Studies on Prostaglandin Control of Practical Bovine Reproduction

This thesis consists of a series of 12 Papers which are related as investigating exogenous hormonal control of bovine reproduction under practical agricultural conditions. In particular the objective was to produce additional information on the use of prostaglandin F2alpha. The methods and techniques which were used take account of the special requirements and restrictions of typical commercial husbandry conditions In PART 1, the need for detection of bovine oestrus as a necessary preliminary to artificial insemination is discussed. This has been identified as a factor responsible for unsatisfactory conception rates. The physiological principles, practical problems, and economic consequences of these difficulties are examined in detail. Methods of improving the effectiveness of oestrus detection, and their limitations, are discussed. The endogenous hormonal control mechanisms of the bovine oestrous cycle are examined in detail with particular regard to how they may be manipulated by administration of exogenous hormones. The development of techniques to control oestrus and ovulation by administration of different hormones is discussed. Detailed discussion of the principles involved, techniques required, and difficulties encountered are illustrated by reference to development trials for a progestagen-implant technique. The principles involved and techniques which were used, are related to bovine oestrous cycle control with prostaglandin F2alpha. (Abstract shortened by ProQuest.)

Progress with the control of oestrus and ovulation in farm livestock

No Abstrac

Application of assisted reproduction technologies on the indigenous Nguni cows and heifers

The aims of the study were to compare superovulatory (SO) response rate and embryo quality recovered; consequently, correlate sperm motility with fertilization rate on superovulated stud Nguni cows and heifers. Furthermore, compare oestrous synchronization response and pregnancy rate of three breed type cows (Brahman, Bonsmara and Nguni) of different body condition scores following timed artificial insemination in KwaZulu-Natal (KZN) and Limpopo provinces. Nguni stud cows (n= 15) and heifers (n= 10) aged 4-6 and 2-3 years were used as embryo donors. Superovulation of donors involved insertion of a controlled internal drug release device (CIDR) and two injections of FSH daily 12 hours apart for 4 days on a decreasing dosage. Fresh Nguni semen was collected from proven Nguni stud bulls and assessed by computer aided sperm analysis (CASA) before artificial insemination (AI). The doses of AI were prepared and conducted twice, 12 hours apart on synchronized and superovulated Nguni cows or heifers. Embryos were flushed 7 days after AI using a non-surgical technique. Embryos were immediately evaluated under stereo microscope and classified according IETS standard codes (C1, C1- and C2). All transferrable embryos were vitrified. Two pilot study sites were chosen in Eastern Cape – Great kei; (n= 5) and Limpopo - Vuvha; (n=5) provinces for Embryo transfer. Each recipient cow was implanted with one frozen-thawed embryo. For oestrous synchronization, cows (Braman, Bonsmara and Nguni type) were selected in different villages, regardless of parity, age, breed and body weight following pregnancy diagnosis. Cows were grouped according to breed type and body condition scores (BCS) on a scale of 1-5. Group 1 had BCS of ≤ 2.5 in KwaZulu-Natal (n=81) and Limpopo n=71), Group 2 had BCS of ≥ 3 in KwaZulu-Natal (n=79) and Limpopo (n=100) cows. Cows were synchronized by inserting the controlled intravaginal drug release (CIDR) and removed on Day 8, followed by administration of prostaglandin. The white heat mount detectors (HMD) were placed on the individual cow’s tail head as an indicator for oestrous response if colour changed to red and inseminated twice at 12 hours interval. Pregnancy diagnosis was performed by an ultra sound scanner and rectal palpation 90 days after TAI in embryos and semen recipient cows. There was no significant difference on the superovulatory response rate between Nguni cows (40%) and heifers (40%). There was a significant difference on the ovary reaction (number of corpus luteum) of cows (11.33±1.41) and heifers (4.00±0.57). There were no significant differences observed on the embryo quality between Nguni cows (2.5±1.00 and 1.25±0.59) and heifers (0.83±0.41 and 1.00±0.36) for excellent (C1) and good (C1-). However, cows had more numbers of unfertilized ova (5.5±1.05 and 1.75±0.47) and degenerate embryos (3.66±1.00 and 1.25±0.39) than heifers. Village cows responded to oestrous synchronization successfully in KZN (100%) and Limpopo (99%) regardless of body conditions and breed type. The lowest pregnancy rate was recorded in Brahman and Bonsmara type cows with BCS of ≤ 2.5 regardless of Province. Interestingly, Nguni type cows with same body condition of ≤ 2.5 had higher average pregnancy rate of 59.5% in Limpopo and 53.5% in KZN. However, cows with BCS of ≥3 had better pregnancy rate regardless of cow breed type, and province. In conclusion, only 40% of both Nguni cows and heifers responded to superovulation. However, Nguni cows had better ovaries reaction compared to heifers. The quality of embryos recovered was similar for both Nguni cows and heifers. Moreover, there was a positive correlation between total sperm motility and fertilization rate bull 1 (93.7%) inseminated the cows (67.5%) and bull 2 (83.5%) inseminated the heifers (53.5%). Higher pregnancy rate (60%) was recorded in Limpopo compared to Eastern Cape (0%). Interestingly, more than 99% of village cows responded to synchronization and inseminated with frozen-thawed semen successfully. Village Nguni type cows were not affected by body condition scoring as they had higher and similar pregnancy rate as those that had body condition of ≥ 3. It is suggested that it is not advisable to breed synchronized Brahman and Bonsmara type cows with the body condition of ≤ 2.5 except in Nguni cow type as more than 57% average pregnancy rate was achieved

