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Strategies for management of transition cows using bovine somatotropin (bST), short dry period and diets to improve their performance during lactation

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Strategies for management of transition cows using bovine somatotropin (bST), short dry period and diets to improve their performance during lactation
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Gulay, Mehmet Sukru, 1970-
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English
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xvii, 288 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Calving ( jstor )
Dry period ( jstor )
Early lactation ( jstor )
Lactation ( jstor )
Mammary glands ( jstor )
Milk ( jstor )
Milk production ( jstor )
Parturition ( jstor )
Plasmas ( jstor )
Postpartum period ( jstor )
Animal Sciences thesis, Ph. D ( lcsh )
Dissertations, Academic -- Animal Sciences -- UF ( lcsh )
City of Gainesville ( local )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 2002.
Bibliography:
Includes bibliographical references (leaves 262-287).
General Note:
Printout.
General Note:
Vita.
Statement of Responsibility:
by Mehmet Åžükrü Gülay.

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STRATEGffiS FOR MANAGEMENT OF TRANSITION COWS USING BOVINE SOMATOTROPIN (bST), SHORT DRY PERIOD AND DIETS TO IMPROVE THEIR PERFORMANCE DURING LACTATION f ^i MEHMET §UKRU GULAY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002

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To my mother Sevil Gulay and my father Ahmet Giilay, for their endless patience and support working toward | this goal and throughout my life. Without their sacrifice, this may never have come about.

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ACKNOWLEDGMENTS I wish to express sincere gratitude to my advisor, Dr. H. Herbert Head. I especially wish to thank him for his advice, kind help, support in solving problems and for giving me the opportunity to begin a new career in my major. I also would like to thank my advisory committee members, Drs. K. C. Bachman, M. B. Hall, R. B. Shireman and F. A. Simmen for their suggestions and support regarding my program and dissertation and for taking time to read it. My deepest gratitude is expressed to our lab chemist, Ms. M. Joyce Hayen, for her help, friendship and knowledge. This study could not have been completed without her. I also appreciate collaboration of the farm crew and the milkers at the Dairy Research Unit (DRU). I thank the department of Animal Sciences for its financial support. I also would like to thank Pam Miles for helping me complete calcium analyses. I would like to express my thanks to my sisters, Ahsev and Giilsev, and brothers in law, ismail Hakki and §ukru Yavuz, who always encouraged me to seek this degree. I would like to express my thanks to Necla who gave me the motivation to go on from one day to another. Thanks go to all my friends, especially Aydm GUzeloglu, §ukru Metin Pancarci, §aban Tekin, Tomas Belloso, Marcio Liboni and all those people who have emotionally supported me during my studies. Finally, I would like to extend my thanks to ALLAH for a faith that has anchored me through the ups and downs of my life. iii

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TABLE OF CONTENTS Y^riV^fi ; \ , . ACKNOWLEDGMENTS iii LIST OF TABLES viii LIST OF FIGURES xii LIST OF ABBREVIATIONS xiv ABSTRACT xvi CHAPTER I 1 GENERAL INTRODUCTION 1 2 USE OF bST IN MANAGEMENT OF THE TRANSITION DAIRY COW TO INCREASE FEED INTAKE, IMPROVE MILK YIELDS AND DECREASE HEALTH PROBLEMS II Introduction 11 Literature Review 12 Regulation of Somatotropin Secretion 12 Secretion Pattern of Somatotropin 15 Somatotropin Gene 16 Structure of Bovine Somatotropin 16 Somatotropin Receptor 19 Responses to Somatotropin Supplementation 22 Effects of bST on the Mammary Gland 23 Effects of Somatotropin in Other Tissues 25 Carbohydrate metabolism 26 Lipid metabolism 28 Amino acid metabolism 30 Mammary blood flow 30 Transition Period 31 Metabolic Adaptations in the Transition Cow 34 Hormonal changes 34 Adipose tissue 36 Liver 37 Muscle 38 Bone and minerals 39 iv

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General Overview 40 Materials and Methods 40 Experimental Design 41 bST Treataments 41 Management 42 Feeding program 42 Body condition scores and body weights 42 Blood collection, handling and storage 44 Milking and milk collection 45 Statistical Analyses 45 Results 46 Changes in Body Weight and Body Condition Scores 47 Milk and 3.5% FCM Yields 54 Hormones, Growth Factor and Metabolites 61 Prepartum 61 Postpartum 71 Discussion 73 Conclusions 84 3 FEEDING MANAGEMENT OF HOLSTEIN COWS GIVEN SHORT (30 d) OR NORMAL (60 d) DRY PERIODS Introduction 86 Dry Matter Intake and the Transition Period 86 Diet and Regulation of Feed Intake 90 bST and Transition Period 93 Anionic Diet 99 Advantages of Short Dry Period on Feed Intake 102 Materials and Methods 105 Experimental Animals 105 Experimental Design 1 06 bST Injections 108 ECP Injections 109 Drying off Procedure 109 Feeding Program 1 09 Body Condition Scores and Body Weights Ill Blood Collection, Handling and Storage Ill Determination of Calcium in Serum Samples 113 Statistical Analyses 114 Results 115 Changes in Body Weights and Body Condition Scores 115 Prepartum period 115 Postpartum period 121 Serum Calcium Concentrations 126 Changes in Dry Matter Intake 128 Postpartum period 132 V

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Discussion 139 Prepartum Period 139 Postpartum Period 145 Conclusions 153 4 IMPROVING MILK PRODUCTION AND HEALTH OF COWS BY SHORTINING THE DRY PERIOD WITH ESTROGEN, USE OF bST DURING THE TRANSITION PERIOD 155 Introduction 155 Dry Period 155 Involution 159 Plasmin and Involution 167 Materials and Methods 169 Milk Samples 169 Plasma Collection, Handling and Storage 169 Second Antibody Preparation 170 Radioimmunoassays 171 lodination and Protein Separation 171 Insulin 171 IGF-I 172 Somatotropin 174 Assays 175 Insulin assay 175 Somatotropin assay 177 IGF-I 178 Determination of Glucose in Plasma Samples 181 Determination of NEFA in Plasma Samples 182 Statistical Analyses 1 84 Results 185 Hormones, Growth Factor and Metabolites 185 Prepartum period 185 Postpartum period 199 Milk, 3.5% FCM and SCM Yields 205 Discussion 216 Hormones, Growth Factor and Metabolites 216 Milk, 3.5% FCM and SCM Yields 227 bST treatment and milk yield 228 Dry period treatments and milk yield 237 Prepartum diets fed and milk yield 241 Conclusions 242 5 GENERAL DISCUSSION 244 vi

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APPENDIX LIST OF SIGNIFICANT TWO-WAY INTERACTIONS 256 I REFERENCES 261 BIOGRAPHICAL SKETCH 288 Hi vn 1.

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LIST OF TABLES Table \ page I 2-1 Dry matter concentrations and chemical composition of CUD ration andTMR 43 2-2 Least squares means and SE of BW and BCS of cows in control and bST injected groups during the prepartum and postpartum periods 49 i 2-3 Least squares analyses of variance for BW and BCS of Holstein cows during prepartum period (-3 to 0 wk) 51 2-4 Least squares analyses of variance for BW and BCS of Holstein cows during early lactation (1 to 10 wk) 52 2-5 Least squares means and SE of milk yield, 35% FCM yield, SCC and percentage of protein, fat and MUN in milk of Holstein cows during early lactation 55 I 2-6 Least squares analyses of variance for milk and 3.5% FCM yields of Holstein cows during early lactation (1-8 wk) 56 2-7 Least squares analyses of variance for daily milk yields of Holstein cows during two early lactation periods 57 2-8 Least squares analyses of variance for concentrations of ST, INS, and IGF-I in plasma of Holstein cows during the prepartum period (-3 to -1 wk) 62 I 2-9 Least squares analyses of variance for concentrations of NEFA and glucose in plasma of Holstein cows during the prepatum period (-3 to -1 wk) 63 2-10 Least squares analyses of variance for concentrations of ST, INS, and IGF-I of Holstein cows during early lactation (1 to 8 wk) 65 I 2-1 1 Least squares analyses of variance for concentrations of NEFA and glucose in plasma of Holstein cows during early lactation (1 to 8 wk) 66 vni

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Table page 212 Least squares means and SE of hormone, growth factor and metabolite concentrations in plasma of Holstein cows during the prepartum and postpartum periods 67 31 Dry matter concentrations and chemical composition of anionic and cationic CUD fed to Holstein cows 107 I 3-2 Dry matter concentrations and chemical composition of TMR with whole cotton seeds (lactation diet) fed to Holstein cows during experiment 112 3-3 Standards for calcium determination flame atomic absorption spectrophotometer 113 3-4 Least squares analyses of variance for BW and BCS of Holstein cows during the prepartum period (wk -8 to wk -1) 116 3-5 Least squares means and SE for BW and BCS of cows during prepartum period (wk -8 to wk -1 ) 117 3-6 Least squares analyses of variance for BW and BCS of Holstein cows during postpartum period (wk 1 to wk 14) 122 3-7 Least squares means and SE for BCS and BW of Holstein cows during the postpartum period (wk 0 to wk 14) 124 3-8 Least squares analyses of variance for DMI of Holstein cows during prepartum period 129 3-9 Least squares means and SE of DMI BW, and BCS during prepartum and postpartum periods for Holstein cows injected or not injected with bST 130 3-10 Least squares means and SE of DMI BW, and BCS during prepartum and postpartum periods for Holstein cows in three dry period groups 133 3-1 1 Least squares means and SE of DMI BW, and BCS during prepartum and postpartum periods for Holstein cows fed anionic or cationic diets prepartum 134 3-12 Least squares analyses of variance of DMI of Holstein cows during postpartum period (d 1 to d 28) 135 ix

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Table page 41 Arrangement of assay tubes for E^JS 1 76 4-2 Arrangement of assay tubes for ST 178 4-3 Arrangement of assay tubes for IGF-I 180 4-4 Standards for glucose determination 1 82 4-5 Standards for NEFA determination 183 4-6 Least squares analyses of variance for concentrations of ST, INS, and IGF-I in plasma of Holstein cows during prepartum period (d -21 to d -1) 188 4-7 Least squares analyses of variance for concentrations of NEFA and glucose in plasma of Holstein cows during the prepartum period (d -21 to d -1) 189 4-8 Least squares means and SE of concentrations of hormone, growth factor, and metabolites in plasma of Holstein cows injected or not withbST(d-21 to d 28) 190 4-9 Least squares means and SE of hormones, growth factor, and metabolites concentrations in plasma of Holstein cows with 30 or 60 d dry periods (d -2 1 to d 28) 1 92 4-10 Least squares means and SE of hormones, growth factor, and metabolites in plasma of Holstein cows fed prepartum anionic or cationic diets during experiment (d -21 to d 28) 193 4-1 1 Least squares analyses of variance for concentrations of ST, INS and IGF-I during postpartum period (d 1 to d 28) 201 4-12 Least squares analyses of variance for concentrations of NEFA and glucose of Holstein cows during postpartum period (d 1 to d 28) 202 4-13 Least squares analyses of variance for milk yields of Holstein cows during postpartum period (1-21 wk) 207 4-14 Least squares analyses of variance for milk, 3.5% FCM and SCM yields of Holstein cows during postpartum period (wk 1 to 10) 208 4-15 Least squares means and SE of milk yield, 3.5% FCM and SCC of Holstein cows during early lactation 209 X

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Table page 4-16 Least squares means and SE of milk yield, 3.5% FCM and SCC of Holstein cows provided different dry periods during early lactation 210 4-17 Least squares means and SE of milk yield, 3.5% FCM and SCC of Holstein cows during early lactation fed anionic or cationic diets during prepartum period 213 xi

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LIST OF FIGURES Figure page 2-1 Three dimensional shape of bST 18 2-2 Signaling and regulation of the transcription factors by ST 21 2-3 Schematic of endocrine changes around parturition. '+' increased concentration or activity, and '-' decreased concentrations or activity 35 2-4 Least squares means of body weight changes of Holstein cows during the prepartum and early postpartum periods (-3 wk through 10 wk) 53 I 2-5 Least squares means of body condition score changes of Holstein cows during the prepartum and early postpartum periods (-3 wk through 10 wk) 53 2-6 Least squares means of weekly milk yields of Holstein cows during the first 8 wk of lactation 59 2-7 Least squares means of weekly 3.5% FCM yields of Holstein cows during the first 8 wk of lactation 59 2-8 Quartic regressions depicting MY of Holstein cows during experiment 60 29 Least squares means of concentrations of ST in plasma during the transition period and throughout 8 weeks of lactation 68 2-10 Least squares means of concentrations of IGF-I in plasma during the transition period and through 8 weeks of lactation 68 2-1 1 Least squares means of concentra,ions of insulin in plasma during the transition period and through 8 weeks of lactation 69 J • * 2-12 Least squares means of concentrations of glucose in plasma during the transition period and through 8 weeks of lactation .... 69 2-13 Least squares means of concentrations of NEF A in plasma during the 'l transition period and through 8 weeks of lactation 70 xii

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Figure j page 3-1 Body weights of Holstein cows during the prepartum and postpartum periods 118 3-2 Body condition scores of Holstein cows during the prepartum and postpartum periods 119 I 3-3 Serum concentrations of Ca in Holstein cows during the prepartum and postparum 127 3-4 Dry matter intake of Holstein cows during the prepartum and postpartum periods 131 35 Regressions depicting changes in dry matter intake of Holstein cows during the prepartum and postpartum periods 138 41 Least squares mean concentrations of ST in plasma during the transition period (-21 d through 28 d) 194 4-2 Least squares mean concentrations of IGF-I in plasma during the transition period (-21 d through 28 d) 195 I 4-3 Least squares mean concentrations of INS in plasma during the transition period (-21 d through 28 d) 119 4-4 Least squares mean concentrations of glucose in plasma during the transition period (-21 d through 28 d) 197 4-5 Least squares mean concentrations of NEFA in plasma during the transition period (-21 d through 28 d) 198 4-6 Milk production of Holstein cows during early lactation 211 4-7 Cubic regressions depicting changes in weekly milk yield of Holstein cows during the experiment 214 xiii

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LIST OF ABBREVIATIONS ACTH ADF AI BCS bST BW CCK CMO CP CRF cs CUD d DA DCAD DIET DIM DMI DRY EB ECP FCM FOD GHRH hr IGF-I INS ; JAK VLDL MUN MY NDF NEB NEFA NEI P4 PC PEPCK PGF,„ PL Adenocorticotropic Hormone Acid Detergent Fiber Artificial Insemination Body Condition Score Bovine Somatotropin Body Weight Cholecystokinin Calving Month Crude Protein Corticotropin Releasing Factor Chorionic Somatomammotropin Close-up Dry Day Displacement of bomasum Dietary Cationic-Anionic Difference Prepartum Diet Treatments Days in Milk Dry Matter Intake Prepartum Dry Period Treatment Energy Balance Estradiol Cypionate Fat Corrected Milk Far-off Dry Growth Hormone Releasing Hormone Hour Insulin-like Growth Factor I Insulin Janus Kinase Very Low Density Lipoproteins Milk Urea Nitrogen Milk Yield Neutral Detergent Fiber Negative Energy Balance Non-Esterfied Fatty Acid Net Energy Intake Progesterone Pyruvate Carboxylase Phosphoenol Pyruvate Carboxy Kinase Prostaglandin Fj^ Placental Lactogen XIV

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PRL Prolactin rlrl Parathyroid Hormone rbSl Recombinant Bovine Somatotropin RDP Rumen Degradable Protem "r>T> RR Reticulo-rumen RUr n TTJ J11T\j.* Rumen Undegradable Protem sec Somatic Cell Count Solids Corrected Milk oiiA beason orlZ " Src Homology Domain 2 CC Somatostatin Si Somatotropin STAT Signal Transducers and Activators of Transcnption T3 Triiodothyronine rp T4 Thyroxine rp/— 1 TG Triglycerides TMR Total Mixed Ration TRT Treatment wk Week Year XV

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy STRATEGIES FOR MANAGEMENT OF TRANSITION COWS USING BOVINE SOMATOTROPIN (bST), SHORT DRY PERIOD AND DIETS TO IMPROVE THEIR PERFORMANCE DURING LACTATION By Mehmet §iikru Giilay August 2002 • . '-'\ ^ Chairperson: H. Herbert Head Major DepartmentrAnimal Sciences , f : Our objectives for first experiment were to evaluate the effects of a low dose of I bST prepartum and postpartum on BCS, BW, MY, ST, IGF-I, fNS and some metabolites in plasma. It was carried out during two consecutive years. Holstein cows were assigned randomly to one of two groups [Control (C)=98; Injected (I)=95 cows]. Biweekly injections of bST were fi-om 28 d before expected calving through 42 d postpartum (C vs. I; 0 vs. 10.2 mg bST/d, POSILAC®). During year 1, ST, IGF-I, INS, NEFA and glucose were measured in plasma of 82 cows. During year 2, BCS and BW of 1 1 1 Holstein cows were evaluated, but cows were not bled. Milk yields through 100 d were analyzed during both years. Prepartum bST increased mean concentrations of ST and INS but not glucose, NEFA or IGF-I. Postpartum treatment increased concentrations of ST and NEFA, but not INS, IGF-I, or glucose. Mean BCS did not differ prepartum or I postpartum, but bST cows maintained better BW postpartum. The MY was greater for

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bST-injected cows during the first 60 d of lactation, but not during the first 100 d. The second experiment included 84 Holstein cows to evaluate DMI, MY, metabolic hormones, metabolites and Ca. The 3x2x2 factorial arrangement of treatments included dry period (30 d dry, 30 d dry+ECP, and 60 d dry), prepartum and postpartum bST (10.2 mg/d), and prepartum anionic or cationic diets. Prepartum bST increased concentrations of ST, IGF-I, INS and glucose. Postpartum bST increased concentrations of ST and IGFI in plasma but not glucose or NEFA. Greater MY was observed for bST cows through the first 21 wk but MY did not differ due to dry period, hijections of ECP at dry-off did not improve MY. Cows with shorter dry period replenished their BW and BCS as well as 60 d dry cows. No effect of prepartum diet on DMI, MY, or blood measures including Ca was detected. Data indicated that bST treatments and short dry period can be used as • management tools to improve the performance of transition cows and their subsequent milk production. XVll

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CHAPTER 1 GENERAL INTRODUCTION Dairy cows undergo gestation and parturition on a regular cycle during the time they are in the producing herd. The change from the nonlactating stage to the lactating stage is very demanding on the cow. Early lactation and peak lactation are the two periods during the lactation cycle when nutrient demands are rapidly increasing and become greatest for cows. Another important period during the lactation cycle is that from 21 d prepartum to 21 d postpartum which has been termed the "transition period" (Drackley, 1999). Cows undergo many physiological changes during the transition period. Pregnant cows usually show reduced DMI during late pregnancy and the early postpartum period. It has been shown that feed intake decreased approximately 30% during the final days before calving (Bertics et al., 1992). Importantly, many metabolic diseases such as fatty liver, ketosis, milk fever, downer cow syndrome, and displaced abomasum (DA) are most likely to occur during the late pregnancy and/or early lactation periods (Goff and Horst, 1997). Lactating dairy cows show peak milk yield between 4 and 12 wk postpartum. During early lactation, cows are deficient in energy and other nutrients (Wheeler et al., 1995), largely because peak DMI is not reached until 10 to 14 wk postpartum (Bertics et al., 1992). High milk secretion associated with high demand for glucose and protein generally gives rise to increased mobilization of body fat and protein reserves. However, cows mobilize relatively more energy than they do amino acids to support milk 1

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2 production because of energy and nutrient deficiencies. This often leads to the health problems seen during early lactation. If the additional energy and protein needed to meet the cow's requirements are not supplied by the diet, then they must be met by tissue mobilization and this maybe a major cause of the development of metabolic diseases during the transition period (Drackley, 1999). Goals for the transition cow are to decrease excessive mobilization of fatty acids from adipose tissue storage and transport to liver, and to minimize the depletion of glycogen stores from the liver. Most important, if feed intake can be maintained or even increased during the transition period and throughout early lactation, when milk yield is also increasing, this will better maintain the energy balance needed to keep cows from tumbling into metabolic disturbances that have bad consequences. Rapid and high rate of increase in DMI during early lactation is essential to provide energy and nutrients to support a rapid increase in milk yield. Knowing that feed intake is a key gives us the opportunity to devise means to keep it high, and to use or develop technologies or feeding strategies to bring about desired effects especially during the last 3 wk of dry period. One of the unique products of biotechnology is recombinant bovine somatotropin (rbST or simply bST). Use of bST during lactation has resulted in exceptional increases in milk production by dairy cows (Bauman, 1999). The development of recombinant biotechnology enabled the development of rbST which provided an unlimited and affordable source of ST for research and, subsequently, for commercial application, hiitially, availability of bST allowed researchers to conduct the first studies to better evaluate its effects. These studies showed that bST increased milk production by 10 to 15 % in dairy cows (Bauman, 1982). Bovine somatotropin has been used extensively for

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more than 10 yr in South America and about 8 yr in the US to improve milk yield of cows in commercial dairy herds. Use of bST during the prepartum period may offer a means to cause positive and beneficial effects on metabolism. This is because of the known positive effects of bST on feed intake during lactation and on health of the injected cow (Eppard et al., 1996). Bovine somatotropin also has the ability to partition nutrients, both ingested and those mobilized fi-om liver and muscle to the mammary gland during lactation. These effects, if exerted during the prepartum period, may allow the close-up cow to better make the transition to high demand for energy and nutrient intake and the active function of the liver and other organs that provide the substrates for many important metabolic pathways. It already has been shown that low doses of bST have positive effects on metabolism, hormones and milk production when injected 14 d postpartum through 60 d (Stanisiewski etal., 1992). Somatotropin is a primary homeorhetic regulator during pregnancy and lactation (Bauman and Vernon, 1993); it regulates partitioning of nutrients (carbohydrates, lipids, proteins, and minerals), and plays an important role in the coordination of various organs and tissues (Bauman, 1 992). To support milk synthesis, metabolism of other tissues is stimulated to provide the necessary precursors. Somatotropin also has a major role in the I regulation of IGF concentrations in the circulation. Insulin like growth factor-I is a local mediator of the mammary epithelial growth and development where it stimulates the cellular activity of mammary gland (Phillips et al., 1990). Increase in IGF-I concentration in peripheral plasma is maintained with continued injections of ST and the increase parallels that of MY during the lactation period.

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Some transition period trials also were performed to evaluate the effects of injecting a full dose of bST (500 mg/14 d); this practice often had negative effects on injected cows. Despite large increases in milk production during the first few weeks after parturition, feed intake did not increase immediately after bST treatments and resulted in severe negative energy balance resulting in initial loss of BCS and subsequent reproductive problems (Moallem et al., 2000). However, it has been hypothesized that use of a low dose of bST injected during the transition period would improve a cow's overall performance, especially during lactation, without the negative effects seen in earlier trials when greater doses were used. Simmons et al. (1994) concluded that when bST (5 and 14 mg/d) was injected into cows during the last 46 d before parturition, DMI tended to be about 3 kg/d greater after parturition. Salfer et al. (1994) reported that postpartum treatment with bST resulted in higher mean DMI after several weeks of injection and that the increase was dose dependent. Garcia (1998) reported that cows injected with 5 mg bST/d during both the prepartum and postpartum periods had greater DMI than uninjected controls, or than cows injected with 5 mg bST/d only during the prepartum or only during the postpartum periods. In addition, reports by Kertz et al. (1991) and Garcia (1998) indicated that cows in the uninjected control group had the greatest loss in BCS and these cows did not begin recovery of body condition until 8 wk of lactation. Maintenance of lactation can be described as maintaining the number of mammary secretory cells and their synthetic activity during a defined time period. Change in the amount of milk produced during a defined time period can be used to measure maintenance of lactation. Along with mammary gland related factors such as removal of the milk, other factors such as environment, management, nutrition, genetics.

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5 and milking frequency affect lactation persistency, a measure of maintenance of lactation. In dairy cows, after peak milk production is reached, gradual involution of mammary tissue occurs during ongoing lactation in spite of the frequent removal of milk. Hence, a decline in daily milk volume follows as the number of functional differentiated epithelial cells in the mammary gland decrease. Part of the reduction in milk yield also may be due to a reduction in the milk secretion rate of each of the functional cells that do remain (Mepham, 1987). On the other hand, dairy cows require a nonlactating dry period between successive lactations. Mammary involution in this period is characterized by a decrease in the total number of alveoli per lobule, a decrease in the total number of alveoli and lobular volume, a decrease in the number of cells per alveolus, and an increase in the height of alveolar cells (Schmidt, 1971). Involution in the dry period is an essential process for the mammary gland so that recovery of body reserves can occur to support subsequent lactation and so that lactation also can be reinitiated at a high level. A period of 45 to 60 d generally has been recommended for the dry period (Smith and Todhunter, 1982). According to Coppock et al. (1974), less than a 40 d nonlactating period decreases milk yield in the subsequent lactation, whereas greater than a 60 d nonlactating period increases feed costs with no associated return and can cause a decrease in the lifetime production of the cow. However, cows do not produce milk during the dry period. One method to increase lactation milk production by individual cows would be by manipulating the length of the dry period. If the dry period could be shortened and if the rate that the mammary tissue involution could be accelerated, then perhaps one could decrease these "unproductive" days, and yet still achieve maximal milk yield during the

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next lactation. In this way total milk yield could be increased but the same number of cows would be milked. Mammary involution is a fairly rapid process that occurs after cessation of milking. Regression of mammary secretory tissue accompanies dramatic changes in composition of secretions during the transition from lactation to involution. As indicated previously, dairy cows require a nonlactating dry period before calving to achieve maximal milk production during the next lactation (Coppock et al., 1974; Hurley, 1989; Klein and Woodward, 1943; Schaeffer and Henderson, 1972). Adequate proliferation I and differentiation of mammary secretory epithelial cells during the nonlactating period are essential for optimal secretory function in the subsequent lactation and the duration of the nonlactating interval is related significantly to milk production (Akers and Nickerson, 1983). Plasmin and its inactive zymogen, plasminogen, two of several significant 1 proteases in bovine milk (Eigel, 1977), have been implicated in the destructive phases during the gradual involution that occurs as lactation advances. Plasmin is an extracellular serine protease which is formed by cleavage of a peptide bond in the single polypeptide chain of the inactive proenzyme plasminogen (Andersen et al., 1990). Plasmin in bovine milk exists mainly in its inactive form. Stage of lactation affects 1 plasmin with late lactation associated with higher concentrations (Politis et al., 1988). They proposed that the increased plasmin activity in milk during late lactation may be involved in subsequent mammary gland involution (Politis et al., 1990). It has been suggested that estrogen administered at cessation of milk removal could accelerate the involution of mammary tissue (Athie et al., 1996) by accelerating the

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activation of plasminogen (Athie et al., 1977). They reported that concentrations of plasminogen, plasmin, and somatic cells in secretions were increased earlier in Holstein cows injected with 15 mg of estradiol17P on each of the 4 d that preceded fmal milk removal than in control cows injected with 4 mL of ethanol. hi addition, the ratio of plasminogen to plasmin in secretions decreased earlier for treated cows than for control cows (Athie et al., 1997). As a result, administration of exogenous estrogen at final milk removal augmented the normal mammary involution process, accelerated active involution and still left enough time for the mammary regrowth phase. These results I suggested that the shorter dry period could be incorporated into a dry period management scheme without any adverse effects on the milk production during the subsequent lactation. To be an effective strategy to improve milk production, shortening the dry period must be coupled with a good nutritional management program to allow cows to maintain body condition and good health after parturition. An important advantage of a superior feeding program during shortened dry periods would be the ability to maintain good rumen function when diets were fed during the shortened dry period that were formulated from constituents similar to those used to formulate the lactation diet. This would encourage maintenance of a population of rumen microbes well suited to the lactation diets fed postpartum. Also, cows would have the ability to maintain good peripartum calcium metabolism whether anionic or cationic diets were fed, thus they would begin transition to lactation into better energy and metabolic status. Total cow numbers in Florida are about 155,000 and average dry period length is 70 d. If it can be shown that cows with a 30-35 d dry period would produce just as much

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milk during the next lactation as those with 60-70 d dry period and with no negative effects of short dry period on health or subsequent milk production, then an opportunity exists for dairy producers to earn extra money. This extra income would arise from the extended number of days in milk during the current lactation. For example, an extra 1 820 kg of milk/d could be sold as a result of extra days in milk that would otherwise be lost if the cows were dry for the full 60 d. Of course, in order to adopt this practice, cows should be producing enough milk at the 60 d dry period mark and have a good BCS (minimum 3.25), and they should be given sufficient nutrients to support their needs during the next 30 d of lactation. The surface of the rumen mucosa is characterized by ruminal papillae, which can be defined as organs of absorption. Their distribution, size and number are closely related to feeding habits, forage availability and digestibility. The typical features of rumen papillae are genetically fixed but may vary considerably under different feeding conditions, resulting in acute and usually temporary or seasonal adaptations (Dirksen et al., 1985). For example, increasing proportions of butyric and propionic acids that increase ruminal blood flow also stimulates mucosal mitosis. This results in vascular budding and epithelial cell proliferation. Thus, there are increases in number and size of papillae within the rumen. Changes in the numbers and development of rumen papillae in response to nutritional changes requires an adaptation period of 2 to 3 wk (Dirksen et al., 1985). Microorganisms in the rumen depend on the ruminant animal to provide the physiological conditions necessary for their existence. In turn, these microorganisms are essential for digestion and fermentation of large amounts of fibrous feeds that the

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ruminant consumes, but otherwise could not use efficiently. Thus, by providing a suitable and consistent environment for these microorganisms, the ruminant is able to use the endproducts of fermentation to meet its own nutritional needs. Comparison of rumen microorganisms shows that there is a high level of variation among cows. The large diversity in the types of microbes found in the rumen is a reflection, to some extent, on the diet. Growth of microorganisms and efficient fermentation of feed by microorganisms depends on a constant and suitable environment (Van Soest, 1982). Changes in feed and feed composition (as well as rumen pH) cause a shift in I microorganisms in the rumen and also a decrease in the efficiency of the fermentation and absorption of products of this fermentation. Changing the diet of the animal provokes a period of transition in the rumen microbial population which causes the proportions of the different species in the rumen to shift until a new balance is established, one which best accommodates the dietary changes. This is referred to as adaptation of the microbial population. Adaptation typically takes several days to weeks to occur (Dirksen et al., 1985; Yokoyama and Johnson, 1988). The current standard 60 d dry period allows nutritional management of dry cows to be organized in two different phases; far-off (FOD) and close-up dry (CUD) periods. During these periods, diets given to animals vary due to the metabolic differences of cows during these short time periods. Diet changes fi-om lactation diet to far-off diet, fi-om faroff diet to close-up dry diet, and from close-up dry diet to lactation diet forces microbes in the rumen to adapt three times during a short time period. It also is likely that feed intake is changing and limited as cows approach calving. These changes in diet offered probably further decrease the feed intake, and likely limit the increase in feed intake

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10 immediately after parturition. This is a concern because early lactation is when greater feed intake and more efficient fermentation/utilization of ingested feed is desired. If length of the dry period can be decreased to -30 d, then a better feeding program can be developed with diets formulated from constituents similar to those that are used to formulate the lactation diet. This will encourage maintenance of a better rumen microbial population, better rumen papillae development, and greater peripartal calcium metabolism. Therefore it should allow greater and more rapid increase in feed intake, should stimulate efficient fermentation and absorption, and should offer better resistance to metabolic disorders during early lactation. The major objectives of this research were to evaluate the use of bST during the transition period and to evaluate dry period length. Thus, two studies were conducted at the Dairy Research Unit (DRU) of the University of Florida. The main objective of the first study was to evaluate the effects of bST on feed intake, BCS and BW preand I postpartum; to evaluate the overall yield of milk during lactation; and determine any adverse or positive effects on the health of the animal. The main objectives of the second study were to evaluate dry period length (60 vs 30 d), the use of ECP injections to enhance speed of dry-off, the types of prepartum transition diets (anionic or cationic), and supplemental injections of bST during the transition period by measuring DMI, BW, BCS, milk production, and cow health during subsequent lactation.

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CHAPTER 2 USE OF bST IN MANAGEMENT OF THE TRANSITION DAIRY COW TO INCREASE FEED INTAKE, IMPROVE MILK YIELDS AND DECREASE I HEALTH PROBLEMS Introduction During the early 20"^ century exceptional discoveries allowed better understanding of the basic biology of growth and development. Biotechnology followed the advances in applied and basic science and had an important impact on agriculture as well. One of the unique products of biotechnology is recombinant bovine . • « ^ *, somatotropin (rbST or ST) which resulted in an exceptional increase in milk production by dairy cows when injected during ongoing lactation. For example, the theoretical annual gain in milk yield with modem reproductive technologies such as artificial insemination (AI), AI and semen sexing, and AI and embryo transfer can be as much as 100, 115 and 135 kg, respectively (Bauman, 1986). However, improvement in milk I yield by administration of bST can result in 2000 kg of additional milk yield per lactation (Bauman et al., 1985). This increase would be equal to that normally achieved by AI and genetic selection over a 10-20 yr period (Bauman, 1999). As early as the 1930s, growth-promoting effects of ST were characterized. Evans and Simpson (1931) showed that crude extracts of bovine pituitary increased the growth rate of rats. In 1937, two Russian scientists, Asimov and Krouze, performed the first bST research using 600 lactating dairy cows. These researchers found that 11

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12 injecting cows with extracts of the pituitaries of slaughtered cows resulted in increased milk production. Research continued during and after World War II as scientists sought an effective means of increasing food production. However, the amount of ST from I each pituitary was small that this source would not be practical for improving total milk production (Young, 1947). As indicated, the arrival of modem technology enabled the development of recombinant bovine somatotrophin (rbST) which provided an unlimited source of ST for research and, potentially, for commercial application. This allowed researchers to conduct many studies using recombinant bST. From these studies it was concluded that exogenous ST could increase milk production of dairy cows at least 10 to 15% (Bauman, 1982). Objective of current research was to evaluate the effects of prepartum and postpartum treatment of cows with bST on milk yield, BCS and BW, and to evaluate some important metabolic hormones during the transition period. This study was conducted at the Dairy Research Unit (DRU) of the University of Florida and used Holstein cows. Literature Review Regulation of Somatotropin Secretion Secretion of ST from the anterior pituitary gland is controlled by a complex neuroendocrine system that triggers neural afferent stimuli. This results in stimulation and/or inhibition of ST secretion through growth hormone releasing hormone (GHRH) and somatostatin (SS) of the hypothalamus, respectively. Electrical stimulation of the

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13 ventromedial hypothalamic nucleus, where secretion of GHRH occurs, results in a marked increase in plasma concentrations of ST. The ventromedial nucleus and ventralbasal hypothalamus are the only regions that are capable of this response. Stimulation of the paraventricular somatostatic area resulted in a decrease in blood concentrations of ST (Martin, 1972). Negative feedback control of the pituitary is exerted at the pituitary level by I IGF-I. Insulin like growth factor-I also acts on the hypothalamus to stimulate secretion of SS, whereas P-adrenergic stimulation inhibits GHRH secretion (Martin, 1972). Norepinephrine, a monoaminergic neurotransmitter, regulates the release of GHRH and SS into the hypophysial portal blood. In rats, inhibition of norepinephrine synthesis or blockade of a2-adrenergic receptors reduces concentrations of ST in plasma and eliminates spontaneous pulses of ST (Muller, 1987). On the other hand, activation of a2-adrenergic receptors stimulates secretion of ST (Terry and Martin, 1981). Somatotropin is secreted from somatotropic cells located in the anterior pituitary gland. Sensory impulses from the periphery terminate in the hypothalamus, activating neurosecretory axons in this area. These secretory axons secrete GHRH or SS into the capillary loops of the median eminence. Capillaries converge into the portal trunks of the neural stalk, pass to the anterior pituitary gland, and break up into venous sinusoids, where they stimulate or inhibit the secretion of ST (Muller et al., 1999). Thus, a variety of neurotransmitters such as norepinephrine, dopamine, and serotonin play a role in the neuroendocrine regulation of ST secretion. Growth hormone releasing hormone and SS act in a somatotropic cell via an adenylate cyclase transduction

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14 system. Growth hormone releasing hormone acts on stimulatory receptors that activate adenylate cyclase via interaction with the regulatory coupling G protein. Activation of adenylate cyclase converts ATP into cAMP, which activates cAMP dependent protein I kinase and this results in secretion of ST. Somatostatin acts via its inhibitory receptor and inhibits the activation of adenylate cyclase. In rat somatotropic cells, low concentrations of Ca caused reduced secretion of ST (Mandell et al., 1988). In addition to this finding, GHRH enhanced intra-cellular Ca levels when SS reduced the level of Ca in somatotropic cells (Holl, 1988). Thus, inositol phospholipids and Ca also were implicated to be involved with somatotropic cell secretion of ST. Evidence suggests that nutritional status plays a major role in determining circulating concentrations of ST. Starvation caused elevated concentrations of ST in I pigs (Atinmo et al., 1978), sheep (Driver and Forbes, 1981), and humans (Merimee, 1980). Underfed cows also had higher concentrations of ST in plasma than well-fed herdmates (Hart et al., 1978). Thus, it was not surprising that non-esterified fatty acids (NEFA), glucose, leptin, and neuropeptide Y also appeared to influence release of ST (Smith et al., 1996). The direct inhibition of ST release from the anterior pituitary gland by NEFA is speculated to complete a feedback loop because ST is known to stimulate lipid mobilization (Imaki et al., 1986; Smith et al., 1996). However, a decrease in plasma NEFA caused a rise in plasma concentrations of ST (Merimee, 1980). Glucose is an important regulator of ST secretion. Hypoglycemia stimulated secretion of ST in humans (Brodows et al., 1973), whereas acute administration of I glucose inhibited secretion of ST (Scanlon et al., 1996). In contrast, INS induced

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15 hypoglycemia or intracellular glucopenia inhibited pulsatile ST secretion in rats through stimulation of SS release from the hypothalamus (Tannebaum and Martin, 1976). Leptin appeared to stimulate ST secretion by altering GHRH and SS release from the hypothalamus (Carro et al., 1 997). Secretion Pattern of Somatotropin ^ The secretion pattern of ST is pulsatile in the species studied. In the adult rat, secretion of ST was synchronous with ST secretory peaks that occurred at 3-4 h intervals (Tannebaum et al., 1976). In male rats, ST secretion occurred in discrete pulses with low interpeak levels, whereas female rats showed less pulsatility and higher interpeak levels (Clark and Robinson, 1988). Women have higher overall concentrations of ST with a higher pulse amplitude and baseline, but the frequency of pulses were the same as in males (Muller et al., 1999). In confrast, secretion of ST in cattle followed an episodic pattern rather than being synchronous. Episodic pulses of ST were seen throughout the 24 h cycle. There appeared to be considerable variation in the secretion pattern of ST among individual cows and no association of time of feeding or time of day was observed (Davis et al., 1977). Bulls exhibited greater amplitude of ST episodes than steers with no significant differences in baseline values (Davis et al., 1977). Basal and pulsatile release of ST were regulated differently by adiposity and steroid hormones. Average concentration of ST in plasma was lower in heifer calves than in bull calves at 5 mo of age and basal secretion of ST was correlated negatively with circulating estrogen, whereas it was correlated positively with circulating testosterone (Muller et al., 1999).

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16 Somatotropin Gene The ST gene is a part of a large gene family including ST, prolactin (PRL), and the placental lactogens (PL) which also are known as chorionic somatomammotropins (CS; Slater et al., 1986). The latter are thought to have arisen as a result of gene duplication. The ST gene has five exons and four introns, covering approximately 2.6 to 3.0 kilobase pairs in most mammalian species including cattle. In chicken and I rainbow trout the gene is 3.5 and 4.5 kilobase pairs, respectively, due to the larger intron sizes (Tanaka et al., 1992; Angellon et al., 1988). In primates, unlike in most mammals, there are multiple ST genes that include ST-N and ST-V. The ST-N gene is expressed in somatotropic cells of the anterior pituitary , whereas the ST-V gene is expressed in placental tissues (Tuggle and Trenkle, 1996). Structure of Bovine Somatotropin Bovine somatotropin is composed of 190 or 191 amino acids and has a molecular mass of approximately 22,000 daltons (Andrews, 1966). The amino acid sequence of bST, which gives it its three-dimensional shape, differs by about 35% from that of human ST (Carr and Friesen, 1976). The amino acid at position number 127 in the sequence can be either leucine or valine. In addition, ST containing only 1 90 amino acids has a phenylalanine at the NH2 terminus rather than alanine that is found in the 191 amino acid form of ST. These differences occur because of a different cleavage of the signal peptide and thus, four different variants of bST are produced naturally (Wood, 1989).

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I 17 Bovine somatotropin has four antiparallel a helices packaged close together which gives the protein a somewhat square appearance with a left-handed twisted handle (Figure 2-1). A bend in a helix number two of 26.2 degrees is caused by amino acid number 89, a proline. This proline consists of 14 atoms and is conserved throughout ST of different species (Carlacci et al., 1991). Bovine somatotropin consists mainly of a hydrophobic core. In the helical region there are 47 hydrophobic side chains. Over 90% of all hydrophobic chains on the helices are buried inside due to either the packaging of the helices or loops (Carlacci et al., 1991). Disulfide bridges are known to contribute significantly to the stability of the molecular structures of many proteins (Havel et al., 1989). The interaction caused by the two disulfide bonds of ST between the helices and loops plays the key role in stabilizing the protein for electrostatic and nonbonded interactions. The disulfide bridges are formed between cysteine residues with both connecting a loop to a helix. The first disulfide bridge connects the hooking middle of one of the long loops (amino acid number 53) with helix 4 (amino acid number 164) which pulls the long loop onto the surface of the bundle (Havel et al., 1989). The second disulfide bridge hooks the small loop of the C-terminal segment (amino acid number 189) to the 4th helix (amino acid number 181) which forces the C-terminal segment to bend toward the helix bundle (Havel et al., 1989). The loops of the ST, which hold the cysteine residues for the disulfide bonds, consist of 32 hydrophobic side chains. Of the 32 side chains 1 7 are completely or partly buried either between the helix and loop or between the loops themselves (Carlacci et al., 1991). The structure of 1

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18 Figure 2-1. Three dimensional shape of bST (Carlacci et al., 1991)

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19 ST is composed of four-helix bundle proteins. The first a helix is composed of 26 amino acids, the second is composed of 21 amino acids, the third is composed of 22 amino acids and the fourth has 30 amino acids (AbdelMeguid et al., 1987). The rest of the protein is composed of two long loops with 40 and 24 amino acids, respectively and a short loop with 9 amino acids. The long loops are between helices 1 and 2 and between 3 and 4. The short loop is between helices 2 and 3 (Carlacci et al., 1991). Six amino acid residues are attached to the N-terminus of a helix number 1 and 8 amino acids are attached to the C-terminus of alpha helix number 4. Somatotropin Receptor Somatotropin exerts cellular actions as a result of its specific membrane bound receptors. These receptors are the first of the class 1 cytokine receptors to be cloned (Leung et al., 1987). The cytokine receptor superfamily is composed of 15 members including PRL, interleukins (2 through 7), erythropoiefin, oncostafin M, and the lepfin receptors. This family of receptors has common features such as a single membrane spanning domain, two pairs of cysteines and a conserved tryptophan adjacent to the cysteine in the N-terminal module, absence of intrinsic protein kinase activity, and a proline-rich region in the cytoplasmic domain (Bazan, 1989). These proline-rich regions are central to signaling because they bind the Janus Kinases (JAKs), the major mediators of this class of cytokine receptor (Waters et al., 1999). I The cytokine receptor superfamily has a three-domain organization including an extracellular ligand binding domain, a single transmembrane segment, and an intracellular domain. The mature ST receptor is 620 residues long, with 246 residues of

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20 the extracellular domain and 350 residues of the intracellular domain. The extracellular region also contains 2 fibronectin p sandwich domains that are closely related to PRL and erythropoietin receptors (Godowski et al., 1989). Human ST receptor consists of 10 exons spanning 85 kb, with the receptor itself being coded by 9 exons that yield an mRNA of 4.2 kb (Godowski et al., 1989). Multiple exons facilitate the regulation of receptor expression in different tissues in response to different stimuli (Adams, 1995). The mechanisms by which ST regulates the transcription of genes required for body growth and its regulation are being delineated. It has been suggested that signal transducers and activators of transcription (STAT) are key contributors to ST signaling and to the mechanisms by which ST activates genes that lead to its physiological actions (Figure 2-2). STATl, 3, 5a and 5b are tightly regulated by ST-ST receptor and JAK2 interactions and participate in the regulation of many genes associated with growth and metabolic effects. Somatotropin uses two different sites to bind identically to two different receptors. Dimerization of receptors by the hormone causes activation by bringing the intracellular domains into close proximity. This dimerization then activates the receptor associated JAK family of tyrosine kinases. JAK2 phosphorylates tyrosines within itself and the ST receptor. These tyrosines form binding sites for a number of signaling proteins, including members of the family of STATs which play very important roles in the regulation of gene transcription (Wells, 1996). STAT proteins are latent, src homology domain 2 (SH2) containing cytoplasmic factors. Studies with ST receptors indicated that JAK2 can activate STAT 1, 3, 5a, and 5b. Activated STAT proteins then yield heteroor homodimers via an SH2 phosphorylated

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Figure 2-2. Signaling and regulation of the transcription factors by ST. Adapted from Herrington et al. (2000).

