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Effects of Induction of Ovulation during Early Lactation on Uterine Health and Fertility in Dairy Cows

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Title:
Effects of Induction of Ovulation during Early Lactation on Uterine Health and Fertility in Dairy Cows
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1 online resource (96 p.)
Language:
english
Creator:
Bittar, Joao Henrique
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Veterinary Medical Sciences, Veterinary Medicine
Committee Chair:
Galvao, Klibs Neblan Alves
Committee Members:
Santos, Jose Eduardo
Thatcher, William W
Risco, Carlos A

Subjects

Subjects / Keywords:
cow -- dairy -- fertility -- gnrh -- ovulation
Veterinary Medicine -- Dissertations, Academic -- UF
Genre:
Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Early ovulation postpartum is fundamental for efficient fertility.Cows having more estrous cycles before matting have higher hazard of pregnancyafter the voluntary waiting period. The objective was to evaluate the effect ofGnRH given early postpartum on induction of ovulation, uterine health andfertility in dairy cows. Holstein cows without corpus luteum (CL) at 17 DIMwere randomly assigned to receive i.m. GnRH (n = 245) at 17 and at 20 DIM orremain as control (n = 245). Ultrasonography (US) of cows’ ovaries wasperformed twice a week totaling four US. Ovulation characterized by appearanceof CL = 20 mm at any US or CL P P = 0.2) or cytological endometritis (30.9%vs. 32.8%, GnRH vs. control respectively; P= 0.6). Prevalence of clinical endometritis increased in cows that hadcalving problems (32.6% vs. 15.9%; P= 0.001) and metritis (40.6% vs. 15.8%; PP P = 0.2) or 74 d after AI (35.0% vs. 31.5%;P = 0.5), but reduced pregnancy loss(6.8% vs. 18.1%; P = 0.002). Interactionbetween GnRH treatment and ovulation showed that GnRH-treated cows that ovulatedhad increased hazard of pregnancy by 300 DIM compared to GnRH and control cowsthat did not ovulate (HR = 2.0 and HR = 1.3, respectively P P = 0.7). GnRH early postpartum inducedovulation without affects uterine health, and improved reproductive performanceby the reduction of pregnancy loss and increase hazard of pregnancy for cowsthat ovulated to the GnRH.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Joao Henrique Bittar.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Galvao, Klibs Neblan Alves.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045997:00001

MISSING IMAGE

Material Information

Title:
Effects of Induction of Ovulation during Early Lactation on Uterine Health and Fertility in Dairy Cows
Physical Description:
1 online resource (96 p.)
Language:
english
Creator:
Bittar, Joao Henrique
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
Publication Date:

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Veterinary Medical Sciences, Veterinary Medicine
Committee Chair:
Galvao, Klibs Neblan Alves
Committee Members:
Santos, Jose Eduardo
Thatcher, William W
Risco, Carlos A

Subjects

Subjects / Keywords:
cow -- dairy -- fertility -- gnrh -- ovulation
Veterinary Medicine -- Dissertations, Academic -- UF
Genre:
Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Early ovulation postpartum is fundamental for efficient fertility.Cows having more estrous cycles before matting have higher hazard of pregnancyafter the voluntary waiting period. The objective was to evaluate the effect ofGnRH given early postpartum on induction of ovulation, uterine health andfertility in dairy cows. Holstein cows without corpus luteum (CL) at 17 DIMwere randomly assigned to receive i.m. GnRH (n = 245) at 17 and at 20 DIM orremain as control (n = 245). Ultrasonography (US) of cows’ ovaries wasperformed twice a week totaling four US. Ovulation characterized by appearanceof CL = 20 mm at any US or CL P P = 0.2) or cytological endometritis (30.9%vs. 32.8%, GnRH vs. control respectively; P= 0.6). Prevalence of clinical endometritis increased in cows that hadcalving problems (32.6% vs. 15.9%; P= 0.001) and metritis (40.6% vs. 15.8%; PP P = 0.2) or 74 d after AI (35.0% vs. 31.5%;P = 0.5), but reduced pregnancy loss(6.8% vs. 18.1%; P = 0.002). Interactionbetween GnRH treatment and ovulation showed that GnRH-treated cows that ovulatedhad increased hazard of pregnancy by 300 DIM compared to GnRH and control cowsthat did not ovulate (HR = 2.0 and HR = 1.3, respectively P P = 0.7). GnRH early postpartum inducedovulation without affects uterine health, and improved reproductive performanceby the reduction of pregnancy loss and increase hazard of pregnancy for cowsthat ovulated to the GnRH.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Joao Henrique Bittar.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Galvao, Klibs Neblan Alves.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Classification:
lcc - LD1780 2013
System ID:
UFE0045997:00001


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1 EFFECTS OF INDUCTION OF OVULATION DURING EARLY LACTATION ON UTERINE HEALTH AND FERTILITY IN DAIRY COWS By JOO HENRIQUE JABUR BITTAR A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFIL LMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Joo Henrique Jabur Bittar

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3 To my parents, Elias and Oneida, my family, friends and colleagues for all the support and encouragement given to me that helped surpass the barriers and challenges of this journey. To God and to one of most hard worker class of the world called farmers. And especially to my wife, Cassandra Bittar, who stood on my side in all moments giving me strength and maturity to act with the adequate manner through all situations.

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4 ACKNOWLEDGMENTS First, I want to express my sincere gratitude to a young professor and researcher that gave me the opportunity and financial support to pursue this MS pr ogram, and to stand as the chairman of my committee, Dr. Klibs N. Galvo. Thanks for the valuable insights, teaching and orientation given during this program, specially the trust and acceptance to have me as his first graduate student. The importance of the member of my graduate committee, composed of Dr. Carlos A. Risco, Dr. Jos Eduardo P. Santos and Dr. William W. Thatcher is well appreciated. Their acceptance to be part of this committee, their valuable inputs, orientation and guidance were of enormou s value to my training Their willingness to share knowledge enhanced even more my respect for them as scientists. Being part of the combine program of residency and graduate student of the Food Animal Reproduction and Medicine Service (FARMS) of the La rge Animal Clinical Sciences Department I would like to express my great appreciation for all of members, Dr. Arthur Donovan, Dr. Carlos Risco, Dr. Fiona Maunsell, Dr. Klibs Galvo, Dr. Owen Rae and Dr. Jorge Hernandez, and also to my house office colleagu es, Dr. Gabriele Meier and Dr. Lucas Ibarbia, as and Mrs. Laura Neumann. Thanks for the opportunity and team work allied with the understanding that provided me the best situation to achieve one of my desired goals in professional life. Special acknowledgments are extended to my graduate student colleagues of the College of Veterinary Medicine, Department of Animal Sciences and Department of Agronomy at the UF, which hel ped me in a multitude of aspects, from data acquisition and analysis, through guidance and learning discussions up to the friendship made and encouragement during the

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5 program. Among them, Rafael Bisinotto, Eduardo Ribeiro, Leandro Greco, Natlia Martinez P atino and Fbio Lima deserv e special thanks. Thanks also to students of the Department of Animal Sciences, veterinary students, and veterinarians Achilles Neto, Carol Cive, Csar Pierobom, Kimberly Hencken, Lauren Stevenson, Manon Vercouteren Mathew Taylor M ohanathas Gobikrushanth; Sabrina Freitas and Stephanie Croyle for their contribution in the realization of this research. And last but not least, my sincere gratitude to the management team and staff of Dairy Unit of the University of Florida represent ed by Eric Diepersloot, and also to North Florida Holstein, represented by the owner Mr. Don Bennink. The recognition of the hard work and commitment to help during the development of the study and for the use of their cows are sincerely appreciated. Speci al thanks to Angel and Jeremias from North Florida Holstein dairy to the commitment and exceptional help during the study.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 7 LIST OF FIGURES ................................ ................................ ................................ ......................... 8 LIST OF ABBREVIATIONS ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................................ ... 11 CHAPTER 1 LITERATURE REVIEW ................................ ................................ ................................ ....... 13 Estrous Cycle ................................ ................................ ................................ .......................... 13 Folliculogenesis ................................ ................................ ................................ ...................... 16 Resumption of Ovarian Cyclicity Postpartum ................................ ................................ ........ 19 Negative Energy Balance Postpartum ................................ ................................ .................... 20 Early Ovulation Postpartum ................................ ................................ ................................ ... 22 GnRH and Ovulation ................................ ................................ ................................ .............. 24 2 INTRODUCTION ................................ ................................ ................................ .................. 34 3 MATERIALS AND METHODS ................................ ................................ ........................... 37 Cows, Housing and Feeding ................................ ................................ ................................ ... 37 Study Groups, BCS and Ultrasonography of the Ovaries ................................ ...................... 38 Uterine Health ................................ ................................ ................................ ......................... 39 Reproductive Management ................................ ................................ ................................ ..... 40 Sample Size and Statistics ................................ ................................ ................................ ...... 42 4 RESULTS ................................ ................................ ................................ ............................... 46 Descriptive Statistics ................................ ................................ ................................ .............. 46 Ovulation ................................ ................................ ................................ ................................ 46 Uterine Health Outcomes ................................ ................................ ................................ ....... 47 Reproductive Outcomes ................................ ................................ ................................ .......... 47 5 GENERAL DISCUSSION AND CONCLUSIONS ................................ .............................. 66 LIST OF REFERENCES ................................ ................................ ................................ ............... 79 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 9 5

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7 LIST OF TABLES Table page 4 1 Descriptive statistics for GnRH treated and control groups ................................ .............. 51 4 2 Effects of GnRH administration at 17 and 20 3 days postpartum on ovulation response in dairy cow s ................................ ................................ ................................ ....... 52 4 3 Effects of GnRH administration at 17 and 20 3 days postpartum and other variables on prevalence of clinical and cytological endometritis in dairy cows ................ 53 4 4 Effects of GnRH administration at 17 and 20 3 days postpartum in dairy cows without a CL at study enrolment on conception rate at PD1 from first service ................. 54 4 5 Effects of GnRH administration at 17 and 20 3 days postpartum in dairy cows without a CL at study enrolment on conception rate at PD2 from first service ................. 55 4 6 Effects of GnRH administration at 17 and 20 3 days postpartum in dairy cows without a CL at study enrolment on pregnancy loss of the first service ............................ 56 4 7 Effects of GnRH administration at 17 and 20 3 days postpartum during the first month of lactation on time to pregnancy up to 300 DIM based on PD2 for Model 1 (Multivariable analysis with all variables included, except ovulation) ............................. 57 4 8 Effects of GnRH administration at 17 and 20 3 days postpartum during the first month of lactation on time to pregnancy up to 300 DIM based on PD2 for Model 2 (Multivariable analysis with all vari ables included, except treatment status) ................... 58 4 9 Effects of GnRH administration at 17 and 20 3 days postpartum during the first month of lactation on time to pregnancy up to 300 DIM based on PD2 for Model 3 (Multivariable analysis with all variables included) ................................ .......................... 59

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8 LIST OF FIGURES Figure page 1 1 Schematic depiction of the patter n of secretion of FSH, LH and progesterone; and pattern of growth of ovarian follicles during the estrous cycle in cattle ............................ 28 1 2 Schematic presentation of factors involved in the growth, ma intenance and regression of the CL ................................ ................................ ................................ ........... 29 1 3 Schematic of follicle growth and FSH for a cow that has two follicular waves during a 21 d estrous cycle ................................ ................................ ................................ ............ 29 1 4 A summary of folliculogenesis in the ewe, developed with data from several sources .... 30 1 5 Diagrammatic scheme of resumption of dominant follicles and ovarian cycles dur ing the postpartum period in dairy and beef suckle d cows not nutritionally stressed ............. 31 1 6 Schematic representation of nutritional status during peripartum, lactation and reproductive function during early postpartum in anovulatory and anovulatory cows at first follicular wave ................................ ................................ ................................ ........ 32 1 7 Metabolic mechanisms linking negative energy balance and oocyte quality in high producing dairy cows ................................ ................................ ................................ ......... 33 3 1 Schematic diagram of study activities ................................ ................................ ............... 45 4 1 Time to ovulati on up to 70 DIM for GnRH treated group ( n = 128) and control group ( n = 127) in Dairy 1 ................................ ................................ ................................ ........... 60 4 2 Time to pregnancy at PD2 up to 300 DIM for GnRH treated group (GNRH; n = 240) and control group (CON; n= 240) ................................ ................................ ...................... 61 4 3 Time to pregnancy at PD2 up to 300 DIM for cows with a CL in the beginning of the study control group and GnRH treated group ................................ ................................ 62 4 4 Time to pregnancy at PD2 up to 300 DIM according to ovulation status for cows that ovulated ( n = 297) and cows that did not ovulate ( n = 183) ................................ .............. 63 4 5 Time to pregnancy at PD2 up to 300 DIM according to ovulation status for cows that entered the study without CL and did not ovulate by 24 DIM for cows t h at entered the study without a CL and ovulated by 24 DIM, and for cows with a CL in the beginning of the study ................................ ................................ ................................ ........ 64 4 6 Time to pregnancy at PD2 up to 300 DIM according to treatment*ovulation status for GnRH treated cows that ovulated or did not ovulate and for control cows that ovulated or did not ovulate by 24 DIM ................................ ................................ ............. 65

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9 LIST OF ABBREVIATIONS AFC Antral follicle count AI Artificial insemination AKT Protein Kinase B AMH Anti M llerian hormone BCS Body condition score B HBA hydroxybutyric acid BMP5 Bone morphogenetic protein 5 CE Clinical endometritis CI Confidence interval CL Corpus luteum Cox 2 Cyclooxygenase 2 CR Conception rate CTE Cytological endometritis DF Dominant follicle DIM Days in milk FSH Follicle stimulating h ormone FOXO3 Forkhead box O3 GC Granulosa cells GDF 9 Growth differentiation factor 9 GH Growth hormone GnRH Gonodo t r opin releas ing hormone HR Hazard ratio IGF 1 Insulin like growth factor 1 LH Luteinizing hormone

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10 LH R Luteinizing hormone receptor mTOR Mamm alian target of rapamycin NEB Negative energy balance NEFA Non esterified fatty acids OR Odds ratio P/AI Pregnancy per artificial insemination PGF 2 Prostaglandin F 2 PL Pregnancy loss PP Postpartum SOP Standard operation procedure TC Theca cells TAI Timed artificial insemination Tbp2 TATA binding protein 2 TaF4b TATA box binding protein associated factor UK United Kingdom US Ultrasonograph y examination VWP Voluntary waiting period

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11 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 EFFECTS OF INDUCTION OF OVUL ATION DURING EARLY LACTATION ON UTERINE HEALTH AND FERTILITY IN DAIRY COWS By Joo Henrique Jabur Bittar August 20 13 Chair: Klibs Neblan Alves Galvo Major: Veterinary Medical Sciences Early ovulation postpartum is fundamental for optimal fertility Cows having more estrous cycles before matting have higher hazard of pregnancy aft er the voluntary waiting period The objective was to evaluate the effect of GnRH given early postpartum on induction of ovulation, uterine health and fertility in dairy cows. Ho lstein cows without corpus l uteum ( CL ) at 17 DIM were randomly assigned to receive i.m. GnRH (n = 24 5 ) at 17 and at 20 DIM or remain as control (n = 2 45 ). scanned by ultrasonography twice a week t otal ing four ultrasonographies (US) Ovul ation was characterized by the appearance of a US or CL < 20mm in two consecutive s US. Clinical and cytological endometritis were diagnosed at 35 DIM. Data were analyzed using the LOGISTIC and PHREG procedure s of SAS adjusting for the eff ects of dairy, parity, calving related problems, me tabolic problems, and metritis. GnRH increased ovulation up to 3.5 d after the last treatment (78.7 % vs. 45.0%; P < 0.001) and did not affect the pr evalence of clinical endometritis (23.9 % vs. 18.6 % GnRH vs. control respectively ; P = 0 .2) or cytological endometritis (30.9 % vs. 32.8 % GnRH vs. control respectively ; P = 0 .6). P revalence of clinical endometritis increased in c ows that had calving problems (32.6 % vs. 15.9%; P = 0 .001) and metritis (40.6 % vs. 15.8%; P < 0.001) Metritis increased prevalence of cytological endometritis (50.7 % vs. 23.5%; P < 0 .001). T reatment with GnR H did not affect

