Induction of Ovulation and LH Response in Cyclic Mares Treated with Gonadorelin Diacetate Tetrahydrate Jennifer Ann Ingwerson Iowa State University

Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

2007 Induction of ovulation and LH response in cyclic mares treated with gonadorelin diacetate tetrahydrate Jennifer Ann Ingwerson Iowa State University

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This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Induction of ovulation and LH response in cyclic mares treated with

gonadorelin diacetate tetrahydrate

Jennifer Ann Ingwerson

A thesis submitted to the graduate faculty in partial fulfillment of the requirement for

the degree of MASTER OF SCIENCE

Major: Animal Physiology (Reproductive Physiology)

Program of Study Committee: Peggy Miller-Auwerda, Co-major Professor Curtis Youngs, Co-major Professor Carolyn Komar Lawrence Evans

Iowa State University

Ames, Iowa

2007 UMI Number: 1449655

UMI Microform 1449655 Copyright 2008 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ii

Dedication

I dedicate this to my dad, James Ingwerson, and my aunt, Linnea Baney, who lead me to horses. I also dedicate this to my mom, Diane Ingwerson, who instilled in me a love of academics. Without them my passion for horses would have never started to lead me down this path in my life.

iii

TABLE OF CONTENTS

LIST OF TABLES...... v

LIST OF FIGURES ...... vi

ABSTRACT...... vii

CHAPTER 1. LITERATURE REVIEW ...... 1

1.1 Introduction...... 1

1.2 Seasonality...... 2

1.2.1 Estrous cycle...... 4

1.2.2 Follicular waves...... 4

1.2.3 Hypothalamic-pituitary-gonadal axis ...... 5

1.3 GnRH ...... 5

1.3.1 GnRH pulsatile release ...... 6

1.3.2 Regulation of GnRH ...... 8

1.4 Endocrinology of the estrous cycle ...... 10

1.4.1 Follicular phase ...... 10

1.4.2 Luteal phase...... 11

1.5 GnRH analogues/hormones used to control estrus...... 12

1.5.1 Cattle...... 12

OvSynch…………………………………………………………………13

CO-Synch ……………………………………………………………....13

Select Synch ……………………………………………………………14

1.5.2 Horses………………………………………………..…………………...15

hCG………………………………………………………………………16 iv

PGF2α (and analogues)……………………………………………….17 Deslorelin Ovuplant™…………………………………………………18

Deslorelin BioRelease………………………………………………...19

Kisspeptin……………………………………………………………....20

1.5.3 Cystorelin™ (gonadorelin diacetate tetrahydrate) …………………...20

1.6 Rationale for experiment...... 22

1.7 Literature Cited ...... 23

CHAPTER 2. INDUCTION OF OVULATION AND LH RESPONSE IN CYCLIC

MARES TREATED WITH CYSTORELIN™…………………………………………28

INTRODUCTION ...... 28

MATERIALS AND METHODS ...... 30

Experimental animals ...... 30

Treatments ...... 30

Blood sample collection...... 31

Assay of plasma LH levels ...... 33

Data Analysis...... 33

RESULTS ...... 34

Days to ovulation ...... 34

LH response ...... 35

DISCUSSION ...... 39

LITERATURE CITED...... 43

ACKNOWLEDGEMENTS ...... 45

LIST OF TABLES

Table 1. Timeline for blood sample collection and treatment with

Cystorelin™……………………………………………...…………………………………32

Table 2. Ovarian status and response to treatment with either Cystorelin™ or saline………………………………………………………………………………………..34

LIST OF FIGURES

Figure 1. Pineal gland control of reproductive seasonality in mares….. ………….....3

Figure 2. Hypothalamic-pituitary-gonadal axis feedback mechanisms in the female……………………………………………………………………………………….10

Figure 3. Methods currently being used to synchronize ovulation in postpartum beef cows: OvSynch, CO—Synch, and Select Synch. …...…………………….…………..15

Figure 4. LH profiles of individual mares treated with Cystorelin™.…………………35

Figure 5. LH profiles of individual mares treated with saline ………….……………..36

Figure 6. Average LH response of treatment and control mares…………………….37

Figure 7. Mean plasma LH increase over time between treatment and control mares…………………………………...... ……………………...38

vii

ABSTRACT

Regulating the time of ovulation in the mare has many practical and beneficial management applications, including use with artificial insemination. The objective of the present study was to test the effectiveness of Cystorelin™ (gonadorelin diacetate tetrahydrate) to elicit increased LH secretion and to induce ovulation in cyclic mares. A total of 24 mares of Thoroughbred and stock type breeding was used in this study. Mares possessing an ovarian follicle 3.5 cm to 4.0 cm in diameter were assigned to either treatment with three 75 µg (1.5 mL) i.m. injections of Cystorelin™ or three 1.5 mL i.m. injections of sterile saline (control). Treatment with Cystorelin™ reduced (P < .05) the mean number of days until ovulation (2.25 ±

0.25 vs 3.23 ± 0.41 for Cystorelin™ and saline, respectively), although variation in the time to ovulation was similar (P>.81). Mean plasma LH levels were calculated over four time periods (-30 to 0 (Period 1), 30-120 (Period 2), 150-240 (Period 3),

270-360 min (Period 4)). Luteinizing hormone response was analyzed by comparing the mean LH value of Period 1, 2, and 3 with the baseline mean. Although the level in Period 2 tended (P<.07) to be higher than baseline, LH levels in Periods 3 and 4 were not different from baseline (P>.15, P>.18 respectively). In summary

Cystorelin™ effectively hastened ovulation in mares. Further research is necessary to develop a protocol that can also reduce variability in the time of ovulation. 1

CHAPTER 1. LITERATURE REVIEW

1.1 Introduction

Regulating the time of ovulation in the mare has many practical and beneficial

applications and the increasing use of artificial insemination technologies in the

equine industry has increased the importance of controlling the time of ovulation in

the mare. Controlling the time of ovulation can increase management and labor

efficiency, optimize the time of breeding when using transported semen, and reduce

the number of times a mare is bred during a given estrous cycle. In cyclic mares,

human chorionic gonadotropin (hCG), prostaglandin F2alpha (PGF2α) (and

analogues), and gonadotropin releasing hormone (GnRH) have been used in attempts to control the time of ovulation. A new product to time ovulation in the

mare, EquiPure-LH (by AspenBio Pharma) has been sold as a specially labeled

reagent to licensed veterinarians since late 2005. AspenBio Pharma is seeking U.S.

Food and Drug Administration (FDA) approval. Apparently, no research has been

published on the efficacy of EquiPure-LH in hastening ovulation in mares.

The repeated use of hCG can result in reduced effectiveness to time

ovulation due to its antigenic effects in the mare (Sullivan et al., 1973; Voss et al.,

1975; Roser et al., 1979; Duchamp et al., 1987; McCue et al., 2004). The use of

PGF2α (and analogues) to time ovulation has resulted in inconsistent responses

(Harrison et al,. 1987; Savage and Liptrap, 1987; Squires et al., 1981). The GnRH

analogue, deslorelin (Ovuplant™), has been reported to cause desensitization to

GnRH and suppressed gonadotropin secretion which leads to an extended interval

between induced ovulation and the subsequent ovulation if the mare is not pregnant 2

when Ovuplant™ is left in place for over 48 hours (McCue et al,. 2000; Johnson and

CartMill, 2002).

1.2 Seasonality

The mare is a seasonally polyestrous breeder. Photoperiod is the most important external factor influencing seasonality in the mare, as is the case for many other species (Ginther, 1992). The pineal gland receives an environmental cue to decrease melatonin secretion during daylight hours; hence, melatonin is secreted during hours of darkness. While the hypothalamic-pituitary-gonadal axis is under the influence of melatonin, GnRH secretion is decreased, resulting in a non-reproductive state (Nagy, 2000). Short daylength is associated with reproductively inactive mares. As daylength increases, melatonin secretion is decreased allowing an increase in the secretion of GnRH and subsequent reproductive cyclicity of the mare

(Figure 1).

Figure 1. Pineal gland control of reproductive seasonality in mares.

