Deslorelin) Implant During and After the Postpartum Summer Heat-Stress Period in Dairy Cattle

Home , Kunisada (crater)

REPRODUCTIVE, OVARIAN AND UTERINE RESPONSES TO A GNRH-AGONIST (DESLORELIN) IMPLANT DURING AND AFTER THE POSTPARTUM SUMMER HEAT-STRESS PERIOD IN DAIRY CATTLE

FLÁVIO TEIXEIRA SILVESTRE

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

UNIVERSITY OF FLORIDA

2003

Flávio Teixeira Silvestre

Dedicated to Juliana, Pai and Mãe, Gustavo, Paula and Daniel

ACKNOWLEDGMENTS

I would like to express my appreciation to Dr. William W. Thatcher for the opportunity to join this graduate program and financial support. Doctor Thatcher’s enthusiasm, knowledge and hard work have been an example for me and influenced me as a person and professional.

I extend my appreciation to Dr. Peter J. Hansen and Dr. Louis F. Archbald, members of my committee, for their valuable suggestions on my experiments during formal and informal discussions.

I would like to thank friends and colleagues in our laboratory who have been supportive and always helpful during my entire program: Dr. Metin Pancarci, Dr. Shun

Kamimura, Dr. Aydin Guzeloglu, Todd Bilby, Alvaro Arteche, Ricardo Mattos and Oscar

Hernandez.

Special thank go to Marie Joel Thatcher for her endless help on computer programming and hormone assays.

I would like to acknowledge friends in the Animal Science Department: Jeremy

Block, Marcio Libone, Maria Padua, Mirian Lopes.

I extend my appreciation to all friends in Gainesville who made Juliana and myself feel closer to home: Eduardo Carlos and family, Steel Vasconcelos and family, Marcelo and Aline Carvalho, Luis Lima and family, Whocely Vitor and family.

I extend my appreciation to Shirley who was my family during the period prior to this program.

I would like to express my appreciation to Peter Gelber and staff at Alliance Dairies for the expert management of experimental cows.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... iv

LIST OF TABLES...... ix

LIST OF FIGURES ...... xi

ABSTRACT...... xiii

CHAPTER

1 INTRODUCTION ...... 1

2 LITERATURE REVIEW ...... 5

Introduction...... 5 Effects of Heat Stress on Reproduction...... 6 Effects on estrus behavior ...... 6 Effects on fertility...... 6 Effects on follicular dynamics...... 7 Effects on Corpus Luteum...... 10 Effects on oocyte ...... 11 Heat-Stress Carry Over Effects ...... 14 Increasing Autumn Fertility...... 16 Embryological Origin of the Pituitary Gland ...... 18 Pituitary Histology and Anatomy ...... 19 Gonadotropin-Releasing Hormone Receptor ...... 20 Regulation of GnRHr...... 22 GnRH Molecule...... 23 GnRH-Agonist Deslorelin ...... 24 GnRH-Agonist Treatments...... 25 Initiation of Follicle Growth...... 26 Follicular Recruitment and Dominance...... 32 Follicular Dynamics Using a GnRH-Agonist...... 35 GnRH-Agonist Recovery...... 36 Follicular Dynamics During GnRH-Agonist Treatment...... 38 Possible Mechanisms of GnRH-Agonist Treatment on Milk Yield...... 39 Role of Growth Hormone in lactation...... 40 Growth Hormone responses to GnRH-Agonists ...... 41 Prolactin responses to GnRH-Agonists...... 42

Estradiol responses ...... 44 Influences of Uterus – Ovarian Interactions and GnRH-Agonist in Postpartum Uterine Involution in Cattle ...... 45 Uterine Physical Involution ...... 46 Uterine Histological Involution ...... 47 Uterine Mechanisms of Involution ...... 48 Uterine Infections ...... 49 Evaluation of Uterine Involution...... 52 Postpartum Ovulation ...... 53 Role of Progesterone...... 56 Role of Estradiol...... 59 Role of Prostaglandin ...... 62 Role of Oxytocin...... 65 Delaying First Ovulation Postpartum ...... 67 Direct Effects of Gonadotrophins on the Reproductive Tract...... 68 Uterine Histology Upon Chronic GnRH-Agonists...... 70

3 REPRODUCTIVE RESPONSES FOLLOWING SUPPRESSION OF FOLLICULAR DEVELOPMENT WITH A DESLORELIN IMPLANT DURING SUMMER HEAT STRESS...... 72

Introduction...... 72 Materials and Methods ...... 74 Implant removal...... 75 Ultrasound (ovarian activity)...... 76 Ultrasound (ovarian recovery)...... 77 Synchronization protocol...... 78 Ultrasound (pregnancy diagnosis)...... 80 Resynchronization protocol...... 80 Body condition score (BCS)...... 81 Blood samples and hormone assays ...... 81 Milk yield ...... 83 Temperature Humidity Index (THI)...... 83 Statistical Analysis...... 84 Results...... 85 Temperature Humidity Index ...... 85 Day 7 postpartum P4 ...... 86 Ovarian responses...... 86 Frequencies of Cyclic and Ovulation Status for All Cows...... 92 First service reproductive responses...... 95 First service reproductive responses accordingly to...... 97 Day 46 pregnancy rates ...... 99 Pregnancy losses...... 99 Second service reproductive responses ...... 100 Survival Analyses for First and Second Service Reproductive Responses...... 101 Milk weights...... 102 Discussion...... 103

vii

4 POSTPARTUM SUPPRESSION OF OVARIAN ACTIVITY WITH A DESLORELIN IMPLANT ENHANCED UTERINE INVOLUTION IN LACTATING DAIRY COW ...... 115

Introduction...... 115 Materials and Methods ...... 118 Experiment 1 ...... 119 Ovarian structures...... 119 Uterine and cervical diameters ...... 120 Vaginoscopy...... 121 Blood samples and hormones assays...... 122 Experiment 2 ...... 125 Statistical Analyses...... 126 Results...... 127 Experiment 1 ...... 127 Ovarian responses...... 127 Hormonal responses ...... 128 Involution of Uterus and Cervix...... 130 Vaginocopy results ...... 133 Correlations ...... 135 Experiment 2 ...... 137 Hormonal responses ...... 137 Uterine horns and cervical diameter...... 137 Cervical discharge and uterine tonus...... 137 Discussion...... 139

5 GENERAL DISCUSSION ...... 149

LIST OF REFERENCES...... 155

BIOGRAPHICAL SKETCH ...... 178

viii

LIST OF TABLES

Table page

3-1 Number of Class 1*, Class 2*, Class 3* follicle and corpora lutea* detected by ultrasound for different periods of treatment in the DESL implant and CON group of cows...... 88

3-2 Mean number of Class 1, Class 2, Class 3 and CL at 22 days after removal of DESL implant for cows implanted with DESL for different lengths of time...... 89

3-3 Mean number of Class 1, Class 2, Class 3 and CL at 29 days after removal of DESL implant for cows implanted with DESL for different lengths of time...... 90

3-4 Cyclic and ovulation responses based on plasma concentrations of P4, for DESL implant cows characterized by ovarian structure at ultrasound on days 22 and 29 after removal of the DESL implant...... 91

3.5 Frequencies of CON and DESL implant cows cycling during the presynchronization period and having a synchronized ovulation following the Ovsynch® protocols...... 93

3-6 Percentage of cows cycling during the presynchronization period as related to duration of DESL implant treatment for DESL implant cows and contemporaneous CON...... 94

3-7 Day 28 pregnancy rates to TAI for primiparous and multiparous cows within the DESL implant and CON groups...... 96

3-8 Pregnancy rates for CON and DESL implant cows that ovulated in response to Ovsynch® as influenced by quartile of accumulated milk yield...... 98

3-9 Pregnancy rates for CON and DESL implant cows that cycled and ovulated in response to presynch and Ovsynch® as influenced by quartile of accumulated milk yield...... 99

3-10 Pregnancy rates for cows in CON and DESL implant group resynchronized after d 28 pregnancy diagnosis...... 101

4-1 Average number of follicles per cow according to class (C) and CL for CON and DESL implant...... 128

4-2 Orthogonal contrasts for size (mm) of PPH during the postpartum period (days) of DESL implant and CON group...... 131

4-3 Frequencies of degree of tone during the postpartum period in CON and DESL implant groups...... 133

4-4 Frequencies of cervical discharge scores during the postpartum period for CON and DESL implant groups...... 134

4-5 Frequencies of cervical os coloration scores during the postpartum period for CON and DESL implant groups...... 135

4-6 Simple and partial correlation among variables measured in the postpartum period...... 136

4-7 Diameter of PPH, PNPH and cervix estimated by rectal palpation at day 30 postpartum...... 138

4-8 Degree of cervical discharge and uterine tonus assessed by rectal palpation at day 30 postpartum ...... 138

LIST OF FIGURES

Figure page

2-1 Proposed seven transmembrane topography of the bovine GnRH receptor ...... 21

2-2 Amino acid sequence of mammalian GnRH...... 24

2-3 Deslorelin Amino acid sequence...... 24

2-4 Putative model for the physiological role of FSH and LH in the uterine tract motility...... 70

3-1 First experimental period consisting of enrollment of DESL implant and CON cows (June 25 to August 8), ultrasound (ovarian activity) for DESL implant and CON sub-sample of cows and DESL implant removal days (August 28 and September 4) in the year of 2001...... 77

3-2 Second experimental period consisting of DESL implant removal days (August 28 and September 4), ultrasound (ovarian recovery) for DESL implant sub-sample of cows at 22 and 29 days after DESL implant removal (September 19 and 26) and onset of presynchronization/Ovsynch® protocol for DESL implant and CON groups of cows in the year of 2001...... 79

3-3 Temperature Humidity Index during the year 2001. A comparison between first period (10 days prior to the initiation of the experiment, June 15, until 10 days after DESL implant removal for the second group, September 14) versus second period (September 15 to 10 days after TAI for the second group of cows). THI differed between periods (P<0.01)...... 86

3-4 Survival curve for percentage of non-pregnant cow along the points in time for the 39 day period after TAI (d0 presynch/Ovsynch® TAI; d 8 to d 26 estrus AI; d 27 to d 38 resynch), non-pregnant cows were censored to day 39 (P< 0.01). ....102

3-5 Average milk yield during the 5-month postpartum period for CON and DESL implant groups for both primiparous and multiparous cows (Group, P<0.03; Parity, P<0.01)...... 103

3-6 Time line for possible number of follicular waves in CON cows during the cool season...... 109

3-7 Time line for possible number of follicular waves in DESL cows during the cool season...... 110

4-1 Ultrasound image of cow uterine horn during different days postpartum. A: Day 7 postpartum; B: Day 14 postpartum; C: Day 21 postpartum. Yellow arrows indicate width of uterine horn cross-section from dorsal to ventral serosa...... 121

4-2 Least squares means for number of follicles according to Class 1 (left Axis), Class 2, Class 3 and CL (right Axis) as measured by ultrasonography on days 23, 30 and 37 postpartum for DESL implant and CON cows...... 127

4-3 Least squares means for plasma concentrations of estradiol (pg/ml) in CON and DESL implant groups of cows during the postpartum period...... 129

4-4 Least squares means for plasma concentrations of progesterone (ng/ml) in CON and DESL implant groups of cows during the postpartum period...... 129

4-5 Least squares means for plasma concentrations of PGFM (pg/ml) for CON and DESL implant groups of cows during the postpartum period. * P<0.03 at 9dpp. .130

4-6 Least squares means for diameter of previous pregnant horn (PPH) in CON and DESL implant groups of cows during the postpartum period...... 131

4-7 Least squares means for diameter of previous non-pregnant horn (PNPH) for CON and DESL implant groups of cows during the postpartum period...... 132

4-8 Least squares means for diameter of cervix for CON and DESL implant groups during the postpartum period...... 133

xii

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

REPRODUCTIVE, OVARIAN AND UTERINE RESPONSES TO A GNRH-AGONIST (DESLORELIN) IMPLANT DURING AND AFTER THE POSTPARTUM SUMMER HEAT-STRESS PERIOD IN DAIRY CATTLE

Flávio Teixeira Silvestre

December 2003

Chair: William W. Thatcher Major Department: Animal Science

Holstein cows received a non-degradable GnRH-agonist implant (Deslorelin, 5 mg) at mean 2 ± 1 days postpartum (dpp), between June 25 to Aug. 8, for comparison to control cows (CON). Enrollment consisted of normal cows with a BCS > 2.75. Cows

were injected with PGF2α 7 d later. DESL implants were removed on Aug. 28 and Sept.

4. Implant exposure ranged from 28 to 67 d of treatment (trt). Ultrasonography (US)

monitored numbers of ovarian follicles and CL at 7, 28, 33, 43, 54 and 64 d of trt or dpp

in sub-samples of cows. At 31 d after implant removal, cows received a

Presynch/Ovsynch protocol: GnRH on d 0, PGF2α on d 7, GnRH on d 17, PGF2α on d 24,

GnRH on d 26 and timed artificial insemination (TAI) 16 h later. Pregnancy rate (PR)

was evaluated at 28 d (US) and 46 d (per rectal palpation) after TAI. Blood samples were

collected at PGF2α of presynch, subsequent GnRH, at TAI and 8 d after TAI. The DESL implant had increased number of Class 1 (3-5 mm) follicles (P< 0.01) and decreased numbers of Class 2 (6-9 mm; P<0.01), Class 3 (> 10 mm) follicles (P<0.01), and CL (P

xiii

<0.01). Follicular development was arrested at < 3 mm in the DESL implant group.

Reduced percent of DESL implant cows was cycling at the beginning of the Ovsynch

(52.2%, 58/111 < 93.7%, 104/111; P<0.01), and PR: overall TAI PR (27.5 %, 33/120 <

53.5 %, 69/128; P< 0.01), TAI PR in cycling cows (30.6 %, 19/62 < 53.7%, 65/121; P<

0.01), and TAI PR in cows that were cycling and ovulated after TAI (39.1%, 18/46 <

62.1%, 54/87; P<0.01). In DESL implant cows cycling and that ovulated after TAI group increased milk yield decreased PR (P<0.06). Pregnancy losses from 28 d to 46 d did not differ between groups. Milk yield was decreased in the DESL implant compared to the

CON cows (P<0.03). DESL implant induced a delay in turnover of postpartum-heat stress damaged follicles that may have contributed to a lower PR.

Postpartum responses were characterized in DESL implant (n= 10) and CON (n= 9) cows: plasma concentrations of P4, E2 and PGFM; ovarian follicular activity, diameters of

uterine horns and cervix; uterine tonicity; vaginoscopy of cervical discharge and color at

16, 23, 30 and 37 d plus at 9 dpp for hormonal responses. The DESL implant arrested

follicular growth to < 3 mm size. Plasma concentrations PGFM at 9 dpp were lower in

DESL implant in comparison to CON (P<0.03) and at all days for P4 and E2 (P< 0.01).

The DESL implant reduced diameters of previous pregnant (P<0.01) and non-pregnant

(P< 0.03) horns and cervix (P<0.01), increased uterine tonicity (P<0.07), reduced frequency of purulent cervical discharge (P< 0.09) and reddish cervical os (P< 0.06) in comparison to CON. Furthermore, diameter of uterine horns (PPH and PNPH; P< 0.01) but not the cervix was reduced at 30 dpp and increased uterine tonicity in DESL implant

(n=77) compared to CON (n=70) cows. The DESL implant treatment enhanced uterine involutionary processes and reduced inflammation of the reproductive tract.

xiv

CHAPTER 1 INTRODUCTION

Life time milk production of cows depends upon re-occurring pregnancy because pregnancy initiates and renews the lactation cycle (Lucy, 2001). Increases in herd size and milk yield per cow, severity of negative energy balance, greater use of free stall housing, postpartum disorders, and a reduction in estrus detection are reported as risk factors for a decrease in pregnancy rates (Lucy, 2001).

Approximately 36% of the annual milk production in the United States originates from herds with more than 500 lactating cows. Associated with the increase in milk production per cow there has been a concurrent decline in reproductive efficiency

(Butler, 2000; Lucy, 2001; Royal et al., 2000).

Summer heat stress is a major contributing factor to low fertility in lactating dairy cattle. It is a worldwide problem, which inflicts heavy economic losses and affects about

60% of the world dairy cattle population (Wolfenson et al., 2000). Stress was first defined as a syndrome produced by diverse nocuous agents that lead to alterations in biological organisms (Selye, 1936). Dobson et al. (2000) used the definition of “stress” for those animals that are exposed to changes in the environment that prevent them from expressing their full genetic potential, for example maximizing reproductive efficiency.

Lactating dairy cows are susceptible to heat stress during periods of high temperatures, because increased metabolic rate associated with lactation elevates internal heat production and decreases the cow’s thermo regulatory ability (Berman et al., 1985).

Kibler and Brody (1953) demonstrated that dairy cows shift body cooling mechanisms

1 2

from nonevaporative (convection, conduction and radiation) to evaporative (sweating and

panting) cooling. During periods of heat stress, milk production, feed intake and

physiological activity are decreased (Fuquay, 1981). At the same time, reproductive

ability is compromised (Thatcher and Collier, 1986).

Fertility in dairy cattle is depressed during months of high temperatures and there is a carry over into the cooler autumn period (Badinga et al., 1984; Al-Katanani et al.,

1999). Moreover, cows with higher milk yields had even greater decreases in fertility during the summer period of heat stress (Al-Katanani et al., 1999). The immediate and delayed effects of heat stress on fertility are multifactorial. Heat stress disrupts follicular development, dynamics of follicular waves, steroidogenic capacity of theca and granulosa cells, corpus luteum development and function, oocyte quality and embryonic survival.

During the early postpartum period, lactating dairy cows undergo a negative energy balance whose magnitude is associated with extended anovulatory periods and decreased fertility (Beam and Butler, 1997; Beam and Butler, 1998). Furthermore, postpartum metritis has a major economic impact for dairy operations because of increased treatment expenses, milk loss, prolonged days open and culling rates (Esslemont and Peeler, 1993).

Moreover, incidence of endometritis is still high at 50 days postpartum and has marked negative effects on fertility (Gilbert et al., 1998; Hammon, 2001). Predisposing factors for metritis are dystocia, retained fetal membranes, deficiencies in hygiene and metabolic imbalances occurring in the periparturient period (Gröhn et al., 1990).

Uterine bacterial contamination during the postpartum period is associated with delayed first ovulation (Sheldon et al., 2002). Also, early ovulation during the period of uterine involution results in prolonged luteal phases, characterized by elevated

progesterone concentrations for more than 25 days (Smith and Wallace, 1998; Opsomer

et al., 2000) in association with pyometra (Etherington et al., 1984).

Based on cultures of uterine fluids and uterine biopsies, the incidence of

endometritis is greater during the first 14 days than during the 29 to 35 days postpartum

(Griffin et al., 1974). The incidence of severe endometritis, which often progresses to

pyometra (Arthur et al., 1989), is increased at approximately the same time that cows

experience their first postovulatory period (i.e., 15 to 28 days postpartum) with increased

progesterone concentrations.

A health-monitoring program has been developed for dairy cows in the postpartum

period (Upham, 1996). In this program, rectal temperature, attitude, appetite and

appearance of manure are used to evaluate whether or not cows appear sick. Treatment is

based on the assumption that the major cause of fever early postpartum is related to

uterine infections and that sick cows have metabolic problems such as ketosis and

hypocalcemia.

Collectively, low fertility in lactating dairy cows results from a complex array of

events and interactions between energy balance, ovarian function, uterine health and overall health status of the cow. Postpartum status of the cow sets the stage for subsequent reproductive performance. Additionally, cooling systems used on farms have not been able to improve fertility substantially during summer heat stress season such that conception rates of lactating dairy cows are appreciably below those of winter (Berman and Wolfenson, 1992).

The objectives of the literature review (Chapter 2) are to summarize: 1) the current understanding of the multifactorial immediate and delayed effects of heat stress on

4 reproductive physiology and the potential technologies to improve fertility during heat stress conditions, and 2) the mechanisms regulating postpartum uterine involution and uterine-ovarian interactions. In the subsequent research chapters, ovarian follicular activity was suppressed with the use of a GnRH-agonist (Deslorelin) implant to evaluate follicle sensitivity to heat stress and associated effects on subsequent fertility during autumn after follicular recrudescence (Chapter 3). Subsequently, the effects of a GnRH- agonist (Deslorelin) implant on ovarian function and involutionary processes of the uterus were evaluated in dairy cows (Chapter 4). In Chapter 5, implications of the research will be addressed in a general discussion.

CHAPTER 2 LITERATURE REVIEW

Introduction

Selye (1936) introduced and popularized stress as a medical and scientific idea. He

was the first to appreciate the crucial role of the adrenal cortex – hypophysis axis in the

stress response and to define the stress-causing agent as a “stressor”. He also made a

distinction between negative (distress) and positive (eustress) reactions to the stressor.

Selye outlined four stages of the stress response: 1) the “alarm reaction” characterized by

an immediate activation of the sympathethic – adrenomedullary axis; 2) a “resistance”

phase characterized by hypothalamic pituitary – adrenal axis activation; 3) a stage of

adrenal hypertrophy and 4) an exhaustion phase and finally death. Therefore Selye’s

studies initiated the notion that a variety of diseases are produced as a result of excessive

or deficient adaptive processes during the stress response.

Stress also can be defined as those animals that are exposed to changes in the

environment that prevent them from expressing full genetic potential, for example

maximal reproductive efficiency (Dobson et al., 2001). Reproductive processes are very

sensitive to disruption by hyperthermia resulting in decreased fertility of females

(Hansen, 1997).

One of the most serious concerns of dairy producers located in tropical and subtropical areas is the seasonal depression of reproductive efficiency (Gwazdauskas et al., 1973). Sub-fertility during summer is considered to be of a multifactorial nature, since hyperthermia impairs various tissues. Furthermore, cattle exposed to heat stress

5 6

undergo responses that can impact indirectly reproductive efficiency. Such responses

include reduction in food intake, respiratory alkalosis, redistribution of blood flow among

body organs (Wolfenson, 2000). Cooling procedures used on farms today have not been

successful to markedly improve fertility and conception rate of lactating cows during

summer and is still appreciably below that of winter (Hansen, 1997).

Effects of Heat Stress on Reproduction

Effects on estrus behavior

High environmental temperatures are reported to reduce estrus behavior. Thatcher and Collier (1986) reported that the percentage of undetected estrus is about 75% during the summer and about 50% during the rest of the year in Florida dairies. Duration of estrus in Guernsey heifers maintained at 18.2oC averaged 17.0h vs. 12.5h for heifers at

33.5oC (Abilay et al., 1975). In another study, number of mounting episodes per estrus

for Holstein cows was decreased during summer in comparison to winter, 4.5 vs. 8.6

mountings per estrus (Nebel, 1997). The mechanisms by which heat stress reduces estrus

behavioral could be a reduction of circulating estradiol (see below).

Effects on fertility

Conception rates to artificial insemination may range from 55% during months of

low temperatures and humidity to only 10% during months of high temperatures and

humidity (Ingraham, 1974).

Breeding records of 12,038 inseminations from a dairy farm located in north

Florida showed that conception rates of lactating cows decreased to approximately 20%

during summer months (June through August) and continued to be low during autumn

(September and October) although temperatures were no longer of heat stress.

Conception rates did not recover to winter levels until November (Badinga et al., 1984).

Al-Katanani et al. (1999), using Dairy Herd Improvement Association records for 8124

lactating Holsteins cows located in Florida and southern Georgia, showed that 90-day

non-return rates to first service were lower during May thought August and did not

achieve normal levels until November and December. The severity of depression was of

greater magnitude as milk yield increased which is likely related to the increased

metabolic rate and decreased thermoregulatory ability for cows with high milk yield

(Berman et al., 1985). These studies have demonstrated clearly an immediate as well as a

delayed effect of summer heat stress on fertility.

Effects on follicular dynamics

Recent studies have indicated that heat stress altered follicular dynamics and depressed follicular dominance. Follicular dominance is associated with a deviation in growth between the dominant and the largest subordinate follicle. Deviation begins at the examination before an apparent change in the differences in diameter between the two largest follicles. Average over several reports, the mean diameter of two largest follicles at the beginning of deviation was 8.5 mm in heifers (Ginther et al., 2003). The mechanisms involved in follicular dominance appear to be closely associated with the acquisition of granulosa cells to express LH receptors, an increase in circulating estradiol

- 17β and a decrease in plasma FSH (Ginther et al., 1996). Follicular estradiol production during follicle deviation is dependent upon expression of granulosa-aromatase activity and androgen substrate from thecal cells. Androgens are produced in thecal cells by the

17α-hydroxylase and C17, 20-lyase activity of the P450c17 enzyme (Xu et al., 1995).

Therefore, provision of sufficient androgen substrate is essential for estradiol production.

Aromatase mRNA and enzyme activity are first expressed in granulosa cells of growing

4-mm follicles and greatly increases when the follicle reaches ≥ 8 mm (McNatty et al.,

1984; Badinga et al., 1992). The final suppression of FSH during the estrous cycle is the

mechanism that causes deviation in growth rates between the resulting dominant and

subordinate follicle. The dominant follicle continues to grow by a shift in a primary

gonadotrophin dependency from FSH to LH. Factors controlling the decrease in

circulating FSH are the increased estradiol secretion close to the moment of deviation and

acquisition of the future dominant follicle to produce inhibin. Inhibin is a protein

component of the follicular fluid that has a striking inhibitory effect on FSH secretion and

follicular growth (Kastelic et al., 1990; Tuzillo et al., 1993).

The first-wave dominant follicle at 8 days of the cycle was smaller and contained

less fluid in heat stressed cows in comparison to controls. Follicular dominance at 7 days

of the cycle was altered as indicated by an increase in the number of medium size follicles and size of the subordinate follicle (Badinga et al., 1993). A greater number of large follicles (≥ 10 mm) also were found in heat stressed cows in comparison to controls during the first follicular wave (Wolfenson et al., 1995) and in heifers during days 17-21 of the cycle (Wilson et al., 1998). Moreover, depression of dominance during heat stress conditions was associated with a 2-3 day earlier emergence of the second wave dominant follicle (Wolfenson et al., 1995) and 1 day earlier in terms of medium sized follicles

(Roth et al., 2000). Earlier emergency was associated with lower plasma concentrations of estradiol and inhibin (Wolfenson et al., 1995) and lower plasma concentrations of inhibin and higher of FSH (Roth et al., 2000). These findings are in agreement with Palta et al. (1997), where reduction of plasma inhibin concentrations was found in cyclic buffaloes during summer. Earlier emergence of the second follicular wave can result in a

9 prolonged dominance phase of the largest follicle since the length of the estrus cycle was not different between heat stress and cool cows (Wolfenson et al., 1995)

Moreover concentration of estradiol in plasma and follicular fluid was lower due to heat stress in late summer (Badinga et al., 1993). The decreased steroidogenic capacity of follicles subjected to heat stress involves reductions in granulosa cell viability, aromatase activity and androstenedione production in thecal cells from day 7 (Lew, 1993) or day 8

(Badinga et al., 1993) of the first wave dominant follicle.

In another study, estradiol in the follicular fluid was lower in the summer in comparison to winter. This decrease was due primarily to a reduction of androstenedione production by theca cells (Wolfenson et al., 1997). In order to characterize the molecular events leading to a decrease in plasma estradiol, the second wave dominant follicle of heat stressed heifers was studied (Wilson et al., 1997). In this study mRNA expression of

17α-hydroxylase was reduced; this enzyme activity is rate limiting for biosynthesis of androgens, serving as the substrate for estradiol synthesis by granulosa cells.

Collectively, the effects of heat stress reduce the availability of androstenedione, leading to a reduction in the production of estradiol. A concurrent reduction in inhibin secretion, leads to a diminished negative feed-back on secretion of FSH from the anterior pituitary.

A system with higher FSH circulating concentrations will result in an increase number of follicles reaching preovulatory sizes.

The reduction of dominance capacity of the large follicle can explain in part the increased percentage of twinning in cows that calve during May to July in hot countries such as Saudi Arabia (Ryan and Boland, 1991). These physiological findings indicate that lower capacity of the dominant follicle to suppress subordinate follicles can result in

increased rate of double ovulations, and an earlier emergency of the preovulatory follicle,

which can result in ovulation of aged follicles.

Effects on Corpus Luteum

The effects of heat stress on corpus luteum (CL) function have been studied by means of plasma progesterone concentrations. This hormone is important for regulation of dominant follicle growth and maintenance of pregnancy. Progesterone concentrations do not depend only on the rates of synthesis and secretion, but also on the rate of clearance from the circulation. Rabbits maintained under heat stress conditions had a

30% lower ovarian luteal blood flow (Lublin and Wolfenson, 1996). Results characterized by plasma progesterone concentrations during heat stress conditions are variable. Some studies show a decreased plasma concentration during heat stress

(Wolfenson et al., 1988), while others reported no differences throughout the estrous cycle between cooled and heat stressed cows (Wise et al., 1988). However, in vitro studies demonstrated a suppression of progesterone secretion from luteal cells incubated at 38oC collected from cows in the summer in comparison with cells collected from cows

in the winter. Also, cells collected in the winter and incubated at 40oC produced 30% less

progesterone than similar cells incubated at 38oC (Wolfenson et al., 1993). Alteration in

preovulatory follicle function, when exposed to heat stress, may also impair subsequent

CL function, and therefore alter the oviductal and uterine environments affecting embryo

development (Breuel et al., 1993). However, Stock et al. (1993) showed that CL formed

from a persistent follicle produced normal concentration of progesterone and concluded

that infertility cannot be attributed to luteal insufficiency.

Effects on oocyte

Low fertility in heat stress cows can result from an aged oocyte from a follicle that emerged earlier during the second follicular wave of the estrus cycle and remained in the dominance phase for a longer period of time. Follicles in prolonged dominance may cause prematuration of the oocyte, because the chromosomes condense and meiosis progresses to methaphase II before the LH surge (Mihm, 1994). Revah and Butler (1996) confirmed that oocytes maintained in a prolonged period of dominance underwent premature nuclear maturation.

Broussard et al. (1996) examined the quality and developmental competence of ova obtained by transvaginally-guided aspiration. They showed that fewer oocytes were identified as normal during periods of heat stress, and for those classified as normal, developmental competence was compromised. In another study using oocytes collected from ovaries of slaughtered cows (predominantly Holsteins), a greater proportion had reduced development to the blastocyst stage during hot months (July to August) in comparison to other months (Rutledge et al., 1999). Similarly, Al-Katanani et al. (2002) using oocytes recovered from Holstein cows at a commercial slaughterhouse in north central Florida, found a reduction in the proportion of cleaved embryos that develop to the blastocyst stage. In contrast, oocytes aspirated from follicles of superstimulated Bos indicus (Brahman) cows exhibited normal morphology and yielded a similar proportion of blastocysts during the hot and cool seasons (Rocha et al., 1998). Collectively, these results indicate differences in heat-sensitivity among cattle breeds.

In a study (Braw-Tal and Yossefi, 1997) using bovine ovaries, follicle diameter and granulosa cell characteristics were analyzed in conjunction with diameter of the oocytes.

Follicles were classified as:

a) primordial: <0.04mm, flattened granulosa cells or mixture of flattened and cuboidal cells;

b) primary: 0.04-0.08mm, one layer of cuboidal granulosa cells;

c) small preantral: 0.08-0.13mm, two or three layers of granulosa cell;

d) large preantral: 0.13-0.25mm, four or more layers of granulosa cells and

e) small antral: follicles with early antral formation.

The analyses of early follicle growth showed a phase in which the shape of the

granulosa cells change from flattened to cuboidal without a significant increase in oocyte

diameter (quiescent oocyte), a second phase was characterized by an increase in the

number of granulosa cells accompanied by a rapid increase in the size of the oocyte.