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12 conception rate at 32 (37.6 % vs. 38.6%; P = 0 .2) or 74 d after AI (3 5 0 % vs. 3 1 5 %; P = 0 5), but reduced pregnanc y loss (6.8% vs. 18. 1 %; P = 0 .0 02 ). I nteraction between GnRH treatment and o v ulation showed that GnRH treated cows that ovulate d had increased hazard of pregnancy by 300 DIM compared to GnRH and control cows that did not ovulate (HR = 2.0 and HR = 1.3 P < 0 .05 respectively ) but hazard of pregnancy was similar to control cows that ovulate d ( P = 0.7 ). GnRH early postpa rtum induced ovulation without a ffect ing uterine health but failed to improve reproductive performance although it reduc ed pregna ncy loss

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13 CHAPTER 1 LITERATURE REVIEW Estrous Cycle The estrous cycle in cattle is a dynamic process represent ing the cyclical pattern of ovarian activity between ovulations t hat allows females to move from a reproductive period of non receptivity to receptivit y given repeated opportunities to the cow or heifer to become pregnant (Forde et al., 2011). The bovine female is non seasonal polyestrous cyclic animals with a uniform distribution of estrous cycles throughout the year that allows them to conceive year ro und. The estrous cycle lasts for approximately 18 to 24 days and compromise the period in between two consecutive estrus events, which normally is composed by two or three follic ular waves (Savio et al., 1988; Ginther et al., 1989; Endo et al., 2012). Its onset occurs at the time of puberty and the cycle is normally divided into two distinct phases, the luteal and the follicular phase. The luteal phase starts right after ovulation with CL formation during the metestrus, characterized by low concentrations o f estrogen and progesterone, until the fully formed CL develops and regresses also known as the diestrus, characterized by the fully functional activity of the CL relative to progesterone production and lasts for approximately 13 15 days (Forde et al., 201 1 ) The follicular phase initiate s with the onset of CL lys i s and decline in concentration of progesterone, also known as proestrus, and is complete during the acceptance of mating, known as estrus (Lee et al., 1988). The anterior pituitary under influence of GnRH will induce release of FSH and LH, and in concert with adequate concentrations of IGF 1 and insulin, follicles are recruited and grow during the luteal phase. However, follicles are not able to ovulate because progesterone exerts a negative feedba ck in the hypothalamus blocking the GnRH/ LH surge naturally induced by high concentrations of estradiol (Figure 1 1; Forde et al., 2011).

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14 Once the main source of progesterone is removed by lysis of the CL the concentration of estradiol, produced by the d ominant follicle, increases and induces estrus behavior and exerts a positive feedback on the hypothalamus to cause the GnRH/ LH surge, which will induce ovulation of the dominant follicle 24 30 hours later (Ke s ner et al., 1981; Vai l es et al., 1992). After ovulation, the newly ovulated follicle undergoes cellular and structural remodeling, giv ing rise to the transient corpus hemorrhagicum, present from right after ovulation up to 3 days later, and finally later the formation of the corpus luteum, in which si gnificant levels of progesterone will be produced about one week after ovulation. There are several process es and factors involved with CL growth, maintenance and regression as described by Skarzynski et al. (2008a) and shown in Figure 1 2. After ovulation the CL forms from the wall of the ruptured follicle, and its growth and vasculariz ation occur s rapidly. In the cow, the weight of the CL 3 days after ovulation averages 640 mg, whereas on day 14, the average is 5.1 g (Fields and Fields, 1996) and most of this rapid mass increase is due to hypertrophy of granulosa (GC) and theca cells (TC) and also TC mitotic division. Endothelial cells and fibroblasts mito sis and growth also contribute to this rapid growth The CL is a complex tissue composed of parenchy mal (small and large steroidogenic) and non parenchymal (fibroblast, vascular smooth muscle, pericytes and endothelial) cells (Reynolds and Redmer 1999). The preovulatory LH surge results in luteinization of GC and TC Within a few hours post ovulation, t he GC give rise to the large luteal cells and the TC give rise to the small luteal cells. With the disruption of the basement membrane, these two cell types will be intermixed and remain in close contact during the reorganization of the follicle into the C L (Stocco et al., 2007). Luteinization of GC and TC alter s their steroidogenic pathway, in which progesterone will be the primary steroid produced. Before ovulation, the GC aromatize androgens produced by the theca cells, and estradiol was the main

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15 steroid being produced (Fortune and Quirk, 1988). Progesterone mainly produced by the CL will influence several organs and tissues preparing the uterus for gestation and maintenance of pregnancy by induction of a quiescen t state in the myom e trium (Csapo a nd Pulkk inen 1978), stimulation of the uterine glands, and suppressi on of the maternal immune response to fetal antigens (Siiteri et al., 1977; McCracken et al., 1999). After exposure to estradiol, exposure to p rogesterone upregulates progesterone receptor s in th e reproductive tract. In contrast, progesterone downregulates receptors for estradiol and thereby blocks many of the actions of estrogens that generally act as mitogenic factors (reviewed by Niswender et al., 2000). L uteinizing hormone and growth hormone p lay an important role in the maintenance of the CL and progesterone production H ypophysectomized ewes treated with growth hormone had circulating progesterone concentrations higher than untreated ewes W hen LH was also replaced the hypophysectomized ewes had similar parameters of luteal function as pituitary intact control ewes ( J uengel et al., 1995 ). The process of the CL lyses initiate s with progesterone losing the ability of block the formation of oxytocin receptors in the uterus, and the rise in estra diol combined with the increase in oxytocin and oxytocin receptors will act through a COX 2 pathway to induce the pulsatile release of the luteolysin ( McCracken et al., 1999) pulses start to increase and through a count ovary and trigger the lysis of the CL ( Thorburn and Nicol, 1971; McCracken et al., 1972). The lyses of the CL follows a fashion in which first there is an inhibition of progesterone synthesis and a reduction o f the blood flow into the CL characterizing the functional luteolysis. In addition, later the structural luteolysis takes place in which the immune response leads to the programmed cell death of the luteal tissue (McCracken et al., 1999). The lifespan of t he CL

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16 normally last for 15 to 17 days in a normal cycle and lasts the duration of gestation in pregnant cows. If, by day 1 5 17 of the oestrous cycle, the maternal recognition of pregnancy signal (interferon tau) has not been detected, luteolysis occurs. W ith the absence or low concentrations of progesterone in blood, the block on LH surge is removed leading to LH surge and ovulation. With the advances of ultrasonography, a better understanding of uterine and ovarian dynamics throughout the estrous cycle a nd pregnancy has been achieved Three functionally critical follicular sizes have been characterized during the stages of antral follicular growth (Figure 1 3): emergence (approximately 4 mm), deviation (approximately 9 mm), and ovulation (from 10 to 20 mm ). These classifications helped to unravel the mechanisms associated with anovulatory condition s and led to corrective interventions (Lucy et al., 1992 ; Thatcher et al., 1996; Wiltbank et al., 2002; Sartori et al., 2004). Folliculogenesis The total reservo irs of available follicles during the entire reproductive life period of the female are the primordial germ cells, which give rise to primordial follicles. Anti Mullerian hormone (AMH) maintains the balance between the number of primordial follicles remain ing in the arrested pool and the follicles being activated by gonadotropins (Snchez and Smitz, 2012). The mechanism involved in the follicular growth from primordial to primary stages is not completely known, although advances in molecular biology ident ified several pathways. Growth factors and Kit ligans will activate the PI3K (phosphatidylinositol 3 kinase) pathway. Through a cascade of events, AKT (a serine/threonine protein kinase that enhances cellular proliferation and survival) is able to phosphor ylate FOXO3 (Forkhead box 3, a transcription factor that leads to apoptosis and cell cycle arrest) and PI3K also phosphorylates and inactivates an inhibitor of mTOR (Mammalian target of rapamycin, a serine/threonine protein kinase that regulates cell growt h). Phosphorylated FOXO3 will not be able to enter into the nucleus and therefore will not

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1 7 be able to promote follicle arrest. C oncomitantly activated mTOR will signal protein translation and follicle development will progress (Snchez and Smitz, 2012). P rogression from primary follicles to secondary follicles is driven by several intraovarian factors produced by the oocyte, GC and TC ( Kol and Adashi 1995) Growth differentiation factor 9 (GDF9) and Bone morphogenetic protein 15 (BMP15) promot e GC prolife ration (i.e., GDF9) in the early stages beyond the primary stage and stimulating the proliferation of GC in a FSH independent manner ( i.e. BMP15; Yan et al., 2001 ). Two other transcription factors are required for the progression of early preantral follicl e development, TATA binding protein 2 (Tbp2) and TATA box binding protein associated factor 4 (Taf4b). Once the secondary follicles are developing, FSH and LH receptors start to be expressed and a rapid follicular growth takes place Insulin like growth fa ctor (IGF) also is important for follicle development (Snchez and Smitz, 2012). With the abrupt proliferation of GC, acti vin production decreases and inhibin production increases to avoid the uncontrolled proliferation of GC (Findlay et al., 2000). The fo llicle at this point of development, becomes an independent unit that is less affected by local stimulus but is now dependent on gonadotrophin hormones. The antral follicle is characteriz ed by differentiation of GC into cumulus cells and mural cell compar tments, and the antrum formation and accumulation of estr adiol. G ranulosa cells utilize androgens produced by theca cells in response to LH stimulation as a substrate for estradiol production gonadotrophin m odel ( Fortune and Armstrong, 1976; Hillier et al., 1994). Elevated FSH levels released by the anterior pituitary induce recruitment of a cohort of follicles from the gonadotrophin sensitive pool within the ovary. E stradiol produced by these antral follicles in conjunction to low levels of inhibin will exert a positive feedback on the hypothalamus pituitary axis to stimulate basal LH secretion. As the follicles grow, inhibin

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18 secretion increases to the point in which combined with estradiol causes a negative f eedback on the anterior pituitary to reduce FSH secretion (Haughian et al., 2013). At this point the deviation of the dominant follicle occurs at approximately 8 mm of diameter (Ramasharma et al., 1981). All the events involved in selection and dominance o f the dominant follicle are not completely understood. The dominant follicle is the faster growing follicle that will express LH receptors in the granulosa cells at approximately 8 mm in diameter By approximately 8 mm in diameter the follicles express LH receptors (Figure 1 4) that will be functionally competent when the follicle reaches the size of approximately 10 mm and is capable of ovulating in response to an LH surge or injection of GnRH that stimulates the release of LH (Sartori et al., 2004; Beg a nd Ginther, 2006). The time from primordial to preovulatory stage is approximately 180 days and the time from the antral to the preovulatory stage is approximately 40 days (Lussier et al ., 1987). In an environment of low progesterone concentrations, high e stradiol lead to a GnRH surge, leading to the preovulatory LH surge, this will lead to disruption of the gap junctions of the oocyte granulosa cells, termination of the meiotic arrest and ovulation (Conti et al., 2012). Alternatively, in a high progesteron e environment, the GnRH/ LH is prevented and the dominant follicle becomes atretic allowing for the release of FSH and recruitment of a new cohort of follicles (Forde et al., 2011 ). Luteinizing hormone (LH) is important for the final growth stages of the fo llicle prior to ovulation and for the ovulation process ( Ireland and Roche, 1983; Ginther et al., 2013). A recent study evaluated the antral follicle count (AFC), which is defined as the number of follicles present in the ovaries with a diameter equal or greater than 3 mm, and is classified as a poor respons e to FSH stimulation compared to follicles in the high AFC group. Consequently,

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19 cows of the low AFC grou p have lower estradiol and Anti Mllerian hormone (AMH) production. This new findings provide insight into the potential mechanisms involved with ovarian dysfunction in females with low AFC (Scheetz et al., 2012 ). AMH is expressed only in the gonads, acts to inhibit the recruitment of primordial follicles into the pool of growing follicles, and avoids premature exhaustion of the ovarian follicular reserve, and AMH expression is high in small healthy follicles. AMH becomes a reliable endocrine marker for th e prediction of a high and healthy population of AFC responsive to gonadotropins (Monniaux et al., 2013). Discovery of more regulatory factors of the follicle growth were made recently and strongly suggest a role of angiotensin II in follicular selection a nd development and ovulation (Gonalves et al., 2012). Resumption of Ovarian Cyclicity Postpartum The first follicular waves postpartum in dairy cows usually start about 5 10 days after parturition and it is followed by clearance of gestational estradiol from blood (Beam and Butler, 1997) which remove s the inhibition on FSH release from the anterior pituitary gland. Consequently FSH leads to follicular recruitment (Adams et al., 1992). Follicles c an then grow in 2 different patterns ( Figure 1 5 ) : 1 with adequate levels of IGF 1 and insulin leading to ovulation ; the follicle will continue to grow beyond 5 mm with the indirect stimulation of GnRH that will induce FSH and LH pulses from the anterior pituitary, and after deviation and selection the rise in E 2 induces the surge of LH from the GnRH surge center in the brain that leads to ovulation (Wiltbank et al., 2002) ; 2 with inadequate levels of IGF 1 and insulin leading to anovulation Anovulation can be subdivided in three categories. In the f irst catego ry, follicles are recruited and grow to the point of acquisition of dominance and ovulatory capacity, but lack GnRH/LH surge due to inadequate levels of estradiol produced by the dominant follicle This condition is associated with lower concentrations of IGF 1, glucose, and high concentrations of

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20 NEFA characteristic of negative energy balance (Chagas et al., 2007). In the second category, follicles do not develop beyond the selection phase This condition is a result of severe undernourishment T he GnRH ne urons become very sensitive to the negative feedback of estradiol GnRH and consequently FSH/LH release is compromised which prevent follicle growth beyond the selection phase (Murphy et al., 1990). In the third category follicles reach ovulatory size but fail to ovulate. This condition is called ovarian cystic degeneration cystic ovarian disease or cystic follicles This pathologic condition affect s approximately 9.5 % to 25 % of the dairy cows (Whitmore et al., 1974) Cystic follicles are believed to be formed after an LH surge that fails to induce ovulation (De Silva and Reeves, 1988; Hamilton et al., 1995; Garverick, 1997; Gmen and Wiltbank, 2002; Kaneko et al., 2002 ; Todoroki et al., 2004) Negative Energy Balance Pos t partum C lose to parturition da iry cows undergo a period of complex endocrine and metabolic changes to adjust to milk production right after calving The nutrient demand s are five fold higher 4 days postpartum compared to 250 days of gestation (Bell, 1995). While the energy demands for production increase rapidly dry matter intake increases slowly which puts cows in a state of negative energy balance (NEB) To compensate for this gap of required nutrient s the re is a massive mob iliz ation of body fat as nonesterified fatty acids (NEFA) i nto the bloodstream. The liver is challenged to produce high amounts of glucose and to oxidize fatty acid s (Butler et al., 2006). The NEFA are utilized to make upwards of 40% of milk fat during the first days of lactation ( Bell, 1995 ). Given that plasma NEFA concentrations increase in response to increased energy needs accompanied by inadequate feed intake, DMI and plasma NEFA concentrations usually are inversely r elated. T he liver takes up NEFA in proportion to their supply ( Pullen et al., 1989 ; Reynolds et al., 2003 ) ; therefore, cows are predisposed to accumulate NEFA as triglycerides within liver when large amounts of NEFA are released from adipose tissue into the