Reproduced with permission from Geisert, R., 2005

Many terms are used to categorize the time of year the mare is able to reproduce such as the breeding, active, estrous, and ovulatory seasons. Anestrous, dormant, anovulatory, acyclic, and nonbreeding season are terms used to describe the time of the year when mares are not able to reproduce (Ginther, 1992). For this thesis the terms estrous season and anestrous season will be used. The majority of mares in the northern hemisphere are in their estrous season during the spring and summer and in their anestrous season during the fall and winter. The early fall and early spring serve as transition periods from estrous to anestrous seasons and from anestrous to estrous seasons, respectively. 4

1.2.1 Estrous cycle

The estrous cycle is defined as the repetitive sequence of events that

prepares the mare for conception (McKinnon and Voss, 1993). The estrous cycle

can be divided into two phases, the follicular phase and the luteal phase. The

follicular phase consists of proestrus and estrus stages of the estrous cycle, and the

luteal phase is comprised of metestrus and diestrus stages. Estrus is the period in

which the mare is sexually receptive to the stallion and during which ovulation

occurs. Diestrus is the period during which the mare is not sexually receptive to the

stallion and it is characterized by the presence of a corpus luteum. The means for

the lengths of estrus, diestrus, and estrous cycle are 6.5, 14.9, and 21.7 days,

respectively (Ginther, 1992).

1.2.2 Follicular waves

Follicular activity is increased during the estrous season versus the anestrous

season. Follicular growth is described in two types of follicular waves, major and

minor waves. A major follicular wave refers to several follicles that initially grow in

synchrony but eventually dissociate (Ginther, 1992). The dissociation of the

synchronized follicles in the major wave is due to the selection of a dominant follicle.

The dominant follicle grows to a diameter greater than 30 mm and then either regresses (in an anovulatory wave) or ovulates (in an ovulatory wave). The remaining follicles from the major wave regress after the dominant follicle emerges

(Ginther, 1992; Samper, 2000).

Major waves can be further categorized into primary or secondary waves.

Primary major waves emerge at mid-diestrus, resulting in ovulation of the dominant 5

follicle at or near the end of estrus. If present, secondary major waves precede

primary major waves and emerge during late estrus or early diestrus. In secondary

major waves the dominant follicle ovulates, becomes hemorrhagic, or regresses

(Ginther, 1992; Samper, 2000).

Minor follicular waves also have been identified. The differences between a minor wave and a major wave are the diameter and ovulation of the dominant follicle. With a minor wave the dominant follicle reaches a diameter of less than 30 mm and then regresses, never reaching ovulation (Samper, 2000). It appears that the minor wave lacks full dominance typically exhibited by a large dominant follicle capable of ovulating.

1.2.3 Hypothalamic-pituitary-gonadal axis

Fertility depends on a functional hypothalamic-pituitary-gonadal axis, and

GnRH from the hypothalamus is the primary regulator of reproduction in vertebrates.

Gonadotropin releasing hormone is released in a pulsatile fashion and travels to the pituitary gland via the portal hypophyseal vasculature. The synthesis and secretion of the two gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone

(FSH), is ultimately regulated by GnRH. These gonadotropins stimulate sex

hormone synthesis and gametogenesis in the gonads to ensure reproductive

competence (Hapgood et al., 2005).

1.3 GnRH

Gonadotropin releasing hormone is a decapeptide with a short endogenous

half life of five to ten minutes (Conn, 1987). The mammalian GnRH structure (pGlu-

. His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly NH2) was first discovered in 1971 (Schally et 6

al., 1971). Gonadotropin releasing hormone and its analogues are valuable in controlling estrous cycles and the timing of ovulation in many mammalian species.

Substitutions with D-amino acids in position six stabilizes conformation of GnRH, increases binding affinity, and decreases metabolic clearance, thus extending the half-life of a GnRH analogue. This substitution is used in all agonist and antagonist analogues. Hydrophobic D-amino acid substitutions in the first, second and third positions at the amino terminus result in antagonist analogues that retain substantial receptor binding affinity and reduced agonist activity (Millar, 2004).

1.3.1 GnRH pulsatile release

The pattern of GnRH secretion dictates the reproductive status of the mare.

GnRH pulse frequency and amplitude vary with season and stage of the estrous cycle. Gonadotropin secretion is influenced by and reflects changes in GnRH pulsatility. Hormonal analyses of jugular blood show high amplitude, synchronous

FSH and LH pulses occur 0-4 times daily during anestrous (Alexander and Irvine,

1991) and 1-4 times daily during diestrus (Irvine and Alexander, 1993). The frequency of these pulses, long half-life, and large amount of available FSH and LH results in low detection of pulses in jugular blood; therefore, it has been found that daily blood samples or long window bleedings are adequate to measure FSH and

LH secretion during the period of estrus (Irvine, 1979; Fitzgerald et al., 1985).

It was not until a new technology was developed that LH and FSH pulses were more adequately measured during estrus. Intercavernous sinus (ICS) cannulation was devised by Irvine and Alexander (1984), allowing for a more accurate measurement of GnRH and gonadotropin secretion. Intercavernous sinus 7 cannulation is a non surgical method used for collecting pituitary venous (PV) blood from horses. The method involves inserting a cannula into the superficial facial vein and then directing the cannula along a venous pathway until the catheter comes to lie in the ICS at the outlet of the paired pituitary veins. Intercavernous sinus cannulation allows measurement of GnRH and gonadotropin secretion that is not detectable in peripheral blood.

Using PV blood samples it was demonstrated that in estrus and diestrus

GnRH pulses are accompanied by LH and FSH pulses with significant coincidence, suggesting a signal-response relationship that GnRH is the primary secretagogue for

LH and FSH (Irvine and Alexander, 1994). The pulse frequency of GnRH affects the relative amounts of FSH and LH secreted. As GnRH frequency increases during estrus, LH secretion rate increases. In contrast, GnRH pulses are less rapid during diestrus while FSH increases. Thus GnRH frequency determines the LH: FSH ratio, being high during estrus and low during diestrus. Pulse frequency of both GnRH and gonadotropins increases during estrus leading to the ovulatory surge.

Gonadotropin releasing hormone secretion has been found to be about three times greater during the ovulatory surge than in mid-diestrus. The response of LH to

GnRH during the periovulatory surge was four times greater than during mid-diestrus phase, suggesting that changes in pituitary responsiveness to GnRH play a major role in generating the mare’s ovulatory LH surge. LuteinIzing hormone and FSH pulses measured in PV blood occur every 60-120 minutes in early estrus, increasing to 20-30 minutes as the peak of the ovulatory surge approaches (Irvine and

Alexander, 1994). 8

1.3.2 Regulation of GnRH

There are various different regulators of GnRH secretion, many of which have been recently discovered and/or are not completely understood. Environmental factors affect GnRH secretion (e.g.; photoperiod). As mentioned previously, melatonin is secreted during hours of darkness in the anestrous season of the mare which lowers GnRH secretion.

Opioids are peptides produced by the brain that modulate GnRH and gonadotropin secretion. Opioids, acting as neurotransmitters, suppress the release of norepinephrine in the vicinity of GnRH-secreting neurons, removing a stimulus of

GnRH secretion (McKinnon and Voss, 1993; Ginther, 1992). The suppression of

GnRH by opioids leads to an affect on gonadotropin release. They also mediate the progesterone-induced suppression of LH secretion. Naloxozone is a potent antagonist of the opioids and is used to test the physiological role of the opioids.

Naloxozone induces small rises in GnRH, LH, and FSH secretion in estrous mares, demonstrating the inhibitory role of opioids (Davison et al., 1998).

