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Reproductive Strategies in the Postpartum Dairy Cow with Reference to Anovulation and Postpartum Uterine Health

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PAGE 1

REPRODUCTIVE STRATEGIES IN THE POSTPARTUM DAIRY COW WITH REFERENCE TO ANOVULATION AND POSTPARTUM UTERINE HEALTH By KATHERINE ELIZABETH MAY HENDRICKS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

PAGE 2

Copyright 2004 by Katherine Elizabeth May Hendricks

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Dedicated to my mother, Elsa, and my husband, Gregory

PAGE 4

ACKNOWLEDGMENTS I would like to express my appreciation and thanks to Dr. Louis F. Archbald for the opportunity to join his graduate program. Dr. Archbalds passion for this subject area, knowledge and dedication as an advisor have been a great motivating force in aiding the completion of this thesis. His professionalism and discipline have been an example that I hope to emulate personally and professionally. I would like to express my thanks to Dr Charles Courtney for funding my program. I would like to take this opportunity to thank the members of my committee, Dr. Peter Hansen, Dr. J. Hernandez, Dr. Pedro Melendez and Dr. Carlos Risco, for their insights and valuable suggestions throughout my study. Special thanks go to my parents, Elsa May Wilmot-Binns and Lloyd Ivanhoe Binns, for believing in my abilities to do anything I set my mind to and for advising me to stick with my life-long dreams of becoming a veterinarian. Thanks go to my Godparents, Dermoth and Merle Bingham, for their sacrifices and invaluable assistance in helping me create the foundation for entry into the University of Florida. I thank my friends who stood by me during my DVM, Merlene Mercano, Dr. Denice Lewis, Dr. Nicole Tull, Dr. Susan McClennon and Dr. Rosemary Murray I would like to acknowledge Dr. Julian Bartolome for his help and support in execution of the project. I would like to express my thanks and appreciation to Ingo Kreig for use of the dairy herd, Roger Rowe and the entire staff at Mecklenberg Dairy. I would like to thank iv

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Don Bennink for the use of the dairy herd and the entire staff at North Florida Holstein Dairy. I thank all who contributed to this study but were not mentioned. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES...........................................................................................................xi ABSTRACT......................................................................................................................xii CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................5 Gonadotropin-releasing Hormone (GnRH)..................................................................5 Chemical Nature of the Hormone..........................................................................5 Neurotransmitters and GnRH Secretion................................................................6 Control of Secretion..............................................................................................7 Function of GnRH...............................................................................................10 Mechanism of Action..........................................................................................12 Prostaglandins.............................................................................................................13 Biosynthesis.........................................................................................................14 Metabolism..........................................................................................................16 Mechanism of Action..........................................................................................16 Function in the Reproductive Tract of the Cow..................................................17 Prostaglandin F2 and its Uses in the Dairy Cow..............................................19 Estrous Cycle of the Cow...........................................................................................21 Follicular Phase...................................................................................................21 Luteal Phase.........................................................................................................22 Hormonal Control of the Estrous Cycle..............................................................23 Effect of Season...................................................................................................25 Folliculogenesis and Ovarian Dynamics in the Dairy Cow........................................29 Follicular Development.......................................................................................29 Follicular Waves..................................................................................................31 Regulation of Follicular Growth by FSH and LH...............................................33 Production of Estrogen........................................................................................34 Corpus Luteum in the Cow.........................................................................................34 Corpus Luteum Development..............................................................................35 vi

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Function of the Corpus Luteum...........................................................................37 The Corpus Luteum and Pregnancy....................................................................38 Luteolysis............................................................................................................40 Involution of the Bovine Uterus.................................................................................44 Macroscopic Changes..........................................................................................44 Microscopic Changes..........................................................................................47 Factors Affecting Uterine Involution..................................................................50 Methods to Assess Uterine Involution................................................................56 Endocrinology of the Immediate Postpartum Period in the Cow...............................58 Resumption of Ovarian Cyclicity Post Partum...........................................................60 Cystic Ovarian Disease in the Dairy Cow..................................................................63 Predisposing Factors............................................................................................64 Pathogenesis........................................................................................................65 Cyst Dynamics.....................................................................................................67 Diagnosis.............................................................................................................68 Treatment.............................................................................................................69 Timed Artificial Insemination Programs in the Dairy Cow.......................................71 Prostaglandin F2................................................................................................73 Combination of Gonadotropin-releasing Hormone (GnRH) and PGF2...........73 Presynch-Ovsynch...............................................................................................75 Postpartum Endometritis............................................................................................75 Pathology.............................................................................................................76 Pathogenesis........................................................................................................78 Diagnosis.............................................................................................................79 3 EXPERIMENT 1: AN EVALUATION OF PRETREATMENT WITH GNRH ON OVARIAN RESPONSE AND PREGNANCY RATE OF LACTATING DAIRY COWS WITH OVARIAN CYSTS SUBJECTED TO THE OVSYNCH PROTOCOL...............................................................................................................84 Introduction.................................................................................................................84 Materials and Methods...............................................................................................85 Statistical Analysis......................................................................................................87 Results.........................................................................................................................88 Discussion...................................................................................................................92 4 EXPERIMENT 2:EFFECT OF SEQUENTIAL ADMINISTRATION OF PGF2 ON THE PREVALENCE OF MUCOPURULENT DISCHARGE, SIZE OF THE CERVIX, SIZE OF THE PREVIOUSLY PREGNANT UTERINE HORN AND FIRST SERVICE PREGNANCY RATE IN LACTATING DAIRY COWS............98 Introduction.................................................................................................................98 Experiment 2 Part A..............................................................................................102 Materials and Methods......................................................................................102 Treatment...........................................................................................................104 Exclusion Criteria..............................................................................................105 Blood Sampling and Hormone Assay...............................................................105 vii

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Vaginoscopy......................................................................................................107 Transrectal Palpation of the Reproductive Tract...............................................107 Statistical Analysis............................................................................................108 Experiment 2 Part B...............................................................................................109 Treatment...........................................................................................................110 Exclusion Criteria..............................................................................................111 Pregnancy Diagnosis.........................................................................................111 Statistical Analysis............................................................................................112 Results.......................................................................................................................112 Experiment 2 Part A.......................................................................................112 Day 8 post partum......................................................................................112 Day 16 post partum....................................................................................112 Day 22 post partum....................................................................................113 Day 36 post partum....................................................................................116 Day 58 post partum....................................................................................117 Model 1......................................................................................................119 Model 2......................................................................................................120 Model 3......................................................................................................123 First service pregnancy rate........................................................................126 Experiment 2 Part B : Conception Rate to First Service................................127 Discussion.................................................................................................................129 5 SUMMARY AND CONCLUSIONS............................................................................134 APPENDIX VAGINOSCOPY AND TRANSRECTAL PALPATION FORM..........138 LIST OF REFERENCES.................................................................................................140 BIOGRAPHICAL SKETCH...........................................................................................163 viii

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LIST OF TABLES Table page 3-1 Baseline data for heifers and cows that were diagnosed with ovarian cysts and successfully completed Experiment 1 for parity (1, 2, 3+), time of year (March-September/October-February), days in milk (DIM) and progesterone concentration (< 0.5 ng/ml / 0.5 ng/ml ) on Day 0................................................89 3-2 Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the risk of finding a CL on Day 7 in cows with ovarian cysts treated with GnRH on Day 0........................................................................................................................90 3-3 Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the risk of finding progesterone concentration > 1.0 ng/ml on Day 7 in cows with ovarian cysts treated with GnRH on Day 0..............................................................90 3-4 Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the risk of pregnancy in lactating dairy cows with ovarian cysts treated with GnRH on Day 0...................................................................................................................91 4-1 Distribution of cows enrolled in the study between June and August 2003 within Groups 1 and 2 based on days in milk (DIM), parity (primiparous/multiparous), dystocia (yes/no) and retained fetal membranes (yes/no)......................................106 4-2 Baseline data for heifers and cows that successfully completed the study for parity (primiparous/multiparous), days in milk, dystocia (no/yes), retained fetal membranes (no/yes) and progesterone concentration ( 1.0 ng/ml / >1.0 ng/ml) at 8 days post partum..............................................................................................113 4-3 Model 1Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy on Day 22 post partum....................................121 4-4 Model 1Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy on Day 58 post partum....................................121 4-5 Model 2Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy and a cervix > 50 mm in diameter by transrectal palpation on Day 22 post partum..........................................................122 ix

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4-6 Model 2Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy and a cervix > 30 mm in diameter by transrectal palpation on Day 58 post partum..........................................................123 4-7 Model 3Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy, cervix > 50 mm in diameter and previously pregnant horn (PPH) 30 mm by transrectal palpation on Day 22 post partum...125 4-8 Model 3Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy, cervix > 30 mm in diameter and previously pregnant horn (PPH) 30 mm by transrectal palpation on Day 58 post partum...126 4-9 Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of pregnancy in dairy cows treated sequentially with PGF2 in the early postpartum period to first service post partum.............................................................................................................127 4-10 Baseline data for cows within Group 1 and Group 2 based on parity (2, 3+), dystocia (yes/no), retained fetal membranes (yes/no) and abnormal calving (RFM and/or dystocia) that successfully completed the study...............................128 4-11 Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of pregnancy in dairy cows treated sequentially with PGF2 in the early postpartum period and subjected to the Presynch-Ovsynch protocol...................................................................................129 x

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LIST OF FIGURES Figure page 3-1 Experiment 1 Experimental Design......................................................................87 4-1 Experimental 2 Part A: Experimental Design.....................................................105 4-2 Experiment 2Part B: Experimental Design..........................................................111 xi

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science REPRODUCTIVE STRATEGIES IN THE POSTPARTUM DAIRY COW WITH REFERENCE TO ANOVULATION AND POSTPARTUM UTERINE HEALTH By Katherine E. M. Hendricks August 2004 Chair: L. F. Archbald. Major Department: Veterinary Medical Sciences In Experiment 1, a total of 155 cows with ovarian cysts was sequentially allocated to 3 groups on the day of diagnosis (Day 0). Cows in Group 1 (n = 55) were treated with GnRH (100 g, im) on Days 0 and 7, PGF2 (25 mg, im) on Day 14, GnRH (100 g, im) on Day 16, and timed inseminated (TAI) 16-20 h later. Cows in Group 2 (n = 49) were treated with GnRH on Day 0, PGF2 on Day 7, GnRH on Day 9, and TAI 16-20 h later. Cows in Group 3 (n = 51) were treated with GnRH on Day 7, PGF2 on Day 14, GnRH on Day 16, and TAI 16-20 h later. Pregnancy was determined by rectal palpation 45-50 d after TAI. On both Days 0 and 7, cows in all groups were subjected to ultrasonography (U/S) and blood samples were obtained for determination of progesterone concentration (P4). Cows in Groups 1 and 2 were more likely to have a CL and high P4 on Day 7 compared to cows in Group 3. There was no significant difference in pregnancy rate (PR) between cows in all groups, but cows with a CL on Day 7 were more likely to become pregnant. It was concluded that administering GnRH to cows with ovarian cysts 7 days xii

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prior to the initiation of the Ovsynch protocol did not increase PR, but cows with a CL on Day 7 were more likely to become pregnant. In Experiment 2 (Part A), a total of 228 postpartum (pp) dairy cows was sequentially allocated to 2 groups between Days 7 and 9 pp. Cows in Group 1 (n=114) were treated twice with PGF2 (25 mg, im) 8 h apart on Days 8 and 15 pp, and once on Days 22 and 36 pp. Cows in Group 2 (n=114) served as untreated controls. Vaginoscopy and rectal palpation were done on Days 22 and 58 pp. Cows in both groups were inseminated at estrus, or TAI approximately 130 to 134 days pp. Pregnancy was determined by rectal palpation between 45-50 d after insemination. It was concluded that sequential administration of PGF2 in the immediate postpartum period reduced the prevalence of a mucopurulent discharge, and both the size of the cervix and previously pregnant uterine horn only on Day 58 pp. There was no difference in PR between cows in both groups. In Experiment 2 (Part B), a total of 418 postpartum dairy cows was sequentially allocated to 2 groups on Day 7 pp. Cows in Group 1 (n=209) were treated twice with PGF2 (25 mg, im) 8 h apart on Days 7 and 14 pp, and once on Days 21 and 35 pp. Cows in Group 2 (n=209) served as untreated controls. All cows were subjected to the Presynch and Ovsynch protocols on Days 49 and 75 pp, respectively. Pregnancy was determined by U/S between Days 29 and 32 after TAI. There was no significant difference in the conception rate to first service between the groups. xiii

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CHAPTER 1 INTRODUCTION The profitability of a commercial dairy farm is based in part on the calving interval of the cows. In order to maximize the economic profitability of the farm, cows must return to ovarian cyclicity, express estrus and be bred within 85 days postpartum. The optimal calving interval is 365 days. There are two physiologic factors which influence reproductive success in the postpartum dairy cow. The first is ovarian cyclicity, and the second is uterine health. Parturition is a very traumatic event, and the ability to control ovarian and uterine events in the postpartum cow could play an important role in achieving subsequent fertility. In this study, two experiments were performed. The first experiment investigated the use of gonadotropin releasing hormone (GnRH) to improve ovarian cyclicity in lactating dairy cows diagnosed with ovarian follicular cysts. The second experiment investigated the use of prostaglandin F2 in the early postpartum period to improve uterine health and improve fertility in lactating dairy cow. Ovarian follicular cysts, also known as cystic ovarian degeneration, cystic ovaries or anovular follicles, is the most common abnormality of follicular function in cattle (Farin and Estill, 1993). It is most often seen in the postpartum dairy cow within the first 60 days post partum (Farin and Estill, 1993). Approximately 6% of dairy cows develop ovarian follicular cysts (McLeod and Williams, 1991; Garverick 1997; Silvia et al., 2002). Cystic ovarian degeneration increases days open by 22 to 64 days and increases the number of cows culled from the herd. Each occurrence of ovarian follicular 1

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2 cysts has been estimated to cost $137 in reduced milk production and veterinary expenses (Silvia et al., 2002). The development of programs to prevent and/or treat cystic ovarian degeneration will benefit the dairy farmer. The resumption of ovarian cyclicity is dependent on a number of factors including clearance of bacterial contamination from the uterus (Sheldon et al., 2002). Bacterial contamination of the uterus occurs within the first week post partum (Elliott et al., 1968) with spontaneous contamination, clearance and recontamination occurring up to seven weeks post partum (Griffin et al., 1974). Some cows have the ability to clear these infections but others do not and the reasons for this variability between cows are unknown. Bacterial contamination of the uterus has a direct effect on the ability of the cow to conceive and maintain a conceptus. Conception and the maintenance of pregnancy are, therefore, dependent on a healthy uterine environment. The incidence of endometritis is greatest during the first 14 days post partum based on cultures of uterine fluids and uterine biopsies (Griffin et al., 1974). Failure to clear bacterial contamination by first ovulation post partum and corpus luteum formation could place the contaminated uterus under the influence of progesterone. Progesterone makes the uterus more prone to uterine infection (Hawk et al., 1964) with the incidence of severe endometritis increasing around Day 15 to Day 21 postpartum. This increase in the severity of endometritis coincides with the time of first post partum ovulation (i.e., 15 to 28 days post partum). The hypothesis of Experiment 1 is based on research done in cows without ovarian cysts. This research demonstrated that cows in the early luteal phase of the estrous cycle at the time of initiation of the Ovsynch protocol had a higher pregnancy rate compared to

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3 cows at other stages of the estrous cycle (Vasconcelos et al., 1999; Moreira et al., 2000a). The hypothesis of Experiment 1 was that GnRH administered to cows at the time of diagnosis of ovarian cysts would induce an early luteal phase and thus increase pregnancy rates to a protocol for synchronization of ovulation and timed insemination (Ovsynch protocol). The Ovsynch protocol consists of a single intramuscular injection of 100 g of GnRH, followed seven days later by PGF2 (25 mg, intramuscularly). The second dose of GnRH (100 g, im) is administered 48 hours after PGF2 with artificial insemination occurring 16 to 24 hours later. The objectives of Experiment 1 were two-fold. The first objective was to evaluate the ovarian response 7 days following diagnosis and treatment of cows with ovarian cysts with GnRH. The second objective was to determine the pregnancy rate of cows with ovarian cysts subjected to treatment with GnRH 7 days prior to the initiation of the Ovsynch protocol. The hypothesis of Experiment 2 was that sequential administration of PGF2 during the early postpartum period would reduce the incidence of mucopurulent discharge, size of the cervix, size of the previously pregnant uterine horn and increase first service pregnancy rates. The objective of Experiment 2, Part A was to determine the effect of sequential administration of PGF2 in the immediate postpartum period on the incidence of mucopurulent discharge, size of the cervix and size of the previously pregnant uterine horn and first service pregnancy rates. The objective of Experiment 2, Part B was to evaluate the effect of sequential administration of PGF2 in the immediate postpartum period on first service conception rate in postpartum dairy cows subjected to a timed insemination protocol consisting of Presynch-Ovsynch protocol. The Presynch portion of the timed insemination protocol consisted of two intramuscular injection of 25

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4 mg PGF2 given 14 days apart. The Ovsynch protocol began 12 days after the second dose of PGF2.

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CHAPTER 2 LITERATURE REVIEW Gonadotropin-releasing Hormone (GnRH) Chemical Nature of the Hormone Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone releasing hormone (LHRH), is a decapeptide produced in the arcuate, suprachiasmatic and preoptic nuclei of the hypothalamus in response to hormonal and integrated neuronal signals. The amino acid sequence of GnRH was first elucidated as early as 1971. The primary structure of GnRH has been established in the pig (Matsuo et al., 1971), sheep (Amoss et al., 1971; Burgus et al. 1972) and human placenta (Tan and Rousseau, 1982). GnRH is synthesized from a precursor molecule, which begins with a 23 amino acid signal sequence, followed by the GnRH decapeptide and ending with a 56 amino acid GnRH-agonist associated peptide. There are between 800 and 2500 GnRH neurons located throughout the brain of vertebrates. These neurons originate in the olfactory placode outside the CNS and migrate inwards during fetal development (Silverman, 1984). Immunohistochemical techniques have been used to elucidate the location of the GnRH containing cell in the bovine hypothalamus and infundibulum. GnRH-positive cell bodies have been located singly or in small clusters in the infundibular nucleus (INFN), and in large discrete clusters in the ventromedial nucleus (VMN) with their axons projecting into the rostral hypothalamus (the grey matter of the lamina terminalis and medial preoptic area). The majority of axons were found within the middle region of the hypothalamus within the 5

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6 dorsomedial nucleus, VMN, INFN, and lateral hypothalamic area, the periventricular nucleus (Dess and McArthur, 1981). Embryonic GnRH neurons in primary cultures of olfactory placodes from ovine embryos begin pulsatile secretion of GnRH by 17 to 24 days, indicating that these neurons must mature prior to the onset of pulsatility (Duittoz and Batailler, 2000). GnRH is synthesized in neuroendocrine cell bodies and transported down axons that terminate in the median eminence where it is released in a pulsatile manner (Silverman, 1984; Rodriquez and Wise, 1989; Naor et al., 1998). In the median eminence, GnRH is released into the surrounding capillaries that drain into the hypothalamo-hypophyseal portal vessels and transported to the pituitary where is stimulates the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from gonadotrophs (Garverick and Smith, 1993). Luteinizing hormone and FSH are released into the systemic circulation, transported to the ovary where they stimulate follicular growth, ovulation, formation of the corpus luteum and steroidogenesis. The ovary synthesizes estrogen, progesterone, inhibin and activin. The gonadotropins and ovarian products regulate the release of GnRH by both negative and positive feedback mechanisms. Neurotransmitters and GnRH Secretion Neurotransmitters, growth-factors, neuromodulatory peptides and ovarian steroids positively and negatively affect GnRH and/or LH release (Kordon et al., 1994;Gore and Roberts, 1997; Gazal et al., 1998; Moenter et al., 2003). Most studies have been carried out in rats and immortalized GnRH neuron (GT1 cell lines). Since some of these substances do not have receptors on the GnRH neuron, it is thought that they influence GnRH gene expression through interneuronal pathways, or by directly binding to a

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7 specific membrane receptor. Receptor binding leads to the activation of second-messenger pathways to cause GnRH and/or LH release (Gore and Roberts, 1997). Glutamate and norepinephrine (NE) have been shown to stimulate GnRH/LH release and gene expression in vivo, and GnRH release in GT1 cells. A single injection of the neurotransmitter glutamate analog, N-Methyl-D-L-aspartic acid (NMDA) causes a 30% increase in GnRH levels within 15 to 60 minutes (Gore and Roberts, 1997). A similar increase in GnRH mRNA and plasma LH is seen in GnRH neurons (Petersen et al., 1991;Liaw and Barraclough, 1993; Gore and Roberts, 1997). The catecholamine, norepinephrine, stimulates GnRH release from intact rats and rabbits and plays a physiological role in the onset of puberty (Gore and Roberts, 1997). Dopamine stimulates and inhibits GnRH release in vivo while dopamine receptor-1 (D1) stimulates GnRH release in GT1 cells (Gore and Roberts, 1997). There are direct connections between gamma-amino-butyric acid-ergic (GABAergic) and GnRH neurons. It is thought that through this direct connection, GABA plays an inhibitory role in GnRH/LH release and causes a blockage of the LH surge (Gore and Roberts, 1997). Opiates have also been shown to inhibit the GnRH/LH release and gene expression in rats and rabbits (Gore and Roberts, 1997). Cannulation of the third ventricle of adult cattle has allowed for sampling, detection and quantification of GnRH secretion and has provided a means to monitor the central regulation of reproduction (Gazal et al., 1998). Gazal et al. (1998) demonstrated that GnRH is secreted into the cerebrospinal fluid in a pulsatile manner. Control of Secretion The GnRH neurosecretory system is diffuse and contains individual neurons located in different areas of the brain (Silverman, 1984). The activity of GnRH neurons is

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8 regulated by integrated signals from the brain and these include photoperiod, steroid hormones, nutrition and stress (Moenter et al., 2003). However, in the absence of other cells types, in pure GnRH neuronal cell cultures, and GT1 cell, GnRH is secreted spontaneously in a rhythmic pattern (Martinez de la Escalera et al., 1992). Martinez de la Escalera et al. (1992) suggested that a model of cell-to-cell communication via intracellular contacts could explain synchronization of pulses in experiments where a single cell-coated coverslip per superfusion chamber was used. It was also suggested that there is a diffusible mediator, which would explain how two separated coverslips synchronized GnRH pulses, and that GnRH itself may be the diffusible mediator (Martinez de la Escalera et al., 1992). The release of GnRH is episodic in both sexes. In females, however, there is an interruption in the episodic release of GnRH by a GnRH surge. This GnRH surge coincides with the pre-ovulatory LH surge, and lasts longer than the LH surge. Yoshioka et al. (2001) demonstrated that there is a preovulatory increase in GnRH secretion into the cerebrospinal fluid (CSF) in the third ventricle following the administration of PGF2 in heifers. The GnRH surge was shown to coincide with the LH surge and also with the onset of standing estrus (Yoshioka et al., 2001). While it is known that GnRH is secreted in a pulsatile manner, little is known about the mechanisms generating this rhythmic secretion. It has been shown that GnRH neurons produce a high-frequency rhythm characterized by oscillations in intracellular calcium levels, burst firing of action potentials, and a low-frequency rhythm with a period corresponding to neurosecretion of GnRH (Moenter et al., 2003). Low frequency

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9 rhythms result in secretion of GnRH and can be modified by the effect of steroids (Moenter et al., 2003). The frequency of GnRH secretion varies during the estrous cycle and the reproductive state of the animal from once every 30 minutes to once every few hours (Moenter et al., 2003). Secretion of GnRH can be controlled at many levels. It may be regulated at the transcriptional, post-transcriptional and post-translational levels (Gore and Roberts, 1997). The biosynthesis and release of GnRH are strictly controlled by afferent neurons to the GnRH neurons in the hypothalamus (Peter and Burbach, 2002). Cell specific expression and biosynthetic regulation rely on transcription from the gene promoter for which the 5-flanking region of the peptidergic gene contains essential elements (Peter and Burbach, 2002). The amount of GnRH secreted from GnRH-containing neurons is controlled by GnRH gene expression. GnRH gene expression is limited by the rate of transcription of GnRH gene, polyadenylation and 5 capping of the RNA, processing of its primary transcript to the mature mRNA, and transport of the mRNA from the nucleus into the cytoplasm. Once in the cytoplasm, mRNA levels are determined by the turnover of the molecule. Further regulation takes place at the level of mRNA translation into proGnRH peptide, maturation of GnRH and the rate of degradation and release of GnRH into the portal circulation, which affect the level of GnRH reaching the gonadotrophs (Gore and Roberts, 1997). Estrogen controls LH and FSH synthesis through a negative feedback loop to the anterior pituitary and hypothalamus. Estrogen has been shown to downregulate mRNA levels and reporter gene activity in immortalized hypothalamic GnRH neurons (Peter and

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10 Burbach, 2002). Progesterone also causes repression of GnRH promoter activity (Peter and Burbach, 2002). Neuropeptide Y (NPY) is a potent orexigenic substance, produced in the hypothalamus, anterior pituitary and adipose tissue. Neuropeptide Y forms a neuromodulatory link between nutritional status and the central reproductive axis (McShane et al., 1992). Infusion of high concentrations of NPY into the third ventricle inhibits GnRH pulses (Gazal et al., 1998). Function of GnRH The number of GnRH receptors in the anterior pituitary changes throughout the estrous cycle. Maximum numbers of the GnRH receptor occur just prior to the preovulatory surge in the cow, and their numbers decline thereafter (Nett et al., 1987; Turzillo and Nett, 1999). Luteolysis and decreasing progesterone concentrations may be the trigger for increased expression of the GnRH receptor gene (Turzillo and Nett, 1999). The GnRH pulse frequency increases when progesterone concentrations decline, and the negative feedback signal due to high concentration of P4 is removed. This increase in GnRH pulse frequency increases the expression of GnRH receptors in the anterior pituitary. The preovulatory follicle produces large amounts of estradiol, which enhance GnRH receptor gene expression. The increasing levels of estradiol and decreasing levels of progesterone increase the numbers of GnRH receptors on the gonadotrophs, and increases pituitary sensitivity to GnRH (Turzillo and Nett, 1999). In the rat, the number of GnRH receptors is less during pregnancy and lactation compared to the estrous cycle. It has been reported that when up to 50% of GnRH receptors are blocked with a GnRH antagonist, ewes still respond fully to GnRH with LH release (Wise et al., 1984).

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11 Ovarian hormones influence the numbers of GnRH receptor. In ewes, estradiol-17 has been shown to increase the number of GnRH receptors (Clarke et al., 1988). In vitro studies using ovine pituitary cell cultures showed an increase in GnRH receptor expression in response to estrogen and decreased expression in response to progesterone (Laws et al, 1990a, 1990b). Inhibin caused an increase in the expression of GnRH receptors and, when combined with estradiol-17, led to greater expression of GnRH receptors that either hormone singly (Laws et al, 1990b). In the immediate postpartum period, the pituitary response to GnRH is reduced. Within the second week post partum, however, the pituitary responds to endogenous and exogenous administration of GnRH with release of LH in dairy cows (Cummins et al 1975). Garverick et al. (1980) showed that administration of GnRH within the second week post partum to dairy cows with large follicles was followed by ovulation and subsequent formation of a CL. Similarly, a single injection of 100 micrograms of GnRH 12 to 14 days post partum initiated cyclic ovarian activity as evidenced by a palpable corpus luteum and plasma progesterone 1.0 ng/ml by Day 9 post-treatment (Zaied et al 1980). Zaied et al. (1980) also suggest that GnRH treatment 12 to 14 days post partum may be useful in reducing abnormal ovarian activity. They found that 30% of non-treated postpartum cows developed ovarian cysts prior to conception compared to 12.5% GnRH-treated cows. Kittock et al. (1973) demonstrated that administration of GnRH to cows with ovarian follicular cysts led to an increase in LH secretion, return to normal cyclicity, and all cows expressed estrus 20 to 24 days after treatment.

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12 Mechanism of Action GnRH binds to receptors in the cell membrane of the gonadotrophs in the anterior pituitary. The GnRH receptor is a member of the seven transmembrane domain receptor coupled to a G-protein (Gq). Receptor binding leads to sequential activation of different phospholipases to provide calcium (Ca2+) and lipid-derived messenger molecules. Initially, phospholipase C is activated followed by activation of phospholipase A2 (PLA2) and phospholipase D (PLD). Following receptor binding, GnRH stimulates a GTP-protein (Gq). Activation of the G-protein results in Ca2+-independent stimulation of phospholipase C (PLC). Activation of PLC leads to the generation of second messenger inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol 4, 5-bisphosphate (PIP2). Phosphatidylinositol 4, 5-bisphosphate is a minor phospholipid constituting approximately 0.5% of the membrane phospholipids. Phosphatidylinositol 4, 5-bisphosphate and DAG are required for Ca2+ mobilization and PKC activation. Calcium is mobilized from intracellular pools stored in the endoplasmic recticulum (ER) and by means of L-type voltage-sensitive Ca2+ channels in the cell membrane (Naor et al., 1998; Ruf et al., 2003). During exocytosis, Ca2+ and phosphokinase C (PKC) act in parallel and exert an additive response on LH and FSH secretion. Calcium and PKC are also required during stimulated mitogen-activated protein kinase (MAPK) activity. Mitogen-activated protein kinase can activate transcription factors such as c-fos to mediate gene transcription with subsequent protein synthesis. In addition, MAPK can lead to stimulation of phosphokinase A2 (PKA2) with subsequent LH and FSH synthesis and release (Naor et al., 1998; Ruf et al., 2003).

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13 The signal cascade leading to the LH and FSH gene regulation is still not fully understood. Activation of the second messenger system leads to protein phosphorylation, gene transcription and biosynthesis of FSH and LH. Pituitary release of LH and FSH is down-regulated in the presence of continuous GnRH secretion (Belchetz et al, 1978). High frequency GnRH pulses favor LH release while low frequency pulses favor FSH release (Wildt et al., 1981). The changes in GnRH pulse frequency are essential for normal reproductive function (Moenter et al., 2003). Prostaglandins Prostaglandins were independently isolated from human seminal plasma in the 1930s by Goldblatt and von Euler. Prostaglandins are formed by most mammalian tissues and by tissues of lower vertebrates and certain invertebrates (Samuelsson et al., 1978). All mammalian cells types have the capacity for converting the membrane bound fatty acids into prostaglandins (Granstrm, 1981; Murray et al., 1996). Prostaglandins act as local hormones, having important physiological and pharmacologic activities (Murray et al., 1996). There are many stimuli (hormonal, nervous, other chemical, mechanical stimuli) known to activate phospholipase and initiate prostaglandin synthesis (Granstrm, 1981). The products formed and the amounts produced will vary within the same tissue under different conditions (Granstrm, 1981). Prostaglandins are C20 carboxylic acids. They have a central five-membrane ring with two side chains, 7 and 8 carbon in length, respectively, attached to adjacent positions on the ring. Depending on the number of double bonds (one, two or three double bonds) on the side chain they are designated to the , or series (Granstrm, 1981). The type of prostaglandin (A, B, C, I) depends on the arrangement of functional groups in the molecule (Champe and Harvey, 1994). The substituents in the

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14 molecule determine its biological activity, most importantly those at position C-9 and C-11 in the ring and C-15 in the side chain (Granstrm, 1981). The prostaglandins that are biologically active compounds have a hydroxyl group (-OH group) at C-15 and a double bond at C-13 (Granstrm, 1981). Prostaglandins belong to a group of unsaturated fatty acids called eicosanoids. Eicosanoids are not stored in cells, but are released upon synthesis and their biosynthesis is limited by the availability of free precursor fatty acid (Katzung, 1995). Eicosanoids include the prostanoids, leukotrienes and lipoxins (Murray et al., 1996). Eicosanoids are produced through two different pathways. Leukotrienes and lipoxins are produced through the lipoxygenase pathway. Prostaglandins and thromboxanes are produced through the cyclooxygenase pathway, and each pathway competes with the other for arachidonic acid (Katzung, 1995). Not all cell types make all of these products (Katzung, 1995). The prostanoids consists of prostaglandins, prostacyclins and thromboxanes. They act as local hormones and function through a G-protein to elicit their biological effects (Katzung, 1995). Biosynthesis The precursor for prostaglandins is arachidonic acid (AA), which is an essential fatty acid that forms part of the glycerophospholipids found in the lipid bilayer of the cell membrane. Cleavage of arachidonic acid from membrane lipids and other lipid esters by phospholipase is activated by both specific and nonspecific stimuli (Katzung, 1995). Arachidonic acid is produced from the interaction of phospholipase A2 (PLA2) with membrane phospholipids (Katzung, 1995). Phospholipase A2 and other phospholipases cleave esterified arachidonic acid from the 2position of glycerophospholipids (Dennis, 1987).

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15 Free AA is converted to PGG2, an unstable product, by oxygenation and cyclization of the pentane ring catalyzed by the enzyme prostaglandin endoperoxide synthase (prostaglandin G/H synthase or cyclooxygenase; COX). Cyclooxygenase is a membrane-bound hemoprotein (Katzung, 1995). The initial unstable product (PPG2) is quickly reduced to PGH2 by COX. Microsomes from cow uteri have been shown to possess strong endoperoxide F2 reductase activity (Wlodawer et al., 1976). The next substance produced from PGH2 depends on the cell in which the reaction occurs (Sun et al., 1977). In tissues containing the cytosolic enzyme, prostaglandin D synthase, PGH2 is converted to PGD2. However, PGE2 is produced if the tissue contains the membrane bound enzyme, prostaglandin E synthase. Reduced glutathione is required for both processes. Prostaglandin E 9-ketoreductase and 15-hydroxyprostaglandin dehydrogenase convert PGE2 into PGF2 (Sun et al., 1977; Katzung, 1995). There are two cyclooxygenases: (I and II) that have a 60% homology and which are pharmacologically different (Katzung, 1995). Cyclooxygenase I is a constitutive form and synthesizes basal levels of prostaglandin. Cyclooxygenase II (COX II) is an inducible form of the enzyme. Cyclooxygenase II is induced by a variety of ligands (Herschman, 1994), and is regulated at transcriptional and posttranscriptional levels (Sirois and Richards, 1992). Its induction is inhibited by glucocorticoids (Herschman, 1994). Cyclooxygenase II is rapidly and transiently expressed, and leads to a bolus of prostaglandin production in response to stimulation (Herschman, 1994). In response to pituitary glycoprotein hormones ovarian granulosa cells induced COX II (Herschman, 1994). Exposure of preovulatory follicles to FSH and LH rapidly and transiently induced the COX II message (Herschman, 1994). Prostaglandin synthesis

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16 in the female reproductive cycle depends on the expression of COX II in response to pituitary glycoprotein hormones (Herschman, 1994). Metabolism Prostaglandins are rapidly inactivated in the body. Enzymes that catabolize prostaglandins are found in the lung, kidney, spleen, adipose tissue and intestines (Katzung, 1995). Oxidation of the secondary alcohol group at C-15 is catalyzed by the enzyme, 15-hydroxyprostanoate dehydrogenase (PGDH). The main sources of PGDH are the lungs, spleen and kidney (Kindahl, 1980). The lungs have the highest enzyme activity and with its vast vascular bed can render large amounts of prostaglandins biologically inactive (Katzung, 1995; Davis et al., 1980). After oxidation at C-15, the 13 double bond is reduced by 13 reductase resulting in 15-keto-13, 14-dihydro compounds, which are the main plasma metabolites (Kindahl, 1980). The main plasma metabolite of PGF2 in male calves is 15-keto-13, 14-dihydro PGF2. Urinary excretion was completed in approximately 6 hours, with recovery of 80% of the injected PGF2 (Kindahl, 1980). In heifers the main metabolite of PGF2 is 15-keto-13, 14-dihydro PGF2 (Kindahl et al., 1976). Mechanism of Action The eicosanoids are short-lived, highly potent local mediators that produce an astonishing array of biological effects by binding to specific cell surface receptors (Katzung, 1995). All binding appears to involve a G-protein linkage (Katzung, 1995). Receptor binding initiates a signal transduction pathway, which links the regulatory substance (PGF2) with its intracellular effect(s).

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17 Prostaglandin F2 is released from the uterus, and transferred from the utero-ovarian vein to the ovarian artery by a countercurrent mechanism. On reaching the ovary, PGF2 binds to high and low affinity-binding sites (receptors) located in the plasma membrane of the corpus luteum (Samuelsson et al., 1978). These receptors are G protein-coupled receptors designated FP and subtyped into FPA and FPB. The high affinity-binding site requires calcium ions in order to be detected (Samuelsson et al., 1978). Prostaglandin F2 receptors are coupled to phospholipase C (PLC). Receptor binding activates PLC and induces hydrolysis of phosphatidylinositol 4, 5-bisphosphate to generate two second messengers, inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). Inositol 1,4,5-triphosphate diffuses into the cytoplasm and is involved in the liberation of intracellular calcium (Ca2+) from the endoplasmic recticulum. Calcium is required for the activation of PKC and DAG increasing PKCs affinity for Ca2+. Activation of PKC leads to the opening of calcium channels. Calcium and PKC promote protein phosphorylation and this eventually leads to the inhibition of progesterone secretion and regression of the corpus luteum (Samuelsson et al., 1978). Function in the Reproductive Tract of the Cow During the bovine estrous cycle, PGF2 is released for 2 to 3 days as rapid pulses with duration of 1 to 5 hours prior to and during luteolysis (Kindahl et al., 1976; Kindahl, 1980). A 9-keto-reductase enzyme that reduces PGE2 to PGF2 can also form PGF2. However, this enzyme is not present in the bovine uterus (Kindahl, 1980). The precise release of PGF2 throughout the bovine estrous cycle presupposes that there is an inhibiting factor in the uterus. This inhibiting factor is important for the

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18 regulation of the physiologic PG biosynthesis and thus regulates its production to prevent premature lysis of the corpus luteum. Wlodawer et al. (1976) noted that an inhibiting factor was found in bovine uterine preparations that suppressed the fatty acid cyclooxygenase. Knickerbocker et al. (1986) noted that the bovine conceptus suppressed uterine production of PGF2 production during pregnancy recognition by what was then called bovine conceptus secretory proteins (CPS) and is now known as interferon tau (INF-). However, the suppression of PGF2 release from the endometrium is regulated by a number of hormones; estrogen, progesterone (Salamonsen et al., 1990; Xiao et al. 1998), oxytocin and endothelin-1 (ET-1). The bovine endometrium contains large quantities of AA and has the ability to metabolize AA into a variety of products (Salamonsen and Findlay, 1990). Ovarian steroids influence the expression of the COX gene in bovine endometrial cells (Xiao et al. 1998). Progesterone directly influenced the basal secretion of PGF2 by the endometrium (Xiao et al. 1998). Progesterone has been shown to stimulate basal PGF2 secretion by bovine endometrial cells and tissues. However, it inhibits oxytocin-induced PGF2 secretion while in luteal cell culture while estrogen stimulated only PGF2 secretion. Prostaglandins have also been shown to have a direct effect on the bovine myometrium. (Patil et al., 1980). In vitro studies carried out on the bovine myometrium indicate that PGF2 has the ability to increase uterine tone and motility (Patil et al., 1980). However, the extent to which motility is enhanced is dependent on the stage of the estrous cycle. During the follicular phase, the myometrium is more active and contractions are more frequent and stronger than in the luteal phase. Contractions occur

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19 on average 9 times per 10 minutes, with mean amplitude of 12 mm. During the luteal phase, the frequency of contractions is less, on average 6 per 10 minutes, with mean amplitude of 5 mm. Under the influence of PGF2, there is a general increase in mean contraction and strength in both stages of the estrous cycle (Patil et al., 1980). Zetler et al. (1969) demonstrated that PFG2 had a similar effect on the fallopian tubes, increasing motility. Prostaglandin F2 and its Uses in the Dairy Cow Prostaglandins are widely used in herd management due to their luteolytic properties. Prostaglandin F2 has been use in cattle as an abortifacient (Johnson, 1981). Vandeplassche et al. (1974) induced abortion in cases of pathological gestation in cattle by intrauterine infusion (into the horn ipsilateral to the ovary with the CL) of 10.5 to 45 mg of PGF2. In cases where the cows carried a mummified fetus, 3 of 5 expelled the fetus. However, in cases of maceration, the fetus was not expelled. There was rapid regression of the CL within 4 to 5 day of intrauterine infusion, followed by estrus within 6 to 7 days. Prostaglandin F2 has also been use in cattle to induce parturition usually from 240 days gestation or within 20 days of predicted term (Johnson, 1981; Schultz and Copeland, 1981). Bosc et al. (1975) compared the use of dexamethasone to an analogue of PGF2 (ICI 79939) for the induction of parturition in cattle. Parturition was successfully induced when prostaglandin was administered in 2 mg intramuscular doses five-hours apart using a dosage which ranged from 4 to 10 mg. Plasma progesterone decreased to a low level before parturition following two successive injections. This decrease in progesterone

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20 concentration was attributed to lysis of the CL of pregnancy. However, cows in which parturition was induced had a higher incidence of retained fetal membranes. Prostaglandin F2 has been used to treat unobserved estrus in lactating dairy cattle with a corpus luteum (Eddy, 1977; Seguin et al., 1978; Seguin, 1981) and for estrus synchronization. Intramuscular administration of the PGF2 analogue, clorprostenol (ICI 80, 996) in dairy cattle with unobserved estrus and a palpable mature CL resulted in cows exhibiting estrus and being inseminated sooner than those treated with saline. In one study (Eddy, 1977), intramuscular administration of 500g clorprostenol resulted in 69% of unobserved estrus cows coming into heat within 8 days of treatment. Prostaglandin F2 has been use in cattle for postpartum infections: pyometra, metritis and endometritis (Ott and Gustafsson, 1981a, 1981b). Pyometra is defined as a condition associated with accumulation of purulent material in the uterus, persistence of a CL and anestrus (Roberts, 1971). The mode of action of PGF2 in the treatment of cows with postpartum infection is based on its luteolytic activity. In cases of pyometra, treatment leads to the regression of the CL resulting in emptying of the uterus. Prostaglandin F2 has been shown to stimulate the myometrium and may aid in the physical evacuation of purulent material from the uterus (Ott and Gustafsson, 1981a, 1981b). Gustafsson et al. (1976) demonstrated that PGF2 administered both intramuscularly and intravenously at various dosages will effect resolution of postpartum and post insemination pyometra in dairy cows not previously treated with antibiotics (systemically or intrauterine) or any other drug. Gustafsson et al. (1976) treated 26 cows (23 with postpartum pyometra and 3 with post insemination pyometra). Eighty-five percent responded by emptying the uterus and exhibited estrus 3 to 4 day following

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21 treatment. Sixty-five percent became pregnant following insemination beginning at the second estrus after treatment. Several other studies have shown the beneficial effect of using PGF2 in cows with pyometra (Jackson, 1977; Fazeli et al., 1980; Ott et al, 1981a; Paisley et al., 1986; Gilbert et al., 1992). Prostaglandin F2 has also been use in cattle with acute and chronic endometritis (Jackson, 1977;Ott et al., 1981a, 1981b;Paisley et al., 1986; Pepper et al., 1987;Gilbert et al., 1992; Sheldon et al., 1998; Heuwieser et al., 2000; Knutti et al., 2000). Estrous Cycle of the Cow There are four stages of the estrous cycle: estrus, metestrus, diestrus and proestrus. The estrous cycle can also be classified as luteal or follicular depending on the dominant structures on the ovaries. In the luteal phase, the CL is the dominant structure, and in the follicular phase the preovulatory follicle is the dominant structure. The average length of the estrous cycle in the heifer is 20 days with a range of 18 to 22 days, and in the cow is 21 days with a range of 18 to 24 days (Roberts, 1971). Follicular Phase The follicular phase consists of proestrus and estrus. It starts with corpus luteum regression and ends with ovulation of the dominant follicle. The follicular phase is approximately 4 to 5 days in the cow. In the event that pregnancy does not occur, the uterus releases prostaglandin F2 (PF2; Kindahl et al., 1976). Following release of PF2 from the endometrium, the corpus luteum of the luteal phase undergoes functional and structural regression. At this time there is a sharp decline in the blood progesterone (P4) concentration (Kindahl et al., 1976). The sudden fall in P4 removes the negative feedback on the developing follicles

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22 leading to the selection and acceleration of preovulatory follicular growth (Savio et al., 1990). The preovulatory follicle produces increasing concentrations of estradiol-17 and this influences the sex centers in the brain to induce estrus. Peak concentrations of estradiol-17 coincide with estrus. At the level of the hypothalamus, estradiol-17 induces a GnRH surge, which induces the preovulatory LH surge and FSH release from the anterior pituitary. There is considerably less estradiol-17 during the 18 to 20 hour period of estrus as compare to proestrus. At the level of the pituitary, estradiol increases the responsiveness of gonadotrophs to GnRH, which results in an increase in the LH pulse amplitude. The duration of estrus is approximately 18 to 20 hours, and ovulation occurs 10 to 12 hours after estrus. During estrus, the cow or heifer will stand to be mounted by the bull or by herd-mates. During estrus, the vulva of the cow sometimes becomes swollen, the mucous membranes of the vagina are hyperemic, and a clear, viscous, mucous discharge may be seen hanging from the vulva. Ovulation is spontaneous and occurs 10 to 12 hours after estrus in cows and 3 hours earlier in heifers (Roberts, 1971). The ovulatory process consists of the following: cytoplasmic and nuclear maturation of the oocyte; disruption of the cumulus cell cohesiveness among the cells of the granulose layer and thinning and rupture of the external follicular wall, with expulsion of the mature oocyte (Hafez et al., 2000). Luteal Phase The luteal phase is the period following ovulation and involves luteinization and development of the CL. It consists of metestrus (Days 1 to 4) and diestrus (Days 5 to 18). In this stage, the corpus luteum develops from the remnants of the pre-ovulatory follicle

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23 and produces increasing quantities of progesterone. Progesterone is usually detectable in blood by day 5 after ovulation and CL formation. The CL also produces oxytocin and estrogen. In the non-pregnant cow, the CL undergoes regression by day 17 after ovulation. Luteolysis occurs as a consequence of the interaction between the CL and prostaglandin F2 (PGF2) released from the uterus. If pregnancy occurs, uterine production of PGF2 is blocked by the action of interferon tau (INF-) on the uterus. The up-regulation of oxytocin receptors in the endometrium is inhibited by the secretion of INFfrom the trophodectoderm from days 12 to 25 in cattle (Farin et al. 1990). The CL is required for the maintenance of pregnancy in the cow. During the luteal phase, the quantity of progesterone produced by the CL increases. The concentration of progesterone impacts LH secretion patterns. When progesterone is low, the LH pulse is characterized by high frequency and low amplitude. When progesterone is high, the LH pattern is characterized by a low frequency and high amplitude. Hormonal Control of the Estrous Cycle Hormones secreted by the hypothalamus, anterior pituitary, ovary and uterus regulate the estrous cycle. The hypothalamus integrates information from higher brain centers, involving internal and external signals, such as nutritional status, season, photoperiod, presence of a male and others (Garverick and Smith, 1993). Gonadotropin-releasing hormone (GnRH) is produced by the neurosecretory cells within the hypothalamus and regulates the release of gonadotropins (luteinizing hormone, LH and follicle stimulating hormone, FSH) from the anterior pituitary (Smith and Jennes,

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24 2001). GnRH is produced in the cell bodies of neurosecretory cells located in the arcuate, suprachiasmatic and preoptic nuclei of the hypothalamus. GnRH is then transported along the axons to the median eminence, where it is released into the hypothalamo-hypophyseal portal circulation and travels to the anterior pituitary (Garverick and Smith, 1993). GnRH is released in a pulsatile manner in response to appropriate physiological cues. Luteinizing hormone and FSH are secreted in a pulsatile manner in response to GnRH secretion (Moenter et al., 1991). Luteinizing hormone is a glycoprotein hormone produced by the gonadotrophs in the anterior pituitary. It consists of alphaand beta-subunits. The alpha-subunit is common to several hormones, including FSH and thyroid stimulating hormone (TSH), and the beta-subunit is unique to the particular hormone. LH is a short-lived molecule, having a half-life of approximately 30 minutes. Follicle-stimulating hormone is a glycoprotein produced in the anterior pituitary by gonadotrophs. It is composed of a common alpha-subunit and specific beta-subunit. FSH stimulates the growth and maturation of ovarian follicles and in the presence of LH stimulates secretion of estrogen from ovarian follicles. Luteinizing hormone and FSH are secreted in a pulsatile manner from the anterior pituitary in response to the pulsatile release of GnRH. Tonic levels are controlled by the negative feedback mechanism from the ovaries. The pulsatile secretion of LH varies throughout the estrous cycle (Rahe et al., 1980; Walters et al., 1984a, 1984b; Schallenberger et al., 1985). Rahe et al. (1980) suggests that the LH pattern throughout the estrous cycle is dependent on the stage of the cycle and ovarian steroids influence the LH pattern.

