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RESPONSE OF RUMEN PROTECTED METHIONINE AND LYSINE SUPPLEMENTATION ON LACTATION AND REPRODUCTIVE PERFORMANCE IN PERIPARTURIENT DAIRY COWS THESIS SUBMITTED TO THE NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL (DEEMED UNIVERSITY) IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN ANIMAL NUTRITION BY AMRUTKAR SUHAS ASHOKRAO (M.V.Sc. Animal Nutrition) DIVISION OF DAIRY CATTLE NUTRITION NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY) KARNAL-132 001 (HARYANA), INDIA 2012 Regn. No. 1080906 Division of Dairy Cattle Nutrition National Dairy Research Institute (Deemed University) Karnal-132001 (Haryana), INDIA _____________________________________________________________________ Dr. S. S. THAKUR Principal Scientist CERTIFICATE This is to certify that the thesis entitled, “RESPONSE OF RUMEN PROTECTED METHIONINE AND LYSINE SUPPLEMENTATION ON LACTATION AND REPRODUCTIVE PERFORMANCE IN PERIPARTURIENT DAIRY COWS" submitted by Amrutkar Suhas Ashokrao towards the partial fulfilment of the award of the degree of Doctor of Philosophy in Animal Nutrition of the National Dairy Research Institute (Deemed University), Karnal (Haryana), India, is a bonafide research work carried out by him under my supervision, and no part of the thesis has been submitted for any other degree or diploma. Dated: 23rd October, 2012 (S. S. Thakur) Major Advisor & Chairman DEDICATED TO MY BELOVED FRIEND Late Dr. Nilesh Wankhede ACKNOWLEDGEMENT _______ ____________ __________________ _______________ _ First and foremost, I bow my head to thank the Almighty for having bestowed me with all what I needed if not what I wanted and showing me the right path at life’s cross roads. I am extremely grateful and profoundly obliged to my major advisor, Dr. S. S. Thakur ,Principal Scientist, Dairy Cattle Nutrition Division, National Dairy Research Institute, Karnal, for his sagacious guidance, invaluable suggestions, constant encouragement and constructive criticism during the entire course of this study, which enabled me to bring the problem in hand to a successful end. My indebtedness is reserved for his goodwill and patience during the entire period of my research work. I would like to express my heartfelt thanks to Dr. (Mrs) Neelam Kewalramani, Principal Scientist, DCN Division and Dr. A. K. Tyagi, Principal Scientist, DCN Division, for their expertise, technical guidance, emphatic help, and pertinent suggestions in planning and critical amendments made during the entire microbiological study of this work. My profound admiration and sincere gratefulness is extended to Dr. Mohinder Singh, Principal Scientist, DCP Division, Dr. S. K. Sirohi, Principal Scientist, DCN Division and Dr. Shivprasad, Principal Scientist, LPM Division for their encouragement and precious, innovative and remedial suggestions during the course of this study. I sincerely pay my regards to Dr. S.S. Kundu, Head, DCN Division for his valuable suggestions, support and providing mandatory facilities during the entire study. I am falling short of words to express my thanks to Dr. T. K. Walli and Dr S. N. Rai, Retd. Head and Principal Scientist, DCN Division and Dr. Chander Datt, Senior Scientist, DCN Division whose cooperation, suggestions, and moral support enabled me in many ways to complete this research work. I am deeply gratified to Dr. A. K. Srivastava, Director, NDRI Karnal for providing requisite facilities and comfortable stay in the NDRI campus. I wish to extend my sincere regards to Dr. G. R. Patil, Joint Director (Academics) and Dr. S. L. Goswami, Joint Director (Research), NDRI Karnal for their valuable inspirations and suggestions for this work. Neither my language nor space is sufficient to express my heartfelt thanks to my wife Dr. Manjushree Amrutkar for giving me moral support and encouragement during the course of this study. The homely atmosphere and cheerful company which I have enjoyed with my friends namely Shivaji, Dr. Bhupendra, Dr. Deepak Sinha, Dr. Prokash Bala, Dr. Sachin, Dr Satish and my seniors Dr. Prokash Bala and Dr Rijusmita Sarma and my ___ __________________ _ _ ____________________________ ABSTRACT _______ ____ __________________________ _______________ ______ This study was conducted to investigate the effect of supplementing rumen protected methionine (RPM) plus lysine (RPL) on milk production and its composition, nutrient utilization, plasma metabolites and reproductive performance in peri parturient crossbred cows (Bos taurus X Bos indicus). Eighteen crossbred cows were selected and divided into two groups (9 each) on the basis of most probable production ability (MPPA) and lactation number. Animals in group 1 (MPPA - 4119 kg) were fed chopped wheat straw, chaffed green maize fodder and concentrate mixture as per requirements (NRC, 2001) whereas, animals in supplemented group 2 (MPPA 4120 kg) were fed same ration as group 1 plus 5 gm RPM and 20 gm RPL, pre partum and 7 gm RPM and 60 gm RPL, postpartum, respectively. The experimental period commenced 40 days before expected date of parturition to 120 days post parturition. During the pre-parturient period, rumen protected methionine and lysine supplemention exhibited higher gross apparent changes in body condition score. Intakes of CP, RDP, RUP, MP, TDN, ME and NE L were similar while duodenal supply of methionine and lysine was increased on supplementation of rumen protected methionine and lysine in pre parturient cows . Plasma triglycerides and VLDL concentration were increased (P<0.05) in prepartum cows on supplementating rumen protected methionine plus lysine. Plasma glucose, phosphatidylcholine, NEFA, vitamin E, cholesterol and BUN were not affected. Plasma amino acids profile of the pre partum cows was not affected on supplementing rumen protected methionine and lysine while plasma methionine, cysteine and lysine concentrations tended to increase on supplementation. During post partum period, fortnightly changes in body weight revealed that there was overall a net loss of 18.59 kg in group 1, whereas there was overall gain of 5.79 kg in group 2. The DM, CP, MP, TDN, ME and NE L intake kg per day as well as per cent body weight was higher in group 2 than that of group 1 lactating cows. Milk yield during supplementation period in group 2 was 17.69 kg/d, which was 11.33 per cent higher (P<0.01) than that of group 1 (15.89 kg/d). There was no effect of supplementation on milk protein, solid not fat (SNF), milk choline and MUN contents in both the groups, whereas milk fat content was higher (P<0.01) by 2.18 per cent in group 2 (4.22%) than that of group 1 (4.13%). The efficiency of utilization of DM, CP, MP, TDN, ME, NE L per kg milk or FCM yield was better in group 2 than that of group 1. The saturated fatty acids, unsaturated fatty acids, mono and poly unsaturated fatty acids in milk fat were similar in group 1 and group 2. Plasma glucose (55.61 and 55.07 mg/dl), phosphatidylcholine (138.57 and 140.98 µg/ml) NEFA (106.80 and 105.77 mg/L), vitamin E (1.01 and 0.90 µg/ ml), cholesterol (193.16 and 198.31 mg/dL) and BUN (18.13 and 18.01 mg/L) concentrations were similar in both the groups. Plasma triglycerides and VLDL concentrations were 13.40 and 16.22 mg/dL and 2.68 and 3.24 mg/dL in group 1 and 2, respectively which were higher (P<0.01) in group 2 than in group 1. Plasma aspartate, glycine, alanine, valine (P<0.05), methionine, cysteine, and lysine (P<0.01) concentrations were increased in cows fed ration supplemented with RPM plus RPL. However, RPM plus RPL supplementation lowered (P<0.05) plasma isoleucine concentration. The plasma prolactin (79.68 and 80.39 ng/L) and growth hormone (6.63 and 6.63 ng/L) concentration were similar in both the groups. The body weight of calves at the time of birth was similar in both the groups. Two cases of premature births were reported in group 1, however, one case was observed in group 2. Four cases of retention of fetal membranes (RFM) were observed in group 1 while only one case was observed in group 2. Higher incidence of metritis in group 1 (3 cases) were recorded whereas only one case was observed in group 2. The time required for commencement of cyclicity was similar (P>0.01) in both the groups. The service period was shorter (P<0.01) by 11.8 days in group 2 than that of group 1, indicating that lesser time was required for the animals in group 2 for conception. AI required for conception was similar in both the groups. The conception rate during the experimental period of 120 days was 55.55 % in group 1 and 66.66 % in group 2. Supplementtion of rumen protected methionine and lysine to high yielding lactating cows during periparturient period was found to be cost effective. The results of the present study showed that the supplementation of rumen protected methionine and lysine improved reproductive performance and lead to efficient use of nutrients at early stage of lactation, which in turn manifested in higher milk yield. In addition, milk fat content was increased, resulting in improvement in the commercial value of milk. Keywords: Crossbred Cows, milk, protected methionine, protected lysine, milk fat lkjka'k bl vuqla/kku dk;Z esa ladfjr nq/kk: xk;ksa dks :esu jf{kr esfFkvksfuu ¼vkj-ih-,e-½ ,oa ykbflu ¼vkj-ih,y-½ laiwjd ds :i esa f[kykuss ls muds nqX/k mRiknu] nqX/k lajpuk] iks"kdrRo mi;ksx] Iyk>ek mikip; rFkk iqujksRiknu laEc/kh {kerk ij izHkko dh tkWap dh xbZA eksLV izkscscy izksMD'ku ,fcfyVh ,oa nqX/kiku la[;k ds vk/kkj ij 18 ladjhr xk;ksa dks nks oxksZ esa ckWaVk x;kA oxZ&1 esa] xk;ksa dks xsgwWa dk Hkwlk] gjk pkjk ,oa nkuk feJ.k mudh vko';drkuqlkj ¼,u-vkj-lh-]2001½ f[kyk;k x;kA tcfd oxZ&2 esa oxZ&1 esa fn, x;s vkgkj ds vfrfjDr xk;ksa dks :esu jf{kr esfFkvksfuu ,oa ykbZflu ¼5 xzke vkj-ih-,e- ,oa 20 xzke vkj-ih-,y- dze'k% C;kus ls iwoZ vkSj 7 xzke vkj-ih-,e- vkSj 60 xzke vkj-ih-,y- C;kus ds mijkUr½ f[kyk;k x;kA ;g v/;;u C;kus ls 40 fnu iwoZ lsa C;kus ds 120 fnu mijkUr rd pykA C;kus ls iwoZ oxZ&2 esa 'kjhj voLFkk vad oxZ&1 ls vis{kkd`r vf/kd ik;k x;kA lh-ih-] vkj-Mh-ih-] vkj-;wih-] ,e-ih-] Vh-Mh-,u-] ,e-bZ- ,oa ,u-bZ-,y- dk vUrxzg.k nksuksa oxksaZ dh xk;ksa esa leku ik;k x;k rFkk vkj-ih-,e,oa vkj-ih-,y- xk;ksa dks f[kykus ls fMoksMhuy esfFkvksfuu vkSj ykbZflu dh vkiwfrZ oxZ&2 esa c<+ xbZA C;kus ls iwoZ Iyk>ek VªkbZXyhljkbZMl~ ,oa oh-,y-Mh-,y- dh ek=k Hkh oxZ&2 dh xk;ksa esas vkj-ih-,e- ,oa vkj-ih-,y- f[kykus ls c<+ xbZ] tcfd Iyk>ek Xyqdkst] QkWLQVhM~kbZy&dksyhu] usQk] ohVkfeu&bZ] dksyLVªksy ,oa Iyk>ek ;wfj;k dh lkUnzrk nksuksa oxksZs esa leku ik;h xbZA C;kus ls iwoZ Iyk>ek vehuks vEy dh :ijs[kk ij nksuksa oxksZ esa dksbZ izHkko ugha iM+k ysfdu Iyk>ek esfFkvksfuu] flLVhu ,oa ykbZflu dh lkUnzrk dh izo`rh oxZ&2 esa oxZ&1 ls vis{kkd`r T;knk ns[kh xbZA C;kus ds 120 fnu ckn rd oxZ&1 dh xk;ksa esa 'kjhj Hkkj 18-59 fd-xzk- de gqvk tcfd oxZ&2 esa 5-79 fdxzk- dh o`f) gqbZ 'kq"d inkFkZ] lh-ih-] ,e-ih-] Vh-Mh-,u-] ,e-bZ- ,oa ,u-bZ-,y- dk vUrxzg.k izfrfnu rFkk izfr 100 fd-xzk- 'kjhj Hkkj ds vk/kkj ij oxZ&2 esa oxZ&1 ls vf/kd vkadk x;kA vkj-ih-,e ,oa vkj-ih-,y- f[kykus ls vkSlr nw/k mRiknu oxZ&2 ¼17-69 fd-xzk-½ dh xk;ksa esa 11-33 izfr'kr oxZ&1 ¼15-89 fd-xzk-½ dh vis{kkd`r vf/kd ¼P<0.01½ ik;k x;kA nw/k izksVhu] yWDVkst] ,l-,u-,Q] nw/k dksyhu ,oa nwX/k ;wfj;k dh ek=k nksuksa oxkZsa esa leku jgh ysfdu oxZ&2 esa nw/k olk dk izfr'kr oxZ&1 dh rqyuk esa 2-18 izfr'kr T;knk jgkA nw/k mRiknu ,oa ,Q-lh-,e- mRiknu ds fy;s 'kq"d inkFkZ] lh-ih-] ,e-ih-] Vh-Mh-,u-] ,e-bZ- ,oa ,u-bZ-,ydh mi;ksx {kerk oxZ&2 esa csgrj ¼P<0.01½ FkhA nksuksa oxksZa esa lar`Ir olk vEy] vlar`Ir olk vEy] eksuks vlar`Ir olk vEy ,oa ikWyh vlar`Ir olk vEy dh nw/k esa lkUnzrk leku ik;h xbZA C;kus ds mijkUr nksuksa oxksaZ esa Iyk>ek Xyqdkst] QkWLQVhMkby dksyhu] usQk] foVkfeu&bZ] dkWyLVªksy ,oa jDr ;wfj;k dh lkUnzrk leku ik;h xbZ tcfd Iyk>ek VªkbZXyhljkbZMl vkSj oh-,y-Mh-,y- dh lkUnzrk oxZ&2 esa oxZ&1 ls vf/kd ¼P<0.01½ ik;h xbZA oxZ&2 esa Iyk>ek ,WlikjVsV] XykbZflu] ,yWfuu] oWyhu ¼P<0.05½] esfFkvksfuu] flLVhu ,oa ykbZflu ¼P<0.01½ dh lkUnzrk oxZ&1 dh vis{kkd`r vf/kd ns[kh xbZ tcfd Iyk>ek vkbZlksyqlhu dh lkUnzrk oxZ&2 esa oxZ&1 ls de ¼P<0.05½ gqbZA Iyk>ek izksysDVhu ,oa xzksFk gkWjeksu dh lkUnzrk nksuksa oxksZa esa leku ik;h xbZA nkuksa oxksZa dh xk;ksa ls txUesa cNM+ks dk 'kjhj Hkkj nksuksa oxksZa esa leku ik;k x;kA dkyiwoZ tUe dh la[;k oxZ&1 esa oxZ&2 ls vf/kd ns[kh xbZA tsj u fxjus ,oa eSVªbZfVl dh n'kk oxZ&1 dh xk;ksa esa vf/kd ns[kh xbZA nksuksa oxkZsa esa C;kus ds ckn en pdz esa vkus ds fy, leku le; yxk ysfdu xHkZ/kkj.k ds fy, oxZ&2 esa 11-8 fnu oxZ&1 ls de yxsA nksuksa oxksZs esa xHkZ/kkj.kk ds fy, d`f=e 'kqdzlspu leku jgkA xHkZ/kkj.kk dh nj oxZ&1 esa 55-55 izfr'kr rFkk oxZ&2 esa 66-66 izfr'kr jghA tc :esu jf{kr esfFkvksfuu ,oa ykbZflu nq?kk: xk;ksa dks f[kykus dk vFkZ'kkL=h; vkadyu fd;k x;k rks Qk;ns ean ik;k x;kA bl v/;;u ls ;g fu"d"kZ fudyrk gS fd vf/kd nw/k nsus okyh xk;ksa dks :esu jf{kr esfFkvksfuu ,oa ykbflu laiwjd ds :i esa f[kykus ls mudk iqujksRiknu rFkk iks"kdrRoksa dh mi;ksx {kerk csgrj gksus ls nqX/k mRiknu c<+kA blds vfrfjDr] nw/k olk dk izfr'kr Hkh c<+k] tks vkSn;ksfxd n`f"V ls Qk;ns ean gSA Contents Chapter Title Page No 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 5 2.1 Sources contributing to metabolizable protein in ruminants 6 2.1.1 Endogenous crude protein 6 2.1.2 Microbial crude protein 7 2.1.3 Rumen-undegradable crude protein 9 2.2 Comparative Amino acid requirements in different species 11 2.3 Rumen Protected Proteins in Ruminant Nutrition 13 2.3.1 Degradation of Proteins in the Rumen 13 2.3.2 Protection of Protein from Ruminal Degradation 14 2.3.2.1 Heat Treatment 14 2.3.2.2 Formaldehyde Treatment 15 2.3.2.3 The Coating of Protein Source Particles with Insoluble 16 Substances 2.3.3 Advantages of Rumen Protection 17 2.4 Limiting amino acids 18 2.5 Amino acid supplementation 19 2.6 Rumen protected amino acids 20 2.6.1 Liquid sources of hydroxy analogs (chemically modified 21 molecules) 2.6.2 Surface coating or matrices of saturated fatty acids and 22 minerals 2.6.3 Surface coating with a fatty acid or pH-sensitive polymer mixture 23 2.7 Responses to rumen protected methionine and Lysine dietary 23 supplementation 2.7.1 Effect on Milk Yield and Milk component yield 23 2.7.2 Effect on plasma amino acids 25 2.7.3 Effect on blood metabolites 26 2.8 Reproductive efficiency of animals fed on protected protein 28 and rumen protected amino acids 3 MATERIALS AND METHODS 33 3.1 Feed and fodder analysis 33 3.1.1 Proximate principles and cell wall constituents 33 3.1.2 Amino acids analysis of feed samples using HPLC 33 3.1.3 Estimation of tryptophan 36 3.2 Rumen protected methionine (RPM) and rumen protected 36 lysine (RPL) product evaluation 3.2.1 Methionine content in RPM 37 3.2.2 Lysine content in RPL 37 3.3 In vitro determination of microbial protein yield 38 3.3.1 Collection of rumen liquor 38 3.3.2 Preparation of substrates in the form of total mixed rations 38 (TMR’s) 3.3.3 In vitro determination of microbial N using nitrogen balance 39 technique 3.4 Separation of rumen bacteria from rumen liquor 42 3.5 Separation of the protozoa from rumen contents 42 3.6 In vitro estimation of RUP intestinal digestibility 42 3.7 Estimation of RDP and RUP of different feed stuffs 43 3.8 Estimation of rumen escape potential of commercial RPM and 44 RPL product 3.9 Location of experiment 45 3.10 Selection and distribution of animals 45 3.11 Estimation of most probable production ability (MPPA) 45 3.12 Feeding, housing and management of experimental animals 47 3.12.1 Green maize fodder 47 3.12.2 Concentrate mixture 47 3.12.3 Housing and management of animals 47 3.12.4 Watering of animals 48 3.13 Observations recorded during pre partum period 48 3.13.1 Body weight 48 3.13.2 Body Condition Score 48 3.13.3 Feed intake 49 3.14 Analysis of blood samples 49 3.14.1 Blood collection 49 3.14.2 Plasma amino acid analysis 49 3.14.2.1 Preparation of sample 49 3.14.3 Plasma choline estimation 50 3.14.4 Plasma glucose estimation 54 3.14.5 Non esterified fatty acids (NEFA) estimation 55 3.14.6 Triglycerides estimation 56 3.14.7 VLDL estimation 56 3.14.8 Estimation of plasma urea 56 3.14.9 Estimation of plasma cholesterol 58 3.14.10 Estimation of plasma vitamins E 58 3.14.11 Plasma growth hormone estimation 59 3.14.12 Plasma prolactin estimation 61 3.15 Reproduction study 64 3.15.1 Calf weight at the time of birth 64 3.15.2 Calf mortality within one month of parturition 64 3.15.3 Occurrence of reproductive disorders 64 3.16 Lactation Study 65 3.16.1 Housing and management 65 3.16.2 Milking of animals 65 3.16.3 Body weight and body condition score 65 3.16.4 Feed intake 65 3.16.5 Milk yield 66 3.16.6 Calculation of fat corrected milk (FCM) 66 3.16.7 Calculation of energy corrected milk (ECM) 66 3.17 Milk Composition 66 3.17.1 Fatty acid analysis of milk 66 3.17.2 Estimation of milk urea 69 3.18 Analysis of blood parameters 70 3.19 Statistical Analysis 70 4 RESULTS AND DISCUSSION 71 4.1 Effect of feeding rumen protected methionine and lysine 71 during pre partum period 4.1.1 Degree of Protection of Rumen Protected Methionine (RPM) 76 and Lysine (RPL) 4.1.2 RDP and RUP content of different feedstuffs 78 4.1.3 RUP Intestinal Digestibility 79 4.1.4 In vitro Microbial protein yield g per kg TDN fermented 80 4.1.5 Body weight changes 81 4.1.6 Body Condition Score 82 4.1.7 Nutrients Intake 83 4.1.8 Overall Plane of Nutrition in prepartum cows 87 4.1.9 Effect of RPM and RPL Supplementation on Certain Blood 87 Parameters 4.2 Effect of supplementing RPM and RPL during post parturient 95 period 4.2.1 Body weight change 95 4.2.2 Body Condition Score 98 4.2.3 Dry Matter Intake 99 4.2.4 Nutrients Intake 101 4.2.4.1 CP intake 101 4.2.4.2 RUP intake 103 4.2.4.3 RDP intake 104 4.2.4.4 MP intake 104 4.2.4.5 Duodenal Methionine supply (% of metabolizable protein) 105 4.2.4.6 Duodenal lysine supply (% of metabolizable protein) 106 4.2.4.7 TDN intake 107 4.2.4.8 ME intake 109 4.2.4.9 NE L intake 110 4.3 Effect of rumen protected methionine and lysine 112 supplementation on milk production and its composition 4.3.1 Milk Production 112 4.3.2 Milk Composition 116 a) Milk fat 116 b) Milk protein 118 c) Milk lactose 121 d) Milk solids not fat 123 e) Milk total solid 123 f) Milk Urea Nitrogen (MUN) 124 g) Milk choline content 125 h) Fatty acid profile of milk 126 4.3.3 Efficiency of nutrients for milk production 131 4.4 Overall Plane of Nutrition in lactating cows 133 4.5 Effect of Rumen Protected Fat and Protein Supplementation on 134 Some Lactation Related Blood Parameters 4.5.1 Blood Glucose Concentration 134 4.5.2 Plasma Phosphatidylcholine Concentration 135 4.5.3 Plasma NEFA Concentration 136 4.5.4 Plasma Triglycerides Concentration 138 4.5.5 Plasma VLDL Concentration 138 4.5.6 Plasma Vitamin Levels 140 4.5.7 Plasma cholesterol Levels 140 4.5.8 Blood Urea Nitrogen Levels 141 4.5.9 Plasma amino acid profile 142 4.5.10 Plasma Prolactin Concentration 150 4.5.11 Plasma Growth Hormone Concentration 151 4.6 Effect of RPM plus RPL supplementation on reproductive 154 performance in crossbred cows 4.6.1 Calving Performance 154 4.6.2 Reproductive abnormalities 154 4.6.3 Reproduction related parameters 157 4.7 Economics of feeding rumen protected methionine plus lysine 158 in crossbred cows 5 Summary and Conclusions Bibliography 162 LIST OF TABLES Table No. Title Page No. 3.1 Composition of TMRs 38 3.2 Details of experimental animals 46 3.3 Ingredients in concentrate mixture (parts) 48 4.1 Chemical composition of feed ingredients offered (% DM basis) 73 4.2 Amino Acid Profile (% of CP) of feed Ingredients 74 4.3 Characteristic of rumen protected methionine (RPM) and rumen 76 protected lysine (RPL) products 4.4 Different protein fractions, RUP (% of CP) of feed Ingredients 78 4.5 RUP Intestinal Digestibility (in vitro) (% DM basis) of feed 79 Ingredients 4.6 Microbial protein yield g per kg TDN fermented of different 80 TMRs 4.7 Fortnightly change in body weights (kg) of crossbred cows fed 81 ration with or without RPM plus RPL 4.8 Fortnightly body condition score of crossbred cows fed ration 82 with or without RPM plus RPL 4.9 Prepartum average nutrient intake of crossbred cows fed ration 84 with or without RPM plus RPL 4.10 Fortnightly average nutrient Intake (kg/100 kg body weight) in 86 crossbred cows fed ration with or without RPM plus RPL 4.11 Plane of nutrition of prepartum cows fed ration with or without 88 rumen protected methinone plus lysine 4.12 Average Plasma metabolites (Prepartum ) of crossbred cows fed ration with or without RPM plus RPL 89 4.13 Average Plasma Amino Acid Profile (µmol/dl) of crossbred 93 cows (prepartum) fed ration with or without RPM plus RPL 4.14 Fortnightly average body weights (kg) of crossbred cows fed 96 ration with or without RPM plus RPL during postpartum period 4.15 Fortnightly change in Body Weight (kg) of crossbred cows fed 97 ration with or without RPM plus RPL 4.16 Percent change in body weights of crossbred cows fed ration 97 with or without RPM plus RPL 4.17 Fortnightly body condition score of crossbred cows fed ration 98 with or without RPM plus RPL 4.18 Fortnightly average dry matter intake (kg/d) in crossbred cows 100 fed ration with or without RPM plus RPL 4.19 Fortnightly average dry matter intake (kg/100 kg body weight) 100 in crossbred cows fed ration with or without RPM plus RPL 4.20 Fortnightly CP intake (kg/d) in crossbred cows fed ration with or 102 without RPM plus RPL 4.21 Fortnightly CP intake (kg/100 kg body weight) in crossbred 102 cows fed ration with or without RPM plus RPL 4.22 Fortnightly RUP intake (kg/d) in crossbred cows fed ration with 103 or without RPM plus RPL 4.23 Fortnightly RDP intake (kg/d) in crossbred cows fed ration with 104 or without RPM plus RPL 4.24 Fortnightly metabolizable protein intake (kg/d) in crossbred 105 cows fed ration with or without RPM plus RPL supplementation 4.25 Fortnightly duodenal methionine supply (% of metabolizable 106 protein) in crossbred cows fed ration with or without RPM plus RPL 4.26 Fortnightly duodenal lysine supply (% of metabolizable protein) 107 in crossbred cows fed ration with or without RPM plus RPL 4.27 Fortnightly TDNI (kg/d) in crossbred cows fed ration with or without RPM plus RPL 108 4.28 Fortnightly TDN intakes (kg/100 kg body weight) in crossbred 108 cows fed ration with or without RPM plus RPL 4.29 Fortnightly ME intake (Mcal/d) in crossbred cows fed ration 109 with or without RPM plus RPL 4.30 Fortnightly ME intake (Mcal/100 kg body weight) in crossbred 110 cows fed ration with or without RPM plus RPL 4.31 Fortnightly NE L intake (Mcal/d) in crossbred cows fed ration 111 with or without RPM plus RPL 4.32 Fortnightly NE L intake (Mcal/100 kg body weight) in crossbred 111 cows fed ration with or without RPM plus RPL 4.33 Fortnightly average milk yield (kg/d) of crossbred cows fed 113 ration with or without RPM plus RPL 4.34 Fortnightly average 4% fat corrected milk yield (kg/d) of 113 crossbred cows fed ration with or without RPM plus RPL 4.35 Fortnightly energy corrected milk yield (kg/d) in crossbred cows 114 supplemented with or without RPM plus RPL 4.36 Fortnightly milk fat content (%) in crossbred cows fed ration 116 with or without RPM plus RPL 4.37 Fortnightly milk fat yields (g/d) in crossbred cows fed ration 117 with or without RPM plus RPL 4.38 Fortnightly milk protein content (%) in crossbred cows fed 119 ration with or without RPM plus RPL 4.39 Fortnightly milk protein yield (g/d) in crossbred cows fed ration 120 with or without RPM plus RPL 4.40 Fortnightly milk lactose content (%) in crossbred cows fed ration 122 with or without RPM plus RPL 4.41 Fortnightly Milk Lactose yield (%) in crossbred cows fed ration 122 with or without RPM plus RPL 4.42 Fortnightly milk SNF content (%) in crossbred cows fed ration with or without RPM plus RPL 123 4.43 Fortnightly TS content (%) in crossbred cows fed ration with or 124 without RPM plus RPL 4.44 Fortnightly MUN (mg/dl) in crossbred cows fed ration with or 125 without RPM plus RPL 4.45 Fortnightly milk choline (mg/dl) in crossbred cows fed ration 126 with or without RPM plus RPL 4.46 Fortnightly Fatty Acid Profile of Milk of crossbred cows fed 128 ration with or without RPM plus RPL 4.47 Overall fatty acid profile of milk of crossbred cows fed ration 130 with or without RPM plus RPL 4.48 Efficiency of utilization of nutrients of crossbred cows fed ration 131 with or without RPM plus RPL 4.49 Plane of nutrition of lactating cows fed with rumen protected 133 methinone plus lysine 4.50 Plasma glucose (mg/dl) concentration in crossbred cows fed 134 ration with or without RPM plus RPL 4.51 Plasma Phosphatidylcholine (µg/ml) concentration in crossbred 136 cows fed ration with or without RPM plus RPL 4.52 Plasma NEFA (mg/L) concentration in crossbred cows fed ration 137 with or without RPM plus RPL 4.53 Plasma triglycerides (mg/dl) concentration in crossbred cows fed 138 ration with or without RPM plus RPL 4.54 Fortnightly plasma VLDL (mg/dl) concentration in crossbred 139 cows fed ration with or without RPM plus RPL 4.55 Plasma vitamin E (µg/ml) concentration in crossbred cows fed 140 ration with or without RPM plus RPL 4.56 Fortnightly plasma Cholesterol (mg/dl) concentration in 141 crossbred cows fed ration with or without RPM plus RPL 4.57 Fortnightly plasma BUN (mg/L) concentration in crossbred cows fed ration with or without RPM plus RPL 142 4.58 Postpartum plasma amino acid (µmol/dl) concentration in 142 crossbred cows fed ration with or without RPM plus RPL 4.59 Overall mean plasma amino acid (µmol/dl) concentration in 148 crossbred cows fed ration with or without RPM plus RPL 4.60 Weekly plasma prolactin (ng/ml) concentration in crossbred 150 cows fed ration with or without RPM plus RPL 4.61 Weekly plasma growth hormone (ng/ml) concentration in 151 crossbred cows fed ration with or without RPM plus RPL 4.62 Condensed information of lactation trial 152 4.63 Calving Performance of crossbred cows fed ration with or 155 without RPM and RPL 4.64 Reproductive abnormalities plus metabolic diseases observed in 156 crossbred cows fed RPM plus RPL 4.65 Reproductive parameters recorded in crossbred cows fed RPM 157 plus RPL 4.66 Economics of feeding rumen protected methionine plus lysine to lactating crossbred cows 159 LIST OF FIGURES Plate No. Title After Page 3.1 Chromatogram of standard amino acid profiile 34 3.2 Standard curve of choline 34 3.3 Standard curve of glucose 54 3.4 Standard curve of NEFA 54 3.5 Standard curve of BUN 56 3.6 Standard curve of cholesterol 56 3.7 Chromatogram of standard α-Tocopherol 58 3.8 Standard curve of milk urea nitrogen 58 3.9 Standard curve of growth hormone 60 3.10 Standard curve of prolactin 60 4.1 RUP Intestinal Digestibility (in vitro) (% DM basis) 80 4.2 Microbial protein yield g per kg TDN fermented of different 80 TMRs 4.3 Average fortnightly body weights (kg) of crossbred cows 96 4.4 Average fortnightly percent change in body weight (kg) of 96 crossbred cows 4.5 Fortnightly average body condition score of crossbred cows 98 4.6 Fortnightly average dry matter intake (kg/d) of crossbred 98 cows 4.7 Fortnightly average crude protein intake (kg/d) of crossbred 102 cows 4.8 Fortnightly average TDN intake (kg/d) of crossbred cows 102 4.9 Fortnightly average milk yield (kg/d) in crossbred cows 114 4.10 Fortnightly average 4% fat corrected milk yield (kg/d) in 114 crossbred cows 4.11 Fortnightly average milk fat (%) of crossbred cows 116 4.12 Average fortnightly milk composition 116 4.13 Average fortnightly fatty acid profile of milk of crossbred 130 cows 4.14 Average fortnightly plasma metabolites of crossbred cows 130 4.15 Average fortnightly plasma triglycerides and VLDL of 138 crossbred cows 4.16 Average fortnightly plasma amino acids of crossbred cows 138 4.17 Average weekly plasma prolactin of crossbred cows 150 4.18 Average weekly plasma growth hormone of crossbred cows 150 4.19 Reproductive abnormalities observed in crossbred cows 156 4.20 Reproductive parameters observed in crossbred cows 156 Abbreviations __________________________________________ AA Amino acid EE Ether extract ACTH Adrenocorticotropic hormone ENS ADF Acid detergent fibre Endogenous nitrogen secretion ADICP Acid detergent insoluble crude protein EN Endogenous nitrogen FA Fatty acids AI Artificial insemination FCM Fat corrected milk Ala Alanine Glu Glutamic acid AP Absorbed protein Gly Glycine Arg Arginine GNC Groundnut cake Asp Aspartic acid His Histidine BCAA Branched chain amino acid HMB BCS Body condition score 2-hydroxy-4methylthiobutanoic acid BHBA ß- Hydroxyl butyric acid HMBi Isopropyl ester of HMB BHT Butylated hydroxytoluene HPLC High performance liquid chromatography BUN Blood urea nitrogen Ile Isoleucine BW Body weight LCFA Long chain fatty acids CNCPS Cornell net carbohydrate and protein system Leu Leucine CP Crude protein LH Luteinizing hormone CPI Crude protein Intake Lys Lysine Cys Cysteine MCP Microbial crude protein DDG Dried distillers grains ME Metabolizable energy DIM Days in milk MEI Metabolizable energy intake DM Dry matter Met Methionine DMB Dry matter basis MP Metabolizable protein DMI Dry matter intake MPPA Most probable production ability DOM Digestible organic matter MUFA Mono unsatutated fatty acids EAA Essential amino acid MUN Milk urea nitrogen ECM Energy corrected milk MY Milk yield ECP Endogenous crude protein NAN Non ammonia nitrogen Abbreviations __________________________________________ NDF Neutral detergent fibre Thr Threonine NDICP Neutral detergent insoluble nitrogen TMR Total mixed ration Trp Tryptophan NDRI National Dairy Research Institute Tyr Tyrosine NEAA Non essential amino acid UFA Unsaturated fatty acids NEFA Non esterified fatty acid UDP Undegradable protein NE L Net energy for lactation Val Valine NE L I NE L intake VFA Volatile fatty acids NPN Non protein nitrogen VLDL Very low density lipoprotein NRC National research council OM Organic matter Phe Phenylalanine Pro Proline PUFA Poly unsaturated fatty acids RDN Rumen degradable nitrogen RDP Rumen degradable protein REP Rumen escape potential RPAA Rumen protected amino acids RPL Rumen protected lysine RPM Rumen protected methionine RPM+L Rumen protected methionine plus lysine RUP Rumen undegradable protein RUP ID RUP intestinal digestibility Ser Serine SFA Saturated fatty acids SG Specific gravity SNF Solid not fat TDN Total digestible nutrients TDNI Total digestible nutrients intake CHAPTER – 1 INTRODUCTION 1. Introduction ________________________________________________ The goal of ruminant protein nutrition is to achieve optimal rumen fermentation efficiency and desired animal productivity (NRC, 2001). But there is still a large amount of uncertainty, especially regarding protein and amino acid (AA) requirements for ruminants. However, nutrient requirements are relatively well defined for most of the domesticated monogastric species. Lysine (Lys) and Methionine (Met) have been identified as first limiting essential amino acids in metabolizable protein (MP) supply to dairy cattle (King et al., 1990; Schwab et al., 1992a and 1992b). Rumen undegradable protein is the second important source of absorbable amino acid (AA) to animal after microbial protein. But relative to concentrations in rumen bacteria, rumen undegradable protein is low in Lys and/or Met. Most feedstuffs have lower amounts of Lys and Met, particularly of Lys whereas contribution of Lys and Met to total essential amino acid in body lean tissue and milk are similar. Production responses of lactating dairy cows to increased supplies of Lys and Met in MP includes variable increase in content and yield of protein in milk, milk yield and feed intake. Feed CP can be divided into rumen-degradable protein (RDP), which is largely incorporated into micro-organisms in the rumen when they synthesize microbial crude protein (MCP) and rumen undegradable protein (RUP) which escapes rumen degradation and passes from the rumen to abomasum and is digested in the small intestine. A lot of the dietary CP that is ingested and absorbed is used for body protein synthesis which, in mature animals, is mostly for replacing tissue (turnover). A part of absorbed CP gets deaminated and is used for glucose synthesis. Therefore faecal and urinary N consists of a mixture of undigested or unabsorbed dietary CP and detritus of metabolic processes. Even though some of the protein is used by the cow for maintenance, growth, health, milk production and reproductive processes, a large amount is excreted in urine and faeces (Lapierre et al., 2002). Poor efficiency of CP use by ruminants may be due to energy limitations, reduced growth of microorganisms in the rumen, catabolism and partitioning of AA’s, imbalances in AA supply to the intestinal absorptive site and genetic limitations (Bequette et al., 2002). __ _________________ ___________ _ ___________ 1 Introduction … _______________________________________________________________________ Ruminants have the unique ability to transform low quality forage based diets, partly indigestible by monogastric species, into high quality consumer products such as milk, meat and fibre. To improve the efficiency of CP use by ruminants, diets need to be balanced according to the specific AA requirements of the animals. Balancing for post ruminal AA delivery could allow use of lower CP rations because they would be balanced to supply individual AA’s to the intestinal absorptive site. Metabolic costs of deamination of excess AA’s and excretion of excess N would be lower, and removal of CP from the ration leaves space to supply other nutrients, such as those that more efficiently supply energy (Lapierre et al., 2002). The nutritional value and chemical composition of feeds are greatly influenced by the vegetative stage of the plant, weather during growth, time of day during harvest, soil fertility, storage and even feed bunk management (Van Soest et al., 1994), and vary widely in their proportions of protein and non-protein N (NPN), rate and extent of CP degradation in the rumen, digestion in the intestine and the AA composition of undegraded feed CP (NRC, 2001). Since 1989, protein requirements have often been expressed in terms of absorbed protein (AP) or total AA reaching the small intestine, which includes protein synthesized by rumen micro-organisms and feed CP that escapes rumen degradation (Dugmore, 1995). Even though the NRC (1989) recognized that intestinal digestion of proteins differed, they used a constant digestibility value of 800 g/kg for RUP in all feedstuffs due to the lack of data to differentiate among feeds. Other shortcomings of this model, as pointed out by several research groups (Satter 1986; Clark 1978), are the prediction of increased milk yield when a protein source high in RDP is substituted by a source high in RUP, when many research studies reported a lack of response. Possible reasons for this, as reviewed by Santos et al (1998a), may be decreased microbial synthesis due to removal of RDP from the diet, a poor AA profile, or low digestibility of the RUP source. Some studies suggested that the source of RUP should have an AA profile to complement that of MCP (Clark et al., 1992; Chen et al., 1993). __ _________________ ___________ _ ___________ 2 Introduction … _______________________________________________________________________ Proteins digested to AA that are actually available for absorption in the small intestine, are largely a combination of RUP, MCP and some endogenous secretions (ECP - proteins secreted into the digestive tract) – collectively known as metabolizable protein (MP) (CNCPS, 2000; NRC, 2001). As milk production increases, the proportion of the total CP requirements met by MCP was predicted to decrease and substantial amounts of dietary CP must escape rumen degradation to meet predicted protein needs (Santos et al., 1998b). Higginbotham et al. (1989) and Taylor et al. (1991) showed that increasing the amount of RUP in the ration could improve milk production, but only up to 30-40% of total CP (Santos et al., 1998b), after which RDP becomes limiting, decreasing MCP production and non ammonia N (NAN) supply to the intestine (Clark et al., 1992; Ferguson et al., 2000). Further increases in RUP could also cause a reduction in diet fermentability, dry matter intake (DMI) and milk production (Olmos Colmenero and Broderick, 2006). Research in poultry (NRC, 1994) and swine (NRC, 1998) revealed that each physiological state in an animal requires a unique profile of absorbed AA. However, these profiles still need to be established for ruminants. Since Lys and Met are generally considered to be the most limiting AA for milk production in ruminants, it is common to feed dairy cows by balancing diets to maximize absorbable Lys and Met delivery. However, many RUP sources are low in Lys and/or Met with AA profiles that are generally inferior to MCP, making it difficult to formulate a ration to achieve the optimum concentration of both Lys and Met in MP, in order to satisfy the animal’s requirement for limiting AA, without oversupplying N (Santos et al., 1998a; Rode and Vazquez-Anon, 2006). Further, Met is source of the methyl donor S- adenosyl Methionine, the metabolite that provides methyl groups in variety of reactions including de novo synthesis of choline from phosphatidylethanolamine, thus Met and choline metabolism are closely associated. As much as 28% of the absorbed methionine is used for choline synthesis (Emmanuel and Kennelly, 1984). Choline is involved in the transport of fat from liver and required for the synthesis of phosphatidylcholine, a phosholipid found in the membranes of very low density lipoprotein. Thus Met plays a role in VLDL synthesis and act to reduce __ _________________ ___________ _ ___________ 3 Introduction … _______________________________________________________________________ plasma ketone bodies during early lactation. Increased percentage of fat in milk has been reported with increased amount of rumen protected methionine and Met+Lys in diet. This reality directed attention toward supplementing only the limiting AA in the diet, leading to numerous studies to determine effects of adding ruminally protected, and free AA to dairy rations, as well as infusing specific AA (or AA mixtures) to the duodenum. Genetic improvements in Indian dairy cows have lead to increased milk production, which requires higher intake of dietary CP to meet the needs of milk protein synthesis. Lys and Met have been suggested to be the most limiting AA for milk protein synthesis. Balancing the concentration of Lys and/or Met in rations shows improvement in efficiency of all the other amino acids. The efficient use of dietary AA in the dairy cow can have immediate benefits such as improving milk production and yield of milk protein and/or fat. Although an increase in milk production, milk protein and/or milk fat in high producing dairy cattle fed rumen protected Met and Lys has been reported, its effect on hormonal responses, plasma metabolites and reproductive performance are not clearly understood. Therefore, the present study has been planned to assess the effect of supplemental rumen protected Met and Lys on feed utilization, milk production and its composition, reproductive performance, plasma metabolites and hormonal responses in periparturient dairy cows under Indian feeding regimen. The objectives of the present study are 1. To evaluate the effect of feeding rumen protected methionine (RPM) and rumen protected lysine (RPL) on plasma metabolites and hormonal response of periparturient dairy cows. 2. To determine the effect of feeding RPM and RPL on feed intake, feed utilization, milk production and its composition and on reproductive performance of dairy cows. 3. __ To calculate cost benefit ratio of RPM and RPL supplementation in dairy cows. _________________ ___________ _ ___________ 4 CHAPTER – 2 REVIEW OF LITERATURE 2. Review of Literature __________________________________________________ Amino acids are the building blocks of all proteins. These are linked by dipeptide bonds to form protein chains. Each protein chain has a specific sequence of AA that determines its integrity and functionality such as production of enzymes, immunoglobins, hormones and milk proteins, making them vital to the maintenance, growth, reproduction and lactation of dairy cattle (Schwab, 1996; Rode and Vazquez-Anon, 2006). The AA that are absorbed, but not used for protein synthesis, are catabolized and serve as a source of metabolic energy when oxidized to CO 2 , while the amino groups are used to synthesize other deficient AA. These can also be converted to fatty acids or serve as precursors of other metabolites in pathways within the body, such as gluconeogenesis (Vanhatalo et al., 2003; Rulquin et al., 2004). An estimated 110-180 g/kg of glucose flux was synthesized from the glucogenic AA’s glutamic acid (Glu), aspartic acid (Asp), serine (Ser) and glycine (Gly). Alanine (Ala) was quantitatively the most important AA (Wolff et al., 1972), but it is much less efficiently used than propionate. Methionine (Met) and lysine (Lys) are considered to be first limiting, but surprisingly little is known of their metabolic fates. Methionine is a precursor for cysteine by donating a sulfur group. As an intermediate in transmethylation reactions, it donates a methyl group to synthesize choline, vitamin B 12 , phospholipids in cell membranes, creatine production for energy storage and transfer, and the carnitine required in lipid metabolism and fatty acid mobilization (Campbell and Farrell, 2003). Lysine is an anomaly since it is almost always taken up by the udder in excess of requirements. Excess Lys is oxidized to produce glutamate, an energy source for intestinal mucosa cells (Windmueller and Spaeth, 1980) and a precursor for de novo Arg and Pro synthesis (Bequette et al., 2002). Amino acids synthesized by cells in the animal body, using metabolites from surplus AA catabolism, are known as non-essential amino acids (NEAA) or dispensable AA and do not necessarily have to be provided in the diet. Ten of the 20 primary AA in proteins are classified as essential or indispensable amino acids (EAA) and need to be supplemented in the diet (in the form of rumen escape protein) since they cannot be synthesized by animal tissues in sufficient quantities to fulfil metabolic requirements for growth and high levels of _______________________________________________________________________ 5 Review of Literature … production. These include Lys, Met, arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) and valine (Val) (NRC, 2001). Classification of AA as essential or non-essential was based on research conducted in nonruminant animals, but was shown to be similar to that of ruminants (Black et al., 1957). Essential AA are the focus of most nutritional studies, since there is little evidence that NEAA profiles are important for efficiency, or that NEAA would ever become more limiting than EAA (Schwab et al., 1976; NRC, 2001). A number of studies, where mixtures of AA were administered post-ruminally, indicated that requirements for NEAA were met before requirements for EAA, and that individual NEAA absorbed below requirements can be synthesized from excess AA in adequate amounts to maintain animal performance (Oldham et al., 1979; Fraser et al., 1991). 