A highly variable yield of viable embryos in superovulated cattle is a major hindrance to the embryo transfer industry. To trace the cause of this problem, investigations were carried out on the intrafollicular steroids and structure of oocytes originating from follicles of follicular stimulating hormone (FSH)-stimulated (superovulated) and unstimulated heifers. Unstimulated heifers were slaughtered at midcycle, or administered cloprostenol (PG) at midcycle and slaughtered after 24, 48, or 72 hr, while superovulated heifers were administered 4 injections of pFSH (2 injections per day) and slaughtered 12 hr later, or administered 6, 7, or 8 injections of FSH in combination with PG at the 5th and 6th injection, and slaughtered 24, 36, or 60 hr, respectively, after the first PG injection. The follicular fluid from the largest (presumptive dominant) follicle of the unstimulated heifers and from potentially ovulatory follicles >> or = 8 mm in diameter) of the superovulated heifers were assayed for estradiol-17 beta (E2) and progesterone (P4), while the oocyte cumulus complexes from such follicles were processed for transmission electron microscopy. The mean E2 and especially P4 concentrations of the potentially ovulatory follicles of the superovulated heifers were lower than similar follicles of the unstimulated animals (83.7 +/- 76.7 ng/ml vs. 208.1 +/- 357.0 ng/ml, P>> 0.05 and 31.1 +/- 38.7 ng/ml vs. 150.3 +/- 202, P < 0.05, respectively). The unstimulated oocytes had, in general, spherical oocyte nuclei and compact nucleoli before PG administration, while after PG, undulation of the nuclear envelope and nucleolus vacuolization was characteristic.(ABSTRACT TRUNCATED AT 250 WORDS)

Twenty-five normally cyclic Holstein heifers were used to examine the effects of oxytocin on cloprostenol-induced luteolysis, subsequent ovulation, and early luteal and follicular development. The heifers were randomly assigned to 1 of 4 treatments: Group SC-SC (n=6), Group SC-OT (n=6), Group OT-SC (n=6) and Group OT-OT (n=7). The SC-SC and SC-OT groups received continuous saline infusion, while Groups OT-SC and OT-OT received continuous oxytocin infusion (1:9 mg/d) on Days 14 to 26 after estrus. All animals received 500 microg, i.m. cloprostenol 2 d after initiation of infusion (Day 16) to induce luteolysis. Groups SC-OT and OT-OT received oxytocin twice daily (12 h apart) (0.33 USP units/kg body weight, s.c.) on Days 3 to 6 of the estrous cycle following cloprostenol-induced luteolysis, while Groups SC-SC and OT-SC received an equivalent volume of saline. Daily plasma progesterone (P4) concentrations prior to cloprostenol-induced luteolysis and rates of decline in P4 following the induced luteolysis did not differ between oxytocin-infused (OT-OT and OT-SC) and saline-infused (SC-SC and SC-OT) groups (P >0.1). Duration of the estrous cycle was shortened in saline-infused heifers receiving oxytocin daily during the first week of the estrous cycle. In contrast, oxytocin injections did not result in premature inhibition of luteal function and return to estrus in heifers that received oxytocin infusion (OT-OT). Day of ovulation, size of ovulating follicle and time of peak LH after cloprostenol administration for oxytocin and saline-treated control heifers did not differ (P >0.1). During the first 3 d of the estrous cycle following luteal regression, fewer (P <0.01) follicles of all classes were observed in the oxytocin-infused animals. Day of emergence of the first follicular wave in heifers treated with oxytocin was delayed (P <0.05). The results show that continuous infusion of oxytocin during the mid-luteal stage of the estrous cycle has no effect on cloprostenol-induced luteal regression, timing of preovulatory LH peak or ovulation. Further, the finding support that an episodic rather than continuous administration of oxytocin during the first week of the estrous cycle results in premature loss of luteal function. The data suggest minor inhibitory effects of oxytocin on follicular growth during the first 3 d of the estrous cycle following cloprostenol-induced luteolysis.

The main objective was to compare pregnancy per AI (P/AI) between sex-selected and conventional semen in cyclic beef heifers subjected to a 5-day Co-synch plus CIDR protocol and evaluated the usefulness of an estrus detection (ED) aid to identify heifers that were most likely to conceive. This study also determined if the expression of estrus before timed-AI (TAI) would be associated with increased P/AI in acyclic heifers inseminated with conventional semen. Heifers (n = 1690; 320-523 kg of body weight, and 13-15 mo of age) at three locations over 2 years were scanned by ultrasonography to determine cyclicity (presence of luteal tissue) and reproductive tract normalcy. Cyclic heifers (n = 1331) received a progesterone releasing device (CIDR) on Day 0, CIDR removal and 500 μg of cloprostenol (PGF) on Day 5, and 100 μg of GnRH along with TAI on Day 8. Acyclic heifers (n = 275) received the same treatment with the addition of GnRH on Day 0. On Day 5, all heifers received ED patches (Estrotect™) that were scored from 0 to 3, based on color change between initial application and Day 8; 0 = unchanged, 1 = ≤ 50% color change, 2 =>> 50% color change, 3 = missing. Estrus was defined to have occurred when an ED patch was scored 2 or 3. Cyclic heifers were inseminated with either frozen-thawed sex-selected or conventional semen from either of three sires available commercially (two per year). Acyclic heifers were inseminated with conventional semen. Pregnancy diagnosis was performed by transrectal ultrasonography 28 or 48 d after TAI, depending on management. The percentage of cyclic heifers was 83.9% and the average estrus response was 63.8%. P/AI was greater (P < 0.01) in cyclic compared to acyclic heifers (53.3 vs. 36.0%) and tended to be greater (P = 0.07) for conventional semen (52.3 vs. 47.6%), despite all acyclic heifers being inseminated with conventional semen. Heifers with an ED patch scored 2 (61.1%) or 3 (58.6%) had greater (P < 0.01) P/AI than those scored 0 (31.8%) or 1 (33.1%), regardless of semen type. Pregnancy per AI was greater (P < 0.01) for heifers detected in estrus (60.6 vs. 32.3%). In cyclic heifers that did not exhibit estrus, P/AI was lower (P < 0.01) in those inseminated with sex-selected semen (27.8 vs. 45.9%), while in heifers that exhibited estrus, P/AI only tended to be lower (P = 0.08; 56.7 vs. 65.5%). In summary, P/AI was greater in cyclic heifers, in those inseminated with conventional semen and in those exhibiting estrus before TAI. The ED patches were considered useful to identify animals for TAI with sex-selected semen and could be used to increase the adoption of this technology in beef herds.