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25 Studies have been done in ovariectomized animals to investigate the effect of steroids on the LH release. Ovariectomy in the cow results in increases in the pulse frequency and amplitude of LH and FSH (Schallenberger and Peterson, 1982). The pattern of secretion is related to circulating concentrations of progesterone. In the early luteal phase when progesterone concentrations are low, the LH pulse frequency is much higher than that seen in the mid-luteal phase; 8.0 pulses/12 hours and 3.6 pulses/12 hours respectively (Walters et al., 1984b). In the early luteal phase the pulse frequency of LH and FSH are similar. However, as the luteal phase progresses, the FSH pulse frequency becomes greater than the LH pulse frequency (Walters et al., 1984b). Luteinizing hormone release is suppressed by exogenous progesterone (Beck et al., 1976). In the luteal phase, progesterone and estrogen are the main factors, which decrease the frequency of the LH pulses in cattle. Individually these hormones can suppress the LH pulse, but when administered together there is a more profound inhibiting effect on LH pulse frequency. Stumpf et al. (1993) showed that when estradiol-17 was administered to ovariectomized cows the LH pulse frequency was 0.97 0.07 pulses/hour and when progesterone was administered the LH pulse frequency was 0.52 0.08 pulses/hour. However, when both estradiol-17 and progesterone were give concomitantly the LH pulse frequency increased to 14 0.07 pulses/hour. Effect of Season The effect of season or time of year on the bovine estrous cycle may be thought of as two fold. There is the effect of photoperiod on the estrous cycle and the effect of heat stress or high environmental temperatures on the estrous cycle. High environmental temperatures reduce estrus behavior. Abilay et al. (1975) demonstrated that the duration of estrus was reduced on average by 5.5 hours in

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26 Guernsey heifers maintained at 18.2 C. It is thought that the reduction in estrus behavior is as a consequence of the decrease in circulating estradiol (Wolfsenson et al., 1997; Roth et al., 2001) noted during periods of heat stress. There is a reduction in the numbers of mounting episodes per estrus during heat stress (Nebel et al, 1997). Badinga et al. (1985) demonstrated that in Florida there is a decrease in conception rate in from 48% in March to 18% in July with recovery occurring in November. Imtiaz Hussain et al. (1992) noted that only 36.8% of cows expressed estrus, even though all cows were cycling based on progesterone concentrations. High environmental temperatures have been shown in affect cortisol levels. There is an increase in cortisol levels in dairy cows that experience heat stress (Wise et al., 1988; Imtiaz Hussain et al., 1992) However, Abilay et al. (1975) demonstrated that there was a decrease in plasma cortisol levels in heat stressed heifers. Cortisol levels have been linked to GnRH release. Increased cortisol levels inhibit GnRH secretion, which in turn decreases LH secretions (Dobson and Smith, 2000). Cortisol has been shown to have a direct effect on the pituitary gland to depress both basal and GnRH-stimulated LH release in the cow (Li and Wagner, 1983; Padmanabhan et al., 1983). High environmental temperatures have been shown to have an effect on the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The effect of high environmental temperatures on LH is inconsistent. Vaught et al. (1977) found no difference in the frequency of preovulatory increase of luteinizing hormone or in the interval between the preovulatory increase and ovulation in either lactating or non-lactating cows exposed to summer heat stress. This is in contrast to findings by Wise et

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27 al. (1988). There was a decrease in the number of LH pulses on Day 5 of the estrous cycle in heat stressed cows compared to controls (Wise et al., 1988). Ovarian follicles are susceptible to high environmental temperatures. Roth et al. (2000) demonstrated that there are alterations in the pattern of growth and development of medium-sized follicles associated with an increase in plasma FSH concentration in the first follicular wave following acute heat stress. Wolfsenson et al. (1997) demonstrated that estradiol concentration in follicular fluid and androstenedione production by thecal cells were lower in dominant follicles collected in autumn than those collected in winter. This finding by Wolfenson et al. (1997), indicate the carry over effect of heat stress. Roth et al. (2001) investigated the delayed effect of heat stress on medium-sized and preovulatory follicles at 20 and 26 days after acute heat exposure. It was noted that the number of medium-sized follicles that emerged during the first follicular wave after heat stress was the same in both control and heat-stressed cows. However, there were more healthy medium-sized follicles in the control than heat-stressed cows (56% vs 38%) but this was not significantly different. In healthy medium-sized follicles, estradiol production by granulosa cells and androstenedione production by thecal cells were lower and follicular fluid progesterone concentration was higher in heat-stressed than in control cows. In preovulatory follicles the viability of the granulose cells, concentration of androstenedione in follicular fluid and androstenedione production by thecal cells were lower in heat-stressed than in control cows (Roth et al., 2001). Guzeloglu et al. (2001) investigated the effect of acute heat stress on long-term follicular dynamics and biochemical characteristics of dominant follicles in non-lactating dairy cows. They found that there was no difference in estradiol or progesterone

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28 concentration in the follicular fluid of the dominant follicle between heat stressed and control cows. However, there was a significant difference in the size of the dominant follicle between the control and heat stressed cows. The dominant follicle size was greater in control cows than in heat stressed cows. Guzeloglu et al. (2001) noted that heat stress reduces follicular dominance during a follicular wave based on the increase in the number of class 3 follicles on Days 7 and 8. Roth et al. (2000) investigated the immediate effect of heat stress on plasma FSH and inhibin in Holstein dairy cows. It was demonstrated that there was a larger cohort of medium sized follicles (6-9 mm) during the second follicular wave of the estrous cycle of heat stressed cows compare to controls. This increase growth was associated with higher plasma concentrations of FSH, which lasted for 4 more days in heat stressed cows than in controls. The increase in plasma FSH was also associated with a decrease in plasma concentrations of inhibin. There is an increase in the length of the estrous cycle in cows experiencing heat stress. Wilson et al. (1998a) noted that the second wave dominant follicle was less likely to ovulate in heat stressed lactating dairy cows. In heat stressed cow, 18% ovulated compared to controls 91%. The average day of luteolysis was delayed by 9 days in heat stressed cows. Similar finding were found in heifers. Heat stress inhibited the growth and function of the dominant follicle, such that heat stressed heifers had three follicular waves and there was a delay in corpus luteum regression (Wilson et al., 1998b). High environmental temperatures have been shown to affect corpus luteum function. These observations were made based on progesterone concentrations. In some studies progesterone concentration was elevated (Abilay et al, 1975; Vaught et al., 1977) in cows experiencing heat stress. However, in other studies progesterone concentration

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29 was unchanged (Wise et al., 1988) and yet in other studied progesterone concentrations were lowered during heat stress (Howell et al., 1994). Wilson et al. (1998a, 1998b) demonstrated that there was delayed regression of the corpus luteum in lactating dairy cows and heifer subjected to controlled heat stress. Heat stress reduced feed intake (Fuquay, 1981; Rensis and Scaramuzzi, 2003). Reduced dry matter intake will prolong the period of negative energy balance in the postpartum dairy cow. Negative energy balance leads to decreased plasma insulin, glucose and insulin-like growth factor I (IGF-I; Rensis and Scaramuzzi, 2003). These factors are required for the normal growth and function of ovarian follicles and thus the estrous cycle. Folliculogenesis and Ovarian Dynamics in the Dairy Cow Follicular Development An ovarian follicle is a spherical aggregation of cells that contain the developing gamete (Banks, 1986). Oogenesis begins during embryologic development and continues throughout fetal development until shortly after birth. Multiplication halts in meiotic prophase. At this stage the germ cell is termed a primary oocyte (Noden, 1985). Follicular development proceeds through further development of the primordial follicle into a primary follicle; primary to secondary follicle and finally a mature follicle. Follicular growth and maturation is under the influence of gonadotropins from the pituitary (Noden, 1985). The primordial follicle contains the primary oocyte. At this stage the primary oocyte has paired homologous chromosomes and each has replicated to form two chromatids (Noden, 1985). At the time the oocyte is undergoing the first stages of meiosis follicular cells surround the primary oocyte to form a primordial follicle. Once

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30 the primordial follicle is activated it becomes the primary follicle. Histologically, the primordial follicle appears as a single layer of flattened follicular cells surrounding the primary oocyte (Banks, 1986) Activation of the primordial follicle results in the development of the primary follicle, involving alterations in the primary oocyte, follicular cells and stromal elements. Histologically, the primary follicle appears as single layer of cuboidal or columnar follicular cells surrounding the primary oocyte. At the stage, accumulation of yolk granules can been seen in the primary oocyte (Banks, 1985). The secondary follicle consists of the primary oocyte separated from the follicular cells by the zona pellucida. The follicular cells are actively dividing and are now know as the membrana granulosa. As the secondary follicle continues to develop, the stromal cells differentiate into theca interna and theca externa, separated from the membrana granulosa by a basement membrane. The theca interna consists of large, epitheliod cells and an extensive vascular network. The theca externa consists of a fibroblastic layer of cells (Banks, 1986). The tertiary follicle develops from the secondary follicle. Histologically, the tertiary follicle consists of cuboidal and columnar follicular cells surrounding the primary oocyte. Within the layers of follicular cells small, fluid filled periodic-acid-Schiff-positive spaces can been seen. Further secretion of follicular fluid by the granulosa cells leads to the formation of a follicular antrum seen in the mature follicle. The mature follicle is also known as the Graafian follicle or preovulatory follicle. The preovulatory follicle ranges form 15 to 17 mm in diameter in the bovine ovary.

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31 Follicles protrude from the ovary and are recognized as a smooth fluctuant structures on palpation. The size of the follicle increases to approximately 20 to 25 mm in diameter by the middle of the estrous cycle. The increase in follicular fluid within the antrum is reflected by the tension in the palpable follicle, which increases up to six to twelve hours before ovulation (Zemjanis, 1970). In terms of the endocrinology, the development of the follicle can be divided into four stages. The initial stage of development is independent of the gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH). In this stage follicles are usually < 3 mm in size. Next follows a stage in which the follicle is dependent on FSH, the follicles range from 3 to 10 mm in diameter. In the thirds stage the follicle is dependent on pulsatile LH and ranges from 10 mm to preovulatory size. The final stage is dependent on the preovulatory LH surge for ovulation (Thatcher et al., 2002). Follicular Waves Ovarian follicles develop in waves. Rajakoski (1960) first put this theory forward after he conducted extensive studies of ovaries taken at slaughter. Pierson and Ginther (1984) later confirmed the existence of follicular waves using ultrasonography. Cattle either have two or three follicular waves during a single estrous cycle. In cows that have 2 follicular waves, recruitment occurs on Day 0 (day of ovulation) and day 10 of the estrous cycle. In cows with 3 follicular wave cycles, recruitment occurs on Days 0, 9 and 16 of the estrous cycle (Ginther et al., 1989a; Ginther et al., 1989b). Follicular waves occur in cyclic cows and heifers and in dairy and beef heifers prior to the onset of cyclicity, in prepubertal calves as early as 2 weeks of age (Evans et al. 1994) and during most of pregnancy (Ginther et al.1996). Follicular growth begins in the fetal ovaries, with the final maturation stages occurring days prior to ovulation.

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32 Follicular dynamics in the cow consists of three distinct phases: recruitment, selection and dominance. Recruitment is the process by which a group of follicles begins to mature and grow under the influence of gonadotropins. In selection, a single follicle is chosen and avoids atresia with the potential to ovulate. In dominance, the selected follicle inhibits the recruitment of a new cohort of follicles (Lucy et al., 1992). Hoak and Schwarts (1980) showed that the secondary increase in FSH levels following the preovulatory LH surge recruits follicles for the next estrous cycle in the rat. Adams et al., (1992) showed that the FSH surge in heifers began 2-4 days before ultrasonically detectable emergence of a follicular wave (follicles 5 mm). FSH peaked 1 or 2 days before emergence and began to decrease at the time of deviation, when the follicles of a wave begin to diverge into a dominant follicle and subordinate follicles (follicle 6-7 mm). One of these follicles becomes dominant and has the potential to ovulate. In a two-wave estrous cycle, a group of small follicles (5 mm) is recruited and begins to grow under the influence of follicle-stimulating hormone (FSH; Adams, et al 1992). Later a single follicle becomes dominant and continues to grow while subordinate follicles will eventually undergo atresia. In the presence of the CL and high progesterone concentration, the dominant follicle of the first wave does not ovulate and regresses. A second follicular wave develops with recruitment of another group of follicles around midcycle. The dominant follicle of this second wave is functional at the time of CL regression and decreasing progesterone concentration and this follicle ovulates after luteolysis.

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33 In a three-wave cycle the second dominant follicle fails to ovulate and a third follicular wave develops. The dominant follicle of the second wave is usually smaller than the first and third dominant follicle (Lucy et al., 1992). Cows with a three-wave cycle have a longer estrous cycle, as the third dominant follicle requires time to develop prior to ovulation (Lucy et al., 1992). Regulation of Follicular Growth by FSH and LH Follicular deviation or selection has been defined as the beginning of the greatest difference in the growth rates between the largest follicle (dominant follicle) and the second largest follicle (largest subordinate follicle) at or before the largest subordinate follicle reaches it maximum diameter (Ginther et al., 1996). The follicle that becomes dominant develops receptors for luteinizing hormone (LH) around the time of deviation, while the subordinate follicles do not. Under the influence of declining FSH concentrations and increasing LH concentrations, the subordinate follicles regress and the dominant follicle continues to develop. In the event that luteolysis does not occur with a resultant fall in progesterone concentrations, the dominant follicle will not ovulate and undergoes atresia. The dominant follicle secretes both inhibin and estradiol. Inhibin is also secreted by follicle greater than 3 mm and also by atretic follicles. Estradiol and inhibin feed back on the pituitary and hypothalamus and decreases the secretion of FSH from the pituitary. Estradiol biosynthesis is dependent on a two-cell system. Theca cells and granulosa cells individually are not equipped with the enzymes necessary for biosynthesis of estradiol from cholesterol. However, together these cells are capable of producing estradiol. Theca cells are the sites of androgen synthesis in follicles. Under the influence of LH androgen secretion is increased. The end product in steroidogenesis in theca cells

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34 is progesterone. Androstenedione produced by the theca cells is transported into the granulosa cells where it is converted into testosterone. Under the influence of FSH, testosterone is converted into estradiol within the granulosa cells (Hansel and Convey, 1983). Production of Estrogen Prior to the LH surge and luteinization of the granulosa and theca cells, the major hormone produced by the ovary is estrogen, estradiol-17. The granulosa and theca cells individually are unable to produce estradiol, since both lack the enzymatic pathways to do so. In order to produce estradiol, cholesterol must be converted to androgens and androgens to estradiol. Thecal cells are capable of the former, but not the latter, while granulosa cells are capable of the latter (Niswender et al., 2000). Corpus Luteum in the Cow The corpus luteum (CL) is a transient endocrine structure, which secretes progesterone, oxytocin (Fields et al., 1992), neurophysin, relaxin and other substances, which act in an autocrine and/or paracrine manner. There is some evidence that the bovine CL may produce estrogen. Kimbal and Hansel (1974) used a dextrand-charcoal adsorption method to show that estrogen-binding proteins (estrogen receptor, ER) were present in the bovine corpus luteum. Recent work by Okuda et al. (2001) demonstrated that estradiol 17 is produced in the bovine CL. The major secretory hormone is progesterone, which is essential for the establishment and maintenance of pregnancy. In the non-pregnant animal, the life span of the CL determines the length of the estrous cycle.

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35 Corpus Luteum Development The corpus luteum is formed from the remnants of the ovarian follicle following ovulation. Simmons and Hansel (1964) postulated that any hormone that could stop oxytocin from shortening the bovine estrous cycle had luteotropic properties. The luteotropic property of luteinizing hormone (LH) was later confirmed by Donaldson et al., (1965b, 1965c). Ovulation is initiated by a preovulatory LH surge. Rupture of the follicular wall is believed to involve enzymatic hydrolyzation of connective tissue by LH-induced collagenases, proteases and plasmins (Banks, 1986). The oocyte and its surrounding follicular fluid are simultaneously ejected from the ruptured follicle. The granulosa and theca cells collapse into the cavity. The hemorrhage associated with ovulation clots, forming the corpus hemorragicum. This stage is a transitory stage. The granulosa and thecal cells undergo hypertrophy and division to become the large and small luteal cells of the corpus luteum, respectively. The production of highly specific antibodies against granulosa and thecal surface antigens proved that as the CL ages the small luteal cells undergo structural and morphological changes to become large luteal cells (Alila et al., 1984). This substantiates earlier work by Donaldson and Hansel (1965a) using histology and histochemical staining techniques. They demonstrated that granulosa cells stop dividing by Day 4 of the estrous cycle and small luteal cells derived from thecal cells continue to respond to luteotropins grow and develop into large luteal cells (Donaldson and Hansel, 1965a). Ursely and Leymarie (1979) identified three types of cells in collagenase dispersed luteal preparations. These included small (10-20m in diameter) steroidogenic

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36 cells, large (>25 m diameter) steroidogenic cells and numerous small (<10 m diameter) non-steroidogenic cells consisting mainly of vascular cells (endothelial cells, erythrocytes leukocytes) and connective tissue cells (Hansel and Blair, 1996). The non-steroidogenic cells of the bovine corpus luteum consist of macrophages and endothelial cells which account for approximately 14% of the volume and approximately 53% of the cells of the mature CL (Parry et al., 1980;OShea et al., 1989). Macrophages are important for their phagocytic activity and for participation in the immune response involved in the regression of the CL (Pate, 1994). Endothelial cells are those that line the microvasculature and are thought to secrete substances which are involved in both luteotropic and luteolytic processes. Fields and Fields (1996) reported the presence of five different phenotypes of endothelial cells from the bovine CL. Type 1 cells are believed to be true endothelial cells, while Type 5 cells displays characteristic of immature granulosa cell, and it has been suggested to be a putative stem cell for renewal of luteal cells (Fields and Fields 1996). Fibroblasts comprise approximately 10% of all luteal cells and approximately 6% of the total volume of the bovine CL (Fields and Fields 1996). The steroidogenic luteal cells make up the majority of the bovine CL. The small luteal cells and the large luteal cells together account for approximately 70% of the volume of the bovine CL (Parry et al., 1980; OShea et al., 1989). The small luteal cells make up approximately 28% of the volume of the CL and are known for its low basal production of progesterone. When stimulated with LH, these cells increase their production of progesterone. The large luteal cells comprise 3% of the luteal cells. However, they comprise approximately 40% of the volume of the CL (OShea et al.,

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37 1989). The large luteal cells do not respond to LH. However, they do secrete oxytocin and are the steroidogenic cells, which produce the majority of basal progesterone. During the early development of the bovine CL, there is rapid growth and increase in size. The CL has a six-fold increase in size during early luteal phase of the estrous cycle (Zheng et al., 1994). Along with an increase in weight, there is also an increase in the amount of progesterone produced and secreted. Function of the Corpus Luteum The major hormone produced by the CL is progesterone. Progesterone functions in determining the length of the estrous cycle and is required for the maintenance of pregnancy in the cow. The preovulatory LH surge results in luteinization of the granulosa and thecal cells to large and small luteal cells, respectively. The luteinization process results in a shifting of the steroidogenic pathway such that the major secretory product is progesterone. This process includes increased expression of enzymes required for conversion of cholesterol into progesterone. There is an increase in cytochrome P-450 side chain cleavage (P-450scc) enzyme and 3-hydroxysteroid dehydrogenase/5,4 isomerase (3-HSD) and decrease expression in the enzymes that convert cholesterol to estradiol17-hydroxylase cytochrome P-450 and aromatase cytochrome (Niswender et al., 2000). In the female reproductive tract, prostaglandin E2 (PGE2) is considered luteotrophic (Pratt et al., 1977). Intrauterine administration of PGE2 protects the CL against induced and spontaneous luteolysis (Pratt et al., 1977; Henderson et al., 1977). Prostaglandin E2 stimulates the production of progesterone (P4) through the activation of cyclic-AMP protein kinase A (PKA) pathway (Kotwica et al., 2003).

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38 Prostaglandin E2 binds to its receptor, a G protein-coupled receptor, (EP) located in the cell membrane. There are four receptor subtypes, EP1, EP2, EP3 and EP4. Only EP2 and EP4 are coupled to adenylate cyclase. Activation of adenylate cyclase leads to an increase in cAMP that in turn activates PKA leasing to a cascade of intercellular signals with the eventual activation of genes for progesterone syntheses. Prostaglandin E2 EP2 receptor is highly expressed in the large luteal cells. The EP2 receptor is the major cAMP-generating PGE2 receptor in the bovine CL (Arosh et al. 2004) and the large luteal cells produce 80% of progesterone (Diaz et al., 2002) The Corpus Luteum and Pregnancy In the event of pregnancy, the secretion of PGF2 from the uterus is blocked, luteolysis does not occur and progesterone concentration is maintained. Between Day 15 and 17 of the estrous cycle, the bovine conceptus produces a signal, which prevents luteolysis induced by the pulsatile release of PGF2 (Kindahl et al., 1981; Asselin et al. 1996). The signal molecule, for the recognition of pregnancy in cows was first identified as bovine trophoblast protein-1 (bTP-1), which was later called interferon(INF-) Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon. (Klemann et al., 1990). In the cow, INFprevents the luteolysis by down-regulation of oxytocin receptors (Meyer et al., 1996). Reducing the number of oxytocin-receptors available for oxytocin binding attenuates oxytocin-stimulated secretion of PGF2 (Meyer et al., 1996). This prevents oxytocin-stimulated PGF2 release and subsequent luteolysis. Recombinant bovine interferon(rbINF-) also causes a decrease in the expression of cyclooxygenase II (COX II) and prostaglandin F synthase (PGFS; Xiao et al., 1998).

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39 Cyclooxygenase II is an inducible rate-limiting enzyme for the conversion of arachidonic acid to PGG2 and PGH2, the precursors of PGF2 and PGE2. Prostaglandin F synthase (PGFS) is responsible for the reduction of PGH2 to PGF2 and PGD2 to 9, 11-PGF2 (a stereoisomer of PGF2). Recombinant bINFhas been shown to decrease COX II mRNA in epithelial cells (the primary source of PGF2) and to increases COX II mRNA and prostaglandin synthesis in stromal cells (the primary source of PGE2; Xiao et al., 1998). Xiao et al. (1998) also demonstrated that there was a reduction in PGFS mRNA in both epithelial and stromal cells, and this was associated with an increase in PGE2: PGF2 ratio. Bovine interferonhas been shown to shift the primary prostaglandin (PG) produced by the endometrium from PGF2 to PGE2 (Asselin et al., 1997: Xiao et al., 1998). Prostaglandin E is thought to be a luteotrophic agent (Pratt et al., 1977). Asselin et al (1997) showed that cultured bovine endometrial cells treated with bovine recombinant INF(rINF-), in the presence and absence of oxytocin had a net PGE2: PGF2 ratio of 3.8 and 7.7 respectively. The conclusions made by Asselin et al. (1997) were rINFregulates PGs by stimulating PGE2 preferentially and rINFtransforms the response to OT from stimulation of PGF2 to stimulation of PGE2. Progesterone is necessary for the maintenance of pregnancy in the bovine. The normal CL produces more progesterone than required to maintain the embryo until Day 15 of pregnancy. Normal embryo development proceeds to Day 15 when the levels of total progesterone production exceed a threshold value by approximately 100 g of total luteal progesterone; 15-day CL normally contain nearly 300 g of progesterone (Hansel and Blair, 1996).

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40 Luteolysis Luteal regression is caused by a pulsatile release of prostaglandin F2 primarily from the intercaruncular region of the surface epithelium of the uterus (Asselin et al. 1996) in the late luteal phase. It is thought that the pulsatile secretion of PGF2 is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF2. Regression of the CL is essential for normal cyclicity, and allows the development of a new ovulatory follicle. However, prevention of luteolysis is necessary for establishment and maintenance of pregnancy (Okuda et al., 2002). Prostaglandin F2 secretion from the bovine endometrium varies during the estrous cycle. During the follicular phase and at estrus, Prostaglandin F2 is at its highest and then declines at early to mid-luteal phase of the estrous cycle (Kindahl et al., 1981). PGF2 is released from the uterus in a series of short pulses 2 to 3 days during and after luteolysis (Kindahl et al., 1981). Schramm et al. (1983) demonstrated that the CL is sensitive to pulsatile administration of PGF2 resulting in luteolysis. Skarzynski and Okuda (1999) demonstrated that long-lasting stimulation with PGF2 desensitizes luteal PGF2 receptors in the cow and luteolysis fails to occur. Oxytocin released from the corpus luteum acts upon the uterus to stimulate production of PGF2, which in turn causes luteal regression. Prostaglandin F2 has the ability to cause further release of oxytocin from the ovine corpus luteum. Oxytocin has been proven to be essential for the initiation of luteolysis in the ewe (Silvia et al., 1991). It has been shown that oxytocin administered during the early stages of the estrous cycle (Days 2-6) causes PGF2 release, which is measurable in uterine venous blood (Milvae

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41 et al., 1980). However, the level of oxytocin in the CL, plasma and oxytocin mRNA in the CL are all low during normal luteolysis (Hansel and Blair, 1996). It has been shown that exogenous administration of PGF2 stimulates the utero-ovarian release of PGF2 in the ewe (Wade and Lewis, 1996). Similarly, Kotwica et al. (1997) and Okuda et al. (2002) reported that administration of exogenous PGF2 increased PGF2 release from the uterus on day 18 of the estrous cycle. It has been demonstrated by Skarzynski and Okuda (1999) that PGF2 activates protein kinase C (PKC) and increases intracellular calcium mobilization, which may in turn stimulate PGF2 production in the endometruim. It has been reported by Schams and Berisha (2002) that exposure to progesterone or inhibition of progesterone action by a progesterone antagonist in early to mid-diestrus regulates the onset of uterine release of PGF2 from endometrium causing shortening or extension of the interestrous interval in sheep and cows. Sheep that were treated with onapristone (progesterone antagonsist) on day 4, 6 and 8 had a longer estrous cycle. The controls had an estrous cycle length of 17.0 0.5 days while those treated with onapristone had a cycle length of 22.0 0.8 days. Endothelin-1 (ET-1), a 21 amino acid peptide is a potent vasoconstrictor originally isolated from porcine aortic endothelial cells (Yanagisawa et al, 1988). Kisanuki et al. (2001) demonstrated using gene-knockout technology in mice that ET-1 is produced primarily by endothelial cells. It has been shown to be a modulator in female reproduction where it serves to inhibit premature luteinization of follicular cells in porcine ovaries (Flores, J.A., 2000) and in the propagation of the luteolytic process in the ewe (Hinckley and Milvae, 2001).

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42 The ET gene is transcribed to produce the preprohormone, preproET-1 (ppET-1). PreproET-1 undergoes posttranslational processing to produce big ET-1, which is further processed to mature ET-1. Big ET has little biologically active while ET-1 is the biologically active form. The final processing stage from big ET-1 to the active form, ET-1 is carried out by endothelin-converting enzyme-1 (ECE-1; Xu et al, 1994). Endothelin-converting enzyme-1 is a membrane bound protein belonging to the zinc-binding metalloendopeptidases (Xu et al, 1994), and has several isoforms (Meidan and Levy, 2002). Endothelin-converting enzyme-1 mRNA is highly expressed in steroidogenic tissues, especially in the ovaries (Xu et al, 1994). Endothelin-1 binds two receptors subtypes, ETA (for aorta) and ETB (type B receptors; for bronchus; Meidan and Levy, 2002). These receptors belong to the seven-transmembrane G protein-coupled receptor superfamily. The ETA receptor has a higher affinity for ET-1, while ETB binds all three endothelins (ET-1, -2 and ) equally (Meidan and Levy, 2002). Hinckley and Milvae (2001) demonstrated that ET-1 has an inhibitory effect of on progesterone synthesis by luteal cells in the ewe. They demonstrated that ET-1 inhibited basal and LH-stimulated progesterone production by dispersed ovine cells and that inhibition was removed by pre-incubation with BQ123 (a highly specific endothelin ETA receptor antagonist). They also showed that intramuscular administration of ET-1 at midcycle reduced plasma progesterone concentrations for the remainder of the estrous cycle. It has been shown that administration of a luteolytic dose of PGF2 stimulates gene expression of ET-1 in ovine CL collected at mid-cycle, and intra-luteal

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43 administration of BQ123 on Days 8 and 9 of the estrous cycle removes the luteolytic effect of PGF2 (Hinckley and Milvae, 2001). Hinckley and Milvae (2001), working with ewes, supported the hypothesis that ET-1 plays an integral part in PGF2-mediated luteolysis. Following pretreatment with a subluteolytic dose of PGF2, intramuscular administration of ET-1 caused a rapid decline in plasma progesterone concentrations and shortened the length of the estrous cycle in the ewe (Hinckley and Milvae, 2001). The ETA receptor gene is expressed in the two luteal cell types enriched from bovine CL and the thecaland granulosa-derived luteinized cells of the CL in vivo. However, the endothelial cells of the CL have higher levels of ETA receptor mRNA levels than in each of the steroidogenic luteal cell types (Mamluk et al, 1999). Little is known about the ETB receptor in the CL. In theca-derived luteal cells, LH and forskolin downregulate ETA receptors, while in granulosa-derived luteal cells, insulin-like growth factor-I (IGF-I) inhibits the ETA receptor mRNA levels (Mamluk et al, 1999). This suggests that during the early stages of luteinization, when both IGF-I and LH are at their peak levels, the expression of ETA receptor is suppressed (Meidan and Levy, 2002). Levy et al. (2003) showed that the ECE-1 gene is expressed by both bovine ovarian endothelial and steroidogenic cells using enriched follicular and luteal cell subpopulations and in situ hybridization techniques. Different ECE-1 isoforms exist based on the different N-terminal cytoplasmic tails, and are located in different intracellular areas. The intracellular ECE-1a isoform is present only in ET-1-expressing endothelial cells (Levy et al. 2003). The membrane-bound ECE-1b isoform is expressed

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44 in both steroidogenic and endothelial cells of the preovulatory follicle and the CL. Insulin and IGF-I upregulate ECE-1 expression when cultured with granulosa cells with concomitant increase in progesterone production while ET-1 and LH down regulate ECE-1 levels in steroidogenic cells. The levels of ECE-1 mRNA are lowest in the bovine CL during the early stages of CL development (Days 2-4). As the CL matures (Days 7-12), ECE-1 levels increase and remain elevated in the late CL (Days13-16). ECE-1 levels decline in the regressed CL (Days 20+; Levy et al. 2003). Levy et al. (2003) suggested the following model for ET-1 biosynthesis in the ovaries. Endothelial cells expressing ppET-1 and the two endothelin-converting enzyme-1 (ECE-1) isoforms are capable of secreting both the precursor, big ET-1 and mature ET-1. Steroidogenic cells only express the cell surface form of ECE-1, ECE-1b, without ppET-1. In order to produce ET-1, steroidogenic cells are dependent on the extracellular supply of big ET-1. Endothelial cells provide the precursor (big ET-1) and the steroid-secreting cells convert it into mature ET-1, ensuring that ET-1 is generated near its site of action, the ETA receptor, to ensure that the short lived ET-1 is active. Involution of the Bovine Uterus Macroscopic Changes The postpartum interval is the period from parturition to the first postpartum estrus accompanied by ovulation. During this period, several processes take place. The uterus involutes to its previously non-gravid state, the endometrium regenerates, bacterial contaminants are cleared from the reproductive tract and ovarian cyclicity resumes. During the process of uterine involution, there is a reduction in the size of the uterus accompanied by loss of tissue and regeneration of the endometrium. The reduction

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45 in the size of the uterus is contributed to by strong muscular contractions that occurs every three to four minutes during the first 24 hours post partum (Moller 1970b). After calving, the uterus decreases in a logarithmic fashion in weight, length and diameter of the previously pregnant horn. The greatest changes in these parameters occur rapidly in the first few days after parturition. At calving, the uterus of a cow weighs approximately 9 kg. At Day 4 to Day 7 post partum, the uterine horns can be palpated cranial and ventral to the pelvis (Morrow et al., 1969c). Over a period of 10 days the uterus rapidly involutes to approximately 3 kg (Gier and Marion, 1968). The rapid reduction in size is accompanied by an increase in uterine tone from Day 10 to Day 14 post partum and the previously pregnant horn decreases from approximately 12 cm to 7 cm in diameter (Morrow et al., 1969c). The period of rapid involution coincided with the first estrus in normal cows and is associated with discharge of uterine lochia (Morrow et al., 1969c). Following the rapid reduction in the size of the uterus, the rate of involution decreases. The rate of involution decreases such that the uterus weighs about 1 kg by 20 to 30 days post partum and 750 g by 50 days post partum (Gier and Marion, 1968). The length of the previously gravid horn decreases to half it length at parturition by Day 15 post partum and by Day 30 it is a third its size at parturition (Gier and Marion, 1968). The postpartum uterus decreases to half its gravid diameter by Day 5 postpartum. Gier and Marion (1968) noted that the cervix in the postpartum cow decreases in diameter from 15 cm on Day 2 postpartum, 9-11 cm by 10 days post partum; 7-8 cm on Day 30 post partum to 5-6 cm 60 days post partum. Morrow et al. (1969c) noted that at Day 4 to Day 7 post partum the cervix was palpated cranial to the pelvis. There is an initial decrease in diameter between days 5 and 10 followed by relaxation of the cervix at

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46 day 10 post partum when final sloughing of the caruncular masses occur (Gier and Marion, 1968). The relaxation of the cervix is interpreted on palpation and a slight enlargement and is associated with discharge of lochia from the uterus and first postpartum estrus (Morrow et al., 1969c). Involution of the cervix continues gradually until Day 30 post partum in normal cows and until Day 35 post partum in abnormal cows (Morrow et al., 1969c). Any other changes taking place after Day 30 to Day 35 post partum could not be detected by transrectal palpation. There is a difference in the rate of reduction between the myometrium and the endometrium. During the first few days post partum, the myometrium reduces in size, while the endometrium becomes edematous during the first day post partum. Endometrial edema then resolves slowly over the next 5 days and by Days 6 to 8 post partum it has disappeared and the endometrium regresses rapidly (Gier and Marion, 1968). Further tissue losses after Day 19 consist of reduction of blood vessels, regression of uterine glands, and contraction of tissue with reduction in cell numbers and cell volume. The caruncles further reduce in size. On Day 19 the caruncles are 15 to 20 mm in diameter. By Day 39 the caruncles appear as smooth knobs 10 to 15 mm in diameter and by Days 50 to 60 the caruncles are reduced to circular cratered cones 8 to 10 mm in diameter across the base and 4 to 6 mm across the crown (Gier and Marion, 1968). At the end of the regression changes the caruncles appear as rows of white disks in a pink endometrium (Gier and Marion, 1968). Involution of the uterus is considered to be complete by 40 to 60 days post partum, when caruncles have regressed to a smooth, oblong, epithelium-covered, avascular knob (Gier and Marion, 1968). Garcia and Larsson

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47 (1982) reported that complete uterine involution occurred between Days 41 to 50 post partum. Uterine lochia is a mixture of normal and degenerate cells from the mucosa, maternal placenta and blood from the shedding of the fetal placenta (Moller, 1970b). A considerable amount of blood is found in the lumen of the uterus following parturition. By Day 4 post partum the blood becomes mixed with sloughed caruncular material. By Day 12 post partum, however, the luminal content changes to a lymph-like fluid that decreases in quantity as the postpartum period progresses (Gier and Marion, 1968). This fluid (lochia) is palpable in the uterus from Days 8 to 13 post partum. Uterine lochial fluid diminishes from 1,400 to 1,600 ml at Day 2 to naught by Day 21 to 25 post partum. The amount that is seen as a vaginal discharge varies from animal to animal and is usually seen between Days 5 to 10 post partum (Moller, 1970b). Microscopic Changes In the cow, fetal cotyledons fuse with maternal caruncles of the uterine mucosa to form placentomes (Hafez, 1993). The fusing of the two tissues forms the primary anchoring system of the placenta, keeping the maternal and fetal tissues in apposition (Eiler, 1997). The secondary anchoring system consists of root-like penetration of caruncle crypts by cotyledon villi and adhesive fluid between fetal and maternal epithelium. The secondary anchoring system functions to hold fetal and maternal epithelia together for physiologic exchange (Eiler, 1997). After parturition, the septa and crypts of the caruncles contain remnants of chorionic villi (Gier and Marion, 1968; Archbald, et al 1972). The remnants of chorioallantoic cells in the maternal crypts undergo necrosis and mineralization and are

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48 phagocytized or expelled in the lochia and are not observed in the caruncles after Day 11 post partum (Archbald et al., 1972). The medium-sized and small arteries of the caruncule undergo progressive vascular degeneration from Days 1 to 19 post partum (Archbald et al., 1972). Gier and Marion (1968) found that within the first 2 days post partum, caruncular blood vessels constrict and the septum and crypts undergo necrosis. The smooth muscle cells of the tunica media undergo hydropic degeneration and pyknosis and the tunica media undergoes fibrinoid necrosis (Archbald et al., 1972). Archbald et al. (1972) noted that sloughing of the superficial areas of the caruncles began on Day 1 post partum and necrotic changes in the stratum compactum were noted on Day 5 post partum with sloughing of the superficial layer occurring by Days 6 and 7 post partum. The stratum compactum is reduced almost to the level of the inter-caruncular area by Day 15 post partum (Archbald et al., 1972). By Day 5 post partum, there is a necrotic layer 1 to 2 mm thick over the stratum compactum and cellular organization is lost leaving only blood vessels and clumps of leukocytes (Gier and Marion, 1968). By Day 10 post partum, most of the necrotic material is removed, and, all of the caruncular mass involved in the placentomes sloughs by Day 15 post partum, leaving stubs of blood vessels extending beyond the stratum compactum. At 19 days postpartum, the arterioles within the stratum compactum and beyond disappear, leaving the caruncle relatively smooth (Gier and Marion, 1968). The leukocytes found in clinically normal cows consist of lymphocytes and plasmacytes. Histiocytes and occasional polycytes are also found in the clinically normal

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49 cow. In cows that have a uterine infection, organized lymphocytic nodules are found in the stratum compactum. Tissue regeneration occurs in all areas of the endometrium. However, the earliest to show full regeneration is the inter-caruncular epithelium, which occurs by 8 days post partum (Gier and Marion, 1968). Archbald et al. (1972) reported that at no time during uterine involution was the inter-caruncular area devoid of an epithelial layer. Tissue regeneration of the uterine epithelium begins just after parturition (Gier and Marion, 1968) with focal areas of epithelial cells found on the caruncular surface on Day 1 post partum (Archbald et al., 1972). These epithelial cells seen on Day 3 and 5 are later sloughed along with the superficial layer of the caruncle due to necrosis of the stratum compactum of the caruncle between Days 5 and 6 (Archbald et al., 1972). The epithelial cells are pleomorphic, and contain large hyperchromatic nuclei and granular cytoplasm. Archbald et al. (1972) observed degenerated and regenerated epithelial cells in the inter-caruncular epithelium on Day 1 post partum. The degenerative cells were confined to the basal area of the epithelium and characterized by pyknosis and vacuolation of the cytoplasm. Regenerated cells were interspersed among degenerated cells and characterized by large, hyperchromatic nuclei and granular cytoplasm. Degeneration and regeneration of the epithelial cells occurred simultaneously until Day 15 when degenerative cells were no longer observed (Archbald et al., 1972). In the event of bacterial contamination, the uterine epithelium may be totally or partially destroyed (Gier and Marion, 1968). Re-epithelialization of the caruncle is complete by Day 25 post partum, 10 days after obvious sloughing is complete (Gier and

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50 Marion, 1968). Archbald et al. (1972) found that a layer of epithelial cells covered the entire caruncular surface by Day 19 post partum. The epithelial cells of the uterine endometrial glands exhibit a pattern of simultaneous degeneration and regeneration similar to that observed in the inter-caruncular area (Archbald et al., 1972). Microscopic changes in the myometrium begin on Day 3 post partum and progress to Day 27 post partum. These changes include granular degeneration of the sarcoplam, vacuolation of the muscle cell and atrophy of the nucleus. By Day 31, the fibers appear normal and the myometrium is greatly reduced in size in comparison to that in the early postpartum period. Necrosis of the muscle fibers was not observed during postpartum myometrial regression (Archbald et al., 1972). Prior to Day 3 post partum the muscle fibers of the uterus contract from 750-800 m to 400 m and by Day 3 are approximately 200 m (Moller, 1970b). These changes in the size of the muscle fibers have been attributed to glycogen formation leading to degeneration and absorption (Moller, 1970b). Factors Affecting Uterine Involution Parity has been show to influence the interval from parturition to complete uterine involution. Marion et al. (1968) found that the average interval from parturition to uterine involution was significantly shorter in primiparous cows (34.0 days) compared to pluriparous cows (40.6 days). Based on rectal palpation, Morrow et al. (1969c) noted that multiparous cows of six lactations or more had larger uteri than primiparous cows, and took a longer time to involute. However, Moller (1970a) and Miettinen (1990) found no difference in the rate of uterine involution between primiparous and multiparous cows. Cervical involution is influenced by parity. Miettinen (1990) found that there was a difference in the size of the cervix with the cervix being larger in multiparous cows.

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51 However, there was no difference in the time the cervix took to involute. In contrast, Otlenacu et al. (1983) found that involution of the cervix occurred earlier in primiparous than in multiparous cows. Based on rectal palpation Morrow et al. (1969c) noted that multiparous cows of six lactations or more had a larger cervix than primiparous cows, and took a longer time to involute. On Day 10 and Day 20 post partum there was a significant difference of 1.2 and 1.0 cm in the diameter of the cervix. However, by Day 30 post partum the cervix was of similar sizes between 3.2 and 4.1 cm in diameter and by Day 50 post partum the cervix was between 2.9 and 3.3 cm in diameter. Otlenacu et al. (1983) noted that the time for complete involution of the cervix in cows with a normal discharge was less than that for cows with an abnormal uterine discharge. The greatest difference in cervix diameter between cows with normal and abnormal discharge was 10 mm at three weeks postpartum (Otlenacu et al. 1983). Time of year, or season, has been shown to influence the rate of uterine involution. Cows calving in the spring and summer have shorter intervals from parturition to uterine involution than cows which calve in the winter, regardless of parity (Marion et al., 1968). Cows calving in the summer have variable intervals from parturition to uterine involution. Marion et al. (1968) found that in cows calving in the summer with an ambient temperature of 38C, the heat stress increased the interval from parturition to uterine involution. Retained fetal membranes have been shown to increase the interval from parturition to the completion of uterine involution. Marion et al. (1968) found that it required 50.1 4.9 days for the uterus to involute in pluriparous cows with retained fetal membranes, and 45.1 3.2 days for primiparous cows. It was also noted that the rate of

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52 uterine involution was affected more in primiparous cows than in pluriparous cows with retained fetal membranes (Marion et al, 1968). Morrow et al. (1969c) defined an abnormal cow as a cow that experienced any of the following: abortion, dystocia, twins, retained fetal membranes, metritis, milk fever, acute mastitis, ketosis or other debilitating disease. Abnormal cows had larger uteri in the early postpartum period, especially cows with retained fetal membranes and metritis. At Day 10 and Day 20 post partum, there was a significant difference of 1.3 cm and 0.8 cm respectively between normal and abnormal cows. Abnormal cows had a combined diameter of both uterine horns on rectal palpation of 14.9 cm on Day 10 and 8.4 cm on Day 20 post partum, respectively. The gross palpable involution of the uterus was notable up to Day 25 post partum in normal cows, and Day 30 post partum in abnormal cows. Any changes that occurred after Day 25 and Day 30 post partum could not be felt on transrectal palpation (Morrow et al., 1969c). Metabolic disturbances can have a negative effect on uterine involution. Cows or heifers that experience hypocalcaemia have a reduction in uterine contractions (Roberts, 1989; Al-Eknah and Noakes, 1989) and rate of uterine and cervical involution (Fonseca et al., 1983). Kamgarpour et al. (1999) investigated subclinical hypocalcaemia in Friesian cows. Subclinical hypocalcaemia was defined as occurring if the plasma total calcium (PTCa) fell below 2.0 mmol/L. Cows calving in the winter and experiencing subclinical hypocalcaemia took longer to complete uterine and cervical involution in comparison to normocalcaemic cows, with hypocalcaemic cows taking 6 more days to reach a mean size. Similar findings were noted in cows that calved in the summer and experienced subclinical hypocalcaemia.

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53 The bovine uterus is invaded and colonized by bacteria within the first two weeks postpartum (Elliot et al., 1968; Griffin et al., 1974). In most cases the uterus clears this infection by the third or fourth week post partum. Not all cows clear the bacterial infection and subsequently develop endometritis. Endometritis has been shown to delay uterine and cervical involution. Endometritis delays the resumption of ovarian cyclicity, increases the interval from calving to ovulation and delays the return of prostaglandin F2 metabolite (PGFM) to basal levels (Lindell et al., 1985; Del Vecchio et al., 1992). Endometritis accounts for approximately 20% of reproductive disorders in postpartum dairy cows (Coleman et al., 1985). Moller (1970a) examined the rate of uterine involution between milked cows and cows suckling three or four calves and found that there was no difference in the rate of involution between milked and suckled cows. Steroid hormones have variable effects on the interval from parturition to uterine involution. In cattle, the first postpartum dominant follicle develops on the ovary contralateral to the previously gravid uterine horn. However, the presence of an estradiol-secreting dominant follicle in the ipsilateral ovary is a marker of subsequent fertility (Sheldon et al., 2000a). Based on their findings, Sheldon et al. (2000b) attempted to promote a dominant follicle on the ipsilateral ovary using equine chorionic gonadotrophin (eCG). Cows treated intramuscularly with 250 iu eCG or 750 iu eCG on Day 14 post partum showed no difference in the rate of uterine involution compared with those treated with a placebo. Treatment with eCG, or the presence of a follicle > 8 mm in diameter in the ovary ipsilateral to the previously gravid uterine horn, did not affect the rate of uterine involution (Sheldon et al. 2000b). The intramuscular administration of estradiol benzoate approximately 48 hours after calving had no effect on the intervals

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54 from calving to the completion of involution or between the intervals from calving to the first ovulation (Tian and Noakes, 1991). Sheldon et al. (2003) infused estradiol benzoate into the previously gravid uterine horn on Days 7 and 10 postpartum, and monitored uterine involution by ultrasonography. There was no effect of estradiol treatment on the diameter of the previously gravid or nongravid uterine horns. They concluded that utero-ovarian signaling in the direction from the uterus to the ovary may be more important during the postpartum period. Administration of oestradiol into the uterine lumen increased uterine pathogenic anaerobic bacterial contamination (Sheldon et al., 2004). Estradiol benzoate was infused into the previously gravid uterine horn on Days 7 and 10 postpartum. Animals treated with estradiol benzoate had higher bacterial load on Day 14, than on Days 7 or 21, attributable to pathogens associated with endometritis, Prevotella melaninogenicus and Fusobacterium necrophorum. These finding by Sheldon et al. (2000a, 2000b, 2003, 2004) and others indicate that estradiol administration in the early postpartum period had no effect on uterine involution (Tin and Noakes, 1991) and may be detrimental by promoting endometritis (Sheldon et al 2004). More first postpartum dominant follicles are selected in the ipsilateral ovary when there is a lower uterine bacterial load (Sheldon et al., 2002). Exogenous progestogens have been administered to cows in an attempt to improve uterine involution. Melengestrol acetate (MGA) was feed to Holstein cows at 1 mg per cow per day for 14 days beginning 14 to 18 days post partum in one group of cows. In a second group MGA was fed in a similar dosage and 500 micrograms of estradiol benzoate was administered intramuscularly 48 hours after the last feeding of MGA. The

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55 third group consisted of control cows that received no treatment. Based on rectal palpation, there was no significant difference in the rate of involution (Britt et al., 1974). Subcutaneous administration of 0.05 mg and 1.0 mg estradiol 17 given every other day from Day 3 post partum was shown to have no effect on the rate of involution. The oral administration of 300 mg medroxyprogesterone acetate every other day from Day 3 postpartum had no effect on the rate of uterine involution. However, daily administration of 30 mg of progesterone has been shown to increase the time for the uterus to involute completely in both intact and ovariectomized cows (Marion et al., 1968). Removal of ovarian hormone by ovariectomy has been shown to significantly reduce the time for uterine involution in pluriparous cows (Marion et al., 1968). The administration of prostaglandin F2 has been shown to decrease the time for complete involution of the uterus as detected by rectal palpation (Lindell and Kindahl, 1983). Lindell (1982) demonstrated that there is a massive release of PGF2 postpartum, which continues for 2 to 3 weeks. It was also deduced from this study that cows that had a shorter interval from parturition to uterine involution had a longer period of postpartum PGF2 release. In a study by Tian and Noakes (1991) five groups of five cows were treated intramuscularly with a single dose of either 100mg progesterone in oil, 25 mg dinoprost trmethamine (PGF2 tromethamine), 5 mg oestradiol benzoate, 1.2 mg long-acting oxytocin analogue, carbonectin or 5 ml sterile water 48 hours after parturition. There was no statistical difference in the rate of uterine involution.