Immunomodulation of reproductive function in domestic ruminants

Active immunisation against GnRH inhibits reproductive function by inducing a hypogonadotropic condition associated with gonadal atrophy. Despite economic, ethical and environmental advantages of GnRH immunisation in cattle over conventional castration methods, the technology has not yet been commercially adopted. Primarily because of the requirement for numerous booster vaccinations because of the reversibility of physiological effects, the commercial efficacy of immunocastration is currently poor. However, neonatal GnRH immunisation in sheep can result in a permanent suppression of reproduction (Brown et al., 1994; 1995; Clarke et al., 1998). These findings and a study in pigs (Molenaar et al., 1993) indicate that, the hypothalamic/pituitary gland unit (HPU) may be particularly susceptible to GnRH antibodies during a specific window of development in the pre-pubertal animal, but no long-term studies in cattle have been conducted. Therefore the primary objective of this project was to determine the effect of neonatal immunisation against GnRH in cattle. Beef cross bull (n=9; Chapter 3) and heifer calves (n=9; Chapter 4) were vaccinated against a newly developed (Pfizer®) GnRH construct vaccine at -2, 6 and 13 weeks of age. Nine calves of each sex served as negative controls, receiving saline injections only. The GnRH vaccine had proved effective (Dr. A.R. Peters, personnel communication 2000) in inducing immune responses and reducing variation between animals in unpublished industrial studies, compared to earlier vaccines, and hence was reasoned to be capable of raising GnRH antibodies despite the relative immaturity of the neonatal immune system. Following vaccination, circulating GnRH antibodies and reproductive hormones, such as FSH (Chapters 3 and 4), testosterone (Chapter 3), progesterone (to assess onset of puberty) and oestradiol (Chapter 4) were measured and additional intensive serial bleeds were carried out to assess LH parameters up to and beyond puberty (puberty defined by testes circumference in bulls). Gonadal (antral follicles and testes growth) and accessory gland development was quantified throughout the trial using ultrasound scanning. Sexual behaviour (Chapter 3) was studied from 38 weeks of age, while an assessment of sperm quality (Chapter 3), and anabolic response to vaccination was also performed post-mortem (Chapters 3 and 4). GnRH immunisation in neonatal calves did not permanently impair reproduction. A temporary suppression in reproductive function was evident through the disruption of pituitary gland function, as indicated by a reduction of LH pulse amplitude and mean plasma LH concentrations (Chapters 3 and 4). In addition, a reduction in medium- sized follicle numbers, testes growth, plasma testosterone concentration, vesicular gland length and juvenile aggression occurred. Some beneficial anabolic effects were observed e.g., carcass composition grades. Changes all occurred subsequent to increased GnRH antibody titres in immunised cattle. Despite some evidence of prolonged effects on LH amplitude and circulating testosterone after anti-GnRH titres had dissipated, all inhibited parameters, except carcass quality, returned to levels comparable to control animals by 72 weeks of age. No treatment effects on FSH concentrations, large follicle numbers, reproductive tracts (post mortem) or peri- and post-pubertal behaviours were observed following treatment. Sperm morphological abnormalities tended to be more prevalent in GnRH immunised bulls. A significant increase in GnRH antibody titres occurred at -23 weeks of age (Chapter 4), this may have been a rebound in antibody titre, possibly caused by an anti-idiotype immune response (antibody response to GnRH antibodies), or due to significant maturational changes in immune function at this time causing a delayed response to vaccination. Alternatively a novel "auto-immune" response may have been detected, which if confirmed/repeatable might be incorporated into an immunisation protocol to act as a "self-booster". However, no previous reports of such an event have been published and further investigation is urgently required. A more prolonged or permanent suppression of reproductive function may be possible following an earlier, greater and more sustained elevation of antibody titres during the neonatal period. Further development of GnRH vaccines and/or protocols (prime-boost, cytokine modulation vaccines, concomitant passive and active immunisation and pregnant cow GnRH vaccination), and studies of performance and GnRH antibody mechanism(s) of action in cattle are required. Chapters 3 and 4 provide a comprehensive study on pubertal development and neonatal GnRH vaccination, thus contributing significantly to knowledge in these fields. Currently, the vaccine used in this trial may be used to delay puberty in older calves or transiently suppress reproductive function to aid management. The economical viability of animal production systems such as beef and lamb are closely related to rates of reproduction. The Fec B gene in ewes increases ovulation rate and litter size, possibly through the development of precocious follicles, which can switch their primary dependence from FSH to LH. As a