The objective was to compare pregnancy per AI (P/AI) between two shortened timed-AI (TAI) protocols in beef cattle. This study also determined whether administration of eCG in heifers and timing of AI in cows would affect P/AI. Cattle were submitted at random to either a modified 5-d Co-synch protocol (Day 0 = progesterone releasing device (CIDR); Day 5 = CIDR removal and 500 μg of cloprostenol (PGF); Day 8 = 100 μg GnRH concurrent with AI) or J-synch protocol (Day 0 = CIDR insertion and 2 mg of estradiol benzoate i.m.; Day 6 = CIDR removal and 500 μg PGF; Day 9 = 100 μg GnRH concurrent with AI). In Experiment 1, 1135 heifers (13-15 mo of age) received an estrus detection patch (Estrotect™) on Day 5 and 579 were selected at random to receive 300 IU of equine chorionic gonadotropin (eCG) at the time of CIDR removal. Patches were scored from 0 to 3 based on color change between initial application and AI; 0 = unchanged, 1 = ≤ 50% change, 2 = > 50% change, 3 = missing. Estrus was defined to have occurred when the patch was scored 2 or 3. In Experiment 2, 399 cyclic, non-lactating beef cows from 1 location were submitted to either the modified 5-d Co-synch or J-synch protocol and within each protocol cows were TAI at either 66 ± 1 (n = 199) or 72 ± 1 h (n = 200) following CIDR removal. Transrectal ultrasonography was used in both experiments to determine presence of a corpus luteum (CL) on Day 0, and to diagnose pregnancy 35 d after TAI. In Experiment 1, eCG increased estrus rate only in heifers without a CL on day 0 that were submitted to the modified 5-d Co-synch protocol (41.9 vs. 69.6%). Heifers submitted to the J-synch protocol had greater (P = 0.03) P/AI compared with those in the modified 5-d Co-synch (48.7 vs. 41.1%) and heifers that expressed estrus before AI had increased (P < 0.0001) P/AI compared to those that did not (53.6 vs. 36.5%). Administration of eCG and presence of a CL tended to affect P/AI (P = 0.13). In Experiment 2, cows submitted to the J-synch protocol tended (P = 0.07) to have greater P/AI compared to those in the modified 5-d Co-synch (74.1 vs. 66.5%). There was no association between P/AI and timing of AI. In summary, the J-synch protocol resulted in greater P/AI than the modified 5-day Co-synch protocol in heifers and cows. Administration of eCG increased estrus rate in heifers without a CL at the start of the protocol and tended to improve P/AI in all heifers. Timing of AI (66 vs. 72 h) had no effect on P/AI in cows subjected to either TAI protocol.

The objective of this study was to evaluate superovulatory programs based on synchronization of follicular waves with GnRH at 2 different stages of the estrous cycle. Sixteen Holstein cows were randomly assigned to 1 of 3 groups and administered GnRH (Cystorelin, 4 ml i.m.) between Days 4 and 7 (Groups 1 and 3) or between Days 15 and 18 (Group 2) of the estrous cycle (estrus = Day 0). Four days after GnRH treatment,>> or = 7-mm follicles were punctured in Groups 1 (n = 6) and 2 (n = 6) or were left intact in Group 3 (n = 4). All cows were superstimulated 2 d later (i.e., from Days 6 to 10 after GnRH treatment) with a total of 400 mg NIH-FSH (Folltropin-V) given twice daily in decreasing doses. The GnRH treatment caused a rapid disappearance of large follicles (P < 0.005), rapid decrease in estradiol concentrations (P < 0.003), and increase in the number of recruitable follicles (4 to 6 mm; P < 0.04), indicative of the emergence of a new follicular wave within 3 to 4 d of treatment. Between 4 and 6 d after GnRH treatment, the mean number of 4- to 6-mm follicles decreased (4.7 +/- 1.8 to 1.5 +/- 3.3) in the nonpunctured group but increased (3.9 +/- 1.0 to 7.3 +/- 1.9) in the punctured group of cows (P < 0.05). In response to FSH treatment, the increase in the number of>> or = 7-mm follicles was delayed by approximately 2 d in the nonpunctured group (P < 0.006). Moreover, the mean number of>> or = 7-mm follicles at estrus was higher (16.9 +/- 1.7 vs 11.5 +/- 3.0; P < 0.1) in the punctured than the nonpunctured group. The increase in progesterone concentration after estrus was delayed in the nonpunctured group (P < 0.1) compared with the punctured follicles. Mean numbers of CL as well as freezable (Grade 1 and 2) and transferable (Grade 1, 2 and 3) embryos were similar (P>> 0.1) in punctured and nonpunctured groups. Spontaneous estrus did not occur prior to cloprostenol-induced luteolysis in any group, and stage of the estrous cycle during which GnRH was given did not affect (P>> 0.1) hormonal and follicular responses in the punctured groups. In conclusion, GnRH given at different stages of the estrous cycle promotes the emergence of a follicular wave at a predictable time. Puncture of the newly formed dominant follicle increases the number of recruitable follicles (4 to 6 mm) 2 d later and, in response to superstimulation with FSH, causes a greater number and faster entry of recruitable follicles into larger classes >> or = 7 mm) and a faster postovulatory increase in progesterone concentrations.