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21 circulation ( Emery et al., 1992 ). Nonesterified fatty acids are metabolized in the liver to more available substrates such as ketone bodies. The three endogenous ketone bodies are acetone, acetoacetic acid, and beta hydroxybut yric acid (BHBA) although BHBA is not technically a ketone but a carboxylic acid. Ketone bodies can be used as an energy source for muscle and nervous tissues ; h owever, its excess is associated with depression in feed intake and increases the risk of clin ical diseases such as fatty liver, ketosis, displaced abomasum, metritis, etc, and subsequent decrease of milk production (Duffield et al., 2009). BHBA administration has been shown to reduce feed intake in pigs (Mller et al., 1984 ) and ewes ( Schlumbohm a nd Harmeyer, 2004). Nonetheless, a recent study fail to show a decrease in feed intake with infusion of BHBA in mid lactation dairy cows (Zarrin et al., 2103). Although feed intake was not reduced, glucose concentrations in blood were reduced because of a decrease in glucagon concentrations. The severity of the NEB is directly related to the decrease in dry matter intake (DMI), and it is associated with lower blood concentrations of metabolic hormones, insulin and IGF 1 (Butler et al., 2006). Growth hormone stimulates IGF 1 which in turn stimulates the development of follicles, whereas high concentration of NEFAs inhibit the proliferation rate of granulosa cells and increase theirs apoptotic rate in vitro (Jorrit s ma et al., 2004; Vanholder et al., 2005 ) Sev ere NEB re duces the pulse frequency of LH, hence compromis ing estradiol production f ollicular selection, maturation and ovulation (Butler, 2006). O vulation occurs on average two weeks after the nadir in NEB (Butler, 2003). Longer periods of NEB are detrim ental to reproduction because of delay in first ovulation (Figure 1 6) and negative effects on oocyte quality (Figure 1 7). The extent of NEB is correlated to body condition score (BCS) loss early postpartum, and cows experiencing higher losses of BCS in the first month of lactation hav e the most severe NEB and consequently hav e longer intervals to first ovulation, reduced risk of conception and

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22 increased risk of culling (Butler, 2012). Another carryover negative effect of NEB is the lower circulating leve ls of IGF 1 that may be associated with reduced fertility, or also lower quality and lower viable embryos recovered from high milk yield cows in early postpartum stages (Sartori et al., 2002). Characterization of metabolic profiles of cows that did or did not ovulate the follicle of the first follicle wave postpartum demonstrated that ovulatory cows had lower concentration of growth hormone and NEFAs, higher concentration of IGF 1 and insulin, and delayed decline in IGF 1 (related to the acquisition of fol licular dominance), compared to cows that fail to ovulate. The ovulatory cows had also higher concentrations of estradiol and LH during the follicular phase (Kawashima et al., 2012). Collectively, these hormonal and metabolic changes reinforce the extreme importance of metabolic status on reproduction. Early Ovulation Postpartum Early postpartum (PP) resumption of ovarian cyclicity leads to higher fertility (Thatcher and Wilcox, 1973; Stevenson and Call, 198 8 ; Staples et al., 1990; Darwash et al., 1997; T anaka et al., 2008; Santos et al., 2009 ; Galvo et al. 20 1 0). Fewer services per conception were necessary for cows to become pregnant when estrus activities occurred in the first month postpartum (Thatcher and Wilcox, 1973) and longer intervals from first ovulation PP to the first service increased pregnancy to the first AI (Darwash et al., 1997). Several studies showed positive effects of resumption of ovarian cyclicity before the first AI on first service conception rate (Darwash et al., 1997; Chebel et al., 2006; Santos, et al., 2009 ; Galvo et al. 20 1 0). Furthermore, c ows ovulating within 21 DIM had higher hazard of pregnancy by 300 DIM than cows that started cycling from 21 to 49 DIM or anovular cows by 49 DIM (Galvo et al., 2010). Kim et al. (2012) showed similar patterns of enhanced reproductive performance in Korean dairy herds, for cows ovulating early postpartum. Increased fertility in

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23 cows that resumed ovulation early postpartum is believed to be influenced by the increased frequency of estrous cycle s before first artificial insemination (Thatcher and Wilcox, 1973) More cycles would provide progesterone priming and uterine cleansing during estrus. F ailure of o vulation of the dominant follicle early postpartum is associated with profound negative energy balance and long interval to its nadir (Beam and Butler 1997, 1998). Uterine diseases such as clinical and cytology endometritis are positively associated with NEFA and BHBA (Hammon et al., 2006); t herefore, early ovulation may be a marker of good overall health status. Resumption of ovulation postpartum is highly variable (Butler and Smith, 1989) and there is still 20% to 40% of anovular cows at the end of the voluntary waiting period (Walsh et al., 2007; Santos et al., 2009; McDougall, 2010; Bisin otto et al., 2010b). Lack of adequate transition period management, inadequate energy balance, and poor health status are directly related to high proportion of anovular cows at the end of the voluntary waiting period. Controversially, e arly ovulation pos tpartum has been hypothesized to have detrimental effects on uterine involution (Padula and Macmillan, 2002) and also on reproducti ve performance. Prolonged luteal phase (defined as elevated progesterone for more than 25 days) is correlated with a greater risk of developing pyometra (Farin et al., 1989) and was associated with lower fertility (Smith a nd Wallace ., 1998). In fact, a side effect of early induced ovulation in response to GnRH injections (15 days postpartum) was an increase in the subsequent inc idence of pyometra and prebreeding anestrous. Thus, treatment with GnRH alone increased calving to first estrous and calving to first breeding intervals, and resulted in a tendency for an increased calving to conception interval (Etherington et al., 1984). Studies performed using continual exposure to a synthetic GnRH implant delayed ovulation postpartum during heat stress periods and successfully increased rates of uterine

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24 involution (Silvestre 2003, 2009) The chronic treatment with a potent and high affi nity GnRH agonist, deslorelin (Karten and Rivier, 1986) first induces an LH surge from the pituitary ; however, downregulates the GnRH receptors on the gonadotroph cells altering the responsiveness of the pituitary to endogenous GnRH. The pituitary gland be comes refractory to gonadotrophin releasing hormones and LH pulsatility secretion will be dimi ni shed resulting in However, there was a high variation in resumption of ovarian cyclicity once the block in ovulation was removed with almost half of the cows still not having ovulated by the beginning of the Ovsynch program. This delay in cyclicity compromised P/AI at first AI and negative ly affected milk production (Silvestre, 2009). Early resumption of postpartum act ivity and enhanced fertility to first service is a challenge when coupled with non optimal management of nutrition during the transition period, inadequate energy balance postpartum, and poor health status. GnRH and Ovulation Gonado tropin releasing hormon e, a decapeptide, produced by the hypothalamus is mainly known for its role in reproduction with an important role in the ovulation process However, the presence of GnRH receptors outside of the hypothalamic pituitary reproductive axis has been demonstrat ed (Skinner et al., 2009) At the molecular level, once GnRH reaches the pituitary gland, it binds to its G coupled protein receptor on the surface of the gonadotrophs cells. This receptor has a unique characteristic because it lacks an intracellular cytop lasmatic tail allowing slower internalization ( Hislop et al., 2001). Once the ligan d is bound to the G receptor, there is activation of the G proteins ( Stanislaus et al., 1997) that subsequently activate its subunits, G and inositol 1, 4, 5 triphosphate (IP3) and the later induces calcium influx to the cytosol from the extracellular fluid an d also from the endoplasmic reticulum. PKC acts through a cascade of

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25 phosphorylation and activates mitogen activated kinases (MAPK), in which MAPK goes to the nucleus to activate the transcription steroidogenic factor 1 (SF 1). Immediate early gene (Egr 1) among SF 1, Egr specific HD protein (Pitx expression in the nucleus Calcium also plays a role on the (Dorn et al., 1999 ; Haisenleder et al., 1997 ). The calcium in the cytosol then interact to calmodulin and activates calcium calmodulin kinases, i n which phosphorilate and activate some of the Haisenleder et al., 2003; Liu et al., 2002) resulting in the synthesis of FSH hormone. FSH is mainly dependent on the calcium calmodulin mediated pathway and FSH is o nly stored in secretory granules in the cytoplasm for short periods whereas LH is s tored for longer periods during the estrous cycle (Farnworth, 1995). GnRH neurons in the cow are located in two different areas of the brain with species variations. The f irst group of GnRH neurons is located at the ventromedial and arcuate nuclei of the hypothalamus, which is known as the tonic GnRH center The tonic center is responsible for the basal secretion, in which various small pulses of different frequencies and a mplitudes of GnRH are released in the female and also in the male. The second group of GnRH neurons the preoptic and suprachiasmatic nuclei center is localized on the superior an terior area of the hypothalamus. This area is called the surge center or p reovulatory center because it is where the preovulatory GnRH surge occurs (Sakakibara et al., 2013). The surge center is developed and active only in the female but not in males. In the female fetuses, alpha fetal protein bind s estradiol and prevent s it fr om reaching and crossing the blood brain barrier ; t he refore, the surge center of the hypothalamus develop s. In male fetuses, t estosterone is converted to estradiol in

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26 brain and defeminizes or prevents the different iation of the GnRH surge center in the hyp othalamus Regulation of GnRH release by steroids is not entirely due to the direct effect of the steroids on GnRH neurons I n fact steroids affect others neurons that are in communication with GnRH neurons. GnRH acts on the pituitary to stimulate the synt hesis and release of FSH and LH, and it is negatively affected by estradiol in the tonic center (Ginther, 2000; Takumi et al., 2012). However, in the surge center, estradiol stimulates the surge in GnRH and ultimately LH. Progesterone suppresses the GnRH s urge center, although the full mechanism is not completely understood. Both estradiol and progesterone act on GnRH neurons through the Kisspeptin pathway E stradiol up regulates the expression of Kiss 1 gene and stimulates GnRH release at the surge center but downregulates the expression of Kiss 1 gene in the tonic center (Alin et al., 2103) P rogesterone down regulat es the expression of Kiss 1 gene and decrease GnRH release in the tonic center (Radovick et al., 2012). Nutritional status also plays an impo rtant role in the control of GnRH production and release (Santos et al., 2010). The neurotransmi tter n europeptide Y (NPY) is an orexigenic peptide that stimulates appetite and is stimulated by low energy levels in the blood associated with low leptin conce ntrations. NPY also is an inhibitor o f GnRH neurons and can be blocked by leptin that is produced by the adipose tissue ( Schwartz et al., 1996) Leptin concentration function s as a sensor of body fat status in the body that ultimately partially integrates metabolic status with GnRH release. Under favorable metabolic condition, leptin upregulates Kiss 1 leading to kispeptin production which stimulate s GnRH release from the GnR H tonic center leading to FSH and LH release by the pituitary, which affects follicle growth leading to increased estradiol concentration which will up regulate the Kiss 1 neurons on the surge center resulting in the GnRH LH surge and ovulation (Garcia Gal iano et al., 201 2 )

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27 Treatment with GnRH is a means to induce ovulation (Britt et al., 1974; McDougall et al., 1995; Gmen and Seguin, 2003; Amaya Montoya et al., 2007) and is used routinely within a variety of synchronization of ovulation protocols. Howev er the use of GnRH to induce ovulation early postpartum does not consistently result in improvement of fertility. In fact, e arly ovulation postpartum has been hypothesized to have detrimental effects on reproduction (Padula and Macmillan, 2002) Etheringt on et al. (1984) reported increased frequency of p yometra and increased calving to first estrus and calving to first breeding intervals for cows treated with GnRH at 15 DIM. Stevenson and Call ( 198 8 ) reported increased calving to conception interval with e arly postpartum GnRH administration in cows with reproductive disorders. Others failed to improve reproductive performance w i t h GnRH administration in healthy postpartum cows (Foote and Riek, 1999). On the other hand, Benmrad and Stevenson (1986), and Nas h et al. (1980) showed a reduction on days to conception and fewer services per conception for GnRH treated cows between 10 1 5 days PP. Cows treated with GnRH between 13 to 15 days PP had increased conception to the first AI (74% versus 56% respectively), better ove rall conception rate (70.6 v s 51.1% respectively) and a lower number of services per conception (1.23 v s 1.74 AIs/pregnancy ) compared to control cows (Nash et al., 1980). Reduction of culling rate because of low reproductive performance was al so achieved with the use of GnRH in early lactation (Britt et all., 1977).

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28 Figure 1 1. Schematic depiction of the pattern of secretion of FSH, LH and progesterone ; and pattern of growth of ovarian follicles during the estrous cycle in cattle. Each wave of follicular growth is preceded by a transient rise in FSH concentrations. Healthy growing follicles are shaded in yellow, atretic follicles are shaded red. A surge in LH and FSH concentrations occurs at the onset of estrus and induces ovulation. Th e pattern of secretion of LH pulses during an 8 h window early in the luteal phase (greater frequency, lesser amplitude), the mid luteal phase (lesser frequency, lesser amplitude) and the follicular phase (high frequency, building to the surge) is indicate d in the inserts in the top panel (Forde et al., 2011).

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29 Figure 1 2. Schematic presentation of factors involved in the growth, maintenance and regression of the CL (Skarzynski et al., 2008) Figure 1 3. Schematic of follicle growth and FSH fo r a cow that has two follicular waves during a 21 d estrous cycle (Wiltbank et al., 2002)

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30 Figure 1 4. A summary of folliculogenesis in the ewe, developed with data from several sources ( Lundy et al. 1999 ; K. P. McNatty, unpubl. data). The upper panel shows the mean (central line) and range (shaded band) time lines of growth of the follicle and oocyte and the number of granulosa cells, from p rimordial to ovulatory stages. The lower panel shows the progressive emergence of several critical functional and morphological characteristics of follicles as they develop. The stages of development have been defined by two different systems one based o n morphology and the other on functional characteristics of follicles (Scaramuzzi et al., 2011).

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31 Figure 1 5. Diagrammatic scheme of resumption of dominant follicles and ovarian cycles during the postpartum period in dairy and beef suckle d cows not nutri tionally stressed. LH pulse frequency is that occurring during an 8 h window where cows are blood sampled every 15 min. Short oestrous cycles do not always occur after first ovulation ( Forde et al., 2011; reprinted from Crowe, 2008).

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32 Figure 1 6. Schema tic representation of nutritional status during peripartum, lactation and reproductive function during early postpartum in anovulatory and anovulatory cows caroteno, IGF 1, glucose and energy balance influencing ovaria n and follicle dynamics that lead or not to ovulation (adapted from Kawashima et al., 2012).

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33 Figure 1 7. Metabolic mechanisms linking negative energy balance and oocyte quality in high producing dairy cows. A status of negative energy balance is hypothe sized to affect the health of the primary follicles which may have a carry over effect on oocyte quality. An altered follicular growth pattern might impair oocyte developmental competence. Biochemical parameters, associated with a negative energy status, a re well reflected in the follicular fluid and can directly affect oocyte competence. NEFA: non hydroxybutyrate (Leroy et al., 2008).

