 1.87±0.09 2.00±0.06
4 1.76±0.03 1.87±0.10 2.00±0.06
5 1.77±0.03 1.88±0.09 1.98±0.06
Mean± SE 1.76c
±0.03 1.86b
±0.01 1.97a
±0.02
Means bearing different superscripts in a row differ significantly (* P < 0.05)
Fig 4.12: Relationship of CP intake (g/kg W0.75) with 6% FCM (kg/kg W0.75)
of Murrah buffaloes
y = 116.05x + 5.0204 R² = 0.4789
2
4
6
8
10
12
14
16
18
0.06 0.07 0.08 0.09 0.1
CP
I (g
/kg
W0.7
5)
6%FCM (kg/kg W0.75)
Results & Discussion
Page | 119
The CP requirement of the maintenance were found to be, 5.42, 2.81
3.43 and 4.87 (g/d) by Paul et al (2002), Pathak and Verma,(1993), Kearl
(1982) and ICAR, (2013), respectively. The values from Pathak and Verma
(1993) are very low because they were derived from non-producing animals. In
current study CP requirements for 1 kg 6% FCM milk was 116.05g which was in
the range of previous reported values viz. 124, 90.30, 110 and 108 g by ICAR,
(2013); Paul et al. (2002); Pathak and Verma, (1993) and Kearl (1982),
respectively.
4.4.12 Comparison of predicted daily energy and protein requirements of
lactating buffaloes with ICAR, 2013 feeding standards
The requirements of energy (ME) and protein (CP, MP or DCP) in
lactating buffaloes was revealed from the developed prediction equations based
on feeding trial. The data are provided in table 4.12
Table: 4.4.12 Comparison of predicted daily energy and protein
requirements of buffaloes with ICAR, 2013 feeding standards
ICAR, 2013 Values obtained in present study for
lactating
Values obtained in present study for
non- lactating buffaloes
Maintenance requirement, (g/kg W0.75)
ME* 0.55 0.53 (-3.6) 0.521
TDN 36.64 35.27(-3.7) 34.45(-6)
MP 2.65 2.57 (-3.0) 2.98(+12)
CP 4.87 5.02 (+3.0) 5.26(+8)
DCP NS 3.19 3.06
Requirements g per kg 6% FCM
ME* 6.61 6.63 --
TDN 440.00 438.51 --
MP 66 .00 66.78 (+1.1) --
CP 124 .00 116.05 (+6.4) --
DCP NS 71.77 --
*(MJ/ kg W0.75), NS: not stated in the feeding standards. Values in parenthesis are % variation from ICAR, (2013)
Results & Discussion
Page | 120
The ME (MJ/kg W0.75) and TDN (g//kg W0.75) requirements for 6% FCM/kg
in present study was 6.63 and 438.51 which was almost similar as
recommended by ICAR, (2013). The maintenance requirement of ME (MJ/ kg
W0.75) and TDN (g//kg W0.75) was 0.53 and 35.27 respectively in present study
which was only 3.6 percent lower than ICAR, (2013). The maintenance
requirement of MP was estimated to be 2.57 g/kg W0.75 whereas MP requirement
for milk production was 66.78 g/kg 6% FCM. The present value of MP
requirements for the maintenance was slightly lower than the ICAR, (2013)
recommendations. The MP requirement for 1 kg 6% FCM milk was marginally
higher than the ICAR, (2013).
The maintenance requirement of CP was estimated to be 5.02 g/kg W0.75
whereas CP requirement for 6 % FCM was 116.05 g/kg FCM. The CP
requirements of the maintenance and 6% FCM/kg in present study were 3.0 and
6.4 percentage higher than ICAR, (2013) respectively.
