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Estrus synchronization protocols for planned breeding and GnRH-agonist after timed insemination for pregnancy… Mohamed, Hirad 2000

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ESTRUS SYNCHRONIZATION P R O T O C O L S F O R PLANNED BREEDING AND GnRH-AGONIST A F T E R T IMED INSEMINATION F O R P R E G N A N C Y R A T E E N H A N C E M E N T IN DAIRY C A T T L E by HIRAD M O H A M E D B. V.Sc, Somali National University, Mogadishu, Somalia, 1984 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T O F T H E REQUIREMENTS FOR T H E D E G R E E O F M A S T E R O F SCIENCE in THE FACULTY OF GRADUATE STUDIES (Faculty of Agricultural Sciences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 2000 & Hirad Mohamed, 2000 In presenting this thesis in partial fulfilment of the requirements for an ^advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date $\1\6<AS\ % | . 3-0° DE-6 (2/88) ABSTRACT The aim of was to develop management protocols to improve reproductive efficiency in dairy cattle. The objective of Experiment 1 was to compare two methods of estrus synchronization. Eighty-nine dairy animals (67 cows and 22 heifers) were randomly assigned to either the Ovsynch or the Prostaglandin F 2 a (PGF 2a) treatment group. Animals in the Ovsynch group received gonadotropin-releasing hormone (GnRH) followed by PGF 2 a on day 7 and second GnRH on day 9. Animals in the PGF 2 a group were given two injections of PGF2 o t 14 days apart. Both synchronization methods were followed by timed artificial inseminations. Milk progesterone (P4) concentrations during the cycle were used to asses corpus Luteum (CL) function. Pregnancy was diagnosed 35 days after insemination. Estrus synchrony, based on Kamar heat mount detector and milk P4 concentrations < lng/ml on the day of breeding, was not significantly different between the two groups. Mean milk P4 during the synchronized cycle was also not significantly different between the two groups. Pregnancy rates were higher (P<0.05) for cows in the Ovsynch group (57.5%) than those in the PGF 2 a group (38.4%). No difference in pregnancy rate was observed in heifers. The objective of Experiment II was to determine the effect of treatment with GnRH at various times after breeding on the induction of accessory CL, P4 concentrations and pregnancy rates. Each month, estrus was synchronized in 12 to 15 postpartum cows using the Ovsynch protocol followed by timed inseminations. Cows were then randomly assigned to receive GnRH on d 7 (n=34), d 14 (n=34), or d 7 and 14 (n=35) after breeding or to serve as controls (n=33). During the first two months, six cows from each group underwent ultrasonographic examination of the ovaries on days, 7, 11, 14, 18, and 21 after insemination to observe the formation of any additional CL. Blood and milk samples were also taken at these times to determine P4 concentrations. Pregnancy was diagnosed 35 days after insemination. Eighty-three percent of cows ovulated in response to GnRH on d 7 as opposed to only 17 percent on d 14. Mean milk P4 ii concentrations in treated cows did not differ from control cows. Pregnancy rates did not differ between the groups. It is concluded from this study that a) pregnancy rates of dairy cows and heifers can be managed effectively without estrus detection by using the Ovsynch estrus synchronization protocol followed by timed artificial inseminations and b) treatment with GnRH following synchronized breeding does not increase pregnancy rates in postpartum dairy cows. iii TABLE OF CONTENTS ABSTRACT _ ii TABLE OF CONTENTS _________________ ___ iv LIST OF TABLES ~ - vii LIST OF FIGURES — — viii ABBREVIATIONS — - - — — — - - - - - ix ACKNOWLEDGMENT — — — XI FORWARD - ix DEDICATION • ix CHAPTER .1 GENERAL INTRODUCTION - 1 1.1. The objectives 3 CHAPTER. 2 LITERATURE REVIEW 5 2.1. The Bovine Estrous Cycle — — — 5 2.1.1 Hormonal control of the estrus cycle _ _ 6 2.1.2. Ovarian follicular wave dynamics in cattle 10 2.2. Estrus Synchronization in Cattle 14 2.2.1. Advantages of Estrus Synchronization __ 14 2.2.2. Methods of Estrus Synchronization 14 2.2.2.1. Long Term Progestogens ._ __ _ - 15 2.2.2.2. Progestogen and Estrogen Combinations _ _ 16 2.2.2.3. Prostaglandins 18 2.2.2.3.1. Single Prostaglandin Injections _ _ _ .18 2.2.2.3.2. Double Prostaglandin Injections __ _ 19 2.2.2.4. Progestogens and Prostaglandin Combination Programs 20 2.2.2.5. Synchronization of Ovulation 21 2.3. Early Embryonic Mortality in Cattle 23 2.3.1. Predisposing Factors of Early Embryonic Mortality 24 2.3.2. Approaches to Enhance Pregnancy in Cattle ~ 28 2.3.2.1. Progesterone Supplementation -—— -28 2.3.2.2. Uterine Infusion of Embryonic Vesicles or Products 29 2.3.2.3. Gonadotropins _ _ 30 2.3.3.4. Gonadotropin-Releasing Hormone 32 iv CHAPTER .3 A COMPARISON OF TWO ESTRUS SYNCHRONIZATION PROTOCOLS FOR PLANNED BREEDING IN DAIRY CATTLE 34 3.1. ABSTRACT 34 3.2. INTRODUCTION ___ ______ 35 3.3. MATERIALS AND METHODS _ _ 36 3.3.1. Animals: General Management Practices — — 36 3.3.2. Estrus Synchronization Treatments _.. 37 3.3.3. Milk Sampling and Progesterone Assay 38 3.3.4. Radioimmunoassay Kit Validation 38 3.3.4. Statistical Analysis — 39 3.4. RESULTS - - - 40 3.4.1. Estrus Synchronization — 40 3.4.2. Corpus Luteum Function — — — — — — 41 3.4.3-.Pregnancy Rates —— 41 3.5. DISCUSSION — - 47 CHAPTER. 4 EFFECTS OF GONADOTROPIN RELEASING HORMONE AGONIST GIVEN ON DAY 7, DAY 14, OR DAY 7 AND 14 After BREEDING ON PROGESTERONE PROFILE, OVULATION AND PREGNANCY RATE 52 4.1. ABSTRACT — - - - 52 4.2. INTRODUCTION - - 53 4.3. MATERIALS AND METHODS- 54 4.3.1. Animals: General Management Practices .._ 54 4.3.2. Treatments _ _ 55 4.3.3. Ultrasound Examination 55 4.3.4. Blood and Milk Sample Collection 57 4.3.5. Radioimmunoassay. _ . 57 4.3.6. Pregnancy Diagnosis 57 4.3.7. Statistical Analysis _._ 57 4.4- RESULTS ~ - ~ 58 4.4.1. Induction of Accessory Corpus Luteum (CL) 58 4.4.2. Plasma Progesterone Profile ~ ~ — 59 4.4.3. Milk progesterone Profile ~ " — 59 4.4.4. Pregnancy Rate — 59 4.5. DISCUSSION 64 CHAPTER 5. GENERAL DISCUSSION ___. 71 REFERENCES. 81 LIST OF TABLES Table 4.1. Number of cows ovulated after GriRH administered on day 7, day 14, day 7 and 14 after breeding in comparison to untreated cows — - 60 Table 4.2. Effect of GnRH given on day 7, day 14, day 7 and 14 or control group on pregnancy rate 63 vi LIST OF FIGURES Figure 2.1. Changes in blood plasma hormone concentrations during the bovine estrous cycle — - 9 Figure. 2.2. Pattern of growth and regression of dominant and subordinate follicles in heifers exhibiting two (A) or three (B) waves of follicular growth during the bovine estrous cycle 13 Figure 3.1. Estrus synchrony based on kamar heat mount detector (A) and milk progesterone profile (B) between animals treated with two injections of PGF 2 a and animals synchronized with Ovsynch. protocol - - - — - 42 Figure 3.2. Effect of treatment and milk production on estrus synchronization between cows synchronized with two injections of PGF 2 a and cows synchronized with Ovsynch protocol 43 Figure 3.3. Milk progesterone profile (Mean + SE) of cows synchronized with two injections of PGF 2 a and animals synchronized with Ovsynch protocol 44 Figure 3.4. Pregnancy rate between lactating dairy cows and heifers synchronized with two injections of PGF 2 a and animals synchronized with Ovsynch protocol 45 Figure 3.5. Pregnancy percentage between cows synchronized with two injections of PGF 2 a and cows synchronized with Ovsynch protocol by milk production (high Vs low) at the time of breeding 46 Figure 4.1. Mean (SE±) plasma progesterone concentrations in control group (G 1) and cows given GnRH on D 7 (G 2), D 14(G 3), D 7&14 (G 4) after breeding 61 Figure 4.2. Mean (±SE) milk progesterone concentrations in control group (G 1) and cows given GnRH on D (G 2)) D 14(G 3), D 7&14 (G 4) after breeding - 62 vii A B B R E V I A T I O N Al = artificial insemination ANOVA = analysis of variance CAP = chlormadionane acetate CIDR = controlled internal drug release CL = corpus luteum cm = centimeter °C = degrees Celsius d = day DF(s) = dominant follicle (s) E 2 = estradiol EPSI = endometrial prostaglandin synthesis inhibitor FSH = follicle stimulating hormone GLM = generalized linear model GnRH = gonadotrpin releasing hormone h = hour hCG = human chorionic gonadotropin INFs = interferons LH = luteinizing hormone MGA = melagesterol acetate MHZ = megahertz min. - minute(s) ml = milliliter(s) mm = millimeter(s) viii N = norgestomet ng = nanogram No = number P4 = progesterone PGF 2 a = prostaglandin F 2 a PRJJD = progesterone releasing intravaginal device SE = standard error SMB = SYNCRO-MATE B TVs = trophoblastic vesicles ix ACKNOWLEDGMENTS I express my indebtedness and thanks to my research supervisor, Dr R. Rajamahendran, for his guidance, understanding, encouragement and especially invaluable suggestions and criticism in writing my thesis and scientific papers independently. Sincere thanks are extended to S. Lisa and W. Patricia Their word-for-word review of the manuscript, coupled with corrections and friendly suggestions, has helped my objective of providing pleasant reading. I remain indebted, too, to management at the collaborating farms for making it possible for me to conduct my thesis project at the farms I appreciate all the technical support offered by G. Gilles, Sylvia. L during my graduate study. I also appreciate to my colleagues with whom I consulted on many occasions: G. Giri, Ming. Y, Maden. P, Mohamed. A. and Negrash. Finally, very special thanks must be addressed to my wife, Deeqa. M, who provided me with a great deal of support. As well as incentive to complete my degree CHAPTER 1 GENERAL INTRODUCTION For dairy cows, a calving interval of 12 to 13 months is generally considered to be economically optimal (Olds, 1979; Hamidakuwanda et al., 1987). An integral component in achieving this calving interval is the incorporation of efficient and accurate estrus detection, proper semen handling techniques, and timely artificial insemination relative to ovulation. Estrus detection has been cited as one of the most important factors affecting the reproductive performance of artificial insemination programs (Everett et al., 1986). Thus, maximizing estrus detection rates can improve overall pregnancy rates in dairy cattle. However, proper control of the time of the onset of estrus is difficult, since the peak in estrus activity often occurs at night and documentation of the actual onset of estrus may be difficult without 24 observation (Henriks et al., 1971). Since thel930s, various estrus synchronization protocols have been developed and used to manage reproduction in dairy cattle. These methods have involved controlling estrus cycle length by administering progestogens and regressing the CL with luteolytic agents. Most of these methods require that the animals be bred over several days (Odde, 1990). Current estrus synchronization programs focus on combining traditional methods of controlling cycle length with the manipulation of follicular development in order to program or select the ovulatory follicle. One of the more commonly used programs for estrus synchronization in dairy cattle is two injections of PGF 2 a at a selected interval (usually 11 and 14 days) and inseminate at a predetermined time without observing estrus (Lauderdale, 1972). This protocol has resulted in the highest conception rates when cattle are bred after an observed estrus, rather than insemination at a predetermined time without reference to estrus (Odde, 1990). 1 Understanding factors that control ovarian folliculogenesis has led to the development of more precise methods of controlling reproductive cycles in cattle. Thatcher et al. (1989) and Macmillan et al.(1991) developed a method that synchronized both follicular development and the CL regression. With this system, an injection of GnRH is followed 7 days later with an injection of PGF 2 a (Badinga et al., 1994; Wolfenson et al., 1994; Schmitt et al., 1995). The GnRH injection is aimed to luteinize or ovulate mature follicles present during the time of treatment without considering the stage of the cycle and to initiate the recruitment and a selection of a new dominant follicle seven days later (Macmillan et al., 1991; Wolfenson et al., 1994). The injection of PGF 2 a initiates the regression or luteolysis of the spontaneous or the one induced by GnRH, or both. After the injection of the PGF 2 a cows can be artificially inseminated at detected estrus. Under this system, a strategy was designed to synchronize ovulation by administering second GnRH 48 h after the PGF 2 a injection (Pursely et al, 1995a). The role of PGF 2 a in CL regression in domestic animals is well established (Lauderdale, 1972). In general, P4 from the CL inhibits the release of the LH surge as well as oxytocin receptor expression during the first 10 days of the luteal phase of the estrous cycle. The release of its negative feedback allows the development of the CL regression mechanisms. However, in pregnant cows conceptus secretes a characteristic protein known as interferons (IFNs). These act by inhibiting the uterine oxytocin receptors necessary for the initiation of the CL regression (Stewart et al., 1989; Roberts et al., 1991, Flint et al., 1991). It has been established that the transfer of these IFNs before maternal recognition periods in cattle (days 11-17) leads to the prolongation of luteal function (Stewart et al., 1989; Roberts et al., 1991, Flint et al., 1991). However, the cost to produce sufficient amounts of IFNs by culture of conceptus, and subsequent purification makes this technique impractical. Recently Mann et al. (1994) showed that treatment of cyclic cows with GnRH during mid-luteal phase has 2 significantly reduced plasma E 2 concentrations from dl2 - dl6 of the estrous cycle. This phenomenon has been suggested to be due to ovulation or atrisia ^ of the dominant follicles (Thatcher, 1989). The authors proposed that suppression of E 2 after the administration of GnRH is transient and if given too soon, E 2 concentrations will have returned! to normal by the time that luteolytic mechanism is potentially developing. Recently Schmitt et L l . (1996) showed that an t injection of GnRH and Human chorionic gonadotropin (hCG) effectively induced accessory CL and increased plasma P4 concentrations. Therefore, it was postulated that a single injection of GnRH given on days 7, 14, or 7 and 14 post breeding can increase the P4 concentrations, extend the CL life span, reduce circulating E 2 , and alter follicular growth pattern either by inducing atresia or ovulation of the large follicles. The ability to eliminate a follicle with high concentrations of estrogen and extend CL life span would increase pregnancy rates of dairy i cattle, by providing additional opportunity for an otherwise viable embryo to establish pregnancy. j I This thesis, therefore, addresses estrus synchronization protocols for planned breeding and hormonal treatments to enhance pregnancy rates after timed breeding in dairy cattle. 1.1. The objectives: J 1. To compare estrus synchronization, CL function, and pregnancy rates following two estrus synchronization protocols for planned breeding in dairy cattle, and j 2. To test the effect of GnRH given after breeding on plasma and milk progesterone concentrations, induction of accessory CL and pregnancy rates after timed breeding in dairy cattle. After this introductory chapter, the second chapter of the thesis provides a review of literature on estrous cycle, follicular dynamics, the importance of estrus synchronization, adopted methods of estrus synchronization in cattle, major factors causing to early embryonic mortality, i and measures available for pregnancy enhancement. Chapter 3 describes the comparison of two 3 estrus synchronization protocols for planned breeding in dairy cattlej In chapter 4 , the role of GnRH given after breeding on ovulation rates, progesterone profile and pregnancy rate will be discussed. Chapter 5 provides discussions and conclusions from the experiments. CHAPTER 2 LITERATURE REVIEW This chapter will briefly describe the bovine estrous cycle, hormonal control of the estrous cycle, ovarian follicular dynamics, methods of estrus synchronization, advantages of estrus synchronization in dairy cattle, early embryonic mortality in cattle, and the methods available for enhancing pregnancy rates after breeding in dairy cattle. 