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tyrosine interaction which enters the nucleus of the cell, binds to DNA, and activates I transcription of target genes (Schindler and Darnell, 1995). Responses to Somatotropin Supplementation Somatotropin is a major regulator of growth in mammals. In the liver ST regulates the expression of a wide range of proteins including hormone receptors and growth factors, secretory products, and enzymes such as cytochrome P450 (Norstedt et al., 1990). Somatotropin also has a unique effect on stimulating mammary gland development (Feldman et al., 1993) and lactation (Barber et al., 1992). Typical MY response to bST is an increase of at least 10-15%. However, the response can be much greater with better care and management of the cows (Bauman et al., 1985; NRC, 1994). After the first few days of bST treatment, MY increases gradually and reaches a peak and the increase is maintained with continuous injections of bST. Milk yield gradually returns to normal pre-injection levels after cessation of treatment (Bauman et al., 1999). Obtaining a milk yield response to bST does not require special diets or different feed ingredients. Substantial milk yield responses have been observed on diets ranging from pasture to the more typical total mixed ration (TMR) diets. However, dry matter intake (DMI) increases in bST treated cows after a few weeks of the supplementation and persists throughout the interval of bST use. Thus, treated cows require adequate amounts of a balanced diet rather than a special diet (NRC, 1994).

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23 Effects of bST on the Mammary Gland In early years of study on actions of ST it was concluded that a direct effect of ST on mammary gland development was unlikely. Studies failed to detect an ST receptor on the mammary epithelial cell of the cow (Gertler et al., 1984). Moreover, infusions of ST directly into mammary artery of sheep did not stimulate milk production (McDowell et al., 1987). However, in rodents, a direct effect of ST on mammary development and function was established (Flint and Gardner, 1994; Kleinberg, 1997). Although most evidence suggested an indirect effect of ST on mammary gland development in ruminants, it was found that ruminants also expressed mRNA for the ST receptor in mammary gland (Glim et al., 1990). Furthermore, immunologic staining of ST receptors in mammary tissue of pregnant and lactating I cows also was reported (Plath-Gabler et al., 2001). Collier et al. (1993) observed a significant effect of ST on mammary growth of pregnant heifers when it was administered through the teat canal. However, as indicated, unilateral close arterial infusion of ST into one-half mammary gland of sheep did not increase milk yield performance over the uninjected half (McDowell et al., 1987). Furthermore, ST administration through the teat canal of lactating goats did not result in increased milk production response (Serjsen and Knight, 1994). This latter response would be expected unless there was uptake of ST across the apical membrane of the epithelial I cells or uptake into the general circulation and subsequent action in mammary gland. The acute rise in MY in response to bST and rapid decline after discontinuing bST injection argues against cell proliferation being caused by ST in the short-term.

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Knight et al. (1994) observed that ST treatment did not affect the amount of mammary parenchyma and BrdU-labeHng of mammary epithehal cells in vivo during the first 6 wk of lactation. On the other hand, a large increase in parenchymal volume was observed in mid-lactation goats treated over a 22 wk period with exogenous ST (Knight et al., 1990). Other studies with cows and goats have established trends and/or significant increases in key enzymes due to bST in the mammary gland such as acetyl I CoA carboxylase, acetyl CoA synthetase and fatty acid synthetase. Thus, bST directly and/or indirectly causes an increase in the rates of milk synthesis per cell and an improved maintenance of secretory cells (Bauman and Vernon, 1993; Etherton and Bauman 1998). One of the mechanisms by which ST affects mammary gland function is via an 1 indirect effect on mammary tissue by action of IGF-I (Cohick, 1998). Concentrations and actions of IGF-I likely are the most important link to tissue response when higher concentrations of ST occur either because it is injected or because of greater secretion fi-om the anterior pituitary. Somatotropin has a major role in regulating the concentrations of IGFs that are in the circulation. Concentrations of IGF-I increase I during bST treatments via both induced release of IGF-I from a hepatic storage pool and because of greater biosynthesis of IGF-I. Biosynthesis is regulated by increased mRNA levels and mRNA stability (Sharma et al., 1994). Increase in IGF-I concentration was maintained with continued injections of ST and the increase in MY was parallel to the increase in IGF-I. Cessation of ST supplementation yielded a parallel decrease in both MY and blood concentrations of IGF-I (Sharma et al., 1994).

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25 Insulin-like growth factor-I has both autocrine and paracrine actions in addition to its endocrine actions (McGuire et al., 1992). At the cellular level, IGF-I locally stimulated amino acid transport, synthesis of RNA and DN A, and synthesis of cellular proteins (Phillips et al., 1990). Receptors for IGF-I were demonstrated in mammary tissue and IGF-I was a local mediator of mammary epithelial growth and development (Forsyth, 1996; Vega et al., 1991). Intralobular stromal cells, small blood vessels, and capillaries contain IGF-I receptors in the mammary gland (Glimm et al., 1992). During ST treatments, IGF-I binds in the cytoplasm and the stroma of epithelial cells. Insulin like growth factor-I stimulates the cellular activity of mammary gland, it increases synthesis of RNA and DNA, and the synthesis of cellular proteins. Therefore, it is a local mediator of mammary epithelial growth and development (Phillips et al., 1990). Interestingly, close arterial infusions of IGF-I into mammary gland of goat, but not systemic infiisions of IGF-I, increased MY dramatically (Prosser et al., 1996). However, lactational response to close arterial infusion of IGF-I was much less than that of systematic bST treatments which suggests that the mechanism of action of bST with regard to stimulatory effects on MY carmot be limited only to the known effects of I IGF-I. , • Effects of Somatotropin in Other Tissues \ , ., ^ Somatotropin exerts many different effects on protein metabolism in both mammary and non-mammary tissues. Bovine somatotropin increases milk yield with no change in milk composition, unless there is energy deficiency. It provokes milk yield increase because of an overall increase in blood supply to mammary gland (water

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26 uptake), alters glucose metabolism (lactose synthesis in the mammary gland), enhances lipolysis and increases lipogenesis in the mammary gland (supply of lipid precurcors), and also modifies protein metabolism (supply of amino acids). As a result, milk yield increases and the composition of milk does not change from normal (Bauman, 1999). Increased MY response due to ST is due mainly to altered partitioning of nutrients in favor of mammary glands and to an increase in the synthetic capacity i and/or longevity of the milk synthesizing cells (Bauman and Vernon, 1993). Somatotropin is a primary homeorhetic regulator during pregnancy and lactation (Bauman and Currie, 1980); it regulates partitioning of nutrients (carbohydrates, lipids, proteins, and minerals) and plays an important role in the coordination of various organs and tissues (Bauman, 1992). To support milk synthesis, the metabolism of V . i r, • other tissues is stimulated to provide the necessary precursors. Carbohydrate metabolism During early lactation glucose is used almost exclusively by the mammary glands (exceptions are nervous system and brain), and MY is heavily dependent upon increased glucose supply to the gland. If bST is to increase milk yield, then it must act in a way to direct more glucose to the mammary gland. This can occur through a variety of individual actions of bST. First, bST increases mammary blood flow so that more blood perfuses the mammary gland and increased uptake of glucose can occur (Davis et al., 1988). A decreased ability of INS to inhibit gluconeogenesis is observed following bST treatments. Both in vivo (Cohick et al., 1989) and in vitro (Knapp et al.,1992) studies demonstrated that hepatic rates of gluconeogenesis were increased

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27 during treatment of dairy cows with bST. Furthermore, ST decreased sensitivity of INS receptors to INS in the peripheral tissues and this resulted in decreased overall uptake of glucose in peripheral body tissues. This minimized the oxidation of glucose to CO2, and as a consequence more glucose was made available to the udder. Somatotropin also stimulated increased feed intake, and as a result, more propionate was produced in the reticulo-rumen and became available for gluconeogenesis (Beauville et al., 1992). In I vitro studies of liver tissue of ST treated cows showed a 60% increase in capacity to use propionate for glucose synthesis (Knapp et al., 1992). Pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) are potential rate-limiting enzymes for hepatic gluconeogenesis during the transition period (Greenfield et al., 2000). It was speculated that exogenous ST would increase mRNA synthesis in the liver that coded for these enzymes (Bauman, 1999). On the other hand, Pershing et al. (2001) concluded bST stimulation of milk production was not mediated through enhanced liver gluconeogensis in cows 80 DIM. Somatotropin also increased lipid mobilization and more glycerol then was available as a precursor for gluconeogenesis. The sum total of these actions would be that higher blood glucose 1 concentrations occurred and this glucose could be directed to the mammary gland to support lactation. In addition to increased glucose production in the liver, glucose usage by other lean body tissues decreased. Because glucose is used as the primary energy metabolite and as a substrate for synthesizing milk constituents, energy needed by other peripheral body tissues would be derived from products of lipolysis or

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metabolism of non-gluconeogenic compounds arising from the rumen and lower I digestive tract. Lipid metabolism Nutritional status plays a major role in the regulation of lipid metabolism. Exogenous bST given to animals alters both lipogenesis and lipolysis in adipose tissue with the net effect being related to energy balance (EB) (Bauman, 1999). When cows are in positive EB, synthesis and deposition of lipids in adipose tissue are reduced by ST which, in turn, increases availability of nutrients and their utilization for milk production (Bauman and Vernon, 1993). Insulin is an important homeostatic control in the regulation of lipid metabolism. Somatotropin reduces the ability of INS to stimulate lipogenesis in adipose tissue. Thus, ST reduces the action of INS, suppresses lipogenic I enzyme activity, and reduces glucose uptake (Bauman and Vernon, 1993). These coordinated changes in INS actions would support production of glucose from available precursors and conservation of glucose for mammary use by shifting peripheral tissues to utilization of other substrates available, in large part, due to combined actions of ST and INS. When cows are in negative EB, ST stimulates lipolysis; it alters the sensitivity of adipose tissue to P-adrenergic agents (Bauman and Vernon, 1993). Therefore, for cows that are in negative EB and are being treated with bST, increased lipid mobilization would be a major source of energy needed to support milk production (Sechen et al., 1990). Lipolysis is regulated by a signal transduction system that includes cAMP, stimulatory G proteins (G^) and inhibitory G proteins (Gj).

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Catecholamines act through the system to stimulate lipolysis, whereas adenosine exerts its antilipolytic affects via the Gj system. When adenosine binds to its receptor it stimulates Gj which uncouples G^ protein activation of adenyl cyclase catalyzed by catecholamines. This leads to the inhibition of the lipolytic pathway that is stimulated by catecholamines. Somatotropin alters lipolysis through an increase in response to catecholamines with no change in sensitivity. Epinephrine challenge following ST treatment dramatically increased NEFA concentrations in plasma. Interestingly, ST treatment resulted in modest changes in P and (f.^ adrenergic receptor numbers. However, the activity of Gj proteins was reduced significantly by ST treatments. As a result, it has been suggested that ST impaired the ability of Gj to interact with adenyl cyclase. This, in turn, would increase the effectiveness of G^ system stimulated by catecholamines (Bauman, 1999). The events described above would dramatically increase mobilization of lipids from the adipose tissue, and increase blood NEFA and glycerol. Thus, there would be greatly reduced fatty acid synthesis or no net synthesis, and hence, less acetate and glycerol use in adipose tissue (Bauman et al., 1988). So, the net result would be a shift in the availability of these metabolites in the mammary gland where they can be used for synthesis of short and medium chain-fatty acids that are themselves used for TG synthesis and milk production. Therefore, lipolysis also must be an important pathway to provide needed precursors in the early postpartum period by cows especially to supply the energy needed for milk production (Baldwin and Knapp, 1993; Bauman, 1999).

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30 Amino acid metabolism Somatotropin treatment increases milk protein synthesis in lactating cows via improved efficiency of amino acid utilization. A reduction in circulating urea nitrogen and in urinary nitrogen loss was reported following bST treatment (Davis and Collier, 1985). During negative energy balance ST will spare protein use as a source of energy in tissues because it increases lipid mobilization and enhances glucose metabolism. I Proteins that are mobilized from the muscles can be used in the liver, in the gut, and in the blood which will increase overall metabolism and efficiency of protein use. Amino acids mobilized have an important role in supporting growth of some organs (liver, heart, digestive tract) rather than to support milk synthesis (Erdman and Andrew, 1989). Amino acids that are oxidized to provide energy will be reduced such that the I protein mobilized can be used for growth of specific tissues and milk protein synthesis. As indicated, ST causes an increase in feed intake which will make more nutrients and amino acids available to support increased milk yield, and this will lessen the need for tissue mobilization. Mammary blood flow It has been established that nutrient supply to the mammary gland is one of the major limitations for the activity of secretory cells and milk synthesis. A lactating mammary gland places a heavy demand on the animal to provide substrates for milk synthesis (Davis and Collier, 1985). With bST injections, along with the increases that occur in MY, cardiac output also increases. Insulin like growth factor-I has a role in the increased blood flow to the mammary gland that appears to be mediated by production

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31 of nitric oxide (Prosser et al., 1996). Somatotropin also increases conversion of T4 to T3 I specifically in the mammary gland and the increase in T3, the tissue active thyroid hormone, increases local metabolism and helps to mediate the galactopoietic response (Capuco et al., 1989). Thus, both increased milk secretion and local metabolism drive more of the blood circulation to mammary gland and this, in turn, supplies greater quantities of water and nutrients needed for milk synthesis. Because of its positive effects on blood glucose, lipids and amino acid concentrations and blood circulation, bST increases milk yield without affecting the overall composition of the milk. In early lactation, amounts of long chain fatty acids available increases relative to short chain fatty acids and generally, during negative energy balance, bST tends to increase milk fat and decrease milk protein. However, the I effect is not great and with positive feed intake this effect disappears. Transition Period Transition Irom pregnancy to lactation is one of the most important challenges faced by dairy cows during a lactation cycle. The cows physiological status during the last 3 wk of the dry period through the first 3 wk of the subsequent lactation can have I significant effects on the lactation and on reproductive performance (Drackley, 1999). Following parturition, nutritional requirements of cows increases greatly due to the increased milk production. Decreased DMI prior to calving carries over into the period immediately following calving and results in slower increase in milk yield. The DMI consumed postpartum is used ahnost totally for milk synthesis. This results in cows

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undergoing a period of NEB because milk yield increase outpaces the increase in nutrient intake (Bell, 1995). Feed consumption by the transition cow decreases by as much as 30% within a I day or two before calving (Bertics et al., 1992; Grummer, 1995). However, this decrease does not occur only during the final weeks preceding parturition. Pregnant dairy heifers showed reduced DMI from wk 26 of pregnancy (1.53% per wk) until 3 wk before calving (Ingvartsen et al., 1992). A similar decline was observed in both heifers and cows during the last 168 d of pregnancy (Ingvartsen et al., 1997) when they were fed a high energy diet (1 1 .6 MJ of metabolizable energy/kg of DM), whereas the decline was less when they were fed less energy dense diets (10.2 or 8.3 MJ of metabolizable energy/kg of DM). I During late pregnancy, fetal metabolic rate increases dramatically at a time when the decline in DMI also is occurring more rapdly. The fetal metabolic rate at this time is approximately 2-fold greater than that of the dam on a BW basis (Reynolds and Ferrell, 1987). The source of carbons for oxidation in the growing fetus is mostly glucose and amino acids. Uterine uptake of glucose (Wieghart et al., 1986), amino I acids (Sniffen et al., 1992), and acetate (Bell, 1995), in relation to maternal supply, is as high as 46, 72 and 12 %, respectively. As a result energy deprived ewes are especially susceptible to hypoglycemia during late pregnancy (Bergman et al., 1974), whereas glucose drain and high plasma concentrations of NEFA predispose cows to ketosis (Littledike et al., 1981).

PAGE 50

33 Fetal energy requirements on d 250 of pregnancy were 2.3 Mcal/d NE, whereas the energy requirements increased to 26 Mcal/d NE in cows averaging 30 kg milk per day (Bell, 1995). Lactating dairy cows reach their peak MY between 5 to 12 wk postpartum, and the lowest DM1 occurs at calving. Peak DMI is not be reached until 8 to 14 wk after parturition (Wheeler et al., 1995). DMI increases by approximately 1.5 to 2.5 kg/wk during the first 3 wk of lactation (Bertics et al., 1992). However, there is great variation among individual cows as to when they reach peak DMI. This time can be affected by prepartum and postpartum rations, and degree of fatness or BCS (Broster et al., 1998). In addition, DMI of primiparous cows is less than that of multiparous cows (Kertz et al., 1991 ). There is great variation in rise to peak DMI in cows. For example, peak DMI of cows has been reported to be 2 and 111% more than DMI of the I same cows at one week postpartum (Bines, 1985). Delayed increase in DMI, with respect to increasing energy requirements, causes a NEB at or soon after parturition. High producing cows show a mean deficit of -10 Mcal/d during their second week of lactation (Chilliard, 1999). High producing cows can mobilize 50 kg of body lipids and 15 to 20 kg of body protein to support the I lactation (Whitelaw et al., 1986), whereas there is a need for more than 3 kg glucose per day for a cow producing 35 to 40 kg of milk daily during early lactation (Vernon, 1988). Cows can mobilize 0.56 kg body fat and 0.04 kg protein/d and the largest part of this mobilization (12% of total fat and 58% of total protein) takes place during the first week of lactation (Tamminga et al., 1997). As a result, the additional energy and

PAGE 51

protein need to be supplied to transition cows to support the great demands for r . metabolites if development of metabolic diseases was to be avoided. Metabolic Adaptations in the Transition Cow During late pregnancy and continuing into early lactation, major changes in metabolism of the cow occur to cope with the increase in nutrient requirements for mammary metabolism. Requirements for lactation are supported, in part, by an I increase in DMI and digestion, as described above. However, "orchestrated or coordinated changes in the metabolism of body tissues are necessary to support a physiological state" (Bauman and Currie, 1980), in this case to support lactation, and it must accompany the increase in DMI and digestion. These homeorhetic changes include hormonal changes, increased hepatic metabolism, lipid and protein I mobilization, and a reduction in the utilization of glucose and amino acids by lower priority organs (Bell, 1995). In this way the nutrient supply to mammary gland can be maintained and enhanced. Hormonal changes Concentrations of various hormones change in a relatively narrow time period around calving (Figure 2-3). Plasma concentrations of P4 peak around d 250 of gestation (7-8 ng/mL). Thereafter, concentrations begin to decline to a 3-4ng/mL and on the day of calving concentrations of P4 are almost undetectable. By midgestation, estrogen concentrations rise from 20 pg/mL to 300 pg/mL. Approximately 7 d before parturition, plasma concentrations of estrogen increase to around 2000 pg/mL, whereas just before calving the total estrogens (free and conjugated) are about 4000-6000

PAGE 52

G7-\ 35 CORPUS LUTEUM (Luleolysis) Progesterone . PITUITARY GLAND Tropic Homiones e.g, ACTH, ST + OTHER ENDOCRINE GLANDS _ e.g. Prolactin + e.g. Cortisol 1+ MAMMARY GLAND PARTURITION Figure 2-3. Schematic of endocrine changes around parturition. '+' increased concentrations or activity, and '-' decreased concentrations or activity. Adapted from Mepham (1987).

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pg/mL (Chew et al., 1977). Plasma Cortisol concentrations increase to 4-8 ng/ml 3 d before calving to peak around calving or the day after calving (1 5-30 ng/mL). The concentrations of Cortisol and estrogen decline to essentially basal levels within days after calving (Tucker, 1985). Around 24-36 h before calving PGF,^ concentrations begin to rise and they peak at calving. Plasma PRL increases rapidly the day before calving. Changes in plasma concentrations of PRL, estrogen and P4 are responsible, in part, for the increased synthesis of colostrum. Circulating concentrations of ST usually increase before calving and remain elevated during early lactation, especially in high producing cows (Bauman and Vernon, 1993). Even though concentrations of INS in circulation remain high during the prepartum period, concentrations decrease rapidly after calving. During the first few days after calving the basal concentration of INS is negatively correlated with ST. Plasma glucagon sometimes increases during early lactation, but changes in the INS to glucagon ratio has more physiological importance because it is one of the factors determining the rate of lipolysis and lipognesis (Sartin et al., 1988). Adipose tissue i Shifting energy balance is the major determinant of the changes that take place in adipose tissue. In ruminants, the major site for tissue lipogenesis is adipose tissue. During early lactation fatty acid synthesis and esterification, plasma TG uptake by adipose tissue, lipogenesis, and the enzymes controlling these events are notably altered (McNamara and Hillers, 1986). There also is a reduction in the utilization of plasma acetate, glucose, and TG by the adipose tissue. These events lead to higher entry of

PAGE 54

37 glycerol and NEFA from the adipose tissue into circulation and to a decrease in the size of adipocytes. Initiation of copious lactation leads to many changes in the activity of a number of important enzymes of adipose tissue which are advantageous to milk production. Activity of lipoprotein lipase declines rapidly in adipose tissue and this leads to reduced uptake of fatty acids. Activity of the most important regulatory enzyme of lipogenesis, acetyl CoA carboxylase, declines dramatically during early lactation as well. The fall in the rate of esterification in adipocytes also is important for lipolysis because it means that less of the fatty acids that are released during hydrolysis of TG, less are reesterified (Vernon et al., 1987). Although capacity for lipid synthesis decreases, the ability to hydrolyze adipose tissue derived TG increases rapidly. Increased activity of hormone-sensitive lipase results in a further increase in lipolysis. As a consequence, there are increased concentrations of NEFA and glycerol in the blood during early lactation. Liver Increased concentrations of NEFA in the blood before and at parturition results I in increased uptake of NEFA by the liver, increased fatty acid esterification, and increased storage of triglycerides (TG) (Grummer, 1993). Liver TG content peaks at or near calving because the rate of TG synthesis is positively associated with plasma concentrations of NEFA (Bell, 1980). Although esterification of NEFA to TG increases during early lactation, discharge of TG from liver in the form of very low density lipoproteins (VLDL) is very slow and limited in cows, which likely causes fatty

PAGE 55

38 liver before and up to calving, and during the early weeks of lactation (Grummer, 1993). Some portion of NEFA taken up by the liver is oxidized to COj and ketone bodies. Ketone body production increases when the ability of the liver to export fatty acids as lipoprotein is exceeded and a glucose drain takes place at a time when concentrations of both glucose and INS in blood are low (Littledike et al., 1981). Because peripheral utilization of ketone bodies is limited and there already is high entry of NEFA into liver, this may predispose animals to ketosis during early lactation. Liver gluconeogenesis and glucose turnover increases greatly to support high demands by mammary gland after calving. Dietary glucose can only account for about 10 to 35% of glucose that is secreted in milk of a cow producing 40 kg milk daily. There is a marked increase in entry of glucogenic substrates such as propionate, amino acids, glycerol and lactate both from the digestive tract and from mobilization of body reserves (Vernon, 1988). The blood flow to the liver, weight of liver, and the activity per unit weight also increase during early lactation. The increase in liver weight, however, is proportional to that of increased protein synthesis (Kelly et al., 1991). Muscle During early lactation a loss of skeletal muscle protein has been reported for cattle (Blum et al., 1985) and sheep (Bryant and Smith, 1982). It was suggested that this was due primarily to the increased degradation of protein with little or no change in protein synthesis (Bryant and Smith, 1982). Amino acids in the peripheral circulation

PAGE 56

39 that arose from muscle can be used either for protein synthesis by the mammary gland or for gluconeogenesis in the liver. Well-fed, high-producing cows can mobilize up to 10 kg of body protein without a health risk during the first 60 d of lactation (Whitelaw et al., 1986). In underfed cows, even though the contribution of muscle protein can be one-half of the total protein utilized, potential mobilization cannot exceed more than 20% of body proteins (Wilson et al., 1988). Altered mobilization of amino acids does not mean, however, that energy requirements of skeletal muscle are decreased. Although energy requirements of the muscle are the same, the source of energy utilized by the muscle shifts fi-om glucose to fatty acids. Glucose uptake by muscle and the proportion of active pyruvate dehydrogenase decreases during lactation (Vernon et al., 1987), whereas there is increased use of fatty acids and ketones for oxidative purposes by muscle during lactation (Pethick and Lindsay, 1982). . • . i , ' -* . Bone and minerals —i . , , Bovine species have the highest calcium turnover per kilogram of B W during milk production (Sciorsci et al., 2001). During the last week of gestation, the fetus requires approximately 5 g Ca and 1.5g P/d. At the onset of lactation, the daily secretion of Ca and P in milk is approximately 30 and 15 g, respectively (Jorgensen, 1974). Up to 60 g Ca/d can be secreted in milk at the onset of lactation suggesting that plasma Ca must be renewed 25-times daily (Horst, 1986). Although intestinal absorption of Ca and its efficiency increased, this is not enough to supply Ca loss due

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to the increased MY. Thus, animals must mobihze their bone Ca and other minerals to meet the requirements or else suffer hypocalcemia. This mobilization represents about 20% of minerals in sheep during early lactation (Marie et al., 1986). Failure to respond to low plasma Ca concentrations results in hypocalcemia (Horst and Reinhardt, 1987). General Overview Important changes occur in metabolism of the dairy cow to support increased nutrient demands of the mammary gland during early lactation. These adaptations are coordinated by changes in the endocrine status of the animals. Prolactin has an important role in the development of the mammary gland and ST has an important role in orchestrating the metabolic adaptations occurring in the body to support lactation. On the other hand, the action of INS is reduced significantly as a consequence of the negative energy balance during early lactation. Although tissue response or responsiveness to INS decreases, the response to catecholamines is enhanced. Thus, changes in both the responsiveness of tissues to hormones and overall circulating concentrations of hormones appear to have important roles in the metabolic adaptations seen during this critical time period during transition and continuing throughout the lactation. ' Materials and Methods One hundred-ninety three multiparous Holstein cows from the University of Florida Dairy Research Unit (DRU) herd were used in an experiment conducted over a 2 yr period and the protocol approved by the Institutional Animal Care and Use Committee of the University of Florida. Cows were assigned randomly about 4 wk

PAGE 58

prior to expected calving date. Ages of the cows ranged from 3 to 6 yr and parity was between 1 and 5. Information on the animals, including parity and age, day and month of expected calving, days dry, and milk yields during previous lactation were obtained from records of the DRU. Actual number of days that cows were sampled prepartum (21±10d) differed from expected because of early or late calving. During year one, plasma samples were collected from 82 Holstein cows [Confrol (C)=41 vs. Injected (I)=41], and all cows calved between October 1998 and January 1999 at the DRU. During year two, effects of bST on BCS and BW of 1 1 1 I Holstein cows (C=57 vs. 1=54) were evaluated, but no blood samples were collected. These cows calved between October 1999 and March 2000. The BCS (1-5, thin to fat, Edmonson et al., 1989) and BW of cows were recorded (8:00-1 1:00 h) before a.m. feeding but after a.m. milking. The BW and BCS of the cows at the time the trial started ranged from 504 to 870 kg, and 3.00 to 4.75, respectively. Experimental Design Cows were assigned randomly to one of two treatment groups. Treatment group I (98 cows) were controls and received no bST treatment, whereas those in group II (95 cows) received injections of 0.4 mL of bST (POSILAC®) biweekly. This volume of POSILAC® contained approximately 142.9 mg bST and provided about 10.2mgbST/d. r • ;^ , • . , bST Treatments A sterile, prolonged-release, injectable formulation of a recombinant DNA derived bovine somatotropin analogue (bST, POSILAC®, 500 mg in= 1.4 mL,

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42 Monsanto, St. Louis, MO) was used for injections. Injections began approximately 4 wk (±3 d) before expected calving dates. Regardless of time of last injection before calving, first postpartum injections were within 24 h of calving and thereafter injections I were at 2 wk intervals. Last injection was at 42 d postpartum. Injections were subcutaneous in the post-scapular region or on either side of the ischiorectal fossa. Injections were administered after blood collection, but were prior to a.m. feeding or milking. No bST injections were given between 42 d postpartum through 100 d postpartum. All cows assigned to TRT I and II received a full dose of bST (500 mg/2wk) beginning at 100 d postpartum. Management ., i Feeding program Starting 4 wk before expected calving dates, cows were fed the close-up dry ration (CUD). The CUD ration was formulated to be anionic (=-10 Meq/lOOg DM) to ! decrease the postpartum risk of hypocalcemia. After parturition, all cows were fed a total mixed ration (TMR) based on com silage, whole cotton seeds (WCS), and grain concentrate (Table 21 ). Clean fresh water was provided in water troughs and was available free-choice in the free-stall bam where they were housed. Bams were equipped with fans and sprinklers which helped cool cows when ambient temperature were above 25 °C. Body condition scores and body weights Body weights and BCS (1-5, thin to fat; Edmonson et al., 1989) of cows were recorded biweekly on the same day each week (Saturday) before a.m. feeding or

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Table 2-1. Dry Matter Concentrations and Chemical Composition of CUD Ration and TMR Bermuda Hay Trace Minerals Dicalcium Phosphate Chemical Composition Ingredients %DM 1 CUD TMR 1 Com Silage 37.12 21.72 Alfalfa Hay 9.14 Cottonseed Hulls , , , 5.57 Citrus Pulp _ 9.94 Hommy 22.24 16.28 Distillers Grains * ^ ^ / 7.44 9.19 Soybean Meal 7.44 7.89 Whole Cottonseeds (WCS) 7.36 14.95 Mineral Mix 5.33 Springer Minerals 6.69 10.70 0.59 0.42 Percentage^ DM 1 56.54 63.77 CP 15.28 17.67 Sol CP ' 34.80 33.43 ADF 22.34 25.33 NDF 37.34 37.16 EE* 4.83 5.83 TDN 68.10 68.29 NEl (Mcal/kg) 1.58 1.68 'Analyses of components from NEDHIA Forage Laboratory, Ithaca, NY.^DM basis. ^Percentage of the CP. " Ether extract.

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milking (8:00 to 12:00 h) during the second year. Measurements began the day cows were assigned to trial and continued up to 100 d postcalving. Blood collection, handling and storage Blood samples were collected from the tail vein of all cows before the a.m. feeding or milking (07:30-10:00 h) during the first year. Cows were bled after elevating the tail without any other restraint. The order in which cows were sampled on a given day was random and differed from bleeding day to bleeding day. Cows were bled the day they were assigned to the trial and biweekly during the prepartum period, the week of calving, and then monthly up to 60 d postpartum. For blood collection Vacutainer® brand needles (2.54 cm, 20 gauge) and tubes containing sodium heparin were used (10 x 100 mm blood collection tubes, Becton-Dickinson. Fairlawn, NJ). Blood samples were placed on ice immediately after collection and processed within 2 h. * All samples of blood were centrifuged at 3000 RPM at 5°C for 30 min in the RC-3B refrigerated centrifuge (6-place swinging basket, H.600A rotor, Sorvall Instruments) to separate plasma. Plasma from each sample was aliquoted into two I labeled 5 mL polyethylene tubes (75x12 mm), capped, and frozen at -20°C until analyzed. The plasma samples were used for analysis of ST, INS, and IGF-I by specific radioimmunoassays (RIAs). In vitro enzymatic colorimetric method of Wako (NEFA C, Wako Pure Chemical Industries, Osaka, Japan) was used for the quantitative determination of NEFA in plasma as described by Johnson and Peters (1993). Sigma I procedure No. 510 (Sigma Diagnostics, St. Louis, MO) was used for the quantitative

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I t-.v 45 enzymatic determination of glucose in deproteinized plasma samples as described by Raabo and Terkildsen (1960). Milking and milk collection All cows were milked in a double12 herringbone milking parlor equipped with 24 DeLaval milking machines and a Galaxy 2000 milk recording system. An automatic cow identification feature was used during the experiment, and individual milk yields were recorded at each daily milking from 3 d after parturition to d 100 postpartum. Cows were milked three times daily (08:30, 15:00, and 01 :30 h). They were brought to the milking parlor holding area before each milking and washed automatically by pulsing sprinklers placed on the floor beneath the cows (3 cycles, about 5 min). After milking was completed, cows were teat dipped using undiluted I Clorox brand bleach and then returned to the free-stall bam. Milk samples were collected using an automatic milk sampling device. Milk samples were collected at three consecutive milkings (08:30, 15:00, and 01 :30 h) on Mondays during the first 10 wk of lactation. A set of 3 milk samples, one for each daily milking for each cow was saved in capped vials (50 mL) containing broad spectrum Microtab™ preservative (D&F control systems. Inc.) and analyzed at Southeast Dairy Lab (McDonough, GA) for contents of fat, protein, milk urea nitrogen (MUN), and the somatic cell count (SCC). Statistical Analyses Data collected during the experiment were analyzed in two sections. The first included data collected during the prepartum period and the second the data from the i

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postpartum period. Data were analyzed using Proc GLM procedure as a nested design by least squares analysis of variance procedures of SAS ( 1 99 1 ). Additionally, Mixed model was used to compare specific least squares means (Littel et al., 2000). Statistical analyses were performed for BW and BCS, milk and 3.5 % FCM yields, and concentrations of ST, INS, IGF-I, glucose and NEFA in plasma. Time periods considered for data analyses were the prepartum period (-4 to -1 wk), overall postpartum period (1 to 8 wk) and the period from 0-100 d postpartum for MY. Models included the main effect of treatment (TRT), calving month (CMO), their interactions (TRT*CMO), cow(TRT*CMO), and weeks or days to the highest order significant for overall prepartum and postpartum periods, as appropriate. Regression analyses were performed to the highest order significant up to quintic order to describe the trends in measures during postpartum period for MY during the overall postpartum period. Tests of heterogeneity of regression were performed to determine whether there was evidence that regression curves were not parallel (Wilcox et al., 1990). In addifion, gross correlations were estimated. Specific models are described in the Results section. Significance was declared at P<0.05, except where noted. Results One hundred-ninety three cows were utilized in the experiment; during year 1 , plasma samples were collected fi-om 82 Holstein cows [Control (C)=41 vs. Injected (I)=41], and plasma samples were analyzed for ST, IGF-I, INS, NEFA and glucose. I During year 2, effects of bST on BCS and BW of 1 1 1 Holstein cows (C-57 vs. 1=54) 1

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were evaluated. During both years, milk yields data were collected and combined and analyzed for the overall MY comparisons. The mathematical model utilized for analyses of dependent variables during the prepartum (-3 and -1 wk for blood measurements; -3 and 0 wk for BCS and BW measurements) and postpartum (1,4 and 8 wk for blood measurements; 2, 4, 6, 8, 10 wk for BCS and B W measurements and 0 to 1 00 d for MY), periods included main effects of treatment (TRT), calving month (CMO), the two-factor interaction of TRT*CMO and cow(TRT*CMO). Weeks to the highest order significant up to cubic order for weekly measurements also were included. The main effect of calving month resulted in ten different groups: CMO 1= those calving in October 1998, CMO 2= those calving in November 1998, CMO 3= those calving in December 1998, CMO 4= those calving in January 1999, CMO 5= those calving in September 1999, CMO 6= those calving in October 1999, CMO 7= those calving in November 1 999, CMO 8= those calving in December 1999, CMO 9= those calving in January 2000, and CMO 10=those calving in February 2000. During the experiment, 15 cows (year 1=6 cows, year 2=9 cows) from TRT I and 12 cows (year 1=5 cows, year 2=7 cows) from TRT II were culled at the end of the lactation because of breeding problems, chronic mastitis, or insufficient milk production. Changes in Body Weight and Body Condition Scores A major objective of this experiment was to evaluate changes in BW and BCS during the prepartum period starting about 3-3wk before parturition and continuing

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48 through 10 wk postpartum to monitor when the decHne in BW and BCS shift to increasing values. During the period from wk -3 to day of calving no differences were detected between the treatment groups for mean BW and BCS (Figures 2-4 and 2-5). Least squares means (LSM) for BW and BCS up to calving are in Table 2-2. No effects due to TRT, CMO or two-factor interaction TRT*CMO were detected for either BW or BCS, whereas significant linear effects of week was detected (P<0001; Table 2-3). The 1 mean BW -3 wk prepartum of cows in TRT I was numerically less than for cows in TRT II (706 vs 727 kg), but the difference was not significant. At the same time, the mean BCS of cows were 3.71 (TRT I) and 3.77 (TRT II) and did not differ due to treatment (Figures 2-4 and 2-5). At the week after calving (wk 0), cows in both groups had lost BW (682 vs 688 kg) and BCS (3.50 vs 3.58)(Table 2-2). Least squares analyses of variance for BW and BCS during the overall postpartum period (1-10 wk) is in Table 2-4. The effect of TRT on BW during this period was significant (P<0.0744); cows in TRT II better maintained their BW during 10 wk postpartum (Table 2-2). No effects were detected for CMO or the two-factor interaction TRT*CMO (Table 2-4). A significant linear effect of wk was detected for BW (P<0.01). Even though cows in both treatments had similar BW the week after calving (wk 0; 682 vs 688 kg), the loss in BW for cows in TRT I was greater during the postpartum period (Figure 2-4). Two weeks following parturition cows in TRT I had lost 8.5% of their BW compared to wk 0, whereas the loss of BW for TRT II was only 5.6%. During the following 2 wk period, BW loss was less by cows in both TRT I groups. There was a slight but not significant increase in BW observed beginning wk 6

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49 00 00 oo IT) o o o IT) o o Q CO -H -H CN 00 00 00 -H -H o 00 s 3 O a, 2 > CO CO o On 00 t-od o VO O -H -W VO (/I c C3 U H 3 cr CO •4-' c3 U CN CO ^ VO ON On CN ^ VO O VO O CO 00 CN I Q S 3 l-i > o CO 00 CO -H -H VO o r00 1^ CN — < CO ^ d -H -H o\ o VO 00 O •« -t^ o o vq o 00 VO CO H Pi H o VO J= ON Moo § 2 CN CO 3§ 00 S X) "O CO W O O ^ CO II ~ a 00 -tt jD CU o 6 II S H CO 0^ ^ H ffl

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o Q 00 o o OS 0\ -H -H o o O C) 00 On O O 00 -H -H VO VO VO -H -W to Q 00 00 00 0\ o VO OS VO CO VO VO O o -H -H CO 3 cr 00 II (73 00 Q Q W 00 00 w 00 00 1-1 oo OS VO o\ VO H H ro 0\ -H -H VO CO VO o fo On -H -H o\ VO H Pi H oo U VO o o o in o -H -H VO o VO ro H rn ro o -H -H o VO ^ OS t30 00 3 OS 00 fa S o 13 r—l II H CO <=^ c H o 0 .5 1 S CO

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51 00 U CQ 00 A o O 00 ON o o O o o r<\ o o O o o d d 00 00 n o o o o ON o o d d d d d On O ^ 00 00 en 00 o o VO (N On oq IT) NO 00 r~ (N ON 00 O NO m 00 0\ rON CN (N m * VO O o f— 1 o o * H H H 00 o c !>0 S s •> ^ 1 H — (SI . 15 p

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52 1 00 U A 00 o — ' >o O 00 ^ — > o o o o o o o O d d d d 00 o On 00 O d d oo' d Oh 00 o 00 ON On 00 On On r~00 ON 00 ON o ON ro d d d d NO rNO o NO o o O o o m o m o d d d d d d oo 00 o m IT) r~in t~o ITl o 5^ o u o u * H H H 00 O c oo C 1-1 C 00 e •> f-' 1 « P

PAGE 70

53 Figure 2-4. Least squares means of body weight changes of Holstein cows during the vi { ' . Prepartumand early postpartum periods (-3 wk through 10 wk). Arrow indicates calving. Weeks Figure 2-5. Least squares means of body condition score changes of Holstein cows during theprepartum and early postpartum periods (-3 wk through 10 wk). Arrow indicates calving.

PAGE 71

54 and at wk 8 for cows in TRT II and TRT I, respectively. Cows in TRT II maintained significantly greater BW throughout the lactation period (Figure 2-4). Trends for BCS of cows in the two treatment groups paralleled that described I for BW (Figure 2-5). The BCS tended to differ during the lactation period (P<0.146). No differences were detected due to CMO or the two-factor interaction TRT*CMO (Table 2-4). Cows in both treatment groups lost BCS beginning at calving (Figure 2-5). Cows in TRT I had greater decline at wk 2 than cows in TRT II. BCS loss from wk 0 to wk 2 was 6.8% and 3.9 % for TRT I and TRT II, respectively. Beginning wk 6 both I groups maintained their BCS; however, they had lost 8.0 % and 7.5 % of their BCS at that time, respectively compared to wk 0. However, BCS of cows in TRT II at wk 10 increased slightly and BCS of cows in TRT II was significantly greater at wk 10 (Figure 2-5). Milk and 3.5% FCM Yields Least square means for MY for period of time that bST was injected (0-60 d) and for overall lactation (0-100 d) periods are in Table 2-5. Significant differences were detected due to treatment during the bST injection period (0 to 60 d). Injected cows had significantly higher mean MY (6.2%) during the first 60 d in milk than control cows (P<0.082, TRT 1=35.9 vs 11=38.3 kg/d). No effects was detected due to the two-factor interaction TRT*CMO. A significant quartic polynominal effect of day was detected (Table 2-7). However, no difference was detected due to treatment for MY during the first 100 d of lactation (Table 2-7).

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Table 2-5. Least Squares Means and SE of Milk Yield, 3.5 %FCM Yield, SCC, and Percentage of Protein, Fat and MUN in Milk of Holstein Cows During Early Lactation. Treatments' Measurements I n Milk Yield (kg /df'' 37.2 ± 0.22 39.7 ± 0.19 3.5% FCM (kg/df ' 37.7 ± 0.27 40.2 ± 0.24 Milk Yield (kg/d)^-" 35.9 ± 0.09 38.3 ± 0.07 Milk Yield (kg/d)" 37.4 ± 0.07 39.07 ± 0.06 SCC 602 ± 33.2 453 ± 29.2 % Protein ^ 3.01 ± 0.01 3.01 ± 0.01 % Fat ^ 3.60 ± 0.02 3.59 ± 0.02 Total MUN 11.6 ± 0.10 11.7 ± 0.09 'Treatment I=No bST, Treatment U=10.2 mg bST/d. ^1-8 wk postpartum. ^0-60 d postpartum. " 0-100 d postpartum. ^SCC=Somatic Cell Count xlOOO. ^MUN=Milk Urea Nitrogen. T<0.1 ^P<0.09

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U A >/~) ON r~oo o o o On vo o o o O O o o o O o O o o o C> vo 00 oo >n q 00 (N (N v6 vd r00 o On 00 NO 00 On 00 NO o w-i o lO OS O 00 q On T3 PL, On ON On NO o o o o 0\ (S 0\ o o o o O m o o o o O O d d d d d vo m m ON m o o NO d NO NO in n (N NO rNO On ON O NO ^ VO ^ r-; NO fS CS in «n On Tj{si NO ^ OS ^<^ NO ^ ^ — <^ ^ ^ m o o CO O U o * o * H 00 o c H 1=: 00 e > — n

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57 O O O O A Pu, 00 A Pi (1. 00 O On o o o o o o o O 00 o o o o o o (N o o o o o o O d d d d d d d d ON O On O On On OO NO o ON 00 NO ro CM On ON o o NO >n On NO o o o o o o 00 00 o o o o o o o o ro o o o o o o d d d d d d d d d ^ o 00 O (N rN O ON (N On On iri OO d d lO o ON NO ir> (N On in ON 1— < fN •-^ o\ o r— < NO OO ro d d cn! r-i 00 00 m (N NO (N (N O CO .— On ON 4> 1 O 00 O o * H o U Q * Q Q * * a Q Q Q * * * a Q Q Q Q * * * CO Q Q Q 1^ g H 00 o c J3 I Q
PAGE 75

58 Least squares analyses of variance for weekly milk and 3.5% FCM yields during the first 8 wk are in Table 2-6. Differences were detected due to treatment for both MY and 3.5% FCM yields (P<0.994 and P<0.0095, respectively). No effects were I detected due to CMO or the two-factor interaction CMO*TRT for either measure of milk production. A significant cubic polynominal effect of wk was detected for each measure of milk production (P<0.0001 and P<0.102, respectively; Table 2-6). Results indicated that cows injected with 10.2 mg bST/d (TRT II) had greater weekly mean milk and 3.5% FCM yields (39.7 and 40.2 kg/d, respectively) than I uninjected cows which were less (37.20 and 37.72 kg/d, respectively; Table 2-5, Figures 2-6 and 2-7). Increases in yields for bST injected over control were 6.6 % for both measures. Least squares means for percent protein, percent fat and MUN for first 8 wk are in Table 2-5. During the first 8 wk no differences due to treatment were detected for percent protein, total fat, percent fat, and MUN. Mean SCC tended to be higher for cows in TRT I (602 vs 453x 1 0^). Quartic regression curves were calculated for the two measures of milk yield to describe the time trends for the individual treatments over 100 d lactation (Figure 2-8). Test of heterogeneity detected evidence that the curves were not parallel (P<0.01). TRT II had higher daily milk yield starting at the beginning of the lactation and it continued greater throughout the 100 d measurement period than that of cows in TRT I. However, discontinuing injections of bST at 60 d resulted in a decrease in MY for cows in TRT II and MY for these cows were similar to the that of cows in TRT I during the last 30 d (Figure 2-8) of the 100 d period.