2.1 Sources contributing to metabolizable protein in ruminants Amino acids utilized by the mammary gland are provided by MP, primarily composed of: • Microbial CP containing an estimated 800 g/kg of true protein (the remainder being nucleic acids), and with 800 g/kg digestibility about 640 g/kg of MCP is therefore converted to MP (NRC, 1989; Verbic, 2002). • Rumen-undegradable protein, assumed to be 1000 g/kg true protein, but the contribution to MP is variable depending on feed type since intestinal digestibilities were assigned to each individual feedstuff range from 500 to 1000 g/kg (NRC, 1989). • Endogenous CP: Data on the proportion and digestibility of true protein in ECP is extremely limited, but its true protein content is estimated to be 500 g/kg and digestibility is assumed to be 800 g/kg, resulting in a 400 g/kg conversion to MP (NRC, 2001). 2.1.1 Endogenous crude protein Endogenous CP originates from various sources (Tamminga et al., 1991): • Mucoproteins in saliva ___________________________________________________________________ 6 Review of Literature … • Epithelial cells from the respiratory tract • Cellular debris abraded from the mouth • Cellular debris from the omasum and abomasum • Enzyme secretions into the abomasum • Enzyme secretions into the ileum The first three don’t contribute to protein passage to the intestine since most is probably degraded by rumen microorganisms (NRC, 2001). A number of studies to identify the sources of endogenous N secretions (ENS) have been reported for sheep but, due to the complexity of N exchanges, these studies are rare for dairy cows (Ouellet et al., 2007). It is technically tedious to distinguish between endogenous, microbial and feed N in the duodenal digesta, hampering attempts to determine passage of ECP to the small intestine. Most studies ignore the contribution from these recycled materials, probably overestimating the ‘true’ AA supply from the diet and MCP. However, some approaches measured the flow of endogenous N (EN) through the rumen and abomasum by using cows fed diets low in CP that were considered to be free of RDP (Hannah et al., 1991; Lintzenich et al., 1995), or ruminants solely nourished by volatile fatty acids (VFA) infused into the rumen (Ørskov et al., 1986). The NRC (2001) adopted an average value of 1.9 g of N/kg of DMI based on data from these studies. 2.1.2 Microbial crude protein Microbial CP synthesis involves degradation of RDP by proteases synthesized by various strains and species of bacteria, protozoa and anaerobic fungi in the rumen, and incorporation of the resulting peptides, AA and ammonia into microbial protein. It also allows ruminants to convert external NPN sources, such as urea into ammonia and subsequently into MCP. Bacteria are the most abundant micro-organism in the rumen and protein degradation occurs through extra-cellular proteolysis, in which soluble or insoluble proteins adsorb to bacteria (Nugent and Mangan, 1981; Wallace, 1985) which hydrolyzes it to small peptides and free AA which are finally absorbed for further degradation and utilization. ___________________________________________________________________ 7 Review of Literature … However, it was found that sequestration of protozoa in the rumen results in a lower protozoa concentration in effluent than in corresponding rumen fluid, contributing too little to outflow protein to significantly affect the composition of the total protein mixture. Unlike bacteria, protozoa make use of intracellular hydrolysis of protein, obtained from ingesting small feed particles, fungi or, primarily, bacteria. Amino acids are incorporated into protozoal protein but they are not able to synthesize AA from ammonia as do bacteria. The contribution of anaerobic fungi to protein degradation is considered negligible, due to relatively low concentrations in rumen digesta (NRC, 2001). Microbial CP is considered to be the most important and least expensive MP source and it is the largest contributor of protein reaching the duodenum, providing about 100-150g MCP/kg of DMI (Verbic, 2002). It has a high quality AA profile (Clark et al., 1992) and apparent intestinal digestibility of about 800 g/kg (NRC, 1989). It has long been recognized that the EAA profile of MCP is fairly constant, because EAA profiles between different micro-organisms, and among predominant strains, vary little (Purser et al., 1966), and their contribution to post-ruminal protein supply is not proportional to the respective rumen biomass fractions (i.e., protozoa, bacteria and endogenous) (Harrison et al., 1979). The AA composition of rumen bacteria was relatively constant regardless of sampling time post feeding (Martin et al., 1996) and diet composition (Prestlokken and Harstad, 2001), but a few studies reported large variations in AA composition of bacteria (Clark et al., 1992) at different levels of DMI (Rodriquez et al., 2000). Regardless, MCP has a relatively high proportion of NPN (200 g/kg nucleic acid-N) (NRC, 1989) and the AA composition of microbial true protein is very similar to that of milk and lean body tissue, ensuring high efficiency of AA utilization (Verbic, 2002). Microbial CP is mainly used for protein synthesis in the mammary gland, but also acts as a precursor in gluconeogenesis for lactose synthesis (Rode and Vazquez-Anon, 2006). The rate of rumen microbial growth and protein synthesis are affected by a number of factors (Yang et al., 2001; Verbic, 2002) including: • Availability of rapidly fermentable carbohydrates: Energy supply in the rumen is usually first limiting factor for microbial growth and the rate of carbohydrate digestion in the rumen is the major factor controlling the amount ___________________________________________________________________ 8 Review of Literature … of energy available (Hoover and Stokes, 1991). At sub-optimal energy input levels, microbial growth will increase with increased energy supply, but an oversupply does not result in extra growth, only reduced efficiency (Dijkstra et al., 1998) due to energy ‘spilling’ (Russell, 2007). • An adequate supply of N compounds: Peptides, AA and ammonia liberated from RDP are incorporated into MCP by rumen bacteria. A deficiency of RDP results in reduced MCP synthesis, fibre digestion, DMI and, ultimately, reduced milk production. (Rode and Vazquez-Anon, 2006). • A suitable rumen environment: Low or high pH values can be deleterious to microbial growth, reduce digestibility of fibre and divert energy in the rumen towards non-growth functions to maintain or correct the pH. • Rumen outflow (turnover) rate: High DMI increases rumen outflow rate, with microbes spending less time in the rumen. A faster turnover rate lowers maintenance costs due to less N recycling. Higher DMI therefore increases efficiency of MCP synthesis (Clark et al., 1992; Rodriquez et al., 2003) with improved N digestion in the rumen (Rode et al., 1985). Dry matter intake has the biggest influence of all dietary factors on passage of microbial N to the small intestine, suggesting that CP in the diet should be determined relative to DMI. The CP content could therefore be reduced in the diet of a cow eating large amounts of DM without affecting microbial AA flow at the intestine or reducing milk yield (Clark et al., 1992). 2.1.3 Rumen-undegradable crude protein Microbial CP has a very high quality AA profile but, alone, it is insufficient to supply adequate amounts of AA for optimum animal production (Rode and Vazquez-Anon, 2006). Factors influencing the rate of passage of digesta include DMI, specific gravity (SG), feed particle size and concentrate to forage ratio (Swanepoel, 2011). An alternative is the use of feeds with naturally protected proteins that are relatively resistant to rumen degradation (Clark et al., 1992) or feeds that have been chemically or physically treated to reduce protein degradability and increase its RUP content. ___________________________________________________________________ 9 Review of Literature … Heat processing causes the carbonyl groups of sugars to combine with the amino groups of protein through the Maillard reaction, forming peptide links (i.e., proteincarbohydrate cross linkages) that are more resistant to enzymatic hydrolysis (Rode and Vazquez-Anon, 2006). However, care should be taken during heat treatment since overheating reduces intestinal digestibility of RUP and leads to the destruction of AA such as Cys, Arg and especially Lys. Categories of chemical treatment include those that • Introduce cross-links by combining with proteins (e.g., aldehydes) • Alter protein structure through denaturation (e.g., acids, alkalis and ethanol) • Bind proteins without altering their structure (e.g., tannins) However, the use of chemical treatments alone was accepted commercially, and leads to combined chemical and heat treatments, which has been more effective in increasing the amount of protein that escapes rumen degradation. One technique involves adding lignosulfonate (i.e., a by-product of the wood products industry) to oilseed meals before heat treatment (Borucki et al., 2007). Most high quality grasses and legumes fed to lactating cows contain adequate amounts of RDP, but are deficient in RUP, moving the focus of protein supplementation to feedstuffs high in RUP (NRC, 2001). Common sources of RUP include animal and marine by-products such as fishmeal and blood meal, dried distillers grains (DDG), brewers dried grains and maize gluten meal (Rode and Vazquez-Anon, 2006). DDG are the solids that remain after fermentation of grains such as maize during the ethanol production process. Another challenge in diet formulation is to optimize the level of RUP reaching the duodenum without reducing MCP synthesis, since low RDP levels have a potentially negative effect on microbial growth due to inadequate available N supplies (Clark et al., 1992; Ipharraguerre and Clark, 2005). No single source of RUP provides a balance of EAA that matches the profile of milk, but proteins with the closest match are regarded as the highest quality with the best nutritive value. In high forage and soybean hull-based diets, where RUP intake is low, or where animal-derived proteins make up most of the dietary RUP, Met is usually first limiting AA ___________________________________________________________________ 10 Review of Literature … (Ahrar and Schingoethe, 1979; Schingoethe et al., 1988). In contrast, Lys has been identified as first limiting when maize and maize by-products provide most of the RUP in the diet (NRC, 2001). Microbial CP is low in Met, but relatively high in Lys, and the level of these AA is lower in most feedstuffs. A deficiency in one of these AA can therefore be exaggerated by feeding high levels of a single RUP source instead of combining several sources with complementary AA profiles (Ferguson et al., 2000; NRC, 2001). Optimum productivity can be achieved with the minimum amount of dietary CP when rations are balanced to provide adequate amounts of RDP and RUP sources with desired, complimentary, AA profiles (Clark et al., 1992; Ferguson et al., 2000; NRC, 2001). The efficiency with which MP is used for protein synthesis depends on the amount of EAA in it and how well the EAA profile in MP matches the AA profile required by animal metabolism (NRC, 2001). Increased milk production and protein yields require an increase in feed CP intake and/or an improved postruminal supply of AA. Feedstuffs with low rumen degradability and/or high quality protein with a well balanced AA profile (such as meat meal, bone meal, fishmeal etc.) can be used to increase postruminal AA supply, but they are expensive and legislatively banned in cattle feed in India. It is therefore becoming more difficult to formulate rations that will provide the desired AA concentrations and ratios in MP. 2.2 Comparative Amino acid requirements in different species There is remarkable consistency across species in amino acid composition of mixed body proteins from fetal, growing and mature animals, suggesting that minimal qualitative requirement for essential amino acids for growth will be similar. However there are few exceptions. In fish, the composition of the tissue is also relatively constant across the fish species, with the exception that lysine and arginine contents are higher in transgenic carp (Fu et al. 2000), which may suggest that requirements for these are higher. In sheep, as expected, wool is higher in the sulphur amino acids than average tissue proteins, but lower in lysine and histidine, supporting recommendations that the sulphur amino acid requirement for wool producing sheep is higher. Egg proteins appear to be similar in composition to body tissues except for low level of lysine in eggs. These appears to be a consistent composition of milk across several species ( human, sows, horses, cows, goat) despite the fact that the whey and ___________________________________________________________________ 11 Review of Literature … casein contents vary across species (Davis et al. 1994). Compared to tissue, the composition of cow and sow milk protein is adequate in most amino acids to support growth rate (Leibolz, 1982; Fligger et al., 1997). Reduced feed intake during the early weaning period may exacerbate these deficiencies leading to the bacterial translocation, gut atrophy, mucosal shedding and weight loss. Supplemental glutamate and glutamate plus arginine appear to reverse these affects by enhancing total gut weight and preventing villus atrophy. Glutathione plays an important role in maintaining the defence mechanisms of the gut mucosa against peroxidative damage. Because glutamate, glycine and cysteine for glutathione synthesis are derived mainly from the gut lumen (Reeds et al., 1997), glutathione synthesis may suffer at the time of weaning when food intake is less than optimal. There are metabolic differences between species that necessitates higher requirement for certain amino acids to support maximum growth, thus these are designated as conditionally essential. One species difference relates to expression of enzyme of the ornithine - urea cycle. This cycle serves to dispose of excess ammonia, and it also plays a critical role in the synthesis of the glutamate family of amino acids (proline, arginine, citrulline, ornithine), in particular arginine, which can be degraded to form urea and ornithine. The high concentration of the arginase, which catalyzes the hydrolysis of Larginine to produce L-ornithine and urea in the liver of rapidly growing animals, coupled with the low rate of intestinal arginine synthesis, limits arginine for protein synthesis (Wu et al., 1997). Arginine is required in cat’s diet because they lack the enzyme to synthesis arginine and ornithine, plus they have the limited ability to convert glutamate into ornithine. In the absence of these precursors, cats become comatose due to the buildup of toxic ammonia. Symptoms can be reversed by supplemenating the diet with the arginine or its precursor ornithione. Chickens also need dietary arginine because they do not have functional urea cycle and the situation is amplified because of the high content of the arginine in the feathers. Fish requires higher amount of arginine in the diet, not because they do not have urea cycle, but because the main route of elimination of excess nitrogen is via ammoniagenesis (transamination and deamination route). As a result, transfer of nitrogen into the ornithine cycle is low with limited synthesis of arginine. In marine cartilagious fish, the arginine requirent is higher and this probably reflects the need to synthesize urea to help maintain buoyancy when salinity is low (Withers, 1998). Another example of species related ___________________________________________________________________ 12 Review of Literature … requirements is taurine and phenylalanine/tyrosine in cats. Due to limited ability to convert cysteine into taurine and the limited conversion of taurine via conjugation with cholic acid, young cat require dietary taurine to prevent renal degeneration. Cat also requires higher dietary levels of phenylalamine/tyrosine for melanine synthesis; otherwise, hair colour turns from black to redish brown. Other species can convert phenylalamine into tyrosine via phenylalanine hydroxylase, but this has not proven to be limitation. Wool growth is limited by the supply of cysteine. Although there is synthesis of cysteine locally within the wool follicle or skin via methionine transsulphuration with serine, the supply of methionine from rumen microbial protein is generally limited. Moreover the transsuphuration pathway in the skin may have to compete with polyamine synthesis and transmethylation reactions for methionine. 2.3 Rumen Protected Proteins in Ruminant Nutrition 2.3.1 Degradation of Proteins in the Rumen Feed proteins are hydrolyzed into peptides and amino acids by rumen micro-organisms. However, most amino acids are rapidly degraded to organic acids, ammonia and carbon dioxide. The ammonia produced is the primary nitrogenous nutrient for bacterial growth. Some species of ruminal bacteria use peptides directly for synthesis of microbial protein. Chalupa (1975) quoting several sources indicated that as little as 40 per cent or as high 80 per cent of dietary protein normally might be degraded in the rumen and transferred into microbial protein. Because rumen microbial protein production is an energy-dependent mechanism, the amount of dietary protein transformed into microbial protein must be an important aspect of nitrogen economy of the animal and should be an important factor in whether or not to decrease ruminal degradation by artificial procedures. Although amino acids are rapidly deaminated in the rumen under practical conditions, the rumen microbial population derives 25-50 per cent of their nitrogen from sources other than ammonia. These presumably intact amino acids or peptides which originate either directly from food protein, from recycled nitrogen to rumen or from turnover of bacterial and protozoal protein within rumen (Oldham, 1981). Ruminal degradation of proteins can be reduced by decreasing retention time in the rumen. Factors known to influence this include level of food intake, specific gravity, particle size of diet, concentrate to roughage ratio and rate of rumen ___________________________________________________________________ 13 Review of Literature … digestion. Similar to other nutrients, the amount of protein that riches the small intestine depends upon food intake. The breakdown of proteins by microorganisms as mentioned earlier, gives rise also to intermediate products such as free amino acids in the rumen. The low concentration of free amino acids in the rumen usually suggest rapid utilization, but increased concentration after feeding imply that proteolysis occurs faster than subsequent utilization of free amino acids (Chalupa, 1975). The free amino acids in the rumen can be assimilated directly by microbes and be absorbed through the rumen but most are deaminated to yield ammonia and other intermediate products (Hoover and Miller, 1991). The microbial protein alone is likely sufficient to meet the needs of cattle at or near maintenance. Young growing cattle and lactating cows need bypass protein in addition to microbial protein to meet their metabolizable protein requirements. The international literature suggests that the requirement for rumen degradable protein is 130 g kg-1 of digestible organic matter (DOM). On the average, the amount of microbial protein synthesized is 130 g/kg of the DOM (Klopfenstein, 1996). 2.3.2 Protection of Protein from Ruminal Degradation It is possible to protect proteins using several procedures such as heat treatment, chemical treatment/modification, and inhibition of proteolytic activity and identification of naturally protected protein (Ferguson, 1975). The use of these techniques in comparison to the usual sources of dietary proteins improves the supply of amino acids without an increase in ammonia production, resulting in a better performance by the animal (Kaufman and Lupping, 1982). 2.3.2.1 Heat Treatment The effect of heat treatment during manufacturing or drying of forages in reducing the rate of microbial fermentation is attributable to reduced solubility of the protein. In feeding trials on heat treated protein, the rumen level of ammonia concentration is usually very low and there is improvement in nitrogen balance or better growth particularly in sheep and in some cases calves (Kaufman and Lupping, 1982). Increases in milk yield have also been reported (Dijk et al.,1983). Heat treatment has been used to increase the undegradable protein of common feedstuffs such as soybeans and grains (McNiven et al., 1994, Robinson and McNiven, 1994, Prestlokken, 1999). However, high temperature and extended heating ___________________________________________________________________ 14 Review of Literature … time have increased the acid detergent insoluble nitrogen content by the Maillard reaction between sugars and amino acids (Satter, 1986, Broderic et al., 1991). Amino acids also form peptide links with asparagine and glutamine (Belitz and Grosch, 1987). This resulting peptide linkages from heating are more resistant to enzymatic hydrolysis. Excessive heating can cause essential amino acids such as lysine, methionine, and cystine to be extensively damaged (Kung, 1996). Although moderate heat may increase the protein flow to the small intestine, excessive heat may decrease the quantity of some amino acids and lower the digestibility of protein in the small intestine (McNiven et al., 2002). The roasting of soybean meal increased the amount of soy protein escaping microbial degradation in the rumen. Reduction of protein degradation occurs because of Maillard type reaction between sugar aldehyde group and free amino groups (Dhiman et al., 1997). Roasting of soybean and soybean meal resulted in the lowest in situ degradation rates of protein 0.037 and 0.029 per hour, respectively (Faldet et al., 1991). Walli, (2005) fine-tuned the heat treatment of GN-cake and soybean-cake and found 1500C for 2hr as the optimum temperature-time combination. Walli and Sirohi (2004) found that the roasting of soybean at 1300C for 30 min protected its protein content from ruminal degradation. However, some disadvantages of dry processing of grains and feeds have also been reported. Ayatse et al. (1983) reported that roasting of maize at 120°C to 130°C decreased its nutritive value with loss of moisture, minerals and vitamins and reduced the essential amino acid index. Roasting regular sunflower seeds tended to decrease the digestibility of fat and energy (Adams and Jensen, 1985). The problem with ‘heat treatment’ is that it may not be cost effective and moreover, it can also over-protect the protein. Formaldehyde treatment has been used by several workers in India to reduce the protein degradability of high degradable cakes and also to study the impact of its feeding on the productive performance of dairy animals (Ramchandran and Sampath, 1995; Chatterjee and Walli, 2003). 2.3.2.2 Formaldehyde Treatment Treatment of proteins with formaldehyde is the most widely used process at the present time and it has been exploited commercially. Treatment of high quality proteins result in the formation of cross-links with amino group and makes the protein less susceptible ___________________________________________________________________ 15 Review of Literature … to microbial attack (Shelke et al., 2012). Such treatments of protein -rich feedstuffs has been shown to increase the protein digested in the intestine and net nitrogen retention. The concentration of amino acids in the plasma is generally increased depending on tissue demands and the balance of amino acids supplied (Ferguson, 1975). The protection of rapidly degradable proteins by either heat or formaldehyde might make more protein or amino acids available for the host animal, but it might reduce the synthesis of microbial matter and actually decrease productivity. Further to this, it has been observed that excessive heating or over-treatment with formaldehyde of dietary protein can have a detrimental effect on the nutritive value of the protein resulting in decreased digestibility (Vallejo, 1996) and poor animal performance. A proportion of plant protein is already protected by its insolubility and deliberate protection is pointless in this case. The excessive heating or overtreatment of protein may also reduce the efficiency of energy utilization due to low ammonia N in the rumen. Antoniewicz et al. (1992) reported that the FT reactions with protein may be an addition or condensation type in which non-ionic bands are formed between the active side chain groups of amino acids like S–H, –OH, NH 3 etc. and the carbonyl (–C=O) group of FT. Secondary cross-linking by methylene bridges may also be possible. Most of these reactions are reversible under the action of diluted acid solution and do not affect amino acid composition or post-abomasal protein digestibility. Formaldehyde is available in several forms and concentrations. The form most often used in feeds is formalin, a solution containing approximately 40 per cent formaldehyde. The level of formaldehyde to be applied varies according to the type of feed and its protein content. Treatment with higher dose of formaldehyde can have an adverse effect in causing overprotection of the cake, which makes protein indigestible even in lower tract and can hardly supply any RDN for rumen microbial growth. Thus, the level of HCHO is very important and should be optimum due to its criticality. 2.3.2.3 The Coating of Protein Source Particles with Insoluble Substances The coating of protein sources with insoluble substances like blood meal (Orskov, 1992) or fats (Sklan and Tinsky, 1993) have been used. The latter has drawn the attention of researchers since fats, beside their effects on protein protection, are energy sources that could ___________________________________________________________________ 16 Review of Literature … be important for high producing dairy cows during the first stage of lactation. The coating soybean meal with lipid substances (Fish oil, beef tallow) is a protective method against microbial degradation in the rumen. The coating of protein with lipid substances decreased the ruminal degradability. The effect of protection increased with increasing amount of coating agent (Manterola et al., 2001). 2.3.3 Advantages of Rumen Protection In India, dairy animals by and large do not get their required energy content through the normal feed which the animals are offered, as the feed is mostly devoid of any energy rich grains. Success achieved in terms of increase in milk yield (volume) through the feeding of bypass protein is essentially due to the supply of more energy to these energy deficient animals, through the same feed, as the extra amino acids supplied through bypass protein feeding are converted to glucose in liver. Thus, essentially the feeding of bypass protein increases the efficiency of protein and energy utilization within the ruminant system. Numbers of studies have been conducted on feeding of naturally occurring bypass protein like cottonseed cake and maize gluten-meal to lactating ruminants, in India with most of these experiments giving positive results (Ramchandra and Sampath, 1995; Chaturvedi and Walli, 2000; Walli, 2005, Shelke et al., 2012). Post ruminal utilization of nutrients eliminates energy losses associated with fermentation and protein losses incurred in the transformation of dietary protein to microbial protein. However, there are suggestions that protein digestion in the small intestine of the ruminants may not be as complete as it is in monogastric, and that alteration of patterns of absorbable nutrients produced by changing sites of digestion could also influence animal’s endocrine status. Protection from ruminal degradation enables more amino acids to reach the intestine and therefore provide more absorbable amino acids per unit of absorbable energy. Heat treatment and the use of chemical treatment such as formaldehyde have been shown to increase the percentage as well as the total of both dietary protein and amino acids escaping degradation in the rumen. Provision of ruminally protected amino acids will depend very much upon whether shortages of many or only a few amino acids are limiting production (Chalupa, 1975). This is because the magnitude of ruminal destruction of dietary proteins varies, and greatest responses can be expected when rumen soluble proteins are protected. To ___________________________________________________________________ 17 Review of Literature … obtain responses with supplements of protected amino acids, one must know that amino acids are in short supply for animals performing a particular physiological function. A lot of studies showed that diet containing higher amounts of ruminally undegradable proteins or ruminally protected amino acids resulted in increased milk production while other studies show little or no response. The lack of response to ruminally undegradable protein is often due to one of following reasons: (1) the rumen undegradable protein may have been bypassed at the expense of ruminal microbial protein synthesis; (2) the ruminally undegradable protein may have been poorly digested postruminally; and (3) the ruminally undegradable proteins may have been deficient in the amino acids that limiting production (Schingoethe, 1996). However, limited numbers of studies have been conducted on feeding of formaldehyde treated cakes to lactating animals. Sampath et al., (1997) reported significantly higher FCM yield in lactating crossbred cows fed formaldehyde treated GNC (7.8 vs. 9.4 kg/d). Walli and Sirohi (2004) also reported 15 % increase in milk yield on feeding of formaldehyde protected mustard cake to crossbred cows. Sahoo and Walli, (2005) reported that milk yield in mustard cake fed goats increased significantly from 1306 g/d in control group to 1439 g/d in formaldehyde treated mustard cake fed group. Positive response on milk production performance as a result of feeding formaldehyde treated proteins has been observed by several workers (Madson, 1982; Morgan, 1985). Garg et al., (2003) while comparing naturally protected protein (30 % UDP) and processed (formaldehyde treated) sunflower seed meal supplement (optimal-bypass with 75 % UDP) in crossbred cows, found a significant increase in milk yield, milk fat and milk protein percent. Yadav and Chaudhary, (2004) reported significantly increased milk yield and FCM yield in medium producing cows on feeding formaldehyde treated GNC. 2.4 Limiting amino acids Limiting AA refer to EAA that are in shortest supply relative to requirements. The efficiency with which absorbed AA are utilized will likewise be determined by the supply of the first limiting AA. Methionine and Lys were identified, more than 30 years ago, as being the most limiting AA for milk production in dairy cattle (Schwab et al., 1976), growth in steers (Burris et al., 1976) and weaned dairy calves (Schwab et al., 1982), followed by Phe, Ile and Thr as ___________________________________________________________________ 18 Review of Literature … most frequently limiting (Vik-Mo et al., 1974; Derrig et al., 1974; Nichols et al., 1998; Liu et al., 2000). These findings have been confirmed by many other infusion studies. His and Arg were recently identified as limiting when cows are fed grass-silage, barley and oat grain based diets (Vanhatalo et al., 1999; Huhtanen et al., 2002) and that His may be the third limiting AA in some maize-based rations. The quantity and ratio of AA from MCP and dietary CP reaching the intestine may determine whether animal production will respond to feeding rumen protected AA (RPAA). Due to the low Lys content in maize products (16.5 g Lys/kg CP), the contribution of Lys to AA passage to the intestine is reduced when higher levels of maize products are included in the diet (Rogers et al., 1989). It has been suggested that large amounts of maize in rations also reduces microbial growth, thereby reducing AA passage to the small intestine. By supplementing, or infusing, various amounts of individual, or combinations, of EAA into the abomasum and duodenum, researchers measured the effects of AA supplementation on N retention to try and increase milk protein production (Pisulewski et al., 1996; Huhtanen et al., 2002; Schei et al., 2007). 2.5 Amino acid supplementation All AA exist as the isomers D and L which are chemically identical, the one being a mirror image of the other. However AA in plant and animal proteins, as well as some produced industrially, such as Lys, Thr and Trp, are in the L-form, while chemically synthesized Met is a mixture of the two (i.e., DL-Met). Rumen protected methionine (RPM) fed in the D-form are absorbed into the plasma but needs to be converted to the L-isomer within tissues before it can be incorporated into animal proteins. Efficiency of conversion of commercial Met products from the D to L form has been of some concern, but studies have shown that the efficiency of use of D-Met, relative to L-Met, was 960 g/kg in growing steers (Campbell et al., 1996). Free AA are very sensitive to microbial degradation in the rumen and, due to extensive deamination of hydrolyzed AA (Lewis and Emery, 1962), concentration of free AA in rumen fluid is low (Velle et al., 1997; Volden et al., 2001). Supplementing free (crystalline) AA to the diet has not been as effective in dairy cows as it has been in pigs and ___________________________________________________________________ 19 Review of Literature … poultry, and studies with growing cattle have shown rapid degradation and minimal passage of free AA to the duodenum when it is introduced into the rumen (Campbell et al., 1997). Many studies have been conducted to increase the amount of AA that escape from the rumen degradation by protecting EAA such as Lys and Met by chemical alteration or physical protection. 2.6 Rumen protected amino acids Use of protected amino acids, in contrast to protected proteins does not reduce the excess of ammonia in the rumen and thus the load on the liver. Their effectiveness depends upon the optimization of amino acids supply and pattern of amino acids reaching the intestine (Kaufman and Lupping, 1982). When protected amino acids are used, response can mainly be expected in the main production parameters such as milk yield and milk content, growth rate etc. (Chalupa, 1975, Ferguson, 1975). For animals of low growth rate or milk production, amino acid needs can be met by mainly microbial protein which has a good amino acid balance. Amino acid deficiencies are only expected to occur in high performing animals. As far as the question of which amino acid are limiting are concerned, this has been a subject of several studies particularly with respect to lactation since amino acids output in milk can be easily measured. Various techniques have been used in these studies, such as comparison of the amino acid pattern of bacterial and milk protein, changes in the amino acid content of blood after casein infusions and arteriovenous differences across the mammary gland. The conclusion of these studies indicated that methionine is always included in the group of most limiting amino acids, often closely followed by lysine, histidine, phenylalanine and branched-chain amino acids (Kaufmann and Lupping, 1982). The primary methods developed to prevent fermentative digestion of amino acids are structural manipulation to produce amino acid analogs and coating with resistant materials (Chalupa et al., 1996). Various analogs of amino acids have been tested for resistance to ruminal degradation (Ayoade et al., 1982). One of the most tested amino acid is methionine hydroxy anolog. Test results have been variable, with occasional improvements in milk production and milk fat (Kung et al. 1996). Methionine and other amino acids can be protected from bacterial degradation in the rumen by mechanical or chemical methods. The use of both processes has led to commercially marketable products. Amino acids have been coated with polymeric ___________________________________________________________________ 20 Review of Literature … compounds, formulized protein, fat, mixtures of fat and calcium, mixtures of fat and protein, and with calcium salts of long chain fatty acids (Chalupa and Sniffen, 1991). Methionine and lysine are limiting for milk yield and milk protein synthesis when cows are fed cornbased diets. Post ruminal supply of specific amino acids can be increased by supplementing the diet with polymerically encapsulated amino acids. In a number of experiments where protected methionine and lysine have been used to increase protein content and milk yield, responses have been small and variable in terms of milk yield. However, there was a small consistent positive response in milk protein concentration (Donkin et al., 1989; Robinson et al., 1992; Kincaid and Cronrath, 1993; Christensen et al., 1992; Martelli et al., 1993). In these situations, the basal supply of amino acids could be different between experiments which could account for the variable responses obtained. However, methionine supplementation has failed on some occasions to produce the expected increase in the level of milk yield and protein content or growth (Weber et al., 1992). Thus supplying protected amino acids to the small intestine appears to offer some scope for increasing milk and protein concentration. However, it may be necessary to supplement a number of the essential amino acids and not just one or two, in order to get a significant response (Murphy and O’Mara, 1993). Significant progress has been made in developing technologies to increase availability and absorption of EAA by ruminants (NRC, 2001). Protection methods currently used can be divided into three categories: 2.6.1 Liquid sources of hydroxy analogs (chemically modified molecules) Analogs differ in chemical structure from their L-AA counterparts. They contain a hydroxyl instead of an amino group and are therefore recognized by rumen microbes as an organic acid (i.e., a fermentation end product) and not an AA. This aids in their rumen escape potential since they are usually relatively reduced acids (compared to lactate which is more oxidized) with only a selective group of microbes capable of extracting energy by fermenting it. The Met analog may be a free acid in an aqueous solution or a Ca salt in a dry solid (Koenig et al., 1999). As a liquid, it is easy to handle and can be incorporated into feed pellets. The most studied Met hydroxy analog is DL-2-hydroxy-4-Methylthiobutanoic acid (HMB) due to its successful use in monogastric animals. Rumen escape of HMB is a function of the passage rate of the liquid phase from the rumen, the extent of degradation by ___________________________________________________________________ 21 Review of Literature … microorganisms and, to some extent, absorption of HMB across the rumen wall (Koenig et al., 1999). Absorption occurs by diffusion across the rumen (the portion degraded by microbes), omasum and intestinal wall (the portion escaping rumen degradation) into the blood stream. Productive tissues such as the mammary gland and muscle remove HMB from circulation and convert it to L-Met (Rode and Vazquez-Anon, 2006). Esterification of HMB to various alcohols, including the isopropyl ester of HMB (HMBi), decreases its rate and extent of rumen degradation (St-Pierre and Sylvester, 2005). Various in vitro (Vazquez-Anon et al., 2001) and in vivo (Koenig et al., 1999) studies showed Met analog, HMB (DL-2-hydroxy-4-Methylthiobutanoic acid) was more resistant to rumen degradation than Met, but controversy exists about its rumen escape rate. Values range from as low as 10 g/kg of fed HMB recovered in the duodenum (Jones et al., 1988) to as high as 500 g/kg, based on serum Met concentrations (Koenig et al., 1999). This variation may be due to differences in dose levels, method of supplementation or physiological status of the cows (Rulquin et al., 2006). Animal responses to HMB has mainly been an increase in milk fat proportion, possibly due to its effect on rumen fermentation and VFA production, increasing acetate production (Noftsger et al., 2003), with less consistent responses in milk yield and none in milk protein (St-Pierre and Sylvester, 2005). Approximately 500 g/kg of HMB escapes rumen degradation, determined by using blood and milk true protein changes as bioavailability indicators (Noftsger et al., 2005), regardless of whether it is supplemented in liquid or dry form (St-Pierre and Sylvester, 2005). It is quickly absorbed in the small intestine, hydrolyzed into HMB and isopropyl and then converted to Met and acetone, delivering about 480 g/kg Met to the cow (Graulet et al., 2005). 2.6.2 Surface coating or matrices of saturated fatty acids and minerals Development of a lipid-protected product requires identification of a process, and a fat, to use for the matrix, or coating, that provides a reasonable degree of protection in the rumen while allowing adequate intestinal release (NRC, 2001). Apparent bioavailability depends on ruminal escape and intestinal release of AA. The products consist of DL-Met in a matrix of Ca salts of long-chain fatty acids (LCFA), lauric acid, and a fatty acid (FA) preservative; butylated hydroxytoluene (BHT). ___________________________________________________________________ 22 Review of Literature … Carbohydrate-protected products has a combination of coating materials applied to ensure slow degradation in the rumen and slow release of Met in the intestine. It has a core, consisting of DL-Met, coated with starch and several thin layers of stearic acid and ethylcellulose. The latter minimizes enzymatic digestion and so the release of Met depends on physical action and abrasion, wearing away the corners of the pellets (Lapierre et al., 2002). 