2.1. The bovine estrous cycle A reciprocal interaction occurs between the hypothalamus, the pituitary gland and the ovary to regulate and maintain hormonal balance and normal reproduction. Altering the natural phenomena, and understanding the consequence of artificially induced conditions are important to the commonly practiced technological advancements in reproductive manipulation including, Al, embryo transfer and estrus synchronization. Thus, to understand estrus synchronization, it will be necessary to briefly describe and discuss the estrous cycle prior to the review of the practical methods of estrus synchronization. Puberty in cattle tends to vary with breed and plane of nutrition. It is defined as the time of the beginning of the reproductive life in females and is usually marked by the beginning of ovarian activity. The average age of puberty in dairy heifers is between 12 to 17 months (Foote, 1974). The cow is a polyestrous animal, therefore once estrous cycle is established they continue unless interrupted by pregnancy. Most have a cycle length of between 17-25 days. In general, estrus cycle is divided into four phases. The period of sexual receptivity or estrus (d 0), being followed by the post-ovulatory period or metestrus (d 1-4), the period of diestrus or luteal phase 5 (d 5-18) and proestrus (d 18 -20) the period just prior to estrus. However, these divisions are not particularly appropriate to the cow, since individual behavioral phases are rather indistinct. It is perhaps better to describe the cycle in terms of ovarian function as two components, the follicular and luteal phase, with behavioral estrus occurring during the follicular phase. Estrus (day 0) typically lasts for between 17 and 30 h with an average of 10 h (Hammond, 1927). The duration of estrus is dependent on several factors including age and season of the year and there also appears to be a diurnal pattern in that cows seem to show estrus mostly at night. During estrus, the cow becomes increasingly restless, the vulva becomes swollen and vaginal mucous membranes become deep red in color. There is often clear string mucous from the vulva. One of the most frequently observed sign of estrus is that the cow tries to mount other cows. The visible estrus changes are used to indicate the appropriate time for AI. 2.1.1. Hormonal Control of the Estrous Cycle In the normal estrous cycle GnRH is synthesized and secreted in a pulsatile pattern by the hypothalamic neurosecretory cells and stimulates the synthesis of the gonadotropins from the anterior pituitary gland. Gonadotropins in turn promote gonadal steroid synthesis and gametogenesis (Peter, 1985). During the luteal phase, FSH from the anterior pituitary gland is the key endocrine hormone responsible for the stimulation of antral follicle growth (from 4-9 mm), while LH pulses are indispensable for follicle development and maturation (Lucy et a., 1992). Follicular growth and steroidogenesis are dependent on the coordinated actions of FSH and LH through their receptors on the granulosa cells and theca cells of ovarian follicles. The most accepted model for follicular growth and steroidogenesis suggests that both granulosa and theca cells are involved in the production of estrogen synthesis (two cell/two gonadotropin model) (Hay and Moor, 1978; Fortune and Quirk, 1988). In this model, granulosa cells contain FSH receptors (FSHr) and thecal cells contain LH receptors (LHr). The production of steroid 6 hormones by the ovarian follicles requires cholesterol that is either derived from de novo cellular synthesis or from plasma lipoprotein cholesterol. Cholesterol is converted to pregnenolone in the mitochondria by cytochrome P-450 cholesterol side chain cleavage (P450scc). The second step is the conversion of pregnenolone to progesterone catalyzed by the microsomal enzyme 3 0-hydroxysteroid deyhydrogenase (3 p HSD). The binding of LH to its receptor on thecal cells stimulates the activity of cytochrome P45017cc-hydroxylase (P45017ot) enzyme necessary for the conversion of progestogens to androstenedione (Fortune, 1986). Androestenodione is then converted to E 2 in the granulosa cells by the action of the aromatize cytochrome P 4 5 0 enzyme (Fortune et al., 1987). Concentrations of E 2 are low in peripheral plasma concentrations for most of the estrous cycle. They rise during the four days before the estrus reaching a peak on the day of or the day before standing estrus (Glencross et al., 1981). The rise in E 2 concentration is correlated with the increasing size of the DF (Hay and Moor, 1978). Estradiol 17p\ in relative absence of progesterone, acts on its receptor cells in the hypothalamus, and induces estrus behavior (Heersche et al., 1974). The high E 2 concentrations during the follicular phase of the estrous cycle induce the preovulatory gonadotropion surges by a positive feed back mechanism. This mechanism acts both to increase the frequency of hypothalamic GnRH secretion and to increase the responsiveness of the anterior pituitary gland (and hence LH and FSH release) to GnRH stimulation (Lobel and Levy, 1968). During early period of estrus the influence of the preovulatory LH surge initiates the nuclear and cytoplasmic maturation of the follicle and oocyte which is destined to ovulate 10-12 h after the end of estrus or 24 h after the LH surge (Heersche et al, 1974). After ovulation, the follicle undergoes dramatic enfolding. Fibroblasts, endothelial cells, and theca interna cells migrate into the central regions of the developing CL (Hansel, 1978). Through 7 the increase in 3-0HSD enzyme activity within the developing CL both theca and granulosa cells produce high levels of P4 during the middle and late luteal phase (Fortune, 1986; Gore-Langton and Armstrong, 1994). The gonadotropin surge is followed by a decrease of FSH from the granulsa cells and decreased secretion of E 2 (Mee et al., 1991). Serum concentrations of P4 are very low during the first 4 days following estrus. As with the actions of E 2 , P4 appears to have an all or none effect on the inhibition of estrus. Therefore, once P4 concentrations increase to threshold level, estrus is inhibited even when estrus-inducing concentrations of E 2 exist. Progesterone production from the CL inhibits the release of GnRH from the hypothalamus, which in turn reduces the release of gonadotropins. Plasma P4 concentrations begin to rise from about d 4 of the estrous cycle (Figure 2.1.), reaching a peak around d 8 and remaining high until d 17. Unless pregnancy occurs these concentrations then drop back to basal levels before the next estrus and ovulation (Murdoh et al., 1983). It is generally recognized that in the cow, as in many other species, the endometrial secretion of PGF 2 a causes the regression of the CL. By the end of the luteal phase, the prolonged exposure of progesterone from the CL and E 2 from the large luteal follicles increases the oxytocin receptors in the endometrium. Oxytocin in turn, acts on the uterus to secrete PGF2 c t The PGF 2 a is released in a pulsatile manner and continue for a 2-3 d period or at least until the P4 concentrations are minimal (Hacket and Hafs, 1975). The fall in P4 concentrations causes an increase in the amplitude and frequency of GnRH release and hence LH release increases which stimulate E 2 production. This eventually results in the onset of estrus, the gonadotropin surges and ovulation (Murdoh et al., 1981). 8 Estrus 18 0 P G Estrus LH+FSH I Ovulation FSH 4 8 12 16 Day of cycle 20 Figure 2.1. Changes in blood hormone concentrations during the bovine estrous cycle: progesterone; , Estradiol 17(5 9 2.1.2. Ovarian Follicular Wave Dynamics in Cattle Ovarian follicular wave development in cattle has been characterized as a dynamic sequence of organized events, which has been described as wave-like (Matton et al., 1981). In 1927, Hammond was the first to report evidence on the morphology and the fate of ovarian follicles in cattle, using slaughterhouse materials. Most researchers agreed with Hammond and the study of follicular dynamics did not advance until 30 years later when a hypothesis that follicular growth occurs in a wave-like pattern was proposed by Rajakoski (1960). Based upon gross and histological examination of the ovaries collected on known days of the estrous cycle, the first wave has been established to start between days 3 and 4 resulting in a large atretic follicle. The second wave begins around day 12 to 14 resulting in a large preovulatory-sized follicle that subsequently ovulates. However, transrectal real-time ultrasonography provided a method for the repeated, direct, non-evasive monitoring and measuring of follicles, regardless of their locations within the ovary. The two-wave hypothesis was supported by the results of studies in which populations of follicles of different size categories were monitored (Pierson and Ginther, 1984 and 1987). Several investigators have used real-time ultrasonography to determine the changes in follicular growth during the bovine estrous cycle (Fortune et al., 1988; Pierson and Ginther 1988; Rajamahendran et al., 1988; Sirois and fortune, 1988; Savio et al 1988; Ginther et al, 1989; Knopf et al., 1989; Lucy et al., 1992; Rajamahendran and Taylor. 1994; Roche et al., 1998; Ginther et al., 1999). Based on this, ovarian follicular development during normal or typical estrous cycles of cattle is characterized by the presence of either two or three follicular growth as shown in Figure 2.2. In general, the emergence of a wave is defined as the time at which a future dominant follicle of 4 mm in diameter is first detected. The defined characteristic of this wave can be detected shortly after estrus (usually on day 2) in cows with either two or three waves during the estrous cycle. The second and third waves are detected around days 9 and 16 of the estrous cycle 10 f o r c o w s w i t h th ree f o l l i c u l a r w a v e s ( G a r c i a et a l , 1 9 9 9 ) a n d o n d a y 12 i n c o w s w i t h o n l y t w o f o l l i c u l a r w a v e s ( G i n t h e r et a l . , 1 9 9 9 ) . D u r i n g e a c h w a v e o f f o l l i c u l a r g r o w t h , a c o h o r t o f f o l l i c l e s ( n o r m a l l y 3 t o 5 m m i n d i a m e t e r ) e m e r g e s a n d b e g i n s t o g r o w . A s i n g l e f o l l i c l e f r o m t h i s g r o u p b e c o m e s l a r g e r t h a n the o the rs a n d a c h i e v e s d o m i n a n c e o v e r t he o t h e r f o l l i c l e s i n t he c o h o r t . T h i s l a r g e f o l l i c l e i s d e s c r i b e d as the d o m i n a n t f o l l i c l e a n d i s r e s p o n s i b l e f o r t h e g rea te r c o n c e n t r a t i o n s o f E 2 f o l l o w i n g o v u l a t i o n . D u r i n g the c y c l e e a c h d o m i n a n t f o l l i c l e h a s a g r o w i n g p h a s e a n d a s ta t i c p h a s e , e a c h l a s t i n g 5 to 6 d a y s ( G i n t h e r et a l . , 1 9 8 9 ; R a j a m a h e n d r a n et a l , 1 9 9 4 ) . U s u a l l y t he f i r s t w a v e d o m i n a n t f o l l i c l e i s a n o v u l a t o r y . It r e m a i n s d o m i n a n t f o r 5 -6 d a y s , a n d u s u a l l y b y d a y 11 o r 12 o f the c y c l e i t l o s e s i ts d o m i n a n c e , b e c o m e s a t re t i c , a n d i s r e p l a c e d b y t h e s e c o n d l a r g e s t f o l l i c l e o f t h e s e c o n d w a v e . T h e s e c o n d l a r g e s t f o l l i c l e b e c o m e s t h e o v u l a t o r y f o l l i c l e i n c o w s w i t h t w o f o l l i c u l a r w a v e s . R e g r e s s i o n o f t he d o m i n a n t f o l l i c l e o f t he f i r s t w a v e c o n s i s t e n t l y o c c u r s f o l l o w i n g the e m e r g e n c e o f a n e w w a v e ( G i n t h e r et a l . , 1 9 8 9 ) . H o w e v e r , i f l u t e a l r e g r e s s i o n o c c u r s d u r i n g the g r o w i n g p h a s e o r w h e n the d o m i n a n t f o l l i c l e o f t he f i r s t w a v e r e a c h e s i ts m a x i m u m d i a m e t e r , i t w i l l o v u l a t e ( G i n t h e r et a l . , 1 9 8 9 ; K a s t e l i c et a l . , 1 9 9 4 ) . S t u d i e s p e r f o r m e d d u r i n g e a r l y p r e g n a n c y h a v e i n d i c a t e d tha t w a v e s o f f o l l i c u l a r g r o w t h c o n t i n u e e v e n d u r i n g p r e g n a n c y i n ca t t l e ( R a j a m a h e n d r a n et a l . , 1 9 9 4 ) . T h e d o m i n a n t f o l l i c l e s p r e s e n t d u r i n g t h e l u t e a l p h a s e o f e a r l y p r e g n a n c y h a v e b e e n s h o w n t o h a v e L H r e c e p t o r s . I t h a s b e e n d e m o n s t r a t e d that f o l l i c l e d i a m e t e r i s o n e o f t he f a c t o r s a f f e c t i n g the p r o b a b i l i t y o f o v u l a t i o n o c c u r r i n g i n r e s p o n s e to t r ea tmen t . D u r i n g the f o l l i c u l a r g r o w t h , t h e l e v e l o f L H r e c e p t o r f o r m R N A e x p r e s s i o n i n c r e a s e s w i t h f o l l i c l e d i a m e t e r ( M a r t i n e z et a l . , 1 9 9 9 ) . S i m i l a r l y ; X U et a l . , ( 1 9 9 5 ) s h o w e d that g r a n u l o s a c e l l s o f f o l l i c l e s c o l l e c t e d o n d a y 7 o f t he e s t r o u s c y c l e h a d a m u c h g rea te r c a p a c i t y to b i n d L H t h a n g r a n u l o s a c e l l s o f f o l l i c l e s c o l l e c t e d o n d a y s 3 a n d 5 . B a s e d o n t h e s e o b s e r v a t i o n s , i t w a s h y p o t h e s i z e d that G n R H c a n b e u s e d to e l i c i t a s u r g e r e l e a s e o f b o t h L H a n d F S H f r o m the p i t u i t a r y g l a n d to i n d u c e o v u l a t i o n i n t he l a t e - g r o w i n g o r e a r l y s ta t i c p h a s e s o f t he d o m i n a n t f o l l i c l e s o n d a y 7 o r 14 o f the c y c l e . S u c h o v u l a t i o n w o u l d 11 result in an extra CL and its presence would result in higher systemic P4 concentrations that would increase the chance of pregnancy. 12 6 20-1 c £ 16-u S .2 12-.2 8-O o 4 -u. (B) o o O O ' O o o o oo B o o Wave 1 W ave 2 W a v e 3 0 1 2 1 5 1 8 2 1 24 25 Days of the cycle Figure. 2.2. Pattern of growth and regression of dominant and subordinate follicles in heifers exhibiting two (A) or three waves (B) of follicular growth during the bovine estrous cycle. Dark dots indicate atretic follicles. Solid arrows indicate the day of ovulation. Adapted from Kastelik (1994). 13 2.2. Estrus Synchronization in Cattle In this section, I will provide a brief introduction about the advantages of estrus synchronization followed by description of the methods of estrus synchronization in cattle. 2.2.1. Advantages of Estrus Synchronization Estrus synchronization techniques for cattle were developed to; a) facilitate the use of artificial insemination in a breeding program designed to accelerate genetic improvement for increased production efficiency; b) provide a useful management aid for detecting estrus; c) benefit the implementation of advanced reproductive technology, such as permitting the reproductive cycle of surrogate cows to be manipulated to allow embryo transfer; d) allow more flexibility in scheduling breeding at a specific date in a group of cattle rather than observing estrus and inseminating cows individually; and e) provide more efficient use of labor in regard to breeding (Beal, 1998). 2.2.2. Methods of Estrus Synchronization The traditional methods of controlling of the ovarian cycle over the past 60 years have involved controlling estrous cycle length either: a) by shortening the life span of the CL, inducing luteolysis, through the use of luteolytic agents such as prostaglandin or estrogen. b) by extending the life span of the CL by the use of progesterone or synthetic analogues in order to mimic CL function (Beal, 1998). 14 2.2.2.1. Long Term Progestogens Progesterone was the hormone originally used in attempts to synchronize the bovine estrous cycle. The administration of exogenous progestogens to control estrus and ovulation has evolved since 1937 when Makepeace et al. demonstrated that daily injections of P4 inhibited ovulation in rabbits. In 1948, Christian and Casida reported that 50 mg of P4 injected daily suppressed estrus and inhibited ovulation during the entire treatment period in cattle. Progesterone has little effect on the CL unless the treatment begins within four days of estrus (Hafez, 1974). Since the exact stage of estrus is usually unknown, P4 is administered for the length of the luteal phase (14-21 days) sufficiently long to allow the CL to regress. All animals usually show estrus 2-6 days after the cessation of treatment. Numerous progestogen compounds administered by different methods were investigated during the 1960s (Berardinelli et al. 1989). Although the synchrony of estrus was effective, there was a marked decrease in fertility. Moreover, the methods involving daily injections were not readily adaptable to field conditions. The development of potent oral synthetic progesterone increased the practicality of estrous cycle control (Pincus et al., 1965). These include medroxy progesterone acetate (MAP), melangesterol acetate (MGA), chloramadionine acetate, (CAP) and dihydoxyprogetserone acetate (DHPA). Feeding these compounds, usually for an 18 day period, resulted in precise synchrony but reduced fertility after the synchronized estrus (De Bois et al., 1970; Zimbleman et al. 1970). In 1962, MGA became the synthetic progesterone used most commonly in North America for control of estrus and ovulation (Gordon, 1976; Hansel et al., 1979). The main disadvantage appeared to be the inability to precisely control the daily dose taken by each animal. The technique of intravaginal administration, as developed for sheep (Robinson, 1964), has been attempted in cattle using CAP, MGA and Flurogestone (FGA) (Carrick and Shelton, 15 1967; Mauleon; 1969; Boyed and Wishart, 1969). However, results were highly variable, and difficulties in sponge retention and vaginal infections occasionally resulted. In the mid-1970s Abbot Laboratories developed a progesterone releasing intra-vaginal device (PRTD). This consisted of a stainless steel coil covered in silastic, which was impregnated with progesterone. After insertion into the vagina, progesterone was absorbed into the systemic circulation; thus controlling the estrous cycle (Maurer et al., 1975; Roche, 1975). Prolonged treatments with progesterone (>14 days) to synchronize estrus resulted in a similar percentage of animals exhibiting estrus within 21 days as compared to untreated control animals. However, fertility rates following the induced estrus were generally far from satisfactory. The low fertility of cows bred at synchronized estrus following long term administration of progesterone is due to the premature resumption of meiosis in the ova or the abnormal development of embryos derived from ova of persistent follicles (Wishart and Youngquest, 1974; Mihm et al 1994; Ahmed et al 1995; Revah & Butler, 1996). However, other factors such as alteration in the uterine environment or reduced sperm transport have been reported to contribute to the low fertility obtained after the use of long term progestin (Quinlivin and Robinson, 1969; Lauderdale and Ericsson, 1970; Butcher and Pobe, 1979). 