Circulating ovarian hormones regulate GnRH secretion as part of a hormonal feedback control mechanism (Figure 2). Progesterone is secreted from luteal structures, primarily the corpus luteum. Progesterone plays an inhibitory role on frequency of GnRH release via an opioid mediator as mentioned above. The role of estradiol in regulation of GnRH secretion is complex and controversial. Estradiol is produced by the maturing ovarian follicle. High estradiol concentrations inhibit

GnRH when progesterone levels are high, but exhibit a positive feedback effect on the hypothalamus, leading to an ovulatory LH surge, when progesterone levels are 9

low. In the mare estradiol administration alone does not lead to either an ovulatory

LH surge or a marked suppression of LH. Combined treatment of estradiol with

GnRH has been designed to mimic hormonal events of the periovulatory period,

when the relative bioactivity of plasma LH increases (Alexander et al., 1984). The combined estradiol-GnRH treatment results suggest that the interaction between

estradiol and GnRH may produce this normal increase in LH bioactivity (McKinnon

and Voss, 1993). Inhibin is a glycoprotein hormone that has a specific effect on the secretion of FSH but does not or only minimally influences LH secretion. Inhibin is

mainly secreted by the granulosa cells and theca cells of large follicles in mares

(Nagamine, 1998). Inhibin concentrations increase prior to the decline of the

follicular wave-stimulating FSH surge, indicating suppression of FSH before and

during selection of the dominant follicle. The circulating concentrations of FSH and inhibin are inversely related during estrous (Roser et al., 1994; Bergefelt et al.,

2001), and it has been shown that passive immunization against inhibin results in

increased plasma FSH concentrations (Nambo et al., 1998; Nagamine et al., 1998;

Donadeau and Ginther, 2001).

Figure 2. Hypothalamic-pituitary-gonadal axis feedback mechanisms in the female.

Reproduced with permission from Geisert, R., 2005

1.4 Endocrinology of the estrous cycle

1.4.1 Follicular phase

The profiles of GnRH, FSH and LH change with the seasonality of the mare’s

reproductive cycle. The start of the breeding season is triggered by the increased

length of daylight per day, resulting in a decrease of melatonin and an increase in

GnRH secretion. This increase in GnRH secretion induces LH and FSH secretion from the anterior pituitary. The estrous cycle is repeated every 22 days in a cycling 11

non-pregnant mare (Samper, 2000). The end of one estrous cycle and the start of another is marked by ovulation, often defined as Day 0. The beginning of the follicular phase is determined by the end of the luteal phase that occurs due to the death of the corpus luteum (luteolysis), and concomitant decrease of progesterone production. The emergence of follicular waves is attributed to a large increase in circulating concentrations of FSH. Follicle stimulating hormone exerts its effect by binding to FSH receptors on the granulosa cells of ovarian follicles. As a dominant follicle is selected increased systemic levels of estrogen and inhibin are released by the follicle, resulting in an FSH decrease via negative feedback at the hypothalamic- pituitary axis. After the selection of a dominant follicle there is a shift of the follicle from FSH responsiveness to LH responsiveness because the dominant follicle changes from primarily having FSH receptors to LH receptors. Increasing LH levels are attributed to positive feedback at the hypothalamic-pituitary axis by estrogen during periods of lower progesterone. Spontaneous ovulation results as the readied preovulatory follicle is exposed to high levels of LH. Following the pattern of the LH surge in spontaneous ovulation, many induced ovulation methods have been created (Ginther, 1992; Samper, 2000).

1.4.2 Luteal phase

The beginning of the luteal phase is attributed to the formation of a corpus luteum following ovulation. The rupture of the ovulatory follicle results in a decreased production of estrogen and inhibin, resulting in a decrease of both systemic LH and FSH. The decrease in LH is due to the combined effect of the declining positive estrogen feedback and the increasing negative progesterone 12

feedback. The transformation of granulosa cells into luteal cells (and the start of the

formation of a corpus luteum) occurs shortly after ovulation. The corpus luteum (CL)

forms approximately 0-5 days in early diestrus, is mature on estrous cycle days 6-

13, and regresses beginning on estrous cycle day 14-16 (Samper, 2000).

Termination of the luteal phase is a cascade of hormonal events with the end result

being synthesis and a peak release of PGF2α by the endometrium. The release of

PGF2α results in luteolysis (death of the corpus luteum). Systemic concentrations of

PGF2-alpha reach peak concentrations by day 14; and PGF2α levels then begin to

decrease concurrent with progesterone levels, signaling the end of the luteal phase

(Ginther, 1992; McKinnon and Voss, 1993). The end of the luteal phase transitions

the mare back into the follicular phase, starting the estrous cycle over again.

1.5 GnRH analogues/hormones used to control estrus

1.5.1 Cattle

The four commercially available GnRH analogues approved by the U.S. FDA for use in cattle are Ovacyst/Fertelin™ (by Phoenix Scientific), Fertagyl™ (by

Intervet), Cystorelin™ (by Merial) and Factrel™ (by Fort Dodge).

Ovacyst/Fertelin™, Fertagyl™, and Cystorelin™ have the same active ingredient, gonadorelin diacetate tetrahydrate, whereas gonadorelin hydrochloride is the active ingredient in Factrel™. These GnRH products have FDA approval for the treatment of ovarian follicular cysts in dairy cattle (Stewart et al., 2004). These products are also commonly used in protocols for synchronization of ovulation in cattle, although their only FDA-approved use is for treatment of ovarian cysts. 13

Reproductive inefficiency is a costly and production-limiting problem that

faces both the dairy and beef cattle industries. Synchronization of ovulation

programs using GnRH analogues have been developed to decrease days open,

optimize labor and service rate, and increase efficiency of artificial insemination (AI).

Three common GnRH protocols have emerged to synchronize ovulation and/or

estrus in cattle: OvSynch, CO-Synch, and Select Synch (Figure 3).

OvSynch

OvSynch (Pursley et al., 1995) was originally created for use in dairy cattle,

but the basic elements of the protocol also have benefits for use in beef cattle.

OvSynch starts with the administration of GnRH followed by PGF2α seven days

after GnRH and administration of a second GnRH injection two days after PGF2α

treatment. Cattle are then inseminated 16-24 hours after the second GnRH

injection. The first GnRH injection is to cause ovulation and the initiation of a new

follicular wave. PGF2α serves to lyse any CL, including those induced to form

following the first GnRH injection. The second GnRH injection is given to cause

ovulation, to be followed by insemination 16-24 hours after the second GnRH

injection (Kesler and Constantaras, 2004).

CO-Synch

The CO-Synch (Geary et al., 1998) protocol utilizes a similar strategy as

OvSynch, but uses a single timed fixed AI at the time of the 2nd GnRH injection. The

CO-Synch protocol involves a GnRH injection followed seven days later by PGF2α.

A second GnRH injection is given two days after PGF2α injection, and cows are 14

artificially inseminated in conjunction with the second GnRH injection (Patterson et

al., 2002; Kesler and Constantaras, 2004).

These GnRH protocols are designed to minimize the number of days that a

cow is left open and to maximize the financial outcome from each cow. Research

has shown the GnRH-based protocols OvSynch and CO-Synch should not be used

in heifers (Kesler and Constantaras, 2004). The protocols have beneficial effects in

cows but not in heifers or anestrous cows because of an inconsistent follicle wave

pattern and a lack of a PGF2α responsive CL (Yaniz et al., 2004).

Select Synch

Select Synch (Geary et al., 2000) includes an injection of GnRH followed by

PGF2α seven days later. Cows synchronized with the Select Synch protocol are

bred based upon the detection of estrus; usually 36 to 72 hours post PGF2α.

Detection of estrus is currently recommended to begin as early as 4d after GnRH injection and continue through 6 d after PGF2α (Kojima et al., 2000). The initial

GnRH injection provokes a preovulatory-like LH surge, and the injection of PGF2α

induces regression of corpora lutea if present (Patterson et al., 2002; Geary et al.,

2000).

Figure 3. Methods currently being used to synchronize ovulation in postpartum beef

cows: OvSynch, CO—Synch, and Select Synch.

Reproduced from Patterson et al., 2000.