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56 Methods to Assess Uterine Involution Uterine involution may be studied by observing the reduction in the size of the vulva, vagina, cervix and the uterus (body and horns). These observations may be made using the following: 1) palpation per rectum of the cows reproductive tract from calving to completion of uterine involution (Marion et al., 1968; Moller, 1970a; Garcia and Larsson 1982); 2) removal of the reproductive tracts at slaughter at predetermined intervals from calving (Gier and Marion, 1968; Moller 1970a); 3) transrectal ultrasonography of the reproductive tract (Mateus et al., 2002; Wehrend et al., 2003; 4) cervical forceps (Wehrend et al., 2003) Garcia and Larsson (1982) performed rectal palpation of the cervix, uterus and ovaries. They assessed uterine size, tone, symmetry and location of the uterus in pelvic cavity as indicators of uterine involution. Uterine involution was considered complete when the uterus was in the normal position in the pelvic cavity, the uterine horns were equal or almost equal in size and there was no enlargement in the thickness of the uterine wall. Marion et al., (1968) used rectal palpation twice weekly to monitor uterine involution, using criteria set by Gier and Marion (1968) as a marker for complete uterine involution. Moller (1970b) refers to Rasbechs (1950) four-stage sequence in involution, which can be followed by rectal palpation. During the first stage (1-8 days post partum), the vagina can be palpated as a band approximately 8 cm in width within 24 hours post partum. The cervix is not distinguishable until Day 3 and by Days 4 and 5 has enough tone to be distinguished from the uterus and is usually located at the anterior edge of the pelvic floor. The surface of the uterus feels hard and corrugated and the uterine caruncles can only be felt through the uterine wall when it is relaxed. In the second stage (8-10 days

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57 post partum) the entire uterus can be palpated. The surface is smooth and soft with fluctuation in the post-gravid horn. The caruncles are palpable as hazel-nut shaped structures and the cervix is firm and lies within the pelvic cavity in younger animals. During the third stage (10-18 days post partum) the uterus feels like a soft plastic body; caruncles and fluctuations are less pronounced; the cervix is firm and continues to decrease in size until it is similar to that of the post-gravid uterine horn. During the fourth stage (18-25 days post partum) there is an increase in uterine tone and the previously gravid horn reduces to a similar size to the non-gravid uterine horn. Moller (1970b) reports that authors on the subject of uterine involution agree that the previously pregnant horn rarely returns to its pre-gravid size. Although the uterus continues to undergo involutional changes after 25-30 days (Gier and Marion, 1968), these changes are small and difficult to detect by rectal palpation (Moller, 1970b). However, Marion et al. (1968) concluded that uterine involution monitored by rectal palpation take 40 days. Gier and Marion (1968) used reproductive tracts collected from slaughtered cows to assess involutional changes. They removed the excess fat and vagina from the cows to access the changes that occur during the process of involution. The remainder of the tract was then measured. The weight, length and diameter of the previously pregnant uterine horn were used to access the rate of regression. The diameter of the cervix was measured to assess uterine involution. Wehrend et al (2003) used a combination of ultrasonography and cervical forceps to measure the rate of uterine and cervical involution. Cervical forceps were positioned in the cervical canal and the position verified by ultrasonography. The distance of the legs

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58 of the forceps was proportional to the opening of the tip of the forceps. A gauge table was used to determine the degree of the opening of the intracervical tip of the forceps by measuring the distance of the extracorporeal legs of the forceps. Endocrinology of the Immediate Postpartum Period in the Cow The corpus luteum of pregnancy secretes progesterone throughout pregnancy and regresses about 2 days prior to parturition (Garverick and Smith, 1993). The placenta also contributes progesterone in the latter part of pregnancy. At the end of pregnancy there is a rapid increase in the secretion of estrogen (Hoffmann and Schuler, 2002). Prior to calving, progesterone and estrogen concentrations are high (Hoffmann and Schuler 2002). The progesterone concentration begins to decrease about two days prior to parturition and is low following calving. Following parturition, there is a rapid decline in circulating concentrations of estrogen. The fall in progesterone and estrogen removes the inhibitory block on the hypothalamic-pituitary axis (Garverick and Smith, 1993; Noakes, 1996) The inhibitory effects of estradiol are thought to operate through inhibition of the expression of mRNA coding for the common -subunit and specific -subunit of FSH and LH (Garverick and Smith, 1993). Messenger RNA expression of the subunits is low following parturition and increases thereafter. During gestation, LH is secreted in a pulsatile manner. However, the pulse frequency and amplitude decrease as gestation progresses. Pituitary LH content and plasma LH concentrations are low shortly after calving and generally increase during the postpartum period (Schallenberger et al., 1982; Garverick and Smith, 1993). Short-term episodic surges of LH increase as time post partum increases and LH responsiveness to

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59 GnRH is low at parturition and increases as time post partum increases (Garverick and Smith, 1993). The frequency of the pulsatile LH secretory pattern increases just prior to the first ovulatory surge of LH (Garverick and Smith, 1993). Ovariectomy in the early postpartum cow has a profound effect on the tonic secretion of gonadotropin from the pituitary. Schallenberger et al. (1982) showed that ovariectomy performed in the early postpartum period caused a two-fold increase in mean LH values as well as the amplitude and frequency of pulsatile release. A similar response was seen for FSH. However, the increase in amplitude was less pronounced than that seen for LH. Following parturition, FSH concentration is within normal range (Garverick and Smith, 1993). Schallenberger et al. (1982) noted pulsatile FSH release during the first day post partum. The frequency of pulsatile LH release coincided with FSH pulses. However, there were additional pulses between the FSH and LH pulses that coincided during the early postpartum period (Schallenberger et al., 1982). Increasing progesterone concentrations caused an immediate suppression of frequency and basal LH, but not FSH secretion. This is similar to that seen during normal cyclicity (Schallenberger et al., 1982). The administration of estrogen has no effect on LH secretion on Days 0 and 5 post partum. However, by Day 10 post partum and beyond, administration of estradiol elicited an LH surge with increasing magnitudes within 12 to 24 hours. Cows possessing a functional CL did not respond to estrogen with an LH surge. However, this was not the case for FSH. FSH surges also occurred in cows at 5 days post partum (Schallenberger et al., 1982).

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60 Schallenberger et al. (1982) showed that first calf heifers could be induced to cycle early in the post partum period when GnRH is administered on an hourly basis at 500 micrograms followed by challenge with 1mg estradiol-benzoate. It has been hypothesized that the following sequence of endocrine events occur in the normal cow after calving based on studies carried out by themselves and others (Peters and Lamming, 1986). Gonadotropin releasing hormone (GnRH) is secreted from the hypothalamus immediately after calving. However, the quantity or frequency of secretion is inadequate to cause sufficient release of LH and FSH from the pituitary to restore normal ovarian cyclicity. The concentration of FSH rises quickly after parturition and stimulates follicular development. The plasma concentration of LH and the frequency of LH pulses increase gradually with time post partum. Secretion of LH and FSH stimulates follicular growth and estradiol production. There is a gradual recovery in the positive feedback mechanism such that by two weeks postpartum normal ovarian cyclicity returns. Resumption of Ovarian Cyclicity Post Partum The interval from parturition to first observed estrus ranges from 30 to 76 days in the dairy cow (Roberts, 1971). However, observation of estrus may not be a reliable method to estimate the resumption of ovarian activity since some cows experience silent estrus. As time progresses post partum, the percentage of cows in which silent estrus occurs decreases (Roberts, 1971). Other methods of determining the onset of cyclicity have evolved which are more accurate. The milk progesterone assay determines the resumption of ovarian cyclicity by the presence of elevated progesterone concentrations (Noakes, 1996). Using milk progesterone concetrations, Bulman and Wood (1980) determined that approximately 50 percent of cows resumed normal

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61 cyclicity by 20 days postpartum and greater than 90 percent by 40 days post partum (Noakes, 1996). Ultrasonography has been used to follow the development of ovarian follicles in the post partum cow. Sheldon et al. (2002) detected follicular development by 7 to 10 days post partum with emergence of a dominant follicle within 14 days of parturition and ovulation of this dominant follicle by 17 to 18 days post partum. This interval was increased in cows with high milk production, in cows nursing calves or being milked four times a day, in cows on a poor or on low plane of nutrition and in older cows with greater than four parturitions (Roberts, 1971). In cows with a normal postpartum period, a mature follicle develops and ovulates with subsequent development of a CL by Days 13 to 15 after calving (Roberts, 1971). The first estrous cycle is usually shorter than the normal 20 to 21 days (Roberts 1971). Terqui et al. (1982) found that the earlier ovarian activity occurs the more likely cows will experience a short luteal phase. The CL associated with the short estrous cycle has a short life span as a result of lack of luteotropic support, failure of the luteal tissue to recognize a luteotropin, or enhanced secretion of a luteolytic agent (Hafez, 1993). The first ovulation post partum usually occurs on the contralateral ovary. Transrectal palpation of the ovaries has shown that approximately 90 percent of corpora lutea formed within 15 days post partum occur on the ovary opposite the previously pregnant horn and 60 percent in cows ovulating between 15 and 20 days after parturition (Roberts, 1971). This has been substantiated by sequential transrectal ultrasonography. The first postpartum dominant follicle is usually found on the ovary contralateral to the previously pregnant uterine horn, that is, opposite the ovary containing the CL of

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62 pregnancy (Foote et al., 1968; Kamimura et al., 1993; Nation et al., 1999). This led many to speculate that the CL of pregnancy may have a local inhibitory effect on folliculogenesis. Dufour et al. (1985) concluded that the CL of pregnancy and/or the conceptus have a carry-over effect on the rate of growth of the antral follicles even after parturition. Bellin et al. (1984) found that the ovary with the CL of pregnancy had smaller follicles and lower follicular estrogen concentrations than those of the ovary without a CL. It was also noted that only 18% of the follicles on the ovary with a CL were healthy compared to 48% of follicles in the ovary without a CL (Bellin et al., 1984). However, recent work done by Sheldon et al. (2002) suggested that the CL of pregnancy does not have a local inhibitory effect on postpartum folliculogenesis. It was further suggested that the previously pregnant uterine horn shortly after parturition may play more of a role. It has been shown that the presence of a large follicle on the ovary ipsilateral to the previously pregnant uterine horn within 4 weeks of parturition was associated with improved subsequent fertility (Sheldon et al., 2000). Bridges et al. (2000a) reported similar findings in beef cows, where less cows ovulated from the ipsilateral ovary. However, ovulation from the ipsilateral ovary tended to increase fertility (Sheldon et al., 2000a). There are several factors that have a negative impact on the resumption of ovarian cyclicity in dairy cows. The interval from calving to first ovulation increased in cows with high milk production, cows nursing calves or being milked four times per day, cows on a poor or on low plane of nutrition, and in older cows with greater than four

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63 parturitions (Roberts, 1971). Bellin et al. (1984) found that suckled cows had smaller and fewer follicles and lower follicular concentrations than nonsuckled cows. There is a close association between the time to first ovulation and negative energy balance in dairy cows (Zurek et al., 1995). The exact mechanism by which energy balance influences reproduction is not completely understood. However, one mechanism may be through the suppression of GnRH and the LH pulse frequency required for follicles to grow to the preovulatory stage (Schillo, 1992). Cystic Ovarian Disease in the Dairy Cow Cystic ovarian degeneration, ovarian follicular cysts, cystic ovarian disease, ovarian cysts and cystic ovaries are terms used to describe the same condition. Ovarian cysts are follicles that fail to ovulate at the time of estrus (Garverick, 1999), and commonly occur in lactating dairy cows. They can also occur in beef cows and dairy heifers. Ovarian cysts have been defined as follicular structures 2.5 cm or greater in diameter that persists for an extended period of time in the absence of a CL (Garverick, 1999). Ovarian cysts can be classified as follicular cysts or luteal cysts. Follicular cysts are thin walled, may be single or multiple and affect one or both ovaries (Elmore, 1986). Higher concentrations of estradiol are usually found in follicular cyst fluid than in fluid from luteal cysts and normal follicles (Odore et al, 1999). Luteal cysts are thick-walled, usually occurring singly, and affect one ovary and have higher concentrations of progesterone in their cystic fluid than follicular cysts and normal follicles (Odore et al, 1999). Bane (1964), in a review of fertility and reproductive disorders in Swedish cattle, quoted Henricson reporting that the risk of ovarian cysts increased from 0.3% for heifers to 8-10% for cows four to five years old and that Henricson found the incidence of ovarian cysts was higher in the winter months than in the summer months. Bane analyzed

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64 the frequency of ovarian cysts in relation to the month of calving and found that cows calving in the winter had a higher risk of developing ovarian cysts in the following reproductive period (Bane, 1964). Lopez-Gatius et al. (2002) indicated that ovarian cysts occur most commonly in cows calving in the summer. Garverick (1999) estimated the incidence of ovarian follicular cysts between 10 % in the United States. The calving date influences the frequency of ovarian cysts. Cows that calved in April-May were less likely (2.2%) to develop ovarian cysts while cows calving in October had a higher frequency (9%) of ovarian cysts (Bane, 1964). In Sweden between 1954 and 1961, the frequency of animals with ovarian cysts in five-year-old cows fell from 10.8 % to 5.1%. This was brought about by the culling of bulls with a high frequency of cystic ovarian degeneration among their daughter (Bane, 1964) suggesting that there may be a hereditary aspect. Spontaneous recovery occurs in some cows that develop ovarian cysts. Lpez-Gatius et al. (2002) found that in cows diagnosed with cysts 43-49 days postpartum, only 38.8% recovered spontaneously while in cows diagnosed with cysts 57-63 days postpartum, 71.4% recovered spontaneously. Erb and White (1981) reported that the highest incidence of ovarian cysts occurs in the first 60 days of lactation, while Morrow et al. (1969b) reported that there was a peak in incidence between 14 and 40 days post partum. Predisposing Factors Lpez-Gatius et al. (2002) reported that ovarian cysts occur most commonly in cows calving in the summer, in high milk producers, in older cows, and in cows that body condition score increased during the prepartum period. The major risk factor for the presence of cysts at the time of insemination was the development of cysts early (43-49

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65 days postpartum) in the postpartum period (Lpez-Gatius et al., 2002). The risk of having a cyst at the time of insemination was 36.6 times higher in cows with early cysts (Lpez-Gatius et al., 2002). Pathogenesis Postpartum uterine infections may increase secretion of PGF2 and cortisol associated with the formation of cystic ovaries in dairy cows (Bosu and Peter, 1987; Peter et al., 1989). In a study carried out on cows that calved normally but subsequently developed ovarian cysts, a correlation between postpartum uterine infections and the development of ovarian cysts was made. In 88.9% and 11.1% of cows that subsequently developed ovarian cysts endometrial swabs yielded bacterial growth densities of +3 and +2 respectively, while 35.1% of cows that did not develop ovarian cysts yielded bacterial growth densities of +1 and +2 (5.4%) (Bosu and Peter, 1987). Further, consistently high cortisol levels were detected prior to cyst detection and in association with high bacterial growth densities in the uterus prior to cyst detection (Bosu and Peter, 1987). Cysts have been experimentally induced using anti-LH serum, estradiol valerate and adrenocorticotropic hormone (ACTH) administration. Cysts have also been induced by daily exogenous injection of 30 mg of estradiol-17 and 150 mg of progesterone dissolved in alcohol for 7 days (Cook et al., 1990; Garverick, 1999). Ovarian cysts have been induced by ACTH administration in the preovulatory period. Adrenocorticotropic hormone has been shown to block the preovulatory LH surge, possibly through cortisol-mediated inhibition of LH release (Refsal et al, 1987). The mode of action of opioids is by inhibiting the release of hypothalamic GnRH. Through their action on GnRH, they are thought to suppress LH secretion from the

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66 pituitary in the prepubertal period and modulate LH during the estrous cycle. It has been established that gonadal steroids suppress LH secretion by negative feedback on the hypothalamic-pituitary axis, and this action may be brought in part by intermediate opioidergic neurons (Brooks et al., 1986). It has been demonstrated that follicular cysts can be induced using estradiol benzoate by stimulating a GnRH/LH surge in the absence of a preovulatory follicle. Subsequent exposure of the cystic cow to progesterone can resolve the cystic condition by reinitiation of GnRH/LH surges in response to estradiol (Gmen et al, 2002). Gmen and Wiltbank (2002) demonstrated in cows that an initial GnRH/LH surge and subsequent ovulation could be induced with high levels of estradiol, but estradiol induction of a subsequent GnRH/LH surge required exposure to progesterone. Follicular cysts were induced using an intravaginal progesterone insert (IPI)/PGF2/estradiol benzoate (EB) protocol, and indicated that the effect is mediated at the level of the hypothalamus (Gmen and Wiltbank, 2002). Odore et al., (1999) suggested that ovarian cysts might be due to alterations in the feedback regulatory mechanism. This hypothesis was based on findings that indicated a difference in the concentration of luteinizing hormone receptors and follicle stimulating receptors in the ovary and pituitary between normal and cystic cows. Silvia et al. (2002) proposed the following model for how intermediate levels of progesterone could lead to the development of ovarian follicular cysts in the dairy cow. During the follicular stage in cows without ovarian cysts, the tonic center of the hypothalamus secretes GnRH at a high frequency stimulating a frequency mode of LH secretion by the anterior pituitary. The high frequency LH secretion promotes maturation

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67 of the dominant follicle. This follicle in turn secretes increasing concentrations of estradiol that eventually reach a threshold adequate to trigger the surge center of the hypothalamus to release GnRH in quantities that will trigger a preovulatory LH surge and induce ovulation of the dominant follicle. However, in cows with ovarian cysts, the intermediate progesterone levels make the surge center of the hypothalamus insensitive to estradiol. The preovulatory GnRH/LH surge is blocked and ovulation fails to occur. However, the tonic center of the hypothalamus is unaffected by intermediate progesterone concentrations and thus the high frequency, tonic pattern of LH is maintained. This tonic pattern of LH provides the cysts with the gonadotropic support it requires to function (Silvia et al., 2002). Cyst Dynamics Ovarian cysts are dynamic structures. They regress and are replaced with other cystic structures (turnover), or spontaneous recovery occurs characterized by ovulation of a new follicle at a site different from that of the original cysts (Cook et al. 1990). Cook et al. (1990), induced ovarian cysts with twice daily subcutaneous injections of 15 mg estradiol-17 and 37.5 mg of progesterone for 7 days starting on Day 15 post partum. Of the 23 cows that became cystic, 7 ovulated (spontaneous recovery), 13 showed cystic turnover and 3 persisted (Cook et al. 1990). In no case did the marked cyst ovulate. In another study carried out on cows that calved normally over a one-year period, 12 out of 47 cows were found to be cystic. Follicular cysts were detected on the ovary as early as 8 days post partum. In 10 cows, serial rectal and ultrasound examination of the cysts indicated an increase in size confirming the dynamic nature of ovarian cysts (Bosu and Peter, 1987). In 4/12 cows (33.3 %) the cyst regressed before Day 28 post partum and in

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68 the remaining 8/12 cows (66.6%) the cyst regressed between Days 29 and 57 post partum (Bosu and Peter, 1987). In a study of 29 heifers undergoing daily transrectal ultrasound in order to follow follicular dynamics, one heifer developed follicular cysts. This heifer regressed the corpus luteum in a normal manner, developed a normal preovulatory size follicle on the ovary, exhibited standing heat, but the follicle failed to ovulate. This follicle lost follicular dominance and a new follicular wave emerged. Two dominant follicles emerged from this wave. When one reached preovulatory size, the heifer once again exhibited estrus but did not ovulate. This process continued through a third follicular wave and finally the heifer was successfully treated using the Ovsynch protocol after emergence of the fourth follicular wave (Wiltbank et al., 2002). Diagnosis Due to the dynamic nature of ovarian cysts, weekly rectal palpation and/or ultrasonic observations will detect those cows with cysts more readily than monthly or bimonthly examinations. The diagnosis of ovarian cysts is usually based on rectal palpation of a fluid filled structure greater than 2.5 cm in diameter on one or both ovaries in the absence of a CL. The diagnosis may be confirmed using transrectal ultrasonography and the type of cysts (luteal or follicular cysts) determined. Luteal cysts may also be confirmed by progesterone assay of milk or serum. The most common sign of cows that have been diagnosed as cystic is a lack of estrus (Farin and Estill, 1993). Nymphomania has also been noted in cows that have follicular cysts (Farin and Estill, 1993). In order to differentiate between cystic follicles and a large preovulatory follicle, the characteristic of the uterus must be evaluated. On rectal palpation a diagnosis of cystic ovaries will be made when the uterus has no tone and is unresponsive to

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69 manipulation. The uterus of a cow in estrus has tone and responds to manipulation by increase coiling of the horns. Treatment Although cows with ovarian cysts have been shown to recover spontaneously, only a portion of cows affected with cystic ovarian disease recovers spontaneously and within a time frame that is economical to the dairy farmer. Morrow et al (1966) reported that only 48% of cows affected with follicular cysts recovered spontaneously. Kesler and Garverick (1982) estimated that even less cows (approximately 20%) with cystic ovaries will spontaneously recover and return to normal cyclicity. One of the earliest methods of resolving ovarian cysts was manual rupture via rectal palpation. Repeated manual rupture of cysts at 6 to 10 day intervals, especially follicular cysts in the early postpartum period, has produced 37% (Roberts, 1971) to 45% recovery rates (Kesler and Garverick, 1982; Ijaz et al., 1987). However, manual rupture can cause injury to the tissue of the ovary and its surrounding structures, causing hemorrhage, promoting adhesions and infertility (Roberts, 1989; Elmore, 1986). Gonadotropin-releasing hormone (GnRH) is commonly used to treat ovarian cysts regardless of the type of cysts. Exogenous administration of GnRH triggers an LH surge that results in ovulation of a LH-responsive follicle at the time of treatment or luteinization of the cyst (Kittok, et al. 1973; Cantley et al. 1975). Repeated use of GnRH intravenously 120 minutes apart has been shown to cause a LH surge similar to the preovulatory LH surge in normal cycling cows; the second dose corresponding to peak LH serum concentrations in cows with cystic follicles (Kittok et al. 1973). The LH surge is induced with 30 minutes of GnRH administration and remains elevated for 4 hours (Cantley et al. 1975). However, the response of the cow with ovarian cysts differs from

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70 that of cows without ovarian cysts. that have an elevated LH concentration for up to 10 hours which may due to luteinization of the cystic structure as a result of ovarian response to GnRH induced LH release (Cantley et al. 1975). Cow treated with GnRH have been shown to return to estrus within 20 to 24 days (Kittok et al. 1973) after treatment. Cows with ovarian cysts treated with a single intramuscular injection of either 50, 100 or 250 g GnRH exhibited estrus by 22 days and most cows exhibited estrus 18 to 23 days post-treatment (Bierschwal et al. 1975). The 100 g dosage had the best response of the three dosages with 82% returning to estrus and 87% of those that returned to estrus conceiving (Bierschwal et al. 1975). In order to shorten the interval between GnRH treatment and estrus, PGF2 has been used 9-14 days after GnRH treatment (Neil, 1991). However, the need for estrus detection has been eliminated with the introduction of the Ovsynch protocol (Pursley, 1995). This protocol uses a GnRH/PGF2/GnRH treatment scheme which has proven successful in treating cystic ovarian degeneration (Bartolome et al., 2000; Gumen et al., 2003). Human chorionic gonadotropin (hCG) is used for its high luteinizing hormone activity (Kesler and Garverick, 1982). It has the same effect as GnRH. However, hCG has the ability to induce antibody production where as GnRH because of its small molecular size is not likely to induce an immune response (Bierschwal, et al. 1975).A dose of hCH between 2,500 and 10,000 IU, given intramuscularly or intravenously will have the same effect as GnRH (treatment response and post treatment fertility). However it can cause undesirable effects (Neil, 1991)

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71 Progesterone has been shown to resolve ovarian cysts. Methods of administering progesterone have included injections, intravaginal devices or ear implants. Zulu et al. (2003) investigated the use of a progesterone releasing intravaginal device (PRID) in the treatment of cows with ovarian cysts. Their study confirmed that follicular and luteal cysts could be successfully treated with PRID; resulting in ovulation 2-4 days after removal of the device with subsequent formation of a CL. Prostaglandin F2 (PGF2) has been used alone in the treatment of ovarian cysts (Chavatte et al., 1993). It is the treatment of choice for luteal cysts or cysts containing luteinized tissue. Luteal cysts respond by regression with subsequent follicular development and estrus in 2-5 days (Kesler and Garverick, 1982; Neil, 1991). It has been shown that there is a higher PGF2 receptor concentration in luteal cysts than in follicular cysts (Odore et al., 1999) and this may explain the success of prostaglandin treatment. Prostaglandin F2 is generally believed to be ineffective in follicular cysts, but some cows with progesterone as low as 0.5 ng/ml will respond positively (like luteal cysts; Neil, 1991). Timed Artificial Insemination Programs in the Dairy Cow One of the factors affecting pregnancy rate in dairy herds is the detection of estrus. In some instances estrous behavior is reduced. Reduced estrus behavior has been noted in cows treated with bovine somatotropin (bST) (Kirby et al., 1997). It has been shown that the physiological state of lactation is associated with lower concentrations of estradiol during proestrus than levels found in non-lactating dairy cows and thus behavioral estrus is reduced in these cows. During lactation progesterone levels are also

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72 lower than in non-lactating dairy cows. In addition it has been shown that cows in lactation have a lower reproductive rate than do heifers. Heat stress is another factor that decreases the expression of estrus behavior (Abilay et al., 1975; Nebel et al., 1997). However, lack of heat detection can play a major role in reducing pregnancy rate. The development of programs for estrus synchronization and timed insemination have eliminated the need for estrus detection, and have been shown to increase conception rates to artificial insemination programs. It should be noted however, that pregnancy rate is affected by a number of factors, such as anestrus, low conception rates and increased embryo mortality. In many regions of the world another factor which influences the dairy cow, especially high producing dairy cows is heat stress. In an attempt to return the high producing lactating dairy cow to normal reproductive function, various pharmacological programs have been devised. These programs manipulate ovarian function. The primary goal of estrous synchronization programs is to synchronize estrus and ovulation effectively, such that cows may be inseminated at a predetermined time without estrus detection and reduction in fertility (Rathbone et al., 2001). Several synchronization protocols have developed over the years for synchronization of estrus. The basis of these protocols rely on one of the following: 1) regression of the CL with PGF2 or its analogue with the cows returning to estrus and ovulating within 2 to 4 days of the (final) injection of PGF2; 2) the use of exogenous progesterone or synthetic progestins to prevent estrus and ovulation for a long enough period to allow for regression of the CL; upon removal of the exogenous progesterone or

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73 synthetic progestins the cows should return to estrus and ovulate (Rathbone et al., 2001). Although these pharmacological substances can be used alone, the conception rates were found to be low and thus they have been used in combination with other reproductive drugs to increase conception rates to timed insemination. Prostaglandin F2 Prostaglandin F2 given randomly during the estrous cycle between Days 5 and 16 will cause luteolysis of the CL and cows should exhibit estrus between 2 and 4 days post treatment. The variability in return to estrus and ovulation depends on the stage of the dominant follicle at the time of luteolysis. If luteolysis occurs when the dominant follicle is viable, then estrus and ovulation will occur in a relatively short period from treatment. However, if luteolysis occurs when the dominant follicle is nonresponsive, then the dominant follicle of the following wave will grow and become the ovulatory follicle and the interval between treatment and ovulation will be longer (Kastelic et al., 1990). For these reasons the use of one luteolytic dose of PGF2 with insemination at a fixed time yielded low conception rates. Combination of Gonadotropin-releasing Hormone (GnRH) and PGF2 Pursley et al. (1995) introduced a new method for synchronizing the time of ovulation in cattle using GnRH and PGF2 (Ovsynch program). In this protocol, GnRH is given at a random stage of the estrous cycle and PGF2 is given seven days later. A second GnRH injection is administered 2 days after PGF2 and the cow or heifer was bred 24 hours later. The first GnRH injection either caused ovulation of the dominant follicle or had no effect. Next, PGF2 caused regression of the CL, which resulted from the initial injection of GnRH. The seven-day waiting period between the initial GnRH

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74 and PGF2 injection gives the CL enough time to mature and become responsive to PGF2. The final GnRH injection caused ovulation of the dominant follicle. The priod of 48 hours between PGF2 and GnRH allowed for a new follicle to emerge, grow to preovulatory size and become sensitive to the LH surge induced by GnRH to cause ovulation. This study demonstrated that the Ovsynch protocol was more suited to lactating dairy cows than heifers. Ninety percent of lactating dairy cows ovulated to the first GnRH injection in comparison to fifty percent of heifers. There are stages within the estrous cycle when the initiation of an Ovsynch protocol will cause a reduction in pregnancy rates. Initiation of the Ovsynch protocol during late luteal phase of the estrous cycle, between Days 13 to 17, may lead to premature lysis and regression of the CL. These cows are asynchronous and exhibit estrus prior to the second GnRH injection. In these cases insemination within 16 to 20 hours after the last GnRH injection will be ineffective and conception is not likely (Moreira et al., 2000a). In the early stages of the estrous cycle, Days 2 to 4, the dominant follicle is not yet sensitive to LH and will not respond to GnRH. These follicles will ovulate to the second GnRH injection. Follicles that fall into this category will range in age from 11 to 13 days, and have been shown to be less fertile. The early luteal phase, between Days 5 and 11 of the estrous cycle, is the optimal time to initiate the Ovsynch protocol to achieve acceptable pregnancy rates. As a result it becomes important to presynchronize lactating dairy cows to an optimal stage in the estrous cycle at which time initiation of the Ovsynch protocol is most effective.

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75 Presynch-Ovsynch In this presynchronization program PGF2 is given twice, 14 days apart, and the Ovsynch protocol is initiated 12 days after the last PGF2 injection. Presynchronization programs have been shown to increase the first-service pregnancy rates in comparison with initiation of the Ovsynch protocol at random stages of the estrous cycle in lactating dairy cows (Moreira et al, 2001). The increase in the pregnancy rate has been attributed to the initiation of the Ovsynch protocol in the most favorable stage of the estrous cycle. Presynchronization beginning on Day 22 post partum with a second injection 14 days later has been shown to lower the incidence of metritis-pyometra, ovarian cysts, improve luteal activity rate on Day 50 post partum, improve estrus detection rate, increase ovulation rate and increase pregnancy rate (Lpez-Gatius et al., 2003). Postpartum Endometritis Endometritis is defined as inflammation of the endometrium. The term is descriptive and refers to the extent and anatomical distribution of the inflammatory process. There are a plethora of papers published on the topic of postpartum uterine infection. However, the definition of uterine infection varies and the terms endometritis and metritis have been used interchangeably in the literature (Bretzlaff, 1987; Lewis, 1997). The following terms have been used to define uterine infection: metritis, endometritis, and pyometra. Postpartum endometritis is a pathologic condition usually diagnosed during the intermediate postpartum period (Ball et al., 1984; Olson et al 1984) during routine postpartum examination of the cow or heifer. It is commonly characterized by the absence of estrus, a vaginal discharge of creamy-white or yellow pus and a large doughy

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76 uterus that fails to involute. Postpartum endometritis is the most common cause of infertility in cows. It delays uterine involution (Tennant and Peddicord, 1968; Griffin et al., 1974), prolongs the time to first estrus, increases the number of services per conception and prolongs the interval to calving (Griffin et al., 1974). Lewis (1997) stated that as many as 40% of postpartum cows within a herd may be diagnosed and treated for uterine infections. Coleman et al. (1985) stated that endometritis was a common reproductive disorder accounting for 20% of the reproductive disorders in dairy cows. However, Ruder et al. (1981) found that the percentage of cows with endometritis could be as high as 67%. It has been reported that each lactating dairy cow with a uterine infection can cost a farmer up to $106. This costs consists of treatment, loss in milk production (associated with the infection and as a consequence of milk discarded due to antimicrobial therapy) and the loss of cows due to culling for reproductive disorders associated with and as a direct cause of uterine infection (Lewis, 1997). Pathology Ball et al. (1984) defined the postpartum period as the period from parturition to complete involution. This period was further divided into the early postpartum period, intermediate period and postovulatory period. The early postpartum period is the period following parturition until the pituitary becomes sensitive to GnRH, and usually lasts 8 to 14 days. The intermediate period begins when the pituitary becomes responsive to GnRH and ends with the first postpartum ovulation. The postovulatory period starts with the first postpartum ovulation and ends with the completion of involution. During the early postpartum period bacteria invade the uterus (Elliot et al., 1968; Griffin et al., 1974; Peter and Bosu, 1987). Elliot et al., (1968) recovered bacteria from

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77 93% of uteri examined between 3 to 15 days post partum. The major groups of bacteria isolated were Streptococcus, Staphylococcus, Micrococcus, Archanobacteria formally known as Corynebacteria, Pseudomonas and Escherichia (Elliot et al., 1968). Griffin et al. (1974) found 92% of uteri sampled infected with bacteria between Days 1 and 7 postpartum, and 96% infected between Days 8 and 14 post partum. The fertility of cows were not affected by the variable uterine flora or endometritis found within the first two weeks post partum. However, cows that had a uterine infection with Archanobacterium pyogenes following Day 21 postpartum developed severe endometritis and were infertile to the first service (Griffin et al., 1974). During the intermediate period and into the postovulatory period, spontaneous clearance of the bacterial contaminant and recontamination occurs. It was noted that the composition of uterine flora changed throughout the first 7 weeks post partum as a result of spontaneous contamination, clearance and recontamination (Griffin et al., 1974). It is likely that as the cow or heifer begins to cycle and the uterus comes under the influence of high concentrations of estrogen, that infections are cleared and there is an increased rate of repair of the endometrium. Rowson et al. (1953) demonstrated that the bovine uterus is more susceptible to bacterial infection during the luteal phase of the estrous cycle when progesterone is the dominant hormone. It was also noted that under the influence of estrogen during the follicular stage of the cycle, the uterus was less susceptible to bacterial infection. Hawk et al. (1960; 1964) demonstrated that the uterus of heifers differed in their leukocytic response during the estrous cycle. The leukocytic response to an inoculum of Escherichia coli was more pronounced and occurred much faster during estrus than in the luteal phase.

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78 During intermediate period and postovulatory period, intrauterine bacterial infection has been associated with short estrous cycles. Peter and Bosu (1987) demonstrated the temporal relationship between infection patterns and serum concentrations of LH, P4 and PGF2 metabolite (PGFM). They noted that cows with a short first estrous cycle length were those with uterine infections. High infection rates were associated with higher concentrations of PGFM before the first postpartum LH surge and ovulation. Following the LH surge, increasing concentration of PGFM was associated with increasing intensity of uterine infection. There was a further increase in PGFM prior to the decrease in P4 and lysis of the CL. However, in cows with a normal estrous cycle length, low PGFM was noted prior to and following the LH surge and only prior to onset of luteolysis (Peter and Bosu, 1987). Pathogenesis Endometritis is a localized inflammation of the endometrium of the uterus associated with chronic postpartum infection with pathogenic bacteria. The primary bacterium associated with endometritis is Archanobacterium pyogenes (Elliot et al., 1968; Griffin et al., 1974; Peter and Bosu, 1987; Bonnett et al., 1991; Dohmen et al., 1995; 2000). Griffin et al. (1974) noted that 80% of the time Archanobacterium pyogenes was isolated form uteri with moderate or severe endometritis. Classification of the severity of endometritis was based on the number of neutrophils, histiocytes, plasma cells and lymphocytes in the biopsy specimen. Moderate endometritis was defined as medium infiltration of the stratum compactum and upper part of the stratum spongiosum and/or three to four foci of cellular reaction per section in a number of sections. Severe endometritis was defined as dense infiltration in the stratum compactum and stratum

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79 spongiosum and/or five or more foci of cellular reaction per section in a number of sections (Griffin et al., 1974). Dohmen et al. (2000) investigated the relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in postpartum cows with dystocia or retained fetal membranes. They found that the presence of an abnormal cervical discharge at Day 14 post partum was higher in cows with retained fetal membranes compared to cows that experiences dystocia. However, Archanobacterium pyogenes was isolated more often in cows with retained fetal membranes on Day 14 post partum than dystocia cows and was positively associated with an abnormal discharge on Days 14 and 28 post partum. Dohmen et al. (2000) suggested that the presence of Escherichia coli and lipopolysaccharides (endotoxins) in lochia early in the postpartum period predisposed the development of uterine infections by A. pyogenes and Gram-negative anaerobes. Reduced neutrophil function plays a role in the development of endometritis. Neutrophils function in phagocytosis and elimination of bacterial contaminants in the uterus. In cows with retained fetal membranes, neutrophil functions such as chemotaxis and bacterial ingestion were reduced in comparison to health cows (Cia et al., 1994). The reduction in neutrophil function may increase the susceptibility of the postpartum dairy cow to infection. Diagnosis Diagnosis of endometritis may be made based using a combination of visual inspection of the vulva and tail, vaginoscopy, rectal palpation, uterine culture or biopsy and/or ultrasonography. Visual inspection may reveal crusts formed on the vulva and/or tail. There may be discharge attached to the ventral commissure of the vulva (Zemjanis,

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80 1970). Visual inspection of the vulva and tail alone is not adequate to diagnose endometritis. Rectal palpation is the most common method used for diagnosing endometritis. However, this has been noted as an insensitive and non-specific method (Bretzlaff, 1987; Lewis, 1997). The results obtained through this method depend on the skill and experience of the individual. The size and consistency of the uterus and cervix along with the fluid content of the uterus are used to ascertain the presence of uterine infection compared to the normal finding for a given time postpartum. Vaginoscopy is seldom used routinely as a diagnostic technique in dairy cows (Zemjanis 1970; Bretzlaff, 1987). Vaginoscopy, visual examination of the vagina, luminal content and external cervical os have been shown to be a superior means of diagnosing endometritis. LeBlanc et al. (2002) found that the prevalence of clinical endometritis was 16.9% and vaginoscopy was required to identify 44% of these cases. Visual and transrectal palpation of the uterus was not sufficient to identify cases oc clinical endometritis. Le Blanc (2002) suggested that the diagnosis of clinical endometritis should be made by the presence of purulent uterine discharge or cervical diameter > 7.5 cm after 20 DIM, or mucopurulent discharge after 26 days in milk. This definition of endometritis is based on the negative effect these criteria have on subsequent fertility. In the absence of vaginoscopy a combination of history, inspection and palpation may be used to diagnose clinical endometritis. In the absence of vaginoscopy the following criteria can be used to classify clinical endometritis (LeBlanc et al., 2002): presence of mucopurulent or

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81 purulent discharge on the perineum, cervical diameter >7.5 cm, and presence of a uterine horn 8 cm in diameter. Griffin et al. (1974) identified cases of endometritis using uterine biopsy and uterine culture. The severity of endometritis was determined on histological findings and the predominant bacterial flora evaluated by uterine culture. Recently, endometrial cytology has been used to identify cows with subclinical endometritis in the postpartum dairy cow (Kasimanickam et al., 2004). Endometrial cytology was performed on clinically normal cows, cows without evidence of an abnormal discharge on visual inspection of the vulva, tail and surrounding areas and vaginoscopy between Days 20 and Day 33 post partum. Clinical endometritis was defined as > 18% polymorphonuclear cells seen in endometrial cytology samples or fluid in the uterus at the first visit (Days 20 and Day 33 post partum) and as > 10% polymorphonuclear cells seen in endometrial cytology samples or fluid in the uterus at the first visit (Days 34 and Day 47 post partum). In a similar fashion to LeBlanc et al (2002) and Kasimanickam et al (2004) defined clinical endometritis based on its impact on subsequent pregnancy rates. Although there was no evidence of abnormal discharge on the day of enrollment, 9.1% had a discharge within 24 hours. This indicated that evaluation of endometritis should be done on more than one occasion. It is interesting to note that 35.1 % of cows were diagnosed with subclinical endometritis by endometrial biopsy on the first visit and 34% on the second visit 2 weeks later. However, 48% were diagnosed with subclinical endometritis by the presence of fluid in the uterus on the first visit and 20.5% on the second visit 2 weeks later. Cows with subclinical endometritis were less like to become pregnant than those without subclinical endometritis (Kasimanickam et al., 2004).

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82 Ultrasonography may be used to aid in the diagnosis of endometritis. One of the sonographic indications of endometritis is the appearance of fluid in the uterine lumen. This fluid accumulation must be distinguished from that which is seen with estrous secretions or the embryonic fluid of the early conceptus. Fluids that accumulate during estrus and early pregnancy are less echogenic than that of endometritis (Fissore et al., 1986). In mild endometritis, there may be no fluid present or few fluid filled pockets found when scanning the uterus (Kahn et al., 1989). In cases of severe endometritis, the uterus may be distended with fluid and involve both horns (Kahn et al., 1989). The echogenicity of the fluid that accumulated during endometritis varies with the products of inflammation. Kahn et al. (1989) described the echogenic pattern displayed by the products of inflammation. Echogenic patterns range from small bright spots in mild cases to a snow-storm effect or, in severe cases, to almost bright white images on the screen. Real-time-scanning may reveal turbulences within the larger collections of fluid. Since plasma levels of 13,14-dihydro,15-keto-PGF2 (PGFM), the stable metabolite of PGF2, are elevated in spontaneous uterine infection, it has been suggested that these levels may aid in the diagnosis of endometritis in the postpartum cow (Del Vecchio et al., 1994). However, Archbald et al. (1998) were unable to correlate plasma levels of PGFM with abnormal uteri identified by per rectum palpation during Days 24 to 29 post partum in lactating dairy cows. In addition, Archbald et al. (1998) demonstrated that the postpartum rate of decline of plasma levels of PGFM was influenced by the presence of a corpus luteum (CL). In cows which had a CL, the rate of decline of PGFM was slower compared to cows without a CL. It has been reported that progesterone plays

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83 an extremely important role in regulating pulsatile secretion of PGF2 from the bovine uterus (Mann et al., 1995) since it can completely restore the number of pulses and partially restore pulse magnitude when administered to ovariectomized ewes. However, a decrease in progesterone appeared to be essential for pulses of PGF2 to reach a maximum magnitude (Silvia et al., 1991).

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CHAPTER 3 EXPERIMENT 1: AN EVALUATION OF PRETREATMENT WITH GnRH ON OVARIAN RESPONSE AND PREGNANCY RATE OF LACTATING DAIRY COWS WITH OVARIAN CYSTS SUBJECTED TO THE OVSYNCH PROTOCOL Introduction Bovine ovarian cysts are follicles that fail to ovulate at the time of estrus (Garverick, 1997; 1999). They are an economic problem in the dairy cow because these cows are infertile as long as the condition persists (Kesler and Garverick, 1982). The exact cause of ovarian cysts is not presently known, but some predisposing factors include age, stress, high milk production and genetics. It appears that an important component in the pathogenesis of this condition is the inappropriate, or lack of, release of hypothalamic gonadotropin-releasing hormone (GnRH) at the time of estrus (Refsal et al., 1987; Gmen et al., 2002; Silvia et al., 2002). One therapeutic approach involves the use of exogenous GnRH, which releases LH from the anterior pituitary and causes ovulation of an ovarian follicle, and/or luteinization of the ovarian cysts (Garverick, 1997; 1999). The occurrence of ovarian follicular waves has been adequately demonstrated in the dairy cow without ovarian cysts (Rajakoski, 1960; Pierson and Ginther, 1984). It has been suggested that ovarian follicular waves also occur in cows with ovarian cysts (Garverick, 1999). The difference is that ovulation of follicles occur in cows without ovarian cysts, but does not occur in cows with ovarian cysts. It has further been suggested that the administration of GnRH to cows with ovarian cysts causes ovulation of a functionally mature follicle of an ovarian follicular wave (Garverick, 1999). An injection 84

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85 of GnRH at random during the estrous cycle of cows without ovarian cysts will either cause ovulation or luteinization of large follicles present in the ovary and synchronize the recruitment of a new follicular wave (Thatcher et al., 1989; Macmillan and Thatcher, 1991). Gonadotropin releasing hormone can advance the follicular wave by increasing the rate of atresia in cows without ovarian cysts (Thatcher et al., 1989; Macmillan and Thatcher, 1991). In cows without ovarian cysts, the initiation of a protocol for synchronization of ovulation and timed insemination (Ovsynch) at a specific stage of the estrous cycle can influence the reproductive responses to this protocol. The stage of the estrous cycle that appears to be the most appropriate for increased pregnancy rates using this protocol is the early luteal phase of the estrous cycle (Vasconcelos et al., 1999; Moreira et al., 2000a). The hypothesis of this study was that GnRH administered to cows with ovarian cysts at the time of diagnosis will induce an early luteal phase of the estrous cycle which will be conducive to an increased pregnancy rate to a protocol for synchronization of ovulation and timed insemination. The objectives of this study were: i) to compare the ovarian response of cows with ovarian cysts to no treatment and ovarian response 7 days after treatment with GnRH; and, ii) to determine the pregnancy rate of cows with ovarian cysts subjected to Ovsynch and pretreatment with GnRH 7 days prior to the initiation of the Ovsynch protocol. Materials and Methods The study was conducted during the period of January 2002 to May 2003 in a dairy herd (approximately 700 milking cows) in north-east Florida. Cows were milked three times per day and were kept in covered, open-sided barns between milking. They were

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86 fed a total mixed ration. The herd was visited weekly, and all reproductive health and management records were computerized. At the time of diagnosis, cows with ovarian cysts were sequentially allocated to one of three groups (Day 0). The diagnosis of ovarian cysts was based on palpation of the ovaries and uterus per rectum, and by ultrasonographic examination of the ovaries. The criteria used on rectal palpation were the presence of multiple follicles on the ovary with at least one follicle being 17 mm diameter (Hatler et al., 2003), the absence of a corpus luteum (CL) on either ovary, and the lack of tonicity of the uterus (Archbald et al., 1991; Bartolome et al., 2000; Bartolome et al., 2002). On ultrasonographic examination of the ovaries, ovarian cysts were recognized by the hypoechogenicity of the structure, and the absence of a CL on either ovary (Pierson and Ginther, 1984). The size of ovarian cysts was determined using ultrasonography and the recorded size was based on the largest diameter observed on ultrasonography. Cows in Group 1 were treated with GnRH (100 g, im; Cystorelin, Rhode Merieux Inc., Athens, GA) on Day 0, and Day 7, PGF2 (25 mg, im; Lutalyse, Pharmacia Upjohn, Kalamazoo, MI) on Day 14, GnRH (100 ug, im) on Day 16, and timed inseminated 16-20 h later. Cows in Group 2 were treated with GnRH (100 ug, im) on Day 0, PGF2 (25 mg, im) on Day 7, GnRH (100 ug, im) on Day 9, and timed inseminated 16-20 h later. Cows in Group 3 were not treated with GnRH on Day 0, but were treated with GnRH (100 ug, im) on Day 7, PGF2 (25 mg, im) on Day 14, GnRH (100 ug, im) on Day 16, and timed inseminated 16-20 h later (Figure 3-1). Pregnancy was determined by rectal palpation of the uterus between 45-50 d after timed insemination using previously described techniques (Zemjanis, 1970).