2.2.2.2 . Progestogens and Estrogen Combinations Roche et al. (1974) showed that suppression of fertility at progestogen compounds induced estrus was greatest when 18-21- day treatments were used, and that normal fertility could be achieved by using a shorter (8-10 day) progesterone treatment. However, short-term progesterone or progestogen treatments are not completely effective in controlling the bovine estrous cycle. For example, if administration begins early in the cycle, the natural CL may outlive the exogenous treatment, therefore, synchronization will not occur. Kaltenbatch et al. (1964) demonstrated that estrogens are luteolytic when administered early in the bovine estrous 16 cycle. Later, Wishart et al. (1964) and Wiltbank et al. (1966) reported that daily injection of exogenous progesterone together with an implant of synthetic progesterone (Norgestomet) plus injection of (E2) at the beginning of treatment suppressed estrus and ovulation. However, both fertility and synchrony were poor. This was followed by the study of Wiltbank et al. (1975) and Spitzer et al. (1978), who reported that a 9 day implant containing 6 mg of norgestomet plus an injection of 5 mg of estradiol valerate and 3 mg of norgestomet (synthetic progesterone) given at the time of implant insertion successfully synchronized estrus in cycling heifers and induced estrus in non cycling heifers as well. There after, combining synthetice progesterone treatment with the administration of estradiol at the initiation of treatment enabled the period of progestogen treatment to be shortened (9-14d) without reducing the percentage of animals exhibiting a synchronized estrus. This treatment now is commercially available as SYNCRO-MATE B (SMB)* and has been approved by the Food and Drug Administration in the United States for use in beef cattle. Other short term progesterone and estrogen treatments that have been widely used for synchronization of estrus in cattle include using a PRID with a gelatin capsule containing 10 mg of estradiol benzoate which is attached to the inside of the coil during the insertion. This protocol is marketed as PRID in the United States and France and EAZI-BREED CIDR® (controlled intravaginal device) in most of Europe, Australia, and New Zealand. In most of the studies, the synchronization rate following synchro-mate B treatment was high during the 5 days following its cessation of treatment (Miksch et al., 1978; Wiltbank et al 1977). However, Spitzer et al (1978) reported a low degree of synchrony when treating cycling cows as compared to untreated cows. The failure to achieve consistently higher rates of synchronization in cyclic cattle treated with SMB depends on the response of animals treated at different stages of the estrous cycle. Estrus synchronization rates of 80-86 %, were reported when treatment with SMB was initiated on d 1 through 8 of the cycle in heifers (Miksh et al., 1978). Pratt et al. (1991) reported that estrus was synchronized in only 48 % of cows treated on d 3 of the cycle, but that 17 synchronization was 100 % when treatment began on d 9 of the estrous cycle. Stage of the estrous cycle at the beginning of treatment has also been reported to influence conception rate after SMB treatment. Authors Brink and Kiracofe (1988) reported higher conception rates for heifers that were on d 12 or higher of the estrous cycle compared to those on d 11 or less of the estrous cycle at the beginning of the treatment (47 % Vs 35%, respectively). Hence, the conception rates of those cattle that began SMB treatment late in the estrous cycle (> d 14) was significantly lower. Therefore, to improve fertility following SMB treatment it is necessary to avoid administering during the early stages of the estrous cycle, suggesting the optimum time for initiation in between d 8 and d 12 of the estrous cycle (Beal, 1998). 2.2.2.3. Prostaglandins Estradiol was initially incorporated into progestogen treatments to promote luteal regression. More direct evidence has shown that estradiol is not a very luteolytic agent nor does it prevent formation of the CL when administered early in the cycle (Wiltbank et al., 1968). However, following the characterization of PGF 2 a as natural luteolytic in ruminants and the development of its analogues, it became the preferred treatment for estrus synchronization in cattle (Lauderdale, 1972). Since then, luteolytic activity of exogenous PGF 2 a in cattle has been documented extensively (Hansel et al., 1973; Hafs et al., 1975; Lauderdale et al., 1974; Thatcher and Chenault, 1976; Rajamahendran , 1976). 2.2.2.3.1. Single Prostaglandin Injection Programs One of the most popular methods of using PGF2 is to detect estrus and inseminate cattle for 4 d then inject those that have not been detected in estrus on d 5, and continue observing for estrus and breed for at observed estrus or timed insemination (Moody, 1979; Lauderdale et al., 1980). Another single-injection system is to inject prostaglandin for 5 d and breed at detected estrus 18 (Lauderdale, 1980). Even though, this system requires less semen, and often results in comparable pregnancy rates to those animals bred after naturally occurring estrus, it needs more labor for detection of estrus and artificial insemination. Stage of the cycle at the time of the prostaglandin treatment has been shown to affect the response. Animals treated with prostaglandin prior to d 5 of the estrous cycle, have been reported to not experience luteolysis (Lauderdale et al., 1974; Jackson et al., 1979). Prior to 1982, it was believed that after d 4 of the estrous cycle, all cows were equally responsive to a luteolytic dose of PGF 2a. However, King et al. (1982), Tenable and Hahn,(1984) and Stevenson et al. (1984) demonstrated that cattle injected with PGF 2 a between 5-9 of the cycle were less responsive than those injected later in the cycle. Larson (1992) summarized the effect of the day of the estrous cycle on percentage of cattle synchronized by a single injection of PGF 2 a. The same author reported that 67 % of cattle showed estrus when treated between d 5 and d 17 of the estrous cycle, while 77 % responded when treated on d 9 through d 12, and the greatest number of animals in estrous was seen when treated after d 12 of the estrous cycle (> 91%). 2.2.2.3.2. Double Prostaglandin Injection Programs Double injection of PGF2 0 11 -14 d apart was developed to circumvent the lower response of PGF 2 a injection during the early part of the estrous cycle. If cattle are distributed equally across the days of the estrous cycle, approximately 70% of the cycling cattle should show estrous after the first injection. These cows and the remaining cycling cattle should be at a stage of the estrous cycle to respond to the second injection (Lauderdale, 1979). Results with this treatment have been inconsistent, especially in milking cows (Macmillan et al., 1977 and 1978). Prostaglandin treatments have tended to be more effective when combined with estrus detection. There was considerable debate over the optimum timing and number of inseminations 19 following PGF 2 a administration during the 1970s (Peter, 1986). Most commercial literature recommended double fixed-time Al at either, 48 and 72 or 72 and 96 h after injection, while Henderson et al. (1981) claimed that a single Al done between 75-80 h after PGF 2 a injection resulted in equivalent fertility as the use of two inseminations. However, this conclusion has not found general acceptance since the onset of estrus in cows following PGF 2 a injection has been shown to be quite variable with a significant proportion of cows showing estrus outside the range of fixed-time insemination recommendations (Donaldson et al., 1982). The average interval from injection of PGF 2 a to estrus is usually 60-72 h. This variation in the timing of estrus is created in part by the differences among cows in the rate of regression of the CL following treatment. Injection of prostaglandin after a single, timed insemination has resulted in acceptable conception rates in some trials; however, there has been significant variation and a greater incidence of very low conception rate following insemination of cattle after PGF2 c t treatment (Macmillan et al., 1982). The lower fertility following timed insemination after of PGF 2 a is most likely related to the variation in the timing of ovulation of dominant follicle following PGF 2 a treatment as compared to other synchronization programs (Beal, 1998). 2.2.2.4. Progestogens and Prostaglandin Combination Programs Combining exogenous synthetic or natural progesterone and PGF2 c t instead of estrogen at the beginning of treatment has been used to synchronize estrus. This was first tested and reported by Heersche et al. (1974) and Wishart (1974). These authors combined a norgestomet implant with an injection of PGF 2 a before or at the time of implant removal. An estrus synchronization method in which a PRID is inserted for 7 d and PGF 2 a is injected on d 6 was developed by Beal, 1983 and Smith et al. (1984). A short-term feeding of MGA combined with an injection of PGF 2 a at the end of the MGA feeding was also proposed by Beal and Good, (1986), and this system 20 induced cyclicity successfully in some non cycling cows. However, after treatment, fertility in cattle known to be cycling prior to the initiation of treatment was reduced relative to controls (Beal et al., 1988). To overcome the reduced fertility following animals bred after being synchronized with progesterone or its derivatives, a program was developed which consisted of feeding MGA for 14 d followed 17 d later by injection of PGF 2 a (MGA 1 4-PGF 2a 17). This protocol was designed to place all cattle in the late luteal phase, since prostaglandin is more effective during the late stage of the estrous cycle (King et al., 1988; Patterson et al., 1989). The rate of synchronization of estrus following this treatment has been reported as being greater than single injection of PGF 2 a alone (Patterson et al., 1995), but may be less than that of SMB (Brown etal., 1988). Synchronization of estrus in cattle based on luteolytic agents alone or in combination with progestin has resulted in variable pregnancy rates of animals bred by AI after estrus detection, but these rates are acceptable in most cases (Beal, 1998). 2.2.2.5. Synchronization of Ovulation The variability in synchrony of estrus and ovulation for fixed-time AI, pointed out the need to develop a method to synchronize follicular development and corpus luteum regression to enable, single, timed insemination, and to enhance conception rates. An increase in the understanding of folliculogenesis in cattle over the past decade has made this possible through the discovery of methods to control follicular development and the time of ovulation. Controlling the time of emergence of a new follicular wave and synchronizing the follicular wave status of animals within a group to be synchronized could improve the synchrony of estrus and ovulation and ensure that the ovulatory follicle has the optimum potential for fertilization and embryo development. 21 The most common methods for altering follicular turnover in conjunction with estrus synchronization have been the administration of GnRH or its analogs (Bo et al., 1995; Beal, 1998) or E 2 administration in conjunction with synthetic progesterones (Bo et al, 1994). The combined use of GnRH and PGF 2 a improved the efficiency and precision of estrous synchronization in beef cows without affecting fertility (Twagiramungu et al., 1992a; Twagiramungu et al., 1992b). This method involves treating the animals with GnRH on day 0 or the day of treatment initiation, and six days later with an injection of PGF 2 a to regress the CL. This protocol eliminates estrus behavior for the periods following the GnRH injection and enables the synchronization of estrus in approximately 80% of females within a period of less than 4 days after the PGF2 --induced luteolysis. Administration of GnRH causes the release of LH and the disappearance of follicles larger than 9 mm in diameter (Giblet et al., 1990) either by ovulation or atresia, depending on the physiological state of the cow (Twagiramungu et al., 1994a). The disappearance of these large estrogenic follicles eliminates the spontaneous occurrence of estrus during the period between the GnRH and the PGF 2 a injection. (Twagiramungu et al., 1992). Regardless of the stage of the estrous cycle or if ovulation is induced, the new dominant follicle is selected from the new GnRH induced follicular wave and becomes the ovulatory follicle three to four days later (Twagiramungu et al., 1994). Pursely et al. (1994) demonstrated that administration of GnRH can induce ovulation within 22-24 h when the CL is regressing. This property of GnRH has been used to develop fixed-time Al with the so-called GnRH-PGF2a-GnRH protocol. This protocol includes the injection of a second GnRH after the PGF 2 a to induce an LH surge, thus synchronizing ovulation (Pursely et al., 1994; Pursely et al., 1995b; Twagiramungu et al., 1995; Roy and Twagiramungu 1996). 22 Pregnancy rates following timed insemination after the ovulation synchronization method (Ovsynch) were similar to those obtained after animals synchronized with 2 injections of PGF 2 a and inseminated 12 h after observed estrus (38.9 vs. 37.8%), (Pursely et al., 1995). Roy and Tawagiramingu (1996) reported a pregnancy rate of 60% after cows were synchronized with Ovsynch method and artificially inseminated after detected estrus. These authors concluded that the development of this new method of synchronization program seems to be the best method of synchronizing estrus and ovulation, and could allow for effective use of fixed-time insemination with acceptable pregnancy rates. 2.3. Early Embryonic Mortality in Cattle Approximately 90% of ova are fertilized following either artificial or natural insemination. However, by d 19 only about 60-65 percent of cows are still pregnant and by d 40 the figure falls to about 55-60 percent. Therefore 30-35 percent of embryos are lost during the first 40 days of pregnancy (Boyd et al., 1961; Rohe et al., 1981; Diskin and Sreenan, 1985; Diskin and Sreenan, 1987; Diskin and Sreenan. 1994). It has been postulated that this disparity between the expected and observed level of fertility results mainly from the loss of embryos or fetuses prior to term (Hammond, 1921: Corner, 1923). Embryonic mortality alluded to in this thesis, strictly interpreted, refers exclusively to losses in embryos that are experienced during the period extending from conception to the initiation of the stage of differentiation. This study used milk P4 concentrations between day 18 to 21 following breeding as being indicative of a return to estrous. Embryonic mortality has been known to occur in almost all mammalian species (Hanly, 1961; Boyd, 1965; Short, 1979). The extent of this loss is generally influenced by the time at which losses in embryos are estimated. Counting CL has been used as one means of estimating ovulation rates and based on which embryonic losses are also assessed (Ayalon, 1978). Using 23 such this method, Ayalon (1978) estimated the incidence of embryonic mortality to range between 25-40 %. Boyd et al. (1969) reported an 8% loss by d 25 after breeding, while Roche et al. (1981) estimated the loss at 23 % also taking the estimates at d 25 after breeding. At d 42 after breeding, Ayalon et al. (1978) as well as Diskin and Sreenan (1980) found the incidence to be 20 and 42 %, respectively. On the other hand, a 5-8 % loss in embryos was reported by Boyd et al. (1969) who took the measurement at 42 d following breeding. Although it has been suggested that most embryos die between conception and 25-30 d of gestation (Robinson, 1951; Sreenan and Diskin, 1983), evidence generated over that last 5-6 years indicates that 75-80 % of these losses take place between d 15-18 after breeding (Sreenan and Diskin, 1994. It has been shown that animals losing their pregnancies on or after d 16, have a slight extension in the estrous cycle length (Northy and French, 1980; Heyman et al., 1984). This difference is due to the fact that day 14-16 has been established to be the time of maternal recognition of pregnancy in the cow (Thatcher et al., 1989). 