result, more follicles are selected to continue growth to an ovulatory size. The precise mechanisms by which these processes occur have recently been shown to involve oocyte follicle interactions (see section 1.1.5). Follicle development is modulated by GHIIGF and inhibin, however attempts to increase follicular development and ovulation through active inhibin immunisation alone have been variable and hence not commercially attractive. To develop successful protocols to induce twin ovulations in cows· and ewes, without superovulation, a clearer, more details understanding of follicullogenesis is required. The objective of the current study was to better understand these mechanisms through investigating interactions of GH/GF and inhibin in the ovary, follicle development, steroidogenesis, and receptor populations using an anoestrous sheep model. Spring born Mule x Charolais ewe lambs were actively immunised (n=8) against porcine inhibin α-C 1-26 peptide conjugated to KLH in NUFCA (primary and 3 boosters (NUFA», while 8 served as negative controls. Seven days following the final booster, the ewes were subdivided to give four groups: (1) controls + saline (n=4); (2) controls + rbGH (4ml s.c; 1mg. mr1; n=4); (3) inhibin immunised + saline (n=4); and (4) immunised + rbGH (n=4). Recombinant bovine growth hormone (rbGH) was given (Lm.) for 6 days. On day 4 GnRH (Receptal®; 1 ml) was injected s.c, to all animals to initiate the beginning of a new follicular wave. Blood samples were collected fortnightly to measure inhibin antibody titres, IGF-I, FSH and steroids. On the seventh day ensuing slaughter serum antibodies and ovaries were harvested. Left ovaries were intended for ISH (mRNA for P450arom) and/or immunohistochemical analysis. Follicles from right ovaries were dissected out, counted, measured and cultured in M199 at 37°C for 2 hours. Culture media was then assayed for oestradiol. Follicle shells were stored at -180°C for LH receptor binding studies. This work reports on the influence of different treatments on follicle populations. All immunised animals produced antibodies, which bound to 1251-inhibin. Using ANOVA to compare treatments it was observed that, Inhibin immunisation significantly (P3.5mm in diameter, but did not affect the smaller 3.5mm follicle numbers. These findings are in agreement with previous research. The molecular studies of left ovaries are not presented herein as due to time constraints the work was not completed and is currently on going. In conclusion, additional results of this study are required to meet the objectives of the experiment. Further research is required on dominant follicle selection if superovulatory programmes in both livestock and humans are to be more precisely controlled and readily accepted. An increased twinning rate in beef suckler cows is acknowledged as an optimal means of improving production efficiency (Diskin et al, 1987), and preliminary observations (Campbell, Webb and Gong, unpublished) suggests that following oestrous cycle synchronisation, a passive inhibin immunisation protocol gives a more predictable twin ovulation response than active inhibin vaccination. Therefore, using inhibin antiserum raised in ewes (Chapter 5), the final thesis objective was to investigate the efficacy of a bolus and booster antiserum administration protocol in heifers and the effects of passive inhibin immunisation on hormone parameters (FSH and progesterone), folliculogenesis (in vivo) ovulation rate and conception. Beef cross heifers (n=18; used earlier in Chapter 4 study) were randomly allocated to receive inhibin antiserum or NSS (controls). Following the Chapter 4 study, and 4 months summer break at pasture, heifers were considered to be exhibiting typical cyclic reproductive function based on hormone measurements and scanning of ovaries. Oestrous cycles' were synchronised using 2 injections (Lm.) of PG 11 days apart and antiserum 'bolus' (108ml) was injected into the jugular vein 4 days after 2nd PG treatment. Subsequently, at 24h intervals for 5 days 14ml booster infusions were administered. Five days after ovulation, as confirmed by twice daily scanning, a 3rd PG was given and 72 and 96h later, heifers were A.1. Blood samples were collected every 8 hours from 2nd PG to 2nd A.1. Dominant follicle number and ovulation rate was unaffected by treatment due to inadequate inhibin antibodies in antiserum compared with the earlier trial (Campbell, Webb and Gong, unpublished), however, recruited follicle numbers were enhanced in inhibin immunised heifers. In addition, further evidence that inhibin antibodies act at the ovarian level to enhance recruitment, or reduce atresia was provided, as FSH concentrations were similar between groups. Higher inhibin antibody titre may be necessary to over-ride the strong dominant follicle selection mechanism in heifers. In addition, the results of this and a previous trial demonstrate that a threshold inhibin antibody titre range is required to obtain a reproducible increase in ovulation rate on which future passive inhibin immunisation studies may be based. Finally, this approach of a bolus followed by subsequent small antiserum injections has successfully been shown to give a steady and consistent concentration of circulating inhibin antibody titres