1.5.2 Horses

Horses are often acknowledged to have lower reproductive efficiency than

that of other domestic animals (Johnson and Becker, 1993; Jones, 2006). The mare

has lower reproductive efficiency than other domestic animals due to natural factors

such as the seasonality of ovulation cycles, the erratic nature of the estrous cycles during transition periods, and variability in the duration of behavioral estrus between estrous cycles. Factors imposed by humans also contribute to this low reproductive inefficiency such as selection based upon a horse’s pedigree and/or performance

(and not on their reproductive performance) and an artificially hastened “breeding season” that precedes the horses’ natural breeding season (Ginther, 1992). 16

Few GnRH or GnRH analogue products are available on the market for horse breeders. The most commonly used GnRH products are deslorelin, a GnRH analogue marketed as Ovuplant™ by Fort Doge Animal Health and BioRelease™ by

BET Pharm.

hCG

Although not a GnRH analogue, human chorionic gonadotropin (hCG) is commonly used for induction of ovulation in the mare because of its ability to bind

LH receptors. Typically, hCG is administered to mares with a minimum ovarian follicle diameter of 35 mm as a single injection of 1500 to 3300 IU to cause ovulation within 48 hours. There is contradictory literature concerning the formation of anti- hCG antibodies after several successive hCG injections, resulting in a decline in the efficacy of hCG in timing or predicting ovulation. Briant et al. (2006) found the stimulatory effect of hCG on ovulation and LH was repeatable in three cycles during the season, suggesting that antibody formation may be functionally insignificant.

Barbacini et al. (2000) attributed the decrease in the ability of hCG to predict the time of ovulation to seasonality and not the repeated use of hCG. In contrast, Voss et al., (1975) showed that hCG treatment lengthened the time of estrus and increased the length of time from the onset of estrus until ovulation when used for three successive cycles. A more recent study by McCue et al. (2004) noted a significant linear trend for a decline in efficacy at inducing a predicted or timed ovulation as the number of hCG treatments increased within a season. Ovulation rate was significantly shorter (0-24 hours) after hCG in mares receiving the hormone on the third through fifth cycle of the year than for mares receiving hCG on the first 17

two cycles of the breeding season (48 hours). Sullivan et al. (1973) found the

efficacy of hCG to decrease in accurately timing ovulation when used more than

twice during a single breeding season. Duchamp et al. (1987) demonstrated that

14/16 mares treated with 3 successive injections of hCG showed a measurable rise

of antibodies. Roser et al. (1979) showed the anti-hCG antibodies can develop and

persist between 30 days to several months after 2-4 injections of hCG. The development of anti-hCG antibodies and decline in efficacy of timing ovulation create a need for a more dependable hormone to induce ovulation in mares.

PGF2α (and analogues)

The use of PGF2α (and analogues) to time ovulation has resulted in

inconsistent responses. Prostaglandin (and analogues) acts indirectly on the

hypophyseal-gonadal axis to affect ovulation. Prostaglandin is administered to lyse

the CL, therefore decreasing progesterone (P4) production. When P4 levels are

decreased GnRH storage is decreased and the GnRH release from the

hypothalamus causes the release of LH and FSH. Estradiol production by the

follicle increases as FSH stimulates follicle growth. This cascade of effects leads to

follicle maturation and ovulation. Squires et al. (1981) reported the increase of

GnRH, LH, and FSH pulses measured in both pituitary venous and jugular blood in

response to a PGF analogue. The inconsistency of PGF2α (and analogues) to time

ovulation was demonstrated in two different research studies. Savage and Liptrap

(1987) found that cycling mares (average follicle size 42.8 mm) treated with a

PGF2α analogue ovulated at an average of 41.8 hours after treatment. Harrison et 18

al. (1987) treated cyclic mares (average follicle size 42.0 mm) with a PGF2α

analogue and reported an average of 4.7 days until ovulation.

Deslorelin Ovuplant™

Ovuplant™ was first approved by the U.S. FDA in 1999 for use in hastening

ovulation in mares. Ovuplant™ is a controlled release subcutaneous implant. The

active ingredient in Ovuplant™ is deslorelin, a nonapeptide analogue of the natural

gonadotropin releasing hormone (European Agency for the Evaluation of Medicinal

Products, 2002). Ovuplant™ is administered in 2.1 mg doses to mares that have a

minimum ovarian follicle diameter of 35 mm. Meinert et al. (1993) reported that the deslorelin implant induced ovulation in 93% of mares 48 hours after insertion and

63% of ovulations occurred 36 to 48 hours after implantation. Endocrine profiles

showed a sharp elevation of LH and FSH with peak levels at 12 hours after

implantation and increased blood concentrations until 48 hours. Similar findings

were reported by Ganheim and Jöchle (1995), McKinnon et al. (1993), and Squires

et al. (1994).

A potential disadvantage to the use of deslorelin implants has been the need

to remove the implants within two days after ovulation to prevent follicular

suppression and delayed return to estrus. McCue et al. (2000) found that mares treated with deslorelin acetate exhibited decreased pituitary FSH secretion during the diestrous period following ovulation. The decreased FSH serum concentration may lead to decreased follicular development and a delay in the emergence of a dominant follicle, increasing the duration of the interovulatory period. Johnson et al.

(2002) obtained further evidence that deslorelin acetate treatment temporarily down 19

regulates the pituitary gland reporting deslorelin administration increased the interovulatory interval, consistently suppressed plasma LH and FSH concentrations, and resulted in a complete lack of responsiveness of anterior pituitary LH and FSH secretion to additional GnRH stimulation. Studies have shown that removal of the

deslorelin implant once ovulation is confirmed leads to no temporary down regulation

of the pituitary gland, thus eliminating follicular suppression and delayed return to

estrus due to the implant (Wendt et al., 2002; McCue et al., 2002). Removal of the

implant, however, may be objectionable and an inconvenience to mare practitioners

and mare owners.

The availability of deslorelin has affected its use in the U.S. equine breeding

industry. Ovuplant™ was withdrawn from the market and no longer available due to

non-compliance with specific FDA regulations. (AspenBio Pharma, Inc, 2007).

Deslorelin BioRelease

Recent research has demonstrated that a short term release deslorelin

product, BioRelease Deslorelin injection by BET Pharm, is effective in inducing

ovulation (Stich et al., 2004). The product is in a biocompatible liquid vehicle that is

administered in a single dose by intramuscular injection. Preliminary studies have

shown that BioRelease Deslorelin injections of 1.5 mg given to mares with ovarian

follicles with a diameter of 35 mm or greater results in ovulation within 48 hours

(Fleury et al., 2003; Stich et al., 2004). Kolling and Allen (2004) demonstrated that

0.75 mg or 1.5 mg doses were both successful in inducing ovulation within 48 hours

in the majority of mares treated with BioRelease Deslorelin injections and suggested

that this compound is a possible alternative to hCG and Ovuplant™. Further studies 20

with larger numbers of mares are needed to confirm the findings of previous

research studies.

Kisspeptin

Recent research has investigated the use of kisspeptin to hasten ovulation.

Kisspeptin acts primarily by regulating GnRH secretion via a G protein-coupled

receptor at the hypothalamic level, causing an increase in gonadotropin release.

Specifically, Kisspepetin-10 has been shown to induce robust LH responses (and to

a lower extent FSH) indicating its potential therapeutic use in pharmacological

manipulation of the female gonadotropic axis (Gutierrez-Pascual, E., et al., 2007;

Roa, J. et al., 2006; Messager, S. et al., 2005). Briant et al. (2006) administered 10

mg of human C-terminal decapeptide Kisspeptin-10, dissolved in sterile saline

intravenously (Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH2) when a follicle

reached 33 mm. Treated mares had shorter days from treatment to ovulation (2.00

± 1.3 days) than control mares (4.25 ± 1.5 days). The proportion of mares ovulating within 48 hours of injection were 6/8 (75%) for treatment mares and 2/8 (25%) for control mares (Briant et al., 2006). This study provides evidence that Kisspeptin induced ovulation in mares, but more research is required before its efficacy can be determined as an effective treatment in inducing ovulation.

1.5.3 Cystorelin™ (gonadorelin diacetate tetrahydrate)

For the purpose of this thesis, Cystorelin™ will be the GnRH product discussed. As mentioned previously the active ingredient of Cystorelin™ is gonadorelin diacetate tetrahydrate. Gonadorelin is a decapeptide composed of the sequence of amino acids 5-oxoPro-His-Trp-Ser-Tyr-Gly-Leu-Narg-Pro-Gly-NH2. 21

Gonadorelin is the hypothalamic releasing factor responsible for the release of gonadotropins from the anterior pituitary. The synthetic gonadorelin is physiologically and chemically identical to the endogenous bovine hypothalamic releasing factor (Merial.com). Cystorelin™ was used in many of the experiments to determine the effectiveness of the OvSynch protocol.