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45 43 41 „ 39 37 ^ 35 > ^ 33 j ^ 31 : 29 ; 27 25 1 ——nobST — — bST 1 1— r 1 1 1 13 4 5 6 7 8 Weeks Figure 2-6. Leas t squares means of weekly milk yields of Holstein cows during the first 8 wk of lactation. Figure 27. Least squares means of weekly 3 .5% FCM yields of Holstein cows during the first 8 wk of lactation.

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Figure 2-8.Quartic regressions depicting MY of Holstein cows during experiment.

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Hormones, Growth Factor and Metabolites . . .. Prepartum . . . , Plasma concentrations of hormones (ST and INS), growth factor (IGF-I) and I metabolites (glucose and NEFA) also were evaluated during the period from -3 wk before calving through 8 wk postpartum (Figures 2-9 through 2-13). No differences in the mean concentrations of IGF-I, NEFA or glucose were detected during the prepartum period due to treatment. However, differences were detected for mean plasma concentrations of ST (P<0.0024) and INS (P<0.078) during the same period (Tables 2-8 and 2-9). The mean concentrations of ST on -2 1 d before expected calving did not differ for the two groups (6.1 vs 6.4 ng/ml). One week before expected calving, which corresponded to the period following start of injections of bST, mean concentrations of ST were greater for cows in TRT II (12.7 ng/ml), whereas concentrations of ST for cows in TRT I were similar to -21 d (6.8 ng/ml; Figure 2-9). Across treatments, significant effects of CMO (P<0.0701) and WK (P<0.0008) were detected but not due to TRT*CMO interaction during prepartum period (Table 2-8). I Plasma concentrations of IGF-I during the prepartum period are in Table 2-8. Mean concentrations of IGF-I did not differ during the prepartum period. Significant effects of CMO (P<0.0029) and WK (P<0.0245) were detected, but not for the TRT*CMO interaction (Table 2-8). Cows in control (TRT I) and bST injected (TRT II) groups had similar concentrations of IGF-I at 3 wk before parturition (170.2 and 171.1 I ng/ml, respectively). Overall concentrations of IGF-I decreased as parturition

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00 1 abl rep H Oh o A CL, 00 as OS o CO (N 00 o o o o o o o o OS O in ro O o m CO oo o 00 iri CN CO fS o od rn OS OS ro o t-H 00 vo m ^ — o o o oo od H 00 o O 00 pi! H O o * H H o * CO oo fo CO ^ o o U II O 60

PAGE 80

0) to o o 3 O < w A 00 PL, CO o o 00 o o o o o o O o o o ^ lO VO <:} >0 (N 00 Tl; O ^ ^ < O m o o <^ o 1— i CN r-< 00 ^ o ° C e t-j C Ml u n B> ^ {!, 03 o E g E > ^ u II II O II s ^ i P o ^ — rj u

PAGE 81

64 approached (wk -1) in both groups (156.4 and 160.9 ng/ml); this corresponded to 8.1 and 5.9% decreases, respectively (Figure 2-10). During the prepartum period, mean concentration of INS in plasma was significantly higher in bST injected cows (TRT II: P<0.0758; Table 2-8). Significant effect of WK (P<0.0546) was detected, but not due to CMO or the two-factor interaction TRT*CMO (Table 2-8). Mean plasma concentrations of INS tended to I increase slightly (10%) from -3 wk to -1 wk in control group, whereas the increase was significantly greater for cows in TRT II on wk -1 (26%; Figure 2-11). Mean concentrations of glucose are in Table 2-12. Mean concentrations of glucose in plasma did not differ due to TRT (66.3 vs 66.4 mg/dl). No effects were detected due to CMO or the two-factor interaction TRT* CMO. A significant effect of WK was detected (P<0.0648; Table 2-9). On wk -3, concentrations of glucose were 66.4 and 64.8 mg/dl for TRT I and II, respectively. However, on wk -1, glucose concentrations had increased slightly to 68.0 mg/dl for cows in bST injected group, 1 whereas they were unchanged for cows in TRT I (66.1 mg/dl; Figure 2-12). Although the slight differences in concentrations between groups on wk -1 were not significant, I increase for bST injected group from wk -3 to wk -1 was significant (P<0.05; Figure 212). " " ' ' During the prepartum period, mean concentrations of NEFA in plasma did not differ between treatments (Tables 2-9 and 2-12). Significant effects were detected due to CMO (P<0.0001) and the two-factor interaction TRT*CMO,(P<0.0051) but not due to WK (Table 2-9). Plasma concentrations of NEFA tended to increase slightly (9.5%)

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65 a 00 A 00 A PL, 00 A Oh 00 o o 00 o o oo VO m O VO (N o o o o d d d d d OS m IT) rs (N OS o 00 00 (N r-; rvq >o OS OS d OS OS 00 OS en OS so o T— < OS OS *— 1 (N o so 00 ON 00 O 00 o (S o o d d d d d 00 fN d CS SO OS o O d d d o\ o d * H Pi H O OS d in o o o o m 00 o o oo oo o cn d d d d d o OS OS so 00 00 00 d d d 00 o so od so so so 00 <400 o ,o H c i

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66 o o 3 O A Pi 00 00 T3 00 CN rf (N 00 o o 00 >n 00 00 (N o o O o o 00 00 q q (N 00 o CN r00 o r~oo o o 1—* 00 o en o 00 00 o o o vo o o o o o o c> o o o o 00 H O u * H H o ^ _ U-1 IT) m On q o6 CN 00 (S ON IT) m On o 00 o O m 00 2 (U o B H i-l A *S ^' -.i

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c £ (L> (/> )-< (L> T3 O O s 00 o s 3 a -*-» (A O o -4-» I £ 3 00 CS o d d d d -H -H -H -H 14.64 0.53 129.2 64.3 399.7 00 O ON d d od d 1— 1 -H -H -H -H in o Ov *n VO (N (N rO m 1 — . t — in d d >n d 00 -H -H -H -H ro ro in o\ d vd VO d oo o m o in d d >n d 00 -H -H -H -H -H r-> 00 d CO VO *— < vd VO CO m on £ 5 £ c O £^ w o o 3 O W

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Figure 2-10. Least squares means of concetrations of IGF-I in plasma during the transition period and through 8 weeks of lactation. Arrow indicates calving.

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Figure 2-11. Least squares means of concentrations of Insulin in plasma during the transition period and through 8 weeks of lactation. Arrow indicates calving. Figure 2-12. Least squares means of concentrations of Glucose in plasma during the transition period and through 8 weeks of lactation. Arrow indicates calving.

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Figure 2-13. Least Squares Means of Concentrations of NEFA in Plasma During the transition period and through 8 weeks of lactation. Arrow indicates calving.

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71 from wk -3 to wk -1 for cows in TRT I (241.1 to 285.4 [lEq/L), whereas concentrations tended to decrease for cows in TRT II (-3.5%, 285.4 to 275.6 |iEq/L; Figure 2-13). Postpartum Another objective of the current study was to evaluate the metabolic response of cows injected with bST from parturition throughout the early postpartum transition period (3 wk) and through wk 8. A second series of analyses was performed to evaluate this time period. During this overall postpartum period, no differences were detected in mean concentrations of INS or glucose due to treatment. Mean concentrations of IGF-I tended to differ postpartum due to treatment (P<0.1083). Significant differences were detected for mean plasma concentrations of ST (P<0.0001) and NEFA (P<0.0085) during the same time period due to treatment (Tables 2-10 and 2-1 1). Least squares mean concentrations of ST differed significantly for TRT groups (P<0.0001; Table 2-10). Cows treated with bST postpartum had greater concentrations of ST (14.6 ng/mL; Table 2-12) than control cows (9.3 ng/mL) and concentrations remained greater throughout this early postpartum period. After calving, mean concentrations of ST in confrol group (TRT I) increased and concentrations remained I greater during this period (Figure 2-9). No effects due to CMO, WK or two-factor interaction TRT*CMO on ST concentrations were detected (Table 2-10). For IGF-I, no significant effects of CMO (P<0.0461) or the two-factor interaction (TRT*CMO; P<0.06020) were detected (Table 2-10). A significant linear effect of WK on IGF-I concentrations was observed (P<0.0230). Overall, mean plasma I concentrations of IGF-I decreased following parturition for cows in both TRT I (-35%)

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and II (-31%) and remained low at wk 4. Differences between the treatments were not significant at wk 1 or wk 4. Mean plasma concentrations of IGF-I increased at wk 8 in TRT I (15 %) and II (32 %) and differed between groups (P<0.09; Figure 2-10). During the postpartum period, mean concentrations of INS in plasma did not differ between treatments (Table 2-12). No significant effects of WK, CMO or the two-factor interacfion (TRT*CMO) were detected for concentrations of INS (Table 2-10). Plasma concentrafions of INS decreased significantly after parturifion in both groups and declined further at wk 4. However, at wk 8, concentrations of INS in plasma increased significantly for cows in both TRT I (P<0.0758) and II (P<0.02; Figure 2-11). ' For glucose, no significant effects of TRT, wk, CMO or the two-factor interaction (TRT*CMO) were detected during the 8 wk postpartum period (Table 2-11). Cows in both treatment groups showed stable or slightly decreased plasma concentrations of glucose after parturition and they remained lower as lactafion advanced (Figure 2-12). Least squares mean concentrations of NEFA during the first 8 wk postpartum for TRT groups differed significantly (P<0.0085). Significant effects were detected on NEFA concentrations due to WK (P<0.0001), CMO (P<0.0610) and the two-factor interacfion TRT*CMO (P<0.0074; Table 2-11). After calving, mean concentrations of NEFA increased for cows in both groups. Cows treated with bST tended to have higher mean concentrafions of NEFA (579.8 ^Eq/L) than control cows (470.0 [lEq/L).

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However, concentrations of NEFA had declined in both groups at wk 8 (TRT 1=249.2 vs TRT 11=222.2 ^Eq/L; Figure 2-13). Discussion A series of metaboHc adaptations normally occurs in cows during late pregnancy and the early lactation period. These adaptations are mainly characterized by increased hepatic gluconeogenesis to provide for increased use of glucose by the mammary gland during lactation, reduced glucose utilization in peripheral tissues, reduced peripheral utilization of acetate, and slightly increased mobilization of NEFA from adipose tissue. Increased availability of glucose to support milk synthesis results from an overall decrease in its use by other tissues. Increased peripheral utilization of NEFA and P-hydroxybutyrate is associated with these changes (Bell, 1995). Reduced glucose utilization and low energy intake, due to the moderate to large decrease in DMI, results in greater concentrations of NEFA and ketones (Petterson et al., 1994). One of the major objectives of this experiment was to evaluate the changes in BW and BCS during ~ -3wk prepartum through 10 wk postpartum. Reports by Kertz et al. (1991) and Garcia (1998) indicated that cows not treated with bST had the greatest I loss in BCS and they did not start recovery of BCS until 8 wk of lactation. However, cows treated with bST prepartum and postpartum had less pronounced losses in BW and BCS losses than cows not treated (Garcia, 1998). In the current study, although prepartum BW and BCS did not differ between the treatment groups, cows in bST treated group maintained their BW and BCS better than cows in control groups following parturition. Recovery of BCS and gain in BW started at wk 6 for both groups

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and cows in TRT II maintained higher BW from wk 2 postpartum through wk 10 postpartum (Figures 2-4 and 2-5). * " ~' ^ ' Rapid and high rate of increase in DMI during early lactation is essential to provide energy and nutrients to support the rapid increase in milk yield. Cows treated with bST (5 and 14 mg/d) during the previous lactation, but not throughout the dry period, had greater DMI during the subsequent early lactation period (Lean et al., 1991). Simmons et al. (1994) concluded that DMI tended to increase about 3 kg/d more in bST freated cows (5 and 14 mg/d) after parturition. Garcia (1998) found that cows injected with -5.1 mg bST/d during both the prepartum and postpartum periods had greater DMI than uninjected controls, or than cows injected with -5.1 mg bST/d only during the prepartum period, or only during the postpartum period. Results of Gulay et al. (2000) also showed that greatest increases in DMI were by cows injected with 15.3 mg bST/d during both the prepartum and the postpartum periods. Even though no direct measure of DMI was made during the current experiment, changes in both BW and BCS following parturition suggested that cows treated with 10.2 mg bST/d better maintained their DMI during the transition period. During current study, greater DMI of cows in bST injected group (TRT II) likely was due to positive effects of bST treatment on DMI, as typically occurs in lactating cows injected with bST. Another objective of this study was to evaluate the effects of bST to determine if there was an effect on milk production due to bST. The galactopoietic response to exogenous injections of bST during lactation confirms that ST has an important role in I many adaptations that occur during the transition from the non-lactating to the

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75 lactating state. In the current study, a significant effect on milk production was detected due to treatment during the 60 d bST injection period. Increase in milk and 3.5% FCM yields were about 6.6% throughout the injection period. This also agreed with the I findings of Stanisiewski et al.(1991). They reported that slightly greater than a 6% increase in 3.5% FCM yields occurred when cows were injected with 5 or 14 mg bST/d from 14 d postpartum through 60 d postpartum. Garcia (1998) reported that after 2 wk of lactation cows that had been injected with low doses of bST (-5.1 mg bST/d) during the prepartum and postpartum periods (d -2 1 through ~d 60) had a greater and sustained increase in production during early lactation compared to cows in the other treatments (no bST, or injected either prepartum or postpartum with 5.1 mg bST/d). In addition to that, although not significant because of small numbers of cows in each of the four treatment groups evaluated, Gulay et al. (2000) found increased mean MY and trends in milk production when cows were injected with 10.2 or 15.3 mg bST/d beginning prepartum and continuing through -60 d postpartum. In another study, cows treated with bST (500 mg over 14 d period) starting 28 d prior to expected calving date through parturition produced 3.3 kg/d more milk than uninjected controls during the first 42 d of lactation (Putnam et al., 1998). Moallem et al. (2000) concluded that bST injected early in lactation increased MY and DMI of cows after treatment with bST was initiated. On the other hand, increase in DMI was not enough to support the increase in MY and injected cows faced an extensive period of NEB which resulted in BW and BCS loss. However, in that study cows received a full dose of bST (500 mg/14d) which resulted in a severe NEB imposed on the injected cows. When cows were injected with

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76 5 or 14 mg bST/d from 14 d postpartum through 60 DIM, they produced more FCM than controls (Stanisiewski et al.,1991). In addition, cows receiving the lower dose had higher pregnancy rate and higher conception rate than all other experimental cows and I they also maintained BCS as well as controls. Eppard et al. (1996) failed to increase MY when they injected Holstein and Jersey cows during the prepartum period with a fiill standard dose of bST. However, because cows also were used for milk fever I induction and plasma concentrations of ST were low for treated cows in this study. This may explain results obtained. Chalupa et al. (1985) reported a 4.7 kg/d increase in MY over control cows, and a 1 .05 point increase in fat percentage when cows were treated with 50 lU bST starting at 4 wk of lactation. In the same study, feed intake of injected cows also tended to increase (P<0. 1 1 ). In the current study, the treatment group that had the greater B W and BCS also had the greatest milk yield. Even though cows in TRT II had the highest milk production, their BW also was significantly greater (P<0.08; Table2-4). Therefore, it seems very likely that milk production was supported to greater extent by increased DMI than by more extensive tissue mobilization to provide the energy to support lactation because they also lost less body condition during this time period. Reduced DMI immediately before and after parturition can limit onset and rise in milk yield during early lactation. This limitation to milk production likely would be least for cows with greater DMI. The group with the greater DMI during the transition period and during the lactation would have greater quantities of energy and other nutrients needed to support maintenance of body tissues and milk production. As a

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77 result, change in BW and BCS for the cows treated with bST would be less and less lipid and protein mobilization likely would occur. The bST injected group had greater BCS and greater production, which supports the conclusion that they had maintained I higher level of DMI during the transition period, especially during the last week of pregnancy and first week of lactation. Injecting low doses of bST had positive effects on concentrations of various hormones and metabolites when injected during ~ -21 d prepartum through ~ 60 d postpartum periods (Garcia et al., 2000; Gulay et al., 2000). Plasma concentrations of hormones (ST and INS), growth factor (IGF-I) and NEFA also were altered during the time period that includes the defined transition period fi-om -3 wk before calving up to 8 wk postpartum in the current experiment. Bauman and Vernon (1993) reported that I plasma concentrafions of ST increased during late pregnancy, with greatest concentrations at calving and during the early postpartum period. In the current experiment, cows treated with bST had higher concentrations of ST than control cows and they remained high throughout the early postpartum period (Figure 2-9). These results agreed with previous findings (Bauman and Vernon, 1993). Lucy et al. (1993) reported that cows injected with bST during the postpartum period had increased concentrations of ST in plasma. Furthermore, cows injected prepartum with 25 mg bST/d had greater plasma concentrations of ST than untreated cows (Bachman et al., 1992). Prepartum injections of 5 and 14 mg bST/d increased plasma concentrations of ST which remained elevated (6.5 vs 22.7 ng/ml) during the prepartum period, whereas !

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78 uninjected cows on the same study had low concentrations (1 .6 ng/ml) during the same time period (Simmons et al., 1994). Somatotropin has a major role in regulation of IGF-I secretion and I concentrations in plasma (Bauman, 1992). Synthesis, release and circulating concentrations of IGF-I are positively correlated with secretion of ST. Injections of bST resulted in increased concentrations of IGF-I during both early and late lactation (Lucy et al., 1993; Staples and Head, 1988). Cows injected with 10.2 or 15.3 mg bST/d had greater concentrations of IGF-I than control cows that were not injected after I parturition (Gulay et al., 2000). In addition, the IGF-I response to bST, as measured by concentrations in plasma, was greater when cows were in positive energy balance (Bachman et al., 1992). The current study failed to detect an increase in concentrations of IGF-I during the prepartum period. However, cows in this study were sampled only once after bST treatments started (wk -1) and this might not be enough time to provoke or even to detect an increase in plasma concentrations of IGF-I, even if it had occurred. Additionally, the sampling time was closer to time of parturition at which time a decline in IGF-I concentration in blood normally is expected. In the cmrent study, plasma concentrations of IGF-I decreased around parturition (Figure 2-9) and after calving in both injected and uninjected groups of cows and this agreed with results of Brier et al. (1988) and McGuire (1992). Although concentrations of ST remained elevated during the last week of pregnancy, plasma concentrations of IGF-I decreased during the prepartum period (wk -3 to wk -1). These results are similar to that of Simmons et al. (1994). They reported

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79 that plasma concentrations of IGF-I did not remain high even though concentrations of bST in plasma were greater from d -21 to d -1 prepartum. Low circulating ; concentrations of IGF-I during this time was associated with low nutrient balances during early lactation (Vicini et al., 1991). Restriction of DMI in growing steers decreased the basal concentration of IGF-I in blood and terminated the positive response of IGF-I to exogenous bST treatment because response was uncoupled from bST due to reduced intake (Brier et al., 1988). Low concentrations of IGF-I in blood during early lactation are associated with low DMI during this period (Ronge et al., 1988). Although a decrease in IGF-I concentrations was observed for both treatment groups in the current study, mean concentrations of IGF-I for cows in TRT II were significantly greater at wk 8 (Figure 2-10). Dechne in plasma concentrations of IGF-I after parturition in both groups might be a response to decreased DMI experienced by the cows around the time of parturition. Thus, the increase in circulating concentrations of IGF-I in bST injected cows after calving might indicate better nutritional status since nutritional status has an important role in the circulating concentrations of IGF-I (Brier etal, 1986). During the prepartum period, mean concentrations of INS in plasma were significantly higher in bST injected cows (Table 2-8). However, Bachman et al. (1992) reported that plasma concentrations of INS decreased as cows approached calving. On the other hand, results of the current study agreed with those of Vicini et al. (1991). They reported increased concentrations of INS during late lactation and the dry period when bST also was injected. Insulin concentrations of cows injected with 10.2 or 15.3

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80 mg bST/d also were greater than in uninjected control cows during the prepartum period (Gulay et al., 2000). High concentrations of INS in blood were associated with positive EB of the cows that had greater DMI; a change that also promoted higher I concentrations of glucose in blood during the dry period. However, during the postpartum period, mean concentrations of INS in plasma declined in two groups of cows that were in negative EB (Vicini et al. 1991). The results of the current study agreed with findings that concentrations of INS declined around parturition (Garcia, 1998; Gulay, 1998; Malven et al., 1987b). Decrease in INS receptors and decrease in I concentrations of INS following parturition result in depression of lipogenesis (Mepham, 1987). Despite the reduced concentrations of INS, INS receptor numbers increase in mammary tissue at parturition. In addition, during late pregnancy, increased resistance to INS causes a decrease in response to INS in adipose tissue such that lipolysis and mobilization of NEFA were increased (Petterson et al., 1994). Overall, actions of ST on INS should result in greater availability of glucose during the dry period to support milk synthesis during the upcoming lactation. Although the current study failed to detect a significant difference in prepartum concentrations of glucose between treatments, the increase seen at wk -1 for cows in TRTII (Figure 2-12) was significant within the TRT group. Somatotropin may have had a posifive effect on plasma glucose concentrations during the Ume period evaluated which coincided with the time period when increased plasma concentrations of INS were seen prepartum. This may have been effected directly via hepatic cells by promoting increased gluconeogenesis or indirectly by antagonistic effects of ST on

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INS. Lactation is characterized by low concentrations of INS and a high ST:INS ratio. Somatotropin has a negative effect on abihty of INS to inhibit gluconeogenesis, and it also inhibits both INS uptake by the cell and the INS protease necessary for the action of INS (Sechen et al., 1990). Somatotropin also has been shown to decrease the ability of INS to suppress gluconeogenesis (Sechen et al., 1990). These changes would increase glucose production via gluconeogenesis and priority use of glucose for mammary tissues then could occur. In the current experiment, no significant effects of TRT on concentrations of glucose in plasma were detected during postpartum period and decreased plasma concentrations of glucose after parturition were observed for cows in both groups. However, MY of cows in TRT II averaged 2 kg/d more than for cows in TRT I. This suggests there was a higher rate of gluconeogenesis and/or higher DMI by the bST injected cows during early postpartum period. These changes would support increased MY by providing energy and precursors needed for milk synthesis. Mean concentration of NEFA for TRT groups did not differ significantly during the prepartum period and no significant increase in concentrations of NEFA in plasma I was observed for either injected or uninjected cows. However, during postpartum period a different pattern was observed. Although cows in both groups had greater mean concentrations of NEFA, cows treated with bST had greater mean concentrations in plasma after calving. However, concentrations declined in both groups of cows after calving and were slightly lower at wk 8 postpartum (Figure 2-13). As mentioned previously, lactation is characterized by low concentrations of INS and a high ST:INS

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82 ratio. Decrease in INS receptors and decrease in concentrations of INS following parturition result in depression of lipogenesis (Mepham, 1987). Despite the reduced concentrations of INS, INS receptor numbers increase in mammary tissue at parturition. In addition, during late pregnancy, there is increased resistance to INS and this causes a decreased effect of INS in adipose tissue resulting in increased lipolysis and NEFA mobilization (Petterson et al., 1994). Bell (1995) concluded that a combination of metabolic changes such as a decrease in de novo synthesis of TG, increased lipolysis, reesterification of fatty acids in the adipose tissue, and reduced intracellular reesterification of fatty acids arising from lipolysis may cause increased mobilization of NEFA. High concentrations of NEFA in plasma also are associated with actions of some metabolic hormones. For example, high concentrations of ST in t plasma during late pregnancy may reduce INS receptors on adipocytes, inhibit the action of a second messenger, or inhibit the INS protease required for action of INS. These changes will decrease rates of lipogenesis. Thus, ST can be considered a major regulator of metabolic adaptations during the transition period (McNamara, 1995). As indicated, ST is a primary homeorhetic regulator during pregnancy and lactation (Bauman and Vernon, 1993); it regulates partitioning of nutrients (carbohydrates, lipids, proteins, and minerals), and plays an important role in the coordination of various organs and tissues (Bauman, 1992). Nutritional status plays a major role in the regulation of lipid metabolism. When cows are in positive EB, synthesis and deposition of lipids in adipose tissue is reduced by ST which, in turn, increases nutrient utilization for milk production (Bauman and Vernon, 1993). Thus, ST reduces INS

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83 action, suppresses lipogenic enzyme activity, and reduces glucose uptake (Bauman and Vernon, 1993). All these changes in actions of INS during the transition period would support production of glucose from available precursors and conservation of glucose for mammary use by shifting peripheral tissues to utilization of other substrates available due to actions of ST and INS. When cows are in negative EB, ST stimulates lipolysis primarily by altering the sensitivity of adipose tissue to P-adrenergic agents (Bauman and Vernon, 1993). Therefore, for cows that are in negative EB and treated with bST, increased lipid mobilization would be a major source of energy needed to t support milk production (Sechen et al., 1 990). In the current study, cows injected with 1 0.2 mg bST/d maintained their BCS and BW better. This suggests that these cows were less dependent upon their body reserves to support lactation than were uninjected cows or those given a low dose of bST. Maintenance of BCS and BW would be one consequence of greater DMI. Therefore, less of the energy and precursors needed to support mammary function during lactation would arise from their body reserves. To support milk synthesis, metabolism of other tissues is stimulated to provide the necessary precursors and energy sources. In addition to increased glucose production in the liver, glucose usage by other lean body tissues decreases. Although glucose is used as the primary energy metabolite and as a precursor for synthesizing milk constituents in the mammary glands, energy needed by these other body tissues can be derived from products of lipolysis. Therefore, lipolysis also must be an important pathway used by cows to

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provide needed precursors in the early postpartum period, especially to supply the ,< V../ ----energy for milk production. . ^, As indicated, ST acting as a homeorhetic controller exerts important control over partitioning of nutrients and metabolism of substrates by various organs and tissues. Importantly, it appears to act on hepatic cells as well as on adipose tissue. Metabolism of proteins, minerals, lipids, carbohydrates and other nutrients is coordinated by ST (Bauman, 1992). Somatotropin exerts many differential effects on protein metabolism in both mammary and non-mammary tissues. I Conclusions Results of this study suggest that use of lower dose of bST during transition period caused no additional negative energy balance in injected cows compared to uninjected cows. Injection of bST resulted in better recovery of BCS during early lactation. Treated cows produced more milk and 3.5% FCM during the injection period. They also had higher concentrations of ST and INS prepartum and higher ST, IGF-I and NEFA postpartum. Thus, the changes in concentrations of metabolic hormones likely had a role in the positive effects on BW, BCS and MY that were seen for cows in TRT II. However, no carryover effects of bST were detected on MY and the increase in MY did not persist after bST injections were discontinued around 42 d postpartum. This could be due to the fact that prepartum injections of bST had no effect or a minimal effect on cell proliferation or amount of parenchymal tissue in the mammary glands. It most likely exerted its effect on the synthetic activity of these cells.

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85 No apparent calving problems or prepartum or postpartum health problems were observed for the cows across the treatments during the transition period. Hence, it appears that cows could be treated with low doses of bST to improve MY, even if they were to be injected with the full dose of bST (500 mg bST/14 d) later in the lactation (after 60 d). No negative treatment effect was observed at the end of lactation, as determined by culling rates (TRT 1=15 vs. TRT 11=12 cows). This suggested that no I detrimental effects occurred during the declining phase of the lactation. As a result, it appears that 10.2 mg bST/d injections could be used during the immediate postpartum period and probably during the prepartum period to improve efficiency of milk production and improve overall milk yields during early lactation. However, the effect of bST injections prepartum must be tested alone to fully evaluate any role it has upon subsequent health and production measures.

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CHAPTER 3 FEEDING MANAGEMENT OF HOLSTEIN COWS GIVEN SHORT (30 d) OR NORMAL (60 d) DRY PERIODS Introduction Dry Matter Intake and the Transition Period Although the maximum capacity to produce milk depends upon the animal's genetic makeup, age, physiological stage and environment, energy intake also is a primary limitation of milk production by animals, especially during early lactation (Illius and Jessop, 1996). A significant decrease in DMI occurs during late pregnancy and continues into early lactation. The DMI is influenced by numerous factors such as physical limitations of rumen capacity, fat mass of animal, and metabolic changes and signals occurring during the transition period (Ingvartsen and Andersen, 2000). It has been suggested that the decline in DMI during late pregnancy is caused by the pressure on the rumen by the growing uterus and contents, and the increasing accumulation of abdominal fat (Forbes, 1968). However, it is unlikely that the decrease in DMI is caused exclusively by rumen volume. Decreased rumen volume actually can be balanced, in part, by increased rate of passage of particles out of the rumen (Kaske and Groth, 1997). In addition, diets having greater amounts of concentrate caused a greater I decline in DMI during late pregnancy compared to a diet containing a low proportion of concentrates (Coppock et al., 1974). Furthermore, (Friggens et al. (1998) did not observe a rapid increase in DMI after calving as might be expected to occur if physical capacity of 86

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87 the rumen were the major factor determining DMI of ruminants. The increase in DMI follows the increase in MY (Friggens et al, 1998). As a result, it can be concluded that rather than physical constraints, metabolic and hormonal changes likely play a major role in regulating DMI. | Increased accumulation of lipids in body reserves during the prepartum period down regulates DMI (Broster and Broster, 1 984). Good body condition at calving is important for high producing dairy cows. On the other hand, overconditioning is not needed and should not occur during the dry period. Postcalving, cows with higher BCS lose more condition than cows with lower BCS, which also return to positive energy balance faster (Gamsworthy, 1988). However, a cow that is dried off with a BCS of 4.5 should be maintained at that level, because losing weight during the dry period increases subsequent incidences of metabolic diseases (Gerloff and Herdt, 1984). Ruegg and Milton (1995) concluded that cows with higher BCS at calving lost weight for a longer time after calving than cows with moderate BCS; average condition lost was 0.80 from d 20 prepartum through d 50 to 90 postpartum. Ingvartsen et al. (1997) concluded that there was a positive relationship between prepartum weight gain and the extent of postpartum mobilization of body tissues. They also argued that more than 40 kg BW gain during the dry period would depress feed intake postpartum and cause excessive mobilization of body tissue. However, association between adipose tissue and metabolic signals that determines appetite has yet to be fully described for cows. Copious milk secretion associated with glucose, protein and lipid drain results in mobilization of body fat and a rise in plasma concentrations of NEFA, glycerol and ketone bodies. In rats, a negative correlation was observed between feed intake and 1

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88 circulating concentrations of NEFA, glycerol and ketone bodies suggesting that these metabolites were potential signals for regulation of feed intake (Carpenter and Grossman, 1983). In dairy cows, 4 h infusion of lipids that provided 16.7 MJ of NE^ resulted in a slight decrease in DM! postinjection (Bareille and Faverdin, 1996). It has been speculated that oxidation of NEFA in the brain and the liver can decrease DMI. However, increased oxidation of NEFA in the ventrolateral hypothalamus for 14 d had no effect on food intake or BW of rats (Beverly and Martin, 1991). When Poxidation was inhibited by mercaptoacetate, which depresses long chain acyl CoA dehydrogenase activity (Singer and Ritter, 1993), or by methylpalmoxirate, which depresses the carnitine palmitoyl acyl transferase-I concentration in the mitochondria (Horn et al, 1999), feed intake was increased in rats having high fatty acid oxidation rates. On the other hand, Choi et al. (1997) observed a substantial decrease in DMI during the first 4 hr postinjection when they blocked fatty acid oxidation in dairy heifers by using 1 sodium mercaptoacetate. It has been postulated that glycerol suppresses feeding and that it influences intake through a central nervous system mechanism. Intracerebroventricular infusions of glycerol in rats decreased their feed intake (Davis et al., 1981). However, only nonphysiological levels of subcutaneous glycerol injection influenced intake in rats (Carpenter and Grossman, 1983). Moreover, portal vein infusion of glycerol did not affect feed intake in castrated male sheep (Forbes, 1995). As indicated previously, it has been suggested that blood metabolites and hormones alter feed intake. In rats, ovariectomy resulted in a temporary increase in feed intake for 3 to 4 wk and this resulted in an increase in BW (Tarttelin and Gorski, 1973).

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89 Injection of physiological doses of estrogen reversed these effects and a reduction of BW was observed as long as estrogen (Ej) treatment continued (Tarttelin and Gorski, 1973). Moreover, in cows, intravenous injections of E2 decreased both DMI and MY (Grummer et al., 1990). Progesterone (P4), on the other hand, reversed the effects of estrogen and stimulated feed intake (Wade, 1975). It has been shown that plasma concentrations of P4 decline rapidly the week of calving and it is almost undetectable the day of parturition. Concentrations of estrogen, on the other hand, rise by midgestation and peak prior to parturition (Chew et al., 1977). Intravenous infusion of physiological amounts of 17 Pestradiol, as seen during estrus and late pregnancy, caused a dose-dependent decrease in feed intake in castrated male sheep and in goats (Ingvartsen and Andersen, 2000). It was suggested that had direct effects in the paraventricular nucleus of the hypothalamus (Butera and Beikirch, 1989). Progesterone has not been reported to have a direct effect on feed intake (Ingvartsen and Andersen, 2000). However, P4 blocks the effects of estrogen on feed intake in cows (Muir et al., 1972). Corticotropin releasing factor (CRF) decreased DMI by central nervous system in cattle (Ruckebusch and Malbert, 1986). Krahn et al. (1986) showed a partial reversal of CRF-induced satiety after central administration of a CRF antagonist. Because hypophysectomy had no effect on feeding or on CRF actions on feeding, ACTH and Cortisol could not be the mediator of the decreased appetite (Levine et al., 1983). Furthermore, exogenous Cortisol did not influence intake in cattle (Head et al., 1976). Because concentrations of CRF are high around parturition, it may play a role in decreased intake of feed around calving (Tucker, 1985). However, it is unlikely that CRF

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90 has an important role in feed intake regulation during the early postpartum period because concentrations of CRF decline after calving. Somatostatin (SS) plays a role in regulating feed intake (higvartsen and Andersen, 2000). Manipulation of the SS center in the brain indicates there is a central depressing effect of SS on feed intake in sheep (Spencer and Fadlalla, 1989). Furthermore, immunization against SS in growing cattle enabled them to consume more DMI with a greater daily gain and improved feed conversion ratio (higvartsen and Andersen, 2000). Insulin also appears to play a role in long-term feed intake and weight regulation in ruminants (McCann et al., 1992). Acute peripheral infusion of INS that caused hypoglycemia also resulted in decreased food intake in monogastrics (Grossman, 1 986) and ruminants (Deetz and Wangsness, 1981). However, injections of glucose prevented INS induced hypophagia (Houpt, 1974). Long-term infusion of INS via hyperinsulinemiceuglycemic clamp techniques applied long-term (4 d) to ruminants generally depressed intake of feed, whereas short-term (4 hr) infusions under euglycemic conditions did not affect intake (Bareille and Faverdin, 1996). It is unlikely that INS plays a central role in DMI during early lactation because concentrations of INS generally decrease after calving t and are maintained at very low concentrations during lactation. Diet and Regulation of Feed Intake Although metabolic signals and hormones play major roles in regulating DMI, physical and chemical characteristics of dietary ingredients and their interactions also are important. Physical regulation of DMI occurs when feed intake is limited by the time required for chewing or by distension of the gastrointestinal tract. Dietary factors that increase eating time could result in decreased ruminating time, which would increase the

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91 filling effect of the diet. The reticulorumen (RR) generally is the site where distention often limits DMI of ruminants (Allen, 2000). Stretch receptors and mechanoreceptors are located in the muscle layer of the wall of the RR and are concentrated in the reticulum I and cranial rumen (Leek, 1986). Epithelial mechanoreceptors are excited by light mechanical and chemical stimuli, whereas tension receptors are stimulated by distension of the RR which provides information to the gastric centers of the medulla oblongata (Leek, 1986). These receptors are very sensitive to distension and can signal brain satiety centers to the end of the meal (Forbes, 1996). I Both volume and weight of digesta in the RR are important for triggering distension. This was demonstrated by an experiment with steers offered a low quality forage diet (Schettini et al., 1999). In their experiment, DMI was reduced 1 12 g for each kilogram of weight and 157 g for each liter of volume that was added to the RR as inert fill. There are multiple mechanisms that regulate DMI. Both the animal's energy I requirement and the filling effect of the diet regulate distension of the RR. However, physical regulation probably becomes a primary factor when the animal's energy requirement and filling effect of the diet increase. Added inert fill in the RR reduced DMI I only when cows were in negative or slightly positive energy balance. Addition of inert fill into the RR had no affect on DMI of cows when energy balance was greater than 3.8 Meal of NEL per day (Allen, 2000). When energy limited intake, NDF concentration was positively correlated with DMI. However, when fill limited intake, NDF was negatively correlated with DMI (Mertens, 1 994). The filling capacity of forages was inversely related to DMI (Balch and Campling, 1962). The NDF content of forages was more closely related to DMI than other chemical

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92 measures (Van Soest, 1965b). Waldo (1986) suggested that NDF content was the best chemical factor to estimate the filling effect of forages. However, NDF alone did not predict the filling effect because other factors such as particle length, particle fi-agility, and rate and extent of NDF digestion were important contributors (Mertens, 1994). Initial density of feeds was related, in part, to NDF content (Mertens, 1980). However, the filling effect of a diet also was dependent upon factors affecting rate of digestion and passage from the rumen. Thus, the size and density of digesta particles, RR motility, fimctional characteristics of reticulo-omasal orifice, and rate of emptying of the abomasum also determined the filling effect (Allen, 1996). Low ruminal pH can decrease fiber digestion and increase filling effect of the diet. Site of starch digestion also can have significant effects on DMI of cows. Increased ruminal starch degradation, as a percentage of DM, resulted in significant depression of daily DMI of cows. This may have been due to increased acid production in the RR. I Increased osmolality could be another reason for reduced DMI. Epithelial receptors in the reticulum and the cranial sac of the rumen are simulated by acids, alkali, and hypoand hyperosmofic solutions. Thus, direct stimulation of receptors by hyperosmotic solutions can stimulate satiety. In addition, increased ruminal starch degradation improved propionate production (Allen, 1996). There is substantial evidence that propionate affects satiety. Anil and Forbes (1988) reported that there were receptors in the liver that were sensitive to propionate. Depression of DMI by propionate was eliminated by splenic blockade, by hepafic vagotomy, and by total liver denervation. Fat can inhibit fiber digestion in the RR and it also can affect distension. High fat diet stimulated cholecystokinin (CCK) secretion,

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93 which suppressed DMI by inhibiting RR motihty and the rate of passage. Added fat also depressed DM digestibiUty via decreased fermentation. The crude protein (CP) of diet, on the other hand, often was related positively to DMI of cows. As dietary CP increased from 13 to 15% and NEL from 1.30 to 1.54 Mcal/kg of DM, there was a 30% increase in DMI before parturition (Emery, 1993). Although CP had a positive effect on DMI, this effect decreased exponentially as the percentage of CP in the diet increased (Allen, 2000). One unit increase in diet CP content equated to nearly a 0.9 kg/d increase in DMI at 12% CP in the diet (Roffler et al., 1986). In the same study, there was only a 0.04 kg/d increase in DMI at 18% CP in diet (Roffler et al., 1986). The positive effect of CP may be due to a reduction in propionate production as protein was substituted for starch in the diets fed. Increased dietary amino acids also can increase the rate of clearance of metabolic fuels from the blood, increasing hunger, and reducing the intermeal interval. Another mechanism that could be involved is from increased RDP effects on digestibility of feeds (Oldham, 1984). Presumably there is a reduction in distension as fiber and DM digestibility increase (Allen, 2000). However, there were no differences in DMI found between feeding RDP and RUP. ' bST and Transition Period Treatment of cows with bST during lactation was approved for use in US during I February 1994 by the Food and Drug Administration (FDA). Since then it has been used extensively. In addition, regulatory agencies in 34 countries have reached similar conclusions as the US agency with respect to food safety, and 24 countries approved use of bST, namely Algeria, Brazil, Bulgaria, Columbia, Costa Rica, Czech Republic, Honduras, Hungary, Jamaica, Kenya, Korea, Malaysia, Mexico, Namibia, Pakistan, Peru,

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94 Romania, Russia, Slovakia, South Africa, Turkey, United Arabic Emirates, Ukraine and Zimbabwe. . . After over a year of use of bST in the US, bST had been given to 335,000 cows in New York (45% of the state total), the major dairy state in the eastern US. USDA data showed that during the first 10 mo of 1994, fluid milk consumption increased by 1% compared to 1993 (pre-bST); milk prices received by farmers did not plummet but increased slightly. Farmers using bST generally increased their productivity and far fi-om large farmers using the technique exclusively and driving small farmers out of business as once was predicted it would, the size of herds that adopted use of bST closely resembled the distribution of herd sizes found in the US. About 55% of all sales of bST have been to farmers with 100 or fewer cows (Hartnell, 1995). In January 1998, of nearly 9 million dairy cows in the US, about 25% were in bST treated herds, and 300 additional dairy farmers a month were reported to be adopting use of bST. The average dairy farmer used the commercial bST product (POSILAC®) to supplement more than 50% of the herd at any one time, depending upon individual herd management practices and stage of the adoption. The approved time during the lactation cycle of cows to start use of bST is at about peak MY (~60±3 d) and its use continued throughout the remainder of the lactation (Chalupa and Gilligan, 1 989). Milk yield gradually increased over the first few days following bST treatment and reached maximum during the first week. Despite large increases in milk production, feed intake did not increase immediately following bST treatment. Early production responses therefore, were due mostly to partitioning of

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95 nutrients away from body tissues to mammary gland use to support increased milk synthesis. Although increased MY responses typically are found when cows are injected with bST beyond 60 d postpartum, no such increase in MY was observed when exogenous bST was injected during prepartum and early postpartum periods, which included the so-called transition period. Bines and Hart (1982) speculated that because of delayed increase in DMI, treatment with bST during the transition period might lead to acute animal health problems such as ketosis, fatty livers, wasting, and increased susceptibility to other diseases, or it could result in lower than expected MY responses. Studies by Eppard et al. (1996) failed to show an increase in MY when they injected Holstein and Jersey cows during the prepartum period with a full standard dose of bST (POSILAC®). Simpson et al. (1991) administered GRF prepartum to beef heifers to increase secretion of ST before parturition and during early lactation. Treated heifers lost more BW and had delayed ovarian activity, whereas no difference was observed in MY. In another trial, Holstein cows received 0, 5 or 14 mg bST/d during the last 46 d before parturition (Simmons et al., 1994). Cows treated with 14 mg bST/d had increased yields of solids-corrected milk (SCM) only during wk 1 . However, they found no differences in SCM among treatments. Except for the cows treated with 5 mg/d of bST during wk 10 of I lactation, EB was negative for all cows during the first 70 d of lactation. Bachman et al. (1992) evaluated whether a dose of 25 mg bST/ d administered prepartum affected postpartum MY of Holstein cows. After covariance adjustment for previous total lactation MY, 3.5% FCM yields of treated and control cows did not differ. They concluded that bST treatments during prepartum period did not have either a positive or negative effect

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on MY during the following lactation. None of the studies that used bST treatments during the transition period have reported acute animal health problems such as ketosis, fatty livers, wasting, and increased susceptibility to diseases in the treated cows. Stelwagen et al. (1991) administered 20 or 40 mg bST daily to Holstein heifers during the last trimester of first pregnancy. They found a significant increase in FCM production of the heifers injected with 20 mg bST/d but only after 90 d of lactation. Putnam et al. (1999) reported a significant effect of prepartum bST treatments on milk production during early lactation which appeared to increase as lactation progressed. The I exogenous bST injections (500 mg over 14 d period) were initiated 28 d prior to expected calving date and were continued until parturition. Cows treated with bST in their trial produced 3.3 kg/d more milk than uninjected controls during the first 42 d of lactation. However, cows in the bST treated group had significantly higher inifial BCS than the controls when they were assigned to the trial. This allowed treated cows to mobilize more body reserves than controls and to lose more BCS without a negative effect due to bST. This is a very important factor because bST use likely increases negative energy balance and greater loss of body weight which supports the greater milk production until the DMI increases. As reported for prepartum treatments, early postpartum injections of bST also gave inconsistent results, de Boer et al. (1991) injected cows with 20.6 mg bST/d starting 4-9 d postpartum. No significant differences were detected for MY of control vs. bST treated cows. Unfortunately, the cows assigned to bST had lower MY potential based on the rate and extent of decline in MY after cessation of bST injection. Thus, bST injections enhanced MY to levels similar to those of controls (de Boer et al., 1991).