Initiation of oocyte growth occurred at the small preantral follicular stage (0.08-0.13mm

of diameter).

The oocyte during early antral stages of follicular development is not a quiescent

cell. Molecules of RNA and proteins are synthesized and stored in the oocyte cytoplasm

that are critical for the development of the embryo until the maternal-embryonic

transition, when the transcription activity of the embryonic genome becomes fully active.

Maternal-embryonic transition in cows occurs at the 8 to 16-cell stage (Telford et al.,

1990). Oocyte biosynthetic activity initiates during early antral stages is referred to as

“oocyte capacitation”, since during this period the oocyte becomes able to sustain

embryonic development (see review Hyttel et al., 1997). During this process the oocyte

has an increasing transcriptional activity of heterogeneous nuclear RNA (a precursor of

messenger RNA) and ribosomal RNA (Fair et al., 1997). In this study 3H-uridine and autoradiography were used to determine transcriptional activity. The primordial follicle was considered to contain an inactive oocyte, but at the secondary follicle stage

(complete or incomplete bilayer of cuboidal granulosa cells), the oocyte was associated with the onset of transcription. Such activity progresses until the oocyte reaches a diameter of 110 µm and is enclosed in a 2-3 mm follicle. After this stage a low level of heterogeneous nuclear RNA is preserved (Fair et al., 1995) which can be detected up to the germinal vesicle stage, but at Metaphase II a sharp decrease is observed (Memili et al., 1998).

The follicular environment is probably one of the most important determinants of oocyte capacitation (Mermillod et al., 1999). Alterations in the physiological characteristics of the follicle can be detrimental to the developmental competence of the oocyte resulting in a reduction of blastocyst development in vitro (Mermillod et al.,

1999). Follicles containing oocytes able to develop to the blastocyst stage had a higher aromatase activity compared with follicles containing oocytes unable to develop beyond the 8-16 cell stage (Driancourt et al., 1998). These authors also reported that concentrations of inhibin were higher in the fluid of follicles containing developmentally competent oocytes. Moreover, heifers subject to heat stress during late stages of oocyte maturation (between the onset of estrus and insemination) yielded a lower number of viable embryos and a higher incidence of embryos with retarded development than their unstressed counterparts (Putney et al., 1989). In addition, the effects of heat stress impaired oocytes during early stages of development (Roth et al., 2001). After the period of high temperature, oocytes obtained by transvaginal aspiration did not recover their quality (percentage of grade I) until approximately six follicular waves or the equivalent period of three estrous cycles. Collectively, their findings indicated that follicles of 0.13 mm contain oocytes that initiate growth, transcription activity and storage processes

14 necessary for early embryo development. This small preantral follicle takes approximately 42 days to achieve a preovulatory size (Lussier et al., 1987). Therefore, it seems plausible that heat stress conditions could impair oocytes not only during later stages of development, but also during early stages of antral follicle development when oocyte development is initiated. Moreover restoration of normal fertility after a period of heat stress takes approximate 40 to 60 days to be achieved (Badinga et al., 1984). In addition to the marked effects of heat stress that alter follicular dynamics and follicular characteristics, heat stress can also affect oocyte quality during the summer.

Heat-Stress Carry Over Effects

Population studies indicated that in addition to the immediate detrimental effects of heat stress on fertility, low fertility of summer is carried-over into the fall season. This could be attributed to effects of summer heat stress on early stages of follicular development. Exposure of follicles during these stages can cause impairment of oocyte quality and future embryo development during the fall. Calculations based on the number of granulosa cells in follicles indicate that a follicle takes 27 days to grow from 0.13 to

0.67 mm, 6.8 days from 0.68 to 3.67 mm and 7.8 days from 3.68 to 8.56 mm, indicating that growth rates varied with the size of the follicle. A period equivalent to 2 estrous cycles or approximately 41 days would therefore be required for a follicle to grow through the antral phase, i.e. from 0.13 mm to preovulatory size (Lussier et al., 1987). In this study growth of follicles could be divided in two phases: first early growth was attributed to an increase in the number of granulosa cells; secondly, follicles larger than

2.5 mm when rate of growth appeared to result from antral development rather than an increase in the number of granulosa cells.

Carry-over effects were seen in both medium-size and preovulatory follicles for a period of 20 and 26 days, respectively after acute heat stress. The alterations were expressed as a decreased secretion of androstenedione in both classes of follicles and a decreased production of oestradiol by the granulosa cells in the medium-size follicles.

These follicles were estimated to be of 0.5-1 mm diameter when the cows were heat stressed, indicating that antral follicles are susceptible of heat stress (Roth et al., 2001). It could be hypothesized that follicles smaller than 0.5 mm also are susceptible to heat stress since fertility is restored after approximated three estrous cycles or the equivalent of 2 months, and during this early stage of development granulosa cells characteristically have the greatest mitotic activity (Lussier et al, 1987). In another study (Roth et al.,

2000), the effects of heat stress were analyzed during the estrous cycle in which cows were submitted to heat stress, followed by the subsequent estrous cycle when the cows were maintained under a cool environment. The immediate effects on follicular dynamics were manifested by a decreased concentration of plasma immunoreactive inhibin and consequently increased FSH concentrations. This altered hormonal concentration led to a depression of follicle dominance with an earlier emergency of the second follicular wave and an increased number of large follicles during the follicular phase. The delayed effects were expressed by increased FSH secretion and reduction in the number of medium sized follicles. This response to heat stress induced a reduction in dominance of the large follicle and is in part the explanation for the rise in twining in cows calving during May and July in hot countries such as Saudi Arabia. This rise in twining is probably due to the insemination of cows that had double ovulations during the transition period of decreased temperature of August and September (Ryan and Boland, 1991). Carry-over effects of

acute heat stress also were detected in cows maintained for 7 days in environmental

chambers (Guzeloglu et al., 2001). Number of class 3 (≥ 10mm) follicles were increased

in the follicular cycle subsequent to the heat stressed one, indicating a reduced follicular

dominance. No other detrimental effects were detected in the following follicular cycles

due to the short-term period of heat stress

In a study conducted during early summer to early autumn (May-September), a timed insemination protocol (TAI) and inseminations following detected estrous (control) were compared (De la Sota et al., 1998). Estrous detection during the experimental period was very low (18.1%) and pregnancy rate also was lower in the control group versus the

TAI group (4.8% vs 13.9%). The TAI group had a higher pregnancy rate due to the fact that more cows of the TAI group were presented for service. However, pregnancy rates were still low since the effects of heat stress on oocyte quality, follicular characteristics and early embryo development were not eliminated. Therefore, technology to improve fertility during autumn to achieve pregnancy rates comparable to winter could be an option to diminish the seasonal effects of heat stress on dairy cattle in subtropical, tropical and arid areas of the world.

Increasing Autumn Fertility

In order to improve autumn fertility, Roth et al. (2001) hypothesized that enhancing removal of the pool of impaired oocytes after summer heat stress could increase the clearance of damage follicles and accelerate the growth rate of healthy follicles.

Transvaginal aspiration of follicles within the range of 3 to 7 mm was performed 4 times within an estrous cycle during the summer, and oocytes were collected at the first aspiration of an estrous cycle for in vitro fertilization. The repeated follicular aspiration resulted in an estrous cycle of five waves. Percentage of eight-cell embryos increased

17 from 5% to 40%, and a 10% increase in blastocyst formation occurred in the third cycle.

Considering that at the first follicular wave of cycle 2 temperatures were no longer in the heat stress range, the quality of oocytes within health follicles had an improvement at the sixtieth follicular wave (Roth et al., 2001) or approximated three estrous cycles. The results of this experiment are in agreement with a recovery in fertility from summer heat stress of approximately 2 months or what is equivalent to 3-4 estrous cycles (Ron et al.,

1984).

Based upon the hypothesis that oocyte quality is improved with removal of impaired follicles, another study (Roth et al., 2002) was performed in which previously heat stressed cows received FSH injections given at days 5 and 12 of the estrous cycle with the objective to stimulate emergence of a maximal number of follicles within a follicular wave. Follicles were aspirated at day 4 after FSH injections of the treated estrous cycle and at the following one for collection and grading of oocytes. The treatment was able to increase the number of medium-size follicles and the percentage of grade I, and cleaved oocytes in the cycle subsequent to the treated cycle. The same procedure was carried out using rBST. In this case, the number of 3 to 5 mm follicles was increased during the treated cycle and the percentage of grade I oocytes increased in the subsequent cycle. However, in both approaches, percent of blastocyst development remained low, therefore requiring a more efficient hormonal treatment.

Delayed effects of summer heat stress that impact autumn fertility can be categorized accordingly to: 1) impairment of oocytes during early stages of follicular development; 2) alteration in follicular hormonal secretion; and 3) alteration in follicular dynamics.

In order to improve fertility after summer and during the autumn, pharmacological approaches that can increase the number of follicles during recruitment stages, and then remove or increase the number of follicular waves within an estrous cycle warrants further investigation. Another approach could be an induced arrest in follicle development at a size of 2 to 3 mm during the summer; this reduces the frequency of antral follicles subjected to late stage alteration in hormonal secretion and follicular dominance. One possible approach could be the desensitization of pituitary gonadotrophs upon chronic treatment with a GnRH-Agonist. The next sections will review embryological and anatomical-histological features of the pituitary as well as the molecular and reproductive mechanisms and responses of animals exposed to chronic

GnRH-Agonist treatment.

Embryological Origin of the Pituitary Gland

The posterior pituitary originates from neural tissue of the brain, while the anterior pituitary originates from the roof of the embryo’s mouth. Tissue in the roof of the mouth, called stomodeal ectoderm, will give rise to the glandular tissue of the anterior pituitary.

Early in embryo development a diverticulum develops from the floor of the brain and grows ventrally towards the roof of the stomodeum. The diverticulum develops from a specific region of the brain called the infundibulum. At the same time another diverticulum (Rathke’s pouch) originates from the roof of the estomodeum and grows dorsally. The cells of Rathke’s pocket differentiate to form the adenohypophyses (Senger,

1999).

The hypothalamus lies on the ventral portion of the diencephalon, and is exposed on the ventral portion of the brain (Jenkins, 1978). The cell bodies within the

hypothalamus send efferent axonal projections into one of four regions: other areas in the

brain, other hypothalamic nuclei, the median eminence and the posterior pituitary.

Pituitary Histology and Anatomy

The pituitary is divided histologically into the adenohypophysis (pars distalis, pars

itermedia and pars tuberalis) and neurohypophysis (Venske, 1975). The epithelial cells

of the pars distalis produce thyroid stimulating hormone (TSH), follicular stimulating

hormone (FSH), luteinizing hormone (LH), prolactin (PRL), growth hormone (GH),

adrenocorticotrophic hormone (ACTH), melanocyte stimulating hormone (MSH) and β-

endorphins. Specific cells produce each hormone. Cells are classified into two unstained

glandular cells (chromophobes) and stained glandular cells (chromophiles).

Chromophiles can be classified in acidophils and basophils. Basophils are thyrotrophs,

gonadotrophs or corticotrophs and acidophils are functionally subdivided into

somatotrophs and lactotrophs that secrete growth hormone and prolactin. The pars intermedia contains cells that produce MSH and ACTH, and function of the pars tuberalis is unclear (Page, 1994).

The neurohypophysis contains the median eminence, the infundibular stem and the neural lobe. Neurones originating from supraoptic and paraventricular nuclei extend along the infundibular stalk and terminate in the neural lobe. Oxytocin and vasopressin are neurohormones released via exocytosis into the perivascular space of fenestrated capillaries (Page, 1994).

Each population of endocrine cells in the adenohypophysis is under the control of a corresponding releasing hormone from the hypothalamus. These releasing hormones are small peptides synthesized by neurons of the hypothalamus and transported by axonal processes to the median eminence. There they are released into capillaries and conveyed

by the hypophyseal portal system to specific endocrine cells in the adenohypophysis,

where each stimulates the rapid release of secretory granules containing a specific

preformed trophic hormone. (Thomson, 1988).

The portal system contains capillaries that carry blood from which oxygen, nutrients and amino acids have been removed and to which metabolic waste products and peptides hormones have been added from the neurohypophysis (i.e., median eminence) to the adenohypophisis. Blood from the neurohypophysis must pass to the adenohypophisis.

Superior and inferior hypophyseal arteries contribute to the blood supply of the neurohypophysis (Page, 1994). The short loop feed-back is postulated to be a small proportion of capillaries that flow up to the pituitary stalk and provide a direct vascular link from the anterior pituitary back to the hypothalamus (Page, 1994).

Gonadotropin-Releasing Hormone Receptor

Gonadotropin-releasing hormone (GnRH) has a central role in regulating the reproductive processes in mammals.

The GnRH receptor (GnRHr) is located in the cell membrane of gonadotrophs where it mediates the release of LH and FSH (Fink, 1998). The bovine GnRHr is a 328- amino acid protein with seven putative membrane-spanning domains, characteristic of the

family of G protein-coupled receptors that utilizes Ca++ as a second messenger. The

GnRHr lacks the typical intracellular carboxyl terminus, making it one of the smallest

receptors with the seven-transmembrane segment motif of G protein-coupled receptors

(Kakar et al, 1993. Figure 2-1).

Figure 2-1. Proposed seven transmembrane topography of the bovine GnRH receptor (Kakar et al., 1993).

The GnRH molecule binds to its receptor, which is coupled to a G protein system, such that Gs phosphorylates GDP into GTP and activates phospholipase C. Subsequently, there is an activation of both phospholipase A2 (PLA2) and phospholipase D (PLD).

Phopholipase C hydrolyses phosphatidylinositol 4,5 biphosphate to generate the second messengers: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). The IP3 leads to mobilization of intracellular pools of Ca++ from the endoplasmic reticulum. The DAG

activates protein kinase C (PKC) that increases the transport of extracellular Ca++ into the cell. Both systems result in an increase of intracellular Ca++ that is coupled with

gonadotropin release through exocytosis (Tse et al., 1993). Arachidonic acid (AA) is

liberated by activated PLA2. Differential cross-talk of Ca++, AA, and selective PKCs generate a compartmentalized signal transduction cascade to downstream elements which are activated during the neuro-hormone action. Among those elements is the mitogen- activated protein kinase (MAPK) cascade that is activated by GnRH in a PKC and Ca++

dependent fashion. GnRH also is coupled to Gs, leading to activation of adenylyl cyclase

and stimulation of cAMP production, which regulates target gene expression. It seems

therefore that cross-talk of Ca++ and PKC is involved in the diverse effects of GnRH upon

gonadotropin secretion and synthesis. (for review Kaiser et al., 1997; Naor et al., 1998).

Regulation of GnRHr

Progesterone, GnRH and estradiol modulate expression of the GnRHr gene in gonadotrophs. GnRHr mRNA is maximal during the preovulatory period (Crowder and

Nett, 1984; Brook et al., 1993; Turzillo et al., 1994) due probably to the decreased circulating concentrations of progesterone at luteolysis. Number of GnRHr’s and GnRHr gene expression are low during the luteal phase (Turzillo et al., 1994), even if estradiol is present. Increased pulsatile GnRH after removal of negative feedback effects of progesterone at the hypothalamus, stimulates expression of the GnRHr gene early in the pre-ovulatory period in preparation for the LH surge (Turzillo et al., 1994). In addition, there are increasing serum concentrations of estradiol produced by the dominant follicle at this stage of the estrous cycle. Estradiol has a stimulatory effect on GnRHr gene expression (Turzillo et al., 1995) and an additive effect in combination with inhibin (Wu et al., 1994).

A decreased number of GnRHr is seen in ovariectomized ewes with a hypothalamic-pituitary disconnection (Clark et al., 1987; Turzillo et al., 1995). It is also seen in ewes deprived of GnRH using non-surgical methods such as GnRH antagonists

(Brooks and McNeilly, 1994). Therefore, continuous stimulation of the pituitary gland by

GnRH is required to maintain GnRHr.

An increase in the number of GnRHr during the preovulatory period is mediated by increased expression of the GnRHr gene, which is regulated by endocrine hormones such

23 as GnRH, progesterone and estradiol (for review Turzillo and Nett, 1999). In contrast exposure to continuous high concentrations of GnRH reduces the response of gonadotrophs to subsequent stimulation with GnRH (homologous desensitization), leading to suppression of gonadotropin secretion (Belchetz et al., 1978). After continuous exposure to GnRH, the anterior pituitary gland becomes refractory to further challenge with GnRH in ewes (Nett et al., 1981) and in cows treated with a GnRH-Agonist implant

(Mattos et al., 2001). This desensitization is marked by decreased tissue concentrations of

GnRHr (Nett et al., 1981; Vizcarra et al., 1997). Amounts of GnRHr mRNA and concentrations of GnRHr in ewes treated continuously with GnRH decreased by 48% and

69%, respectively (Turzillo et al., 1998). Similar results where found in cows infused continuously with GnRH; GnRHr and GnRHr mRNA were reduced (Viscarra et al.,

1997). Also, chronic treatment of ewes with GnRH agonist causes downregulation of

GnRHr and reduces GnRH receptor gene expression (Brooks and McNeilly, 1994; Wu et al., 1994).

Hazum et al. (1980) demonstrated that GnRHr is internalized after binding to the

GnRH molecule. Moreover, the reduction of GnRHr induced by continuous GnRH treatment leads to a reduction in GnRHr gene expression. Therefore pituitary desensitization reflects the internalization of existing GnRH receptors on the gonadotrophs in addition to the reduction of de novo synthesis of GnRH receptors characterized by a decrease GnRH receptor gene expression.

GnRH Molecule

Gonadotropin-releasing hormone is a central neuropeptide produced by a small number of neurons in the hypothalamus. GnRH is synthesized as a portion of a large precursor molecule which begins with a 23 amino acid signal sequence, followed by the

GnRH decapeptide, and a 56 amino acid GnRH-Agonist associated peptide (Jennes and

Conn, 1994).

The primary structure of the GnRH is a decapeptide with NH2- and COOH-

terminal domains (Figure 2-2). These terminal domains are most important in receptor

binding and activation, with the residues in the NH2-terminal domain predominantly

responsible for receptor activation. Substitution of residues outside of the NH2-terminal

domain can affect receptor activation (Sealfon et al., 1997).

1 2 3 4 5 6 7 8 9 10 pyroGlu – His – Trp – Ser – Tyr – Gly – Leu – Arg – Pro – Gly – NH2

Figure. 2-2. Amino acid sequence of mammalian GnRH.

GnRH-Agonist Deslorelin

Deslorelin is a synthetic GnRH – agonist, formed by nine peptides. The amino acid

sequence is shown in Figure 2-3.

pyroGlu1 – His2 – Trp3 –Ser4 –Tyr5 – D – Trp6 –Leu7 –Arg8 – Pro9 – NHethylamide

Abbreviated to

[D-Trp6, Pro9-NEt]GnRH

Figure 2-3. Deslorelin Amino acid sequence.

This structure is typical of GnRH-Agonists. Glycine at position 6 of the peptide is substituted with a D-amino acid such as D-tryptophan in the case of Deslorelin. This feature increases the half-life of Deslorelin in the circulation. Another characteristic is the removal of glycine at the amino terminus, which increases the affinity of the GnRH – agonist (i.e., Deslorelin) to its receptor (Karten and Rivier, 1986).

GnRH-Agonist Treatments.

The effects of continuous treatment with GnRH -agonist are characterized by an acute stimulatory phase followed by a chronic depression gonadotrophin release.

Cows implanted with Deslorelin at day 7 of the estrous cycle had an acute increase in plasma LH, with maximum concentrations detected at 6 h. Most of the cows had associated increases in concentrations of plasma progesterone, possible due to the induction of an accessory CL or due to the luteotropic actions of released LH (D’Occhio et al, 1995), FSH was not measured in this study. Plasma concentrations of both LH and

FSH increased within 1h of Buserelin infusion in heifers treated at day 5 of the estrous cycle, and concentrations at 2 h were consistent with a preovulatory surge (Gong et al.,

1996). In this study the LH surge induced ovulation of dominant follicles in all heifers.

Furthermore, the CL had life span equal to the control animals. A Deslorelin biodegradable implant (2.1 mg) inserted at day 7 postpartum induced ovulation in five of eight cows (Mattos et al., 2001) as a consequence of an induced LH surge. In another study pre-pubertal heifers also responded with an acute increase in immunoreactive LH after insertion of a Deslorelin implant (Bergfeld et al, 1996). Cyclic Holstein cows also responded with an acute release of LH within 1h after GnRH-Agonist treatment injection.

The LH response was greater to the Deslorelin implant than to an injection of Buserelin

(Rajamahendran et al., 1998).

The effect of a chronic GnRH-Agonist on gonadotrophin responses was well described by Gong et al. (1996). Cyclic heifers were treated continuously with a GnRH-

Agonist (Buserelin) for 48 days. After a surge at the beginning of infusions using osmotic mini-pumps, LH concentrations fell at day 8 of infusion to basal levels until the termination of GnRH-Agonist treatment. The levels of FSH remained elevated for 3 days

26 following the start of infusions and returned to normal basal levels characteristic of the control group. However, FSH levels decreased to levels below the controls after 28 d of treatment. In this study suppression of pulsatile secretion of LH was achieved earlier than

FSH. Somewhat similar results were obtained when heifers were injected with Buserelin twice a day, although the day of LH suppression was different due to twice a day injection as opposed to continuous infusion. In the later study, LH suppression was achieved after 16-17 days of treatment and FSH remained at constant higher concentration than the control group (Gong et al., 1995). In summary, these studies have shown that complete suppression of gonadotrophs is achieved within 28 days of chronic treatment of GnRH-Agonist. Suppression in LH secretion was achieved earlier because it is necessary to have a higher number of GnRH receptors to result in a surge release of LH

(Turzillo et al., 1994), whereas FSH secretion can still occur with a lower number of

GnRH receptors.

Initiation of Follicle Growth

The initiation of follicle growth is defined as the transition of primordial follicles from the quiescent to the growth phase. Primordial follicles are the source from which follicles will be gradually recruited to grow to life. Two distinct stages were observed during early follicle growth in the bovine (Braw-Tal and Yossefi, 1997). A first stage, involving primordial follicles, was characterized by a change in the shape of the single layer of granulosa cells from flattened to cuboidal in association with an increase in the number of cells without a significant increase in oocyte diameter. In a second stage, an increase in the number of granulosa cells is associated with a linear and positive increase in the size of the oocyte. This later is stage initiated at a critical point of 40 granulosa cuboidal cells.

The mechanisms involved in the growth of follicles from the primordial to the

primary stage are not well understood. Development of primordial follicles to the primary

stage was achieved in organ cultures of whole mouse ovaries maintained in medium that

contained serum but no pituitary hormones (Eppig and O'brien, 1996). Also, bovine

ovarian cortical explants can initiate growth in vitro in serum-free medium, with a decline

in the number of primordial follicles and concomitant increase in the number of primary

follicles within 2 days of culture (Wandji et al., 1996). In this study, further observations

demonstrated that during 7 days of culture, diameter of primordial and primary follicles

increased, as well as the diameter of oocytes in primary follicles. A higher rate of

primordial follicle activation in the later study compared to the former, raises the

possibility that in the intact ovary, an inhibitor in the central part of the ovary keeps most

follicles in a quiescent stage.

Studies of regulation of early preantral and antral follicular development have

increased in the past decade as it became recognized that the primordial pool of follicles

represents a resource that could potentially increase the reproductive efficiency of domestic animals and endangered species.

Follicle stimulating hormone (FSH) is unlikely to be a critical factor for initiation of primordial follicle growth. In sheep and cows the gene for the FSH receptor is not expressed until the follicle reaches the primary to the small preantral stage (reviewed by

McNatty et al., 1999). At this and subsequent stages of development, FSH receptor mRNA is localized exclusively in granulosa cells. It is likely that the FSH receptor is functionally active during preantral development as characterized by an increased number of granulosa cells and incorporation of 3H-thymidine into DNA of bovine preantral

follicles in vitro (Wandji at al., 1996) that are cultured in medium containing only FSH.

Moreover, granulosa cells in large preantral and early antral mouse follicles can

synthesize cAMP or lactate in response to FSH in vitro (Boland et al., 1993).

Although it can be demonstrated that FSH has stimulatory effects on granulosal cell

proliferation and function of preantral follicles, FSH is not an essential factor for these

events. Many preantral follicles of hypophysectomized rats showed evidence of

continued cell proliferation despite the absence of gonadotropins (Hirshfield, 1983). In

long term (i.e. 70 days) hypophysectomized ewes no reduction in the total number of

antral follicles of < 2 mm were detected and follicular growth was arrested to < 3 mm in

follicle size (McNatty at al., 1990). However, a question raised by the authors was

whether some of the small antral follicles (< 3 mm) developed after hypophysectomy or were present at the time of hypophysectomy and simply persisted after gonadotrophin withdraw.

Driancourt et al. (1987) studied follicular dynamics in ovaries surgically removed from ewes at 1, 2, 4 and 8 days after hypophysectomy. Total number of follicles > 2 mm underwent a steady decrease from day 0 to day 8. At day 8, follicles > 2 mm were not present in the ovaries. There was no effect of treatment (i.e. hypophysectomy) in the overall total (healthy + atretic) population of follicles > 0.8 mm in diameter. At day 4 and day 8, the proportion of healthy follicles > 0.8 mm and the mitotic index of granulosa cells of these follicles did not differ between hypophysectomized and control ewes.

Therefore, this study demonstrated that follicles > 2 mm in diameter are dependent on gonadotrophins and that follicular turn over (i.e. growth and atresia) in follicles < 2 mm in diameter is very active in short term (i.e., 8 days) hypophysectomized ewes.

In contrast, the number of follicles and the percentage of atretic follicles of < 2 mm

was decreased and increased, respectively in ewes hypophysectomized for a long period

(i.e., 70 days) in comparison to short term hypophysectomized (i.e., 4 days) and control

ewes. Moreover, the mitotic index of granulosa cells was reduced in the long-term treated

ewes compared to short term and controls ewes (Dufour et al., 1979).

Wandji et al. (1996) studied the responses of preantral bovine follicles to FSH and growth factors in vitro. Survival rate of preantral follicles was greatly increased when incubated with FSH, basic fibroblast growth factor (bFGF) and epidermal growth factor

(EGF) in comparison to control media. The survival rate was highest in media containing bFGF with or without the presence of FSH. In contrast, transforming growth factor-β

(TGF-β) dramatically decreased the rate of survival of preantral follicles and reduced the stimulatory effects of FSH and bFGF. The increase in diameter of bovine preantral follicles was stimulated by the presence of FSH and bFGF alone or in combination to a similar magnitude. Labeling index of granulosa cells based on 3H-thymidine in bFGF

media was twice as effective as FSH. However, TGF-β inhibited the stimulatory effect of

bFGF in both follicular diameter and labeling index. These observations suggested that

FSH can stimulate early follicular development, but its presence is not essential,

suggesting that bFGF may act as an autocrine modulator of preantral follicular growth in

catlle. Moreover, medium-sized preantral follicles isolated from bovine and maintained in

culture media with FSH, EGF, IGF-I separately or in association stimulated follicle

growth and antrum formation in comparison to control media without hormone treatment

(Gutierrez et al., 2000). However, coculture with granulosa cells inhibited the FSH/IGF-I

stimulatory growth effect, indicating that inhibitory substances secreted within the

follicle also regulate preantral follicular growth

Control of early follicular development also is controlled by locally produced

growth factors or receptors including the tyrosine kinase c-kit and its ligand, stem cell

factor (SCF), members of the transforming growth factor superfamily (i.e., βA

inhibin/activin, α-inhibin, GDF-9) and follistatin (McNatty et al., 1999).

In sheep, c-kit protein can be localized to oocytes of primordial and primary follicles and SCF to granulosa cells and oocytes of both primordial and primary follicles.

These findings are consistent with the view that activation of the c-kit tyrosine kinase system by SCF is an important factor in the growth of primordial follicles. Yoshida et al.

(1997) determined that granulosal cells ceased to proliferate after the administration of c- kit antibody to mice.

Activin is composed of two inhibin-β subunits, either βA or βB, which combined to

form activin A, B, or AB. Inhibins and activins are dimeric growth factors of the transforming growth factor β superfamily. In sheep ovaries, the mRNA and peptide for βB

inhibin/activin mRNA subunit is first detected in granulosa cells of primordial primary

and early preantral follicles (McNatty et al., 1999). In contrast, mRNA for inhibin/activin

βA in granulosal cells are not detected until follicles reached the small antral stage of growth (Torney et al., 1989; Braw-Tal 1994). Inhibin α subunit mRNA was first observed in granulosal cells of developing ovine small preantral follicles. Thereafter, the hybridization signal increased exclusively in granulosal cells of both preantral and antral follicles (Braw-Tal, 1994).

Factors produced by the oocyte such as growth differentiation factor 9 (GDF-9) and bone morphogenic protein 15 (BMP-15) are related structurally to TGFβ and expressed exclusively in the oocyte (McGrath et al., 1995). Deletion of GDF-9 gene in mouse and mutation of BMP-15 in sheep results in the arrest of granulosa cell proliferation with no effect on oocyte growth. In sheep with mutation of BMP-15, no normally developing follicles beyond the primary stage were detected (Braw-Tal et al., 1993).

Follistatin gene expression was first observed in granulosa cells of small preantral follicles (Braw-Tal et al., 1994). Co-culture of medium size preantral follicles and larger follicles (0.3-0.35 mm) from adult mice blocked the stimulatory effects of FSH on the smaller follicles and the suppression could be overcome by removing the larger follicle or by adding follistatin (which binds to activin) to the culture medium (Mizunuma et al.,

1999).

Collectively, Braw-Tal (2002) proposed a hypothetical model for early follicular growth. The proliferation of primordial flattened granulosa cells to enter the primary stage of growth could be inhibited by activin A secreted by preantral follicles, while bFGF and stem cell factor secreted by surface epithelium may act as stimulators.

Therefore the fate of individual follicles might depend on the local relationship between inhibitors and stimulators. When the follicle reaches the primary stage it secretes follistatin, which protects it from possible inhibitory effects of activin A. At this stage, the oocyte starts to secrete GDF-9 and BMP-15 that are essential for granulosa cell proliferation. The oocyte and granulosa cells now form an autonomous unit, and their further growth depends on follicle-produced factors rather than local intraovarian signals.

Follicular Recruitment and Dominance

After ovulation or turn over of the dominant follicle, follicles begin to grow larger

than 4 mm in diameter. This event is described as the recruitment phase of a follicular

wave (Fortune, 1994). The recruitment phase or follicular wave emergence also is

defined as the initiation of gonadotrophin-dependent folliculogenesis by a cohort of

healthy follicles (Driancourt, 2001). Lack of follicular development beyond 4 mm in

diameter in cows with low basal levels of plasma concentrations of FSH indicates a

dependency to circulating FSH for follicular recruitment beyond 4 mm (Gong et al.

1996).

In cattle follicular recruitment is acutely dependent on FSH. An emergence of small

antral follicles (3-4 mm) detected by ultrasound is always preceded by transient FSH

rises (Adams at al., 1992). Afterwards, the cohort of recruited follicles increases in

diameter beyond 5 mm, along with a gradual elevation in intrafollicular amounts of

estradiol and higher molecular weight inhibins in the largest follicle (Austin et al., 2001).

These changes contribute to the decrease in FSH secretion. The group of follicles

recruited, a single follicle is selected to continue growth; whereas, other follicles of the follicular wave undergo atresia. Due to the presence of a functional CL and high progesterone concentrations, this first dominant follicle does not induce a LH surge from by the pituitary. Thus, no ovulation occurs. The first dominant follicle will become non- functional and a second follicular wave begins at about mid cycle (day 10 of estrus cycle). Again a dominant follicle is selected from this second follicular wave, and this follicle continues growth until ovulation because this dominant follicle is functional at the time of CL regression. Some estrous cycles have three follicular waves such that the

dominant follicle of the third wave is functional at the time of luteolysis and therefore is

capable to ovulate.