Pursley (1995) used Cystorelin™ as the GnRH analogue in an OvSynch protocol stating that the OvSynch protocol “could have a major impact on managing reproduction in lactating dairy cows, because it allows for AI to occur at a known time of ovulation and eliminates the need for detection of estrus”. Pursley et al.

(1997) demonstrated with Cystorelin™ that the OvSynch protocol reduced the median days to conception by 19 days; thus, synchronization of ovulation provided an effective way to manage reproduction in lactating dairy cows by eliminating the need for detection of estrus.

Martinez et al. (2003) evaluated the effects of Cystorelin™, Fertagyl™ and

Factrel™ on dairy and beef cattle. Cystorelin™ induced a greater LH release, resulting in a higher ovulatory rate in dairy cows, but not in beef heifers. Cystorelin™ caused a greater total LH release than Factrel™ (Stevenson et al., 2000). These results can be contributed to the fact that OvSynch protocol synchronizes follicular and luteal development in lactating cows but not in heifers as shown by Anderson

(1998) in which Cystorelin™ was the GnRH analogue used. Cystorelin™ has also been successfully used in Select-Synch (Stevenson et al., 2000; Geary et al., 2000; 22

Dejarnette et al., 2001), Co-Synch (Geary et al., 2001), and OvSynch (Pursley et al.,

1995) protocols.

1.6 Rationale for experiment

The mare is a domestic species with many challenges pinpointing the exact time of ovulation. The length of the period of estrus is highly variable, and the timing of ovulation within the period of estrus is also unpredictable. The use of hCG and deslorelin, as mentioned above, has adverse consequences and presents a challenge to the equine breeding industry. The cattle industry has many different

GnRH analogues available to potentially control the time of ovulation. At present, it seems that no one has tested the effect of Cystorelin™ in horses to control the time of ovulation. This experiment is designed to test the effectiveness of a cattle GnRH analogue, Cystorelin™, on inducing ovulation in the mare.

1.7 Literature Cited

Alexander, S.L., Irvine, C.H., 1991. Control of onset of breeding season in the mare and its artificial regulation by progesterone treatment. J. Reprod. Fertil. Suppl. 44, 307-318. Alexander, S.L., Riving, C.G.G., 1984. Alteration of relative bio:immonopotency of serum LH by GnRH and estradiol treatment in the mare. Proceedings of the Tenth International Congress of Animal Reproduction and Artificial Insemination, vol. 2, 1-4. Anderson, L.L., 1998. Strategies for artificial insemination of cattle at synchronized ovulation or synchronized estrus. Beef Research Report - Iowa State University, A.S. Leaflet R1550. AspenBio Parma, Inc., 2007. http://www.aspenbioinc.com/rdproduct/equine/index.html. Retrieved July, 2007. Barbacini, S., Zavaglia, G., Gulden, P., 2000. Retropspective study on the efficacy of hCG in an equine artificial insemination programme using frozen semen. Equine Vet Edu. 2:404-410. Bergfelt, D.R., Gastal, E.L., Ginther, O.J., 2001. Response of estradiol and inhibin to experimentally reduced luteinizing hormone during follicle deviation in mares. Biol. Reprod. 65, 426-432. Briant, C., Schneider, J., Gullaume, D., Ottogalli, M., Duchamp, G., Bruneau, B., Caraty, A., 2006. Kisspeptin induces ovulation in cycling Welsh pony mares. Anim. Reprod. Sci 94, 217-219. Conn, P.M., McArdle, C.A., Andrews, W.V., Huckle, W.R., 1987. The molecular basis of gonadotropin-releasing hormone (GnRH) action in the pituitary gonadotrope. Biol. Reprod. 36, 17-35. Davison, L.A., McManus, C.J., Fitzgerald, B.P., 1998. Gonadotropin response to naloxone in the mare: effect of time of year and reproductive status. Biol. Reprod. 59, 1195-1199. DeJarnette, J.M., Day, M.L., House, R.B., Wallace, R.A., Marshall, C.E., 2001. Effect of GnRH pretreatment on reproductive performance of postpartum suckled beef cows following synchronization. J. Anim. Sci. 79, 1675-1682. Donadeu, F.X., Ginther, O.J., 2001. Effect of number and diameter of follicles on plasma concentrations of inhibin and FSH in mares. Reproduction 121, 897- 903. Duchamp, G., Bour, B., Conbarnous, Y., Palmer, E., 1987. Alternative solutions to hCG induction of ovulation in the mare. J. Reprod. Fert., Suppl. 35, 221-228. European Agency for the Evaluation of Medicinal Products. Committee for Veterinary Medicinal Products. Deslorelin Acetate. Summary Report. 2002, 1- 3. Fitzgerald, B.P., I’Anson, H., Legan, S.J., Loy, R.G., 1985. Changes in patterns of luteinizing hormone secretion before and after the first ovulation in the postpartum mare. Biol. Reprod. 33, 316-323. 24

Fleury, P., Alonso, M.A., Alvarenga, M.A. Douglas, R.H., 2003. Intervals to ovulation after treatment with estradiol cypoinate (ECP) or biorelease deslorelin (BRT- Des). http://www.betpharm.com/doc/Fleury%20abstract.doc. Abstract Retrieved July, 2007. Gånheim, A., Gånheim, A., Jöchle, W., 1995. Acceleration and timing of fertile ovulation in cyclic mares with a deslorelin implant. Acta. Vet Scand. 36, 393- 400. Geary, T.W., Downing, E.R., Bruemmer, J.E., Whittier, J.C., 2000. Ovarian and estrous response of suckled beef cows to the Select Synch estrous synchronization protocol. Prof. Anim. Sci. 16, 1-5. Geary, T.W., Salverson, R.R., Whittier, J.C., 2001. Synchronization of ovulation using GnRH or hCG with the CO-Synch protocol in suckled beef cows. J. Anim. Sci. 79, 2536-2541. Ginther, O.J. Reproductive biology of the mare: basic and applied aspects, 2nd ed. Cross Plains, WI: Equiservices, 1992. Gutierrez-Pascual, E. Mariniez-Fuentes, A.J., Pinilla, L., Tnea-Sempere, M., Malagon, M.M., Castano, J.P., 2007. Direct pituiraty effects of Kisspeptin: Activation of gonadotrophs and somatotrophs and stimulation of luteinising hormone and growth hormone secretion. J. Neuroendocrinol. 19, 521-530. Hapgood, J.P., Sadie, H., van Biljon, W., Ronacher, K., 2005. Regulation of expression of mammalian gonadotrophin-releasing hormone receptor genes. J. Neuroendocrinol. 17, 619-638. Harrison, L.A., Squires, E.L., McKinnon, A.O., 1987. Acute effects of luprostiol on LH and FSH during estrus in cycling mares. Proceedings of the 10th Equine Nutrition and Physiology Symposium, 265-69. Irvine, C.H., 1979. Kinetics of gonadotrophins in the mare. J. Reprod. Fertil. Suppl. 27, 131-141. Irvine, C.H.G., Alexander, S.L., 1993. Secretory patterns and rates of gonadotropin- releasing hormone, follicle-stimulating hormone, and luteinizing hormone revealed by intensive sampling of pituitary venous blood in the luteal phase mare. Endocrinology 132, 212-218. Irvine, C.H.G., Alexander, S.L., 1994. The dynamics of gonadotrophin-releasing hormone, LH and FSH secretion during the spontaneous ovulatory surge of the mare as revealed by intensive sampling of pituitary venous blood. J. Endocrinol. 140, 283-295. Jöchle, W., Irvine, C.H.G., Alexander, S.L., Newby, T.J., 1987. Release of LH, FSH, and GnRH into pituitary venous blood in mares treated with a PGF analogue, luprostiol, during the transition period. J. Reprod. Fert., Suppl. 25, 261-267. Johnson, C.A., Thompson, D.L., Cartmill, J.A., 2002. Pituitary responsiveness to GnRH in mares following deslorelin acetate implantation to hasten ovulation. J. Anim. Sci. 80:2681-2687. Johnson, A.L., Becker, S.E., 1993. Hormonal control of ovulation in the mare. Anim. Rep. Sci. 33, 209-226. 25