2.6.3 Surface coating with a fatty acid or pH-sensitive polymer mixture This system involves protection of an AA core by coating it with a lipid/pH-sensitive polymer. Release of AA do not depend on digestive enzymes, but rather a change in pH between the rumen (i.e., pH 6.2 + 0.7), and the abomasum (i.e., pH 2.5 + 0.3). Here, DL-Met is protected by a layer of ethylcellulose covered with a coating of stearic acid. It has improved resistance to rumen degradation due to the presence of a copolymer, poly (2vinylpyridine-co-styrene), altering the steriochemistry of stearic acid. The copolymer solubilizes at low pH, rapidly releasing Met in the abomasum. 2.7 Responses to rumen protected methionine and Lysine dietary supplementation 2.7.1 Effect on Milk Yield and Milk component yield Low concentrations of Met in MP limited the responses to increasing concentrations of Lys in MP and that low concentration of Lys in MP limited the responses to increasing concentration of Met in MP (NRC, 2001). Amino acid concentrations in plasma, measured at different time intervals after feeding the ruminally-protected product can be used to assess and to compare AA availability among product. (Bach and Marshall, 2000). Due to the high rumen protection and intestinal release coefficient of AA in the pH sensitive products, it seems to be the most effective technology with the largest increases in blood AA concentrations, but according to Watanabe et al., (2006), AA protected by a fat coating are just as capable of improving production performances as the pH sensitive polymer products. Lara et al. (2006) reported no treatment effects of supplemented ruminally protected methionine on DM intake (20.38 kg d-1), body weight (599.78 kg), body condition score (2.51 units). However, milk production (35.8 kg d-1) and protein yield (3.16 kg d-1) were ___________________________________________________________________ 23 Review of Literature … increased with addition of RPM. Milk production responded quadratically to methionine levels. The quadratic response in milk protein output was also reported by Guinard and Rulquin (1995), effects which have been associated to a major synthesis of casein and a reduction in urea nitrogen (Wu et al., 1997a,b; Dinn et al., 1998). Holstein cows with a mean production of 35 kg d-1 milk require addition of ruminally protected methionine (16 g d-1) to improve milk production (Lara et al., 2006). Noftsger and St-Pierre, (2003) observed significant improvement in milk production on dietary supplementation of rumen protected methionine (23.6 kg d-1) as compared to that of control (21.7 kg d-1). Similarly, Bach and Marshall (2000) demonstrated an increase in milk yield on supplementing rumen protected methionine to lactating Holsteins cows (45.9 vs 47.7 kg d-1). Similarly, Broderick et al. (2009) reported that supplementation of rumen protected methionine and lysine enhanced the milk yield in crossbred cows (41.5 kg d-1) to that of control (39.4 kg d-1) while Yang et al. (2010) reported increase (18.95 vs 21.55 kg d-1) in milk yield on supplementation of rumen protected methionine and lysine. While Socha et al. (2009) found that there was increase in milk production on abomasal infusion of methionine and lysine during peak and early lactation while there was no difference in mid lactation. Multiparous cows showed increase in milk yield, milk protein and fat yield and efficiency of dry matter utilization for milk production on rumen protected methionine supplementation as compared to primiparous cows (Davidson et al. 2008). Broderick et al. (2009) found that supplementation of rumen protected methionine in diet with reducing CP level by replacing dietary soybean meal with high moisture corn, had no effect on DMI but there was increase in milk yield, milk fat and protein yield. However there are very few published articles involving supplementation of RPL without concurrent supplementation of RPM. Rogers et al. (1989) fed three levels of RP Lys (5.9, 13.5, and 21.1 g/d) to 3 groups of cows at different stages of lactation and reported improved milk and milk protein production when cows were fed maize based diets, but not when soybean meal was added to the ration. Plasma concentrations of Met and Lys were also increased. ___________________________________________________________________ 24 Review of Literature … Xu et al. (1998) reported a positive response in milk yield and milk protein, that was consistent through different stages of lactation, and an increase in milk fat content during early lactation when RPL and RPM were supplemented to a ration limiting in metabolizable Lys and Met. Socha et al. (2005) studied the effect of RPM (10.2 g) and RPL (16.0 g) supplementation at two level of dietary CP i.e. 18.5 and 16.0% and found that there was increase in milk yield, milk fat, milk protein yield in both treatment groups while group receiving diet with 16% CP and supplemented with RPM and RPL performed with same efficiency compared to the group receiving 18.5% CP. Other studies also found that there was increase in milk yield, milk fat yield and milk protein yield without affecting dry matter intake on RPM and RPL supplementation. (Wu et al., 1997; Armentano et al., 1997; Robinson et al., 1997). In contrasts, many researchers Berthiaume et al. (2001), Misciattelli et al. (2003), Girard et al. (2004), Noftsger et al., (2005), Broderick and Muck, (2008), Davidson et al. (2008), Benefield et al. (2009) did not found any effect on milk production on supplementing rumen protected methionine while Swanepoel et al. (2011) did not find any effect on milk production on supplementing rumen protected lysine alone. 2.7.2 Effect on plasma amino acids Increased supply to the small intestine of any AA is expected to change its concentration in blood plasma and, possibly, improve availability of that AA for milk protein synthesis. This was demonstrated by Blauwiekel et al. (1997) in an experiment in which lysine supplementation increased duodenal lysine flow, as well as the concentration of lysine in plasma, leading to an increased milk and milk protein yield. Baumrucker (1985) explained that the transport systems for absorption of AA depend on transport specificity and competition among AA. As feeding RPL provides more lysine to cells with a Y+ (cationic) transport system, the uptake of AA sharing the same transport system (i.e., arginine and isoleucine) may be reduced through competitive inhibition with lysine. ___________________________________________________________________ 25 Review of Literature … Supplementation with RPM caused an elevation in arterial concentration of Met (Berthiaume et al., 2001, Overton et al., 1996, 1998; Blum et al., 1999) or the results were same when Met infused postruminally (Guinard and Rulquin, 1995; Pisulewski et al., 1996; Varvikko et al., 1999). It is reported that there were decrease in plasma methionine concentrations (Rogers et al., 1987) or decrease in most non-essential AA (NEAA) as well as threonine, leucine and tryptophan (Swanepoel et al., 2011) when RPL was fed. Swanepoel et al. (2011) reported that the plasma lysine concentration was only numerically increased with RPL feeding. Results of Weekes et al. (2006) suggested that a decreased concentration of plasma AA (especially histidine) without a positive milk protein response could be due to a more efficient catabolism or deposition of AA into body protein, which might be supported by the trend for lower BCS loss on RPL supplementation. 2.7.3 Effect on blood metabolites Postruminal infusion of a basal amount of Lys and incremental amounts of Met resulted in a linear increase in plasma BHBA (ß- Hydroxyl Butyric Acid) of cows entering the second 100 d of lactation (Socha, 1994), but had no effect on plasma BHBA of cows assigned to the treatments before 50 DIM (Pisulewski et al., 1996; Socha, 1994) or after 150 DIM (Socha, 1994). In these same infusion studies, increasing the intestinal supply of Met linearly reduced plasma NEFA concentrations when the cows were assigned to the studies before 50 DIM (Pisulewski et al., 1996; Socha, 1994) but not when they began receiving treatments as they approached 100 DIM or after 150 DIM (Socha, 1994). One potential reason why blood concentrations of energy metabolites such as NEFA, BHBA, and glucose are not affected by improved Lys and Met nutrition in production studies (Chow et al., 1990; Chapoutot et al., 1992; Xu et al., 1998) is that the effect may be transitory. In the production studies, the experimental periods were 21 d or longer, with blood samples usually being collected several weeks after initiation of treatments. This is in contrast to the infusion experiments where the length of experimental periods was 10 to 14 d and all blood samples were taken less than 2 wk after initiation of treatments (Socha, 1994; Pisulewski et al., 1996). In contrast, Socha et al., (2005) found that there were no effects of RPM and RPM+L supplementation on postpartum plasma NEFA and BHBA concentrations. While cows fed ___________________________________________________________________ 26 Review of Literature … RPM+L tended to have lower plasma glucose concentrations than cows fed only the basal diet and RPM supplemented group. Feeding RPM reduced serum urea concentrations compared with feeding on feeding RPM+L. They also reported that the effect of RPAA supplementation on plasma glucose concentrations was dependent upon week postpartum. Between wk 1 and 4 postpartum, plasma glucose concentrations of cows receiving RPM+L declined at a more rapid rate than cows receiving either the basal diet or RPM. However, between wk 4 and 7 postpartum, plasma glucose concentrations of RPM+L supplemented cows increased, whereas plasma glucose concentrations of cows receiving no RPAA or RPM remained essentially unchanged. Immediately following parturition, glucose concentrations decreased in cows receiving 16 % CP in diet and 15 gm of RPM supplemented group, 16% CP in diet and 6 gm of RPM plus 40 g/d of a RPM+L supplemented group, and 18.5% CP in diet and 6 gm of RPM plus 40 g/d of a RPM+L supplemented group and increased in cows receiving 18.5% CP and 15 gm of RPM supplemented group. Similarly, in other reports, plasma NEFA, glucose and BHBA were not affected when lactating dairy cows were fed RPM+L (Chow et al., 1990; Chapoutot et al., 1992; Xu et al., 1998). Further, Misciattelli et al. (2003) did not found any effect on plasma NEFA concentration on supplementation of rumen protected methionine and lysine, and Davidson et al. (2008) on supplementation of rumen protected methionine. There was no significant effect of abomasal infusion of methionine and lysine on plasma NEFA during mid lactation (Socha et. al., 2008). However, decrease in plasma nonesterified fatty acids concentrations in preruminant calves (Auboiron et al., 1995; Chilliard et al., 1994) and lactating cows (Pisulewski et al., 1996; Rulquin and Delaby, 1997) with increased amounts of Met in MP had been reported. Similarly Socha et al. (2008) reported that there was decrease in plasma NEFA concentration on abomasal infusion of rumen protected methionine and lysine during early lactation and Bach et al. (2000) reported decreased plsma NEFA concentration on supplementing rumen protected methionine and lysine. Socha et al., (2005) found that there was no effect on BUN level on abomasal infusion of methionine and lysine. While Berthiaume et al. (2001) and Bach et al. (2000) ___________________________________________________________________ 27 Review of Literature … found increased blood urea nitrogen concentration on supplementation of rumen protected methionine. Davidson et al., (2008) reported increased plasma triglyceride and VLDL level on supplenentation of rumen protected methionine. The increase in milk fat percent may relate to the role of AA in the intestinal and hepatic synthesis of chylomicrons and VLDL. Required substrates for the synthesis of chylomicrons and VLDL, in addition to the presence of the long-chain fatty acids that stimulate their formation, include apolipoproteins and phospholipids (Bauchart et al., 1996). The synthesis of apolipoproteins requires AA. The synthesis of phosphatidylcholine (lecithin), the most abundant phospholipid, requires choline. It has been demonstrated that a portion of the dairy cows’ requirement for Met is as a methyl donor for choline synthesis (Sharma and Erdman, 1989; Erdman, 1994), choline can be a limiting nutrient for milk fat synthesis. That Met and Lys may sometimes be limiting for the synthesis of chylomicrons or VLDL such that the availability of long chain fatty acids for milk fat synthesis is reduced has not been demonstrated. However, there is limited evidence that formation or secretion of these lipoproteins can be enhanced with improved Met and Lys nutrition (Auboiron et al., 1995; Durand et al., 1992). 2.8 Reproductive efficiency of animals fed on protected protein and rumen protected amino acids Studies conducted at NDRI have shown that the feeding bypass protein to dairy animals improves reproductive efficiency in both males and females. Feeding bypass protein as such leads to higher growth rate in cattle/buffalo calves and goat kids. This improved growth rate as such results in early maturity of both male and female animals, which has also an over all impact on lifetime production of the animals. Apart from that, the feeding of bypass protein improved the conception rate and reduced the number of days open in crossbred cows (Kaur and Arora, 1995). Similarly, feeding bypass protein increased the sexual behavior and seminal attributes in Mehsana buffalo bulls and also in crossbred bucks (Walli, 2008). Thus, the improvement in reproductive efficiency of dairy animals is yet another distinct advantage of feeding bypass protein to such animals. Protein in excess of lactation requirements has been shown to have negative effects on reproduction. Several workers have reported that feeding diets containing 19 percent or ___________________________________________________________________ 28 Review of Literature … more CP in diet DM lowered conception rates (Bruckental et al., 1989; Canfield et al., 1990; Jordan and Swanson, 1979; McCormick et al., 1999). Others have observed that cows fed 20 – 23 percent CP diets (as compared to 12– 15 percent CP) had decreased uterine pH, increased blood urea, and altered uterine fluid composition (Jordan et al., 1983; Elrod and Butler, 1993). In a majority of the studies reviewed by Butler (1998), plasma progesterone concentrations in early lactation cows were lower when diets contained 19– 20 percent CP vs. lower concentrations of CP. Feeding more than the recommended amounts of protein or inclusion of excess degradable protein in the diet adversely affects animal reproduction. On such diets, it is likely that relatively more ammonia is released in the rumen through the process of deamination by bacterial enzymes. The excess ammonia or urea produced in the rumen can diffuse from the alimentary tract to the peripheral circulation and appear in the uterine secretions (Tamminga, 2006), thereby disturbing endocrine functions (Ferguson et al., 1986) and the corpus luteum (Garwacki et al., 1979), and may also reduce motility and survival of sperms as a result of a changed uterine environment. In a review on effects of protein on reproduction, Butler (1998) concluded that excessive amounts of either RDP or RUP could be responsible for lowered reproductive performance. However, intakes of ‘‘digestible’’ RUP in amounts required to adversely affect reproduction without a coinciding surplus of RDP would be uncommon. In most of the studies, reviewed by Butler (1998), excessive RDP rather than excessive RUP was associated with decreased conception rates. Canfield et al. (1990) showed that feeding diets containing RUP to meet requirements while feeding RDP in excess of requirements resulted in decreased conception rates. Garcia-Bojalil et al. (1998) reported that RDP fed in excess (15.7 percent of DM) of recommendations decreased the amount of luteal tissue in ovaries of early lactation cows. Although most studies have indicated an adverse effect on reproductive performance of feeding high CP diets, others indicate no effect of diet CP on reproduction. Carroll et al. (1988) observed no differences in pregnancy rate or first service conception rates of dairy cows fed 20 percent CP and 13 percent CP diets. Howard et al. (1987) reported no difference in fertility between cows in second and greater lactation fed 15 percent CP or 20 percent CP ___________________________________________________________________ 29 Review of Literature … diets. There are many theories as to why excess dietary CP decreases reproductive performance (Barton, 1996a, 1996b; Butler, 1998; Ferguson and Chalupa, 1989). The first theory relates to the energy costs associated with metabolic disposal of excess N. To the extent that additional energy may be required for this purpose, this energy may be taken from body reserves in early lactation to support milk production. Delayed ovulation (Beam and Butler, 1997; Staples et al., 1990) and reduced fertility (Butler, 1998) have been associated with negative energy status. Another effect of negative energy status is decreased plasma progesterone concentrations (Butler, 1998). Another theory is that excessive blood urea N (BUN) concentrations could have a toxic effect on sperm, ova, or embryos, resulting in a decrease in fertility (Canfield et al., 1990). High BUN concentrations have also been shown to decrease uterine pH and prostaglandin production (Butler, 1998). High BUN may also reduce the binding of leutinizing hormone to ovarian receptors, leading to decreases in serum progesterone concentration and fertility (Barton, 1996a). Ferguson and Chalupa (1989) reported that byproducts of N metabolism may alter the function of the hypophysealpituitary-ovarian axis, therefore decreasing reproductive performance. And last, high levels of circulating ammonia may depress the immune system and, therefore, may result in a decline in reproductive performance (Anderson and Barton, 1988). Milk urea nitrogen (MUN) and blood urea nitrogen (BUN) are both indicators of urea production by the liver. Milk urea N concentrations greater than 19 mg/dl have been associated with decreased fertility (Butler et al., 1995). Likewise, BUN concentrations greater than 20 mg/ dl have been linked with reduced conception rates in lactating cows (Ferguson et al., 1988). Bruckental et al. (1989) found that BUN levels increased when diet CP was increased from 17 to 21.6 percent and pregnancy rate decreased by 13 percentage units. In a case study, Ferguson et al. (1988) observed that cows with BUN levels higher than 20 mg/dl were three times less likely to conceive than cows with lower BUN concentrations. Although high BUN concentrations have been associated with decreased reproductive performance, others have reported no adverse effects on pregnancy rate, services per conception, or days open with BUN levels above 20 mg/dl (Oldick and Firkins, 1996). ___________________________________________________________________ 30 Review of Literature … Studies by Carroll et al. (1988) and Howard et al. (1987) indicated that maintaining a strict reproductive management protocol can reduce the negative effects of excess protein intake on reproduction. Barton (1996a) demonstrated that an intense reproductive program could be used to reach reproductive success regardless of diet CP level or plasma urea N concentrations. These studies highlight the idea that dietary protein is just one of many things that have an effect on reproductive performance. Protein intake, along with other factors such as reproductive management, energy status, milk yield, and health status all have an effect on reproductive performance in dairy cattle. The toxic effects of ammonia, urea or other unidentified nitrogenous compounds influence the survival of ova, sperms and early developing embryos (Jordan et al., 1983; Sahlu et al., 1992). Excess protein, irrespective of protein source or degradability, decreases uterine pH on day 7 of the estrous cycle (Elrod and Butler, 1993) by decreasing the uterine Mg, K and PO 4 concentrations during the luteal phase (Jordan et al., 1983) which can in turn inhibit the endometrial carbonic anhydrase activity (Rowlett et al., 1991). The decrease in pH in the uterus adversely affects the activity and survivability of sperms in the reproductive tract (White, 1974). Ferguson et al., (1988) suggested that dietary protein intake producing serum urea-N greater than 200 mg/l were indicative of excess protein intake, particularly RDP. High protein intake may also influence the reproductive system as a result of activation of different mechanisms in the liver for hepatic detoxification and the increase of energy demand for deamination of excess amino acids (Visek, 1984). Excess ammonia may also disturb intermediary metabolism and thus increase blood concentrations of urea, glucose, non-esterified fatty acids and insulin. Smith (1986) reported that some nitrogenous end products might alter the functioning of the hypophyseal-pituitary-ovarian axis by decreasing LH pulse frequency and amplitude. Scanty literature is available on the effect of supplemental rumen protected Met and Lys on reproductive performance of cows. Berthiaume et al. (2001) and Bach et al. (2000) reported decreased blood urea nitrogen on supplementation of rumen protected methionine, but they did not study its effect on reproduction. ___________________________________________________________________ 31 Review of Literature … Ardalan et al. (2009) reported that there was significant decrease in days to first oestruos, open days, services per conception and increase in pregnancy rate in Holstein dairy cows fed 18 gm RPM from 4 week prepartum to 20 weeks postpartum. Xu et al. (1998) reported the overall incidence of health related disorders were numerically lowest for the dairy cows fed high amount of rumen protected methionine and lysine. In contrast, Polan et al. (1991) reported that supplementation of rumen protected form of Methionine and Lysine had no significant effect on days to first service, services per conception and calving interval in dairy cows. Ardalan et al. (2010) reported that there was increase in milk yield while incidences of postpartum reproductive and metabolic diseases viz. retained placenta, mastitis, uterine problem, dystokia, were significantly reduced in Holstein dairy cow fed RPM 18 g/d from 4 week prepartum to 14 weeks postpartum. Reviewing the recent information on the protein nutrition of transition cows suggest that high producing dairy animals, especially during early lactation, DMI is decreased which adversely affects the productivity. Where as high milk yield during early lactation retards development of ovarian follicles, prolonging the postpartum interval to first ovulation. High yield is antagonistic to the expression of estrus and is associated with reduced conception rate. Reproductive disorders are one of the major factors reducing the milk yield and affecting the production potential of cows. Therefore, this research project was planned to incorporate the rumen protected methionine and lysine in the ration of transition cows to avoid the negative N balance and to enhance the milk productivity with desirable composition, which will have far reaching benefits on their reproductive performance. ___________________________________________________________________ 32 CHAPTER – 3 MATERIALS AND METHODS 3. Materials and Methods _________________________________________________ PHASE – I – Feed, fodder and product evaluation 3.1 Feed and fodder analysis 3.1.1 Proximate principles and cell wall constituents Feeds, fodders and total mixed rations were dried to constant weight at 80oC and ground (1 mm) using a Willey mill. The ground samples were stored in air tight polyethylene bags for further analysis. Dry matter, proximate analysis of feed i.e. crude protein (CP), ether extract (EE), crude fibre (CF) and ash was carried out as per AOAC (2005) and cell wall fractions i.e. neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin were done as per modified method of Van Soest et al. (1991) using amylase. TDN of feeds and fodders was calculated as per formulae given in NRC (2001). For feeds that are not animal proteins or fats and that contain some NDF (forages, many byproducts, concentrates), the following equations are used: TDN = tdNFC + tdCP + (tdFA X 2.25) + tdNDF - 7 Where, tdNFC (Truly digestible NFC) = 0.98 (100 - [(NDF-NDICP) + CP + EE + Ash]) X PAF tdCPf (Truly digestible CP for forages) = CP X exp[ -1.2 X (ADICP/CP)] tdCPc (Truly digestible CP for concentrates) = 1- (0.4 X (ADICP/CP))] X CP tdFA (Truly digestible FA) = FA Note: If EE < 1, then FA = 0 tdNDF (Truly digestible NDF) = 0.75 X (NDFn – L) X [1 – (L/NDFn) 0.667] 3.1.2 Amino acids analysis of feed samples using HPLC (AOAC, 2005) Amino acids profile of common feedstuffs were analysed using High Performance Liquid Chromatography (HPLC) except tryptophan. _______________________________________________________________________ 33 Materials and methods … A. Reagents a) Redrying solution: In a ratio of 2:2:1 (v/v) methanol, water and triethylamine was mixed to formulate the redrying solution. b) Derivatization reagent: This was prepared by mixing methanol: triethylamine: water: phenyl isothiocyanate in a 7: 1: 1:1 ratio (v/v). c) Sample diluents: Disodium hydrogen phosphate 710 mg was dissolved in 1 litre of HPLC grade water. pH of the solution was adjusted to 7.4 by adding 10% aqueous phosphoric acid. Then acetonitrile was added @ 5% by volume (v/v) to the resultant solution. d) Eluent A: Sodium acetate trihydrate 19.0 g was dissolved in 1 litre of HPLC grade water, to this 0.5 ml triethylamine was added and mixed properly. The pH of the solution was adjusted to 6.4 by adding glacial acetic acid and was filtered through 0.45 µ Millipore filter paper. From this solution, 940 ml was taken in a 1 litre volumetric flask and volume was made to 1 litre by adding HPLC grade acetonitrile. It was degassed by sonicating few second under vacuum. e) Eluent B: Acetonitrile 600 ml and HPLC grade water 400 ml were mixed and filtered through 0.45 µ Millipore filter paper and degassed by sonicating under vacuum pressure. B. Preparation of protein hydrolysate Feed sample (75 mg) was taken in duplicate in 30 ml capacity vials and 10.0 ml 6N HCl was added. The tube was sealed under constant supply of nitrogen gas. The sealed vial was kept at 1100C for 24 h for complete hydrolysis of protein. At the time of analysis, the seal was broken and filtered through whatman no. 1 filter paper and the filtrate was filtered through 0.45 µ Millipore filter paper. C. Derivatization An aliquot of 5 µl of protein hydrolysate was taken in a reaction vial and mixed with 20 µl redrying solution. It was dried under vacuum and several additional drying were carried out. After drying, 20 µl derivatization solution was added and mixed gently. The tube was kept at room temperature for 20 minutes and then again dried under vacuum using ______________________________________________________________________ 34 Fig. 3.1 Chromatogram of standard amino acids profile Fig 3.2 Standard curve of choline Materials and methods … PICO tag assembly. Then the sample was diluted to 100 µl using sample diluents and was used for amino acids analysis. D. Standard preparation In a sample vial, 5 µl of amino acid standard was taken and dried, redried and derivatized as described above for sample. E. Analytical procedure An aliquot of 10 µl of derivatized sample was injected in to the HPLC system fitted with absorbance detector adjustable at 254 nm and PICO tag column (15.0 cm × 3.9 mm). The gradient controller programming for eluents with respect to time is in table given under. Gradient controller programming for eluents used for amino acids analysis Time (min) Flow Eluent A Eluent B Initial 1.0 100 % 0% 10.0 1.0 54 % 46 % 10.5 1.0 0% 100 % 11.5 1.5 100 % 0% 12.0 1.5 0% 100 % 12.5 1.5 100 % 0% 20.0 1.5 100 % 0% 20.5 1.0 100 % 0% The derivatized standard mixture of amino acids and unknown sample was injected under identical condition and their areas were compared with the standard (Fig 3.1). ______________________________________________________________________ 35 Materials and methods … 3.1.3 Estimation of tryptophan Tryptophan was analysed by colorimetric method given by Mertz et al. (1975). Composition of reaction mixtures A. Papain solution Dissolve 400 mg technical grade papain in 100 ml 0.1 N sodium acetate buffer pH 7.0. Fresh solution was prepared every day. B. Reagent A Dissolved 135mg FeCl 3 .6H 2 O in 0.25 ml water and diluted to 500 ml with glacial acetic acid containing 2% acetic anhydride. C. Reagent B 30 N H 2 SO 4 D. Reagent C Mixed equal volumes of reagent A and B about one hour before use. E. Standard Tryptophan Dissolved 5 mg tryptophan in 100 ml water (50 ug/ml). Air-dried, powdered and defatted feed samples (100 mg) were weighed into a small test tubes. 5 ml papain solution was added, shaked well. The tube were closed and incubated at 65°C overnight. The digested sample was cooled to room temperature, centrifuged and collected the clear supernatant. 1ml supernatant was taken in test tube and 4ml reagent C was added. Then it was mixed in a vortex mixer and incubated at 65°C for 15 min. the digest was cool to room temperature and read the orange-red colour at 545 nm. A blank was set with 5ml papain alone. 0, 0.2, 0.4, 0.6, 0.8 and 1ml standard tryptophan were pipetted out and made up to 1 ml with water. 3.2 Rumen protected methionine (RPM) and rumen protected lysine (RPL) product evaluation The RPM and RPL were purchased from market. Both products were in the form of ______________________________________________________________________ 36 Materials and methods … encapsulation with fatty acids and prepared by spray freeze drying technology. 3.2.1 Methionine content in RPM Methionine in RPM product was estimated by Kjeldahl method. 0.5 gm RPM sample in triplicate was digested in the digestion tube with conc. H 2 SO 4 in presence of digestion mixture (Na 2 SO 4 and CuSO 4 in the ratio of 9:1) over a hot plate. After completion of digestion, the contents were cooled and transferred to 100 ml volumetric flask and total volume was made up to the mark with distilled water. An aliquot (10 ml) was transferred to Micro kjeldahl apparatus and sufficient amount of 40 % of NaOH solution was added to make the contents alkaline. The distillate was collected in a conical flask containing 20 ml of 2 % boric acid with mixed indicator (20 g boric acid powder, 0.1 percent solutions of each methyl red and bromocresol green in 2:1 proportion was added and the volume was made to 1000 ml in distilled water). The distillate was titrated against standard 0.01 N H 2 SO 4 . This was the total nitrogen content. The Methionine content in the sample was calculated by multiplying nitrogen content with 10.65. Volume of H2SO4 used x Normality x 0.0014 x Volume made Methionine % = x 10.65 x 100 Weight of sample x Volume taken 3.2.2 Lysine content in RPL The total nitrogen was estimated in 3.2.1. The Lysine content in the sample was calculated by multiplying nitrogen content with 6.52. Volume of H2SO4 used x Normality x 0.0014 x Volume made Lysine % = x 6.52 x 100 Weight of sample x Volume taken ______________________________________________________________________ 37 Materials and methods … PHASE – II – In vitro study 3.3 In vitro determination of microbial protein yield 3.3.1 Collection of rumen liquor The rumen liquor was collected from three rumen fistulated cattle before feeding and watering in the morning after removing the ice cap. Rumen liquor was collected from different sides of rumen and at different depths by to and fro movement of the plastic tube in order to get a representative and homogenous sample. The samples were mixed and strained through four layered muslin cloth into a thermos flask which was previously flushed with CO 2 to maintain anaerobic conditions and brought to laboratory immediately for in vitro studies. 3.3.2 Preparation of substrates in the form of total mixed rations (TMR’s) Concentrate mixture, green maize and wheat straw were dried in hot air oven at 70oC for overnight. Samples were ground separately to reduce particle size (1mm) and mixed weighed quantities of concentrate and roughage in the different proportions. The sample was again mixed by a mixer to avoid sample variation. Three TMRs were prepared by taking concentrate to roughages ratio as 60:40, 50:50 and 60:40 respectively for three groups as shown in table 3.1. In roughage, wheat straw and green maize were taken in the ratio of 40:60. Table 3.1 Composition of TMRs Ingredients TMR-1 TMR-2 TMR-3 Wheat Straw 16 parts 20 parts 24 parts Maize fodder 24 parts 30 parts 36 parts Concentrate Mixture 60 parts 50 parts 40 parts 19.8 parts 16.5 parts 13.2 parts Maize ______________________________________________________________________ 38 Materials and methods … GNC 3.3.3 12.6 parts 10.5 parts 8.4 parts Mustard cake 7.2 parts 6.0 parts 4.8 parts Deoiled rice bran 6.6 parts 5.5 parts 4.4 parts Wheat bran 12.0 parts 10.0 parts 8.0 parts Mineral Mixture 1.2 parts 1.0 parts 0.8 parts Salt 0.6 parts 0.5 parts 0.4 parts In vitro determination of microbial N using nitrogen balance technique The incubations were carried out in 100 ml calibrated Glass syringes (Leur make) as described by Menke and Steingass (1988). The syringes were incubated in incubator at 39 ± 0.5oC. The substrate (500 mg) was weighed on a plastic boat with removable stem and placed into the bottom of the glass syringe without sticking to the sides of the syringes. The piston was lubricated with petroleum jelly and pushed inside the syringe. The syringes were incubated at 39 ± 0.5oC up to 48hr. The medium was prepared by mixing 400 ml distilled water, 200 ml rumen buffer solution and 200 ml macro minerals solution, 1 ml Resazurine solution, 0.1 ml micro minerals solution and 40 ml reducing solution (prepared fresh and added just prior to incubation). The medium was pre-warmed to 39oC and bubbled with CO 2 till the blue color of the medium vanished. The details of solutions and the order in which these were added prior to the filling in syringes. Items 30 syringes 45 syringes 60 syringes Distilled water 365 ml 550 ml 730 ml Micromineral solutiona 0.1 ml 0.15 ml 0.185 ml Solution-I ______________________________________________________________________ 39 Materials and methods … Rumen buffer solutionb 183 ml 275 ml 365 ml Macromineral solutionc 183 ml 275 ml 365 ml Resazurine solutiond 0.95 ml 1.45 ml 1.90 ml 1N NaOH 1.6 ml 2.4 ml 3.1 ml Na 2 S.7H 2 O 220 mg 330 mg 440 mg Distilled water 37 ml 55 ml 73 ml 330 ml 500 ml 660 ml Solution-IIe Solution-III Rumen liquor a Micromineral solution CaCl 2 .2H 2 O 13.2 g MnCl 2 .4H 2 O 10.0 g CoCl 2 .6H 2 O 1.0 g FeCl 3 .6H 2 O 8.0 g Dissolved in 100 ml of water. b Rumen buffer solution NH 4 HCO 3 NaHCO 3 4.0 g 35.0 g Dissolved in 1000 ml of water. c Macromineral solution Na 2 HPO 4 anhydrous 5.70 g KH 2 PO 4 anhydrous 6.20 g MgSO 4 .7H 2 O 0.60 g Dissolved in 1000 ml of water. ______________________________________________________________________ 40 Materials and methods … d Resazurine solution e 0.10 % w/v Reducing solution 1N NaOH Na 2 S, 9H 2 O Water 4.0 ml 625 mg 95 ml Rumen liquor was collected from three donor bulls, fitted with permanent rumen fistula. Donor animals were fed on roughage and concentrate based diet (2.0 kg concentrate mixture and wheat straw ad libitum). Rumen liquor was collected before feeding the donor animal into a pre-warmed thermo-flask and brought to the laboratory. The rumen liquor was bubbled with CO 2 for about 2 minutes and filtered through 4 layers of muslin cloth. After medium became colourless, the required amount of strained rumen liquor (SRL) was added. The ratio of medium to rumen liquor was 2:1. Then, 30 ml of incubation medium was injected to each syringe using auto pipette. The syringes were shaken gently, residual air or air bubble, if any, was removed and the outlet was closed. The level of piston was recorded and the syringes were placed in an incubator (39 ± 0.5oC). The syringes were shaken every 30 minutes for first 2 h from the start of the incubation and thereafter every 2 h up to 24 h of incubation. After 24 hr incubation, the content of the syringe was centrifuged at 8000 rpm for 15 min at 4oC. N content of the pellet were estimated and corrected for N in blank. The pellet obtained from second tube was treated with NDS for 24 hr. Neutral Detergent insoluble nitrogen were estimated by Kjeldahl method. MN = Pellet N (24 hr) – Pellet N in blank (0 hr) – NDIN Where MN – Microbial Nitrogen NDIN – Neutral Detergent Insoluble Nitrogen ______________________________________________________________________ 41 Materials and methods … 3.4 Separation of rumen bacteria from rumen liquor The strained rumen liquor was centrifuged in duplicate at 1000 rpm for 2 min. to remove feed particles. The supernatant were centrifuged at 8000 rpm for 15 min at 4oC. Supernatant was discarded to separate bacterial pellet. Bacterial pellets were washed with Bridge detergent solution and centrifuged. And then pellet was treated with 10% TCA solution and centrifuged. N and amino acids content of the bacteria were estimated as discussed above in 3.1. 3.5 Separation of the protozoa from rumen contents The strained rumen liquor was strained through six layers of surgical gauze without squeezing, then scraped the deposit from the gauze, resuspended it in distilled water and restrained through clean gauze. The combined filtrates were then allowed to stand for atleast 1 hr. in boiling-tubes filled to the top, when the larger, whiter, and heavier protozoa gradually sank to the bottom. The supernatant was carefully decanted, the deposit washed several times by decantation with 0.003 M phosphate buffer (pH 6.8) and finally with distilled water. The final deposits consisted almost entirely of protozoa. Again supernatant was carefully decanted and protozoa were dried at 550C in hot air oven. N and amino acids content of the bacteria were estimated as discussed above in 3.1. 3.6 In vitro estimation of RUP intestinal digestibility A three step in vitro procedure developed by Calsamiglia and Stern (1995) were adopted for estimating intestinal digestibility of the RUP fraction of feed proteins. Ruminally undegraded feed residue containing about 15 mg residual N were incubated for 1 hr in 10 ml 0.1 N HCl solution containing 1 g/L of pepsin. The mixture were neutralized with 0.5 ml 1N NaOH and 13.5 ml of pancreatin solution followed by 24 h incubation. The undigested protein as precipitated with TCA solution and RUP intestinal digestibility was calculated as follows, TCA Soluble N x 100 RUP Intestinal Digestibility = Undegraded N ______________________________________________________________________ 42 Materials and methods … PHASE –III - in sacco Study 3.7 Estimation of RDP and RUP of different feed stuffs RDP and RUP of different feed stuffs were estimated by in sacco nylon bag technique as described by Orskov and McDonald (1979). Three rumen cannulated bulls (18 to 24 months of age) were stalled in the well ventilated animal shed of Cattle Yard, NDRI, Karnal. The animals were kept on a constant diet of wheat straw and concentrate mixture (65:35) for one month of preliminary feeding period, before commencing the experiment. The concentrate mixture consisted of maize, barley, GNC, mustard cake, DORB, wheat bran, mineral mixture and salt in proportion same as listed in Table 3.2. The animals were also offered clean drinking water ad libitum twice a day. The method of Mehrez and Orskov (1977) was used for suspension and removal of nylon bags in the rumen. Pre-weighed nylon bags (9 x 15 cm with 40 µ pore size) containing 3 g of different ground feedstuffs were tied with nylon threads and fastened tightly to three iron chains. The chains containing the bags were then placed in the rumen of three fistulated animals so that the observation for each ingredient could be recorded, in triplicate, in three animals. The bags were taken out at regular intervals of time viz., 0, 2, 4, 6, 8, 12, and 24h. After removal from the rumen, the bags were washed thoroughly by swirling vigorously in plastic trough filled with a continuous supply of fresh water, till the water in the trough was clear. The bags were drained off to get rid of excess water and then dried in oven at 550C for 48 h. The lumps formed inside the bags were broken manually and the material was thoroughly mixed, followed by drying at 550C till constant weight. The dried bags after desiccation were weighed again in order to determine the dry matter loss at different hours for each of the samples. The residue left in each bag was subjected to nitrogen estimation. From the degradability data obtained at various hours, the time versus degradation curves were drawn and the constants a, b and c was measured by computer analysis using ______________________________________________________________________ 43 Materials and methods … the following expression given by Mehrez and Orskov (1977): P = a + b (1- e –ct) Where as, P = Degradability t = Time a = Intercept on Y-axis b = Potentially degradable fraction and c = Degradation rate or rate constant RDP and RUP were calculated as follows. RDP = A + B [kd / (kd + kp)] RUP = B [kp / (kd + kp)] + C Here, Fraction A is the percentage of total CP that is NPN (i.e., assumed to be instantly degraded) and a small amount of true protein that rapidly escapes from the in situ bag because of high solubility or very small particle size. Fraction B is the rest of the CP and includes the proteins that are potentially degradable. Fraction C is the percentage of CP that is completely undegradable; this fraction generally is determined as the feed CP remaining in the bag at a defined end-point of degradation. 