CHAPTER – 5
Summary and Conclusions
Page | 121
SUMMARY AND CONCLUSIONS
Buffalo is the preferred milch animal of the farmers in many regions of
India. She is major contributor (>51%) of the milk produced in Asia especially
India. If their nutrient needs are not met, they will not reach their optimum milk
production capacity. Productivity of buffalo is low and enhancing the productivity
of animals is a major concern which can be solved by developing proper feeding
systems for genetically improved animals. Dietary energy and protein are the
most limiting factor in milk production. Murrah is established as a top milk
producing buffalo breed so this breed was selected for current study. The
lactating animals must receive sufficient nutrients to supply the nutrient secreted
in their milk, and for maintenance. There is no separate feeding standard and
standards meant for cattle are applied for them. Thus the present study was
targeted to estimate the protein and energy requirement of lactating buffaloes
fed silage based rations, likely to emit less methane. Previously used DCP and
TDN systems have drawbacks since DCP system did not consider the use of
rumen degradable N by microbes and TDN was determined using crude fibre,
which suffered from analytical errors, as well as TDN did not account for the
urinary and gaseous losses in ruminants. The metabolizable protein (MP) and
metabolizable energy (ME) system overcome the above limitations and are more
precise. Thus in the present study requirement of energy and protein were
estimated in terms of ME and MP. In ruminants a significant part of energy is lost
through gaseous emission of methane which creates risk to environment as it is
a green house gas. So there is need of balanced and precise feeding to
buffaloes, which may also reduce methane emission.
The present study has been carried out in three phases; first; in vitro
study, second; in vivo methane trial and third; two separate lactation and
metabolic trials. During Phase-I, preparation of silage in lab and its evaluation in
terms of silage quality, chemical composition, in vitro rumen fermentation
parameters and estimation of utilizable crude protein (uCP), metabolizable
protein in feeds were undertaken. Phase-II included estimation of methane
emissions from dry buffaloes fed on oat silage or oat hay by SF6 technique. In
Summary & Conclusions
Page | 122
phase III, two separate experiments were conducted on lactating buffaloes to
estimate ME and MP requirements, respectively.
Maize and oat fodders were selected for preparation of silage in plastic
jar. The chopped fodder material was tightly packed in the plastic jar and
covered with cap and sealed the cover with paraffin wax to maintain anaerobic
condition. Silages after prserving for two month were analyzed for pH, colour,
texture and dry matter.
5.1 Chemical composition and organoleptic characteristics of maize
silage, oat silage and fodders before ensiling
The CP (%), TDN (%) and ME (MJ/kg DM) contents of maize fodder,
maize silage, oat fodder, oat silage and oat hay were 8.59, 8.86, 11.85, 11.97
and 9.20; 57.80, 56.78, 63.15,62.68 and 53.10; 8.89,8.70,9.88,9.80 and 8.01
respectively. Maize silage was greenish yellow in color while oat silage was
golden yellow in colour. Both silages were soft, non viscous in texture and had
slightly acidic, vinegar smell.
5.1.2 Fermentation characteristics of silages
The TVFA contents of maize and oat silages were 37.20 and 35.11
mM/100g DM, respectively which was non significantly different (P>0.05). The
propionate (mM/100g DM) was 4.48 and 3.78 in maize silage and oat silage,
respectively. The acetate content was 32.01, 30.77 and butyrate content 0.71,
and 0.49 mM/100g DM, respectively. There was variation in nitrogen (%DM) of
maize and oat silages. The average value of total nitrogen (%DM) content of
maize and oat silage was 1.43 and 1.92, respectively, which was significantly
(P<0.05) higher in oat silage.
5.1.3 In vitro total gas, methane production of maize, oat silages and
respective fodders
The In vitro organic matter digestibility (IVOMD) was significantly higher in
oat fodder and silage (80.53 and 88.07) than maize fodder and silage. The mean
values of in vitro methane production (g/ kg IVDMD) of maize fodder and its
silage; oat fodder and its silage were 38.47 and 35.24; 39.23 and 36.52,
respectively. It was higher (P<0.05) in oat fodder and lower in maize silage. The
methane production in silages were significantly lower (P<0.05) than respective
Summary & Conclusions
Page | 123
fodders. In vitro study revealed 8.40 and 6.91% lesser (P<0.05) methane
production (g/ kg IVDMD) in maize and oat silages compared to their respective
fodders
5.1.4 Estimation of utilizable crude protein (uCP), intestinal digestibility of
uCP and metabolizable protein
Among the analysed grains the uCP (%DM) content was highest in oat
(9.96) and lowest in pearl millet (5.21) while the MP content was 8.24, 8.11, 7.36
and 4.26 %DM in maize, barley, oat and pearl millet. Among the grains MP
content was lowest in pearl millet. Among the cakes, intestinal digestibility (%)
was lower side in DOMC (73.19) than the CSC and SBM. The MP contents were
27.88, 14.82 and 19.15%DM in DOMC, CSC and SBM respectively and differed
among each other significantly (P<0.05). The uCP and MP (%DM) in the wheat
bran and rice bran were similar. The MP content among the fodders ranged from
the 6.36-8.46 % DM.