Comparison of three reproductive management strategies for lactating dairy cows using detection of oestrus or synchronisation of ovulation and Fixed-Timed Artificial Insemination

The most common dairy cattle reproductive management strategies combine oestrus detection with hormonal protocols for synchronisation of ovulation for breeding. However, approximately 50% of oestrus cycles are missed in commercial dairy farms due to human error and poor expression of oestrus behaviour. Furthermore, around 20% of cows experience prolonged postpartum anovulation. Synchronisation protocols may have variable synchronisation rates, producing suboptimal pregnancies per artificial insemination (P/AI). The aim of this study was to compare the reproductive performance of three commercial reproductive management strategies in lactating dairy cows: a combination of oestrus detection (OD) followed by ovulation synchronisation protocol for fixed timed artificial insemination (FTAI) either using Ovsynch or PRID-synch, or Double Ovsynch at FTAI. Cows (n = 1681) were randomly assigned to one of three different reproductive strategies at calving: Oestrus detection - Ovsynch (OD-Ov), Oestrus detection - PRIDsynch (OD-PR) and Double Ovsynch (DO). Cows enrolled in OD-Ov, and OD-PR were eligible to be inseminated after observed oestrus between 50 and 70±3 days in milk (DIM). Cows in which oestrus was not detected between 50 and 70±3 DIM (OD-Ov, n = 541; OD-PR, n = 562) received their respective hormonal treatments at 70±3 DIM. In these two groups, cows that returned to oestrus within the period for first FTAI (<83DIM) had more than one opportunity for AI. Cows enrolled in DO were subjected to FTAI only. Postpartum disorders were recorded between 1 and 7 DIM; and lameness, mastitis and bovine respiratory disease were recorded until first AI. Body condition score (BCS) was recoded at calving, 43±3 and 70±3 DIM. Ovarian monitoring was performed by transrectal ultrasonography (US) at 43±3 and 50±3 DIM, and at 70±3 and 77±3 DIM only for synchronised cows. Effects of treatments were assessed with multivariable statistical methods relevant for each outcome variable. Pregnancy 32±3 d after first AI was similar among treatment groups (OD-Ov = 43.2%, ODPR = 41.6%; DO = 45.7%) and proportion of cows pregnant by 83 DIM was also similar among treatment groups (OD-Ov = 46.5%, OD-PR = 46.6%; DO = 45.7%). Farm, parity, BCS at 43±3 DIM and breeding sire were associated with reproductive performance. Pregnancy loss (PL) was significantly higher in the OD-Ov (9.7%) than in the OD-PR (4.1%). In conclusion, no difference in reproductive performance among reproductive strategies was observed in this study, suggesting that reproductive performance is influenced by farm-specific factors such as oestrus detection rate and P/AI, and overall cow health