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34 CHAPTER 2 INTRODUCTION It is well documented that reproductive performance affects profitability of the dairy enterprise (De Vries et al., 2006; Galvo et al., 2013) Having high reproductive performance (i.e. high pregnancy p er AI and low pregnancy loss) will increase the proportion of cows conceiving immediately after the voluntary waiting period which can increase the herd average daily mi lk yield by increasing the proportion of cows i n the early stages of lactation in the subsequent lactation ( Ribeiro et al., 20 12 ) Furthermore, high reproductive performance lead to reduce d culling rates due to reproductive failure (Pinedo and de Vries, 20 10) and maximize farm revenue (Risco et al., 1998 ; De Vries et al., 2006; Galvo et al., 2013 ). Reports using the average herd producing of 9 ,000 Kg of milk in the United States of America showed that an extra day open beyond 90 days costs o n average of $ 3 .00 and this value increases with increased days to conception (De Vries, 2008). Treatment with GnRH is not a new tool to induce ovulation (Britt et al., 1974; McDougall et al., 1995; Gmen and Seguin, 2003; Amaya Montoya et al., 2007) and it is used ro utinely within a variety of synchronization of ovulation protocols. However, effects of GnRH administration early postpartum on fertility are not consistent. Fernandes et al ( 1978 ) and Kesler et al (1977) recommended the use of GnRH after 7 to 10 days po stpartum when the pituitary had restore responsiveness to GnRH. Padula and Macmillan (2002) and Etherington et al. (1984) reported detrimental effects on reproductive performance (ie. increased frequency of pyometra, increased calving to first estrus and t o the first breeding interval) with the use of GnRH at 15 DIM. Administration of GnRH between 18 and 25 DIM to cows with reproductive disorders resulted in increased calving to conception interval (Stevenson and Call, 198 8 ). Others failed to improve reprod uctive performance w i t h GnRH administration at 13 or 14 days postpartum in

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35 healthy postpartum cows (Foote and Riek, 1999). Nonetheless there are beneficial effects demonstrated by GnRH use in early postpartum as demonstrated with an increase in conception rate, a reduction i n days to conception and in services per conception when GnRH treat ment occurred between 10 to 1 5 days postpartum (Benmrad and Stevenson, 1986; Nash et al. 1980). Reduction of culling rate because of low reproductive performance was als o achieved with use of GnRH between 8 to 23 days of lactation (Britt et al., 1977). Studies had shown increased ovulation rates for cows treated with GnRH early in lactation. In one study (Benmrad and Stevenson, 1986) 75% of the cows that received GnRH bet ween 10 to 14 DIM ovulated and only 28% of the non treated cows ovulated within 7 days post treatment. Britt et al. (1974) obtained 90% of ovulation rate in GnRH treated dairy cows at 14 DIM, however, less than 20% of control cows that received saline, ovu lated within 7 days post treatment. More recently a study performed in Korea (Jeong et al., 2013) showed that GnRH administration around 30 days postpartum limited to cows t hat did not experience any peripartum or metabolic problem such as dystocia, retain ed placenta, abo rtion, metritis or endometritis, increased ovulation rate compared to non treated cows (ovulation was measured in a subset of cows 14 days after GnRH injection; 70.9% vs. 53.0%; P < 0.001). The GnRH group also had increase d hazard of pregna ncy by 210 DIM (HR = 1.3; 95 % CI = 1.06 to 1.61; P = 0.01) compared to control cows. None of the previous studies used modern synchronization methods such as the Presynch Ovsynch as part of the reproductive management The inconsistent results, the small sample sizes combined to the inconsistent characterization of uterine health in the previous studies evaluating the use of GnRH during the first month postpartum justify further investigation in this field and the novel of the proposed research is the tre atment with GnRH performed in cows that had protocol of ovulation synchronization to be time d artificially inseminated. The hypothesis of

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36 the study was that administration of exogenous GnRH early postpartum in cows without a CL would induce ovulation witho ut detrimentally affect ing uterine health and would induce resumption of cyclicity in anovular cows which is expected to increase fertility. The main objective of the study was to evaluate the effects of administration of GnRH at 17 and 20 3 DIM in Hols tein dairy cows without a CL on induction of ovulation, uterine health, and reproductive performance

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37 CHAPTER 3 MATERIALS AND METHODS Cows H ousing and Feeding A randomized clinical experiment was conducted on two freestall dairy farms located in North Ce ntral Florida from December 2010 through August 2012 All animal procedures were approv ed by the University of Florida Institutional Animal Care and Use Committee (IACUC UFL). Dairy 1, the Dairy Unit from the University of Florida, milked approximately 45 0 Holstein cows twice daily with a rolling herd average of ~10,500 Kg/milk/cow. A single lactating total mixed ration (TMR) was fed twice daily to match or exceed the requirements of second lactation Holstein cows with the following characteristics: 6 5 0 Kg of body weight, producing 4 5 Kg /day with 3.7% fat corrected milk and 3.15% of protein at 60 DIM; fresh water was available ad libitum. The freestall barns were cleaned twice daily and freestalls bedded twice a week with sand. Fans with misters and sprinkl ers over the feed line were present in the barns. The AfiMilk system was comprised of AfiMilk Pedometer, AfiLab and AfiFarm software (AfiMilk System, SAE Afikim; Kibbutz, Israel) to monitor cow health, occurrence of estruses and milk measurements (i.e., mi lk weight; % fat; % protein; % lactose, somatic counting cell; etc.) daily of individual cows. Cows were restrained in head lock gates along the feed line when all experimental procedures were conducted. The second dairy (Dairy 2) was a private owned dair y milking approximately 4,800 Holstein cows thrice daily with a rolling herd average of ~11,500 Kg/milk/cow. Cows had ad libitum fresh water available, and TMR offered twice a day formulated to meet or exceed the requirements of a second lactation Holstein dairy cow with the following characteristics: 680 Kg of body weight producing 5 2 Kg of milk /day with 3.5% fat corrected milk and 3.0% of protein at 60 DIM. In Dair y 2, cows were housed in tunnel ventilated

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38 freestall barns, freestall were cleaned thrice da ily and bedded twice a week with sand. PCDart software (Dairy Records Management Systems, Raleigh, NC) and Allflex RFID ear tags (Allflex USA, Dallas, TX) were used as software program and electronic devices for identification, collection and recording of milk yield and health/reproductive events. All procedures were performed while cows were restrained at a palpation rail after the milking on the afternoon shift. In both dairies primiparous and multiparous were housed separately. Cows were vaccinated and t reated for common diseases according to standard operating procedures (SOP) developed with participation of the veterinarians from University of Florida/College of Veterinary Medicine (Food Animal Reproduction and Medicine Service). Study Groups, BCS and U ltrasonography of the Ovaries Once weekly on Tuesdays, a cohort of lactating cows within 17 3 DIM was enrolled in the study for 65 consecu tive weeks in Dairy 1 and for 16 c onsecutive weeks in Dairy 2. A total of 637 Holstein lactating dairy cows were initially examined by ultrasonography (US) at day 17 3 of lactation and 23.4% of them had a CL (n = 147). Cows with a corpus luteum (CL) detected on the first US at 17 3 DIM were excluded from enrollment in the experiment, although used in some univari able analyses. The remaining cows with no detected CL (n = 490) were stratified by parity and randomly allocated (by the tossing of a coin) to one of the two treatments : GnRH (n = 245) i.m. injection of 100 g gonadotropin releasing hormone (gonadorelin hy drochloride, Factrel, Zoetis, Madison, NJ) at 17 3 DIM (GnRH 1) and ag ain 3 .5 days later (Friday afternoon) at 20 3 DIM (GnRH 2) ; or c ontrol (n = 245) no GnRH treatment. Figure 3 1 shows the schematic diagram of study activities. Body condition (BC S), based on a 5 points scoring system (Ferguson et al., 1994) was scored three times during the study, at calving, at enrolment on day 17 3 DIM and at 35 DIM. D escriptive statistic of variables of interest shown in Table 4 1.

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39 Ovarian ultrasonography we re performed twice a week using a portable ultrasonography scanner (Ease Scan, BCF Technology, Livingston, UK) with a 7.5 MHz linear transducer, starting at 17 3 DIM and continued until ovulation was detected or the cow began an ovulation synchronization protocol in Dairy 1. In Dairy 2, ultrasonography was also done twice a week for up to 28 3 DIM Size of follicles and corpus luteum /corpora lutea (CL) were measured and when CL < 20 mm appeared in two consecutive US. Ovulation to GnRH 1 was characterized by appearance of a CL in the US at 20 3 DIM or the disappearance of a follicle > 10 mm present in the US at 17 3 DIM but absent in the US at 20 3 DIM and the appear ance of a CL in the US at 24 3 DIM. After excluding cows that had already ovulated to GnRH 1, o vulation to GnRH 2 was evaluated follow ing the same criteria as ovulation to G nRH 1 ; however, evaluation started on the day of the G nRH 2 and cows either had t o have the appearance of a CL in the US at 24 3 DIM or the disappearance of a follicle > 10 mm that was present in the US at 20 3 DIM but absent in the US at 24 3 DIM and the appearance of a CL in the US at 28 3 DIM. Therefore, ovulation to GnRH 1 or GnRH 2 was the overall ovulation from 17 3 DIM up to 24 3 DIM. Uterine Health Clinical endometritis (CE) was diagnosed in both dairies at 5 weeks post partum. Cytological endometritis (CTE) was only diagnosed in Dairy 1. Clinical endometritis was determined using a Metricheck device ( Simcro Tech Ltd. Hamilton, New Zealand) After cleaning of the vulva, t he Metricheck device a 50 cm lo ng stainless steel rod with a silicon cup in one end (4 cm in diameter) was inserted into the vagina until the tip reached the fornix of the vagina The device was then retracted. Material adhered on the internal surface of the silicon cup

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40 was assessed visually and classified according to the method of Williams et al. (2005) on a scale from 0 to 3. Clear or translucent mucus was score d as 0 ; score 1 was described as mucus containing flecks of white or off white pus ; score 2 is a discharge containing less than 50% of white or of f white mucopurulent ma terial and sanguineous d ischarge or d ischarge composed by more than 50% of white or yellow pus was s core d 3 ( Williams et al., 2005). Scores equal or greater than 2 were considered positive for CE (Sheldon et al., 2006). Cytological (Sheldon et al., 2006). Basically, after cleaning and disinfecting the external vulva with alcohol 70%, a Cytobrush Plus Cell collector ( CooperSurgical, Inc. Trumbull, CT) attached to a stainless steel modified AI gun and protected by a plastic sheath protector (Continental Plastic Corp.; Delavan, WI) was carefully introduced in the vagina, through the cervix and into the body of the uterus, then the brush was inserted past the shea th protector and pressed against the endometrium while being rotated for three times The brush was retrieved into the stainless steel AI gun and remov ed from the animal The brush was then disconnected and smeared onto a glass slide and left to air dry. S meared slides were then Wright Geimsa stained with Camco Stain Pak 702 (Cambridge Diagnostic Products Inc.; Fort Lauderdale, FL) and left in room temperature to dry R eading of the slides was done in a manner so that investigator was unaware of the treatme nts. A total of 200 cells per slide were counted including all leukocyte types and epithelial cells, but excluding erythrocytes The proportion of PMN was determined and as described previously ( Kasimanickam et al., 2004; Sheldon et al., 2006 ). Reproductive Management Cows were managed under the respective dairies reproductive programs (Figure 3 1) : in

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41 (PreSynch) at 41 and 55 3 DIM, followed 12 days later (67 3 DIM) by Ovsynch 56 TAI protocol (GnRH, 7 days later PGF 56 hours later GnRH followed by ti med artificial insemination 16 20 hours). Pregnancy diagnosis was made by transrectal ultrasonography at day 32 after AI (i.e., pregnancy was characterized by the presence of an amniotic vesicle containing a live embryo heart beat present during ultrason ography) and reconfirmation of the pregnancy by transrectal palpation was performed on day 74 after conception. Cows diagnosed not pregnant at the time of pregnancy diagnosis were resynchronized on the same day using the Ovsynch 56 TAI program described an d inseminated 10 days later. Dairy 2 had distinct reproductive protocols based on parity. Cows in their first and second lactation DIM, followed 12 days later (76 3 DIM) by Ovsynch 56 TAI protocol. Cows in their t hird or greater lactation 5 5 and 69 3 DIM, followed 12 days later ( 8 1 3 DIM) b y Ovsynch 56 T AI protocol Nonetheless if cows were detected in estr of the PreSynch AI was performed and cows were automatically removed from the synchronization of ovulation program. Pregnancy diagnosis was performed at 40 3 days after AI and reconfirmation at 85 3 days after AI by palpation per rectum of the gravid uterine horn. Cows diagnosed not pregnant at pregnancy diagnosis were resynchronized on the same day using the Ovsynch 56 program. Cows detected in estrus were considered non pregnant and were immediately AI Periparturient disease s such as abo rtion (gestation less than 260 days), s tillbirth ( born dead from a gestation of 261 days or more), dystocia (calving ease score 2 on the 5 points scale system ; 1 = calving without assistance; 2 = light help with use of obstetric chains; 3 = moderate force to deliver the calf; 4 = extreme force to deliver the calf; 5 = cesarean section or

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42 fetotomy ), hypocalc emia (clinical disea ses characterized by hypothermia and incapability to stand up ruled out other diseases) retained fetal membranes (fetal membranes retained for after at least 12 h rs after parturition), metritis (reddish, fetid fluid vaginal discharge within 2 weeks postpa rtum ), and ketosis (ketones body detected on urine before and/or after study enrolment) were diagnosed by the farm personnel and recorded in an on farm computer software for on farm retrieval from both dairies. Sample Size and Statistics A sample size of 2 40 cows per group was calculated (Minitab Inc, State College, PA) for = 0.05 and = 0.2, to detect differences in pregnancy per artificial insemination (P/AI) of 8% when P/AI varies from 32% to 40%. Outcomes of interest were: o vulation to the GnRH 1 an d/or GnRH 2; prevalence of clinical and subclinical endometritis at 7 weeks postpartum; pregnancy to the first service diagnosed between 32 and 42 days post service (PD1) and between 74 to 90 days post service (PD2), pregnancy loss from the first service ( defined as the number of cows pregnant from the first service that were not pregnant on the pregnancy reconfirmation divided by the number of cows diagnosed pregnant on the first pregnancy diagnosis) and hazard rate of pregnancy up to 300 DIM (measured 74 to 90 days after breeding) Explanatory variables included in the model were treatment status (GnRH treated, c ontrol), calving season (cool [November to May] vs. warm [May to September]), dairy (Dairy 1 vs. Dairy 2), parity (primiparous vs. multiparous), c alving related problems (group of the following events: abortion, stillbirth, dystocia, twins and RFM), met abolic problems (hypocalcaemia and ketosis), metritis, body condition score at study ovulation to GnRH 1 or GnRH 2 ( yes or no), and two way interactions between GnRH treatment and other covariates Treatment wi th GnRH was the main effect of interest and was forced in all the models