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87 Group 1 (GnRH + Ovsynch)Group 2 (Ovsynch)Group 1 (NoTx+ Ovsynch) Day 0 7 14 16 17/18 62-68 Day 0 7 9 10/11 54-61 Day 0 7 14 16 17/18 62-68 GnRH GnRH PGF2GnRH TAI Pregnancy DiagnosisGnRH PGF2GnRH TAI Pregnancy DiagnosisNoTx GnRH PGF2GnRH TAI Pregnancy Diagnosis Figure 3-1. Experiment 1 Experimental Design On both Days 0 and 7, cows in all groups were subjected to ultrasonographic examination of the ovaries. On both Day 0 and Day 7, blood samples for progesterone (P4) concentration were obtained from all cows and placed on ice immediately after collection. Samples were centrifuged at 5000 x g for 15 minutes, and serum was stored at -20C until assayed for progesterone using a solid-phase, no-extraction RIA previously described (Srikandakumar et al., 1986). Serum progesterone concentration on Day 0 was classified as high (> 0.5 ng/ml) or low ( 0.5 ng/ml). A serum progesterone concentration >1 ng/ml on Day 7 was used to confirm the presence of a functional corpus luteum. Statistical Analysis Baseline data for parity, DIM, time of year and P4 on Day 0 were compared using Chi-square (P<0.05). The outcomes of interest for this experiment were ovarian response 7 days after treatment with GnRH, and pregnancy rate. Data for ovarian response on Day

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88 7 were analyzed using logistic regression (Proc Logistic, SAS; SAS 9.0, 2002) adjusting for days in milk (DIM), time of year (October to February, and March to September), parity (1, 2, 3+) and P4 on Day 0. Data for pregnancy rate were analyzed using logistic regression (SAS 9.0, 2002), adjusting for DIM, time of year, parity, P4 on Day 0 and Day 7, and ovarian response on Day 7. The explanatory variables were evaluated using the backward elimination procedure and variables that significantly affected the outcomes remained in the model (Agresti, 1996). The level of significance was set at P 0.05. Results A total of 176 cows were diagnosed with ovarian cysts and enrolled on Day 0. Fifteen cows failed to complete assigned treatment or were culled prior to completion of the study, and six cows were inseminated to the wrong date and were removed from the study. A total of 155 cows completed the study; Group 1 (n=55), Group 2 (n=49), Group 3 (n=51). The following statistical analysis is based on the 155 cows that completed the study. The baseline comparison for parity, time of year, day in milk, and P4 on Day 0 is shown in Table 3-1. On Day 0, data for P4 were present for 150/155 (96.8%) cows enrolled in the study. There was no significant difference (P > 0.12) in any of the variables among the groups. On Day 7, data for ultrasonographic examination of the ovaries were available for 153/155 (98.7%) cows enrolled in this study. The percentage of cows in Group 3 (4%) with a CL on Day 7 was significantly less (P < 0.0001) than that of cows in Group 2 (72.9%) and Group 1 (50.9%). On Day 7, P4 was present for 145/155 (93.5%) cows enrolled in this study. The percentage of cows in Group 3 (40.8%) with a P4 > 1.0 ng/ml

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89 on Day 7 was significantly less (P < 0.03) than that of cows in Group 2 (63.6%) and Group 1 (63.5%). The adjusted odds ratios (AOR) and 95% confidence interval (CI) for the risk of the presence of a CL on Day 7 in lactating dairy cows with ovarian cysts treated with GnRH on Day 0 are shown in Table 3-2. Cows in Group 1 and Group 2 (treated with GnRH on Day 0) were more likely to have a CL on the ovary 7 days later (AOR: 21.30; 95% CI: 4.67-97.04; P = 0.04; AOR: 61.92; 95% CI: 13.11-292.41; P < 0.0001, respectively) compared to cows in Group 3 (non-treated cows). There was no effect of DIM, time of year, parity and P4 on Day 0 for this outcome. Table 3-1. Baseline data for heifers and cows that were diagnosed with ovarian cysts and successfully completed Experiment 1 for parity (1, 2, 3+), time of year (March-September/October-February), days in milk (DIM) and progesterone concentration (< 0.5 ng/ml / 0.5 ng/ml ) on Day 0. Group 1 Group 2 Group 3 P-value Variable n % n % n % Parity 0.85 1 17/55 30.9 13/49 26.5 13/51 25.5 2 22/55 40.0 23/49 47.0 26/51 51.0 3+ 16/55 29.1 13/49 26.5 12/51 23.5 Time of year 0.17 March-September 34/55 61.8 35/49 71.4 40/51 78.4 October-February 21/55 38.2 14/49 28.6 11/51 21.6 Days in Milk 0.42 88 16/55 29.1 13/49 26.5 9/51 17.7 89-134 12/55 21.8 10/49 20.4 17/51 33.3 135-223 16/55 29.1 10/49 20.4 12/51 23.5 >224 11/55 20.0 16/49 32.7 13/51 25.5 P4 on Day 0 0.12 < 0.5 ng/ml 34/52 65.4 33/49 67.4 24/49 49.0 0.5 ng/ml 18/52 34.6 16/49 32.6 25/49 51.0

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90 Table 3-2. Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the risk of finding a CL on Day 7 in cows with ovarian cysts treated with GnRH on Day 0. Variable CL AOR 95% CI P value n % Group 1 28/55 50.9 21.30 4.67-97.04 0.04 2 35/48 72.9 61.92 13.11-292.41 < 0.0001 3 2/50 4.0 1.00 Referent NA The adjusted odds ratios (AOR) and 95% confidence interval (CI) for the risk of having a progesterone concentration greater than 1.0 ng/ml on Day 7 in lactating dairy cows with ovarian cysts treated with GnRH on Day 0 are shown in Table 3-3. Cows in Group 1 and Group 2 (treated with GnRH on Day 0) were more likely to have P4 > 1.0 ng/ml 7 days later compared to cows in Group 3 (non-treated cows). Cows with P4 < 0.5 ng/ml on Day 0 were more likely to have P4 1.0 ng/ml on Day 7 regardless of treatment. There was no association between DIM, time of year and parity with P4 concentration on Day 7. Table 3-3. Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the risk of finding progesterone concentration > 1.0 ng/ml on Day 7 in cows with ovarian cysts treated with GnRH on Day 0. Variable High (>1.0 ng/ml) AOR 95% CI P value n % Group 1 33/52 63.5 3.52 1.43 8.71 0.12 2 28/44 63.6 3.74 1.46 9.59 0.09 3 20/49 40.8 1.00 Referent NA P4 on Day 0 < 0.5 ng/ml 38/86 44.2 0.22 0.10 0.48 0.0002 0.5 ng/ml 41/56 73.2 1.00 Referent NA

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91 Data for risk of pregnancy were available for 127/155 (81.9%) cows that successfully completed the study. The risk of pregnancy at 45 to 50 days after timed insemination for cows in each group is shown in Table 3-4. There was no difference in the risk of pregnancy at 45 to 50 days between the groups. The adjusted odds ratios (AOR) and 95% confidence interval (CI) for the risk of pregnancy in lactating dairy cows with ovarian cysts treated with GnRH on Day 0 are shown in Table 3-4. Cows with ovarian cysts were 0.20 times less likely to become pregnant if inseminated between the periods of March to September compared to cows inseminated during the period of October to February, regardless of treatment. Cows with a CL on Day 7 were more likely to become pregnant compared to cows without a CL on Day 7. There was no effect of group, progesterone concentrations on Day 0 and Day 7 on risk of pregnancy. Table 3-4. Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the risk of pregnancy in lactating dairy cows with ovarian cysts treated with GnRH on Day 0. Variable Pregnancy AOR 95% CI P value n % Group 1 8/43 18.6 1.41 0.25 8.33 0.77 2 8/41 19.5 1.43 0.21 10.00 0.82 3 3/43 7.0 1.00 Referent NA Season March-September 8/89 9.0 0.20 0.06 0.61 0.005 October-February 11/38 28.9 1.00 Referent NA Ovarian Response Follicle 5/72 7.3 0.19 0.04 0.86 0.03 CL 14/53 26.4 1.00 Referent NA

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92 Discussion The hypothesis of this study was that GnRH administered to cows with ovarian cysts at the time of diagnosis will induce an early diestrual stage of the estrous cycle which will be conducive to an increased pregnancy rate to a protocol for synchronization of ovulation and timed insemination. This hypothesis was based on previous research in cows without ovarian cysts, which showed that cows in early diestrus at the time of initiation of the Ovsynch protocol had a higher pregnancy rate compared to cows subjected to this protocol at other stages of the estrous cycle (Vasconcelos et al., 1999; Moreira et al., 2000). However, the results of this study did not fully substantiate this hypothesis. It appeared that while pretreatment with GnRH induced an early diestrual stage of the estrous cycle (cows in Group 1), initiation of the Ovsynch protocol at this time (early diestrus) did not increase pregnancy rate compared to cows which were subjected to this protocol at the time of diagnosis (cows in Group 2). Our interpretation of these results is that the progesterone concentration in early diestrus when the Ovsynch protocol was initiated may not be an important factor in determining the response to GnRH 7 days later. This interpretation is supported by another significant finding in this study, which indicated that the level of progesterone at the time of initiation of the Ovsynch protocol (Day 0; cows in Group 2) was not associated with the presence of CL on the ovaries on Day 7. It is speculated that the presence of an ovarian follicle that can respond to the effect of the GnRH-induced LH surge may be more important than the concentration of progesterone at the time of initiation of the Ovsynch protocol. The presence of ovarian follicular waves has been shown to occur in cows with ovarian cysts (Garverick, 1999).

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93 However, it is impossible to determine the stage of these ovarian follicular waves when the diagnosis of ovarian cysts is made, and treatment with GnRH is initiated in these cows. Therefore, it may simply be a matter of chance that treatment with GnRH at the time of diagnosis coincides with the presence of an ovarian follicle, which can respond to the effects of the GnRH-induced LH surge. A similar situation may exist when GnRH is administered to initiate the Ovsynch protocol in cows without ovarian cysts. It is interesting to note that not all cows responded, as determined by the presence of a CL 7 days later, to the initial treatment with GnRH. In fact, this response was in the range of 50.9 to 70.9%. These values fall within the range of heifers and cows that ovulated to the first GnRH of the Ovsynch protocol (heifers: 54.2%; cows: 90%; Pursley et al., 1995). In the present study, pre-treatment with GnRH 7 days prior to initiation of the Ovsynch protocol did not increase the risk of pregnancy. The risk of pregnancy achieved in Group 1 (cows pre-treated with GnRH) was 18.6% compared to Group2 (Ovsynch) 19.5% and Group 3 (no treatment and Ovsynch started 7 d following diagnosis) 7.0%. Combining Groups 2 and 3, the risk of pregnancy is 13.25%. It is tempting to simply accept these percentages. However, due to the low power of this study, it is more likely to make a Type II error; a higher chance of saying that there is no difference between the treatments when there is. The risk of pregnancy for Group 2 and 3 in this study is lower that those obtained by Bartolome et al. (2000; 23.6% in cystic cows subjected to Ovsynch). However, the risk of pregnancy in Group 1 (18.6%), cystic cows pretreated with GnRH is similar to a conception rate of 15.1% obtained with a larger sample size (Bartolome et al., 2003) and higher than the 9% obtained by Gmen et al. (2003).

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94 In the present study, cows with ovarian cysts treated with GnRH on Day 0 were more likely to have a CL 7 days later compared to untreated cows. This response was independent of the progesterone concentration on the day of diagnosis, days in milk, parity and time of year. The presence of a CL on Day 7 appeared to have a beneficial effect on fertility of these cows following the subsequent use of the Ovsynch protocol. In fact, cows with a CL on Day 7 were more likely to become pregnant compared to those without a CL on Day 7. Nevertheless, treatment with GnRH on Day 0 did not have any effect on the risk of pregnancy, even though it had a positive effect on the presence of a CL on Day 7. Therefore, it is tempting to suggest that ovulation occurring without the use of exogenous GnRH (spontaneous ovulation) may also be an important factor affecting fertility in cows with ovarian cysts, subsequently subjected to the Ovsynch protocol. In support of this statement, it should be noted that spontaneous ovulation occurred in untreated cows with ovarian cysts (Group 3) since 2/50 (4%) possessed a CL on Day 7, and 3/43 (7%) of these cows in Group 3 conceived following subsequent treatment with the Ovsynch protocol. This is in agreement with previous research (Bierschwal et al 1975), which showed that spontaneous ovulation occurred in 6/28 (21.4%) untreated cows with naturally-occurring ovarian cysts 14 days after diagnosis, and 4/6 (66.7%) of these cows conceived to artificial insemination at estrus. Also, it has been reported that 1/8 (12.5%) untreated cows with experimentally-induced ovarian cysts possessed a CL 10 days after charcoal marking of these cysts (Cook et al., 1990). However, pregnancy was not an outcome in that study.

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95 In the present study, since the rate of spontaneous ovulation was relatively low compared to that obtained through the use of exogenous GnRH, it would seem clinically prudent to administer GnRH at the time of diagnosis of this condition. In this way, it is more likely that most cows with ovarian cysts will respond with a CL on the ovaries 7 days later. In fact, the authors suggest that cows with ovarian cysts be treated with exogenous GnRH on the day of diagnosis, and that ultrasonographic examination of the ovaries be performed 7 days later. If there is a CL on the ovaries at this time, these cows should be treated with a luteolytic dosage of PGF2, GnRH 2 d later, and timed inseminated 16-20 h after treatment with GnRH. However, if there is no CL on the ovaries on Day 7, these cows should be judiciously re-treated with GnRH until the presence of a CL is evident. The results of this study showed that cows with ovarian cysts treated with GnRH at the time of diagnosis (Day 0) were more likely to have a P4 concentration greater than 1 ng/ml on Day 7. However, cows with P4 0.5 ng/ml on Day 0 were more likely to have a P4 concentration > 1 ng/ml on Day 7. This was observed in cows in all groups without regard to treatment. This phenomenon could probably be explained by the occurrence of spontaneous ovulation, and the presence of luteinized, anovulatory follicles in cows in Group 3 (non-treated), and the response to exogenously administered GnRH to cows in Group 1 and Group 2. It was also observed that cows with a P4 concentration < 0.5 ng/ml on Day 0 were more likely to have a P4 concentration on Day 7 1.0 ng/ml. This could probably be explained by lack of response to GnRH by these cows. Although P4 concentrations in cows in Group 1 and Group 2 were significantly higher (P4 > 1 ng/ml) than that observed in cows in Group 3, pregnancy rate was not

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96 affected by P4 concentrations on Day 7 in cows in all groups. This was in contrast to the observation that the presence of a CL on Day 7 (detected by ultrasonography) positively influenced subsequent pregnancy following timed insemination of cows in all groups. It is assumed that the presence of a mature CL on the ovary 7 days after the initiation of the Ovsynch protocol is an important component in the success of this protocol since this would determine the effectiveness of PGF2 administered at this time. Therefore, a possible explanation for the apparent discrepancy between the presence of a CL and P4 concentrations on Day 7 with respect to pregnancy may be a reflection of the presence of luteinized anovulatory follicles, developing corpora hemorrhagica, and mature CL on the ovaries at this time. It is, therefore, speculated that these luteinized anovulatory follicles and developing corpora hemorrhagica were not as responsive to the luteolytic dosage of PGF2 administered on Day 7 as would the mature CL present at this time. This speculation is supported by previous research (Nanda et al., 1988) which demonstrated that luteinized follicular cysts showed a poor response to PGF2 administered 7 days after treatment with GnRH, and that developing corpora lutea (corpora hemorrhagica) are unresponsive to the luteolytic effects of PGF2 (Lauderdale, 1975). A valid criticism of this study revolves around the ability and accuracy of the investigators to make a diagnosis of ovarian cysts using per rectum palpation of the ovaries on one occasion. The criteria used to determine the presence of ovarian cysts were the presence of multiple follicles on one or both ovaries, the absence of a CL on the ovaries, and the lack of uterine tonicity. The palpable characteristics used in this study to identify a CL have been previously described (Zemjanis, 1970). In the opinion of the

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97 authors, the criteria used to identify ovarian cysts using per rectum palpation of the ovaries and uterus can be justified, since a CL is not present in cows with ovarian cysts (because of a lack of ovulation), and the uterine tone that accompanies a functional follicle at the time of estrus is also not present in this condition. In addition, the absence of a CL was substantiated using ovarian ultrasonography and concurrent determination of plasma progesterone concentrations. From the results of this study, it was concluded that administering GnRH to cows with ovarian cysts 7 days prior to the initiation of the Ovsynch protocol increased the proportion of cows with a CL on Day 7 but did not increase pregnancy rate. However, independent of treatment, the presence of a CL on Day 7 had a beneficial effect on pregnancy rate.

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CHAPTER 4 EXPERIMENT 2: EFFECT OF SEQUENTIAL ADMINISTRATION OF PGF2 ON THE PREVALENCE OF MUCOPURULENT DISCHARGE, SIZE OF THE CERVIX, SIZE OF THE PREVIOUSLY PREGNANT UTERINE HORN AND FIRST SERVICE PREGNANCY RATE IN LACTATING DAIRY COWS Introduction It has been reported that the uterus of all postpartum dairy cows is invaded by opportunistic bacteria within the first 21 days post partum (Elliot et al., 1968). While most cows spontaneously eliminate these bacteria from the uterus, some do not, and the presence of these bacteria in the uterus could predispose these cows to periods of subsequent infertility. It is not known why the uterus of some postpartum dairy cows resist infection by these opportunistic bacteria. It has been shown that the ovarian sex hormones (estrogen and progesterone) could modulate the ability of the uterus to respond to bacterial invasion. In fact, estrogen has been shown to have a protective effect while progesterone has been shown to make the uterus more susceptible to infection (Rowson et al., 1953). There are relatively high blood concentrations of estrogen at parturition and during the immediate postpartum period. However, little is known about the estrogen concentrations at the level of the uterus at this time. The first dominant ovarian follicle postpartum is usually formed on the ovary opposite to the previously pregnant uterine horn (contralateral ovary; Foote et al, 1968). With ovulation of this dominant follicle, usually between 10-12 days post partum, the uterus becomes subjected to physiologic concentrations of progesterone. In the presence of any intrauterine fluid or subclinical endometritis at this time, there is the possibility of 98

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99 exacerbating the subclinical endometritis and this could lead to development of subsequent pyometra. In fact, it has been shown that treatment with GnRH in the immediate postpartum period could exacerbate a subclinical endometritis (Etherington et al., 1984). Therefore, while progesterone may be needed for proper uterine function, it would appear that there is the potential for normal or prolonged exposure of the uterus to progesterone to have a detrimental effect on the uterus at this time. It has been reported that the presence of a large follicle in the ovary on the same side as the previously pregnant uterine horn (ipsilateral ovary) by 9 days post partum is a marker of subsequent improved fertility (Sheldon et al., 2000a). In addition, it has been suggested that elimination of bacterial contamination of the postpartum uterus may cause the selection of a dominant follicle in the ipsilateral ovary (Sheldon et al., 2003) in the early postpartum period. There are conflicting reports on the effectiveness of exogenously administered prostaglandin F2 alpha (PGF2) to increase the rate of uterine involution, cause evacuation of bacterial contamination from the uterus, and subsequently improve conception rate (Young et al., 1984; Etherington et al., 1988; Archbald et al., 1990; Risco et al., 1994). These reports indicate the use of PGF2 on either one or 2 occasions on random days postpartum without regard to the presence or absence of a functional corpus luteum (CL). Nevertheless, it has been suggested that exogenously administered PGF2 in the early postpartum period could have a direct beneficial effect on the uterus of cows that calved normally (Lindell et al., 1982) or abnormally (Risco et al., 1994), and this effect could occur in the absence of a CL.

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100 However, we speculate that exogenous PGF2 would be more consistently effective in causing evacuation of bacterial contamination of the postpartum uterus if administered when there is a CL on the ovary. In most postpartum dairy cows, this would be approximately 20-24 days post partum since ovulation usually occurs within 10-12 days post partum. It is further speculated that consecutively lysing the CL with exogenous PGF2 at specific times post partum (sequential luteolysis) will result in exposing the uterine environment to normal concentration of progesterone for a reduced period of time. This reduced exposure of the uterine environment to normal concentration of progesterone in the early postpartum period could be beneficial to uterine health since progesterone has been shown to increase the susceptibility of the uterus to infection (Rowson et al., 1953). However, even though some cows may not have a CL on the ovary at the time of administration of PGF2, it is speculated that a beneficial effect of exogenous PGF2 would occur through its direct effect on the uterus (Lindell et al., 1982; Risco et al., 1994). Recent research (Lewis, 2003) in the ewe has suggested that a method for increasing uterine production of PGF2 could enhance the immune function of the uterus and its ability to resist uterine infections. However, the mechanism(s) by which this could be accomplished in the postpartum dairy cow are presently unknown. It has been shown that luteal oxytocin can stimulate the synthesis and release of endogenous uterine PGF2 at the time of luteolysis (Silvia et al., 1989). Nevertheless, it appeared that while administration of exogenous oxytocin was unable to compliment the effect of uterine PGF2 during diestrus, exogenous administration of PGF2 appeared to enhance the effect of endogenous PGF2 at this time (Archbald et al., 1994).

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101 LeBlanc et al. (2002) defined clinical endometritis based on its impact on pregnancy rate. When vaginoscopy was performed clinical endometritis was defined as the presence of a purulent, foul-smelling or fetid discharge, or cervical diameter >7.5 cm between 20 and 33 DIM, or as a mucopurulent discharge after 26 DIM. In the event that vaginoscopic examination was not performed, the presence of mucopurulent or purulent discharge on the perineum, cervical diameter >7.5 cm and the presence of a uterine horn 8 cm in diameter were used to define clinical endometritis. The Ovsynch protocol introduced by Pursley et al. (1995) uses a combination of GnRH and PGF2 to synchronize ovulation such that insemination may occur at a fixed time. Momcilovic et al. (1998) showed that the reproductive performance of lactating dairy cows was better when time-inseminated without the need for estrus detection was used compared with estrus detection and insemination at estrus. In the Ovsynch protocol GnRH is administered at a random stage of the estrous cycle followed seven days later by PGF2. A second GnRH injection is administered 2 days after PGF2 and insemination occurs 16 to 20 hours following the second GnRH. However, it was noted that different pregnancy rates were achieved when the first GnRH was administered at different stages of the cycle. Reduced pregnancy rates occurred when GnRH was administered between Days 1 to 4 and 13 to 17 of the estrus cycle (Vasconcelos et al., 1999; Moreira et al., 2000a). Pre-synchronization (Presynch) with two injection of PGF2 14 days apart has been shown to increase the pregnancy rate to the Ovsynch protocol. The Presynch-Ovsynch protocol, in which PGF2 is given 14 days apart and the Ovsynch protocol initiated 12 days after the second injection of PGF2 has been shown to increase

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102 pregnancy rate by 18 percentage units (25% to 43%; Moreira et al., 2000a). This increase in the pregnancy rate was attributed to starting the Ovsynch protocol during the early luteal phase, Days 5-11 of the estrous cycle. The hypothesis of Experiment 2 was that sequential administration of PGF2 in the immediate postpartum period would decrease the prevalence of mucopurulent discharge, size of the cervix and previously pregnant uterine horn (PPH) and increase first-service conception rate in postpartum dairy cows. The objective of Experiment 2, Part A was to determine the effect of sequential administration of PGF2 in the immediate postpartum period on the prevalence of mucopurulent discharge, size of the cervix and previously pregnant uterine horn (PPH) and first-service pregnancy rate in postpartum dairy cows. The objective of Experiment 2, Part B was to evaluate the effect of sequential administration of PGF2 in the immediate postpartum period on first-service conception rate in postpartum dairy cows subjected to a timed insemination protocol consisting of Presynch-Ovsynch protocol. Experiment 2 Part A Materials and Methods The study was conducted during the period June to September 2003 in a commercial dairy herd of approximately 3,000 milking cows in north central Florida. These cows were milked 3 times per day and were kept in shaded areas between milking. They were fed a total mixed ration to meet or exceed the recommendations of National Research Council (National Research Council, 2001). The herd was visited weekly and all reproductive health and management records were computerized.

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103 The prevalence of mucopurulent discharge by 50 days post partum in this herd was 20%. Mucopurulent discharge was detected by visual inspection of the perineum, vulva and tail. This was later confirmed by transrectal palpation of the reproductive tract with expulsion of purulent material from the vaginal canal and/or presence of fluid in the uterine horn(s). It was anticipated that the proposed treatment would reduce this prevalence to approximately 8%. A total of 102 cows per group provided a 95% confidence and 80% power to declare a difference in the prevalence of mucopurulent discharge between 20% and 8% as statistically significant. A total of 228 cows was enrolled in this study. Cows were between 7 to 9 days post partum (pp). On the day of initiation of the study, cows were allotted to two groups using sequential randomization. All cows entered the study between the months of June and August 2003. For convenience, the day of initiation of the study was designated as Day 8 post partum. Group 1 (n=114) formed the treated group and Group 2 (n=114) formed the control group. In Group 1, 45/114 (39.5%) were enrolled on Day 7 post partum, 31/114 (27.2%) were enrolled on Day 8 post partum and 38/114 (33.3%) were enrolled on Day 9 post partum. Similarly in Group 2, 38/114 (33.3%) were enrolled on Day 7 post partum, 44/114 (38.6%) were enrolled on Day 8 post partum and 32/114 (28.1%) were enrolled on Day 9 post partum (Table 4-1). In addition, information concerning parity, dystocia (yes/no) and retained fetal membranes (yes/no) were recorded. Cows enrolled in the study were grouped into primiparous (first lactation) and multiparous (second lactation and higher; Table 4-1).

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104 Dystocia was defined as heifers or cows requiring medium to heavy assistance to deliver their calf (two or more persons assisting for more than 15 minutes). Eighty-nine percent (203/228) of animals enrolled in the study required no assistance and were classified as calving normally with 106/114 (93.0%) and 97/114 (85.1%) in Group 1 and Group 2 respectively. A total of 25 required assistance and was classified as experiencing dystocia. Cows that underwent a Cesarean section or fetotomy were excluded from the study. Retained fetal membranes (RFM) were defined as the failure to expel the placenta within 24 hours of parturition. In Group 1 and Group 2, 32/114 (28.1%) and 25/114 (21.9%) had RFM respectively (Table 4-1). Treatment Cows in Group 1 (n=114) were treated with two luteolytic dosage of PGF2 (25 mg intramuscularly: im) 8 hours apart on Days 8 and 15 post partum, and one luteolytic dosage of PGF2 (25 mg, im) once on Days 22 and 36 post partum. Cows in Group 2 (n=114) were not treated with PGF2 (25 mg, im) on Days 8, 15, 22 and 36 post partum and served as untreated controls (Figure 4-1). At the termination of the treatment protocol cows followed the normal herd reproductive management practices. At the time reproductive management consisted of a voluntary waiting period of 100 days and detection of estrus using visual observation and a computerized system that utilized increased walking activity and an accompanied decrease in milk production (Afimilk, S.A.E. Afikim, Israel). A specific individual was assigned the duty of observing these cows for estrus during each 8-hour shift. Cows that failed to show heat by 100 days post partum were subjected to the Ovsynch protocol.

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105 Group 1 (PGF2; n=114)Group 2 (Control; n=114) Day 8 pp 15 pp 22 pp 36 pp 58 pp PGF2PGF2Vaginoscopy IVaginoscopy II 2 X PGF2; 8 hours apartDay 8 pp 15 pp 22 pp 36 pp 58 pp Vaginoscopy IVaginoscopy II Pregnancy was determined using rectal palpation 42-49 days after artificial insemination by on staff veterinarians. Figure 4-1. Experimental 2 Part A: Experimental Design. Exclusion Criteria Animals were excluded or withdrawn from the study if they had any of the following: failed to complete the treatment scheme, were not examined at Day 22 post partum, were in the hospital barn and received systemic antibiotic therapy during the study period, underwent surgery (such as correction of displaced abomasum, Cesarean section, fetotomy), controls treated with PGF2 during the study period, and culled from the herd or died during the study period. Blood Sampling and Hormone Assay Blood samples were collected on Days 8, 15, 22, 36 and 50 post partum, to measure progesterone (P4) concentrations. Blood samples were collected from the coccygeal vessels by venipuncture into 10 ml vacutainers without any anticoagulant and

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106 placed on ice immediately after collection. Samples were centrifuged at 5000 x g for 15 minutes, and serum was stored at -20C until assayed for progesterone. Table 4-1. Distribution of cows enrolled in the study between June and August 2003 within Groups 1 and 2 based on days in milk (DIM), parity (primiparous/multiparous), dystocia (yes/no) and retained fetal membranes (yes/no). VARIABLE GROUP 1 (Treated) GROUP 2 (Control) n % N % DIM 7 45/114 39.5 38/114 33.3 8 31/114 27.2 44/114 38.6 9 38/114 33.3 32/114 28.1 Parity Primiparous 36/114 31.6 33/114 28.9 Multiparous 78/114 68.2 81/114 71.1 Dystocia Yes 8/114 7.0 17/114 14.9 No 106/114 93.0 97/114 85.1 Retained Fetal Membranes No 82/114 71.9 89/114 78.1 Yes 32/114 28.1 25/114 21.9 Serum progesterone (P4) concentrations were determined using a Coat-A-Count Kit (DPC Diagnostic Products Incorporation, CA, USA) using solid-phase, no-extraction radioimmunoassay previously described (Srikandakumar et al., 1986). A standard curve was prepared using plain uncoated polypropylene tubes for total counts and non-specific binding and coated tubes. A volume of 100 l of calibrators was added to each tube in increasing concentrations of 0.1, 0.25, 0.5, 1, 2, 5, 10 and 20 ng/ml. Reference samples, ovariectomized, low and high (6.0 -7.0 ng/ml representative of luteal phase P4 concentrations) were also used. The calibrators and reference samples were

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107 performed in duplicate. A 100 l serum sample was added to coated tubes and 1 ml of 125I-labelled P4. Every sixth sample was done in duplicate. The tubes were incubated at room temperature for 3 hours, decanted, dried for 15 minutes and counted for 1minute in a gamma counter. The gamma count for each tube was converted using the calibration curve to give the P4 concentration of the serum samples. Vaginoscopy On Days 22 and 58 post partum, vaginoscopy was performed to determine the presence or absence of a mucopurulent vaginal discharge. The vulva and surrounding area were washed with water to remove fecal matter. Next the vulva was washed with iodine and water and dried with a paper towel. Lubricant was applied to a sterile disposable foil-lined cardboard vaginal speculum. The speculum was inserted into the vagina up to the level of the external cervical os and a penlight was used to visualize the vaginal walls, cervix and content of the vagina. The cervix was evaluated to be either open or closed. Vaginal discharge was classified as normal, clear, mucoid (clear mucus), mucopurulent (presence of flecks of pus and mucus to purulent thick creamy to cheesy exudates) or serous mucopurulent (pus, red-brown and/or foul smelling). The quantity of the discharge was also classified as slight, moderate or copious. The color of the vaginal wall and the external os of the cervix was evaluated and classified as pink, red and red and inflamed. These finding were recorded (Appendix A). Transrectal Palpation of the Reproductive Tract Prior to vaginoscopy, transrectal palpation of the reproductive tract was performed and the findings recorded (Appendix A). The data were noted as follows:

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108 Size of cervix (diameter of cervix: <20, 25, 30, 35, 40, 45, 50, >50mm) Previously pregnant horn (right/left) Size of the left horn (diameter of uterine horn: <20, 25, 30, 35, 40, 45, 50, >50mm) Size of the right horn (diameter of uterine horn: <20, 25, 30, 35, 40, 45, 50, >50mm) Fluid in right horn (absent/present) Fluid in left horn (absent/present) Structures on right and left ovaries (no significant structures (NSS), follicle and follicle size, CL present/absent) Statistical Analysis All analysis was performed with SAS, version 9.0 (2001). Baseline comparisons for parity (primiparous and multiparous), DIM, dystocia (yes/no), retained fetal membranes (yes/no) and P4 (1.0 ng/ml or > 1.0 ng/ml) on Day 8 post partum were compared using Chi-square (P0.05). LeBlanc et al. (2002) defined clinical endometritis based on its impact on pregnancy rate. When vaginoscopy was performed clinical endometritis was defined as the presence of a purulent, or foul-smelling or fetid discharge, or cervical diameter >7.5 cm between 20 and 33 DIM, or as a mucopurulent discharge after 26 DIM. In the event that vaginoscopic examination was not performed, the presence of mucopurulent or purulent discharge on the perineum, cervical diameter >7.5 cm and the presence of a uterine horn 8 cm in diameter were used to define clinical endometritis. Based on these definitions, three models were used to assess the effect of treatment on the criteria used to define clinical endometritis. Model 1 consisted of assessing the effect of treatment on the presence of purulent or foul-smelling or fetid discharge at Days 22 and 58 post partum. A purulent or foul-smelling discharge is defined here as a mucopurulent (presence of flecks of pus and mucus to purulent a thick creamy to cheesy exudates) or serous mucopurulent (pus, red-brown and/or foul

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109 smelling) discharge. Model 2 consisted of assessing the effect of treatment on the presence of purulent or foul-smelling discharge and cervix > 50 mm at Day 22 and the presence of purulent or foul discharge and cervix > 30 mm at Day 58 post partum. Model 3 consisted of assessing the effect of treatment on the presence of purulent or foul-smelling discharge, cervix > 50 mm and the presence of a uterine horn 30 mm in diameter at Day 22 and the presence of purulent or foul-smelling discharge, cervix > 30 mm and the presence of a uterine horn 30 mm in diameter at Day 58 post partum. The diameter of the cervix and uterine horn were chosen based on the median value on Day 22 and Day 58 post partum. The outcomes of interest for this experiment were the effect on treatment on Models 1, 2, and 3 on Days 22 and 58 post partum. Data for Models 1, 2, and 3 on both Day 22 and Day 58 post partum were analyzed using logistic regression (Proc Logistic, SAS 9.0) adjusting for parity (primiparous/multiparous), dystocia (yes/no) and RFM (yes/no) and a value of P 0.05 was considered statistically significant. The association between treatment and first service conception rate was evaluated using logistic regression (Full Model; Proc Logistic, SAS 9.0) adjusting for group, parity (primiparous/multiparous), dystocia (yes/no), RFM (yes/no), and days to first service (130 days/>130 days). A value of P 0.05 was considered statistically significant. Experiment 2 Part B This study was performed between October 2003 and March 2004 in a the same dairy herd as Experiment 1. The conception rate to timed insemination in this dairy herd was approximately 30%. It was anticipated that the proposed treatment would result in a 15% increase in conception rate to approximately 45%. A total of 127 cows per group

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110 will provide 95% confidence and 80% power to declare a difference in conception rates between 30% and 45% statistically significant. A total of 418 cows was used in this study, and cows were enrolled at 7 days post partum. At this time, 2 experimental groups were formed using sequential randomization. In addition, information concerning retained fetal membranes (yes/no), dystocia (yes/no) and parity (2, 3+) were recorded. No primiparous or first-calf heifers were enrolled in the study based on management discussions. Treatment Cows in Group 1 (n=209) were treated with two luteolytic dosage of PGF2 (25 mg intramuscularly: im) 8 hours apart on Days 7 and 14 post partum, and one luteolytic dosage of PGF2 (25 mg, im) once on Days 21 and 35 post partum. On Days 49 and 63 post partum, cows were treated with one luteolytic dosage of PGF2 (25 mg, im; Presynch). Cows were subjected to the Ovsynch protocol on Day 75 post partum and time inseminated on either Days 85 or 86 post partum (Figure 4-2). Cows in Group 2 (n=209) were not be treated with PGF2 on Days 7, 14, 21 and 35 post partum and served as untreated controls. However, cows in Group 2 were treated with one luteolytic dosage of PGF2 (25 mg, im) once on both Days 49 and 63 post partum (Presynch). Cows in Group 2 were also subjected to the Ovsynch protocol starting on Day 75 post partum, 12 days following the last PGF2 injection of Presynch, and time inseminated on either Days 85 or 86 post partum (Figure 4-2).

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111 Group 1 (PGF2; n=209)Group 2 (Control; n=209) Day 7 pp 14 pp 21 pp 35 pp 49 pp 63pp 75 pp 85 pp 86/87 pp 115-118d pp PGF2PGF2OvsynchPre-Synch TAIUS for Pregnancy 2 X PGF2; 8 hours apartDay 7 pp 14 pp 21 pp 35 pp 49 pp 63 pp 75 pp 85 pp 86/87 pp 115-118d pp Figure 4-2: Experiment 2Part B: Experimental Design Exclusion Criteria Animals were excluded or withdrawn from the study for any of the following reasons; failure to complete the treatment scheme; animals with RFM or dystocia which later experienced metritis and were aggressively treated with penicillin, cephalosporin and/or PGF2; animals that underwent surgery (correction of displaced abomasum); animals placed in the hospital barn that received systemic antibiotic therapy during the study period; animals assigned as controls treated with PGF2 during the study period; animals culled from the herd or which died during the study period. Pregnancy Diagnosis Pregnancy was determined using ultrasonography (ALOKA 500 with a 5 MHz probe) 29-32 days after timed insemination of cows in both groups. The presence of intrauterine fluid and a viable embryo were the criterion used to evaluate pregnancy status. The presence or absence of a heartbeat was the criterion used to determine viability of the embryo.

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112 Statistical Analysis Baseline comparison for parity (2, 3+), retained fetal membranes (yes/no), dystocia (yes/no), and abnormal calving (dystocia and/or RFM) was carried out to establish comparability of the groups using Chi-Square test. A value of P0.05 was considered statistically significant. The outcome of interest was conception to first service. The effect of treatment (group) on conception to first service was evaluated using both Chi-Square and logistic regression (Proc Logistic, SAS 9.0). Included in the model were parity (2, 3+), retained fetal membranes (yes/no) and dystocia (yes/no). A value of P0.05 was considered statistically significant. Results Experiment 2 Part A Day 8 post partum A total of 203 cows successfully completed the study with distribution as follows: Group 1, 101/114 (88.6%) and Group 2 102/114 (89.5%). The baseline comparison for parity (primiparous/multiparous), days in milk (DIM), dystocia (no/yes), retained fetal membranes (no/yes) and P4 concentration ( 1.0 ng/ml / >1.0 ng/ml) at 8 days post partum are shown in Table 4-2. Day 16 post partum Blood was obtained and analyzed for P4 for 97/101 (96.0%) of cows in Group 1, and 98/102 cows (96.1%) in Group 2. Group 1 had 5/97 cows (5.2%) with P4 levels 1.0 ng/ml on Day 16 post partum, and Group 2 had 9/98 cows (9.2%) with P4 levels 1.0 ng/ml on Day 16 post partum. Progesterone levels ranged from 0.1 ng/ml to 6.26 ng/ml

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113 for cows in Group 1, and from 0.1 ng/ml to 3.11 ng/ml for cows in Group 2. The mean and standard error of the mean were: Group 1: 0.29 0.07 ng/ml; Group 2: 0.29 0.06 ng/ml, (P>0.05). Table 4-2. Baseline data for heifers and cows that successfully completed the study for parity (primiparous/multiparous), days in milk, dystocia (no/yes), retained fetal membranes (no/yes) and progesterone concentration ( 1.0 ng/ml / >1.0 ng/ml) at 8 days post partum. VARIABLE GROUP 1 (Treated) Group 2 (Control) P-Value n % n % Parity 0.61 Primiparous 32/101 31.7 29/102 28.4 Multiparous 69/101 68.3 73/102 71.6 Day in Milk 0.09 7 40/101 39.6 32/102 33.4 8 27/101 26.7 42/102 41.2 9 34/101 33.7 28/102 27.4 Dystocia 0.11 No 94/101 93.1 88/102 86.3 Yes 7/101 6.9 14/102 13.7 Retained Fetal Membranes 0.17 No 73/101 72.3 82/102 80.4 Yes 28/101 27.7 20/102 19.6 P4 on Day 8 post partum 0.56 >1.0 ng/ml 1/101 1.0 2/101 2.0 1.0 ng/ml 100/101 99.0 99/101 98.0 Day 22 post partum On Day 22 post partum, blood was taken for P4 evaluation, vaginoscopy and transrectal palpation of the reproductive tract were performed. P 4 evaluation : Blood was obtained and analyzed for P4 for 96/101 (95.0%) of cows in Group 1, and 99/102 (97.1%) in Group 2. Cows in Group 1 had 20/96 (20.8%) with P4

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114 levels 1.0 ng/ml on Day 22 post partum, and cows in Group 2 had 27/99 (27.3%) with P4 levels 1.0 ng/ml on Day 22 post partum. Progesterone levels ranged from 0.1 ng/ml to 5.47 ng/ml in cows in Group 1 and from 0.1 ng/ml to 7.94 ng/ml in cows in Group 2. The mean and standard error of the mean were: Group 1: 0.78 0.14 ng/ml; Group 2: 0.83 0.15 ng/ml, (P>0.05). Vaginoscopy : Of the 228 cows enrolled at 7-9 days post partum, one cow died (1/228; 0.4%), two cows were culled (2/228; 0.9%) from the herd, and 8 cows were placed in the hospital barn (8/228; 3.5%) for various ailments prior to vaginoscopy and transrectal palpation of the reproductive tract. At Day 22 post partum, 14 cows were not examined by vaginoscopy (14/228; 6.1%). Data for vaginoscopic examination were available for 203/228 (89.0%) of the cows enrolled in the study. However in Group 1, 99/101 cows (98.0%) and Group 2, 100/102 cows (98.0%) were completely examined and all the variables were recorded. Group 1 (Treated) : The cervix was visualized in 100/101 (99.0%) cows. In one cow, the cervix could not be seen due to the large volume of fluid in the lumen of the vagina. The cervix was open in 53/100 cows (53.0%). The vaginal wall and external os of the cervix were pink in color in 69/101 cows (68.3%), red in 24/101(23.8%), and red and inflamed in 8/101 (7.9%). In one cow, discharge type and discharge quantity were not noted. An abnormal discharge (a mucopurulent discharge with presence of flecks of pus and mucus, purulent, thick creamy to cheesy exudate or serous mucopurulent with pus, red-brown and/or foul-smelling) was seen in 57/100 cows (57.0%) with 54/100 cows (54.0%) classified as having a mucopurulent and 3/100 cows (3.0%) with a serous mucopurulent discharge.

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115 Group 2 (Control) : The cervix was visualized in 100/102 (98.0%) cows. In one cow, vaginoscopy was not performed, and in a second cow the cervix was not classified as open or closed. The cervix was open in 58/100 cows (58.0%). The vaginal wall and external os of the cervix were pink in color in 72/101 cows (71.3%), red in 25/101 (24.7%), and red and inflamed in 4/101(4.0%). An abnormal discharge was seen in 53/101 cows (52.5%) with 51/101 (50.5%) classified as having a mucopurulent and 2/101 (2.0%) with a serous mucopurulent discharge. Transrectal palpation : Data for transrectal palpation of the reproductive tract were available for 203/228 (89%) of the cows enrolled in the study. However in Group 1, 93/101 (92.1%) cows, and Group 2, 94/102 (92.2%) cows were completely examined and all the variables were recorded. Group 1 (Treated) : The size of the cervix was classified as greater than or less than or equal to the median value on Day 22 post partum. The median value was 50 mm in diameter with a range of <20 mm to >50 mm. There was a total of 38/101 cows (37.6%) with the cervix > 50 mm in diameter on rectal palpation. The previously pregnant horn (PPH) was identified as the larger of the two uterine horns. The majority of pregnancies appeared to have occurred in the right horn (67/101; 66.3%), and in 7/101 (7.0%) the horns were estimated to be the same size and were designated as non-distinguishable. The size of the PPH was classified as greater than or less than or equal to the median value. The median value on Day 22 post partum was 30 mm in diameter with a range of <20 to >50 mm in diameter. Both categories were almost evenly distributed with 47/100 (47.0%) > 30 mm in diameter. Fluid was present in the uterine horns of 25/92 cows (27.2%) and a palpable CL was identified in 37/97 cows (38.1%).

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116 Group 2 (Control) : The size of the cervix was classified as greater than or less than or equal to the median value on Day 22 post partum. The median value was 50 mm in diameter with a range of <20 mm to >50 mm. There was a total of 40/102 cows (39.2%) with the cervix > 50 mm in diameter on rectal palpation. The previously pregnant horn (PPH) was identified as the larger of the two uterine horns. The majority of pregnancies appeared to have occurred in the right horn (69/102; 67.7%), and in 9/102 cows (8.8%) the horns were estimated to be the same size and were designated as non-distinguishable. In one animal (1/102; 0.9%) there were adhesions of the uterine horns and the size could not be determined. The size of the PPH was classified as greater than or less than or equal to the median value. The median value on Day 22 post partum was 30 mm in diameter with a range of <20 to >50 mm in diameter. Both categories were almost evenly distributed with 49/101 (48.5%) > 30 mm in diameter. Fluid was present in the uterine horns of 18/90 (20.0%) cows and a palpable CL was identified in 58/97 cows (59.8%). Day 36 post partum Blood was obtained and analyzed for P4 for 94/101 (93.1%) of cows in Group 1 and 95/102 (93.1%) in Group 2. Group 1 had 36/94 cows (38.3%) with P4 levels 1.0 ng/ml, and Group 2 had 42/95 cows (44.2%) with P4 levels 1.0 ng/ml. Progesterone levels ranged from 0.1 ng/ml to 12.90 ng/ml in Group 1 and from 0.1 ng/ml to 9.22 ng/ml in Group 2. The mean and standard error of the mean were: Group 1: 1.80 0.23 ng/ml; Group 2: 1.80 0.22 ng/ml, (P>0.05).

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117 Day 58 post partum On Day 58 post partum, blood was taken for P4 evaluation, and vaginoscopy and transrectal palpation of the reproductive tract were performed. P 4 evaluation : Blood was obtained and analyzed for P4 for 92/101 (91.1%) of cows in Group 1 and 96/102 cows (94.1%) in Group 2. Group 1 had 45/92 cows (48.9%) with P4 levels 1.0 ng/ml, and Group 2 had 47/96 cows (49.0%) with P4 levels 1.0 ng/ml. Progesterone levels ranged from 0.1 ng/ml to 8.97 ng/ml in cows in Group 1, and from 0.1 ng/ml to 7.56 ng/ml in cows in Group 2. The mean and standard error of the mean were: Group 1: 2.45 0.24 ng/ml; Group 2: 1.95 0.21 ng/ml, (P>0.05). Vaginoscopy : Of the 228 cows enrolled on Day 58 post partum, two cows died (2/228; 0.9%); three cows were culled (3/228; 1.3%) from the herd, four cows were placed in the hospital barn (4/228; 1.8%) for various ailments prior to transrectal palpation of the reproductive tract and vaginoscopic examination, and 7 were missed on Day 22 post partum which were removed from the study due to failure to complete the treatment regime. On Day 58 post partum a total of 9 cows was not examined by vaginoscopy (9/228; 3.9%). Group 1 (Treated) : A total of 93 animals was available for vaginoscopy. The cervix was visualized in 90/93 (96.8%) cows. The cervix was open in 46/90 cows (51.1%). The vaginal wall and external os of the cervix were pink in color in 60/92 (65.2%), red in 27/92 (29.4%), and red and inflamed in 5/92 (5.4%). An abnormal discharge was seen in 26/92 cows (28.3%), and in 24/92 cows (26.1%) this was classified as mucopurulent and 2/92 (2.2%) as serous mucopurulent.

