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Effects of unilateral ovariectomy of follicular development, plasma gonadotropin, progesterone, IGF-I… Mohan, Mahesh 1998

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EFFECTS OF UNILATERAL OVARIECTOMY ON FOLLICULAR DEVELOPMENT, PLASMA GONADOTROPIN, PROGESTERONE, IGF-I PROFILES, OVULATION AND PREGNANCY RATES IN C A T T L E by MAHESH MOHAN B . V . S c , Tamil Nadu Veterinary and Animal Sciences University, Madras, India, 1994 A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF MASTER OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Animal Science) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA March, 1998 ®Mahesh Mohan, 1998 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 of JWMC<L The University of British Columbia Vancouver, Canada Date Apr\), 2**, M8T DE-6 (2/88) A B S T R A C T In cattle, during the estrous cycle, two to three DFs develop. Since cattle are monoovular, follicle deviation occurs particularly in an expeditious fashion resulting in the development of a single DF from a recruited pool, thereby, preventing multiple DFs in any single wave. The controlled emergence of a single DF in cattle is to a great extent made possible by the negative and positive feedback effects exerted by the various ovarian secretions (inhibin and E2) on the release of FSH from the pituitary. All growing follicles are capable of becoming dominant if adequate concentrations of FSH is available during the recruitment phase of follicular development. Initiation of FSH treatment during the emergence of a follicular wave stimulates many follicles to attain a diameter greater than 10 mm. Therefore, FSH seems to be a primary limiting factor in the attainment of follicular dominance. The focus of this thesis was to investigate follicular development, hormonal profiles, ovulation and pregnancy rates following removal of one ovary (ULO) in cattle. A bilateral ovariectomy completely eliminates the negative feedback effects of ovarian secretions resulting in a tremendous increase in the output of pituitary FSH and LH. Examination of the physiological changes associated with bilateral ovariectomy prompted a hypothesis that removal of one ovary would limit the inhibitory feedback to the pituitary resulting in increased FSH secretion sufficient for the growth, development and ovulation of more than one follicle. Non-lactating cycling cows and cycling heifers were used in a total of three experiments. The availability of sonographic procedures proved to be of immense help in tracking follicular development before and after ULO. In both cycling cows (Expt I) and cycling heifers (Expt II), ULO resulted in the development of co-dominant follicles in 6 out of 8 cycles and 4 out of 6 cycles, respectively. The co-dominant follicles had an altered size distribution with the largest follicle i i smaller in diameter than the intact animals and the second largest follicle larger than the subordinate follicles in intact animals. In both cows and heifers no significant increases were recorded in plasma gonadotropin (FSH and LH), and P 4 concentrations following U L O . In pubertal heifers, U L O increased pregnancy but not twinning rates. In experiment III, plasma FSH, L H and IGF-I concentrations following U L O were determined by performing serial blood samplings during the early follicular and mid-luteal phases of the estrous cycle. Even though mean concentrations after U L O were not significantly different from those before U L O , two of four heifers showed a two-fold increase in the magnitude of both FSH and L H surges after U L O . I conclude that U L O resulted in the development of co-dominant follicles and is a good method to obtain twin ovulations and possible twin pregnancies without causing any significant increases in plasma FSH, L H , P 4 and IGF-I concentrations. Therefore, subtle endocrine alterations at both the pituitary and ovarian levels might be the stimulus for the development and ovulation of twin follicles following U L O . iii T A B L E O F CONTENTS ABSTRACT ii T A B L E OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii ABBREVIATIONS x A C K N O W L E D G E M E N T S xii FOREWORD xiii DEDICATION xv C H A P T E R 1 G E N E R A L INTRODUCTION 1 1.1 The objectives 4 C H A P T E R 2 REVIEW OF LITERATURE 5 2.1. Ovarian Follicular Dynamics 5 2.1.1. Follicular Waves 5 2.1.2 . Regulation and turnover of Follicular waves 7 2.1.3. Follicular dominance 8 2.1.4. FSH and follicular dominance 11 2.2. Steroid and Peptide feedback 13 2.2.1. Progesterone Feedback 13 2.2.2. Estrogen feedback 14 2.3. Ovarian peptides 15 2.3.1. Inhibin 15 2.3.2. Activin 16 2.4. Ovarian Peptide growth factors 16 2.4.1. Insulin like growth factors 16 2.5. Twinning in cattle 17 2.5.1. Twinning by Genetic selection 17 2.5.2. Twins through the administration of exogenous Gonadotropins 20 2.5.3. Twinning by immunizaton against certain ovarian steroids and peptides 25 2.5.3.1. Passive and Active immunization against ovarian steroids 26 iv 2.5.3.2. Immunization against Inhibin 28 2.5.3.2.1. Native inhibin immunogens 29 2.5.3.2.2. Synthetic inhibin a-chain fragments 30 2.5.3.2.3. Recombinant inhibin and inhibin a-subunits 30 2.5.4. Twinning by Embryo transfer 31 2.6. U L O as a method to produce twins in cattle 36 2.6.1. Effects of U L O on species with short estrous cycles ..37 2.6.2. Effects of U L O in species with Long estrous cycles 38 2.6.3. Effects of U L O on livestock species that are normally monoovular 39 2.6.4. Long term effects of U L O 40 CHAPTER- 3 EFFECTS OF UNILATERAL OVARIECTOMY ON F O L L I C U L A R DYNAMICS, PLASMA GONADOTROPIN A N D PROGESTRONE CONCENTRATIONS IN D R Y C Y C L I N G COWS 42 3.1. ABSTRACT 42 3.2. INTRODUCTION 43 3.3. MATERIALS A N D METHODS 44 3.3.1. Animals and Ovarian examination 44 3.3.2. Blood sampling and analysis for plasma FSH, L H and P 4 44 3.4. RESULTS 45 3.4.1. Ovarian follicular dynamics during the control cycle 45 3.4.2. Ovarian follicular dynamics following U L O 46 3.4.3. Plasma FSH and L H concentrations before and after U L O 47 3.4.4. Plasma P 4 concentrations before and after U L O 51 3.5. DISCUSSION 51 CHAPTER- 4 EFFECTS OF U N I L A T E R A L OVARIECTOMY O N F O L L I C U L A R D E V E L O P M E N T , PLASMA GONADOTROPIN, PROGESTERONE CONCENTRATIONS, O V U L A T I O N A N D PREGNANCY RATES IN C Y C L I N G HEIFERS 55 4.1. ABSTRACT 55 4.2. INTRODUCTION 56 4.3. MATERIALS A N D METHODS 57 4.3.1. Animals and Ovarian examination 57 4.3.2. Blood sampling and analysis for plasma FSH, L H and P 4 57 4.4. RESULTS 58 4.4.1. Ovarian follicular dynamics during the control cycle in heifers 58 4.4.2. Ovarian follicular dynamics following ULO 59 4.4.3. Plasma FSH and L H concentrations before and after U L O 60 4.4.4. Plasma P 4 concentrations before and after U L O 60 4.5. DISCUSSION 64 CHAPTER - 5 PLASMA FSH, L H A N D IGF-I CONCENTRATIONS BEFORE A N D A F T E R U N I L A T E R A L OVARIECTOMY IN HEIFERS 71 5.1. ABSTRACT 71 5.2. INTRODUCTION 72 5.3. MATERIALS A N D METHODS 73 5.3.1. Animals and Blood sampling before unilateral ovariectomy 73 5.3.2. Animals and Blood sampling after unilateral ovariectomy 74 5.3.3. Hormone Assays 75 5.3.4. Statistical Analysis 76 5.4. RESULTS 76 5.4.1. Plasma concentrations of FSH during the early follicular and mid-luteal phases before and after U L O 76 5.4.2. Plasma concentrations of L H during the early follicular and mid luteal phases before and after U L O 77 5.4.3. Plasma IGF-I concentrations before and after U L O 77 5.5. DISCUSSION 84 CHAPTER 6 G E N E R A L DISCUSSION 90 REFERENCES 99 V I LIST OF TABLES Table 2.1. Summary of superovulation studies using PMSG to induce twinning in cattle 23 Table 2.2. Summary of superovulation studies using FSH to induce twinning in cattle 24 Table 3.1. Number of ovulatory follicles, follicular waves, mean (±SE) cycle length, plasma gonadotropin (FSH & LH), progesterone concentrations in cows before and after U L O 49 Table 4.1. Number of ovulatory follicles, follicular waves, mean (±SE) cycle length, plasma gonadotropin (FSH & LH), progesterone concentrations in six heifers before and after U L O 60 Table 5.1. Mean (+ SE) FSH and L H concentrations and pulse frequencies during the early follicular phase and mid-luteal phase, respectively before and after U L O . Also shown in the table are mean IGF-I concentrations during the last 9 days of the cycle and P 4 concentrations during the entire cycle before and after U L O 84 vii LIST OF FIGURES Figure 3.1: A representative pattern of follicular development and progesterone profile observed before unilateral ovariectomy 46 Figure 3.2: A representative pattern of follicular development and progesterone profile observed after unilateral ovariectomy 47 Figure 3.4: Ovary of cow #9923 removed 7 days after induced estrus after completion of the second experimental cycle following unilateral ovariectomy. Twin corpora lutea (CL) measuring 18 and 17 mm in diameter along with two large follicles (f) measuring 15 and 10 mm in diameter can be seen 48 Figure 3.5: Cut section of the retained ovary (cow # 8809) that was removed 7 days after the second experimental cycle following unilateral ovariectomy. The growth, development and ovulation of three follicles is evidenced by the presence of three corpora lutea measuring 15, 10 and 7mm in diameter, two of them sectioned and the third intact. ..48 Figure 3.3: Sequential growth and regression of follicles and corpus luteum in four dry cycling cows [8809 (a), 9111 (b), 9209 (c), 9233 (d)] observed before (one cycle) and after (two cycles) unilateral ovariectomy. The termination of the control cycle and removal of one ovary is indicated by O V U / U L O . The ovulatory follicles are indicated as ovu and corpus luteum as C L 50 Figure 4.1: A representative pattern of follicular development and progesterone profile observed before unilateral ovariectomy 58 Figure 4.2: A representative pattern of follicular development and progesterone profile observed during trial ii after unilateral ovariectomy 59 Figure 4.3a : Sequential growth and regression of follicles and corpus luteum in heifer (#s 9417 & 9418) observed for one cycle before and after unilateral ovariectomy. The termination of the control cycle and removal of one ovary is indicated by O V U / U L O . The ovulatory follicles are indicated as ovu and corpus luteum as C L 61 Figure 4.3b: Sequential growth and regression of follicles and corpus luteum in heifer (ft 9449 & 9450) observed for one cycle before and after unilateral ovariectomy. The termination of the control cycle and removal of one ovary is indicated by O V U / U L O . The ovulatory follicles are indicated as O V U and corpus luteum as C L .62 Figure 4.3c: Sequential growth and regression of follicles and corpus luteum in heifer (tt 9501 (white), 9501 (yellow) observed for one cycle before and after unilateral ovariectomy. The termination of the control cycle and removal of one ovary is indicated by O V U / U L O . The ovulatory follicles are indicated as O V U and corpus luteum as C L . . 63 viii Figure 5.1: Circulating concentrations of FSH in heifer numbers 9555 (a, upper left panel), 9553 (b, upper right panel), 9554 (c, lower left panel), 9555 (d, lower right panel) from blood samples taken at 4 h intervals for 72 h during the early follicular phase of the estrous cycle before and after unilateral ovariectomy (ULO- Unilateral ovariectomy) 78 Figure 5.2: Circulating concentrations of FSH in heifer numbers 9555 (a, upper left panel), 9553 (b, upper right panel), 9554 (c, lower left panel), 9555 (d, lower right panel) from blood samples taken at 20 minute intervals during the mid-luteal phase of the estrous cycle before and after unilateral ovariectomy (ULO- Unilateral ovariectomy) 79 Figure 5.3: Circulating concentrations of L H in heifer numbers 9555 (a, upper left panel), 9553 (b, upper right panel), 9554 (c, lower left panel), 9555 (d, lower right panel) from blood samples taken at 4 h intervals for 72 h during the early follicular phase of the estrous cycle before and after unilateral ovariectomy (ULO- Unilateral ovariectomy) 80 Figure 5.4: Circulating concentrations of L H in heifer numbers 9555 (a, upper left panel), 9553 (b, upper right panel), 9554 (c, lower left panel), 9555 (d, lower right panel) from blood samples taken at 20 minute intervals for 6 h during the mid-luteal phase of the estrous cycle before and after unilateral ovariectomy (ULO- Unilateral ovariectomy) .....81 Figure 5.5: Mean IGF-I concentrations during the last nine days of the estrous cycle in heifer numbers 9552 (1), 9553 (2), 9554 (3), 9555 (4) from daily blood samples taken before (blank bars) and after (cross bars) unilateral ovariectomy 82 Figure 5.6: Mean + SE concentrations of FSH (a, left panel) and L H (b, upper right panel) during the early follicular phase (blood sampling at 4h intervals) and mid-luteal phase (20 minute blood sampling) of the estrous cycle, respectively 83 ix A B B R E V I A T I O N S A C T H = Adrenocorticotropic hormone BFF = Bovine follicular fluid CIDR = Controlled internal drug release C L = Corpus luteum cm = Centimeter ° C = degrees celcius C L = Corpus luteum d = day DF(s) = Dominant follicle(s) eCG or PMSG = Equine chorionic gonadotropin or Pregnant mare serum gonadotropin Ej = Estradiol-17 p FSH = Follicle stimulating hormone g = gauge GnRH = Gonadotropin releasing hormone h = hour H C G = Human chorionic gonadotropin IGF-I & II = Insulin-like growth factor I & II IVMFC = In vitro maturation fertilization and culture IGF-I = Insulin-like growth factor IGFBP(s) = Insulin-like growth factor binding protein(s) h = hour L H = Luteinizing hormone min = minute(s) ml = millilitre(s) mm = millimetre(s) N = Norgestomet ng = nanogram P 4 = Progesterone No = number P G F 2 a = Prostaglandin SE = standard error U L O = Unilateral ovariectomy u.1 = microlitre(s) xi ACKNOWLEDGEMENTS I am very grateful to my research supervisor Dr. R. Rajamahendran, for stimulating my interest in this thesis research, supporting my M.Sc program and making me capable of pursuing independent work. His suggestions and criticisms were very valuable in writing my thesis and scientific papers. I am thankful to all my supervisory committe members, Dr. J.A. Shelford, Dept of Animal Science, Dr. K . M . Cheng, Dept of Animal Science, Dr. Lorne. J. Fisher, Pacific Agricultural Research Station, Agassiz for their guidance to the research program and reading through my thesis for editorial corrrections. I appreciate the comradeship and ideas of fellow graduate students Lisa Johnson, Ming Yang, Giritharan, Suleman Bhatti, Marcus Abraham, Hephzibah shanthi. Special thanks also go to Mohan Mannikkam and his wife Jayanthi, Divakar Ambrose and Jamal Kurtu for their guidance and immense help rendered during my earlier days at UBC. I am thankful to J. Rajeswaran for helping me in the statistical analysis. I am thankful to Dr. Rawlings, Dept of Veterinary Physiology, Universtiy of Saskatchewan and Dr. Joanne Fortune, Dept of Veterinary Physiology, Cornell University for doing the FSH and L H assays. I appreciate all the technical help offered by Gilles Galzy, Sylvia Leng, Luz Aquino during my graduate study. I am grateful to K. Sivakumaran for his constant help during my graduate program. I am thankful to the Faculty of Graduate Studies for the travel grant and the Department of Animal Science for the Graduate Teaching Assistantships. xii F O R E W O R D This thesis comprises the following peer-reviewed scientific papers and conference presentations which have been accepted or submitted for publication. 1. M . Mohan and R. Rajamahendran 1996. Effects of unilateral ovariectomy on follicular and corpus luteum dynamics in cows. In Proceedings of the 4th Annual research workshop of the Reproductive and developmental sciences programme held at B .C. Womens hospital, (abstract #11). 2. M . Mohan and R. Rajamahendran (1997). Unilateral ovariectomy in Cattle: Potential for twin ovulations and twin pregnancies. Proceedings of the 19th Annual Winter Workshop of the Canada West Society for Reproductive Biology held at Calgary. 3. M . Mohan and R. Rajamahendran (1997). Ovulation rates, gonadotropin and progesterone levels following unilateral ovariectomy in cattle. Presented at the 47th annual meeting of the Canadian Society for Animal Science meeting held at Montreal, Quebec: July 24-26, 1997. (Abstract # 97M7) p-218. 4. M . Mohan and R. Rajamahendran (1996). Effects of unilateral ovariectomy on follicular development and ovulation in cattle. Presented at the 1997 Annual Meeting of the International Embryo Transfer Society held in Nice, France January 12-14, 1997. (Abstract # 235). xii i 5. M . Mohan and R. Rajamahendran. 1998. "Effects of unilateral ovariectomy on follicular development and ovulation in cattle". Theriogenology.49(5): 1059-1070 (Chapter 3 and 4). 6. M . Mohan and R. Rajamahendran. 1997. "Twinning in Cattle- Methods and Applications". Accepted for publication. Canadian Veterinary Journal. (Chapter 2). 7. M . Mohan and R. Rajamahendran. 1998. " Plasma gonadotropin (FSH & LH), IGF-I concentrations following unilateral ovariectomy in cattle, (in preparation; chapter 5). X I V D E D I C A T I O N This thesis is dedicated to my loving parents and to my sister for their constant help, encouragement and support. X V CHAPTER 1 GENERAL INTRODUCTION Transrectal ultrasonography has proven to be a simple, rapid, non-invasive, innocuous, repeatable and reliable device for studying ovarian follicular growth and regression in cattle. It is also more reliable than transrectal palpation (Pierson and Ginther, 1984; Fortune et al., 1991). The earlier predicted "two wave hypothesis" of Rajakoski (1960) regarding growth, development and regression of follicles during the bovine estrous cycle remained untested and languished for nearly two decades until the introduction of transrectal imaging technology into bovine reproductive research (Pierson and Ginther, 1984). An enormous amount of information has accumulated during the last decade which has considerably improved our understanding of ovarian physiology and enabled successful manipulation of follicular growth in the bovine to optimize reproductive efficiency and various reproductive technologies. As soon as the wave hypothesis was clarified, subsequent ultrasound tracking studies helped characterise the composition of follicular waves (Ginther et al., 1989). The emergence of the first follicular wave of an estrous cycle can be detected as a cohort of follicles in the 4 mm size range immediately after ovulation. In the next few days one follicle functionally and morphologically overrides the others becoming the DF, and the others become subordinates. Around day 10 after ovulation (Day 0= ovulation) a second follicular wave emerges and in a three wave cycle is followed by a third follicular wave around day 16 (Taylor and Rajamahendran, 1991a). Thus two to three potential ovulatory DFs emerge during the bovine estrous cycle. The ovulatory DF is always the one that is present at the time of luteolysis while the DF(s) that develop during the luteal phase always succumb(s) to atresia. l Reports on the occurrence of a wave-pattern of follicular growth and atresia until about d 60 was available in pregnant cows (Taylor and Rajamahendran, 1991b) and recently the continuation of this pattern in an undisrupted fashion until parturition was clarified (Ginther et al., 1996a). Prepubertal heifers also exhibit a wave pattern of follicular growth and regression (Adams et al., 1994; Evans et al., 1994). This is also the case with postpartum dairy cows (Savio et al., 1990a; Rajamahendran and Taylor, 1990) and suckling beef cows (Murphy et al., 1990). The mechanism underlying follicular turnover in prepubertal heifers and postpartum cows may be different and our understanding is far from complete in these two reproductive states as turnover of DFs takes place in the absence of a CL. The information available on the growth and regression of follicles in the bovine ovary until now came from intact animals with both ovaries. Whether the same pattern can be observed in cattle if one of the ovaries is removed (ULO) is not known and has not been tested. Will ULO alter estrous cycle length, number of follicular waves, number of ovulatory follicles, plasma gonadotropin (FSH & LH), P 4 concentrations ? Can subtle changes in the availability of FSH increase the number of ovulatory follicles ? If yes, how can such changes in hormonal milieu be brought about ? What effects would ULO have on pregnancy ? These questions and a few others are important for several reasons that are outlined at the end of this chapter. Studies on the effects of ULO began almost two centuries ago (Hunter, 1787) and have been intensively investigated on several polytoccus laboratory and livestock species. Some studies in cattle were initiated even before the elucidiation of the dynamics of follicular growth in cattle. The results observed in several of the polytocous species is interesting as the retained ovary compensates for the ovulations that would have occurred on the other ovary in an intact animal. Even though a few studies were performed on cattle earlier, these endeavours 2 proved unsuccessful as they were deprived of a monitoring device to track the sequential growth of follicles in the retained ovary. Moreover, results of an early study (Saiduddin et al., 1970) predicted the possible occurrence of twin ovulations in unilaterally ovariectomized heifers. This thesis therefore, addresses the effects of ULO in cattle on follcular dynamics, plasma FSH & LH, IGF-I and P4 concentrations and pregnancy rates. As mentioned earlier, the studies on ULO in most livestock and laboratory species have enabled us understand some of the mechanisms controlling follicular growth. ULO has been used to investigate mechanisms regulating the control of gonadotropin secretion from the pituitary by the gonads. Following ULO there is a transient, selective rise in FSH that has been demonstrated in rats (Butcher, 1977), hamsters (Bast and Greenwald, 1977), gilts (Redmar et al., 1985 ), cattle (Johnson et al., 1985). This increase in FSH has been shown to occur immediately after ULO in cattle but it is not known if this increase would continue into subsequent cycles. Results from an old study in cattle (Saiduddin et al., 1970) tells us that ULO could serve as a good model to study follicular dominance. It could also serve as a model for other monotoccus species like humans since treatment for ovarian cancer in women invariably is ovariectomy and if it is ULO, the pattern of follicular growth and accompanying hormonal changes in ovarian function may be extrapolated from bovines. Chapter 2 of this thesis provides a review of literature on follicular development, follicular dominance, secretions of the follicle, studies on the effects of ULO in various polytoccus laboratory species and currently available methods to induce twinning in cattle. Chapter 3 describes the effects of ULO on follicular dynamics, ovulation rate, plasma FSH, LH and P 4 concentrations in dry cycling cows. Investigations into the effects of ULO in cycling pubertal heifers on follicular dynamics, plasma gonadotropin, P 4 concentrations, ovulation rate and pregnancy rates are described in Chapter 4. Whether or not U L O causes changes in plasma concentrations of FSH, L H and IGF-1 are described in Chapter 5. Chapter 6 provides a summary of findings and conclusions from the experiments and gives directions for future research in this area. 1.1 The objectives 1. To examine the effects of ULO on follicular dynamics, plasma FSH, L H and P 4 concentrations and ovulation rate in dry cycling cows. 2. To investigate the effects of U L O on follicular dynamics, plasma FSH, L H and P 4 concentrations and pregnancy rates in pubertal heifers. 