3.8 Estimation of rumen escape potential of commercial RPM and RPL product Rumen escape potential of the RPM and RPL were determined using an in situ nylon-bag technique (Mehrez and Orskov, 1977; Osuji et al, 1993; Nozière and MichaletDoreau, 2000). Pre-weighed nylon bags (9 x 15 cm with 40 µ pore size) containing 3 g of RPM or RPL were tied with nylon threads and fastened tightly to three iron chains. The chains containing the bags were then placed in the rumen of three fistulated animals so that the observation for each ingredient could be recorded, in triplicate, in three animals. After ______________________________________________________________________ 44 Materials and methods … removal of bags, these were rinsed with cold water, dried at 550C for 48 hours and weighed. Contents of the bags were subjected to N analysis (AOAC, 2005) by Kjeldahl Method. Data obtained at various hours from the degradability, the time versus degradation curves were drawn and the constants a, b and c was measured by computer analysis using the following expression given by Mehrez and Orskov (1977). Effective degradability was calculated as per equations in 3.7. PHASE – IV - In vivo Study Eighteen crossbred cows (Karan Fries) in second to fourth lactation with most probable production ability (MPPA) of around 4500 lit milk production were selected from the cattle herd maintained at NDRI, Karnal. The experimental animals were randomly divided into two dietary groups of 9 each in control and experimental group on the basis of MPPA and lactation number. 3.9 Location of experiment The study was conducted in the experimental cattle shed of National Dairy Research Institute, Karnal, India located at 29° 42″ 20 sec N and 76° 58″ 52.5 sec E at an altitude of 834 feet amsl. Minimum and maximum ambient temperature ranged from near freezing point in winter to 45° C in summer with annual rainfall of 700 mm. The experiment was conducted in May, 2011 to April, 2012 with daily minimum and maximum temperature averaging 5.6° C and 40° C. 3.10 Selection and distribution of animals The animals were put on experiment around 40 days before parturition and given adaptation period of ten days. The duration of experiment was thirty days before calving to 120 days after parturition. The details of the experimental animals are given in table 3.2. 3.11 Estimation of most probable production ability (MPPA) The Most Probable Production Ability (MPPA) or Expected Producing Ability (EPA) was computed on the basis of formula given by Lush (1945) as follows: nr MPPA = A + x (P-A) 1 + (n-1) ______________________________________________________________________ 45 Materials and methods … Where, A – Population mean n – Total number of animals r – Repeatability of lactation milk record P – Milk yield in previous lactation Table 3.2 Details of experimental animals Group 1 (Control) Group 2 (RPM plus RPL supplementation) Sr. Animal No. No. 1 KF-6918 2 Lactation Sr Animal No. No. No. No. 5034 3 1 KF-6774 4757 4 KF-7068 4234 2 2 KF-6969 3952 1 3 KF-6997 3949 1 3 KF-7083 3975 1 4 KF-7034 4770 1 4 KF-7045 4010 1 5 KF-6960 4037 1 5 KF-7088 4643 1 6 KF-7013 4160 1 6 KF-6944 3985 2 7 KF-7085 3394 1 7 KF-7000 3654 1 8 KF-7106 4028 1 8 KF-7052 4111 1 9 KF-6998 3569 1 9 KF-7108 3992 1 Avg. MPPA 4119 ± 173 Avg. MPPA Lactation 4120 ± 117 ______________________________________________________________________ 46 Materials and methods … 3.12 Feeding, housing and management of experimental animals All the eighteen cross bred cows were fed as follows: A. Control group (T1) Animals in control group fed chopped wheat straw (particle size- 1.5 to 2.0 cm), chaffed green maize fodder (particle size- 2.0 to 2.5 cm) and compounded concentrate mixture as per NRC (2001) requirements. Before diet formulation; the concentrate mixture, wheat straw and maize fodder were analyzed for proximate composition, ADF and NDF. The daily ration was offered to individual cows three times a day before each milking. A. Treatment group (T2) Animals in group 2 (treatment group) were fed same ration as control group plus 5 gm RPM and 20 gm RPL prepartum and 7 gm RPM and 60 gm RPL postpartum, respectively. RPM and RPL is mixed in concentrate feed at the time of feeding of individual animal. 3.12.1 Green maize fodder Green maize was supplied daily by Farm Maintenance Section, National Dairy Research Institute, Karnal. After chopping on the chaff cutter, it was fed to experimental animals. The dry matter contents of green maize were estimated at weekly intervals through out the experimental period. 3.12.2 Concentrate mixture The various ingredients of compounded concentrate mixture fed to cows of both groups were shown in Table 3.3. 3.12.3 Housing and management of animals All the crossbred cows were housed in well-ventilated shed in Cattle Yard of NDRI, Karnal, having the arrangement for individual animal feeding. The animals shed were washed twice daily and thoroughly cleaned to remove faeces and dirt. All the animals were maintained under clean and hygienic conditions. Antiseptic solution containing phenyl was applied at regular intervals on the floor of the shed to keep the animals away from infection. ______________________________________________________________________ 47 Materials and methods … Table 3.3 Ingredients in concentrate mixture (parts) Feed ingredients Parts Maize grain 33 GNC (Exp) 21 Mustard cake 12 DORB 11 Wheat Bran 20 Mineral mixture 02 Salt 01 DORB= Deoiled rice bran; Exp= Expeller; GNC= Ground nut cake 3.12.4 Watering of animals The animals were provided with fresh and clean tap water free of choice three times daily at 8.00 hrs, 12.00 hrs and 18.30 hrs. PRE PARTUM STUDY 3.13 Observations recorded during pre partum period 3.13.1 Body weight Body Weight of the animals was recorded at fortnightly intervals using computerized weight management system from Leotronic Scales Pvt. Ltd. The maximum capacity of the system was 1500 kg and minimum capacity of 4 kg, the error was ± 200 g. The animals were weighed for two consecutive days in the morning before offering feed and water. The average of two days was considered as body weight for that fortnight. 3.13.2 Body Condition Score For recording the body condition of the animals, following points were taken into ______________________________________________________________________ 48 Materials and methods … account: i. Vertebral column (chine, lion and rump) flesh covering at the spinous processes of these regions. ii. Spinous processes: their prominence and sharpness. iii. Tail head region: prominence of depression between backbone and pins and between pin and hook bones. iv. Ribs: their flesh covering. Considering the above points, NRC (2001) presented a score chart, which was adopted in the present study. Body condition score of experimental cows were scored on every fortnight up to parturition. The condition-scoring chart has been presented in Annexure I. 3.13.3 Feed intake Daily DM intake was calculated by recording the daily feed offered and residue from 40 days before parturition to calving day. The DM of different feed ingredients was recorded once every week. Fortnightly intake of nutrients viz CP, RDP, RUP, MP, TDN, ME, NE L of each cow was calculated by using formulae of NRC (2001) (Annexure II). 3.14 Analysis of blood samples 3.14.1 Blood collection Blood Samples were collected at fortnightly intervals (-30 and -15th day) for estimation of phosphatidylcholine, amino acid, VLDL, NEFA, glucose and vitamin E and at weekly intervals (-15 and -7th day) before calving for estimation of prolactin and growth hormone, respectively. The blood samples from individual animals were collected by jugular vein puncture into heparinized vaccutainer tubes, 16 X 100 mm (Becton Dickinson, Rutherford, NJ). The plasma was separated by centrifugation of the blood samples at 2400 rpm for 15 min and it was stored in plasma vials at -200 C. 3.14.2 Plasma amino acid 3.14.2.1 Preparation of sample ______________________________________________________________________ 49 Materials and methods … Plasma samples (0.1 ml) were mixed with ice-cold methanol (400 μl) while vortexing and allowed to stand for 15 minutes in an ice bucket before centrifuging (5000 rpm / 15 minutes) and the supernatant was collected. The protein free supernatants were processed immediately for assaying total amino acids by HPLC analysis as mentioned above in 3.1.7. 3.14.3 Plasma choline estimation A. Principle The test portion was acid digested at 70°C, thereby releasing the majority of the predominantly bound choline. Following pH adjustment, residual choline phospholipids were cleaved with phospholipase D and free choline moiety subjected to choline oxidase with consequent liberation of hydrogen peroxide. In the presence of peroxidase, phenol is oxidized and a quinoneimine chromophore formed with 4-aminoantipyrine. Both enzymatic and color-forming reactions are formatted to occur concurrently. Absorbance was measured at 505 nm and choline content calculated by interpolation from a multilevel calibration. For this procedure, choline is defined and reported as the hydroxide. B. Apparatus (a) Spectrophotometer.—Digital readout to 0.001 absorbance at 505 nm. Glass cuvettes (10 mm) or flow-through cell. (b) Covered water baths.—37° ± 2°C and 70° ± 4°C. (c) pH meter.—2 dp, with calibration buffers, 4.0 and 7.0. (d) Pipettes.—Calibrated glass, or preferably auto-pipettes for 0.100 and 3.00 ml. (e) Volumetric flasks.—10, 50, and 100 ml and 1 L. (f) Digestion vessels.—Conical flasks, 100 or 150 ml, with stoppers. (g) Test tubes.—10 ml with glass stoppers, plus rack. Disposable polypropylene or polystyrene plastic tubes fitted with screw-capped lids may also be used for convenience. (h) Filter paper.—Medium speed, porosity 1.4–2.9 µm, Whatman No. 2 or equivalent. ______________________________________________________________________ 50 Materials and methods … C. Reagents All the reagents used were of AR grade. All enzymes and 4-aminoantipyrine were stored at 4°C or as instructed by the supplier. (a) Water.—Purified to >18 MS resistivity. (b) Hydrochloric acid (HCl)—Concentrated (37% w/v). (c) Hydrochloric acid (1.0 M).—85 ml of concentrated HCl was taken into 1 L volumetric flask and filled to mark with water. Standardization was not required. (d) Sodium hydroxide (NaOH, 50% w/v).— 50 g of NaOH was introduced into 100 ml graduated cylinder. It was carefully dissolved in 80 ml water. After cooling, it was filled to mark with water. (e) Choline bitartrate.—Sigma C-2654 or equivalent. About 0.7–1.0 g of choline bitartrate was dried at 102°C, to constant weight and stored in desiccator at ambient laboratory temperature. (1) Stock choline standard (2500 µg/ml as choline hydroxide). Into l00 ml volumetric flask, 523 mg dry choline bitartrate was introduced. It was dissolved in water and filled to mark. Stock solution was refrigerated at 4° ± 2°C. (2) Working choline standard (250 µg/ml as choline hydroxide). 10 ml of stock solution was pipetted in 100 ml volumetric flask and filled up to l00 ml with distilled water. Working solution was prepared fresh daily. (f) Tris (hydroxymethyl) aminomethane (Trizma) buffer (0.05M, pH 8.0). Into 1 L volumetric flask containing 500 ml water, 6.057 g tris (hydroxymethyl) aminomethane (Sigma T-1503 or equivalent) was introduced. The pH was brought to 8.0 with 1M HCl, and filled to mark with water. It was stored at 4 0 C. This solution was stable for 1 month. (g) Phospholipase D, Sigma Type VI, P-8023, from Streptomyces chromofuscus, 150 units/mg. Unit definition: one unit will liberate 1.0 µmol choline from L-"-phosphatidyl ______________________________________________________________________ 51 Materials and methods … choline (egg yolk)/h at pH 5.0 at 30°C. This form of phospholipase D was preferred to others due to its activity for sphingomyelins and lysophospholipids. (h) Choline oxidase.—Sigma, C-5896, from Alcaligenes species, 10 units/mg. Unit definition: one unit will form 1.0 µmol H2O2 with oxidation of 1.0 µmol choline to betaine aldehyde per min at pH 8.0 at 37°C. (i) Peroxidase.—Sigma Type I, P-8125, from horseradish, 80 units/mg. Unit definition: one unit will form 1.0 mg purpurogallin from pyrogallol in 20 s at pH 6.0 at 20°C. (j) 4-Aminoantipyrine.—Sigma A4382 or equivalent. (k) Phenol. (l) Chromogenic reagent Into l00 mL volumetric flask, sequentially weighed 75–100 units phospholipase D, 100–120 units choline oxidase, 250–280 units peroxidase, 15 mg 4-aminoantipyrine, and 50 mg phenol. It was dissolve and filled to volume with 0.05M Trizma buffer. D. Procedure All steps of the assay were performed under incandescent lighting in the absence of fluorescent lighting or direct sunlight. (a) Preparation of test sample 5.00 g sample was weighted into 100 ml conical flask (or boiling tube). 30 ml of 1.0 M HCl was added, immediately stoppered, and mixed by shaking until well dispersed. The flasks were placed into water bath at 70°C for 3 h, shaked occasionally. The stoppers were loosened or removed during early heating stage to avoid excessive pressure build-up. Further periodic pressure release was necessary. After 3 h, it was cooled to ambient temperature. The pH was adjusted to 3.5 – 4.0 with 50% NaOH, and quantitatively transferred discolored solution to 50 ml volumetric flask, filled to mark with water. The hydrolysate was filtered through Whatman filter paper No 2, and filtrate was collected after discarding the first 5 – 10 ml. It was ensured that filtrate was free of particulates, otherwise re-filtered. This filtrate at this stage was stable and could be stored in the dark at 4°C for up ______________________________________________________________________ 52 Materials and methods … to 3 days prior to analysis. The filtrate was allowed to return to ambient temperature before proceeding with the assay. Two 10 ml test tubes were separately labeled for each sample as Sample Test Solution (tube 1) and Sample Blank (tube 2). Into each tube, 0.100 ml aliquot of sample filtrate was dispensed. (b) Preparation of standard curve 2, 4, 6, and 8 ml aliquots of working standard (250 µg/mL) were pipetted into four 10 ml volumetric flasks and filled to mark with water. The fifth level calibrant was the undiluted working standard. The 5-level standard curve (50, 100, 150, 200, and 250 µg/ml) was prepared by dispensing 0.100 ml of each standard into separately labeled 10 ml test tubes. A Reagent Blank tube (0 µg/mL) was also included by substituting 0.100 ml water in place of choline. (Table 3.2) (c) Enzymatic determination To each Sample Blank (tube 2), 3.00 ml water was added. 3.00 mL chromogenic reagent was added to each Sample Test Solution (tube 1), the 5 standards, and the Reagent Blank. The test tubes were placed in covered water bath at 37° ± 2°C for 15 min during which time a pink/red color was developed. After 15 min, tubes were removed from water bath and cooled to ambient temperature for 15 min. Without delay, contents of each tube were transferred into 10 mm optical cells, or a flow-through cuvette. The spectrophotometer was set to zero at 505 nm against water and measured the absorbance of Standards (Astd), Sample Test Solutions (Asmp), Sample Blanks (Abl), and Reagent Blank (Areag). E. Calculation Standard calibration of net absorbance (Astd –Areag) vs choline concentration (µg/ml) was created using linear least mean squares regression analysis, with forced origin. Absorbance of Sample Blanks and Reagent Blank was subtracted from each Sample Test Solution to eliminate nonspecific spectral background: Net absorbance of sample (A) = Asmp – Abl – Areag The choline concentration in test sample was determined and expressed as choline hydroxide: ______________________________________________________________________ 53 Materials and methods … Choline hydroxide, mg/dL = where , A x S V x 100 W 1000 A = net absorbance of sample; S = slope of standard graph; V = volume in ml of hydrolysates (50 ml); W = weight in grams of sample (5.00 g); 100 = conversion to l00 g basis; 1000 = conversion µg to mg. The data was reported to one decimal place. 3.14.4 Plasma glucose Principle: Glucose is reduced in the presence of O-toluidine solution to emit a bluish-green color which can be measured at 625 nm. Reagent: 1. Trichloroacetic acid solution (3% w/v) with distilled water. 2. O-toluidine solution (6% v/v) with glacial acid. 3. Distilled water (DW) 3. Glucose standard solution. 4. Working standard = 1.2 mg/ml Procedure: The plasma samples, standard and blank were run according to the protocol given below. Reagents Blank Sample (ml) Plasma - 0.20 Standard 1 2 3 4 5 - - - - - ______________________________________________________________________ 54 Fig 3.3 Standard curve of glucose Fig 3. 4 Standard curve of NEFA Materials and methods … Standard - - 0.04 0.08 0.12 0.16 0.20 DW 0.20 - 0.16 0.12 0.08 0.04 0.00 O-toluidine 3.00 3.00 3.00 3.00 3.00 3.00 3.00 reagent Tubes were boiled in water bath for 8 minutes and then cooled under tap water. Absorbance was read at 625 nm against reagent blank. Standard curve (Fig 3.3) was plotted and read the concentration of unknown sample. Calculation: Glucose (mg/100ml) = OD unknown OD standard x concentration of standard x dilution fact.x 100 3.14.5 Non esterified fatty acids (NEFA) The non-esterified fatty acids (NEFA) concentration in the plasma samples was standardized using the extraction method of Itaya and Ui (1965) and the colour development procedure of Duncombe (1963) as follows: Reagents used i) Redistilled chloroform of Analar grade. ii) Phosphate buffer, pH 6.4, 33 mmol/ 1 was prepared by mixing 2 volumes of potassium dihydrogen phosphate (4.53 g/1) and 1 volume of disodium hydrogen phosphate dehydrate (5.938 g/l) solutions. iii) Copper-ethanolamine solution: 1 M triethanolamine; 1 N acetic acid and 6.45 per cent of Cu (NO 3 ) 2 .3H 2 O in 9:1:10 (v/v/v). iv) 0.5 per cent Na-diethyl dithiocarbamate solution in n-butanol. v) Standard palmitic acid (MW 256.43) was dissolved in chloroform to prepare ______________________________________________________________________ 55 Materials and methods … standard solution in the range of 20 to 200 mg/1. Method To 0.2 ml plasma samples in glass stoppered test tubes (15 x 150 mm), 6.0 ml of chloroform and 1 ml of phosphate buffer (pH 6.4), were added and the mixture was shaken for 2 min. After a settling period of 20 min, the upper layer along with the protein precipitate was aspirated with a fine tipped glass syringe and was discarded. Thereafter 3 ml of Cu-Triethanolamine solution was added in the tube and the mixture was hand shaken again for 2 min. The tubes were thereafter centrifuged for 5-10 min at 1,000 g to separate the phases clearly. The Cu-triethanolamine solution was aspirated with a fine tipped syringe taking care that all the Cu-reagent was removed from the solution. Three ml of chloroform solution was pipetted into clean dry tube; care was taken not to touch the inner wall of the tube as traces of Cu-containing aqueous phase might be transferred. Then 0.5 ml of diethyl dithiocarbamate reagent was added to chloroform solution and the solutions after mixing were read immediately at 440 nm in 1 cm light path cell against the blank (Fig. 3.4). 3.14.6 Estimation of plasma Triglycerides Estimation of triglycerides was done using GPO-PAP liquid Gold Kit purchased from Cogent Span Diagnostic Ltd., Surat, India. 3.14.7 Estimation of plasma VLDL Estimation of VLDL was calculated from the value of triglycerides as per the formula given by Friedewald et al. (1972). VLDL (mg/dL) = triglycerides (mg/dL) ÷ 5 3.14.8 Estimation of plasma urea The plasma urea was estimated according to the procedure described by Rahmatullah and Boyde (1980). Principle Urea forms a complex with the Diacetyl monoxime reagent under hot (90oC, acidic conditions). The chemical reaction is stimulated by ferric ions and is stabilized by ______________________________________________________________________ 56 Fig 3.5 Standard curve of BUN Fig 3.6 Standard curve of cholesterol 1.4 Absorbance 1.2 1 0.8 0.6 0.4 0.2 0 0 0.5 1 1.5 Concentration (ug/ml) 2 2.5 3 Materials and methods … thiosemicarbazide. Urea + DMAB H3PO4 (90oC) Thiosemicarbazide + Fe+ Red colour complex Colour was determined by spectrophotometer at 525 nm. Reagents a) Acid ferric solution Added 100 ml concentrated phosphoric acid to 300 ml of concentrated Sulphuric acid and 600 ml distilled water. Dissolved 100 mg ferric chloride in this solution. b) Diacetyl monoxime (DAMO) thiosemicarbazide (TSC) solution 500 mg DAMO and 10 mg TSC were dissolved in distilled water and diluted to 100 ml volume. c) Chromogenic reagent Mixed two parts of reagent 1 with one part of reagent 2 immediately before use. Procedure a) Deproteinisation of samples To 0.2 ml of serum sample, added 2 ml of TCA solution of 5 per cent concentration in a centrifuge tube. Mixed well and centrifuged the samples for 5 to 10 minutes at 1,500 to 2,000 rpm. After centrifugation, removed the precipitate and collected the deproteinised samples in test tube. b) Plasma urea concentration Took 0.2 ml of serum sample and added 3 ml of chromogenic reagent. Mixed well and boiled in water bath for 5 minutes. Cooled it room temperature and read absorbance at 525 nm against a blank comprised of distilled water (0.2 ml) and chromogenic reagent (3 ml). ______________________________________________________________________ 57 Materials and methods … c) Preparation of standard curve Stock standard urea solution (500 µg/ ml) was prepared by dissolving 50 mg urea in 100 ml distilled water. Working standard (50 µg) was prepared by diluting 1 ml of stock with 9 ml of distilled water. Different volumes of working standard 0.02 ml, 0.04 ml, 0.06 ml, 0.08 and 0.10 ml were taken separately in test tube and distilled water 0.18 ml, 0.16 ml, 0.14 ml, 0.12 ml and 0.10 ml, respectively was added to make the volume 0.2 ml. Then 3 ml of chromogenic reagent was added into each test tube and mix well. The blank contained 0.2 ml distilled water and 3 ml chromogenic reagent. Tubes are incubated in boiling water bath for 5 minutes exactly and then cooled to room temperature and absorbance was read against the blank at 525 nm. Absorbance was plotted against concentration to prepare the standard curve (Fig 3.5). 3.14.9 Estimation of plasma cholesterol Plasma cholesterol concentration of blood samples was done using CHOD-PAP, Liquid Gold Kit purchased from Cogent, Span Diagnostic Ltd., Surat, India. (Fig 3.6) 3.14.10 Estimation of plasma vitamins E A. Extraction of plasma samples: 0.5 ml of plasma was deproteinized with an equal volume of 95% ethanol containing 5% ascorbic acid. Three extractions were carried out using 2 ml petroleum ether (40-600C) each time by keeping the test tube in the ice salt mixture. The ether layer was decanted to amber coloured test tubes. B. Preparation of Standards Stock solutions of α-tocopherol (Sigma) 2 mg/ml were prepared in 100 % ethanol. Requisite aliquots of individual stock solutions were taken in amber coloured tubes and were dried under nitrogen at room temperature. The dried standards were reconstituted in mobile phase. A working standard solution containing 20 µg/ml α-tocopherol was prepared at the time of use. C. HPLC system and procedure: The mobile phase consisted of acetonitrile and HPLC water in the ratio of 95:5. The HPLC system (Waters) consisted of a model 510 pump, UV visible absorbance detector ______________________________________________________________________ 58 Fig. 3.7 Chromatogram of standard α-Tocopherol Fig 3.8 Standard curve of milk urea nitrogen 1.4 1.2 Absorbance 1 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 0.8 1 Concentration (ug/ml) 1.2 1.4 1.6 Materials and methods … 486, rheodyne injector with 20 µl loop, using multiwavelengh detector. A reverse phase Discovery C-18 (15 cm x 4.6 mm) column was used. The flow rate was 1.5 ml/minute. 20 µl of standard/ sample was injected in HPLC column for chromatographic separation and the run time was 10 minutes per sample (chromatograph for vitamin E given in Fig 3.7). A specific programme was adopted for the separation of α-tocopherol using Millennium software. Program for HPLC analysis of α-tocopherol on was as follows: Wavelength Change time Retention time (nm) (min) (min) 290 4.00 6.70 Vitamin α-tocopherol 3.14.11 Plasma growth hormone estimation Estimation of plasma growth hormone was done using Bovine Growth Hormone Elisa Test Kit manufactured by Endocrine Technologies Inc., USA. Principle: The rGH quantitative test kit is based on the principle of a solid phase enzyme linked immunosorbent assay (ELISA). The assay system utilizes a mouse anti- rGH specific antibody for solid phase (microtiter wells) immobilization and high affinity anti-mousebGH antibody in the antibody-enzyme (horseradish peroxidase) conjugate solution. The test sample was allowed to react simultaneously with the antibodies, resulting in bGH molecules being sandwich between the solid phase and enzyme linked antibodies. After 3 hours of incubation at 37oC, the wells were washed with water to remove unbound-labelled antibodies. A solution of TMB was added and incubated for 20 minutes, resulting in the development of a blue colour. The colour development was stopped with the addition of stop solution, and the colour was changed to yellow and measured spectrophotometrically at 450 nm. The concentration of bGH is directly proportional to the colour intensity of the test samples. Materials provided: 1. Antibody coated micro-titer wells, 96 well plate. ______________________________________________________________________ 59 Materials and methods … 2. Enzyme conjugate reagent (12 ml). 3. Reference standards (0, 1.0, 2.5, 5.0, 10, 25 ng/ml). 4. TMB colour reagent (12 ml). 5. Stop solution (2N HCl) 6 ml. 6. 20x washing buffer (20 ml). Material required: 1. Precision pipettes: 50 µl, 100 µl, 200 µl and 1 ml. 2. Disposable pipette tips 3. Distilled water 4. Glass tubes or flasks to prepare TMB solution 5. Vortex mixture or equivalent 6. Absorbent paper 7. Graph paper 8. Microtiter plate reader Reagent preparation: All reagents in kit were brought to room temperature (18-250 C) before use. Ready to use reference standards were provided with the kit. Assay procedure: 1. Secure desire no of coated wells in the holder 2. Dispensed 100 µl of standards, specimens and controls into appropriate wells 3. Dispensed 100 µl of enzyme conjugate reagent into each well. Shook the plate for 30 seconds. It was very important to shake the plate very well at this step. 4. Incubated at room temperature (18-250 C) for 3 hours. 5. Removed the incubation mixture by dumping plate contents into a waste container. ______________________________________________________________________ 60 Fig 3.9 Standard curve of growth hormone 4.5 4 Absorbance 3.5 3 2.5 2 1.5 1 0.5 0 0 5 10 15 20 25 30 Concentration (ng/ml) Fig 3.10 Standard curve of prolactin Concentration (ng/ml)) C 2500 2000 1500 1000 500 0 0 0.5 1 1.5 Abosorbance 2 2.5 3 Materials and methods … 6. Rinsed and dumped the microtititer wells five (5) times (200-300 µl) with dilute wash buffer. 7. Dispensed 100 µl of TMB solution into each well. Gently mixed for 10 seconds. 8. Incubated at room temperature for 20 minutes in the dark. 9. Stopped reaction by adding 50 µl of stop solution (2N HCl) to each well. 10. Gently mixed for 30 seconds. It was important to observe a colour change from blue to yellow. 11. Read optical density at 450 nm with a microtiter well reader. Calculations of the results Calculated the mean absorbance value (A450) for each set of reference standards, specimens, controls and patient samples. Construct the standard curve by plotting the mean absorbancy value on the vertical or Y axis and the concentration on the horizontal or X axis (Fig 3.9). Use the mean absorbency values for each specimen to determine the corresponding concentration of bGH (ng/ml) from the standard curve. 3.15.12 Plasma prolactin estimation Estimation of blood prolactin was done by using enzyme-linked immunosorbent assay kit for bovine prolactin (PRL) purchased from the Uscn Life Science Inc. Principle The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific to prolactin. Standards or samples were then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for PRL. Next, Avidin conjugated to horseradish peroxidase was added to each microplate well and incubated. After TMB substrate solution was added, only those wells that contains PRL, biotin conjugated antibody and enzyme conjugated Avidin will exhibit a change in colour. The enzyme substrate reaction was terminated by the addition of sulfuric acid solution and the colour change was measured spectrophotometrically at a wavelength 450 ± 10 nm. The ______________________________________________________________________ 61 Materials and methods … concentration of PRL in the sample is then determined by comparing the optical density (OD) of the samples to the standard curve. Material required Microplate reader 450 ± 10 nm filter Precision single and multichannel pipettes and disposable tips Eppendorf tubes for diluting samples Deionized or distilled water Absorbent paper for blotting the microtiter plate Container for wash solution PBS solution (pH = 7.0 to 7.2) Reagent preparation All kit components and samples were brought to room temperature (18 to 290 C) before use. 1. Standard preparation The standard was reconstituted with 1 ml of Standard Diluent (kept for 10 min. at room temperature) and shaken gently (not to foam). The concentration of the standard in the stock solution was 2000 pg/ml. Seven tubes were prepared containing 0.5 ml Standard Diluent and produced a double dilution series. Each tube was thoroughly mixed before the next transfer. 7 points of the diluent standards such as 2000, 1000, 500, 250, 125, 62.5, 31.2 pg/ml were set and at the last EP tube with the standard diluent was kept the blank as 0 pg/ml. 2. Assay Diluent A and Assay Diluent B: Assay Diluent A or B concentrate (2X) were diluted with 6 ml of deioniozed water to prepare 12 ml of Assay Diluent A or B. The prepared working dilution was used immediately. 3. Detection Reagent A and Detection Reagent B ______________________________________________________________________ 62 Materials and methods … The stock Detection A and Detection B were briefly vortexed before use. It was diluted to the working concentration with working Assay Diluent A or B, respectively (1:100) 4. Wash solution 20 ml of wash solution concentration (30X) was diluted with 580 ml of deionized water to prepare 600 ml of wash solution (1X). 5. TMB substrate The needed dosage of the solution was aspirated with sterilized tips. Sample preparation Plasma samples were diluted by 100 fold. 10 µl of sample was mixed with 990 µl PBS (0.02 mol/L PBS). Assay procedure 1. 7 wells for standard and 1 well for blank were prepared. 100 µl each of dilutions of standard, blank and samples were added into the appropriate wells. Plate was covered with the plate sealer and incubated for the 2 hours at 37 0 C. 2. The liquid of each well was removed. 3. 100 µl of Detection Reagent A working solution was added to each well and incubated for 1 hour at 37 0 C after covering it with the plate sealer. 4. The solution was aspirated and washed with 350 µl of 1X wash solution to each well using multichannel pipette and allowed to stand for 1-2 minutes. The remaining liquid from all wells was removed completely by snapping the plate onto absorbent paper. Totally there were 3 washings. After the last wash, remaining any wash buffer was removed by aspirating or decanting. The plate was inverted and blotted it against absorbent paper. 5. 100 µl of Detection Reagent B working solution was added to each well and incubated for 30 min at 37 0 C after covering it with plate sealer. 6. The aspiration/wash process was repeated for total 5 times as conducted in step 4. ______________________________________________________________________ 63 Materials and methods … 7. 90 µl of substrate solution was added to each well and covered with new plate sealer. The microplate was incubated for 15-25 minutes at 370C (did not exceed 30 minutes). The liquid turned blue by the addition of substrate solution in each well. 8. 50 µl of stop solution was added to each well. The liquid turned yellow by the addition of the stop solution. The liquid was mixed by tapping the side of the plate for proper mixing. 9. Any drop of water and fingerprint on the bottom of the plate were removed and conformed that there was no bubble on the surface of the liquid. Then, the microplate was measurement at 450 nm immediately. Calculation The readings for each standards, control and samples subtracted from the average zero standard OD. Create the standard curve on log-log graph paper, with prolactin concentration on the Y axis and absorbance on the X- axis (Fig 3.10). Draw a best fit strait line through the standard points and it can be determined by regression analysis. 3.15 Reproduction study All animals was transferred to calving pens 10 days before the expected calving date in calving shed of Cattle Yard, NDRI, Karnal. Separate calving pen were provided to each animal and feeding and management of all animals was same as described earlier during prepartum study. Following parturition related parameters were noted during the study: 3.15.1 Calf weight at the time of birth The Body weight of the calves was measured at the time of birth using Avery Platform balance with round display panel. The maximum capacity of the balance was 300 kg and error was ± 500 g. 3. 15.2 Calf mortality within one month of parturition 3.15.3 Occurrence of reproductive disorders The occurrences of reproductive disorders like dystokia, retained placenta, metritis, ______________________________________________________________________ 64 Materials and methods … mastitis were recorded. For this the help of experts in the field of veterinary medicine and veterinary surgery in the Cattle Yard, NDRI was obtained for disease diagnosis and treatment. POSTPARTUM STUDY The postpartum study was divided into four parts: A. Lactation study B. Reproduction study 3.16 Lactation Study 3.16.1 Housing and management Housing, management and watering of animals was same as described in pre partum studies. 3.16.2 Milking of animals Animals were hand milked trice a day i.e. early in morning (5.00 am), noon (11.30 am) and in the evening (6.00 pm). 3.16.3 Body weight and body condition score Body Weight of the animals was recorded at fortnightly intervals by using computerized weight management system from Leotronic Scales Pvt. Ltd as described in the pre partum study. The animals were weighed for two consecutive days in the morning before offering feed and water and after milking. The average of two days was considered as body weight for that fortnight. Body condition score was recorded as per described in annexure I. 3.16.4 Feed intake Daily DM intake was observed by recording the daily feed offered and residue throughout the experiment period of 60 days pre-partum to 210 days post-partum. The DM of different feed ingredients was recorded once every week. Fortnightly intake of nutrients viz CP, RDP, RUP, MP, TDN, ME, NE L of each cow was calculated by using formulae of NRC (2001) (Annexure II). ______________________________________________________________________ 65 Materials and methods … 3.16.5 Milk yield Daily milk yield was recorded for individual animal by using circular dial type spring balance, with the capacity of 20 kg and an accuracy of ± 0.05 kg, at each milking time. 3.16.6 Calculation of fat corrected milk (FCM) For the conversion of whole milk into 4 per cent FCM, the following equation was adopted. 4% FCM = 0.4M + 15F Whereas, M = Milk yield (kg) F = Milk fat (Kg) 3.16.7 Calculation of energy corrected milk (ECM) For the conversion of whole milk into ECM, the following equation given by Sjaunja et al. (1990) was adopted. ECM (kg/d) = Milk Yield (kg/d) x ((38.3 x Fat (g/kg) + 24.2 x Protein (g/kg) + 16.54 x Lactose (g/kg) + 20.7))/3140 3.17 Milk Composition Milk samples from individual animals were collected and analyzed for milk composition at fortnightly intervals throughout the experimental period. The samples collected twice from milking of the day for each animal were proportionately pooled to represent milk sample of that animal. Representative samples were analyzed for its composition (fat, Protein, lactose and SNF) using precalibrated Milk Analyzer (LactoStar, FUNKE GERBER, Article No 3510, Berlin). The total solids in milk were calculated by adding milk fat % and SNF %. 3.17.1 Fatty acid analysis of milk From the pooled milk samples of individual animal, during early, mid and late lactation the fatty acid analysis of milk was done using saponification method (Gulati and Ashes, 2000) as follows: ______________________________________________________________________ 66 Materials and methods … 1. Preparation of methyl esters a) Saponification 1. Pipetted 2 ml of milk sample into a 25 ml stoppered test tube 2. Added 1 ml of Ethanol 3. Added 1 ml of 5 N sodium hydroxide (NaOH) 4. Shook well 5. Covered with foil 6. Placed into oven at 80oC for 1.5 – 2 h Removed from oven and allowed to cool a little b) Acidification 1. Added 5 N Hydrochloric Acid (HCL) approx. 2 ml. 2. Inverted test tube with care 3. pH was checked for each sample using pH paper (must be pink) 4. Cooled the samples sufficiently 5. Extract Fatty Acids – added 4 ml of Petroleum Ether shaked well, pipetted the supernatant into a labeled 15 ml test tube 6. Repeated step-4 – pooling the extracts 7. Evaporated PE extracts to dryness in a warm water bath under a stream of Nitrogen c) Methylation 1. To the dried sample, added 3 ml of 1% Sulphuric acid in methanol 2. Refluxed on a heating block at 50-60oC for 1.5 h 3. Added 3 ml of 5% NaCl (salt solution) 4. Added 3 ml of PE 5. Cooled, shook well ______________________________________________________________________ 67 Materials and methods … 6. Centrifuged at 1500 rpm 7. Decanted supernatant into GLC vial and capped the vial 2. Reagents needed 1. Ethanol (absolute) 2. 5 N Sodium Hydroxide (NaOH) (200 g of Na OH pellets/ 1 litre of distilled water) 3. 5 N Hydrochloric Acid (HCL) (500 ml of concentrated acid added to 500 ml of distilled water) 4. Petroleum Ether (PE) (Boiling range 40-60) Hexane can be used as an alternative 5. 1% sulphuric acid in Methanol: 6. 5% salt solution (Na Cl) (50 g to 1 litre of distilled water) 3. Fatty acid analysis by gas liquid chromatography Analysis was carried out on GLC fitted with Flame ionization detector and 50 m length of capillary column. Initial temperature of the column was 140ºC. The RAMP rate was 2 ºC / min. The other conditions were as follows: Stationary Phase - Fused silica column Injector temperature - 210 ºC Delay Time - 0.00 Sampling Rate - 12.50pts/sec Carrier Gas - Helium Flow Rate - 11.2 psig Ignition Gas - Air plus H 2 Identification of peaks was made through retention time of the reference standards purchased from Supelco, Bellefonte PA, USA. ______________________________________________________________________ 68 Materials and methods … 3.18.2 Estimation of milk urea Milk urea content was determined by a modified colorimetric DMAB (pDimethylaminobenzaldehyde) assay (Bector et al., 1998). Principle Urea forms a yellow complex with the p-Dimethylaminobenzaldehyde reagent in an alcohol low acidic solution at room temperature. The intensity of colour was measured by spectrophotometer at 425 nm. The standard curve is presented in Fig. 3.8. Alcohol Urea + DMAB Yellow color complex HCl Reagents a) Trichloroacetic acid (TCA) 12% (s/v) solution 120 gm TCA was dissolved in the distilled water and the volume was made to 1000 ml. b) Phosphate buffer, pH 7.0 Anhydrous potassium dihydrogen orthophosphate (3.403 gm) and anhydrous dipotassium monohydrogen orthophosphate (4.355 gm) were dissolved in distilled water and volume was made to 1000 ml. c) Diluting reagent Equal volumes of 12% (w/v) TCA and phosphate buffer (pH 7.0) were mixed to make the diluting reagent. d) Coloring reagent (1.6% DMAB solution) 1.6 gm DMAB dissolved in ethyl alcohol, 10 ml concentrated HCL was added into it and the volume was made to 100 ml. Procedure a) Preparation of milk samples ______________________________________________________________________ 69 Materials and methods … Milk samples were warmed at room temperature (27oC to 30oC) and mixed well. 10 ml of milk was mixed with 10 ml of 12% TCA solution and filtered through Whatman filter paper no. 1. Filtrates were then centrifuged at 3,000 rpm for 15 to 20 min. and supernatants were collected. b) Estimation of urea 2 ml of coloring reagent (1.6% DMAB solution) was added to 2 ml of supernatant to develop the yellow color complex. The absorbance was read at 425 nm against a blank containing 2 ml of diluting reagent and 2 ml of coloring reagent (1.6% DMAB solution). c) Preparation of standard curve Standard urea solution (1 mg/ml) was prepared in phosphate buffer (pH 7.0). Different concentrations of urea solution (0.25 mg, 0.50 mg, 0.75 mg, 1.00 mg, 1.25 mg and 1.50 mg) were separately taken into different test tubes and the total volume was made to 2 ml of each case with diluting reagent solution. Two ml of 1.6% DMAB solution was added to each test tube to develop the yellow colour complex. The absorbance was read at 425 nm against a blank containing 2 ml of diluting reagent and 2 ml of 1.6% DMAB solution. The values were plotted against concentration (Fig. 3.3). 3.18 Analysis of blood parameters Blood Samples were collected at fortnightly intervals up to 45 days of parturition and thereafter at monthly intervals up to end of experiment. The blood samples from individual animals were collected by jugular vein puncture into heparinized vaccutainer tubes, 16 X 100 mm (Becton Dickinson, Rutherford, NJ). The plasma was separated by centrifugation of the blood samples at 2400 rpm for 15 min and it is stored in plasma vials at -20 0 C for subsequent analysis of amino acids, choline, glucose, non esterified fatty acid (NEFA), triglycerides, blood urea nitrogen (BUN) and VLDL as described in pre partum study section. 