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118 Group 2 (Control) : A total of 98 animals was available for vaginoscopy. The cervix was visualized in 97/98 (99.0%) cows. The cervix was open in 51/98 cows (52.0%). The vaginal wall and external os of the cervix were pink in color in 45/97 (46.4%), red in 47/97 (48.5%), and red and inflamed in 5/97 (5.1%). Abnormal discharge was seen in 34/98 cows (34.7%) with all abnormal discharge classified as mucopurulent. Transrectal palpation : Data for transrectal palpation of the reproductive tract was available for 191/228 (83.7%) of the cows enrolled in the study. Group 1 (Treated) : A total of 92 animals was available for transrectal palpation of the reproductive tract. The size of the cervix was classified as greater than or less than or equal to the median value on Day 58 post partum. The median value was 30 mm in diameter with a range of <20 mm to >50 mm. There was a total of 38/92 (41.3%) with the cervix > 30 mm in diameter on rectal palpation. The previously pregnant horn (PPH) was identified as the larger of the two uterine horns. The majority of pregnancies occurred in the right horn (67/101; 66.3%), and in 7/101 cows (7.0%) the horns were estimated to be the same size and were designated as non-distinguishable. The size of the PPH was classified as greater than or less than or equal to the median value. The median value on Day 58 post partum was 30 mm in diameter with a range of <20 to >50 mm in diameter. Both categories were almost evenly distributed with 13/92 (14.1%) > 30 mm in diameter. Fluid was present in the uterine horns of 5/89 (5.6%) animals and a palpable CL was identified on an ovary in 64/92 cows (69.6%). Group 2 (Control) : A total of 99 animals was available for transrectal palpation of the reproductive tract. The size of the cervix was classified as greater than or less than or equal to the median value on Day 58 post partum. The median value was 30 mm

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119 in diameter with a range of <20 mm to >50 mm. There was a total of 50/99 cows (50.5%) with the cervix > 30 mm in diameter on rectal palpation. The previously pregnant horn (PPH) was identified as the larger of the two uterine horns. The majority of pregnancies occurred in the right horn (69/102; 67.7%), and in 9/102 cows (8.8%) the horns were estimated to be the same size and were designated as non-distinguishable. In one animal (1/102; 0.9%), there were adhesions of the uterine horns and the size could not be determined. The size of the PPH was classified as greater than or less than or equal to the median value. The median value on Day 58 post partum was 30 mm in diameter with a range of <20 to >50 mm in diameter. Both categories were almost evenly distributed with 30/98 (30.6%) > 30 mm in diameter. Fluid was present in the uterine horns of 10/95 (10.5%) animals and a palpable CL was identified in 66/97 cows (68.0%). Model 1 In Model 1, the effect of treatment on the presence of purulent or foul-smelling discharge at Days 22 and 58 post partum was evaluated. Progesterone concentrations (<1.0 ng/ml/1.0 ng/ml) on Days 8 and 15 post partum were not associated with the presence of purulent or foul-smelling discharge on Day 22 post partum (Chi-square: P = 0.66 and P = 0.41 respectively). However, P4 concentrations on Day 22 (Chi-square: P = 0.02) were associated with the presence of purulent or foul-smelling discharge on Day 58 post partum, while P4 concentrations on Day 36 post partum tended to be associated with the presence of purulent or foul-smelling discharge on Day 58 post partum (Chi-square: P = 0.08). There was no effect of treatment on the presence of purulent or foul-smelling discharge at Days 22 and 58 post partum (P = 0.904 and P = 0.134, respectively)

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120 adjusting for parity (primiparous/multiparous), RFM (yes/no) and dystocia (yes/no; Table 4-3 and Table 4-4). Primiparous cows (AOR: 2.87; 95%CI: 1.43 5.71; P = 0.003) and cows with retained fetal membranes (AOR: 8.40; 95%CI: 3.45 20.41; P < 0.0001) were more likely to have a purulent or foul-smelling discharge at Day 22 post partum regardless of treatment. There was a tendency for cows that experienced dystocia (P = 0.064) to have a purulent or foul-smelling discharge at Day 22 post partum regardless of treatment (Table 4-3). Similarly, cows with retained fetal membranes (AOR: 6.13; 95%CI: 2.92 12.99; P < 0.0001) and cows that experienced dystocia (AOR: 4.07; 95%CI: 1.47 11.24; P = 0.007) were more likely to have a purulent or foul-smelling discharge at Day 58 post partum regardless of treatment (Table 4-4). Parity was not associated with the presence of a mucopurulent vaginal discharge on Day 58 post partum (P = 0.397). Model 2 In Model 2, the effect of treatment on the presence of purulent or foul-smelling discharge and cervix > 50mm at Day 22, and the presence of purulent or foul discharge and cervix > 30mm at Day 58 post partum were evaluated. There was no association between progesterone concentrations (<1.0 ng/ml/1.0 ng/ml) on Days 8 and 15 post partum with the presence of purulent or foul-smelling discharge on Day 22 post partum (Chi-square: P = 0.31 and P = 0.76, respectively). There was a tendency for P4 concentrations (<1.0 ng/ml/1.0 ng/ml) on Day 22 (Chi-square: P = 0.08) to have an association with the presence of purulent or foul-smelling discharge and cervix > 30mm at Day 58 post partum while P4 concentrations on Day 36 post

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121 partum had no association with the presence of purulent or foul-smelling discharge on Day 58 post partum (Chi-square: P = 0.14). Table 4-3. Model 1Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy on Day 22 post partum. VARIABLE MUCOPURULENT DISCHARGE COR AOR 95% CI P-VALUE n % Group Treated 56/100 56.0 1.15 1.04 0.56 1.94 0.904 Control 53/101 52.5 1.00 1.00 Referent NA Parity Primiparous 42/61 68.9 2.41 2.87 1.43 5.71 0.003 Multiparous 67/140 47.9 1.00 1.00 Referent NA RFM Yes 41/48 85.4 7.32 8.40 3.45 20.41 <0.0001 No 68/153 44.4 1.00 1.00 Referent NA Dystocia Yes 17/21 61.9 4.07 3.13 0.94 10.42 0.064 No 92/180 25.8 1.00 1.00 Referent NA Table 4-4. Model 1Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy on Day 58 post partum. VARIABLE MUCOPURULENT DISCHARGE COR AOR 95% CI P-VALUE n % Group Treated 26/101 25.7 0.74 0.59 0.30 1.18 0.134 Control 34/102 33.3 1.00 1.00 Referent NA Parity Primiparous 20/61 32.8 1.24 1.37 0.66 2.84 0.397 Multiparous 40/142 28.2 1.00 1.00 Referent NA RFM Yes 28/48 58.3 5.38 6.13 2.92 12.99 <0.0001 No 32/155 20.6 1.00 1.00 Referent NA Dystocia Yes 13/21 61.9 4.66 4.07 1.47 11.24 0.007 No 47/182 25.8 1.00 1.00 Referent NA

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122 There was no effect of treatment on the presence of purulent or foul-smelling discharge and a cervix >50 mm in diameter at Day 22 post partum (P = 0.913; Table 4-5) adjusting for parity, RFM and dystocia. Cows with retained fetal membranes (AOR: 4.31; 95%CI: 2.10-8.85; P < 0.0001) and cows experiencing dystocia (AOR: 2.93; 95%CI: 1.07-8.00; P = 0.037) were more likely to have a purulent or foul-smelling discharge at Day 22 post partum regardless of treatment. There was no association between parity and a mucopurulent vaginal discharge on Day 22 post partum (P = 0.612; Table 4-5). Table 4-5. Model 2Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy and a cervix > 50 mm in diameter by transrectal palpation on Day 22 post partum. VARIABLE MUCOPURULENT DISCHARGE AND CERVIX > 50 mm COR AOR 95% CI P-VALUE n % Group Treated 26/100 26.0 0.99 0.96 0.48 1.91 0.913 Control 26/101 25.7 1.00 1.00 Referent NA Parity Primiparous 14/61 23.0 0.80 0.82 0.39 1.75 0.612 Multiparous 38/140 27.1 1.00 1.00 Referent NA RFM Yes 24/48 50.0 4.46 4.31 2.10 8.85 <0.0001 No 28/153 18.3 1.00 1.00 Referent NA Dystocia Yes 10/21 47.6 2.99 2.93 1.07 8.00 0.037 No 42/180 23.3 1.00 1.00 Referent NA There was no effect of treatment on the presence of purulent or foul-smelling discharge and a cervix >30 mm in diameter at Day 58 post partum (P = 0.692; Table 4-6) adjusting for parity, RFM, and dystocia. However, multiparous cows (AOR: 0.15; 95%CI: 0.03-0.62; P = 0.010) were less likely to have a purulent or foul-smelling discharge and a cervix > 30 mm in diameter at Day 58 post partum regardless of treatment. Cows with retained fetal membranes (AOR: 6.49; 95%CI: 2.53-16.67; P <

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123 0.0001) and cows that experienced dystocia (AOR: 11.66; 95%CI: 3.22-50.00; P = 0.0003) were more likely to have a purulent or foul-smelling discharge and a cervix > 30 mm in diameter at Day 58 post partum regardless of treatment (Table 4-6). Table 4-6. Model 2Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy and a cervix > 30 mm in diameter by transrectal palpation on Day 58 post partum. VARIABLE MUCOPURULENT DISCHARGE AND CERVIX > 30 mm COR AOR 95% CI P-VALUE n % Group Treated 12/92 13.0 0.71 0.83 0.32 2.11 0. 692 Control 17/98 17.3 1.00 1.00 Referent NA Parity Primiparous 3/56 5.4 0.23 0.15 0.03 0.62 0.010 Multiparous 26/134 19.4 1.00 1.00 Referent NA RFM Yes 16/42 38.1 6.39 6.49 2.53 16.67 <0.0001 No 13/148 8.8 1.00 1.00 Referent NA Dystocia Yes 9/19 47.4 6.79 12.66 3.22 50.00 0.0003 No 20/171 11.7 1.00 1.00 Referent NA Model 3 In Model 3, the effect of treatment on the presence of purulent or foul-smelling discharge, cervix > 50mm and the presence of a uterine horn 30 mm in diameter on Day 22 post partum and the presence of purulent or foul-smelling discharge, cervix > 30mm and the presence of a uterine horn 30 mm in diameter on Day 58 post partum were evaluated. Progesterone concentrations (<1.0 ng/ml/1.0 ng/ml) on Days 8 and 15 post partum had no association with the presence of purulent or foul-smelling discharge,

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124 cervix > 50mm and the presence of a uterine horn 30 mm in diameter at Day 22 post partum (Chi-square: P = 0.42 and P = 0.79, respectively). There was no effect of treatment on the presence of purulent or foul-smelling discharge, a cervix >50 mm in diameter and previously pregnant horn 30 mm in diameter at Day 22 post partum (P = 0.508; Table 4-7) adjusting for parity, RFM and dystocia. There was a tendency for primiparous cows (AOR: 0.38; 95%CI: 0.14 1.06; P = 0.065) to be less likely to have a purulent or foul-smelling discharge, a cervix > 50 mm in diameter and the previously pregnant horn 30 mm at Day 22 post partum regardless of treatment (Table 4-7). Cows with retained fetal membranes (AOR: 7.58; 95%CI: 3.34 13.89; P < 0.0001) and cows experiencing dystocia (AOR: 4.22; 95%CI: 1.28 13.89; P = 0.018) were more likely to have a purulent or foul-smelling discharge, a cervix > 50 mm in diameter and the previously pregnant horn 30 mm at Day 22 post partum regardless of treatment (Table 4-7). There was a tendency for P4 concentrations (<1.0 ng/ml/1.0 ng/ml) on Days 22 post partum to be associated with the presence of purulent or foul-smelling discharge, cervix > 30mm and the presence of a uterine horn 30 mm in diameter at Day 58 post partum (Chi-square: P = 0.07) while P4 concentrations (<1.0 ng/ml/1.0 ng/ml) on Day 36 post partum were not associated with the criteria for Model 3 (Chi-square: P = 0.59).

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125 Table 4-7. Model 3Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy, cervix > 50 mm in diameter and previously pregnant horn (PPH) 30 mm by transrectal palpation on Day 22 post partum. VARIABLE MUCOPURULENT DISCHARGE, CERVIX > 50 mm and PPH 30 mm COR AOR 95% CI P-VALUE N % Group Treated 20/99 20.2 1.33 1.33 0.57 3.06 0.508 Control 16/100 16.0 1.00 1.00 Referent NA Parity Primiparous 6/60 10.0 0.40 0.38 0.14 1.06 0.065 Multiparous 30/139 21.6 1.00 1.00 Referent NA RFM Yes 22/48 45.8 8.28 7.58 3.34 13.89 <0.0001 No 14/151 9.3 1.00 1.00 Referent NA Dystocia Yes 8/21 38.1 3.30 4.22 1.28 13.89 0.018 No 28/178 15.7 1.00 1.00 Referent NA There was an effect of treatment for the variables in Model 3 on Day 58 post partum. Cows in Group 1 (treated) were less likely to have a purulent or foul-smelling discharge, a cervix >30 mm in diameter and the previously pregnant horn 30 mm in diameter on Day 58 post partum (AOR: 0.30; 95%CI: 0.09 0.98; P = 0.047; adjusting for parity, RFM and dystocia). Cows with retained fetal membranes (AOR: 8.47; 95%CI: 2.84 25.00; P = 0.0001) were more likely to have a purulent or foul-smelling discharge, a cervix > 30 mm in diameter and the previously pregnant horn 30 mm in diameter at Day 58 post partum regardless of treatment (Table 4-8). There was a tendency for cows that experienced dystocia (AOR: 3.92; 95%CI: 0.98 15.63; P = 0.054) to be more likely to have a purulent or foul-smelling discharge, a cervix > 30 mm in diameter and the previously

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126 pregnant horn 30 mm in diameter at Day 58 post partum regardless of treatment (Table 4-8). Table 4-8. Model 3Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of finding a mucopurulent vaginal discharge by vaginoscopy, cervix > 30 mm in diameter and previously pregnant horn (PPH) 30 mm by transrectal palpation on Day 58 post partum. VARIABLE MUCOPURULENT DISCHARGE, CERVIX > 30 mm and PPH 30 mm COR AOR 95% CI P-VALUE n % Group Treated 5/92 5.4 0.34 0.30 0.09 0.98 0.047 Control 14/97 14.4 1.00 1.00 Referent NA Parity Primiparous 2/55 3.6 0.26 0.24 0.05 1.23 0.088 Multiparous 17/134 12.7 1.00 1.00 Referent NA RFM Yes 12/42 28.6 8.00 8.47 2.84 25.00 0.0001 No 7/147 4.8 1.00 1.00 Referent NA Dystocia Yes 5/19 26.3 3.98 3.92 0.98 15.63 0.054 No 14/170 8.2 1.00 1.00 Referent NA First service pregnancy rate A total of 33 cows was either bred and culled prior to examination for pregnancy, or not bred for various management and reproductive reasons. The median day to first service was 131.80 1.19 days for cows in Group 1 (treated) and 132.52 1.04 days for cows in Group 2 (control; P>0.05). Data for first service pregnancy rate were available for 170/203 (83.7%) cows, and this included 86 and 84 cows in Groups 1 and 2, respectively. The first service pregnancy rate was 23.3% (20/86) and 22.6% (19/84) for cows in Groups 1 and 2, respectively. There was no effect of treatment on first service pregnancy rate (AOR: 1.10; 95%CI: 0.52-2.30; P = 0.81), and there was no association

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127 between parity (P = 0.24), dystocia (P = 0.61), RFM (P = 0.09) or days to first service (P = 0.20) on the outcome of pregnancy regardless of treatment (Table 4-9). Table 4-9. Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of pregnancy in dairy cows treated sequentially with PGF2 in the early postpartum period to first service post partum. VARIABLE PREGNANCY COR AOR 95% CI P-VALUE n % Group Treated 20/86 23.3 1.04 1.10 0.52 2.30 0.81 Control 19/84 22.6 1.00 1.00 Referent NA Parity Multiparous 29/114 25.4 1.57 1.65 0.72 3.79 0.24 Primiparous 10/56 17.9 1.00 1.00 Referent NA Dystocia Yes 3/19 15.8 0.60 0.71 0.19 2.67 0.61 No 36/151 23.8 1.00 1.00 Referent NA RFM Yes 5/38 13.2 0.44 0.41 0.15 1.15 0.09 No 34/132 25.8 1.00 1.00 Referent NA Days to First Service 130 days 15/80 18.8 0.63 0.61 0.29 1.29 0.20 >130 days 24/90 26.7 1.00 1.00 Referent Na Experiment 2 Part B : Conception Rate to First Service One hundred and sixteen (116) cows were removed from the study. Sixty (60) cows were not bred and culled for various management and reproductive reasons prior to completion of the experiment, 13 cows were bred to the incorrect date, and 43 cows were removed from the experiment for failing to complete treatment and/or Presynch-Ovsynch protocol. A total of 302 animals successfully completed Experiment 2, Part B (Group 1; n=145 and Group 2; n=157). All cows were enrolled at 7 days in milk (DIM or 7 days post partum). Table 4-10 represents the baseline comparison of Group 1 and 2. There was no difference in parity (2, 3+), retained fetal membranes (yes/no), dystocia (yes/no) and abnormal calving (RFM and/or dystocia) between the groups.

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128 Table 4-10. Baseline data for cows within Group 1 and Group 2 based on parity (2, 3+), dystocia (yes/no), retained fetal membranes (yes/no) and abnormal calving (RFM and/or dystocia) that successfully completed the study. VARIABLE GROUP 1 (Treated) GROUP 2 (Control) P-value N % N % Parity 0.20 2 67/145 46.2 61/157 38.9 3+ 78/145 53.8 96/157 61.1 Dystocia 0.76 No 120/145 82.8 132/157 84.1 Yes 25/145 17.2 25/157 15.9 Retained Fetal Membranes 0.14 No 128/145 88.3 129/157 82.2 Yes 17/145 11.7 28/157 17.8 Abnormal Calving 0.33 No 108/145 74.5 109/157 69.4 Yes 37/145 25.5 48/157 30.6 A total of 116/302 (38.4%) cows was pregnant to first service. This included 50/145 (40.0%) in Group 1, and 58/157 (36.9%)in Group 2. There was no significant difference between the conception rates to first service between the groups (Chi-square: P = 0.59; Logistic regression: AOR: 1.13; 95% CI: 0.71 1.81; P = 0.60; Table 4-11). There was no association between parity, dystocia or retained fetal membranes on the conception rate to first service (Table 4-11).

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129 Table 4-11. Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence interval (CI) and P-value for the risk of pregnancy in dairy cows treated sequentially with PGF2 in the early postpartum period and subjected to the Presynch-Ovsynch protocol. PREGNANT P-value VARIABLE N % COR AOR 95% CI Group 1 (Treated) 58/145 40.0 1.14 1.13 0.71 1.81 0.60 2 (Control) 58/157 36.9 1.00 1.00 Referent NA Parity 2 51/128 39.8 1.11 1.11 0.69 1.77 0.68 3+ 65/174 37.4 1.00 1.00 Referent NA Dystocia No 95/252 37.7 1.20 1.19 0.64 2.22 0.57 Yes 21/50 42.0 1.00 1.00 Referent NA Retained Fetal Membranes No 98/257 38.1 1.08 1.08 0.56 2.08 0.81 Yes 18/45 40.0 1.00 1.00 Referent NA Discussion The hypothesis of Experiment 2, Part A, was that sequential administration of PGF2 in the immediate postpartum period would decrease the prevalence of mucopurulent discharge, size of the cervix and previously pregnant uterine horn (PPH), and increase first-service conception rate in postpartum dairy cows. In Experiment 2, Part A, PGF2 treatment had no effect on Model 1 (presence of a mucopurulent discharge) on Day 22 or Day 58 post partum, Model 2 (presence of a mucopurulent discharge and a cervix >50 cm on Day 22 post partum and presence of a mucopurulent discharge and a cervix >30 cm on Day 58 post partum), or Model 3 (presence of a mucopurulent discharge, cervix >50 cm and previously pregnant horn 30 mm) on Day 22 post partum. However, PGF2 treatment had an effect on the prevalence of the variables in Model 3 on Day 58 post partum. Cows treated with PGF2 in the early postpartum period were less likely to have a mucopurulent discharge, cervix >30 mm and previously pregnant

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130 horn 30 mm on Day 58 post partum. However, treatment was not associated with an increase in risk of pregnancy in Experiment 2 Part A. The rationale behind the administration of PGF2 was based on studies which showed that the administration of prostaglandin F2 decreased the time for complete involution of the uterus as detected by rectal palpation (Lindell and Kindahl, 1983). This may be due to a direct effect of PGF2 on the bovine myometrium by increasing uterine tone and motility (Patil et al., 1980) with reduction in size of the previously pregnant uterus and the evacuation of uterine content. The results of these studies substantiate the findings in Experiment 2, Part A, which suggested that PGF2 reduced the size of the previously pregnant horn by Day 58 post partum in Model 3. There was no effect of treatment on the variables of Model 3 at Day 22 post partum. There is a massive release of PGF2 post partum in both normal and abnormal cows and the blood level of PGF2 remains high for 2 to 3 weeks post partum (Lindell et al., 1982). Cows that had a shorter interval from parturition to uterine involution had a longer period of postpartum PGF2 release (Lindell et al., 1982). It is possible that the high endogenous levels of PGF2 in Group 2 (control) were sufficient to cause the uterus to involute at a similar rate as cows in Group 1 (treated). Cows with endometritis and retained fetal membranes have elevated PGF2 for the first five days post partum which return to normal levels by 7 days post partum (Thompson et al., 1987). Continuous administration of PGF2 has been shown to cause down-regulation of the PGF2 receptor. It is questionable if the administration of PGF2 had any effect on the uterus prior to ovulation and subsequent development of a corpus luteum. This theory is substantiated by the results of Experiment 2, Part A which showed

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131 that treatment had no effect on the variables of Models 1, 2 or 3 at Day 22 post partum. This was probably due to the absence of a CL when PGF2 was administered on Days 8 and 15 post partum. The positive effect of treatment on prevalence of the variables of Model 3 at Day 58 post partum may be due to the luteolytic effect of PGF2 on Day 22 and Day 36 post partum. This probably resulted in the emergence of a new follicular wave with a resultant increase in estrogen secretion by the dominant follicle. It has been established that estrogen is beneficial to clearance of bacterial contaminants from the uterus (Black et al., 1953;1954) and is effective in preventing experimental infection (Rowson et al., 1953; Hawk et al., 1960). It is speculated that lysis of the CL reduced the length of time the uterus was under the influence of progesterone, increased the time when estrogen was the dominant hormone and led to clearance of uterine contamination and improvement of uterine involution. There was no effect of treatment on pregnancy rate to first service in Experiment 2, Part A, and to conception rate to first service following the Presynch-Ovsynch protocol in Part B. This is in contrast to several studies which have indicated that the administration of PGF2 in the early postpartum period had a positive effect on reproductive performance (White and Dobson, 1990; Pankowski et al. 1995; Nakao et al., 1997; Kristula and Bartholomew, 1998; Sheldon and Noakes, 1998; Schofield et al., 1999; Heuwieser et al., 2000). In Experiment 2, Part A, the first postpartum insemination of cows in both Group 1 and Group 2 occurred at a mean of 130 to 134 days post partum. Cows were inseminated at detected estrus after the 100-day voluntary waiting period or subjected to

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132 the Ovsynch protocol. The first service occurred at a mean of 100 days post treatment with PGF2. It is difficult to say that treatment had no effect on the outcome of first service conception rate. The time lapse between treatment and insemination makes evaluation of the effect of treatment speculative. The decision to not inseminate cows earlier was made by management, based on the high number of dystocia and retained fetal membranes experienced in cows calving in the summer. In Experiment 2, Part B, attempts were made to objectively investigate the effect sequential administration of PGF2 on the first conception rate. There was no effect of treatment on first service conception rate (Group 1: 40% vs Group 2: 36.9%). This is in contrast to earlier work which showed that the use of PGF2 in the early postpartum period increased subsequent fertility (White and Dobson, 1990; Pankowski et al. 1995; Nakao et al., 1997; Kristula and Bartholomew, 1998; Sheldon and Noakes, 1998; Schofield et al., 1999; Heuwieser et al., 2000). In cows calving normally, Pankowski et al. (1995) found that cows receiving two doses of PGF2 14 days apart, with the first dose administered between 25 to 32 days post partum, had 10% higher rate of pregnancy than cows not receiving PGF2. A possible explanation for the lack of difference between pregnancy rate in Group 1 and Group 2 is the use of Presynch-Ovsynch protocol. Pre-synchronization (Presynch) with two injections of PGF2 14 days apart increases the pregnancy rate to the Ovsynch protocol. In a group of 100 cows with a 20-day estrous cycle, 40% of cows would be in the ideal stage of the estrous cycle (Days 5 to12) to begin the Ovsynch protocol. However, administration of PGF2 14 days apart places 90% of cows between Day 5 to 10 of the estrous cycle and an expected pregnancy rate of 45% to the Ovsynch protocol

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133 (Thatcher et al., 2001). The Presynch-Ovsynch protocol, in which PGF2 is given 14 days apart and the Ovsynch protocol initiated 12 days after the second injection of PGF2 have been shown to increase pregnancy rate by 18 percentage units compared to Ovsynch alone (25% to 43%; Moreira et al., 2000a). The pregnancy rates obtained in the present study are similar to that predicted by Thatcher et al. (2001) and actual percent pregnancy obtained by Moreira et al. (2000a). From the results of this experiment, it was concluded that sequential administration of PGF2 early post partum only had an effect on the prevalence of a mucopurulent discharge, size of the cervix and size of the previously pregnant horn at Day 58 post partum. In addition, there was no effect on pregnancy either following insemination at estrus, or timed insemination.

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CHAPTER 5 SUMMARY AND CONCLUSIONS There are two physiological factors which influence reproductive success in the postpartum dairy cow. The first is ovarian cyclicity, and the second is uterine health. In this study, 2 experiments were conducted using postpartum dairy cows. In Experiment 1, exogenous hormones were used to initiate normal cyclicity and improve pregnancy rate in cows with ovarian cysts. In Experiment 2, exogenous hormones were used to reduce the incidence of a mucopurulent discharge, the size of the cervix, the size of the previously pregnant uterine horn, and increase pregnancy rate. The objective of Experiment 1 was to determine the ovarian response and pregnancy rate of lactating dairy cows with ovarian cysts treated with GnRH and subjected to the Ovsynch protocol 7 days later. A total of 155 cows with ovarian cysts was sequentially allocated to 3 groups at the time of diagnosis (Day 0). Cows in Group 1 (n = 55) were treated with GnRH on Day 0 followed by the Ovsynch protocol on Day 7. Cows in Group 2 (n = 49) were subjected to the Ovsynch protocol on Day 0, and cows in Group 3 (n = 51) were not treated with GnRH on Day 0, but were subjected to the Ovsynch protocol on Day 7. Pregnancy was determined by per rectum palpation of the uterus between 45-50 days after TAI. On both Days 0 and 7, cows in all groups were subjected to ultrasonographic examination of the ovaries and blood samples were obtained from the coccygeal vessels for determination of progesterone concentration (P4) using a solid-phase, no-extraction RIA. Baseline data for parity, time of year, days in milk (DIM) and P4 on Day 0 were compared using Chi-square (P 0.05). Data for 134

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135 ovarian response on Day 7 were analyzed using logistic regression adjusting for DIM, time of year, parity and P4 on Day 0. Data for pregnancy rate were analyzed using logistic regression adjusting for DIM, time of year, P4 on Day 0 and Day 7, and ovarian response on Day 7. There was no significant difference in the baseline data for cows in all groups. Cows in Groups 1 and 2 were more likely to have a CL and high P4 on Day 7 compared to cows in Group 3. There was no difference in pregnancy rate between cows in all groups. However, cows with a CL on Day 7 were more likely to become pregnant compared to cows without a CL on Day 7. From the results of Experiment 1, it was concluded that administering GnRH to cows with ovarian cysts 7 days prior to the initiation of the Ovsynch protocol did not increase pregnancy rate. However, the presence of a CL on Day 7 had a beneficial effect on pregnancy rate. There were 2 parts to Experiment 2 (Part A and Part B). The objective of Part A was to determine the effect of sequential administration of PGF2 in the immediate postpartum period on the incidence of mucopurulent discharge, size of the cervix and previously pregnant uterine horn (PPH) and first-service pregnancy rate in postpartum dairy cows to either insemination at estrus or timed insemination. Postpartum dairy cows between Day 7 to 9 post partum were sequentially allocated to 2 groups. Cows in Group 1 (n=114) were treated with one luteolytic dosage of PGF2 8 hours apart on Days 8 and 15 post partum, and one luteolytic dosage of PGF2 once on Days 22 and 36 post partum. Cows in Group 2 (n=114) were not treated with PGF2 on Days 8, 15, 22 and 36 post partum and served as untreated controls. Vaginoscopy and transrectal palpation were performed on Days 22 and 58 post partum. Cows in both groups were either inseminated at estrus, or timed inseminated approximately 130 to 134 days post partum.

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136 Pregnancy was determined by per rectum palpation of the uterus between 45-50 days after TAI. Baseline data for parity, days in milk (DIM), dystocia, retained fetal membranes and P4 on Day 8 were compared using Chi-square (P 0.05). Data for the incidence of mucopurulent discharge, size of the cervix and previously pregnant uterine horn (PPH) were analyzed using logistic regression adjusting for parity, dystocia and retained fetal membranes. Data for first-service pregnancy rate were analyzed using logistic regression adjusting for parity, dystocia, retained fetal membranes and days to first service. There was no significant difference in the baseline data for cows in both groups. Sequential administration of PGF2 in the immediate postpartum period had no effect on the incidence of mucopurulent discharge, the size of the cervix, and the previously pregnant uterine horn on Day 22 post partum. However, this treatment scheme reduced the incidence of mucopurulent discharge, size of the cervix and previously pregnant uterine horn on Day 58 post partum. However, there was no difference in pregnancy rate between cows in both groups. The objective of Part B was to determine the effect of sequential administration of PGF2 in the immediate postpartum period on first-service conception rate in postpartum dairy cows subjected to a timed insemination protocol consisting of Presynch-Ovsynch protocol. Postpartum dairy cows at 7 days post partum were sequentially allocated to 2 groups. Cows in Group 1 (n=209) were treated with one luteolytic dosage of PGF2 8 hours apart on Days 7 and 14 post partum, and a single luteolytic dosage of PGF2 on Days 21 and 35 post partum. Cows in Group 2 (n=209) were not treated with PGF2 on Days 7, 14, 21 and 35 post partum and served as untreated controls. Cows in both groups were subjected to Presynch and Ovsynch protocols on Day 49, and TAI on either Day 85

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137 or Day 86 post partum. Pregnancy was determined by ultrasongraphy on Days 29 to 32 after TAI. Baseline data for parity, dystocia, and retained fetal membranes were compared using Chi-square (P 0.05). Data for first-service conception rate were analyzed using Chi-square and logistic regression adjusting for parity, dystocia and retained fetal membranes. There was no significant difference in the baseline data for cows in both groups. There was no significant difference between the conception rates to first service between cows in both groups. From the results of Experiment 2, it was concluded that sequential administration of PGF2 in the early postpartum only had an effect on the incidence of mucopurulent discharge, size of the cervix, and size of the previously pregnant horn at Day 58 post partum. In addition, there was no effect on pregnancy either following insemination at estrus, or timed insemination.

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APPENDIX VAGINOSCOPY AND TRANSRECTAL PALPATION FORM Date: Cow Number: Group Assignment: Group Blood Calving date: / /2003 Season: Parity: Calving Status:_________ Normal = 0 Abnormal (assisted by vet/personnel) = 1 Retained Fetal Membranes (failure to pass fetal membranes within 24 hours of calving) = 2 Vaginoscopic Examination: Cervix Open = 1 Color of vaginal wall Pink = 0 Closed = 0 Red = 1 Red and inflamed = 2 Discharge Lochia = 0 Discharge Quantity Normal_______ Slight = 1 Moderate = 2 Copious = 3 Clear_________ Slight = 1 Moderate = 2 Copious = 3 Mucoid________ Slight = 1 Moderate = 2 Copious = 3 Mucopurulent_________ Slight = 1 Moderate = 2 Copious = 3 Serous mucopurulent ________ Slight = 1 Moderate = 2 Copious = 3 138

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139 Transrectal Palpation of the Reproductive Tract & Ultrasound Size of cervix: Previously pregnant horn Right horn = 1 Size of Right horn ______________ Left horn = 2 Size of Left horn _____________ Fluid in right horn Fluid in left horn Present Present Absent Absent Structures on ovary Left ovary Right ovary NSS = 0 NSS = 0 Follicle(s) = 1 Size(s) Follicle(s) = 1 Size(s) CL present = 2 CL present = 2 CL absent = 3 CL absent = 3

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141 Archbald LF, Tsai I-F, Thatcher WW, Tran T, Wolfsdorf K, Risco C. Use of plasma concentrations of 13,14-dihydro,15-keto-PGF2 alpha (PGFM) in the diagnosis of sub-clinical endometritis and its relationship to fertility in the postpartum dairy cow. Theriogenology 1998;49:1425-1436. Arosh JA, Banu SK, Chapdelaine P, Madore E, Sirois J, Fortier MA. Prostaglandin biosynthesis, transport, and signaling in corpus luteum: a basis for autoregulation of luteal function. Endocrinology 2004;145:2551-2560. Asselin E, Goff AK, Bergeron H, Frointer MA. Influence of sex steroids on the production of prostaglandin F2 and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium. Biol Reprod 1996;54:371-379. Asselin E, Fuller WB, Frointer MA. Recombinant ovine and bovine interferon tau regulate prostaglandin production and oxytocin response in cultured bovine endometrial cells. Biol Reprod 1997;56:402-408. Badinga L, Thatcher WW, Diaz T, Drost M, Wolfenson D. Effect of environmental heat stress on follicular development and steroidogenesis in lactating Holstein cows. Theriogenology 1985;39:797-810. Ball L, Olson JD, Mortimer RG Bacteriology of the postpartum uterus. In: Proc Soc Theriogenology. Hastings, NE. 1984 p. 164-169. Bane, A. Fertility and reproductive disorders in Swedish cattle. Brit Vet J 1964;20:431-441. Banks, WJ. Female Reproductive System. Chapter 7. In: Stamathis, G. (Ed). Applied veterinary histology. 1986,Williams & Williams, Baltimore. USA. Bartolome JA, Archbald LF, Morresey P, Hernandez J, Tran T, Kelbert D, Long K, Risco CA, Thatcher WW. Comparison of synchronization of ovulation and induction of estrus as therapeutic strategies for bovine ovarian cysts in the dairy cow. Theriogenology 2000;53:815-825. Bartolome J, Hernandez J, Landaeta A, Kelleman A, Sheerin P, Risco CA, Archbald LF. The effect of interval from day of administration of bovine somatotropin (bST) to synchronization of ovulation and timed-insemination on conception rate of dairy cows with and without ovarian cysts. Theriogenology 2002;57:1293-1301. Bartolome J, Hernandez J, Sheerin P, Luznar S, Kelbert D, Thatcher WW, Archbald LF. Effect of pretreatment with bovine somatotropin (bST) and/or gonadotropins-releasing hormone (GnRH) on conception rate of dairy cows with ovarian cysts subjected to synchronization of ovulation and timed insemination. Theriogenology 2003;59:1991-1997.

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142 Beck TW, Smith VG, Seguin BE and Convey EM. Bovine serum LH, GH and prolactin following chronic implantation of ovarian steroids and subsequent ovariectomy. J Anim Sci 1976;42:461-468. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E. Hypophysial responses to continuous and intermittent delivery of hypopthalamic gonadotropin-releasing hormone. Science 1978;202:631-633. Bellin ME, Hinshelwood MM, Hauser ER, Ax RL. Influence of suckling and side of corpus luteum or pregnancy on folliculogenesis in postpartum cows. Biol Reprod 1984;31:849-855. Bierschwal CJ, Garverick HA, Martin CE, Youngquist RS, Cantley TC, Brown MD. Clinical response of dairy cows with ovarian cysts to GnRH. J Anim Sci 1975;41:1660-1665. Black WG, Ulberg LC, Kidder HE, Simon J, McNutt SH, Casida LE. Inflammatory response of the bovine endometrium. Am J Vet Res 1953;14:179-188. Black WG, Simon J, Kidder HE, Wiltbank JN. Bactericidal activity of the uterus in the rabbit and the cow. Am J Vet Res 1954;15:247-251. Bonnett BN, Martin SW, Gannon VP, Miller RB, Etherington WG. Endometrial biopsy in Holstein-Friesian dairy cows. III. Bacteriological analysis and correlations with histological findings. Can J Vet Res 1991;55:168-173. Bosc MJ, Fevre J, Vaslet de Fontaubert Y. A comparison of induction of parturition with dexamethasone or with an analogue of prostaglandin F2 (A-PGF) in cattle. Theriogenology 1975;3:187-191. Bosu WTK, Peter AT. Evidence for a role of intrauterine infections in the pathology of cystic ovaries in postpartum dairy cows. Theriogenology 1987; 28:725-736. Bretzlaff K. Rational for treatment of endometritis of endometritis in the dairy cow. Vet Clin North Am Food Anim Pract 1987;3:593-607. Bridges PJ, Taft R, Lewis PE, Wagner WR, Inskeep EK. Effect of the previously gravid uterine horn and postpartum interval on follicular diameter and conception rate in beef cows treated with estradiol benzoate and progesterone. J Anim Sci 2000;78:2172-2176. Britt JH, Morrow DA, Kittok RJ, Seguin BE. Uterine involution, ovarian activity, and fertility after melengestrol acetate and estradiol in early postpartum cows. J Dairy Sci 1974;57:89-92. Brooks AN, Lamming GE, Haynes NB. Endogenous opioid peptides and the control of gonadotrophin secretion. Res Vet Sci 1986;41:285-299.

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BIOGRAPHICAL SKETCH Katherine Elizabeth May Hendricks was born on June 4, 1972, as the first of two daughters to Elsa May Binns and Lloyd Ivanhoe Binns in St. Andrew, Jamaica. In 2002, she received her degree in veterinary medicine from the University of the West Indies, St. Augustine campus, located in the Republic of Trinidad and Tobago. In January of 2003 she started her masters program at the University of Florida under the supervision of Dr. Louis Archbald. After completion of her program, she plans to continue her education in the animal molecular and cell biology PhD program under the supervision of Dr. Peter Hansen. 163


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REPRODUCTIVE STRATEGIES IN THE POSTPARTUM DAIRY COW WITH
REFERENCE TO ANOVULATION AND POSTPARTUM UTERINE HEALTH

















By

KATHERINE ELIZABETH MAY HENDRICKS


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Katherine Elizabeth May Hendricks
































Dedicated to my mother, Elsa, and my husband, Gregory















ACKNOWLEDGMENTS

I would like to express my appreciation and thanks to Dr. Louis F. Archbald for the

opportunity to join his graduate program. Dr. Archbald's passion for this subject area,

knowledge and dedication as an advisor have been a great motivating force in aiding the

completion of this thesis. His professionalism and discipline have been an example that I

hope to emulate personally and professionally. I would like to express my thanks to Dr

Charles Courtney for funding my program.

I would like to take this opportunity to thank the members of my committee, Dr.

Peter Hansen, Dr. J. Hernandez, Dr. Pedro Melendez and Dr. Carlos Risco, for their

insights and valuable suggestions throughout my study.

Special thanks go to my parents, Elsa May Wilmot-Binns and Lloyd Ivanhoe

Binns, for believing in my abilities to do anything I set my mind to and for advising me

to stick with my life-long dreams of becoming a veterinarian. Thanks go to my

Godparents, Dermoth and Merle Bingham, for their sacrifices and invaluable assistance

in helping me create the foundation for entry into the University of Florida. I thank my

friends who stood by me during my DVM, Merlene Mercano, Dr. Denice Lewis, Dr.

Nicole Tull, Dr. Susan McClennon and Dr. Rosemary Murray

I would like to acknowledge Dr. Julian Bartolome for his help and support in

execution of the project.

I would like to express my thanks and appreciation to Ingo Kreig for use of the

dairy herd, Roger Rowe and the entire staff at Mecklenberg Dairy. I would like to thank









Don Bennink for the use of the dairy herd and the entire staff at North Florida Holstein

Dairy.

I thank all who contributed to this study but were not mentioned.















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ....................... .. .. .. ................................ ix

LIST OF FIGURES ........................................ .............. xi

ABSTRACT ................................................... ................. xii

CHAPTER

1 IN T R O D U C T IO N ................................................. .............................................. .

2 L IT E R A TU R E R E V IE W .................................................................. ..................... 5

Gonadotropin-releasing Hormone (GnRH) ............................................................5...
Chem ical N ature of the H orm one..................................................... ...............5...
Neurotransmitters and GnRH Secretion..........................................................6...
C control of Secretion .............. .................. ................................................ 7
Function of G nR H .............. .................. ............................................... 10
M echanism of A action .................. ............................................................ 12
P ro stag lan d in s ............................................................................................................. 13
B io sy n th e sis ......................................................................................................... 14
M etab o lism .......................................................................................................... 16
Mechanism of Action ........................................... ......... ............... 16
Function in the Reproductive Tract of the Cow ............................................. 17
Prostaglandin F2u. and its Uses in the Dairy Cow ........................................19
E strou s C y cle of th e C ow ...........................................................................................2 1
F ollicular P hase .......................................................................................... 2 1
L u te al P h a se ......................................................................................................... 2 2
Hormonal Control of the Estrous Cycle .........................................................23
Effect of Season.................................................. ..................... ........ .... ........ ..... 25
Folliculogenesis and Ovarian Dynamics in the Dairy Cow...................................29
F ollicular D ev elopm ent .......................................................................................2 9
Follicular Waves ............ .. .......... ..... ................................ 31
Regulation of Follicular Growth by FSH and LH.........................................33
P rodu action of E strog en ........................................................................................34
C orpus L uteum in the C ow ......................................... ........................ ................ 34
C orpus Luteum D evelopm ent......................................................... ................ 35









Function of the C orpus Luteum ...................................................... ................ 37
The Corpus Luteum and Pregnancy ............................................... ................ 38
L uteolysis ...................................................................................................... 40
Involution of the Bovine Uterus ......................................................... 44
M acroscopic C hanges.......................................... ........................ ................ 44
M icroscopic C changes ..................................................................... ................ 47
Factors A affecting U terine Involution ............................................. ................ 50
M ethods to A ssess U terine Involution ........................................... ................ 56
Endocrinology of the Immediate Postpartum Period in the Cow...............................58
Resumption of Ovarian Cyclicity Post Partum............... .....................................60
Cystic Ovarian D disease in the D airy Cow ............................................. ................ 63
P redisposing F actors......................................... .. ....................... .. ........... .. 64
P ath o g en e sis ........................................................................................................ 6 5
C y st D y n am ics..................................................................................................... 6 7
D ia g n o sis ............................................................................................................. 6 8
Treatm ent................... .... .... ......... ..... .. ......... .................. 69
Timed Artificial Insemination Programs in the Dairy Cow ..................................71
P ro staglan din F 2 a ............... ... ............................ ............ ..... .... .............. 73
Combination of Gonadotropin-releasing Hormone (GnRH) and PGF2a ...........73
Presynch-Ovsynch ................................................................... 75
Postpartum Endom etritis ................... .............................................................. 75
P ath o lo g y ............................................................................................................. 7 6
P ath o g en e sis ........................................................................................................ 7 8
D ia g n o sis ............................................................................................................. 7 9

3 EXPERIMENT 1: AN EVALUATION OF PRETREATMENT WITH GNRH ON
OVARIAN RESPONSE AND PREGNANCY RATE OF LACTATING DAIRY
COWS WITH OVARIAN CYSTS SUBJECTED TO THE OVSYNCH
P R O T O C O L ............................................................................................................... 8 4

In tro d u ctio n ............................................................................................................... .. 8 4
M materials and M ethods .. ..................................................................... ................ 85
Statistical A n aly sis...................................................................................................... 87
R e su lts....................................................................................................... ....... .. 8 8
D isc u ssio n ............................................................................................................... ... 9 2

4 EXPERIMENT 2:EFFECT OF SEQUENTIAL ADMINISTRATION OF PGF2ca
ON THE PREVALENCE OF MUCOPURULENT DISCHARGE, SIZE OF THE
CERVIX, SIZE OF THE PREVIOUSLY PREGNANT UTERINE HORN AND
FIRST SERVICE PREGNANCY RATE IN LACTATING DAIRY COWS ............98

In tro d u ctio n ................................................................................................................ 9 8
Experim ent 2 Part A .................... .............................................................. 102
M materials and M ethods ................. ......................................................... 102
T re atm en t........................................................................................................... 10 4
Exclusion Criteria .................................................. ........ ................. 105
Blood Sampling and Horm one Assay ....... .......... ...................................... 105









V aginoscopy .............. ..... ... .... ... ............... ....................... 107
Transrectal Palpation of the Reproductive Tract.................... .................. 107
Statistical A analysis .............. .... ............. .............................................. 108
E xperim ent 2 P art B .. ..................................................................... ............... 109
T re atm en t........................................................................................................... 1 10
E exclusion C riteria ......................................................... .... .. .............. 111
Pregnancy D iagnosis .. ..................................................... ... ... ............ .. 111
Statistical A analysis .............. .... ............. .............................................. 112
R esults...................................................................................................... . 112
E xperim ent 2 Part A ..................................... ....................... ............... 112
D ay 8 post partum ...................................................... .... ....... ................ 112
D ay 16 post partum ........................................................ 112
D ay 22 post partum ........................................................ 113
D ay 36 post partum ........................................................ 116
D ay 58 post partum ........................................................ 117
M o d e l 1 ...................................................................................................... 1 1 9
M o d e l 2 ...................................................................................................... 12 0
M odel 3 ........................................................... .. ............... 123
First service pregnancy rate................................................... ............... 126
Experiment 2 Part B : Conception Rate to First Service............................. 127
D isc u ssio n ............................................................................................................... .. 12 9

5 SUM M ARY AN D CON CLU SION S ............................................................................134

APPENDIX VAGINOSCOPY AND TRANSRECTAL PALPATION FORM .......... 138

L IST O F R E F E R E N C E S ................................................................................................. 140

BIOGRAPHICAL SKETCH ................................................................................ 163























viii















LIST OF TABLES


Table page

3-1 Baseline data for heifers and cows that were diagnosed with ovarian cysts and
successfully completed Experiment 1 for parity (1, 2, 3+), time of year (March-
September/October-February), days in milk (DIM) and progesterone
concentration (< 0.5 ng/ml / > 0.5 ng/ml ) on Day 0........................... ................ 89

3-2 Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the
risk of finding a CL on Day 7 in cows with ovarian cysts treated with GnRH on
D ay 0 ................................................................................................................. . 9 0

3-3 Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the
risk of finding progesterone concentration > 1.0 ng/ml on Day 7 in cows with
ovarian cysts treated with GnRH on Day 0......................................... ................ 90

3-4 Percentage, adjusted odds ratios (AOR) and 95% confidence interval (CI) for the
risk of pregnancy in lactating dairy cows with ovarian cysts treated with GnRH
o n D a y 0 ............................................................................................................. .. 9 1

4-1 Distribution of cows enrolled in the study between June and August 2003 within
Groups 1 and 2 based on days in milk (DIM), parity (primiparous/multiparous),
dystocia (yes/no) and retained fetal membranes (yes/no) ................................106

4-2 Baseline data for heifers and cows that successfully completed the study for
parity (primiparous/multiparous), days in milk, dystocia (no/yes), retained fetal
membranes (no/yes) and progesterone concentration (< 1.0 ng/ml / >1.0 ng/ml)
at 8 days post partum ......................................................................... 113

4-3 Model 1-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy on Day 22 post partum...............................121

4-4 Model 1-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy on Day 58 post partum...............................121

4-5 Model 2-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy and a cervix > 50 mm in diameter by
transrectal palpation on Day 22 post partum ....... ... ...................................... 122









4-6 Model 2-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy and a cervix > 30 mm in diameter by
transrectal palpation on Day 58 post partum ....... ........................................ 123

4-7 Model 3-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy, cervix > 50 mm in diameter and previously
pregnant horn (PPH) > 30 mm by transrectal palpation on Day 22 post partum... 125

4-8 Model 3-Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95%
confidence interval (CI) and P-value for the risk of finding a mucopurulent
vaginal discharge by vaginoscopy, cervix > 30 mm in diameter and previously
pregnant horn (PPH) > 30 mm by transrectal palpation on Day 58 post partum... 126

4-9 Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence
interval (CI) and P-value for the risk of pregnancy in dairy cows treated
sequentially with PGF2ca in the early postpartum period to first service
post partum .............. ............................................................................ 127

4-10 Baseline data for cows within Group 1 and Group 2 based on parity (2, 3+),
dystocia (yes/no), retained fetal membranes (yes/no) and abnormal calving
(RFM and/or dystocia) that successfully completed the study.............................128

4-11 Percentage, crude odds ratio (COR), adjusted odds ratio (AOR), 95% confidence
interval (CI) and P-value for the risk of pregnancy in dairy cows treated
sequentially with PGF2ca in the early postpartum period and subjected to the
Presynch-Ovsynch protocol. ........................................................ 129















LIST OF FIGURES

Figure page

3-1 Experim ent 1 Experim ental D esign ................................................. ................ 87

4-1 Experimental 2 Part A: Experimental Design.......................... ...................105

4-2 Experiment 2- Part B: Experimental Design........................................................ 111















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

REPRODUCTIVE STRATEGIES IN THE POSTPARTUM DAIRY COW WITH
REFERENCE TO ANOVULATION AND POSTPARTUM UTERINE HEALTH

By

Katherine E. M. Hendricks

August 2004

Chair: L. F. Archbald.
Major Department: Veterinary Medical Sciences

In Experiment 1, a total of 155 cows with ovarian cysts was sequentially allocated

to 3 groups on the day of diagnosis (Day 0). Cows in Group 1 (n = 55) were treated with

GnRH (100 [g, im) on Days 0 and 7, PGF2ca (25 mg, im) on Day 14, GnRH (100 [g, im)

on Day 16, and timed inseminated (TAI) 16-20 h later. Cows in Group 2 (n = 49) were

treated with GnRH on Day 0, PGF2ca on Day 7, GnRH on Day 9, and TAI 16-20 h later.