Follicular deviation has been defined as the beginning of the greatest difference in

growth rates (diameter changes between successive ultrasound examinations) between

the largest follicle (i.e., dominant follicle) and the second largest follicle (i.e., largest

subordinate follicle) at or before the examination when the second largest follicle reached

the largest diameter (Ginther et al., 1996). At the time of deviation, the diameter of the future dominant follicle averages 8.5 mm and the future largest subordinate follicle averages 7.2 mm (Ginther et al., 1996). The circulating FSH concentration reaches a nadir near the time of follicular deviation, and this decrease is probably essential for selection of a single dominant follicle.

One critical interaction occurring during the estrous cycle is between secretion of pituitary FSH and secretion of ovarian inhibitors of FSH. This has been termed two-way functional coupling between FSH and follicle (Ginther et al., 2000). In the absence of any follicles following ovariectomy in cattle, there is a rapid increase of circulating FSH that reaches a maximum by 24 h. Moreover, aspiration of all follicles larger than 3 mm results in a similar increase in FSH indicating that follicular FSH inhibiting activity is coming from follicles greater than 3 mm (Gibbons et al., 1997). The two primary inhibitors of

FSH that are secreted by the follicle are inhibin and estradiol. Maximum FSH concentrations are observed at the time of emergence of a new follicular wave due to the low circulating concentrations of inhibin and estradiol. As the follicular wave progresses, larger follicles produce greater FSH inhibitory activity by increased dominant follicle secretion of inhibin and estradiol. On average, the nadir in circulating FSH is reached at

the time of follicular deviation with the dominant follicle at approximately 8.5 mm in

diameter (Ginther et al., 1997). Circulating estradiol increases from ~0.2 to ~1 pg/ml near the time of follicle selection (Kulick et al., 1999) and this contributes to the final suppression of circulating FSH. Estradiol synergizes with inhibin at the time of dominant follicle selection to strongly suppress FSH secretion (Armstrong and Webb, 1997).

Near the time of deviation LH action in follicular growth becomes critical. Beg et al. (2001) compared the diameter of the largest and second largest follicles from the first follicular wave using slaughterhouse ovaries to estimate diameter deviation and determined granulosa cell LH receptor mRNA. The increased difference in LH receptor mRNA expression between the two largest follicles occurred, on average, an equivalent of 8 hours before any increased difference (i.e., deviation) in follicle diameters or increased follicular fluid concentrations of estradiol (Beg et al., 2001). In another study

(Xu et al., 1995), follicular diameter deviation in individual cows was not determined but similar changes in LH receptors mRNA or binding have been noted near the time of follicular selection. There was an increase in LH receptor mRNA on granulosa cells from non-detectable in follicles at day 2 (average of 6.7 mm) to highly expressed levels on day

4 (average of 10.8 mm; day 0 = day of wave emergence). Near the time of follicular selection, there was a 4-fold increase in LH receptor mRNA in thecal cells.

Follicle growth past the time of deviation as well post deviation appears to be regulated by LH pulses. Gong et al. (1995) found that follicles failed to grow beyond ~9 mm in diameter (size of largest follicle at deviation) in cows in which LH pulses had been suppressed by chronic treatment with a GnRH-Agonist. Moreover, inhibition of LH concentration by treatment with progesterone did not alter the time or diameter

characteristics at the time of follicular selection (Ginther et al., 2001). However, the

growth rate of the developing dominant follicle was reduced when the follicle reached ~

10 mm in diameter. Thus, follicle growth past deviation appears to require LH pulses.

Collectively, follicular growth before follicular selection requires FSH, but after

follicular selection, LH pulses are required. The emergence of the follicular wave and

growth until the time of follicular selection is primarily regulated by circulating FSH. The

FSH concentration is progressively inhibited until they reach a nadir at follicular

selection/deviation. At this time, continued growth of the follicle and follicular estradiol

production requires LH pulses. The dominant follicle continues to grow until sufficient

circulating estradiol is achieved to induce an LH surge and ovulation of the dominant

follicle.

Follicular Dynamics Using a GnRH-Agonist

Treatment with a GnRH-Agonist can be used to control ovarian follicular development and atresia in cattle. Heifers receiving chronic GnRH-Agonist treatment for

3 weeks did not develop dominant follicles beyond 9 mm in diameter during approximately 30 days of treatment due to a suppression of LH release. No follicles larger than 4 mm of diameter were observed after 30 days consistent with the suppression of FSH concentrations (Gong et al., 1996). Similar results were observed after 10 days of twice daily injections of Buserelin such that follicles did not develop beyond 9 mm in size due to the suppression of LH. In this second study, a 21 day injection sequence was unable to suppress FSH (Gong et al., 1995). Postpartum cows treated with Buserelin had induced ovulations with a short CL lifespan, but then seemed to have a delay in additional ovulations over a period of 28 days (MacLeod et al., 1991). A degradable

Deslorelin implant inserted at day 7 postpartum reduced the rate of accumulation of class

2 and 3 follicles for approximately 40 days, increased the accumulation rate of class 1 follicles (Mattos et al, 2001), and suppressed ovulation during a 28 day period using a non-degradable implant (Padula and Macmillan, 2002). An experiment was conducted to investigate the capacity of a commercial Buserelin implant to suppress estrous cycles in cattle. Post-pubertal heifers received a 6 mg or a 12 mg implant at random stages of the estrous cycle (implants were not removed). Non-luteal phase plasma progesterone was maintained for 48 ± 4 and 87 ± 17 days in heifers treated with 6 and 12 mg implants, respectively (D’Occhio et al., 1996). Heifers treated with 5 and 10 mg of Deslorelin implants maintained basal plasma progesterone for an average of 203 and 170 days, respectively. There was a wide variation among heifers such as 111 to 280 days for 5 mg group and 70 to 280 days for the 10 mg (D’Occhio et al., 1996).

GnRH-Agonist Recovery

Few studies have determined accurately the physiology of recovery following pituitary desensitization. Variability among species, length of treatment, dose, delivery and type of GnRH-Agonist used make interpretation and comparison of data difficult.

Cows were treated on day 7 of the estrous cycle with Deslorelin implants (20 mg) for 28 or 56 days (D’Occhio et al., 1995). Occurrence of ovulation was monitored by weekly plasma progesterone. Days after implant removal to first estrus were similar for

28 and 56 days of treatment (23.6 ± 2.1 and 21.5 ± 3.3). Clear conclusions about progesterone rises after implant removal were not addressed. However, examination of progesterone profiles indicated that five of six cows treated for 56 days had a rise in progesterone at 28 days after implant removal. In cows treated for 28 days, two of six did not have a rise in progesterone during 10 weeks after removal of implants, and the

37 remaining four cows had a rise in progesterone between 14-28 days after removal

(D’Occhio et al., 1995). There appears to be appreciable cow to cow variability in recovery from a GnRH agonist induced desensitization of the gonadotroph.

Gong et al. (1995) reported that ovulation occurred approximatly 10 days after cessation of Buserelin treatment in heifers, but it is important to stress that those 21 days of treatment was not enough to cause complete pituitary desensitization as described previously. In another study (Gong et al., 1996), the authors reported that all seven heifers expressed estrus after 8 to11 days after termination of a 58 day continuous infusion of Buserelin. However no ovulation was detected, since no pre-ovulatory surge of LH was detected within 22 days after treatment in any animal. Apparently, LH secretion occurred to support development of estrogenic follicle, but a LH surge mechanism was not in place and heifers become cystic.

In a study with ewes, Buserelin was infused for 29 days, and a suppression of FSH and LH was achieved. After cessation of treatment, estrus behavior was observed within

7-11 days but no rise in plasma progesterone was detected (McNeilly et al., 1987), suggesting a failure to generate a pre-ovulatory surge of LH.

Secretion of LH was monitored in pre-pubertal heifers treated with Deslorelin for

28 days and then challenged with GnRH injections at 0, 2, 4, 8, 12, 16 and 20 days after removal of implants. A significant increase in estradiol was detected at day 12 and at the following GnRH challenges, which coincided with rises in plasma LH. A sequence of

GnRH injections may have stimulated an earlier resumption of LH release (Bergefeld et al., 1996).

A non-degradable Deslorelin implant (6 mg) was inserted in 41 cows within 7 days postpartum, and the implants removed after 28 days of treatment. The mean interval from implant removal to first heat was 21 days. However, 42.5% of the cows failed to ovulate.

Data for occurrence of ovulation based on progesterone plasma concentrations is unclear.

However, approximately 50% of the cows had ovulated within 30 days after implant removal (Padula and Macmillan, 2000).

In summary, behavioral estrus after termination of chronic GnRH-Agonist treatment is not a reliable indication of a restoration in normal cyclicity. Future studies focused on recovery from pituitary desensitization must rely on sequential plasma progesterone concentration or ultrasonografic determination of CL to document complete recovery. Also, variability of time for recover within treatments appears to create a challenge for the use of GnRH-Agonists.

Follicular Dynamics During GnRH-Agonist Treatment.

No characterization of follicular wave patterns has been undertaken following long term chronic GnRH-Agonist treatment has been done. A close scenario of reduced release of gonadotrophins could be the late stages of pregnancy when estradiol secretion is increased (Larsson et al., 1981; Knickerbocker et al., 1986; Schallenberger et al.,

1985). Ginther et al. (1996) monitored follicular activity by ultrasound in heifers from day 90 of pregnancy to the emergence of the first follicular wave postpartum. In the last month of gestation, number of defined FSH surges was reduced in comparison to the previous months, and the days between FSH peaks was increased. Such responses are indicative of a partial suppression in FSH secretion. Follicular activity was associated with FSH concentrations during the last month of gestation; number of follicles reaching

5mm was significantly reduced as well as number of follicles reaching more than 6, 8 and

10mm. Moreover number of follicular waves in the last month of gestation was reduced.

Clearly the partial suppression of FSH was accompanied with a reduction in follicular activity as indicated by a reduction of number of follicles recruited and number of follicular waves. It is possible that GnRH-Agonist treatments may follow the same scenario but in a more severe fashion. Suppression of FSH in late pregnancy is more likely due to high levels of placental estrogen. However, increased pituitary secretion of

FSH occurs rather quickly following the periparturient decrease in estradiol. An immediate rise of plasma FSH occur within 5 days postpartum (Beam and Butler, 1997).

Another feature that could be reducing occurrence of follicles is the fact that cows under chronic GnRH-Agonist treatment may have reduced concentration of growth hormone and insulin-like growth factor I (see below). In contrast, studies have shown that animals treated with recombinant bovine somatotropin (rbST) have an increased number of follicles smaller than 5mm in cows and heifers (Roth et al., 2002; Kirby et al., 1997;

Gong et al., 1997) in associated with follicular recruitment and increased number of follicles of 6-9 mm in lactating cow (De La Sota et al., 1993). The increased of follicles smaller than 5 mm was dependent upon increased serum concentrations of IGF-I and insulin (Gong et al., 1997).

Possible Mechanisms of GnRH-Agonist Treatment on Milk Yield

Literature on whether chronic treatment with a GnRH-Agonist affects milk yield in domestic animals is not yet available. Most of the information available is in humans and deals with direct and indirect effects of GnRH-Agonists on hormones (GH, Prolactin and steroids) that regulate milk production. These aspects of literature review will be based on physiological findings of other species and when possible in ruminants.

Role of Growth Hormone in lactation

Briefly, growth hormone (GH) has an effect on lactational performance via a homeorhetic control that results in a coordinated series of changes involving both nutrient supply and mammary utilization (Terry, 1998). The mechanism by which GH affects mammary gland function appears to be indirect, involving the insulin-like growth factors

(IGF) system. This is supported by the abundant concentration of type I and type II IGF receptors in the mammary gland (Burton et al., 1994; Dehoff et al., 1988) and the increased milk yield response after close arterial infusion of IGF I and IGF II (Prosser et al., 1994; Prosser et al., 1990). In contrast attempts to detect GH receptors in bovine mammary tissue have been unsuccessful (Akers, 1985; Keys and Djiane, 1988), and closed arterial infusion of the mammary gland with GH had no effect on milk yield

(McDowell et al., 1987). GH has an important role in the stimulation of IGF-I. Low GH concentrations or low GH activity, results in depressed IGF-I concentrations (Underwood et al., 1994). It has been long recognized that members of the IGF family stimulate cell hypertrophy (i.e., elevate nutrient transport, protein synthesis, and RNA synthesis; Zapf et al., 1984). The liver is thought to be the primary source of circulating IGF-I upon GH stimulation; IGF-I may act in an endocrine manner and directly affect target tissues, such as the mammary gland. IGF-I is potent mitogen for mammary epithelial cells (Cohick et al., 1997). Also, IGF-I mRNA is expressed in the bovine mammary gland, suggesting that local production of IGF-I also may be important (Glimm et al., 1992; Sharma et al.,

1994).

The chronic administration of GnRH-Agonist is used in cows with the purpose to induce suppression of LH and FSH through mechanisms involving downregulation and desensitization of pituitary gonadotrophin receptors. Consequently a suppression of

follicular development and estradiol production occurs (D’Ochio et al., 2000; Mattos et

al., 2001; Rajamahendran et al., 1998). The GnRH-Agonist is used in the treatment of

various gynecological and endocrine disorders in humans (Forti, 1998). However,

chronic treatment with GnRH-Agonist can affect other aspects of pituitary function that

could lead to important changes in physiological responses such as milk production.

Growth Hormone responses to GnRH-Agonists

Healthy, normal women treated with a long-acting GnRH-Agonist (d-Trp-6-

LHRH) for 2 months showed a significantly lower growth hormone (GH) release response to growth hormone releasing hormone (GH-RH) stimulation than before treatment with the GnRH-Agonist (Kaltsas et al., 1998). The reduction was significant at

30 and 90 minutes after GH-RH stimulation. Similar results were found in another study

(Word et al., 1990) where premenopausal women were treated for 1 to 6 months with a

GnRH-Agonist. The plasma concentration of GH after injection of GH-RH was reduced in GnRH-Agonist treated women in comparison to non-treated. Basal GH concentrations were not affected by the GnRH-Agonist treatment in these studies. However, blood samples were not collected across a series of days during the period of GnRH-Agonist treatment or during nocturnal sleep.

Long term GnRH-Agonist administration has been the treatment of choice for central precocious puberty (CCP). This condition occurs in children and is characterized by increased growth rate, advanced skeletal maturation and impaired final height due to premature hardening of epiphyseal plates (Sigurjonsdottir et al., 1968). Studies conducted with children diagnosed with CPP and treated with GnRH-Agonist (leuprolide acetate) showed a mean decrease in growth velocity and nocturnal GH secretion after 6 months of treatment. The decrease in nocturnal GH secretion was identified as an amplitude

42 modulated phenomenon, as the number of secretory peaks remained unchanged

(DiMartino-Nardi et al., 1991). Harris et al. (1985) obtained similar results in a study in which 4 hours of nocturnal GH determinations demonstrated a decline in mean GH after

6 months of GnRH-Agonist therapy. In another study, 2 years of gonadal suppression using GnRH-Agonist treatment in girls with CPP, also resulted in a decrease of nocturnal serum GH, IGF-I and decrease of growth rate (Mansfield et al., 1988). Similar results were found by Sklar et al. (1991), where children diagnosed with CPP were treated with a

GnRH-Agonist until pituitary-gonadal suppression was achieved (5-17 months).

Nocturnal mean basal GH release and mean peak level was decreased upon injection of

GH-RH after treatment with GnRH-Agonist.

Prolactin responses to GnRH-Agonists

Various studies indicated no significant alteration in prolactin (PRL) concentrations in children with CPP during treatment with GnRH-Agonist (Sklar et al., 1991; Kreiter et al., 1990; Bourguignon et al., 1987) or in young men (Stoffel-Wagner et al., 1995).

Similar results were found when women were treated with GnRH-Agonist (leuprolide acetate) and then stimulated with TRH. Basal levels of PRL were not altered (Chantilis et al., 1995). However, an in vitro study using human prolactinomas, showed that GnRH receptors of three adenomas had specific and high affinity binding to a GnRH-Agonist.

Furthermore, stimulation increased in vitro PRL secretion in a subset of prolactinomas

(Brandi et al., 1995). In a Japanese study, a GnRH-Agonist (Buserelin) was administered continuously to five patients until inhibition of the secretion of LH and FSH was achieved. Prolactin was inhibited markedly in the patients, and the degree of inhibition appeared to be dependent on the dose and duration of Buserelin administration. No correlation between estradiol and PRL was observed during Buserelin administration.

The findings suggested that Buserelin inhibited PRL secretion by direct effect on the

CNS, not indirectly through inhibition of estradiol production (Inoue et al., 1986). The

TRH receptor and GnRH receptor are both members of the family of G protein-coupled

receptors, and both are coupled to G proteins of the Gq/11 family that respond by

enhancing phosphoinositide turnover, calcium mobilization, and PKC activation (Naor et al., 1990). Moreover, GnRH-Agonist is able to stimulate PRL release from somatolactotropic cells transfected with the GnRH receptor, whereas it does not in the

parental GH3 cells or in control cells. This confirms that this response is mediated by the

GnRH receptor, rather than by cross-activation of the TRH receptor by GnRH (Kurphal et

al., 1994). In addition, chronic treatment with GnRH agonist may indirectly affect PRL

secretion through E2 suppression. In a study of short-term administration of a GnRH-

Agonist in normal male rats, circulating PRL levels were not altered but PRL content of

the pituitary gland was depleted by 24, 49 and 73% after 2, 3 and 4 days, respectively. A

75% depletion after 4 days occurred in normal male rats (Lamberts et al., 1986). This

type of response is similar to FSH and LH secretion during chronic GnRH-Agonist

treatment in heifers (Gong et al., 1996). In another study, rats were treated continuously

with 5 µg/day of a GnRH-Agonist using an osmotic minipump on days 7 to 11 of

pregnancy. The magnitude of nocturnal PRL surges decreased significantly after 24 h of

GnRH-Agonist treatment in comparison to controls, and this concentration remained low

through day 11 of pregnancy. The non-surge levels of PRL in the treated group were not

different than the control at day 8, but were significantly lower at days 9, 10 and 11

(Sridaran et al., 1988). In another study with pregnant rats, animals were treated with 200

µg of GnRH-Agonist administered daily over days 1 to 7 of pregnancy. The treatment

reduced serum concentration of PRL during this same time period (Beattie et al., 1977).

In contrast, in a study which hypogonadal women were subjected to ethinyl estradiol

treatments (1 µg/Kg/day), the treatment induced a significant elevation of serum PRL

levels during 4 weeks of the study (Yen et al., 1974). These findings were in agreement

with other studies where serum PRL concentrations increased after exogenous estrogen

treatment in rats (Meites et al., 1972; Ajika et al., 1972). In a study conducted in

ovariectomized ewes, an injection of estradiol benzoate induced a greater nocturnal rise

of PRL with peak concentration approaching 400ng/ml in comparison to 200 ng/ml in the

control group (Davis and Borger, 1974).

Estradiol responses

In a study using a method to disperse bovine anterior pituitary cells in static culture, perfusion of estradiol to hypothalamic slices in series with anterior pituitary tissue resulted in an increased release of GH. Estradiol increased the baseline of GH release by 54% and the peak frequency by 67% above control. Treatment was associated with 27% increase of GHRH and a 33% decrease in somatostatin. In contrast, perfusion of estradiol directly to anterior pituitary cells did not affect GH release from somatotropes. This study showed that estradiol does not directly induce GH release from somatotropes but only via regulation of GHRH and somatostatin release from hypothalamic neurons (Hassanab et al., 2001). In another study conducted in ovariectomized Angus and Brahman cows treated with an estradiol ear-implant (24mg estradiol 17β) for 45 days increased anterior pituitary weight after 45 days of treatment in comparison to controls (Simpson et al., 1997). Plasma concentration of GH was greater from day 7 to day 42 in estradiol treated animals. During the 45 day study, plasma concentrations of IGF-I were greater for estradiol treated than control animals, as well as

total IGFBP activity. Treated cows also had greater concentrations of plasma glucose

during the 45 day study period. In a study with bovine anterior pituitary cell monolayers

cultured and preincubated with 10-8 M of estradiol or not, estradiol did not alter basal GH

secretion. However, it increased the bovine GH secretory response to GHRH in

comparison to non-preincubated controls (Silverman et al., 1988). Collectively estrogen

acts via hypothalamus to stimulate the secretion of GH from the anterior pituitary but E2

also is necessary to enhance the somatotrophs responsiveness to GHRH.

Influences of Uterus – Ovarian Interactions and GnRH-Agonist in Postpartum Uterine Involution in Cattle

Postpartum metritis has a great economic impact for dairy operations; this includes

treatment expenses, milk loss, prolonged days open and culling rates (Esslemont and

Peeler, 1993). Approximately 90% of cows have bacterial contamination during the first

10 days postpartum (Morrow et al., 1980). The lactational incidence rate of metritis has

been reported by Dohoo et al. (1983), Martin et al. (1983), Erb and Martin (1980) and

Bartlett et al. (1986) to be 18.2%, 11.1%, 13.8% and 18%, respectively. Metritis was more frequently diagnosed between 11 and 20 days postpartum and 6.1% of culled cows were due to metritis (Bartlett et al., 1986).

Factors such as retained placenta, dystocia, routine postpartum examinations, herd size, and parity were detected as being associated significantly with the incidence of metritis in a large epidemiological study (Kaneene and Miller, 1994). Data collected from

15,320 Holstein cows during a period of 3 years were used to determine the risk factors for conception; cows with retained placenta, metritis or ovarian cysts had 14%, 15% and

21% reduced likelihood of conceiving than normal cows, respectively (Grohn and Rajala-

Schultz, 2000). The risk of early metritis was reported to be higher in cows calving

during winter (Grohn et al., 1995).

Uterine Physical Involution

The time of complete involution of the uterus postpartum has a wide range of

intervals depending on the report. Marion et al. (1968) reported an average of 39 days

was required for a dairy cow to complete uterine involution. Kiracofe (1980) estimated a period of 28 to 56 days is required for uterine involution, and uterine size approximates the pre-gravid state by 25 to 30 days postpartum (Hussain and Daniel, 1991).

A sequence of events was proposed to occur during involution of the uterus: reduction in size, loss of tissue, and tissue repair (Marion et al., 1968). The rate of involution of the uterus decreases with time, and it is greater in the gravid than in the non-gravid uterine horn (Lauderdale et al., 1968). The cervix normally involutes at a lower rate than the uterus, but both organs (cervix and previous pregnant horn) are expected to reach a diameter smaller than 5 cm by 25 days postpartum in normal cows

(Morrow et al., 1966). Reduction of uterine size occurred on a log scale, with most of the changes occurring within a few days of calving (Gier et al., 1968).

Uterine involution involves contraction of uterine musculature that aids in sloughing of excess caruncular tissue (Olson et al., 1986). This process occurs during distinct phases: a) Puerperal period: is defined as the period extending from calving until the pituitary gland becomes responsive to GnRH at 7 to 14 days postpartum. b) Intermediate period: extend from the end of the puerperal period until the first ovulation, which varies depending on nutritional and energy status. c) Post-ovulatory period: extends from the moment of first ovulation until approximately 45 days postpartum.

Uterine Histological Involution

The intercaruncular luminal surface is replaced within 8 days postpartum in the cow (Gier et al., 1968). Although, Archbald et al. (1972) indicated there was no loss of intercaruncular epithelium at any stage postpartum (Archbald et al., 1972).

Remnants of chorioallantoic cells in the caruncular maternal crypts are observed at day 1 postpartum. These remnants undergo necrosis and mineralization, and are either phagocytized by macrophages or expelled with the lochia (Archbald et al., 1972). Gier and Marion (1968) observed necrosis and cellular disorganization of the caruncle on day

5 postpartum. Sloughing of the superficial layer of the caruncle begins around day 6 and

7 postpartum and the stratum compactum reduces in size to almost the intercaruncular level by day 15 postpartum (Archbald et al., 1972). A sequence of events in the maternal caruncle was proposed to be the following: degenerative vascular changes, peripheral ischemia, necrosis and sloughing. The results of these regressive changes are a central crater on a vascular bed after completed sloughing of caruncle (Gier and Marion, 1968).

Cuboidal-type ephitelial cells were present at focal areas of the caruncle by day 15 postpartum (Wagner and Hansel, 1969; Archbald et al., 1972), and an epithelial layer covered the entire caruncle surface by day 19 postpartum. Gier and Marion (1968) found that the caruncular surface was covered with epithelium by day 25 postpartum.

Degeneration and death of many glandular epithelial cells were observed on the day after parturition in ewes, and glandular tissue regeneration began at 8 days postpartum with complete restoration by day 15 (O’Shea et al., 1984).

Both Gier and Marion (1968) and Wagner and Hansel (1969) suggested that recovery of the exposed caruncular vascular bed occurs by cells originating from the edges of the caruncles. Moreover, O’Shea et al. (1984) showed that in ewes the epithelial

cells present at the base of the maternal septa of placentomas contributed to the

regeneration of the caruncular epithelium following shedding of plaques of degenerated

placental tissue from the caruncles. This process commenced after 8 d and was completed

before 31 days. However, in some caruncles, regeneration of the epithelium was not

completed until after 31 d postpartum. In another study, histological assessment showed

that completed recovery of the caruncular areas required approximately 30 days in cattle

(Wagner and Hansel, 1969).

Granular degeneration of the sarcoplasm, vacuolization of the muscle cell, and atrophy of the nucleus characterized histological changes in the myometrium. These processes were observed to begin at day 3 postpartum, and by day 31 the fibers appeared normal and the entire myometrium was reduced to a normal size. Overall necrosis of the myometrial cells was not observed (Archbald et al., 1972).

Uterine Mechanisms of Involution

The fetus plays a major role to trigger the onset of parturition. At parturition, it is

believed that the fetal mass approaches a space limitation in the uterus, which results in a

“stressed” fetus. Such a stress causes the release of adrenocorticotropic hormone (ACTH)

from the anterior pituitary of the fetus. ACTH is responsible for stimulation of fetal

adrenal corticoids. Elevated fetal corticoids promote the synthesis of enzymes

(hydroxylases, lyases and aromatase) within the cotyledons and increase the capacity of

the bovine placenta to convert C-21 steroids (progesterone, pregnenolone) into a C-19

estrogen precursor (i.e., androstenedione) and androstenedione converted into estrogen by

increased aromatase activity. This placenta maturation processes results into a dramatic

drop in progesterone and increase in estradiol. Collectively, all these endocrine events

leads to the removal of the “progesterone block”, by converting placental progesterone

into estradiol and by synthesis of PGF2α that also regresses the corpus luteum of pregnancy. During parturition pressure of the fetus on the cervix stimulates the secretion of oxytocin to facilitate myometrial contractility (Senger, 1999).

Uterine involution involves a continuation of contractions and peristalsis as rhythmical waves of the uterine musculature occurs after parturition (Roberts, 1986).

Myometrial contractions are greater at the tip of the uterine horn than at the body of the uterus, resulting in a peristaltic tubo-cervical contraction pattern. This pattern intensifies when cows experience retained fetal membranes (Burton et al., 1987). Persistent uterine infection is associated with the presence of a flacid, atonic uterus resulting in a delay of involution. In fact, nutritional related disorders such as hypocalcemia are correlated with a delay in uterine involution (Risco et al., 1994; Pelissier, 1976). Plasma concentrations of calcium were lower within 24 hours after parturition and throughout the first 8 days postpartum in cows that developed retained placenta and uterine prolapse.

Impediment of uterine motility during manipulation to manage a dystocia is related to pain and fear, which causes release of endorphins and epinephrine (Ruesse, 1982). In fact, relaxation of the uterus, determined by the softening of the uterine wall, is detected within 2 minutes after initiation of an intravenous infusion of epinephrine (Guilbault et al., 1984). The same inhibitory response on myometrial activity is observed when epinephrine is administered intravenously in cows during estrous (Ruckebusch and

Bayard, 1975).

Uterine Infections

Uterine infection is the term to indicate that the uterus is contaminated with pathogenic organisms and is classified accordingly to clinical signs and degree of severity

(Youngquist and Little, 1988). Uterine infections are strongly associated with calf

mortality and dystocia. Twinning and dystocia are important predictors of calf mortality.

Moreover, retained fetal membranes, twins, calf mortality and dystocia, in that order of

importance, are risk factors for abnormal vaginal discharge (Peeler et al., 1994).

Metritis is characterized by an inflammation of all layers of the uterine wall, palpable ballottement of fluid, a possible crepitant feel, lack of myometrial tone, and presence of an abnormal discharge (Callahan and Horstman, 1987). A life-threatening situation can occur when metritis becomes septic and toxic (Morrow, 1986).

Endometritis is characterized by an inflammation of the endometrial lining of the uterus without systemic signs, and is common during during the first 2 weeks of the postpartum period (Hussain, 1991). Endometritis is associated with a chronic infection,

involving primarily Arcanobacterium pyogenes (previously designated as

Corynebacterium pyogenes and Actinomyces pyogenes) (Lewis, 1997).

Pyometra is characterized by an accumulation of purulent exudates, growth of

predominantly Arcanobacterium pyogenes and gram-negative anaerobic bacteria (i.e.,

Fusobacterium necrophorum and Bacteroides melaninogenicus) in the uterine lumen in

association with the presence of a CL. Pyometra often results from after an early

ovulation postpartum (15 to 22 days), and during chronic endometritis or metritis (Arthur

et al., 1989; Olson et al., 1994).

Bacteriological data obtained after 14 days postpartum from uterine swabs of cows

with subacute-chronic endometritis, revealed a positive correlation between the presence

of Arcanobacterium pyogenes, Bacteroides spp., and Fusobacterium necrophorum with

mucopurulent, purulent and hemorrhagic-foul cervical discharge scores (Dohmen et al.,

1995). These bacteria are the most common organisms associated with uterine diseases.

In a large epidemiological study that included approximately 1865 cows, palpation

of the reproductive tract and vaginoscopy examination of cervical discharge were used to

determine criteria for clinical diagnosis of endometritis and associated reproductive

performance (LeBlanc et al., 2002). Cervical diameter larger than 7.5cm and a uterine

horn diameter larger than 8 cm at any time between 20 to 33 days postpartum were

associated significantly with reduced fertility by 120 days postpartum. The category of

clear mucus with flecks of pus was not associated with reduced pregnancy at any day

from 20 to 33 days postpartum. Therefore, cows in this category became part of the

reference group (clean and no discharge). A mucopurulent discharge tended (P=0.09) to increase time from calving to pregnancy, and purulent or foul discharge was associated significantly with a 20% reduction in pregnancy rates. However, an interaction of discharge and cervical diameter by week of examination was detected. Specifically, mucopurulent discharge only affected pregnancy rates if found from 27 to 33 days postpartum. Cows with purulent or foul discharge between 27 and 33 days postpartum were significantly associated with 25% reduction in pregnancy rates. A reduction of 16% in pregnancy rates was detected when this category of discharge was detected from 20 to

26 days postpartum. However, this decline was not significant due to the reduced sample size. Therefore, clinical endometritis was defined during the period of 20 to 33 days postpartum, as the presence of purulent or foul discharge, or a cervical diameter larger than 7.5 cm between 20 and 33 days postpartum, or mucopurulent discharges after 26 days postpartum (LeBlanc et al., 2002). Similarly, cows that had uterine infection after 3 weeks postpartum were more likely to develop a severe endometritis with a delay in conception rate (Hartigan et al., 1974; Griffin et al., 1974).