A single injection (100 mug i.m.) of Estrumate (I.C.I. 80996) was used to induce luteal regression on day 8 of the estrous cycle in 3 sheep. Progesterone levels in the utero-ovarian vein and femoral artery had fallen within 6 h to less than 50% of the concentrations seen before injection of the analogue. Luteolysis was not associated with endogenous production of PGF. The concentration of PGF in the uteroovarian vein began to increase 27-39 h after the administration of Estrumate, reaching a mean maximum concentration of 1455pg/ml 48 h after Estrumate. The mean concentration of PGF in the utero-ovarian vein between 36-69 h after Estrumate was significantly greater than during the 24 h before Estrumate (control period) or during the 0-30 h immediately after injection (both P less than 0.001). The maximum secretion of estradiol and the pre-ovulatory LH peak occurred during the period of elevated PGF concentrations in the utero-ovarian veins. The possible importance of endogenous PGF production at this time is discussed.

The first of 2 injections of 0.5 mg cloprostenol (PG1 and PG2) eleven days apart was given to 19 Friesian-Hereford cross heifers between days 8-14 of their cycle (Treatment A) and 16 similar animals between days 0-4 (Treatment B). Oestrus show was monitored by Kamar Heat Mount detectors and vasectomised bulls with chin-ball markers. Blood samples taken at PG1, six days later, at PG2 and four days later were assayed for progesterone to confirm that luteolysis had occurred as expected. Four hourly rectal examinations of the ovaries were carried out from 56-112 hours after PG2 and four hourly blood samples from 36-96 hours after PG2 were collected for FSH and LH assay. Mean time in hours from PG2 to oestrus onset, LH peak and ovulation respectively was 57.4 +/- 2.9, 60.2 +/- 2.0, 91.7 +/- 1.8 for Treatment A and 64.9 +/- 4.1, 68.9 +/- 2.4, 96.7 +/- 1.3 for Treatment B. Treatment A animals showed significantly higher (p<0.01) FSH levels at PG2 than Treatment B. Time from PG2 to LH peak was significantly shorter in animals treated either on days 7 and 8 (p<0.01) or days 15-16 (p<0.05) of their cycle compared with treatment on days 12-14 and it is suggested that these shorter response times correspond to an early and late cycle wave of follicular growth. Secondary FSH peaks some 28 hours after that occurring synchronously with the pre-ovulatory LH peak were observed to be significantly (p<0.01) higher at oestrus associated with the early cycle follicular growth wave as compared with that later in the cycle which may argue a difference in endocrine control of the two periods of follicular maturation.

This study was designed to compare two timed insemination protocols, in which progesterone, GnRH and PGF2alpha were combined, with the Ovsynch protocol in presynchronized, early postpartum dairy cows. Reproductive performance was also evaluated according to whether cows showed high or low plasma progesterone concentration, at the onset of treatment. One hundred and six early postpartum dairy cows were presynchronized with two cloprostenol treatments given 14 days apart, and then assigned to one of the three treatment groups. Treatments for the synchronization of estrus in all three groups started 7 days after the second cloprostenol injection, which was considered Day 0 of the actual treatment regime. Cows in the control group (Ovsynch, n=30) were treated with GnRH on Day 0, PGF2alpha on Day 7, and were given a second dose of GnRH 32 h later. Cows in group PRID (n=45) were fitted with a progesterone releasing intravaginal device (PRID) for 9 days, and were given GnRH at the time of PRID insertion and PGF2alpha on Day 7. In group PRID/GnRH (n=31), cows received the same treatment as in the PRID group, but were given an additional GnRH injection 36 h after PRID removal. Cows were inseminated 16-20 h after the administration of the second GnRH dose in the Ovsynch group, and 56 h after PRID removal in the PRID and PRID/GnRH groups. Ovulation rate was determined on Day 11 postinsemination by detecting the presence of a corpus luteum in the ovaries. Lactation number, milk production, body condition at the onset of treatment and treatment regime were included as potential factors influencing ovulation and pregnancy after synchronization. Logistic regression analysis for cows with high and low progesterone concentration on treatment Day 0 revealed that none of the factors included in the models, except the interaction between progesterone and treatment regime, influenced the risk of ovulation and pregnancy significantly. In cows with high progesterone concentration at treatment onset, Ovsynch treatment resulted in a significantly improved pregnancy rate over values obtained following PRID or PRID/GnRH treatment. In cows with low progesterone concentration, PRID or PRID/GnRH treatment led to markedly increased ovulation and pregnancy rates with respect to Ovsynch treatment. These findings suggest the importance of establishing ovarian status in early postpartum dairy cows before starting a timed AI protocol, in terms of luteal activity assessed by blood progesterone.