5.2 Estimation of methane emissions from the dry buffaloes fed on oat
hay or silage
The dry Murrah buffaloes were fed on oat hay or oat silage solely and
CH4 production was measured using SF6 technique. There existed no difference
in the DM intake (kg and % BW) among the groups fed on oat hay or silage.
There was no significant difference observed in the DM, EE intake but NDF and
ADF intake was significantly (P<0.05) higher in oat hay group as compared to
the oat silage group.
The digestibility coefficients of all nutrients were similar between the
groups except that of CP. Crude protein digestibility was higher in oat silage
followed by oat hay, which followed similar trend as that of the CP intake. The
DM and CP digestibility coefficients in oat hay and oat silage fed groups were
68.87 to 69.09% and 58.28 to 60.59%, respectively.
5.2.1 Energy loss from dry buffaloes through methane emissions
Enteric CH4 emissions (L/d) was significantly higher (P<0.05) in oat hay
fed group (341.35) than oat silage (317.86) group. Methane loss as percentage
of DE and ME energy intake was 13.51,
16.41 and 11.61, 13.97 in oat hay and
oat silage groups respectively which is significantly (P<0.05) differed between
Summary & Conclusions
Page | 124
the group. The highest methane emission (g/kg DM, MJ/kg DM and g/kg NDF
intake) was in oat hay (24.36, 1.36 and 35.68) compared to oat silage group. The
overall methane production was significantly lower (P<0.05) in oat silage group
than oat hay fed group. Significantly high correlation coefficients were observed
between methane emissions and NDF intake (R2 = 0.61, p<0.05). So, it is
suggested that feeding of silage in ruminant can reduces methane production
(6.86%) compare to feeding of hay.
5.2.3 Nutrient requirements of non-lactating Murrah buffaloes
Based on the ME, TDN, CP, MP and DCP intake of the non-lactating
buffaloes fed on oat hay or silage and their body weight change (BWC),
following regression equations were developed to predict the requirements of
energy and protein from the respective BWC
Y1 = 32.651 X +521.27 (R2 = 0.867, P<0.01, n = 48) eqn.1
Y2 = 2.1581X + 34.455 (R2 = 0.867, P<0.01, n = 48) eqn.2
Y3 = 0.3614X + 5.2642 (R2 = 0.8721, P<0.01, n = 24) eqn.3
Y4 = 0.2106X + 3.068 (R2 = 0.8571, P<0.01, n = 24) eqn.4
Y5 = 0.185X + 2.9866 (R2 = 0.8571, P<0.01, n = 24) eqn.5
Where,
Y1 = ME intake (kJ/kg W0.75),
Y2 = TDN intake (g/kg W0.75)
Y3 = CP intake (g/kg W0.75)
Y4 = DCP intake (g/kg W0.75)
Y5 = MP intake (g/kg W0.75) and
X = BWC (g/kg W0.75)
5.2.4. Energy and protein requirements for maintenance in non lactating
Murrah buffaloes
Based on equation 1, ME intakes at zero BWC was 521.27 kJ/kg W0.75
which would be the ME requirements for maintenance of non-lactating buffaloes
per day. TDN requirement was estimated to be 34.455 g/kg W0.75 for
maintenance. Similarly, the maintenance requirement of CP, DCP and MP for
non-lactating buffaloes was as revealed from equation 3, 4 and 5 were 5.2642 g/
kg W0.75, 3.068 g/kgW0.75 and 2.9866 g/kg W0.75, respectively. The present
Summary & Conclusions
Page | 125
requirement of MEm of Murrah buffaloes was comparable with the ICAR, (2013)
and Paul et al. (2002) reported values.