2.3.1. Predisposing Factors of Early Embryonic Mortality Genetic factors have been suggested as one of the major contributors to early embryonic mortality in all species (Boyd, 1965). These genetic factors could be as a result of lethal genes being expressed early in development or due to structural (including chromosomal) abnormalities in the gametes (Bishop, 1964). However, Boyd, (1965); Eddy (1969); Garverick (1983); and Moore (1985) proposed that the extent of the losses cannot be accounted for solely by genetic abnormalities and that various other factors are likely involved in embryonic mortality. The extent of chromosomal abnormalities in the cow has not been yet quantified though recently Burton et al., (1996) reported a 10 % incidence of gross abnormalities. According to Bishop (1964), the genetic factors involved in embryonic death are not inherited by parents, but rather arise from the parent generation in definitive gametes. Therefore, 24 a considerable part of embryonic death is unavoidable and should be regarded as a normal elimination of unfit genotypes at low biological cost and that little could be done to reduce its magnitude. The presence of bacteria in the uterus, such as Actinomyces pyogens is generally considered to cause embryonic mortality in cattle (Alder, 1959). These infections in the uterus result in conditions, such as retained placenta and prolapse of the reproductive tract, which in dairy cows can depress pregnancy rates as much as 40%. Attempts to demonstrate the existence of low grade, non-specific infections as a cause of embryonic death in repeat-breeder cows has not been successful, either by direct culture or by response to uterine infusions of antibodies, (Ulberg et al., 1952; Shelton et al., 1990). High ambient temperatures have been reported to increase the incidence of embryonic mortality by acting directly on the embryo or by altering the hormonal status of the mother (Scott and Williams, 1962). Badiga et al. (1993) found that summer heat stress might alter the timing of the appearance and duration of the dominant follicle, resulting in a long lasting detrimental effects on the quality of the ovarian follicles through a reduction in FSH secretion leading to a low level of E 2 Wolfenson et al. (1993) conducted studies during the summer in California indicated that in pregnant heifers, heat stress antagonized the suppressive effects of the embryo on the uterine secretion of PGF 2 a However, Bar-Anan et al. (1980) contended that the main effect of thermal stress was to reduce fertilization rate rather than increase the rate of embryonic mortality. Sreenan and Diskin (1983) concluded from their studies that the complex interactions of many factors including climate, nutrition, endocrine often make it difficult to experimentally determine their individual effects on embryo mortality. Severe under nutrition and stress (Edey, 1969) have also been shown to increase the incidence of embryonic mortality. Morrow, (1980) Sreenan, (1983) and Hillet al. (1970) observed a reduced fertilization rate and lower circulating plasma P4 levels but no clear effect on 25 embryo mortality in heifers subjected to under nutrition. Similarly, Eddy, (1969), and Spitzer et al. (1978) concluded that the reduced pregnancy rates in heifers on restricted energy intakes were not due to fertilization failure but that some other factors may contribute to embryonic mortality. In a later study, it was reported food quality has an influence on fertility (Blanchard et al., 1990). Sreenan and Diskin (1994) concluded from their studies that fertilization failure or early degeneration of the embryo might occur when cows are fed excess rumen-degradable protein. Similarly, Elrod and Butler (1993) concluded that excess degradable protein acts through some undefined mechanism by decreasing the uterine pH during the luteal phase of the estrous cycle that may be associated with the reduction in fertility. Deficiencies in dietary constituents such as minerals have been associated with reproductive failure. For example, inadequate selenium in the diet predisposes the retention of the placenta at parturition (Trinder and Renton, 1973). This in turn reduces the chances of an animal establishing and maintaining it pregnancy (Bouters, 1965). It has also been reported that cows inseminated on the day after estrus show a greater tendency to undergo embryonic mortality than those inseminated on the day of the estrus (Ayalon, 1978). This suggests that aging ova are capable of being fertilized, but that some abnormality develops so that the resultant embryo is incapable of developing into a full term fetus. In the absence of specific causes a higher proportion of older cows (particularly after their fourth lactation) appear to lose their embryos (Peters, 1995). In these cattle, many of the losses occur around d 30 after conception, when implantation should be almost complete, possibly suggesting a uterine problem. It has been suggested that the older uteri that have carried several previous pregnancies need longer for involution before it is capable of supporting a further implantation. In non-pregnant cows, the role of uterine PGF2 c t in the regression of the CL during the early diestrus is well-established (Sreenan and Diskin, 1983). Estradiol from the large anovulatory follicles stimulates the synthesis of oxytocin receptors in the endometrium, which in turn stimulates the luteolytic pulses of PGF 2 a from the endometrium (MacCraken et al., 1984). 26 Early recognition of pregnancy in cattle occurs approximately on days between 15-16 after fertilization (Sreenan and Diskin, 1983). It has been postulated that maternal recognition of pregnancy can be considered as the interaction between maternal units and the products of the conceptus that signal its presence to the mother the need to maintain pregnancy (Lamming et al., 1989). This dialogue can occur at various critical stages during pregnancy, mainly on days 15 to 17 of gestation (Northy and French, 1980; Johns et al., 1979). Disruption of this signal (i.e. failure of the embryo to communicate with its mother) can result in the loss of the fetus in the pregnancy (Johns et al., 1979; Bazer et al., 1983). The need for the continuos secretion of P4 by a normal CL in early pregnancy is well established (Heap et al., 1972; McDonald et al., 1953). During the first luteal phase after service, cows that remain pregnant tend to have higher peripherial plasma P4 levels than those do not (Henricks et al., 1970; Holness et al., 1977). This secretion has been reported as early as d 4 after breeding, and must continue beyond the time when luteal regression normally occurs in non-fertile reproductive cycles (Butler et al., 1996) or d 6 (Erb et al., 1976; Shelton et al., 1990). As a consequence, insufficient circulating P4 during the early or mid luteal phase is often suggested as a cause of early embryonic mortality (Hawks et al., 1973, Maurer et al., 1983). It has been suggested that in animals with low P4 concentrations during the early period of pregnancy the CL may not function optimally causing substantial losses in reproduction (Hernicks et al., 1970; Erb et al., 1976; Luaszeswska and Hansel 1980; Wilmut et al., 1985). Subnormal CL function can be categorized into two groups. The first group includes CL having a short life span (Odde et al., 1980; Colman and Daily, 1983; Copelin et al., 1987). The second group includes CL having normal life spans but reduced P4 secretion (DiZerga et al., 1981; Pratt et al., 1982; Coleman and Daily, 1983; Copelan et al., 1987; Lamming et al., 1989). Low levels of P4 during early gestation is considered to lead to an impairment or a decrease in the rate at 27 which the luteal function of the pregnant cow approximates the time of transport of the embryo from the oviduct to uterus (Thibault et al., 1972). This is an event that links the early influence of P4 to the musculature of the isthmus (Chang, 1966). 2.3. 2. Approaches to Enhance Pregnancy in Cattle The majority of hypotheses developed from the purpose of addressing the problems imposed by an adequate CL have mostly evolved around trying the following options; a) since the assumption is that the CL is not functioning optimally, exogenous P4 has been provided in order to supplement endogenous P4 secretion. b) believing that a bovine embryo does influence the secretary capacity of the CL, various laboratories have appropriately concentrated on infusing into the uterus of the cow either viable embryos or homogenates of embryonic vesicles, c) believing, also that CL function is dependent on continued support from the anterior pituitary secretary products, namely LH, gonadotropins, e.g. human chorionic gonadotropins (hCG) have duly been utilized. d) Since the pituitary is stimulated by secretions originating from higher centers (Hypothalamus), an alternative rationale has involved the use of gonadotropin releasing hormone (GnRH). 2.3.2.1. Progesterone Supplementation The increase and decrease in P4 levels during the normal estrous cycle reflect the development, maintenance and regression of the CL. If, after breeding, fertilization and normal embryo development occur, and then the plasma P levels remain at approximately the mid-luteal level indicating the retention of the corpus CL in a functional state (Sreenan and Diskin, 1983). Establishment and maintenance of early pregnancy in most mammals depends on P4 originating from this functional CL. The level of P4 must remain relatively elevated in order for 28 pregnancy to be maintained (Heap, 1972; Robinson et al., 1989). Progesterone is also essential for the establishment and maintenance of a quiescent uterus during pregnancy, thus permitting implantation to occur. Insufficient P4 during the early post-insemination period may contribute to the high level of embryonic mortality, which mainly occurs between d 8-16 after service (Diskin et al., 1980). Various investigators have examined the use of progesterone after breeding. Peripheral levels of P4 were increased in some trials (Northy et al., 1985; Robinson et al., 1989;) while others have not reported any detectable difference in P4 concentrations between cows treated with P4 after insemination and control cows (Diskin and Sreenan, 1980). Progesterone supplementation was found to increase pregnancy rates in lactating dairy cows (Robinson et al., 1989; Larson et al, 1995). In contrast to these results, authors Zimbelman, (1959); Loy et al., (1960); Clef et al., (1991); Peters, (1995) reported no detectable improvement in pregnancy rates following P4 supplementation. 2.3.2.2. Uterine Infusion of Embryonic Vesicles or Products In the pregnant cow, only embryos beyond or equal to days 16 of age have been shown to be luteotropic (Lamming et al., 1989). Hence one viable option for extending the duration of the luteal phase and enhancing CL function in the cow has been the use of embryonic vesicles (Macmillan et al., 1986). Starting from day 16 after fertilization, embryos secrete a protein initially termed bovine trophoplastic protein-I, but is now known as bovine interferon (INFs). These secretary products can either have an antiluteolytic or luteotropic effect (Lamming et al., 1989). These products (INFs) act on the secretion of PGF 2 a , at least in part, by the stimulation of an endometrial prostaglandin synthesis inhibitor (EPSI) which is present in the endometrium of pregnant cattle (Helmer et al., 1986; Basu et al., 1987; Danet et al., 1993). Structures known as trophoblastic vesicles (TVs), which are similar in many ways to blastocysts without the embryonic disc, can be formed by cutting the elongated blastocyst (13-14 29 days old) into several pieces and culturing for 24 h or so until they reform into vesicles. Uterine infusions of these embryonic vesicles have been demonstrated to extend length of the estrous cycle in the cow (Betteridge et al., 1980; Northy and French, 1980; Heyman et al., 1984). Extracts from blastocysts between d 16-18 have been shown to increase P4 production by dispersed luteal cells in culture (Beal et al., 1981). Embryonic vesicles have also been deemed to amplify the signals from the conceptus (Thatcher et al., 1989). However, administration of these products at certain dose levels caused a hypothermic response that was temporally associated with a decrease in P4 (Maurer et al., 1985). Therefore, technical problems make it difficult to achieve successful application in farm animals (Mauer et al., 1985). 2.3.2.3. Gonadotropins In 1966, Donaldson and Hansel provided evidence that LH is the bovine luteotropin hormone and suggested that using it after breeding in cattle should prolong the life span of the CL and increase pregnancy rates. Human placental gonadotropins (hCG) have also been shown to be luteotropic in the bovine, and have been administered as an alternate to P 4 supplementation during the early and mid-luteal phase. (Wiltbank et al., 1961; Christie et al., 1979; Sreenan et al., 1979). Human chorionic gonadotropin is a glycoprotein in which its beta sub-unit bears a 90% homology with the corresponding sub-unit of LH. Due to this homology, hCG has been shown to mimic the actions and effects of LH (Flint et al., 1990). Human chorionic gonadotropin promotes ovulation of the first wave DF and formation of an accessory CL when administered on d 4 (Breuel et al., 1989; Price and Webb, 1989), d 5 (Walton et al 1990; Schismet et al, 1996; Diaz et al 1998), d 6 (Rajamahendran and Sianangama, 1992), d 7 (Sianangama and Rajamahendran, 1992, 1996) d 10 (Breuel et al., 1989), and d 14 to 16 (Price and Webb, 1989) of the estrous cycle. 30 The majority of these studies report dramatic increases in circulating P4 levels that arise from the induction of the accessory CL. However, the effect of this increase may be somewhat dependent on the presence of a functional CL, since administration of hCG on day 1 (early luteal phase) or d 17 (late luteal phase) of the estrous cycle had no effect on serum P4 concentrations (Sequin et al., 1977; Breuel et al., 1989). Results obtained with the use of hCG in cows have been variable. Some reports indicate no beneficial effect on pregnancy rates resulting from using hCG (Wiltbank et al., 1961; Holness et al., 1979), Other workers have shown only a slight increase in pregnancy rates among treated effects (Santoz-Valadez, 1984; Looney et al., 1984 Sianangama and Rajamahendran, 1992). Walton et al. (1989) treated cows with hCG on d 5 after breeding and reported a significant difference in both plasma and milk P4 profiles between hCG treated and control. These significant differences were noted after d 8 for plasma and at d 18 and 21 of milk samples. Treatment with hCG as a method of increasing peripherial levels of P4 was found to be beneficial in this case. However, the same authors noted a 3 day extension in the estrous cycle length in non-fertile inseminations. Wiltbank et al. (1961) injected hCG daily starting at d 14 until d 34. These authors reported a 6 % improvement in pregnancy rates in cows receiving the treatment. Similarly, Wagner et al. (1973) reported an 11% improvement in pregnancy rates after cows were given hCG on a 3 d period starting at d 15 after insemination. Massey et al. (1983) administered hCG on d 7 after embryo transfer found a 5.7 % improvement in pregnancy rates. However, Ecthernkamp and Maurer, (1983) reported a 10.2 % reduction in pregnancy rates. When treatment was given to twice open cows, a 5.5 % loss in pregnancy rates was detected among cows, but not heifers. It is clear that the results obtained with the use of hCG in cows have been variable, moreover, note should be taken that one consequence of its administration might be the production of antibodies (Thatcher et al., 1993). 31 2.3.2.4. Gonadotropin Releasing Hormone Gonadotropin-releasing hormone is a decapeptide, synthesized in the cell bodies of neuro secretary neurons located in the mediobasal hypothalamus and is secreted into the primary capillary bed of the median eminence. This peptide is responsible for the release of LH and FSH from the pituitary. Through an understanding of GnRH, its effect is hypothesized to be either indirect through the synthesis and release of the gonadal steroids (Chenault et al., 1990) or perhaps direct through its effects on reproductive tissue (Macmillan et al., 1989). Based on these, GnRH has been used in attempts to improve pregnancy rates in cattle (Lee et al., 1985). The application of GnRH was subsequently aimed at the synchronization of ovulation and insemination in repeat breeder cows. It has been reported that the use of GnRH at the time of insemination can increase the pregnancy rate as well as P4 levels (Macmillan et al., 1986; Mee et al., 1993). Injection of a GnRH, Buserelin, between d 11 and 13 after estrus in inseminated, lactating dairy cows resulted in higher pregnancy rates, extended inter-estrus intervals, and elevated concentrations of serum P4 (Macmillan et al., 1985; and Thatcher, 1986). These authors proposed that GnRH injected during the mid-luteal phase had an antiluteolyitc role by inducing surge of LH from the pituitary gland, which in turn causes atresia or luteinization of these large mid-luteal follicles. This in turn, alters the follicular secretion of E 2 , which is necessary to initiate uterine changes in oxytocin receptors and serum concentrations of PGF 2 a proceeding luteolysis (McCracken et al., 1984). Drew and Peters (1994) and Shelton and Dobson (1993) have reported similar findings. Ryan et al. (1991) reported that the use of GnRH after insemination increased the pregnancy rate by 12.5%. In contrast, Macmillan et al. (1985), Ryan et al. (1991), Jub et al. 1991; Ryan et al. (1992), Thatcher et al., (1992), and Harvey et al., (1994), used GnRH at different times after insemination without recording any significant effect on conception rates and P4 levels. 32 In the cow, luteal cells were not shown to have GnRH receptors. This indicates that GnRH does not have direct stimulatory or inhibitory effects on the CL (Ireland et al., 1991; Brown and Reevis, 1989). These authors demonstrated that the absence of this specific receptor in bovine ovarian tissue; thus, the potential effects must be mediated indirectly through alterations in endogenous gonadotropin secretion or directly via a non-receptor mediated mechanism 33 CHAPTER 3 A COMPARISON OF TWO ESTRUS SYNCHRONIZATION PROTOCOLS FOR PLANNED BREEDING IN DAIRY C A T T L E 3.1. ABSTRACT The objective of this experiment was to compare the use of GnRH ("Ovsynch") to two injections of PGF 2 a for planned breeding in dairy cattle. Sixty-seven dairy cows and twenty-two virgin heifers eligible for breeding were palpated per rectum and randomly assigned to two estrus synchronization treatment groups. Each treatment group was subdivided into high and low producing cows. Animals in the Ovsynch group (n=44) received injections of GnRH (day 0), PGF 2 c t (day 7) and GnRH (day 9). Animals in the prostaglandin group (n=45) were given PGF 2 a 14 days apart. Kamar heat mount detectors were placed during the PGF 2 a injection in the Ovsynch group and the second PGF 2 a injection in the PGF 2 a group to determine estrus. Timed inseminations were done 12 and 36 h after the second GnRH injection in the Ovsynch group, and 60 and 84 h after the last PGF 2 a injection in the PGF 2 a group. Milk samples were collected on DO (day of the breeding), D 7, D14 and D 21 for progesterone analysis. All animals were subjected to ultrasound examination for on D 35 for pregnancy diagnosis. Estrus synchrony based on Kamar heat mount detector and milk progesterone concentrations (less than 1 ng/ml) on the day of breeding was not significantly different between the two groups (P>0.05). Mean milk progesterone was also not different (P>0.05) between the two treatment protocols. Pregnancy rates were significantly higher (P<0.05) for Ovsynch treated cows (57.5%) than for the PGF 2 a group (38.4%). No difference in pregnancy rate was observed in heifers. Pregnancy rates of high producing cows treated with Ovsynch (63%) were higher 34 can be concluded that the pregnancy rate of cows and heifers could be managed effectively without estrus detection by using the Ovsynch. 