3.19 Statistical Analysis Statistical analysis was carried out by Three Way ANOVA using SigmaStat for Windows version 3.10. ______________________________________________________________________ 70 CHAPTER – 4 RESULTs AND DISCUSSION 4. Results and Discussion _______________________________________________ To investigate the effect of supplementing rumen protected methionine (RPL) and lysine (RPL) on pre and post parturient performance such as calving, lactation, nutrient utilization and reproduction, eighteen crossbred cows were selected and divided into two groups (9 each) on the basis of Most Probable Production Ability (MPPA) and lactation number (Table 3.2). Animals in group 1 (control group) were fed chopped wheat straw, chaffed green maize fodder and compounded concentrate mixture as per requirements (NRC, 2001). However, animals in group 2 (treatment group) were fed same ration as control group plus 5 gm RPM and 20 gm RPL prepartum and 7 gm RPM and 60 gm RPL postpartum, respectively. The experimental period was from 40 days before expected date of parturition to 120 days post parturition. The results obtained during the course of this study have been presented and discussed in respective sections. 4.1 EFFECT OF FEEDING RUMEN PROTECTED METHIONINE AND LYSINE DURING PRE PARTUM PERIOD The physical composition of concentrate mixture used in the experiment is presented in Table 3.3. Percent composition of the concentrate mixture was: maize 33, groundnut cake 21, mustard cake 12, wheat bran 20, deoiled rice bran 11, mineral mixture 2 and common salt 1. Chemical composition of feeds and fodders used in the experiment is presented in Table 4.1. Concentrate mixture contained 21.00, 3.75, 35.96, 11.23, 24.73, 3.97, 0.94, 4.1, 8.74 and 69.24 per cent CP, EE, NDF, ADF, hemicellulose, NDICP, ADICP, lignin, Ash and TDN, respectively. The corresponding values for green maize fodder were 10.52, 1.67, 48.64, 25.26, 23.38, 1.38, 1.08, 2.89, 4.36 and 65.26; for wheat straw these values were 3.50, 1.32, 73.45, 50.65, 22.80, 2.18, 1.42, 9.30, 7.66 and 46.57, respectively. The chemical composition of green maize forage and wheat straw was within the normal range (Ranjhan, 1998). Maize contained 9.63, 5.50, 10.21, 3.24, 6.97, 0.83, 0.40, 1.11, 2.54 and 85.22 per cent CP, EE, NDF, ADF, hemicellulose, NDICP, ADICP, lignin, Ash and TDN, respectively. The corresponding values for ground nut cake were 45.06, 7.03, 22.42, 12.54, 9.88, 5.82, 1.11, 4.71, 5.76 and 83.79. The corresponding values for mustard cake __________________________________________________________________ 71 Results and Discussion … _______________________________________________ were 35.44, 9.50, 29.54, 21.60, 7.94, 6.23, 2.51, 7.86, 7.29 and 74.94. The corresponding values for deoiled rice bran were 14.38, 1.25, 26.86 , 4.70, 12.16, 3.6, 0.45, 4.71, 10.53 and 66.53. The corresponding values for wheat bran were 14.94, 4.30, 43.05, 15.75, 27.30, 2.91, 1.47, 4.42, 6.36 and 69.05. Amino acid profile of feed and fodder used in the experiment is presented in Table 4.2. Essential amino acid conetnts of concentrate mixture viz. arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine were 5.82, 0.89, 1.64, 2.92, 5.71, 3.43, 1.50, 3.68, 2.74, 1.07 and 4.23 per cent of CP, respectively. The corresponding values for green maize fodder were 4.43, 0.71, 1.24, 3.83, 6.62, 3.60, 1.45, 4.26, 3.29, 0.52 and 5.04; for wheat straw these values were 5.53, 1.18, 1.66, 3.11, 5.10, 3.11, 1.23, 4.03, 2.64, 1.57 and 4.48, respectively. Non - essential amino acid profile of concentrate mixture viz. alanine, aspartic acid, glutamic acid, glycine, proline, serine and tyrosine was 3.68, 7.36, 11.50, 3.78, 6.10, 3.65 and 3.56 per cent of CP, respectively. The corresponding values for maize green fodder were 5.15, 7.94, 8.79, 4.09, 1.24, 5.19, 3.46 and 3.72; for wheat straw these values were 3.42, 5.37, 14.97, 3.80, 1.66, 6.76, 3.27 and 3.35, respectively. The amino acid profile of other common feed stuffs which are commonly used in feed formulation is presented in table 4.2. Essential amino acid contents of bacteria viz. arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine were 4.48, 0.91, 0.63, 3.39, 3.76, 7.23, 2.65, 3.18, 4.30, 2.58 and 4.72 per cent of CP, respectively. The corresponding values for protozoa were 3.43, 2.01, 1.57, 2.27, 4.40, 5.16, 2.62, 3.06, 3.15, 1.87 and 3.22. Both bacteria and protozoa are the good source of methionine and lysine. But all the feed stuffs are either difficient in methionine and/or lysine. __________________________________________________________________ 72 Table 4.1 Chemical composition of feed ingredients offered (% DM basis) Ingredient CP EE NDF ADF Maize fodder 10.52 1.67 48.64 25.26 Wheat straw 3.50 1.32 73.45 Concentrate 21.00 3.75 Maize grain 9.63 GNC Hemicellul- NDICP ADICP Lignin Ash TDN%* 23.38 1.38 1.08 2.89 4.36 65.26 50.65 22.8 2.18 1.42 9.3 7.66 46.57 35.96 11.23 24.73 3.97 0.94 4.1 8.74 69.24 5.50 10.21 3.24 6.97 0.83 0.40 1.11 2.54 85.22 45.06 7.03 22.42 12.54 9.88 5.82 1.11 4.71 5.76 83.79 Mustard Cake 35.44 9.50 29.54 21.6 7.94 6.23 2.51 7.86 7.29 74.94 Deoiled Rice Bran 14.38 1.25 26.86 14.70 12.16 3.6 0.45 4.71 10.53 66.53 Wheat Bran 14.94 4.30 43.05 15.75 27.3 2.91 1.47 4.42 6.36 69.05 ose * Calculated as per formula given in NRC (2001) 73 Table 4.2 Amino Acid Profile (% of CP) of feed Ingredients Feed Essential Amino Acids (% of CP) ARG Non Essential Amino Acids (% of CP) CYS HIS ILE LEU LYS MET PHE THR TRP VAL ALA ASP GLU GLY PRO SER TYR Experimental Diet Wheat Straw 5.33 1.18 1.66 3.11 5.10 3.11 1.23 4.03 2.64 1.57 4.48 3.42 5.37 14.97 3.80 6.76 3.27 3.35 Maize Fodder 4.43 0.71 1.24 3.83 6.62 2.60 1.45 4.26 3.29 0.52 5.04 5.15 7.94 8.79 4.09 5.19 3.46 3.72 Concentrate 5.82 0.89 1.64 2.92 5.71 3.43 1.50 3.68 2.74 1.07 4.23 3.68 7.36 11.50 3.78 6.10 3.65 3.56 Common Feed Ingredients Oat fodder 2.13 0.78 1.97 5.53 6.61 3.51 1.87 4.74 4.10 1.58 4.10 8.90 7.01 1.60 9.32 12.29 4.02 10.10 fodder 4.13 1.07 2.14 3.93 7.05 4.17 1.43 4.86 4.06 1.51 4.96 7.28 8.63 5.74 7.43 13.35 5.77 6.99 Maize grain 4.40 0.94 1.46 2.48 6.27 1.35 1.63 3.43 2.35 0.81 4.14 3.75 5.48 9.29 2.73 6.79 3.03 3.89 Barley grain 5.40 2.19 2.31 3.44 6.85 3.59 1.66 5.00 3.35 1.37 4.84 6.40 6.00 7.36 6.37 9.14 9.44 6.25 Pearl Millet 4.56 1.85 4.00 4.31 4.18 5.44 2.04 5.31 3.54 1.39 5.92 6.55 5.39 8.39 8.13 7.63 10.2 5.25 Oat grain 6.79 2.84 2.43 3.73 7.27 4.16 1.70 5.14 3.45 1.21 5.08 10.38 5.53 6.96 6.02 8.00 8.18 4.58 GNC 8.54 0.49 1.70 2.75 4.91 3.94 1.55 4.09 2.14 1.07 3.36 2.79 8.83 13.78 4.30 4.10 3.76 3.46 Berseem Continued…. Feed Essential Amino Acids (% CP) Non Essential Amino Acids (% CP) ARG CYS HIS ILE LEU LYS MET PHE THR TRP VAL ALA ASP GLU GLY PRO SER TYR Mustard Cake 7.12 1.15 2.28 4.05 7.16 5.13 1.48 4.48 4.14 1.57 5.29 3.72 6.44 16.02 4.91 7.38 4.06 4.06 Raw Soybean 7.05 1.45 2.77 4.38 7.32 5.92 1.46 4.92 3.91 1.32 4.35 5.02 9.12 6.08 4.24 2.71 7.76 10.35 Extr. Soybean 6.62 1.42 2.63 4.15 6.95 5.87 1.39 4.23 3.65 1.24 4.52 5.02 8.90 5.96 4.71 2.54 7.65 10.49 Soybean meal 7.11 1.59 2.61 4.27 7.93 6.46 1.38 4.85 4.69 1.29 6.42 3.82 6.89 5.73 3.51 1.64 7.39 10.99 Whole Cott. S. 6.88 2.79 3.62 3.51 5.57 4.88 1.63 5.07 3.69 1.29 4.45 4.96 9.52 3.77 10.92 5.40 7.02 4.83 Cotton S. C. 7.92 1.75 2.88 3.07 6.29 4.08 2.74 5.44 3.07 1.28 4.28 9.59 4.59 10.00 5.22 7.94 2.49 5.79 Clusterbean M 5.86 1.24 2.63 3.94 5.29 4.30 2.67 4.36 3.97 1.11 3.40 2.77 4.55 3.86 12.90 4.38 3.20 2.19 Maize gluten 3.21 1.80 2.14 4.12 16.83 1.69 2.38 6.37 3.39 0.50 4.65 3.03 4.63 2.13 4.22 5.73 3.17 14.69 Rice Bran 7.95 1.08 2.11 3.58 6.79 3.75 1.11 4.70 3.75 1.33 5.92 5.28 8.62 12.53 4.88 6.88 4.42 4.22 Wheat Bran 4.21 1.11 1.46 3.24 5.02 3.97 1.67 3.19 3.02 1.52 4.39 4.15 9.89 11.18 4.27 6.79 4.43 2.98 Rumen microbes Protozoa 3.43 2.01 1.57 2.27 4.40 5.16 2.62 3.06 3.15 1.87 3.22 4.05 11.25 3.96 15.68 4.37 4.39 0.97 Bacteria 4.48 0.91 0.63 3.39 3.76 7.23 2.65 3.18 4.30 2.58 4.72 9.45 7.29 5.01 4.46 2.92 3.76 1.26 Results and Discussion … 4.1.1 Degree of Protection of Rumen Protected Methionine (RPM) and Lysine (RPL) The total methionine content in the supplemental RPM was 52.68 per cent and the ether extract content was 44.90 percent. The rumen escape potential of RPM from rumen hydrolysis was 75.20 per cent. The total lysine content in the supplemental RPL was 40.20 per cent and the ether extract content was 49.00 percent. The rumen escape potential of RPL from rumen hydrolysis was 54.97 per cent. The effective degradability of RPM and RPL was 24.80 and 45.03 per cent , respectively. (Table 4.3). Table 4.3 Characteristic of rumen protected methionine (RPM) and rumen protected lysine (RPL) products Particular RPM RPL 52.68±0.04 0 0 40.20±0.46 44.90±0.32 49.00±0.50 0 hr 0.77 ± 0.28 15.34 ± 0.88 3 hr 20.25 ± 0.91 44.64 ± 0.52 6 hr 22.01 ± 0.60 46.41 ± 0.16 12 hr 28.37 ± 0.68 48.04 ± 0.25 24 hr 44.17 ± 2.28 48.99 ± 0.16 ED 24.80 ± 0.57 45.03 ± 0.22 REP 75.20 ± 1.04 54.97 ± 0.79 Methionine (% of DM) Lysine (% of DM) Fat (% of DM) Rumen Escape Potential (REP) For Met in the RPM and Lys in the RPL to have an impact on milk protein synthesis, it needs to be delivered to the small intestine without being degraded in the rumen. To determine how much of the Met and Lys would escape from the rumen _________________________________________________________________ 76 Results and Discussion … hydrolysis, it is necessary to consider the rate at which the particles are degraded and the amount of time that they spend in the rumen. Rumen degradability can then be calculated as Kp/(Kd + Kp) (McDonald et al, 2002) where Kp = rate of passage and Kd = rate of digestion. Using the results obtained from the degradation study on the RPM and RPL particles, the kd was 0.045/h. If a rate of passage of 0.05/h is assumed, which is the passage rate used for animals fed a diet between maintenance (0.02/h) and high production (0.08/h), 75.20 of the RPM and 54.97% of the RPL particles would have been delivered to the duodenum. 4.1.2 RDP and RUP content of different feedstuffs This section deals with the estimation of RDP and RUP content (% of CP) from nylon bag over a period of time and degradation constants (Protein – A, Protein – B and Protein -C values). Protein Fraction A is the percentage of total CP that is NPN (i.e. assumed to be instantly degraded) and a small amount of true protein that rapidly escapes from the in situ bag because of high solubility or very small particle size. As presented in table 4.4, protein A was found highest in GNC (62.11) followed by mustard cake (39.74) and found lowest in wheat straw (9.39). Fraction B is that percent of the proteins which are potentially degradable. Protein B fraction was found highest in maize (69.26) and lowest in GNC (35.60). Only the B fraction is considered to be affected by relative rates of passage; all of fraction A is considered to be degraded and all of fraction C is considered to pass to the small intestine. The amount of fraction B that is degraded in the rumen was determined by the fractional rate of degradation that was determined in the study for fraction B and an estimate of fractional rates of passage. Both A and B were calculated by computer analysis using the expression given by Mehrez and Orskov (1977) i.e. P = a + b (1- e –ct). While Protein Fraction C is the percentage of CP that is completely undegradable; this fraction generally is determined as the feed CP remaining in the bag at a defined end-point of degradation was calculated by difference. Fraction C was found highest in wheat straw (36.00) and lowest in GNC (2.29) and mustard cake (5.46). The RDP and RUP values for a feedstuff (percent of CP) using this model were computed using the equations RDP= A + B [kd / (kd+ kp)] and RUP = B [kp / _________________________________________________________________ 77 Results and Discussion … (kd + kp)] + C. GNC had the highest RDP (89.27% of CP), followed by mustard cake (83.58) and lowest for wheat straw (31.88). While highest RUP per cent observed in wheat straw (68.12) followed by maize (44.82) and deoiled rice bran (40.01). Numerous factors affect the amount of CP in feeds that will be degraded in the rumen. The chemistry of feed CP is the single most important factor. The two most important considerations of feed CP chemistry are: (1) the proportional concentrations of NPN and true protein, and (2) the physical and chemical characteristics of the proteins that comprise the true protein fraction of the feedstuff. Non protein N compounds are degraded so quickly in the rumen (>300%/h) that degradation is assumed to be 100 percent (Sniffen et al., 1992). Table 4.4 Different protein fractions, RUP (% of CP) of feed Ingredients Feed Protein – A Protein – B Protein – C RDP RUP Wheat straw 9.39 ± 0.42 54.61 ± 0.65 36.00 ± 0.73 31.88 ± 0.47 68.12 ± 0.47 Maize fodder 34.39 ± 1.16 52.43 ± 0.47 13.18 ± 0.75 60.61 ± 0.95 39.39 ± 0.95 Concentrate 33.00 ± 1.02 51.64 ± 0.82 15.36 ± 0.67 69.41 ± 0.69 30.59 ± 0.69 Maize 20.90 ± 0.35 69.26 ± 0.50 9.84 ± 0.44 55.18 ± 0.31 44.82 ± 0.31 GNC 62.11 ± 0.55 35.60 ± 0.83 2.29 ± 0.64 89.27 ± 0.51 10.73 ± 0.51 Mustard Cake 39.74 ± 0.33 54.80 ± 0.76 5.46 ± 0.79 83.58 ± 0.66 16.42 ± 0.66 Deoiled Rice Bran 34.11 ± 0.66 51.75 ± 0.83 14.13 ± 0.80 59.99 ± 0.60 40.01 ± 0.60 Wheat bran 32.82 ± 0.76 56.08 ± 1.09 11.10 ± 0.88 77.68 ± 0.74 22.32 ± 0.74 Differences in 3-dimensional structure and chemical bonding (i.e., cross-links) that occur both within and between protein molecules and between proteins and carbohydrates are functions of source as well as processing. These aspects of structure affect microbial access to the proteins, which apparently is the most important factor affecting the rate and extent of degradation of proteins in the rumen. Proteins that _________________________________________________________________ 78 Results and Discussion … possess extensive cross-linking, such as the disulfide bonding in albumins and immunoglobulins or cross-links caused by chemical or heat treatment, are less accessible to proteolytic enzymes and are degraded more slowly (Ferguson, 1975; Hurrell and Finot, 1985; Mahadevan et al., 1980; Mangan, 1972; Nugent and Mangan, 1978; Nugent et al., 1983; Wallace, 1983). 4.1.3 RUP Intestinal Digestibility RUP intestinal digestibility values of wheat straw, maize fodder and concentrate were 64.40, 70.47 and 79.84 percent, respectively. While maize, groundnut cake, mustard cake, rice bran and wheat bran had 89.60, 73.73, 91.47, 75.13 and 65.33 percent RUP intestinal digestibility, respectively. (Table 4.5; Fig 4.1) Table 4.5 RUP Intestinal Digestibility (in vitro) (% DM basis) of feed Ingredients Ingredients RUP ID % Wheat straw 64.40 ± 0.67 Maize fodder 70.47 ± 0.29 Concentrate 79.84 ± 1.34 Maize 89.60 ± 1.83 GNC 73.73 ± 2.38 Mustard Cake 91.47 ± 0.89 Rice Bran 75.13 ± 0.57 Wheat bran 65.33 ± 1.57 Ruminally undegraded feed CP is assumed to be 100 percent true protein (National Research Council, 1989). Estimates of intestinal digestibility have been assigned to the RUP fraction of each feedstuff; assigned values vary from 50 to 100 percent (NRC, 2001). Therefore, the contribution of RUP to MP is variable and dependent on feed type. _________________________________________________________________ 79 Results and Discussion … 4.1.4 In vitro Microbial protein yield per kg TDN fermented The microbial protein yield per kg TDN fermented was 127.84, 119.62 and 110.04 g in different TMRs containing concentrate to roughage ratio of 60:40, 50:50 and 40:60, respectively (Table 4.6; Fig 4.2). The microbial protein yield (g) per kg TDN fermented was higher (P<0.01) in T1 than T2 and T3 and in T2 than T3 (P<0.01). The result of the present in vitro study revealed that the relative availability of nitrogen for fermentation is an important factor affecting efficiency of microbial protein synthesis. Table 4.6 Microbial protein yield g per kg TDN fermented of different TMRs Trials C:R 60:40 C:R 50:50 C:R 40:60 (TDN – 64.66%) (TDN – 63.51%) (TDN – 62.37%) 1 125.46 117.79 106.29 2 124.91 124.38 109.98 3 128.32 114.43 110.29 4 128.93 120.65 114.35 5 130.46 119.56 105.42 6 128.95 120.90 113.93 Average 127.84 ± 0.89 119.62 ± 1.36 110.04 ± 1.52 a, b,c Means having different superscripts in a row differ significantly (P<0.01) In roughage portion, wheat straw and maize fodder was in the ratio 40:60 on DMB. Microbial protein is the protein of the ruminal bacteria, protozoa, and fungi that pass to the small intestine. Bacteria provide most of the microbial protein leaving the rumen. Protozoa contribute significantly to the microbial biomass of ruminal contents. However, because they are more extensively recycled in the rumen than bacteria (Ffoulkes and Leng, 1988; Leng et al., 1986; Punia et al., 1992,), protozoa do not contribute to postruminal protein supply in proportion to their contributions to the total microbial biomass in the rumen (NRC, 2001). Ruminally synthesized microbial _________________________________________________________________ 80 Fig 4.1 RUP Intestinal Digestibility (in vitro) (% DM basis) Fig 4.2 Microbial protein yield g per kg TDN fermented of different TMRs Results and Discussion … protein typically supplies a majority of the AA flowing to the small intestine of growing cattle (Titgemeyer and Merchen, 1990) and dairy cows (Clark et al., 1992). 4.1.5 Body weight changes Fortnightly changes in body weights of pre parturient cows are presented in Table 4.7; Fig 4.3 and Fig 4.4. In the first fortnight (from day -30th to -15th days pre parturient), the experimental animals gained 7.6 and 6.7 kg body weight in groups 1 and 2, respectively, while in the 2nd fortnight (from day -15th to the partuirition), the loss in weight was 58.2 and 56.1 kg, respectively due to parturition and release of the foetal membranes. The changes in body weight were not different between the groups. Table 4.7 Fortnightly change in body weights (kg) of crossbred cows fed ration with or without RPM plus RPL Day Group 1 Group 2 -30 509.8 ± 22.9 498.6 ± 10.1 -15 517.4 ± 23.0 505.3 ± 9.6 1* 459.2 ± 20.3 442.7 ± 11.5 -15 + 7.6 ± 0.6 + 6.7 ± 0.9 1* -58.2 ± 4.3 -56.1 ± 3.1 -15 + 1.5 ± 0.1 + 1.4 ± 0.2 1* -11.2 ± 0.7 -11.2 ± 0.7 Change in Body weight % Change in body weight Group 1: Cows fed control diet Group 2: Cows fed control diet + RPM 5 gm (Effective Methionine was 1.98 g ) & RPL 20 gm (Effective lysine was 4.42 g) * : Day of parturition During the transition period, feed intake decreases at a time when the protein and energy requirements increase due to rapid growth of the conceptus, leading to the _________________________________________________________________ 81 Results and Discussion … increased demand of methionine and lysine. Consequently, in order to prevent a change in N balance, the deodenal flow of methionine and lysine should be optimum. Thus, due to supplementation of rumen protected methionine and lysine to the treatment group, the duodenal supply of methionine and lysine from the consumed ration were increased. But in the present study, there were no significant effect on body weight change while decreasing trend in body weight loss was observed in treatment group. Similar findings were also observed by Socha et al. (2005) on supplementation of rumen protected methionine. 4.1.6 Body Condition Score Body condition score (BCS) is a logistic tool for assessment of nutritional status of dairy cows and their management for optimal performance. The maintenance of an optimal body condition score relative to lactation stage, milk yield, nutrition and health status is perhaps the most important aspect of dairy cow management that facilitates a healthy transition from pregnancy to lactation. Table 4.8 Fortnightly body condition score of crossbred cows fed ration with or without RPM plus RPL Day Group 1 Group 2 -30 3.8 ± 0.05 3.8 ± 0.06 -15 3.9 ± 0.05 4.0 ± 0.04 1 3.5a ± 0.11 3.7b ± 0.06 Body condition score reflected changes in the body weight in both supplemented and control groups. The body condition score during pre parturient period ranged from 3.84 to 3.88 in group 1, and from 3.81 to 4.00 in group 2 (Table 4.8; Fig 4.5). The initial BCS of group 1 and 2 was 3.84 and 3.81, respectively. The pre partum changes in body weights were not conspicuously reflected in BCS changes which were higher in supplemented group than control group before parturition. However, supplemented group exhibited higher gross apparent difference of changes in body condition score than control group due to higher energy intake. Body condition score at calving is a reliable indicator of reproductive performance _________________________________________________________________ 82 Results and Discussion … (Baruselli et al., 2001). Cows that are having high body condition score at calving or those losing excess body weight are more likely to have a prolonged interval to first oestrus; thereby prolonging days open (Roche, 2006). Major increases in or loss of BCS has been found to be undesirable and the optimum reproductive efficiency observed when BCS loss was at or below 0.5 units during the transition period (Roche, 2006). The findings of the present study are in agreement with those of Socha et al. (2005) who studied the effect of rumen protected methionine and lysine in transition cows. 4.1.7 Nutrients Intake The average DMI (Table 4.9, Fig, 4.6) in the first fortnight (from day -30th to -15th days pre parturient) was 9.33 and 8.87 (kg/d) and in the second fortnight (day 15th to the day of parturition) it was 8.21 and 7.75 kg/d in group 1 and 2, respectively. However, DMI/100 kg body weight (Table 4.10, Fig. 4.6) in first and second fortnight was 1.83 and 1.59 kg/d in group 1 and 1.78 and 1.53 kg/d in group 2, respectively. There was no difference (P>0.05) among two groups in average DMI/ 100 kg body weight before parturition. The results of present study indicated that there was no effect of rumen protected methionine and lysine supplementation on DMI of prepartum cows. The average CPI (Table 4.9, Fig, 4.7) in the first fortnight (from day -30th to 15th days pre parturient)) was 1.17 and 1.14 (kg/d) and in the second fortnight (day 15th to the day of parturition) it was 1.09 and 1.04 (kg/d) in group 1 and 2, respectively. However, CP intake/ 100 kg body weight (Table 4.11, Fig. 4.6) in first and second fortnight was 0.23 and 0.21 kg/d in group 1 and 0.23 a 0.23 kg/d in group 2, respectively. There was no difference (P>0.05) between two groups in average prepartum CP intake/ 100 kg body weight. The average RUP and RDP intakes (Table 4.9) in the first fortnight (from day -30th to -15th days pre parturient) were 0.40 and 0.39; 077 and 0.75 (kg/d) and in the second fortnight (day -15th to the day of parturition) they were 0.36 and 0.34 and 0.72 and 0.70 (kg/d) in group 1 and 2, respectively. _________________________________________________________________ 83 Results and Discussion … Table 4.9 Prepartum average nutrient intake of crossbred cows fed ration with or without RPM plus RPL Parameter Fortnight Group 1 Group 2 DM (kg/d) -2 9.33 ± 0.50 8.87 ± 0.28 -1 8.21 ± 0.54 7.75 ± 0.24 -2 1.17 ± 0.04 1.14 ±0.04 -1 1.09 ± 0.05 1.04 ±0.03 -2 0.40 ± 0.02 0.39 ± 0.01 -1 0.36 ± 0.02 0.34 ± 0.02 -2 0.77 ± 0.03 0.75 ± 0.02 -1 0.72 ± 0.03 0.70 ± 0.03 -2 0.76 ± 0.03 0.74 ± 0.02 -1 0.70 ± 0.03 0.67 ± 0.03 Methionine Duodenal -2 1.90 a ± 0.03 2.22 b ± 0.02 Flow (% of MP) -1 1.97 a ± 0.02 2.30 b ± 0.02 Lysine Duodenal -2 6.44a ± 0.03 7.09b ± 0.04 Flow (% of MP) -1 6.57 a ± 0.06 7.35b ± 0.07 TDN (kg/d) -2 5.90 ± 0.20 5.67 ± 0.18 -1 5.14 ± 0.31 4.66 ± 0.18 -2 21.78 ± 0.90 21.28 ± 0.63 -1 19.78 ± 1.00 18.50 ± 0.80 -2 13.59 ± 0.55 13.33 ± 0.39 -1 12.36 ± 0.61 11.60 ± 0.50 CP (kg/d) RUP (kg/d) RDP (kg/d) MP (kg/d) ME (Mcal/d) NE L (Mcal/d) a, b Means having different superscripts in a row differ significantly (P<0.01) _________________________________________________________________ 84 Results and Discussion … The average MP Intake (Table 4.9) in the first fortnight (from day -30th to 15th days pre parturient) was 0.76 and 0.74 (kg/d) and in the second fortnight (-15th to the day of parturition) it was 0.70 and 0.67 (kg/d) in group 1 and 2, respectively. The average TDNI (Table 4.9; Fig 4.8) in the first fortnight (from day -30th to -15th days pre parturient) was 5.90 and 5.67 (kg/d) and in the second fortnight (-15th to the day of parturition) it was 5.14 and 4.66 (kg/d) in group 1 and 2, respectively. TDN intake/100 kg body weight (Table 4.10) in first and second fortnight was 1.14 and 1.01 kg/d in group 1 and 1.14 and 0.97 kg/d in group 2, respectively. There was no difference (P>0.05) among two groups in average TDN/ 100 kg body weight before parturition. The average ME Intake (Table 4.9) in the first fortnight (from day -30th to 15th days pre parturient) was 21.78 and 21.28 (Mcal/d) and in the second fortnight (15th to the day of parturition), it was 19.78 and 18.50 (Mcal/d) in group 1 and 2, respectively. ME intake/ 100 kg body weight (Table 4.10) in first and second fortnight was 4.28 and 3.83 Mcal/d in group 1 and 4.28 and 3.67 Mcal/d in group 2 respectively. There was no difference (P>0.05) among two groups in average prepartum ME intake/ 100 kg body weight. The average NE L Intake (Table 4.9) in the first fortnight (from day -30th to 15th days pre parturient) was 13.59 and 13.33 (Mcal/d) and in the second fortnight (15th to the day of parturition) it was 12.36 and 11.60 (Mcal/d) in group 1 and 2, respectively. NE L intake/ 100 kg body weight (Table 4.10) in first and second fortnight was 2.67 and 2.40 Mcal/d in group 1 and 2.68 and 2.30 Mcal/d in group 2, respectively. There was no difference (P>0.05) among two groups in average prepartum NE L intake/ 100 kg body weight. The nutrient intake was within the normal range as per NRC (2001) and no difference was observed between the two groups. The average duodenal methionine flow (Table 4.9) in the first fortnight (from day -30th to -15th days pre parturient) was 1.90 and 2.22 (% of MP/d) and in the second fortnight (-15th to the day of parturition) was 1.97 and 2.30 (kg/d) in group 1 and 2, respectively. The average duodenal lysine flow (Table 4.9) in the first fortnight (from day -30th to -15th days pre parturient) was 6.44 and 7.09 (kg/d) and in the _________________________________________________________________ 85 Results and Discussion … second fortnight (-15th to the day of parturition) was 6.57 and 7.35(kg/d) in group 1 and 2, respectively. Duodenal methionine and lysine flow in group 2 was higher (P<0.01) than group 1 due to supplementation of RPM and RPL. Table 4.10 Fortnightly average nutrient Intake (kg/100 kg body weight) in crossbred cows fed ration with or without RPM plus RPL Parameter DM (kg/100 kg body weight) TDN (kg/100 kg body weight) CP (kg/100 kg body weight) ME (Mcal/100 kg body weight) NE L (Mcal/100 kg body weight) Fortnight Group 1 Group 2 -2 1.83 ± 0.05 1.78 ± 0.06 -1 1.59 ± 0.08 1.53 ± 0.03 -2 1.14 ± 0.03 1.14 ± 0.04 -1 1.01 ± 0.04 0.97 ± 0.05 -2 0.23 ± 0.01 0.23 ± 0.01 -1 0.21 ± 0.01 0.21 ± 0.01 -2 4.28 ± 0.09 4.28 ± 0.15 -1 3.83 ± 0.14 3.67 ± 0.19 -2 2.67 ± 0.06 2.68 ± 0.09 -1 2.40 ± 0.08 2.30 ± 0.12 The diets formulated in this experiment was designed to cover energy and protein requirements of dairy cows as per NRC (2001) except for Met and Lys as a percentage of Metabolizable protein. Based on calculations made as suggested by NRC (2001), it was estimated that Met represented 1.90 % of MP in the small intestine in control group, which represented 79.2 % of the recommended level (2.30% of MP) and Lysine supply was 6.44 % of MP which was lower than the recommended level (7.2 % of MP) by 10.55 %. The addition of RPM (5 g/d) and Lys (20 gm) fulfilled the requirement of methionine and lysine as per NRC (2001). The results of the present study indicated that there was no effect on DMI in RPM plus RPL supplemented group in the period before calving in cows. These _________________________________________________________________ 86 Results and Discussion … results are in agreement with those of Socha et al. (2005) who reported there was no effect of pre partum rumen protected methionine supplementation on DMI of cows. In most of the cases, e.g. Christensen et al., 1994; Piepenbrink et al., 1998; Polan et al., 1991, DMI was depressed by feeding RPM, but this depression was reversed when RPL was also supplemented. In contrast, RPL supplementation depressed DMI by 0.68 kg/d on being fed @ 16 g of lysine/day (Watanabe et al., 2006). Reductions in DMI might also be attributed to lower microbial activity, and therefore digestion in the rumen. However, in the present study analysis of the RPM and RPL products didn’t reveal any contaminants that could affect microbial growth. 4.1.8 Overall Plane of Nutrition in prepartum cows The overall plane of nutrition of prepartum cows is presented in Table 4.11. The actual nutrient intake was compared with nutrient requirement as per NRC (2001). DM intake was undersupplied by 10.51 percent and 13.44 percent than requirements in group 1 and 2, respectively. The CPI was 7.62 percent higher than requirement in group 1 and 3.81 percent higher in case of group 2. TDN intake was 12.65 percent and 5.51 percent higher than requirements in group 1 and 2, respectively whereas NE L intake was 13.77 percent and 11.25 percent higher in group 1 and group 2, respectively. The plane of nutrition of cows of both the groups indicated that both the groups were adequately fed, thereby fulfilling the requirements as per NRC (2001). 4.1.9 Effect of RPM and RPL Supplementation on Certain Blood Parameters a) Blood glucose concentration The blood glucose concentration (Table 4.12) on -30th day of parturition was 57.19 and 56.06 (mg/dL) and on -15th day of parturition, it was 56.55 and 54.14 (mg/dL) in group 1 and 2, respectively while it was decreased (P<0.05) at the day of parturition. The blood glucose concentration remained within the normal range and no difference was observed at any stage of experiment. The reason may be a high metabolic rate of utilization of glucose and homeostatic mechanism of animal body that does not allow appreciable change in glucose level. _________________________________________________________________ 87 Results and Discussion … In contrast, Socha et al. (2005) reported decrease in blood glucose level on supplementing rumen protected methionine and lysine to prepartum cows. Table 4.11 Plane of nutrition of prepartum cows fed ration with or without rumen protected methinone plus lysine Parameter DM CP TDN NE L Group 1 Group 2 Intake (kg/d) 8.77 8.31 Requirement * (kg/d) 9.80 9.60 Deficit in DMI % 10.51 13.44 Intake (kg/d) 1.13 1.09 Requirement * (kg/d) 1.05 1.05 Extra CP intake (%) 7.62 3.81 Intake (kg/d) 5.52 5.17 Requirement * (kg/d) 4.90 4.90 Extra TDN intake (%) 12.65 5.51 Intake (Mcal/d) 12.97 12.46 Requirement * (Mcal/d) 11.4 11.2 Extra NE L intake (%) 13.77 11.25 * Requirements as per NRC (2001) b) Plasma phoshatidylcholine levels The blood phosphatidylcholine concentration (Table 4.12) on -30th day of parturition was 152.10 and 149.39 (µg/ml) and on -15th day of parturition, it was 122.35 and 133.26 (µg/ml) in group 1 and 2, respectively. There was increase (P<0.05) in plasma phosphatidylcholine levels on the day of parturition on supplemention of rumen protected methionine and lysine. The plasma phosphatidylcholine concentration remained within the normal range and no difference was observed on -30th and -15th of parturition. _________________________________________________________________ 88 Results and Discussion … The increase in plasma phosphatidylcholine on the day of parturition may be due to utilization of the absorbed methionine for de nova synthesis of choline (Emmanuel and Kennely, 1984). C) Plasma NEFA level The plasma NEFA concentration (Table 4.12) on -30th day of parturition was 69.53 and 67.63 (mg/L) and on -15th day of parturition, it was 91.89 and 77.58 (mg/L) in group 1 and 2, respectively. While the plasma NEFA concentration on the day of parturition was 115.94 and 112.14 in group1 and group 2, respectively. The plasma NEFA concentration remained within the normal range and no difference was observed in both the groups at any stage of experiment. These results are in agreement with that of Socha et al. (2005). NEFA concentration increased in both the groups as the pregnancy advanced. Higher plasma NEFA concentration observed in multifarious cows towards parturition suggested that they were mobilizing more stored energy to support increasing demand of growing conceptus. Table 4.12 Average Plasma metabolites (Prepartum ) of crossbred cows fed ration with or without RPM plus RPL Parameter Day Group 1 Group 2 Glucose, mg/dL -30 57.19 ± 1.20 56.06 ± 0.86 -15 56.55 ± 0.85 54.14 ± 0.82 1 46.02 ± 1.36 45.86 ± 0.53 Phosphatidylcholine -30 152.10 ± 5.49 149.39 ± 5.80 , µg/ml -15 122.35 ± 2.54 133.26 ± 7.26 1 95.58 a ± 2.31 109.00 b ± 5.57 -30 69.53 ± 1.14 67.63 ± 1.05 -15 91.89 ± 1.32 77.58 ± 2.89 1 115.94 ± 1.74 112.14 ± 2.55 NEFA, mg/L _________________________________________________________________ 89 Results and Discussion … Triglycerides, mg/dL VLDL, mg/dL Vitamin E, µg/ml Cholesterol, mg/dL BUN, mg/L a, b -30 17.30 ± 0.84 18.66 ± 0.31 -15 16.27a ± 0.58 18.24b ± 0.23 1 16.12a ± 0.67 17.89b ± 0.43 -30 3.46 ± 0.17 3.73 ± 0.05 -15 3.25a ± 0.12 3.65b ± 0.05 1 3.22a ± 0.13 3.58b ± 0.09 -30 1.07 ± 0.11 1.03 ± 0.03 -15 0.81 ± 0.07 0.79 ± 0.04 1 0.68 ± 0.03 0.66 ± 0.03 -30 166.80 ± 6.13 167.37 ± 5.66 -15 167.25 ± 5.90 172.74 ± 4.57 1 171.33 ± 5.53 173.88 ± 5.97 -30 14.08 ± 0.20 13.38 ± 0.42 -15 19.39 ± 0.87 19.03 ± 0.55 1 20.34 ± 0.90 20.75 ± 0.34 Means having different superscripts in a row differ significantly (P<0.05) D) Plasma Triglycerides levels The plasma triglycerides concentration (Table 4.12) on -30th day of parturition was 17.30 and 18.66 (mg/dL) and on -15th day of parturition, it was 16.27 and 18.24 (mg/dL) in group 1 and 2, respectively. There was increase (P<0.05) in plasma triglycerides on the -15th day of parturition and on the day of parturition in RPM plus RPL supplemented group. In contrast, Fahey et al. (2002) and Delbecchi et al. (2001) reported no difference in plasma triglyceride concentration in Holstein cows fed TMR supplemented with 4.8% canola meal or 4.8% formaldehyde protected canola seeds. _________________________________________________________________ 90 Results and Discussion … E) Plasma VLDL levels The plasma VLDL concentration (Table 4.12) on -30th day of parturition was 3.46 and 3.73 (mg/dL) and on -15th day of parturition, it was 3.25 and 3.65 (mg/dL) in group 1 and 2, respectively. There was increase (P<0.05) in plasma VLDL on the 15th day of parturition and on the day of parturition in RPM plus RPL supplemented group. F) Plasma vitamin E levels The plasma vitamin E concentration (Table 4.12) on -30th day of parturition was 1.07 and 1.03 (µg/ml) and on -15th day of parturition, it was 0.81 and 0.79 (µg/ml) in group 1 and 2, respectively. The plasma vitamin E concentration remained within the normal range and no difference was observed at any stage of experiment. G) Plasma cholesterol levels The plasma cholesterol concentration (Table 4.12) on -30th day of parturition was 166.80 and 167. 37 (µg/ml) and on -15th day of parturition, it was 167.25 and 172.74 (µg/ml) in group 1 and 2, respectively. The plasma cholesterol concentration remained within the normal range and no difference was observed at any stage of experiment. Higher cholesterol concentration is associated with better reproductive performance in high yielding dairy cows, as it acts as a precursor of steroid hormones (Son et al. 1996). However in the present experiment, higher cholesterol concentrations were not reported between two groups. H) Blood urea nitrogen levels The blood urea nitrogen concentration (Table 4.12) on -30th day of parturition was 14.08 and 13.38 (mg/dL) and on -15th day of parturition, it was 19.39 and 19.03 (mg/dL) in group 1 and 2, respectively. The concentrations of plasma urea N did not differ in response to dietary treatment, suggesting that overall N utilization was similar in both the groups. But there was increasing trend in plasma BUN concentration as the cows progressed towards parturition. It may be due to the decreased dry matter intake. _________________________________________________________________ 91 Results and Discussion … Feeding of rumen protected methionine and lysine not only results in more supply of methionine and lysine, but also saves energy wasted in urea synthesis due to optimum use of amino acids. Blood urea concentration is an indicator of efficient protein balance (Campanile et al., 1998; Dhali et al., 2006) and is typically increased in cows deficient in energy. Similar results were reported by Tiwari and Yadava, (1994) on feeding formaldehyde treated mustard cake to buffalo calves and Sahoo and Walli, (2005) to lactating goats. Similarly, non significant difference in BUN on supplementing rumen protected methionine and lysine was reported by Socha et al. (2005) in prepartum cows. I) Plasma amino acid profile Prepartum fortnightly plasma amino acids viz. aspartate, glutamate, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cysteine, leucine, isoleucine, Phenylalanine and lysine concentrations are presented in Table 4.13. The plasma concentration of different amino acids on -15 day of parturition and on the day of parturition were 20.28 and 21.97 µmol/dl in group 1 and 21.99 and 22.91 µmol/dl in group 2 (aspartate), 24.12 and 23.58 µmol/dl in group 1 and 23.85 and 24.48 µmol/dl in group 2 (glutamate), 13.37 and 12.59 µmol/dl in group 1 and 13.38 and 13,72 µmol/dl in group 2 (serine), 19.46 and 18.68 µmol/dl in group 1 and 20.13 and 20.72 in group 2 (glycine), 4.95 and 4.66 µmol/dl in group 1 and 4.41 and 4.31 µmol/dl in group 2 (histidine), 7.84 and 7.63 µmol/dl in group 1 and 8.01 and 8.29 µmol/dl in group 2 (arginine), 12.26 and 11.84 µmol/dl in group 1 and 12.45 and 12.50 µmol/dl in group 2 (threonine), 23.14 and 22.82 µmol/dl in group 1 and 22.83 and 24.03 µmol/dl in group 2 (alanine), 8.85 and 9.14 µmol/dl in group 1 and 8.81 and 9.04 µmol/dl in group 2 (proline), 7.01 and 7.49 µmol/dl in group 1 and 7.17 and 7.58 µmol/dl in group 2 (tyrosine), 15.66 and 15.21 µmol/dl in group 1 and 16.32 and 16.20 µmol/dl in group 2 (valine), 5.54 and 5.39 µmol/dl in group 1 and 5.95 and 5.93 µmol/dl in group 2 (methionine), 1.68 and 161 µmol/dl in group 1 and 2.43 and 2.11 µmol/dl in group 2 (cysteine), 11.24 and 10.79 µmol/dl in group 1 and 9.44 and 9.54 µmol/dl in group 2 (isoleucine), 15.19 and 15.11 µmol/dl in group 1 and 15.36 and 16.18 in group 2 (leucine), 7.74 and 8.26 µmol/dl in group _________________________________________________________________ 92 Results and Discussion … 1 and 7.92 and 9.40 µmol/dl in group 2 (phenylalanine) and 13.26 and 13.28 µmol/dl in group 1 and 14.49 and 14.25 µmol/dl in group 2 (lysine), respectively. Table 4.13 Average Plasma Amino Acid Profile (µmol/dl) of crossbred cows (prepartum) fed ration with or without RPM plus RPL Amino Acid Day Group 1 Group 2 Aspartate -30 21.24 ± 1.56 21.22 ± 2.71 -15 20.28 ± 1.17 21.99 ± 1.33 1 21.97 ± 1.11 22.91 ± 0.76 -30 25.81 ± 1.94 21.52 ± 2.78 -15 24.12 ± 1.50 23.85 ± 0.87 1 23.58 ± 1.18 24.48 ± 1.00 -30 13.10 ± 0.93 13.05 ± 0.42 -15 13.37 ± 0.88 13.38 ± 0.32 1 12.59 ± 0.69 13.72 ± 0.35 -30 19.31 ± 1.05 19.66 ± 0.56 -15 19.46 ± 1.16 20.13 ± 0.51 1 18.68 ± 0.98 20.72 ± 0.56 -30 4.91 ± 0.56 4.59 ± 0.30 -15 4.95 ± 0.55 4.41 ± 0.38 1 4.66 ± 0.49 4.31 ± 0.43 -30 7.69 ± 0.68 8.02 ± 0.45 -15 7.84 ± 0.52 8.01 ± 0.24 1 7.63 ± 0.56 8.29 ± 0.21 -30 11.84 ± 0.91 11.14 ± 0.92 Glutamate Serine Glycine Histidine Arginine Threonine _________________________________________________________________ 93 Results and Discussion … Alanine Proline Tyrosine Valine Methionine Cysteine Isoleucine -15 12.26 ± 0.71 12.45 ± 0.70 1 11.84 ± 0.61 12.50 ± 0.85 -30 22.85 ± 1.77 23.03 ± 1.18 -15 23.14 ± 1.88 22.83 ± 1.00 1 22.82 ± 1.79 24.03 ± 0.62 -30 9.15 ± 0.73 8.28 ± 0.38 -15 8.85 ± 0.45 8.81 ± 0.37 1 9.14 ± 0.59 9.04 ± 0.38 -30 6.90 ± 0.61 6.86 ± 0.19 -15 7.01 ± 0.51 7.17 ± 0.36 1 7.49 ± 0.66 7.58 ± 0.41 -30 14.93 ± 1.15 14.46 ± 0.91 -15 15.66 ± 1.11 16.32 ± 0.41 1 15.21 ± 0.91 16.20 ± 0.53 -30 5.34 ± 0.54 5.63 ± 0.11 -15 5.55 ± 0.50 5.95 ± 0.20 1 5.39 ± 0.32 5.93 ± 0.27 -30 1.57 ± 0.43 1.66 ± 2.32 -15 1.68 ± 0.42 2.43 ± 0.40 1 1.61 ± 0.45 2.11 ± 0.29 -30 11.15 ± 0.94 8.24 ± 1.17 -15 11.24 ± 1.08 9.44 ± 0.69 1 10.79 ± 0.83 9.54 ± 0.72 _________________________________________________________________ 94 Results and Discussion … Leucine Phenylalanine Lysine -30 14.67 ± 1.13 13.43 ± 1.95 -15 15.19 ± 1.25 15.36 ± 0.84 1 15.11 ± 1.08 16.18 ± 0.61 -30 8.04 ± 0.78 7.11 ± 0.94 -15 7.74 ± 0.63 7.92 ± 0.42 1 8.26 ± 0.62 9.40 ± 0.87 -30 12.97 ± 1.06 13.09 ± 1.94 -15 13.36 ± 1.18 14.49 ± 0.73 1 13.28 ± 0.86 14.25 ± 0.84 There were no effect of supplementation of rumen protected methionine and lysine on prepartum plasma amino acid profile of cows while increasing trends had been observed in plasma methionine, cysteine and lysine concentration in cows supplemented with RPM plus RPL. 4.2 EFFECT OF SUPPLEMENTING RPM PLUS RPL DURING POST PARTURIENT PERIOD 4.2.1 Body weight changes The initial body weights of animals at day one after parturition were 459.2 and 442.7 kg in group 1 and 2, respectively and after 120 days of feeding, final body weight were 440.6 and 448.4 kg (Table 4.14, Fig 4.3). There was a declining trend in body weights of cows of both the groups due to mobilization of body reserves to support lactation. This trend was evident up to fourth fortnight in both the groups (Table 4.15). Perusal of the fortnightly changes in body weight revealed that there was overall a net loss of 18.59 kg in group 1, whereas there was overall gain of 5.79 kg in group 2. There was a net loss of 15.5 kg in group 1 at first fortnight, whereas it was 10.6 kg in group 2 owing to decrease in DMI during first fortnight and mobilization of body reserves to support lactation. In first fortnight, there was decrease in DMI owing to calving stress. _________________________________________________________________ 95 Results and Discussion … Table 4.14 Fortnightly average body weights (kg) of lactating crossbred cows fed ration with or without RPM plus RPL during postpartum period Fortnight Group 1 Group 2 1 day* 459.2 ± 20.3 442.7 ± 11.5 1 443.7 ± 20.8 432.0 ± 12.2 2 431.5 ± 20.8 423.8 ± 12.6 3 420.4 ± 20.2 416.8 ± 12.7 4 418.6 ± 21.3 415.1 ± 12.7 5 422.7 ± 21.7 423.7 ± 13.2 6 427.9 ± 21.2 432.8 ± 13.1 7 434.2 ± 20.9 441.0 ± 12.7 8 440.6 ± 20.6 448.4 ± 12.4 Group 1: Cows fed control diet Group 2: Cows fed control diet + RPM 7 gm (Effective Methionine was 2.77 g ) & RPL 60 gm (Effective lysine was 13.26 g) * : Day of parturition Perusal of body weight changes as per cent change in body weight (Table 4.16, Fig. 4.5) revealed that there was overall a net loss of 4.07 percent in group 1, whereas there was overall net gain of 1.32 per cent in group 2. The impact of negative energy balance appeared to be less severe in group 2 as the loss in weight was 2.4 kg in group 2 as compared to 3.5 kg loss in group 1 in the first fortnight. Cows in group 1 and group 2 attained positive energy balance from fourth fortnight onwards as was evident from increasing trend in body weights. Supplementation of RPM and RPL to group 2 cows was effective in reducing the extent and duration of body weight loss as compared to group 1 cows. Socha et al. (2005) reported higher post partum body weight and body condition scores of cows supplemented with rumen protected methionine and lysine. _________________________________________________________________ 96 Fig 4.3 Average fortnightly body weights (kg) of crossbred cows Fig 4.4 Average fortnightly percent change in body weight (kg) of crossbred cows Results and Discussion … Table 4.15 Fortnightly change in body weight (kg) of lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 -15.5a ± 1.3 -10.6b ± 1.4 2 -12.1a ± 1.8 -8.2b ± 1.0 3 -11.1a ± 2.6 -7.0b ± 1.6 4 -1.9 ± 3.0 -1.8 ± 2.2 5 4.1a ± 1.4 8.7b ± 1.7 6 5.2a ± 1.2 9.0b ± 1.1 7 6.4a ± 1.8 8.3b ± 1.6 8 6.4a ± 1.5 7.4b ± 1.4 Overall change -18.59a ± 6.32 + 5.79b ± 5.11 Means having different superscripts in a row differ significantly (P<0.01) Table 4.16 Percent change in body weights of lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 -3.5a ± 0.4 -2.4b ± 0.4 2 -2.8a ± 0.5 -1.9b ± 0.3 3 -2.5a ± 0.6 -1.7b ± 0.4 4 -0.5 ± 0.7 -0.4 ± 0.6 5 1.0a ± 0.3 2.1b ± 0.4 6 1.3a ± 0.4 2.2b ± 0.3 7 1.6a ± 0.5 2.0b ± 0.4 8 1.5 ± 0.4 1.7 ± 0.3 Overall change -4.07a ± 1.30 + 1.32b ± 1.16 Means having different superscripts in a row differ significantly (P<0.01) _________________________________________________________________ 97 Results and Discussion … However, many researchers found that on supplementation of rumen protected methionine alone, there was no effect on postpartum body weight change. (Noftsger and St-Pierre, 2003, Berthiaume et al., 2001, Girard et al., 2005, Noftsger et al., 2005, Benefield et al., 2009 and Davidson et al., 2008). 