5.2.5. Energy and protein requirements for body weight change in Murrah
buffaloes
The requirement for body weight change was derived from the prediction
equations. Equation 1 suggests a requirement of 32.651 kJ of ME for g BWC/ kg
W0.75/day. Similarly equation 2, 3, 4 and 5 suggest requirement of 2.1581 g TDN,
0.3614g CP, 0.2106g DCP and 0.185g MP for g BWC/ kg W0.75/day of Murrah
buffaloes.
Phase III
Estimation of metabolizable energy requirements of Murrah buffaloes fed
on silage based diet
5.3.1 Effect of varying metabolizable energy level in diet on body weight
and nutrient intake of lactating buffaloes
After 75 days of feeding the final body wt. were 569.39, 571.24 and
570.32 in ME -10, ME0 and ME +10 groups. A non significant difference was
observed between the mean body weights of ME -10, ME0 and ME +10,
respectively. The average DMI in 75 days was 14.72, 14.81 and 14.74 kg/d in
ME -10, ME0 and ME +10 groups respectively. The average CPI was 1.84, 1.83
and 1.85 kg/d in ME -10, ME0 and ME +10 groups respectively which were did not
differ among the groups. The overall mean fortnightly metabolizable energy
intake (MJ/d) were 129.22, 135.87 and 143.13 in ME -10, ME0 and ME +10
respectively, which differ significantly (P<0.05) among the groups.
5.3.2 Nutrient digestibility and nitrogen balance in lactating buffaloes fed
on varying ME in diets
The digestibility coefficients of OM, CP, EE, NDF and ADF were 68.93,
60.83, 70.50, 57.51 and 44.14 in ME -10; 69.57, 61.13, 72.64, 58.01 and 44.76 in
ME0; 70.11, 60.76, 73.29, 58.73 and 45.10 in ME +10 respectively, which did not
differ significantly among the groups. N excretion in faeces and nitrogen
excretion via urine and N outgo in milk was not affected by the different level of
ME in diet. Nitrogen balance (g/d) was comparable among the groups.
Summary & Conclusions
Page | 126
5.3.3 Effect of varying ME in diets on milk production, composition and %
feed efficiency in lactating Murrah buffaloes
The overall mean milk yield was 8.48, 8.84, 8.74 kg/d in ME -10 , ME0 and
ME +10 respectively, decreasing trends in milk production with advancing
lactation observed among all the groups but significantly low milk production
observed in the ME -10 as comparison to ME0 and ME +10. The overall mean 6%
FCM (kg/day) were 9.64, 9.99 and 9.88 in ME -10, ME0 and ME +10 respectively
which was significantly (P<0.05) lower in ME -10 as compare to the ME0 and ME
+10. The milk composition i.e. protein, fat, lactose, total solid and SNF were did
not differed among the groups.
5.3.4 Nutrient requirements of lactating Murrah buffaloes
5.3.5 Energy requirements of lactating Murrah buffaloes
Based on the ME and TDN intake of lactating buffaloes fed on varying
metabolizable energy in diets and 6% FCM, following regression equations were
developed to predict the requirements of energy from the respective 6% FCM.
Y1 = 6634.2 X +533.65 (R2 = 0.4898, P<0.01, n = 50) eqn.1
Y2 = 438.51X + 35.27 (R2 = 0.4916, P<0.01, n = 50) eqn.2
Where,
Y1 = ME intake (kJ/kg W0.75),
Y2 = TDN intake (g/kg W0.75)
X = 6% FCM (g/kg W0.75)
The TDN requirement of lactating buffaloes for maintenance was 35.27 g/
kg W0.75 per day when the body weight change was zero and TDN requirement
for 6%FCM in buffaloes was 438.51 g per kg 6%FCM. The present energy
requirements for maintenance (g TDN/kg W0.75) of lactating buffaloes was
comparable with ICAR, 2013 and Paul et al., (2002). The ME intake 533.65 kJ/kg
W0.75 which was the ME requirements for maintenance of lactating buffaloes per
day and the ME requirement for per kg 6% FCM in was 6634.2 kJ.