Cows in Group 3 (n = 51) were treated with GnRH on Day 7, PGF2ca on Day 14, GnRH

on Day 16, and TAI 16-20 h later. Pregnancy was determined by rectal palpation 45-50 d

after TAI. On both Days 0 and 7, cows in all groups were subjected to ultrasonography

(U/S) and blood samples were obtained for determination of progesterone concentration

(P4). Cows in Groups 1 and 2 were more likely to have a CL and high P4 on Day 7

compared to cows in Group 3. There was no significant difference in pregnancy rate (PR)

between cows in all groups, but cows with a CL on Day 7 were more likely to become

pregnant. It was concluded that administering GnRH to cows with ovarian cysts 7 days









prior to the initiation of the Ovsynch protocol did not increase PR, but cows with a CL on

Day 7 were more likely to become pregnant.

In Experiment 2 (Part A), a total of 228 postpartum (pp) dairy cows was

sequentially allocated to 2 groups between Days 7 and 9 pp. Cows in Group 1 (n= 114)

were treated twice with PGF2ca (25 mg, im) 8 h apart on Days 8 and 15 pp, and once on

Days 22 and 36 pp. Cows in Group 2 (n= 114) served as untreated controls. Vaginoscopy

and rectal palpation were done on Days 22 and 58 pp. Cows in both groups were

inseminated at estrus, or TAI approximately 130 to 134 days pp. Pregnancy was

determined by rectal palpation between 45-50 d after insemination. It was concluded that

sequential administration of PGF2ca in the immediate postpartum period reduced the

prevalence of a mucopurulent discharge, and both the size of the cervix and previously

pregnant uterine horn only on Day 58 pp. There was no difference in PR between cows in

both groups.

In Experiment 2 (Part B), a total of 418 postpartum dairy cows was sequentially

allocated to 2 groups on Day 7 pp. Cows in Group 1 (n=209) were treated twice with

PGF2ca (25 mg, im) 8 h apart on Days 7 and 14 pp, and once on Days 21 and 35 pp.

Cows in Group 2 (n=209) served as untreated controls. All cows were subjected to the

Presynch and Ovsynch protocols on Days 49 and 75 pp, respectively. Pregnancy was

determined by U/S between Days 29 and 32 after TAI. There was no significant

difference in the conception rate to first service between the groups.














CHAPTER 1
INTRODUCTION

The profitability of a commercial dairy farm is based in part on the calving interval

of the cows. In order to maximize the economic profitability of the farm, cows must

return to ovarian cyclicity, express estrus and be bred within 85 days postpartum. The

optimal calving interval is 365 days.

There are two physiologic factors which influence reproductive success in the

postpartum dairy cow. The first is ovarian cyclicity, and the second is uterine health.

Parturition is a very traumatic event, and the ability to control ovarian and uterine events

in the postpartum cow could play an important role in achieving subsequent fertility. In

this study, two experiments were performed. The first experiment investigated the use of

gonadotropin releasing hormone (GnRH) to improve ovarian cyclicity in lactating dairy

cows diagnosed with ovarian follicular cysts. The second experiment investigated the use

of prostaglandin F2uc in the early postpartum period to improve uterine health and

improve fertility in lactating dairy cow.

Ovarian follicular cysts, also known as cystic ovarian degeneration, cystic ovaries

or anovular follicles, is the most common abnormality of follicular function in cattle

(Farin and Estill, 1993). It is most often seen in the postpartum dairy cow within the first

60 days post partum (Farin and Estill, 1993). Approximately 6-19% of dairy cows

develop ovarian follicular cysts (McLeod and Williams, 1991; Garverick 1997; Silvia et

al., 2002). Cystic ovarian degeneration increases days open by 22 to 64 days and

increases the number of cows culled from the herd. Each occurrence of ovarian follicular









cysts has been estimated to cost $137 in reduced milk production and veterinary expenses

(Silvia et al., 2002). The development of programs to prevent and/or treat cystic ovarian

degeneration will benefit the dairy farmer.

The resumption of ovarian cyclicity is dependent on a number of factors including

clearance of bacterial contamination from the uterus (Sheldon et al., 2002). Bacterial

contamination of the uterus occurs within the first week post partum (Elliott et al., 1968)

with spontaneous contamination, clearance and recontamination occurring up to seven

weeks post partum (Griffin et al., 1974). Some cows have the ability to clear these

infections but others do not and the reasons for this variability between cows are

unknown. Bacterial contamination of the uterus has a direct effect on the ability of the

cow to conceive and maintain a concepts. Conception and the maintenance of pregnancy

are, therefore, dependent on a healthy uterine environment.

The incidence of endometritis is greatest during the first 14 days post partum based

on cultures of uterine fluids and uterine biopsies (Griffin et al., 1974). Failure to clear

bacterial contamination by first ovulation post partum and corpus luteum formation could

place the contaminated uterus under the influence of progesterone. Progesterone makes

the uterus more prone to uterine infection (Hawk et al., 1964) with the incidence of

severe endometritis increasing around Day 15 to Day 21 postpartum. This increase in the

severity of endometritis coincides with the time of first post partum ovulation (i.e., 15 to

28 days post partum).

The hypothesis of Experiment 1 is based on research done in cows without ovarian

cysts. This research demonstrated that cows in the early luteal phase of the estrous cycle

at the time of initiation of the Ovsynch protocol had a higher pregnancy rate compared to









cows at other stages of the estrous cycle (Vasconcelos et al., 1999; Moreira et al., 2000a).

The hypothesis of Experiment 1 was that GnRH administered to cows at the time of

diagnosis of ovarian cysts would induce an early luteal phase and thus increase

pregnancy rates to a protocol for synchronization of ovulation and timed insemination

(Ovsynch protocol). The Ovsynch protocol consists of a single intramuscular injection of

100 pg of GnRH, followed seven days later by PGF2a (25 mg, intramuscularly). The

second dose of GnRH (100 rg, im) is administered 48 hours after PGF2a with artificial

insemination occurring 16 to 24 hours later. The objectives of Experiment 1 were two-

fold. The first objective was to evaluate the ovarian response 7 days following diagnosis

and treatment of cows with ovarian cysts with GnRH. The second objective was to

determine the pregnancy rate of cows with ovarian cysts subjected to treatment with

GnRH 7 days prior to the initiation of the Ovsynch protocol.

The hypothesis of Experiment 2 was that sequential administration of PGF2a

during the early postpartum period would reduce the incidence of mucopurulent

discharge, size of the cervix, size of the previously pregnant uterine horn and increase

first service pregnancy rates. The objective of Experiment 2, Part A was to determine the

effect of sequential administration of PGF2a in the immediate postpartum period on the

incidence of mucopurulent discharge, size of the cervix and size of the previously

pregnant uterine horn and first service pregnancy rates. The objective of Experiment 2,

Part B was to evaluate the effect of sequential administration of PGF2a in the immediate

postpartum period on first service conception rate in postpartum dairy cows subjected to

a timed insemination protocol consisting of Presynch-Ovsynch protocol. The Presynch

portion of the timed insemination protocol consisted of two intramuscular injection of 25






4


mg PGF2a given 14 days apart. The Ovsynch protocol began 12 days after the second

dose of PGF2a.














CHAPTER 2
LITERATURE REVIEW

Gonadotropin-releasing Hormone (GnRH)

Chemical Nature of the Hormone

Gonadotropin-releasing hormone (GnRH), also known as luteinizing hormone

releasing hormone (LHRH), is a decapeptide produced in the arcuate, suprachiasmatic

and preoptic nuclei of the hypothalamus in response to hormonal and integrated neuronal

signals. The amino acid sequence of GnRH was first elucidated as early as 1971. The

primary structure of GnRH has been established in the pig (Matsuo et al., 1971), sheep

(Amoss et al., 1971; Burgus et al. 1972) and human placenta (Tan and Rousseau, 1982).

GnRH is synthesized from a precursor molecule, which begins with a 23 amino acid

signal sequence, followed by the GnRH decapeptide and ending with a 56 amino acid

GnRH-agonist associated peptide.

There are between 800 and 2500 GnRH neurons located throughout the brain of

vertebrates. These neurons originate in the olfactory placode outside the CNS and

migrate inwards during fetal development (Silverman, 1984). Immunohistochemical

techniques have been used to elucidate the location of the GnRH containing cell in the

bovine hypothalamus and infundibulum. GnRH-positive cell bodies have been located

singly or in small clusters in the infundibular nucleus (INFN), and in large discrete

clusters in the ventromedial nucleus (VMN) with their axons projecting into the rostral

hypothalamus (the grey matter of the lamina terminalis and medial preoptic area). The

majority of axons were found within the middle region of the hypothalamus within the









dorsomedial nucleus, VMN, INFN, and lateral hypothalamic area, the periventricular

nucleus (Dess and McArthur, 1981).

Embryonic GnRH neurons in primary cultures of olfactory placodes from ovine

embryos begin pulsatile secretion of GnRH by 17 to 24 days, indicating that these

neurons must mature prior to the onset of pulsatility (Duittoz and Batailler, 2000). GnRH

is synthesized in neuroendocrine cell bodies and transported down axons that terminate in

the median eminence where it is released in a pulsatile manner (Silverman, 1984;

Rodriquez and Wise, 1989; Naor et al., 1998). In the median eminence, GnRH is released

into the surrounding capillaries that drain into the hypothalamo-hypophyseal portal

vessels and transported to the pituitary where is stimulates the secretion of luteinizing

hormone (LH) and follicle stimulating hormone (FSH) from gonadotrophs (Garverick

and Smith, 1993).

Luteinizing hormone and FSH are released into the systemic circulation,

transported to the ovary where they stimulate follicular growth, ovulation, formation of

the corpus luteum and steroidogenesis. The ovary synthesizes estrogen, progesterone,

inhibin and activin. The gonadotropins and ovarian products regulate the release of

GnRH by both negative and positive feedback mechanisms.

Neurotransmitters and GnRH Secretion

Neurotransmitters, growth-factors, neuromodulatory peptides and ovarian steroids

positively and negatively affect GnRH and/or LH release (Kordon et al., 1994;Gore and

Roberts, 1997; Gazal et al., 1998; Moenter et al., 2003). Most studies have been carried

out in rats and immortalized GnRH neuron (GT1 cell lines). Since some of these

substances do not have receptors on the GnRH neuron, it is thought that they influence

GnRH gene expression through interneuronal pathways, or by directly binding to a









specific membrane receptor. Receptor binding leads to the activation of second-

messenger pathways to cause GnRH and/or LH release (Gore and Roberts, 1997).

Glutamate and norepinephrine (NE) have been shown to stimulate GnRH/LH

release and gene expression in vivo, and GnRH release in GT1 cells. A single injection of

the neurotransmitter glutamate analog, N-Methyl-D-L-aspartic acid (NMDA) causes a

30% increase in GnRH levels within 15 to 60 minutes (Gore and Roberts, 1997). A

similar increase in GnRH mRNA and plasma LH is seen in GnRH neurons (Petersen et

al., 1991;Liaw and Barraclough, 1993; Gore and Roberts, 1997). The catecholamine,

norepinephrine, stimulates GnRH release from intact rats and rabbits and plays a

physiological role in the onset of puberty (Gore and Roberts, 1997). Dopamine stimulates

and inhibits GnRH release in vivo while dopamine receptor-1 (D ) stimulates GnRH

release in GT1 cells (Gore and Roberts, 1997).

There are direct connections between gamma-amino-butyric acid-ergic

(GABAergic) and GnRH neurons. It is thought that through this direct connection,

GABA plays an inhibitory role in GnRH/LH release and causes a blockage of the LH

surge (Gore and Roberts, 1997). Opiates have also been shown to inhibit the GnRH/LH

release and gene expression in rats and rabbits (Gore and Roberts, 1997).

Cannulation of the third ventricle of adult cattle has allowed for sampling, detection

and quantification of GnRH secretion and has provided a means to monitor the central

regulation of reproduction (Gazal et al., 1998). Gazal et al. (1998) demonstrated that

GnRH is secreted into the cerebrospinal fluid in a pulsatile manner.

Control of Secretion

The GnRH neurosecretory system is diffuse and contains individual neurons

located in different areas of the brain (Silverman, 1984). The activity of GnRH neurons is









regulated by integrated signals from the brain and these include photoperiod, steroid

hormones, nutrition and stress (Moenter et al., 2003). However, in the absence of other

cells types, in pure GnRH neuronal cell cultures, and GT1 cell, GnRH is secreted

spontaneously in a rhythmic pattern (Martinez de la Escalera et al., 1992). Martinez de la

Escalera et al. (1992) suggested that a model of cell-to-cell communication via

intracellular contacts could explain synchronization of pulses in experiments where a

single cell-coated coverslip per superfusion chamber was used. It was also suggested that

there is a diffusible mediator, which would explain how two separated coverslips

synchronized GnRH pulses, and that GnRH itself may be the diffusible mediator

(Martinez de la Escalera et al., 1992).

The release of GnRH is episodic in both sexes. In females, however, there is an

interruption in the episodic release of GnRH by a GnRH surge. This GnRH surge

coincides with the pre-ovulatory LH surge, and lasts longer than the LH surge. Yoshioka

et al. (2001) demonstrated that there is a preovulatory increase in GnRH secretion into

the cerebrospinal fluid (CSF) in the third ventricle following the administration of

PGF2uc in heifers. The GnRH surge was shown to coincide with the LH surge and also

with the onset of standing estrus (Yoshioka et al., 2001).

While it is known that GnRH is secreted in a pulsatile manner, little is known about

the mechanisms generating this rhythmic secretion. It has been shown that GnRH

neurons produce a high-frequency rhythm characterized by oscillations in intracellular

calcium levels, burst firing of action potentials, and a low-frequency rhythm with a

period corresponding to neurosecretion of GnRH (Moenter et al., 2003). Low frequency









rhythms result in secretion of GnRH and can be modified by the effect of steroids

(Moenter et al., 2003).

The frequency of GnRH secretion varies during the estrous cycle and the

reproductive state of the animal from once every 30 minutes to once every few hours

(Moenter et al., 2003). Secretion of GnRH can be controlled at many levels. It may be

regulated at the transcriptional, post-transcriptional and post-translational levels (Gore

and Roberts, 1997).

The biosynthesis and release of GnRH are strictly controlled by afferent neurons to

the GnRH neurons in the hypothalamus (Peter and Burbach, 2002). Cell specific

expression and biosynthetic regulation rely on transcription from the gene promoter for

which the 5'-flanking region of the peptidergic gene contains essential elements (Peter

and Burbach, 2002). The amount of GnRH secreted from GnRH-containing neurons is

controlled by GnRH gene expression. GnRH gene expression is limited by the rate of

transcription of GnRH gene, polyadenylation and 5' capping of the RNA, processing of

its primary transcript to the mature mRNA, and transport of the mRNA from the nucleus

into the cytoplasm. Once in the cytoplasm, mRNA levels are determined by the turnover

of the molecule. Further regulation takes place at the level of mRNA translation into

proGnRH peptide, maturation of GnRH and the rate of degradation and release of GnRH

into the portal circulation, which affect the level of GnRH reaching the gonadotrophs

(Gore and Roberts, 1997).

Estrogen controls LH and FSH synthesis through a negative feedback loop to the

anterior pituitary and hypothalamus. Estrogen has been shown to downregulate mRNA

levels and reporter gene activity in immortalized hypothalamic GnRH neurons (Peter and









Burbach, 2002). Progesterone also causes repression of GnRH promoter activity (Peter

and Burbach, 2002). Neuropeptide Y (NPY) is a potent orexigenic substance, produced in

the hypothalamus, anterior pituitary and adipose tissue. Neuropeptide Y forms a

neuromodulatory link between nutritional status and the central reproductive axis

(McShane et al., 1992). Infusion of high concentrations of NPY into the third ventricle

inhibits GnRH pulses (Gazal et al., 1998).

Function of GnRH

The number of GnRH receptors in the anterior pituitary changes throughout the

estrous cycle. Maximum numbers of the GnRH receptor occur just prior to the

preovulatory surge in the cow, and their numbers decline thereafter (Nett et al., 1987;

Turzillo and Nett, 1999). Luteolysis and decreasing progesterone concentrations may be

the trigger for increased expression of the GnRH receptor gene (Turzillo and Nett, 1999).

The GnRH pulse frequency increases when progesterone concentrations decline, and the

negative feedback signal due to high concentration of P4 is removed. This increase in

GnRH pulse frequency increases the expression of GnRH receptors in the anterior

pituitary.

The preovulatory follicle produces large amounts of estradiol, which enhance

GnRH receptor gene expression. The increasing levels of estradiol and decreasing levels

of progesterone increase the numbers of GnRH receptors on the gonadotrophs, and

increases pituitary sensitivity to GnRH (Turzillo and Nett, 1999). In the rat, the number

of GnRH receptors is less during pregnancy and lactation compared to the estrous cycle.

It has been reported that when up to 50% of GnRH receptors are blocked with a GnRH

antagonist, ewes still respond fully to GnRH with LH release (Wise et al., 1984).









Ovarian hormones influence the numbers of GnRH receptor. In ewes, estradiol-17P3

has been shown to increase the number of GnRH receptors (Clarke et al., 1988). In vitro

studies using ovine pituitary cell cultures showed an increase in GnRH receptor

expression in response to estrogen and decreased expression in response to progesterone

(Laws et al, 1990a, 1990b). Inhibin caused an increase in the expression of GnRH

receptors and, when combined with estradiol-17p3, led to greater expression of GnRH

receptors that either hormone singly (Laws et al, 1990b).

In the immediate postpartum period, the pituitary response to GnRH is reduced.

Within the second week post partum, however, the pituitary responds to endogenous and

exogenous administration of GnRH with release of LH in dairy cows (Cummins et al

1975). Garverick et al. (1980) showed that administration of GnRH within the second

week post partum to dairy cows with large follicles was followed by ovulation and

subsequent formation of a CL. Similarly, a single injection of 100 micrograms of GnRH

12 to 14 days post partum initiated cyclic ovarian activity as evidenced by a palpable

corpus luteum and plasma progesterone > 1.0 ng/ml by Day 9 post-treatment (Zaied et al

1980). Zaied et al. (1980) also suggest that GnRH treatment 12 to 14 days post partum

may be useful in reducing abnormal ovarian activity. They found that 30% of non-treated

postpartum cows developed ovarian cysts prior to conception compared to 12.5% GnRH-

treated cows. Kittock et al. (1973) demonstrated that administration of GnRH to cows

with ovarian follicular cysts led to an increase in LH secretion, return to normal cyclicity,

and all cows expressed estrus 20 to 24 days after treatment.









Mechanism of Action

GnRH binds to receptors in the cell membrane of the gonadotrophs in the anterior

pituitary. The GnRH receptor is a member of the seven transmembrane domain receptor

coupled to a G-protein (Gq). Receptor binding leads to sequential activation of different

phospholipases to provide calcium (Ca2+) and lipid-derived messenger molecules.

Initially, phospholipase C is activated followed by activation of phospholipase A2 (PLA2)

and phospholipase D (PLD). Following receptor binding, GnRH stimulates a GTP-

protein (Gq). Activation of the G-protein results in Ca2+ -independent stimulation of

phospholipase C (PLC). Activation of PLC leads to the generation of second messenger

inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol 4,

5-bisphosphate (PIP2). Phosphatidylinositol 4, 5-bisphosphate is a minor phospholipid

constituting approximately 0.5% of the membrane phospholipids. Phosphatidylinositol 4,

5-bisphosphate and DAG are required for Ca2+ mobilization and PKC activation. Calcium

is mobilized from intracellular pools stored in the endoplasmic recticulum (ER) and by

means of L-type voltage-sensitive Ca2+ channels in the cell membrane (Naor et al., 1998;

Ruf et al., 2003).

During exocytosis, Ca2+ and phosphokinase C (PKC) act in parallel and exert an

additive response on LH and FSH secretion. Calcium and PKC are also required during

stimulated mitogen-activated protein kinase (MAPK) activity. Mitogen-activated protein

kinase can activate transcription factors such as c-fos to mediate gene transcription with

subsequent protein synthesis. In addition, MAPK can lead to stimulation of

phosphokinase A2 (PKA2)with subsequent LH and FSH synthesis and release (Naor et

al., 1998; Ruf et al., 2003).









The signal cascade leading to the LH and FSH gene regulation is still not fully

understood. Activation of the second messenger system leads to protein phosphorylation,

gene transcription and biosynthesis of FSH and LH. Pituitary release of LH and FSH is

down-regulated in the presence of continuous GnRH secretion (Belchetz et al, 1978).

High frequency GnRH pulses favor LH release while low frequency pulses favor FSH

release (Wildt et al., 1981). The changes in GnRH pulse frequency are essential for

normal reproductive function (Moenter et al., 2003).

Prostaglandins

Prostaglandins were independently isolated from human seminal plasma in the

1930s by Goldblatt and von Euler. Prostaglandins are formed by most mammalian tissues

and by tissues of lower vertebrates and certain invertebrates (Samuelsson et al., 1978).

All mammalian cells types have the capacity for converting the membrane bound fatty

acids into prostaglandins (Granstrom, 1981; Murray et al., 1996). Prostaglandins act as

local hormones, having important physiological and pharmacologic activities (Murray et

al., 1996). There are many stimuli (hormonal, nervous, other chemical, mechanical

stimuli) known to activate phospholipase and initiate prostaglandin synthesis (Granstrom,

1981). The products formed and the amounts produced will vary within the same tissue

under different conditions (Granstrom, 1981).

Prostaglandins are C20 carboxylic acids. They have a central five-membrane ring

with two side chains, 7 and 8 carbon in length, respectively, attached to adjacent

positions on the ring. Depending on the number of double bonds (one, two or three

double bonds) on the side chain they are designated to the '1', '2' or '3' series

(Granstrom, 1981). The type of prostaglandin (A, B, C, .. .1) depends on the arrangement

of functional groups in the molecule (Champe and Harvey, 1994). The substituents in the









molecule determine its biological activity, most importantly those at position C-9 and C-

11 in the ring and C-15 in the side chain (Granstrom, 1981). The prostaglandins that are

biologically active compounds have a hydroxyl group (-OH group) at C-15 and a double

bond at C-13 (Granstrom, 1981).

Prostaglandins belong to a group of unsaturated fatty acids called eicosanoids.

Eicosanoids are not stored in cells, but are released upon synthesis and their biosynthesis

is limited by the availability of free precursor fatty acid (Katzung, 1995). Eicosanoids

include the prostanoids, leukotrienes and lipoxins (Murray et al., 1996).

Eicosanoids are produced through two different pathways. Leukotrienes and

lipoxins are produced through the lipoxygenase pathway. Prostaglandins and

thromboxanes are produced through the cyclooxygenase pathway, and each pathway

competes with the other for arachidonic acid (Katzung, 1995). Not all cell types make all

of these products (Katzung, 1995). The prostanoids consists of prostaglandins,

prostacyclins and thromboxanes. They act as local hormones and function through a G-

protein to elicit their biological effects (Katzung, 1995).

Biosynthesis

The precursor for prostaglandins is arachidonic acid (AA), which is an essential

fatty acid that forms part of the glycerophospholipids found in the lipid bilayer of the cell

membrane. Cleavage of arachidonic acid from membrane lipids and other lipid esters by

phospholipase is activated by both specific and nonspecific stimuli (Katzung, 1995).

Arachidonic acid is produced from the interaction of phospholipase A2 (PLA2) with

membrane phospholipids (Katzung, 1995). Phospholipase A2 and other phospholipases

cleave esterified arachidonic acid from the 2- position of glycerophospholipids (Dennis,

1987).









Free AA is converted to PGG2, an unstable product, by oxygenation and cyclization

of the pentane ring catalyzed by the enzyme prostaglandin endoperoxide synthase

(prostaglandin G/H synthase or cyclooxygenase; COX). Cyclooxygenase is a membrane-

bound hemoprotein (Katzung, 1995). The initial unstable product (PPG2) is quickly

reduced to PGH2 by COX. Microsomes from cow uteri have been shown to possess

strong endoperoxide F2a reductase activity (Wlodawer et al., 1976).

The next substance produced from PGH2 depends on the cell in which the reaction

occurs (Sun et al., 1977). In tissues containing the cytosolic enzyme, prostaglandin D

synthase, PGH2 is converted to PGD2. However, PGE2 is produced if the tissue contains

the membrane bound enzyme, prostaglandin E synthase. Reduced glutathione is required

for both processes. Prostaglandin E 9-ketoreductase and 15-hydroxyprostaglandin

dehydrogenase convert PGE2 into PGF2c, (Sun et al., 1977; Katzung, 1995).

There are two cyclooxygenases: (I and II) that have a 60% homology and which are

pharmacologically different (Katzung, 1995). Cyclooxygenase I is a constitutive form

and synthesizes basal levels of prostaglandin. Cyclooxygenase II (COX II) is an inducible

form of the enzyme. Cyclooxygenase II is induced by a variety of ligands (Herschman,

1994), and is regulated at transcriptional and posttranscriptional levels (Sirois and

Richards, 1992). Its induction is inhibited by glucocorticoids (Herschman, 1994).

Cyclooxygenase II is rapidly and transiently expressed, and leads to a bolus of

prostaglandin production in response to stimulation (Herschman, 1994).

In response to pituitary glycoprotein hormones ovarian granulosa cells induced

COX II (Herschman, 1994). Exposure of preovulatory follicles to FSH and LH rapidly

and transiently induced the COX II message (Herschman, 1994). Prostaglandin synthesis









in the female reproductive cycle depends on the expression of COX II in response to

pituitary glycoprotein hormones (Herschman, 1994).

Metabolism

Prostaglandins are rapidly inactivated in the body. Enzymes that catabolize

prostaglandins are found in the lung, kidney, spleen, adipose tissue and intestines

(Katzung, 1995). Oxidation of the secondary alcohol group at C-15 is catalyzed by the

enzyme, 15-hydroxyprostanoate dehydrogenase (PGDH). The main sources of PGDH are

the lungs, spleen and kidney (Kindahl, 1980). The lungs have the highest enzyme activity

and with its vast vascular bed can render large amounts of prostaglandins biologically

inactive (Katzung, 1995; Davis et al., 1980). After oxidation at C-15, the A13 double bond

is reduced by A13 reductase resulting in 15-keto-13, 14-dihydro compounds, which are

the main plasma metabolites (Kindahl, 1980). The main plasma metabolite of PGF2a in

male calves is 15-keto-13, 14-dihydro PGF2a. Urinary excretion was completed in

approximately 6 hours, with recovery of 80% of the injected PGF2a (Kindahl, 1980). In

heifers the main metabolite of PGF2a is 15-keto-13, 14-dihydro PGF2a (Kindahl et al.,

1976).

Mechanism of Action

The eicosanoids are short-lived, highly potent local mediators that produce an

astonishing array of biological effects by binding to specific cell surface receptors

(Katzung, 1995). All binding appears to involve a G-protein linkage (Katzung, 1995).

Receptor binding initiates a signal transduction pathway, which links the regulatory

substance (PGF2ca) with its intracellular effectss.









Prostaglandin F2ca is released from the uterus, and transferred from the utero-

ovarian vein to the ovarian artery by a countercurrent mechanism. On reaching the ovary,

PGF2ca binds to high and low affinity-binding sites (receptors) located in the plasma

membrane of the corpus luteum (Samuelsson et al., 1978). These receptors are G protein-

coupled receptors designated FP and subtyped into FPA and FPB. The high affinity-

binding site requires calcium ions in order to be detected (Samuelsson et al., 1978).

Prostaglandin F2ca receptors are coupled to phospholipase C (PLC). Receptor

binding activates PLC and induces hydrolysis of phosphatidylinositol 4, 5-bisphosphate

to generate two second messengers, inositol 1,4,5-triphosphate (IP3) and 1,2-

diacylglycerol (DAG). Inositol 1,4,5-triphosphate diffuses into the cytoplasm and is

involved in the liberation of intracellular calcium (Ca2+) from the endoplasmic

recticulum.

Calcium is required for the activation of PKC and DAG increasing PKC's affinity

for Ca2+. Activation of PKC leads to the opening of calcium channels. Calcium and PKC

promote protein phosphorylation and this eventually leads to the inhibition of

progesterone secretion and regression of the corpus luteum (Samuelsson et al., 1978).

Function in the Reproductive Tract of the Cow

During the bovine estrous cycle, PGF2ca is released for 2 to 3 days as rapid pulses

with duration of 1 to 5 hours prior to and during luteolysis (Kindahl et al., 1976; Kindahl,

1980). A 9-keto-reductase enzyme that reduces PGE2 to PGF2ca can also form PGF2ca.

However, this enzyme is not present in the bovine uterus (Kindahl, 1980).

The precise release of PGF2ca throughout the bovine estrous cycle presupposes that

there is an inhibiting factor in the uterus. This inhibiting factor is important for the









regulation of the physiologic PG biosynthesis and thus regulates its production to prevent

premature lysis of the corpus luteum. Wlodawer et al. (1976) noted that an inhibiting

factor was found in bovine uterine preparations that suppressed the fatty acid

cyclooxygenase. Knickerbocker et al. (1986) noted that the bovine concepts suppressed

uterine production of PGF2ca production during pregnancy recognition by what was then

called bovine concepts secretary proteins (CPS) and is now known as interferon tau

(INF-'c). However, the suppression of PGF2ca release from the endometrium is regulated

by a number of hormones; estrogen, progesterone (Salamonsen et al., 1990; Xiao et al.

1998), oxytocin and endothelin-1 (ET-1).

The bovine endometrium contains large quantities of AA and has the ability to

metabolize AA into a variety of products (Salamonsen and Findlay, 1990). Ovarian

steroids influence the expression of the COX gene in bovine endometrial cells (Xiao et al.

1998). Progesterone directly influenced the basal secretion of PGF2ca by the

endometrium (Xiao et al. 1998). Progesterone has been shown to stimulate basal PGF2ca

secretion by bovine endometrial cells and tissues. However, it inhibits oxytocin-induced

PGF2ca secretion while in luteal cell culture while estrogen stimulated only PGF2ca

secretion.

Prostaglandins have also been shown to have a direct effect on the bovine

myometrium. (Patil et al., 1980). In vitro studies carried out on the bovine myometrium

indicate that PGF2ca has the ability to increase uterine tone and motility (Patil et al.,

1980). However, the extent to which motility is enhanced is dependent on the stage of the

estrous cycle. During the follicular phase, the myometrium is more active and

contractions are more frequent and stronger than in the luteal phase. Contractions occur









on average 9 times per 10 minutes, with mean amplitude of 12 mm. During the luteal

phase, the frequency of contractions is less, on average 6 per 10 minutes, with mean

amplitude of 5 mm. Under the influence of PGF2ca, there is a general increase in mean

contraction and strength in both stages of the estrous cycle (Patil et al., 1980). Zetler et

al. (1969) demonstrated that PFG2ca had a similar effect on the fallopian tubes, increasing

motility.

Prostaglandin F2a and its Uses in the Dairy Cow

Prostaglandins are widely used in herd management due to their luteolytic

properties. Prostaglandin F2a has been use in cattle as an abortifacient (Johnson, 1981).

Vandeplassche et al. (1974) induced abortion in cases of pathological gestation in cattle

by intrauterine infusion (into the horn ipsilateral to the ovary with the CL) of 10.5 to 45

mg of PGF2ca. In cases where the cows carried a mummified fetus, 3 of 5 expelled the

fetus. However, in cases of maceration, the fetus was not expelled. There was rapid

regression of the CL within 4 to 5 day of intrauterine infusion, followed by estrus within

6 to 7 days.

Prostaglandin F2a has also been use in cattle to induce parturition usually from 240

days gestation or within 20 days of predicted term (Johnson, 1981; Schultz and Copeland,

1981). Bosc et al. (1975) compared the use of dexamethasone to an analogue of PGF2ca

(ICI 79939) for the induction of parturition in cattle. Parturition was successfully induced

when prostaglandin was administered in 2 mg intramuscular doses five-hours apart using

a dosage which ranged from 4 to 10 mg. Plasma progesterone decreased to a low level

before parturition following two successive injections. This decrease in progesterone









concentration was attributed to lysis of the CL of pregnancy. However, cows in which

parturition was induced had a higher incidence of retained fetal membranes.

Prostaglandin F2a has been used to treat unobserved estrus in lactating dairy cattle

with a corpus luteum (Eddy, 1977; Seguin et al., 1978; Seguin, 1981) and for estrus

synchronization. Intramuscular administration of the PGF2a analogue, clorprostenol (ICI

80, 996) in dairy cattle with unobserved estrus and a palpable mature CL resulted in cows

exhibiting estrus and being inseminated sooner than those treated with saline. In one

study (Eddy, 1977), intramuscular administration of 500g clorprostenol resulted in 69%

of unobserved estrus cows coming into heat within 8 days of treatment.

Prostaglandin F2a has been use in cattle for postpartum infections: pyometra,

metritis and endometritis (Ott and Gustafsson, 1981a, 1981b). Pyometra is defined as a

condition associated with accumulation of purulent material in the uterus, persistence of a

CL and anestrus (Roberts, 1971). The mode of action of PGF2a in the treatment of cows

with postpartum infection is based on its luteolytic activity. In cases of pyometra,

treatment leads to the regression of the CL resulting in emptying of the uterus.

Prostaglandin F2a has been shown to stimulate the myometrium and may aid in the

physical evacuation of purulent material from the uterus (Ott and Gustafsson, 1981 a,

1981b). Gustafsson et al. (1976) demonstrated that PGF2ac administered both

intramuscularly and intravenously at various dosages will effect resolution of postpartum

and post insemination pyometra in dairy cows not previously treated with antibiotics

systemicallyy or intrauterine) or any other drug. Gustafsson et al. (1976) treated 26 cows

(23 with postpartum pyometra and 3 with post insemination pyometra). Eighty-five

percent responded by emptying the uterus and exhibited estrus 3 to 4 day following









treatment. Sixty-five percent became pregnant following insemination beginning at the

second estrus after treatment. Several other studies have shown the beneficial effect of

using PGF2ca in cows with pyometra (Jackson, 1977; Fazeli et al., 1980; Ott et al, 1981a;

Paisley et al., 1986; Gilbert et al., 1992). Prostaglandin F2a has also been use in cattle

with acute and chronic endometritis (Jackson, 1977;Ott et al., 1981a, 1981b;Paisley et al.,

1986; Pepper et al., 1987;Gilbert et al., 1992; Sheldon et al., 1998; Heuwieser et al.,

2000; Knutti et al., 2000).

Estrous Cycle of the Cow

There are four stages of the estrous cycle: estrus, metestrus, diestrus and

proestrus. The estrous cycle can also be classified as luteal or follicular depending on the

dominant structures on the ovaries. In the luteal phase, the CL is the dominant structure,

and in the follicular phase the preovulatory follicle is the dominant structure. The average

length of the estrous cycle in the heifer is 20 days with a range of 18 to 22 days, and in

the cow is 21 days with a range of 18 to 24 days (Roberts, 1971).

Follicular Phase

The follicular phase consists of proestrus and estrus. It starts with corpus luteum

regression and ends with ovulation of the dominant follicle. The follicular phase is

approximately 4 to 5 days in the cow.

In the event that pregnancy does not occur, the uterus releases prostaglandin F2ca

(PF2ca; Kindahl et al., 1976). Following release of PF2ca from the endometrium, the

corpus luteum of the luteal phase undergoes functional and structural regression. At this

time there is a sharp decline in the blood progesterone (P4) concentration (Kindahl et al.,

1976). The sudden fall in P4 removes the negative feedback on the developing follicles









leading to the selection and acceleration of preovulatory follicular growth (Savio et al.,

1990).

The preovulatory follicle produces increasing concentrations of estradiol-170 and

this influences the sex centers in the brain to induce estrus. Peak concentrations of

estradiol-170 coincide with estrus. At the level of the hypothalamus, estradiol-170

induces a GnRH surge, which induces the preovulatory LH surge and FSH release from

the anterior pituitary. There is considerably less estradiol-170 during the 18 to 20 hour

period of estrus as compare to proestrus. At the level of the pituitary, estradiol increases

the responsiveness of gonadotrophs to GnRH, which results in an increase in the LH

pulse amplitude.

The duration of estrus is approximately 18 to 20 hours, and ovulation occurs 10 to

12 hours after estrus. During estrus, the cow or heifer will stand to be mounted by the

bull or by herd-mates. During estrus, the vulva of the cow sometimes becomes swollen,

the mucous membranes of the vagina are hyperemic, and a clear, viscous, mucous

discharge may be seen hanging from the vulva.

Ovulation is spontaneous and occurs 10 to 12 hours after estrus in cows and 3 hours

earlier in heifers (Roberts, 1971). The ovulatory process consists of the following:

cytoplasmic and nuclear maturation of the oocyte; disruption of the cumulus cell

cohesiveness among the cells of the granulose layer and thinning and rupture of the

external follicular wall, with expulsion of the mature oocyte (Hafez et al., 2000).

Luteal Phase

The luteal phase is the period following ovulation and involves luteinization and

development of the CL. It consists of metestrus (Days 1 to 4) and diestrus (Days 5 to 18).

In this stage, the corpus luteum develops from the remnants of the pre-ovulatory follicle









and produces increasing quantities of progesterone. Progesterone is usually detectable in

blood by day 5 after ovulation and CL formation. The CL also produces oxytocin and

estrogen.

In the non-pregnant cow, the CL undergoes regression by day 17 after ovulation.

Luteolysis occurs as a consequence of the interaction between the CL and prostaglandin

F2ca (PGF2ca) released from the uterus. If pregnancy occurs, uterine production of

PGF2ca is blocked by the action of interferon tau (INF--T) on the uterus. The up-regulation

of oxytocin receptors in the endometrium is inhibited by the secretion of INF-T from the

trophodectoderm from days 12 to 25 in cattle (Farin et al. 1990). The CL is required for

the maintenance of pregnancy in the cow.

During the luteal phase, the quantity of progesterone produced by the CL increases.

The concentration of progesterone impacts LH secretion patterns. When progesterone is

low, the LH pulse is characterized by high frequency and low amplitude. When

progesterone is high, the LH pattern is characterized by a low frequency and high

amplitude.

Hormonal Control of the Estrous Cycle

Hormones secreted by the hypothalamus, anterior pituitary, ovary and uterus

regulate the estrous cycle. The hypothalamus integrates information from higher brain

centers, involving internal and external signals, such as nutritional status, season,

photoperiod, presence of a male and others (Garverick and Smith, 1993).

Gonadotropin-releasing hormone (GnRH) is produced by the neurosecretory cells

within the hypothalamus and regulates the release of gonadotropins luteinizingg hormone,

LH and follicle stimulating hormone, FSH) from the anterior pituitary (Smith and Jennes,









2001). GnRH is produced in the cell bodies of neurosecretory cells located in the arcuate,

suprachiasmatic and preoptic nuclei of the hypothalamus. GnRH is then transported along

the axons to the median eminence, where it is released into the hypothalamo-hypophyseal

portal circulation and travels to the anterior pituitary (Garverick and Smith, 1993). GnRH

is released in a pulsatile manner in response to appropriate physiological cues.

Luteinizing hormone and FSH are secreted in a pulsatile manner in response to GnRH

secretion (Moenter et al., 1991).

Luteinizing hormone is a glycoprotein hormone produced by the gonadotrophs in

the anterior pituitary. It consists of alpha- and beta-subunits. The alpha-subunit is

common to several hormones, including FSH and thyroid stimulating hormone (TSH),

and the beta-subunit is unique to the particular hormone. LH is a short-lived molecule,

having a half-life of approximately 30 minutes.

Follicle-stimulating hormone is a glycoprotein produced in the anterior pituitary by

gonadotrophs. It is composed of a common alpha-subunit and specific beta-subunit. FSH

stimulates the growth and maturation of ovarian follicles and in the presence of LH

stimulates secretion of estrogen from ovarian follicles.

Luteinizing hormone and FSH are secreted in a pulsatile manner from the anterior

pituitary in response to the pulsatile release of GnRH. Tonic levels are controlled by the

negative feedback mechanism from the ovaries.

The pulsatile secretion of LH varies throughout the estrous cycle (Rahe et al., 1980;

Walters et al., 1984a, 1984b; Schallenberger et al., 1985). Rahe et al. (1980) suggests that

the LH pattern throughout the estrous cycle is dependent on the stage of the cycle and

ovarian steroids influence the LH pattern.









Studies have been done in ovariectomized animals to investigate the effect of

steroids on the LH release. Ovariectomy in the cow results in increases in the pulse

frequency and amplitude of LH and FSH (Schallenberger and Peterson, 1982). The

pattern of secretion is related to circulating concentrations of progesterone. In the early

luteal phase when progesterone concentrations are low, the LH pulse frequency is much

higher than that seen in the mid-luteal phase; 8.0 pulses/12 hours and 3.6 pulses/12 hours

respectively (Walters et al., 1984b). In the early luteal phase the pulse frequency of LH

and FSH are similar. However, as the luteal phase progresses, the FSH pulse frequency

becomes greater than the LH pulse frequency (Walters et al., 1984b).

Luteinizing hormone release is suppressed by exogenous progesterone (Beck et

al., 1976). In the luteal phase, progesterone and estrogen are the main factors, which

decrease the frequency of the LH pulses in cattle. Individually these hormones can

suppress the LH pulse, but when administered together there is a more profound

inhibiting effect on LH pulse frequency. Stumpf et al. (1993) showed that when estradiol-

170 was administered to ovariectomized cows the LH pulse frequency was 0.97 0.07

pulses/hour and when progesterone was administered the LH pulse frequency was 0.52 +

0.08 pulses/hour. However, when both estradiol-170 and progesterone were give

concomitantly the LH pulse frequency increased to 14 0.07 pulses/hour.

Effect of Season

The effect of season or time of year on the bovine estrous cycle may be thought of

as two fold. There is the effect of photoperiod on the estrous cycle and the effect of "heat

stress" or high environmental temperatures on the estrous cycle.

High environmental temperatures reduce estrus behavior. Abilay et al. (1975)

demonstrated that the duration of estrus was reduced on average by 5.5 hours in









Guernsey heifers maintained at 18.2 C. It is thought that the reduction in estrus behavior

is as a consequence of the decrease in circulating estradiol (Wolfsenson et al., 1997; Roth

et al., 2001) noted during periods of heat stress. There is a reduction in the numbers of

mounting episodes per estrus during heat stress (Nebel et al, 1997). Badinga et al. (1985)

demonstrated that in Florida there is a decrease in conception rate in from 48% in March

to 18% in July with recovery occurring in November. Imtiaz Hussain et al. (1992) noted

that only 36.8% of cows expressed estrus, even though all cows were cycling based on

progesterone concentrations.

High environmental temperatures have been shown in affect cortisol levels. There

is an increase in cortisol levels in dairy cows that experience heat stress (Wise et al.,

1988; Imtiaz Hussain et al., 1992) However, Abilay et al. (1975) demonstrated that there

was a decrease in plasma cortisol levels in heat stressed heifers. Cortisol levels have been

linked to GnRH release. Increased cortisol levels inhibit GnRH secretion, which in turn

decreases LH secretions (Dobson and Smith, 2000). Cortisol has been shown to have a

direct effect on the pituitary gland to depress both basal and GnRH-stimulated LH release

in the cow (Li and Wagner, 1983; Padmanabhan et al., 1983).

High environmental temperatures have been shown to have an effect on the

gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The

effect of high environmental temperatures on LH is inconsistent. Vaught et al. (1977)

found no difference in the frequency of preovulatory increase of luteinizing hormone or

in the interval between the preovulatory increase and ovulation in either lactating or non-

lactating cows exposed to summer heat stress. This is in contrast to findings by Wise et









al. (1988). There was a decrease in the number of LH pulses on Day 5 of the estrous

cycle in heat stressed cows compared to controls (Wise et al., 1988).

Ovarian follicles are susceptible to high environmental temperatures. Roth et al.

(2000) demonstrated that there are alterations in the pattern of growth and development

of medium-sized follicles associated with an increase in plasma FSH concentration in the

first follicular wave following acute heat stress. Wolfsenson et al. (1997) demonstrated

that estradiol concentration in follicular fluid and androstenedione production by thecal

cells were lower in dominant follicles collected in autumn than those collected in winter.

This finding by Wolfenson et al. (1997), indicate the carry over effect of heat stress. Roth

et al. (2001) investigated the delayed effect of heat stress on medium-sized and

preovulatory follicles at 20 and 26 days after acute heat exposure. It was noted that the

number of medium-sized follicles that emerged during the first follicular wave after heat

stress was the same in both control and heat-stressed cows. However, there were more

healthy medium-sized follicles in the control than heat-stressed cows (56% vs 38%) but

this was not significantly different. In healthy medium-sized follicles, estradiol

production by granulosa cells and androstenedione production by thecal cells were lower

and follicular fluid progesterone concentration was higher in heat-stressed than in control

cows. In preovulatory follicles the viability of the granulose cells, concentration of

androstenedione in follicular fluid and androstenedione production by thecal cells were

lower in heat-stressed than in control cows (Roth et al., 2001).