3.2. INTRODUCTION The ability to synchronize the onset of estrus, and hence the time of breeding and calving offers economic and management benefits to dairy producers. Approximately 80-90 % of dairy farmers in North America currently uses artificial insemination (AI). Most of these farmers inseminate animals in the afternoon if they observe the signs of estrus in the morning, and inseminate animals in the next morning if the animals show estrus in the afternoon. This system is known as AM/PM breeding rule. It has been reported that almost half of the estrus period in normal cycling dairy cows may have not been detected (Lauderdale et al., 1974; Macmillan et al., 1984) and several studies have linked poor estrus detection to the lower fertility obtained after breeding (Bar, 1975; Rousavile et al., 1979). This inefficiency results in longer than optimal calving intervals, loss of milk yield, and can limit reproductive performance in commercial dairy herds (Pursely et al., 1995). In order to improve estrus detection, various estrus synchronization protocols that focus on controlling the life span of CL have been developed and used in many dairy herds. Estrus synchronization of lactating dairy cows has primarily been limited to the use of PGF 2 a to regulate estrus (Daily et al., 1993; Momcilovic et al., 1998; Pankowiski, 1995; King et al., 1992). Numerous studies indicated that the fertility rate of cattle inseminated after PGF 2 a induced estrus is similar (Lauderdale et al., 1974; Pursely et al., 1995b) or superior (Kastelk et al., 1990; Jenks et al., 1998) than that cattle inseminated after naturally observed estrus. However, most estrus synchronization programs involving timed AI with two injections of PGF 2 c t are associated with a reduction in fertility, because the interval from PGF 2 a to estrus is often too variable (Daily et al., 3 5 1993; Gracia et al., 1991). It is now clear that successful use of a planned breeding program with a fixed-time Al requires not only CL control but accurate control of follicular development in relation to ovulation. Recently, a new protocol using GnRH, PGF 2 a followed by another GnRH dose has been developed (Pursely et al., 1995). This protocol could improve the pregnancy rate of lactating cows without the need of estrus detection. The objectives of the present study were to compare estrus synchrony, CL function and pregnancy rates between the ovulation synchronization method (Ovsynch) and two injections of PGF 2 a. 3.3. MATERIALS AND METHODS 3.3.1 Animals: General Management Practices Animals included in this experiment were part of the dairy herd of Holstein at the UBC Dairy Farm at Oyster River, BC. The experiment commenced in March 1998 and was concluded in June 1998. Cows were milked twice daily at 03:00 and 14:00 in a herring bone milk parlor. These cows were produced, on average, 8436 kg during the period of this experiment was conducted. The average percentage of fat and protein were 3.20 and 2.96, respectively. During the beginning of the experiment, the cows were on pasture and concentrations for their nutrition. The pasture consisted of orchard grass while the concentrate portion of the feed (16% protein) was made up 3 kg of barley mash grain. Each animal was allocated 4 kg per day to meet its maintenance requirements. Extra grain, at a ratio of approximately 1 kg of grain to 4 kg of milk produced, was given to each animal in order to meet the requirements for production. This concentrate ration also contained mineral supplement but did not include copper, excluded due to a toxicity problem experienced by the farm. It was standard practice on the farm to give each animal an intramuscular injection of selenium at the time of dry in off. Dry cows were not provided with any mineral supplement were mostly fed diet consisting of grass silage and corn 36 silage. All animals eligible for the experiment were palpated per rectum before final inclusion in order to exclude those that were cystic, not cycling, or had detectable uterine pathology. Milk yield per day follows a predictable curvilinear function that peaks at 6 to 9 weeks of lactation and then declines at a constant rate (Lean et al., 1989). During the first two months following calving, Individual milk production were recorded for all cows at monthly production tests during which milk yield, protein, and fat production were measured and recorded by the farm management personnel. Peak milk yield, the highest recorded 3.5% fat corrected milk yield (FCM) given by a cow on the herd-recording test days during the cow's lactation averaged 32.2 kg/day. Based on deviation of peak milk production record from the average of herd mates, cows were classified into high (>32.2 kg/day) or low (<33.2 kg/day) milk yield. 3.3.2. Estrus Synchronization Treatments (Ovsynch Protocol) Thirty-three lactating dairy cows between 60-90 days postpartum, and 11 virgin heifers, 13-22 months old were synchronized with GnRH (2 ml, i.m. Factrel; Ayerst Laboratories, Montreal, QB) on day 0 (day 0 = day of the treatment initiation), seven days later animals were given PGF 2 a (25 mg, i.m. Lutalyse; Upjohn; Kalamazoo, MI). At the time of the PGF 2 a injection, a Kamar heat mount detector (Kamar INC, Steamboat Springs, Co.) was glued to the hair over the mid-line just in front of the tail-head in order to determine estrus. This device is designed to record evidence that a cow has been mounted; the herd is checked at least twice for every 4 to 6 hrs in order to observe whether the device has been activated. The Kamar heat mount detector consists of an outer white tube and an inner phial of red dye that is squeezed out by the mounting cow. The brisket of the top cow squashes the device and changes color into red when the cow stands to be mounted for more that few seconds. A second injection of GnRH was given 48 h after the PGF2 o t. All animals were inseminated twice, 12 and 36 h after the second GnRH 37 (PGF 2 ( X Protocol) Animals assigned to the PGF 2 a group (n = 45; 34 cows and 11 heifers) were given two injections of PGF2 o t (25 mg Lutalyse; Upjohn Co.: Kalamazoo, MI) 14 d apart. At the time of the second PGF 2 c t injection, Kamar heat mount detectors were placed as described before. Timed Al was done 60 and 84 h after the second injection of PGF 2 a 3.3.3. Milk Sampling and Progesterone assay To determine estrus and CL function after ovulation, milk samples were collected through a tail vessel puncture from each experimental cow. The samples were taken on day 0 (the day of the insemination), 7, 14, and 21 after breeding, using 20 gauge vacutainer needles, holders and heparinized vacutainer tubes (Becton & Dickinson Vacutainer System, B & d Co., Rutherford, NJ, USA). Milk P4 concentrations were measured with a solid phase radioimmunoassay Kit (Coat-A Count, Diagnostic Products Corporation, Los Angeles, CA, USA). At each sampling assay an aliquot of 100 ul of standard, was transferred into the appropriate antibody (AB) coated tubes (labeled A l , A2, B l , Gl and G2). Tubes containing standards A through G corresponded to P4 concentrations of 0, 0.1, 0.5, 2.0, 10.0, 20.0, and 40.0 ng/mL"1 progesterone Buffered I125 - labeled P4 (1.0 ml) was added to all tubes. Mixed and left to incubate at room temperature (20°c) for 3 h. Tubes were aspirated with a Pasteur pipette under vacuum, after the incubation period. Tubes were counted for 1 min in gamma -counter (Packard Auto gamma 500, Packard Instruments, Downers Grove, IL, USA). 3.3.4. Radioimmunoassay Kit Validation The concentrations of P4 in milk were quantified as described above. While blood plasma has been the main biological material utilized in the assessment of the reproductive status of 3 8 livestock, the discovery in the 1970's that progesterone is detectable in milk (Darling et al., 1972) pointed to the potential use of such a material in routine diagnostic procedures. It was proposed that the progesterone concentrations in milk are one means of diagnosing animals in estrus as well as those in early pregnancy (Laing and Heap, 1971). A simple and rapid RIA for quantifying progesterone in milk soon followed (Heap et al., 1973). RIA in the present experiment was performed using commercially available kits. These kits have since been validated (Gowan and Etches, 1979; Srikandakumar et al., 1986; Robinson et al., 1989) and found to be a reliable tool for monitoring luteal function in the cow (Srikandakumar et al., 1986). Data from research undertaken in the department of Animal sciences at University of British Columbia using the Coat-A-CountR P4 RIA kit indicated that P4 levels lower than 1.00 ng/ml, in both plasma and milk samples, are indicative of the presence of a functional CL (Rajamahendran et al., 1989). These findings are in agreement with those obtained by other laboratories (Srikandakumar et al., 1986, Gowan and Etches, 1986). A P4 concentration of 1.00 ng/ml was, therefore, used to discriminate between cows in estrus versus non-estrus at the time of breeding. 3.3.5. Statistical Analysis Estrus synchronization, Based on Kamar was defined as that number of animals detected in estrus either within 4 d following in the second injection of PGF 2 a in the PGF 2 a treated group or within 4 d period following the only PGF2 o t upon in Ovsynch group as the proportion of animals treated. Similarly, estrus synchrony based on milk P4 was defined as the number of cows with milk P4 value less than 1 ng/mL on the day of breeding following normal increase in P4 concentrations (>2 ng/mL) on d 7 after breeding. CL function was measured by determining P4 concentration on d 0, 7, 14 and 21 after breeding. Pregnancy rate referred to the number of cows and heifers that were diagnosed pregnant 35 days after insemination. Data on milk P4 concentrations were statistically analyzed by the least square analysis of 39 variance (ANOVA), using IMP procedures of the Statistical Analysis System Institute Inc. (SAS) (version 1997). Contingency tables in chi-square analysis were used to analyze data; 1) To test the treatment effects on estrus synchronization rates (the number of animals showed estrus by Kamar and the number of animals showed estrus in milk P4 levels less than I ng/ml on the day of the breeding) between two different synchronization. 2) To test the treatment effects on pregnancy rate between the two estrus synchronization methods of two different age groups of animals (cows or heifers). 3) To test the effect of milk production on estrus synchronization rates between two different estrus synchronization methods. 4) To test the effect of milk production on pregnancy rates between two different estrus synchronization methods. Unless stated otherwise, all comparisons were made at the 5 % level of significance (P< 0.05). 3.4. RESULTS 3.4.1 Estrus Synchronization Based on kamar heat mount detector, estrus was observed in 19 of 45 PGF 2 a treated group and 22 of 44 Ovsynch treated animals (Figure 3.1), and did not differ between the two treatments. Similarly the number of cows that showed estrus by milk P4 concentrations were not different between cows treated with PGF 2 a and Ovsynch (23 of 30) and (23 of 31), respectively (Figure 3.1). Milk Production (high or low producing cows) did not affect (P>0.05) the estrus synchronization rate between the two treatment groups (Figure 3.2). However, the number of cows detected in estrus by the analysis of P4 was higher than the number detected by the Kamar (Figure 3.1). 40 3.4.2 Corpus luteum function Analysis of milk P4 concentrations on day 0 (day of the breeding), 7, 14, and 21 after breeding allowed to compare CL status between cows synchronized with Ovsynch or PGF 2 a protocol. The least square means of milk P4 concentrations indicating CL status on day 7, 14, 18, and 21 after breeding was not significantly different between the two estrus synchronization protocols (P> 0.05) (Figure 3.3). 3.4.3. Pregnancy rates As shown in Figure 3.3. Heifers had similar pregnancy rates following synchronization with two injections of PGF 2 a or Ovsynch (P>0.05). However, cows synchronized by the Ovsynch method had a higher 19 of 33 (57.5%) (PO.05) pregnancy rate than cows treated with two injections of PGF 2 a 13 of 34 (38.4%) (Figure 3.4). Also, the pregnancy rate was higher (P <0.001) for PGF2 o t heifers than for cows treated with the PGF 2 a However, there was no difference in pregnancy rate between cows and heifers treated with Ovsynch protocol (Figure 3.4) . Ovsynch treated cows with high milk producing cows had a pregnancy rate higher (P< 0.01) than PGF 2 a treated cows with high milk production, but there was no significant difference (P > 0.05) in pregnancy rate between Ovsynch and PGF 2 a cows with low milk production (Figure 3.5) . Also, there was no difference in the pregnancy rate between high and low milk producing cows treated with PGF 2 a (Figure 3.5). 41 o (—4 < N t - H O Pi o oo oo P i H oo W „ ( A ) ( B ) 8 0 - i 7 0 -6 0 -5 0 -4 0 -3 0 -2 0 -1 0 -0 -• '1 l b . i l l • m • : : _ J > -P G F illlllllli illlltllll I l l l llllillill iiiiiii • iiiiiiiiii jfljjjjjjl lllHlill Illllllllil IIIIIIIIII i i i IIIIIIIIII • v.-iKhll f,:; u l G n R H P G F G n R H Figure 3.1. Estrus synchrony based on kamar heat mount detector (A) and milk progesterone (B) between animals synchronized with two injections of PGF 2 a and animals synchronized with Ovsynch protocol. 42 P G F Ovsynch Figure 3.2. Effect of treatment and milk production on estrus synchronization between synchronized with two injections of PGF2aand cows synchronized with Ovsynch protocol. 43 44 Cows Heifers Figure 3.4. Pregnancy percentage between lactating dairy cows and heifers synchronized with two injections of PGF2aand animals synchronized with Ovsynch protocol. 45 P G F G n R H Figure 3.5. Pregnancy percentage between cows synchronized with two injections of PGF 2 a and cows synchronized with Ovsynch protocol by milk production (high vs. low) at the time of breeding. Percentages with different letters are significantly different (P< 0.05) 46 3.5 DISCUSSION This study compared the estrus synchrony, CL function and pregnancy rate for timed-AI in lactating dairy cows and heifers after two different synchronization strategies. The ability to achieve acceptable fertility after timed insemination could have a major impact on reproductive management of lactating dairy cows. Therefore, timed artificial insemination relative to estrus and ovulation would allow more control in AI programs and would remove dependence on estrus detection. No method has been available to dairy producers that will consistently synchronize estrus and allow AI of the cattle to be fertilized by a single fixed time insemination strategies that could yield pregnancy rate equivalent to those achieved by cows inseminated after observed estrus (Larson etal., 1992). In the present study, there was no significant difference in estrus detection rate between the two treatment protocols. However, the percentage of animals which showed estrus based on Kamar, was significantly lower than the percentage of animals detected in estrus by milk P4 concentrations, determined on the day of breeding. In a large experiment conducted by Lean et al. (1989), which compared the relationship between signs of estrus at insemination and error of estrus detection based on milk progesterone concentrations, it was reported that a number of animals (15.5%) show estrus by a fully triggered Kamar heat mount detector had a P4 concentration more than 10.2 ng/mL. Although low milk P4 concentration alone is not a positive indicator of estrus, high milk P4 reliably confirms the non-estrus condition. During their experiment, the same authors found a lower conception rate in cows inseminated after estrus detected by fully triggered Kamar heat mount detector, compared to the conception rates obtained after standing estrus (42 Vs 53 %). Britt, (1978) and Folman et al. (1990) reported an error and loss rate of 15-20% with heat mount detectors as well. Therefore, in large herd of cows, 47 Kamar heat mount detectors are not sufficient means for the detection of estrus if they are not used in conjunction with heat detection records and good management, while the use of milk progesterone analyses can be an accurate indicator of identifying individual as well as groups of cows in estrus and their ovarian function as well In this study, estrus synchronization rate was not affected by milk production. Schapper et al. (1993) and Daubinger (1994) reported that the intensity of estrus synchronization decreased significantly with increasing milk production. These authors found that cows with high milk production were associated with reduced estrus displays, while Fonseca et al. (1983) reported that estrus detection rates were highest in cows producing slightly above the mean herd milk production. In my study, estrus synchronization pregnancy rate was affected by milk production. Ovsynch treated cows with high milk production had a higher pregnancy rate (P<0.001) than PGF 2 a treated cows with high milk production. A study conducted by Lean et al. (1989) demonstrated that cows with higher milk production were associated with reduced reproductive performance, suggesting that the metabolic demands of higher production may reduce fertility. However, other authors have found no significant relationship between milk production and fertility (Lauderdale et al, 1974; Macmillan et al., 1982). Stevenson et al. (1987) reported high milk producing cows resumed ovarian cyclicity earlier than low producing cows. These authors also noted that quiet ovulations were associated with high producing cows than low producing cows. In my study, high-producing cows synchronized with Ovsynch protocol had a higher pregnancy rate than low producing cows. It would appear that treatment of cows with Ovsynch method resulted in the ovulation of oocytes with a relative high potential for fertilization and embryo development competence. Previously, few comparisons have been made between the Ovsynch protocol and PGF 2 o synchronization methods. Most Ovsynch synchronization protocols reported a pregnancy rate equivalent to those animals bred after observed or detected estrus 48 (Burke et al., 1996; Pursely et al., 1997; Momilovic et al., 1998; Rouse et al., 1998). However, Jenks et al. (1998) consistently obtained higher pregnancy rate with cows synchronized with Ovsynch protocol than cows synchronized with PGF 2 a injected after the diagnosed the presence of CL through rectal exploration (Jenks et al., 1998). This is the first comparison made between PGF 2 o and Ovsynch methods per timed insemination. It has been reported that the ability of prostaglandin to induce estrus improves during the luteal phase, specifically from day 8 of the estrous cycle (Stevenson et al., 1983; Lauderdale et al., 1974). Many authors obtained a lower fertility rate for cows treated with PGF 2 a on the early days of the cycle (d 4, d5 and d 6) than days 12-16 (Larson et al., 1992; Smith et al., 1996). Treatment with PGF 2 a between d 5-8 of the estrous cycle has resulted in less variable intervals than treatment from dl 1-16 (Kastelik et al., 1990; Smith et al., 1996). To find an explanation for this would seem important, since the pregnancy rate may be highly dependent on the degree of estrus synchrony, particularly if fixed time AI is used. A partial explanation may be that the intra-ovarian processes associated with various stages of luteal development play a significant role in determining the variability in the interval to estrus and the degree of synchronization after CL regression (Folman et al., 1988; and Garcia et al., 1991). Much of the variation in the time to ovulation is related to variation in the stage of the preovulatory follicle present at the time of treatment; thus, in some cows the dominant follicle takes longer to mature prior to ovulation. Early workers proposed that that fertility response of PGF 2 a treated animals depend on the stage of the cycle. Stevenson et al. (1983) and Smith et al. (1993) reported a lower conception rate when PGF2(X was injected during the early stages rather than the late stages of the bovine estrous cycle. 