The objective of this study was to determine whether periovulatory treatments with PGF2alpha affects the development of the CL, and whether the treatment was detrimental to the establishment of pregnancy. Reproductively sound mares were assigned randomly to one of the following treatment groups during consecutive estrus cycles: 1. 3,000 IU hCG within 24 hours before artificial insemination and 500 microg cloprostenol (PGF2alpha analogue) on Days 0, 1, and 2 after ovulation (n=8), 2. 2 mL sterile water injection within 24 hours before artificial insemination and 500 microg cloprostenol on Days 0, 1, and 2 after ovulation (n=8); 3. 3,000 IU hCG within 24 hours before artificial insemination and 500 microg cloprostenol on Day 2 after ovulation (n=8); or 4. 3,000 IU hCG within 24 hours before artificial insemination and 2 mL of sterile water on Days 0, 1, and 2 after ovulation (controls; n=8). Blood samples were collected from the jugular vein on Days 0, 1, 2, 5, 8, 11, and 14 after ovulation. Plasma progesterone concentrations were determined by the use of a solid phase 125I radioimmunoassay. All mares were examined for pregnancy by the use of transrectal ultrasonography at 14 days after ovulation. Mares in Group 1 and 2 had lower plasma progesterone concentrations at Day 2 and 5, compared to mares in the control group (P < 0.001). No difference was detected between group 1 and 2. Plasma progesterone concentrations in group 3 were similar to the control group until the day of treatment, but decreased after treatment and were significantly lower than the control group at Day 5 (P < 0.001). Plasma progesterone concentrations increased in all treatment groups after Day 5, and were comparable among all groups at Day 14 after ovulation. Cloprostenol treatment had a significant effect on pregnancy rates (P < 0.01). The pregnancy rate was 12.5% in Group 1, 25% in Group 2, 38% in Group 3, and 62.5% in Group 4. It was concluded that periovulatory treatment with PGF2alpha has a detrimental effect on early luteal function and pregnancy.

The antigonadotrophic action of a prostaglandin F2 alpha analogue, cloprostenol, has been investigated in human granulosa cells obtained from cycles stimulated for in-vitro fertilization and induced to secrete luteal quantities of progesterone by culture in serum-supplemented medium. Cells were exposed to conditions which may mimic those occurring in early pregnancy to establish the roles of human chorionic gonadotrophin (hCG) versus LH and that of cyclic AMP (cAMP) in the anti-gonadotrophic action of cloprostenol. When human granulosa cells were cultured in the absence of treatment for 3 days, exposure to cloprostenol had no effect on basal progesterone production but inhibited hCG-stimulated progesterone (60% decrease; P less than 0.01), hCG-stimulated cAMP (40% decrease; P less than 0.05) and the progesterone response to dibutyryl cAMP (dbcAMP; 70% decrease; P less than 0.01), suggesting pre- and post-cAMP sites of cloprostenol action. The inhibitory actions of cloprostenol were prevented when the granulosa cells were either continuously exposed to treatment from the start of culture or pre-exposed for 3 days to maximum concentrations of LH, hCG, dbcAMP or 8-bromo-cAMP. We conclude that prior exposure either in vivo or in vitro to LH or hCG prevents the subsequent antigonadotrophic action of cloprostenol via a cAMP-dependent mechanism. Prevention of the antigonadotrophic action of cloprostenol after exposure to hCG may be a mechanism through which CG prevents regression of the corpus luteum in early pregnancy, while the suppressive effect of LH pretreatment may account for the refractory response of the early corpus luteum to cloprostenol following the midcycle LH surge.

The mu, delta and kappa opioid receptor subtypes were measured across the oestrous cycle of the ewe and in ovariectomised (OVX) ewes treated with oestrogen and/or progesterone. We have used a subtype-specific opioid receptor binding assay, in which [3H]diprenorphine non-preferentially labelled each receptor subtype in the presence of blocking concentrations of site-specific opioid analogues. The density and affinity of each receptor subtype was measured in the preoptic area (POA) of the hypothalamus and the mediobasal hypothalamus (MBH). Normally cycling ewes were killed during the luteal phase of the oestrous cycle and at various times after an injection of a synthetic prostaglandin (cloprostenol) to synchronise the onset of the follicular phase. OVX ewes were either untreated as controls (n = 4) or treated with oestrogen (n = 4), progesterone (n = 4) or oestrogen and progesterone combined (n = 4). The total number of opioid receptors did not alter across the oestrous cycle or with steroid hormone treatment. In the POA, the mean (+/- S.E.M.) number of delta receptors was significantly (P < 0.05) greater during the luteal phase than 24 h into the follicular phase (133 +/- 45 vs 35 +/- 8 fmol/mg protein). A significantly (P < 0.05) greater number of delta receptors was also found in the OVX progesterone-treated ewes compared with the control animals (172 +/- 9 vs 39 +/- 4 fmol/mg protein). In the MBH, the number of delta receptors was significantly (P < 0.01) greater in ewes killed 56 h after prostaglandin than luteal-phase ewes (184 +/- 40 vs 51 +/- 7 fmol/mg protein). The number of mu receptors in both the POA and the MBH was also significantly (P < 0.05) higher in the 56-h group than in the 12-h group. A similar trend was also observed in the steroid-treated animals, although differences did not reach statistical significance. The delta:mu ratio in the POA was significantly (P < 0.05) higher in the luteal-phase animals than any of the other groups killed after a cloprostenol injection that causes luteolysis. Similarly the ratio of delta receptor density to mu receptor density was greater (P < 0.05) in the OVX progesterone-treated ewes than in the OVX control ewes. No differences were found in the kappa receptor density across the cycle or with different steroid treatments. These data suggest that the relative proportions of the delta and mu subtypes of the opioid receptor in the hypothalamus change during the oestrous cycle. Regulation appears to be due to the feedback effects of ovarian steroids with progesterone altering the delta:mu ratio. In the MBH, there was a general increase in both delta and mu subtypes during the follicular phase of the oestrous cycle. This may explain, in part, how the responsiveness of the GnRH/LH axis to opioid peptides and antagonists changes across the cycle.