Endocrine control of ovulation rate in the cow

Techniques to increase prolificacy in cattle have met with limited success; the aim of these studies was to investigate the potential of actively immunising cattle against certain gonadal hormones, and to examine the physiological basis of these treatments. Eight heifers were immunised against 8mg of a testosterone conjugate in Freund's Incomplete Adjuvant, & nine animals served as controls. These heifers were given one priming and two booster injections at four-month intervals. After the last booster injection, 7/8 animals had become anoestrous, and displayed significantly raised blood progesterone & mean LH concentrations,increased LH pulse frequency, & decreased mean FSH concentrations.Seven months after this treatment, 3/7 anoestrous heifers resumed ovarian cyclicity, with a mean ovulation rate of 2.7+0.7.To determine if the different ovarian responses observed above could be obtained by changes of ovarian steroid feedback seen during the oestrous cycle, groups of 6 heifers were implanted with large,medium or small sized oestradiol capsules during the luteal phase of the cycle. Five control heifers received empty implants. During the luteal phase of the cycle following implantation, all heifers were ovariectomised. The effect of the treatment on ovarian function and gonadotrophin secretion in the presence or absence of progesterone (PRID) was then determined. Increasing physiological concentrations of oestradiol reduced the number of large antral follicles and corpora lutea, but not the total number of antral follicles >lmm diameter. A combination of progesterone and oestradiol were fully effective in maintaining luteal-phase concentrations of LH and FSH, and follicular-phase concentrations of oestradiol alone were able to maintain LH and FSH concentrations within the physiological range. Thus changes of blood steroid levels similar to those seen during the oestrous cycle may interrupt ovarian function.Cattle were therefore immunised against a non-steroidal, partly purified fraction of ovine follicular fluid (PPFF) enriched ininhibin-like activity as measured in vivo and in vitro. Active immunisation against 0.4mg and 4mg ovine PPFF produced 1/5 & 3/5 heifers with multiple ovulations, respectively; this was not associated with changes of FSH secretion. To examine in more detail the endocrine responses to this treatment, and to investigate possible comparative aspects, cows were immunised against 4mg ovine, porcine or equine PPFF. No treatment increased ovulation rate, but the porcine-PPFF immunised heifers showed a 7-fold increase in mean LH secretion that could not be explained by alterations in pulsatile secretion or in steroid feedback.Collectively, these results suggest that the cow does not respond consistently to treatments so far designed to alter gonadotrophin secretion, that inhibin is not a major feedback hormone in this species, and that the heifer may possess an influential intra-ovarian control mechanism which ultimately determines ovulation rate

Monitoring and improving reproductive performance of crossbred dairy cattle in Tigray Region, Ethiopia