3. To characterise changes in plasma FSH and L H during the follicular and mid-luteal phase and IGF-1 concentrations following ULO in cattle. 4 CHAPTER 2 REVIEW OF LITERATURE In this chapter, a brief description to the reader is provided about the dynamics of ovarian follicular growth in cattle, the maintenance and turnover of DFs, and the exertion of dominance. The role played by FSH in controlling follicular growth, a brief account on the methods available to induce twinning in dairy cattle, effects of U L O on follicular development in various farm and laboratory species is also reviewed. In addition the potential for U L O in cattle as a method to induce twinning in cattle is also examined. 2.1. Ovarian Follicular Dynamics 2.1.1. Follicular Waves Researchers have used three different methods to study follicle dynamics in cattle; a) Counts of follicles in ovaries obtained at slaughter alone or coupled with measurement of steroid hormones in follicular fluid, b) following individual follicles by marking them with dye; c) observations of the regrowth of large follicles following destruction of large follicles on the ovary. All these methods proved futile and unreliable until the advent of transrectal ultrasonography (Pierson and Ginther, 1984; Fortune et al., 1991). Histological examination of the ovaries obtained at slaughter revealed contradictory ideas about the pattern of development of large bovine follicles. Earlier, Rajakoski (1960) postulated the existence of 2 waves of growth of follicles >5 mm in diameter during the bovine estrous cycle, one occurring between days 3 & 4 of the cycle producing a large non-ovulatory atretic follicle and the second beginning around day 12-14 and culminating in ovulation. In contrast, Marrion et al. (1968) asserted that the growth of follicles is continuous 5 and constant and that follicles develop to > 10 mm independant of the stage of the estrous cycle after examining ovaries of 700 dairy cows of known reproductive history. In a review, Marrion and Gier, (1971) hypothesized that an average of 11 follicles per cycle grew larger than 8 mm in diameter so that there was an almost constant progression of large follicles. Donaldsen and Hansel (1968) also presented evidence against waves of follicular growth. Studies involving dye marking of selected large follicles or cauterization of all follicles > 5mm lead to the conclusion that the growth and replacement of the largest follicle on the ovary is more rapid at the end of the cycle. These authors found that the preovulatory follicle was the largest present on each pair of ovaries 3 days prior to estrus, but before that, the preovulatory follicle was not consistently the largest follicle. Examination of follicular populations on days 3, 5, 7, 11 and 13 of the estrous cycle and based on their E2 concentrations, Ireland and Roche (1983) hypothesized the existence of 3 successive phases of follicular growth in cattle. They suggested that during the estrous cycle one follicle is selected, achieves dominance and either ovulates or becomes atretic, depending on plasma P 4 concentrations during its dominance phase. The three different approaches discussed above have certainly provided valuable information about follicle populations in cattle, but they suffered from a major limitation that individual follicles could not be followed over time. The advent of real-time ultrasonography has enabled the visualization of the ovaries with rectal transducers and its widespread use since then by several researchers has clearly established the wave pattern of follicular development that was proposed earlier by Rajakoski (1960). During the estrous cycle in cows, follicular development is characterised by two or three successive waves of follicular growth (Ireland and Roche, 1987; Savio et al., 1988; Rajamahendran and Walton 1988; Knopf et al., 1989). A follicular wave is characterised by 6 the synchronous development of a group of follicles, one of which becomes dominant and achieves the greatest diameter, suppressing the growth of the other smaller follicles. In prepubertal heifers, growth and regression of large follicles occur in a wave-like pattern, with characteristics and associated patterns of gonadotropin secretion similar to those seen in adult cyclic animals (Evans et al., 1994). Postpartum cows similar to cycling and prepubertal heifers show waves (ranging from 2 to 3) of follicular growth with a greater number of anovulatory DFs developing, leading to a protracted interval to first ovulation (Murphy et al., 1990; Rajamahendran and Taylor, 1990; Savio etal., 1990 a&b). 2.1.2 . Regulation and turnover of Follicular waves Several independant investigations have been conducted to study the regulation of follicular waves in prepubertal, pubertal heifers and postpartum cows. Basal concentrations of gonadotropins permit the emergence of follicular waves every 7-9 days, with the time of luteolysis determining whether two or three waves of follicular growth would occur (Ginther et al., 1989; Savio et al., 1990a). Another school of thought is that the length of the luteal phase would determine the number of waves of follicular growth (Taylor and Rajamahendran, 1991a). Additional evidence that basal concentrations of FSH and L H would permit regular follicular waves came from studies during pregnancy (Ginther et al., 1989; Savio et al., 1990a; Taylor and Rajamahendran, 1991b) which showed continued emergence of follicular waves. Treatment of cows with P 4 equivalent to mid-luteal phase concentrations in serum reduced L H pulse frequency (Roberson et al., 1989) and produced turnover of follicular waves (Sirois and Fortune, 1990). On the other hand low doses of P 4 increased L H pulse frequency and favoured persistence of the largest follicle. Evidence came from studies that 7 utilised an implant containing 6 mg of N that probably mimiked subluteal phase concentrations of P 4 (Roberson et al., 1989) thereby, increasing L H pulse frequency (Garcia-Winder et al., 1986) causing the largest follicle to persist while blocking the L H surge and ovulation. These studies revealed the regulatory role played by P 4 in causing turnover of follicular waves in cattle. In two other independant studies, treatment of heifers with charcoal extracted BFF rich in inhibin suppressed the FSH surge, delaying the emergence of follicular waves (Turzillo and Fortune, 1990) and estrus (Quirk and Fortune, 1986). Treatment with BFF caused regression of the supposed ovulatory follicle initiating a new wave from which the ovulatory follicle emerged, implying that normal basal concentrations of FSH are critical for follicular development during the estrous cycle. 2.1.3. Follicular dominance Even though 3-6 follicles are recruited during each wave of follicular growth, only a species-specific number of follicle(s) attain(s) dominance and continues its growth for more than a few days to reach ovulatory size (Fortune, 1994). In cattle the initiation of each wave of follicular development is characterised by the contemporaneous emergence from the pool of small follicles of a number of (5-7) larger, growing follicles >5 mm in diameter called the recruitment phase. During the next phase called the selection phase, one follicle from this group rapidly emerges as the DF and grows through the dominance phase, while the others become atretic and regress. In a two wave cycle, the first wave DF normally reaches a diameter of 10-15 mm and remains dominant for a few days, until it becomes atretic and regresses, to be replaced within approximately five days by a second DF from the second wave or by a third DF in a three wave cycle. The DF during its growth or early dominance phase will continue to develop to preovulatory size (up to 20 mm) and will eventually trigger 8 the hormonal cascade leading to ovulation under two conditions, a) if freed from the restrictive hormonal environment imposed by the C L on the hypothalamus and the pituitary following luteolysis. b) DF(s) that have reached an advanced stage of development before becoming atretic could ovulate in response to an ovulatory dose of 1500 iu of H C G (Price and Webb, 1989; Rajamahendran and Sianangama, 1992) or 500 u.g of synthetic GnRH on d 6 of the cycle (Rusbridge et al., 1991). Several workers have investigated the mechanism of dominance (Lavoir and Fortune, 1990; Fortune et al., 1991; Kastelic and Ginther, 1991). A DF is said to be morphologically dominant since it is the largest follicle on a pair of ovaries. It is also denoted as being functionally dominant as it has the ability to suppress the growth of subordinate follicles, the emergence of a new wave and ovulate in response to a surge in gonadotropins. The first wave DF has been studied to explore the relationship between morphological and functional dominance in non-ovulatory DFs (Lavoir and Fortune, 1990; Fortune et al., 1991). A luteolytic dose of P G F 2 a was given while the DF was still growing, 2 d after it reached a plateau (early in plateau) and 5-6 d when it stopped growing (late in plateau) (Lavoir and Fortune, 1990). The DF ovulated only when luteolysis was induced during its growing and the early plateau stage. These results confirmed that DFs retained morphological dominance longer than it retained functional dominance. Also the ability of the first wave DF to ovulate and to suppress the emergence of the second wave depended on the day of emergence of the second wave or in other words was linked in time. In their study, Kastelic and Ginther (1991) recorded ovulation of the first wave DF in 82% of the heifers following luteolysis on d 8 of the cycle, excepting when the second wave emerged earlier than expected. Confirmation that the DF suppresses the appearance of the next wave came from studies that employed CIDR or N to prolong the lifespan of the DF. CIDR or N permit L H pulses at an increased frequency thereby, promoting follicular E2 sysnthesis which in turn suppressed appearance of smaller follicles (Sirois and Fortune, 1990; Rajamahendran and Taylor, 1991; Savio et al., 1993; Stock and Fortune, 1993; Taylor et al., 1993). Extension of the lifespan of the DF resulted in larger than normal size follicles capable of secreting several fold higher concentrations of E2 than normal (Sirois and Fortune, 1990). A number of other studies have demonstrated that the growth of medium size follicles occur only when the large follicle is either regressing or has been destroyed (Matton et al., 1981; Staigmiller and England, 1982). This phenomenon was further proved by Ko et al. (1991) who showed that cauterization of the DF delayed both the regression of the largest subordinate and an emergence of the second wave further corroborating the fact that the DF suppresses the subordinates in addition to suppressing the emergence of the next wave. It is assumed that the DF suppresses the growth of the subordinates by depriving them of gonadotropins by inhibition of its output from the pituitary through secretions of E2 and /or inhibin. Plasma FSH concentrations have been shown to fall precipitously immediately following the selection of the DF (Adams et al., 1992a), which is in concurrence with the above hypothesis. Exposure of the DF maintained by a CIDR or N to luteal concentrations of P 4 or high concentrations of progestins caused reduction in size and shorter persistence (Bergfelt et al., 1991; Adams et al., 1992b; Savio et al., 1993; Stock and Fortune, 1993; Burke et al., 1994; Taylor et al., 1994; Taylor and Rajamahendran 1994). Sawyer et al. (1995) following the administration of P 4 at different stages of the estrous cycle before superovulation treatments recorded higher embryo yields from the superstimulatory treatments timed at wave emergence 10 before the DF was selected. Treatment of early weaned postpartum cows with progestins in the form of a N implant resulted in more ovulations with silent estrus than controls suggesting that N partially synchronizes follicle maturation and the L H surge. 2.1.4. FSH and follicular dominance Adams et al. (1992a) investigated the temporal relationship between growth of the DF and the pattern of FSH release following treatment with BFF and follicle cauterization. The association between an FSH surge and emergence of a follicular wave has been confirmed independently for the estrous cycle (Sunderland et al., 1994; Gong et al., 1995) and has been shown in calves as young as 6-8 months (Evans et al., 1994) and during pregnancy and postpartum period (Ginther et al., 1996a). The peak of the FSH surge occurs at or near the time when the future DF or the resultant DF of the resulting wave has a mean diameter of only 4 mm (Bergfelt et al., 1996). The first appearance of growing 3 mm follicles of a wave occurs during the incline in the FSH surge and continues until the FSH declines to basal concentrations (Ginther et al., 1996b). There are several factors that control increasing and decreasing concentrations of circulating FSH during an FSH surge. The proteinaceous components of follicular fluid which include inhibin, has a striking inhibitory effect on FSH and follicle growth when administered to cattle (Kastelic et al., 1990; Turzillo and Fortune, 1990), and inhibin antiserum increased the circulating FSH concentrations (Kaneko et al., 1995). The initial decline in FSH concentrations after the peak of the FSH surge occurs when the future DF and its largest companions are approximately 6 mm. Aromatase, an enzyme in the E2 synthetic pathway, is present in 4-mm follicles (McNatty et al., 1984), and low levels of E 2 are present in 5-7 mm follicles (Echternkamp et al., 1994). From the above discussion it 11 is clear that a negative feedback effect predominantly of inhibin or other proteinaceous factors potentially play a role in regulating the declining portion of the FSH surge. The final supression of the FSH surge is assumed to be the cause for deviation in growth rates between the resulting dominant and subordinate follicles. Deviation in growth rates between the dominant and subordinate follicles can be explained in two ways. Firstly, the growing DF through increased secretion of E2 and inhibin maintains FSH at basal levels, which assures the loss of FSH needed by the subordinate follicles. Secondly, the DF continues to grow and thrive by a shift in primary gonadotropin dependency from FSH to L H , whereas the FSH-dependant subordinate follicles are deprived of FSH (Ginther et al., 1996b). Chronic treatment of cattle with a GnRH agonist suppressed the pulsatile secretion of L H , and the largest follicle did not grow beyond 7-9 mm, indicating the necessity of L H for post-deviation development (Gong et al., 1995). Since cattle are monoovular, it is particularly critical that follicle deviation occur in an expeditious fashion to prevent multiple DFs which is to a great extent made possible by the factors discussed in this section. Following the FSH surge, rapid synchronous expression of L H receptors in granulosa cells and possibly thecal cells of the future DF may be pivotal event in the follicle deviation process (Xu et al., 1995). On the other hand, it is now clear that all growing follicles or viable follicles are capable of becoming dominant if provided the proper stimuli with what we have learnt from the aforementioned studies is probably FSH. Initiation of an FSH treatment protocol early in the wave stimulates many follicles to attain the diameter of the DFs (Adams et al., 1993) or a follicle randomly selected from a pool of 5 mm follicles at the beginning of a wave can be directed towards dominance by destroying all other 5 mm follicles (Gibbons et al., 1996). Finally, even after deviation in growth rates between the two 12 largest follicles, the subordinate follicle can remain viable for a day or two and can assume dominance, if the original DF is destroyed (Ko et al., 1991). Cauterisation or manual rupture of the DF is followed by a surge in FSH which is the reason why the subordinate follicles start growing and attain dominance if selected. Therefore, we can conlcude that FSH is necessary for intiating the recruitment phase and if available in higher concentrations will result in a greater number of follicles attaining dominance and L H thereafter is responsible for determining the size of ovulatory follicle. 2.2. Steroid and Peptide feedback The synthesis and secretion of pituitary gonadotropins is regulated by sex steroids produced by growing ovarian follicles and consequently by the resultant C L . Sex steroids most importantly, F^ and P 4 produced by the ovarian follicle and the C L , respectively exert a negative feedback effect on the release of FSH and L H by the pituitary. Elimination of this negative feedback loop by gonadectomy leads to a significant increase in circulating FSH & L H concentrations (Gay and Midgley, 1969; Rajamahendran et al., 1979). 2.2.1. Progesterone Feedback In both sheep (Goodman and Karsch, 1980) and cow (Ireland and Roche, 1982; Roberson et al., 1989) P 4 has been demonstrated to selectively inhibit the secretion of L H by decreasing the L H pulse frequency by acting at the level of the hypothalamus. Treatment of ovariectomized ewes with P 4 has shown that this inhibitory effect of P 4 does not extend to FSH (Nettetal., 1981). 13 Evidence also exists for the effect of P 4 at the pituitary level as ovine pituitary cell cultures treated with P 4 have an increase in FSH a and p subunit mRNA's and a decrease in responsiveness to GnRH (Miller et al., 1990). 2.2.2. Estrogen feedback It appears that E2 has both negative and positive feedback effects. An acute fall in circulating FSH and L H with little or no effect on GnRH secretion from the hypothalamus occurs following a single injection of E2 in the ewe (Nett et al., 1974) and cow (Beck and Convey, 1977) followed several hours later with an ovulatory-like surge of both FSH and L H . The positive feedback effect of E2 that permits the preovulatory surge of FSH & L H , is assumed to result from its effect on the hypothalamus. An increase in GnRH secretion in association with the L H surge has been shown to occur in rats (Sarkar et al., 1976), monkeys (Neill et al., 1977) and women (Miyake et al., 1980). In ewes, approximately 12 hours following the administration of Ej , an increase in GnRH pulse frequency occurs in the hypophyseal portal blood (Clarke and Cummins, 1985). Surges in GnRH preceeding E2 induced L H surges (Moenter et al., 1990), as well as preceeding natural L H surges (Moenter et al., 1990), have been described in the ewe. Increased hypothalamic secretion of GnRH leads to the secretion of elevated levels of FSH and L H , producing the ovulatory surges of gonadotropins, resulting in depletion of the pituitary gonadotropin stores (Roche et al., 1970; Nett et al., 1990). Initially, E 2 appears to increase the induction of GnRH receptors in the pituitary (Gregg and Nett, 1989) thereby, increasing the sensitivity of the pituitary to increases in GnRH. In this way, the positive feedback effects of E2 is enhanced by its action on the pituitary. 14 The effects of E2 switch from a positive to a negative one if serum concentrations remain elevated following the gonadotropin surges. Long term effects of E2 is due to its actions on the hypothalamus decreasing GnRH secretion (Karsch et al., 1987) and consequently a decrease in gonadotropin secretion. 