4.2.2 Body Condition Score Body condition score (BCS) is a tool to adjust feeding and management practices in order to maximize the potential for milk production and minimize reproductive disorders. In early lactation, dairy cows frequently produce far more milk than can be supported by feed intake alone. They do this by drawing on body reserves that were built up before calving. BCS influences productivity, reproduction, health, and longevity of the dairy cattle and buffaloes. Table 4.17 Fortnightly body condition score of lactating crossbred cows fed ration with or without RPM plus RPL a, b Day Group 1 Group 2 1 3.5a ± 0.11 3.7b ± 0.06 15 3.2a ± 0.12 3.3b ± 0.08 30 3.0 ± 0.09 3.1 ± 0.04 45 2.9 ± 0.07 3.0 ± 0.07 60 2.8 ± 0.08 2.9 ± 0.07 75 2.7a ± 0.07 2.9a ± 0.06 90 2.7a ± 0.07 2.9a ± 0.06 105 2.8 ± 0.06 2.9 ± 0.04 120 3.0 ± 0.03 3.1 ± 0.04 Overall Mean 3.0a ± 0.08 3.1b ± 0.06 Means having different superscripts in a row differ significantly (P<0.01) Thinness or fatness can be a clue to underlying nutritional deficiencies, health problems, or improper herd management. Thus, BCS of the lactating cows on a _________________________________________________________________ 98 Fig 4.5 Fortnightly average body condition score of crossbred cows Fig 4.6 Fortnightly average dry matter intake (kg/d) of crossbred cows Results and Discussion … routine basis is an excellent tool to help manage the herd more effectively and reduce the incidence of metabolic disorders during early lactation. Body condition score reflected changes in the body weight in both supplemented and control groups (Table 4.17; Fig 4.5). The BCS after parturition was 3.47 and 3.69 in group 1 and 2, respectively. The body condition score during post parturient period ranged from 2.72 to 3.16 in group 1, and from 2.92 to 3.33 in group 2 during different fortnights. In first fortnight, BCS of group 1 and 2 was 3.16 and 3.33, respectively. There was higher body weight change (Table 4.16, Fig 4.4) during first fortnight which reflected in BCS of this fortnight. Overall average BCS of different fortnights was 2.95 and 3.09 in group 1 and 2, respectively, which was slightly higher in group 2 due to higher energy intake by this group that led to less negative energy balance. The results of present study indicated that supplementation of rumen protected methionine and lysine during early lactation lead to less mobilization of body fat to support milk production. 4.2.3 Dry Matter Intake The DMI (Table 4.18, Fig 4.6) ranged from 11.24 to 13.59 kg/d in group 1 and 11.26 to 14.39 kg/d in group 2 during different fortnights. In 2nd, 7th and 8th fortnight, the DMI per day was higher in group 2 than that of group 1. The overall mean DMI was 13.01 kg and 13.36 kg/d in group 1 and 2, respectively, and higher (P<0.05) DMI was recorded in group 2. The highest DMI was observed in group 2 during eighth fortnight (14.39 kg/d) but in case of control group, it was 13.59 kg/d. It appears that animals in group 2 were able to withstand the effect of post parturient stress in a better way due to supply of rumen protected methionine and lysine. DMI/100 kg body weight (Table 4.19) ranged from 2.48 to 3.23 kg/d in group 1 and 2.55 to 3.30 kg/d in group 2. In all fortnights except 6th forthnight, the DMI per 100 kg body weight was higher in group 2 than that of group 1. The overall average DMI was 3.03 and 3.13 kg/100 kg body weight in group 1 and 2, respectively. The overall mean DMI/100 kg body weight was higher (P< 0.05) in group 2 than group 1, due to higher body weight recorded in group 2 (Table 4.19). _________________________________________________________________ 99 Results and Discussion … Table 4.18 Fortnightly average dry matter intake (kg/d) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 11.24 ± 0.50 11.26 ± 0.43 2 12.67a ± 0.51 12.85b ± 0.44 3 12.88 ± 0.53 13.38 ± 0.50 4 13.16 ± 0.59 13.64 ± 0.48 5 13.43 ± 0.60 13.69 ± 0.44 6 13.58 ± 0.63 13.60 ± 0.56 7 13.49a ± 0.64 14.08b ± 0.56 8 13.59a ± 0.64 14.39a ± 0.51 Overall Mean 13.01a ± 0.28 13.36b ± 0.34 Means having different superscripts in a row differ significantly (P<0.01) Table 4.19 Fortnightly average dry matter intake (kg/100 kg body weight) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 2.48a ± 0.14 2.55b ± 0.08 2 2.86 a ± 0.11 2.98b ± 0.10 3 3.01a ± 0.12 3.16b ± 0.08 4 3.15a ± 0.12 3.27b ± 0.06 5 3.22a ± 0.07 3.30b ± 0.05 6 3.23 ± 0.09 3.24 ± 0.07 7 3.17a ± 0.09 3.28b ± 0.07 8 3.14a ± 0.08 3.30b ± 0.04 Overall Mean 3.03a ± 0.09 3.13b ± 0.09 Means having different superscripts in a row differ significantly (P<0.01) _________________________________________________________________ 100 Results and Discussion … The results of the present study are in agreement with Benefield et al. (2009) and Broderick et al. (2009) who found that there was increase in DMI on supplementation of rumen protected methionine. In contrast, Girard et al. (2005) supplemented rumen protected methionine (18 g/head/day) to lactating dairy cows and observed no effect on DMI. Similarly, Berthiaume et al. (2001) observed non-significant effect on DMI on supplementing rumen protected methionine to dairy cows. Similarly, Bach et al. (2000), Noftsger and St-Pierre (2003), Davidson et al. (2008) and Swanepoel et al. (2011) reported that there was no effect on DMI on supplementing rumen protected methionine and lysine to lactating cows. Socha et al. (2008) observed no effect of abomasal infusion of methionine and lysine to lactating cows on DMI in peak and mid lactation while in there was increase in DMI in early lactation. Lara et al. (2006) reported no treatment effects of supplemented ruminally protected methionine on DM intake (20.38 kg d-1), body weight (599.78 kg), and body condition score (2.51 units). The results of the present study indicated that there was significant increase in DMI in lactating cows on rumen protected methionine and lysine supplementation. 4.2.4 Nutrients Intake 4.2.4.1 CP intake The CP intake ranged from 1.86 to 2.20 kg/d in group 1 and 1.96 to 2.26 kg/d in group 2 (Table 4.20, Fig 4.7). In 1st, 3rd and 8th fortnight, CP intake was higher (P<0.05) in group 2 than that of group 1. The overall average mean CPI was 2.11 and 2.17 kg/d in the group 1 and group 2, respectively, which was higher (P< 0.01) in group 2 than that of group 1, due to higher intake of DMI. The lowest CPI within the group was observed in first fortnight in both the groups which may be due to reduced DMI in the first fortnight. Average CP intake on percent body weight basis (Table 4.21) ranged from 0.41 kg to 0.52 kg/d in group 1 and 0.44 to 0.53 kg/d in group 2. Fortnightly average CP intake per 100 kg body weight was higher in group 2 than that of group 1 except 6th and 7th fortnight. _________________________________________________________________ 101 Results and Discussion … Table 4.20 Fortnightly CP intake (kg/d) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 1.86a ± 0.08 1.96b ± 0.06 2 2.06 ± 0.11 2.12 ± 0.07 3 2.10a ± 0.08 2.24a ± 0.07 4 2.16 ± 0.10 2.19 ± 0.07 5 2.18 ± 0.10 2.20 ± 0.08 6 2.20 ± 0.11 2.17 ± 0.09 7 2.16 ± 0.11 2.23 ± 0.08 8 2.16a ± 0.11 2.26a ± 0.07 Overall Mean 2.11a ± 0.10 2.17b ± 0.07 Means having different superscripts in a row differ significantly (P<0.01) Table 4.21 Fortnightly CP intake (kg/100 kg body weight) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 0.41a ± 0.02 0.44b ± 0.01 2 0.47a ± 0.02 0.49b ± 0.01 3 0.49a ± 0.02 0.53b ± 0.02 4 0.51a ± 0.01 0.53b ± 0.01 5 0.52a ± 0.01 0.53b ± 0.01 6 0.52 ± 0.01 0.52 ± 0.01 7 0.51 ± 0.01 0.52 ± 0.01 8 0.50a ± 0.01 0.52b ± 0.01 Overall Mean 0.49a ± 0.01 0.51b ± 0.01 Means having different superscripts in a row differ significantly (P<0.01) _________________________________________________________________ 102 Fig 4.7 Fortnightly average crude protein intake (kg/d) of crossbred cows Fig 4.8 Fortnightly average TDN intake (kg/d) of crossbred cows Results and Discussion … The overall average CP intake/ 100kg BW during the experimental period was 0.49 and 0.51 kg/d in group 1 and group 2, respectively, which was higher (P<0.05) in group 2 than that of group 1. The faster regain in body weight of the cows in group 2 and higher DMI might be responsible for the higher CP intake observed in group 2. 4.2.4.2 RUP intake The RUP intake (Table 4.22) ranged from 0.61 to 0.76 kg/d in group 1 and 0.64 to 0.80 kg/d in group 2 during different fortnights. In 1st, 3rd, 7th and 8th fortnight, RUP intake was higher (P<0.05) in group 2 than that of group 1. The overall average mean RUP intake was 0.72 and 0.74 kg/d in the group 1 and group 2, respectively, which was higher (P< 0.05) in group 2 than that of group 1, due to higher intake of DMI and CPI. The lowest RUP intake within the group was observed in first fortnight in both the groups which may be due to reduced DMI in the first fortnight. Table 4.22 Fortnightly RUP intake (kg/d) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 0.61a ± 0.03 0.64b ±0.02 2 0.68 ± 0.04 0.68 ± 0.03 3 0.71a ± 0.03 0.77b ± 0.03 4 0.73 ± 0.03 0.75 ± 0.03 5 0.75 ± 0.03 0.76 ± 0.03 6 0.76 ± 0.04 0.75 ± 0.03 7 0.74a ± 0.04 0.78b ± 0.03 8 0.74a ± 0.04 0.80b ± 0.03 Overall Mean 0.72a ± 0.04 0.74b ± 0.03 Means having different superscripts in a row differ significantly (P<0.05) _________________________________________________________________ 103 Results and Discussion … 4.2.4.3 RDP intake The RDP intake (Table 4.23) ranged from 1.25 to 1.44 kg/d in group 1 and 1.32 to 1.47 kg/d in group 2 during different fortnights. In 1st, 3rd and 8th fortnight, RDP intake was higher (P<0.05) in group 2 than that of group 1. The overall average mean RDP intake was 1.40 and 1.43 kg/d in the group 1 and group 2, respectively, which was higher (P< 0.05) in group 2 than that of group 1, due to higher intake of DMI and CPI. The lowest RDP intake within the group was observed in first fortnight in both the groups which may be due to reduced DMI in the first fortnight. Table 4.23 Fortnightly RDP intake (kg/d) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 1.25a ± 0.06 1.32b ± 0.04 2 1.37 ± 0.07 1.41 ± 0.04 3 1.41a ± 0.07 1.47b ± 0.04 4 1.43 ± 0.07 1.44 ± 0.05 5 1.44 ± 0.07 1.44 ± 0.05 6 1.44 ± 0.07 1.42 ± 0.05 7 1.42 ± 0.07 1.45 ± 0.05 8 1.42a ± 0.07 1.47b ± 0.05 Overall Mean 1.40a ± 0.07 1.43b ± 0.05 Means having different superscripts in a row differ significantly (P<0.05) 4.2.4.4 MP intake The MP intake (Table 4.24) ranged from 1.15 to 1.37 kg/d in group 1 and 1.19 to 1.44 kg/d in group 2 during different fortnights. The MP intake was higher in group 2 than that of group 1 in 3rd, 7th and 8th fortnights. The overall average mean MP intake was 1.31 and 1.35 kg/d in the group 1 and group 2, respectively, which was higher (P< 0.01) in group 2 than that of group 1, due to higher intake of DMI and CPI. _________________________________________________________________ 104 Results and Discussion … The lowest MP intake within the group was observed in first fortnight in both the groups which may be due to reduced DMI in the first fortnight. Table 4.24 Fortnightly metabolizable protein intake (kg/d) in lactating crossbred cows fed ration with or without RPM plus RPL supplementation a, b Fortnight Group 1 Group 2 1 1.15 ± 0.05 1.19 ± 0.04 2 1.26 ± 0.06 1.29 ± 0.05 3 1.30a ± 0.05 1.40b ± 0.05 4 1.33 ± 0.06 1.36 ± 0.05 5 1.35 ± 0.06 1.38 ± 0.05 6 1.37 ± 0.07 1.36 ± 0.09 7 1.36a ± 0.07 1.41b ± 0.05 8 1.36a ± 0.07 1.44b ± 0.05 Overall Mean 1.31a ± 0.06 1.35b ± 0.05 Means having different superscripts in a row differ significantly (P<0.01) 4.2.4.5 Duodenal Methionine supply (% of metabolizable protein) The fortnightly duodenal methionine supply (Table 4.25) ranged from 1.78 to 1.90 % of metabolizable protein, in group 1 and 2.26 to 2.33 % of metabolizable protein, in group 2 during different fortnights. The overall mean duodenal methionine supply was 1.86 % and 2.27 % of metabolizable protein per day in the group 1 and group 2, respectively, which was higher (P< 0.01) in group 2 than that of group 1, due to supplementation of rumen protected methionine in group 2. (Table 4.25) Estimation of duodenal amino acid flow suggests that lysine was the first limiting amino acid and methionine the second, however after addition of RPL, methionine may be the first one. In the experiment conducted by Wu et al. (1997), increased lysine supplied to 15.2% of essential amino acids improved performance in cows during the first stage of lactation. Schwab (1996) and Schwab et al. (1992a,b) _________________________________________________________________ 105 Results and Discussion … estimated the requirements as a percentage of essential amino acids, at 5.3% methionine and 15.2% lysine during the first third of lactation. Table 4.25 Fortnightly duodenal methionine supply (% of metabolizable protein) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 1.90a ± 0.01 2.33b ± 0.01 2 1.88 a ± 0.01 2.29 b ± 0.01 3 1.88 a ± 0.00 2.27 b ± 0.01 4 1.87 a ± 0.01 2.27 b ± 0.01 5 1.87 a ± 0.00 2.27 b ± 0.01 6 1.87 a ± 0.00 2.28 b ± 0.01 7 1.87 a ± 0.09 2.26 b ± 0.01 8 1.87 a ± 0.01 2.26 b ± 0.01 Overall Mean 1.86 a ± 0.02 2.27 b ± 0.01 Means having different superscripts in a row differ significantly (P<0.01) 4.2.4.6 Duodenal lysine supply (% of metabolizable protein) The fortnightly duodenal lysine supply (Table 4.26) ranged from 6.11 to 6.27 % of metabolizable protein (MP), in group 1 and 7.04 to 7.33 % of MP in group 2 during different fortnights. The overall mean duodenal lysine supply was 6.15 and 7.12 % of metabolizable protein per day in the group 1 and group 2, respectively, which was higher (P< 0.01) in group 2 than that of group 1, due to supplementation of rumen protected lysine in group 2. (Table 4.26) _________________________________________________________________ 106 Results and Discussion … Table 4.26 Fortnightly duodenal lysine supply (% of metabolizable protein) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 6.27a ± 0.03 7.33b ± 0.05 2 6.19 a ± 0.03 7.18 b ± 0.05 3 6.14 a ± 0.02 7.06 b ± 0.06 4 6.12 a ± 0.02 7.10 b ±0.05 5 6.11 a ± 0.01 7.08 b ± 0.05 6 6.12 a ± 0.02 7.11 b ± 0.05 7 6.14 a ± 0.02 7.06 b ± 0.05 8 6.14 a ± 0.02 7.04 b ± 0.04 Overall Mean 6.15 a ± 0.02 7.12 b ± 0.05 Means having different superscripts in a row differ significantly (P<0.01) 4.2.4.7 TDN intake TDN intake (Table 4.27, Fig. 4.8) ranged from 7.75 to 9.18 kg/d in group 1 and 7.97 to 9.70 kg/d in group 2. In 3rd, 7th and 8th fortnight, TDN intake was higher (P<0.05) in group 2 than that of group 1. Overall average TDNI was 8.76 kg and 9.05 kg/d in group 1 and 2, respectively. The average TDNI was higher (P<0.05) by 3.31 per cent in group 2 over that of group 1. The TDN intake on percent body weight basis (Table 4.29) ranged from 1.71 to 2.18 kg/d in group 1 and 1.80 to 2.22 kg/d in group 2. The TDN intake per 100 kg body weight was higher (P<0.01) in group 2 than that of group 1 in all the fortnights except in 6th fortnight. The overall average TDN intake/ 100 kg BW (Table 4.28) was 2.04 kg/d in group 1 and 2.12 kg/d in group 2. TDN intake/100 kg BW was higher (P<0.05) by 3.92 % in group 2 than that of group 1. _________________________________________________________________ 107 Results and Discussion … Table 4.27 Fortnightly TDNI (kg/d) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 7.75 ± 0.29 7.97 ± 0.24 2 8.47 ± 0.41 8.61 ± 0.31 3 8.65a ± 0.36 9.30b ± 0.33 4 8.84 ± 0.40 9.08 ± 0.32 5 9.00 ± 0.42 9.21 ± 0.31 6 9.18 ± 0.45 9.09 ± 0.41 7 9.09a ± 0.45 9.48b ± 0.36 8 9.10a ± 0.46 9.70b ± 0.31 Overall Mean 8.76a ± 0.40 9.05b ± 0.32 Means having different superscripts in a row differ significantly (P<0.05) Table 4.28 Fortnightly TDN intakes (kg/100 kg body weight) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 1.71a ± 0.08 1.80b ± 0.04 2 1.92a ± 0.08 2.00b ± 0.06 3 2.02a ± 0.08 2.20b ± 0.08 4 2.11a ± 0.06 2.18b ± 0.04 5 2.16a ± 0.05 2.22b ± 0.04 6 2.18 ± 0.06 2.16 ± 0.06 7 2.13a ± 0.07 2.21b ± 0.05 8 2.10a ± 0.06 2.22b ± =0.04 Overall Mean 2.04a ± 0.07 2.12b ± 0.05 Means having different superscripts in a row differ significantly (P<0.05) _________________________________________________________________ 108 Results and Discussion … 4.2.4.8 ME intake ME intake (Table 4.29) ranged from 28.98 to 33.71 in group 1 and 29.76 to 35.41 Mcal/d in group 2. In 3rd, 7th and 8th fortnight, ME intake was higher (P<0.05) in group 2 than that of group 1. Table 4.29 Fortnightly ME intake (Mcal/d) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 28.98 ± 1.06 29.76 ± 0.86 2 31.44 ± 1.47 31.88 ± 1.07 3 32.01a ± 1.27 34.21a ± 1.11 4 32.55 ± 1.39 33.40 ± 1.11 5 33.11 ± 1.50 33.76 ± 1.08 6 33.71 ± 1.58 33.38 ± 1.39 7 33.39a ± 1.58 34.66b ± 1.24 8 33.49a ± 1.65 35.41b ± 1.07 Overall Mean 32.33a ± 1.44 33.31b ± 1.12 Means having different superscripts in a row differ significantly (P<0.05) Overall average ME intake was 32.33 and 33.31 Mcal/d in group 1 and 2, respectively. The average ME intake was higher (P<0.05) by 3.03 per cent in group 2 over that of group 1. The ME intake on percent body weight basis (Table 4.30) ranged from 6.37 to 8.01 Mcal in group 1 and 6.73 to 8.14 Mcal/d in group 2. The ME intake per 100 kg body weight was higher (P<0.01) in group 2 than that of group 1 in all the fortnights except in 6th fortnight. The overall average ME intake/ 100 kg BW was 7.53 Mcal/d in group 1 and 7.82 Mcal in group 2. ME intake/100 kg BW was higher (P<0.01) in group 2 than that of group 1. _________________________________________________________________ 109 Results and Discussion … Table 4.30 Fortnightly ME intake (Mcal/100 kg body weight) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 6.37a ± 0.28 6.73b ± 0.13 2 7.13a ± 0.27 7.40b ± 0.22 3 7.48a ± 0.28 8.11b ± 0.28 4 7.78a ± 0.22 8.01b ± 0.12 5 7.94 ± 0.16 8.14 ± 0.13 6 8.01 ± 0.20 7.94 ± 0.20 7 7.83a ± 0.22 8.08b ± 0.16 8 7.73a ± 0.19 8.12b ± 0.13 Overall Mean 7.53a ± 0.19 7.82b ± 0.18 Means having different superscripts in a row differ significantly (P<0.01) 4.2.4.9 NE L intake NE L intake (Table 4.31) ranged from 18.19 to 22.21 in group 1 and 18.69 to 22.51 Mcal/d in group 2. In 3rd, 7th and 8th fortnight, NE L intake was higher (P<0.05) in group 2 than that of group 1. Overall average NE L intake was 20.58 and 21.00 Mcal/d in group 1 and 2, respectively. The average NE L intake was higher (P<0.01) by 2.04 per cent in group 2 over that of group 1. The NE L intake on percent body weight basis (Table 4.32) ranged from 4.00 to 5.01 Mcal in group 1 and 4.23 to 5.08 Mcal in group 2. The NE L intake per 100 kg body weight was higher (P<0.01) in group 2 than that of group 1 in all the fortnights except in 6th fortnight. The overall average NE L intake/ 100 kg BW was 4.72 Mcal in group 1 and 4.89 Mcal in group 2. NE L intake/100 kg BW was higher (P<0.01) in group 2 than that of group 1. _________________________________________________________________ 110 Results and Discussion … Table 4.31 Fortnightly NE L intake (Mcal/d) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 18.19 ± 0.67 18.69 ± 0.53 2 19.71 ± 3.20 19.99 ± 0.67 3 20.09a ± 0.81 22.51b ± 1.34 4 20.44 ± 0.89 20.92 ± 0.69 5 20.74 ± 0.94 21.13 ± 0.67 6 21.10 ± 0.99 20.89 ± 0.87 7 20.93 ± 0.99 21.70 ± 0.78 8 20.95a ± 1.03 22.16b ± 0.68 Overall Mean 20.58a ± 1.19 21.00b ± 0.78 Means having different superscripts in a row differ significantly (P<0.05) Table 4.32 Fortnightly NE L intake (Mcal/100 kg body weight) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 4.00a ± 0.18 4.23b ± 0.08 2 4.47a ± 0.17 4.64b ± 0.13 3 4.69a ± 0.17 5.07b ± 0.17 4 4.88a ± 0.13 5.02b ± 0.08 5 4.97 ± 0.10 5.10 ± 0.08 6 5.01 ± 0.12 4.97 ± 0.13 7 4.91a ± 0.14 5.06b ± 0.10 8 4.83a ± 0.12 5.08b ± 0.08 Overall Mean 4.72a ± 0.12 4.89b ± 0.11 Means having different superscripts in a row differ significantly (P<0.01) _________________________________________________________________ 111 Results and Discussion … The results of the present study indicated that there was significant increase in DMI on rumen protected methionine and lysine supplementation in lactating cows. The result of the present study are in agreement those of with Benefield et al. (2009) and Broderick et al. (2009) who also found that there was increase in DMI on supplementation of rumen protected methionine. This lead to an increased TDN, ME or NE L intake in RPM plus RPL supplemented group with that of control group 4.3 EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE SUPPLEMENTATION ON MILK PRODUCTION AND ITS COMPOSITION 4.3.1 Milk Production Average daily milk production (Table 4.33, Fig 4.9) ranged from 14.23 to 16.48 kg/d in group 1 and 16.70 to 18.12 kg/d in group 2 in different fortnights on supplementation of rumen protected methionine and lysine. Milk production was higher in group 2 than that of group 1 in all the fortnights. Average milk production during supplementation period was 15.89 kg/d in group 1 and 17.69 kg/d in group 2, which was 11.33 per cent higher (P<0.01) in group 2 over that of group 1. Fortnightly average 4 % FCM yield (Table 4.34; Fig 4.10) ranged from 14.52 to 16.91 in group 1 and 17.40 to 18.67 in group 2 in different fortnights on supplementation of rumen protected methionine and lysine. 4 % FCM yield was higher in group 2 than that of group 1 in all the fortnights. Average daily 4 % FCM yield was 16.21 in group 1 and 18.24 kg in group 2. Group 2 had 12.52 per cent higher (P<0.01) FCM yield over that of group 1. Fortnightly average energy corrected milk (ECM) yield (Table 4.35) ranged from 14.55 to 16.88 in group 1 and 17.40 to 18.63 in group 2 in different fortnights on supplementation of rumen protected methionine and lysine. ECM yield was higher in group 2 than that of group 1 in all the fortnights. Average daily ECM yield was 16.20 in group 1 and 18.11 kg in group 2. Group 2 had 11.79 per cent higher (P<0.01) ECM yield over that of group 1. _________________________________________________________________ 112 Results and Discussion … Table 4.33 Fortnightly average milk yield (kg/d) of lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 14.23a ± 1.81 16.70b ± 1.11 2 16.17a ± 1.87 18.01b ± 1.18 3 16.46a ± 1.60 18.12b ± 1.15 4 15.99a ± 1.37 17.99b ± 1.07 5 16.15a ± 1.64 18.09b ± 1.16 6 15.47a ± 1.99 17.93b ± 1.37 7 16.48a ± 1.48 17.62b ± 1.18 8 16.19a ± 1.27 17.02b ± 1.30 Overall Mean 15.89a ± 0.26 17.69b ± 0.19 Means having different superscripts in a row differ significantly (P<0.01) Table 4.34 Fortnightly average 4% fat corrected milk yield (kg/d) of lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 14.52a ± 1.91 17.40b ± 1.12 2 16.54a ± 2.00 18.67b ± 1.21 3 16.73a ± 1.66 18.60b ± 1.18 4 16.17a ± 1.40 18.46b ± 1.14 5 16.44a ± 1.68 18.60b ± 1.21 6 15.76a ± 2.03 18.44b ± 1.40 7 16.91a ± 1.52 18.19b ± 1.20 8 16.65a ± 1.30 17.65b ± 1.36 Overall Mean 16.21a ± 0.27 18.24b± 0.17 Means having different superscripts in a row differ significantly (P<0.01) _________________________________________________________________ 113 Results and Discussion … Lara et al. (2006) observed increased milk production (31.4 vs 35.8 kg d-1) and protein yield (3.35 vs 3.58 kg d-1) on addition of RPM in diet. Milk production responded quadratically to added methionine levels. The quadratic response in milk protein output was also reported by Guinard and Rulquin (1995), effects which have been associated to a major synthesis of casein and a reduction in urea nitrogen (Wu et al., 1997a,b; Dinn et al., 1998). Holstein cows with a mean production of 35 kg d-1 milk require addition of ruminally protected methionine (16 g d-1) to improve milk production (Lara et al., 2006). Dairy cows fed with alfalfa hay and heat treated soybean meal may meet lysine requirement but not of methionine (Armentano et al., 1997); diets with soybean meal or cotton seed are deficient in methionine and lysine, therefore, a positive response in milk production and composition can be observed on ruminally protected amino acids supplementation (Rulquin and Delaby, 1997). The same response can be expected in rations with moderate amounts of blood meal, fishmeal, or meat meal, which presumably are low in bypass methionine (Xu et al., 1998). Table 4.35 Fortnightly energy corrected milk yield (kg/d) in lactating crossbred cows supplemented with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 14.55a ± 1.89 17.40b ± 1.03 2 16.54a ± 1.98 18.63b ± 1.12 3 16.69a ± 1.63 18.57b ± 1.10 4 16.18a ± 1.40 18.45b ± 1.05 5 16.39a ± 1.67 18.55b ± 1.14 6 15.73a ± 2.05 18.14b ± 1.29 7 16.88a ± 1.53 17.99b ± 1.10 8 16.62a ± 1.30 17.11b ± 1.27 Overall Mean 16.20a ± 0.27 18.11b ± 0.20 Means having different superscripts in a row differ significantly (P<0.01) _________________________________________________________________ 114 Fig 4.9 Fortnightly average milk yield (kg/d) in crossbred cows Fig 4.10 Fortnightly average 4% fat corrected milk yield (kg/d) in crossbred cows Results and Discussion … Maximum milk yield was recorded in seventh fortnight in group 1 and in third fortnight of group 2 (16.48 kg in group 1 and 18.12 kg/d in group 2). The two major reasons for higher milk production in group 2 may be, higher fortnightly TDN intake observed in this group due to fortification of the diet with rumen protected methionine and lysine and occurrence of less number of cases of RFM and metritis in this group (Table 4. 61). The lesser post-parturient stress was also manifested by trend observed in fortnightly body weight changes (Table 4.21) as animals in group 2 had lower body weight change. The significant improvement in milk production on supplementation of rumen protected methionine and lysine is in line with the findings of many researchers. Noftsger and St-Pierre, (2003) observed significant improvement in milk production on dietary supplementation of rumen protected methionine (23.6 kg d-1) as compared to that of control (21.7 kg d-1). Similarly, Bach et al. (2000) demonstrated an increase in milk yield on supplementing rumen protected methionine to lactating Holsteins cows (45.9 vs 47.7 kg d-1). Similarly, Broderick et al. (2009) reported that supplementation of rumen protected methionine and lysine enhanced the milk yield in crossbred cows (41.5 kg d-1) to that of control (39.4 kg d-1) while Yang et al. (2010) reported increase (18.95 vs 21.55 kg d-1) in milk yield on supplementation of rumen protected methionine and lysine. While Socha et al. (2008) found that there was increase in milk production on abomasal infusion of methionine and lysine during peak and early lactation while there was no difference in mid lactation. In contrast, many researchers (Berthiaume et al., 2001; Misciattelli et al., 2003; Girard et al, 2005; Noftsger et al., 2005; Broderick and Muck, 2001; Davidson et al., 2008 and Benefield et al., 2009) did not found any effect on milk production on supplementing rumen protected methionine while Swanepoel et al. (2011) did not find any effect on milk production on supplementing rumen protected lysine alone. Christensen et al. (1994) concluded that addition of fat along with ruminally protected amino acids to the diet of lactating cows, increased the production of milk and 4% FCM yield. _________________________________________________________________ 115 Results and Discussion … In the present study, the higher incidences of calving related abnormalities delayed the cows in group 1 to reach the peak yield and also lowered milk production at peak, which in turn reduced the milk production during experimental period. Thus, the present study establishes that supplementation of rumen protected methionine and lysine to high yielding crossbred cows was beneficial as it increased milk and FCM production. 4.3.2 Milk Composition a) Milk Fat The milk fat content (Table 4.36, Fig 4.11) ranged from 4.07 to 4.19 per cent in group 1 and 4.17 to 4.29 per cent in group 2 in different fortnights. Milk fat per cent was higher (P<0.01) in group 2 than that of group 1 in all the fortnights. The overall average milk fat per cent was higher (P<0.01) by 2.18 per cent in group 2 (4.22%) than that of group 1 (4.13%). Table 4.36 Fortnightly milk fat content (%) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 4.11 a ± 0.07 4.29 b ± 0.05 2 4.12a ± 0.07 4.25b ± 0.06 3 4.10a ± 0.05 4.18b ± 0.05 4 4.07a ± 0.05 4.17b ± 0.05 5 4.12a ± 0.05 4.18b ± 0.05 6 4.13a ± 0.04 4.22b ± 0.05 7 4.17a ± 0.02 4.24b ± 0.03 8 4.19a ± 0.03 4.27b ± 0.03 Overall Mean 4.13 a ± 0.05 4.22 b ± 0.05 Means having different superscripts in a row differ significantly (P<0.01) _________________________________________________________________ 116 Fig 4.11 Fortnightly average milk fat (%) of crossbred cows Fig 4.12 Average fortnightly milk composition * Results and Discussion … The milk fat yield (Table 4.37) ranged from 588.50 to 687.59 g/d in group 1 and 714.43 to 764.51 g/d in group 2 in different fortnights. Milk fat yield was higher (P<0.05) in group 2 than that of group 1. The overall average milk fat yield was 657.18 g/d in group 1 and 745.32 g/d in group 2 which was higher (P<0.01) by 13.41 per cent in group 2 than that of group 1. In this experiment, feeding RPM plus RPL increased milk fat percentage over the entire treatment period; however, differences in milk fat yield were not significant when compared for each fortnight. Reported effects of Met and Lys supplementation on milk fat percentage and yield have been inconsistent. Socha et al. (2008) reported that duodenal infusion of Met increased milk fat percentage and yield in cows during early lactation (~15 wk of lactation), but not during peak (~5 wk of lactation) or mid lactation (~21 wk of lactation). Misciatteilli et al. (2003) also concluded that early lactation cows fed RP-Met had increased milk fat percentage compared with control cows; however, supplementation did not affect milk fat yield. Overton et al. (1996) reported that RP-Met supplementation during the transition period and early lactation increased yield of 3.5% FCM during the first 105 d of lactation. Table 4.37 Fortnightly Milk Fat yields (g/d) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 588.50a ± 78.92 714.43b ± 45.20 2 671.29a ± 83.63 764.51b ± 49.47 3 676.52a ± 67.99 757.11b ± 48.31 4 651.57a ± 57.32 750.58b ± 47.56 5 665.14a ± 68.64 757.25b ± 49.96 6 638.36a ± 82.32 745.00b ± 56.96 7 687.59a ± 62.13 740.06b ± 48.54 8 678.43a ± 53.11 705.99b ± 56.03 Overall Mean 657.18a ± 65.38 745.32b ± 46.29 Means having different superscripts in a row differ significantly (P<0.01) _________________________________________________________________ 117 Results and Discussion … Moreover, other research showed that supplementing diets with rumenprotected methionine (RPM) improved yields of milk, fat, and protein (Bach et al., 2000; Noftsger and St-Pierre, 2003; Misciattelli et al., 2003; Broderick et al., 2008, 2009; Benefield et al., 2009; Yang et al., 2010; Swanepoel et al., 2011). In contrast, in several other studies (Berthiaume et al., 2001; Leonardi et al., 2003; Noftsger and St-Pierre, 2003; Noftsger et al., 2005; Davidson et al., 2008; Broderick et al., 2008; Preynat et al., 2009) RP-Met supplementation did not affect milk fat percentage or yield. It is not clear why increased amounts of Met and Lys in MP may sometimes increase fat content of milk. One reason may relate to the role of AA in the intestinal and hepatic synthesis of chylomicrons and very low density lipoproteins (VLDL). Required substrates for the synthesis of chylomicrons and VLDL, in addition to the presence of the long-chain fatty acids that stimulate their formation, include apolipoproteins and phospholipids (Bauchart et al., 1996). The synthesis of apolipoproteins requires AA. The synthesis of phosphatidylcholine (lecithin), the most abundant phospholipid, requires choline. It has been demonstrated that a portion of the dairy cows’ requirement for Met is as a methyl donor for choline synthesis (Sharma and Erdman, 1989; Erdman, 1994), choline can be a limiting nutrient for milk fat synthesis. That Met and Lys may sometimes be limiting for the synthesis of chylomicrons or VLDL such that the availability of long chain fatty acids for milk fat synthesis is reduced has not been demonstrated. However, there is limited evidence that formation or secretion of these lipoproteins can be enhanced with improved Met and Lys nutrition (Auboiron et al., 1995; Durand et al., 1992). In the present experiment, plasma VLDL concentration had been increased and that may be the reason for the higher time-related average milk fat content of milk exhibited by the experimental group 2 than in the group 1 (P<0.01). b) Milk Protein Fortnightly milk protein content is presented in Table 4.38 and the same is graphically presented in Fig 4.12. The milk protein content ranged from 3.21 to 3.32 per cent in group 1 and 3.22 to 3.35 per cent in group 2 in different fortnights. The _________________________________________________________________ 118 Results and Discussion … overall average milk protein was 3.27 and 3.28 per cent in group 1 and group 2 respectively which was similar. The milk protein yield (Table 4.39) ranged from 470.12 to 538.73 g per day in group 1 and 539.57 to 594.77 g/d in group 2 in different fortnights. Milk protein yield in all the fortnights was significantly higher (P<0.01) than that of group 1.The overall average milk protein yield was 518.68 g/d in group 1 and 578.63 g/d in group 2 exhibiting higher (P<0.01) milk protein by 11.56 per cent in group 2 than that of group 1. Table 4.38 Fortnightly milk protein content (%) in lactating crossbred cows fed ration with or without RPM plus RPL Fortnight Group 1 Group 2 1 3.32 ± 0.03 3.35 ± 0.04 2 3.29 ± 0.03 3.30 ± 0.02 3 3.25 ± 0.03 3.28 ± 0.02 4 3.27± 0.03 3.31 ± 0.02 5 3.24 ± 0.02 3.26 ± 0.02 6 3.21 ± 0.03 3.22 ± 0.01 7 3.26 ± 0.02 3.24 ± 0.02 8 3.28 ± 0.02 3.26 ± 0.03 Overall Mean 3.27 ± 0.03 3.28 ± 0.02 The results of the present experiment are is in agreement with those of Berthiaume et al. (2001); Girard et al. (2005); Noftsger et al. (2005); Davidson et al. (2008) and Yang et al. (2010) who reported no effect of rumen protected methionine fortification on milk protein content while Swanepoel et al. (2011) found that there was no effect of rumen protected lysine on milk protein percent. In contrast, several other workers (Noftsger and St-Pierre, 2003; Bach et al., 2000; Misciattelli et al., 2003; Brydl et al., 2005; Broderick and Muck, 2008; _________________________________________________________________ 119 Results and Discussion … Benefield et al., 2009; Broderick et al., 2009) reported that RP-Met supplementation increased milk protein percentage or its yield. Studies have suggested that Lys and Met are the most limiting AA for milk protein synthesis in dairy cows fed corn silage and alfalfa-based commercial diets typically used in the United States and that optimal concentrations of these limiting AA are required in MP to increase milk protein synthesis (Schwab et al., 1976). Thus, balancing the ration for these AA is of practical significance as a means to enhance milk protein production and to improve efficiency of milk protein synthesis (Cho et al., 2007). Table 4.39 Fortnightly milk protein yield (g/d) in lactating crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 470.12a ± 58.38 557.47b ± 33.13 2 532.12a ± 61.64 593.70b ± 37.26 3 533.87a ± 50.42 593.61b ± 36.73 4 522.02a ± 43.87 594.77b ± 34.65 5 521.64a ± 52.57 590.79b ± 38.47 6 500.02a ± 66.96 569.92b ± 43.18 7 538.73a ± 50.18 565.90b ± 36.03 8 530.90a ± 42.23 539.57b ± 44.13 Overall Mean 518.68a ± 50.30 578.63b ± 35.43 Means having different superscripts in a row differ significantly (P<0.01) In contrast, the infusion of graded amounts of Met (0, 3.5, 7.0, 10.5, and 16.0 g/d) into the duodenum of post peak lactation cows fed a corn-based diet supplemented with soybean products and blood meal increased percentages in milk of both fat (3.73, 3.86, 3.78, 3.91, and 4.15) and true protein (3.00, 3.07, 3.09, 3.13, and 3.15) (Socha et al., 1994b). However, when the same cows fed the same feedstuffs _________________________________________________________________ 120 Results and Discussion … were infused with similar amounts of Met during peak lactation (Socha et al., 1994c) or mid lactation (Socha et al., 1994a), percentages of fat in milk did not change but protein in milk increased. As reviewed by the NRC (2001) and generally accepted, Met is the most limiting AA or is co-limiting with Lys for milk protein production when cows are fed green maize fodder and alfalfa diets. Furthermore, the NRC (2001) reported that relationships of both with content and yield of milk protein with digestible Met supply, expressed as a percentage of MP, were best described by rectilinear models (straight line at lower digestible Met supplies leading to a breakpoint as digestible Met supply increases). Leonardi et al. (2003) determined that equal amounts of RPMet increased milk protein percentage for cows fed both high- and low-CP diets, although milk production and milk protein yield were not affected by treatments. In the present study, supplementation with RPM plus RPM did not increase milk protein but milk protein yield was significantly increased due to increased milk production. c) Milk Lactose Fortnightly milk lactose content is presented in Table 4.40 and the same is graphically depicted in Fig 4.12. The milk lactose content ranged from 4.86 to 4.89 per cent in group 1 and 4.85 to 4.87 per cent in group 2 in different fortnights. The overall average milk lactose was 4.88 and 4.86 per cent in group 1 and group 2, respectively which was similar in both the groups. The milk lactose yield (Table 4.41) ranged from 694.38 to 803.13 g per day in group 1 and 802.34 to 881.69 g/d in group 2 in different fortnights. Milk lactose yield in all the fortnights was significantly higher (P<0.01) than that of group 1.The overall average milk lactose yield was 774.60 g/d in group 1 and 858.08 g/d in group 2 which was significantly higher (P<0.01) by 10.77 per cent in group 2 than that of group 1. This was due to increased milk yield. _________________________________________________________________ 121 Results and Discussion … Table 4.40 Fortnightly milk lactose content (%) in lactating crossbred cows fed ration with or without RPM plus RPL Fortnight Group 1 Group 2 1 4.88 ± 0.01 4.86 ± 0.01 2 4.87 ± 0.01 4.86 ± 0.01 3 4.87 ± 0.01 4.87 ± 0.01 4 4.87 ± 0.01 4.85 ± 0.02 5 4.88 ± 0.01 4.87 ± 0.01 6 4.89 ± 0.01 4.87 ± 0.01 7 4.88 ± 0.01 4.87 ± 0.02 8 4.86 ± 0.01 4.86 ± 0.02 Overall Mean 4.88 ± 0.01 4.86 ± 0.02 Table 4.41 Fortnightly Milk Lactose yield (%) in crossbred cows fed ration with or without RPM plus RPL a, b Fortnight Group 1 Group 2 1 694.38a ± 88.44 812.15b ± 54.01 2 787.41a ± 90.98 875.78b ± 57.26 3 801.05a ± 77.09 881.58b ± 56.42 4 779.51a ± 67.48 872.21b ± 51.91 5 787.57a ± 79.70 881.69b ± 57.08 6 756.95a ± 97.86 862.86b ± 66.57 7 803.13a ± 70.78 851.69b ± 57.94 8 786.80a ± 61.13 802.34b ± 61.99 Overall Mean 774.60a ± 74.83 858.08b ± 53.95 Means having different superscripts in a row differ significantly (P<0.01) _________________________________________________________________ 122 Results and Discussion … d) Milk Solids-not-fat (SNF) content The SNF content (Table 4.42; Fig 4.12) ranged from 8.83 to 9.00 per cent in group 1 and from 8.86 to 8.98 per cent in group 2. Mean SNF content was the same (8.91) in groups 1 and 2. There was no difference in average SNF contents between the groups. Table 4.42 Fortnightly milk SNF content (%) in lactating crossbred cows fed ration with or without RPM plus RPL e) Fortnight Group 1 Group 2 1 9.00 ± 0.03 8.98 ± 0.04 2 8.97 ± 0.03 8.94 ± 0.03 3 8.87 ± 0.02 8.91 ± 0.02 4 8.93 ± 0.03 8.93 ± 0.03 5 8.88 ± 0.02 8.91 ± 0.03 6 8.83 ± 0.05 8.86 ± 0.02 7 8.90 ± 0.02 8.88 ± 0.03 8 8.90 ± 0.02 8.88 ± 0.03 Overall Mean 8.91 ± 0.03 8.91 ± 0.03 Milk Total Solids contents Total solids content of milk is given in Table 4.43 and graphically represented in Fig 4.12. The total solids ranged from 12.91 to 13.10 in group 1 and from 13.09 to 13.27 per cent in group 2 in different fortnights. The milk TS content was higher (P<0.05) in group 2 than that of group 1 in all the fortnights. The mean total solids content in group 1 and 2 were 13.03 and 13.14 per cent respectively. The total solid content of group 2 was higher (P<0.05) than that of group 1. The higher milk total solid was mainly due the increased milk fat content in dairy cows fed ration supplemented with RPM plus RPL. _________________________________________________________________ 123 