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43 Binary outcomes were analyzed by logistic regression using the LOGISTIC procedure of SAS version 9. 3 ( SAS Institute Inc., Cary, NC ) and backward elimination was performed by removin g explanatory variables from the model with P > 0.05 according to Wald statistics criterion. Hazard of ovulation up to 70 DIM and hazard to pregnancy up to 300 DIM at PD2 in which 255 cows were included in this analysis Three models were generated; first model with all variables included, except ovulation status by 2 4 DIM because GnRH treatment affected ovulation by 2 4 DIM and early ovulation is known to affect fertility; th erefore, ovulation by 2 4 DIM would become an intermediate variable The second model was composed by all variables except treatment status to evaluate the effect of ovulation by 2 4 DIM on hazard of pregnancy The third model included GnRH treatment, ovulat ion by 2 4 DIM, and interaction between GnRH treatment and ovulation by 2 4 DIM besides other covariates. Hazard ratio (HR) was characterized as the daily probability of a given event (ovulation or pregnancy) The variable time was the interval in days from calving to ovulation or to pregnancy. Ten cows (5 from each group) were excluded from the study because they were sold died or became recumbent before 28 DIM ; therefore, ovulation could not be confirmed or cows did not receive their treatment as assigned Hence, only 480 cows (240 per group) were i ncluded in the multivariable analysis. Cows that were sold or died after 28 DIM or did not ovulated by 70 DIM were censored in the analysis of time to ovulation. Cows that were sold or di e d after 28 DIM or did not conceive by 300 DIM were censored in the analysis of time to pregnancy. For the analysis of time to pregnancy, cows were conside red pregnant if they were confirmed pregnant at PD2 and 480 cows were included in the analysis In the analysis of clinical endometritis, 435 cows were included in the analysis (217 and 215 for GnRH and control groups, respectively), and 245 cows

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44 were included in the cytological endometritis analysis (123 and 122 for GnRH and control groups, respectively). Twenty one cows were called do not breed due different reasons and were excluded of the analysis of conception rate at PD1 and PD2, leaving 459 cows in the analysis (231 and 228 cows for GnRH and control groups, respectively). One hundred and fifty five cows conceived of the first service and were included in the analysis of pregnancy loss. Kaplan Meier plots and median days to ovulation by 70 DIM and days to pregnancy by 300 DIM were generated using M edCalc version 12.7 for Windows (MedCalc Software, Mariakerke, Belgium). Whe n interaction between two dichotomous variables was detected, a new variable containing all the 4 combinations (dummy variables between variables 1 and 2 for example would be: [variable 1= No, variable 2 = No]; [variable 1 = Yes, variable 2 = No]; [variabl e 1 = No, variable 2 = Yes] and [variable 1 = Yes, variable 2 = Yes]) was created and the model was run again including only the new variable. Univariable survival analysis was generated to evaluate time to pregnancy up to 300 DIM according to GnRH treatme nt and ovulation includ ing cows that had a CL at the first US Statistical significance was considered when P value 0 .05.

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45 Figure 3 1 Schematic diagram of study activities. Weekly a cohort of cows without a corpus luteum at 17 3 DIM was stratified by parity and randomly assigned to GnRH g roup (100 g i.m. of ; Factrel 50 g/mL, Zoetis Ltd., Madison NJ ) or re main as c ontrol (no GnRH injection). DIM = days in mi lk. BCS = body condition score US = ultrasonography. Metr icheck = evaluation of the vaginal discharge using m etricheck device Cyto brush = uterine cytology. Pre S injection 14 da ys apart. Ovsynch 20 hrs later TAI. TAI = timed artificial insemination

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46 CHAPTER 4 RESULTS Descriptive Statistics Descriptive statistics is shown in Table 4 1. O f the 480 cows used in the analysis 255 w ere from Dairy 1 and 225 were from D a i ry 2 ; 216 were primiparous and 264 were multiparous ; 75 at 17 3 DIM; 321 calved in the warm season (May to September) and 159 calved in the cool season (October to April) Preva lence of calving problems, metabolic problems and metritis were 32.9 %, 27.1 % and 20.6% respectively None of the variables was significantly diff erent between GnRH treated and c ontrol cows. Ovulation Treatment with GnRH significantly increased ( P < 0.00 1) ovulation rates within 3.5 days from GnRH 1 or GnRH 2. Ovulation within 3.5 days of GnRH 1 was 44.6% for GnRH treated cows and 20.9% for c ontrol cows. GnRH treated cows had 2.9 times the odds of ovulation to G nRH 1 compared to c ontrol cows (95 % CI = 1. 9 to 4.4; Table 4 2). BCS at study enrollment also affected ( P = 0.01) ovulation within 3.5 days of G nRH 1, and 36.3% of the cows with BCS > 2.75 ovulated C ows with BCS > 2.75 had 1.9 times the odds of ovulati on within 3.5 days of G nRH 1 compared to cows with (95 % CI = 1.1 to 3.1). Ovulation within 3.5 days of GnRH 2 was 61.7 % for GnRH treated cows and 30.5 % f or c ontrol cows Ovulation within 3.5 days of GnRH 2 was also influenced by calving seaso n and ovulation was higher in the warm season than in the cool season (49.5 % vs. 32.2 %; OR = 2.8; 95% CI = 1.6 to 4.9; P < 0.001). Overall ovulation by 24 3 DIM ( response to either GnRH 1 or GnRH 2, measured 3.5 days after treatment ) was significantly higher for GnRH treated cows than c ontrol cows (78.7% vs. 45.0%; OR = 4.7; 95% CI = 3.2 to 7.5; P < 0.001) Cows that calved in the warm season also had higher overall ovulation by 24 3 DIM

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47 than cows that calved in the cool season (67.3% vs. 50.9%; OR = 2.2.; 95% CI = 1.4 to 3.3; P < 0.001). Median days to ovulation up to 70 DIM was decreased by 8 days ( P = 0 .003) for GnRH treated cows compared to c ontrol cows (24 d vs. 32 d; Figure 4 1). Uterine Health Outcomes Administration of GnRH early postpartum d id not affect the prevalence of CE (Table 4 3). Prevalence of CE was 23.9 % for GnRH treated cows and 18.6 % for c ontrol cows The prevalence of CE w as significantly higher for cows that had calving problems ( 32.6 vs. 15.9%; OR = 2.2; 95% CI = 1.3 to 3.6; P = 0.001) and metritis ( 40.6 vs. 15.8%; OR = 3.1 ; 95% CI = 1. 9 to 5.4 ; P = 0.001) compared to cows that did not have these condition s The prevalence of CTE was also not different between GnRH treated and c ontrol cows (30.9% vs. 32.8% ; P = 0.61). Cows tha t had metritis had increased prevalence of CTE ( 50.7 vs. 23.5%; OR = 3.4; 95% CI = 1.9 to 5.9; P < 0.001). Reproductive Outcomes C onception to the first service diagnosed at PD1 was similar for GnRH and c ontrol groups (37.6% vs. 38.6%; OR = 0.8; 95% CI = 0 .5 to 1.2; P = 0. 29; Table 4 4). Cows that ovulated by 24 3 DIM ( 40.9 vs. 33.4%; OR = 1.6; 95% CI = 1.1 to 3. 0 ; P = 0.04 ), cows in Dairy 1 ( 45.4 vs. 30.1%; OR = 2 .0; 95% CI = 1.4 to 2 .9; P < 0.001 ) and cows with BCS > 2.75 at study enrollment ( 41.6 vs. 2 6.6%; OR = 1.8; 95% CI = 1.1 to 3.1; P = 0.01 ) had higher conce ption at first service. Table 4 5 shows conception to the first service diagnosed at PD2 in which only dairy (39.6 vs. 26.5% for Dairy 1 and Dairy 2, respectively; OR = 1.8; 95% CI = 1.2 to 2. 7; P = 0.004) and BCS at study enrollment (36.5 vs. 22.8% for BCS > 2.75 an respectively; OR = 1.8; 95% CI = 1. 1 to 3 1 ; P = 0.015 ) had significant effect. Pregnancy loss from the first service diagnosed at PD2 (Table 4 6) was significantly hi gher for control group compared to GnRH treated group (18.1% vs. 6.8%; OR = 6 25 ; 95% CI = 2.1 to 20.2; P < 0.01 );

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48 for cows that had metritis compared to cows that did not have m etritis (21.5% vs. 9.7%, OR = 3 6 ; 95% CI = 1. 3 to 10.2 ; P = 0.02 ); and for cows that ovulated from G1G2 compared to cows that did not ovulate from G1G2 (14.5% vs. 8.6%, OR = 3.9 ; 95% CI = 1. 2 to 14 2 ; P = 0.02 ). In the CL group (cows with detectable CL at study enrollment day), the pregnancy loss was 6.9%. Figure 4 2 shows time to p regnancy up to 300 DIM at PD2 according to treatment status for the GnRH treated and control groups that respectively had median days to pregnancy and proportion of cows pregnant by 300 DIM of 122 78.8 % and 136 76.3 % (univariable analysis containing only treatment status, P = 0.9). Yet Figure 4 3 shows similar analysis as the previous however with the CL group included with median days to pregnancy of 120 days and 78.3 % of cows pregnant by 300 DIM (univariable analysis containing only treatment stat us, and with CL group included; P = 0.5). Figure 4 4 shows time to pregnancy up to 300 DIM at PD2 according to ovulation status for cows without CL at study enrollment that did or did not ovulate by 2 4 DIM. The respective median days to pregnancy and propo rtion of cows pregnant by 300 DIM for cows that ovulated and for cows that did not ovulate were 121 81.8 % and 155 70.5 % (univariable analysis containing only ovulation status, P = 0.001). In Figure 4 5, similar analysis performed in Figure 4 4 is sho wed, although including the CL group (cows with a detectable CL at 17 3 DIM) in which the median days to pregnancy and proportion of cows pregnant by 300 DIM were similar as showed above for cows that ovulated and for cows that did not ovulate by 2 4 3 DIM, and 120 78.3 % for the CL group (univariable analysis containing only ovulation status which CL group is included ; P = 0.003). Hazard of pregnancy up to 300 DIM at PD2 for the first model (multivariable analysis with all variables except ovulation status by 24 DIM; treatment status composed only by GnRH treated and c ontrol groups ) was influenced only by BCS in which cows with BCS > 2.75 had

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49 to 1.6 ; P = 0.03 ) T reatment status did not have effect in the h azard of pregnancy in the first model ( P = 0.91 ; Table 4 7 ) In the second model (composed with all variables except treatment status) only ovulation had significant effect ( P < 0.01 ; Table 4 8) in h azard of pregnancy up to 300 DIM at PD2 in which cows that ovulated by 2 4 DIM had higher hazard of pregnancy compared to cows that did not ovulated by 2 4 DIM ( HR =1.4, 95% CI = 1.1 to 1.8). Regarding the third model (composed with all variables) treatment status and the interaction treatment with ovulation had significant effects in the hazard of pregnancy up to 300 DIM at PD2 ( P = 0.03 and P = 0.05, respectively). Ovulation had no effect on hazard of pregnancy in the third model ( P = 0.17).Table 4 9 shows the int eraction of treatment with ovulation for the third model. In which GnRH treated cows that ovulated to G1G2 had 2 times the hazard of pregnancy compared to GnRH treated cows that did not ovulate (95% CI = 1.3 to 2.9; P < 0.001), and 1.3 times the hazard of pregnancy compared to control cows that failed to ovulate by 2 4 DIM (95% CI = 1.0 to 1 7 ; P = 0.05). There was no difference in hazard of pregnancy for GnRH treated cows that ovulated compared to control cows that also ovulated ( P = 0. 7 0 ). Control cows tha t ovulated and that did not ovulate had no difference in the hazard of pregnancy among themselves ( P = 0.17), and both groups had higher hazard of pregnancy than GnRH treated cows that did not ovulate by 2 4 DIM ( HR = 2 0 ; 95% CI = 1.2 to 2.8; P = 0.002 an d HR = 1.7 ; 95% CI = 1.03 to 2.5; P = 0.03, for control cows that ovulated and control cows that did not ovulate, respectively) Median days to pregnancy and proportion of cows pregnant by 300 DIM for GnRH treated cows that ovulated, GnRH treated cows that did not ovulate, control cows that ovulated and control cows that did not ovulated by

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50 2 4 DIM were 119 d 80.9 %, 168 d 58.9 %, 133 d 83.4 %, and 140 d 74.9 %, respectively (Figure 4 6).

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51 Table 4 1. Descriptive statistics for G nRH treated and c ontrol groups. Variables level All cows n GnRH 1 n (%) Control 1 n (%) P value Location Dairy 1 255 127 (49.8%) 128 (50.2 % ) 0 .9 1 Dairy 2 225 112 (49.8 % ) 113 (50.2 % ) Parity Primiparous 216 110 ( 50.9% ) 106 ( 49.1% ) 0 .7 1 Multiparous 264 130 (49.2 % ) 134 (50.8 % ) Calving season 2 Warm 321 161 ( 50.1% ) 160 ( 49.9% ) 0 .9 2 Cool 159 79 ( 49.7% ) 80 ( 50.3% ) Calving problems 3 Yes 158 83 ( 52.5% ) 75 ( 47.5% ) 0 .4 3 No 322 157 ( 48.7% ) 165 ( 51.3% ) Metabolic problems 4 Yes 130 60 ( 46.1% ) 70 ( 53.9% ) 0.30 No 350 180 ( 51.4% ) 170 ( 48.6% ) Metritis 5 Yes 99 53 (53.5 % ) 46 (46.5%) 0.43 No 381 187 (49.1 % ) 194 (50.9%) BCS enrollment 6 114 48 (42.1%) 66 (57.9%) 0 .0 6 > 2.75 366 192 (52.4%) 174 (47.6%) 1 GnRH treated group, cows received 100g i.m. of gonadorelin hydrolochloride at 17 3 and 20 3 DIM (n = 240) and control group had no hormonal treatment (n = 2 40). 2 Calving season characterized as warm (May to September) or cool (November to May). 3 Calving problems (i.e. abortion, dystocia, retain fetal membranes, twins or stillbirth). 4 Metabolic problems (i.e. ketosis, hypocalcemia). 5 Metritis, characterized for malodorous reddish fluid vaginal with or without fever, diagnosis following the dairy standard operation procedures. 6 Body condition score evaluation based on Ferguson et al., 1994 (5 points scale varying from 1 = thin to 5 = fat) evaluated at study da y enrollment (17 3 DIM).

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52 Table 4 2. Effects of GnRH administration at 17 and 20 3 days postpartum on ovulation response in dairy cows. Outcome variable Outcome level Stratum Cows, n % OR 1 95% CI 2 P value Ovulation G1 3 Treatment Groups GnRH 240 44.6 2.9 1.9 to 4.4 < 0.001 Control 240 20.9 referent BCS enrolment 4 > 2.75 366 36.3 1.9 1.1 to 3.1 0.01 114 21.7 referent Ovulation G2 3 Calving season 5 W arm 208 49.5 2. 6 1.5 to 4. 6 < 0.001 Cool 115 32.2 referent GnRH*Parity Prim*GnRH 110 78.2 3.0 1.6 to 5.7 < 0.001 Mult*GnRH 130 79.2 2.3 1.2 to 4.2 0.008 Prim*Control 40 37.7 0.5 0.2 to 0.9 0.02 Mult*Control 134 50.7 referent Ovulation G1 & G2 3 Treatment Groups GnRH 240 78.7 4.7 3.2 to 7.5 < 0.001 Control 240 45.0 referent Calving season Warm 321 67.3 2. 2 1.4 to 3.3 < 0 .001 Cool 159 50.9 referent 1 OR = Odds ratio. 2 CI = 95% confidence interval. 3 Cows in the GnRH group received an i.m. injection of 100g of gonadorelin hydrochloride at 17 3 DIM (G1) and at 2 0 3 DIM (G2). Ovulation to G1 or G 2 was evaluated up to 3.5 days post administration. 4 Body condition score evaluation based on Ferguson et al., 1994 (5 points scale varying from 1 = thin to 5 = fat) evaluated at study day enrolment (17 3 DIM). 5Calving season characterized as warm (May to September) or cool (November to May).