Guzeloglu et al. (2001) investigated the effect of acute heat stress on long-term

follicular dynamics and biochemical characteristics of dominant follicles in non-lactating

dairy cows. They found that there was no difference in estradiol or progesterone









concentration in the follicular fluid of the dominant follicle between heat stressed and

control cows. However, there was a significant difference in the size of the dominant

follicle between the control and heat stressed cows. The dominant follicle size was

greater in control cows than in heat stressed cows. Guzeloglu et al. (2001) noted that heat

stress reduces follicular dominance during a follicular wave based on the increase in the

number of class 3 follicles on Days 7 and 8. Roth et al. (2000) investigated the immediate

effect of heat stress on plasma FSH and inhibin in Holstein dairy cows. It was

demonstrated that there was a larger cohort of medium sized follicles (6-9 mm) during

the second follicular wave of the estrous cycle of heat stressed cows compare to controls.

This increase growth was associated with higher plasma concentrations of FSH, which

lasted for 4 more days in heat stressed cows than in controls. The increase in plasma FSH

was also associated with a decrease in plasma concentrations of inhibin.

There is an increase in the length of the estrous cycle in cows experiencing heat

stress. Wilson et al. (1998a) noted that the second wave dominant follicle was less likely

to ovulate in heat stressed lactating dairy cows. In heat stressed cow, 18% ovulated

compared to controls 91%. The average day of luteolysis was delayed by 9 days in heat

stressed cows. Similar finding were found in heifers. Heat stress inhibited the growth and

function of the dominant follicle, such that heat stressed heifers had three follicular waves

and there was a delay in corpus luteum regression (Wilson et al., 1998b).

High environmental temperatures have been shown to affect corpus luteum

function. These observations were made based on progesterone concentrations. In some

studies progesterone concentration was elevated (Abilay et al, 1975; Vaught et al., 1977)

in cows experiencing heat stress. However, in other studies progesterone concentration









was unchanged (Wise et al., 1988) and yet in other studied progesterone concentrations

were lowered during heat stress (Howell et al., 1994). Wilson et al. (1998a, 1998b)

demonstrated that there was delayed regression of the corpus luteum in lactating dairy

cows and heifer subjected to controlled heat stress.

Heat stress reduced feed intake (Fuquay, 1981; Rensis and Scaramuzzi, 2003).

Reduced dry matter intake will prolong the period of negative energy balance in the

postpartum dairy cow. Negative energy balance leads to decreased plasma insulin,

glucose and insulin-like growth factor I (IGF-I; Rensis and Scaramuzzi, 2003). These

factors are required for the normal growth and function of ovarian follicles and thus the

estrous cycle.

Folliculogenesis and Ovarian Dynamics in the Dairy Cow

Follicular Development

An ovarian follicle is a spherical aggregation of cells that contain the developing

gamete (Banks, 1986). Oogenesis begins during embryologic development and continues

throughout fetal development until shortly after birth. Multiplication halts in meiotic

prophase. At this stage the germ cell is termed a primary oocyte (Noden, 1985).

Follicular development proceeds through further development of the primordial follicle

into a primary follicle; primary to secondary follicle and finally a mature follicle.

Follicular growth and maturation is under the influence of gonadotropins from the

pituitary (Noden, 1985).

The primordial follicle contains the primary oocyte. At this stage the primary

oocyte has paired homologous chromosomes and each has replicated to form two

chromatids (Noden, 1985). At the time the oocyte is undergoing the first stages of

meiosis follicular cells surround the primary oocyte to form a primordial follicle. Once









the primordial follicle is activated it becomes the primary follicle. Histologically, the

primordial follicle appears as a single layer of flattened follicular cells surrounding the

primary oocyte (Banks, 1986)

Activation of the primordial follicle results in the development of the primary

follicle, involving alterations in the primary oocyte, follicular cells and stromal elements.

Histologically, the primary follicle appears as single layer of cuboidal or columnar

follicular cells surrounding the primary oocyte. At the stage, accumulation of yolk

granules can been seen in the primary oocyte (Banks, 1985).

The secondary follicle consists of the primary oocyte separated from the follicular

cells by the zona pellucida. The follicular cells are actively dividing and are now know as

the membrana granulosa. As the secondary follicle continues to develop, the stromal cells

differentiate into theca internal and theca externa, separated from the membrana granulosa

by a basement membrane. The theca internal consists of large, epitheliod cells and an

extensive vascular network. The theca externa consists of a fibroblastic layer of cells

(Banks, 1986).

The tertiary follicle develops from the secondary follicle. Histologically, the

tertiary follicle consists of cuboidal and columnar follicular cells surrounding the primary

oocyte. Within the layers of follicular cells small, fluid filled periodic-acid-Schiff-

positive spaces can been seen. Further secretion of follicular fluid by the granulosa cells

leads to the formation of a follicular antrum seen in the mature follicle. The mature

follicle is also known as the Graafian follicle or preovulatory follicle. The preovulatory

follicle ranges form 15 to 17 mm in diameter in the bovine ovary.









Follicles protrude from the ovary and are recognized as a smooth fluctuant

structures on palpation. The size of the follicle increases to approximately 20 to 25 mm in

diameter by the middle of the estrous cycle. The increase in follicular fluid within the

antrum is reflected by the tension in the palpable follicle, which increases up to six to

twelve hours before ovulation (Zemjanis, 1970).

In terms of the endocrinology, the development of the follicle can be divided into

four stages. The initial stage of development is independent of the gonadotropins, follicle

stimulating hormone (FSH) and luteinizing hormone (LH). In this stage follicles are

usually < 3 mm in size. Next follows a stage in which the follicle is dependent on FSH,

the follicles range from 3 to 10 mm in diameter. In the thirds stage the follicle is

dependent on pulsatile LH and ranges from 10 mm to preovulatory size. The final stage is

dependent on the preovulatory LH surge for ovulation (Thatcher et al., 2002).

Follicular Waves

Ovarian follicles develop in waves. Rajakoski (1960) first put this theory forward

after he conducted extensive studies of ovaries taken at slaughter. Pierson and Ginther

(1984) later confirmed the existence of follicular waves using ultrasonography.

Cattle either have two or three follicular waves during a single estrous cycle. In

cows that have 2 follicular waves, recruitment occurs on Day 0 (day of ovulation) and

day 10 of the estrous cycle. In cows with 3 follicular wave cycles, recruitment occurs on

Days 0, 9 and 16 of the estrous cycle (Ginther et al., 1989a; Ginther et al., 1989b).

Follicular waves occur in cyclic cows and heifers and in dairy and beef heifers prior to

the onset of cyclicity, in prepubertal calves as early as 2 weeks of age (Evans et al. 1994)

and during most of pregnancy (Ginther et al. 1996). Follicular growth begins in the fetal

ovaries, with the final maturation stages occurring days prior to ovulation.









Follicular dynamics in the cow consists of three distinct phases: recruitment,

selection and dominance. Recruitment is the process by which a group of follicles begins

to mature and grow under the influence of gonadotropins. In selection, a single follicle is

chosen and avoids atresia with the potential to ovulate. In dominance, the selected

follicle inhibits the recruitment of a new cohort of follicles (Lucy et al., 1992).

Hoak and Schwarts (1980) showed that the secondary increase in FSH levels

following the preovulatory LH surge recruits follicles for the next estrous cycle in the rat.

Adams et al., (1992) showed that the FSH surge in heifers began 2-4 days before

ultrasonically detectable emergence of a follicular wave (follicles <5 mm). FSH peaked 1

or 2 days before emergence and began to decrease at the time of deviation, when the

follicles of a wave begin to diverge into a dominant follicle and subordinate follicles

(follicle 6-7 mm). One of these follicles becomes dominant and has the potential to

ovulate.

In a two-wave estrous cycle, a group of small follicles (<5 mm) is recruited and

begins to grow under the influence of follicle-stimulating hormone (FSH; Adams, et al

1992). Later a single follicle becomes dominant and continues to grow while subordinate

follicles will eventually undergo atresia. In the presence of the CL and high progesterone

concentration, the dominant follicle of the first wave does not ovulate and regresses. A

second follicular wave develops with recruitment of another group of follicles around

midcycle. The dominant follicle of this second wave is functional at the time of CL

regression and decreasing progesterone concentration and this follicle ovulates after

luteolysis.









In a three-wave cycle the second dominant follicle fails to ovulate and a third

follicular wave develops. The dominant follicle of the second wave is usually smaller

than the first and third dominant follicle (Lucy et al., 1992). Cows with a three-wave

cycle have a longer estrous cycle, as the third dominant follicle requires time to develop

prior to ovulation (Lucy et al., 1992).

Regulation of Follicular Growth by FSH and LH

Follicular deviation or selection has been defined as the beginning of the greatest

difference in the growth rates between the largest follicle (dominant follicle) and the

second largest follicle (largest subordinate follicle) at or before the largest subordinate

follicle reaches it maximum diameter (Ginther et al., 1996). The follicle that becomes

dominant develops receptors for luteinizing hormone (LH) around the time of deviation,

while the subordinate follicles do not. Under the influence of declining FSH

concentrations and increasing LH concentrations, the subordinate follicles regress and the

dominant follicle continues to develop. In the event that luteolysis does not occur with a

resultant fall in progesterone concentrations, the dominant follicle will not ovulate and

undergoes atresia.

The dominant follicle secretes both inhibin and estradiol. Inhibin is also secreted by

follicle greater than 3 mm and also by atretic follicles. Estradiol and inhibin feed back on

the pituitary and hypothalamus and decreases the secretion of FSH from the pituitary.

Estradiol biosynthesis is dependent on a two-cell system. Theca cells and granulosa

cells individually are not equipped with the enzymes necessary for biosynthesis of

estradiol from cholesterol. However, together these cells are capable of producing

estradiol. Theca cells are the sites of androgen synthesis in follicles. Under the influence

of LH androgen secretion is increased. The end product in steroidogenesis in theca cells









is progesterone. Androstenedione produced by the theca cells is transported into the

granulosa cells where it is converted into testosterone. Under the influence of FSH,

testosterone is converted into estradiol within the granulosa cells (Hansel and Convey,

1983).

Production of Estrogen

Prior to the LH surge and luteinization of the granulosa and theca cells, the major

hormone produced by the ovary is estrogen, estradiol-17P3. The granulosa and theca cells

individually are unable to produce estradiol, since both lack the enzymatic pathways to

do so. In order to produce estradiol, cholesterol must be converted to androgens and

androgens to estradiol. Thecal cells are capable of the former, but not the latter, while

granulosa cells are capable of the latter (Niswender et al., 2000).

Corpus Luteum in the Cow

The corpus luteum (CL) is a transient endocrine structure, which secretes

progesterone, oxytocin (Fields et al., 1992), neurophysin, relaxin and other substances,

which act in an autocrine and/or paracrine manner. There is some evidence that the

bovine CL may produce estrogen. Kimbal and Hansel (1974) used a dextrand-charcoal

adsorption method to show that estrogen-binding proteins (estrogen receptor, ER) were

present in the bovine corpus luteum. Recent work by Okuda et al. (2001) demonstrated

that estradiol 17P3 is produced in the bovine CL.

The major secretary hormone is progesterone, which is essential for the

establishment and maintenance of pregnancy. In the non-pregnant animal, the life span of

the CL determines the length of the estrous cycle.









Corpus Luteum Development

The corpus luteum is formed from the remnants of the ovarian follicle following

ovulation. Simmons and Hansel (1964) postulated that any hormone that could stop

oxytocin from shortening the bovine estrous cycle had luteotropic properties. The

luteotropic property of luteinizing hormone (LH) was later confirmed by Donaldson et

al., (1965b, 1965c).

Ovulation is initiated by a preovulatory LH surge. Rupture of the follicular wall is

believed to involve enzymatic hydrolyzation of connective tissue by LH-induced

collagenases, proteases and plasmins (Banks, 1986). The oocyte and its surrounding

follicular fluid are simultaneously ejected from the ruptured follicle. The granulosa and

theca cells collapse into the cavity. The hemorrhage associated with ovulation clots,

forming the corpus hemorragicum. This stage is a transitory stage.

The granulosa and thecal cells undergo hypertrophy and division to become the

large and small luteal cells of the corpus luteum, respectively. The production of highly

specific antibodies against granulosa and thecal surface antigens proved that as the CL

ages the small luteal cells undergo structural and morphological changes to become large

luteal cells (Alila et al., 1984). This substantiates earlier work by Donaldson and Hansel

(1965a) using histology and histochemical staining techniques. They demonstrated that

granulosa cells stop dividing by Day 4 of the estrous cycle and small luteal cells derived

from thecal cells continue to respond to luteotropins grow and develop into large luteal

cells (Donaldson and Hansel, 1965a).

Ursely and Leymarie (1979) identified three types of cells in collagenase

dispersed luteal preparations. These included small (10-20[tm in diameter) steroidogenic









cells, large (>25 |tm diameter) steroidogenic cells and numerous small (<10 [tm

diameter) non-steroidogenic cells consisting mainly of vascular cells (endothelial cells,

erythrocytes leukocytes) and connective tissue cells (Hansel and Blair, 1996).

The non-steroidogenic cells of the bovine corpus luteum consist of macrophages

and endothelial cells which account for approximately 14% of the volume and

approximately 53% of the cells of the mature CL (Parry et al., 1980;O'Shea et al., 1989).

Macrophages are important for their phagocytic activity and for participation in the

immune response involved in the regression of the CL (Pate, 1994). Endothelial cells are

those that line the microvasculature and are thought to secrete substances which are

involved in both luteotropic and luteolytic processes. Fields and Fields (1996) reported

the presence of five different phenotypes of endothelial cells from the bovine CL. Type 1

cells are believed to be true endothelial cells, while Type 5 cells displays characteristic of

immature granulosa cell, and it has been suggested to be a putative stem cell for renewal

of luteal cells (Fields and Fields 1996). Fibroblasts comprise approximately 10% of all

luteal cells and approximately 6% of the total volume of the bovine CL (Fields and Fields

1996).

The steroidogenic luteal cells make up the majority of the bovine CL. The small

luteal cells and the large luteal cells together account for approximately 70% of the

volume of the bovine CL (Parry et al., 1980; O'Shea et al., 1989). The small luteal cells

make up approximately 28% of the volume of the CL and are known for its low basal

production of progesterone. When stimulated with LH, these cells increase their

production of progesterone. The large luteal cells comprise 3% of the luteal cells.

However, they comprise approximately 40% of the volume of the CL (O'Shea et al.,









1989). The large luteal cells do not respond to LH. However, they do secrete oxytocin

and are the steroidogenic cells, which produce the majority of basal progesterone.

During the early development of the bovine CL, there is rapid growth and increase

in size. The CL has a six-fold increase in size during early luteal phase of the estrous

cycle (Zheng et al., 1994). Along with an increase in weight, there is also an increase in

the amount of progesterone produced and secreted.

Function of the Corpus Luteum

The major hormone produced by the CL is progesterone. Progesterone functions in

determining the length of the estrous cycle and is required for the maintenance of

pregnancy in the cow.

The preovulatory LH surge results in luteinization of the granulosa and thecal cells

to large and small luteal cells, respectively. The luteinization process results in a shifting

of the steroidogenic pathway such that the major secretary product is progesterone. This

process includes increased expression of enzymes required for conversion of cholesterol

into progesterone. There is an increase in cytochrome P-450 side chain cleavage (P-

450soc) enzyme and 3p3-hydroxysteroid dehydrogenase/A ,A4 isomerase (3P3-HSD) and

decrease expression in the enzymes that convert cholesterol to estradiol-17a-hydroxylase

cytochrome P-450 and aromatase cytochrome (Niswender et al., 2000).

In the female reproductive tract, prostaglandin E2 (PGE2) is considered

luteotrophic (Pratt et al., 1977). Intrauterine administration of PGE2 protects the CL

against induced and spontaneous luteolysis (Pratt et al., 1977; Henderson et al., 1977).

Prostaglandin E2 stimulates the production of progesterone (P4) through the activation of

cyclic-AMP protein kinase A (PKA) pathway (Kotwica et al., 2003).









Prostaglandin E2 binds to its receptor, a G protein-coupled receptor, (EP) located in

the cell membrane. There are four receptor subtypes, EP1, EP2, EP3 and EP4. Only EP2

and EP4 are coupled to adenylate cyclase. Activation of adenylate cyclase leads to an

increase in cAMP that in turn activates PKA leasing to a cascade of intercellular signals

with the eventual activation of genes for progesterone syntheses. Prostaglandin E2 EP2

receptor is highly expressed in the large luteal cells. The EP2 receptor is the major

cAMP-generating PGE2 receptor in the bovine CL (Arosh et al. 2004) and the large

luteal cells produce 80% of progesterone (Diaz et al., 2002)

The Corpus Luteum and Pregnancy

In the event of pregnancy, the secretion of PGF2ua from the uterus is blocked,

luteolysis does not occur and progesterone concentration is maintained. Between Day 15

and 17 of the estrous cycle, the bovine concepts produces a signal, which prevents

luteolysis induced by the pulsatile release of PGF2ua (Kindahl et al., 1981; Asselin et al.

1996). The signal molecule, for the recognition of pregnancy in cows was first identified

as bovine trophoblast protein-1 (bTP-1), which was later called interferon-'c (INF-'c)

Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon. (Klemann et al.,

1990).

In the cow, INF-'c prevents the luteolysis by down-regulation of oxytocin receptors

(Meyer et al., 1996). Reducing the number of oxytocin-receptors available for oxytocin

binding attenuates oxytocin-stimulated secretion of PGF2a (Meyer et al., 1996). This

prevents oxytocin-stimulated PGF2a release and subsequent luteolysis.

Recombinant bovine interferon-'c (rblNF-'c) also causes a decrease in the expression

of cyclooxygenase II (COX II) and prostaglandin F synthase (PGFS; Xiao et al., 1998).









Cyclooxygenase II is an inducible rate-limiting enzyme for the conversion of arachidonic

acid to PGG2 and PGH2, the precursors of PGF2 and PGE2. Prostaglandin F synthase

(PGFS) is responsible for the reduction of PGH2 to PGF2 and PGD2 to 9ac, 11 3-PGF2 (a

stereoisomer of PGF2a). Recombinant bINF-'c has been shown to decrease COX II

mRNA in epithelial cells (the primary source of PGF2a) and to increases COX II mRNA

and prostaglandin synthesis in stromal cells (the primary source of PGE2; Xiao et al.,

1998). Xiao et al. (1998) also demonstrated that there was a reduction in PGFS mRNA in

both epithelial and stromal cells, and this was associated with an increase in PGE2: PGF2a

ratio.

Bovine interferon-'c has been shown to shift the primary prostaglandin (PG)

produced by the endometrium from PGF2a to PGE2 (Asselin et al., 1997: Xiao et al.,

1998). Prostaglandin E is thought to be a luteotrophic agent (Pratt et al., 1977). Asselin et

al (1997) showed that cultured bovine endometrial cells treated with bovine recombinant

INF-'c (rINF-'c), in the presence and absence of oxytocin had a net PGE2: PGF2a ratio of

3.8 and 7.7 respectively. The conclusions made by Asselin et al. (1997) were rINF-'c

regulates PGs by stimulating PGE2 preferentially and rINF-'c transforms the response to

OT from stimulation of PGF2a to stimulation of PGE2.

Progesterone is necessary for the maintenance of pregnancy in the bovine. The

normal CL produces more progesterone than required to maintain the embryo until Day

15 of pregnancy. Normal embryo development proceeds to Day 15 when the levels of

total progesterone production exceed a threshold value by approximately 100 |tg of total

luteal progesterone; 15-day CL normally contain nearly 300 |tg of progesterone (Hansel

and Blair, 1996).









Luteolysis

Luteal regression is caused by a pulsatile release of prostaglandin F2ca primarily

from the intercaruncular region of the surface epithelium of the uterus (Asselin et al.

1996) in the late luteal phase. It is thought that the pulsatile secretion of PGF2ca is

generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and

uterine PGF2ca. Regression of the CL is essential for normal cyclicity, and allows the

development of a new ovulatory follicle. However, prevention of luteolysis is necessary

for establishment and maintenance of pregnancy (Okuda et al., 2002).

Prostaglandin F2ca secretion from the bovine endometrium varies during the estrous

cycle. During the follicular phase and at estrus, Prostaglandin F2ca is at its highest and

then declines at early to mid-luteal phase of the estrous cycle (Kindahl et al., 1981).

PGF2au is released from the uterus in a series of short pulses 2 to 3 days during and after

luteolysis (Kindahl et al., 1981). Schramm et al. (1983) demonstrated that the CL is

sensitive to pulsatile administration of PGF2ca resulting in luteolysis. Skarzynski and

Okuda (1999) demonstrated that long-lasting stimulation with PGF2ca desensitizes luteal

PGF2au receptors in the cow and luteolysis fails to occur.

Oxytocin released from the corpus luteum acts upon the uterus to stimulate

production of PGF2ca, which in turn causes luteal regression. Prostaglandin F2ca has the

ability to cause further release of oxytocin from the ovine corpus luteum. Oxytocin has

been proven to be essential for the initiation of luteolysis in the ewe (Silvia et al., 1991).

It has been shown that oxytocin administered during the early stages of the estrous cycle

(Days 2-6) causes PGF2ca release, which is measurable in uterine venous blood (Milvae









et al., 1980). However, the level of oxytocin in the CL, plasma and oxytocin mRNA in

the CL are all low during normal luteolysis (Hansel and Blair, 1996).

It has been shown that exogenous administration of PGF2ca stimulates the utero-

ovarian release of PGF2ca in the ewe (Wade and Lewis, 1996). Similarly, Kotwica et al.

(1997) and Okuda et al. (2002) reported that administration of exogenous PGF2ca

increased PGF2ca release from the uterus on day 18 of the estrous cycle. It has been

demonstrated by Skarzynski and Okuda (1999) that PGF2ca activates protein kinase C

(PKC) and increases intracellular calcium mobilization, which may in turn stimulate

PGF2ca production in the endometruim.

It has been reported by Schams and Berisha (2002) that exposure to progesterone or

inhibition of progesterone action by a progesterone antagonist in early to mid-diestrus

regulates the onset of uterine release of PGF2ca from endometrium causing shortening or

extension of the interestrous interval in sheep and cows. Sheep that were treated with

onapristone (progesterone antagonsist) on day 4, 6 and 8 had a longer estrous cycle. The

controls had an estrous cycle length of 17.0 0.5 days while those treated with

onapristone had a cycle length of 22.0 0.8 days.

Endothelin-1 (ET-1), a 21 amino acid peptide is a potent vasoconstrictor originally

isolated from porcine aortic endothelial cells (Yanagisawa et al, 1988). Kisanuki et al.

(2001) demonstrated using gene-knockout technology in mice that ET-1 is produced

primarily by endothelial cells. It has been shown to be a modulator in female

reproduction where it serves to inhibit premature luteinization of follicular cells in

porcine ovaries (Flores, J.A., 2000) and in the propagation of the luteolytic process in the

ewe (Hinckley and Milvae, 2001).









The ET gene is transcribed to produce the preprohormone, preproET-1 (ppET-1).

PreproET-1 undergoes posttranslational processing to produce big ET-1, which is further

processed to mature ET-1. Big ET has little biologically active while ET-1 is the

biologically active form. The final processing stage from big ET-1 to the active form, ET-

1 is carried out by endothelin-converting enzyme-1 (ECE-1; Xu et al, 1994). Endothelin-

converting enzyme-1 is a membrane bound protein belonging to the zinc-binding

metalloendopeptidases (Xu et al, 1994), and has several isoforms (Meidan and Levy,

2002). Endothelin-converting enzyme-1 mRNA is highly expressed in steroidogenic

tissues, especially in the ovaries (Xu et al, 1994).

Endothelin-1 binds two receptors subtypes, ETA (for aorta) and ETB (type B

receptors; for bronchus; Meidan and Levy, 2002). These receptors belong to the seven-

transmembrane G protein-coupled receptor superfamily. The ETA receptor has a higher

affinity for ET-1, while ETB binds all three endothelins (ET-1, -2 and -3) equally

(Meidan and Levy, 2002).

Hinckley and Milvae (2001) demonstrated that ET-1 has an inhibitory effect of on

progesterone synthesis by luteal cells in the ewe. They demonstrated that ET-1 inhibited

basal and LH-stimulated progesterone production by dispersed ovine cells and that

inhibition was removed by pre-incubation with BQ123 (a highly specific endothelin ETA

receptor antagonist). They also showed that intramuscular administration of ET-1 at

midcycle reduced plasma progesterone concentrations for the remainder of the estrous

cycle. It has been shown that administration of a luteolytic dose of PGF2Uc stimulates

gene expression of ET-1 in ovine CL collected at mid-cycle, and intra-luteal









administration of BQ123 on Days 8 and 9 of the estrous cycle removes the luteolytic

effect of PGF2ca (Hinckley and Milvae, 2001).

Hinckley and Milvae (2001), working with ewes, supported the hypothesis that ET-

1 plays an integral part in PGF2a-mediated luteolysis. Following pretreatment with a

subluteolytic dose of PGF2ca, intramuscular administration of ET-1 caused a rapid

decline in plasma progesterone concentrations and shortened the length of the estrous

cycle in the ewe (Hinckley and Milvae, 2001).

The ETA receptor gene is expressed in the two luteal cell types enriched from

bovine CL and the thecal- and granulosa-derived luteinized cells of the CL in vivo.

However, the endothelial cells of the CL have higher levels of ETA receptor mRNA

levels than in each of the steroidogenic luteal cell types (Mamluk et al, 1999). Little is

known about the ETB receptor in the CL.

In theca-derived luteal cells, LH and forskolin downregulate ETA receptors, while

in granulosa-derived luteal cells, insulin-like growth factor-I (IGF-I) inhibits the ETA

receptor mRNA levels (Mamluk et al, 1999). This suggests that during the early stages of

luteinization, when both IGF-I and LH are at their peak levels, the expression of ETA

receptor is suppressed (Meidan and Levy, 2002).

Levy et al. (2003) showed that the ECE-1 gene is expressed by both bovine ovarian

endothelial and steroidogenic cells using enriched follicular and luteal cell

subpopulations and in situ hybridization techniques. Different ECE-1 isoforms exist

based on the different N-terminal cytoplasmic tails, and are located in different

intracellular areas. The intracellular ECE-la isoform is present only in ET-1-expressing

endothelial cells (Levy et al. 2003). The membrane-bound ECE-lb isoform is expressed









in both steroidogenic and endothelial cells of the preovulatory follicle and the CL. Insulin

and IGF-I upregulate ECE-1 expression when cultured with granulosa cells with

concomitant increase in progesterone production while ET-1 and LH down regulate ECE-

1 levels in steroidogenic cells.

The levels of ECE-1 mRNA are lowest in the bovine CL during the early stages of

CL development (Days 2-4). As the CL matures (Days 7-12), ECE-1 levels increase and

remain elevated in the late CL (Daysl3-16). ECE-1 levels decline in the regressed CL

(Days 20+; Levy et al. 2003). Levy et al. (2003) suggested the following model for ET-1

biosynthesis in the ovaries. Endothelial cells expressing ppET-1 and the two endothelin-

converting enzyme-1 (ECE-1) isoforms are capable of secreting both the precursor, big

ET-1 and mature ET-1. Steroidogenic cells only express the cell surface form of ECE-1,

ECE-lb, without ppET-1. In order to produce ET-1, steroidogenic cells are dependent on

the extracellular supply of big ET-1. Endothelial cells provide the precursor (big ET-1)

and the steroid-secreting cells convert it into mature ET-1, ensuring that ET-1 is

generated near its site of action, the ETA receptor, to ensure that the short lived ET-1 is

active.

Involution of the Bovine Uterus

Macroscopic Changes

The postpartum interval is the period from parturition to the first postpartum

estrus accompanied by ovulation. During this period, several processes take place. The

uterus involutes to its previously non-gravid state, the endometrium regenerates, bacterial

contaminants are cleared from the reproductive tract and ovarian cyclicity resumes.

During the process of uterine involution, there is a reduction in the size of the

uterus accompanied by loss of tissue and regeneration of the endometrium. The reduction









in the size of the uterus is contributed to by strong muscular contractions that occurs

every three to four minutes during the first 24 hours post partum (Moller 1970b). After

calving, the uterus decreases in a logarithmic fashion in weight, length and diameter of

the previously pregnant horn. The greatest changes in these parameters occur rapidly in

the first few days after parturition. At calving, the uterus of a cow weighs approximately

9 kg. At Day 4 to Day 7 post partum, the uterine horns can be palpated cranial and ventral

to the pelvis (Morrow et al., 1969c). Over a period of 10 days the uterus rapidly involutes

to approximately 3 kg (Gier and Marion, 1968). The rapid reduction in size is

accompanied by an increase in uterine tone from Day 10 to Day 14 post partum and the

previously pregnant horn decreases from approximately 12 cm to 7 cm in diameter

(Morrow et al., 1969c). The period of rapid involution coincided with the first estrus in

normal cows and is associated with discharge of uterine lochia (Morrow et al., 1969c).

Following the rapid reduction in the size of the uterus, the rate of involution

decreases. The rate of involution decreases such that the uterus weighs about 1 kg by 20

to 30 days post partum and 750 g by 50 days post partum (Gier and Marion, 1968). The

length of the previously gravid horn decreases to half it length at parturition by Day 15

post partum and by Day 30 it is a third its size at parturition (Gier and Marion, 1968). The

postpartum uterus decreases to half its gravid diameter by Day 5 postpartum.

Gier and Marion (1968) noted that the cervix in the postpartum cow decreases in

diameter from 15 cm on Day 2 postpartum, 9-11 cm by 10 days post partum; 7-8 cm on

Day 30 post partum to 5-6 cm 60 days post partum. Morrow et al. (1969c) noted that at

Day 4 to Day 7 post partum the cervix was palpated cranial to the pelvis. There is an

initial decrease in diameter between days 5 and 10 followed by relaxation of the cervix at









day 10 post partum when final sloughing of the caruncular masses occur (Gier and

Marion, 1968). The relaxation of the cervix is interpreted on palpation and a slight

enlargement and is associated with discharge of lochia from the uterus and first

postpartum estrus (Morrow et al., 1969c). Involution of the cervix continues gradually

until Day 30 post partum in normal cows and until Day 35 post partum in abnormal cows

(Morrow et al., 1969c). Any other changes taking place after Day 30 to Day 35 post

partum could not be detected by transrectal palpation.

There is a difference in the rate of reduction between the myometrium and the

endometrium. During the first few days post partum, the myometrium reduces in size,

while the endometrium becomes edematous during the first day post partum. Endometrial

edema then resolves slowly over the next 5 days and by Days 6 to 8 post partum it has

disappeared and the endometrium regresses rapidly (Gier and Marion, 1968).

Further tissue losses after Day 19 consist of reduction of blood vessels, regression

of uterine glands, and contraction of tissue with reduction in cell numbers and cell

volume. The caruncles further reduce in size. On Day 19 the caruncles are 15 to 20 mm

in diameter. By Day 39 the caruncles appear as smooth knobs 10 to 15 mm in diameter

and by Days 50 to 60 the caruncles are reduced to circular cratered cones 8 to 10 mm in

diameter across the base and 4 to 6 mm across the crown (Gier and Marion, 1968). At the

end of the regression changes the caruncles appear as rows of white disks in a pink

endometrium (Gier and Marion, 1968). Involution of the uterus is considered to be

complete by 40 to 60 days post partum, when caruncles have regressed to a smooth,

oblong, epithelium-covered, avascular knob (Gier and Marion, 1968). Garcia and Larsson









(1982) reported that complete uterine involution occurred between Days 41 to 50 post

partum.

Uterine lochia is a mixture of normal and degenerate cells from the mucosa,

maternal placenta and blood from the shedding of the fetal placenta (Moller, 1970b). A

considerable amount of blood is found in the lumen of the uterus following parturition.

By Day 4 post partum the blood becomes mixed with sloughed caruncular material. By

Day 12 post partum, however, the luminal content changes to a lymph-like fluid that

decreases in quantity as the postpartum period progresses (Gier and Marion, 1968). This

fluid (lochia) is palpable in the uterus from Days 8 to 13 post partum. Uterine lochial

fluid diminishes from 1,400 to 1,600 ml at Day 2 to naught by Day 21 to 25 post partum.

The amount that is seen as a vaginal discharge varies from animal to animal and is

usually seen between Days 5 to 10 post partum (Moller, 1970b).

Microscopic Changes

In the cow, fetal cotyledons fuse with maternal caruncles of the uterine mucosa to

form placentomes (Hafez, 1993). The fusing of the two tissues forms the primary

anchoring system of the placenta, keeping the maternal and fetal tissues in apposition

(Eiler, 1997). The secondary anchoring system consists of root-like penetration of

caruncle crypts by cotyledon villi and adhesive fluid between fetal and maternal

epithelium. The secondary anchoring system functions to hold fetal and maternal

epithelia together for physiologic exchange (Eiler, 1997).

After parturition, the septa and crypts of the caruncles contain remnants of

chorionic villi (Gier and Marion, 1968; Archbald, et al 1972). The remnants of

chorioallantoic cells in the maternal crypts undergo necrosis and mineralization and are









phagocytized or expelled in the lochia and are not observed in the caruncles after Day 11

post partum (Archbald et al., 1972).

The medium-sized and small arteries of the caruncule undergo progressive

vascular degeneration from Days 1 to 19 post partum (Archbald et al., 1972). Gier and

Marion (1968) found that within the first 2 days post partum, caruncular blood vessels

constrict and the septum and crypts undergo necrosis. The smooth muscle cells of the

tunica media undergo hydropic degeneration and pyknosis and the tunica media

undergoes fibrinoid necrosis (Archbald et al., 1972).

Archbald et al. (1972) noted that sloughing of the superficial areas of the caruncles

began on Day 1 post partum and necrotic changes in the stratum compactum were noted

on Day 5 post partum with sloughing of the superficial layer occurring by Days 6 and 7

post partum. The stratum compactum is reduced almost to the level of the inter-

caruncular area by Day 15 post partum (Archbald et al., 1972). By Day 5 post partum,

there is a necrotic layer 1 to 2 mm thick over the stratum compactum and cellular

organization is lost leaving only blood vessels and clumps of leukocytes (Gier and

Marion, 1968).

By Day 10 post partum, most of the necrotic material is removed, and, all of the

caruncular mass involved in the placentomes sloughs by Day 15 post partum, leaving

stubs of blood vessels extending beyond the stratum compactum. At 19 days postpartum,

the arterioles within the stratum compactum and beyond disappear, leaving the caruncle

relatively smooth (Gier and Marion, 1968).

The leukocytes found in clinically normal cows consist of lymphocytes and

plasmacytes. Histiocytes and occasional polycytes are also found in the clinically normal









cow. In cows that have a uterine infection, organized lymphocytic nodules are found in

the stratum compactum.

Tissue regeneration occurs in all areas of the endometrium. However, the earliest

to show full regeneration is the inter-caruncular epithelium, which occurs by 8 days post

partum (Gier and Marion, 1968). Archbald et al. (1972) reported that at no time during

uterine involution was the inter-caruncular area devoid of an epithelial layer.

Tissue regeneration of the uterine epithelium begins just after parturition (Gier and

Marion, 1968) with focal areas of epithelial cells found on the caruncular surface on Day

1 post partum (Archbald et al., 1972). These epithelial cells seen on Day 3 and 5 are later

sloughed along with the superficial layer of the caruncle due to necrosis of the stratum

compactum of the caruncle between Days 5 and 6 (Archbald et al., 1972). The epithelial

cells are pleomorphic, and contain large hyperchromatic nuclei and granular cytoplasm.

Archbald et al. (1972) observed degenerated and regenerated epithelial cells in the

inter-caruncular epithelium on Day 1 post partum. The degenerative cells were confined

to the basal area of the epithelium and characterized by pyknosis and vacuolation of the

cytoplasm. Regenerated cells were interspersed among degenerated cells and

characterized by large, hyperchromatic nuclei and granular cytoplasm. Degeneration and

regeneration of the epithelial cells occurred simultaneously until Day 15 when

degenerative cells were no longer observed (Archbald et al., 1972).

In the event of bacterial contamination, the uterine epithelium may be totally or

partially destroyed (Gier and Marion, 1968). Re-epithelialization of the caruncle is

complete by Day 25 post partum, 10 days after obvious sloughing is complete (Gier and









Marion, 1968). Archbald et al. (1972) found that a layer of epithelial cells covered the

entire caruncular surface by Day 19 post partum.

The epithelial cells of the uterine endometrial glands exhibit a pattern of

simultaneous degeneration and regeneration similar to that observed in the inter-

caruncular area (Archbald et al., 1972). Microscopic changes in the myometrium begin

on Day 3 post partum and progress to Day 27 post partum. These changes include

granular degeneration of the sarcoplam, vacuolation of the muscle cell and atrophy of the

nucleus. By Day 31, the fibers appear normal and the myometrium is greatly reduced in

size in comparison to that in the early postpartum period. Necrosis of the muscle fibers

was not observed during postpartum myometrial regression (Archbald et al., 1972). Prior

to Day 3 post partum the muscle fibers of the uterus contract from 750-800 tm to 400

lm and by Day 3 are approximately 200 [im (Moller, 1970b). These changes in the size

of the muscle fibers have been attributed to glycogen formation leading to degeneration

and absorption (Moller, 1970b).

Factors Affecting Uterine Involution

Parity has been show to influence the interval from parturition to complete uterine

involution. Marion et al. (1968) found that the average interval from parturition to uterine

involution was significantly shorter in primiparous cows (34.0 days) compared to

pluriparous cows (40.6 days). Based on rectal palpation, Morrow et al. (1969c) noted that

multiparous cows of six lactations or more had larger uteri than primiparous cows, and

took a longer time to involute. However, Moller (1970a) and Miettinen (1990) found no

difference in the rate of uterine involution between primiparous and multiparous cows.

Cervical involution is influenced by parity. Miettinen (1990) found that there was a

difference in the size of the cervix with the cervix being larger in multiparous cows.









However, there was no difference in the time the cervix took to involute. In contrast,

Otlenacu et al. (1983) found that involution of the cervix occurred earlier in primiparous

than in multiparous cows. Based on rectal palpation Morrow et al. (1969c) noted that

multiparous cows of six lactations or more had a larger cervix than primiparous cows,

and took a longer time to involute. On Day 10 and Day 20 post partum there was a

significant difference of 1.2 and 1.0 cm in the diameter of the cervix. However, by Day

30 post partum the cervix was of similar sizes between 3.2 and 4.1 cm in diameter and by

Day 50 post partum the cervix was between 2.9 and 3.3 cm in diameter.

Otlenacu et al. (1983) noted that the time for complete involution of the cervix in

cows with a normal discharge was less than that for cows with an abnormal uterine

discharge. The greatest difference in cervix diameter between cows with normal and

abnormal discharge was 10 mm at three weeks postpartum (Otlenacu et al. 1983).

Time of year, or season, has been shown to influence the rate of uterine involution.

Cows calving in the spring and summer have shorter intervals from parturition to uterine

involution than cows which calve in the winter, regardless of parity (Marion et al., 1968).

Cows calving in the summer have variable intervals from parturition to uterine

involution. Marion et al. (1968) found that in cows calving in the summer with an

ambient temperature of > 380C, the heat stress increased the interval from parturition to

uterine involution.

Retained fetal membranes have been shown to increase the interval from

parturition to the completion of uterine involution. Marion et al. (1968) found that it

required 50.1 4.9 days for the uterus to involute in pluriparous cows with retained fetal

membranes, and 45.1 3.2 days for primiparous cows. It was also noted that the rate of









uterine involution was affected more in primiparous cows than in pluriparous cows with

retained fetal membranes (Marion et al, 1968).

Morrow et al. (1969c) defined an abnormal cow as a cow that experienced any of

the following: abortion, dystocia, twins, retained fetal membranes, metritis, milk fever,

acute mastitis, ketosis or other debilitating disease. Abnormal cows had larger uteri in the

early postpartum period, especially cows with retained fetal membranes and metritis. At

Day 10 and Day 20 post partum, there was a significant difference of 1.3 cm and 0.8 cm

respectively between normal and abnormal cows. Abnormal cows had a combined

diameter of both uterine horns on rectal palpation of 14.9 cm on Day 10 and 8.4 cm on

Day 20 post partum, respectively. The gross palpable involution of the uterus was notable

up to Day 25 post partum in normal cows, and Day 30 post partum in abnormal cows.

Any changes that occurred after Day 25 and Day 30 post partum could not be felt on

transrectal palpation (Morrow et al., 1969c).

Metabolic disturbances can have a negative effect on uterine involution. Cows or

heifers that experience hypocalcaemia have a reduction in uterine contractions (Roberts,

1989; Al-Eknah and Noakes, 1989) and rate of uterine and cervical involution (Fonseca

et al., 1983). Kamgarpour et al. (1999) investigated subclinical hypocalcaemia in Friesian

cows. Subclinical hypocalcaemia was defined as occurring if the plasma total calcium

(PTCa) fell below 2.0 mmol/L. Cows calving in the winter and experiencing subclinical

hypocalcaemia took longer to complete uterine and cervical involution in comparison to

normocalcaemic cows, with hypocalcaemic cows taking 6 more days to reach a mean

size. Similar findings were noted in cows that calved in the summer and experienced

subclinical hypocalcaemia.









The bovine uterus is invaded and colonized by bacteria within the first two weeks

postpartum (Elliot et al., 1968; Griffin et al., 1974). In most cases the uterus clears this

infection by the third or fourth week post partum. Not all cows clear the bacterial

infection and subsequently develop endometritis. Endometritis has been shown to delay

uterine and cervical involution. Endometritis delays the resumption of ovarian cyclicity,

increases the interval from calving to ovulation and delays the return of prostaglandin F2a

metabolite (PGFM) to basal levels (Lindell et al., 1985; Del Vecchio et al., 1992).

Endometritis accounts for approximately 20% of reproductive disorders in postpartum

dairy cows (Coleman et al., 1985). Moller (1970a) examined the rate of uterine involution

between milked cows and cows suckling three or four calves and found that there was no

difference in the rate of involution between milked and suckled cows.

Steroid hormones have variable effects on the interval from parturition to uterine

involution. In cattle, the first postpartum dominant follicle develops on the ovary

contralateral to the previously gravid uterine horn. However, the presence of an estradiol-

secreting dominant follicle in the ipsilateral ovary is a marker of subsequent fertility

(Sheldon et al., 2000a). Based on their findings, Sheldon et al. (2000b) attempted to

promote a dominant follicle on the ipsilateral ovary using equine chorionic

gonadotrophin (eCG). Cows treated intramuscularly with 250 iu eCG or 750 iu eCG on

Day 14 post partum showed no difference in the rate of uterine involution compared with

those treated with a placebo. Treatment with eCG, or the presence of a follicle > 8 mm in

diameter in the ovary ipsilateral to the previously gravid uterine horn, did not affect the

rate of uterine involution (Sheldon et al. 2000b). The intramuscular administration of

estradiol benzoate approximately 48 hours after calving had no effect on the intervals









from calving to the completion of involution or between the intervals from calving to the

first ovulation (Tian and Noakes, 1991).

Sheldon et al. (2003) infused estradiol benzoate into the previously gravid uterine

horn on Days 7 and 10 postpartum, and monitored uterine involution by ultrasonography.

There was no effect of estradiol treatment on the diameter of the previously gravid or

nongravid uterine horns. They concluded that utero-ovarian signaling in the direction

from the uterus to the ovary may be more important during the postpartum period.

Administration of oestradiol into the uterine lumen increased uterine pathogenic

anaerobic bacterial contamination (Sheldon et al., 2004). Estradiol benzoate was infused

into the previously gravid uterine horn on Days 7 and 10 postpartum. Animals treated

with estradiol benzoate had higher bacterial load on Day 14, than on Days 7 or 21,

attributable to pathogens associated with endometritis, Prevotella melaninogenicus and

Fusobacterium necrophorum.

These finding by Sheldon et al. (2000a, 2000b, 2003, 2004) and others indicate that

estradiol administration in the early postpartum period had no effect on uterine involution

(Tin and Noakes, 1991) and may be detrimental by promoting endometritis (Sheldon et al

2004). More first postpartum dominant follicles are selected in the ipsilateral ovary when

there is a lower uterine bacterial load (Sheldon et al., 2002).

Exogenous progestogens have been administered to cows in an attempt to improve

uterine involution. Melengestrol acetate (MGA) was feed to Holstein cows at 1 mg per

cow per day for 14 days beginning 14 to 18 days post partum in one group of cows. In a

second group MGA was fed in a similar dosage and 500 micrograms of estradiol

benzoate was administered intramuscularly 48 hours after the last feeding of MGA. The









third group consisted of control cows that received no treatment. Based on rectal

palpation, there was no significant difference in the rate of involution (Britt et al., 1974).

Subcutaneous administration of 0.05 mg and 1.0 mg estradiol 17P3 given every

other day from Day 3 post partum was shown to have no effect on the rate of involution.

The oral administration of 300 mg medroxyprogesterone acetate every other day from

Day 3 postpartum had no effect on the rate of uterine involution. However, daily

administration of 30 mg of progesterone has been shown to increase the time for the

uterus to involute completely in both intact and ovariectomized cows (Marion et al.,

1968). Removal of ovarian hormone by ovariectomy has been shown to significantly

reduce the time for uterine involution in pluriparous cows (Marion et al., 1968).

The administration of prostaglandin F2a has been shown to decrease the time for

complete involution of the uterus as detected by rectal palpation (Lindell and Kindahl,

1983). Lindell (1982) demonstrated that there is a massive release of PGF2a postpartum,

which continues for 2 to 3 weeks. It was also deduced from this study that cows that had

a shorter interval from parturition to uterine involution had a longer period of postpartum

PGF2a release.

In a study by Tian and Noakes (1991) five groups of five cows were treated

intramuscularly with a single dose of either 100mg progesterone in oil, 25 mg dinoprost

trmethamine (PGF2ac tromethamine), 5 mg oestradiol benzoate, 1.2 mg long-acting

oxytocin analogue, carbonectin or 5 ml sterile water 48 hours after parturition. There was

no statistical difference in the rate of uterine involution.









Methods to Assess Uterine Involution

Uterine involution may be studied by observing the reduction in the size of the

vulva, vagina, cervix and the uterus (body and horns). These observations may be made

using the following: 1) palpation per rectum of the cow's reproductive tract from calving

to completion of uterine involution (Marion et al., 1968; Moller, 1970a; Garcia and

Larsson 1982); 2) removal of the reproductive tracts at slaughter at predetermined

intervals from calving (Gier and Marion, 1968; Moller 1970a); 3) transrectal

ultrasonography of the reproductive tract (Mateus et al., 2002; Wehrend et al., 2003; 4)

cervical forceps (Wehrend et al., 2003)

Garcia and Larsson (1982) performed rectal palpation of the cervix, uterus and

ovaries. They assessed uterine size, tone, symmetry and location of the uterus in pelvic

cavity as indicators of uterine involution. Uterine involution was considered complete

when the uterus was in the normal position in the pelvic cavity, the uterine horns were

equal or almost equal in size and there was no enlargement in the thickness of the uterine

wall. Marion et al., (1968) used rectal palpation twice weekly to monitor uterine

involution, using criteria set by Gier and Marion (1968) as a marker for complete uterine

involution.

Moller (1970b) refers to Rasbech's (1950) four-stage sequence in involution, which

can be followed by rectal palpation. During the first stage (1-8 days post partum), the

vagina can be palpated as a band approximately 8 cm in width within 24 hours post

partum. The cervix is not distinguishable until Day 3 and by Days 4 and 5 has enough

tone to be distinguished from the uterus and is usually located at the anterior edge of the

pelvic floor. The surface of the uterus feels hard and corrugated and the uterine caruncles

can only be felt through the uterine wall when it is relaxed. In the second stage (8-10 days









post partum) the entire uterus can be palpated. The surface is smooth and soft with

fluctuation in the post-gravid horn. The caruncles are palpable as hazel-nut shaped

structures and the cervix is firm and lies within the pelvic cavity in younger animals.