We studied the secretory function of the corpus luteum (CL) in cows following different estrus synchronization protocols. Estrus was synchronized using one (n=4) or two injections (n=5) of prostaglandin F(2alpha) (PGF(2alpha); dinoprost), two injections of different analogues of PGF(2alpha) (aPGF(2alpha)), luprostiol (n=5) and cloprostenol (n=5), at eleven-day intervals, a gestagen implant (norgestomet, n=5, for 10 days) or norgestomet together with a subsequent dinoprost injection on the day of implant removal (n=5). CL samples were collected by ovariectomy on Day 7-8 of the estrous cycle. Luteal strips were stimulated with LH (100 ng/ml) or prostaglandin E(2) (PGE(2), 10(-6)M) for 24 h in culture media. The progesterone (P(4)) and PGE(2) concentrations in the media were measured by enzyme immunoassay. In the control CL (spontaneous estrus; n=5), LH and PGE(2) stimulated P(4) and PGE(2) (P<0.001). The effects of both factors on P(4) were reduced in the CL following dinoprost- and cloprostenol-synchronized estrus (P<0.05) and were absent in the luprostiol-synchronized CL (P>0.05). In the norgestomet-synchronized CL, the stimulatory effects of LH and PGE(2) were higher compared with the CL synchronized by aPGF(2alpha) (P<0.05). Pharmacological manipulation of the estrous cycle using aPGF(2alpha) may cause lower P(4) secretion. Estrus synchronization inhibited CL sensitivity to luteotropic factors. Therefore, attention should be focused on the estrous synchronization method in both in vivo and in vitro studies of CL functions in cattle.

The objective was to determine the effects of exogenous prostaglandin F(2α) (PGF), with or without progesterone treatment, on first ovulation in prepubertal heifers. We tested the hypothesis that PGF has a luteolysis-independent ovulatory effect in cattle. Crossbred Angus heifers (12 to 14 mo old, 250 kg body weight, and an average body condition score of 3 out of 5) were examined by transrectal ultrasonography on two occasions, 11 days apart. Heifers in which a CL was not detected at either examination were considered prepubertal. Heifers were assigned randomly to three experimental groups: (1) PG group (N = 14); heifers were treated with a PGF analog (500 μg cloprostenol im) 5 days after the emergence of a spontaneous (i.e., naturally occurring, noninduced) follicular wave; (2) PPG group (N = 12); heifers were given an intravaginal progesterone-releasing insert (CIDR; Pfizer Animal Health, Montreal, QC, Canada), and a follicular wave was induced with 50 mg of progesterone + 2 mg of estradiol benzoate im, and a PGF analog was given at the time of CIDR removal, on day 5 of the follicular wave (on average, 8.6 ± 0.5 days after CIDR insertion); and (3) control group heifers were given no treatment (N = 14). Heifers were examined daily by transrectal ultrasonography from the start of the experiment to confirmation that ovulation had occurred, or to 5 days after PGF injection (PG and PPG groups) or until dominant follicles of the next follicular wave reached 8 mm (control group). The percentage of heifers that ovulated within 10 days after wave emergence was higher in PPG (10/12; 83.3%) and PG (11/14; 78.5%) groups than in control (1/14; 7.1%; P < 0.0001). Ovulations occurred 69.6 ± 6 h and 93.8 ± 5 h after PGF treatment in PPG and in PG groups, respectively, whereas only one heifer in the control group ovulated 96 h after day 5 of follicular wave (P = 0.13). In summary, PGF treatment was associated with ovulation in prepubertal heifers whether or not exogenous progesterone was used as a pretreatment. The hypothesis that PGF will induce ovulation by a luteolysis-independent mechanism was supported.

The objective was to investigate the ovarian response of Brahman heifers to two modified ovulation synchronisation protocols developed to increase the proportion of normal synchronous ovulations. Experiment 1 characterised the growth of the ovulatory follicle in heifers (n=19) treated with an intravaginal progesterone releasing device (IPRD) and oestradiol benzoate (ODB), to determine the optimal time to induce ovulation. Using the findings from Experiment 1, Experiment 2 investigated the effect of reducing the duration of IPRD insertion and increasing the interval from IPRD removal to ODB treatment (modified protocol 1 - OPO-6; n=20), and omitting ODB treatment at the time of IPRD insertion (modified protocol 2 - PO-6; n=20). An IPRD (0.78 g progesterone) was inserted at Day 0 (OPO-8) or Day 2 (OPO-6 and PO-6) and all heifers also received 1 mg ODB i.m. Day 8: IPRD removed + 500 μg cloprostenol i.m. At 24 h (OPO-8) and 36 h (OPO-6 and PO-6) post IPRD removal: 1 mg ODB i.m. Fixed-time AI (FTAI) occurred at 54 h for OPO-8 and 72 h for OPO-6 and PO-6, post IPRD removal. After IPRD treatment all OPO-6 and OPO-8 heifers initiated a new follicular wave whereas 25% of PO-6 heifers failed. Diameter of the dominant follicle was larger at FTAI in the PO-6 (11.34 ± 0.50 mm) compared to the OPO-8 protocol (9.74 ± 0.51 mm; P<0.05), but similar to the OPO-6 protocol (10.52 ± 0.51 mm). Proportion of ovulations occurring 12 h prior and 24 h post FTAI was similar for the PO-6 (80%) and OPO-6 (75%) protocols but numerically lower in the OPO-8 heifers (60%). The apparent improvement in ovarian response in heifers treated with the modified protocols needs to be confirmed in larger field studies.