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53 Table 4 3. Effects of GnRH administration at 17 and 20 3 days postpartum and other variables on prevalence of clinical1 (CE) and cytological endometritis2 (CTE) in dairy cows. Outcome variable Outcome level Str atum Cows, n % OR 3 95% CI 4 P value Clinical endometritis Treatment g roups GnRH 217 23.9 1.3 0.8 to 2.1 0.23 C ontrol 215 18.6 referent Calving pr oblems 5 Yes 138 32.6 2.2 1.3 to 3.6 0.001 No 294 15.9 referent Metritis 6 Y es 91 40.6 3.1 1.9 to 5.4 < 0.001 No 341 15.8 referent Cytological endometritis Treatment g roups GnRH 123 30.9 0.9 0.5 to 1.5 0.61 C ontrol 122 32.8 referent Metritis Yes 75 50.7 3.4 1.9 to 5.9 < 0.001 No 170 23.5 refer ent 1 2 iry 1. 3 OR = Odds ratio. 4 CI = 95% confidence interval. 5 Calving problems (abortion, dystocia, retain fetal membranes, twins or stillbirth). 6 Metritis, characterized for malodorous reddish fluid vaginal with or without fever, diagnosis following the dairy standard op eration procedures

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54 Table 4 4 Effects of GnRH administration at 17 and 20 3 days postpartum in dairy cows without a CL at study enrolment on c onception rate at PD1 from first service. 1 OR = Odds ratio. 2 CI = 95% confidence interval. 3 Ovulation response to G1 or G2, monitoring for ovulation up to 3.5 days post GnRH administration (i.m. injection of 100g of gonadorelin hydrochloride at 17 3 DIM and at 2 0 3 DIM respectively). 4 Body condition score evaluation based on Ferguson et al., 1994 (5 points scale varying from 1 = thin to 5 = fat) evaluated at study day enrolment (17 3 DIM). Outcome level Stratum Cows, n % OR 1 95% CI 2 P value Treatment g roups GnRH 231 3 7.6 0.8 0 5 to 1.2 0.29 Control 228 38.6 referent Dairy 1 240 45.4 2.0 1.4 to 2.9 < 0.001 2 219 30.1 referent Ovulation G1G2 3 Yes 286 40.9 1.6 1 1 to 3.0 0.044 No 173 33.4 referent BCS enrol l ment 4 > 2.75 354 41.6 1.8 1.1 to 3.1 0.014 105 26.6 referent

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55 Table 4 5. Effects of GnRH administration at 17 and 20 3 days postpartum in dairy cows without a CL at study enrolment on c onception rate at PD2 from first service. 1 OR = Odds ratio. 2 CI = 95% confidence interval. 3 Body condition score evaluation based on Ferguson et al., 1994 (5 points scale varying from 1 = thin to 5 = fat) evaluated at study day enrolment (17 3 DIM). Outcome level Stratum Cows, n % OR 1 95% CI 2 P value Treatment g roups GnRH 231 35.0 1.1 0 7 to 1.6 0.53 Control 228 33.5 referent Dairy 1 240 39.6 1.8 1.2 to 2.7 0.004 2 219 26.5 referent BCS enrol l ment 3 > 2.75 354 36.5 1.8 1.1 to 3.1 0.015 105 22.8 referent

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56 Table 4 6. Effects of GnRH administration at 17 and 20 3 days postpartum in dairy cows without a CL at study enrolmen t on p regnancy loss of the first service 1 1 Calculated as [(No. of pregnant cows on PD1from first service that were not pregnant on PD2 from first service / No. of pregnant cows on PD1 from first servi ce)*100]. 2 OR = Odds ratio. 3 CI = 95% confidence interval. 4 Metritis, characterized for malodorous reddish fluid vaginal with or without fever, diagnosis following the dairy standard op eration procedures. 5 Ovulation response to G1or G2, monitoring for o vulation up to 3.5 days post GnRH administration (i.m. injection of 100g of gonadorelin hydrochloride at 17 3 DIM and at 2 0 3 DIM respectively). Outcome level Stratum Cows, n % OR 2 95% CI 3 P value Treatment g roups GnRH 87 6.8 0.16 0.05 to 0.5 < 0.01 Control 88 18.1 referent Metritis 4 Yes 42 21.5 3.6 1.3 to 10.2 0.02 No 133 9.7 refer ent Ovulation G1G2 5 Yes 117 14.5 3.9 1.2 to 14.2 0.02 No 58 8.6 referent

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57 Table 4 7 Effects of GnRH administration at 17 and 20 3 days postpartum during the firs t month of lactation on time to pregnancy up to 300 DIM based on PD2 for M odel 1 (M ultivariable analysis with all variables included except ovulation ). Outcome level Stratum Cows, n HR 1 95% CI 2 P value Treatment GnRH 3 240 1.0 0.8 to 1.2 0.91 Control 240 referent BCS enrollment 4 > 2.75 365 1.3 1 02 to 1.6 0. 03 115 referent 1 HR = Hazard ratio. 2 CI = 95% confidence interval. 3 GnRH treated group, cows received 100g i.m. of gonadorelin hydrolochloride at 17 3 an d 20 3 DIM and control group had no hormonal treatment 4 Body condition score evaluation based on Ferguson et al., 1994 (5 points scale varying from 1 = thin to 5 = fat) evaluated at study day enrolment (17 3 DIM).

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58 Table 4 8 Effects of GnRH administration at 17 and 20 3 days postpartum during the first month of lactation on time to pregna ncy up to 300 DIM based on PD2 for M odel 2 (M ultivariable analysis with all variables included except treatment status) Outcome level Stratum Cows, n HR 1 95% CI 2 P value Ovulation G1G2 3 Yes 297 1.4 1.1 to 1.8 < 0.01 No 183 referent 1 HR = Hazard ratio. 2 CI = 95% confidence interval. 3 Ovulation response to G1or G2, monitoring for ovulation up to 3.5 days post GnRH ad ministration (i.m. injection of 100g of gonadorelin hydrochloride at 17 3 DIM and at 2 0 3 DIM respectively).

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59 Table 4 9 Effects of GnRH administration at 17 and 20 3 days postpartum during the first month of lactation on time to pregna ncy up to 300 DIM based on PD2 for Model 3 (M ultivariable analysis with all variables included). Outcome level Stratum Cows, n HR 1 95% CI 2 P value Treatment*ovulation 3 0.05 GnRH*Ov 4 189 1.3 1.0 to 1.7 0.05 GnRH*NoOv 4 5 1 0.6 0.4 to 1.0 0.03 Control*Ov 132 1.2 0.9 to 1.6 0.17 Control*NoOv 108 referent 1 HR = Hazard ratio. 2 CI = 95% confidence interval. 3 Treatment interaction with ovulation characterized in four groups: GnRH*Ov = GnRH treated cows that ovulat ed by 28 DIM; GnRH*NoOv = GnRH treated cows that did not ovulate; Control*Ov = control cows that ovulated by 28 DIM; and Control*NoOv = control cows that di d not ovulate. 4 Ovulation response to G1or G2, monitoring for ovulation up to 3.5 days post GnRH adm inistration (i.m. injection of 100g of gonadorelin hydrochloride at 17 3 DIM and at 2 0 3 DIM respectively).

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60 Figure 4 1 Time to ovulati on up to 70 DIM for GnRH t reated group (dashed line; n = 12 8) and control group (solid line; n = 12 7) in Dairy 1. GnRH treated group (cows received 100 g i.m. injection of g onadorelin h ydrochloride at 17 3 and 20 3 DIM) and c ontrol group (no further injection) had median days to ovulation and proportion of cows that ovulated by 70 DIM of 24 days 9 6.8 %, and 32 days 97.6 % respectively ( P = 0 .003).

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61 Figure 4 2. Time to pregnancy at PD2 up to 300 DIM for GnRH treated group (GNRH; dashed line; n = 240) and c ontrol group (CON; solid line; n= 240) GnRH treated group (cows received 100g i.m. in jection of gonadorelin hydrochloride at 17 3 and 20 3 DIM) and c ontrol group (no hormonal injection) had median days to pregnancy and proportion of cows pregnant by 300 DIM of 122 days 78.8 % and 13 6 days 76.3 %, respectively ( Univariable survival analysis; P = 0.93 ).

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62 Figure 4 3 Tim e to pregnancy at PD2 up to 300 DIM for cows with a CL in the beginning of the study (CL1; solid line, n = 147), c ontrol group (CON; dashed line; n= 240) and GnRH treated group (GNRH; dotted line; n = 240) Cows in the GnRH treated group received 100g i.m. injection of gonadorelin hydrochloride at 17 3 and 20 3 DIM, and c ontrol group no hormonal injection. M edian days to pregnancy and proportion of cows pregnant by 300 DIM of 120 day s 78.3 %; 136 day s 76 .3 % and 122 days 78.8 % for CL group, c ontrol group and GnRH treated group respectively ( Univariable survival analysis; P = 0.5 ).

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63 Figure 4 4 Time to pregnancy at PD2 up to 300 DIM according to ovulation status for cows that ovulated ( O vulated; d ashe d line; n = 297 ) and cows that did not ovulate ( Did not ovulate; solid line; n = 1 83). Ovulation was characterized from cows enrolled in the study without a CL and evaluated up to 24 DIM ; and ovulation group and not ovulation group had median days to pregnancy and proportion of cows pregnant by 300 DIM of 121 day s 81.9 % and 155 days 70.5 % respectively ( Univariable survival analysis; P = 0.001 ).

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64 Figure 4 5 Time to pregnancy at PD2 up to 300 DIM according to ovulation status for cows that entered the study without CL and did not ovulate ( Did not ovulate; solid line; n = 1 83 ) for cows that entered the study without CL and ovulate d by 28 DIM ( Ovulated; d ashe d line; n = 297) and for cows with a CL in the beginning of the study (CL1 at US1; d otted line; n = 147). Ovulation was characterized only in cows enrolled in the study without a CL, and evaluated up to 2 4 DIM. M edian days to pregnancy and proportion of cows pregnant by 300 DIM of 155 day s 70.5 %; 121 day s 81.9 % and 120 days 78.3 % for cows that did not ovulate by 24 DIM cows that ovulated by 24 DIM and cows with CL at study enrollment, respectively (Univariable survival analysis; P = 0.003 ).

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65 Figure 4 6 Time to pregnancy at PD2 up to 300 DIM according to treatment*ovulation status for GnRH treated cows that ovulated (GOv; dashed line; n =189) or did not ovulate (GNoOv; solid line; n = 51) and control cows that ovulated (NoGOv; dash dotted line; n = 108) or did not ovulate (NoGNoOv; dotted line; n = 132) ( Multivariable analys is; Treatment*Ovulation; P = 0.045). GOv had increased hazard of pregnancy (HR) compared to GNoOv (HR = 2.0; 95% CI = 1.4 to 2.9 ; P < 0.001), or NoGNoOv (HR = 1.3; P = 0.05), and similar to NoGOv (HR = 1.1; P = 0.70). Control cows that did or that did not ovulate had similar hazard of pregnancy among themselves (HR = 1.2; P = 0.17) and higher hazard of pregnancy than GnRH treated cows that did not ovulate ( P = 0.002 and P = 0.033, respectively). Median days to pregnancy and proportion of cows pregnant by 30 0 DIM of 119 days 80.9 % ; 168 days 58.9 %; 133 days 83.4 %; 140 days 74.9 % for GOv, GNoOv, NoGOv, NoGNoOv cows respectively.

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66 CHAPTER 5 GENERAL DISCUSSION AND CONCLUSIONS GnRH usage in dairy cows has been evident since the 0 and early 80 when it was used for the treatment of ovarian follicular cysts in dairy cows (Kittok et al., 1973; Britt et al., 1977 ; Troxel and Kesler, 1984) an d for induction of ovulation in early postpartum (Britt et al., 1974; Fernandes et al., 1978; Kesler et al., 1978). Nowadays, GnRH is used mostly in association with prostaglandins (Richardson et al., 1983; Benmrad and Stevenson, 1986; Thatcher et al., 1993; Pursley et al., 1997) and or progestins (Thatcher et al., 1993; Lynch et al., 1999; Xu et al., 2000; Lima et al. 2011; Bisinotto et al., 2013) for the purpose of synchronization of ovulation as part of TAI protocol. The randomized clinical trial reported herein was designed to evaluate the effect of GnRH agonist administration at 17 3 and 20 3 DIM on ovulation responses by the end of the fourth week postpartum, prevalence of CE and CTE at 35 DIM and reproductive outcomes from the first service and up to 300 DIM. O ur results showed that both the GnRH 1 and GnRH 2 increased ovulation within 3 to 4 days of adminis tration compared to control cows which resulted in overall greater ovulation by 2 4 DIM (78.7% vs. 45.0%) This findings support the thought that the pituitary gland responsiveness to GnRH (Fernandes et al., 1978 ; Alam and Dobson, 1987; Bosu et al., 1988 ) i s restored withi n 14 days postpartum and agree with others that showed that exogenous GnRH can induce ovulation from 10 to 30 DIM (Britt et al., 1974; McDougall et al., 1995; Gmen and Seguin, 2003; Amaya Montoya et al., 2007) Interestingly, Benmrad and S tevenson (1986) reported similar ovulation rate (75%) with only a single GnRH injection (200 g) between 10 and 14 DIM while only 28% of the controls ovulated in the same period ( with in 3 to 5 days post treatment). Beam and Butler, (1999) reported that vi rtually all Holstein dairy cows have the first wave of follicle growth starting two

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67 weeks postpartum ; therefore, by administering GnRH 1 between 14 and 20 DIM, our aim was to increase ovulation of the dominate follicle of the first follicular wave postpart um. Nonetheless, at the time of GnRH 1, 23.4% of the cows already had a CL; therefore, it is possible that earlier administration of GnRH at a narrower interval (b etween 10 and 14 DIM ) could have achieved higher ovulation rate than administration between 1 4 and 20 DIM Because follicular growth is not tightly synchronized, a s it can be observed by the pattern of ovulation in the control group, a two injection treatment scheme was used herein. If only one injection was administered, only 44.6% of the cows wo uld have ovulated within 7 days of GnRH administration, which is a little over the double of the ovulation in the c ontrol group (20.9%) or an increase of 23.7 percentage points This improvement is in agreement with a recent report where only one injection of 100 g of GnRH was given at 29.2 5.2 DIM and ovulation was improved by 17.9 percentage units (70.9% vs. 53.0%). One overt difference from the report herein and the re p ort by Benmrad and Stevenson, (1986) is the higher dose (200 g ) of GnRH used by th e latter. Nonetheless, it has been shown that only 50 g of GnRH are needed to induce ovulation ( Fricke et al., 1998); t herefore it seems plausible that two injections of GnRH are needed to achieve a greater increase in ovulation rate. The interval of 3.5 days would allow follicles that did not respond to the first GnRH to grow another 5 to 9 mm (growth of 1.5 to 2. 6 mm/day ; Ginther et al., 1997 ), which would allow them to gain ovulatory capacity if they were at least 6 mm at GnRH 1 (Sartori et al., 2001). It is possible that waiting another day or two would have increased ovulatory response even further; however, this needs to be investigated. In a studied done by McDougall et al. (1995) ovulation rate of 90 % was achieved with the use of a synthetic GnRH agonist (250 g) in cows with a dominant follicle (follicles of at least 10 mm in diameter ) detected after 14 days post partum while only 10% of the control s

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68 cows (receiving only saline solution) that also had dominant follicles ovulated in the same peri od ( up to 4 days post treatment) It is expected that administering GnRH to only cows that had a dominate follicle would increase ovulatory response; however, it is intriguing that only 10% of the controls would ovulate spontaneously In our herds, spontan eous ovulation was high; 23.4% already ha d a CL between 14 and 20 DIM (day of GnRH 1), 20.9% of the control cows ovulat ed within 7 days of GnRH 1 and another 30.5% of the remaining anovular control cows ovulated within 7 days of GnRH 2. It is possible that differences in metabolic state among herds used herein and the herd used by McDougall et al., (1995), such as deeper NEB, could contribute to lower ovulation rate in control cows. Body condition score at 7 weeks postpartum had a significa n t effect on ov ulation to G1 2.75 at 17 3 DIM. Higher BCS loss is associated to longer and pronounced negative energy balance (NEB) and it is extensively showed to delay resumptio n of postpartum ovarian cyclicity and ovulation (Heuwieser et al., 1994; Beam and Butler, 1999; Butler 2005; Santos et al., 2009; Kim et al., 2012). In situation of negative energy balance high concentration s of NEFA and low IGF 1 are detected in bloodstr eam NEFA can be inhibitory of granulosa cells proliferation and also increase their apoptotic rate (Jo rr itsma et al., 2004; Vanholder et al., 2005) IGF 1 in turn stimulates the development of follicles maximizing steroidogenesis in which positively feed s back in LH secretion that further stimulates ovulation However, BCS loss are associated to NEB that compromise the pulse frequency of LH, hence compromis ing estradiol production, follicular selection, maturati on and ovulation (Butler, 2006). Nonetheless effort to minimize the body condition loss postpartum can minimize the negative effects of NEB on resumption of ovarian cyclicity and ovulation postpartum and subsequent pregnancy/AI.