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Moallem et al. (1996) studied the mechanisms by which Ca soaps of fatty acids and bST affected production and reproduction of high producing cows. They injected 500 mg of bST every 14 d from 10 to 150 DIM. Milk yield during the first 60 d did not differ between treatments. However, bST treatment significantly enhanced MY beyond 60 d and peak milk production increased for injected cows. On the other hand, the effect of treatment on BCS was severe. The BCS of injected cows decreased more and was considerably less for treated cows and postpartum conception rate was affected adversely compared to uninjected cows. Santos et al. (1999) compared effects of bST on the performance of early lactation cows fed diets differing in ruminally degradable starch. Holstein cows received biweekly injections of 500 mg bST for 90 d starting at 5 DIM. A positive MY response was observed during the first 45 d and for the total treatment period (90 d). Interestingly, MY response to bST was less from 7 tol3 wk than from 1 to 6 wk. Neither EB nor BCS were determined in this study. Authors concluded that the response to bST was less than usually observed for cows when bST injections began at peak MY. In another study, the same dose of bST was injected every 14 d fi-om 10 to 150 I DIM (Moallem et al., 2000). They concluded that bST injected early in lactation increased MY but at the expense of an extensive period of NEB and BW and BCS decrease despite an increase in DM1 after treatment with bST was initiated. Richard et al. (1985) reported a 6% increase in MY when cows were injected with 50 lU of bST starting 20 d postpartum; milk fat also was elevated by 25%. In the same trial, when cows were injected beginning 0 d, MY response was greater (12%) with no change in milk fat. Chalupa et al. (1985) treated cows with 50 lU bST starting at 4 wk of lactation using cows fed a diet that contained 0 or 1.2 % sodium bicarbonate. They

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98 reported that bST treatment increased MY by 4.7 kg over control cows and there also was a 1.05 point increase in milk fat percentage. Feed intake of injected cows tended to increase (17.6 kg vs. 16.1, P<0.11). In one of the largest trials, Stanisiewski et al. (1991) i injected 5 mg or 14 mg bST/d from 14 d postpartum through 60 DIM. Cows that received either 5 or 14 mg of bST/d produced more FCM than controls, but PCM of the two bST treated groups did not differ. However, cows receiving the lower dose (5 mg/d) had higher pregnancy rate and higher conception rate than all other experimental cows. Cows receiving 5 mg bST/d also maintained BCS as well as uninjected controls. Variable results within and among trials that evaluated use of bST either preor postpartum period may have been due, in part, to differences among the doses, diets the animals were fed, or due to differences in BW and BCS of animals. Usually, a high dose of bST increased the length of time cows were in NEB; loss of BW and BCS occurred even though an increase in DMI might have occurred. Thus, adequate BCS (3.5 3.75, I Nocek et al., 1983) for cows injected with bST preand/or postpartum is required because the cows require good management and adequate nutrition to produce and reproduce well. Treatment with bST during both prepartum and postpartum periods likely caused metabolic changes after parturition that were beneficial to health and performance of the cows (Gulay et al., 2000). In their study, preand postpartum injections of 15.3 mg bST/d I increased DMI of cows after parturition, and there was less decrease in BCS and BW. This allowed the cows to recover to satisfactory BW and BCS more rapidly during early lactation. Cows also produced numerically greater daily MY and 3.5% FCM. In addition, Garcia et al. (2000) reported that injections of 5.1 mg of bST/d before and after

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' I 99 parturition increased DMI, MY and efficiency of milk production during the early lactation period (60d). It also is likely that treatment with bST during both prepartum and postpartum periods caused metabolic changes after parturition that were beneficial to cows during the lactating phase. Evidence suggests that changes in circulating concentrations of hormones, growth factor and glucose were beneficial. Cows injected with 10.2 or 15.3 mg bST/d before and after parturition showed increased concentrations of ST, IGF-I and Tj in plasma (Gulay et al., 2000). Thus, the changes in concentrations of metabolic I hormones likely had a role in the positive effects on DMI, BCS, BW and MY of these cows. I Anionic Diet Milk fever is a common metabolic disorder in dairy cattle that generally affects older, higher producing cows. The majority of milk fever cases occur within 48 to 72 hr after calving, although some may occur later in lactation. It was estimated that 5 to 10% of cows are affected by this disease with some herds having a prevalence as high as 60% (Eppard et al., 1996). On the other hand, the incidence rate of milk fever among lactating Friesian-type cows is less than 7% (Erb and Crohn, 1988). The onset of milk production decreases the cow's blood Ca levels and she is unable to rapidly or completely replace this Ca. The body has reduced capacity to mobilize reserves of Ca in bone and therefore is dependent upon the ability to absorb Ca from the gastrointestinal tract. As a result, hypocalcemia occurs and decreases the cow's muscle contractions and rumen motility (Goffet al., 1997).

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100 The key to prevention of milk fever is management of the close-up dry cow group. Recommendations for the prevention of milk fever traditionally have included the proper feeding of Ca and P, especially during the late lactation and dry periods. More recently, however, dietary acidity and alkalinity have been associated with controlling the incidence of milk fever. Increasing dietary K from 1.15% to 2.1% increased the incidence of milk fever from 10.5 to 50% in Jersey cows. Potassium concentration in forage plays an important role in incidence of milk fever (Goff et al., 1997). Addition of strong cations to prepartum diets causes a metabolic alkalosis and this suggests that bone resorption of Ca is inhibited in cows fed high K or Na diets because feeding these diets results in increased blood and urine pH (Goff et al., 1997; Horst et al., 1984). Mild metabolic acidosis caused by feeding anionic diets increases the absorption of Ca from the intestinal tract (Goff et al., 1997). Research into the dietary cation-anion difference (DCAD) of pre-calving dairy I rations has been extensive during recent years. A major conclusion has been that acidifying the diet can allow feeding of high Ca diets without causing hypocalcaemia (Beede et al., 1991). Calculation of the DCAD is based on the levels of cations (K* and Na^) and anions (Cf and ) in the diet DM (Horst et al., 1984). The most commonly used formula to calculate DCAD is the sum of the positively-charged ions (K* and Na^) I minus the sum of the negatively-charged ions (CI" and S^") in the diet, as shown in the following formula: , ' * ^ . • ' i . . * i. mEq (K" + Na ) (CI' + S^t ' /' ' ' -J lOOgmDM

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101 Positive DC AD diets can have a systemic alkalinizing effect on the animal. High levels of K and Na in the ration cause the blood to be slightly alkaline and this reduces the effectiveness of parathyroid hormone (PTH). Diets with a negative DCAD, on the 1 other hand, are acidogenic. Acidogenic diets fed to dry cows have significantly decreased the incidence of milk fever (Block, 1984; Horst et al., 1984). For dry cows, lowering the DCAD level to -10 to -15 mEq/100 g DM results in an inflow of negatively charged ions (chloride and sulfate). Initially the cow attempts to maintain electrical neutrality at the expense of acid-base balance. Hydrogen ions are generated to neutralize these negative 1 ions, resulting in a mild metabolic acidosis. With chronic subclinical metabolic acidosis, bone is mobilized because it is a reserve for carbonate ion, which serves to buffer the pH. In the process of releasing CO3, Ca (and P) are mobilized from the bone and also Ca absorption from the GIT is enhanced. Chronic subclinical metabolic acidosis also increases urinary excretion of Ca, and as a result Ca retention decreases, which causes formation of l,25(OH)2D3 and release of PTH to further stimulate bone mobilization. The mechanisms used to increase blood Ca are therefore in an activated state at time of calving, and the end result is a higher level of Ca in blood (Gaynor et al., 1989). Beede et al. (1991) recommended that cows should not be on the anionic diet for more than 4 wk before calving and the DCAD should be returned to the normal positive postpartum levels after calving. With variations in forage and concentrate intakes, urine pH monitoring has become an essential tool to determine if the laboratory analyses and calculated DCAD of the ration, in fact, is correct. Alkaline diets produce urine pH of 8.0-8.5, whereas acidic

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102 rations cause this to fall to 6.0 or even less. If the pH falls to 5.5 and below, there is the danger of metabolic acidosis (Goff, 1999). hi order to formulate a diet with -10 to -15 mEq/100 g DM, it is essential to analyze all feeds for macromineral content (Ca, P, Mg, Na, K, CI, and S). Feed ingredients with a low DCAD, especially forages, should be selected. If the basal DCAD is greater than +20 mEq/100 g DM, large amounts of anionic salts must be incorporated into diet and this can result in reduced feed intake problems because the ration is less palatable (Horst et al., 1994). Adding appropriate anionic salts (magnesium sulfate, ammonium chloride, ammonium sulfate, calcium chloride, calcium sulfate) is very important (Moore et al., 2000). Magnesium sulfate is recommended as the first addition because it appears to be the most palatable, and because it can be used to meet the cow's requirement for magnesium. Doses between approximately 1,900-3,400 mEq should be added from salts, because there is a risk of acute acidosis when doses exceeds 3,500 mEq (Block, 1984). Finally, chloride sources (ammonium, calcium, or magnesium chloride) can be added to bring the DCAD to -10 to -15 mEq/100 g DM. Formulation for phosphorus intake should be considered and done carefully and also should ensure that requirements for other nutrient are met (energy, protein, vitamins, and minerals) (Oetzel, 1993). I Advantages of Short Dry Period on Feed Intake The surface of the rumen mucosa is characterized by ruminal papillae which can be defined as organs of absorption (Van Soest, 1982). Their distribution, size and number are closely related to feeding habits, forage availability and digestibility. The typical features of rumen papillae are genetically fixed but may vary considerably under different

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103 feeding conditions. This can result in acute and usually temporary or seasonal adaptations (Van Soest, 1982). For example, increasing proportions of butyric and propionic acids increases the ruminal blood flow which stimulates mitosis in the mucosa resulting in i vascular budding and epithelial cell proliferation. Thus, there are differences in number and size of papillae within the rumen. Lower energy diets fed during the early dry period may reduce the absorptive area of rumen as much as 50% during the first 7 wk of the dry period. Changes in the numbers of ruminal papillae occur in response to nutritional changes. Complete adaptation of ruminal papillae requires a period of 2 to 3 wk (Dirksen et al., 1985; Goff and Horst, 1997). Microorganisms in the rumen depend upon the animal to provide the physiological conditions necessary for their existence. In turn, these microorganisms are essential for digestion and fermentation of large amounts of fibrous feeds which the ruminant consumes, but otherwise cannot utilize efficiently. Thus, by providing a suitable and consistent environment for these microorganisms, the ruminant is able to utilize the end-products of fermentation to meet it's own nutritional needs. Comparison of rumen microorganisms shows that there is a high level of variation. The large diversity in the types of microbes found in the rumen reflects, to some extent, the diet the animal consumes (Van Soest, 1982). Growth of microorganisms and efficient fermentation of feed by microorganisms depends upon a constant and suitable environment. Changes in feed and feed composition, as well as rumen pH, not only cause a shift in microorganisms in the rumen but also a decrease in the efficiency of the fermentation and absorption of end-products of the fermentation.

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104 Changing the diet of the animal provokes a period of transition in the rumen microbial population. The proportions of the different species in the rumen will shift to a new balance, one which best accommodates the dietary change and is referred to as adaptation of microbial population. For example, ingestion of a less energy dense ration upon drying off causes a shift in microbial population and the population of bacteria that are capable of converting lactate to acetate, propionate, or long-chain fatty acids declines. Adaptation of the lactate converting bacterial population is slow and may take several weeks to occur (Yokoyama and Johnson, 1988). The current standard dry period of 60 d essentially requires dairy managers to feed dry animals in different phases; most recently termed as far-off dry period (F0D>3 wk prepartum) and close-up dry period (CUD<3 wk prepartum). During these periods, diets fed to animals will vary because of metabolic differences of cows during these short time periods. The changes in diets fed as cows progress from the lactation diet to POD, from POD to CUD, and CUD to early lactation diet forces the rumen and its microbes to adapt three times during a relatively short time period when these changes in feed intake are occurring. It is likely that these changes may, in part, be a reason for reduced DM1 and will lead to further decrease in the DM1 that occurs during the later CUD period especially during the time close to parturition. This also may limit the increase in feed intake that should occur shortly after parturition. Early lactation is the time period when more rapid increase feed intake and more efficient fermentation /utilization of ingested feed are desired (Drackley, 1999). If the length of dry period can be decreased to -30 d, then it may be possible to develop a better feeding program so cows can be fed a diet formulated using the same constituents as in the lactation diet. This would require making 1 ..

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105 fewer changes in the diets that are fed and could adversely affect papillae development and prevent complete adaptation of the microbial population of the rumen near to calving (Grummer, 1995). This will encourage maintenance of the desired rumen population, better rumen development, and greater peripartum metabolism of Ca. Thus, it is possible that cows will increase feed intake faster, have better efficiency of fermentation and absorption of end products of fermentation, and better resistance to metabolic disorders during early lactation. The major objectives of this research were to evaluate dry period length, the types of prepartum transition diets (anionic or cationic), and supplemental injections of bST during the transition period to improve DM1, BCS, milk production and cow health during lactation. Materials and Methods Experimental Animals Eighty seven multiparous Holstein cows were utilized in this experiment. Cows were selected randomly from the Dairy Research Unit (DRU) herd of the University of Florida approximately 8-9 wk before expected calving dates. Cows were assigned to one of two treatment groups; 60 or 30 d dry period length. Cows with longer dry periods were dried off and moved to the dry herd of the DRU and fed the herd FOD diet, whereas cows with shorter dry period remained in the milking herd. Approximately -30 d before expected calving day, cows in the shorter dry period groups were dried off Cows in both groups were housed and managed in a free-stall bam and trained to use electronic feed gates (American Calan, Inc., Northwood, NH). They were assigned to the individual gates randomly, fed cationic diet (Table 3-1) then trained to use gates for one week before

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106 intake measurements were recorded. Three weeks before expected calving, cows were assigned to either an anionic CUD diet or remained on the cationic CUD diet (Table 3-1). Cows assigned to trial completed the experiment and were removed from trial over a 300 d period from September 2000 through June, 2001 . The body weight (BW) and BCS of the cows at the time they were assigned ranged from 575 to 870 kg and 3.00 to 4.75, respectively. When cows were assigned to treatments, no differences were detected among treatment groups for mean BW (-650 kg) and BCS (-3.25). Experimental Design Cows were assigned randomly to a 3 x 2 x 2 factorial arrangement of treatments. Treatment group I (27 cows) was a 60 d dry control group that received no estradiol cypionate treatment (ECP; Pharmacia & Upjohn, Kalamazoo, MI), cows in groups II (28 cows), and III (29 cows) had 30 d dry period and received IM injections of ECP or cotton seed oil (no ECP), respectively at the time they were dried off Each of these three treatment groups were further divided into two groups; one-half of each was injected with bST (0.4 mL bST; POSILAC®)/ biweekly) and the other half not injected. This quantity of POSILAC® provided approximately 10.2 mg bST/d, and injections were continued up to 60 d postpartum. After 60 d, all cows on experiment received the full dose of POSILAC® (500 mg) biweekly. One half of each of the three main groups were fed either anionic or cationic diet during last 3 wk prepartum. Urine pH of the cows was measured routinely (3xwk) during the prepartum period. One cow from 60 d dry period bST injected and fed cationic diet prepartum, one cow from 30 d dry no ECP, no bST injected and fed anionic diet prepartum, and one cow from 30 d dry ECP injected, no bST and fed anionic diet prepartum were removed from the experiment due to

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paratuberculosis, severe mastitis and displacement of abomasum, respectively. A total of 84 cows completed the experiment. Actual days dry for 30 d dry no ECP, 30 d dry ECP and 60 d dry cows were 30.6, 28.9 and 61.1 d, respectively. I The 3x2x2 arrangement of treatments resulted in twelve treatment groups. The experimental arrangement of treatments (T), dry periods, diets and cow numbers in groups follows: Treatment I Treatment II Treatment III Treatment IV Treatment V Treatment VI Treatment VII Treatment VIII Treatment IX Treatment X Treatment XI Treatment XII bST Injections 7 cows, 60 d dry period, 0 bST, anionic diet prepartum. 7 cows, 60 d dry period, 0 bST, cationic diet prepartum. 7 cows, 60 d dry period, + bST, anionic diet prepartum. 6 cows, 60 d dry period, + bST, cationic diet prepartum. 7 cows, 30 d dry period, ECP, 0 bST, anionic diet prepartum. 7 cows, 30 d dry period, ECP, 0 bST, cationic diet prepartum. 8 cows, 30 d dry period, ECP, +bST, anionic diet prepartum. 7 cows, 30 d dry period, ECP, + bST, cationic diet prepartum. 7 cows, 30 d dry period. Cottonseed oil, 0 bST, anionic diet prepartum. 7 cows, 30 d dry period. Cottonseed oil, 0 bST, cationic diet prepartum 7 cows, 30 d dry period, Cottonseed oil, + bST, anionic diet prepartum. 7 cows, 30 d dry period, Cottonseed oil, + bST, cationic diet prepartum. A sterile, prolonged-release, injectable formulation of a recombinant DNA derived bovine somatotropin analogue (bST, POSILAC®, 500 mg in^ 1 .4 mL, Monsanto, St. Louis, MO) was used for injections. Injections of bST (10.2 mg bST/d) began approximately 4 wk (±3 d) before expected calving dates. Regardless of time of last injection before calving, first postpartum injections were within 24 h of calving and *

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109 thereafter injections were at 2 wk intervals. Last injection was at 42 to 44 d. All bST I injections were subcutaneous in the post-scapular region or on either side of the ischiorectal fossa, kijections were administered after blood collection, but were prior to a.m. feeding or milking. All cows received a full dose of bST beginning 56 to 60 d postpartum and injections then were continued biweekly during the remainder of their lactation, according to DRU herd practice. ECP Ejections All 30 d dry cows received a single injection of either 7.5 mL ECP (2 mg ECP/mL in cotton seed oil; 15 mg ECP; Pharmacia & Upjohn, Kalamazoo, MI) or cotton seed oil (CSO). The ECP and CSO were injected IM at the time they were dried off Drying off Procedure Cows in both 30 d and 60 d dry groups were completely milked out at the last milking prior to drying off A syringe containing Quartermaster® (penicillin and dihydrostreptomycin; Pharmacia & Upjohn, Kalamazoo, MI) was warmed and the plastic end of syringe inserted into teat canal and entire contents were slowly infused. The same procedure was applied to each quarter. The syringe was discarded after use. Teats then were dipped into Stronghold® (West Agro, Inc., Kansas City, MO) teat sealant to protect against any acute bacterial entry into the quarters via the teat canal. ! Feeding Program Cows were housed and managed in an open-sided free-stall bam equipped with Calan electronic feeding gates (American Calan Inc., Nortwood, NH) beginning approximately 4 week before expected calving day. Cows were assigned to the individual gates randomly, then each cow was fitted with a transponder key for her specific gate.

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Transponders were placed around the neck of cows so each could open the assigned gate and they were trained to use their gates for one wk before feed intake measurements were recorded. The bam, including feeding area, was covered with a metal roof for protection from rain and sun. Bams were equipped with fans and sprinklers which helped cool cows when ambient temperatures increased above 25 °C. Before calving, all cows had access to a dirt loafing lot where they could calve. Clean fresh water was provided in water troughs for ad libitum consumption. After calving, cows were moved to an adjacent freestall bam also equipped Calan gates, fans and sprinklers. They remained in this bam for I 28 d after which they were moved to the DRU milking herd and fed the same lactation TMR free choice but intake was not measured. ' During the time cows were in the free-stall bams, they were fed once daily (10:0012:00 h) and feed adjustments were made daily. Cows were fed ad libitum to allow 510% daily feed refusal. During the first week of the trial, when they were being trained to use Calan gates, they were fed a cationic CUD diet (=+20 mEq/lOOg DM) ration which was formulated for the average weight of the cows. Starting 3 wk before expected calving date, cows were switched to the anionic CUD (-10 to -15 mEq/lOOg DM) or they remained on the same cationic CUD (Table 3-1). Because feed was offered only one time each day, it was pushed back into the I Calan gate feed area several times daily to ensure that they had access to all their feed. After parturition, all cows were fed a total mixed ration (TMR) based on com silage, whole cotton seeds (WCS), and grain concentrate. This met the requirements of highproducing lactating cows (Table 3-2) (NRC 1988,1 989).

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Ill Body Condition Scores and Body Weights Body condition scores (1-5, thin to fat; Edmonson et al., 1989) and BW of cows were recorded during the experiment. Each cow was weighed and BCS was estimated at 1 60 and 30 d before expecting calving and weekly on the same day each week (Saturday) before a.m. feeding or milking (8:00 to 12:00 h) through 28 d postpartum. Thereafter, BCS and BW were estimated biweekly until they completed the experiment (~ 100 d postpartum). Blood Collection. Handling and Storage Blood samples were collected from the tail vein of all cows three times weekly before the a.m. feeding or milking (07:30-10:00 h). Cows were bled in the free-stall bam from the tail vein after elevating the tail without any other restraint. For serum collection Vacutainer® brand needles (2.54 cm, 20 gauge) and tubes containing no anticoagulant were used (10 x 100 mm blood collection tubes, Becton-Dickinson. Fairlawn, NJ). Serum samples were allowed to clot at room temperature for ~1 h after collection, then placed on ice and processed within 2 h. The order in which cows were sampled on a given day was random and differed among days. After sampling, cows were milked and then returned to the free-stall bam. All blood samples taken to harvest semm were centrifuged at 3000 RPM at 5°C for 30 min in RC-3B refiigerated centrifiige (6-place swinging basket, H.600A rotor, Sorvall Instmments) to separate the semm from the clot. Semm from each sample was aliquoted into 2 labeled 5 mL (75x12 mm) polystyrene tubes, capped, and frozen at 20°C until analyzed. The semm samples were used only for the analysis of Ca. ' "

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Table 3-2. Dry Matter Concentrations and Chemical Composition of TMR with Whole Cottonseeds (Lactation Diet) Fed to Holstein Cows During Experiment.' Ingredients TMR Com Silage 22.38 Alfalfa Hay 11.69 Cottonseed I iulls 5.07 Citrus Pulp 9.94 Hominy 16.08 Distillers Grains 10.42 Soybean Meal 8.38 Whole Cottonseed 10.71 Mineral Mix 5.33 Chemical Composition Percentage' DM 62.35 CP 17.18 Sol CP ' 32.66 ADF 22.58 NDF 34.66 EE'' 3.56 TDN 67.83 NEl (Mcal/kg) 1.56 'From NEDHIA Forage Laboratory, Ithaca, NY, analyses of components. ^ DM basis' Percentage of the CP. Ether extract.

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113 Determination of Calcium in Serum Samples Calcium standard solution (1000 ppm, Fisher scientific) was diluted 10-fold in a volumetric flask to prepare 100 ppm Ca stock solution. From the stock Ca, 5 and 15 ppm standards were prepared (Table 3-3). To prepare 5% Lanthanum (La), 1 17.3 g Lanthanum Oxide (LajOj) was added to a 2 liter pyrex beaker and dissolved in 500 mL concentrated HCl in a fume hood. After the solution released O2 and Clj and all La was in solution, it was transferred to a 2 L volumetric flask and brought to 2 L with deionized water to prepare a 5% La solution. To prepare a 50% TCA solution 500 g of trichloracetic I • Table 3-3. Standards for calcium determination flame Atomic Absorption Spectrophotometer. Ingredient Standard, ppm 0 5 15 1 00 ppm Ca stock, mL 0 5 15 50% TCA, mL 18 18 ' 18 5% La, mL 18 18 I? • TCA=Trichloracetic acid, La=Lanthanum acid (TCA) was dissolved in deionized water in a 1 L volumetric flask. Then, 200 mL 5% La and 200 mL 50% TCA were transferred to a 1 L volumetric flask and 600 mL deionized water were added to give a 1% La-10%TCA solution. Five hundred ^L of serum samples were pipetted into plastic tubes (Sarstedt Inc. Newton, NJ). To precipitate proteins, 4.5 mL of the 1% La10% TCA solution was added to each sample. Tubes were vortexed for 20 sec and then centrifuged at 3000 RPM for 20

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114 min at 4°C (RC-3B H.600A rotor, refrigerated centrifuge, Sorvall Instruments). About 4 mL of filtrate were transferred into plastic tubes (Sarstedt Inc. Newton, NJ) and samples were analyzed for concentrations of Ca using a Flame Atomic Absorption Spectrophotometer (Perkin-Elmer Model 5000; Miles et al., 2001). Statistical Analyses Data from this experiment were analyzed in two sections. The first section included data collected for BW and BCS during the final 8 wk prepartum, and during the final -21 d prepartum for DMI. The second set of analyses included data collected during the first 28 d postpartum for DMI and during 14 wk postpartum for BW and BCS. Data were analyzed as a nested design by least squares analysis of variance procedures of SAS (1991). Proc Mixed procedure of SAS was used to estimate individual daily and/or weekly least squares means for specific variables and treatments (Littel et al.,2000). Statistical analyses were performed for BW, BCS and DMI. Time periods considered for data analyses were the overall prepartum period (-3 to 0 wk), days and weeks within this period, overall postpartum period (1 to 14 wk) for BW and BCS, and the time period 0-28 d postpartum for DMI. In addition, gross correlations were estimated Models included the main effect of bST treatment (bST), effect of dry period length (DRY), effect of prepartum diet (DIET), season (SEA; 1= cows with dry periods during hot months {September, October, March, April, and May}, n= cows with dry periods during cool months {November, December, January, and Febaruary}), interactions among the treatments and SEA, cow(bST*DRY*DIET*SEA), and weeks or days to the highest order significant for overall prepartum and postpartum periods. '

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115 Results The recorded data were analyzed as two separate data sets. The data obtained during these periods included BW, BCS and DMI. The first set included data collected I during the final 8 wk prepartum for BW and BCS, and during the final -21 d prepartum for DMI. The second set of analyses included data collected during the first 28 d postpartum for DMI and 14 wk postpartum for BW and BCS. Changes in Body Weight and Body Condition Scores Prepartum period ' Least squares analyses of variance for BW and BCS during the overall prepartum period (wk -8 to wk -1) are in Table 3-4. No significant effects of bST, DRY, SEA or DIET were detected; however the interaction DIET*SEA was significant for BW (P<0.0369) and BCS (P<0.0588) and the interaction SEA*DRY was significant for BCS (P<0.0330) but not for BW. There was a significant quadratic effect of WK detected for both BW (P<0.0006) and BCS (P<0.0459). During the period from -8 wk prepartum to day of calving no differences were detected among the treatment groups for mean BW or BCS. Least squares means for BW and BCS during the prepartum period are in Table 3-5. The mean prepartum BW of cows in bST injected and untreated cows were 688 and 682 kg, respectively. Both groups of I cows gained BW fi-om -8 wk to parturition to -1 wk (Figure 3-1). At -8 wk, the control group cows weighed only 4 kg more than cows injected with bST. During the final week before calving increases in BW compared to that at -8 wk were 9 and 10% for control and bST injected groups of cows, respectively. The mean prepartum BCS for the same treatment groups also followed the same pattern (Table 3-5; Figure 3-2). No differences

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116 Table 3-4. Least Squares Analyses of Variance for BW and BCS of Holstein Cows During Prepartum Period (wk -8 to wk-1). ^ Source BW BCS df MS F P>F MS F P>F bST' 1 4956.25 0.21 0.6518 0.195 0.32 0.5762 SEA' 1 3088.76 0.13 0.7216 0.335 0.54 0.4642 1 DRY' 2 29681.59 1.23 0.2990 0.317 0.51 0.6003 DIET * 1 20186.75 0.84 0.3637 0.371 0.60 0.4414 bST*DIET 1 7241.45 0.30 0.5856 1.325 2.14 0.1482 bST*SEA 1 8160.06 0.34 0.5628 0.011 0.02 0.8926 bST*DRY 2 23891.54 0.99 0.3770 0.403 0.65 0.5245 SEA*DIET 1 109727.17 4.55 0.0369 2.291 3.71 0.0588 DRY*DIET 2 99003.51 4.11 0.0212 0.927 1.50 0.2310 DRY*SEA 2 31378.65 1.30 0.2795 2.231 3.61 0.0330 bST*DRY* SEA 2 7467.31 0.31 0.7347 0.219 0.36 0.7019 bST*DRY*DlET 2 9373.86 0.39 0.6795 0.107 0.17 0.8414 DRY*SEA*DIET 2 2348.66 0.10 0.9073 0.009 0.02 0.9842 bST*SEA*DIET t 1 5850.89 0.24 0.6240 0.060 0.100 0.7557 bST*SEA*DRY*DIE T 2 9919.61 0.41 0.6644 0.002 0.00 0.9966 Cow (bST*DRY*DIET*SEA) 61 24103.10 66.47 0.0001 0.617 37.44 0.0001 WK 1 218496.36 602.53 0.0001 3.038 184.13 0.0001 WK*WK' 1 4382.33 12.08 0.0006 0.066 4.01 0.0459 Frrnr* 390 362.62 0.016 •bST=Bovine somatotropin treatments (I=No bST, 11=10.2 mg bST/d) SEA=Season ( " ^o^vs v.th periods during hot months {September, October, March, April, and May} 11= cows wift dry penods dS^g coZfonths {November. December, January, and February}), 'pRY=Dry period trea« S 5Z period 11= 30 d dry period + ECP, 111= 60 d dry period), ^DIET=Prepartum diet treatments Urcv^^^ZoJcDici, iSeparum Cationic Diet), 'WK=week, ' Type I Sums of Squares for WK term, others are Type III.

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H +H -H -H -H -H VO ^ ^ S C; 2 S ^ o JT) P rrrttts r— t -H -H -H -H -H -H -H Ln ^ ^ VO — O O Ov fN O S r^ r^ fs I* 2 ^ ^ 4^ 41 41 -H -« -H -H 0 ON to ^ S rVO ov t~oo £ S VO VO VO VO VO ^ ^ r> in Ov >n oo 00 in r m o VO O VO O d d d -H -H •tl oo O r V) O C 1^ II cx o. o s> i3 p: S E U ^P CO 1) ' .S ^5i. ° PQ J) , P 2-3 n u _) II -4 Ov 00 Ov

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Figure 3-1. Body weights of Holstein cows during the prepartum and postpartum periods. Arrow indicates calving.

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3.9 3.7 3.5 CO O 3.3, 3.1 2.9 2.7 1 no bST — — bST t -1 1 1 1 1 1 1 1 1 1 1 1 1 -8 -5 -4 -3 -2 -1 calving 1 2 3 4 6 8 10 12 14 Weeks -8-5-4-3 -2 -1 calving 1 Weeks -1 1 1 1 1 1 1 1 2 3 4 6 8 10 12 14 Figure 3-2. Body condition scores of Holstein cows during the prepartum and postpartum periods. Arrow indicates calving.

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120 were observed in mean BCS of untreated and bST treated cows at -8 wk (3.28 for both groups). The BCS of both groups increased during prepartum period and highest BCS was one week before calving (control=3.50 vs. treated=3.55). The mean BW of cows in 60 d dry period group was significantly greater (678 kg) when these cows were dried off at 8 wk than those of cows in 30 d dry (636 kg) and 30 d dry + ECP (638 kg) groups of cows (Figure 3-1; Table 3-5). However, at the time of 30 d drying off difference was less and not significant (694 kg vs 667 and 672 kg, respectively). Cows in all treatment groups continued to gain BW during weeks following I dry off and at 1 wk before calving increases were 9%, 11%, and 10% for cows in 60 d dry, 30 d dry and 30 d dry + ECP groups, respectively (Table 3-5). hi contrast to BW, the mean BCS for groups of cows did not differ at 8wk (Table 3-5). The mean BCS of cows I in 30 d dry +ECP group was numerically greater (3.31) than those in 30 d dry (3.27) and 60 d dry cows (3.26). Mean BCS increased 0.06, 0.10 and 0.08 points for cows in 60 d dry, 30 d dry and 30 d dry +ECP cows at wk -5 and continued to increase during the prepartum period (Figure 3-2). The increases in mean BCS during the last 30 d of the dry period for cows in 30 d dry, 30 d dry +ECP and 60 d dry cows were 0.22, 0.15 and 0.13 points, respectively. No differences in mean BW for diet treatments were detected (Table 3-4). Cows fed the anionic diet had numerically lower BW (646 kg) than cows fed cationic diet (656 kg) at -8 wk (Table 3-5). Cows in both groups increased their BW during the weeks of the prepartum period. The mean increase from wk -8 to wk -1 was 9.9 % and 1 1.3 % for anionic and cationic diet groups, respectively (Figure 3-1). Similarly, no differences were observed in mean BCS by diet treatment during the prepartum period (Table 3-4). The

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121 mean BCS of cows fed anionic and cationic diets at wk -8 were 3.26 and 3.30, I respectively. Both groups of cows increased their BCS from -8 wk to parturition (Figure 3-2). Increase in mean BCS was numerically but not significantly greater for cows in 1 cationic group (0.28 points) than cows in anionic diet group (0.22 points). Postpartum period Another objective of the current study was to evaluate the changes in BW and BCS throughout the early postpartum period (wk 1 to wk 14) and to determine if prepartum treatments had effects on postpartum BW or BCS. To evaluate this time period a second series of analyses was performed. Least squares analyses of variance for BW and BCS during the overall postpartum period are in Table 3-6. There were no significant effects of bST, DRY, DIET or SEA observed for BW. However, the interaction SEA*DIET (P<0.0793) was significant. Cubic effect of WK on BW also was significant. There were no significant bST, DIET or SEA effects observed for BCS during the same I time period. Significant effect of DRY treatment on postpartum BCS was detected (P<0.0372). The interaction SEA*DRY (P<0.0501) and cubic effect of WK also were significant. The birth weights of calves also were analyzed. The birth weights of calves of cows injected or not injected with bST did not differ significantly (38.3 ± 1.2 vs 36.5 ± 1 .2 kg, respectively). The calf birth weights were 37.0 ± 1 .4, 36.0 ± 1 .4 and 39.2 ± 1 .5 kg for cows in 30 d dry, 30 d dry + ECP and 60 d dry groups, respectively. No differences in birth weights of caves were detected for cows fed prepartum anionic (37.1 ± 1.4 kg) or cationic diets (37.7 ± 1 .4 kg). * •

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122 Table 3-6. Least Squares Analyses of Variance for BW and BCS of Holstein Cows During Postpartum Period (wk 1 to wk 14). ^ Source BW BCS df MS F P>F MS F r>r 1 1640.60 0.05 0.8182 0.001 i\ f\f\ 0.00 1 41753.58 1.36 0.2488 0.026 0.03 2 14977.69 0.49 0.6173 2.964 3.47 A Am 0.03 11 1 16470.63 0.53 0.4674 0.408 0.48 0.49 lo 1 8723.57 0.28 0.5965 1.467 1.72 0.1946 1 840.14 0.03 0.8694 0.019 0.02 0.8814 2 3039.88 0.10 0.9062 0.181 0.02 r\ c\nc\c\ o.y /yu 1 98102.42 3.19 0.0793 0.096 0.11 f\ T2Q 1 U. 1 ioi 2 99648.94 3.24 0.0462 0.126 0.15 U.OOZD 2 18735.91 0.61 0.5475 2.683 TIC 3.15 A A
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During the postpartum period, mean changes in BW did not differ due to bST treatment (Table 3-6). Mean BW decreased sharply after parturition in both groups. The decrease in BW was observed up to 6 wk postpartum. After this point, cows in both I groups maintained their BW (Figure 3-1; Table 3-7). No differences were detected in mean BCS between bST injected and uninjected cows following parturition. Changes in BCS generally followed the same pattern as BW. The decrease in BCS lasted to 6 wk postpartum and thereafter remained slightly above 3.0 for cows in both treatment groups (Figure 3-2). Least squares means and SE for BW during the postpartum period (wk 0 through 14) for dry period treatment are in Table 3-7. Mean BW of cows during the postpartum period did not differ due to dry period treatment. Cows in all three treatment groups showed an acute decrease in mean BW after calving, as would be expected. Decrease in BW were similar from wk -1 prepartum to wk 6 postpartum when cows stopped losing I BW (Figure 3-1). Losses in BW for cows in 60 d dry group (15.7%), 30 d dry no ECP (14.6%) and 30 d dry ECP groups (13.1%) were similar from wk -1 through wk 8. During the postpartum period, changes in mean BCS differed significantly (P<0.0372) among dry period treatments (Table 3-6). Decrease in BCS was detected in all three groups following calving. Changes in BCS were sharper for cows in 60 d dry cows and remained significantly less than cows given shorter dry period after wk 2 postpartum. The BCS reached lowest values around wk 6 postpartum for cows in the 30 d dry groups, whereas cows in 60 d dry treatment lost BCS up to wk 10 (Figure 3-2). Mean BCS at wk 8 were greater than 3.0 for cows in 30 d dry (3.10) and 30 d dry +ECP (3.17) cows, but less than 3.0 (2.89; Table 3-7) for cows with a 60 d dry period.

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124 2 W 00 J 00 oo w 00 00 w 00 CO JN o — f2 o o 4^ ^ 41 -H -H -H _» ro VO t~ ET". s S 3 s s s s ^ o ^ <^ <^ o 2 -H -H -H -H -W 1/-^ T* U-) m p O ^ ^ i) Co vo \c> VO ^ 41 4^ 41 -H -H -H O
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126 No differences in mean BW or BCS during the postpartum period (wkl through 14) were detected between prepartum anionic and cationic diet treatments. Mean BW and BCS for cows in the two prepartum diet treatments followed the same pattern (Figures 3I 1 and 2). Decreases in BW and BCS were seen through the first 6 wk after calving but after that they maintained their mean BW and BCS (Table 3-7). Serum Calcium Concentrations . i n >• Concentrations of Ca in serum did not differ significantly due to bST, dry period, or prepartum diet treatments. Serum concentrations declined around parturition and were I least the day following calving (Figure 3-3). However, only 16 of 80 cows had serum concentrations of Ca less then 7 mg/dL the day following calving. Concentrations of Ca during the 2 wk before through 2 wk after calving in uninjected cows were 9.41mg/dL and 9.28 mg/dL for cows injected with bST. On the other hand, 11 of 16 cows that had Ca concentrations intermediate than 7 mg/dL on the day following calving were in the I uninjected cows. Overall (-14 d through +14 d) serum concentrations of Ca were numerically greatest for 60 d dry cows (9.46 mg/dL), less for 30 d dry cows (9.37 mg/dL) and least for cows in 30 d dry +ECP (9.20 mg/dL). Five cows in 60 d dry group, 6 cows in 30 d dry no ECP group and 5 cows in 30 d dry ECP group had Ca concentrations less than 7 mg/dL following calving. Feeding the anionic diet did not significantly improve prepartum or postpartum concentrations of Ca. Cows fed the prepartum anionic diet had similar serum concentrafions of Ca (9.35 mg/dL) to those in cows fed prepartum cationic diet (9.34 mg/dL), and 8 out of 16 cows fed the anionic diet had serum concentrations of Ca lower than 7 mg/dL the day following calving. However, no cases of clinical hypocalcemia was observed in any cows irrespecfive of diet fed.

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;ure 3-3. Senim concentrations of Ca in Holstein cows during the prepartum and postpartum. Arrow indicates calving.

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128 Changes in Dry Matter Intake A major objective of this experiment was to evaluate changes in DMI during the transition period starting -21 d before parturition and continuing through +28 d postpartum. Therefore, data were divided into prepartum (-21 to -1 d) and postpartum (0 to 28 d) sets for analyses. During the prepartum period, no differences were detected in DMI for bST, DRY or DIET treatments. The two-factor interaction of SEA* DIET was significant (P<0.0521) and cubic effects of days (P<0.0001) and quadratic effects of BCS (P<0.0004) also were significant. No differences in DMI were detected for bST treatment groups before parturition. Least squares analysis of variance for DMI in Table 3-8 and least squares means for DMI during the prepartum period are in Table 3-9. Average DMI are 3 wk before calving was greater than 22 kg/d for cows in both bST treatment groups. Eight days before calving cows showed no decrease in DMI and still had similar DMI (24 kg/d for both groups; Figure 3-4). From this point, decline in DMI was observed for cows not injected with bST and 2 d before calving the DMI for this group was around 19 kg which was a 20 % decrease. On the other hand, DMI of cows in bST treated group had declined to -21 kg/d, a 13% decrease in DMI. One day before parturition for cows in uninjected and bST treated groups of cows the DMI was reduced to 14 kg and 15 kg, respectively. Reductions in DMI were 40% for uninjected and 37% for bST treated cows from d -8 to d -1 prepartum (Figure 3-4). Least squares analysis of variance for DMI of the cows in the three dry period treatment groups during the overall prepartum period is in Table 3-8. No significant effects of dry period length were detected for DMI. Average DMI 3 wk before calving

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129 Table 3-8. Least Squares Analyses of Variance for DMI of Holstein Cows During Prepartum Period. DMI Source bST ' df MS F PR>F 1 307.80 1.20 0.2781 SEA1 528.25 2.06 0.1570 DRY' 2 142.74 0.56 0.5763 DIET^ 1 0.96 0.00 0.9513 bST*DIET 1 116.57 0.45 0.5030 bST*SEA 1 315.98 1.23 0.2719 bST*DRY 2 69.62 0.27 0.7632 SEA*DIET 1011.87 3.95 0.0521 DRY*DIET ^ " -' i t ' i ^ : 51.92 0.20 0.8172 DRY*SEA 2 4 1.69 0.01 0.9934 bST*DRY* SEA 2 63.64 0.25 0.7810 bST*DRY*DIET 2 107.78 0.42 0.6589 DRY*SEA*DIET 2 157.36 0.61 0.5450 bST*SEA*DIET 1 107.59 0.42 0.5198 bST*SEA*DRY*DIET 2 22.36 0.09 0.9166 Cow (bST*DRY*DIET*SEA) 53 256.30 10.99 0.0001 BCS I 321.33 10.99 0.0002 BCS*BCS 295.33 13.78 0.0004 DAY 2663.81 114.26 0.0001 DAY*DAY 3296.11 141.38 0.0001 DAY*DAY*DAY 777.59 33.35 0.0001 Error* 1599 23.31 'bST=Bovme somatotropin treatments (I=No bST. 11=10.2 mg bST/d), -SEA=Season (1= cows with dry periods during hot months {September. October, March, April, and May} .11= cows xvith dry periods during cold months {November. December. January, and February}) DRY-Diy penod Latments (1= 30 d dry period, 11= 30 d dry period + ECP, ni= 60 d dry period). ^DIET=Prepartum diet treatments (I=Prepartum Anionic Diet. II=Preparum Cationic Diet). Type I Sums of Squares for DAY terms, others are Type III.

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130 T3 OO c U 00 O O -H 00 o -H -H ON — fN O -H -H o 00 rn VO o -H -H IT) Vt od r«i 00 so IT) o OO CO 00 u CQ OO OO -H o s o o -H -H 00 O 00 O o O o o IT) C7\ iri -H -H fN SO SO o OS fN OO so 41 -H fN (N Q Q fN I •o to c '5 n '5b u a 00 x> E (N O T a 4>>> C ni C 00 x> o c

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131 Figure 3-4. Dry matter intake of Holstein cows during the prepartum and postpartum periods. Arrow indicates calving.

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132 was about 23 kg for cows in all dry period treatment groups (Table 3-10). Cows showed no decrease in DMI 8 d before calving and still had similar DMI to that at d -21 (Figure 3-4). However, cows in all dry period treatment groups showed decreases in mean DMI 1 during the last few days prepartum with decreaes in DMI of 31, 37 and 45% for cows in 30 d dry, 30 d dry +ECP and 60 d dry cows, respectively (Figure 3-4). The least squares analysis of variance for DMI of cows fed anionic and cationic diets prepartum did not differ during the prepartum period (Table 3-8). The mean DMI were 23.2 kg/d and 23.4 kg/d for cows fed prepartum anionic and cationic diets, respectively (Table 3-11). Cows in both diet groups maintained their intake through d -8 with DMI greater than 23 kg/d. Cows in both treatment groups showed a dramatic decrease the day before calving (Figure 3-4). The decreases in mean DMI from d -8 to d 1 were similar (37% and 39% for cows fed anionic and cationic diets, respectively). Postpartum period For DMI data collected after parturition (0 to 28 d), statistical analyses included the main treatments, their interactions, days significant up to quadratic order, and linear effect of BW of cows. During the postpartum period, no differences were detected in mean DMI for bST, DRY or DIET treatments. Significant effects of SEA and the interaction bST*DRY were detected (Table 3-12). When the DMI of cows was expressed as a percentage of BW (%BW), no effects were detected due to the main treatments bST and DIET, however, effects due to DRY treatments were detected significant. Least squares analysis of variance for DMI of bST treatment during the overall postpartum period is in Table 3-12. No differences in mean DMI were observed due to bST treatment. Cows in both bST injected and uninjected groups showed increased DMI

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r .1 133 c '53 00
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134 to o o o o o »— * E Fed -H -H +1 -H -H lartu BCO O O U -CO o E D > w .a c S .2 atus (Mc rep; l.Le rCat tI=P ^ o MI (%B c able 3Jiionic w (kg) CS Energy S MI (kg/ 'Treatme period. PQ PQ Q Q i

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135 Table 3-12. Least Squares Analysis of Variance of DMI of Holstein Cows During Postpartum Period (d 1 to d 28). DMI Source bST' df MS F PR>F 1 33.46 0.08 0.7804 SEA1 2365.09 5.55 0.0222 DRY' . 2 627.40 1.47 0.2387 DIET* 1 182.04 0.43 0.5162 bST*DIET 1 62.07 0.15 0.7043 bST*SEA 1 1016.66 2.39 0.1284 bST*DRY 2 2250.25 5.28 0.0081 SEA*DIET 1 338.96 0.80 0.3766 DRY*DIET 2 11.04 0.03 0.9744 DRY*SEA 2 361.57 0.85 0.4339 bST*DRY* SEA 2 116.85 0.27 0.7613 bST*DRY*DIET W ; ' 2 123.76 0.29 0.7492 DRY*SEA*DIET U T 2 681.24 1.60 0.2118 bST*SEA*DIET 1 58.33 0.14 0.7129 bST*SEA*DRY*DIET 2 90.19 0.21 0.8100 Cow (bST*DRY*DIET*SEA) 53 426.24 19.95 O.OOOl BW 1 8211.63 10.08 0.0015 DAY' 1 14669.71 659.15 0.0001 DAY*DAY 1 1617.40 72.67 0.0001 Error* 1997 22.25 'bST=Bovine somatotropin treatments (I=No bST, 11=10.2 mg bST/d), 'SEA=Season (1= cows with dry periods during hot months {September, October, March, April, and May} 11= cows with dry periods during cold months {November, December, January, and February}). pRY=Dry penod tteatments (1= 30 d dry period, 11= 30 d dry period + ECP. 111= 60 d dry period^ DIET=Prepartum diet treatments (I=Prepartum Anionic Diet, II=Preparum Cationic Diet), 'DAY, Type I Sums of Squares for DAY terms, others are Type III.