Evaluation of Uterine Involution

Rectal palpation has been used as a practical method to evaluate uterine and cervical involution (Morrow et al., 1969). Estimated reductions of uterine horn and cervical diameters were similar between retained placenta and normal cows. Uterine involution seemed to be completed by approximatly 30 days postpartum and cervical size remained constant by day 40 postpartum (Morrow et al., 1969).

However, palpation per rectum of the reproductive tract is neither a sensitive nor a specific method for accurate diagnosis of endometritis. In one study (Miller et al., 1980),

157 cases of endometritis were diagnosed by palpation but bacteria were isolated from uterine fluid from only 22% of them (Miller et al., 1980). Therefore, use of a vaginoscopy may be an important aid for clinical diagnosis of subacute and chronic endometritis and evaluation of treatment responses (Dohmen et al., 1995; LeBlanc et al.,

2002).

Oltenacu et al. (1983) indicated an association among conditions at calving with type of discharge early postpartum and diameter of cervix obtained by rectal palpation from 12 to 26 days postpartum. Cows with a normal parturition had a significantly greater percentage of normal vaginal discharges (clear or mucoid) and a smaller cervical diameter (< 4 cm). In contrast, cows that experienced abnormal parturitions had an increased percentage of abnormal discharges (ranging from presence of flakes of pus to a purulent fetid appearance) and an increased cervical diameter. The rate of cervical involution was greater for cows with normal vaginal discharge. Moreover, cows with an abnormal discharge had a delayed time to first estrous, and first service cows with a large cervical diameter (> 60mm) had a lower pregnancy rate at first service and consequently greater days open.

An ultrasonic linear scanner can be used for characterizing postpartum uterine and

cervical involution in the cow (Okano and Tomizuka, 1987). Ultrasonic evaluation of

uterine involution was performed twice a week until the sixth week postpartum (Mateus

et al., 2002). In this study, uterine infection significantly retarded uterine involution as

assessed by diameter of the uterine body and determination of intrauterine fluid volume

(IUFV). Uterine body diameter was greater until the fourth week postpartum in cows with severe puerperal endometritis in comparison to controls and cows with mild endometritis. By 6 weeks the uterus had regressed to pregravid size, but IUFV remained significantly higher, indicating that a chronic endometritis was established. The IUFV score was correlated positively with uterine swab bacterial growth density. Additionally, ovarian activity measured by ultrasound scanning of the ovaries and plasma progesterone concentrations was more abnormal (prolonged anoestrous, prolonged luteal phases and ovarian cysts) in cows with severe endometritis than in controls.

Postpartum Ovulation

After parturition, first ovulation is expected to occur by 21-30 days postpartum

(Beam and Butler, 1997; Beam and Butler, 1998) at a time in which uterine involution has not been completed. Previous studies in sheep indicated that ovulation induced during the period of uterine involution results in a normal mature oocyte and in embryos that are viable when recovered on Day 3 after oestrous and transferred into a normal uterine environment with successful pregnancies (Wallace et al., 1989a). However, embryos rarely survive beyond the duration of a normal estrous cycle if transferred or returned to an involuting uterus postpartum, even though recipient ewes have normal luteal function.

Pregnancy failure after embryo transfer may be caused by an inappropriate uterine environment (Wallace et al., 1989b).

Cows that ovulated prior to day 21 (47% [41/87]) postpartum had a prolonged

calving to conception interval, more services per conception, lower conception rate and increased culling rate for failure to conceive in comparison to cows that ovulated after 21 days (Smith et al., 1998). The incidence of prolonged luteal function (elevated progesterone for more than 25 days) was greater in the cows with early ovulation and was associated with a lower fertility (Smith et al., 1998; Ball et al., 1998).

Similarly, resumption of ovarian activity measured by means of twice a week concentration of milk progesterone, showed that cows ovulating between 19 to 24 days postpartum had a higher risk to develop a prolonged luteal cycle (progesterone elevated for more than 20 days) than cows ovulating later than 32 days postpartum. The risk increased if the cows had an ovulation before 19 days postpartum. Moreover, high incidence of prolonged luteal phase was observed (21.5%, 72/334) when the time of ovulation was within a range in which uterine involution was not completed (Opsomer et al., 2000). A prolonged luteal phase is correlated with a greater risk of developing pyometra (Farin et al., 1989). In fact, a side effect of early induced ovulation in response to GnRH injections (15 days postpartum) was an increase in the subsequent incidence of pyometra and prebreeding anestrous. Thus, treatment with GnRH alone increased calving to first estrous and calving to first breeding intervals, and a tendency for an increased calving to conception interval (Etherington et al., 1984).

Lamming and Darwash (1998) using concentrations of milk progesterone in a large data set, identified that atypical ovarian patterns (delayed first ovulation, prolonged luteal phase) were associated with delayed conception, higher number of services per conception, lower first service conception rate, reduced total conception rate, and a

higher incidence of embryonic loss. In another study, approximately 25% of first luteal

phases during the postpartum period were classified as being longer than 25 days, and

early ovulation was associated with reduced conception rates (Royal et al, 2000).

In contrast, cows with abnormal calving, abnormal discharge and clinical ketosis had a greater risk of having low concentrations of progesterone during the first 50 days postpartum (anovulatory condition). This anovulatory category of progesterone profile accounted for 21.5% (72/334) of the cows (Opsomer et al., 2000). Puerperal diseases have long been implicated in retarding ovarian activity (Marion and Gier., 1968).

Morrow et al. (1966) reported averages of 15 days from parturition to first ovulation in cows having normal parturition and 34 days in abnormal cows.

Reduced diameter of the first dominant follicle, slower growth rate and decreased secretion of estradiol resulted in a delayed first ovulation postpartum. These events were associated with high uterine bacterial contamination (Sheldon et al., 2002, Peter et al.,

1988). Similarly, postpartum follicular activity in the ovary ipsilateral to the previous gravid uterine horn is lower than that in the contralateral ovary (Saiduddin et al., 1968;

Kamimura et al., 1993; Guilbault et al., 1987). The mechanisms involved in this condition could be associated with endotoxins and cytokins resulting from inflammatory processes, which could disrupt hypothalamic GnRH release and pituitary LH secretion

(Peter et al., 1989; Rivest et al., 1993).

Lower conception rates have been associated with delayed return to estrus after calving (Menge et al., 1962). Similarly, Thatcher and Wilcox (1972) proposed that early and frequent estrus activity during the postpartum period was associated with increased reproductive performance due to greater restoration of uterine environment. In this study,

cows expressing 0 or 1 estrus within 60 days postpartum had reduced fertility resulting in

a higher percentage of non-pregnant cows sold and required more services per pregnancy

per cow. These responses decreased linearly as the number of estrous increased, i.e. cows that had 2, 3 or 4 estruses were of greater fertility, in which most of the variability was associated with occurrence of estrus during the first 30 days postpartum. The conclusion that spontaneous estrous cycles during the postpartum period can enhance uterine involution and subsequent fertility is debatable. Animals with no estrus could represent the cows with dystocia, retained placenta, metabolic disorders and metritis, and the cows expressing one estrous could represent the animals with a prolonged luteal phase (failure to turn-over CL). These cows generally have lower fertility. The animals expressing 2 or more estrous periods represent the cows with a normal postpartum that were able to ovulate once and continue to do so. Therefore, a sequential occurrence of events from the moment of parturition to the time of first service is seen. Early abnormal events in the postpartum period (i.e. dystocia, retain fetal membranes, metabolic diseases, metritis) are related with uterine health and involution, which will dictate time of first ovulation and subsequent fertility.

Role of Progesterone

The rise of progesterone during the postpartum period results in delayed uterine involution, which is possibly due to reduced myometrial contraction, a reduction in the

immunoresponse, and development of pyometra associated with a prolonged luteal phase.

Treatment with 30 mg of progesterone beginning 3 days postpartum until uterine

involution was completed resulted in a significant increase in the interval to complete

uterine regression (Marion et al., 1968). Similar results were obtained when cows were injected with 100 mg of progesterone for 22 alternate days, beginning on the day of

calving (Fosgate et al., 1962). Rabbits treated with 25 mg of progesterone every 4 hours immediately after parturition had a significant reduction in its rate of uterine involution, with or without estradiol-17β added to the treatment, when compared to placebo controls

(Goodall, 1966)

A Similar phenomenon was observed during the estrous cycle. Rodriguez-Martinez et al. (1987) used miniature pressure transducers to measure tone and uterine contractility of cows during the estrous cycle. Uterine activity was minimal during diestrus, increased during proestrus and reached maximum values during estrus. Additionally, uterine contractility assessed by transrectal ultrasonography revealed a decrease in contractility from the day of ovulation (d –1 to 0) to day 11 (mid-diestrous) of the estrous cycle

(Bonafos and Ginther, 1995). In the same study, uterine tone was assessed by rectal palpation and scored from 1 (flaccid) to 5 (turgid). Uterine tone was high during the periovulatory period and decreased on days 3 and 4. In women treated with vaginal progesterone starting on the day of oocyte retrieval, uterine contraction frequency, assessed by transvaginal ultrasonography, was decreased on the day of embryo transfer as compared with preovulatory values (Fanchin et al., 2001).

The mechanisms by which progesterone prevents myometrial contraction are related to the ‘’progesterone block” that prevents the development of oxytocin receptors.

Fuchs et al. (1983) found that estrogen increased and progesterone inhibited the estrogen- induced rise in oxytocin receptor concentrations in the myometrium. Number of oxytocin receptors induced by estrogen was reduced approximately 60% by 24 hours after addition of progesterone to the myometrium explants (Soloff et al., 1983). Additionally uterine oxytocin receptors induced by estradiol declined soon after progesterone replacement

(Vallet et al., 1990), which supports that the removal of the “progesterone block” must be

more important then the estradiol stimulation.

Another feature involving progesterone and uterine contraction is the formation of

gap junctions in the muscle cells. Studies demonstrated that a decrease in progesterone levels followed by increases in estradiol close to parturition are coincident with the formation of gap junctions, which in turn may coordinate the increased uterine activity required for parturition (Puri et al., 1982). These gap junctions are intercellular connections that enhance the movement of electrolytes and small molecules between adjacent myoepithelial cells to increase contractility (Bengtsson, 1982).

Progesterone also is associated with increased severity to uterine infections.

Ovariectomized cows treated with continuous injections of progesterone had similar frequency of positive intrauterine cultures of Arcanobacterium pyogenes in comparison to controls at 17 days after intrauterine inoculation (Carson et al., 1988). However, recovery from infection was slower judged by occurrence of thicker and larger uteri. In another study, neutrophil phagocytic capacity was lower in diestrous in comparison to ovariectomized cows treated with estradiol (Subandrio et al., 2000). Leucocytic and phagocytic activity were reduced in a progesterone environment (Chacin et al., 1990); also progesterone delayed the onset of leucocytic response in uterine flushings of heifers inoculated with Eschericia coli (Hawk et al., 1964). Moreover, physiological concentrations of progesterone have been shown to affect polymorphonuclear nuclear leukocyte activity in the systemic circulation (Roth et al., 1983).

Immunosuppressive uterine luminal protein (ULP) is known to suppress lymphocyte blastogenesis in mixed lymphocyte cultures (Murray et al., 1972). Sergerson

et al. (1986) found that a combination of estradiol and progesterone treatment increased

the quantity of ULP in ovariectomized cows; this treatment combination could mimic a

diestrous situation. When ovariectomized rats were infected with Chlamydia trachomatis

via the intrauterine route, histopathological examination showed severe inflammation in

the uterus and vagina of progesterone-treated animals with a large numbers of

chlamydiae found in vaginal secretions (Kaushic et al., 2000). Authors concluded that

progesterone increased susceptibility to intrauterine chlamydial infection in this rat

model.

In summary, progesterone can inhibit the physical clearance of intrauterine

bacterial infections trough a mechanism involving oxytocin as well as cell morphology.

Moreover, progesterone has an immunosuppressive activity that can reduce uterine host

defense mechanisms.

Role of Estradiol

Saiduddin (1968) reported that 80% of first ovulations up to 20 days postpartum occurred on the ovary contra-lateral to the previous gravid uterine horn. In a population study, cows were more likely to have a CL on the contralateral ovary of the previous gravid uterine horn. Also, a shorter calving to conception interval was associated with normal vaginal mucus, a smaller diameter of the previously gravid uterine horn, and with the presence of a dominant follicle on the ovary ipsilateral to the previous gravid horn

(Sheldon et al., 2000). Therefore, attempts to increase follicular activity associated with an elevation of estradiol during the puerperium have been tested in a series of experiments by Sheldon and colleagues (Sheldon and Dobson, 2000; Sheldon et al.,

2001; Sheldon et al., 2002; Sheldon et al., 2003) in order to improve uterine involution.

Injection of equine chorionic gonadotrophin (eCG) at day 14 postpartum in cows increased follicular activity on the ovary ipsilateral to the previous gravid uterine horn, but it did not result in a faster rate of uterine involution. Additionally, plasma concentrations of PGFM after treatment were increased in the cows with abnormal vaginal mucus and were not affected by eCG (Sheldon and Dobson, 2000).

The hypothesis that the regressing CL of pregnancy suppresses folliculogenesis on the ipsilateral ovary after parturition was tested. The administration of PGF2α at 200 days

of pregnancy caused luteolysis without disturbance of the fetus. However, it did not

improve follicular growth and function, or timing and location of ovarian events after

parturition (Sheldon et al., 2002). Thus, it seems that uterine size and degree of uterine

inflammation regulates ovarian activity in the early postpartum period rather than the

ovary.

An attempt was made to test the possible beneficial effects of an estrogenic dominant follicle during the early postpartum period of uterine involution and on inflammatory factors such as PGFM and acute phase proteins (Sheldon et al., 2003).

Acute phase proteins are hepatocyte-derived substances that limit tissue damage and promote tissue repair (Baumann and Gauldie, 1994) and are associated with uterine bacterial contamination (Regassa and Noakes, 1999; Sheldon et al., 2001). Cows were infused with estradiol benzoate or saline into the previous gravid uterine horn between day 7 and 10 postpartum. No treatment effect was detected on uterine diameters.

However, uterine horns were larger in the cows with greater bacterial contamination and in animals in which the first dominant follicle regressed compared to cows that ovulated.

Plasma PGFM, haptoglobin and ceruloplasmin did not differ between groups, but plasma

α-1 acid glycoprotein was reduced in treated cows. Similar results were obtained in ewes in which silastic implants containing estradiol or no eatradiol were surgically placed within the bursa of the ovary ipsilateral to the previous gravid uterine horn for the first 17 days postpartum. Although constant higher concentrations of plasma estradiol were achieved during the duration of treatment, no consistent differences were detected in tissue size (i.e., uterine horns and cervix), and concentrations of acute phase proteins

(Sheldon et al., 2003). Increased estradiol concentrations, pharmacologically or by follicular activity, are unable to improve uterine involution and reduce bacterial contamination. Also, early ovulation after calving followed by continual estrous cycles is dependent on normal uterine involution and low bacterial contamination of the uterus.

The pathway uterus to ovaries seems to prevail over ovaries to uterus.

The effect of 5 mg of estradiol cypionate (ECP) on myometrial activity was measured using strain gauge transducers during the early postpartum period. Treatment with ECP led to a reduction in uterine motility and formation of a sustained contraction in which all parts of the uterus contracted simultaneously (Burton et al., 1990). In another study, uterine motility was recorded during the estrous cycle by means of two ultraminiature pressure transducers located in the middle of the uterine horn and near the body of the uterus. The lowest uterine activity was during diestrous, and a peak of estradiol-17β preceded the highest activity during estrus (Rodrigues-Martinez et al.,

1987). The pattern of motility at estrus was characterized by a cervico-tubal wave. In contrast, contractions located at the tip of the previous gravid uterine horn are greater than at the body of the uterus resulting in a tubo-cervical wave propagation of contraction during the early postpartum period in a low estrogenic environment (Burton et al., 1987).

Inhibition of spontaneous myometrial activity also is seen in the rats (Downing et al.,

1978) and in the bitch (Wheaton et al., 1986) when administered with estradiol benzoate

and estradiol cypionate, respectively. The estrogen-induced anti-peristaltic effect is

probably the reason for a higher incidence of salpingitis when estradiol cypionate was

used to treat metritis (Gustafsson and Ott, 1981, Youngquist and Shore, 1997).

Role of Prostaglandin

Native prostaglandin F2α is metabolized almost entirely into 15-keto-13, 14-

dihydro-prostaglandin F2α (PGFM) upon a single passage to the lungs (Maule Walker and

Peaker, 1981).

The bovine uterus is a primary source of F series prostaglandins during the early postpartum, and secretion increased from the day of parturition to approximately 4 days postpartum followed by a decline to basal levels by 14 days. The caruncular endometrium has a greater ability to synthesize PGF2α and to metabolize it into PGFM (Guilbault et al.,

1984).

Massive release of PGF2α also is a result of an inflammatory process in the uterus.

Del Vecchio et al. (1992) transcervically inoculated Actinomyces pyogenes and

Escherichia coli or sterile PBS alone into the uterine horns on three consecutive days, ranging from d 8 to 14 of the first postpartum estrous cycle. Based on clinical observations and results of bacterial cultures, all treated cows developed acute uterine

infections, but not the PBS infused control cows. Bacterial infusions increased mean

PGFM concentration in plasma indicating that induced uterine infection promoted the

release of PGF2α.

In cows with normal puerperium, those showing a relatively longer duration of elevated plasma concentrations of PGFM needed a shorter period for postpartum uterine

involution than the cows showing a shorter duration of elevated PGFM; however, no such

relationship was observed in cows with abnormal puerperium (Lindell et al., 1982; Nakao

et al., 1997). Animals experiencing dystocia and retained placenta have elevated PGFM

profiles during the first 4 days postpartum followed by a precipitous decline from day 7

to 10. During this period, treatment with a long acting PGF2α analogue (fenprostalene)

did not alter plasma PGFM concentrations. The precipitous drop of PGFM in cows with

dystocia and retained placenta could be due to a massive loss of caruncular tissue

connected to a fetal cotyledon after a more severe necrosis of the caruncle stalk in cows

with intensive bacterial infections. Cows with normal puerperium had lower levels at day

1 to 4 postpartum, but these levels were maintained constant until 7 days postpartum

(Nakao et al., 1997). In this later study, fenprostalene treatment between 7 to 10 days

postpartum reduced time for uterine involution and increased conception rate at first

service.

The in vivo uterotonic nature of PGF2α was demonstrated in cycling lactating cows.

The total work of the uterus increased up to 250% of base-line value as the dose of PGF2α

was increased (i.e., 0.1 to 32.0 mg), but it was followed by a partial refractoriness of the

uterus (Eiler et al., 1981). The same author, using an intrauterine balloon technique,

showed no effect of luteolytic PGF2α dose on uterine motility when given at 48 to 72 hours after parturition (Eiler et al., 1984). Luteolytic doses of PGF2α (25 mg of

Dinoprost) administered by rapid intravenous bolus injections were uterotonic in postpartum cows, increasing the frequency of contractions and the amount of tubo- cervical wave propagation (Gajewski et al., 1999). In the bitch, uterine motility was increased by more than 100% after intravenous administration of PGF2α and by 52% after

intramuscular PGF2α administration (Wheaton et al., 1986). In contrast, an early

postpartum i.m. injection of fenprostalene did not increase myometrial activity

determined by strain gauge transducers (Burton et al., 1987). Guilbault et al. (1988) were

unable to detect an improvement in uterine involution after continual infusion of

exogenous PGF2α.

Uterine motility responses after PGF2α administration seems to be dependent on the

stage postpartum, since early postpartum concentrations of PGFM are already high

(Guilbault et al., 1984) such that there is no additive motility. Additionally, responses

could be dependent on route of administration, such that intravenous administration

seems to result in increased motility in comparison to an intramuscular injection.

Differences also could be explained by the very short half-life of PGF2α, reported to be

less than 1 minute (Samuelsson et al., 1975).

Flunixin meglumine is a potent non-steroidal anti-inflammatory drug, which

reduces the biosynthesis of PGF2α through the inhibition of cyclooxygenase enzymes in

the arachidonic acid cascade. Administration of flunixin meglumine (i.e., during the first

10 days postpartum) decreased plasma concentrations of PGFM, but did not affect the

rate of uterine involution (Guilbault et al., 1987). Moreover, twice daily injections (08:00 and 16:00 h) of flunixin meglumine (2.2 mg/Kg of b.w.) for the first 10 days postpartum inhibited endogenous PGF2α by more than 80%, but treatment did not interfere with uterine involution, return to cyclicity and the first postpartum cycle length (Thun et al.,

1993). In another study, cows were treated intravenously with flunixin meglumine, at a dosage of 2.2 mg/kg, on six occasions (i.e., twice daily on the first 2 days and once daily for the subsequent two). No adverse effects of flunixin meglumine on the process of

uterine involution and subsequent ovarian resumption were detected. In fact, flunixin-

treated cows had a faster uterine involution than controls and showed the first postpartum

estrus earlier than controls. (Amiridis et al., 2001). No adverse effects were obtained in a

study in which cows were injected with flunixin meglumine four times daily or twice

daily for a period of 14 days beginning at parturition. The PGF2α synthesis and release

were decreased by the treatment (Odensvik and Fredriksson, 1993). However, flunixin

meglumine did not affect the time period to complete uterine involution, not even when a

very intensive drug dosage was used. Therefore, the suppression of PGF2α does not interfere in the processes of uterine involution. In cases in which anti-inflammatory therapy improved the rate of uterine involution (Amiridis et al., 2001), it could be through a reduction of the inflammatory process and ameliorating overall clinical signs in the cows.

Role of Oxytocin

The close association between myometrial oxytocin receptor (OTR) concentrations and sensitivity to oxytocin during the estrous cycle suggests that the myometrial response to oxytocin in vivo is regulated at the receptor level by circulating oxytocin concentrations (Soloff and Fields, 1989). Incubation of uterine explants from immature rats with estradiol resulted in approximately a five-fold increase in the number of OTR per milligram of protein within 48 h. However, addition of progesterone and estrogen to explants with elevated receptor levels resulted in almost a 60% reduction in OTR concentration by 24 h, with no change in affinity of the receptor for oxytocin (Soloff et al., 1983).

In cows, the period from 2 days before to 2 days after estrus, small intravenous doses (2.5 IU) of oxytocin caused the proximal ends of the uterine horns to respond

within 30 to 50 seconds as monitored with an extra-cellular multi-electrode assembly.

The increased frequency of myometrial activity persisted for up to 80 minutes

(Ruckebush et al., 1975).

During the first 6 days postpartum of cows, intravenous doses of oxytocin can

increase the frequency of tubo-cervical myometrial contractions while maintaining

unchangeable contraction intensiveness, duration and propagation time. The magnitude

of myometrial response to oxytocin is dependent on dose and day of treatment, and

response did not differ when oxytocin was given prior or after administration of estradiol

cypionate (Burton, 1990; Kundig et al., 1990). Larger doses of oxytocin produced

significantly greater increase in contraction frequency, ranging from one contraction

every 6 minutes for a 2 USP dose, to one contraction every 3 minutes for a 40 USP dose.

Uterine contractions are no longer detectable to doses of 2, 5, 10, 20, and 40 USP of

oxytocin at 6, 7, 8, 9 and 10 days postpartum, respectively. Oxytocin doses of 25 USP

consistently caused an increased contraction frequency associated with higher tubo- cervical wave propagation on days 1 to 5 postpartum (Burton, 1986). Oxytocin given intravenously provoked increased uterine contractions until day 4 postpartum. However, high doses such as 40 USP during the first 3 days postpartum resulted in a tetanal uterine effect rather than a peristaltic contraction with the duration of response decreasing from 2 to 6 days postpartum. Thus, an efficacious oxytocin therapy must orchestrate dose and period postpartum in order to accomplish potential optimal results.

Oxytocin receptors (OTR) also were found in mucosal and muscle layers of the cervix. All regions of the cervix from cows at estrus had high concentrations of OTR; in the luteal phase, OTR was abruptly down-regulated. At estrous the mucosal layer had

67 about 30-fold higher concentrations than the muscle layer (Fuchs et al., 1996). Oxytocin was found to stimulate PGE2 output in vivo from bovine cervical tissues and to cause softening of the cervix (Fuchs et al., 2002). Therefore, oxytocin may have a novel physiological function to cause softening of the bovine cervix mediated by the release of

PGE2.

Delaying First Ovulation Postpartum

Delayed first ovulation in suckled cows has been well reviewed (Yavas and

Walton, 2000). The interval from calving to first ovulation in non-suckled beef cows was

10.2 days compared to 34.7 for suckled cows (Carter et al., 1980). Delayed first ovulation in suckled cows has been associated with increased rate of uterine involution in dairy

(Riesen et al., 1968) and beef cows (Lauderdale et al., 1968). Others (Wagner and

Hansel, 1969) have found no effect of suckling rate on uterine involution. Presence of the calf thoughtout the postpartum period may represent a natural defense mechanism to prevent early ovulation. Also, frequent suckling by the calf is related with increased secretion of oxytocin in comparison to machine milking (Bar-Peled et al., 1995).

Cows ovariectomized between 3 to 5 days postpartum had a significantly shortened uterine regression interval in comparison to untreated controls and ovariectomized progesterone treated cows (Marion et al., 1968). In contrast, interval between parturition and occurrence of uterine involution was not affected by ovariectomy in comparison with control cows (Oxenreider, 1968). Perhaps the absence of gonadal hormones enhanced for the physical involutionary process of the uterus. Moreover, presence of the calf after parturition may represent a natural phenomenon to protect the involuting uterus against the relaxation and immunosuppressive effects of progesterone and to stimulate a higher secretion of oxytocin.

Chronic treatment with GnRH-Agonists can induce downregulation of GnRH receptors on the gonadotroph cells, desensitize the anterior pituitary gland to endogenous

GnRH, and abolish pulsatile LH secretion leading to a block of ovulation (D’Occhio et al., 2000). These features of GnRH-Agonists could be used to block ovulation during the period of uterine involution, such that this involutionary process could be concluded prior to the first ovulation postpartum. The GnRH-Agonist may “mimic” the natural mechanisms offer by presence of the calf.

High doses of estradiol during the early postpartum period could be another approach for delaying the onset of ovulatory cycles. A single intramuscular injection of

10 mg of estradiol cypionate (ECP) at 7 days postpartum delayed the resumption of ovulation until at least 40 days postpartum in association with a suppression of follicle development (<10 mm) until 29 days postpartum. However, there appeared to be considerable variation in the interval to resumption of ovulation after ECP treatment, for example, 35% of the cows had ovulated by day 50 postpartum and 94% by day 90 postpartum (Haughian et al., 2002). Moreover, in the same study, a reduction in conception rate for cows injected with 4 mg of ECP was of potential concern; anti- peristaltic effects of estradiol in association with increased salpingitis should be considered.

Direct Effects of Gonadotrophins on the Reproductive Tract

The LH receptors (LHr) have been found in the myometrium of pigs (Ziecik et al.,

1986), women (Reshef et al., 1990), rats (Bonnamy et al., 1990), rabbits (Jensen and

Odell, 1988), and cows (Shemesh et al., 2001). Expression of LHr is dependent on stages of the cycle. Expression of LHr is high during the luteal phase and expressed weakly during the follicular phase.

The LHr may have a role in regulating uterine motility. Binding of LH to its receptor activates signaling pathways that increase expression of cyclooxygenase and production of PGE2 in the myometrium that results in relaxation (Shemesh et al., 2001).

Uterine relaxation seen during early pregnancy may be regulated in part by pituitary LH in pigs (Flowers et al., 1991). Therefore, increased receptor binding of LH and increased cAMP may serve to maintain quiescence of the uterus during the luteal phase. Also, the relaxing effect of hCG on uterine motility was observed in women, and the action of hCG is tissue specific and appears to be mediated by decreasing intracellular free Ca++

concentrations in myometrial smooth muscle cells (Eta et al., 1994).

In the cervix, LH binding initiates signaling pathways that increase expression of

cyclooxygenase and the production of PGE2, which could be related to the softening of

the cervix during the proestrous and estrous stages (Shemesh, 2001). Administration of

PGE1 to cows intracervically resulted in a decrease of cervical resistance within 24h

(Graddy et al., 1998).

Studies in gilts revealed that LH caused relaxation of the oviduct, especially during the periovulatory stage of the estrous cycle (Gawronska et al., 1999). Thus LH action may facilitate the passage of embryos through the isthmus towards the uterus.

In summary, it is proposed that activation of adenylate cyclase is associated with relaxation; whereas, activation of COX-2 can lead to either a release of PGE, to relax the muscles, or of PGF2α, which may contract them (Figure 2-4). Therefore, suppression of

gonadotrophins may eliminate some of its relaxation effects on the reproductive tract,

such that increased uterine tone is a default.

Figure 2-4. Putative model for the physiological role of FSH and LH in the uterine tract motility (Shemesh, 2001).

Studies using reverse transcription-PCR revealed that myometrium and isolated myometrial smooth muscle cells express GnRH receptor mRNA in rats (Chegini et al.,

1996) and in endometrium of humans (Raga et al., 1998; Imai et al., 1994). However the actions of GnRH on these tissues are still not clear

GnRH-Agonists are widely used in the treatment of women with symptomatic leiomyomas (Broekmans, 1996), endometriosis (Rumore and Rumore, 1989) and endometrial carcinoma (Dessole et al., 2000). These conditions are considered to be estrogen-dependent. Thus, GnRH-Agonist treatment is an efficient and reversible pharmacological method to achieve hypoestrogenism, which leads to reduction in tumor volume. Also hysterectomy is preceded by GnRH-Agonist treatment in order to decrease uterine volume and facilitate either abdominal or vaginal procedures (Broekmans, 1996).

Uterine Histology Upon Chronic GnRH-Agonists

Ultrasound measurement of endometrial volume and thickness is being currently used in medical practice as a predictor of pituitary suppression following chronic administration of a GnRH-Agonist. The relationship between oestradiol status and endometrial thickness has been proven (Child et al., 2002; Nakamura et al., 1996).

Ultrasound measurement of three-dimensional volume is used for predicting pituitary

down-regulation in human IVF-embryo transfer programs. It is a tool for diagnosing

relative hypo-estrogenism or down-regulation after chronic treatment with GnRH-

Agonists, and eliminates the need for blood sample collection and estradiol radioimunoassays.

Evidence from ultrasound studies has shown a decrease in myometrial volume of

40% in response to GnRH-Agonist therapy (Carr et al., 1993). Doppler ultrasound of the uterine artery showed that the uterine vasculature vasoconstricts when patients are treated with GnRH-Agonists (Matta et al., 1988). In one study, premenopausal women were treated with a GnRH-Agonist for 8 weeks before hysterectomy. Postoperatively, two independent pathologists examined the myometrium. The uterine volume of women

treated with GnRH-Agonist decreased by 28%, as opposed to no changes of volume in

the control group. The cellularity (cell/mm2) of the GnRH-Agonist-treated myometrium was higher than the controls with less stromal edema. The arteries in the GnRH-Agonist- treated uteri underwent atrophy of the tunica media and had significantly more perivascular fibrosis. The number of vessels per 100 myocytes also was decreased.

Therefore, the authors concluded that the hypoestrogenism as a secondary response to

GnRH-Agonist treatment led to myocyte atrophy, decreased stromal edema, atrophy of the arcuate arteries, and decreased myometrial vascularity (Weeks et al., 1999).