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69 There was an interaction between GnRH treatment and parity on ovul ation to GnRH 2 This interaction was mainly because of a lower spontaneous ovulation in primiparous control cows compared to multiparous control cows Previous showed low er postpartum resumption of ovulation for primiparous compared with multiparous ( Gme n and Seguin, 2003; Santos et al., 2009) Primiparous are known to have greater incidence of metritis ( Goshen and Shpigel, 2006) and to experience greater degree of negative energy balance (Wathes et al., 2007; Meikle et al., 2004) which are known to affe ct resumption of ovulation postpartum ( Peter et al., 1989; Beam and Butler, 1999; Williams et al., 2007, 2008). Santos et al. (2009) hypothesized that primiparous cows are more sensitive to metabolic and endocrine signals in the transition period that migh t delay resumption of ovarian cyclicity postpartum It is speculated in our study that the main deficiency in primiparous cows compared to multiparous was the inability to induce a GnRH/LH surge and not a lack of dominant follicles since administration of GnRH was able to induce similar ovulatory response in primiparous and multiparous but spontaneous ovulation was lower in primiparous compared to multiparous. Season of calving had a significant effect on overall ovulation rate and cows that calved in the warm season had higher odds of ovulation than cows that calved in the cool season. Th e s e findings agree with previous reports where ovulation was increased in the warm season ( Hansen and Hauser 1983; Opsomer et al., 2000; Walsh et al., 2007; Santos et al ., 2009; Kim et al., 2012). Santos et al., 2009 hypothesized that photoperiod stimulation and or nutritional changes are influencing this seasonal pattern of increased ovulation on during the warm season Photoperiod may impact follicle development because melatonin is decreased with increased day length, and melatonin has been shown to have a negative impact on IGF 1 secretion in cows (Dahl et al., 2000, 2002) Hence, a decrease in IGF 1 would negatively impact estradiol

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70 production by granulosa cells and i mpair follicle development (Beam and Butler, 1998; Beam and Butler, 1999) Furthermore, melatonin has been shown to decrease LH concentrations in ovariectomized cows (Rhodes et al., 1979). The change from negative feedback to a positive feedback of estradi ol on LH is important for resumption of cyclicity (Legan et al., 1977; Schillo et al., 1982b). In ovariectomized heifers, it has been observed that release of LH induced by estradiol was greater for heifers exposed to 18 h of light and 6 h of dark than for heifers exposed to short days (Hansen et al., 1982) Furthermore primiparous cows that calved in the winter but were exposed to 1 8 h of light and 6 h of dark had reduced intervals to first ovulation and to first estrus compared to cows exposed to natural day light cows; although, no difference was observed for multiparous (Hansen and Hauser, 1984) Uterine b lood flow in the warmer season of the year is de creased I t is believed that during the final trimester of the gestation, the reduction of nutrie nts delivered to the calf due the reduction in uterine blood flow, the conceptus adaptively increase the branching of the vessels and capillary density within the placentomes. This adaptive situation carry over effects after parturition in which the rich v ascularized caruncles are capable to produc e high amounts of prostaglandins early postpartum Among these prostaglandins, PGF2 is being produced in large amounts hasten ing uterine involution (Lewis et al., 1984), which could explain the lower rate of metritis in cows that calved in summer observed in our study (17.4 % vs. 27.8 % for cows that calved in the warm and cool season r espectively) and showed elsewhere (Benzaquen et al., 2006) The combination of these factors may support the association in which calving in warmer time of the year favor the higher overall ovulation rate achieved in the current studies and showed elsewher e (Opsomer et al., 2000; Walsh et al., 2007; Santos et al., 2009; Kim et al., 2012).

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71 Etherington et al (1984) demonstrated that uterine health was negatively impacted by the early use of GnRH postpartum in lactating dairy cows due to an increased frequency of pyometra. However, our findings showed no statistical difference on the frequency of either clinical or cytological endometritis (a less subjective diagnosis of uterine health status done by cytology of the uterus), the proportions of this condition we re similar in both group (30.9% vs. 32.8% of cytological endometritis positive diagnosis for GnRH treated and c ontrol groups respectively, P = 0.61). O ur results agree with Stevenson and Call (1988) where no evidence of negative effect s (such as high frequ ency of pyometra) of GnRH administration early in lactation (between 11 to 25 DIM) on uterine health was observed Calving related problems significantly increased the odds of having CE and metritis increased the odds of having both CE and CTE. The occurr ence of CTE in our study (31.9 %) was lower than report ed by Gilbert et al. ( 2005 ), r ang ing from 43 % to 73 % however, in his study, sampling was performed between 40 to 60 DIM and cutoff point for CTE was lower ( 5 % PMN in the endometrial) in which could explain the higher prevalence obtained compared to our study that used a higher cutoff point ( 10 % ) Indeed, ours results are in agreement with s tudies that us ed similar cutoff point for PMN count ( 10 % ) i n which the mean prevalence of CTE was 25.9% (Cheong et al., 2011). Although Rutigliano et al. (2008) showed lower prevalence of CTE ( prevalence of 1 8.2 % using higher cutoff point for CTE PMN 18 %) together, ours findings are in agree ment that cows wi th peripartum uterine disorders (i.e. metritis, dystocia, retained fetal membranes) ha ve increased odds of CTE. Postpartum diseases such as dystocia, retain fetal membranes and metritis predispose cows to endometritis by reduce d uterine involution and disr upt ion of the balance between the immune response of the cow and the bacteria load present in the lumen of the uterus N egative energy status induced from the reduction of dry matter intake on the cows

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72 affected by th o se disorders (Huzzey et al., 2007) comp romise the availability of energy to the neutrophils ( the main leukocyte type involved in bacteria clearance in uterine infection) that impair it s function, such phagocytosis and killing capacity (Cai et al., 1994; Kehrli and Goff 1989; Gilbert et al., 199 3) given course for the chronic infection of the uterus post 21 days postpartum. Pregnancy to the first service was not different for GnRH treated and control groups In a recently study ( Jeong et al., 2013) in which GnRH was injected at 29 5 DIM in health cows without a CL, GnRH increased conception rate to the first AI (38.7% vs. 29.1 %) compared to control cows even though the increase in ovulatory response reported herein ( ~ 34 percentage points) was considerably greater than what was reported i n the study by Jeong et al., (2013) ( ~ 18 percentage points) The main differences from the study by Jeong et al., (2013) were the time of GnRH administration and the reproductive management of the cows. In the study herein, cows received GnRH at 17 3 and 20 3 DIM and both farms used TAI programs as part of their reproductive management while in the study by Jeong et al., cows received GnRH at 29 5 DIM and the farm relied exclusively on estrus detection for the first AI. Because GnRH was administered ea rlier it is possible that c ontrol cows had enou gh time to resume cyclicity and reestablish fertility to the level of Gn R H treated cows. In fact, survival curves showed that by 60 DIM, the proportion of cows that were cycling was similar between GnRH treat ed and control cows. When ovulation occurred spontaneously and AI was performed upon estrus detection cows that were cycling by 21 DIM had increased fertility compa red to cows that started cycling from 21 to 49 DIM (Galv o et al., 2010) Likewise, others have observed higher fertility in cows that experienced an earlier resumption of cyclicity and were submitted to AI upon estrus detection (Thatcher and Wilcox, 1973; Darwash et al., 1997) It is not clear why TAI would

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73 mask any beneficial effect of early c yclicity because w hen cows are not cycling at the start of the Ovsynch program, conception rate s are indeed decreased (Galv o et al., 2004; Moreira et al., 2001; Santos et al., 2004) Nonetheless, since a similar proportion of cows were cycling in the GnRH treated and control groups approximately one week before the start of the Ovsync h program, it is possible that differences in fertility because of earlier cyclicity in the GnRH treated group and control group were attenuated Nonetheless, when evaluating fertility of cows that were already cycling at 17 3 DIM, cows that started cycling by 2 4 3 DIM and cows that remained anovulatory, it is clear that cows that were cycling at 17 3 DIM and cows that were induced to cycle from 17 to 24 3 DIM had impro ved fertility than cows that remained a novulatory up to 24 3 DIM. BCS at study enrollment also affected the likelihood of pregnancy to the first service and cows with BCS > 2.75 at time of the study enrollment had 1.8 times the odds of conceiv ing compar ed to c positive association of high BCS enhancing fertility can be explained by the fact in which cows in better BCS are also known to be in better metabolic status with adequate concentrations of glucose, insulin and IGF 1 in blo od that increase rate s of ovarian follicle growth. The consequence is the enhance d steroidogenesis with high follicular estradiol production that positively feed s back on the hypothalamus pituitary axis up to the point to induces LH surge and ovulation (Be am and Butler, 1999). Several studies have shown improved cyclicity and fertility for cows with higher BCS ( Pryce et al., 2001; review by Lpez Gatius et al., 2003; Butler et al., 2006; Roche et al., 2007; Santos et al., 2009). Herein, cows with BCS > 2.75 had increased ovulation within 3.5 d of GnRH 1 (36.3% vs. 21.7%), which probably contributed to improve ment of fertility.

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74 Dairy had an effect in conception rate at first service, in which Dairy 1 had higher c onception rates than Dairy 2. Differences in c onception rates between dairies may be because of a multitude of cow level and herd level factors that goes beyond the scope of this study. Our main goal in including Dairy as a fixed effect was to be able to test for interactions between GnRH treatment an d Dairy; however, one was not observed, indicating that GnRH treatment was equally effective in both dairies. Therefore, it is safe to assume that similar response to GnRH treatment may be expected in dairies with similar characteristics to the ones used h erein. One important component of the reproductive performance is the ability of the pregnant cow to maintain and carry the conceptus to term. Therefore, pregnancy loss is an important component of herd reproductive performance In the present study, GnRH treated cows had lower pregnancy loss compared to c ontrol cows To the authors knowledge this is the first time that GnRH treatment early postpartum is associated to reduction in late pregnancy loss A potential explanation for decreased pregnancy loss in the GnRH group would be the increased ovulation rate by 24 3 DIM because cows that remain anovulatory by the end of the voluntary waiting period have an increased pregnancy loss (Galvo et al., 2004; Santos et al., 2004) Nonetheless, p regnancy loss was actually higher in c ows that ovulated by 2 4 3 DIM In fact, all the pregnancy losses in the GnRH treated group came from the cows that ovulated [8% ( 6 /7 5 ) in GnRH treated that ovulated and 0 (0/12) in GnRH treated that did not ovulate] and in the contro l group most of the pregnancy losses also came from the cows that ovulated [26.1% (11/42) in control that ovulated and 10.8% (5/46) in c ontrol that did not ovulate]. In cows that had a CL at 17 3 pregnancy loss was low compared to cows that ovulated betw een 17 3 and 24 3 DIM [6.9% (4/58) vs. 14.5 (17/117)] ; therefore, at this point it is not clear why GnRH administration resulted in decreased pregnancy loss and why cows that ovulated by 24 3 DIM

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75 had increased pregnancy loss since spontaneous early o vulation d id not have the same negative effect on pregnancy loss. There is always a chance for type 1 error; therefore, further research is needed to confirm the beneficial effect of GnRH administration on pregnancy loss and the negative effect of ovulatio n between 17 3 and 24 3 DIM on pregnancy loss compared to cows that had ovulated by 17 3 DIM and cows that failed to ovulate by 24 3 DIM. Metritis in our study was also associated with increased incidence of pregnancy loss and interestingly there a re not much relates of this association. Few studies showed results of the negative effect of metritis in pregnancy loss (Ribeiro et al., 2013, unpublished). In fact, the complete mechanism in which metritis leads to the interruption of the pregnancy is no t well understood or demonstrated. In an epidemiologic study evaluating fertility of grazing dairy cows, metritis increased the odds of pregnancy loss by 4 times compared to no metritis cows (Ribeiro et al., 2013, unpublished ). What it is known it is the r ole of metritis reducing reproductive performance (review by Fourichon et al., 2000 ; Williams et al., 2008 ) Infection of the uterus also disturb ovarian follicle growth and function, and in cows that ovulated, reduced CL size and lower progesterone produc tion occur affecting reproduction however, the mechanism is not yet elucidated (Williams et al., 2007). M oreover metritis be ing associated to development of endometritis as demonstrated in our study and elsewhere ( Galvo et al., 2009, 2011) Lpez Gatius et al. (1996) showed association of uterine diseases such as retained fetal membranes and pyometra in pregnancy loss, in which cows that underwent to these pathologies had 1.8 and 2.6 odds of pregnancy loss compared to non disease cows, respectively. This research group speculated that the alterations in the uterine environment caused by those diseases could be critical during the implantation of the conceptus, compromising the attachment and survival of the embryo although it was just a hypothesis of poss ible mechanism and it was not proved The

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76 overall effect of uterine diseases such as metritis and endometritis is damage t o the uterine glands and activation of the inflammat ory cascade with release of pro inflammatory cytokines. Among the cytokines, tumor necrosis factor release of PGF2 from the endometrium and luteal cells, hence inducing luteolysis (Skarzynski et al., 2005) ; therefore if the inflammation persists the pregnancy may be lost A lthough metritis occurs within the first 3 weeks postpartum (Sheldon et al., 2006) and breeding usually takes place after 60 DIM, the direct association of metritis with pregnancy loss is through its association with CE and CTE as observed herein. The inflammatory process associated with uterine diseases lead to occlusion of the endometrial glands, dilation of underlying glands with deposit of connective tissue and formation of scar tissue in the uterus which may affect embryonic implantation and maintenance of gestation Clinical e ndometritis (Galv o et al., 2009) and CTE (Lima et al., 2013 ) have been show n to increas e the risk of pregnancy loss In vitro studies showed th at culturing embryos in a medium containing non specific inflammatory products (Hill and Gilbert, 200 8) or pro inflammatory cytokines such as TNF (Soto et al., 2003) impair early embryo development. More recently, it was demonstrated that expression of genes in volved in maintenance of membrane stability and the cell cycle were downregulated in embryos from cows with CTE (Hoelker et al., 2012) Fur thermore, embryos from cows with CTE had developmental retardation, which corroborates with the findings from previous studies (Hill and Gilbert, 2008; Soto et al., 2003) Hazard of pregnancy by 300 DIM based on PD2 was evaluated and an interaction betwee n GnRH treatment and ovulation from 17 3 DIM to 24 3 DIM was observed The hazard of pregnancy for GnRH treated cows that ovulated was 2 times the hazard for GnRH treated cows that did not ovulate and 1.3 times greater than control cows that did not ov ulate,