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136 after parturition. The first day following calving, cows had their lowest DM1 (~ 1 7 kg/d). Thereafter, a gradual increase in daily DMI was observed and one week after calving the DMI of cows in both treatment groups was greater than 23 kg/d; these were similar to greatest intakes seen prepartum (Table 3-9). At the end of the measurement period (d 28), mean DMI for the cows was greater than 30 kg/d (Figure 3-4). No differences in mean DMI were detected for the three dry period treatment groups after parturition (Table 3-12). However, cows in 60 d dry group tended to have lower mean DMI after calving (Figure 3-4). Cows in the two 30 d dry groups reached I DMI that were seen prepartum (-23 kg/d) 7 d after parturition but 60 d dry cows DMI was ~ 21 kg/d. In general, trends for mean DMI of 60 d dry cows tended to be less throughout the 28 d period than for cows in the two 30 d dry groups (Table 3-10). The mean postpartum DMI was greatest for cows in 30 d dry +ECP cows (27 kg/d), lower for cows in 30 d dry cows (25.7 kg/d) and least for 60 d dry cows (24.7 kg/d). However, at d 28 postpartum cows in three dry treatment groups mean DMI was greater than 30 kg/d DMI (Figure 3-4). On the other hand, DMI expressed as a percentage of BW differed among groups (P<0.01). Similar to results for DMI (kg/d), the greatest DMI expressed as a percentage of BW was for cows in 30 d dry ECP group (1.95%), intermediate for cows in 30 d dry no ECP (1.86%) and least for cows in 60 d dry group (1.75%; Table 3-10). The mean DMI during the overall postpartum period (28 d) were similar (26. 1 kg/d and 25.5 kg/d, respectively) for cows fed cationic and anionic diets prepartum (Table 3-11). Cows fed cationic diet tended to have slightly numerically greater DMI during the first week following parturition. However, the DMI of cows in both diet groups was greater than 23 kg/d 8 d postpartum (Figure 3-4).

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137 Energy status of the cows during the first 4 wk postpartum was negative and did not differ significantly for bST and non-bST groups (-18.25 vs -16.07 Mcal/d, respectively; Table 3-9). The energy status of cows fed anionic or cationic diets prepartum did not differ during the first 4 wk postpartum (-16.36 vs-17.97Mcal/d, respectively; Table 3-10). Cows in 30 d dry group had significantly less negative energy status (-13.77 Mcal/d) than cows in 30 d dry + ECP group (-17.04 Mcal/d) or than cows in 60 d dry group (-20.68 Mcal/d; P<0.06; Table 3-11) during the first 4 wk postpartum. Regression analyses were performed for DM1 to describe trends in consumption throughout the transition period (-28 to 28 d). Regression curves indicated that no differences were observed between bST injected or uninjected cows as well as between prepartum anionic diet and cationic diet treatments. On the other hand, 30 d dry groups appeared to have greater DMI at the beginning of the lactation. Heterogeneity tests of the prepartum and postpartum curves for dry period treatments provided no evidence that curves were not parallel. Results presented in Figure 3-5 showed almost identical trends for bST injected and uninjected control cows from the beginning of the trial through 28 d postpartum. Cows in prepartum cationic diet treatment tended to start with greater DMI at the beginning of the experiment, but no differences were observed during the final weeks prepartum. In addition, cows in prepartum cationic diet treatment also started with greater DMI during the first week following parturition, but DMI of cows in prepartum anionic diet treatment tended to be greater at the end of the experiment. Heterogeneity tests of the I prepartum curves provided no evidence that curves were not parallel and all cows in dry period groups had similar trends during the prepartum period.

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Figure 3-5. Regressions depicting changes in Dry matter intake of Holstein cows during the prepartum and postpartum periods.

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139 Discussion Prepartum Period ^ High yielding dairy cows undergo a rapid and extensive metabolic challenge I between late pregnancy and early lactation. A significant fall in DMI occurs during late pregnancy and it continues through early lactation (Bertics et al., 1992; Coppock et al., 1972; Lodge et al., 1976). Bertics et al. (1992) described two pronounced phases that can affect cows health and lactational performance. The first phase takes place during the last week prepartum and is characterized by a 30% or greater decrease in DMI. The second phase takes place during the first 3 wk postpartum, a time when DMI should increase rapidly to support milk production. Rate of increase in DMI is the primary determinant of energy intake and energy balance during early lactation cows and it is important that it occurs during this early postpartum period. In the current study, limited decline in DMI was observed in all treatment groups before calving. At the end of the Calan gate training period (—21 d) cows in all treatment groups had essentially achieved their greatest DMI and it remained high for 2 wk except for the cows in 30 d dry EC? group. These cows showed a decline in DMI —2 Id which lasted about 1 wk. However, cows in all treatment groups had mean DMI greater than 22 kg/d 1 wk before calving. Decrease in mean DMI from d -14 to d -2 was about 15 % across all treatment groups and overall decrease seen at d -1 was about 30%. Thus, even though a gradual decline was seen during the last week prepartum, the major decrease in I feed consumption took place just before or on the day of calving. The DMI of cows was much greater than previously reported. Average DMI reported 3 wk before calving was around 15 kg/d for Holstein cows (Bertics et al., 1992; Garcia et al., 2000; Grum et al.,

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140 1996; Lodge et al., 1975; Zamet et al, 1979). They also described a 20 to 40 % decrease in DMI during the final week prepartum. Although our data confirmed previous reports that DMI decreased during the final week prepartum, the decrease in DMI was less than that reported by others (Bertics et al., 1992; Garcia et al., 2000; Grum et al., 1996; Zamet et al., 1979), but agreed with Gulay (1998). In the latter study, decreases in DMI for cows treated with bST (5.1, 10.2 or 15.3 mg/bSTd) and uninjected control cows were between 17 to 22% at 1 wk before calving compared to that at wk -3 before calving (Gulay, 1998). On the other hand, Garcia (1998) reported that there was about 16 % decrease in mean DMI 1 wk before calving for cows injected and uninjected with bST. On the day before calving the DMI of both injected or uninjected groups of cows was about 65 % of that seen on d-7 (Garcia, 1998). Greater decrease in DMI for cows injected with bST (5 and 14 mg/d) was reported before calving (Simmons et al., 1994). Zamet et al. (1979) reported that DMI decrease I during the last week before partuition was associated with high incidence of metabolic diseases during early lactation, which differed from the current study. During the present study, no clinical ketosis or clinical hypocalcemia (milk fever) was observed. Only two cows (one cow from 60 d dry, no bST, anionic diet group and one cow from 60 d dry, bST cationic diet group) presented displacement of abomasum (DA). The DA occurrence rate for this experiment was about 2.5% which was much less than the average observed in the DRU herd and often reported for cows (up to 14%; Drackley, 1999; Erb and Grohn, 1988). hi current experiment, the relatively high DMI during the prepartum period also affected the BW and BCS for the cows and from wk -8 to wk -1 . The BW and BCS of

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141 the cows increased on a week to week basis, and it was not affected by the decline in DMI during the final week prepartum. Body Weight and BCS were positively correlated (P<0.001; r=0.5758) during the prepartum period. In addition, DMI was correlated positively with BW (P<0.0255; r=0.04875) but not with BCS (P<0.2421; r=-0.0255). No differences in DMI were observed due to any of the treatments. Putnam et al. (1999) reported no significant differences in prepartum BCS between control cows and those injected with a fiall dose of bST (500 mg bST/d) when injections began -28 d prepartum. Similarly, no effects of low doses of bST injected prepartum (5.1, 10.2 or 15.3 mg/d) were observed on BW and BCS compared to control cows (Gulay, 1998). Garcia (1998) also reported no effect of low bST dose injection on BW and BCS (5.1 mg bST/d). Our results indicated that cows in both uninjected control and bST injected groups had similar DMI during the overall prepartum period which led to similar BW and BCS trends for both treatment groups. If a difference exists, perhaps a 3 wk time period was too short to detect a difference in DMI due to bST, if any effects were provoked during prepartum period. In the current experiment, there was a decrease in mean DMI beginning around d 21 which lasted about a week in cows that had been injected with ECP and given a 30 d dry period. Schairer (2001) reported that injecting dry cows with ECP resulted in increases in plasma concentrations of compared to control cows and that peak concentrations of were reached 48 h post-injection and they persisted for up to 96 h. hi cows, intravenous injections of 17 p-estradiol caused a decrease in feed intake (Grummer et al., 1990). Studies in rats indicated that estrogen has negative effects upon feed intake due to direct actions on the brain (Butera et al., 1989). In addition, Forbes (1974) also

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142 reported depressed feed intake in castrated male sheep when intraventricular injections of estradiol benzoate greater than 60 |ag were given. Ovariectomy resulted in a temporary increase in feed intake for 3 to 4 wk and this resulted in elevation in BW in rats (Tarttelin and Gorski, 1973). Injection of physiological doses of estrogen reversed this effect and a reduction of BW was observed as long as estrogen treatment continued (Tarttelin and i Gorski, 1973). Moreover, in cows, intravenous injections of decreased both MY and DM1 (Grummer et al., 1990). Thus, the expected additional increase in plasma concentrations of estrogen due to ECP injections may have had an acute but short-term negative effect on DMI, as seen in ECP injected cows (Schairer, 2001). No differences in BW or BCS were observed due to dry period treatment. Cows in all three dry period treatment groups showed increased BW and BCS from wk -8 to wk -4, which continued through the final week before calving. Although cows in both 30 d dry with and without ECP were not dried off in this -8 wk to -4 wk time period, they gained about the same BCS (0.1 point) as 60 d dry cows (0.06 point) which were not being milked during this time. Cows in 60 d dry group had significantly higher BW at the time of drying off (-8wk) than cows in 30 d dry groups. However, although BW still was numerically greater during the remaining prepartum period, no significant differences in BW were observed between 60 d dry and both groups of 30 d dry cows. No significant differences in mean DMI were detected between prepartum diet treatment groups. Cows in anionic and cationic diet treatments showed gradual decline in I DMI during the last week prepartum with the major decline in mean DMI occurring within 24 h of parturition for cows in both groups. No effect of prepartum diet was detected for prepartum BW and BCS in current study. The results of current study agreed

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143 -I with others (Block, 1984; Moore et al., 2000; Oetzel et al., 1991). Moore et al. (2000) reported no differences in prepartum DMI between cows fed cationic and anionic diets, hi the same study, anionic salt treatments did not change BW and BCS compared to cows fed the control diet that did not contain salts. In another study, anionic salt treatments did not cause a decrease in DMI compared to cows fed the control diet without salts, and they reported no differences in prepartum BCS of cows fed the different diets (Oetzel et al., 1991). Block (1984) also failed to detect a difference in DMI of cows fed anionic or cationic diets. On the other hand, Vagnoni and Oetzel (1998) reported that when diets were fed for one week periods that the daily DMI increased as the time of access to the anionic diet increased during the first 5 d. However, mean DMI over d 5 to 7 was reduced when dietary anionic salts were included due to a mild metabolic acidosis. Because feeding periods were relatively short in their trial, no differences could have been detected if reduced DMI occurred or persisted for a greater length of time. Results of the current experiment indicated that DMI was maintained at a high level in both anionic and cationic diet groups of cows such that there was no effect on BW and BCS prepartum and no negative effects of feeding diet that contained anionic salts were evident. Early studies demonstrated that a major decline in DMI is initiated in late pregnancy irrespective of diet fed and extending early lactation (Drackley, 1999; Ingvartsen and Andersen, 2000). It has been suggested that the decline in the DMI during late prepartum period is caused by the pressure on the rumen exerted by the growing uterus and also by the increasing accumulation of abdominal fat during late pregnancy (Forbes, 1968). However, decreased rumen volume actually can be balanced by an increase in the passage rate of particles out of the rumen (Kaske and Groth, 1997). As a

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result, it is more likely that decreased DMI during the late prepartum period is due to metabolic adaptations that occur before calving. It has been proposed that increased accumulation of lipids in the body reserves during the late prepartum period down regulates DMI (Broster and Broster, 1984). Increased glucose uptake by the growing fetus occurs with an associated reduction in the use of glucose by maternal tissues. Thus, maternal tissues rely on metabolism of NEFA and ketone bodies (Bell, 1995). Increased lipid drain results in mobilization of body fat and a rise in plasma concentrations of NEFA, glycerol and ketone bodies. It has been speculated that oxidation of NEFA in the brain and the liver can decrease DMI (Horn et al., 1999). In dairy cows, a 4 h infusion of lipids that provided 16.7 MJ of NEl resulted in a slight decrease in DMI postinjection (Bareille and Faverdin, 1996). Moreover, Choi et al. (1997) observed a substantial decrease in DMI during the first 4 hr postinjection after they had blocked fatty acid oxidation in dairy heifers by using sodium mercaptoacetate. It was suggested that the increased plasma glycerol also may influence intake through a central nervous system mechanism. Intracerebroventricular infusions of glycerol in rats decreased their feed intake (Davis et al., 1981). Along with metabolites in blood, it has been suggested that hormones also altered feed intake. Metabolic adaptations that affect feed intake are regulated, in part, by INS. I Thus, reduced uptake and reesterification of fatty acids, decrease in de novo synthesis of TG, and increased lipolysis most likely result from decreased ability of INS to promote lipogenesis and oppose lipolysis (Bell, 1995). Moreover, concentrations of estrogen rise by midgestation and peak prior to parturition (Chew et al., 1977). The increased concentrations of Ej in the peripheral circulation may have direct effects in the

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145 paraventricular nucleus of the hypothalamus (Butera and Beikirch, 1989) and this has been implicated to reduce DMI before calving (Forbes, 1987). In addition to E2 , concentrations of ST begin to increase during late pregnancy. Somatotropin reduces the ability of INS to stimulate lipogenesis in adipose tissue and actively stimulates lipolysis; ST alters the sensitivity of adipose tissue to P-adrenergic agents (Bauman and Vernon, 1993). These effects would dramatically increase mobilization of lipids from the adipose tissue and increase concentrations of NEFA and glycerol in blood. Thus, there would be greatly reduced fatty acid synthesis or, at least, no net synthesis, and hence less acetate and glycerol use in adipose tissue (Bauman et al., 1988). For these reasons, ST is considered the major regulator of metabolic adaptations that starts during the transition period, or earlier, and continues throughout the lactation (McNamara, 1995). Postpartum Period Rate of increase in DMI during early lactation is the primary determinant of I energy balance along with the rate of increase in milk yield. Thus, the first 3 wk postpartum is important because DMI should increase rapidly to provide the energy and precursors to support and sustain milk production. The current study detected no differences in mean DMI during the first 28 d postpartum due to bST treatment. Cows in both groups showed increased DMI after parturition (Figure 3-4). The first day following calving, cows had the lowest DMI (~ 17 kg/d). Thereafter, rate of increase in DMI for cows in both bST injected and uninjected control groups were similar. At the end of the sampling period (d 28), mean DMI for the cows was greater than 30 kg/d. Also no differences in BW or BCS were seen due to bST effects. This agreed with results of others where low, intermediate or high doses of bST had been injected prepartum.

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146 Putnam et al. (1999) reported no significant differences in postpartum BCS of cows in control or prepartum bST injected groups (500 mg/14 d). In the same study, the DMI was significantly greater at 28 d after parturition. Others also reported no increases in DMI or efficiency of milk production by cows injected with bST during early lactation (Gallo and Block, 1990; Schneider et al., 1999). In addition, postpartum DMI of cows treated with lesser doses of bST (5 and 14 mg bST/d) during the prepartum period did not differ (Simmons et al., 1994). Eppard et al. (1996) reported no differences in DMI, BCS or BW during the first 9 wk of lactation of cows treated with full dose of bST (500 mg/14 d) prepartum compared to uninjected control cows. When cows were injected with 20.6 mg bST/d (de Boer et al., 1991) or 5 and 14 mg bST/d (Stanisiewski et al., 1992), postpartum DMI, BW (de Boer et al., 1991) or BCS (Stanisiewski et al., 1992) were not affected by bST treatments. Moallem et al. (2000) concluded that bST injected very early in lactation (d 10) increased DMI after injections were initiated. On the other hand, increase in DMI was not enough to support the increase in MY and injected cows faced an extensive period of NEB and, therefore, BW and BCS decreased. However, in that study cows received a full dose of bST (500 mg/14d) which resulted a severe NEB for the injected cows. Additional reports also suggested no increase in DMI of cows when bST was injected during mid to late lactation (Lucy et al., 1993; Remond et al., 1991). On the other hand, prepartum and postpartum injections of 15.3 mg bST/d increased DMI after parturition, and there was less decrease in BCS and BW allowing the cows to recover BW and BCS in less time during early lactation (Gulay et al., 2000). In addition, Garcia et al. (2000) reported that a low dose of bST (5.1 mg/d) injected into cows before and after

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147 parturition increased numerical DMI during the early weeks of the lactation period, however this was not significant. In current study, no severe NEB was seen, as indicated by the changes in BW and BCS, even though MY increased. Good BCS at calving is important for high producing cows and dry cows should achieve an adequate BCS (3.25-3.75) at calving (Nocek et al., 1983). In current study, cows in bST injected and uninjected groups had BCS of -3.50 at calving and also had high DMI. These two factors might have enabled cows to recover their BCS earlier during postpartum period which would agree with the report that cows having BCS of 3 to 3.5 had better recovery of BCS at 10 wk postpartum than cows with BCS of 4 (Pedron et al., 1993). Importantly, low dose injections of bST during prepartum and early postpartum periods did not have a negative effect on BW or BCS, although the bST injected group was producing greater quantities of milk suggesting there was more efficient production of milk and 3.5% FCM. Thus, the low dose of bST during the transition period caused less problems for the cows compared to full dose of bST because higher doses resulted in severe NEB and BCS loss for these cows. In the current study, it is important to note that no negative or positive effects of bST on BW or BCS were observed any time postpartum. Previous reports where full dose of bST following parturition suggested that the effect of treatment on BCS was severe and cows were affected adversely compared to uninjected cows (Moallem et al., 1996; Moallem et al., 2000). In another study, when full dose of bST injections were initiated 28 d prior to expected calving date and were continued until parturition, cows treated with bST produced 3.3 kg/d more milk than uninjected controls during the first 42 d of lactation (Putnam et al., 1999). Because cows in bST treated group had significantly

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higher initial BCS than controls when they were assigned to the trial, these authors were unable to conclude if injecting a full dose bST had a negative effect on postpartum BCS. hi the current study, overall mean BCS was maintained greater than 3.0 for cows in both bST injected and uninjected groups and no differences in DMI, BW and BCS were observed between treatment groups. No differences in mean DMI or BW were observed due to dry period treatment after parturition. Following parturition, mean DMI increased for cows in all treatments (30 vs. 60 d dry), even though there was a slight numerical increase in DMI for 30 d dry cows compared to 60 d dry. Although mean BW were similar, cows in 60 d dry period tended to lose more BW after parturition. Cows in 60 d dry group gained about 60 kg during the dry period. Although cows in 30 d dry groups gained about 70 kg BW during same time, only -30 kg of the 70 kg was gained during the dry period, with the remaining 40 kg gained during the extra 30 d they still were milking. Perhaps the greater increase (40 kg) in BW during dry period depressed DMI postpartum. A positive relationship has been shown previously between weight gain during dry period and the extent of postpartum mobilization of body tissues (Ingvartsen and Andersen, 2000). When DMI was expressed as a percentage of BW, it was significantly greater for 30 d dry cows with or without ECP than 60 d dry cows. Furthermore, the mean BCS of I the cows in 60 d dry group was significantly less than cows in both 30 d dry groups during the weeks following parturition and they also lost more BCS for a longer time period (Figure 3-2). The average BCS gain for 30 d dry cows and 60 d dry cows was -0.27 and 0.20 points, respectively during the prepartum period. Moreover, although 60 d dry cows were not being milked from wk -8 to wk -4, they essentially did not gain more

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149 compared to those 30 d dry cows that still were milking (0.06 vs 0.10 point, respectively). Apparently, the 60 d dry cows consumed less DM during FOD period because BCS and BW did not increase and yet they were not using nutrients to produce milk, as were the 30 d dry cows. . i . ,_. : • It has been concluded that body reserves replenished during late lactation occurs more efficiently than that replenished during the dry period (Moe et al., 1971). Armstrong and Blaxter (1969) suggested that efficiency of lipogenesis during the ongoing lactation was higher than occurs during the dry period. If BCS increased during late lactation, the efficiency of replacing BCS was -74%, however, if replaced during the dry period, the efficiency of replacing BCS was only -59% (Moe et al., 1971). Thus, they concluded that BCS lost during early lactation could be replaced more efficiently during late lactation than during the dry period. The data indicated that 30 d dry period with or without ECP injections did not have a negative effect on postpartum BCS or lactational performance in the current experiment. On the contrary, results suggested better maintenance of BCS with 30 d dry period than 60 d dry period because BCS of the cows in shorter dry periods were greater (>3.0) than BCS of cows given the longer dry period (< 3.0). As a result, more persistent BCS was seen in 30 d dry cows, because short-term dry cows gained more of their BCS when they were still lactating. Moreover, the greatest DMI, expressed as a percentage of BW was for cows in 30 d dry ECP group, intermediate for cows in 30 d dry no ECP and lowest for cows in 60 d dry group. Thus, injecting prepartum ECP did not reduce DMI. Cows in 30 d dry groups experienced only one diet change during their dry period, whereas diet for 60 dry cows changed two times. Making changes in diet might adversely

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150 affect development of papillae and incomplete adaptation of the microbial population of the rumen. Changes in the numbers of ruminal papillae occurs in response to nutritional changes. Complete adaptation requires a period of 2 to 3 wk (Dirksen et al., 1985; Goff and Horst, 1997). Making fewer changes, on the other hand, might encourage maintenance of the desired rumen population and better rumen papillae development. Thus, it is possible that cows given shorter dry periods would have better efficiency of fermentation and absorption of end products of fermentation during early lactation even though absolute DMI did not increase faster or to much greater amounts, yet the changes in fermentation and absorption would result in greater maintenance of BCS during early lactation, as was found. Although mean DMI of cows fed anionic and cationic diet treatments did not differ during the first week postpartum, cows fed the cationic diet prepartum tended to have slightly higher DMI following parturition. Urine of the animals was collected and pH measured routinely during the prepartum period. Cows in anionic diet group did have a lower range of urine pH than cows in cationic group (5.6 to 6.0 vs 7.0 to 7.6). Typically, cows fed anionic diets show lower rumen pH and this causes a mild ruminal acidosis (Moore et al., 2000; Oetzel et al.,1991). It is likely that in current study, anionic diet resulted in decreased rumen pH during the prepartum period. Lower pH in the rumen has been reported to decrease rate of fiber digestion and increase filling effect of the diet, which might increase distension in the reticulo-rumen (Allen and Mertens, 1988). Consequently, it is possible that lower rumen pH immediately after calving due to prepartum anionic diet might have influenced the DMI of the cows that were fed the anionic diet prepartum and caused the slightly lower increase in DMI for the cows in this

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group. However, the differences in DMI for the cows in the two diet groups were small and remained similar during the transition period. Milk fever is a conmion metabolic disorder in dairy cattle and the incidence rate of milk fever among lactating Friesian-type cows is less than 7% (Erb and Crohn, 1988). Manipulating the DCAD below zero by including anionic salts in prepartum diets has been suggested to improve Ca metabolism by increasing intestinal absorption of dietary Ca and/or by increasing bone Ca mobilization (Block, 1984). Cows fed anionic diets tend to have greater circulating concentrations of Ca than cows fed cationic diets prepartum (Oetzel, 1988; Goff et al., 1991). However, in the current study no effect on serum Ca was detected due to prepartum feeding of an anionic diet. More recently, dietary K also has been shown to have a role in the incidence of milk fever (Goff et al., 1997). Diets 1 with a high K content can increase the risk of hypocalcemia more than high dietary Ca during the last weeks of the prepartum period. For example, increasing dietary K from 1.15% to 2.1% increased the incidence of milk fever from 10.5 to 50% in Jersey cows. Addition of strong cations to prepartum diets causes a metabolic alkalosis and this suggests that bone resorption of Ca is inhibited in cows fed high K or Na diets because feeding these diets results in increased blood and urine pH (Goff et al., 1997; Horst et al., 1984). In the current study, dietary K was calculated to be 1 .02 % of diet DM for anionic and 1.14% of diet DM for cationic diet. These low quantities of K in the diets fed might have helped cows fed cationic diet group to maintain serum concentrations of Ca at an acceptable level and reduced the risk of hypocalcemia (milk fever). High producing dairy cows are unable to consume sufficient amounts of energy during early lactation because MY usually peaks between 5 to 7 wk postpartum, while

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152 maximum DMI is reached between 8 to 22 wk after calving (Ingvartsen and Andersen, 2000). Cows often need to replenish their body reserves during the dry period. Thus, good body condition (3.25 to 3.75) at calving is important to high producing cows (Nocek et al., 1983). On the other hand, overconditioning is not needed and should not occur during the dry period. In the current study, BCS of cows in all treatment groups were at acceptable levels (3.45 3.59) at calving and postpartum DMI was correlated negatively with BCS (P<0.0001 ; r^O.0876). It was reported that postcalving, cows with higher BCS lose more condition than cows with lower BCS and they reach positive energy balance faster (Gamsworthy, 1988). Increased accumulation of lipids in body reserves during the prepartum period down regulates DMI (Broster and Broster, 1984). Moreover, losing weight during the dry period increases incidence of metabolic diseases (Gerloff and Herdt, 1984). There was a positive relationship between prepartum weight gain and the extent of postpartum mobilization of body tissues (Ingvartsen et al., 1997). If BW gain was more than 40 kg during the dry period, this caused a depressed feed intake postpartum and caused excessive mobilization of body tissue (Ingvartsen and Andersen, 2000). Furthermore, cows with higher BCS at calving lost weight longer after calving than cows with moderate BCS (Ruegg and Milton, 1995). In the current experiment there was a high positive correlation between BW and BCS during the postpartum period (P<0.01; r=0.5177). This supports the view that BCS is indeed a practical, if not quantitative, method of evaluating nutritional status and energy reserves of cows (Ruegg and Milton, 1995). The rate of BCS loss was greatest during the first 2 wk postpartum for cows in all treatment groups and the difference in BCS (from -0.25 to -0.35 point) reflected rapid tissue mobilization that occurred after

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• ' • 153 calving. Cows reached their minimum BCS by about 42 to 49 DM. These results were similar to those reported earlier. Ruegg and Milton (1995) described a 0.25 point loss in BCS during the first week after calving. Pedron et al. (1993) reported that recovery of cows that had BCS of 3.0 to 3.5 at calving started at 10 wk postpartum. Moreover, cows with higher BCS at calving (>3.5) appeared to lose BCS for a longer period of time (>80 DIM) than cows with lower BCS at calving (<3.25; 50 DIM)(Gamsworthy and Jones, 1987). Energy balance is most negative during wk 1 postpartum (Harrison et al., 1990). Thus, the BCS loss in early lactation appears to be rapid. As mentioned above, continued BCS loss from 50 to 90 d was reported (Ruegg and Milton, 1995; Pedron et al., 1993). The data from current study showed greatest BCS loss during the first 2 wk postpartum and cows reached their minimum BCS by 6-7 wk postpartum. Thus, no negative effect of prepartum or postpartum treatments of bST, dry period length or prepartum diet treatment were observed. The observed the rapid increase in postpartum DM! shortened the time of postpartum loss in BCS Conclusions Results of this study indicated that use of low doses of bST during prepartum period (d -28 to 0 d) caused no negative or positive effect on the treated cows. Cows treated or not treated with bST appeared equally capable of replenishing their body reserves during early postpartum period. Injections of low doses of bST during the early lactation (d 1 to d 60) did not have a negative or positive effect on rate of increase in DMI, because cows had the same rate of mobilization of body tissues and loss of BCS irrespective of prepartum or postpartum treatment.

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154 No effects of prepartum diet treatments were observed on either prepartum or postpartum DMI, BW or BCS. No clinical hypocalcemia was observed in this trial and serum concentrations of Ca before and after calving did not differ in cows fed anionic and 1 cationic diets. Thus, there were no obvious advantages to support view that anionic diets should be fed to cows during the close-up dry period as compared to a properly formulated cationic diet when provided K in diet was regulated. Serum concentrations of Ca in Holstein cows during the current trial and diet fed prepartum had little or no negative or positive effects on postpartum DMI. Dry period length did not have a significant effect on DMI, BW or BCS of the cows prepartum. Cows in the shorter dry period groups were able to replenish their BW and BCS as well as cows in the longer dry period group. Short dry period cows (~ 30 d) gained more of their BW and BCS while they still were lactating, whereas 60 d dry cows mostly gained BW and BCS after drying off During postpartum period, short dry period cows lost less BCS postpartum than 60 d dry cows and had more DMI as % BW. Thus, it might be advantageous to allow cows to replenish their BCS before they are dried off. In conclusion, it appears that injections of bST during the prepartum and postpartum periods, cationic diet during prepartum period, or shorter (~ 30 d) dry periods had no negative influences that caused detrimental effects on DMI, BW or BCS changes or health problems during postpartum period.

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CHAPTER 4 IMPROVING MILK PRODUCTION AND HEALTH OF COWS BY SHORTENING THE DRY PERIOD WITH ESTROGEN, AND USE OF bST DURING THE TRANSITION PERIOD Introduction Dry Period The lactation cycle begins with a period of mammary gland development followed by lactogenesis. Milk synthesis and secretion occurs after parturition. After the peak in milk production, a declining phase follows until milk removal is stopped either at weaning or due to the management practices enforced by the dairy producer. This lactation cycle of the female continues several times during her reproductive life (Hurley, 1989). The period between successive lactations after periodic milk removal is stopped is called the dry period. This dry period allows remodeling of mammary tissue and the reinitiation of lactation at a maximal level after next calving (Hurley, 1989). Many factors must be considered to determine the appropriate length of the dry period for individual cows. In a genetic and environmental study, Schaeffer and Henderson (1972) examined the lactation records of Holstein cows in the New York area. They concluded that age and month of calving significantly affected length of dry period. O'Connor and Oltenacu (1988) included parity, month of calving and time of conception as factors that affect optimum time of drying off. Dias and Allaire (1982), after analyzing data fi-om 8981 Holstein cows, reported that the length of time required for dry period I decreased as the lactation number increased from first through fourth lactations. They ' 155

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156 stated that cows with calving intervals greater than 365 d required fewer days dry than cows with shorter calving intervals except those in first lactation. Effect of dry period length on milk yield was greater for younger cows and the optimal number of days dry I declined from 65 to 23 d as age at calving increased from 24 to 83 mo in the same study. They also stated that calving interval and daily MY at 100 d before calving were significant factors affecting total days needed for the dry period. Establishing optimum length of the dry period is critical to achieve maximum milk production during the next lactation. Since 1936 many observational and experimental data have been generated to establish an optimal drying off time for cows. Fifty-five to 60 d dry period length has been recommended based on the fact that this would maximize producfion in the following lactation (Coppock et al., 1974; Dias and Allaire, 1982; Klein and Woodward, 1943; Schaeffer and Henderson, 1972). Arnold and Becker (1936) evaluated Jersey cows with dry periods of 30 d or less (10 cows), 31 to 60 I d (54 cows), 61 to 90 d (45 cows) and 91 d or more (56 cows) preceding the lactation. In their study a dry period of 3 1 to 60 d allowed the maximum MY in the subsequent lactation. Klein and Woodward (1943) surveyed 1 139 lactafion records from Dairy Herd Improvement Associafion (DHIA). They found optimum dry period was 55 d for cows yielding -5000 kg of 4% FCM with 12 mo calving interval. Smith et al. (1967) evaluated the effect of milking throughout pregnancy to time of next calving compared to a dry period of 8 to 9 wk was for effects on milk yield within the same cows . Two quarters of the udder of 2 cows were milked continuously while the other two quarters were dried off for ~ 60 d before expected calving. The quarters allowed a dry period of 8 to 9 wk produced 40% more milk in the subsequent lactation.

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157 Swanson (1965) took another approach and used five pairs of identical twin dairy cows to evaluate the need for the dry period. One of each pair of identical twins was dried off to give at least an 8 wk dry period, whereas other pairmates were milked continuously for two consecutive lactations. Average milk yield of the continuously milked twins in the second and third lactations was 75 and 62% of the control twins that had -60 d dry period. Results of both studies indicated that the mammary gland benefitted from a dry I period. To evaluate the effects of dry period length on later milk production, Coppock et I al. (1974) conducted a 42 mo field trial. Cows (n=1019) were assigned to treatments of 20, 30, 40, 50 and 60 d dry periods. Dry period lengths were allowed to have ±10 d range in each group; 305 cows completed the 42 mo study. Cows that averaged less than a 40 d dry period produced 450 to 680 kg less milk in the subsequent lactation compared to cows having dry periods of 40 d or longer. Cows with 40 d dry period produced as much milk as cows in 50 d dry period. When cows in shorter dry period groups were allowed to have longer dry periods during the next lactation, no carry over effect was observed. Schaeffer and Henderson (1972), in a survey based analysis, reported that cows with dry periods of 50-59 d had the highest production in the subsequent lactation. Yet, average milk production for 40 to 49 and 60 to 69 d dry periods did not differ I significantly. In another study, Funk et al. (1987) analyzed data collected for 6 yr fi-om over 84,000 cows. They concluded that cows given dry periods longer than 70 d produced only moderately less than cows dry for 60-69 d. Moreover, cows dry for 40 d or less produced significantly less (-459 kg) in the subsequent lactation than cows dry for 60 to 69 d which produced the most milk.

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-> 1 158 In a more recent study, Sorensen and Envoldsen (1991) evaluated the effect of different dry periods on subsequent milk yield. Cows were dried off at 4, 7 or 10 wk before expected calving. They found a decrease in yield of 2.8 kg of 4% FCM/d when dry period length was decreased from 7 to 4 wk, whereas there was a 0.4 kg/d increase in milk production when dry period was increased from 7 to 10 wk. In contrast to the study by Dias and Allaire (1982), Sorensen and Envoldsen (1991) failed to detect an interaction between the dry period length and lactation number. In another study, effects of days dry on milk yields of Holsteins from Zimbabwe and North Carolina were evaluated (Makuza and McDaniel, 1996). In their study first (n=l 1583), second (n=7143) and third (n=6102) lactations for cows were evaluated. In both locations shorter dry periods had detrimental effects on milk yield. Milk yields of cows dry for 30-39, 40-49 and 50-59 d were 610, 633 and 202 kg less than for 60 d dry periods. On the other hand, little advantage was observed for dry periods longer than 60 d. The dry period length is very important because it is directly related to subsequent milk production and income. During the dry period mammary glands undergo a number of changes that are necessary to stimulate maximal milk production during the subsequent lactation. The time period needed for involution is the major factor determining the optimum length of the dry period. This is a very important topic. Yet, few studies have been completed and published to evaluate the problem. Nonexperimental and experimental data suggested that 7 to 10 wk dry periods were necessary to maintain maximal production. Thus, it is important to understand the changes that occur during the involution process that leads to the necessity for a dry period, especially for a longer dry period.

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I 159 Involution After frequent periodic milk removal from the mammary gland is discontinued, the individual mammary glands undergo involution. Three types of involution have been described (Lascelles and Lee, 1978). Gradual involution occurs during the declining phase of lactation after the peak milk yield has been reached. Initiated involution describes regression of the lactation function with sudden cessation of milk removal, either natural or indicated by the dairy producer. Finally, senile involution occurs at the end of the reproductive life of the animal. Initiated involution of mammary glands occurs after cessation of milking. Regression of mammary secretory tissue accompanies dramatic changes in secretion composition during the transition fi-om lactation to a non-functional gland. As described above, it has been shown that dairy cows require a nonlactating period prior to the next lactation to achieve maximal milk production during that lactation (Coppock et al., 1974). Adequate proliferation and differentiation of mammary secretory epithelium during the nonlactating period are essential for optimal secretory function during the subsequent lactation and duration of the nonlactating interval is related significantly to milk production (Akers and Nickerson, 1983). Smith and Todhunter (1982) suggested that there are three important stages during a typical dry period. The first stage is one of active involution which begins with cessation of milking and is completed within 21 to 28 d. This stage is characterized by engorgement of cisternal spaces, ducts and alveoli with milk constituents, gradual changes in mammary secretion composition, and regression of mammary tissue. The second stage is that of steady state involution representing fially involuted mammary gland. The final stage represents colostrum formation and initiation

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160 of lactation which begins about 14 d before parturition. Near parturition, mammary glands undergo significant changes characterized by intense growth, rapid differentiation of secretory epithelial cells, and synthesis and secretion of proteins, fat and carbohydrates leading to accumulation of colostrum. Thus, according to this view, a 45 to 60 d dry period would represent an active period of involution until the involution phase was completed, followed by redevelopment of mammary gland beginning 21 to 28 d prior to parturition (Hart and Morant, 1980; Nickerson and Akers,1984). Much of the information on involution has been based on research conducted using laboratory animals. In rodents, continuous milk production during lactation is dependent upon a complex interplay of lactogenic hormones and the suckling stimulus exerted by the young. Involution can be initiated in the mouse mammary gland at any stage of lactation by removing the pups (Richards and Benson, 1971). Cessation of milking causes accumulation of milk in alveoli and ducts, and this increases I intramammary pressure that causes degeneration of secretory cells and subsequent disruption of alveolar and lobular structures. In rat, involution is associated with a massive engorgement of the gland with milk followed by apoptosis of secretory epithelial cells and destruction of the gland. Involution remains reversible for about 30 to 36 h after it has been initiated (Richards and Benson, 1971). After weaning, the decline in lactogenic hormones and milk stasis leads to involution, a process that is mainly characterized by three events: (i) downregulation of milk protein gene expression, (ii) loss of epithelial cells by apoptosis, and (iii) tissue remodeling and preparation of the gland for a new lactation. Each of these processes is likely to depend upon the activity of specific sets of transcription factors in the mammary

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161 epithelium and stroma that ensure the timely and spatially coordinated expression of critical gene products (Marti et al., 2000). In the mammary gland, secretory epithelial cells are removed by apoptosis during involution. A number of transcription factors were found to control apoptosis. These include c-Fos, c-Jun (Colotta et al., 1992), p53 (Yonish-Rouach et al., 1991), E2F (Qin et al., 1994), Myc/Max (Amati et al., 1993) and STATS (Liu et al., 1996). STATS was reported to be activated during pregnancy and lactation at the start of involution. This activation was characterized by removal of epithelial cells by apoptosis (Liu et al., 1996; I Walker et al., 1989). STATl also is activated during latter stages of involution when there is remodeling of the mammary gland (Liu et al., 1996). The timely breakdown of extra-cellular matrix is essential for remodeling (Nagase and Woessner. 1999). The cell loss coincides with matrix metalloproteinase (MMP) activation and basement membrane degradation in rats. Using in vitro culture, it was demonstrated that first passage epithelial cells isolated from pregnant mouse mammary gland die by apoptosis (Pullan et al., 1996). On the other hand, cell death was suppressed by basement membrane suggesting the requirement of basement membrane for cell survival (Pullan et al., 1996). In the same experiment, blocking of integrin with anti-beta 1 integrin antibody doubled the rate of apoptosis, whereas expression of Bcl-2 did not correlate with cell survival. However, increased levels of Bax were associated with apoptosis. Thus, basement membrane provides a survival stimulus for epithelial cells in vivo and loss of interaction between cells and this type of matrix may act as a control point for cell deletion during mammary gland involution (Pullan et al., 1996).

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162 In rat, mouse and rabbit, autophagocytic and heterophagocytic mechanisms play a very important role in tissue degeneration during mammary involution (Hoist et al., 1987) . Formation of lysosomes and structures containing cytoplasmic organelles (cytosegrosomes) have been interpreted as evidence of autophagocytosis of alveolar cells. This often was accompanied by dissociation of epithelial cells from the basement membrane (Richards and Benson, 1971a), whereas infiltration of mononuclear leukocytes into involuting tissue has been associated with heterophagocytosis of degenerating cells and cellular debris (Richards and Benson, 1971b). Thus, involution in the rodent mammary gland is distinguished by the separation of epithelial cells from the basement membrane and extension of myoepithelial cells to fill the gaps left by the discarded epithelial cells (Hoist et al., 1987). Ruminant mammary epithelial cells, on the other hand, apparently do not regress to the same extent as occurs in rodent mammary glands. They appear to maintain some synthetic and secretory activity throughout the nonlactating period. This conclusion is based upon conditions of mammary involution in dairy cows that generally are considerably different from laboratory species (Hoist et al., 1987; Sordillo and Nickerson, 1988) . Dairy cows still are producing large quantities of milk and most often they are pregnant at the time of milk cessation by initiated involution or stoppage of milk removal I (Oliver and Sordillo, 1989). Even in the absence of pregnancy, mammary involution in dairy animals occurs at a slower rate than in rodents (Li et al., 1999). Alveolar structure is maintained for several weeks and lactation can be reinitiated after 4 wk or more of involution. Although apoptosis in the mammary tissue of ewes appears to be initiated within a similar time frame to that in rodents, beginning about 2 d after cessation of

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I 163 milking or postweaning with a peak at about 4 d, the maximum proportion of apoptotic epithelial cells appears to be less than in rodents, and apoptosis may be accompanied by an initial increase in cell proliferation (Capuco and Akers, 1 999). Alveolar structure of cows is largely maintained and little or no loss of cells occurs with cessation of milking. On the other hand, there is increased apoptosis and cell proliferation in freshly dried off mammary glands, relative to that in lactating glands during the same stage of gestation. Thus, it appears that a nonlactating period serves to promote cell turnover prior to the next lactation (Capuco and Akers, 1999). In contrast to the view that suggests an active involution process during dry period requiring 45 to 60 d in dairy cows (Smith and Todhunter, 1982), more recent studies (Capuco et al, 1997; Capuco and Akers, 1999; Li et al., 1999) imply that the dry period is important for replacement of damaged cells before the next lactation starts but not for extensive degeneration of mammary gland structure and apoptosis. This suggests that 45 to 60 d dry period may not be the optimal time interval for maximum production of dairy cows and that the length of the dry period may be shortened without a negative effect on the cow's subsequent lactation performance. Sordillo and Nickerson (1988) examined the morphologic changes in the mammary glands of 5 cows during mammary involution. Mammary tissue samples were obtained weekly beginning the day milking was discontinued through next parturition. As involution progressed, a gradual reduction in synthetic and secretory activity of alveolar epithelium was noted under light and electron microscopic evaluation. During the first 2 wk of involution they observed increased stroma and nonactive secretory epithelium with concomitant decreases in epithelium, lumen, and fully active secretory epithelium. There

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164 was a decreased number of organelles associated with milk synthesis and secretion at the alveolar epithelium level. These changes were gradually reversed beginning 2 wk before parturition and by the time of calving occurred cell structure was typical of the lactating mammary gland. Free fatty acid levels in milk increased more than 10-fold in cows during mammary involution (Thompson, 1988). Their appearance did not immediately follow the cessation of milking but followed the increase in permeability of the mammary epithelium which paralleled changes in the electrolyte content of the milk. However, the 1 concentration of free fatty acids did not remain high throughout the dry period but declined to low levels before the change in permeability was reversed at the next parturition. They concluded that the high level of free fatty acids in milk during mammary involution most likely arises from breakdown of triglycerides remaining in the gland and this may be accelerated in some manner by the increase in permeability of the mammary epithelium (Thompson, 1988). Lactating gland morphological studies in goat by Li et al. (1999) showed tightly packed secretory alveoli with columnar shaped alveolar cells that retained a large apical secretory vesicle. Secretory alveoli were separated by small amounts of interstitial connective tissue. After drying off, the unmilked gland showed a lactating morphology I for 3 d with reduced number of alveolar cells and more intralobular stromal tissue around the alveolus. However, less than 1% of alveolar epithelial cells underwent apoptosis as determined by both presence of TUNNEL-positive cells and DNA fragmentation in tissue extracts. The lumen of unmilked alveoli contained residual secretion and polymorphic neutrophils that infiltrated the parenchyma. Morphology of the unmilked gland changed 7

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165 d after milk removal ceased. Alveolar cells lost their columnar shape and the cytoplasm contained a large apical vesicle with a compressed ill-defined nucleus. Apoptic bodies were observed next to alveolar cells (2-3% of alveolar cells) within some alveoli. During the second week, the myoepithelial cells were pronounced and formed a band around each alveolus. Body defense cells were present among the structurally deficient alveolar cells which had indistinct cell membranes with an intensely stained pyknotic nucleus. Apoptic cell rate was estimated to be 5%. Three weeks after the cessation of milking, fibrocollagenous tissue separated the ducts and ductules. Apoptotic bodies were present in most ductal structures in and around the mammary parenchyma with masses of body defense cells. In this study (Li et al., 1999), apoptosis was not limited to the dry glands. Infrequent TUNNEL-positive cells and low level of DNA fragmentation also were observed in milked glands suggesting apoptic cells could have accounted for the net decrease in mammary cell numbers of the goat during declining lactation. Capuco et al. (1997) concluded that, in contrast to rodents, no net loss of mammary cells occured during the dry period in dairy cows. In their experiment, dry and lactating cows were sacrificed on days corresponding to different number of days into the dry period and mammary tissues were sampled for total DNA and RNA and morphological analyses. Neither parenchymal weight nor DNA content differed in glands of lactating or dry cows. Seven days into the dry period, DNA content was identical in glands of dry and lactating cows and subsequently increased more rapidly in dry glands than in lactating glands. More epithelial cells (96%) were labeled with [^H]Tdr in dry glands than in lactafing glands (86%). Based upon morphology, 62% of epithelial cells in lactating glands contained secretory vesicles and lipid droplets and these were not i

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166 affected by day of gestation. However, if cows had been dry for 7 d, 25% of epithelial cells appeared to be secretory. At 35 d prepartum (cows dry for 25 d), none of the epithelial cells were secretory. Cells showing secretory activity increased to 78% and 98% after d -35 and d -7, respectively. They concluded that processes of proliferation and cell turnover seemingly increased the percentage of epithelial cells in dry mammary glands prior to parturition (Capuco et al., 1997). Earlier studies suggested that along with the changes in composition of mammary secretions, mammary gland was fully involuted during the steady state involution phase in cows (Smith and Todhunter, 1982). During this process, it was believed that total destruction of the gland occurred. However, as a result of more recent studies, it has been suggested that mammary involution is an inappropriate term to describe changes that occur in the bovine mammary gland during the dry period. Current results do not support a net loss of mammary cells during the characteristic 60 d dry period in dairy cows (Capuco et al., 1997). Apparently, the bovine mammary gland does not degenerate to the virgin state as it does in rodents during the dry period. The retention of alveolar structures still can be seen 30 d following milk stasis (Capuco et al., 1997; Hoist et al., 1987). Rather, it has been suggested that the dry period is necessary to allow replacement of damaged or senescent epithelial cells prior to the succeeding lactation (Capuco et I al.,1997). Because mammary gland completed involution by 25 d dry (Capuco et al., 1997), it may not be necessary to have 60 d dry period in dairy cows. Thus, it is possible that the dry period could be shortened to 30 to 35 d without an effect on subsequent milk production. However, this conclusion implies that they have regained adequate BCS and replenishment of body tissue reserves that will be needed during the subsequent lactation.