2.3. Ovarian peptides 2.3.1. Inhibin Inhibin is a glycoprotein hormone in follicular fluid and its biosynthesis has been localized to granulosa cells using immunoblot and immunocytochemistry tehniques (Erickson and Hseuh, 1978). Inhibin is a heterodimer consisting of two subunits a and (3 linked by disulphide bonds. Two forms of pA, pB form active inhibin A (ctpA) and inhibin B (apB). The most important action of Inhibin is to inhibit the release of FSH from the pituitary through a classic negative feedback loop. FSH stimulates granulosa cells to produce inhibin. LH and HCG also has a similar effect on granulosa cells pretreated with FSH to induce LH receptors (Bicsak et al., 1986). On the other hand, FSH stimulated inhibin production is blocked by GnRH (Lapolt et al., 1990). Therefore, during the time of ovulation, the hypothalamic GnRH secretion shuts down inhibin secretion permitting the secondary surge of FSH. This secondary FSH surge recruits new follicles and stimulates inhibin production which again suppresses the output of FSH. Active immunization of heifers against inhibin has resulted in elevated serum FSH concentrations, ovulation rate (Hillard et al., 1995) and twin pregnancies (Morris et al., 1993). Inhibin selectively suppresses the release of FSH and not LH from the pituitary. This is evidenced by the ability of inhibin to suppress the synthesis of FSH p subunit at the pretranslational level (Mercer et al., 1987; Attardi et al., 1989). 15 2.3.2. Activin In contrast to inhibin, activin stimulates the release of FSH from the pituitary. Activin is a dimer composed of two inhibin p subunits; either p Ap B called activin A or p Bp B called activin B and immunostaining techniques have enabled its localization to the granulosa cell layer (Ogawa et al., 1991; Robinovici et al., 1992). Activin-A stimulates pituitary FSH synthesis and secretion in vitro and vivo probably due to an increase in FSH p subunit biosynthesis (Carroll et al., 1989). Activin has both paracrine and autocrine activities. Activin thus has a role in promoting folliculogenesis during preantral and antral stages through acquisition and propagation of FSH receptors in granulosa cells (Findlay, 1993). The actions of activin are primarily at the ovarian level rather than at the level of the pituitary unlike inhibin. 2.4. Ovarian Peptide growth factors 2.4.1. Insulin like growth factors IGF-1 & II are low molecular weight single chain polypeptides with a 45 % aminoacid sequence similarity to insulin (Rinderknecht and Humble 1978a&b). Concentrations of immunoreactive IGF-I was much greater in the follicular fluid than in serum establishing the ovary as a predominant production site (Hammond, 1981). In situ hybridization studies using ovarian cells from rats have enabled localization of granulosa cells of growing follicles as the site of production of IGF-I but not atretic follicles nor the C L (Oliver et al., 1989). IGF-II appears to be synthesized exclusively in the theca (Hernandez et al., 1990). In cattle, IGF-I is known to stimulate proliferation of granulosa cells and mitogenesis and promotes FSH-induced granulosa cell steroidogenesis (Spicer et al., 1993). A 2-4 fold 1 6 increase in cell number has been obtained which exemplifies the stimulatory effect of IGF-I on granulosa cell proliferation and DNA synthesis (Chakravorthy et al., 1993; Spicer et al., 1993). The mitogenic effect of IGF-I in granulosa cells from small (<5mm) and not large follicles (> 10 mm) is enhanced by FSH and L H . IGF-I increases FSH stimulated induction of granulosa cell L H receptor (Adashi et al., 1985), FSH stimulated and basal inhibin synthesis (Bicsak et al., 1986) in addition to FSH stimulated proteoglycan synthesis (Adashi et al., 1986) which indicates that IGF-I plays an important role in follicle selection, maturation and atresia. The role of IGF-I in follicle selection is further proved by the fact that cattle genetically selected for twinning had 47% more IGF-I concentrations in serum and follicular fluid than those ovulating a single follicle (Echternkamp et al., 1990b). IGFBPs comprise a class of six binding proteins that transport IGFs and present them to the cell surface receptor ultimately either potentiating or nullifying the actions of IGFs. They are regarded as the potential regulators of follicular development (Margot et al., 1989). Progressive follicular growth is associated with a reduction in the concentrations of low molecular weight IGFBPs, while their concentrations tend to increase in atretic follicles (Echternkamp et al., 1994). The biologic activity of IGF-I is regulated by IGFBPs which by either inhibiting E2 synthesis or through altering the availability of IGF-I causes a reduction in the number of ovulations (Ui et al., 1989; Shimasaki et al., 1990). 2.5. Twinning in cattle 2.5.1. Twinning by Genetic selection Selection of cattle for twinning is the oldest method and has a longer standing history than any other method of obtaining multiple births. It has an additional advantage over other methods of presumably being an inherent character. A detailed account on a higher propensity 17 for multiple births to run in certain cow families has been reported (Erb et al., 1960) as well as an excellent review by Rutledge (1975) on extraordinary fecundity in select individual cows. Several breeding schemes have been undertaken for this purpose, but very little success in the form of twin production has been achieved. During the last three decades several experiments have been conducted to investigate the practicality of increasing twin production in beef cattle through selection (Morris and Day, 1986; Gregory et al., 1988). Gregory et al (1990) reviewing the effects of genetic and environmental effects on twinning rate reported that the realized heritability of single-parity twinning rate estimated from selection of parents and response in daughters of foundation females was 0.06. Similarly, Maijala and Syvajarvi (1977) reported an average heritability of 0.028 from several studies. This method suffers from several limitations and it is the contention of most authors who have reviewed the subject, that inheritance of twinning has a low heritability of 0.02 to 0.06 (Gregory et al., 1990) and a low repeatability of 0.06 to 0.12 (Maijala and Syvajarvi, 1977; Gregory et al., 1990). Cattle, being a monoovular species have a low reproductive rate and a longer generation interval, a major limiting factor that slows the response to selection. To observe differences in twinning rate it is necessary to retain a greater percentage of females in large numbers for several parities so that a constant herd size is maintained. Echternkamp et al (1990a) suggested an indirect selection criterion for twinning rate in pubertal heifers based on the ovulation rate before 18 to 21 months of age. They found that heritability was 0.07 ± 0 . 0 3 for single observations and 0.34 ± 0.18 for the mean of 7.9 estrous cycles. Genetic progress however, has been made to a moderate extent by identifying and gathering together twin ovulating cows from the industry and segregating their germplasm in a nucleus herd (Morris, 18 1994). The largest herd selected for twinning is in the USA at The Clay Center, Nebraska (Gregory et al., 1990) where a twinning rate of 30% has been reported. Emphasizing ovulation rate as a primary constraint to increasing productivity in cattle, Echternkamp et al (1990a) suggested that "use of ovulation rate in pubertal heifers should permit effective indirect selection for twinning rate among yearling heifers based on individual performance and among young sires based on ovulation rate of sibs and daughters". Subsequent studies in USA have made similar recommendations, that repeated measurements of ovulation rate in pubertal heifers may be an effective way of selecting cattle for the twinning trait (Leymaster and Bennett, 1990; van Vleck et al., 1990) over a period of several cycles. Further, Echternkamp et al (1990) have provided evidence suggesting the association of natural twinning in cattle with increased concentrations of IGF-1 in both blood and follicular fluid. Twin producing cows had 47% greater concentrations of IGF-1 in both peripheral blood and the two largest follicles than control cows which is consistent with the hypothesis that IGF-1 is an important mediator of a genetic component of twinning in cattle. Based on a selection experiment established in 1981 at The Clay Center, Nebraska it was recently reported that the twinning rate increased in all cows born in the project from 3.4% in 1982 to 28.5% in 1993, a phenotypic increase of 25.1% (van Vleck and Gregory, 1996). These authors were of the opinion that the rate of twinning may be on the verge of reaching the range needed for widespread commercial use of the technology. A steady increase in calf weight of 53.1%, 54.7% and 58.4% at birth, 150 d and 200 d respectively, was recorded in individual twin calving cows compared to those producing singles (Gregory et al., 1996). Twin calving cows had 65.2% more live calves at 200 d than 19 cows producing singles (Gregory et al., 1996). The same study reported greater dystocia and lower calf survival as major constraints to twinning technology. Selection of bulls for the twinning trait has also been attempted for twinning in dairy cattle (Stolzenberg and Schonmuth, 1990a&b). The long generation interval seems to be the biggest constraint here. Beerpoot and Dykhuizen (1992) reporting on the economics of naturally occurring twinning in cattle discouraged selection of cattle for twinning as the returns from extra calves failed to balance the additional costs incurred due to twinning. A greater frequency of twin ovulations is not likely to result in a dramatic increase in twinning unless the ovulations occur on separate ovaries. This is evident from the studies of Rowson et al (1971), Scanlon (1974) who were the first to show that twinning rate is low when two embryos are transferred to a single uterine hornT In conclusion, genetic progress can be made for twinning, but this trait in addition to having a low heritability and a low repeatability, requires a greater percentage of females for several parities to comprise a large herd size and a large data base for screening animals. 2.5.2. Twins through the administration of exogenous Gonadotropins As an accidental discovery, a fourfold increase in ovulation rates occurred following the intramuscular implantation of anterior pituitary tissue in mice and rats during the days when reproductive endocrinology was in its incipient stages. Subsequently, it was demonstrated that serum of pregnant mares could induce multiple ovulations in rats which eventually became the gonadotropin of choice for superovulating cattle. The gonadotropins that have been used hitherto are PMSG or FSH of porcine or ovine origin. On account of its 20 ready availability, low cost, effectiveness and ease of use, requiring only one injection, PMSG has been used extensively to superovulate cattle. A moderate to high degree of multiple ovulations and multiple births have been induced in cattle by the exogenous administration of gonadotropins. These studies began with a single injection of PMSG given during the follicular phase of the cycle (Gordon et al., 1962) (Table 1). This regimen was soon modified by the inclusion of two injections given on days 3-6 (1500 iu) and 16-18 (2000 iu) of the estrous cycle (Schilling and Holm, 1963; Laster et al., 1971; Turman et al., 1971; Johnson et al., 1975; Mulvehill and Sreenan, 1977; Sreenan, 1981) (Table 2.1). To improve ovulation responses and multiple births in cattle, PMSG was replaced with partially purified follicle stimulating hormone (FSH-P) of either porcine (Vincent and Mills, 1972; Smith et al., 1973; Bellows et al., 1974; Wildt et al., 1975; Davis et al., 1992) or ovine origin (Bindon and Hillard, 1992) (Table 2.2). Results have been inconsistent due to excessive ovarian stimulation in a high proportion of cows producing triplets, quadruplets, quintuplets leading to high fetal mortalities, low birth weights and survival rates. Attempts to curtail the circulating half life of PMSG through administration of its anti-sera have been successful to the extent of reducing the duration of the estrus period without any control over ovulation rate (Yang et al., 1992). High abortion rates as experienced in earlier studies were recorded here too. A maximum of three fetuses per uterine horn and a total of five fetuses have been reported following the administration of FSH-P to 379 lactating and non-lactating cattle beyond which a greater number of abortions occurred (Echternkamp, 1992), thus clearly showing that ovulation rate is the most limiting factor to increasing productivity in cattle. On the other hand, increases in ovulation rate should be modest since the limited uterine space offered by the bovine uterus is only sufficient for sustaining twin 21 fetuses beyond a certain stage of gestation. Failure to comply to these limitations is the prime reason why fetal wastage is greater following gonadotropin induced multiple births. Despite the use of various treatment combinations, the occurrence of twin pregnancy has not been consistent. The half-life of PMSG greatly exceeds that of FSH in cattle and it involves rapid (to5 40 to 50 h) and slow (t 0 5 118 to 123 h) clearance components (Vos P L A M et al., 1994). With the existing knowledge, arriving at an effective dose for either of the gonadotropins that would foster the growth of just two ovulatory follicles is highly unlikely. Idiosyncrasy is a common occurrence as a given dose that has no effect on an individual animal (i.e., the animal produces a single ovulation) may cause more than the expected two, at times even three, ovulations in certain others. Gonadotropin induced multiple births have invariably resulted in the production of single calves, twins, triplets and sporadically, quadruplets and quintuplets. Because of the high mortality associated with litters of three or more calves, it is highly necessary to control the number of ovulations to a maximum of two. Laster (1973) reported that ovulation occurred on only one ovary in 75% of the gonadotropin treated heifers having two or three corpora lutea. Therefore, another critical requirement which makes this procedure more exacting is that one egg must be shed from each ovary if a high twin pregnancy rate is to be achieved (Gordon et al., 1962). Confirmation of this observation came later from surgical embryo transfer studies made by Rowson et al. (1971) who showed that a high twin-pregnancy rate was achieved (73%) after bilateral embryo transfer of a single embryo to each uterine horn, but not after the unilateral transfer of two embryos to the uterine horn ipsilateral to the corpus luteum (45%). From the above results it is quite clear that multiple birth induction is possible by exogenous gonadotropins, although it is difficult to restrict the resulting litters to the 22 acceptable size of two calves. Hence, understanding the feedback and intraovarian regulatory substances responsible for the control of ovarian function in cattle is necessary before any advancements in improving the efficacy of this technique could be made. With the current knowledge available it is difficult to foresee any major improvements in this technique. Table 2.1. Summary of superovulation studies using PMSG to induce twinning in cattle. Authors Estrus Treatment Results 1. Gordon et al (1962) Unsynchronized (Cows). PMSG administered at 800, 1000, 1200, 1600 or 2000 IU on either day 16 or 17 after estrus. Six weeks post breeding 20.8, 25.2, 31.3, 41.7 and 42.7% respectively, were carrying multiple fetuses. At higher dosage triplet and quadruplets occurred frequently. 2. Turman et al (1971) Unsynchronized (Beef cows). PMSG administered at 1500 IU on day 3, 4, 5 or 6 and 2000 IU on day 16, 17 or 18 followed by an intramuscular injection of 2500 IU ofhCG on day of estrus Of 81 cows treated 52 conceived at the first estrus following the second PMSG injection and produced 29 singles and 23 multiple births (12 sets of twins, 8 sets of triplets, 2 sets of quadruplets and one set of quintuplets. 3. Johnson et al (1975) Synchronized with orally feeding CAP (Beef cows). Unsynchronized PMSG administered at 1080 IU to 50 cows on day-5 and 1400 IU on day-17 of the cycle, with an intravenous injection of 2500 IU of hCG given at the time of insemination. Of 12 sets of multiple births produced, eight were from cows in the synchronized estrus group (5 sets of twins and 3 sets of triplets). Remaining 4 sets included (2 sets of twins, 1 set of triplet and 1 set of quadruplet). 4 Mulvehill & Sreenan (1977) Progesterone intravaginal sponges for 9 days (Beef cows). PMSG administered at 750 IU to 62 cows at the time of sponge removal. 2 sets of twins. Cronolone PMSG administered at 750 11 sets of twins. 23 intravaginal sponges for 9 days. IU to 59 cows at the time of sponge removal. 5. Sreenan (1981) Progesterone Injected cows with 600 to 750 IU of PMSG. Obtained twinning percentages of 15.9, 11.2 and 8.6% in three consecutive years. 6. Yang et al (1992) Unsynchronized. PMSG administered at 7 IU/kg to 29 cows on day 9 of the cycle followed by 3 mg of PGF 2 ( X two days later. Of 7 sets of multiple births (3 sets of twins, 2 sets of triplets, 2 sets of quadruplets). Unsynchronized. PMSG administered at 7 IU/kg to 23 cows on day 9 of the cycle followed by 3 mg of P G F 2 a two days later along with 2 ml of rabbit antiserum against PMSG at the onset of post-treatment estrus. Of 11 sets of multiple births (8 sets of twins, 3 sets of triplets). Table 2.2. Summary of superovulation studies using FSH to induce twinning in cattle. Authors Estrus Treatment Results 1. Vincent and Mills (1972) Norethandrolone (Beef cows). Administered FSH-P to 90 cows either once or twice daily at 4 dosage levels totalling 6.3 to 12.5 mg. 46 of 90 cows calved from the first breeding with 24% having multiple births (10 sets of twins and one set of triplets). Norethandrolone. Administered FSH-P to 55 cows on day 10 as a single subcutaneous injection. 10 of the 30 cows calved producing one set of twins and one set of triplets. 2. Smith et al (1973) Norethandrolone (Beef cows). Administered in C M C or PVP to 262 cows used in 4 experiments. Nine sets of twins and one set of triplets were born. 3. Bellows et al (1974) Unsynchronized. Expt: 1- Administered FSH to 43 beef heifers twice daily on days 8 through 12 of the estrous cycle (total dosage-6.2 mg). Thirty-nine calves were born which included five twin sets. 24 4. Davis and Bishop (1992) 5. Bindon and Hillard (1992). Unsynchronized. Prostaglandin F 2 a (Beef cows) Expt: 2- Fifty-five lactating 2 yr old dams were divided into groups of 28 and 27 cows that received 6.2 and 9.4 mg of FSH, respectively. Injected FSH to 166 cows at 2 mg on days 9 and 10 and 1 mg on days 11 and 12 followed by P G F 2 a on day 12 in three replicates. Injected 109 beef cows with a fixed dose (8 mg) of ovine FSH over 4 days. 21 calves resulted from the first group with one twin set. The second group produced 30 calves which consisted of 4 sets of twins and one set of triplets. Replicate I- Multiple birth rate was 27% (6 sets of twins, 1 set each of triplet and quadruplet. Replicate II- Multiple birth rate was 10% (7 sets of twins and 1 set of triplet). 34 calvings included 15 multiple births (12 sets of twins and 3 sets of triplets). 2.5.3. Twinning by immunizaton against certain ovarian steroids and peptides Vaccines have been and are still being employed widely in the livestock industry for the control and prevention of infectious diseases. Subsequent technologies that emerged in the early and mid-eighties permitted the development and use of vaccines against hormones that controlled and regulated reproduction, growth and lactation (Reeves et al., 1989). In the current discussion emphasis will be given to hormone vaccines that were developed to enhance the reproductive efficiency of domestic livestock, more importantly, cattle. Cattle have been immunized against certain ovarian steroid and peptide hormones, notably androstenedione, and inhibin. Immunization against each of these hormones for increasing ovulation rate in cattle and the results obtained from these studies will be briefly discussed in the following section. 25 2.5.3.1. Passive and Active immunization against ovarian steroids Very few successful attempts have been made to immunize cattle against steroids to induce twinning and highly variable ovarian responses have been observed in majority of these studies. Active immunization of cattle against E2 have yielded variable results with animals either continuing to cycle and become pregnant (Wise and Schanbacher, 1983) or failing to exhibit estrus and producing large anovulatory cystic/luteinized follicles (Martin et al., 1978). The results from the latter study indicated that immunoneutralization of E2 abolished the preovulatory L H surge leading to the development of large cystic and luteinized follicles. Among the other ovarian steroids, immunizing cattle against androstenedione has been successful. In their study, Wise and Schanbacher (1983) recorded twin ovulations in four of 15 (29%) androstenedione-immunized Simmental cross heifers, three (21%) of which had twin fetuses at ~46 d of pregnancy. However, these results were not repeated in subsequent similar studies conducted (Sreenan, 1984; Walton, 1985; Hoskinson et al., 1986) on beef or dairy cattle which instead encountered various degrees of ovarian dysfunction. An increase in ovulation rate has been obtained in testosterone immunized cattle treated with either equine chorionic gonadotropin (Boland et al., 1985) or human chorionic gonadotropin (Hoskinson et al., 1986). Seven out of a total 8 immunized heifers had measurable antibody titres after immunization against testosterone, following which they became anestrus and developed ovarian cysts (Price et al., 1987b). Three of the 7 anestrus heifers resumed cyclicity 25 weeks following the second booster. One and two consecutive twin ovulations were recorded in the first and second heifer, respectively, while the third heifer had quadruple ovulations (evidenced by four corpora lutea) which later became anestrus. Active immunization against dehydroepiandrosterone produced twin ovulations in 26 three out of 4 treated heifers (Sreenan et al., 1987). Even though immunization against testosterone increased ovulation rate, treatment was accompanied by long periods of acyclicity due to the hyperimmune state. These divergent ovulatory responses observed in cattle following androgen immunization has been attributed to subtle changes in the antibody titre (Hoskinson et al., 1986). With the objective of exerting a more controlled immunomodulation of the negative feedback mechanism, Morris et al. (1988) examined the feasibility of using passive immunization using xenogenic ovine testosterone antisera. Preliminary results were not encouraging as treatment increased ovulation rate in some animals, blocked ovulation in certain others and the majority remained refractory to the treatment. Later in a separate study the same authors inspite of using increased doses of anti-testosterone antiserum confronted disappointing results. The cows that received the higher doses of anti-serum (6.5 and 7.5 standard dose) had two large follicles. Immunization of heifers against inhibin alone or in combination with androstenedione resulted in an increase in the development of multiple of dominant follicles without an increase in ovulation rate (Pvhind et al., 1993). Recent findings show that androstenedione immunity functions through a local intra-ovarian mechanism (Scramuzzi et al., 1993 ). Antibodies to steroids have been detected in both follicular fluid and in other extracellular fluid. Both P 4 and dehydrotestosterone have been shown to inhibit the induction of L H receptors by FSH in granulosa cells. Further, androgen treatment appears to exert an anti-estrogenic effect by decreasing the ovarian estrogen receptor content. A high androgen to estrogen ratio in the follicular fluid of atretic follicles extends more support to this observation. It is therefore, suggested that immunoneutralizing androgens would be ideal to protect growing follicles from the atretogenic 27 effects of locally produced androgens. Androgens also exert a wide variety of autocrine and paracrine effects, a thorough understanding of which may be key to understanding the underlying mechanisms of immunoneutralization. Taking into account these undesirable ovarian responses following steroid immunization, the likelihood for the development of a product for increasing prolificacy in cattle similar to "Fecundin" that was developed for sheep is highly remote without further research. 