During the third stage (10-18 days post partum) the uterus feels like a soft plastic body;

caruncles and fluctuations are less pronounced; the cervix is firm and continues to

decrease in size until it is similar to that of the post-gravid uterine horn. During the fourth

stage (18-25 days post partum) there is an increase in uterine tone and the previously

gravid horn reduces to a similar size to the non-gravid uterine horn.

Moller (1970b) reports that authors on the subject of uterine involution agree that

the previously pregnant horn rarely returns to its pre-gravid size. Although the uterus

continues to undergo involutional changes after 25-30 days (Gier and Marion, 1968),

these changes are small and difficult to detect by rectal palpation (Moller, 1970b).

However, Marion et al. (1968) concluded that uterine involution monitored by rectal

palpation take 40 days.

Gier and Marion (1968) used reproductive tracts collected from slaughtered cows

to assess involutional changes. They removed the excess fat and vagina from the cows to

access the changes that occur during the process of involution. The remainder of the tract

was then measured. The weight, length and diameter of the previously pregnant uterine

horn were used to access the rate of regression. The diameter of the cervix was measured

to assess uterine involution.

Wehrend et al (2003) used a combination of ultrasonography and cervical forceps

to measure the rate of uterine and cervical involution. Cervical forceps were positioned in

the cervical canal and the position verified by ultrasonography. The distance of the legs









of the forceps was proportional to the opening of the tip of the forceps. A gauge table was

used to determine the degree of the opening of the intracervical tip of the forceps by

measuring the distance of the extracorporeal legs of the forceps.

Endocrinology of the Immediate Postpartum Period in the Cow

The corpus luteum of pregnancy secretes progesterone throughout pregnancy and

regresses about 2 days prior to parturition (Garverick and Smith, 1993). The placenta also

contributes progesterone in the latter part of pregnancy. At the end of pregnancy there is

a rapid increase in the secretion of estrogen (Hoffmann and Schuler, 2002). Prior to

calving, progesterone and estrogen concentrations are high (Hoffmann and Schuler

2002).

The progesterone concentration begins to decrease about two days prior to

parturition and is low following calving. Following parturition, there is a rapid decline in

circulating concentrations of estrogen. The fall in progesterone and estrogen removes the

inhibitory block on the hypothalamic-pituitary axis (Garverick and Smith, 1993; Noakes,

1996) The inhibitory effects of estradiol are thought to operate through inhibition of the

expression of mRNA coding for the common ac-subunit and specific P-subunit of FSH

and LH (Garverick and Smith, 1993). Messenger RNA expression of the subunits is low

following parturition and increases thereafter.

During gestation, LH is secreted in a pulsatile manner. However, the pulse

frequency and amplitude decrease as gestation progresses. Pituitary LH content and

plasma LH concentrations are low shortly after calving and generally increase during the

postpartum period (Schallenberger et al., 1982; Garverick and Smith, 1993). Short-term

episodic surges of LH increase as time post partum increases and LH responsiveness to









GnRH is low at parturition and increases as time post partum increases (Garverick and

Smith, 1993). The frequency of the pulsatile LH secretary pattern increases just prior to

the first ovulatory surge ofLH (Garverick and Smith, 1993).

Ovariectomy in the early postpartum cow has a profound effect on the tonic

secretion of gonadotropin from the pituitary. Schallenberger et al. (1982) showed that

ovariectomy performed in the early postpartum period caused a two-fold increase in

mean LH values as well as the amplitude and frequency of pulsatile release. A similar

response was seen for FSH. However, the increase in amplitude was less pronounced

than that seen for LH.

Following parturition, FSH concentration is within normal range (Garverick and

Smith, 1993). Schallenberger et al. (1982) noted pulsatile FSH release during the first day

post partum. The frequency of pulsatile LH release coincided with FSH pulses. However,

there were additional pulses between the FSH and LH pulses that coincided during the

early postpartum period (Schallenberger et al., 1982). Increasing progesterone

concentrations caused an immediate suppression of frequency and basal LH, but not FSH

secretion. This is similar to that seen during normal cyclicity (Schallenberger et al.,

1982).

The administration of estrogen has no effect on LH secretion on Days 0 and 5 post

partum. However, by Day 10 post partum and beyond, administration of estradiol elicited

an LH surge with increasing magnitudes within 12 to 24 hours. Cows possessing a

functional CL did not respond to estrogen with an LH surge. However, this was not the

case for FSH. FSH surges also occurred in cows at 5 days post partum (Schallenberger et

al., 1982).









Schallenberger et al. (1982) showed that first calf heifers could be induced to cycle

early in the post partum period when GnRH is administered on an hourly basis at 500

micrograms followed by challenge with lmg estradiol-benzoate.

It has been hypothesized that the following sequence of endocrine events occur in

the normal cow after calving based on studies carried out by themselves and others

(Peters and Lamming, 1986). Gonadotropin releasing hormone (GnRH) is secreted from

the hypothalamus immediately after calving. However, the quantity or frequency of

secretion is inadequate to cause sufficient release of LH and FSH from the pituitary to

restore normal ovarian cyclicity. The concentration of FSH rises quickly after parturition

and stimulates follicular development. The plasma concentration of LH and the frequency

of LH pulses increase gradually with time post partum. Secretion of LH and FSH

stimulates follicular growth and estradiol production. There is a gradual recovery in the

positive feedback mechanism such that by two weeks postpartum normal ovarian

cyclicity returns.

Resumption of Ovarian Cyclicity Post Partum

The interval from parturition to first observed estrus ranges from 30 to 76 days in

the dairy cow (Roberts, 1971). However, observation of estrus may not be a reliable

method to estimate the resumption of ovarian activity since some cows experience

"silent" estrus. As time progresses post partum, the percentage of cows in which silent

estrus occurs decreases (Roberts, 1971). Other methods of determining the onset of

cyclicity have evolved which are more accurate. The milk progesterone assay determines

the resumption of ovarian cyclicity by the presence of elevated progesterone

concentrations (Noakes, 1996). Using milk progesterone concentrations, Bulman and

Wood (1980) determined that approximately 50 percent of cows resumed normal









cyclicity by 20 days postpartum and greater than 90 percent by 40 days post partum

(Noakes, 1996).

Ultrasonography has been used to follow the development of ovarian follicles in

the post partum cow. Sheldon et al. (2002) detected follicular development by 7 to 10

days post partum with emergence of a dominant follicle within 14 days of parturition and

ovulation of this dominant follicle by 17 to 18 days post partum. This interval was

increased in cows with high milk production, in cows nursing calves or being milked four

times a day, in cows on a poor or on low plane of nutrition and in older cows with greater

than four parturitions (Roberts, 1971).

In cows with a normal postpartum period, a mature follicle develops and ovulates

with subsequent development of a CL by Days 13 to 15 after calving (Roberts, 1971).

The first estrous cycle is usually shorter than the normal 20 to 21 days (Roberts 1971).

Terqui et al. (1982) found that the earlier ovarian activity occurs the more likely cows

will experience a short luteal phase. The CL associated with the short estrous cycle has a

short life span as a result of lack of luteotropic support, failure of the luteal tissue to

recognize a luteotropin, or enhanced secretion of a luteolytic agent (Hafez, 1993).

The first ovulation post partum usually occurs on the contralateral ovary.

Transrectal palpation of the ovaries has shown that approximately 90 percent of corpora

lutea formed within 15 days post partum occur on the ovary opposite the previously

pregnant horn and 60 percent in cows ovulating between 15 and 20 days after parturition

(Roberts, 1971). This has been substantiated by sequential transrectal ultrasonography.

The first postpartum dominant follicle is usually found on the ovary contralateral to

the previously pregnant uterine horn, that is, opposite the ovary containing the CL of









pregnancy (Foote et al., 1968; Kamimura et al., 1993; Nation et al., 1999). This led many

to speculate that the CL of pregnancy may have a local inhibitory effect on

folliculogenesis. Dufour et al. (1985) concluded that the CL of pregnancy and/or the

concepts have a carry-over effect on the rate of growth of the antral follicles even after

parturition. Bellin et al. (1984) found that the ovary with the CL of pregnancy had

smaller follicles and lower follicular estrogen concentrations than those of the ovary

without a CL. It was also noted that only 18% of the follicles on the ovary with a CL

were healthy compared to 48% of follicles in the ovary without a CL (Bellin et al., 1984).

However, recent work done by Sheldon et al. (2002) suggested that the CL of pregnancy

does not have a local inhibitory effect on postpartum folliculogenesis. It was further

suggested that the previously pregnant uterine horn shortly after parturition may play

more of a role.

It has been shown that the presence of a large follicle on the ovary ipsilateral to the

previously pregnant uterine horn within 4 weeks of parturition was associated with

improved subsequent fertility (Sheldon et al., 2000). Bridges et al. (2000a) reported

similar findings in beef cows, where less cows ovulated from the ipsilateral ovary.

However, ovulation from the ipsilateral ovary tended to increase fertility (Sheldon et al.,

2000a).

There are several factors that have a negative impact on the resumption of ovarian

cyclicity in dairy cows. The interval from calving to first ovulation increased in cows

with high milk production, cows nursing calves or being milked four times per day, cows

on a poor or on low plane of nutrition, and in older cows with greater than four









parturitions (Roberts, 1971). Bellin et al. (1984) found that suckled cows had smaller and

fewer follicles and lower follicular concentrations than nonsuckled cows.

There is a close association between the time to first ovulation and negative energy

balance in dairy cows (Zurek et al., 1995). The exact mechanism by which energy

balance influences reproduction is not completely understood. However, one mechanism

may be through the suppression of GnRH and the LH pulse frequency required for

follicles to grow to the preovulatory stage (Schillo, 1992).

Cystic Ovarian Disease in the Dairy Cow

Cystic ovarian degeneration, ovarian follicular cysts, cystic ovarian disease, ovarian

cysts and cystic ovaries are terms used to describe the same condition. Ovarian cysts are

follicles that fail to ovulate at the time of estrus (Garverick, 1999), and commonly occur

in lactating dairy cows. They can also occur in beef cows and dairy heifers. Ovarian cysts

have been defined as follicular structures 2.5 cm or greater in diameter that persists for an

extended period of time in the absence of a CL (Garverick, 1999). Ovarian cysts can be

classified as follicular cysts or luteal cysts. Follicular cysts are thin walled, may be single

or multiple and affect one or both ovaries (Elmore, 1986). Higher concentrations of

estradiol are usually found in follicular cyst fluid than in fluid from luteal cysts and

normal follicles (Odore et al, 1999). Luteal cysts are thick-walled, usually occurring

singly, and affect one ovary and have higher concentrations of progesterone in their

cystic fluid than follicular cysts and normal follicles (Odore et al, 1999).

Bane (1964), in a review of fertility and reproductive disorders in Swedish cattle,

quoted Henricson reporting that the risk of ovarian cysts increased from 0.3% for heifers

to 8-10% for cows four to five years old and that Henricson found the incidence of

ovarian cysts was higher in the winter months than in the summer months. Bane analyzed









the frequency of ovarian cysts in relation to the month of calving and found that cows

calving in the winter had a higher risk of developing ovarian cysts in the following

reproductive period (Bane, 1964). Lopez-Gatius et al. (2002) indicated that ovarian cysts

occur most commonly in cows calving in the summer. Garverick (1999) estimated the

incidence of ovarian follicular cysts between 10 -13% in the United States.

The calving date influences the frequency of ovarian cysts. Cows that calved in

April-May were less likely (2.2%) to develop ovarian cysts while cows calving in

October had a higher frequency (9%) of ovarian cysts (Bane, 1964).

In Sweden between 1954 and 1961, the frequency of animals with ovarian cysts in

five-year-old cows fell from 10.8 % to 5.1%. This was brought about by the culling of

bulls with a high frequency of cystic ovarian degeneration among their daughter (Bane,

1964) suggesting that there may be a hereditary aspect.

Spontaneous recovery occurs in some cows that develop ovarian cysts. L6pez-

Gatius et al. (2002) found that in cows diagnosed with cysts 43-49 days postpartum, only

38.8% recovered spontaneously while in cows diagnosed with cysts 57-63 days

postpartum, 71.4% recovered spontaneously. Erb and White (1981) reported that the

highest incidence of ovarian cysts occurs in the first 60 days of lactation, while Morrow

et al. (1969b) reported that there was a peak in incidence between 14 and 40 days post

partum.

Predisposing Factors

L6pez-Gatius et al. (2002) reported that ovarian cysts occur most commonly in

cows calving in the summer, in high milk producers, in older cows, and in cows that body

condition score increased during the prepartum period. The major risk factor for the

presence of cysts at the time of insemination was the development of cysts early (43-49









days postpartum) in the postpartum period (L6pez-Gatius et al., 2002). The risk of having

a cyst at the time of insemination was 36.6 times higher in cows with early cysts (L6pez-

Gatius et al., 2002).

Pathogenesis

Postpartum uterine infections may increase secretion of PGF2a and cortisol

associated with the formation of cystic ovaries in dairy cows (Bosu and Peter, 1987; Peter

et al., 1989). In a study carried out on cows that calved normally but subsequently

developed ovarian cysts, a correlation between postpartum uterine infections and the

development of ovarian cysts was made. In 88.9% and 11.1% of cows that subsequently

developed ovarian cysts endometrial swabs yielded bacterial growth densities of +3 and

+2 respectively, while 35.1% of cows that did not develop ovarian cysts yielded bacterial

growth densities of +1 and +2 (5.4%) (Bosu and Peter, 1987). Further, consistently high

cortisol levels were detected prior to cyst detection and in association with high bacterial

growth densities in the uterus prior to cyst detection (Bosu and Peter, 1987).

Cysts have been experimentally induced using anti-LH serum, estradiol valerate

and adrenocorticotropic hormone (ACTH) administration. Cysts have also been induced

by daily exogenous injection of 30 mg of estradiol-17P3 and 150 mg of progesterone

dissolved in alcohol for 7 days (Cook et al., 1990; Garverick, 1999).

Ovarian cysts have been induced by ACTH administration in the preovulatory

period. Adrenocorticotropic hormone has been shown to block the preovulatory LH

surge, possibly through cortisol-mediated inhibition of LH release (Refsal et al, 1987).

The mode of action of opioids is by inhibiting the release of hypothalamic GnRH.

Through their action on GnRH, they are thought to suppress LH secretion from the









pituitary in the prepubertal period and modulate LH during the estrous cycle. It has been

established that gonadal steroids suppress LH secretion by negative feedback on the

hypothalamic-pituitary axis, and this action may be brought in part by intermediate

opioidergic neurons (Brooks et al., 1986).

It has been demonstrated that follicular cysts can be induced using estradiol

benzoate by stimulating a GnRH/LH surge in the absence of a preovulatory follicle.

Subsequent exposure of the cystic cow to progesterone can resolve the cystic condition

by reinitiation of GnRH/LH surges in response to estradiol (Gumen et al, 2002).

Gumen and Wiltbank (2002) demonstrated in cows that an initial GnRH/LH surge

and subsequent ovulation could be induced with high levels of estradiol, but estradiol

induction of a subsequent GnRH/LH surge required exposure to progesterone. Follicular

cysts were induced using an intravaginal progesterone insert (IPI)/PGF2a/estradiol

benzoate (EB) protocol, and indicated that the effect is mediated at the level of the

hypothalamus (Gumen and Wiltbank, 2002).

Odore et al., (1999) suggested that ovarian cysts might be due to alterations in the

feedback regulatory mechanism. This hypothesis was based on findings that indicated a

difference in the concentration of luteinizing hormone receptors and follicle stimulating

receptors in the ovary and pituitary between normal and cystic cows.

Silvia et al. (2002) proposed the following model for how intermediate levels of

progesterone could lead to the development of ovarian follicular cysts in the dairy cow.

During the follicular stage in cows without ovarian cysts, the tonic center of the

hypothalamus secretes GnRH at a high frequency stimulating a frequency mode of LH

secretion by the anterior pituitary. The high frequency LH secretion promotes maturation









of the dominant follicle. This follicle in turn secretes increasing concentrations of

estradiol that eventually reach a threshold adequate to trigger the surge center of the

hypothalamus to release GnRH in quantities that will trigger a preovulatory LH surge and

induce ovulation of the dominant follicle. However, in cows with ovarian cysts, the

intermediate progesterone levels make the surge center of the hypothalamus insensitive to

estradiol. The preovulatory GnRH/LH surge is blocked and ovulation fails to occur.

However, the tonic center of the hypothalamus is unaffected by intermediate

progesterone concentrations and thus the high frequency, tonic pattern of LH is

maintained. This tonic pattern of LH provides the cysts with the gonadotropic support it

requires to function (Silvia et al., 2002).

Cyst Dynamics

Ovarian cysts are dynamic structures. They regress and are replaced with other

cystic structures (turnover), or spontaneous recovery occurs characterized by ovulation of

a new follicle at a site different from that of the original cysts (Cook et al. 1990). Cook et

al. (1990), induced ovarian cysts with twice daily subcutaneous injections of 15 mg

estradiol-17P3 and 37.5 mg of progesterone for 7 days starting on Day 15 post partum. Of

the 23 cows that became cystic, 7 ovulated (spontaneous recovery), 13 showed cystic

turnover and 3 persisted (Cook et al. 1990). In no case did the marked cyst ovulate. In

another study carried out on cows that calved normally over a one-year period, 12 out of

47 cows were found to be cystic. Follicular cysts were detected on the ovary as early as 8

days post partum. In 10 cows, serial rectal and ultrasound examination of the cysts

indicated an increase in size confirming the dynamic nature of ovarian cysts (Bosu and

Peter, 1987). In 4/12 cows (33.3 %) the cyst regressed before Day 28 post partum and in









the remaining 8/12 cows (66.6%) the cyst regressed between Days 29 and 57 post partum

(Bosu and Peter, 1987). In a study of 29 heifers undergoing daily transrectal ultrasound in

order to follow follicular dynamics, one heifer developed follicular cysts. This heifer

regressed the corpus luteum in a normal manner, developed a normal preovulatory size

follicle on the ovary, exhibited standing heat, but the follicle failed to ovulate. This

follicle lost follicular dominance and a new follicular wave emerged. Two dominant

follicles emerged from this wave. When one reached preovulatory size, the heifer once

again exhibited estrus but did not ovulate. This process continued through a third

follicular wave and finally the heifer was successfully treated using the Ovsynch protocol

after emergence of the fourth follicular wave (Wiltbank et al., 2002).

Diagnosis

Due to the dynamic nature of ovarian cysts, weekly rectal palpation and/or

ultrasonic observations will detect those cows with cysts more readily than monthly or

bimonthly examinations. The diagnosis of ovarian cysts is usually based on rectal

palpation of a fluid filled structure greater than 2.5 cm in diameter on one or both ovaries

in the absence of a CL. The diagnosis may be confirmed using transrectal

ultrasonography and the type of cysts (luteal or follicular cysts) determined. Luteal cysts

may also be confirmed by progesterone assay of milk or serum. The most common sign

of cows that have been diagnosed as cystic is a lack of estrus (Farin and Estill, 1993).

Nymphomania has also been noted in cows that have follicular cysts (Farin and Estill,

1993).

In order to differentiate between cystic follicles and a large preovulatory follicle,

the characteristic of the uterus must be evaluated. On rectal palpation a diagnosis of

cystic ovaries will be made when the uterus has no tone and is unresponsive to









manipulation. The uterus of a cow in estrus has tone and responds to manipulation by

increase coiling of the horns.

Treatment

Although cows with ovarian cysts have been shown to recover spontaneously,

only a portion of cows affected with cystic ovarian disease recovers spontaneously and

within a time frame that is economical to the dairy farmer. Morrow et al (1966) reported

that only 48% of cows affected with follicular cysts recovered spontaneously. Kesler and

Garverick (1982) estimated that even less cows (approximately 20%) with cystic ovaries

will spontaneously recover and return to normal cyclicity.

One of the earliest methods of resolving ovarian cysts was manual rupture via

rectal palpation. Repeated manual rupture of cysts at 6 to 10 day intervals, especially

follicular cysts in the early postpartum period, has produced 37% (Roberts, 1971) to 45%

recovery rates (Kesler and Garverick, 1982; Ijaz et al., 1987). However, manual rupture

can cause injury to the tissue of the ovary and its surrounding structures, causing

hemorrhage, promoting adhesions and infertility (Roberts, 1989; Elmore, 1986).

Gonadotropin-releasing hormone (GnRH) is commonly used to treat ovarian cysts

regardless of the type of cysts. Exogenous administration of GnRH triggers an LH surge

that results in ovulation of a LH-responsive follicle at the time of treatment or

luteinization of the cyst (Kittok, et al. 1973; Cantley et al. 1975). Repeated use of GnRH

intravenously 120 minutes apart has been shown to cause a LH surge similar to the

preovulatory LH surge in normal cycling cows; the second dose corresponding to peak

LH serum concentrations in cows with cystic follicles (Kittok et al. 1973). The LH surge

is induced with 30 minutes of GnRH administration and remains elevated for 4 hours

(Cantley et al. 1975). However, the response of the cow with ovarian cysts differs from









that of cows without ovarian cysts. that have an elevated LH concentration for up to 10

hours which may due to luteinization of the cystic structure as a result of ovarian

response to GnRH induced LH release (Cantley et al. 1975). Cow treated with GnRH

have been shown to return to estrus within 20 to 24 days (Kittok et al. 1973) after

treatment. Cows with ovarian cysts treated with a single intramuscular injection of either

50, 100 or 250 [tg GnRH exhibited estrus by 22 days and most cows exhibited estrus 18

to 23 days post-treatment (Bierschwal et al. 1975). The 100 tg dosage had the best

response of the three dosages with 82% returning to estrus and 87% of those that returned

to estrus conceiving (Bierschwal et al. 1975).

In order to shorten the interval between GnRH treatment and estrus, PGF2ca has

been used 9-14 days after GnRH treatment (Neil, 1991). However, the need for estrus

detection has been eliminated with the introduction of the Ovsynch protocol (Pursley,

1995). This protocol uses a GnRH/PGF2a/GnRH treatment scheme which has proven

successful in treating cystic ovarian degeneration (Bartolome et al., 2000; Gumen et al.,

2003).

Human chorionic gonadotropin (hCG) is used for its high luteinizing hormone

activity (Kesler and Garverick, 1982). It has the same effect as GnRH. However, hCG

has the ability to induce antibody production where as GnRH because of its small

molecular size is not likely to induce an immune response (Bierschwal, et al. 1975).A

dose of hCH between 2,500 and 10,000 IU, given intramuscularly or intravenously will

have the same effect as GnRH (treatment response and post treatment fertility). However

it can cause undesirable effects (Neil, 1991)









Progesterone has been shown to resolve ovarian cysts. Methods of administering

progesterone have included injections, intravaginal devices or ear implants. Zulu et al.

(2003) investigated the use of a progesterone releasing intravaginal device (PRID) in the

treatment of cows with ovarian cysts. Their study confirmed that follicular and luteal

cysts could be successfully treated with PRID; resulting in ovulation 2-4 days after

removal of the device with subsequent formation of a CL.

Prostaglandin F2a (PGF2a) has been used alone in the treatment of ovarian cysts

(Chavatte et al., 1993). It is the treatment of choice for luteal cysts or cysts containing

luteinized tissue. Luteal cysts respond by regression with subsequent follicular

development and estrus in 2-5 days (Kesler and Garverick, 1982; Neil, 1991). It has been

shown that there is a higher PGF2ca receptor concentration in luteal cysts than in

follicular cysts (Odore et al., 1999) and this may explain the success of prostaglandin

treatment. Prostaglandin F2a is generally believed to be ineffective in follicular cysts, but

some cows with progesterone as low as 0.5 ng/ml will respond positively (like luteal

cysts; Neil, 1991).

Timed Artificial Insemination Programs in the Dairy Cow

One of the factors affecting pregnancy rate in dairy herds is the detection of

estrus. In some instances estrous behavior is reduced. Reduced estrus behavior has been

noted in cows treated with bovine somatotropin (bST) (Kirby et al., 1997). It has been

shown that the physiological state of lactation is associated with lower concentrations of

estradiol during proestrus than levels found in non-lactating dairy cows and thus

behavioral estrus is reduced in these cows. During lactation progesterone levels are also









lower than in non-lactating dairy cows. In addition it has been shown that cows in

lactation have a lower reproductive rate than do heifers.

Heat stress is another factor that decreases the expression of estrus behavior

(Abilay et al., 1975; Nebel et al., 1997). However, lack of heat detection can play a major

role in reducing pregnancy rate. The development of programs for estrus synchronization

and timed insemination have eliminated the need for estrus detection, and have been

shown to increase conception rates to artificial insemination programs. It should be noted

however, that pregnancy rate is affected by a number of factors, such as anestrus, low

conception rates and increased embryo mortality. In many regions of the world another

factor which influences the dairy cow, especially high producing dairy cows is heat

stress.

In an attempt to return the high producing lactating dairy cow to normal

reproductive function, various pharmacological programs have been devised. These

programs manipulate ovarian function. The primary goal of estrous synchronization

programs is to synchronize estrus and ovulation effectively, such that cows may be

inseminated at a predetermined time without estrus detection and reduction in fertility

(Rathbone et al., 2001).

Several synchronization protocols have developed over the years for

synchronization of estrus. The basis of these protocols rely on one of the following: 1)

regression of the CL with PGF2a or its analogue with the cows returning to estrus and

ovulating within 2 to 4 days of the (final) injection of PGF2a; 2) the use of exogenous

progesterone or synthetic progestins to prevent estrus and ovulation for a long enough

period to allow for regression of the CL; upon removal of the exogenous progesterone or









synthetic progestins the cows should return to estrus and ovulate (Rathbone et al., 2001).

Although these pharmacological substances can be used alone, the conception rates were

found to be low and thus they have been used in combination with other reproductive

drugs to increase conception rates to timed insemination.

Prostaglandin F2a

Prostaglandin F2a given randomly during the estrous cycle between Days 5 and 16

will cause luteolysis of the CL and cows should exhibit estrus between 2 and 4 days post

treatment. The variability in return to estrus and ovulation depends on the stage of the

dominant follicle at the time of luteolysis. If luteolysis occurs when the dominant follicle

is viable, then estrus and ovulation will occur in a relatively short period from treatment.

However, if luteolysis occurs when the dominant follicle is nonresponsive, then the

dominant follicle of the following wave will grow and become the ovulatory follicle and

the interval between treatment and ovulation will be longer (Kastelic et al., 1990). For

these reasons the use of one luteolytic dose of PGF2a with insemination at a fixed time

yielded low conception rates.

Combination of Gonadotropin-releasing Hormone (GnRH) and PGF2a

Pursley et al. (1995) introduced a new method for synchronizing the time of

ovulation in cattle using GnRH and PGF2a (Ovsynch program). In this protocol, GnRH

is given at a random stage of the estrous cycle and PGF2a is given seven days later. A

second GnRH injection is administered 2 days after PGF2a and the cow or heifer was

bred 24 hours later. The first GnRH injection either caused ovulation of the dominant

follicle or had no effect. Next, PGF2a caused regression of the CL, which resulted from

the initial injection of GnRH. The seven-day waiting period between the initial GnRH









and PGF2ca injection gives the CL enough time to mature and become responsive to

PGF2ca. The final GnRH injection caused ovulation of the dominant follicle. The period of

48 hours between PGF2ca and GnRH allowed for a new follicle to emerge, grow to

preovulatory size and become sensitive to the LH surge induced by GnRH to cause

ovulation.

This study demonstrated that the Ovsynch protocol was more suited to lactating

dairy cows than heifers. Ninety percent of lactating dairy cows ovulated to the first

GnRH injection in comparison to fifty percent of heifers.

There are stages within the estrous cycle when the initiation of an Ovsynch

protocol will cause a reduction in pregnancy rates. Initiation of the Ovsynch protocol

during late luteal phase of the estrous cycle, between Days 13 to 17, may lead to

premature lysis and regression of the CL. These cows are asynchronous and exhibit estrus

prior to the second GnRH injection. In these cases insemination within 16 to 20 hours

after the last GnRH injection will be ineffective and conception is not likely (Moreira et

al., 2000a). In the early stages of the estrous cycle, Days 2 to 4, the dominant follicle is

not yet sensitive to LH and will not respond to GnRH. These follicles will ovulate to the

second GnRH injection. Follicles that fall into this category will range in age from 11 to

13 days, and have been shown to be less fertile.

The early luteal phase, between Days 5 and 11 of the estrous cycle, is the optimal

time to initiate the Ovsynch protocol to achieve acceptable pregnancy rates. As a result it

becomes important to presynchronize lactating dairy cows to an optimal stage in the

estrous cycle at which time initiation of the Ovsynch protocol is most effective.









Presynch-Ovsynch

In this presynchronization program PGF2ca is given twice, 14 days apart, and the

Ovsynch protocol is initiated 12 days after the last PGF2ca injection. Presynchronization

programs have been shown to increase the first-service pregnancy rates in comparison

with initiation of the Ovsynch protocol at random stages of the estrous cycle in lactating

dairy cows (Moreira et al, 2001). The increase in the pregnancy rate has been attributed

to the initiation of the Ovsynch protocol in the most favorable stage of the estrous cycle.

Presynchronization beginning on Day 22 post partum with a second injection 14

days later has been shown to lower the incidence of metritis-pyometra, ovarian cysts,

improve luteal activity rate on Day 50 post partum, improve estrus detection rate,

increase ovulation rate and increase pregnancy rate (L6pez-Gatius et al., 2003).

Postpartum Endometritis

Endometritis is defined as inflammation of the endometrium. The term is

descriptive and refers to the extent and anatomical distribution of the inflammatory

process. There are a plethora of papers published on the topic of postpartum uterine

infection. However, the definition of uterine infection varies and the terms endometritis

and metritis have been used interchangeably in the literature (Bretzlaff, 1987; Lewis,

1997). The following terms have been used to define uterine infection: metritis,

endometritis, and pyometra.

Postpartum endometritis is a pathologic condition usually diagnosed during the

intermediate postpartum period (Ball et al., 1984; Olson et al 1984) during routine

postpartum examination of the cow or heifer. It is commonly characterized by the

absence of estrus, a vaginal discharge of creamy-white or yellow pus and a large doughy









uterus that fails to involute. Postpartum endometritis is the most common cause of

infertility in cows. It delays uterine involution (Tennant and Peddicord, 1968; Griffin et

al., 1974), prolongs the time to first estrus, increases the number of services per

conception and prolongs the interval to calving (Griffin et al., 1974). Lewis (1997) stated

that as many as 40% of postpartum cows within a herd may be diagnosed and treated for

uterine infections. Coleman et al. (1985) stated that endometritis was a common

reproductive disorder accounting for 20% of the reproductive disorders in dairy cows.

However, Ruder et al. (1981) found that the percentage of cows with endometritis could

be as high as 67%. It has been reported that each lactating dairy cow with a uterine

infection can cost a farmer up to $106. This costs consists of treatment, loss in milk

production (associated with the infection and as a consequence of milk discarded due to

antimicrobial therapy) and the loss of cows due to culling for reproductive disorders

associated with and as a direct cause of uterine infection (Lewis, 1997).

Pathology

Ball et al. (1984) defined the postpartum period as the period from parturition to

complete involution. This period was further divided into the 'early postpartum period',

'intermediate period' and 'postovulatory period'.

The 'early postpartum period' is the period following parturition until the pituitary

becomes sensitive to GnRH, and usually lasts 8 to 14 days. The 'intermediate period'

begins when the pituitary becomes responsive to GnRH and ends with the first

postpartum ovulation. The 'postovulatory period' starts with the first postpartum

ovulation and ends with the completion of involution.

During the early postpartum period bacteria invade the uterus (Elliot et al., 1968;

Griffin et al., 1974; Peter and Bosu, 1987). Elliot et al., (1968) recovered bacteria from









93% of uteri examined between 3 to 15 days post partum. The major groups of bacteria

isolated were Streptococcus, Staphylococcus, Micrococcus, Archanobacteria formally

known as Corynebacteria, Pseudomonas and Escherichia (Elliot et al., 1968). Griffin et

al. (1974) found 92% of uteri sampled infected with bacteria between Days 1 and 7

postpartum, and 96% infected between Days 8 and 14 post partum. The fertility of cows

were not affected by the variable uterine flora or endometritis found within the first two

weeks post partum. However, cows that had a uterine infection with Archanobacterium

pyogenes following Day 21 postpartum developed severe endometritis and were infertile

to the first service (Griffin et al., 1974).

During the intermediate period and into the postovulatory period, spontaneous

clearance of the bacterial contaminant and recontamination occurs. It was noted that the

composition of uterine flora changed throughout the first 7 weeks post partum as a result

of spontaneous contamination, clearance and recontamination (Griffin et al., 1974). It is

likely that as the cow or heifer begins to cycle and the uterus comes under the influence

of high concentrations of estrogen, that infections are cleared and there is an increased

rate of repair of the endometrium. Rowson et al. (1953) demonstrated that the bovine

uterus is more susceptible to bacterial infection during the luteal phase of the estrous

cycle when progesterone is the dominant hormone. It was also noted that under the

influence of estrogen during the follicular stage of the cycle, the uterus was less

susceptible to bacterial infection. Hawk et al. (1960; 1964) demonstrated that the uterus

of heifers differed in their leukocytic response during the estrous cycle. The leukocytic

response to an inoculum of Escherichia coli was more pronounced and occurred much

faster during estrus than in the luteal phase.









During intermediate period and postovulatory period, intrauterine bacterial

infection has been associated with short estrous cycles. Peter and Bosu (1987)

demonstrated the temporal relationship between infection patterns and serum

concentrations of LH, P4 and PGF2ca metabolite (PGFM). They noted that cows with a

short first estrous cycle length were those with uterine infections. High infection rates

were associated with higher concentrations of PGFM before the first postpartum LH

surge and ovulation. Following the LH surge, increasing concentration of PGFM was

associated with increasing intensity of uterine infection. There was a further increase in

PGFM prior to the decrease in P4 and lysis of the CL. However, in cows with a normal

estrous cycle length, low PGFM was noted prior to and following the LH surge and only

prior to onset of luteolysis (Peter and Bosu, 1987).

Pathogenesis

Endometritis is a localized inflammation of the endometrium of the uterus

associated with chronic postpartum infection with pathogenic bacteria. The primary

bacterium associated with endometritis is Archanobacterium pyogenes (Elliot et al.,

1968; Griffin et al., 1974; Peter and Bosu, 1987; Bonnett et al., 1991; Dohmen et al.,

1995; 2000). Griffin et al. (1974) noted that 80% of the time Archanobacterium pyogenes

was isolated form uteri with moderate or severe endometritis. Classification of the

severity of endometritis was based on the number of neutrophils, histiocytes, plasma cells

and lymphocytes in the biopsy specimen. Moderate endometritis was defined as medium

infiltration of the stratum compactum and upper part of the stratum spongiosum and/or

three to four foci of cellular reaction per section in a number of sections. Severe

endometritis was defined as dense infiltration in the stratum compactum and stratum









spongiosum and/or five or more foci of cellular reaction per section in a number of

sections (Griffin et al., 1974).

Dohmen et al. (2000) investigated the relationship between intra-uterine bacterial

contamination, endotoxin levels and the development of endometritis in postpartum cows

with dystocia or retained fetal membranes. They found that the presence of an abnormal

cervical discharge at Day 14 post partum was higher in cows with retained fetal

membranes compared to cows that experiences dystocia. However, Archanobacterium

pyogenes was isolated more often in cows with retained fetal membranes on Day 14 post

partum than dystocia cows and was positively associated with an abnormal discharge on

Days 14 and 28 post partum. Dohmen et al. (2000) suggested that the presence of

Escherichia coli and lipopolysaccharides (endotoxins) in lochia early in the postpartum

period predisposed the development of uterine infections by A. pyogenes and Gram-

negative anaerobes.

Reduced neutrophil function plays a role in the development of endometritis.

Neutrophils function in phagocytosis and elimination of bacterial contaminants in the

uterus. In cows with retained fetal membranes, neutrophil functions such as chemotaxis

and bacterial ingestion were reduced in comparison to health cows (Cia et al., 1994). The

reduction in neutrophil function may increase the susceptibility of the postpartum dairy

cow to infection.

Diagnosis

Diagnosis of endometritis may be made based using a combination of visual

inspection of the vulva and tail, vaginoscopy, rectal palpation, uterine culture or biopsy

and/or ultrasonography. Visual inspection may reveal crusts formed on the vulva and/or

tail. There may be discharge attached to the ventral commissure of the vulva (Zemjanis,









1970). Visual inspection of the vulva and tail alone is not adequate to diagnose

endometritis.

Rectal palpation is the most common method used for diagnosing endometritis.

However, this has been noted as an insensitive and non-specific method (Bretzlaff, 1987;

Lewis, 1997). The results obtained through this method depend on the skill and

experience of the individual. The size and consistency of the uterus and cervix along with

the fluid content of the uterus are used to ascertain the presence of uterine infection

compared to the "normal" finding for a given time postpartum.

Vaginoscopy is seldom used routinely as a diagnostic technique in dairy cows

(Zemjanis 1970; Bretzlaff, 1987). Vaginoscopy, visual examination of the vagina,

luminal content and external cervical os have been shown to be a superior means of

diagnosing endometritis. LeBlanc et al. (2002) found that the prevalence of clinical

endometritis was 16.9% and vaginoscopy was required to identify 44% of these cases.

Visual and transrectal palpation of the uterus was not sufficient to identify cases oc

clinical endometritis.

Le Blanc (2002) suggested that the diagnosis of clinical endometritis should be

made by the presence of purulent uterine discharge or cervical diameter > 7.5 cm after 20

DIM, or mucopurulent discharge after 26 days in milk. This definition of endometritis is

based on the negative effect these criteria have on subsequent fertility. In the absence of

vaginoscopy a combination of history, inspection and palpation may be used to diagnose

clinical endometritis. In the absence of vaginoscopy the following criteria can be used to

classify clinical endometritis (LeBlanc et al., 2002): presence of mucopurulent or









purulent discharge on the perineum, cervical diameter >7.5 cm, and presence of a uterine

horn >8 cm in diameter.

Griffin et al. (1974) identified cases of endometritis using uterine biopsy and

uterine culture. The severity of endometritis was determined on histological findings and

the predominant bacterial flora evaluated by uterine culture. Recently, endometrial

cytology has been used to identify cows with subclinical endometritis in the postpartum

dairy cow (Kasimanickam et al., 2004). Endometrial cytology was performed on

clinically normal cows, cows without evidence of an abnormal discharge on visual

inspection of the vulva, tail and surrounding areas and vaginoscopy between Days 20 and

Day 33 post partum. Clinical endometritis was defined as > 18% polymorphonuclear

cells seen in endometrial cytology samples or fluid in the uterus at the first visit (Days 20

and Day 33 post partum) and as > 10% polymorphonuclear cells seen in endometrial

cytology samples or fluid in the uterus at the first visit (Days 34 and Day 47 post partum).

In a similar fashion to LeBlanc et al (2002) and Kasimanickam et al (2004) defined

clinical endometritis based on its impact on subsequent pregnancy rates. Although there

was no evidence of abnormal discharge on the day of enrollment, 9.1% had a discharge

within 24 hours. This indicated that evaluation of endometritis should be done on more

than one occasion. It is interesting to note that 35.1 % of cows were diagnosed with

subclinical endometritis by endometrial biopsy on the first visit and 34% on the second

visit 2 weeks later. However, 48% were diagnosed with subclinical endometritis by the

presence of fluid in the uterus on the first visit and 20.5% on the second visit 2 weeks

later. Cows with subclinical endometritis were less like to become pregnant than those

without subclinical endometritis (Kasimanickam et al., 2004).









Ultrasonography may be used to aid in the diagnosis of endometritis. One of the

sonographic indications of endometritis is the appearance of fluid in the uterine lumen.

This fluid accumulation must be distinguished from that which is seen with estrous

secretions or the embryonic fluid of the early concepts. Fluids that accumulate during

estrus and early pregnancy are less echogenic than that of endometritis (Fissore et al.,

1986).

In mild endometritis, there may be no fluid present or few fluid filled pockets found

when scanning the uterus (Kahn et al., 1989). In cases of severe endometritis, the uterus

may be distended with fluid and involve both horns (Kahn et al., 1989). The echogenicity

of the fluid that accumulated during endometritis varies with the products of

inflammation. Kahn et al. (1989) described the echogenic pattern displayed by the

products of inflammation. Echogenic patterns range from small bright spots in mild cases

to a "snow-storm effect" or, in severe cases, to almost bright white images on the screen.

Real-time-scanning may reveal turbulences within the larger collections of fluid.

Since plasma levels of 13,14-dihydro,15-keto-PGF2a (PGFM), the stable

metabolite of PGF2a, are elevated in spontaneous uterine infection, it has been suggested

that these levels may aid in the diagnosis of endometritis in the postpartum cow (Del

Vecchio et al., 1994). However, Archbald et al. (1998) were unable to correlate plasma

levels of PGFM with abnormal uteri identified by per rectum palpation during Days 24 to

29 post partum in lactating dairy cows. In addition, Archbald et al. (1998) demonstrated

that the postpartum rate of decline of plasma levels of PGFM was influenced by the

presence of a corpus luteum (CL). In cows which had a CL, the rate of decline of PGFM

was slower compared to cows without a CL. It has been reported that progesterone plays






83


an extremely important role in regulating pulsatile secretion of PGF2a from the bovine

uterus (Mann et al., 1995) since it can completely restore the number of pulses and

partially restore pulse magnitude when administered to ovariectomized ewes. However, a

decrease in progesterone appeared to be essential for pulses of PGF2a to reach a

maximum magnitude (Silvia et al., 1991).














CHAPTER 3
EXPERIMENT 1: AN EVALUATION OF PRETREATMENT WITH GnRH ON
OVARIAN RESPONSE AND PREGNANCY RATE OF LACTATING DAIRY COWS
WITH OVARIAN CYSTS SUBJECTED TO THE OVSYNCH PROTOCOL

Introduction

Bovine ovarian cysts are follicles that fail to ovulate at the time of estrus

(Garverick, 1997; 1999). They are an economic problem in the dairy cow because these

cows are infertile as long as the condition persists (Kesler and Garverick, 1982). The

exact cause of ovarian cysts is not presently known, but some predisposing factors

include age, stress, high milk production and genetics. It appears that an important

component in the pathogenesis of this condition is the inappropriate, or lack of, release of

hypothalamic gonadotropin-releasing hormone (GnRH) at the time of estrus (Refsal et

al., 1987; Giimen et al., 2002; Silvia et al., 2002). One therapeutic approach involves the

use of exogenous GnRH, which releases LH from the anterior pituitary and causes

ovulation of an ovarian follicle, and/or luteinization of the ovarian cysts (Garverick,

1997; 1999).

The occurrence of ovarian follicular waves has been adequately demonstrated in

the dairy cow without ovarian cysts (Rajakoski, 1960; Pierson and Ginther, 1984). It has

been suggested that ovarian follicular waves also occur in cows with ovarian cysts

(Garverick, 1999). The difference is that ovulation of follicles occur in cows without

ovarian cysts, but does not occur in cows with ovarian cysts. It has further been suggested

that the administration of GnRH to cows with ovarian cysts causes ovulation of a

functionally mature follicle of an ovarian follicular wave (Garverick, 1999). An injection









of GnRH at random during the estrous cycle of cows without ovarian cysts will either

cause ovulation or luteinization of large follicles present in the ovary and synchronize the

recruitment of a new follicular wave (Thatcher et al., 1989; Macmillan and Thatcher,

1991). Gonadotropin releasing hormone can advance the follicular wave by increasing

the rate of atresia in cows without ovarian cysts (Thatcher et al., 1989; Macmillan and

Thatcher, 1991).

In cows without ovarian cysts, the initiation of a protocol for synchronization of

ovulation and timed insemination (Ovsynch) at a specific stage of the estrous cycle can

influence the reproductive responses to this protocol. The stage of the estrous cycle that

appears to be the most appropriate for increased pregnancy rates using this protocol is the

early luteal phase of the estrous cycle (Vasconcelos et al., 1999; Moreira et al., 2000a).

The hypothesis of this study was that GnRH administered to cows with ovarian

cysts at the time of diagnosis will induce an early luteal phase of the estrous cycle which

will be conducive to an increased pregnancy rate to a protocol for synchronization of

ovulation and timed insemination. The objectives of this study were: i) to compare the

ovarian response of cows with ovarian cysts to no treatment and ovarian response 7 days

after treatment with GnRH; and, ii) to determine the pregnancy rate of cows with ovarian

cysts subjected to Ovsynch and pretreatment with GnRH 7 days prior to the initiation of

the Ovsynch protocol.

Materials and Methods

The study was conducted during the period of January 2002 to May 2003 in a dairy

herd (approximately 700 milking cows) in north-east Florida. Cows were milked three

times per day and were kept in covered, open-sided barns between milking. They were









fed a total mixed ration. The herd was visited weekly, and all reproductive health and

management records were computerized.

At the time of diagnosis, cows with ovarian cysts were sequentially allocated to one

of three groups (Day 0). The diagnosis of ovarian cysts was based on palpation of the

ovaries and uterus per rectum, and by ultrasonographic examination of the ovaries. The

criteria used on rectal palpation were the presence of multiple follicles on the ovary with

at least one follicle being > 17 mm diameter (Hatler et al., 2003), the absence of a corpus

luteum (CL) on either ovary, and the lack of tonicity of the uterus (Archbald et al., 1991;

Bartolome et al., 2000; Bartolome et al., 2002). On ultrasonographic examination of the

ovaries, ovarian cysts were recognized by the hypoechogenicity of the structure, and the

absence of a CL on either ovary (Pierson and Ginther, 1984). The size of ovarian cysts

was determined using ultrasonography and the recorded size was based on the largest

diameter observed on ultrasonography.

Cows in Group 1 were treated with GnRH (100 rtg, im; Cystorelin, Rhode Merieux

Inc., Athens, GA) on Day 0, and Day 7, PGF2ca (25 mg, im; Lutalyse, Pharmacia Upjohn,

Kalamazoo, MI) on Day 14, GnRH (100 ug, im) on Day 16, and timed inseminated 16-20

h later. Cows in Group 2 were treated with GnRH (100 ug, im) on Day 0, PGF2ca (25 mg,

im) on Day 7, GnRH (100 ug, im) on Day 9, and timed inseminated 16-20 h later. Cows

in Group 3 were not treated with GnRH on Day 0, but were treated with GnRH (100 ug,

im) on Day 7, PGF2ca (25 mg, im) on Day 14, GnRH (100 ug, im) on Day 16, and timed

inseminated 16-20 h later (Figure 3-1). Pregnancy was determined by rectal palpation of

the uterus between 45-50 d after timed insemination using previously described

techniques (Zemjanis, 1970).












Group I (GnRH + Ovsynch)
GnRH GnRH PGF2a GnRH TAI Pregnancy Diagnosis

Day 7 14 16 17/18 62-68


Group 2 (Ovsynch)

GnRH PGF2a GnRH TAI Pregnancy Diagnosis

Day 0 7 9 10/11 54-61


Group 1 (No Tx + Ovsynch)
No Tx GnRH PGF2a GnRH TAI Pregnancy Diagnosis


Day 0 7 14 16 17/18 62-68

Figure 3-1. Experiment 1 Experimental Design

On both Days 0 and 7, cows in all groups were subjected to ultrasonographic

examination of the ovaries. On both Day 0 and Day 7, blood samples for progesterone

(P4) concentration were obtained from all cows and placed on ice immediately after

collection. Samples were centrifuged at 5000 x g for 15 minutes, and serum was stored at

-200C until assayed for progesterone using a solid-phase, no-extraction RIA previously

described (Srikandakumar et al., 1986). Serum progesterone concentration on Day 0 was

classified as high (> 0.5 ng/ml) or low (< 0.5 ng/ml). A serum progesterone concentration

>1 ng/ml on Day 7 was used to confirm the presence of a functional corpus luteum.

Statistical Analysis

Baseline data for parity, DIM, time of year and P4 on Day 0 were compared using

Chi-square (P<0.05). The outcomes of interest for this experiment were ovarian response

7 days after treatment with GnRH, and pregnancy rate. Data for ovarian response on Day