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167 Plasmin and Involution Throughout lactation, milk contains a number of proteases such as leukocyte derived proteases, milk acid protease and plasmin (Aslam and Hurley, 1998). Plasmin has been implicated in the destructive phases during gradual involution because most proteolytic activity found in milk is stimulated by plasmin (Politis et al., 1989). Plasmin is an extracellular serine protease that is formed by cleavage of a peptide bond in the single polypeptide chain of the inactive proenzyme plasminogen (Andersen et al., 1990). Plasmin generation in the extracellular space is initiated by cellular release of the single1 chain forms of the plasminogen activators (PA) and their subsequent activation is responsible for degradation of fibrin (Thorsen et al., 1984). Components of the plasmin system often are associated with casein miscelles but also are present in the serum and cream phases of milk (Politis et al., 1992). Plasmin appears to be responsible for fibrinolysis and thrombolysis, as well as for biological processes involving breakdown of cellular matrix and basement membranes such as cell migration, invasion, organ involution, tissue remodeling and destruction (Akers et al., 1990). The plasmin system is believed to have a role in the mammary gland during involution. Plasmin and its inactive zymogen, plasminogen, are two of the several significant proteases in bovine milk (Eigel, 1977). Plasmin in bovine milk exists mainly in its inactive form. Plasminogen activators in milk convert plasminogen to plasmin (De Rham and Andrews, 1982). Stage of lactation affects plasmin with late lactation associated with higher concentrations of plasmin (Politis et al., 1989). Potential mechanisms responsible for increased milk plasmin include an inflow of plasminogen from blood (Politis and Hang, 1989). ki the rodent mammary gland, PA converts

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168 plasminogen to plasmin during late lactation and this is associated with the onset of involution (Ossowki et al., 1979). Activation of plasminogen in bovine milk increases as lactation progresses and plasminogen activity increases further by d 3 after drying off (Politis et al., 1990). Elevated plasmin activity during involution of the bovine mammary gland is responsible for increased hydrolysis of casein and lactoferrin and protease activities other than those of plasmin do not seem to play a major role in protein I hydrolysis during involution (Aslam and Hurley, 1998). It has been suggested that the increased plasmin activity seen during late lactation may be involved in subsequent mammary gland involution (Politis et al., 1990) and estrogen accelerated plasmin activity in the mammary gland (Athie et al., 1996). Increased plasmin and PA in milk are correlated during the declining phase of lactation. It has been suggested that mammary gland involution can be partially reversed by bST administration via modulation of the plasmin-plasminogen system (Politis et al., 1990). Treatment with bST interferes with conversion of plasminogen to plasmin and this prevents the increase of plasmin in milk (Politis, 1996). Baldi (1999) studied the effect of bST treatment on the plasminogen system in late lactation dairy ewes. Even though I plasmin level was not affected, there was a significant reduction in plasminogen activity in these bST treated ewes. Moreover, the plasmin:plasminogen ratio decreased in treated I ewes, suggesting a retardation of conversion of plasminogen to plasmin in those animals. It has been suggested that bST may preserve the integrity of tight junctions in late lactation because the plasminogen in milk is derived mostly from the blood (Baldi, 1999)

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169 Materials and Methods The second phase of the study reported in Chapter 3 was designed to evaluate concentrations of hormones (ST and INS), growth factor (IGF-I), and metabolites (glucose and NEFA) in plasma of dairy cows during the period beginning 28 d before parturition and extending through 28 d postpartum. Blood samples were collected from 80 Holstein cows from the research herd at the DRU. Distribution of experimental animals, management, feeding program, drying off times and bST injections were described in detail in chapter 3. Plasma harvested from blood samples collected throughout the experiment were frozen at 20 °C until analyzed. Milk Samples Milk samples were collected weekly during three consecutive milkings (08:30, 15:00, and 01 :30 h) on same day of the week for analyses of milk constituents during the first 10 wk of lactation. Samples (50 mL) were analyzed for fat, protein and SCC contents I at Southeast Milk Laboratory, Inc. (Belleview, Fl). Milk yield was recorded at each daily milking from 3 d after parturition through 150 d postpartum. Plasma Collection, Handling and Storage Blood samples were collected from the tail vein of all cows three times weekly before the a.m. feeding or milking (07:30-10:00 h). Cows were bled from the tail vein I in the free-stall bam after elevating the tail without any other restraint. For blood collection, Vacutainer® brand needles (2.54 cm; 20 gauge) and tubes containing sodium heparin were used (10 x 100 mm blood collection tubes, Becton-Dickinson. Fairlawn, NJ). Blood samples were placed on ice immediately after collection and processed within 2 h. The order in which cows were sampled on a given day was random and differed from

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170 bleeding to bleeding. After sampling, cows were milked and then returned to the freestall bam. All samples of blood were centrifuged at 3000 RPM at 5°C for 30 min in the RC3B refrigerated centrifuge (6-place swinging basket, H.600A rotor, Sorvall Instruments, Wilmington, DE) to separate plasma. Plasma from each sample was aliquoted into 2 labeled 5 mL (75x12 mm) polypropylene tubes, capped, and frozen at -20°C until analyzed. The plasma samples were used for analysis of ST, E^JS, IGF-I, glucose and NEFA. Second Antibody Preparation Second antibody for use in radioimmunoassays was prepared in four Florida Native sheep managed at the DRU. Guinea pig gamma globulin (20-25 mg, Sigma Chemical Co, St. Louis, MO. # R-9135) and 30-40 mg of rabbit gamma globulin (Sigma Chemical Co, St. Louis, MO. # R-9133) were weighed into separate 25 mL Erlenmeyer flasks, then 5 tol5 mL of distilled water were added to each. After protein had dissolved an equal amount of Freud's complete adjuvant (P' injection only) or incomplete adjuvant (2"*^ and greater injections) was added to each flask and it was mixed in a microblender for about 1 min on high speed until the mixture had the consistency of whipped cream. Sheep were injected over the shoulder on both left and right sides with about 5 mL of a mixture of the two proteins. After 14 d, each was bled from the jugular vein to obtain about 400 mL blood. This was done using a 1 6-gauge butterfly needle inserted into jugular vein; a 60 cc syringe was attached to the tubing attached to the needle to withdraw the blood. Blood was transferred to 40 mL screw-top centrifiige tubes and placed on ice as soon as it was collected. Tubes then were refrigerated for 24 h at 4 °C to allow blood

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171 to clot. The clotted blood was centrifuged at 5000 RPM for 30 min in the RC-3B refrigerated centrifuge (6-place swinging basket, H.600A rotor, Sorvall Instruments, Wilmington, DE) to obtain serum. Serum was frozen until used in assays as the second antibody at appropriate dilutions. Sheep were bled again at 4 wk or greater intervals. They were reinjected with mixture of gamma globulins at about 4-6 mo intervals. Radioimmunoassays Double antibody radioimmunoassay procedures were used to determine concentrations of INS, ST, and IGF-I in plasma. All samples from individual cows were assayed in duplicate in a single assay. lodination and Protein Separation Insulin Bovine INS (100-300 ^ig; Sigma Immunochemicals, St. Louis, MO; #82F-0453) was weighed and mixed with an equal quantity of 5 mM HCl. Then, an equal amount of 0.01 M borate buffer (pH 8.0) was added to the solution to give a final concentration of 0.5 \ig INS /\iL buffer; 10 |iL of this solution were frozen in microcentrifuge vials (Fisher Scientific, 1.0 mL. flat top) The column used to separate the iodinated protein from free iodine was prepared by cutfing off the mouthpiece of a disposable 10 mL glass pipette. A small glass wool plug or glass bead was placed into the column before adding the Sephadex G-50 packing (Sigma Chemical; dispersed in 0.01 M phosphate buffer, pH:7.5) until the Sephadex filled the column. Subsequently, the column was washed with 2 mL 0.5 M phosphate buffer which cointained 0.5 % BSA followed by 10 mL of 0.01 M phosphate buffer. Phosphate buffer was retained at the top of the Sephadex bed until used for separation.

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172 Immediately prior to the iodination, 3 mg chloramine-T and 5 mg sodium metabisulfite were weighed into individual tubes and just prior to use each was dissolved in 1 mL of 0.5 M phosphate buffer. Then, 1 mCi (10 \iL) I'" was transferred to a borosilicate tube (12 x 75 mm) containing 10 INS and then 10 [iL of 0.5 M phosphate buffer were added. After mixing, 10 [xL of chloramine-T were added to the reaction tube, contents were mixed with finger tapping for 20 sec after which 10 p.L of sodium metabisulfite were added to stop the reaction. Sequentially, the solution containing INSl'^^ was transferred to the top of the Sephadex column, the reaction tube was rinsed with 50 [iL of 0.0 IM phosphate buffer and this also was transferred to the top of the column and allowed to flow into the Sephadex bed. Borosilicate tubes (15 x 100 mm), numbered 1-40, that contained 500 |j,L of 0.5 M borate-BSA buffer, were used to collect 20 drop fractions using a fraction collector (model FC-80 K micro fracUonaUon. Gilson Medical, Middleton, WI). The fractions collected were mixed by finger-tapping , then 1 0 [i,L of each eluted fraction were transferred to a second set of tubes (12 x 75 mm) to identify radioactive peaks by counting radioactivity using a Tracor Analytic Gamma Counter (NuclearChicago, Gamma Trac 1191, G.D. Searle and Co., Des Plaines, IL). Tubes fi-om the first large peak corresponding to I'" bound INS were saved and stored at 4°C until used in the radioimmunoassays. IGF-I One vial, containing 1 ^g of IGF-I (0.1 \ig/ |iL), was allowed to thaw at room temperature. The mouthpiece of a 10 mL disposable glass pipette was cut off with a glass tubing cutter, and then a small glass wool plug or glass bead was placed into the bottom

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173 of the glass pipette. Sephadex G-50 (Sigma Chemical, St Louis, MO) dispersed in 0.01 M phosphate buffer was transferred into the pipette until the Sephadex was between the 0 and 1 mark. Subsequently, the column was rinsed with 2 mL of 0.5 M phosphate buffer containing 0.5 % BSA, followed by 10 mL of 0.01 M phosphate buffer. Phosphate buffer was retained at the top of the Sephadex bed until the column was used for separation. Polypropylene microcentrifuge tubes (Fisher Scientific, 1 .0 mL, flat top) were washed with glacial acetic acid, rinsed with deionized water, and allowed to dry. One mg of lodogen (Pierce Chemical Co., Rockford, IL) was weighed and dissolved in chloroform to a final concentration of 100 ^ig/ml, then 20 ^iL of lodogen solution were transferred directly into the bottom of the acid-washed microcentrifuge tubes followed by 40 |iL of chloroform. The chloroform then was dried under a stream of air to leave the lodogen coated on the inside of the tube. For the iodination, 10 ^L of 1 .0 ^ig IGF-I, followed by 50 |iL 0.1 M phosphate buffer were added to the polypropylene microcentrifuge tube and mixed with 10 |j.L 1'" (1 mCi) and allowed to react for 5 min. Subsequently, IGFI-l'^^ solution was transferred to the top of the column. The reaction tube was rinsed with 50 \iL 0.01 M phosphate buffer, and it also was transferred to the I top of the column and allowed to flow into the Sephadex bed. Borosilicate tubes (15 x 100 mm), numbered 1-40, containing 500 ^L 0.5 M phosphate buffer were used to collect 20 drop fractions using a fraction collector (model FC-80 K micro fractionation, Gilson Medical, Middleton, Wl). The fractions were mixed by finger-tapping, then 10 ^L of each eluted fracfion were transferred to a second set of tubes (12 X 75 mm) to identify elution peaks by counting radioactivity using a Tracor analytic gamma counter (Nuclear-Chicago, Gamma Trac 1 191, G. D. Searle and Co., Des

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174 Plaines, IL). Tubes from the first large peak corresponding to l'" -bound IGF-I were saved and stored at 4°C until used for radioimmunoassays. Somatotropin [ /• ' ' * Bovine ST (100-300 ng; USDA Reproduction Lab. AFP 5200) was weighed and diluted with an equal quantity of 0.01 M NaHCOj. Then an equal amount of O.OIM phosphate buffer was added to the solution to yield a final concentration of 0.5 fig ST/ |j.L solution; 10 [iL of the hormone solution were frozen in microcentrifuge vials (Fisher Scientific, 1.0 mL. flat top) for use in iodinations. The column used to separate the iodinated ST fi"om free iodine was prepared by cutting off the mouthpiece of a 10 mL disposable glass pipette. A small glass wool plug or glass bead was placed into the bottom of the glass pipette. Sephadex G-75 (Sigma Chemical) dispersed in 0.01 M phosphate buffer was transferred into the pipette until the sephadex was between the 0 and 1 mark on barrel of the pipette. Subsequently, the column was rinsed with 2 mL of 0.5 M phosphate buffer containing 0.5% BSA followed by 10 mL of 0.01 M PO4 buffer. Phosphate buffer was retained at the top of the Sephadex column bed until column was used for ST separation. Three mg cloramine-T and 5 mg sodium metabisulfite were weighed into individual tubes and each was dissolved in 1 mL of 0.5 M phosphate buffer immediately I prior to the iodination. One mCi (10 |iL) l'^' was transferred into a conical plastic centrifuge tube containing 10 ^iL of ST solufion and then 10 p-L of 0.01 phosphate buffer were added. After mixing, 10 ^L of chloramine-T were added to the reaction tube and contents mixed with finger tapping for 20 sec after which 10 fiL of sodium metabisulfite were added to stop the reaction. Sequentially, ST1'-^ solution was transferred to the top

PAGE 191

175 of column, the reaction tube was rinsed with 50 ^iL O.OIM phosphate buffer and this also was transferred to the top of the column, and transferred solutions were allowed to flow into the Sephadex bed. Borosilicate tubes (15 x 100 mm), numbered 1-40, were filled with 500 \iL of 0.5 M phosphate-BSA buffer and then 20 drop fractions were collected in the tubes using the fraction collector (model FC-80 K micro fractionation. Gilson Medical, Middleton, WI). The fractions were mixed by finger-tapping, then 10 |j,L of I each eluted fraction were transferred to a second set of tubes (12 x 75 mm) to identify elution peaks by counting radioactivity using a Tracor Analytic Gamma Counter (NuclearChicago, Gamma Trac 1191, G.D. Searle and Co., Des Plaines, IL). Tubes from the first large peak corresponding to I'^-^-ST were saved for radioimmunoassays and stored at 4°C until used in the assays. Assays . Insulin assay ' | i* ' A double antibody radioimmunoassay procedure, as described by Soeldner and Sloane (1965), and modified by Malven et al. (1987b), was used for assay of INS in plasma. Highly purified INS (Sigma Immunochemicals, St. Louis, MO. 1 1 8F-4826) was weighed (^ 100 \ig), then dissolved in 30 mM HCl (pH 2.5). The INS was (10 \ig/\0 \iL) aliquotted into 1 mL microfuge vials and frozen unfil used. Stock INS was diluted in borate/BSA assay buffer (0.133 M Borate, 0.01% Merthiolate and 0.5% BSA , pH=8.0) to give a final concentration of 100 ng/ml for preparation of standards. Twelve standards containing 0.3, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10, 15, 20, 25, and 30 ng INS/ml were prepared and frozen in 5 mL aliquots. For preparation of first antibody, Guinea pig anti-bovine INS (Sigma Chemical CO., St. Louis, MO) was dissolved in borate/BSA buffer

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176 (1:20,000). Second antibody (anti guinea pig and rabbit sheep serum) was diluted 1 :4 in borate /EDTA buffer (1 .86 g EDTA in 100 mL borate buffer). Arrangement of assay tubes is in Table 4-1. Plasma samples (150 |iL) were assayed in duplicate along with 150 |iL borate buffer (0.5% BSA) in 12 x 75 mm borosihcate tubes, and 100 |iL first antibody (except the tubes for total count, NSB-B, and NSB-P) were added. Then, 12 h later, 100 [iL iodinated INS (^ 25,000 CPM) were pipetted into all tubes. After a 24 h incubation at 4°C (starting from addition of first antibody), 100 diluted sheep anti-guinea pig second antibody (SAGP, 1 :4 dilution) and 100 \iL normal guinea pig serum ( 1 : 1 OOdilution) were added to all tubes except the total count tubes. Tube contents were mixed then allowed to stand for 1 0 min. Table 4-1 . Arrangement of Assay Tubes for INS Tubes* Samples Buffer** F' Antibody I'" INS Plasma TCT 100 NSB-B 400 100 NSB-P 250 100 150 ZERO 300 100 100 STANDARDS 100 200 100 100 SAMPLES 150 150 100 100 *TCT=total activity count tube, NSB-B=non-specific binding for buffer, NSB-P=nonspecific binding for plasma, ZERO=Teference, no INS added. **borate/0.5% BSA buffer. Volumes are in |iL. Following the 10 min incubation, 750 [iL of 15% polyethylene glycol (PEG; Sigma Immunochemicals, St. Louis, MO.) in borate buffer were added to all tubes except for the total count tubes and tubes were vortexed for 1 min. Tubes then were centrifuged

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177 at 3000 RPM for 30 min at 4°C (RC-3B refrigerated centrifuge with 6 place swinging basket, H.600A rotor, Sorvall Instruments) decanted, and allowed to dry in inverted position. Bound radioactivity in the dry tubes was measured with a Packard® auto gamma counter (model B-5005). Final results were calculated using the spline radioimmunoassay data processing procedure for a coded assay to correct for any differences in NSBs for plasma and buffer. Somatotropin assay Highly purified bovine ST supplied by USDA (AFP-5200) was used for preparation of standards: one mg bovine ST was diluted in 10 mL of 0.01 M NaHCO^ to give a concentration of 100,000 ng/ml, then 2.0 mL of this solution were diluted to 20 mL with PBS/1.0% BSA to give a concentration of 10,000 ng/ml. Finally, 1.0 mL of this solution was diluted with 99 mL PBS/1 .0% BSA to give a final concentration of 100 ng/ml for preparation of standards. A set of 12 standards was prepared to contain 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 ng ST/lOO^l. For preparation of first antibody. Rabbit anti-ovine ST (National Hormone and Pituitary Program) was diluted in 0.01 M phosphate /BSA buffer (1 :40,000). Second antibody (anti guinea pig and rabbit sheep serum) was diluted 1 :4 in phosphate/EDTA buffer. Plasma samples (100 ^L) were assayed in duplicate and were diluted with 200 jxL 0.01 phosphate buffer containing 0.25% BSA in 12 x 75 mm borosilicate tubes. Immediately after samples were pipetted, 100 |j,L first antibody (except for total count, NSB-B and NSB-P tubes) and 100 |iL iodinated ST were pipetted into all tubes. After incubation for 24 h at room temperature, 100 |iL diluted second antibody (1 :4) and 100 jiL normal rabbit serum (NRS, 1:100 dilution) were added to all tubes, except the total

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178 count tubes, and mixed (Table 4-2). Then, 1 .0 mL of 6% PEG (Sigma Immunochemicals, St. Louis, MO.) in 0.01 M phosphate buffer was added to all tubes, except total count tubes, and vortexed for 1 min, reactants were incubated for 10 min, then all tubes were centriftiged at 3000 RPM at 4°C for 30 min (RC-3B refrigerated centrifuge with 6 place swinging basket, H.600A rotor, Sorvall Instruments) decanted, and allowed to dry in inverted position. Bound radioactivity in the dry tubes was measured with a Packard® auto gamma counter (model B-5005). Final results were calculated using the spline radioimmunoassay data processing procedure as a coded assay to correct for any i differences that may exist in NSB-B and NSB-P. Table 4-2. Arrangement of Assay Tubes for ST. Tubes* Samples Buffer** r' Antibody jl25 sj Plasma TCT 100 NSB-B 400 100 NSB-P 300 100 100 ZERO 300 100 100 STANDARDS 100 200 100 100 SAMPLES 100 200 100 100 *TCT=total activity count tube, NSB-B=non-specific binding for buffer, NSB-P=nonspecific binding for plasma, ZERO=reference, no ST added. **PBS/0.25%BSA buffer. Volumes are in \iL. IGF-I A double antibody radioimmunoassay, as described by Abribat et al. (1990) and modified for sample extraction by method of Enright et al. (1989) and Daughaday et al. (1980), was used for IGF-I determination in plasma samples.

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179 Extraction of IGF-I from binding proteins. The method of Enright et al. (1989) was used for the extraction of IGF-I from its binding proteins in plasma samples. An extraction mixture of ethanol, acetone, and acetic acid (EAA 60:30:10 by volume) was used for extraction. Exactly 100 |iL plasma (100 \iL distilled water for NSB-P) and 400 |iL of extraction mixture were pipetted into 12 x 75 polystyrene tubes. Tube contents were mixed for 15 sec on a vortex and then were allowed to stand for 30 min at room temperature. Tubes then were centriftiged at 3000 RPM for 30 min at 4°C (RC-3B refrigerated centrifuge with 6 place swinging basket, H.600A rotor; Sorvall Instruments). Then 250 |iL of the supernatant were transferred to polystyrene tubes (12 x 75), and 100 \iL of 0.855 M trizma base and 350 |j.L of the assay buffer were added to make the final dilution 1:14. Assay. Highly purified Bovine Insulin-like growth factor-I (IGF-I), supplied by Upstate Biotechnology (Lake Placid, NY. Cat# 01-189) was dissolved (10 ^lg) in 100 of 0.1 M acefic acid to give stock 0 (100 ng/mL); this was aliquoted into microcentrifuge tubes (10 nL/tube) and frozen. To make stock 1, 10 |iL of stock 0 were added to 490 \iL of assay buffer. Stock 2 was made by adding 10 |iL of stock 1 to 990 |xL of assay buffer to give a final concentration of 20 pg IGF-I/ mL. Standards were prepared from stock 2 to contain 50, 100, 200, 300, 600, 800, 1000, 1200, 1500, and 2000 pg IGF-I/ml. The I first antibody, rabbit anti-bovine IGF-I, (Lot #AFP4892898), was dissolved (1:160000) in assay buffer (200 mg protamine, 4.4 g sodium monobasic phosphate, 10 mL of 2% sodium azide, 3.72 g EDTA and 2.5 g BSA in 1 L). The SAR was diluted 1:3 in EDTA. Twenty [iL of plasma extract were mixed with 180 nL assay buffer. Then, 100 \iL iodinated IGF-I were added to all tubes, and 100 jiL of first antibody were added to

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180 reference, standards and samples but not to NSB tubes. All tubes were incubated for 24 h at 4 °C. After incubation, 50 \iL second antibody diluted 1 :3 in assay buffer and 50 normal rabbit serum (1:50) were added to all tubes except the total count tubes. Samples were allowed to stand 30 min and then 1 mL of 6% PEG (Sigma Immunochemicals, St. Louis, MO.) in assay buffer was added, tubes were vortex ed and allowed to stand 1 5 min, and then they were centrifiiged at 3000 RPM for 30 min at 4°C (RC-3B refrigerated centrifiige H.600A rotor, Sorvall Instruments). Finally, supernatant in tubes was decanted and tubes were inverted on absorbent paper to dry. Finally, bound radioactivity in the dry tubes was measured using a Packard® auto gamma counter (model B-5005). Final results were calculated using the spline radioimmunoassay data processing procedure for a coded assay to correct for any differences in NSBs for plasma extract and buffer. ' ' ' Table 4-3. Arrangement of Assay Tubes for IGF-I. Tubes* Samples Buffer** P' Antibody I'^' PRL Plasma Extract TCT 100 NSB-B 300 100 NSB-P 280 100 20 ZERO 200 100 100 STANDARDS 100 100 100 100 SAMPLES 20 180 100 100 *TCT=total activity count tube, NSB-B=non-specific binding for buffer. NSB-P=nonspecific binding for plasma extract, ZERO=reference, no IGF-I added. **phosphate/1.0%BSA buffer. Volumes are in |iL.

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181 Determination of Glucose in Plasma Samples Sigma procedure No. 510 (Sigma Diagnostics, St. Louis, MO) was used for the quantitative enzymatic determination of glucose in deproteinized plasma samples as described by Raabo and Terkildsen (1960). Enzyme solution for the assays was prepared by adding one capsule of PGO enzymes, which contained 500 units of glucose oxidase (catalog no. 510-6) to 100 mL deionized water in an amber bottle. Color reagent solution was prepared by reconstituting one vial of o-Dianisidine Dihydrochloride (catalog no 510-50) with 20 mL deionized water. Then, 100 mL of enzyme solution and 1 .6 mL of color reagent solution were mixed by mild shaking. Glucose standards (0, 25, 50, 75, and 1 00 mg of glucose/dL) were prepared by diluting the glucose standard solution provided (100 mg/dL) with deionized water to achieve the desired standard concentrations (Table 4-4). Along with standards, 100 \lL of unknown plasma samples were pipetted into borosilicate tubes (Fisher Scientific, Pittsburg, PA). Then, 900 |iL of distilled water were added to bring volume to 1 mL. Plasma was deproteinized by adding 500 [iL barium hydroxide solution (0.3N) and 500 [iL zinc sulfate solution (5%) into all tubes, including standards. All tubes were vortexed for 30 sec and centrifuged at 3000 RPM for 30 min at 3 ° C (RC-3B refrigerated centrifuge with H.600A rotor, Sorvall Instruments). Ninety six-well flat bottom polypropylene micro-plates (0.50 mL capacity, Sarstedt Inc. Newton, NJ) were used to complete assays. Standards and samples were assayed in triplicates and duplicates, respectively. Twenty microliters of supernatant (standards and plasma) were added to wells followed by 200 |iL of combined enzyme-

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I 182 color reagent solution and plates were incubated for 30 min at 37 °C in a constant temperature oven (model DN-41, American Scientific Products). After incubation, the absorbance was read in an Automated Microplate Reader (Model EL 309, Bio-tek ,, • « Instruments, INC., Laboratory Division, Winooski, VT) using blank as reference at 450 nm wavelength. Linear regression of absorbence and glucose concentration was used to determine the concentration of glucose in plasma samples. Table 4-4. Standards for Glucose determination. Standards (mg/dl) Glucose Stock Deionized water Final Volume Solution (l.OmM) 0 0\lL 400 liL 400 ^L 25 100 \iL 300 |iL 400 |iL 50 200 \iL 200 \iL 400 \iL 75 300 \iL 100 |iL 400 \iL 100 400 ^L 0 [lL 400 [iL Determination of NEFA in Plasma Samples In vitro enzymatic colorimetric method (NEFA C, Wako Pure Chemical Industries, Osaka, Japan) was used for the quantitative determination of NEFA in plasma as described by Johnson and Peters (1993). Color reagent A solution was prepared by adding 1 0 mL of dilution solution A and 13.3 mL 50 mM phosphate buffer (4.6 g Sodium phosphate monobasic, 14.2 g Sodium phosphate dibasic in 500 mL distilled water, pH 6.9) to one vial of dry color reagent A. Color reagent solution B was prepared by adding 20 mL of diluent for color

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183 reagent B and 33.3 mL of 50 mM phosphate buffer to one vial of dry color reagent B. Vials containing reagents A and B were mixed gently and stored at 5 °C for up to 2 wk. Specific concentrations of standards (0, 200, 400, 600, 800 and 1000 ^Eq NEFA/L) were prepared by diluting the NEFA standard solution provided (1000 p,Eq/L) with 0.9% saline solution (9 g of NaCl in 1000 mL deionized water) to achieve the desired standard concentrations (Table 4-5). Ninety six-well flat bottom polypropylene micro-plates (0.50 mL capacity, Sarstedt Inc. Newton, NJ) were used. Standards and samples were assayed in triplicates and duplicates, respectively. Then, 2.5 \iL of , Isupematant (standards and plasma) were added to wells followed by 50 [iL of Wako Reagent A and these were incubated for 30 min at 25 °C in a constant temperature oven (model DN-41, American Scientific Products). After incubation, 100 ^L of Wako Table 4-5. Standards for NEFA determination. Standards (|iEq/L) 0 200 400 600 800 1000 Oleic Stock Solution (l.OmM) 0 |iL 100 ^L 200 [iL 300 \xL 400 nL 500 |iL 0.9% Saline 500 \iL 400 \iL 300 ^L 200 \iL 100 [iL 0 [iL Reagent B were added to the wells and placed in the oven for an additional 30 min at 25 °C. Microplate then was allowed to sit for 5 min on the bench at room temperature after which absorbance was read in an Automated Microplate Reader (Model EL 309, Bio-tek histruments, INC., Laboratory Division, Winooski, VT) using blank as reference at 550

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184 nm wavelength. Linear regression of absorbence and NEFA concentration was used to determine the concentration of NEFA in plasma samples. Statistical Analyses ' Data collected during the experiment were analyzed in two sections. The first section included data collected during the 21 d prepartum period. The second section included data collected during the 28 d postpartum period and during 150 d for MY. Data were analyzed using Proc GLM procedure as a nested design by least squares analysis of variance procedures of SAS (1991). Additionally, Mixed model was used to 1 compare specific least squares means (Littel et al., 2000). Statistical analyses were performed for BW and BCS, milk and 3.5 % PCM yields, and concentrations of ST, fNS, IGF-I, glucose and NEFA in plasma. Time periods considered for data analyses were the prepartum period (-21 to Id), overall postpartum period (1 to 28 d) and 0-150 d postpartum period for MY. Models included the main effect of bST treatment (bST), 1 effect of dry period length (DRY), effect of prepartum diet (DIET), season (SEA; 1= cows with dry periods during hot months {September, October, March, April, and May}, 11= cows with dry periods during cool months {November, December, January, and Febaruary}), interactions among the treatments and SEA, cow(bST*DRY*DIET*SEA), and weeks or days to the highest order significant for overall prepartum and postpartum periods. Regression analyses was performed to the highest order significant up to cubic order to describe the trends in measures during postpartum period and for MY during the overall postpartum period. Tests for heterogeneity of regression was performed to

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185 determine whether there was evidence that regression curves were not parallel (Wilcox et al., 1990). Specific models are described in the Results section. Significance was declared at P<0.05, except where noted. Results Objectives of this study were to evaluate changes in plasma concentrations of ST, INS, IGF-I, glucose and NEFA during the period of blood sampling from -21 d before calving through +28 d postpartum, and MY through 150 DIM. Plasma concentrations of ST, INS, IGF-I, glucose and NEFA and MY were analyzed for treatment effects [Somatotropin treatments (control and injected), dry period treatments (30 d dry, 30 d dry + ECP or 60 d dry), prepartum diet treatments (anionic diet or cationic diet), and season effects]. Data were obtained from 84 Holstein cows for MY and 80 of those cows for blood measures (4 cows were not bled) as described in Chapter 3. Hormones, Growth Factor and Metabolites Prepartum period Least squares analyses of variance for all blood plasma measures are in Tables 4-6 and 4-7. No differences between bST treatment groups were detected for mean concentrations of glucose and NEFA during the overall prepartum period from d -21 to d -1, but differences were detected for the other measures. Significant effects of bST treatment were detected for ST (P<0.0065), IGF-1 (P<0.0001), and INS (P<0.0125) during prepartum period (Table 4-6). Least squares means and SE for all dependent blood plasma variables during the overall prepartum period are in Table 4-8. Mean concentrations of ST during the overall

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186 prepartum period were greater for bST treated cows (8.19 vs 5.51 ng/mL) and increased concentrations were maintained throughout the prepartum period (Figure 4-1). Plasma concentrations of IGF-I during the prepartum period (from d -21 to d-1) are in Table 4-8. Mean concentrations of IGF-I during the last 3 wk prepartum differed due to treatment (P<0.01). Cows in bST treated group had greater mean plasma concentrations of IGF-I than cows in untreated group (318.7 vs 235.2 ng/mL; 35.5%). Overall, plasma concentrations of IGF-I decreased progressively from d -21 to parturition in both groups of (Figure 4-2); means on d -1 for the treatments were 160.7 ng/ml (untreated) and 244.2 ng/ml (bST), which corresponded to decreases of 31.7 and 23.7% from d -2 1 , respectively. Mean concentrations of INS also were significantly greater (24.7%) for bST treated cows during the overall prepartum period (0.85 vs 1.06 ng/ml, respectively; Table 4-8). Concentrations of INS declined in both groups as they approached calving but concentrations remained less for cows in untreated compared to bST injected throughout the prepartum period. However, the decline appeared sharper begirming d -3 for cows in the uninjected group because concentrations declined from greater to essentially the same during the week before calving (Figure 4-3). Although overall prepartum mean concentrations of glucose did not differ due to bST treatment (Table 4-8), plasma concentrations of glucose increased significantly at d 14 in bST injected cows and tended to stay higher through calving (Figure 4-4). Increase in mean plasma concentrations of glucose for the bST injected group at d -1 was about 6.4% greater than d -12 and this was significant (P<0.001).

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187 Mean concentrations of NEFA are in Table 4-8. Considering the overall prepartum period (from d -21 to -1), mean concentrations of NEFA in plasma did not differ between the two bST treatment groups (265.0 vs 273.5 |iEq/L, respectively). Even I though mean plasma concentrations of NEFA were steady during the prepartum period, a significant increase was observed beginning d -3 prepartum within groups and mean j concentrations were greatest around calving for both groups (Figure 4-5). > j £ | No differences in mean concentrations of ST and NEFA were detected among dry period groups during the overall prepartum period. However, treatment differences were detected for IGF-I (P<0.0037), INS (P<0.0644) and glucose (P<0.0188). Plasma concentrations of ST for the three dry period treatments did not differ during prepartum period. Cows in 60 d dry period had the lowest mean concentration of ST (5.91 ng/mL), whereas cows in 30 d dry and 30 d dry +ECP groups had greater concentrations of ST but they did not differ significantly during the overall prepartum period (7.17 ng/mL vs 7.48 ng/mL). Although cows in 30 d dry +ECP group had numerically greater mean concentrations of ST at d -21 and d -18, they did not differ significantly. Moreover, no differences were detected among the three treatment groups for any days of the prepartum period (Figure 4-1). On the other hand, prepartum mean concentrations of IGF-I were greatest (13%) for cows in 30 d dry ECP group (309.8 I ng/ml), whereas cows in 60 d dry and 30 d dry groups had lower mean concentrations (269.7 and 251.5 ng/mL, respectively; Table 4-9). Cows in 30 d dry + ECP group had highest plasma concentrations of IGF-1 on d -2 1 , but plasma concentrations of IGF-I decreased progressively for all three treatment groups as cows approached parturition.

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188 S GO C •c Q O u .c '5 «-* o c O C 173 I CS C u u c o U § •c > o IT. 2 -o ^ 2 dj ^ fa cd I — — r~ o o o d o d o d o d o d d d * — — moo — o o c^ o o odd o d r^ 00 ^ — ^ — \i O — so Tt n o (N "O 00 so r*^ r— — 00 00 ri c> o r*N »n 00 O o oo < z o o rON o On t >n * u. o^ ON o On ON o o o 00 ON 00 n m p rn »n d ON d NO ON d in V-i d ON ON ON o rNl (N fN ON VfNj NO •o (N CNI rN) *N rsi nO CU u >-l I H o 'C u cd a u < ^ E w S S W3 Q Q UJ < S UJ Q CO J3 (UJ < ttJ CO tu > ai Q < UJ > a: Q < UJ oo * > a5 Q * H C/5 UJ 5 « < UJ CO * >• Q < lUJ > OS D < UJ CO * !UJ > ai D * co > < a > < * > < a < a * > < a * > < < « ^ S 2 .> it H ^ t3 IO-a 5 ° to « PI CO ^ 1 o o w E E gcg o 5 u J= « p. 00 S I? c « Sc -o 3 O •o c c II o Si 5 1°i S ^ o. o 5 £ " 3 a. JL -o JL O "O 4> CO CO UJ CO II S = < 3 .9 UJ c c CO ^ < ^ u i ;^ to ^ a — PC ^ i i « £ . S >i E ^ 2 t O Lu 03 eg ^ ^ ^ -2 CO ^ tS c u -no.

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189 d 00 — m o r— Tt (N — r-^ — 00 OO O ^ 1^ fN 00 — CN O O O O TT rn O (N O O d: d d d> <6 d — 00 rj— O tN — d * i~ o _ >o — d d — o — ^ sd — 2 d u. A 0. 00 00 o 00 ON t 00 1-* 00 r>o ro o o o o vo O o m 00 (N IN o o o »n o o o r-i o o ao o o o d d d d d d d d d d d d d d d d d d d z d o d o — d n Ov p rIN 00 00 v\ OV (> 00 00 vd vd P^ o o IN 5t 00 vO IN Ov a^ o vO tON m fS l~ vO "O rvo vo IN O 00 IN CO vO a < on (>a « (tu < UJ H ID < UJ 00 * >• a: Q < UJ • > O • H (A < UJ (/3 < UJ » UJ 5 P > OS Q < UJ C/J * >• Q H UJ D * o U > < * >• < D < O * > < Q * >< D

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190 oo n d d d >n VO m VO in o Ov 00 40.8 d d vd d -H -H -H n ' 1 r ' *

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Overall, on d -1, concentrations were about 40% less for all cows relative to concentrations on d -21 (Figure 4-2). Least squares means and SE of INS for cows in the three dry period treatment groups are in Table 4-9. Mean concentrations of INS during the overall prepartum period were greatest for cows in 30 d dry that had been injected with ECP (1.09 ng/mL). Plasma concentrations of INS were similar for cows in 30 d dry group (0.89 ng/mL) and 60 d dry groups (0.88 ng/mL). Overall, concentrations of INS tended to decrease slightly from d 21 to d -1 in both 30 d dry period treatment groups (Figure 4-3). Mean concentrations of glucose in plasma during the same time period tended to parallel that of INS and concentrations also were greatest for cows in 30 d dry + ECP group (73.5 mg/dL), and least for cows in the 30 d dry (69.1 mg/dL) and 60 d dry groups (68.9 mg/dL; Figure 4-4). On the other hand, plasma concentrations of NEFA did not differ among the dry period treatment groups during the prepartum period. All treatment groups had steady plasma concentrations of NEFA during the prepartum period until d -3. Beginning at d -1, there was a significant increase in concentrations of NEFA was observed within each of the three groups, mean concentrations were greatest and similar around calving for all three treatment groups, but there was about a 3-fold increase around calving (Figure 4-5). No significant effects of prepartum diet were detected for ST, IGF-I. However, plasma concentrations of NEFA were significantly higher (34 %, Table 4-10) for cows in cationic group (P<0.0018), especially around calving. Mean concentrations of ST during overall prepartum period did not differ due to diet (anionic or cationic) fed prepartum (Table 4-6). Cows fed the anionic diet had slightly, but not significantly, greater mean concentrations of ST than those fed caUonic

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, . . 192 I I c lU e P o Q 4J o o 5 ON p cn O o o 00 -H -H -H » n OO o\ 00 rON iri o od m V o 00 o o O 00 m (0 to 2 -H -H -W -H H stpartu OO rO o oq OO OS O CO cn r-fN in p ON o O OO -H -H -H « n r-ON u 00 in o ON in in VO OO o o 3 aW u i-< H T3 O 'C Ph Q ON >0 rjo o o ^ p ON ^ -H -H -ti -H od m ^ ON >n 00 VO 'ao d d p ON ^ -H -H -H -« ^ m vq 00 d OO CO -H m o >n o -H On m od -H O ^ H 00 60 E c O VO 0\ d m in NO E, u tn O o 3 VO m -H VO — 00 O VO 00 -H -H On VO 00 m cr W il. < w

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193 00 O B 3 v. w £ O 0I 3 cs ex C ID l-c H Q S 3 d C3 P. c as i5 6 3 C3 P. U 3 O O 5 oo in O O O o in -H o -H 00 o -H d o o d o o -H m d — • ON oo d d ^ p d -H -H -« -H 0\ ON m ? i 00 60 c O o "fcb u o o 3 -H -H in O VO -H -H -H -H 00 o CO p p p --^ d od in -H d m W 3. < w c 5 S C 3 O -p o Ou, cd U e 3 ^ V. oJ O O. 4> E cu B II I c H ^ S 'g. o o !& E § ed o JJ^ E c U V

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Figure 41 . Least squares mean concentrations of ST in plasma during the transition period (-21 d through 28 d). Arrow indicates calving.

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Figure 4-2. Least squares mean concentrations of IGF-I in plasma during the transition period (-21 d through 28 d). Arrow indicates calving.

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Figure 4-3. Least squares mean concentrations of INS in plasma during the transition period (-21 d through 28 d). Arrow indicates calving.

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Figure 4-4. Least squares mean concentrations of Glucose in plasma during the transition period (-21 d through 28 d). Arrow indicates calving.

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-21 -18-16-14-11 -9 -7 -3 -1 1 4 6 9 12 14 16 19 21 23 25 28 Days 1100 -21 -18-16-14-11 -9 -7 -3 -1 1 4 6 9 12 14 16 19 21 23 25 28 Days Figure 4-5. Least squares mean concentrations of NEFA in plasma during the transition period (-21 d through 28 d). Arrow indicates calving.

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199 diet (7.29 ng/mL vs 6.42 ng/mL, respectively). Although plasma concentrations of IGF-I did not differ between cows fed the anionic (274.9 ng/mL) or cationic diet (279.7 ng/mL) during the prepartum period, plasma concentrations decreased significantly from d -21 to d -1 for both groups (Figure 4-2). However, there were no differences in the final concentrations between treatment groups achieved before calving (204 vs 200 ng/mL, respectively). Mean concentrations of ESfS also were tended to be greater on d -21 for cows in both groups. Concentrations of INS in plasma declined as they approached calving in cows fed anionic or cationic diets (Figure 4-3). However, cows in anionic diet group tended to have numerically greater (not significant) mean concentrations of INS than cows fed cationic diet during the overall prepartum period (1 .00 vs 0.91 ng/mL, respectively; Table 4-10). Mean concentrations in glucose of cows fed the two diets are in Table 4-10. Mean glucose concenfrations of cows did not due to diet fed prepartum (70.7 vs 70.2 mg/dL). However, mean concentrations of NEFA in plasma were greater (34%) for cows fed cationic diet (308.4 ^lEq/L) than cows fed anionic diet (230.2 [xEq/L), although * -' concentrations of NEFA increased sharply in both groups of cows during the last days of the prepartum period (Figure 4-5). ' --^iji^. ^ Postpartum period Another objective of the current study was to evaluate the metabolic response of cows during the early postpartum period (from 1 to 28 d) which included 21 d of the postpartum phase of the transition period. To accomplish this, a second series of analyses was performed to evaluate data collected during this postpartum time period.