Jones, S.M., Troxel, T.R., 2006. Understanding reproductive physiology and anatomy of the mare. University of Arkansas, Division of Agriculture, Cooperative Extension Service, FSA3039, pp. 1-4. Kesler, D.J., Constantaras, M., 2004. Estrus synchronization systems: GnRH. Proc. Applied Reproductive Strategies in Beef Cattle Symposium pp. 41-52. Kojima, F.N., Salfe, B.E., Bader, J.F., Ricke, W.A., Lucy, M.C., Smith, M.F., Patterson, D.J., 2000. Development of an estrus synchronization protocol for beef catle with short-term feeding of melengestrol acetate: 7-11 synch. J. Anim. Sci. 78: 2186-2191. Kollin, M. Allen, W.R., 2005. Ovulation induction for embryo transfer: hCG versus GnRH analogue. International Equine Gametes Group Workshop II. Rostock, Germany. Martínez, M.F., Mapletoft, R.J., Kastelic, J.P., Carruthers, T., 2003. The effects of 3 gonadorelin products on luteinizing hormone release, ovulation, and follicular wave emergence in cattle. Can. Vet J. 44, 125-131. Messager, S. Chatzidaki, E., Ma, D., Hendrick, A., Zahn, D., Dixon, J., Thresher, R., Malinge, I., Lomet, D., Carlton, M., Colledge, W., Caraty, A., Aparicio, S., 2005. Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54. Proceedings of the National Academy of Sciences of the United States of America, Feb 1, vol. 102, no.5, 1761-1766. McCue, P.M., Hudson, J.J., Bruemmer, J.E., Squires, E.L., 2004. Efficacy of hCG at inducing ovulation: a new look at an old issue. In: 50th Annual Convention of the American Association of Equine Practitioners, Denver, Colorado (Ed.) Publisher: American Association of Equine Practitioners, Lexington, KY, pp. 1492-1204. McCue, P.M., Farquhar, V.J., Carnevale, E.M., Squire, E.L., 2002. Removal of deslorelin (Ovuplant™) implant 48 h after administration results in normal interovulatory intervals in mares. Therio.58, 865-870. McCue P.M., Farquhar, V.J. Squires, E.L., 2000. Effect of the GnRH agonist deslorelin acetate on pituitary function and follicular development in the mare. AAEP Proceedings, 2000, Vol., 46 355-356. McKinnon, A.O., Nobelius, A.M., del Marmol Figueroa, S.T., Skidmore, J., Vasey, J.R., Trigg, T.E., 1993. Predictable ovulation in mares treated with an implant of the GnRH analogue deslorelin. Equine Vet J. 25, 321-323. McKinnon, A.O., Voss J.L. Equine Reproduction. Philadelphia: Lea & Febiger, 1993. Meinert, C., Silva, J.F., Kroetz, I., Klug, E., Trigg, T.E., Hoppen, H.O., Jochle, W., 1993. Advancing the time of ovulation in the mare with a short-term implant releasing the GnRH analogue deslorelin. Equine Vet J. 25, 65-68. Millar, R.P., Lu, Z.L., Pawson, A.J., Flanagan, C.A., Morgan, K., Maudsley, S.R., 2004. Gonadotropin-releasing hormone receptors. Endocrine Rev. 25, 235- 275. Mumford, E.L., Squire, E.L., Jochle, E., Harrison, L.A., Net, T.M., Trigg, T.E., 1995. Use of deslorelin short-term implants to induce ovulation in cycling mares during three consecutive estrous cycles. Anim. Reprod. Sci. 39, 129-140. 26

Nagamine, N., Nambo, Y., Nagata, S., Nagaoka, K., Tsuno, N., Taniyama, H., Tanaka, Y., Tohei, A., Watanabe, G., Taya, K., 1998. Inhibin secretion in the mare: localization of inhibin α, βA, and βB subunits in the ovary. Biol. Reprod. 59, 1392-1398. Nagy, P., Guillaume, D., Daels, P., 2000. Seasonality in mares. Anim. Reprod. Sci. 60-61, 245-262. Nambo, Y., Kaneko, H., Nagata, S., Oikawa, M., Yoshihara, T., Nagamine, N., Watanabe, G., Taya, K., 1998. Effect of passive immunization against inhibin on FSH secretion, folliculogenesis and ovulation rate during the follicular phase of the estrous cycle in mares. Theriogenology 50, 545-557. Patterson, D.J., Kojima, F.N., Smith, M.F., 2003. A review of methods to synchronize estrus in replacement beef heifers and postpartum cows. J. Anim. Sci. 81, E166-E177. Pursley, J.R., Kosorok, M.R., Wiltbank, M.C., 1997. Reproductive management of lactating dairy cows using synchronization of ovulation. J. Dairy Sci. 80, 301- 306. Pursley, J.R., Mee, M.O., Wiltbank, M.C., 1995. Synchronization of ovulation in dairy cows using PGF2α and GnRH. Theriogenology 44, 915-923. Roa, J., Vigo, E., Castellano, J.M., Navarro, V.M., Fernandez-Gernandez, R., Casaneueva, F.F., Dieguez, C., Aguilar, E., Pinilla, L., Tena-Sempere, M., 2006. Hypothalamic expression of KiSS-1 system and gonadotropin-releasing effects of Kisspeptin in different reproductive states of the female rat. Endocrin. 147(6), 2864-2878. Roser, J.F., Kiefer, G.L., Evans J.W., Neely, D.P., Pacheco, D.A., 1979. The development of antibodies to human chorionic gonadotrophin following its repeated injection in the cyclic mare. J. Reprod. Fertil. Suppl. 27,173-9. Roser, J.F., McCue, P.M., Hoye, E., 1994. Inhibin activity in the mare and stallion. Dom. Anim. Endocrinol. 11, 87-100. Samper, J.C. Equine Breeding Management and Artificial Insemination. Philadelphia: W.B. Saunders Company, 2000. Savage, N.C. and Liptrap, R.M., 1987. Induction of ovulation in cyclic mares by administration of a synthetic prostaglandin , fenprostalene, during oestrus. J. Reprod. Fertil., Suppl., 35, 239-243. Schally, A.V., Arimura, A., Baba, Y., Nair, R.M., Matsuo, H., Redding, T.W., Debeljuk, L., 1971. Isolation and properties of the FSH and LH-releasing hormone. Biochem. Biophys. Res. Comm. 43, 393-399. Squires, E.L., Moran, D.M., Farlin, M.E., Jasko, D.J., Keefe, T.J., Meyers, S.A., Figueiredo, E., McCue, P.M., Jochle W., 1994. Effect of dose of GnRH analogue on ovulation in mares. Theriogenology 41, 757-769. Squires, E.L., Wallace, R.A., Boss, J.L., Picken, B.W., Shideler, R.K., 1981. The effectiveness of PGF2alpha, HCG and GnRH for appointment breeding of mares. J. Equine. Vet Sci. 1, 5-9. Stevenson, J.S., Thompson, K.E., Forbes, W.L., Lamb, G.C., Grieger, D.M., Corah, L.R., 2000. Synchronizing estrus and (or) ovulation in beef cows after 27

combinations of GnRH, norgestomet, and prostaglandin F2α with or without timed insemination. J. Anim. Sci. 78, 1747-1758. Stewart, S., Rapnicki, P., Fricke, P.M., 2004. Dairy reproductive synchronization notes. Proc. Minnesota Dairy Herd Health Conference. May 20, Minneapolis, MN. Stich, K.L., Wendt, K.M. Blandchard, T.L., Brinsko, S.P., 2004. Effects of a new injectable short-term release deslorelin in foal-heat mares. Theriogenology. 62, 831-836. Sullivan, J.J., Parker, W.G., Larson, L.L., 1973. Duration of estrus and ovulation time in nonlactating mares given human chorionic gonadotropin during three successive estrous periods. J. Am. Vet Med. Assoc. 162, 895-898. Voss, J.L., Sullivan, J.J., Pickett, B.W., Parker, W.G., Burwash, L.D., Larson, L.L., 1975. The effect of HCG [sic] on duration of oestrus, ovulation time and fertility in mares. J. Reprod. Fertil., Suppl. 23, 297-301. Wendt, K.M., Stich, K.L., Blanchard, T.L., 2002. Effects of deslorelin administration in vulvar mucosa, with removal in 2 days, in foal-heat mares. AAEP proceedings, vol. 48, 61-64. Yániz, J.L., Murugavel, K., López-Gatius, F., 2004. Recent developments in oestrous synchronization of postpartum dairy cows with and without ovarian disorders. Reprod. Dom. Anim. 39, 86-93.