49 Fifteen percent of cows not synchronized by PGF 2 a had low levels of P4 in during the period of sampling, and fourteen out of eighteen cows in the PGF 2 a group that did not become pregnant had a P4 concentration of < 1 ng/mL during the sampling period. This indicates that the cows either had a long anestrous period after calving or had a disturbance in the normal development and functional of the CL. Therefore, the lower pregnancy rates observing in animals synchronized with two injections of PGF 2 a might have resulted from either the lack of proper timing or failure of normal CL function. Silcox et al. (1993) proposed that disturbances to ovarian follicular distribution may predispose suboptimal function of luteal tissue after ovulation when injections of PGF 2 a were used. However, milk P4 samples taken to asses corpus luteal status after ovulation indicated that there were no differences between the two groups mean. Achieving acceptable fertility after fixed time insemination requires close synchronization of luteolysis with the final growth and maturation of the preovulatory follicle. The protocol used for cows and heifers synchronized with Ovsynch in this study was previously reported by Burke et al. (1996) and Pursely et al. (1997). This involved the administration of a GnRH followed by an injection of PGF 2 c t 7 days later and then a second injection of GnRH 48 hr after PGF 2 o This method was designed to maximize the potential for ovulation of the dominant follicle present at the time of treatment and to induce premature turnover of new follicles, thus, ensuring their availability for ovulation after the second GnRH injection. To achieve successful estrus synchronization by using Ovsynch protocol, it is particularly critical to induce ovulation in a follicle when the first GnRH is given. Pursely et al. (1992) and Rouse et al. (1996) demonstrated that 87-100 % of Ovsynch treated cows ovulated after the second GnRH injection, while the same authors reported 60-78% of heifers responded. This difference of ovulation resulted in lower pregnancy rates of heifers with Ovsynch than heifers treated with the PGF 2 a during their experiments (35 % vs. 75, respectively). However; our study 50 does not favor PGF 2 a treatment over Ovsynch for heifers. This difference between the studies may be attributed to the small sample of heifers used in the current experiment. It can be concluded from the present study that precision of estrus synchronization by using PGF 2 a is inadequate to warrant timed insemination for dairy cows, while the Ovsynch method can provide acceptable fertility. 51 CHAPTER 4 EFFECTS OF GONADOTROPIN-RELEASING HORMONE GIVEN ON DAY 7, DAY 14, OR DAY 7 AND 14 AFTER BREEDING ON PROGESTERONE PROFILE, OVULATION AND PREGNANCY RATE 4.1 ABSTRACT The objective of this study was to determine the effect of treatment with GnRH at various times after breeding on the induction of accessory CL, P4 concentrations and pregnancy rates. Each month, estrus was synchronized in 12-15 postpartum cows by using the Ovsynch protocol followed by timed artificial insemination. Cows were then randomly assigned to receive GnRH on d 7 (n=34), d 14 (n=34), or d 7 and 14 (n=35) after breeding or to serve as controls (n=33). During the first two months, total of 24 cows, six cows from each treatment group under went ultrasononographic examinations on the ovaries on days 7, 11, 14, 18, and 21 after breeding to observe the formation of any additional CL. Blood and milk samples were also taken on days 0 (day of the breeding), 7, 14, 16, 18, and 21 after breeding to determine P4 concentrations. Pregnancy was diagnosed 35 days after breeding. The rest of the experimental cows (n=l 12) did not go through ultrasonographic examinations, blood and milk samples were not taken, but pregnancy diagnosis was conducted similarly 35 days after breeding. Eighty-three percent of cows ovulated in response to GnRH on d 7 as opposed to only seventeen percent on d 14. Mean plasma P4 concentrations in treated cows did not differ from control cows. Gonadotropin releasing hormone after breeding did not lead any significant increase (P > 0.05) in pregnancy rates for cows treated with GnRH on days 7, 14, and 7 and 14, (50 %, 41.1 %, 37.1 %), compared to the control (39.4%). 52 4.2 . INTRODUCTION The continued secretion of P4 from a viable CL is essential for the establishment and maintenance of early pregnancy in cattle (Manning et al., 1989). Cows that become pregnant have been reported to have consistently higher peripheral P4 concentrations than non-pregnant cows (Shelton, 1990; Gersot, 1992; Butler, 1996; Shelton, 1997). Insufficient P4 during early pregnancy may contribute to a high level of early embryonic mortality in cattle (Diskin, 1980). Although other factors may also contribute to this early embryonic loss, most of it has been attributed to an inadequate CL function (Weilbold, 1988; Laming, 1989). Various treatments have been used in attempt to control inadequate CL function, including the use of progesterone supplements (Northy et al., 1985; Robinson et al., 1989) administered at various times after insemination. The results from these studies were inconsistent regarding to on pregnancy rate. Other methods designed to increase inadequate CL function in cattle include the use of gonadotropins such as Luteinizing hormone (LH) (Donaldson et al., 1965; Laming et al., 1981) or Human chorionic gonadotropin (hCG) (Holness et al., 1982; Wielbold et al., 1988; Lewis et al., 1990; Bruel et al., 1990; Sianangama et al., 1992) at various times following insemination. The use of hCG in cattle improved pregnancy rates in some studies (Wiltbank et al., 1961; Holness et al., 1982; Sianangama et al., 1992), but other workers have shown only a slight increase in pregnancy rates among treated cows (De Los-Santos-Valdez et al., 1982) or no beneficial effect at all (Looney et al., 1984). Recent interest in commercial dairy herds has centered on the use of GnRH at various times after breeding to enhance pregnancy rates. Experimental studies have shown that GnRH (Buserelin) and hCG are equally effective in inducing an accessory CL when administered on d 5 of the cycle, but the subsequent increase in plasma P4was greater in hCG- treated heifers. Higher P4 concentrations following hCG may be attributed to the long half-life of hCG and its action on 53 both induced an accessory CL. One draw back to repeated use of hCG in cattle is the formation of antibodies (Thatcher et al., 1993). Therefore, it would be more desirable to use GnRH in cows with compromised CL function. E 2 from the large follicles during the luteal phase has been demonstrated to be essential for the activation of the luteolytic mechanism (Mann et al., 1994). Accordingly, this study was based on the premise that administration of GnRH on d 7, d 14, or d 7 and 14 after breeding will eliminate DF present either first or second wave, thereby increasing pregnancy rate. Therefore, the objectives of this study were to determine ovulatory response, plasma and milk P4 concentrations, and pregnancy rate following administration of GnRH on d 7, d 14, or d 7 and 14 after breeding. 4.3. MATERIALS AND METHODS 4.3.1 Animals: General Management Practices This experiment was conducted at the University of British Columbia Dairy Education and Research Center, in Agassiz, BC, during the months of August 1998 to May 1999. Animals were in between 60-90 days postpartum. Rectal palpation of the reproductive tract was conducted before the start of the experiment in order to assure complete reproductive organs and their normal physiological status. Cows were milked twice 4:00 A.M and again 15:00 p.m. The experiment commenced in August, 1998. This month was part of a period (April to October) during the summer when cows were maintained on pasture. This pasture consisted of a mixture of clovers, orchard grass rye. During this period, the cows were also provided with grain for both maintenance and production. The criteria used in deriving the production ration was on a 1 part of grain to each 4 kg of milk produced. Five kg of this portion of the feed was presented to the animal in the milking parlour, split in equal portions given to the animal at each milking time, the balance was given to each animal in barn. 54 4.3.2. Treatments Each month, estrus was synchronized in 12-15 postpartum cows by using the Ovsynch protocol followed by timed artificial insemination. Cows were then randomly assigned into four treatment groups: Group 1 (n=33) received saline (control) on day 7 and 14, Group 2 (n=34) received GnRH (Fertiline) on d 7, Group 3 (n=34) received GnRH (Fertiline) on d 14, Group 4 (n=35) received GnRH (Fertiline) on d 7 and 14 after breeding. During the first two months, a total number of 24 cows, six cows from each treatment group under went ultrasononographic examinations on the ovaries on days 7, 11, 14, 18, and 21 after breeding to observe the formation of any additional CL. Blood and milk samples were also taken on days 0 (day of the breeding), 7, 14, 16, 18, and 21 after breeding to determine P4 concentrations. Pregnancy was diagnosed 35 days after breeding. The rest of the experimental cows (n=112) did not go through ultrasonographic examinations, blood and milk samples were not taken, but pregnancy diagnosis was similarly conducted 35 days after breeding. 4.3.3. Ultrasound Examination During the first two months, a total number of 24 cows, Six cows from each treatment group were conducted with ultrasound scanning on d 7, 14, 18, and 21 after breeding. The ultrasound equipment used was a "Toshiba" real time ecograph model Aloka 500 linear scanner with impermeable transrectal probe capable of producing and receiving ultrasound at a frequency range 7.5 MHz. To start each examination the rectum was evacuated of all fecal materials and a made a quick note on the uterine tone and the location of the ovaries was established by palpating through the rectum. During the ultrasound examination, internal structures in the body of cows fall under two categories. The probe emitted sound waves. The characteristics of specific tissues determined what proportion of the sound beam were reflected. Images of the reflected 55 portions registered on the screen as shades of gray, extending from black to white. All fluid-filled body structures being non-ecogenic (absorb ultrasound waves emitted by the probe) appeared black. Follicles for example were visualized as black. On the other hand, dense tissues such as bone or cervix appeared white. Tissues in between these extremities were seen as different shades of gray, depending upon their degree of echogenecity (Pierson et al., 1988). I n order to keep a permanent record and to facilitate the measurement of the structures of interest, once the optimum scan was obtained, these were frozen on the screen. The dimensions of CL and follicles measuring more than 3 mm in diameter were obtained. Measurements were taken at the widest poles using a built- in system of calibrated calipers on the ultrasound machine. Information on individual cow was recorded on the frozen/images using an alpha-numeric key board. Such data included the date and time of scanning, the cow identification number, number of days after breeding. Hard copies of the frozen images of the CL and follicles more than 3 mm were obtained using a Mitsubishi Video copy processor connected to the ultrasound scanner. Ovulations resulting from treatment with genre were verified by observing for an acute disappearance of one or more of the dominant or pre-ovulatory size (antral) follicles present at the time of administering the treatment. The emergence of an induced CL was characterized by luteal tissue appearing on a site previously occupied by an antral follicle. The growth of such induced CL was followed by determining the diameter of the new luteal tissue at the widest poles. This, in some cases, was only possible until the induced CL reached the size of the spontaneous CL. The functional capacity of induced CL could not be established from this in-vivo experiment. It was, however, hoped that increased levels of circulating P4 would be indicative of an additive effect of an increase in spontaneous CL production of P4 and P4 originating from the induced CL. 56 4.3.4. Blood and Milk Sample Collection For each experimental animal, 10 mL of blood was collected via the coccygeal vein in vacutainer tubes containing sodium heparin (Becton, Dickinson, Vacutainer Systems, and Ruthfield New Jersy, USA). Samples were immediately centrifuged at 1500xg for 20 minutes, then the plasma was removed and stored at -20°c until P4 analysis. Twenty ml of milk samples were also taken from each cow in each treatment group. Milk samples were frozen at -20°c and thawed only when assaying for P4 Milk and blood were collected on day 0 (d 0= the day of the breeding), day 7, day 14, day 16, day 18, and day 21 after breeding. The purpose of this sampling was to asses the P4 profile after GnRH induced ovulation 4.3.5. Radioimmunoassay Concentrations of milk and plasma P4 were determined by using a commercial, solid phase I125 label radioimmunoassay (Coat-A-Count, Diagnostic Products Corp., Los Angles, CA) as described in the first experiment. This kit has been validated in our laboratory for the measurement of P4 in cow's milk and plasma by Rajamahendran et al. (1989). 4.3.6. Pregnancy Diagnosis In cattle, ultrasound examinations have been shown an efficient and direct means of pregnancy diagnosis 35 days after breeding (Rajamahendran et al., 1994). Therefore, pregnancy was verified using ultrasonography conducted on d 35 after breeding. 4.3.7. Statistical Analysis Data for milk and plasma P4 concentrations were statistically analyzed by the least squares analysis of variance (ANOVA), using JMP procedures of the Statistical Analysis System Institute Inc. (SAS) (version 1997). Least square analysis was used to test the difference between 57 control and the GnRH treated cows. Chi-square analysis was employed to test in the number of cows with induced CL and the number of cows diagnosed pregnant. 4.4. RESULTS 4.4.1. Induction of Accessory Corpus Luteum Results of cows to GnRH treatment are summarized in Table 4.1. No CL were induced in control cows not injected GnRH at any stage after breeding. Among the six cows treated with GnRH on d 7, 4/6 (67%) formed an induced CL. Among the cows treated with GnRH on dl4 after breeding, the largest and the second largest follicles present at the time of giving the GnRH ranged between 13 to 22.9 mm in diameter. Among these cows only 1/6 (17%) responded to the GnRH injection on d 14 and formed secondary CL. Among cows given GnRH on day 7 and 14 post insemination, the dominant follicles present at the time of treatment were between 13.3 mm and 17.8 mm in diameter, all of the six cows 6/6 (100%>) responded on the day 7 injection and each cows had 2 CL. However, only one cow responded to the GnRH treatment on day 14 after breeding. The diameter of the dominant follicle measured on day 14 in this cow was 14 mm and was located on the right ovary. After ovulation, this cow also had 3 CL two on the right ovary and one on the left ovary. The induced CL, were clearly visible by ultrasound by day 3 to 4 after breeding in all experimental cows. No differences were observed in the maximum diameter of either spontaneous or induced CL among treatment groups. Based on the size, it was not possible to distinguish between the existing and induced CL after d 7 of the ultrasound examination. 58 4.4.2. Plasma progesterone Profile Plasma P 4 concentrations are presented in Figure 4.1. Mean plasma P4 concentrations of cows given GnRH on day 7, 14, or 7 and 14 after breeding and the control group were not significantly different (P> 0.05). Maximum P4 concentrations were observed on d 14 and 18 (7.7 ng/ml and 7.9 ng/ml, respectively), for cows treated with GnRH on days 7, however, this deviation did not influence the mean P4 profile. 4.4.3. Milk Progesterone Profile Milk P4 concentrations are presented in Figure 4.2. Mean milk P4 concentrations of cows given GnRH on day 7, 14, or 7 and 14 after breeding and the control group were not significantly different (P> 0.05). Maximum P4 concentrations were observed on d 14 and 16 (10.08 ng/ml and 10.4 ng/ml, respectively), for cows treated with GnRH on days 7, however, this deviation did not influence the mean P4 profile. 4.4.4. Pregnancy Rate Treating with GnRH after timed breeding did not lead to any significant increase (P>0.05) in pregnancy rates Table 4.2. Ultrasound examinations to diagnose pregnancy on day 35 after breeding showed pregnancy rates of 50 %, 41.2 %, 37.1 %, 39.4 % for cows treated on d 7,14, or 7 and 14, and the control group, respectively. 