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200 No differences were detected for mean concentrations of E^S, glucose or NEFA or trends in concentrations due to bST treatment during the overall postpartum period (d 1 to 28; Tables 4-8 and Figures 4-3 through 4-5). On the other hand, treatment differences were detected for plasma concentrations of ST and IGF-I. Least squares analyses of variance are in Tables 4-1 1 and 4-12. Results showed significant effects of bST for both ST (P<0.0001) and IGF-I (P<0.0091) during the 28 d postpartum period. No differences among the three dry period groups were detected for mean concentrations of ST, IGF-I, EMS, glucose or NEFA during the overall postpartum period. No significant postpartum effect of prepartum diet was detected during the postpartum period for concentrations of ST, IGF-I, INS, glucose or NEFA. However, there were significant two-factor interactions bST*DRY for INS (P<0.0538) and NEFA (P<0.0044), and DRY*SEA (P<0.0 1 44) for IGF-I. J '>{ /\ ^ * ! Mean plasma concentrations of ST during the early postpartum period (from d 1 to 28) are in Table 4-8. Mean concentrations of ST during the first 4 wk postpartum period differed due to treatment (P<0.01). Cows in uninjected control group had lower mean concentrations of ST in plasma (5.52 ng/mL) than bST injected cows (10.33 ng/mL). The cows treated with bST had greater mean concentrations of ST (48%) and they remained greater throughout the early postpartum period than in control cows during the same time period (Figure 4-1); concentrations of ST in control cows remained essentially constant throughout the 28 d period. Least squares means and SE of IGF-I concentrations during the overall postpartum period are in Table 4-8. Mean concentrations of IGF-I during the overall postpartum period also were greater concentrations for the bST treated group (150.2 vs 1 1 7.4 ng/mL)

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201 u. A O C/5 2 11. A a. Z 5 (A u ° ^ -o to O U u ^ E I es o H P^ o o 4323 2229 7614 9196 2368 1794 3561 .5678 .0144 .2762 .9796 .9284 .4876 .6015 1000'' .0001 1.0645 nd o d d d d o o o o o o o o o o o April, a l + ECP o vO ov o O o 1^ 1^ oo vr> VI (N O o ov v% 1vO ch, riod III. d d d d d * d d d d vd vd oo ber.M d dry l ireTyp oo o ov Ov ov ov Ov 00 vO vO vO Cv OV OO vO O; CV O Go" o <^ S2 214658.. 18399.1 45341. 2738. oo VO 42044. 52096. 25460. 16811, 134515. 38715 1-^ o vO 2188 14357 15083 29404 154829 6125 NA 17X7 mber, O od , 11= rm; othe epte peri Yte 52. t< £ -o & c o o m Ov 00 p VO p ro »o o r~l V) ov o cs vO vO 9 o o vO h dry period =Dry period t), 'Type I S d d d d d d d d d d d 00 v-i VO o Ov CN 00 »n pn o 00 o oo VO >o Ov n (N vO ov oo r-^ d oo d od 00 5O vO — — — so < u CO D < in 1/3 > D [5 < CO UJ > O < UJ « > (Si Q < UJ c/1 * > Bi D * H 00 UJ < UJ on « >• a: D < UJ CO * UJ >• a: Q • fco H UJ Q « H CO e o U < a * < a > < D < « >• <

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202 A Oi a. o d A OS CL d < UJ £1 < t/3 1d o o o d oo — o — o^ordo (N roo o o fM o oo O "O ON fS »n fN d d o d 00 — O; ^ ^ »o ~ — OO ^ — >• a: UJ < H >Q » (Zl 3 — _ o < Hi 00 u 5 * >• Q d o d < * > Q t — — < LU 1/3 * >• ai Q uj (< >• Q < CO « 1/^ — — < UJ on » UJ >• a * C/3 o o o d < UJ on * UJ > OS Q » on o o o o d < > < >< * >< Q > < Q * < * < a a 4° ^ ^ "u u. 0 u ,o S.J!. ° ''^ ^ c — i2 zi c = S Ji 1 s & 00 fe .2 = o-Q St" Q c X II -2 ^ ^iJ^ .2 ego ^'^ 1 J= t g* ^ II s C3 S < a o ou p; Q ji_ on uT ^ 00 -g c EC 0) rs > £ o a 2 Z H i I o c u Q. & : + P I -g „ ; o c 1 -c a (gab .C ^ ±; •5 o

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203 and they were maintained at greater than in injected cows throughout the early postpartum period (+27.9%). Cows in both treatment groups had greater plasma concentrations of IGF-I prepartum, and there was a continuous decrease up to parturition with slightly lower plasma concentrations observed during the first 2 wk following parturition (Figure 4-2). During the early postpartum period, mean concentrations of INS in plasma did not differ between bST treatments (0.62 vs 0.58 ng/mL). Plasma concentrations tended to decline further during the first week following parturition and a slight linear increase was observed in concentrations during the remainder of the 28 d postpartum period (Figure 43). Concentrations of glucose in plasma followed the same trend as INS and no differences were observed in mean plasma concentrations after calving (61.9 vs 62.1 mg/dL). Mean concentrations decreased following calving for first 2 weeks and then increased slightly during the last 2 wk of the 28 d sampling period (Figure 4-4). However, postpartum concentrations of glucose in plasma were significantly lower than prepartum concentrations. Mean concentrations of NEFA during the early postpartum period are in Table 4-8. Plasma concentrations of NEFA in both groups followed the same trend and no differences were observed due to bST treatment (584.3 vs 634.2 \iEq/L). Concentrations •: V . ft were greatest around time of calving and remained greater for both bST injected and uninjected cows during the first 2 wk following calving, but decreased continuously through the 28 d sampling period (Figure 4-5). Mean plasma concentrations of ST, IGF-I, INS, glucose and NEFA did not differ during the 28 d postpartum sampling period among the three dry period treatments. Mean

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204 plasma concentrations of ST were least for cows in 30 d dry without ECP (7.0 ng/ml), whereas cows in 30 d dry + ECP and 60 d dry groups had similar and numerically (but not significantly) greater mean concentrations of ST (8.4 and 8.3 ng/mL, respectively). Mean plasma concentrations of IGF-I were greatest for the cows in 30 d dry + ECP group (148.7 ng/ml), whereas cows in 30 d dry and 60 d dry groups had lower mean i concentrations (121.2 and 131.5 ng/mL). Concentrations were least around parturition and remained low during the 28 d postpartum period (Figure 4-2). Neither mean concentrations of INS nor glucose differed during the first 28 d postpartum differed due to dry period treatment (Table 4-9). Cows in 30 d dry + ECP group had greatest mean concentrations of INS (0.65 ng/mL) and glucose (63.6 mg/dL) in plasma, whereas cows in 30 d dry group had intermediate concentrations of fNS (0.59 ng/mL) and glucose (62.9 mg/dL) in plasma, and cows in 60 d dry group had the lowest concentrations of both INS (0.56 ng/mL) and glucose (59.5 mg/dL) in plasma. Plasma concentrations of both INS and glucose tended to decrease during the first 2 wk following parturition with a slight increase observed during the following week for glucose (Figures 4-3 and 4-4). Decrease in concentrations of glucose in plasma of 60 d dry cows was more pronounced but did not differ significantly from the other two groups (Figure 4-4). Mean concentrations of NEFA in plasma did not differ among dry period treatments during the early postpartum period. Plasma concentrations of NEFA were greatest around calving but decreased for the three dry treatments afterwards (Figure 4-5). Mean concentrations of ST during early postpartum period did not differ due to diet fed prepartum. Cows fed the anionic diet had slightly greater mean concentrations of ST than cows fed the cationic diet (8.75 ng/mL and 7.10 ng/mL, respectively). Plasma

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205 concentrations of IGF-I during early postpartum period were about 50% of prepartum concentrations and did not differ between cows fed anionic (131.1 ng/mL) or cationic diets (136.5 ng/mL) during the postpartum period. Mean concentrations of INS also were less (-60%) during early postpartum period than during prepartum period (Figure 4-3), although concentrations were maintained slightly, but not significantly, greater for cows fed anionic diet (0.63 vs 0.57 ng/mL). Mean concentrations of glucose for cows fed the two diets are in Table 4-10. Mean plasma concentrations of glucose did not differ for cows fed anionic or cationic diets. However, cows in anionic diet group tended to have slightly greater mean concentrations of glucose then cows fed cationic diet during the early postpartum period. (63.0 vs 60.9 mg/dL) about 12 and 15% less, respectively, than concentrations during prepartum period. Plasma concentrations of NEFA followed the same pattern in cows fed the two diets during the postpartum period (Figure 4-5). Plasma concentrations of NEFA were greatest around calving but decreased essentially linearly during the 28 d postpartum sampling period, although mean concentrations still were about double the mean prepartum concentrations (d -21 to -1). Milk. 3.5% FCM and SCM Yields Least squares analyses of variance for MY for the first 21 wk, and milk, 3.5 % FCM, and SCM yields during the first 10 wk are in Tables 4-13 and 4-14, respectively. A trend toward significant differences in milk, 3.5% FCM and SCM was detected due to bST treatment during first 10 wk (P<0.0731, P<0.0985, and P<0.0921), respectively, and for first 21 wk for MY (P<0.027). Untreated cows had significantly greater SCC during the first 10 wk (P<0.044; Table 4-15). No significant differences were detected for dry

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206 period and prepartum diet treatments for milk, 3.5 % FCM, or SCM yields during first 10 wk period. Mean daily yields of milk for dry period and prepartum diet treatments did not differ during overall lactation period (1 to 21 wk). Results indicated that cows treated with bST had greater mean milk, 3.5 % FCM, and SCM yields (39.6 kg/d, 42.1 kg/d and 40.5 kg/d, respectively) than control cows (36.7 kg/d, 38.9 kg/d and 37.5 kg/d, respectively) during first 10 wk (Table 4-15). No differences were observed in percentages of protein (2.86 vs 2.87%) or fat ( 3.93% vs 3.96 %) between bST treatments. However, cows in uninjected control group had significantly greater SCC than cows treated with bST during the first 10 wk in lactation (527 vs 323x10^). Milk yields during the for first 21 wk also were greater for bST treated than cows those uninjected (40.1 vs. 37.0 kg/d, respectively; Figure 4-6). To evaluate the trends in mean MY during the first 21 wk postpartum, regression curves also were plotted using coefficients obtained firom cubic regression analyses for bST injected and uninjected cows (Wilcox et al., 1990). Test of heterogeneity indicated there was no evidence to indicate that curves were not parallel. Cubic order regression curves for milk yield (Figure 4-7) indicated cows injected with bST prepartum and postpartum had greater and more sustained increase in milk production compared to cows in control group that were uninjected and daily yield continued greater during the overall postpartum period even though both groups were injected with full dose of bST beginning 60±2 d postpartum. Least squares means for milk, 3.5%FCM and SCM yields for dry period treatments during first 10 wk are in Table 4-16. No significant differences among dry period treatments were detected for any measure of MY evaluated. Cows in 60 d dry

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207 Table 4-13. Least Squares Analyses of Variance for Milk Yields of Holstein Cows During postpartum period (1-21 wk). Source AC UI IVIO F PR>F bST' 1 Oj / j.DyJ 5 12 0 0270 SEA' 1 1 8A7 9fi 1 oO / .ZO 1 46 0 2321 DRY' 1 7 "il 0 9905 DIET" 1 1 A7 1 jO.O/ 0 7279 bST*DIET 1 10o4.// bST*SEA 1 1 1 C 71 1 1 o. / J 0 no bST*DRY 2 Zy.Vi n 09 0 0776 SEA*DIET 1 14/6.71 1 1 1 0 2953 DRY*DIET 2 U.J-/ 0 7089 DRY*SEA 2 no ZOO.U7 0 92 0 8008 bST*DRY* SEA 2 1847.97 1 .44 A 0/1/1/; U.Z440 bST*DRY*DIET 1761 72 1.37 0.2608 DRY'obA'iJlt 1 2 461.62 0.36 0.6993 bST*SEA*DIET 1 0.92 0.00 O.v/oU bST*SEA*DRY*DET 2 3350.58 2.61 0.0813 Cow (bST*DRY*DIET*SEA) 64 1283.30 658.15 0.0001 1 678.25 25.97 0.0001 WK*WK 1 26804.02 1026.48 0.0001 WK*WK*WK 1 11348.42 434.60 0.0001 Error* 3407 26.11 'bST=Bovine somatotropin treatments (I=No bST, Treatment 11=1 0.2 mg bST/d), SEA=Season (1= cows with dry periods during hot months {September, October, March, Apnl, and May} , 11= cows with dry periods during cold months {November, December, January, and February}), 'DRY=Dry period treatments (1= 30 d dry period, 11= 30 d dry period + ECP, 111= 60 d dry period), ''DIET=Prepartum diet treatments (I=Prepartum Anionic Diet, II=Preparum Catiomc Diet), 'WK, *Type I Sums of Squares for WK term; others are Type III.

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208 o •c (X B « I/) o D. 60 C •c D Q CO O U c o o S u IS u c2 u o B •c > 3 cr 00 •*-» CO CO V 4 S ,0 M 2 u — — e^ (N O C 00 00 O O -£> O O CD IN t-~ 00 ^ f*) ri o o CD o o o o o d d o d o o d d o d — CN — S 00 (N 00 o6 00 CN Tt t-; O m — U. A in 00 CN •>t 00 «o o•o C5V VO CTi o 00 r>00 00 o o o o o o o •n 00 C^ 00 rVO 00 o o o o d d d d d d d d d d d d d d d d d d d 2 u u. (^ o ^ — d d c/5 2 ro r1-^ Tf so r*^ r— o 0^ n o r-; o CN CN O CN £> o vo CN >o rcn 00 o> cn o O i-~ r~ en CN CN cO o o o t1o »n 00 00 o t VO O O 00 O o o o o O -o cn rm en oo CO t-CN o o o o d d d d d d d d d d d d d d d d d d d * cn CN o 00 CN r-j CN en ON CN CN CN o r-; o r~ r~ 'aCM CN o CN o Ov od •i 5 «o ro > 2 CO 2 ^ tc^ CS O «0 CN O — — t~ VO Ov c*> 00 — wi r» — 1I— CN O O — 00 — — f-l Ov VO r— — IS E E 3 O H < .o • Q >• D CO >oi O co < u CO c» >Q r\ • > Q CO • (O CO Q » H CO o O ~ = w •= •8 3 2 b " 1u S S ° Oil K. oS t/1 C „ i2 "O C3 = O 3O II CO c O w w CO M E to & i« "D [— •D O < O -r • o V II S > * Q .a JL O (J t C3 ^ CL 11 g <• JL w t = £5 n 5 c V < JL ^ o. £ 2 g E g HiCO .O _ £ E II .Eg.!: o. £ 8 b 21 =

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209 Table 4-15. Least Squares Means and SE of Milk Yield, 3.5 % FCM and SCC of Holstein Cows During Eariy Lactation. bST Treatments' Measurements I II Milk Yield (kg Idf -' 36.7 ± 0.27 39.6 ± 0.29 3.5% FCM (kg/d)2-" 38.9 ± 0.34 42.1 ± 0.37 SCM (kg/d)2 ' 37.5 ± 0.34 40.5 ± 0.35 Milk Yield (kg/d)^ '' 37.0 ± 0.49 40.1 ± 0.56 305d Milk Yield (adjusted; kg)"' 9512 ± 266 10071 ± 296 Previous ME Milk Yield (kg) ' "^ 10423 ± 254 10588 ± 283 Current ME Milk Yield (kg)^ ' 9420 ± 284 10076 ± 317 Apparent Efficiency * 1.06 ± 0.01 1.21 ± 0.02 Gross Efficiency (kg/Mcal)* *" 0.68 ± 0.01 0.77 ± 0.01 SCC ^•'^^ 527 ± 39.0 323 ± 43.0 % Protein ^ 2.86 ± 0.01 2.87 0.01 % Fat ^ 3.93 ± 0.03 3.96 ± 0.03 'Treatment I=No bST, Treatment 11=10.2 mg bST/d.; through 60±2 d postpartum. ^During 1 1 0 wk postpartum. ^During 1-21 wk postpartum. "Adjusted for previous actual 305 d milk yield. '305 d Mature Equivalent milk yield. ''During 1 -4 wk postpartum. ^SCC=Somatic Cell Count xlOOO. ='P<0.1, ''P<0.03, T<0.09,''P<0.05.

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210 OO m 00 OO so o ro ro 00 d d so d d -H -H -H -H -H -H -H -H -H -H -H -H CO 2 is W CO 2 I c u fc 3 u (J a u o iS W C Si < u U 00 o a

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50 20 T 1 1 1 1 1 1 1 — I — I ' 1 1 1 ' 1 ' ' ' ' ' ' 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Weeks Figure 4-6. Milk production of Holstein cows during early lactation. Arrow indicates the time full dose of bST injection started

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212 treatment did have slightly greater numerical milk (39.4 kg/d) 3.5% FCM (41.6 kg/d) and SCM (39.8 kg/d) yields than cows in 30 d dry with no ECP (37.4 kg/d, 39.3 kg/d and 37.9 kg/d, respectively) and than cows in 30 d dry + ECP (37.5 kg/d, 40.7 kg/d and 39.1 kg/d, respectively), but, as indicated, they did not differ significantly. Furthermore, MY during first 21 wk did not differ significantly due to dry period length or treatment (Table 4-6). No significant differences in percentages of protein (2.78, 2.93 and 2.89 %), fat (3.90, 4.09 and 3.84 %) or SCC (477, 489 and 309 xlO^) were detected due to prepartum dry treatments (60 d dry , 30 d dry + ECP and 30 d dry, respectively) during the first 10 wk lactation period (Table 4-16). Cubic order regression curves were calculated for MY to describe the time trends for the individual treatments over the 21 wk lactation period (Figure 4-7). Tests of heterogeneity detected evidence that curves were not parallel (P<0.01 ;Wilcox et al., 1990). Cows in 60 d dry had highest milk yield that were greater at the beginning of lactation but had lowest milk yield after 10 wk. Cows in 30 d dry with and without ECP had similar milk yields after parturition. No differences were observed in mean milk, 3.5 % FCM, and SCM yields and percentages of protein or fat or SCC for the cows fed different prepartum diets (anionic or cationic) during the first 10 wk period. Similarly, mean MY during the first 21 wk did not differ significantly due to prepartum diet fed the cows. Milk yield of cows fed anionic group were about 1 kg greater during first 10 wk (38.7 vs 37.5 kg/d). However, over the first 21 wk period, the small numerical difference disappeared and mean daily MY were similar for the two prepartum diet treatments (38.6 vs 38.5 kg/d; Table 4-17).

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213 Table 4-17. Least Squares Means and SE of Milk Yield, 3.5 % FCM and SCC of Holstein Cows During Early Lactation Fed Anionic or Cationic Diets During Prepartum Period. Prepartum Diet Treatments' Measurements I n Milk Yield (kg/d)38.7 ± 0.28 37.5 ± 0.29 3.5% FCM (kg/d)' 40.9 ± 0.35 40.2 ± 0.36 SCM (kg/d)' 39.4 ± 0.32 38.6 ± 0.34 Milk Yield (kg/df 38.6 ± 0.51 38.5 ± 0.54 305d Milk Yield (adjusted; kg)* 9593 ± 266 9990 ± 288 Previous ME Milk Yield (kg)' 10444 ± 255 10567 ± 275 Current ME Milk Yield (kg)' 9592 ± 290 9905 311 Apparent Efficiency* 1.09 ± 0.02 1.18 ± 0.02 Gross Efficiency (kg/Mcal)* 0.70 ± 0.01 0.75 ± 0.01 SCC 429 ± 40.0 421 ± 42.0 % Protein ^ 2.84 ± 0.01 2.89 ± 0.01 % Fat ^ 3.88 ± 0.03 4.01 ± 0.03 'Treatment 1= Anionic diet prepartum. Treatment II=Cationic diet prepartum. ^During 1-10 wk postpartum. ^During 1-21 wk postpartum. * Adjusted for previous actual 305 d milk yield. ' 305d Mature Equivalent milk yield. ^During 1 -4 wk postpartum. 'SCC=Somatic Cell Count xlOOO.

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50 20 -1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 — 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Weeks 50 45 20 T 1 1 1 1 1 1 1 — ^ ' ' ' ' ' ' ' ' ' ' ' I 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Weeks Figure 4-7. Cubic regressions depicting changes in weekly milk yield of Holstein cows during the experiment. Arrow indicates the time full dose of bST injection started.

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215 Using coefficients obtained from cubic regression analyses, the trends in mean MY were evaluated during the first 21 wk postpartum period for prepartum anionic and cationic diets (Wilcox et al., 1990). For prepartum diets, there was no evidence detected to indicate that individual MY curves were not parallel during the early postpartum ' periods (21 wk), as determined by heterogeneity test (Figure 4-7). Although MY of cows fed anionic diet initially showed greater daily production, decline in MY also started earlier than that of cows fed cationic diet and overall result was that there was no change in mean MY during the first 21 wk in lactation. Apparent efficiency (MY kg/DMl kg) and gross efficiency (MY kg/NEl Meal) of milk production were calculated for all treatment groups (Tables 4-15, 4-16, 4-17). Apparent efficiency of milk production was greater for cows injected with bST than for control cows (1 .21 vs. 1 .06, P<0.03). No differences in apparent efficiency of milk production were detected among dry period treatment groups or for the prepartum diet treatment groups (Tables 4-16 and 4-17). Gross efficiency of milk production followed the same trend. Gross efficiency of milk production by cows injected with bST was significantly greater, therefore these cows were more efficient than cows not injected with bST (0.78 kg/d vs. 0.68 kg/Mcal, respectively; P<0.03; Table 4-15). Gross efficiencies were 0.68, 0.75 and 0.75 kg/Mcal for cows in 30 d dry, 30 d dry + ECP and 60 d dry groups, respectively (P<0.24; Table 4-16). No difference in gross efficiency of milk production was detected for cows fed prepartum anionic (0.70 kg/Mcal) or cationic diets (0.75 kg/Mcal; Table 4-17). The previous lactation 305-d mature equivalent (ME) MY of cows did not differ significantly in bST and non-bST groups (10423 ± 254 vs 10588 ± 283 kg, respectively;

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216 Table 4-15). Cows in 30 d dry (10674 ± 307 kg), 30 d dry + ECP (10448 ± 329 kg) or 60 d dry groups (10396 ± 343 kg; Table 4-16) had similar previous lactation 305-d ME milk yields. The previous 305-d ME milk production of cows fed anionic or cationic diets prepartum also did not differ significantly (10444 ± 255 vs 10567 ± 275 kg respectively; Table 4-17). The current lactation 305-d ME yields of cows also were analyzed. The current 305-d ME yields of cows injected with bST (10076 ±317 kg) was significantly greater than cows not injected with bST (9420 ± 284 kg; P<0.09; Table 4-15). The current 305-d ME were 9586 ± 343, 9959 ± 375 and 9700 ± 385 kg for cows in 30 d dry, 30 d dry + ECP and 60 d dry groups, respectively (Table 4-16). No differences in current 305-d ME milk production were detected for cows fed prepartum anionic (9592 ± 290 kg) or cationic diets (9905 ±311 kg; Table 4-17). The current 305-d ME milk yields also were adjusted for actual 305-d MY in the lactation that preceded the experimental dry period. The adjusted 305-d ME milk production was significantly greater for bST injected (10071 ± 266 kg) than non-bST injected cows (9512 ± 296 kg, P<0.1; Table 4-15). No significant differences in the adjusted 305-d ME milk producfion of cows assigned to 30 d dry, 30 d dry + ECP and 60 d dry groups were detected (9580 ±321, 9960 ± 344 and 9836 ±358 kg, respectively; Table 4-16). The adjusted 305-d ME milk production of cows fed prepartum anionic or cationic diets also did not differ significantly (9593 ± 266 vs 9990 ± 288 kg; Table 4-17). Discussion Hormones, Growth Factor and Metabolites . , A series of orchestrated changes must occur for cows to have a healthy transiUon into lactation and satisfactory milk production throughout the lactation. It was

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217 hypothesized that bST injection prepartum and postpartum would have its primary effect on directing the use of absorbed nutrients in all classes by altering the metabolism of different tissue types directly (e.g., liver, adipose) and indirectly (e.g., mammary gland) mediated in large part by IGF-1 (Bauman, 1992). Somatotropin decreases sensitivity of INS receptors to INS in the peripheral tissues and decreases overall uptake of glucose in peripheral body tissues. Thus, this minimizes the oxidation of glucose to CO2 in these peripheral tissues and more glucose is available for milk synthesis. Somatotropin also increases lipolysis in adipose tissue. Because glucose is used as the primary energy metabolite and as a substrate for synthesizing milk constituents in mammary tissue, energy needed by other peripheral body tissues would be derived from products of lipolysis or metabolism of non-gluconeogenic compounds arising from the rumen and lower digestive tract. Thus, more glucose would be available to the mammary gland. During negative energy balance, ST also will spare protein use as a source of energy in tissues because it increases lipid mobilization and inhibits glucose oxidation in the whole body. Proteins that are mobilized from the muscles can be used in the liver, in the gut, and in the blood which will increase overall metabolism and efficiency of protein use. Adjustments of these orchestrated changes would be especially important during the early lactation because of lower DMI at this time. Both prepartum and postpartum injections of bST did result in elevated concentrations of ST in the peripheral circulation of treated cows. Concentrations of ST tended to be greater during the postpartum period than during the prepartum period. Although ST concentrations increased around parturition, no net increase in plasma concentrations of ST were observed in untreated cows as they moved from the prepartum

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218 to the postpartum period. Generally, the results of this study agreed with others (de Boer et al., 1985; Koprowski and Tucker, 1973; Vicini et al., 1991). Somatotropin is a homeorhetic hormone that is involved in the regulation of the metabolic adaptations that occur during the transition from the nonlactating to the lactating state. Hence, ST concentrations begin to increase during late pregnancy and rise during early lactation and then fall during the latter stages as MY declines (Collier et al., 1984). Through these changes, ST can exert a powerful galactopoietic influence once lactation is established; it decreases rates of lipogenesis and activities of lipogenic enzymes and promotes glucose utilization for milk synthesis (Bauman and Vernon, 1993). Changes in concentrations of ST following injections of bST are seen over a wide range of injection doses and times within the lactation cycle. Simmons et al. (1994) reported elevated concentrations of ST in plasma of cows when prepartum injections of 5 and 14 mg bST/d were administered. It also was shown that cows that were injected with 25 mg bST/d had higher concentrations of ST than untreated cows (Bachman et al., 1992). Lucy et al. (1993) reported an increase in plasma concentrations of ST following injection of cows with bST during both mid and late lactation. Additionally, concentrations of ST were elevated throughout the entire transition period when cows were injected during both prepartum and postpartum periods (Garcia et al., 2000; Gulay et al., 2000). The amount chosen (10.2 mg bST/d) to inject during the current studies was based on previous studies (Gulay, 1998) that indicated this dose was the least of the amount evaluated that brought about desired effects on ST and IGF-I. Thus, increase in plasma ST concentrations in current studies were expected and increase in ST

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219 concentrations were appropriate for the amount of bST injected either prepartum or postpartum. No differences were observed in concentrations of ST in the plasma of cows during the prepartum or postpartum periods in any of the three dry period groups or in groups fed the prepartum anionic and cationic diets. Consequently, no differences were detected in the milk yields of cows due to prepartum diet fed. Increase in concentrations of ST in plasma was not limited to only a type of diet or dry period length, rather the effect was more general. Thus, positive effect of bST treatment may be expected to work under a wide variety of management strategies. Concentrations of ST during the transition period were reported to follow the same pattern in a number of published studies. The concentrations of ST during prepartum period were less than postpartum period and injecting bST did not change this. However, ST concentrations were elevated during early lactation (de Boer et al., 1986; Koprowski et al., 1973; Vicini et al., 1991). Normal physiological concentrations of ST in cows are altered after parturition, most likely, to facilitate the important changes occurring during early lactation (Sartin et al., 1988). As mentioned previously, ST has a very important role as a homeorhetic controller of metabolism. Typically, concentrations of ST in cows begin to increase during late pregnancy and are elevated at calving. Moreover, the galactopoietic response to exogenous injections of ST during lactation suggests that ST has an important role in many adaptations that occur during the transition period and that increased concentrations of ST due to bST injection hkely enhance these adaptations. Physiological processes such as increase in hepatic rates of gluconeogenesis, reduction in muscle uptake and oxidation of glucose, regulation of adipose tissue, and increase in lipolysis and increased uptake of

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220 nutrients used for milk synthesis by mammary gland are altered in various tissues including liver, adipose, muscle, kidney, intestine and mammary. As indicated, these changes likely are enhanced by ST treatments and also because of increases in endogenous synthesis and secretion of ST (Bauman, 1992). Increased concentrations of ST also decreases the ability of INS to inhibit gluconeogenesis, inhibits actions of INS second messenger, and decreases uptake of INS into the cell. These and other alterations in mammary and extra-mammary metabolism are especially important during the early prepartum period because milk production is initiated and amount of milk produced increases rapidly but DMI is not sufficient to support this increase. In the current study, low dose of bST injection before and after parturition increased concentrations of ST and it is likely this rise had many beneficial effects because of the linked rise in concentrations of ST and IGF-I and decrease in INS and this favored increased MY. In general, bST injected cows had higher IGF-I concentrations than control cows during prepartum and postpartum periods of this study, although increase in IGF-I concentrations after parturition were less than the increase that occurred during the prepartum period (-36% vs -28%). In addition, cows in all three dry period treatments followed the same trend with high plasma concentrations of IGF-I during prepartum period and a continuous decrease through parturition with the lowest plasma concentrations observed during the first 2 wk after parturition. These results indicated a relationship between the decrease in energy and specific nutrient intake because of the decrease in DMI around calving and decreased concentrations of IGF-I during this time. Adverse effects of negative energy balance and diet on plasma concentrations of IGF-I were reported previously (Ronge et al., 1988; Elsasser et al.,1989). Vicini et al. (1991)

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221 also reported that when bST was injected during late lactation, dry period and early lactation, increase in concentrations of IGF-I were greater during the late lactation and the dry period (when DMI was greater), than during early lactation when DMI is limited. Although plasma concentrations of IGF-I are under control of ST, nutritional status plays an important role in the regulation of the somatotropic axis (Brier et al., 1986). When a diet was fed to steers for a 2 wk period that resulted in severe energy deficiency, the lGF-1 response to bST declined markedly (Elsasser et al., 1989). Cows in early lactation typically are in NEB and they have greater circulating concentrations of ST but low basal concentrations of IGF-I. Synthesis and secretion of IGF-I in response to ST is affected by the energy balance (Phillips et al., 1990) and the ST/IGF-I axis is attenuated by nutritional status (Bauman and Vernon, 1993). Restriction of DMI in growing steers decreased the basal concentration of IGF-I in blood and terminated the positive response of IGF-I to exogenous bST treatment (Brier et al., 1988). Moreover, the IGF-I response to bST treatments was greatest when cows were fed high protein/high energy diets (McGuire et al., 1992). Similarly, in the current study, the IGF-I response to bST was less after parturition than it was during the prepartum period. Nutritional status also regulates the concentrations of IGFBP and changes in concentrations of IGF-I. Vicini et al. (1991) suggested that these changes were the result of a change in specific circulating binding proteins. About 75% of IGF-I are found bound to IGFBP-3 and circulating concentrations of IGFBP-3 are positively correlated with those of ST (Cohick et al., 1992). Administration of ST to lactating cows (Cohick et al., ,1992), humans (Blum et al., 1990) and pigs (Walton and Etherton, 1989) causes increases

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222 in concentrations of IGFBP-3. Moreover, infusion of IGF-I into normal adults increases IGFBP-3 (Zapfet al., 1990). It seems that IGFBP-2 concentrations are higher and IGFBP-3 concentrations are lower during negative nutrient balance (Vicini et al., 1991). In contrast to increases in concentrations of IGFBP-3, concentrations of IGFBP-2 decline in circulation following bST injections in cows (McGuire et al., 1992; Cohick et al., 1989). The IGFBP-2 inhibits IGFs in various cell lines in vitro (Yang et al., 1989). Basal concentrations of IGFBP-2 were greatest during early lactation and least during the dry period when cows were in positive EB. Conversely, IGFBP-2 concentrations were least during the dry period when IGF-I was high, and were reduced during bST administration when IGF-I concentrations were elevated (Vicini et al., 1991). Thus, lower IGF-I concentrations during early lactation probably occurs because of the NEB observed following parturition (Vicini et al., 1991). Although there was a reduced increase in IGF-I during bST administration during early lactation, greater concentrations of IGF-I found in cows in the bST injected group than in the uninjected group did suggest that increased concentrations of ST in blood, as a result of bST injections, might have caused a shift in proportions of the various IGFBPs in plasma and changes in the binding of IGFs to these proteins (Armstrong et al., 1996). Although concentrations of IGFBPs were not measured during the current study, because of greater concentrations of ST, shifts in the proportions of IGFBPs in plasma and changes in the binding of the IGF-I to these proteins might have occurred resulting in greater circulating concentrations of IGF-I of the bST injected cows. Because IGF-I acts as a local mediator of mammary epithelial growth and development.

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greater concentrations in plasma might have increased the lactational performance of the bST treated cows via increased cell numbers. Another important hormone that regulates metabolism in cows is INS. hisulin is considered to be an anabolic hormone. It regulates fat deposition and mobilization from adipose tissue. Moreover, INS causes a decrease in the concentrations of blood glucose and stimulates uptake and utilization of glucose by the liver, muscle, and adipose tissue, as well as inhibiting gluconeogenesis and glycogenolysis, which are two main sources of peripheral blood glucose (Hart et al., 1978). In the current study, concentrations of INS were high for cows in all treatment groups during the prepartum period. Greater prepartum concentrations of INS were expected because of the greater amounts of circulating glucose and the positive energy balance of the cows during late lactation and throughout much of the dry period (Vicini et al., 1991). On the other hand, cows treated with bST had significantly greater concentrations of INS. As cows approached calving, INS concentrations typically would be decreasing and would be further reduced after parturition; concentrations would remain lower throughout the lactation sampling period (+28 d). Vicini et al. (1991) also reported that FNS was elevated when bST was injected during late lactation and the following dry period, but concentrations declined during early lactation. Bachman et al. (1992) also reported that plasma concentrations of INS decreased as cows approached calving. Thus, results of many studies indicate that the changes in INS observed were the expected physiological response. In dairy cows after calving and during early lactation, there is a strong positive relationship between concentration of INS in plasma and energy balance (Lucy et al., 1991). Increased concentrations of INS in plasma are associated with positive EB of the cows. This also

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224 would be expected to cause higher concentrations of glucose in blood during the dry period. Decreased concentrations after parturition likely occurred because of the rapid onset of the negative EB during early lactation (Vicini et al., 1991), as lactation demands for energy and precurcors for milk synthesis rapidly increase. In current experiment, plasma concentration of INS declined, beginning 3 d before parturition. These results agreed with findings that concentrations of INS declined around parturition (Garcia, 1998; Gulay, 1998; Malven et al., 1987), even in bST injected cows. Lactation is characterized by low concentrations of INS and a high ST:INS ratio. Decrease in INS receptors and decrease in concentrations of INS following parturition result in a decrease in of lipogenesis (Mepham, 1987). Despite the reduced concentrations of INS and reduced INS receptor numbers in liver and adipose tissue, INS receptor numbers increase in mammary tissue at parturition (Petterson et al., 1994). During late pregnancy, increased resistance to INS in adipose tissue causes decreased INS response, expressed as decreased adipose tissue lipolysis and FFA mobilization (Petterson et al., 1994). Somatotropin has a negative effect on ability of DSfS to inhibit gluconeogenesis and it also inhibits both INS uptake by the cell and INS protease activity that is necessary for the action of INS. The sum of these changes would lead to an increase in glucose production via gluconeogenesis and priority use of glucose by mammary tissues then could occur. Treatment of cows with bST during postpartum period stimulates glucose metabolism in cows. Typical responses include decreased whole body oxidation of glucose (Cohick et al., 1989), increased hepatic rates of gluconeogenesis (Bauman et al., 1988), and decreased glucose response to INS (Bauman and Vernon, 1993). In the current

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study, mean concentrations of glucose did increase prepartum in bST treated cows. Concentrations of glucose also were greater during the prepartum period, whereas, after parturition there was a decline observed in glucose concentration. Furthermore, cows treated with a much greater dose of bST during prepartum period had significantly higher glucose concentrations (Putnam et al., 1999). Although no prepartum increase in DMI was observed in bST injected compaired to uninjected groups of cows in the current experiment, decreases in glucose oxidation and a reduction in the use of glucose in peripheral tissues likely was the reason for the greater concentrations of glucose prepartum in the bST injected group (McDowell et al., 1987). McDowell (1991) also reported that hepatic glucose production fi-om propionate was enhanced during bST treatment probably because the ability of INS to inhibit liver gluconeogenesis is negatively affected by bST treatments (Sechen et al., 1990). As a result, it was expected that glucose concentrations of bST treated cows would be greater than in controls. A decrease in use of glucose by non-mammary tissues increases availability of glucose for mammary use mainly as a source of energy and milk component synthesis. Increased concentration of glucose also would be expected where greater concentrations of INS were seen, as during bST injections of cows during the prepartum period. Greater availability and higher concentrations of glucose due to bST injections during the dry period without a change in glucose extraction by the mammary gland may have resulted, either directly or indirectly, in greater concentrations of INS in cows of the same treatment group. Because more glucose was available from synthesis or reduced peripheral utilization (due to reduced peripheral tissue response to INS), more INS might have been synthesized and secreted to regulate blood glucose. On the other hand, decrease

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226 in concentrations of glucose, and no change in concentration of INS during early lactation might have been due to less availability of glucose in peripheral circulation because of the extremely high demands by the mammary gland cells for synthesis of milk lactose. Although bST injected cows produced significantly more milk during early lactation, these cows still were able to maintain nearly stable concentrations of glucose in plasma after parturition. Moreover, the additional increase in MY in bST injected cows did not cause a decline in concentrations of glucose in the circulation compared to control group. Under most feeding situations, energy is the major limiting nutrient for high producing cows. High yielding dairy cows are in negative energy balance for up to 8 to 10 wk postpartum and there is mobilization of body fat to provide the equivalent of about 30% of the energy found in the secreted milk (Mepham, 1987). This occurs even when cows are fed adequately formulated diets ad libitum. Because of the increase in demand for energy and nutrients after calving, it is important that preparation for the lactation begins prepartum. The maternal tissues, especially during the last trimester of pregnancy, mostly rely on the metabolism of NEFA and ketone bodies because glucose is used largely for conceptus metabolism (Bell, 1995). Thus, increased mobilization of NEFA during the late prepartum period is essential. Decreased ability of INS to promote lipogenesis and to oppose lipolysis results in decreased de novo synthesis of TG and reduction in the re-esterification of fatty acids arising from the TG (Vernon et al., 1990). Reduced DMI of ruminants before calving also is associated with greater occurrence of NEFA mobilization from adipose tissue before calving (Forbes, 1986). In the current study, low concentrations of NEFA in plasma were detected up to the final week of pregnancy. During this final week of pregnancy a sharp increase in concentration of ' '

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227 NEFA in plasma was seen for cows in all treatment groups (Figure 4-5). During the overall postpartum sampling period, concentrations of NEFA declined after parturition but still were more than double the prepartum concentrations. Concentrations of NEFA tended to be higher during the first week following parturition in cows injected with bST but then concentrations declined, as they did in untreated cows. Typically, lower utilization of glucose and low energy intake results in greater blood concentrations of NEFA and ketones during late dry period (Petterson et al., 1994). Transition from pregnancy to lactation is characterized by a sharp increase in flow of NEFA into plasma from adipose tissue and concentrations of NEFA in plasma and adipose tissue mobilization are directly correlated (Bauman et al., 1988). The reason for the high release of NEFA may be due to increased lipolysis driven by adrenergic stimulation around parturition (Grummer, 1993). Furthermore, the metabolic pathways of de novo fatty acid synthesis, plasma TG uptake and fatty acid esterification, and the activities of the enzymes controlling them are greatly reduced during late prepartum and early lactation periods (McNamara, 1991). In addition, reduced FNS receptors, second messenger or lower activity of INS protease on adipocytes also may be responsible for the further increase in plasma concentrations of NEFA during this time period (McNamara, 1995). So all these actions would favor availability of NEFA for use by peripheral tissues (rather than use of glucose) because glucose is conserved for mammary use during the lactating stage. Milk. 3.5% FCM and SCM Yields It is important to improve milk yield of cows in commercial dairy herds. One of the purposes of this study was to modulate or perturb the metabolism of cows in order to

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228 increase milk production. Thus, this research was done to study the effects of i) low dose of bST, ii) 30 d and 60 d dry periods, and iii) prepartum anionic and cationic diets to determine if any or combination of these treatments were beneficial for the health and productivity of cows. bST treatment and milk yield Exogenous ST enhances lactational performance not only in dairy cows but also in sheep, goats, pigs, rats and humans (Etherton and Bauman, 1998). However, most research has involved the dairy cow because of its greater importance as a producer of milk and the commercial importance of milk. Even though typical MY responses of cows injected with POSILAC® are increases of 10-15 % (~4 to 6 kg/d), up to 40% in MY was reported (Bauman, 1999). hi earlier studies, response to bST treatments were negligible during early lactation and use of bST was recommended for the last 80 % of the lactation of dairy cows (Etherton and Bauman, 1998). A few studies reported on use of a full dose of bST (POSILAC®) that started as early as 10 d following parturition. Yet, when a fiill dose of bST was injected into cows when the EB was negative, MY still increased but it did result in a severe loss in BCS compared to uninjected similarly managed control cows (Moallem et al., 1996). Furthermore, treating high yielding dairy cows with bST during early lactation extended the duration NEB and BCS loss by 25-28 d (Moallem et al., 2000). Thus, NEB was more severe when a full dose of bST (500 mg bST/14d) was injected into dairy cows during early lactation. However, when Stanisiewski et al. (1991) injected cows with 5 mg or 14 mg bST/d from 14 d postpartum through 60 DIM, the injected cows produced more FCM than controls and the cows receiving 5 mg bST/d maintained BCS as well as the uninjected controls. In another study a lower dose of bST

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229 was injected prepartum and postpartum (15.3 mg bST/d) that resulted in increased DMI of cows after parturition (Gulay et al., 2000). Also, there was less decrease in BCS and BW and injected cows also produced numerically greater daily MY and 3.5% FCM than uninjected controls (Gulay et al., 2000). Thus, use of different amounts of bST during prepartum and early postpartum periods appears to have the potential to improve lactational performance during early lactation and the results of this study (Gulay et al., 2000) encouraged use of low dose of bST during the transition period in an attempt to positively affect metabolism and increase subsequent milk production of transition cows. In the current study, cows injected withlO.2 mg bST/d prepartum and during the early postpartum period did produce 6.6% more milk and 7.6% more 3.5% FCM than uninjected herdmates in the control group during the first 70 DIM (Figure 4-6). Treated cows started lactation with higher daily yields of milk which continued throughout the first 70 DIM. Prepartum and/or postpartum changes in circulating concentrations of ST, IGF-I, INS and glucose were beneficial to the cow during the transition period and during the lactating phase. Somatotropin is a homeorhetic controller affecting numerous target tissues and it shifts the partitioning of nutrients among various tissues. Somatotropin t treatment also reduces the tissue responsiveness to INS. It also reduces the use of glucose for fat deposition and fatty acid synthesis in adipose tissue to support the increase in milk synthesis in lactating animal. Furthermore, ST stimulates cell proliferation, an effect mediated by IGF-I. As suggested previously, increased concentrations of IGF-I might have increased mammary cell numbers and/or increased cell differentiation during prepartum and/or early lactation periods (Putnam et al., 1999). Long term injection with bST increases voluntary intake of DM and this increase persists during the time interval

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230 that bST is supplemented. Although bST injected cows had greater amounts of milk and/or 3.5% FCM, these cows lost the same amount of BW and BCS compared to uninjected cows. This suggests that there was more efficient production of MY and greater DMI during the lactation, especially after cows had achieved neutral or positive energy balance. Thus, the changes in concentrations of metabolic hormones, endogenous, and as a consequence of bST injection, had positive effects on DMI, BCS, BW and MY of cows. . -^wi • . Early postpartum treatment of dairy cows (d 14 after calving) with 5 or 14 mg bST/d stimulated >6% increase in FCM yields (Stanisiewski et al., 1992). Richard et al. (1985) also reported a 6% increase in MY when cows were injected with 50 RJ of bST starting 20 d postpartum. In the same trial, milk fat also was elevated by 25 %. Moallem et al. (2000) reported over a 12% increase in MY when full dose of bST (500 mg for every 14 d) was injected from 10 to 150 d postpartum. However, Eppard et al. (1996) did not see an increase in MY when they injected Holstein and Jersey cows during the prepartum period with a full standard dose of bST (POSILAC®). Maybe the latter result occurred because cows also were used for milk fever induction and plasma concentrations of ST and IGF-I in treated cows failed to increase more than occurred in uninjected control cows. In the current study, all cows received the full dose of bST at -60 d in lactation, but bST injected cows still had greater daily MY at 150 d. Cows in both injected and control groups showed increased persistency in yield over the sampling period (150 DIM), and were still producing over 35 kg/d milk at this time. However, cows injected with this relatively low prepartum and postpartum dose of bST responded

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231 to the full dose of prepartum and postpartum bST injections better than cows in the control group because yield of milk for the treated cows still were greater (Figure 4-6). The mechanism by which ST affects mammary gland function likely involves the IGF-I system. Administration of bST increases circulating concentrations of IGF-I and IFGBP-3 and these parallel the increase in MY (Bauman and Vernon, 1993). One of the effects that is mediated by IGF-I is increased cell proliferation (Rechler and Nissley, 1990). Baumrucker and Stemberger (1989) reported that IGF-I stimulated DNA synthesis in cultured mammary cells obtained from both pregnant and lactating cows. Thus, increased concentrations of IGF-I in bST treated cows during the prepartum and postpartum periods might have increased mammary cell numbers during prepartum and/or early lactation period (Putnam et al., 1999). As a result, increase in secretory cell numbers would have resulted in greater MY when activity of these cells was further enhanced by the full dose of bST, even though it followed a lower dose. Furthermore, when DMI increases during later stage of lactation, more nutrient supply would have been available for the cells which would further support milk production. Results of the current study indicated that cows treated prepartum and postpartum with bST did have greater mean milk, 3.5 % FCM, and SCM yields than control cows during first 10 wk (Figure 4-6). However, no differences were observed in percentages of protein or fat in the milk. The two treatment groups of cows had essentially the same DMI throughout the first 4 wk postpartum and no differences in their BW or BCS were detected. Simpson et al. (1992) administered GRF prepartum to beef heifers to increase secretion of ST before parturition and during early lactation. Treated heifers lost more BW and had delayed ovarian activity, whereas no difference was observed in MY. When

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232 cows were injected with 20.6 mg bST/d starting 4-9 d postpartum, no significant differences were detected in MY of control or bST treated cows. Unfortunately, the cows assigned to bST had lower MY potential than controls based on the rate and extent of decline in MY after cessation of bST injection (de Boer et al., 1991). In another trial, when Holstein cows received 0, 5 or 14 mg bST/d during the last 46 d before parturition, no differences were detected among treatments in SCM during subsequent lactation (Simmons et al., 1994). Except for the cows treated with 5 mg/d of bST during wk 10 of lactation, EB was negative for all cows during the first 70 d of lactation. On the other hand, studies ft-om our laboratory suggested positive metabolic changes that were beneficial to health and performance of the cows injected with bST during both prepartum and postpartum periods (Garcia et al. 2000; Gulay et al., 2000). Garcia et al. (2000) reported that prepartum and postpartum injections of only 5.1 mg of bST/d increased DMI, MY and efficiency of milk production during the first 60 DIM. Injections of 15.3mg bST/d before and after parturition increased DMI of cows following calving allowing treated cows to recover BW and BCS more rapidly during early lactation even though these cows also produced numerically greater daily MY and 3.5% FCM (Gulay et al., 2000). During treatment with bST, DMI increase typically occurs within 3 to 8 wk and supports the cows ability to increase MY. In the current study, the relative increase in MY of bST treated cows was greater than the increase in DMI compared to untreated cows. Moreover, BW and BCS loss was not affected by bST treatment. This implies, but does not prove, there was an increase in feed efficiency especially, during the early weeks of the postpartum period in the bST injected cows and this was the source of energy and nutrients needed to allow for this increase in milk production.