Summary & Conclusions
Page | 127
5.4 Estimation of metabolizable protein requirements of Murrah
buffaloes fed on silage based diets
5.4.1 Effect of varying metabolizable Protein level in diet on body weight
and nutrient intake of lactating buffaloes
Overall final body wt. was 545.92, 548.64 and 547.92 in MP 90, MP100 and
MP110 groups respectively. A non significant difference was observed between
the mean body weights of MP -10, MP0 and MP +10 groups, respectively. Average
DMI was 13.24, 13.29 and 13.26 kg/d in MP -10, MP0 and MP +10 groups,
respectively. The MEI (MJ/d) was 134.70, 135.74 and 135.92 in MP -10, MP0 and
MP +10, respectively which did not differed significantly among the groups. CP
and MP intake (kg/d) were 1.12, 1.23, 1.32 and 0.88, 0.94, 0.99 in MP -10, MP0
and MP +10 groups, respectively which differ significantly (P<0.05) among the
groups.
5.4.2 Nutrient digestibility and nitrogen balance in lactating buffaloes fed
on varying MP in diets
The digestibility coefficients of DM were 62.36, 62.62 and 63.57 per cent
in MP -10, MP0 and MP+10 groups respectively which did not differ significantly
among the groups. Similar trend was observed in the digestibility of other
nutrients except CP in which was significantly higher in MP0 (66.23) and MP+10
(66.65) than that of MP -10 (63.61). N intake (g/d) was 284.43, 298.11 and 309.42
in MP -10, MP0 and MP+10, respectively which differed significantly (P<0.05)
among the groups. N excretion in faeces and N outgo in milk was not affected
by the different level of MP in the diet but the urinary excretion of nitrogen
increased with the increase in N intake in the diet. Urinary nitrogen excretion
(g/d) was 94.29, 106.27 and 114.70 in MP -10, MP0 and MP+10, respectively
which differ significantly (P<0.05) among the groups. While the overall N balance
(g/d) were comparable among groups i.e. 23.18, 24.31 and 24.48 in MP -10, MP0
and MP+10, respectively.
5.4.3 Effect of varying MP in diets on milk production, composition and %
feed efficiency in lactating Murrah buffaloes
Overall average milk production was 7.30, 7.28 and 7.31 kg/d in MP -10,
MP0 and MP +10 groups respectively which was non-significant (P>0.05) among
Summary & Conclusions
Page | 128
the groups. Average 6%FCM was 8.98, 8.98 and 9.05 kg/d in MP -10, MP0 and
MP +10 groups respectively. Percent feed conversion efficiency of milk
production was 55.13, 54.79 and 55.13 in MP -10, MP0 and MP +10 respectively
which was comparable among the groups. Similarly no changes were observed
on milk composition of buffaloes fed on varying MP in the diets.
5.4.4 Effect of dietary protein levels on urinary purine derivatives,
creatinine and microbial N production in lactating Murrah buffaloes
There was no significant effect of varying protein levels on allantoin, uric
acid, creatinine, total purine derivatives and microbial N production. Allantoin
constituted the principal PD in the urine. Allantoin and uric acid ranged from 4.51
to 4.62 mmol/l and 2.32 to 3.49 mmol/l, respectively. Total PD varied from
203.38 to 214.06 mmol/day. The microbial N productions (g/d) were 135.47,
141.83 and 142.94 in MP -10, MP0 and MP +10 respectively.
5.4.5 Protein requirements of lactating Murrah buffaloes
Based on the MP, DCP and CP intake of lactating buffaloes fed on
varying metabolizable protein in diets and 6% FCM, following regression
equations were developed to predict the requirements of energy from the
respective 6% FCM.