CHAPTER 2. INDUCTION OF OVULATION AND LH RESPONSE IN CYCLIC

MARES TREATED WITH CYSTORELIN™

INTRODUCTION

Regulating the time of ovulation in the mare has many practical and beneficial applications. The increasing use of artificial insemination technologies in the equine industry has increased the importance of controlling the time of ovulation to improve management and labor efficiency, optimize the time of breeding when using transported semen, and reduce the number of times a mare is bred during a given estrous cycle. In cyclic mares, human chorionic gonadotropin (hCG), prostaglandin

F2alpha (PGF2α), and gonadotropin releasing hormone (GnRH) analogues have been used in attempts to control the time of ovulation. A new product to time ovulation in the mare, EquiPure-LH (by AspenBio Pharma), is being investigated in the U.S. but research data on its efficacy has not been published.

The repeated use of hCG to control the time of ovulation has yielded inconsistent results, largely due to its antigenic effects in the mare (Voss et al., 1975;

McCue et al., 2004; Sullivan et al., 1973; Duchamp et al., 1987; Roser et al., 1979).

The use of PGF2α (and analogues) to time ovulation has also resulted in inconsistent responses (Harrison et al.,1987; Savage and Liptrap, 1987; Squires et al.,1981). The GnRH analogue deslorelin (Ovuplant™) reportedly causes suppressed gonadotropin secretion and desensitization to GnRH, which leads to an extended interval between induced ovulation and the subsequent ovulation, when

Ovuplant™ is left in place for over 48 hours and the mare does not become pregnant

(Johnson et al., 2002; McCue et al., 2000). 29

In other species such as cattle, exogenous GnRH has been used to effectively induce ovulation (Pursley et al., 1995; Geary et al., 2000). The objective of the present study was to determine the effect of Cystorelin™ (gonadorelin diacetate tetrahydrate) on induction of ovulation and LH response in cyclic mares.

MATERIALS AND METHODS

Experimental Animals

A total of 24 Iowa State University mares was used in this study. Mares were of Thoroughbred (n=9) and Stock type (Paint and Quarter Horse; n=15) breeding.

The first trial (Trial A) was performed during the 2006 breeding season (May-June)

using 6 mares, and the second trial (Trial B) was performed during the 2007

breeding season (February-May) using 18 mares. Throughout each breeding

season mares were teased to a stallion to identify estrus. When mares showed

signs of estrus they were evaluated daily or every other day via transrectal

ultrasonography to monitor ovarian follicle size. Mares were kept in individual stalls

during treatment periods with free choice hay and water. All procedures performed

with animals were reviewed and approved by the institutional animal care and use

committee.

Treatments

Mares possessing an ovarian follicle 3.5 cm to 4.0 cm in diameter were

randomly assigned to either treatment with gonadorelin diacetate tetrahydrate

(Cystorelin™, Merial)1 or with sterile saline (control). Treatment mares were given a

75 µg (1.5 mL) i.m. injection of Cystorelin™ in the neck at 0, 120, and 240 minutes.

Control mares were given three 1.5 mL injections of sterile saline at 0, 120, and 240

minutes. After the start of treatment, ultrasonography was performed daily until

1 Cystorelin™ is not approved by the U.S. Food and Drug Administration (FDA) for use in horses, and FDA has not determined that the product is safe and effective in horses. 31 ovulation was confirmed. Across trials 17 treatment mares and 7 control mares were used.

Blood sample collection

Blood samples were collected from treatment and control mares via an indwelling jugular catheter according to the timetable shown in Table 1. Ten mL of blood was drawn 30 minutes prior to the initial injection (GnRH or saline), immediately prior to the initial injection, and at 30-minute intervals after the initial injection for six (Trial A) or 10 (Trial B) hours to measure LH. In trial B, an additional blood sample was collected daily starting the day after treatment until ovulation was confirmed. Blood samples were placed in heparinized tubes and were refrigerated briefly until centrifugation for 25 min at 3,000 RPM. Plasma was harvested and stored frozen until assayed for LH.

Table 1. Timeline for blood sample collection and treatment with Cystorelin™

Day 1 (start of treatment)

Time of day Cystorelin™ injection Blood sample Time line (min)

1130 1 (prior to Cystorelin™) -30

1200 1 2 (prior to Cystorelin™) 0

1230 3 30

1300 4 60

1330 5 90

1400 2 6 120

1430 7 150

1500 8 180

1530 9 210

1600 3 10 240

1630 11 270

1700 12 300

1730 13 330

1800* 14 360

1830 15 390

1900 16 420

1930 17 450

2000 18 480

2030 19 510

2100 20 540

2130 21 570

2200 22 600

* last blood sample for Trial A

Day 2 and daily afterwards until ovulation was documented

Time of day Blood sample number Time line (min)

0900 23 1260

0900 24 2700

0900 25 4140

Assay of plasma LH levels

Luteinizing hormone was measured in plasma by radioimmunoassay

(Thomspon et al., 1983). Intra-and interassay coefficients of variation and assay sensitivities were 6%, 9%, and 0.2 ng/mL, respectively.

Data Analysis

Data were analyzed using the GLM procedure of SAS for a completely randomized design (SAS Inst. Inc., Cary, NC) to test the effect of treatment and breed on days to ovulation and plasma LH levels. LH means were calculated over four time periods [-30 to 0 (Period 1 (baseline mean)), 30-120 (Period 2), 150-240

(Period 3), 270-360 min (Period 4)], and the LH response was analyzed by comparing the mean LH value of Period 2, 3, and 4 with Period 1 (baseline mean).

Levene’s test (Levene, 1960) was used to analyze the variability in days to ovulation between treatment and control mares.

RESULTS

Days to ovulation

The effect of year was tested and found to be non significant (P>.57), so

data were pooled across years. The diameter of the largest follicle at the time of

treatment was not different (P>.42) between treatment (3.8 ± .03 cm) and control

(3.8 ± .06) mares. Mares treated with Cystorelin™ ovulated 2.25 ± .25 days after

treatment which was one day earlier (P<.05) than control mares (3.25 ± .41). Of the

treatment mares, 71% (12/17) ovulated within 48 hours after treatment compared with 14% (1/7) of control mares (Table 2). Treatment and control mares were not different (P>.81) for variability in days to ovulation.

Table 2. Ovarian status and response to treatment with either Cystorelin™ or saline.

Variable Cystorelin™ Control mares mares Follicle size (cm) 3.8 ± .03 3.8 ± .06 at treatment*

Days to 2.25 ± .25a 3.25 ± .41b ovulation*

Hours to ovulation (post treatment) 24 6% (1/17) 0% (0/7) 48 65% (11/17) 14% (1/7) 72 6% (1/17) 43% (3/7) ≥96 12% (2/17) 43% (3/7) No ovulation 12% (2/17) 0% (0/7) *Mean ± standard error of the mean (SEM) a,b Means with unlike superscripts differ (P<.05)

LH response

Plasma LH values from time -30 to 360 minutes for Trial A and Trial B were analyzed for statistical analysis. Mean plasma LH levels were calculated over four time periods [-30 to 0 (Period 1 (baseline mean)), 30-120 (Period 2), 150-240

(Period 3), 270-360 min (Period 4)], and LH response was analyzed by comparing the mean LH value of Period 2, 3, and 4 with Period 1 (baseline mean). Although the level in Period 2 tended (P<.07) to be higher than baseline, LH levels in Periods

3 and 4 were not different from baseline (P>.15, P>.18 respectively) (Figure 7). The

LH profiles of individual mares are shown in Figures 4 and 5, and mean LH values averaged across treatments are illustrated in Figure 6.