Ovarian follicular dynamics and steroid secretion patterns were monitored in postpartum beef cows that were synchronized for estrus with melengestrol acetate (MGA) or prostaglandin F(2alpha) (PGF) prior to superovulation. Twenty-four muhiparous Angus cows were stratified by number of days postpartum to an MGA or PGF treatment prior to superovulation. Cows in the MGA group were fed 0.5 mg MGA/d for 14 d in a grain carrier. Superstitnulatory treatments began 14 d after withdrawal of MGA from feed or 11 d after administering a single injection of 500 microg <em>cloprostenol</em> (PGF). Supersthnulatory treatments (FSH) were administered twice daily in decreasing doses (7.5, 5, 5, 2.5 mg) over 4 d. Sixty and 72 h after initiating the superstimulatory treatments, all cows were treated with 750 microg and 500 microg PGF, respectively Cows were inseminated at 0, 12, and 24 h from the onset of standing estrus with semen from 2 proven sires. Cows within treatment were inseminated with 1, 2 and 1 (single) or 2, 4 and 2 units (double) of semen at the designated insemination times. Blood sampling and transrectal ultrasonography of ovaries were performed daily beginning 2 d prior to the initiation of FSH treatment and were continued through embryo recovery. Ovaries were examined daily to determine the number and size of follicles. Plasma samples were analyzed for progesterone and estradiol. Follicles were counted and categorized based on a 5 to 9 mm range or>>/= 10 mm. At the end of superovulatory treatment there were more (P </= 0.01) follicles>>/= 10 mm among cows that were estrus synchronized with MGA (75 +/- 1.2) than with PGF (3.9 +/- 1.2) These differences were reflected in higher (P </= 0.05) subsequent concentrations of plasma progesterone, suggestive of differences in ovulation rate between treatments (MGA = 8.5 ng/ml; PGF = 5.6 ng/ml). There were no significant differences between treatments in concentrations of estradiol, total number of ova (MGA = 11; PGF = 9), fertilized embryos (MGA = 9; PGF = 7), or transferable embryos recovered (MGA = 8; PGF = 6) Furthermore, double insemination offered no significant improvement in the number of recovered fertilized ova. These data identify differences in follicular and endocrine response when cows were estrus synchronized with MGA versus PGF prior to superovulation.

The objective of Experiment 1 was to compare the effects of estradiol benzoate (EB) given 0 or 24h after the end of a progestagen treatment on ovulation and CL formation in anestrous cows. Twenty cows were treated with an intravaginal sponge containing 250 mg of medroxiprogesterone acetate (MPA). At sponge insertion, each cow received 3 mg EB and 10 mg MPA im. At device removal, cows received 0.7 mg EB either at that time (EB0) or 24h later (EB24). Ultrasound examinations and blood sampling to determine plasma progesterone concentrations were performed to detect ovulation and CL formation. Ovulation occurred in 77.8 and 81.8% cows in the EB0 and EB24 groups, respectively. Diameter of the ovulatory follicle (EB0 = 10.9 +/- 0.5mm; EB24 = 12.1 +/- 0.8 mm; P = 0.26) and the interval from sponge removal to ovulation (median = 3 days; P = 0.64) did not differ between treatments. Among the cows that ovulated (n = 16), short-lived CL were present in 2/7 and 2/9 cows in the EB0 and EB24 groups, respectively. Plasma progesterone concentrations and CL area did not differ between treatments (P>> 0.05). In Experiment 2, cows were treated with the same protocol as in Experiment 1, but at sponge withdrawal all cows received 250 microg cloprostenol and timed artificial insemination (TAI) was performed 48 h after sponge removal. In Replicate 1 (n = 204 multiparous cows), pregnancy rates were 45.0 and 47.5% for EB0 and EB24, respectively (P>> 0.05). In Replicate 2 (n = 69 primiparous cows) pregnancy rate did not differ between EB0 and EB24 (51.4% versus 52.9%). In conclusion, EB given 0 or 24h after the end of a progestagen treatment had the same effect on ovulation rate, time to ovulation, diameter of the ovulatory follicle, incidence of short-lived CL, luteal tissue area, and plasma progesterone concentrations of normal lifespan CL, and pregnancy rate after TAI in suckled beef cows.

The objective of the experiment was to compare the use of a PGF2α analogue (Cloprostenol) IM, with an intravaginal progestagen sponge, flurogestone acetate (FGA), and equine chorionic gonadotropin (eCG) IM application protocol. A total of 30 cyclical hair ewes (54.07 ± 0.5 kg live weight, body condition score 3.5 ± 0.5, and age 3 ± 1 years) were used. For the control group ewes (n = 15), intravaginal sponges (IS) impregnated with 20 mg of FGA were inserted for 12 days with 500 IU of eCG IM at sponges withdrawal. For the PG group ewes (Treatment group n = 15), two injections of Cloprostenol (75 mcg) were given 12 days apart. The presence of estrus was detected using two rams with 8 h interval beginning at the end of the treatment. Progesterone concentrations in blood were measured by solid phase radioimmunoassay. A student's t test was performed to analyze the duration of estrus and the interval between the ends of the treatment and the onset of estrus (ET-OE) presentation. Progesterone levels were compared with two-way ANOVA, with treatment, and day of menstrual cycle as fixed factors. Treatment costs ratio was calculated by dividing the total costs of FGA IS application between total costs of Cloprostenol application. Significant differences (P < 0.05) were found in the (ET-OE) interval and estrus duration. For the control group, estrus was presented at 30 + 8.2 h; in treatment group, at 44 h after the last application, duration of estrus was 54.9 + 8.34 h, and 41 + 1.83 h for the control and treatment group, pregnancy rates were 53.3 and 60.0 %, respectively. Significant differences (P < 0.001) were found from days 9 to 13 on Progesterone levels in both treatments. Treatment costs of Cloprostenol protocol were 2.63 cheaper than FGA including disposable material, biological products, and labor. It was concluded that Cloprostenol could be an effective tool in estrus synchronization in hair sheep in tropical areas.