Ethiopia maintains an extensive livestock population; however, reproductive performance of cattle and their breeding management are unsatisfactory. Currently, the sole diagnostic tool in the country is rectal palpation, which is inaccurate for early pregnancy in cattle. The study assessed reproductive performance and major reproductive problems using questionnaire survey, and evaluated simple, cost-effective alternative monitoring approaches using on-farm diagnostic tools to determine milk and serum progesterone (P4) and evaluate reproductive status. There were 177 dairy farms (range 1-115 cattle per farm) included in the questionnaire survey. Of these, 47 participated in the quantitative determination of P4 and estradiol profiles that used an enzyme-linked immunosorbent assay (ELISA) and in the on-farm diagnostic trial that used qualitative ELISA (Target P4 and Dipstick (P4 Rapid), and the reproductive status of 319 crossbred [Holstein Friesian (HF) X Zebu] dairy cattle was assessed. Questionnaires indicated that heifers in the study area reach puberty at older age and calve late, and cows have long postpartum estrus and calving interval. Anestrus, repeat-breeding, dystocia, retained fetal membranes (RFM), endometritis and abortion as the major reproductive problems in dairy cattle in the study area. Serum, milk, saliva and urine P4 and serum estradiol profiles of cattle at different reproductive status was determined using quantitative laboratory ELISA. High P4 levels was detected in pregnant and diestrus cattle than cattle that were anestrus and in-heat. Estradiol level was higher in cattle that were in-heat than cattle in other reproductive conditions. On-farm P4 ELISA indicated in-heat (estrus) 10 (3.1%), anestrus 77 (24.2%), repeater (follicular cyst) 9 (2.8%), normally cycling 69 (21.6%) and pregnant 154 (48.3%). The field P4 ELISA findings were validated using quantitative laboratory P4 ELISA, and similar results were obtained. The sensitivity and specificity of on-farm and laboratory P4 ELISA tests for diagnosing pregnancy were 88.6 & 99.4% and 98.1 & 100%, respectively. Once reproductive problems were identified using on-farm P4 ELISA and per rectal palpation, along with reproductive history, 122 cattle (75 cows and 47 heifers) were assigned to a 10-day Controlled Internal Drug Release (CIDR) in combination with prostaglandin F2-alpha (PGF2α) and equine chorionic gonadotrophin (eCG) based estrus synchronisation protocol to study the estrus response and conception rate. The overall estrus response and conception rates were 97.5% and 78.3%, respectively, with no significant differences in parity, pre-treatment reproductive status (anestrus or cycling (repeat-breeding/silent-estrus)) and farming system (smallholder vs organised commercial farms). The study has shown high estrus response and conception rate. Hence, this protocol is highly recommended to enhance fertility of dairy cattle in the study area and other regions. Finally, the study has determined the macronutrient composition of milk and assessed the effect of reproductive status, farm (nutritional) management, stages of lactation, parity and breed on milk composition in 246 dairy cows. This consisted of 184 crossbred cows from smallholder (n= 76: 36 non-pregnant and 40 pregnant) and organised commercial dairy farms (n= 108: 62 non-pregnant and 46 pregnant) in and around Mekelle, Ethiopia and by way of comparison, 62 HF cows (25 non-pregnant and 37 pregnant), either managed indoors or outdoors in a commercial farm in Edinburgh, United Kingdom. The mean milk fat, protein, lactose, total solid (TS) and solid-no-fat (SNF) recorded were 2.36%, 3.46%, 4.37%, 10.39% and 7.82%, in crossbred dairy cows, compared with 5.05%, 3.71%, 4.72%, 13.68% and 8.43%, in HF cows, respectively. Significantly lower (p<0.05) level of macronutrient was recorded in milk from crossbred cows than HF cows. Milk fat in both breeds was affected by reproductive status, farm (nutritional) management and stages of lactation, but not by parity. In both cattle breeds, the milk fat content was significantly higher (p<0.05) in pregnant than non-pregnant cows. Milk protein content was significantly (p<0.05) affected by reproductive status (in crossbreds) and stages of lactation (in both breeds), but not by farm management or parity. Reproductive status (in crossbreds), stages of lactation (in both breeds) and parity (in crossbreds) affected lactose level; however, farm management had no effect on lactose level in both breeds. Milk fat was the most affected macronutrient content in both breeds. Low milk fat level in crossbred cows could be as a result of samples obtained from early milking coupled with nutritional management. In conclusion, the present study has determined the major reproductive problems in crossbred dairy cattle, assessed their actual reproductive status using rapid, cost effective, simple and applicable on-farm P4 tests, and established P4 and estradiol profiles at different reproductive status. The major breeding problem was poor estrus detection evidenced when animals reported anestrus were confirmed normally cycling using on-farm as well as laboratory P4 assays. These studies offer opportunities for establishing simple field reporting of reproductive status in these crossbred dairy cattle, which can have a major impact on breeding management and productivity