2.5.3.2. Immunization against Inhibin Inhibin is a peptide hormone and it is known beyond doubt that the granulosa cells of the ovarian follicle is the major site of production. Inhibin is a heterodimer consisting of two subunits namely a and p linked by disulphide bonds. The Inhibin p- subunit exists in two different forms, termed p A and pB sharing aproximately 70% homology in aminoacid sequence. Two mature forms of inhibin oc-pA and a-pB have been isolated and both exert similar biological action of suppressing pituitary output of FSH. Our understanding of the physiology of inhibin as a peripheral hormone regulating FSH secretion during the estrous cycle in cattle is well documented (Taya et al., 1991). The secondary surge of FSH that follows luteolysis is assumed to initiate the next wave of follicle growth. As the DF emerges from the cohort the increasing production of inhibin and could decrease FSH secretion thereby, suppressing the growth of the subordinate follicles from the contemporary cohort of recruited follicles present at the onset of the follicular phase. Increasing concentrations of FSH have been shown to increase the secretion of inhibin (Bicsak et al., 1986; Tsonis et al., 1989; McNeilly et al., 1989). Therefore, inhibin by exerting a suppressive effect on FSH secretion 28 from the pituitary plays an important role in maintaining the species specific number of one ovulatory follicle in cattle. It was postulated that interference with this negative feedback system will be necessary to increase the ovulation rate in cattle. However, the original idea that laid all the ground work came from the observation that the ovary of the prolific breed of sheep boorola Merino was deficient in bioactive inhibin (Cummins et al., 1983). Being deficient in inhibin the booroola had elevated plasma concentrations of FSH and accordingly exceptional ovulation rates. Several groups have used either native inhibin or synthetic peptide production to produce inhibin fragments for immunization purposes, the details of which will be briefly discussed in the rest of this section. 2.5.3.2.1. Native inhibin immunogens Cummins et al. (1986) reported multiple ovulations in the range of 2-6 in 50% of the treated cows following active immunization against a crude native ovine inhibin. Price et al. (1987a) recorded a moderate increase in ovulation rate in 4 of 10 heifers immunized with 4 mg of a partly purified ovine inhibin at first estrus following a second booster injection. However, results were not repeatable following the booster injection. Immunization of pubertal heifers with an immunoaffmity-purified preparation of ovine follicular fluid produced a more consistent and significant multi-ovulatory response after booster vaccinations (O'Shea et al., 1994). Immunization of Hereford cows against ovine follicular fluid inhibin followed by mild superovulation using exogenous ovine FSH increased ovulation rates yielding more transferrable embryos than control animals (Bindon et al., 1994). 29 2.5.3.2.2. Synthetic inhibin a-chain fragments Immunization with synthetic inhibin a-chain fragments (conjugated with appropriate carrier protein for increased immunogenicity) have produced significant increases in ovulation rate in beef and dairy heifers subjected to multiple (2-8) booster vaccinations with different bovine inhibin a-chain fragments (Glencross et al., 1992; Morris et al., 1993; Scanlon et al., 1990). Glencross et al. (1992) recorded multiple ovulations in 19 out of 56 cycles monitored which comprised 14 twin ovulations, 4 triple ovulations and 1 quadruple ovulations, without any significant increase in plasma FSH concentrations in inhibin immunized heifers. Morris et al. (1993) actively immunized cattle against three peptide sequences of inhibin a-subunit and was the first to report twin pregnancies and to suggest peptide immunization as a promising approach to induce twinning in cattle. Unilaterally ovariectomized cows immunized against an a-chain fragment of inhibin alone or in combination with FSH had larger diameter follicles than untreated unilaterally ovariectomized and untreated intact cows (Rocha et al., 1996). However, in the same study none of the treatments used increased oocyte recovery rate compared to untreated intact control cows. 2.5.3.2.3. Recombinant inhibin and inhibin a-subunits Continuing efforts in Australia to develop a prototype bovine inhibin vaccine have identified recombinant human inhibin and four inhibin-a fusion proteins for use in cattle (Hillard et al., 1995). Early evaluation of the prototype vaccine showed that the recombinant ovine inhibin-a.3 with Montanide:Marcol was extremely potent and ovulatory responses in immunized cattle depended on the timing of the booster vaccination within the preceding ovarian cycle. Significantly higher ovulation rates were recorded in cows boosted on either d 2 or d 9 than d 16 of a synchronized estrous cycle. Booster vaccination on d 2 was more 30 effective in the induction of multiple pregnancy (Bindon et al., 1994). However, pregnancy data were not encouraging, as 40-50% of pregnant cows were carrying multiple fetuses at days 40-45 of gestation and consequently high fetal mortalities were recorded in cows with greater than 3 ovulations resulting in variable number of calves born (Hillard et al., 1995). Difficulties in restricting the ovulation rate strictly to two using the immunization technique presents it with disadvantages similar to that observed with gonadotropin treated cows. As a concluding remark, for this procedure to be accepted commercially, the treatment effects following inhibin immunization should be optimally maintained and extended over several estrous cycles and considering the restricted uterine space available, should produce a modest increase in ovulation rate, ideally two. 2.5.4. Twinning by Embryo transfer Apart from genetic selection, treatment with exogenous gonadotropins and immunoneutralization against ovarian steroids and peptides, embryo transfer offers an alternative approach to the artificial induction of twin pregnancy in cattle. Just as artificial insemination increases the reproductive potential of the male, so does embryo transfer increase female reproductive potential. This technology has such tremendous potential that it could very easily change a monotoccus species such as a cow into a polytoccus species like the sow. It has several merits over the other methods in that it makes possible an increase in the number of calves without a greater number of dams and always ensures that the cow carries no more than two fetuses. Accordingly, opportunities to increase beef production were created and two eggs originating from beef cows were transferred to the lesser yielding recipient dairy cows and helped increase the calf crop through twinning. 31 Twinning through embryo transfer could be accomplished by introducing in vivo or in vitro produced embryos either into the contralateral horn of bred animals presumed to be pregnant or into both horns of a non-bred animal: Under the circumstances mentioned above these animals will either produce twins in the form of their own calf and another calf that originated in a suitable donor animal or both calves originating from a donor animal. Majority of the estrous cycles in cattle culminate in a single ovulation resulting in the formation of one C L . Skepticism remained in the early days as to whether or not this single C L would maintain the development of an embryo introduced into the contralateral horn. This uncertainity was soon resolved when a higher percentage of viable twins were obtained (73%) in heifers following bilateral embryo transfer thereby disproving the existence of a unilateral utero-ovarian-embryo relationship. Following the initial success of Rowson et al. (1971), several studies performed later have confirmed that transfer of one embryo can be employed successfully to induce twin pregnancies in a previously bred recipient (Boland et al., 1975; Sreenan and McDonagh, 1979; Renard et al., 1979; Sreenan, 1981; Holy et al., 1981). Suggestions are available in the literature that concentrations of P 4 > 3ng/ml on the day of embryo transfer may be required for successful implantation of the transferred embryo (Sreenan and Diskin, 1983; Goto et al., 1988). As a result, better ways to increase P 4 concentrations to successfully maintain a twin pregnancy have recently been proposed. Induction of multiple corpora lutea in the previous cycle using exogenous gonadotropins significantly boosted the P 4 pool throughout gestation following the transfer of two embryos bilaterally into unbred recipients (Patel et al., 1995). A low dose of FSH administered to induce a single ipsilateral C L on each of the ovaries however did not succeed (Kojima et al., 1995). Unfortunately, evidence indicates that supraphysiologic P 4 concentrations (Lamond, 32 1974) following induction of multiple corpora lutea is unlikely to improve fertility (Britt and Holt, 1988) and hence favor embryonic development (VanCleeff et al., 1992). An ovulatory dose of H C G given on d 7 post insemination to ovulate the first wave DF has increased P 4 concentrations and improved pregnancy rates in cows carrying singletons (Sianangama and Rajamahendran, 1992). This very well could be an alternative approach to ensure the availability of adequate P 4 levels at the time when a second embryo is transferred into the contralateral uterine horn. To our knowledge no study has attempted to translate this idea practically to increase plasma P 4 concentrations for successful embryo transfer twinning. The technical success of twin production from embryo transfer with synchronized and inseminated recipients will depend on the conception rate achieved from the induced estrus. Accurate heat detection and the use of semen from highly fertile bulls will be required to attain higher conception rates so that pregnancy is not compromised. Preliminary investigations on embryo transfer twinning began with superovulation, non-surgical embryo recovery and following evaluation, non-surgical transfer to recipient cows. Superovulating a cow for the procurement of embryos for use in commercial embryo transfer programmes is feasible but the response is highly unpredictable and is also expensive (Wall et al., 1992). Therefore, a more flexible way to achieve optimum twinning rates with reduced expenses would be to employ IVMFC embryos obtained from abattoir ovaries or by a more sophisticated means such as ultrasound guided follicular aspiration from ovaries of live high yielding animals. It is less expensive and provides the possibility of producing a large number of embryos enabling evaluation and manipulation during development and production of twin calves with desirable characters from superior cows with desirable characters that are infertile for non-genetic reasons (Reichenbach et al., 1992). 33 Embryo and sperm manipulation has resulted in the production of identical twins of predicted sex (Ushijima et al., 1995) which even though laborious could help avoid freemartinism. Numerous reports on live births are available following the transfer of IVMFC embryos (Goto et al., 1988; Reichenbach et al., 1992; Lu et al., 1988; Xu et al., 1990; Takada et al., 1991; Sinclair et al., 1995b; Penny et al., 1995). Attempts to produce twin calves by implanting IVMFC embryos into dairy cows resulted in twinning rates consistently at the lower end of the range reported for in vivo produced embryos (Reichenbach et al., 1992; McCutcheon et al., 1991; McEvoy et al., 1995 ). However, in vivo produced embryos resulted in high pregnancy, twinning rates at d 56, and higher calving and twin calving rates than did in vitro produced embryos (Sinclair et al., 1995a). Until some of the problems encountered in these studies such as low pregnancy rates, higher than expected early embryonic losses, fetal oversize and increased fetal deaths and abortions are overcome it is far from reality that IVF technology will find widespread acceptance in commercial embryo transfer programmes. Despite the considerable success, a myriad of factors influence the effectiveness of embryo transfer twinning. Higher pregnancy rates have resulted following the transfer of one embryo to a bred recipient as against two embryos ipsilaterally to a non-bred recipient (Johnson et al., 1989; Suzuki et al., 1989). It may be assumed that the recipient's own embryo creates ideal conditions in the uterus for the maternal recognition of pregnancy which may then permit the survival of the transferred embryo. A recent study (Sinclair et al., 1995b) extended support to this contention where inseminated recipients (AI+Bred) had slightly greater pregnancy (61.6% v 51.6%), twinning (36.9% vs 28.7%) and calving (54.3% vs 42.0%) rates than non-bred recipients recieving two embryos. 34 Unilateral or bilateral placement of twin embryos to a great extent is assumed to determine the establishment and outcome of an induced twin pregnancy. Several studies have been conducted to examine the effect of twin embryo distribution (unilateral vs bilateral) on embryo and fetal survival through to term in cattle. High to moderately high twinning rates have been obtained following the transfer of two embryos into the ipsilateral uterine horn of unbred recipient cattle (McCutcheon et al., 1991; Newcomb et al., 1980; Williams and Evans, 1985; Sreenan and Diskin, 1989; Sakakibara et al., 1996; Schmidt et al., 1996). However, there are suggestions in the literature that twin pregnancies successfully terminate in twin calvings provided the twin pregnancy is bilateral. Higher embryonic survival and twinning rates have been reported following bilateral transfer of embryos (Rowson et al., 1971; Reichenbach et al., 1992; Suzuki et al., 1989; Davis et al., 1989). Transuterine migration of embryos that frequently occurs in the sow (Niemann and Elsaesser, 1986) and ewe (Nephew et al., 1989 & 1992) is a rare phenomenon in cows. Consequently, overcrowding of two embryos within one uterine horn may lead to competition for space and nutrients resulting in the death of one of the embryos in majority of the cases (Rowson et al., 1971). Higher abortion and still birth rates have been observed following unilateral distribution of twin embryos (Reichenbach et al., 1992; Tachikawa et al., 1993). Opinion remains divided whether cows or heifers would serve as better recipients. Success has been attained using heifers as recipients but higher abortions and still birth rates have been reported frequently in heifers when used as embryo recipients (Sreenan and Diskin, 1989). Unless heifers are well grown it is questionable if they will successfully be able to establish placental area sufficient to sustain twin calves till term (Rowson et al., 1971). Breed differences have been shown to exist among cattle in their ability to gestate twin pregnancies 35 to term successfully following transfer of frozen thawed embryos (Sakakibara et al., 1996). Comparing Holstein with Japanese black cows, these workers concluded that the average birth weight, placental weight and placentome numbers was influenced by the breed of the dam in case of twin births. In addition, Holstein cows calving twins had a higher number of placentomes. In conclusion, the induction of twins by embryo transfer is an ideal method to improve the reproductive efficiency in cattle. It may be asserted that the deleterious effects commonly known to accompany the production of twins by natural or by hormonal methods can be precluded owing to the greater control over pregnancy and parturition. The importance of the embryo transfer approach to twinning would be particularly far-reaching in the beef industry with advancements in in vitro embryo production coupled with development of methods to transfer eggs non-surgically. The production of embryos in vitro as against in vivo has in some ways reduced the high expenditures involved in using embryo transfer as a method to induce twinning in cattle. Further, the advent of transvaginal ultrasound guided oocyte aspiration has ensured availablity of good quality oocytes from superior cows on a regular basis in surplus numbers without the incurrment of additional costs. 2.6. ULO as a method to produce twins in cattle From the previous section it is evident that much research has gone into developing techniques for the induction of twinning in cattle. While embryo transfer seems to be highly successful method of producing twins, it involves extensive labour and high costs. Exogenous gonadotropin treatment and inhibin immunization even though successful, have many inconsistencies. The number of ovulations induced could range anywhere from two to ten which is undesirable when considering the limited space offered by the bovine uterus. 36 FSH seems to the limiting factor in the development of number of ovulatory follicles as administration of low doses of FSH in the early part of the cycle has been shown to increase the number of follicles that develop to the ovulatory size (Rajamahendran et al., 1987). The output of FSH from the pituitary is known to be regulated by ovarian secretions (steroids and peptides) (Findlay and Clarke, 1987). A significant increase in the pituitary release of gonadotropins occur following bilateral ovariectomy (Rajamahendran et al., 1979). We therefore, hypothesize that U L O would lower the inhibitory effects of ovarian secretions on FSH release to an extent that would be sufficient for the selection, growth and ovulation of more than one follicle and exploit it as a novel method to induce twinning in cattle. "Unilateral ovariectomy is a time-honoured procedure that has been useful in elucidating follicular kinetics in a diverse variety of species" (Greenwald and Terranova, 1988). In the immediate period following U L O the remaining ovary undergoes compensatory hypertrophy and has been attributed to the formation of increased number of corpora lutea and enhanced follicular activity. ULO has served as an important tool to elucidate information on follicular development as to how late in the cycle successful follicular recruitment and hence increased ovulation rate can be elicited. The first experiments on U L O dates back to 1897 when Hunter following the farrowing records of two sows, one intact and the other semi-spayed reported that over the first eight litters, the intact sow produced 87 young while the sow semi-spayed produced 76. 2.6.1. Effects of ULO on species with short estrous cycles In the hamster, U L O at 9:00 am for the first 3 days of the 4-day estrous cycle resulted in doubling of the number of ovulations from the remaining ovary (Greenwald, 1961). U L O 37 on d 3 within 4 h has been shown to mobilize preantral follicles with six to seven layers of granulosa cells and converts them into small antral follicles (Chiras and Greenwald, 1978). The effects of U L O in cyclic rats depends on the length of the estrous cycle. In rats with 4-day cycles, U L O as late as day 3 results in doubling of the number of ovulations from the retained ovary. In rats with 5-day cycles, follicular compensation results following removal of one ovary as late as 2:00 am of diestrus two (Peppier and Greenwald, 1970a). Doubling in the number of large follicles due to increased proliferation of smaller follicles following U L O serves to maintain the normal ovulatory quota in rats (Peppier and Greenwald, 1970b). U L O in mice performed at random times during the cycle within 3 days doubles the number of ova shed after the procedure. In induced ovulators like rabbits, a two to three fold increase in follicles in the size range of ^ 1 mm in the remaining ovary occurs within 48 hrs after U L O and an ovulatory dose of H C G given 4 days later resulted in ovulation of 11.4 ova (Fleming etal., 1984). 