Mares (n = 37) were treated from 4h after breeding through 2 days post-ovulation with oxytocin or cloprostenol. Oxytocin (20 units i.m.) was administered every 6 h and cloprostenol (250 mcg i.m.) daily. Luteal function was impaired for several days following treatment, however, lower progesterone levels among cloprostenol treated mares in this study did not result in decreased pregnancies. Pregnancy outcome at 15 days post-ovulation was not different between the oxytocin (13/18) and cloprostenol (13/19) treatment groups, respectively (P = 0.80). The results of this study indicate cloprostenol can be used to treat post-breeding mares through the second day following ovulation without decreasing pregnancy outcome.

The insulin-like growth factors, IGF-I and -II, have been shown to play a key role in luteal function in some species. The IGF binding proteins, IGFBP-2 and -3, have been shown to inhibit binding of IGF-I and -II to bovine luteal cells and decrease progesterone production. We have recently shown that equine follicles have the genetic capacity to produce IGFBP-2, and that levels decrease in healthy preovulatory follicles. In the present study expression of mRNAs encoding IGFBP-2, as well as the rate-limiting steroidogenic enzyme, P450scc, were studied in equine corpora lutea to investigate whether IGFBP-2 might be involved in luteolysis. Corpora lutea were collected from mares in mid-luteal phase (day 10), at early regression (day 14), late regression (day 17), and 12 and 36 h after intramuscular administration of the PGF(2alpha) analogue, cloprostenol (0.5 microg/kg). During early natural regression, and 12 h after administration of cloprostenol on day 10, steady state levels of mRNAs encoding P450scc had decreased significantly compared with day 10 of dioestrus (P < 0.001). Levels of mRNA encoding IGFBP-2 increased significantly between mid-diestrus and early (P < 0.01) and late (P < 0.001) regression, and 36 h after cloprostenol administration (P < 0.001). We conclude that the genetic capacity for increased IGFBP-2 production in the early stages of natural luteolysis in the mare may act to sequester IGF-I in the CL, assisting in inhibition of progesterone production. However the delay in increase in mRNA encoding IGFBP-2 after cloprostenol administration, combined with the sharp fall in expression of P450scc mRNA, suggests that the luteolytic action of a pharmacological dose of cloprostenol may not be mediated via IGFBP-2 in the mare.

The aim of this study was to determine whether endogenous progesterone regulates synthesis and secretion of luteal oxytocin. In Expt 1, mature ewes (n = 5 per group) were assigned randomly to control or mifepristone (RU486) treatment groups. Ewes were injected s.c. twice a day with vehicle or 10 mg RU486 on days 5-7 of the oestrous cycle (oestrus = day 0). On day 8, after an i.v. injection with prostaglandin F(2alpha) (250 microg cloprostenol), venous blood samples were collected at frequent intervals to determine plasma oxytocin concentrations. Plasma oxytocin concentrations of RU486-treated ewes were not significantly different from those of control ewes. In Expt 2, ewes were injected s.c. each day with vehicle or 175 mg RU486 on days 2-5 of the oestrous cycle followed by administration of prostaglandin F(2alpha) on day 6. Four of five RU486-treated ewes showed 'split-oestrus' (oestrous behaviour for 36 h and then again at 84-108 h after the onset of initial oestrus). There was no significant difference in mean plasma oxytocin or progesterone concentrations between treatment groups. The mean masses of mature corpora lutea from control and RU486-treated ewes on day 6 of the oestrous cycle did not differ significantly (394.8 +/- 28.8 versus 319.5 +/- 48.3 mg). RU486-treated ewes contained mature corpora lutea, new corpora lutea (two of four ewes) and preovulatory follicles >>or= 10 mm, two of four ewes). The average interoestrous interval for RU486-treated ewes was 9 days more than that for control animals (26.2 +/- 2.9 versus 17 +/- 0.5 days; P < 0.025).

Human granulosa cells, from women undergoing ovum collection for in-vitro fertilization (IVF), will luteinize in vitro and provide a model for investigating the antigonadotrophic action of a prostaglandin F2 alpha (PGF2 alpha) analogue, cloprostenol, on granulosa-derived luteal cells. The granulosa cells were cultured in a defined medium and exposed to treatments during a preincubation period of 0 to 3 days and a final incubation with low density lipoprotein (LDL) from days 3 to 4. In the absence of human chorionic gonadotrophin (HCG), progesterone production was low, whereas exposure to HCG in the final incubation resulted in a 10-fold increase in progesterone concentrations. The inclusion of cloprostenol with HCG in the final incubation significantly (P less than 0.05) inhibited HCG-stimulated progesterone production. Exposure to HCG during the preincubation prevented the antigonadotrophic action of cloprostenol in the final incubation. The antigonadotrophic action of cloprostenol was retained when the granulosa cells were exposed to cloprostenol during the preincubation. Omission of LDL from the final incubation lowered the production of progesterone but the pattern of responses to HCG and cloprostenol were similar. Prevention of the antigonadotrophic action of cloprostenol after exposure to HCG may be a mechanism through which chorionic gonadotrophin can prevent regression of the corpus luteum in early pregnancy. Cloprostenol does not appear to inhibit LDL-stimulated steroidogenesis in human granulosa cells.