Figure 4. LH profiles of individual mares treated with Cystorelin™.

Injection @ 0 Injection @ 120 Injection @ 240 20.0

18.0 A B C 16.0 D E 14.0 F G 12.0 H I 10.0 J K LH, ng/mL LH, 8.0 L M N 6.0 O P 4.0 Q

2.0

0.0 -30 0 30 60 90 120 150 180 210 240 270 300 330 360 Time (min) 36

Figure 5. LH profiles of individual mares treated with saline.

20.0 Injection @ 0 Injection @ 120 Injection @ 240

18.0

16.0

14.0 R 12.0 S T 10.0 U V LH, ng/mL 8.0 W X 6.0

4.0

2.0

0.0 -30 0 30 60 90 120 150 180 210 240 270 300 330 360 Time (min)

Figure 6. Average LH response of treatment and control mares.

Injection @ 0 Injection @ 120 Injection @ 240 4.5

4.0

3.5

3.0

2.5 Control Treatment 2.0 LH ng/mL

1.5

1.0

0.5

0.0 -30 0 30 60 90 120 150 180 210 240 270 300 330 360 Time (min)

Figure 7. Mean plasma LH increase over time between treatment and control mares.

3.5

2.5

2 Treatment Control

LH ng/mL 1.5

0.5

0 Period 2 - Period 1 Period 3 - Period 1 Period 4 - Period 1 Differences in LH from Period 1 (baseline)

DISCUSSION

Follicle diameter at the start of treatment (3.8 cm ± .03) in our experimental mares was comparable to that reported by others when using deslorelin (Ovuplant™) to induce ovulation (Squires et al., 1994; Mumford et al., 1995; McCue et al., 2000;

Wendt et al., 2002). Our Cystorelin™ protocol was effective in hastening ovulation in mares by one day. The result that 71% of treated mares ovulated within 48 hours after the Cystorelin™ injections is comparable to results obtained by others who investigated hCG (Sullivan et al., 1973; McCue et al., 2004) and deslorelin

BioRelease™ (Stich et al., 2004). Variability in days to ovulation was similar between Cystorelin™ treated and control mares (P>.81), but this may be due to the relatively small number of experimental animals used in this study.

Recent research has investigated the use of Kisspeptin to hasten ovulation

(Briant et al., 2006). Treatment mares had shorter days from treatment to ovulation

(2.00 ± 1.3 days) than control mares (4.25 ± 1.5 days). The proportion of mares ovulating within 48 hours of injection was 6/8 (75%) for treatment mares and 2/8

(25%) for control mares, similar to our results. Kisspepetin-10 has been shown to induce robust LH responses (and to a lower extent FSH) indicating its potential therapeutic use in pharmacological manipulation of the female gonadotropic axis

(Gutierrez-Pascual et al., 2007; Roa et al., 2006; Messager et al., 2005).

Luteinizing hormone secretion patterns were similar between control and treatment mares, yet 71% of Cystorelin™ treated mares ovulated within 48 hours of treatment compared with 14% for control mares. Although LH response was not statistically significant between treatment and control mares, there was a tendency 40

(P<.07) for elevated LH level after the first Cystorelin™ treatment (Figure 6). The lack of a significant increase in plasma LH responses could be due to the low dose of gonadorelin, individual variation among mares, or the relatively small number of

experimental animals used in our study. Interestingly, inconsistencies in LH response have been previously demonstrated. Harrison et al. (1987) administered

7.5 mg of luprostiol or placebo to mares on the first day estrus was exhibited, but

gonadotropin levels did not significantly change in response to luprostiol injection.

Jöchle et al. (1987) administered 7.5 mg of luprostiol to mares in late anestrus or the

transitional phase. In 7 out of the 8 mares treated with luprostiol there was an

increase in gonadotropin concentrations in jugular blood. Evans et al. (2006)

performed an hCG dose-response study (2500, 1500, 500, or 0 IU hCG) for three

successive cycles. Human chorionic gonadotropin or saline was administered when

the largest follicle reached ≥ 35 mm, and no effect of treatment was found on mean

LH level. Treatment with 2500 and 1500 IU of hCG, however, advanced the onset of

the endogenous LH surge, and the profile of the advanced LH surge was similar to that associated with spontaneous ovulation. These results imply that flexibility is present for the timing of the normal cascade of endocrine events which result in the ovulatory LH surge. Furthermore, those researchers demonstrated that an endogenous LH surge may not be needed to cause ovulation of the dominant follicle.

Briant et al. (2003) performed two experiments on the use of a GnRH

antagonist with or without the use of hCG to control ovulation in mares. For

Experiment 1, mares were treated with vehicle or antarelix for 3 days when the 41 largest follicle reached 32 mm. For Experiment 2, mares were treated with vehicle or antarelix for 6 days, and hCG (1600 IU, i.v.) was injected one day after start of treatment. Fifty percent of mares treated with antarelix for three days ovulated an average of one day earlier than controls, but the other 50% did not ovulate. All mares treated with antarelix for 6 days ovulated the same time as control mares (2.8

± 0.2 days after the beginning of treatment). In both experiments mares treated with antarelix had suppressed LH after start of treatment, and the LH surge was completely abolished compared to controls, this despite the fact that ovulation occurred in some of the antarelix mares. Briant et al. (2003) concluded that short and small elevations in plasma LH concentration in mares are sufficient to resume terminal follicular growth and ovulation. Our data supports the findings of Evans et al. (2006) and Briant et al. (2003).

Data from this experiment suggest that Cystorelin™ could be used in equine breeding management to hasten ovulation in mares. Although we did not test this in our study, Cystorelin™ treatment potentially could be used to overcome problematic hCG antibody formation (as well as availability issues with deslorelin products). This treatment protocol is practical for horse breeders to use and eliminates the inconvenience of needing a veterinarian to inject and remove Ovuplant™. Further studies with increased mare numbers need to be conducted before recommending the use of Cystorelin™ in inducing ovulation in cyclic mares. For example, few of the mares used in our study were bred due to the timing of the experiment and farm breeding management decisions, so data on post-treatment pregnancy rates are 42 needed. Additional studies with slightly different protocols aimed at reducing variability on the time of ovulation are also warranted.

In conclusion, three 75 µg doses of Cystorelin™, administered at two-hour intervals to mares possessing an ovarian follicle between 35 and 40 mm in diameter are effective in hastening ovulation.

LITERATURE CITED

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ACKNOWLEDEGMENTS

Cystorelin™ was kindly provided by Dr. Joe Dedrickson at Merial. The LH assay was kindly provided by Dr. Don Thompson at Louisiana State University.

Thank you to all of my friends and family. Without your love and support this would not have been possible.

I would like to thank my fiancé, Josh Benton, for his help and support during my strive to continue my education.

Thank you to my major professors Dr. Peggy-Miller Auwerda and Dr. Curtis Youngs for their time, leadership, and dedication to my master’s program. Thank you to my committee as a whole, Dr. Peggy Miller-Auwerda, Dr. Curtis Youngs, Dr. Carolyn Komar, and Dr. Lawrence Evans, for your guidance and advice through my master’s program.

Thank you to Dr. Phillip Dixon for his aid with statistical analysis.

Thank you to the horse barn manager, Angela Chandler, for her time and her support of my project.

Thank you to the undergraduate students who contributed their time to this project: Alex Novotny, Kirsty Husby, Chelsey Messerschmidt, Megan Anderson and Justin Bisinger.

I would also like to thank Dr. Maynard Hogberg for his dedication to seeing me finish my master’s program.

A special thank you Dr. Lawrence Evans, DVM. It was his idea that made this project a reality, and his time and dedication to the project that made it possible.