Results and Discussion … Table 4.43 Fortnightly TS content (%) in lactating crossbred cows fed ration with or without RPM plus RPL a, b f) Fortnight Group 1 Group 2 1 13.10a ± 0.07 13.27b ± 0.07 2 13.09a ± 0.09 13.19b ± 0.08 3 12.99a ± 0.06 13.09b ± 0.05 4 13.00a ± 0.07 13.10b ± 0.04 5 12.91a ± 0.11 13.10b ± 0.06 6 12.96a ± 0.08 13.09b ± 0.05 7 13.07a ± 0.03 13.13b ± 0.06 8 13.10a ± 0.05 13.17b ± 0.07 Overall Mean 13.03 a ± 0.07 13.14 b ± 0.06 Means having different superscripts in a row differ significantly (P<0.01) Milk Urea Nitrogen (MUN) Milk urea nitrogen ranged from 17.76 to 18.95 in group 1 and 17.06 to 19.79 mg/dL in group 2 (Table 4.44) in different fortnights. The overall average of MUN levels was 18.57 and 18.44 mg/dL in group 1 and 2, respectively. The average fortnightly MUN was similar in group 1 cows to that of group 2. Milk urea nitrogen (MUN) measurement can be a useful diagnostic tool in evaluating the N efficiency of diets. Milk urea nitrogen is an indicator of ammonia levels in the rumen. High MUN levels would suggest excess ruminal ammonia, perhaps due to overfeeding of degradable protein and/or not enough fermentable carbohydrate, while low MUN levels would indicate low ruminal ammonia levels. Also, there is an additional energy cost for excreting excess unusable ammonia, which can reduce milk production. It has been suggested that milk urea nitrogen levels might provide guidance about proper ration formulation. Thus BUN is the major end product of N metabolism in ruminants and high concentrations of it are indicative of _________________________________________________________________ 124 Results and Discussion … an inefficient utilization of dietary nitrogen. It is well established that urea equilibrates rapidly with body fluids including milk (Brodrick and Clayton, 1997; Hof et al., 1997). Variance in MUN has been shown to be related to extent of CP degradation in rumen and the amount of ammonia in excess of microbial nitrogen requirements (Ropstad et al., 1989; Hof et al., 1997). However, Bach et al. 2000, reported greater MUN concentration on the 18% CP rations than the 15% CP rations, which paralleled arterial urea concentrations. In the present study, there were no differences in CP percent of the ration of both control as well as treatment group. Similar results had been reported by Noftsger et al. (2005); Benefield et al. (2009) and Swanepoel et al. (2011). Table 4.44 Fortnightly MUN (mg/dl) in lactating crossbred cows fed ration with or without RPM plus RPL g) Fortnight Group 1 Group 2 1 18.59 ± 0.55 18.68 ± 0.71 2 18.39 ± 0.68 19.61 ± 0.55 3 18.68 ± 0.61 17.63 ± 0.29 4 18.94 ± 0.81 18.84 ± 0.62 5 18.82 ± 0.72 17.06 ± 0.53 6 18.42 ± 0.67 18.22 ± 0.62 7 17.76 ± 0.42 17.72 ± 0.57 8 18.95 ± 0.76 19.79 ± 0.51 Overall Mean 18.57 ± 0.65 18.44 ± 0.55 Milk choline content Milk choline levels ranged from 83.25 to 91.75 in group 1 and 83.25 to 92.75 mg/dL in group 2 (Table 4.45) in different fortnights. The overall average of milk choline was 87.69 and 88.63 mg/dL in group 1 and 2, respectively. The average fortnightly choline was similar in group 1 cows to that of group 2. _________________________________________________________________ 125 Results and Discussion … Table 4.45 Fortnightly milk choline (mg/dl) in lactating crossbred cows fed ration with or without RPM plus RPL h) Fortnight Group 1 Group 2 1 84.25 ± 3.62 87.25 ± 3.66 2 90.25 ± 2.81 90.75 ± 1.70 3 91.75 ± 2.50 88.25 ± 3.59 4 86.75 ± 1.77 88.75 ± 2.42 5 83.25 ± 3.02 87.75 ± 2.69 6 85.75 ± 1.70 90.25 ± 3.02 7 91.25 ± 2.15 83.25 ± 1.27 8 88.25 ± 3.76 92.75 ± 1.70 Overall Mean 87.69 ± 2.67 88.63 ± 2.51 Fatty Acid Profile of Milk The fortnightly fatty acid profile of milk of group 1 and group 2 cows are presented in Table 4.46, table 4.47 and Fig 4.13. Milk saturated fatty acids ranged from 66.89 to 70.11 % of fatty acids in group 1 and 66.63 to 70.68 % of fatty acids in group 2 (Table 4.47) in different fortnights. The overall average of milk saturated fatty acid was 68.83 and 68.33 % of fatty acids in group 1 and 2, respectively. The average fortnightly saturated fatty acid contents was similar in group 1 cows to that of group 2. Milk unsaturated fatty acid (as per cent of total FA) ranged from 29.91 to 33.11 % in group 1 and 29.33 to 33.39 % in group 2 (Table 4.47) in different fortnights. The overall average of milk unsaturated fatty acid contents were 31.30 and 31.67 % of fatty acid in group 1 and 2, respectively. The average fortnightly saturated fatty acids was similar in group 1 cows to that of group 2. Mono unsaturated fatty acid (MUFA) (as per cent of total FA) ranged from 27.12 to 30.12 % in group 1 and 26.26 to 30.39 % in group 2 (Table 4.47) in different fortnights. The overall average of milk _________________________________________________________________ 126 Results and Discussion … mono unsaturated fatty acid contents were 28.21 and 28.70 % of fatty acid in group 1 and 2, respectively. The average fortnightly mono saturated fatty acid contents were similar in group 1 cows to that of group 2. Poly unsaturated fatty acid (PUFA) (as per cent of total FA) ranged from 2.44 to 4.48 % in group 1 and 2.42 to 4.47 % in group 2 (Table 4.47) in different fortnights. The overall average of milk poly unsaturated fatty acid was 3.09 and 2.97 % of fatty acid in group 1 and 2, respectively. The average fortnightly poly un saturated fatty acid was similar in group 1 cows to that of group 2. The findings of the present study are in agreement with those of Casper et al., 1987; Chow et al., 1990; Karunanandaa et al., 1994; Kowalski et al., 1999; Rulquin and Delaby, 1997; Varvikko et al., 1999 who also did not observe any effect of increased postruminal supplies of Met on fatty acid composition of milk fat. In contrast, Pisulewski et al. (1996) demonstrated that the infusion of Met into the duodenum of early lactation cows increased the proportions of short- and mediumchain fatty acids and decreased proportions of long-chain fatty acids in milk fat. Christensen et al. (1994) reported a similar trend in the fatty acid composition of milk when lactating cows were fed ruminally protected Met and Lys and suggested that increased in milk fat percent may involve a possible effect of Met on de novo synthesis of short- and medium-chain fatty acids in the mammary gland. _________________________________________________________________ 127 Table 4.46 Fortnightly Fatty Acid Profile of Milk of lactating crossbred cows fed ration with or without RPM plus RPL 1 Fatty Acid (% of fatty acid) 2 3 4 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Caproic Acid (C6:0) 0.95 ± 0.07 1.23 ± 0.23 1.01 ± 0.10 0.91 ± 0.06 1.11 ± 0.07 1.27 ± 0.13 1.10 ± 0.11 1.02 ± 0.18 Caprylic Acid (C8:0) 2.96 ±0.37 2.75 ± 0.61 3.09 ± 0.16 3.19 ± 0.25 2.57 ± 0.44 2.88 ± 0.91 2.87± 0.08 3.18 ± 0.16 Capric Acid (C10:0) 3.75 ± 0.12 3.56 ± 0.14 3.32 ± 0.35 3.31 ± 0.36 3.39 ± 0.35 3.88 ± 0.94 3.53 ± 0.08 3.92 ± 0.28 Lauric Acid (C12:0) 3.65 ± 0.08 3.53 ± 0.08 3.59 ± 0.10 3.58 ± 0.09 3.74 ± 0.19 3.59 ± 0.11 3.42 ± 0.13 3.18± 0.15 Myristic Acid (C14:0) 9.86 ± 0.32 10.56 ± 0.30 9.88 ± 0.19 10.10 ± 0.28 9.75 ± 0.18 9.60 ± 0.43 10.40± 0.08 9.34± 0.17 Myristoleic Acid (C14:1) 1.64 ± 1.03 0.73 ± 0.24 2.82 ± 1.34 2.81 ± 1.34 1.61 ± 0.46 1.53 ± 0.39 1.44 ± 0.47 2.47 ± 0.00 Palmitic Acid (C16:0) 28.04 ± 0.74 27.64 ± 0.46 25.66 ± 1.05 26.17 ± 1.41 27.79 ± 0.49 29.91± 0.68 28.45 ± 0.56 26.47 ± 0.62 Palmitoleic Acid (C16:1) 1.50 ± 0.11 1.36 ± 0.19 1.44 ± 0.17 1.43 ± 0.16 1.54 ± 0.06 1.48 ± 0.04 1.47 ± 0.13 1.21± 0.04 Hepiadecanoic Acid (C17:0) 0.56 ± 0.06 0.50 ± 0.09 0.62 ± 0.03 0.62 ± 0.03 0.52 ± 0.01 0.50 ± 0.01 0.68 ± 0.11 0.54± 0.09 Stearic Acid (C18:0) 14.16 ± 0.18 14.51 ± 0.30 13.52 ± 0.23 13.25 ± 0.26 14.14 ± 0.64 12.11 ± 0.93 14.11 ± 0.50 13.73± 0.17 Oleic Acid (C18:1) 23.04± 0.81 22.19 ± 0.87 21.19 ± 0.20 21.12 ± 0.26 20.43 ± 0.63 19.77 ± 1.09 20.94 ± 0.52 23.49 ± 0.21 Elaidic Acid (C18:1) 1.87 ± 0.27 2.14 ± 0.22 1.97 ± 0.18 1.82 ± 0.17 2.29 ± 0.04 2.20 ± 0.04 1.84 ± 0.12 2.09 ± 0.14 Linoleic Acid (C18:2) 1.77 ± 0.12 1.81 ± 0.09 1.87 ± 0.18 1.86 ± 0.18 1.93 ± 0.07 1.84 ± 0.05 1.76 ± 0.40 1.87 ± 0.00 α-linolenic Acid (C18:3) 0.72 ± 0.12 0.63 ± 0.07 0.62 ± 0.33 2.61 ± 2.03 0.90 ± 0.21 0.87 ± 0.22 0.62 ± 0.08 0.87 ± 0.08 Arachidic Acid (C20:0) 0.45 ± 0.03 0.46 ± 0.03 0.46 ± 0.07 0.45 ± 0.07 0.46 ± 0.11 0.44 ± 0.11 0.48 ± 0.02 0.40± 0.04 ∑SFA 68.92 ± 1.66 70.16 ± 0.68 66.89 ± 0.88 67.15 ± 0.99 69.61 ± 0.40 70.68 ± 1.08 70.11 ± 0.85 66.63 ± 0.53 ∑MUFA 29.04 ± 0.30 27.39 ± 0.72 28.63 ± 1.32 28.39 ± 1.23 27.30 ± 0.13 26.36 ± 0.75 27.12 ± 0.90 30.39 ± 0.32 ∑PUFA 3.12 ± 0.65 2.44 ± 0.05 4.48 ± 2.20 4.47 ± 2.21 3.09 ± 0.28 2.97 ± 0.34 2.79 ± 0.51 3.00 ± 0.31 ∑UFA 32.17 ± 0.57 29.83 ± 0.67 33.11 ± 0.89 32.86 ± 1.00 30.39 ± 0.40 29.33 ± 1.09 29.91 ± 0.84 33.39 ± 0.54 ∑UFA/∑SFA 0.47 ± 0.02 0.43 ± 0.01 0.50 ± 0.02 0.49 ± 0.02 0.44 ± 0.01 0.42 ± 0.02 0.43 ± 0.02 0.50 ± 0.01 (Table continued……) 5 Fatty Acid (% of fatty acid) 6 7 8 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 Caproic Acid (C6:0) 1.09 ± 0.05 1.11 ± 0.08 0.95 ± 0.02 1.04 ± 0.08 0.98 ± 0.10 1.06 ± 0.07 1.18 ± 0.13 1.36 ± 0.49 Caprylic Acid (C8:0) 2.47 ± 0.57 3.15 ± 0.21 3.02 ± 0.13 3.24 ± 0.10 3.22 ± 0.07 2.97 ± 0.21 2.58 ± 0.55 3.01 ± 0.26 Capric Acid (C10:0) 2.81 ± 0.58 3.64 ± 0.10 3.08 ± 0.26 3.42 ± 0.13 4.34 ± 0.21 3.58 ± 0.13 3.48 ± 0.09 3.62 ± 0.10 Lauric Acid (C12:0) 3.60 ± 0.22 3.30 ± 0.14 3.62 ± 0.07 3.42 ± 0.20 3.60 ± 0.04 3.21 ± 0.16 3.47 ± 0.03 3.57 ± 0.06 Myristic Acid (C14:0) 10.46 ± 0.55 9.67 ± 0.05 9.84 ± 0.27 9.73 ± 0.36 10.05 ± 0.75 10.04 ± 0.67 10.87 ± 0.25 10.39 ± 0.45 Myristoleic Acid (C14:1) 0.97 ± 0.02 1.98 ± 0.49 0.93 ± 0.04 1.47 ± 0.50 0.99 ± 0.01 1.99 ± 0.49 0.98 ± 0.01 0.75 ± 0.25 Palmitic Acid (C16:0) 26.24 ± 0.72 27.70 ± 0.49 28.36 ± 1.08 27.99 ± 1.43 27.54 ± 0.20 27.02 ± 0.12 27.08 ± 0.18 27.38 ± 0.62 Palmitoleic Acid (C16:1) 1.50 ± 0.27 1.48 ± 0.19 1.11 ± 0.26 1.12 ± 0.26 0.88 ± 0.25 1.28 ± 0.10 1.30 ± 0.15 1.53 ± 0.06 Hepiadecanoic Acid (C17:0) 0.57 ± 0.04 0.55 ± 0.06 0.88 ± 0.40 0.47 ± 0.05 0.53 ± 0.09 0.56 ± 0.06 0.48 ± 0.08 0.59 ± 0.02 Stearic Acid (C18:0) 14.20 ± 0.28 13.65 ± 0.15 13.38 ± 0.38 13.86 ± 0.11 13.65 ± 0.28 13.61 ± 0.23 14.26 ± 0.55 13.57 ± 0.27 Oleic Acid (C18:1) 23.25 ± 0.35 22.63 ± 0.74 22.28 ± 0.71 22.77 ± 0.83 22.95 ± 0.79 23.65 ± 0.25 22.46 ± 1.01 23.48 ± 0.21 Elaidic Acid (C18:1) 2.06 ± 0.06 2.13 ± 0.17 2.12 ± 0.26 2.03 ± 0.17 2.01 ± 0.13 1.92 ± 0.08 2.13 ± 0.22 2.00 ± 0.14 Linoleic Acid (C18:2) 1.90 ± 0.15 1.66 ± 0.13 2.08 ± 0.25 1.86 ± 0.10 2.00 ± 0.03 1.83 ± 0.07 1.73 ± 0.06 1.78 ± 0.09 α-linolenic Acid (C18:3) 1.07 ± 0.36 0.87 ± 0.08 0.82 ± 0.15 0.93 ± 0.12 0.71 ± 0.13 0.87 ± 0.08 0.71 ± 0.01 0.64 ± 0.08 Arachidic Acid (C20:0) 0.53 ± 0.04 0.41 ± 0.04 0.35 ± 0.06 0.30 ± 0.02 0.46 ± 0.04 0.52 ± 0.03 0.49 ± 0.04 0.46 ± 0.04 ∑SFA 66.90 ± 0.25 67.56 ± 0.44 69.17 ± 0.69 68.20 ± 1.43 69.34 ± 0.52 67.37 ± 0.55 69.74 ± 0.88 68.86 ± 0.45 ∑MUFA 30.12 ± 0.55 29.89 ± 0.40 27.74 ± 1.01 28.57 ± 1.48 27.92 ± 0.40 29.90 ± 0.42 27.83 ± 0.92 28.72 ± 0.47 ∑PUFA 2.97 ± 0.44 2.53 ± 0.14 3.09 ± 0.38 3.23 ± 0.42 2.73 ± 0.18 2.71 ± 0.15 2.44 ± 0.06 2.42 ± 0.01 ∑UFA 33.09 ± 0.25 32.42 ± 0.45 30.83 ± 0.67 31.81 ± 1.43 30.66 ± 0.54 32.61 ± 0.56 30.27 ± 0.87 31.13 ± 0.46 ∑UFA/∑SFA 0.49 ± 0.01 0.48 ± 0.01 0.45 ± 0.01 0.47 ± 0.03 0.44 ± 0.01 0.48 ± 0.01 0.43 ± 0.02 0.45 ±0.01 Table 4.47 Overall fatty acid profile of milk of lactating crossbred cows fed ration with or without RPM plus RPL Fatty Acid Group 1 Group 2 Caproic Acid (C6:0) 1.04 ± 0.03 1.13 ± 0.05 Caprylic Acid (C8:0) 2.85 ± 0.10 3.05 ± 0.06 Capric Acid (C10:0) 3.46 ± 0.16 3.62 ± 0.07 Lauric Acid (C12:0) 3.59b ± 0.04 3.42a ± 0.06 Myristic Acid (C14:0) 10.14 ± 0.14 9.93 ± 0.15 Myristoleic Acid (C14:1) 1.42 ± 0.23 1.72 ± 0.26 Palmitic Acid (C16:0) 27.40 ± 0.36 27.54 ± 0.40 Palmitoleic Acid (C16:1) 1.34 ± 0.08 1.36 ± 0.05 Hepiadecanoic Acid (C17:0) 0.60 ± 0.05 0.54 ± 0.02 Stearic Acid (C18:0) 13.93 ± 0.12 13.54 ± 0.24 Oleic Acid (C18:1) 22.07 ± 0.38 22.39 ± 0.48 Elaidic Acid (C18:1) 2.04 ± 0.05 2.04 ± 0.04 Linoleic Acid (C18:2) 1.88 ± 0.04 1.81 ± 0.03 α-linolenic Acid (C18:3) 0.77 ± 0.23 0.80 ± 0.23 Arachidic Acid (C20:0) 0.46 ± 0.02 0.43 ± 0.02 ∑SFA 68.83 ± 0.44 68.33 ± 0.52 ∑MUFA 28.21 ± 0.35 28.70 ± 0.48 ∑PUFA 3.09 ± 0.22 2.97 ± 0.24 ∑UFA 31.30 ± 0.46 31.67 ± 0.52 ∑UFA/∑SFA 0.46 ± 0.01 0.46 ± 0.01 (% of fatty acids) a, b : Means having different superscripts in a row differ significantly (P<0.05) __________________________________________________________________130 Fig 4.11 Fortnightly average milk fat (%) of crossbred cows Fig 4.12 Average fortnightly milk composition * Results and Discussion … 4.3.3 Efficiency of nutrients for milk production Efficiency of utilization of DM, CP and TDN for milk production is presented in Table 4.48. The efficiency of utilizing DMI, kg per kg milk production, was 0.82 and 0.76 and for 4 % FCM, it was 0.80 and 0.73 for group 1 and 2, respectively. The DM was utilized better (P<0.01) for milk production on supplementation of rumen protected methionine and lysine in group 2 than that of group 1. The rumen protected methionine and lysine supplementation increased the methionine and lysine supply in metabolizable protein and hence resulted in more milk production which may be the reason for increased efficiency for DM utilization for milk production. Table 4.48 Efficiency of utilization of nutrients of lactating crossbred cows fed ration with or without RPM plus RPL Parameter Group 1 Group 2 DMI, kg/kg Milk Yield 0.82a ± 0.01 0.76b ± 0.02 DMI, kg/kg FCM 0.80 a ± 0.02 0.73 b ± 0.02 CPI, g/kg Milk Yield 132.81 a ± 1.72 122.18 b ± 1.79 CPI, g/kg FCM 130.18 a ± 1.75 128.99 b ± 1.76 MPI, g/kg Milk Yield 82.37 a ± 1.19 76.62 b ± 1.56 MPI, g/kg FCM 80.73 a ± 1.18 74.73 b ± 1.53 TDN, kg/kg Milk Yield 0.55 a ± 0.01 0.51 b ± 0.01 TDN kg/kg FCM 0.54 a ± 0.01 0.50 b ± 0.01 MEI, Mcal/kg Milk Yield 2.04 a ± 0.03 1.88 b ± 0.04 MEI, Mcal/kg FCM 2.00 a ± 0.03 1.83b ± 0.03 NE L I, Mcal/kg Milk Yield 1.28 c ± 0.02 1.19d ± 0.02 NE L I, Mcal/kg FCM 1.25 a ± 0.02 1.15b ± 0.02 a, b Means having different superscripts in a row differ significantly (P<0.01) c, d Means having different superscripts in a row differ significantly (P<0.05) __________________________________________________________________131 Results and Discussion … The CP intake, kg per g milk production, was also better (P<0.05) in group 2 than that of group 1. These values for milk production were 132.81 and 122.18 g and for 4 % FCM production, these were 130.19 and 128.99 g in group 1 and 2, respectively. The CP intake (kg) per kg 4 % FCM production was also better (P<0.01) in group 2 than that of group 1. The better CP utilization for milk production in group 2 may be due to higher availability of methionine and lysine from rumen protected methionine and lysine. The rumen protected methionine and lysine supplementation in group 2 increased supply of these amino acids in the intestine for absorption and subsequently their uptake by mammary gland with a greater efficiency, which resulted in an increased efficiency of protein utilization in the group 2. The metabolizable protein intake, g per kg milk yield, was 82.37 g in group 1 and 76.62 g in group 2. MPI, g per kg FCM yield, was 80.73 and 74.73 in group 1 and 2, respectively. Group 2 exhibited better utilization (P<0.01) of metabolizable protein for milk and FCM production than that of group 1. The TDN intake, kg per kg milk yield, was 0.55 in group 1 and 0.51 in group 2 and TDNI, kg per kg FCM yield, was 0.54 and 0.50 in group 1 and 2, respectively. Group 2 exhibited better utilization (P<0.01) of TDN for milk and FCM production than that of group 1. The metabolizable energy intake, Mcal per kg milk yield, was 2.04 in group 1 and 1.88 in group 2 and MEI, Mcal per kg FCM yield, was 2.00 and 1.83 in group 1 and 2, respectively. Group 2 exhibited better utilization (P<0.01) of metabolizable energy for milk and FCM production than that of group 1. The NE L intake, Mcal per kg milk yield, was 1.28 in group 1 and 1.19 in group 2 and MEI, Mcal per kg FCM yield, was 1.25 and 1.15 in group 1 and 2, respectively. Group 2 exhibited better utilization (P<0.01) of metabolizable energy for FCM production than that of group 1. Thus, the results of the present study on efficiency of utilization of nutrients exhibited that rumen protected methionine and lysine supplemented group could utilize the available nutrients towards milk production more efficiently. __________________________________________________________________132 Results and Discussion … 4.4 Overall Plane of Nutrition in lactating cows The overall plane of nutrition of cows is presented in Table 4.49. The actual nutrient intake was compared with nutrient requirement as per NRC (2001). Table 4.49 Plane of nutrition of lactating cows fed with rumen protected methinone plus lysine Parameter DM CP TDN NE L Group 1 Group 2 Intake (kg/d) 13.01 13.36 Predicted * (kg/d) 13.60 14.40 Deficit in DMI 4.34 7.22 Intake (kg/d) 2.10 2.17 Requirement * (kg/d) 1.77 1.92 Extra CP intake (%) 18.64 13.02 Intake (kg/d) 8.76 9.05 Requirement * (kg/d) 8.21 8.75 Extra TDN intake (%) 6.70 3.43 Intake (Mcal/d) 20.58 21.00 Requirement * (Mcal/d) 19.70 21.30 Extra NE L intake (%) 4.47 - 1.41 * Requirements as per NRC (2001) The DMI was undersupplied by 4.34 percent than requirement in group 1 and 7.22 percent in group 2. The CPI was 18.64 percent higher than requirement in group 1 and 13.02 percent higher in case of group 2. TDN intake was 6.70 percent and 3.43 __________________________________________________________________133 Results and Discussion … percent higher than requirements in group 1 and 2, respectively whereas NE L intake was 4.47 percent higher in group 1 while 1.41 percent less in group 2. The plane of nutrition of cows of both the groups indicated that both the groups were adequately fed, thereby fulfilling the requirements as per NRC (2001). 4.5 Effect of Rumen Protected Methionine and Lysine Supplementation on Some Lactation Related Plasma Parameters 4.5.1 Blood Glucose Concentration The blood glucose concentration (Table 4.50, Fig 4.14) in different fortnights varied from 46.02 to 58.71 and 47.15 to 57.91 in group 1 and 2, respectively. The overall average was 55.61 and 55.07 mg/dl in group 1 and 2, respectively. The lowest level of blood glucose was found at the day of parturition in both the groups. It may be due to parturition related stress and decreased dry matter intake, but it did not fall below the lower limit of the physiological range (40-60 mg/dL) in either group. Table 4.50 Plasma glucose (mg/dl) concentration in lactating crossbred cows fed ration with or without RPM plus RPL Day Group 1 Group 2 1 46.02 ± 1.36 47.15 ± 0.66 15 56.80 ± 1.07 56.55 ± 1.50 30 57.20 ± 1.04 54.70 ± 1.00 45 57.91 ± 0.62 57.27 ± 1.33 75 57.03 ± 1.23 56.87 ± 0.77 105 58.71 ± 1.26 57.91 ± 0.35 Overall Mean 55.61 ± 1.10 55.07 ± 0.92 The blood glucose concentration remained within the normal range and no difference was observed at any stage of experiment. The reason may be a high metabolic rate of utilization of glucose and homeostatic mechanism of animal body __________________________________________________________________134 Results and Discussion … that does not allow appreciable change in glucose level. The results of the present study are similar to that of Bach et al. (2000) on supplementing rumen protected methionine and Socha et al. (2008) on supplementing rumen protected methionine and lysine in peak and mid lactation but increase was observed in early lactation. However, Berthiaume et al. (2001) found increased blood glucose level on supplementing rumen protected methionine. Socha et al., (2005) also reported that the effect of RPAA supplementation on plasma glucose concentrations was dependent upon period after parturition. Between wk 1 and 4 postpartum, plasma glucose concentrations of cows receiving RPM+L declined at a more rapid rate than cows receiving either the basal diet or RPM. However, between wk 4 and 7 postpartum, plasma glucose concentrations of RPM+L supplemented cows increased, whereas plasma glucose concentrations of cows receiving no RPAA or RPM remained essentially unchanged. Immediately following parturition, glucose concentrations decreased in cows receiving 16 % CP in diet and 15 gm of RPM supplemented group, 16% CP in diet and 6 gm of RPM plus 40 g/d of a RPM+L supplemented group, and 18.5% CP in diet and 6 gm of RPM plus 40 g/d of a RPM+L supplemented group and increased in cows receiving 18.5% CP and15 gm of RPM supplemented group. 4.5.2 Plasma Phosphatidylcholine Concentration The plasma phosphatidylcholine concentration (Table 4.51, Fig 4.14) in different fortnights varied from 95.58 to 164.16 and 109.00 to 163.90 µg/ml in group 1 and 2, respectively. The overall average was 138.57 and 140.98 µg/ml in group 1 and 2, respectively. Met is source of the methyl donor S- adenosyl Methionine, the metabolite that provides methyl groups in variety of reactions including de novo synthesis of choline from phosphatidylethanolamine, thus Met and choline metabolism are closely associated. Choline is a nutrient involved with the transport of fat from liver. Choline is synthesised in part from methionine and required for the synthesis of phosphatidylcholine, a phospholipid found in the membrane of VLDL. As much as 28% of the absorbed methionine is used for choline synthesis (Emmanuel and Kennely, 1984). Thus, Methionine plays a direct role in VLDL synthesis in bovine __________________________________________________________________135 Results and Discussion … and acts to reduce plasma ketone bodies during early lactation. (Durrand et al., 1992). Methionine is also involved in many pathways including the synthesis of carnitine, creatinine, polyamines and energy metabolism. Table 4.51 Plasma Phosphatidylcholine (µg/ml) concentration in lactating crossbred cows fed ration with or without RPM plus RPL a, b Day Group 1 Group 2 1 95.58 a ± 2.31 109.00 b ± 5.57 15 121.19 a ± 3.81 129.52 b ± 2.79 30 133.32 ± 2.99 135.71 ± 1.82 45 155.19 ± 5.07 147.19 ± 3.37 75 161.97 ± 3.46 160.55 ± 1.25 105 164.16 ± 4.09 163.90 ± 2.73 Overall Mean 138.57 ± 3.72 140.98 ± 3.83 Means having different superscripts in a row differ significantly (P<0.05) In the present study, there was increase in plasma phoshatidylcholine during first fortnight on supplementation of rumen protected methionine and lysine which might be due to contribution of methionine in de novo synthesis of choline. 4.5.3 Plasma NEFA Concentration The plasma NEFA concentration (Table 4.52, Fig 4.14) in different fortnights varied from 96.14 to 118.17 and 100.28 to 112.14 mg/L in group 1 and 2, respectively. The overall average was 106.80 and 105.77 mg/L in group 1 and 2, respectively. The NEFA concentration was higher in both the groups at the day of parturition (P=0.3626). The NEFA concentration decreased (P<0.05) during first __________________________________________________________________136 Results and Discussion … fortnight on supplementation of rumen protected methionine and lysine. But overall plasma NEFA average was statistically similar in both the groups. Table 4.52 Plasma NEFA (mg/L) concentration in lactating crossbred cows fed ration with or without RPM plus RPL Day Group 1 Group 2 1 115.94 ± 1.74 112.14 ± 2.55 15 118.17 a ± 2.03 109.64 b ± 3.81 30 103.61 ± 1.45 102.67 ± 1.18 45 103.78 ± 1.13 106.39 ± 0.80 75 103.17 ± 0.95 103.50 ± 0.66 105 96.14 ± 1.78 100.28 ± 1.14 Overall Mean 106.80 ± 3.46 105.77 ± 1.83 Comparative decreases in plasma nonesterified fatty acids concentrations in first fortnight in RPM plus RPL supplemented group reflected reduced mobilization of fatty acids from body reserves. Similarly, Socha et al. (2008) reported that there was decrease in plasma NEFA concentration on abomasal infusion of rumen protected methionine and lysine during early lactation. Bach et al. (2000) reported decreased plsma NEFA concentration on supplementing rumen protected methionine and lysine. Whereas, Misciattelli et al. (2003) did not found any effect on plasma NEFA concentration on supplementation of rumen protected methionine and lysine, and Davidson et al. (2008) on supplementation of rumen protected methionine. There was no effect of abomasal infusion of methionine and lysine on plasma NEFA level during mid lactation (Socha et. al., 2008). __________________________________________________________________137 Results and Discussion … 4.5.4 Plasma Triglycerides Concentration The plasma triglycerides concentration (Table 4.53, Fig 4.15) in different fortnights varied from 11.87 to 16.12 and 13.77 to 17.89 mg/dL in group 1 and 2, respectively. It was higher (P<0.01) in group 2 in all the forthnights. The overall average was 13.40 and 16.22 mg/dL in group 1 and 2, respectively, which was higher (P<0.01) in RPM plus RPL supplemented group. Table 4.53 Plasma triglycerides (mg/dl) concentration in lactating crossbred cows fed ration with or without RPM plus RPL Day Group 1 Group 2 1 16.12c ± 0.67 17.89d ± 0.43 15 14.62 a ± 0.26 17.89 b ± 0.43 30 12.81 a ± 0.33 16.84 b ± 0.72 45 12.58 a ± 0.24 15.79 b ± 0.66 75 12.42 a ± 0.23 15.16 b ± 0.62 105 11.87 a ± 0.14 13.77 b ± 0.53 Overall Mean 13.40 a ± 0.34 16.22 b ± 0.54 a, b Means having different superscripts in a row differ significantly (P<0.01) c, d Means having different superscripts in a row differ significantly (P<0.05) 4.5.5 Plasma VLDL Concentration The plasma VLDL concentration (Table 4.54, Fig 4.15) in different fortnights varied from 2.37 to 3.22 and 2.75 to 3.58 mg/dL in group 1 and 2, respectively. The overall average was 2.68 and 3.24 mg/dL in group 1 and 2, respectively, which was higher (P<0.01) in RPM plus RPL supplemented group. The increase in milk fat percent may relate to the role of AA in the intestinal and hepatic synthesis of chylomicrons and VLDL. Apolipoproteins and phospholipids __________________________________________________________________138 Fig 4.13 Average fortnightly fatty acid profile of milk of crossbred cows Fig 4.14 Average fortnightly plasma metabolites of crossbred cows Results and Discussion … are the required substrates for the synthesis of chylomicrons and VLDL, in addition to the presence of the long-chain fatty acids that stimulate their formation (Bauchart et al., 1996). The synthesis of apolipoproteins requires AA. The synthesis of phosphatidylcholine (lecithin), the most abundant phospholipid, requires choline. It has been demonstrated that a portion of the dairy cows’ requirement for choline was accomplished by Met as Met is as a methyl donor for choline synthesis (Sharma and Erdman, 1989; Erdman, 1994), choline can be a limiting nutrient for milk fat synthesis. Table 4.54 Fortnightly plasma VLDL (mg/dl) concentration in lactating crossbred cows fed ration with or without RPM plus RPL Day Group 1 Group 2 1 3.22c ± 0.13 3.58d ± 0.09 15 2.92 a ± 0.09 3.58b ± 0.06 30 2.56 a ± 0.07 3.37 b ± 0.14 45 2.52 a ± 0.05 3.16 b ± 0.13 75 2.48 a ± 0.05 3.03 b ± 0.12 105 2.37 a ± 0.03 2.75 b ± 0.11 Overall Mean 2.68 a ± 0.11 3.24 b ± 0.06 a, b Means having different superscripts in a row differ significantly (P<0.01) c, d Means having different superscripts in a row differ significantly (P<0.05) That Met and Lys may sometimes be limiting for the synthesis of chylomicrons or VLDL such that the availability of long chain fatty acids for milk fat synthesis is reduced has not been demonstrated. However, there is limited evidence that formation or secretion of these lipoproteins can be enhanced with improved Met and Lys nutrition (Auboiron et al., 1995; Durand et al., 1992). __________________________________________________________________139 Results and Discussion … 4.5.6 Plasma Vitamin E levels Plasma vitamin E concentration (Table 4.55, Fig 4.14) in different fortnights ranged between 0.68 to 1.33 µg/ml in group 1 and 0.66 to 1.17 µg/ml in group 2. The average values were 1.01 and 0.90 µg/ml in group 1 and 2, respectively, which were similar in both the groups. Table 4.55 Plasma vitamin E (µg/ml) concentration in lactating crossbred cows fed ration with or without RPM plus RPL a, b Day Group 1 Group 2 1 0.68 ± 0.03 0.66 ± 0.03 15 0.82 ± 0.06 0.75 ± 0.03 30 0.94 ± 0.10 0.82 ± 0.05 45 1.05 ± 0.08 0.96 ± 0.02 75 1.25a ± 0.06 1.06 b ± 0.05 105 1.33 a ± 0.04 1.17 b ± 0.03 Overall Mean 1.01 ± 0.07 0.90 ± 0.04 Means having different superscripts in a row differ significantly (P<0.05) 4.5.7 Plasma cholesterol levels Plasma cholesterol concentration (Table 4.56, Fig 4.14) in different fortnights ranged between 171.33 to 237.07 mg/dL in group 1 and 173.29 to 229.27 mg/dL in group 2. The average values were 193.16 and 198.31 mg/dL in group 1 and 2, respectively, were similar in both the groups. __________________________________________________________________140 Results and Discussion … Table 4.56 Fortnightly plasma Cholesterol (mg/dl) concentration in lactating crossbred cows fed ration with or without RPM plus RPL Day Group 1 Group 2 1 171.33 ± 5.53 173.88 ± 5.97 15 171.86 ± 5.31 173.29 ± 5.24 30 183.92 ± 6.66 191.42 ± 5.90 45 184.45 ± 7.34 203.80 ± 8.41 75 210.32 ± 7.57 218.19 ± 4.78 105 237.07 ± 9.17 229.27 ± 5.32 Overall Mean 193.16 ± 6.93 198.31 ± 5.94 4.5.8 Blood urea nitrogen levels Blood urea nitrogen (BUN) concentration (Table 4.57 Fig 4.14) in different fortnights ranged between 15.28 to 20.34 mg/L in group 1 and 14.33 to 21.51 mg/L in group 2. The average values were 18.13 and 18.01 mg/L in group 1 and 2, respectively. The plasma urea concentration observed in the present study was within the normal range in cows (Borghese, 2005). Norton et al. (1979) reported lower blood urea concentrations in cattle than buffaloes. The BUN level remained similar in both the groups. Similar results were reported by Socha et al., (2005) by abomasal infusion of methionine and lysine. While Berthiaume et al. (2001) and Bach et al. (2000) found decreased blood urea nitrogen on supplementation of rumen protected methionine. But no significant difference was observed on BUN level in present study on supplementation of rumen protected methionine plus lysine. __________________________________________________________________141 Results and Discussion … Table 4.57 Fortnightly plasma BUN (mg/L) concentration in lactating crossbred cows fed ration with or without RPM plus RPL Day Group 1 Group 2 1 20.34 ± 0.90 20.75 ± 0.34 15 19.58 ± 0.80 21.51 ± 1.29 30 18.11 ± 0.31 17.19 ± 0.39 45 17.77 ± 0.27 17.47 ± 0.30 75 17.67 ± 0.38 16.80 ± 0.58 105 15.28 ± 0.47 14.33 ± 0.27 Overall Mean 18.13 ± 0.52 18.01 ± 0.53 4.5.9 Plasma amino acid profile Fortnightly plasma amino acids viz. aspartate, glutamate, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cysteine, leucine, isoleucine, phenylalanine and lysine concentration are presented in Table 4.58, 4.59 and Fig 4.16. Table 4.58 Postpartum plasma amino acid (µmol/dl) lactating concentration in crossbred cows fed ration with or without RPM plus RPL Amino Acid Aspartate Day Group 1 Group 2 1 21.97 ± 1.11 22.91 ± 0.76 15 21.53 ± 0.97 23.24 ± 1.48 30 24.77 ± 1.08 24.00 ± 0.85 __________________________________________________________________142 Results and Discussion … Glutamate Serine Glycine 45 21.34 ± 1.89 23.07 ± 1.18 75 20.56 ± 1.48 22.02 ± 1.64 105 20.72 ± 1.20 22.20 ± 1.05 1 23.58 ± 1.18 24.48 ± 1.00 15 22.58 ± 1.04 24.60 ± 1.00 30 24.83 ± 0.49 24.12 ± 1.10 45 22.99 ± 2.31 24.29 ± 0.83 75 22.83 ± 1.89 22.14 ± 0.94 105 24.00 ± 1.63 23.20 ±1.17 1 12.59 ± 0.69 13.72 ± 0.35 15 12.99 ± 0.56 13.45 ± 0.42 30 13.69 ± 0.82 13.06 ± 0.47 45 12.43 ± 1.01 13.32 ± 0.39 75 12.26 ± 0.61 12.94 ± 0.65 105 13.92 ± 1.14 12.59 ± 0.52 1 18.68 ± 0.98 20.72 ± 0.56 15 19.39 ± 0.53 20.35 ± 0.68 30 20.11 ± 0.71 19.93 ± 0.71 45 18.13 ± 1.55 20.26 ± 0.60 75 16.71 ± 2.09 19.76 ± 1.04 __________________________________________________________________143 Results and Discussion … Histidine Arginine Threonine Alanine 105 19.28 ± 0.96 19.15 ± 0.81 1 4.66 ± 0.49 4.31 ± 0.43 15 4.69 ± 0.52 4.44 ± 0.39 30 4.98 ± 0.61 4.79 ± 0.29 45 4.16 ± 0.34 4.55 ± 0.39 75 4.12 ± 0.32 4.24 ± 0.46 105 4.62 ± 0.48 4.60 ± 0.57 1 7.63 ± 0.56 8.29 ± 0.21 15 7.66 ± 0.51 8.02 ± 0.22 30 8.28 ± 0.63 7.93 ± 0.30 45 7.72 ± 0.33 8.02 ± 0.22 75 7.34 ± 0.30 7.54 ± 0.25 105 7.50 ± 0.54 7.51 ± 0.33 1 11.84 ± 0.61 12.50 ± 0.85 15 11.28 ± 0.59 11.46 ± 0.71 30 12.16 ± 0.86 11.69 ± 0.66 45 11.15 ± 0.97 11.82 ± 0.94 75 11.53 ± 0.61 11.90 ± 0.76 105 11.88 ± 0.83 11.34 ± 0.68 1 22.82 ± 1.79 24.03 ± 0.62 __________________________________________________________________144 Results and Discussion … Proline Tyrosine Valine 15 23.81 ± 3.33 23.68 ± 0.85 30 23.27 ± 1.56 24.04 ± 1.16 45 22.64 ± 3.28 23.63 ± 0.98 75 22.41 ± 2.16 22.79 ± 1.47 105 21.13 ± 1.15 21.79 ± 0.97 1 9.14 ± 0.59 9.04 ± 0.38 15 9.03 ± 0.48 9.02 ± 0.41 30 8.87 ± 0.56 8.83 ± 0.39 45 9.14 ± 0.47 8.99 ± 0.36 75 8.41 ± 0.40 8.76 ± 0.49 105 8.65 ± 0.56 8.76 ± 0.43 1 7.49 ± 0.66 7.58 ± 0.41 15 7.36 ± 0.50 7.02 ± 0.32 30 6.80 ± 0.45 6.87 ± 0.31 45 7.03 ± 0.46 7.06 ± 0.29 75 7.09 ± 0.35 7.13 ± 0.46 105 6.82 ± 0.54 6.82 ± 0.28 1 15.21 ± 0.91 16.20 ± 0.53 15 15.55 ± 0.79 16.53 ± 0.60 30 16.42 ± 0.98 16.33 ± 0.56 __________________________________________________________________145 Results and Discussion … Methionine Cysteine Isoleucine 45 14.29 ± 1.09 15.52 ± 0.63 75 15.04 ± 0.85 16.00 ± 0.85 105 15.15 ± 0.71 15.63 ± 0.72 1 5.39a ± 0.32 5.93b ± 0.27 15 6.11 ± 0.53 6.40 ± 0.18 30 5.92 ± 0.24 6.10 ± 0.22 45 5.71 ± 0.33 6.04 ± 0.21 75 5.57 ± 0.18 5.90 ± 0.13 105 5.60 ± 0.36 5.94 ± 0.19 1 1.61a ± 0.45 2.11b ± 0.29 15 1.83 ± 0.32 2.21 ± 0.21 30 1.84 ± 0.48 2.10 ± 0.31 45 2.01 ± 0.43 2.24 ± 0.36 75 2.09 ± 0.46 2.17 ± 0.30 105 1.85 ± 0.43 1.92 ± 0.22 1 10.79 ± 0.83 9.54 ± 0.72 15 10.63 ± 0.76 10.23 ± 0.74 30 11.34 ± 1.00 10.18 ± 0.75 45 10.65 ± 0.75 10.23 ± 0.80 75 10.50 ± 0.88 10.30 ± 0.87 __________________________________________________________________146 Results and Discussion … Leucine Phenylalanine Lysine a, b 105 11.66 ± 0.90 11.20 ± 0.96 1 15.11 ± 1.08 16.18 ± 0.61 15 14.22 ± 1.77 15.52 ± 1.00 30 14.40 ± 2.05 15.11 ± 1.03 45 12.68 ± 1.92 13.14 ± 0.93 75 13.20 ± 1.65 14.64 ± 0.80 105 14.74 ± 1.06 14.91 v 0.68 1 8.26 ± 0.62 9.40 ± 0.87 15 8.66 ± 0.78 9.12 ± 1.01 30 8.93 ± 0.82 9.14 ± 1.02 45 7.51 ± 0.47 9.14 ± 0.99 75 7.84 ± 0.37 7.95 ± 0.37 105 8.60 ± 0.75 8.81 ± 1.01 1 13.28 ± 0.86 14.25 ± 0.84 15 13.50 ± 0.93 14.00 ± 0.68 30 15.47a ± 0.92 16.63b ± 0.88 45 14.39a ± 0.68 16.68b ± 1.01 75 13.93 ± 1.83 14.93 ± 0.51 105 12.89 ± 0.78 13.97 ± 0.86 Means having different superscripts in a row differ significantly (P<0.01) __________________________________________________________________147 Results and Discussion … Table 4.59 Overall mean plasma amino acid (µmol/dl) concentration in lactating crossbred cows fed ration with or without RPM plus RPL Amino acid Group 1 Group 2 Asparate 21.81a ± 1.31 22.91b ± 1.37 Glutamate 23.47 ± 1.50 23.80 ± 1.21 Serine 12.98 ± 0.83 13.18 ± 0.44 Glycine 18.72 c ± 1.13 20.03 d ± 0.68 Histidine 4.54 ± 0.48 4.41 ± 0.40 Arginine 7.69 ± 0.51 7.88 ± 0.28 Threonine 11.64 ± 0.76 11.79 ± 0.78 Alanine 22.68 c ± 2.12 23.33 d ± 1.03 Proline 8.88 ± 0.53 8.90 ± 0.40 Tyrosine 7.10 ± 0.51 7.08 ± 0.33 Valine 15.28 c ± 0.95 16.04 d ± 0.65 Methionine 5.71 a ± 0.38 6.05 b ± 0.19 Cysteine 1.87 c ± 0.43 2.13 d ± 0.55 Isoleucine 10.93d ± 0.89 10.28 c ± 0.84 Leucine 14.06 ± 1.49 14.92 ± 0.98 Phenylalanine 8.30 ± 0.65 8.93 ± 0.83 13.91 a ± 1.03 15.08 b ± 0.93 Lysine a, b c, d Means having different superscripts in a row differ significantly (P<0.01) Means having different superscripts in a row differ significantly (P<0.05) __________________________________________________________________148 Results and Discussion … Overall average of free plasma amino acid concentrations are presented in Table 4.59; Fig 4.16. Aspartate, glycine, alanine, valine, methionine, cysteine, and lysine concentrations were increased (P<0.01 or P<0.05) in cows fed ration supplemented with RPM plus RPL. However, RPM plus RPL supplementation lowered (P<0.05) plasma isoleucine concentration. All other free plasma amino acids i.e. glutamate, serine, histidine, arginine, threonine, proline, tyrosine, leucine and phenylalanine were unaffected by the treatment. Increased supply to the small intestine of any AA is expected to change its concentration in blood plasma and, possibly, improve availability of that AA for milk protein synthesis. This was demonstrated by Blauwiekel et al. (1997) in an experiment in which lysine supplementation increased duodenal lysine flow, as well as the concentration of lysine in plasma, leading to an increase in milk and milk protein yield. Baumrucker (1985) explained that the transport systems for absorption of AA depend on transport specificity and competition among AA. As feeding RPL provides more lysine to cells with a Y+ (cationic) transport system, the uptake of AA sharing the same transport system (i.e., arginine and isoleucine) may be reduced through competitive inhibition with lysine. Supplementation with RPM caused an elevation in arterial concentration of Met (Berthiaume et al., 2001, Overton et al., 1996, 1998; Blum et al., 1999) or the results were same when Met infused postruminally (Guinard and Rulquin, 1995; Pisulewski et al., 1996; Varvikko et al., 1999) It is reported that there were decrease in plasma methionine concentrations (Rogers et al., 1987) or decrease in most non-essential AA (NEAA) as well as threonine, leucine and tryptophan (Swanepoel et al., 2011) when RPL was fed. Swanepoel et al. (2011) reported that the plasma lysine concentration was only numerically increased with RPL feeding. Results of Weekes et al. (2006) suggest that a decreased concentration of plasma AA (especially histidine) without a positive milk protein response could be due to a more efficient catabolism or deposition of AA into body protein, which might be supported by the trend to lower BCS loss on RPL supplementation. __________________________________________________________________149 Results and Discussion … Abomasal Met infusions of 56.5 g/d (Seymour et al., 1990) and duodenal Met infusions of 0, 6, 12, 18, and 24 g/d (Pisulewski et al., 1996) elevated blood and plasma Met concentrations linearly. Similarly, dietary supplementation with rumenprotected Met generally is associated with higher blood or plasma Met concentrations (Nichols et al., 1998; Blum et al., 1999) but not in all instances (Colin-Schoellen et al., 1995; Xu et al., 1998). Sylvester et al. (2003) reported increased plasma Met concentration on diets supplemented with HMBi. Bequette et al. (1997) compared unidirectional fluxes of AA across the mammary gland to their secretion in milk protein. Based on their results, milk protein synthesis must compete with other metabolic processes within the mammary gland for the use of some AA. 4.5.10 Plasma prolactin levels Plasma Prolactin concentrations (Table 4.60 Fig 4.17) in different weeks ranged between 13.50 to 215.41 ng/ml in group 1 and 13.29 to 216.26 ng/ml in group 2. The average values were 18.13 and 18.01 ng/L in group 1 and 2, respectively. There was no effect of rumen protected methionine and lysine supplementation on plasma prolactin concentration. Table 4.60 Weekly plasma prolactin (ng/ml) concentration in lactating crossbred cows fed ration with or without RPM plus RPL Day Group 1 Group 2 -15 25.58 ± 4.60 26.08 ± 4.63 -7 118.31 ± 14.00 120.10 ± 14.32 1 215.41 ± 23.85 216.26 ± 23.99 7 25.59 ± 2.27 26.22 ± 2.22 15 13.50 ± 0.65 13.29 ± 0.63 Overall Mean 79.68 ± 9.07 80.39 ± 9.16 The hormones involved in lactogenesis include increased secretion of prolactin, adrenocorticotropic hormone (ACTH) and estrogen, and a decrease or virtual absence of progesterone. Prolactin concentration in cattle does not change __________________________________________________________________150 Fig. 4.17 Average weekly plasma prolactin of crossbred cows Fig. 4.18 Average weekly plasma growth hormone of crossbred cows Results and Discussion … appreciably during gestation, but a major increase occurs just before parturition. Prolactin induces gene expression in mammary tissue for casein synthesis. Once the lactation has been established in cows, the concentration of basal circulating prolactin and the release of prolactin at milking can be reduced to low level without affecting milk yield. (Reece, 2004) The results of the present study indicated that there was increase in level of prolactin on day of parturition and thereafter sudden decrease in prolactin concentration with the progression of lactation. 