CHAPTER 3 REPRODUCTIVE RESPONSES FOLLOWING SUPPRESSION OF FOLLICULAR DEVELOPMENT WITH A DESLORELIN IMPLANT DURING SUMMER HEAT STRESS

Introduction

One of the most serious concerns of dairy producers located in tropical and

subtropical areas is the seasonal depression of reproductive efficiency (Gwazdauskas et al., 1973). Conception rates to artificial insemination may range from 55% during months of low temperatures and humidity to only 10% during months of high temperatures and humidity (Ingraham et al., 1974). Conception rates of lactating cows located in north

Florida showed a decrease to approximately 20% during summer months (June through

August) and continued to be low during autumn (September and October) when temperatures were no longer stressful. Conception rates did not recover at normal levels of winter until November (Badinga et al., 1984). Similarly, Al-Katanani et al. (1999) observed a depression of 90-day non-return rates to first service for lactating dairy cows in Florida and southern Georgia during May thought August, and normal levels were not achieved until November and December. These studies have demonstrated clearly an immediate as well as a delayed effect of summer heat stress on fertility.

Ovarian follicles are susceptible to heat stress (Badinga et al., 1993). Follicular dominance at day 7 of the cycle was reduced as indicated by an increase in the number of medium size follicles and size of the subordinate follicle (Badinga et al, 1993). A greater number of large follicles (≥ 10 mm) also were found in heat stressed cows in comparison to controls during the first follicular wave (Wolfenson et al., 1995) and in heifers during

72 73

days 17 to 21 of the cycle (Wilson et al., 1998). Heat stress decreased the number of

viable granulosa cells, reduced granulosa aromatase activity and suppressed thecal

androstenedione production at day 7 from the first wave dominant follicle (Wolfenson at

el., 1997).

Heat stress also has a delayed effect on ovarian function as characterized by alterations in follicular steroidogenesis, follicular dynamics and altered concentrations of

FSH and inhibin (Badinga et al., 1994; Wolfenson et al., 1997; Roth et al., 2000;

Guzeloglu et al., 2001).

A seasonal effect during hot months is reported to reduce oocyte quality and to reduce development of in vitro-fertilized (IVF) oocytes to blastocysts in dairy cattle

(Rutledge et al., 1999; Al-katanani et al., 2002). The effects of heat stress impaired oocytes during early stages of development (Roth et al., 2001). After a period of high temperature, oocytes obtained by transvaginal aspiration and that underwent parthogenic activation did not recover their ability to produce percentage of grade I embryos (i.e., blastocyst) until approximately six follicular waves or the equivalent period of three estrous cycles. Such an effect in part accounts for the delayed effects of heat stress on fertility. Restoration of normal fertility after a period of heat stress is achieved in approximately 40 to 60 days (Badinga et al., 1984).

Chronic treatment with GnRH-Agonists can induce down-regulation of GnRH receptors on gonadotrophs, desensitize the anterior pituitary gland to GnRH, and abolish the pulsatile release of LH (D’Occhio et al., 2000). Such effects lead to suppression of follicular growth and an arrest of follicles at 2-3 mm in diameter after a chronic treatment for 28 days (Gong et al., 1996).

It was hypothesized that suppressing follicular development using a GnRH-Agonist

(Deslorelin [DESL]) implant during the postpartum period and summer time may serve as a experimental model to determine if follicle sensitivity to heat stress is impaired at ≤ 3 mm follicle size. We further hypothesized that follicles > 3 mm will be affected adversely by heat stress such that fertility of cows chronically treated with Deslorelin would be higher than the non-treated cows during the synchronized breeding period in the fall following the summer heat stress period.

The objective of this study was to evaluate pregnancy rate (PR) to a timed artificial insemination protocol in the autumn for cows that received a GnRH-Agonist implant

(DESL) between 1 to 4 days postpartum, during the summer heat stress period in comparison to non-treated control cows.

Materials and Methods

The study was conducted on a commercial dairy farm located in Trenton, north central Florida, where 3,500 cows are milked three times a day. Cows were kept in free- stalls barn with sand bedding and self-locking stanchions. Cows were fed three times per day a TMR consisting of 46% forage (corn silage, rye silage, alfalfa and oat hay) and

54% concentrate (hominy, cotton seed, citrus pulp, soy bean meal, corn gluten, brewer condensed and molasses) containing 1.60 Mcal of NEL/Kg and 18% CP.

The experiment was designed as a randomized complete block with the main effect

of treatment. Enrollment consisted of clinically normal (no retained fetal membranes,

milk fever, dystocia or stillborns) postpartum cows with a body condition score (BCS)

equal or greater than 2.75. Primiparous and multiparous cows were blocked before

assignment to treatments.

A total of 300 cows were assigned, from June 25 to August 8, 2001. Due to deaths,

culling and missing cows at specific days of data collection, analysis of variables

affecting pregnancy rates were reduced to 247 cows. Cows were assigned randomly to

treatments twice a week (Mondays and Fridays) between 1 to 4 days postpartum (dpp).

The two groups were:

DESL implant: A total of 120 cows received a 5 mg, sub-cutaneous, non- degradable Deslorelin implant (Peptech Animal Health, North Ryde, Australia) in the right ear. Implants were placed in the outer surface of the ear using an implanter device after intensive cleaning of the area with alcohol gauze. Blood vessels were avoided during implantation.Control (CON): A total of 127 cows did not receive a DESL implant.

All cows were enrolled with a mean of 2 ± 1 dpp. Therefore, day of enrollment will be considered as experimental day 0 (d0). Frequency of cows allocated to both groups remained balanced throughout the weeks of enrollment. All cows, DESL implant and

CON, were injected with PGF2α (25 mg, i.m., Dinoprost Tromethamine; Lutalyse®;

Pharmacia Upjohn, Kalamazoo, MI) at 7 days after enrollment in order to regress any possible corpus luteum induced by the GnRH-Agonist implant.

Implant removal

Implants were removed after an incision was made under the fascia tissue below the implant using a surgical blade. The implant area was cleaned previously with alcohol gauze. Implants were encapsulated totally in fascia tissue; after removal, the area was flushed with alcohol and bleeding minimized by manual pressure with alcohol gauze.

The experiment was designed in order to maintain cows under heat stress conditions, which ends around September 15th (Figure 3. 1 and Figure 3. 2). The CON

cows could express normal follicular activity, and DESL implant cows would undergo

follicular suppression due to the DESL implant. To facilitate management, DESL implant

cows were divided into two groups to remove the implants, on August 28 (n=56) and

September 4 (n=64). Since it takes around 10 days to recover from desensitization (Gong

et al, 1996; Padula and Macmillan, 2002), the treatment was expected to suppress ovarian

follicular development thought out the summer season. Animals that had parturitions

during the summer would be expected to have suppressed follicular development during

late pregnancy prior to calving (Ginther et al, 1996). The duration of treatment was dependent of the day that the cows calved, since the implants were removed at a common date at the end of the summer heat stress season. The implants were in place for a minimum of 28 days and a maximum of 67 days of treatment depending upon date of parturition. Duration of treatment was categorized by number of weeks in which the cows were treated with the DESL implant and will be described as weeks of treatment.

Ultrasound (ovarian activity)

A real time Ultrasound Aloka 500 scanner (Aloka Co., Ltd, Tokyo, Japan) equipped with a 7.5 MHz linear rectal transducer was used to monitor ovarian structures thought out the experimental period. Size, location and number of follicles were recorded onto ovarian maps and categorized into Class 1 (2 – 5 mm), Class 2 (6 – 9 mm) and Class

3 (≥10 mm) follicular classes (Lucy et al, 1992). Presence or absence of CL and its location were recorded. Size of CL was determined by measurement of length and width and analyzis of CL size was performed using the average of both measurements

(Pancarci, 1999)

A sub-sample of cows was ultrasounded at d 7 or dpp10 ± 1 in CON (n= 15) and

DESL implant (n= 16) groups. Ovarian scanning continued in the CON group at d 28 (n=

14; dpp 30 ± 1), d 34 (n= 10; dpp 36 ± 1), d 41 (n= 8; dpp 44 ± 1), d 53 (n= 11; dpp 55 ±

1) and d 60 (n= 10; dpp 63 ± 1). The measurements were performed at 5 to 4 days prior

to the first day of implant removal (August 28) at various intervals postpartum. Cows in

the DESL implant group also were ultrasounded at d28 (n= 15; dpp 30 ± 1), d 33 (n=6;

35 ± 1 dpp), d 43 (n= 9; 45 ± 1 dpp), d 54 (n= 7; 56 ± 1 dpp) and d64 (n= 6; 66 ± 1 dpp).

The measurements were conducted at 3 days prior to implant removal at various intervals

postpartum (Figure 3-1). All cows examined were sub-samples of the experimental cows.

The same cows were not necessarily repeated at all stages of post enrollment.

Implant Implant Removal Removal DES / no DES ( 2 ± 1) st US 1 group 2nd group Ovarian Activity Enrollment 44 d period ( n = 300 ) 17 d 2 d 3 d 7 d

Jun/25 Aug/08 Aug/23-24 Aug/25 Aug/2 Sep/04 US Ovarian Activity

CON DES implant d 7 ( n= 15) d 7 ( n= 16) d 28 ( n= 14) d 28 ( n= 15) CON DES implant d 34 ( n= 10) d 33 ( n= 6) d 41 ( n= 8) d 43 ( n= 9) d 53 ( n= 11) d 56 ( n= 7) d 60 ( n= 10) d 66 ( n= 6)

Figure 3-1. First experimental period consisting of enrollment of DESL implant and CON cows (June 25 to August 8), ultrasound (ovarian activity) for DESL implant and CON sub-sample of cows and DESL implant removal days (August 28 and September 4) in the year of 2001.

Ultrasound (ovarian recovery)

To verify time necessary for resumption of ovarian activity after implant removal, a

sub-sample of DESL implant cows (n=7) was examined. The cows were enrolled on June

25 and had the implants removed on July 23 such that they had the implants for 28 days.

Ovarian structures were recorded on a weekly basis until the presence of a Class 3 follicle

(≥10 mm) was detected and at this time a Pre-synchronization/Ovsynch® protocol (see

78 below) was initiated and cows underwent an ultrasound examination on each injection day.

Sub-samples of cows were examined at 22 days after implant removal (September

19) in order to evaluate ovarian activity. Sub-samples of DESL implant cows were selected randomly within treatment durations of 32 d (n= 5; 56 dpp), 36 d (n= 5 ; 60 dpp),

43 d (n= 6; 67 dpp), 46 d (n= 5; 70 dpp), 53d (n= 5; 77 dpp) and 57 d (n= 5; 81 dpp).

Cows that did not have Class 3 follicles (≥10 mm) at 22 days after implant removal were ultrasounded again 7 days later. On September 26, at 29 days after DESL implant removal, additional sub-samples of cows were selected within 32 d (n= 3; 63 dpp), 36 d

(n= 4; 67 dpp), 41 d (n= 1; 72 dpp); 43d (n= 2; 74 dpp), 46 d (n= 2; 77 dpp), 53 d (n= 6;

84 dpp), 57 d (n= 5; 88 dpp) and 66 d (n= 7; 97 dpp) of DESL implant treatment to evaluate follicular status (Figure 3-2).

Synchronization protocol

At 31 days after DESL implant removal, on September 28 (CON= 54; DESL implant= 56 [77 ± 12 dpp]) and October 5 (CON= 74; DESL implant= 64 [75 ± 13 dpp]) cows started a presynchronization (presynch) followed by a Ovsynch® protocol for their first service as follows: 100 µg, i.m., GnRH (Gonadorelin Diacetate Tetrahydrate,

Cystorelin, Merial Ltd., Athens, GA) on day 0; 25 mg, i.m., PGF2α on day 7; 100 µg, i.m., GnRH on day 17; 25 mg, i.m., PGF2α on day 24; 100 µg, i.m., GnRH on day 26 and time artificial insemination (TAI) 16 - 20h later. Timed artificial insemination was performed on October 25 (CON= 54; DESL implant= 56 [104 ± 12 dpp]) and November

1 (CON= 74; DESL implant= 64 [102 ± 12 dpp]). A presynch GnRH injection followed with PGF2α 7 days later was chosen as opposed to two injections of PGF2α 14 days apart

(Moreira et al., 2001). The intent was to decrease the number of cows in an anovulatory and cystic condition with the use of GnRH in the presynch phase.

The farm staff gave hormone injections at fixed times in the afternoon (~ at 16:00h) and cows were inseminated in the morning (~ at 8:00h). An experienced veterinarian examined frozen semen from two sires and quality was assured as far as sperm concentration, motility and morphology. One sire was used for the majority of cows (only four cows were inseminated to the second sire) for the first synchronized service. Four inseminators participated in the experiment.

Second service cows seen in estrus within 26 days after TAI were artificially inseminated (AI) 12h after onset of estrus (CON=29; DESL implant=49 [122 ± 14 dpp]).

Ovarian recovery – 31 d

Onset Implant Implant Presynch/Ovsynch Removal Removal US 1st group 2nd group Ovarian Recovery 7 d 15 d 7 d 2 d

Sep/04 Aug/2 Sep/19 Sep/26 Sep/28 Oct/05

DES implant/ 22dAR DES implant/ 29dAR d 32 ~ 56 dpp (n= 5) d 32 ~ 63 dpp (n= 3) d 36 ~ 60 dpp (n= 5) d 36 ~ 67 dpp (n= 4) d 43 ~ 67 dpp (n= 6) d 43 ~ 74 dpp (n= 2) d 46 ~ 70 dpp (n= 5) d 46 ~ 77 dpp (n= 2) d 53 ~ 77 dpp (n= 5) d 53 ~ 84 dpp (n= 6) d 57 ~ 81 dpp (n= 5) d 57 ~ 88 dpp (n= 5) d 65 ~ 88 dpp (n= 9)

Figure 3-2. Second experimental period consisting of DESL implant removal days (August 28 and September 4), ultrasound (ovarian recovery) for DESL implant sub-sample of cows at 22 and 29 days after DESL implant removal (September 19 and 26) and onset of presynchronization/Ovsynch® protocol for DESL implant and CON groups of cows in the year of 2001.

Ultrasound (pregnancy diagnosis)

At 28 days after first and second service, a real time Ultrasound scanning unit

(Aloka Co., Ltd, Tokyo, Japan) equipped with a 5.0 MHz linear rectal transducer was used to identify presence of embryonic fluid, appearance of embryo and an embryonic heart beat. Cows diagnosed pregnant at ultrasonography were reexamined by rectal palpation at 46 d after TAI to evaluate pregnancy losses. All pregnant cows after the first service and pregnant or open cows after the second service were returned to the regular herd management.

Resynchronization protocol

Cows diagnosed non-pregnant by ultrasonography at 28 days after TAI and had not received a second service to a spontaneous estrus were resynchronized for a second service. Non-pregnant cows were resynchronized accordingly to stage of the estrous cycle determined by the presence of ovarian structures and uterine tone.

Diestrus. Presence of corpus luteum and flaccid uterus. Quicksynch: 25 mg, PGF2α

® was injected i.m. at the day of a non-pregnancy diagnosis; ECP (Pharmacia Corp; 1mg) was injected i.m. 1 day later, and TAI was conducted 48 hours later (CON=11; DESL implant=5 [131 ± 13 dpp]). Cows presenting estrous behavior within 24 hours after ECP injection were inseminated 12 hours after the onset of estrus.

Proestrus and metestrus. Absence of corpus luteum, presence of ovulatory follicle and uterine tonicity; or absence of dominant follicle, presence of corpus hemorrhagic and uterine edema, respectively. Heatsynch: 100 µg GnRH was injected i.m.

® followed by a PGF2α injected 7 days later; and ECP was injected 24 h after PGF2α and cows were inseminated 48 h later (CON=8; DESL implant=19 [141 ± 13 dpp]). Cows

81 presenting estrus behavior within 24 hours after ECP injection were inseminated 12 hours after the onset of estrus.

Follicular cyst and anovulatory. Absence of corpus luteum, no uterine tonicity, presence of one or multiple follicles > 25 mm; or absence of corpus luteum, no uterine tonicity and presence of only follicles < 10 mm; respectively. Ovsynch®: 100 µg GnRH injected i.m. followed by PGF2α 7 days later; a 100 µg GnRH injection at 48h PGF2α and cows were inseminated 16 - 20 h later (CON=0; DESL implant=9 [136 ± 13 dpp]).

Body condition score (BCS)

Cows were examined for BCS at 2 ± 1 dpp (enrollment day), 44 ± 1 dpp, 77 ± 12 dpp (onset of presynch), 104 ± 12 dpp (day of TAI) and at pregnancy diagnosis at 28 and

46 days after TAI. Scores were given by one veterinarian based on a 1 (thin) to 5 (obese) scale using a quarter point system (Edmonson et al., 1989). Changes in body condition score were obtained by subtracting BCS at 44 dpp from enrollment day BCS, day of TAI

BCS from 44 dpp BCS, and 28 d after TAI from TAI BCS.

Blood samples and hormone assays

Blood samples were scheduled to be collected for all cows at 9 ± 1 dpp (7 days after enrollment, just prior to the PGF2α injection). Moreover, a set of blood samples was collected at 84 ± 12 dpp (just prior to the PGF2α of the presynch) and 94 ± 12 dpp (just prior to the first GnRH injection of the Ovsynch®). With this set of blood samples, cows were considered to be cyclic if they had plasma concentration of progesterone (P4) greater than 1 ng/ml in at least one of the two samples (cyclic) or anovulatory if both samples were below 1 ng/ml (non-cyclic). Another set of blood samples was collected at 104 ± 12 dpp (day of TAI) and at 112 ± 12 dpp (8 days after TAI); and cows were considered to have ovulated if plasma concentration of P4 was below 1 ng/ml at the TAI and greater

than 1 ng/ml 8 days later (ovulated), other combinations of plasma concentrations of P4 were considered failed to ovulate (non-ovulated).

Blood samples were collected from coccygeal vessels by venipuncture into heparinized vacutainer tubes. Samples were cooled immediately in ice until centrifugation for 20 minutes at 2619 x g. After centrifugation, plasma was harvested and stored frozen at –20°C until assayed. Because of missing cows on certain days of blood collection, not all experimental cows had both sets of blood samples. Consequently, not all animals were included in the analyses to evaluate separate effects of cyclic status or ovulation status on PR. However, cows having both samples of each set were included in analyses to evaluate cyclic and ovulation effects on PR within the same model.

Concentrations of plasma progesterone (P4) were determined using a Coat-A-Count

Kit (DPC® Diagnostic Products Incorporation, CA, USA) solid phase 125I radioimmunoassay designed for the quantitative measurement of progesterone in plasma.

(6.0 ng/ml) was also used. Experimental plasma samples (100 µl) were added to coated tubes and 1ml of 125I – labeled progesterone to all tubes. In every 6th sample duplication was performed. Incubation was for 3 hours followed by a discard of supernatant and

83 drying tubes for 15 minutes. Tubes were transferred to and counted in a gamma counter.

An Intra-assay coefficient of variation was calculated from reference samples (luteal phase) and duplicated samples obtained from all assays. Duplicated plasma concentrations of P4 were categorized into high (≥3.0 ng/ml) and medium (≥1.0 and <3.0 ng/ml) samples. High and medium samples had intra-assay coefficients of variation of

8.43 % and 12.3 %, respectively. Inter- and intra-assay coefficient of variation for the luteal phase reference samples were 5.8% and 10.5%, respectively. Sensitivity of the assay was 0.1 ng/ml.

Milk yield

Milk weights were recorded once a month for all the cows. First measurements occurred at different times after calving for the first monthly sample depending upon the day of parturition and the day of the monthly milk test for the herd. Since cows were assigned to the treatments randomly on weekly basis, the first sample postpartum was balanced between treatments. The single measurement of milk production for the month was considered as the average for the month. Data for the first 5 months of lactation were obtained from the Dairy Herd Improvement (DHI) association (Raleigh, NC).

Temperature Humidity Index (THI)

The temperature (oC) and relative humidity (%) data were obtained from the

Florida Automated Weather Network (http://fawn.ifas.ufl.edu/scripts/reportrequest.asp) for the year of 2001. The weather station is located in Alachua, FL, approximatly 40 miles from the farm. Average daily temperature was converted from Celsius to

Fahrenheit using the equation oF = (oC x 9/5) + 32 and THI was calculated as described by West (1994): THI = Temp (oF) - [0.55-(0.55 x RH)] x (Temp -58).

Statistical Analysis

Pregnancy rates were analyzed by logistic regression analyses of SAS (SAS

Institute Inc; 1999; Version 8). The logistic regression stepwise selection procedure was used for all independent variables and possible interactions with pre-determined significance levels set for a variable to be entered with a P ≤ 0.30 and retained with P ≤

0.20 in the model. Mathematical model for pregnancy responses included group, parity, weeks of treatment, cyclic status (cyclic or non-cyclic), ovulation status (ovulated or

NON-ovulated), inseminator, 150 days accumulated milk weight, BCS, changes in BCS and all higher order interactions. Milk weight and BCS data were categorized in quartiles using the Univariate procedure of SAS. Analyses of cyclic and/or ovulated effects, separately or in association, were possible by maintaining in the data set only the animals with complete combinations within sets of blood samples (see above). After identifying variables that were significant, the model was reduced and analyzed in Gen model of

SAS to obtain odds ratios, confidence intervals and probability values.

Proc Lifetest of SAS was used to estimate and compare survival curves for pregnancy rates between CON and DESL implant groups. In this analysis, pregnancy rates included the results from the first synchronized service, and second service (estrus or resynchronization). The Log-Rank test was used to determine differences between groups, because it is a more sensitive test to determine differences that occur along the points in time (Allison, 1995)

Numbers of Class 1, Class 2, Class 3 follicles and CL recorded on ovarian maps during the treatment period were analyzed using the method of least squares ANOVA using the general linear model (GLM) procedure of SAS. The mathematical model included group, day and group by day interaction. Analysis of ovarian recovery for sub-

85 samples of DESL implant cows examined at 22 and 29 days after implant removal were also conducted using the GLM procedure, and the mathematical model included days of treatment and days after removal.

Milk weight data for the first 5 months were analyzed using the repeated measures analyses of the mixed model of SAS (Littell et al., 1999). Five covariance structures were tested in order to determine the best relative goodness of fit based upon penalty criteria

(Bayesian criterion). Variance Components was the least penalized structure and was therefore used for analyses of milk weights. The statistical model consisted of group, parity, weeks of treatment, month and higher order interactions. Cow was considered as the experimental unit nested within group-parity-weeks of treatment.

Analysis for plasma concentrations of P4 collected at 7 days after enrollment was conducted using the method of least squares using the GLM procedure of SAS.

Mathematical model consisted of group, days postpartum at enrollment and the first order interaction.

Temperature humidity index results also were analyzed using the GLM procedure of SAS. Data from June 15th to September 14th was grouped (period 1) and compared to results from September 15th to November 15th (period 2) using the contrast function.

Results

Temperature Humidity Index

Data of THI for the year 2001 is shown in Figure 3-3. The first period consisted of

10 days prior to the initiation of the experiment (June 15) until 10 days after implant removal of the second group (September 14). The second period consisted from

September 15 to 10 days after TAI for the second group. Mean THI values were higher for the first period in comparison to the second (75.81 ± 0.6 > 66 ± 0.74, P<0.01). The

86 first period THI range from 68.1 to 79.7 with a coefficient of variation of 2.9%, and second period range from 51.2 to 75.1 with a coefficient of variation of 10.7%.

100.00 75.81 ± 0.6 66 ± 0.74 80.00

THI 60.00

40.00 nd 1st Period 2 Period 20.00 TA

0.00 1 Jan22 Feb43 64 Mar 85 April106 127 May148 Jun169 190July211 Aug232 253Sep 274 Oct 295 Nov316 337 Dec3 5 Month Month Figure 3-3. Temperature Humidity Index during the year 2001. A comparison between first period (10 days prior to the initiation of the experiment, June 15, until 10 days after DESL implant removal for the second group, September 14) versus second period (September 15 to 10 days after TAI for the second group of cows). THI differed between periods (P<0.01).

Day 7 postpartum P4

Mean plasma concentrations of P4 at 7 days after enrollment did not differ between

CON (n=145; 0.11 ± 0.07 ng/ml) and DESL implant (n=150; 0.23 ± 0.07 ng/ml) groups

(P>0.35), and were not affected by dpp at implant insertion (2 ± 1 dpp; range 1 to 4 dpp;

P> 0.53). No interaction of group by dpp at enrollment was detected. Only two cows had

plasma concentrations of P4 higher than 1ng/ml at d 7; both of these cows were in the

DESL implant group and received the implants at 4 dpp.

Ovarian responses

Ovarian activity. The DESL implant clearly suppressed ovarian activity during all days of treatment (Table 3-1). Mean numbers of Class 2, Class 3 and CL were diminished in the DESL implant group in comparison to the CON (P< 0.01). Mean numbers of Class

1 follicles were increased in the DESL implant group during the time of treatment

(P<0.01). Although Class 1 follicles were defined as 2-5 mm size, only follicles of 2-3 mm were detected in DESL implant group during the period of treatment.

Ovarian recovery. A sub-sample of cows (n= 7) had the DESL implants removed after 28 days of treatment. Ultrasonic examination of the ovaries conducted on a weekly basis determined the appearance of Class 3 follicles at different days after removal (dAR) of the DESL implant. Class 3 follicles were detected at 14 dAR (4/7; 57.14%), 28 dAR

(2/3; 66.6%), and at 53 dAR (1/1; 100%). Detection of Class 3 follicles occurred at a mean 23.57 ± 14.55 dAR. The presynch GnRH injection, given at detection of a Class 3 follicle, induced a CL at PGF2α injection in 3/6 (50%) of the cows (n=6, because one cow was not injected). Overall 2/6 (33.33%) cows responded to the presynch with induced ovulations and subsequent synchronized ovulations to the Ovsynch®, based on the presence of CL determine by ultrasound examination of the ovaries.

Cows in the DESL implant group (n=30) treated for different periods of time were ultrasounded at 22 dAR of DESL implant (Table 3-2). Presence of Class 3 follicles was detected in 17/30 (56.6%) cows. Mean number of Class 3 follicles was significantly increased after implant removal for cows treated for the shorter intervals of 32 and 36 days (P<0.04).

Table 3-1. Number of Class 1*, Class 2*, Class 3* follicle and corpora lutea* detected by ultrasound for different periods of treatment in the DESL implant and CON group of cows. Days 1 Class 1** Class 2*** Class 3 CL CON DESL implant CON DESL implant CON DESL implant CON DESL implant

7 15.7 ± 1.1 21. 3 ± 0.8 2.7 ± 0.3 0.4 ± 0.3 1.5 ± 0.2 0 0 0

28 14.3 ± 0.9 22.1 ± 0.9 2.9 ± 0.2 0 1.8 ± 0.1 0 0.7 ± 0.1 0

33 8.8 ± 1.5 24 ± 2.0 2.5 ± 0.1 0 1.6 ± 0.2 0 0.9 ± 0.1 0

43 7.1 ± 1.7 20 ± 1.6 1.1 ± 0.4 0 2.0 ± 0.3 0 0.9 ± 0.2 0

54 10.8 ± 1.4 17.3 ± 1.8 1.81 ± 0.4 0.1 ± 0.5 1.4 ± 0.2 0 0.8 ± 0.1 0

88 64 10.6 ± 1.4 21.5 ± 2.0 1.9 ± 0.4 0 2.3 ± 0.2 0 0.9 ± 0.1 0

Overall 11.2 ± 0.6 21.0 ± 0.7 1.9 ± 0.1 0.1 ± 0.2 1.8 ± 0.1 0 0.7 ± 0.1 0 1Days in which DESL implant cows had implant and contemporaneous cows for the CON group. * Differences between groups for each follicle class and CL numbers (P<0.01) ** Group by Day interaction for Class 1 follicles (P<0.01) *** Day effect for Class 2 follicles (P<0.04)

Table 3-2. Mean number of Class 1, Class 2, Class 3 and CL at 22 days after removal of DESL implant for cows implanted with DESL for different lengths of time. Days 1 Class 1 Class 2 Class 3 CL (%) 2 (%) (%) (%)

32 + 36 22.9 ± 2.3 (100) 0.6 ± 0.4 (40) 1.1 ± 0.2 (80) a 0.2 ± 0.1 (20) (n= 10)

43 + 46 18.9 ± 2.4 (100) 1.7 ± 0.4 (60) 0.6 ± 0.2 (50) b 0.3 ± 0.1 (20) (n= 10)

53 + 57 23.4 ± 2.3 (100) 0.6 ± 0.4 (40) 0.4 ± 0.2 (40) b 0 (n= 10)

Overall 21.8 ± 2.4 (100) 0.9 ± 0.4 (46.6) 0.7 ± 0.2 (56.6) 0.2 ± 0.2 (13) (n=30) 1 Days of DESL implant treatment. 2 Percentage of cows detected with follicles or CL within days. a b Differences between days of mean number class 3 follicles; P < 0.04.

Among the 17 cows that had Class 3 follicles, one cow had a CL (d32) and an additional three cows without Class 3 follicles were detected with a CL. Therefore, a total of 20/30 (66.6%) cows were considered recovered from the pituitary desensitization at 22 dAR. No significant differences among days of treatment were observed for numbers of

Class 1 and Class 2 follicles and number of CL.

Although these 20 cows were considered recovered from pituitary desensitization, only 12/20 (60.0%) responded to the presynch protocol initiated at 31 dAR, and were classified as cyclic based upon plasma P4 responses. Overall 12/30 (40.0%) of cows recovered from desensitization of the DESL implant were cyclic during the presynch period.

Another sub-sample of DESL implant cows (n=30) treated for different periods of time were ultrasounded at 29dAR of DESL implant (Table 3-3). Presence of a Class 3 follicle was detected in 16/30 (53.3%) cows. Mean number of follicles did not differ

90 among the days of treatment (P>0.10). Presence of CL was detected in four of the 16 cows with Class 3 follicles (2 cows d 32, 1 cow d 53, 1 cow d 66) in addition to 3 more cows with a CL without Class 3 follicles. Thus 23.3% of cows (7/30) had a CL. Presence of CL was dependent on the duration of treatment. Cows treated for 32 and 36 days had higher mean number of CL, (P<0.01), indicating that these animals were in a more advanced stage of follicular development and were more able to have a spontaneous ovulation by 29 dAR of DESL implant (Table 3-3).

Table 3-3. Mean number of Class 1, Class 2, Class 3 and CL at 29 days after removal of DESL implant for cows implanted with DESL for different lengths of time. Days 1 Class 1 Class 2 Class 3 CL (%) 2 (%) (%) (%)

32 + 36 18.3 ± 2.5 (100) 0.3 ± 0.3 (43) 0.8 ± 0.4 (57) 0.7 ± 0.2 (71) a (n= 7)

41 + 43 + 46 13.5 ± 3.1 (100) 0 0 0 b (n= 5)

53 + 57 15.8 ± 1.9 (100) 0.8 ± 0.2 (45) 0.8 ± 0.3 (55) 0.2 ± 0.1 (9) b (n= 11)

66 12.6 ± 2.3 (100) 0.7 ± 0.3 (57) 1.71 ± 0.4 (86) 0.14 ± 0.2 (14) b (n= 7)

Overall 15.2 ± 2.4 (100) 0.6 ± 0.4 (36.3) 0.9 ± 0.2 (53.3) 0.3 ± 0.1 (23.3) (n= 30) 1 Days of DESL implant treatment. 2 Percentage of cows detected with follicles or CL within days. a b Differences between days of mean number of CL; P < 0.01.

Number of cows considered recovered was 19/30 (63.33%), which was similar to the first group of cows described in Table 3.2 at 22 dAR of DESL implant. Among the 19 cows recovered, 12 (63.15%) were classified as being cyclic based upon plasma P4 responses. Overall 12/30 cows (40.0%) recovered from desensitization of the DESL implant were cycling during the presynch period.

Both sub-samples of cows (i.e., 22 and 29 dAR of DESL implant) were pooled to examine cyclic and ovulated status (Table 3-4). All cows detected with a CL responded to the presynch/Ovsynch® protocol. However a lower frequency of response occurred in cows detected with Class 3 follicles and a minimal response in cows with follicles <10 mm (Table 3-4).