59 Table 4.1. Number of cows ovulated after GnRH administered on day 7, day 14, day 7 and 14, after breeding comparison to untreated cows. Group no* Total Days after breeding Number of cows with accessory CL on d 11 Number of cows with accessory CL on d 14 1 6 0 0 2 6 4(66,7%) 0 3 6 0 1 (16.7%) 4 6 6 (100 %) 1 (16.7%) Total 24 10/12a (83%) 2/10b (17%) * Group 1: Control cows Group 2: Cows given GnRH on d 7 after breeding Group 3: Cows given GnRH on d 14 after breeding Group 4: Cows given GnRH on d 7& 14 after breeding abValues in the same row with different superscript are significantly different (P< 0.05). 60 Figure. 4.1. Mean (± SE) plasma Progesterone concentrations in control group (Gl) and cows given GnRH on d 7 (G2),d 14 (G3), d 7 and 14 (G4) after breeding, 61 Figure 4.2. Mean (± SE) milk progesterone concentrations in control group (G 1) and cows given GnRH on d 7 (G 2), d 14 (G 3), d 7 and d 14 (G 4), after breeding 62 Table 4.2. Effect of GnRH given either on day 7, day 14, day 7 and 14 or control group on pregnancy rate. Group noa Total number of cows inseminated Number of cows Pregnant Pregnancy % 1 33 13 39.4 2 34 17 50 3 34 14 41.2 4 35 13 37.1 Total 136 57 41.9 a Group 1: Untreated cows Group 2: Cows given GnRH on d 7 after breeding Group 3: Cows given GnRH on d 14 after breeding Group 4: Cows given GnRH on d 7&14 after breeding. A b Values in the same row with different superscript are significantly different (P< 0.05). 63 4.5 . DISCUSSION In the present study, the dominant follicle present on day 7 ovulated in response to GnRH and formed an accessory CL in 10 out of 12 cows. In contrast, of the 12 cows treated on day 14 only 3 cows responded. In cattle, the ovarian follicular development of normal estrous cycle is characterized by the presence of either two or three follicular wave (Ginther et al., 1989). The first wave, and the second wave in animals with three waves per cycle produce a large non ovulatory follicle that undergoes atresia, where as the last follicular wave produces the ovulatory follicle (Sirois et al., 1988; Ginther et al., 1989; Sirois et al., 1990). The DF in each follicular wave has three phases, growing, static and regressing (Matton et al., 1981; Fortune et al., 1987; Pierson et al., 1987; Sirois et al., 1987 Savio et al., 1988 and Rajamahendran et al, 1988). Based on these follicular dynamics, the beneficial effects of GnRH on fertility derived from inducing ovulation of otherwise late-maturing ovulatory follicles. Several experiments conducted by Chenault et al. (1992), Tawagiramingu et al. (1995), Kohran et al. (1998) suggested that GnRH can induce preovulatory LH release which could ovulate the dominant follicle during its growing stage of development. However, ovulation did not occur in all cases after GnRH treatment because the ability of a follicle to ovulate depends on the acquisition of LH receptors on its granulosa cells (Garcia et al., 1999). Silcox et al. (1993) reported that GnRH treatment altered the diameter of the large DF during its growing or plateau phase, but not during its regressing phases as atresia has already been initiated (Chenault et al., 1993). During the mid-luteal phase, the negative effects of P4 produced by the CL also reduces the pulses of LH frequency from the pituitary gland and hence, ovulation does not occur, and the large follicles present at the time of GnRH treatment regressed (Ginther et al., 1989). It has been reported that, during a two wave cycle, the DF of the first wave present around day 12 of the cycle is in regressing phase, and the process of atresia has already been 64 initiated, this leads to the emergence of the second DF (Guibault, 1993). The time of deviation in growth rates between the future dominant and subordinate follicles occurs on an average of 2.8 days (Vasconcelos, 1999). The LH mRNA is not expressed in the granulosa cells of the growing follicle during the first 2 days of the follicular deviation (Ireland and Roche, 1981). In my experiment, it is possible that the majority of the experimental animals had two follicular waves during the period of the GnRH administration. Therefore, follicles present on day 14 of the cycle were unlikely to respond to the administered GnRH because LH receptors have not yet been expressed in their granulosa cells. Based on follicular growth during early and late luteal phase, the increase in the responsiveness of GnRH at different periods observed in the present study appears to coincide with the stage of the DF, and the lower ovulatory response of animals given GnRH on d 14 may reflect an absence of follicles at an adequate stage of maturity. Establishment and maintenance of pregnancy in domestic ruminants is dependent upon the secretion of P4 from a viable CL (Bazer and First, 1983; Wiltmut et al., 1985). An insufficiency of P4 may originate from an abnormal CL development that leads to an early embryonic mortality (Bulman and Lamming, 1978). The focus of this study was to increase the P4 concentrations by giving GnRH on d 7 and 14 after breeding in order to induce ovulation of the DF and form secondary CL. However, administration of the GnRH did not show any significant increase either in milk or plasma P4 concentrations. Similarly, Macmillan and Thatcher (1985), Lucy and Stevenson (1986), Rodger and Stormshak (1990), Lewis et al. (1994) did not report any progesterone increase after cows and heifers were given GnRH at different stages after breeding. In contrast, Mee et al. (1993), Macmillan et al. (1985) and Macmillan et al. (1991), Rayon et al. (1992), Mee et al. (1993) and Schmitt et al. (1996) reported increases in serum P4 concentrations after administration of GnRH at different times of the estrous cycle in cattle. 65 Administration of GnRH has been reported to induce an LH surge (Lucy et al., 1985; Thatcher et al., 1989; Lamming et al., 1989). The increase of P4 concentration depends on the ability of LH released by GnRH to stimulate bovine luteal synthesis of P4 (Mason et al., 1962). Hooley et al. (1974) and Rodger et al. (1986) reported that the quantity of LH released in response to GnRH administered to cows on d 5 did not differ from that released on d 5 to d 12 of the estrous cycle. This indicated that the stage of the estrous cycle did not affect the quantity of LH released. However, the preovulatory LH surges of cattle induced by GnRH differed in terms of duration and magnitude (Chenault et al., 1985) In general, the normal duration of the LH secretion associated with the preovulatory surge is 10 h (Chenault et al., 1993; Rahe et al., 1993). It has been suggested that the normal LH surge induces more complete stimulation of the ovulatory follicle that is necessary for the development of a fully functional CL (Burke et al., 1996). Schmitt et al. (1993) and Schmitt et al. (1994) demonstrated that administration of GnRH agonist or hCG induced ovulation of the first wave dominant follicle, however, the resultant CL had a reduced capability to secrete P4 when compared to CL induced by hCG. The authors concluded that administration of GnRH elicited an LH release for 5 hours which is approximately half the duration of a naturally occurring LH surge (Chenault et al., 1975; Roche et al., 1980), while administration of hCG induced an increase in LH-like activity for up to 30 h (Sequin et al., 1977; Schmitt et al., 1996). The later authors reported that GnRH induced CL were lighter than the hCG induced CL, and supported the hypothesis of Smith (1994) that the larger the accessory CL induced, the more luteal cells are present, which in turn secrete more P4. These authors also suggested that the persistence of hCG in plasma may provide more optimal gonadotropic support for greater growth of the induced CL. In contrast, Rajamahendran et al. (1998) reported greater basal concentrations of LH and maintained for 48 h in cows implanted GnRH compared to those injected with GnRH. The authors reported also an increase in plasma 66 concentrations of P4 following GnRH implant during the estrous cycle. Increased of P4 synthesis could have resulted from the hypertrophy of the luteal cells in the spontaneous CL (Sianangama and Rajamahendran, 1992). The present study clearly demonstrated that single injection of GnRH given on d 7 of the estrous cycle is effective in inducing accessory CL formation. The induced ovulations resulted in the development of structures similar in appearance to spontaneous CL. The induced CL grew so rapidly that at one point it was difficult to distinguish between spontaneous and induced CL. However, these observations of induced CL in animals treated with GnRH were not followed by any detectable increase of P4 concentrations when compared to the control group or cows not induced formation of accessory CL by giving single injection of GnRH. These results are in agreement with Britt et al., 1975 and Sequin et al., 1977 who suggested that GnRH had no effect on P4 secretion when given single (100-200 u.g) or daily, repetitive injections (400 u.g/day) during mid-or late luteal stages of the estrous cycle. During the process of the differentiation of the follicular cells into luteal cells, granulosa cells differentiate into large luteal cells. No mitosis occurs during the differentiation of granulosa cells (Donaldson et al., 1965), therefore, the number of the granulosa cells in the preovulatory follicle is the same as the number of the large luteal cells after the CL formation (Smith et al., 1994). However, intense luteal activity is observed in theca cells from d 1 to d 4 of the estrous cycle (Donaldson et al., 1965) and the theca cells differentiate into small luteal cells. These processes of differentiation are controlled by the LH stimulation (Fitz et al., 1982). The small luteal cells contain the majority of LH receptors (Farin et al., 1988). Based on these mechanisms, Mee et al., 1992; Rajamahendran et al., 1998; Ambrose et al., 1998 suggested that induced release of LH by exogenous GnRH or hCG promoted the conversion of small luteal cells into large luteal. Because approximately 85% of basal P4 production is attributed to large luteal cells (Niswender et al., 1985), suggesting the increased P4 concentrations properly resulted from the 67 altered ratio of large to small luteal cells. In contrast Ford and Stormshack, 1978; Lynn and Stormshack 1985 found that GnRH injected into heifers during d 2, 8 and 10 of the estrous cycle caused a reduction in serum P4 concentration, but these authors did not report whether ovulation had occurred or not. These researchers proposed that administration of the GnRH might have attenuated the normal functioning of the CL causing down regulation. Diskin and Sreenan (1984) have been reported that among fertilized eggs, a certain number of embryos are lost during the first 8 to 19 days after conception. Most of these animals seem to be clinically normal, without showing any apparent physical abnormalities of the reproductive tract. However, these animals may often show abnormal estrous cycles length (i.e. long intervals between the estrous cycles). Pregnancy rates following administration of GnRH after breeding have been reported with variable results (Lee et al., 1987). The present study does not indicate any increase of pregnancy rate from GnRH administration after breeding. Similar incidences were reported by Macmillan et al. (1986); Jub et al. (1990); Trayon et al. (1991), Thatcher et al. (1992); Rayan et al. (1994) and Small et al. (1999). In contrast, Macmillan et al. (1985) reported higher pregnancy rates associated with high level of P4 concentration during the luteal phase after giving GnRH at mid-cycle, either days 11,12, or 13 after breeding. The onset of CL function has been reported to be essential for the normal embryonic development (Macmillan et al., 185; Thatcher et all., 1989). Recent work (Larson et al., 1997), demonstrated that pregnant cows show a steady increase in luteal P4 concentrations from d 10-24 after breeding, and the time of the luteal onset was delayed in non pregnant cows versus pregnant cows. This increase of P4 concentrations has been suggested to be the initiation of luteotropic support from the embryo (Helmer et al., 1988 and Thatcher et al., 1989). However, in the present study, It could not be determined if any embryonic loss had occurred during the period of experiment, since continuos blood samples after ovulation were not taken. It appears, therefore, 68 that the action of exogenous GnRH on fertility is mediated by some means other than augmenting P4 in treated animals. A depression in milk P4 concentration on day 7 and a subsequent increase on d 10 in the mated non-pregnant cows has also been reported by Lamming et al. (1989). A biological explanation has not been established for this phenomenon, except that it might signal the onset of a sustained luteolytic effect in animals that are failing to maintain pregnancy (La France et al., 1988 and Laming et al., 1989). The present study did not indicate that cows losing fertilized ova or embryos had a deficiency in progesterone secretion on d 7 post breeding, therefore, it appears that ovulation was followed by normal development of the CL in terms of P4 production for up to d 21 after breeding. However, four out of twelve cows (30%) found not pregnant at the time of the pregnancy diagnosis showed high levels of P4 concentrations on d 21 after insemination. This finding may indicate an early embryonic mortality occurred before day 21 after insemination, and is supportive to that suggested by Laming etal. (1989). The presence of high affinity binding sites for GnRH in ovaries of the rat (Clayton et al., 1979 and Howard et al., 1980) implicated a possible direct action of GnRH on the mammalian ovary (Huseuh and Jones, 1986). In addition, a GnRH-like ovarian hormone that binds to rat GnRH receptors was isolated from the ovaries of the ewe and cow (Alan et al., 1987). However, direct action on the bovine ovary is unlikely because GnRH receptors have not yet been detected in ovarian tissue of the cow. In this species, observed effects of GnRH on luteal steroidogenesis in some studies may be mediated indirectly via increased release of LH, and hence inducing ovulation of otherwise late-maturing ovulatory follicles. Although LH is luteotropic in the cow (Donaldson et al., 1965) brief exogenous of the CL to large amounts of LH may cause down regulation of the receptors (Conti et al., 1976). Thus, in the cow, luteal response evoked by exogenous GnRH may depend upon such variables as frequency and/ or route of injection of the GnRH, quantity of LH released and/or stage of development of the CL. 69 In the present study administration of GnRH post breeding induced ovulation of the dominant wave follicles present on day 7, increased the estrous cycle length of inseminated non-pregnant cows, but, pregnancy rates were not affected by its administration. The inconsistent increase of P4 and pregnancy rates observed in some experiments and a failure to detect a response in others suggest that unidentified environmental factors could have impinged on the response to GnRH treatment. 70 CHAPTER 5 GENERAL DISCUSSION Reproductive efficiency is dependent upon service and conception rates. Since the development of estrus synchronization protocols, various methods have been used in dairy cattle, predominately double injection of PGF 2 a . It has been reported when animals synchronized with P G F 2 a and inseminated 12 h after detecting estrus result in comparable pregnancy rates to those animals bred after a naturally observed estrus. However, since estrus detection rates are generally low in lactating dairy cows, producers have attempted to increase the service rate by using timed A l after two injections of PGF 2 c ( (Pursely et al., 1995). The difficulty with this approach is that timed A l of animals synchronized by P G F 2 a has resulted in reduced conception rates in lactating dairy cows (Lucy et al., 1986; Stevenson et al., 1987; Archibald et al., 1992). In the first experiment, the pregnancy rates following timed artificial inseminations after animals were synchronized with two injections of P G F 2 a were significantly lower than those animals synchronized with Ovsynch. The reduced fertility reported in this experiment is most likely related to the greater variation in the timing of ovulation following P G F 2 a treatment compared to the Ovsynch method. It has been proposed that the average interval from the P G F 2 a injection to estrus is usually between 60-72 h. However, studies conducted by King et al. (1982), Stevenson et al. (1984), Tenable et al. (1984), and Walt and Faquay, (1985), reported that estrus occurred an average of 48-59 h after animals were treated with P G F 2 a between d 5 to d 8 of the estrous cycle. In contrast, the average time of estrus was 53 to 73 h when heifers in the same studies were treated with P G F 2 a between d 12 and 15 of the estrous cycle. Thus estrus occurred within a 5 day period after the P G F 2 a treatment, regardless of the stage of the estrous cycle at the time of the treatment, but the exact day in which the cow show on estrus cannot be predicted due to 71 differences in the stage of the follicular development. It has been hypothesized that this variability may directly relate to the size of the future ovulatory follicle at the time of PGF 2 a injection. Altering the development of the ovulatory follicle can affect the timing of estrus and fertility of the ovulated ovum (Larson et al., 1997). Clearly, if a herd of cows is randomly distributed throughout the estrous cycle, a follicular wave would be emerging in some animals while others would have dominant follicles capable of immediate maturation and rapid ovulation. Unless follicular development in such a mixed group is controlled, the timing of synchronized estrus and ovulation would be variable. In order to overcome this, it is necessary to develop a method of synchronization that eliminates the growing or static dominant follicles, controls the time of the emergence of a new follicular wave, and allows the follicular turnover of the majority of animals to be synchronized in a fixed period. Development of such protocols will lead to the improvement of estrus synchronization and