The Effect of Triptorelin on Ovulation Rate and Conception Rate in Gilts and the Endocrine Profile in Non-Pregnant and Early pregnant Gilts

Triptorelin is a gonadotropin releasing hormone agonist that has been shown to be effective in weaned sow single fixed time artificial insemination protocols. Administration of triptorelin 96 h after weaning has been shown to be effective in synchronizing sows to be inseminated one time 24 h later without reducing pregnancy rate or the number of pigs born. In the current experiment, 23 crossbred gilts (249 d, 123 kg) were administered an intramuscular injection of PG600. Nine days after PG600 administration, gilts were fed 15 mg of altrenogest once daily for 16 d. Twelve gilts (OVU) were administered 200 μg triptorelin 96 h after the last altrenogest feeding. A single artificial insemination was then performed regardless the expression of estrus 126 h after the last altrenogest feeding. Eleven gilts (CON) were inseminated upon the expression of standing estrus and received a second insemination 24 h later. Blood was collected on d 0 (day of triptorelin administration), 1, and 2 after the administration of triptorelin for the measurement of serum concentrations of estrogen and progesterone by RIA. Estrus detection was performed daily beginning 3 d following the last altrenogest administration and ended 7 d after the last altrenogest administration. Gilts were slaughtered on d 33 when reproductive tracts were collected. Reproductive tracts were evaluated for the number of fetuses in the uterus and the number of CL on the ovary. No differences (P \u3e0.05) were found in pregnancy rate, number of fetuses, number of CL, and the ratio of fetuses to CL in all animals. When non-pregnant gilts were removed from the analysis there was a trend (P=0.08) for the CON group to have a greater ratio of fetuses to CL. Expression of standing estrus was also greater (P\u3c 0.05) in the CON group than OVU group. No differences (P \u3e0.05) were found in serum concentrations of estrogen between OVU and CON gilts. The CON group’s expression of estrus occurred around the time when progesterone was at the lowest in the group. The OVU had its lowest serum concentration of progesterone earlier than CON group. This may indicate that the OVU group could have ovulated earlier than the CON group. Data from the first experiment has demonstrated that even though conception rates of fixed timed AI protocols involving GnRH agonists are similar to standard AI, the conception rate versus ovulation rate may show differences in the two protocols. When comparing the ovulation rate with conception rate, our experiment has shown that GnRH agonists, accompanied by a single fixed timed AI, may not be effective in maximizing the number of conceptuses in gilts. Many of the gilts in this experiment failed to become pregnant even though standing estrus was exhibited in the CON group. Anterior pituitary and blood samples from these gilts were collected to determine serum concentrations of insulin like growth factor (IGF) and luteinizing hormone. Anterior pituitaries were also used to determine the expression of IGF receptor, gonadotropin releasing hormone (GnRH) receptor, luteinizing hormone beta (LH-β), IGF binding protein -2, 3, and 5. Anterior pituitaries and serum samples were collected from 15 pregnant and 8 non-pregnant gilts on d 33 after AI. Serum concentrations of estradiol and progesterone and AP concentrations of LH and IGF-1 were determined by RIA. Relative expression of GnRHR, LH-β, IGF-1, IGFBP-2, IGFBP-3, and IGFBP-5 were determined using real time reverse transcriptase PCR. Fold changes in relative expression were determined using the Relative Expression Software Tool. Non-pregnant gilts were assumed to be undergoing the luteal phase of their reproductive cycle. Our data supported this because no differences (P \u3e0.05) were determined in serum concentrations of progesterone or estradiol between non-pregnant and pregnant gilts. Mean AP concentrations of LH were greater (P0.05) between pregnant and nonpregnant gilts. Mean relative expression of LH-β was .8 fold lower (P\u3c 0.05) and IGFBP- 2 tended to be 0.8 fold lower (P=0.095) in pregnant gilts compared to non-pregnant gilts. No differences were found (P \u3e0.05) between pregnant and non-pregnant gilts in relative expression of AP GnRHR, IGF-1, IGFBP-3, and IGFBP-5. Data from the second experiment have shown that changes in multiple endocrine factors, such as LH and the IGF system, may play a crucial part in maintaining early pregnancy but further investigation is needed