If cows were classified as cyclic among the three categories of ovarian structures, then ovulation followed in response to the last GnRH injection of the Ovsynch® protocol

(Table 3-4).

Table 3-4. Cyclic and ovulation responses based on plasma concentrations of P4, for DESL implant cows characterized by ovarian structure at ultrasound on days 22 and 29 after removal of the DESL implant. Ovarian Cyclic ovulated Structure % (n=) % (n=)

CL 1 100 (11/11) a 100 (11/11) a

Class 3 2 57 (16/28) b 57 (16/28) b

Others 3 14.3 (3/21) c 14.3 (3/21) c

Overall 50 (30/60) 50 (30/60) 1 Cows detected with CL 2 Cows detected with only class 3 follicles 3 Cows detected with follicles < 10 mm ab P<0.01; bc P<0.01. Differences determined by chi-square analysis

Experimental responses indicated that the effects of the DESL implant remained for periods longer than 29 after the removal of the implant. Fifty percent (30/60) of the sample population did not respond to the presynch protocol. Presence of Class 3 follicles was not a valuable predictor of pituitary GnRH responsiveness since 43% of cows with a

Class 3 follicle failed to ovulate to the Ovsynch program following a presynch period

(Table 3-4). Furthermore, the longer the implant was in place, the greater was the number of cows still desensitized to the DESL implant (Table 3-2 and 3-3).

Frequencies of Cyclic and Ovulation Status for All Cows

Cyclic status. Stepwise procedure of logistic regression indicated that group

(P<0.01) and weeks of treatment by group interaction (P<0.08) affected cyclic status.

Cows in the DESL implant group had a lower chance (OR=0.05, CI=0.019 – 0.155,

P<0.01) to be cyclic [58/111 (52.2%)] in comparison to CON group [104/111 (93.7%)],

Table 3-5.

Cyclic status did not change along the weeks of treatment for the CON group.

However, there was a tendency for a decreased percentage of cyclic cows as the duration of the treatment increased for the DESL implant group (P<0.08; Table 3-6).

Cyclic status was not affected by 150 d accumulated milk weight expressed in quartiles, BCS, changes in BCS and higher order interactions.

Table 3.5. Frequencies of CON and DESL implant cows cycling during the presynchronization period and having a synchronized ovulation following the Ovsynch® protocols. Status CON DESL implant Cyclic Non-cyclic Overall Cyclic Non-cyclic Overall % (n=) 1 % (n=) % (n=) % (n=) 1 % (n=)

Ovulated 84.7 (94) e 5.4 (6) 90 (100) c 44.1 (49) f 27.0 (30) 71.1 (79) d

Non-ovulated 9.0 (10) 1 (1) 10 (11) 8.1 (9) 20.7 (23) 28.9 (32)

Overall 93.7 (104) a 6.3 (7) 111 52.2 (58) b 47.7 (53) 111

1 Percentage of cows within CON and DESL implant groups that were cyclic/non-cyclic and ovulating/non-ovulating. 93 a b OR=0.05, CI=0.019 – 0.155, P<0.01 (within row, group by cyclic status). c d OR=0.47, CI=0.205 – 1.116, P=0.08 (within row, group by ovulated status). e f OR=0.142, CI=0.075 – 0.270, P=0.01 (within row, group by cyclic by ovulated status).

Table 3-6. Percentage of cows cycling during the presynchronization period as related to duration of DESL implant treatment for DESL implant cows and contemporaneous CON. Weeks of CON a DESL implant b Treatment % (n=) % (n=)

4 + 5 91.1% (41/45) 57.2% (34/59)

6 +7 96.8% (30/31) 62.5% (10/16)

8 93.8% (15/16) 40.7% (11/27)

9 + 10 + 11 94.7% (18/19) 33.3% (3/9)

Overall 93.69% (104/111) 52.3% (58/111) ab (OR=0.05, CI=0.019 – 0.155, P<0.01); the GENMODEL procedure of SAS is modeling the probability of DESL implant to be cyclic in comparison to CON.

Ovulated status. When using the stepwise procedure of logistic regression to model the probability of cows ovulating to the Ovsynch® protocol as a dependent variable, cyclic status was the only significant independent variable (P<0.01) influencing ovulated status. A tendency for a group effect (P<0.08) was detected. It is intuitive that when cyclic status is left in the model the effect of group is no longer significant, because the percentage of cyclic cows was reduced due to the DESL implant. The non-ovulated condition due to the DESL implant treatment indicates that the cows were still suffering the effects of pituitary desensitization and were unable to respond to the Ovsynch® protocol with an induced ovulation. If the analysis is performed only with the cows that are cyclic the frequency of ovulated cows does not differ between the CON [90.38 %

(94/104)] and the DESL implant [84.48 % (49/58)] group, (OR=0.57, CI=0.221 – 1.519,

P>0.26).

Cyclic cows had a higher chance to ovulate [143/162 (88.3%)] in comparison with cows that were non-cyclic [36/60 (60.0%)], (OR=3.54, CI=1.60–7.82, P<0.01).

Cows in the DESL implant [79/111 (71.2%)] group tended to have a lower ovulation rate in comparison to cows in the CON [100/111 (90.1%)] group, (OR=0.47,

CI=0.205 – 1.116, P=0.08).

Ovulation was not affected by weeks of treatment, 150 d accumulated milk weight expressed in quartiles, BCS, changes in BCS and higher interactions.

Cyclic and ovulated status. Stepwise procedure of logistic regression modeled the probability of cows cycling during the presynch period and ovulating to the Ovsynch® protocol as a dependent variable, group was the only significant independent variable

(P<0.01). Cows in the DESL implant [49/111 (44.1%)] group had a lower frequency of cyclic and ovulated in comparison to cows in the CON [94/111 (84.6%)] group,

(OR=0.142, CI=0.075 – 0.270, P<0.01), Table 3-5.

First service reproductive responses

Analysis of pregnancy rates for the TAI included only the cows that received all injections for the presynch/Ovsynch® protocol and were ultrasounded for pregnancy diagnosis. Average day of pregnancy diagnosis was 28 ± 1 days after TAI. One cow was not examined by ultrasonography for pregnancy, but was found pregnant when palpated at 46 days after TAI. This cow was then considered pregnant at 28 days after TAI.

Analysis of pregnancy rates to the first synchronized service using stepwise logistic regression detected ovulation status and a interaction of parity by group as being significant (P<0.01). The parity by group interaction was due to the greater reduction in

PR from primiparous to multiparous cows in the DESL implant group than between primiparous and multiparous cow of the CON group (Table 3-7). The interaction was significant even though the sample size of primiparous cows was lower than the multiparous cows.

Table 3-7. Day 28 pregnancy rates to TAI for primiparous and multiparous cows within the DESL implant and CON groups. Parity CON DESL implant Overall % (n=) % (n=) % (n=)

Primiparous 62.1 (18/29) 48.0 (12/25) 55.5 (30/54)

Multiparous 51.5 (51/99) 22.1 (21/95) 36.8 (71/193)

Overall 53.5 (69/128) 27.5 (33/120) 41.1 (102/248) P< 0.01 for Group by Parity interaction.

An additional analysis was done in the stepwise procedure of logistic regression, which included all previous variables and higher order interactions but excluding parity.

This was done since parity was a blocking factor in the experimental design. After removing parity from the analysis, ovulation following to Ovsynch® continued to be significant as well as the main effect of treatment (CON [53.3%] > DESL implant

[27.5%]; P<0.01).

Pregnancy rates for the first synchronized service was not affected by cyclic status,

AI technician, weeks of treatment, BCS, accumulated 150 d milk weight expressed in quartile and higher order interactions. Further analyses were then conducted using Gen

Model procedure of SAS with ovulation status and group in the mathematical model in order to obtain odds ratio, confidence intervals and probability values. Pregnancy rates were higher for cows that ovulated [51.67% (93/180)] compared to cows that did not ovulate [2.27% (1/44)], (OR=36.36, CI=4.81 - 274.94, P=0.001). Cows in CON group had a higher pregnancy rate [53.54 % (69/128)] in comparison to the DESL implant group [27.5 % (33/120)], (OR=2.36, CI=1.30 – 4.28, P=0.006).

First service reproductive responses accordingly to

Cyclic status. When pregnancy rates were analyzed only for the animals that were classified as cyclic, the stepwise procedure of logistic regression detected ovulation status and group as being significant (P<0.01). Among the cows that were classified as cyclic, as expected the animals that ovulated had a higher pregnancy rate [52.45% (75/143)] than the animals that failed to ovulate [5.26% (1/19)], (OR=20.1191, CI=2.5972 – 155.85,

P<0.001). When comparing the two treatment groups in cyclic cows, a higher pregnancy rate was detected for the CON group [53.72% (65/121)] than the DESL implant [30.65%

(19/62)], (OR=2.462, CI=1.2240 – 4.9520, P<0.01) for cows classified as cyclic, regardless of ovulation status. It is important to recognize that in the analysis for only cyclic cows, ovulation rate did not differ between CON [90.38 % (94/104)] and the

DESL implant [84.48 % (49/58), P>0.26]. The DESL implant cows that were cycling still had a lower pregnancy rates following TAI. First service pregnancy rates for cyclic cows was not affected by 150 d accumulated milk weight expressed in quartiles, weeks of treatment, BCS, changes in BCS and higher interactions

Ovulated cows. Pregnancy rates were also analyzed only for the cows that ovulated, regardless of cyclic status. The stepwise logistic regression procedure detected an interaction of group and accumulated 150 d milk weight (P<0.01; Table 3-8)

Table 3-8. Pregnancy rates for CON and DESL implant cows that ovulated in response to Ovsynch® as influenced by quartile of accumulated milk yield. Quartile CON DESL implant Milk Yield % (n=) % (n=)

1 (Lowest) 70.6 (12/17) 56.5 (13/23) a

2 62.0 (13/21) 35.3 (6/17) b

3 64.3 (18/28) 35.7 (5/14) b

4 (Highest) 60.0 (15/25) 26.3 (5/19) b

Overall 63.7 (58/91) 39.7 (29/73) a b OR= 3.9247, CI=1.2251 – 12.5731, P<0.02; the GENMODEL procedure of SAS is modeling the probability of pregnancy only for DESL implant that ovulated for quartile 1 versus 2,3 and 4.

Analyses of the interaction using the GENMODEL procedure of SAS showed a significant interaction between groups and quartiles of 150 d accumulated milk weights.

Analyses for only CON cows and comparing quartile 1 [70.58% (12/17)] versus 2,3,4

[62.16% (46/74)] indicated no statistical difference in pregnancy rates, (OR= 1.6911,

CI=0.4529 – 6.3142, P<0.43); however, analyses of only the DESL implant cows and comparing quartile 1 [56.52% (13/23)] versus 2,3,4 [32% (16/50)] indicated that pregnancy rates were suppressed in the animals with higher milk production, (OR=

3.9247, CI=1.2251 – 12.5731, P<0.02; Table 3-8).

Cyclic and ovulated. The stepwise procedure of log regression for only cows that were cyclic and that ovulated detected a treatment by quartile milk weight interaction

(P<0.01; Table 3-9). Analyses of the interaction using the Genmodel procedure of SAS showed a significant interaction between groups and quartiles of 150 d accumulated milk weights. Analyses of only the CON group and comparing quartile 1 [70.59% (12/17)] versus 2, 3, 4 [60.0% (42/70)] revealed no statistical difference in pregnancy rates (OR=

1.5406, CI=0.4601 – 5.1583, P<0.48); however, analyses of only DESL implant cows and comparing quartile 1 [60% (9/15)] versus 2, 3, 4 [29.03% (9/31)] indicated pregnancy rates were suppressed in cows with higher milk production (OR= 3.6734,

CI=0.9467 – 14.2539, P<0.06; Table 3-9).

Table 3-9. Pregnancy rates for CON and DESL implant cows that cycled and ovulated in response to presynch and Ovsynch® as influenced by quartile of accumulated milk yield. Quartile CON DESL implant Milk Yield % (n=) % (n=)

1 (Lowest) 70.6 (12/17) 60.0 (9/15) a

2 62.0 (13/21) 36.7 (4/11) b

3 65.4 (16/26) 37.5 (3/8) b

4 (Highest) 56.32 (13/23) 16.7 (2/12) b

Overall 62.1 (54/87) 39.1 (18/46) a b OR= 3.6734, CI= 0.9467 – 14.2539, P< 0.06; the GENMODEL procedure of SAS is modeling the probability of pregnancy only for DESL implant that was cyclic and ovulated for quartile 1 versus 2,3 and 4.

Day 46 pregnancy rates

Pregnancy rates measured at 46 days after TAI by means of rectal palpation were influenced by treatment. Cows in the CON group continued to have a higher pregnancy rates [47.24% (60/127)] in comparison to the DESL implant [23.33% (28/120)],

(OR=2.12, CI=1.159 – 3.903, P<0.01).

Pregnancy losses

Pregnancy losses from day 28 to day 46 did not differ between the two groups. The

CON cow pregnancy loss was 13.43% (9/69) and pregnancy loss for the DESL implant group was 15.15% (5/33); (OR=1.09, CI=0.333 – 3.575, P<0.88). Pregnancy loss was not

100 affected by weeks of treatment, cyclic status, inseminator, quartile 150 d accumulated milk weight, BCS and changes in BCS.

Second service reproductive responses

AI at estrus. An increased number of DESL implant cows (40.8% [49/120]) in comparison to the CON cows (22.6% [29/128]) were in estrus within 26 days after TAI

(χ2 =9.49; P< 0.01). Pregnancy rates for cows inseminated within 26 days after TAI did not differ between the DESL implant [32.65% (16/49)] and CON [27.6% (8/29)] groups,

(OR=2.387, CI=0.7051 – 8.082, P<0.16). Therefore, from the total population of DESL implant cows, recovery from pituitary desensitization was still occurring and fertility to their detected estrus was the same as the CON group detected in estrus following TAI.

However, the conception rate to these detected estrus period was less than the conception rate to the timed inseminated CON group.

Two sires were used for cows inseminated in estrus, and they differed in pregnancy rate. Sire number one had a higher fertility [38.46% (15/39)] than number two [14.71%

(5/34)], (OR=4.22, CI=1.290 – 13.804, P>0.01). Pregnancy rates for cows inseminated within 26 days after TAI were not affected by cyclic status, ovulation status, AI technician, weeks of treatment, milk weight quartiles and higher interactions.

Resynch. Overall pregnancy rates for resynchronized DESL implant and CON cows did not differ (P<0.13; Table 3-10).

101

Table 3-10. Pregnancy rates for cows in CON and DESL implant group resynchronized after d 28 pregnancy diagnosis. Group Quicksynch Heatsynch Ovsynch Overall % (n=) % (n=) % (n=) % (n=)

CON a 36.4 (4/11) 12.5 (1/8) - 26.3 (5/19)

DESL implant b 40.0 (2/5) 42.1 (8/19) 44.4 (4/9) 42.4 (14/33)

Overall 37.5 (6/16) 33.3 (9/27) 44.4 (4/9) 34.6 (18/52) a b Odds ratio: 2.76; Confidence interval: 0.75-10.15; P<0.13.

Survival Analyses for First and Second Service Reproductive Responses

Survival curve for proportion of non-pregnant cows during a 39 days period is depicted in Figure 3-4. Day 0 represents the day of TAI, the period from day 8 to day 26 represent the cows that were artificially inseminated when seen in estrus (AI) and the period from day 27 to day 38 are representative of cows artificially inseminated after resynchronization. Non-pregnant cows were censored to day 39.

There was an immediate decrease in the proportion of non-pregnant cows associated with the synchronized TAI with an increased number of CON cows becoming pregnant in comparison to the DESL implant group. Survival curve detected differences along the points in time for the 39 day period in CON [63.28% (81/128)] and DESL implant [52.5% (63/120); P< 0.01] groups (Figure 3-4). The survival curve indicates that the difference in proportion of non-pregnant cows occur at the first TAI service and that fertility at detected estrus and resynch services was the same between groups and that the initial difference in pregnancy rate was sustained throughout the period.

102

DESL

w Implant AI at Estrus Resynch Co nt a Control egn r n-P No f % o

Interval after TAI (days) P< Figure 3-4. Survival curve for percentage of non-pregnant cow along the points in time for the 39 day period after TAI (d0 presynch/Ovsynch® TAI; d 8 to d 26 estrus AI; d 27 to d 38 resynch), non-pregnant cows were censored to day 39 (P< 0.01).

Milk weights

Average milk yield during the 5-month postpartum period was decreased in the

DESL implant group compared to the CON group. Mean milk production in the CON group (n=127) was 34.05 ± 0.94 Kg and for the DESL implant group (n=120) 31.11 ±

0.96 Kg (P<0.03; Figure 3-5). This represents a 10% decrease in milk yield due to the insertion of the DESL implant at parturition.

Milk yield was consistently lower in primiparous cows in comparison to multiparous cows regardless of group. Mean milk production for primiparous was 29.40

± 1.22 Kg and multiparous 35.77 ± 0.58 Kg (P< 0.01). The DESL implant was associated with a decrease in milk production for both primiparous and multiparous cows (Figure 3-

5).

103

45.00

) 40.00 g K ( 35.00 d l e i 30.00 Y k

l 25.00

Mi 20.00 15.00 12345 Months Post-Partum Control Primiparous Control Multiparous Implant Primiparous Implant Multiparous

Figure 3-5. Average milk yield during the 5-month postpartum period for CON and DESL implant groups for both primiparous and multiparous cows (Group, P<0.03; Parity, P<0.01).

Discussion

Plasma concentration of P4 at 7 days after enrolment did not differ between the two groups. Enrolment at 2 dpp was a time that ovulation was not induced by the GnRH-

Agonist, which is in agreement with Fernandes et al. (1978) who reported that plasma concentrations of LH did not increase after GnRH injection on 3 dpp. The first 5 dpp is characterized by low stores of pituitary LH (Saiduddin et al., 1968). Thus, ovulation induced by an exogenous GnRH would not be possible. The two cows that had plasma concentration of P4 ≥ 1 ng/ml were animals that received the implant at 4 dpp, which could be a time that the first follicular emergence had occurred for these animals.

Follicular emergence occurred in a range of 2 to 7 days postpartum in dairy heifers

(Ginther et al., 1996). No rise in plasma concentration of P4 was found in CON cows at 7 days after enrollment.

The objective of maintaining the DESL implant group to the GnRH-Agonist implant during the hot season was achieved. The weather results indicated that cows

104 enrolled into the experiment were experiencing heat stress temperatures with mean THI above 72 (Johnson, 1976; West et al, 2003). The first period from 10 days before the initiation of the study (June 15) to 10 days after the second DESL group had the implant removed (September 14) reflected a THI average of approximately 76. During the first period of the heat stressed environment, DESL implant cows had follicular activity suppressed while in the CON group follicular recruitment, deviation, dominance and ovulation occurred as characterized by the increased numbers of Class 2, Class 3 follicles and CL. The DESL implant group had follicular activity suppressed during the entire period of DESL implant treatment coincident with the summer heat stress season (Figure

3-3 and Table 3-1). During the time of follicular suppression, an accumulation of Class 1 follicles occurred. Despite the definition of Class 1 follicle used (2-5 mm), follicles larger than 3 mm were not detected in the ovaries of the sub-sample of DESL implant cows, which are the sizes known to be sensitive to the effects of heat stress.

During the second period, from September 15 (10 days after removal of DESL implants) to November 15 (10 days after second group TAI), THI decreased to a mean value of approximately 66, which is a level no longer typical of heat stress (Figure 3-3).

During the second period a higher variation in THI was observed in contrast to the steady high THI of the first period. The higher variation during the second period possibly did not result in harmful heat stress condition to the cows since TAI pregnancy rates of CON cows were of comparable values of the cool season. During the second period, the DESL implant cows were expected to return to normal follicular activity for TAI.

Reestablishment of follicular growth after chronic DESL treatment had a wide variation in time for the first sub-sample of DESL implant cows treated for 28 days (n=

105

7). Class 3 follicles were detected at a mean of 23.6 ± 14 dAR (range 14 to 53 dAR).

Padula and Macmillan (2002) described that 50% of ovulations occurred within 30 dpp in cows treated with DESL for 28 days during the postpartum period.

In the sub-sample of DESL implant cows, presence of Class 3 follicles and/or a CL, as detected by ultrasound was 66.6% (20/30) at 22 dAR and 63% (19/30) at 29 dAR. At

22 dAR a greater number of Class 3 follicles were detected in animals treated for 32 and

36 days compared to retention of DESL implants for more than 41 days. When ovaries where evaluated at 29 dAR (i.e., an additional 7 days), cows treated for 32 and 36 days appeared to be in an advanced stage of follicular activity characterized by the increased number of CL compared to retention of DESL implants for more than 41 days. Amounts of GnRHr mRNA and concentrations of GnRHr in ewes treated continuously with GnRH decreased by 48% and 69%, respectively (Turzillo et al., 1998). Such suppression also occurred in cows infused continuously with GnRH (Viscarra et al., 1997). Moreover, number of follicles and the percentage of atretic follicles of < 2 mm were decreased and increased, respectively in ewes hypophysectomized for a long period (i.e. 70 days) in comparison to either short term hypophysectomy (i.e., 4 days) or control ewes (Dufour et al., 1979). Also, the mitotic index of granulosa cells was reduced in the long-term treated ewes compared to short term and controls ewes. Perhaps, long-term GnRH-Agonist treatment, via implant, induces a greater reduction in GnRHr expression and GnRH protein in the pituitary associated with a more severe suppression of small antral follicles

(i.e., < 2 mm) resulting in longer periods for resurgence of follicular growth beyond the 2 mm follicle diameter.

106

In DESL implant cows, presence of Class 3 follicles (≥ 10 mm), at 22 or 29 dAR was not a good indicator of complete reestablishment of pituitary responsiveness to exogenous GnRH. Cyclic status, based on plasma concentrations of P4, was reestablished in 57% of cows with Class 3 follicles detected at days 9 (22 dAR) and 2 (29 dAR) prior to the presynch protocol. In contrast, all cows detected with a CL were classified as cyclic and had synchronized ovulations following TAI. If cows with CL or Class 3 follicles ovulated during the presynch period then these same cows had a synchronized ovulation following the Ovsynch® protocol. Presence of Class 3 follicles may result from a gradual increase in FSH and LH secretion associated with increased GnRHr gene expression. However, when cows are detected with Class 3 follicles, the concentration of

GnRHr or post receptor signal transduction systems may not be fully recovered. Thus, a

GnRH induced surge of LH does not occur in approximately half of the DESL implanted cows. This could explain the high frequency of cows that are detected in estrus but failed to ovulate after a 28 days treatment with Deslorelin (Padula and Macmillan, 2002).

Based on plasma concentration of P4, the frequency of cyclic cows for the DESL implant group ultrasounded before initiation of the presynch/Ovsynch® protocol was similar to the overall frequency of cyclic cows used in the study. Approximately half of total population of DESL implant cows (47.7%, [47/111]) was in non-cyclic condition prior to the Ovsynch® protocol. The 31 day interval from the removal of the implant to presynchronization was not enough time for reestablishment of responsiveness to exogenous GnRH, with a tendency for exacerbation of pituitary desensitization in cows treated for longer periods of time. In contrast, a high frequency of CON cows was cyclic.

107

The average 76 dpp at the initiation of the presynch is a time postpartum well beyond the nadir in negative energy balance.

The overall frequency of cows having a synchronized ovulation following the

Ovsynch® protocol was decreased in the DESL implant group. Once the DESL implant cows were cycling during the presynchronization period, the frequency of ovulation after

Ovsynch® did not differ between DESL implant and CON groups.

Pregnancy rates to the first synchronized service were reduced in the DESL implant group and were related to prior cycle status and subsequent ovulatory response to the

Ovsynch® protocol. Cows that failed to cycle during the period of presyncronization and were non-ovulatory to Ovsynch® had reduced fertility in both groups, but the proportion of these cows was greater in the DESL implant. Pregnancy rates to TAI were reduced in the DESL implant when only cyclic cows were analyzed. Since a higher proportion of cyclic cows ovulated to the Ovsynch® protocol in CON (90.4% [94/104]) and DESL implant (84.4% [49/58]) groups, then the reduction in fertility of the DESL implant group is perhaps due to quality of the follicle ovulated. Cows that were cyclic in the DESL implant had progesterone exposure prior to the initiation of the Ovsynch protocol similar to the CON cows; thus, the differences in fertility may not be related necessarily to P4 exposure. The difference in fertility is not due to DESL implant desensitization since the response is restricted to only cyclic cows that ovulated to the Ovsynch® protocol.

Therefore, the quality of the ovulatory follicle and its oocyte may be responsible and still subjected to heat stress damage.

It is important to recognize that number of follicles emerging that reached 5 mm and ≥ 6 mm in a follicular wave as well as the number of follicular waves was reduced in

108 the last month of pregnancy in heifers (Ginther et al., 1996). This follicular pattern was associated with a reduction of number of defined surges of FSH. The diminished follicular activity and FSH secretion near the end of pregnancy is due to the negative feedback of placental estrogens at this time.

A more severe suppression of gonadotrophin secretion is achieved when cows are treated chronically with GnRH-Agonist. Plasma concentrations of FSH and LH were suppressed to low basal levels after 28 days of GnRH-Agonist treatment and it was associated with suppression in follicular growth that was limited at 2 to 3 mm follicles

(Gong et al, 1996). Likewise, the DESL implant group, during chronic treatment with a

GnRH-Agonist, probably led to a total suppression of FSH based on follicular growth to

≤ 3 mm in size. In contrast, the cows in the CON group were able to have a higher rate of follicle turn over in comparison to the DESL implant group throughout the summer time experimental period.

After implants were removed, a 58 days interval occurred during the fall season until TAI. This interval corresponds to 31 days between removal of implants and presynchronization and an additional 27 days to complete the presynchronization/

Ovsynch® protocol. Considering that one follicular wave takes approximately 7 to 10 days based on three and two follicular wave estrous cycles, respectively (Savio et al,

1993; Diaz et al, 1998), we estimated length of a follicular wave to be 8.5 days.

Therefore, the CON cows were able to have four follicular waves during the 31d period of recovery, a fifth wave induced by the first GnRH of the presynch, a sixth wave during the intervening period just prior to implementing the Ovsynch® protocol and a final 7th

109 follicular wave that is induced to ovulate and release an oocyte for subsequent fertilization to a TAI (Figure 3-6).

THI = 66 GnRH Implant Removal

Wave 1 Wave 2 Wave 3 Wave 4

Sep 04 8.5d 17d 25d 31d Pre-synch

GnRH GnRH PGF2α PGF GnRH 2α TAI

Wave 5 Wave 6 Wave7

31d 38d 48d 55d 57d Pre-synch Ovsynch

Figure 3-6. Time line for possible number of follicular waves in CON cows during the cool season.

When comparing the number of estimated follicular waves in the CON group with

DESL implant cows classified as cyclic and that ovulated at TAI (optimal situation for

DESL implant cows), the CON cows probably had additional 2 to 3 follicular waves during the period prior to the initiation of the presynch protocol. This is based on the observation that DESL implant cows that were cycling appear to initiate follicular development at approximated 22 days after removal of the DESL implants (Figure 3-7).

110

THI = 66 GnRH Implant Removal

Down regulated followed by follicular growth Wave 1

Sep 04 23d 31d Pre-synch

GnRH GnRH PGF2α PGF2α GnRH TAI

Wave 2 Wave 3 Wave 4

31d 38d 48d 55d 57d Pre-synch Ovsynch

Figure 3-7. Time line for possible number of follicular waves in DESL cows during the cool season.

Accordingly, CON cows had the seventh follicular wave as the candidate dominant follicle containing the oocyte involved in fertilization for first service. Seven follicular waves is one more wave than the six waves necessary to have 40% increase in number of eight-cell embryo and a 10% increment in blastocyst development after summer heat stress (Roth et al., 2001). In contrast, cows in the DESL implant group, classified as cyclic and that ovulated to the Ovsynch® possibly had an oocyte from the candidate dominant follicle belonging to the fourth follicular wave that was still impaired from the hot season and was not optimal. This estimation for the DESL implant group of cows is a liberal interpretation, keeping in mind that 43.7% of DESL implant cows were not yet cyclic at the time the Ovsynch® protocol was initiated. For those DESL implant cows subsequently cycling after TAI, the recruited follicle may have been previously impaired due to heat stress when they where less than 3 mm in size. Each follicular wave

111 comprises in average of about 24 small (3 to 4 mm) antral follicles (Ginther et al, 1996).

Seven follicular waves result in the emergence and removal of approximately 170 follicles. Bovine ovaries contain approximately 250 follicles of 0.9 to 1.15 mm in diameter (Miyamura, 1996) which are sizes reported to be susceptible to heat stress (Roth et al, 2001). Therefore, due to a reduction in follicular turn over in the DESL implant group during the second period of the study with a lower THI index (Figure 3-3), it is possible that the ovulatory follicle present at TAI for the DESL implant group belonged to a cohort of antral follicles that suffered the effects of the heat stress. This would result in a lower pregnancy rate. Additionally, post removal effects of the GnRH-Agonist implant on follicular growth patterns also could be associated with reduced fertility in cyclic and ovulated cows. Rate of follicular growth is increased in cows after chronic treatment with GnRH-Agonist compared to non-treated control cows leading to the development of larger and aged pre-ovulatory follicles of lower fertility (Silvestre and

Thatcher, unpublished). Possible confounding effects of post GnRH-Agonist treatment in this study could be eliminated by using the same experimental design during the cool season.

In DESL implant cows that were cyclic and ovulated, pregnancy rates were higher in cows with a lower milk yield. Perhaps, DESL implant cows with lower milk production had a higher plasma concentration of P4 during follicular recruitment of

Ovsynch® that sensitized the follicle and/or uterus/oviduct environment to increase pregnancy rate that did not occur for the higher producing category for DESL implant cows. High producing dairy cows appear to increase liver blood flow and the metabolic clearance rate of P4 (Sangsritavong et al., 2002). This phenomenon appears to be

112 associated with sensitization interrelated with the DESL implant since it was not detected in the CON group. Another explanation could be that high milk yield exacerbated the effects of heat stress on reproduction during summer and delayed achievement of normal fertility for a longer period of time in cows with higher milk yield (Al-Katanani et al.,

1999). Increased metabolic rate associated with lactation elevates internal heat production and decreased thermal regulatory ability of cows (Berman et al., 1985). Since the CON group had an increased follicular turn over and depletion of damage follicles during the cool season, the effect of heat stress was dissipated for all categories of milk yield of

CON cows. In contrast, the DESL implant group was TAI during a period that heat stressed follicles were still present in the ovaries such that higher producing cows expressed an increased effect of summer heat stress in the subsequent fall season.

Since pregnancy rates at estrus, did not differ between the two groups, it is possible that within the 26 d after TAI the DESL implant group of cows were ovulating follicles that no longer had suffered the detrimental effects of heat stress during the summer.

Service sire affected pregnancy rates following insemination to a detected estrus.

Sire one of higher fertility was also the sire used for the majority of cows at TAI. Semen from this sire was examined by an experienced veterinarian as described previously.

Semen from sire two had not been examined. Service sire quality have been reported to play an important role in determining dairy cattle pregnancy rates (Gwazdauskas et al.,

1973; Stevenson, 1983; Donovan, 2003), and it is becoming an important issue for producers and sire companies.

Although fertility appeared to be recovered after first service for the DESL implant group, the survival curve indicated that the percentage of cows diagnosed pregnant

113 continued to differ during the 39 day period. The high pregnancy rates to first TAI service in the CON group contributed to the sustained difference between the DESL implant and CON groups.