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77 however similar to control cows that ovulated. Cows that responded and ovulated from GnRH injection are more ready to conceive then cows that did not ovulate, which agrees with previous findings that precocious resum ption of ovulation postpatum posi tively effects fertility (Darwash et al., 1997; Santos et al., 2009; Galvo et al. 2010; Kim et al., 2012). Recently a study performed in Korean dairy herds (Jeong et al., 2013) demonstrated increased hazard of pregnancy by 210 DIM (HR = 1.3; 95 % CI = 1. 055 to 1.61; P = 0.01) for GnRH treated cows around one month postpartum compared to control cows. The higher conception rate obtained with the use of GnRH, shorter voluntary waiting period combined to the enrolment of only health cows in the study may exp lain the success in the GnRH use postpartum in the Korean study. We can speculate that this group of cows may be in a better metabolic status that leads to a higher hazard of pregnancy. The low reproductive performance of the GnRH treated cows that failed to ovulate may be explained by the overall worse uterine health in this group. It has been hypothesized that early ovulation is a marker for uneventful transition into lactation (Galv o et al., 2010), with cows ovulating early having better uterine health (i.e decreased prevalence of CTE) tha n cows that fail to ovulate. Probably cows that ovulate are in a better health status, which would lead to higher IGF 1, higher LH pulse frequency and greater follicle development than cows that fail to ovulate. To help confirm our hypothesis, GnRH treated cows that failed to ovulate had greater prevalence of C T E than cows that ovulated ( 43 vs. 25%). Interestingly, prevalence of metritis (22 vs. 22%) and CE (25 vs. 23%) did not differ between GnRH treated cows that ovula ted and did not ovulate. This data shows that probably there were differences in metabolic state in cows that did and did not ovulate to GnRH independently of disease state (i.e. metritis), that later translate into improved uterine health (i.e. decreased CTE) in cows that ovulate to GnRH In the control group, the prevalence of metritis (18 vs 20%), CE ( 20 vs. 17 % ),

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78 or CTE (32 vs. 33%) did not differ for control cows that did and did not ovulate respectively I t was also observed that prevalence of metri tis (11%), CE (11%) and CTE (26%) for cows that had a CL at 17 3 DIM were considerably lower than cows that did not have a CL, again confirming our hypothesis of an overall better health status in cows with early ovulation (Galv o et al., 2010). In con clusion, the administrations of two doses of GnRH agonist at 17 and at 20 3 DIM in lactating Holstein cows successfully induce d ovulation up to 2 4 3 DIM. GnRH treatment did not affect uterine health status and pregnancy to the first service although si gnificantly decreased pregnancy loss to t he first service. Time to pregnancy was not affected by GnRH treatment although hazard of pregnancy was increased in cows that responded to GnRH treatment but was decreased in cows that did not respond to GnRH trea tment. In summary, the use of GnRH in early lactation dairy cows lead to increased ovulation but does not affect long term fertility. The effect of GnRH administration on pregnancy loss needs further investigation.

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79 LIST OF REFERENCES Adams, G. P., R. L. Matteri, J. P. Kastelic, J. C. Ko, and O. J. Ginther. 1992. Association between surges of follicle stimulating hormone and the emergence of follicular waves in heifers. J. Reprod. Fertil. 94:177 188. Alam, M. G. S., and H. Dobson. 1987. Pituitary response s to a challenge test of GnRH and oestradiol benzoate in postpartum and regularly cyclic dairy cows. Anim. Reprod. Sci. 14:1 9. Al in, E. A. Sahu, S. Ramaswamy, E. D. Hutz, K. L. Keen, E. Terasawa, C. L. Bethea, and T. M. Plant 2013 Ovarian regulation o f kisspeptin neurones in the arcuate nucleus of the rhesus monkey ( Macaca mulatta ). J. Neuroendocrinol. 25:488 496 Amaya Montoya, C., M. Matsui, C. Kawashima, K. G Hayashi, G. Matsuda, E. Kaneko, K. Kida, A. Miyamoto, and Y. I. Miyake. 2007. Induction of ovulation with GnRH and PGF2alpha at two different stages during the early postpartum period in dairy cows: ovarian response and changes in hormone concentrations. J. Reprod. Dev. 53:867 875. Beam, S. W., and W. R. Butler. 1997. Energy balance and ovarian follicle development prior to the first ovulation postpartum in dairy cows receiving three levels of dietary fat. Biol. Reprod. 56:133 142. Beam, S. W., and W. R. Butler. 1998. Energy balance, metabolic hormones, and early postpartum follicular development in dairy cows fed prilled lipid. J. Dairy Sci. 81:121 131. Beam, S. W., and W. R. Butler. 1999. Effects of energy balance on follicular development and first ovulation in postpartum dairy cows. J. Reprod. Fert il 54:411 424. Beg, M. A., and O. J. Ginther. 2006. Follicle selection in cattle and horses: role of intrafollicular factors. Reproduction. 132:365 377. Bell, A. W. 1995. Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation J. Anim. Sci. 73:2804 2819. Ben mrad, M., and J. Stevenson. 1986. Gonadotropin for postpartum dairy cows: estrous, ovulation, and fertility traits. J. Dairy Sci. 69:800 811. Benzaquen, M. E. 2006. Evaluation of rectal temperature and calving relate d factors on the incidence of puerperal m etritis in postpartum dairy cows. M S thesis University of Florida Gainesville Bisinotto, R. S., R. C. Chebel, and J. E. P. Santos. 2010b. Follicular wave of the ovulatory follicle and not cyclic status influence s fertility of dairy cows. J. Dairy Sci. 93:3578 3587.

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90 Ribeiro, E. S., F. S. Lima, L. F. Greco, R. S. Bisinotto, A. P A. Monteiro, M. Favoreto, H. Ayres, R. S. Marsola, N. Martinez, W. W. Thatcher, and J. E. P. Santos. 2013. Prevalence of periparturient diseases and impacts on fertility of seasonally calving grazing dairy cows supplemented with concentrate J. Dairy Sci I n press Richardson G. F., L. F. Archbald D. M. Galton and R. A. Godke 1983. Effect of gonadotropin Theriogenology 9:763 770. Risco, C. A., P. J Chenoweth, B. I. Smith, J. S. Velez, and R. D. Barker. 1998. Management and economics of natural service bulls in dairy herds. Comp. Cont. Edu. 20:385 390. Roche, J. R. K. A. Macdonald, C. R. Burke, J. M. Lee, and D. P. Berry. 200 7. Associations a mong b ody c ondition s core, b ody w eight, and r eproductive p erformance in seasonal c alving d airy c attle J. Dairy Sci. 90:376 391 Rhodes R. C., P. K. Forrest, and R. D. Randel 1979. T he effect of melatonin upon luteinizing hormone in ovar iectomized Brahman cows J. Anim. Sci. 49 : 330 Rutigliano, H. M., F. S. Lima, R. L. A. Cerri, L. F. Greco, J. M. Vilela, V. Magalhes, F. T. Silvestre, W. W. Thatcher, and J. E. P. Santos. 2008. Effects of method of presynchronization and source of seleniu m on uterine health and reproduction in dairy cows. J. Dairy Sci. 91:3323 3336 Sakakibara, M ., C. Deura S. Minabe Y. Iwata Y. Uenoyama K. I. Maeda and H. Tsukamura 2013. Different critical perinatal periods and hypothalamic sites of oestradiol action in the defeminisation of luteinising hormone surge and lordosis capacity in the rat. J. Ne uroend. 25:251 259 Santos, J. E. P., W. W. Thatcher, R. C. Chebel, R. L. A. Cerri, and K. N. Galvo. 2004. The effect of embryonic death rates in cattle on the efficacy of estrus synchronization programs. Anim. Reprod. Sci. 82 83:513 535. Santos, J. E. P ., H.M. Rutigliano, and M. F. S Filho. 2009. Risk factors for resumption of postpartum estrous cycles and embryonic survival in lactating dairy cows. Anim. Reprod. Sci. 110:207 221. Santos, J. E. P, R. S. Bisinotto, E.S. Ribeiro, F.S. Lima, L.F. Greco, C .R. Staples, and W.W. Thatcher. 2010. Applying nutrition and physiology to improve reproduction in dairy cattle. Soc. Reprod. Fertil. Suppl 67:387 403. Snchez F., and J. Smitz 2012. Molecular control of oogenesis. Bioch im Bio phys. Acta 1822:1896 1912. Sartori, R., R. Sartor Bergfelt, S. A. Mertens, J. N. Guenther, J. J. Parrish, and M. C. Wiltbank. 2002. Fertilization and early embryonic development in heifers and lactating cows in summer and lactating and dry cows in winter. J. Dairy Sci. 85:2803 2812.

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93 Thatcher, W. W., R. L. de la Sota, E. J. Schmitt, T. C. Diaz, L. Badinga, F. A. Simmen, C. R. Staples, and M. Drost. 1996. Control and management of ovarian follicles in cattle to optimize fertility. Reprod. Fertil Dev. 8:203 217. Thissen, J. P ., J. M. Ketelslegers and L. E. Underwood 1994. Nutritional regulation of the insulin like growth factors. Endo cr Rev. 15:80 101. Thorburn, G. D., and D. H. Nicol. 1971. Regression of the ovine corpus luteum after infusion of prostaglandin F2a into the ovarian artery and uterine vein. J Endocrinol. 51:785 786. Todoroki, J., J. Noguchi, and K. Kikuchi. 2004. Plasma concentrations of inhibin A in cattle with follicular cysts: relationships with turnover of follicular waves and plasma levels of gonadotropins and steroid hormones. Domest. A nim. Endocrinol. 27:333 344. Troxel, T. R., and D. J. Kesler. 1984. The effect of progestin and GnRH treatments on ovarian function and reproductive hormone secretions of anestrous postpartum suckled beef cows. Theriogenology 21:699 711. Vailes, L. D., S. P. Washburn, and J. H. Britt. 1992. Effects of various steroid milieus or physiological states on sexual behavior of Holstein cows. J Anim. Sci. 70:2094 2103. Vanholder, T., J. L. Leroy, A. V. Soom, G. Opsomer, D. Maes, M. Coryn, and A. Kruif. 2005. Effect of non esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro. Anim. Reprod. Sci. 87:33 44. Wa lsh, R. B., D. F. Kelton, T. F. Duffield, K. E. Leslie, J. S. Walton, and S. J. LeBlanc. 2007. Prevalence and risk factors for postpartum anovulatory condition in dairy cows. J. Dairy Sci. 90:315 324. Wathes, D. C., Z. Cheng, N. Bourne, V. J. Taylor, M. P. Coffey, and S. Brotherstone. 2007. Differences between primiparous and multiparous dairy cows in the inter relationships between metabolic traits, milk yield and body condition score in the periparturient period. Dome st Anim. Endo crinol 33:203 225. Whit more, H. L., W. J. Tyler, and L. E. Casida. 1974. Incidence of cystic ovaries in Holstein Friesian cows. J. Am. Vet. Med. Assoc. 165:693 694. Williams, E. J., D. P. Fischer, D. U. Pfeiffer, G. C. W. England, D. E. Noakes, H. Dobson, and I. M. Sheldon. 2005 Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle. Theriogenology. 63:102 117. Williams E J D P Fischer, D E Noakes, G C England, A Rycroft, H Dobson, and I M Sheldon. 200 7. The relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow. Theriogenology 68:549 559. Williams E J K. Sibley, A. N. Miller, E. A. Lane, J. Fishwick, D. M. Nash, S. Herath, G. C. England, H. Dobson, an d I. M. Sheldon. 2008. The effect of Escherichia coli

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94 lipopolysaccharide and tumour necrosis factor alpha on ovarian function. Am J Reprod Immunol 60:462 473. Wiltbank M. C., A. Gmen, and R. Sartori. 2002. Physiological classification of anovulatory c onditions in cattle. Theriogenology. 57:21 52. Xu, Z. Z., L. J. Burton S. McDougall and P. D. Jolly 2000 .Treatment of noncyclic lactating dairy cows with progesterone and estradiol or with progesterone, GnRH, prostagla ndin and estradiol. J. Dairy Sci. 83: 464 470. Yan, C., P. Wang, J. DeMayo, F. J. DeMayo, J. A. Elvin, C. Carino, S. V. Prasad, S. S. Skinner, B. S. Dunbar, J. L. Dube, A. J. Celeste, and M. M. Matzuk. 2001. Synergistic roles of bone morphogenetic pro tein 15 and growth differentiation factor 9 in ovarian function. Mol. Endocrinol. 15:854 866. Zarrin, M., L. De Matteis, M. C. M. B. Vernay, O. Wellnitz, H. A. van Dorland, and R. M. Bruckmaier 2013. Long hydroxybutyrate in dairy cows through infusion: Effects on feed intake, milk production, and metabolism. J. Dairy Sci. 96:2960 2972.

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95 BIOGRAPHICAL SKETCH Joo Henrique Jabur Bittar was born in Goinia, the capital the state called Gois, Brazil in 1980 He is the youngest o f three siblings of a Syrian Lebanese descend ant father born in Brazil and his wife, who was born and raised in a rural area of Minas Gerais state. Since his childhood Joo Henrique has b een interested in livestock, especially farm animals and led him to pursue a degree of Veterinary Medicine in the Federal University of Gois in 1999. Throughout his veterinary studies he became very active participating in several internship programs relat ed to beef and dairy production and equine as well as in a pharmaceutical company, wh ich enhanced his knowledge and network of professional contacts. After graduation he work ed as a veterinarian with beef and dairy cattle in Gois and Mato Grosso, both states consider ed one of the larger producers of livestock and agriculture in Brazil. In 2006 he joined the SENAR (an educational institution funded with agriculture commodity tax revenues ) where his duties included development of hoof care education and training progra ms of equine for farmers and farmer workers in the state of Gois. In 2007, he moved to the United States to learn more about dairy farming in this country, especially in the state of New York and to enhance his English communication skills. In 2008 he st arted work ing for a large dairy farm group also in New York that was client of the Ambulatory Veterinarian Service of Cornell University I n this position, he had the opportunity to interact with university veterinarians, residents, and interns Ambulatory Veterinarian Service of Cornell University inspired him to pursue advanced education in the United States. I n the summer of 2011, he was offered and accepted the position of Resident in Food Animal Reproductive and Medicine at the University of Florida College of Veterinary Medicine, Department of Large Animal Clinical Sciences In summer 2012, he was admitted to the Masters of Science Program in Veterinary Medical Sciences at the sa me

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96 University, and he is expecting to graduate in summer 2013. Joo Henrique complete his residency training by July 2014, and later to pursue a doctoral degree in the United States or go back to veterinary practice in the United States or in his home country.