2.6.2. Effects of ULO in species with Long estrous cycles Unilateral ovariectomy performed as late as day 10 of the cycle results in doubling of the ovulation rate from the remaining ovary in the guinea pig due to an increased rate of transformation of smaller sized follicles into larger ones (Hermreck and Greenwald, 1964). Hemi-ovariectomy of Finn-dorset sheep on day 2, 8 or 14 does not affect the number of ova shed (measured by number of copora lutea) at the very next estrus (Land, 1973). Leicester-Merino ewes hemi-ovariectomized on d 14 results in compensatory ovulation by the next cycle (ULO, 2 corpora lutea; intact. 1.70) but is only partially effective when hemi-ovariectomy is deferred to day 16 (Findlay and Cunrming, 1977). In a breed of sheep with 38 high ovulation rate (2.4 corpora lutea), removal of one ovary on day 10 of the cycle results in an overall ovulation rate of 2.2 from the remaining ovary (Land, 1973). In pigs U L O on day 2 of the estrous cycle results in a compensatory increase in the number of large follicles in the size range from 5 - 12.9 mm in diameter by day 13. U L O in sows on day 2 of the estrous cycle results in compensatory ovulation by the remaining ovary of as many ova in the next estrus as intact sows (18.1 & 16.0 corpora lutea, respectively). Even though the ewe increases its ovulation rate per ovary, the overall lamb production over 2 years is significantly less than intact ewes : 1.35 vs 1.61 (Sundaram and Stob, 1967). 2.6.3. Effects of ULO on livestock species that are normally monoovular In the cow, U L O on day 8 of the estrous cycle results in an increase in the number of follicles of the 9-16 mm class range in the retained ovary than controls. In hemi-ovariectomized group, the largest follicle was smaller than in the control, albeit the next largest follicle was consistently larger in the U L O group (Saiduddin et al., 1970). In the heifer, a significant increase in the number of follicles 5 to 6 mm and > 9 mm in diameter that occurs 7 days after U L O could be blocked by daily injections of BFF (Johnson et al., 1985). These studies clearly indicated that inhibin was the crucial component that was probably eliminated following U L O . From the above discussion it is clear that follicular compensation by the remaining ovary (maintenance of number of ovulations characteristic of the species) depends on rapid recruitment of small follicles as an acute response to U L O . In the earlier days when radioimmunoassays were not available it was assumed that U L O did not result in an increase in FSH secretion from the pituitary, instead had a "sparing effect" meaning that the amount of hormone normally available to two ovaries now exerted its actions on the retained ovary. The 39 current school of thought is that a transient increase in the concentrations of FSH occurs within 12 hrs following U L O . This acute increase has been observed in hamster (Bast and Greenwald, 1977), rat (Welschen et al., 1978), rabbit (Fleming et al., 1984), gilt (Redmer et al., 1985), cow (Johnson et al., 1985), ewe (Findlay and Cummins, 1977) etc. This increase in FSH concentrations is probably due to a reduction in inhibin concentrations. Follicular compensation following ULO on diestrus 2 is inhibited by administration of inhibin. Similarly, daily treatment of hemi-ovariectomized gilts (Redmer et al., 1985) and heifers (Johnson et al., 1985) with porcine and BFF, respectively inhibited compensatory hypertrophy. 2.6.4. Long term effects of ULO Compensatory ovulation following U L O has been recorded for up to 14 and 10 cycles in the cyclic hamster (Chattergee and Greenwald, 1972) and rat (Peppier, 1971), respectively. At proestrus hemiovariectomized rats had twice as many follicles > 450 pm than did intact rats which resulted in compensatory ovulations. Twice the number of follicles were observed in the semi-spayed rats by metestrus and diestrus and this was assumed to be due to a reduction in follicular atresia. In sheep approximately 70 days after U L O , there appears to be a significant increase in the number of small pre-antral follicles (< 0.06 to 0.07 mm in diameter) and antral follicles, in the range of 2.0 to 3.6 mm (Land, 1973). In both the rat (Butcher, 1977) and hamster (Bast and Greenwald, 1977) an extension of the secondary FSH surge into the second day of the cycle is proposed to be the signal for recruiting additional follicles (300 - 450 pm in the rat) into the ovulatory size or range. 40 It can therefore be concluded that an increase in the concentrations of FSH following U L O (both short and long term effects) can be accounted for by the increased follicular recruitment or reduced follicular atresia. Even though compensatory ovarian hypertrophy along with increased follicular recruitment and ovulation has been observed in most of the polytoccus laboratory and livestock species, data available for the cow is limited. Ovarian compensatory hypertrophy following U L O has been reported in cyclic and postpartum cows (Saiduddin et al., 1970; England et al., 1973; Grass and Hauser, 1981) and in prepubertal heifers (Johnson et al., 1985; Moser et al., 1989). In some of these studies, hormonal reponses were only partially evaluated and there were no devices to monitor follicular development during the compensatory response. During the last decade considerable amount of information on ovarian follicular growth and development has been generated and it is now clear that follicles in the bovine ovary grow in waves ultimately leading to the development and ovulation of one follicle. However, in litter bearing species it is well documented that the retained ovary following U L O compensates by ovulating additional follicles thereby unaltering the litter size. Cattle are monovular and gestate only a single calf with only a few exceptions. Therefore, it is interesting to investigate the response to ULO in cattle on its effect on follicular wave number, hormonal changes, ovulation and pregnancy rates. 41 CHAPTER- 3 EFFECTS OF UNILATERAL OVARIECTOMY ON FOLLICULAR DYNAMICS, PLASMA GONADOTROPIN AND PROGESTRONE CONCENTRATIONS IN DRY CYCLING COWS 3.1. ABSTRACT This study investigated the effects of U L O in four cycling dry cows on follicular dynamics, plasma FSH & L H and P 4 concentrations. Ovarian activity in all four cows was monitored daily using transrectal ultrasonography until the day of standing estrus during which period daily blood samples were also taken from the tail vein for FSH, L H and P 4 determination. U L O was performed on the day after ovulation. The ovary that ovulated was left behind in two of the cows and surgically removed in the other two. Thereafter, ovarian activity was monitored daily (ultrasonography and blood sampling for FSH, L H and P 4 ) for two consecutive cycles (eight cycles in all). Mean cycle length, FSH, L H and P 4 concentrations before and after U L O were compared using a paired sample-t test. Results show that U L O neither altered the cycle length nor the number of follicular waves in the cows. U L O increased the number of ovulatory follicles; two follicles developed and ovulated in six of the eight cycles. The mean diameter of the largest follicle was 16.1 ± 0 . 9 mm and the second largest 12 .5±0 .9 mm. No significant (P > 0.05) differences were observed in FSH (0 .72±0 .09 vs 0 .71±0 .07) , L H (0 .42±0.1 vs 0 .37±0.07) and P 4 ( 2 .8±0 .6 vs 2 .6±0 .4 ) levels before and after U L O . Removal of the retained ovary in three cows on day 7 following induced estrus after completion of the second experimental cycle revealed two and three corpora lutea, respectively in two cows. Therefore, given the small number of cows involved 42 in this study I conclude that U L O may be a possible method to obtain twin ovulations in dry cows. 3.2. INTRODUCTION In monotocous species like cattle antral follicular growth and development is characterized by two or three successive waves of follicular development (Taylor and Rajamahendran, 1991a). Each of these waves consists of a recruitment phase followed by a selection phase during which one follicle is selected, enters the dominance phase and either regresses or ovulates at the end of the cycle. FSH plays a pivotal role in the recruitment and selection phases and determines the number of ovulatory follicles. An association is known to exist between the surge of FSH and the number of follicles recruited during each wave of follicular growth in cattle (Adams et al., 1992a). A priming dose of FSH given early in the cycle has been shown to increase the number of follicles that develop to the ovulatory size (Rajamahendran et al., 1987). Ovarian secretions (E 2 & inhibin) are known to regulate the output of FSH from the pituitary (Findlay and Clarke, 1987). A significant increase in serum FSH and L H occurs following bilateral ovariectomy in cattle (Rajamahendran et al., 1979). Accordingly, this study was based on the premise that U L O would reduce the negative feedback effects of ovarian secretions on pituitary output of FSH to an extent that is sufficient for the recruitment, selection, growth and ovulation of more than one follicle. Therefore, the objectives of this study were to investigate the effects of U L O on plasma gonadotropin levels, P 4 profiles, follicular development and ovulation rates. 43 3.3. M A T E R I A L S AND M E T H O D S 3.3.1. Animals and Ovarian examination Four dry cycling Holstein cows were used in this study. The animals were obtained from the Agriculture Canada Research Station, Agassiz, BC and were housed at The University of British Columbia Dairy Teaching and Research Unit and cared for according to the guidelines of the Canadian Council of Animal Care. The cows were fed a standard ration of alfalfa cubes (approximately 16% crude protein, CP) and timothy hay (13%). Ovarian activity in the cows was monitored daily using transrectal ultrasonography until the day of standing estrus. The day of estrus was also confirmed by the ultrasonic detection of a large ovulatory follicle, and by rectal palpation for uterine turgidity. U L O was performed one day after ovulation using an ecraseur (colpotomy) under epidural anaesthesia (Drost et al., 1992). Following U L O , ovarian follicular development in the retained ovary was monitored for two complete cycles. After completion of the second experimental cycle after U L O , estrus was induced in three cows and 7 days post-estrus the retained ovary was surgically removed to assess the number of ovulations. 3.3.2. Blood sampling and analysis for plasma FSH, LH and P4 Blood samples (7ml) were collected daily through a tail vessel puncture from all four cows before and after U L O just before each ultrasound examination for determining plasma FSH, L H and P 4 concentrations. Plasma P 4 concentrations were measured using a commercially available solid-phase radioimmunoassay kit (Coat-A-Count, Diagnostic Products Corp., Los Angels, CA). This kit has been previously validated in our laboratory for the measurement of P 4 in cow's milk and plasma (Rajamahendran et al., 1989). Quantification of plasma FSH was done by a double antibody radioimmunoassay validated by Rawlings et al. 44 (1984). All samples were measured in a single assay. Plasma L H concentration was assayed by the method of Sanford (1987). Mean cycle length, plasma P 4 , FSH and L H data before and after U L O in both cows and heifers were compared using a paired sample -t test. 3.4. RESULTS 3.4.1. Ovarian follicular dynamics during the control cycle Estrus was not synchronized and ovarian activity was monitored in all four cows till the day of standing estrus. Among the four cows one of them was in estrus on the day of first ultrasound scanning and therefore it was possible to monitor ovarian follicular growth in this cow for a complete control cycle. This cow had a cycle length of 21 days, two waves of follicular growth and ovulated twin follicles before U L O . The remaining three cows had one large follicle that developed to the ovulatory size at the end of the cycle (Table 3.1). A representative pattern of follicular development and P 4 profiles profile observed in the cows prior to U L O is shown in Figure 3.1. 45 OVULATION CL 2 4 6 8 10 12 14 16 18 20 22 Day o f e s t r o u s cycle Figure 3.1. A representative pattern of follicular development and progesterone profile observed before unilateral ovariectomy. 3.4.2. Ovarian follicular dynamics following ULO A representative pattern of follicular growth and development observed in six of eight cycles (Trial I) is shown in Figure 3.2. All cows had a mean cycle length of 21 days after U L O . Follicular wave pattern remained the same in all the animals following U L O . The number of ovulatory follicles increased in the interovulatory intervals observed after ULO in cows. Codominant follicles developed and ovulated in six of the eight cycles followed (Figure 3.3). An altered size distribution was observed between the two preovulatory follicles. The mean diameter of the largest follicle was 16.1+0.9 mm and next to largest 12.5+0.9 mm (Table 3.1). All four cows had two waves of follicular growth during the cycle after ULO. Removal of the retained ovary on d 7 of the third cycle revealed more than one ovulation evidenced by the presence of more than one corpora lutea (Figures 3.4 & 3.5). 46 3.4.3. Plasma FSH and LH concentrations before and after ULO Even though the cows did not have a complete control cycle, all cows had a minimum interval of nine days between start of the experiment and the day of estrus before ovariectomy. Hence the mean FSH and L H concentrations in the samples taken during the last seven days before they were observed in estrus served as the control levels and were compared with the concentrations during the last seven days of the cycles after ULO. No significant differences in mean plasma FSH (0.72+0.09 vs 0.71+0.07) and L H (0 .42±0 .1 vs 0.37+0.07) were observed in all cows before and after ULO (Table 3.1). OVULATION CL Day of estrous cycle Figure 3.2. A representative pattern of follicular development and progesterone profile observed after unilateral ovariectomy. 47 Figure 3.4. Ovary of cow # 9923 removed 7 days after induced estrus after completion of the second experimental cycle following unilateral ovariectomy. Twin corpora lutea (CL) measuring 18 and 17 mm in diameter along with two large follicles (F) measuring 15 and 10 mm in diameter can be seen. Figure 3.5. Cut section of the retained ovary (Cow # 8809) that was removed 7 days after the second experimental cycle following unilateral ovariectomy. The growth, development and ovulation of three follicles is evidenced by the presence of three corpora lutea measuring 15, 10 and 7mm in diameter, two of them sectioned and the third intact. 48 Cows (n=4) Comparisons Before ULO After ULO Cycle length (days) 21 21 Follicular waves (No.) 2 2 Ovulatory follicles (No.) 1 (3/4), 2 (1/4) cycles 2 (6/8 cycles) P 4 concentrations (ng/ml) 2 . 8 ± 0 . 6 a 2 . 6 ± 0 . 4 a FSH concentrations (ng/ml) 0.72 ± 0.09a 0.71 ± 0.07a LH concentrations (ng/ml) 0 . 4 2 ± 0 . 1 a 0.37 ± 0.07a Means in a row with the same superscript do not differ significantly (p > 0.05). Table 3.1. Number of ovulatory follicles, follicular waves, mean (SE±) cycle length, plasma gonadotropin (FSH & LH) and progesterone concentrations in cows before and after ULO. (A) COW# 8809 0 l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l T - i o o ) C T h - T - n r > - T - i n o ) c o T t o o m o > o T - T - C N I T - T - T - CN T - T - C M Day of estrous cycle (B) COW# 9111 OVU/ULO 0-1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 T - T - r- r- T - CM CM Day of estrous cycle 49 Figure 3.3. Sequential growth and regression of follicles and corpus luteum in in four dry cycling cows [8809 (A), 9111 (B), 9209 (C), 9233 (D)] observed before (one cycle) and after (two cycles) unilateral ovariectomy. The termination of the control cycle and removal of one ovary is indicated by O V U / U L O . The ovulatory follicles are indicated as O V U and corpus luteum as C L . 50 3.4.4. Plasma P4 concentrations before and after ULO The mean plasma P 4 concentration before ULO was 2.8+0.6. After ULO the mean plasma P 4 concentration was 2.6+0.4 (Table 3.1). 3 .5 . DISCUSSION Some 210 years ago, Hunter (1787) first demonstrated that the litter size in swine was unaltered following ULO. Later, in similar studies ULOs performed in various livestock and laboratory species during the estrous cycle have demonstrated compensatory ovulation by the retained ovary thereby maintaining the species specific litter size (Jones and Krohn, 1960; Hermreck and Greenwald, 1964; Peppier and Greenwald, 1970b; Dailey et al., 1970; Land 1973). Although polytoccous livestock species have responded with a compensatory increase in ovulation rate, no such reports on ovulation rate are available for monoovular species like cattle, leaving open the question as to how would cattle respond to such a procedure. In this study involving cattle ULO resulted in doubling of the normal ovulatory quota further adding credibility to an old question as to whether or not twin ovulations could be induced in cattle following ULO (Saiduddin et al., 1970). During the 21 d estrous cycle in cattle, antral follicular growth and development is characterized by two or three successive waves of follicular development (Taylor and Rajamahendran, 1991a). Each of these waves consists of a recruitment phase followed by a selection phase from which one follicle is selected, enters the dominance phase. The dominance phase is characterized by the growth of one large follicle (> 10 mm) (Fortune, 1994). ULO neither altered the interovulatory interval nor the number of follicular waves in all cows. But following ULO, an altered pattern of follicular growth and development was observed in all cows (Figure 3.2). 51 Earlier Saiduddin et al. (1970) had observed an altered size distribution of follicles following U L O on d 8 of the estrous cycle in cattie. At the time of estrus the largest follicle was smaller and the next to largest follicle larger in unilaterally ovariectomized than those in intact animals. Unfortunately, their study suffered from the limitation that individual follicles could not be followed over time until ovulation. The use of modern techniques such as ultrasound scanning has allowed to document an interesting effect of U L O in this study. In the light of my results, codominant follicles developed and either regressed or ovulated during each wave and this pattern was observed in six out of eight cycles following U L O (Figure 3.2). In all cows following U L O , an altered size distribution and pattern of follicular growth and development was observed. The largest ovulatory follicle in the retained ovary following U L O was smaller in diameter than its counterpart observed during the control cycle. The second largest follicle, however, was still smaller in size (Figure 3.2). This is the first observation of follicular dynamics measured ultrasonically after compensatory ovarian follicular growth and development induced by U L O . The morphological basis for this increased ovulation rate by the retained ovary is still not clear and is believed to be due to a decrease in the amount of follicular atresia (Hirshfield, 1982) and/or through an increase in the recruitment of developing follicles (Meijs-Roelofs et al., 1984). Actively growing follicles are dependant upon gonadotropin support for their growth and development. This study was based on the precept that U L O would lower the inhibitory effects of ovarian secretions on pituitary output of FSH to an extent that is sufficient for the recruitment, selection, growth and ovulation of more than one follicle. A transient increase in serum FSH following U L O may stimulate additional follicular growth and the concomitant ovarian hypertrophy (Butcher, 1977; Findlay and Gumming, 1977; Welschen et al., 1978; 52 Redmar et al., 1985; Johnson et al., 1985). In the study reported here, no significant differences (P > 0.05) were observed in serum FSH & L H levels before and after U L O . Since only one cow had a full control cycle the number of days available in the control cycle were not sufficient to make effective comparisons. As blood samples were not collected frequently this transient increase in FSH following U L O could have gone undetected. An increase in the plasma concentrations of P 4 has been reported following H C G induced ovulation of the first (d 7) or second (d 14) wave DF during the estrous cycle in cattle (Rajamahendran and Sianangama, 1992). However in the same study, equivalent elevations in plasma P 4 were not observed when twin corpora lutea resulted through administraton of H C G on the day of estrus (day- 0) (Rajamahendran and Sianangama, 1992). In the study reported here there was no significant effect of U L O on plasma concentrations of P 4 before and after U L O . Since in my study, the formation of twin corpora lutea were spontaneous, a lack of correlation observed between P 4 concentrations and twin corpora lutea is in agreement with findings of Rajamahendran and Sianangama. (1992). A higher metabolic clearance of the hormone could be a probable reason for the failure to observe increased concentrations following U L O (Rajamahendran et al., 1976 & 1982). To summarize, U L O caused no change in cycle length, follicular wave pattern, plasma gonadotropin (FSH & LH) and P 4 concentrations. The development and ovulation of codominant follicles following U L O in six of the eight cycles monitored was the most important finding in this study. However, it is not clear as to how long or for how many cycles will the twin follicular development and ovulation continue. Since the growth, development of codominant follicles followed by ovulation was recorded up to three cycles following U L O , a greater possibility for this pattern to continue in subsequent cycles is 53 envisioned. Hence, we conclude that U L O is a possible method to obtain twin ovulations in cattle. Cycling cows are good candidates for this procedure and it is highly necessary that the same procedure be applied to cattle of different age groups or reproductive stages such as pubertal heifers and postpartum cows and evaluate their response. Since U L O produced a modest increase in ovulation rate in the majority of the cycles monitored a new possible method for the induction of twinning in cattle apart from four existing methods became promising. Combining the above two conclusions it is necessary that the exact stage at which U L O has to be performed be determined in order for it be used as a method to induce twinning in cattle. The expected increases in plasma FSH concentrations were not observed after U L O in any of the cows. Therefore, we may have to perform a detailed study with a complete control cycle so that comparisons before and after performing U L O could be made with the same animal. 