Pregnancy losses did not differ between groups, emphasizing that once the DESL implant cows ovulated a health oocyte and a positive diagnosis was made at day 28 of pregnancy, embryo losses were similar between groups.

Milk yield for the first 5 months was suppressed in the DESL implant and it was evident in both primiparous and multiparous cows. These results contradict a previous report where no differences were found between animals treated with a lower dose of a biodegradable 2.1 mg DESL implant (Mattos et al., 2001). Literature on chronic treatment with GnRH-Agonist affecting milk yield in domestic animals is not extensively available. Most of the information available is in humans and deals with direct and indirect effects of a GnRH-Agonist on hormones that regulate milk production (i.e., GH,

Prolactin and steroids). Chronic treatment with GnRH-Agonists reduced growth hormone

(GH) release in response to growth hormone releasing hormone (GH-RH) (Kaltsas et al.,

1998; Word et al., 1990), and decreased nocturnal GH secretion and reduced growth velocity in children diagnosed with central precocious puberty (Harris et al., 1985;

DiMartino-Nardi et al., 1991). A decrease of nocturnal serum GH was associated with a reduction in serum concentrations of IGF-I in humans (Mansfield et al., 1988). Prolactin was reduced during chronic treatment with a GnRH-Agonist in humans (Sklar et al.,

1991; Kreiter et al., 1990; Bourguignon, 1987). Moreover, the suppression of follicular activity and possibly hypoestrogenism may play important role in the reduced milk yield, since estradiol indirectly induces GH release from somatotrophes via stimulation of GH-

114

RH and somatostatin release from hypothalamic neurons obtained from cows (Hassanab et al, 2001).

In conclusion, chronic treatment with the GnRH-Agonist (Deslorelin, 5 mg) administered via implant suppressed follicle growth. Recrudescence of ovarian follicular growth of class 3 follicles after termination of treatment has a wide variation of time among cows regarding pituitary responsiveness to exogenous GnRH injection to induce ovulation, as part of a TAI/Ovsynch® protocol

We rejected our hypothesis that heat stress damage to follicles is limited exclusively to follicles > 3 mm. Follicles less than 3 mm appear to be susceptible to effects of heat stress. Increased rates of follicle turn over for heat stress damaged follicles appears to be more important than arresting follicular development. Possible effects of

GnRH-Agonist on follicular growth patterns after Deslorelin implant removal could have contributed to the low first service pregnancy rates of DESL implants cows. Chronic

GnRH-Agonist treatment suppressed milk yield, probably due to reduction in GH, IGF-I and prolactin due to direct action of GnRH-Agonist on the pituitary or indirectly through induced hypoestrogenism. However, these associated effects were not examined in the present experiment and warrant further investigation.

CHAPTER 4 POSTPARTUM SUPPRESSION OF OVARIAN ACTIVITY WITH A DESLORELIN IMPLANT ENHANCED UTERINE INVOLUTION IN LACTATING DAIRY COW

Introduction

Postpartum metritis has a great economic impact for dairy operations. This includes treatment expenses, milk loss, prolonged days open and culling rates (Esslemont and

Peeler, 1993). The lactational incidence rate of metritis has been reported by Dohoo et al.

(1983), Martin et al. (1983), Erb and Martin (1980) and Bartlett et al. (1986) to be 18.2%,

11.1%, 13.8% and 18%, respectively. Metritis was more frequently diagnosed between

11 and 20 days postpartum and 6.1% of culled cows were due to metritis in a large epidemiological study (Bartlett et al., 1986).

Metritis is associated with dystocia, retained placenta and herd size (Kaneene and

Miller, 1994). Data collected from 15,320 Holstein cows during a period of 3 years was used to determine the risk factors for conception; among other factors, cows with metritis had a 15% reduced likelihood of conceiving than normal cows (Grohn and Rajala-

Schultz, 2000).

In early studies, evaluation of uterine involution was based on rectal palpation

(Marion et al., 1968; Studer and Morrow, 1978) or post mortem anatomical-histological observations (Gier and Marion, 1968; Archbald et al., 1972). Rectal palpation has been used as a practical method to determine rates of uterine and cervical involution (Morrow et al., 1969). The ultrasonic linear scanner is a device that provides a non-invasive

115 116 method to precisely characterize postpartum uterine and cervical involution in the cow

(Okano and Tomizuka, 1987).

Complete gross involution of the uterus occurs over a wide range of 25-30 days postpartum, in which uterine dimension approximates the pre-gravid state (Hussain and

Daniel, 1991). Most of the changes are occurring within a few days after calving (Gier et al., 1968).

Uterine involution involves contraction of uterine musculature for sloughing of excess of caruncular tissue (Olson et al., 1986). Sloughing of the superficial layer of the caruncle is initiated around day 6 and 7 postpartum, and the stratum compactum reduces in size to almost the intercaruncular level by day 15 postpartum. Also a granular degeneration of the sarcoplasm, vacuolization of the muscle cell and atrophy of the nucleus occurs without necrosis of the myometrial cells (Archbald et al., 1972).

Metritis is characterized by an inflammation in all layers of the uterine wall, palpable ballottement of fluid, possible crepitant feel, lack of myometrial tone, and presence of abnormal discharge (Callahan and Horstman, 1987). Endometritis is characterized by an inflammation of the endometrial lining of the uterus without systemic signs, it is quite common during the first 2 weeks after calving (Hussain, 1991) and associated primarily with chronic infection of Arcanobacterium pyogenes (Lewis, 1997).

Pyometra is characterized by an accumulation of purulent exudate, growth of predominantly Arcanobacterium (Corynebacterium) pyogenes, and gram-negative anaerobic bacteria (Fusobacterium necrophorum and Bacteroides melaninogenicus) in the uterine lumen, which is associated with the presence of a CL on the ovary. Pyometra

117 often results from an early ovulation postpartum (i.e., 15 to 22 days), and during chronic endometritis or metritis (Arthur et al., 1989; Olson et al., 1994).

Visualization and scoring of cervical discharge using vaginoscopy, was associated strongly with a uterine bacterial infection (Dohmen et al., 1995) and visualization is highly recommended as a tool for diagnosing postpartum endometritis (LeBlanc et al.,

2002).

Uterine infection also has been correlated with a delay in the first postpartum ovulation (Peter et al., 1988; Opsomer et al., 2000; Sheldon et al., 2002). The mechanisms involved in this condition could be associated with endotoxins and cytokines resulting from inflammatory processes, which could disrupt hypothalamic GnRH release and pituitary LH secretion (Peter et al., 1989; Rivest et al., 1993).

If first ovulation occurs in the presence of a heavy contaminated uterus, it can lead to prolonged luteal phases (Smith et al., 1998; Opsomer et al., 2000; Royal et al., 2000), which is associated with pyometra (Farin et al., 1989) and lower fertility (Smith et al.,

1998; Royal et al., 2000; Opsomer et al., 2000). Indeed, a side effect of an early induced ovulation in response to GnRH injections (i.e., at 15 days postpartum) was an increase in the incidence of pyometra and prebreeding anestrus (Etherington et al., 1984).

Delayed first ovulation in suckled cows has been associated with increased rates of uterine involution in dairy (Riesen et al., 1968) and beef cows (Lauderdale et al., 1968).

Presence of the calf may represent a natural phenomenon to protect the involuting uterus against the relaxation (Rodriguez-Martinez et al., 1987; Bonafos and Ginther, 1995) and immunosuppressive effects of progesterone (Chacin et al., 1990; Subandrio et al., 2000).

118

Chronic treatment with GnRH-Agonists can induce down-regulation of GnRH receptors on gonadotroph cells, desensitization of the anterior pituitary gland to endogenous GnRH, and abolishment of pulsatile release of LH (D’Occhio et al., 2000), which collectively prevent ovulation and CL formation.

The objective of this study was to investigate the effects of a chronic GnRH-

Agonist treatment on ovarian follicular activity as well as uterine and cervical involution in association with occurrence of uterine infection during the postpartum period as a repeated measurement response in Experiment 1 and in a large group of cows as a single measurement on d 28 (Experiment 2).

Materials and Methods

The study was conducted at Alliance Dairy at Trenton, Florida, which is comprised of 3,500 cows that are milked three times a day. Cows were kept in free-stall barns, fed three times per day a TMR consisting of 46% forage (corn silage, rayelage, alfalfa and oat hay) and 54% concentrate (hominy, cotton seed, citrus pulp, soy bean meal, corn gluten, brewer condensed and molasses) containing 1.60 Mcal of NEL/Kg and 18% CP.

This study was performed within a larger experiment (see Chapter 3). Only clinically normal (i.e., no retained fetal membranes, milk fever, dystocia and stillborns) postpartum cows with a body condition score (BCS) equal or greater than 2.75 were enrolled. Recording of BCS occurred on the day of enrollment and at 42 days postpartum.

Scores were given by two veterinarians based on a 1 (thin) to 5 (obese) scale using a quarter point system (Edmonson et al., 1989). In Experiment 1 only multiparous cows were used. Primiparous and multiparous cows were blocked before assignment.

119

Experiment 1

All cows were enrolled within a mean of 2 ± 1 days postpartum (dpp). All cows were enrolled on July 30, 2001 to one of the two experimental groups:

DESL implant: a total of ten cows received a non-degradable implant containing 5 mg of the GnRH-analog Deslorelin implant (Peptech Animal Health, North Ride,

Australia) in the right ear. Implants were placed sub-cutaneously in the outer surface of the ear using an implanter device after intensive cleaning of the area with alcohol gauze and avoiding blood vessels. All cows had the implants removed on the same day,

September 4, 2001. Briefly, implants were removed after an incision under the fascia tissue located just below the implant using a surgical blade; the area was cleaned previously with alcohol gauze. Implants were removed in totality within the encapsulated fascia tissue; the implanted area was then flushed with alcohol and hemorrhage contained by manual pressure to the area with alcohol gauze. Cows had the implants for 36 days, which would cover the entire period necessary for complete physical involution of the uterus estimated to be up to 30 dpp (Hussain et al.; 1991).Control (CON): a total of nine cows did not receive a Deslorelin implant.

All cows, DESL implant and CON, were injected with PGF2α (25 mg, i.m.,

Dinoprost Tromethamine; Lutalyse® Pharmacia Upjohn) at 7 days after enrollment (9 dpp) in order to regress any possible corpus luteum induced by the GnRH-Agonist implant.

Ovarian structures

A real time Ultrasound (US) scanning Aloka 500 (Aloka Co., Ltd, Tokyo, Japan) equipped with a 5.0 MHz linear rectal transducer was used at 23 dpp, 30 dpp and 37 dpp to record ovarian structures. Size, location and number of follicles were recorded onto

120 ovarian maps and categorized into class 1 (2 – 5 mm), class 2 (6 –9 mm) and class 3 (≥10 mm) follicular classes (Lucy et al, 1992); presence or absence of a corpus luteum (CL) was recorded on all days described previously (23, 30, 37 dpp) in addition to day 16 postpartum in order to determine induction of CL by the Delorelin implant. Size of CL was determine by measurements of length and width, and analyzed as the average.

Uterine and cervical diameters

Previous pregnant uterine horn (PPH) was determined by rectal palpation as the longer horn with the greater diameter compared to the previously non-pregnant uterine horn (PNPH) at 16 dpp. Immediately prior to each US scanning, uterine tonus was estimated and classified as no tone, moderate tone, and intense tone. Uterine tone was assessed without knowledge of treatment groups.

A real time US scanner equipped with a 5.0 MHz linear rectal transducer was used to determine uterine horn and cervical diameters at 16, 23, 30, 37 dpp. The diameters were obtained by placing the transducer in a transverse position in relation to the horns, at approximately 4 cm past the bifurcation of the horns. When the transducer was positioned and the horns could be seen clearly, the image was fixed. Pressure with the transducer on the uterine horns was avoided in order to obtain a circular cross-section image of the horns. Built in machine calipers were activated such that a vertical line was extended from serosa to serosa of the uterine cross-section (Figure 4-1).

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Figure 4-1. Ultrasound image of cow uterine horn during different days postpartum. A: Day 7 postpartum; B: Day 14 postpartum; C: Day 21 postpartum. Yellow arrows indicate width of uterine horn cross-section from dorsal to ventral serosa.

Cervical diameter was measured by placing the transducer in a transversal position in relation to the cervix at its middle section; distance between to points was obtained as described above.

Vaginoscopy

The vulva was first cleaned using an iodine solution and dried with a paper towel.

A sterile disposable foil-lined cardboard vaginal speculum was inserted into the vagina until the external cervical os could be seen; visualization was performed using illumination from a penlight.

Cervical discharge was classified as clear mucus (or absence of discharge), mucupurulent (presence of flecks of approximately 50% pus and 50% mucus) and purulent (>50% pus to brown and foul smell). Color of the external cervical os was

122 classified as red (inflammatory), intermediary (pink with scattered red regions) and pink

(no inflammation).

Two veterinarians performed and agreed with the procedures described above, and were unaware of the experimental groups.

Blood samples and hormones assays

Blood samples were collected on a weekly basis beginning at 9 dpp just prior to injection of PGF2α and at 16, 23, 30, 37 dpp. Blood samples were collected from coccygeal vessels by venipuncture into heparinized vacutainer tubes and kept in ice until centrifugation (2619 x g for 20 minutes). After centrifugation, plasma was harvested and stored frozen at –20°C until assayed.

Concentrations of plasma progesterone (P4) were determined using a Coat-A-Count

Kit (DPC® Diagnostic Products Incorporation, CA, USA) solid phase 125I radioimmunoassay designed for the quantitative measurement of progesterone in plasma.

The 125I – labeled progesterone competes for a fixed time with the progesterone contained in the plasma sample. The antibody is immobilized to the wall of the polypropylene tube and removing the supernatant terminates the competition and isolates the antibody-bound fraction of the radiolabeled P4. Standard curve dilution was prepared using plain tubes for total counts and non-specific binding and coated tubes. A 100 µl volume of increasing concentrations of calibrators (P4), 0.1, 0.25, 0.5, 2, 5, 10, 20 and 40 ng/ml were added to the tubes. Reference sample containing 100 µl of volume of high concentrations of P4 (6 ng/ml) were also used. Experimental plasma samples (100 µl) were added to coated tubes and 1ml of 125I – labeled progesterone to all tubes. In every 6th sample duplication was performed. Incubation was for 3 hours followed by discarded of supernatant and drying

123 for 15 minutes. Tubes were transfer to and counted in a gamma counter. An Intra assay coefficient of variation was calculated from duplicated samples obtained from all assays.

Duplicated plasma concentrations of P4 were categorized into high (≥3.0 ng/ml) and medium (≥1.0 and <3.0 ng/ml) samples. High and medium samples had intra assay coefficients of variation of 8.43 % and 12.3 %, respectively. Inter and intra coefficient of variation for the luteal phase reference samples was 5.8% and 10.5%, respectively.

Sensitivity of the assay was 0.1 ng/ml.

A single antibody radioimmunoassay procedure was used for quantifying plasma estradiol-17β concentrations (Kirby et al., 1997). Duplicate plasma samples (0.3 ml) were extracted in 4 ml of methyl-tert-butyl ether (HPLC grade; Fischer Chemical Co., Fair

Lawn, NJ) for 2 minutes on a multitube vortexer. Extractions were frozen at -20 oC in methanol-dry ice bath. The solvent fraction was decanted into 12 x 75 mm borosilicate glass tubes and dried in the 37 oC water bath under air.

The plasma extracts and estradiol standards (0.25, 0.5, 1.0, 2.5, 5.0, 7.5, 10.0 and

20.0 pg/tube), obtained from a serial dilution of an E2 stock solution into ethanol (1 ng/ml), were incubated with 0.1 ml of 1.0% BSA (1g of BSA dissolved in 100 ml of

PBSG), 0.1 ml of estradiol-17β antisera (ICN Pharmaceuticals Inc. Costa Mesa CA; diluted into PBSG/EDTA at a dilution of 1:400,000 vol/vol), and 7,000 to 9,000 cpm 0.1 ml of 3-iodo [125I]-estradiol-17β (ICN Pharmaceuticals Inc. Costa Mesa CA; 2000

µCi/µg). Total binding was approximately 40%. Tubes were vortexed and then incubated at 4 oC for 20 hours. Separation of bound and free estradiol was performed by addition of

500 µl of dextran-coated charcoal solution in distilled water (0.0025 g/ml of charcoal and

0.01 g/ml of dextran) followed by 10 minutes of incubation at 4 oC, and centrifugation at

124

2619 x g for 10 minutes at 4 oC. Supernatant (0.3 ml) was counted in a gamma counter for 1 minute per tube. A 0,3 ml reference sample containing 300 µl of volume of high concentrations of E2 (8.95 pg/ml) were used. An Intra and inter assay coefficient of variation was 10.6 % and 6.3%, respectively. Sensitivity of the assay was 0.25 pg/ml.

Plasma samples (300 µl) were used to analyze the concentration of PGFM as an index of uterine PGF2α secretion (Knickerbocker et al., 1986). A polyethylene glycol radioimmunoassay procedure described by Meyer et al. (1995) was used. Briefly, the

PGFM standard solutions were made by serial dilutions in buffer of stock solution (1

µg/ml of Tris-HCL buffer) of authentic PGFM. Final PGFM standard concentrations were 15, 30, 50, 100, 250, 500, 1000, 2500, 5000 and 10000 pg/ml in duplicate. Each tube contained 300 µl of prostaglandin-free plasma and 100 µl of PGFM standard solution. Experimental plasma samples (300 µl) were added to 100 µl of buffer. A 100 µl aliquot of 1:4,000 rabbit antiserum to PGFM was added to each tube and incubated at 22

°C for 30 minutes. Approximately 18,000 dpm [3H] PGFM in 100 µl of buffer solution

(Tris-HCL) was added to give final volume of 600 µl. Tubes were incubated for 1 h at

22°C and then 12 h in 4°C. Separation of free and bound was accomplished by precipitation of protein (plasma protein, human gamma globulin, and rabbit anti-PGFM) with 750 µl of cold 40% solution of polyethylene glycol-8000 in distilled water.

Following centrifugation at 3000 x g for 30 minutes at 4°C, the supernatant was discarded, and the pellet resuspended in 750 µl of buffer and 750 µl of polyethylene glycol. A second centrifugation was performed as before, and the pellets were resuspended in 1 ml of buffer (Tris-HCL) and then transfer to a scintillation vial.

Scintiverse-BioHP (4 ml; Fischer Scientific, Fair Lawn, NJ) was added to each

125 scintillation vial. Inter- and intra assay coefficients of variation were calculated from reference samples. Mean values for first reference was 47.1 pg/ml. Inter and intra assay coefficients of variation were 9.96% and 8.3%, respectively. Mean value for second reference was 87.5 pg/ml. Inter and intra assay coefficients of variation were 12.3% and

7.9%, respectively. Sensitivity of the assay was 1.5 pg/ml.

Experiment 2

A total of 147 cows were assigned randomly to two experimental groups as described for Experiment 1. All procedures for Deslorelin implant insertion and clinical condition of the cows were the same as in Experiment 1. Cows were enrolled within 2 ± 1 dpp in the experiment from July 2 to August 5, 2001. All cows, DESL implant and CON, were injected with PGF2α (25 mg, i.m.) at 7 days after enrollment (9 dpp).

Cows were examined at 30 dpp [DES (n=77) and Con (n=70)]. Rectal palpation of the reproductive tract was performed to determine PPH and PNPH as previously described. Uterine tonus was first estimated and classified as no tone, moderate tone, and intense tone.

Diameter of the uterine horns was estimated at 4 cm past the bifurcation of the horns, and cervical diameter was measured at mid-cervix. Diameter of cervix and uterine horns were estimated by rectal palpation at increments of 1.5, 2.5, 3.5, 4.5 and 5.5 cm.

Two veterinarians agreed on location of measurements and estimation of the diameters at the start of the study and were unaware of the experimental groups.

Vaginoscopic examination for cervical discharge and clinical appearance of the cervical os (color) was the same as previously described for Experiment 1.

126

Blood samples were collected only at 9 dpp, just prior to the PGF2α injection.

Plasma progesterone and PGFM concentration were analyzed as described previously for

Experiment 1.

Statistical Analyses

In Experiment 1 follicle numbers, CL numbers, diameter of uterine horns and cervix as well as plasma concentrations of progesterone, estradiol and PGFM were analyzed using repeated measures of the mixed model procedure of SAS (SAS Institute

Inc; 1999; Version 8). For each dependent variable, the covariance structure that had the best relative good of fitness based upon penalty criteria (Bayesian criterion) was used.

The mathematical model contained group, day and group by day interaction with cow nested within group.

Logistic regression (GENMOD procedure of SAS) was used to analyse categorical data such as uterine tone, cervical discharge and color in Experiments 1 and 2 as well as diameter of uterine horns (PPH and PNPH) and cervix estimated by rectal palpation at 30 dpp in Experiment 2. When a variable contained two levels of response a binomial distribution was used as opposed to a multinomial distribution for variables with more than two levels of response. The mathematical model included group and day in

Experiment 1 and group and parity in Experiment 2. When variables were measured in repeated days (uterine tonus, cervical discharge and color) such as in Experiment 1, cow was used as a repeated subject.

The GLM procedure for multivariate analyses of variance (Manova procedure of

SAS) was used to determine partial correlation coefficients among response variables of interest and their corresponding probability values adjusted for group. Simple correlations were obtained by the correlation procedure (PROC CORR) that calculates

127

Pearsons product-moment correlations in which no adjustments for group were made.

Spearman statement is used when categorical responses are correlated (SAS Institute Inc;

1999; Version 8).

Results

Experiment 1

BCS did not differ between DESL implant and CON groups. Overall mean and median BCS were 3.0 ± 0.31 and 3.0 at day of enrollment, and 2.90 ± 0.23 and 2.87 at 44 dpp.

Ovarian responses

The DESL implant group increased the accumulation of Class 1 follicles (10.6 +

0.51 > 5.3 + 0.52; P< 0.01), and decreased the number of Class 2 (0.0 + 0.19 < 0.9 + 0.2

P<0.01) and Class 3 follicles (0.0 + 0.19 < 1.3 + 0.20; P<0.01) that are dependent upon gonadotrophin secretion (Figure 4. 2). Ovulation was suppressed in cows in the DESL implant group resulting in a complete absence of CL (0.0 + 0.09 < 0.45 + 0.1; P <0.01;

Figure 4-2).

12 2 r

e 9 1.5 b 6 1 m 3 0.5 Nu 0 0 Class 1 Class 2 Class 3 CL DESL Implant Control

Figure 4-2. Least squares means for number of follicles according to Class 1 (left Axis), Class 2, Class 3 and CL (right Axis) as measured by ultrasonography on days 23, 30 and 37 postpartum for DESL implant and CON cows.

128

Average number of follicles and CL at 23, 30 and 37 dpp is described in Table 4-1.

At day 16 postpartum Class 2, Class 3 follicles and CL were recorded for DESL implant group, whereas only CL was recorded for CON group.

In the DESL implant group, 0/10 (0.0%) had a CL during the days of ultrasography measurements. In contrast, 6/9 (66.6%) of the CON group had visible CL at ultranography at mean of 25.3 + 7.2 dpp.

Table 4-1. Average number of follicles per cow according to class (C) and CL for CON and DESL implant. Days CON (n=9) DESL implant (n=10) Postpartum C1 C2 C3 CL C1 C2 C3 CL

16 NR NR NR 0 NR 0 0 0

23 6.2 1.5 1.05 0.38 10.42 0 0 0

30 4.5 1 1 0.44 10.16 0 0 0

37 5.8 0.5 1.72 0.44 11.05 0 0 0

Overall 5.3 0.9 1.3 0.45 10.6 0 0 0 Class 1 (C1); Class 2 (C2); Class 3 (C3) and Corpus Luteum (CL). NR: not recorded

Hormonal responses

Estradiol. Plasma concentrations of estradiol were suppressed in the DESL implant group in comparison to CON cows (1.07 + 0.13 pg/ml < 1.68 + 0.13 pg/ml; P<0.01) throughout the days of sample collection. (Figure 4-3).

129

3 l

/m 2 g p

2 1 E 0 9 16233037 Days postpartum Control DESL Implant

Figure 4-3. Least squares means for plasma concentrations of estradiol (pg/ml) in CON and DESL implant groups of cows during the postpartum period.

Progesterone. Plasma concentrations of progesterone were suppressed in the

DESL implant group compared to CON cows (0.15 + 0.31 ng/ml < 1.40 + 0.32 ng/ml;

P<0.01). A group by day interaction was detected (P<0.02); the DES group of cows maintained a low basal plasma concentration of progesterone during the experimental days (Figure 4-4). In contrast, the CON group had an elevation of progesterone (>1 ng/ml) at 23, 30, 37 dpp characteristic of the presence of functional CL (Figure 4-4).

l 4

/m 3 ng

4 2 P 1 0 9 16233037 Days postpartum Control DESL Implant

Figure 4-4. Least squares means for plasma concentrations of progesterone (ng/ml) in CON and DESL implant groups of cows during the postpartum period.

130

PGFM. Plasma concentrations of PGFM did not differ between the DESL implant and CON groups (45.26 + 21.68 pg/ml < 75.01 + 22.37 pg/ml; P<0.35) during all days of sample collection. However, at 9 dpp PGFM plasma concentration was lower in DESL implant group in comparison to CON (122.27 + 33.02 pg/ml < 224.37 + 34.77 pg/ml;

P<0.03; Figure 4-5).

300 ) l 250

g/m 200 p

( 150

FM 100 G

P 50 0 9 16233037 Days postpartum

Control DESL Implant

Figure 4-5. Least squares means for plasma concentrations of PGFM (pg/ml) for CON and DESL implant groups of cows during the postpartum period. * P<0.03 at 9dpp.

Involution of Uterus and Cervix

Previous pregnant horn. Diameter of the previous pregnant horn (PPH) was reduced in the DESL implant group in comparison to CON (2.28 + 0.08 cm < 2.86 + 0.8 cm; P<0.01). The PPH was smaller in diameter during days 16, 23, 30 postpartum

(P<0.01; Figure 4-6).

Orthogonal contrasts determined the time in which physical involution was completed for PPH. A single day of measurement was compared with subsequent days and involution was considered completed when no significant differences were found in the contrast. Diameter of PPH at 16 dpp was significantly greater than subsequent days of measurement (23, 30 and 37 dpp) and measurements at 23 dpp were no longer significant

131 different with the subsequent days (30 and 37 dpp) for both groups. However, DESL implant group had a smaller diameter of PPH at all days of measurement except at 37 dpp when CON cows had a PPH size approximately the DESL implant group (Figure 4.6 and

Table 4-2). r

e 3.5 t e

) 3 m m a 2.5 (c

H Di 2 P

P 1.5 16 23 30 37 Days postpartum

Control DESL Implant

Figure 4-6. Least squares means for diameter of previous pregnant horn (PPH) in CON and DESL implant groups of cows during the postpartum period.

Table 4-2. Orthogonal contrasts for size (mm) of PPH during the postpartum period (days) of DESL implant and CON group. Contrast Means P-value Control 16 vs 23, 30, 38 3.25 + 0.20 vs 2.73 + 0.18 0.05 23 vs 30, 37 2.91 + 0.20 vs 2.65 + 0.20 0.30 30 vs 37 2.75 + 0.20 vs 2.54 + 0.20 0.41

DES Implant 16 vs 23, 30, 38 2.60 + 0.19 vs 2.18 + 0.14 0.01 23 vs 30, 37 2.15 + 0.19 vs 2.19 + 0.15 0.71 30 vs 37 2.16 + 0.19 vs 2.23 + 0.19 0.54

Previous non-pregnant horn. Diameter of previous non-pregnant horn (PNPH) was reduced in the DESL implant group in comparison to CON (1.87 + 0.8 cm < 2.21 +

0.08 cm; P<0.03). However, there was no day effect (P<0.26), suggesting that physical involution of PNPH was completed at 16 dpp for both groups (Figure 4-7), but physical size of the involuted PNPH was smaller for the DESL implant group of cows.

132

er 3 et 2.5 ) am i m 2 (c D

H 1.5 P

N 1 P 16 23 30 37 Days postpartum

Control DESL Implant

Figure 4-7. Least squares means for diameter of previous non-pregnant horn (PNPH) for CON and DESL implant groups of cows during the postpartum period.

The ratio of the horns were obtained (i.e., PPH/PNPH) and further analyzed. No significant differences were detected between groups (P<0.24). The group by day interaction also was not significantly different between groups. Thus, the smaller uterine horns (PPH and PNPH) for the DESL implant group had a relative proportional difference between horns that was the same for both groups.

Uterine Tone. The DESL implant group tended to have higher chances of expressing intense tone during days of measurement in comparison to CON (P=0.07) and no interaction of group, tone score and days was detected. Therefore tonus was consistently increased during all days of data collection (P<0.72; Table 4-3).

133

Table 4-3. Frequencies of degree of tone during the postpartum period in CON and DESL implant groups. CON a DESL implant b Days No Mod Intense Days No Mod Intense postpartum % (n) % (n) % (n) postpartum % (n) % (n) % (n)

16 44 (4) 22 (2) 33 (3) 16 30 (3) 30 (3) 40 (4)

23 44 (4) 33 (3) 22 (2) 23 10 (1) 10 (1) 80 (8)

30 22 (2) 44 (4) 33 (3) 30 30 (3) 0 70 (7)

37 44 (4) 11 (1) 44 (4) 37 30 (3) 0 70 (7)

Overall 39 (14) 28 (10) 33 (12) 25 (10) 10 (4) 65 (26) a (OR=3.00, CI=0.89 – 10.08; P=0.07); the GENMODEL procedure of SAS is modeling the probability of DESL implant cows to have higher ordered value of tone in comparison to CON cows.

Cervix. Diameter of the cervix tended to reduce in DESL implant group in comparison to CON (2.87 + 0.09 cm < 3.12 + 0.09 cm; P<0.08; Figure 4-8).

) 4 m c (

er et am

i 3 D cal i v r e

C 2 16 23 30 37 Days postpartum

Control DESL Implant

Figure 4-8. Least squares means for diameter of cervix for CON and DESL implant groups during the postpartum period.

Vaginocopy results

Cervical discharge. The DESL implant group tended to have a higher chance of having a clean discharge in comparison to CON (P= 0.09) during the days of measurement (Table 4-4). No significant interaction of group, discharge and days was

134 detected between groups (P> 0.30). Also, cows in DESL implant group tended to have higher chances of having a lower discharge score at 37 dpp in comparison to day 16 postpartum (P= 0.06), but no day differences were detected in the CON group (Table 4-

4).

Table 4-4. Frequencies of cervical discharge scores during the postpartum period for CON and DESL implant groups. CON a DESL implant b Days C 1 M P Days C 1 M P postpartum % (n) % (n) % (n) postpartum % (n) % (n) % (n)

16 33 (3) 33 (3) 33 (3) 16 c 70 (7) 0 30 (3)

23 56 (5) 11 (1) 30 (3) 23 80 (8) 20 (2) 0

30 60 (6) 10 (1) 20 (2) 30 70 (7) 30 (3) 0

37 50 (5) 40 (4) 0 37 d 90 (9) 10 (1) 0

Overall 50 (19) 24 (9) 21 (8) 78 (31) 15 (6) 7.5 (3) 1 C (clean mucous), M (mucopurulent) and P (purulent). ab (OR=3.21, CI=0.80 – 12.82; P=0.09); the GENMODEL procedure of SAS is modeling the probability of DESL implant to have a lower degree of discharge in comparison to CON. cd (OR=5.46, CI=0.92 – 32.46; P=0.06); the GENMODEL procedure of SAS is modeling the probability of DESL implant to have lower degree of discharge on d 35 in comparison to d 14.

