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


94


silage

4.2.6 CP intake at fortnight intervals and prediction of its



silage

96

4.2.7 DCP intake at fortnight intervals and prediction of its



silage

97

4.2.8 MP intake at fortnight intervals and prediction of its



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


buffaloes

107

4.4. Phase III: Estimation of metabolizable protein

requirements of Murrah buffaloes fed on silage

based diets

108


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


buffaloes

115

4.4.10 DCP intake at fortnight intervals and prediction of its


buffaloes

116

4.4.11 CP intake at fortnight intervals and prediction of its


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


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


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


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


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


107


metabolizable protein level concentrates fed to lactating

buffaloes

109*

4.4.2 Fortnightly body weight of lactating Murrah buffaloes fed


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


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


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


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


6 | P a g e

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


7 | P a g e

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


8 | P a g e

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


9 | P a g e

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


10 | P a g e

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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11 | P a g e

(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



























12 | P a g e

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.


13 | P a g e

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


14 | P a g e

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.


15 | P a g e

(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


16 | P a g e

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.


17 | P a g e

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


18 | P a g e

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


19 | P a g e

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


20 | P a g e

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


21 | P a g e

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.


22 | P a g e

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


23 | P a g e

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.


24 | P a g e

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)


25 | P a g e

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.


26 | P a g e

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


27 | P a g e

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.


28 | P a g e

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,


29 | P a g e

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


31 | P a g e

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.


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


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


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


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


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


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


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


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


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


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,


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,


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


35.0 g

Macro mineral solution

Na

c

2HPO4

KH

anhydrous 5.70 g

2PO4

MgSO

anhydrous 6.20 g

4.7H2


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


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

Page | 66

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


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


Page | 79

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


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)


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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

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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.


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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)


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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.


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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



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


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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


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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



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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



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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

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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.


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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)


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


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


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)


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



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


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


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


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).


Page | 96

4.2.6 CP intake at fortnight intervals and prediction of its requirement for



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


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


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



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



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


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


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)


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 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


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.


Page | 99

4.2.8 MP intake at fortnight intervals and prediction of its requirement for



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


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


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 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



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)


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


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


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


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.


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


Table 4.3.5 : Productive performances and feed efficiency for milk


energy level


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


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


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


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


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


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


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


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


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


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


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)


Page | 108



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.


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


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


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


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


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


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


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


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


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


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


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


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


Page | 116



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


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.



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)


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


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.



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)


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


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)


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)


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


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


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


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.


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.


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


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.


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.


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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doctor of philosophy in animal nutrition by · his constant oasis of ideas and passions...

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