The endocrine and ovarian responses to prolonged adrenal stimulation at the time of corpus luteum (CL) regression were studied in non-lactating non-pregnant Friesian cows. Cows were synchronized with two cloprostenol (PG) injections 11 days apart (second PG referred as time 0). Experiment 1 was carried out on five animals in two phases with a resting period in between. Between -48 and 84 h, animals received 12 injections of either saline (CTR) or adrenocorticotrophic hormone (ACTH) agonist (Synacthen; SYN) every 12 h. Cortisol (C), progesterone (P4), oestradiol (E2) and LH were analysed in the blood samples collected every 8-12 h between days -3 and 4. Pulsatile LH release was studied 4 h before and 4 h after naloxone administration beginning at 96 h. Experiment 2 was carried out on four cows in a cross-over experimental design (two phases, with a resting period in between). Treatments were performed by administering either saline (CTR) or Synacthen (SYN) every 12 h between -36 and 24 h. The concentrations of C, P4 and E2 were measured in blood plasma every 4-12 h from days -3 to 3, then every day from days 5 to 9. In both experiments, ovaries were examined by ultrasonography every 1-3 days. ACTH administration induced a significant increase (p < 0.001) of plasma C lasting for 7 days (experiment 1), and for 3-4 days (experiment 2). Plasma C returned to baseline levels within 6 days (expt 1) or 36 h (expt 2) after treatment interruption. During the SYN phase, LH pre-ovulatory surge was not detectable. During the CTR phase, naloxone administration induced a significant increase (p < 0.05) of average LH concentrations that was not evident during the SYN phase. The dominant follicle development was retarded and mean plasma E2 concentrations were significantly lower during the SYN phase (p < 0.01). Luteolysis was completed within 2 days. However, P4 decline between 0 and 4 h was slower (p < 0.01) during the SYN phase. Our results indicate that, under prolonged adrenal stimulation, follicular development is delayed and LH release is impaired, which are independent of CL function.

The objective of this study was to assess the effect of ovine follicular fluid (FF) treatment (with or without FSH replacement) during the late follicular phase on plasma concentrations of gonadotrophins and the development of the ovulatory follicle. Ovarian steroid secretion and expression of mRNA encoding inhibin alpha and beta A, beta B subunits, P450 aromatase and P450 17 alpha-hydroxylase were used as endpoints. After induction of luteolysis by injection of 100 micrograms cloprostenol on days 10-12, Scottish Blackface ewes were allocated to one of three groups: (1) control (n = 7): no further treatment; (2) FF (n = 9): subcutaneous injections of 3 ml steroid-free ovine follicular fluid at 9 h intervals, 18 and 27 h after cloprostenol injection; (3) FF + FSH (n = 8): injections of follicular fluid as above plus subcutaneous injections of 0.36 iu ovine FSH at 6 h intervals, 18, 24, and 30 h after cloprostenol injection. Jugular venous blood samples were obtained via indwelling cannulae at 6 h intervals from 0 to 36 h after cloprostenol injection, and at 10 min intervals from 12 to 18 h (control phase) and from 30 to 36 h after cloprostenol injection (treatment phase). At laparotomy, 36 h after cloprostenol injection, ovarian venous blood was collected and ovaries were removed and processed for in situ hybridization. Plasma concentrations of FSH, luteinizing hormone (LH) and oestradiol were determined by radioimmunoassay. Follicular fluid treatment resulted in a decrease (P < 0.001) in FSH concentrations associated with an acute decrease in ovarian steroid secretion (P < 0.01) and a specific depression in P450 aromatase, (P < 0.001), inhibin-activin beta B subunit (P < 0.05) and thecal LH receptor (P < 0.001) expression. Follicular fluid treatment had no effect on inhibin-activin alpha and beta A, subunit or P450 17 alpha-hydroxylase expression. FSH co-treatment with follicular fluid restored circulating FSH concentrations to normal values and reversed some of the effects of follicular fluid (androstenedione, testosterone and progesterone secretion, and inhibin beta B and thecal LH receptor expression) but not oestradiol secretion or P450 aromatase expression. It was concluded that the actions of follicular fluid are mediated via both central effects on pituitary FSH secretion and by direct ovarian effects on granulosa cell aromatase activity. The results indicate that follicular fluid contains a factor that inhibits aromatase activity of granulosa cells directly and may play a role in the selection of the dominant follicle.

This experiment was undertaken in order to investigate the production of inhibin, oestradiol and androstenedione by ovarian follicles at different stages of the oestrous cycle in sheep. Twenty-four Scottish Blackface ewes were allocated to four groups of six ewes, i.e. those operated on during the luteal phase (day 10), and those operated on during the follicular phase 24-30, 36 and 60 h after the induction of luteal regression by an injection of 125 micrograms cloprostenol on day 10 of the luteal phase. Samples of jugular and ovarian venous blood were collected under anaesthesia and ovaries were then removed and all follicles larger than 3 mm diameter dissected out and incubated in medium for 2 h. After injection of cloprostenol, luteal regression occurred as indicated by a fall in the secretion rate of progesterone. The ovarian secretion rate of inhibin was similar at all stages of the follicular phase and during the luteal phase while, in contrast, the secretion rate of oestradiol was significantly (P less than 0.05) elevated in the group 24 h after injection of cloprostenol. There was good correlation between the in-vivo ovarian secretion rate and production rate during incubation in vitro for both inhibin (r = 0.57) and oestradiol (r = 0.60). When follicle diameter was compared with in-vitro hormone production there was good correlation for inhibin (r = 0.72) with larger follicles producing more inhibin, while the value for oestradiol was somewhat lower (r = 0.57) owing to the presence of large atretic follicles with low oestradiol production. Androstenedione production showed a lower correlation with follicle diameter (r = 0.39). When the four time periods were compared separately, there were significantly (P less than 0.05) more follicles with high in-vitro oestradiol production (greater than 90 fmol/min) in the group at 36 h than in the other three groups, while inhibin release in relation to follicle size was similar in the four groups. Large oestrogenic follicles were responsible for 90% of the total oestradiol production during culture while only providing 55% of the total inhibin production, with large non-oestrogenic and small follicles contributing 33% and 12% of inhibin production respectively. From the results of this study we conclude that while oestradiol is mainly produced by the large oestrogenic follicles, a considerable amount of inhibin is also produced by large non-oestrogenic and small follicles.(ABSTRACT TRUNCATED AT 400 WORDS)