Cervical Color. The DESL implant group also tended to have a higher chance of a lower score for color of the cervix in comparison to CON (P=0.06) during the days of measurement (Table 4-5).

135

Table 4-5. Frequencies of cervical os coloration scores during the postpartum period for CON and DESL implant groups. CON a DESL implant b Days 1 2 3 Days 1 2 3 postpartum % (n) % (n) % (n) postpartum % (n) % (n) % (n)

16 78 (7) 0 22 (2) 16 100 (10) 0 0

23 22 (2) 56 (5) 22 (2) 23 90 (9) 0 10 (1)

30 67 (4) 44 (4) 11 (1) 30 60 (6) 20 (2) 20 (2)

37 67 (6) 11 (1) 22 (2) 37 80 (8) 10 (1) 10 (1)

Overall 59 (19) 28 (10) 19 (7) 83 (33) 7.5 (3) 10 (4) 1 (pink), 2 (intermediary) and 3 (reddish). ab (OR=3.80, CI=0.92 – 15.70; P=0.06); the GENMODEL procedure of SAS is modeling the probability of DESL implant to have lower ordered value of color in comparison to CON.

Correlations

A significant simple and a significant partial correlation were integrated to indicate that two variables were associated (+ or -) and this association was present after adjustment for treatment groups. In contrast, a significant simple correlation, but a non- significant partial correlation for two variables was indicative that the association was not inherent, but due to differences induced by treatments. Diameter of the PPH, diameter of the cervix, plasma concentrations of PGFM and cervical discharge were significant and positively correlated regardless of treatment (P<0.05; Table 4-6). Therefore, these responses are indeed associated with one another regardless of whether cows were treated with a DESL implant. Based on previous analyses of mean responses, the DESL implant appeared to induce a lower degree of bacterial infection and inflammation of the reproductive tract. This is suggested since responses such as plasma concentrations of

136

PGFM, degree of discharge, diameter of PPH and diameter of cervix were of lower magnitude in the DESL implant group.

Degree of tone had a negative tendency to be correlated with plasma P4 in simple correlations, but it became no longer significant when adjusted for DESL implant and

CON groups. This supports the concept that the DESL implant increased tonus. Degree of uterine tonus was significantly and negatively correlated to diameter of PPH and cervix when simple correlations were analyzed (P<0.05). However, they were no longer significant when adjusted for treatment (P>0.10). Therefore, the Deslorelin implant had an effect of increasing uterine tonicity, which possibly induced a smaller diameter of the

PPH. Such an association may possibly increase rate of uterine bacterial clearance and consequently lower inflammatory processes as well as a smaller cervix. Additionally, the absence of P4 in the Deslorelin treated group may also have contributed to a higher tonicity of the uterus during the postpartum period.

Table 4-6. Simple and partial correlation among variables measured in the postpartum period. Correlations Responses Simple 1 Partial 2 PPH – Cervix 0.54 (P<0.05) 0.50 (P<0.05) PPH – PGFM 0.38 (P<0.05) 0.36 (P<0.05) PPH - Discharge 0.51 (P<0.05) 0.45 (P<0.05) Cervix – PGFM 0.23 (P<0.05) NS Cervix - Discharge 0.41 (P<0.05) 0.38 (P<0.05) PGFM - Discharge 0.28 (P<0.05) 0.33 (P<0.05) PPH – Tonus - 0.24 (P<0.05) NS Tonus - P4 - 0.21 (P<0.05) NS Color – PGFM 0.24 (P<0.05) 0.28 (P<0.05) Color - Discharge NS 0.31 (P<0.05) 1 Simple correlations: not adjusted for group; 2 Partial correlations: adjusted for group (i.e., DESL implant and CON)

137

Experiment 2

BCS did not differ between DESL implant and CON groups. Overall mean and median BCS were 2.94 ± 0.22 and 3.0 at at day of enrollment, and 2.87 ± 0.22 and 2.75 at

44 dpp.

Hormonal responses

Progesterone. Mean plasma concentrations of progesterone did not differ between

DESL implant (0.10 + 0.08 ng/ml) and CON (0.14 + 0.04 ng/ml) (P>0.10) group at 9 dpp. None of the cows treated with the Deslorelin implant had an induced ovulation.

PGFM. Plasma concentrations of PGFM (n=140) measured at 9 dpp enrolment were significantly lower for cows in the DESL implant group compared to CON cows

(133.72 + 18.32 pg/ml < 211.07 + 19.40 pg/ml; P<0.01).

Uterine horns and cervical diameter

The DESL implant group of cows had higher chance of having a small PPH

(P<0.01) and PNPH (P<0.01) in comparison to CON at day 30 postpartum as assessed by rectal palpation. Diameter of cervix at 30 dpp did not differ between groups (P<0.20;

Table 4-7).

Cervical discharge and uterine tonus

Cervical discharge did not differ between groups at d30 dpp (OR=1.28, CI=0.61 –

2.66; P<0.50). Degree of uterine tonus was increased in DESL implant cows in comparison to CON (P<0.01; Table 4-8).

138

Table 4-7. Diameter of PPH, PNPH and cervix estimated by rectal palpation at day 30 postpartum. Score (cm) Group 1.5 2.5 3.5 4.5 5.5 % (n) % (n) % (n) % (n) % (n) Control PPH a (n=76) 9 (7) 54 (41) 28 (21) 4 (3) 5 (4) PNPH c (n=64) 38 (24) 53 (34) 8 (5) 0 2 (1) Cervix (n=70) 3 (2) 27 (19) 50 (35) 14 (4) 6 (4)

DESL implant PPH b (n=79) 9 (7) 75 (59) 14 (11) 3 (2) 0 PNPH d (n=73) 63 (46) 37 (26) 1 (1) 0 0 Cervix (n=77) 3 (2) 32 (25) 55 (42) 5 (4) 5 (4) ab (OR=3.14, CI=1.56 – 6.40; P<0.01); the GENMODEL procedure of SAS is modeling the probability of DESL implant to have lower ordered value of PPH diameter in comparison to CON. cd (OR=3.16, CI=1.59 – 6.30; P<0.01); the GENMODEL procedure of SAS is modeling the probability of DESL implant to have lower ordered value of PNPH diameter comparison to CON.

Table 4-8. Degree of cervical discharge and uterine tonus assessed by rectal palpation at day 30 postpartum Score C M P Discharge % (n) % (n) % (n)

CON (n=68) 75 (51) 13 (9) 12 (8)

DESL implant (n=75) 68 (51) 28 (21) 4 (3) No Mod Intense Tonus % (n) % (n) % (n)

CON a (n=67) 16 (11) 61 (41) 22 (15)

DESL implant b (n=77) 9 (7) 32 (19) 66 (51) C (clean mucus), M (mucopurulent) and P (purulent). ab (OR=4.71, CI=2.37 – 9.37; P<0.01); the GENMODEL procedure of SAS is modeling the probability of DESL implant to have higher ordered value of uterine tone in comparison to CON.

139

Discussion

None of the cows inserted with the 5 mg Deslorelin implant in Experiments 1 and 2 responded with a rise in progesterone 7 days after implant insertion (9 dpp), and no corpus luteum was detected on the first day of ultrasound (16 dpp) in Experiment 1. The

DESL implant group was implanted at 2 ± 1 dpp, and at this stage stores of LH are depleted in the anterior pituitary (Saiduddin et al., 1968). Release of LH from pituitary upon exogenous stimulation with GnRH is minimal at day 5 (Fernandes et al., 1978) and not fully restored until 10 to 20 days postpartum (Fernandes et al., 1978; Mattos et al.,

2001; Padula and Macmillan, 2002).

Chronic pituitary desensitization was achieved resulting in long term suppression of follicular growth in the DESL implant, which was also associated with a suppression of estradiol concentrations in plasma. Although the definition of Class 1 follicles used in this experiment represents follicles with a diameter less than 5 and larger than 2 mm, actual follicles larger than 3 mm were not seen in the DESL implant group at d 14, d 21, d 28 and d 35. Follicular growth was arrested at 2-3 mm in the Deslorelin treated group, and the dynamics of follicle development below these sizes could not be evaluated due to the sensitivity of the ultrasound transducer.

Follicular recruitment is dependent on FSH, and follicular growth is arrested at 2-3 mm stage when cows are chronically treated with GnRH-Agonist associated with low basal plasma concentrations of FSH (Gong et al., 1996). The absence of follicular growth in the DESL implant group resulted in no ovulations, which was characterized by the absence of corpora lutea and decreased plasma concentrations of progesterone during the period of data collection (9 to 37 dpp). In contrast, CON cows had normal follicular growth characteristics of the postpartum period, with a mean elevation in plasma

140 concentrations of estradiol at approximately 23 ± 2 dpp followed by rise in plasma concentrations of progesterone and the presence of corpora lutea as detected by ultrasound. Ovulation occurred in 66% (6/9) of CON cows at approximately 25.3 + 7 dpp during the postpartum period. After parturition, first ovulation is expected to occur by 21-

30 dpp (Beam and Butler, 1997; Beam and Butler, 1998). Five cows had ovulations in the contra lateral ovary of the PPH and only one cow ovulated on the ipsilateral ovary of the

PPH. The skewed ovulation to the contra lateral ovary of the PPH is agreement with other authors (Saiduddin et al., 1968; Kamimura et al., 1993)

The bovine uterus is a primary source of F series prostaglandins during the early postpartum period with secretion increasing from day of parturition to approximately 4 days postpartum. This increase is followed by a progressive decline to basal concentrations at day 14 postpartum (Guilbault et al., 1984). In Experiment 1, plasma concentrations of PGFM measured at 16, 23, 30 and 37 dpp were already low for both groups of cows and no differences were detected. However, at day 9 postpartum the

DESL implant group of cows had a lower plasma concentration of PGFM, which may reflect a greater decrease in uterine size and/or lower degree of inflammation in these cows. Induction of uterine infection promotes the release of PGF2α (Del Vecchio et al.,

1992). Cows experiencing dystocia and retained placenta, which are associated with subsequent uterine infections, have elevated plasma concentrations of PGFM in the postpartum period (Nakao et al., 1997). A lower plasma concentration of PGFM also was detected in cows of Experiment 2 reflecting a smaller uterine size and/or lower degree of uterine inflammation at day 9 postpartum. The lower plasma concentrations of PGFM could also reflect a more rapid sloughing of the caruncular tissue at that time of sample

141 collection on day 9 postpartum. The caruncular endometrium is the major source of

PGF2α synthesis (Guilbault et al, 1984), and the gradual reduction of plasma concentrations of PGFM during the postpartum period occurs in association with the observed necrosis and cellular disorganization of the caruncle on day 5 postpartum (Gier and Marion, 1968). This is followed by sloughing of the superficial layer of the caruncle, that begins around day 6 and 7 postpartum, and a reduction in caruncular size to the intercaruncular level by day 15 postpartum (Archbald et al., 1972). Therefore, the lower levels of PGFM observed at 9 dpp appear to reflect the enhanced physical clearance of uterine contents with a possible lower degree of bacterial contamination and inflammation. Moreover, the DESL implant may possibly lead to an earlier histological involution that was not evaluated in this study.

Diameter of PPH decreased during the postpartum period for both groups (P<0.01).

Physical involution of the uterus was completed at 23 dpp since the diameter of the horns did not change in subsequent days of measurement in both groups to 37 dpp size (Table

1). Uterine involution is completed by 25-30 days postpartum (Hussain and Daniel,

1991). Earlier involution in both groups could have resulted from the fact that only cows with no dystocia and retained fetal membranes were used in this experiment. However, the DESL implant cows had a smaller horn at the initiation of measurements on day 16 postpartum and continued to be smaller on 23 and 30 postpartum. The CON cows achieved the size of the DESL implant group only by 37 ± 2 dpp. No significant differences were detected when the ratio of PPH and PNPH were analyzed. Therefore, the reduction in diameter for PPH and PNPH were proportionally the same in both groups, indicating an effect of the treatment on the entire reproductive tract. The reduced

142 size of the uterine horns on such early stages of the postpartum could have an important impact on the reduction and abolition of bacterial contamination and inflammation within the uterine environment. Metritis is more frequently diagnosed during the first 20 dpp

(Dohoo et al., 1983; Bartlett et al., 1986).

Mechanisms involving a more rapid involution of reproductive tract of DESL implant group of cows are most likely multifactorial. The reduced diameter of the PPH at the first ultrasound measurements (i.e., 16, 23 and 30 dpp) can be associated with direct effects of the GnRH-Agonist on the uterus, reduced basal concentrations of estradiol and decreased of pituitary secretion of gonadotrophins.

The expression of GnRH receptor mRNA has been found in myometrial cells of rats (Chegini et al., 1996) and in the endometrium of humans (Raga et al., 1998; Imai et al., 1994). However, the actions of GnRH on these tissues are still not clear.

Chronic GnRH-Agonist treatment is used widely in the treatment of women with symptomatic leiomyomas (Broekmans, 1996), endometrioses (Rumore and Rumore,

1989) and endometrial carcinoma (Dessole et al., 2000). These conditions are considered to be estrogen-dependent. Thus GnRH-Agonist is an efficient and reversible pharmacological method to achieve hypoestrogenism, which leads to a reduction in tumor volume. Moreover, hysterectomy is preceded by chronic GnRH-Agonist treatment in order to decrease uterine volume and facilitate either abdominal or vaginal surgical procedure (Broekmans, 1996).

The reduction in uterine size after chronic GnRH-Agonist treatment in women is a result of induced atrophy of the myocyte and arcuate arteries, and a decrease in stromal edema (Weeks et al., 1999). The spontaneous atrophy of the myometrium also occurs in

143 postpartum cows, and is initiated at 3 days postpartum (Archbald et al., 1972). In addition, endometrial volume and thickness were reduced during GnRH-Agonist treatment in women (Child et al., 2002; Nakamura et al., 1996). These processes seem to occur in a low estrogenic environment such as in the DESL implant group during the period of uterine involution. In addition, binding of FSH and LH to its receptors is associated with a direct activation of adenylate cyclase to produce cAMP and activation of signaling pathways that increase expression of COX-2 and production of PGE2 in the myometrium and cervix (Shemesh et al., 2001). Both cAMP and PGE2 induce relaxation of these tissues.

Although, plasma FSH and LH concentration were not measured in this study, the chronic GnRH-Agonist treatment most likely suppressed secretion of both FSH and LH in the DESL implant group based on suppression of follicle development, which would reduce its relaxation effects on the uterus. In contrast, the CON cows initiated secretion of FSH and LH in the early postpartum period following the delivery of the calf and placenta and the abrupt reduction in both progesterone and estrogens.

Follicular growth into Class 2 follicles was detected during the period of uterine involution of the control group. Therefore, the smaller uterine size during the first 21 days (23 ± 2 dpp) of GnRH-Agonist treatment could be an additive effect of a low estrogenic environment and the absence of uterine relaxation compounds leading to a more efficient atrophy of myocites, a reduction in endometrial volume, and a lower degree of inflammation than the CON cows. Perhaps, the increased uterine tonus observed in the DESL implant group reflects the improved atrophy of myometrial cells, increased reduction in size of the uterus and reduction of uterine luminal content.

144

Physical involution seemed to be completed at approximately 23 dpp for both groups. However, at approximately 25 dpp, ovulation and formation of the corpus luteum was detected by ultrasonography in CON cows. Consequently, plasma progesterone concentrations increased from basal levels compared to the DESL implant group. The rise of progesterone during the postpartum period results in delayed uterine involution

(Marion et al., 1968; Fosgate et al., 1962), which is possibly due to reduced myometrial contraction as seem during the diestrus stage of the cycle (Rodriguez-Martinez et al,

1987; Bonafos and Ginther, 1995). Reduction of myometrial contraction is due to the inhibitory effects of progesterone on the number of oxytocin receptor concentrations in the myometrium (Fuchs et al., 1983; Vallet et al., 1990) and a reduction in formation of gap junctions (Puri et al., 1982). Gap junctions are intercellular connections that enhance the movement of electrolytes and small molecules between adjacent myoepithelial cells to increase contractility (Bengtsson, 1982). Cows in the control group had a first ovulation occurring at a mean of 23.3 + 7.2 days postpartum. The concomitant rise in progesterone may have decreased myometrial contraction causing the PPH of the CON group not to reach the DESL implant size in the earlier stages postpartum.

Diameter of the PNPH was smaller in DESL implant group of cows (P<0.03) throughout the postpartum period, and no differences between days was detected

(P<0.26). Therefore, physical involution of the PNPH was completed by day 16 postpartum for both groups.

The smaller diameter of the cervix in the DESL implant group also reflected effects detected on the overall reproductive tract. The reduced diameter of the cervix in DESL implant cows may also be an indication of lower inflammatory processes resulting from

145 uterine bacterial infection. Increased size of cervix is associated with abnormal discharges in early postpartum period (Oltenacu et al., 1983).

The results obtained by rectal palpation at 28 dpp in Experiment 2 were in agreement with the ultrasound measurements of Experiment 1. The DESL implant group of cows had a smaller PPH at palpation compared to the CON (P<0.01). It is possible that at this time the DESL implant cows had completed the physical process of involution and the CON cows were still progressing in the involutionary process. Difference also were detected between groups for PNPH, reflecting, perhaps the induced atrophy effect due to chronic treatment with GnRH-Agonist, since at 28 dpp physical involution of PNPH was likely completed for both groups.

The enhanced physical involution of the uterus obtained in the DESL implant group increased the mechanical clearance of uterine contents. Concomitantly, with possible earlier occurrence of events associated with histological involution such as degenerative vascular changes, peripheral ischemia, necrosis and sloughing of caruncle tissue, there was a tendency for a lower frequency of abnormal cervical discharge in

Experiment 1. This also was associated with a lower degree of inflammation characterized by lower frequency of reddish coloration in the cervical os. Due to the improvement in all aspects of involution measured in the DESL implant group, it seems clear that a significant difference in cervical discharge could be detected by increasing the sample size and thus statistical power. Although bacterial analyses using endometrial biopsies or swabs were not performed in these experiments, there is a positive correlation between uterine bacterial load and cervical discharge (Dohmen et al., 1995). An argument that a low plasma estradiol concentration during the processes of uterine

146 involution could impair immunoresponses against bacterial contamination in the uterus is not supported by this study. Results of these experiments support the new concept that supplemental estradiol therapy does not enhance physical uterine involution and immunoresponses (Sheldon et al., 2003a; Sheldon et al., 2003b). Estradiol may not play a major role during the postpartum period regarding uterine involution. In Experiment 2, a single vaginoscopy exam at d 28 failed to detect differences between groups in uterine discharge. However it is important to restate that enrollment consisted only of clinically normal (no retain fetal membranes, no milk fever, no dystocia and stillborns) postpartum cows, and that at d 28 most of the CON cows were in the process of completing involution of the uterus. Thus abnormal uterine discharge was more likely not to be seen.

After parturition, first ovulation is expected to occur by 21-30 days postpartum

(Beam and Butler 1997; 1998) at a time in which uterine involution has not been completed. Early ovulation in the postpartum period (< 24 dpp, Opsomer et al., 2000; <

21dpp, Smith et al., 1998) is associated with a higher risk to develop a prolonged luteal phase (progesterone elevated for more than 20 days), prolonged calving to conception interval, more services per conception, and lower conception rate in comparison to cows that ovulated after 21 days (Smith et al., 1998; Royal et al., 2000). Moreover, a prolonged luteal phase has been report to be correlated with a greater risk of developing pyometra

(Farin et al., 1989).

On the other hand, cows experiencing puerperal diseases (abnormal calving, abnormal discharge, retained fetal membrane and clinical ketosis) have long been implicated in retarding ovarian activity and delayed first postpartum ovulation (Morrow et al., 1966; Marion and Gier, 1968; Opsomer et al., 2000). These events were associated

147 with high uterine bacterial contamination (Sheldon et al., 2002, Peter et al., 1988). The mechanisms involved in delaying ovulation could be associated with endotoxins, and cytokines resulting from the inflammatory processes, which could disrupt hypothalamic

GnRH release and pituitary LH secretion (Peter et al., 1989; Rivest et al., 1993).

In a classical study, Thatcher and Wilcox (1972) proposed that early and frequent estrus activity during the postpartum period was associated with increased reproductive performance due to greater restoration of the uterine environment. In this study, cows expressing 0 or 1 estrus within 60 days postpartum had reduced fertility resulting in a higher percentage of non-pregnant cows sold and required more services per pregnancy.

These responses decreased linearly as the number of estrous increased, i.e. cows that had

2, 3 or 4 estrous were of greater fertility.

The conclusion that spontaneous estrus cycles during the postpartum can enhance uterine involution and subsequent fertility is debatable. Animals with no estrus periods could represent a greater proportion of cows with dystocias parturition, retained placenta, metabolic disorders and metritis. Cows expressing one estrus could represent the animals with prolonged luteal phase (failure to turn-over CL) and with possible uterine bacterial contamination these cows would have lower fertility. The animals expressing two or more estrous periods, are representative of cows with a normal postpartum period. These cows are able to ovulate once and continue to do so. Therefore, a sequential occurrence of events from the moment of parturition to the time of first service is seen. Early events in the postpartum (i.e. dystocia, retained fetal membranes, metabolic diseases, metritis) are related with uterine health and involution, which will dictate time of first ovulation and subsequent fertility. Therefore, we propose a uterus to ovarian pathway, i.e.,

148 completion of physical involution and clearance of the uterus must occur in a short period of time in the early postpartum period without the occurrence of ovulations. After completion of uterine involution, sequential ovulations would be a goal towards normal fertility.

In conclusion, chronic treatment with non-degradable GnRH-Agonists (Deslorelin,

5 mg) during the period of uterine involution can: suppress follicular development to 2-3 mm diameter, enhance physical involution of the uterus and cervix, increase tonicity of the uterine wall, reduce frequency of abnormal cervical discharges and reduce inflammatory processes of the reproductive tract. Duration of treatment could be restricted to the first three weeks of the postpartum period in order to avoid complete and sustained desensitization of the pituitary (Gong et al., 1996). Dose effects of the

Deslorelin implant and practical routes of treatment need further investigation.

CHAPTER 5 GENERAL DISCUSSION

Increases in herd size, milk yield per cow in association with increasing severity of negative energy balance, greater use of free stall housing, postpartum disorders, and reduction in estrous detection are reported as risk factors for decreased pregnancy rates

(Lucy, 2001). Furthermore, the reproductive physiology of dairy cows is altered during periods of thermal stress that carry over into subsequent cool season. Alterations in ovarian follicular physiology and oocyte competence are among the reasons for subfertility during early fall. Global warming has increased the regions predisposed to seasonal effects that compromise reproductive efficiency. Not only sub-tropical and tropical areas are at risk (Badinga et al., 1984; Al-Katanani et al., 1999) but also temperate regions (Rutledge et al., 1999). Therefore, there is a need for development of technologies to lessen the adverse effects of the summer heat stress season in order to maintain high fertility in lactating dairy cattle throughout the year.

In chapter 3, we hypothesized that limiting follicular growth to follicular size of 2 to 3 mm in dairy cows, during the summer and postpartum period would protect follicles and oocytes from the deleterious effects of heat stress. Consequently, once follicular growth is reestablished during the fall season healthy follicles containing normal oocytes would result in fertility levels equivalent to those of winter.

Pharmacological suppression of follicular growth from the antral stage of 2-3 mm in size to the pre-ovulatory stage is achieved successfully when cows are chronically treated with a GnRH-Agonist (Delorelin, 5 mg) implant in the postpartum period.

149 150

Deslorelin implants, which are non-degradable and require removal, provide the capability to precisely initiate and terminate treatment at pre-determined days to control duration of treatment.

In chapter 3, ovarian follicular growth was arrested at follicle size of 2 to 3 mm size during the heat stress season in the DES implant group of cows in contrast to the control group that underwent normal follicular activity. At the beginning of the cool season when

DES implants were removed, a proportion of treated cows (58%) had prompt recovery from pituitary desensitization with the first follicular wave formed at approximately 23 days after withdraw of the implant. Indeed these cows responded to the presynchronization/Ovsynch® protocol with synchronized ovulations following TAI.

However, pregnancy rates were reduced in comparison to the control cows.

Normal pregnancy rates were observed in the control group of cows that were TAI approximately 60 days after the heat stress period. This delay in insemination allowed several spontaneous and induced follicular waves to occur that deplete damage follicles containing impaired oocytes such that TAI was performed in cows ovulating follicles that were not previously damage by heat stress (Figure 3-6). In contrast, treated cows had less follicular waves or follicular turn over such that TAI was performed in cows ovulating oocytes originating from small heat sensitive antral follicles that were present in the ovaries during the summer heat stress (Figure 3-7). The hypothesis that follicles > 3 mm are exclusive sensitive to heat stress and that blocking their development during summer period of heat stress would improve fertility is rejected. Pregnancy rate in the DES implant group with suppressed follicular development and turn over was reduced. Results support the concept that follicles ≤ 3 mm are damage during heat stress, and it is critical

151 that these follicles be eliminated (i.e., recruited and undergo subsequent atresia). Roth et al. (2002) demonstrated that repeated aspiration of follicles (> 3 mm) hastened recruitment of follicles that were not heat damaged and which had oocytes of higher quality in terms of embryo development following parthenogenetic activation.

Additionally, post removal effects of the GnRH-Agonist implant on follicular growth patterns also could be associated with reduced fertility in cyclic and ovulated cows. Rate of follicular growth is increased in cows after chronic treatment with GnRH-Agonist compared to non-treated control cows leading to the development of larger and aged preovulatory follicles of lower fertility (Silvestre and Thatcher, unpublished). Possible confounding effects of post GnRH-Agonist treatment in this study could be eliminated by using the same experimental design during the cool season.

Chronic treatment with a GnRH-Agonist (Deslorelin) implant for a minimum of 28 days during the postpartum period is an effective pharmaceutical method to suppress follicular growth. Initiation of treatment within 4 dpp did not induce an ovulation and maintained a block in follicular growth to sizes that are not dependent on pituitary FSH and LH (i.e., ≤ 3 mm). Induction of a desensitization of pituitary gonadotrophs to GnRH is achieved via down-regulation of GnRHr gene expression and protein synthesis

(Turzillo et al., 1998; Viscarra et al., 1997).

After termination of treatment, restoration of follicular waves or follicular turn over occurs unpredictably at different periods of time among cows. Cows with longer periods of DES implant treatment required more days to develop the first follicular wave.

Moreover, presence of a class 3 follicle (≥ 10 mm) is not indicative of complete reestablishment of pituitary responsiveness to exogenous GnRH. For example, half of the

152

DES implant cows (57%) with follicles ≥ 10 mm at 22 and 29 days after implant removal failed to cycle following presynchronization with GnRH and PGF2α treatment. However, if CL formation had occurred by 22 and 29 days after implant removal, cows underwent successful presynchronization and subsequent ovulation to Ovsynch® protocol. When evaluating all experimental cows, 90% of the CON cows ovulated following the

Ovsynch® protocol and 94% of these cows were cycling prior to initiating the Ovsynch® protocol. In contrast, 71.1% of the DES implant cows ovulated following the Ovsynch® protocol and only 49% of these cows were cycling prior to initiation of the Ovsynch® protocol (Table 3-5).

A reduction in the number of estrus periods and ovulations in the postpartum period is associated with reduced first service fertility (Thatcher and Wilcox, 1972; Smith et al,

1998; Opsomer et al; 2000). The rise in plasma concentrations of progesterone during the time of postpartum uterine involution can delay the processes of uterine involution and induce persistent infection of the reproductive tract. Postpartum metritis has a great economic impact for dairy operations, which include treatment expenses, milk loss, prolonged days open and culling rates (Esslemont and Peeler, 1993).

In chapter 4, we investigated the effects of suppression of ovarian follicular activity on uterine and cervical involution in association with occurrence of uterine infection for dairy cows that received a non-degradable GnRH-Agonist (Deslorelin) implant during the postpartum period. The GnRH-Agonist treatment induced suppression of follicular growth and plasma concentrations of both estradiol and progesterone during the period of uterine involution. The DES implant treatment had a major impact in all measured responses associated with the processes of uterine involution.

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Deslorelin treatment enhanced physical involution of the uterine horns and cervix as characterized by smaller diameters. The reduction of uterine horn diameter was detected as early as 16 days postpartum. The reduction in size was associated with increased tonicity of the uterine wall, reduced frequency of abnormal cervical discharges early postpartum, and reduced inflammation of the reproductive tract as characterized by lower plasma concentrations of PGFM.

Improvement of early postpartum uterine involution is a multifactorial process.

Possible direct effects of the GnRH-Agonist on the uterus, reduced basal concentrations of estradiol and increased tonicity due to decreased secretion of pituitary gonadotropins

(FSH and LH) may be additively associated. In addition, a suppression of ovulation until after the period of uterine involution may avoid the potential immunosuppressive effect of progesterone from the CL. A suppression of early ovulation reduces the risk of pyometra and prolonged luteal phases.

The improvement in physical clearance of uterine contents (i.e., lochia), with a reduced inflammation and possibly lower degree of bacterial contamination in the early postpartum period associated with ovarian inactivity partially contradicts former concepts that re-occurring estrous cycles are beneficial to postpartum health and subsequent fertility.

A prolonged anovulatory condition due to the GnRH induced desensitization of pituitary gonadotrophs may be overcome by applying the treatment for less than 28 days, which is the period described to be necessary for total down-regulation of gonadotrophs

(Gong et al., 1996). Yet this period may be sufficient to augment the uterine involutionary process as described and warrants further investigation. Such options of

154 lower doses of the DES implant for shorter periods of time need to be evaluated. Cows need to undergo ovarian recrudescence following withdraw of the implant such that fertility is restored at the designated breeding period.

The lack of pharmacological treatments that consistently reduce reproductive tract dimensions, coupled with increased sloughing of uterine tissue and a reduction in bacterial contamination of the postpartum uterus are of major concern to dairy producers.

The positive effects of a Deslorelin treatment on uterine involution can potentially have a great impact in the dairy industry to reduce cost associated with antibiotic treatment, milk withhold, labor and culling rates due to reproductive failure. Moreover, reduction of uterine infections in the postpartum period can reduce calving to conception interval, services per conception, and increase first service conception rates. However, this technology with the DES implant need to be further developed (dose and duration of treatment) to overcome the induced decrease in milk production and fertility to first service.

In summary, arresting follicular growth to follicle ≤ 3 mm did not protect follicles and their containing oocytes from the deleterious effects of heat stress. Practical technology to improve the depletion rate of heat stressed damaged follicle of ≤ 3 mm is required as an alternative method for reestablishment of normal fertility of lactating dairy cows during early autumn. Chronic treatment with a GnRH-Agonist in the postpartum period in association with a suppression of follicular growth, can improve overall involution of the postpartum reproductive tract. Studies to improve fertility after GnRH-

Agonist warrant further investigation and would include such factors as dose, duration of treatment and rout of administration

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BIOGRAPHICAL SKETCH

Flávio Teixeira Silvestre was born on November 11, 1973, to Paulo Augusto Alves

Silvestre and Maria Angela Teixeira Silvestre in Campinas, São Paulo, Brazil. He is the second son of four. In 1997, he received his degree in veterinary medicine from the

Universidade Federal de Minas Gerais, located in Belo Horizonte, Minas Gerais. After his degree, he worked as a practitioner for a dairy cooperative located in Espírito Santo

State during three months. He terminated his work in Brazil to join the University of

Florida’s International Dairy Farm Fellows Program. He spent two years as a training student at North Florida Holsteins Dairy Farm. After this program he worked for one year as an agent in a finance loan office. In the summer of 2001 the author started his master’s program at the University of Florida under supervision of Dr. William W. Thatcher. After completion of his program he will continue his education in the Ph.D. program under supervision of Dr. William W. Thatcher.

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