54 CHAPTER- 4 EFFECTS OF UNILATERAL OVARIECTOMY ON FOLLICULAR DEVELOPMENT, PLASMA GONADOTROPIN, PROGESTERONE PROFILES, OVULATION AND PREGNANCY RATES IN CYCLING HEIFERS 4.1. ABSTRACT The purpose of this study was to investigate the effects of U L O on follicular dynamics, plasma gonadotropins (FSH & LH), P 4 , ovulation and pregnancy rates in cycling heifers. Estrus was synchronized using two injections of P G F 2 a given 12 days apart. Following estrus and ovulation, ovarian follicular development in six heifers was monitored for one complete control cycle using transrectal ultrasonography. Daily blood samples were also taken for FSH, L H and P 4 determination. Following U L O and a brief period of rest, ovarian follicular development in the retained ovary was monitored for one complete cycle using transrectal ultrasonography. Again daily blood samples were taken for FSH, L H and P 4 determination. All animals were inseminated at standing estrus after completion of the second experimental cycle following U L O . Out of the six heifers, five had two and one had three waves of follicular growth during the control cycle and this pattern did not change after U L O . Mean cycle length (20 + 0.9 vs 21 ± 0.9) did not differ before or after U L O . No significant differences were observed in plasma FSH, L H and P 4 concentrations before and after U L O . Four out of six heifers ovulated twin follicles following U L O . The mean diameter of the largest follicle follicle was 14.5 ± 0.7 and the second largest was 12.1 ± 0.8 mm. Three of the six heifers were identified as carrying twin fetuses through transrectal ultrasonography 35 days post-breeding. Three of the heifers gave birth to single calves and the remaining three aborted. 55 4.2. INTRODUCTION In my previous study conducted to investigate the effects of U L O on follicular development and ovulation rate in dry cycling cows it was found that ovulation rate was increased following U L O . This increase in ovulation rate was observed for at least 2 cycles in all four cows following U L O . In two of the four cows following induced estrus using a single injection of P G F 2 a a similar increase in ovulation rate was observed following the second experimental cycle. Even though I hypothesized that plasma FSH concentrations would be elevated following U L O to an extent sufficient for the development and ovulation of more than one follicle, no significant increases were observed. One probable reason could be that not all cows had a complete control cycle as a result of which we were unable to make effective comparisons for the different ovarian parameters before and after U L O . Further, my primary objective of the study using dry cycling cows was to see whether or not probable alterations in ovarian activity in cattle occurred after U L O because subsequent investigations in this area depended on results that would be obtained from this study. From experiment I it was clear that parous cows were good candidates for this procedure and that ovulation rates could be increased following U L O . Based on the conducive results obtained in the previous study and to offset the deficiencies of my previous experiment it was decided to perform another similar but detailed experimentation with cattle from a lower age group ie., pubertal heifers. When dry cycling cows could respond with an increase in ovulation rate it was intriguing to understand how heifers would react to such a procedure. Therefore, the purpose of this study was to determine the effects of U L O in pubertal heifers on ovarian follicular dynamics, plasma concentrations of FSH, L H and P 4 , ovulation rates. Since it was evident from my previous study that twin ovulations occurred in the majority of the ovulatory cycles monitored after 56 U L O the possibility of obtaining twin pregnancies became promising. Hence, the current study incorporated determination of pregnancy rates as an additional objective. 4.3. MATERIALS AND METHODS 4.3.1. Animals and Ovarian examination Six cycling heifers were included. The animals were cared for as mentioned in chapter-1. The estrous cycles in all heifers were synchronized using two injections of P G F 2 a ' given 12 days apart. Following ovulation, ovarian follicular development in the heifers was monitored daily using transrectal ultrasonography for one complete cycle (control cycle) until subsequent ovulation. A laporotomy was performed under paravertebral anaesthesia and the left ovary in all heifers was removed using an ecraser. After U L O , all heifers were given a period of rest and their estrous cycles were synchronized using P G F 2 a injections. Following estrus and ovulation, ovarian follicular development in the retained ovary was monitored for one complete cycle. At the completion of the second experimental cycle all six heifers were inseminated and pregnancy rates were assessed 35 days post-breeding using transrectal ultrasonography. 4.3.2. Blood sampling and analysis for plasma FSH, LH and P4 Blood samples (7ml) were collected daily through a tail vessel puncture from all heifers before and after U L O just before each ultrasound examination for determining plasma FSH, L H and P 4 concentration. Plasma P 4 concentrations were measured using a commercially available solid-phase radioimmunoassay kit (Coat-A-Count, Diagnostic Products Corp., Los Angels, CA). This kit has been previously validated in our laboratory for the measurement of P 4 in cow's milk and plasma (Rajamahendran et al., 1989 ). Quantification of plasma FSH was 57 done by a double antibody radioimmunoassay validated by Rawlings et al. (1984). All samples were measured in a single assay. Plasma L H concentration was assayed by the method of Sanford (1987). Mean cycle length, plasma P 4 , FSH and L H data before and after ULO in both cows and heifers were compared using a paired sample-t test. 4.4. R E S U L T S 4.4.1. Ovarian follicular dynamics during the control cycle in heifers Mean cycle length of all heifers was 20.7+0.9 days before U L O . Out of the six heifers, five had two waves of follicular growth and one had three follicular waves. All six heifers had one preovulatory follicle that developed and ovulated at the end of the cycle (Table 4.1). A representative pattern of follicular development and P 4 profiles profile observed in cows and heifers prior to U L O is shown in Figure 4.1. O V U L A T I O N C L 2 4 6 8 10 12 14 16 18 20 22 Day of estrous cycle Figure 4.1. A representative pattern of follicular development and progesterone profile observed before unilateral ovariectomy. 58 4.4.2. Ovarian follicular dynamics following ULO A representative pattern of follicular growth and development observed in four of six cycles after U L O is shown in Figure 4.2. The number of ovulatory follicles increased in the interovulatory intervals observed after ULO. Following ULO in the heifers, no alterations were observed in the mean cycle length (21+0.9 d). Out of a total of six cycles (one cycle per animal) observed in the heifers (trial II) after U L O , twin ovulations were recorded in four cycles. The mean diameter of the largest preovulatory follicle was 14.5+0.7 mm and next to largest 12.1+0.8 mm. The follicular wave pattern essentially remained the same in all animals after U L O . Figure 4.2. A representative pattern of follicular development and progesterone profile observed during Trial II after unilateral ovariectomy. OVULATION CL 5 9 4.4.3. Plasma FSH and LH concentrations before and after ULO Unlike the previous study, all heifers had one complete cycle and it was possible to make comparisons for mean plasma FSH and L H concentrations before and after U L O . Plasma FSH and L H concentrations in all heifers before ULO were 0.16+0.02 and 0.11+0.03, respectively and did not differ significantly from values after U L O (0.21+0.03 and 0.15+0.04) (Table 4.1). 4.4.4. Plasma P4 concentrations before and after ULO The mean plasma P 4 concentration for all heifers before and after U L O was 3.6+0.26 and 3.8+0.29, respectively (Table 4.1). A slight increase in P 4 concentration was observed in the heifers after U L O on account of twin corpora lutea arising from twin ovulations that occurred after the rest period but was not significantly different from control levels. Heifers (n=6) Comparisons Before U L O After U L O Cycle length (days) 20 ± 0.9 21 ± 0 . 9 Follicular waves (No.) 2(n=5), 3 (n=l) 2 (n=5), 3 (n=l) Ovulatory follicles (No.) 1 (6/6) cycles 2 (4/6 cycles) P 4 concentrations (ng/ml) 3 . 6 ± 0 . 2 6 a 3 . 8 ± 0 . 2 9 a FSH concentrations (ng/ml) 0.16 + 0.023 0.21 ± 0 . 0 3 a L H concentrations (ng/ml) 0 . 1 1 ± 0 . 0 3 a 0.15 ± 0 . 0 4 a No of twin pregnancies - 3/6 Means in a row with the same superscript do not differ significantly (p > 0.05) Table 4.1. Number of ovulatory follicles, pregnancy rates, mean (±SE) cycle length, number of follicular waves, plasma gonadotropin (FSH & LH) and progesterone concentrations in six heifers before and after U L O . 60 Heifer #9417 30 T 0 I I I I I I I I I I I I I I I I I I I I I I I I I I I | | | | T - C O U > l ^ - O v - C O W h . O ? v - l O O } < O e O C N | Day of es t rous C y c l e Heifer #9418 0 I I I I I I I I I I I I I I I I I I I I I I l l l l l l | | | | | | T - r- T - CM Day of es t rous C y c l e Figure 4.3a. Sequential growth and regression of follicles and corpus luteum in heifer (#s 9417 & 9418) observed for one cycle before and after unilateral ovariectomy. The termination of the control cycle and removal of one ovary is indicated by O V U / U L O . The ovulatory follicles are indicated as O V U and corpus luteum as C L . 61 Heifer # 9449 Heifer # 9450 o I I I I I I I I I I I I I I I I I I I I I I I I I I | | | i i i | t - c o i n h « . 0 ) T - f o i o r > . o > T - T - i n o > f o o o c M T - T - T - T - T - C M T - T - C M Day of estrous Cycle Figure 4.3b. Sequential growth and regression of follicles and corpus luteum in heifer (# 9449 & 9450) observed for one cycle before and after unilateral ovariectomy. The termination of the control cycle and removal of one ovary is indicated by O V U / U L O . The ovulatory follicles are indicated as O V U and corpus luteum as C L . 62 Heifer #9501 (Y) 30 T OVU OVU Day of estrous Cycle Heifer #9501 (W) 30-r E 3 OVU/ULO OVU I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I at T -T- CM OO CM T - CM Day of estrous Cycle Figure 4.3c. Sequential growth and regression of follicles and corpus luteum in heifer {ft 9501 (White), 9501 (Yellow) observed for one cycle before and after unilateral ovariectomy. The termination of the control cycle and removal of one ovary is indicated by O V U / U L O . The ovulatory follicles are indicated as O V U and corpus luteum as C L . 63 4.5. D I S C U S S I O N This study identified cycling heifers as suitable responders to U L O . Similar to the results obtained in my previous study with dry cycling cows, four of the six heifers developed codominant follicles which either regressed or ovulated at the end of the second experimental cycle. No attempt was made during the experimentation to compare the number of small follicles (<5mm) recruited during the interovulatory intervals followed before and after U L O . As in the study performed with dry cycling cows my objectives were restricted to monitoring the number of ovulatory follicles and determining if any increases resulted in the concentrations of the most crucial follicular growth regulating peptide hormone, namely, FSH. An additional objective incorporated in this study was to examine if pregnancy rates could be increased following U L O . All six heifers had one full control cycle before they were unilaterally ovariectomized. As ultrasound examinations were carried out daily two of the heifers developed proctitis by d 16 of the control cycle as a result of repeated rectal tenesmus. Therefore, ultrasonographic examinations were suspended for the rest of the cycle excepting that daily blood samples were taken and the P 4 profiles levels served as an indicator of cycle length. However, the dominant ovulatory follicle was identifiable by d 16 and ovulation was confirmed by the presence of corpora lutea at the time of laporotomy. During the control cycle five of the heifers [#s 9417, 9418, 9449, 9450, 9501 (Yellow)] had two waves of follicular growth and the sixth heifer [9501 (White)] had three waves of follicular growth. Looking at Figure 4.3, it can be seen that the second wave DF had started to regress by d 16 and the drop in P 4 concentrations occurred by d 23 based on which 64 we assumed that this heifer had three waves of follicular growth. Further, the three wave pattern was again observed in this heifer after U L O which confirmed my assumption made during the control cycle. Heifer #9418 had a very short cycle of 8 days duration and ovulated the first wave DF (Figure 4.3). Thereafter, she had a complete cycle and the second wave DF was present on her left ovary which was removed at the end of the control cycle. Heifer # 9449 (Figure 4.3) had a shorter cycle length of about 17 days and the same duration was observed following U L O . At the completion of the control cycle it was decided to remove the left ovary in all six heifers for two reasons. First, it was more convenient and comfortable for the operator to scan the right ovary. Secondly, it has been well documented that the right ovary is more active than the left and that 60% of the ovulations occurred from the right ovary (Casida et al., 1935; Rajakoski et al., 1960). This experiment was initiated with the idea that removal of one ovary would create a conducive endocrine milieu that would favor an increase in follicular selection and ovulation. Since the right ovary is known to be more active than the left we assumed that retaining the right ovary and providing a favourable endocrine environment would be more likely to satisfy our objectives. Following U L O , an altered size distribution and pattern of follicular growth and development was observed in four of the six heifers, namely, 9417, 9418, 9449, and 9501 (yellow). The largest ovulatory follicle in the retained ovary following U L O was smaller in diameter than its counterpart observed during the control cycle. The second largest follicle, however, was still smaller in size (Figure 4.2). Unfortunately, heifer #s 9449 and 9450 did not ovulate soon after the rest period when their cycles were synchronized. This was evident from their low P 4 levels observed during the cycle after U L O . The results observed here are similar 6 5 to the results observed in the previous experiment. Again it is assumed that a subtle increase in plasma FSH though undetected in this study could have served as a follicular survival factor thereby decreasing follicular atresia (Hirshfield, 1982) and increasing the selection of developing follicles from each recruited pool during the cycle (Meijs-Roelofs, 1984). The mean plasma FSH and L H levels in all heifers were comparatively lower than the cows. However, there was a slight increase in FSH and L H levels after U L O , but this increase was not statistically significant. This suggests that a relatively small increase in circulating FSH concentration might be sufficient to increase ovulation rate by increasing the number of developing follicles exposed to their individual "threshold" concentration of FSH, thereby escaping atresia and allowing full maturation followed by ovulation (Ireland, 1987). As blood samples were not collected frequently this transient increase in FSH following U L O went undetected. Data on plasma concentrations of FSH presented in this study are from daily sampling and since more frequent sampling was not done, any transient increase in plasma FSH if it occurred went undetected. In the study reported here, some of the heifers had twin corpora lutea as a result of twin ovulations following induced estrus immediately after the rest period. However, mean plasma P 4 concentrations after U L O , even though slightly higher than the levels prior to U L O , were not significantly different. Since in our study, the formation of twin corpora lutea were spontaneous, a lack of correlation observed between P 4 concentrations and twin corpora lutea is in agreement with findings of Rajamahendran and Sianangama, (1992). A higher metabolic clearance of the hormone could be a probable reason for the failure to observe elevated concentrations following U L O . An increase in the plasma concentrations of P 4 has been reported following H C G induced ovulation of the first (d 7) or second (d 14) wave D F during 66 the estrous cycle in cattle. On the other hand, equivalent elevations in plasma P 4 profiles were not observed when twin corpora lutea resulted through administraton of H C G on day of estrus (day 0) (Rajamahendran and Sianangama, 1992). In order to fulfill an additional objective of possibly increasing pregnancy rates, all six heifers were bred following estrus induction using 2 injections of P G F 2 a and pregnancy diagnosis carried out echographically on d 35 postbreeeding identified three [Heifer #s 9417, 9418, 9501 (yellow)] of the six heifers as carrying twin fetuses. Heifer #9417 delivered a single bull calf, 9418 gave birth to a single heifer calf one month early and 9501 (yellow) expelled a dead heifer calf 2 months in advance. The remaining two heifers (9449 & 9450) aborted during the first trimester of pregnancy and no fetus was found. The death of the second fetus in all three heifers identified earlier as gestating twins is in agreement with the results obtained following the unilateral placement of twin embryos into the ipsilateral uterine horn of heifers through embryo transfer for the artificial induction of twin pregnancies. Heifers compared with cows generally have less room to accomodate twin fetuses and competition between embryos for space could have possibly led to the demise of one of the embryos. Further, the development of a septic reaction cannot be discounted. This leads us to conclude that adequate uterine space is an important requirement for the gestation and maintenance of twin pregnancies successfully to term. Such a requirement could be met by cows as the uterus would have undergone sufficient hypertrophy during previous pregnancies. The frequency of natural twinning is known to increase as parity increased (Hendy and Bowman, 1970, Cady and Van Vleck, 1978). Gordon et al. (1962) suggested that the ovulation rate increased with the age of the cow and that multiple pregnancies are maintained successfully to term as the age of the dam increases. 