ovulation by assuring that the ovulatory follicle has an optimum potential for fertilization and embryo development. To attempt these, various methods have been studied including the Ovsynch protocol. Researchers (Brown et al., 1988; Patterson, 1990; Jaeger et al., 1992) reported that the synchrony of estrus following administration of GnRH followed by a luteolytic dose of PGF2 o t is more precise than when PGF 2 a is administered twice, 11 or 14 days apart. The precise estrus synchrony following GnRH-PGF2a treatments make it a possible candidate for a single, timed insemination breeding protocol that can be adopted in the field. Recently, Pursely et al. (1995b) reported that the range in timing of ovulation in lactating dairy cows following GnRH-PGF2a treatment extended from 84 to 120 h after the PGF 2 a. The same author demonstrated that the range of ovulation in cows could be reduced to 8h (72-80 h if a second GnRH injection is administered 48 h after the PGF 2 t x injection. This method has been termed "Ovsynch" regimen. This treatment 72 was devised to provide an alternative system for inducing estrus and ovulation, because PGF2 o t alone has been shown to be too variable to induce fixed-time insemination reliably in lactating cows (Stevenson et al., 1996). However, various pregnancy results following Ovsynch protocol have been reported depending if cows were bred at 8, 16, 24 or 32 h after the second injection of GnRH (Pursely et al., 1997). Higher fertility can be achieved if there is a follicle with a viable oocyte capable of ovulating in response to GnRH treatment and if cattle are inseminated during a relatively short period, approximately 12 hours before ovulation to a few hours after ovulation. The highest conception rate was reported by Pursely et al. (1995b) after dairy cows were synchronized by the Ovsynch method and bred 16 h after the second GnRH injection (40 %), but tended to be lower (P<0.1) when AI was done 32 h after the second GnRH injection (32%). The same authors demonstrated that the majority of the animals treated with this regime ovulated between 24 and 32 h after the second GnRH injection. Pursely et al. (1995b, 1997) reported similar pregnancy rates between animals synchronized with Ovsynch and those synchronized with PGF 2 a and inseminated 12 h after observed estrus (38.9 vs. 37.8 %). Similarly, pregnancy rates following synchronization by Ovsynch and timed AI were not different from those bred 12 h after observed estrus 40.8% Vs 46.4 %, respectively. In the first study, pregnancy rates after GnRH ~ PGF 2 a and a second GnRH treatment 48 h after the PGF 2 a were comparable to those reported by Jenks et al. (1998) but higher than those reported by Pursely et al. (1995a, 1997). Results of the present study support the hypothesis that the administration of GnRH or one of its potent agonists induces the release of gonadotropins from the pituitary gland. The production of these hormones in turn alters the pattern of the existing follicular wave dynamics either by inducing ovulation or atresia of dominant follicles present during the time of administration (Thatcher et al., 1989). This in turn permits the recruitment of new follicles; the injection of PGF 2 a 7 days later induces the regression of the CL, and giving second GnRH 73 i n j e c t i o n 4 8 h a f te r t he P G F 2 a c a n i n d u c e a m o r e p r e c i s e t i m e d o v u l a t i o n w i t h i n 2 4 a n d 3 2 h a f te r t h e las t G n R H i n j e c t i o n . T h i s r e s u l t s i n t he r e l e a s e o f a n o v u m , a n d i f t he c o w i s i n s e m i n a t e d 16 to 3 6 h a f te r t h e s e c o n d G n R H i n j e c t i o n a n d a c c e p t a b l e p r e g n a n c y ra tes c a n b e o b t a i n e d as i n d i c a t e d b y the f i r s t e x p e r i m e n t o f t h i s t h e s i s . H o w e v e r , m a n y f a c t o r s s u c h as e n d o g e n o u s e n v i r o n m e n t , m a g n i t u d e o f g o n a d o t r o p i n r e l e a s e , c l i n i c a l c o n d i t i o n s o f t he a n i m a l , d o s a g e a n d t i m e o f a d m i n i s t r a t i o n o f G n R H , a n d e n e r g y b a l a n c e c a n a l l a f f ec t the r e s u l t s o f t h e G n R H o r i ts a g o n i s t o n e n h a n c i n g p r e g n a n c y ra tes i n d a i r y ca t t l e . H o w e v e r , t he c h a n g e s tha t t a k e p l a c e i n l a c t a t i n g d a i r y c o w s f r o m p a r t u r i t i o n to c o n c e p t i o n are c o m p l e x ( B u t l e r et a l . , 1 9 8 9 ; T h a t c h e r et a l . , 1 9 9 3 ; I n s k e e p , 1 9 9 5 ; G a r v e r i c k , 1 9 9 7 ) . T h e r e f o r e , t he s u c c e s s o f a n y es t rus s y n c h r o n i z a t i o n p r o g r a m d e p e n d s u p o n the c o w s c y c l i n g n o r m a l l y b e f o r e a n y t r ea tmen t . A f t e r p a r t u r i t i o n , the i n i t i a t i o n o f c y c l i c a c t i v i t y , a c o m p l e t i o n o f s e v e r a l c y c l e s b e f o r e b r e e d i n g h a s b e e n p r o p o s e d to b e a k e y f a c t o r e n a b l i n g c o w s to m a i n t a i n a h i g h r e p r o d u c t i v e p e r f o r m a n c e ( B u t l e r a n d S m i t h , 1 9 8 9 ) . I n t h e p r e s e n t s t u d y , s e v e r a l v a r i a b l e s that m i g h t r e s u l t i n d i f f e r e n t r e s p o n s e s to G n R H w e r e e l i m i n a t e d . A r e s e a r c h h e r d o f m u l t i p a r o u s c o w s b e t w e e n 6 0 - 9 0 d a y s p o s t p a r t u m a n d v i r g i n h e i f e r s f e d c a r e f u l l y a c c o r d i n g to p r o d u c t i o n r e q u i r e m e n t s a n d w i t h g o o d r e p r o d u c t i v e m a n a g e m e n t w a s u s e d . H o w e v e r , i r r e s p e c t i v e o f t h e s t a g e o f t h e f o l l i c u l a r w a v e , G n R H h a s b e e n r e p o r t e d to r e s u l t i n o c c u r r e n c e o f a n e w d o m i n a n t f o l l i c l e ( D F ) w i t h i n 1 to 8 d a y s w h i c h m i g h t r e s u l t i n t h e l o w p r e g n a n c y ra tes r e p o r t e d b y P u r s e l y et a l . ( 1 9 9 7 ) a n d K o h r a n et a l . ( 1 9 9 8 ) . D u r i n g t h e s t u d y p e r i o d , the hea t d e t e c t i o n a i d s u s e d i n d i c a t e d that 5 4 % o f t h e c o w s w e r e i n es t rus . H o w e v e r , m i l k P 4 l e v e l s i n d i c a t e d that 9 4 % o f t he c o w s w e r e a c t u a l l y c y c l i c a n d 7 5 % o f t h e s e a n i m a l s s h o w e d es t rus w h e n e x p e c t e d b a s e d o n the P 4 p r o f i l e t a k e n at t he d a y o f t he b r e e d i n g . T h i s i n d i c a t e s that b a s e d o n P 4 c o n c e n t r a t i o n s , K a m a r hea t m o u n t d e t e c t o r s w e r e a c c u r a t e o n l y 7 1 % o f t he t i m e i n i n d i c a t i n g es t r us . T h e r e f o r e , t h i s v a r i a t i o n i n t h e n u m b e r o f a n i m a l s t h o u g h t to b e i n es t rus b a s e d o n the K a m a r hea t m o u n t d e t e c t o r w h e n c o m p a r e d to P 4 74 concentrations, reflect the poor conception rates result resulting from estrus synchronization programs on many farms, and their reliability for breeding cows may lead to poor reproductive management in the field unless combined with 24 h observation Pregnancy rates of lactating dairy cows following AI have decreased from 66% in 1951 to about 50 % in 1975, and to about 40 % in the 1990s (Nebel et al., 1993; Pursely et al., 1995 and Beal 1998). Factors including negative energy balance, toxic concentration of urea, heat stress, lack of vitamins or minerals, and many other internal endocrine factors such as abnormal CL functions limit the achievements of high pregnancy rates in dairy cattle (Larson et al., 1997). This study focused on low pregnancy rates resulting from low P4 concentrations from an inadequate CL function. In order to overcome these, various hormonal treatments have been used including GnRH given at different days after breeding. In the normal cycling animals, a single injection of GnRH has been reported to induce a peak of LH within 2 to 3 h after its administration (Williams et al., 1982; Chenault et al., 1990, Rajamhendran et al., 1998, Ambrose et al., 1998). Gonadotropin releasing hormone injection late in the luteal phase of the estrous cycle has been reported to have a protective effect on CL function and maintains elevated luteal P4 (Macmillan et al., 1985). During the early or mid luteal phase, its administration causes an alteration of follicular distribution in the ovary by increasing the number of medium sized follicles, and inducing the large follicles either luteinization or atresia (Thatcher et al., 1989; Guilbault et al., 1990). Further study has shown that single injection of GnRH on d 11 and d 14 after breeding increased pregnancy rates in virgin beef heifers (Rettmer et al., 1992). Similarly (Macmillan et al., 1986 and Macmillan et al., 1989) reported higher pregnancy rates, extended estrous cycle length, and elevated serum concentrations of P4 in animals given single injection of GnRH on d 11 and 13 after breeding when compared to untreated animals. Furthermore, Macmillan et al. (1982) and Thatcher et al. (1989) proposed that the treatment with GnRH enabled the CL to temporarily rebut the luteolytic effect of PGF 2 a. These researchers concluded 75 that the antiluteolyitc role of a mid-luteal injection of GnRH can be achieved, because it induces an LH like release from the pituitary, which in turn either luteinize or ovulates follicles present during the mid luteal phase of the estrous cycle. This alters the follicular secretion of E 2 necessary for the initiation of the uterine oxytocin receptors and serum concentration of PGF 2 a. Recently, numerous studies have been conducted to test whether administration of GnRH after breeding could improve conception rates. Results of the present study indicated that injection of GnRH on day 7 or 14 after breeding did not increase milk or plasma P4 profile, and did not increase the pregnancy rates when compared to those untreated. Lewis et al. (1990) concluded from their study that the effects of GnRH on P4 are varied, as are the effects of GnRH on pregnancy rate, and these effects are difficult to understand. GnRH given 12 days after breeding, after animals were synchronized with GnRH-PGF2ot and inseminated after detected estrus, failed to enhance conception rate (33.3% Vs 37%) but tended to extend the mean inter-estrus interval by 2-5 days (Pursely et al., 1995). These authors concluded that an early embryonic mortality from the devastating effects of heat stress precludes the ability of GnRH to improve embryonic survival. Thatcher et al. (1986), Jubb et al. (1990), Lewis et al. (1990), Drew et al. (1992), Ryan et al. (1992), Ryan et al. (1994), Mann et al. (1994) also reported no improvement on pregnancy rates following GnRH administration given d 11,13, and 14 after breeding. It has been demonstrated that mid-cycle follicles respond similarly to an induced LH surge and hence, provide a luteotrophic stimulus to the existing CL (Berthold et al., 1978; Farine et al., 1988). Thatcher et al. (1989) proposed that the luteotrophic effects of exogenous LH or GnRH-induced LH release increased P4 synthesis by the CL that might be a luteoprotective mechanism to prevent CL regression. Flint et al. (1988) and Flint et al. (1992) also reported a luteotrophic effect of GnRH given in sheep. These findings were supported by Rettmer et al. (1992) who found that progesterone secretion by the CL was increased not only shortly after administration of GnRH, but also for up to d 12 in pregnant heifers. This suggests that the 76 luteoprotective and antiluteolyitc effects of GnRH increased pregnancy rates by allowing a developmentally retarded embryo additional time to establish the signals for maternal recognition of pregnancy. Injection of GnRH on day 5 (Alila et al., 1984; Schmitt et al, 1996; Kohran et al., 1998) or day 6 (Webb et al., 1992) caused ovulation of the dominant follicle of the cycle and formed an accessory CL, this was followed by a sudden increase in plasma progesterone concentration. Authors Schmitt et al. (1996) concluded from their study that the injection of GnRH on d 5 of the estrous cycle followed by the formation of the secondary CL is responsible for the subsequent increase in plasma P4. Recently, an implant of GnRH on d 12 resulted in elevation in serum concentrations of LH and P4, and extended the estrous cycle among cows not become pregnant and returning to estrus (Rajamahendran et al., 1998; Ambrose et al., 1998). However, none of these physiological phenomena following GnRH given on d 12 was associated by any increase of pregnancy rates. In the present study, only two cows were induced accessory CL formation by giving GnRH on dl4 after breeding indicating that the effect of exogenous GnRH depends on the stage of the DF and the wave length of the animal (i.e. whether two or three follicular waves). It has been reported that the DF of the first wave in each estrous cycle reaches its maximum diameter on day 7 after ovulation (Knopf et al., 1989) or between d 6 and 7 (Ginther et al., 1989) and this maximum size is maintained for 3 to 6 days. During this period the follicle has been experimentally shown to have more receptors for LH and therefore be more responsive to GnRH treatment (Kohran et al., 1998.) According to Garverick (1997), for cows with two follicular waves, the second wave starts at around day 9 and after 5 to 7 days this dominant follicle reaches its maximum diameter and acquires the highest concentration of receptors for endogenous LH. In cows with three follicular waves, the second wave can be detected on around d 16 after ovulation. Therefore, giving exogenous GnRH around day 14 seems too early to 77 induce ovulation because of the lack of LH receptors. This difference between two or three wave cycles reduces the probability of increasing the number of animals having a full compliment of LH receptors on their granulosa cells and might led the lower response to GnRH in inducing ovulation as reported in this thesis. Current with the last 25 years of research on controlling estrous cycle length has been the development of a better understanding of follicular development. Methods of interrupting or manipulating the wave-like pattern of follicular growth and controlling ovulation have been developed. However, pharmacological hormones to synchronize estrus without estrus detecting aids and at the same time obtain consistent high pregnancy rates have not yet been achieved through fixed time Al programs. To synchronize a new wave it is necessary to cause the demise of the existing follicular wave within a specific time span, and this must be followed by recruitment of a new healthy ovulatory DF capable of inducing high pregnancy rates. Non-hormonal regiments to obtain effective synchronization of follicular waves have been integrated in studies in which an earlier FSH surge and the emergence of the second follicular wave were observed after cauterization of the growing follicle (Adams et al., 1993). These results suggested that the removal of the suppressive effects of the DF enhanced subsequent follicular development. Based on these observations, the physical removal of the DF or aspiration of follicles greater than 5 mm in diameter have been developed. This resulted in the synchronous emergence of a new follicular wave two days later (Bergfelt et al., 1994; Garcia et al., 1998), but is still impracticable. Through their suppressive effects on gonadotropins, steroids (i.e. estradiol and progestogens) become an alternative method to synchronize follicular wave dynamics. However, dose, route of administration, variation of plasma concentrations after their administration created to halt their practical applications. Moreover, when given alone they did not consistently hasten the demise of the DF in all phases of the follicular development (Bo et al., 1995a and Bo et al., 1994). 78 The pregnancy rates observed in the first study were comparable to those reported by Jenks et al. (1998) (51%), but higher than the 37.7 and 38.9% reported by Pursely et al. (1995) and Burke et al. (1996.), respectively. The better response in pregnancy rates may be due to the number of animals responding to the first GnRH injection. This depends on the percentage of mature follicles ovulated in any of the various waves during the estrous cycle. Even though animals synchronized with GnRH-PGF2a-GnRH in the second experiment more reliably resulted in acceptable pregnancy rates, pregnancy rates were not significantly improved by administering of GnRH on days 7, 14, 7 and 14 after breeding. The current state of estrous cycle length has been elevated by the discovery of methods to regress an existing DF and to initiate a new wave of follicular development. Administration of GnRH synchronizes the follicular development among cows prior to the luteolytic dose of PGF 2 a Induction of an LH surge by a second dose of GnRH in a group of animals that have had follicular development and CL regression synchronized, enables timed ovulation. In cattle, the (Ovsynch) method seems to be a better method for planned breeding than the PGF 2 a program. The fact remains, however, that despite advances in the development of the Ovsynch method, there is still some way to go in meeting the needs of farmers seeking high pregnancy rate after a single, fixed time insemination. From this thesis I conclude that: a) The administration of GnRH on day 0, PGF 2 a on day 7, and second GnRH on day 9 followed by timed insemination at 16 and 36 h more reliably resulted in acceptable pregnancy rates than two injections of PGF 2 a b) Single injection of GnRH after breeding resulted in ovulation in most animals, but this was not followed by any increases in P4 concentration or pregnancy rate. These results suggest that 79 further studies are needed to understand the mechanisms that control ovarian follicular dynamics and to enhance conception rates and embryonic survival. 80 REFERENCES Adams, G.P., Kot, K., Smith, C.A. and Ginther, O.J. 1993. Selection of a dominant follicle and suppression of follicular growth in heifers. Anim. Reprod. 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