4.5.11 Plasma growth hormone levels Plasma growth hormone concentration (Table 4.61; Fig 4.18) of different weeks ranged between 6.25 to 7.01 ng/ml in group 1 and 6.22 to 6.97 ng/ml in group 2. The average values were similar (6.63 ng/L) in group 1 and 2, respectively. There was no effect of rumen protected methionine and lysine on plasma growth hormone concentration. Similarly Misciattelli et al. (2003) also did not observe any effect of rumen protected methionine on plasma growth hormone level. Table 4.61 Weekly plasma growth hormone (ng/ml) concentration in lactating crossbred cows fed ration with or without RPM plus RPL Day Group 1 Group 2 -15 6.36 ± 0.07 6.33 ± 0.08 -7 6.25 ± 0.08 6.22 ± 0.09 1 6.84 ± 0.10 6.95 ± 0.06 7 6.67 ± 0.08 6.67 ± 0.07 15 7.01 ± 0.06 6.97 ± 0.07 Overall Mean 6.63 ± 0.08 6.63 ± 0.07 Growth hormone is more important in the maintenance of cow’s lactation as it is galactopoeitic in cattle. A surge in growth hormone from the anterior pituitary occurs just before parturition, perhaps assuming its role of directing nutrients to the __________________________________________________________________151 Results and Discussion … mammary gland for milk synthesis. It has been shown that plasma growth hormone concentration is significantly higher in high yielding cows than in low yielding cows, and that a significant reduction occurs when high yielding cows cease lactating. (Reece, 2004). The results of the present study showed that there was increase in concentration on plasma growth hormone after parturition in both the groups. Table 4.62 Condensed information of lactation trial Sr. No. 1 2 Parameter Average Initial Body Weight (After Calving) Average Final Body Weight (at 120 Days) Group 1 Group 2 459.2 ± 20.3 442.7 ± 11.5 440.6 ± 20.6 448.4 ± 12.4 3 BCS (After Calving) 3.47 ± 0.11 3.69 ± 0.06 4 BCS (at 120 days) 2.97 ± 0.03 3.06 ± 0.04 5 Average Nutrients Intake (kg/d) a DMI 13.01a ± 0.28 13.36b ± 0.34 b CPI 1.91a ± 0.09 1.95b ± 0.07 c TDNI 8.76a ± 0.40 9.05b ± 0.32 6 Milk Yield (120 Days) a Average MY (kg/d) 15.89a ± 0.26 17.69b ± 0.19 b Average FCM Yield (kg/d) 16.21a ± 1.59 18.24b± 1.13 c Average ECM yield (kg/d) 16.20a ± 0.27 18.11b ± 0.20 __________________________________________________________________152 Results and Discussion … 7 Average Milk Composition a Total Solids (%) 13.03 a ± 0.07 13.14 b ± 0.06 b Milk Fat (%) 4.13 a ± 0.05 4.22 b ± 0.05 c Milk Protein (%) 3.27 ± 0.03 3.28 ± 0.02 d Milk Lactose (%) 4.88 a ± 0.01 4.86 b ± 0.02 e Solid-Not-Fat (%) 8.91 ± 0.03 8.91 ± 0.03 f Milk Urea Nitrogen (mg/dl) 18.57 ± 0.65 18.44 ± 0.55 g Milk Choline 87.69 ± 2.67 88.63 ± 2.51 8 Efficiency of Nutrient Utilization per kg milk yield a DM (kg/kg MY) b CP (g/kg MY) c TDN (kg/kg MY) 9 Efficiency of Nutrient Utilization per kg FCM a DM (kg/kg FCM) b CP (g/kg FCM) c TDN (kg/kg FCM) 10 Blood Parameters a Blood Glucose (mg/dl) b Vitamin E (µg/ml) 0.82a ± 0.01 0.76b ± 0.02 132.81 a ± 1.72 122.18 b ± 1.79 0.86 ± 0.01 0.80 a ± 0.02 0.85 ± 0.01 0.73 b ± 0.02 130.18 a ± 1.75 128.99 b ± 1.76 0.54 a ± 0.01 0.50 b ± 0.01 55.61 ± 1.10 55.07 ± 0.92 1.01 ± 0.07 0.90 ± 0.004 __________________________________________________________________153 Results and Discussion … c NEFA (mg/l) 106.80 ± 3.46 105.77 ± 1.83 d Phosphatidylcholine (µg/ml) 138.57 ± 3.72 140.98 ± 3.83 e Triglycerides (mg/dl) 13.40 a ± 0.34 16.22 b ± 0.54 f VLDL (mg/dl) 2.68 a ± 0.11 3.24 b ± 0.06 g Cholesterol (mg/dl) 193.16 ± 6.93 198.31 ± 5.94 h BUN (mg/L) 18.13 ± 0.52 18.01 ± 0.53 a, b Means having different superscripts in the same row differ significantly (P<0.01) 4.6 EFFECT OF RPM PLUS RPL SUPPLEMENTATION ON REPRODUCTIVE PERFORMANCE IN CROSSBRED COWS 4.6.1 Calving Performance The effect of feeding RPM and RPL on the calving performance of animals is presented in Table 4.63. The average body weight of calves at the time of birth was 25.72 kg in group 1 and 26.00 kg in group 2 which was not different. Incidence of premature births was higher in group 1 than that in group 2. As maximum growth of the foetus takes place in the last trimester of the pregnancy, adequately supply of energy and protein are crucial for the normal development of foetus. 4.6.2 Reproductive abnormalities The reproductive abnormalities observed during the course of experiment are presented in Table 4.64; Fig 4.19. There were two cases of premature births in group 1, However, one case was observed in group 2. No case of still birth was reported in both the groups. Four cases of retention of fetal membranes (RFM) were observed in group 1 while one case was observed in group 2. Generally, the RFM leads to metritis, endometritis and pyometra in animals. It may be reason behind higher incidence of metritis in group 1 in the post parturient period than that of group 2 cows. Dystokia and milk fever cases did not occur in both the groups. Two case of mastitis was observed in group 1, whereas, one such case was observed in group 2 cows during post partum experimental period of 120 days. The major problem __________________________________________________________________154 Results and Discussion … associated with such calving related abnormalities is delay in reaching the peak yield and also lower milk production at peak, which in turn reduced the milk production during experimental period as observed in present study. Table 4.63 Calving Performance of crossbred cows fed ration with or without RPM and RPL Dam No. Date of Calf No. Calf sex Parturition Calf Birth weight (kg) Group 1 6918 11.06.2011 7500 Female 26 7068 16.06.2011 7597 Male 22.5 6997 20.06.2011 7598 Male 38 7034 27.06.2011 7504 Female 32 6960 02.08.2011 7606 Male 28 7013 10.08.2011 Died Male 8 7085 18.08.2011 7510 Female 29 7106 08.11.2011 Died Female 18 6998 03.01.2012 7632 Male 30 Average 25.72 ± 2.91 Group 2 6774 22.06.2011 7502 Female 27 6969 24.06..2011 7600 Male 25 7083 24.07..2011 Died Female 14 __________________________________________________________________155 Results and Discussion … 7045 09.08.2011 7608 Female 28 7088 18.08..2011 7511 Female 25 6944 25.08..2011 7612 Male 28 7000 02.09.2011 7513 Female 26 7052 22.09.2011 7617 Male 31 7108 02.01.2012 7631 Male 30 Average 26.00 ± 1.65 Table 4.64 Reproductive abnormalities plus metabolic diseases observed in crossbred cows fed RPM plus RPL S. No. Parameter Group 1 Group 2 1 Still Birth 0 0 2. Premature Birth 2 1 3. RFM 4 1 4. Metritis 3 1 5. Dystokia 0 0 6. Milk fever 0 0 7 Mastitis 2 1 RFM= Retention of fetal membranes Four cases of retention of fetal membranes (RFM) were observed in group 1 while one case was observed in group 2. Generally, the RFM leads to metritis, endometritis and pyometra in animals. It may be reason behind higher incidence of __________________________________________________________________156 Fig 4.19 Reproductive abnormalities observed in crossbred cows Fig 4.20 Reproductive parameters observed in crossbred cows Results and Discussion … metritis in group 1 in the post parturient period than that of group 2 cows. Dystokia and milk fever cases did not occur in both the groups. Two case of mastitis was observed in group 1, whereas, one such case was observed in group 2 cows during post partum experimental period of 120 days. The major problem associated with such calving related abnormalities is delay in reaching the peak yield and also lower milk production at peak, which in turn reduced the milk production during experimental period as observed in present study. The results of the present study are similar to that of Ardalan et al. (2010) who reported that incidences of postpartum reproductive and metabolic diseases viz. retained placenta, mastitis, metritis, dystokia, were significantly reduced in Holstein dairy cows fed RPM 18 g/d from 4 week prepartum to 14 weeks postpartum. 4.6.3 Reproduction related parameters The reproduction related parameters are presented in Table 4.65. The average duration for commencement of cyclicity was 65.13 days in group 1 and 64.11 days in group 2. The time required for commencement of cyclicity was similar (P>0.01) in both the groups. Table 4.65 Reproductive parameters recorded in crossbred cows fed RPM plus RPL Parameter Group 1 Group 2 Commencement of cyclicity (Days ) 65.13 ± 2.26 64.11 ± 1.02 Days Open (Days ) 164.2 ± 9.53 152.4 ± 6.64 AI/Conception 2.60 ± 0.81 2.67 ± 0.51 Conception rate 55.55 66.66 The days open or service period was calculated on the basis of five animals that conceived in group 1 and six animals in group 2 during the experimental duration of 120 days. It was 164.2 and 152.4 days in group 1 and 2, respectively. The service period was shorter by 11.8 days in group 2 than that of group 1, indicating that lesser __________________________________________________________________157 Results and Discussion … time was required for the animals in group 2 for conception. Similarly, Ardalan et al. (2009) reported that there was significant decrease in days to first estrous and open days in Holstein dairy cows fed 18 gm RPM from 4 week pre partum to 20 weeks postpartum. Cows of Group 1 required 2.60 AIs per conception while group 2 required 2.67 AIs per conception. There were no effect of supplementation of RPM plus RPL on no of AI required for conception. The conception rate during the experimental period of 120 days was 55.55 % in group 1 and 66.66 % in group 2. The results of the present study indicate that RPM plus PRL supplementation increased the conception rate in cows. Similar results were reported Ardalan et al. (2009) on supplementing rumen protected methionine and lysine in the diet of high yielding lactating dairy cows. Xu et al. (1998) reported the overall incidence of health related disorders were numerically lowest for the dairy cows fed high amount of rumen protected methionine and lysine. In contrast, Polan et al. (1991) reported that supplementation of rumen protected form of methionine and lysine had no significant effect on days to first service, services per conception and calving interval in dairy cows. 4.7 ECONOMICS OF FEEDING RUMEN PROTECTED METHIONINE PLUS LYSINE IN CROSSBRED COWS The economics of feeding rumen protected methionine and lysine to lactating cows throughout the trial period (120 days) has been shown in Table 4.66. The cost of rumen protected methionine and lysine was Rs 450 and Rs 250 per kg, respectively. RPM and RPL used as a supplement in the concentrate at the rate of 7 and 60 gm, respectively, the additional cost of supplement was Rs 18.15 per day postpartum. Based on the prevalent price of different ingredients, the cost of concentrate mixtures worked out to be Rs. 1640. Net return over feed cost of milk yield per animal per day in control and treatment group was Rs. 192.1 and 215.8, respectively, indicating that it was higher by Rs. 23.7 in treatment group over that of control group. The feed cost (Rs.) per kg __________________________________________________________________158 Results and Discussion … FCM produced was Rs. 10.94 and Rs. 10.92 respectively in group 1 and group 2. While cost benefit ratio was similar in both the groups. Table 4.66 Economics of feeding rumen protected methionine plus lysine to lactating crossbred cows throughout the whole experimental period (120days) Attributes Group 1 Group 2 13.01 ± 0.28 13.36 ± 0.34 33 34 35640 36720 1 1.2 1080 1296 Concentrate intake (kg/d/animal) 8.62 8.75 Total concentrate intake in 120 days trial by 9 9310 9450 Total cost for green maize (@ Rs. 100.00/qtl.) 35640 36720 Total cost for wheat straw (@ Rs. 300.00/qtl.) 3240 3888 Total cost for concentrate (@ Rs. 1640.00/qtl.) 152677.4 154980.00 Total feed cost (Rs.) 191557.4 195588.00 - 3.15 DMI (kg/day) Feed intake (fresh basis) Green maize intake (kg/d/animal) Total green maize intake in 120 days trial by 9 animals (kg) Wheat straw intake (kg/d/animal) Total wheat straw intake in 120 days trial by 9 animals (kg) animals (kg) Feed cost (Rs.) Cost of postpartum supplementation of rumen __________________________________________________________________159 Results and Discussion … protected Methionine (7 g/d/animal for 120 days @ Rs. 450/kg) Cost of postpartum supplementation of rumen - 15 - 3402.0 - 16200.0 191557.4 215190.0 Feed cost (Rs/animal/day) 177.37 199.25 Average milk yield (kg/d/animal) 15.89 17.69 Average FCM milk yield ((kg/d/animal) 16.21 18.24 Average milk fat % 4.13 4.22 Average Milk SNF % 8.91 8.91 Total milk yield in 120 days by 9 animals (kg) 17161.2 19105.2 Total FCM yield in 120 days by 9 animals (kg) 17506.8 19699.2 10.94 10.92 23.25 23.46 399072.6 448249.9 protected Lysine (60 g/d/animal for 120 days @ Rs. 250/kg) Additional cost of Rumen protected Methionine supplementation Additional Cost of Rumen protected Lysine supplementation Total feed cost with RPM plus RPL (Rs.) Feed cost (Rs.) per kg FCM produced Market price per kg of milk (@ Rs. 230.93/ kg milk fat and Rs 153.95/ kg of milk SNF) Gross income (Rs.) from selling of milk (@ Rs. 230.93/ kg milk fat and Rs 153.95/ kg of milk SNF) __________________________________________________________________160 Results and Discussion … Net return in 120 days from 9 animals (Rs.) Net return (Rs./animal/day) Additional profit in 120 days from 9 animals on 207515.2 233059.9 192.1 215.8 - supplementation of RPM plus RPL (Rs) Additional profit per day per cow on 25544.6 - supplementation of RPM plus RPL (Rs) Benefit cost ratio 1.08 23.7 1.08 The net return from experimental ration was markedly higher by 12.30% than that of the control ration. As a result, additional profit in 120 days from 9 cows on supplementation of rumen protected methionine and lysine was Rs. 25544.6. Thus, based on the results of the present study, it could be concluded that feeding rumen protected methionine and lysine to high yielding lactating cows during early lactation was cost effective. __________________________________________________________________161 CHAPTER – 5 SUMMARY AND CONCLUSIONs 5. SUMMARY AND CONCLUSIONS _________ _______ To investigate the effect of supplementing rumen protected methionine (RPL) and lysine (RPL) on pre and post parturient performance such as calving, lactation, nutrient utilization and reproduction, eighteen crossbred cows were selected and divided into two groups (9 each) on the basis of Most Probable Production Ability (MPPA) and lactation number. Animals in group 1 (control group) were fed chopped wheat straw, chaffed green maize fodder and compounded concentrate mixture as per requirements (NRC, 2001), whereas, animals in group 2 (treatment group) were fed the same ration as to control group plus 5 gm RPM and 20 gm RPL during prepartum period and 7 gm RPM and 60 gm RPL during postpartum period, respectively. The experimental period started from 40 days before expected date of parturition to 120 days post parturition. The results obtained during the course of this study have been summarized. 5.1 In vitro and in sacco study 5.1.1 Product evaluation Rumen escape potential of RPM and RPL from rumen hydrolysis was 75.20 and 54.97 per cent, respectively. 5.1.2 RDP and RUP contents of feed ingredients RDP content of wheat straw, maize fodder and concentrate were 31.88, 60.61 and 69.41 per cent of CP, respectively. Whereas RUP content of wheat straw, maize fodder and concentrate were 68.12, 39.39 and 30.59 per cent of CP, respectively. Groundnut cake had the highest RDP (89.27% of CP), followed by mustard cake (83.58) and lowest for wheat straw (31.88). While highest RUP was observed in wheat straw (68.12) followed by maize (44.82) and deoiled rice bran (40.01%). 5.1.3 Microbial protein yield The microbial protein yield g per kg TDN fermented was 127.84, 119.62 and 110.04 g in different TMRs containing concentrate to roughage ratio 60:40, 50:50 and 40:60, respectively. 5.1.4 RUP digestibility RUP intestinal digestibility values of wheat straw, maize fodder and 162 Summary and Conclusions… ___________________ ______________________________________ concentrate were 64.40, 70.47 and 79.84 percent, respectively. 5.2 EFFECT OF FEEDING RUMEN PROTECTED METHIONINE AND LYSINE DURING PRE PARTURIENT PERIOD 5.2.1 Body weight In the first fortnight (from day -30th to -15th day pre parturient), the experimental animals gained 7.6 and 6.7 kg body weight in groups 1 and 2, respectively, while in the 2nd fortnight (from day -15th to the day of parturition), the loss in weight was 58.2 and 56.1 kg, respectively due to parturition and release of the foetal membranes. The changes in body weight were not statistically different between the groups. 5.2.2 Body condition score The body condition score during pre parturient period ranged from 3.84 to 3.88 in group 1, and from 3.81 to 4.00 in group 2. The initial BCS of group 1 and 2 was 3.84 and 3.81, respectively. The pre partum changes in body weights were not conspicuously reflected in BCS changes which were higher in supplemented group than control group before parturition. 5.2.3 Nutrients intake The average DMI in the first fortnight (from day -30th to -15th day pre parturient) was 9.33 and 8.87 (kg/d) and in the second fortnight (day -15th to the day of parturition), it was 8.21 and 7.75 kg/d in group 1 and 2, respectively. DMI/100 kg body weight in first and second fortnight was 1.83 and 1.59 kg/d in group 1 and 1.78 to 1.53 kg/d in group 2 respectively. There was no difference among two groups in average DMI/d as well as DMI/100 kg body weight before parturition. The average CPI and TDNI in the first fortnight (from day -30th to -15th day pre parturient) was 1.17 and 1.14 (kg/d) and 5.90 and 5.67 (kg/d) and in the second fortnight (day -15th to the day of parturition) was 1.09 and 1.04 (kg/d) and 5.14 and 4.66 (kg/d) in group 1 and 2, respectively, which were similar. CP intake/100 kg body weight and TDN intake/100 kg body weight in first and second fortnight was 0.23 and 0.21 kg/d and 1.14 and 1.01 kg/d in group 1 and 0.23 and 0.23 kg/d and 1.14 and 0.97 kg/d in group 2 respectively, which were also similar in both the groups before parurition. __ _____________________________ _ _ __ 163 Summary and Conclusions… ___________________ ______________________________________ The average RUP and RDP intakes in the first fortnight (from day -30th to 15th day pre parturient) were 0.40 and 0.39 (kg/d) and 077 and 0.75 (kg/d) and in the second fortnight (day -15th to the day of parturition) they were 0.36 and 0.34 and 0.72 and 0.70 (kg/d) in group 1 and 2, respectively. The average MP Intake in the first fortnight (from day -30th to -15th day pre parturient) was 0.76 and 0.74 (kg/d) and in the second fortnight (-15th to the day of parturition), it was 0.70 and 0.67 (kg/d) in group 1 and 2, respectively. It was statistically similar. The average ME and NE L Intake in the first fortnight (from day -30th to -15th day pre parturient) was 21.78 and 21.28 (Mcal/d) and 13.59 and 13.33 (Mcal/d) and in the second fortnight (-15th to the day of parturition), these were 19.78 and 18.50 (Mcal/d) and 12.36 and 11.60 (Mcal/d) in group 1 and 2, respectively. ME and NE L intake/ 100 kg body weight in first and second fortnight was 4.28 and 3.83 Mcal/d and 2.67 and 2.40 Mcal/d in group 1 and 4.28 and 3.67 Mcal/d and 2.68 and 2.30 Mcal/d in group 2, in group 2 respectively. There was no difference among two groups in average prepartum ME and NE L intake per day aswelll as per100 kg body weight. The average duodenal methionine flow in the first fortnight (from day -30th to -15th day pre parturient) was 1.90 and 2.22 (% of MP/d) and in the second fortnight (15th to the day of parturition) was 1.97 and 2.30 (kg/d) in group 1 and 2, respectively. The average duodenal lysine flow in the first fortnight (from day -30th to -15th day pre parturient) was 6.44 and 7.09 (kg/d) and in the second fortnight (-15th to the day of parturition) was 6.57 and 7.35(kg/d) in group 1 and 2, respectively. Duodenal methionine and lysine flow in group 2 was higher (p<0.01) than group 1 due to supplementation of RPM and RPL. 5.2.4 Overall plane of nutrition in prepartum cows On comparison of the actual nutrient intake with that of nutrient requirement as per NRC (2001), DM intake was deficient by 10.51 percent and 13.44 percent than requirements in group 1 and 2, respectively. The CPI was 7.62 percent higher than requirement in group 1 and 3.81 percent higher in case of group 2. TDN intake was 12.65 percent and 5.51 percent higher than requirements in group 1 and 2, respectively and NE L intake was 13.77 percent and 11.25 percent higher in group 1 __ _____________________________ _ _ __ 164 Summary and Conclusions… ___________________ ______________________________________ and group 2, respectively. The plane of nutrition of cows of both the groups indicated that both the groups were adequately fed, thereby fulfilling the requirements as per NRC (2001). 5.2.5 Effect of rumen protected methionine and lysine supplementation on certain blood parameters of prepartum cows The blood glucose concentration on -30th day of parturition was 57.19 and 56.06 (mg/dL) and on -15th day of parturition, it was 56.55 and 54.14 (mg/dL) in groups 1 and 2, respectively while it was decreased (p<0.01) at the day of parturition in both the groups. The blood phosphatidylcholine concentration on -30th day of parturition was 152.10 and 149.39 (µg/ml) and on -15th day of parturition, it was 122.35 and 133.26 (µg/ml) in groups 1 and 2, respectively. There was increase in plasma phosphatidylcholine level on the day of parturition on supplemention of rumen protected methionine and lysine. The plasma NEFA concentration on -30th day of parturition was 69.53 and 67.63 (mg/L) and on -15th day of parturition, it was 91.89 and 77.58 (mg/L) in group 1 and 2, respectively. Plasma NEFA concentration on the day of parturition was 115.94 and 112.14 in group1 and group 2, respectively. No difference was observed in both the groups at any stage of experiment. The plasma triglycerides and VLDL concentration on -30th day of parturition was 17.30 and 18.66 (mg/dL) and 3.46 and 3.73 (mg/dL) and on -15th day of parturition was 16.27 and 18.24 (mg/dL) and 3.25 and 3.65 (mg/dL) in group 1 and 2, respectively. There was increase (P<0.05) in plasma triglycerides and VLDL on the 15th day of parturition and on the day of parturition in RPM plus RPL supplemented group. The plasma vitamin E and cholesterol concentration on -30th day of parturition was 1.07 and 1.03 (µg/ml) and 166.80 and 167. 37 (µg/ml) and on -15th day of parturition, it was 0.81 and 0.79 (µg/ml) and 167.25 and 172.74 (µg/ml) in group 1 and 2, respectively. The plasma vitamin E and cholesterol concentration remained within the normal range and no difference was observed at any stage of experiment. __ _____________________________ _ _ __ 165 Summary and Conclusions… ___________________ ______________________________________ The blood urea nitrogen concentration on -30th day of parturition was 14.08 and 13.38 (mg/dL) and on -15th day of parturition, it was 19.39 and 19.03 (mg/dL) in group 1 and 2, respectively. The concentrations of plasma urea N did not differ in response to dietary treatment, suggesting that overall N utilization was similar in both the groups. 5.2.5 Plasma amino acid profile Prepartum fortnightly plasma amino acids viz. aspartate, glutamate, serine, glycine, histidine, arginine, threonine, alanine, proline, tyrosine, valine, methionine, cysteine, leucine, isoleucine, phenylamine and lysine concentration were not affected by supplementation of rumen protected methionine and lysine while increasing trends were observed in plasma methionine and lysine concentration in cows supplemnted with RPM plus RPL. 5.3 EFFECT OF FEEDING RUMEN PROTECTED METHIONINE AND LYSINE DURING POST PARTURIENT PERIOD 5.3.1 Body weight changes The initial body weights of animals at day one after parturition were 459.2 and 442.7 kg in group 1 and 2, respectively and after 120 days of feeding, final body weight were 440.7 and 448.4 kg. Perusal of the fortnightly changes in body weight revealed that there was overall a net loss of 18.59 kg in group 1, whereas there was overall gain of 5.79 kg in group 2. 5.3.2 Body condition score BCS after parturition was 3.47 and 3.69 in group 1 and 2, respectively. The body condition score during post parturient period ranged from 2.72 to 3.16 in group 1, and from 2.92 to 3.33 in group 2 during different fortnights. Overall average BCS of different fortnights was 2.95 and 3.09 in group 1 and 2, respectively, which was higher (P<0.01) in group 2 due to higher energy intake by this group that lead to less negative energy balance. 5.3.3 Nutrient Intake The overall mean DMI was 13.01 kg and 13.36 kg/d in group 1 and 2, respectively, and higher (P<0.05) DMI was recorded in group 2. The average DMI/100 kg body weight was 3.03 and 3.13 kg/d in group 1 and 2, respectively. The __ _____________________________ _ _ __ 166 Summary and Conclusions… ___________________ ______________________________________ average CPI was 2.11 and 2.17 kg/d in the group 1 and group 2, respectively, which was higher (P< 0.01) in group 2 than that of group 1. The overall average CP intake/ 100 kg BW during the experimental period was 0.49 and 0.51 kg/d in group 1 and group 2, respectively, which was higher in group 2 than that of group 1. Average TDNI was 8.76 kg and 9.05 kg/d in group 1 and 2, respectively. The average TDNI was higher (P<0.01) by 3.31 per cent in group 2 over that of group 1. The overall average TDN intake/ 100 kg BW was 2.04 kg/d in group 1 and 2.12 kg/d in group 2. The average RDP and RUP intake was 1.40 and 1.43 kg/d and 0.72 and 0.74 kg/d in the group 1 and group 2, respectively, which were higher (P<0.05) in group 2 than that of group 1. The average MP intake was 1.31 and 1.35 kg/d in the group 1 and group 2, respectively, which was higher (P< 0.01) in group 2 than that of group 1. The overall mean duodenal methionine and lysine supply was 1.86 and 6.15% and 2.27 and 7.12 % of metabolizable protein per day in the group 1 and group 2, respectively, which was higher (P<0.01) in group 2 than that of group 1, due to supplementation of rumen protected methionine and lysine in group 2. Average ME and NE L intake was 32.33 and 20.58 Mcal/d in group 1 and 33.31 and 21.00 Mcal/d in group 2, respectively. The average ME and NE L intake was higher (P<0.05) by 3.03 and 2.04 per cent in group 2 over that of group 1, respectively. Average ME intake/ 100 kg BW was 7.53 and 4.72 Mcal/d in group 1 and 7.82 Mcal and 4.89 in group 2. ME and NE L intake/100 kg BW was higher (P<0.05) in group 2 than that of group 1. 5.4 EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE SUPPLEMENTATION ON MILK PRODUCTION AND COMPOSITION 5.4.1 Milk production Average milk production during supplementation period was 15.89 kg/d in group 1 and 17.69 kg/d in group 2, which was 11.33 per cent higher (P<0.01) in group 2 over that of group 1. Average daily 4 % FCM yield was 16.21 in group 1 and 18.24 kg in group 2. Group 2 had 12.52 per cent higher (P<0.01) FCM yield over that of group 1. Average daily ECM yield was 16.20 in group 1 and 18.11 kg in group 2. __ _____________________________ _ _ __ 167 Summary and Conclusions… ___________________ ______________________________________ Rumen protected methionine and lysine supplemented group exhibited 11.79 per cent higher (P<0.01) ECM yield over that of control group. 5.4.2 Milk composition The overall average milk fat per cent was higher (P<0.01) by 2.18 per cent in group 2 (4.22%) over that of group 1 (4.13%). Average milk fat yield was 657.18 g/d in group 1 and 745.32 g/d in group 2 which was higher (P<0.01) by 13.41 per cent in group 2 than that of group 1. The average milk protein and lactose content was 3.27 and 3.28 per cent and 4.88 and 4.86 per cent in group 1 and group 2, respectively. Average milk protein and lactose yield was 518.68 and 578.63 and 774.60 and 858.08 g/d in groups 1 and 2, respectively showing higher (P<0.01) milk protein and lactose yield by 11.56 and 10.77 per cent in group 2 than that of group 1, respectively. Mean SNF content was similar (8.91 vs 8.91 %) in both the groups. Mean total solids content in group 1 and 2 were 13.03 and 13.14 per cent, respectively which were higher (P<0.01) in group 2 than that of group 1. The overall average of MUN and milk choline contents were 18.57 and 18.44 mg/dL and 87.69 and 88.63 mg/dL in group 1 and 2, respectively, which were similar in groups 1 and 2. Average milk saturated fatty acids contents were 68.83 and 68.33 % of fatty acids in group 1 and 2, respectively. Average milk unsaturated fatty acids contents were 31.30 and 31.67 % of fatty acids in group 1 and 2, respectively. The average milk mono unsaturated fatty acids content were 28.21 and 28.70 % of fatty acids in group 1 and 2, respectively. Average milk poly unsaturated fatty acids content was 3.09 and 2.97 % of fatty acids in group 1 and 2, respectively. The saturated acids, unsaturated acids, MUFA and PUFA content of the milk were similar in group 1 and group 2. 5.4.3 Efficiency of milk production The efficiency of utilizing DMI (kg) per kg milk production was 0.82 and 0.76 and for 4 % FCM, it was 0.80 and 0.73 for groups 1 and 2, respectively. The CP intake (kg) per kg milk production was also better (P<0.01) in group 2 than that of group 1. The TDN intake kg per kg milk yield was 0.55 in group 1 and 0.51 in group __ _____________________________ _ _ __ 168 Summary and Conclusions… ___________________ ______________________________________ 2 and TDNI kg per kg FCM yield was 0.54 and 0.50 in group 1 and 2, respectively. Group 2 exhibited better utilization (P<0.01) of TDN for milk production as well as FCM production than that of group 1. The ME and NE L intake, Mcal per kg milk yield, was 2.04 and 1.28 in group 1 and 1.88 and 1.19 in group 2, respectively. ME and NE L Intake, Mcal per kg FCM yield, was 2.00 and 1.83 Mcal and 1.25 and 1.15 Mcal in group 1 and 2, respectively. Group 2 exhibited better utilization (P<0.01) of ME and NE L for milk and FCM production than that of group 1. 5.5 EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE SUPPLEMENTATION ON SOME BLOOD PARAMETERS Average blood glucose levels were similar in both the groups (55.61 and 55.07 mg/dl). Average phosphatidylcholine levels were 138.57 and 140.98 µg/ml in group 1 and 2, respectively, which were also similar in the two groups. Average plasma NEFA and vitamin E concentrations were 106.80 and 105.77 mg/L and 1.01 and 0.90 µg/ ml in group 1 and 2, respectively. The average values of plasma cholesterol and BUN were 193.16 and 198.31 mg/dL and 18.13 and 18.01 mg/L in group 1 and 2, respectively. Plasma NEFA, vitamin E, cholesterol and BUN concentrations in both the groups were similar. Average plasma triglycerides and VLDL concentrations were 13.40 and 16.22 mg/dL and 2.68 and 3.24 mg/dL in group 1 and 2, respectively which were higher (P<0.01) in group 2 than in group 1. 5.6 EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE SUPPLEMENTATION ON PLASMA AMINO ACIDS Aspartate, glycine, alanine, valine, methionine, cysteine, and lysine concentrations in plasma were increased (P<0.05) in cows fed ration supplemented with RPM plus RPL. However, RPM plus RPL supplementation lowered (P<0.05) plasma isoleucine concentration. All other free plasma amino acids i.e. glutamate, serine, histidine, arginine, threonine, proline, tyrosine, leucine and phenylamine were unaffected by RPM plus RPL supplementation. 5.7 EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE SUPPLEMENTATION ON PLASMA HORMONAL PROFILE __ _____________________________ _ _ __ 169 Summary and Conclusions… ___________________ ______________________________________ The average weekly plasma prolactin and growth hormone concentration were 79.68 and 80.39 ng/L and 6.63 and 6.63 ng/L in group 1 and 2, respectively. There was no effect of rumen protected methionine and lysine supplementation on plasma prolactin and growth hormone concentration. 5.8 EFFECT OF RUMEN PROTECTED METHIONINE AND LYSINE SUPPLEMENTATION ON REPRODUCTIVE PERFORMANCE IN COWS 5.8.1 Calving performance The average body weight of calves at the time of birth was 25.72 kg in group 1 and 26.00 kg in group 2 which was not affected on supplementing rumen protected methionine and lysine. 5.8.2 Reproductive abnormalities There were two cases of premature births in group 1, however, one case was observed in group 2. Four cases of retention of fetal membranes (RFM) were observed in group 1 while one case was observed in group 2. Higher incidence of metritis in group 1 (3 cases) were recorded whereas only one case was observed in group 2. Two cases of mastitis were observed in group 1, whereas, one such case was observed in crossbred cows of group 2 during experimental period of 120 days post partum. 5.8.3 Reproduction related parameters The average duration for commencement of cyclicity was 65.13 days in group 1 and 64.11 days in group 2, which was similar in both the groups. The service period was 164.2 and 152.4 days in group 1 and 2, respectively. The service period was shorter (P<0.01) by 11.8 days in group 2 than that of group 1, indicating that lesser time was required for the animals in group 2 for conception. Cows of Group 1 required 2.60 AIs per conception while group 2 required 2.67 AIs per conception. There were no effect of supplementation of RPM plus RPL on no of AI required for conception. The conception rate during the experimental period of 120 days was 55.55 % in group 1 and 66.66 % in group 2. 5.9 ECONOMICS OF FEEDING RUMEN PROECTED METHIONINE AND LYSINE IN COWS __ _____________________________ _ _ __ 170 Summary and Conclusions… ___________________ ______________________________________ Net return over feed cost of milk yield per animal per day in control and treatment group was Rs. 192.1 and 215.8, respectively, indicating that it was higher by Rs. 23.7 in treatment group over that of control group. The feed cost (Rs.) per kg FCM produced was Rs. 10.94 and Rs. 10.92, respectively in group 1 and group 2. While cost benefit ratio was similar (1.08) in both the groups. The net return on experimental ration was markedly higher by 12.30% than that of the control ration. As a result, net profit in 120 days from 9 cows on supplementation of rumen protected methionine and lysine was Rs. 25544.6 over that observed in group1. CONCLUSIONS The following conclusions were drawn on the basis of results obtained in the present study on supplementation of rumen protected methionine and lysine to crossbred cows during pre and post parturient period. Prepartum • Rumen protected methionine (5g/d) and lysine (20g/d) supplementation exhibited higher gross apparent difference of changes in body condition score. • Intakes of CP, RDP, RUP, MP, TDN, ME and NE L in prepartum cows were similar while duodenal supply of methionine and lysine was increased on supplementation of rumen protected methionine and lysine. • Plasma triglycerides and VLDL concentration were increased in prepartum cows on supplementating rumen protected methionine and lysine. Plasma glucose, phosphatidylcholine, NEFA, vitamin E, cholesterol and BUN were not affected. • Plasma amino acids profile of the prepartum cows was not affected on supplementing rumen protected methionine and lysine while plasma methionine, cysteine and lysine concentrations tended to increase on supplementation. Postpartum • Body weight loss during early lactation was reduced on supplementation of rumen protected Methionine (@ 7 gm/d/animal) and rumen protected lysine (@ 60 gm /d/animal). • Improvement in CP, RDP, RUP, MP, TDN, ME and NE L intakes were observed on supplementation of rumen protected methionine and lysine. __ _____________________________ _ _ __ 171 Summary and Conclusions… ___________________ ______________________________________ • Average daily milk, FCM and ECM yields were increased on supplementation of rumen protected methionine and lysine. • The efficiency of utilization of DM, CP and TDN for milk and FCM production was improved in rumen protected methionine and lysine fortified cows. • Milk fat and total solid content was increased, whereas milk protein, SNF, lactose, BUN and milk choline content was similar in both groups on supplementation of rumen protected methionine and lysine. • The proportions of saturated fatty acids, unsaturated fatty acids, MUFA, PUFA in milk fat were not affected in cows fed ration fortified with rumen protected methionine and lysine. • Plasma triglycerides and VLDL concentration were increased in cows on supplementating rumen protected methionine and lysine. • Plasma aspartate, glycine, alanine, valine, methionine, cysteine, and lysine concentrations were increased on supplementation of rumen protected methionine and lysine whereas plasma isoleucine concentration was lowered. • Periparturient plasma prolactin and growth hormone concentration were not affected on supplementing rumen protected methionine and lysine. • The birth weight of calves was not affected while cases of premature births were lowered on supplementation of rumen protected methionine and lysine. • Occurrence of parturition related abnormalities like RFM and metritis were reduced on supplementing rumen protected methionine and lysine. • The initiation of cyclicity was not affected but service period was reduced on supplementing rumen protected methionine and lysine. • Feeding rumen protected methionine and lysine to high yielding lactating cows during early lactation was cost effective. __ _____________________________ _ _ __ 172 Bibliography BIBLIOGRAPHY Adams, K.L. and A.H. 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TDN Calculation for feed and forages For feeds that are not animal proteins or fats and that do contain some NDF (forages, many byproducts, concentrates), the following equations are used: TDN = tdNFC + tdCP + (tdFA X 2.25) + tdNDF - 7 Where, Truly digestible NFC (tdNFC) = 0.98 (100 - [(NDF-NDICP) + CP + EE + Ash]) X PAF Truly digestible CP for forages (tdCPf) = CP X exp[ -1.2 X (ADICP/CP)] Truly digestible CP for concentrates (tdCPc) = 1- (0.4 X (ADICP/CP))] X CP Truly digestible FA (tdFA) = FA Note: If EE < 1, then FA = 0 Truly digestible NDF (tdNDF) = 0.75 X (NDFn – L) X [1 – (L/NDFn) 0.667] ME and NE L for feed and fodders If Fat ≥ 3 and if the animal is a dry cow or a lactating cow, then ME = (1.01 X DiscDE) - 0.45 + (0.0046 X (Fat - 3)) Net energy for lactation for feeds having more than 3% fat is computed. NE L = (0.703 X ME) - 0.19 + ((((0.097 X ME) + 0.19) /97) X (Fat - 3)) If the feeds have < 3% fat, the equation to compute ME for lactating and dry cows is ME = (1.01 X DiscDE) - 0.45 The equation to compute the NE L of low fat feeds is: Annexure II NE L = (0.703 X ME) - 0.19 Calculations for Protein Supply Microbial yield (MCP Total) is calculated as a percentage of discounted TDN (TDN_Act_Total): MCP_Total = 0.13 X TDN_Act_Total The following equation is used to calculate the amount of crude protein from each feed. CP X = (Feed X CP /100) X (DMFed X 1000) To calculate the site of digestion of protein, both passage (kp) and digestion (kd) rates are needed. Separate passage equations are used for concentrates, dry forages, and wet forages. Concentrate Kp = 2.904 + (1.375 X BW_DMI) - (0.02 X PercentConc) Dry Forage Kp = 3.362 + (0.479 X BW_DMI) -(0.017 X Feed X NDF) - (0.007 X PercentConc) Wet Forage Kp = 3.054 + (0.614 X BW_DMI) The amount of RDP in a specific feed is calculated using the following equation. It is assumed that all of Protein A is ruminally available and that none of Protein C is degraded in the rumen.Thus, only Protein B is affected by digestion and passage rates. If (Feed X .Kd + Kp) > 0 Then RDP X = ((Feed X .Kd /(Feed X .Kd + Kp)) X ((((Feed X .PrtB /100) X (Feed X CP /100)) X Feed X DMFed))) +(((Feed X PrtA /100) X (Feed X CP /100)) X Feed X DMFed) Otherwise, RDP X _ 0 The amount of ruminally-undegraded proteinis obtained by subtraction: RUP X = (CP X - (RDP X X 1000)) /1000 If RUP_Total > 0, then DietRUPDigest= Total Digested RUP /RUP_Total Otherwise, Diet RUPDigest = 0. Annexure II The supply for RDP and RUP is calculated in the following equation. RDPSup =TotalDMFed X 1000 X DietCP X CP_RDP RUPSup = CP Total - RDPSup The efficiency of microbial crude protein synthesis can not exceed 0.85. If MCP_Total > (0.85 X (RDP_Total X 1000)), then MCP_Total = (0.85 X (RDP_Total X 1000)) MPBalance = (((MPFeed X 1000) + MPBact + MPEndo) - (MPMaint + MPPreg + MPLact + MPGrowth)) Amino Acids supply The amino acid supply is calculated using the following equation with Methionine (Met) as an example. The structure of this equation is similar for all of the amino acids that are considered in the model. TMet = TMet + (((DMFed /TotalDMFed) X (CP /100) X ((RUP X X 1000) /CP X ) x (Met /100) X TotalDMFed) X 1000) Where TMet = Total Metionine, DMFed = quantity of feed X fed, TotalDMFed =Total dry matter fed, CP = % Crude Protein, RUP X = RUP in feed X , CP X = Crude protein in feed X. The next step is to calculate the total digestible supply of each amino acid. Below is the equation for Dig TMet. The equations for the other amino acids have the same format. Dig TMet = Dig_TMet + (((DMFed /TotalDMFed) X (CP /100) X ((RUP X X 1000) / CP X ) X (Feed X RUPDigest /100) X (Met /100) X TotalDMFed) X 1000) Where Dig_TMet = Total digestible Methionine, RUPDigest = RUP digestibility of feed X Annexure II The total essential amino acid supply before the contribution of the microbial protein has been added (EAATotal Before MP) is calculated. EAA Total Before MP = (TArg+This+Tile+Tleu + TLys + TMet + TPhe + TThr + TTrp + TVal) The variables x1and x2 areused in the following sets of calculation softhe total amount of each amino acid supplied. The equations tocal culate the total amounts of each amino acid follow. In all equations, it is assumed that: If EAATotal BeforeMP > 0 then x1= ((TMet (or other amino acid)/EAATotal Before MP X 100) Otherwise x1 = 0 If ((RUP_Total X 1000) + EndCP + MCP_Total) > 0 then x2 = ((RUP_Total X 1000) /((RUP_Total X 1000) + EndCP + MCP_Total)) x 100 Otherwise, x2 = 0 TotalArg = 7.31 + (0.251 X x1) TotalHis = 2.07 + (0.393 X x1) + (0.0122 X x2) TotalIle = 7.59 + (0.391 X x1) - (0.0123 X x2) TotalLeu = 8.53 + (0.41 X x1) + (0.0746 X x2) TotalLys = 13.66 + (0.3276 X x1) - (0.07497 X x2) TotalMet = 2.9 + (0.391 X x1) - (0.00742 X x2) TotalPhe = 7.32 + (0.244 X x1) + (0.029 X x2) TotalThr = 7.55 + (0.45 X x1) - (0.0212 X x2) TotalVal = 8.68 + (0.314 X x1) The total essential amino acid supply is calculated below: Total EAA = 30.9 + (0.863 X EAATotal Before MP) + (0.433 X MCP_Total) Total flows of RUP of specific amino acids are calculated below: TotalRUPArgFlow = 0.863 X TArg Annexure II TotalRUPHisFlow = 0.863 X THis TotalRUPIleFlow = 0.863 X TIle TotalRUPLeuFlow=0.863 X TLeu TotalRUPLysFlow = 0.863 X TLys TotalRUPMetFlow = 0.863 X TMet TotalRUPPheFlow = 0.863 X TPhe TotalRUPThrFlow = 0.863 X TThr TotalRUPTrpFlow = 0.863 X TTrp TotalRUPValFlow = 0.863 X TVal Duodenal flow(g/day) is calculated using an equation of the form below for each amino acid. Methionine is given as an example. Met Flow = (TotalMet /100) X TotalEAA The contribution of microbial crude protein and endogenous protein to the amino acid supply is calculated as follows. The form of this equation is similar for all amino acids. TotalMCPEndMetFlow = Met_Flow - TotalRUPMetFlow The next step is to calculate the supply of each amino acid in RUP that is digestible. The form of the equation for each amino acid is similar to that given for Metinine below: If TMet > 0, then dTotalRUPMet=TotalRUPMetFlow X (Dig_TMet /TMet) Otherwise, dTotalRUPMet = 0 The amount of a specific amino acid that is digestible and is of microbial or endogenous origin then is calculated. Metinine is used as the example but similar calculations are made for all amino acids. dTotalMCPEndMet = 0.8 X TotalMCPEndMetFlow The flow of digestible Metinine, or other amino acids, then is calculated. Dig_Met _Flow = dTotalRUPMet + dTotalMCPEndMet Annexure II The protein in the duodenum must be converted from crude protein to a metabolizable protein basis. Microbial crude proteinis converted to metabolizable protein with an efficiency of 0.64: MPBact = 0.64 X MCP_Total MPFeed = TotalDigestedRUP MPEndo = 0.4 X EndCP The next computation is to determine the percent of a specific amino acid of metabolizable protein.The Methionine equation is similar to those of the other amino acids. If (MPBact+(MPFeedX1000)+MPEndo) > 0, then MetPctMP = 100 X (Dig_Met_Flow /(MPBact+ (MPFeed X 1000) + MPEndo)) Otherwise, MetPctMP = 0