67 Bellows et al. (1974) suggested that cows are more likely to carry multiple pregnancies to term than heifers. Considering this fact the utilization of cows from large commercial farms may be justified as suitable candidates for U L O at the termination of their milking period. In Canada cows are culled when their reproductive capabilities are compromised, or as a result of untreatable mastitis, lameness or when their production goes down drastically. Hence if economically feasible a few cows after their fourth or fifth lactation may be selected as candidates for U L O . I assume that these cows would be able to satisfy the prerequisites necessary for successfully gestating twin fetuses till term in one uterine horn. Increasing productivity in cattle has been one of the major goals of livestock research. Reproductive potential is low in cattle as most of the bovine females release only one oocyte per estrous cycle and, thereby, produce one progeny each year. Ovulation of additional eggs at natural estrus in cattle would provide a simple method for increasing productivity. An increased incidence of twin or multiple births has been reported following the administration of exogenous gonadotropins-PMSG:(Turman et al., 1968) or FSH:(Echternkamp et al., 1992), through the introduction of a second embryo into the uterus of an already bred cow (Diskin et al., 1987) or through immunoneutralization against the follicular peptide, inhibin (Morris et al., 1993). Given the limited capacity of the bovine uterus a modest increase in ovulation rate (two) would be ideal. Absence of an effective methodology to regulate ovulation rate is the major limiting factor to achieving increased productivity. Since I observed twin follicular growth and ovulations in both experiments after U L O the possibility of U L O being used as a method to induce twinning in cattle became promising. A positive relationship has been shown to exist between twinning in cattle and increased concentrations of IGF-I in both serum and follicular fluid (Echternkamp et al., 1990b). In their study, cattle that were selected for twin 68 births had 47% higher concentration of IGF-I than those nonselected for twinning. From these findings a possible role for intraovarian peptides like IGF-I in regulating folliculogenesis following U L O in cattle cannot be ignored. The basis for expecting plasma IGF-I concentrations to increase in cattle following ULO is not clear. Even if twin pregnancies are not desired or if twin pregnancies do not result at least the availability of two eggs following twin ovulations would increase the chances of fertilization. Twinning has not been attractive to the dairy farmers due to higher incidences of freemartinism, calving difficulties, prolonged postpartum intervals and poor performance in subsequent lactations. The problem of freemartinism can be overcome as there is ample scope for the refinement of new technologies (Johnson et al., 1994) in semen separation procedures so that twin calves of the same sex can be obtained. A recent study from Europe (Penny et al., 1995) showed that the adverse effects of twinning on the dam can be prevented by adopting proper management measures such as early identification of twin bearing cows, their separate management during gestation and providing proper veterinary assistance at the time of parturition. To summarize, U L O caused no change in cycle length, follicular wave pattern, plasma gonadotropin (FSH & LH) and P 4 concentrations. Following U L O , codominant follicles developed and ovulated in four of the six heifers. Twin pregnancies resulted in 50% of the heifers. Hence, I conclude that ULO is a possible method to obtain twin ovulations in cattle. However, further enquiry using more frequent plasma sampling, will be needed to address the hypothesis that increasing concentrations of plasma FSH following U L O is responsible for the development and ovulation of twin follicles. Whether intraovarian peptides like IGF-I are involved in regulating folliculogenesis following U L O awaits determination. Detailed enquiries 69 using parous cows are necessary to ascertain if twin pregnancies would successfully result and be maintained till term. 70 C H A P T E R - 5 P L A S M A FSH, L H AND IGF-I C O N C E N T R A T I O N S B E F O R E A N D A F T E R U N I L A T E R A L O V A R I E C T O M Y IN HEIFERS 5.1. A B S T R A C T This study was designed to determine plasma FSH, L H and IGF-I concentrations before and after U L O in cattle. Six pubertal cyclic heifers were included in this study. The estrous cycles of all 6 heifers were synchronized using 2 injections of P G F 2 a given l i d apart. Blood samples were collected from the tail vein for 72 h at 4 h intervals beginning 24 h after the second injection of P G F 2 a to determine the concentrations of FSH and L H . In addition, more frequent blood samples were taken during d 11 of the estrous cycle (4 heifers) to determine both L H and FSH pulse frequencies. Blood samples were also taken daily for one complete cycle to determine plasma IGF-I concentrations. U L O was performed 1 day after the subsequent ovulation and all animals were rested for 2 weeks. After the rest period the estrous cycles were synchronized and blood sampling was performed as per the control cycle for FSH, L H and IGF-I concentrations. Heightened FSH and L H surges were observed in 2 out of 4 heifers during the early follicular phase after U L O . However, no significant differences (p > 0.05) were observed in mean FSH (13.56 ± 0.36 ng/ml vs 13.00 ± 0.45 ng/ml) and L H (2.52 ± 0 . 4 8 ng/ml vs 3.51 ± 1.1 ng/ml) concentrations before and after U L O . FSH and L H pulse frequencies during the mid-luteal phase were not altered following U L O , [FSH: (2.0 ± 0 . 4 1 ) vs (1.75 ± 0.48) pulses/6 h] [LH: (2.75 ± 0.25) vs (3.25 ± 0.63) pulses/6 h]. Plasma IGF-I averaged (Mean ± S.E) 174.99 ± 28.08 and 187.91 ± 28.22 ng/ml before and after U L O , respectively. We conclude that no significant increases in mean plasma concentrations of FSH, L H and IGF-I in subsequent cycles were detected following U L O and that some other subtle 71 interactive mechanism among gonadotropins and growth factors may be involved in the selection, development and ovulation of co-dominant follicles. 5.2. INTRODUCTION In experiments I and II involving dry cycling cows and post pubertal heifers, U L O increased the number of ovulatory follicles at the subsequent estrus, caused no significant increases in plasma FSH, L H and P 4 profiles concentrations following U L O . Increased serum FSH concentrations immediately following ULO has been observed in the hamster (Bast et al., 1970), rat (Welschen et al., 1978), rabbit (Fleming et al., 1984), gilt (Redmar et al., 1985), cow (Johnson et al., 1985) and ewe (Findlay and Cumming, 1977). This increase in FSH has been attributed to be the cause for the compensatory follicular development by the retained ovary following U L O , thereby maintaining the number of ovulations characteristic of the species. Gonadotropins in comparison to other pituitary hormones are secreted in small doses and not at a constant rate and therefore, frequent blood sampling is necessary to demonstrate changes in the patterns of secretion of this hormone. Failure to observe an increase in plasma FSH concentrations in our earlier study could be due to infrequent blood sampling. Even though a significant increase in serum FSH has been observed immediately following U L O in cattle (Johnson et al., 1985; Lussier et al., 1994), to our knowledge there are no studies that have tested the significance of this increase in terms of its effect on follicular development in the subsequent cycles. Again it is not known whether this increase is restricted to the immediate period after U L O or if it continues into the ensuing cycles. In the rat (Butcher, 1977) and hamster (Bast et al., 1970), the second FSH surge that is normally restricted to the estrus is extended into the second day of the cycle and appears to be the signal that recruits 72 additional follicles into the ovulatory range. Substantial evidence is available to show the key role played by gonadotropins in controlling folliculogenesis in cattle (Quirk and Fortune, 1986). However, follicular development is also controlled by a myriad of other factors (steroids and growth factors) of endocrine and paracrine origin. As the importance of putative intraovarian regulators has become widely recognized, a greater part of the attention has been directed on insulin-like growth factors (IGFs). Currently, there is enough evidence suggesting the existence of an intraovarian IGF system inclusive of ligands, receptors and binding proteins (Monget and Monniaux, 1995). Therefore, this study was undertaken with the objective of unravelling the endocrine changes especially, those of plasma gonadotropins and IGF-I during the estrous cycle before and after U L O in cattle. 5.3. MATERIALS AND METHODS 5.3.1. Animals and Blood sampling before unilateral ovariectomy Six cyclic pubertal heifers were included in this study. Experimental heifers from the Agriculture Canada Research Station, Agassiz, BC were housed at the University of British Columbia Dairy Teaching and Research Unit and cared for according to the guidelines of the Canadian Council of Animal Care. The heifers were fed a standard ration of alfalfa cubes approximately 16% crude protein and timothy hay (13%). The estrous cycles of all 6 heifers were synchronized using 2 injections of P G F 2 a (Lutalyse; Upjohn, Kalamazoo, MI, USA) given 11 days apart. The blood sampling was begun 24 h after the second injection of P G F 2 a to characterise the preovulatory FSH/LH surge. Blood samples were collected at 4 h intervals beginning 24 h after the second injection of P G F 2 a as follows: 1600, 2000, 2400, 0400, 0800, 1200 h for a period of 72 h. Each sample was collected through a tail vessel puncture using a vacutainer containing heparin as an anticoagulant. The samples were then centrifuged and 73 plasma was separated and stored at - 20° C until assayed for plasma FSH and L H . Ultrasound examinations were performed to confirm ovulation. Two heifers failed to ovulate and therefore were eliminated from the study. Ovulated heifers (four) were also fitted with an indwelling catheter for more frequent blood sampling on day 11 of the estrous cycle. Silastic tubing was used as the catheter, and about 50 cm was threaded through a 13-g trocar and into the jugular vein. The trocar was removed and the external end of the catheter was fitted with an 18-g blunt end needle, flushed with saline containing heparin (60 units/ ml) to maintain patency and capped. The external length of the catheter (about 115 cm) was lightly coiled and placed in a cloth pouch taped to the neck of the animal. Blood samples were collected at 20 min intervals. Each sample was collected by withdrawing an initial 4 ml of blood, which was discarded, and withdrawing a separate 6 ml sample for transfer to a tube containing heparin. Blood samples were centrifuged immediately and plasma separated and stored at - 2 0 ° C until assayed for plasma FSH and L H . In addition daily blood samples were also taken through a tail vessel puncture for the determination of plasma IGF-1 concentrations. 5.3.2. Animals and Blood sampling after unilateral ovariectomy U L O was performed one day after ovulation through laparotomy and the right ovary was removed in all 4 animals. Following laparotomy all 4 heifers were given a brief period of rest following which their estrous cycles were synchronized and the blood sampling protocol as mentioned earlier was repeated. 74 5.3.3. Hormone Assays Both plasma FSH and L H concentrations were assayed in the laboratory of Dr. Joanne Fortune (Cornell University). Plasma concentrations of L H were measured by RIA using anti-ovine L H antiserum, highly purified ovine L H for radioiodination and bovine L H as standard. The second antibody was goat anti-rabbit immunoglobulin (Quirk and Fortune, 1986). Plasma concentrations of FSH were measured by RIA (Quirk and Fortune, 1986) using reagents obtained from USD A Animal Hormone Program. Reagents included specific antiserum directed against the (3-subunit of highly purified bovine FSH (USDA-FSH-B1, with biological potency 1.7 x NIH-FSH-B1). The second antibody was the same as that used in the RIA for plasma L H . The intra and inter assay coefficient of variation was 7.6% and 8.4%, respectively. L H pulse detection was done manually from individual L H profiles, based on at least 2 of the following criteria of Cook et al. (Cook et al., 1991): 1) an L H pulse peak was one having an increase equal or greater than one standard deviation from the mean or previous reading 2) a peak reading one standard deviation from the mean or subsequent reading and 3) two consecutive decreasing readings after the peak. Frequency of L H pulses was calculated as the mean number of pulses detected in all serial blood samples from all 4 heifers before and after U L O . The intra and inter assay coefficient of variation was 9.3% and 12.8%, respectively. Plasma IGF-I was assayed using a immunoradiometric assay kit supplied by Diagnostic systems laboratory, Texas. Briefly, 50 pi of the plasma was taken in a microcentrifuge tube to which 200 pi of the extraction reagent was added. The tubes were vortexed and incubated for 30 min and centrifuged for 3 minutes at 12,000 rpm. Following centrifugation 100 pi of the 75 supernatant was withdrawn into a fresh microcentrifuge tube. To this tube 500 pi of the neutralising solution was added and the tubes were stored at - 2 0 ° C until assayed for plasma IGF-I. For the Immunoradiometric assay 50 pi of the neutralised sample was drawn into antibody coated tubes. This was followed by addition of 1 2 5 I- IGF-I (tracer). The tubes were incubated for three hours on a shaker. The tubes were then decanted and washed thrice with 3 ml of deionized water and counted on a gamma counter for 300 seconds. All samples were run in one assay. The intra assay coefficient of variation was 1.5%. 5.3.4. Statistical Analysis A 3-way analysis of variance for sequential data was performed using MINITAB statistical software to determine the effect of treatment, time and animal on mean changes in circulating concentrations of L H and FSH during the early follicular phase before and after U L O . Mean number of L H and FSH pulses, for samples collected during the midluteal phase on day 11 of the estrous cycle and mean IGF-1 concentrations before and after U L O were compared using a paired sample t-test. 5.4. RESULTS 5.4.1. Plasma concentrations of FSH during the early follicular and mid-luteal phases before and after ULO Two of the four heifers (# 9552, 9555) had a two fold increase in the magnitude of FSH surge following U L O (Figure 5.1). A surge in FSH was not observed in one heifer (#9553) after U L O . Similarly a surge was undetected in another heifer (#9554) before U L O . Time of blood sampling did not have a significant effect on mean changes in FSH and L H 76 before and after U L O . Mean plasma concentrations of FSH during the early follicular phase of the control cycle was about 13.56 ± 0.36 ng/ml and did not differ significantly (p > 0.05) from the levels after U L O (13.00 ± 0.45 ng/ml) (Table 5.1). No significant differences (p > 0.05) were observed in the FSH pulse frequency during the mid-luteal phase (Table 1) before and after U L O (2.0 + 0.41 vs 1.75 ± 0.48 pulses/6 h) (Figure 5.2). 5.4.2. Plasma concentrations ofLH during the early follicular and mid luteal phases before and after ULO A two fold increase in the height of the L H peak was observed following U L O in two heifers (9552, 9555) (Figure 5.3). Time of blood sampling did not have a significant effect on mean changes in FSH and L H before and after U L O . Mean plasma concentrations of L H during the early follicular phase of the control cycle was about 2.52 ± 0.48 and was not statistically different (p > 0.05) from the cycle followed after U L O (3.51 ± l.lng/ml) (Table 5.1). A surge in L H was not observed in heifer #9554 before U L O and in heifer #9553 after U L O . An overall 39% increase in L H concentrations was observed during the early follicular phase following U L O . Similarly, L H pulse frequency during the mid-luteal phase (Figure 5.4) of the control cycle (2.75 ± 0.25 pulses/6 h) did not differ from the cycle after U L O (3.25 ± 0.63 pulses/6 h) (Table 5.1) . 5.4.3. Plasma IGF-I concentrations before and after ULO There was no significant effect of U L O (p>0.05) on plasma concentrations of IGF-I (Figure 5.5). Plasma IGF-I averaged (Mean ± S.E) 174.99 ± 28.08 and 187.91 ± 28.22 before and after U L O , respectively (Table 5.1). 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I P po 1-1 Ct> M P -P B 0 0 g CD ft" ^  2 o* CD O o o rT o o S3 cr a CD < &5 CD o o o CD* o I — • o" CJ K> O B CD r-—• B CD 1-1 < 1:00. 8:00 , 16:00 . 24:00 32:00 . 40:00 48:00 56.00 64:00 72:00. 10:50 11:30 12:10 12:50 1:30 2:10 2:50 3:30 4:10 4:50 11:00 . . 11:40 . . 12:20 X 1:00 . 1:40 . . 2:20 . . 3:00 X 3:40 . 4:20 J . 5:00 I H 1 1 1- H—I—I—I—I—I—t-Heifers (n=4) Comparisions Before ULO After ULO Mean FSH concentrations (ng/ml) (Early follicular phase) 13.56 ±0.36a 13 ±0.45a Mean LH concentrations (ng/ml) (Early follicular phase) 2.52 ± 0.48a 3.51 ± 1.1* Mean FSH pulse frequency (Mid-luteal phase) (pulses/6 hr) 2.0 ± 0.41a 1.75 ±0.48a Mean LH pulse frequency (Mid-luteal phase) (pulses/6 hr) 2.75 ± 0.25a 3.25 ±0.63a Mean IGF-I concentrations (ng/ml) 174.99 +28.08a 187.91 ±28.22a Means in a row with the same superscript do not differ significantly (p>0.05) Table 5.1. Mean (± SE) FSH and LH concentrations and pulse frequencies during the early follicular phase and mid-luteal phase, respectively before and after ulo. Also shown in the table are mean IGF-I concentrations during the last 9 days of the cycle and P4 concentrations during the entire cycle before and after ULO. 5.5. DISCUSSION Investigations into the effects of ULO on follicular development in experiments I and II revealed increases in ovulaton rate. What hormonal changes triggered these altered patterns of follicular development were not clear from our previous study (Mohan and Rajamahendran, 1998). Both FSH and LH are essential for growth and maturation of ovarian follicles (Richards, 1980). In cattle and sheep final growth and maturation of preovulatory follicles occur during the follicular phase extending a few days post luteolysis (Hansel and Convey, 1983). After luteolysis basal plasma concentrations of LH and frequency of pulsatile LH release increases (Rahe et al., 1980). Increased stimulation by LH is thought to trigger preovulatory follicular development (McNatty et al., 1981). It has also been shown that FSH 84 may be critical to support the growth and development of the preovulatory follicle after luteolysis in cows (Quirk and Fortune, 1986). Even though we observed an altered pattern of follicular development following U L O in our early studies (Mohan and Rajamahendran, 1998) it was not clear whether increased concentrations of gonadotropin continued into subsequent cycles to promote twin follicular development and ovulation following U L O . A transient increase in plasma FSH has been reported in the cow 24 h after U L O (Johnson et al., 1985, Lussier et al., 1994, Badinga et al., 1992). In the study reported here, in two of the four heifers (#9552, 9555) the peak of the FSH surge observed following U L O was greater than that observed before U L O (Figure 5.1 (a, d)). Reduction or elimination of the ovarian inhibitory secretions, especially those of inhibin could be the possible reason for observing an increase in the height of the FSH surge in ovariectomized animals. Injections of BFF during the follicular phase of the estrous cycle in heifers and ewes lengthened the interval from luteolysis to estrus (Miller et al., 1979). Treatment of heifers with injections of BFF delayed the normal preovulatory increase in plasma concentrations of E2 and the onset of estrus indicating that both development and function of preovulatory follicles was inhibited during treatment (Quirk and Fortune, 1986). These studies lended support to the fact that FSH is most critical for follicular development and that inhibin present in the follicular fluid was capable of inhibiting FSH secretion from the pituitary. Increase in the magnitude of the FSH surge suggests that concentrations of FSH early in the estrous cycle might be related to later growth of follicles and possibly could be a reason for the development and ovulation of co-dominant follicles following U L O in cattle. The magnitude of the L H peak after U L O was greater in two heifers (#9552,9555) than that occurred prior to U L O (Figure 5.3 (a, d)). Enhanced positive feedback effects exerted on the pituitary and hypothalamus by increased 85 secretion of E 2 by the developing co-dominant follicles could account for the increased peak of the L H surge. A surge in both FSH and L H was not detected following U L O in heifer #9553 (Figure 5.1 (b)). Similarly, a gonadotropin surge was not observed in heifer #9554 before U L O (Figure 5.3 (c)). However, ovulation was confirmed in these two heifers using ultrasound scanning. Stress is a likely factor that could be implicated for the failure to observe a surge as stress has been shown to prolong the L H and FSH surges in cattle. In cattle, infusion of Cortisol for 90 h during the follicular phase prevented the L H surge in 75% of treated heifers (Stoebel and Moberg, 1982). It is possible that stressful factors such as presence of the person taking blood samples, repeatedly bringing animals into the holding area for blood sampling could have prolonged the LH/FSH surge and could have occurred after 72 h of blood sampling. Two heifers were eliminated from this study as they became cystic. Stress could again be the likely reason as administration of A C T H during the follicular phase of the estrous cycle results in the formation of cystic follicles and emergence of other follicles is suppressed for upto 10 to 15 days (Dobson and Smith, 1995). An increased height observed in the FSH and L H surge following U L O in two of the four heifers suggests that this is probably the instigating factor for the final development to ovulatory size and ovulation of co-dominant follicles. However, FSH and L H concentrations averaged over all four heifers during the early follicular phase before were no different from the levels recorded after U L O . However, a 39% increase in mean L H concentrations was observed during the early follicular phase following U L O (Table 5.1). Similarly, elevated L H concentrations has been reported in prolific sheep in comparison to breeds with lower ovulation rates (Land et al., 1973; McNatty et al., 1987). Another complicating feature in this study is that FSH immunoreactivity was monitored. Since the radioimmunoassay reflects 86 immunoreactive concentrations of FSH, it cannot be determined if the residual circulating concentrations of FSH after U L O has an altered bioactivity (Wilson et al., 1990; Robertson et al., 1991). Further, the plasma clearance of FSH obtained from ovariectomized sheep is delayed compared to pituitary FSH from gonadal-intact sheep (Robertson et al., 1991). Removal of gonadal steroids from circulation following castration is thought to activate pituitary enzymes important for the sialylation of the FSH molecule (Chappel et al., 1983). Greater amounts of sialic acid are found in the pituitary gland following castration and it is suggested that this modified FSH has a longer half-life in serum and therefore increases its biological effectiveness in vivo (Chappel et al., 1983). It remains unknown as to what changes would hemicastration bring about to the FSH molecule. During the mid-luteal period, under high P 4 environment the pulses have a high amplitude and low frequency (Rahe et al., 1980). In the study reported here the pattern of pulsatile L H secretion (Figure 5.4) before U L O was no different from that observed after U L O . Same was the case with FSH (Figure 5.2). Mean L H and FSH pulse frequencies during the mid-luteal phases were no different from those observed after U L O (Table 5.1). A surge in FSH is known to precede the emergence of a follicular wave in cattle (Adams et al., 1992). Since blood samples in our study were taken only on one occasion during the mid-luteal phase, the impact of U L O on the FSH surges that normally precede the emergence of each follicular wave remains undetermined. Recently, much attention has been diverted to the effects of growth factors on follicular development, the most important being IGF-I. From numerous in vitro studies it is clear that IGF-I is an important mediator of follicular development (Adashi et al., 1985; Spicer et al., 1995). In addition, in vivo studies have shown systemic and ovarian IGF-I concentrations to 87 be inherently high and responsible for multiple ovulations in cattle selected for twins (Echternkamp et al., 1990) supporting the idea that plasma levels of IGF-I may regulate follicular growth and differentiation in cattle. There is evidence to show that administration of exogenous E2 increases hepatic mRNA in rats (Norstedt et al., 1989) and concentrations of IGF-I in blood of animals on an adequate diet (Bass et al., ; Richards et al., 1991). On the other hand, ovariectomy has been shown to decrease serum concentrations of IGF-I suggesting that the ovaries produce a substance, probably E 2 that enhances secretion of IGF-I by the liver. Since we observed twin follicular growth and development in cattle following U L O (Mohan and Rajamahendran, 1998) we assumed that the E2 produced by twin follicles might be sufficient to stimulate the liver to increase IGF-I secretion. In the present study, a 7.4% increase in plasma IGF-I concentrations following U L O , though insignificant, could have enhanced the stimulatory effects of FSH within the ovaries resulting in the selection of two instead of the usual one ovulatory follicle(s). The E D 5 0 for the effects of IGF-I on mitogenesis indicates IGF-I as a highly potent mitogen (Spicer and Echternkamp, 1995) which extends further support to the conclusion made above. A recent study (Gutirrez et al., 1997) reported enhanced estradiol production and proliferation of bovine granulosa cells in vitro following the addition of IGF-I at concentrations as low as 1 ng/ml. In addition, these stimulatory effects were observed even in the absence of gonadotropic support highlighting the gonadotropic action and importance of this peptide as an intraovarian regulator of follicular function. Determining the concentrations of IGF-I and IGFBPs in the follicular fluid of twin follicles could lend more support to the above conclusion. Moreover in this study, IGF-I concentrations were measured only from daily blood samples and we assume that more information could have been available if concentrations were determined during the early follicular phase. 