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^ ^ ' "'^Tsa^str-i 233 As mentioned earlier, when studies were conducted that used different doses of bST during prepartum and/or early postpartum period results differed with respect to metabolic responses. The variable results within and among trials that evaluated use of bST during either prepartum or postpartum periods may have been due, in part, to differences among the doses, diets the cows were fed, or due to differences in BW and BCS of the cows during the early phase of lactation, hi some studies, bST failed to increase concentrations of ST and /or IGF-I in the peripheral circulation (Bachman et al.,1992; Eppard et al., 1996). Eppard et al. (1996) did not observe increased MY of prepartum bST injected Holstein and Jersey cows possibly because cows also were fed diets to induce milk fever. Furthermore, a high dose of bST during early postpartum period usually increases the length of time cows are in NEB, and there is greater loss of BW and BCS even though an increase in DMI probably occurs (Moallem et al., 1996; Moallem et al., 2000). If part of the increase in MY results from greater mobilization of body tissue reserves, then good BCS (3.25-3.75; Nocek et al., 1983) is critical if they are to be injected with bST prepartum and/or postpartum. Clearly, cows injected with bST require good management and adequate nutrition to produce and reproduce well because the increase in DMI and hence nutrients available to the cow to support lactation is delayed. On the other hand, treatment with bST during both prepartum and postpartum periods likely causes metabolic changes, such as increased lipolysis and gluconeogenesis and increased plasma concentrations of ST, IGF-I and T3 after parturition that are beneficial to health and performance of the cows (Gulay et al., 2000). In their study, prepartum and postpartum inj ections of 1 5 .3 mg bST/d increased DMI of cows after

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234 parturition, and there was less decrease in BCS and BW. Cows recovered BW and BCS more rapidly during early lactation which indicated they reached positive EB more quickly and, in part, this allowed injected cows to produce numerically greater daily milk and 3.5% FCM yields. In addition, Garcia et al. (2000) reported that injections of 5.1 mg of bST/d before and after parturition increased MY and calculated efficiency of milk production during the early lactation period (60d). Hence, low dose of bST during prepartum and postpartum periods might have the potential to increase MY with no negative effect on BW or BCS of injected cows. Typically, the gross composition of milk is not altered during bST treatment of cows (Bauman, 1992; Chalupa and Galligan, 1989). However, the changes in fat percentage seen in response to bST treatments will vary with the energy status of the injected animals. When animals are in NEB during periods of somatotropin injections, percentage of fat in milk also tends to increase as MY increases. Thus, if use of bST during early lactation increases energy deficit to a greater extent and for a longer duration, it likely would resuU in increase in fat percentage in milk (Bauman and Vernon, 1993). In the current study, no changes in milk composition (fat or protein %) were detected up to 70 DIM. After this time period, cows likely would be in positive energy balance. This fact alone suggests that apparent differences in the energy status of the injected and uninjected cows were not great during the milk sampling period (110 wk postpartum), and agree with the facts there were no changes in mean BW and BCS during this time period. As a result, the low dose of bST treatment that increased MY seemed to be without negative effects on the energy status of the treated cows and implied that DMT increased.

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235 The amount of body fat estimated by BCS is a good indicator of the energy that is available during lactation to support body maintenance and milk production. This indirectly describes supply of energy potentially available to provide for the difference between dietary energy intake and the requirements for body maintenance and milk production (Gearhart et al., 1990). Energy intake to increase accretion of body tissues and thus BCS, or energy arising from tissue mobilization to support milk production will ' depend upon the starting and ending BCS (NRC, 1996). One unit change in BCS (scale thin=l, moderate=3, fat=5) was estimated to be equivalent to 56 kg (Otto et al., 1991) or 41 .6 kg (Seymour and Polan, 1986 ) live body weight change. Similar decrease in BW and BCS and similar recovery of these in bST injected and uninjected cows during the current study suggests that increase in MY in the bST injected cows follows an increase in DMI and/or efficiency of utilization of ingested nutrients (DMI) in these cows later in lactation. Although fat and protein percentages in milk of bST treated cows did not differ in current study, cows treated prepartum and postpartum with bST did show lower SCC levels in milk. Increased MY of cows usually is associated with increases in SCC in milk (White et al., 1994). Somatic cell count is higher in mastitic glands due to damage of tight junctions of cells due to inflammation (Burvenich et al., 1999). Thus, increases in milk SCC can be used as an indicator of bovine mastitis. Various types of stress have been implicated as causing increases in SCC. However, attempts to experimentally induce stress in uninfected cows has shown only modest or no effects on SCC. Although SCC of milk from heat-stressed cows increases, some of this increase may be due to decreased milk production because of the heat stress (Paape et al., 1973; Paape et al., 1979).

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236 Somatic cell counts generally are lowest during the winter and highest during the summer. High temperature and humidity do not directly cause increases in SCC. Rather, the increase in SCC is due to greater exposure of teat ends to pathogens, which results in more new infections and clinical cases during the summer months (Harmon, 1994). hi addition, cows undergoing significant heat stress tend to have reduced immunity, resulting in greater SCC and higher rates of clinical mastitis (Wagner et al., 1976). Although it has been demonstrated that there is a slight increase in mastitis in lactating cows treated with bST, this increase is associated primarily with the increased MY (White et al., 1994). Actually, it has been suggested that ST has a potential role in preventing mastitis in ruminants. Increased concentrations of endogenous ST are seen in cows during experimentally induced mastitis (Burvenich et al., 1999). Moreover, treatment of cows with bST during 10 consecutive days starting 2 d after experimentally induced E. coli mastitis showed they had better ability to recover from coliform mastitis than placebo cows, and recovery was more pronounced in the treated cows (Vandepute et al., 1993). However, beneficial effects were limited to severe mastitis (Burvenich et al., 1999) which suggests that ST seems to protect the blood-milk barrier and restore the integrity of the tight junctions in the mammary epithelium of an inflamed mammary gland. This may occur because of positive effects of ST and IGF-I on the cytoskeleton, tubular mRNA and for cytoskeletal organization as observed in rats (Goh et al., 1997; Berfield et al., 1997). In addition, administration of bST to lactating cows increases absolute leukocyte counts in blood (Elvinger et al., 1991). hi the current study, SCC of cows treated prepartum and postpartum with a low dose of bST was significantly less even though MY of the treated cows was greater. This implies there was a positive effect

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237 of bST on total SCC in the milk, perhaps because of some unmeasured combination of effects, as described previously. Dry period treatments and milk yield Dry period length has been addressed in a few designed trials. The most critical problem for the evaluation of different dry period lengths is the relationship between days dry and subsequent milk production (Sorensen et al., 1993). It is assumed that when a cow is dried off, the loss in the current lactation will be compensated for by greater milk production during the following lactation. However, the process of parturition and initiation of lactation are extremely important events that are associated with many problems that may result in removal of the individual cow from the herd or in greatly reduced milk production, especially early in the lactation. So, shortest possible dry period that would allow maximum milk production after parturition must be identified and evaluated, hideed these are only a limited number of studies, experimental and observational, that have been conducted to establish the association between minimum days dry and maximum milk yield in the lactation that follows the dry period. Fifty-five to 60 d dry period length has been recommended for use based on the fact that this would maximize production in the following lactation (Coppock et al., 1974; Dias and Allaire, 1982; Klein and Woodward, 1943; Schaeffer and Henderson, 1972). However, the current study did not detect either a benefit in setting the dry period at 60 d. Cows in 30 d dry group (5950±1 17 kg) and 30 d dry + ECP group (5857±150 kg) produced as much milk as cows in 60 d dry group (5835±144 kg) at 150 DIM. Moreover, 30 d dry groups produced an additional -5 10 kg milk during the extended 30 d lactation period before they were dried off. These results agreed with those previously reported

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238 (Bachman, 2002; Schairer, 2001). In their studies cows (n=15; 34 d dry) produced as much milk as their herdmates that had 57 d dry period (n=l 9). The overall milk yields for both short and long dry periods were about 9125 and 8986 kg at 305 DIM (Bachman, 2002). Moreover, 10 cows produced about 1 1,194 kg milk following a 32 d dry period, whereas 9 cows with 61 d dry period from the same herd produced 10,551 kg (Schairer, 2001). These authors indicated that shorter dry periods can be a profitable practice for dairy farmers. Between lactations, a nonlactating period is necessary for optimal milk production during the following lactation. On the other hand, follow up research should be done to study the effect of short dry period on long time health and longevity of these cows. Earlier recommendations were that dry periods should not be less than 50 d. Klein and Woodwork (1943) utilized 1 139 lactation records from Dairy Herd Improvement Association (DHIA) to study dry period length. They found that the optimum dry period was 55 d; cows producing -5000 kg of 4% FCM with 12 mo calving interval (CI). They made this recommendation even though average milk production for 40 to 49 and 60 to 69 d dry periods did not differ significantly from the 55 d dry period. Schaeffer and Henderson (1972) concluded that cows with dry periods of 50-59 d had the highest milk production during the subsequent lactation. Moreover, Funk et al. (1986) reported that cows dry for 60 to 69 d produced significantly more milk (-459 kg) in the subsequent lactation than cows dry for 40 d or less. Effects of days dry on milk yields of first (n=l 1583), second (n=7143) and third (n=6102) lactation Holstein cows from Zimbabwe and North Carolina were evaluated by Makuza and McDaniel (1996). Milk yields for 30-39, 40-49 and 50-59 d dry cows were 610, 633 and 202 kg less than for 60 d

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239 dry periods in both locations and there was little advantage observed for dry periods longer than 60 d. Observational data will be affected by many factors, in addition to dry period length, that are highly related to subsequent milk production. For example, data from existing records often will not include the reason why a specific cow was dried off earlier than other cows or why cows were dried off late (<60 d). Some cows cease lactation spontaneously or the dairy producer will dry off cows early because of insufficient milk production. Thus, the reason why cows had shorter dry periods most often cannot be learned from the milk yield records. Cows with short dry periods also may include those cows that calved early due to physiological problems, sickness or exposure to heat stress, among others. This would bias the estimated effect of days dry on milk yield in the subsequent lactation because of potential or actual problems during early lactation associated with early calving; this would affect the lactational performance. As a result, flaws in record analysis may produce a bias in the milk production records and this may result in insufficient information to adequately estimate the true effects of dry period length. On the other hand, early experimental studies also recommended a 50 to 60 d dry period. In one study, Swanson (1965) used five pairs of identical twin dairy cows. One of each pair of identical twins was given at least an 8 wk dry period, whereas other pairmates were milked continuously for two consecutive lactations. Average milk yield of the continuously milked twins in the second and third lactations was 75 and 62% of the control twins that had -60 d dry period. In another study, two quarters of each mammary gland of 2 cows were milked continuously while the other two quarters within the same cow were dried off for ~ 60 d before expected parturifion (Smith et al., 1967). The

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quarters allowed the dry period of 8 to 9 wk produced 40% more milk in the subsequent lactation (Smith et al., 1967). However, both trials had too few cow numbers and these only showed the need for a dry period not the dry period length. The only conclusion from many of these studies would be that mammary gland benefits from a dry period. Studies using a designed experimental protocol and utilizing larger numbers of cows have been conducted. For example, Coppock et al. (1974) conducted a 42 -mo field trial to evaluate the effects of dry period length on later milk production. Cows were assigned to treatments of 20, 30, 40, 50 or 60 d dry periods. Although the cow numbers were high (n=1019), only 305 cows (-30 %) completed the 42-mo study. At the end of 42 mo they concluded that cows averaging less than a 40 d dry period produced 450 to 680 kg less milk in the subsequent lactation compared to cows having dry periods of 40 d or longer. However, dry period lengths were allowed to have ±10 d range in each group. Therefore, a cow in 30 d dry group could have had a dry period ranging from 20 to 40 d. Importantly, the average length of days dry between cows assigned to 20 and 50 d dry could have been only 10 d. This could have seriously biased the estimated effect of the dry period length on subsequent milk production because the actual days dry varied greatly within the individual groups (Coppock et al., 1974). As described, there is a little doubt that cows need a dry period if they are to reach maximum possible MY that is determined by genetics and management. The exact length of time needed for the dry period has not been established definitively and likely is importantly influenced by the time needed for mammary involution. The time course and degree of mammary involution that occurs in cows differs noticeably from that seen in rodents (Capuco et al., 1997) which makes it difficult to model and evaluate the correct

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241 dry period length for cows by using a rapidly reproducing short lactation species, hivolution of the mammary gland occurs at a slower rate and alveolar structure is maintained for a greater portion of the period of involution in dairy cows than in rodents (Capuco and Akers, 1999). Moreover, it has been proposed that the process of mammary involution is completed by 25-d into the dry period in dairy cows. A nonsecretory state was achieved at 35 d prepartum (no epithelial cells containing secretory vesicles or fat droplets and mammary luminal area decreased to its minimum)(Capuco et al., 1997). This finding differs greatly from previously held view on speed of and extent of involution in dairy cows but does support the results obtained during the current study based upon lactation performance. Shorter dry periods did not negatively affect subsequent lactational performance compared to cows provided a 60 d dry period. Although estradiol 17p injections have been suggested as a way to increase rate of mammary involution in cows (Athie et al., 1996), no benefits of ECP injection were seen in the current study. Also, no significant differences among dry period treatments were detected for any measure of MY evaluated in the current study. This observation suggests that mammary gland involution and remodeling apparently can be completed within ~ 30 d . Thus, 30 d dry period should be long enough to allow cows to produce milk following parturition similar to that of cows that had essentially double the dry period length. Prepartum diets fed and milk yield Anionic or cationic diets fed during the prepartum period did not affect MYduring the first 21 wk postpartum, or MY or milk components during the first 10 wk postpartum. Moore et al. (2000) indicated that when cows are fed prepartum diets that had DC AD of 15, 0 or -15 mEq/100 g of dry matter, no observed effect on MY or any milk constituents

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242 are observed from 2 to 10 wk postpartum. On the other hand, MY increases when low DCAD diets are fed (-24.7 mEq/lOOg DM) prepartum compared with high DCAD diets (+5 mEq/lOOg DM) (West et al., 1991). In contrast, MY was not significantly improved when a negative DCAD diet (-7 mEq/lOOg DM) was offered 3 wk prepartum compared to high DCAD diets (+30 and +35 mEq/lOOg/ DM) (Joyce et al., 1997). Furthermore, lactation performance of cows was greatest when DCAD was between +30 and +50 mEq/lOOg dietary DM during lactation (Sanchez et al., 1994). In the current experiment, diet treatments effected no differences in DMI during prepartum or postpartum period in serum concentrations of Ca. Therefore, it seems that either positive or negative DCAD diets can be fed prepartum to Holstein cows as long as K in diet is below 1 .2% as percent of diet dry matter (Goff et al., 1997). Conclusions Injections of a low dose of bST (10.2 mg bST/d) during late prepartum and early postpartum periods (-21 to +28 d) caused increased prepartum concentrations of ST, IGF-I, INS and glucose and also postpartum concentrations of ST and IGF-I but no changes in postpartum concentrations of glucose and NEFA. Treated cows produced more milk, 3.5% FCM, and SCM from parturition through 10 wk, and milk through 21 wk. When both treated and untreated cows received a full dose of bST (500 mg bST/14 d) starting about 60 DIM the increase in milk production was maintained better through 21 wk in the bST cows. The mechanism(s) by which greater MY was stimulated by the low dose was not identified but undoubtedly was the result of a complex interplay of hormones, growth factor and metabolites on various organs and tissues, as well as other factors including increased DMI. Low dose of bST may have improved maintenance of

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243 mammary cell activity and/or numbers. However, it is likely the effect is more wide spread than just in the mammary gland. There was no evidence that shortening the dry period to -30 d caused a reduction in milk production. All cows were in adequate BCS before drying off (>3.25) and producing sufficient amounts of milk (>15 kg/d). Cows assigned to the three dry period treatments had almost identical total milk yields at the end of the 21 wk observation period. Providing ECP at time of dry off did not improve milk production of 30 d dry group through 150 d. Based upon milk production responses a ~ 30 d dry period was sufficient time for the mammary gland to involute, for epithelial cells to redifferentiate, and for a new lactation to be established. Prepartum diet treatments did not affect prepartum or postpartum DMI and subsequent milk production or milk composition. Prepartum anionic diet treatment did not have a significant effect on plasma concentrations of Ca and the cationic diet was just as effective as the anionic diet for maintaining plasma concentrations of Ca before and after calving.

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CHAPTER 5 GENERAL DISCUSSION After the termination of pregnancy, initiation of lactation necessitates a high degree of integration between the mammary gland and rest of the body. Lactation makes demands on the body of such a magnitude that the physiology of the mother differs greatly from that of the nonlactating state. The metabolic changes that occur ensures that the mammary gland is supplied with nutrients adequate to sustain an appropriate level of secretory activity. The metabolic requirements are especially demanding in highly selected dairy animals. A Holstein cow yielding 40 kg of milk daily secretes around 2 kg of lactose, 1.4 kg of fat and 1.2 kg of protein. Thus, daily feed intake (DMI) increases by more than 40-50 % in high yielding cows during lactation compared to nonlactating state. However, peak DMI does not occur immediately following parturition and therefore, increased nutrient demand of the lactating mammary gland cannot be met exclusively by increased DMI. High level of milk secretion associated with high demand for glucose, fatty acids and amino acids generally gives rise to increased hepatic gluconeogenesis, and increased mobilization of body fat and protein reserves. In addition, the utilization of these limiting nutrients (glucose, amino acids and lipid precursors) is reduced in low priority organs to support increased mammary gland activity. All these orchestrated changes in the metabolism of various organs and tissues ensure the adequate supply of needed by nutrients to the mammary gland. These modifications in the partitioning of nutrients during lactation are considered to be the consequence of homeorhetic control. 244

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245 Metabolic adaptations of organs and tissues are closely regulated by the alteration of responses to homeostatic controls. During early lactation there is a diminished whole body utilization of glucose and a decreased responsiveness of the lipolytic system and NEFA mobilization to INS. Reduced responsiveness to INS with the onset of lactation decreases the ability of fNS to inhibit gluconeogenesis in the liver and to stimulate lipogenesis in adipose tissue. In addition, glucose uptake in skeletal muscles and glucose oxidation in the whole body is decreased. Thus, a moderate degree of INS resistance in adipose tissue and lean muscle mass promotes the mobilization of NEFA and amino acids and spares glucose for other priority needs. Homeorhetic hormones are mostly responsible for regulating the metabolic adaptations that start during the transition period including partitioning of nutrients to adipose tissue, liver and skeletal muscle. Somatotropin is an important homeorhetic hormone. Along with its powerful galactopoietic effects, ST also alters tissue responsiveness to INS and catecholamines. Reduced responsiveness to INS decreases rates of lipogenesis and the activities of key enzymes such as acetyl CoA carboxylase, the rate limiting enzyme in fatty acid synthesis from acetate or glucose. In addition, ST dramatically increases the lipolytic response to catecholamines. High plasma concentrations of ST during late pregnancy may reduce INS receptors on adipocytes, inhibit the action of a second messenger, or inhibit the INS protease required for action of INS. Thus, ST has a pivotal role as a homeorhetic control on metabolism and nutrient partitioning (carbohydrates, lipids, proteins, and minerals) in the cow during the transition period.

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246 Bovine somatotropin (bST) is one of the unique products of biotechnology that has been developed and used on commercial dairy farms with a resultant exceptional increase in milk production by dairy cows when injected during an ongoing lactation. Obtaining a milk yield response to bST does not require special diets or different feed ingredients. However, treated cows do require adequate amounts of a balanced diet that contains all nutrients needed to support expected milk production. Major objectives of an efficient dairy farm operation include a successful lactation, high milk yield relative to the feed costs, reproductive competence, and finally the return of the cow to the BCS that existed before lactation so she will be prepared for another lactation. In farm animals a milk yield response to bST treatments has been well studied and fully documented (Bauman, 1999). Milk production responses to bST occur because of its known effects on partitioning of nutrients and because a greater proportion of the nutrient intake is used for milk synthesis. It increases liver glucose output, cardiac output, blood flow to the mammary gland and uptake of nutrients used for milk synthesis by the mammary gland among other effects. M addition, ST decreases the rate of oxidation of amino acid and glucose and glucose clearance. Treatment with bST results in coordinated changes of various organs and tissues which naturally occurs during the transition fi-om a nonlactating to lactating state when circulating concentration of ST is high. Because of the known effects of bST described above, use of bST during the transition period offers a means to cause positive and beneficial effects supporting of milk synthesis prior to parturition. These posifive effects of bST would allow dry cows to make better transition to lactation when a high demand for nutrient intake occurs. Consequently, injection of a low dose of bST (10.2 mg/d) during the transition period

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247 (-28 d prepartum through +28 d postpartum) and throughout the early lactation period (28 d through 56 d) was considered to have the potential enhance lactational performance. It was hypothesized that increasing concentrations of ST would augment the metabolic changes that favor the mammary gland and have a positive effect on DMI. Data from both the first and second studies suggest that use of 10.2 mg bST/d during late prepartum and early postpartum periods caused no apparent negative effects on the treated cows. Although EB was measured only in the second study during first 4 wk postpartum, NEB was not greater in injected cows than in uninjected cows in either study. This observation is inferred indirectly because the increase in MY and BW and the changes in BCS were equal or better in injected relative to in uninjected cows. Injection of bST resulted in better recovery of BW and BCS during early lactation, especially after injections were discontinued around 42 d postpartum. During the second study, low dose of bST did not provoke a greater loss of BW or a faster rate of decrease in BCS compared to untreated cows. Cows in both groups appeared equally capable of replenishing their body reserves even though all cows started injection of a fiill dose bST around 60 d postpartum; a daily dose that was three fimes greater than that injected before d 42. Bovine somatotropin treatments did not adversely or positively affect the rate of increase in DMI during the first 28 d postpartum; increase in DMI was same for both treated and untreated cows. In both studies, bST treated cows produced more milk and 3.5% FCM during the injection period. In the first study, no carryover effects of bST were detected on MY as evidenced by the fact that the increase in MY did not persist after bST injections were discontinued around 42 d postpartum. This suggested that positive effects on epithelial cell activity occurred because of prepartum and early postpartum bST

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248 injections. It is less likely that the amount of tissue in the gland, or at least number of epithelial cells in the gland were increased due to the low dose of bST injected. Clearly injections should be continued in order to maintain the synthetic activity of these cells and gain further benefits. On the other hand, results obtained during the second study contrast those just described. Treated cows showed similar increase in milk production through 60 d. However, treated cows also produced more milk during the time period when all cows (controls and treated) were injected with full dose of bST (>60 d of lactation). They also had higher concentrations of ST, INS and IGF-I prepartum and higher ST and IGF-I postpartum. While somewhat contradictory, resuhs of both studies indicate that low doses of bST injected prepartum and postpartum had measurable effects on the mammary gland and very likely on other physiological functions and organs to support the lactation. Although no specific data were collected to allow definitive answer as to what those changes were, it is likely that it was the sum of many small physiological changes that occurred and which stimulated greater milk production. Because MY of cows in the second study was maintained greater in the prepartum and early postpartum injected cows whereas, MY response was lost if all bST was discontinued after 60 d postpartum, this suggests that gland lost the stimulatory action of bST but likely had potenfial to respond to a greater level if it had been present. So, while results seem contradictory, they support a general and likely more diverse effect of bST injection which results in greater MY and DMI without apparent negative effects. In a broad view of these actions, bST perturbed the system in a positive way and made it possible for cows to respond better later in lactation without apparent negative effects on health. Indeed, results indicate the overall effects of prepartum and early postpartum bST

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249 were beneficial since BW and BCS were better maintained. Changes in concentrations of metabolic hormones, likely coupled with effects on various organs, suggest strongly a beneficial effect of bST during the transition period that affected the subsequent milk production whether or not the cows were injected with the full dose of bST (500 mg bST/14 d) later on in the lactation (>60 d in lactation). Importantly, no negative treatment effects on calving or prepartum or postpartum health status were observed during transition period for the cows across the treatments. This has been interpreted to indicate that injections of 10.2 mg bST/d can be used during the postpartum period and probably the prepartum period, to improve metabolic status and improve overall milk yields during early lactation. In current study, the low dose bST injections (10.2 mg bST/d) also were continued during early lactation period. Putnam et al. (1999) and Bachman et al. (1992) evaluated effects of use of bST during late gestation on subsequent milk production. Although Bachman et al. (1992) failed to detect improved MY for the prepartum bST injected cows, Putnam et al. (1999) reported a positive galactopoietic response when a full dose bST was injected prepartum beginning 28 d before expected calving. Garcia (1998) also reached the similar conclusion when a much smaller dose was used. He injected 5.1 mg bST/d before and after parturition, and saw greater MY response than treating cows only prepartum or only during the postpartum period. One possible explanation for improved MY response due to the prepartum bST treatment may be via enhanced IGF-I production in the treated cows, which, in turn, might have increased overall mammary cell numbers during prepartum injection period and/or early lactation. The potential of IGF-I to act as mammary mitogen is known (Cohick, 1 998). It increases mammary cell numbers and

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250 increases cell differentiation during prepartum and early lactation period. Because numbers of secretory cells in the mammary gland is a very important determinant of lactational performance, increased cell numbers would be beneficial. However, there is no conclusive evidence that cell numbers were increased. To fully understand any role ST has upon subsequent health and milk production measures prepartum, low dose of bST treatment effect must be tested alone. Another way to improve MY, without exogenous injections, would be to increase length of the lactation period. Dairy cows require a nonlactating period between successive lactations for optimal milk production during the subsequent lactation. Clearly, the dry period also allows cows to recover body reserves essential to support subsequent lactation. However, body reserves replenished during late lactation occurs more efficiently than that replenished during the dry period (Moe et al., 1971), so cows should maintain or gain BCS before they are dried off. In the current study, although dry period length did not have a significant effect on DMI, BW or BCS of the cows prepartum, cows provided the short dry period regained more BW and BCS when they were still lactating. In addition, cows given 60 d dry lost more BCS than 30 d dry cows and had less DMI as aper percent BW during the postpartum period. Although not quantified during the current experiment, incorporating fewer dietary changes in the dry cow management program may be beneficial to rumen function via the maintenance of a desired population of rumen microbes. The large diversity in the types of microbes found in the rumen is a reflection, to some extent, of the ruminants diet. Growth of microorganisms and efficient fermentation of feed by the microorganisms depends upon a constant and suitable environment. Changes in

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251 components fed or feed formulations cause a shift in microorganisms in the rumen and also decreases the efficiency of the fermentation and absorption processes. Changing the diet of the animal provokes a period of transition in the rumen microbial population such that the proportions of the various microbial species in the rumen will shift to a new balance, one which best accommodates the dietary change. This is referred to as adaptation of the microbial population. Adaptation may require several days to weeks. hi the current experiment, diet changes in 60 d dry cows (from lactation diet to FOD diet, from FOD diet to CUD diet and from CUD diet to lactation diet) likely would have required the rumen and its microbes to adapt three time during a short time period. These changes would probably limit the increase in feed intake immediately after parturition. On the other hand, lesser changes in diet of 30 d dry cows might have encouraged maintenance of a more stable rumen microbial population and better rumen papillae development. Thus, it might be advantageous to have fever diet changes prepartum and to allow cows to replenish body condition before they are dried off Our results suggested that if an adequate BCS can be achieved before drying off (>3.25), there were no advantages, based upon subsequent milk production, of providing cows with a 60 d dry period compared to a 30 d short dry period. In fact, cows might have better ability to maintain body condition and good health following parturition if shortening of the dry period is coupled with a good nutritional management program. One of the most important objectives of the dairy producer is to keep cows producing milk as much as possible throughout the year. The change from nonlactating to lactating stage is very demanding on the cows and results in important metabolic changes and loss of milk production during the time they are not milking. These metabolic

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252 changes may lead to increased incidence of metabolic problems which may affect their health and/or productivity and cause the cows to be removed from the herd. In addition, during the dry period, cows do not produce milk and achieving maximal milk production during the next lactation with the least number of days dry becomes important. Establishing optimum length of the dry period is critical to achieve maximum milk production during the next lactation. Cows given 30 d dry periods yielded essentially the same amounts of milk at the end of 21 wk period compared to the traditionally managed 60 d dry herdmates. Similar results also were reported by Bachman (2002) and Schairer (2001). Furthermore, an additional 500 kg of milk was obtained from 30 d dry cows by shortening dry period to only 30 d and milking the cows an additional 30 d. If one can verify that cows with -30 d dry periods will produce just as much milk as those with 6070 d dry periods during the next lactation, with no other negative effects on the cow as a consequence of reducing dry period length, then there is an opportunity of extra milk income being generated for each cow during a lactation. Although the current study did not attempt to quantify economics of a 30 d dry period, it is possible to roughly estimate potential economic benefits of incorporating a 30 d dry period in dairy management. For example, an additional 1 5-20 kg milk/d might be expected from the increased days in milk (-30 d); this will milk income by as much as $160.00 (30 d* 16 kg/d*$0.33/kg) per cow. Statewide, 24 million dollars ($160.00*150,000 cows in producing milk in Florida) would otherwise be lost. Furthermore, an additional 4,500,000 DIM (150,000 cows*30 d) would be achieved without the addition of any more cows to the current population. Of course, in order for us to perform this short dry period practice, cows should be producing adequate amounts of milk at -60 d prepartum when they traditionally would be dried off

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253 They also should have a good BCS (minimum 3.25), and should be provided needed nutrients via an excellent feeding program to support their needs. On the other hand, there is an extra feed cost to be accounted for if cows were to have shorter dry periods and it is very unlikely that all cows would qualify for a shorter dry period. Lactation diets are relatively costly, greater than for dry cows. Thus, income coming from the increased milk yield and the cost of additional feed needs to be determined carefully to achieve economically sound management. In the current experiment, shortening the dry period did not decrease the yield of milk in the subsequent lactation or during 21 wk postpartum. Moreover, no evidence was detected to suggest that ECP injection at the time of drying off is necessary to achieve maximal milk production during the next lactation. Based upon milk production, it appears that ~ 30 d dry period is sufficient time for the mammary gland to involute and subsequently remodel with differentiation of the epithelial cell population. Certainly, favorable results of this and other studies (Bachman, 2002; Schairer, 2001) strongly supports need for further research efforts in this area to evaluate potential health effects, any changes in calving intervals, and cow turnover (culling rates) that may occur. Similarly, it must be determined whether the practice can be used during hotter summer months when gestation length may already be shorter and cows would be at risk of too short a dry period if they calved earlier than expected. On the other hand, none of the cows used in current studies on dry period length had dry periods during the hot summer days. This was purposely avoided because of the potential for earlier than expected calving. Heat stress during the summer is known to induce early calving. As a result, employing shorter dry periods may result in cows

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254 having dry periods less than 20 d. Although ECP injections did not have positive effects on the lactational performance, there may be benefits of using ECP for cows that would have shorter dry periods, even less than 30 d, because estrogen has been found to increase speed of mammary involution. Administration of estrogen was associated with induction of serine protease, plasminogen and plasmin activity within the gland and activation of these serine protease are known to accelerate mammary involution (Athie et al., 1997). Finally, although cow numbers were too few to critically evaluate health status, no apparent health and/or calving problems or benefits were observed for the cows across the dry period treatments. Importantly, no effect of ECP on early calvings and/or abortions was observed during the experiment, not even for cows that calved during the months of September through May, which were somewhat more stressful time periods. Because of the risk of early calvings due to heat stress, use of ECP and short dry period (<30) combinations should be tested during the summer months to evaluate effects on subsequent health and production and if drt periods are actually reduced to less than 30 d.. Hypocalcemia is a metabolic disorder of Ca homestasis that affects fresh cows. This disease is related to the metabolic turnover of Ca. This is especially important for dairy cows around calving because metabolic turnover of Ca is the greatest for cows per kg of BW at calving and during early lactation. As a result, feeding diets that have a negative DCAD has been recommended and used to prevent onset of milk fever during the last 21 d prepartum and shortly after calving. However, in recent years it has been suggested that high potassium contents of ingredients utilized in close-up rations is as important or even more important than acidifying blood by feeding negative DCAD diet to prevent hypocalcemia in dairy cows (Goff et al., 1997). No clinical hypocalcemia was

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255 observed during this study and the overall serum concentrations of Ca did not differ in cows fed anionic (-10 Meq/lOOg) or cationic diets (+ 20 Meq/lOOg) before, during, or around calving. Prepartum anionic diet treatment did not have a significant effect on plasma concentrations of Ca and the cationic diet was just as effective as the anionic diet for maintaining plasma concentrations of Ca before and during the 28 d immediately after calving. Thus, feeding anionic diet did not result in better maintenance of serum concentrations of Ca in Holstein cows. Additionally, no effects of prepartum diets fed were observed on prepartum or postpartum DMI, BW or BCS, and had little, if any, effect on postpartum DMI. In fact, cows fed anionic and cationic diets prepartum maintained DMI greater than typically observed during the transition period. The DMI were greater than 23 kg/d 8 d postpartum. High DMI around the calving also might have helped cows to maintain adequate concentrations of Ca around parturition which , in turn, prevented onset of hypocalcemia (milk fever) and allowed cows to transition into lactation in good health and with greater availability of ingested nutrients. In conclusion, results of these studies suggest that injections of 10.2 mg of bST/d before and after parturition increased plasma concentrations of ST and IGF-I with a significant increase in milk production during treatment in early lactation. Furthermore, non-significant differences in MY of cows provided 30 and 60 d dry periods suggested that a 30 d dry period is sufficient time for mammary gland to involute and redifferentiate. Finally, because no strong adverse effects of bST or short dry period length were evident, these practices have potential to improve management of transition cows.

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APPENDIX LIST OF SIGNIFICANT TWO-WAY INTERACTIONS Table A-1. Prepartum significant two-way interactions between diet treatments and season for BW. Treatments DIET-I*SEA-I DIET-PSEA-II DIET-II*SEA-I DIET-IPSEA-II LSM±SD 668.6 ± 11.1 696.9 ± 18.9 714.4 ± 17.3 679.7 ±15.1 DIET-PSEA-I DIET-I*SEA-II DIET-II*SEA-I DIET-II*SEA-II NS P<0.04 NS NS NS NS P<0.04 NS NS NS NS NS DIET-I=prepartum anionic diet, DIET-II=Prepartum cationic diet, SEA-I= cows with dry periods during hot months (September, October, March, April, and May), SEA-II= cows with dry periods during cold months (November, December, January, and February). Table A-2. Prepartum significant two-way interactions between diet treatments and season for BCS. Treatments DIET-PSEA-I DIET-PSEA-Il DIET-II*SEA-I DIET-IPSEA-II LSM ± SD 3.35 ± 0.07 3.41 ± 0.09 3.52 ±0.09 3.35 ±0.08 DIET-PSEA-I DIET-PSEA-II DIET-IPSEA-I DIET-II*SEA-II NS P<0.1 NS NS NS NS P<0.1 NS NS NS NS NS DIET-I=prepartum anionic diet, DIET-II=Prepartum cationic diet, SEA-I= cows with dry periods during hot months (September, October, March, April, and May), SEA-I1= cows with dry periods during cold months (November, December, January, and February). 256

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257 Table A-3. Prepartum significant two-way interactions between dry period treatments and season for BCS. Treatments DRY-I *SEA-I DRY-I *SEA-II DRYII*SEA-I DRYII*SEA-II DRY-III *SEA-I DRY-III *SEA-II LSM ±SD 3.52 ± 0.08 3.38 ± 0.09 3.31 ±0.11 3.56 ± 0.08 3.48 ± 0.08 3.21 ±0.12 DRY-I *SEA-I — NS NS NS NS P<0.05 DRY-I SEA-II NS — NS NS NS NS DRYII*SEA-I NS NS P<0.1 NS NS DRYIISEA-II NS NS P<0.1 NS P<0.03 DRY-III *SEA-I NS NS NS NS P<0.08 DRY-III *SEA-II P<0.05 NS NS P<0.03 P<0.08 DRY-I=30 d dry period with no ECP, DRY-II=30 d dry period + ECP, DRY-III= 60 d dry epriod, SEA1= cows with dry periods during hot months (September, October, March, April, and May), SEA-II= cows with dry periods during cold months (November, December, January, and February). Table A-4. Postpartum significant two-way interactions between diet treatments and season for BW. Treatments DIET-PSEA-I DIET-PSEA-II DIET-II*SEA-I DIET-IPSEA-II LSM ± SD 23.6 ±0.5 23.3 ±0.5 21.4 ±0.6 25.4 ±0.5 DIET-I*SEA-I DIET-PSEA-II DIET-II*SEA-I DIET-IPSEA-II NS P<0.01 P<0.01 NS P<0.05 P<0.02 P<0.01 P<0.05 P<0.01 P<0.01 P<0.02 NS DIET-I=prepartum anionic diet, DIET-II=Prepartum cationic diet, SEA-I= cows with dry periods during hot months (September, October, March, April, and May), SEA-II= cows with dry periods during cold months (November, December, January, and February).

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Table A-5. Postpartum significant two-way interactions between dry period treatments and season for BCS. Treatments DRY-I *SEA-I DRY-I *SEA-II DRYII*SEA-I DRYII*SEA-II DRY-III *SEA-I DRY-III SEA-II LSM ± SD 3.21 ±0.08 3.14 ±0.09 3.10 ±0.10 3.34 ±0.08 3.09 ±0.07 2.88 ±0.11 DRY-I *SEA-I — NS NS NS NS P<0.02 DRY-I *SEA-II NS NS NS NS P<0.07 DRYII*SEA-I NS NS P<0.07 NS NS DRYIISEA-II NS NS P<0.07 P<0.02 P<0.01 DRY-III SEA-I NS NS NS P<0.02 NS DRY-III *SEA-II P<0.02 P<0.07 NS P<0.01 NS DRY-I=30 d dry period with no ECP, DRY-II=30 d dry period + ECP, DRY-III= 60 d dry epriod, SEA1= cows with dry periods during hot months (September, October, March, April, and May), SEA-II= cows with dry periods during cold months (November, December, January, and February).

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1 259 Table A-6. Postpartum significant two-way interactions between bST and dry period treatments for ENS. Treatments DRY-I *bST-I DRY-I *bST-II DRYII*bST-I DRYII*bST-II DRY-III *bST-I DRY-III *bST-II LSMiSD 0.56 ±0.07 0.63 ± 0.07 0.78 ± 0.07 0.50 ± 0.06 0.52 ± 0.07 0.63 ±0.08 ; DRY-I *bST-I — NS NS NS NS NS DRY-I *bST-II NS NS NS NS NS DRYII*bST-I P<0.02 NS P<0.01 P<0.01 NS DRYII*bST-II NS NS P<0.01 NS NS DRY-III *bST-I NS NS P<0.01 NS NS DRY-III *bST-II NS NS NS NS NS DRY-I=30 d dry period with no ECP, DRY-II=30 d dry period + ECP, DRY-III= 60 d dry epriod, SEA1= cows with dry periods during hot months (September, October, March, April, and May), SEA-n= cows with dry periods during cold months (November, December, January, and February).

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260 I Table A-7. Postpartum significant two-way interactions between bST and dry period treatments for NEFA. Treatments DRY-I *bST-I DRY-I *bST-II DRYII*bST-I DRYII*bST-II DRY-III *bST-I DRY-III *bST-II LSM ± SD 565.5 ±47 597.3 ± 45 441.4 ±47 773.9 ±57 740.7 ± 50 530.8 ± 56 DRY-I *bST-I NS P<0.07 P<0.01 P<0.02 NS DRY-I *bST-II NS P<0.03 P<0.02 P<0.04 NS DRYII*bST-I P<0.07 P<0.03 P<0.01 P<0.01 NS DRYII*bST-II P<0.01 P<0.02 P<0.01 NS P<0.01 DRY-III *bST-I P<0.02 P<0.04 P<0.01 NS P<0.01 DRY-III *bST-II NS NS NS P<0.01 P<0.01 DRY-I=30 d dry period with no ECP, DRY-II=30 d dry period + ECP, DRY-III= 60 d dry epriod, SEA1= cows with dry periods during hot months (September, October, March, April, and May), SEA-II= cows with dry periods during cold months (November, December, January, and February).

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261 Table A-8. Postpartum significant two-way interactions between dry period treatments and season for IGF-I. Treatments DRY-I *SEA-I DRY-I *SEA-II DRYII*SEA-I DRYII*SEA-II DRY-III *SEA-I DRY-III *SEA-II LSMiSD 107.6 ±11 135.9 ±12 132.2 ±15 163.7 ± 11 151.0± 10 104.9 ± 16 DRY-I *SEA-I P<0.09 NS P<0.01 P<0.01 NS DRY-I *SEA-II P<0.09 NS P<0.1 NS NS DRYII*SEA-I NS NS P<0.1 NS NS DRYII*SEA-II P<0.01 P<0.1 P<0.1 NS P<0.01 DRY-III *SEA-I P<0.01 NS NS NS p<0.02 DRY-III *SEA-II NS NS NS P<0.01 P<0.02 DRY-I=30 d dry period with no ECP, DRY-II=30 d dry period + ECP, DRY-III= 60 d dry epriod, SEA1= cows with dry periods during hot months (September, October, March, April, and May), SEA-II= cows with dry periods during cold months (November, December, January, and February).

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BIOGRAPHICAL SKETCH I was bom in Antalya, Turkey in 1970. I completed my elementary and middle school education in Antalya and Iskenderun, respectively. Because of the changes in my father's business position, we moved to Egirdir where I graduated from high school. I sat for a national exam right after my graduation and was admitted into Ankara University, College of Veterinary Medicine. I started my college education there in 1987 and graduated from the University in 1992 as a veterinarian. I worked for 1 year as a veterinarian and then took another nationwide exam for the opportunity to study in the USA for both the Master's and PhD degrees. I spent 6 months at the University of Delaware for English education and started my master's degree program at Clemson University in January 1996. Then, I transferred to the University of Florida, Department of Dairy and Poultry Sciences in August, 1996 to continue to my master's degree and received an MS degree in August 1998. 1 have been working on my PhD studies since then. 288

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I certify' that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. H. Herbert Head. Chairman Professor of Animal Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fuliy adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. K^rmit C. Associate Professor of Animal Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Mary Befh Hall Assistant Professor of Animal Sciences I certifV' that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. "Rachel B. Shireman Professor of Food Science and Human Nutrition This dissertation was submitted to the Graduate Faculty of the College of Agriculture and Life Sciences to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of.DQClQj_ot-PMosophy. August 2002 ( X Dean. College of Agricuihurkl | and Life Sciences Dean. Graduate School