Y1 = 66.781X +2.5691 (R2 = 0.4967, P<0.01, n = 75) eqn.1
Y2 = 71.774X + 3.191 (R2 = 0.478, P<0.01, n = 75) eqn.2
Y3 = 116.05X + 5.0209 (R2 = 0.4789, P<0.01, n = 75) eqn.3
Where,
Y1 = MP intake (g/kg W0.75),
Y2 = DCP intake (g/kg W0.75)
Y3 = CP intake (g/kg W0.75)
X = 6% FCM (g/kg W0.75)
The MP requirement of lactating buffaloes for maintenance was 2.5691 g/
kg W0.75 per day and MP requirement for 6%FCM in buffaloes was 66.781 g per
kg 6%FCM. The present MP requirement for maintenance (g/kg W0.75) of
lactating buffaloes was comparable with ICAR, (2013).DCP intake 3.191
g/kgW0.75 was the DCP requirement for maintenance of lactating Murrah
buffaloes and the DCP requirement for 6% FCM was 71.774 g per kg.
Summary & Conclusions
Page | 129
Based on the prediction equation the CP intake 5.0204 g/ kg W0.75 which
was the CP requirements for maintenance of lactating Murrah buffaloes and the
CP requirement for 6% FCM of buffaloes was 116.05 g per kg 6% FCM.
5.5. Conclusions
The present study was aimed to find out the energy (ME, TDN) and
protein (CP or MP) requirements of lactating buffaloes fed on silage based diet
and compare methane emissions from buffaloes fed on hay or silage based diet.
The following conclusions can be drawn from the present studies.
1. In vitro study revealed 8.40 and 6.91% lesser (P<0.05) methane
production (g/ kg IVDMD) in maize and oat silages compared to their
respective fodders.
2. Among the grains MP (% DM) was comparable in maize, oat and barley.
Whereas, among cakes DOMC had lowest (19.15%) and SBM had
highest MP (27.88%). Among forages, MP (% DM) was highest in oat
fodder (8.46) and lowest in oat hay (6.36).
3. Enteric CH4 emissions (L/d) was significantly higher (P<0.05) in oat hay
fed group (341.35) than oat silage (317.86) group.
4. Methane loss as percentage of ME intake was higher (P<0.05) in oat hay
group (16.41) than oat silage group (13.97).
5. Total CH4 production was depressed by 6.86% in dry buffaloes fed on oat
silage instead of oat hay.
6. Feeding of lactating Murrah buffaloes on 10% less ME than ICAR, (2013)
recommendation resulted into the decreased milk production and feed
efficiency while feeding 10% ME higher had no benefits for production
performance.
7. Milk production and composition were not affected by varying MP levels
i.e. 10% more or less than ICAR, (2013) recommendation in the diet.
However, better nitrogen balance was observed in buffaloes fed MP as
per ICAR, 2013.
8. The ME and TDN requirements for maintenance in dry buffaloes were
521.27 kJ, 34.455 g per kg W0.75, respectively.
9. The ME and TDN requirements for body weight change (BWC) in dry
buffaloes was 32.651kJ and 2.158 g for g BWC per kg W0.75, respectively.
Summary & Conclusions
Page | 130
10. The ME and TDN requirement for maintenance during lactation were 533
KJ and 35.27g per kg BW0.75, respectively.
11. The ME and TDN requirement for milk production were 6.6 MJ and
438.51g per kg 6% FCM, respectively.
12. CP, DCP and MP requirements for maintenance in dry buffaloes were
5.2642 g/ kg W0.75, 3.068 g/kgW0.75 and 2.9866 g/kg W0.75, respectively.
13. CP, DCP and MP requirements for body weight change in dry buffaloes
were 0.3614 g, 0.2106 g and 0.185 g for g BWC/ kg W0.75/day,
respectively.
14. CP, DCP and MP requirements for maintenance in lactating buffaloes
were 5.02 g, 3.19 g and 2.56 g per kg W0.75, respectively.
15. CP, DCP and MP requirements for milk production in buffaloes were
116.05 g, 71.77 g and 66.78 g per kg 6% FCM, respectively.
The outcome / findings of project can be applied on the farm by feeding
oat silage or maize silage based rations, and thereby resulting a decrease in
about 7% CH4 emission. The data generated can be used to further refine
existing feeding standards, and develop newer standards for buffaloes in terms
of metabolizable energy and metabolizable protein. The study also revealed that
buffaloes use feed nutrient very efficiently.
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Page | i
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