88 A large body of evidence now sugggests the involvement of a neuroendocrine mechanism wherein the ovarian nerves are thought to play a modulatory role on the response of the remaining ovary to gonadotropins thereby, regulating compensatory contralateral ovarian hypertrophy (Gerendai et al., 1978; Flores et al., 1990; Chavez and Dominguez, 1994). Treatment of hemiovariectomized rats with an ovarian autograft using guanethidine, a drug that inhibits the release of norepinephrine from the nerve endings blocked compensatory ovulation but not compensatory ovarian hypertrophy (Ayala and Dominguez, 1988). These results indicate the likely involvement of adrenergic neural elements of the ovary in the development of compensatory ovulation and ovarian hypertrophy by the remaining ovary following U L O . In conclusion, heightened preovulatory gonadotropin (FSH & LH) surges were distinct in two out of four heifers following ULO. However, no significant elevations in mean plasma concentrations of FSH and L H during the pre-ovulatory period were observed in the current study. Similarly, no significant increases were observed in plasma IGF-I concentrations following U L O . One possible explanation for the development of co-dominant follicles could be that during the period after ULO subtle increases in plasma FSH though, undetected in the current study could have favoured the selection of co-dominant follicles. Augmentation of the role of these subtle increases in FSH on follicular selection following U L O could have been provided by the the minute increases observed in the plasma IGF-I concentrations. There are suggestions in the literature on the participation of ovarian adrenergic nerves in compensatory contralateral ovarian development and this area needs further investigation before we conclude its involvement in regulating folliculogenesis after U L O in cattle. 89 CHAPTER 6 GENERAL DISCUSSION The dynamics of follicular growth during the estrous cycle is interesting and it is still not clear why one follicle becomes dominant in spite of the fact that there were another 4-8 similar sized follicles in the cohort at the time of wave emergence. During the estrous cycle the emergence of each follicular wave is preceeded by a surge in FSH. It is assumed that the FSH surge that occurs before each wave nurtures about 2-3 follicles from the cohort which attain finally a diameter of about 7-9 mm and are visible on ultrasound by 2-4 days postovulation. During this period of growth, these 7-9 mm follicles actively produce and secrete another peptide hormone called inhibin which negatively feeds back on to the pituitary to inhibit FSH release thereby maintaining basal concentrations of FSH. Amidst such a harsh endocrine environment all other follicles from the cohort start regressing due to the unavailability of FSH in adequate concentrations. In the meantime, surprisingly, one follicle gets selected, because it now, through a still unresolved process has gained L H receptors and is thereafter completely L H dependant for further growth to ovulatory size. The selected follicle becomes dominant and actively secretes E2 and inhibin which feeds back on the pituitary to further inhibit the secretion of FSH as a result of which the subordinate follicles now start regressing. When the subordinate follicles are regressing the granulosa and thecal cells of the selected follicle secretes certain other local hormones collectively called peptide growth factors. Noteworthy among these is IGF-1 produced locally by the granulosa cells. IGF-1 potentiates the actions of FSH, as a result, the selected follicle even though now predominantly L H dependant is still able to thrive in a reduced FSH environment. This is one of the proposed and widely accepted theories for the selection of a DF. 90 What is clear from the above discussion is that FSH is essential for follicular growth in all mammalian species and is crucial for it regulates follicular growth in cattle. The endogenous levels of this hormone released from the pituitary is so critically regulated at the ovarian level that optimum concentration for growth and development is available only to one follicle which ultimately becomes the dominant ovulatory follicle. Support to the above conclusion comes from the fact that FSH from an exogenous source if provided in divided doses over a period of three to four days in synchrony with the time of wave emergence results in the development of multiple follicles finally attaining ovulatory size. Therefore, the limited bio-availability of FSH and consequently the development and ovulation of a single follicle in majority of the estrous cycles in cattle is the reason why this species has a low reproductive potential. So unless reproductive potential of cattle is enhanced the likelihood of increasing animal productivity is remote. Instead of providing FSH from an exogenous source to induce multiple follicular development, another approach would be to increase endogenous levels of FSH through manipulation of the gonads. Removal of both ovaries completely eliminates feedback effects of ovarian secretions on the pituitary resulting in an uninterrupted increase in the output of both FSH and L H from the pituitary. Accordingly, I hypothesized that U L O would reduce the feedback effects of gonadal secretions to an extent that would result in a concomitant increase in the ouput of FSH sufficient for the development and ovulation of more than one follicle. Unilateral ovariectomy in cattle has resulted in a compensatory increase in the size of the ovary (Saiduddin et al., 1970; England et al., 1973; Grass and Hauser, 1981), an increase in total follicular surface area in the retained ovary (Saiduddin et al., 1970), and an increase in the number of follicles >4mm, with a concomitant elevation in serum FSH concentrations 91 (Johnson et al., 1985). The potential for twin ovulations following U L O in cattle was proposed by Saiduddin et al. (1970). A combination of U L O and exogenous gonadotropin treatment resulted in an increase in follicular size (Moser et al., 1989). Even though transient increases in FSH concentrations have been demonstrated following U L O in cattle it is not known if this increase would continue to occur in subsequent cycles. Further, there is no information on spontaneous ovulation rates in cattle following U L O which is available for most of the laboratory and livestock species. Alterations in follicular populations have been reported in some of these earlier studies. However, these studies were performed even before the wave pattern of follicular growth was elucidiated. Rocha et al. (1996) recently reported a preponderance of large follicles (>10 mm) following U L O in combination with inhibin immunization in cattle. Therefore, it is clear that the dynamics of follicular growth following U L O is incomplete and undefined. Today with the availability of non-invasive monitoring devices and abundance of information on the patterns of follicular growth in cattle of varied reproductive states it is necessary to investigate whether these patterns would change or remain the same following unilateral reomoval of the ovary and fill in the information gap. This is the first study to report the tracking of follicular dynamics in cattle ultrasonically following induction of compensatory follicular growth after U L O . Results of Expt-I showed that ovulation rates could be increased by U L O . The results I observed in cattle are in agreement with those reported for other polytoccus laboratory and livestock species. The results I observed in cattle were different in certain aspects. In polytoccus species following U L O , the retained ovary compensated by increasing the ovulations thereby, maintaining the litter size. On the contrary, U L O increased the ovulation rate in cattle and 92 increases were observed in six out of a total of eight cycles. In Expt-II comprising six pubertal heifers four of the six cycles monitored had twin ovulations. Carefully analysing the results of these two experiments it not clear as to what caused twin follicles to develop and ovulate. Concentrations of both gonadotropic hormones (FSH & L H ) before U L O were no different from concentrations after U L O in both experiments. However, owing to the fact that both F S H and L H are released in a pulsatile fashion and due to our infrequent blood sampling, no clear conclusion was reached. In spite of an insignificant increase in plasma F S H concentrations following U L O we were able to sonographically track the emergence of codominant ovulatory follicles. Interestingly, there was an altered size distribution between the co-dominant follicles which was a major deviation from what is observed in a normal fertile cow with both ovaries intact. The presumptive largest ovulatory follicle that developed following U L O was smaller in diameter than the ovulatory follicle usually observed in an intact animal and the second ovulatory follicle following U L O was larger in diameter than the subordinate follicles that are normally seen in intact animals. One likely reason for this could be a lack of L H availability in sufficient concentrations as L H is the predominant gonadotropic hormone that stimulates final follicular growth to preovulatory size. Moreover, insertion of a N implant subcutaneously prevents ovulation of the follicle and instead stimulates the growth of the follicle to diameters beyond normality and also depends on the duration of the period the implant is in place. N permits high frequency L H pulses and as a result there is constant L H stimulation on the thecal cells resulting in the production of more androgens. Thecal androgens serve as substrates for the granulosa cells so that they can be aromatised to estrogens and as a result, this follicle remains dominant both morphologically and functionally. Recently, it was shown that continuous infusion of G n R H into heifers for 93 prolonged periods completely inhibited L H but not F S H release and as a result follicles failed to grow beyond 7-9 mm. These authors concluded that antral follicles in the diameter ranges of 3-4 m m require F S H for growth until they attain a size of about 7-9 m m and beyond which they are dependant on L H for subsequent growth to ovulatory size. Collectively, it can be concluded that L H is also a limiting factor to follicular growth and could be the reason why co-dominant follicles with altered size distribution resulted in this study. Numerous studies in the past have focussed on one main objective of increasing reproductive potential or in other words increasing ovulation rate. Initial studies began with injecting F S H either of bovine, porcine or ovine origin or P M S G to induce multiple follicle development and ovulation. As soon as the role of inhibin was realized attempts were made to immunoneutralize its effect. Immunoneutralization resulted in a corresponding increase in plasma F S H concentrations and concomitant increase in follicular development and ovulation rate. The phrase "increase in ovulation rate" has literally no limits and can have any numerical ranges. Unfortunately, uterine space provided by the bovine female is sufficient to comfortably accomodate and gestate two fetuses successfully to term. Therefore, it is highly necessary to devise a method that is repeatable and would increase the ovulation rate to a maximum of two. Embryo transfer is the only available procedure to ensure that the dam only bears two and not more than two fetuses. Embyro transfer has a few disadvantages as it requires plentiful supply of embryos, is labour intensive and is therefore an expensive procedure on the whole. Results on follicular dynamics in Experiments I and II prompted us to use U L O as a method to induce twinning in cattle, mainly for three reasons. First this method caused a modest increase in ovulation rate. Secondly, increases in ovulation rate were observed in subsequent cycles (3 cycles after U L O ) . Thirdly, it is a simple procedure and is therefore, not 94 expensive and is practically feasible for free-range system of farming. In Experiment II all six heifers were inseminated at the end of the second experimental cycle and three of them were diagnosed as carrying twin fetuses approximately day 35 post-breeding. Unfortunately, the twin pregnancies were not succesfully carried to term. In cattle transuterine migration of embryos seldom occurs in comparison to the pig and sheep. When twin ovulations occur from the same ovary both fetuses would develop in the same horn. Since these animals were heifers uterine space offered by a single horn for twin gestations is limited. I assume that the induction of twin pregnancies and subsequent twin calvings in experiment II would have improved if we had used parous cows. The uteri of cows of greater parity on account of experiencing a few pregnancies would have undergone sufficient hypertrophy and should be large enough to accomodate twin fetuses in one horn. In addition, ovulation rate has been shown to increase as parity increases so that the chances of twin ovulations occurring in parous cows following U L O in future cycles are bright. As I did not see an increase in plasma FSH and L H following U L O in Experiments-I and II, more frequent blood samplings were conducted during the early follicular phase and mid-luteal phase of the estrous cycle in Experiment-Ill. The magnitude of the gonadotropin surges (both FSH and LH) was two fold greater in two of the four heifers sampled indicating that subtle changes in gonadotropin secretion patterns could alter follicular selection and ovulation following U L O without any elevations in mean FSH and L H concentrations. This is in contradiction with previous studies in other species which demonstrated elevated FSH concentrations extending into subsequent cycles. In some of the heifers either the L H or FSH surges went undetected. Stress could be one main reason. A recent review on "Stress and Reproduction" (Dobson and Smith, 1995) recommends that the animals be restrained in a 95 holding area for at least 45 minutes before collecting frequent blood samples as elevated plasma Cortisol levels during stress return to baseline levels after about 45 minutes. Similarly, no significant increases in IGF-I concentrations were detected following U L O . IGF-I profiles following U L O have received no attention in any species studied earlier. Twin calvings did not result in this study. Before I recommend additional studies on utilising parous cows to fulfill this objective it is necessary to address a few questions on animal ethics. Even though the results reported in this study on follicular dynamics demonstrates interesting scientific facts, such a method being invasive cannot be justified ethically as a technique in countries like the UK for use in animal production. Further, induction of twinning itself is considered stressful to the dam as it drains a lot of body reserves in order to nourish the twin fetuses. Fortunately, these adverse effects on the dam can be overcome to a great extent if these cows are identified early in pregnancy and managed separately. Freemartins can be avoided as soon as sexed semen becomes available. What do we do with the twin calves ? Additional income can be obtained through sales or they can be used as replacers. If it is not economically feasible to maintain these heifer calves they can be used for beef purposes as is the case in most European countries. By releasing two eggs during each cycle we are also increasing the chances of fertilization . The observation that ULO resulted in the development of twin follicles may have implications in studying follicular dominance. Recently (Rocha et al., 1996) it was shown that a combination of U L O and inhibin immunization resulted in the growth of larger diameter follicles (> 10 mm). Unfortunately, that study did not provide information on ovulation rate. Inhibin is well known for exerting a preferential inhibitory effect on FSH. It is interesting to readdress the question of whether inhibin immunization in combination with U L O is a superior 96 method to increase FSH concentrations compared to U L O alone. Cystic ovarian degeneration is an important condition causing infertility and serious economic losses to the cattle industry. The pathology seems to lie in the hypothalamo-pituitary regions. Affected animals have high concentrations of L H but a surge in L H is absent and accordingly treatment regimens were designed to provide L H exogenously in the form of H C G or indirectly stimulate endogenous L H release by way of GnRH injections. In recent years it has been observed that some of these animals remain refractory to treatment and the cysts continue to enlarge in size and remain anovulatory. A few cows identified as cystic at the Agriculture Canada's Research station at Agassiz were treated by removing the ovary bearing the anovulatory cystic follicle. All these cows resumed normal cyclicity, conceived and gave birth to healthy calves. This research has further created a few questions that could be addresed in future. Did concentrations of L H influence the altered size distribution observed between co-dominant follicles. Whether FSH or L H is a limiting factor in attaining final ovulatory size is still a major question. N implants can increase L H pulse frequency. Will increased pulse frequency following N implantation result in normal sized co-dominant ovulatory follicles following U L O contrary to what we observed in this study. How can nutrition influence folliculogenesis following ULO? What effects would improved nutrition have on serum IGF-I concentrations would be interesting to know. From this thesis research I conclude that ovulation rates increased following U L O in both cows and heifers without any significant increases in plasma concentrations of FSH and L H . Pregnancy rates increased following U L O while twinning rates did not. In spite of the insignificant increases in plasma FSH and L H the development of twin follicles resulted which implies that the hormonal regulation of ovarian follicular growth is highly intricate and 97 elucidation of hormonal mechanisms controlling ovarian follicular growth is crucial to improving our understanding of follicular dominance. Attempts to induce twinning in this study failed, not only did the cows abort but also compromised their reproductive health and milk production which finally lead to their being culled. Currently, in North America most of the dairy farmers do not consider twinning as a profitable endeavour and as a result are disinclined towards induction of twinning in cattle on account of the adverse effects it has on both the dam and the twin calves. Much research has gone and is still being undertaken on a large scale to develop a simple reliable method to induce twinning in cattle, especially in European countries where meat production is predominantly from dairy calves. In Canada, here in the province of British Columbia conception rates from first service in cattle is low, about 40% (personal comminucation- Dr. Fisher. L). 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